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By George W. Corner,

Assistant Professor of Anatomy in the University of California.


With two plates.


ON THE WIDESPREAD OCCURRENCE OF RETICULAR FIBRILS PRODUCED BY CAPILLARY ENDOTHELIUM.


By George W. Corner.


In adding his contribution to those here gathered, the writer deems it most appropriate to present a study which not only took origin during the course of an investigation suggested by Dr. Mall, but which led back into another field that he had made particularly his own, and in which his interest and advice would have been most eagerly sought, had a happier providence allowed.

In one of his best-known and most important papers Dr. Mall, in 1891, announced his discovery that the framework of many organs and tissues of the mammaUan body is composed neither of white fibrous nor of yellow elastic con- nective tissue, but of a third tj^De of supporting substance composed of fine inter- lacing fibrils wliich not only differ from the white fibers in appearance, but are more resistant to both acid and alkahne solvents and are not so readily attacked by digestive ferments. He applied the name "reticulum" to the new tissue because the fibrils of the lymph-nodes, already bearing this name, were the first which he found to present the characteristics just mentioned. The supporting fibrils of the spleen, gastric and intestinal mucosa, Uver, lung, thyroid, heart-muscle, the base- ment membranes of the testis, and the entire supporting structure of the kidney, including the basement-membranes, were all demonstrated in tliis first paper to be of the same type. That the internal connective tissue of many other organs falls in the same category was later shown in pubhcations by various of ^Mali's pupils, of which the most interesting in the present connection are those upon the corpus luteum by J. G. Clark (1898),- and the adrenal gland by J. M. FUnt (1900). In 1902 Mall pubhshed his important account of the development of the connective tissues, showing that in the intestine, and presumably in other organs, the reticular fibrils are developed within the cytoplasm of the mesenchymal syncytium.

To this last statement, however, he noted one striking exception. In the fiver the reticulum arises from von Kupfler's endothelial cells :

"The observations upon the development of the reticulum of the liver are entirely out of harmony with those of the development of connective tissue elsewhere. In all other places the syncytium arises from the mesenchyme, but here it is from the endothelial

lining of blood vessels The fibrils are in no way connected with the liver cells and

true mesenchyme cells are not present at all."

This discovery was confirmed by J. Kon in 1908,\ A denial by ]\Iadame Schum- kow-Trubin (1909) would seem to be erroneous; the present writer's preparations agree with the description of Mail and Kon.

After the work of several investigators had proved the identity of the fibrils shown in the Uver by the digestion method with the "Gitterfasern" long since observed by Henle and Kupffer, for which Oppel had in 1890 discovered a method of selective impregnation by a silver-chromate method, further proof of Mall's conclusions as to the difference between reticulum and white connective tissue was afforded by the application of Bielsehowsky's silver-nitrate method to the staining of these tissues. As shown by Ferguson (1912), successful impregnation of adult tissues gives a totally distinct coloration of the two kinds of fibers.

CORPUS LUTEUM.

The present writer's interest in this subject began during liis study of the origin of the corpus luteum (Corner, 1919). It will be remembered that Sobotta,in his well-known account of the formation of the corpus luteum of the mouse, states that the connective-tissue framework is formed by the migration of the theca interna cells inward among the granulosa and their simultaneous conversion into "Spindelzellen." In the sow's ovary the author did not observe such a change of the theca cells, but found that thej^ remain interspersed among the granulosa lutein cells of the fully formed corpus luteum, and Uke the latter are merely enveloped by the interlacing fibrils of the connective tissue. Nor is there sufficient participa- tion of the theca externa to provide fibroblasts to lay down the supi)orting frame- work; indeed, in the fully developed corpus luteum one does not see, except perhaps in the septa clo.se about large vessels, any spindle-shaped cells which can be definitely stated not to be components of capillary walls. As before mentioned, Clark, using Mall's methods, has shown the connective tissue within the corpus luteum to possess the characteristics of reticulum, and this observation is readily confirmed by the Bielschowsky stain. In the presence of such a dilemma — an organ plenti- fully supplied with reticular connective tissue, yet without connective-tissue cells- one could not fail to recall Mall's account of the condition in the liver. The endo- thelium is the only possible source of the reticulum when we exclude the granulosa and theca lutein cells.

