DETECTION OF VASCULAR ENDOTHELIAL GROWTH-FACTOR (VEGF) PROTEIN IN VASCULAR AND NONVASCULAR CELLS OF THE NORMAL AND OXYGEN-INJURED RAT RETINA

Vascular endothelial growth factor (VEGF) is a potent and specific end othelial cell cytokine that can be up-regulated by hypoxia. There is e vidence that VEGF is a significant mediator in retinal neovascular dis eases and other disorders in which hypoxia is believed to influence th e pathogenesis. Here we demonstrate the spatial relationships among ar eas of retinal non-perfusion, VEGF protein and vascular endothelial ce lls throughout the retina, and relate these results to cellular distri bution of VEGF in cross section. Newborn albino rats were oxygen-injur ed by cycles of alternating 50% and 10% oxygen for 14 days and then pl aced in room air. On days 16, 21 and 26, oxygen-injured and control (r aised in room air) rats were sacrificed, enucleated and retinas were d issected and fixed for whole mount immunostaining for VEGF or embeddin g in glycol methacrylate for VEGF immunohistochemistry. Intact eyes ta ken on days 16 and 20 were processed similarly. Vascular endothelial c ells were demonstrated by staining whole-mounted retinas for adenosine diphosphatase (ADPase) activity. Preretinal neovascular growths (i.e. , abnormal vessels extending from the retina into the vitreous) were V EGF-positive. There was also a pan-retinal distribution of non-endothe lial cells that were VEGF-positive in both room air and oxygen-injured rats, with stronger immunostaining in day 16 oxygen-injured retinas. In cross-section, VEGF staining was confirmed in preretinal growths, n ormal retinal vessels, cells in the inner nuclear layer (primarily Mul ler cells) and ganglion cells. Retinas which had been incubated with n onimmune IgG or absorbed anti-VEGF antibody showed little or no staini ng. In conclusion, we have identified cells of the inner retina which express VEGF. The production of VEGF by these cells - in particular, M uller cells - may promote preretinal neovascularization in oxygen-inju red eyes. We have found, moreover, that the combination of immunohisto chemistry and ADPase staining of,whole mount preparations is a unique and powerful tool for evaluating relationships between presumed areas of retinal ischemia, VEGF (and other cytokines) and retinal blood vess els, within an entire retina. This approach can be used to study any p roliferative retinal disorders in which VEGF is a potential component of the pathogenesis.