脊髓冲击伤后应用低剂量FK506抑制iNOS表达及神经细胞凋亡

Inhibitive effect of low-dose Tacrolimus on expression of inducible nitric oxide synthase and neuronal apoptosis in rats following contusion spinal cord injury

目的观察大鼠脊髓冲击伤后应用低剂量FK506对诱导型一氧化氮合酶(iNOS)表达及神经细胞凋亡的抑制作用。方法45只成年雄性Wistar大鼠,根据改良Allen法制备大鼠脊髓冲击伤模型。暴露脊髓T10节段,以重量为10g的铁锤从4cm高处自由落下,造成T10段脊髓冲击伤。假手术组5只、对照组和实验组各20只。实验组在脊髓损伤后5min一次性经尾静脉注射FK506(0.3mg/kg),其余两组以相同方法给予0.9%的生理盐水。于伤后不同时间点取材,行苏木精-伊红染色观察损伤脊髓组织病理变化,原位末端脱氧核糖核苷酸转移酶介导dUTP标记技术检测神经细胞凋亡,逆转录聚合酶链反应(RT-PCR)和免疫组织化学染色检测iNOSmRNA和蛋白的表达,同时采用BBB评分法进行后肢运动功能评价。结果实验组病理学观察和行为学评分明显优于对照组(P<0.05,P<0.01);神经细胞凋亡在实验组较对照组明显减少(P<0.05);i-NOSmRNA和蛋白的表达均于伤后7d达高峰,两者表达实验组明显低于对照组(P<0.05,P<0.01)。结论FK506能抑制大鼠脊髓损伤后iNOS表达及神经细胞凋亡,减轻继发性损害从而改善脊髓损伤后的功能恢复。

[Objective] To examine the inhibitive effect of low-dose Tacrolimus on expression of inducible nitric oxide synthase （iNOS） and neuronal apoptosis in rats following contusion spinal cord injury （SCI）. [Methodsl Forty- five male Wistar rats were divided randomly into sham-operation group, control group and experiment group. The rat contusion SCI model was prepared according to the modified Allen method. After exposure of T10 spinal cord, the T10 segment was injured by a 10 g weight falling down from 4 cm high place. The experiment group was injected with Tacrolimus 5 minutes after SCI, while the other groups received 0.9% saline likewise. The BBB scales were used to assess hind limb neurological function at 3, 7, 14 and 21 days after SCI. The expression of iNOS mRNA and protein was detected by using reverse transcription polymerase chain reactions （RT-PCR） and immunohistochemistry staining. The terminal deoxynucleotidyl transferase-mediated deoxyuredine triphosphate-digoxin nick end labeling （TUNEL） method was employed to determine neuronal apoptosis. The pathological changes in injured spinal cord were observed with hematoxylin and eosin staining. [Results] The pathological outcomes and behavioral score of the experiment group were significantly superior to those of the control group. The neuronal apeptosis in the experiment group was significantly less than that in the control group. The peak expressions of iNOS mRNA and protein were si- multaneously observed at 7 days after SCI and the expressions were significantly lower in the experiment group than in the control group. [Conclusion] Tacrolimus could inhibit expression of iNOS and neuronal apeptosis, mitigate seeondary spinal cord damage and thus ameliorate function recovery following SCI.