| Backgrounds:Brain stroke has been a critical disease threatening human lives and health. Of the patients with it, 70-80 % of them suffered from ischemic stroke. It is reported that the disease occurs in 15 million people in the world annually and 5 million of them are Americans. In China, its morbidity is about 120-180/100000 and the mortality 60-120/100000. A percentage of 75% of the surviving patients loss their labor and 40% of them are severely disabled. The annual direct and indirect economic loss is as high as several hundred billion yuan.In particular, the human aging of society in the world and China is increasing, thus the mobidities of stroke and senile dementia are also increasing. The prevention and treatment of stroke and senile dementia becomes a great challenge in the fields of geriatrics and neurology. Such disease is difficult to recover. Therefore, the early prevention is the major principle.Large numbers of researches show that chronic hypoperfusion injury is more likely to be the early stage of stroke and senile dementia. Meanwhile, about 2/3 of the middle-aged and aged people are in the state of it. Thus chronic hypoperfusion injury is called a potential killer for life and health of the middle-aged and aged.Chronic hypoperfusion injury, clinically called chronic cerebral hypoperfusion(CCH), refers to chronic and extensive ischemia of cerebral tissue due to various reasons resulting in cerebral ischemia and hypoxia to cause chronic cerebral injury presenting as a series of cerebral functional disorders. And it is usually reversible at the early stage, which gives hope to treatment of chronic hypoperfusion injury, and prevention of stroke and senile dementia, and brings more valuable points for studies.For a long time, researchers have paid close attention to neurobiological features, post-injury repair of nurons. Glial cells are considered to have the effects of supporting and noruishing. As such, the studies on glial cells have been ignored. In fact, the effects of glial cells in cerebral nervous network are more complicated and important than we have expected. In addition, few studies have been conducted to investigate the effects of glial cells after cerebral ischemic injury.Consequently, the model of chronic hypoperfusion injury was established in aged rats by partial narrowing of bilateral common carotid arteries. Then pathological and ultrastructural examinations were employed to observe and evaluate the damage in axon, myelin sheath and oligodendrocyte. The relevant critical strucutures of oligodendrocytes in white matter such as axon-myelin sheath connecting Ranvier's reagion and its associated structures were observed and morphological, cognitive and behavorial as well as electrophysiological features determined to primarily study and evaluate the chronic hypoperfusion. The results of which might be of great guidance for judgment of chronic hypoperfusion injury degree and reversion and prevention and treatment of stroke and VD . This study was divided into the following 3 parts:Part I Establishment of model of chronic hypoperfusion injury by controllable partial stenosis of bilateral common carotid artery and pathological and ultrastructural evaluation of cerebral tissueMethodsThe suturing method was used and aged rats employed. Under the asepsis condition, the bilateral common carotid arteries were separated. A syringe needle was bound with the common carotid arteries at a lower place about 1.5cm to the bifurcation of internal and external carotid arteries .The syringe needle was then with drawn to cause partial narrowing of bilateral common carotid arteries and reduction in blood infusion of cerebral tissue at a controllable degree. Two weeks and 1 and 3 months after the hypoperfusion, pathomorphological and electron microscopic examinations of the cerebral tissue were performed.Results1. There was no death of the rats except for anesthesia-induced one. The model had good stability and repeatability and the hypoperfusion was of reliable results.2. Pathological examination showed that there was no focal and diffusive cerebral infarction. However, the fibrotic components in the white matter became rare. In 3 months after the chronic hypoperfusion, the fibrous components were relatively mixed. There was loss of cell compoents, in cortical and hippocampus neurons , especially CA1, and they were significantly more obvious than in the control group (P<0.05).3. Electron microscopy revealed that there were significant changes in axons, myelin sheath and oligodendrocytes. a) Vesicles and dense granule intensive particles of opaque structure, axon decomposition, destruction and mitochondrial vocuolation were seen in the axons. The layer structure of the myelin sheath was disarranged in mass. There were fusion, local deformation and vacuolation of myelin sheath, The gap increased distance between the sheath and axons widened,and partial loss of the sheath was observed.. b) The electronic density of the oligodendrocytes increased and there were corpuscles in cytoplasm and cellular