脊髓损伤后延迟给予腹腔注射依那西普的疗效

Efficacy of delayed administration of etanercept after spinal cord injury

目的:观察大鼠在脊髓损伤后延迟给予依那西普的后肢运动功能恢复和脊髓组织凋亡相关基因表达情况,并观察脊髓组织的病理改变。方法:72只成年雄性SPF级SD大鼠,随机分为6组(实验组为E1组、E2组、E3组,对照组为C1组、C2组、C3组),每组12只,采用70 g动脉瘤夹钳夹脊髓1 min制作钳夹型损伤模型,分别在损伤后即刻、1 h、8 h对实验组(E1组、E2组、E3组)予以腹腔注射依那西普1.25 mg/kg,对照组(C1组、C2组、C3组)同样时间点予以腹腔注射生理盐水1 mL,每组取6只大鼠在损伤24 h后处死取脊髓组织,用Western blot法测定损伤处Bax和Bcl-2表达情况,其余大鼠饲养14 d,期间每日予以Basso Beattie Bresnahan(BBB)运动功能评分,并在第14天处死取脊髓组织制成HE染色切片,光镜下观察。结果:(1)Bax和Bcl-2表达情况:E1组Bax表达灰度值为165.423±2.946,E2组为135.391±3.045,E3组为108.543±6.999,对照组为69.054±0.774;E1组Bcl-2表达的灰度值为58.854±3.592,E2组为84.315±2.138,E3组为125.091±2.699,对照组为156.304±2.490,以上各组数据差异均具有统计学意义(P<0.01)。(2)BBB评分结果:损伤后即刻或1 h给予依那西普可以明显改善大鼠BBB评分的恢复情况,进一步比较第14天BBB评分数据,E1组评分平均值为13.000±1.095,C1组为7.167±0.753,E2组为9.833±1.472,C2组为7.000±0.632,以上各组数据差异均有统计学意义(P<0.05),而E3组为7.333±0.516,C3组为6.833±0.753,两组差异无统计学意义(P>0.05)。(3)病理切片结果:光镜下观察E1组和E2组大鼠脊髓病理切片可见散在神经元细胞体,淋巴细胞、成纤维细胞浸润较少,组织坏死程度轻,而E3组和对照组可见大量淋巴细胞和成纤维细胞浸润,组织坏死严重。结论:脊髓损伤后注射依那西普可能会起到抑制脊髓组织凋亡的作用,但给药时间延迟超过8 h会显著降低其凋亡抑制作用,无法显著改善大鼠脊髓病理改变和促进后肢功能的恢复,依那西普用于脊髓损伤的实际临床价值仍有待进一步验证。

ABSTRACT Objective： To observe the effect of delayed administration of etanercept on the motor func- tion, the expression of apoptosis-related genes and the pathological alterations of spinal cord in vivo in ex- perimental murine model of spinal cord injury （SCI）. Methods： Seventy-two male adult SD rats were randomly divided into 6 groups, which were subjected to SCI induced by the application of vascular clips （force of 70 g） to the dura. Experimental groups （El, E2, and E3 group） were given administration of etanercept immediately, 1 h, and 8 h after SCI. The control groups （ C1, C2, and C3 group） were given administration of saline at the same time as experimental groups. Six rats of each group were killed 24 h after SCI in order to collect the samples for testing the expression of Bax and Bcl-2 by Western blot. The rest were killed 14 d after SCI for observing the pathological alteration using light microscopy. The recovery of motor function was graded using the modified murine Basso, Beattle, and Bresnahan （BBB）. Results： （ 1 ） The results of the expressions of Bax and Bcl-2 by Western blot ： the gray value of the ex- pression of Bax of E1 group was 165. 423 ±2.946, of E2 group 135. 391 ±3. 045, of E3 group 108. 543 ± 6.999, and of the control group 69.054 ± 0.774, and the gray value of the expression of Bcl-2 of E1 group was 58. 854 ±3. 592, of E2 group 84. 315 ±2. 138, of E3 group 125. 091 ±2. 699, and of the control group 156. 304 ±2. 490. （2） The results of BBB score： etanercept given immediately or 1 h after SCI could improve the recovery of the rats. There were significant differences in BBB score 14 d after SCI be- tween E1 group （13.000 ±1.095） and C1 group （7. 167 ±0.753）, E2 group （9.833 ±1.472） and C2 group （7. 000 ± 0. 632） while there were no significant difference between E3 group （7. 333 ± 0. 516 ） and C3 group （6. 833 ± 0. 753 ）. （3） The result of histological alteration ： histological alterations, suchas necrosis, infiltration of lymphocytes and fibroblast and loss of nerve cells, were found attenuated in E1 and E2 groups, compared with C1 and C2 groups. There was no obvious difference between E3 and C3 groups. Conclusion： Administration of etanercept may inhibit the apoptosis after SCI, but this kind of effect may be too weak to improve the BBB score and histological alterations obviously when administration of etanercept is delayed 8 h after SCI. The clinical value of etanercept to SCI needs to be further vali- dated.