The Neuroprotective Effects And Underlying Mechanisms Of Melatonin In Secondary Injury Following Experimental Traumatic Brain Injury

Secondary brain injury plays a key role in the outcome of patients suffering traumatic brain injury (TBI). There are several pathological processes responsible for the neuronal death in the secondary damage of TBI, including oxidative stress, inflammation, excitotoxicity, loss of ionic homeostasis, and apoptosis. Previous treatment only focus on one pathway to alleviate the neurological degeneration following TBI, regardless the inner self protective mechanism. Thus, until now there have no satisfactory curative effect has been achieved in the therapy for TBI. Melatonin has been proved to be a protective agent in a variety of neurological diseases. It can alleviate several pathological processes of brain injury and also activate the internal variety of self protective mechanism. Therefore, this study propose the application of melatonin in TBI, which can expand the protective mechanism of melatonin and provide a new direction for the clinical treatment of TBI.Part I Role of melatonin in the oxidative stress following traumatic brain injury.Oxidative stress is involved in secondary injury after TBI, this part is designed to evaluate the effect of melatonin on oxidative stress after traumatic brain injury, and explore its inner mechanism. This part includes two experiments.Experiment1Melatonin active the Nrf2-ARE signaling pathway and alleviate oxidative stress following traumatic brain injury.Object:The goal of this study was to evaluate the potential involvement of melatonin in the activation of the nuclear factor erythroid2-related factor2and antioxidant responsive element (Nrf2-ARE) signaling pathway and the remission of oxidative stress in an experimental model of traumatic brain injury (TBI).Methods and Results:ICR mice were divided into four groups:sham group, TBI group, TBI+vehicle group, and TBI+melatonin group (n=38per group). Melatonin (10mg/kg) was administered via an intraperitoneal (i.p.) injection at0,1,2,3, and4h post TBI. The administration of melatonin following TBI significantly ameliorated the effects of the brain injury, such as oxidative stress, brain edema, and cortical neuronal degeneration. Melatonin markedly promoted the translocation of Nrf2protein from the cytoplasm to the nucleus, increased the expressions of Nrf2-ARE pathway-related downstream factors, including hemeoxygenase-1(HO-1) and NAD(P)H:quinone oxidoreductase1(NQO-1), and prevented the decline of antioxidant enzyme activities, including superoxide dismutase (SOD) and glutathione peroxidase (GPx).Conclusion:Melatonin administration may increase the activities of antioxidant enzymes and attenuate brain injury in a TBI model, potentially via the mediation of the Nrf2-ARE pathway.Experiment2The role of the Nrf2-ARE signaling pathway in melatonin’s alleviating oxidative stress following traumatic brain injury.Object:The goal of this study was to evaluate the role of the Nrf2-ARE signaling pathway in melatonin’s alleviating oxidative stress in an experimental model of traumatic brain injury (TBI).Methods and Results:Nrf2wild-type (Nrf2(+/+) group) and Nrf2knock out (Nrf2(-/-) group) mice received a TBI insult followed by melatonin administration (10mg/kg, i.p.) at the corresponding time-points (n=35per group). Knock out of Nrf2significantly ameliorated the protective effects of melatonin on the brain injury, such as oxidative stress, and cortical neuronal degeneration. Knock out of Nrf2also declined the activities of antioxidant enzyme, including superoxide dismutase (SOD) and glutathione peroxidase (GPx).Conclusion:Melatonin administration attenuate oxidative stress in a TBI model via the mediation of the Nrf2-ARE pathway.Part II Role of melatonin in the inflammatory reaction following traumatic brain injury.Object:Activated microglia is the major source of proinflammatory cytokines following traumatic brain injury (TBI). Melatonin has been proved to be a neuroprotective agent in TBI. However, there is a lack of data regarding an influence of melatonin on microglial activation following TBI and the underlying molecular mechanisms of melatonin inhibiting inflammatory response remains unclear. Hence, the current study was designed to detect the modulation manner of melatonin on microglial activation and explore herein possible involvement of mammalian target of rapamycin (mTOR) pathway following TBI.Methods and Results:Male ICR mice were randomly divided into four groups:sham group (n=35), TBI group (n=35), TBI+sal group (n=45), and TBI+melatonin group (n=45). Melatonin (10mg/kg) was administered via an intraperitoneal (i.p.) injection at0,1,2,3, and4h post TBI. A weight drop TBI model was induced and the brain tissues were harvested at24h following injury. Neurological severity score (NSS) tests were performed to measure behavioral outcomes. Nissl staining was conducted to observe the role of melatonin in the neuronal degeneration after TBI and wet-to-dry weight ratio indicated brain water content. The effect of melatonin treatment on neuroinflammation was analyzed by immunofluorescence analysis of microglial activation and enzyme-linked immunosorbent assay (ELISA) was employed to evaluate proinflammatory cytokine levels (interleukin-beta (IL-1β), tumor necrosis factor-alpha (TNF-a)) in ipsilateral brain. The expressions of phosphorylated protein of mammalian target of rapamycin (mTOR), p70ribosomal S6kinase (p70S6K) and S6ribosomal protein (S6RP) in ipsilateral cortex were detected by western blotting. Melatonin administration after TBI was associated with a decreased activated microglia and degenerative neuron at the peri-injury site, reduced levels of proinflammatory cytokines and increased neurobehavioral function, possibly mediated by inactivation of mTOR pathway.Conclusions:The results of this study suggest that melatonin have the ability to decrease microglial activation and the expression of proinflammatory cytokines in TBI-induced cerebral inflammatory responses. This study presents a new insight into the mechanisms responsible for the anti-neuroinflammation of melatonin, with possible involvement of mTOR pathway.Part Ⅲ Role of melatonin in apoptosis following traumatic brain injury.Apoptosis play a important role in the process of secondary damage following traumatic brain injury (TBI). Melatonin has been proved to be a neuroprotective agent in TBI. However, there is a lack of data regarding an influence of melatonin on apoptosis following TBI and the underlying molecular mechanism of this effect of melatonin remains unclear. Hence, the current part was designed to detect the modulation manner of melatonin on apoptosis and explore herein possible involvement of mitochondrial pathway following TBI.Experiment1Melatonin stimulate autophagy following traumatic brain injury.Object:This study was designed to detect the modulation manner of melatonin on autophagy following TBI.Methods and Results:Male ICR mice were randomly divided into four groups:sham group (n=11), TBI group (n=11), TBI+sal group (n=21), and TBI+melatonin group (n=21). Melatonin (10mg/kg) was administered via an intraperitoneal (i.p.) injection at0,1,2,3, and4h post TBI. A weight drop TBI model was induced and the brain tissues were harvested at24h following injury. Neurological severity score (NSS) tests were performed at1h,1d,3d and7d to measure behavioral outcomes. The expressions of LC3and Beclin-1in ipsilateral cortex were detected by western blotting. Immunofluorescence analysis for LC3and NeuN was undertaken to measure autphagy. Melatonin administration after TBI was associated with a enhanced autophagy and a decreased apoptotic cells following TBI. Conclusions:The results of this study suggest that melatonin can stimulate autophagy and improve neurological performance following traumatic brain injury.Experiment2Melatonin alleviate neuronal apoptotic death via mitochondrial pathway following traumatic brain injury.Object:This study was designed to detect the modulation manner of melatonin on the mitochondrial apoptotic pathway following TBI.Methods and Results:Male ICR mice were randomly divided into five groups:sham group, TBI+sal group, TBI+3MA, TBI+melatonin group and TBI+melatonin+3MA group (n=26per group). Melatonin (10mg/kg) was administered via an intraperitoneal (i.p.) injection at0,1,2,3, and4h post TBI. A weight drop TBI model was induced and the brain tissues were harvested at24h following injury. Neurological severity score (NSS) tests were performed at1h, Id,3d and7d to measure behavioral outcomes. The effect of melatonin treatment on apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick3’-end labeling (TUNEL). The expressions of LC3, Beclin-1, cleaved caspase-3, mitochondrial Bax and out-mitochondrial cyt-c were detected by western blotting.3MA reversed the up-regulated autophagy induced by melatonin. Melatonin administration after TBI was associated with a decreased expressions of mitochondrial Bax and out-mitochondrial cyt-c.3MA reversed the above change.Conclusions:The results of this study suggest that melatonin can alleviate the mitochondrial apoptotic pathway via up-regulated autophagy following traumatic brain injury.