American Journal of Anatomy 15 (1913-14)

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THE AMERICAN JOURNAL OF ANATOMY

Editorial Board
Charles R. Bardeen

University of Wisconsin

Henry H. Donaldson

The Wlstar Institute

Simon H. Gage

Cornell University

G. Carl Huber

University of Michigan

J. Playfair McMurrich

University of Toronto

George S. Huntington

Columbia University

Franklin P, Mall

Johns Hopkins University

Henry McE. Knower, Secretary

University of Cincinnati

Charles S. Minot

Harvard University

George A. Piersol

University of Pennsylvania

VOLUME 15 1913-1914

THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA, PA.

THE WAVERLY PRESS BALTIMORE. U. S. A.

Contents

1913-1914

No. 1. JULY, 1913

Jackson CM. Postnatal growth and variability of the body and of the various organs in the albino rat. Amer. J Anat. 15(1): 1-68.

C. M. Jackson. Postnatal growth and variability of the body and of the various organs in the albino rat. Seven figures 1

.1. A. Badertschkr. Muscle degeneration and its relation to the origin of eosinophile leucocytes in amphibia (Salamandra atra). Seven figures 69

Shinkishi Hatai. On the weights of the abdominal and the thoracic vicera, the sex glands, ductless glands and the eyeballs of the albino rat (Mus nor vegicus albinus) according to body weight. Twelve charts 87

y^ Edward F. AIaloxk. The nucleus cardiacus nervi vagi and the three distinct types of nerve cells which innervate the three different types of muscle. Three figures 121

No. 2. SEPTEMBER

Adam M. Miller. Histogenesis and morphogenesis of the thoracic duct in the chick ; development of blood cells and their passage to the blood stream via the thoracic duct. Twenty-eight figux'es (seventeen plates) 131

William H. F. Addison and Harold W. How\ On the prenatal and neonatal lung. Eight figures 199

Chester H. Heuser. The development of the cerebral ventricles in the pig. Twenty-six figures 215

No. 3. NOVEMBER

Charles R. Stockard. An experimental study of the position of the optic anlage in Amblystoma punctatum, with a discussion of certain eye defects. Nine figures 253

W. B. Kirkham and H. S. Burr. The breeding habits, maturation of eggs and ovulation of the albino rat. Eighteen figures 291

Wallin IE. A human embryo of thirteen somites. (1913) Amer. J Anat. 15(3): 319-331.

Ivan E. Wallin. A human embryo of thirteen somites. Seven figures 319

Walter E. Dandy. The nerve supply to the pituitary body. Three figures 333

Template:Ref-Kingsbury1913

B. F. Kingsbury. The morphogenesis of the mammalian ovary : Felis domestica. Thirty-two figures 345

No. 4. JANUARY, 1914

E. V. CowDRY. The development of the cytoplasmic constituents of the nerve cells of the chick. Five plates 389

J. F. GuDERNATSCH. Feeding experiments on tadpoles. II. A further contribution to the knowledge of organs with internal secretion. Two double plates 431

Charles H. Swift. Origin and early history of the primordial germ-cells in the chick. Fifteen figures 483


Postnatal Growth And Variability Of The Body And Of The Various Organs In The Albino Rat

C. M. Jackson

The Anatomical Laboratory of the University of Missouri

SEVEN FIGURES

CONTENTS

Introduction 2

Material and methods 3

Growth and variability of the body 8

1. Growth in body weight 8

2. Variability in body weight 11

3. Fraternal variability 16

Growth and variability of the individual organs 20

1 . Head 20

2. Central nervous system 21

3. Eyeballs -23

4. Thyroid gland 25

5. Thymus 26

6. Heart 27

7. Lungs J 31

8. Liver , 33

9. Spleen 34

10. Stomach and intestines 35

11. Suprarenal glands 36

12. Kidneys 38

13. Gonads 39

a. Testes 39

b. Ovaries 40

14. General considerations 41

Summary 48

Bibliography 50

Tables 53


THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 1 JULY, 1913


C. M. JACKSON


INTRODUCTION


The present paper includes statistical data upon certain phases of the growth of the body as a whole and more especially the relative growth and variability of the individual organs in the albino rat. A study of this kind may be of value in two respects. In the first place, it should give a better insight into the nature of growth, a fundamental biological phenomenon worthy of more study for its own sake. In the second place, a more complete knowledge of the process of growth, including the limits of normal variation, should be of value for reference in experunental work of various kinds, for which the rat is often used.

For both these purposes there is great need of a series of complete growth norms, comparable to KeibeFs morphological 'Normentafeln.' A complete growth norm for any given species would involve adequate data upon the prenatal and postnatal growth of the body as a whole, and of its component parts, organs, tissues and cells. It would include a determination of the extent of variability due to intrinsic or hereditary factors and to extrinsic or environmental factors. It is evident that a single fixed or absolute growth norm for a given species does not exist. We can, however, discover the norm and Imiits of variation within a group of animals homogeneous in constitution and in an environment as constant as possible. Furthermore, it is possible to determine to what extent the norm and variability are changed by varying the different factors involved.

Such a complete growth norm has not as yet been even approximately determined for any species, although numerous observations have been made upon different phases of growth in various animals. For the rat, extensive data on the growth of the body and of the central nervous system are available in a series of papers by Donaldson and Hatai. The relative growth of the principal parts and systems of the rat has recently been studied by Jackson and Lowrey ('12); and formulas for the growth of the individual viscera have been determined by Hatai ('13) and are published in the present number of this journal. Further data upon the growth and especially the variability of the body and of the various organs are included in the present paper.


MATERIAL AND METHODS

The following data are utilized in the present paper. For the weight of the whole body of the albino rat (Mus norvegicus albinus) at various ages a series of 570 original observations is given in table 1. For the individual organs, 344 animals were killed and dissected at various ages, and the weights of the organs were observed (tables 3 to 15).

Of data available for comparison, there should be mentioned first the extensive series of observations by Donaldson on the growth of the whole body ('06) and of the brain and spinal cord ('08) of the albino rat. I am furthermore indebted to Professor Donaldson and Dr. Hatai, of The Wistar Institute of Anatomy, for unpublished observations upon the weight of the principal viscera in about 200 albino rats. With the exception of the central nervous system, however, these data are not included with my own, but were utilized merely for comparison. . The albino rats used by me (table 1) were reared from stock obtained partly from M. Cattell, Garrison-on-Hudson, New York, and partly from B. F. McCurdy, a dealer in Chicago. The litters from each source were kept separate, but no constant difference in growth and variability was noticeable between them. The 68 litters included in table 1 are for the most part not closely related (although in a few cases observations upon the body weight of the same litter are repeated at successive ages).

The albino rats were kept in stationary cages in an animal house and were well cared for. A supply of chopped corn was kept constantly in the cages. A liberal amount of wheat bread soaked in whole milk was supplied daily, and fresh meat (beef) once a week. Water in abundance was provided.

From the age of about six weeks onward, the cexes of each litter were kept separate, and (with the exception of a part of the females at one year) therefore represent unmated animals. Watson ('05) has shown that weights of the body and of the central nervous system are somewhat increased in the female through bearing young. It is probable that other organs, also, may be thereby more or less affected (especially the reproductive system), although data are wanting to determine this point. The external characters whereby the sexes may be distinguished in young rats have been described by Jackson ('12).

In general, the albino rat remains healthy and thrives in captivity. The chief exception noted is the frequent tendency to lung disease, especially in the older rats and more rarely in the younger. Two forms of disease were observed. The first is an acute pneumonitis, which is usually fatal. This form appeared but once in a period of three years. It occurred as an epidemic which in a short time destroyed nearly the entire colony. This epidemic was caused by a specific bacterium, bacillus muris, as determined by Mitchell ('12). The second form of lung disease noted is a chronic disturbance which is common in the wild Norway rat at Columbia as well as in the domesticated albino. It appears to develop first in the form of multiple small clear spots scattered over the surface of the lung. Later these spots may form small abscesses, or lead to the consolidation of one or more lobes. An associated catarrhal condition of the respiratory mucosa causes an audible snuffling or wheezing, through which the disease may usually be recognized when well developed. It usually is not fatal, or at least not immediately so, and in some cases does not appreciably affect the growth or the general state of nutrition. Generally, however, it tends to produce more or less emaciation. This is probably the same disease as the pneumonia mentioned by King ('11) and by Currie ('10). Mitchell ('12) failed in repeated attempts to find in this disease the specific bacterium causing the first form, and its etiology appears uncertain.

Animals affected with lung disease were excluded from the present data except in some cases in which the lesions were slight and it appeared necessary to exclude only the lungs. This was the case in 3 of the 43 albino rats dissected at the age of ten weeks, 11 of 41 at five months, and 20 of 25 at one year. Even though the lesions were slight, however, it is probable that the average body weight at five months, and especially at one year, is somewhat below the normal. »

Since the number of observations was somewhat limited, it was thought better, especially for the study of variation with age, to restrict them to certain definite ages, rather than to scatter them over the entire period. Seven ages were chosen for this purpose, namely, newborn (one day or less), seven days, twenty days, six weeks, ten weeks, five months, and one year. These ages were selected for the following reasons. At seven days, the body weight at birth has about doubled. At twenty days, the weight has approximately doubled again, and at this age the albino is usually weaned. At six weeks, the body weight has somewhat more than doubled again, and the animal is well established upon the new diet. At ten weeks, the rat has again about doubled its weight, and sexual maturity is reached. At one year, the weight has again approximately doubled, and at this age, according to the observations of Slonaker ('12), the albino rat (in stationary cages) has nearly reached its maximum body weight. Five months was arbitrarily selected as a point intermediate between ten weeks and one year.

A few observations upon the body weight at fourteen days and at thirty days are also recorded -in table 1. The continuous growth curves for the absolute weight of the various organs, in terms of body weight, are given by Hatai ('13). It is hoped that the age periods selected for the present paper are sufficiently close together so that no material change in variability, correlation, and so forth, will be overlooked.

In some cases, as previously mentioned, only the gross body weight was observed. This weight was always taken in the forenoon, before feeding. In the cases where the animal was to be dissected, it was killed by chloroform, and the gross body weight, and lengths of trunk and tail recorded. The head was then removed on a plane just posterior to the cranium and anterior to the larynx, and was weighed; while the trunk was suspended by the tail, allowing the blood (unmeasured) to escape. The eyeballs and in some cases the brain were then removed and placed in a closed jar upon moist filter paper. Next the trunk was dissected and the following organs successively removed: thyroid gland; thymus (dissected out of surrounding fat); heart (cavities opened and blood-clots removed) ; lungs (right and left separately); liver; spleen; stomach and intestines, including contents, mesentery and pancreas; same, without contents; suprarenal glands ; kidneys ; ovaries or testes (including epididymis) ; spinal cord (in a few cases). The extremities, skin, skeleton and musculature were also weighed in some cases, as described in a separate paper by Jackson and Lowrey ('12).

The organs were weighed in closed containers, and loss by evaporation was avoided so far as possible. The organs were usually weighed to 0.1 mgm. (0.0001 gm.) excepting some of the larger organs in the older rats, which were weighed to 1 mgm. The observations were recorded on printed cards, and any unusual conditions carefully noted. The calculations were made by the aid of a Burroughs adding machine, Crelle's Rechentafeln, and the tables in Davenport's 'Statistical Methods.' The calculations were carefully checked independently to eliminate errors.

I am greatly indebted to Dr. S. Hatai of The Wistar Institute, for valuable aid and criticism, especially on the mathematical phases of the work.

In calculating the various .statistical constants — mean, standard deviation, coefficients of variation and of correlation and probable errors — the usual formulas (Pearson's) were employed as given by Davenport ('04). The ungrouped data were used in all cases.

Since the present paper is concerned largely with variability as measured by the coefficient of variation, a brief discussion of this coefficient may be desii'able. The coefficient of variation is one hundred times the ratio which the standard deviation bears to the mean; or the percentage of the mean which the standard deviation forms, as expressed by the formula:

C = ^100(%) For the standard deviation, the formula is as follows:

(X =

n


POSTNATAL GROWTH IN THE ALBINO EAT 7

where d represents the deviation from the mean, and n the number of variates. The standard deviation is therefore a concrete number which serves to measure the dispersion from the mean. If the variates tend to diverge greatly from the mean, the standard deviation will of course be large; while if they are concentrated closely around the mean, the standard deviation will be small. It is further clear that the standard deviation is not dependent upon the number of cases, but merely upon the manner of their distribution.

Since the standard deviation is a concrete number, however, it will in general vary with the absolute size of the variate. It is therefore often difficult to judge the relative variability of different objects by comparing their standard deviations, and where different units of measurement are used the comparison is meaningless. On this account the coefficient of variation is preferable for measuring the relative magnitude of variations, since it is independent of the size or character of the unit of measurement.

It is of course obvious that from the statistical point of view the number of observations is small and the results cannot be considered final. Even although the probable errors are relatively large, however, it will be found that certain conclusions of importance may be drawn with a fair degree of certainty; and others, more or less strongly indicated, may point the way to further investigation with more adequate data.

To economize space, the individual data are not included in the tables, but only the averages and ranges are given. The cards containing the original individual data are, however, deposited in The Wistar Institute of Anatomy in Philadelphia whence they may be obtained if desired by anyone interested.

GROWTH AND VARIABILITY OF THE BODY

1. Growth in body weight

The data presented in this paper are inadequate for a complete discussion of the growth of the body as a whole. Certain phases, however, may be noted. Donaldson ('06) has made numerous observations upon the postnatal growth of the albino rat, on the basis of which he has drawn certain conclusions, with which the present data may be compared. Table A shows the average

TABLE A Average gross body weight of albino rat at various ages, sexes separated^


Jackson's larger

SERIES


Jackson's smaller

SERIES


DONALDSON S SERIES

(1906)


newborn <

7 days

3 weeks

6 weeks

10 weeks

5 months

1 year


(63 m.

(66 f. (36 m. (64 f. (53 m. (59 f. (45 m. (50 f . (23 m, (25 f.


5.13

4.89

10.53

10.29

23.99

21.50

63.72

64.25

130.4

108.9


(44 m. (43 f. (30 m. (27 f. (24 m. (25 f. (22 m. (20 f. (20 m. (23 f. (20 m. (21 f. ( 5 m. (20 f.


5.06 4.82 10.61 10.48 22.20 17.01 52.89 54.86 121.9 103.3 167.5 142.1 213.0 163.7


(40 m.

(17 f. (11m. ( 8f. (19 m. (17 f. (19 m. (17 f. (19 m. (11 f. (19 m. (11 f. (6 m. ( 7f.


5.4

5.2

9.2

8.7

21.2

22.6

46.3

47.9

106.6

99.8

225.4

184.6

279.0

226.4


1 Jackson's observations listed under '3 weeks' were nearly all at 20 days, and those of Donaldson under '6 weeks' were at 43 days. The females were unmated, excepting all of Donaldson's series at 1 year, and a part of Jackson's series at 1 year.

gross body weight in each sex at various ages in my larger series (recorded in table 1), in my smaller series (dissected for the organs), and in those observed by Donaldson at corresponding ages.

From table A it is evident that the rats used in the present study averaged somewhat smaller at birth for both sexes than those of Donaldson, but show a more rapid growth during the first week, so that at seven days their average weight is greater. At three weeks the males in Donaldson's series average somewhat smaller than in mine but the females are heavier. At six weeks and ten weeks both sexes average smaller in Donaldson's series, but at five months and one year they are decidedly larger.

In general, therefore, it is evident that Donaldson's rats remained smaller than mine up to the age of six weeks, but surpassed them in the later stages. The probable errors were not calculated for the gross body weights (except at six weeks, smaller series) ; but as will be seen later the variation for the gross weight is not materially different from that of the net body weight, the probable errors of which, in the smaller series, are given in table 2. The differences between Donaldson's results and mine are greater than the probable errors and therefore appear significant. The explanation of these differences will be considered later under variability. 1

The relative growth of the sexes of the albino rat was noted by Donaldson ('06), who found that, beginning about the end of the first week, the female grows more vigorously, overtaking and usually exceeding the male in body weight from the fifteenth to the fifty-fifth day of age. My data (table 1) show that in 16 newborn litters containing both sexes the males averaged greater in every case; at seven days, the males exceed in 11 litters, the females in 5 ; at fourteen days, the males exceed in 3, the females in 1; at twenty days, the males exceed in 10, the females in 3; at thirty days; the males exceed in 2, females in none; at six weeks, the males exceed in 10, the females in 2; at ten weeks, the males exceed in 6, the females in 1 ; at five months the males exceed in 4, females in 1; at one year, the males exceed in 2, the females in 1. According to litters, therefore, the excess of average weight was invariably in favor of the male at birth, and also in the majority of cases at all succeeding ages.

The ratio of the average gross body weight in the male to that in the female at corresponding ages in my data is given in table B.

1 It may be pointed out in this connection that whereas my rats represent for the most part a 'random sample' of the general population at each age, those of Dr. Donaldson were taken while young, and used throughout the period of observation.


According to table B, it is seen that the average body weight of the male was greater at every age noted, except at six weeks, when the female was slightly larger. At seven days, however, the male was but slightly the greater.

The extent of the variation in the ratio of body weights between males and females of the same litter may be noted from data in table 1. Further information of interest is afforded by table


TABLE B


AGE


NUMBER OF EACH SEX


AVERAGE GROSS BODY WEIGHT


AVERAGE GROSS BODY WEIGHT


RATIO OP MALE TO



Males


Females


Male


Female



newborn


63 56 53 45 23 20 5


66 64 59 50 25 21 20


grams

5.13

10.53

23.99

63.72

130.40

167.50

213.00


grams

4.89

10.29

21.50

64.25 108.90

142.10

163.70


1 05


7 days


1 02


20 days

6 weeks


1.12 99


10 weeks

5 months


1.20 1 18


1 year


1 30




TABLE C

Ratio of average male to female gross body weight in individual litters at

successive ages


LITTER AND NUMBER OF EACH SEX


A 32 M 9 M 9 M 6 MIO M 8


M M M M


A 29

A 28


(3 m. 5f.) (5 m. 3f.) (3 m. 3 f.) (5 m. 3 f.) (2 m. 4f.) (3 m. 5f.) (2 m. 4f.) (Im. 4f.) (4 m. 1 f.) (4 m. 1 f.) (2 m. 4f.) (4 m. 2f.)


1.05 1.12


1.02 1.08


1.03 1.061 1.06 1.04 1.05 1.06 0.98


1.16 0.99


14 D.4.YS


20 DAYS


30 DAYS


1.04


1.05



1.08


1.03



0.99


1.00



1.06


1.05 0.863

0.95 1.08 1.04 1.01


1.09 1.11*


1.11

1.222

1.00* 1.04 1.10 1.08 0.90


1 Two males of this litter were killed at 7 days.

In this litter, the ratio at 10 weeks was 1.39. ' One male, a 'dwarf,' died before the age of 30 days.

One female a 'dwarf at these ages.


POSTNATAL GROWTH IN THE ALBINO RAT 11

C showing the ratios in a few cases where the gross body weight was observed in the same litters at successive ages.

It is clear that there is considerable variation among individual litters, and evidently observations upon a much larger number of litters would be necessary to determine the typical growth relations by sexes. The available data seem in general to confirm the conclusion of Donaldson ('06) that during the first two months of postnatal life the growth of the body of the albino rat is more vigorous in the female. In the majority of litters, however, the average body weight of the females does not appear to reach that of the niales, at least at the ages observed.

2. Variability in body weight

It is evident from the foregoing that there is in some respects considerable difference between my data and those of Donaldson on the growth of the body of the albino rat. The question naturally arises as to the significance of this difference, as both series are presumably normal (excepting the possible effect of lung disease in the older rats) . This brings up the general question as to the nature and extent of normal variation in the growth of the body. It is known that this variation is greatly influenced by external factors, such as the quantity and quality of food, temperature and so forth. Slonaker ('12) has recently demonstrated that the growth of the albino rat in bod}^ weight may be greatly retarded by exercise, and that it is also much less with vegetarian than with mixed diet.

These factors, however, were nearly alike in the case of Donaldson's rats and mine, though unavoidable differences in environment may account in part for their difference in growth. But even when the environment is kept constant, there still remains the variability due to intrinsic factors, which probably varies somewhat according to the ancestral strain from which the animals were derived, as well as according to litter, sex and individual. Pearson ('00, p. 473) states that The individual contains in itself, owing to a bathmic (i.e., intrinsic) law of growth, a variability which is quite sensible, being 80 to 90 per cent of the variability of the race." As before mentioned, it is therefore


12 C. M. JACKSON

evident that in general there is no such thing as a single 'normal' course of growth and variation for a given species. We may, however, determine the process under a given set of conditions, and also learn the effect produced by varying the individual factors. It is probable that some variation in the intrinsic growth factors is largely responsible for the differences noted between Donaldson's data and mine. The possibility of unrecognized pathological factors must also be kept in mind.

We may now consider the variability in body weight found in the present series.- The variability is best measured by the coefficient of variation, which expresses the percentage of the mean formed by the standard deviation. For the net body weight (table 2), it is seen that the coefficient of variation, taking both sexes together, increases from 12.3 at birth to 15.6 at seven days and to a maximum of 28.4 at twenty days. Thereafter it decreases, being 21.3 at six weeks, 19.9 at ten weeks, and 19.1 at five months. On account of the small nmnber of observations and probable abnormalities at one year, no calculations were made for the total at this age; but the coefficient of variation in body weight for the 20 females at this age was slightly greater than for those at five months. Even when allowance is made for accidental variations due to the small number of observations (as indicated by the relatively large probable errors), the approximate extent of variation, and the general trend according to age are clearly evident. Hatai ('08) in mature albino rats (over 150 days old) found the coefficient of variation for the gross body weight in 53 males, 25.076 ±2.675 and in 51 females, 12.235 ±0.974.

The difference in variability between the sexes of the rats used in the present study is indicated in table 2. It is seen that the coefficient of variation appears greater in the males at every age, except at twenty days. No great stress can be laid upon

^ The variability in body- length was not calculated. According to Donaldson ('09), however, the body length of the albino rat is less variable than the body weight; but the two are closely correlated, the coefficient of correlation being 0.90. In the human species, the body length (height) is only about one-third as variable as the body weight (measured by the coefficient of variation).


POSTNATAL GROWTH IN THE ALBINO RAT 13

this indication, however, as it must be remembered that the nmnber of observations is comparatively small, and the differences are usually within the limits of probable error. It is noteworthy, however, that Hatai ('08) likewise found for the body weight and for various skull measurements of the albino rat a tendency to greater variability in the male.

At the ages of twenty days and six weeks, the standard deviation and coefficient of variation were also calculated for the corresponding gross body weights. At twenty days, the coefficient of variation was, for the males 28.7 (as compared with 28.4 for the net body), and for the female 18.4 (17.5 for the net) ; at six weeks, for the male 17.7 (17.4 for the net), and for the female 24.6 (24.7 for the net). It is therefore evident that there is no material difference between the coefficients of variation for the gross and for the corresponding net body weights.

For a larger series of 56 males and 36 females newborn, corresponding only in part to those given for net body weight, the coefficient of variation for the gross body weight of the male was 14.0 (13.6 for the net), and of the feniale 9.2 (9.9 for the net). A larger series was also calculated for the gross body weight at twenty days and six weeks, including all those in table 1. In this larger series (which was about twice as large as the smaller) the coefficient of variation at twenty days for the male was 24.4 (28.7 in the smaller series) and for the female, 29.4 (18.4 in the smaller series). At six weeks, the coefficient of variation in the larger series for the male was 20.8 (17.7 in the smaller series) and for the female, 24.2 (24.6 in the smaller series). This would seem to indicate that the great difference in the coefficient of variation between the sexes of the smaller series at twenty days and at six weeks is due to an abnormally small variation among the females at twenty days and among the males at six weeks. This should be remembered in considering the variability of the organs, which correspond to the smaller series and are, as will be shown later, all more or less closely correlated with the body weight. The necessity for caution in drawing conclusions as to variability from a comparatively small number of observations is thus emphasized.


14 CM. JACKSON

The foregoing data indicate clearly that the coefficient of variation in body weight is smaller in the younger rats (newborn and seven days) and larger in the older (twenty days, six weeks, ten weeks and five months). Less conclusive is the evidence that the maximum variation, for the ages observed, occurs at twenty days, and that the variation is in general greater in the male than in the female. The changes in variation cannot be ascribed to a selective death rate, as there were in the litters observed very few deaths before the age of five months.

For the variation in the weight of the human body at different ages, fairly complete data are available for comparison. For the English newborn, Pearson ('99) finds the coefficient of variation for the male to be about 15.7, and for the female 14.2. For Cambridge University students, nineteen to twenty-five years of age, he finds the coefficient for 1030 males to be about 10.8, and for 160 females 11.2. For Oxford students (male) of eighteen to twenty-three years, Schuster ('11) similarly finds the average coefficient of variation in body weight to be 10.8, varying from 10.2 to 11.1 for different years of age.

For the period from birth to the age of sLx years, no data are available. But from six years onward we have the extensive observations of Porter ('95) on St. Louis school children. Porter uses the 'probable deviation' (which equals approximately 0.6745 times the standard deviation) as a measure of dispersion, but for convenience of comparison this has been reduced to the standard deviation for calculating the coefficient of variation in table D.

Porter concludes that variation is correlated with rapidity of growth, a conclusion previously reached by Thoma ('82). Boas ('97, '04) reaches similar conclusions. This relation will not hold good for the human newborn, however; for although the growth rate is then far more rapid than at any subsequent period, the coefficient of variation is slightly less than at the age of puberty. In the rat, as we have seen, the coefficient of variation is smaller in the younger animals although the growth rate is far more rapid then than later.


POSTNATAL GROWTH IN THE ALBINO RAT


15


We may therefore conclude that for the human (British and American) the coefficient of variation for the body weight in the newborn male is about 15.7. At six years, it has diminished to a little below 11, and fluctuates in this neighborhood until the twelfth year. During the acceleration of growth from thirteen to sixteen years, the coefficient of variation increases to 16 or 17, thereafter diminishing rapidly to about 11 in the young adult. For the female newborn the coefficient of variation (14.2) is somewhat below that of the male. From six years onward, the coefficient of variation appears to be correlated with the relative rapidity of body growth, being somewhat greater in the female up to the age of fourteen, after which the male exceeds it.

In comparison with the human species, the coefficient of variation in the body weight of the rat at birth appears somewhat smaller. Although in all subsequent stages (except at the age of puberty) the variation in the human body is smaller than at birth, in the rat it appears constantly greater, and shows no evident correlation with the rate of growth of the body.

TABLE D Body weight of St. Louis school children {from Porter's data)


AGE AT NEAREST BIRTHDAY


NUMBER OF OBSERVATIONS


COEFFICIENT OP VARIATION


REL.VTIVE ANNUAL

INCREASE IN AVERAGE

BODY WEIGHT



Male


Female


Male


Female


Male


Female







■per cent


per cent


6


707


798


10.7


11.3




7


1814


1714


11.6


13.3


9.7


10.0


8


2188


2147


12.2


12.6


9.7


9.9


9


2188


2055


11.9


13.2


9.6


9.6


10


2064


1947


11.7


12.4


8.7


9.6


11


1743


1708


12.4


14.2


9.5


9.7


12


1644


1676


10.8


14.5


8.1


11.6


13


1242


1343


15.7


16.3


9.3


14.3


14


946


1082


16.7


16.3


10.5


9.9


15


498


690


16.3 12.9


14.3


10.4


16


203


420


17.8 12.4


11.6


7.6


17


71


230


11.7 10.4


7.9


4.7


18



155


10. .2




19



81


' 10.7




20



68


10.4




16


C. M. JACKSON


3. Fraternal variability

It is a matter of common knowledge that yariation within fraternities is always less than that of the general population. In the various litters of rats used for the present study, it is in most cases evident from mere inspection that variation in the body weights within the litter is much less than the variation in the total population of the same age. To measure the variation within the litter, three methods were used and the results are summarized in table E.

TABLE E

Coefficient of variation in body weight for total population by ordinary method, and on litter basis {fraternal variation) estimated by various methods


Total population (ordinary method)

Litter basis (average of lit-1 ters, calculated by ordinary > method J

Litter basis (calculated from Yule's formula)

Litter basis (from Kellogg's / formula) \



NEW

7


20


6


10



BORN


DATS


DAYS


WEEKS


WEEKS


male


13.61


16.91


24.42


20.82


18.81


female


9.9'


13.71


29.42


24.22


16.81


male


7.0


6.1


5.7


6.6


5.8


female


4.4


5.4


4.0


5.9


12.0


male


6.8


7.6


6.8


7.1


6.1


female


5.2


4.4


4.5


7.9


12.2


male


7.3


8.4


6.0


7.2


6.7


female


5.2


4.5


4.1


8.5


12.0


18. 51 15.31

7.4 10.4

8.1 9.3 8.5 9.0


1 For net body weight.

2 For gross body weight, larger series.

The first method of determining fraternal variation consists simply in calculating the standard deviation and coefficient of variation for each separate litter by the usual formula. The sexes were calculated separately, only those litters with four or more of either sex being used. The average coefficient for each sex at each age is given in the preceding table. On account of the small number of individuals in each litter, the results can of course be considered only as grossly approximate.

The second method of calculating the fraternal variability is based upon a formula by Yule ('11) who demonstrates (p. 142) that if a series 'of (N) observations consists of r component series with standard deviations (Xi, a^, ■ ■ ■ • o"r, and means diverging from the general mean of the whole series by di,


POSTNATAL GROWTH IN THE ALBINO RAT 17

di, . . . . dr, the standard deviation a of the whole series is given (using m to denote any subscript) by the equation: N.a'- = Z (.Vn,. (fj) + 2 (iVn,. rfn,2)." If We let dm represent the mean standard deviation of the component series (which may be assumed to be fairly constant) then

3 (iV„ a J)

becomes

and

N<7'-i:(Nr.dj)


(Tm'- =


N


whence the value of am is obtained.^ In applying this method to the rat data A^ represents the total number of rats at a given age; o-, the standard deviation of the body weight of all the rats; dm, the deviation of any litter mean from the total mean, Nm, the number of individuals in the corresponding litter;' and cm, the average standard deviation within a litter. The sexes are considered separately. The results, as shown by the preceding table, do not differ materially from those by the first and third methods. In the third method^ of determining fraternal variation, all the litters at each age (sexes separately) are reduced to a common litter basis. This is accomplished by multiplying the body weight of each member of a given litter by the factor

Mean of total population (at given age) Mean of given litter

^ Since the formula may be written in the form: crm=a- .

and since ^ "^ — is necessarily a positive quantity, it is evident that according to this formula the standard deviation of the individual fraternity is usually (and on the average necessarily) less than that of the total population. The formula gives no indication as to the magnitude of the difference, however.

For this method, and for aid otherwise, I am indebted to my colleague Prof. O. D. Kellogg, of the Department of Mathematics. He adds the caution that under some circumstances the method might tend to give a spurious reduction in the standard deviation and coefficient of variation, but this does not appear to be the case with the present data.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 1


18 C. M. JACKSON

Since the coefficient of variation for each Utter and also the mean of the total population remains unchanged by this procedure, the products may be considered as forming a single large litter, whose coefficient of variation may be calculated in the usual way. The results by this method are seen to correspond closely to those by the preceding methods. In fact all three methods agree much more closely than might have been anticipated, considering the relatively small number of litters and of individuals.

Comparing the coefficients of fraternal variation, calculated on the litter basis, with those of the total population calculated by the ordinary method at corresponding ages, it is evident that (excepting only the unusually large fraternal variation for the female at ten weeks) the litter variation is always very much smaller, being usually only from one-third to one-half as large as the total variation. It also appears that the increased variability in the body weight of the total population from twenty days onward is due to an increased variability between litters more than to increased variability among the individuals within the litter. Dunn ('12) states (p. 137) that if conditions of growth are favorable the rats of unlike initial weight within a litter tend to approach each other in weight as growth goes on. If this be true, the fraternal (intra-litter) variation should decrease with age.

Table F includes my observations upon the variation in individual litters at successive ages. The sexes are here taken together. It is evident that there is considerable variation, both among different litters at the same age and in the same litter at different ages. The data suggest that there is a tendency for intra-litter variation to decrease up to the age of about twenty days, followed by a tendency to increase. This is supported to certain extent by table E, showing the coefficients of variation on the litter basis.

It may be concluded from all the evidence available that in general the variation in body weight within a given litter of albino rats is probably less than half that of the general population of the same age under similar environment. Nevertheless, even within the litter, variations are sometimes so great that it


POSTNATAL GROWTH IN THE ALBINO RAT


19


is never safe to draw conclusions from a single litter, and there is some risk even with several litters. These conclusions are of practical importance in selecting animals for experimental work, and in interpreting the results of such work. Although fraternal variability was not directly calculated for the individual organs, the same principle doubtless holds good with them; for, as will be shown later, all the organs are more or less closely correlated with the bod}^ weigh J;.

In the human species, fraternal variation appears to be relatively greater than in the rat. For example, Galton ('94) found the mid-stature for the human adult (male) population to be 68.2 inches. The quartile (or probable) deviation for the whole adult population he found to be about 1.70 inches, and that for brothers (average of four methods) about 1.06 inches. Thus for human stature by this method the fraternal variability appears to be only about 62 per cent of the racial variability. On the basis

TABLE F Coefficient of variation in individual litters at different ages



NUMBER OF








LITTER NO.


EACH SEX


NEWBORN


7 DAYS


14 DATS


20 DAYS


30 DAYS


6 WEEKS



Males


Females



A 32


. 3


5


5.81


3.82






M 9


. 5


3


5.44


4.85






M 91


3


3



4.19


2.92


4.45




M 6


5


3



10.202


4.11


5.61




MIO


2


4


8.18


7.61


7.47


7.64



11.8


M 8


3


5


8.78


4.82


3.94


2.42


6.76


12. 4»


A 33



7



12.00



6.67




A 331



5





7.34


4.70


6.94


M 7


2


4



3.95



18.00




M 71


1


4






21.30


30.60


M 1


4


1





2.83



7.91


M 2


4


1





4.11



6.. 57


A 29


2


4





4.47



4.95


A 28


4


2





5.06



6.86


^ Two males killed in litter M 9 at 7 days, and 2 females in A 33 at 20 days; one (a 'dwarf') in litter M 7 died between 20 and 30 days.

2 The large variation here was due to a 'dwarf which caught up with the others in body weight before the age of 14 days.

' At 10 weeks the coefficient of variation in this litter was 17.0, the increase being due chiefly to sexual differentiation in body weights.


20 C. M. JACKSON

of more recent biometric investigations in various lines by Pearson and others, it appears (cf. Encyclopaedia Britannica, ISth edition, vol. 27, p. 911) that in general the fraternal S. D. =

racial S. D. X — , reckoning the fraternal correlation at 0.50.

According to this formula, the fraternal variability would amorunt to about 87 per cent of the racial variability. Compared with these figures the fraternal variability of the rat, which is less than half of the racial, appears relatively small. This, however, is intra-litter variability, which may be somewhat less than fraternal variability in general. The latter could be determined for the rat only by comparing numerous litters born from the same pair of rats at different times.

GROWTH AND VARIABILITY OF THE INDIVIDUAL ORGANS

1. Head

In a previous paper by Jackson and Lowrey ('12), the relative growth of the head was considered. It was pointed out, as may be observed in table 3, that the head (sexes together) of the albino rat increases in relative size from an average of about 23 per cent of the body at birth to a maximum of nearly 26 per cent at seven days, after which it decreases gradually to about 10 per cent in the adult. The figures in the table for the percentage at one year are probably somewhat high, as they include some slightly emaciated individuals, in which the head is always relatively large. In very large rats (above 300 grams in body weight) the head continues to decrease in relative weight and may reach 8 per cent or less.

As is furthermore evident from table 3, the variability of the head is less than that of the body as a whole, the coefficient increasing from about 9 in the newborn to about 13 in the adult. The variation also appears slightly greater in the male than in the female (except at six weeks), as is also the case with the whole body.

The coefficient of correlation between the head and the total body weight varies at different ages from 0.76 to 0.95 (sexes together). These figures are somewhat too high, however, the


POSTNATAL GROWTH IN THE ALBINO RAT 21

true correlation being augmented by a 'spurious correlation,' as will be explained later. There appear no constant differences according to age and sex. If such exist, as is probably the case, they are obscured by accidental variations in the comparatively small number of observations.

The close correlation between the weights of the head and of the whole body is also evident from the slight variability in the percentage weight which the head forms of the whole body (table 3). If the head at each age formed a constant per cent of the body, the variabihty of the percentage weight would be zero. The coefficient of variation of the percentage weight of the head is, however, usually not very much smaller than that of the absolute weight, and in a few cases even appears slightly larger. That is to say, at any given age the absolute weight of the head can be predicted almost as accurately as the percentage weight. This is not the case with most of the individual organs, as will be seen later.

Whether reckoned upon the absolute or upon the percentage basis, it is evident the head of the rat exhibits slight variability when compared with most other organs as well as with the body as a whole. This agrees with the observations of Hatai ('08) upon the variability of the skull bones of the rat, as well as with what has been found in the human body. Quetelet ('71) and other anthropometrists have observed that the head is the least variable portion of the human body, a conclusion supported by extensive and careful measurements on the skeleton of different parts of the body by various observers.

2. Central nervous system

Since the growth of the brain and spinal cord has been carefully worked out by Donaldson ('08) from extensive data, it was thought unnecessary to include these organs in the present investigation. Incidentally, however, some observations were made upon the brain, and a few upon the spinal cord. From these, together with those published by Donaldson ('03) and some unpublished data from The Wistar Institute, a few additional conclusions may be drawn.


22 C. M. JACKSON

In regard to the brain, it may be noted that the maximum postnatal relative size occurs, not at birth, but a short time later. Using the data derived from Donaldson's formula expressing the brain weight in terms of (gross) body weight ('08, table 1), calculations show that with a body weight of 5 grams (approximately that at birth) the brain weight forms 4.6 per oent of the body weight. This increases to 6.7 per cent at a body weight of 15 grams. From this (apparent) maximum, the relative size of the brain decreases to 5.0 per cent at 25 grams, 2.7 per per cent at 55 grams, 1.5 per cent at 115 grams, 1.2 per cent at 155 grams, 0.9 per cent at 205 grams, and 0.62 per cent at 315 grams. In this case, the sexes are not separated, although the male brain is relatively slightly heavier than that of the female.

The foregoing figures for the relative size of the brain, as derived from Donaldson's formula, agree fairly well with the present data grouped according to age. We find (combining Donaldson's data with my own) that in 92 newborn albino rats (56 males, 36 females) the brain averaged 4.8 per cent of the (gross) body weight; in 22 (10 males, 12 females) at seven days, 6.4 per cent; in 31 (28 males, 3 females) at three weeks, 5.6 percent; in 30 (27 males, 3 females) at six weeks, 3.1 per cent. Only scattering observations are available at the later stages. It therefore appears probable that the brain reaches its maximum relative size about the second or third week, when the body weight is between 10 and 15 grams.

The combined data for the brain are sufficient for estimation of variability and correlation with body weight only at birth, three weeks, and six weeks. The coefficient of variation for the brain was found to be at birth, male, 13.9; female, 9.1, total (sexes together) 12.2. The coefficient of variation for the corresponding (gross) body weights was 12.8. At three weeks the coefficient of variation of the brain, for males only, was 6.8, which, excepting the eyeballs (table 4) was the lowest variability found for any organ at any age. The corresponding coefficient of variation for total body weight was 14.8. At six weeks, the coefficient of variation for the brain, males only, was 12.2, the coefficient for the corresponding total body weights being 36.2.


POSTNATAL GROWTH IN THE ALBINO RAT 23

For the same data, the coefficient of correlation between brain weight and (gross) body weights was, at birth, male, 0.736; female, 0.566, total, 0.690 ±0.037; at three weeks, male only, 0.783 ± 0.049 ; at six weeks, male only, 0.883 ± 0.020. These high figures are due in part to 'spurious correlation' (as will be explained later). In general, Donaldson ('08) found (in 680 records at various ages) the coefficient of correlation between brain weight and body weight to be 0.7639± 0.0108.

Hatai ('08) found for the cranial capacity of adult albino rats a coefficient of variation of about 6.7 in the male, and 7.2 in the female. The coefficient of correlation between cranial capacity and (gross) body weight was 0.516 ±0.074 in the male and 0.692 =t 0.058 in the female.

For the spinal cord, the relative weight (according to Donaldson's formula) increases from about 0.66 per cent of the body at 5 grams to a maximum of 0.77 per cent at 10 to 15 grams, declining to 0.28 per cent at 205 grams and 0.22 per cent at 315 grams. From the combined data the coefficient of variation was calculated for the newborn only (48 males, 29 females) and was found to be as follows: male, 17.8 (for total body, 13.1); female 12.6 (total body, 9.4) ; total (sexes combined) 16.0 (for total body 12.5). The corresponding coefficient of correlation with the body weight was 0.666 ±0.043, which is somewhat lower than that found by Donaldson ('08) for the spinal cord of the rat in general (0.8564 ±0.0071).

In comparing these results with those for other organs, it must be remembered that the rats from which the brain and spinal cord were derived correspond only in part to those for all the other organs.

3. Eijeballs

The relative or percentage weight of the eyeballs for various body weights, when calculated from Hatai's ('13) formula, is shown by the curve in figure 1 b. According to this curve, it is seen that the eyeballs increase in relative weight from about 0.59 per cent of the body weight at 5 grams to 0.61 per cent at 10 grams, thereafter decreasing steadily, reaching 0.48 per cent


24 CM. JACKSON

at 20 grams, 0.29 per cent at 50 grams, 0.18 per cent at 120 grams, 0.15 per cent at 170 grams, 14 per cent at 200 grams, and 0.11 per cent at 300 grams.

In my data grouped according to age periods (table 4, fig. 1 b) the average weight of the eyeballs at birth forms about 0.52 per cent of the (net) body weight in the male, and 0.54 per cent in the female. The relative weight increases to a maximum at seven days of about 0.60 per cent in the male, and 0.64 per cent in the female.^ Thereafter the eyeballs decrease steadily in relative weight, excepting a slight rise at one year (which, like that for the head, is probably abnormal). It will be noted that the percentage weight of the eyeballs is larger in the female at every age except at six weeks. Whether this expresses a true difference according to sex is uncertain, however, as the difference is small and the data perhaps inadequate to determine this point.

In connection with this difference in relative weight according to sex, it is noteworthy (table 4) that the absolute weight of the eyeballs is nearly the same in both sexes at every age. Since the average body weight of the female is smaller at each of the ages noted (except six weeks), it follows that the relative weight of the eyeballs must be correspondingly larger in the females.

The similarity in the absolute weight of the eyeballs in both cases at various ages suggests the possibihty that the growth of these organs may be somewhat independent of influences affecting the growth of the body as a whole. This idea is to a certain extent confirmed by the coefficient of variation in the absolute weight of the eyeballs (table 4). Although at birth this coefficient (15.5) appears larger than that of the whole body (12.3), after the age of seven days the variation in the absolute weight of the eyeballs is exceedingly low (7.45 to 13.3), only the brain approaching it in this respect.

5 In comparing the observed with the calcuhxted values in this and the other organs, it should be pointed out that even slight fluctuations in absolute weight which may lie within the experimental error, may produce a much greater percentage deviation in the youngest animals, owing (1) to the small absolute size of the organ, and (2) to the relatively greater weight of the organ in the earlier periods.


POSTNATAL GROWTH IN THE ALBINO RAT 25

Furthermore, the idea of an independent growth of the eyeballs is supported by the variation in the percentage weight relations (table 4). While in the newborn the coefficient of variation in the percentage weight is less than that for the absolute weight (the usual relation in other organs at all ages), it is a remarkable fact that after seven days the variation in the eyeballs is decidedly greater for the percentage weight. Finally, the coefficient of correlation between body weight and weight of the eyeballs, as might be expected from the foregoing, is comparatively low and very irregular (0.21 to 0.67).

4. Thyroid gland

When the calculations are made according to Hatai's formula, the relative weight of the thyroid (fig. 3 a) is greatest in the earliest stages, forming about 0.029 per cent of the body weight at 5 grams. The relative weight decreases slowly to 0.028 per cent of the body at 10 grams, 0.026 per cent at 20 grams, 0.022 per cent at 50 grams, 0.019 per cent at 100 grams, 0.016 per cent at 200 grams, and 0.015 per cent at 300 grams.

On account of certain difficulties, the number of observations upon the thyroid gland in my own series is somewhat limited. The gland is small and difficult to separate accurately from the adjacent muscles, especially in the younger stages. It was also sometimes injured in the process of decapitation. The observations recorded, arranged according to age (table 5, fig. 3 a), indicate that the thyroid is relatively largest in the newborn, decreasing from about 0.04 per cent of the body weight to 0.018 per cent at one year. In comparison with Hatai's theoretical curve of growth, there is in my data a lagging behind during the first week. The thyroid apparently increases but slightly during this period while the body weight doubles. This causes a considerable drop in the percentage weight of the thyroid at seven days. No difference according to sex is apparent. On account of the limited data, the coefficients of variation and correlation were not calculated.

Watson ('10) finds the thyroid (and parathyroid) glands usually enlarged in rats fed upon meat, and especially oatmeal, diet.


26 C. M. JACKSON

They were also found relatively much larger in wild rats, due probably to difference in exercise and diet.

5. Thymus

On account of inadequate data, Dr. Hatai was unable to construct a satisfactory fonnula for the growth in absolute weight of the thymus in the albino rat.

In my own data grouped according to age periods (table 6, fig. 2 b), it appears that the thymus increases, in the male, from about 0.15 per cent of the body weight at birth to 0.24 per cent at seven days, and to a maximum of 0.38 per cent at twenty days. Thereafter it decreases, and at one year forms an average of only 0.02 per cent of the body. The relative weight undergoes a similar change in the female with no significant difference according to sex.

A unique feature of the thymus is its decrease in absolute as well as in relative weight. As shown in table 6, the average absolute weight at five months is slightly smaller than at ten weeks, and at one year it has undergone a very striking decrease. This is of course in connection with the process of involution, following the age of puberty, whereby the thymus is largely transformed into a mass of adipose tissue.

As would necessarily follow from the decrease in the absolute weight of the thymus during the process of involution, there is actually a negative correlation at five months between the thymus and body weights. During the earlier life, on the other hand, there is a well marked positive correlation, the coefficient increasing from 0.668 at birth to 0.904 at six weeks (the high figures being partly due to 'spurioUs correlation').

The coefficient of variation in absolute weight is high, increasing from 30.9 at birth to 50.4 at six weeks. Later, however, when a greater variation might naturally be expected during the involution process, it is actually less, the coefficient being 25.3 and 22.2. For the percentage weight, the coefficient of variation, though still high, is considerably smaller than that for the absolute weight, excepting the periods at ten weeks and five months.


POSTNATAL GROWTH IN THE ALBINO RAT


27


6. Heart

According to the curve of relative (percentage) weight (fig. 1 a) constructed from Hatai's formula, the heart increases from 0.64 per cent of the body weight at 5 grams to a maximum of 0.70 per cent at 10 grams. Thereafter it gradually decreases to 0.67 per cent at 20 grams, 0.56 per cent at 50 grams, 0.45 per cent at 120 grams, 0.42 per cent at 170 grams, and 0.40 per cent at 200 grams, 0.37 per cent at 300 grams and 0.35 per cent at 400 grams.

When grouped according to age periods, my own data (table 7, fig. 1 a) show an increase from an average in the male of 0.65 per cent at birth to 0.68 per cent at seven days and to a maximum of nearly 0.73 per cent at twenty days; thereafter it decreases to about 0.45 per cent at one year. In the female the relations are similar, although the average relative weight is greater at every age (except twenty days). This apparently greater relative weight of the heart in the female, however, is probably without significance. In the first place, the relative weight of the heart in the female at any given age beyond six weeks should be slightly greater on account of the smaller body weight. More


%



(


> ®


D









.60 a So


a




\



Fig. 1 a HEART Percentage of body weight

Curve derived from Hatai's formula (for males).

Means at seven age penods from Jackson's datai males.®, females,®.


<


1




V



e Q






1





\



"^



e


9 G



o


o





\



Fig.l b


EYEBAl Cur> Mea


.LS Perc e derived ns at sev


entage of from Hata ■n age per


body weic i's formul ods from



CD

c e .30


hL

3 (for males).

Jackson's data ; males, 0, females, 9.














"^--^l


e




Q














5 10 20


150 Body weight m grams


28


C. M. JACKSON


2.00



(5











<











°>1.25 1



i











\



Fig. 2 a LUNGS Percentage of body weight

Curve derived from Hatai's formula (for males).

Means at seven age periods from Jackson's data; males, ©, females, ®.


c




V






ffl



O


o .75

a.





%



®


®


- Q







.25




Q


Fig. 2


b THY^ Mei


/lUS Pen ns at sev


entage of jn age pe


body wei iods from


3ht. Jackson's


data; males.©, females,®.


«!i


^"



^



« 


©


o e



O



5


10- ?


E



10



i;


>0


200 300


Body weight in grams


over, the differences between the sexes in percentage weight shown in the table are comparatively slight, usually well within the limits of probable error. It is noteworthy that my data give percentage weights which are slightly, but almost constantly, higher than those of the curve derived from Hatai's formula (fig. la).

The heart shows a considerable degree of variability in absolute weight (table 7), the coefficient increasing from 18 at birth to 33.7 at twenty days and 29.7 at six weeks. At ten weeks and five months the coefficient decreases (18.4 and 21.3) to near that at birth. For the relative weight, however, the relations are quite different. At birth and seven days, the coefficient of •variation for the percentage weight of the heart is rather high (15 to 18), though still somewhat less than that for the absolute weight. From twenty days onward, however, there is remarkably little variation in the percentage weight of the heart, the coefficient being usually less than 10. This corresponds to the high coefficient of correlation between the weight of the heart and the body weight after twenty days (0.840 to 0.968), which


POSTNATAL GROWTH IN THE ALBINO RAT 29

at birth and seven days is much lower (0.583 and 0.504). However, the high figures for the older rats are partly due to a 'spurious correlation/ as will be explained later.

7. Lungs

According to calculations made from Hatai's ('13) formula, the lungs, as shown by the curve in figure 2 a, decrease in relative (percentage) weight from a maximum of 1.60 per cent of the entire body weight at 5 grams to 1.32 per cent at 10 grams, 1.06 per cent at 20 grams, 0.80 per cent at 50 grams, 0.64 per cent at 120 grams, 0.59 per cent at 200 grams, 0.55 per cent at 300 grams, and 0.54 per cent at 400 grams.

