Objective: To observe the effect of electroacupuncture(EA) on activities of A2 type astrocytes(A2s)and A1 type astrocytes (A1s) , expressions of neurofilament protein 200 (NF-200, a marker of axon regeneration), nexin 1(NL1, a marker of synaptic regeneration), and regeneration of Nissl bodies in rats with spinal cord injury (SCI), so as to explore its mechanisms underlying improvement of SCI.
Methods: A total of 75 male SD rats were rando-mized into sham operation, model, antibody neutralizing (AN), EA and EA+AN groups, with 15 rats in each group. The SCI model was established by using an infinite field impactor to deliver an about 200 k dyne weight onto the exposed spinal cord after making a dorsal laminectomy at vertebral level T10. EA (2 Hz, 1 mA) was applied to"Dazhui"(GV14) and "Mingmen"(GV4) for 20 min, once daily for 28 days. After modeling, intraspinal injection of neutralizing antibodies IL-1α, TNF-α and complement 1q (C1q, 2 μL) to the injured spinal locus for inhibition of A1 type astrocytes (A1s) was conducted on the 1st, 7th , 14th and 21st day for rats of AN and EA+AN groups. BBB rating scale was used to evaluate hindlimb locomotor function on day 1, 7, 14, 21 and 28 after modeling. The activation of A2s (its specific marker S100a10), astrocyte (its specific marker glial fibrillary acidic protein, GFAP), and A1s (its specific marker C3) in the spinal cord was detected by immunofluorescence, and the protein expressions of NF-200 and NL1 in the spinal cord detected by Western blot and immunohistochemistry, separately, and the neuronal regeneration was observed after Nissl staining.
Results: After SCI, the BBB scores at 1 , 7, 14, 21 and 28 day, and the immunoactivity of NL1 and NF-200 were significantly decreased (P<0.01), and the fluorescence intensity of double labelled S100a10 (A2s)/GFAP and C3, and the expression of NF-200 were considerably increased in the model group (P<0.05, P<0.01). In contrast to the model group, the BBB scores at 7, 14, 21 and 28 day, and the immunoactivity of NL1 and NF-200, and the fluorescence intensity of A2s/GFAP in the AN, EA and AN+EA groups, and the expressions of NL1 in the EA and AN+EA groups, and expression of NF-200 protein in the AN+EA group were evidently increased (P<0.05, P<0.01), and the fluorescence intensity of C3 was strikingly decreased in the EA group (P<0.01). The effect of AN+EA was significantly superior to that of single AN and EA in increasing BBB scores at 14, 21 and 28 day, and in up-regulating the immunoactivity of NF-200(P<0.01, P<0.05). Nissl staining showed damaged structure of the gray matter of the spinal cord, atrophy of the Nissl body, and pyknosis of neurons, which was milder in the AN and EA groups, particularly in the AN+EA group.
Conclusion: EA at GV14 and GV4 may promote activation of A2s and promote regeneration of axons and synapses in SCI model rats.
目的：通过观察督脉电针对脊髓损伤（SCI）大鼠脊髓中A2型星形胶质细胞（A2s）、A1型星形胶质细胞（A1s）和轴突再生标记物神经丝蛋白200（NF-200）、突触再生标记物神经连接蛋白1（NL1）表达及尼氏体生成情况的影响，探讨督脉电针治疗SCI的可能机制。方法：SD大鼠随机分为假手术组、模型组、中和抗体组、电针组、电针+中和抗体组，每组15只。采用无限视野撞击法建立SCI大鼠模型。中和抗体组与电针+中和抗体组在大鼠造模损伤后即刻注射白细胞介素（IL）-1α、肿瘤坏死因子（TNF）-α、C1q 3种中和抗体。电针组与电针+中和抗体组电针“大椎”“命门”，每次20 min，每日1次，连续治疗28 d。分别于第1、7、14、21、28天进行BBB评分；免疫荧光染色法检测各组大鼠脊髓组织A2s特异性标记物S100a10与GFAP共表达情况，A1s特异性标记物C3表达情况；Western blot法和免疫组织化学法检测大鼠脊髓组织NF-200及NL1表达情况；尼氏染色法观察大鼠脊髓神经元再生情况。结果：与假手术组比较，各时点模型组大鼠BBB评分下降（P<0.01）；与模型组比较，造模第7、14、21、28天，中和抗体组、电针组、电针+中和抗体组BBB评分显著升高（P<0.05，P<0.01）。与模型组比较，中和抗体组、电针组、电针+中和抗体组脊髓组织中GFAP与A2s标记物S100a10双标荧光表达强度升高（P<0.05，P<0.01）；电针组A1s标记物C3荧光强度显著降低（P<0.01）。与假手术组比较，模型组脊髓组织NF-200蛋白表达升高（P<0.05）；与模型组比较，电针组与电针+中和抗体组脊髓组织中NL1蛋白表达升高（P<0.05，P<0.01），电针+中和抗体组中NF-200蛋白表达升高（P<0.01）。与假手术组比较，模型组脊髓组织NL1、NF-200阳性表达降低（P<0.01）；与模型组比较，中和抗体组、电针组、电针+中和抗体组NL1、NF-200阳性表达升高（P<0.01）；与中和抗体组比较，电针组和电针+中和抗体组中NF-200阳性表达升高（P <0.01）。与假手术组比较，模型组脊髓组织尼氏体数目减少；与模型组比较，中和抗体组、电针组、电针+中和抗体组尼氏体数目增加。结论：督脉电针能够促进A2s激活，这可能是其促进SCI后大鼠轴突、突触的再生的原因之一。.
Keywords: A2 type astrocyte; Electroacupuncture; Nerve regeneration; Neurofilament protein 200; Nexin 1; Spinal cord injury.