Prolonged survival of fragments of liver transplanted to an ectopic site in the mouse.код для вставкиСкачать
Prolonged Survival of Fragments of Liver Transplanted to a n Ectopic Site in the Mouse MARY J. SELLER Paediatric Research Unit, Guy’s Hospital Medical School, Loiidon, S . E . 1 . Fragments of liver were successfully transplanted to an ectopic site in the kidney cortex of the mouse. Survival was followed for varying periods of time. In some cases both syngeneic grafts and allogeneic grafts placed in mice previously made immunologically tolerant to the genotype of the donor were still alive one year later. However, extensive amounts of liver tissue would not survive, at the best, strips of about 30 cells thick grew, and various measures tried to promote an increase in this amount were not successful. ABSTRACT Transplantation of small grafts of liver to ectopic sites has been easily achieved in invertebrates such as the garden snail, and in some of the lower vertebrates, for example the roach, toad, lizard (Grigorev, ’SS), the newt (Williams, ’61) and the bullfrog (Koppanyi and Hsi Chung Chang, ’ 2 5 ) , but in mammals, such attempts usually have been unsuccessful. Indeed, as recently as 1970, Tavassoli and Crosby stated categorically that “fragments of liver tissue cannot be maintained in ectopic sites” following a series of experiments in the rat. They had transplanted autologous liver to several sites: under the skin, in the kidney cortex, and also back into the liver, and had found a similar fate for each transplant. There was necrosis of the liver cells, a polyniorphonuclear leucocyte invasion and an eventual replacement by connective tissue. In this paper, I wish to report the results of experiments in the mouse, in which, using a technique devised by Wheeler, Corson and Dammin ( ’ 6 6 ) , I have successfully transplanted liver tissue to a n ectopic site, with survival in some cases for one year - the longest period of observation so far. Wheeler et al. (’66) showed that a bed cut in the kidney cortex of the mouse proved to be an extremely satisfactory site for the survival of syngeneic grafts of spleen, lymph nodes, thymus and kidney. They also transplanted liver to this posiANAT. REC., 172: 149-156. tion, but only assessed its survival for 28 days, and although there were some viable parenchyma cells present at that time, there were also areas of necrosis, and so there was no certain indication as to the eventual fate of the graft. For the purposes of other experiments (the results of which will be published a t a later date), I sought to get grafts of liver fragments to survive for long periods, and also to transplant successfully allogeneic a s well as syngeneic tissue. MATERIALS AND METHOD Mice. For the syngeneic experiments, the mice used were a pure line strain of CBA/H mice. Each donor and recipient pair were of the same sex, and in all, 56 mice were grafted. For the allogeneic transplantation, the donors were the CBA/H pure line mice, and the recipients were mice of the W-series - either the genotype Wv/+ which are slightly anaemic, or completely normal. Donors and recipients were matched for sex. There are several ways of overcoming the immunological barrier in mice and one is to render the recipient mice tolerant to the genotype of the prospective donor before grafting. In this experiment this was achieved by injecting the W mice at birth with 5 X lo6 CBA lymphoid cells (Seller, ’68). When these mice were adult, they were challenged with a CBA skin graft, +/+, Received July 20, ’71. Accepted Sept. 20, ’71. 149 150 MARY J. SELLER TABLE 1 and those which accepted the skin were classified as having been rendered im- Results of transplanting syngeneic liver t o a n ectopic site in the kidney cortex munologically tolerant, and were then used of the mouse in the liver grafting experiments. Fifteen Number with of these mice were transplanted and will viable liver tissue Time after transplant be referred to as “allogeneic tolerant” mice. Number transplanted when graft was assessed As controls, four completely untreated, and 28 therefore immunologically competent W 4 weeks a mice, also received a liver graft. These are 4 allogeneic grafts where no attempt has 5 weeks 6 been made to immunosuppress the recipients. 