| Objective: Intracerebral hemorrhage(ICH) occurs when a diseased blood vessel ruptures, allowing blood to leak into the surrounding brain. ICH accounts for 10–15% of all strokes and is associated with high mortality and morbidity. Secondary brain injury(SBI) plays a critical role in neurological deterioration in patients with ICH. Excitatory amino acids toxicity has an adversenegative effect on the occurrence and progress of SBI following ICH. Membrane associated protein is a kind of calcium dependence of phospholipids binding protein which can promote membrane protein fusion, recent studies have shown that it play an important role in the occurrence, development and outcome in a wide variety of tumor. However, the effects ANXA7(annexin A7) under ICH condition was still not well known. This study was designed to evaluate the potential effects of ANXA7 on ICH-induced secondary brain injury and the underlying mechanisms.Methods: In Experiment 1, 42 health male Sprague-dawley(SD) rats were assigned randomly to 7 groups: sham, ICH 6h, ICH12 h, ICH24 h, ICH72 h and ICH1 w groups(n=6). All rats were executed and the brain was removed at each time point. The brain tissue adjacent to the hematoma cavity were this study following ICH. The gene level and protein expression of ANXA7 were detected by Semi-quantitative RT PCR,western blot(WB) analysis and immunofluorescence(IF), brain edema and blood–brain barrier(BBB) permeability were detected by wet/dry method respectively EB(Evans blue) extravasation. In Experiment 2, 48 male SD rats were assigned randomly into following groups: sham group, ICH group(n=12), ICH +ANXA7-protein group(n=12)and ICH+ANXA7 antibody(n=12). All ICH animals were subjected to injection ofcollagenase into the basal ganglia region once. The intervention rats were given injections of recombinant ANXA7 protein and ANXA7 antibody at post-ICH 12 h via intracerebroventricular injection. All rats were executed at 48 h after ICH. The brain tissue adjacent to the hematoma cavity were this study following ICH. We measured glutamate content in cerebrospinal fluid by high performance liquid chromatography(HPLC) method, brain edema by wet/dry method, blood–brain barrier(BBB)permeability by EB extravasation and cortical apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) and FLUORO-JADEB staining method.Results: Experiment 1: The mRNA levels of ANXA7 was up-regulated early after ICH(6h), peaked at 24 h post-ICH, start to down-regulated at 48 h post-ICH still significantly higher than those in sham at 1 w. The protein levels of ANXA7 appeared an obvious rise at 12 h, peaked at 24 h post-ICH and an slight rise at 1 w.;The binding activitives between ANXA7 and synaptosomal-associated protein 23(SNAP23)/SNAP25 significantly increased compared to sham group. At the same time, the brain water content and EB content significantly increased and reached to 82.4±0.44% at 48 h and reached to3.8 ug/g. Experiment 2: The apoptosis and necrosis percentage of neurons of ICH rats were all significantly higher than those of the sham rats, the elevated apoptosis and necrosis percentage of neurons was aggravated and restrained after intraperitoneal ANXA7 protein and ANXA7 antibody administration separately. Ultimately, brain edema, BBB impairment and glutamate content was significantly ameliorated compared with ICH rats and reversed by ANXA7 antibody intervention.Conclusion: Excitatory amino acids toxicity probably participated in the process of SBI fowllowing ICH, the high expression of ANXA7 promoted the release of excited amino acid and result in neurons damage. Brain edema, BBB impairment and glutamate content and the apoptosis and necrosis percentage of neurons of ICH were alleviated via resisting ANXA7 expression. Therefore, a new treatment strategy supplied to treat SBI after ICH via the suppression of nervous excitability toxicity route triggered by ANXA7 highexpression. |
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