Paper - A study of pathological cat embryos (1909)

Jordan HE. A study of pathological cat embryos. (1909) Anat. Rec. 3(4): 212-220.

A Study Of Pathological Cat Embryos

By H. E. Jordan. From the Anatomical Laboratory of the Vmversiiy of Virginia.

The science of descriptive teratology is founded mainly on facts relating to the embryonic pathology of man. Recently, Denison (1) made a study of ten abnormal pig embryos ajid reported results in harmony with the conclusions of Mall (2) regarding the origin of merosomatous human monsters. The results of a microscopic study, which I am making of diseased cat embryos, are thus far also strikingly consonant with recent opinion respecting the etiology of human terata.

Schwalbe (3) regards "amniotic constrictions and bands" as among "the most abundant of anomalies of the amnion" and states that ' "abnormalities thus produced are manifold" (p. 192-193). Mall in his article on "The Origin of Human Monsters" says, "Xo amniotic bands are found in any of the 169 specimens which I have studied." Denison likewise finds no amniotic bands in pig embryos, but the "amnion is often thickened, rough and smaller than normal." Ballantyne (4) in summarizing his chapter on "Amniotic Diseases in Teratogenesis" says that in the case of some of the terata at least, "the amnion would seem to act by pressure, and so delay, or altogether stop the progress of events in ontogenesis."

In one horn of the uterus of a cat I found two embryos measuring 12 mm. and 9 mm., respectively, and in the other horn one embryo measuring 7 mm. The first two embryos with their adnexa appeared normal. The third embryo was enveloped in a closely fitting amnion which was adherent to the uterus over a wide area (Fig. 1). The amnion had formed a band extending across the body between the two limb-buds, and the constriction associated with the band had produced a partial separation of the posterior from the anterior portion of the embryo. Three additional minor constrictions of the amnion produced adhesions with the ectoderm of the head, fore-limb and the body-wall in the region of the heart. The eyes were faintly visible externally. No sign of somites could be recognized. The head appeared considerably swollen. The embryos and a portion of the uterine wall were immediately fixed in Zenker's fluid.

The first striking fact is the great variation in length between embryos from the same uterus and probably of the same age. Professor McClure, who has studied many cat embryos, in his work on the development of the venous system and lymphatics, writes me that from 1 to 1.5 mm. is the greatest diflFerence he has ever noted. He says, moreover, that he has not found many embryos with amniotic bands. In the case under consideration one naturally infers that the amniotic band interfered with the nutrition of the smallest

Fio. 1. — Photograph of 7 mm. cat embryo showing the area of fusion between the closely-fitting amnion and the wall of the uterus; also the deep amniotic constriction between the limb-buds. Magnification about 3 diametei-s. (Made by Prof. Theo. Hough.)

embryo and prevented normal growth. However, the difference of 3 mm. between the larger embryos indicates the influence of a more primary malevolent factor. I undertook a comparative study of the pathological embryo with the amniotic band and the two apparently normal embryos from the same uterus. The sectioned material showed that all three embryos were similarly pathological though in varying degrees, thus indicating a common fundamental inciting cause.

Fio 2. Semidiagrammatic drawing of transverse section through region of fore-brain showing solid cord and brain (the unstippled areas represent an acidophile coagulum) ; also the engorged right anterior cardinal vein (V. A. C).

The above facts combined with others even more obvious, make it improbable that the union between amnion and chorion represents an incomplete original separation. The union is intimate (Fig. 6), but is probably due to secondary fusion following amnionitis. A better understanding of the early development of the cat would aid in distinguishing a primary from an acquired adhesion. The problem is complex, since, in early stages, there is an allantois-amnion, an allantois-chorion, and a yolk-sac placenta. (O. Schultze) (5).

Both amnion and chorion have become much thickened in places, and both may perhaps be best described by the term "fibro-cystic" adopted by Denison for apparently similar changes. Cells with large fragmenting nuclei lie in the cyst-like interstices of the chorionic mesodermal tissue. The cells of the amnion are for the most part smaller, the fibrous tissue is less compact, and the lacunae are wanting. Sections of the uterine wall show that chorionic villi are present and apparently normal in some regions, while in others they are covered with an exfoliating epithelium or are absent. In short there is evidence of necrosis, but no sign of inflammation.

