电针夹脊穴和督脉穴对急性脊髓损伤家兔后肢神经功能的影响及其作用机制

Effects of electroacupuncture at Point JIAJI( EX-B2) and Point governor vessel on neural function of hindlimbs in rabbits with acute spinal cord injury and the mechanism of action

目的：观察电针夹脊穴和督脉穴对急性脊髓损伤家兔后肢神经功能的影响，并探讨其作用机制。方法：采用随机数字表将60只新西兰家兔随机分为假手术组、模型组、电针组，每组20只。假手术组咬除T13-L1棘突及全部椎板，暴露0．5cm宽硬膜，不损伤脊髓；模型组和电针组采用改良Allens法建立急性脊髓损伤家兔模型，并行脊髓MRI扫描鉴定模型。模型组和假手术组不给予治疗，电针组给予电针刺激受损脊髓节段上下两对夹脊穴及上下督脉穴（筋缩、命门）治疗，每次30min，每日1次，共7次。电针干预开始后1d、3d、7d采用改良Tarlov评分法评价家兔后肢神经功能。干预结束后每组取10只家兔，以4％多聚甲醛行心脏灌注后剖取损伤脊髓约1cm，制成切片后经HE染色于显微镜下观察脊髓组织形态，并采用免疫组织化学法观察脊髓细胞增殖情况和巢蛋白（Nestin）表达水平。将每组剩余的10只家兔麻醉，分离坐骨神经并注入5肛L的30％辣根过氧化物酶（horse—radishperoxidase，HRP），24h后取脊髓横断面背侧靠近损伤处脊髓，采用HRP逆行示踪法标记红核神经元细胞数。结果：①模型鉴定结果。MRI片上假手术组脊髓横断面信号正常，可见清晰的脑脊液通过。模型组和电针组脊髓横断面信号增强，脑脊液受压；受伤节段的脊髓在矢状位MRI片T2WI上呈明显的高信号，T1WI上呈低信号；提示脊髓断端周围充血、水肿，证明造模成功。②后肢神经功能评分。电针干预后不同时间点Tarlov评分比较，差异有统计学意义，即存在时间效应（F=6．920，P=0．001）；除假手术组外，干预后模型组和电针组Tarlov评分均逐渐升高。3组Tarlov评分总体上比较，组间差异有统计学意义，即存在分组效应（F=426．78，P=0．000）。干预开始后1d，电针组Tarlov评分高于模型组[（0．30±0．47）分，（0．20±0．41）分，q=7．010，P=0．486]，低于假手术组[（0．30±0．47）分，（4．70±0．47）分，q=30．843，P=0．000）]；模型组低于假手术组（q=31．544，P=0．000）。干预开始后3d，电针组Tarlov评分高于模型组[（1．55±0．60）分，（1．05±0．53）分，q=2．877，P=0．006]，低于假手术组[（1．55±0．60）分，（4．80±0．41）分，q=20．775，P=0．000]；模型组低于假手术组（q=23．651，P=0．000）。干预开始后7d，电针组Tarlov评分高于模型组[（2．65±0．59）分，（1．40±0．50）分，q=8．004，P=0．000]，低于假手术组[（2．65±0．59）分，（4．85±0．41）分，q=14．081，P=0．000]；模型组低于假手术组（q=22．093，P=0．000）。时间因素和分组因素之间存在交互效应（F=70．786，P=0．000）。③脊髓组织形态。假手术组脊髓形态结构完整，神经元结构正常、分布均匀，细胞膜、细胞核及组织间隙均正常，尼氏体显示清晰；模型组脊髓组织正常结构丧失，可见组织充血、水肿，神经元细胞数量减少，部分尼氏体消失，灰质中部分区域甚至有较大的空腔；电针组与模型组相比，脊髓形态结构基本完整，神经元细胞结构基本正常，空泡变性减少，组织充血、水肿消失。④脊髓细胞增殖情况。电针干预开始后7d，200倍显微镜下观察，每个视野下3组家兔脊髓增殖细胞数比较，组间差异有统计学意义[（43．67±8．19）个，（18．77±3．55）个，（6．30±2．32）个，F=382．413，P=0．000]；电针组脊髓增殖细胞多于模型组（q=20．889，P=0．000）和假手术组（q=31．348，P=0．000），模型组多于假手术组（q=10．459，P=0．000）。⑤脊髓细胞Nestin表达水平。干预开始后7d，200倍显微镜下观察，每个视野下3组家兔脊髓细胞Nestin阳性表达细胞数比较，组间差异有统计学意义[（23．37±5．10）个，（10．77±3．26）个，（4．43±1．33）个，F：218．084，P=0．000]；电针组Nestin阳性表达细胞数多于模型组（q=15．770，P=0．000）和假手术组（q=23．696，P=0．000），模型组多于假手术组（q=7．926，P=0．000）。⑥红核神经元细胞数。干预开始后7d，200倍显微镜下观察，每个视野下3组红核神经元细胞数比较，组间差异有统计学意义（513．40±46．79，417．90±43．03，578．40±43．91，F=32．760，P=0．000）；电针组红核神经元细胞数多于模型组（q=6．771，P=0．000）、少于假手术组（q=4．608，P=0．000），模型组少于假手术组（q=11．379，P=0．000）。结论：电针刺激夹脊穴和督脉穴能明显改善急性脊髓损伤家兔的后肢神经功能，其作用机制可能是通过促进神经干细胞增殖分化和抑制红核神经元细胞凋亡，从而使损伤节段神经组织修复及神经通路再通。

