| White matter lesions(WML) appeared to be associated with attenuated performance in immediate and delayed memory, processing speed, and executive function.WML has been proved to be a serious risk factor of the conversion from mild cognitive impairment to AD. Cerebrovascular disease and hypoperfusion caused by cerebrovascular disease were the major causes of WML. In central nervous system, the neuropathological changes in WML were characterized by diffuse demyelination, loss of the axons, and mild gliosis. Myelin, mainly consisting of oligodendrocytes, plays an important role in nutrition and protection of axons. Protecting oligodendrocytes from damage and loss can be a hopeful way for the treatment of WML. It is not clear the mechanism of oligodendrocytes damage and loss in WML. Under the pathological condition, accumulation of byproducts as well as local acidification caused by energy metabolism disorder finally activates ASIC1 a, a kind of cation channels-acid sensitive ion channel1 a. Few researches have been reported about function of ASIC1 a in the formation of WML. Therefore, the aim of this study is to clarify its expression, mechanism and the function in WML. This research will provide new in- sights and lay experimental basis for clinical treatment of WML in the future.The first part the expression of ASIC1 a in cerebral white matterObjective: to observe the expression of ASIC1 a in cerebral white matter.Methods: In vivo, it was tested double marks between NG2 of OPC marker with ASIC1 a, and between CNPase and APC of mature oligodendrocytes markers with ASIC1 a by immunofluorescence double staining. In vitro, as the same, it was observed the double marks between A2B5 of OPC marker with ASIC1 a, and between CNPase of mature oligodendrocytes markers with ASIC1 a. Results: In vivo, it was observed the expression of ASIC1 a in NG2 positive cells, CNPase positive cells and APC positive cells in the corpus callosum; In vitro, double immunofluorescent staining were revealed co-localization between A2B5 positive cells and CNPase positive cells with ASIC1 a. Conclusion: Oligodendrocyte lineage cells could be expressed ASIC1 a whether in vivo or in vitro.The second part the function of ASIC1 a in WML model ratObjective: to study the function of ASIC1 a in WML model rat.Methods: WML were made by CCAO method, intraperitoneal injection of different doses(2,10mg/kg) of the ASIC1 a channels inhibitors amiloride, 30 d. Then the rats are randomly divided into normal group, the WML model control group, 2 mg/kg/d amiloride treatment group(AMI2), 10 mg/kg/d amiloride treatment group(AMI10), each group 5 only.Luxol fast blue(LFB) method is used to test the morphology of myelin in corpus callosum. Diameter of myelin axon, thickness of myelin sheath and G-ratio were observed by ultrastructure electron microscope. In vitro, Acidosis induced OPC injury. Then, it was observed its effects on OPC differentiation.Western blot method was used to detect the expression of MBP, CNPase, caspase3 and ASIC1 a. Results: 1) the LFB staining showed that compared with normal group, WML control group became shallow, optical density value decreased, and axonal injury obviously(P < 0.05, vs the normal group); compared with the control group, AMI2 and AMI10 groups became relatively more dense, LFB staining increased, the difference was statistically significant(P < 0.05 vs. control). 2) Diameter of myelin axon, thickness of myelin sheath and G-ratio were observed by ultrastructure electron microscope. Compared with the normal group, demyelination and reduction of myelin thickness and axon diameter were observed in WML control group by ultrastructure electron microscope. Compared with the control group, AMI2 and AMI10 groups attenuated demyelination and reduction of myelin thickness and axon diameter, and an attenuated G-ratio. 3) immunohistochemistry:Compared with the normal group, the number of APC positive cells was significantly fewer; Compared with control group, in AMI2 and AMI10 groups the number of APC positive cells were increase.4) Compared with the normal group, the expression of MBP, CNPase and ASIC1 a in WML control group were significantly reduced(P < 0.05, vs. normal); The expression of caspase3 was increased(P < 0.05, vs. normal). Compared with control group, AMI2 and AMI10 groups can increase the expression of MBP, CNPase and ASIC1a(P < 0.05, vs.control); the expression of caspase3 was decreased(P < 0.05, vs.control). 5)effects of acidosis contributes to OPC differentiation in vitro:Compared with p H7.0 physiological conditions, in p H6.0 acidosis, the number that OPC were differentiated into CNPase positive cells was significantly fewer( P<0.05, vs. p H7.0).Under the condition of acidosis, the numbers of CNPase positive cells dramatic were increased after amiloride suppress ASIC1 a. Compared with p H6.0 acidosis, the difference was statistically significant(P<0.05, vs. p H 6.0).Conclusion: the use of amiloride inhibit ASIC1 a channels can improve WML, and have the function of the myelin sheath protection。The third part the effect of ASIC1 a on the cognitive function of WML model ratObjective: to study the effect of ASIC1 a on the cognitive function of WML model rat Methods: WML were made by CCAO method, intraperitoneal injection of different doses(2,10mg/kg) of the ASIC1 a channels inhibitors amiloride, 30 d. Then the rats are randomly divided into normal group, the WML model control group, 2 mg/kg/d amiloride treatmentgroup(AMI2), 10 mg/kg/d amiloride treatment group(AMI10), each group 5 only.Morriswater maze test was used to evaluate the ability of learning and memory in rats. Then, werecorded each rat escape latency and escape latent track. Results: During the four days ofthe navigation training, compared with escape latency(10.74±1.469s) in the normal group,the time(22.16±1.978s) in the control group became longer(p<0.05,vs. normal)Compared with the control group, the time(22.16±1.978s) in AMI2 group was revealedshorter but the difference was no statistically difference(p>0.05,vs. control).The time inAMI10 groups is 18.79±1.195 s.It was obviously showed shorten escape latency. The difference is statistically difference(p< 0.05, vs. control). Conclusion: the inhibition ofASIC1 a can improve the ability of learning and memory in WML rats. |
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