The Role And Mechanism Of Pannexin-1 In Arterioles Hypercontractility After Subarachnoid Hemorrhage

Background and Objectives:Cerebral microcirculation(CM)dysfunction is the main cause for cerebral blood flow(CBF)decrease after subarachnoid hemorrhage(SAH).As an important component of CM dysfunction,arterioles contractility plays a key role in CBF regulation.Hypercontractility is a form of CM dysfunction,but the fundamental mechanism for arterioles hypercontractility after SAH remains unclear.ATP is an important purinergic signaling molecule and has a significant vasoconstrictor function,which is most likely related to arterioles hypercontractility after SAH.Based on the above theoretical analysis,we propose a scientific hypothesis that Pannexin-1-mediated ATP release from arterioles smooth muscle cells(SMCs)are closely related to arterioles hypercontractility and CBF decrease after SAH.In this study,we propose to test this hypothesis to confirm the role and molecular mechanism of Pannexin-1-mediated ATP release in causing arterioles hypercontractility after SAH,and to provide a new theoretical basis and therapeutic target for improving CM dysfunction and CBF decrease after SAH.Methods:1.Preparation of SAH rat model and SAH cell model1.1 SAH rat model establishment:The SAH rat model was established by blood injection into the suprasellar pool with assistance from a stereotactic instrument.1.2 SAH cell model establishment:Oxygenated hemoglobin was co-cultured with arterioles SMCs to simulate the SAH cell model.2.Pannexin-1 localization and expression in arterioles SMCs after SAH2.1 Pannexin-1 expression quantification:Divided into Sham,SAH-3d,SAH-5d and SAH-7d groups,and arterioles were collected by arterioles isolation method and then Pannexin-1 expression was measured by Western blot.2.2 Pannexin-1 expression localization and quantification:Divided into Sham and SAH-3d groups,and Pannexin-1 expression in arterioles SMCs was measured by immunofluorescence.3.Investigation of Pannexin-1 channel function in arterioles SMCs after SAH3.1 ATP release assay:arterioles SMCs cultured were divided into Control,OxyHb-12h-5μM,OxyHb-12h-10μM,OxyHb-24h-5μM,OxyHb-24h-10μM,Control+Pannexin-1 inhibitor Carbenoxolone(CBX),OxyHb-12 h-10μM+CBX groups,and the concentration of ATP released from arterioles SMCs was measured by ATP concentration assay.3.2 Pannexin-1 macroscopic current recording:arterioles SMCs cultured were divided into Control,Control+CBX,mock SAH and mock SAH+CBX groups,and the density of Pannexin-1 macroscopic current in arterioles SMCs was recorded by whole cell patch clamp.4.Investigation of Pannexin-1 and arterioles contractility and CBF after SAH4.1 Arterioles contractility assay:Divided into Sham and SAH groups,and after different concentrations of CBX were applied to both groups,the arterioles diameters in active brain slices were measured by single photon microimaging and arterioles contractility was calculated.4.2 CBF measurement:Divided into Sham,Sham+CBX,SAH and SAH+CBX groups,and CBF was measured by in vivo blood flow fluorescence imaging.Results:1.Pannexin-1 expression were significantly increased in arterioles SMCs after SAH1.1 Pannexin-1 were expressed in arterioles SMCs in all groups;the expression levels were higher in the SAH-3d and SAH-5d groups than in the Sham group(P<0.01).1.2 The expression level of Pannexin-1 in SAH-3d group were significantly higher than that in Sham group(P<0.001).2.Pannexin-1-mediated ATP release from arterioles SMCs and the density of macroscopic currents significantly increased after SAH2.1 The release of ATP from arterioles SMCs in the OxyHb-12h-5μM and OxyHb-12h-10μM groups was significantly higher than that in the Control group(P<0.0001);the concentration of ATP release from arterioles SMCs in the OxyHb-12h-10μM group was significantly higher than that in all other groups(P<0.0001).There was no significant difference in the concentration of ATP released from SMCs by arterioles in the Control+CBX group compared with the Control group;the concentration of ATP released from SMCs by arterioles in the OxyHb-12 h-10 μM+CBX group compared with the OxyHb-12 h-10 μM group was significantly decreased(P<0.0001).2.2 The density of Pannexin-1 macroscopic currents in the Control+CBX group was significantly lower than that in the Control group(P<0.0001),and the density of Pannexin-1 macroscopic currents in the simulation SAH group was significantly higher than that in the Control group(P<0.0001);the density of Pannexin-1 macroscopic currents in the simulation SAH+CBX group was significantly was significantly lower than that of simulation SAH group(P<0.0001).3.Pannexin-1 was strongly correlated with arterioles hypercontractility and CBF decrease after SAH3.1 Arterioles contractility was higher in the SAH group than in the Sham group(P<0.05);contractility gradually decreased in both the Sham and SAH groups with increasing CBX concentration.3.2 The CBF in the SAH group was significantly lower than that in the Sham group(P<0.01);the CBF in the SAH+CBX group was significantly higher than that in the SAH group(P<0.001);there was no significant difference between the CBF in the Sham group and the Sham+CBX group.Conclusions:Pannexin-1-mediated ATP release after SAH is strongly correlated with arterioles hypercontractility and CBF decrease.