Talk:Paper - The corpus luteum in the ovary of the chicken (1918)
Sox studies. X. THE CORPUS LUTEUM IN THE OVARY OF THE DOMESTIC FOWL
RAYMOND PEARL AND ALICE M. BORING
SIX TEXT FIGURES AND NINE PLATES
I. INTRODUCTION
The corpus luteum is one of the clearly recognized sources of an internal secretion in the mammal. Various functions have been ascribed to it. Its function in connection with secondary sex characters has been discussed by Pearl and Surface ('15), with one piece of clear cut evidence. The case was that of a cow which developed cystic ovaries and took on male secondary sex characters. The ovaries were compared histologically with those of a normal cow and the two were found to resemble each other in all respects except that the cystic ovaries had no corpora lutea. The interstitial cells were the same in both so that the difference in secondary sex characters could not be attributed to them. The implication of the facts is that the corpus luteum has an inhibitory influence in the female which prevents maleness from developing and that when no corpus luteum is formed, male characters appear.
The chief difficulty with such a view has been that its application is very limited, as the corpus luteum has been supposed to be a structure occurring only among mammals. The secondary sex characters of birds are particularly pronounced and the results of ovariotomy experiments, such as those of Goodale, ('16) show the possibility of changing these characters experimentally. Also the many cases of hermaphrodite birds (to be
' Papers from the Biological Laboratory of the Maine Agricultural Experiment Station, No. 115.
1
THE AMERICAN JOURNAL OF ANATOMY, VOL. 23, NO. 1 JANUARY, 1918
2 RAYMOND PEARL AND ALICE M. BORING
considered in Study XI of this series), with varying degrees of maleness and femaleness indicate the presence of some sex regulating substance in birds. Is this substance entirely different from the corpus luteum probably connected with it in mammals, or is there a corpus luteum or its homologue in birds? An investigation of this question has been undertaken in this study. We consider that we have successfully demonstrated the presence of the corpus luteum in the domestic chicken. Further discussion of the bearing of this fact on the whole question of secondary sex characters will be deferred until a later paper of this series, which will unfortunately probably be delayed for some time, as one of the authors (R. P.) has been called upon by the government to turn his attention to practical problems during the war.
A careful examination of the ovary of a bird which has been actively laying shows three kinds of structures: the yolks of various .sizes indicating different stages of development, the discharged follicles in various stages of regression, and the atretic follicles or degenerating eggs of different sizes. These are all easy to identify when they are large enough to protrude far from the surface of the ovary, that is, when they are larger than 2 or 3 mm. in diameter. Under this size, it is impossible to distinguish the discharged follicle from the atretic. Both of them show a yellow or orange spot in the center. The question naturally arises whether these yellow spots are homologous in structure and origin with the mammalian corpus luteum. They never develop into a large mass like the corpus luteum of the mammal. They have the color of the spots on the cow ovary which indicate remains of old corpora lutea. In order to interpret these yellow spots, a study has been undertaken of the progressive and regressive changes in the cell structure of egg follicles in different conditions, undischarged, discharged and atretic.
The material used came chiefly from four birds, an actively laying Bantam, a Barred Plymouth Rock in the same condition, an old Compine past the laying condition, and a guinea-hen with a large ovary containing several large yolks. Material
CORPUS LUTEUM IN OVARY OF THE CHICKEN 6
from a number of other Ijirds was used in the study of special points. These are some of the same birds used in Study IX. The ovaries were fixed in Gilson and McC'lendon. In the Barred Plymouth Rock ovary the different discharged follicles were sectioned separately and arranged in a series, according to size and consequent order of age since ovulation. After the study of this series, it was easy to judge of the condition of various follicles in pieces of the other ovaries cut at random. Various stains were tried, iron haematoxylin and Delafield's haematoxyhn for general histology and Mallory's and Mann's stains for secretion granule tests.
II. UNDISCHARGED FOLLICLES OF THE HEN'S OVARY
A study of the follicles of large undischarged oocytes shows them to consist of an epithelial layer, the granulosa, and two connective tissue layers, the inner and the outer theca folliculi (fig. 1). In the inner theca are located groups or nests of epithehal cells {I, figs. 1 and 2). They have been described by many authors, notably Ganfini, Sonnenbrodt and Poll, but have been called interstitial cells. Poll calls them Kornzellen at first, describes their collection into the internal theca and then implies their function by saying that the biological role of the theca interna in the formation of the corpus luteum still needs to be worked out. That he also confuses them with interstitial cells is shown by his statement that the theca interna fills up the atretic follicle with groups of Kornzellen, which is the same thing as an interstitial gland. These nests of cells in the bird are not anything like the usual glandular interstitial cells of the ovary in structure. They are about three times as large (compare fig. A and C). The nucleus is bigger and plumper, the cytoplasm is usually clear and vacuolated in appearance, only occasionally containing a few acidophile granules which stain with the fuchsin in Mallory's stain or the eosin in Mann's stain; while the real interstitial cells are crowded with granules. These large clear cells are seldom found alone, but are usually grouped into nests of various shapes, as already mentioned. The cytoplasm of these cells usually will not take
4 RAYMOND PEARL AND ALICE M. BORING
up an acid stain. They remain strikingly clear, when the connective tissue all around them is highly colored. So great is the contrast that they show distinctly even at low magnification in a section such as figure 1. Furthermore, they are found in
Fig. A Part of follicle of wall of medium sized oocyte in hen ovary. (X i)50.) Compare figure 2.
Fig. B Part of thoca interna of sixth discharged follicle in hen ovary, showing many vacuolated lutear cells. (X 9.50.) Compare figure 0.
CORPUS LUTEUM IN OVARY OF THE CHICKEN 5
different parts of the ovary, mostly in the thcca interna, while the interstitial cells he in the general stroma, and especially on the periphery.
Figure 3 shows several very young oocytes from the same ovary as figure 1. In these, the follicle consists only of a single layer of epithelial or granulosa cells (g). The connective tissue layers are not yet formed. But there are nests of clear cells (I) in the stroma nearby. Presumably these are included with the connective tissue when the theca interna is formed.
III. DISCHARGED FOLLICLES OF THE HEN'S OVARY
In the largest follicles before ovulation, the three layers are stretched out very thin by the pressure of the large yolk within them. After o^oilation, there is a shrinkage of the follicle walls, probably due to the elasticity of the connective tissue recoiling at the sudden release of pressure from inside. On the Barred Plymouth Rock ovary, the ripe yolk measured about 40 mm. in diameter, and the last discharged follicle measured 20 mm. in length from base to tip, while the next to last was 12 mm., and the fourth in the series was 7 mm. As this shrinkage in length takes place, the walls thicken until finally a small oval mass results having no resemblance to a hollow follicle. The ruptured place through which ovulation took place, becomes gradually closed up, by the growing together of the edges, and the filling of cells into the cavity. Sometimes this mass of cells proti-udes shghtly from the cavity at the old place of rupture, thus somewhat more resembling a miniature mammalian corpus luteum. Yellow pigment forms in the puckered edges of the follicle and also in the central mass.
The microscopic study of sections through discharged follicles of various ages shows that the increase of thickness of walls is due chiefly to a thickening of the theca interna. Figure 4 is a section of the last discharged follicle of the Barred Plymouth Rock ovary. It shows the thickened theca interna (i) and in addition the remnants of the granulosa (g). The latter seems to loosen from the follicle after ovulation, and the cells collect in masses in the cavity and degenerate.
6 RAYMOND PEARL AND ALICE M. BORING
The first subsequent discharged folHcle in the series to show any new microscopic features is the sixth (fig. 5), where there appears a marked increase in the number of nests of vacuolated cells in the theca interna (l). They are concentrated toward the cavity. The closeness of nests together may be partlj^ due to the shrinkage of the cavity after discharge of the egg. But as this does not seem sufficient to account entirely for the increase, the number must be added to either by di\dsion or migration. The fact that division plays some part in the process is proven by the observation of several mitotic spindles. The character of these cells shows better in greater magnification, as in figure 6 and figure B.
The further progress of the increase of vacuolated cells in the theca interna is shown in figure 7, a section of a discharged follicle too small to have been placed in the series as to time of discharge. Here the whole internal theca looks full of holes, due to vacuolated cells (I). The central ca\dty is nearly obliterated, almost as though the edges had been pulled up by a gathering string. There are, however, a few cells in the central cavity (p). These get in there by migi-ation from the internal theca.
Figure 13 shows the process in an atretic follicle where it is more conspicuous, but it is true to a more limited extent in the discharged follicles. The cells concerned have a speckled appearance in figiue 13 (d). They are abundant in the follicle wall, some are scattered among the yolk spheres in the central cavity and some are on the border line between the follicle wall and the cavity, indicating that the cells actually migrate into the cavity. Occasionally a very large central plug is formed which protrudes from the spot of rupture. Figure 7 shows a small plug of this kind (p).
The cavity usually becomes finally obhterated by the thickening of the internal theca and the formation of large masses of vacuolated cells from the original nests. In figure 8, the chief tissue consists of the masses in the internal theca (i). The line between the theca interna and externa is marked by the irregular spaces and blood vessels. The connective tissue in the
CORPUS LUTEUM IN OVARY OF THE CHICKEN 7
center (c) shows where the edges of the internal theca have drawn together and obhterated the cavity.
We have traced thus far the general histological changes involved in the shrinking and lilling up of the discharged follicle. We must consider next in more detail, the cytology of these particular cells involved. Figure 2 and figure A show them in their original condition from a large undischarged follicle. We have earlier in this paper pointed out their especial characteristics in distinction to the interstitial cells. By the time they are close enough together to cause the vacuolated appearance of the whole inner part of the theca interna, the nuclei are somewhat shrunken and pushed to the side of the cell, suggesting active elaboration of secretion material (fig. 6 and fig. B). By the time the closing in of the follicle has neared completion (figs. 8 and 9), the character of the cells is decidedly modified (fig. C). The cell boundaries in any one small mass of cells are indistinguishable. The cells seem to have melted together so that the outlines of the vacuoles are the evidently visible lines rather than the cell outlines. The vacuoles also are much larger than previously. The nuclei are smaller and less regular in outline, they stain darker, in fact, they look shrunken. These figures show nicely the contrast between the cells which fill up this discharged follicle and the interstitial cells. The interstitial cells lie in the connective tissue of the external theca and of the internal theca in between the masses of transformed epithelial nest cells. They are entirely unchanged from their usual appearance. They show clearly because the granules with which they are packed stain vividly with acid stains. A homologous mass of cells from an older solidly filled follicle (fig. 10) is shown in figure 11 and figure D. Here the nuclei show still further signs of degeneration and the general network of the cytoplasm contains clumps that look like secretion material. These secretion particles are yellow in color. They look amorphous in character, and they vary greatly in size (fig. 20). They can not be fatty, for they have not dissolved in the clearing oils. They cannot be of the protein nature of the secretion granules of the interstitial cells, as they retain their distinct yellow color
8
EAYMOND PEARL AND ALICE M. BORING
no matter how the preparation may be stained. They make a fine contrast with iron haematoxyhn, acid fuchsin, eosin, methyl blue, and still show their own characteristic yellow even with
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Fig. C Masses of lutear cells from older discharged follicle, with interstitial cells lying in connective tissue between masses. (X 950.) Compare figure 9.
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Fig. D Mass of lutear cells from discharged hen follicle, with pigment particles developed in the network. (X 950.) Compare figure 11 and figure 20.
CORPUS LUTEUM IN OVARY OF THE CHICKEN 9
orange (1. The cell masses finally become nearly filled with this yellow material, some of it collecting in clumps several times larger than the degenerated nuclei.
Further tests of the character of the cell contents in these cell masses were made with Sudan III. Hand sections were made of material in McClendon's fluid. Although these could not be cut very thin, they showed that the inner lining of the early discharged follicles contains fatty material. In an old follicle with central yellow mass the cells of the yellow mass take the red of the Sudan III, but the yellow amorphous particles show in the mid^t of the red. They can be squeezed out of broken cells and isolated from the red fatty background, showing they are still yellow, unaffected by the Sudan III, and therefore not of a fatty nature. The fatty substance indicated by the Sudan III reaction in both young and old folhcles is probably contained in the vacuoles so conspicuous in paraffin sections. The xylol would have dissolved out all the fat lea\dng the vacuoles in which it had been contained.
IV. DEGENERATION OF CORPUS LUTEUM IN COW OVARY
In order to show the significance of the yellow mass formed in the center of discharged follicles in the hen ovary, we have made a brief study of the degeneration of the corpus luteum in the cow ovary for comparison. There is an extensive literature on mammalian corpus luteum, but this deals chiefly ^vith the development and early involution. Now the bird quite evidently has no structure similar to the large corpus luteum which fills up half the ovary of a cow at its full development. The small yellow spot on the bird ovary resembles the small yellow spots on the cow ovary which mark the old remains of former corpora lutea. Ovulation in the cow alternates between the two ovaries. So by studying the two largest corpora lutea on both ovaries we can arrange a series of four involution stages. Beyond that, they all seem equally shrunken and therefore can not be arranged in a further series. Such a series of four involution stages has been studied for two cows, and in addition several older corpus luteum remains.
