Effects Of Combination Therapy With Nogo-A Monoclonal Antibody And Methylprednisolone On Repair Of Spinal Cord Injury

Part I The animal model establishment and the spinal cord histopathologyobservationObjective:To construct an animal model of spinal cord dorsal hemisection in adult rats, and to observe the differences of spontaneous functional recovery and histopathological changes after spinal cord injury(SCI) under different gender. Methods: 40 Wistar rats were randomized into two groups:male group; female group. Constructing SCI dorsal hemisection model(T9). Observing the hindlimb spontaneous function recovery features after injury.12h,Id,3d,7d postoperation, these rats were all killed, the spinal cord including the iniured site were taken out, and HE stain technique was used to observe the histopathological changes with light microscope in different groups. Results:There was significantly haemorrhage, edema, degeneration, vacuole and so on in spinal cord in rats with T9 dorsal transaction. The spontaneous hindlimb function BBB score have no significant difference between male and female rats. There are very close about hindlimb BBB score in all the time among all animals. Hindlimb BBB score of all animals reached about 8 postoperative 4th week, and at later time maintained at the level of 8. But the postoperative mortality and the urinary system complications in female rats were significantly less than male rats. The postoperative care on female rats is more facilitated than male rats. Conclusion:According to change rule of the common organization pathology change in the spinal cord, the acute spinal cord damage animal model established successfully, may be applied to the experimental study of the SCI. Spontaneous rats hindlimb motor function recovery BBB scores have no relation to animal gender. The postoperative care on female rats is more facilitated than male rats, so it is a good choice that applicate female rats to model.spinal cord injury in ratsObjective:To investigate the expressions of a receptor of myelin-associated inhibitory molecule Nogo(NgR) at the mRNA and protein levels after SCI in rats, and evaluate the effect of Nogo antagonistic peptide(NEP1-40) on the treatment of SCI of adult rats model. Methods:108 Female Wistar rats were randomized into three groups:a sham operated group, a trauma control group, a NEP1-40 group. Constructing SCI dorsal hemisection model(T9). NEP1-40 was given through sub-meninx fibrosa tube at a dose of 12.5 ug/PBS (PH7.4),20μl/d for 28 days. the control group was treated with saline injection.24h,3d,7d,14d,28d,42d postoperation, these rats were all killed, the spinal cord including the injured site were taken out. RT-PCR was used to determine the change of NgR mRNA expressions while the immunofluorescence and Western Blot were used to detect the NgR protein in different groups. Results:no significant change of the level of NgR-mRNA and protein was detected at any time points mentioned above in the sham operation group(P>0.05). The level of NgR mRNA started to decrease at the 24th hour after injury and increased again at the 3th day, and increased to the normal level at the 7rd day(P>0.05 vs sham group); Then the expression reached the peak at the 14th day. During the 14th to the 28th day after injury, the expression of NgR mRNA kept high(P<0.05 vs sham group) and decreased to the normal level(at the 42th day) step by step. The level of NgR protein started to decrease at the 24th hour after injury, decreased again at the 3th day, and increased to the normal level at the 7rd day(P >0.05 vs sham group); Then the expression reached the peak at the 14th day. During the 14th to the 28th day after injury, the expression of NgR protein kept high(P<0.05 vs sham group) and decreased to the normal level(at the 42th day) step by step. At the 14th and 28th day the expression of NgR mRNA and protein decreased significantly in NEPl-40 group as compared with a trauma control group(P<0.05). Conclusion:NgR expression has dynamic changes after SCI, and NgR could be as one of the targets for neuroprotective therapy after SCI. NEP1-40 may play a pivotal role in regeneration of the nerve.PartⅢEffect and mechanism of methylpredllisolone on apoptosis andTNF-αexpression after spinal cord injury in ratsObjective:To investigate the effect of high dose Methylprednisolone(MP) on TNF-αcell apoptosis and correlative genes after acute spinal cord injury(ASCI). Methods:124 healthy Female