| Traumatic brain injury (TBI) is one of the most common diseases inneurosurgery, featured with high incidence, high morbidity and high mortality.It bring huge emotional and economic burden to their families and the society,is become one of global social issues. In early stage, the study of neurologicaldysfunction follow TBI focused on cell necrosis and apoptosis. Recently, as anew form of eukaryotic cell death, autophagy is drawing more and moreattentions, which is become a hot topic in the scientific community.Recent studies have confirmed that the presence of neurons autophagyis in the pathological process following TBI. The investigation of theregulation of autophagy may provide new drug targets on the treatment ofsecondary neuronal injury and repair following TBI. Thus, find out neuronsautophagy occurring and regulation targets and stating final death pathway ofneurons after trauma, is important for discussion and avoid damage and deathof neurons, while giving targeted therapy following TBI.Previous studies in our laboratory found that autophagy after TBI occursmainly in neurons rather than the glial cell, and we also investigate themechanism of neurons autophagy induced by ERK1/2and JNK signalingpathway, but use the neurons autophagy as drug targets need furtherdiscussion. On the other hand, glial cells are the most abundant in brain tissuecells. And could neurons autophagy be regulated by an important factorglutamate transporter subtype (GLT-1) in glial cells after TBI? This alsorequires us to further experimentation and exploration.Objective: This study was applied with autophagy inhibitor Chloroquine(CQ) and GLT-1activator ceftriaxone (CTX) following TBI, detect theexpression of GLT-1, IL-1and LC3in hippocampus subregion in rats. The aim of our study is to open up new ideas on neuronal autophagy as a therapeutictarget, and we also provide an experimental basis on clinical treatment of TBI.Method: A total of160male Sprague-Dawley (SD) rats were randomlydivided to four groups: Sham group (n=40), TBI group (n=40), CQ group(n=40) and CTX group (n=40). Furthermore, each group was divided into12h,24h,48h and72h. We reproduce the TBI model by Marmarou’s falling method.We observe these following indexes: H﹠E stains was used to observemorphological changes in hippocampus subregion. Wet/dry method was usedto measure brain water content. The cellular localization of LC3was observedby immunofluorescence microscope. The expression of GLT-1, IL-1and LC3were evaluated by Western blotting. Another40rats were selected to detecttheir ability of learning and memory at7-11day following TBI by Morriswater maze test. The western blot was quantified by using the Gel-Docanalysis software. SPSS18.0was used to analysis experimental data. Q-testwas selected to determine the significance in two groups. P＜0.05wasconsidered as statistically significant.Result:1Pathological changes observed by H﹠E: Neurons in the hippocampusof Sham group have a larger number, structure intact, arrangement rules, withno significant pathological changes. In the TBI group, brain tissue is loose andedema. The gap surrounding cells and capillaries widened significantly aftertrauma. Neurons in the hippocampus structure damage, the number have asignificant reduction, nuclear condensation, nuclear Jen part disappears. In CQgroup, the tissue is mild edema, the gap surrounding cells and capillaries onlyslightly widened. In hippocampus, neuronal structures are relatively intact, thenumber of neurons is slightly increased compared with TBI group. In CTXgroup, the tissue is slightly edema, the gap surrounding cells and capillariesonly slight widened. In hippocampus, neuronal structures are relatively intact,the number of neurons is significantly increased compared with TBI group.2Results of spatial learning and memory ability: TBI group performednavigation test7-10days after trauma, rats escape relatively significant increase in the average latency compared with the Sham group (P<0.05).Compared with TBI group, the average escape latency of CQ group wassignificantly shorter in9,10days following TBI (P<0.05). The average escapelatency of CTX group was significantly shorter in7-10days following TBIcompared with TBI group (P<0.05). In the search space experiment in11dayspost-TBI, the number of rats across the platform, the platform quadrant lengthand time ratio of the TBI group rats were significantly reduced (P<0.05).Compared with the TBI group, the number of rats across the platform and thetwo ratios were significantly increased in CQ group and CTX group (P<0.05).3Brain tissue water content detect: Sham group have no difference intissue water content at each time point. Brain tissue water content of TBIgroup increases with time, was significantly higher than Sham group in12-72h. CQ group was significantly lower than the TBI group rats in24-72h(P<0.05). CTX group were significantly lower than the TBI group rats in12-72h (P <0.05).4Expression of GLT-1by western blot: GLT-1has a strong expression inSham group, the expression have no difference at all time points. Comparedwith the Sham group, GLT-1protein expression was significantly lower12hafter TBI (P<0.05), and reached its lowest point at24h, although GLT-1hasbeen raised later, the expression of it still lower than the Sham group72h posttrauma (P<0.05). The expression levels of GLT-1in CTX group issignificantly higher than the TBI group in12-72h (P<0.05).5Expression of IL-1by western blot: Only a small amount of IL-1expression in Sham group, and the expression level have no difference at alltime points. Compared with the Sham group, the expression of IL-1wasincreased12h after injury (P<0.05), peaked at24h (P<0.05), continuous highexpression at48h (P<0.05),72h after TBI IL-1protein expression decreased,but still significantly higher than the Sham group (P<0.05). In CQ group, theexpression of IL-1is slightly lower than the TBI group in24-48h (P<0.05).The expression levels of IL-1in CTX group is significantly lower than theTBI group in12-72h (P<0.05). 6Result of LC3and NeuN by immunofluorescence. The greenfluorescent of LC3accumulated in cytoplasm labeled by FICT, the redfluorescent of NeuN was labeled by TRITC in neuronal nucleus. At24h afterTBI, LC3(green) and NeuN (red) were collocated in hippocampus.7Expression of LC3by western blot: In Sham group, we could not seethe change of LC3Ⅱ/LC3Ⅰin all points. The value of LC3Ⅱ/LC3Ⅰisgradually raising from12h and peaked at24h after trauma compares with theSham group(P<0.05). Although LC3Ⅱ/LC3Ⅰ has been decreased later, it isstill higher than the Sham group at72h post trauma (P<0.05). The changes ofLC3Ⅱ/LC3Ⅰ expression in CQ group is significantly lower than the TBIgroup in12-48h (P<0.05). The changes of LC3Ⅱ/LC3Ⅰ expression in CTXgroup is slightly lower than the TBI group in24-48h (P<0.05).Conclusion: Following TBI in rats, the autophagy inhibitor CQ andGLT-1activator CTX could affect the expression of autophagy-relatedproteins LC3, thereby regulating neurons autophagy. It played aneuroprotective effect via anti-inflammatory, anti-excitotoxicity, andimproving brain learning and memory following TBI in rats. |
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