The relative weight of the lungs in the male according to my data increases from about 1.59 per cent of the body weight at birth to 1.93 per cent at seven days. From this maximum it decreases to 0.68 per cent at six weeks, and 0.57 per cent at ten weeks. The apparent increase at five months and one year is probably due to the inclusion of unrecognized pathological cases. Except at twenty days, the relative weight of the lungs appears slightly higher in the female than in the male. This is chiefly because the body weight is usually greater in the male. Any sexual difference aside from this is very doubtful. It will be observed that the curve derived from Hatai's formula does not show the preliminary increase in percentage weight which is indicated by my data, and which is found so characteristic of the viscera in general.

The variability of the lungs in absolute weight at birth (23.3) is much greater than that of the whole body, but is later approximately equal to it in most cases (16.6 to 23.9). The variation in percentage weight is usually much less than in absolute weight ; and the correlation with the body weight is correspondingly high (73.6 to 94.3 except at ten weeks, 62.3). The high figures are partly due to a 'spurious correlation.'

The weights of the right and the left lungs were also taken separately, although not recorded in the table. The right lung is always much larger than the left, the ratio being approxi


30


C. M. JACKSON


mately 2:1, which does not change appreciably according to age. The variabiUty in absolute weight of right and left lungs taken separately, is about the same as for both together, as shown by table G.


TABLE G



Coefficient of variation for the lungs {sexes


together)





NEWBORN


7 DATS '


20 DAYS


6 WEEKS



44 m. 4.3 f. ' 30 m. 27 f.


24 m. 25 f.


22 m. 17 f.


Both lungs

Right lung

Left lung


23.3*1.3

24.2

22.9


16.6*1.1 17.0

17.8


23.9*1.7

24.9

22.3


19.4*1.5

19.7

19.4


The coefficient of correlation between the body weight and weight of lungs, including right and left lung separately, is given in table H. While the coefficients are too high (due to 'spurious correlation') those at any given age may be safely compared with each other.

TABLE H

Coefficient of correlation between the lungs and the net body weight (sexes together)



NEWBORN


7 DAYS


20 DAYS


6 WEEKS



44 m. 43 f .


30m.27f. 1 24m.25f.


22 m. 17 f.


Both lungs

Right lung

Left lung


0.736*0.033 0.739*0.033 0.693*0.038


0.799*0.032 0.868*0.024 0.813*0.030 0.854*0.026

0.687*0.047 0.876*0.022

1


0.943*0.012 0.949*0.011 0.902*0.020


From this table it is evident that the correlation between body weight and right lung is practically the same as between body weight and both lungs; and that the correlation between body weight and left lung is usually but slightly less. These figures show no evidence of the existence between the right and left lungs of any compensatory regulation in size, for deviations within the limits of normal variation. This question may be complicated by other factors, however, so that it is unsafe to draw any definite conclusion regarding the matter.


POSTNATAL GROWTH IN THE ALBINO RAT 31

8. Liver The curve representing the relative (percentage) weight of the liver, according to calculations from Hatai's formula, is shown in figure 6 a. It is seen that the relative weight of the liver increases from 4.87 per cent of the body weight at 10 grams to a maximum of 7.80 per cent at 25 grams, after which it gradually decreases to 7.17 per cent at 50 grams, 5.75 per cent at 120 grams, 5.02 per cent at 200 grams, 4.55 per cent at 300 grams, and 4.27 per cent at 400 grams.

When grouped according to age periods, my own data (table 9, fig. 6 a) show that in the male the relative weight of the liver forms an average of 4.74 per cent of the body weight at birth. Unlike the organs previously considered, it apparently decreases at seven days, to 3.39 per cent, increasing to 4.64 per cent at twenty days and reaching a maximum of 6.78 per cent at six weeks. Thereafter it decreases to an average of 4.42 per cent at one year. The female exhibits a similar course of growth; but is slightly larger relatively than the male at birth and seven days, and smaller at all later periods.* My data therefore indicate a sexual difference, although none was found by Dr. Hatai in his data.

It will also be noted that the liver in my data is at all periods relatively small, when compared with the formula derived from Hatai's data. It is possible that the discrepancy may be due to a slight difference in the diet. Chalmers Watson ('10) finds a marked decrease in the relative size of the liver in captured wild rats, and ascribes the decrease to a diminution in the protein of the bread and milk diet during captivity. If the lower figures in my data were due to this cause, however, we should expect to find a similar condition in the kidneys, which Watson finds also to be larger with rich protein diet. As will be seen later, however, my data usually show for the kidneys a higher relative weight than that according to the formula calculated from Hatai's data. The difference, therefore, can hardly be explained on this basis.


32


C. M. JACKSON


The large but irregular variability of the liver in the present data is shown in table 9. The coefficient of variation in absolute weight appears at different periods from 18.6 to 40.8, but with no definite change according to age or sex. The variation in the percentage weight is seen to be, as a rule, very much less. Accordingly, the coefficient of correlation between liver and body weight is high, from 73.6 to 96.8 (high figures, however, partly due to 'spurious correlation').



Fig. 3 a THYROID GLAND Percentage of body weight Curve derived from Hatai's formula (both sexes). Means at seven age periods from Jackson's data; males, 0, females,


Fig. 3 b SPLEEN Percentage of body weight

Curve denved from Hatai's formula (males, enlarged spleens excluded). Means at seven age periods from Jackson's data; males. 0, females,®.


5 10 20


100 150

Body weight m grams


9. Spleen


The relative (percentage) weight of the spleen, calculated from Hatai's ('13) formula, is represented by the curve in figure 3 b. It is seen that the spleen increases in relative size from 0.16 per cent of the body weight at 5 grams to 0.30 per cent at 10 grams, and a maximum of 0.32 per cent at 20 grams, thereafter decreasing slowly to 0.30 per cent at 50 grams, 0.28 per cent at 120 grams, 0.27 per cent at 200 grams, and 0.26 per cent at 300 to 500 grams.


POSTNATAL GROWTH IN THE ALBINO RAT 33

When grouped according to age periods (table 10, fig. 3 b) my own data show an increase in the relative size of the (male) spleen frdm 0.22 per cent of the body weight at birth to a maximum of 0.41 per cent at seven days. For succeeding ages, the percentage weight agrees fairly with that derived from the formula, excepting the unusually high figure (0.396 per cent) at one year. There is no distinct difference between the sexes, or change in relative size according to age (after the first week).

It may be noted that my data for the spleen tend to run somewhat higher than the curve derived from Hatai's formula. This may be because Hatai's formula is based upon data from which the 'enlarged' spleens had been excluded. His figures for all spleens average very much higher than mine.

The spleen is known to be an unusually variable organ and this is certainly true for the rat. As shown in table 10, the coefficient of variation in absolute weight is from 25 to 51 (excepting at five months, 18.9) The coefficient of variation in percentage weight is usually but slightly less than that in absolute weight. The coefficient of correlation between spleen and body weight is accordingly somewhat low, varying from 0.406 to 0.542 with an exceptionally high correlation (partly 'spurious') of 0.967 at twenty days.

10. Stomach and intestines

From the curve of relative (percentage) weight constructed from Hatai's ('13) formula (fig. 5), it appears that the empty alimentary canal increases from about 3.0 per cent of the body at 10 grams, to 7.5 per cent at 20 grams, and a maximum of 8.0 per cent at about 35 grams, after which it decreases to 6.3 per cent at 100 grams, 5.0 per cent at 200 grams, and 4.5 per cent at 300 grams.

Observations were made upon the empty intestinal canal in only a part of my own series. These observations, grouped according to ages (table 11, fig. 5) indicate that the relative weight of the empty tract increases from an average of about 2.4 per cent of the body weight in the newborn to a maximum of about 8 per cent at six weeks, decreasing to 5 per cent at one year.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO 1


34 C. M. JACKSON

They therefore agree fairly well with the curve from Hatai's formula. The data are too few for calculation of the coefficients of variation and correlation at the various ages.

A complete series of observations is available for the stomach and intestines plus contents, however, a summary of which is given in table 12 and figure 5. The increase in relative weight runs somewhat parallel to that for the empty tract, increasing from an average of about 6 per cent of the body weight in the newborn to a maximum of about 16 per cent at six weeks, decreasing thereafter to about 7 per cent at one year.

The coefficient of variation for the canal ivith contents (calculated only to six weeks) is somewhat high, 28.6 to 42.1, but not so high as was anticipated. It is, as might be expected, highest in the newborn (only a part of which had suckled) and at twenty days (weaning period). The coefficient of correlation between body weight and canal with contents is low in the newborn (0.29) but comparatively high later (0.59 to 0.84), partly due to 'spurious correlation.' Although one naturally thinks of the alimentary canal with contents as exceedingly variable in size, it is interesting to note that it is actually less variable than some other viscera. In general, after the newborn age, it is usually less variable and more closely correlated with the body weight than are the ovaries, spleen and suprarenals. It is on account of this degree of correlation between body weight and alimentary canal with contents that in the rat the gross weight serves almost as well as the net body weight as a basis for estimating the relative weight of the various organs.

1 1 . Suprarenal glands

From the curves of relative (percentage) weight (fig. 4) calculated according to Hatai's formulas, it is seen that the relative weight of the suprarenals in the male increases from 0.031 per cent of the body at 5 grams to a maximum of about 0.040 per cent at 10 to 15 grams, thereafter decreasing to 0.038 per cent at 20 grams, 0.030 per cent at 50 grams, 0.021 per cent at 120 grams, 0.018 per cent at 160 grams, and 0.014 per cent at 300 grams. In


POSTNATAL GROWTH IN THE ALBINO RAT


35


%


a;











.030 .020


^


r^


\



F.g,4


SUPRARf Curve der Means at


nal gu ved from seven age


^NOS P(

Hatai's for

periods f


'reentage mula, mal om Jacks


of body weight.

es, , females,

on's data; males, 0, females, ©.



3)




\


e


""""©

. ®__










O



Q


~


r — 17:47, 5 August 2019 (AEST)














5


2


} 5



IC



IE



2C


300


Body weight m gran


16





+








12

1,10 1

ti

















+■







+






+






^


1



^

-^


©




+




i

ff i



e


D






©0



-^


»,




2


%


i



Fig. 5 STOMACH-INTESTINE Percentage of body weight

Curve, for empty canal, derived from Hataj's formula (for males). Means at seven age penods from Jackson's data.






For emp For can


ty canal, r 3l with cor


nales, 0, f tents; ma


jmaleS, ® es, •, tern


iles,+



15)10 .20


150 Body weight in grams


36 CM. JACKSON

the female the relative weight increases from 0.037 per cent of the body at 5 grams to a maximum of about 0.042 per cent at 10 grams, decreasing to 0.038 per cent at 20 grams, 0.032 per cent at 50 grams, 0.029 per cent at 100 grams, 0.027 per cent at 160 grams, and 0.026 at 300 grams. Thus the suprarenal bodies appear relatively much larger in the female, especially for a body weight above 100 grams.

■ Grouped according to age periods, my own data (table 13, fig. 4) likewise show the relative growth of the suprarenals to be quite different in the two sexes. In the male, the maximum relative size, 0.038 per cent of tlie body weight, apparently occurs in the newborn, decreasing to 0.023 per cent at seven days, but increasing again to 0.036 per cent at twenty days, which is nearly as large as the maximum. Thereafter the relative weight decreases to 0.027 per cent at six weeks, 0.018 per cent at ten weeks, and 0.016 per cent at five months and one year. The course of relative growth is somewhat similar in the female, but the maximum (0.043 per cent) occurs at twenty days instead of at birth (0.041 per cent) . After six weeks, the relative size is much greater in the female, being 0.026 per cent to 0.028 per cent of the body weight. Even the absolute weight of the suprarenals (after six weeks) is considerably greater in the female, although the body weight is much less.

When compared with the theoretical curve of growth, a marked discrepancy may be noted in the present data (table 13) at seven days. For some unaccountable reason, the suprarenals in my series appear to lag behind during the first week, increasing in weight only about 20 per cent, while the body weight is doubled. There is also a corresponding irregularity in correlation with the body weight at seven days, which is very low, especially in the male.

The coefficient of variation in absolute weight is somewhat high and irregular (20.4 to 38.7) with variations showing no definite relation to age or sex. The coefficient of variation in percentage weight is usually but little, if any, lower. The coefficient of correlation between suprarenals and body weight is cor


POSTNATAL GROWTH IN THE ALBINO RAT 37

respondingly low, 0.347 to 0.583 (except at seven days) for the sexes combined. In the sexes, separately, however, the correlation is usually somewhat higher, as might be expected, since the sexes show a characteristic difference in the relative weight of the suprarenals.

12. Kidneys

The relative (percentage) weight of the kidneys, reckoned according to Hatai's formula, is shown by the curve in figure 6 b. The kidneys appear in the newborn male to form 0.90 per cent of the body weight at 5 grams, which increases to a maximum of 1.53 per cent at 10 grams, thereafter decreasing to 1.38 per cent at 20 grams, 1.10 per cent at 50 grams, 0.91 per cent at 130 grams, 0.85 per cent at 200 grams, 0.82 per cent at 300 grams, and 0.80 per cent at 400 grams.

When grouped according to age periods, my own data (table 14, fig. 6 b) show the relative (percentage) weight of the kidneys to increase in the male from 0.96 per cent of the body weight at birth to 1.29 per cent at seven days and to a maximum of 1.44 per cent at twenty days. Thereafter it decreases to 1.27 per cent at six weeks, 1.03 per cent at ten weeks, 0.93 per cent at five months, and 0.95 per cent at one year. In the females, the average percentage weight of the kidneys is usually slightly higher than in the male of the same age, but nearly the same as that of a male, with the same body weight. The agreement of my data with the curve derived from Hatai's formula is remarkably close (fig. 6 b) .

The coefficient of variation of the kidneys in absolute weight (table 14) is greater than that of the body in the earlier periods, reaching a maximum of 33.7 at twenty days. Later, however, the kidneys become less variable than the body weight, the coefficient being 15 to 19. The variation in the percentage weight is low, especially from seven days onward. The coefficient of correlation between kidneys and body weight is correspondingly high, being 0.703. at birth, and 0.788 to 0.955 at later age periods, (partly due to 'spurious correlation'). No constant difference in variation is noticeable according to age or sex.


38


C. M. JACKSON



Fig- 6 a LIVER Percentage of body weight.

Curve derived from Hetai's formula (for males).

I^eans at seven age periods from Jackson's data; males. 0, females, 0.


Fig. 6 b KIDNEYS Percentage of body weight.

Curve derived from Hatai's formula (for males'.

Means at seven age penods from Jackson's data; males, 0, females, (


ZSEL


150 Body weight in grams



Fig. 7 a TESTES Percentage of body weight

Curve derived from Hatai's formula (epididymis not included).

Means at seven age periods from Jackson's data (epididymis included).


o (U.04C


Fig. 7 b OVARIES Percentage Of body woight Curve derived from Hatai's formula. Means at seven age periods from Jackson's data<.


- 150 Body weight in grams


POSTNATAL GROWTH IN THE ALBINO RAT 39

13. Gonads

a. Testes. The relative (percentage) weight of the testes, calculated from Hatai's (13) formulas, is represented by the curve in figure 7 a. This gives a relative weight of 0.40 per cent of the body weight at 10 grams and about the same at 20 grams. Unlike that of other organs, the relative weight of the testes continues to increase for a long time (up to age of puberty), being 0.80 per cent at 50 grams and reaching a maximum of 1.30 per cent from 90 to 100 grams. Thereafter it decreases to 1.28 per cent at 120 grams, 1.16 per cent at 170 grams, 1,09 per cent at 200 grams, 0.90 per cent at 300 grams, and 0.76 per cent at 400 grams.

Unfortunately, in my own data the epididymis is included with the testes and the results are therefore not strictly comparable with those of the formula above, which do not include the epididymis. A few special ijieasurements indicate that the epididymis fomis one-fifth or one-sixth of the weight of the testes proper in the younger rats, which however increases (with irregular variations) to one-third after the age of puberty, at about ten weeks. In extreme cases, the weight of the epididymis is one-half that of the testis proper. The weight of the testes proper, as well as of accessory sexual apparatus (epididymis, seminal vesicles, et cetera) , probably undergoes considerable change during cycles of sexual activity. This has been noted by Disselhorst' ('98, '08) in various mammals and especially in birds, for the gonads in both sexes.

My own data (table 15, fig. 7 a) indicate that the testes (including the epididymis) form an average of about 0.13 per cent of the body at birth, increasing gradually to a maximum of 1.50 per cent at ten weeks, and decreasing somewhat at later periods. The variability in absolute weight is quite high, the coefficient varying from 25.3 at birth to 40.7 at five months (excepting at seven days, when it is only 17.7). The coefficient of variation in percentage weight is much lower (10.3 to 32.8) and the correlation with the body weight higher than might be expected (0.67 to 0.95, except at ten weeks, 0.48). These figures are somewhat too high, however, due to 'spurious correlation.'


40 C. M. JACKSON

b. Ovaries The growth of the ovaries is quite complex. In the relative (percentage) growth curve (fig. 7 b) constructed from data according to Hatai's formulas, two distinct phases are noticeable. In the first phase, beginning at birth, the ovaries increase in relative size from about 0.017 per cent to a maximum of about 0.030 per cent at a body weight of 10 to 15 grams. Thereafter the ovaries decrease to about 0.015 per cent of the body weight at 60 grams. Then begins the second period of acceleration, corresponding to the advent of puberty, during which the ovaries increase to a second maximum of about 0.037 per cent of the body weight at 110 to 120 grams. Thereafter the ovaries lag behind steadily in relative growth, and form only about 0.017 per cent of the body weight at 300 grams.

When grouped according to age periods, my data (table 15, fig. 7 b), show considerable irregularity in the relative size of the ovaries. From an average of about 0.017 per cent of the body at birth they increase (after an apparent drop at seven days) to a first maximum of about 0.022 per cent at twenty days. Then they decrease to about 0.020 per cent at six weeks, increasing to a second maximum of 0.034 per cent at ten weeks, the age of puberty. Thereafter they decrease, averaging 0.025 per cent of the body weight at one year.

The extremes recorded in the size of the ovaries (table 15) show a remarkable range. This is due partly to difficulty during the earlier stages in dissecting out the ovaries accurately, and partly to fluctuations in size in the later stages, probably on account of cycles of ovulation. Coefficients of variation were not calculated for the earliest stages, as the data were considered inadequate. For periods from twenty days onward, the highest coefficient of variation in absolute weight, 50.9, was found at ten weeks (the age of puberty), and the lowest, 32.7, at five months. The coeflScients of variation in percentage weight are somewhat lower (25 to 39); and the coefficient of correlation between ovaries and body weight varies from 0.64 to 0.82 (somewhat too high, due to 'spurious correlation').


POSTNATAL GROWTH IN THE ALBINO RAT 41

IJj.. General considerations

We may now review briefly certain phases of growth and variabihty of the viscera. First should be noted the growth of the viscera, taking the group as a whole (including brain), in comparison with the remainder of the body. As found by Jackson and Lowrey ('12), the visceral group of the rat at birth forms about 18 per cent of the body weight. It increases in relative weight to an average of about 19.2 per cent at seven days, and to a maximum of about 21.3 per cent of the body weight at three weeks. At six weeks, it has decreased slightly, to 20.4 per cent, but continues to decrease in relative weight to about 16 per cent at ten weeks, 14.8 per cent at five months, and 13.3 per cent at one year.

Data for comparison of the relative size in other animals are cited by Jackson and Lowrey ('12), who point out that in general the smaller mammals have a relatively larger visceral apparatus, probably correlated with a more intense metabolism. The rat occupies a somewhat intermediate position, the relative weight of the viscera being less than that of most of the smaller mammals, but greater than that of the larger mammals.

Since the visceral group forms a comparatively small part of the entire body, its relative size will evidently be influenced greatly by the rate of growth of the other parts of the body. . Jackson and Lowrey ('12) have shown that in the rat the relative increase in the weight of the viscera during the first week is accompanied by a similar slight increase in the skeleton and a very marked increase in the relative weight of the integument. The relative increase in these three systems is apparently balanced by a small decrease in the relative weight of the musculature, and by a remarkable decrease in the 'remainder,' due chiefly to a disappearance of excess body liquids. The slight further increase in the relative weight of the viscera at three weeks is accompanied by an increase in the musculature, and is balanced by decreases in the skin, skeleton and remainder. Thereafter the slow gradual decrease in the relative weight of the visceral group is accompanied by a similar decrease in the skin and skeleton. This is


42 C. M. JACKSON

balanced by a corresponding increase in the musculature, which by its large size virtually dominates the further growth of the body as a whole.

It is further evident that the growth of tiie body as a whole is the resultant of unlike growths of its various component systems. This principle applies likewise to the growth of the visceral group, the various organs having each its own characteristic mode of growth. As a matter of fact, only three individual organs, thymus, heart, and kidneys (and perhaps also the suprarenals) have their apparent maximum relative weight at the age of three weeks, when the visceral group as a whole appears relatively largest. The stomach and intestines and the liver apparently reach their maximum relative weight at the latter period of six weeks, and the gonads at ten weeks. Of those reaching their maximum at an earlier period, the brain, spinal cord, eyeballs, lungs, and spleen appear to be relatively heaviest in the second week while the thyroid gland appears relatively heaviest in the newborn.

Somewhat similar relations as to the relative growth of the various organs were found by Kellicott ('08) in the dogfish. Of the various organs observed, only the brain and rectal gland appear relatively largest at birth. The heart, pancreas, spleen and liver increase rapidly so as to reach their maximum relative size a short time after birth, and thereafter decline steadily (with a secondary increase in the liver, due to accumulation of fat). The gonads reach their maximum relative size at sexual maturity in the dog-fish.

Scattering data for the growth of the human viscera are recorded by various authors (for references, see paper by Jackson '09), but they are scarcely adequate to determine the question as to the course of postnatal relative growth for the individual organs. In general, so far as may be judged from the data available, the human visceral group appears relatively larger at birth than at any subsequent age. The lungs, however, appear to increase somewhat in relative weight so as to reach a maximum after birth. This is perhaps also true of the heart, kidneys and gonads, and it is quite possible that more extensive data would


POSTNATAL GROWTH IN THE ALBINO RAT 43

show this to be true also for other organs. Some difference between man and rat might naturally be expected, however, since the rat is born in a more immature condition.

The variability in the weight of the body must also depend ultimately upon the variability of its component parts. As to the variability of the component parts of the rat, we have data for the head and viscera only, A brief summary of the coefficients of variation in round numbers (from tables 2 to 15; sexes together, excepting gonads and brain) is given in table I.

The difference in variation between the sexes is usually not very marked (as may be noted in tables 2 to 15) ; although there appears to be a tendency to greater variability in the male, both in body weight and in weight of the indi^ddual viscera. In the table above it may be noted that the head and head organs (brain, eyeballs) form a group of small variability (average coefficient 10 to 12) which is usually far below that for the body as a whole (average 19). The other organs are more variable than the body. The lungs, kidneys, heart" liver, and suprarenals form a moderately variable group (average 21 to 26) while the gonads, thymus, spleen and intestinal canal (with contents) are exceedingly variable (average 29 to 43) .

The average coefficient of variation for the viscera examined is 25. It is noteworthy, however, that the average coefficient of variation is lowest at birth and one week, and highest at three weeks. This agrees with what is found also for the body as a whole.

A brief summary of the coefficients of correlation between the body weight and the weight of the individual viscera (from tables 3 to 15, sexes together, excepting brain and gonads) is given in round numbers in table J.

In this table likewise the differences according to sex are disregarded, but even when these are taken into account (cf. tables 3 to 15) the general relations are not materially changed. In about two-thirds of the cases, however, the coefficient of correlation is higher in the males than in the corresponding females;

^ The blood, the percentage weight of which in adult rats, according to Chisolm ('11), has a coefficient of variation of 10.7, possibly also belongs in this group.


44


CM. JACKSON


TABLE I Coefficients of variation in weight: albino rat


NEWBORN


1 WEEK


3 WEEKS


6 WEEKS


10 WEEKS


5 MONTHS


AVERAGE


Braini

Eyeballs

Head

Total body. . .

Lungs

Kidneys

Heart

Liver


12 16 10 12 23 24 18 22 24 25 31 39

38


15 11 16 17 22 20 19 20 18 32 34

29


7

13 15 28 24 34 34 41 33 30 43 51

42

42


12

10 21 19 15 30 19 22 27 50 26

30

47


11

14 20 21 17 18 33 21 35 25 38

51


9 13 19

19 21 25 39 41 22 19

33


10 12 12 19 21 22 24 26


Suprarenals . .

Testes

Thymus

Spleen

Intestinal canal (plus contents) . .

Ovaries


26 29 34 34

35

43


Average of viscera. . .


23


22


31 •


24


26


24


25


1 From combined data as explained under Central nervous system.


TABLE J Coefficients of correlation with the body weight: albino rat



NEWBORN


1 WEEK


3 WEEKS


6 WEEKS


10 WEEKS


5 MONTHS


AVERAGE


Head ] 0.76

Kidneys , 0.70

Liver | 0.76

Lungs i 0.74

Brain 0.69

Heart 0.58

Testes 0.67

Ovaries

Intestinal

canal (plus

contents) . . 0.29

Thymus 0.67

Spleen 0.54

Eyeballs ' 0.67

Suprarenals..! 0.51


0.89 0.79 0.76 0.80

0.50 0.75

0.59 0.74 0.44 0.52 0.13


0.93 0.96 0.97 0.87 0.78 0.91 0.95 0.73

0.84 0.89 0.97 0.67 0.58


0.95 0,92 0.84 0.94 0.88 0.97 0.75 0.64

0.76 0.90 0.50 0.31 0.41


0.75 0.90 0.74 0.62

0.86,

0.48

0.82

0.51 0.41 0.22 0.41


0.85 0.91 0.87

0.84 0.88 0.81

-0.09 0.46 0.32 0.35


0.86 0.86 0.83 0.80 0.78 0.78 0.75 0.75

0.62 0.60 0.55 0.45 0.40


Average 0.63


0.63


0.85


0.75


0.62


0.70


0.70


POSTNATAL GROWTH IN THE ALBINO RAT 45

SO there appears to be a tendency to closer correlation, as well as to greater variability, in the males. It will be observed, on comparing this table with the preceding, that (excepting the head) the organs most closely correlated with the body weight are not those of least variability in weight, but a group which is somewhat more variable than the body as a whole (kidneys, liver, lungs). In these organs, the average coefficient of correlation varies from 0.80 to 0.86.

Next come the brain and heart (0.78). The gonads are more closely correlated (0.75) with the body weight than might be anticipated from their variability in weight; while the eyeballs (0.45) and suprarenals (0.40) are lowest in the scale.

The average coefficient of correlation for the organs studied is 0.70. It will be noted that there seems to be a direct relation between the variability of the viscera and their correlation with the body weight. In both it is evident that the average coefficient is lowest at birth and seven days, and highest at three weeks.

As a matter of fact, however, a higher coefficient of correlation is to be expected during growth at times when the body weight and organ weight are most variable. This is due to a 'spurious correlation,' which has been referred to repeatedly in connection with the discussion of correlation of the individual organs with the body weight at the definite age periods. When all the rats at a given age are grouped together there is naturally, on account of their unequal growth, a considerable scattering of the body weights and corresponding organ weights. This causes an augmentation of the real coefficient of correlation, in accordance with the general principle of correlation due to heterogeneity of material" (Yule '11, p. 214). To determine the true correlation between the body weight and organ weight at any given age, it would be necessary to have a sufficient number of animals so that they could be separated into groups of approximately the same body weight or organ weight. The present data are unfortunately inadequate for this purpose. It is evident, however, that all the coefficients of correlation above given are somewhat too high, and that no conclusions can be drawn from them as


46 CM. JACKSON

to change in correlation with age. Coefficients of correlation in different organs at the same age may be compared with each other, however, since the body weights and the 'spurious correlation' factor would be the same for all.

The coefficient of variation of the relative or percentage weight is usually much lower than that of the absolute weight of the various organs.^ The difference is inconstant, however, and the eyeballs form a marked exception to the rule. In most cases, therefore, the percentage weight of an organ can be predicted much more accurately than the absolute weight of the organ at any given age. The data agree in general with the theory that the growth of the individual organs is correlated with the growth of the whole body more closely than with age, as Donaldson ('08) has found for the brain of the rat.

For comparison of the variability in the human viscera, a few data are available. Pearson ('97) from data of Reid and Peacock calculates the coefficients of variation for the human heart (absolute weight), male, 19.8, female, 20.7; liver, male, 14.5, female, 22.2; kidneys, male, 20.5, female, 22.5. Greenwood ('04) from more extensive data finds the coefficients of variation for healthy organs (males only) approximately as follows: heart, 17.7; liver, 14.8; kidneys, 16.8; spleen, 38. From the foregoing it would therefore appear that the heart, liver and kidneys are less variable in man than in the rat. The spleen is more nearly alike in the two forms, and in both is by far the most variable of the organs compared. Pearl ('05) classifies the human bodily characters with reference to variability in three groups: (1) those with coefficient of variation above 10, viscera in general, whose weight depends largely upon the general metabolic condition of the body and in which natural selection is concerned with functional ability rather than with size: (2) those below 7, chiefly skeletal dimensions, in which the conditions of (1) are reversed; and (3) those with coefficient from 7 to 10, including brain and skull capacity, in which intermediate conditions are found.

' Chisolm ('11) finds the coefficient of variation for the percentage weights in adult rabbits, for the spleen, 46.7; kidneys, 16.5; liver, 32.5 This would indicate that these organs are more variable in the rabbit than in the albino rat.


POSTNATAL GROWTH IN THE ALBINO RAT 47

For various skull measurements, Hatai ('08) finds the variability in the adult albino rat slightly less than in man.

Finally, the question may be raised as to the significance of the relative size of an organ. Why does it vary so greatly in different species and even in individuals of the same species, especially at different stages of development? While a satisfactory answer to this question is impossible in the present state of our knowledge, certain features of interest in this connection may be noted. There are several conditions according to which a priori differences in the relative size of organs might be expected; even though, to a certain extent, a change in the relative size of any part tends, through physiological correlation, to produce a corresponding change in other parts of the body.

1. The relative size of an organ may vary according to the size of the whole body. On account of mechanical principles, with changes in the ratio of dimensions to surface and mass of the body, and so forth, corresponding changes in the relative size of skin, skeleton, musculature, et cetera, might be expected, as has been pointed out by Welcker and Brandt ('03).

2. Unequal growth of any (especially of a large) part of the body necessarily involves an inverse change in the relative size of the remaining parts of the body. For example, the relative increase in the musculature during the later periods of growth involves a corresponding decrease in the relative size of other organs. Fluctuations in the relative amounts of intestinal contents, body fat, hair, and so forth, may likewise produce changes in the relative size of the various organs, as has been emphasized by E. Voit ('05).

3. Differentiation in histological structure may result in more efficient physiological activity, so that a relatively smaller organ may suffice to perform a given function. For example, it is doubtless partly on this account that the embryonic heart and other organs are relatively larger than later.

4. Changes in functional activity, as is well known, occasion an atrophy or hypertrophy of the corresponding organs, with resultant changes in their relative size. Thus an increase of protein in the diet throws more work upon the liver and kidneys,


48 C. M. JACKSON

causing them to become relatively larger. This principle of 'use and disuse' is widespread in its application as an explanation of changes in the relative size of organs. Joseph ('08) suggests that the relatively large heart in the smaller mammals may be due to a 'physiological hypertrophy,' correlated with the more rapid rate of pulsation.

5. Within certain limits, however, an organ is normally somewhat larger than is necessary to perform the functional activity usually demanded. Fluctuations in the amount of this excess of structure (which Melzer has termed the 'factor of safety') may account for a part of the individual variations in relative size found to a greater or less extent in every organ.

6. Various pathological conditions may be associated, either directly or indirectly, with changes in the relative size of individual organs or parts of the body.

The foregoing illustrate conditions associated with changes in the relative size of organs, and representing for the most part teleological explanations, rather than actual causes of varying growth rates in the different organs and parts. For any given organ the immediate cause of its characteristic rate of growth is, like that for the whole body, a complex of factors. These may be divided into (a) intrinsic structure and chemical composition of the organ and (b) its environment, especially the conditions affecting the vascular supply, which transmits the respiratory, nutritive and excretory materials, as well as specific hormones or substances which may stimulate the organ to growth and activity.

SUMMARY

Although the extensive data presented cannot satisfactorily be summarized, some of the more important general conclusions concerning growth and variability of the albino rat are included in the following :

1. At birth the males invariably exceed the females of the same litter in average body weight. Although growth is in general more vigorous in the females during the first six weeks, they fail to overtake the males in the majority of litters (at observed ages). At six weeks, however, the total average body weight of


POSTNATAL GROWTH IN THE ALBINO RAT 49

the females approaches or slightly exceeds that of the males. After six weeks, growth is more vigorous in the males and the females lag behind.

2. Variability in body weight is lowest at birth (the coefficient being about 12) and is not much higher at seven days (16). It appears highest at three weeks (28), and at later periods varies from 19 to 21. The average coefficient, taking all ages together, is 19. There is in the rat no evident correlation between variability and rapidity of growth. The coefficient of variation is practically the same for the gross as for the net body weight. The coefficient usually appears higher in the male, but the difference is slight and of doubtful significance.

3. Fraternal variability (within the litter) in body weight is very low, usually less than half that of the total population of the same age.

4. As the growth rate of the whole body is a resultant of the varying growth rates of the component systems (musculature, skeleton, viscera, and so forth) so the growth rate of the visceral group is a resultant of the different growth rates of the individual organs. While the visceral group as a whole reaches its maximum relative (percentage) weight at about the age of three weeks, the thyroid gland apparently is relatively largest at birth, the brain, spinal cord, eyeballs, lungs and spleen about the second week, the thjonus, heart, suprarenals (?) and kidneys at three weeks, intestinal canal and liver at six weeks, and the gonads at ten weeks. Differences according to sex, aside from the gonads, are most marked in the eyeballs (?) and suprarenal glands.

5. Similarly the variability in the weight of the body as a whole depends upon the variability of its component parts. In this respect the individual organs may be classified in three groups: (1) the head and head organs (brain, eyeballs) form a group of comparatively slight variability (average coefficient 10 to 12) ; (2) the heart, lungs, liver, suprarenals and kidneys form a group of moderate variability (average coefficient 21 to 26) ; (3) the thymus, spleen, gonads and intestinal canal (with contents) form an extremely variable group (average coefficient above ,29).

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 1


50 C. M. JACKSON

The average coefficient of variation for the viscera, taking all ages together, is about 25. The average however is lowest at birth and one week (22 or 23) and highest at three weeks (31), resembling in this respect the variability in the weight of the body as a whole.

6. The coefficient of variation for the relative or percentage weight is usually much lower than that for the absolute weight of the various organs, as the growth of the organs is correlated with that of the whole body more closely than with age. The eyeballs, however, form a conspicuous exception to this rule.

7. The highest degree of correlation with the body weight (average coefficient 0.80 to 0.86) is found, not among the organs of least variability (excepting the head), but among those of moderate variability (kidneys, liver, lungs). Next come the brain, heart and gonads (0.75 to 0.78). The remaining organs are much less closely correlated with the body weight, the lowest being the eyeballs and suprarenals (0.40 to 0.45). The average coefficient of correlation, all ages together, is about 0.70. The figures for coefficient of correlation are somewhat too high, being augmented by a 'spurious correlation' due to the heterogeneity of the weights when grouped at the age periods. Like the variability, the correlation appears lowest at birth and one week (0.63) and highest at three weeks (0.85). Similarly the coefficient of correlation appears usually higher in the male, as is also the case with the coefficient of variation.

BIBLIOGRAPHY

Boas, F. 1897 The growth of Toronto school children, Report of U. S. Commissioner of Education, II.

Boas, 0.0., and Wissler, O. O. 1904 Statistics of growth. Report U. S. Commissioner of Education, I.

Chisolm, R. a. 1911 On the size and growth of the blood in tame rats. Quarterly Jour. Exp. Physiol., vol. 4.

CuRRiE, D. H. 1910 The rat and its relation to the public health (by various authors). Public Health and Marine Hospital Service of the U. S.

Davenport, C. B. 1904 Statistical methods with especial reference to biological variation. 2nd ed.. New York.

DissELHORST, R. 1898 tjber Asymetrien und Gewichtsunterschiede der Gesahlechtsorgane, Archiv f. wiss. u. prakt. Thierheilk. Bd. 24.


POSTNATAL GROWTH IN THE ALBINO RAT 51

DissELHORST, R. 1908 Gewichts- und Volumszunahme der mannlichen Keimdriisen bei Vogeln und Saugern in der Paarungszeit; Unabhangigkeit des Wachstums. Anat. Anz., Bd. 32, S. 113-117.

Donaldson, H. H. 1906 A comparison of the white rat with man in respect to the growth of the entire body. Boas Memorial Volume, New York.

1908 A comparison of the albino rat with man in respect to the growth of the brain and of the spinal cord. Jour. Comp. Neur., vol. 18.

1909 On the relation of the body length to the body weight and to the weight of the brain and of the spinal cord in the albino rat. Jour. Comp. Neur., vol. 19.

Dunn, Elizabeth H. 1912 The influence of age, sex, weight and relationship upon the number of medullated nerve fibers and on the size of the largest fibers in the ventral root of the second cervical nerve of the albino rat. Jour. Comp. Neur., vol. 22.

Galton, Francis 1894 Natural inheritance. New York.

Greenwood, M. 1904 A first study of the weight, variability and correlation of the human viscera, with special reference to the healthy and diseased heart. Biometrika, vol. 3.

Hatai, S. 1908 Studies on the variation and correlation of skull measurements in both sexes of mature albino rats. Amer. Jour. Anat., vol. 7. 1913 On the weight of the abdominal and thoracic viscera, and sex glands, ductless glands, and the eyeballs of the albino rat according to body weight. Amer. Jour. Anat., vol. 15.

Jackson, C. M. 1909 *0n the prenatal growth of the human body and the relative growth of the various organs and parts. Amer. Jour. Anat., vol. 9. 1912 On the recognition of sex through external characters in the young rat. Biol. Bulletin, vol. 23.

Jackson, C. M., and Lowrey, L. G. 1912 On the relative growth of the component parts (head, trunk and extremities) and systems (skin, skeleton, musculature, and viscera) of the albino rat. Anat. Rec, vol. 6.

Joseph, D. R. 1908 The ratio between the heart weight and body weight in various mammals. Jour. Exper. Med., vol. 10.

Kellicott, W. E. 1908 The growth of the brain and viscera in the smooth dog-fish (Mustelus canis, INIitchill). Amer. Jour. Anat., vol. 8.

King, Helen D. 1911 The effects of pneumonia and of post-mortem changes on the percentage of water in the brain of the albino rat. Jour. Comp. Neur., vol. 21.

MiTgHELL, O. W. H. 1912 Bacillus muris as the etiological agent of pneumonitis in white rats and its pathogenicity for laboratory animals. Jour. Infect. Diseases, vol. 10.

Pearl, R. 1905 Biometrical studies on man. Variation and correlation in brainweight. Biometrika, vol. 4. (Also, Some results of a study of variation and correlation in brain-weight. Jour. Comp. Neur., vol. 25. 1905.)

Pearson, Karl 1897 The chances of death and other studies in evolution. 2 vols.; London and New York.

1899 Data for the problem of evolution in man. Proc. Roj^al Soc, London, vol. 66.

1900 Thegrammar of science. 2nd ed., London.


52 C. M. JACKSON

Porter, W. T. 1905 Growth of St. Louis children. Trans. St. Louis Acad. Science, voL 6. (Cf. also, The relation between the growth of children and their deviation from the physical type of their sex and age. Ibid.)

QuETELET, A. 1871 Anthropometrie, ou mesure des differents facultes de I'homme. Bruzelles et Paris.

Schuster, E. 1911 First results from the Oxford anthropometric laboratory. Biometrika, vol. 8.

Slonaker, J. R. 1912 The effect of a strictly vegetable diet on the spontaneous activity, the rate of growth, and the longevity of the albino rat. Leland Stanford Junior University Publications, University Series. (Cf . also. The normal activity of the albino rat from birth to natural death, its rate of growth and the duration of life. Jour. Animal Behavior, vol. 2, 1912.)

Thoma, R. 1882 Untersuchungen liber die Grosse und das Gewicht der anatomischen Bestandtheile des menschlichen Korpers im gesunden und im kranken Zustande. Leipzig.

VoiT, Erwin 1905 Welchen Schwankungen unterliegt das Verhaltnis der Organgewichte zum Gesamtgewichte des Tieres? Zeitschr. f. Biol., Bd. 46.

Watson, Chalmers 1910 Food and feeding in health and disease. Edinburgh and London. (In the Appendix the results of several feeding experiments and investigations are republished from the Journal of Physiology, etc.)

Watson, J. B. 1905 The effect of the bearing of young upon the body-weight, and the weight of the central nervous system of the female white rat. Jour. Comp. Neur., vol. 15.

Welcker, H. AND Brandt, A. 1903 Gewichtswerte der Korperorgane bei dem Menschen und den Tieren. Archiv f. AnthropoL, Bd. 28.

Yule, G. U. 1911 An introduction to the theory of statistics. London and Philadelphia.


TABLE 1 Litters of albino rats used


6



MALES


FEMALES


m


AGEi OF LITTER



Average gross


Average net


Average gross


Average net


a



No


weight


weight


No. weight


weight


J




(and range)


(and range)


1 (and range)


(and range)



1



grams


grams


grams


grams


20 1 newborn


2


4.12 (3.67-4.57)


4.04 (3.59-4.48)


2 : 3.71 (3.56-3.86)


3.65 (3.50-3.79)


22 1 newborn


5


4.78(3.48-5.56)


4.69 (3.43-5.17)


3 , 4.43 (4.0.3-4.70)


4.24 (4.01-4.62)


23 1 newborn


1


4.44


4.30


oi


!


24 1 newborn


5


5.00 (4.68-5.25)


14.90 (4.56-5.17)


4 1 4.74 (4.62-4.91)


4.63 (4.50-4.84)


25 I newborn


1


5.19


4.98


1 4.28


4.07


47


1 newborn


3


6.50 (6.40-6.62)


6.03 (5.88-6.25)





48


newborn


3


5.73 (5.53-5.97)


5.42 (5.24-5.59)


3


5.47 (5.29-5.59)


5.05 (4.90-5.20)


52


newborn


4


3.96 (3.41-4.75)


3.82 (3.30-4.60)


1


3.88


3.72


53a i newborn


1


5.27


5.09


5


4.93 (4.67-5.14)


4.77 (4.52-4.98)


58 1 newborn


4


5.48 (5.30-5.62)


5.32 rs. 16-5.45)


3 j 4.95 (4.23-5.41)


4.82 (4.12-5.26)


61 newborn


4


5.49 (5.23-5.66)


5.35 (5.10-5.52)


5


5.27 (5.00-5.53)


5.13 (4.91-5.44)


64 1 newborn


8


4.77 (4.35-5.09)


4.46 (4.06-4.74)


3


4.37 (4.24-4.55)


4.07 (3.96-4.22)


M9 newborn


5


5.42 (5.1-5.8)



3


5.13 (4.9-5.4)



MIO newborn


2


5.85 (5.7-6.0)



4


5.58 (4.8-6.3)



A28 1 newborn


4


4.71



2


4.37



A29 j newborn


2


4.70



4


4.63



A32


newborn


3


5.11 (4.95-5.38)



5


4.73 (4.50-4.91)



M8


1 day


3


6.08 (5.75-6.35)



5


5.43 (4.70-6.05)



62


1 day


3


5.41 (5.30-5.56)


5.13 (4.97-5.33)


8 j 4.96 (4.45-5.19)


4.74 (4.27-4.98)


63


1 day





5


4.90 (4.68-5.07)


4.53 (4.15-4.78)


18


7 days


5


9.92 (8.54-10.46)


9.56 (8.29-10.02)


3


9.13 (8.88-9.57)


8.78 (8.58-9.11)


19


7 days


6


9.42 (9.11-9.58)


9.15 (8.85-9.33)


3 1 8.04 (7.56-8.72)


7.71 (7.19-8.32)


42


7 days


2


11.62 (10.80-12.43)


10.59 (9.84-11.33)


4 12.07 (11.32-12.70)


11.02 (10.04-11.77)


46


7 days


1


15.56


14.95


1 Il4.13


13.23


49


7 days


7


12.36 (10.58-13.06)


11.77 (9.94-12.41)


3


11.60 (10.80-12.17)


11.07 (10.33 11-68;


51


7 days


2 11.95 (11.89-12.00)


11.16(11.04-11.27)


3


11.67 (11.50-11.77)


10.93 (10.71-11.07)


56a


7 days


!



3


10.46 (9.87-10.96)


10.01 (9.48-10.43)


59


7 days


4 9.39 (8.07-10.13)


8.78 (7.40-9.55)


3


10.12 (9.78-10.42)


9.44 (9.08-9.85)


60


7 days


3


8.48 (6.46-10.46)


8.24 (6.32-10.15)


4


9.40 (9.05-9.78)


9.10 (8.70-9.63)


M6


7 days


5


12.01 (9.10-12.95)



3


11.55 (11.15-12.15)



M7


7 days


2


10.75 (10.20-11.30)



4


10.96 (10.55-11.40)



M8


7 days


3


12.22 (12.05-12.55)



5


11.52 (10.55-12.25)



M9


7 days


5


9.26 (8.8-9.7)



3


8.7 (8.4-9.1)



MIO


7 days


2 j


11.3 (11.1-11.5)



4


10.75 (9.2-11.8)



A28


7 days


4


7.52



2


7.56


,


A29


7 days


2 12.42



4


10.42



A32


7 days


3


9.83 (9.44-10.14)



5


9.50 (9.04-10.08)



A33


7 days





7


9.44 (6.70-10.12)



M6


14 days


5 ,21.0 (20.4-21.9)



3


19.5 (19.1-19.9)



M8


14 days


3 18.8 18.3-19.2)



5


17.8 (16.9-18.7)



M9


14 days


3 18.7(18.5-19.0)



3


17.9 (17.5-18.6)



MIO


14 days


2 17.7 (17.0-18.4)



4


17.9 (15.3-19.3)



26


20 days


5 16.66 (13.92-18.52)


16.12 (13.82-17.74)


3


16.98 (15.39-17.82)


16.20(14.99-17.03)


43


20 days


3 22.68 (21.90-23.15)


21.67 (20.98-22.04)


3


22.58 (22.45-22.65)


21 59 (21.52-21.71)


45


20 days


3 !14.02 (13.49-14.56) '


13.55 (13.00-14.09)


8


13.31 (12.71-13.76)


12.85 (12.34-13.23)


53b


20 days


5 29.42 (27.25-30.94)


28.31 (25.80-29.79)





56b


20 days


2 33.84 (29.70-37.97)


32.05 (28.20-35.90)




57


20 days


4 22.11 (21.16-22.43)


20.50 (19.64-21.90)


3


19.46 (18.30-20.22)


18.05 (17.34-18.60)


Ml


20 days


4 23.7 (22.9-24.1)



1


25.0



M2


20 days


4 26.9 (25.8-27.9)



1


24.8



M6


20 days


5 27.2 (23.7-28.8)



3


26.3 (25.8-26.8)



M7


20 days


2 21.0 (15.7-26.4)



4 24.4 (19.7-27.0)



53


TABLE 1 (Continued)


d



MALES



TEMALKS


a


AGE OF

LITTER


No.


Average gross

weight

(and range)


Average net

weight (and range)


No.


Average gross

weight

(and range)


Average net

weight (and range)





grams


grams



grams


grama


M8


20 days


3


26.2 (25.8-26.5)



5


25.1 (24.7-25.5)



M9


20 days


3


26.9 (25.6-27.9)



3


25.8 (24.3-26.6)



MIO


20 days


2


24.7 (22.6-26.8)



4


24.7 (21.8-26.5)



A28


20 days


4


25.4 (23.5-27.0)



2


25.2 (24.3-26.2)



A29


20 days


2


24.1 (23.4-24.8)


i *


23.2 (21.5-24.1)



A33


20 days




1 7


24.6 (21.0-26.1)



15


21 days


2


18.22 (17.71-18.72)


17.77 (17.4S-18.08)j 8


17.72 (15.27-19.19)


16.95 (14.77-17.89)


M7


30 days


1


45.2 (Idied)


4


40.8 (24.1-48.7)



M8


30 days


3


47.9 (44.8-50.2)



5


44.0 (41.1-48.1)



M9


30 days



(killed)



3


49.9 (47.2-51.4)



29


41 days


2


54.55 (52.9-56.2)


49.65 (48.9-50.4)


6


49.65 (42.8-53.7)


46.0 (40.5-48.6)


30


41 days


4


56.58 (55.1-58.4)


50.78 (49.6-52.2-)


3


52.67 (40.8-58.7)


48.43 (39.4-53.9)


M2


41 days


4


73.5 (70.2-80.9)



1


66.7



M9


42 days



(killed)



3


82.5 (75.2-86.6)



MIO


42 days


2


89.0 (83.0-95.0)



4


80.0 (66.0-92.0)



A28


42 days


4


60.8 (57.3-63.5)



2


65.3 (59.8-70.7)



A29


42 days


2


66.7 (66.1-67.4)



4


61.1 (59.6-63.6)



A33


42 days





5


62.1 (58.4-69.8)


(2 killed)


31


42 days


10


48.02 (39.4-53.4)


44.63 (38.1-49.6)


1


42.4


39.5


82


42 days


5


51.78 (43.1-57.4)


49.24 (41.3-54.9)


4


46.27 (42.9-48.2)


44.05 (39.6-46.3)


50a


42 days


1


89.0


82.5


3


84.57 (83.5-85.6)


78.07 (76.8-78.8)


M6


42 days


3


62.7 (55.0-69.0)



1


66.0



33


43 days





3


49.83 (42.9-60.8)


45.4 (40.1-54.5)


Ml


43 days


4


73.1 (64.4-81.6)



1


70.0



M7


44 days


1


57.1



4


56.8 (23.5-71.8)



MS


48 days


3


105.9 (93.5-114.2)



5


86.5 (81.6-95.0)



4


69 days


3


117.1 (107.4-125.8)


112.5 (103.6-120.8)'




5


70 days




3 |100.7 (86.1-127.3)


97.2 (83.2-122.2)


12


70 days


3


89.9 (77.5-97.9)


86.2(74.1-93.7) i 2:100.2(98.2-102.1)


89.2 (87.2-91.1)


40


70 days


1


151.1


145.3 ; 1 ,101.9


97.6


41


70 days


4


117.8 (110.1-124.8)


112.7 (105.4-119.1) 4 91.5 (82.2-106.9)


88.1 (78.2-102.3)


50b


70 days


1


182.2


175.2 2 :i29. 6 (123.9-135.4)


125.3 (120.3-130.2)


1 j 71 days


2


149.9 (140.8-159.)


