The Neuroprotective Effect And Its Mechanism Of Peroxisome Proliferator Activated Receptor-γ Sci In Rats

Neuroprotection treatment is applied before or after nerve injury by means of medicine or some special procedure to minimize the degree of nerve injury.There are many aspects of prevention and cure for spinal cord injury (SCI),including prevention for secondary lesion,neuroprotection for subacute and chronic stage,neuraxis and neuron regeneration and reinnervation obtaining etc.,and there are many issues remained unclear in each aspect.In the past two decades, researchers in and abroad did a lot experimental and clinical studies to investigate the biology mechanism of SCI,which led to a more profound understanding of SCI on cellular and molecular level.Based on these researches,this study is to explore new neuroprotection treatment for improving the prognosis of SCI. The ability for spinal cord to autochthonous renovation is much limited after spinal cord injury(SCI),the dead neurons can't be replaced by the self-poietic or the adjacent neurons. The cilinical treatment at present is to contorl the aggravation of the secondary lesion. How to promot the anagenesis and renovation of the injuried spinal cord effectually has been the puzzle of the neuroscience realm. The research of the neural stem cells (NSCs)recently has changed the standpoint.The neural stem cells can differentiate into various composition of nervous system, such as neuron,astrocyte,oligodendrocyte etc.,which can promote the regeneration and renovation of spinal cord. The therapy of SCI by NSCs is an important method for neuroprotection and renovation,which is different from the traditional methods, it has provoked the people's interesting and attention more and more in recent years, it has been the focus of the neuroscinece research realm. The research found that the activation of peroxisome proliferator activated receptor y(PPARy)can promote the differentiation and ripening of the neural stem cells[1],it has showed favourable perspective in the therapy of central nervous system injury. The research of PPARγin the neuroprotection after SCI is in exploration stage at present. This study utilized SD rat as a SCI model, to research the chan-ges of PPARy expression following the secondary injury after SCI in rats,and the influence of hyperbaric oxygen precondition(HBOP) to PPARy expression,to research the neuropro-tective effect and its mechanism of PPARy on SCI, and also evalutaed the curative effect of HBOP and PPARy excitomotor therapy for SCI.There are four parts in this study as follows:1 The changes of PPARy expression following the secondary injury after SCI in rats,and the influence of HBOP to PPARy expression.ObjectTo establish an animal model for SCI and HBOP, investigate the changes of PPARy expression following the secondary injury after SCI in rats,and the influence of HBOP to PPARy expression.Method1) Animal model establishment for HBOP:SD male rats were divided into theree groups randomly,and preconditioned for 5 days in hypobaric chambers.2) SCI animal model establishment:Micrurgy were applied on the rats under anaesthesia to expose canalis spinalis and dural sac,utilizing a 30g lash stick to strike on the spinal cord from a height of 5cm.The inferior extremity of the lash stick is 3 mm in diameter,with a shock energy of 150 gcf.3) Observation items: quantitative observation for PPARymRNA and protein.4) Data were analyzed by using SPSS 16.0 statistics software.Result1) The experimental animal model established is reliable and stable,presenting a good repeatability.2) PPARy expression began to increase at 1d after SCI, and reached the peak at 14d, which is higher than control group,the statistic difference is significant.3) PPARy expression began to increase at 1d after SCI in HBOP rats, and reached the peak at 14d,which is higher than SCI group,the statistic difference is still significant at 28d.Conclusion1)the rat SCI model is resemblant with human SCI,which is reliable with an insignificant individual difference and a good reproducibility,and a simple preparation process.2) PPARyexpression increased obviously after SCI,and keep high lever in long time.3)HBOP promoted the expression of PPARy obviously, the time of high lever expres-sion is prolong obviously.2. Neuroprotective effect of PPARy on SCI in rats ObjectTo investigate the impact of PPARy on the adult rats in different preriod after acute SCI,and evaluate the neuroprotective effect of PPARy in SCI.Method1) Adult SD male rats was divided into spinal cord injury group,rosiglitazone theraphy group,GW9662 theraphy group.2) The treatment group was treated with PPARy excitomotor or antagon respectively one week by intraperitoneal injection.3) Observation items:(1) Behavioral assessment:We applied BBB motor function score system in this study, and assessed the score at 1d,1w,2w,4w,and 8w after SCI.(2) Evoked potential examination:Somatosensory evoked potentia(SEP) and motor evoked potential(MEP)were detected at different time after SCI in each group.(3) HE dyeing,Nissl's staining and observation under light microscope:the samples of spinal cord were obtained at different time after SCI, made paraffin section in anteropos-terioraxes.(4)Nerve-tract tracing:anterogradely traceing were performed using biotin dextran amine(BDA),observed the regenerated neuraxis ascendingly and descendingly,so as to identify the effect of PPARy excitomotor and antagon on neuraxis regeneration.4) Data were analyzed by using SPSS 16.0 statistics software.Result1)The experimental animal model established and intraperitoneal injection is reliable and stable,presenting a good repeatability.2) The BBB score was dramatically dropped right after SCI,and then gradually increased afterward.The score of rosiglitazone therapy (RT) groups were higher than the spinal cord injury(SCI) groups,but the GW9662 therapy(GT) groups were lower than SCI groups, the statistic difference among them were significant.3) SEP and MEP showed a prolonged lantency after SCI in all groups,the lantency in RT groups were shorter compared with SCI groups, GW9662 groups were longer compared with SCI groups and there were significant differences among them.The SEP and MEP were relatively stable 4w after SCI.4) light microscope observation:In SCI group, the central gray of injured region appeared