| Background and objiectiveChronic cerebral ischemia is a pathological physiological state that a existence of long period of low perfusion in the brain, it can cause the shortage of oxygen and glucose to brain cells, causing a series of neurological dysfunction. As the rising percentage of elderly population in the world, chronic cerebral ischemia and associated diseases gradually increased, causing cognitive decline and degenerative nervous system disease which cause significant impact on quality of life. Therefore, it is urgent for us to explore the pathogenesis of chronic cerebral ischemia and propose effective measures for prevention and treatment.Transient receptor potential melastain 2 (TRPM2) channel is a new discoverednon-glutamate dependent cation channels in recent years, it expresses in neurons and glial cells of the central nervous system. In the effect of ischemia injury and various harmful factors, TRPM2 channel is activated and then mediate the activities of calcium, leading to calcium overload, and induce cell apoptosis. As a newly discovered cation channel, TRPM2 and its roles in nervous system diseases are one of the focuses of current research.Endonuclease G is a calcium-dependent enzymes, its induction of apoptpsis is independent of caspases pathway. In chronic cerebral ischemia, the intracellular calcium overload and tBid, Bax produced during ischemia can induce the EndoG release from mitochondria to the package slurry, and then translocate to the nucleus, causing mitochondrial rupture, leading to cell Apoptosis.Dl-butylphthalide(NBP) have a good therapeutic effect on acute ischemic cere brovascular disease. But whether dl-butylphthalide could exert neuroprotective effec ts by affecting the expression of TRPM2 and EndoG after chronic cerebral ischemi a in rats was not reported. Our purpose is to explore the general and specific mechan isms of dl-butylphthalide exerting neuroprotective effects on chronic cerebral tissue s in aged rats by observing the effects of dl-butylphthalide on the expressions of TRP M2 and EndoG in the cortex and hippocampus after chronic cerebral ischemia in rats, providing a new vision of effective prevention and treatment of chronic cereb ral ischemia.Materials and methods80 clean Wister rats, whose quality were between 450-550g, were selectde for the experiments. After 7 days adaptation-feeding and were tested by passing the water maze, rats were randomly divided into 4 groups, and each group include 20 rats. The 4 groups are A, B, C and D, and corresponding are control group (sham operation), model group (surgery+solvent), ischemic dl-butylphthalide low dose treatment group (surgery+Low dose+solvent) and dl-butylphthalide ischemia high-dose treatment group (surgery plus high-dose+solvent). Establish animal model of chronic cerebral ischemia by ligating bilateral common carotid artery permanently (BCAL),2 months after surgery, morris water maze was used to test the learning and memory function of rats. Rats in group A and group B were treated with solvent (sterile peanut oil) by gastric irrigation, while rats in group C and group D were giving dl-butylphthalide at the dose of 60mg/kg/d and 120mg/kg/d by gastric irrigation respectively. Each group were fed with dl-butylphthalide or solvent successivly for 1 month.1 moth later, all rats alive underwent the Morris water maze test, then, rats were anesthetized and thoracic cavity were cuted to expose the heart, sterile saline and paraformaldehyde were perfused through heart respectively so as to geting rid the blood of vessels. Brains were moved out, placed in 4% paraformaldehyde for 24 hour. Then brains were dehydrated, transparented, dipped in wax and embedded conventionally. Brains were cuted at 4μm coronally. The changes of morphology of brain were observed by HE staining and the expressions of TRPM2 and EndoG were observed by immunohistoc-hemically staining. Data were presented as mean±SEM(x±s), and analyzed by SPSS 13.0 for windows software. A one-way analysis of variance(ANOVA) was performed for differences among the 4 groups, LSD-t test was conducted between every two groups. Values of P<0.05 were considered to be significant.Results1. Observation of the general behavior of experimental animalsBefore operation, all the animals were breezy, responsive and able to self-feeding and drinking. After operation, rats in group B, C and D were depressed, lose interest in food, behave Irritabily. While group A haven't significant changes compared with the preoperative.7 days later, each group slow improvement.2. The results of learning-memory abilityBefore BCAL the differences between group A and group B were not significant (p>0.05). After BCAL two months the differences were significant (p<0.05). The learning-memory ability of group C and D were improved comparing with group B, moreover, group D was more obvious than group C. All the difference were significant (p<0.05).3. HE stainingThe brain tissue of the temporal cortex and hippocampus regions of each group's rats were not obvious lesions. Neurons in hippocampus and temporal cortex of group A have several levels, and most of the neurons'shape are normal, while few necrotic cells were observed occasionally. In hippocampus and temporal cortex of group B, most of neurons were disappeared and only few normal neurons were observed. Compared with group B, the number of dead neurons decreased while ascent of normal neurons in group C, The degeneration and death of neurons in hippocampus and cortex of group D were significantly restored compared with group B and group C.4. The immunoreaction of TRPM2 proteinThe immune reaction production of TRPM2 protein appears as brown or yellow-brown color, most of production located in the capsule and cytoplasm of neurons. Only a small amount of TRPM2 protein immunopositive cells were seen in the cortex and hippocampus of group A. The number of TRPM2 protein immunopositive cells were significant increase in group B. The number of TRPM2 protein immunopositive cells were decrease in group C compared with group B, while in group D, this decreasion is more obvious. In the hippocampus group, the value of F was 127.115, P<0.01, the different between groups was significant. In the cortex group, the value of F was 310.046, P<0.01, the different between group A, B, C and D was significant.5. The immunoreaction of EndoG proteinThe immune reaction production of EndoG protein appears as brown or yellow-brown color, which located in the cytoplasm and nuclei of neurons. Only a small amount of EndoG protein immunopositive cells were seen in the cortex and hippocampus of group A. The number of EndoG protein immunopositive cells were significantly increase in group B. The number of EndoG protein immunopositive cells were decrease in group C compared with group B, while in group D, this decreasion is more significant. In the hippocampus group, the value of F was 80.292, P<0.01, the different between groups was significant. In the cortex group, the value of F was 49.959, P<0.01,the different between groups was significant.Conclusion1. Dl-butylphthalide can reduce brain tissue morphology result from chronic ischemic damage to brain tissue morphology. 2. Dl-butylphthalide may plays an active role in protecting ischemic brain by reducing the expression of TRPM2 protein and EndoG protein, inhibiting the death and degeneration of cortical and hippocampal neurons.3. Dl-butylphthalide at the high dose maybe more effectively to the brain tissue of chronic cerebral ischemia... |
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