10
RAYMOND PEARL AND ALICE M. BORING
The last formed corpus luteum is of a salmon pink color, due to a combination of the blood color and the lutein color. Sections show it composed of large plump cells with rounded nuclei, as described by Corner. These luteum cells are scattered in the midst of an areolar connective tissue groundwork (fig. 18 and fig. E). In dehydrating for embedding, the absolute alcohol and xylol become very yellow, indicating that the cells contain something soluble in these reagents. This is of course one chemical character of lutein.
Fig. E Cells from youngest corpus luteum of cow. (X 950.) Compare figure 18.
Fig. F Cells from older corpus luteum of cow, showing pigment developed in cells. (X 950.) Compare figure 19 and figure 21.
The next to last corpus luteum is much reduced in size. Its color has lost the pinkish shade and it appears a solid bright yellow. This is also soluble in absolute alcohol and xylol as in the first stage. The cells and nuclei both look a little shrunken. In one cow, this second corpus luteum contained a few amorphous yellow particles like those described foi the hen.
CORPUS LUTEUM IN OVARY OF THE CHICKEN 11
111 the third oldest corpus luteum, the tissue is shrunken so that a mere speck shows on the surface. This is the stage resembhng the yellow spots of the hen's ovary. Dissection shows that it is reduced in all diameters. That part of this decrease in size is due to cell shrinkage is well demonstrated by comparison of figures E and F, which are drawn to the same scale. Not only the nucleus but the cell body is at least halved in size. The color now is darker, being a brick red. This is not due to blood vessels, as sections do not show any more than formerly. It is due to the development of a dark yellow pigment, the same substance which appeared in small quantity in the younger corpus luteum and in large quantity in the hen ovary. In this stage of involution it is developed in large quantities, practically fiUing up many of the lutear cells (figs. 19 and 21 and fig. F). In unstained sections it gives a yellow color to most of the section.
In the fourth oldest corpus luteum of the two series and in the scattered older ones sectioned, the structure is similar to that in the third oldest, the yellow amorphous masses being possibly larger and more distinct.
This yellow material certainly looks the same as that in the hen ovary. The chief structural difference is that it is all confined within cells with distinct cell walls in the cow, while in the bird, the cells forming it, lose their boundaries and the particles are formed in a vacuolated network with scattered shrunken nuclei (cf. figs. 20 and 21).
Sudan III reacts similarly with hand sections of formalin material from both cow and hen ovary. All four stages in the cow series take the red color showing the presence of a fatty substance in the cell. This corroborates the evidence from the solvent action of absolute alcohol and xylol. But in the third and fom-th stages, yellow amorphous pigment particles can be seen glistening in the red background. The pigment is not of fatty nature in the cow, any more than it is in the hen. In fact, this substance is so similar in the two animals, that we shall from now on speak of a corpus luteum in the hen, and call the cells forming this pigment, lutear cells.
12 RAYMOND PEARL AND ALICE M. BORING
This development of a non-fatty pigment in the mammalian lutear cells has been already described by Mulon as occuiTing in atretic follicles. He speaks of the lipocholesterine as changing over to an indelible pigment. This same substance certainly forms in the involution of the corpus luteum of a discharged follicle as shown in this present work.
It is of especial interest to find that Blair Bell's description of the corpus luteum in Ornithorhynchus, a primitive oviparous mammal, shows it very much like that in the hen. It often remains hollow, it never becomes very large. It consists chiefly of a thickened theca interna. Sometimes it becomes a solid fibrous mass. One of Bell's figures almost exactly resembles figure 4 of this paper. One would like to know whether the yellow pigment is found in Ornithorhynchus thus making its resemblance to the bird even more striking.
V. BIOCHEMICAL CHARACTER OF PIGAIEx\T OF CORPUS LUTEUM
The identity of this yellow amorphous pigment in the corpus luteum remains in the ovary of the hen and of the cow has been put to chemical tests as well as morphological; first of a microchemical nature, as already partially described, and secondly by various special chemical solvents. The work of Escher and of Palmer and Eckles on animal pigments has been consulted in selecting the reagents to use.
The microchemical tests have been discussed in previous sections, but will be summarized here. Microscopical technique processes have shown the identical beha\'ior of the pigment in hen and cow. It does not dissolve in alcohol or oils. It will not stain with basic nuclear stains such as haematoxylin and Kresylviolet, or with acid counterstains, such as eosin, methyl blue, anihn blue, orange G, or with such a stain as iron haematoxyhn. Neither does it stain with the fat stain, Sudan III, although there may be much fatty material in the cell in which it lies. As normal secretion granules of a protein nature take acid stains and secretion granules of a fatty nature take Sudan III, this pigment is neither protein nor fat in composition.
CORPUS LUTEUM IN OVARY OF THE CHICKEN 13
A further test of its chemical nature was made by ti-ying some of the various solvents used by Escher and by Palmer. Sections were cut in paraffine and mounted on slides and then the paraffine removed b}^ xylol and the sections treated with different chemicals. This pigment is not the carotin described by Palmer, but we could not reach any conclusion as to its chemical nature, as nothing could be found to dissolve it. But the fact of the identity of this pigment in the hen and cow is proven beyond a doubt. Concentrated HCl, HNO3 and H2SO4 were tried and had no effect except that the H2SO4 turned the particles dark brown and made them even more distinct than before. For an alkali solvent, strong KOH was used; it turned the pigment bright orange but did not ehssolve it. In adeUtion to these various other solvents were tried after consultation with the chemistry department, petroleum ether, sulphuric ether, acetone, carbon bisulphide, and carbon tetrachloride, but none of these had the slightest solvent effect on the pigment. Acetone cleared the background and this made the particles stand out more sharply. Carbon bilsulphide was allowed to act for several hours, but the preparations still contained the pigment at the enei of that time in undiminished degree. We conclude that any two substances which can withstand the action of as many well known solvents of as many different properties as this list includes must be of very similar chemical nature. This gives us one more proof that the yellow particles in the hen ovary are the same as those in involuted mammalian corpora lute a.
VI. CHANGES IX ATRETIC FOLLICLES IN THE HEN'S OVARY
Among the developing yolks and discharged follicles of the hen ovary are many degenerating eggs. They can be distinguish d from developing eggs by the shrunken appearance as though the contents did not ejuite fill out the foUicle. Eggs may start to degenerate at different stages. Tbe largest one on the Barred Plymouth Rock ovary was 12 mm. in diameter. Many of them show dark spots which are masses of coagulated blood. Mostly they are smaller than this when involution begins. The
14 RAYMOND PEARL AND ALICE M. BORING
degree of shrinkage shows whether the involution process had recently begun or not. When these degenerating eggs are cut open, the contents is found to be in a more or less fluid state. When these atretic follicles have become reduced in size to 2 or 3 mm., it is no longer possible to distinguish them externally from the discharged follicles; the same kind of a yellow pigment appears in the center.
Studied microscopically, the chief difference between atretic and discharged follicles is that the former have a more distinct cavity which becomes obliterated chiefly by migration of lutear cells into it instead of by shrinkage of the walls. The granulosa is shed similarly. There must frequently be hemorrhage as corpuscles are often found ia the cavity. The varying quantity of yolk spheres is one indication of the degree of involution, also the number of lutear cells in the cavity. Figure 12 is an atretic follicle with considerable yolk still unabsorbed. A few lutear cells have filled in to the cavity (fig. 13, 1). It is particularly clear here that the cells inside of the inner mar^n of the theca interna are the same in structure as those of epithelial nature in the interna theca. This is just as Benthin describes it for the atretic mammalian follicles. Figures 14 and
15 show a later stage where the yolk is almost all absorbed and the ca\dty is filled with lutear cells.
Not until the cavity is filled with lutear cells does the yellow pigment already described in discharged follicles, make its appearance. It forms in the lutear cells of atretic follicles in a similar way to that in the discharged follicle. The cell boundaries are possibly not obliterated so completely, so that the morphological resemblance to the cow corpus luteum remains is even more striking than in the case of the discharged follicles. Figure 16 is part of an atretic follicle where the cells are filled with pigment. The amorphous character of this matedal shows in figure 17 a part of figure 16 under higher magnification.
It is of interest to notice that the lutear cells in the hen in both discharged and atretic follicles originate entirely from the theca interna. In mammals the origin of the lutear cells is a mooted question. Some authors, as Niskoubina, hold that they
CORPUS LUTEUM IN OVARY OF THE CHICKEN 15
have a double origin, from granulosa and theca interna, while others such as B(>nthin and Hegar, claim that they all come from the theca interna. This point is perfectly clear in birds due to the ease with which one can distinguish these peculiar cells in the internal theca of undischarged follicles and follow them to the thickened mass in the center of the discharged follicles, and see them migrating out into the cavity of the atretic follicles.
The formation of a corpus luteum in atretic as well as discharged follicles makes it possible to identify ovarian tissue in ovaries too abnormal to have ovulated any eggs. Most of the literature of the mammalian ovary considers the involution of the atretic follicle as something distinct from that of the discharged follicle. The mass forming in the atretic follicle is called the corpus atreticum or fibrosum in contradistinction to the corpus luteum. However, Hegar says that it is hard to tell one from the other. They are practically identical in the hen,
VII. SUMMARY
We are now in a position to sum up the points proving the homology of the corpus luteum in the hen and in the cow. There has been much discussion about the origin of the corpus luteum in mammals. In the hen there is no question but that the origin is simply from the theca interna.
The course of development in the hen corpus luteum is an abbreviation or fore-shortening of that in the cow. It corresponds directly to the late involution stages of the cow corpus luteum. They both contain a yellow fatty substance, as shown by the Sudan III, absolute alcohol and xylol reactions. There develops in both a yellow amorphous pigment in the cells containing the fatty substance. This pigment is similar chemically in that it will not stain with basic or acid stains; also in that it will not dissolve in any of the usual solvents, acid alkali or oil.
In the hen, a corpus luteum forms m both discharged and atretic follicles. «
16 RAYMOND PEARL AND ALICE M. BORING
VIII. LITERATURE CITED
Bell, W. Blaik 1917 The sex complex. Wood and Company, New York. Bknthin, W. 1910 Ueber Follikelatresie in kindlichen Ovarien. Arch. f.
Gyniikologie, Bd. 91, p. 2.
1911 Ueber Follikelatresie in Siiugetier Ovarien. Arch. f. Gynakol ogie, Bd. 94, p. 599. BouiNT ET Ancel 1912 Sur la nature lipoidienne, d'une substance active se cretee par le corps jaune des mammifere. C. R., T. 151, p. 1391. Corner, G. W. 1915 Corpus luteum of pregnancy as it is in swine. Carnegie
Inst. Washington, 222, p. 69. DuBAissoN, H. 1906 Contribution a I'etude du vitellus. Arch, de Zool. Exp.
et Gen. T. 4. Series 5, p. 153. EscHER, H. H. 1913 Ueber den Farbstoff des Corpus Luteum. Zeitschr.
Physiol. Chem., Bd. 83, p. 198. Fraenkel, L. 1910 Neue Experimente zur Function des Corpus Luteum.
Arch. f. Gynakologie, T. 91, p. 705. Ganfini, C. 1908 Sulla strutturo e sviluppo delle cellule interstiziali dell'
ovajo. Arch, di Anat. e di Emb., S. 7, p. 373. GooDALE, H. D. 1916 Gonadectomjr in relation to the secondary sex characters of some domestic birds. Carnegie Inst. Washington, 243. Hegar, K. 1910 Studien zur Histogenese des Corpus luteum und seiner Riick bildungsproducte. Arch. f. Gynakologie, Bd. 91, p. 530. Henneguy, L. F. 1894 Recherches sur I'atresie des foUicules de Graaf chez
les mammiferes et quelques autres vertebres. Jour, de I'Anat. et
Phys., T. 30, p. 1. Miller, J. W. 1910 Die Riickbildung des Corpus luteum. Arch. f. Gynakologie, Bd. 91, p. 263. MuLON AND Jong 1913 Corps jaunes atresiques de la femme. Leur pigmentation. C. R. Soc. Biol., T. 74. NiSKOUBiNA 1909 Recherches sur la morphologie et la fonction du corps
jaune de la grossesse. Dissert, de la facultc de med. de Nancy. Palmer, L. S. and Eckles, C. H. 1914 Carotin. The principal natural yellow
pigment of milk fat. I, II, III, IV. Research Bui., nos. 9, 10, 11, 12,
Univ. of Mo., Agr. Exp. Stat. Pearl, R. and Surface, F. M. -1915 Sex Studies. VII. On the assumption
of male secondary characters by a cow with cystic degeneration of
the ovaries. Ann. Rept. Me. Agr. Expt. Stat., 1915, p. 65. Poll, H. 1911 Mischlingstudien VI: Eierstock und Ei bei fruchtbaren u.
unfruchtbaren Mischlingcn. Arch. f. Mikr. Anat., Bd. 78, II, p. 63. Sonnenbrodt 1908 Die Wachstuuisperiode der Oocyte des Huhnes. Arch.
f. Mikr. Anat., Bd. 72, p. 415.
PLATES
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THE AMERICAN JOURNAL OF ANATOMY, VOL. 23, NO. 1
DESCRIPTION OF PLATES
We wish to take this occasion to acknowledge our indebtedness to Mr. Royden Hammond for all the photomicrographs, and to Mrs. Maud DeWitt Pearl for the paintings on plate 9.
PLATE 1
EXPLANATION OF FIGURES
1 Medium sized oocyte in hen ovary (X 40), showing three layers to the follicle, the granulosa {g), theca interna ({), and theca externa (e), with nests of lutear cells in the theca interna {I).
