Purpose: The method of counting cell nuclei above the internal limiting membrane in histologic sections is considered the standard when quantifying neovascularization (NV) in rodent oxygen-induced retinopathy (OIR). An alternative, more rapid method of counting clock hours in flatmounted adenosine diphosphatase (ADPase)-stained rat retinas is analogous to clinically scoring retinopathy of prematurity (ROP). In the present study, the validity of counting clock hours was evaluated by a direct comparison of these techniques. The intereye correlation of NV score and retinal vascular area were also studied.
Methods: Newborn Sprague-Dawley rats were exposed to cycles of O2 (80-10%) for 7 days, followed by 5 days of room air recovery. Preretinal NV was quantified by three masked observers counting clock hours in flatmounted ADPase-stained retinas of both eyes. Retinal vascular and total retinal areas were calculated using computer-assisted analysis. Representative retinas that had been scored positive (n = 10) and negative (n = 3) for NV and room air control retinas (n = 3) were embedded in paraffin. Each entire peripheral retinal quadrant was serially sectioned at 6 microm and stained with hematoxylin and eosin. Nuclei above the internal limiting membrane were then counted in a masked manner. The total number of nuclei counted per retina was defined as the nucleus count (704-938 sections per retina; 12,900 sections). Correlations were evaluated using Spearman rank coefficients.
Results: The nucleus count was 0 to 44 in room air control retinas, 0 to 40 in negative OIR retinas, and 250 to 5634 in positive OIR retinas. The nucleus count was highly correlated with the clock hour score (r(s) = 0.95, P = 0.0001). For the paired retinas, there was a significant correlation between right and left eyes in the severity of NV (clock hours; r(s) = 0.76, P = 0.0001) and the ratio of retinal vascular area to total retinal area (r(s) = 0.81, P = 0.0001).
Conclusions: The more rapid method of counting clock hours in flatmounted ADPase-stained retinas is valid for quantifying NV in rat models of ROP. Incidence and severity of NV and vascularized areas were similar between left and right eyes, which permits the use of paired retinas for complementary research techniques.