| Objective：To investigate the effects of Necrostatin-1on SCI in rats.Methods：1. Ninety female SD rats were randomly assigned into4groups.The4groups weresham-operation group （Sham group, n=21）,SCI group(spinal cord injury group, T10injury,n=24),NS group(0.9%Nacl group,T10injury+0.9%Nacl,20ul,intrathecal injection,n=21),Nec-1group(Necrostatin-1group,T10injury+20ug/ul Nec-1,20ul,intrathecalinjection,n=24).The10thoracic vertebra (T10) was beaten and injuried by improvedAllen’s Method.2.Observation items:(1)Behavioral assessment:We applied BBB motor function score system in this study,and assessed the score at different times after SCI.(2)Evoked potential examination: the MEP were detected at1d,3d and7d after SCI ineach group.(3)HE dyeing:the samples of spinal cord were obtained at7d after SCI, made frozensection in anteroposterior axes,performed HE dyeing, observed and measured thenecrotic areas of injured spinal cord of the slices.(4)Ponceau2R-brilliant green stain:the samples of spinal cord were obtained at7dafter SCI, made frozen transverse section,performed Ponceau2R-brilliant green doublestain,observed and measured the survival number of neurons and the relative integraloptical density(IOD) of myelin.(5) PI fluorescent staining method:the samples of spinal cord were obtained at24h afterSCI, made frozen transverse section,observed and measured the number of necrosis cell. (6)Receptor interacting protein kinase3(RIP3) concentration:The expression of RIP3inspinal cords removing from rats at24h after SCI was tested by Western blotting.(7) Proinflammatory cytokines(IL-1β、IL-6、TNF-α)concentration:the IL-1β、IL-6、TNF-α concentrationin in spinal cords removing from rats at24h after SCI wasdetected by means of ELISA.(8)Malonyldialdehyed (MDA) concentration: the MDA concentrationin in spinal cordsremoving from rats at24h after SCI was determined with TBA.Results：(1)The experimental animal model established is stable and reliable,presenting agood repeatability.(2)The BBB score was dramatically dropped right after SCI,and then graduallyincreased afterward.After treatment with Nec-1, the score was higher than in the SCIgroup and NS group, as early as the7day（P＜0.001）.It was also higher than in the SCIgroup and NS group at14day、21day and28day after SCI,No statistical differences inSCI group and NS group （P>0.05）.(3)The MEP of all groups had no obvious main reaction within three days after spinalcord injury,which were like the nearly horizontal wavy lines.Compared to the SCI group,the latency of MEP decreased, and the amplitude increased in the Nec-1groups at7dayafter SCI.No statistical differences in SCI group and NS group.(4)Compared with SCI group, the necrotic areas of injured spinal cord decreased in theNec-1groups at7day after SCI（P＜0.01）.In the Nec-1group, the pathological changewere less severe within7day after SCI compared with SCI group,especially thesurrounding area of injured region.(5)Compared with SCI group, the relative IOD of myelin and the survival number ofneurons both increased in the Nec-1groups at7day after SCI（P＜0.01,P＜0.001）(6)Compared with Sham group,the number of PI positive cells and the expression ofRIP3were significantly more in the spinal cord tissues of SCI rats (P<0.001).It wasreduced after Nec-1treatment (P<0.001). Spinal cord tissues RIP3expression and PInumber of positive cells was significantly positively related（r=0.915，P<0.001）.No statistical differences in SCI group and NS group.(7) Low levels of these cytokines（L-1β、IL-6、TNF-α） in the lesion site weredetected in the Sham group.The levels of these cytokines were increased in SCI group,(P<0.001).They were significantly decreased after Nec-1treatment (P<0.001)，althoughstill higher than Sham group(P<0.001). No statistical differences in SCI group and NSgroup.(8)The concentration of MDA was low in Sham group.It was greatly increased in SCIgroup(P<0.001). After treatment with Nec-1, it decreased significantly（P<0.01）, butstill higher than Sham group（P<0.01）.No statistical differences in SCI group and NSgroup.Conclusion：Nec-1has neuroprotective effect on SCI in rats, and it promotes motor function andMEP recovery in rats.Nec-1plays an important role in rats spinal cord injury repair by inhibitingNecroptosis. |
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