丙戊酸对大鼠急性脊髓损伤后神经功能恢复的影响及作用机制

Effect and mechanism of valproic acid on neurofunctional recovery after acute spinal cord injury in rats

目的:探讨丙戊酸(VPA)对大鼠脊髓损伤(SCI)后运动功能恢复的影响及作用机制。方法:60只雄性SD大鼠随机均分为3组:假手术组(C组)、损伤组(SCI组)和丙戊酸保护组(VPA组)。SCI组和VPA组采用改良Allen法制作大鼠T10 SCI模型。VPA组术后即刻及其后每12h皮下注射VPA 300mg/kg,至取材;C组和SCI组在相应时间点注射等体积的生理盐水。伤后6h,每组取5只大鼠处死取材,其余大鼠在伤后24h、48h和72h每组取5只先行后肢运动功能BBB评分,随后处死取材。切片后分别行HE染色观察脊髓组织病理变化,免疫荧光双标法在激光共聚焦显微镜下观察核因子κB(NF-κB)途径的激活状态,免疫组化法检测白介素1β(IL-1β)的表达。结果:C组大鼠各时间点BBB评分均为21分,VPA组和SCI组各时间点的评分均低于C组(P<0.05),但VPA组各时间点的评分均高于同时间点SCI组,在伤后48h和72h两组差异有显著性(P<0.05)。病理检查显示C组脊髓组织形态正常,VPA组和SCI组伤后6h损伤中央区即可见明显出血灶,灰质中神经元肿胀坏死,白质中神经纤维肿胀;伤后24h、48h出血灶界限更明显,并可见空洞形成和炎症细胞浸润;伤后72h上述病理变化仍明显;VPA组各时间点的病理变化与SCI组相似,但炎症细胞浸润减少。C组偶见或未见NF-κB核阳性细胞和IL-1β表达,与C组相比,SCI组和VPA组NF-κB核阳性细胞百分比和IL-1β表达量从伤后6h即显著性增高,24h达高峰,以后逐渐减少,72h仍显著性高于C组(P<0.05);VPA组各时间点NF-κB核阳性细胞百分比和IL-1β表达量均低于同时间点SCI组(P<0.05)。结论:VPA可促进大鼠SCI后运动神经功能恢复,其机制可能与抑制炎症反应有关。

Objectives： To investigate the effect and mechanism of valproic acid（VPA）on neurofunctional recovery after acute spinal cord injury（SCI） in rats. Methods： Sixty adult male SD rats were randomly divided into three groups： sham operation group, injury group and VPA treated group. Spinal cord injury model at T10 was maded by modified Allen technique. VPA（300mg/kg） was administrated in rats through hypodermic injection immediately after injury, then repeated per 12h until killing; while sham operation group and injury group received the same dose of normal saline at the same time point. The rats of each group （n=5） were killed at 6h after injury. The recovery of the locomotor function of each group（n=5） was evaluated with Basso,Beattie and Bresnahan（BBB） scale at 24h, 48h, 72h after injury, then the rats were killed. The sections were stained with hematoxylin and eosin（HE） for pathological analyses. The activation of nuclear factor kappa B（NF-κB） was examined by fluorescence double-labeling staining technique and laser scanning eonfocal microscope. The expression of interleukin 1β（IL-1β） was detected with immunohistochemistry. Results： The BBB score was 21 in sham operation group, and the score in VPA treated group or injury group was significantly lower than that in sham operation group（P〈0.05）, but the score in VPA treated group was higher than that in injury group at each time point after injury, which showed significant difference at 48h and 72h after injury （P〈0.05）. The pathological analyses showed the rat spinal cord in sham operation group was normal. In VPA treated group and injury group at 6h after SCI, necrotic neurons and hemorrhagic zone were observed in gray matter, and white matter tracts appeared swollen and edematous. At 24h and 48h after SCI, cystic cavities and inflammatory cell invasion were observed, and the hemorrhagic zone became well defined. At 72h after SCI, the pathological changes were still obvious. Compared with injury group, the pathological changes were similar in VPA treated group at each time point, but inflammatory cell invasion was suppressed in VPA treated group. The NF-KB positive nuclei-stained cell and the expression of IL-1β had never been found or occasionally been found in sham operation group. Compared with sham operation group, the percentage of NF-KB positive nuclei-stained cell and the expression of IL-1β both increased obviously at 6h after SCI （P〈 0.05）, which reached peak at 24h, then decreased, and were still significantly higher at 72h after injury（P〈 0.05）; the percentage of NF-KB positive nuclei-stained cell and the expression of IL-l[3 evidently decreased in the VPA treated group compared with the injury group at each time point after injury （P〈0.05）.Conclusions： VPA can promote neurofunctional recovery after spinal cord injury by attenuating inilammatory reaction.