The symptomatology and gross 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 I. THE SYMPTOMATOLOGY AND GROSS MORPHOLOGY OF EXPERIMENTAL SCURVY IN THE GUINEA PIG I. THE SYMPTOMATOLOGY1 AND GROSS MORPHOLOGY OF EXPERIMENTAL SCURVY IN THE GUINEA PIG' By Antrrun W. Mrvpn CoNDITToNS oF TrrE Expenrrvrex:r A so-called synthetic, basic diet was not used in these experiments, partly because my original object was to ascertain, if possible, the causes for the appearance of peculiar phenomena in guinea pigs kept on ordinary feeds and used in experimental anatomy. Among these phenomena \ryere marked nervous manifestations and permanent, as well as profound temporary, locomotor disabilities. Competent investigators, both from at home and from abroad, who had seen some of these animals, were of the opinion that, if they suffered from experimental scurv¡ the signs certainly were anomalous. A competent bacteriologist who saw some of them and inspected a number of the necropsies was of the opinion that death was due to bronchisepticus pneumonia. Upon my mentioning this to a prominent European endocrinologist, he stated that rabbits which he and a colleague had used years ago, and which had suffered similarly to my guinea pigs, were believed to have died from infection with an organism called "Bacillus cunnisepticus" by his colleague. However, some of my animals, which were lost after unavoidable exposure to cold, did not seem to me to have suffered from pneumonia. Moreover, the behavior of a few that survived longest fortunately reminded me of the pictures of Cowgill's '21 dogs, and this supplied the clue. Hence, I injected tomato juice subcutaneously after the method of Cowgill, and began the feeding of tender lettuce leaves to some of the younger animals. They recovered as marvelously as had Cowgill's dogs. I do not mean to imply, however, that Cowgill's dogs did not suffer from polyneuritis, or that some of the animals in our two large lots, lost after exposure, did not die of pneumonia. 1 Since the occurrence of symptoms in an animal always is a matter of inference, I use this word in the title with some reluctance. It might seem preferable to use the word "signatology," but one hesitates to add another term-especially a hybridto the already grievous number. Yet there is no question that the common confusion of the words "symptoms" and "signs" often leaves an author's meaning obscure. This is true also in the literature on scurvy. Moriquand, Michel, and Bernheim '24, for example, state that they produced experimental scurvy in guinea pigs with all the symptoms of human scurvy, and Smith '27 reported. that "two guinea-pigs showed none of the recognized s1¡mptoms of scurvy." 2 I gratefully acknowledge generous support for this investigation from the Committee on Research of the American Medical Association, t1331 17 8STUDIES ON SCURVY I had not previously become suspicious regarding the dietary of our animals because we had used the same feed for almost a decade without difficulty, and during all that time every visitor had remarked upon the fine appearance and the large size of our experimental guinea pigs. But upon inquiry I found that the fresh lawn-clippings which formed a part of the feed had not been received regularly for some time. Until vitamines are available in chemical purity,"for experimental purposes, it will, to be sure, remain impossible to ascertain the exact dietary deficiency to which particular disabilities and lesions observed in experimental animals are due. And as long as the exact vitamine content of food, the need of a particular animal for vitamines, and the exact number of vitamines remain unknown, no one can be certain regarding his conclusions. To date, all our knowledge is largely empirical, and one might gladly wait upon the chemist were the subject not of such surpassing interest and outstanding importance, and did it not have such direct bearings upon all other experimental work. It was the latter fact that compelled me to turn aside from other investigations in order to determine, if possible, what cytological changes in our experimental animals had resulted from deficiencies in diet. Ever since the possible rôle of vitamines has become known it has seemed to me that qualitative rather than quantitative defrciencies in diet might have been responsible for some of the changes reported in starvation. I have in mind especially changes similar to those reported by Meyer '17, although f have no evidence whatever supporting the view that the lesions of scurvy are due to ordinary inanition. It would be interesting to ascertain what cytological changes would result from a diet quantitatively sufficient, but lacking wholly in vitamines. The guinea pigs used in these experiments were kept under controlled temperature, recorded three times a day, and were given a basic diet of raw alfalfa hay, rolled badey, and water, in abundance' In some series the afiaffa hay and barley were heated to 100o C. for an hour, with gas, with electricity, or with steam, separately and also together. Lawn-clippings, lettuce, orange juice, wheat germ, and evaporated milk were added singly, or in combination, to the diet of some of the series in order to determine experimentally the identity of the vitamine from the lack of which the animals had suffered. The only noticeable effect of the presence of wheat germ or evaporated milk in the diet was a slight prolongation of the life of the pigs. Although 10 grams of one of these feeds, or of both, were fed daily, mixed with moistened rolled barley, it is not possible to say just how much of each ingredient the pigs ate. However, some animals which ate the mixture well survived no longer than those that ate very little. This would seem to indicate that the latter apparently received enough of other vitamines. t134I GROSS MORPHOLOGY Bn¡revron oF TrrE ANrlr¡r-s The time at which the signs of illness appeared varied with the age of the pigs, although some fairly mature animals failed to succumb after several months on the raw basic diet. This agrees with the observations on human scurvy and with the splendid pioneering observations of Holst and Frölich '12. Jackson and Moore '16 also reported individual variations, although it is difficult to understand how they could produce scurvy with a diet containing green vegetables, unless the latter were given in exceedingly small quantities, or were of a kind of which the animals ate an inadequate amount. The native difierence in resistance to scurvy, noticed also by Furst '12, suggested the possibility of developing a particularly resistant strain of pigs, which, in itself, might throw some light on the question under investigation. It is possible, however, that the differences in resistance noted are not due to hereditary differences in resistance to scurvy, but to differences in predilection for food on the part of the animals. If the bacterial flora of the intestine is a factor in this matter, the habits of the pigs also might play a r.