FUNCTIONAL HORIEOPLASTIC GRAFTS O F THE: ADEENAL GLAND O F NlW7BOBN RATS GEORGE 31. HIGGINB A S D DWIGIIT J. INGLE Division of Experimental Medicine and Division of Biochemistry, T h e klago Foziwdatioii, Rochester, Minncaofa FIVE FIGURES Autotransplantation of the adrenal gland has been successfully pcrformed on small animals, especially rats (Jaffc, ' 2 6 ; Poll, 1898; Wymaii and turn Sndeii, '32) and guinea pigs (Jaffe, '27, '27 a ) , but results have been uniformly uiisuccessful with dogs (Blodinger, I<lebaiioff and Laurens, '26 ; Johnson and Johnson, '31 ; Oldberg, ,as). Surviving cortical grafts of adrenal glands of rabbits have been reported (Busch, Leonard and Wright, '08). For review of the work on transplantation, attention is directed to the articles of Jaffe ('27), Brittoii ('30), and Loeb ('30). Homeotransplaiitation of the gland has heeii less successful than autotransplantation. Schmieden ( '03) reported some success with rabbits and Jaffe ( ' 2 7 ) , working with rats, reported four successful honieografts out of fifteen trials. Wyrnan and turn Sudeii ('32) secured successful homeografts when they exchanged glands between litter mates. They showed that such homeografts grew equally as well as autografts. I n a large series of homeografts between unrelated rats of our own strain we (Inglc, IGggins and Nilson, unpublished data) found that only an occasional graft survived and functioned. I f , on the other hand, glands were cxchangcd between rats of a strain closely inbred for many generations, successful homeografts were obtained in a high percentage of the animals (Iiigle, Iliggiiis and Nilson, unpublished data). 146 146 G. M. IlIGGINY A N i ) I). J. I N G LE Lux, Higgins and Mann ('37) attempted to acclimatize adrenal glands of newborn rats to unrelated adult rats by growing fragnents of the glands in mcdia containing the body fluids of the animals selected as hosts. After the tissue had grown in vitro for 2 weeks, the fragments were grafted to the adrenalectomized hosts. Successful grafts were obtained in half of their grafted animals but the data were not convincing that any adaptation of the biologic constituents of the grafts to those of the hosts was made by 'in vitro' growth of the glands prior to transplantation. They concluded that successful grafts were owing to similarities between the genetic constitutions of the donors and those of the hosts rather than to changes in the tissue during the growth in vitro. Because of the success of Lux, Higgins and Mann ('37)' with adrenal glands of newborn rats, v e grafted adrenal glands of newborn rats to unrelated adults without first grow4 number of attempts have ing them in tissue culture media. , been made to graft embryonic tissues o r tissues of newborn rats, and a review of these studies was given by May ( ' 3 3 ) . May has reported successful homeografts of the thyroid and parathyroid glands of newborn rats, grafted t o the anterior chamber of the eye. May ('35) likewise grafted the pituitary bodies of newborn rats and secured successful grafts. Salmon and Severinghaus ( '36) likewise have reported successful results with homeografts of thyroid glands of newborn rats. Considerably after our work had begun, Rutishauser and Guye ('36) reported that they secured viable homeografts of the adrenal glands of newborn rats, grafted t o the renal parenchyma of adult rats. However, percentages of surviving fiirictional grafts were not included in their report. METHODS A S D PROCEDURES The adrenal glands were removed from newborn rats and iinmediately placed in the fascia along the femoral veins of unrelated rats. Ether anesthesia was used, and satisfactory surgical technic was employed. One group of forty healthy, male, adult rats, previously adrenalectomized, were used as GCAFTS O F ADBENAT, GLAKDS IN NEWBORN RATS 147 hosts for grafts. I n another group, consisting of ien healthy adult, malc rats, the same procedure of grafting was followed and the same kind of material was grafted but these recipient animals were not adreualcctomized. Of a third group, consisting of forty-five healthy adult male, previously adrcnalcctomized rats, fifteen received adrenal glands removed from rats 2 wceks old; fifteen, adrenal glands from animals 1month old and