144.1 (134.1-154.) 7


110.7 (85.4-126.9)


107.1 (82.7-123.5)


44 1 71 days


.6


119.0 (111.8-134.1)


114.0 (108.4-128.6) 4


93.0 (75.5-100.)


88.2 (71.1-94.8)


M8 70 days


3


187.3 (177.-193.)


5 1


135.0 (124.-145.)



10


149 days




2 137.8 (126.-149.6)


134.4 (122.5-146.3)


6


150 days


5


158 1 (142.5-167.8)


154.7 (139.4-164.6) 4 145.3 (120.6-159.9)


142.1- (115.9-156.7)


11


150 days


4


157.2 (150.7-166.2)


151.7 (145.9-157.1) 3 il60.0 (150.-164.0)


151.9 (140.1-158.1)


36


150 days


3


172.6 (163.3-189.8)


168.0 (156.3-186.5) 3


127.9 (111.8-136.3)


125.2 (110.3-133.)


38


150 days


6


152.0 (120.6-176.8)


149.7 (118.8-176.) ' 4


114.9 (97.3-130.3)


111.9 (94.6-126.2)


28


151 days


2


247.2 (241.8-252.5)


243.1 (239.4-246.8) 5 '160.8 (133.5-174.1)

i


158.2 (131.8-171.5)


2


1 year




2 184.3 (183.5-185.5)


181.9 (181.-182.7)


3


1 year




j 3 Il92.0 (156.2-209.7)


189.1 (154.-207.3)


4


1 year





2 195.8 (188.7-202.8)


190.6 (182.6-198.5)


7


1 year


1


290.5


287.3


2 ,120.9 (107.3-134.5)


119.5 (105.8-133.1)


8


1 year


1


142.8


139.6


1 1188.3


186.0


13 1 year





2


174.8 (142.-207.5)


172.5 (141.-204.)


34 1 1 year





1


115.8


114.2


37 1 year


1


281.1


276.4





39 1 year


2


175.3 (118.4-2.32.1)


172.4 (115.6-229.2)


3 !ll3.8 (86.1-165.0)


110.8 (83.4-161.1)


35 13 mo.




4 il75.0 (167.2-181.3)


170.7 (161.9-178.6)


54


POSTNATAL GROWTH IN THE ALBINO RAT


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MUSCLE DEGENERATION AND ITS RELATION TO THE

ORIGIN OF EOSINOPHILE LEUCOCYTES IN

AMPHIBIA (SALAMANDRA ATRA)

J. A. BADERTSCHER

Department of Histology and Embryology, Cornell University, Ithaca

SEVEN FIGURES

The investigation presented in this paper has a twofold object: Primarily, to offer an explanation for the source of large numbers of free eosin-staining granules and eosinophile leucocytes found in connection with degenerating muscle and with the absorption of the gills taking place during the period of transformation of Salamandra atra; and, secondarily, to present briefly the manner of muscle degeneration as it occurs in this form. In Salamandra atra an intimate relation exists between the origin of the eosinophile leucocytes and degenerating muscle tissue so that the one cannot be studied without considering the other. ^

An investigation into the origin of the eosinophile leucocytes seemed proper for the reason that the source and nature of the granules in the granular leucocytes is still a much debated question. Also, as far as I was able to determine, no work has yet

1 The material used was prepared by Dr. B. F. Kingsbury to whom it was kindly furnished by Professor MoUier of the Histological-Embryological Institute, Munich. I wish to express my thanks to Professor Kingsbury for the use of the material and for the valuable suggestions given me on this work. Twenty-seven series were examined, of which six were of animals still in the larval condition, ranging in length from 12.5 to 38 mm.; thirteen in the period of transformation, ranging in length from 44 to 48 mm.; and seven in the adult condition. Different approved fixers (Helly's, Zenker's, Tri-chlor-acetic) were used in fixing the material. Only the head and anterior part of the body, including the heart, were sectioned. The sections were cut from 8 to 15 micra in thickness. The greater number of the series were stained with iron or copper (Weigert's) hematoxylin, and eosin or orange-G was used as a counter stain.

69


70 J. A. BADERTSCHER

been done on amphibians with a view of tracing out the origin of eosinophile leucocytes.

Brown ('98) is of the opinion that the products of degenerated muscle tissue are a source of eosinophile granules. In cases of trichinosis in man, he observed that the muscle fibers invaded by Trichina spiralis undergo a granulation in the immediate neighborhood of the parasite. All through the affected parts of a muscle are found large numbers of leucocytes, many of which are of the eosinophile type. The latter are very numerous in areas of marked degeneration. Though there were no evidences that the leucocytes ingested bodily fragments of muscle tissue, he thinks that possibly the degenerated substance is taken up in a soluble form and transformed into eosinophile granules.

Weidenreich ('08, '11), who worked with mammals, is the chief exponent of the theory that the eosinophile granules are hemoglobin, containing products of degenerated erythrocytes. According to him, leucocytes may ingest entire erythrocytes which then undergo disintegration, resulting in the formation of eosinophile granules, or the disintegration of the red cells may take place extracellularly and the eosinophile granules thus formed may be taken up, in their fully formed condition, by the leucocytes. In referring to the work of Brown he says Til, p. 635) : " . auch die Mogiichkeit ist nicht ausgeschlossen, dass das Hamoglobin der Muskelfasern, .... eine Rolle bei der Bildung der Granula spielt."

The amphibians furnish most suitable material to test the correctness of this theory, for during the period of metamorphosis marked disintegration of both erythrocytes and muscle tissue takes place. Also the degeneration of muscle tissue in this class of animals, in contrast to the pathological degeneration in cases of trichinosis, is a normal process that regularly occurs while they are adapting themselves to a different mode of life. While Brown ('98) pointed out a possible relation of the origin of eosinophile cells to pathological degenerating muscle, no work apparently has yet been done on any form to show a relation of the origin of those cells to normally degenerating muscle.


MUSCLE DEGENERATION IN AMPHIBIA


71


Among some of the muscles that undergo degeneration in this salamander are the gill muscles, some of the muscles of the lower jaw (fig. 1) and some of the dorsal muscles along the spinal column (fig. 4). In the specimens studied, muscle degeneration was most pronounced in those far along in their period of transformation or in the young adult, thus indicating that even after the external signs of metamorphosis have been obliterated, there are still morphological changes taking place internally. In the degenerating



Fig. 1 Part of a transverse section through the head of a 51 mm. young adult, showing the position in the lower jaw of a muscle well advanced in degeneration. M.c, Meckel's cartilage; d.ni., degenerating muscle; t., tongue. Photo. X 30.


muscles and gills there is also a degeneration of blood capillaries. As the capillaries are broken down there is a marked destruction of erythrocytes which can be satisfactorily demonstrated especially in the degenerating gills. If the eosinophile granules are derived from degenerating red cells and degenerating muscle tissue, both granules and eosinophile leucocytes should be found in connection with those tissues. On examining the different series from the larval to the adult condition the evidence is conclusive that the free eosinophile granules are products of the degenerating



jfid .



Fig. 2 Section of a part of the degenerating muscle represented in Fig. 1, showing a large accumulation of eosinophile leucocytes in connection with the degenerating muscle fibers. The section being rather thick the granules in the eosinophile cells could not be photographed sharply. Photo. X 370.

Fig. 3 Degenerating muscle taken a few sections posterior to that represented in figure 1. Free eosinophile granules, represented by black dots about the size of an ordinary pin head; some a little smaller, can be seen scattered promiscuously along two degenerating muscle fibers and in the spaces not occupied by cells, most of which are eosinophile leucocytes. On account of the section being rather thick not all parts could be focused sharply. Photo. X 700.

72


MUSCLE DEGENERATION IN AMPHIBIA 73

tissues above named and are taken up by the leucocytes, transforming them into the eosinophile type. These observations extend the scope of Weidenreich's views to the amphibia.

During the larval period only an occasional eosinophile leucocyte can be found in the blood stream. A few cells containing eosinophile granules can also be demonstrated in the connective tissue. The number of eosinophile cells at this stage of development is so small that only after considerable searching could one be found. During the period of transformation, eosinophile cells can invariably be demonstrated in connection with degenerating muscle, the number of those cells corresponding approximately with the advancement of degeneration. Where degeneration is at its greatest height a large accumulation of eosinophile cells is present (fig. 2) . Also more eosinophile cells can be demonstrated in the blood stream and especially in the connective tissue in the neighborhood of the degenerating parts during the period of transformation than before or after this period. No differential counts were made of the leucocytes in the blood stream. The greater number of eosinophile cells during the perod of metamorphosis was evident by the great frequency in their occurrence when searching for them in the sections.

In the section from which figure 3 was photographed can be seen free eosinophile granules lying among the debris of degenerating muscle. That these granules lie free in the regions mentioned is beyond doubt for when the sections are followed along in serial order they are not found to lie in the bodies of leucocytes but lie loose in the intercellular spaces. These granules, apparently formed from the degenerating muscle and degerating erythrocytes, vary somewhat in size, but the majority closely approximate in size and hue those found in the eosinophile leucocytes. They have various positions in respect to the degenerating muscles fibers. They may be found among lymphocytes or eosinophile leucocytes some distance away from a muscle fiber, scattered promiscuously' among them, strung along in a row between two adjacent fibers, or often in small recesses of a muscle fiber. Some muscle fibers can be seen, the central portions of which are completely degenerated, the gap between the two remaining ends being filled in



sp.c.






Fig 4 Transverse section through the neck of a 47 mm. adult, showing the position of a muscle in which degeneration has just begun; sp.c, spinal cord; d.m., degenerating muscle. Photo. X 30.

Fig. 5 Portion of the degenerating muscle represented in figure 4, showing the accumulation of lymphocytes among the muscle fibers in the first stage of muscle degeneration. Photo. X 450.

74


MUSCLE DEGENERATION IN AMPHIBIA 75

with numerous eosinophile granules and while blood cells, thus suggesting the origin of the granules from the degenerating muscle fiber. Disintegrated erythrocytes can also be seen in connection with degenerating muscle tissue. However, from the material studied the majority of eosinophile granules found in degenerating muscle are products of muscle degeneration while the smaller portion is contributed by degenerating red cells. Eosinophile leucocytes can often be seen in connective tissue in the neighborhood of degenerating muscle. These through their amoeboid movements have perhaps wandered away from their source of origin.

In the degeneration of the gills numerous blood vessels are necessarily broken down, producing in some cases stasis in the small vessels resulting in the disintegration of the erythrocytes in those parts. Degenerating red cells can be demonstrated especially where the degeneration of the gills is well advanced. They can also be found isolated or in smaller and larger groups outside of the walls of blood vessels. The degeneration of some is evident, as can be seen from the appearance of their nuclei which stain intensely, lose their oval shape and become irregular in outline and a few were seen apparently about to be extruded from the cell body. The cell envelope also becomes very irregular in outline and in some cases even ruptured. In some the cytoplasmic material is coarsely granular, losing its seemingly homogeneous nature. In others globular masses of varying sizes are apparently about to be detached from the cell. Many of these large masses or coarse granules together with smaller granules can often be seen lying free in close approximation to degenerating erythrocytes. These larger masses and granules stain with eosin, the smaller granules staining more deeply than the coarse granules. A difference readily observed between the eosinophile granules formed from the degenerating erythrocytes and degenerating muscle tissue is that the granules formed from the former vary much in size as stated above while those formed from the latter, while varying somewhat in size, approximate more nearly the dimensions of those found in the eosinophile cells. That these eosinophile granules are taken up by cells of the character of leucocytes is indicated b}^ the presence of such cells, sometimes in large numbers, in the immediate


76


J. A. BADERTSCHER


neighborhood or in contact with them (fig. 6). I have been unable to find leucocytes ingesting entire or large fragments of erythrocytes as was observed by Lewis ('04) who worked with the ungulates and by Weidenreich ('08) who worked with guineapigs. A possible reason for amphibian leucocytes not ingesting entire erythrocytes is found in the large size of the red blood cells in this class of animals.



7a



7b



Fig. 6 Lymphocytes among free eosinophile granules found in degenerating muscle. Camera lucida drawing.

Fig. 7 Series of diagrams, a, b, c, to show the formation of eosinophile leucocytes from lymphocytes. The limit of the cytoplasm is marked by a dotted line. Camera lucida drawings.


The first evidences of muscle degeneration are marked by a collection of leucocytes among the muscle fibers. The number of leucocytes that accumulate between the fibers is so great that the width of the intercellular spaces is increased to several times their normal extent (fig. 5) . In this accumulation of white blood cells the great majority are the smallest of the leucocytes. Their nuclei are large, taking up almost the entire cell body. The outline of the nuclei which vary, may be round, shghtly elongated, gently dented on one side or irregularly crescent-shaped, indicat


MUSCLE DEGENERATION IN AMPHIBIA 77

ing amoeboid movement. The rim of cytoplasm around the nucleus is very narrow, in some cells so scanty in its amount that it is difficult to see. The minority of cells in this accumulation are larger than the ones described. They have large nuclei which may vary somewhat in outline as those of the smallest white cells. They have a wide rim of cytoplasm around the nuclei to which the large size of the cell is due. According to Friedsohn's work ('10) on the amphibian blood, the smaller cells appear to be the small lymphocytes and the larger ones the large lymphocytes. An occasional eosinophile leucocyte can also be seen, while partially and completely degenerated erythrocytes are thinly scattered throughout the accumulation of leucocytes. No perceptible change in the muscle fiber itself has yet taken place in this stage of degeneration.

After the degeneration of the muscle tissue has advanced to the stage in which the degeneration of the fibers is readily perceptible the number of white blood cells is as numerous as at the time when the degeneration was first evident, but the lymphocytes ha\'e greatly decreased, while the eosinophile leucocytes have increased in number. In this stage of degeneration numerous eosinophile granules can be seen lying free in the intercellular spaces and often in contact with lymphocytes and eosinophile leucocytes. The number of eosinophile granules varies greatly in the eosinophile cells, from only a few granules to the gorged condition of some of the cells (fig. 7), thus suggesting again that the lymphocytes take up eosinophile granules. The presence of the large number of lymphocytes during the first stages of degeneration and a subsequent reduction of their number and replacement by large numbers of eosinophile leucocytes as degeneration advances, clearly indicates that the eosinophile granules are taken up b}'- the lymphocytes. The point in case is that the lymphocytes are the main cells that take up the granules and are thus converted into eosinophile cells.

The majority of the eosinophile leucocytes found in connection with degenerating muscle and erythrocytes have round or nearly round nuclei. In some the nuclei are of the transitional type


78 J. A. BADERTSCHER

while in others they are of the polymorphic type. In studying the eosinophile leucocytes with the various types of nuclei, one can find sufficient evidence — as can be indicated by a series of gradual transitions from one type of nucleus to another — to confirm the conclusion that all are derived fi^om the round nucleated type of eosinophile cells. This transformation takes place by a gradual change in the shape of their nuclei. They are thus genetically related, the round nucleated type being the forerunner of the transitional and polymorphic types.

No attempt was made to determine the chemical nature of the eosinophile granules. The material was not prepared for that purpose. However, the hemaglobin nature of the eosinophile granules as held by Weidenreich and others, is indicated by circumstantial evidence in that the free eosinophile granules and groupings of eosinophile leucocytes are found only in connection with tissue containing hemoglobin, namely, muscle tissue and erythrocytes. The hemoglobin-containing material of degenerating muscle and erythrocytes apparently does not break up directly into the eosinophile granules, that is, this compound is altered in some way before it is taken up by the lymphocytes. This is indicated by the fact stated above that the very coarse granules or masses are stained more faintly than the smaller granules, which are often stained as deeply as the eosinophile granules in the cells.

Although the evidences are strongly in favor of a hemoglobinnature of the granules, yet when their complex chemical source (erythrocytes and muscle tissue) is considered, it would not be unreasonable to assume that other elements besides hemoglobin also enter into their composition. In considering the chemical nature of the granules Weidenreich ('11, p. 622) says: "Der von Sherrington angeblich festgestellte Phosphorgehalt der Granulation braucht nun keineswegs, wie dieser Autor annimmt, flir eine Nucleinnatur zu sprechen ; denn wir wissen heute, dass die roten Blutkorperchen auch aus dem phosphorhaltigen Lecithin aufgebaut werden, so dass also unter der Voraussetzung, die Granula sind Zerfallsprodukte der Erythrocyten, auch Phosphor in ihnen enthalten sein konnte." If the granules do contain both phosphorous and hemoglobin then the term 'phosphorous-nature'would


MUSCLE DEGENERATION IN AMPHIBIA 79

represent the chemical nature of the granules as well as 'hemoglobin-nature,' for both muscle tissue and erythocytes contain phosphorous-containing compounds. The term 'hemoglobinnature' does not seem to be comprehensive enought to express their chemical nature. The simple phrase " Zerfallsprodukte der Erythrocyte!!" to which I add 'products of degenerating muscle tissue,' would express a greater probability of the granules containing other elements beside hemoglobin, than does the term ' Hiimoglobinnatur . '

To give a detailed account of the different theories relative to the degeneration of muscle tissue in different classes of animals is beyond the scope of this paper. Observations by different investigators of various animal groups indicate that the processes involved in the degeneration of muscle tissue vary considerably. Briefly stated, the interpretations of different workers of the manner by which a muscle degenerates and the part played by the leucocytes in this phenomenon has resulted in the establishment of three general views: (1) a purely aphagocytic process by which the muscle fibers undergo liquefaction, either through the activity of internal conditions of the fiber or through the action of the fluids surrounding them; (2) a purely phagocytic process by which the phagocytes break up the muscle fibers into fragments which are taken up and removed by them ; (3) by a combination of the aphagocytic and phagocytic processes. The views enumerated above are stated only in a general way and the theories as worked out by different investigators have detailed modifications.

Barfurth ('87), in his work on the frog larvae, came to the conclusion that a muscle degenerates independently of the action of phagocytes. The myofibrillae break up into comparatively long fragments, bundles of which make up the sarcolytes. The crossstriations are present in the sarcolytes when they are first formed but gradually disappear as the muscle fragments undergo dissolution. The liquefaction of the fragments takes place within the sarcolemma, which is comparatively resistent to the action of the liquefacient, and disappears only after the sarcoh^tes have been reduced to granules — the debris of degeneration. The initiatory causes of degeneration are unknown to him but he thinks


80 J. A. BADERTSCHER

that they are extrinsic in their nature and is of the opinion that a lack of nutrition or atrophy through disuse after the appearance of the forelegs, are factors to be considered.

According to Katz ('00), who worked with transforming toads, the initiatory causes of degeneration appear to be intrinsic in the muscle itself. Sarcolytes are formed only in some fibers. In the course of degeneration a homogeneous material is produced, which disappears by liquefaction in situ.

Looss ('92) arrived at practically the same conclusions as those of Barfurth, with the exception that a small portion (less than 10 per cent) of the sarcolytes are taken up and destroyed by the phagocytes. The remaining large proportion undergo dissolution in place through the activity of the surrounding fluids.

Metchnikoff ('84), in his work on larval frogs, held that muscle degeneration was effected entirely through the action of phagocytes which collected in large numbers among the degenerating fibers. The source of the numerous phagocytes he then ascribed to the blood. Further investigations ('92) led him to modify the phagocytosis theory of muscle degeneration. In his later work he holds that the phagocytes, which play the important part in muscle degeneration, are formed in the muscle fiber. The first perceptible changes in degeneration are a proliferation of muscle nuclei and an increase in the amount of sarcoplasm. The muscle nuclei and protoplasm then differentiate into cells, the muscle phagocytes, which find their way among the fibrillae, breaking them up into fragments. These fragments (sarcolytes) are ingested and removed by them. The usual type of phagocytes takes no part in muscle degneration, that work being entirely limited to the muscle phagocytes.

Muscle degeneration in Salamandra atra is, to all appearances, a purely aphagocytic process. However, on comparing the degenerating jaw muscles in this class of animals with those in the tails of larval toads, a marked point of difference is recognized in that the formation of sarcolytes does not occur in the former. On examining a series of toads from the larval to the adult condition, sarcolytes in the degenerating tail muscles were found as described by the investigators mentioned above. In the Sala


MUSCLE DEGENERATION IN AMPHIBIA 81

mander the mj^ofibrillae, instead of forming sarcolytes, break up into fragments at short intervals in their course, thus obUterating the cross-striations and giving the affected part of a muscle fiber a granular appearance. These fragments or granules fade away as if by liquefaction. Their dissolution does not take place uniformly along the course of a fiber, so that in the extent of a well advanced degenerating fiber indefinitely outlined groups of dismembered fibrillae can be seen. Coincidently with the liquefaction of the fibrillae appear numerous spherical granules that stain intensely with eosin, the eosinophile granules, which are taken up by the leucocytes in the manner described above. According to Barfurth, in the frog larvae degeneration begins at one end and gradually advances to the opposite end of the fiber. While this is the case in some of the degenerating fibers in Salainandra atra, it is not so with all, for in some fibers can be seen a large gap, produced by degeneration, and containing numerous eosinophile granules and leucocytes, while the ends of the fibers appear quite normal. The sarcolemma degenerates simultaneously with the fibrillae. There are no evidences indicating that it spans the gap of a degenerated portion of a fiber, which would be the case if it were more resistant to the agencies bringing about the liquefaction of the fibrillae. The muscle nuclei and sarcoplasm are not transformed into phagocytes as is the case, according to Metchnikoff, in larval frogs. No perceptible increase in the amount of protoplasm or in the number of nuclei was noticeable. They are, however, very resistant to the agencies that cause the liquefaction of the fibrillae. During the last stages of degeneration they can be seen lying free among the numerous leucocytes and debris of degenerated muscle. On account of their large size they cannot be mistaken for lymphocytes or for connective tissue cells. Their fonn varies. Some retain their natural rod shape, some are crescent in outline, some are bent double so that the two ends are in close approximation, while others are bent and twisted. A considerable number show marked signs of degeneration by the massing of their chromatin. Their fate is unknown to me but from the appearance of some I am of the opinion that they undergo degeneration.

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 1


82 J. A. BADERTSCHER

The origin of the lymphocytes found in a muscle during its first stages of degeneration can be attributed to two sources, namely, connective tissue cells and the blood. The connective tissue cells in the affected part of a muscle lose their stellate form, become rounded, their nuclei grow denser, thus acquiring all the appearances of nongranular leucocytes. This process is much like the transfonnation into lymphocytes of the mesenchymal cells which surround the epithelial anlage of the thymus in Axolotol as described by Maxunow ('12). Metchnikoff ('84) also holds that in Triton larvae transformed connective tissue cells assume phagocytic functions. It is difficult to determine to just what extent the formation of lymphocytes takes place in muscle. However, I am confident that the connective tissue cells do not transform rapidly enough to account for the large numbers of lymphocytes present. No mitotic figures were seen and only an occasional dumbbell-shaped nucleus suggested amitosis. It is, therefore, necessary to assume that the blood also is a source of a part of the lymphocytes. According to an hypothesis of Metchnikoff ('84) the endothelial walls of the blood capillaries are acted upon chemically, rendering them more permeable to leucocytes and permitting of a 'passive diapedesis' of the red blood cells. Although this is only an hypothesis, it seems to be a very reasonable way to account for the presence of such large numbers of leucocytes and also of red blood cells which are scattered in small numbers among them, as was stated above. Since muscle degeneration in Salamandra atra is an aphagocytic process it seems probable that the cells of the delicate endothelial walls of capillaries may be among the first elements to be affected. Another view, however, may be taken. Dantschakoff ('08), in her work on the development of the blood in birds, gives evidence that the cells of the vascular endothelium have the power of transforming into lymphocytes. This genetic relationship of the leucocytes to the vascular endothelium has also been held by other investigators. If this view be correct, is it not probable that the vascular endothelial cells- are transformed into leucocytes? This process also would permit leucocytes and erythrocytes to escape into the intercellular spaces.


MUSCLE DEGENERATION IN AMPHIBIA 83

The only apparent role of the leucocytes in the degenerating muscles of Salamandra atra is the taking up and removal of the products of degeneration. They take no perceptible active part in breaking down the muscle tissue. According to a theory of Anglas ('00), the leucocytes produce an enzyme which acts upon the tissues, causing their dissoliltion. The only indication of the correctness of this view is the large accumulation of leucocytes in the first stages of degeneration, even before any changes of the fibers are apparent under the microscope.

CONCLUSIONS

The conclusions of this work are summarized as follows:

1. The eosinophile granules are exogenous in their nature, that is, they are derived from material outside of the cell containing them and not directly a product of the activity of the leucocyte itself. The presence of free eosinophile granules found to be so plentiful among degenerating erythrocytes and muscle tissue and later taken up by lymphocytes, support this conclusion.

2. The eosinophile granules are products of degenerated muscle tissue and degenerated erythrocytes. This is supported by the fact that they are found only in connection with the above named degenerating tissues. The fact also that more eosinophile leucocytes are found in the blood during the period of metamorphosis than in the lar^^al or in the adult condition, supports this conclusion.

3. As they are found onl}*^ in connection with tissue containing a relatively large amount of hemoglobin, it is believed that hemoglobin is a part of their chemical composition. This conclusion by no means excludes the probability that the eosinophile granules contain chemical compounds besides hemoglobin.

4. The eosinophile granules are taken up by the lymphocytes (large and small) which are thus converted into eosinophile cells. The eosinophile leucocytes are, therefore, the white blood cells ingested with the products of degenerated erythrocytes and degenerated muscle tissue.

5. The eosinophile cells with different types of nuclei are genetically related to one another.


84 J. A. BADERTSCHER

6. Muscle degeneration in Salamandra atra is a process of liquefaction (a purely aphagocytic process), and is apparently brought about through the activity of the surrounding fluids.

7. The only apparent function of the leucocytes in degenerating muscle is the removal of the products of degenerated muscle tissue and erythrocytes. They apparently play no part in the processes bringing about the degeneration of those tissues.

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MUSCLE DEGENERATION IN AMPHIBIA 85

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86 J. A. BADERTSCHER

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1909 Zur Morphologie und morphologischen Stellung der ungranulierten Leucocyten-Lymphocyten des Blutes und der Lymphe. Arch, f . mikr. Anat., Bd. 73.

1911 Die Leucocyten und verwandte Zellformen. Ergebn. d. Anat. u. Entwickelungsge.


ON THE WEIGHTS OF THE ABDOMINAL AND THE THORACIC VISCERA, THE SEX GLANDS, DUCTLESS GLANDS AND THE EYEBALLS OF THE ALBINO RAT (MUS NORVEGICUS ALBINUS) ACCORDING TO BODY WEIGHT

SHINKISHI HATAI

The Wistar Institute of Anatomy and Biology

TWELVE CHARTS

Complete quantitative data on the various anatomical components of the body are important not only for the study of growth, but also for cross reference. A lack of such reference data interferes in many cases with a clear recognition of alterations which are taking place in the animal body under various experimental conditions. Fortunately the following data on the growth of the albino rat are already available :

Growth of body in weight in respect to age (Donaldson '06) and body length (Donaldson '09) ; weight of the brain and spinal cord (Donaldson '08) ; weight of total amount of blood and hemoglobin content (Chisolm '11), the growth of the head, trunk, extremities, skin, skeleton, musculature and viscera in weight according to age (Jackson and Lowrey '12) ; and finally the growth of the dry substance in the albino rat (Lowrey '13). Besides these we have several other series of data on the albino rat, though not so comprehensive as those just cited.

T];ie addition of the present data on the weight of individual organs to the records above cited will make available observations on nearly all the important anatomical components of the albino rat in a form useful either for the study of growth or for cross reference. Indeed in no other mammal are such adequate data available at the present moment.

Since the object of the present paper is to report the objective findings, mainly for reference purposes, many interesting points

87


88 SHINKISHI HATAI

relating to growth will not be discussed at length, but merely mentioned as they come, in connection with the presentation of the formulas and charts.

The data used were secured mainly from two colonies of albino rats: (1) a colony kept at The Wistar Institute (during 1911 and 1912), and (2) a colony kept at the University of Missouri (during 1910 and 1911). For the latter, the present writer is under great obligations to Professor Jackson, who not only supplied numerous rats for the purpose of control examination, but has also granted the free use of his entire collection of data on the weight of the viscera.

I take this opportunity therefore to acknowledge my indebtedness to Professor Jackson for his courtesy.

In addition, I have secured some rats from both New Haven and Chicago for the purpose of determining the range of variability under different climatic and nutritional conditions. For these rats I am indebted to Professor Mendel of New Haven and Professor Carr of Chicago, and I desire to thank both of these gentlemen for their generous assistance.

For the interpretation of the results which follow, it is of the greatest importance to bear in mind that the animals examined were nearly all less than one year old — a very few having attained this age. If we take the nonnal span of life of the albino rat as three years, then it is plain that we are dealing with rats in the first third of their life and that the end of the records gives the conditions at the close of the growing period only, leaving untouched the changes which may be expected to occur during the next two years. For the discussion of the span of life in the albino rat, the reader is referred to Donaldson ('08, p. 368), and to Slonaker ('12).

TECHNIQUE

Although most of the organs are clearly marked off from the surrounding tissues, and thus may be readily removed in an exact manner, nevertheless I shall describe my method of dissection in detail so that others may be fully informed concerning it.


ALBINO RAT VISCERA, GLANDS, EYEBALLS.* WEIGHTS 89

Method of dissection

The animal was removed from the colony in the morning before feeding and killed by chlorofonn. The gross body weight (to one-tenth of a gram) and the linear measurements were next taken. For the latter determinations, the animal was placed with the abdominal side up and the following measurements made with the calipers: (1) Total length, the distance between the tip of the nose and the tip of the tail; (2) body length, distance between the tip of the nose and anal opening; (3) tail length, obtained by the difference between measurements (1) and (2). The linear measurements were m,ade to the nearest millimeter.

The animal was then at once opened with the scissors along the abdominal line from the anus to the level of the diaphragm. Thus the thorax was left closed to prevent evaporation from the thoracic viscera while the abdominal organs were being removed.

All the dissections were made beneath a glass hood designed to protect the operator against draughts and so prevent a loss of moisture from the organs during their removal.

The details of the procedure for each organ will be given in the order of removal. The following entries give the names of the organs in this order, unless otherwise stated. Each organ was prepared rapidly and then put alone .or with others into a closed weighing bottle. Only the special treatment (if any) of an organ is noted in each case:

1. Liver: The blood which fills the main vessels was gently squeezed out before the liver was placed in the weighing bottle.

2. Spleen: The blood vessels were cut close to the hilum.

3. Testes: Accessory organs such as the epididymis, are not included.

4. The alimentary tract was removed from the level of the diaphragm to the anus, together with the pancreas and mesentery, and any other adherent structures, such as fat, were left intact. The oesophagus was therefore not included. The stomach was cut open and the contents removed. The contents of the small intestine were removed by gentle pressure along its entire course


90 SHINKISHI HATAI

from above downward. In the case of the rectum and large intestine, it was usually necessary to cut them open in order to remove the contents. The entire group of the structures above named but minus contents, is here called 'alimentary tract.' The removal of contents can be accomplished easily and uniformly after short practice. The body weight was not corrected for the contents of the alimentary tract.

5. The suprarenal glands were carefully separated from the surrounding structures. These glands are usually imbedded within some fatty tissue but a little familiarity will enable one to dissect them out without difficulty.

6. The ovaries were carefully dissected out of the capsule. The fallopian tube was not included. On account of its minuteness, the ovary is often difficult to remove when the animal is young. A dissecting microscope is sometimes necessary.

7. The kidneys: All blood vessels were cut close to the hilum and any masses of fat carefully removed.

All these organs having been removed and placed in weighing bottles, the thoracic cavity was next opened along the median ventral line by a cut extending as far as the upper end of the neck. Care was taken not to injure the thyroid gland, which lies close to the trachea.

8. Thymus gland: Large lymphatic glands together with fat lie close to this organ but were not included. Because the weight of this gland is so closely correlated with age, the data already obtained will not be presented at this time but reserved until the study of an extended series of animals of known age has been completed.

9. The heart was removed by cutting all the vessels close to their proximal ends. The heart was next cut open by longitudinal slits and any blood clots carefully removed.

\0. The lungs were severed from the trachea. The esophagus which lies near to the lungs must be removed. The infected lungs of rats suffering from so-called 'pneumonia' are more or less filled with pus. Such lungs were weighed as removed but these data were not used in computing the formula for lung weight.


ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 91

Associated with the infected lung a pathological condition of the alimentaiy tract is usually found. The tract is diminished in weight.

11. Thyroid gland: The removal of the thyroid is rather difficult on account of the minute muscles, similar in coloration to the gland, which adhere very closely to it. A little experience enables one to avoid this difficulty.

This completes the list of viscera removed by the ventral incisions.

The animal was now turned with the dorsal side up, an incision made along median dorsal line, from the root of the tail to the tip of the nose, and the skin laid back on each side.

12. The eyeballs were removed free from all the muscles.

13. The vertebral canal was then opened and the spinal cord removed, free from the spinal nerves, which were severed at the point of their emergence from the cord. The cord was separated from the brain at the tip of calamus scriptorius, equivalent to the level of the first spinal nerve.

14. The brain: Care was taken not to injure the olfactoiy bulbs or the flocculus; the latter is embedded in a bony capsule.

15. The hypophysis was removed last. Removal of this organ is very simple in the rat as it can be lifted from the floor of the cranium with a fine forceps without further dissection. Both the glandular and infundibular portions were included.

For weighing, these fifteen organs were placed as removed in four closed bottles. Bottle 1 contained liver, spleen, kidneys, testes, heart and lungs. Bottle 2 contained alimentary tract, ovaries, suprarenals, thymus, thyroid, eyeballs and hypophysis. Bottle 3 contained brain. Bottle 4 contained spinal cord.

In each instance the bottle with all the organs in it was first weighed, then the organs were successively removed, one at a time, and the bottle reweighed after each removal. The emptied bottle with such small quantities of fluid as drained from the organs was finally weighed, and from these data the weight of each viscus was computed. It is seen from this that each organ was weighed with its contained blood. The weighings were all made on delicate balances to one-tenth of a milligram.


92 SHINKISHI HATAI

In making these weighings I had the help of Miss Wolfe and Miss Conrow and desire to acknowledge here my indebtedness to both of them for their accurate work.

The formulas and their application

By means of a mathematical formula the relations between the weight of the body and that of the organ have been expressed in each case. Such formulas have an evident use for the purpose of interpolation, while for the study of various growth phenomena, such as relative rate of growth, form of graph during various stages of life, and so forth, they give a proper basis for discussion.

In expressing the weight relations mentioned above, I have employedatypeof formula (Hatai'll) which may bewritten as follows:

Y = aX + 6 log X + c

or in some cases a simpler form was used

F = 6 log X + c

where Y = weight of organ; X = body weight; a, h, c = constants; to be detennined from the observed data.

In the case of the sex glands it was necessary to use the two formulas in order to express the several phases of growth, that is

Y ^ a + hX H- cX- for one phase and

Y = h log X + c for the other phase.

In detemiining the constants of the formulas, I have used as far as possible the data obtained from the rats reared in The Wistar Institute. This procedure was important in order to avoid the mixture of data.

In some instances however, I have made use of the data obtained by Dr. Jackson, when my own observations were too few for an adequate treatment. Dr. Jackson's data were used, as follows : ^ 1. For studying the eyeballs and ovaries I have used only the data obtained by Dr. Jackson. None of my own observations were employed.

2. For studying the suprarenals, thyroid and alimentary tract, I have used the data obtained by Dr. Jackson to fill the interval


ALBINO EAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 93

between 5 and 50 grams in body weight. I have however, tested with my own Umited data the weights of the organs belonging to the rats of these body weights (5 to 50 grams) and found that the two sets of data agree perfectly.

In fact, in this period all the organs determined by both of us agree closely, though I have not used the data obtained by Dr. Jackson, except in the cases named. Thus in the cases of the three organs mentioned above, the observed data are those furnished by Dr. Jackson and none of my own have been addad, while after 50 grams in body weight, all the data are from my own observations made on the rats from the colony at The Wistar Institute. In the case of all of the other organs, the formulas are based on my own data alone.

The total number of The Wistar Institute rats examined by me for the presfent purpose was 271 (220 males and 51 females) while Dr. Jackson examined 344 (165 males and 179 females) at the University of Missouri. The actual number of rats used in the case of each organ will be stated when the formula is presented.

It should be added that when a rat with infected lungs was found — the rest of the organs except the alimentary tract being apparently nomial — the records for the lungs and alimentary tract were discarded, while the dat^ on the remaining organs were emplo^^ed. Furthermore, in order to simplify the matter, the determinations of the constants were based on the male data alone, as in the case of most of the viscera, there is no clear evidence of a sex difference.

When however the sex differences are evident, the constants were determined separately for each sex. As will be seen later, there are no distinct differences between the two sexes, except in the case of some of the ductless glands. The formula deduced for the males can therefore also be used for the females unless the contrary is explicitly stated.

It should be noted also that in determining the form of the graphs, especially at the upper end where both my data and those of Dr. Jackson were scanty, I have been much helped by


94 SHINKISHI HATAI

making cross references to the data on a series of inbred albino rats which have been grown here by Dr. King for several years past. There are already on hand data for over 800 inbred rats of various body weights — mostly very large. In addition to these, the records for several hundred Norway and hybrid rats were also employed for cross reference. Therefore, although our data on the stock albino rats are not very extensive, nevertheless, by the aid of the various cross references, it has been possible to produce graphs which are believed by the present writer to be nearly correct in their essential features.

In table 1 the calculated weights of the organs for various values of the body weight are given. This table has been used in making the charts and for the purpose of graphic interpolation. Complete tables giving the calculated weights of all the individual organs for every millimeter of the body length will be published later for the purpose of laboratory reference. Table 2 gives the mean values of the observed weights for all the organs examined.

We now pass to a brief description of the formulas, the graphs based on them and the relations between the observed and computed values.

In charts 1 to 12 the growth of organs in weight for increasing body weight is illustrated. Since I have not taken the age into consideration, any given body weight may be represented by individuals of various ages. In general, the weight of the organs follows closely the body weight, and therefore in rats growing normally, the weight of the organs may be considered as a function of the body weight. We notice in all cases that the greater fluctuations occur among the larger individuals. This is due, in part at least, to the fact that the relation between body weight and body length varies much more in the larger than in the smaller animals, owing either to abnormal fat deposition or emaciation. Such fluctuations may be reduced to a minimum by computing the normal body weight from the observed body length (Donaldson '09) and substituting the computed value of the body weight. However, I have not adopted this method except in the case of a few observations where the emaciation was very considerable.


ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS


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SHINKISHI HATAI


TABLE 2


Shoifing the mean values of the observed loeights for all the organs examined. The

weights are given in grams


BODY


HEART


i

NO.


BODY


KIDNEYS


NO.


BODY


LIVER


NO. I

1


BODY LUNGS



BQDY [spleen



■WEIGHT


&


WEIGHT


WEIGHT


cf


WBIGHTi d'


NO.


WEIGHT, ^


NO.


5 3


0.039


2


5.3


0.044


2


5.3


0.271


2


5.3 ! 0.081


2


5.3


0.012


2


14.3


0.103


4


14.3


0.199


4


H.3


0.711


4


14.3 0.156


4i


14.3


0.036


4


26.7


0.140


r


26.7


0.370


1|


26.7


1.782


li


26.7 0.243


1'


26.7


0.081


1


37 6


0.241


6


37.6


0.431


6


37.6


2.600


6


37.6 0.329


6


37.0 , 0.134


4


44.2


0.252


5


44.2


0.472


5


44.2


3.066


5i


44.2 0.386


5


46.9 0.139


2


54.8


0.286


7


54.8


0.558


7!


54.8


3.385


7'

54.8 0.439


7


54.5 : 0.208


5


65.1


0.339


6


65.1


0.673


6


65.1


4.739


6


65.1 1 0.478


6


66.0 0,186


3


76.3


0.387


7


76.3


0.775


7


76.3


5.111


7|


76.3 0.563


7


77.6 0.200


4


83.0


0.432


4


83.0


0.955


4


83.0


5.999


4


83.0 0.620


4


80,6 0.218


2


93 4


0.487


21


93.4


1.005


2


93.4


5.980


2


94.9 0.584


1


94.9


0.229


1


107.7


0.493


4


107.7


1.090


4


107.7


6.325


4


107.1 0.614


3


107.1


0.294


2


115.9


0.571


2


115.9


1.119


2


115.9


7.784


2


113.2 0,680


1


115.9 j 0.368


2


124 4


0.575


11,1 124.4


1.357


11


124.4


7.611


'11


124.4 0.805


7


124.0 1 0.350


7


138.1


0.587


3 138.1


1.232


3


138.1


7.165


3


138.4 ' 0.983


2


138.4 : 0.432


2


145,2


0.616


2


145.2


1.292


2


145.2


8.005


2


145.2 ' 1.141


2


141.9 0.288


1


156.8


0.669


3


156.8


1.496


3;


156.8


10.206


3


155.2 1 0.944


2


157.1 0.487


2


164.9


0.706


6


164.9


1.496


6


164.9


9.666


6


164.9 i 0.982


6


165.4 : 0.484


2


173.6


0.705


8


173.6


1.425


8


173.6


9.025


8


175.4


1.040


2


173,9 I 0.508


7


185.1


0.757


4


185.1


1.599


4


185.1


10.385


4


180.5


1.198


1


185.1 1 0.499


4


195.0


0.786


4


195.0


1.624


4

195.0


9.955


4


199.5


1.137


1


197.3 1 0.496


1


204.9


0.853


4


204.9


1.547


4


204.9


8.967


4


209.4 i 1.202


1


205 1 ' 0.591


3


215 7


834


1


215.7


1.647


1


215.7


11.174


1


226.4 1.200


4


224.0 0.438


2


226


0.840


5


226.0


1.649


5


226.0


10.916


5


237.6 1.356


2


236.4 0.571


5


236.4


0.884


5


236.4


2.035


5


236.4


9.717


5


240,6 1.175


1


254.7 0.804


3


240.6


0.929


1


240.6


1.982


1


240.6


13.699


1


260,5 ! 1.507


3


260.5 0.598


2


254.2


0.938


5


254.2


2.166


5


254.2


11.698


5


279.7 1 1.760


1


273.2 0.429


1


263.5


0.988


3


263.5


2.264


3


263.5


12.246


3


296.3


1.104


1


294,6 0,758


3


274.4


1.016


3


274.4


2.098


3


274.4


12.871


3


314.7


1.685


2


315.4 i 0.933


4


294.6


1.011


3, 294.6


2.200


3


294.6


12.159


3


324.4


1.713


2


323.8 0.612


3


314.6


1.171


5 314.6


2.550


5


314.6


12.580


5


331.4


1.820


1


358.7 0.729


1


323.8


1.150


3 323.8


2.855


3


323.8


12.794


3


397.0


1.697


1


373.4 ■ 1.042


1


332.0


1.132


2


332.0


2.722


2


332.0


15.276


2


454.0


2.376


1


454.0 1 1.286


1


343.7


1.299


1


343.7


2.867


.


343.7


13.628





90



87


358.7


1.194


1


358.7 373.4


2.805 3.007


.


358.7 373.4


16.175 16.152









397.0


1.439


1


397.0


3.179



397.0


16.964









454.0


1.504


1 135


454.0


3.954


136


454.0


21.786


136









ALI








1






BODY WEIGHT


MENTARY TRACT


NO.


BODY WEIGHT


TESTES


NO.


BODY WEIGHT


OVARIES

9


NO.


BODY WEIGHT


EYEBALLS


NO.


BODY WEIGHT


THYROID

9


NO.


5.0


0.120


37


5.3


0.004


2


5.2


0.0008


13


5.4


0.026


34


5.2


0.0019


12


10.4


0.310


25


14.3


0.095


4


9.8


0.0009


3


7.2


0.045


7


1 9.8


0.0021


3


15.9


0.734


12


26.7


0.096


1


14.2


0.0025


30


9.5


0.057


8


13.2


0.0033


12


23.5


1.640


13


37.6


0.272


6


22.0


0.0054


4 11.4


0:061


17


22.2


0.0063


5


36.1


2.261


3


44.2


0.311


5


45.2


0.0093


11 15.9


0.087


12


! 45.2


0.0090


4


46.7


3.800


8, 55.3


0.451


6


55.0


0.0088


5' 25.2


0.116


12


66.1


0.0162


2


54.3


4.253


14 1- 65.1


0,691


6


60.8


O.0O80


1 36.1


0.124


3


95.5


0.0236


3


ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS


97


TABLE 2— Continued


BODY

WEIGHT


ALIMENTARY TRACT

Cf


)| BODY WEIGHT


64.0 75.8 91.0 105.0 115.0 124.7 142.1 154.2 164.5 186.3 245.5 259.5 293.2 347.8


4.779 5.146 6.485 5.740 7.609 7.647 8.062 8.921 9.796 8.889 10.853 11.683 13.878 14.721


5 7 5 2 11 11 5 11 7 6 2 6 2 2 194


76.3 82.1 93.4 107.7 115.9 124.4 138.1 145.2 156.8 164.9 173.6 185.1 195.0 204.8 215.7 226.0 236.4 240.6 255.1 263.5 274.4 294.6 314.6 323.8 332.0 358.7 373.4 397.0 454.0


0.886 0.822 1.323 1.460 1.452 1.455 1.512 1.873 2.207 2.030 2.072 2.138 2.069 2.238 1.854 2.291 2.144 2.495 2.355 2.503 2.687 2.545 2.872 2.616 2.714 2.901 2.852 2.583 3.429


BODY WEIGHT


75.5

85.7 95.0 104.3 113.9 124.2 135.1 144.2 155.7 165.1 171.8 184.7 207.5


OVARIES

9


0.0140 0.0194 0.0216'

0.0318;

0.04821 0.0442 0.0431 0.0464 0.0457 0.0519 0.0423 0.0508 0.0455


BODY WEIGHT


45.8

54.3 77.1 99.8 114.9 124.6 142.1 153.6 164.0 181.0 259.6


BYEBALLS


0.153 0.159 0.179 0.209 0.194 0.207 0.252 0.240 0.255 0.241 0.318


BODY WEIGHT


120.4

146.1 155.2 164.5 177.8 185.2 193.9


THYROID

9


0.0196 0.0216 0.0268 0.0268 0.0288 0.0305 0.0319


BODY WEIGHT


5.5 7.0

11.8

14.1

23.5

34.5

47.6

64.0

74.5

86.6

105.0

115.2

124.5

136.1

154.3


THYROID

&


0.0018 0.0022 0.0025 0.0041 0.0071 0.0103 0.0103J

o.oiiej

0.0126 0.0163 0.0207, 0.0193J 0.02511 0.0274 0.0263


BODY WEIGHT


SUPBA I RENALS NO.;


5.4

9.4 15.9 23.5 36.1 46.7 64.0 74.5 86.6 105.0 115.2 124.5 136.1 154.3 186.4


0.0020 0.0022 0.0053 0.0085 0.0120 0.0122 0.0176 0.0255 0.0218 0.0212 0.0191 0.0266 0.0317 0.0296 0.0323


BODY

WEIGHT


SUPBA BENALS 9


13 22 45 66 95 120 146 156 164 177 185 193


0.0021 0.0030 0.0049 0.0080 0.0115 0.0222 0.0243 0.0344 0.0405 0.0494 0.0403 0.0406 0.0507 0.0493


20:

12

36

5

lli

2;

3

3l 2

3' 41 3| 4' 5 113


BODY

WEIGHT


HYPOPHYSIS


5.4

13.7

15.0

19.0

47.6

64.0

83.3

95.2

105.0

115.7

123.9

137.7

154.5

178.8

202.5


0.0005 0.0012 0.0009 0.0013 0.0030 0.0035 0.0033 0.0040 0.0047 0.0053 0.0050 0.0057 0.0062 0.0065 0.0082


BODY WEIGHT


HYPOPHYSIS NO.

9


9.2

16.2

21.2

64.3

90.0

104.3

116.0

127.4

136.6

147.2

155.5

163.7

174.2

184.5

202.5


0.0009 0.0010 0.0018 0.0032 0.0057 0.0062 0.0074 0.0073 0.0096


0.0088


0.0114


0.0106


0.0112


0.0127


0.0162


THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 1


98


SHINKISHI HATAI


TABLE 2— Continued


BODY WEIGHT


THY- 1 ROID 1


NO.


BODY

WEIGHT


SUPRA RENALS


NO.


BODY SUPRA {WEIGHT «^^-^


NO.


BODY WEIGHT


HYPOPHYSIS

cf


NO.


BODY WEIGHT


HYPOPHYSIS

9


NO.


186.4


1

0.0313i


3


202.5


0.0370


2


I

i




214.6


0.0077


2


242.0


0.0153


2


202.5


0.0373


2


211.4


0.0400


2





235.3


0.0083


4




80


211.4


0.0306


2


224.4


0.0339


2





244.9


0.0104


2





224.4


0.0374


2


240.8


0.0404


3





284.0


0.0109


2





240.8


0.0376


3


252.6


0.0444


2





310.4


0.0104


2





252.6


0.0331


2


269.5


0.0490


2





356.8


0.0125


3





' 269.5


0.0390


3


284.2


0.0353


3






78





284.2


0.04491


2


293.9


0.0445


4










293.9


0.0466|


4


348.6


0.0450


4











348.6


0.0477J


4 91




145











In all charts the observed mean values are indicated by either black dots (male) or clear circles (female) while those calculated from the formulas are represented either by continuous (male) or discontinuous (female) lines. Since the calculated values represent the observed data verj^ closely the formulas may be used not only for the purpose of interpolation, but as the basis for the comparison of the growth of the various organs.

In connection with the amount of fluctuation, it should be recalled that the number of individuals was not large, particularly in the case of the heavier rats, in which a greater fluctuation is to be expected, so that with a larger number of cases, the fluctuations would probably be much diminished.

To those using the formulas which are given below, the following practical suggestions are offered:

1. When its value differs markedly from the normal, the body weight should be corrected to the observed body length, as the body length is least modified in animals exhibiting emaciation or abnormal fat deposition. The proper correction can be made according to the following formula (Donaldson '09).


Body length = 143 log (Bd. wt. + 15) - 134


ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 99

Or expressing the body weight in terms of body length, we have

Body length +134

Body weight = 10 ^^^ _ 15

2. Under some conditions the growth of rats is considerably accelerated in both body weight and body length for given ages. Such rats show generally a marked disproportion in the weight relation between body and organs; that is, the viscera are, as it were, overgrown by the remainder of the body. In such cases the body weight normal for the viscera may be estimated from the weight of any one of the organs, such as the weight of the heart. Thus one determines the body weight from the observed heart weight, and then compares the weights of the other organs as observed with those computed for the body weight thus obtained.

