Tan Cheng-Fang, Huang Si-Qin, Tang Cheng-Lin, Zhang An-Ning, Zhao Dan-Dan, Wu Meng-Jia, An Hui-Yu, Qiu Li, Dai Ni, Dai Pan
College of Traditional Chinese Medicine, Chongqing Medical University, Chongqing 400016, China.
Zhen Ci Yan Jiu. 2019 Jun 25;44(6):391-8. doi: 10.13702/j.1000-0607.180780.
To observe the effect of electroacupuncture (EA) combined with transplantation of Schwann cells (SCs) on limb locomotor, myelin sheath repair and expression of CD4 and CD8 in compressed spinal cord injury (CSCI) rats, so as to explore its mechanisms underlying improvement of CSCI.
A total of 45 female SD rats were randomly divided into normal control, model, EA, Schwann cell (SC) transplantation, and EA＋SC transplantation groups (＝9 rats in each group). The CSCI model was established by laminectomy at T12-L2 and clip compression. Rats of the SC transplantation group accepted injection of the cultured SC suspension (2×10/6 µL) into the central, upper and lower sites of the injured spinal cord (5 mm in depth) 7-8 days after CSCI modeling. EA (2 Hz) was applied to bilateral "Zusanli" (ST36) and "Sanyinjiao" (SP6) for 10 min, once daily and 6 days a week for 3 weeks. The Basso, Beattie and Bresnahan locomotor rating scale (BBB scale) was used to evaluate the function state of CSCI. Morphological changes of the regional injured tissue were observed under light microscope after H．E. staining. The myelin sheath repair state and survival of SCs were detected by Luxol fast blue (LFB) staining and immunofluorescence histochemistry, and the expression of CD4, CD8 and P0 of the injured spinal cord was detected by Western blot.
Compared with the normal control group, the BBB scores at the time-points of 0 d, and 1, 2, and 3 weeks were significantly decreased in the model group (<0.001), and those of the EA＋SC transplantation group at the 2 and 3 week were significantly higher than those of the model group (<0.05). No significant changes of BBB scores were found after EA and SC transplantation relevant to the model group (>0.05). LFB staining showed a disordered arrangement of the nerve fibers in the white matter, myelinociasis and obvious decrease of the medullated fibers in the model group, and these situations were relatively milder in both EA and SC transplantation groups and obviously milder in the EA＋SC transplantation group. H．E. staining displayed that the structure of the injured region of the spinal cord was incomplete, accompanied with a large number of defect cavities and neuronal karyopyknosis in the model group, while the structure was relatively clear, with an increase of the normal neurons and fewer neuronal karyopyknosis in the EA＋SC transplantation group. Compared with the normal control group, MBP in the model group was significantly decreased (<0.001)，and P0 was significantly increased (<0.001). Compared with the model group, the expressions of MBP and P0 were significantly increased in the EA, SC transplantation, and EA＋SC transplantation groups (<0.01, <0.001), and was significantly higher in the EA＋SC transplantation group than in both EA and SC transplantation groups (<0.001). The average immunofluorescence intensity of Hoechst33342-labeled SCs was significantly higher in the EA＋SC transplantation group than in the SC transplantation group (<0.05). After CSCI, the expression levels of spinal CD4, CD8 and P0 proteins had no significant changes in comparison with the normal control group (>0.05), while after the intervention and in comparison with the model group, the expression levels of P0 protein were significantly increased in the EA, SC transplantation and EA＋SC transplantation groups (<0.05), and was significantly higher in the EA＋SC transplantation group than in both EA and SC transplantation groups (<0.05). The expression levels of CD4 and CD8 proteins were significantly lower in the EA＋SC transplantation group than in the SC transplantation group (<0.05)．.
EA＋SCs transplantation can improve the locomotor function in CSCI rats, which may be related to its effects in increasing the survival of transplanted SCs to promote the remyelination and in reducing the immune rejecting reaction.
观察电针（EA）联合雪旺细胞（SCs）移植对脊髓压迫性损伤（CSCI）大鼠肢体运动功能、髓鞘修复及CD4和CD8表达的影响，以探讨其改善CSCI的机制。
将45只雌性SD大鼠随机分为正常对照组、模型组、电针组、雪旺细胞移植组和电针＋雪旺细胞移植组（每组9只）。通过T12-L2椎板切除术和夹闭压迫建立CSCI模型。雪旺细胞移植组大鼠在CSCI建模后7-8天，于损伤脊髓的中央、上下部位（深度5 mm）注射培养的雪旺细胞悬液（2×10/6 μL）。采用2 Hz电针刺激双侧“足三里”（ST36）和“三阴交”（SP6），每次10分钟，每日1次，每周6天，共3周。采用Basso、Beattie和Bresnahan运动评分量表（BBB量表）评估CSCI大鼠的功能状态。苏木精-伊红（H.E.）染色后在光镜下观察局部损伤组织的形态学变化。采用Luxol固蓝（LFB）染色和免疫荧光组织化学检测髓鞘修复状态和雪旺细胞存活情况，采用蛋白质免疫印迹法检测损伤脊髓中CD4、CD8和P0的表达。
与正常对照组相比，模型组在0天、1周、2周和3周时的BBB评分显著降低（<0.001），电针＋雪旺细胞移植组在2周和3周时的BBB评分显著高于模型组（<0.05）。电针组和雪旺细胞移植组与模型组相比，BBB评分无显著变化（>0.05）。LFB染色显示，模型组白质神经纤维排列紊乱、髓鞘脱失，有髓纤维明显减少，电针组和雪旺细胞移植组上述情况相对较轻，电针＋雪旺细胞移植组明显更轻。H.E.染色显示，模型组脊髓损伤区域结构不完整，伴有大量缺损空洞和神经元核固缩，而电针＋雪旺细胞移植组结构相对清晰，正常神经元增多，神经元核固缩减少。与正常对照组相比，模型组髓鞘碱性蛋白（MBP）显著降低（<0.001），P0显著升高（<0.001）。与模型组相比，电针组、雪旺细胞移植组和电针＋雪旺细胞移植组MBP和P0的表达显著升高（<0.01，<0.001），且电针＋雪旺细胞移植组显著高于电针组和雪旺细胞移植组（<0.001）。电针＋雪旺细胞移植组Hoechst33342标记的雪旺细胞平均免疫荧光强度显著高于雪旺细胞移植组（<0.05）。CSCI后，与正常对照组相比，脊髓CD4、CD8和P0蛋白表达水平无显著变化（>0.05），而干预后与模型组相比，电针组、雪旺细胞移植组和电针＋雪旺细胞移植组P0蛋白表达水平显著升高（<0.05），且电针＋雪旺细胞移植组显著高于电针组和雪旺细胞移植组（<0.05）。电针＋雪旺细胞移植组CD4和CD8蛋白表达水平显著低于雪旺细胞移植组（<0.05）。
电针＋雪旺细胞移植可改善CSCI大鼠的运动功能，可能与其提高移植雪旺细胞的存活率以促进髓鞘再生及减轻免疫排斥反应有关。
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