Effects And Mechanism Of Melatonin On The Secondary Brain Injury After Intracerebral Hemorrhage In Rats

PARTⅠObjective:To observe the activation of microglia and the level of SOD and MDA in brain and study the effect of melatonin on activation of microglia and oxidative stress after intracerebral hemorrhage in rats.Methods:150 SD rats were randomly divided into four groups:normal group, sham-operated group (Sham group), intracerebral hemorrhage model group (Model group) and melatonin intervention group (MT group). Each group was divided into 5 subgroups respectively at 12h, 1d,2d,4d and 7d after establishment of animal model. The models of ICH were created by Rosenberg methods. Melatonin intervention group was injected intraperitoneally with melatonin (1mg/ml, 10ml/kg). The morphology of microglia was observed through electron microscope. OX42-positive cells were detected by immunohistochemical (ABC) methods. The contents of MDA or the activity of SOD was measured respectively by thiobarbituric acid or xanthine oxidation method.Results:Electron microscope showed that the proliferation and activation of microglia cell and the morphology was more about ameboid displayed in hemocortex in Model group, the activation of microglia cell of the same cortex was insignificant in MT group at 2d after ICH. Immunohistochemistry assay showed that the little expression of OX42-positive microglia appeared in normal group and sham group, OX42-positive microglia had already appeared in perihematoma region at 12h after ICH, peaked at 1h, lasted at 7d. OX42-positive microglia at different time points after ICH in MT group were significantly lower than in Model group (P<0.05). The content of MDA increased significantly after ICH in Model group, peaked at 1d, then lowered gradually, and higher than normal group at 7d, the activity of SOD changes in the opposite. Contrast to Model group, the content of MDA were lower and the activity of SOD were higher than that at different time points after ICH in MT group (P<0.05).Conclusion:Melatonin could inhibit the activation and proliferation of microglia and improve the damage of nerve cell after ICH in rats, and The mechanism was related to melatonin by reducing the level of oxidative stress in brain tissue, delayed the activation of microglia, then alleviated the secondary brain injury after ICH. PARTⅡObjective:To observe the effects of melatonin on the neuronal apoptosis, the expression of inflammatory factors and apoptosis-related genes after intracerebral hemorrhage (ICH) in rats, and discuss the effect of inflammatory reaction and apoptosis on the mechanism of secondary nerve injury after ICH. Then explore the protective effect of melatonin on delayed neuronal apoptosis after ICH.Methods:The model of ICH were created by Rosenberg methods, animals were randomly divided into four groups:normal group, sham-operated group(Sham group), intracerebral hemorrhage model group(Model group) and melatonin intervention group(MT group).The morphology of neuron was observed through electron microscope. The apoptosis of neuron was detected by TUNEL. The morphology of neuron and the microvascular structure were observed through electron microscope. Expression of NF-κB, Caspase-3 were observed by immunohistochemical (ABC) methods. The dynamic changes of Bax mRNA, Bcl-2 mRNA, MMP-2 mRNA and MMP-9 mRNA were measured by real-time Polymerase chain reaction (RT-PCR). Expression of Caspase-3 protein and TNF-a protein were determined by Western bloting.Results:Apoptotic cells were not detected in normal group and sham-operated group, a number of apoptotic cells around hematoma were detected at 12h and peaked at 4d, until 7d there had still been more apoptotic neuronal cells in Model group, the number of apoptotic cells in MT group were lower than in Model group at different time points after ICH (P<0.05). Electron microscope showed that chromatins gathered to the rim of nucleus, vacuoles were formed in most neuron and the edema around microvascular was obviously in Model group at 2d after ICH, but the morphology of neuron was normal in MT group at same time. The expression of NF-κB positive cell raised significantly in Model group at 12h after ICH, peaked at 4d, the expression of NF-κB positive cell were lower in MT group than in Model group at different time points after ICH (P<0.05). The expression of Caspase-3 positive cell appeared in perihematoma region at 12h after ICH, peaked at Id, then decreased gradually, but was still high at 7d, the expression of Caspase-3 positive cell were lower in MT group than in Model group. The results of real time quantitative PCR showed that the expression of Bax mRNA raised rapidly in Model group after ICH, peaked at1d, and were still higher than in normal group from 4d to7d. The expression of Bax was lower in MT group than in Model group. The expression of Bcl-2 mRNA increased at 12h after ICH in MT group, peaked at1d, and were higher than in Model group