Erythropoietin protects the inner blood–retinal barrier by inhibiting microglia phagocytosis via Src/Akt/cofilin signalling in experimental diabetic retinopathy

Abstract

Aims/hypothesis Microglial activation in diabetic retinopathy and the protective effect of erythropoietin (EPO) have been extensively studied. However, the regulation of microglia in the retina and its relationship to inner blood-retinal barrier (iBRB) maintenance have not been fully characterised. In this study, we investigated the role of microglia in iBRB breakdown in diabetic retinopathy and the protective effects of EPO in this context. Methods Male Sprague Dawley rats were injected intraperitoneally with streptozotocin (STZ) to establish the experimental model of diabetes. At 2 h after STZ injection, the right and left eyes were injected intravitreally with EPO (16 mU/eye, 2 μl) and an equivalent volume of normal saline (NaCl 154 mmol/l), respectively. The rats were killed at 2 or 8 weeks after diabetes onset. Microglia activation was detected by ionised calcium binding adaptor molecule (IBA)-1 immunolabelling. Leakage of the iBRB was evaluated by albumin staining and FITC-dextran permeability assay. BV 2 cells and primary rat microglia under hypoxic conditions were used to model microglial activation in diabetic retinopathy. Phagocytosis was examined by confocal microscopy in flat-mounted retina preparations and in microglia and endothelial cell cocultures. Protein levels of IBA-1, CD11b, complement component 1r (C1r), and Src/Akt/cofilin signalling pathway components were assessed by western blotting. Results In diabetic rat retinas, phagocytosis of endothelial cells by activated microglia was observed at 8 weeks, resulting in an increased number of acellular capillaries (increased by 426.5%) and albumin leakage. Under hypoxic conditions, activated microglia transmigrated to the opposite membrane of the transwell, where they disrupted the endothelial cell monolayer by engulfing endothelial cells. The activation and phagocytic activity of microglia was blocked by intravitreal injection of EPO. In vitro, IBA-1, CD11b and C1r protein levels were increased by 50.9%, 170.0% and 135.5%, respectively, by hypoxia, whereas Jingfa Zhang and Guo-Tong Xu contributed equally to this study.

An in vitro cell model to study microglia activation in diabetic retinopathy

“…For immunostaining, the sections were immobilized in 2% PFA for 3 h. After permeabilizing and blocking in the solution (1% BSA, 0.1% Triton X‐100) for 1 h at ambient temperature, the sections were incubated with the corresponding antibodies, listed in Table S1. The methods for preparation and examination were as described in our recent publication (Xie et al, 2021).…”

The Role of PI3K/AKT and MAPK Signaling Pathways in Erythropoietin Signalization

“…This effect of EPO is partly dependent on the activation of PI3K and MAPK pathways. During diabetic retinopathy, inner blood-retinal barrier (BRB) breakdown occurs while EPO prevents this damage via the inhibition of microglial activation and phagocytosis mediated by the SRC/AKT/COFFILIN signaling pathway [ 108 ]. Furthermore, BRB integrity was maintained by EPO-regulated downregulation of HIF-1α, JNK signaling, and thus by the up-regulation of ZO-1 and occludin expression [ 109 ].…”

C1q/TNF-Related Protein 3 Prevents Diabetic Retinopathy via AMPK-Dependent Stabilization of Blood–Retinal Barrier Tight Junctions

“…Retinal capillary leakage assay was analyzed by previously reported methods with modifications [ 15 , 16 , 17 , 18 ]. Briefly, the mixture of tetramethylrhodamine isothiocyanate conjugate Concanavalin A (TRITC-ConcanavalinA) (indicating vascularity, 25 μg/g, Cat#C860, Thermo Fisher Scientific, MA, USA) and FITC-dextran (indicating vascular leakage, 50 μg/g, Cat#46945, Sigma-Aldrich, St. Louis, MO, USA) were injected into the superior vena cava.…”