Receptor Dependent Activation Of PAF Signaling In Reactive Gliosis After SCI

Reactive gliosis, also known as glial scar (GS) formation, is a reactive cellularprocess that occurs after injury in the central nervous system (CNS), involvingreactive astrocyte, activated microglia, fibroblast, endothelial cells, infiltratingimmune cells, and extracellular matrix surrounding the damaged region. Theinflammation seems to be a critical step in secondary degeneration after the CNSinjury and causal to the GS formation. The glial scar forms a physical and molecularbarrier to isolate the injured area from adjacent normal nervous tissue forre-establishing the integrity of the CNS. It prevents the further spread of cellulardamage but represents an obstacle to regrowing axons. Even we paid much emphasisof reactive gliosis on SCI; still know little about the molecular mechanism ofreactive gliosis in SCI.Platelet activating factor (PAF) is a unique phosphoglycerine that mediates thephysiological and pathological process. Under pathological stimulus condition, PAFis released by a wide variety of cells and conduct cellular signal transduction bybinding with PAF receptor in target cells. Previous studies showed that expression ofPAF increase dramatically after SCI, with high level expression of cytokines,intensify local inflammation response that result to severe pathological damage. Atthe same time, application of PAF antagonist can depress the PAF high level andinflammation response, further decrease pathological damage. the newest studyshowed that PAF stimulate GFAP up-regulation in focal brain ischemia model,meanwhile PAF antagonist can reverse these changes.Based on these research background, in this project, we use PAF receptorknock-out mice to set up SCI model in vivo,explore immunofluorescencestaining,axon trace,western blot,Rotarod test technique to observe the effect of PAF signaling on the gliosis activation,glial scar formation,behavior restore from theaspects of morphological changes,protein expression,functional evaluation.Thecompletion of the project will provide new theoretical basement and treatmentpathway for SCI and bring important theoretical and economical value.Method:6-7weeks old wild-type and PAFR-ko mice will be divided into SCI and shamgroups, the SCI group mice will be performed spinal cord dorsal hemisection at C5-6level with0.75mm depth and1.25mm width using the Vibraknife. The spinal cordtissue surrounding hemisection lesion will be dissected and collected as the fresh andfrozen tissue respectively at3,7,14,21,28,42days post injury(dpi).We use IHC andwestern blot technique to detect changes of the microglia and astrocyte activationafter SCI at morphological and protein level. To further evaluate functional recoverybetween the two groups, we use Rotarod test device to detect the locomotive abilityafter SCI at different time point.Result:Plenty of microglia activated in the surrounding of lesion after SCI, theexpression level up to peak at7dpi, and then decrease slowly. Compared to Wt group,PAFR-ko group has more activated microglia surrounding the lesion, at the sametime, Western blot results show that IL-6level has been depressed in PAFR-ko groupat7dpi,it has significant difference compared with Wt group. At42dpi,IL-6leveldecrease dramatically in the two groups, there is no significant difference betweenthe two groups.Astrocyte activated continuously after SCI, GFAP up-regulate and ECMformation increase significantly. At7dpi, compared with Wt group, GFAP expressiondepressed dramatically in PAFR-ko group, there is no significant difference betweenthe two groups. At42dpi, GFAP expression depressed dramatically in PAFR-kogroup, there is no significant difference compared with the sham group, meanwhileGFAP expression still at high level in the Wt group, there is significant differencecompared with the PAFR-ko group. In addition, compared with Wt group at the same timepoint, the CS56immune positive strength decrease obviously, there issignificant difference compared with the Wt group.Rotarod test evaluation between the two groups shows that Rotarod test valuedecline to “0”level compared with sham group, and increase slowly. From14dpi,compared with Wt group, Rotarod test value of PAFR-ko group increase quickly, upto42dpi, Rotarod test performance of PAFR-ko group still better than that of Wtgroup, there is significant difference compared with the Wt group.Conclusion:In CNS, PAF involved in microglia and astrocyte activation process in SCI viabinding PAF receptor. Blocking the PAF signaling depress the activation ofmicroglia and astrocyte, reduce the inflammation response, decrease the glial scarformation and promote functional recover.