The minute morphology of experimental scurvy in the guinea pig

t'o*u STANFORD UNIVERSITY PUBLICATIONS UNÏVERSITY SERIES MEDICAL SCIENCES Vo¿uun II Nu¡rr¡nn 2 Srudies on By ARTHUR TT. MEYER and LELS M. McCORMICK STÁ,NFORD UNIVERSITY PRESS STANFORD UNIVERSITY, CALIFORNIA tmg CONTEI{TS pAtT ?¡tcE I. Tse Svuprol¡¿tor-ocy AND Gnoss MonpgoLocv or. ExPERTMENIAL Scunvy rN TEE GurrvsA Prc, by Arthu'r W. Meyer.. 7 REFERENCES n P¿erns I axo II, AND E¡rÞLANATToN oF P¡.¿rps. , .. .. .foÜoøíng 44 II. Tss M¡¡¡urp Monpnor-ocv or Exppn¡upnr¡r, Scunvv ¡N TEE Gungæ¡ PrgbyArtlwrl4/.Meyer. ........47 Rornnp¡¡cBs 69 P¡.¡rns III-IX, ¡¡.¡o Exp¡,¡¡v¡.uoN oF P¿¿tBs ..foUcuôtus 70 III. Sor¡n Cs¡n¡,cran¡st¡cs oF TEE Br.ooo oF TED Gurr¡re P¡c ¡¡¡ Exppn¡- ldErvrlr. Scunw, by Lewìs M. McCormôcþ...... 73 Ror¡npncns ......... 106 P¿ern I ¿uo Expr.¿¡¡¿troN oF P¡-¡tn. ....foUauíng tß TL?gI l3 PART II. THE MINUTE MORPHOLOGY OF EXPERIMENTAL SCURVY IN THE GUINEA PIG II. THE MINUTE MORPHOLOGY OF EXPERIMENTAL SCURVY IN THE GUINEA PIC+ By Anrnun W. MBvBn The microscopic study of intensely hemorrhagic subcutaneous tissue or perivesical fat confirmed the presence of both hemorrhage and some edema evident at the time of autopsy. However, often only relatively few erythrocytes \4/ere present in tissue that had looked very hemorrhagic to the unaided eye. The absence, as a rule, of active phagocytosis by polymorphonuclears also was striking. Most of the phagocytes \ryere macrophages, and in some specimens of subcutaneous tissues almost all the connective tissue cells either had undergone, or apparently were undergoing, such a conversion. In one specimen a fair percentage of these contained pigmentary, or other inclusions indicative of phagocytosis. There also was but little phagocytosis in areas of hemorrhage in the skeletal or visceral musculature, and the connective tissue in the areas of subcutaneous and the fat in cases of perivesical hemorrhage never looked well preserved, except in cases of very recent hemorrhage. They looked degenerate and stained, as though they had undergone incipient maceration, althougfi removed immediately after death and placed in Bouin. The presence of few blood cells in the darkJooking subcutaneous tissue, and other matters to be noted later, caused me to surmise at first that intravascular hemolysis had taken place, and that the highly pigmented plasma had stained the tissues after extravasation; but my associate, McCormick, could not establish the presence of intravascular hemolysis by appropriate tests; but, unless phagocytosed, the extravasated erythrocytes must have undergone lysis in the tissues which they had infiltrated. It does not seem improbable to me that such lytic action might be responsible for the absence of marked phagocytosis in many specimens and that phagocytic action may be depressed through the existence of a lowered general vitality. If the hemorrhages themselves are due to lytic processes which cause an increased permeability of the vascular walls, it would seem that the lysin concerned must be produced in the walls of the vessels themselves, or it must gain entrance to them from the surrounding tissues. It also is possible, I presume, for lysis of the extravasated blood to occur through lysins produced or present in the subcutaneous tissues themselves, although I do not assume that the presence of lysins is hereby established. Appearances merely suggest their presence. Since identical cytologic changes are not present in cases of starvation, 1 I gratefully acknowledge generous support for this investigation fiom the Committee on Research of the American Medical Association. 11731 lu 48 STUDIES ON SCURVY it might seem to follow that the scorbutic changes are due to the fact that food deficient in vitamines cannot be properly metabolized. Animals fed a deficient diet, however, not merely fail to gain in weight, or lose weight because of the depletion of body fat, but actually suffer disintegration in such hard structures as cartilage, bone, and teeth. I never have met epiphyseal separation, loose teeth, absorption of bone, desquamation of epithelium, etc., in marked cases of starvation, and the fact that guinea pigs dying of scurvy frequently are fat is of great significance in this connection. Evidence of lytic processes in the walls of blood vessels is frequently seen. The appearance of the small, disintegrating blood vessels was much the same, no matter where they were located. In the earlier stages of change the endothelium often was cubical, with the nuclei radially arranged and projecting markedly into the lumen' They sometimes were vacuolated, and in some vessels a portion, or all, of the endothelium had been desquamated and lay clumped in the lumen. Not infrequently the musculature of the vessels also was vacuolated or defrcient, as shown in Figure 14, or the entire disorganized wall of an arteriole lay among a mass of blood contained in an edematous area, as represented in Figure 15. No proliferative reaction was noticed at many of the costochondral junctions, but at others the cartilage was thickened, producing the grossly well-known beading, shown in microscopic section in Figure 16. It is the proliferation of the cartilage and not a bony reaction which forms the scorbutic rosary. Some distance from some junctions an ingrowth of connective tissue into the costal cartilages from the perichondrium was taking place into the cartilage, as shown in Figure 17. This ingrowth occurred only where areas of degeneration .rvere present, and I never observed the presence of isolated masses of fibrous tissue in degenerate areas in the interior of the costal cartilages, no matter how large these areas ï/ere, or how complete the degeneration. It may seem unnecessary to state this, but anyone familiar with the frbrous transformation of hyaline articular cartilage will realize that emphasis on this fact is not needless. Since I did not observe frbrosis in all the costal cartilages of every scorbutic guinea pig from which specimens were taken, I was at first inclined to doubt its relation to scurvy; but since I did not find it in about a score of specimens from costal cartilages from normal animals, I could not help but infer that the degeneration and fibrosis were ¡elated to scurvy. It is well to remember, however, that I never examined all of