In order to test this hypothesis it was necessary to demonstrate the reticulum by methods which do not at the same time destroy the other elements of the organ, but wliich on the contrary give the sharpest possible pictures of the fibrils in their relatif)n to surrounding tissues. To this end the Bielsehowsk3'-]\Iaresch silver- nitrate method jjroved best adapted. It was used exactly as directed by Fergusony^- (1912), after fixation in 10 per cent formol, alcohoUc formol, Bouin's fluid, or Zenker's fluid. Sections were cut in paraffine at 4 microns. After impregnation they were usually counterstained with alcohoUc carmine. The Bielschowsky- Maresch method has been extremely capricious in the author's hands, but when successful the jet-black fibrils show against the red counterstain in the clearest pos.sible manner. Occa.sionally the cytoplasm is colored a deep golden brown by the silver, and counterstaining is unnecessary. In order to display the capillary net, vascular injections were made, but these for various reasons proved chemically incompatible with the reagents used in impregnating the sections. Hence it was necessary to rely upon the presence of erythrocytes in the capillaries, in cases where the exact relations of the endothelium would otherwise have been dubious. Tliis


RETICULAR FIBRILS PRODUCED BY CAPILLARY ENDOTHELIUM. 89

was secured, in the preparation of some of the specimens illustrated in this paper, either by selecting engorged tissues, by ligating the vena cava under anesthesia, or (in the case of the fetal kidney) by injecting oxalated blood of the same species into the arterial sj'stem under considerable pressure.

The test of our hj^pothesis is shown in figures 1' and 2, which display the condition found in the fully developed corpus luteum of the cow and sow respective- ly. There can be no doubt that in tliis organ the endotheliaTcytoplasm itself con- tains llieiibrils of reticulum which support the tissues by embracing each lutein cell in their basket-Uke meshes. The deUcate complexity of the network, well seen in figure 2, is explained by the richness of the capillary bed, which touches every cell in the whole gland. As in the figures, the fibrils rarely if ever embrace the endotheUal nuclei, but usually pass between them and the lutein cells, lea\dng the nuclei to bulge into the capillary lumen. Fibrils are never seen in the endotheUum of vessels whose walls are more than one cell-layer thick, but such arterioles and venules are provided with a perivascular reticulum.

ADRENAL, HYPOPHYSIS, THYROID.

T he adrenal gland js^another organ whose framework is known to consist of reticular fibrils (Fhnt, 1900), yet contains no fibroblasts. Professor Evans's studies of vital staining with benzidene dj-es provide a deUcate test for the presence of fixed as well as wandering connective-tissue cells, and they show that there are no fibro- blasts in the adrenal cortex (personal communication). Figure 3 illustrates the condition shown by the Bielschowsky method in the zona reticularis of the rat's adrenal; figure 4, in the zona fasciculata. Here again there can be no doubt that it is the capillarxsndotheUum that subserves the function of reticulum formation.

In the anterior lobe^oOhe liypophysis the relation between the circulating blood and the epitheUal cells is so close that not only is there no space for fibro- blasts, but some have even doubted the continuity of the capiUarj^ tubes, suggesting incomplete walls, as in the liver. In this gland the fibroblastic acti\aty of the endothelium is very readily demonstrated, for it is seen in the walls of relatively large sinusoids (fig. 5) .

