Administration Of Valproate Sodium Protects The Integrity Of Blood-brain Barrier After Intracerebral Hemorrhage In Mice

Objective: Secondary damage after ICH is a major factor contributing to the aggravation of ICH’s condition and poor prognosis.Studies have demonstrated that valproate(VPA)protects the nervous system through multiple signaling pathways.We speculated that VPA also had a protective effect on the bleed brain barrier(BBB)of ICH model.In this study,the spontaneous ICH model induced by collagenase was used to explore the protective effect of different doses of VPA on the integrity of the BBB after ICH and its potential mechanism.Methods: The content of this study is divided into five parts: In experiment 1,the effects of VPA on short-term nerve function deficit and brain edema in mice after ICH were studied by neurobehavioral score measurement and brain edema measurement.The mice were divided into five groups,Sham group,ICH group,ICH+VPA(100 mg/kg)group,ICH+VPA(300 mg/kg)group,ICH+VPA(600 mg/kg)group.There are six in each group.Short-term neurological function was measured at72 h after ICH using a modified Garcia score,anterior limb extension test,and Corner test.The cerebral edema was measured at 72 h after ICH,and the data were obtained by wet and dry weighing method and analyzed statistically.In experiment 2,the effect of VPA on the integrity of the BBB after ICH in mice was studied by immunofluorescence staining and Evans blue staining.Mice were divided into 3groups,Sham group,ICH group,ICH+VPA(300 mg/kg)group.There are 12 mice in each group.Evans blue staining was performed at 72 h after ICH.After the cerebral supernatant of the hematoma side was extracted,the data were measured by spectrophotometer and finally analyzed statistically.Fluorescence excitation test was carried out 72 h after ICH.Frozen brain sections were taken and the extravasation around the hematoma was observed under the excitation of fluorescence microscope red light.The final fluorescence extravasation image density was calculated using Image J software.Immunofluorescence staining was performed on mice at 72 h after ICH.Experiment 3,the effect of different doses of VPA on hematoma volume after ICH in mice was studied by measuring hematoma volume.Mice were divided into 3groups,Sham group,ICH group,ICH +VPA(300 mg/kg)group.There are six in each group.Hematoma volume was measured at 72 h after ICH.The data were measured by spectrophotometer.In experiment 4,the effect of VPA on the expression of related proteins in the brain of mice at 72 h after ICH was studied by western blot.Mice were divided into 3 groups,Sham group,ICH group,ICH +VPA(300 mg/kg)group.There are six in each group.Western blot was performed 72 h after ICH.Experiment 5: to determine the effect of different doses of VPA on long-term neurobehavioral scores.Mice were divided into 3 groups,Sham group,ICH group,ICH +VPA(300 mg/kg)group.There are 8 mice in each group.At frist week,second weeks,and third weeks after ICH,the left forelimb stomping test and rotation acceleration test were performed.A water maze test,including swimming distance and escape latency,was conducted 21 to 25 days after ICH.Results: The total animal mortality rate in this study was 8.16%(8/98).A total of 136 mice were enrolled,38 in the sham group,and 98 in ICH.8 mice died.There was no significant difference in mortality between the two groups.After 72 h of ICH,VPA treatment reduced neurobehavioral deficits(p < 0.05).300 mg/kg and 600mg/kg VPA improved neurobehavioral scores and reduce brain water content at 72 h after ICH(p < 0.05).300 mg/kg VPA significantly reduced ICH induced breakdown of BBB(p < 0.05).After 72 h of ICH,there was no statistically significant difference in the volume of hematoma between the 300 mg/kg VPA treatment group with the ICH group(p > 0.05).VPA intervention decreased the expression of p-NFκB,MMP-9,IL-6 and TNF-α(p < 0.05),increased the expression of endothelial connexin(p < 0.05).Conclusion: Both VPA 300 mg/kg and 600 mg/kg could improve short-term behavioral score of ICH model in mice and reduce brain edema.VPA protects BBB integrity after ICH in mice.VPA treatment inhibited NFκB activation and reduced expression of downstream target such as MMP-9.The down-regulation of MMP-9helps to weaken the damage of BBB,thus reducing the infiltration of neutrophils and monocytes,reducing the expression of inflammatory factors,upregulating endothelial ligand,and maintaining the integrity of BBB.