ôle in these apparent native differences in resistance to scurvy. Pregnant and fairly young animals, those weighing about 400 grams, suffered and succumbed eadier, although some difference in resistance was noticeable among all ages. However, Nabel '23 stated that pregnant animals die before they become scorbutic, and that even when the mothers are fed as long as 39 days on oat bran and milk boiled for two hours, the young show no evidences of scurvy. The non-pregnant in Nabel's series, on the other hand, all became scorbutic. Although I did not investigate this point especially, the few pregnant pigs among my series all suffered from scurvy both clinically and anatomically, and so did the young, and all succumbed from this disease. The older animals usually \ryere more resistant, and none of these became absolutely helpless in the posterior half of the body, as did the young animals, which suggests that such helplessness as that shown in Figure 13 may not be due to a true paralysis. However, Sffiith'27 stated that in "chronic scurvy" the guinea pigs were "paralyzed" so completely that they could not stand, and lay in one position so long that decubitus developed. This paralysis was said to have extended from the pectoral girdle "down," yet Smith suggested that the lack of movement in these animals may have been due "to the great pain occasioned thereby." I never saw decubitus and none of my animals lived long enough after they became helpless to develop it. Helplessness always indicated death within a few days at most, s The figures for Parts I and II appear, consecutively numbered, on half-tone ptates, those for each Part immediately following its text. ll3sl 9 10 STUDIES ON SCURW and I do not comprehend Srnith's statement that great pain prevented, paralyzed, animals from walking. According to Smith, "The first manifestations of vitamine deficiency in the guinea-pig are swollen and tender joints." I never saw swollen nor found tender joints in mature animals, and the first change noticed in the behavior of my animals was a lessened activity and a reduced interest in the feed and feedings. Poupart '99 mentioned weakness in man, and, according to Boerhaave'76, van Swieten stated that lassitude is one of the first symptoms of scurvy in men. Since our cages \Mere only about two feet square, it is not easy to determine whether the animals felt lethargic first, but it seems a fair presumption that they dicl. Cohen and Mendel 'LB also mentioned the presence of lethargy in their animals. There may also have been some anorexia, for the animals soon began to manifest little, and finally no, interest, not even in green feed exhibited before their cages. This reduced interest in the feed was noticed because the pigs first called less loudly and less insistently for it, and then gradually desisted altogether. The decrease in activity gradually increased until the animals sat still. After several weeks on the basic diet, they showed some tendency to pick out the tender constittrents of the hay and the smaller particles of the rolled barley, suggesting the presence of tender or painful teeth or difficulty in chewing, or both. Schepilewskaja and Jarussova '26 also stated that the pigs used by them ceased to shell out the oats after a time. Some of our animals were found apparently chewing most of the time during the later stages, and frequently were found to have a small morsel of chewed food in their mouths at the time of death. I did not, however, find that the pigs "were hungry and ate voraciously" till death, as reported by Smith of paralyzed animals in "chronic scurvy," and hemorrhagic, ulcerated, or gasdistended, fou'l stomachs would certainly make that unlikely. However, Poupart spoke of a canine hunger to the end, in man. It is possible that the tendency to continued chewing movements in guinea pigs may be due to pain rather than hunger, for, as noticed also by Heymann '26, they do not seem to swallow. Since Poupart stated that the gums of children itched so intensely that they tore them into shreds with their finger nails, it is possible, but unlikely, that guinea pigs seek relief from a similar sensation by chewing. When the sensory effects of the presence of hemorrhage and edema in the periodontium are considered it will not be any more difficult to understand why the guinea pig ,chews incessantly than to account for a similar tendency on the part of a teething child. All the animals, except some that died early, evinced some difficulty in chewing, and preferred soaked, or otherwise softened, food in this stage. Many of them, however, chewed at hard dry alfalfa stems even in late l136l GROSS MORPHOLOGY 11 stages, and pressure against or traction upon the incisors did not seem to cause pain. It is, of course, difficult to examine the molars, but I never discovered any sensitiveness, although it would seem that it must have been present. The eyes of some animals lost their luster as the disease progressed, and a slight roughening of the hair, especially about the anterior portion of the body, was sometimes present in the later stages. This roughening became more general as a rule near exitus, although it often was slight or absent altogether. Only two animals among several hundred had glued lids for a few days, and in these neither the lids nor the eyeballs showed gross structural changes with a hand 1ens, although an excess of secretion was present. In the later stages the animals seemed drowsy and closed the eyes more frequently and continuously. They also felt cool to the touch, and crawled under the surplus hay or any protecting material that was placed in the cages. Some of them were soiled with blood and urine about the anus and vulva, or with feces, or with all three, and in some a large soft mass of foul feces protruded from the anus for a week or ten days before death occurred. A few passed very soft or hemorrhagic feces or suffered from vesical incontinence during the later stages, probably due to hemorrhages into the musculature, the mucosa, or the cavity of the bladder. Since vesical hemorrhages were present in a pig that had lost only 8.4 per cent in weight, it is evident that they can occur in otherwise well-nourished animals. My observations confrrm McCarrison's'21 in this regard, and I am certain that microscopic examination of the urine would reveal the presence of hematuria in a large percentage of cases. The lower jaw and throat of some pigs were wet with a greenish fluid, and a foul odor was relatively common in the later stages of the disease; but diarrhoea or melena were infrequent. The condition of the stomach at autopsy, of gassed pigs, as well as the condition of the mouth and lungs, shows conclusively that the foul odor is largely gastric in origin. Even in cases in which gastric distension was moderate this odor was rather "unsupportable," as emphasized by Poupart in scurvy in man. It certainly is an odor characteristic of putrefaction, although Smith holds that there is no increased putrefaction. The presence of a small amount of foul discolored food thickly coated with mucus was common in the stomach. Instead of sitting quietly in a corner, as most of the animals did, a few became very nervous and seemed apprehensive. They acted as if frightened and startled easily at attempts to take them up or upon the occurrence of a sudden noise. Some of these pigs stood upon their hind legs against the sides of the cage when alone or as soon as they \ryere approached, and looked toward the solid ceiling as if they hoped to escape through it. That ttsT l t2 STUDIES ON SCURVY these animals frequently assumed this attitude when alone was shown by the