3. In any experiment where the controls are compared with experimented animals, modifications in the experimented group^ can be determined in the following manner: First determine the percentage deviation of the controls from the calculated values as given by the formulas and then the corresponding percentage deviation of the experimented animals. The algebraical difference of the two values thus obtained will be the difference between the experimented and controls. This method is important when the body weights of the two groups of rats (experimented and controls) differ widely since the weight relations between body and organs are not constant but vary with the change in body weight.

We now turn to a brief description of the formulas^ for the weights of the viscera and the graphs which represent them.

1 In order to bring together all the formulas which so far have been worked out in connection with the albino rat the following additional formulas are inserted here. This addition will, I believe, be very convenient for reference.

Weight of braiii = 0.569 log (Bd. wt. - 8.7) + 0.554 [Bd. wt. > 10]

Weight of spinal cord = 0.585 log (Bd. wt. + 21) - 0.795

Tail length, male = 0.853 Bd. wt. + 38.8 log Bd. wt. - 90.5

Tail length, female = 0.874 Bd. wt. + 43.2 log Bd. wt. - 98.1


100


SHINKISHI HATAI


1.7^


1 M 1 1 1 1 1 1 1








- —




-■









n


^


n











HEART

EIGHT GRAM





























































'


























^



1.5


























^































'


^

































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^'
































.^






























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f


^




'






























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^



















'














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1.0














, ■

































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0.6






,


i"^

































/





























0.5







•.'•


































/^'^



































































/•'


































/




































































/


























!








I















1




' '




'


'


'























BODY WEIGHT GRAMS




i 1
































100


200 250 300 350 400


Chart 1 Showing the heart weight of the male albino rat according to body weight. The observed weights are represented by 134 male rats. • Observed weight. — — Calculated weight.


1. Heart? The growth of the heart in weight is represented by the following formula: Weight of heart =

0.0026 (body weight + 14) + 0.249 log (Bd. wt. + 14) - 0.336

The constants of the fomiula were determined from 134 male rats and the relation between observed and calculated values is shown in chart 1. The greater fluctuation which occurs in the rats of larger size has already been noted and explained. As has been mentioned earlier, variations in body growth alter more or less the relation between heart weight and body weight. The heart weight is usually high when the rat is small for a given age

- The weight relation between heart and body has been examined by numerous investigators at various times. A list of important papers, as well as a table which illustrates this relation found in the various mammals is given by Joseph ('08) including his own extensive observations on dogs of various sizes.


ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 101


3.5


r











^








-.



"










^






\ ■







h-t


/



I]


3.5


KIDNEYS 2

WEIGHT GRAMS















t




\

































y



































y































'














/









3.0










1




























.y




































i








y










































1






/i














































,,


y












































1



/

















2.5































1


• '

















2.5






























/













































/












































1 •


/■












































i'-'




















2.0


























/













































->


' 1









L— 1^


















































































/


















1





























■i\


















1





1




1.5




















■'















1













1 5


















/









1









1






























/^










. !



















































































/




































1.0












1


/















































y















































/





































































,

































































0.5






/


'•








































n 5





,,














































r*




























1





1









1






/































BODY WEIGHT GRAMS





'





























1


































1




(


)





5






1C


30





1


30





2(


30





2


30





300





350





400





45C




Chart 2 Showing the weight of kidneys of the male albino rat according to body weight. The observed weights are represented by 136 male rats. • Observed weight. Calculated weight.


and it is usually low when the rat is large. A change in the relative weight of the heart is usually accompanied by a corresponding change for all the other organs.

2. Kidneys. The growth of the kidneys in weight is represented by the following formula: Weight of kidneys =

0.00718 (Bd. wt. - 3) +0.132 log (Bd. wt. - 3) - 0.009

The constants of the formula were determined from 136 male rats and the relation between observed and calculated values is shown in chart 2. We notice in this case a much greater fluctuation than in the case of the heart, but at the moment no adequate explanation for this can be given. Possibly it is due to the neglected factor of age.


102


SHINKISHI HATAI



"



'




















...







i i '


LIVER

WEIGHT GRAMS





















1





















A 1 1













!








j/\\\\































1


























,^





1





















1































1




y








1























.^









1






















• /










1





















1


^






























[ y




















'









^_



i/














1

















.^


























































\


<


.




























M




























,





i










1










j








r*























!




,




.





1






















,



p>




^












■ 1



1 ; '■




\














1











1




\



1




/


























1



i-f

























1




,


y




























.


/








1











1 !











j/












(


.








! i











•1









1












1 1









y





























/












i

















/




























/





























/
















1









'





/

















1









1



I








1











1






1







/
























1




































1




















BODY WEIGHT GRAMS 1


,
























n ! 1 1 ■





















I


100


250


300 350 400 450


Chart 3 Showing the weight of liver of the male albino rat according to bodyweight. The observed weights are represented by 136 male rats. • Observed weight. ' Calculated weight.

3. Liver. The growth of the liver in weight is represented by the following formula: Weight of liver =

0.0303 (Bd. wt. + 5) + 3.340 log (Bd. wt. + 5) - 3.896 [Bd. wt. > 10]

The constants of the formula were determined from 136 male rats and the relation between the observed and calculated values is shown in chart 3. We notice here a much greater fluctuation of the observed values than in the two previous cases. Since the liver is the seat of food storage, as well as performing several other important and complex functions, one might naturally expect a high variability as the consequence of its varied physiological activities. It is possible also that the weight of the liver may


ALBINO RAT VISCERA, GLANDS, EYEBALLS! WEIGHTS 103


2.5


M 1 M 1 1 1 1





rn


p










-■







m



~








'~



"


~\





~



LUNGS 2

WEIGHT GRAMS


































Y





































^






















1















r^







2.0





































^











































■^













































,-■'











































J



,








a





































,^
























































1








1.0



























i^














1






















































































































,






^





























10



















,














































•'





























































































K^















































^















































,•















































.'






























































































.J































BODY WEIGHT GRAMS

1 1 I 1 J 1 1 1 1 1 1 1 1 1




•'
































































I


)





5






la






IE


)0





2C






2:


)0





300





JbO





400





4b0



2,5


Chart 4 Showing the weight of lungs of the male albino rat according to body weight. The observed weights are represented by 90 male rats. • Observed weight. Calculated weight.


alter according to the nature of the diet as well as according to other conditions not yet studied. It is interesting to note that a heavy liver is usually associated with a heavy spleen.

.4. Lungs. The growth of the lungs in weight is represented by the following formula: Weight of lungs =

0.00471 (Bd. wt. +2) +0.122 log. (Bd. wt. +2) -0.056.

The constants of the formula were determined from 90 male rats and the relation between the observed and calculated values is shown in chart 4. All evidently pathological cases were eliminated from the records used. Nevertheless on account of difficulty in deteraiining the infection at an early period, some diseased cases may have been retained. This pulmonary trouble, commonly called 'pneumonia,' seldom appears in rats less than 100 grams in body weight, but after this period almost 90 per cent of the ordinary rat population is affected. This is true not only for the albino rat kept in captivity but for the Norway rat when freshly trapped. Although an elimination of the pathological records was difficult on account of the great frequency of


104


SHINKISHI HATAI


l.b

SPLEEN







~


~
















r—




p



p



p



p


p




r

p


p



p


14


WEIGHT GRAMS











































































1 1





1























































































1 2[





























































































1 1











































>














































^^






1








































^














































^













































A











































^












































^













































i"



















07




























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^










i




r~


































y







^








































^



























05




















^














































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04















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ft
































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,








































r







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BODY WEIGHT GRAMS




^
































-■*














































1


200 250 300 350 400 450


Chart 5 Showing the weight of spleen of the male albino rat according to body weight. The observed weights are represented by 87 male rats.

Observed weight. Calculated weight. infection, nevertheless when this was made as far as possible, the resulting data were quite uniform as will be seen from the chart.

5. Spleen. The growth of the spleen in weight is represented by the following fonnula: Weight of spleen =

0.00245 Bd. wt. + 0.0301 log Bd. wt. - 0.025

The constants of the formula were determined from 87 male rats and the relation between observed and calculated values is shown in chart 5. The weight of the spleen is highly variable owing to a- great frequency of cases of 'enlarged' spleen. In treating the records I have excluded all such plainly altered spleens. Such enlarged spleens are usually darker in color, soft to the touch, and exhibit all over the surface dark or grayish patches. A slight acquaintance with this organ will remove any difficulty in distinguishing the 'enlarged' from the normal spleen.


ALBINO RAT VISCERA, GLANDS, EYEBALLS! WEIGHTS 105



Al IMFNTARY


xd&otI


"

















\










^




WEIGHT GRAMS











1
















^



1




























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1



16.



































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15.



































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Chart 6 Showing the weight of alimentary tract of the male albino rat according to body weight. The observed weights are represented by 112 (Jackson) rats, below 50 grams in body weight, and 82 (Wistar) rats, above 50 grams in body weight.

• Observed weight. Calculated weight.

The cause of this enlargement, as found in the rat, has not been studied.

6. Alimentary tract. The growth of the aHmentary tract in weight is represented by the following formula: Weight of alimentary tract =

0.0245 Bd. wt. +4.720 log (Bd. wt. +7) -5.753

The constants of the formula were determined from 194 male rats and the relation between observed and calculated values is shown in chart 6. Despite the fact of some difficulty in removing this organ, the weight of the aHmentary tract is highly uniform in the rats up to 150 grams in body weight, but beyond this period it becomes decidedly variable owing to the greater frequency of intestinal disturbance. This intestinal disturbance seems to


106


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Chart 7 Showing the weight of testes of the male albino rat according to body weight. The observed weights are represented by 121 male rats. • Observed weight. Calculated weight.


be associated mainly with the. infected lungs. It is quite safe to conclude that the alimentary tract is more or less altered in cases where pulmonary infection is well advanced. This means that nearly 90 per cent of adult rats are unfitted for this determination. The alteration in question causes a loss of weight. The present formula was determined from the rats in apparently good health and thus practically free from this infection. Consequently the upper end of the graph will naturally be higher than when based on the values obtained from a random sampling of the general population without regard to the condition of the alimentary tract.

7. Testes. Owing to dissimilar rates of growth during the earlier days of life, the graph illustrating the growth of the testes has three distinct phases. The first phase however occupies such a brief interval (5 to 10 grams in body weight) that it was thought, from a practical standpoint, not worth while to work


ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 107


out a* special formula for it. Consequently the following formulas represent onl}^ the two remaining phases. The weight of the testes on body weight between 5 and 10 grams may be calculated with a sufficient degree of accuracy from the values given by a straight line which joins the observed weight at 5 grams with the calculated weight at 10 grams in body weight.

Testes =0.043 -0.000966 Bd. wt. +0.000163 Bd. wt.^

[10 < Bd. wt. < 80] Testes = 2.910 log Bd. wt. - 4.520 [Bd. wt. > 80]

The constants of the formula were determined from 121 male rats, and the relation between observed and calculated values is shown in chart 7.

Although I have not considered such factors as the season and age, yet from the functions of these organs, it seems probable that data controlled for both those factors would be less variable than that here presented.

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Chart 8 Showing the weight of ovaries of the female albino rat according to body weight. The observed weights are represented by 136 (Jackson) rats. O Observed weight. Calculated weight.

8. Ovaries. The growth of ovaries in weight is even more complicated than that of the testes. We notice in the ovaries


108 SHINKISHI HATAI

three distinct phases of growth. The first and last phases are represented by the logarithmic curves, while the second is represented by a parabolic curve. Weight of ovaries :

(Phase 1) = 0.010 log (Bd. wt. + 3) - 0.0082

[Bd. wt. < 50] (Phase 2) = 0.0425 - 0.00121 Bd. wt. + 0.0000108 Bd. wt. [50 < Bd. wt. < 80] (Phase 3) = 0.007 log (Bd. wt. - 105) + 0.0352

[Bd. wt. > 110]

The constants of the formulas were determined from 136 female rats and the relation between observed and calculated values is shown in chart 8. The data used here were obtained by Dr. Jackson from the rats kept at the University of Missouri. These rats were all unmated excepting a few of the oldest (at age of one year). It is therefore possible that the ovaries belonging to the mated females may show some deviation from the values given by the present formulas.

9. Suprarenal glands. In this gland a sexual difference is clearly shown. Consequently the growth of the suprarenals in weight is treated separately according to sex and is represented by the following formulas respectively. Weight of suprarenals:

Male = 0.0000855 (Bd. wt. + 3) +0.0113 log (Bd. wt. + 3) - 0.0093 Female = 0.00023 Bd. wt. + 0.00388 log Bd. wt. - 0.0020

[Bd. wt. > 30]

The constants of the formulas were determined from 145 males and 113 females respectively. The relation between observed aild calculated values is shown in chart 9. As is shown in the chart, the sex difference becomes clearly marked in the rats of about 30 grams in body weight. This difference becomes greater as the rats increase in weight. The calculated value of the female glands is represented by the discontinuous line, and the observed values, by circles. The male values are given by the continuous line record and the black dots. The physiological significance of the greater weight of the suprarenals of the female has still to be investigated.


ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS


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Chart 9 Showing the weight of suprarenals of the albino rat according to body weight. The observed weights are represented by 92 (Jackson) male rats, below 50 grams in body weight, and 53 (Wistar) male rats, above 50 grams in body weight; and 84 (Jackson) female rats, below 50 grams in body weight, and 29 (Wistar) female rats, above 50 grams in body weight.

• Observed weight, male. O Observed weight, female.

Calculated weight, male. Calculated weight, female.


10. Hypophysis. Like the suprarenal glands, the hypophysis also shows a distinct sex difference in weight. This appears in rats weighing more than 50 grams. Consequently the growth of this gland in weight is represented after 50 grams in body weight by the two formulas, one for each sex. Weight of hypophysis :

Male = 0.0000257 (Bd.wt. +3) +0.00140 log. (Bd. wt. +3) -0.00097 Female = 0.00205 + 0.000081 Bd. wt. - 0.00196 log Bd. wt.

[Bd.wt. >50]


110


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Chart 10 Showing the weight of hypophysis of the albino rat according to body weight. The observed weights are represented by 78 male and 80 female rats.

• Observed weight, male. O Observed weight, female.

Calculated weight, male. Calculated weight, female.


The constants of the formulas were determined from 78 males and 80 females. The relation between observed and calculated values is shown in chart 10.

• The sex difference is clearly noticeable in the rats at and after ' 50 grams in body weight, and it becomes greater as the rats increase in weight. Before 50 grams in body weight the sex difference is not evident, consequently the weight of the female hypophysis prior to 50 grams should be calculated from the male formula. This sex difference in the weight of the hypophysis has not been shown previously in any mammal.


ALBINO EAT VISCERA, GLANDS, EYEBALLS! WEIGHTS 111



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• Observed weight, male. Calculated weight for both se.xes.

O Observed weight, female.


11. Thyroid gland. Unlike the two ductless glands already mentioned, the thyroid gland does not exhibit any weight difference between the two sexes. The growth of the thyroid gland in weight is represented by the following formula: Weight of thyroid =

0.0000973 (Bd. wt. +27) + 0.0139 log (Bd. wt. + 27) -0.0226.

The constants of the formula were determined from 91 males and 63 females. The relation between calculated and observed values is shown in chart 11. We notice in the chart that the two sexes do not differ fom each other, as the observed mean values for the two sexes are not segregated and those for both sexes cluster round the theoretical line.

The thyroid gland is the most variable among all the ductless glands treated in the present paper. The variation is due to cases


112


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of enlargement. In some instances this enlargement amounts to almost ten times the average value. From the data here used, all hypertrophied cases were excluded. The enlargement of the gland can be easily recognized not only by the size, but also by the color. The color of the normal gland is pink while that of the abnormal gland is milky owing perhaps to an excessive accumulation of the colloidal material. A slight acquaintance removes any difficulty in distinguishing the abnormal from the normal gland,

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The constants of the formula were determined from 149 male rats. The relation between observed and calculated values is shown in chart 12. The entire data used were furnished by Dr. Jackson and obtained by him from the rats belonging to the colony kept at the University of Missouri.


ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 113

For the weight of the Brain and of the Spinal Cord, formulas have already been published (Donaldson '08) (Hatai '09).

As already stated, in the case of the Tkymiis no treatment of the data has yet been attempted.

Types of graphs

A comparison of the graphs reveals several interesting points touching the form of growth characteristic for each organ. A little study of the graphs makes it evident that we can class them imder three general types.

Tijpe 1. The graph represented by the growth of eyeballs. This is characterized by very rapid growth in weight at an early period and after this period the rate of growth is much reduced. This type is characteristic for the growth of the brain and the spinal cord in weight (Donaldson '08).

Type 2. The graphs representing the growth of all the other viscera except the sex glands. These graphs are characterized by a relatively rapid rise at an earlier period followed by an almost straight line which makes an angle of varying degree with the base line; an angle always much greater than that of Type 1.

Type 3. The graphs representing the growth of the sex glands. These graphs are characterized by fluctuating rates of growth between birth and sexual maturity. For example, in the case of the ovaries, the first phase (from birth to 50 grams) is represented by a logarithmic curve. This is followed by a phase of rapid growth represented by a parabolic curve (50 to 110 grams). This second phase is followed in turn by a phase of slower growth represented again b}' a logarithmic curve. Similarly, the growth of the testes has three phases, though the time relations of the corresponding phases are not the same as in the case of the ovaries.

The difference between Type 1 and Type 2 is very clear when the graphs are plotted to an equal scale on one sheet. When this is done the graph for Type 1 appears almost parallel to the base line soon after the very rapid early rise is completed, while the graph of Type 2 continues to rise at a more rapid rate. When

THE AMERICAN JOURNAL OF ANATOMl", VOL. 15, NO. 1


114 SHINKISHI HATAI

however, animals lower in the zoological scale are taken, Type 1 tends to resemble Type 2.

This can be seen from the graphs for the growth of the brain weight in the fish (Kellicott '08) and in the frog (Donaldson '03). Type 3, however, seems to be quite characteristically differentiated from the rest even in the fish, as shown by the graph for the gonads. The relative growth of the organs i-epresented by these three types of graphs is represented in table 3.

The data giving the percentage growth of the brain and spinal cord (Donaldson '08) are included for comparison. In the case of each organ in table 3 the percentages were calculated by taking the final weight of the organ, at a body weight of 450 grams for the male, as 100 and then expressing the preceding weights as percentages. In the case of the female, the final organ weight (100) was taken from the rats of 300 grams in body weight. This is about the average maximum body weight the female can attain.

Examination of table 3 reveals many interesting points. First of all the three types of growth are clearly shown; that is the eyeballs and the nervous system are characterized by a precocious growth in an earlier stage. Fifty per cent of the final weight is attained by the brain in rats when still below 20 grams in body weight — in the case of the spinal cord, below 100 grams and in the case of the eyeballs, below 130 grams. In the viscera and ductless glands on the other hand, 50 per cent is attained at a body weight of about 200 grams or more. In the case of the sex glands, half of the final weight is attained at about the same stage as the eyeballs 130 grams. Nevertheless the characteristic double or triple phases of growth serve to distinguish clearly the type of the gonads from that of the eyeballs or nervous system.

It is highly interesting to notice the similarity of growth rate of all the abdominal and thoracic viscera and ductless glands, especially after the 50 per cent increment has been attained.

This similarity of the growth rate of the organs just named throughout this first third of the span of life suggests a close quantitative interrelation between these various organs and their


ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 115


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116 SHINKISHI HATAI

relative independence of both the gonads and the nervous system. A direct comparison between the two sexes with respect to the relative growth rate, as here given, cannot be made because the final body weight taken in the case of the female is different from that taken for the male. Nevertheless a study of both table 3 and the charts shows that both the ductless glands and the ovary grow in the same manner as the corresponding organs in the male.

GENERAL CONSIDERATIONS

The usual view that the viscera are highly variable, in man for example, seems to have arisen largely (1) from the use of individuals past the prime of life, that is, more than thirty years of age; (2) from confusing normal and pathological material, and (3) from treating the data without proper regard for the controlling factors of race, age, body weight, stature and sex. Indeed from a standpoint of curve fitting the viscera are probably no more variable than other parts of the body.

This optimistic view is based on the data presented in this paper. It will be noticed that the total number of rats used is certainly not large, when the range of body weight is considered. Nevertheless, the resulting data show a considerable uniformity and the observed values do not usually deviate to any great extent from those calculated.

It should be possible, however, to get even a better agreement between the observed and computed values for the albino rat were it possible to control certain important modifying conditions. What these conditions are may be briefly stated.

Factors modifying the relative weights of the organs

1. Food, care and physical condition of rats. It is generally assumed that the rat can eat anything and can grow and multiply under almost any conditions. It is true that one can raise rats with an ordinary bread and milk diet, or e\ en with some artificial diet, for one or. two generations if the original strain was thoroughly sound.


ALBINO RAT VISCERA, GLANDS, EYEBALLS I WEIGHTS 117

Our experience with rats for maii}^ years, however, shows that they can not be kept in normal health and reared for any length of time without great care and a highly complex diet. For a time rats may grow and multiply under almost any condition, but if one takes the trouble to record the fertility, death rate, growth rate, and so forth, the importance of the proper food and surroundings will at once be recognized. The relations between the body and organs in weight are clearly modified according to whether the growth of the individual was retarded, accelerated, or normal. Although I have not arranged the data to demonstrate such modifications, the records for such a demonstration are at hand. Recent work by Watson ('10) shows more or less histological alteration of various organs following the different kinds of food given to the rats.

2. Different strains. Another cause of fluctuations in the records may be due to the use of different strains of rats. How many strains are there, and how these strains originated or further how stable such strains will be under varying conditions we do not yet know.

Nevertheless, it has come to our notice that certain strains, represented by rats coming from a new source, tend to show constantly some degree of deviation always in the same sense. For example, we had a strain called the Ridley Park strain." The rats belonging to this strain had for a given body weight a relatively shorter tail and body and possessed a markedly heavier brain. We had again a strain called the summer strain" — animals which had been born and reared under unfavorable summer conditions. The rats belonging to this strain had a small body weight for their age and an unusually small nervous system. Such strains may occur under various climatic, nutritional or other environmental conditions. It is important therefore to consider the conditions of the particular colony of rats which may be used for any series of observations. If the fluctuations found are not merely statistical, but deviate constantly in one direction, this fact must be given its proper weight when comparing these animals with those represented in the tables here presented.


118 SHINKISHI HATAT

We found by comparing our data with those obtained by Dr. Jackson from his rats that our rats had a constantly, though slightly, smaller heart and kidneys, but a heavier liver. Just what the cause of these differences may have been was not determined.

3. Influence of age and season. A third cause of disturbance may be due to the age and season. As has been already stated, I have not taken either age or season into account, but have purposely taken the rats at random in order that my data may meet the usual conditions. However, there can be little question that age and season influence the weight of some of the organs at least. For instance, the weight of the heart is slightly larger in older than in younger rats of the same body weight. The weight of the sexual glands also may vary slightly according to the season. Recent w^ork by Seidell and Fenger ('13) demonstrates a seasonal variation of the iodin content of the thyroid gland.

4-. Sex differences. The sex differences are so marked in the case of some of the organs that different formulas were needed to represent the two sexes. However, in the majority of instances, we failed to distinguish any difference according to sex, so far as weight relations are concerned, and consequently only one formula for each organ, namely, that based on the male data, was worked out. It may however so happen that certain organs react differently according to sex under similar conditions. For instance, in the albino rat removal of the sex glands from the male produces an enlargement of the hypophysis, while in the spayed female, the hypophysis is not affected (Hatai).

Doubtless a more precise analysis of the environmental conditions would enable us to extend the list of factors modifying the growth of the organs, but those which have just been enumerated are evidently worth consideration and the control of them will doubtless go far to reduce the variability of the data.


ALBINO RAT VISCERA, GLANDS, EYEBALLS: WEIGHTS 119

LITERATURE CITED

Chisolm, R. A. 1911 On the size and growth of the blood in tame rats. Quat. Jour. Exp. Physiol., vol. 4, no. 3. pp. 207-229.

Donaldson, H. H. 1903 On a formula for determining the weight of the central nervous system of the frog from the weight and length of its entire body. University of Chicago, Decennial Publications, Series 1, vol. 10.

1906 A comparison of the white rat with man in respect to the growth of the entire body. Boas Anniversary Volume, New York.

1908 A comparison of the albino rat with man in respect to the growth of the bi-ain and of the spinal cord. Jour. Comp. Neur., vol. 18, no. 4, pp. 34.5-392.

1909 On the relation of the Updy length to the body weight and to the weight of the brain and of the spinal cord in the albino rat (Mus norvegicus var. albus). Jour. Comp. Neur., vol. 20, no. 2, pp. 119-144.

Hatai, S. 1909 Note on the formulas used for calculating the weight of the brain in the albino rats. Jour. Comp. Neur., vol. 19, no. 2, pp. 169-173. 1911 An interpretation of growth curves from a dynamical standpoint. Anat. Rec, vol. 5, no. 8, pp. 373-382.

Jackson, C. M., and Lowrey, L. G. 1912 On the relative growth of the component parts (head, trunk, and extremities) and systems (skin, skeleton, musculature and viscera) of the albino rat. Anal;. Rec, vol. 6, no. 12, pp. 449-474.

Kellicott, W. E. 1908 The growth of the brain and viscera in the smooth dog fish (Mustelus canis, Mitchill). Amer. Jour. Anat., vol. 8, no. 4, pp. 319-353.

JOSEPH; D. R. 1908 The ratio between* the heart weight and body weight in various animals. Jour. Exp. Med., vol. 10, pp. 521-528.

Watson, C. 1910 The influence of diet on the structure of the tissues. London. Appendix to "Food and feeding in health and disease," pp. 555615.

Slonaker, J. R. 1912 The normal activity of the albino rat from birth to natural death, its rate of growth and the duration of life. Jour. Anim. Behavior, vol. 2, no. 1, pp. 20-42.

Seidell, A., and Fenger, F. 1913 Seasonal variation in the iodine content of the thyroid gland. Jour. Biol. Chem., vol. 13, no. 4, pp. 517-526.


THE NUCLEUS CARDIACUS NERVI VAGI AND THE THREE DISTINCT TYPES OF NERVE CELLS WHICH INNERVATE THE THREE DIFFERENT TYPES OF MUSCLE

EDWARD F. MALONE

The Anatomical Laboratory of the University of Cincinnati

THREE FIGURES

When one has carefully and critically studied in series of Nissl preparations the brains of various mammals, there is revealed the presence of constant cell groups whose cells invariably possess certain definite characteristics as to size, form and structure. A separation of two groups of cells based merely upon differences in their histological characters is justified in the present state of our knowledge, only when such differences are constant and striking. When these conditions are fulfilled we may conclude that such constant and striking differences in cell character correspond to a difference in cell activity, just as in other portions of the body. I have pointed out elsewhere that very real differences in cell character have been neglected by experimental workers, and that their results have been rendered thereby of less value. Since the dorsal motor (sympathetic) nucleus of the vagus is known to contain centers for the control of both heart muscle and smooth muscle, one would suppose that any real difference in the cell character of various portions of this nucleus would at once claim the attention of the experimental worker and that he would attempt to inform us as to the relation of these different types of cells to the various functions of the vagus nerve. But such is not the case; we are informed casually that some cells are large and others small, and thereafter the cells are considered as if they were all of the same type.

121


122 EDWARD F, MALONE

The discovery of two different types of cells in the sympathetic vagus nucleus was not accidental; I was led to look for this difference on the following grounds. In the first place, I had recently shown that all cells concerned in transmitting efferent impulses to striated muscle possess a fundamental similarity of structure, whether the axone of the cell be in direct relation to the muscle or whether the cell act on the muscle through the mediation of one or more efferent neurones. This observation naturally strengthened my belief in the significance of the relation of cell character to cell function. In the second place, I had observed the striking difference between the cells supplying striated and those supplying smooth muscle. Since in a recent paper Molhant had shown that all fibers of the vagus supplying heart and smooth muscle arise from the sympathetic vagus nucleus, I concluded that in cells having such diverse functions there must exist a fundamental difference in histological character. As was anticipated, two different types of cells were found; the evidence in favor of ascribing to the cells of one type the innervation of heart muscle, and on the other hand, to cells of the other type the innervation of smooth muscle, will be considered later.

The material available consisted of two complete series. The first was a series of the brain of a lemur, while the second was of the brain of macacus rhesus. Both brains were fixed in 95 per cent alcohol, and after the usual treatment with absolute and chloroform, were imbedded in paraffin. Serial sections were stained in a 1 per cent aqueous solution of toluidin blue (Griibler), differentiated in 95 per cent alcohol, dehydrated in absolute, cleared in xylol, and mounted in Canada balsam. Series of brains of other forms will have to be prepared and studied before I feel justified in committing myself upon many points, and the present article has therefore been limited, especially as to the exact location and distribution of the different types of cells.

The efferent fibers of the vagus nerve arise from two distinct columns of cells. From the nucleus ambiguous arise the fibers which supply striated muscle, while from the so-called dorsal motor nucleus arise fibers which innervate heart muscle and


NUCLEUS CARDIACUS NERVl VAGI 123

smooth muscle. This fundamental difference as to function, which has been proved beyond doubt by the recent investigation of Molhant, had not been clearly recognized; this obscurity was probably favored by the fact that the nucleus ambiguous, together with the motor nuclei of the eleventh, seventh and fifth cranial nerves have often been regarded as visceral, regardless of the fact that their cells cannot be distinguished either histologically or functionally from the cells of the other motor nerves supplying striated muscle. Thus this classification giving undue emphasis to a condition which in mammals no longer exists, has contributed to the general lack of appreciation that the dorsal motor nucleus of the vagus is composed of cells which differ radically both histologically and functionally from those of the nuclei supplying striated muscle, regardless of whether the striated muscle be of somatic or of visceral origin. The name dorsal motor nucleus" does not indicate the true function of this cell group, and I shall use the name "sympathetic or visceral nucleus of the vagus."

The location and extent of the sympathetic nucleus of the vagus is well known and will not be considered in this paper, except to call attention to the fact that it extends as a long column of cells dorso-lateral to the hypoglossus nucleus from the lowest portion of the medulla to almost the level of the oral pole of the inferior olive. An excellent description of the location of this nucleus is given in Jacobsohn's monograph. The oral portion of the nucleus is composed .of small cells of the type shown in figure 2; this is true both in the case of the lemur and the monkey. As one follows the nucleus caudally a second type of cell begins to appear (fig. 1). The portion of the nucleus in which both types of cells occur is at the level of the oral portion of the hypoglossus nucleus, and here the sympathetic nucleus attains its greatest diameter. The cells of each type are partly separated from each other, although no sharp line of separation is evident. In the lemur the large cells (fig. 1) form a fairly compact group dorsal from the small cells, whereas in the monkey their relative position is reversed. Proceeding further in a caudal direction, the small cells become rapidly less numerous


124 EDWARD F. MALONE

and finally disappear. After the disappearance of the small cells the sjTnpathetic nucleus, consisting now entirely of the large cells (fig. 1) proceeds caudally as a well developed and definite group. In the most caudal portion of the medulla the sympathetic nucleus is much reduced; only a few cells are seen in each section, and these cells become smaller and have the appearance of the smallest cell in figure 1 ; in this portion of the nucleus (the caudal end) are probabl}^ also cells of the type shown in figure 2, that is, similar to those in the oral portion of the nucleus, but at present I cannot be absolutely sure of this, as the surrounding cell groups have not been sufficiently studied. The smallest cell shown in figure 1, is probably a transition type between the other cells of figure 1, and those of figure 2. To sum up, the sjTiipathetic vagus nucleus consists of three portions: (a) an oral portion whose cells are of the type in figure 2; (b) a middle portion whose cells are shown in figure 1; and (c) a caudal portion composed of cells shown in figure 2 (same as oral portion) and also of cells such as the smallest cell in figure 1 (probably a transition type).

It is not my intention to present in this paper a detailed description of the types of cells in the vagus sympathetic nucleus, but rather to point out the fact that there are very definite differences in histological character between the cells of the various groups; a study of the illustrations will make this evident. Since these differences in cell character exist, and since such differences must necessarily be an indication of corresponding differences of cell activity, we may now consider whether these different cell groups of different character may be brought into relation with definite functions. In the first place, it has been shown by Molhant, in his excellent and extensive work on the vagus nerve, 'that the sympathetic nucleus of the vagus gives origin to all the fibers of the ^-agus which supply smooth and heart muscle, and that all its cells give origin to such fibers. Further, he has shown that the oral portion supplies smooth muscle (stomach, lungs), the function of the extreme caudal portion is doubtful (possibly connected with the trachea and bronchi), while the intermediate portion supplies


NUCLEUS CARDIACUS NERVI VAGI 125

heart muscle, but he has failed to connect these different functions with different types of cells. Concerning the function of the caudal portion of the nucleus, which is composed of cells of the type shown in figure 2, together with cells resembling the smallest cell of figure 1, we can draw no definite conclusion. The oral portion consists exclusively of the type of cells shown in figure 2, and we may conclude that this type of cell supplies smooth muscle; of course this does not justify us in concluding that this type of cell (fig. 2) is the only type of cell which may supply smooth muscle, or that this type may not in other regions have a different function. Overlapping thecells supplying smooth muscle (fig. 2) and extending caudally unaccompanied by other types of cells is the type of cell shown in figure 1, and this portion of the sympathetic nucleus has been shown (Molhant) to supply heart muscle.

It is evident therefore that the cells of figure 1 supply heart muscle, while those of figure 2 supply smooth muscle (stomach and lui^gs). In addition there is purely histological evidence to support the functional relations of these two types of cells (figs. 1 and 2), since the cells supplying heart muscle (fig. 1) are a type intermediate in histological structure between those supplying smooth muscle (fig. 2) and those supplying striated muscle (cells of hypoglossiis nucleus, fig. 3). The relative size of the Nissl bodies in the three types of motor cells illustrated in figs. 1 to 3 is especially worthy of notice. The fact that nerve cells supplying heart muscle are of a type intermediate betw^een those supplying striated and smooth muscle constitutes one of the strongest arguments in support of the importance of the relation of cell character to cell function, since heart muscle is histologically intemiediate between the two other types of muscle.

The cell group which supplies heart muscle, composed of the characteristic cells shown in figure 1, I shall name provisionally nucleus cardiacus nervi vagi." I do not feel justified in assigning any name to the other portions of the vagus sympathetic nucleus, but shall be content with pointing out that the cells of the oral portion which supply smooth muscle are of a definite type (fig. 2). A further division is at present not advisable


126 EDWARD F. MALONE

because the functional relations of the caudal group are not understood, and because the pigmented cells described by Jacobsohn in man, have not been identified and studied (of course, in lower animals pigment is wanting, although homologous non-pigmented cells may exist). Further subdivision of the sympathetic nucleus, together with a detailed description of the location and extent of the various cell types, the consideration of transition types, and of the relations of the nucleus to the cells of surrounding nuclei, must await a thorough study of numerous series of various animals (including man).

CONCLUSIONS

1 . The -histological character of a nerve cell is an indication of its function. Differences in connections with portions of the organism which differ merely in spatial relations do not involve a difference in the character of the nerve cells, but are associated merely with the location of the nerve cell; for instance, arm and leg muscles, flexors and extensors are all innervated by the same type of cell, although such differences in peripheral connections correspond to differences in the position of the corresponding nerve cells.

2. The three types of muscle are innervated by three distinct types of nerve cell, which, however, are related to one another in such a manner that the cell innervating heart muscle is of a type intermediate between the other two types of cells. Heart muscle, smooth muscle, and striated muscle are innervated by cells such as are illustrated in figures 1, 2 and 3 respectively, the cells of figure 1 constituting a type intermediate between the other two.

3. The nucleus cardiacus nervi vagi is situated in the middle portion of the sjTnpathetic nucleus of the vagus and is composed of cells shown in figure 1.

4. The time has passed when experimental workers can afford to neglect to inform themselves of the existence of definite types of cells situated in the region under investigation, and to attempt to bring cell character into relation with cell function.


NUCLEUS CARDIACUS NERVI VAGI 127

BIBLIOGRAPHY

Jacobsohn, L. 1909 tJber die Kerne des menschlichen Hirnstamms. Aus dem Anhang zu den Abhandlungen der konigl. preuss. Akad. d. Wiss.

Malone, E. 1910 tJber die Kerne des menschlichen Diencephalon. Aus dem Anhang zu den Abhandlungen der konigl. preuss. Akad. d. Wiss.

1913 Recognition of members of the somatic motor chain of nerve cells, etc. Anat. Rec, vol. 7, no. 3.

MoLHANT, M. 1910 Le nerf vague (primiere partie). Le nevraxe, torn. 11.

MoLHANT, M., ET Van Gehuchten, A. 1912 Contribution a I'etude anatomique du nerf pneumo-gastrique chez I'homme. Le nevraxe, torn. 13.


PLATE 1


EXPLANATION OF FIGURES


1 to 3 The cells illustrated in the three figures were all drawn from the same section with the aid of the .camera lucida, and for all cells the magnification is 580 diameters. I have attempted to reproduce as nearly as possible the actual appearance of the cells, combining to a certain extent different levels of focus. These three figures clearly show that the cells supplying heart muscle (fig. 1) are histologically intermediate between the cells supplying smooth muscle (fig. 2) and those supplying striated muscle (fig. 3).

1 Cells from nucleus cardiacus nervi vagi of lemur. The smallest cell represents probably a transition type to the cell type of figure 2, and this type occurs more frequently in the caudal portion of the vagus sympathetic nucleus where it is found together with the cells of the type shown in figure 2. 580 diameters.

2 Cells of vagus sympathetic nucleus of lemur which innervate smooth muscle. In the oral portion of the sympathetic vagus nucleus these cells occur alone; more caudally they occur together with the cells of the nucleus cardiacus (fig. 1) in the most oral portion of this nucleus. In the caudal portion of the sympathetic nucleus such cells probably reappear and are accompanied by the small cell type shown in figure 1 (smallest cell). 580 diameters.

3 Cells from hypoglossus nucleus of lemur, innervating striated muscle. 580 diameters.


128


NUCLEUS CARDIACUS NERVI VAGI

EDWARD F. MALONE


PLATE I








^'1


THE AMERICAN JOURNAL OF AXATOMY, VOL. 15, NO. 1


129


HISTOGENESIS AND MORPHOGENESIS OF THE THORACIC DUCT IN THE CHICK; DEVELOPMENT OF BLOOD CELLS AND THEIR PASSAGE TO THE BLOOD STREAM VIA THE THORACIC DUCT

ADAAI M. MILLER

The Analomical Laboratory of Columbia Univernily

TWENTTY-EIGHT FIGURES (SEVENTEEN PLATEs)

I. INTRODUCTION

The study of the development of the jugular lymph sac in the chick, the result of which was published in this Journal/ (1) led to the investigation also of the developing thoracic duct and the means whereby its communication with the lymph sac is established. This investigation has been carried on with the advice and under supervision of Dr. Huntington and in the light of his recent work on reptiles (2) and the cat (3).

Within the past few years different investigators have shown that in the frog (4), (5), the chick (1), (8), the rabbit (6), the cat (9), and in man (7) each jugular l>Tnph sac develops directl}^ from a venous capillar}^ network adjacent to the junction of the early precardinal with the postcardinal vein to form the duct of Cuvier. It has also been pointed out by Huntington (14) that the jugular lymph sacs, regarded as of venous origin, constitute the connecting links between the hemal vascular system and the general system of lymphatic vessels.

The origin of the systemic h'mphatic vessels is a problem on which investigators are sharply divided. A summary of the different views, including the bibliography, can be found on pages 10-13 of Huntington's monograph in the Memoirs of The Wistar Institute (3).

^ References, by number, will be found on page 162.

131

THE AMERICAN- JOURNAL OF ANATOMY, VOL. 15, NO. 2 SEPTEMBER, IQl."?


132 ADAM M. MILLER

As regards the particular case of the thoracic duct, Sala (10) in 1900 published the results of his work on the chick in which he holds that it develops by canalization of solid mesenchymal cords. In 1902 Sabin (11), after working with the injection method, published her account of the development of the lymphatic system in pig embryos. In this she reinforces and extends the view of Langer (12) and Ranvier (13), that lymphatics arise from veins by a process of sprouting and centrifugal growth, maintaining that the system as a whole is developed by blind ducts that 'bud off' from the veins of the cervical and inguinal regions, widen out to form sacs from which lymphatics grow to the skin, stating also that "at the same time a growth of ducts occurs along the dorsal line following the aorta to make a thoracic duct from which lymphatics grow to the various organs." The two views expressed by Sala and Sabin are thus diametrically opposed, the one being that the lymphatics arise in the mesenchyme independently of the veins, the other that the lymphatics are outgrowths from the veins.

In 1905 Lewis (6) expressed the view that in the rabbit the lymphatic system is derived directly from the embryonal veins, multiple detached portions of these becoming confluent to form the permanent systemic lymphatics, stating that the thoracic duct "arises from a plexus of lymphatics surrounding the aorta" (p. 109).

In 1907 Huntington and McClure (25), studying the development of lymphatic vessels in their relation to the veins in embryos of the cat, found that "the lymphatics begin as extra-intimal spaces along the course of the primitive embryonal veins. They* subsequently become confluent and form continuous vascular channels" (p. 42).

Huntington, in 1908 (14), while retaining this view of the genesis of the systemic lymphatic vessels, as distinguished from the jugular lymph sacs, defined the latter as the connecting links between the hemal vascular system and the general system of the lymphatic vessels, which "arise, not by transfonnation of veins, but by the formation of spaces lying outside the intimal lining of the veins, which spaces, becoming confluent, form the general lymphatic channels of the body" (p. 25).


THORACIC DUCT IN THE CHICK 133

In the same year (1908) McClure (21) abandoned the view previously held jointly by him and Huntington as regards the development of the thoracic and right lymphatic ducts in cat embryos, and states:

The anlages of the thoracic and right lymphatic ducts consist of a series of independent outgrowths which first appear along the common jugular and innominate and then along the azygos veins exactly in the line subsequently followed by these ducts; these outgrowths are subsequently split off from the veins, by a process of fenestration, in the form of a series of isolated, more or less spindle-shaped spaces which later become confluent with one another and with a process of the jugular lymph sac to form a continuous system disconnected from the veins, .... (p. 542).

In 1909 Sabin (7) reiterates her original view, based on the study of injected pig embryos, stating that the "presumption seems to lie on the side that the thoracic duct develops in the same manner as all other ducts," namely, "from endothelial sprouts from the sacs" (p. 58).

McClure in 1910 (23), after further studies of critical stages in lymphatic development, retracted his former view and stated :

The venous line .... along which the cat's thoracic duct develops is topographically replaced by the lymphatic channel, not directly, as assumed by me (in 1908), but secondarily by extra-intimal lymphatic space development, .... the 'extra-intimal theory,' as originally outlined by Huntington and myself (in 1906), establishes a fundamental principle of development for the main systemic lymph channels in mammals (p. 105).

Sabin, in a later article ('11) (22), states that "the thoracic duct develops in part as a down growth of the jugular sac and in part, especially its dilated portion or cisterna chyli, as a direct transformation of the branches of the azygos veins" (p. 424) . This expression of opinion seems to be a correction of her earlier statements and a partial adoption of Lewis's view. In place of her former concept of an uninterrupted centrifugal lymphatic growth from the sacs, she now appears to hold that in addition a portion of the thoracic duct develops as the result of direct transformation of azygos venous tributaries into lymphatics.


134 ADAM M. MILLER

Huntington again in 1911 confirmed and elaborated his former view by extensive observations on reptiles (2) and the cat, stating specifically on page 13 of the first number of the Memoirs of The Wistar Institute (3) that "the entire extensive system of lymphatic vessels proper of the adult animal, including the thoracic and right l}miphatic ducts and their tributaries, is formed by the confluenceof the extravenous intercellular mesodermal spaces," and that "these spaces are lined by a lymphatic vascular endothelium which is Jiot derived from the hemal vascular endothelium, but develops independently of the same," and giving his summary and conclusions in remarkably clear terms on pages 153-171 of the same publication. He also points out that the systemic lymphatic development in the mammalian embryo is "by no means confined to the immediate environment of degenerating embryonic veins. The same field, which shows the above described histogenetic processes in the development of extra-intimal lymphatic spaces surrounding and replacing a decadent venule, will at the same time contain numerous equivalent lymphatic mesenchymal clefts and spaces which continue to develop independently of any association with retrograding veins" (p. 49).

Sabin in 1912 (24) still maintains that "the thoracic duct (in the pig) arises in part as a downgrowth from the left jugular sac and in part from a plexus of lymphatics which buds off from the veins of the Wolffian body" (p. 336).

Recently Kampmeier (15), after studying serial sections of both uninjected pig embryos and one of Sabin's injected specimens, concluded that

the actual genesis of the thoracic duct is initiated by the appearance of blind mesenchymal Ij^mphatic spaces either around or not immediately in contact with the venous derivatives, or veno-lymphatics, which become detached from their venous trunks and break up into degenerating segments .... During their inception and growth the walls of the discontinuous thoracic duct anlagen are composed of mesenchymal cells .... Injected specimens of the early lymphatic stages certify the reality of blind uninjectible anlagen beyond the farthest points to which the injecta have penetrated, demonstrating that discontinuities in a developing lymphatic channel are not 'appearances' found only by the study of uninjected embryos (pp. 463-464).


THORACIC DUCT IN THE CHICK 135

Stromsten (16) in his account of the development of the thoracic duct in turtles arrived at a like conclusion, stating that "the development of the peri-aortic lymphatic plexus in the loggerhead turtle is immediately preceded by the formation of isolated, independent spaces. They (the spaces) cannot be injected . . . ." (p. 354). "The intercellular spaces thus formed enlarge and fuse together to form lymphatic lacunae. At a later stage the lacunae acquire an endothelial lining and become the isolated anlagen of the thoracic duct" (p. 356).

As regards the thoracic duct in the chick, it^will be my object in this article (1) to demonstrate its origin by confluence of intercellular spaces in the mesenchyme, independent of the veins, and to reconsider the significance of the mesenchymal cords described by Sala in their relation to the developing lymphatics; (2) to discuss the establishment of the morphological drainage line of the thoracic duct and the means by which this duct and the jugular lymph sacs communicate; and (3) to show that the organization of the avian thoracic duct corresponds in type with that established in reptiles and mammals.

II. MATERIAL

Embryos of the domestic fowl (Gallus gallus) have been used chiefly on account of the certainty in procuring the critical stages. Some embryos of the English sparrow (Passer domesticus) have also been used. Of this material I have examined thirty-two individual embryos in serial sections, comprising twenty-seven chicks and five sparrows. Four of the chicks were injected with India ink through the umbilical vein (table 1).

Unfortunately the sparrow embryos, of which series nos. 123, 124, 126, 154 and 509 were examined, were not measured and could be judged as to stages of development only by comparison with the chicks.

The embryos were fixed in vom Rath's, Bouin's, or Zenker's fluid (some in Zenker-formol), Bouin's fluid giving the best results with the least shrinkage. The sections were cut in paraffin and stained on the slide by one or the other of the following methods. After fixation in vom Rath's mixture, the staining was


136


ADAM M. MILLER

TABLE 1


CHICK EMBRYOS SERIES NO.


TIME OF INCUBATION IN HOURS


GREATEST LENGTH IN MM AFTER FIXATION


415


96




7.0


371


108




6.75


336


120




9.0


355


120




11.5


356


120




12.0


326


130




12.5


411


132




9.0


410


132




9.25


370


132




9.5


357


132




10.75


412


145




10.75


340


145




11.0


428


145




11.0


414


145




11.25


339


145




13.5


426


160




13.5


465


165




14.0


463


166




16.0


464


166




16.5


520


171


inj


ected



521


192


inj


ected



522


192


inj


ected



519


206





523


216


inj


ected



320


230





524


240





483


260





done with a much diluted Delafield's hematoxylin, followed by a weak solution of picric acid in alcohol. After all the other fixatives the sections were overstained in Weigert's hematoxylin, decolorized in water acidulated with HCl, and counterstained in a weak solution of Orange G in distilled water! The blood cells' are clearly differentiated by either method; the cytoplasm of those containing even a trace of hemoglobin shows some tinge of yellow. Developing muscle tissue and nerve fibers also are yellow. Other elements are stained by the hematoxylin, the delicate processes of the irregular mesenchymal cells showing especially well.


THORACIC DUCT IN THE CHICK 137

III. HISTOGENESIS

Sala, in his account of the development of the thoracic duct in the chick (10), states that the anlagen of this lymph channel appear as isolated mesenchymal spaces which become clothed with endothelial cells derived from the mesenchyme, and which subsequently coalesce to form continuous vessels. He thus implies a denial that the lymphatics in this region arise from veins and that the lymphatic endothelium is derived from hemal vascular endothelium. He also calls attention to accumulations of mesenchymal cells which contrast clearly with the surrounding tissue. They appear to consist in large part of elements exhibiting all the characteristics of young connective tissue cells, with roundish forms, no processes and large intensely colorable nuclei. Among these elements appear red corpuscles in larger or smaller numbers. The accumulations or clumps of cells develop first on the mesial aspect of the superior vena cava, and then extend caudad to the level of the celiac artery. Without describing the histogenesis of these masses of cells, Sala states further that within them in large part the anlagen of the thoracic duct are excavated or 'hollowed out.' The details of the 'hollowing' process are not given.

In the main I can confirm the results of Sala's observations so far as he has carried them. As for the isolated mesenchymal spaces which he describes as the anlagen of the thoracic duct my study of their histogenesis in the closely graded series of chick embryos leads to the conclusion that they do arise independently of the veins and that their endothelial lining is derived from the indifferent mesenchymal cells bordering upon them. The details of the process I shall attempt to demonstrate in the following pages. After tracing the development of the accumulations of cells in the mesenchyme and their subsequent history in relation to the developing thoracic duct, it seems to the writer that a different and greater significance can be attached to them than was given by Sala. Since the cells composing them correspond so closely in their history to the developing blood cells as described by Dantschakoff (17) in the extraembryonic area of the chick,


138 ADAM M. MILLER

undoubtedly they should be considered as collections of intraembryonic developing blood cells.

These clumps of cells, as noted by Sala, develop in the vicinity of the aortic arches, especially the sixth, and along the dorsal aortic roots and the aorta as far as the exit of the superior mesenteric artery. The early stages in the formation can be clearly seen in embryos of 100 to 110 hours.

