The Effect Of Chronic Cerebral Hypoperfusion On White Matter Function And Related Molecular Mechanism

Chronic cerebral hypoperfusion(CCH) can lead to cerebral ischemia and hypoxia, it participates in vascular dementia and age-related diseases such as Alzheimer’s disease and other diseases. In the past the white matter was considered a high tolerance to ischemia, but recent studies have shown that white matter in chronic ischemia and hypoxia is more sensitive than the cortex, especially in the white matter with rich-myelin, easy to cause demyelination, which lead to irreversible damage of the axon. This may be a key structural base of brain function degeneration such as cognitive disorder.The study showed that the extensive small arteries pathological changes in deep cerebral hemispheric white matter or the stenosis of carotid artery and their intracranial branches reduced cerebral blood flow. The persistent low-perfusion state may result in white matter rarefaction that causes neurological function disorder and leads significant clinical symptoms. The molecular pathological mechanism of ischemic white matter lesion induced by CCH remains unclear and is not recognized clearly.In the interaction between glia and axons, adhesion molecule changes in the juxtaparanode region affects the function of myelin and axon. Cell adhesion molecules plays an important role on the contact in interface between glia and axon. Its changes can cause axonal degeneration. The Caspr2(Contactin-associated protein2) in the juxtaparanode region have adhere effect between the axon and glial cells. They all constitute juxtaparanode region of myelinated nerve fibers. The connection plays an great important role in the normal myelin lamellar formation of myelin and the saltatory conduction of the nerve impulse. Its changes made interaction between glia and axons decrease, the myelin structure changes and nerve conductive velocity slow. Caspr2 changes cause the boundary between Na+ channels in the nodal region and K+ channels in juxtaparanode region disappear, which result in the changes of white matter function. The effect of Caspr2 in white matter damage have not been studied and recognized during chronic hypoperfusion. Therefore, we first observed the functional changes of white matter in CCH, including the conductive function, the transporting function and cytoskeleton changes. Then further investigation was done on the effect of Caspr2 on the white matter changes and ion channels in order to explore the molecular mechanism of white matter changes in chronic hypoperfusion.This study was divided into the following 3 parts:Part I Establishment of of CCH animal models and pathological evaluation of cerebral tissueMethodsThe suturing method was used at SD rats. Under the asepsis condition, the bilateral common carotid arterys were separated. A syringe needle was bound with the common carotid artery at a lower place about 1.5cm to the bifurcation of internal and external carotid artery. The syringe needle was then withdrawn to cause partial narrowing of bilateral common carotid artery and reduction in blood infusion of cerebral tissue at a controllable degree. 2 weeks,4 weeks and 12 weeks after the hypoperfusion, HE pathomorphological examinations of the cerebral tissue were observed.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. After bilateral carotid artery partial ligation in rats, the hippocampus pyramidal cell has undergone from ischemia, edema at early stage to gradually develop into a serious neuron loss, nuclear light staining and the micro-cavity formation in cytoplasm, which was consistent with the pathological manifestations in chronic persistent decline of cerebral blood flow. The model is suitable for experimental study on the chronic cerebral hypoperfusion.Part II Observation of the NF200 and Aβ1-40 protein expression after CCHMethodsThe motor evoked potentials was used to detect the axon conduction function of rats in each group(control group and after 2 weeks, 4 weeks and 12 weeks). The expression changes of NF200 and Aβ1-40 in hippocampal CA1 region was studied by immunohistochemistry during CCH.ResultsThe dysfunction of white matter in CCH as follow:(1) In conductive function of white matter, MEP latency was significantly prolonged in the groups at 2 weeks, 4 weeks and 12 weeks in chronic hypoperfusion. The latency was more extended with the persistence of hypoperfusion. That indicates the axon conductive function lesion in cerebral ischemia. The permanent hypoperfusion aggravates conductive function.(2) In the transport function of axon, it was observed that Aβ1-40 expressed rarely and NF200 obviously in hippocampus CA1 in the control. In 2 weeks group, Aβ1-40 immunostaining-positive cells were seen in hippocampus CA1. Their immunostaining was shallow. The NF200 expression decreased as compared to the control group. At 4 weeks of hypoperfusion, there were more Aβ1-40 immunostaining-positive cells in CA1. NF200 immunoreaction was more weak than that at 2 weeks group. At 12 weeks of cerebral ischemia, the number of Aβ1-40-specific immunostaining positive cells in hippocampus CA1 area increased progressively and NF200 expression decreased continually. The results reveals that CCH can injure axon transport function, so that Aβ1-40 can not be transfer to the distal end by axon and aggregated near cell body. As a kind of cytoskeletal protein, the decline of NF200 expression may lead axon damage, changes of axon transport function and axon degeneration.Part III Observation of the Caspr2 and Na+ channel protein expression after CCHMethodsThe changes of Caspr2 and Na+ channes in the rat corpus callosum were observed by immunofluorescence and laser confocal microscopy and western blot.ResultsThe expression changes of Caspr2, Na+ channel protein. The techniques of immunofluorescence and laser scanning confocal microscopy were used to observe Caspr2 expression. The results showed that Caspr2 expression weakened in CCH. The number of the positive particles and the intensity of decreased significantly, moreover, the intensity of stain tended gradually to reduce as the time went by. At the same time, the distribution pattern of Caspr2 expression had changed. which had disorder in the distribution of strip and point in normal fibers. It could be seen that the Caspr2 positive particles gathered around in part area at 12 weeks. It was detected that the channel protein had the same decline trend. The results suggest that adhesion molecule Caspr2 had the expressive changes in cerebral chronic hypoperfusion(CCH), and the expression of channel prote in of Na+ also did as Caspr2. The decline of the expression of Na+ channel protein made the Na+ inflow decrease and progressively influence the conducting function. The changes of Caspr might be the important reason for myelin sheath separation, myelinocla sis, the decline of Na+ channel protein expression and the descent of the conducting function.The animal model of CCH was produced by the bilateral carotid partial ligation. It is one of the ideal animal model to study the cerebral white matter’s ischemic pathologic mechanism and treatment under the hypoperfusion state. The low-perfusion can influence the conduction and transmission of axon. The latency of motor evoked potential gradually extended as the gradual appearance of the ischemia. The Aβ1-40 displayed aggregation due to the disconnection to the distal end of the process after expression. the NF 200 low-expression affected the axon structure and transmission. The white matter’s changes might be related to the Caspr 2 low-expression and the abnormal distribution, and the Na+ channel protein’s low-expression. The adhesion molecule Caspr may play an important part in the the pathological and physiological process of the white matter’s damage in the hypoperfusion state.