fact that the walls of the wooden cages were soiled and scratched at the level at which their heads and front feet touched them and by the packed condition of the sawdust and alfalfa. Some of these pigs executed what Kihn '22 called, "Reitbahnbewegungen," if I understand the word correctly; anything that startled them caused them to run around the periphery of the rectangular cages, and that they often did so when not startled by any one was shown by. the presence of a definite path in this region of the cages. The sawdust and feed were trampled down smoothly especially here, and somewhat also elsewhere, indicating that the animal had spent most of its time walking or running around' A few of the animals were so nervous that a sudden noise startled them so that they ran wildly around bumping their heads against the corners, until they fell exhausted and lay panting and helpless wherever they had dropped. These pigs ran around quite frantically, and one of them remained in a comatose condition and for an hour it could not maintain its equilibrium after it had been set upon its feet. Aside from the statement of Hess and Fish 'l4,that the knee kicks of children may be increased in scurvy, I know of no observations such as those here reported, except the tremors in men mentioned by Boerhaave, the frightened look, the headaches, and the convulsions spoken of by Poupart, and the statement of Howe '20 and'21 that, in a dietary condition unidentified by him, the pigs were said to have been very nervous at one stage. Boerhaave also wrote of faintings and anxieties which often were suddenly fatal to men, of a contraction of the limbs, and of palsy and convulsions. He thought, however, that it was not a true palsy, because patients always recovered completely from it. Poupart also stated that the muscles in man may be rigid as boards, and Euglenus is said to have stated that the ham strings became contracted. Hart, Steenbock, and Smith '19 noted an "inability to use the hind legs which finally developed into an apparent paralysis of those parts," and Cohendy and Wollman'22 also spãt e of i paralysis following the feeding of a sterilized diet. It is very interesting that the behavior of many of the guinea pigs in my series reminded rne of the behavior of Hofmeister's '22 rats fed on polished rice alone. Indeed, Hofmeister's description could be adopted with but slight changes for the nervous animals in my series, and although rats apparently do ttãt suffer from scurvy I cannot help but wonder if Hofmeistef's rats suffered only from polyneuritis. The anatomical report of Kihn '22 seems fully to justify my doubt. bohen and Mendel '18 and Cohendy and Wollman'22 mentioned stiffening in the hind legs in guinea pigs on a scorbutic diet and the occurrence of an apparent or a pseudoparalysis. Morgen and Beger '15 and Bauman [138] GROSS MORPHOLOGY t9 less, actually gained 41.5 per cent, although kept on the same diet for 136 days, or more than twice as long. Pig No. 6 followed a similar course. However, had these two animals been kept on this diet long enough, they no doubt would eventually have sufiered a greater loss and succumbed in the end. Taar.n II, Spn¡es 2å Comment of Pls: No I 2 3 4 M M r' M M r' F F r' x' r r' 790 970 975 910 827.5 Heated rolled barley and heated alfalfa hav and water ad rib. 0 n 42 42 34 1& 43 31 4l 56 47 34 u 49 49 58 34 52 34 39 31 42. 32.9 32.9 24.0 31.6 + 0.3 33.5 23.5 27.3 21.3 10 m.6 31.1 28.9 31.5 n.6 34.2 26.6 3û.4 29.9 34.4 28.48 530 650 741 622 830 608 656 745 645 684 758 689 737 623 713 608 697 667 597 600 655. 915 858 t0?Ë' 820 7ñ ItT7 1000 1037 910 985 925 9&7 815 842 915 9L9.2 Died Gassed Gassed Gassed Died of intrapericardial hemorrhage 56. 7. 8. 9. 10. 11. t2. 13. 14, 15. 16. 17. 18. 19. 24. Died Died Died Died Died Died Died Died Died Died Died Died Died Died Died Died t Number 5 was not included in the averages' l{o. ol Pis Terr-n III, Snnrrs 3ø u.7 32.t 29.7 36.1 n.6 32.3 30.3 Conment Gassed Died Gassed Gassed Gassed Gassed Gassed 1 4 l) 8 11 18 2t A à o(l. ñã= -ôr¡- 'O dt ßd I'tr rr Ë-gü k(dÍs ¡c9F ËeË OHCI HG ¡¡r 4 39 Ðo Ðq 36 34 32 2L 32 26.9 õb/ 522 390 507 468 430 372 4æ.7 763 722 575 7n nÐÐ 593 550 665.4 Sex Inltla,l Weight trtinel Wetght Percentsge of f,oss ,n Days Nûtu¡e of Diet percentage of I Duration I Loss I ln Days I Nature of Dieü Sex rnifisl I ¡lnal weiehtl \Teleiht ll4sl 20 STUDIES ON SCURVY Since none of the females rnere pregnant, Series 3ø strikingly illustrates the native differences in resistance in the same sex. This is illustrated by those that succumbed as well as by those that survived. Although it is possible that some of these pigs got alÍ"alfa which \ryas not as pure as that received by others, it is very unlikely indeed that this is responsible for the observed differences. It should not be forgotten, however, that the freshness of the alfalfa, and perhaps also of the barley, has much to do with the duration of survival of animals on this diet, at different times of the year. Tear,r IV, SBnrrs 4 No. oi Natu¡e of Dl€t Comment Pic 15....... 16....... 17....... 18.... .. . Averageg., 1.. . .... 2....... 19...... . 20....... Averages,. 3.. ... . . ¿, 5....... 6.. . .... Averages.. 7....... 8.. ... . . 9..... .. 10.. .... . Averages.. r' F rì I' M I' F I| rì M M t[ F I' F F 400 540 962 ffi 40 4 4L 43 a 270 ó¡¿) 278 330.5 315.9 32.5 30.5 n.2 n.3 28.3 ÐÐo zt.2 41.0 29.0 32.6 Raw rolled barley, raw alfalfa ha¡ and water ad lib. Gassed Died Gassed Gassed 4 9.25 592 530 653 514.6 557.3 4y¿ 515 480 457 468.5 355 385.5 385 365 372.6 43 43 óÐ 32 38.25 Gas-heated rolled barle¡ raw alfalfa hay, and water ad lib. Gassed Gassed Gassed füssed &2. ÐÐ tftf . 22. 30. 32. 26. &2. x3. 28. 285 345 32t 353 326 600 560 505 559 556 894 762 752 7n 783.75 4 0 1 7 3 8 5 I 1 8 12 33 4L 30 m.0 22 22 2t 2l 21.5 Electrically heated rolled barley, raw alfatfa ha¡ and water ad lib. Gassed Gassed Died Gassed Electrically heated rolled barley and Gassed Gassed Died Gassed That the younger animals, as noticed also by Holst and Frölich, usually succumbed sooner is shown by the first group in Table IV. Although the average weight of these pigs was only 439.3 grams, their average loss in weight was only 28.3 per cent. The average duration was 42 days. On the other hand, the five animals in Table I, Series 1, with an average weight oÍ. 7t6.4 grams, lost 42 per cent and lived 76 days. Other animals in other groups lost but slightly, while some maintained their weight. However, if the alfalfa hay or the rolled barley, or both, was heated to 100" C. for an hour, by gas, electricity, or steam, the period of survival was [146] Sex Ioltial Weisht trlin¿l Iüeisht Percentage of f,oss Durstlon in Days GROSS MORPHOLOGY 2L greatly shortened, even if the loss in weight at the time of death was less. Reference to Tables II, III, and IV will make this evident. The third group of Series 4 (1able IV) had almost the same weight as Group I, but tJrey lost somewhat more, although they lived only twothirds as long as those in Group I. Group II of this series, which was 118 grams heavie¡ lost still more, although they lived only 7l per cent as long; but those in Group IV, which were almost twice as heavy at the start as those in Group I, lost but little more in weight and lived only a little more than half as long. They received heated barley and alfalfa. Tarr.n V, Snnrss 5 No. of Initial 'lVeight Comment Pig Aver¿ges. 5. 