Intrathymic islet transplantation in the canine: I. Histological and functional evidence of autologous intrathymic islet engraftment and survival in pancreatectomized recipients.

Abstract

Although an attractive alternative to daily insulin therapy, allogeneic pancreatic islet transplantation has yielded suboptimal results in clinical trials, in contrast to islet allotransplantation in animal models, which have demonstrated consistent success. The successful transplantation of isolated islets to the thymus, with a single concomitant dose of antilymphocyte serum, has been demonstrated in rodents, and more significantly, such intrathymic islet allografts have been shown to induce recipient tolerance toward subsequent extrathymic donor strain islet allografts. Intrathymic islet autotransplantation has been pursued, as a prelude to studies of allogeneic IT islet transplantation and tolerance induction, in canine, porcine, and non-human primate models, to assess the large animal thymus as a site capable of supporting a viable islet graft. However, little functional or histological evidence has established definitive survival of islets transplanted within the thymus of a phylogenetically advanced species, which may be requisite to tolerance induction. This study describes the successful intrathymic autotransplantation of isolated islets using a canine model. Purpose-bred juvenile dogs, aged 4-6 months, underwent partial (n=4), or total pancreatectomy (n=11), and transplantation of autologous islets. The pancreas (or pancreatic limb) was distended with collagenase solution, and digested using a modification of the semiautomated system of Ricordi. Islets were purified by discontinuous gradient centrifugation, using Euroficoll (ficoll in Euro-Collin's kidney preservation solution). Partially pancreatectomized canines underwent IT transplantation of purified autologous islets (8000+/-4000 IEs), and were killed 8 weeks posttransplant. Totally pancreatectomized canines underwent transplantation of autologous islets to the liver (via portal vein embolization, n=5, IPO group) or the thymus (via direct IT injection, n=6, IT group), and were serially evaluated for a period of 8 weeks posttransplant to assess fasting blood glucose (FBG), serum insulin (SI) levels, and i.v. glucose tolerance (IVGTTs). K values (defined as the %-decrease/minute of the log(e) of blood glucose values) were calculated from IVGTT results. After autotransplantation in this cohort of animals, five of five IPO, and three of six IT islet recipients, remained normoglycemic (mean FBG< or =250 mg%) immediately posttransplant, and all recipients exhibited significantly elevated SI levels compared to apancreatic controls (n=10, followed 72 hr postpancreatectomy). Normal k values (=-1.1) were observed in two of five IPO, and in one of six IT recipients, 8 weeks after transplantation, and thymic tissue insulin content was increased compared to non-islet-bearing thymi (93.7+/-48.6 ng/g tissue vs. 0.7+/-0.4 ng/g tissue). At 8 weeks posttransplantation thymi from both partially and totally pancreatectomized animals were resected and processed for histological examination. Microscopic analysis of islet-bearing thymi revealed positive staining for islet-specific hormones (insulin and glucagon) within all IT recipients., Identification of islets within thymi of hyperglycemic IT recipients was problematic as islet beta cells were highly degranulated as a result of the recipients glycemic state. These results indicate that autologous islets, transplanted to the canine thymus, engraft, function, and survive for up to 8 weeks after islet autotransplantation to the canine thymus and establish the feasibility of intrathymic islet transplantation in a phylogenetically advanced animal model. The ability of islets to survive within the thymic environment for a period of at least 8 weeks after transplantation suggests that the successful induction of specific unresponsiveness secondary to intrathymic transplantation will not be impaired or limited by the inability of a viable islet mass to survive within the thymus for a sufficient period.