Assessment of Longterm Changes in Retinal Vasculature in the InsAkita+/- Diabetic Mouse Using Two-Photon Microscopy

Abstract

Purpose: : Diabetes causes neuronal degeneration and vascular damage in the human retina. Here we assess longterm changes of retinal morphology in the diabetic mouse model Ins^Akita+/- using two-photon microscopy.

Methods: : Whole-mounted retinal explants of wildtype mice (C57bl/6) and Ins^Akita+/-mice (6 months after onset of diabetes) were life-stained with sulforhodamine 101 (SR101) and then analyzed morphologically using two-photon microscopy. Three independent observers analyzed changes in layer thickness and retinal vasculature, as well as tissue integrity and cell viability. For the latter we made use of the fact that in the retina SR101 stains the somata of damaged neurons but not healthy ones.

Results: : Overall retinal morphology was intact in all animals. Retinal thickness was significantly reduced in diabetic mice as compared to wild type mice, predominantly in the inner retinal layers. Qualitatively, vascular occlusions and acellular capillaries were found in the intermediate and deep vascular layers of diabetic mice, but not in the superficial vascular layer. In wildtype mice, these abnormalities were not observed. After 6 months of diabetes, we could not observe an increase in the number of SR101-positive cells, suggesting that neurons are not substantially damaged.

Conclusions: : We demonstrate a detailled analysis of retinal architecture in Ins^Akita+/- mice with longstading diabetes. While diminished layer thickness mainly in the inner (vascularized) layers suggest a vascular degeneration resulting in capillary occlusion, no increase in damaged cells was seen at this stage. Further studies shall clarify the time course of neuroglial degeneration and the relation to microangiopathy in this prototype model.

Keywords: diabetic retinopathy • inner retina dysfunction: biochemistry and cell biology • microscopy: confocal/tunneling