2 Part of follicle wall in figure 1 at greater magnification (X 352). Labels the same as in figure 1.
18
CORPUS LUTRUM TM OVARY OF THE CHICKEN
liAVMOND PEAIili AN'I) AI.UK M. IIORINO
PLATE 1
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PLATE 2
EXPLANATION OF FIGURES
3 Young oocytes in hen ovary (X 352), with follicles consisting of a single layer of granulosa (g). Nests of lutear cells in the stroma nearby (l).
4 Portion of last discharged follicle in hen with thickened theca interna(0 and granulosa (g) being sloughed off into the cavity. (X 40.)
20
CORPUS LUTEUM IN OVARY OF THE CHICKEN
nAYMOND PKARL AND ALICE M. BOHING
PLATE 2
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PLATE 3
EXPLANATION OF FIGURES
5 Portion of sixth from last discharged follicle, showing large number of lutear cells (Z) in the theca interna (X 40).
6 Part of figure 5 enlarged (X 176).
7 Small discharged follicle with cavity nearly obliterated. Small plug of cells (p), filling in the cavity. Theca interna filled with masses of lutear cells H). (X 40.)
22
CORPUS LUTEUM IN OVARY OF Till'; CHICKEN
KAYMOND PEAHL AND AMCH M. liOIUXG
PLATE 3
23
PLATE 4
EXPLANATION OF FIGURES
8 Discharged follicle with cavity completely obliterated. The chief component is masses of lutear cells (l). The connective tissue center represents original location of cavity (c). X 40.
9 Part of figure 8 at greater magnification (X 176), showing interstitial cells (i.e.) in connective tissue between lutear masses.
24
CORPUS LUTEUM IN OVARY OF THE CHICKEN
RAYMOND PEARL AND ALICE M. UOIUNO
PLATE 4
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PLATE 5
EXPLANATION OF FIGURES
10 Later stage of solid discharged follicle, showing large development of yellow pigment in lutear masses (X 40).
11 Part of figure 10 at greater magnification (X 176), showing pigment particles.
12 Atretic follicles in hen ovary, with yolk spheres in central cavity (X -10).
13 Part of figure 12 at greater magnification (X 176). Lutear cells (/) show in theca interna and also among yolk spheres in the cavity.
26
CORPUS LUTEUM IN OVARY OF TIIi: CIHC^KEX
RAYMOND PEARL AND ALICK M. IIOUIVC
PI, ATI; 5
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PLATE 6
EXPLAXATIOX OF FIGURES
14 Later stage of atretic follicle (X 40). Only a few yolk spheres remain in cavity. Cavity is practically filled with lutear cells.
15 Part of figure 14 at greater magnification (X 176), showing lutear cells in theca interna {i), as well as the central cavity.
28
CORPUS LUTEUM IN OVARY OF THE CHICKEN
RAYMOND PEAUL AND ALICE M. DOUINfi
PLATE (i
29
PLATE 7
EXPLANATION OF FKiURES
16 Atretic follicle in which the pigment particles have developed in the lutear cells (X 40).
17 Part of figure l(i (X 176).
31)
CORPUS LUTKL'.M IN 0\AI{V OF Till'; ClirCKKN
KAVMOMl TKAHL AND AI.ICIO M. HOUINC
PLATE 7
Orrf
31
PLATE 8
EXPLANATION OF FIGURES
18 Section of youngest corpus luteum of cow (X 176).
19 Section of older corpus luteum of cow (X 176), showing cells filled with pigment particles.
32
CORPUS LUTEUM IN OVARY OF THE CHICKEN
RAYMOND PEAUL AND ALICE M. BORING
PLATE 8
i9kJt.MMLl .MM
33
PLATE 9
EXPLAXATIOX OF FIGURES
20 Section of discharged follicle of hen ovary, stained in Alallory's stain. Connective tissue = blue. Corpuscle = red. Interstitial cells = purple. Lutear pigment = yellow.
21 Section of older corpus luteum of cow, stained in Mallory's stain. Tissues colored as in figure 20.
34
CORPUS LUTEUM IN OVARY OF TIIK CllICKKN
RAYMOND PEAUI, AND AI.ICK M. BOUINC
PLATE 9
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35
STUDIES ON THE GROWTH OF BLOOD-VESSELS IN THE TAIL OF THE FROG LARVA— BY OBSERVATION AND EXPERIMENT ON THE LIVING ANIMAL
ELIOT R. CLARK
■' Department of Anatomy, University of Missouri
SIXTEEN FIGURES
These studies were begun and part of them were made in the laboratory and under the inspiration of my beloved teacher and master, the late Professor Franklin P. Mall, and it is with a sense of the deepest gratitude and reverence that I acknowledge the immeasurable debt which I owe to him.
INTRODUCTION
The development of the vascular system falls broadly into two stages: (1) the stage of primary differentiation, or histogenesis, and (2) the stage of extension and elaboration of arteries, veins, and capillaries. The exact^ manner and place, in which the primary differentiation occurs is an unsettled problem, and is, at the present time, the subject of spirited controversy. It has not been satisfactorily decided whether blood-vessel endothelium differentiates from entoderm, or mesoderm — and if from mesoderm, whether from mesenchyme generally or from the mesothelial lining of the coelom. Nor has it been determined whether this primary differentiation occurs on the walls of the yolk sac alone, or in the embryo proper, or whether it may take place both on the yolk sac and in the embryo. Another unsettled point is the extent of time over which the primary differentiation takes place. Recent discussions and observations supporting one or another of these views may be found in the following: Minot ('12), Evans ('12), Ruckert and Mollier ('06), Schulte ('14), Bremer ('14), Stockard ('15 A),Reagan ('17), Sabin ('17).
The second stage of vascular development includes the further
37
THE AMERICAN JOURNAL OF ANATOMY, VOL. 23, NO 1 ,
38 ELIOT R. CLARK
extension and development of the system after the primarydifferentiation has taken place and after the circulation has been established, and it is with this second stage that the studies here reported are concerned. In this stage, which continues throughout life, the vascular endothelium spreads through the growing organism, arteries and veins develop, until the extensive and complicated vascular system of the adult is perfected. It is principally characterized by the formation of new vessels by the sending out of sprouts from the vessels already present, instead of by the transformation of mesenchyme, of other undifferentiated cells, as in the first stage, and by the action on the vessels of the mechanical and chemical factors concerned with the circulation of blood and interchange of substances through the wall.
In spite of the abundant evidence in favor of this mode of spreading of the vascular endothelium, after its primary differentiation, there ■ are observers who adhere to the view that at any time throughout life, mesenchyme (or other undifferentiated ceUs) may be transformed into vascular endothelium. This view is held by Maxinow, Weidenreich, and Mollier (cf. discussion in Schulte, '14,) who believe that, not only may reticulum cells and leucocytes be transformed into blood-vessel endothelium, but that the reverse transformation may take place — in brief, that vascular endotheUum is not a specific tissue, but is interchangeable with the other tissues mentioned. The evidence for this view has in no case been conclusive. It is clear, however, that the manifestation of the property of sprouting does not form a sharp boundary line in time of development between stages, for apparently sprouting commences before the differentiation of endothelium is everywhere complete (cf. Stockard '15, B and Sabin '17). It is probable that the period of overlapping is very short.
It is also clear, particularly from the studies of Miss Sabin ('17), that the development of arteries and veins takes place to some extent before the circulation is established. In chick embryos she found that, before circulation starts, part of the aortae, the two vitelline veins next the heart, parts of the cardinal veins and the duct of Cuvier are clearly present as definite vessels.
GROWTH OF BLOOD-VESSELS IN FROG LARVAE 39
That there is a secondary stage in the development of the blood-vascular endothelium, in which the endothelium spreads by sprouting, instead of by the transformation of indifferent cells, has been proven by direct observation. In the transparent fin expansion of the tail of the tad-pole, this process has been watched during life by several observers, especially Golubew ('69), J. Arnold ('71) and Rouget ('73), who have seen blood capillaries send out sprouts, which extended until they met and anastomosed with other sprouts or capillaries and into which a lumen advanced — all without the interposition of outside cells. This view is supported also, among others, by J. Meyer ('53), Bobritzky ('85), His ('69), KoUiker ('86), R. Thoma ('93), Marchand ('01), Ziegler ('05) and Evans ('09 A). While this mode of growth has not been proven by direct observation for all vessels in all animals, and while the existence of other modes of extension is perhaps not necessarily excluded, it is a fair hypothesis that this is the universal mode of spreading of the vascular endothelium, once it has differentiated, and cannot be abandoned until more convincing objections are brought than have been produced up to the present time.
It is not the primary purpose of the present study to enter either into the problem as to the time, in embryonic development, at which the second stage begins, nor the problem whether growth by sprouting is the universal mode of spreading during this period. It is rather to consider the problem as to how, in a region where, and at a time in development when growth by sprouting has been repeatedly verified, and after the circulation has become established, the capillaries are transformed into arteries and veins; to study the modes of action and reaction of endothelium — the laws which regulate its growth.
Such a study is by no means new, for it has been, through many years of fruitful investigations, the object of W. Roux and particularly of R. Thoma and numerous coworkers to discover the factors which regulate the growth of vessels, while many others, including Nothnagel ('84), Mall ('03), Evans ('09, A and B, '12) have studied the same problem less extensively.
40 ELIOT R. CLARK
The initial stimulus to this study was given by W. Roux ('79), in his Inaugural Dissertation, in which he studied the '"angle of branching" in relation to the relative size of the branch, and the shape of vessels in the neighborhood of a branch. He found that this angle, which lies between a line continuing the axis of the main stem and the axis of the branch, varies with the relative size of the branch — that, in general, the larger (relatively) the branch, the smaller the angle, and the smaller the branch, the larger the angle. He also found that the lumen of an artery shows a widening with subsequent narrowing immediately after branching, and that the opening of the branch is oval rather than round. By experiments with openings made in vessels and in tubes and with the use of malleable substances such as lard placed in such openings and on the interior of tubes, he found that the direction taken and the shape found is, in the case of the artery, practically the same as the shape and direction of the stream of fluid emerging from openings in vessels and tubes. He concluded that the shape and direction of arteries at the place of branching are determined by the action of hemodynamical factors; that the blood-vessel wall responds by taking the shape which allows a minimum of fric-. tion. The general and important conclusion was that the size and shape of arteries and veins, in the growing and adult animal, are regulated, not by heredity, but by the action of mechanical factors.
Thoma's conclusions were based mainly on studies made on the extra-embryonic yolk sac vessels of chick embryos. From a series of injections he found that there is formed, first, an indifferent plexus of capillaries, interposed between the aorta and the venous end of the heart, and that out of this plexus, those vessels which are so placed as to have the greatest amount of blood flowing through them enlarge to become arteries and veins, while others remain capillaries, or atrophy.
The results of these and other studies by Thoma ('11) may be briefly summarized. He finds that blood-vessels are regulated in their growth by mechanical factors, which he expresses in the form of 'laws' ('Histomechaniche Principien'), as follows:
GROWTH OF BLOOD-VESSELS IN FROG LARVAE 41
1. Das Wachstuin ties qucrcn Durchinessers, also des Gefasslichtung ist abliiingig von dcr Geschwindigkeit dcs Blutstromes. Dasselbe l)egmnt, sowie die Stronigeschwindigkcit der nahe an der Gefiisswand stromenden Blutschichten einen Schwellenwert iiberschreitet, den ich mit U bezeichnen will, und i.st innerhalb gewisser Grenzen ein um so raschcres, je so mehr die Stronigeschwindigkeit liber den Schwellenwert U, hinaus zunininit. Dagegcn tritt ein negatives Wachstum, eine Abnanie des Gefiissumfanges ein, wenn die Geschwindigkeit der nahe an der Gefiisswand stromenden Blutschichten kleiner wird als der Schwellenwert v.
2. Das Liingenwachstum der Gefiisswand ist abhiingig von den Zugwirkungen der das Gefass umgebenden Gewebe und zwar sowohl von denjenigen Zugwirkungen welche das Langenwachstum der umgebenden Gewebe erzeugt als von denjenigen Zugwirkungen, welche bei "Anderungen der Gelenkstellungen eintreten," etc.
3. "Wird das Wachstum der Wanddicke bestimmt durch die Spannung der Gefiisswand." This is determmed by the l)lood pressure and the size of the vessel.
4. (proposed as an hypothesis, not yet proven). Die Umbildung von Kapillaren ist abhiingig von dem in den Kapillaren herrschenden Blutdruck und stellt such an denjenigen Stellen der Kapillarbezirke ein, an welchen der zwischen dem Kapillarinhalte und der Gewebsfliissigkeit bestehende Druckunterschied einen gewissen Schwellenwert p iiberschreitet. Dieser Schwellenwert ist jedoch in den verschiedenen Kapillarbezirken je nach den Eigenschaften der die Kapillaren umgebenden Gewebe verschieden gross.
Expressed more simply they are:
1. Increase or decrease in the size of a vessel is regulated by the rate of the blood flow. 2. Increase or decrease in the length of a vessel is governed by the tension exerted on the vessel wall in a longitudinal direction by tissues and organs outside of the vessel.
3. Increase or decrease in the thickness of the vessel w^all is dependent upon the blood pressure.
4. New^ formation of capillaries depends upon increase of pressure in the capillary area (proposed as an " hypothesis — not yet proven).