6. 7. 8. 9. .... .. 10....... 11....... 12...... . AYereges.. aÐ 14.. ..... Ib....... 16... .... Averages.. t7 ....... 18....... 19.. ..... 20.. .. .. . Averogeð.. 910 830 105õ 940 933.7 795 873 740 873 8n.2 79t t04 1140 1051 1005.2 865 Ðl/ 765 790 7U.2 590 910 560 618 669.5 zI.4 28.5 27.0 27.1 27.5 26.0 n.3 %.3 25.1 ?Ã.9 26.8 +37 24.3 ooo 26.4 (of 3) 37.6 22.3 28.5 28.4 m.2 Sex M M M M M M M M M M M M M M M M 33 .f .l 36 33 ði). ¡ 33 35 33 36 34.2 58n 48', 45 47 49.5 660 593 770 685 677 Raw alfalfa hay and raw barley ad lib. Raw alfalfa hay, raw barley, and wheatgerm ad lib. Raw alfalfa hay, barley, wheat germ, and evaporated milk, ad lib. Died Died Gassed Gassed Gassed Gassed Died Gassed Gassed Died Gassed Gassed 1170 732 998 1055 988 7 583 805 820 752 740 47" 4 4L 42 43.7 Gassed Gassed Gassed Died Raw alfalfa hay, barley, and evaporated milk, ad lib. Duration ln Days Nature of Diet ¡'inal 'lVeight Percent&ge of Loss a These pigs would have died sooner but for a change in diet. D Death accidental. The still older and heavier group of nineteen animals, comprising Series 2b (Table II), which averaged 919.2 grams, lost only a little more (0.1 per cent) in weight and lived no longer. That is, animals more than twice as heavy as the young pigs in Group I, Series 4, lived only a day longer, in spite of the fact that the average loss in weight was only 0.1 per cent larger. This splendidly illustrates the effect of differences in the degree of deprivation of vitamines. 11471 22 STUDIES ON SCURVY The existence of marked individual variations in resistance toward deficiencies in vitamine C is evident also in this group of pigs. Since the exact ages of these animals were not known, although none \ryere over two years old, it is possible, although nbt probable, that slight age differences may be a factor in the differences in resistance of the immature animals, but I only feel justified in concluding that immature, senile, and pregnant animals yield sooner. Groups I and II of Series 5 (Table V), which were composed almost wholly of mature males ranging from 732 to 1,170 grams in weight, showed almost no differences in resistance. One of these two groups of four pigs each received the basic diet only, while the other received wheat germ in addition. All except one of these pigs were full gro\r/n at the time that they were placed upon the deficient diet, but their weight differed greatly, as indicated above. Their ages also differed by several months, yet the periods of survival of these eight pigs ranged only from 33 to 36 days and the loss of weight fuom23.3 to 29.3 per cent. The first four, the weight of which ranged from 830 to 1,055 grams, had survival periods ranging from 33 to 36 days and a loss in weight from 27 to 28.5 per cent. It would perhaps be difficult to obtain other groups of animals with such variations in size and with so slight a difference in the times of death and total loss of weight, but nothing could better illustrate the old rule that like causes produce like results, and that the addition of vitamine B had little or no efiect. This is probably due to the fact that the diet used was not deficient in this respect. The weights of the pigs in the second group of this series varied from 732 to 1,170 grams; the periods of survival showed the same differences, but there was a somewhat larger difference-6 per cent-in loss of body wqight. The striking uniformity in these results seems to imply rather uniform conditions as causes of death, and were it not for the fact that some of the animals in the other groups survived very much longer, I might have questioned */o*t earlier results. Since I know of no reason other than that of a difference in resistance, for the greater length of survival of some of the animals in the previous groups, I am encouraged to believe that such differences really exist. That this is probable is indicated by pigs in Groups III and IV of Series 5 which survived those in Groups I and II of this series and also some of those in Groups III and V which had exactly the same diet. Gnoss Srnucruner, Cn¡rces Full-term fetuses and young pigs only a few days old had the same gross lesions as older animals, except that the bony changes were more pronounced. This agrees with the observations of Ingier '15, although it 11481 GROSS MORPIIOLOGY 23 must not be ove¡looked that the duration of the scorbutic affection of the mothers is a crucial factor in the extent of the lesions. No gross changes \ryere encountered in the gonads, and marked, general subcutaneous edema or ascites was not encountered in this series. Among several hundred animals examined during the last few years, there was only one in which a decided anasarca \¡/as present. A slight amount o{ edema always accompanies hemorrhage, however, and some local edema is not uncommon in the internal organs and is especially common in the perivesical fat. According to Smith "the parts first to evidence actual impairment in the intact animal are the joints and tendons." I took particular pains to examine carefully the articulations in a large series of mature animals at autopsy but never found them abnormal. Nor did I ñnd the tendons affected, although I must add that I have not made an extensive microscopic study of them, and Smith supplies no evidence. The only lesion I saw near the larger articulations was hemorrhage into the periosteum in the immediate proximity of the articular cartilages, and while it is probable that extravasations into the articular cavities may occur from these regions, I never obseryed them in the knee, elbow, and other joint cavities. {Jlcers, or infections of the mouth grossly evident, intra vitam or post mortem, and gross evidences of hemorrhage into or from the gums themselves, and the presence of loose incisors never \ryere encountered; but subcutaneous and deep hemorrhages were common and sometimes extensive and surprisingly symmetrical even as to detail. However, the occurrence of hemorrhages was very erratic. This appears also from the literature, as recordedin the experiments of Bauman and Howard'l7, Lor example. Subperitoneal or retrobulbar hemorrhages in the orbit were not observed. The nature of the hemorrhages corresponded very well to that recorded in the literature on scurvy. Sometimes they were very slight indeed. Intracutaneous hemorrhages also occurred a few times. In these instances the appearance of the under surface of the skin of the abdomen and thorax suggested the presence of numerous small petechiae. They 'were rare, however, as reported also by Holst and Frölich, and Jackson and Moore. Abels'24 stated that skin hemorrhages are present only where infection occurs, but microscopic evidences of infection were not present in these areas in my pigs. Neither Ìtras edema or rise in temperature present, and although no bacteriological examination was made of the hemorrhagic areas, no evidence of infection was noticed in the animals while alive or during the microscopic examination of the material. However, cultural methods were not used, and it is possible that the few animals affected by t14el 24 STUDIES ON SCURVY hydrothorax suffered from pleuritic infection and that some may have died of pneumonia. Almost all subcutaneous hemorrhages occurred on