The Inhibitory Effects Of Valproate On Ischemic Stroke-induced Glial Scar Formation And Its Mechanisms

Aim: After cerebral ischemia injury, inflammatory reaction induces reactive proliferation of astrocytes to form a glial scar. As a physical barrier, glial scar impedes patients functional recovery in the late stage of ischemic injury. Valproate(VPA) is commonly used to treat seizures and bipolar disorder in clinical. Previous reports have suggested that VPA may play a role in the treatment of stroke. This study investigated whether VPA has a beneficial effect on ischemic stroke-induced glial scar formation and its underlying mechanisms in rats,models of middle cerebral artery occlusion(MCAO)-reperfusion and OGD-reperfusion model in primary cultured astrocytes.Methods: We established transient middle cerebral artery occlusion(tMCAO) and oxygen-glucose deprivation(OGD)-reperfusion model to verify the hypothesis in vivo and in vitro, respectively. Cell death was detected by a lactate dehydrogenase dectection kit. The number of necrotic and apoptotic cells were stained with propidium iodide(PI) and Annexin-V. The changes in the expression of GFAP, neurocan, phosphacan, acetyl-histone H3, H4 and autophagy-related proteins(LC3â…¡, p-ULK1, Cathepsin D and p62) were detected with Western Blotting and immunofluoresence. Lentiviral-delivered shRNA against ATG5(shRNA ATG5) or rapamycin was used to specifically inhibit or activate autophagy.Results: The results showed that intraperitoneal injection of VPA(250mg/kg/day) for one month could significantly reduce brain atrophy volume and improve behavioral deficits in rats,models of MCAO-reperfusion. And also, In an OGD-reperfusion-induced glial scar formation model, VPA protected OGD-reperfusion-induced astrocytic cell death. In the meantime, VPA could reduce the levels of the glial scar markers GFAP, neurocan and phosphacan protein. Further investigation revealed that VPA up-regulated acetyl-histone H3, acetyl-histone H4 and ac-ULK1 protein level. More importantly, we found that the autophagy level was reduced during the formation of glial scar, and VPA enhanced the autophagy. In order to further confirm whether the activation of autophagy can inhibit the formation of glial scar, we used rapamycin and shRNA Atg5 to activiate and inhibit autophagy, respectively. The results showed that activation of autophagy can inhibit glial scar information and VPA fails to inhibit glial scar formation while autophagy pathway was blocked.Conclusion: These results suggest that VPA can improve brain atrophy and behavioral deficits in the late stage of ischemic stroke rats, and this effect may be associated with inhibition of the formation of glial scar via up-regulation of acetyl-histone H3, acetyl-histone H4 and ac-ULK1 protein level and activation autophagy.