| Background:Traumatic spinal cord injury(SCI)is a devastating disease with high morbidity and high disability rate,and effective treatment is not yet available.The pathophysiology of SCI is complex and includes both primary and secondary injury mechanisms.As oligodendrocytes(OLs)injury and demyelination progress continuously during both the acute and chronic phase of SCI,it is an important contributor to dysfunction in SCI.Reducing OLs damage and demyelination have been the subject of exploring therapeutic approaches for SCI.Bone morphogenetic proteins(BMPs)belong to the transforming growth factor-β(TGF-β)superfamily.As a common subtype,BMP7 is widely involved in the development of the central nervous system(CNS).The abundant expression of BMP7 and its receptors in the CNS of adult animals suggests that BMP7 plays an important role not only in embryonic neural development but may also in the adult CNS.Studies have shown that the BMP7 is regulated and plays a neuroprotective role in some CNS diseases,but the specific mechanism is unclear.Our previous study has revealed that BMP7 inhibits OLs apoptosis induced by TNF-α.However,studies on the protective effect of BMP7 on OLs are limited to cellular experiments,animal experiments are lacking to prove the effect in vivo.Therefore,our study intends to explore the expression of BMP7 and its regulatory effect on OLs damage and demyelination in the spinal cord of normal and SCI rats.Our study provides one of the theoretical basis to explore BMP7 as a novel target for OLs damage and demyelination treatment,which could benefit SCI patients.Aim:1.To investigate the expression of BMP7 in the spinal cord of healthy adult rats and explore the role of BMP7 on the OLs survival and myelin sheath.2.To detect the expression changes of BMP7 in the spinal cord of SCI rats and clarify the therapeutic effect of BMP7 on OLs damage,demyelination,and neurological dysfunction caused by SCI.Methods and Results:First part: Downregulation of BMP7 causes oligodendrocytes damage and demyelination in the spinal cord of adult ratsIn this part of the experiment,we firstly took spinal cord sections from healthy adult rats for immunofluorescence experiments to examine the expression and distribution of endogenous BMP7 and p-smad1//5/9 in OLs.Then we injected AAV in the T9 spinal cord of healthy adult rats to mediate sh RNA-BMP7 to downregulate BMP7.After verifying the knockdown efficiency of sh RNA-BMP7,24 rats were randomly divided into the sh RNA-control and sh RNA-BMP7 groups,with 12 rats per group.AAV-sh RNA-control and AAV-sh RNA-BMP7 were injected into the dorsal spinal cord,respectively.After four weeks,animals were sacrificed and the spinal cord was harvested.WB and q PCR were performed to assess the expression of CNPase and MBP.Ultrastructural changes of myelin sheath were evaluated by transmission electron microscopy(TEM).WB and immunofluorescence were used to detect changes in the expression and density of NF200 to define the axon damage.In addition,the animal’s locomotor function was evaluated at indicated times before and after injection of AAV,using Basso,Beattie,and Bresnahan(BBB)scores.Results:1.OLs in the spinal cord of healthy adult rats express both BMP7 and p-smad1/5/9.2.AAV-mediated sh RNA-BMP7 effectively reduced BMP7 expression compared with sh RNA-control.Accompanied by the decreased expression of CNPase and MBP,and thinner myelin with abnormal G-ratios.3.The sh RNA-BMP7 significantly decreased the expression level and density of NF200 in the spinal cord,compared to the control group.Moreover,the BBB scores of animals in the sh RNA-BMP7 group were also significantly decreased at 21 days and 28 days after the injection.Second part: Overexpression of BMP7 alleviated oligodendrocytes damage and demyelination caused by SCIMethods:1.In this part of the experiment,48 rats were randomly divided into the sham group(only laminectomy)and SCI groups,which included 5groups(1d、7d、14d、21d and 28d).Animals in the sham group were allowed to survive for 28 days after the laminectomy.Animals in the SCI groups were sacrificed at 1day、7days、14days、21days and 28 days after SCI.The spinal cord was collected for WB experiments,assessing the relative expression of CNPase and BMP7,to clarify the damage of OLs and the trends in BMP7 changes.2.Then we used AAV to regulate the expression of BMP7 and validated the overexpression of BMP7 mediated by AAV in the spinal cord of SCI rats by q PCR and WB.48 rats were randomly divided into sham group、SCI group、SCI+AAV-GFP group and SCI+AAV-BMP7 group,with 12 rats per group.28 days later,the T9 spinal cord of the four groups was taken and q PCR,WB,and IHC were performed to assess the expression of CNPase、MBP,and APC,to clarify the damage of OLs.Further,the ultrastructural changes of the myelin sheath were observed under a TEM.We next detected the expression changes of the key downstream signaling molecules of BMP7 in the four groups.WB assay was used to determine the changes of the phosphorylation level of smad1/5/9 and STAT3.The expression and location of p-smad1/5/9 and p-STAT3 in OLs were examined by immunofluorescence double staining.Finally,changes of NF200-labeled axonal were detected by WB and immunofluorescence,and the recovery of locomotor function of animals was evaluated by the BBB scores.Results:1.Compared to those in the sham group,the expression of CNPase in the spinal cord of SCI rats was reduced consistently after injury,and the expression of BMP7 was also gradually downregulated with the duration of the injury.2.Compared to the AAV-GFP injected group,AAV-BMP7 injection upregulated the expression level of BMP7 in the spinal cord of SCI rats and increased the expression of CNPase and MBP,and the number of APC-positive cells.Furthermore,the myelin sheath in the spinal cord of AAV-BMP7 injected animals was tighter and thicker,with a smaller G-ratio.3.Compared to those in the SCI+AAV-GFP group,the expression of p-smad1/5/9 and p-STAT3 were both significantly increased in the spinal cord of animals in the SCI+AAV-BMP7 group,accompanied by more p-smad1/5/9 and p-STAT3 positive OLs.4.Rats in the SCI+AAV-BMP7 group showed increased amount and density of NF200,and higher BBB scores at 21 days and 28 days after the AAV injection,compared to the SCI+AAV-GFP group.Conclusion:1.Abundant expression of BMP7 signaling in OLs of healthy adult rats was involved in maintaining the survival of OLs and myelination formation.Knockdown of BMP7 resulted in OLs damage and demyelination,and caused a degree of injury to axons and neurological function.2.After SCI,the OLs damage persisted for at least 28 days,and the expression of BMP7 continued to decrease with the duration of injury.3.Overexpression of BMP7 attenuated OLs damage and demyelination,and the protection of BMP7 to OLs may be achieved by inducing phosphorylation of smad1/5/9 and STAT3 in OLs.Moreover,overexpression of BMP7 alleviated axonal damage and motor dysfunction caused by SCI.These results suggest that,in the spinal cord of SCI animals,BMP7 was also shown to have a protective role to OLs and myelin sheath,indicating that,as a novel target for protection of OLs injury and demyelination,BMP7 has a potential application in the therapy of SCI. |
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