In text-book descriptions of the thyroid gland it is stated or implied that the reticular framework, mentioned by IVIall (1891) and described by Fhnt (1903), is laid down by connective tissue in interfollicular strands which carrj' the blood- vessels; yet careful study of thin sections of the thyroid readily confirms the state- ment of Fhnt that "interfolhcular connective tissue is scant save in the neighbor- hood of the great vessels." Major (1909) has pointed out the exceedingly close contact between the perifollicular capillaries and the folUcle-cells. In preparations of the rat's thjToid, made to illustrate this paper, the strands of connective tissue along the larger vessels are composed of collagenous fibers, with, only here and there a cell which may be interpreted as a fibroblast containing reticular fibrils. Away from the arteries and veins, between the follicles, the only cellular elements present

' Figure 1 is from a preparation of Mr. J. F. Cobb, for whose assistance, cut short by his entrance into the military service, I am much indebted.


90 RETICULAR FIBRILS PRODUCED BY CAPILLARY ENDOTHELIUM.

are the endothelial walls, and these contain the reticular fibrils. Figure 6 well represents the condition.

RENAL BASEMENT MEMBRANES.

Besides the glands of internal secretion, there are other organs in wliich fixed interstitial connective-tissue cells are known to be scarce or absent, yet which possess well-developed supiwrting fibers, and in wliich the capillaries are intimatelj' applied to the secreting cells whose functions they subserve. These conditions exist in the cortex of the kidney, and here again the point of departure for our investiga- tion is found in the work of Dr. Mall. In his first paper of 1891 he showed that after digestion of sections of the kidney with pancreatin the entire structure, from capsule to pelvis, including the basement membranes, is a single mass of anastomos- ing fibrils which possess all the characteristics of reticulum. This statement, amply confirmed by Riihle (1897), was in disagreement with the older belief in a homo- geneous basement membrane. The perplexity was cleared up in 1901 by IMall's discovery that both structures exist, the structureless membrane appUed closelj- to the bases of the epithelial cells, and itself intimatelj^ invested without by the cylinder of interlacing fibrils. In sections prepared with connective-tissue stains it is the outer coat that gives the traditional appearance of a basement membrane; in the macerated and teased preparations of older histologists probably the inner coat was most obvious. Mall's whole conception has recently been confirmed and extended by von Frisch (1915).

It is now generally held, therefore, that there is an intertubular stroma through- out the kidnej^ composed of interwoven fibrils, some of which are condensed against the tubules to form the membrana propria. They are said to be produced by flattened nucleated cells which are more common in the kidneys of young animals; according to Disse (1902), who has given a full description of the "renal stroma," in adult life the cells are found chiefly in the neighborhood of the papillae and the fibrils become independent of the cells.

Apphcation of the Bielschowsky method with counterstaining permits us to observe the exact relation of the fibrils to surrounding cells with a clearness not known to former observers, and it at once becomes evident that we shall have to revise the conception of the stroma of the kidney as well as of the endocrine glands previously described. In the renal cortex, between the convoluted tubules and about the glomeruU, the "stroma" is no more nor less than a network of reticular fibrils imbedded in the cytoplasm of the capillary endotheUum or deposited by the latter against the tubules and Bowman's capsules. True fibroblasts are very infre- quent, i)erhaps altogether absent. Figure 7 shows the renal cortex of the adult rat.

In the papillie and along the medullar}- rays there is, on the other hantl, a true stroma consisting (jf fibrol)lasts which produce reticular fibrils. P'igure 9 is taken from a medullary ray in the renal cortex of a fetal pig 145 mm. long, and well illus- trates the contrasting condition. The medullary fibrils are in general much thicker than those of the cortex, but no chemical difference has as yet developed, such as miglit be expected from the difi'crent origin of the two types.


