The Effect And Mechanism Of P53 In Demyelination Induced By CSCI

BackgroundCompressed spinal cord injury(CSCI)is a kind of common disorder in nervous system.It is a intraspinal space occupying lesions which is usually caused by ligamenta flava rigidification,vertebral fracture,intramedullary tumors,etc.The clinic manifestations show sensory,motor and musculi sphincter partial or total dysfunctions which will lead to the irreversible loss of function without timely treatment.It not only brings physical and psychology agony to the patient's but also heavy burden to the family and the society.Oligodendrocytes(OLs)are myelin-formation cells in the central nervous system.Myelin sheath which wrapped the neuronal axons is a myelin composed membrane.It supports the axons and surrounding tissues in the nervous system;besides,it can also speed up the action potential with ?saltatory conduction? and guide axonal regeneration when the axon was damaged.Once demyelination occurs,?saltatory conduction? can be blocked,which further influences the function of the nervous system thus affecting a patients' quality of life.So,it is urgent to explore the pathogenesis of demyelination,for seeking an effective preventive measures and restoring the conduction of nerve impulse.To this end,on the basis of modeling CSCI animal and cell models,using transmission electron microscopy(TEM),immumofluorescence,western blot and flow cytometry(FCM)methods,the mechanism of p53 on CSCI is elucidate,for further clarifying the mechanism of demyelination after CSCI,and providing a reliable experimental basis for clinical treatment.Aims1.To explore whether p53 plays a role in demyelination in CSCI.2.Specific p53 inhibitor(PFT-?)was used to intervene the CSCI animal and cell models,for research the possible mechanism of PFT-? in alleviating pathological process during demyelination and further clarify the effect of p53 after CSCI.Methods1.A total of 63 adult female Sprague-Dawley(SD)rats were randomly divided into normal group(n=7),sham group(n=7)and CSCI group,while CSCI group subdivided into CSCI 1h group(n=7),CSCI 3h group(n=7),CSCI 12 h group(n=7),CSCI 1d group(n=7),CSCI 3d group(n=7),CSCI 7d group(n=7)and CSCI 14 d group(n=7).In the normal group,rats received no treatment.In the sham group,rats were subjected to laminectomy without compression.Rats in the CSCI group were subjected to CSCI surgery and compression injury using a custom-made stainless steel compressor for 2h.The Basso,Beattie and Bresnahan(BBB)rating scale was used to assessing rats' locomotor function on day 1,3,7,14 after CSCI,and the osmic acid staining and TEM was used to observed the changes of myelinated nerve fibers in each group.Western blot assays were applied to detected the expression changes of myelin basic protein(MBP),p53,E2F1,active caspase-3,caspase-12 and cytochrome C.Co-expression of p53,E2F1,active caspase-3 and caspase-12,cytochrome C in OLs were detected by immunofluorecence.2.The B104CM(the conditioned medium prepared from B104 neuroblastoma cells)proliferation and EDTA digestion mechanical pipetting methods was applied to culture the oligodendrocyte progenitor cells(OPCs)use neonatal SD rat cortex.The OPCs were then cultured for three days in differentiate solution so as to let OPCs differentiate into OLs.3.After the establishment of CSCI cell model,we devided the cultured OLs into 3 groups: normal group,CSCI cell model group and PFT-? group.Apoptosis and mitochondrial membrane potential were detected by FCM.Immunofluorescence and western blot assays were applied to detected the changes of E2F1,active caspase-3,caspase-12,cytochrome C and CNPase.4.A total of 45 adult female SD rats were randomly divided into sham group(n=15),PFT-? group(n=15)and vehicle group(n=15).The BBB locomotor rating scale method was used to detection locomotor function on day 1,3,7,14 after CSCI of each group.The osmic acid staining and the TEM were used to detected the pathological changes and the number of myelinated nerve fibers on day 7 after CSCI in each group.Immunofluorescence and western blot assays were applied to detected the expression difference of MBP,E2F1,active caspase-3,caspase-12,cytochrome C and CNPase.5.The data were statistically analyzed with SPSS software.Results1.The scores of neurological function assessment in the normal and sham groups were 21,whereas the scores in the CSCI groups were decreased gradually with an increase in time after injury.Compared with the normal and sham groups,the difference is statistically significant(P<0.05).Osmic acid staining and TEM detection revealed normal morphology of spinal cord white matter in the normal and sham groups;myelinated nerve fibers were distributed evenly and myelin sheaths were densely arranged in a regular manner.However,in the CSCI group,the myelinated