the costal cartilages or all of any cartilage in any scorbutic animal, but, as far as I know, no one has observed such changes in the costal cartilages of normal animals. The first evidences of degenerative changes in hyaline cartilage appear in the cartilage cells themselves. The cytoplasm becomes vacuolated and tt74l MINUTE MORPHOLOGY 49 rarefied. Later degenerative changes may also appear in the nuclei and finally in the matrix, as shown in Figure 1$ until only a noncellular acidophile detritus remains. Since this granular debris contains large clear spaces, it is evident that complete lysis of the cartilage finally occurs, and that this is in entire harmony with observations made on the peribronchial cartilages and on other tissues. Since hyaline cartilages are avascular, the occurrence of this degeneration is especially interesting and is striking proof of the presence of metabolic activities in mature hyaline cartilage. Aside from the presence of a very cellular immature form of cartilage near the place of beading, I have observed no other changes, and I have not been able to find a satisfactory explanation for the contradictory phenomena of destruction of cartilage in the epiphyseal region of young bones and of degenerative changes in the interior of the adjacent costal cartilages on the one hand, and the presence of proliferative changes at the costochondral junctions on the other. However, it is possible that the latter areas are stimulated by increased tension due to weakening of the junction, and later also by increased motion here. I have not examined many costochondral junctions microscopically, but I have never observed hemorrhage in or detachment of the perichondrium itself. Perhaps its relative avascularity and the composition of the cartilage are partly responsible for this fact. The proliferative process at the costochondral junction seems to arise {rom the perichondrium, and in some instances enough additional cartilage is formed to cover some remnants of the dissolving compacta in the adjacent portions of the rib. Proliferative changes u/ere not present at a1l costochondral junctions, however, nor rri/ere they always observed around the entire perimeter of a particular junction, and they were absent in mature pigs. Although I did not make a careful tabulation, I got the impression that the beading is more common on the outer than on the inner perimeter of the costochondral junction. It is possible that the greater strain present at the former during respiration is related to this fact. The fibrosis of the costal marro'r¡r', or perhaps better the frbrous invasion of it, begins in close proximity to the costal cartilage, probably because it is at the costochondral junction that disunion begins, and, although separation of bone and cartilage may occur first at the center of these junctions, the fibrous changes occur latest at this place. It is particularly interesting that degenerative changes in the costal cartilages should be accompanied by proliferative changes so near by when the latter always are absent in the bones. I never found the proliferating connective tissue in the medulla of the ribs and other bones to be nonvascular, as reported by \Molbach and Howe'26, in cases of wound-healing in scurvy, and it would seem that not t17s1 50 STUDIES ON SCURVY much regeneration can take place in the entire absence of blood vessels, especially in such a condition as experimental scurvy. Although small groups of marrow cells still are found among the fibrous areas in the osseous medulla, the region of contact between them and the cellular marfow is so well defined as a rule that one scarcely can avoid the conclusion that the connective tissue slowly displaces the marrow, gradually forcing it back. This hyperplasia of the connective tissue could be regarded as a protective reaction in the region of mechanical weakening rather than a general or local fibrosis of the marrow. The appearance is not that of a general and progressive increase in the reticulum of the mafrow, but rather that of the advance of a solid fibrous wall of connective tissue from the region of the cartilage into the marrow. Hemorrhage into the periosteum and also beneath it was very evident in some portions of the sections of the femur, tibia, and mandible. An intense hemorrhage occurfing under the periosteum of the tibia is represented in Figure 19. Phagocytosis or infiltration suggestive of infection was always absent. The periosteum is not thickened by proliferative changes, and since intensely hemorrhagic periosteum may lie in close contact with the compacta, it is clear that its detachment is not due to hemorrhage alone. Wherever the compacta of the diaphysis has become deficient, an ingrowth of connective tissue occurs, and I have the impression that in the guinea pig the so-called Gerüstmark, or framework marrow, really does not result from a fibrosis of the marro\ry, but is due to an invasion of connective tissue at the epiphyseal zone or elsewhere where the compacta has become deficient. Although absorption of bone is so marked in scurvy, osteoclasts are rarely present; and, although some osteoblasts remain, I never have encountered bone formation. Large multinucleated, degenerate masses simulating osteoclasts are sometimes seen near affected chondrocostal junctions, in close contact with the cartilage, but these may be remnants of the disintegrating cartilage or, possibly, degenerate myeloplaxes' Very large clear âreas, such as represented in Figures 20 and 2I, ate quite common in the marrow, but I do not understand their origin. They suggest edema, but it is diffrcult to see why the accumulation of fluid should be so localized. They may be so large as to occupy most of the medullary cavity. Cellular changes very likely are present in the marrow, but I have not as yet made a careful cytologic study, and routine preparations reveal nothing abnormal. Congestion, collapse, and autolysis are very striking in the lungs. In the completely collapsed areas the characteristic alveolar structure is lost, 11761 MINUTE MORPHOLOGY 51 its place being taken by a loose, degenerate connective tissue containing erythrocytes and much desquamated alveolar epithelium, as represented in Figure 22. Since these areas are not infiltrated and show no other signs of hepatization, they