The Potential Effects Of Bone Morphogenetic Protein Signaling On The Development And Roles Of Neuroglia In Rat Spinal Cord

Section I The expression pattern of the major molecules relevant to BMP signaling pathway in neuroglia during the later embryonic and postnatal rat spinal cord.Objective To observe the expression pattern of the major molecules relevant to BMP signaling pathway during the later embryonic and postnatal spinal cords.Methods Obtained the spinal cord of Fisher344rat at embryos18th day and postnatal0,7,14,21,28,56days. The expression of BMPs, BMPR and pSmad1/5/8on oligdendrocytes and astrocytes in spinal cord was performed by immunohistochemistry.Results1.Embryos18th:BMPR IA mainly expressed in the3CB2marked radial glial which located at central canal and subpial of spinal cord. BMP2and pSmadl/5/8were detected in the neurons of gray matter and central canal.2.Postnatal0-7days:BMPR IA still mainly expressed in the radial glial which located at central canal and subpial of spinal cord, while the intensity was decreased with time. Meanwhile, BMPR IA also emerged faintly in other cells of spinal cord. The expression of BMP2and pSmad1/5/8was increased obviously, mainly present in oligodendrocytes and part of astrocytes.3. Postnatal7-21days: BMPR IA, BMP2and pSmadl/5/8were all highly expressed in mature oligodendrocytes. Among of them, BMPR IA even overexpressed during postnatal14-21days. As for astrocytes, the expression of these molecules was complex:BMP2presented in the majority astrocytes of white matter while BMPR IA only faintly emerged in a small part of cells; pSmad1/5/8stongly expressed in astrocytes of subpial while faintly in other position.4.Postnatal28days up to adult:The expression of almost molecules decreased obviously and maintained at some intensity.Conclusion1.The postnatal period is critical for the development and functional building of spinal cord glia. The high expression of major molecules relevant to BMP pathway on oligdendrocytes and astrocytes indicated BMP signaling pathway play the essential roles on neuroglia genesis of rat spinal cord.2. Meanwhile, these molecules were expressed at low level in adult rat spinal cord, which implied BMP signaling pathway may participate in keeping the biological function of adult glial. Section Ⅱ The effects on glia genesis to interrupt the BMP signaling transductionObjective To explore the effect on glia genesis to interrupt the transduction of BMP signaling pathway via in vitro or in vivo.Methods In vitro:Dissect spinal cord of rat pups and cultured the oligodendrocytes precursor cells (OPCs), and then passaged them for usage. The interruption of BMP pathway was performed through increase the concentration of BMP extracellular or blocked the BMP pathway intracellular by infected with adenovirus carrying Smad6/Smad7. To observe the effects on differentiation, maturation and mylination of oligodendrocytes, the immunocytochemistry and western-blot were performed.In vivo:The selected knockout BMPR1Ain oligodendrocytes related cells in mouse was also selected. To observe the effect of BMP pathway defect on differentiation and maturation of oligodendrocytes, the immunohistochemistry and in situ hybridization were administered.Results In vitro:Treated with0.1ng/ml BMP4protein didn’t alter the percentage of01positive cells. While the proportion of O1positive oligodendrocytes decreased accompanied the increase of GFAP positive astrocytes with the BMP4protein concentration increasing. Infected with adenovirus carrying inhibitor Smad (Smad6or Smad7) to block the BMPs signaling transduction through intracellular, there were no obvious difference on OPCs differentiated into01positive oligodendrocytes when compared with the nomal control and virus control groups. While the MBP protein in oligodendrocytes was decreased. When cocultued with the DRG neurons, the myelin cells were less in Smad6and Smad7groups than the nomal control and virus control groups.In vivo:Selective knockout BMPR1A in oligodendrocytes, the distribution and numbers of olig2or PDGFR a mRNA marked OPCs had no obvious changes. While the MBP and MBP mRNA staining cells or area were decreased in the early stage of postnatal spinal cord.Conclusion1. Increasing extracellular BMP protein concentration inhibited OPCs differentiation and induced the genesis of astrocytes.2. The BMP pathway signaling normal transduction is indispensable to the development and function of oligodendrocytes. Section Ⅲ The effects of different type astrocytes induced by BMP on the survival and neurites outgrowth of DRG neuronsObjective Through observing the effects of different type astrocytes induced by BMP on the survival and neurites outgrowth of DRG neurons, further to discuss the relationships between BMP and the "Two sides" of astrocyte in spinal cord injury.Methods Cultured glial restricted precursor cells (GRPs) and OPCs derived from embryonic and adult rat spinal cord respectively. Differentiated them with BMP4protein and achieved two types of astrocytes, type Ⅰ derived from GRPs (GDA) and type Ⅱ derived from OPCs (ODA). Western-blot was used for detect the expression of fibronectin. And cocultured GDA or ODA with DRG neurons, the immunocytochemistry was performed to observe the survival neurons and outgrowth of neurites.Results The results from immunocytochemistry and western-blot indicated that the expression of fibronectin was significantly higher than GRPs on type Ⅰ astrocyte GDA, while there no obvious singnal was detected on type Ⅱ astrocyte ODA. When cocultured with DRG neurons, the survival neurons in GDA group were much more than the ODA and other groups. At the same time, the neurites outgrowth was also longer than the ODA and other groups.Conclusion BMP induced different precursor cells to generate two types of astrocytes. The promotion effects of type Ⅰ astrocyte on neuronal survival and neurite outgrowth were much better than type Ⅱ astrocyte.