Quantification of Retinal Microvascular Apoptosis

Retina was isolated from the formalin-fixed eye, and rinsed overnight with the running water. The microvasculature was isolated by incubating the retina with 3% crude trypsin (Invitrogen-Gibco, Grand Island, NY) containing 200 mM sodium fluoride for 45 to 70 minutes at 37°C, and the neuro-retinal tissue was gently brushed away. The apoptotic vascular cells were detected by incubating the preparation with terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling stain (TUNEL; In Situ Cell Death kit; Roche Molecular Biochemicals, Indianapolis, IN). In each experiment, a positive control was run by exposing the retinal vessels to DNAse before initiation of the TUNEL reaction [9 (link),10 (link)]. TUNEL positive cells were identified in a masked fashion, and each trypsin digest was surveyed systematically under a Zeiss Axiophot photomicroscope.
After TUNEL staining, the microvasculature was stained with periodic acid-Schiff and hematoxylin for histologic evaluation. The number of acellular capillaries was counted in multiple mid-retinal fields (one field adjacent to each of the 5–7 retinal arterioles radiating out from the optic disc) and expressed as total acellular capillaries per retina [9 (link),10 (link)].