Evans blue staining to detect deep blood vessels in peripheral retina for observing retinal pathology in early-stage diabetic rats.

Aim: To observe and compare the statistical significance of superficial and deep vascular leakage in the pathological changes of the diabetic rats retina after the Evans blue (EB) perfusion, and utilize the modified whole-retina spreading method to make the slides while protecting the periphery of the retina.

Methods: The Sprague-Dawley (SD) rats were randomly divided into 6 groups. Each group named as the normal groups for 4, 8, and 12wk and the diabetic groups for 4, 8, and 12wk. The EB was injected into the cardiovascular system of the rats at the different time points. The retina of each group was obtained for observation.

Results: The superficial vascular leakage was found in all 6 groups. The size of leakage area of superficial retinal blood vessels was (0.54±0.23)%, (0.65±0.11)%, and (0.58±0.10)% in normal group. No notable leakage was found in the deep blood vessels [(0.03±0.04)%, (0.03±0.05)%, and (0.03±0.05)%]. The deep retinal vascular leakage was found in the peripheral retina of diabetic rats. The size of leakage area of superficial retinal blood vessels in diabetic group were (0.53±0.22)%, (0.69±0.16)%, and (0.52±0.11)%. The leakage areas of deep blood vessels were (0.54±0.50)%, (1.42±0.16)%, and (1.80±0.07)% at 4, 8, and 12wk, respectively. There was a statistically difference of the leakage area between the 8th week and the 4th week of diabetes group (=0.003). The statistically significant difference between the diabetes and the control groups was noted at 4wk and 8wk (<0.001).

Conclusion: The main retinal pathological changes of early-stage diabetic rats are the vascular leakage of the periphery of deep retina. Diabetic rats modeled after 8wk have semi-quantitative statistical difference compared with the normal rats, thus early intervention treatment research can start at this time point.