Some of the stellate elements of the mesenchymal reticulum (syncytium) become differentiated from their neighbors. Their processes are retracted and separated from the general reticulum, the cells thus becoming rounded (figs. 1 and 2). The cytoplasm acquires a more strongly basophilic character, increases in amount and becomes more homogeneous than that of the true mesenchyme. The nuclei contain a relatively small amount of chromatin and one or two, usually two, distinct nucleoli. Mitotic figures are occasionally seen (fig. 2). While the cell contours are generally regular, there are sufficient irregularities to denote an ameboid character. These cells appear to be identical with the large mononuclear cells which Dantschakoff describes as differentiating from the blood islands in the area vasculosa of the blastoderm and later in the capillaries of the yolk sac, and which she calls lymphocytes.

The cells here under consideration increase in number not only by their own proliferation but also by continued differentiation from the mesenchymal syncytium (fig. 2). For the most part they lie in compact groups, which gradually increase in size as the cells increase in number, but some cells usually appear in the vicinity of the groups (figs. 4, 5, 7, 8 and 10, 16) and not infrequently at some distance from them. After their differentif,tion and separation from the mesenchymal syncytium all the cells, both members of the groups and isolated, lie free in the spaces among the stellate elements of the mesenchymal tissue (figs. 3 and 4). No blood vessels or lymphatics are present in the immediate vicinity of the cell groups when the latter first develop and consequently the cells are extravascular (figs, 1, 2, 3 and 4). It will be shown later that after the lymphatics develop in this region the cells for the most part are included within them and thus become intravascular elements.


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The application of the term 'blood islands' to the accumulations of cells has been avoided because there is no real condensation of the mesenchyme whereby the differentiating elements fuse to form a solid protoplasmic mass. Each stellate element differentiates individually (figs. 1 and 2) and condensation occurs only as a subsequent aggregation of the differentiated cells. The resulting large mononuclear cells, however, so closely correspond to the first lymphocytes that develop in the area vasculosa of the blastoderm, the subsequent histories of the cells arising in the two localities being identical, the conclusion is warranted that the differentiated mesenchymal elements in question serve as an intraembryonic source of blood cells.

While the present investigation has not been of such a nature as to determine whether the mononuclear basophilic cells which have been described give rise to granular leucocytes, they certainly give rise to red blood cells.. The cytoplasm of some of the cells gradually loses its basophilic character and acquires a stronger affinity for the plasma dyes, at the same time becoming quite homogeneous. These changes are concomitant with the addition of hemoglobin. The nuclear changes comprise an increase in the amount of chromatin, its arrangement into a rather heavy reticulum and the disappearance of the nucleoli (fig. 2).

Cells thus modified and containing a moderate amount of hemoglobin, as indicated by the reaction to plasma stains, are erythrobalsts. Without further visible structural changes these acquire more hemoglobin until eventually they are indistinguishable from the red blood cells in the general circulation. They have therefore become erythrocytes. It is probable also that they are definitive rather than the larger primitive erythrocytes described by Dantschakoff. The changes described above occur both in the cells composing the groups and in the isolated cells, sd that fully developed red blood cells as well as the earlier de\'elopmental stages are seen not only in the groups but also in the mesenchymal spaces more or less remote from the groups.

Among the developing erythi'oblasts, especially in the later stages, are manj^ small cells which apparently go through the same processes of differentiation as the red blood cells themselves and


140 ADAM M. MILLER

resemble the latter in every respect except size. These are undoubtedly microcytes, the earlier stages being microblasts.

After the middle of the fifth day the masses of developing blood cells increase rapidly in size by proliferation of the component cells and continued differentiation of the branching elements of the mesenchymal syncytium. They increase in number also by the same processes of differentiation in other localities. Up to about the beginning of the sixth day all the developing blood cells in question remain extravascular, that is, free in the intercellular mesenchymal spaces which are not lined by endothelium.

By the end of the seventh day the aggregations of cells reach the height of their development. At this time they extend in two main lines from the level of the aortic arches along the dorsal aortic roots to the confluence of the latter to form the aorta (figs. 6, 20 and 22, 16). At this level, or a little further caudal, the two main lines unite to form a single line which extends about to the level of the superior mesenteric artery (figs. 7, 8, 10, 20 and 22, 16). At their greatest development the larger groups together form an almost continuous mass of the cells which in places is greater in diameter than the aorta. Usually there are also numerous smaller outlying groups which belong to the same general line (figs. 7 and 8).

While the main lines above described are established in all the embryos examined, yet there is a wide range of variability in the form of the groups and their arrangement in the lines. In most of the embryos examined a large mass of cells or a collection of smaller groups had developed in the region dorsal to the aortic roots and esophagus (figs. 6 and 22, 16a). One of the most interesting and one of the most important features of the masses of developing blood cells is the fact that the main lines in their general arrangement correspond with the lines of the thoracic duct (figs. 20, 22 and 24, 16, 17 ,17a).

To anticipate, it may be stated here that as the multiple anlagen of the thoracic duct develop the masses of developing blood cells are in a large part included within them, and thus become strictly intravascular (see fig. 10, 16, 17). The details of this process will


THORACIC DUCT IN THE CHICK 141

be considered in the subsequent description of the formation of the lymphatic spaces and channels. After the seventh day, as the lymphatic channels in this region develop and establish communication with the jugular lymph sacs, the intravascular masses of blood cells decrease and, by about the eleventh da}^ are reduced to a few groups scattered through the lymphatic plexus (cf. figs. 24, 27 and 28). A considerable number of extra vascular groups persist, however, until the fourteenth or fifteenth day, or even later. In the later stages the vast majority of the cells in these groups are practically mature erythrocytes lyiiig free in the mesenchymal spaces in the vicinity of the lymphatics (fig. 11).

Inasmuch as the reduction in the masses of blood cells in the lymphatics follows the coalescence of the lymphatic spaces to form continuous channels and the establishment of communication between the latter and the jugular lymph sacs, and since the lymph sacs open into the great veins, the blood cells in question eventually reach the general circulation by way of the thoracic duct and jugular lymph sacs. The thoracic duct at one period acts, therefore, as conveyor of the erythrocyte series of hemal cellular elements which have developed from the indifferent mesenchyme along the line of the duct.

A consideration of the histogenesis of the lymph channels constituting the anlagen of the thoracic duct leads on to controversial ground. As stated earlier in this paper, the controversy hinges upon the question whether the lymphatics, exclusive of the lymph sacs or hearts, arise as sprouts or outgrowths from pre-existing vascular channels or de novo from the intercellular mesenchymal spaces.

It is the opinion of the writer that in the chick the lymph channels which constitute the anlagen of the thoracic duct arise through enlargement and coalescence of intercellular spaces in the mesenchymal tissue, and that the endothelial lining of these channels is derived directly from indifferent mesenchymal cells^ that chance to border upon the spaces. In the material studied there

^ It should be understood that when 'mesenchymal cells' are spoken of, they are considered as the irregularly stellate masses of protoplasm the processes of which anastomose to form the mesenchymal syncytium, or reticulum.


142 ADAM M. MILLER

is no evidence of any growing out, budding or sprouting from the endothelium of pre-existing blood vessels, the tissue in which the thoracic duct develops being non-vascular. The lymphatics in question, and their endothelial lining, arise independently of pre-existing vascular channels.

Prior to the appearance of any specialized spaces or channels in the mesenchyme in the region of the future thoracic duct, the mesenchymal syncytium consists of irregularly stellate protoplasmic elements the slender processes of which anastomose freely with like processes of neighboring elements. The cytoplasm is finely granular and the nuclei, while relatively large and vesicular, contain little chromatin and one or two distinct nucleoli. Among the protoplasmic components of this tissue are the correspondingly irregular interstices or spaces which also are continuous with one another. These are called the mesenchymal intercellular spaces. The tissue as a whole might be compared with a sponge, the anastomosing protoplasmic parts representing the parenchyma of the sponge and the intercellular spaces the pores. While some of the protoplasmic elements during this time differentiate into blood cells, as previously described, our conception of the general syncytium and its spaces is in no way invalidated.

The first changes in the mesenchyme leading toward the formation of definite channels occur during the latter half of the sixth day of incubation. These changes, instead of involving the mesenchyme generally, begin in several localities. In one of the localities, for example, the intercellular spaces increase in size and coalesce, most of the protoplasmic elements of the syncytium being pushed farther apart or broken. In this manner a considerably larger space is formed out of a number of the original smaller mesenchymal spaces (fig. 9, 17). For the most part the smaller spaces of the surrounding tissue open freely into the larger space, although in places along the edge of the latter the protoplasmic elements lose some of their stellate character and become flattened on the side toward the larger space.

The phenomena in general indicate the accumulation of the fluid filling the mesenchymal spaces, the cells and their processes


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being subjected to pressure and friction incident to the flow of the interstitial substance. Thus there arises in the mesenchyme a space larger than the original interstitial spaces but derived directly from them by their enlargement and coalescence (cf. figs. 12, 13 and 14). So far as there is any definite lining for the new space, it is formed by the partiall}^ or wholly flattened mesenchymal cells upon which the fluid in the space impinges (fig. 13).

The further changes in one of these larger spaces consists in the main of its elongation through the enlargement and addition to it of other intercellular mesench3aiial spaces, a progressive flattening of the cells along its sides, and an approximation of the edges of the flattened cells to form a definite lining of endothelium. There is thus formed a distinct channel in the mesenchyme. For the most part it is lined and its lumen is separated from the surrounding interstitial mesenchymal spaces by a layer of endothelial cells which represent metamorphosed stellate elements of the mesenchyme (figs, 15 and 16). At or near its ends the channel opens freely into the adjoining mesenchymal tissue spaces which in the further course of development are added to the lumen of the channel and thus become a part of it (figs. 15, 16, 17 and 18). The endothelial lining already formed merges near the ends of the channel with the mesenchymal syncytium which in turn, as the channel elongates, gives rise to more endothelium by differentiation of certain of its elements (figs. 15, 16, 17 and 18).

As stated in a previous paragraph, the larger spaces appear in several localities in the mesenchyme. Consequently the channels resulting from their further development are for a time isolated; that is, they are not directly connected with one another or with the jugular lymph sacs or any part of the hemal vascular system. These isolated spaces and channels constitute the multiple anlagen of the thoracic duct (fig. 21),

In succeeding stages each of the channels in question increases in size, especially in length, until it meets and coalesces with its neighbors. The increase in length is due in the main to the addition of more mesenchymal tissue spaces to its ends and the concomitant transformation of more stellate mesenchvmal cells into


144 ADAM M. MILLER

endothelial cells. There is probably also some proliferation of the endothelial cells, although in the study of the sections mitotic figures were not seen.

The coalescence of the originally unconnected lymphatics results in a network or plexus of channels (cf. figs. 21, 22 and 24). This is constantly being augmented by the coalescence of other independently formed spaces and channels with one another and with the previouly established plexus. Most of the vessels composing the plexus lie longitudinally in the embryo along the line of the aorta and dorsal aortic roots, and out of this plexus is e\'entually crystallized the main drainage lines of the thoracic duct. The establishment of these lines is best considered, however, in the section on morphogenesis.

The correlation of groups of developing blood cells in this region, the formation of which has already been described, and the lymph spaces and channels constituting the anlagen of the thoracic duct now remains to be discussed. As stated earlier in this paper, the blood cells that are differentiated from the mesenchymal syncytium, whether they are arranged in groups or isolated, lie free in the tissue spaces. In case the tissue spaces enlarge and coalesce in the region where the blood cells are situated the latter are then allowed to become free also in the larger space resulting from the enlargement and coalescence. The larger space become^ lined with endothelium, in the manner previously described, to form a definite vessel or channel. The blood cells, therefore, which were originally free in the tissue spaces are included in an}" of the lymphatics developing in that particular locality (fig. 10, 16, 17).

In that manner some of the blood cells become intravascular elements during the earliest stages of lymphatic development. Occasionally an entire group of cells is included in a lymph channel; in other cases only part of a group or a few scattered cells. It is true also that a great many lymphatics develop in the mesenchyme quite apart from the blood cells (figs. 15, 16 and 17).

In the earlier stages of lymph vessel formation there are great numbers of blood cells, in various degrees of differentiation, in the mesenchymal tissue spaces in the vicinity of or more or less


THORACIC DUCT IN THE CHICK 145

remote from the lymph spaces and channels. In part at least these cells become intravascular when new lymphatics are formed out of the tissue spaces in which they lie and join the general plexus of previously formed vessels.

Thus far, therefore, the admission of the blood cells to the lymph vessels depends merely upon the topographical relationship in the development of the two sets of structures. There are, however, other factors which in all probability enter into this process. Two are of especial interest and importance in the case under consideration.

It has been pointed out by Dantschakoff that the primitive blood cells in the area vasculosa of the blastoderm and the large mononuclear cells derived from them are capable of ameboid movement. The developing blood cells in the region of the thoracic duct anlagen are demonstrably of the same type as those in the area vasculosa. Hence it is reasonable to conclude that some of the developing blood cells pass from the tissue spaces into the vessels by virtue of their ameboid character.

The other factor has hitherto, so far as the writer is aware, been considered only in the study of living tissues. It seems justifiable, however, to extend the conclusions drawn therefrom to fixed tissues. In their study of chick blastoderms in vitro, McWhorter and Whipple (18) have observed the to and fro movement, synchronous with the heart-beat, of blood cells not only in the isolated, endothelium-lined spaces which eventually coalesce to form blood vessels but also in the tissue spaces. Furthermore, they have observed the entrance of blood cells into the general circulation following then- to and fro movements in the tissue spaces. These phenomena certify the pulsation of the fluid substance in the tissue spaces in response to the heart-beat. It is not uiu-easonable, therefore, to conclude that some of the blood cells in the region of the developing thoracic duct are driven or sucked into the lymph spaces or channels which, as pointed before, open freely into the mesenchymal tissue spaces.

As a corollary to the phenomena mentioned in the preceding paragraph, an additional factor in the formation of endothelium might be suggested. Granting that the blood cells lying free in


146 ADAM M. MILLER

the tissue spaces move to and fro in response to the heart-beat, as has been clearly observed in the living chick blastoderm, the assumption is justifiable that their movement, with the resultant friction and pressure upon the adjacent protoplasmic elements of mesenchymal tissue, assists in the flattening of these elements and the consequent formation of endothelial cells.

The hydrodynamic factors of pressure and friction of the interstitial fluid substance upon the protoplasmic elements of the mesenchyme, which were first discussed by Thoma (19) in connection with blood vessel formation and have already been noted in this particular case of development of lymphatics, together with the additional factor of pressure and friction of oscillating blood cells would, in the opinion of the writer, afford adequate mechanical means of changing the irregular plastic elements of the mesenchymal syncytium into the endothelial cells of the vessel wall.

IV. MORPHOGENESIS

Up to this point we have been considering the histogenetic changes which occur in the mesenchj^mal tissue, resulting in the formation of lymph channels and their endothelial lining and of blood cells. It has been demonstrated that aggregations of the developing blood cells, identical with the mesenchymal 'cords' described by Sala, appear along the lines of the future thoracic duct. It has been shown also that the rudiments of the thoracic duct develop as isolated spaces and channels in the mesenchyme, that the endothelial lining of the channels is derived directl}^ from the mesenchymal cells forming their borders and that the channels coalesce to form a plexus. It is our object, under the head of morphogenesis, to trace the subsequent history of the isolated channels and the plexus formed therefrom.

Chick embryo of six days and sixteen hours, 13.5 mm. {Columbia Embryological Collection, series no. 4^6). Reconstruction, ventral view. Figure 20. The incipient stages of thoracic duct development are shown about this time. The lymph spaces {17) are three in number, two on the right side and one on the left, situated in the mesenchyme ventro-lateral to the aorta {I) about midway between the exits of the celiac (-5) and superior mesenteric arte


THORACIC DUCT IN THE CHICK 147

ries. They are isolated, for absolutely not any divect connection with any other vessel can be traced even with high powers of magnification. They open freely into the surrounding intercellular spaces, as show in figures 12, 13 and 14, which are photographic reproductions of three successive sections of the embryo.

The spaces fit the previous description, given in the section on histogenesis, of accumulations of an intercellular fluid in the mesenchyme. The objection that they may be shrinkage spaces is nullified by the fact that the preservation of the tissue is practically perfect, and that in this and in other stages the more nearly perfect the preservation the more clearly defined are the spaces. Furthermore, in cases of poorer preservation where there are obvious shrinkage spaces in the mesenchyme the boundaries of such spaces are almost invariably ragged and irregular and do not anywhere exhibit a smooth endothelial lining.

The masses of developing blood cells in this particular embryo (six days and sixteen hours) are perhaps unusually extensive (fig. 20, 16). They extend in irregular groups from about the level of the superior mesenteric artery forward along the ventral and ventro-lateral aspect of the aorta (1) to the level of the celiac artery (5), with a tendency to cluster around the last named vessel (cf. fig. 7, 16). They then divide into two general lines, one on each side, which bend laterad and extend forward along the mesial aspect of the ducts of Cuvier (12) and precardial veins (10), ending rather abruptly in a large mass which hes at the level of the sixth aortic arches (3) and extends across the mesial line ventral to the dorsal aortic roots (2).

The three spaces representing the first anlagen of the thoracic duct (17) bear no particular relation to the large groups of developing blood cells (16), although a few isolated blood cells lie in the tissue spaces around the larger rudimentary lymph spaces.

Chick embryo of six days and twenty-one hours, llf. 7nm. (Columbia Collection, series no. ^65). Reconstruction, dextro-ventral view. Figure 21. In this embryo there is a considerable increase in the size and number of lymph spaces and channels which constitute the early anlagen of the thoracic duct (17). They are situated for the most part in the mesenchymal tissue ventral and ventro THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 2


148 ADAM M. MILLER

lateral to the aorta (i) near the exit of the celiac artery (5). Other spaces and channels of a similar nature are seen along the dorsal aortic root (2), thus continuing the same general line of lymphatics toward the jugular lymph sac (15) with its thoracic duct 'approach' {15a). Some of these early lymphatics are of considerable length and have acquired a distinct endothelial lining. Others are simply enlarged spaces in the mesenchyme exactly like those described in the preceding stage (figs. 13 and 14). There is not yet any plexiform arrangement of the channels.

The principle underlying the formation of the longer channels is in the main the enlargement and coalescence of two or more of the original smaller spaces or channels. For instance, two isolated spaces or channels lying near each other in the mesenchyme increase in size until eventually they flow together to form a single space or channel. Or, the same process occurs in several spaces or channels in a discontinuous series until all the members of the series flow together and thus form a continuous channel.

There is also in all probability some proliferation of the endothelium lining the channels. In fact there is no valid reason for not assuming that, after the inception of a given vascular channel, either hemal or lymphatic, along with its increase in size there is a concomitant proliferation of its endothelial cells. The crucial point, however, is the origin of the channels. The evidence at hand points clearly to the origin of the lymphatics constituting the rudiments of the thoracic duct directly from the mesenchymal interstitial spaces in the manner previously described, the endothelium of the lymphatics representing mesenchymal cells which are modified in accordance with the new conditions of pressure and friction.

In this embryo (series no. 465) the rudiments of the thoracic duct (fig. 21, 17) are isolated. There is no connection between these and the hemal vascular system; nor on the other hand is there any communication with the jugular lymph sacs (15). The thoracic duct 'approach' of the lymph sac, which the duct eventually joins, is well developed at this stage (15a) but a considerable distance intervenes between the 'approach' and the rudimentary duct.


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The masses of developing blood cells in this embryo are unusually scarce. A few small groups (16) are situated in the mesenchymal tissue ventral to the aorta (1), and three larger groups (also marked 16) are seen between the dorsal aortic root (2) and the jugular vein (10).

Chick embryo of six days and twenty-two hours, 16 mm. (Columbia Collection, series no. 463). Reconstruction, ventral view. Figure 22. In this embryo the lymphatics (17) are in approximately the same stage of development as in the preceding embryo. They are situated for the most part along the ventral aspect of the aorta (1). A few spaces are situated ventro-lateral to the dorsal aortic roots (2).

The masses of developing blood cells (16) are much more extensive than in the preceding embryos of about the same stage, thus exhibiting the variability of the structures. The groups associated with the rudimentary thoracic duct (17) lie ventrolateral to the aorta (1), with some tendency to cluster around the celiac artery (5) . The rest of the groups extend in a continuous mass on each side along the ventro-lateral aspect of the dorsal aortic roots (2) and along the mesial aspect of the jugular veins (10) (cf. fig. 6, 16) as far forward as the fourth aortic arch (4)A large aggregation of developing blood cells (16a) also lies between and somewhat dorsal to the aortic roots (2) and the arches (3, 4) (cf. fig. 6, 16a), being connected with the left lateral groups by cords extending ventral and dorsal to the roots and arches. Associated with this large mass are a few lymphatics (19) (cf. fig. 6, 19) which subsequently join the thoracic duct and may also communicate with the cephalic end of the jugular lymph sac.

One of the most interesting features of this particular stage is the well developed thoracic duct ^approach' of the jugular lymph sac (fig. 22, 15a). A reconstruction to show this structure was made on a larger scale and is illustrated in figure 23. The lymph sac iself (13) lies dorsal to the jugular vein (10) and fits into the angle between the latter and the subclavian vein (11). The thoracic duct 'approach' (15a) is situated on the mesial aspect of the j ugular-subclavian angle and thence extends a short distance caudad along the mesial side of the precaval vein. It lies for the


150 ADAM M. MILLER

most part between a mass of developing blood cells {16) and the vein [10) and ends blindly (see also fig. 19, 16, 15a). A short distance farther caudad are two spaces in the mesenchyme which represent the extreme cephalic end of the rudimentary thoracic duct (fig. 23, 17). These spaces are isolated, for with high magnification there cannot be discerned any connection between them and the thoracic duct 'approach' {15a) or the other rudiments of the thoracic duct lying still farther caudad.

The thoracic duct 'approach' of the jugular lymph sac in the chick is without doubt the homologue of a similar structure" described by Huntington and McClure (9) in the cat. Like the lymph sac itself, it is of venous origin in the chick as in the mammal, and forms an integral part of the sac. It arises from some of the more mesially and caudally situated components of the early venous plexus in the region of the jugulo-subclavian angle. When fully formed it extends caudad and mesad for some distance along the mesial aspect of the precaval vein, and terminates blindly (figs. 19 and 23, 15a). Subsequently when it is joined by the thoracic duct, it serves as the portal of entry of this duct into the lymph sac.

Chick embryo of seven days {Columbia Collection, series no. 512). Reconstruction, ventral view in figure 24-; view from left side, figure 25. In this embryo there is considerable advance in the development of the rudiments of the thoracic duct. The spaces and channels, which in the preceding stage were unconnected, have here coalesced to form an extensive plexus {17) of channels extending from the level of the junction of the dorsal aortic roots {2) nearly to the exit of the superior mesenteric artery. The plexus lies for the most part ventral to the aorta {!), but a few of its components extend around on the lateral aspect of this vessel. Most of the channels in the network are much larger than the original spaces and channels, leaving but small areas of mesenchymal tissue among them; in part they have even fused to form irregular sinuses. The celiac artery (5) in most of its longitudinal course penetrates the plexus {17). The plexus as a whole is still isolated, being connected neither with any part of the hemal vascular sj^stem nor with the thoracic duct 'approach' of the jugular lymph sac {15a).


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Along the dorso-lateral aspect of the aorta (1) a number of spaces and channels have also developed (fig. 25, 20). In addition, a few smaller lymphatics have appeared along the lateral aspect of the aorta. The dorsal aorta is thus almost completely encircled by a group of spaces and channels comprising the large ventral lymphatic plexus described in the preceding paragraph and the dorsal and lateral sets of lymphatics.

These encircling lymphatics are in the aggregate homologous with the dorsal set of peri-aortic sinuses in reptiles, as described by Huntington (2). They are also homologous, in all probability, with the azygos portion of the thoracic duct in the cat, as described by Huntington (3), and in Tragulus, as described by Tilney (20). A comparison with Stromsten's (16) description and figures indicates, too, that the lymphatics associated topographically with the dorsal aorta in the chick are collectively the homologue of the peri-aortic network of h'mph vessels in the loggerhead turtle, or, more specifically, with that portion of the network surrounding the dorsal aorta. In addition, the lymphatics around the dorsal aorta in the chick, as previously described, may be placed in the same phylogenetic line as the postcardinal and supracardinal divisions of the thoracic duct in the pig, as recently worked out by Kampmeier (15).

Returning to a further consideration of conditions in the chick at this stage (seven days, fig. 24), it is seen that a few small Ijnmph spaces {17a), also isolated, have appeared along the ventrolateral aspect of the dorsal aortic roots (2) in the interval between the plexus previously described {17) and the thoracic duct 'approach' of the jugular l}Tnph sac {15a). These small spaces represent the beginning of the connection between the thoracic duct 'approach,' on each side, and the unpaired portion of the thoracic duct itself, here composed of the large plexus {17).

The conditions in the chick thus correspond so closely to those in other forms that it is possible to draw a clear homology between the lymphatics joining the thoracic duct 'approach' to the unpaired portion of the duct itself in the chick and the preazygos segment of the thoracic duct in the cat (Huntington) and Tragulus (Tilney), with the cephalic portion of the peri-aortic lymph


152 ADAM M. MILLER

plexus in the loggerhead turtle (Stromsten), and with the precardinal division of the thoracic duct in the pig (Kampmeier).

In the mesenchyme between and dorsal to the aortic roots and arches in this embryo (fig. 24) a number of lymph spaces have developed. Some of these lie within the masses of developing blood cells in this region {16a) while others are situated at some distance from them. A few have coalesced to form a distinct channel (19). The subsequent history of these lymphatics will be given in the discussion of later stages.

A few isolated lymph spaces are also found in and near the root of the dorsal mesentery at the level where the celiac artery enters the mesentery (figs. 24 and 25, 21). These belong to the category of mesenteric lymphatics, but do not yet communicate with the developing thoracic duct.

The masses of developing blood cells in this embryo are extensive (figs. 24 and 25, 16, 16a). Those associated with the main portion of the developing thoracic duct lie either in the meshes of the lymph plexus (17) or are already included within the vessels composing the plexus. A plate-like mass extends over the mesial aspect of the pleural cavity on the right side. The blood cells still, as in preceding stages, tend to cluster about the celiac artery (5). There is no connection in this stage between the masses just mentioned and those which lie farther forward along the mesial aspect of the great veins (10, 12), and which are associated with the thoracic duct 'approach' of the jugular lymph sac (15a). In the region between and dorsal to the dorsal aortic roots (2) and aortic arches (3) there is seen a large mass (16a) with which certain lymphatics (19), previously referred to, are associated. Situated farther caudad are also several masses associated with the lymphatics which lie along the dorso-lateral aspect of the aorta (fig. 25, 16a).

A feature shown in this reconstruction, and not in any other, is a portion of the splanchnic plexus of veins (figs. 24 and 25, IJf).^

' Splanchnic venous plexus is the name given by Dr. A. J. Brown, in his yet unpublished work on the development of the pulmonary veins, to the network of venous capillaries in the wall of the alimentary tube in the earlier stages of development.


THORACIC DUCT IN THE CHICK 153

This is seen near the cephalic end of the large ventral lymph plexus {17) but has no connection with the latter.

At this point it may be well to state that in the chicks of the Columbia Collection I have found no evidence of extra-intimal or perivenous origin of the lymphatics that make up the thoracic duct, such as described by Huntington in the cat and Kampmeier in the pig. This mode of development appears to be a strictly mammalian specialization. In fact Huntington (3), speaking of the extra-intimal replacement of veins by developing lymphatics, states (p. 155) :

The association of these (Ij^mphatic) channels, in the mammalian embryo, with certain embryonal venous lines is purely a secondarj^ mechanical and topographical relationship, expressed by the condensed term of 'extra-intimal' development of mammalian systemic lymphatic vessels, and absolutely devoid of genetic significance. This is, without reference to other vertebrate classes, proved by the development within restricted areas in the mammalian embryo of systemic lymphatic channels through the direct confluence of intercellular mesenchymal clefts, not related topographically or in any other sense to the embryonal veins. It is true that in the mammal this independent lymphatic genesis is extremely limited, and that the majority of the lymphatic vessels develop in close association with embryonal veins, as products of the confluence of perivenous extra-intimal spaces. But this is merely, as shown by comparison with other amniote embryos, the expression of the peculiar relations obtaining in the mammal between the venous and lymphatic circuits of the vascular system, developed independently of each other.

Further, Huntington (2), describing the independently formed system lymphatic channels of the reptilian embryo, by confluence of intercellular mesenchymal spaces, states concerning the latter:

They are not complicated by close topographical relations to adjacent temporary embryonic venous plexuses, as in the mammal, but develop independently by themselves in mesenchymal territory not occupied by hemal vascular elements In both lacertilian and chelonian embryos the greater part of the enormously enlarged systemic lymphatic channels develop without any reference whatever to embryonic veins, in mesenchymal areas where the latter are extremely scanty or entirely wanting (pp. 271-273).

Stromsten (16) has reached similar conclusions in studying the development of the prevertebral peri-aortic sinuses in chelonian embryos.


154 ADAM M. MILLER

These finding in reptiles, and my own results in the bird, warrant the conclusion that the sauropsids agree absolutely genetically with the mammals in the development of the main axial lymphatic lines bj^ confluence of independently formed intercellular mesenchymal spaces, but that the latter are characterized by close topographical association of these spaces with temporary embryonic veins which they in large part replace, while the former present no such association. In them the progress of thoracic duct development is not, as is the case in the mammal, complicated by the presence of an extensive azygos venous system, and axial lymphatic development, especially in the reptile, occurs chiefly by confluence of mesenchymal spaces surrounding the main arterial trunks.

Chick embryo of eight days {Columbia Collection, series no. 513). Reconstruction, ventral view. Figure 26. The next important change in the lymphatics constituting the thoracic duct comprises the further development of certain isolated spaces situated along the ventro-lateral aspect of the dorsal aortic roots in the interval between the cephalic end of the large ventral plexus and the thoracic duct 'approach' of the jugular lymph sac. The incipient stage in the formation of these spaces was illustrated in flgure 24, 17a. In the embryo now under consideration they have enlarged and coalesced to form a continuous channel (figure 26, 17a). This in turn has united with the ventral unpaired portion of the thoracic duct {17) and with the thoracic duct 'approach' of the lymph sac {15a). There is thus established a direct and free communication between the ventral lymph plexus {17), which had arisen as an independent and isolated structure, and the jugular lymph sac {15). Therefore, as clearly shown in figure 26, the thoracic duct 'approach' {15a), which was previously described as an integral part of the jugular Ijanph sac (see fig. 23, 15a), serves as the portal of entry of the thoracic duct into the lymph sac (fig. 26, 15).

The writer has in a previous article (1) shown and will here again point out in a later stage that in the chick, as in reptiles and mammals, a communication is established between the jugular lymph sac and the great veins in this region through one or more


THORACIC DUCT IN THE CHICK 155

taps. And inasmuch as the thoracic duct opens into the lymph sac, the latter serves as the portal of entry of the systemic lymphatics into the venous system, a point upon which emphasis has already been laid by Huntington (2) (3) in his work on reptiles and the cat.

In the embryo of eight daj's the large ventral lymph plexus of the preceding stage (cf. fig. 24, 17) has undergone further coalescence of its component channels to form a large irregular sinus with a few fenestrae (fig. 26, 17). The sinus lies ventral to the aorta (1) and extends from the junction of the dorsal aortic roots {2) to the level of the exit of the superior mesenteric artery {9). At its cephalic end it branches off into the two slender trunks, one on each side, which extend cephalad and laterad to join the 'approaches' {15a) of the lymph sacs {15). These trunks, which are the last components of the Ijrmphatic drainage line to develop, constitute in the bird, according to the anatomical terminology, the right and left thoracic ducts.

The lymphatics (19) situated between and dorsal to the aortic roots {2) and arches {3, Jf) are here seen to comprise a plexus and a few outlying isolated spaces and channels (cf. fig. 24 and 26, 19). This plexus now communicates with the chain of lymphatics {20) lying dorso-lateral to the aorta ( / ) , which in* turn communicate with the large ventral plexus through channels formed by coalescence of spaces lateral to the aorta (cf. fig. 25, 20, 17). The entire group of lymphatics in the region of the dorsal aortic roots and the dorsal aorta as far back as the superior mesenteric artery, with the exception of the mesenteric lymphatics (fig. 26, 21), which have not yet joined the thoracic duct, drain into the jugular lymph sacs.

The masses of developing blood cells were omitted from the reconstruction of the eight day stage (fig. 26). A careful study of the serial sections showed that they were fewer and smaller than in the preceding stage. This may be due to the variability characteristic of the masses, or it may be due to actual reduction of the masses since the blood cells, as stated in in the section on histogenesis, now have access to the jugular lymph sacs through the recent connection established between these structures and


156 ADAM M. MILLER

the chain of lymphatics with which the blood cells have for the most part been associated.

Chick embryo of nine days and fourteen hours {Columbia Collection, series no. 320). Reconstruction, ventral view and viewfrorn left side. Figures 27 and 28. At this stage the main features of the adult thoracic duct have been established. The large plexus {17) ventral to the aorta {1) now drains through the right and left thoracic ducts {17a) into the 'approaches' {15a) of the jugular lymph sacs {15) and then through the sacs into the great veins {10, 12).

The ventral plexus {17), the isolated anlagen of which were the first lympathics to develop in the thoracic duct line, is relatively smaller and the component channels less dilated than in the preceding stages. This is probably due to the outflow of the contents into the lymph sacs and veins through the more recently formed channels which have been called the right and left thoracic ducts {1 7a) . The mesenteric lymphatics {21 ) have increased in size and for the most part coalesced to form sinus-like channels. Between these and the thoracic duct chain no connection can be detected at this stage, although little tissue intervenes. Farther cephalad another isolated group of lymphatics {22) is associated with the esophagus.

The right and left thoracic ducts {17a) are longer than in the preceding stage (cf. fig. 26, 17a). The left is considerably greater in diameter than the right. Each opens into the corresponding 'approach' {15a) of the jugular lymph sac. The 'approach' on each side is still patent as a branch of the main portion of the sac.

There is now free communication between the lymph sacs {15) and the great veins through recently formed taps, of which two are present on the left side of the embryo and one on the right. Of the two on the left side, one is situated on the dorsomesial side of the superior vena cava {12) just below the level of the jugulo-subclavian junction {10, 11); the other is situated farther forward on the mesial side of the jugular vein {10). The tap on the right side of the embryo is located on the dorso-mesial side of the superior vena cava at the level of the jugulo-subclavian junction.


THORACIC DUCT IN THE CHICK 157

In a previous article on the development of the jugular lymph sac in birds (1) the writer stated, in the description of tap formation, that "it is not improbable that a study of later stages will reveal a homologue of the common jugular tap in the mammal" (loc. cit,, p. 486). There is little doubt that the tap on the mesial side of the left jugular vein, referred to in the preceding paragraph, fulfils the requirement. Moreover, the other tap on each side near the jugular-subclavian junction in the chick is in all probability homologous with the jugulo-subclavian tap in the mammal (26), although it is slightly different in position.

The previously described lymphatics situated dorsal to the aortic roots and arches and dorso-lateral to the aorta here constitute a long chain of continuous channels reaching from the level of the jugular lymph sac to the level of the celiac artery (figs. 27 and 28, 19, 20; cf. figs. 24, 25 and 26, 19, 20). At the extreme cephalic end one of the channels of this series opens into the right lymph sac. The probable significance of this opening will be considered in the subsequent discussion of the masses of developing blood cells. The plexus [19) in the region of the aortic arches is considerably reduced as compared with preceding stages. The portion of the dorsal plexus {20) associated with the dorsal aorta communicates with the large ventral plexus {17) through small channels which curve around the lateral aspect of the aorta. It is seen, therefore, that the entire group of lymphatics associated with the aorta and dorsal aortic roots, with the exception of a few still isolated spaces and channels, can now discharge the contents of the channels into the great veins.

The most interesting and, in fact, from the standpoint of the hemophoric* function of the thoracic duct, the most important feature of this stage is the great reduction in size and number of the masses of developing blood cells. It has been shown in the foregoing pages that the differentiating blood cells associated with the developing lymphatics are admitted or gain access to the lymph spaces and channels and that when the communication is established between the thoracic duct and the jugular lymph sacs the blood cells can thus reach the sacs. Now since the taps

Hemophoric — blood bearing or carrying — is a term suggested by Dr. Schulte as a coordinate with hemopoietic — blood producing.


158 ADAM M. MILLER

between the lymph sacs and great veins have been formed, the blood cells are admitted to the hemal vascular system.

In this connection there are two aspects of the question which are especially worthy of consideration. In the first place there are few blood cells within any of the channels forming a part of the thoracic duct system, while in earlier stages, before the thoracic duct had open communication with the lymph sacs and great veins, many of the lymph channels were filled with the hemal cellular elements. In view of this, and the fact that there are no other means of egress for the blood cells formerly contained in the lymphatics, it must be concluded that the blood cells reach the hemal vascular system via the thoracic duct, the jugular lymph sacs and the recently formed taps between the sacs and the great veins.

The other feature is the great reduction in the size and number of the extravascular masses of developing blood cells. The diminished masses are shown in yellow in figures 27 and 28, the conditions in which should be compared with those in figures 22 and 24. A few fairly extensive groups are found in the mesenchymal tissue among the channels composing the large ventral plexus [17). These are for the most part quite closely associated with the lymphatics. A few small groups still are found in the region of the thoracic duct 'approach' (fig. 28, 15a). The extensive masses associated in earlier stages with the lymph plexus dorsal to the aortic arches have almost wholly disappeared. This plexus itself is considerably reduced, and it is not unreasonable to assume that the connection between the cephalic end of the plexus and the jugular lymph sac, previously alluded to, is in some way associated with the discharge of the numerous blood cells differentiated in this region into the lymph sac.

In view, therefore, of the intimate relationship between the developing thoracic duct and blood cells in the same general region, and of the sudden and marked reduction in number of these blood cells, both intravascular and extravascular, following the establishment of communication between the duct and the jugular lymph sacs and great veins, the importance of the thoracic duct as a carrier of hemal cellular elements for a period of embryonic life in the bird can scarcely be doubted.


THORACIC DUCT IN THE CHICK 159


V. SUMMARY


Prior to the appearance of lymphatics in the region of the future thoracic duct, namely, along the aorta and dorsal aortic roots, the mesenchyme comprises a syncytium of irregular strands with correspondingly irregular interstitial spaces. The tissue is nonvascular.

The initial change in conditions is manifested in the appearance of distinct lacunae in the mesenchymal tissue along the ventro-lateral aspect of the aorta at the level of the celiac artery. The lacunae are bounded by unmodified protoplasmic elements of the mesenchymal syncytium, and open freely into the adjacent intercellular — interstitial — spaces. Obviously the lacunae represent enlarged intercellular spaces, and the inference is justifiable that they are filled with the intercellular fluid.

In a slightly advanced stage of development in general there is a greater number of lacunae in the same region in the embryo and also an increase in size and a difference in the appearance of some of the lacunae. The increase in size certainly depends in part upon actual dilatation and in part upon addition of more of the adjacent mesenchymal intercellular spaces, for every possible gradation can be seen between the smallest and the largest. The difference in appearance is observed to be due to the presence of flat cells which form a distinct boundary or wall, although not usually complete. Morphologically these cells are equivalent to endothelial cells. Inasmuch as they shade by invisible gradations into the unmodified mesenchymal cells bordering upon the rest of the lacuna, we conclude that they are derived directly from the indifferent mesenchymal cells. The differentiation, we may also infer, is due in part to pressure and friction incident to the flow of the tissue fluid. Another factor in the differentiation may also be pressure and friction incident to the to and fro motion of blood cells in the tissue spaces and lacunae in response to the heart-beat, a phenomenon observed in living blastoderms.

Studies of later stages show these isolated lacunae to be the rudiments of the thoracic duct, and the conclusions that they are direct derivatives of the mesenchymal intercellular spaces and


160 ADAM M. MILLER

that the flat cells forming their endothelial walls are differentiated in situ from the mesenchymal cells are based upon prolonged and thorough studies, with high magnification, of serial sections of chick embryos in practically perfect states of preservation. The writer is therefore forced to ally himself unequivocally with the advocates of the view that the thoracic duct originates independently of the veins and lymph sacs.

Studies of subsequent stages also show that the numerous isolated lacunae, or rudiments of the thoracic duct, enlarge still further, principally in a longitudinal direction, and coalesce with one another to form a plexus of lymph channels which lies ventral to the aorta. Other similar isolated lymphatics develop along the dorso-lateral aspect of the aorta and in the region dorsal to the aortic roots and arches, and then coalesce to fonii plexuses. Eventually all the plexuses intercommunicate.

In the meantime a connection is established between the large ventral plexus and a branch of each jugular lymph sac known as the thoracic duct 'approach,' All the components of the thoracic duct system thus drain into the jugular lymph sacs. Communications, or taps, are established between the lymph sacs and the great veins, and the thoracic duct then drains into the hemal vascular system, the lymph sacs serving as portals of entry.

In the region of the developing thoracic duct, namely, along the aorta and dorsal aortic roots, and also in the region dorsal to the aortic arches, a great number of blood cells arise. The genesis of these cells is indicated by certain changes in some of the irregular elements of the mesenchymal syncytium, comprising a marked increase in the basophilia of the cytoplasm, a rounding of the cell body and a separation from the general mesenchymal reticulum. The resulting cells thus lie free in the interstitial spaces, and structurally are similar to the large mononuclear cells (lymphocytes of Dantschakoff) in the area vasculosa of the blastoderm. They increase in number both by mitosis and by constant differentiation from the mesenchymal syncytium.

Many, at least, of these basophilic cells are transformed into erythrocytes through the addition of hemoglobin to the cytoplasm and certain nuclear modifications comprising the


THORACIC DUCT IN THE CHICK 161

disappearance of the nucleoli and the rearrangement of the chromatin into a heavy reticulum.

These developing blood cells, at first scattered in the mesen-chymal intercellular spaces, become aggregated, following the increase in their number, into extensive masses which lie along the line of the thoracic duct and also in the region dorsal to the aortic arches.

When the lymphatics comprising the rudiments of the thoracic duct develop, some of the developing blood cells, even some of the smaller groups, are seen to be contained within them, while others are still free in the mesenchymal intercellular spaces, that is, extravascular. Subsequently more and more of the cells are observed to be intravascular. It may be concluded that the developing blood cells become intravascular by simple inclusion as the lymph channels develop, or by virtue of their ameboid character, or as a result of their motion to and fro in the tissue fluid in response to the heart-beat.

The blood cells that are admitted to the lymph channels constituting the thoracic duct system during its development rapidly diminish in number after communication is established between the thoracic duct and the jugular lymph sacs and between the latter and the great veins. It can be inferred then that the blood cells which develop along the line of the thoracic duct reach the blood stream via this duct and the lymph sacs. Considering the vast number of hemal cellular elements, especially erythrocytes, arising in this region and the probability that they reach the general circulation via the thoracic duct, this duct assumes an additional phase of importance in the chick in that it performs a hemophoric, or blood carrying, function.

In conclusion, I wish to thank Dr. Huntington and Dr. Schulte for their valuable criticism and suggestions. Dr. McWhorter for his painstaking work in making the photomicrographs, and Mr. Petersen for his careful execution of the color plates.


162 ADAM M. MILLER

REFERENCES

1) Miller, A. M. 1912 The development of the jugular lymph sac in birds. Amer. Jour. Anat., vol. 12.

2) Huntington, George S. 1911 The development of the lymphatic system in reptiles. Anat. Rec, vol. 5.

3) 1911 The anatomy and development of the systemic lymphatic vessels in the domestic cat. Memoirs of The Wistar Institute of Anatomy and Biology, no. 1, May.

4) HoYER, H. 1905-1908 Untersuchungen iiber das Lymphgefasssystems der Froschlarven. Bull, de I'acad. des sciences de Cracovie, classe des sciences math, et nat., I Theil, 1905; II Theil, 1908.

5) Knower, H. McE. 1908 The origin and development of the anterior lymph hearts and the subcutaneous lymph sacs in the frog. Anat. Rec, vol. 2.

6) Lewis, F. T. 1905 The development of the lymphatic system in rabbits. Amer. Jour. Anat., vol. 5.

7) Sarin, Florence R. 1909 The lymphatic system in human embryos, with a consideration of the morphology of the system. Amer. Jour. Anat., vol. 9.

8) MiERZEjEWSKY, L. 1909 Beitrag zurEntwicklung desLymphgefiisssystems der Vogel (vorlaufige Mitteilung). E.xtrait du bull, de I'acad. des sciences de Cracovie.

9) Huntington, G. S., andMcClure, C. F. W. 1910 The anatomy and devel opment of the jugular lymph sac in the domestic cat. Amer. Jour. Anat., vol. 10.

(10) Sala, L. 1900 Sullo sviluppo dei cuori limfatici e dei dotti toracici nell'

embryone di polio. Ricerche fatta nel Laboratorio di Anatomia Normale della R. Univ. di Roma, vol. 7.

(11) Sarin, Florence R. 1902 On the origin of the lymphatic system from

veins, and the development of the lymph hearts and thoracic duct in the pig. Amer. Jour. Anat., vol. 1.

(12) Langer, C. 1868 tJber d^s Lymphgefiisssystem des Frosches. Sitz. d.

Akad. d. Wissensch., Bd. 57.

(13) Ranvier, L. 1897 Morphologie et developpement des vaissaux lympha tiques chez les mammiferes. Arch, d'anatomie microscopique, tome 1.

(14) Huntington, George S. 1908 The genetic interpretation of the develop ment of the mammalian lymphatic system. Anat. Rec, vol. 2.

(15) Kempmeier, Otto F. 1912 .The development of the thoracic duct in the

pig. Amer. Jour. Anat., vol. 13.

(16) Stromsten, Frank A. 1912 On the development of the prevertebral (tho racic) duct in turtles as indicated by a study of injected and uninjected embryos. Anat. Rec, vol. 6.

(17) Dantschakoff, W. 1908 Untersuchungen iiber die Entwickelung des

Blutes und Bindegewebes bei den Vogeln. I. Die erste Entstehung der Blutzellen beim Hiihnerembryo und der Dottersack als blutbildenea Organ. Anatomische Hefte, 113 Heft (37. Band, Heft 3).


THORACIC DUCT IN THE CHICK 163

(18) McWhorter, J. E., AND Whipple, A. (). 1912 The development of the blastoderm of the chick in vitro. Anat. Rec, vol. 6.

(1^9) Thoma, R. 1893 Untersuchungen iiber die Histogenesc vind Histomechanik des Blutgefjisssystems. Stuttgart.

(20) TiLXEY, F. 1912 The development of the veins and lymphatics in Tragu lus meminna. Amer. Jour. Anat., vol. 13.

(21) ^IcClure, C. F. W. 1908 The development of the thoracic and right lym phatic ducts in the domestic cat. Anat. Anz., Bd. 32, nos. 21 and 22.

(22) Sabix, Florence R. 1911 A critical study of the evidence presented in

several recent articles on the development of the lymphatic system. Anat. Rec, vol. 5. .(23) ]\IcClure, C. F. W. 1910 The extra-intimal theory and tlie development of the mesenteric lymphatics in the domestic cat (Felis domestica). Anat. Anz., Erganz. z. Bd. 37.

(24) Sabix, Florexce R. 1912 On the origin of the abdominal lymphatics in

mammals from the vena cava and the renal veins. Anat. Rec, vol. 6.

(25) HuxTixGTON, Geo. S., and McClure, C. F. W. 1907 The development of

the main lymph channels of the cat in their relations to the venous system. Anat. Rec, vol. 1, no. 3.

(26) McClure, C. F. W., and Silvester, C. F. 1909 A comparative study of

the lymphatico-venous communications in adult mammals. Anat. Rec, vol. 3.


THE AMERICAN .lOURNAI, OK ANATOMY, VOL. 15, NO.


PLATE 1


EXPLAXATIOX OF FIGURES


1 From a section of a chick embryo of 108 hours, 6.75 mm. (series no. 371; slide II, section 80). Outlines drawn under Edinger projection apparatus, X 1500; reduced to XlOOO. In the upper left corner is a portion of the aorta with a few erythrocytes. Outside of the aortic wall is an area of mesenchyme in which some elements show stages of differentiation leading toward large basophilic cells, two of the latter lying free in the tissue spaces at the lower left corner of the figure. These are apparently identical with the developing blood cells (lymphocytes) in the area vasculosa of the blastoderm as described by Dantschakoff.

2 From the same embryo as figvu-e 1 (slide II, section 97). Outlines drawn under Edinger i^rojection apparatus, X 1500; reduced to X 1000. A small part of the aorta is shown in the upper right corner. Some of the protoplasmic elements of the mesenchymal syncytium are shown in various stages of differentiation leading toward the free basophilic cells (lymphocytes of Dantschakoff). In the lower left corner is seen one of these cells in the anaphase of mitosis. At the right of this is a practically mature erythrocyte lying free in a mesenchymal intercellular space.

3 From a section of a chick embryo of 132 hours, 9 mm. (series no. 411; slide IV, section 16). Outlines drawn under Edinger projection apparatus, X 1500; reduced to X 1000. This figure, taken just ventral to the aorta, shows a number of the rounded basophilic cells lying free in the mesenchymal intercellular spaces.


164


THORACIC DUCT IN THE CHICK

ADAM M. MILLER




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165


PLATE 2


EXPLANATiOX OF FIGURES


4 From a section of a chick embryo of o (lays, 12 mm. (series no. 336; slide IV, section?). Photomicrosi'aph, X 166.

1, aorta 10, preeardinal vein

3, aortic arch VI 16, grouj) of developing blood cells

4, aortic arch 1\

5 From a section of a cfiick embryo of 6 days, 13.5 mm. (series no. 339; slide VIII, section 35). Photomicrojirajjh, X 166.

2, dorsal aortic root 16, groups of developing blood cells

3, aortic arch VI 27. vagus nerve


166


THORACIC DUCT IN THE CHICK

ADAM M. MILLER






PLATE 3


EXPLANATION OF FIGURES


6 From a section of a chick embryo of 7 days (series no. 512; slide III, section 15). Photomicrograph, X SO.

2, dorsal aortic root 19, lymphatics dorsal to aortic arches

3a, pulmonary artery and esophagus

10, precardinal vein 27, vagus nerve

16, groups of developing hhjod cells esophagus in center of figure

along precardinal vein bronchi at lower border of figure

16a, groups of developing blood cells

dorsal to esophagus

7 From a section of a chick embryo of 6 days and 16 hours, 13.5 mm (series no. 426; slide XI, section 21). Photomicrograph, X 133.


1, aorta

5, celiac artery

8, dorsal somatic artery

16, groups of developing blood cells


23, celom (pleural cavity)

25, mesonephros

26, lung anlage


168


THORACIC DUCT IN THE CHICK

ADAM M. MILLER


PLATE 3


16a







23


— 2 6


169


PLATE 4


EXPLANATION OF FIGURES


8 From a section of a chick embryo of 6 daj's and 22 hours, 16.5 mm. (series no. 464; slide XXII, section 22). Photomicrograph, X 133.

