Progress in clinical and experimental islet cell transplantation. Renal Subcapsular Islet Cell Transplantation

Transcription

1 Renal Subcapsular Islet Cell Transplantation LUIS H. TOLEDO-PEREYRA, KARL 0. BANDLIEN, DEBRA A. GORDON, GERALD H. MACKENZIE, AND THEODORE A. REYMAN SUMMARY Islet cell transplantation has been associated with ultimate graft rejection. This preliminary study investigates the use of the renal subcapsular region as a site for placement of canine islet cell a I log rafts. A new noncollagenase mechanical technique was used for preparation of the allografts. Animals in group I (N = 6) died of hyperglycemia in 4.0 ± 1.89 days (X ± SD) after pancreatectomy without subsequent islet cell transplant. Normoglycemia and excellent survival (>60 days) was obtained in pancreatectomized animals in group II (N = 6) and in group III (N = 6), who received an islet cell allograft to the renal subcapsular site. Group II recipients were given no immunosuppression, and animals in group III received minimal immunosuppression with azathioprine. Dependence on the islet cell allograft for maintenance of normoglycemia was confirmed in the majority of the recipients by nephrectomy, to remove the graft, with resulting hyperglycemia and death. One normoglycemic animal in group II died on day 6 from peritonitis. One recipient in group II was normoglycemic at >1 mo after removal of the first graft by nephrectomy, followed by retransplantation of islet cells from a third-party donor. Two other recipients are being studied on a long-term basis, and have been normoglycemic for >6 mo and >4 mo after transplantation. These studies encourage further investigation in this area for application of islet cell transplantation in man, and elucidation of the possible mechanisms for prolongation of islet cell allograft survival at the renal subcapsular site. DIABETES 1984; 33: Progress in clinical and experimental islet cell transplantation has been slowed by many obstacles. Various implantation sites have been attempted for long-term islet cell engraftment; however, graft failure and/or rejection have usually been the result. 1 ~ 3 Depending on the method of preparation, the condition of the pancreas, and species used, a sufficient islet cell yield, which will ameliorate diabetes, also has often been difficult to obtain. 1 " 3 In an effort to improve survival and function after transplantation, we have investigated a new simplified method of pancreas preparation together with islet cell allograft placement into the renal subcapsular region. This article analyzes these preliminary findings. MATERIALS AND METHODS Islet cell preparation. Pancreata were carefully dissected from unrelated, adult mongrel dogs, flushed with cold (4 C), heparinized (10,000 U/L) Ringer's lactate until the venous effluent was clear, and placed in saline ice. The pancreatic tissue was then cut into 1-cm cubes and placed in cold (4 C) Hank's balanced salt solution (HBSS). The plunger of a 50- cc syringe was used to press the pieces of pancreatic tissue through a stainless steel screen (1.5-mm mesh). Processed tissue was then briefly washed twice in a refrigerated centrifuge (2000 rpm, 4 C) with HBSS. The resulting tissue pellet (10-15 g) was placed in a syringe for injection and the supernatant was discarded. Subcapsular implantation. After total pancreatectomy, a small incision was made in the lower pole of the left kidney of the recipient animal. A specially constructed instrument was inserted to separate the renal capsule from the underlying renal cortex, thereby creating a pocket around the kidney. The preparation of islet cell fragments was then injected into the subcapsular pocket and the incision was closed with a purse-string suture (Figures 1 and 2). Experimental design. Three groups of experimental animals were studied. All animals underwent total pancreatectomy. Group I (N = 6) animals served as apancreatic controls and were not transplanted. Recipients in group II (N = 6) were From the Department of Surgery (L.H.T.-P., K.O.B., D.A.G., G.H.M.), Sections of Transplantation and Surgical Research, and the Department of Pathology (T.A.R.), Mount Carmel Mercy Hospital, Detroit, Michigan. Address reprint requests to Dr. Luis H. Toledo-Pereyra, Department of Surgery, Section of Transplantation, Mount Carmel Mercy Hospital, 6071 W. Outer Drive, Detroit, Michigan Received for publication 30 April 1984 and in revised form 23 May DIABETES, VOL. 33, SEPTEMBER 1984

