Immunohistochemical staining and morphometric analysis of the monkey choroidal vasculature

The purpose of this study was to develop a method for staining non-human primate choroidal blood vessels for combined whole mount and histologic analysis. Ten monkey eyes were used in this study. Diabetic and non-diabetic aged rhesus monkeys ranging in age from 16 to 31 years were killed and eyes were obtained immediately after death. Anterior segments were removed and retinas dissected from the posterior eyecup. Eyecups were soaked in 0.1% EDTA for 1hr to remove the retinal pigment epithelium (RPE). The whole choroids were dissected from the sclera, fixed in 2% paraformaldehyde and processed for immunohistochemical demonstration of perlecan, a heparan sulfate proteoglycan, using a streptavidin alkaline phosphatase technique. The alkaline phosphatase reaction product was developed using Histomark Red. Following immunohistochemical staining, choroids were incubated for the enzyme histochemical demonstration of non-specific esterase to label granulocytes. Anti-perlecan was localized to the endothelial cells of viable choroidal blood vessels and provided excellent staining of the choriocapillaris in all regions. Morphometric measurements of choriocapillaris were made in wet preparations using digitized images and image analysis software. The staining was absent in degenerated choroidal capillaries (lacking endothelial cells), thus allowing for sites of pathology to be identified and analysed. The reaction product was retained within the tissue throughout the flat-embedding process and allowed for precise site-specific identification of angiopathic lesions en bloc during sectioning. This technique provides a method of staining monkey choroidal blood vessels in situ and allows for identification and analysis of pathologic changes associated with aging or diseases.