An improved method of fluorescent dual insulin and endothelial staining allows visualisation of the revascularisation of intraportally transplanted islets.

BACKGROUND

Intraportally transplanted islets are avascular at the time of transplantation and take up to 14 days to fully revascularize, during which time up to 60% of islet mass may be lost. To investigate and improve islet revascularization, a robust method for the visualization and quantification of this process is required.

METHODS

Islets isolated from Lewis rats were transplanted intraportally into the liver of diabetic syngeneic Lewis recipients. The animals were humanely killed either on the day of transplant or at 3, 5, 7, or 14 days posttransplant. The harvested livers were sectioned and stained with Bandeiraea simplicifolia lectin (for endothelial cells) and anti-insulin antibody and counterstained with DAPI. The slides were visualized with a fluorescent microscope.

RESULTS

Islets were visualized over the whole time course. Insulin and endothelial staining was clearly visualized on the day of transplantation, but by day 3 endothelial staining was scarce within the islet. By day 5, early vessel formation could be seen within the islet, but insulin staining was patchy and associated with apoptotic nuclei. By day 7, vessels could be seen throughout the islet and insulin staining had returned. Day 14 sections showed a fully revascularized islet.

CONCLUSIONS

The staining provided good delineation of islet endothelium and beta-cell location, with clear observation of the revascularization process. This technique also suggests that days 3 through 5 may be a critical period for islet survival and provides a good model for studying the effects of manipulating the revascularization process.