Abstract
Purpose: :
Pannexin1 (Panx1), a gap junction protein expressed in neurons and glia, can form mechanosensitive hemichannels that are opened by ATP in physiological Ca2+ concentrations. In vitro studies demonstrated that Panx1 is essential for the inflammasome formation, and suggested that the opening of the pannexin1 hemichannels may play a role in neuronal death from ischemia. To directly test this hypothesis, we investigated whether the Panx1 deficiency influence the survival of primary neurons challenged by ischemia in vitro and in vivo, in the mouse model of retinal ischemia-reperfusion.
Methods: :
We designed the Panx1fl/fl conditional knockout mouse line, where Cre-mediated recombination inactivates the Panx1 gene by the deletion of the floxed exons 3 and 4. Two germline knockout mouse strains were obtained independently by crossing these mice with either Thy1-Cre or CMV-Cre lines. We explored the effects of the Panx1 deficiency on neuronal tolerance to the oxygen/glucose deprivation (OGD) in vitro and to retinal ischemia/reperfusion (IR) injury and in vivo in retinal ischemia induced for 1 hour by unilateral elevation of intraocular pressure via corneal canulation. Primary RGCs, acutely purified by immunopanning from the retinas of Panx1fl/fl and Panx1-/- animals were exposed to the OGD for 4 hours, in a hypoxia chamber. Cell death rate was assessed in vitro using Annexin V and propidium iodide (PI) labeling 24 h post OGD, or in vivo by a direct counting of neurons in the ganglion cell layer (GCL) of flat-mounted retinas 7 days post-reperfusion.
Results: :
The absence of the Panx1 transcript in the Panx1-/- mice was confirmed by real time PCR, the lack of corresponding protein- by Western Blot and immunohistochemistry. Following OGD, we observed 30% increase in survival of the primary RGCs derived from the Panx1-/- vs. Panx1fl/fl mice (71% vs. 40%). The percentage of apoptotic and necrotic cells decreased 13 % and 11%, respectively, in the OGD-challenged RGCs from the Panx1-/- relative to the Panx1fl/fl retinas. The Panx1 deficiency significantly (26% vs 64% in the Panx1fl/fl control) reduced the GCL neuron death rate 7 days after reperfusion, and preserved structural integrity of retinal layers.
Conclusions: :
Our results is the first direct evidence that Panx1 activity is implicated in ischemic death of RGCs and the inactivation of this channel represents potential new target for therapy of retinal ischemia and stroke.
Keywords: ischemia • cell survival • ganglion cells