4 6 weeks Thus there are three groups of mice, 4 the syngeneic group where there are no 5 8 weeks immunological complications, and sur6 vival of the graft should depend on the 5 effectiveness of the transplantation tech6 months 6 nique ; the allogeneic tolerant series where an attempt has been made to overcome the 4 1 year 4 immunological barrier, and graft survival should be similar to the first group; and the truly allogeneic group where persistTABLE 2 ence of the graft should be determined by Results of transplanting allogeneic h e r to an the immunological capabilities of the reectopic site in the kidney cortex of mice cipient, regardless of the efficacy of the previously made immunologically tolerant transplantation technique. Number with Liver grafting. All the mice underwent viable liver tissue Time after transplant when graft was assessed a similar operation, and the technique was Number transplanted exactly as described by Wheeler et al. 3 4 weeks (’66). Under ether anaesthesia, and using 4 semi-sterile precautions, the recipient kid1 ney was exteriorised and the blood vessels 3 months 2 clamped. A graft bed was cut in the cortex. 1 A sliver of the donor liver, no more than 4 months 1 1 mm thick, was placed on the bed, and held there for about thirty seconds for ad1 5 months hesion to take place between the cut sur1 faces of the two tissues. The kidney and 3 graft were then gently restored to the 7 months 5 peritoneum, after removal of the clamp. Assessment of graft survival. Initially, 2 1 year mice were killed after four weeks, and the 2 kidney on which the grafting had been performed was removed. It was inspected macroscopically and then fixed in formalin, Fig. 1 Healthy liver graft (left) on allogeneic sectioned, and stained with haematoxylin kidney (right). The graft has survived for seven and eosin and examined under the light months, the recipient previously having been tolerant to the donor genotype. X 238. microscope. As a result of good graft sur- rendered Fig. 2 Syngeneic liver graft of two months vival at this time, other mice were left duration on the kidney ( K ) . Two mitoses for longer intervals before the condition of (arrowed) are visible in the liver ( L ) . X 467. Fig. 3 Syngeneic liver graft ( L ) on kidney their grafts was assessed. Good graft survival was continually achieved, and so ( K ) at one year. The macrophages in the liver have engulfed particles (arrowed) from Indian eventually i t was decided to terminate the ink which was injected several hours before experiment one year after the transplant. sacrifice. x 467. LIVER FRAGMENTS TRANSPLANTED ECTOPICALLY Figures 1-3 151 152 MARY J. SELLER This would often extend much of the lcngth of the kidney: the thickness varied from about seven parenchyma cells to 3 maximum of about 30 cells. Unfortunately, n m e of the measures resorted to, in an attempt to encourage proliferation of the liver cells, such as partial hepatectomy of the recipient, was successful in increasing the thickness or overall volume of the graft. The detailed microscopic picture of the fate of the grafts with the passage of time was the same in both the syngeneic and “allogeneic tolerant” mice. One month after grafting, there were many viable liver cells, but also present were browncoloured, necrotic cells. These were found especially along the cut edge of the liver where it was apposed to the kidney (fig. 5). Small areas of these necrotic cells were also within the graft. The grafts sometimes contained a few mononuclear cells, and small foci of polymorphonuclear leucocytes. At two to three months there RESULTS were fewer necrotic liver cells, which had Tables 1 and 2 show the numbers of been further reduced by six to seven mice in which there was healthy and viable months to a small cluster at the terminal liver tissue in the kidney bed, when as- ends of the graft. However, by this time sessed at various time intervals from four in some cases, they were completely absent. weeks to one year after grafting, in both The junction between the kidney and liver the syngeneic combination and when an was marked in some cases by a dense allogeneic graft was introduced into mice band of mononuclear cells, which was repreviously made immunologically tolerant. duced as time went on, but they were nor In all, 50 out of 56 (89% 1 syngeneic always present. By one year after the grafts were successful, and 11 out of 15 transplant there was no necrosis of the (73% ) “allogeneic tolerant” grafts sur- liver cells and no mononuclear cells where vived. Thus, consistently in both combina- the liver and kidney cells were apposed. tions there was a high degree of prolonged Occasional grafts had very small pockets engraftment of fragments of liver tissue Fig. 4 Allogeiieic liver grafted onto a n imin this one ectopic site (see fig. 1 ) . Bear- munologically competent recipient one month ing in mind the healthy appearance of previously. The graft is being rejected. The kidgrafts which had survived for one year ney bed ( K ) has a layer of connective tissue (fig. 3 ) it is tempting to suggest that such ( C ) between it and the degenerating liver cells (D). Mononuclear cells (arrowed) are present grafts may last permanently. the liver cells. X 467. Macroscopically, on removal of the kid- zround Fig. 5 The state of the liver graft a t one ney and its graft from the animal, there month i n a syngeneic recipient. The liver tissue was no definite sign of the presence of any ( L ) is healthy except at the interface with ihe liver tissue. The site of the possible graft kidney ( K ) where there i s a line of degenerating ( D ) . X 297. could be identified by a depression in the cells Fig. 6 The picture one month after graftinq kidney cortex where the bed had been cut, which was recorded as having been “unsuccessbut the presence or absence of grafted liver fully grafted.” It is thought that the graft became could not be determined until microscopic separated from the kidney soon after the graftprocedure. There is no evidence on any decxamination. Then, it could be seen that ing generating liver cells or cellular infiltration. Cona successful graft comprised a very thin nective tissue ( C ) h a s been laid down on the strip of liver tissue apposed to the cortex. kidney bed ( K ) . x 467. Other procedures. In view of the acknowledged great proliferative ability of the liver, attempts were made in some mice to enlarge the size of grafts by various means. The measures tried were: ( 1 ) the liver graft was taken 72 hours after the donor had been injected subcutaneously with 0.2 ml of 40% carbon tetrachloride in olive oil; ( 2 ) the donor liver was taken from newborn, or 21 day old juvenile mice; ( 3 ) the recipient was partially hepatectomised after i t was estimated that the graft should have taken; (4) similar recipients were injected intravenously, with serum from mice partially hepatectomised two days previously. In addition, in order to see whether the macrophage elements of the liver graft were functioning normally, two mice which had borne grafts for one year were injected intravenously with a solution of Indian ink in saline several hours before sacrifice. LIVER FRAGMENTS TRANSPLANTED ECTOPICALLY Figures 4-6 153 154 MARY J. SELLER of polymorphonuclear leucocytes at various times after one month, but there was never a large or consistent cellular infiltration of the grafts. These observations were in marked contrast to the allogeneic control mice which were immunologically competent to reject the tissue. After one month the whole graft was densely invaded by mononuclear cells. Nearly all the liver cells were degenerating, although there were a very few parenchyma cells which appeared to be viable. A layer of connective tissue was present on both sides of the graft (fig. 4). In turn, this picture of rejection contrasted with the situation in several of the syngeneic and “allogeneic tolerant” mice which were recorded as being unsuccessrully grafted. For, in all cases examined, at one month, the graft site bore no sign of liver cells, degenerate or otherwise, there was only a thin layer of connective lissue present. Mononuclear cells were present in the cut layer of the kidney cortex, but they did not extend into the connective tissue (fig. 6 ) . It is thought that in these cases; the graft did not adhere properly to the kidney, and was displaced shortly after the kidney was restored to the peritoneum. In one case, noted as a failure in the “allogeneic tolerant” series at three months, the whole kidney was atrophied. Possibly, the blood supply had not been effectively restored after clamping for the grafting procedure. Despite the fact that large scale proliferation of the graft could not be encouraged by any of the means tried, it was common to observe at least one mitosis in a graft (fig. 