Autophagy in diabetic retinal ischemia/reperfusion injury

Abstract

Purpose: Retinal ischemia/reperfusion (I/R) injury is a common cause of retinal cell death. Despite increasing interest in autophagy modulation as a therapeutic strategy, the effect of retinal I/R injury on autophagy (up or downregulation) remains controversial. We investigated the effect of retinal I/R injury on autophagy using both an in vivo model and an in vitro model. Methods: In vivo: A middle cerebral artery occlusion (MCAO) stroke model was used to induce unilateral retinal I/R injury in wild-type and Akita (type I diabetic) mice. Ischemia was maintained for two hours, followed by 2 or 22 hours of reperfusion. Autophagy markers, microtubule-associated light chain protein (LC3) and lysosomal associated membrane protein (LAMP1), were assessed via immunohistochemical staining on retinal sections. In vitro: R28 cells (a retinal precursor cell line) were cultured in low glucose DMEM medium. One group of cells was incubated with additional glucose to mimic hyperglycemia. Hypoxia was chemically induced using cobalt (II) chloride for 24 hours. Cell viability and autophagy was assessed via the MTS assay and the detection of LC3 by immunoblotting, respectively. Results: In vivo: After 2 hours of reperfusion, LC3 was significantly upregulated in the ganglion cell layer (GCL) in MCAO-injured Akita retinae as compared with sham-treated Akita retinae. After 22 hours of reperfusion, LAMP1 immunoreactivity was increased in the GCL and inner nuclear layer of MCAO-injured retinae as compared with their sham-treated counterparts. In vitro: Hypoxic cells had lower cell viability than untreated cells. Hypoxia also resulted in significant LC3 upregulation. Conclusion: Retinal I/R injury and CoCl2-induced hypoxia induces autophagy activation in mouse retinae and R28 cells, respectively. Both our in vivo and in vitro results suggest that retinal I/R injury-induced autophagy may be exacerbated by elevated glucose levels.