1, aorta 16, groups of developing blood cells

5, celiac artery 23, celom (pleural cavity)

8, dorsal somatic artery 24, sympathetic nerves

9 From a section of a chick embryo of 6 days and 16 hours, 13.5 mm. (series no. 426; slide XVIII, section 3). Photomicrograph, X 233.

1, aorta 17, lymph spaces, rudiments of thoracic

16, developing blood cells duct

23, celom (abdominal cavity)


170


THORACIC DUCT IX THE CHICK

ADAir M. Mll.I.fH

24


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16


-23


8



9

171


PLATE 5


EXPLANATION OF FIGURES


10 From a section of a chick embryo of 7 days (series no. 512; slide VII, section 1). Photomicrograph, X 233.

1, aorta 17, lymphatics, rudiments of thoracic

5, celiac artery duct

16, groups of developing blood cells 23, celom (abdominal cavity)

11 From a section of a chick embryo of 14 days (series no. 518; slide XIII, section 9). Photomicrograph, X 133.

1, aorta 17, lymphatics (part of thoracic duct

16, blood cells, mostly mature erythro- system)

cytes, in tissue spaces


172


THORACIC DUCT IN THE CHICK

ADAM M. MILLER


PLATE 5








17


II


173


PLATE 6


EYPLAXATIOX OF FIGURES


12, 13, 14 From tliree saccessive .sections of a chick einbryo of (i days and 16 hours, 13.5 mm. (series no. 42(i: slide XII. sections 9, 10 and 11). Photomicrographs, X 350.

In figure 13 the arrow (/?) points to a lymph space in the mesenchyme. At its right side the lymph space is seen clearly to open into the adjacent mesenchymal intercellular spaces. The preceding section in the series (fig. 12) shows no space (the arrow points to the same locality as in fig. 13). In figure 14 the arrow points to a light area in the mesenchyme which represents the opening of the space of figure 13 into the adjacent mesenchymal intercellular spaces; this can be seen much more clearly with the microscope by changing the focus. The lymph space shown in figure 13 is the same one represented in figure 20. 17 at the right of the aorta. Aorta. /; celom (abdominal cavity), 23.


174


THORACIC DUCT IX THK CHICK

ADAM M. MII.I.EH


PLATE 6



12







13



14

175


PLATE 7

EXPLANATION OF FIGURES

15, 16, 17 and 18 From four successive sections of a chick embryo of 7 days (series no. 512; slide VII, sections 21 and 22; slide VIII, sections 1 and 2). Photomicrographs, X 250.

In figure 15 four lymph spaces, in part lined with endothelium, are seen at the left of the aorta (1). The one nearest the aorta, while in part lined with endothelium, opens freely into the adjacent mesenchymal intercellular spaces. The largest space also opens below in a similar manner. In figure 16, from the succeeding section in this series, only one of the spaces appears, opening above into tissue spaces. The others have simply become continuous with tissue spaces and therefore do not appear as distinct lacunae; this can be clearly demonstrated with the microscope by changing the focus on the rather thick sections (20 micra). In figure 17 the arrow points to the termination of the distinct lacuna of figure 16; the free communication with the tissue spaces is quite obvious. In figure 18 there are no lacunae, all those of the preceding sections having opened into the mesenchymal intercellular spaces. Aorta, 1; part of sympathetic nervous system, 24-


176


THORACIC DUCT IN THE CHICK

ADAM M. MILLER


PLATE 7




PLATE S

EXPLAXATIOX OF FIGURE

Ul From a section of a chick embi\vo of 6 days and 22 hours, 16 mm. (series no. 463; sliiU' X\'I, section 9). Photomicrograph, X 160.

£, dorsal aortic root 15, jugular lymph sac

3(1. pulmonary artery 15a, thoracic duct 'approach'

12. precaval vein 27, vagus nerve

13, vertebral vein


178


THOKACIC DUCT IN THE CHICK

ADAM M. MILLEIJ


PLATE S



179


THE AMERICAN JOUBNAL OF ANATOMY, VOL. 15, NO. 2


PLATE 9


EXPLANATION OF FIGURE


20 Drawn from a reconstruction of a chick embryo of 6 days and 10 liOLirs, 13.5 mm. (series no. 426). Ventral view.


1, aorta

2, dorsal aortic roots

3, aortic arch VI

3(1, pulmonary artery

4, aortic arch IV

5, celiac artery

10, precardinal vein


11, subclavian vein

12, duct of Cuvier

13, vertebral vein

15, jugular lymph sac

16, groups of developing blood cells

17, first rudiments of thoracic duct


180


THORACIC DUCT IN THE CHICK

ADAM M. MILLER


PLATE 9



20


181


PLATK 10


EXPLANATIOX OF FIGURE


21 Dniwn from a reconstruction of a chick cnihrvo of (i days and '_M hours, 14 mm. (series no. 465). Ventro-mesial view.

1, aorta ^■^. duct of Cuvier

2, dorsal aortic roots ^.?, vertebral vein

3, aortic arch VI 15, jugular lymjih sac

3n, pulmonary artery 15a, thoracic duct 'approach' of jugular

5, celiac artery lymph sac

6, notocord 16, groups of develo))ing blood cells

10, precardinal vein 17, rudiments of thoracic duct

11, subclavian vein


182


HORACIC DUCT IN THE CHICK

\l) \M \t. Mrl.T.KR


-12



183


PLATE 11


EXPLANATION OF FIGURE


22 Drawn from a reconstruction of a chick embryo of 6 days and 22 hours, 16 mm. (series no. 463). Ventral view.


I , aorta

£, dorsal aortic root

3, aortic arch VI

3a, pulmonary artery

4, aortic arch IV

5, celiac artery

10, precardinal vein

II, subclavian vein 1°2, duct of Cuvier 13, vertebral vein

15, jugular lymph sac


15a, thoracic duct 'approach' of jugular lymph sac

16, groups of developing blood cells 16a, groups of blood cells dorsal to

aortic roots and arches

17, rudiments of thoracic duct

18, lymphatics along aortic arches

19, lymphatics dorsal to aortic roots and arches

21, mesenteric lymphatics


184


THORACIC DUCT IN THE CHICK

ADAM M. MILLER


PLATE 11



PLATE 12


EXPLANATION OF FIGURE


23 Drawn from a reconsti-uftion of a chick embryo of 6 days and 22 hours, 16 mm. (series 463). Mesial view.

10, prccardiiial vein 15a, thoracic duct 'apiiroach' of jugular

11, suhclavian vein lymph sac

12, duct ofCuvicr 16, groups of developing blood cells 15. jugular lymi)h sac 17, rudiments of thoracic; duct, extreme

cephalic end


use.


IHOKACIC DUCT IN THE CHICK

ADAM M. MIt.I.ER


PLATE 12



23


187


PLATE 13


EXPLANATION OF FIGURE


24 Drawn from a reconstruction of a chick embryo of 7 days (series no. 512) Ventral view.


1, aorta

2, dorsal aortic roots

3, aortic arch VI

3n, pulmonary artery 5, celiac artery 7, carotid artery

10, precardinal vein

11, subclavian vein

12, duct of Cuvier

14, part of splanchnic plexus of veins

15, jugular lymph sac

15a, thoracic duct 'approach' of jugular lymph sa:'


16, groups of developing blood cells 16a, blood cells dorsal to aortic roots

and arches

17, thoracic duct, ventral plexus (homologue of azygos segment)

17a, thoracic duct (homologue of i)reazygos segment)

18, lymphatics along aortic arches

19, lymphatics dorsal to aortic roots and arches

21, mesenteric lymphatics


188:


THORACIC DUCT IN THE CHICK

ADAM M. MILLER


PLATE 13



189


PLATE 14


EXPLANATION OF FIGURE


25 From same reeonstnu-tioii as in fig. 24. View from left side.


I, aorta

3, aortic arch VI 5, celiac artery

7, carotid artery

8, dorsal somatic arteries 10, ])recardinal vein

II, subclavian vein 12, duct of Cuvier IS, vertebral vein


14, part of splanchnic plexus of veins

15, jugular lymph sac

16, groups of developing blood cells 16a, blood cells dorsal to aortic roots

17, thoracic duct, ventral plexus

18, lymphatics along aortic arches 20, lymphatics dorso-lateral to aorta ?/, mesenteric lyniphatic^s


190


THORACIC DUCT IN THE CHICK

ADAM M. MILLEn


I'LATE It


10



PLATE 15


EXPLANATION OF FIGURE


26 Drawn from a reconstruction of a chick embryo of 8 days (series no. 513). Ventral view.


1, aorta

2, dorsal aortic roots

3, aortic arch VI

3a, pulmonary artery

4, aortic arch IV

5, celiac artery

6, notocord

7, carotid artery

9, superior mesenteric artery

10, precardinal (jugular) vein

11, subclavian vein

12, duct of Cuvier 15, jugular lymph sac


15a, thoracic duct 'approach' of jugular lymph sac

17, thoracic duct, ventral plexus (homologue of azygos segment

17a, thoracic duct (homologue of preazygos segment)

18, lymphatics along aortic arches and duct of Cuvier

19, lymphatics dorsal to aortic roots and arches

20, lymphatics dorso-Iateral to aorta

21, mesenteric lymphatics


192


THORACIC DUCT IN THE CHICK

ADAM M. MILLER


PLATE 15



193


PLATE 16


EXPLANATION OF FIGURE


27 Drawn from a reconstruction of a chick embryo of days and 14 hours (series no. 320). Ventral view.


1, aorta

^, dorsal aortic roots

3, aortic arch VI

3a, puhnonary artery

4, aortic arch IV

5, celiac artery 7, carotid artery

9, superior mesenteric artery

10, p ecardinal (juguhir) vciii //, subchivian vein

12, duct of Cuvier 15, juguhir lymph sac 15(1, thoracic duct 'approach' of jugular lymph sac


16, groups of developing blood cells

17, thoracic duct, ventral plexus (honiologue of azygos segment.

17a, thoracic duct (homologue of preazygos segment.

18, lymphatics along aortic arches

19, lymphatics dorsal to aortic roots and arches

£1, mesenteric lymphatics ^^, lymphatics associated with esophagus


194


THORACIC DUCT IN THE CHICK

ADAM M. MILI.ER


PLATE 16



195


THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 2


PLATE 17


EXPLAXATION OF FIGURE


28 From same reconstruction as fijjuro 27. View from left side.


I, aorta

3, dorsal aortic roots 3, aortic arch VI

7, carotid artery

8, dorsal somatic arteries

.9, superior mesenteric artery 10, precardinal (jugular) vein

II, subclavian vein

12, duct of Cuvier

13, vertebral vein

15, jugular lymph sac


15a, thoracic duct 'approach' of jugular lymph sac

16, groups of developing blood cells

17, thoracic duct

18, lympathics along aortic arches

19, lymphatics dorsal to aortic roots and arches

20, lymphatics dorso-lateral to aorta £1, mesenteric lymphatics

£2, lymphatics associated with esophagus


196


THORACIC DUCT IN THE CHICK

ADAM JI. MILLER

7 10


PLATE 17



28 197


ON THE PRENATAL AND NEONATAL LUNG

WILLIAM H. F. ADDISOX AND HAROLD W\ HOW

The Anatomical Laboratory of (he University of Pennsylvania

EIGHT FIGURES

IXTRODUCTIOX

Of the various changes occurring in the lung at birth, we have studied especially the increase in size of the spaces within the respiratory lobules, and the change in the character of the contents of these spaces. Whenever this portion of the lung's history is considered, the statement is made that, with the first pulmonary inspirations after birth, the spaces become distended, but we have found no data expressing the amount of this distention. As to the question of the existence of fluid contents in the spaces of the prenatal lung, it is seldom mentioned in current literature, although it would seem to be of importance, especially in connection with the beginning of breathing.

In making comparisons, we have used material from experimentally controlled animals, in order to obtain specimens of prenatal and neonatal lungs, which would be comparable in all respects. This was done by using animals of the same litter, of which some had breathed, and others had not breathed.

LITERATURE

Measurements made on the minute parts of the lung have been recorded by a comparatively small number of investigators. Friedrich Merkel, in Bardeleben's Handbuch ('02), reproduces from Rossignol ('46) a table of sizes of lung alveoli at different ages. The first item in the list gives the mean size of alveoli of newborn children which have not breathed, or have breathed

199


200 WILLIAM H. F. ADDISON AND HAROLD W. HOW

only a few hours. It is exactly between these two classes of individuals that we have sought to study the differences. Kolliker made numerous lung measurements but apparently none directly contrasting the two conditions seen in the prenatal and postnatal lung. ^Miller's ('95) table of sizes of the various air-passages in dogs is from material of one stage only. Concerning the phenomenon of expansion from a more general point of view, there are many observations. For instance, the fact that all parts of the neonatal lung do not expand equally has been recorded a number of times. Dohrn ('91) says that at least two days are required to unfold the lungs completely. Jalan de la Croix ('83), found in the lung of a child which lived seven days groups of alveoli which persisted airless. In dealing with the lung of children which have survived only a few days after birth, there is the possibility, however, of the organ being prematiu'e or pathological, and either of these conditions would affect its expansion. The question of the contents of the spaces in the fetal lung has for the most part been referred to only incidentally, by observers interested in premature respiratory movements. The experiments recorded by Preyer ('85) in his "Specielle physiologie des embryo," are the most definite we have found, and these will be referred to later.

A complicating factor in studying the appearance of the normal fetal lung is the possible occurrence of intrauterine respiratory movements. These have been described in the study of living human fetuses by a whole series of clinical observers (for instance, Ferroni '99) and have even been recorded by means of tracings. The current opinion as expressed by Howell ('11) is that the mammalian fetus under normal conditions makes no respiratory movements while in utero. Ballantyne ('02) in his Antenatal pathology and hygiene" accepts the results of the clinicians, but says it is doubtful if such movements are strong enough to draw liquor amnii into the lungs. It would also seem from our experiments that if these movements are really similar to respiratory movements, the}^ are not to be compared in point of intensity with the postnatal efforts, and do not affect the structure of the lung to any extent.


PRENATAL AND NEONATAL LUNG 201

METHOD OF PRESERVATION OF MATERIAL

In order to preserve, as nearly as possible, the normal relations of the lung to surrounding structures, and to prevent its collapse, all animals were injected with 5 or 10 per cent formalin, either through one of the umbilical vessels, or through the aorta, without opening the thoracic cavity. After the lungs had hardened in situ, portions were removed, embedded in paraffin or celloidin and sectioned.

CONTEXTS OF THE SPACES \MTHIX THE FETAL LUNG

When a living fetus, near full-term, is exposed within the uterus, with the amniotic sac still unruptured, it can readily be stimulated to make respiratory movements. The angles of the mouth begin to twitch, and are drawn slightly upwards, the abdomen enlarges, evidently due to the descent of the diaphragm, and almost simultaneously the nostrils dilate, and the mouth slightly opens in a yawning manner. The result is the drawing into the respiratory tract of the amniotic fluid. We found that the mere manipulation necessary to expose the fetus is sufficient to bring about these movements, if the animal is very near the end of gestation. A surer method is to clamp the umbilical cord, but neither method will act if the animal is not sufficiently advanced in its development. Several have previously recorded the above or similar observations. Winslow is quoted by Preyer as having written in 1787, Liquorem amniirespirare videntur." Leclard ('15) clamped the neck of a still living fetus, and on opening the trachea found there a fluid analogous to amniotic fluid. When a colored fluid had been injected previously into the amniotic fluid that in the bronchi was likewise colored. Preyer ('85, p. 148) repeated and verified Leclard's experiment with a guinea-pig near the end of gestation. He found that the fuchsin which he injected into the amniotic sac not only colored the lips, tongue, palate and all the pharynx, but also the lungs and the inside of the stomach. Geyl ('80) added to the experiment in the following manner. With all aseptic precautions, he injected aniline blue into the amniotic sacs of seven fetuses of a


202 WILLIAM H. F. ADDISON AND HAROLD W. HOW

rabbit, which was nearly three weeks pregnant. Three days later the seven young were born, three dead and four alive. The three former had their lungs colored blue, as had also one of the latter. While there is no doubt that liquid is present within the lungs after these inspirations of amniotic fluid, none of the observers have directed their attention especially towards seeing the contents before such inspirations had taken place. One can easily deduce that liquid is present all the time during the de\'elopment of the fetal lung, but in order to obtain, if possible, direct evidence several simple experiments were performed. The first series was with large sheep fetuses, obtained from an abattoir, with membranes and uterus intact. In two cases the following procedure was followed. The uterus was opened, and the fetus, 35 cm. in length, exposed within the unruptured amniotic sac. By means of a needle carefully passed through the amnion, a strong ligature was drawn through the tissues of the neck of the fetus, behind the trachea, and out of the amnion again at the point of entrance. With the head of the fetus covered by the amniotic fluid, the ligature was tied tightly and the trachea constricted. Our special aims were to prevent liquid escaping from the amniotic sac and air from entering it. The trachea and lungs were carefully dissected out, without injuring the ^'isceral pleura, and placed in a large jar of water, from which all air-bubbles had been previously removed. After they had been allowed to sink to the bottom of the jar they were agitated in order to remove adherent air. Different parts of the lung were then cut with scissors and crushed. A yellowish-red fluid escaped from the crushed masses of tissue and diffused through the water but no bubbles were seen to escape.

In a third sheep fetus the trachea was ligated at its upper and lower ends and then dissected out. The closed segment of trachea was carefully cleaned and dried, before being opened over a dry glass plate. It was found to contain a faintly yellowish slightly viscid fluid, which was pressed out on the plate. When this fluid was tested with acetic acid there was a reaction showing the ]>resence of mucin. The bronchi contained a similar fluid, and when the lungs were compressed this fluid was forced out.


PRENATAL AND NEONATAL LUNG 203

In order to secure histological material for a study of the normal appearance of these lungs, another fetus of approximately the same size was injected through the umbilical vein with 10 per cent formalin. Later the thorax was opened and after the relations of the lung were observed, pieces of the lung were taken for histological sections. The appearance as evidenced by characters to be described later, was that of a lung which had not made premature respiratory movements.

The second series of experiments were repetitions of the foregoing, performed on a litter of living dog fetuses lying within the uterine horns of the mother. The parent animal was anesthetized and the young carefully exposed one at a time without unnecessary manipulation. In order to prevent the entrance of amniotic fluid, due to premature respiratory movements, the trachea of each fetus used was clamped with an artery forceps as soon as it was exposed and before the amnion was ruptured. As a result of this stimulation the animal at once began to make violent efforts to breathe, but if the trachea was well clamped these were ineffectual and the animal soon died of asphyxiation. The lungs were removed as described for the sheep fetuses, and experiments carried out in a parallel way. The results were similar to those of the first series, so it may be concluded that normally in the fetal trachea, bronchi and lungs there exists a liquid which resembles the amniotic fluid in appearance. The question arises as to what becomes of this liquid when breathing begins. From obser\'ations on fetuses removed from the uterus, the first act in l^reathing is always inspiration. As the lung enlarges, due to the contraction of the diaphragm and other respiratory muscles part of the liquid in the upper parts of the tract is drawn downwards and part remains distributed along the walls of the air-passages. When the fetus inspires amniotic fluid before its removal from the sac, it is always much handicapped. It does not breathe so strongly, and its efforts become weaker, and farther apart until they cease altogether. If in these circumstances, as we tried with young dogs, the animal be held with head downwards, and the thorax compressed at short intervals, the difficulty in l^reathing is often relieved, and the animal im


204 WILLIAM H. F. ADDISON AND HAROLD W. HOW

proves and lives. As is known from both clinical medicine and exjierimental studies, the lung has a marked capacity for the absorption of liquids, and the quantity of fluid present in normal birth is readily disposed of.

AL\TERIAL FOR BREATHIXG AND XON-BREATHIXG LUNG

In order to secure examples of the breathing and non-breathing lung which would be strictly comparable, we used pregnant dogs near the end of the gestation period. By the manner described in experiment on the contents of the prenatal lung, some of the fetuses were removed and not allowed to breathe. Others were removed from the membranes as quickly as possible, before any attempt at breathing had been made. After the umbilical cord was tied and severed, they were laid in a warm place. They soon showed signs of activity, crying and crawling about. These were etherized at the end of an hour, and injected with formalin. Fetal material was also obtained by etherizing the parent animal after several fetuses had been taken out and allowing the remainder of the young to die before removing them. For comparison with the new-born, pups two days old were used, and these were injected with formalin through the abdominal aorta.

STUDY OF PRENATAL LUNG

The appearance of sections from the fetal lung obtained in the above described manner is shown in figures 1 and 2, The texture of the organ is quite gland-like, with the mesenchyme framework present in large amount. The spaces are variable in size and shape on account of the structures being cut in different planes of section, but the distribution of the spaces is fairly uniform. In order to study the relative area occupied by lung tissue and the intervening open spaces, drawings were made with the Edinger drawing apparatus, on cross-ruled millimeter paper. Various magnifications were used, varying from 60 to 220. The results of a series of such drawings showed that the percentage of area occupied by lung tissue was 70 to 80 per cent and by the intervening spaces 20 to 30 per cent. In view of this condition it does not seem correct to call the fetal lung 'solid,' as do some


PRENATAL AND NEONATAL LUNG


205





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Fig. 1 Fetal lung of dog. at end of gestation. X 62.

Fig. 2 ' Fetal lung of dog, at end of gestation, with the granular reticular substance in the spaces, and two of the free mononuclear cells. X 280.


206 WILLIAM H. F. ADDISON AND HAROLD \V. HOW

of the present-day text-books of physiology and pathology. On the other hand, it is very easy to increase the size of the spaces artificially. In J. AI. Flint's paper on the development of the lungs ('06), he has an illustration of the lung of a fetal pig 27 cm. long, which is a stage shortly before birth. For the purpose of preservation the lung was injected intra-tracheally with the fixing fluid and this fact is responsible for the appearance of the lung in section. The appearance resembles very closely that of a lung which has breathed, as one can see by comparing it with his next figure, no. 29, that of a two-day-old pig. This is because the spaces were distended by the injection of the fixing fluid and indeed, as we found with sheep fetuses, relatively little force is required to distend the fetal lungs when introducing fluid through the trachea. In consequence of this distention the lining cells of the respiratory lobules are artificially stretched and flattened and no longer show the normal condition.

When the spaces are examined they are seen not to be entirely empty, but to contain here and there light pink-staining irregular masses of a finely granular substance. This is apparently a precipitate derived from the licjuid existing within the spaces, and was clearly seen at the period just before birth in all well-preserved fetal lungs not only of dog, but also of cat, rat and man. The origin of the mucin constituent of the fluid is at least partly from the goblet cells of the trachea, for in sections of the latter we found them numerous and characteristically stained. Mention may also be made of conspicuous large rounded cells lying free within the spaces. They are distributed rather evenly but not in large numbers, and usually occur singly. They measure 11 to 14 At in diameter, and have a single nucleus which is eccentrically placed. The nucleus may be round, oval or indented, and varies from 6 to 7 /x in its greatest dimension. If flattened in shape, the width is 3 to 4 ix. These cells in the dog have a distinctly granular cytoplasm, which stains well with eosin. They probably belong to the same variety of cells as those present in the air-spaces of the normal breathing lung which take part in removing carbon particles and other foreign matter from the alveoli. In the fetal lung they may have the similar function of


PRENATAL AND NEONATAL LUNG 207

removing cellular debris, from the liquid-filled spaces. They also resemble the 'heart-failure cells' of the lung which are seen in certain pathological conditions. Several views are held as to the nature of these phagocytic cells in the breathing lung, but the study of these very similar appearing cells in the fetal lung, leads one to agree with Kolliker ('02 vol. 3, p. 310), that they are a form of 'Wanderzellen,' and not desquamated epithelium.

NEONATAL LUNG ONE HOUR OLD

When the respiratory muscles first begin to act at the beginning of neonatal life, the thoracic cavity is enlarged. In consequence of the relation between thorax and pleural sacs the lung is likewise enlarged and inspiration is the result. With the increase in size of the lungs to occupy a larger volume, the framework of the organ is stretched and put on a tension, while the spaces become larger. This is easily apparent when one compares figures 4 and 3, showing the lung of a dog which had breathed one hour, and a fetal lung respectively. By examining sections across the entire lung and sections from different regions, it can be seen that expansion does not take place equally in all parts of the lung, nor in any particular area do all the alveoli and airsacs increase alike. As a result it was not easy to arri\'e at a quantitative estimation of the area of lung-tissue and of the airpassages, as seen in the sections. In regions, which had not been much inflated, and did not appear much more open in character than fetal lung, the framework still occupied 60 per cent of the total area. But in regions where the respiratory channels were more dilated, it occupied only 40 per cent or in restricted areas even less. Thus there is a variation in the ratio, but in general it may be said that the tissue occupies 40 to 60 per cent of the area of the cross-section. The fact that the lung does not expand at once after birth was seen also in newborn albino rats. These were obtained from The Wistar Institute of Anatomy with the aid of Dr. Stotsenburg. These young were taken immediately after birth, before they were dry and before they had begun to feed. Examination of their lung (fig. 6) shows that some areas have been inflated very little, in contrast with other parts, which are c^uite expanded.




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Fig. f) Lung of albino rat, onc-lialf hour after birth, showing partial expansion. X 25.


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210 AVILLIAM H. F. ADDISON AND HAROLD W. HOW

is seen in the peripheral portions of the lobes, immediately under the pleura, as if more of the liquid had found its way to the ends of the lobules.

The large mononuclear cells which were seen scattered about in the fetal lung are now difficult to find, although they may still be seen by searching. In comparing the prenatal and neonatal lungs of kittens and other litters of dog, this same condition was noted.

LUNG OF TWO-DAY-OLD DOG

The structure of the lung at this stage resembles, in general, the mature lung. Reference to figure 5 shows the texture to be very open in character. By means of outline drawings on crossruled millimeter paper the framework is found to be only 20 to 30 per cent of the entire area, the open spaces constituting the remainder 70 to 80 per cent. As the lung enlarges with the further growth of the animal, the spaces still further increase. As it is generally expressed, the growth of the lungs does not keep pace with the growth of the thorax, as a whole, and consequently the framework of the lungs becomes more and more stretched, and the spaces are thereby also enlarged.

In the study of the lungs of these prenatal and postnatal animals other points are seen. The behavior of the lungs when the thorax is opened is different in animals which have breathed, and those which have not breathed. In the former the lung retracts quickly into the dorsal parts of the thoracic cavity, while in the latter little change takes place. The best method for comparing the positions of the lungs within the chest cavity, in these two conditions, is to inject the blood vessels with formalin without opening the thorax. It is true that the formalin causes a swelling of the tissues of the organs, but the relations are wellpreserved. When one opens the thorax under these conditions, the fetal lung is found not to extend so far forward and not to cover as much of the heart as the postnatal lung, but in general the relations are very similar. The differences are the result of the increase in size of the lungs due to inspiration, while the size of the other organs remains the same.


PRENATAL AND NEONATAL LUNG 211

The fetal lung is opaque, and dense-looking, being often compared in consistency with the thymus. Its color is dull grayish-red. The breathing lung has a translucent pinkish color due to the greatlj' increased flow of blood, and to the presence of air. It floats upon water in contrast to the non-breathing lung, and this is utilized as a medico-legal test. When the breathing lung is moved between thumb and finger, the characteristic crepitations are elicited.

In sections the postnatal lung shows congested blood-vessels, in striking contrast with the fetal lung, which shows but little blood. It w^ould seem from examples of lung, which we obtained from still-born animals, that the increased flow of blood begins immediately, when the thoracic cavity is enlarged under the action of the muscles. These examples were from kittens, which were born at full-term. Tvvo of them were found with the fetal membranes intact, and had never breathed air, while one of the litter had breathed freely. In the lungs of the non-breathing animals, the spaces were distended slightly more than in the normal fetal lung and the blood-vessels were much congested. The animals had evidently tried to begin to breathe air, but had only succeeded in inspiring the liquor amnii. It would appear that, as soon as the negative pressure commences, more of the blood stream is deflected into the pulmonary arteries, less going through the ductus arteriosus, and this occurs irrespective of what is being drawn into the lung spaces.

APPEARANCE OF LUNG OF PREMATURELY BORN ANIMAL

An important factor in the expansion of the lung is the stage of development of the fetus at birth, that is, whether it is prematurely born or not. In a kitten born one week before the remainder of the litter, we had an opportunity of seeing this condition. This kitten had the appearance of a healthy normal annual, although somewhat undersized. It crawled about and cried vigorously w^hen disturbed but lived only about twelve hours. Sections from the lung of this animal showed evidence of the struggle to start and maintain respiration. The general appearance is given in the outline drawing, figure 7. The bronchi are

THE AMERICAN JOURNAL OP ANATOMY, VOL. 15, NO. 2


212


WILLIAM H. F. ADDISON AND HAROLD W. HOW





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PRENATAL AND NEONATAL LUNG 213

all much distended, but only a few of the respiratory spaces have been affected. Those which have been distended, however, are often o\'er-expanded, as may be seen by comparison with figure 8, from a kitten of the same litter, born at normal time, and which had breathed twelve hours. The remainder of the lung is denser than the ordinary fetal lung, and has apparently been compressed by the over-distention of a few of the spaces. Evidently respiration was begun before the lungs were sufficiently developed to assume their noraial function. A point of interest seen in examining the sections is that the alveoli which are situated directly on the bronchioles are distinctly distended. No doubt it was these alveoli which played a large part in aerating the blood of the animal during the period that it survived, and indeed it must be these which function first at the beginning of normal respiration.

We here wish to express our thanks to Professor Piersol for helpful advice and criticism and to Mr. E. F. Faber for assistance with certain of the drawings.

SUMMARY

1. During prenatal life the future respiratory passages are filled with a liquid. With the first inspirations in air, the thoracic cavity is enlarged by the action of the respiratory muscles, the lung is thereby enlarged, and the Uquid in the trachea and bronchi is drawn down into the lung, and is distributed along the walls of the alveolar and other spaces.

2. In microscopic sections of lung taken from fetal animals, there is seen a finely granular substance (a precipitate from the fluid present) widely scattered through the spaces; and large mononuclear cells, probably phagocytic in function are found uniformly distributed in small numbers.

3. In sections of lung of neonatal animals the finely granular substance is still found, usually close to the w^alls of the airspaces and fewer mononuclear cells are seen.

4. Before breathing, the lining epithelial cells of the alveoli are irregularly cuboidal with rounded nuclei; but after breathing, with the increased area of the walls of the alveoli, the nuclei are


214 WILLIAM H. F. ADDISON AND HAROLD W. HOW

spaced farther apart, the cytoplasm is drawn out and the cells are very thin and flat. After breathing has begun, the mesenchjone appears denser, with its nuclei more compacted, and the bloodvessels are distended and more conspicuous.

5. Measurements on cross-ruled millimeter paper show that the lung tissue in sections of fetal lung of dog constitute 70 to 80 per cent of the entire area; in sections of lung of dog which has breathed one hour, 40 to 60 per cent; and in sections of lung of dog which has breathed two days, 20 to 30 per cent.

LITERATURE CITED

Ballantyne, J. W. 1902 Antenatal pathology and hygiene. William Wood,

New York. DE LA Croix, J. 1883 Die Entwickelung des Lungenepithels beim menschlichen

Foetus und der einfluss der Atmung auf dasselbe. Archiv fiir mikros.

Anatomic, Bd. 22. DoHRN 1891 tjber den Alechanismus der Respiration des Neugeborenen. Abstract in Archiv fiir Kinderheilkunde, Bd. 13. Ferroni, E. 1899 Osservazioni e ricerche sui movimenti ritmici fetali intra uterini. Annali di Ostetricia e Ginecologia, T. 21. Flint, J. M. 1906 The development of the lung. Amer. Jour. Anat., vol. 6. Geyl, a. 1880 Die Aetiologie der sogenannten 'puerperalen Infection' des

Fotus und des neugeborenen. Archiv fur Gynakologie. Bd. 15. Howell, W. H. 1911 . Text-book of physiology; 4th Edition. W. B. Saunders,

Philadelphia. KoLLiKER, A. 1902 Handbuch der Gewebelehre des Menschen. Bd. 3, p. 310.

Edited by V. von Ebner. Lecl.\rd 1815 Abstract in Intelligenzblatt of Deutsches Archiv fiir die Phy siologie, Bd. 1, p. 154, under heading 'Untersuchungen, welche zu

beweisen scheinen, dass der Fotus das Schafwasser athmet.' Merkel, F. 1902 'Atmungsorgane,' in Bardeleben's Handbuch der Anatomie

des Menschen. Miller, W. S. 1893 The structure of the lung. Jour. Morph., vol. 8. Preyer, W. 1885 Specielle physiologie des embryo. Leipzig.


THE DEVELOPMENT OF THE CEREBRAL VENTRICLES

IN THE PIG

CHESTER H. HEUSER

Harvard Medical School, Boston, Massachusetts

TWENTY-SIX FIGURES

In this study of the cerebral ventricles of pig embryos three methods have been employed — wax reconstruction, dissection, and the making of minute casts. Wax reconstructions of the ventricles have the advantage of large size. The volume of the various cavities of the brain can best be estimated by immersing portions of such reconstructions in water, and observing the displacement. From these observations the actual size of the ventricles can be readily calculated. But dissections and casts are more accurate for showing details, as, for example, the neuromeral grooves; and most of the drawings have been made from such preparations.

Brains of pig embryos measuring from 12 to 45 mm. were dissected under the binocular microscope, with finely ground instruments. Some of the dissections were designed to give a median sagittal view of the right half of the entire brain. The embryo, fixed preferably in Zenker's fluid, was held in a mass of cotton wet with alcohol, between the thumb and index finger of the left hand; and under a small stream of alcohol, a longitudinal cut was made with a sharp safety razor blade. Very perfect cuts can thus be prepared, such as will pass between the hemispheres and thence backwards, sectioning the infundibulum, corpus mamillare and other divisions of the brain almost exactly in the median plane. Mesenchyma which covers the hemisphere, olfactory lobe, or other portions which it is desired to expose, can be easily removed with delicate needles and forceps.

215


216 CHESTER H. HEUSER

Casts of the ventricles, in embryos measuring from 12 to 22 mm., were made after the following method. Longitudinal sections of the entire embryo, made slightly to the right side of the median plane, were prepared as above described. Then, under the binocular microscope, obstructing portions of the brain walls were dissected off, and the ventricles were cleaned of all precipitation with a small brush and syringe. Next the embryo was taken from the alcohol and pinned down to a piece of cork and with narrow pointed strips of filter paper all the alcohol was removed from the cavities of the brain. In a large measure the success of the cast depends upon the completeness of the removal of the alcohol. The cast was made by filling the cavities with black wax with the aid of a fine electrically heated needle. A small piece of wax was carried with a cold needle to the proper place and then melted with the electric needle. This process was repeated until the ventricles had been filled. Then if desired, white wax was melted over the upper surface of the dissection for holding the parts more closely together. Moreover, the white background thus provided contrasts sharply with the black cast. Lastly the preparation was turned upside down and the brain wall was carefully dissected away from the wax. For making a permanent preparation of the cast, it is best to mount it in a dry cell on a microscopic slide. Casts thus made show a little more than one-half the ventricles. They can be studied with the binocular microscope, and drawings can be made quite as satisfactorily as from larger models. After embryos exceed 20 mm. in length the interventricular foramen is so small that it is difficult to get the lateral ventricles completely filled, and after 24 mm. the lateral recesses can hardly be cast.

For the electric needle a piece of no. 18 copper wire is coated, except for about 15 mm. at one end, with a thin layer of some insulating paste which will harden and be resistant to heat. The commercial 'caementium' is very satisfactory. The bare end is ground down to a fine point which is the working part of the instrument. Behind this, covering a length of about 20 mm., a piece of no. 32 German silver wire, about 20 cm. long, is wrapped around the insulated copper wire, the coils being well insulated from each other and covered with caementium. Attached to the ends of the German silver wire are small copper wires which run


CEREBRAL VENTRICLES IN THE PIG 217

to a lamp board. The lamp board, with the sockets connected in parallel, serves as a rheostat, and any desired temperature can be obtained by turning on the proper lights. One 50 e.p. globe allows about the desired amount of current to flow. A larger electrically heated smoother for wax models and an electrically heated knife for cutting wax plates, which were used in making the models, will be described at a future date.

Those who study the nervous system from the standpoint of comparative physiology regard the "functional system of neurones" as the real unit of the nervous system. Thus Herrick ('08), tin discussing the morphological subdivisions of the brain, refers to the influence of metamerism as primitive, dominating the subdivision of the nervous system of lower vertebrates. He further states that transverse divisions, such as the diencephalon and mesencephalon, are not ' natural regions' because the primary metamerism has ceased to be an important factor. But the anatomical subdivisions exist no less than the physiological, and although their significance may not be profound, they form a convenient basis for description. In the following account the primary divisions of the brain will be considered in this order — fore-brain, mid-brain, hind-brain. Reference will be made especially to the casts and models of the ventricles, but the wall of the brain must also be studied as the changing structure which modifies the cavities within.

FORE-BRAIN

A model of the cerebral ventricles of a 5.1-mm. embryo, the youngest one studied, shows that the three divisions of the brain are distinctly marked off from each other, and that the fore-brain is aheady subdivided into telencephalon and diencephalon. Johnston ('09) states that the boundary between the telencephalon and the diencephalon is determined in mammals, as well as in lower vertebrates, by the velum transversum above and the primitive optic groove or postoptic recess below. This subdivision is distinct in the young pig studied as seen in figure 4. In the model very slight lateral swellings from the telencephalon indicate the cavities of the rudimentary * cerebral hemispheres. The diencephalon shows a division into two parts or neuromeres, the first


218 CHESTER H. HEUSER

or parencephalon, and the second or synencephalon of Kupffer ('03). The relatively large cavities of the optic vesicles extend out from the antero-ventral part of the diencephalon. Almost one-fourth of the length of each vesicle, representing the optic stalk, extends nearly straight out laterally, and the remainder bends sharply toward the mid-brain.

In an embryo of 12 mm. the lateral ventricles have expanded considerably and they now slope outward forward and slightly upward from the interventricular foramen. The foramen is almost circular in outline and has an area of about 0.428 sq. mm. In the cast (fig. 5) the rounded mass of wax filling the lateral ventricle shows a slight concavity below, which has been produced by the developing corpus striatum. This body arises as a thickening of the ventro-lateral wall of the hemisphere, as seen in the dissection, figure 6. This figure, it may be noted, bears a very striking resemblance to the reconstruction of the brain of Anguis fragilis (40-50 somites), published by Kupffer ('03, p. 220, fig. 224). In both, the parencephalon and synencephalon are sharply marked off from each other. Two swellings, which correspond with these subdivisions, are seen in the cast (fig. 5). The parencephalon is larger than the synencephalon, and its cavity produces a more extensive swelling, which however, is quite low.

The mamillary recess and the infundibulum below it, neither of which had appeared in the 5.1-mm. embryo, are now very distinct. The velum transversum, a portion of which is shown in figure 7, is well developed, producing a deep inward bend of the anterior wall of the fore-brain above the hemisphere.

The brain of a 17-mm. pig embryo, as seen in median section (fig. 8), shows a prominent corpus striatum, above which is the interventricular foramen. The foramen is now crescentric, since it has been invaded from below by the corpus striatum, but notwithstanding the growth of the entire embryo, its area has become actually reduced. It measures about 0.315 sq. mm. As seen in the dissection, the lower portion of the corpus striatum is bounded behind by a deep groove which is continuous with the optic recess. This groove appears as a ridge upon the cast (fig. 9), and in front of it the position of the corpus striatum is indicated by an excava


CEREBRAL VENTRICLES IN THE PIG 219

tion. Abo^-e this hollow is seen the inferior horn of the lateral \'entricle. The outline of the ventricle is no longer round, as in the 12-mm. embryo. It is prolonged anteriorly or downward to form the first indication of the olfactory lobe, and posteriorly or above, it is somewhat flattened. This upper portion terminates in the inferior or descending horn. Fraser ('94) has referred to this horn in an abnormal human adult brain as in ' ' reality ascending" and Thompson ('08) similarly states that "from the developmental point of view, this descending horn is in reality an ascending horn." In pig embryos, however, as seen in figures 5 and 9, its primary direction is apparently ventral to the cerebral axis so that the inferior horn may be said to descend, even in early stages. The cavity of the lateral ventricle is seen laid open in figure 10. It differs from that of the 12-mm. pig embryo (fig. 6) since it has been invaded by a prominent chorioid fold. This fold is a lateral extension of the velum transversum, and it consists of vascular mesenchyma covered by the thin wall of the brain. At this stage its ventricular surface is perfectly smooth, but in a 22mm. embryo the chorioid fold has developed a vascular fringe which appears as a plaited frill. This is shown in figure 13.

The walls of the diencephalon in the 17-mm. embryo have thickened considerably so that the external depression between the parencephalon and the synencephalon has become almost imperceptible. Their cavities however, can be distinctly seen when the dissection of the brain is studied (figs. 8 and 9). The mamillary recess now projects out some distance from the third ventricle. Midway between this recess and the infundibulum the floor of the brain has thickened to form the tuber cinereum. The optic thalami, represented by the thickenings of the lateral walls of the diencephalon, have developed to such an extent that they have already partially fused with the corpus striatum. The place of fusion appears as an interruption of the groove between the interventricular foramen and the optic recess.

The median section of a brain of a 22-mm. embryo (fig. 11), as compared with that of the 17-mm. specimen, shows several new features. Along the cut edge dorsally, the pineal body has appeared as a slight elevation. Within the third ventricle,


220 CHESTER H. HEUSER

the thalamus has enlarged, and the corpus striatum appears as if pushed forward before it. Between the corpus striatum and the upper part of the thalamus, the interventricular foramen is seen as a slit, which arches up over the corpus striatum, being widest immediately under the velum transversum. The area of the aperture has become further reduced. Between the corpus striatum and hypothalamus, the optic recess appears as in the 17-mm. specimen. Somewhat further back, and parallel with it, is the recessus infundibuli, and between the two grooves made by these recesses, is the pars optica hypothalami. This is convex toward the ventricle. Between the hypothalamus below and the thalamus above is the sulcus hypothalamicus (B.N.A.) or sulcus limitans (His '93) — a groove which continues from the mid-brain downward and forward becoming somewhat indistinct above the pars optica hypothalami. Reichert ('61) described this groove in the brain of a pig embryo (also in cat and human embryos) as extending from the foramen of Monro to the entrance of the aqueduct of Sylvius, and he named it the sulcus of Monro. In fig. 12 an extension may be traced from the sulcus to the lower end of the interventricular foramen; another extension proceeds toward the recessus postopticus. But the main continuation is probably that which ends in the optic recess. This accords with the opinion of His ('93, p. 177), and also of Johnston ('09, p. 517). The sulcus limitans is indicated in the embryo of 17 mm. and in younger specimens, but it is more distinct at 22 mm.

The median section of the 22-mm. embryo shows the uncut median surface of the right hemisphere, ending below in a rounded olfactory lobe. Extending from the lower border of this lobe toward the notch below the velum transversum, is a groove, which, with the lamina terminalis, bounds a triangular portion of the hemisphere. Smith ('03) named this triangular thickening the ' paraterminal body'. Herrick ('10) proposes to divide this body into a ventral component, the 'corpus precommissural,' and a dorsal component, the 'primordium hippocampi,' but these subdivisions do not appear externally in the 22-mm. embryo.

In the dissection of a 45-mm. embryo (fig. 14) most of the paraterminal body together with the adjacent medial wall of the right


CEREBRAL VENTRICLES IN THE PIG 221

hemisphere have been cut off, thus displaying the structures within the ventricle. The corpus striatum is seen to be divided by a vertical groove into two portions. The groove is deep below but fades out above. Thompson ('09) has described a similar groove in a cat embryo 20 mm. in length, and he refers to the larger lateral and the smaller median subdivisions which are set apart by the groove as 'roots' of the corpus striatum. His ('89) described the corpus striatum as being composed of three limbs. In the 45-mm. pig there are also three limbs, but only two are seen in figure 14. The small median limb, which is not shown, fuses with the lamina terminalis just below the interventricular foramen. The groove is shown at x in figure 18, which is a frontal section through the head of a 45-mm. pig. The plane of this section is indicated in figure 14. The groove is best seen in the right side of the section, where the median subdivision may be observed to pass down toward the paraterminal body. In a section further back (fig. 19) the groove is quite shallow. Above the corpus striatum (figs. 14 and 19) the chorioid plexus is seen projecting into the lateral ventricle. The fold, surmounted by a frill, which was seen in the 22-mm. embryo, has given place to a very thin layer with many reduplications and subdivisions. Villi are nowhere present, but in thin sections the smaller processes extending out from the main folds may simulate them. Under the binocular microscope it is clearly seen that this plexus consists only of folds with secondary subdivisions. The plexus is attached along a fissure measuring 2.1 mm. in length, which extends backward from the interventricular foramen.

The caudal portion of the plexus — about one-fourth of the entire length — is very much attenuated. It forms a short free projection extending 0.18 mm. beyond the end of the fissure, and the free portion shows no secondary folds (fig. 21). The anterior portion of the plexus forms a larger free projection which extends 0.72 mm. past the front end of the fissure.

In the 22. mm. embryo (fig. 13) a slight invagination of the medial wall of the hemisphere, above the chorioid plexus and parallel with it, extends from the interventricular foramen backward into the inferior horn. This represents the hippocampus,


222 CHESTER H. HEUSER

which is better developed in the 45-mm. embryo. It is seen in figures 19, 20, and 21 (marked +).

Considered as a whole, the lateral ventricle has expanded greatly and may readily be divided into three parts — the anterior horn, which is in front of the corpus striatum; the body, which is above it; and the inferior horn, descending behind it. A cast of the left lateral ventricle is shown in side view in figure 16, and in ventral view in figure 17. The latter shows the large excavation made by the corpus striatum. Toward the olfactory lobe this concavity is bisected by a ridge, which lies between the large roots of the corpus striatum, as described in connection with figure 14. Below the concavity, in figure 17, there is a ridge which is fissured, posterior to the interventricular foramen, to receive the chorioid plexus. This ridge separates the hollow for the corpus striatum above, from that for the hippocampus below.

The lateral ventricle communicates with the third ventricle by an interventricular foramen which is smaller than in the preceding stages. The third ventricle has become reduced to a slitlike space, owing to the great thickening of the walls of the diencephalon. A portion of it has become practically obliterated. This is where the thalami have grown against each other (figure 21). They have not yet fused, however, to form the massa intermedia; that is the ependymal layer over each thalamus is still uninterrupted. Along the dorsal margin of the cleft, which represents the ventricle, there is a thin lateral expansion on either side (fig. 15). The expansion begins at the interventricular foramen, where it is broadest, and it diminishes backward, ending a short distance in front of the pineal recess. Thus the expansion is wedge-shaped when seen from above. The brain-wall which overlies this expansion is the tela chorioidea, which has a corrugated surface in relation with the vascular mesench^ma. Below and behind the thalamus is a deep groove — the sulcus limitans — ■ which, as already described, sends prolongations to the interventricular foramen, recesses postopticus and recessus opticus. The sulcus is seen in section in figure 20, with the thalamus above and the pars optica hypothalami below.


CEREBRAL VENTRICLES IN THE PIG 223

The oldest embryo studied measured 260 mm. Its brain was dissected out, embedded in celloidin, and cut into sections 0.2 mm. thick. From these sections a wax model of the ventricles was constructed, as shown in figures 25 and 26. The three subdivisions of the lateral ventricle have become highly developed. The anterior horn, which in the 45-mm. embryo ended in a short and slightly pedunculated olfactory bulb, now extends through the olfactory stalk and terminates in the expanded ventricle of the olfactory bulb. The body of the lateral ventricle is corrugated above, w^here it is in relation with bundles of fibers in the corpus callosum. The inferior horn not only descends, but extends forward as a slender prolongation, which ends blindly in the temporal lobe of the brain. As seen from below, the cast of the lateral ventricle shows the concavities for the corpus striatum and the hippocampus respectively, separated by a ridge into which the chorioid plexus is invaginated. The ridges bounding the corpus striatum correspond with those seen in the 45-mm. embryo.

In the 260-mm. specimen, the interventricular foramen has expanded considerably, and its area was found to be 6.92 sq. mm. Extending backward from the foramen, between the region occupied by the thalamus below and the tela chorioidea above, there is a tubular expansion of the third ventricle, somewhat flattened dorso-ventrally. This corresponds with the much smaller expansion, T-shaped in section, which was described in the 45-inm. embryo. In the 260-mm. embryo there is a long caudal extension of the dorsal part of the third ventricle which curves slightly upward and forms the suprapineal recess. It measures about 5.6 mm. in length and extends backward with quite a uniform diameter of 1.4 mm. This conspicuous feature is not represented in the 45-mm. specimen. The cast of the cavity (fig. 25) is deeply corrugated on all sides by longitudinal folds. These accomodate branches of the medial cerebral vein. A large branch of the vein on either side produces a deep groove along the dorsal expansion of the third ventricle. Between the suprapineal recess above and the pineal recess below there is a well marked lateral compression of the ventricle, produced by the hab


224 CHESTER H. HEUSER

enular ganglion. The conspicuous ridge at the lower margin, of this habenular concavity continues caudally into the pineal recess. Between the pineal recess and the groove for the posterior commissure there are two slight projections of the third ventricle (infrapineal recesses). The very extensive fusion of the thalami, forming the massa intermedia, has produced a hole in the model of the third ventricle. It is oval in outline, measuring about 4.6 mm. by 3 mm.

MID-BRAIN

It is well known that in young embryos the cavity of the midbrain is very large, forming the middle cerebral vesicle. In the adult it is generally described as '^ reduced to a narrow slit— the aqueduct of Sylvius." Cunningham states that the mid-brain "is tunnelled by a narrow passage, the aqueduct of Sylvius, which extends between the fourth and third ventricles, "and Bensley ('10) writes: The mesencephalon is noteworthy in a mammal as lacking a ventricle. Its cavity is a narrow canal, the aqueduct of Sylvius . . . . " But Retzius ('00) described a spindle-shaped expansion in the middle portion of the cavity of the adult human mid-brain, and named it 'ventriculus mesencephali.' It will be shown that a distinct cavity exists in the mid-brain of the adult pig, comparable with that found by Retzius in the human brain.

In the 5.1-mm. pig (fig. 4) a slight constriction separates the caudal part of the diencephalon from the cavity of the mid-brain. Another slight constriction marks the position of the isthmus between the mid-brain and the hind-brain. The angle formed by a line passing through the axis of the diencephalon with another through the isthmus and cavity of the medulla, is very acute (30°) in this stage. In the adult it is very wide.