the ventral surface of the body. They \ryere most common in the upper tibial, the anterior femoral, and the lateral, mid-thoracic regions; but I am unable to state as yet whether they tend to appear first in some particular region. Since the hemorrhages are, after all, erratic as to location, I surmise that they do not constantly occur frrst at any one place. The ventral hemorrhages seldom extended up the throat and ovef the mandible and down the whole abdomen, but dorsal hemorrhages also occurred. The distribution of the former corresponds in general with that in human scurvy as reported by Aschoff and Koch'19. They found the legs affected in 80 per cent of their cases, and it is on the anterior surface of the legs and on the thorax that I found hemorrhages most common in the guinea pig. I never found them in the feet, although they were present at the carpal and tarsal regions of young animals in which epiphyseal separation had occurred. They occur especially in places of pressure and movement such as the axilla, abdomen, and chest. This, to be sure, does not contradict the suggestion of Aschoff and Koch that gravity is a deciding factor in their production in man. The implications of the "capillary resistance test" of Hess '21 make both inferences probable. Were it not for the fact that guinea pigs sit still so much, and that the anterior tibial and femoral regions are not the most dependent parts, one might reach a similar conclusion regarding the efiect of gravity as to most hemorrhages in the guinea pig. Since the pigs ate barley out of cups, it is not improbable that pressure and friction from the edges of the cups upon the throat and lower jaw may have been factors in determining the presence of sub'cutaneous hemofrhages there, and it seems probable to me that obstruction to the blood flow from contact with the bedding, or with other parts of the body, as in case of the legs and head, may be responsible for the characteristic localization of most of the hemorrhages in the guinea pig. The only dorsal hemorrhages observed were seen over the spines of the lower thoracic and upper lumbar vertebrae and in the dorsal scapular, brachial, femoral, carpal, and tarsal regions. Those over the lumbar spines were seldom more than 2 mm. or 3 mm' in size, but they occurred in a very large per.centage of the animals. It does not seem unlikely to me that tension and movement at these points, and possibly also exposure with increasing loss of weight, may be factors in their production, and it is interesting that extensive ventral subcutaneous hemorrhages may be present in a mature pig that still is gaining in weight. I never observed intra-articular hemorrhages' Muscle hemorrhages, on tlsol GROSS MORPHOLOGY 25 the other hand, practically always accompanied subcutaneous hemorrhages, although the latter rarely were grossly absent; but I never found it to be true, as reported by Ingier '15, that there may be no lesions, but hemorrhages, in scurvy. The muscles most affected were those concerned in respiration, in locomotion, and in masti'cation, but I did not make a detailed, comprehensive examination regarding the particular muscles affected throughout the entire body of the animals. Extensor muscles, such as the triceps, also were involved. The subscapulares were frequently slightly hemorrhagic, especially near their origins, and sometimes they were extensively so. In a few cases the hemorrhage into the adductor muscles of the thighs and the flexors of the legs was in the form of striations as shown in the Roentgen-ray photograph, Figure 6. Hemorrhages into the intercostals at the costochondral regions were very common, and when restricted were confined to the fourth and fifth spaces. They usually involved the intercostal muscles, and very commonly were exceedingly intense and extensive. Srrbperiosteal hemorrhages v¿ere especially common here and over the processes of the mandible and the tibiae. While describing changes at the costochondral junctions, Hess '21 wrote: "On closer examination a yellowish-white transverse line may be seen at the epiphyseal junction of the ribs." Since no epiphyses are found in the region of the distal extremities of the ribs, I presume that Hess was referring to the costochondral junctions. I surmise that the line referred to here is the "yello\ry bar" mentioned by Chick, Hume, and Skelton '18 as being surrounded by hemorrhage, and which they frequently found present at the costochondral junctions. It will be recalled that Fraenkel '04 also spoke of the presence of a reddish-yellow zone proximal to the epiphyseal line but not on the ribs. Although changes at the epiphyses of the ribs probably also occur, so far as I know no one has described any. Even young guinea pigs, weighing only about 200 grams, may die of scurvy without the presence of very evident gross hemorrhages, or other gross lesions, except loose teeth, fatty livers, and thickening in the carpal or tarsal regions or at the costochondral junctions. There may be no gross hemorrhage in these regions, and there may be an entire absence of edema, and perhaps also of pain. As will appear later, the thickenings at the costochondral regions are clue only to the reaction in the cartilages. This so-called beading of the ribs was particularly marked in prolonged cases in relatively young animals, i.e., in cases in which there was only a partial deprivation of the antiscorbutic vitamine. In young animals, in which the deprivation was complete, the interference r,vith growth was so marked that the thickening of the costochondral junctions was wholly absent and in older animals the cartilaginous separation did not occur. [lsl] 26 STUDIES ON SCURVY The condition in the carpal regions was quite difierent in cases in which epiphyseal separation had occurred at the distal extremities of the radius and ulna. In these cases considerable hemorrhage and some edema v/ere present at the site of the lesion. If this separation is complete, tfauma from pressure in sitting, and from movement in locomotion, also becomes a factor in the production of further hemorrhage, edema, and deformity. I do not imply, however, that hemorrhage or edema, or both, are never present before epiphyseal separation is complete. Both periosteal and subcutaneous hemorrhages are, in fact, frequently present before separation of the epiphyses has occurred in fairly young animals. Bleeding gums \ryere never observed. This agrees with the observations of Bauman and Howard. Holst and Frölich, too, found only one case in which the gums near the incisors bled easily, and noticed gingival ulcers near the molars but once. I never encountered them. In a few cases the gingival margins afound the posterior molars were found bloody at neciopiy, the blood very clearly having come up from around the teeth. This also was the conclusion of Holst and Frölich. It was hence an alveolar or periodontial, rather than an intragingival hemorrhage. However, Talbot ãnd Petersen'13, who fed only oats, bread, and water to guinea pigs, found that the gums bled at the slightest touch. Likewise, Howe, who, however, did not pfonounce upon the nature of the condition concerned in his experiments, stated that the gums were infected and bled copiously in some instances. Jackson and Moore '16 found petechial hemorrhages near the lower incisors