The ultimate controlling factor Thoma considers to lie in the metabohsm of the organs ('93, pp. 49-51). It is this which regulates, primarily, the increase or decrease in capillaries, which, in turn, sets in motion the mechanism which results in the increase or decrease in the size of arteries and veins, the increase in strength of heart beat, etc.
42 ELIOT P.. CLARK
Roux, in his later writings, discusses, mainly in a theoretical way, the factors involved in the increase in size of vessels, and the new formation of capillaries. His views as to the new formation of capillaries, expressed briefly in 1895, repeated more fully in 1910, and again repeated, in a controversial article in 1911, are perhaps most completely expressed in 1910, p. 88, where he says:
1st der Verbrauch in dem Parenchym, welches cine Kapillare umgibt, eiiiige Zeit dauernd derartig gesteigert, dass aiis den vorstehend erorterten Griinden mehr Stoff als normal liindurchtritt, so wird wohl die an der Stelle stiirksten Durchtritts gelegene Wandungszelle diirch die verstarkte Leistimg in der Richtung des Austritts zur Sprossimg angeregt. Dasselbe geschieht natlirlich auch an der denselben grosseren Parenchj'mtheil von der andern Seite der umschliessenden und ernahrenden Kapillare. Diese noch nicht als Kapillarenfungierenden sprossen treffen, Avohl diii'ch chemotropisch vermittelten Cjd^otropism, aiifeinander, also in ahnlicher Weise wie ich es an von mir isolirten Furchungszellen sah, einerlei ob diese Zellen noch freilagen oder schon wieder an etwas anderem (an der Zellen oder am Boden des Gefasses) hafteten. Der vererbte gestaltende Reaktionsmechanismus der Kapillarwand, der zum Hohlwerden und zur weiteren Ausbildung der neuen Kapillaren mit Bildung von Nerven und kontraktilen Elementen fiihrt, wird auf diese Weise aktiviert und so eine neue funktionsfabing KapiUare gebildet.
Like Thoma, Roux considers the metabolism of the tissue the primarj^ factor in new growth of capillaries. As for the specific stimulus, however, he disagrees. According to Thoma, increased metabolism causes increase in blood pressure in the capillary area, to which the endothelium is thought to respond by sending out sprouts, while Roux' view is that the new sprout is sent out as a direct response on the part of the endothelial cell to the passage through it of an increased amount of substances. In criticism of Thoma's hypothesis, Roux ('11, p. 201) calls attention to the absence of any noticeable new formation of capillaries in tricuspid or mitral insufficiency, in which conditions there is a rise in blood-pressure in the capillaries.
Thoma's first histomechanical law that the size of the vessel is regulated by the rate of blood flow, is criticized by Roux chiefly because he can see no way in which the moving stream can affect the wall, since, as first shown by Helmholtz, there is
GROWTH OF BLOOD-VESSELS IN FROG LARVAE 43
a thin layer of fluid next the wall which is immovable. His explanation for growth in size of vessels is that it is brought about through the agency of the vasomotor nervous mechanism; that, following increased metabolism and formation of new capillaries, there is a reflex widening of the arteries and possibly also of the veins of the affected region. This widening, if continued long enough, results in a permanent adaptation of the vessel wall to the increased volume of blood by growth processes.
Roux apparently agrees with Thoma's law as to the increase in thickness of the vessel wall.
Mall ('00), in an extensive review and discussion of Thoma's histomechanical laws, finds support for Thoma's first law, in his studies on the growth of glands. Like Roux, however, he disagrees with Thoma in his hypothesis that the formation of capillaries is dependent on increase in blood-pressure in the capillary area. In reality," he says (p. 250), we can only state definitely that with the new formation of tissue new blood-vessels may grow into it, for all new tissue does not have bloodvessels." The precise stimulus for the formation of capillaries is unknown. Again (p. 251), he says, "The first and guiding blood-vessel is the capillary, which grows in all directions, forming a plexus. Secondary changes made arteries and veins of them and their laws of growth have been discovered and clearly stated by Thoma."
It has been shown by a series of investigators — among them — Erick Miiller ('03, '04), Rabl ('07), Bremer ('12) and H. Smith ('09), and particularly Evans ('09, A and B) that many of the larger arteries and veins in the body of the developing embryo are first formed as capillaries, which grow as irregular plexuses, and out of which certain ones are differentiated to form arteries and veins. Evans, who has made the most extensive studies in this field, has described the caudal portion of the aorta, the chief veins, the pulmonary, subclavian and sciatic arteries as developing in this manner. He concludes that the histomechancal laws of Thoma are the factors which govern the process.
A number of investigators have suggested that new capillaries are formed as the result of the action of specific 'chemiotactic'
44 ELIOT R. CLARK
(better 'hemangiotactic') substances outside the capillaries. According to Marchand ('01, p. 148), Leber ('88) first suggested this explanation, to which Marchand is slightly inclined. It was suggested again by J. Loeb ('93) as an explanation for the growth of vessels in fish embryos whose heart action was eliminated by the action of chemical substances. Evans makes a similar suggestion. In each case it has been proposed merely as a tentative hypothesis and has not been tested.
Over against this group of investigators whose studies have gone to show that blood-vessels are regulated in their growth by the action of mechanical and chemical factors, and some of whom have attempted to define this regulation in terms of specific laws of growth, there are others who have supported the view that mechanical factors play little if any part in determining the formation of arteries and veins, and who attribute it rather to the action of hereditary influences. Possibly the strongest adherent of this view is Hochstetter, who has made so many important studies on the comparative anatomy of the vascular system. His view is probably most concisely presented by his pupil, Elze ('12) in an article criticizing the conclusions of Evans and Thoma. In brief, it is that the primitive form of the" vascular system is not a capillary plexus, but a single artery and vein, such as is formed in the limbs and digits of amphibians, and also in the segmental arteries ; while capillary plexuses are secondary formations.
Now it is interesting that support for this view has come in part from the two men who have been most prominent in advocating the regulating action of mechanical factors, namely, Thoma and Roux. Thoma ('93, p. 28) mentions that the aorta is developed as a definite vessel before the heart commences to beat, while Roux emphasizes a first stage in the development of the vascular system, as of other systems, in which differentiation and growth take place as a result of heredity (preformation) — a stage which includes the formation of ' the anlage of the typically laid down chief vascular stems' ('95, pp. 326-7, footnote). Roux bases this conclusion on chance observations made on the area vasculosa of chick embryos, in which the embryo failed to
GROWTH OF BLOOD-VESSELS IN FROG LARVAE 45
develop, but in which vessels, including the border vein and some others differentiated in situations corresponding with the normal. That growth of capillaries and larger vessels in embryos is regulated not entirely by the metabolism of the tissues, but in part at least by hereditary influences, is shown, he believes, by the richness of the capillary plexus in the lung and the relatively great size oJf the pulmonary arteries and veins, which, ' according to Wiener,' are, before birth, four to six times as large as the weight of the lung tissue justifies, in comparison with other organs. Wiener studied the proportion between size of artery and weight of organ.
Support is lent to this view by the results of studies made on embryos whose heart-beat has been eliminated experimentally either by mechanical removal or by chemical inhibition. Dareste (77) J. Loeb ('93), Patterson ('09), Knower ('07) and Stockard ('15 A) agree in finding certain typical arteries and veins formed in such embryos, in which the mechanical action of the circulation has been eliminated — in fish, frog and chick embryos.
The indications are that the truth lies between the two extreme views; that what we are forced to call hereditary factors do play a part, not only in the primary differentiation of bloodvascular endothelium and its capacity for gro^vth by sprouting, but in the formation of some of the main vessels in the embryo (how great a part and how long exerted in embryonic life, has not yet been cleared up, cf. Miss Sabin, '17, previously referred to) that, on the other hand, the vascular system does become, at an early stage, dependent, at least to a very great extent, upon the regulative action of mechanical and chemical forces.
Were it found that arteries and veins in latter stages are completely regulated as regards diameter, length, thickness of wall, and position by the action of mechanical and chemical factors, it would be quite compatible with our knowledge of the development of other tissues and organs, to find that a crude pattern of such mechanically controlled structures should reappear in the embryo (Thoma, '93, p. 28).
As to the precise nature of the mechanical and chemical factors which regulate the growth of the vascular endothelium,
'46 ELIOT R. CLARK
there is, as the foregoing review and discussion shows, difference of opinion sufficient to justify further observation and experiment.
METHODS USED IN PRESENT STUDIES
Since most of the studies referred to were made on successive stages, usually of injected embryos, in fixed preparations, it seemed that it would be worth while to study the changes in the same vessels of the same living embryo, following certain vessels through the critical stages in their development, keeping records of the circulatory conditions, and of all changes in the size of the vessels, and the direction of the angle of branching, et cetera. For such a study the transparent fin expansion of the tail of frog larvae is admirably adapted, for a larva can, by the use of chloretone as an anesthetic, be kept under observation over a period of weeks, and careful camera lucida records made as frequently as desired. Since the chloretone interferes but little with the heart beat, records can also be kept of the circulatory conditions in each of the vessels which is being watched. (For details of the method used see E. R. Clark ('12).) In the most extensive series of studies made on a single tad-pole, the observations were started when the larva (rana sylvatica) first became transparent enough to enable the course of the vessels in the dorsal fin to be made out, and records were made at daily intervals, at first, when new formation of vessels was most rapid, later, when changes were slower, at considerably longer intervals. During the observations the larva increased in length from 10.5 mm. to 29 mm. There was thus procured a record giving the vascular changes, with notes as to the condition of circulation in each vessel for a considerable section of the fin, from a stage at which the entire system consisted of a few capillary loops, to a stage in which a fairly complicated system of arterioles, capillaries and venules had developed. In addition to this series of studies, numerous shorter studies were made, on larvae of r. sylvatica, r. palustris and r. catesbiana. Brief reference has been made in an earlier paper (E. R. Clark ('09) ) to the bloodvessel changes in the tail of the frog larva, and some of the matter
GROWTH OF BLOOD-VESSELS IN FROG LARVAE 47
included in the present study was presented at the meeting of the Am. Ass. of Anat. in 1914 (E. R. Clark ('15), ) where drawings were shown.
DESCRIPTION or FINDINGS
When the blood-vessels in the dorsal fin of r. sylvatica larvae first become clearly visible, owing to the absorption of some of the yolk and pigment present in young larvae, they form a system of capillary loops, making an irregular meshwork of rather wide vessels, all connected with one another. On the arterial side they are connected with the main caudal artery, and on the venous side with the main caudal vein, which are located ventral to the notocord, and between the two layers of myotomes. The vessels reach the dorsal fin by passing dorsally between the notocord and spinal cord in the center and the layers of myotomes on either side. With the low power of the microscope their course may be easily follow^ed from the main caudal vessels to their emergence ^ from between the myotomes. With the higher power this is more difficult, and in most of the studies made, only the vessels in the dorsal fin proper, after their emergence from between the myotomes, are drawn.
While in many of the studies all the vessels iu the dorsal fin have been followed, a small area is selected for closer study, and for reproduction, because any section illustrates the fundamental principles involved in blood-vessel development. In the series which is reproduced an area was chosen which included an arteriole and a venule and the region between the two, as well as a part of the regions on either side. This area is sufficiently large to make it possible to follow the changes introduced by the development of new capillaries on the vessels already present.
The changes which occur in such a selected area are shown in figures 1 to 8, and will now be taken up in detail and analyzed.
There is present in the first record a very simple type of circulation. An arteriole, or, perhaps better, an arterial capillary is seen toward the left. Two branches are given off from this vessel on the right, and two on the left, through which blood corpuscles are circulating. In addition there is a third branch
48
ELIOT E. CLARK
APR^ 15
Figs. 1 to 8 Camera lucida drawings of blood-vessels in a section of the dorsal fin expansion of the tail of a rana sylvatica larva, on April 15, 16, 17, 18, 20, May 12, 20 and 31. Length of larva April 15, 10.5 mm., May 31, 29 mm. Arrows indicate direction of circulation. Relative rate of circulation indicated as FAST, MOD., moderate, SLOW, NO C, no circulation. Corresponding vessels are numbered. Vessels present in one drawing which have been retracted in the next are cross-hatched. The positions formerly occupied by vessels which have retracted are indicated by dotted lines. In figure 8, the vessels which were present in figure 1 are stippled, enl. (1: 50).
CxROWTH OF BLOOD-VESSELS IN FROG LARVAE
49
on the left, with a continuous hunon, but without circulation, and a fourth which ends blindly. The arteriole ends with a bend to the right, from which a long thread extends to another non-circulating vessel. Following the two branches to the right, it is seen that the first pursues a winding course, while the second passes fairly directly to the main venule or venous capillary, near the right. Between the two branches are three communicattng vessels, the last of which forms a non-circulating loop. Below the venule there is a rather elaborate plexus of vessels
APR 17
APR 18
50 ELIOT R. CLARK
containing, for the most part, lumina, but without circulation. Extending peripherally from them are several blind-ending projections. As regards the rate of circulation, through branch 6 it is relatively 'fast,' while through branch 10 it is 'slow.' To summarize the condition of the vessels for the small area selected, there is a simple irregular plexus of capillaries with wide and rather irregular lumen, some of them with and some without circulation. For convenience, the principal afferent and effefent capillaries have been called arteriole, or arterial capillary, and venule, or venous capillary, though they are of capillary size and appearance.