The cavity of the mid-brain continues to expand and becomes sharply marked off from that of the hind-brain at the isthmus, as shown in the 12-mm. embryo (fig. 5). Between the mid-brain and fore-brain, however, the cavity shows only a slight constriction, which is limited to the lateral surface. As seen in the cast


CEREBRAL VENTRICLES IN THE PIG 225

neither the mid-dorsal nor the mid-ventral line is indented at this point.

Figure 9, of a 17-mm. embryo, shows that the dorsal surface of the mid-brain has become flatter than before and its caudal end projects further toward the hind-brain. This projection, on either side, marks the position of the future inferior colliculus. The ventricle of the mid-brain is somewhat quadrilateral in cross section, and in the cast it shows a prominent longitudinal ridge corresponding with the line of maximum width. The groove in the wall of the brain corresponding with the ridge may be referred to as the lateral sulcus (sulcus lateralis internus). It must not be confounded with the sulcus limitans which, in the cast, is a much less conspicuous ridge, more ventrally placed. The sulcus limitans is more evident in the dissection, figure 8. It separates a small thick-walled ventral zone from an extensive thin-walled dorsal zone. The ventral zones, on either side of the mid-ventral sulcus, form low rounded eminences projecting into the cavity of the ventricle which constitutes the tegmental fold of His. The lateral sulcus passes longitudinally through the dorsal zones, terminating on either side, in the projection which forms the inferior colliculus.

In a slightly older embryo (22 mm.) the dorsal wall of the mid-brain has become relatively thinner, and its ventricle has expanded greatly. At this stage it forms a very conspicuous portion of the whole ventricular system. Its volume is estimated at 4.1 cu. mm. Separate recesses for the inferior colliculi can now be recognized. In earlier stages the posterior overhanging portion of the mid-brain ended abruptly in a straight transverse line. In the 45-mm. embryo this border shows a deep median cleft, and each arm of the bifurcation thus formed represents the cavity of an inferior colliculus. In the 22-mm. specimen the cavities projecting backward from the dorso-lateral corners of the midbrain ventricle have just begun to develop.

In the 45-mm. embryo the third ventricle is connected with the cavity of the mid-brain by a more elongated constricted portion than in previous stages. At the anterior end of the midbrain ventricle there is a median dorsal extension 1.2 mm. caudad


226 CHESTER H. HEUSER

to the pineal recess. This outgrowth (figs. 14 and 15) which Ues just behind the posterior commissure is the incisura postcommissurahs. It is not present in the 20-mm. pig but is very distinct in the 45-mm. embryo. The ependymal and mantle layers but not the ectoglial layer of the brain-wall make a distinct outward bend above it. Caudad to the incisure, the dorsal surface of the mid-brain cavity slopes gradually upward to a height of 0.6 mm. and then extends nearly straight backward over the body of the cavity. The inferior collicular recesses are very much more distinct than in younger pigs.

The length of the mid-brain, including its collicular recesses is 5.0 mm. and- the width, in the widest part is 2.6 mm. The volume of the cavity is very much greater than in preceding stages and is now about 6.9 cu. mm.

A cast of the cavities of the mid-brain in an embryo measuring 110 mm. is of an irregular prismatic form as shown in figure 24. It presents a median dorsal ridge which extends from the incisura postcommissuralis posteriorly, ending in a median depression between the eminences representing respectively^ the cavities of the right and left inferior colliculi. The outer margin of the cavity of each inferior colliculus is continued forward as a prominent ridge, corresponding with the sulcus lateralis. This ridge flattens out rather sharply and completely a short distance behind the posterior commissure. At the base of the cavity of the inferior colliculus, a prominent ridge ascends from the isthmus, but it can be followed only a short distance behind the posterior commissure into the territory of the mid-brain, where it terminates. This ridge represents the sulcus limitans of the isthmus. Ventrally there is another ridge which may be regarded as the sulcus limitans of the mid-brain, although it is not continuous with the structure just described as the sulcus limitans of the isthmus. It arises beneath the latter in or near the median ventral sulcus. It then passes laterallj', diverging from its fellow on the opposite side. The point of widest separation is soon reached, after which the two sulci gradually converge, coming together in the mid-ventral line beneath the incisura postcommissuralis. The surfaces between the ridges on the cast are


CEREBRAL VENTRICLES IN THE PIG 227

all concave, and in the central part of the mid-brain there are five surfaces — two dorso-lateral, bounded by the median dorsal ridge and the lateral ridges; two ventro-lateral, extending downward to the sulci liniitantes; and a ventral surface between these sulci.

The form of the mid-brain ventricle in the embryo measuring 110 mm. has been described at length, since subsequently it undergoes only slight modifications. This was determined by modelling the ventricles in the 260-mm. pig (figs. 25 and 26) and by making dissections of the adult. The ventricle of the mid-brain continues to increase in size but it does not keep pace with the growth of the adjoining cavities. The grooves remain as described, and the ventral surfaces, between the sulci liniitantes, are always subdi^dded posteriorly by a forward extension of the median ventral sulcus.

HIND-BRAIN

In the 5.1-mm. embryo the cavity of the hind-brain or third cerebral vesicle is elongated and quite straight. Behind the ventricle of the mid-brain (fig. 4) a well marked constriction indicates the future isthmus. Caudad to this, the cavity, or fourth ventricle, widens somewhat, and then slopes gradually through the medulla to the spinal cord. The low ridge which follows the line of greatest width corresponds at this stage with the line of attachment of the thin roof-plate, and not with the sulcus limitans. Along the ventral surface of the cast there is a sharp median ridge which represents the sulcus medianus of the rhomboid fossa.

Seven neuromeral grooves can be identified. The first produces a low but broad ridge on the cast anterior to the widest part of the rhombencephalon. Dorsally it flattens out before reaching the line of maximum width and ventrally it does not extend quite to the median sulcus. The second neuromeral groove is situated opposite the widest portion of the fourth ventricle. The next four are about equalh' spaced. They are very prominent in the ^-entral zone and some of them appear to reach upward a short distance into the dorsal zone. The last one is

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 2


228 CHESTER H. HEUSER

represented on the cast by a prominent mound, but the elevation does not extend as far dorsally or ventrally as do those immediately preceding it. Caudally it blends with the sulcus limitans. Bradley ('04 and '05) described seven neuromeres in a nineteen-day embryo. ^

The pontine flexure soon becomes definitely established as shown in figure 5, which is a cast from a 12-mm. embryo. In this embryo the median ventral edge of the cast is very sharp around the flexure and backward toward the spinal cord. At the pontine flexure there are six neuromeral grooves. The relation of the grooves to the median sulcus is clearly shown in the dissection, figure 7, The former seventh groove has been taken up by the sulcus limitans so that it is no longer recognizable. Above the grooves the A^entricle has become quite wide. In dorsal view the body of the ventricle slopes out laterally quite rapidly behind the isthmus, attaining its maximum width in the region of the future lateral recesses, which become distinct in embrj^os of about 15 mm.

In the 17-mm. embryo the isthmus leading from the midbrain to the body of the fourth ventricle is diamond-shaped in cross section; the ventral zones are here more extensive than the dorsal, which is the reverse of the condition in the mid-brain. The ventral median sulcus is narrow and deep, so that in the cast there is a sharp edge on the ventral surface of the fourth i^entricle extending around the pontine flexure, and onward caudally to the spinal cord. There are now five neuromeres in the hind-brain. The body of the fourth ventricle has expanded a great deal since the last stage described, and the lateral recesses are now well indicated. Slight concavities on either side of the body of the ventricle between the isthmus and the lateral recesses, in the dorsal zones, are caused by the thickening of the brain in this region to form the lateral portions of the cerebellum. Behind the lateral recesses the thin roof of the hind-brain has become deeply invaginated to form the chorioid plexus. About 100 villi are developed on the fold.

1 According to Keibel's Normentafel, pig embryos of nineteen days measure from 4.5 to 8 mm.


CEREBRAL VENTRICLES IN THE PIG 229

In a 22-nim. embryo (figs. 11 and 12) the fundamental arrangement of parts has not been altered. The cerebellar thickenings have enlarged considerably, producing correspondingly greater depressions in the dorsal zones along the body of the fourth ventricle. The sulcus limitans, for the same reason, has been made verj^ much more evident. The lateral recesses extend further to each side and are comparatively much narrower than in the younger stage considered. The chorioidal lamina extends out to the ends of the lateral recesses, and the number of villi springing from it has increased to about 150. Caudad to the chorioid plexus the dorsal part of the cavity is slightly distended or puffed up dorsally, thus forming the 'caudal protrusion' (Blake '00). In advanced embryos of dogs, cats, pigs, sheep and also the chick, according to Blake, there is a marked caudal protrusion. He wrote, This protrusion is completely closed and resembles the finger of a glove." It can hardly be described in the pig as 'finger-like' since, as shown in figure 11, it forms a rounded dome.

Taken as a whole, the fourth ventricle in the 45-mm. pig (figs. 14 and 15) has the same parts as seen in the 22-mm. embryo. The cavity of the isthmus has increased in dorso-ventral diameter from 0.75 to 0.85 mm. As seen by comparing figures 11 and 14, it has become relatively low, but laterally it has become further expanded. Its thin lateral edge, as seen in the cast (fig. 15), extends backward and becomes continuous with the body of the ventricle a short distance in front of the lateral recess. Where this thin edge fuses with the body there are three small wrinkles — the lateral remnants of the neuromeral grooves. The two halves of the cerebellum have become very much thicker and meet at an angle of about 130°. The medial part of the cerebellum is also thicker than before, so that all together the broad median fissure of younger stages has been appreciably reduced. The lateral recess, while absolutely larger than before, appears much slimmer. The floor and roof of the recess have almost come together, the cavity being largely filled with the chorioid plexus. In figure 23, in the left recess, is seen a long stretch of the chorioid fold. Sections a short distance caudad


230 CHESTER H. HEUSER

to this one show the fold to be continuous from one recess to the other. The marked villous character of the chorioid plexus is illustrated well in this section (fig. 23). It shows also the rhomboid fold projecting into the cavity of the hind-brain below the chorioidal lamina. The rhomboid fold appears in the model (fig. 15) as a sharp rim, extending along the upper part of the body of the ventricle behind the lateral recess. It extends to the caudal protrusion of the fourth ventricle, which has expanded to a very great size; its volume is more than one-half that of the remainder of the ventricle. This protrusion, from above and from behind, appears almost spherical. The posterior boundary slopes downward making almost a right angle with the narrow cavity of the caudal part of the medulla. When the body of the fourth ventricle is viewed from below, the ventral median sulcus and the two sulci limitantes are seen converging towards the cervical flexure. The ventral median groove is nearly straight and produces with the slim cavity of the cord an angle of about 125°.

In the 260-mm. embryo (figs. 25 and 26) the cavity of the isthmus is very broad and flat. On the ventral surface a deep ridge — the median ventral sulcus — is continuous with the similar ridge of the mid-brain ventricle. On account of the extensive growth of the cerebellum the dorsal surface of the body of the ventricle has become deeply concave. Only a very slight caudal protrusion is now present. Bradley ('05) described the first appearance of openings in the lateral recesses — foramina of Luschka — ^in embryos of about 100 mm. Because of these openings and on account of the growth of the chorioid plexus the cavities of the lateral recesses, as seen in the model, have been reduced to slim irregular bodies.

MEASUREMENTS OF THE BRAIN

In order to show the change in the volume of the cavities during development, measurements of wax models were made by the immersion method. The results of these measurements, reduced to cubic milhmeters, are given in table 1.


CEREBRAL VENTRICLES IN THE PIG


231


TABLE 1

Volume of the cerebral ventricles in cubic millimeters


EMBBYO


FOREBRAIN


THIRD VENTRICLE


LATERAL VENTRICLE


MIDBRAIN


HINDBRAIN


TOTAL CAVITY OP BRAIN


mm. 5.1


0.0948




0.022


0.206


0.3227


12


1.716


1.374


0.1712


0.357


3.38


5.453


20


7.11


2.148


2.481


2.96


3.407


13.477


24


9.893


2.357


3.768


5.181


3.57


18.644


45


30.49


4.19


13.15


6.904


5.84


43.234


260


711.58


94.6


308.4


10.07


35.5


757.15


From table 1 it is seen that each part of the entire ventricular system increases in size continually during development. The increase is relatively greater at first, and as would be expected, it becomes gradually less toward the adult. For a time — until embryos measure between 12 and 20 mm. — the fourth ventricle is the largest part of the ventricular sj^stem. After the lateral ventricles have expanded appreciably the cavity of the fore-brain is the largest part.

The cross-sectional area of the interventricular foramen was determined from wax models by measuring the area of the foramen cut through the narrowest portion.

The cross-sectional area of the interventricular foramen becomes progressively greater up to about the 12-mm. stage after which it becomes both relatively and absolutely smaller for some time. In later stages it again expands considerably; in the adult it is very much larger than in younger stages.

TABLE 2 Area of the foramen interventriculare


EMBRYO


FORAMEN INTERVENTRICULARE


Length in mm.


Area in square millimeters



12


0.42



20


0.26



45


0.18



260


6.92



The mid-brain angle in early stages is very acute. For measuring the angle in the different embryos during development it


232


CHESTER H. HEUSER


is difficult to establish lines whose loci will be absolutely comparable throughout the series. The apex of the angle has been located in the middle point of the mid-brain ventricle; one line has been drawn from it to the optic recess, and the other to the middle point in the cavity of the central canal opposite the cervical bend



Fig. 1 Outline from figure 5, showing the position of the mid-brain angle

TABLE 3 Increase in the mid-hrain angle


EMBRYO


MID-BRAIN ANGLE


EMBRYO


MID-BRAIN ANGLE


mm.


degrees


mm.


degrees


5.1


30.0


45


84.0


12


48.5


260


146.0


17


59.5


Adult


152.0


22


68.0




The angle increases from 30° in the 5.1-mm. embryo to 152° in the adult. The curve of growth of this angle rises up relatively rapidly at first and slowly in later stages to the adult.


CONCLUSIONS

The well-known series of models of the developing human brain made by His and the papers which they illustrate make it possible to compare the conditions which have been described in the pig with those in man. Comparisons with the embryonic brain of other mammals cannot be made, since it appears that no one has attempted to extend the work of His here referred to, or even to repeat it critically. The first impression derived


CEREBRAL VENTRICLES IN THE PIG 233

from comparing His's models of the interior of the human embryonic brain with those of the pig is that they are strikingly similar, especially in the earlier stages; but more careful study shows very considerable differences. The 6.9-mm. human embryo (His, '89, Taf. 1, Fig. 2) is somewhat more advanced than the pig embryo of 5.1 mm., as indicated by the greater development of the hemisphere, and of the pontine flexure. In His's model the cavities of the parencephalon and synencephalon are not indicated and the neuromeres of the hind-brain are not shown. It may be questioned however, 'whether these structures are actually absent from the human brain at this stage. On the other hand, the model of the human brain shows a prominent tegmental fold passing from the mid-brain toward the fore-brain, but no such fold occurred in the pig embryo. In a model of a pig embryo of 5 mm. Johnston ('09) has shown the neuromeres of the fore-brain and hind-brain but there is no tegmental fold. This fold is the portion of the tegmentum which projects dorsally into the cavity of the mid-brain; the fold on either side extends from the median ventral sulcus to the sulcus limitans. Altogether Johnston's model of the brain of the 5-mm. pig agrees very closely with the first model described in the present paper, and it is evident that we have more detailed and accurate knowledge of the shape of the brain in the pig embryo than in the human embryo of corresponding stage.

His's model of the human embryo of 10.2 mm. corresponds quite closely with the pig embryo of 12 mm. and his 13.6-mm. specimen is comparable with the pig of 17 mm.; the human embryo of the third month, which completes His's series of models, is the stage intermediate between those dissected in pigs of 22 and 45 mm. In all of His's models, except the oldest, the tegmental fold is very prominent; and in the embryo of three months it is perfectly distinct. The sulcus limitans is accordingly well defined in the mid-brain, but it does not form a distinct continuous longitudinal groove from the spinal cord to the optic recess in any of His's models. In fact. His described the sulcus as becoming leveled off in the territory of the isthmus


234


CHESTER H. HEUSER


by the growth of the walls — being interrupted at the junction of the isthmus with the mid-brain.

In the casts of the ventricles of the pig, in early stages, there is a broad line of maximum width corresponding with the sulcus limitans.



s.i.



Fig. 2 Sections through the hind-brain of pig embryos, {A, 3.9 mm. ; B, 10 mm. ; C, 23.6 mm.), showing the change in position of the sulcus limitans.

This groove which is lateral in the cord becomes a ventral groove in the medulla as described by His, and at the junction of the isthmus with the mid-brain it comes to an end. On account of the expansion of the roof-plate in the hind-brain the sulcus limitans soon falls below the line of maximum width. Thus in the diagram (fig. 2, B) the sulci are ventrally placed, and the width of the fourth ventricle between them is less than it is slightly further dorsally. In an older embryo (fig. 2, C) the sulci are close together and on the floor of the ventricle. In the mid-brain there is early a line of greatest width, which becomes shifted so as to form the outer border of the tegmental fold, as shown in the diagram, figure 3.


■ S. I.




Fig. 3 Sections through the mid-brain of pig embryos, (A, 3.9 mm.; B, 23.6 mm.; C, 110 mm.); showing the change in position of the sulcus limitans.


CEREBRAL VENTRICLES IN THE PIG 235

This shifting in the mid-brain is brought about by the expansion in the dorsal zone to form the cavities in the colHcuh. The line of maximum width begins behind the constriction between the mid-brain and fore-brain in the superior collicular cavity and terminates at the caudal end of the mid-brain in the inferior collicular recess. Anteriorly the hypothalamic sulcus is seen to connect with the tegmental sulcus. This connection is most distinct in the 22. mm. embryo.

His first called attention to the dorsal and ventral zones of the brain and cord. A demarcation of the sensory from the motor portion of the central nervous system is obviously of fundamental importance and Johnston ('09) has described the sulcus limitans as the most important landmark in the brain. Diagrams have appeared which show an uninterrupted line, extending from the optic recess backward through the brain and spinal cord. However, there is no real picture which shows a sulcus continuous throughout the brain. It is distinct in each division of the brain, but at the cephalic end of the isthmus it is interrupted.

The hemisphere of course is very much larger in the human embryos than in approximately corresponding stages of the pig. The thalamus and habenular region are also further advanced in the human stages. The tegmental folds never form conspicuous projections into the ventricle of the mid-brain as they do in early human embryos. The olfactory lobe becomes progressively more conspicuous in the pig series — ^the reverse of the condition in human embiyos, of later stages especially. In the latter, the olfactory lobe is covered over by the rapidly expanding pallium. The olfactory lobe in the pig always extends some distance in front of the end of the hemisphere. The roof of the fourth ventricle is not present in aii}^ of the His models, hence there is no indication of a caudal protrusion of the fourth ventricle. This protrusion is first well indicated in pig embryos about 22 mm. long. It expands very rapidly until the cerebellum grows downward into this region. There being no foramen of Majendie in the pig, the posterior medullary velum stretches nearly straight across the caudal part of the fourth ventricle so that the caudal protrusion is just recognizable in the adult.


236 CHESTEK H. HEUSER

The form of the ventricles in the adult pig has apparently never been studied bj^ means of casts or models. Figures 25 and 26, representing the cerebral ventricles in an embryo of 260 mm., are however very similar to those which would be obtained from an adult and they ma}^ be compared with Dexler's figure of the cavity of the brain in the horse, and with any one of several figures of the ventricles in the human brain. The first of these were published by Welcker (78) who filled the ventricles with wax, injecting through the infundibulum, and published figures and a brief description of the model thus obtained. Testut ('97) figured and described a plaster cast of the ventricles. Barratt ('02) constructed a wooden model from measurements obtained from thick sections (12.5 mm.). From the method employed accuracy of detail could scarcely be expected. By far the most delicate and satisfactory' figures are those of Retzius ('00) who made casts by the use of Wood's metal. Harvey ('10) has recently used Wood's metal for the same purpose with similar results.

In the human brain immediately above the pineal body there is a backward extension of the cavity of the third ventricle which forms the supra-pineal recess. This term was introduced by Reichert ('61, Bd. 2, S. 69) who described the structure and showed its relations in median sections of the brain. In his figures the recess measures nearly 5 mm. in length. In Welcker's plaster cast it appears to be somewhat larger and in the cast by Retzius it measures 10 mm. But Testut and Harvey show onl}^ very slight protrusions. The recess may be variable in the human brain. In Dexler's model of the ventricles in the horse it is a very conspicuous object, nearly 25 mm. in length. In the adult pig it measures 5.5 mm. In proportion to the size of the third ventricle it is very much longer than in man but much shorter than in the horse. The significance of this recess has not been determined.

A very conspicuous feature of the casts of the third ventricle is the aperture made by the massa intermedia. In the several models of the human brain this aperture varies considerably in size, but in no case is it as large as in the pig or the horse. In


CEREBRAL VENTRICLES IN THE PIG 237

the pig it appears to be somewhat smaller than in the horse, but in both these animals the opening is very large. The area of fusion in the 260-mm. pig measures about 8.4 sq. mm., and in the adult pig about 61.6 sq. mm.

The lateral ventricles in pig, horse and man differ very greatly. In man there is a posterior horn which is absent in the horse and pig. But the latter both show an extension of the ventricle into the olfactory lobe, whereas in man, the anterior horn ends bluntly. In the horse the pedicle of the cavity of the olfactory stalk is very long and markedly concave dorsally, it ends in an elongated irregular ventricle. The pedicle in the pig is nearly straight and much shorter. It terminates in a flattened expansion which in the anterior end is compressed dorso-ventrally.

In connection with the lateral ventricle it may be noted that its chorioid plexus in the pig is not provided with villi. Meek ('07) in his discussion of the general morphology of the plexus writes: "Villi are scarce in the chicken, duck and pigeon, but more abundant in the hog, while they reach a considerable development in the, horse, ox, and especially among porpoises, crocodiles, and some of the selachians (Pettit '02-'03)." Findlay ('99) writes similarly of this plexus: The surface of the chorioid plexus is beset with a large number of highly vascular villous projections. These are of all sizes, and the largest may branch and subdivide many times before the ultimate villi are formed." The study of the brain of the pig has shown that the chorioid plexus of the lateral ventricle first develops as an extension of the velum transversum into the lateral ventricle. The free border rapidly becomes much longer than the attached part so that it is early thrown into folds. These primary folds increase in number and give rise to secondary folds, but as shown by means of the binocular microscope, villi are not developed in this plexus.

In pig embryos of 35 to 40 mm. and in all older stages, there is a median dorsal recess behind the posterior commissure. This postcommissural recess is shown by Retzius who labels it the 'incisura postcommissuralis.' It is shown also in Harvey's lateral view. None of the other authors have an indication of it in their figures. In the adult pig the recess is very prominent.


238 CHESTER H. HEUSER

Finally, it should be noted that the aqueduct of Sylvius remains as a distinct ventricle of the mid-brain in the adult pig. One of Retzius's figures alone gives a good lateral view of the midbrain of man. This he described as 'ventriculus mesencephali.' The cavity of the mid-brain in the horse stands out also as a distinct ventricle. In the pig the mid-brain has throughout development and in the adult a well defined ventricle which constantly increases in size as long as the brain grows.

BIBLIOGRAPHY

Barratt, J. O. W. 1902 The form and form relations of the human cerebral

ventricular cavity. Journ. Anat. and Physiol., vol. 36, pp. 106-125. Bensley, B. a. 1910 Practical anatomy of the rabbit. Philadelphia. Blake, J. A. 1900 The roof and lateral recesses of the fourth ventricle, considered morphologically and embryologically. Jour. Comp. Neur., vol.

10, pp. 79-108. Bradley, O. C. 1904 Neuromeres of the rhombencephalon of the pig. Rev.

Neurol, and Psych., vol. 2, pp. 625-635. Dexler, H. 1904 Beitrage zur Kenntnis des feineren Baues der Zentralnerven systems der Ungulaten. Morphol. Jahrb., Bd., 32, s. 288-389. FiNDLAY, J. W. 1899 The choroid plexus of the lateral ventricle of the brain.

Brain, vol. 22, pp. 161-202. Harvey, R. W. 1910 A cast of the ventricles of the human brain. Anat. Rec,

vol. 4, pp. 369-384. Herrick, C. J. 1908 The morphological subdivisions of the brain. Jour. Comp.

Neur., vol. 18, pp. 393-408.

1910 The morphology of the fore-brain in Amphibia and Reptilia.

Jour. Comp. Neur., vol. 20, pp. 413-547. His, W. 1889 Die Formentwickelung des menschlichen Vorderhirns vom Ende

des ersten bis zum Beginn des dritten Monats. Abh. math.-phys.

Classe Kgl. Sachs. Gesellsch. Wiss. Nr. 8, Bd.'lo, S. 675-736.

1890 Die Entwickelung des menschlichen Rautenhirns vom Ende dea

ersten bis zum Beginn des dritten Monats. I. Verlungertes Mark. Abh.

math.-phys. Classe Kgl. Siichs. Gesellsch. Wiss., Bd. 17, S. 1-74.

1892 Zur allgemeinen Morphologie des Gehirns. Archiv. f. Anat. u. Physiol. Anat. Abt., S. 346-383.

1893 Vorschlage zur Eintheilung des Gehirns. Archiv. f. Anat. u. Physiol. Anat. Abt., S. 172-179.

1904 Die Entwicklung des menschlichen Gehirn wahrend der ersten

Monate. Leipzig. Johnston, J. B. 1909 The morphology of the fore-brain vesicle in vertebrates.

Jour. Comp. Neur., vol. 19, pp. 457-539. Keibel, F. 1897 Normentafel zur Entwicklungsgeschichte des Schweines.

Jena.


CEREBRAL VENTRICLES IN THE PIG 239

VON KuPFFER, C. 1!)03 Die Moiphogenie clos Xorvensj'stems. In Oskar Hert wig's Handbueh des vorgleichenden ii. experimentellen Eutwicklungs lehre der Wirbelthiere, Bd. 2, Th. 3, S. 1-272. Jena. Le"V\'IS, F. T. 1903 The gross anatomj' of a r2-mm. pig. Amer. Jour. .\nat., vol.

2, pp. 211-225. .Meek, \V. J. 1907 A study of the choroid plexus. Jour. C'omp. Near., vol. 17,

pp. 286-306. MiHALKOVics, V. 1877 Entwicklungsgeschichte des Gchiriis. Leijjzig. MiNOT, C. S. 1892 Human embr3^ology. New York. Norman, C, and Fraser, A. 1894 A ease of porencephaly. Journ. Mental

Science, vol. 40, pp. 649—665. Reichert, C. B. 1859-1861 Der Bau des Menschlichen Gehirns. Leipzig. Retzius, G. 1900 Die Gestalt der Hirnventrikel des Menschen, nach Metallaus gussen dargestellt. Biol. Untersuch. von G. Retzius. Bd. 9. N:o.

3 u. 4. Jena. SissoN, S. 1910 A text-book of veterinary anatomy. Philadelphia. S.MiTH, G. E. 1903 On the morphologj^ of the cerebral commissures in the verte brata, with special reference to an aberrant commissure found in the

fore-brain of certain reptiles. Trans. Linn. Soc. London. July, pp.

455-500. Testut, L. 1897 Traite d'anatomie humaine. Paris. Thompson, P. 1909 Description of a model of the brain of a foetal cat, 20 nuu. in

length. Journ. Anat. and Physiol., vol. 43, pp. 134-145. Welcker, H. 1878 Wachsausguss der Gehirnventrikel. Virchow's Archiv. f.

Pathol. Anat., Bd. 74, S. 500-503.


THE AMERICAN JOURXAL OF ANATOMY, VOL. 15, NO. 2


ABBREVIATIONS


B.olf., bulbus olfactorius

C.pr., caudal protrusion

CbL, cerebellum

Ch.op., chiasma opticum

Coll. %., colliculus inferior

Coll.s., colliculus superior

C.inf., cornu inferius

C.par., corpus paraterminale

C.pin., corpus pineale

C.str., corpus striatum

For., foramen interventriculare

F.plx., fissure for plexus chorioideus

Hah., habenula

Hip., hippocampus

Inc.pc, incisura postcommissuralis

/., infundibulum

Is., isthmus

L.ch., lamina chorioidea epithelialis

Mes., mesencephalon

A'^i — Nt, neuromeres


N.op., nervus opticus

N^.tr., nervus trigeminus

Pa7\, parencephalon

Plx., plexus chorioideus

R.i., recessus infundibuli

R.I., recessus lateralis

R.m., recessus mamillare

R.np., recessus opticus

R.pin., recessus pinealis

R.post., recessus postopticus

R.sup., recessus suprapinealis

Rf., rhomboid fold

S.I., sulcus limitans

Syn., synencephalon

T.ch., tela chorioidea

Th., thalamus

V.b.olf., ventriculus bulbi olfactorii

V.I., ventriculus lateralis

Vel.fr., velum transversum

V.op., vesicula optica


PLATE 1

EXPLANATION OF FIGURES

4 Wax model of the cerebral ventricles of a 5.1-mm. pig. H.E.C. series 1904. X 20 diam.

5 Cast of the cerebral ventricles of a 12-mm. pig. X 15 diam.

6 Dissection of the brain of a 12-mm. pig. X 15 diam.

7 Dissection of the brain of a 12-mm. pig. X 15 diam. The plane of the cut edge of this dissection is indicated by arrows in figure 5.


240


THE DEVELOPMENT OF THE CEREBRAL VENTRICLES IN THE PIG

CHESTER H. HEUSER

Is., Mes.^



C^tr.


Fi.i. n



Vel.tr.


Fig. 7


241


PLATE 2

EXPLANATION OF FIGURES

8 Dissection of the brain of a 17-mm. pig. X 10 diam.

9 Cast of the cerebral ventricles of a 17-mm. pig X 10 diam. 10 Dissection of the brain of a 17-mm. pig. X 10 diam.


242


THE DE^TSLOPMENT OF THE CEREBRAL VENTRICLES IN THE PIG

CHESTER H. HEUSER


PLATE 2



Par



C. str.



L.ch.


Fiq.10


243


PLATE 3

EXPLANATION OP FIGURES

11 Dissection of the brain of a 22-mm. pig. X 10 diam.

12 Cast of the cerebral ventricles of a 22-mm. pig. X 10 diam.

13 Dissection of the brain of a 22-mm. pig. X 10 diam.


244


THE DEVELOPMENT OF THE CEREBRAL M3NTRICLES IN THE PIG

CHESTER H. HEUSER


S.l.


PLATE 3


Vel. tr. ^


C.str.



C. pa r.


Fig. 11



pi n.


PLATE 4

EXPLANATION OF FIGURES

14 Dissection of the brain of a 45-mm. pig. X 6.5 diam.

15 Wax model of the cerebral ventricles of a 45-mm. pig. X 6.5 diam.

16 Left lateral ventricle of the model shown in Fig. 15.

17 Ventral view of the same ventricle.


246


THE DEVELOPMENT OF THE CEREBRAL VENTRICLES IN THE PIC. PLATE 4

CHESTER H. HEUSEU


T. ch.


C. ))ln. .Mt^sS^Z


P!x,~^


Cstr.:


ColU.



Jnc. pc.



V.holf. a inf.

Fig.16


24;


PLATE 5

EXPLANATION OF FIGURES


18 to 23 Alicrophotographs of frontal sections through the head of a 45-mm. pig. H.E.C. series 1826. X 5 diam. The position of section 563 is indicated by


arrows in figure 14.


18


Section 563.


19


Section 629.


20


Section 687.


21


Section 755.


22


Section 1067.


23


Section 1115.


248


THE DEVELOPMENT OF THE (^EREBRAL VENTRICLES IN THE PIG PLATE 5

CHESTKR H. HEDSER



Fig. 18


Fig. 19



Fig. 21



Fiq. 21


Fig. 23


249


PLATE 6

EXPLANATION OF FIGURES

24 Wax model of the mid-brain ventricle of a 110-mm. pig, viewed slightly from below and behind. X 15 diam.

25 Wax model of the cerebral ventricles of a 260-mm. pig. X 3 diam. 20 Ventral view of the same model.


250


THE DEVELOPMENT UF TEE CEREBRAL VENTRICLES IN THE PIG

CHESTER H. HEUSER


Colli .


PLATE U



.Fiii.24


R.sup.



Fiy.25



V. b.olf.


y^


F. plx.



V. b. olf.


Fig.2G


251


AN EXPERIMENTAL STUDY OF THE POSITION OF THE OPTIC ANLAGE IN AMBLYSTOMA PUNCTATUM, WITH A DISCUSSION OF CERTAIN EYE DEFECTS

CHARLES R. STOCKARD

Department of Anatomy, Cornell University Medical College, Neiv York City


NINE FIGURES


INTRODUCTION


Numerous investigations have recently been directed towards an analysis of the processes concerned in the development of the vertebrate eye. Both mechanical and chemical methods of experiment have been employed and at the present time most of the results seem open to explanation. Various theories and speculations, however, have been advanced which will probably form a source of contention until the experimental results are better or more uniformly interpreted.

By way of introduction to the experiments recorded in the present paper and a discussion of their significance, it may be well to outline briefly the status of the main problems concerned.

The writer has previously recorded the results of experiments bearing upon an analysis of the manner of origin and development of the optic cup and the optic lens. It was demonstrated that in the- species studied the crystalline lens can arise entirely independent of any influence from other eye parts. It seems also equally clear from the many cases observed that the optic vesicle or cup can at some stage during its development induce the formation of a lens from the ectoderm with which it comes in contact.


253

THE AMERICAN JOURNAL OF ANATOMY, VOL. 15, NO. 3 NOVEMBER, 1913


254 CHARLES R. STOCKARD

even though this ectoderm is not exactly that from which the normal lens would have arisen. Lenses were induced to arise only from head ectoderm and independent lenses invariably arose in the head region, it was thus concluded that the head ectoderm principally possessed the independent lens forming power.

A consideration of numerous eye abnormalities which occurred in these experiments seemed to throw light on the earlier conditions of origin of the optic anlage from the medullary tissues. A number of the abnormal eyes appeared to lend themselves to a common interpretation and for this reason I advanced in an entirely hypothetical manner the view that these conditions might be considered as arrests in eye development.

Spemann in two more recent contributions to the development of the eye has confirmed all of my observations that were touched upon by his experiments. In an equally general manner he has disagreed with most of the deductions which were drawn from my experimental results. ' An attempt to satisfy these disagreements will be made in the body of the present paper.

It is now concluded from a study of abnormal eyes and the experiments below that the eye anlage in the medullary plate is primarily median and single and normally separates or spreads into two almost equal growth regions which develop in lateral directions reaching further and further out until finally the optic vesicles come in contact with the ectoderm at the sides of the head. Provided this view is correct cyclopia is then an arrest in eye formation.

Spemann., on the contrary, holds that the eye anlagen originally arise lateral in position along the borders of the medullary plate. The cyclopean defect according to him is due to a failure of central medullary tissue to develop so that the lateral eye anlagen slump towards the median plane, fuse and form a single cyclopean eye. Spemann, however, presents no experimental evidence to show that the eye anlagen do occupy lateral positions since all of his operations included the median medullary tissue as well as the lateral.


POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 255

The only evidence to draw upon is the interesting experiments of Lewis, in which it was found that cyclopia sometimes resulted in Fundulus embryos when the anterior end of the embryonic shield was injured by pricking. Lewis also interpreted these defects as due to a fusion of the optic anlagen, and had suggested, as Spemann now does, that the chemicals used in my experiments suppressed the development of niedian tissue in the medullary plate and thus caused the eye anlagen to come together, fuse and produce cyclopia. There are a great many strong objections to this hypothesis of Lewis and Spemann which have been enumerated in my previous papers and to which I shall take occasion to refer briefly in the following discussion.

An objection of primary importance to the idea of cyclopia as a result of the coming together of lateral anlagen through a failure of intermediate tissue to form is the fact that cyclopean eyes are rarely in size and extent equal to the sum of the two normal eyes combined. A cyclopean eye is, as a rule, little if any larger than one normal lateral eye and in fact is often much reduced or actually minute in size as compared with a normal eye. This fact indicates most decidedly that eye material, as such, has been, injured or arrested in its development and differentiation. One is then scarcely warranted in assuming that the defect is solely due to a failure in formation of material between the eyes,

Spemann has found, although he locates the anlagen in the wrong place, that not only is the eye anlage definitely localized in the open medullary plate but actually the tapetum nigrum is distinct from the other retinal layers. How then could absence of material between the lateral eye anlagen cause less eye material to arise?

The differentiation of these prelocalized anlagen require definite amounts of energy. Any treatment that weakens the developing embryo at certain periods in a definite way renders the eye anlagen incapable of differentiation so that they do not arise from the brain.

The entire problem is readily open to experimental test. The contention may resolve itself into the question: Where are the


256 CHARLES R. STOCKARD

optic anlagen originally located in the medullary plate or tube? The present communication will present the results of experiments aimed towards an answer to this question. Certain interpretations put forward by Spemann regarding the origin and development of the primary components of the eye will also be considered.

MATERIAL AND METHOD

The material used in all the experiments has been the developing embryos of the salamander, Amblystoma punctatum. Amblystoma eggs are surrounded by masses of jelly-like material from which they may readily be separated. The eggs live perfectly without the jelly mass, provided they are well covered with fresh water.

These eggs are of sufficient size to render it possible to cut out with fair accuracy definite regions or groups of cells from the medullary plate or groove.

The method of experiment has been entirely by mechanical operation. This method is particularly useful for the problem in hand since definite areas may be removed and the results studied. The operations were made under a binocular microscope. Fine steel needles and the smallest scissors were used as instruments.

The embryos were kept from three to five days after the operation in water which had been previously boiled. They were killed in a mixture of one part formalin to three parts saturated corrosive sublimate, left for three hours, rinsed in water and put into 70 per cent alcohol with iodine. Others were fixed for three hours in Bouin's fluid, formalin and picroacetic. The sectional embryos were stained with Delafield's hematoxylin and eosin.


POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 257

CONSIDERATION OF THE EXPERIMENTS

1. Sticking the future brain portion of the medullary plate with

needles

The anterior portion of the medullary plate was stuck with steel needles in such a manner as to disturb the cells over an area extending from the anterior border of the medullary fold back to the constricted portion of the plate and laterally from fold to fold. The needle was inserted below the outer layer of cells and raised so as to push the cells apart; this was done a number of times with each specimen. The needles were also swung to the right and left in the medullary tissue until the cells were considered to be disturbed to quite an extent. The object in such an experiment was to determine how severe an injury to the cells was necessary in order to prevent the development of the optic vesicles.

Twenty-three embryos were treated in this manner, and all were killed four days after the operation. Under a high power binocular microscope most of them distinctly showed that the optic cups were well pushed out laterally and in contact with the ectoderm at the sides of the head. Both eyes were clearly seen in seventeen of the individuals, while six seemed to have eyes yet not so well developed. These six doubtful specimens were sectioned and studied microscopically.

Both eyes were present and apparently normal in structure in five of the six embryos. The sixth individual showed eyes which were slightly irregular in form and poorly developed, yet both eyes were distinctly present.

The experiment would indicate that a disturbance, of the type employed, of the cells constituting the eye anlage in the medullary plate was not sufficient to prevent the normal development of the eyes in these embryos.

Another point of interest might have been attacked, by such an experiment provided the embryos had been allowed to develop sufficiently long after the operation. That is, whether or not the cells destined to form the tapetum nigrum layer might be


258 CHARLES R. STOCKARD

intermixed with those cells destined to form other retinal layers, ^nd so produce an eye with the pigment cells scattered irregularly through the retina. Spemann's recent experiments on cutting out and reversing certain areas of the open medullary plate indicate that the tapetum cells are fully localized and separate from the other retinal cells in certain amphibian embryos. The embryos in my experiments had not differentiated the retinal layers sufficiently far to determine with certainty whether there was a persistent disarrangement of the cells, yet the general appearance of the eyes seemed perfectly normal.

2. The median region of the anterior part of the 7nedullary plate cut out, reversed and transplanted in the medullary plate

This experiment is similar to 'fehose performed b}^ Spemann on several amphibian embryos. Spemann found that pieces of the medullary plate when cut out and turned around continued to develop with their original orientation undisturbed, thus indicating the early prelocalization of certain future parts of the brain and eyes. When the operation chanced to cut the eye anlage so that part of the future eye material was anterior to the cut and remained in position, while part was contained in the cut-out piece which was then turned around and transplanted, carrying the future eye cells to a more posterior position, two eye regions developed on each side. One arose from the anterior undisturbed cells and the other from the transplanted posterior cells.

The reversed pieces in the present experiment were not long enough to carry the eye back to a distant posterior position, and the cut extended so far foreward that the eye anlage was not divided transversely as in Spemann's operations. The operations were done chiefly to test whether the eye anlage in Amblystoma was well localized and would develop after such reversal of tissues.

Eight embryos were studied after having had antero-median pieces of the medullary plate cut out, reversed and transplanted. Seven of the eight developed both eyes, many of which showed


POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 259

indications of their misplacement. One individual showed one abnormal eye while the other was probably indicated by a doubtful eye structure situated within the archenteron. The general structure of the eyes seemed normal and only slight indication of their reversed origin was shown by a study of these early stages.

Thus if the eye anlage was contained within these cut-out pieces the reversal and transplantation of the pieces did not show any very detrimental effects on the future development of the eyes, though in one of the eight cases one eye was abnormal and the other was greatly misplaced and indistinctly indicated.

The experiments of sticking and disturbing the cells in the anterior end of the medullary plate without actually removing the cells does not prevent the subsequent development of the optic vesicles in an apparently normal manner. Cutting out rectangular pieces of the medullary plate which contain the eye anlage, reversing and replanting them merely cause the eyes to develop in misplaced positions. These two experiments demonstrate the fact that unless the future eye material is well removed by the operation the optic vesicles may later form; this is important in estimating the value of the results in the experiments to follow.

3. Lateral regions cut from the anterior part of the medullary -plate

Four embryos were operated upon, as shown in figure 1. The indicated region of the right lateral portion of the flat medullary plate was cut entirely away with fine scissors. The area removed did not extend quite to the median line except that in one case it may have or probably did remove tissue beyond the median line. The embryos were killed three days later, cut into sections and studied microscopically.

One of the four lacked both eyes or had eyes so small and poorly formed that they could not be recognized, and the brain showed on one side indications of the operation. Both eyes had thus been removed by an operation which extended at most


260 CHARLES R. STOCKARD

slightly beyond the median line and certainly did not include the left lateral medullary tissue.

The other three embryos had both eyes present, though one eye in one specimen and both eyes in another were abnormal or defective. In these cases the cut did not extend close enough to the median line to remove all of the eye substance of that side; 3^et if the eye anlagen at this time had been lateral in position, one would have been completely removed.

A similar experiment was performed on seven somewhat earlier embryos. In these cases where the medullary plate was younger and wider in extent the removed area was confined to a lateral position and did not extend close to the median line, yet it extended laterally into the medullary fold (fig. 2).

Three days later the embryos were killed. On studying the sections of the head region six of the seven embryos were found to possess two perfect eyes of normal proportions. The seventh individual had only one well defined eye while the other eye was absent.

The removal of this lateral area of tissue which has been considered the position of the early eye anlage by certain investigators gives no effect on the development of the future eye unless the cut be made to extend very close to the median line. In eleven operations of this type nine individuals did not suffer the loss of either eye, while one specimen lacked both eyes and another one had only one eye. The case in which both eyes were absent might have been due to the fact that the cut extended a little way beyond the median line and removed cells destined to form the material of both eyes. Experiments recorded below would indicate that this was the case. A second possibility is that the operation so weakened the individual — • which may have been below normal in vigor, though apparently perfect specimens were selected for all the operations — that it lacked the power to differentiate the eyes from the medullary tissue.

Six of the nine specimens which possessed both eyes showed no effects of the removed material in either the size or form of their eyes. The other three embryos had one or both eyes some


POSITION OF OPTIC ANLAGE IN AMBLYSTOMA


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262 CHARLES R. STOCKARD

what smaller than usual. The cases with only one eye defective are perhaps clearly understood on the ground that the cut encroached upon the material destined to take part in the development of this eye. In the cases with both e3^es defective it scarcely seems possible that the opposite eye could have been injured by these cuts without entirely removing the eye on the side where the cut was so extensive. The only plausible possibility is that the material for both eyes was mediallj^ located and the two eye anlagen closely connected. Any injury to this region might later be shown by both eyes if the injury were at all extensive, and if less extensive and confined entirely to one side of the median line only one eye might show the effect.

I realize that this explanation might be interpreted in part as opposed to Spemann's experuuents in which he found the future eye materials so consistently definite in the medullary plate. When the eye anlage was cut transversely and the posterior portion reversed and planted in a distant position it was found that if the forward part of one eye was small the part which had been cut from this and placed posteriorly was large. This fact does not indicate entirely that the eye stuffs are separate; only that when the eye anlage, even if it be median and single, is cut across obliquely and the posterior part removed and transplanted it continues development in a normal consistent manner, the small eye part coming off on the side where less material exists and vice versa.

4. The anterior lateral part of the 7ieural fold and a portion of the medullary wall cut from one side

These experiments were performed on embryos older than those used above in order to test whether the eye anlagen shortly before the coming together of the neural folds might be situated laterally along the border of the folds. In other words, do the eye anlagen primarily arise in the median line or region of the medullary plate and later occupy more lateral positions?

The operation consisted in cutting away one side of the anterior part of the medullary fold and also including in the removed


POSITION OP OPTIC ANLAGE IN AMBLYSTOMA 263

part some of the material constituting the lateral wall of the medullar}^ groove (fig. 3).

Fom- embryos were operated upon in this manner. Three days after the operation they were killed and prepared for study. A careful examination showed that three of the four specimens possessed both eyes, while one had no eye, or a questionable formation, on the operated side although a perfect eye was present on the unoperated side. Thus in three-fourths of the few cases used, neither of the future eyes had been injured by the operation.

Six other embryos were operated upon in a similar fashion. These were killed two days after the operation. Studying the six embryos in cross section it was found that three possessed both eyes in perfect condition, one individual had two eyes, yet one eye was small and defective on the operated side, and two of the specimens had no eye on the operated side. Thus four of the six specimens had two eyes each, and two of the six had only one eye each.

Combining the results of both experiments it is shown that, of the ten operated embryos seven possessed both eyes and three lacked an eye on the operated side. One of the seven which had both eyes present showed a defective eye on the operated side.

A comparison of these experiments with those recorded in the preceding section in which the operation was performed on earlier embryos would indicate that the eye anlage has possibly widened out so as to extend into more lateral regions in these later stages. The weight of evidence, however, would indicate that the position is not directly lateral along the edge or border ol the medullary plate, as Spemann has assumed.

It must be remembered that in the folding process which converts the medullary groove into a tube the original borders or edges of the medullary plate come to meet in a middorsal line. After the medullary tube is thus formed the optic vesicles push out from a lateral or really ventro-lateral region and certainly do not in any sense come from the original borders of the medullary plate which are now dorso-median in position.


264 CHARLES R. STOCKARD

5. Both anterior lateral parts of the neural fold cut away just before the folds close together

The operation consisted in clipping away with scissors the lateral wall of the neural groove from both sides; that is, the raised folded parts and the dorsal portion of the lateral neural wall indicated in figure 4. The actual crest of the neural folds is of course not future lateral material as in the closure of the furrow to form the neural tube the crest becomes dorsomedian as just mentioned.

Four embryos were successfully operated upon in the given manner. Three days after the operation they were killed and prepared for study. All of the embryos were found to possess both eyes in well developed conditions.

Five other embryos in a similar stage of development were operated upon so as to remove the lateral neural folds as indicated in figure 4.

Four days after the operation the embryos were killed. Studying these embryos in section showed that four of the five possessed both eyes in a well formed condition giving no indication of the injury while the fifth individual possessed only one eye. This last specimen was evidently cut too near the median line on one side so that the future eye material of that side was destroyed.

Thus in nine specimens with both sides operated upon, eighteen operations, only a single eye was missing. The other seventeen operations did not cause smy noticeable effect in either the development or nature of the resulting eye.

These experiments clearly demonstrate that the eye anlage is not located along the lateral edge or border of the medullary plate or groove, as Spemann holds. The importance of such facts in connection with opposing explanations of certain eye abnormalities will be fully considered in a following section of this paper.

The next experiments to be presented were performed in order further to substantiate the fact that the eye parts do occupy a


POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 265

median position in the medullary plate or groo\'e. I have been led to think from a study of a number of eye abnormalities that the eye anlage is more or less median in its original position.

6. The removal of anterior median strips of cells from the wide

medullary plate

Tn these operations a narrow median strip was cut from the anterior region of the wide medullary plate. The strip in all cases was narrow, being in fact only about one-third or one-fourth the entire width of that portion of the medullary plate lying between the medullary ridges, as shown in figure 5.

Four embryos with wide medullary plates were operated upon in the above manner. Four days later they were preserved for study.

Three of these four specimens were eyeless, showing that the operation had removed the entire eye anlage from the middle of the medullary plate. The remaining one of the four embryos had one poorly formed eye while the other was only slightly indicated, so that in this case almost all of the ability to develop eyes had been lost as a result of the operation.

Five other embryos were subjected to a similar operation to that indicated in figure 5 except that the median strip removed was still narrower than in the preceding experiment. These embryos were of different stages but all showed the medullary folds far apart so that the medullary groove was wide open. Three days after the operation the embryos were prepared and studied.

One of the specimens was eyeless, one had two poorly developed eyes, two had one well formed eye while the other eye was questionable or absent, one had both eyes present. Thus the removal of a very narrow median strip gave less decided effects than the removal of somewhat wider strips in the experiment above. Yet four out of the five embryos showed eye defects, two having both eyes affected and two having one normal eye and one eye questionable or absent.


266 CHARLES R. STOCKARD

Combining the results of the two experiments it is found that the removal of narrow median strips from wide medullary plates exerts the following influence on the future development of the eyes: Of nine embryos thus operated upon four failed entirely to develop either eye. Two showed two defective eyes. Tivo individuals developed one perfect and one defective or questionable eye. Only one of the nine embryos showed two apparently normal eyes.

Since six of the nine embryos had the development of both eyes either entirely suppressed or decidedly affected, and two of the remaining three had one eye affected, it seems most certain that cells destined to take part in eye formation are located in the median region of the medullary plate and are removed by the operation employed. One must conclude that the median optic anlage occupied at least one-fourth or one-third of the width of the medullary plate in the anterior region.