2 EXPERIMENTAL DESIGN DONOR ANIMAL RECIPIENT ANIMAL Pancreatectomy Total Pancreatectomy Flush with heparinized Ringer's lactate (4*C) Cut into 1 cm cubes and press through stainless steel sieve Injection of islet cell preparation into renal subcapsular region Centrifugal washing 4"C, HBSS FIGURE 1. Steps involved in the preparation of pancreatic islet cell fragments for transplantation. The technique is very simple since no collagenase is used. As soon as the islet cell fragments are prepared, they are implanted into the kidney capsule of a pancreatectomized recipient. Islet cell preparation ready for injection transplanted with the islet cell preparation placed under the renal capsule. No immunosuppression was given to this group after transplantation. Animals in group III (N = 6) received a renal subcapsular islet cell allograft as in group II, but were given minimal immunosuppression with azathioprine ( mg/kg/day until death or sacrifice). All animals were followed with daily determinations of plasma glucose for the first week after transplantation, and biweekly or weekly thereafter. Renal function was periodically assessed via serum creatinine determinations. In several animals, insulin determinations (RIA, Cambridge Medical Diagnostics, Billerica, Massachusetts) were obtained from blood from the right and left renal veins, portal vein, and jugular vein in order to determine the site at which the pancreatic islet cell fragments were producing the insulin. Nephrectomies of the kidneys that received the pancreatic islet cell fragments were performed in several animals. In one case, after nephrectomy, the pancreatic islet cell fragments from a third-party donor were transplanted subcapsularly in the remaining kidney. Insulin was determined in this case in blood obtained from the right renal, portal, and jugular veins. Intravenous glucose tolerance tests were periodically performed in transplanted animals by administration of 0.5 g glucose/kg body wt. Blood samples for glucose and insulin were taken every 10 min for the first hour and at 2 h. K- values were calculated using the method of Lundbaek. 4 Student's Mest was used to compare the functional response of the transplanted groups (II and III) with the nontransplanted pancreatectomized group (I). RESULTS Allograft function. Table 1 shows the mean plasma glucose values after surgery. All pancreatectomized, nontransplanted animals in group I became hyperglycemic by the first postoperative day and survived between 2 and 7 days after pancreatectomy (X ± SD = 4.0 ± 1.89 days). All transplanted animals in both groups II and III were normoglycemic on the first postoperative day and continued to DIABETES, VOL. 33, SEPTEMBER

3 SUBCAPSULAR CELL TRANSPLANTATION STEP1 ANTERIOR DISSECTION STEP 2 POSTERIOR DISSECTION STEP 3 R4NCREATIC FRAGMENTS INJECTION STEP 4 COMPLETED PANCREATIC FRAGMENTS SUBCAPSULAR TRANSPLANT FIGURE 2. Renal subcapsular transplantation of pancreatic fragments: detailed aspects of the subcapsular implantation. Careful dissection is important to prevent bleeding when the capsule is lifted for the transplant. have plasma glucose values that were within normal range and significantly less than those of the pancreatectomized group (P < 0.001). Several animals in each group underwent removal of the islet cell transplant, which was accomplished by removal of the kidney containing the allograft. These animals subsequently became hyperglycemic as described below. In group II, one animal died on the sixth postoperative day of peritonitis, but maintained normoglycemia until death. Two of the other five animals survived >60 days with normoglycemia and in the remaining three animals, left nephrectomies were done at >4 mo after transplant to remove the islet cell allograft. In these nephrectomized recipients plasma glucose values rose to hyperglycemic levels on the following day. Two of the nephrectomized group II recipients died on the second day after islet cell allograft removal and one recipient was retransplanted with an islet cell allograft from a second canine donor into the subcapsular region of the right kidney. This recipient has continued to be normogly- cemic without immunosuppression for >1 mo after transplantation. Four of the five animals in group III had their islet cell allografts removed at >3 wk, >2 mo, >4 mo, and >4 mo via left nephrectomy, became hyperglycemic on the next postoperative day, and died within the next 1-2 days from hyperglycemic coma. The remaining two animals in this group have been normoglycemic for >6 mo and >4 mo and will be studied on a long-term basis. Intravenous glucose tolerance tests. The K-values for the intravenous glucose tolerance tests in animals transplanted between 1 and 5 mo showed a normal response to the glucose challenge and ranged between 2.6 and 3.4. Normal, nontransplanted, nonpancreatectomized control animals had K-values between 2.3 and 2.6. Differential insulin determinations. Insulin levels obtained from the left renal vein adjacent to the islet cell transplant were considerably higher ( xu/ml) than the insulin levels noted on the contralateral side ( xu/ml), in the 912 DIABETES, VOL. 33, SEPTEMBER 1984