Xeither umbilical cord nor vesicle is present. The embryo appears attached directly to the amnion and chorion (Figs. 3 and 4). Extreme strangulation evidently obtained, centering about the point of entrance of the blood supply of the embryo. Since there was no endometritis, the pathological condition may be the result of the "faulty implantation" (Mall) or some other elusive cause producing "disorderly ontogenesis". (Ballantyne.)

Sections of the 7 mm. embryo reveal the following points: The embryo is much deformed in the facial region. A portion of the head has fused with the wall of the thorax thus obliterating the mouth and involving the base of the tongue (Fig. 2). However, the mandible, maxilla and two gill-arches can be distinguished. No distinct epidermal ectoderm can be recognized. The left forelimb has turned upward (dorsal ward, Fig. 3) and rests over a thickened area of the chorion. Caudalward from the fore-limb, the bodywall is much deformed (Fig. 5). In the region where the amniotic band has. not cut through the entire body-wall, the internal organs are much compressed and misshapen (Figs. 4 and 5); they have been invaded by blood cells. Still more posteriorly the remains of chorionic villi, appear with exfoliating epithelium and necrotic areas.

The brain and spinal cord are enlarged and almost solid. Their cavity is filled with a mass of coagulum and round cells, probably derived from the dissociating nervous elements. No wandering blood cells are present. The nerves are merely masses of pale disintegrating fibres, and the ganglia are in process of dissociation.

Fig. 3. — Semidlagrammatic drawing of transverse section through the region of the fore-limbs and heart, showing a necrotic area (N) in the chorion; also the area of fusion between body-wall and amnion (X) and the fusion of heart with body-wall and the amnion with the right forelimb. X 20.

The epithelium of the ear has broken down and the cells are disintegrating. The eye appears as a confused mass of broken elongate cells (the prbduct of the dissociating and disintegrating

Fig. 4. — ^Drawing of region 85 sections posterior to the last, showing enlarged area of fusion (X) between amnion and body-wall, and the strangnlated condition of the blood vessels and intestines in the region of the umbiUcus. X 20.

lens), surrounded by a layer of dissociating retinal cells, and by large pigment granules (the residue of disintegrating choroid cells).

The epithelium of the trachea, oesophagus, stomach, duodenum and mesonephros is also detached from its basement membrane and dissociating. The liver is represented merely by an amorphous mass of round hepatic cells mixed with blood cells (Fig. 4). The pharynx is small ; the infundibulum, thyroid gland and thymus are dissociating.

Fig. 5. — Drawing of section through the mesonephron and the associated posterior cardinal veins (V. P. C), showing also tlie thickened character of the amnion and the malformation due to pressure of the amniotic band. X 20.

The aortic arches are very small and filled with dissociating endothelial cells. The blood vessels and the heart are filled with blood. The right anterior and posterior cardinal veins are much dilated and engorged with blood.. (Fig. 2.) In a few cases the walls of the blood vessels have disappeared and erythroblasts have wandered

Fio. 6. — Photomicrograph of region of fusion between amnion and chorion. X 300. (Made by Dr. Frank P. Smart.)

into the surrounding tissues. Many of these have fragmented nuclei. The heart appears almost normal, though the atria are small and irregular and there are signs of tissue dissociation.

In the head region the mesenchymal tissue seem oedematous and the nuclei of the cells are fragmented. In other regions the mesenchyme appears generally in healthy condition. At points of fusion between the embryo and the amnion the mesoderm seems

D 220 The Anatomical Record.

continuous from one to the other. In the region of the heart the body-wall has fused with the amnion over a wide area (Fig. 3), and the heart has fused with the mesenchyme of the body-wall.

Myoblasts are sparsely scattered here and there through the dorsal regions of the body. Precartilage and cartilage everywhere appear normal. The notochord is in process of dissociation.