A general statement of the results of certain of the experiments described above may be expressed as follows: Thirty embryos studied after various operations in which lateral portions of the medullary plate were removed at slightly different developmental stages (sections 3, 4, 5) showed in twenty-four individuals, or 80 per cent of the cases, subsequent development of both eyes, while only six specimens or 20 per cent of the cases, showed absence of the eye. In one case the presence of the eye is questionable, in five cases one eye and in one case both eyes were absent. The absence of eyes in the latter cases is possibly due to the cut having been made in a more median position than was intended.

Nine embryos studied after having been operated upon so as to remove a narrow median strip of cells from the anterior portion of the medullary plate (section 6) showed in four cases, or about 45 per cent of the specimens, entire absence of eyes. In four other individuals the eyes were highly defective, one specimen having one poorly formed eye while the other was questionably present. In only one of the nine embryos did both eyes approach the normal condition, from this specimen an extremely narrow piece had been cut away. The optic anlage in this case might have been sufficiently wide at the time of the


POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 267

operation to allow its median portion to be removed and yet enough material remain on either side of the cut to give origin to the two eyes. According to the views of several investigators the removal of this median material should have caused cyclopia, yet it did not. I shall presently attempt to show that cyclopia is not due to the coming together of lateral material in the median line but to a failure of median material to spread laterally.

When the results of these two classes of experiments are contrasted one must conclude that: The eye anlage in the medullary plate occupies an antero-median position as shown by the various abnormalities incurred when this region is cut away. The failure to injure the development of the eyes in the great majority of cases when the lateral portions of the medullary plate are removed by operation indicates further that the eye anlagen do 7iot occupy lateral positions during this stage of development.

A DISCUSSION OF PREVIOUS IDEAS REGARDING THE GENESIS OF CERTAIN EYE ABNORMALITIES IN THE LIGHT OF THE ABOVE

RESULTS

There has lately been considerable discussion regarding the way in which the cyclopean defect occurs. The experiments described above may serve to concentrate the case on a definite developmental period and, to my mind, settle the question as far as the medullary plate stage is concerned. Whether it is possible to carry any question of vertebrate eye abnormalities, as such, further back in development than this stage seems doubtful, since here it is that the localization of the eye anlage is first knOwn to exist. It is certain, of course, that this anlage does come from cells which are present before the medullary plate has formed. Whether these cells are localized and are entirely future eye anlagen cells, and not indifferent ectoderm cells which might have the power of forming any portion of the brain or central nervous system, one is at present unable to state.

As the case stands, it seems possible to explain the entire genesis of the cyclopean defect from the earliest time at which the optic anlage is definitely known to be localized. This as


268 CHARLES R. STOCKARD

sumption is not made solely from the material presented in the present paper, but from the facts furnished by these experiments together with the observations made upon the large number of Cyclopean eyes and brains which the writer has studied during the past several years.

Various authors have at different times thought that cyclopia was due to a fusion of the eyes after they had arisen from the brain. The earlier in development the fusion occurred the more intimately associated the two eye components became. This view has been proven incorrect by actual observation on Cyclopean monsters where it is found that the cyclopean condition of the eye, whether large and hour-glass shaped or of small size resembling a normal eye, is present from the earliest appearance of the optic vesicle from the brain. ' In other words, the several degrees of the cyclopean eye come off from the brain in their final conditions.

The idea of the fusion of the eye parts was deep rooted, however, and now exists in the recent views of Spemann in a refined form. Spemann believes, as several others had previously suggested, that cyclopia is due to an absence of non-ophthalmic tissue in the median region of the medullary plate or groove. This lack of median tissue allows the eye anlagen which he holds to be lateral in position, near the borders of the medullary plate, to come together and fuse iii the median plane and later give rise to a cyclopean eye. Cyclopia, according to this idea, occurs in a more or less passive manner, and is, after all, actually a fusion of the eye anlagen of the two sides during development.

I am certain that this fusion explanation which has now been forced entirely back into the medullary plate, is as false as* its bolder predecessor which assumed the fusion to take place outside of the brain tissues after the optic vesicles or cups had arisen. Spemann did not advocate this late-fusion view, but claimed from his beautiful experiments on Triton that the cyclopean eye arose out of the medullary tissues in its final condition. He now, however, assumes the role of a most ardent supporter of the fusion of the optic anlagen within the medullary plate.


POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 269

The present writer's opinion of the cause of cyclopia, which was advanced in 1909, was to the effect that this deformity is the result of a weakened development. Spemann has termed this view the 'laming: hypothesis,' since my assumption was that the A^arious chemical substances employed in producing Cyclopean defects in fish embryos have a tendency to lower the developmental energies and so reduce the power necessary to accomplish the processes involved in the outpushing of the optic vesicles from the brain.

Considering the probable manner in which the cyclopean defect occurs, Adami ('08, p. 241) has theoretically concluded that it is due to developmental arrest or lack of vigor. While I am unable to agree with the details in Adami 's argument of a primary growth point at the anterior tip from which is budded off successively the paired parts of the two sides, the anterior ones necessarily arising last after the other parts had been left in more posterior positions, the final conclusion that a weakening of particular developmental processes results in cyclopia is confirmed by all my experiments.

The different . types or degrees of the cyclopean defect depend upon the stage in development at which the arrests occur as well as upon the strength or severity of the treatment employed. I shall now attempt to defend this position with the evidence at hand, and in so doing shall as decidedly prove the mistake in considering the defects as the result of any failure to arise of median medullary tissue (other than future eye tissue) and the subsequent fusion of the lateral optic anlagen. There is no median tissue between the eye anlagen. The median tissue is the eye anlage itself and will subsequently go to form some portion of the eye, either optic cup or optic stalk, depending largely upon its position and the extent of normal development attained.

The writer had ('07, '09, '10) recorded a number of eye conditions which are considered to be different degrees of cyclopia using the term in a general sense. At any rate, these several conditions differ only in degree and grade perfectly into a continuous series. There is no qualitative difference between them. Spemann has objected to including among these defects certain

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270 CHARLES R. STOCKARD

modified types which are at least closely related to the series in manner of origin, though they may not actually intergrade. (We are now considering only the cyclopean series, not the monster monophthalmica asymmetrica which will be dealt with later.)

One finds on referring to my paper of 1909 (p. 293) that the cyclopean series, A to G, passes from the normal individual through different degrees of association of the two eyes to a median cyclopean eye only as large as one normal lateral eye, then to a cyclopean eye of smaller dimensions until it is extremely minute and may finally be deeply buried beneath the brain as a small pigmented vesicle, as is shown in figure 52, page 321. Only one step further and the eyes fail to arise entirely so that eyeless individuals exist which with a slightly greater power of differentiation or more developmental energy might have given cyclopean monsters. The last assumption is warranted since these eyeless specimens actually resemble the cyclopean monsters in other structures; for instance, the mouth is a narrow proboscis similar to that in the cyclopean monster instead of the usual laterally spread mouth of the normal embryo.

Why should every step and gradation in this series exist if several, or any of the conditions are of a different quality or type? It seems certain that one examining the large number of cyclopean fish on which* my study was based would be forced to admit the correctness of the statement that these individuals exhibit different degrees of one and- the same defect.

The question then follows, if cyclopia were due, as Lewis, Spemann and others assume, to a failure to develop of median medullary tissue so allowing the eye anlagen to come together in the median plane and fuse, why is not e^^ery cyclopean eye equal in mass to the two normal eyes fused? Spemann does not suggest in any place that eye material also fails to arise. He shows in his recent experiments that the future eye is fully laid down in the medullary plate. Not only is the eye present in the medullary plate but the cells destined to form different layers are distinct. Spemann found that certain cells cut out of the medullary plate and planted in more posterior positions formed only the tapetum nigrum layer. If the eye is thus so


POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 271

definitely predetermined in the medullary plate and Spemann believes that cyclopia is due to failure of median medullary cells other than future eye cells then cyclopean eyes ought always to be large or double in size.

The fact is that these predetermined eye cells in the medullary plate in most cases of cyclopia are incapable of perfect differentiation on account of insufficient energy, so they remain in the brain, or only part of them is capable of difTerentiation and thus small defective cyclopean eyes result.

The actual 'Lahmungs' or suppression is of the eye material itself. Cyclopia is an eye defect, and an injury of the eye forming material is the cause. The brain may also be defective as an accompaning abnormality, although in some cases the brain, with the exception of the optic tracts and parts, may be structurally and functionally perfect, as is indicated by the normal life and behavior of many of the cyclopean Fundulus embryos as well as by the existence of the huge cyclopean ray, Myliobates noctula, reported by Paolucci in 1874.

The chemical substances employed by the author in producing the cyclopean defect and a number of others with which McClendon has obtained similar results, all tend to suppress or arrest the development of the eye material in the brain. This future eye material is assumed to occupy a median position. When the arrest is complete, and necessarily taking place at early stages, no eye parts arise from the brain nor are any differentiated within the brain substance itself. Thus a completely eyeless individual is produced.

Spemann states that the eye is capable of differentiation even though it be contained within the brain substance as he has found in amphibia and as MencI recorded in a teleost. It must be realized that these are exceptional cases. A certain amount of energy is necessary for differentiation of the eye to take place even within the brain and when only this amount of energy is present the eye may differentiate within the brain, but when the required energy for any reason is not available the eyes are incapable of any differentiation. Many eyeless individuals have been observed in my experiments which have no indication what


272 CHARLES R. STOCKARD

ever of eye parts within the brain. Could any one ask, whethei this be due to the failure of non-ophthalmic parts of the medullar}^ plate to arise or, on the other hand, to a failure of the future eye forming cells to arise, or to differentiate after they have arisen?

It is not meant to convey the idea that all eyeless brains are related to the cyclopean series, as this is not the case. The future eye forming cells may in some cases have been absent from the start. In other specimens the future optic vesicles might have been in positions to arise normally and laterally and for some reason were incapable of outpushing or of differentiation.

Certain eyeless brains, however, such as those in individuals having the proboscis-shaped mouth, do belong to the series and must be caused in the same way as are the various cyclopean conditions. They have merely responded to a more exaggerated degree.

The most extreme cases of cyclopia with actual eye structures are those in which a small pigmented vesicle arises from a ventromedian part of the brain, as is shown in my figure 52, 1909. This pigment la^^er of the retina seems to be its most persistent portion, as it may appear when all other recognizable retinal parts fail to arise. There is a possibility that the small tapetum nigrum groups of cells which in some cases formed from Spemann's transplanted portions of eye anlagen may not be due to the fact that only the anlagen qf such cells were transplanted, but that all other retinal cells except these were incapable of differentiation when so small a piece of future eye tissue was isolated by the operation.

The next degree of cyclopia is exhibited by an individual having a median eye that is much smaller than one normal lateral eye. We then have a median cyclopean eye of about the same size as one normal lateral eye. The latter case has been termed the 'perfect' cyclopean condition.

All these abnormalities are best explained as follows: The future eye forming cells occupy a median position in the medullary plate and the cells destined to form the two eyes are arranged in one group. This median group of future eye cells


POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 273

normally widens or spreads laterally while two centers of active growth become established which gradually assume more lateral positions until they push out as the two optic vesicles. In the degrees of cyclopia mentioned in the preceding paragraphs the median eye anlage does not widen or spread laterally but is arrested in its primary condition; thus the two growth centers are not sufficiently separated and only a single center exists, and even more than this, the arrest is to such an extent that the entire or normal amount of optic material does not differentiate. Hence, one finds a median cyclopean eye consisting of an amount of eye material far below that normally present.

Other individuals are found in which greater masses of eye material have succeeded in differentiating, and development has been vigorous enough to allow the early eye anlagen to spread to a greater or less degree and establish the two eye forming centers. Such specimens finally present a cyclopean eye showing distinctly its double composition, the two retinae are more or less distinct and the eye large consisting of a greater amount of material than a normal lateral eye, yet less bulky as a rule than the sum of the two normal eyes of the species.

The hour-glass eye or incomplete cyclopiq, is commonly observed in the experiments. This case is due to a later or less complete arrest in development than those mentioned above. Both eyes have differentiated out of the medullary tissue but the embryo was not vigorous enough to permit their normal separation and outpushing. Thus the eyes come off from their original ventro-median position and remain in close contact or actual union.

Finally one observes individuals in which the eyes are separate and distinct yet unusually close together. Such embryos are able to differentiate their eye material and this material is capable of pushing out from the brain but a slight weakness or arrest has occurred on account of which the optic stalks are short and the optic cups are unable to take a normal positionso that they remain unusually near together and look in an .abnormal direction.


274 CHARLES R. STOCKARD

In the ordinary individual the future eye forming material is first located in a median position in the medulla^-y plate. This material becomes more extensive or widens laterally, and two growth centers are established, the material between the centers finally becomes the median ventral layer of cells of the optic stalks. Later the incipient optic vesicles begin to evaginate or push in a ventro-lateral direction and finally turn dorsally and laterally to reach their usual places at the sides of the head. The optic stalks, however, still lead back to the ventro-median position and there in the fish the optic fibers, following the optic stalks as paths, cross and in higher vertebrates form the optic chiasma always in the ventro-median plane below the brain floor and from here the optic tracts proceed to their centers in the brain.

The median position of the optic chiasma outside and below the brain is an important structural fact in the present consideration. Figure 6 is a diagram of a transverse section through an early brain with the optic cups in their usual position. The optic stalks connect with the brain and the median ventral cell layer is actually part of the stalks. As development proceeds the optic fibers arising from the cells in the retina follow along the optic stalks to reach the brain. The investigations on the development of the optic nerve have shown that the optic stalks become solid and form the supporting paths or neuroglial scaffolding along which the optic nerve fibers grow. The fibers from one retina meet those from the other in the median plane below the brain and in the fish the fibers from the two retinae cross directly while in higher forms partial crossing takes place and the optic chiasma is formed outside the brain. Figure 7 is a sketch representing a cross section of the actual condition of the early optic nerves in a fish embryo. This position of the optic cross is only possible if the median tissue be optic stalk tissue.

• Suppose, on the other hand, that the eyes primarily originate from lateral medullary tissues and between the two eyes other brain tissue is present. The optic stalks are then attached to. the lateral regions of the brain from which the optic vesicles


POSITION OF OPTIC ANLAGE IN AMBLYSTOMA


275



Fig. 6 A diagrammatic cross section through the brain and optic cups of an early embryo, with all of the median ventral medullary tissue represented as being part of the optic stalks. The future fibers of the optic nerves, shown in red, follow the optic stalks to the median plane where they cross and afterward enter the brain to pursue their course as the optic tracts. The optic cross is entirely beneath and outside the brain.

Fig. 7 A sketch representing the actual outlines of a cross section through the brain and eyes of a late fish embryo. The optic nerves cross below and outside the brain and are surrounded by cells derived from the original optic stalks.


276


CHARLES E. STOCKARD


pushed out. Figures 8 illustrates diagrammatically a cross section of this condition. In the course of development the fibers of the optic nerve following the stalk reach the lateral position and must enter the brain and continue within its tissue in order to meet the nerve of the opposite side and form the cross or chiasma. Brain tissue would lie beneath the optic chiasma and the chiasma would necessarily be within the brain. This condition is never found in any normal vertebrate.



Fig. 8 A diagrammatic cross section through the brain and optic cups in an imaginary case in which the optic connections are lateral with median brain tissue originally lying between the eye anlagen. The future optic fibers gr-owing along the optic stalks as paths reach the lateral points on the brain from which the stalks arose and then enter the brain tissue before having formed the cross. Continuing to grow, the optic nerves meet and cross in the median plane. The cross is within the brain itself and lies above the median mass of tissue which has always existed between the eyes. No vertebrate brain exhibits such a condition.


One might claim that the optic fibers on reaching the brain ran ventrally and formed the cross beneath, but no such change in direction is seen at any stage of their development. The structural relationships seem to depend upon a median origin and connection of the optic stalks.

The fact that a cyclopean eye may have no well formed optic stalk and is entirely median in position not necessitating the


POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 277

absence of any other brain part is in entire accord with the above facts.

It is believed, therefore, that the various degrees of the typical Cyclopean condition from eyes unusually close together, to median double or hour-glass eyes, to the large oval eye, to a median round eye of usual size, to the median eye smaller than one normal eye, and finally to complete failure of eye material to arise from the brain are probably all due to developmental arrest. The arrest in development is the result of some influence which has reduced the developmental vigor below the normal so that the energy is not available to carry out the usual processes of differentiation and growth.

The author has figures and described other cases of eye defects which do not fall exactly into the series of cyclopia as considered above; yet these cases are modified conditions which are closely related to the Cyclopean series both in point of origin and in their final condition. The curtain-like eyes which face the median plane and often have a single lens between them might be considered as delayed cases of cyclopia. The eye anlage widened, the eyes became separated and continued their diff"erentiation, yet they were unable to turn out and assume their normal lateral positions so that they faced in a ventromedian dkection with their anterior walls closely approximated as is shown in figure 38 ('09) and figures 1, 4, 5, 6, 13, 14 ('10 a). Such eyes often excite a single stimulus upon a more or less ventral ectodermal region which responds by forming a single lens lying between the two eyes.

The writer has illustrated by a diagram (fig. 15, A and B '10 a,) the difference between these eyes with their choroid surfaces against the lateral ectoderm of the head and their concave retinal surfaces facing medially, and the eyes of a normal individual ('10 a p. 380). As was then stated, the experiments did not give a definite clue to indicate the position of the optic anlagen in the early brain, thus my explanation, or 'laming hypothesis,' referred mainly to the pushing out and lateral development of the optic vesicle and cup. All these cases are decidedly


278 CHARLES R. STOCKARD

of a type which would suggest lack of developmental energy necessary to attain the normal.

Another group of eye anomalies were extremely common under the same experimental conditions which caused the cyclopean series. These individuals possessed one normal eye in the usual lateral position while the eye of the opposite side in the numerous specimens showed various degrees of imperfection from a condition slightly below the normal in size to complete absence of the eye. Such anomalies were termed 'monophthalmica asymmetrica' in contrast to the symmetrical one-eyed monsters with a median cyclopean eye.i

The genesis of the asymmetrical defects is not entirely clear, yet they also are probably due to developmental arrest or suppression of the one eye. The growth centers representing the two future eyes of an individual are rarely equally vigorous and it is frequently noticed that one eye arises slightly before its mate and develops at a little faster rate. It might be that at some critical point in development one of the future eye centers is affected after the growth centers had begun to localize in more or less lateral positions.

In treating the eggs with alcohol a number of embryos occurred in which both eyes were small and defective even though they arose from the brain and attained more or less lateral positions. One might assume this to occur as a result of an arrest in development which affected both eye forming centers after the centers became separate or distinct from one another. Part of the eye forming material is suppressed or its differentiation is prevented so that each eye is decidedly under size and defective. In some of the cases of cyclopia mentioned above the eye was also very small and defective, in these cases the two growth centers which would give rise to the future lateral eyes did not become sufficiently separated so that only a single median eye arose and the reduced vigor permitted this to form only as a small and poorly differentiated structure.

In some instances where an embryo possessed only one member of the normal eye pair this eye was unable to attain its usual


POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 279

lateral position so that it faced the median plane, and if the lips or peripher}' of the optic cup failed to reach the ectoderm the eye was without a lens.

In none of the specimens, although many were old with the central nervous tissue highly differentiated, was I able to detect any material within the brain which might be considered as representing the missing eye.

Eye conditions such as these are doubtless due to the action of some inhibitory influence which prevents the complete origin and differentiation of eye material, or when it does arise allows it to develop onlj^ in a weakened or defective manner. Since monophthalmica asymmetrica occurs so persistently in the same experiments with cyclopean individuals as Lewis, McClendon and I have all found, it is not improbable that the cause is the same in the two cases.

The above considerations have been entirely from the standpoint that the ophthalmic defects under discussion originate during the medullary plate stage. The cause of cyclopia or the tendency to produce such a defect might of course have its origin much earlier in development. It might, in fact, go back to the germ cells themselves or finally it might possibly occur as an hereditary variation. All experimental cyclopia, however, furnishes evidence directly contrary to the latter possibilities. This point I have considered in a previous paper and have clearly demonstrated as have several other investigators that the condition is not due to a germinal variation but may be induced as the result of external stimuli applied during the early development of the eggs.

The cyclopean abnormality may be caused in Fundulus embryos by subjecting the eggs to various chemical stimuli after they have developed normally for as long as fifteen hours. A fifteen-hour Fundulus embryo has the germ ring beginning to form and descend over the yolk sphere, the embryonic shield is scarcely indicated but appears very soon afterwards. Embryos of later stages subjected to the same treatment develop normally, or do not show cyclopia, while stages younger than fifteen hours and


280 CHARLES R. STOCKARD

as early as the first cleavage are much more readily affected in such a manner as to cause the cyclopean defect. The optic vesicles appear at about thirty hours after fertilization, but the stimulus must be applied at a time sufficiently long before this process occurs, since a number of important steps in eye formation are doubtless taking place before the visible signs of optic vesicles are present.

The fact that cyclopia may be produced after the beginning of the germ ring and embryonic shield in the teleost embryo indicates directly that the defect may occur in what would be the medullary plate stage of amphibian embryos. Thus explanations of the cause of cyclopia must consider it as occurring in the medullary plate stage.

Spemann ('12 b, pp. 38-39) has taken exception to my statement regarding the non-occurrence of cyclopia when eggs are treated later than fifteen hours after fertilization (although the optic vesicles do not arise until about the thirtieth hour) "since insufficient time exists for the substances to act on the eye anlagen.^' The paragraph following this statement in my paper (p. 388, '10a) is : The solutions are effective up to a stage in development preceding the formation of the germ ring and embryonic shield, and the action of the Mg on the eye anlagen probably takes place while the embryonic shield and outline of the embryo are forming J^^

This statement seems to me perfectly direct and clear; Yet Spemann intimates in one sentence that I mean to infer that fifteen hours are necessary for the substances used to penetrate the egg membrane! He then states that I showed in 1907 that KCl would penetrate within a few minutes and stop the embryonic heart beat. The Mg and other solutions used may pass through the membrane equally as rapidly, this I have not fully tested. The fact that it does has no bearing on the fact that stimuli do not have sufficient time to act upon the optic anlagen to induce cyclopia when applied to the eggs at later periods in normal development than fifteen hours after fertilization since they must act on the anlagen in the embryonic shield, and to act later is

1 The last clause was not originally in italics.


POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 281

too late. The reason for the nonoccurrence of cyclopia when eggs were treated at periods later than fifteen hours after fertilization or fifteen hours before the appearance of the optic vesicles is that the optic anlagen and all other embryonic parts are constantly changing during development and have passed beyond the critical stage. It is not only a question of how quickly one may stimulate but in what condition the anlagen are at the time of stimulation. It is safe to say that cyclopia can not be produced after the optic anlagen have proceeded to some definite stage in their normal development. Even if Mg should penetrate all the membranes within a few seconds its action could never induce cyclopia in an embryo with the two optic vesicles visibly formed. There was no question of the time necessary for penetration, but an important question of the embryonic condition to be acted upon, the critical condition of the optic anlagen.

In the above connection it may be mentioned that such substances as alcohol and ether, when administered to an early embryo may cause it to develop into a decided monster. A late embryo or foetus may respond to the same treatment by producing an individual exhibiting no gross morphological defects yet showing decidedly abnormal nervous reactions, while a similar treatment might exert little or no effect upon a fully formed individual. A stimulus could not cause the mature individual to change into a structural monster. The developmental period of administration is of as high importance in determining the result as is the nature of the stimulus used, unless of course the stimulus be entirely destructive.

Again Spemann misinterprets a statement regarding the action of Mg. It was remarked that the cyclopean embryos developing in the Mg solutions were, except for the eye defects, more perfect than those arising from treatments with alcohol, ether, and so forth. The Mg cyclops often had apparently normal brains, could swim in normal fashion, took food and reacted to stimuli much as normal embryos did, while those embryos treated with alcohol and ether had various defects of the brain and cord. In this connection it was cited as of interest that Mayer had re


282 CHARLES E. STOCKARD

cently found in studying nerve muscle preparations that Mg salts seemed to prevent activity by affecting the muscle directly without apparently affecting the nerve. There is, of course, no direct connection between these facts and cyclopia; the mention was made merely in a general way, and most decidedly did not intend to convey the notion that muscle contractibility and the outpushing of the optic vesicles were phenomena of similar nature. They are similar only in that both are dynamic processes and require energy for their accomplishment.

There is no necessity for further discussing the fact that a number of eggs when subjected to the same solution do not all respond in a like manner (Spemann '12 b, p. 37)'. This is a typical case of differences in individual resistance and vigor which is observed among any one hundred individuals of any living species. It is equally true that the two sides of a so-called bilateral individual are rarely, if ever, identical.

Spemann is no doubt correct in stating that the relationship between cause and effect in my chemical experiments on cyclopia is not clear. Yet it seems to me that it is not entirely dark, the entire relationship between cause and effect in biological experiments is rarely if ever clear step for step.

I should like, however, to point out that the chemical experiments did one thing in proving that cyclopia could be caused from normal embryos through the action of the environment. This fact did away with all theories of germinal origin of the defect, one of which was strongly presented by Wilder about the same time. The experiments also make clear the stage in development at which cylopia may occur, and they further supply the richest amount of material yet available for the study of this defect. Finally, they prove to my mind that cyclopia is a developmental arrest and may be due to any cause which lowers developmental vigor at certain critical stages in the formation of the eye anlagen. These important points in the study of this defect have certainly not been made clear by the mechanical experiments though I do not deny that they might possibly have been.

To quote again from Spemann:


POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 283

Gegen diese Defekthypothese erhebt Stockard (1909b, p. 172) die Fragen: warum sollte bei den ]\Iagnesiumembryonen gerade das Gewebe zwischen den Augen ausf alien, und keine anderen Gewebe?; warum sind die Riechgruben bei Cyclopie manchmal verschmolzen und manchmal getrennt?; . . . . ist bei den asymmetrisch-einaugigen Missbildungen der einseitige Augendefekt etwa auf die Abwesenheit der einen ersten Augenanlage zuriickzufiihren? — ^Darauf mochte ich mit der Gegenfrage antworten : sind denn all diese Tatsachen verstandlicher bei Zugrundelegung der Stockard 'schen Lahmungshypothese?

I should answer now, as it was inferred above, that they most decidedly are, and an attempt to show this fact in some detail has been made in the preceding pages.

The title of the recent paper by Spemann Zur Entwicklung des Wirbeltierauges" might better have been '^The development of the vertebrate lens," as little or almost no attention is given to other parts of the eye. The problem of the lens formation is very fully considered.

"Cyclopia of the lens" is discussed from the same standpoint as cyclopia of the optic cups. In the case of the lens median ectodermal cells may be missing so that the two normally lateral lenses fuse in the median line. It seems to me that this is the one straw too much for the 'Defekthypothese.' The imagination which pictures the falling out of just the exact amount of median ectodermal tissue which would allow the primary lens forming cells of the ectoderm to fuse towards the center and keep proper pace with the movements and final position of the various cyclopean eyes which myFundulus material presents must be most vividly active. An even more plausible possibility out of this imaginary dilemma is to consider the lens anlage as a single median group of cells that divides into two parts which come later to lie in lateral positions. This view would at least have the advantage that in cyclopia of the eye cyclopia of the lens" would maintain the lens forming cells in a fairly median region, and in normal development the lens cells would have a more or less definite path to follow and place to reach. The pineal eye in many forms possesses a fairly definite lens which must have arisen medially and the present lens may have been somewhat more anterior yet also median. These suggestions are of the


284 CHARLES R. STOCKARD

most speculative nature and are intended merely as such. In my experiments many of the embryos which possess supernumerary lenses show, as Spemann has called attention to, that the accessory lens may actually lie more anterior than the eye. Nevertheless, others, show free lenses in lateral positions.

The presence of a lens in the cyclopean eye is explained by the fact, well established for several species, that an optic vesicle or cup possesses the power to stimulate lens formation from any region of the head ectoderm with which it comes in contact. There would be no necessity of imagining a condition of cyclopia of the lens" even though a median lens should be observed in an anophthalmous monster. Normal lateral lenses have been observed in anophthalmic monsters (see figs. 1 and 5, and fig. 3, plate II, 1910 b).

The embryo from which my figure 4 ('10 b), was taken is of new interest in connection with a hypothetical case called for by Spemann (p. 81 '12 a). He states as a possibility that

der cyclopische Defekt nur die Epidermis betrifft, so dass Riechgruben undprimare Linsenbildungszellen median zusammenriicken, wahrend die Augenbecher, wie normal seitlich gelegen, sich ihre Linsen aus der dortigen Epidermis bilden. Durch solche Falle wiirden in der Tat an einem und demselben Kopf beide Fahigkeiten demonstriert, die der Linse zur Selbstdifferenzierung und die des Auges zur Linsenerzeugung. Bis jetzt liegen aber derartige Falle nicht vor.

The case however, was, recorded at the time and seems to. fill the requirements set forth. The independent origin and differentiation of the lens is demonstrated in a median position slightly more anterior than the eyes, and the more or less lateral eyes have derived lenses from the ectoderm with which they came in contact as is shown by figs. 9 and 10, 1910 b, yet this ectoderm is part of the usual region from which an optic cup has the power to derive a lens. The power to form lenses, as Speman and I have claimed, is possessed by the ectoderm of the head.

According to Spemann's assumption, the embryo (fig. 4) presents true cyclopia of the lens." The condition, however, may better be interpreted as an illustration of the high lens-forming


POSITION OF OPTIC ANLAGE IN AMBLYSTOMA


285


capacity possessed by the ectoderm at the anterior tip of the head.

Lens-forming power seems to diminish from the anterior tip of the head backward until trunk ectoderm no longer possesses the capacity to form a lens, as Spemann found in transplantation experiments. For this reason independent lenses arise, as a rule, far anterior to and often in front of the more or less lateral eyes (my figs. 3, 4, 7, 9, 10, 11, 12, 21, etc. '10 b). In fewer cases independent lenses are found in more posterior lateral regions approximately in the normal lateral eye position (figs. 1 and 2



Fig. 9 Two diagrams indicating the primary lens-forming power of various portions of the head ectoderm. The lens-forming tendency is considered to be greatest at the anterior end and gradually decreases towards the trunk ectoderm until the ability to form a lens is lost where the trunk begins. The rows of circles indicate the magnitudes of lens-forming tendency in different regions and do not signify the size of the lenses. Posterior lenses may be as large as anterior ones, yet they occur less frequently as independent structures. Free lenses usually occur near the anterior tip.

and 3, plates I and II '10 b). Figure 9 may serve to illustrate diagrammatically the extent and gradation of the lens-forming power possessed by the head ectoderm. This rather diffuse localization of lens-forming cells in the general head ectoderm as demonstrated by numerous experiments seems sufficient to account for all phenomena of lens formation in cyclopia, as well as the supernumerary lenses which the writer has reported.

The median position in cyclopia of normally bilateral organs involves one other part. The nose or nasal pits in the cyclopean


THE AMERICAX JOURNAL OF ANATOMY, VOL. 15, NO. 3


286 CHARLES R. STOCKARD

fish are sometimes median and single, or occasionally bilateral and more or less normal in position. I might also add that the nasal pits are often absent. Again the 'defect hypothesis' must expect median cells to fail and so allow the two nasal pits to fuse medially. In this case the evidence is still stronger for the primary median origin of the ectodermal anlage. Dohrn's studies on Ammococtes have shown the median position and relationship of the nasal and hypophyseal invaginations. The question of monorhiny in the cyclostomes is not fully determined but evidence is certainly available to indicate a median nose anlage. These, however, are phylogenetic considerations which would only serve to prolong the present discussion.

The experimental results presented by Spemann relative to questions of lens formation agree almost entirely with the conclusions which were presented in my paper of 1910 b. He disagrees, however, with many of my interpretations, yet I believe the disagreement is not as complete as it often seems.

I had suggested that the power of the ectoderm to form a lens without the presence of an optic cup was less vigorous or efficient than when the optic cup combined its stimulus with the tendency to lens formation possessed by the ectoderm. For this reason when the ectoderm was injured by many of the mechanical operations which have been employed in the study of lens formation the injured ectoderm was unable to form independent lenses although normally it would have had such power. Attention was called to the different results of Lewis and Miss King on Rana palustris.

Spemann ('12 a, p. 49) rejects this idea although he produces evidence in his paper to prove its very probable correctness. Compare the results he obtained in the origin of independent lenses after cutting out the optic anlagen from medullary plates with glass needles, in which case the ectoderm in the primary lens-forming region was uninjured, with the results following a burning out of the eye anlagen with hot needles, in which case neighboring tissues were necessarily injured. After the latter operation only one well formed lens occurred in five cases. A


POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 287

further comparison between the glass needle operations on earl}^ stages with open medullary plates and later stages where the ectoderm was raised and the optic vesicles cut out from beneath it show a difference in the response of the ectoderm in producing free lenses in favor of the less injured or less disturbed ectoderm.

A few of the early lenses figured by Spemann are at least questionable. For example, figure 44 ('12 a) shows on the operated side an irregular thickening within a single cell layer. Such a thickening might readily have resulted from the disturbance to which the portion of ectoderm had been subjected. This specimen is cited as evidence that the accessory or supernumerary lenses in my experiments may have arisen as buds from a common origin. The possibility of early lenses constricting or budding into two or more is freely admitted. Figures 14, 17, 18 and 20 ('10 b) show constricted or double lenses. If the constriction had been carried further two lenses might have resulted. Yet the relative positions occupied by several other of the lenses figured are difficult to account for on the above basis.

Finally, there is no question of the fact that in numbers of the fish monsters which I have figured and described small and ill-formed eye vesicles or fragments are associated with large well-formed lenses. The fact of the constant association of such optic structures with lenses whenever the optic parts chance to lie near the ectoderm makes it practically certain that the defective e3'"es have stimulated the lenses to arise in these positions. The size of the lens in Fundulus is not regulated by the size of the optic cup. This is further proven* by the small lenses in large eyes and by large protruding lenses in rather well-formed but small eyes. The error in logic which Spemann ('12 a, p. 81) claims to exist on page 405 of my 1910 b paper in discussing these eye fragments I am unable to detect and several other embryologists have been unsuccessful in pointing it out.


288 CHARLES R. STOCKARD

SUMMARY

1 . Experiments in which certain regions are removed by mechanical operations from the medullary plate of Ambly stoma punctatum seem to show that the earliest optic anlage is median in position.

Thirty embryos from which lateral portions of the medullary plate and the anterior lateral part of the medullary fold were removed at slightly different stages gave in twenty-four cases, or in 80 per cent of the individuals, subsequent development of both eyes. In five individuals one eye was absent and in one specimen both eyes failed to arise. The absence of eyes in the latter cases was probably due to the cut having been made in a more median position than was intended.

Nine individuals were operated upon so as to remove narrow strips of cells from the anterior median portion of the medullary plate. Four of these cases, or about 45 per cent of the specimens, failed entirely to develop eyes. According to Spemann and others they should have given some degree of cyclopia. Four other individuals possessed highly defective Qyes, one embryo having one eye poorly formed while the other was questionably present. Only one of the nine specimens so operated upon was capable of developing both eyes to an extent approaching the normal.

2. When the cells in the anterior portion of the open medullary plate are disturbed by being stuck and scraped in various ways with steel needles they do not loose their power of giving rise to optic vesicles and cups which are normal in appearance during the early stages, later stages were not studied.

3. If the optic anlage be cut out of the medullary plate and reversed in position and then transplanted in the medullary plate it still retains the power to give rise to optic vesicles and cups which are abnormal in position to an extent depending upon the distance the anlagen were shifted by the operation.

The facts furnished by these experiments are considered in connection with recent views regarding the genesis of certain ophthalmic defects.


POSITION OF OPTIC ANLAGE IN AMBLYSTOMA 289

LITERATURE CITED

Adami, J. G. 1908 Principles of pathology, vol. 1, p. 241, Lea and Febiger, New York.

DoHRN, A. 1883 Studien zur urgeschichtc des Wirbelthierkorpers III. Die Entstehung und Bedeutung der Hypophysis dei Petromyzon planeri. Mitt. Zool. Stat. Neapel., Bd. 4, pp. 172-189.

King, II. D. 1905 Experimental studies on the eye of the frog. Archiv f. Entw.-¥ech., Bd. 19.

Lewis, VV. H. 1909 The experimental production of cyclopia in the fish embryo (Fundulus heteroclitus). Anat. Rec, vol. 3, pp. 175-181.

Mayer, A. G. 1909 Rhythmical pulsation in Scyphomedusae. Carnegie Institution Pub., no. 102, pp. 113-131.

McClendon, J. F. 1912 An attempt toward the physical chemistry of the production of one-eyed monstrosities. Am. Jour. Physiol., vol. 29, pp. 289-297.

Mencl, E. 1903 Ein Fall von beiderseitiger Augenlinsenausbildung wiirhrend der Abwesenheit von Augenblasen. Arch. f. Antw.-IMech., Bd. 16.

1908 Neue Tatsachen zur SelbstdifTerenzierung der Augenlinse. Arch, f. Entw.-Mech., Bd. 25.

Paolucci, L. 1874 Sopra una forma mostruosa della myliobatis noctula. Atti

della societa Italiana di Sc. Naturali, tom. 17, pp. 60-63. Spemann, H. 1912 a Zur Entwicklung des Wirbeltierauges. Zool. Jahrb., Bd.

32. Abt. f. allg. Zool. u. Physiol., pp. 1-98.

1912 b tJber die Entwicklung umgedrehter Hirnteile bei Amphibien embryonen. Zool. Jahrb., Suppl. 15. (Festschrift fiir J. W. Spengel,

Bd. 3) pp. 1-48. Stockard, C. R. 1907 The artificial production of a single median cyclopean

eye in the fish embryo bj means of sea-water solutions of magnesium

chloride. Arch. f. Entw.-Mech., Bd. 23.

1909 The development of artificially produced cyclopean fish, "The magnesium embryo." Jour. Exp. Zool., vol. 6, pp. 285-338.

1910 a The influence of alcohol and other anaesthetics on embryonic development. Am. Jour. Anat., vol. 10, pp. 369-392.

1910 b The independent origin and development of the crystalline lens. Am. Jour. Anat., vol. 10, pp. 393-423. Wilder, H. II. 1908 The morphology of cosmobia. Am. Jour. Anat., vol. 8, pp. 355-440.


THE BREEDING 'HABITS, MATURATION OF EGGS AND OVULATION OF THE ALBINO RAT

W. B. KIRKHAM and H. S. BURR

Sheffield Biological Laboratory, Yale University

EIGHTEEN FIGURES

INTRODUCTION

The present work was started by the senior author under the supervision of Professor W. R. Coe in the spring 1907, and in 1908 Professor Coe published in Science a brief statement of what had been found. In the summer of 1911 the junior member, Mr. Burr, took up the work. In the interim the literature of the subject had been enriched by three papers, and since then two additional ones have appeared. Lantz in 1910 contributed to a United States government report, on the economic importance of the rat, a short paper on the natural history of the animal. The author describes the different species of rats, their distribution, and general habits, but pays little attention to the details of their reproduction.

Sobotta and Burckhard ('10) made a careful study of the maturation and fertilization of the egg of the albino rat, and they describe and figure the ovarian egg in the stages of the first polar spindle, and first polar body with the second polar spindle, and the tube egg in the stages of the second polar spindle, fertilization, second polar body, and the pronuclei. Ovulation is stated to occur independent of pairing within thirty-six hours after the birth of a litter, and the eggs fertilized nine to twelve hours after copulation. Sobotta and Burckhard found the mature rat egg, in the ovary, to measure in preserved material 0.06 to 0.065 mm. in diameter; practically the same as the

291


292 W. B. KIKKHAM AND H. S. BURR

mouse egg. These investigators never saw a definite first polar body associated with an egg in the tube.

Newton Miller's paper ('11) on reproduction in the brown rat is based solely upon observations of the living animals. He found that both sexes become sexually mature at least by the end of the fourth month," that the litters contain from six to nineteen young apiece, and that these animals breed the year, round.

Mark and Long ('12) devote most of their contribution to an extended description of the elaborate warm chamber they have devised for the study of living mammalian eggs. When it comes to the results obtained with their apparatus they have, at present, little to say. Living eggs of rats and mice obtained in a manner similar to that described by one of us (Kirkham '07) were placed on the stage of the microscope in the warm chamber and spermatozoa added, the mouse eggs underwent no change, but the rat eggs within five minutes to two hours began the formation of the second polar cell. Cleavage has never been observed, and after twelve hours the eggs begin to degenerate.

The latest contribution to the literature on the subject of rat breeding is by Helen Dean King ('13) who records for the albino rat somewhat the same phenomena previously observed by Daniels ('10) in mice. The normal period of gestation for the albino rat, according to Miss King, is twenty-one to twentythree days. If six or more young are being carried while a previous litter of five or less are still suckling the period of gestation may be prolonged, while if more than six young are suckling the period is always prolonged, regardless of the number being carried. Unlike the mouse, the albino rat appears not to exhibit any exact relation between the number of young either suckling or borne and the extent of prolongation of the gestation period. This paper also contains evidence that the eggs of a given oestrus cycle in the albino rat may be discharged from the ovaries in two sets, with an interval of two to three days, and also that in very rare instances this interval may be extended to two weeks. Miss King would like to interpret the latter cases as instances of a distinct oestrus cycle occurring during pregnancy.


BREEDING HABITS OF ALBINO RAT 293

The present paper has as its object the filling in, as far as possible, of such stages as have not previously been described, and the presentation of evidence regarding the time relations in the development of individual eggs. The authors' thanks are due to Prof. W. R. Coe for the use of the notes and drawings of the eggs of the brown rat, and to Dr. T. B. Osborn of the Connecticut Agricultural Experiment Station for the designs of the cages used and for the animals with which the work was started.

BREEDING HABITS

About 150 albino rats were under observation at different times during the investigation. One' large cage was used for all rats not at the time under special care. For individuals two types of cages were employed, one, a cylindrical cage of wire netting of sufficient size to accommodate two rats at a time, and the other a much larger, rectangular cage of galvanized iron, with wire netting only on the front and bottom. This second type of cage was designed primarily as a breeding cage and was large enough to house a mother rat and a litter of the largest size until the latter were sexually mature.

The food of the animals consisted of oats, corn, wheat, sunflower seeds, and dog-biscuit, together with bits of lettuce, string beans, bread, and various kinds of cooked meat and fish.

All cages were kept as clean as possible, but except when absolutely necessary litters less than two weeks old were never disturbed. At the times when we were inspecting them the rats were encouraged to come out of their cages and run about the room, and to this faixdliarity with us as well as to the additional exercise thus secured we attribute much of our success in rearing large litters without their being maimed or eaten by the parents.

Usually females were isolated in breeding cages as soon as they were seen to be pregnant, but in the few instances when males were left with such females until several days after the birth of the litter no mortalitv occurred. This fact leads us to


294 W. B. KIRKHAM AND H. S. BURR

agree with Miller ('11) and King ('13) that mother rats, unless they are in an unhealthy condition, or have been frightened in some way, rarely if ever kill or maim their young.

Albino rats give birth to young in all seasons of the year, but it is only from April to October that ovulation as a rule occurs within 48 hours after parturition; during the remaining months they are apt to skip oestrus cycles, ovulation not occurring until some three weeks after parturition.

The senior author showed in 1910 that the albino rat o\tilates regardless of whether pairing has previouslv taken place, and when males are continuously present copulation may occur before the ripest eggs in the ovaries have formed the first polar spindles. On several different occasions we have seen the actual pairing. It differs markedly from the condition described by Sobotta ('95) for the mouse, since the male albino rat is not prostrated by the sexual act, but walks slowly away. When a previously isolated female who is in heat is placed in a cage wdth several males they will all pair with her in rapid succession.

The period of gestation in the albino rat is twenty-two days when the female is not nursing a previous litter, in which event the period may be lengthened as found by King ('13). The litters varied in number from four to twelve and the birth usually took place in the late afternoon or the early evening, although probably it may occur at any hour of the day, since we have observed it at noon. The process of parturition is briefly as follows : The female in order to aid in the expulsion of the foetus flattens herself against the bottom of the cage while a series of wave-like muscular movements pass posteriorly along the body starting just behind the shoulder. As soon as the young rat is free from her body, the female rises up on her haunches, seizes in her forepaws the button-like placenta? which is still attached to the offspring by the umbilical cord, and devours first it and then the cord, cutting off the latter as close to the body of the young animal as she can get with her teeth. The female then again flattens herself out against the bottom of the cage preparatory to the appearance of the next young rat. The process is repeated until all have been brought forth. Then, and not


BREEDING HABITS OF ALBINO RAT 295

before, does the mother assemble the young, cleaning them up with her tongue, after which they lie close together under her to keep warm. From this time on until the young are able to crawl around by themselves the mother never leaves the nest until she has carefully covered her litter. On returning she always looks around for any that may have rolled or crawled out in her absence, and such offenders are quickly seized in her jaws and hauled back into the nest.

The albino rat becomes sexually mature, at least in some cases, as early as fifty-five days after birth, since in one instance a litter was born to rats that were only seventy-seven days old.

MATURATION AND OVULATION

The paper by Sobotta and Burckhard ('10) on the maturation and fertilization of the albino rat is by far the most complete account of the subject that has so far been published. However this report left a number of things to be cleared up.

Working with material from 81 rats we have attempted to investigate and make clear the following points: (1) the early development of the egg previous to the formation of the first polar spindle, (2) the formation of the first polar body, (3) the condition of the egg at o\nilation, (4) the process of fertilization and second polar body formation.

At first the rats were watched and killed at short intervals up to forty-eight hours after pairing. This gave no data that could be depended upon for determining the stage which either the ovarian or the tube eggs had reached. However, by relating the time of killing the female to the time of parturition it was found that tiie approximate development of the egg could be predicted without much difficulty. We say approximate because even though the exact hour of parturition be known it is impossible to say that at a given interval of time the eggs are in a given stage of development.

The parturition of a female caged with a male having been observed, she was killed twenty-four hours later. This female yielded unfertilized tube eggs, indicating that ovulation had re


296 W. B. KIRKHAM AND H. S. BURR

centh^ occurred. Cases such as this show that ovulation usually occurs about twenty-four hours after parturition. The individual variation is so great that any complicated apparatus for determining the exact date of parturition is valueless. We have obtained the best results by killing the females at half hour intervals, beginning in the later afternoon and continuing through the early evening. By doing this practically all the stages of maturation can be obtained.

In a number of instances the senior author dissected out the Fallopian tubes, and after placing them in warm salt solution, by slitting the tubes he was enabled to obtain two eggs fertilized but unsegmented, three eggs in the two cell stage and three eggs so obscured by follicle cells as to prevent any exact information as to their condition. The technique of this operation is so simple, requiring only a binocular microscope, two needles, some warm physiological salt solution, and a female rat that has given birth to a litter at least twenty-four hours before and not more than five days previously, that we recommend the rat as highly as the mouse for obtaining live mammalian eggs for class demonstration.

In all other instances the ovaries (and also the tubes, wherever ovulation was thought to have occurred) were fixed in either Zenker's 'fluid or in a strong solution of Flemming, imbedded in paraffin, cut serially into sections 0.010 mm, thick and stained in Delafield's haematoxylin. Such sections as were found on subsequent examination to be worthy of detailed study were later decolorized with acid alcohol and restained with Heidenhain's iron-haematoxylin.

A study of the ovaries of the above rats showed that there is a progressive development of the egg until it is ready to leave the ovary at ovulation. The developing eggs of any adult ovary can be readily divided into six groups. The first of these (fig. 1) includes all those eggs that are in the resting condition. These vary considerably in size, as do also their follicles. The earlier stages show a small egg with a follicle consisting of from one to three layers of radially arranged follicle cells with scattered cells lying between the layers, the later stages lie in larger follicles


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with many more layers of cells. The egg nucleus presents a constant appearance, a clearly defined nuclear membrane, scattered chromatin and a deeply staining nucleolus.

The second group includes those eggs which differ from those of Group I only in their size and in the fact that they lie in much larger follicles, the latter consisting of a large number of cells closely packed but showing no radial arrangement except in the layer immediately surrounding the egg. Such an egg is shown in figure 7.

The third group includes a much smaller number of eggs which lie in follicles similar to the preceding, except that the cells lying



Fig. 1 Normal resting follicle. X 630


in or near the center of the follicle show a marked tendency to separate, leaving a clear space. This condition may, however, be found in follicles belonging to eggs of Group II, for the factors governing the growth of the follicle are not, according to our observation, constant, since growth may set in when the egg has reached the stage of development included in either Groups II, III or IV (figs. 2, 3 and 4). The nuclei of the eggs of this third group show a marked change. The nuclear membrane is still distinct, but the chromatin is less scattered and the nucleolus has become partially vacuolated, since it shows much less affinity for the stain. Figure 8 shows an egg of this group.

The fourth group shows further modifications. It is at this point that the maximum growth in the size of the follicle takes


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Fig. 2 Earliest observed maturation phenomena — increase in size of follicle. X 90.

Fig. 3 Follicle of egg with first polar spindle shown in figure 10 of Plate II. X90.

Fig. 4 Follicle of egg with first polar body and second polar spindle shown in figure 11 of Plate III. X 90.


BREEDING HABITS OF ALBINO RAT 299

place. While growth may have started in either of the two preceding groups, the greatest growth occurs with the egg in this stage of development. Eggs of this group have been observed with follicles similar to those of the two preceding groups, and also with follicles of nearly the maximum size. The nuclei of these eggs show a diminution in the amount of chromatin present and a complete vacuolization of the nucleoli, the latter showing no affinity whatever for the stain. Such an egg is shown in figure 9.

The fifth group consists of the eggs with first polar spindles. The follicles here are typical, showing a slight tendency to be thinner in the region where the follicle is nearest to the surface of the ovary. The nucleus of the egg has disappeared, and in its place lies the first polar spindle (fig. 10).

The sixth group shows no change in the size of the follicle. The first polar body has been extruded and a second polar spindle formed (fig. 11).

In all the above divisions, with the exception of the sixth group, wherever a distinct zona radiata can be seen, very fine protoplasmic bridges can readily be distinguished crossing froni the follicle cells to the egg. The presence of these very distinct filamentous processes of the follicle cells seems to have been entirely overlooked by previous investigators.

One striking thing is to be noted with regard to the above divisions — never were all six found together in one ovary at a given time. As was to be expected. Group I, since it included all the resting eggs, was present in all ovaries. Group II, on the other hand, was seen to drop out on the appearance of Group

IV and to reappear on the disappearance of the latter. Group

V also appeared on the disappearance of Group IV. When Group V dropped out, Group VI appeared. Group III was found in all ovaries.

From the fact that perfectly normal eggs of Gr