RECTICULAR FIBRILS PRODUCED BY CAPILLARY ENDOTHELIUM. 91

The relations of the fibrils to the elements of the glomeruli are naturally of much interest. At favorable points where the epitheUal layer of Bowman's capsule is fairly thick, or where it has become separated from the adjacent tissues, as in figure 8, it is seen that the fibrils are related to the capsule exactlj^ as to the thicker epitheUum of the convoluted tubules. These fibrils are at some points so far away from capillaries that one hesitates to make the statement that they are produced by endothelial cells here also. In some species wandering connective-tissue cells are found at the point of entrance of the arteriole into the glomerulus, and it is possible that fibroblasts are also present and form the reticulum immediately surrounding the glomeruli. The endotheUum of the glomerular tuft is in no case provided with reticulum, affording a marked contrast to that of the intertubular capillaries.

At first thought it seems impossible that the capillary network, with its open meshes, could Ue against the renal tubules sufficientlj^ to cover their surface at all points with a fine network of fibrils, but two considerations remove the force of this objection. (1) Even in thin sections of kidney in which the vascular sj^stem has been completelj^ injected with a color-mass, it is found that the capillaries touch the convoluted tubules at practicalh' all points. The familiar figure of an injected human kidney given by Disse (1902, p. 79) illustrates the point. The renal capillary network must be so rich and the individual vessels so flattened against the tubules by the pressure of neighboring tissues as to make the meshes very small and thus practically to complete the surface of endothelium which rests upon the bases of the epithehal cells. (2) Of late we are beginning to think of the capillary endothelial cells not as parts of a rather inert, fixed conducting tube, but rather as dynamic elements, in some organs activel}- phagocj^tic, in others perhaps engaged in elaborate chemical processes, at all points ever ready to vary their pattern by putting out and withdrawing sprouts in response to the circulatory needs of the tissues.

GENERAL CONSIDERATIONS.

We shall need to await a more complete exploration of the capillaries of the whole body in their relation to the reticulum before attempting to discuss the general significance of the facts here fragmentarily reported. For the present we can merely say that in certain organs where true connective tissue is absent, and the blood-capillaries come into direct contact with actively secreting epitheUal cells, the capillary endothehal cells themselves are able to lay down the supporting framework of the gland. Where the support of tissues is provided by fibroblasts and their products, the endothelium seems devoid of reticular fibrils. It will be important to determine next, if possible, whether there are chemical differences between endothehal and fibroblastic reticulum, and whether the endothehal cells which produce fibrils are otherwise different from those which do not possess such a function.

Already, however, our observations begin to throw Ught upon certain problems of endothehal physiology and pathology. Since the studies of vital staining demon- strated the phagocytic properties of the cells which fine the peripheral sinuses of


92 RETICULAR FIBRILS PRODUCED BY CAPILLARY ENDOTHELIUM.

lymph-nodes, the exact nature of these cells has been in doubt. Evans (1914, 1915) placed them in the category of endotheUal phagocytes. Downey (1915) gives a good account of the two prevailing opinions, pointing out (juite correctly that the isame cells produce the reticulum; but his further assumption that by that fact they can not be endothehal, would seem to be invahd when we know that endothe- lium in other places has the power to lay down fibrils. In the spleen and lymph- node we may assume that the endothehal cell and that which forms reticulum are one and the same.

Our results would also seem to bear on the discussion of pathologists as to the connective tissue in endotheUal neoplasms. Wooley (1903) has reported the fre- quent presence of "intercellular fibrils" in these tumors, wliich he ascribes to rever- sion of the prohferating endothelial cells to a more primitive mesoblastic character. We have shown that such a function is in some localities characteristic of fully differentiated endotheUum.

DESCRIPTION OF FIGURES.

All the preparations illintrate;! are from sections cut at 4 microus, and stained by Ferguson's modification of the Bielschowsky-Maresch method. .\ll except those sliowii in figures 1, 7. and S were counterstained with alcohoUc carmine. Tlie plates were drawn by Ralph W. Sweet.

Plate L

Fio. 1. Corpus luteum of cow, fetuses 210 mm. long. (Preparation by Mr. J. F. Cobb, jr.) X 1,10().

cap a bloo<l-capillary in longitudinal section.

retic reticular fibrils in an endothelial cell of the capillary wall.

