| Research BackgroundAlong with the social development and the changing of human dietary structure, the prevalence of cerebral vascular disease has increased gradually. The worldwide incidence of stroke is 150-200/100,000, up to 85% of which is ischemic. There are about 2 million cases of new-onset stroke in our country annually, and 1.5 million domestic patients die of CVD every year. Cerebral ischemic injury are also found extensively in the neurosurgical practice, such as brain trauma complicated with cerebral ischemia, vasospasm after the aneurysm incarcerated operation and wrongly clipping of the parent artery or inappropriate controlled hypotension during the operation. The acute ischemia usually suggests a timely repatency, which can aggravate the ischemia in return.The mechanism of cerebral ischemic/reperfusive injury is mainly associated with the energy exhaustion of neurocytes, toxicity of excitatory neurotransmitter glutamate, calcium overload, delayed cell death, free radical damage, release of different cytokines, depolarization of ischemic penumbra and inflammatory reaction. All those complicated pathological changes lead to the activation of CaMKs, ERK, p38 and JNK (the MAPKs signal transduction pathway consists of the latter three).Epidemiological studies show that the prevalence of dementia in ischemic stroke patients is remarkably higher than in control subjects. Many of these dementias develop progressively, and cerebral damage is not the direct cause of the subsequent dementia in over half of these cases. Alzheimer's Disease (AD) is a degenerative change and the most common cause of senile dementia, which is mainly characterized by the progressive memory loss and cognitive handicap of the patients. neurofilament tangles(NFTs) formation, loss of cortical neurons and sinile plaques are the major pathological features of AD.So far AD remains to be idiopathic. Multiple gene locus abnormalities were found in familial AD cases, whereas the mechanism of sporadic cases may be associated with cerebral I/R injury. Cerebral ischemia, hypoxia and free radical damage may be the main causes of AD[7].Vascular risk factors of stroke can also increase the risk of AD, and adequate perfusion of brain can relieve the symptoms of AD remarkably. Moreover, AD and cerebral vascular diseases (CVD) are found to share similar neuropathological features such as aberrant hyperphosphorylation of tau protein and neuron necrosis. However, it is not yet clear whether the neuropathological changes of AD take place after cerebral ischemia occurs, so it has special importance to clarify whether AD-like pathological changes can be induced by cerebral ischemia/reperfusion.Tau protein is an microtubule-associated protein widely expressed in nervous system, which plays an important role in stabling microtubules, promoting microtubule assembly,maintaining neuronal morphology and advancing the axonal transportation. The modification of tau protein may be the essential link in neuronal dysfunction and death, for the balance between phosphorylation and dephosphorylation of which is the critical factor to maintain the stability of microtubule. The hyperphosphorylation of tau protein is the characteristic patholobical change in certain nervous system degenerative diseases such as AD,Pick disease. Exceptionally in AD, paired helical filaments (PHFs) are found to contain a significant amount of tau antibodies specific for a phosphorylated form, and then aggregate into NFTs(neurofilament tangles), the hallmark of the disease. It is learned that there are more than 21 phosphorylation sites in tau protein, however,the majority are Thr231, Ser396, Ser404, Ser199, Ser202, Thr205, Ser262 and so on. The hyperphosphorylation of these sites are mainly regulated by protein kinase GSK-3β, cdk5 and MAPK.JNK is one kind of stressor-activated Serine/threonine protein kinases which can phosphorylate the amino activation area of nuclear transcription factor c-Jun. JNK/ SAPK is one of the important signal transduction pathways of MAPKs, which plays an important role in the pathogenesis of AD.It is now found that the pathway can phosphorylate the sites of tau protein mainly through Thr205, Ser396, Ser422, Ser202,Ser404,Ser199,Thr212,Thr231. The hyperphosphorylation of tau protein is considered the early event of AD, which is the key to the formation of NFTs, and is concerned with the neuron loss of the disease. The JNK/SAPK signal transduction pathway is actived during after cerebral ischemia-reperfusion injury. However, it is still unclear what is the function of JNK in tau protein abnormal hyperphosphorylation after focal cerebral ischemia.When cerebral Infarcted,the regional center of cerebral blood vessels dominant will form the core area and the surrounding ischemic penumbra, neuronal necrosisis are mainly in the former, while the latter is composed of neuronal apoptosis and is the key point to protect. The expression of apoptosis related genes, bcl-2 and bax, is correlated with hyperphosphorylated tau protein positive cells. bcl-2 and bax expression changes after cerebral ischemia. JNK is the