plainly were not due to pneumonia, although their gross appearance suggested this. Autolytic changes are quite evident in the lung, and the walls of some of the arteries have undergone almost complete lysis, as shown in Figure 15. The desquamated, swollen epithelium of the bronchial mucosa often completely fills the lumen, as shown in Figure 23, and some of the cartilage cells in the peribronchial cartilages may have disappeared completely, as shown in an early stage in Figure 24, The matrix of the cartilage not only is less dense, but it has become acidophile in some areas. cartilage cells in various stages of lysis are common, and the slvollen, degenerate nuclei often lie in the clear spaces formerly occupied by the cartilage cells. The most striking thing, however, was the presence of abundant vacuolation, especially near the subpleural region of some collapsed areas, as shown in Figure 25. These vacuoles were often much larger than the adjacent cerls, and also were very numerous. They were not confined to this region of the lung, however, but were contained also in the septa of otherwise apparently normal areas. Although the presence of this vacuolation suggested a fatty infiltration or degeneration from the beginning, stains for fat were at first negative. Later, however, frozen sections from several lungs were found compretery studded with the red areas which had a Sudan III color and which were plainly visible under low magnification. The staining of these areas with sudan III leaves no doubt that they contained fat, but the most surprising thing to me was the presence of fat in the peribronchial cartilages. The cytoplasm of the cartilage cells was studded with red granules and small droplets. That this fat was not introduced accidentally was indicated very clearly by the arrangement of the droplets, by the wide distribution of the cartilages containing fat, and also by the appearance of the cells themselves. It should be said, however, that I found the cartilage cells in the peribronchial cartilages from normal animals to contain granules that stain with Sudan III. As far as I know this has not been observed heretofore, and it is significant that the amount of material which stains with Sudan III is larger in the cartilages from the scorbutic animals. It was relatively easy to determine that the parenchyma cells containing the substances which stained with sudan III were not degenerate alveolar epithelial cells, and that the droplets were not contained in capillaries. small vacuoles were not found in the intact alveolar epithelium in apparently normal areas, but they were contained in reticulum cells, the nuclei of many of which were greatly swollen and forced to one side by tt77l 52 STUDIES ON SCURVY the large fat droplets which were contained in the cytoplasm. In areas of complete collapse one cannot be certain that alveolar cells may not also be affected by the fatty change, but I never found desquamated alveolar cells, which had undergone a fatty change, lying free in the alveoli. Often they could be identified quite positively by means of the pigment they contained. As in the areas of subcutaneous hemorrhage, so also in the lungs, there was little evidence of the existence of phagocytosis in spite of the presence of some free erythrocytes in the collapsed areas. However, some areas contained quite a number of eosinophiles. Vacuolation lüas never observed in the mesothelium of the pleura, but in cases of hydrothorax it had changed from a pavement to a cubical and even to a columnar form over considerable areas. The depressed areas noticed on several livers, and represented in Figure 10, were found to be areas of beginning necrosis. The parenchyma here was composed of a solid mass of swollen, ill-defined cells and nuclei, without the characteristic arrangement into cords. Blood vessels ruere not evident in these areas, which did not contain fat. This suggests that the fatty change around them may have resulted in interference with the blood supply and that it may thus have brought on necrosis. However, since the fatty change is universal elsewhere, and since these areas of necrosis were but seldom observed, this explanation does not seem very plausible. It is possible that they represent mere coincidences, but similar small areas of necrosis occur in the interior of the liver and were overlooked at first, largely because they were not evident on the surface. The superficial areas did not extend far into the liver, and it is possible that they have no direct relationship to the scorbutic change. Sections of extremely fatty hepatic areas, some of which looked absolutely white to the unaided eye, did not reveal the presence of much re' maining cytoplasm. As shown in Figure 26, no substance stainable with hematoxylin and eosin was present in some regions, not even the cell walls being preserved. Since all the cytoplasm, and also the nuclei, is gone from these areas, it is evident that the change concerned involved not merely a Í.atty infiltration, but a degeneration as well. Moreover, it follows that the cells destroyed could not undergo a regenerative change, and that if regeneration occurs it must proceed from the better-preserved areas, or from the bile ducts. The degree of degeneration varies considerably in microscopic specimens, as it does in the gross. It varies not only in different portions of the liver, but in different portions of a lobule, and of the individual cells even. An earlier though very marked stage of fatty degeneration is shown in Figure 27. This is a photograph of a section stained with Sudan III. However, fatty and necrotic changes are not the only ones that were oblr78l MINUTE MORPHOLOGY 59 diet for a considerably longer period, I cannot accept them as final or as indicated by the changes in the other organs. In the thyroid, as in other glandular organs, degenerative changes in the cytoplasm easily could cause an increase in the size of the follicular epithelial cells; and, although hyperplasia of the connective tissue occurs in the bones and probably also in the cartilages, no evidences of fibrosis have been observed in any other organ. Moreover, the use of the terms "acute" and "chronic" to differentiate scurvy in animals that have been kept for less or rnore than 33 days, respectively, on a partially deficient