Endoplasmic Reticulum Stress-regulated CXCL10/CXCR3 pathway mediates inflammation and neuronal injury after retinal ischemia

Abstract

Purpose: Retinal ischemia causes many vision threatening diseases including glaucoma, diabetic retinopathy, retinal vascular occlusion, etc. Mechanisms of neuronal injury caused by retinal ischemia remain to be defined. CXCL10 is a chemokine which induces recruitment and activation of inflammatory cells after binding to its receptor CXCR3. The CXCL10/CXCR3 is involved in tissue injury but its role in retinopathy is unknown. This study is to determine the role of CXCL10/CXCR3 pathway in neuronal injury after retinal ischemia.

Methods: Retinal ischemia-reperfusion (IR) was induced by increasing the intraocular pressure up to 110 mm Hg for 45 mins to block the retinal blood flow. Eye and retina were collected at 1 hr to 7 days after retinal ischemia and analyzed by qRT-PCR, immunohistochemistry and western blotting. 1°GCs were isolated from mice pups and were treated with 1, 10 and 100 ng/ml of CXCL10 for 24 hrs. Cell death was evaluated by TUNEL analysis. To investigate the relation between ER stress and CXCL10 expression, qRT-PCR was performed in in PBA or TUDCA treated retina after IR.

Results: Expressions of CXCL10 mRNA and protein were significantly increased at 6 hours after retinal ischemia. Blockade of the CXCL10/CXCR3 pathway by deleting CXCR3 gene significantly attenuated IR-induced upregulation of inflammatory molecules (IL-1β and E-selectin), inhibited recruitment of microglia/monocyte to the superficial retina, reduced peroxynitrite formation, and prevented loss of retinal ganglion cells. Treatment of 1°GCs to various doses of CXCL10 (1, 10 and 100 ng/ml) resulted in the dose dependent cell deaths (19.38±0.97%, 23.74±1.11% and 26.15±0.83 respectively). Additionally, IR-induced CXCL10 upregulation was associated with increases in ER stress related genes (GRP78, ATF4 and CHOP). Inhibition of ER stress with the PBA and TUDCA significantly decreased IR-induced CXCL10 expression in the retina.

Conclusions: These results indicate that activation of CXCL10/CXCR3 pathway has an essential role in retinal inflammation, oxidative stress and neuronal injury after retinal ischemia. ER stress is implicated in IR-induced upregulation of CXCL10.

Keywords: 572 ischemia • 426 apoptosis/cell death • 531 ganglion cells