Diabetic retinopathy is a major cause of visual impairment, which continues to increase in prevalence as more and more people develop diabetes. Despite the importance of vision, the retina is one of the smallest tissues in the body, and specialized techniques to study the retinopathy have been developed. This chapter will summarize several methods used to (i) induce diabetes, (ii) maintain the diabetic animals throughout the months required for the development of typical vascular histopathology, (iii) evaluate vascular histopathology of diabetic retinopathy, and (iv) quantitate abnormalities implicated in the development of the retinopathy.

PurposeRecent studies suggest that photoreceptor cells regulate local inflammation in the retina in diabetes. The purpose of this study was to determine if photoreceptor cells themselves produce inflammatory proteins in diabetes and if soluble factors released by photoreceptors in elevated glucose induce inflammatory changes in nearby cells.MethodsLaser capture microdissection was used to isolate the outer retina (photoreceptors) from the inner retina in nondiabetic and diabetic mice. Diabetes-induced changes in the expression of inflammatory targets were assessed by reverse transcription polymerase chain reaction and immunohistochemistry. Cell culture experiments were carried out to determine if photoreceptors in vitro and ex vivo release soluble mediators that can stimulate nearby cells. Photoreceptor contribution to leukocyte-mediated endothelial cell death was tested using coculture models.ResultsMessenger ribonucleic acid and protein expression levels for inflammatory proteins intercellular adhesion molecule 1 (ICAM1), inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX2) were increased in photoreceptors cells in diabetes. In vitro and ex vivo studies show that photoreceptor cells in elevated glucose release mediators that can induce tumor necrosis factor-α in leukocytes and endothelial cells, but not in glia. The soluble mediators released by photoreceptor cells in elevated glucose are regulated by transforming growth factor β-activated kinase 1 and nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) signaling. In contrast to enhanced leukocyte-mediated killing of endothelial cells by leukocytes from wild-type diabetic mice, leukocytes from diabetic mice lacking photoreceptor cells (opsin−/−) did not kill endothelial cells.ConclusionsThese data indicate that photoreceptor cells are a source of inflammatory proteins in diabetes, and their release of soluble mediators can contribute to the death of retinal capillaries in diabetes.

PurposeLoss of photoreceptor cells is associated with retinal vascular degeneration in retinitis pigmentosa, whereas the presence of photoreceptor cells is implicated in vascular degeneration in diabetic retinopathy. To investigate how both the absence and presence of photoreceptors could damage the retinal vasculature, we compared two mouse models of photoreceptor degeneration (opsin−/− and RhoP23H/P23H ) and control C57Bl/5J mice, each with and without diabetes.MethodsRetinal thickness, superoxide, expression of inflammatory proteins, ERG and optokinetic responses, leukocyte cytotoxicity, and capillary degeneration were evaluated at 1 to 10 months of age using published methods.ResultsRetinal photoreceptor cells degenerated completely in the opsin mutants by 2 to 4 months of age, and visual function subsided correspondingly. Retinal capillary degeneration was substantial while photoreceptors were still present, but slowed after the photoreceptors degenerated. Diabetes did not further exacerbate capillary degeneration in these models of photoreceptor degeneration, but did cause capillary degeneration in wild-type animals. Photoreceptor cells, however, did not degenerate in wild-type diabetic mice, presumably because the stress responses in these cells were less than in the opsin mutants. Retinal superoxide and leukocyte damage to retinal endothelium contributed to the degeneration of retinal capillaries in diabetes, and leukocyte-mediated damage was increased in both opsin mutants during photoreceptor cell degeneration.ConclusionsPhotoreceptor cells affect the integrity of the retinal microvasculature. Deterioration of retinal capillaries in opsin mutants was appreciable while photoreceptor cells were present and stressed, but was less after photoreceptors degenerated. This finding proves relevant to diabetes, where persistent stress in photoreceptors likewise contributes to capillary degeneration.

Diabetic retinopathy is a prevailing diabetes complication, and one of the leading causes of blindness worldwide. IL-17A is a cytokine involved in the onset of diabetic complications. In the current study, we examined the role of IL-17A in the development of retinal inflammation and long-term vascular pathology in diabetic mice. We found IL-17A expressing T cells and neutrophils in the retinal vasculature. Further, the IL-17A receptor was expressed on Muller glia, retinal endothelial cells, and photoreceptors. Finally, diabetes-mediated retinal inflammation, oxidative stress, and vascular leakage were all significantly lower in IL-17A −/− mice. These are all clinically meaningful abnormalities that characterize the onset of diabetic retinopathy.