diet hardly seems justifiable; and a comparison between the thyroids taken from two guinea pigs which succumbed from starvation in eight to ten days with others from animals that were kept on a partially deficient scorbutic diet from 5I to 97 days is of very doubtful validity. Medes '26 reported the occurrence of congestion and degeneration of the germinal epithelium "in some of the tubules" of the testes, and stated that cells in the early stages of spermatogenesis were especially affected. She further found complete recovery within seventeen days from a condition of "chronic scurvy" produced by thirty to forty days'deficient dieting. I never found congestion, but the usual degenerative and lytic changes were present in both testes and ovaries in my series. Some of the germ cells were profoundly affected and others probably completely destroyed. The severer cases suffered complete destruction of the spermatozoa and the germinal epithelium, as shown in Figures 4I and 42; and in view of the presence of desquamated epithelium in the alimentary canal, bladder, kidneys, and the biliary and pancreatic ducts, I was not surprised to find degenerate testicular epithelium and remnants of spermatozoa in the ductus deferentes. Large multinucleated masses, probably fusion products, were frequently present in the lumina of degenerate testes, and vacuolation was observed in the interstitial cells. Many testicular tubules often contained only a few or only degenerate spermatozoa, the heads apparently surviving longest. All the epithelium might be profoundly affected, not even the Sertoli cells being preserved. Here, too, one may find the contrasted phenomena of rarefaction of the cytoplasm and condensation of the nuclei seen elsewhere. The epithelium of some tubules is studded with cells with pycnotic acidophile nuclei almost hyaline in appearance and with denser cytoplasm which also shows an acidophile reaction. Hyaline fragments may lie among these degenerate epithelial cells even when the tubules contain fairly well-preserved spermatozoa. The ovaries also seemed to contain an inc¡ease in the amount of fat, but since I did not make a special study of this matter and since normal ovaries contain a large amount of fat, a more definite statement must wait I18sl 60 STUDIES ON SCURVY upon a more comprehensive examination. In view of the changes in the testis, I should expect to find similar changes in the ovary. The lymph nodes showed no very marked changes, except that those draining heÍrorrhagic areas contained many free erythrocytes. Some of these nodes are typical of what used to pass for hemolymph nodes, for their sinuses are engofged with blood, but they contained almost no evidence of phagocytosis, while the sinuses of others which contained only a few erythrocytes were crammed with eosinophiles, only a few of which had segmented or polymorphous nuclei. Others contained moderate numbers of free shadow forms of erythrocytes and large numbers of macrophages filled with degenerate erythrocytes. I found only moderate amounts of pigment and relatively few nodes u/ere congested. Microscopic preparations of the cardiac musculature show the same lack of definition observed in other tissues. The flner architecture becomes obscured and the stain is uneven. The nuclei Seem mofe numerous than normal, and the myofibrils, the cross striations, and intercalated discs are less clearly defined of not fecognizable at all, except in areas of rarefaction. In these, especially the myofrbrils and cross striations, when seen in longitudinal section, may be more evident than normal and the safcoplasm as seen in cross sections apparently is increased. The nuclei, too, are less constant in size and many of them are s\4tollen, rarefied, and vesicular, and areas of vacuolation appear in the fibers, the myoplasm of which may undergo lysis so that the nuclei become free. Frequently these nuclei lie approximately in a ro\ry and fuse to form nuclear complexes. The latter may be composed of only t\4/o or of as many as a dozen nuclei, and give a very striking appearance to the areas so affected. However, since some of the smaller of these linear nuclear complexes afe sometimes found in the muscle fibers, still surrounded by cytoplasm, it is difficult to account for them on the assumption of fusion. I found no convincing evidence of the production of these multinuclear masses by direct division, although such a possibility cannot be excluded with certainty, and I never found areas of cardiac muscle which contained such an increase in the number of nuclei as were found in the hemorrhagic skeletal muscle undergoing waxy degeneration. Although a condition suggesting beginning hyaline degeneration occurs, I never encountered waxy degeneration, which ìMas sometimes so pronounced in the skeletal musculature. ' fn some areas the cardiac musculature is quite degenerate and in the more lytic regions the outlines of the muscle frbers are incomplete, and the swollen nuclei have undergone more or less complete solution. However, in the areas in which a granular disintegration of the muscle has occurred, the nuclei are usually more pycnotic and are contained in a mass of granu- [186] MINUTE MORPIIOLOGY 6t lar debris. Vacuolation and dissolution of the walls of the veins and arteries also occur. The changes in the musculature of the urinary bladder were similar to some of those in the intestine and ducts, and my findings confirm those of McCarrison '21. Hemorrhage into the mucosa often was intense and not infrequently seemed confined to it and the subserosa in gross sections at autopsy, so that the vesical wall had a very thin outer and a much thicker and darker inner red layer. Hemorrhage into the mucosa often also was very intense, as shown in Figure 43. The mucosa not only was hemorrhagic and showed evidences of lysis, but not infrequently had been desquamated partly or wholly over considerable a¡eas. Large plaques of cells lay in the cavity. Sometimes these plaques lay near the place of detachment, as shown in Figure 44,but at other times they were included in a mass of semen and urinary sediment, not rarely found in the bladders of male guinea pigs. \Mhen one considers the profound changes present in the mucosa and musculature, and probably also in the nerves of the bladder, one scarcely can