fifteen, adrenal glaiids from animals 2 months old. The basic diet of the hosts was our regnlar, standard ration. Following transplantation, all adrenalectomized rats, that is, rats of the first and third groups, wcre given 0.8% sodium chloride in their drinking water and were fed a ration containing 1.5% sodium chloride. Rats of the first and third groups received the added sodium chloride for 1 week after transplant ation. Tn the first group, five rats were killed at intervals in the first five weeks following grafting, to dctermine the histologic appearance of the graft. a11 other grafted rats of this group which lived 5 months were explored and the adrenal tissue ~ 7 a sremoved from the groins. Duration of life of each animal following removal of the grafted glands was noted. -4411 grafts were fixed, sectioned and stained with hematoxylin and eosin, with Illallory’s connective tissue stain, and for €at and chromaffin reaction. Of the second group of ten adult rats, two were explored each week for 5 weeks. The grafts were removed, fixed, sectioned and stained. Concerniiig the third group, careful data were kept on the length of time the animals lived and the number of functional grafts recovered. RESTILTS Of the forty rats comprising thc first group, twenty-four survived with functional cortical grafts in the groin. Of these twenty-four animals7 five were killed during the first 5 weeks after transplanting, as has been said, and nineteen lived f o r 5 moilths. Each of the iiinctecii had a cluster of small cortical nodules in the groin closely adjacent t o the femoral rcssels (fig. 1). The number and size of these nodules TTIE ANATOMICAL RECORn, VOL 70, SO 2 AN D S U P P L X > l I ~ h TNU 2 148 G . M. H I G G I N S A N D 1). J. INGLE varied. Although ten small glands were grafted, yet the number recovered m r e r exceeded seven. The size of these cortical nodules bore some relationship to the number that regenerated and survived. If three or four survii-ed, the size of each approximated that of a normal gland but if more survived, tlie size of each nodule was correspondingly rcdnced. The total amount of restored cortical tissue was slightly in excess of the combined weight of the adrenal glands that had been reniowd from tlie host. Sixteen of the forty animals x Fig. 1 Cluster of adrenal glands iu groin of host, 5 months after grafting. 5. mThic2i werc grafted with glands froin nemhorn r a t s died of adreiial iiisufliciciicy within 2 to 3 weeks after the salt cow tent of their drinking water and ration liacl been reduced to normal. The grafts in these aiiimals had failed to snrvivc. All nineteen aiiiinals which had viable grafts after 5 months died within 3 weeks after grafts had been removed from the groin, indicating that they were functioning and delivering to the organism the activc cortical principle. In the scconcl group, the grafts which were renioved at weekly intervals from tlie p o i n s of rats, which liad not been GBAFTS O F ADRENAL G I A K D S I N FEWBORN RATS 149 adrenalectomized, were always necrotic. We did not find a functioning graft in this entire series. Soon after transplantiiig they were infiltrated with leukocytes arid were eitlier resorbed or fibrotic when the last pair of animals were killed at 5 weeks. Observations on the third group of adult animals were continued for 10 weeks after the salt content of their chinking water and ration had been reduced. There was a considerable difference in the n u n h e r of animals which survived in each of the three subgroups. Of the fifteen adult r a t s grafted with glands reinovecl from unrelated rats 2 nionths old, only one animal survived for the 10 weeks with a mass of functiouing cortical tissue. Of the fifteen grafted with glands of rats 1 month old, three were alive, with functioning grafts at 10 weelrs. Of the fiftecn grafted with glands of rats 2 weeks old, seveii were alive and h r d functional homeografts at 10 weeks. When contrasting these figures with those given above, wherein twenty-four of the forty rats grafted with aclrenals of newborn rats survived with functional grafts, i t seems clear that age of the transplanted tissue is a factor in the surviral of a homeograft. OBSERVATION3 Oh' ?'HE GXA ITS When a11 adrmal gland of a ncwboru r