in spite of the fact that they fed green vegetables and raw milk to produce scufvy, but Cohen and Mendel '18 did not encounter them. None of my animals showed evidences of hemorrhages into the gums or directly from them. Slight intestinal hemorrhages lryefe commonly present, especially in the walls of the duodenum and cecum. Hemorrhages into the stomach and gall-bladder were rare, but they wefe very common in the urinary bladder. They were especially common in the paravesical fat, and often were so intense that the bladder was imbedded in very dark edematous fat. I encountered only two cases of blood in the gall-bladder. Hematuria, as determined by microscopic findings, apparently is common in scurvy in man, but Pestalozza'25 regatded the gross presence of blood in human urine so uncommon that he reported such a case in a boy ten months old, and pointed out that various investigators found blood in the urine upon microscopical examination in about one-fourth of all the cases of scurvy examined. Hematuria was present 10 days before death in one of my guinea pigs that had lost only 8.4 per cent in weight during 42 days. f 1s2l GROSS MORPIÍOLOGY 27 Although the uterus sometimes seemed congested, gross hemorrhages lüere never encountered in it. Fresh blood seen on the vulva had a vesical or intestinal origin. It might also have been renal. The incisor teeth of none of the animals were loose, and in some pigs all the teeth were still quite firmly set. In others only the last molars could be depressed with much force although they usually were loosened somewhat. It is possible that Holst and Frölich always found the molars loose because they used a different diet and differences in diet and age probably account for many contradictory observations. However, in advanced cases the last molars almost always were loose and hemorrhagic at the root and could be depressed easily. They seemed somewhat looser in the mandible than in the maxillae. The loosening, and also the extent of absorption of the rootsa always was progressively less from back forward, a fact which may be related to the decreasing amount of use arrd pressure to which these teeth are subjected.in chewing. Moreover, as the incisors become longer from disuse, in spite of retardation of their normal growth by the deficient diet, the molars would be protected from normal pressures and use in a progressively decreasing way from forward back. If the rate of growth of the guinea pig incisors is at all comparable to that of those of rats, as revealed by Addison and Appleton '15, it is easy to see that this growth would not be a negligible factor in the course of four to six weeks or longer, in spite of the fact that lessened use would tend to affect the molars similarly as the incisors. It is possible that the incisors would become looser and that hemorrhages from the gums near them would occur more frequently, were the animals given food demanding greater use of these teeth than is required by the diet provided in these experiments. However, since the incisors are set so much more deeply into the jaws than the molars, they undoubtedly would not become loose so quickly. If the very singular method of fixation of the molar teeth in the jaws of the guinea pig is considered in this connection, it is evident that hemorrhage into or edema of the periodontium alone could not detach them. Only absorptive changes capable of dissolving bone or cartilage can do so for the osteodentine and the "Knorpelzement" form a continuous osteoid, if not osseous, union in places. The posterior molars were so loose in some cases that they offered practically no resistance whatever to extraction, and they could often be rAlthough guinea pigs have holodont teeth, I am using the word root because it is unequivocal and because o{ the lack of a convenient term for the implanted portion of such teeth. In the absence of another term constant circumlocution would be unavoidable. [1s3] 28 STUDIES ON SCURVY depressed not only belorv the surface of the gums but below that of the alveolar processes, with the greatest ease. In many of the animals they v/ere so loose and the roots were so greatly shortened by absorption that it was difficult to see why they were not depressed during chewing. However, no teeth ever had fallen out during life, as reported by Robb, Mecles, McClendon, et al.'21. Although some animals died before the teeth became loose, one can use the looseness and degree of absorption of the roots of the teeth as fairly reliable indices of the stage of the scorbutic process. The only reference to the absorption of the roots of teeth which I have found occurs in Poupart. He stated that the teeth of a scorbutic boy of ten years "étøi,ent rongéøs à Ia raci.ne st ne tewient þlus," IJnfortunately the age of this patient raises the question of the normal absorption in the temporary teeth regarding which-and infection-Poupart is silent. If a very loose molar tooth is extracted from a scorbutic guinea pig, a drop of gelatinous material usually is found adhering to what remains of the root, or occupies the place of ii. After this semifluid material is wiped away, the free portion of the root is seen to have a thin irregular border, as shown in Figure 7. Although it is very evident that a considerable portion of the root, sometimes almost all of it, must have been absorbed, the portion that remained always seemed as hard as the rest, or as a normal root, except in tfre immediate neighborhood of the affected end. It is significant that molars that are still very firmly implanted indeed may show considerable shortening by absorption. They were not as strong merely because they had been thinned, by absorption. No eviden'ce was obtained indicating the presence of demineralization of the exposed portions, or crowns of the teeth, or of the implanted portions, as reported by Howe. Since Howe found the "tips of the teeth" to soften first, it is clear that this softening must have been due to external influences. This inference is strengthened by the fact that Morel and Mouriquand '2I found no change in the chemical composition of the bones of the guinea pigs in scurvy. Moreover, Howe found cavity formation, and according to him the teeth in the unknown condition reported by him, could be penetrated easily with a sharp instrument, and "large portions could be brushed away in the process of cleaning the jaws." I never could establish that the teeth \4/ere more brittle, by attempts to break them, nor did I observe broken teeth more commonly in my series of scorbutic than in the stock animals. I never saw teeth chipped at the crown, nor broken off at the root, or elsewhere, although the loss of substance was not limited to the "cementum" as observed by Robb, Medes, and McClendon. Cohendy and Wollman'22 spoke only of a "loosening of the molars and osseous fragility characteristic of scurvy." Since the loss t1s4I GROSS MORPHOLOGY 35 drop out of the jaws, but they also drop into the alveoli in cleaned specimens, because of the shortening of their roots by absorption. The posterior molars may sink below the level of the alveolar processes. They never seemed more curved than normal and the loosening apparently did not interfere with normal occlusion. The presence of tenderness may have prevented wear from taking place in the customaty way