wonder that urinary incontinence occurs in some of these animals. The hemorrhage and desquamation alone would imply irritability. The first change obse¡ved so far, in stained sections of voluntary muscle, was the unevenness of the staining and a blurred appearance of the sections, as shown in Figtrre 45. In some instances a portion of the cross section of a frber had lost the uniform appearance and had taken a basic stain. The cytoplasm of others contained several nuclei, as many as twenty-five having been observed in the cross section of a single fiber. In other fibers some of the nuclei of the sarcolemmae were missing, their place being represented by clear areas. Hemorrhagic muscle may be changed so extensively. as to be scarcely recognizable in microscopic preparations, and I found it true, as stated by Moore and Jackson '16, that "certain portions of muscle fibers are completely disintegrated." This is represented in Figure 46 and was also observed by Holst and Frölich 'I2, who spoke of fatty degeneration and of a disintegration of the muscles into irregular hyaline "lumplets" which did not stain like normal flbers. They stated that small collections of sarcolemma nuclei lay between these lumps. Such a change is shown in Figure 47. I observed only slight fatty infiltration in a few specimens, although many were stained with Sudan III, Scharlach R I and II, and osmic acid. Marked hydropic degeneration frequently was present, some of the fibers being completely fenestrated, as shown in Figure 48. Sometimes these clear areas, although numerous, were scarcely visible with an oil-immersion lens, while in other fibers they occupied the greater portion of the cross section. Some of the smaller areas are shown in Figure 49. I have not t1871 62 STUDIES ON SCURVY been able to obtain a reaction for fat in these clear areas by means of special stains, and since Holst and Frölich dicl not state how they determined that fat r¡ras present I surmise that it \ryas an inference made on the basis of the microscopic appearance of material not specifically stained for fat. Sections of hemorrhagic muscle showed the presence of a pronounced waxy change. Usually there was a great increase in the nuclei in these areas, as represented in Figure 47. 'fhe appearance of these nuclei, and the absence of infiltration suggests that they probably arose from the sarcolemma, although I doubt that their increase in number represents an attempted regeneration. Sometimes a group of nuclei lay in the center of a clegenerated mass, and at other times they formed a circle near the periphery. Some of these areas were small and contained much evidence of lysis, but others were large with little evidence of lysis. Although near-by fibers seldom were wholly unchanged, they often were remarkably well preserved, and I am entirely at a loss for an explanation for this peculiar selective action of the scorbutic process. Although it will be interesting to follow the reparative changes in such areas as these, it does not seem that restitutio ad integrum is likely to occur after such extensive changes as those shown in Figures 46 and 47. Just as in the case of the glandular organs, so also here, nothing but a foamlike residue remained in some areas, as shown in Figure 50. Not infrequently the degeneration was very marked locally, so that adjacent fibers might be but slightly affected. This focal degeneration often took the form of a skein, as represented in Figure 51 and to a lesser degree also in Figure 52. Although previous investigators had spoken merely of the occurrence of a pseudoparalysis, it seemed that the lack of a constituent in the diet, which is indispensabie to an organism and which affects so many other systems, should also affect the nervous system. Since hemorrhages occur in such widely scattered areas of the body, it seems that the vascular supply of the nervous system should also 'be affected or that the nerve sheaths at least would be infiltrated. Although I have not as yet made a thorough investigation of changes in the nervous systems, I have found cerebral hemorrhages, such as represented in Figure 53, and I also have encountered extravasations into the posterior root ganglia and lumbar motor roots, as represented in Figures 54 and 55. In case of the cerebral hemorrhage, remnants of the arteúal wall lie among the erythrocytes, and the hemorrhage is separated from the cortex by a clear area. There is no indication of phagocytosis, although the hemorrhage apparently is a relatively recent one. Evidences of lysis are present, however. In the posterior root ganglion from the lumbar region (F'igure 54) only scattered erythrocytes are present, but degenerative changes also occur in 11881 MTNUîE MORPHOLOGY 63 the ganglion cells, the nerve roots, the peripheral and central nervous systems, and also in the sympathetic ganglia. The nerve roots of the cauda equina reveal the complete destruction of some of the medullary sheaths and axis cylinders. They also contain very small vesicular oval nuclei, regarding the origin of which I still am uncertain; they may come from the neurotremma. The medullary sheath may undergo lysis, as indicated by vacuolation, and when lysis is complete the place of the fiber is occupied by a larger clear area just as in the case of the muscles. Sometimes several of these areas lie close together, and when they are near the periphery of the section they protrude beyond the normal outline of the root, just as did the degenerate fatty cells at the periphery of the liver. Sometimes an extremely attenuated crescentic nucleus lies against the wall of a clear area, and at other times a greatly swollen and rarefied axis cylinder is surrounded by only a narrorvr¡ rim of clear cytoplasm, or a shriveled axis cylinder lies eccentrically in a clear area. In other instances an apparently normal axis cylinder is surrounded by a fair amount of acidophile cytoplasm and this in turn by a clear zone bounded by the neurolemma. Large, clear areas containing only remnants of nerve fibers also are present within the nerves and, although not all the roots of the cauda equina of this case are equally affected, none are unaffected. Portions of cutaneous nerves included in hemorrhagic subcutaneous tissue and muscle also are profoundly affected. An earlier stage of degeneration is