胃黏膜下层胰岛移植物的生存率与功能的实验研究

目的 探讨胃黏膜下层胰岛移植的可行性.方法 应用雄性Wistar-Furth大鼠,STZ介导糖尿病大鼠模型.同种同基因大鼠体外胰岛分离纯化,分别移植进入糖尿病大鼠胃黏膜下层或肾被膜下,移植后不同时间段评估移植物生存率与代谢功能.结果 移植后早期移植胰岛被有效确认,并迅速分泌胰岛素逆转大鼠高血糖状况,糖耐受功能良好,与肾被膜下移植组比较,差异无显著意义.移植后4周,胃黏膜下移植组胰岛移植物胰岛素分泌功能逐渐丧失.结论 胃黏膜下胰岛移植技术具有良好的开发前景,如何提高其远期效果有待进一步考证.     

胃黏膜下层胰岛移植物的生存率与功能的实验研究

目的 探讨胃黏膜下层胰岛移植的可行性.方法 应用雄性Wistar-Furth大鼠,STZ介导糖尿病大鼠模型.同种同基因大鼠体外胰岛分离纯化,分别移植进入糖尿病大鼠胃黏膜下层或肾被膜下,移植后不同时间段评估移植物生存率与代谢功能.结果 移植后早期移植胰岛被有效确认,并迅速分泌胰岛素逆转大鼠高血糖状况,糖耐受功能良好,与肾被膜下移植组比较,差异无显著意义.移植后4周,胃黏膜下移植组胰岛移植物胰岛素分泌功能逐渐丧失.结论 胃黏膜下胰岛移植技术具有良好的开发前景,如何提高其远期效果有待进一步考证.     

胃黏膜下层胰岛移植物的生存率与功能的实验研究

目的 探讨胃黏膜下层胰岛移植的可行性.方法 应用雄性Wistar-Furth大鼠,STZ介导糖尿病大鼠模型.同种同基因大鼠体外胰岛分离纯化,分别移植进入糖尿病大鼠胃黏膜下层或肾被膜下,移植后不同时间段评估移植物生存率与代谢功能.结果 移植后早期移植胰岛被有效确认,并迅速分泌胰岛素逆转大鼠高血糖状况,糖耐受功能良好,与肾被膜下移植组比较,差异无显著意义.移植后4周,胃黏膜下移植组胰岛移植物胰岛素分泌功能逐渐丧失.结论 胃黏膜下胰岛移植技术具有良好的开发前景,如何提高其远期效果有待进一步考证.     

Grafting of mitomycin C-treated islet xenograft under the kidney capsule produces a clear bleb

We have previously shown that mitomycin C (MMC) treatment of donor tissue resulted in significant prolongation of graft survival in allo- and xenotransplantation models. However, the mechanisms involved in this prolongation are not clearly understood. This study aims to shed light on the immune responses to MMC-treated islet xenografting under the kidney capsule. Collagenase-digested WS (RT1k) rat islets incubated for 30 min with MMC and subsequently cultured for 20 h were transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 (B6;H-2b) mice. The grafts were harvested on postgrafting day 7 and sections were prepared and stained by hematoxylin and eosin (H&E). Histological study of the grafts in a group not treated with MMC showed marked cellular infiltration and destruction of islet clusters, whereas that of MMC-treated grafts demonstrated a bleb formation under the kidney capsule, in which islet cell clusters were reorganized, creating a layer of cells fixed to the interior of the bleb. Minimal invasion by inflammatory cells was observed only at the edge of the bleb, and most islet cells were protected from these infiltrating cells. In conclusion, MMC treatment induces remodeling of islet structure and forms a bleb under the kidney capsule, where no inflammatory cell infiltration occurs, suggesting that this site is a kind of immunologically privileged environment for xenografted islets.     

Donor islet endothelial cells participate in formation of functional vessels within pancreatic islet grafts

Pancreatic islet transplantation has emerged as a therapy for type 1 diabetes and is today performed using both freshly isolated and cultured islets. Islet blood vessels are disrupted during islet isolation; therefore, proper revascularization of the transplanted islets is of great importance for islet graft function and survival. We have studied intraislet endothelial cells after islet isolation, during islet culture, and following islet transplantation. By isolating islets from the transgenic Tie2-GFP (green fluorescent protein) mouse, characterized by an endothelial cell-specific expression of GFP, living endothelial cells could be studied in intact islets utilizing two-photon laser-scanning microscopy (TPLSM). Intraislet endothelial cells were found to survive islet transplantation but to rapidly disappear during islet culture. By transplanting freshly isolated Tie2-GFP islets and applying a novel ex vivo model for simultaneous perfusion and TPLSM imaging of the graft-bearing kidneys, GFP fluorescent endothelial cells were found to extensively contribute to vessels within the islet graft vasculature. Real-time imaging of the flow through the islet graft vasculature confirmed that the donor-derived vessels were functionally integrated. Hence, intraislet endothelial cells have the capability of participating in revascularization of pancreatic islets subsequent to transplantation. Therefore, preservation of intraislet endothelial cell mass may improve long-term graft function.     

