HRP/trypsin technique for studies of the retinal vasculature

The HRP/trypsin technique is a new histologic method for the light microscopic study of the retinal blood vessels. A two-stage procedure, the first step results in a retinal whole amount preparation which permits visualization of the three-dimensional architecture of perfused vessels and their relationship within the retina. This allows analysis of gross vessel morphology and differentiation of deep and superficial vascular beds. The second step involves digesting the whole mount with trypsin and staining with hematoxylin. This permits detailed evaluation of the density of retinal capillary endothelial cells and pericytes, recognition of basement membrane ghosts, microaneurysms, and other intraretinal microvascular abnormalities, with intravascular horseradish peroxidase as a perfusion marker. This technique has been employed with success in studies of the retinal vasculature in both normal and RCS rat retinas, and in a monkey model of branch retinal vein occlusion.