4 TABLE 1 Plasma glucose values (mean ± SD) noted at various times after pancreatectomy and renal subcapsular islet cell transplantation Group Group Group N Treatment after pancreatectomy Immunosuppression Mean plasma glucose (mg/dl, X ± SD) 1 day 3 days 5 days 7 days 2-3 wk 4-6 wk >8 wk None no transplant ± ± ± Renal subcapsular islet cell transplantation None 85.8 ± ± ± ± ± ± ± 5.2 Renal subcapsular islet cell transplantation Azathioprine 77.7 ± ± ± ± ± ± ± 8.5 portal vein ( jill/ml), or in the jugular vein ( ml). In the animal who underwent left nephrectomy and retransplantation of islet cell fragments to the contralateral kidney, the insulin levels were higher in the right renal vein (17.0 xu/ml) and near the islet cell transplant than at the portal vein (1.0 xu/ml) or jugular vein (7.6 xu/ml) sites. Histologic findings. The islet cell preparation for injection appeared as pancreatic fragments of various sizes with acinar tissue surrounding islet cells. Early histologic studies showed an inflammatory reaction in the renal subcapsular region. By 3-4 mo the inflammatory exudate had cleared and was replaced by a proliferating fibrous tissue layer. Underlying this layer, insulin- and glucagon-producing cells were identified by specific immunoperoxidase staining techniques. These cells were frequently arranged in close proximity to capillaries (Figure 3). Cells with secretory granules of appropriate size and configuration typical of alpha and beta islet cells were also identified by transmission electron microscopy studies. Renal function. No compromise of renal function was seen after islet cell transplantation as evidenced by serum creatinine values. In addition, renal biopsies taken at death or sacrifice confirmed normal kidney histology of the renal cortex after islet cell implantation. DISCUSSION Our study demonstrates the feasibility of the renal subcapsular region as a site for normal function of transplanted pancreatic islet cell fragments (prepared without collagenase). Many techniques have been attempted experimentally in an effort to improve islet cell allograft survival. These include various methods of islet cell preparation, graft and donor pretreatment, 56 cryopreservation of islets, 7 islet cell culture before transplantation, 8 utilization of fetal tissue, 9 and placement of islets in special diffusion chambers to protect them from immune recognition. 10 Although the intraportal, intrasplenic, and intrap'eritoneal sites have been commonly used for islet cell allotransplantation, several others have also been described. 1 These include the testis, anterior chamber of the eye, the hamster cheek pouch, the mammary fat pads, and the omentum. The kidney subcapsular region has also been explored as a potential site for islet cell transplantation in small animals. However, our work is the first to identify the feasibility FIGURE 3. Using immunoperoxidase staining techniques, specific for insulin, positive staining cells (beta cells) were observed. In this photomicrograph, one is located adjacent to a capillary (C), within an area of connective tissue. (Immunoperoxidase, 40 x.) DIABETES, VOL. 33, SEPTEMBER

5 of performing subcapsular islet cell transplantation (without collagenase) in large animals. Bowen et al. 11 successfully transplanted isogeneic fetal pancreas tissue beneath the kidney capsule and produced reversal of experimental diabetes in the rat. However, Garvey and associates 12 observed that similar allograft preparations were rejected rapidly at this site. Pi-Sunyer and associates 13 made similar observations with neonatal pancreas tissue. Reece-Smith et al. 14 attempted renal subcapsular implantation of adult rat islet allografts and found that although survival was somewhat prolonged, this type of islet preparation was also rejected. Recent work by Reece-Smith et al. 14 has indicated that transplantation of islet cell allografts beneath the renal capsule of diabetic rat recipients, immunosuppressed with cyclosporine (Cy A), was not prolonged. The capsule in their studies was not sutured; thus, possible spillage of islets from beneath the renal capsule into the peritoneal cavity during and immediately after transplantation could have caused sensitization of the recipients. Furthermore, there might be species differences. Improved survival was only obtained by transplantation of syngeneic islet cell-kidney composite grafts to histoincompatible diabetic recipients given Cy A. 15 Subsequent studies have shown that prolonged islet cell allograft survival may be obtained by preliminary culture of the islet cells before implantation under the renal capsule. 