| Global cerebral ischemia very commonly occurs in myocardial infarction, shock, suffocation caused by low blood brain perfusion in clinical practice. Cerebral ischemia reperfusion injury refers to the brain cells are damaged after cerebral ischemia, the recovery of blood reperfusion, not only can not make the recovery of tissue and organ function, on the contrary the phenomenon that further increases the weight of of its ischemia injury.The study have shown that the hippocampus is related to the formation of emotion, visceral activity and recent memory. Therefore, it is particularly important for using mouse cerebral ischemia and reperfusion model to simulate human cerebrovascular disease research,. Recently, with the rising of occurrence rate of ischemic cerebrovascular disease, animal model has been widespreadly concerned. Bilateral carotid artery occlusion,because of its simple operation, less damage, lower mortality rate, high success rate model, easy control and ischemia-reperfusion, much favored by scholars.Cerebral ischemia/reperfusion injury is a more complex mechanisms involved in the pathophysiological process, and interaction between the various aspects of factors 。neuron apoptosis is the most common form of injury, and gene regulated the process of apoptosis.Mitogen-activated protein kinase p38 (p38 MAPK) can participate in various physiological processes such as cell growth, propagation, differentiation, dead and intercellular function be synchronous.After cerebral ischemic neuronal damage, p38 MAPK is activated early stage and then increase in the expression of glial cells and neuron ischemic area, and showed a time-dependent, suggesting that p38 MAPK signal pathway play an important regulatory role during in cerebral ischemic neuronal death. Blocking the signal path level and regulation of p38 MAPK expression and activity might be a new way to treat ischemic stroke. It has established global ischemia and reperfusion in mice, observe the changes of p38 MAPK expression and neuronal apoptosis and to investigate mechanisms of neuronal apoptosis,and observe protective effect of p38 MAPK inhibition after global ischemia and reperfusion, in order to provide the guidance for the treatment of clinical cerebral ischemic injury.Our study include the following three parts:1. The establishment of cerebral ischemia reperfusion model in mice2. The study of p38MAPK expression after cerebral ischemia and reperfusion3. The study of p38MAPK inhibition in cerebral ischemia and reperfusion injuryPart 1. The establishment of cerebral ischemia reperfusion model in miceObjective:Genotype identification, breeding mice, mice global cerebral ischemia reperfusion model, the survival rateMethods:(1)the mice kept in SPF animal room, get mouse tail and extract DNA, confirm the mouse genotype by PCR, gel electrophoresis, gel imaging system after taking determine genotype mice, the mouse genotype that evaluation needs is classified and the scale breeding. (2) Application BCCAO (bilateral common carotid artery occlusion) to study after global ischemia and reperfusion model, the specific steps are as follows:Before each organizes the preceding fasting 12h of mouse art, forbidden to drink 4h,1%amyl bar will duly mice were anesthetized by intraperitoneal injection of sodium,80mg/kg body weight, post-injection anesthesia attention to closely observe the spontaneous breathing test mice, waiting righting reflex, supine fixed on homemade mouse plate under a dissecting microscope to operate along the midline of the neck midline incision, blunt dissection of muscle and connective tissue, isolated bilateral common carotid artery and embedding, during the operation to avoid damage to the vagus nerve, with no mycelium line sequentially ligating bilateral common carotid artery, observes the vital sign of mouse;, such as breathing and heartbeat are normal mice after reperfusion can be stitched layers of muscle and skin, can sew up each layer muscle and skin after 20min pours into again, and wiping tincture of iodine is protected from infection, surgery using electric heaters to keep them warm, after a single cages to prevent cross-infection. A sham operated rats separating muscle and connective tissue are sewed up the incision behind the exposure bilateral common carotid arteries. (3) mice in each group after treatment, the survival rate of each group statistics, making survival curve.Results:(1) PCR amplification products electrophoresis, detected by photographing the gel imaging system. Control DNA marker has 328bp,195bp two strips Ⅱ〠is transgenic p38KI/+ mice, only 195bp bands for the wild-type C57BL/6 mice (WT), referring to the results of gene identified and kept separate in some cage.(2) ischemia-reperfusion model was prepared after the general situation. the sham group:a few days after surgery to restore good mental state in mice, self-activity and more agile, responsive variety, eating drinking normally. Ischemia-reperfusion model group:the mice after surgery most apathetic, unresponsive, activity reduces,eating into the water is also significantly reduced, and is slow in action; there will be a small part of the mice increased activity, irritability, unsteady gait, such as performance, and self-cleaning ability of mice decreased, hair thinning, dry, lack luster hair shaft or, occasionally there are transient paroxysmal seizures in mice. (3) SHAM group of mice survival rate of 100%.compared with WT mice, transgenic p38KI/+ mice survival rate of ischemia-reperfusion model group increased.Conclusions:Utilize PCR successfully to identify the genotype of transgenic mice, the I/R model was made by BCCAO method, and some symptoms were also showed.Part2. The study of p38MAPK expression after cerebral ischemia and reperfusionObjective:To establish a mouse globle brain cerebral ischemia and reperfusion model was observed p38MAPK expression changes in different times and the corresponding time point of apoptosis related protein after injury,also at different time points were morphological study on neural function defect research and organization to further define the research content and the period of research..Methods:(1) three months old male mice were randomly divided into SHAM group, ischemia-reperfusion