Role And Mechanism Of Valproic Acid In Early Brain Injury After Experimental Subarachnoid Hemorrhage

BackgroundSubarachnoid hemorrhage accounts for about5%to10%of all strokes and mostly due to rupture of intracranial aneurysms. Over the past few decades, cerebral vasospasm and aneurysm rebleeding have been considered the most determinant of poor outcome. However, many multi-center clinical trials showed delayed vasospasm alleviation did not improve the prognosis of patients with subarachnoid hemorrhage. Therefore, some scholars have proposed early brain injury is the main cause for death and disability after subarachnoid hemorrhage.The term "early brain injury" has been generated referring to the immediate injury within72h and includes secondary events to SAH before the development of cerebral vasospasm. Currently, the possible mechanisms of early brain injury have been systematically studied, including elevation of intracranial pressure, reduction of cerebral blood flow, suppression of cerebral perfusion pressure, fall in brain oxygenation, blood-brain barrier breakdown, brain edema, and neuronal cell death. However, definitive mechanisms underlying early brain injury remain incompletely understood to date. Valproic acid is the first-line drugs to treat epilepsy and bipolar disorder. Recently, studies show that it has neuroprotective effect.Valproic acid can reduce damage and improve neurological function in several models of acute central nervous system injuries, such as stroke, traumatic brain injury, and spinal cord injury. Its possible neuroprotective mechanisms including, alleviating blood-brain barrier (BBB) disruption, inhibiting expression of inflammatory cytokines and reducing the number of apoptosis cell.In this study, we investigated the neuroprotective effects of valproic acid in the rat subarachnoid hemorrhage model. Furthermore we explored valproic acid’s neuropretective mechanism by detecting whether it preserve the blood-brain barrier and reduce the number of apoptosis cells.Experimental Methods1. SAH model was performed using endovascular perforation technique. Adult male Sprague Dawley rats were randomly assigned into sham group, SAH+Vehicle group and SAH+VPA group. Mortality, neurological deficit scores and brain edema were evaluated after24h SAH.2. Adult male rats were randomly divided into sham group, SAH+Vehicle group and SAH+VPA group. After24h SAH, BBB permeability was quantitatively evaluated by Evans blue extravasation and IgG staining. MMP9cellular localization is performed by double fluorescence labeling. Examining the effect of VPA treatment on the blood cells infiltration by immunohistochemistry against ED-1for macrophage. The mRNA of inflammatory cytokines (MMP9, IL-1β, IL-6, TNF-α) were evaluated by RT-PCR. Protein (pERKl/2, MMP9, Occludin, Claudin-5) were evaluated by western blot analysis. The activity of MMP9detected by gel zymography3. Adult male rats were randomly divided into sham group, SAH+Vehicle group and SAH+VPA group. After24h SAH,Apoptosis related protein (Bcl-2, cleaved caspase3) and HSP70, pAkt were evaluated by western blot analysis. Apoptosis were evaluated by TUNEL.Result1. VPA treatment improved neurological score and reduced water content, compared with SAH+Vehicle group (P<0.05). but the mortality was no sgnificant difference between the groups.2. MMP9and pERK1/2mainly located in neuron and endothelia cells, respectively.Treatment with VPA significantly decreased EB extravasation, IgG staining, macrophage infiltration and the mRNA of inflammatory cytokines (MMP9, IL-1β, IL-6, TNF-α) compared with the SAH+vehicle group (P<0.05). Gel zymography experiments showed that MMP9activity was significantly lower in SAH+VPA group compared with SAH+Vehicle group (P<0.05). Furthermore, VPA treatment significantly reduced the proteins pERK1/2and MMP9expression and increased tight junction proteins occluding and claudin-5expression.3. Valproic acid treatment increase the protein HSP70, pAkt, Bcl-2expression, decrease caspase-3activity and the number of TUNEL-positive cells after SAH.Conclusion1. Valproic acid teatment reduced brain edema and improved neurological function after subarachnoid hemorrhage.2. Valproic acid treatment preserved the blood brain barrier and inhibited macrophage infiltration.3. Valproic acid treatment reduced cell apoptosis by stimulating Akt surviving passway and increasing HSP70protein levels.