and at the usual rate, however, and under conditions of intermittent deficiency in the diet they may stand more obliquely and become worn abnormally. The entire jaws, as well as the rest of the skeleton also suffered from absorption. Small portions of the alveolar processes were sometimes gone. I encountered partial absorption, but I never saw a case of complete absorption of the alveolar processes such as mentioned by Howe. It is clear, however, that this might occur if the defrciency lasted for a sufficiently long period of time. This absorption frequently produced defects in the outer walls of the mandibles opposite the posterior alveoli as reported by Holst and Fröli'ch and as shown in Figure 8. \Mhen sufificiently marked it exposed the roots of the lower molars, but I never observed roughening which looked like erosion, on the mandible or on other parts of the skeleton as observed by them. This may be due to the fact that absorption was slower in my series, or that I did not clean a large series of entire skeletons and thus missed it. According to Howe, a condition like senile atrophy results from slow absorption, and a condition like caris from rapid absorption. A similar condition of absorption was evident also on the lateral wall of the maxillae opposite the roots of the upper molars where the overlying bone is normally very thin. That absorption occurs over the entire skeleton is indicated by the reduction in, or the absence of the customary relief, as well as by thinning. The latter is evident particularly on the coronoid and condyloid processes and on the coxal bones. A1l the large bones are smoother and look somewhat glassy. In young animals complete separation of the epiphyses occurs and the proximal portions of the shafts of the tibiae may be defective. If the duration of the deficient feeding is sufficiently long, or if the pigs are sufficiently young, spontaneous fractures might hence occur, but I never saw any. It is easy, however, to produce fractures and I surmise that epiphyseal separation has been mistaken for fracture in the living. Cohen and Mendel '18 stated that in young animals the "joints," especially those of the \4/rist, may fracture spontaneously, but this location would seem to suggest epiphyseal separation on the radius and ulna, rather than fracture o{ the carpals. In older animals epiphyseal separation is especially common at the proximal end of the tibia. The late union of this epiphysis and the marked strain to which the proximal portion of the bone t16rl 36 STUDIES ON SCURVY is subjected in locomotion may partly account for this fact and the late persistence of a fibrous area in the diaphysis opposite the epiphyseal cartilage is mainly responsible for detachment of this epiphysis in relatively mature animals. The detachment of the molar teeth and of the periosteum apparently is not due only to the hemorrhages, for they may be slight or absent in some areas. The same thing applies to separation of epiphyses. Since an actual decrease in caliber of the bones and of the roots of the teeth occurs, and since the latter also become shortened, it is evident that the process which is responsible for removal of bone is not merely a demineralizatioî, a halisteresis. According to Pelkan '25 the deposition of calcium at the zone of temporary calcification is chemical and therefore not interfered with, while resorption is cellular and therefore interfered with, thus causing a broadening of the calcified zone. Although I do not fully comprehend this explanation, all the evidence I have obtained so far indicates that a chemical and not a directly cellular activity is responsible for the absorption on bones and teeth. X-ray examination of living or of dead animals, or of the partly cleaned skeleton, even in marked cases of scurvy in mature animals, did not reveal Fraenkel's line. The only line noticed is characteristic, but not of the deficiency, and is present in normal bones. It is shown in Figure 13. Talbot and Petersen '13 also failed to frnd Fraenkel's line during life, but stated that they nevertheless found X-ray evidence of scurvy in the bones after death. Except for the better exposure that can be obtained, it is difficult to see why this should be so. However, if the living animal could be placed in a good position, the shortening of the roots of the teeth by absorption should, to be sure, be revealed by the X-ray. The same thing applies to detached epiphyses, but Pelkan '25 stated that he could not, by this means, detect acute, or chronic scurvy in pigs weighing 250 grams. This may be due to the fact that the animals died before the effects were sufficiently pronounced, for Talbot and Petersen '13 found that pigs weighing less than250 grams died in less than ten days, without "symptoms"õ of scurvy, after having been fed on oats, bread, and water. However, on another diet they well might live longer and show more extensive change. Yang'24 published two skiagrams, one of a normal and a second of a scorbutic guinea pig, which he says show the characteristic changes in the bones. But since he does not say what particular changes are supposed to show, and since the photographs are very small, the thickenings at the costochondral junctions are not evident in the reduced figures. The pubõ One cannot but wonder whether these animals really died without symptoms, and signs no doubt were present. 11621 GROSS MORPHOLOGY, 37 lished skiagram of the scorbutic skeleton actually is lighter than the normal, but it is probable that rarefaction of the skeleton would be evident in skiagrams taken under prope¡ safeguards if the s'corbutic condition had been maintained long enough. The periosteum usually can be stripped with the greatest ease. The entire mandible, for example, can {requently be shelled out as easily as though it were merely wrapped in a non-adherent covering. The membrane usually is loosest over the condyloid and coronoid processes and is separated from the bone by a small amount of hemorrhagic fluid or by blood. I am certain, however, that the hemorrhages do not cause the detachment. The gums and the soft parts over the entire hard palate can be detached with equal ease in many cases. That such conditions as these furnish abundant opportunity for infection is self-evident, yet I never encountered any in this series, or found evidences of them upon the bones themselves or in an elevation of temperature. It is likely that the loosening of the periosteum is due to bone absorption, for the latter would necessarily release many perforating fibers of Sharpey, either wholly or partially. I never found the perichondrium detached or hemorrhagic, however, although degenerative changes occur in the costal cartilages. This probably finds its explanation in the differences in chemical composition of bone and cartilage. Except for the fact that nerves commonly are included in hemorrhagic areas, and that changes in the nervous system were indicated by the behavior of many of the animals, I failed to find gross lesions in the nervous system. I inspected a fair number of spinal cords and brains, either in whole or in part, and also made some free-hand sections without encountering instances of gross hemorrhage into the meninges or nerve tissue itself. However, microscopic changes were present in both the central and the peripheral