a t (fig. 2) was removed and grafted to the groin of an adiilt, adrenal rat, it degeiierated exactly in the manner of an autografted adult gland ( Pencharz, Olmstecl and Giragossintz, '31). Kecrosi< first appeared in the center of the gland, the niethdla TrT'as dcstroyecl and gradually the entire gland, except f o r a narrow margin of the glomei*ularzone, just below the capsule, degenerated (fig. 3 ) . During this timc blood vessels penetrated through the capsule from adjacent tissues and polpmorphonuclear leukocytes invaded the necrotic portion. Ruhseyuently, regeneration of new cortical cells took place. This occurred not only from cells in the persistent portion of the glomerular zone but from the adjacent capsule as well. This is in accord with Z~~..emer's ('36) finding that regeneration of 150 G . M. H I G G I N S A N D D. J. IXGLE GRAFTS O F ADRENAL GLANDS IN NEWBORN BATS 151 zoiia gloinerulosa takes place from the capsule in a normal adrenal gland. Regeneration of cortical tissue proceeded from the capsule and extended into the center of the gland as rapidly a s the old necrotic tissue was removed. Sections of grafts removcd a t 3 month showed evidence of considerable regeneration of cortical tissue, with some necrotic material still persisting (fig. 4). I n sectioiis of grafts removed 5 months after grafting were found vascularized masses of cortical tissue cornpri sed of smaller nodules which had regenerated from tho separate capsules of the grafted glands (fig. 5). The characteristic cellular response of the host against a homeograft occurred in the sixteen aiiirnals which died of adrenal insufficiency soon after grafting. L:mphocytes infiltrated the gland and foreign body giant cells were commoulv seen. Here were host reactions against a graft such as Loeb ('30) has described. COMMEXT The adrenal gland of the newhorn rat may be grafted to an iinrelatecl adult r a t and a large percentage of successful functional grafts may survive. Our data show that the results were someit-hat better than when the technic of tissue culture was employed to acclimatize the adrenal fragments of one animal t o the body fluids of the host before grafting. It seems, therefore, that the succcssful grafts obtained by Lux, Higgins and Maim ( ' 3 7 ) were owing to the fact that they were grafting undifferentiated tissue of newborn animals rather than t o changes which they may have induced in the biologic differentials of the grafts by growing them in host media. The chance of securing successful homeografts were greater when tissues of newborn animals were used. It seems probable that the age and the degree of differentiation attained by a grafted tissue may be factors which control or modify its survival and function in the host. I n our experience, the older the donor from which a gland to be grafted mas removed, the less likely werc we to secure viable grafts. T H E AA+ATOVICAL RECORD, VOL 70, NO 2 -4ND SUPPLEMENT N O 2 152 G . M. HIGGIKS A N D D. J. IFGLE Homeografts of an adrenal gland of an adult only occasionally survived; and yet of forty homeografts of adrenal glands of newborn animals, 60% survived and functioned. But age and degree of differentiation of a gland do not constitute all that is involved in its survival and function. Of forty animals which receired grafts of adrenal glands of newborn rats, as stated above, sixteen died. The grafts in all of these had been destroyed by an aggressive cytologic reaction of the host. Since all grafts in tliese sixteen hosts were placed along the femoral vein in exactly the same way as that employed in placing the grafts in the entire group, failure of the grafts to survive cannot have been owing to mechanical or to oper nt’ive procedures. Furthermore, the age of the donors could not have been cause for failure since all sixteen were grafted with glands removed from donors, of the same age as those from whom were taken grafts which survived; that is the grafts were removed at birth oi- very soon thereafter. We believe that our failures mere owing to marked biologic or genetic differences between the donors ancl the hosts. A s far as we know there was only a remote relationship between the donors providing the glands and the adults which received them. All were taken at random