hemorrhage,cellular swelling,tigroid body abolition,and a great quantity of mononuclear or polynuclear inflammatory cells infiltration at 1d after SCI;Necrosis focuses appeared in injured region at lw after SCI,and the inflammatory reaction was stronger;the inflammatory cell infiltration began to abate at 2w after SCI in injured and peri-injured region;capsular space-like change occurred in injured myeloid tissue at 4w after SCI, and at 8w after SCI glial scar became the major component. In RT groups,the pathological change was less severe within 2w after SCI compared with SCI group,especially the surrounding area of injured region,and no significant difference were found after 4w after SCI,but in GT groups,the pathological changes were more severe within 2w after SCI compared with SCI groups,the changes were obvious after 4 weeks.5) Nerve tract tracing revealed that the number of regeneration neuraxis ascending and descending increased in RT groups, glial scarwas much lesser than SCI groups,but the number of regeneration neuraxis ascending and descending decreased obviously in RT groups, glial scarwas was much more than that of SCI groups.ConclusionCompared with the SCI group,rats in the RT groups present a higher BBB score,a shorter lantency of SEP and MEP,a less severe pathological change, there was more survival Nissl body and, an increased regeneration neuraxis etc., which indicate a neuropro-tective effect of PPARγon rat SCI,but the GT group was contrary, which indicate a aggrevation effect of PPARγon rat after SCI.3.Mechanism of the neuroprotective effect of PPARγin rat SCI.ObjectTo investigate the neuroprotective mechanism of PPARγin rat SCI,so as to provide rationale basis for its prevention and cure.MethodAfter divided into different groups preparation for SCI model, samples were collected at different time after SCI,then immunofluorescence stain were performed to research the influence of PPARγto stem cell multiplicationll differentiate into neuroprecur -oprecursor cell,astrocyte,oligodendrocyte,neuron.Data were analyzed by using SPSS 16.0 statistics software.Observation items:1)immunofluorescence stain of Nestin+Brdu for neuroprecuroprecursorcell obser-vati-on.2) immunofluorescence stain of MBP+Brdu for oligodendrocyte observation.3) immunofluorescence stain of GFAP+Brdu for astrocyte observation. 4) immunofluorescence stain of NSE+Brdu for neuron observation.Result1) In SCI group,a few nestin immunostaining positive cells were observed at 1w after SCI in spinal cord parenchyma,and at 2w after SCI,a large amount of nestin-positive cells were seen.Most nestin-positive cells were polygon or orbicular-ovate in cell body with a lot of processes, which extended coincidently with the cell long axis;a minority of nestin-positive neurons were found in the vicinal gray matter of injured region.Nestin-positive cells were obviously decreased at 4w after SCI.In the RT groups, nestin expression was stronger than that in control group after SCI,but in the GT groups the result is contrary.2) we observed that neogenetic oligodendrocyte gradually increased in paren chyma-tous of spinal cord injury in SCI group at 3d, and it increased obviously at lw,and reached the peak at 2w.The neogenetic oligodendrocyte in RT group is much more than SCI group, but the GT group is contrary.3) GFAP expresses in both gray matter and white matter in normal spinal cord.In the SCI group,GFAP expression began to increase surrounding the injured region at Id till 2w after SCI,especially in the ependyma of spinal medullary canal.Capsular space began to form 4w after SCI,and GFAP expression decreased and mainly centered in the glia cell body and process surrouding the capsular space.GFAP expression in RT group increased in the first and the second week after spinal cord injury than SCI group, but the GT group is contrary.4) Few of neogenetic neurons were found in perimeter region of pars affecta,but there was no neogenetic neruons in the injuried part. The neogenetic neruons of RT group increased obviouly at 3d after SCI,but the GT group is contrary,the statistic difference is significant among them. The expression of neogentic neuron reached the peak at 2w afer SCI, and it gradually decreased at 4w,but there still was statistic difference.Conclusion1) Rosiglitazone has an effect of neuroprotetion by promoting the differen-erentiation of neuro stem cells,but GW9662 inhibited the process,aggrevated the injury of the spinal cord.2) In the early state after SCI(within 4weeks after SCI), rosiglitazone could promote nerve precurosor cell regeneration so as to protect neuron,but GW9662 could inhibit the process.3) In the early state after SCI(within 4weeks after SCI), rosiglitazone could promote oligdendrocyte regeneration so as to protect succession of nerve conduction,but GW9662 could inhibit the process.4) In the early state after SCI(within 2weeks after SCI), rosiglitazone could promote neogenerate astrocyte so as to protect neuron.5) In the early state after SCI(within 4weeks after SCI), rosiglitazone could promote neogenesis nuron generate,but GW9662 could inhibit the nerve recovery after SCI.4.Evaluation of curative effect of HBOP and Rosiglitazone on experimental rat SCIObjectTo evaluate the curative effect of HBOP and rosiglitazone on rat SCI and provide reliable evidence for its clinical useage.Method1)Rats were divided into 3 groups:SCI with HBOP group, SCI with rosiglitazo-ne therapy group,SCI with HBOP and rosiglitazone therapy group.2) Observation items:(1) Behavioral assessment:BBB motor function score system was utilized in this study, and assessed the score at 1 w,2w,4w,and 8w after SCI.(2) Evoked potential examination:Somatosensory evoked potentia(SEP) and motor evoked potential(MEP)were detected at different time after SCI in each group.3) Data were analyzed by using SPSS 16.0 statistics software.Result1)BBB score in SCI with HBOP and rosiglitazone therapy groups are higher than the other two group,and the difference between them is significant.2) SEP and MEP showed a prolonged lantency after SCI in all groups, the lantency in HBOP and rosiglitazone therapy groups is shortened compared with the other two group,and the difference in group is significant.ConclusionThe neuroprotect effect of HBOP and rosiglitazone therapy after SCI is obviouly,is an important strategy for prevention and cure of SCI.