nervous systems and will be considered in the following section. fn some pigs the peritoneal surfaces of the transversus abdomines were puckered slightly in a most peculiar way so as to roughen the internal surface. I had never seen this before, do not know how to account for it, and do not mean to imply that this phenomenon necessarily is peculiar to scurvy. However, the profound degenerative changes present in the musculature in this disease suggest that it may well be associated with them, especially since the surface of the liver sometimes has a similar rough appearance. It is very likely that the reports of different investigators of experimental scurvy in the guinea pig do not wholly agree regarding the behavior of the animals and the lesions, because of differences in the age of the animals and in the diets used. The basic diets used in these series unt1631 38 STUDIES ON SCURVY doubtedly contained some antiscorbutic, at least in the fresh raw alfalfa hay, even if not in the barley, and it is unlikely that all of vitamine C was destroyed by heating to 100" C. for an hour. This is indicated by the work of Barsikow'13, Daniels and McClurg'19, Davery'2l,Yagliano '25, etc. In my experiments the lesions were always the same, no matter whether the raw or heated basic diet was used alone or in supplemented form. They varied only in intensity, and this is what one should expect. However, if very young pigs are fed oats and water alone they apparently succumb before very pronounced gross lesions can occur, although it was by the feeding of oats that Holst and Frölich obtained the pioneering results. It is particularly interesting that they stated that pigs over 600-700 grams in weight are not suitable. I have not tried their diet, but with the diet which I used they were especially suitable. Jackson and Moore stated that some diets produce more general scorbutic lesions than others, and Höjer and \Vestin '25 claimed that there is a distinct difference between the lesions produced in the teeth and the jaws by a complete and by a partial lack of C ; but that was not my experience. That my guinea pigs suffered from scurvy was established experimentally as well as anatomically. Those that received wheat germ in addition to the basic diet succumbed just the same, and none which were scorbutic could be saved by either yeast or wheat germ. That a lack of A in the basic diet was not the cause of the difficulty was established by the fact that the addition of dried whole milk neither prevented nor cured the disease. Jackson and Moore claimed that neither raw nor boiled milk prevented scurvy in their series, and Hess and Unger '17-'18 found that the addition of cod liver oil or abolene to a diet of oats, hay, and water did not prolong life. Mouriquand an<l Michel likewise found that the addition of butter, or of olive or cod liver oil to a diet of barley and hay (they do not state what kind of hay) did not prevent scurvy, and Findlay '21 found that autoclaved milk, caseinogen, cane sugar, and cod liver oil added to a basic diet of bran and oats did not modify the lesions of scurvy. Hence it could not have been the lack of A which 'vvas responsible for the lesions produced, although I do not thereby imply that the lack of this vitamine does not produce osseous lesions such as described by Hame'21 and Tozer '21. I did, not control the presence of an adequate amount of A in the diet by the method of vaginal smears, because of the infrequency of oestrus and because it seemed to me that animals which suffer such profound disturbances as those of scurvy might well experience irregular oestrus even in the presence of an adequate amount of A. However, the behavior of many of the pigs suggested that the oestrus cycle had not been wholly suppressed. t1641 GROSS MORPHOLOGY 39 Sumunnv So far as the gross lesions regarded as diagnostic of experimental scurvy in the guinea pig are concerned, I can add little except that the presence of fatty degeneration in the liver and absorption on the ¡oots of the molar teeth, with the possibility of depressing the posterior or all of the molars, are constant morphologic signs. The extent of depression that is possible always is greatest in the last molars and decreases from back forward. Outwardly evident lesions in the bones and joints usually are absent in the mature guinea pig, and such animals may show evident signs of pain without the presence of swollen or tender joints and without hemorrhage in the extremities. They also may be very active and apparentþ unaffected and yet have extensive hemorrhages and have suffered considerable absorption on the roots of the molars and in the alveoli. Some subcutaneous and muscular hemorrhage is almost always present somewhere at the time of autopsy, and duodenal, cecal, and vesical (urinary) and paravesical hemorrhages are common. The stomach, and in fact the entire intestinal canal, usually contains little food, and I have come to regard the lack of appetite and interest in food and also the lessened activity among the earliest signs of the existence of the scorbutic condition. An entire lack of interest in food always lryas an absolute indication of death within a few days no matter how well the animal seemed or how active it was. Increasing inactivity and weakness and increased loss in weight and appetite usually enabled one to forecast the approximate time of death except for the advent of sudden complications' The new observations upon the gross pathology of experimental scurvy in guinea pigs here reported are: 1. The highly nervous condition of some of the animals. 2. The recovery of health of young pigs after a condition of complete helplessness in the posterior half of the body. 3. The occurrence of permanent locomotor disabilities. 4. Shortening of the roots of the teeth by absorption so that the molars can be depressed below the level of the gums. 5. The occurrence of pronounced gaseous distension of the stomach in some animals, and 6. Pseudopneumonic lesions in the lungs of others. 7, Fatty change, even to whiteness, especially of the liver, to a lesser degree of the adrenals, and microscopically also in other organs as will appear in Part II. 8. Necrotic areas in both liver and adrenals. 9. 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"some Morphorogicar Effects of prolonged rnanition,', Journal, of Med,ical Reseørck (Boston),36: 51. Monrr-, A', et MounrguAND, G. (et ør,.), L921. "sar l'absence de troubles électifs du métabolisme du calcium osseux dans le scorbut expérimental,,, Comþtes rendus, Socíété de Biol,ogie (paris), gS: 469. [1ó8] GROSS MORPHOLOGY 43 Moonr, J. J., and Jecxsow, L., t916. "Experimental Scurvy Produced in Guinea-Pigs by Milk and Milk Products," Jou,rnal' of th.e Americøn Med,ical' Associøtion (Chicago), 67 z 1931. MoncrN, 4., and Becun, C., 1915. "Ueber den schädlichen, auf eine Säurevergiftung zuruckzuf.ihrenden Einfluss einer ausschliesslichen Haferfütterung," Zeítschrift für þhysiol,ogiscke Ckemie (Stuttgart), 94 z 324. Mounrgu*No, G., and Mrcuer., P.,7922. "De l'action de certains aliments gras sur le métabolisme osseux adjuvants et antagonistes de la substance antiscorbutique," Comþtes rendus, Société de Biologie (Paris), 86: 1170. Mounrguexo, G., and Mtcnrr-, P., and BonNunru, 7924. 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