Pannexin1 Channel Controls Membrane Permeability And Inflammasome Activation In Retinal Ischemia

Abstract

Purpose: : Membrane channels formed by pannexin1 (Panx1) are opened by ATP, mechanical stress, and ischemia. In vitro studies suggested that Panx1 is essential for the inflammasome formation in neurons and glia, and proposed that the opening of the pannexin1 hemichannels may play a role in neuronal death. In this study, we utilized Panx1knockout mice to directly test this hypothesis in a mouse model of retinal ischemia-reperfusion.

Methods: : Panx1-LoxP mice were generated and crossed with Thy1-Cre line to obtain conditional neuron-specific (nsKO) Panx1 gene inactivation and with CMV-Cre for global (gKO) inactivation. Retinal ganglion cell (RGC) tolerance to ischemia was tested in models of oxygen/glucose deprivation (OGD) and retinal ischemia/reperfusion (IR). Cell death was measured using Annexin V/propidium iodide labeling in primary cells, or by counting the beta-III Tubulin-labeled neurons in the ganglion cell layer (GCL) in flat mounted retinas. Membrane permeability was assessed using calcein-AM leakage assay. Pathway activation was measured by PCR, western blot and immunohistochemistry.

Results: : Panx1 deficiency resulted in a 25% increase in survival of primary RGCs from gKO and nsKO vs. wild type (WT) retinas in the OGD model. The percentage of apoptotic and necrotic cells in post-ischemic gKO vs. WT retinas decreased by 15 % and 17%, respectively. Seven days post-reperfusion, the GCL neuron death was reduced by 38% in both the gKO and nsKO compared to the WT. Ischemia-induced membrane permeability to calcein-AM dye observed in WT RGCs was blocked in both KO models or by a hemichannel blocker carbenoxolone in the WT retinas. Analysis of ischemic WT retinas revealed activation of caspase-1 and IL-1β production in the inner retina and RGCs. This activation was blocked in gKO and nsKO mice, suggesting that Panx1-dependent IL-1β production is involved in neurotoxicity.

Conclusions: : Panx1 channel activity is implicated membrane permeability, IL-1β production, and facilitates ischemic death of RGCs. Panx1 represents potential new target for therapy of retinal ischemia and stroke.

Keywords: ganglion cells • ischemia • gap junctions/coupling