| Cerebral white matter lesion induced by chronic cerebral ischemia is one of the important factors leading to cognitive function impairment in elderly patients,during which the main pathological changes are demyelination and oligodendrocyte loss.Our latest study showed that aspirin,a traditional nonsteroidal anti-inflammatory drug,can promote oligodendrocyte precursor cell proliferation,differentiation and maturation in the corpus callosum through ERK and Rho A pathway sina rat white matter lesion model,but underlying mechanism(s)is still uncovered.Aspirin can reduce the production of prostaglandin by inhibiting cyclooxygenase-1.Prostaglandin was reported to activate the interaction of Ephrin ligand and Eph receptor,one of members of receptor tyrosine kinase family,consequently triggering inflammatory reaction.Additionally,Eph-Ephrin is a joint upstream molecule for regulating ERK and Rho A pathways.Based on these facts,we hypothesize that aspirin may promote oligodendrocyte precursor cell proliferation,differentiation and maturation through inhibiting Eph-Ephrin signaling pathway.Therefore,in this project we will use the morphological and molecular biological methods to study the expression of various Eph and Ephrin molecules on OLs and their precursors,and employ drug intervention and virus infection techniques to investigate whether aspirin can inhibit Eph-Ephrin signal pathway and consequently promote oligodendrocyte precursor cell proliferation,differentiation and myelination in vivo and in vitro.Our results are expected to provide a theoretical basis for the application of aspirin in the treatment of cerebral white matter lesion.Experiment 1 Expression of Eph Receptors in Rat Corpus Callosum and OPC CellObjective: To study the expression of different types of Eph receptors in corpus callosum and OPC cell;Methods: Expression of 14 types of Eph receptors in corpus callosum and multitype Eph receptors in OPC cell were examined in normal rats by real-time PCR;Results: 1)In corpus callosum,all 13 examined Eph receptors(Eph A1-8、Eph B1-4、Eph B6)were found expression except Eph A10.Expression of Eph A4 was the highest,Eph B6、Eph B1 and Eph A7 were also on the high level.While Eph B4 expressed very low.2)In OPC cell,we examined and compared the expression of Eph A4、Eph B6、Eph B1、Eph A7 which were high expression in corpus callosum with Eph A4,the results showed the expression of Eph A4、Eph B6、Eph B1、Eph A7 were still on high level as in corpus callosum and Eph B4 were very low.Expression of Eph A4 was still the highest;Conclusion: The results showed that there are multiple expressed Eph receptors,and the expression of Eph A4 was the highest both in corpus callosum and cultured OPC cell.Which suggest that Eph A4 plays important role in Eph-Ephrin pathway in oligodendrocytes.Experiment 2 Location of Eph A4 in Rat Corpus Callosum and OligodendrocyteObjective:To study the expression and location of Eph A4 receptor in rat corpus callosum and cultured OPC cell,Ols and astrocyte;Methods: Frozen slices of rat corpus callosum were prepared before.Immunohistochemical double-staining was carried out to examine co-location of Eph A4 and OPC marker NG2,mature oligodendrocytes marker MBP or astrocyte marker GFAP in rat corpus callosum.The same experiment also conducted in cultured OPC cell,Ols and astrocyte respectively;Results: In vivo experiment,results showed Eph A4 were co-located with OPC marker NG2,mature oligodendrocytes marker MBP or astrocyte marker GFAP respectively.In vitrohe experiment,the results showed Eph A4 were co-located with OPC marker NG2,OLs marker CNPase or astrocyte marker GFAP respectively.Immunohistochemical double-staining results showed that Eph A4 staining was on neurite and cellular body in oligodendrocyte progenitor cells,mainly on cellular body.Eph A4 coud be stained on both of neurite and cellular body in mature oligodendrocytes.Eph A4 could be dispersed homogeneously on neurite and cellular body in astrocytes;Conlusion: Eph A4 receptor is expressed in OPC,mature oligodendrocytes and astrocytes.Experiment 3 Effect of Aspirin on Expression Eph A4 Receptor in lesions of cerebral white matterObjective: To study the effect of aspirin on expression of Eph A4 receptor in lesions of cerebral white matter;Methods: Rats with WML model using bilateral common carotid artery ligation were treated with low dose(25mg/kg/d)and high dose(100mg/kg/d)of ASA for 28 days.The expression of Eph A4,OPC marker NG2 and OLs marker CNPase in corpus callosum were examined before and after treatment by immunohistochemical double-staining.The protein level of Eph A4,OPC marker PDGFaR and OLs marker MBP in corpus callosum were examined before and after treatment by Western blot;Results: 1)Compared with the normal group,the number of Eph A4 positive cells and NG2 positive cells in corpus callosum significantly increased(p<0.05),while the number of CNPase positive cells significantly decreased in CCAO group.After treated with 25mg/kg/d aspirin in CCAO group,the number of Eph A4 positive cells in corpus callosum significantlydecreased(p<0.05),but NG2 positive cells more increased(p<0.05).Compared with the normal group,the number of CNPase positive cells in corpus callosum significantly decreased(p < 0.05)in CCAO group.The number of Eph A4 positive cells in corpus callosum significantly decreased(p<0.05)after CCAO group were treated with 100mg/kg/d aspirin,while the number of CNPase positive cells remained similar.2)Western blot results showed that the epxression of Eph A4 and PDGFaR at protein level were significantly increased in rat corpus callosum of CCAO group(p<0.05).CCAO with 25mg/kg/d aspirin treated showed lower Eph A4(p<0.05)and more higher PDGFaR(p<0.05).Compared with the normal group,MBP was lowered in rat corpus callosum in CCAO group(p<0.05).Compared with the CCAO group,Eph A4 was significantly decreased(p<0.05)and MBP increased(p<0.05)in CCAO group treated with 100mg/kg/d aspirin;Conlusion: Based on the above results,we could conclude that in lesions of cerebral white matter,the expression of Eph A4 and the number of OPC was significantly increased,while the number of mature oligodendrocytes significantly decreased.ASA could down-regulate the expression of Eph A4.Low dose ASA could increase the number of OPC,but the number of OLs did not changed in high dose ASA.Experiment 4 The mechanism of OPC proliferation affected by Aspirin via Eph A4 ReceptorObjective: To investigate whether aspirin affect OPC proliferation via Eph A4 Receptor and act on directly;Methods: The purified OPC were treated with low dose(0.1mM)ASA and 250 n M Eph A4 inhibitor for 24 h,then counted the number of positive OPCs by immunohistochemical staining.Firstly,a ligand of Eph A4 receptor,radioactive isotope was incubated with isolated Eph A4 receptor in vitro,then ASA or prostaglandin E2 was added,and radioactive isotope values was recorded to caculate binding rate;Results: 1)The results of NG2 immunohistochemical staining showed the total number of OPC was increased significantly in 250nmol/L Eph A4-Fc(p<0.05),and more increased after treated with ASA(0.1mM ASA).2)In ligand receptor binding assay,the binding rate of control group(Ephrin A4,15mM)was 100%,while the binding rate of ASA(10mM)was only 2%,and PGE2(0.5mM)was also only 4.7% which was almost the baseline value;Conlusion: The number of OPC was increased after treated with Eph A4 inhibitor.ASA could increase the number of OPC much more significantly.These evidences suggested that ASA is not bind with Eph A4 receptor directly,but affect the OPC proliferation via other indirect means. |
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