Quantification of Photoreceptors' Changes in a Diabetic Retinopathy Model with Two-Photon Imaging Microscopy.

Emerging evidence suggests that retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy (DR), preceding the development of microvascular abnormalities. Here, we assessed the impact of neuroinflammation on the retina of diabetic-induced rats. For this aim we have used a two-photon microscope to image the photoreceptors (PRs) at different eccentricities in unstained retinas obtained from both control (N = 4) and pathological rats (N = 4). This technique provides high-resolution images where individual PRs can be identified. Within each image, every PR was located, and its transversal area was measured and used as an objective parameter of neuroinflammation. In control samples, the size of the PRs hardly changed with retinal eccentricity. On the opposite end, diabetic retinas presented larger PR transversal sections. The ratio of PRs suffering from neuroinflammation was not uniform across the retina. Moreover, the maximum anatomical resolving power (in cycles/deg) was also calculated. This presents a double-slope pattern (from the central retina towards the periphery) in both types of specimens, although the values for diabetic retinas were significantly lower across all retinal locations. The results show that chronic retinal inflammation due to diabetes leads to an increase in PR transversal size. These changes are not uniform and depend on the retinal location. Two-photon microscopy is a useful tool to accurately characterize and quantify PR inflammatory processes and retinal alterations.