Sections of the 9 mm. embryo show similar pathological changes, though it is plain that the embryo has attained a slightly later stage of development. There is a smaller area of fusion between amnion and chorion. The umbilical cord is very short and compressed, and the yolk-sac has disappeared. The embryo is misshapen and flattened in the region of the umbilicus. In the oral region and the spinal cord the embryo is more nearly normal. But again the brain is solid, enlarged and filled with round cells; the epidermal ectoderm is lacking; ganglia, nerves, eyes and epithelial linings are dissociating.

The liver is a confused mass of large round cells. The walls of the blood vessels have very generally disappeared and the blood cells have invaded the tissues. The aorta and aortic arches are filled with dissociating and probably proliferating endothelial cells. Myoblasts are more numerous, but never aggregated in myotomes. Mesenchyme and cartilage again appear perfectly normal.

The 12 mm. embryo has attained a considerably later stage of development, but similar diseased conditions prevail in the tissues, apparently with great severity. There is no blood in the heart or vessels. The walls of the vessels have disappeared. The tissues including the brain are invaded with nucleated blood cells. Here also the brain and cord are solid. The area of adhesion of amnion to chorion is very small; but there is undoubted strangulation and consequent interference with nutrition. The face in the region of the mouth is much misshapen. All the tissues except cartilage and mesenchyme have dissociated as in the other embryos. Myoblasts are very numerous and individually they appear in good condition. There are regions where mucoid degeneration has taken place. All the organs belonging to this stage of development are present, but dissociating, the liver, brain and blood vessels being most seriously affected.

D Proceedings of the Association of American Anatomists. 221

Summary. The stage of development is inversely proportional to the area of adhesion between amnion and chorion or to the degree of strangulation. Barring the deformity due to the pressure of the amnion and the presence of an amniotic band in the 7 mm. embryo, the degree of abnormality varies slightly but directly as the development, as indicated more especially by the character of the blood vessels. Since the three embryos of the same uterus are similarly diseased and since only one has an amniotic band, the latter can only have been secondary to some underlying more primary cause. This was not endometritis, but some other pathologic agent producing necrotic areas in the placenta, destruction of chorionic villi, fusion of amnion and chorion, strangulation of the cord and interference with nutrition. Another interesting fact is the selective influence of the disease-producing factor on the various tissues. Liver, brain, cord, nerves and blood vessels show progressively less susceptibility to the morbid agent in the order named. Mesenchyme and cartilage seem most resistant. The pathological cat embryos agree among themselves and with certain human and pig embryos in being hydrocephalic and oedematous, with tissue dissociation and local histolysis. The results of this study support the position of Mall respecting human embryos that amniotic bands are secondary factors in the production of merosomatous monsters.

BIBLIOGRAPHY.

1. H. S. Denison, Anat. Record, Vol. 2, No. 7, 1908.

2. F. P. Maix, Jour. Morph., Vol. 19, No. 1, 1908.

3. E. ScHWALBE, Die Morph ologie der Missbildungen des Menschen und der Thiere, Jena, Pt. 1, 1906.

4. J. W. Ballantyne, Manual of Antenatal Pathology and Hygiene* 2 Vols., Edinburgh, 1904.

5. O. ScHULTZE, Grundriss der Entwickelungsgeschichte des Menschen und der Sttugerthiere, 2 Vols., Leipzig, 189G-*97.

REMARKS ON THE DYES USED IN THE HISTOLOGICAL LABORATORY

By Robert Retzer, Anatomical Laboratory, John Hopkins University ,

An effort to arrange the dyes used in this laboratory, so that not only the members of the staff, but also the errand boy should have no difficulty in finding them and putting them back to their proper places, was found to be a more difficult task than it seems at first sight. The dyes are known by English, French and German names and translating them into the terms of one language is but the first step. Most dyes have compound names and the question then arises, "Under which name shall they be classified?" The problem was solved by arranging them alphabetically according to the colors indicated on the bottle. Where the color was not mentioned (dahlia, fuchsin, etc.) the bottles were placed in an alphabetical order interspersed between the other bottles. With this method of classification, the dyes were not misplaced and a great deal of annoyance ob\"iated.