2). This was so, even a year after the transplant. Following the injection of Indian ink into a mouse several hours before sacrifice, black particles from the ink were found in the cytoplasm of the macrophages of both the recipient’s own liver and also in those of the liver graft (fig. 3 ) . DISCUSSION Successful and prolonged survival of fragments of liver tissue can be achieved if they are transplanted in close apposition to the cut surface of the renal cortex. The extreme vascular nature of this area must be of paramount importance in the success of grafts of tissues, such as the liver, which require constant oxygenation. The fact that so few cells survived compared with the size of the initial graft may reflect this, as the survivors could be those cells originally placed nearest to the blood supply in the graft bed. Organs less vulnerable to anoxia fare better (Wheeler et al., ’66; Tavassoli and Crosby, ’70; Seller, unpublished) : I have grafted spleen successfully and permanently many times, the size of the graft is comparatively large, usually about one-fourth the size of the recipient kidney. However, despite the fact that so few liver cells survive, the ones which do seem to be quite viable and healthy. The evidencc for this in the present experiments is taken from the regular observation of parenchymal mitoses in the grafts, and the fact that the macrophages phagocytose particles from Indian ink quite normally. There is no certainty whether the macrophages are the ones contained in the original graft or whether they have immigrated from the host, since it is known that there is a turnover of macrophages in the liver. Nevertheless, whatever their derivation, the fact that they are functioning normally by phagocytosing particles suggests that they are in a favourable environment. The finding that none of the ways tried would encourage the proliferation of the liver graft was disappointing, and limits certain possible applications for the technique in the future (Seller, in preparation). It does, however, emphasize how complex the regulatory system is for liver growth and regeneration. The reason for the unsuccessful grafting in a minority of cases in the syngeneic and “allogeneic tolerant” mice was thought to be due to the graft becoming detached from the kidney soon after it was replaced in the peritoneum, rather than other possibilities such as the whole graft dying from anoxia, or a rejection process occurring. For, in each case, no necrotic liver cells were seen in the graft bed - there was only connective tissue present, and a true rejection reaction with massive liver cell necrosis and mononuclear cell infiltration was seen only in the allogeneic trans- LIVER FRAGMENTS TRANSPLANTED ECTOPICALLY plants to immunologically competent mice. However, no observations were made during the early days after the transplant to prove this point. Leduc and Wilson ('63) also got small syngeneic implants of liver tissue to survive in the mouse by transplanting them to the spleen; however, unlike the present work they were unsuccessful with adult liver, succeeding only with foetal and neonatal liver. They too obtained long lasting survival, but did not observe any proliferation of the grafts. ACKNOWLEDGMENTS This work was supported by The Spastics Society and the M.R.C. Miss A. R. Harcourt is thanked for able technical assistance, and Mr. L. Kelberman for help with photography. 155 LITERATURE CITED Grigorev, N. I. 1963 Changes in transplantcd hepatic tissue in various vertebrate animals. Trudy Ieningr. sanit. gig. med. Inst., 75: 141-156. Koppanyi, T., Hsi Chung Chang 1925 Experiments on liver transplantation. Endocrinol., 9: 513-514. Leduc, E. H., J. W.Wilson 1963 Production of transplantable hepatomas by intrasplenic implantation of normal liver i n the mouse. J. Natl. Cancer Inst., 30: 85-95. Seller, M. J. 1968 The transplantation of allogeneic haemopoietic tissue in adult anaemic mice of the W-series previously made immunologically tolerant. Transplant., 6: 856860. Tavassoli, M., and W. H. Crosby 1970 The fate of fragments of livar implanted in ectopic sites. Anat. Rec., 166: 143-152. Wheeler, H. B., J. M. Corson and G. J. Dammin 1966 Transplantation of tissue slices i n mice. Ann, N.Y. Acad. Sci. 129: 118-129. Williams, D. D. 1961 Homografting of liver in the newt Triturus viridescens. Transplant. Bull., 27: 100-103.