摘要
目的:观察FTY720对大鼠急性脊髓损伤(ASCI)后神经功能的影响,并探讨其相关机制。方法:168只雌性SD大鼠,随机分成A、B、C三组,每组56只,A组(假手术组)大鼠麻醉后仅切除T9椎板,不打击脊髓,缝合后立即以0.3ml生理盐水灌胃。B、C组采用Allen′s法制作T9脊髓损伤模型,B组(对照组)以0.3ml生理盐水灌胃,C组(治疗组)以FTY720按3mg/kg生理盐水稀释至0.3ml灌胃。每组取8只大鼠分别于术后1d、3d、7d、14d、21d行斜板试验及BBB评分。分别于术后6h、12h、24h、48h、72h、7d、21d处死大鼠,每个时间点每组8只,取损伤段(A组取相应部位)脊髓行超薄切片,HE染色观察各组脊髓坏死情况、炎细胞浸润情况、胶质瘢痕形成情况及脊髓空洞大小,并计数各组术后12h淋巴细胞数、术后12h与72h炎性细胞、术后7d胶质瘢痕区细胞,计算伤后21d脊髓空洞面积与脊髓面积比值;取术后6h、12h、24h、72h的切片行SP免疫组化染色观察caspase-3表达及Tunel染色观察细胞凋亡情况,计算相应时间点免疫组化染色阳性细胞比值和凋亡指数。所有数据以SPSS 13.0进行统计学分析。结果:B、C两组各时间点斜板实验及BBB评分均较A组同时间点差(P<0.05),在术后1d时B、C组之间无显著性差异(P>0.05),术后3d、7d、14d、21d时C组优于B组(P<0.05)。HE染色结果显示A组各时间点脊髓形态正常;B、C组脊髓术后6h可见脊髓内出血、血肿形成,术后12h^48h脊髓进行性水肿、损伤中心区出现液化坏死,伴有炎细胞浸润,以中性粒细胞、淋巴细胞、单核细胞为主,至术后72h,损伤中心区形成无组织结构空洞,空洞周围有大量炎细胞浸润,以小胶质细胞/单核细胞为主;术后12h及72h,B组炎细胞浸润程度明显重于C组(P<0.05),术后12h C组淋巴细胞浸润程度相对B组明显减少(P<0.05),术后7d,脊髓水肿减轻,空洞周围形成胶质瘢痕,胶质瘢痕细胞计数B组明显大于C组(P<0.05),术后21d脊髓空洞形成,脊髓空洞比值B组明显大于C组(P<0.05)。SP免疫组化染色和Tunel染色结果显示A组各时间点几乎见不到caspase-3和细胞凋亡表达阳性细胞,B、C组脊髓损术后6h即可见凋亡细胞,到术后24h达高峰,而后随术后时间延长而逐渐减弱,但是仍然保持在较高水平;caspase-3表达与细胞凋亡同步,各时间点C组caspase-3表达阳性细胞比值和细胞凋亡指数均显著低于B组(P<0.05)。结论:FTY720可以显著改善大鼠ASCI后神经功能,其可能是通过抑制脊髓损伤后的炎症反应,减少caspase-3的表达及神经细胞凋亡,从而减轻脊髓继发性损伤。
Objectives： To observe the effect of FTY720 on acute spinal cord injury （ASCI） and explore its possible mechanism. Methods： One hundred and sixty-eight pre-numbered Sprague-Dawley rats were randomly divided into three groups of 56 each as group A, B and C. In group A（sham operation group）, rats were administered with 0.3ml saline by garage after laminectomy but without contusion. In group B and group C, animal ASCI model was established by Allen＇s WD method at the T9 level of spinal cord. After operation, rats in group B（control group） were also administered with saline at same dose, and rats in group C（treated group） were administered by garage with 0.3ml FTY720-saline （3mg/kg body weight）. For 8 rats of every group, functional recovery was evaluated after ASCI via inclined plane test and standardized Basso, Beattie and Bresnahan（BBB） locomotor scoring system at ld, 3d, 7d, 14d, 21d. Rats of each group were sacrificed at 6h, 12h, 24h, 48h, 72h, 7d, 21d（n=8 per different time point） after spinal cord injury, and spinal cord tissues were collected and sections were made to do HE stain to observe the necrosis of injured spinal cord, infiltra- tion of inflammatory cells, formation of glial sear and the size of syringomyelia cavities. Then, the numbers of lymphoeytes（12h）, inflammatory cells（12h,72h）, sear cells（7d） and syringomyelia cavity size（21d） were calculated or measured. Sections of the time point at 6h, 12h, 24h, 48h, 72h were made to do SP immunohistochemical stain to observe expression of caspase-3 and Tunel stain to observe apoptosis of cells, then immunostain positive cells were calculated. All the data were statistically analyzed using SPSS 13.0 computer package. Resuits： The BBB scores and the resuhs of inclined plane at the different time points in group B and C were both lower than those in group A（P〈0.05）, but no significant difference between group B and group C at ld after spinal cord injury（P〉0.05）, therefore functions of group C were better than group B at 3d, 7d, 14d, 21d （P〈0.05）. HE stain showed normal in group A at each time point; in group B and C, it showed bleeding and hematoma at 6h, gradual hydroncus, liquefaction necrosis combined with inflammatory cells infiltrating（neutrophil, lymphocyte, mononuclear cells mainly） at 12h to 48h, syringomyelia cavities forming surrounded by lots of inflammatory cells （gitter cells/mononuelear cells mainly） at 72h, the number of inflammatory ceils in group B was significantly more than that in group C at 12h and 72h（P〈0.05）, the number of lymphoeytes in group C was less than that in group B at 12h（P〈0.05）; at 7d, hydroneus decreased, glial scar formed around syringomyelia cavities, the glial scar density in group B was significantly higher than that in group C（P〈0.05）; at 21d, the syringomyelia cavities in group B were significantly larger than those in group C（P〈0.05）. Positive caspase-3 expression or neural apoptosis was not observed in group A at the different time points. The number of apoptotie cells increased with time increasing of acute spinal cord injury, peaked at 24 hours following injury, and then gradually reduced. However, neural apoptosis remained at a high level. Caspase-3 expression positively correlated with neural apoptosis. Caspase-3 expression and neural apoptosis in group C were significantly lower than those in group B （P〈0.05）. Conclusions： FTY720 can inhibit the expression of caspase-3 and the apoptosis of cells after ASCI and provide neuroprotective effects, so as to reduce secondary spinal cord injury and improve spinal nerve recovery.
出处
《中国脊柱脊髓杂志》
CSCD
北大核心
2012年第4期339-345,共7页
Chinese Journal of Spine and Spinal Cord
基金
辽宁省教育厅资助课题(编号:L2010105)
