Research On The Mechanisms Of Melatonin Alleviates Secondary Brain Damage And Neurobehavioral Dysfunction After Experimental Subarachnoid Hemorrhage

PartⅠ: Research on the effects of Melatonin on the secondary braindamage and neurobehavioral dysfunction after experimentalsubarachnoid hemorrhageObjective: To investigate the effects and mechanisms of melatonin on the secondarybrain damage and neurobehavioral dysfunction after experimental subarachnoidhemorrhage （SAH）.Methods:80adult SD rats were divided into four groups: control group （n=20）,SAH group （n=20）, SAH+vehicle group （n=20）, and SAH+melatonin group （n=20）.The rat SAH model was induced by injection of0.3ml fresh arterial, nonheparinized bloodinto the prechiasmatic cistern in20s. In SAH+melatonin group, melatonin wasadministered i.p. at150mg/kg at0,12,24and36hr after the induction of SAH. Part of ratsin each group were killed on48hr after SAH.The brain sample was removed and keepedin10%formalin solution.Cognitive and memory changes were investigated in the Morriswater maze at48hr after the induction of SAH with the rest of rats in each group.Results: The behavior and activity scores, escape latency and swimming distance inMorris water maze task, apoptosis and necrosis percentage of neurons of SAH rats were allsignificantly higher than those of the contol rats （P<0.01）, while the above index ofSAH+melatonin group were significantly lower than those of the SAH+vehicle rats （P<0.05）, but no significant difference was found in the above index between SAH andSAH+vehicle rats （P>0.05）.Conclusion: Melatonin may improve the neurobehavioral dysfunction after SAH anddecrease the apoptosis and necrosis of neurons. Part II: Research on the mechanisms of Melatonin on the secondarybrain damage and neurobehavioral dysfunction after experimentalsubarachnoid hemorrhage: possible involvement of TLR4-mediatedinflammatory pathwayObjective: To establish subarachnoid hemorrhage model and observe the expressionchanges of TLR4pathway-related agents when treated with melatonin after experimentalsubarachnoid hemorrhage （SAH）.Methods:80adult SD rats were divided into four groups: control group （n=20）,SAH group （n=20）, SAH+vehicle group （n=20）, and SAH+melatonin group （n=20）.The rat SAH model was induced by injection of0.3ml fresh arterial, nonheparinized bloodinto the prechiasmatic cistern in20s. In SAH+melatonin group, melatonin wasadministered i.p. at150mg/kg at0,12,24and36hr after the induction of SAH. Each groupof rats were killed on48hr after SAH.The brain sample was removed and keeped in10%formalin solution and liquid nitrogen.The expressions of TLR4pathway-related proteinwere measured by western blot, immunohistochemistry,EMSA,ELISA and RT-PCR.Results: The protein levels of TLR4pathway-related agents were detected by Westernblot.These proteins were expressed at a low level in the rat brains of control group. Thelevels of TLR4, NF-κB, iNOS, HMGB1, and MyD88were significantly increased in thecortex in SAH group as compared to that of control group （P <0.01）. The protein expressions did not differ significantly between SAH group and SAH+vehicle group （P>0.05）. The expressions of TLR4, NF-κB, iNOS, HMGB1, and MyD88in the brains ofSAH+melatonin group were significantly lower than those of the SAH+vehicle group （P<0.05）. EMSA autoradiography of NF-κB DNA-binding activity of the brain sampleswas shown that low NF-κBbinding activity （weak EMSA autoradiography） was foundin the control group. Compared with control group, NFκB-binding activity in the injuredbrain was significantly increased （P<0.01） in SAH and vehicle-treated groups.InSAH+melatonin group, the NF-κB-binding activity wassignificantly down-regulated （P<0.05） in the brain area surrounding the blood clot site after SAH.To assess thelocalization of TLR4pathway, immunohistochemistry for TLR4, NF-κB P65, and iNOSwas performed.A few TLR4, NF-κB, or iNOS positive cells were observed in the controlgroup, which indicates the constitutive activity of TLR4/NF-κB pathway in the controlbrain of rats. Remarkably increased positive cells in the SAH groups could be found in thebrain samples （P <0.01）. These proteins’ immunoreactivity was mainly present in bothneurons and glia cells. In SAH+melatonin group, the number of positive cells wasdecreased significantly （P <0.05）.The mRNA levels of three genes, IL-1b, TNF-a, andIL-6, were detected by quantitative real-time PCR. The mRNA of these proteins wasexpressed at a low level in brains of the control group. The levels of IL-1b, TNF-a, andIL-6mRNA were significantly increased in the cortex in SAH and SAH+vehicle groupscompared with the control group （P <0.01）. The mRNA expressions had no significantdifference between SAH group and SAH+vehicle group （P>0.05）. The mRNAexpressions of IL-1b, TNFa and IL-6in the brains of the SAH+melatonin group weresignificantly down-regulated versus those of the SAH+vehicle group （P <0.01）.Concentrations of IL-1b, TNF-a, and IL-6were detected by quantitative real-timePCR show that low in the rat brains of control group. Compared with control group,cortical levels of the three inflammatory cytokines were greatly elevated after SAH （P <0.05）. Melatonin administration after SAH could lead to significant reduction in IL-1b,TNF-a, andIL-6concentrations （P <0.05）. Conclusion: the up-regulated cortical levels of these agents related to TLR4-mediatedinflammatory signaling pathway were attenuated when treated with melatonin in level ofprotein synthesis after experimental SAH.