In figure 2 (a day later) a 'slow' circulation has started in a numlDer of vessels which had been non-circulating on the previous day, and several new sprouts and connections between sprouts have formed. In figure 3 this has continued, and has been accompanied by an increase in the rate of circulation in some of the vessels. On the other hand, there are some vessels in which the circulation has diminished, or ceased altogether. In figures 4 and 5 the same processes have continued — a slight formation of new vessels, with modification of the rates of circulation in many of the vessels, an increase in some and a decrease in others, in addition a new change has become marked, namely, the disappearance of certain capillaries, in which the circulation had ceased, or in which the circulation had never started.
Owing to the fact that the prolonged use of chloretone had caused a slowing of growth processes, the larva was allowed to develop in fresh water, with observations at less frequent intervals, in order to see the fate of the vessels being watched, after a considerable amount of growth had taken place. A record was made on April 22, which showed very little change from the record of April 20. The succeeding records were made May 12, May 20 and May 31. "While these later records are not close enough together to show all the growth changes, they show very well the new capillary areas which have developed, their relation to the vessels already present, and the changes which the earlier formed vessels have undergone, in consequence. Thus it will be
GROWTH OF BLOOD-VESSELS IN FROG LARVAE
51
seen that in figure (j and 7 the no n- vascular zone toward the margin of the fin has become much reduced in .extent by the formation of new vessels, until only a very narrow non-vascular zone is left. It will also be noted that there has been a general expansion of the tail, so that the meshes between the vessels are not
A ^ R 2,
52 ELIOT R. CLARK
iceably larger, and the vessels longer. The last record shows this enlargement of the tail most markedly. ' The tail has increased not only in length and height, but also in thickness, and with the enlargement there has been a very great development of new capillaries, in the widened spaces of the blood-vessel meshwork. In the half of the fin next the muscle, where the growth in thickness has been most pronounced, many of the new capillaries are in new planes, more superficial than the earlier formed vessels. As regards the fate of the vessels present in the earlier stages, it is seen that there has been a marked differentiation. In figure 1 the vessels present are nearly all of a uniform diameter. In each successive record there is a progressive differentiation, in which certain capillaries increase in size, others remain of the same, or slightly diminished caliber, while others disappear. In the last stage this differentiation is seen at its maximum; definite arterioles and venules have formed, which supply and drain considerable capillary areas. In this elaborated system there are present many of the same vessels and parts of vessels which w^ere present in the first stage recorded. Some have been incorporated as parts of the larger vessels, others are still capillaries, while others have disappeared.
Considered as a whole, then, this series shows strikingly that arterioles and venules develop, at least in this region in tadpoles, not by a steady outgrowth of a single vessel, which grows straight ahead into a new region, giving off branches where they are needed, and fulfilling its predetermined destiny to grow in a particular place, but rather by the sending out of numerous capillaries, in various directions, which anastomose, adding new loops of circulating capillaries to those already present. Of these new loops some are so placed that a circulation is never established through them, and they disappear; others are incorporated as parts of arteriole or venule or remain as capillaries. The effect of the addition of new capillaries on the system already present depends upon the relation' which the older parts bear to the new; thus a vessel which is at one stage the chief vessel of the region may entirely disappear, while another vessel, w^hich is small, and has a slow circulation at one stage, may later be a
GROWTH or BLOOD-VESSELS IN FROG LARVAE
53
part of the main arteriole or venule of the region. It is impossible to predict at one stage, which way a capillary will go — whether it will increase in size, remain the same, or atrophy and disappear; it all depends upon the relation which it bears to the other vessels in existence at the time, and to those which are developed later. The endothelium is equipotential, then, and its differentiation into arteries, veins and capillaries is determined by factors outside the endothelial wall or in the lumen.
Further evidence for this view is found in the following experiments on chick embryos. They were performed to test another point, but the results are sufficiently interesting in their bearing on the problem of blood-vessel growth to deserve brief mention.
The anterior cardinal view of one side, from a point anterior to the otic vesicle to and including a part of the duct of Cuvier, was dissected out from chicks of two and one-half to three days incubation. The method employed is as follows : Berlin Blue is injected into the vein through a very fine glass cannula. As
MAY 20-2!
THE AMERICAN JOURNAL OF ANATOMY, VOL. 23, NO. 1
54 ELIOT R. CLARK
soon as the Berlin Blue mixes with the blood it forms a precipitate which plugs the vessel and sticks to the endothehal wall, outlining the position of the vein. Using this as a guide, the vessel is dissected out, with considerable of the surrounding tissue, to make certain that all is removed. The egg is then sealed and the chick allowed to develop further.
This experiment was performed successfully six times and in every case, there was found to be a large vein in the place of the one removed. In one case, the vein on the operated side was larger than the one on the unoperated side. The chicks were examined four to eight days after the operation.
The conclusion seems justified that the secondary development of a large vein in the neck, in the place of the one removed, indicates that the mechanical conditions of the circulation favor the growth of a large vein in this region. Surely the new vein can hardly be considered as due to inheritance.
What are the laws which govern the growth of the endothelium, making the differentiation of such an elaborate system possible? What, in other words are the modes of reaction of bloodvascular endothelium?
THE FORMATION OF NEW CAPILLARIES
The first property to be noted is the capacity of the endothelium to send out sprouts. This process has been frequently observed in the transparent tails of living frog larvae, and verified by other studies, and is the generally accepted mode of spreading of the vascular system, after its primary differentiation. The sprout consists of an elevation of the endothehum which is sent out, usually starting at right angles to the vessel wall, and with a lumen continuous with the lumen of the parent vessel. The end of the sprout consists of a solid process of varying length, which may be in the form of a single thread, or of a thread with one or more branches. This process usually extends in a straight line from the parent vessel, for a varying distance, and may then curve. Sooner or later it reaches a similar sprout, or approaches a fully formed capillary, when it shows itself possessed of a prop
GROWTH OF BLOOD-VESSELS IN FROG LARVAE
55
erty most important for the development of a system of anastomosing vessels, namely, that blood-vascular endothelium has an affinity for blood-vascular endothelium, of such a nature that if two processes of blood-vascular endothelium draw near one another in their growth, a union will be formed between them ('cytotropism,' Roux). Equally important is the fact, readily observable in the tail of the frog larva, that blood-vessel endothelium avoids, in its growth, the cells of other tissues among which it grows, such as mesenchyme, and lymphatic endothelium. As a rule, the lumen eventually extends through the entire extent of this new sprout, it widens, and after a varying amount of time
56 ELIOT R. CLARK
the circulation of blood cells commences, and a new circulating capillary has been added to the system. This whole process may, however, not be completed, for some sprouts grow out a short distance and are retracted, while some in which the lumen has been formed, never have a circulation, but retrogress — becoming solid, and disappearing. Throughout this process the endothelium remains complete, the lumen being separated from the tissue fluid outside by a complete investment of endothelium.
The facts concerning the morphological changes which take place in the formation of sprouts are clear enough; the question then arises as to why sprouts are sent out, to what sort of stimulus the endothelium responds when it sends out a sprout. The answer to this question is not entirely clear, yet certain facts together with certain general considerations justify the proposal of an hypothesis.
A study of the positions at which sprouts are formed and of the general direction taken in their growth shows that they are preceded in their formation by the growth of the other tissues and that they extend into regions where the amount of tissue not yet vascularized is greatest in amount. In the tad-pole's tail, at early stages, vessels develop first along the muscle — the thickest part of the tail. Later they grow out into the fin expansions, which attain a considerable size before vessels reach them. Growth of new capillaries continues in a general direction toward the dorsal and ventral margins of the fin, until eventually the plexus reaches nearly to the margin. During the growth of this first set of vessels, the fin remains thin, and the capillaries — save for the thickest part next the muscle — are all in a single plane. Later, the fin becomes much thicker and there occurs a corresponding new growth of capillaries, from the older parts of the plexus, which pass toward the epidermis, and form plexuses in two new planes.
In both cases it is clear that the growth of new blood-capillaries has been secondary to the growth of the outside tissue. It has been suggested by Thoma as an hypothesis that the stimulus responsible for sprout formation lies in an increase in bloodpressure. If this wxre so, one would expect to find them growing
GROWTH OF BLOOD-VESSELS IN FROG LARVAE 57
out from the arterial rather than the venous end of the capillary, since, obviously, the pressure is higher in the arterial end. This, however, is not the case — at least, in the tad-pole's tail new sprouts grow out as frequentl}^ from the venous as from the arterial ends. Loeb ('93) suggests that the explanation of the new growth of blood-capillaries must l^e sought in the stimulus exerted by specific chemical substances outside the capillary. A similar suggestion is made by Evans ('09 B, note, p. 296), who says in discussing vascular and non- vascular areas in embryos : ^Ve have to do here, perhaps, with a matter of cell chemistry or tropisins, for endo.thelium apparently avoids certain areas in the embryo — the non-vascular areas." In discussing this paper (see also '12, p. 584), Thoma ('11) argues that the findings of Evans fit in wdth his hypothesis, explaining nonvascular areas in the embryo as areas in which the pressure is high, due to the compactness of the tissues in such areas. As a result of this, according to Thoma, the difference in pressure betw^een the blood inside the capillary and the fluid outside is less in such areas than in looser tissues where he supposes the pressure to be lower. There appears, however, to be a valid objection to the suggestion of Loeb and of Evans, which is found in the variations in the richness of the capillary plexus in the different organs and tissues of the adult. This is summarized as follows in Kolliker's Gewebelehre ('02, p. 670) :
Bestimmend ftir die Anordnung der Kapillaren ist die physiologische Leistung, und ergiebt sich als allgemeines Gesetz, dass, je grosser die Thatigkeit eines Organes, beziehe sie sich nun auf Bewegung oder Empfindung, auf Ausscheidung oder Aufsaugung, vor allem in den Lungen, der Schilddriise, der Leber, den Nieren, dann in den Hiiiiten und den Schleimhaiiten, viel weiter in den Organen, die nur behufs ihrer Ernahrung imd zu keinen anderen Zwecken Blut erhalten, wie*in den Muskeln, Nerven, Sinnesorganen, serosen Hauten, Sehnen imd Knochen.
Thus we find that richness of capillary plexus may occur where the chief factor concerned is the passage of substances through the w^all of the blood-capillary from the lumen outward, as in the kidney ; again where the absorption- of substances is apparently the chief factor, as in the intestine; and again, where removal
58
ELIOT R. CLARK
and absorption are approximately equal, as in the liver. It seems almost inconceivable that the great richness of capillaries in each of these cases can be due to the presence of an unusual amount of tropic substances.
The only common factor that can be discovered would seem to be the total amount of passage of substances through the endothelial wall, whether the direction be to or from the lumen of the capillary. That it is the quantity of substances passing through, and not some specific chemical body is strongly indicated by the fact that, in different organs, the substances which pass through the capillary wall are, in many cases, of a widely different nature.
Fig. 9 Diagram to represent the modification in amount of tissue supplied by a capillary as the result of the addition of a new capillary. The dotted area indicates the area supplied by the portion of capillary DBF; the lined area that supplied by the new capillary DEF.
This, then, would seem to me to be the most likely hypothesis as to the nature of the stimulus which is chiefly responsible for the formation of new sprouts — that it is the total quantity of passage of substances through the endothelial wall. This conclusion is in agreement with that of Roux ('95) which has previously been fully quoted. According to this hypothesis, when the amount of fluid passing through any part of the endothelial wall exceeds a certain point, the endothelium reacts by sending out a sprout, which eventually becomes a new capillary, thereby increasing the endothelial surface and diminishing the relative amount of interchange through any part of the wall. Figure 9 has been constructed as a diagram to show how such a law, re
GROWTH OF BLOOD-VESSELS IN FROG LARVAE 59
duced to its simplest terms, would operate. The square (fig. 9 A) represents, diagrammatically, the amount of tissue supplied by the capillary ABC. The large area, supplied by the portion DBF, shown by stippling, causes the amount of interchange through the wall near D and F to become excessive, and new sprouts are sent out which form a new capillary, DEF (fig. 9 B). The relatively diminished area supplied by this new capillary is shown by cross hatching, while the greatly diminished areS, left for DBF is shown by stippling.
Let us see how this hypothesis accords with the observations of the present study. When capillaries first enter the fin expansion there is an extensive, growing non-vascular area, for the fin attains a considerable development before the blood-capillaries reach it. The formation of new sprouts at this stage is extremely rapid, a rapidity which may easily be accounted for by the excessive amount of interchange involved in the relatively enormous non-vascular area. The new formation takes place in one plane — the sprouts being sent out toward the dorsal and ventral margins of the fin, in the direction to and from which there is clearly the greatest amount of interchange, for the fin^ at this stage, is quite thin. New sprout formation continues, in this plane, until the plexus of capillaries reaches nearly to the fin borders. As the borders are approached, sprout formation diminishes in rapidity, and this may be explained by the relatively smaller amount of tissue beyond the furthermost capillaries. Later, the tissue through the fin increases in all dimensions — in thickness, as well as in length and height, the capillaries increase in length, and a secondary formation of new sprouts takes place in the interstices of the old plexus. Many of the capillaries of this secondary set are in new planes, nearer the surface, especially in the thicker portions of the fin, near the muscle. It is significant that, in this secondary formation, new capillaries may grow out at places in the wall where vessels have been present but have been retracted, at an earlier stage. This secondary formation is best explained by the increase in exchange of substances due to the increase in amount of tissue.
