| Traumatic brain injury (traumatic brain, injury, TBI) is a common lethal and disabling disease which cause cognitive dysfunction and abnormal behavior changes.Now it is the worldwide difficult problem to human health. With the rapid development of China’s economy and traffic, the prevalence of traumatic brain injury also increased. Although the treatment system and therapy method of traumatic brain injury continues to improve, but the mortality rate is still high, and the mechanism of the traumatic brain injury is still unclear, therefore, further study and improvement of traumatic brain injury and pathological molecular mechanism will become the focus of research in the field of neuroscience.Recently, autophagy (Autophagy) as nerve cells of another kind of programmed death mode has attracted more and more attention, but the specific mechanism is not clear. Autophagy is a lysosome by the interaction mechanism and the microtubule system such as a metabolic pathway. It is clear and the cell components in neurons, and participate in many physiological and pathological processes. After traumatic brain injury, start autophagy can protect the injured nerve cells, to avoid the tendency of death, the timely removal of damaged cells, toxicity of excitatory amino acid, free radical and metabolic waste, playing an important role in the supporting the survival of neural cells after injury or stress.Rapamycin (Rapamycin) is a classical mTOR receptor inhibitors, activation, by inhibiting the mTORC1 specific and mediated autophagy signaling pathways downstream of the receptor, mTOR plays a key role in the regulation of autophagy. In recent years, autophagy rapamycin excited in protective effect of nerve cells after injury has gradually been recognized and attracted many researchers attention.In this study:(a)、Construction of the lateral fluid percussion brain injury model in rats, 6 weeks after TBI, the cognitive dysfunction of rats hippocampus injury, autophagy related protein significantly reduced; (b), giving the autophagy agonist drugs,6 weeks after TBI, the cognitive function of the rats has been improved; (b). the ipsilateral hippocampus enhanced autophagy rapamycin after TBI in rats, inhibited neuronal apoptosis.Part I、The damage of spatial learning and memory function and changes of autophagy in rats post moderate traumatic brain injuryObjective:(1) The construction iniury model in rats and the lateral fluid percussion brain injury; (2) The change of cognitive behavior in rats after the functional verification of TBI; (3) The change of autophagy side injury in hippocampus of rats were observed after TBI;Materials and methods:1、Adult male SD rats were purchased from the animal experimental center of Second Military Medical University, weight (350+20) g, reared in standard animal room, the rats underwent craniotomy drilling operation, drilling center is located in the anterior midline open after 4.5mm, left 3mm, the hole diameter 0.5cm, scalp incision, remove the bone window, to avoid damaging the dura mater with zinc phosphate cement, dental fixation material fluid percussion, connected with the hydraulic hammer, (2.12±0.03) times against the atmospheric pressure strength; the sham operation control group (Sham) only after drilling suture skin incision. After injury, the body weight of rats and the daily record describing curve for weight loss more than 40% of their body weight or serious infection or dying from experimental rats.2、Adult male SD rats (n=28) were randomly divided into injury group (TBI, n=15), sham operation group (Sham, n=10).3、Six weeks (thirty-sixth and 40 days) after injury, Morris water maze test, the main test injury in rats after short-term memory and ability to explore space, is used to evaluate the learning and memory storage function changes in cognitive TBI rats.4、Behavioral test at the end of the day (40 days after injury), randomly selected rats in different groups, to be satisfied with the anesthesia after rapid decapitation, ice material (60s), cortical tissue range (the radius of the hole±0.3cm), total protein was extracted and detected for further. The configuration of 15% separation gel, SDS-PAGE gel electrophoresis, transferred to PVDF membrane humidity, into the closed solution, the PVDF film with a reference in anti beta -Actin (1:5000), LC3 (1:1000), Beclin-1 (1:500) hybrid bag, placed 4 degrees rotation overnight, washed with PBST film three times each after incubation with 5min, the fluorescence of two anti 1.5h, once again light membrane washing 3 times after fluorescence scan film color. Observation of autophagy related protein differential expression.Results:1、Morris water maze data suggest that moderate traumatic brain injury may lead to cognitive dysfunction of rats (P< 0.05), compared to the TBI group and Sham group, with the test of the last day, the latency of P (the time needed to find the escape platform into the water) was significantly shortened (19.75s VS 7.75s, P< 0.05). The results suggest