The Pannexin-1 hemichannel and neuroinflammation in acute focal cerebral ischemia
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The pannexin-1 (panx1) hemichannel is associated with the Nod-Like Receptor Protein-1 (NLRP1) inflammasome following MCAO in a mouse model of focal ischaemic stroke. We investigated how blocking panx1 using probenecid affects post-stroke neuroinflammation and the formation of the NLRP1 inflammasome complex. We first investigated the expression levels of panx1, NLRP1 and interleukin-1β (IL-1β) using semi-quantitative Western blotting of brain tissue lysates. We investigated the presence of a spatial distribution overlap between NLRP1 and panx1 using confocal microscopy on acute brain slices taken from the brains of mice that had had strokes. To determine whether or not NLRP1 and panx1 for a direct protein-protein interaction, we also performed co-immunoprecipitation on brain tissue lysates collected from mice that had been given strokes. We make several key findings. The first is that the normalized optical density of IL-1β Western blot bands were increased in the ipsilateral hemisphere of the post-stroke brain relative to the contralateral one (n=6; meanODipsilateral=0.94±0.1; meanODcontralateral=0.81±0.1; p=0.04). Further, we found that the normalized OD of NLRP1 Western blot bands was also increased in the ipsilateral hemisphere relative to the non-ischaemic hemisphere (n=6 per hemisphere; meanODipsilateral=0.27±0.05; meanODcontralateral =0.21±0.04; p=0.04). Band optical densities for panx1 protein did not differ between hemispheres of mice given strokes. Importantly, probenecid attenuated the interhemispheric difference between IL-1β and NLRP1 levels after stroke, displaying an anti-inflammatory effect in the post-stroke brain. We performed co-immunoprecipitation o brain tissue lysates from mice that had strokes and found that NLRP1 did not coimmunoprecipitate with panx1. We draw the conclusion that they do not form a direct protein-protein interaction in acute stroke. Confocal imaging revealed demonstrated an overlap in the spatial distribution patterns of pannexin-1 and NLRP1 that occurred after stroke, but only in ischaemic tissue. The results of Western blot protein quantification studies, co-immunoprecipitation and confocal imaging together suggest that panx1 and NLRP1 are related by function but not by a direct protein-protein relationship. The anti-inflammatory effects of probenecid that we observed make it an attractive target for future studies on the prevention of post-ischaemic brain damage after stroke.