model group (I/R group), application of two vascular Act (bilateral common carotid artery occlusion, BCCAO) establish global cerebral ischemia-reperfusion model mice, Western blot assay in mice hippocampus p38 MAPK phosphorylation level changes. Study on the change of p-p38 and c-caspase-3 expression after determining the change time point. (2) after BCCAO model was made in mice, TTC staining respectively 1d,3d,5d, and several time points was carried out, and the ischemia was observed at. Calculation of ischemic infarction area ratio and volume ratio. (3) after reperfusion,NDS score (25-point scale) and modified NSS scores (18-point scale) to assess the situation in mice modeling Id,3d,5d,7d. With a score of "Virginia in the United States modeling Mice were trained before the 3d and record of value before ischemia 1h, after modeling Id,3d 5d,7d,scores were measured twice, recording time and aking the mean. (4) Take male C57 mice were randomly divided into three groups, each group 6.after modeling 1d arid 3d.after BCCAO model was made in mice,it using respectively saline and 4%paraformaldehyde perfusion, TUNEL staining calculate the number of positive cells in reperfusion of ischemic CAl region, and calculate the apoptotic index. Results: (1) western blot analyzes shows that 3d experimental group is more higher than the expression of p38 protein after Id,5d and the 7d experimental group respectively,And then select choose the 3d time point,and knew that I/R model group and p-p38 protein expression increased compared to SHAM group (p<0.05). I/R model group c-caspase-3 and increased expression compared to SHAM (p<0.05). (2)Compared with the SHAM group, 1d (55.344+3.325, p<0.05) than the 3d (44.047+2.099) and 5d (34.985+5.435) infarct volume ratio greater. (3) stroke NDS index score higher than the model group, SHAM group score. Modified NSS score in model group 1d,3d,5d and 7d score higher than the SHAM group respectively. In the point system of Virginia score, compare with the sham group, the model group score value is high, there were significant differences (p<0.05). (4) After TUNEL staining, there were significant differences (p<0.05) for CA1 region apoptotic index and sham group behind the model group 3d, but apoptotic index that CA1 distinguishes behind the Id and SHAM do not have significant difference (p< 0.05),Conclusions:After 3d cerebral ischemia reperfusion, relevant factor p38 protein increased and apoptotic protein factor caspase3 increased, after cerebral ischemia and reperfusion in mice and it showed that neurological defects occurred, after 3d the occurrence of apoptosis,TUNEL staining showed that the experimental group apoptotic index was significantly increase.After TTC staining 1d, infarct volume is significantly increase. NDS score results, consistent with NSS scores and Virginia score score results, the experimental group and the sham group was obvious,it has taken place damaged.Part 3:The study of p38MAPK inhibition in cerebral ischemia and reperfusion injuryObjective:To investigate the protective effect of p38MAPK inhibition in cerebral ischemia and reperfusion.Methods:(1) The month-old male C57 mice were randomly divided into sham group, I/R model group, join transgenic p38KI/+mice as I/R+KI model group application method two vessels (bilateral common carotid artery occlusion, BCCAO) establish global cerebral ischemia. Western blotting was used to detect changes of mouse hippocampus p38MAPK(p-p38/p38)phosphorylation and caspase-3 expression (c-caspase3/caspase3) apoptotic proteins. (2) After BCCAO in mise, TTC staining was carried out in 1d,ischemic area and infarct volume was calculated. (3) After cerebral ischemia reperfusion,NDS score (25 point scale) and modified NSS scores (18-point scale) in mice modeling Id,3d,5d,7d after to assess the situation. With a score of Virginia in the United States modeling Mice were trained before the 3d and record of value before ischemia lh, after modeling 1d,3d 5d,7d,scores were measured twice, recording time and taking the mean. (4) Take male C57 mice and transgenic p38KI/+mice were randomly divided into three groups, each group 6. Take male C57 mice and transgenic p38KI/+mice were 5.After BCCAO model was made at 3d in mice,it using respectively saline and 4%paraformaldehyde perfusion, TUNEL staining calculate the number of positive cells in reperfusion of ischemic CA1 region, and calculate the apoptotic index.Results:(1) I/R+KI model group and the sham group, I/R model group p38MAPK expression (p-p38/p38) decreased, a significant difference (p<0.05). I/R+KI model group and I/R model group caspase-3 expression (c-caspase3/caspase3) decreased, a significant difference (p<0.05). (2) TTC staining,compared with I/R model group, I/R+KI model group infarct volume was reduced, there were significant differences(p <0.05). (3)Stroke index score, I/R model group (12+1.414) higher than the I/R+KI model group (7.75+1.500) scores. Improved NSS score in sham group, I/R model group and I/R+KI model group scored. I/R model group 1d,3d,5d and 7d than the I/R+KI model group,3d,5d and 7d score high, there were significant differences(p <0.05).Virginia score, I/R model group score higher than V R+KI model group, the difference was significant (p<0.05). (4) After TUNEL staining, the sham group hippocampus complete nuclear membrane, nucleolus clearly visible even after the degeneration of pyramidal cells, and I/R model group and I/R+KI model group nuclear membrane disintegrate; Chromatin concentrates, marginalisation; After birth has the formation of vesicle shape, chromatin to be divided into bulk etc., I/R model group 3d after CA1 region of apoptotic index and I/R+KI model group were significantly different, (p<0.05).Conclusions:The I/R+KI model group.compared with the I/R model group, p38MAPK suppresses the protein expression to be reduced, and apoptosis-related protein caspase3 expresses reduction. TUNEL staining showed that the experimental group apoptotic index was significantly increase. NDS score results, consistent with NSS scores and Virginia score score results, Compared with I/R model group and SHAM model group, the I/R+KI model group was obvious minimizes, neurological function results has improved and has a protective effect. |
