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Valproic Acid Reduces Vasospasm through Modulation of Akt Phosphorylation and Attenuates Neuronal Apoptosis in Subarachnoid Hemorrhage Rats.

ABSTRACT: Aneurysmal subarachnoid hemorrhage (SAH) is a devastating emergent event associated with high mortality and morbidity. Survivors usually experience functional neurological sequelae caused by vasospasm-related delayed ischemia. In this study, male Sprague-Dawley rats were randomly assigned to five groups: sham (non-SAH) group, SAH group, and three groups with SAH treated with different doses of valproic acid (VPA) (10, 20, 40 mg/kg, once-daily, for 7 days). The severity of vasospasm was determined by the ratio of cross-sectional areas to intima-media thickness of the basilar arteries (BA) on the seventh day after SAH. The BA showed decreased expression of phospho-Akt proteins. The dentate gyrus showed increased expression of cleaved caspase-3 and Bax proteins and decreased expression of Bcl-2, phospho-ERK 1/2, phospho-Akt and acetyl-histone H3 proteins. The incidence of SAH-induced vasospasm was significantly lower in the SAH group treated with VPA 40 mg/kg (p < 0.001). Moreover, all groups treated with VPA showed reversal of the above-mentioned protein expression in BA and the dentate gyrus. Treatment with VPA upregulated histone H3 acetylation and conferred anti-vasospastic and neuro-protective effects by enhancing Akt and/or ERK phosphorylation. This study demonstrated that VPA could alleviate delayed cerebral vasospasm induced neuro-apoptosis after SAH.

SUBMITTER: Wu CH

PROVIDER: S-EPMC8198375 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The diagnosis of cerebral vasospasm after Subarachnoid-Hemorrhage is currently very difficult, additional tools such as blood biomarkers are necessary. We tested the ability of gene expression profiles of blood cells to predict vasospasm. 32 patients suffering subarachnoid-hemorrhage were included in this prospective monocentre study. They were grouped according to have a complicated cerebral vasospasm (Vasospasm) or not (Control) and Paired according to age (+/- 10 years), sex, Fisher grade (+/- 1), location, smoking (at least 3 first parameters). Gene expression profiles of blood cells were determined using 25,000~gene microarray. Blood sample: 2.5 mL harvested in PAXgene® Blood RNA tubes (PreAnalytix) RNA extraction: PAXgene® Blood RNA kit (Qiagen). We used a Universel Reference RNA (Stratagene). RNA amplification and labelling: kit Amino Allyl MessageAmp II (Ambion). We hybridized 4 microarrays per patient using pangenomic microarrays from the "Réseau National des Génopôles" (Illkirch, France). 2 slides were hybridized with reference RNA labelled Cy3 and patient RNA labelled Cy5, and 2 slides were hybridized with reference RNA labelled Cy5 and patient RNA labelled Cy3. Hybridation : Agilent protocol with few modifications : 750 ng of each labelled RNA were hubriddized at 60°C during 17 hours in an Aglient hybridization oven. After washings, Slides were scanned with a GenePix 4000B scanner (Molecular Devices). Image intensity data were extracted with GenePix Pro 6.0 analysis software. Quantification of Cy3 and Cy5 and selection of good spots were performed using the MAIA software (Novikov E and Barillot E. Software package for automatic microarray image analysis (MAIA). The ACUITY software was then used to normalize log ratios Cy3/Cy5 with Lowess non linear normalization, to filter out genes not present in at least 3 slides out of 4, to evaluate the reproducibility of the 4 microarrays of each patient (hierarchical clustering, Self Organizing Maps). Statistical analyses to insure reproducibility was performed using Excel (correlation coefficients, ANOVA). Only slides that passed all reprocubility tests were validated.

Project description:BackgroundNeuroinflammation and neuronal apoptosis are closely associated with a poor prognosis in patients with subarachnoid hemorrhage (SAH). We investigated the role of C-C motif chemokine receptor 2 (CCR2) in SAH.MethodsPre-processed RNA-seq transcriptome datasets GSE167110 and GSE79416 from the Gene Expression Omnibus (GEO) database were screened for genes differentially expressed between mice with SAH and control mice, using bioinformatics analysis. The endovascular perforation model was performed to establish SAH. RS504393 (a CCR2 antagonist) and LY294002 (PI3K inhibitor) were administered to explore the mechanism of neuroinflammation after SAH. SAH grading, neurological scoring, brain water content and blood-brain barrier (BBB) permeability determination, enzyme-linked immunosorbent assay (ELISA), western blotting, and immunofluorescence were performed. An in vitro model of SAH was induced in H22 cells by hemin treatment. The protective mechanism of CCR2 inhibition was studied by adding RS504393 and LY294002. Clinical cerebrospinal fluid (CST) samples were detected by ELISA.ResultsExpression of CCR2 was upregulated in both datasets and was identified as a hub gene. CCR2 expression was significantly upregulated in the cytoplasm of neurons after SAH, both in vitro and in vivo. RS significantly reduced the brain water content and blood-brain barrier permeability, alleviated neuroinflammation, and reduced neuronal apoptosis after SAH. Additionally, the protective effects of CCR2 inhibition were abolished by LY treatment. Finally, the levels of CCR2, inflammatory factors, and apoptotic factors were elevated in the CSF of patients with SAH. CCR2 levels were associated with patient outcomes at the 6-month follow-up.ConclusionCCR2 expression was upregulated in both in vitro and in vivo SAH models. Additionally, inhibition of CCR2, at least partly through the PI3K/AKT pathway, alleviated neuroinflammation and neuronal apoptosis in vivo and in vitro. CCR2 levels in the CSF have a moderate diagnostic value for 6-month outcome prediction in patients with SAH.

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Valproic Acid Reduces Vasospasm through Modulation of Akt Phosphorylation and Attenuates Neuronal Apoptosis in Subarachnoid Hemorrhage Rats.

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