interstitia. c) Part of CA1 neurons in hippocampus were damaged, which is presented as nucleolus disappearance or reduction in size, increase in electronic density of the cytoplasm, dilatation of the reticulum and Golgi body, disorder and vacuolation of mitochondria. Meanwhile, some mitochondria were malformed to have contents.Part II Effects of chronic cerebral hypoperfusion on expression of Caspr2 in cerebral tissue and cognitive and electrophysiological functions in aged ratsMethods:The aged rats were divided into the CCH group (n=15) and control group (n=12). The model of chronic hypoperfusion was established in the experimental group while no partial narrowing of the bilateral common carotid arteries was performed in the control. In 2 weeks and 1 and 3 months after hypoperfusion, cognitive and behavorial , central conductive function, perforant-pathway transduction speed, expression of Caspr2 in Ranvier's node were determined with behavioral method, electrophysiological method, immunofluorescent method and immune blotting, respectively.Results:1.The escape latency was significantly prolonged, times of crossing platform in spatial probing markedly decreased in the experimental group as compared with the control (P<0.05) ,The first time passing hidden platform also prolonged significantly(P<0.05).2. The perforant-pathway conductive time was remarkably longer in the expenimental group than in the control (P<0.05). Meanwhile, the transduction speed was more rapidly decreased with time prologation in the experimental group.3. The Caspr2 expression in the white matter was significantly down-regulated in the experimental group as compared with the control (P<0.05). Furthermore, there was marked difference between the 2 groups at different phases.4 .The level of Caspr2 expression was significantly correlated to escape latency, crossing platform latency and central conductive latency (P<0.05).Part III Changes in expression of myelin sheath protein MBP and PLP and activation of oligodendricyte precursor cells during chronic cerebral hypoperfusion in aged ratsMethods:We established the aged CCH groups , aged CCH control groups, young experimental and young control groups. The changes in MBP and PLP levels and expression of NG2 and 04 protein were investigated.Results:1. In aged and young experimental groups, the NG2-positive cells distributed in cortex, hippocampus and subcontical white matter, which is consistent with distribution of 04 protein. However, the cells were significantly decreased in the 2 experimental groups after chronic hypoperfusion (P<0.05). There was also maked difference between the aged and young experimental group (P<0.05). The proliferation of NG2-positive cells was more obvious in the young experimental group.2. After the chronic hypoperfusion injury, the expression of both MBP and PLP in the white matter was markedly decreased and this decrease was more and more rapid along with time prolongation. PLP was significantly lower in the aged experimental group than in the aged control (P<0.05). There was significant difference between the 2 groups at different phases (P<0.05).The major conclusions in this study are as follows:1. The established model is the same as clinical cases of chronic cerebral hypoperfusion in imaging features. Meanwhile, it has good stability and repeatability and can be used to simulate chronic cerebral hypoperfusion2. The pathological and ultrastructural examinations show that the oligodendrocytes are highly sensitive and susceptible during chronic hypoperfusion injury, which suggests that both neurons and oligodendrocytes should be protected in clinical practice.3. The Caspr2 expression in Ranvier's node is significantly decreased after chronic hypoperfusion injury and the decrease is significantly correlated to decrease in cognitive and central conductive functions, which suggests that change in Caspr2 might be the critical link to and possible molecular basis for cerebral functional damage after chronic hypoperfusion injury.4. The finding that cognitive and central transduction functions are markedly reduced after chronic hypoperfusion injury in animals suggests that very early determination of cognitive function and evoked potentials in middle-aged and aged patients are of the role of alert for judgment of severity of chronic hypoperfusion injury, and possible development to cerebral infarction and senile dementia.5. MBP and PLP expression decreased, which suggested that oligodendrocytes was marked injuried during chronic hypoperfusion. The result also showed that MBP and PLP may be used as sensitive parameter for judgeing white damage.6. There is obvious proliferation and activation of Oligodendrocyte precursor cells (OPCs), OPCs and the proliferation is remarkably reduced in aged rats as compared with the young ones, which might be a mechanism of compensation or repair after chronic hypoperfusion injury. The activation of the OPCs might also be affected by aging, the mechanism of which needs to be further clarified. |
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