60 ELIOT R. CLARK
It would seem difficult to explain the formation of new sprouts — especially the exact location at which they are sent out from the older capillaries — on the basis of the action of specific chemical substances. Such substances, if present, should act equally on all parts of the endothelium, resulting in streams of sprouts, sent out by each endothelial cell affected. We find, however, that excessive sprout formation occurs only in early stages, when the atnount of tissue entirely non-vascular is very great. Later, when the tissue has received its primary supply, and when new sprouts are clearly associated with the general enlargement of the organs, the formation of new sprouts occurs in a much more orderly fashion, a single new sprout here, another there, a condition which seems much better explained by the hypothesis that the new formation is due to increase in interchange of substances beyond a certain point, than by supposing the presence of specific chemical substances.
On more general grounds, also, the proposed hypothesis seems the most plausible. It has been brought out especially by Roux that the growth, maintenance and atrophy of tissues is to a considerable extent regulated by the extent of their performance of certain functions in the body as a whole. Increased or diminished function results in increased or diminished growth. The endothelium of blood capillaries functions as a membrane through which substances pass to and fro between the lumen and the fluid outside. It would be in harmony with Roux' general conclusions, if it were found that the new growth, maintenance and atrophy of capillaries is regulated by the intensity of this passage of substances through their endothelium.
To be sure, it is impossible to go with certainty beyond the conclusions of Mall that, with the new formation of tissue new blood-vessels may grow into it"— that it is "the growth of the tissue which leads the way," and that into this new-formed tissue the capillaries grow." Nevertheless the proposed hypothesis as to the precise formative stimulus seems to the author to be more in accordance with the facts than the other hypotheses which have been suggested.
GROWTH OF BLOOD-VESSELS IN FROG LARVAE 61
INCREASE IN SIZE OF CAPILLARIES TO FORM ARTERIOLES AND
VENULES
It is obvious, in looking over the series of changes which take place in the capillaries, that, while some remain capillaries or are retracted, others gradually increase in size to form arterioles and venules. A study of the position of those which increase in size, shows that the increase takes place in the capillaries or parts of capillaries which are so placed that they form vessels for the suppty or drainage of larger and larger capillary areas, so that their endothelium is subjected to the action of the passage of an increasing amount of blood. It would seem, then, that the conclusion is justified that increase in the size of the lumen of a capillary is regulated by the amount of blood flow. This is somewhat similar to the conclusion of Thoma, expressed in his first histomechanical law, that the size of the lumen depends upon the rate of the blood flow, at a minute distance from the wall. There is, however, this difference, that according to Thoma the rate of flow is the determining factor, while the present studies indicate that it is the total amount rather than the rate.
The capillaries in early stages — that is, during their early extension into the fin — are markedly wider than a few days later. At the same time the rate of flow in all vessels, at the earlier stages, is decidedly less than later — so that, coincidently with the increase in rate, there is, at this stage, a general diminution in the size of the lumen of all vessels. Moreover, in later stages, new capillaries are, for a time, relatively wide, with a slow circulation, and become narrower as the rate of circulation through them increases. Again, many instances may be seen, in any growing tad-pole's tail, of vessels remaining the same size or even diminishing in size until the lumen is obliterated, through which the rate of blood flow is relatively rapid, but, because of frequent complete stoppage of the flow, with the total amount very small, while, side by side with them, new capillaries, with a decidedly slower circulation, though they may diminish slightly in size, are not obliterated.
Another fact which must be referred to here is the well-known one that veins, in general, have larger diameters than arteries,
62 ELIOT R. CLARK
and yet the rate of circulation in veins is less than in arteries, while the amount of blood flowing through the two sets of vessels is the same.
It is clear, then, that the size of the lumen is not solely dependent upon either the rate or the amount of blood-flow. The chief difference in the condition existing in arteries as compared with veins, aside from the rate of blood flow, is the difference in blood-pressure. It would, therefore, seem 'that, with the same amount of blood to be propelled, the size of the lumen varies inversely as the pressure, providing the resistance is such that, with the greater pressure, there is a higher rate of blood-flow.
The diminution in caliber of blood-capillaries, after the early stage, is probably to be explained in this way. At the early stage the strength of the heart-beat is relatively small, as is shown by the slow rate of the circulation. Later the heart-beat evidently becomes stronger, for the rate of circulation increases markedly in all vessels. With this increase in rate there is diminution in caliber of all vessels.
If, however, we consider the changes which take place in a number of vessels which are subjected to approximately the same pressure conditions and rate of circulation, we find that the lumen varies with the amount of blood flowing through. Given a sufficient amount of blood to fill all vessels, and a sufficient strength of heart-beat to keep the capillary circulation up to the necessary standard, and it is found that the size of the vessel varies with the amount of blood flowing through. Thus, of two capillaries near one another, the one so placed that it forms a pathway for the supply or drainage of an enlarged capillary area has an increased circulation and increases in caliber, while the one not so situated remains the same size or becomes smaller.
The objection might be raised that the movement of the blood is not a formative factor — that the vessel merely fits the stream, and that its size is solely the result of the mechanical distention. That this is not valid is shown by the fact that vessels in which the circulation ceases altogether, or in which the circulation never starts, grow smaller and smaller until their lumen is entirely
GROWTH OF BLOOD-VESSELS IN FROG LARVAE 63
obliterated. (This will be taken up more fully later.) Thoma has made similar observations on ligated vessels — finding that, in spite of the mechanical distention due to blood-pressure, the lumen of ligated vessels becomes reduced to zero between the point where the last branch is given off and the point of ligature. We are, then, in agreement with Thoma, on this point, that the endothelium responds to the action of a moving fluid. To us, however, it appears that Thoma' s claim that it is the rate of flow is not justified. Rather, it appears that it is the amount of blood flow through a vessel which determines the size of its lumen. To be sure the factor of rate of flow, as well as the closely related factor of blood pressure, cannot be disregarded. To a certain extent, however, their action appears to be the opposite of that claimed by Thoma, for with increased rate there may be a diminution in size.
THE REGRESSION OF VESSELS
It has already been mentioned that in the growth of blood vessels many capillaries and parts of capillaries disappear. This process was referred to briefly in a former paper (2), '09 and will now be examined more in detail. On the series of records shown, there are many instances of the disappearance of vessels, in some cases of vessels which had never attained a circulation, in others of vessels which had had an active circulation.
First let us see what are the morphological changes which take place in the 'disappearance' of a vessel. The process has been watched carefully many times, and two sets of records are reproduced to show the details (figs. 10 and 11). The first change to be noted is a narrowing of the lumen. Next there appears an interruption of the lumen by the formation of a solid portion. The solid part may start near the middle of the capillary or nearer one end, and it gradually increases in extent toward the two ends. As it increases, the solid portion becomes narrower and narrower, until a varying amount of the former capillary is represented by only a fine thread. This thread becomes thinner until it is barely visible, and eventually disappears, leaving the two ends forming blind-ending projections from the
64
ELIOT R. CLARK
vessels to which the former capillary was connected. These remains of the capillary shorten by the retraction of the endothelium into the vessels with which they are connected, until they form onl}^ a slight swelling on the surface. This eventually disappears, and there is nothing left to mark the site of the former capillary. In figure 11 may be seen the movement back into the connecting vessel of a nucleus and a small pigment granule, from the capillary which is undergoing retrogression.
In this process of retrogression of blood-vessels as it is seen in the living animal, there is nothing that even remotely suggests the
Maijl^ / /' Mai^l5 |/^ Max^II j 1^
Maui/l
Mdi|Z.
11
Fig. 10 Several stages in the narrowing and retracting of a vessel in the tad-pole's tail (rana palustris). NO C, no circulation.
transformation of an endothelial cell into a mesenchjnme cell, or of any other type of cell. The impression is gained that the entire protoplasm is withdrawn into the parts of the system which persist, and that none of it is lost. Certainly some of it is withdrawn, as for example, in the case of the nucleus mentioned above. If any part fails to be withdrawn, it must be that it is dissolved, for the last that can be seen is a thread so minute that it is barely visible with high power lenses.
In the tail of the frog larva parts of blood-vessels rarely become completely isolated from the rest of the system, as has been de
GROWTH OF BLOOD-VESSELS IN FROG LARVAE 65
>^-^}=
vV_JL
Fig. 11. Stages in the retraction of a capillar}- in the tail fin of a rana pipiens larva.
66 ELIOT R. CLARK
scribed for vessels elsewhere. In order to observe the fate of such a capillary I completely isolated a small section of a capillary by cutting through its connections. Much to my surprise, this isolated capillary was found, two day.s later, to have formed an anastomosis with one of the circulating capillaries (figs. 12). Thus a capillary which has become isolated, does not lose its blood-vascular-endothelial properties, and may be reincorporated in the vascular system. It is perhaps worth noting that this vessel was in a region, near the margin of the fin, where active new formation of vessels was taking place.
What, then, are the factors which lead to the regression of capillaries? In keeping a record of the developing vessels, a record was also kept of the presence or absence of circulation in each capillary, and of the approximate rate of the circulation. On looking over the previous records of the circulatory conditions of any vessel which has undergone retrogression it is found that this process is preceded by a period in which the circulation has ceased. This period is usually preceded in turn, by a period in which the circulation has diminished in quantity. This latter, of course, applies only to the vessels in which a circulation has been established, for, as has been said, some vessels are withdrawn before any circulation has been established in them. Thus many definite records have been obtained in which the retrogression of a capillary has been associated with the stoppage or absence of circulation. The conclusion, therefore, seems justified that a vessel which is connected with the rest of the circulating system of vessels, retrogresses and disappears if the circulation within it ceases for a sufficient length of time.
The finding that the diminution in size of lumen which precedes the retraction of capillaries involves the property, on the part of the endothelium, of reacting to the amount of blood flowing through the vessel, agrees, in part, with Thoma's first histomechanical law. According to this law, however, it is the rate ('Geschwindigkeit') of blood flow which is the determining factor ('93, p. 37 ff). In these studies it appears that, in capillaries, at least, it is not the rate but the amount of blood flow which is
GROWTH OF BLOOD-VESSELS IN FROG LARVAE
67
12 a
12 b
Fig. 12 Drawing to illustrate fate of an isolated portion of blood-vessel. The portion A was cut through at the two points labeled X in drawing a. Two days later (b) this isolated portion had formed a connection with the rest of the blood-vascular system at B.
68 ELIOT R. CLARK
important. For while in some capillaries there is diminution in rate before retraction, others become smaller and have the lumen reduced to zero through which the rate never has become slower. The only common factor between these two is the total amount of blood flowing through. In the second type this is shown by the fact that though the flow is rapid, it is scanty and intermittent. The question naturally arises why the circulation becomes slower and even ceases altogether in certain capillaries, in a region where an active new growth of capillaries is taking place. In the case of many of the capillaries, the answer is pretty clear. With the continuous formation of new vessels, the pressure conditions in the vessels already present are changed, and a capillary which formerly may have been the only vessel between vein and artery, may later form merely a cross connection between two parallel vessels, in which the pressure is equal. With an equal pressure in the two ends of the capillary, the flow of blood through the capillary ceases. The majority of the cases are evidently of this character, as may be seen by looking over the records (cf. branches 12 and 16, figs. 1 to 5). There are, however, other vessels to which this does not seem to apply, particularly branches of the larger arterioles (cf. branch 3, figs. 1 to 6). A considerable percentage of the branches of each vessel which differentiates into an arteriole disappear. In the case of these capillaries the retraction is preceded by a period during which there is no flow through the vessel, but the period of absence of circulation is not preceded by a slowing of the circulation. Instead of a slowing there is a diminution in the amount of blood passing through, while the rate remains rapid. Periods of total absence of circulation alternate with periods in which a few cells pass through at a rapid rate. In this condition the capillary branch affected leaves the arteriole at a right angle, and is relatively small as compared with the arteriole. Frequently the entrance to the branch becomes plugged by an erythrocyte or a leucocyte, which causes a stoppage of the circulation. The retrogression of such vessels takes place usually much later than in the case of the other retrogressing vessels, such a vessel often remaining for several days, with a gradual increase in the
(iltOWTH OF BLOOD-VESSELS IN FROG LARVAE 69
length of the periods of no circulation before retrogression takes ])lacc. I have no entirely satisfactory explanation to offer to account for the stoppage of cinuilation in these branches. It is possible that the increasing thickness and elasticity of the arteriole causes a constriction a])()ut the opening of the branch, until it becomes so small that a blood cell cannot pass through. Another possil^ility which has suggested itself is that the narrowing is due to a suction on the branch caused by the rapid passage of fluitl past an opening into a branch going off at a right angle. A\'hate\er the explanation, however, the important fact remains that the retrogression of these branches is associated with a diminution in the total amount of blood which passes through them.