from 4d to7d after ICH (P<0.05). The expression of MMP-2 mRNA increased gradually form 12h to 4d after ICH, until at 7d that the expression of MMP-2 mRNA in MT group was lower than in Model group (P<0.05). The expression of MMP-9 mRNA in Model group increased significant and peaked at 2d after ICH, then decreased rapidly, and increased again at 7d. Compared to Model group, early appearance of peak expression at Id in MT group, then decreased to the level of normal, but increased significant again at 7d and higher than the corresponding time point in Model group(P<0.05). Western bloting showed significant increases in the expression of Caspase-3 protein and TNF-a protein in Model group than in MT group at different time points after ICH(P<0.05).Conclusion:The apoptosis around hematoma were related to the degree of inflammation reaction leaded by microglia activation after ICH. Melatonin alleviated significant the activation and release of inflammatory factors and inflammatory mediators, reduced vasogenic brain edema, decreased delayed neuronal apoptosis after ICH. The mechanism might be related to melatonin by reducing activation of NF-κB and release of TNF-a, regulating the transcription of matrix metalloproteinase, alleviating inflammation reaction, meanwhile increasing the expression of Bcl-2 gene and decreasing the expression of Caspase-3. PARTⅢObjective:To observe the expression of glial derived neurotrophic factor (GDNF), neuroglobin(NGB), mitochondrial transcription factor A (mtTFA) of brain and the effects of melatonin intervention after intracerebral hemorrhage (ICH). And discuss the self-repair function of neuron and mitochondrial on the secondary brain injury after ICH. To explore promote effect and mechanism of melatonin on endogenous protection and functional recovery of neuron after ICH.Methods:The model of ICH were created by Rosenberg methods, animals were randomly divided into four groups:normal group, sham-operated group(Sham group), intracerebral hemorrhage model group(Model group) and melatonin intervention group(MT group). Neurological function of rats was evaluated with modified Neurological Severity Scores (NSS) on 12h, Id,2d,4d and 7d after ICH respectively. Expression of GDNF and NGB were observed by immunohistochemical methods.The dynamic changes of GDNF mRNA, NGB mRNA and mtTFA mRNA were measured by real-time Polymerase chain reaction (RT-PCR). Expression of GDNF was determined by Western bloting.Results:There was no significant difference on the volume of hematoma in each group. The NSS score in MT group were lower than Model group (P<0.05). Immunohistochemistry assay showed that the little expression of GDNF and NGB-positive cells appeared in normal group and sham group. The expression of GDNF positive cells was significantly increased at 12h after ICH, and still higher than the control group at 7d after ICH(P<0.05). Expression of GDNF positive cells in MT group was significantly higher than in Model group, the difference were significant in statistics (P<0.05). The expression of NGB in Model group increased at 12h after ICH, then decreased at Id after ICH, and increased gradually from 2d to 7d after ICH. Expression of NGB positive cells in MT group was higher than in Model group (P<0.05). The results of real time quantitative PCR showed that the expression of GDNF mRNA in MT group increased and peaked at 12h after ICH, then decreased gradually, but still higher than normal group at 7d after ICH. Expression of GDNF mRNA in Model group increased and peaked at 1d after ICH, then decreased gradually, still higher than normal group at 7d after ICH. The expression of NGB mRNA in MT group increased and peaked at 12h after ICH, then decreased gradually, but still higher than in normal group from 1d to 4d after ICH, the expression of NGB increased significantly again at 7d after ICH. Expression of NGB mRNA in Model group peaked at 1d, decreased rapidly to the level lower than normal group at 2d, and increased again from 4d to 7d after ICH. The expression of mtTFA mRNA in MT group increased significantly from 12h to 1d, the expression of mtTFA mRNA in Model group increased and peaked at 1d after ICH, Expression of mtTFA mRNA increased in both two groups form 2d to 7d after ICH, there was significant difference between two groups on statistics(P<0.05). At 2d after ICH, there were the swelling of mitochondria in the cortical neurons, the number of mitochondria decreased significantly and the remaining mitochondria translocated to the cell wall in the Model group, the morphology of mitochondria was normal in MT group. The expression of GDNF protein in MT group was significantly higher than in Model group at 12h,4d and 7d points after ICH (P<0.05).Conclusion:Melatonin could promote functional recovery of Nerve cells after ICH. Melatonin might have the effects of neuroprotective and recovery by inducing early expression of GDNF and NGB and promoting functional recovery of mitochondria.