Fig. 2. Corpus luteum of sow, fetiLses 20 mm. long, showing reticular fibrils in the capillary endothelium. X 1,160.

cap a blood-capillary in cross-section, containing an erythrocyte.

nuc nucleus of an endothelial cell.

retic reticular fibrils which can be traced to a capillary, but are not themselves actually in a

vessel- wall. Fjo. 3. Adrenal gland of rat, showing reticular fibrils in the endothehal cells of a sinusoidal blood-vessel in the zona

reticularis near the point of exit of central vein from the medulla. X 1,100.

sin sinusoid.

fibr fibrils connected with the endothelium but not actually in the Vivscular wall.

Fig. 4. .\drenal gland of rat, showing reticular fibrils in the endothelium of a capillury in the zona fasoiculata. Some

of the erythrocytes are impregnated hghtly, others are intensely blackened. X 1,100. Fio. .'). Hypophysis of beef, showing reticular fibrils in the endothelial cells of a sinusoidal blood-vessel. X 1,100.

sin sinusoid.

fibr interlacing reticular fibrils displayed where a portion of the vascular wall is cut tangentially

Plate 2.

Fio. 6. Thyroid gland of rat, showing reticular fibrils in the endothelium of a capillary between two alveoli. X 1,100.

nuc nucleus of an endothelial cell.

eryth erythrocytes in the capillary lumen.

Fio. 7. Kiiiney of julult rat. Small areji of cortex, showing that the reticular fibrils, which here form b.asement-

ineinbranes for the convoluted tubules, are imbedded in the enilothelium of the capillary blood-vessels.

The figure also shows a portion of a very small venule, in which endotlu^lium is free from fibiils. X 880.

vein venule of the smallest order.

eryth erythrocytes in capillary.

Fia. 8. Kidney of adult rat, showing relations of the fibrils to the epithelial elements of Bowman's capsule. X 760.

epith epithelial element of Bowman's capsule, which is slightly wrinkled, so that its cytopkism

is clearly visible and is se<!n to be distinct from the reticular fibrils. Fio. 9. Kidney of fetal pig Wt mm. long. .\ri'a of medullary ray showing portions of a collecting tubule and of a

stHjreting tubule (probably a distal convoluted tubule near the ascending Umb of Henle's loop). The

field contains reticular fibrils produced both by fibroblasts and by endothelial cells. X 700.

bl. vesa. . . .blood-vessels.

retic reticular fibrils pnxluced by endothelium, forming the biisement-membrane of a secreting

tubule.

fibrobl. . . .fibroblasts producing reticulum and forming supporting sheath of a collecting tubule.


PLATE 2



R W S^eet de


BIBLIOGRAPHY.


Clark, J. G., 1898. Ursprung, Wach.stum, und Enile

des Corpus luteum nach Beobachtungen am Ovarium des Schweine-s und des Men.schen.

Arch. f. Anat. u. Physiol. Anat. Abt., p. 9.'>-l,34. Corner, G. W., 1919. On the origin of the corpas luteum

of the sow from both granulosa and theoa interna.

Am. Jour. Anat., vol. 26. DissE, J., 1902. Ham- und Geschleehtsorgane. In

von Bardeleben, Handbuch der Anatomie des

Menschen, Jena, Gustav Fischer, Bd. 7, T. 1. Downey, H., 1915. The so-called "endothelioid" cells.

Anat. Record, vol. 9, p. 73-77. Evans, H. M., 1914. The physiology of endothelium.

Anat. Record, vol. 8, p. 99-101. 1915. The macrophages of mammals. .\m.

Jour. Physiol., vol. 37, p. 243-258. Ferguson, J. S., 1912. The application of the silver

impregnation method of Biclschowsky to reticular

and other connective tissues. Am. Jour. Anat.

vol. 12, p. 277-296. Flint, J. M., 1900. The blood-vessels, angiogenesis,

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