16 Our preliminary studies using a simplified collagenasefree method for preparation of canine islet cell allografts, with subsequent transplantation into the renal subcapsular region, are promising. Using this new method of pancreas processing, normoglycemia was obtained after transplantation in all recipient animals. No compromise of renal function was observed in any of the recipient animals. It is difficult to assess the factors that may have contributed to the improved survival, without evidence of rejection, in our studies. Possibly, the renal subcapsular site has more of an immunologically privileged character in the dog, thus allowing for islet cell allograft survival with minimal or no immunosuppression. It is evident that further studies will be necessary to fully answer these questions and provide for future application of these techniques to improve human islet cell transplantation. We are currently conducting studies to determine the survival of collagenase-prepared islet cell allografts placed at the renal subcapsular site. In addition, intrasplenic transplantation of the noncollagenase preparation is being evaluated. In summary, these preliminary studies indicate that prolonged survival of canine islet cell allografts may be obtained using a collagenase-free technique for preparation and subsequent implantation under the renal capsule. These results may suggest that this site may be immunologically privileged, since no evidence of rejection was observed. ACKNOWLEDGMENT This work was supported by an institutional research grant from the Mount Carmel Research and Education Corporation. REFERENCES 1 Sutherland, D. E. R.: Pancreas and islet transplantation. I. Experimental Studies. Diabetologia 1981; 20: Sutherland, D. E. R.: Pancreas and islet transplantation. II. Clinical trials. Diabetologia 1981; 20: Toledo-Pereyra, L. H: Islet cell transplantation. In Pancreas-Basic Concepts of Medical and Surgical Practice. In press. New York, John Wiley & Sons, Lundbaek, K.: Intravenous glucose tolerance as a tool in definition and diagnosis of diabetes mellitus. Br. J. Med. 1962; 1: Toledo-Pereyra, L. H., Gordon, D. A., and MacKenzie, G. H.: Prolongation of pancreatic islet cell allograft survival by graft pretreatment with antilymphoblast globulin (ALG). Am. Surg. 1982; 48: Toledo-Pereyra, L. H., Gordon, D. A., and MacKenzie, G. H.: Transplantation of islet cells. Surg. Gynecol. Obstet. 1984; 158: Toledo-Pereyra, L. H., Gordon, D. A., and MacKenzie, G. H: Cryopreservation of islets of Langerhans. Cryobiology 1981; 18: Gordon, D. A., Toledo-Pereyra, L. H., and MacKenzie, G. H.: Preservation for transplantation: a review of techniques of islet cell culture and storage. J. Surg. Res. 1982; 32: Eloy, R., Doillon, C, anddubois, P.: Fetal pancreatic transplantation review of experimental data. Transplant. Proc. 1980; 12 4 (Suppl. 2): Theodorou, N. A., Easterbrook, P., Tyhurst, M., and Howell, S. L: Islets of Langerhans implanted in diffusion chambers do not initiate antibody production. Transplantation 1981; 31: Bowen, K. M., Andrus, L, and Lafferty, K. J.: Successful allotransplantation of mouse pancreatic islets to nonimmunosuppressed recipients. Diabetes 1980; 30: Garvey, J. F. W., Klein, C, Millard, P. R., and Morris, P. J.: Rejection of organ-cultured allogeneic fetal rat pancreas. Surgery 1980; 87: Pi-Sunyer, F. X., Woo, R., Weber, C, Hardy, M. A., and Reemtsma, K.: Selection of a practical method of pancreatic islet transplantation in the rat. Surg. Forum. 1979; 30: Reece-Smith, H., DuToit, D. F., McShane, P., and Morris, P. J.: Prolonged survival of pancreatic islet allografts transplanted beneath the renal capsule. Transplantation 1981; 31: Reece-Smith, H, Homan, W. P., DuToit, H., McShane, P., and Morris, P. J.: A technique for transplanting pancreatic islets as a vascularized graft and prevention of rejection with cyclosporine A. Transplantation 1981; 31: Lacy, P. E., Finke, E. H., Janney, C. G., and Davis, J. M.: Prolongation of islet xenograft survival by in vitro culture of rat megaislets in 95% 0 2. Transplantation 1982; 33: DIABETES, VOL. 33, SEPTEMBER 1984