Before coming to the decision of adopting this manner of arrangement, it was considered whether it would not be advisable to place synonymous dyes together, with a view of thus utilizing the old stock. To my surprise, this turned out to be an impossible labor.

What according to one author is a synonymous dye means according to another an entirely different one. This, in turn, led me to look into the literature more thoroughly and study the origin of the names and the constitution of the dyes we use. For reference, I used the Encvklopadie der mikroskopischen Technik, Mann's Physiological Histology, Bernthsen's Lehrbuoh der organ ischen Chemie, Xietzke's Organische Farbstoffe, Beilstein, the chemical dictionaries of Watts and Thorpe, and the catalogues of Griibler and of Merck.

Even the cursory examination of a few of these books will reveal the lamentable state of our knowledge of the dyes. No two authors seem to agree, a fact readily understood when we consider that one is a chemist, another a histologist, another a manufacturer or dealer. The confusion seems to arise with the manufacturer who caters to the demand of the dyer and calls the product he sells by the name of the dye from which it is derived. For instance, Echtgriin is dinitroresorcin to the chemist, while one manufacturer places the sodium salt and another the potassium salt on the market as Echtgriin, Frequently it is still more confusing and complicated. The chemist applies the name to the base while the histologist buys a salt of its sulphonic acid under the same name. I might have stated that the histologist not alone suffers from the impositions of the manufacturer. If the chemist wants pure methyl alcohol in his laboratory he calls for Columbian spirits, if he asks for methylalcohol he is not sure to get a pure product.

In tabulating^ the synonyms of the dyes used by histologists, and there are about three hundred names to be found in biological literature, one is beset by a great many obstacles due to the inaccuracy of the authors. Each one stretches the synonyms a little further, until by comparing the first with the fifth author we find two entirely separate and distinct dyes called by the same name.

So, for instance, helianthin becomes crocein 3 B, dahlia (sol. in water) becomes anilin violet (insol. in water) and alcohol blue becomes primula. The confusion of terms is partly due to the histologist who will dissolve a dye in water that is according to all the authors insoluble. Evidently he used a different dye from the one he mentioned. In this paper, I intend to present but a few of the most commonly used dyes and point out some of the errors we fall into.

Haematoxylin, the dye obtained from the wood of Haematoxylon campechianum (logwood), is spoken of by the older German authors as Blauholzextrakt or Campeschaholzextrakt. The crystals are colorless and are but little soluble in cold water. They oxidize very rapidly by exposure and become haematein and some other oxidization products, that are more soluble. It follows from this that the solubility depends upon the age and exposure of the hjematoxylin and when making up a saturated solution it must be taken into consideration.

'The tables contain : First, tlie name of the dye ; second, all of its synonyms and pseudonyms; third, the chemical formulae of each of these; fourth, the manufacturer; and fifth, the process of manufacture or Its derivation. Rows 1, 2, and 3 of the tables are fairly complete, but 4 and 5 are necessarily incomplete, because the manufacturers* catalogues were inaccessible. The size and incompleteness of the tables prevent me from having them published. They form, however, the basis of this study, which has extended over a period of six months.

Eosin is probably the next most commonly used dye and is manufactured by a great many German, English and American factories. Whenever this is the case the products differ in their physical and chemical properties, because these dyes are not made for the chemical laboratories, but for dyeing and staining. Eosin is derived from fluorescein, the sodium salt of which is occasionally used in histology under the name of uranin. When fluorescein is treated with bromium it forms among the bromium compounds tetra-bromfluorescein, called by chemists eosin. We buy the sodium or potassium salt under the name of eosin, Eosin gelblich, Eosin w. gelb, or water-soluble eosin. The pure tetra-bromfluorescein is practically insoluble in water, however.