represented in the photograph of a cross section of a nerve in skeletal muscle, shown in Figure 56. Some superficial nerves are edematous in the center so that degenerate cytoplasm surrounds a central, clear area, but the most marked degeneration was noticed in peripheral nerves lying in a small piece of voluntary muscle excised from the leg, and represented in Figures 57 and 58. In one instance the degeneration was so marked and so peculiar that the nerve is hardly recognizable as such. A large portion of the cross section was formed by waxy, basophile masses of various sizes, and the rest was composed of granular acidophile materials containing small pycnotic and vesicular nuclei. Only three nerve frbers can be identified positively in the section. The epineurium is represented by a faintly staining, hyaline mass, containing a few nuclei. In earlier stages of this apparently waxy degeneration, as revealed in cross sections, the fibers lose their circular outlines, become indistinct, and fuse more or less completely into larger masses. The axis cylinders are greatly swollen, the medullary sheaths are less evident, and the epineurium is separated from the contained fibers by a clear space which may result from edema. It will be recalled that Holst and Frölich stated that they found Vy'al- [18e] 64 STUDIES ON SCURVY lerian degeneration sometimes accompanied by swelling and fragmentation of the axis cylinders. Although I have not examined many specimens, r have found degenerative changes in peripherar nerves which are evident by the use of the Marchi method. A section of an ischiadic nerve with this degeneration is shown in Figure 59, and it must be quite evident that such changes as reported here easily explain the occurrence of paralyses, although they do not account for their appearance in the posterior Lxtremities.Hemorrhage occurs also into the spinal cord, as shown in Figure 60, and degenerative changes are present in the large motor cells of the anterior horn of the lumbar cord. In these cells lysis and vacuolation also occur and shadow forms are not uncommon. The outlines of some of the cells are very irregular and the. cytoplasm is surrounded by a clear area. other cells are represented by degenerate masses of cytoplasm with disintegrated nuclei. changes observed in cross sections of the spinar cord remind me very decidedly of those observed by Miller in swine-Hart, Miller, anã Mccollum '16-and of those reported by myself (Meyer 'rz) in a case of voluntary fatal starvation in man. This would seem to suggest that the lack of different vitamines may result in similar morphologic changes. Hyaline areas of degeneration are seen also in the fiber tracts, and rarely such a degenerated mass contains several nuclei, thus reminding one of similar changes in the skeletal muscles. Some sections of both cord and brain were studded throughout by clear areas which one might call vacuoles if they were intracellular. These areas were exceedingly numerous, as shown in Figure 61, and because of their size it seems màre correct to speak of fenestration. As shown in Figure 62, it was present in the cerebrum also. It does not seem unlikely to me that this degenerative change, as well as those in the cortex of the adrenal, may be responsible for the highly nervous and restless state characteristic of some of the animals. changes in the number and the size of the Nissr granules, even to their complete disappearance, also were noticed, and it apparently is the failure of recovery from the central lesions which is responsible for the permanent spastic condition observed in the posterior extremities of somè animals. Although this condition fully simulates ankylosis at the knee, a complete celloidin series of the right knee of the pig represented in Figures z and,3 showed that there 1ryas no union of the opposed articurar surfaces, and gross examination of the left knee showed that the articular cartilages on the femur, tibia, and patella had undergone no marked changes. yãt this pig had what seemed to be ankylosed knee joints for a period. of over two years, until killed. upon microscopic examination of the right knee, the articular [1e0] MINUÎE MORPTTOLOGY 65 cartilages were atrophic in some places where they were not in direct contact, but in all regions of contact and also upon the patella they were quite well preserved. Since relatively slight motion is sufficient to preserve the articular cartilages over long periods of time, this is not surprising, but the entire absence of any evidence of ankylosis is indeed so. Moreover, it leaves one without an explanation for this permanent stiffening in the hind legs except lesions in the nervous system, and perhaps to a minor degree in the muscles, which in this case, however, did not seem to have undergone frbrosis. The earliest microscopic changes observed in the teeth were congestion, hemorrhage, and lysis at the base of the pulp, as shown in the premolar represented in sagittal section in Figure 63. Hemorrhage into, and separation of, the periodontium also occurred early, but edema and hemorrhage alone do not seem to result in detachment of it from the tooth. In case of the incisors this hemorrhage always rvr/as most marked opposite the enamel. Later a more intense hemorrhage, as illustrated in the pulp of the incisor shown in Figure 64, and also cavity formation and lysis, such as shown in Figure 65, were found. These cavities in the pulp of incisors are diffrcult to interpret, and some of them may represent cysts, but since none of them were found to contain any stainable content or lining epithelium this seems rather unlikely to me. Some contained a faintly evident coagulum, however. They varied greatly in size, and sometimes extended only through a few colloidin sections cut twelve micra thick. Others were very much larger and looked more as though they had been formed through local edema, and the surrounding connective tissue condensed by pressure. According to Cohn '27 such changes as these, among other changes, described by Höjer and \Mestin '24, occur in the teeth of caged guinea pigs and only are more pronounced in scorbutic animals. The sections of teeth from normal guinea pigs which I have so far examined do not support Cohn's statement, although a larger series may do so. It should be stated, however, that