Revascularization and remodelling of pancreatic islets grafted under the kidney capsule

Abstract

The revascularization and the structural changes resulting from interactions between the graft and the host were investigated in transplanted pancreatic islets under the kidney capsule. Islets were isolated from mice pancreata and transplanted in syngeneic diabetic animals. Graft-bearing kidneys were collected on different days post-transplant and processed for light microscopy, immunohistochemistry and transmission electron microscopy. A numerical analysis was performed in order to compare the percentage number of the different types of cells in native islets and at different time points after the transplant. Recipient animals reversed diabetes within 4 days. An intraperitoneal glucose tolerance test was performed to determine islet functionality under stressful conditions. During the initial few days post-transplant, the islets showed peculiar shapes and the graft tended to aggregate along the vessels. Starting at days 4-7 post-transplant, islets were revascularized from vessels connected to both the cortical and the capsular vascular network of the kidney. From day 7-14 post-transplant, the vessels progressively appeared more similar in features and size to those of in situ pancreatic islets. Both the percentage number of the different cell types and the distribution of Alpha, Beta and Delta cells inside the graft were significantly different as compared with intact islets, demonstrating quantitative and structural changes after the engraftment. No concomitant proliferation of Beta cells was detected using a bromodeoxyuridin staining method. Despite the fact that quick revascularization preserved a large mass of tissue, the remodelling process of the graft and the newly formed vascularization led to a different organization of the endocrine tissue as compared with intact in situ islets. This constitutes the morphological basis for alterations of the normal intercellular interactions and may explain the altered secretory cell function often observed in transplant.

Fig. 1

In vivo functionality of transplanted pancreatic islets. (a) Blood glucose level determined before the injection of streptozotocin (STZ), just before the transplant (day 0) and in the post-transplant follow-up. Note the induction of diabetes, the stable normalization of glycaemia approximately 3 days after the transplant, and the re-induction of diabetes after the nephrectomy (⇑). (b) Intraperitoneal glucose tolerance tests (IPGTTs) performed on six animals for each time point (see also Table 2). After the intraperitoneal injection of glucose, blood glucose levels were measured at 15, 30, 45, 60, 90 and 120 min. An abnormal response was observed in the immediate post-transplant period (2 and 4 days). Animals at 21 days after the transplant showed the best response with lower levels of glycaemia at 45 min post-glucose infusion and normal levels after 120 min, while at 7 and 14 days an intermediate response to the test was observed.

Fig. 2

Graft at day 2 post-transplant. A large, marginally located islet shows a necrotic core (*). Soft tissue appears largely oedematous, with infiltration of blood cells and congestion of blood (arrow) and lymphatic vessels. Fragments of islets and residual exocrine tissue are also present. Haematoxylin and eosin stain, 125×, scale bar = 200 µm.

Fig. 3

Fragment of islet at day 4 post-transplant. These cells are disposed in a single or double layer close to pre-existing blood vessels (arrowhead) from which vascular proliferation is also visible (arrow). Semithin section, methylene blue stain, 400×, scale bar = 50 µm.

Fig. 4

Graft at day 4 post-transplant. Transplanted islets showing irregular shape tend to aggregate forming clusters. Necrotic zones inside the connective tissue of the kidney capsule are almost completely repaired by macrophagic cells and proliferating fibroblasts. Some residual exocrine ducts are also present. Immunoperoxidase stain for insulin, 80×, scale bar = 260 µm.

Fig. 5

Periphery of an islet at day 7 post-transplant. Long and slender processes of fibroblasts (arrow) secreting collagen fibrils (arrowheads) surround the islet and a vessel of the kidney capsule. TEM, scale bar = 2.5 µm.

Fig. 6

Islet at day 7 post-transplant. Arising from the vessels running along the outer surface of this large islet, many new vessels (arrows) are growing and penetrating from all directions towards the inner part of the endocrine parenchyma. Some vessels, lined by endothelial cells, are also visible in the islet parenchyma (arrowheads). Semithin section, methylene blue stain, 200×, scale bar = 100 µm.

Fig. 7

Angiogenesis at day 7 post-transplant. Two proliferating endothelial cells assume a sprout feature penetrating between B cells. They do not present a basal lamina and their processes are encircling a lumen that appears as a thin cleft between the two cells. TEM, scale bar = 3 µm.

Fig. 8

Capillary from the core of an islet at day 7 post-transplant. This capillary is lined by a single endothelial cell containing cytoplasm. A basal lamina surrounds the capillary; a perivascular cell (p) is also visible. TEM, scale bar = 2.5 µm.

Fig. 9

Detail of fenestrations (arrowheads) closed by a very thin diaphragm in the thinner part of the capillary wall. TEM, scale bar = 1 µm.

Fig. 10

Consecutive sections of islets re-aggregated in a cluster. A large number of cells show positive staining for insulin (a). Also, the peripheral layer of the cluster is largely represented by B cells. Some glucagon-stained A cells are singly scattered peripherally and also in the inner part of the cluster (b). Immunoperoxidase stain for (a) insulin, (b) glucagon, 200×, scale bar = 150 µm.

Fig. 11

Microvascular network of an islet at day 14 post-transplant. The endocrine cells form cordonal-like structures disposed radially around capillaries (*) located inside the islet parenchyma, in most of the cases completely surrounded by endocrine cells. The vessels supplying this islet arise from host vessels located at the periphery of the islet. They are connected to the microvascular network of both the renal parenchyma (arrowheads) and the capsular district (arrows). Semithin section, methylene blue stain, 400×, scale bar = 100 µm.

Fig. 12

Consecutive sections of in situ islets. In (a) the insulin-stained B cells occupy the core of the islet. They are surrounded by a nearly complete crown of unstained cells that are positive for glucagone, as shown in (b). Immunoperoxidase stain for (a) insulin, (b) glucagon, 200×, scale bar = 200 µm.

Fig. 13

BrdU incorporation for detection of proliferating islet cells. No evidence of proliferating cells within islets transplanted under the kidney capsule is detected (a). For comparison, proliferation (arrows) is clearly observed in native pancreatic islets of pregnant mice as positive control (b).