| ObjectiveCerebrovascular dysplasia and selective vulnerability of oligoendrocytes are closely related to the occurrence of white matter injury(WMI)in premature infants.However,it is not clear whether endogenous angiogenesis and oligodendrogenesis and the expression of nutritional factors are time-dependent after WMI.Our study aims to establish a neonatal rat model of hypoxic-ischemic WMI to clarify the dynamic change trend of endogenous angiogenesis and oligodendrogenesis,which can help to provide theoretical and experimental basis for further exploring the pathogenesis of WMI and developing therapeutic targeted drugs.Methods1.3-day-old SD rats were randomly divided into WMI group and Sham group.In WMI group,bilateral common carotid artery electrocoagulation combined with hypoxia was used to establish a WMI animal model.In Sham group,bilateral common carotid arteries were only separated without electrocoagulation and hypoxia.2.The growth and development of rats were observed by weight test.The white matter damage of rats after hypoxia-ischemia was determined by MBP immunofluorescence staining and Western Blot.3.Oligodendrocyte lineage markers(Olig2,PDGFRα and APC)antibodies and Ki67(cell proliferation marker)were used for immunofluorescence staining,combined with DAPI for nuclear staining.The number and proliferation of oligodendrocytes at different time points after operation were counted by Image J 6.0software to determine the dynamic changes of oligodendrocytes.4.Vascular endothelial specific markers-platelet endothelial cell adhesion molecule(PECAM 1/CD31)antibody and Ki67 were used to perform immunofluorescence staining,combined with DAPI for nuclear staining.The total amount of vascular endothelial cells and newborn vascular endothelial cells were counted at different time points after WMI to analyze the dynamic changes of the angiogenesis.5.Enzyme linked immunosorbent assay(ELISA)was used to determine the content of BDNF protein in white matter to define the dynamic change of endogenous nutrient factors,which are related to both angiogenesis and oligodendrocyte development.Results1.Evaluation of WMI modelCompared with Sham group,the growth and development of rats in WMI group were delayed,and the time of opening eyes was longer.The weight of rats at post operation day 14(POD 14)was lower than that in the Sham group(P < 0.05).Immunofluorescence staining and Western Blot showed that the expression of MBP in WMI group was significantly lower than that in Sham group at POD 28(p < 0.05).2.Dinamic changes of oliodendrocyte lineagesThe total number of oliodendrocyte lineage in Sham group increased gradually,reached the peak at POD 14,then began to decline,and became stable after POD 28.And the total number of cells at POD 28 was similar to that at POD 3.The number of oligodendrocyte lineages in WMI group increased to a peak at POD 3 and then maintained at this level.The number of oligodendrocyte lines in WMI group was significantly higher than that in Sham group at POD 1 and 3,but was lower than that in Sham group at POD 7 and 14.The change trend of proliferating oligoendrocytes in WMI and Sham group were basically the same with time,which showed a trend of first decreasing,then increasing,decreasing again and finally becoming stable.The number of proliferating oligodendrocytes in WMI group was higher than that in Sham group at POD 1,but significantly lower than that in Sham group at POD 3 and 7.And the number of proliferating oligodendrocytes in both groups reached a peak at POD 7.3.Dinamic changes of oliodendrocyte precursor cellsBoth the number of total OPCs and proliferating OPCs in Sham group decreased at POD 3,increased significantly to the peak at POD 7,then decreased at POD 14,and remained stable at a low level.The change trend of the number of total OPCs and proliferating OPCs in WMI group was consistent with that in Sham group.However,the number of total OPCs and proliferating OPCs in WMI group were significantly higher than those in Sham group at POD 1,but significantly lower at POD 3 and 7.4.Dinamic changes of mature oliodendrocytesThe total number of mature oliodendrocytes in Sham group showed an overall upward trend.But the total number of mature oliodendrocytes in the WMI group was significantly less than that in the Sham group at POD 3,14,28,and it reached the level of the Sham group at POD 56 and 84.In Sham group,the proliferation of mature oligodendrocytes was the most obvious at POD 7,followed by POD 3 and POD 28.However,the proliferation of mature oligoendrocytes in WMI group was less than that in Sham group,and there was no significant change at each time point..5.Dinamic changes of the expression of MBP in white matterAt POD 56 and POD 84,the immunofluorescence intensity of MBP in the corpus callosum in WMI group was significantly lighter than that in Sham group,and WB showed that the level of MBP protein was significantly lower than that in Sham group.6.Changes in total number of blood vessels and neovesselsThe total number of vascular endothelium in Sham group reached the peak at POD 14 and then decreased gradually.While it reached the peak at POD 28 in WMI group and then decreased gradually.At POD 3,the total number of vascular endothelium in WMI group was significantly lower than that in Sham group.The number of neovascular endothelium in Sham group increased at first and then decreased,and reached the peak at POD 14.In WMI group,there was no significant change of neovascular endothelium within 14 days after surgery,which gradually increased from POD 14,reached the peak at POD 28,and then decreased.At POD 1,POD 3,POD 7,POD 14,and POD 56,the number of neovascular endothelium in WMI group was significantly lower than that in Sham group.7.Dinamic changes of BDNF in white matterThe level of BDNF in Sham group showed a overall decreasing trend with time.The level of BDNF in WMI group decreased at first,and then increased,and reached the lowest level at POD 14.The level of BDNF in WMI group was lower than that in Sham group at POD 1,POD 3 and POD 14,but higher than that in Sham group at POD 56 and POD 84.Conclusions1.The oligoendrocyte lineage in the white matter(CC)reached a developmental peak from POD 3 to POD 14(P6～P17).The proliferation and development of OPCS also occurred mainly within 14 days after operation(before P17).The number of mature oligodendrocytes showed an upward trend and their proliferation changed dynamically with time.2.Hypoxia-ischemia affected the process of oligodendrogenesis.It could down-regulate the number of oligodendrocyte lineages.Transient hypoxia-ischemia(1day)could promote the proliferation of OPCs,but hypoxia-ischemia(> 3 days)could inhibit the proliferation of OPCs,and the total number of OPCs decreased significantly after hypoxia-ischemia for 3 to 14 days.Hypoxia-ischemia(< 28 days)reduced the number of mature oliodendrocytes,while long-term hypoxia-ischemia(56days,84 days)restored the number of mature oliodendrocytes,but the myelination was still impaired.3.The total number and proliferation of vascular endothelial cells reached the peak at POD 14(P17).Hypoxia-ischemia delayed the peak of angiogenesis to POD 28,and the angiogenesis was significantly inhibited(within P17).4.The down-regulation of BDNF expression within 14 days of hypoxia ischemia may be an important reason for endogenous angiogenesis and oligodendrogenesis deficiency,while the up-regulation of BDNF expression after 14 days of hypoxia ischemia may be beneficial to oligodendrogenesis and angiogenesis. |
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