that the spatial probe test, TBI group compared with the sham operation group, the retention time in target quadrant was significantly shortened, showing regional preference difference or does not exist. The sham operation group (Sham) and blank control group (CTR) in short term memory and spatial probe test showed no obvious difference (P> 0.05).2、The Western-blot results suggest that traumatic brain injury in rats 6 weeks after injury, injury side of hippocampus tissue autophagy protein Beclin-1 and LC3, compared with the sham operation group and blank control group decreased significantly (P< 0.05).Conclusion:1、The hippocampus of rats after moderate got structural damaged after TBI, along with cognitive dysfunction.2、The level of autophagy in injury side of hippocampus decreased after TBI(40 days after injury).Part Ⅱ、The effect of autophagy induced by rapamycin on spatial learning and memory function in rats post moderate traumatic brain injuryObjective:By using the mTORCl receptor inhibitor rapamycin, enhanced autophagy of ipsilateral hippocampus to observe the changes of cognitive function in rats. Materials and methods:1、Construction of adult male SD rats were moderate traumatic brain injury model, with the former method.2、SD rats (84 rats) were randomly divided into 6 groups of first coding, group (brain injury group,2mg/kg+rapamycin group (second, n=17); brain injury group and solvent group DMSO, n=18); the third group (sham operation+rapamycin,2mg/kg, n=12); the fourth group (sham operation+solvent in group DMSO, n=12); the fifth group (blank control group 2mg/kg+rapamycin, n=13); the sixth group (blank control group and solvent group DMSO, n=12), given 30 minutes after injury first intraperitoneal injection administration, after 2 weeks of continuous interval 24 hours of administration.3、thirty-sixth to forty days after traumatic brain injury in Morris water maze test, with the former method.4、The behavioral tests, the last day (40 days after injury), anesthesia were randomly selected each half of the rats were taken out after ipsilateral hippocampus were detected with Western-blot method with the change of autophagy; the other half, extraction of intact brain tissue, fixed with 4% paraformaldehyde and embedded in paraffin after, from the 2.31mm to the anterior fontanel 4.9mm posterior continuous slices, thickness of 10 m. Histological observation area is defined as injury hippocampus ipsilateral brain tissue (CA3) area. By immunohistochemistry, immunofluorescence and apoptosis of TUNEL were observed in different groups the number of damage zone CA3 injuries nerve cells autophagy and apoptosis cells in hippocampus.Results:Morris water maze test data show that:after brain injury in rats in rapamycin treatment group, solvent group, the latency of P was significantly shortened (13.82s VS 6.0s, P< 0.05); damage solvent group showed significant cognitive impairment (19.75s VS 7.75s, P< 0.05); and the pseudo operation group and the blank control group showed no significant the difference (6.75s VS 7.5S, P> 0.05).Conclusion:After the excitement of autophagy rapamycin, the cognitive function of the rats was improved.Part Ⅲ、Effects of rapamycin in hippocampal CA3 area of rats in autophagy and apoptosis post the moderate traumatic brain injury.Objective:By using the mTORCl receptor inhibitor rapamycin, the autophagy of ipsilateral hippocampus was enhanced and observe the pathological changes and apoptosis of nerve cells in CA area of hippocampus.Materials and methods:1、the behavioral tests, the last day (40 days after injury), anesthesia were randomly selected each half of the rats were taken out after ipsilateral hippocampus were detected with Western-blot method with the change of autophagy; the other half, extraction of intact brain tissue, fixed with 4% paraformaldehyde and embedded in paraffin after, from the 2.31mm to the anterior fontanel 4.9mm posterior continuous slices, thickness of 10 m. Histological observation area is defined as injury hippocampus ipsilateral brain tissue (CA3) area. By immunohistochemistry, immunofluorescence and apoptosis of TUNEL were observed in different groups the number of damage zone CA3 injuries nerve cells autophagy and apoptosis cells in hippocampus.Results:1、Western-blot, immunohistochemistry and immunofluorescence results showed moderate traumatic brain injury after 6 weeks, the level of autophagy in hippocampal tissue injury side were significantly decreased (P< 0.01), rapamycin treatment group can improve the level of autophagy in injuried hippocampal;2、Western-blot and immunohistochemistry, immunofluorescence, TUNEL apoptosis detection:the hippocampus ipsilateral increased autophagy, and inhibit neuronal apoptosis, apoptosis in hippocampus CA3 neurons number decreased (P< 0.05).Conclusion:Rapamycin enhanced autophagy played an important role TBI by reducing the injury and inhibiting the apoptosis in CA3 area nerve cells of hippocampus. |
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