Purpose: To investigate the effect of a selective aquaporin 4 (AQP4) inhibitor, 2-(nicotinamide)-1,3,4-thiadiazole (TGN-020), on the expression of vascular endothelial growth factor (VEGF) and reactive oxygen species (ROS) production, as well as on the retinal edema in diabetic retina. Methods: Intravitreal injections of bevacizumab, TGN-020, or phosphate-buffered saline (PBS) were performed on streptozotocin-induced diabetic rats. Retinal sections were immunostained for anti-glial fibrillary acidic protein (GFAP), anti-AQP4, and anti-VEGF. Protein levels of VEGF from collected retinas were determined by Western blot analysis. In addition, retinal vascular leakage of Evans Blue was observed in the flat-mounted retina from the diabetic rats in the presence or absence of TGN-020. Volumetric changes of rat retinal Müller cells (TR-MUL5; transgenic rat Müller cells) and intracellular levels of ROS were determined using flow cytometry analysis of ethidium fluorescence in the presence or absence of TGN-020 or bevacizumab under physiological and high glucose conditions. Results: In the diabetic retina, the immunoreactivity and protein levels of VEGF were suppressed by TGN-020. AQP4 immunoreactivity was higher than in the control retinas and the expressions of AQP4 were co-localized with GFAP. Similarly to VEGF, AQP4 and GFAP were also suppressed by TGN-020. In the Evans Blue assay, TGN-020 decreased leakage in the diabetic retinas. In the cultured Müller cells, the increase in cell volumes and intracellular ROS production under high glucose condition were suppressed by exposure to TGN-020 as much as by exposure to bevacizumab. Conclusion: TGN-020 may have an inhibitory effect on diabetic retinal edema.

Microvascular changes are the earliest adverse events in diabetic retinopathy, but recent studies have shown that oxidative stress induced by photoreceptors is associated with the development of the retinopathy. The purpose of this study was to determine the roles played by superoxides formed by photoreceptors under hyperglycemic conditions on autophagy. To accomplish this, we cultured 661 W cells, a transformed murine cone cell line, with 5.5 or 25 mM glucose in the presence or absence of 3 methyl adenine (3MA) or rapamycin. The superoxides were determined by flow cytometry using hydroethidine as a fluorescence probe. The autophagy activity was determined by changes in the expression of LC3B2 and P62 by immunoblotting. The degree of mitophagy was determined by the accumulation of mitochondria and lysosomes. Apoptotic changes of 661 W cells were determined by the caspase 3/7 activities. Our results showed higher levels of P62 and superoxides in cells cultured in 25 mM glucose than in 5.5 mM glucose. Addition of 3MA caused a significant increase of P62, superoxides, and caspase 3/7 activities in the 661 W cells cultured in high glucose but not in low glucose. These findings suggest that autophagy is important for the functioning and survival of 661 W cells under hyperglycemic conditions.

The fovea centralis, an anatomically concave pit located at the center of the macula, is avascular, hypoxic, and characteristic of stem-cell niches of other tissues. We hypothesized that in the fovea, undifferentiated retinal-stem-cell-like cells may exist, and that neurogenesis may occur. Hence, we performed an immunohistological study using cynomolgus monkey retinas. After preparing frozen tissue sections of the retina including the foveal pit, immunostaining was performed for glial fibrillary acidic protein (GFAP), nestin, vimentin, neuron-specific class III β-tubulin (Tuj-1), arrestin 4, neurofilament, CD117, CD44, Ki67, and cellular retinaldehyde-binding protein (CRALBP), followed by fluorescence and/or confocal microscopy examinations. Immunostaining of the tissue sections enabled clear observation of strongly GFAP-positive cells that corresponded to the inner-half layer of the foveolar Müller cell cone. The surface layer of the foveal slope was partially costained with GFAP and vimentin. Tuj-1-positive cells were observed in the innermost layer of the foveolar retina, which spanned to the surrounding ganglion cell layer. Moreover, colocalization of Tuj-1 and GFAP was observed at the foveal pit. The coexpression of CD117 and CD44 was found in the interphotoreceptor matrix of the fovea. The foveolar cone stained positive for both nestin and arrestin 4, however, the photoreceptor layer outside of the foveola displayed weak staining for nestin. Colocalization of nestin and vimentin was observed in the inner half of the Henle layer, while colocalization of nestin and neurofilament was observed in the outer half, predominantly. Scattered Ki67-positive cells were observed in the cellular processes of the outer plexiform layer and the ganglion cell layer around the foveola. Immunostaining for CRALBP was negative in most parts of the GFAP-positive area. The Müller cell cone was divided into GFAP-strongly positive cells, presumably astrocytes, in the inner layer and nestin-positive/GFAP-weakly positive radial glia-like cells in the outer layer. These findings indicated that groups of such undifferentiated cells in the foveola might be involved in maintaining morphology and regeneration.

The pathophysiologic mechanism of diabetic retinopathy is not yet fully elucidated; however, the apoptotic death of microvascular pericytes and endothelial cells is one of the hallmarks of diabetic retinopathy.¹ The loss of pericytes seems to play an important role in the progression of diabetic retinopathy.² Many studies have examined retinal blood flow in diabetes.^3,4 As the disease progresses before vasoproliferation, retinal blood flow was found to be above normal.^5-7 However, some researchers have reported reduced retinal blood flow in patients with diabetes mellitus without diabetic retinopathy or with background diabetic retinopathy.^8,9 These changes in retinal blood flow might be related to the dysfunction or loss of microvascular pericytes.