IN-VIVO ANALYSIS OF ANGIOGENESIS AND REVASCULARIZATION OF TRANSPLANTED PANCREATIC-ISLETS USING CONFOCAL MICROSCOPY

A technique to measure angiogenesis and revascularization in pancreati c islets transplanted at the renal subcapsular site in the rat has bee n developed. In-vivo imaging of the microcirculation of transplanted p ancreatic islets was conducted using a confocal scanning laser microsc ope (CSLM) to achieve optical sectioning through the graft in order to perform a computer reconstruction of the three-dimensional neovascula r morphology. Individual islets were harvested by enzymatic digestion of excised pancreas from Fischer 344 rats. Isolated islets were cultur ed for 24 h, and approximately 300-350 islets were transplanted at the renal subcapsular site of the left kidney in an anaesthetized rat. Si x to 14 days post-transplantation, the animal was anaesthetized and pr epared for in-vivo imaging of the microvasculature on a Zeiss LSM-10. Optical contrast of the microvasculature was enhanced by the administr ation of fluorescein-labelled dextran into the circulating blood. The transplant site was identified and serial sections were obtained throu gh the vascular bed at varying z-intervals. Complementary fluorescence video images were also obtained via a silicon intensifier tube camera mounted on the CSLM. At completion of the imaging procedure, the kidn ey was returned into the body cavity, the area was sutured and the ani mal was allowed to recuperate for subsequent examinations. Image proce ssing algorithms, such as grey-level thresholding, median filtering, s keletonization and template matching, were applied to compute the vess el density and diameters and extrapolated to measure 3-D vessel length s and the tortousity index of the neovasculature.