Abstract 3709: Acute Stroke opens Neuronal Pannexin1 Channels

Abstract

Introduction: Oxygen-glucose deprivation induces neuronal uptake of the sulforhodamine-based dye SR101 via pannexin1 channels in cultured neurons and brain slices. We assessed the hypothesis that pannexin1 channels are opened during acute stroke. Methods: Focal cerebral ischemia was induced in male C57BL/6 mice (20 to 25g, n=5) by middle cerebral artery occlusion (MCAO) for 30 minutes using the intraluminal suture occlusion method. Brain slices (2mm thickness) were taken immediately after stroke and incubated at room temperature in oxygenated sucrose-modified artificial cerebral spinal fluid for 60 minutes in 3 μ M Texas Red hydrazide (TRH), a structurally similar SR101 analog selectively taken up by astrocytes under non-ischemic conditions. Five brain slices co-incubated with 1 mM probenecid (a specific pannexin1 channel blocker) and seven brain slices without were assessed for pannexin1 channel-mediated dye uptake. Following cryosectioning and NeuN immunofluorescent labelling, the proportion of neurons showing TRH uptake (mean percentage ± S.E.M.) was determined in both hemispheres by fluorescence microscopy from 114 regions within the dorsomedial and lateral cortex, and 54 regions within the striatum. In a separate mouse, 2-photon laser scanning microscopy was used to assess in vivo neuronal uptake of sulforhodamine B (an SR101 analog) during acute stroke. Detailed neuronal morphology following 30 minutes of MCAO was assessed in a separate group of transgenic mice (n=3, C57 background) expressing yellow fluorescent protein in a subpopulation of neocortical pyramidal neurons. Results: Neuron morphology was unaltered after 30 minutes MCAO in the ipsilateral and contralateral hemisphere. TRH uptake was dramatically increased in the ischemic hemisphere in the lateral neocortex (ipsilateral vs contralateral 28% ± 1.8% vs 14% ± 1.6%, p < 0.0001) and striatum (ipsilateral vs contralateral 55% ± 3.0% vs 38% ± 3.1%, p = 0.005). The pannexin1-specific blocker probenecid reduced ipsilateral dye uptake to the same level seen contralaterally: (ipsilateral vs contralateral) lateral cortex 14% ± 2.5% vs 16% ± 2.6% (p = 0.58), dorsomedial cortex 13% ± 2.0% vs 16% ± 2.1% (p=0.31), and striatum 40% ± 3.2% vs 36% ± 2.2% (p=0.31). In vivo uptake of sulforhodamine B was demonstrated in neocortical and striatal neurons only in the ipsilateral hemisphere by 2-photon laser scanning microscopy. Conclusion: The opening of neuronal pannexin1 channels is widespread specifically in ischemic tissue within 30 minutes of MCAO onset. Pannexin1 channel opening appears to precede and possibly contribute to early neuronal membrane breakdown during acute stroke.