The alcohol soluble eosins are the ethylethers or methylethers of tetra-bromfluorescein. The former are but little used in histology, while the latter we buy under the name of methyl eosin, primerose and Spriteosin. Kaiserroth, saffrosin, phloxin, Bengal rose, Eosinscharlach and Lutetienne have been called synonymous, but wrongly. They are iodine, chlorine or other compounds of tetra-bromfluorescein.

By erythorosin is meant an alkali salt of either tetra- or di-iodide fluorescein. We may get a sodium salt, potassium salt, or ammonium salt of tetra-iodide fluorescein, and the same salts of di-iodide fluorescein, and mixtures of all. The difference in results we notice by solubility and color reaction. Unless we get the stain from the same bottle we are never sure to get the same result the second time, because we deal with arbitrary mixtures and not chemical compounds.

1. Basic Fuchsin. G. RosaniUn. 11. Anilin Red.

2. Neutral Fuchsin. 7. Rosanllln hydrochloride. 12. Azalelne.

3. Fuchslclenne. 8. Solferlno. 13. Harmallne.

4. Magenta. 9. Rubin. 14. Rublanlte.

5. Magentaroth. 10. Erythrobenzlne.

These are the names which are given as synonyms of fuchsin. The one most commonly used in this country and England is frequently spoken of as a separate and distinct dye. Histologists recommend magenta for staining the inner substance of elastic fibres, but fuchsin for a nuclear stain. The dealers naturally make stock of this and we find the two names in their catalogue. The original magenta was an English manufacture, while fuchsin is made in Berlin and Elberfeld. We find frequent errors, especially in every-day parlance, made in the opposite direction. Histologists will speak of sudan, when they mean sudan III, pyronin when pyronin G' or pyronin B is meant, and methyl violet when they mean any one of six or seven dyes that are put on the market.

A word about the methyl violets and the use of numerals and letters. Methyl violets are oxidization products and are the mixtures of hexa-methyl-para-rosanilin with penta, tetra, or even di and nonomethylpararosanilin. The salts are called methyl violet 6B, 5B, 4B, etc., the numerals indicating respectively the preponderance of 6, 5, 4, etc., methyl groups. Methyl violet 6B, therefore, is frequently called hexamethyl violet. Just as the methyl violets are the methylated rosanilins (or fuchsins) so the anilinblues are the phenyl ated rosanilins. With one or two phenyl groups the color is violet, with three or more blue. There are more than twenty anilinblues mentioned in histological literature. Most authors do not state which anilinblue they have used, and it is, therefore, little wonder that other histologists fail to get the same results.

Every person who has worked with methylene blue knows of the inconstancy of the results. The main cause of this lies in the impurities. There are various methods of manufacture and each method brings in a diflFerent impurity. Knowing what chemists can do, I am convinced they can put a pure methylene blue on the market for the. use of histologists. As soon as we get a pure stain we must discard all the others, because the results obtained from them are but empirical. I have frequently heard the term methyl blue used synonymously for methylene blue, more from carelessness than from ignorance, but as it is just such carelessness that brings the vast number of pseudonyms into literature, it is worth while calling attention to it.

What conclusions must we draw from these observations ? What can be done to give us more uniformity in the matter of dyes?

It seems a hopeless state of affairs and even an optimist can find little pleasure in the contemplation of the problem. First of all, we must be more careful in the use of names. When we use eosin we must state what kind of eosin, that is, we must be explicit and not use class names such as methylene blue, anilin blue, etc. Secondly, we must state whether it is the sodium, potassium or ammonium salt. If we do not know, then stating the manufacturer's or dealer's name will be of some use. To-day we know the process of manufacture of most of our dye-stuffs, as the patents have long expired, and when we find out the manufacturers' name we can also conclude what the most likely impurities are. I may here say that it is immaterial whether we consider staining a chemical . or a physical action. We well know that the solubility of a salt is not dependent on the chromofore radical, but upon a number of undetermined factors, and where some stains are done with a watch in the hand, the concentration of a solution is very important.

To-day nearly every histologist is a dyer, as soon as he uses stains he belongs to the same class as the silk manufacturer and dyer. Histological staining is to-day unscientific, or if we dignify it by the name, let us call it an empirical science. As long as we do not know the nature of the reagents we are dealing with, histological staining is to-day where medicine was a hundred years ago. We must do away with empiricism if we want to learn something about the principles underlying all structures.