There remain vessels in which the blood-flow diminishes and the lumen decreases, with resultant retraction, for which none of the factors thus far suggested seems to offer a satisfactory explanation. It is .quite a striking fact that, in most new capillaries the blood flow is at first slow — usually slower than in older capillaries — that the circulation through the newer capillaries increases while that through some of the older ones diminishes. It would seem that here, as in the case of the formation of new sprouts, a regulating factor must be looked for in the rate of interchange of substances through the wall. A glance at the diagram (fig. 9) will illustrate the significance of this suggestion. While the portion of the capillary DBF, in figure 9 A, is so placed that it forms the medium of interchange for the large stippled area; in figure 9 B, the new capillary DEF, which has developed peripherally, has taken over the greater portion of this area, leaving only the small stippled area for capillary DBF. It seems logical to suppose that, if the area . supplied by such a capillary is sufficiently reduced so that the amount of interchange through the wall falls below a certain point, the capillary wdll diminish in caliber and eventually retract.
The capillary is concerned chiefly with this interchange of substance, and it is difficult to escape the conclusion that the growth processes of endothelium are regulated by it. Were they not, it seems impossible to conceive of how an organ becomes
THE AMERICAN JODRXAL OF ANATOMY. VOL. 23, NO. 1
70 ELIOT R. CLARK
sufficiently vascularized and how supernumerary capillaries are disposed of — in fact, how any equilibrium is established between the extent of metabolism of the various organs and the richness of their blood capillary supply.
The precise nature of the stimulus may well be conceived as a physical one — the frictioii produced by the passing through the endothelium of fluid substances, just as the other regulating factor — the blood-flow over the interior of the endothelium — is a physical character. With a • certain (undetermined) amount of interchange, the endothelial cell remains unchanged; with increased interchange beyond a certain (undetermined) point, the endothelial cell sends out a sprout; with diminished interchange below a certain (undetermined) point the endothelial cells constrict the lumen and are eventually withdrawn into the active capillaries, leaving no trace of the capillary which had fallen into disuse. It is probable, if this hypothesis is correct, that the retraction of capillaries through which circulation has ceased as the result of equalized pressures at the extremities or of plugging of the vessel, is due to the operation of this factor of endothelial response to diminished interchange.
These two factors, then, amount of interchange through and amount of flow over the inner surface of the endothelial wall of capillaries appear to be the chief ones concerned in the regulation of the new growth of capillaries, their maintenance, increase in diameter to form arterioles and venules, or decrease in diameter with e^'entual solidification and retraction. Of these two factors, the amount of interchange is primary and the amount of flow secondary, since increased or diminished blood-flow depends in the main upon changes incident to the formation of new capillaries.
Endothelium subjected to either of these factors will survive, grow, or retract according to the intensity of the stimulus. If the blood-flow is increased, there is increase in the diameter of the vessel. If the rate of interchange is increased there results sprout formation. Diminution of blood-flow causes diminution in diameter, and diminution of interchange, narrowing, solidification and retraction of endothelium.
(JROWTH OF BLOOD-VESSELS IN FROG LARVAE
71
.>'P
Fig. 13 From series shown in figures .1 to 8, showing vessels of stage of May 31 (dotted lines) superimposed on vessels of stage of April 15 (solid linesj. The corresponding vessels are lettered. Enlargement the same for both stages.
Fig. 14 Same vessels as shown in figure 13. The vessels from stage of May 31 (dotted lines) have been reduced, by means of the pantograph, until they have the same dorso-ventral length as vessels from stage of April 15 (solid lines).
72 ELIOT R. CLARK
THE INCREASE IN THE LENGTH OF VESSELS
With the general growth expansion of the tissue, the question arises as to what effect this has on the blood vessels present in the midst of the organ. In figures 13 and 14 there have been superimposed capillaries and vessels which were present at the earliest and at the latest records. In one case (fig. 13), they are both drawn at the same degree of enlargement. In the other (fig. 14) the drawing of the older stage has been reduced, by means of the pantograph, enough so that the two sets of vessels have the same dorso-ventral dimensions. Measurements show that the increase has been greater in the antero-posterior than in the dorsoventral direction. The increase in the dorso-ventral measurement of the fin expansion has been in the proportion of 1 to 2.22; while in the antero-posterior measurement (the total length of he tadpole) the increase has been in the ratio of 1 to 2.73. Thus the ratio of the dorso-ventral to the antero-posterior increase is about 7: 10. Almost the same proportion is found to exist between the measurements of corresponding parts of the capillary plexus at the same two stages. Thus the ratios obtained are, for the dorso-ventral increase: 1. to 2.2, for the antero-posterior increase: 1. to 2.9. It is possible that the agreement between the two sets of antero-posterior measurements would be even closer, had the measurement been made of the increase in length of the tail, instead of the increase in the length of the entire larva, including the head. It is obvious that the growth of the tissue has caused a proportionate growth in the length of the blood capillaries, and the size of the capillary mesh-work.
There would seem to be but one possibility as to the factors responsible for this increase in length of vessels, namely, the one proposed by Thoma ('11), as his second histomechanical law, that increase in length of vessels results from a tension exerted in a longitudinal direction on the vessel wall, by the surrounding tissue. In the tail of the frog larva, the space between the blood capillaries is occupied mainly by branched mesenchyme cells, from which fine fibrillae in great abundance are given off in all direction, surrounding and supporting the blood-capillaries, lym
GROWTH OF T3LOOD-VESSELS IN FROG LARVAE 73
phatics and nerves, and extending between the right and left layers of epiderniis. As the tail grows there is an increase in the number of these cells, and in the size and complexity of their l)rocesses. It is clear that such an increase in the tissue filling up the space formed by a blood-capillary mesh- work, the fine processes of the tissue being in contact with the blood-vessels, would lead to a pushing and pulling on the capillary wall, and it seems (luite safe to infer, as Thoma has done, that the growth in length of vessels is due to the response of the vessel wall to this mechanical tension.
CHANGE IN THE ANGLE OF BRANCHING
It was somewhat surprising to find how closely the direction and pattern of the capillaries which persisted resembled, in the latest stage, their pattern in the earliest stage. Thus, bends, present in the capillary at its first formation, although they may diminish, or disappear entirely, may be retained throughout. To a considerable extent, also, the angles between branches remained nearly the same as when first formed. It is apparent that the capillaries, once formed, are held fairly rigidly by the surrounding network of fine connective tissue fibrillae. There is, howe\'er, a marked change in the angle of branching, in the case of the arterioles. Here one sees the working out, in a general way, of the laws of branching discovered by Roux, for the smaller the branch, relative to the main stem, the nearer its angle of branching approaches a right angle, and the larger the branch, the more acute — relatively — its angle of branching. Figure 15 illustrates the change. On March 18, the two branches — B and C — into which A divides are approximately equal, and the angle of branching of each is nearly 90°. This stage is an early one — the capillaries are newly formed, and are characteristically wide, and the angles are those which happened to form as a result of the direction taken by the new sprouts. March 20, branch B is slightly larger than C, and there is a tendency for its angle of branching to become slightly more acute. March 24, April 13, and April 26, however, show a progressive increase in the size of branch C over branch B, and a corresponding dimi
74
ELIOT R. CLARK
nution of its angle of branching, as compared with that of branch B, until, April 26, it forms, as it leaves the main stem A, almost a direct continuation of A, while the angle formed by B is much nearer a right angle. It will be noted that, in the later stages, there is a reduction in the size of all the vessels. With this reduction, however, there is marked increase in rate of flow. Other examples may be seen by comparing, in the successive stages of the main series shown, the branches from the chief arteriole.
Marl^'
Mar.ZO
Mar, li
Apr. \3
A}}r.Z6
Fig. 15 Several stages of blood-vessels in the tail of rana sylvatica larva, to show changes in angle of branching. In x, stage of April 26 is shown in dotted lines, superimposed on stage of March 18.
In these cases, the angle of branching is found to vary according to the relative size of the branch — the larger the branch the smaller the deviation from the line continuing the axis of main stem, while the smaller the branch, the larger the angle — until, when the disproportion is great, the angle reaches 90°.
These results corroborate the results of Roux' studies, which were based on measurements of arterial branches in adult animals. Roux stated his results in the form of a law, namely, that the size of the branch divided by the size of the main stem gives a series of figures which vary about as the cotangent of the angle of branching. He considered that the size of the angle represents
GROWTH OF BLOOD-VESSELS IN FROG LARVAE 75
the response of the tissue to hydrodynamical factors, and that the angle formed is always the one by which the minimum of friction is permitted.
The same problem is discussed by Thoma, ('11, p. 26) who agrees in the main, with Roux' findings, but considers that the blood-vessel, in assuming this shape, is merely responding to the rate of blood-flow, according to his first histomechanical law.
It would seem that Thoma's explanation (if amount of flow is substituted for rate) is sufficient, for, if fluid flowing through any tube tends to leave at a greater and greater angle the smaller the opening, then, if the blood vessel did not correspond with the direction naturally taken by the fluid stream, part of its wall would be subjected to the action of a greater flow of blood, than other parts, and would enlarge, while other parts would retract from the opposite cause. By this, the branch would be, remodelled until its angle of branching represented that in which the blood flowed in even amounts over all parts of the wall.
My observations on the angle of branching add nothing new to the results obtained by Roux, except that they represent studies on the same vessels at different stages, and give a picture of the actual changes in shape going on, hand in hand with the changes in the relative sizes of the branches, together with an approximate record of the relative amount of blood flow.
STUDY OF VESSELS IN TAD-POLES WITHOUT HEARTS
Since it has been found that a considerable development takes place in embryos deprived of a circulation by eliminating the heart-beat, it would seem, at first sight, that an opportunity offered itself here to test certain of the hypotheses dealt with in this paper — to find out whether any, and, if any, how much development of arteries, veins and capillaries takes place — particularly in the portion of the tad-pole's tail studied.
Brief reference has been made to such studies, and they will now be referred to more fully.
Roux ('95, p. 83) refers to a picture given by Dareste ('77, PI. VII, fig. 6) of a chick embryo in which the embryo proper failed to develop. In commenting, Roux says:
76 ELIOT K. CLARK
Nach dicser Abbildung Dareste's und einigcn von mir zufallig aufgefiindenen, weit ausgebildeten Fallen treten in dieseni Capillarnetz schon einige den normalen grosseren Gefassen entsprechende Richtigungen deutlich hervor, wie ich sehe, die entsprechende Erweiterung derselben und die Verdickung ihrer Wandung f ehlt ; der Sinus terminalis ist jedoeh ausgebildet und beweist so allein schon die vererbte localisierte Anlage eines typischen Gef asses.
Embryos without circulation were produced by J. Loeb ('93) who found that, by growing embryos of fundulus, a salt water minnow, in the proper concentration of potassium chloride, the beat of the heart could be entirely eliminated, or so diminished that all circulation of the blood was absent. The development of the embryo, however, continued for several days — about half the normal hatching time. In such embryos Loeb found an extensive development of blood-vessels which, except for irregu"larities in caliber, resembled in distribution the vessels in normal embryos. He concluded that:
Die mechanischen Ursachen fiir das Wachstum der Gefasswande sind deshalb nicht im Gefasslumen zu sucheii, sondern in alien oder einzelnen Zellen der Gefasswande und die Abgabe von Aesten ist bestimmt durch innere Ursachen in den Zellen der Gefasswande oder durch Reizursachen, die von der Umgebung ausgehend, diese Zellen treffen, ahnlich wie im Falle der Stolonenbildung von Hydroidpolypen.
Patterson ('09, pp. 87-88) studied the area vasculosa of chick embryos in which the development of the embryo proper had been prevented by operation upon the unincubated blastoderm. He describes the finding of vessels which radiate toward the remains of the embryo, and which he interprets as omphalomesenteric arteries.
Knower ('07) studied the development of frog larvae from which the heart anlage had been removed before pulsation had started. In a brief summary he states that — the aorta, the large veins and the segmental vessels are laid down." "Both arteries and veins are very abnormal and have a few well-defined branches. All vessels become much distended and follow very irregular courses." He found no vessels in the fin expansions of the tail.
GROWTH OF BLOOD-VESSELS IN FROG LARVAE i i
Stockard found in fundulus embryos, in which the circulation of blood was inhibited from the start by the use of alcohol, that the vessels of the yolk sac and many of the vessels of the embryo, including the two aortae, are formed, that some of them become much distended, and that they may persist without circulation for many days. While he gives no detailed or careful study of the exact amount of development of the vascular system, or the amount of retraction of vessels, he states ('15, B, p. 586) with reference to the aorta:
Tlie aorta in old embryos that never had their blood to circulate and in which the heart is actually a solid stream of tissue, grows and attains a well-developed lumen and a wall .lined with endothelium and surrounded by concentric fibers of connective tissue, as is shown in figure 4 a in the previous paper, drawn from such a specimen. This vessel is very slow to degenerate, in fact, it shows no sign of degeneration and actually persists as long as the embryo is able to exist without a circulation, for 30 days or more." .... The function of the vessel as a blood conductor, therefore, seems in these embryos of Fundulus, to have little if anything to do with its early development and not much effect on its ability to survive These facts are most significant in a consideration of the influence of function on growth and development, auto-differentiation. Here it is seen that the structure both grows and develops in entire absence of function.
The descriptions of the extent of vascular development which takes place without a blood circulation, as given by these investigators, w^hile meagre and incomplete, agree in the finding of an extensive vascular system, in which at least some of the main vessels appear to have developed sufficiently to give the impression of being fairly similar to the vessels in normal embryos. Thoma ('93, p. 28) recognized, in chick embryos, that there is not only an extensive development of capillaries in the extraembryonic area, but part of the aorta is well developed before the heart beat commences.