from our colony, which is not closely inbred, but which has been developed from a stock derived from tlie T i s t a r strain some time ago. It seems likely that some of the donors may have resembled, genetically more closely than others, the hosts t o which their aclrenal glands were grafted. Likewise, others may have been gcnetically urililre tlie liosts. And it is likely that viability of grafts depended entirely on the degree of the differences that existed bctsveeii the genetic constitutions of those animals serving as hosts and those serving as ~lonors. MTe are repeating- this experiment, using rats of a closely inbred strain in which brother to sister matings have been made f o r thirty-eight generations. GRAFTS O F ADREKAL GLANDS IN NEWBORN BATS 153 CONCLUSIONS 1. Adrenal glands of newborn animals, when grafted to adult animals, will regenerate, grow and function iii a high percentage of cases. 2. Regeneration of cortical tissue proceeds from the capsule, hut medulla does not survive nor regencrate. 3. A considerable pcrccntage of such grafts will not survive. Eost reactions against these grafts resemble the wellknown reactioii against homeografts. 4. The siiccess or failure of a graft probably depends on the degree of genetic identity between donor and host, . 5. Glands of newborn animals will not regenerate, t o any extent, in the presence of the host’s ow11 adrenal glands. 6. It seems apparent that another factor in successful homcotransplantatioii is the age of the donor ; the younger the donor the greater is the likelihood of survival of the graft. LITERATURE CITED BLODINGLR, 1926 Suprarenal transI., H. F:. KLEBANOFF A N D HBNRYLAUXENS plantation i n the dog. Am. J. Phjsiol., vol. 76, pp. 151-157. BRITTOK, 8. W. 1930 Adrenal insuficiency arid related considerations. Physiol. Rev., vol. 10, pp. 617-682. Buscn, F. C., T. X. LEONBKD AND T. WRIGHT 1908 Further results in suprarenal transplantations. J. Am. Ned. Awn., vol. 51, pp. 640-642. INGLE, D. J., G. 11. HJGGINS A N D H. W. NILSON ITnpublished data. JAFFE, H. L. 1927 On t h c transplantation of the guinea pig suprarenal and the functioning of the grafts. 6. Ex?. Xed., vol. 45, pp. 587-594. 1927 a Thc suprarenal gland. Arch. Path., vol. 3, pp. 414-453. JAFFE, H. L., AND ALEXANDRA PLAT SKA 1926 Functioning autoplastic suprar e n d transplants. Proc. Soc. Exp. Biol. and Xed., vol. 23, pp. 528-530. JOHNSON, ADELAIDEAND VICTY~R, JOENSOX 1931 Attempted autotraiisplaiitation of the adrenal cortex. 9m. J. Phpsitrl., vol. 97, pp. 392-395. Lorn, L. 1930 Transplantation arid inciividuality. PhysioI. Rev., vol. 10, pp. 547-61 6. LUX, LYDIA, G. &f. I I I G G I N S AND F. c. I h X N 19.37 Functional homeografts of the rat adrenal grown in vitro. Anat. h e . , rol. 70, pp. 29-43. MAY, R. 11. 1933 Action Tiearlante durable rle la greffe intraoculaire de thyrolde de raton nouveau-n6 sur le d~veloppeuientdu r a t blaiic ethyroitle. Arch. de Hiol., T. 44, pp. 149-178. -____ 1935 La greffe brhphoplastique cle l’hypophyse chez le rat. Compt. rend Soc. de Biol., T. 120, pp. 867-870. OLDBERG,ERIC 1929 A n attempt t o auto-transplant thc adrenal. Bm. J. Phgsiol., vol. 91, pp. 275-283. 154 G. M. HIGGINS AND D. J. INGLE PENCIIARZ,R. I., J. 31. D. OLMSTEDAKD G. GIRAGOSSINTZ1931 The survival of rats after total and partial adreiialectoniy aiid adrenal transplantation. Physiol. Zool., vol. 4, pp. 501-514. POLL, H. 1898 Ueber das Sehicksal der verpflanzten R’ebenniere. Centralblat. f. Physiol., Bd. 12, S. 321-326. RUTISHAUSER, ERWIN, AND PIERREGUYE 1936 La br6phoplastic surr6nalieiine ehez le rat. Compt. rend. Roc. de biol., T. 121, pp. 1046-1049. SALMON, THEODORA N., AND A. E. SEVERRINGIIAUS 1936 Functional auto- and homoplastic: thyroid grafts in the rat. Proc. Roc. Exp. Kiol. a n d Ned., rol. 34, pp. 251-253. SCHMIEDEN, VIKTOR 1903 Erfolgreiche, experimentelle Vrrlagerung von Nebennierengewebe, eiii Britrag zur Lehre von den Strumae suprareiiales aberratae. Deutseh. Ztschr. f . chir., Bd. 70, S. 453-504. WYMAN, L. C., AND CAROLINET i m STJDEN 1932 Studics ou suprarenal iiisufficiency. XI. The growth of transplanted cortical tissue i n the rat. S m . J. Physiol., vol. 101, pp. 662-GG7. ZTTEMER,R. L. 193G 9 study of adrenal cortex morphology. Am. J. Path., vol. 12. pp. 107-114.