Sympathetic reinnervation of rat kidney grafts

BACKGROUND: Reinnervation occurs in many transplanted tissues and organs. Sympathetic reinnervation in rat kidney grafts was investigated. METHODS: Rats were syngeneically transplanted with a kidney and bilaterally nephrectomized. Reinnervation was assessed by immunohistochemical staining for neuron-specific protein gene product 9.5 (PGP 9.5) and by tissue norepinephrine measurements in grafts removed 1.5 (n=6), 3 (n=7), 6 (n=8), and 9 (n=7) months after transplantation. RESULTS: PGP 9.5-positive neural structures were significantly reduced in grafts removed 1.5 and 3 months after transplantation compared with native kidneys with slightly increased numbers at 6 and 9 months after transplantation. Median transplant norepinephrine concentrations remained at approximately 3% compared with native kidneys until 9 months after transplantation. CONCLUSIONS: In transplanted rat kidneys, some reinnervation occurs in the hilum within 9 months after transplantation. This is not accompanied by a significant recovery of norepinephrine concentration in renal tissue indicating persistent sympathetic denervation.     

Prevention of recurrent autoimmune diabetes in the BB rat by islet transplantation under the renal capsule.

Pancreatic islet grafts transplanted into subjects with spontaneous autoimmune diabetes are threatened by two immune responses, allograft rejection and the recurrence of autoimmune insulitis. To examine the recurrent autoimmune response to transplanted islets it is necessary to exclude islet allograft rejection. The BB rat is a unique model of spontaneous diabetes with clinical and pathological characteristics identical or similar to those found in human insulin dependent diabetes mellitus (IDDM). In this study we demonstrate permanent acceptance of histocompatible islet grafts in chemically induced diabetes and a lack of intracolony tissue antigen rejection in our BB rat colony. Therefore the vigorous destruction of transplanted BB islets in the liver of spontaneously diabetic BB rats is due to recurrence of diabetes. This recurrence can be prevented by transplantation of islets under the renal capsule. This may be important for clinical application in IDDM, particularly with regard to host and donor tissue matching.     

Development of thymus autografts under the kidney capsule in the pig: A new "organ" for xenotransplantation

Abstract: Ten piglets, 7 to 16 weeks old, were partially thymectomised and 1 to 4 cm³ of minced thymic fragments autografted under the renal capsule. They were sacrificed, respectively, after 2, 4, 6, 8, 12, and 20 weeks. After 2 weeks, irregular whitish zones are present under the renal capsule. They were composed principally of two cell types: the first type was characterized by small round basophilic nuclei and little cytoplasm typical of lymphocytes; the second cell type had larger ovoid nuclei and a large vacuolised cytoplasm. Each cell type could be found in separate lobules or mixed in variable proportion in the same structure. The thymic autografts grew to form a layer up to 4 mm thick after 20 weeks. In the meantime, at the beginning of 4th week, the lobular structure became well organized with the cell type presenting large nuclei and cytoplasm being restricted to the center of the lobules while lymphocytes composed a peripheral layer. Hassal corpuscles (HC) appeared in the center of the lobules. Immunohistochemical labeling with anti-cytokeratin mono- and poly- clonal Ab and with anti-neurophysin polyclonal Ab displayed all the characteristics of normal functional thymic microenvironment. It is proposed that this novel experimental preparation ending up as a neo-organ (thymo-kidney) be used for xenotransplantation in an attempt to produce specific xenotolerance.     

Chronically decreased oxygen tension in rat pancreatic islets transplanted under the kidney capsule

BACKGROUND: A factor of potential importance in the failure of islet grafts is poor or inadequate engraftment of the islets in the implantation organ. This study measured the oxygen tension and blood perfusion in 1-, 2-, and 9-month-old islet grafts. METHODS: The partial pressure of oxygen was measured in pancreatic islets transplanted beneath the renal capsule of diabetic and nondiabetic recipient rats with a modified Clark electrode (outer tip diameter 2-6 microm). The size of the graft (250 islets) was by purpose not large enough to cure the diabetic recipients. The oxygen tension in islets within the pancreas was also recorded. Blood perfusion was measured with the laser-Doppler technique. RESULTS: Within native pancreatic islets, the partial pressure of oxygen was approximately 40 mm Hg (n=8). In islets transplanted to nondiabetic animals, the oxygen tension was approximately 6-7 mm Hg 1, 2, and 9 months posttransplantation. No differences could be seen between the different time points after transplantation. In the diabetic recipients, an even more pronounced decrease in graft tissue oxygen tension was recorded. The mean oxygen tension in the superficial renal cortex surrounding the implanted islets was similar in all groups (approximately 15 mm Hg). Intravenous administration of glucose (0.1 gxkg(-1)x min(-1)) did not affect the oxygen tension in any of the investigated tissues. The islet graft blood flow was similar in all groups, measuring approximately 50% of the blood flow in the kidney cortex. CONCLUSION: The oxygen tension in islets implanted beneath the kidney capsule is markedly lower than in native islets up to 9 months after transplantation. Moreover, persistent hyperglycemia in the recipient causes an even further decrease in graft oxygen tension, despite similar blood perfusion. To what extent this may contribute to islet graft failure remains to be determined.     