nerve fibers became swollen,degeneration,and even broken-down.The number of myelinated nerve fibers decreased with an increase in the duration after injury(P<0.05).Immunofluorescence staining confirmed high immunoreactivity of MBP in the sham group.However,MBP immunoreactivity became weak 7 days after CSCI.Immunofluorescence also revealed that the co-expression of p53,E2F1,and active caspase-3 was observed in random locations in the sham group,but it was markedly elevated in the CSCI group on day 7 post injury.Caspase-12 and cytochrome C immunoreactive OLs were rarely distributed in the sham group,but in the CSCI group it was abundant in the entire white matter on day 7.For western blot analysis,MBP expression in the CSCI group was gradually decreased after injury(P<0.05).Expression levels of p53 and E2F1 increased at first and then decreased,though the expression leves were decreased afterward,it was still higher when compared with the normal and sham groups(P<0.05).Western bolt analysis also showed a significant increase of active caspase-3,caspase-12 and cytochrome C at the corresponding time points after CSCI when compared to the sham group(P<0.05).2.Immune-staining of NG2 were applied to assess the purity of OPCs after cultured with B104 CM proliferation and EDTA digestion mechanical pipetting methods and the purification was more than 95%.After induced and cultured in differentiation medium,the purification of OLs was more than 90% after labeled by MBP.3.Apoptosis and mitochondrial membrane potential of OLs in each group were assessed by FCM.The result showed that though the apoptosis rate of cells in PFT-? group was higher than the normal group,it was obviously lower when compared with CSCI cell model group(P<0.05).Mitochondrial membrane potential assessment found that fluorescence intensities in both CSCI and PFT-? groups became weakened when compared with the normal group,but the fluorescence intensities in the PFT-? group was higher than the CSCI group(P<0.05).Western bolt analysis showed a significant increase of active caspase-3,caspase-12 and cytochrome C in both the CSCI and PFT-? group,but the expression levels of these proteins in the PFT-? group grew less than the model group(P<0.05).Immunofluorescence revealed that both caspase-12 and cytochrome C positive CNPase cells were wide-spread in the CSCI group,but their distribution was sparse in the PFT-? group;expression of active caspase-3 in OLs distributed widely in the CSCI group but barely in the PFT-? group.4.After intervened with PFT-?,though the BBB scores was still lower than the sham group,it was obviously higher when compared with the vehicle group(P<0.05);moreover,the number of myelinated nerve fibers was visibly higher than the vehicle group(P<0.05).TEM revealed that,in the sham group,the lamellar myelin that wraps around the axon in the center of the myelinated nerves were routinely found to have the same thickness.However,in the vehicle group,the myelin sheaths became disordered,thin,and even broken.In contrast,the degree of swelling of the myelin sheaths in the PFT-? group was milder;the layers of myelin sheaths were more compact and most of the myelin layers were normal.Western blot analysis showed that MBP expression in the PFT-? group was lower than the sham group,but it was significantly higher when compared with the vehicle group on day 7 after CSCI(P<0.05).Though the levels of E2F1,active caspase-3,caspase-12 and cytochrome C increased in both PFT-? and vehicle groups on day 7 after injury,the growth in the PFT-? group was milder than the vehicle group(P<0.05).Immunofluorescence staining indicated that co-expression of E2F1 and active caspase-3 distributed widely in the vehicle group but barely in the PFT-? group.We also found that both caspase-12 and cytochrome C positive CNPase cells were wide-spread in the vehicle group,but their distribution was sparse in both the sham and PFT-? groups 7 days after CSCI.Conclusions1.Demyelination occured after CSCI,and with the duration after injury,the pathological changes became more and more severe,result in motor dysfunction in rats.The expression levels of apoptosis-related proteins p53,E2F1,active caspase-3,caspase-12 and cytochrome C also up-regulated,with increased OLs apoptosis.2.After intervened with PFT-?,the expression levels of p53,E2F1,active caspase-3,caspase-12 and cytochrome C were down-regulated with reduced OLs apoptosis and alleviated demyelination.3.It was manifested that over-expression of p53 was involved in demyelination after CSCI.Over-expression of p53 enhanced E2F1 expression which lead to apoptosis.It could also enhance ER stress,cytochrome C mediate mitochondrial apoptosis pathway and ER-mitochondria interactions at the same time,and then initiates apoptosis among OLs,which finally causes demyelination.