Effect Of Myelin Debris On The Phenotypic Transformation Of Astrocytes After Spinal Cord Injury In Rats

Objective:Oligodendrocytes died after traumatic injury of the central nervous system,and myelin debris were released from myelin sheath,while the central nervous system could not effectively remove myelin debris as the peripheral nervous system.The myelin debris continue to exist as inflammatory substances,producing local inflammation,inhibiting axon growth,and causing continuous damage to the central nervous system.There are specialized phagocytes in the central nervous system include microglia in situ and macrophages derived from bone marrow.Ho wever,after spinal cord injury,specialized phagocytes often fail to deal with myelin well: their phagocytosis and removal of myelin debris are limited,and the foam cells formed often further create a more serious inflammatory microenvironment.Astrocytes,as the most abundant glial cells in the central nervous system,have phagocytic function,but the pathophysiological results and mechanism after phagocytosis are still unclear.Astrocytes can be divided into A1s/A2 s or Naive astrocytes(NAs)/Reactive astrocytes(RAs)/ Scar-forming astrocytes(SAs)according to different classification criteria.This study uses the spinal cord injury model in rat to explore the phenotypic changes of astrocytes under spinal cord injury,the difference and connection of different phenotypes of astrocytes and the possible role of astrocytes with different phenotypes after spinal cord injury.Methods: In vivo experiment:1.Healthy female Sprague Dawley(SD)rats with a weight of 180-220 g and 6-8weeks were selected.The non-injured(Pre)group did not undergo experimental intervention.The spinal cord injury(SCI)group used the T10 thoracic spinal cord clamp model.The samples were taken at 4 days post-injury(dpi),7 dpi,14 dpi and 28 dpi.2.The ability of astrocyte to engulf myelin debris after SCI was detected by tissue immunofluorescence technique,and the location and quantity of myelin debris at various time points in the spinal cord injury group were analyzed,as well as the morpholog y and location of astrocyte engulfing myelin debris relative to the lesion core.3.The expression of marker proteins(C3/S100A10)and the percentage of positive cells at each time point after SCI were analyzed by tissue immunofluorescence technique.4.The effect of myelin debris on the transformation of A1s/A2 s was analyzed by tissue immunofluorescence and oil red O(ORO)staining.5.The expression of marker proteins(Nestin/N-cadherin)and the percentage of positive cells at each time point after SCI were analyzed by tissue immunofluorescence technique.6.Analyze the difference and connection between astrocytes of different phenotypes.7.The spatial relationship between astrocytes and neuronal axons at different time points and effects of A1 s and A2 s on the growth of neurons were analyzed by tissue immunofluorescence technology.In vivo experiment:1.Primary astrocytes from rats were cultured to the 3rd to 5th generation(P3-P5),and the cell morphology was observed under inverted microscope.2.Set the control group and the myelin concentration gradient groups of 0.125mg/ml,0.25 mg/ml and 0.5 mg/ml,and observe whether astrocytes can phagocytose myelin by immunofluorescence labeling.Whether the percentage of phagocytic cells and the amount o f phagocytic myelin were related to concentration were analyzed.3.The apoptosis of cells in each group was analyzed by TUNEL fluorescence labeling.Results: After SCI,the myelin sheath was broken,and a large number of myelin debris were present at the injured cord at 4 dpi(P<0.001),while the myelin debris at 7 dpi were significantly reduced(P<0.001).With the passage of time,the area of myelin debris in the injured cord continued to decrease.Astrocytes migrated to the lesion core participating in engulfing myelin debris and the percentage of astrocytes in Z1 and Z3 zones participating in phagocytosis at different time points was significantly different(P<0.001).The phagocyte-related molecule was ATP binding cassette transporter A1(ABCA1).In vitro,the amount of myelin phagocytosis and the number of engulfed astrocytes were correlated with the concentration of myelin(P<0.001).And the conditioned medium of myeli n could induce astrocyte apoptosis(P<0.001).After SCI,astrocytes showed a variety of phenotypes,including: the A1 s expressing C3 existed at 4-28 dpi and reached the peak at14 dpi(P<0.001).It was rapidly induced by myelin debris after injury,which may participate in local inflammatory reaction and guide the growth direction of neurons.The RAs expressing Nestin was abundant at 4 and 7 dpi(P<0.001).As a skeleton protein,Nestin can help us clearly observe that at the early stage after SCI,most astr ocytes were oriented perpendicular to the lesion core,and their arrangement may be related to the early migration to the lesion core.The A2 s expressing S100A10 was abundant at 14 and 28 dpi(P<0.001).As neurotrophic astrocytes,A2 s,whose cell direction was perpendicular to the lesion core and slightly far from the glial scar,may participate in promoting the growth of neurons.SAs was abundant at 14 and 28 dpi(P<0.001).SAs lost its migration ability because of expressing N-cadherin and formed a dense glial scar at the edge of the injured cord.Conclusion:The phenotype of astrocytes after SCI is closely related to the time of injury and relative to the location of lesion core.After SCI,astrocytes do not convert to a certain phenot ype.In general,it may be related to the microenvironment of system.For example,myelin debris can induce the transformation of A1 s.The two classification methods A1/A2 and RA/SA are not contradictory but complement each other.It is impossible to simply identify the state of astrocytes after SCI by a single classification method or even two extremes of one typing method,However,it is significant to clarify the characteristics and functions of each phenotype for future treatment.