Wistar rats were randomized into three groups:a sham operated group, a trauma control group, a MP group. Constructing SCI dorsal hemisection model (T9). MP was given through vena caudalis at a dose of 30mg/kg within 30 min after the operation, the control group was treated with saline injection.1h,4h,12h,24h,3d,7d postoperation, these rats were all killed, the spinal cord including the injured site were taken out. The terminal deoxynucleotide transferase-mediated DUTP-biotin nick end labeling(TUNEL) was used to observe neural apoptosis; RT-PCR was used to determine the change of Bcl-2,Bax,TNF-αmRNA expressions while the immunohistochemistry and Western Blot were used to detect the Bcl-2,Bax,TNF-αprotein in different groups.Results:It was found that the apoptosis cell can be seen clearly 24h after SCI(P<0.05 vs sham group), which can be prevented after treatment with MP(P<0.05 vs trauma control group). The results of image analysis of Bcl-2 and Bax mRNA and protein showed that the index of Bcl-2/Bax of MP group increased at 4h,12h,3d,7d after SCI(P>0.05 vs trauma control), While at 24h after MP treatment, the index of Bcl-2/Bax of MP treated group increased significantly(P<0.05 vs trauma control group). At 12h after SCI, amount of TNF-αmRNA and protein expression was at the peak value (P<0.05 vs sham group). With times go on, the amount of TNF-αexpression were lower gradually, and at 14d after SCI, the amount of TNF-αexpression were access to the normal level. While after MP treatment was applied, TNF-αexpression decreased significantly(P<0.05 vs trauma control group).Conclusion:Apoptosis is an important event of SCI; Bax/Bcl-2 family take part in the development of SCI through control the process of apoptosis. MP can inhibit the process of apoptosis, regulate the expression of TNF-αafter SCI in rats.monoclonal antibody promotes axonal regeneration and functional recoveryfollowing spinal cord injury in ratObjective:The purpose of this study was to investigate the effects of combination therapy with MP and NEP1-40 on morphological and functional recovery in adult rats subjected to thoracic dorsal hemisection SCI, and discusses its potential mechanisms. Methods:108 Female Wistar rats were randomized into four groups:A trauma control group, an MP group, a NEP1-40 group, an combined treatment group. Constructing SCI dorsal hemisection model (T9). MP was given through vena caudalis at a dose of 30 mg/kg within 30 min after the operation, NEP1-40 was given through sub-meninx fibrosa tube at a dose of 12.5 ug/PBS(PH7.4),20μl/d for 28 days, the control group was treated with saline injection. At the different time point, immunofluorescence was used to detect the GAP-43,MBP,GFAP protein. RT-PCR was used to determine the change of GAP-43,MBP mRNA expressions while Western Blot was used to detect the GAP-43,MBP protein in different groups. The regenerated axons and the myelin sheaths were determined by BDA anterograrle tracing technique and transmission electron microscopy. Hindlimb functional recovery of rats in each group was evaluated by BBB locomotor functional scale. Results:In comparing with other groups, the results of image analysis of GAP-43,MBP protein of the combined treatment group increased at 14d,28d after SCI(P>0.05), the activated astrocyte number(labed with GFAP) and its activating duration of the combined treatment group decreased obviously after SCI(P<0.05). Electron microscopic analysis showed much more axons and better integrity of the myelin sheaths preservation in rats receiving combination therapy(P<0.05). The regenerated nerve fiber stained by BDA was seen in the defected position in MP group and NEP1-40 group, especially in combined treatment group(P<0.05), but almost nothing in the injured site in trauma control group. The effect of MP treatment on BBB score was apparent the day after SCI whereas the effect of NEP1-40 was not apparent until 2 weeks after SCI. Combined treatment with MP and NEP1-40 had a more pronounced effect on recovery of function and axonal growth compared with either treatment alone(P<0.05). Conclusion: These in vitro data support the hypothesis that MP and NEP1-40 will enhance SCI recovery in a sequentially effective manner and suggest that they may have complementary and synergistic neuroprotective effects. Combination therapy with MP and NEP1-40 promotes significantly remyelination and functional recovery following SCI and is an effective approach to treatment of SCI.