Histo-morphological analyses after xenogeneic Transplantation mikroencap-sulated Islets of Langerhans in the Model „Pig-in-Rat“

Summary

The aim of this study was the detailed histological and immunohistochemical analyzation of microencapsulated xenogeneic islet grafts in previously streptozotozin (STZ)-diabetic Wistar WU rats. Microencapsulated porcine islets of Langerhans (mpLI) were transplanted intraperitoneal and under the left renal capsule of these rats without any medical immune suppression. New insights were to be gained into the cell-mediated reactions of the recipi-ent to the microencapsulated foreign cells and into the fibrosis of the grafts and their ne-ovascularization with the goal of establishing an effective therapy for type I diabetes with this form of xenogeneic islet cell transplantation (xITX). Complex immunohistochemical assays were established to accomplish this goal. When no species-specific antibodies or antisera were available, the cross reaction of human-specific reagents was used. This work analyses in retrospect the data of 18 animals following the implantation of empty micro-capsules and 65 animals that underwent xenotransplantation. The following questions were examined:
1. How long will non-encapsulated and alginate-encapsulated porcine islets of Langerhans function in STZ-diabetic Wistar WU rats?
2. How is the biocompatibility of empty alginate microcapsules in vivo in different transplantation sites and with different exposure times?
3. How do alginate-microencapsulated porcine islets of Langerhans behave in differ-ent transplantation sites and with different exposure times?
Special attention was given to the vitality of the islet cells, the fibrosis and neovasculariza-tion of the microcapsules, and the inner cell infiltrates. The most important results were:
1. As expected, 20 of the 64 transplanted rats lost their non-encapsulated grafts by day +5. The 44 microencapsulated grafts evolved into two groups; those with short-term function (n=30; up to day +40) and those with long-term function (n=14; up to day +502). Criteria for the graft function were normoglycemia (daily testing of the blood glucose levels) and body weight.
2. Empty alginate microcapsules showed an excellent biocompatibility up to 200 days after implantation; they kept their original form and size; few inflammatory cells developed (no more than in a normal wound healing process); no neovasculariza-tion or fibrosis was evident on the empty capsules.
3. The microencapsulated islets of Langerhans were found, without exception, in both transplantation sites: “new islet organs” developed in the peritoneum, mainly in the spleen, liver, pancreas, small intestine, and omentum. Some of the capsule aggre-gates that evolved were embedded in the “guest organ”. Each microcapsule was surrounded with a dense net of capillaries (recipient vessels). Despite the normo-glycemia, only a few insulin-positive cells were present on the microcapsules. Every capsule maintained its original round, firm form and size. No broken, crushed or damaged microcapsules were observed. Partial islet cell regeneration was shown in 30% of the recipient-specific pancreata. This was more distinctive in the rats with long-term function than in those with short-term function of the graft. mpLI showed different degrees of fibrosis depending on the transplantation site and the function. The fibrosis was much stronger in the rats with short-term function of the graft; we attribute this to the lower vitality and purity of the graft. Fibroblasts encased the mpLI, but didn’t infiltrate the alginate. Fibrotic and non-fibrotic mpLI were repeatedly observed alongside each other even after long exposure in vivo. The reasons for this are unknown. The strong neovascularization of each and every mpLI was surprising; this was never observed in the empty microcapsules. The capillaries lay solely in the inner capsule on the periphery of the fibrotic ring. They did not infiltrate the ring or grow into the microcapsules. Therefore, we conclude that the mpLI stimulate the regeneration of the vessels and support the nutrition of the graft. Even after 502 days the immunohistochemical tests of the mpLI showed several insulin-positive beta cells, mainly single cells and smaller cell aggregates, few larger, intact cells. The immunohistochemical tests of the mpLI inner capsule showed cell infiltrates which were not seen in empty capsules and which were ob-viously activated by the microencapsulated cells: Infiltrates from CD4*, CD8+, ED1+, and HIS48+ cells were found more often in grafts with short-term function than in those with long-term function. Both graft types contained weak NK+ cell infiltrates but no TNF+ cells were observed. The cell infiltrates were more dis-tinctive in the grafts under the kidney capsules than in the grafts in the peritoneum.
In our experimental STZ-diabetic rat model we were able to show that mpLI induce long-term normoglycemia, even without immunosuppression. Despite the effective prevention of acute rejection (by the alginate microcapsule), a slight specific and non-specific immune response to the foreign cells develops which must be further investi-gated. The quality and function of the porcine islets of Langerhans continues to im-prove so that we expect a much higher percentage of normoglycemic rats with long-term graft function in this model. All in all, the xITX appears to be a very promising method to replace the absent endocrine function of the pancreas on a long-term basis.