Experimental Study On Abnormal Myelination In Newborns After Hypoxia-Ischemia Brain Damage

OBJECTIVE:To clarify the dynamic changes of OLs during the early development of C57BL/6J mice brain and the spatiotemporal changes of myelination and to provide a laboratory evidence for the mechanism of abnormal myelination after Hypoxia-Ischemia Brain Damage(HIBD).METHODS:1.The neonatal C57BL/6J mice were divided into 7 groups,as P 1,P 3,P 5,P 7,P 10,P 14 and P 28 time points.Immunofluorescence staining was used to observe the expression of Olig2 marker in OLs lineage and of MBP marker in mature OLs.2.Luxol Fast Blue staining was used to observe the changes of brain myelination in each group.3.The HIBD model of C57BL/6J mice was established by the classic Vannucci modified method,and the damage degree of brain tissue after operation was evaluated by gross observation,TTC staining,laser speckle cerebral blood flow imaging and HE staining results.4.Luxol Fast Blue staining was used to observe the changes of myelination in the brain tissue after HIBD.5.Immunofluorescence staining and Western-blotting were used to observe the possible causes of changes in myelination in the brain tissue after HIBD.6.After extracting and culturing mixed cells from cerebral cortex of neonatal mice d0,OPCs were isolated and purified and cultured in primary culture.The morphology of purified cells was observed under optical microscope.7.Carry out OGD experiment on the purified primary OPCs,and observe the morphology of primary cultured OPCs after OGD experiment by microscope.8.OLN93 cells were subjected to OGD experiment,and the proliferation capacity of cells in the normal group and the OGD experimental group was detected by flow cytometry.RESULTS:1.Olig2 positive cells were observed d1 after birth,aggregated in the corpus callosum,and almost evenly distributed in other locations;The expression of MBP first occurred d7 after birth.MBP staining of corpus callosum manifested that the array of MBP fibers were incompact and extends to the cortex outside the corpus callosum,while MBP was spotted in the striatum.At 28 days after birth,MBP at the corpus callosum was densely flaky,short and uniformLy distributed in the cortex.2.The staining of Luxol Fast Blue only occurred d28 after birth in these groups and always tight and strong in corups callosum and also spotted in striatum.No myelination formed at other time points.3.After the first and third day of HIBD,compared with the normal group(N),on the ipsilateral side of the brain tissue of a group of mice,white opaque areas can be seen in gross observation,white infarcts can be seen by TTC staining,laser speckle cerebral blood flow imaging showed a significant decrease in cerebral blood flow(p<0.05)and HE staining showed significant liquefaction necrosis,which we classify as severely injured HIBD group(HI-S).Another group of mice did not show any significant difference on the ipsilateral side of brain tissue,then we classified it as a mild injury HIBD group(HI-M).4.After the eighteenth day and the ninth month of HIBD,the brain tissue of HI-M and HI-S groups mice showed ruptures of myelin sheath of corpus callosum on the ipsilateral side.5.After the first and third day of HIBD,there was no significant change in the number of Olig2 positive cells and in MBP morphology on the ipsilateral side of brain tissue in the HI-M group.And in the HI-S group,the number of Olig2 positive cells increased(p<0.0002)in the ipsilateral side,MBP was partially broken,and the arrangement was loose and disordered.After the ninth month of HIBD,there was no significant change in the number of Olig2 positive cell on the ipsilateral side of the brain tissue in HI-M group and HI-S group,but the morphology of MBP changed,and the degree of MBP morphological change in HI-S group was severe than that in HI-M group.6.After the first day of HIBD,there was no significant change in the morphology of F-actin on the ipsilateral side of brain tissue in HI-M group,and also no significant change around the infarct area on the ipsilateral side of brain tissue in HI-S group.7.After the third day of HIBD,the MBP expression in the corpus callosum of Hl-M and HI-S groups was gradually decreased,the expression of Tau5 was gradually increased and pT205 was gradually increased.8.Most of the purified OPCs were bipolar,and a few were tripolar or multipolar by microscopy.9.OLN93 cells of the proliferation cycles in OGD group had fewer than those in normal group.CONCLUSION:1.Immature OLs develop from 1 to 7 days after birth,the OLs matured after the seventh day of birth,and the mature myelin sheath can be observed in the brain after the twenty-eighth day of birth.2.The normal formation of myelin sheath is affected after HIBD of C57BL/6J mice.3.Tau5 and pT205 in C57BL/6J mice are involved in the process of abnormal myelination after HIBD of C57BL/6J mice.4.The primary cultured OPCs were damaged by oxygen-glucose deprivation experiment,so it is speculated that this affects the normal myelination process.5.Increase of Olig2 positive cells failed to change the abnormality of myelination.