Yet, staining has once and for all time become a necessary adjunct to our histological course and our own researches, and it is not my intention to suggest a sweeping reform, but to advocate the elimination of a great many evils connected with it. Every teacher knows that it is difficult to demonstrate to a student the form of it when the nucleus or cell is unstained, yet it can be done and it trains the student's power of observation. We must not forget that there are so many artefacts connected with staining that we do not always get a true picture of the structure when we study it stained. Staining has very little to do with morphology, it deals with far more complicated phenomena, which will never be approached until the histologist learns that he must not only know that a given dye will stain a nucleus blue, and another protoplasm red, but that he must also know what its chemical constitution is. There are a great many dyes that have formed the basis of chemical work, of whose physical properties we know something. The histologist can have more, if he demands more. How can he learn anything about the physical properties of a gland or of a cell if he is" ignorant of the essential properties of the reagents he uses 'i

To repeat, staining has very little to do with morphology. Sufficient evidence is given by the progress of histology. We can use Strieker's Handbuch to-day, and learn all of the essentials; indeed, a number of facts we find in it have been forgotten and rediscovered. And this book was written before the introduction of anilin dyes. It is hard to recall any great discovery with the exception of Ehrlich's and his followers, that has been based on the reaction of cells to dyes. The discoveries were made by the physiologist, who pointed out certain physiological actions of cells, glands or tissues, and these discoveries were only verified by the histologist. With all the complicated methods for staining the nervous system we have only managed to verify the work or suggestions of the physiologist. The dispute about the structure of ganglion cells and nerve-endings will not be settled until we put staining on a sound physical basis.

This seeming digression was necessary in order to make clear the conclusions. It is every evident that the histologist is being imposed upon by the manufacturer of dyes, and this imposition is due mostly to his ignorance and stands in the way of progress. The study of the synonyms has led us to get at the root of the evil. There are, as far as I have been able to find out, about ten factories from which we get our dyes. Each one of these factories turns out a product which is intended to stain blue, green, violet, etc., but not to have definite chemical reactions, and the name which the manufacturer gives, means something to him but nothing to us. We have, chemically speaking, a number of synonyms for a great many dyes, but practically — practically to the histologist — there are no two dyes alike. This evil must be remedied.

We owe to Weigert and Ehrlich the science of histological staining, which is still in its infancy. Because it is a difficult task it needs the comhined efforts of all histologists to place this science in the same rank with the others. The efforts must at first be directed towards the obtaining of pure products. As soon as we have these, and not before then, can further discussions be legitimately introduced. Isot so many years ago the chemists were in the same predicament and had to purify practically every reagent with which they worked. They could purify them because they had the appliances and the knowledge, but the histologist cannot do so to-day. It may mean that eventually the histological laboratory will have its chemical division, but in all events that lies in the far future. We must consider the present, and so the histologist should use only those dyes of which the chemist has found the constitution, and of which the most likely impurities and their influence upon the reaction are known. Even those histologists who do not believe in the importance of chemistry must admit that to-day it is difficult, sometimes impossible, for a man to obtain the same results in this country as another gets in France or Germany. This applies especially to results obtained from the newer stains, with which the market is being flooded. It may be unscientific to consider the protection of American industry, but we certainly could get products fresher and quicker by patronizing American dye manufacturers. Competition is great enough to make the insistence upon purity tenable. To-day we get impure products because there is no demand for other. We must therefore —

1. Exercise more care in the use of names, because carelessness is the prime cause of misleading pseudonyms.

2. Insist upon the name of the manufacturer, if not his, then the dealer's, because each manufacturer means a definite dye when he puts it on the market, and does not consider that other dyes carry similar names.

3. Give the chemical formula, so that there be no misunderstanding and our work can be repeated and verified by other observers.

4. Require pure products, made for the use of the chemist and histologist and not the dyer, and following this, require a standardization of dyes.