Changes Of MDA,NF-κB,TNF-α In Brain Tissue And Plasma Following Intracerebral Hemorrhage In Rats And The Intervention Of Edaravone

Background and objectiveIntracerebral hemorrhage(ICH) is spontaneous hemorrhage in brain parenchyma,which is associated with high incidence,high mortality and poor recovery in survivors.The brain edema and secondary neural injury are important factors of unfavourable prognosis.There is still no effective therapeutic measure to ICH only because it's pathologic mechanisms are still uncertain.Up to now, it is believed that there exists an area around haematoma which resembles ischemic stroke,namely"penumbra".Complicated pathologic courses happened in the"penumbra"which contributed to brain edema and secondary neural injury.With the approach of experiments and researches,more and more attentions are being payed to the effect and mechanism of free radical and inflammatory response.It is necessary to make further investigation for it so that more effective therapeutic measures could be found. MCI-186 (3-methyl-1-phenyl-2-pyrazoline-5-ketone,edaravone),one of the free radical scavengers,has been used to treat acute cerebral infarction extensively.But it is not clear whether it was effective to treat ICH.This study intended to explore the therapeutic efficacy and possible mechanisms of edaravone in ICH treatment,and to provide theoretical basement for ICH research of pathophysiologic mechanism and clinical treatment by observing the changes of free radical and inflammatory factors in brain tissue and plasma.Methods1. Animal preparation:120 male and female Sprague-Daley rats involved in the present study. They were divided into three groups by random digits table: Control group,ICH group and Therapy group.Each group was further divided into four subgroups according to different phase.Each subgroup had 10 rats:5 rats for brain water content testing and histopathology analyses,other 5 rats for MDA, TNF-α, NF-κB detection.Blood was collected from inferior caval vein when the rats were killed. 2. Animal ICH model:By stereo-orientation technique.The scalp was incised through the midline,skull and Begma exposed.At the 0.2mm-front and 3mm-right point from the middle of Bregma,a needle was inserted 6mm in depth through the skull and into the deep cerebrum nearby caudatum,and 50μl of autoblood was injected in twice.3. Rats in control group were administrated the same as ICH group,but no blood was injected;Rats in therapy group were treated with edaravone (3mg/Kg)30min before operation and once per 12h after operation through intraperitoneal injection;Rats in ICH and therapy group were both treated with normal saline in tales doses.4. Neurologic impairment score:To test the rats by Longa standard(1989) before killing.5. Brain water content(BWC) test:Refered to the dry-wet weight method.The water content was determined as (wet weight?dry weight)/ wet weight×100%.6. Malondialdehyde(MDA)assaying:By thio-barbituric acid method,Unit: nmol/mgprot.7. Tumor necrosis factor-alpha (TNF-α)assaying:By enzyme-linked immunosorbent assay (ELISA),Unit:ng/ml.8. Nuclear factor-kappa B(NF-κB)detection:By electrophoretic mobility shift assay(EMSA),Unit:DU.9. Histopathology analyses:By Hematine-Eosine dyeing(HE),observed by optical microscope.Results1. Neurologic impairment score of rats in ICH group was obviously higher than that in control group;Compared with ICH group,the score was much lower in therapy group (P<0.05).2. Brain water content(BWC) of rats in ICH group increased more clearly at 6h,24h and 72h(P<0.05)than that in control group,but it showed no difference between two groups at 168h(P>0.05);Compared with ICH group,BWC in therapy group was significantly lower at 6h,24h and 72h (P<0.05),but it showed no difference between two groups at 168h(P>0.05).3. Content of MDA and TNF-αin brain homogenate in ICH group was significantly higher than that in control group(P<0.05);Compared with ICH group, content of MDA and TNF-αin brain homogenate was much lower in therapy group (P<0.05).4. Correlation test showed BWC in ICH and therapy group is closely correlated with TNF-αin brain homogenate,coefficient of correlation: R2=0.934, (p<0.01).5. NF-κB activity in brain tissue in ICH group showed much higher than that in control group(p<0.01);Compared with ICH group,NF-κB activity in brain tissue showed much lower in therapy group (p<0.01).6. Content of MDA and TNF-αin plasma in ICH group was significantly higher than that in control group(P<0.01);Compared with ICH group,content of MDA and TNF-αin plasma was much lower in therapy group (P<0.01).7. Light microscope assessment showed severe brain edema and obvious neutrophilic leukocytes infiltration in the tissue around hematoma in ICH group;This situation showed relatively relieved in therapy group.Conclusion1. Free radical and TNF-αrelease closely correlated with pathophysiologic mechanism of formation of brain edema and neural injury after intracerebral hemorrhage.2. Inflammatory response following intracerebral hemorrhage may due to over generation of free radical which activated NF-κB and resulted in TNF-αrelease.3. Edaravone had protective effect for tissue with ICH probably related to it's ability to antagonize lipid peroxidation by scavenging free radical and restrict inflammatory response indirectly.