Cohn drew his conclusion from the examination of the incisor teeth of only three animals which had been infected for other purposes ! Moreover, since Cohn did not examine these animals further and spoke of the presence of regenerative processes in the incisors of scorbutic animals, his statement, as well as his evidence, is very unconvincing. Since the pulp sometimes was surrounded wholly or partly by a clear area which contained a coagulum, it is evident that separation of it probably was not the result of shrinkage during fixation. A definite odontoblast layer, as usually represented, was present in none of the teeth examined, and I never saw any evidences of bone formation in the incisors described by Höjer and Westin. As hemorrhage, edema, and especially lysis increase in extent, the extremities of the roots of the teeth become greatly thinned and finally tlell 66 STUDIES ON SCURVY completely absorbed beginning from the extremity of the root. Near the end of the root only a few remnants of the osteodentine may remain. It should be remembered, however, that the osteodentine may form only an extremely thin layer as compared with the overlying dentine and enamel farther up. Since the osteodentine has more the structure and probably also the chemical composition of bone, it is not surprising that it is affected early by absorption, and I always found a decrease, not an increase, of it in the roots of the teeth. Hence, I am wholly at a loss to account for the observation of Robb et al.'2I that it was increased one hundred times in the teeth of scorbutic guinea pigs. No evidence of the presence of an inflammatory reaction was observed in any of the teeth examined, and only occasional osteoclasts and osteoblasts were noted on the alveolar walls or elsewhere on the jaws. Hence absorption is not accomplished through them, and the portions of the teeth and jaws which remained always had a normal structure. I saw no evidence of an iucreased porosis of the bone or osteodentine, nor did I note any such changes in the dentine. Since the enamel usually is completely removed in the processes of decalcification, one cannot be absolutely certain how large a gap should normally be present between the dentine and the periodontium in histologic preparations; or between the latter and the osteodentine; or between the chondroid processes (Knorpelzement) or the cement pearls and the rest of the tooth. However, by comparing sections from opposite extremities of the same molar tooth, as shown in Figures 66 and 67, one can easily see that an increasingly large space is present the nearer the extremity of the root is approached. Had all this space been occupied by enamel, the latter must have been three or four times as thick near the end of the root as upon the free portion of the tooth. Moreover, since the periodontium often is very hemorrhagic and hence apparently thickened, it follows that the actual absorption upon the implanted portion of the tooth and upon the walls of the alveoli must have been greater than is indicated by the spaces found in celloidin sections of affected teeth. Since the amount of shrinkage in the tooth, during preparation, is probably not greater than that in the surrounding tissues, I do not think that much account need be taken of it. In the earlier stages of scurvy, some of the chondroid processes, or organs rather, and of the cement pearls (von Brunn) remain closely in contact with the tooth, but later a wide gap may exist between them. That the molar teeth may also be dislocated somewhat after they have been reduced in size and the alveoli have become enlarged by absorption, is indicated by the fact that the toe of the boot-shaped anterior chondroid organ is not always found in the corresponding region of the surrounding space, as it normally should be; and it is the tiiting of the loosened molars in the Í tezl MINUTE MORPHOLOGY 67 enlarged alveoli which is responsible for irregularities in alignment and wear, and not a bending after softening from demineralízation. On the basis of an extensive study of scurvy in man, Aschoff and Koch surmised that the scorbutic condition is due to a lack of, or to a failure in the formation of, cement substance (Kittsubstanz), but this study of experimental scurvy in the guinea pig does not offer any evidence in support of this supposition. The predominant picture is one of destruction and the earliest demonstrable morphological changes were intracellular and concerned the epithelia of many organs, muscles-both striated and unstriated-and nerve cells and fibers as well. After observations and experiments on guinea pigs, rily'olbach and Howe also wrote: "\Me characteÅze the condition of scorbutus as inability of the supporting tissues to produce and maintain intercellular substances. . . . Direct proof of this conclusion has been obtained in a study of teeth in regard to dentine, in the study of growth and repair of bone, in regard to bone matrix, and in the study of repair of soft tissue in regard to collagen of connective tissue. Our proof in regard to cartilage is incomplete. . . . . The proliferative power of the epidermis, endothelium, fibroblasts, and osteoblasts is not diminished in scorbutus. We are reasonably certain that it is augmented in the case of osteoblasts, which, however, undergo striking morphologic change." I have not experimented with repair after injury in scurvy, but all the evidence I have seems to contradict these conclusions, and since bone and teeth undergo resorption, it is difficult to believe that osteoblasts can be more active under such circumstances or under those of epiphyseal separation. Moreover, since Kittsubstanz, or intercellular substances, whatever they are and wherever they may be, must be cell products, it is evident that the cytoplasm must be first affected, and that this is quite generally the case is inclicated by the evidence here presented. I never found that normal glandular, muscular, nerve cells, etc., became separated from each other; but I did find that the cytoplasm of these celis underwent degenerative changes of the profoundest sort u'hile the cells still lay in their normal positions. Plaques of epithelium of various sorts were detacired, but these never were composed of normal cells, and since they were detached as plaques the hypothetical intercellular substance rnust manifestly have held even these degenerated cells together. Sulvrlvrenv l. Hemorrhages were noted rarely in the skin, almost universally somewhere in the subcutaneous tissues, very commonly in the muscles, the urinary bladder, the periostea, the periodontia, many glandular organs, lungs, stomach, and intestine. They were also seen in the gail-bladder, the brain, [1e3] 68 STUDIES ON SCURVY spinal cord, posterior root ganglia, and the nerve trunks. Intragingival and intra-articular hemorrhages were not observed. 