critical regulator of neuronal apoptosis after cerebral ischemia-reperfusion injury. bax is the main pro-apoptotic genes which JNK regulats to induce apoptosis of neural cells during cerebral ischemia-reperfusion injury. Cerebral ischemia induces the aberrant hyperphosphorylation of tau protein as well. So we presume that the post-ischemic neurocyte apoptosis may have correlation with the aberrant hyperphosphorylation of tau protein, and they may share the same process with AD by regulating the apoptosis related genes bcl-2 and bax. JNK may induce AD's progression through hyperphosphorylation of tau protein and neurocyte apoptosis after cerebral ischemia.This study we established the model of middle cerebral artery occlusion of rats and examined specific phosphorylation sites of tau protein and the degree of phosphorylation, the solubility of phosphorylated tau protein, all of which was to discuss the relationship between brain stroke and AD. Furthermore, we studied the function of JNK in hyperphosphorylation of tau protein and expression of bcl-2, bax after cerebral ischemia, and the relationship between tau phosphorylation and post-ischemic neuronal apoptosis. In the last, we observed the effects of JNK in the infraction volume after cerebral ischemia-reperfusion by blocking the activity of JNK, The aims were to clarify the function of JNK in the phosphorylation of tau protein and neuronal apoptosis after brain ischemia. Objective:To establish rodent models of local cerebral ischemia/reperfusion through the modified thread occlusion method of MCAO and to evaluate the validity and stability of this model for the following research. Methods:A 3-0 monofilament nylon suture was introduced into the internal carotid artery (ICA) lumen through a puncture at the common carotid artery (CCA). With a depth of (20±3)mm, the suture could reach the initiation part of the anterior cerebral artery (ACA) and embolize this vessel, leading to local cerebral ischemia. The validity and stability of this model were evaluated though TTC staining and neurological grading scale. Results:The general conditions of successful models were bad. The infarction focus was obvious under TTC staining. Results were stable and little variation was observed. Excluding the minority of failed models, all rodents were scored 1-3 according to neurological grading scale. Conclusion:Modified MCAO method has relatively mild damage, more stable results and a higher repeatability, which provides a reliable guarantee for the following research.Objective:To explore the changes of tau protein phosphorylation in rat parietal cortex neurons after focal cerebral ischemia-reperfusion (I/R) and to clarify the relationship between cerebral infarction and Alzheimer's disease. Methods:The rat focal cerebral ischemia-reperfusion model was induced by occlusion of the right middle cerebral artery using the intraluminal suture method. The level of tau hyperphosphorylation at Ser199/202, Ser396, Ser404 and Thr231 sites and total tau in rat parietal cortex during focal cerebral ischemia/reperfusion were detected by and Western blot. Results:The levels of tau hyperphosphorylation at Ser199/202, Ser396, Ser404 and Thr 231 sites were significantly higher in I/R. group than those in the sham group, but the total tau did not increase. Conclusion:The cerebral infarction may contribute to Alzheimer's disease occurrence and development.Objective:To investigate the function of JNK in hyperphosphorylation of tau protein and expression of bcl-2, bax after cerebral ischemia, and the relationship between tau phosphorylation and post-ischemic neuronal apoptosis. In the last, we observed the effects of JNK in the infraction volume after cerebral ischemia-reperfusion by blocking the activity of JNK, and to clarify the function of JNK in the phosphorylation of tau protein and neuronal apoptosis after brain ischemia. Methods:The rat focal cerebral ischemia-reperfusion model was induced by occlusion of the right middle cerebral artery using the intraluminal suture method. The level of JNK activity, tau hyperphosphorylation at Ser396 and Thr231,Serl 99/202,Ser404 sites and the expressions of bcl-2, bax during focal cerebral ischemia/reperfusion were detected by Western blot. The relationship between the phosphorylated tau protein and the exression of bax were checked out by immunofluorescence method. The effect of JNK activity on infarction volume was tested by TTC method. Results:The activity of JNK were significantly higher in I/R groups than that in the control groups after ischemia-reperfusion.When the activity of JNK was inhibited by SP600125,the level of tau hyperphosphorylation at Ser396 and Thr231,Ser199/202,Ser404 sites,the expression of bax and the infarct volume notably reduced after ischemia-reperfusion. bax expression and the tau protein hyperphosphorylation were co-localized. Conclusion:JNK plays an important role in Alzheimer's like disease pathology change after cerebral ischemia and reperfusion. It can be used as as a therapeutic target of dementia after cerebral ischemia.
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