Effect of prolonged warm ischemia and pneumoperitoneum on renal function in a rat syngeneic kidney transplantation model

Background Laparoscopic donor nephrectomy is associated with several advantages for the donor. However, graft function may be impaired due to use of pneumoperitoneum and prolonged warm ischemia. This study investigated the impact of pneumoperitoneum and prolonged warm ischemia on long-term graft function in a syngeneic rat renal transplant model. Methods A total of 27 Brown Norway rats were randomized for transplantation of kidneys after three different procedures: no insufflation and no warm ischemia (group 1), no insufflation with 20 min of warm ischemia (group 2), and CO₂ insufflation and 20 min of warm ischemia (group 3). Glomerular filtration rate (GRF), serum creatinine, urine volume, urine creatinine, and proteinuria were determined monthly for 1 year. One year after transplantation, the grafts were removed for histomorphologic analysis. Results No significant differences in GRF, serum creatinine, urine volume, and proteinuria were found among the three groups. Histologic analysis also showed no differences between the groups. Conclusion Warm ischemia in combination with CO₂ pneumoperitoneum, as used in laparoscopic donor nephrectomy, does not result in a negative effect on long-term graft function. Paper presented at the annual meeting of the European Association for Endoscopic Surgery (EAES), 2004.     

Progressive deterioration of endocrine function after intraportal but not kidney subcapsular rat islet transplantation

In inbred streptozocin-induced diabetic rats, the long-term function of different endocrine pancreatic isografts was compared. Isolated islets transplanted into the portal vein showed a progressive deterioration of function over time. In contrast, islets under the kidney capsule sustained a constant long-term function controlling all clinical signs of diabetes. Recipients of kidney subcapsular islets displayed normal growth rate, peripheral serum glucose and insulin levels, and metabolic parameters. However, their functional reserve was markedly reduced as revealed by diminished glucose tolerance and reduced insulin-secreting capacity after an intravenous glucose challenge. Vascularized whole-organ pancreatic grafts with portal venous drainage led to complete normalization of all parameters determined in this study. This study showed that the long-term function of islets transplanted under the kidney capsule is superior compared with islets transplanted into the portal vein.     

Follow-up study of the revascularization process of purified rat islet beta-cell grafts

The revascularization of islets of Langerhans transplanted in heterotopic sites like the liver by portal vein embolization or the renal subcapsular space is a major process necessary for the viability of grafted cells. This process has been extensively studied by different techniques and the results have shown that islet revascularization is an early phenomenon that takes place soon after transplantation. In this report we have analyzed by a double indirect immunofluorescence technique, the revascularization process of purified endocrine islet beta-cells transplanted in the renal subcapsular space of syngeneic rats. Lewis rats were grafted with islets cultured for 24 h, with a suspension of purified beta-cells cultured for 24 h, and with a suspension of purified beta plus nonbeta-cells cultured for 24 h. Rats were killed at different days after implantation and the kidney bearing the grafts were snap frozen and immunohistochemically stained with a rabbit anti factor VIII antiserum (which labels endothelial cells). Immunocytochemical analysis revealed that cultured islets completed revascularization by days 3–5 after transplantation, as shown by the detection of capillary endothelial cells within and surrounding the islets. Within purified endocrine beta-cell grafts, the presence of numerous endothelial cells was not observed until days 10–14, indicating that revascularization of beta-cells with host vessels is not such an early phenomenon as it takes place in whole isolated islets. Conversely, the addition of a population of endocrine nonbeta-cells to the purified islet cell grafts, partially accelerated the revascularization of pure beta-cell grafts, which showed the presence of abundant capillary endothelial cells already at day 7 after transplantation, indicating that some other unidentified factors besides the absence of endothelial cells may explain the retardation of beta-cell grafts revascularization.     

Transplantation of newborn rat testis under the kidney capsule of adult host as a model to study the structure and function of Leydig cells

Newborn rat testis was transplanted under the kidney capsule of adult castrated and uncastrated male rats to develop and characterize a model system for studies on Leydig cell development. Two weeks after transplantation, the number of Leydig cells and the size of their nuclei in the transplants had increased. Secretion of testosterone was indicated by increased seminal vesicle weights and decreased pituitary LH in the castrated host animals. Pituitary FSH content increased significantly in the uncastrated animals with transplants, which suggested production of an FSH-stimulating factor. Cells with the morphologic features characteristic of fetal- and adult-type Leydig cells were observed in the transplants. The seminiferous tubules with spermatocytes, incipient lumina, and significantly larger average diameters showed more advanced development than those in the normal 2-week-old testis. By the present morphologic and functional criteria, the kidney subcapsular transplantation technique provides a suitable model for studies of fetal and adult Leydig cell development.