2. The marked fatty infiltration and degeneration evident grossly in the liver and sometimes also in the kidney were found present microscopically in the kidney, the adrenal, and even in the lung and the peribronchial cartilages, the pancreas, some skeletal muscles, and the walls of a blood vessel. 3. Degenerative changes other than fatty, resulting in the complete loss of substance, \4/ere observed in the cartilages, bones, teeth, muscles, many glandular organs, and also in the central, the peripheral, and the sympathetic nervous systems. 4. This widely distributed liquefaction of the cytoplasm and cell walls results in destruction of cartilage cells and of the cartilaginous matrix, at least in the costal cartilages; in the detachment of the periostea and periodontia; in reduction in caliber of the bones, and in both the caliber and the length of the implanted portions of the teeth. It may efiect not only the separation of ununited epiphyses and loosening of the teeth, but the complete destruction of the parenchyma of some areas in the glandular and other organs, in desquamation of the mucosa and of renal epithelium, and in complete disintegration of the walls of blood vessels. 5. In addition to the fatty and lytic changes, coagulative changes, such as extreme waxy degeneration in the múscles, ï¡ere also noted in glandular and nervous tissues. 6. The only proliferative changes noted occurred in the costal cartilages and concerned an increase in caliber at the region of the costochondral junctions and an invasion of connective tissue into areas of degeneration. 7. Necrotic areas on the surface and in the substance of the liver were occasionally observed, but may be wholly unrelated to scurvy. 8. Vacuolation was common in many organs, and {enestration up to a marked degree was observed, especially in muscle, bone rnarrow, the pulp of the teeth, and also in the cord and brain. [1e4] Pr..r.Ta V, Pan'r. II FIGURE PÂcD 30. Disintegration of the wall of a large hepatic vein. ........... 53 31. Fatty degeneration in the kidney. Sudan III. . ..... . . 53 32. Plaques of desquamated renal epithelium ir-r the collecting tubules of the kidney 53 33. A ma¡kedly degenerate area in the cortex of the adrenal, showing the complete effacement of the cell outlines. .... ..... . 54 34. Yery degenerate area in the mecfulla of the adrenal, showing some cortical shadow cells in a foamlike residue and some pycnotic, .degenerate, medullarycells ........55 35. Degenerate changes in the cortex and medulla of the adrenal .... ... .... 55 36. A lytic portion of the gastric musculature. Only remnants of the latter remain 56 37. Lipomatosis o{ the pancreas. Note the small pancreatic islands. ...... 57 \1 ¡:nrt ,u. St n.:rt r.:s. \''or-. I I. No. 2Itr.,tl r.: \r. I',rlll I I Pr-aru VI, P¡nr II FI GURE 38. A huge, almost isolated pancreatic isla'd. Note the normally sized island a little distance below.. 39. Fatty infiltration of the pancreas ancl of the wall of a large artery. The der-rsely black areas represent body fat. Sudan III. 40. Degenerate area in the spleen. 41. Degeneration of the seminiferous tubules. 42. Almost complete degeneration of a seminiferous tubule. Note the large fusion products, simulating giant cells. 43. Hemorrhage into the mucosa of the urinary.bladder. 44. A plaque of desquamated mucosa in the lumen of the urinary bladder. 45. Cross section of a skeletal muscle, showing uneven staining, complete lysis of some fibers, and destruction of the sarcolemma of others. P.{GE 57 57 57 59 59 61 6l 6l M¡inr<:¡r, Scrnxcns, Vol,. II, No. 2Pr-,rrr.: VI, P¡nr II Pra:rri VII, Panr II FIGURD P.\6E 46. Markecl waxy degeneration of skeletal muscle. 47. Increase in nuclei and pseuclo-giant cell formation in a very degenerate area of a skeletal muscle. 48. Marked hydropic degeneration of skeletal musculature. 49. Earlier hydropic degeneration of the same. 50. Foam-like residue in a degenerate area oÍ. a longitudinal section of a skeletal muscle., .....i. 51. Local skein-like degeneration in a skeletal muscle. 52. Local degeneration in skeletal muscle, the striations in which are still preserved . 53. Hemorrhage into frontal lobe of the cerebrum...... 6t 6l 61 6t 62 62 62 62 { ''.1b- .: xt:. ir r ^t 7. = .; : 7 = a 'f. j a. : : /. .çr..,...1, Pr-etn VIII; P¡nt II FIGURE PÀGD 54. Hemorrhage into a posterior root ganglion. Scatteied erythrocytes and degeneration, especially in the large nerve cells. 55. Hemorrhage into a lumbar motor root. ........ 56. Early degenerative changes in a peripheral nerve. (tì i'rtCro.r sections of peripheral nerves illustrating marked degeneration. Jð} 59. Marchi degeneration in an ischiadic nerve. . 60. A small area of hemorrhage in the spinal cord.. . 61. Fenestration in the lumbar region of the spinal cord... 62 62 63 63 64 64 64 ,\lr.:rrt .rt. St r.:rt r..s. \'or.. I I. ,\o. fI'r. r'r'r: \' I I I. I'.rrll I I l ä Pr,em IX, P¡nr II FICURE PAGE 62. Fenestration in the frontal lobe of the cerebrum........... .:..;. .,,........ ó3. sagittal section of a right premolar (on the left) and cross section of the lower incisor (on the right). Some hemorrhage into the base of the pulp of the ' former and cavity formation in that.of the latter. 64. Intense hemorrhage into the pulp of lower incisor. 65. Cross section of the mandibles showing cavity formation in the pulp of the lèft lower incisör and lysis of the pulp of the right. 6ó. A section of the same premolar somewhat deeper down, showing the effect of lytic processes as indicated by the widening of the peridental space..... .. . 67. Entfte cross section of a lower right premolar and a pôrtion of a longitudinal section of a right lower incisor, showing cavity formation in the pr_rlp of the latter 64 65 ó5 65 66 65 Mrorc,t Scrnxces, Vor. II, No. 2Pr¡re IX, P¡nr II I