He offers t\vo possible explanations of the early development of the aorta. One is that there is an inheritance of an anatomical structure which is in agreement with the structure resulting from the action of mechanical forces. He says (p. 28) :
Man kann somit nur feststellen, dass die vererbte Form sich in Uebereinstimmung befindet mit jenem allgemeinen von mir aufgestell
78 ELIOT R. CLARK
ten Gesetze, welches das Wachstum der Gefasswand von den Stromungsverhaltnissen des Blutes abhangig erscheinen lasst.
At another place, however, (p. 32) he suggests that the development of large vessels in the site of the two primitive aortae may be due to favorable mechanical or nutritional conditions.
That other main vessels develop, in chick embryos, before circulation commences, has been shown by Miss Sabin ('17) and referred to earlier in this paper.
In order to study the character of the blood-vessels in the tail fin of frog embryos deprived of a circulation, the type of plexus formed, and the mode of growth, I have employed Knower's method on frog larvae, and studied the vessels in the fin expansion of the tail. The heart was removed after it was sufficiently developed to be clearly visible, under the binocular microscope, but before it had started to beat, by making an opening through the skin, into the pericardial cavity grasping the heart with a pair of forceps, and dissecting it loose with a needle. In some cases a small pulsating fragment was left but there was no bloodcirculation. Embryos operated on in this way rarely live more than seven days, if the weather is warm, though they survive for ten to twelve days in cool weather. As Knower had described them, they become greatly swollen, due to the accumulation of fluid in the body cavities. They are very active, swim about the dish restlessly, and respond quickly to stimulation.
Unlike Knower, I found a considerable vascular development in the tail fins. Since the tail is opaque in early stages, due to the yolk and pigment, it was not possible to obtain very striking records of the growing blood-vessels. In several cases, however, the tail became sufficiently clear to permit records to be made. In one case records were made covering three days of growth of the vessels in the dorsal fin, and a considerable amount of growth was observed. The blood-vessels in the dorsal fin, in these larvae without hearts, form a primitive close-meshed plexus, of delicate vessels. In some cases the vessels are distended with blood cells, in others they are distended with a clear fluid, while in others they are very narrow. The blood cells are pushed about in the vessels by the movements of the embryos, and are
GROWTH OF BLOOD-VESSELS IN FROG LARVAE
79
Fig. 16 Vessels from a portion of the tail of Rana Pipiens larva from which the heart was removed on April 27, before pulsations had started. Drawings made May 6, 7 and 9 of same region of dorsal fin. A, B, C, D— indicate the same blood-vessels. Lymphatics are dotted.
80 ELIOT R. CLARK
also moved about to some extent by the action of gravity. Thus, vessels which have been empty, have a little later been found to be packed with cells, and vessels filled wdth cells, may later be quite empty, or filled only with a clear fluid. Figure 16 shows some of the vessels in the dorsal fin of such a larva, on three different days.
On comparing the three records, it is obvious that growth has taken place by the formation of sprouts, which are at first narrow threads, but which later acquire a lumen. Anastomoses form between neighboring sprouts, and no additions are made by outside cells. In fact the vascular plexus extends in essentially the same manner as in the normal tail. There is, however, a larger number than in the normal, of fine solid processes. In this particular specimen the vessels are very narrow, and contain no blood cells.
It is to be noted that the vessels, once formed, show no tendency to differentiate further — into arterioles or venules. All diminish somewhat in caliber. In other specimens all the vessels are considerably distended. While three days is not a sufficient time, even in the embryo with a circulation, for much differentiation, still, in stages as early as this, at least a beginning differentiation is noticeable, as may be seen in the series shown in figures 1 to 3.
It is of interest to note the growth of Ijrmphatics in the embryos deprived of blood circulation. Knower's observation that the anterior lymph hearts in such embryos are larger and beat more strongly than in normal embryos, was confirmed. In the tail fin, lymphatics grow out often beyond the bloodvessels, although in the normal embryos at this stage and in this species (Rana pipiens) the blood-vessels in this region grow out well in advance of the lymphatics. The lymphatics are somewhat wider than in normal embryos. The mode of growth of lymphatics is the same as in normal embryos, by the extension outward of sprouts, and there is no tendency for the lymphatic and blood- vascular endothelium to form anastomoses with one another.
The enlarged caliber of the lymphatics is of interest, especially in connection with the enlarged lymph hearts, and with the ob
GROWTH OF BLOOD-VKSSKLS IN FROG LARVAE 81
servation made by Knower, and confirmed by myself, that there is movement of lymph, as shown by the passage through the l>'ni])h h(>art of an occasional blood-cell, for it shows that passage of lymph into lymphatics is not dependent upon the maintenance of a certain amount of l^lood pressure.
The studies on embryos without circulation show that without the action of the mechanical factors concerned with circulation an extensive development of blood-vessels takes place ; that some vessels — at least the aorta in chick and fundulus embryos — differentiate bej^ond the capillary stage. My finding that, in such embryos, growth may occur by the usual process of sprouting, indicates that this property of endothelium is not dependent for its earh^ manifestation upon the action of the specific mechanical or chemical factors which seem to regulate it in later stages. 80 far as they bear on the main problem of this investigation, these results are important as indicating the extent and character of development of the vascular system which may take place without the mechanical factors concerned with the circulation of blood. It has been clearly brought out, especially by Roux, that there are two chief stages in the development of each organ or tissue. The first stage is the stage of 'auto-differentiation,' in which, by virtue of what our ignorance forces us to call heredity, or as Noel Paton expresses it, 'hereditary inertia,' each tissue differentiates and develops to a certain point. The second stage is the stage of 'functionelle anpassung,' functional adaptation, in which the further grow^th is regulated mainly by factors concerned in a quantitative way with the especial function of the organ or tissue. It is, therefore, consonant with our knowledge of many other organs and tissues to find that bloodvessel endothelium differentiates and grows for a certain period, and even that a vessel such as the aorta develops, as the results of 'heredity,' and without the action of mechanical forces. Such a finding affords no objection to the thesis that, in their later growth, blood-vessels are subject to the regulative action of the moving blood stream, the blood-pressure, the mechanical tension exerted on the wall by outside tissues, and the amount of passage of substances through the vessel wall. In fact, were it
82 ELIOT R. CLARK
not true of blood-vessel, as it is of other tissues, that two such phases exist, the vascular system would furnish a marked exception — at least, so far as our present knowledge of other tissues and organs goes. The question as to how far the vascular system might develop without heart-beat has not yet been satisfactorily worked out and presented.
SUMMARY AND DISCUSSION
The results of these studies on living blood-vessels are:
a. An extensive vascular development takes place, in the early embryonic stages, which is independent of the mechanical factors concerned with the circulation of the blood and the interchange of substances through the endothelial wall. During this stage, which to some extent precedes the inauguration of cardiac pulsation, it has been found that the aorta develops beyond the capillary stage (Thoma ('93) ) and that a number of other main arteries and veins are formed (Miss Sabin, '17) while several observers, by producing embryos with the heart beat eliminated, have found, apparently, that a number of the other main vessels develop. My own studies on frog embryos without hearts, show that extension of the blood-vascular system during this primary stage takes place by sprouting, and by the formation of anastomoses between sprouts.
Thus the vascular system, like other systems about which we have knowledge, differentiates and is carried a considerable distance on its developmental course — manifesting the property of extension by sprout formation, and forming some of the larger arteries and veins — as a result of 'hereditary inertia,' or 'self development.'
This stage, however, comes to an end relatively early; and the vascular system, for its further development into the complicated and nicely balanced system of the adult animal comes to be dependent upon the mechanical factors concerned with the pull and push of outside tissues, with blood-pressure and bloodcirculation, and with the interchange of substances through the wall. The picture presented in the tails of a-cardiac tad-poles
GliOWTH OF BLOOD-VESSELS IN FROG LARVAE 83
by the irregular indifferent plexus with no tendency toward transformation into arterioles, venules and capillaries, showing little more than the ability to send out and withdraw sprouts, is in marked contrast to the picture presented by the vessels in the same region, in tad-poles with a healthy circulation.
b. In normally developing tad-poles, the establishment of circulation brings into play, on the endothelium, the distending action of the blood-pressure, the mechanical friction of the moving blood stream, the mechanical (and possibly chemical) action produced by the passage of substances through the endothelial wall, in the interchange between the blood and the tissue fluid, and the pull and push of the enlarging and shifting outside tissues and organs. These studies, which have been principally confined to this stage, indicate, in general agreement with Thoma, Roux, Mall and Evans, that the new factors come to play a predominant part in the regulation of the growth of the vascular system, a part so important that the vascular endothelium may be said to depend for its growth on its response to the action of these forces.
The morphological changes are as follows:
The blood-vascular system extends by the well-known method of sprout formation. Blood-vascular endothelium has an affinity for other blood-vascular endothelium, cytotropism, (Roux), and avoids cells of other types of tissue, so that connections form between one sprout and another, or between a sprout and a fully formed capillary, while no connections form betw^een a sprout and foreign cells. Thus the vascular system growls as a continuous network.
A sprout once formed may have one of several fates; it may enlarge to form part of an arteriole or venule, which may become an artery or vein, or it may remain a capillary, or it may retrogress, losing its lumen, separating in the middle, and retracting into the capillaries with which it is connected at its two ends.
Changes take place in the angle of branching, and vessels increase in length.
The mechanical conditions which regulate these morphological changes are the following :
84 ELIOT R. CLARK
The new formation, enlargement, maintenance and atrophy of capillaries is dependent upon two factors (1) the amount of blood flow and (2) the amount of interchange of substances through the wall.
1. A capillary or part of a capillary so located that an increased amount of blood passes through it, increases in diameter until it may form part of an arteriole or venule; one so located that there is no especial increase or decrease in the amount of blood passing through remains a capillary; while to decrease or absence of blood-flow the capillary reacts by a diminution in lumen to final solidification and complete retraction. This, in a general way, agrees with Thoma's first histomechanical law, according to which the diameter of the vessel responds to the action of the moving blood stream. A difference, however, lies in this, that, while Thoma assigns chief importance to the rate of the blood stream, I find, in capillaries, that it is rather the amount of blood flow.
The changes in the amount of flow through different capillaries are brought about in various ways. The addition of new capillaries beyond, may cause an increase in a capillary so placed as to help supply or drain the new area. Again, the opening up of a new capillary may place an older capillary in such a position that it forms merely a cross connection between two parallel vessels, in which the pressure is equal, bringing about a slowing or stoppage of circulation in the older capillary. In certain cases of stoppage of circulation the cause is more puzzling — that is, in case of branches of capillaries which enlarge to form arterioles. It is suggested that the stoppage, here, is due to the constriction about the beginning of the branch, resulting from the increased thickness and elasticity of the arteriole. Some support is lent to this explanation by the fact that blood cells are often seen to plug the entrance to such branches often for long periods.
It is possible, however, that the narrowing of such vessels, as well as the slowing of circulation in the case of other capillaries, is due to the second factor mentioned.
2. The amount of interchange through the wall. This is pro
(iROWTH OF BLOOD-VESSELS IN FROG LARVAE 85
]:)()scd as an hypothesis, because it seems to fit the facts better than any other. According to this hypothesis, the endothehum of blood-capillaries responds to the passage through it of various substances, in the interchange which takes place between the blood and the outside tissues. To an increase, beyond a certain maximum, the endothelium is thought to react by sending out a sprout; to a diminution, beyond a certain minimum, in a capillary which is not so placed as to form part of an arteriole or venule, the capillary is thought to react by narrowing its lumen; while for the maintenance of a capillary, a certain intensity of interchange is thought to be necessary.
The formulation of this hypothesis, which agrees, in general with Roux' conception, is merely carrying the explanation for the new formation of capillaries a step further than Mall, who recognized, as did Thoma, that the ultimate cause for new growth of capillaries lies in the growth and metabolism of outside tissues. It seems to fit more facts than the suggestion of Loeb and Evans that the cause lies in the action of specific substances outside the capillaries, or Thoma's hypothesis that it results from increase in capillary blood-pressure. It needs, however, further proof, before it can be accepted as a law of growth."
The changes in the angle of branching, which were observed, represent a response to the relative amount of blood-flow through the branch, as compared with the main stem. If a branch remains or becomes relatively large, as compared with the stem vessel, the angle between the two approaches 0°, if relatively small, 90°. This is, in general, in agreement with the studies of W. Roux who has made elaborate mathematical estimations of the angle of branching, and finds that the relation is so precise that it can be expressed within limits, as a mathematical formula.
The growth in the length of vessels goes hand in hand with the increase in outside tissue, and is clearly, as Thoma has expressed in his second histomechanical law, brought about by the reaction of the vessel to the mechanical pull, exerted in a longitudinal direction on the vessel.
Thoma's third law, according to which the thickness of the
THE AMERICAN JOURNAL OF ANATOMY, VOL. 23, NO. 1
86 ELIOT R. CLARK
4
vessel-wall is regulated by blood-pressure, has not been tested in these studies.
In general, my findings are that, while blood- vascular endothelium differentiates, develops the power to grow by sprouting, and forms a primitive system of arteries, veins and capillaries as the result of hereditary factors, it very early becomes dependent, for its complete and orderly w^orking out into the elaborate and beautifully proportioned adult vascular system, upon the regulative action of outside factors, to which it reacts in definite ways and upon which it comes to be completely
dependent.
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88 ELIOT R. CLARK
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author's adsthact of this papeh issued by the binliographic service, september 28
