| Objective:.The present experiment is to characterize the effects of mild hypothermia(MHT) and erythropoietin(EPO) therapy on traumatic brain injury rats,and identify their possible targets and protective signaling pathways in traumatic brain injury brain.Methods:. The experiment includes two parts. Experiment one was used to identify the neuropective effects of mild hypothermia(MHT) and erythropoietin(EPO) therapy and experiment two was used to evaluate the effects of mild hypothermia(MHT) and erythropoietin(EPO) therapy on the expression of AKT, phosphor-AKT, GSK3β,phosphor-GSK3β, Claudin-5, Caspase-3in traumatic brain injury brain. In experiment one, fourty male Wistar rats were randomly divided into five groups (eight rats in each group):normothermic uninjuried group(Sham group), normothermic treatment TBI group(NT group), mild hypothermic treatment TBI group(MHT group), erythropoietin(EPO) treatment TBI group(EPO group) and combined group(MHT+EPO group),EPO group and combined group(MHT+EPO group) were given intra-abdominal injection of erythropoietin(EPO)(5000IU/kg), the rest groups received equal volume0.9%NaCl at the same time. Neurological deficit was evaluated using a modified neurological severity score(mNSS)48h after fluid percussion injury (FPI).The changes of brain water content and permeability of blood-brain barrier were measured by the methods of wet and dry weight and Evans blue fluorometry.In experiment two, fifty male Wistar rats were randomly divided into five groups(10rats in each group):normothermic uninjuried group(Sham group), normothermic treatment TBI group(NT group), mild hypothermic treatment TBI group(MHT group), erythropoietin(EPO) treatment TBI group(EPO group) and combined group(MHT+EPO group), EPO group and combined group(MHT+EPO group) were given intra-abdominal injection of EPO(5000IU/kg), the rest groups received equal volume0.9%NaCl at the same time.48h after FBI all rats were decapitated, The size of injury lesion were evaluated by hematoxylin and eosin stain (HE stain), Western blotting, Immunohistochemistry, and RT-PCR were used to analyze the expression of AKT、pAKT、GSK3β、pGSK3β、Claudin-5and caspase-3.Results:1.The result of neuological deficit:Compared with the NT group, MHT group, EPO group, MHT+EPO group all get better result of nerve functional defect(P＜0.05,P＜0.01), while it seems that combining hypothermia treatment with EPO treatment has the best effect(#P＜0.05).2. Brain water content measurement:Water content of ipsilateral brain in the Sham group was77.16±2.11%, while83.23±1.99%in the NT group,Compared with Sham group, brain water content was significantly increased in the NT group; Compared with NT group, MHT group, EPO group, MHT+EPO group show an intense decline in the percentage of brain water content(NT vs.EPO,83.23±1.99%vs.80.43±2.38%; NT vs. MHT,83.23±1.99%vs.79.85±1.78%; NT vs. MHT+EPO,83.23±1.99%vs.78.12±1.34%)(P＜0.05,P＜0.01).;Compared with MHT group, EPO group, MHT+EPO group have better effect in atteunating brain water content (MHT vs. MHT+EPO,79.85±1.78%vs.78.12±1.34%; EPO vs. MHT+EPO,80.43±2.38%vs.78.12±1.34%)(#P＜0.05).3.The permeability of blood-brain barrier measurement:Compared with NT group, MHT group, EPO group, MHT+EPO group showed effect of changing the blood brain barrier in TBI rats according the Evans-blue test(P＜0.05,P＜0.01), there is no significant difference between MHT group and EPO group in changing the permeability of blood-brain barrier, while, combining MHT treatment with EPO treatment has the best effect(#P＜0.05,##P＜0.05)in attenuating the impairment in blood-brain barrier.4.The results of HE stain:. In NT group the slice showed brain damage in the cortical and subcortical regions, a severe astrocytic reaction in the area surrounding the lesion; Compared with NT group,MHT group,EPO group,MHT+EPO group attenuate the impairment of cortical and subcortical regions,with slight astrocytic reaction in the area surrounding the lesion.5.The expression of pAKT、pGSK3β、Claudin-5and Caspase-3at protein and mRNA level:Compared with NT group, The expression of pAkt, pGSK3P and Claudin-5were siginificantly upregulated at protein level in MHT group, EPO group, MHT+EPO group(P＜0.05), The expressions of pAkt, pGSK3β and Claudin-5in MHT group, EPO group, MHT+EPO group was significantly increased at protein level vs. NT group(P＜0.05,P＜0.01),and the MHT+EPO group have the best effect in upregulating the expression of pAkt, pGSK3(3and Claudin-5(#P＜0.05,##P＜0.01). The expression of Claudin-5was significantly increased at mRNA level,while the expression of Caspase-3was significantly decreased in MHT group, EPO group, HT+EPO group vs. NT group(P＜0.05,P＜0.01),compared with MHT group, EPO group, MHT+EPO group can upregulate the expression of Claudin-5and downregulate the expression of Caspase-3at mRNA level(#P＜0.05,##P＜0.01).Conclusions:In conclusion, our present study provides beneficial evidences for MHT and EPO treatment and its underling mechanisms after traumatic brain injury, Both MHT treatment and EPO treatment can lessen neurological deficit scores, the brain water content, lesion size and attenuate BBB permeability. Specifically, the protective effect of MHT treatment and EPO treatment against traumatic brain injury may through PI3K/Akt/GSK signaling activation, downregulation of Caspase-3and upregulation of Claudin-5. When combining MHT treatment with EPO treatment, we can obtain an optimized result than the single application of above mentioned two treatments, So combining MHT treatment with EPO treatment should be one of the strategic targets for Traumatic Brain injury therapies. |
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