Objective ：To observe the effects of electroacupuncture at Point JIAJI （ EX - B2） and Point governor vessel on neural func- tion of hindlimbs in rabbits with acute spinal cord injury and to explore its mechanism of action. Methods：Sixty New Zealand rabbits were randomly divided into sham - operated group, model group and electroacupuncture group, 20 cases in each group. The random digits table was used for grouping. The spinous processes and ertebral plates of T13 - L1 of rabbits in sham - operated group were removed to expose 0.5 cm of endorachis in width without spinal cord injuries, while the models of acute spinal cord injury were built in rabbits in model group and electroacupuncture group by using improved Aliens method and the models were identified by spinal cord MRI scanning. The rabbits in model group and sham - operated group were untreated,while the rabbits in electroacupuncture group were treated with electroacupuncture at two pairs of Point JIAJI（EX- B2） and upper and lower Point governor vessel（ Point JINSUO and Point MINGMEN）at the injured seg- ments of spinal cord,30 minutes at a time, once a day for consecutive 7 times. At 1,3 and 7 days after the beginning of electroacupuncture intervention, the neural function of hindlimbs of rabbits were evaluated by using improved Tarlov scoring method. After the end of interven- tion, 10 rabbits were randomly selected from each group and were treated by perfusing their hearts with 4% paraformaldehyde. Then approx- imate 1 cm injured spinal cord were fetched out and sectioned for HE staining. The spinal cord tissue morphology were observed under the microscope and the proliferations of spinal cord cells and Nestin expression levels were detected by using immunohistochemical method. The sciatic nerves of the remaining 10 rabbits in each group were separated under anesthesia and 30% horseradish peroxidase（HRP） were injec- ted into the sciatic nerves in dosage of 5 μL. Twenty - four hours later, the dorsal spinal cord tissues near the injuried spinal cord were fetched out and the numbers of red nucleus neuronal cells were marked by using HRP retrograde tracing technique. Results：The MRI sig- nals of spinal cord in the transverse plane were normal and clear cerebrospinal fluids were found in sham -operated group;while enhanced MRI signals of spinal cord in transverse plane and compressed cerebrospinal fluids were found in model group and electroacupuncture group, and the injured segments of spinal cord presented with obvious high signal on T2 - weighted MRI and low signal on TI - weighted MRI in the sagittal plane, which suggested that there were engorgement and edema arround the broken ends of spinal cord and the models were successfully built. There was statistical difference in Tarlov scores between different time points after electroacupuncture intervention, in other words, there was time effect（ F = 6. 920,P = 0.001 ）. The Tarlov scores increased gradually in model group and electroacupuncture group after intervention. There was statistical difference in Tarlov scores between the 3 groups in general, in other words, there was group effect （ F = 426. 78, P = 0. 000 ）. At 1 day after the beginning of intervention, the Tarlov scores were higher in electroacupuncture group com- pared to model group （0.30 ＋/- 0.47 vs 0.20 ＋/- 0.41 points, q = 7.010, P = 0. 486） and were lower in electroacupuncture group compared to sham - operated group（0.30 ＋/- 0.47 vs 4.70 ＋/- 0.47 points, q = 30. 843, P = 0. 000 ） and were lower in model group compared to sham- operated group（ q = 31. 544 ,P = 0.000）. At 3 days after the beginning of intervention, the Tarlov scores were higher in electroacu- puncture group compared to model group（ 1.55 ＋/-0. 60 vs 1.05 ＋/- 0.53 points, q = 2. 877, P = 0. 006） and were lower in electroacu- puncture group compared to sham - operated group （ 1.55 ＋/- 0. 60 vs 4.80 ＋/- 0.41 points, q = 20. 775, P = 0.000 ）, and the Tarlov scores were lower in model group compared to sham - operated group（ q = 23.651, P = 0.000）. At 7 days after the beginning of intervention, the Tarlov scores were higher in electroacupuncture group compared to model group（2.65 ＋/- 0.59 vs 1.40 ＋/- 0.50 points, q = 8. 004, P = 0.000） and were lower in electroacupuncture group compared to sham - operated group （2.65 ＋/-0.59 vs 4.85 ＋/-0.41 points, q = 14. 081, P = 0. 000）, and the Tarlov scores were lower in model group compared to sham- operated group （q = 22. 093, P = 0.000 ）. There was interaction between time factor and group factor （ F = 70. 786, P = 0. 000）. In sham - operated group, the shape and structure of spinal cord were complete and the neurons were uniformly distributed in normal structure ; the cellular membrane, cellular nucleus and tissue spaces were normal and Nissl bodies were clear. In model group, the normal structure of spinal cord tissue disappeared, and hyperaemia and edema in the tissues and decrease of number of neurons were found ; some Nissl body disappeared and there were big cavity in some areas of grey matter. In electroacupuncture group, the shape and structure of spinal cord were basically complete and the structure of neuron was ba-sically normal;the vacuolar degeneration decreased and hyperaemia and edema in the tissues disappeared. At 7 days after the beginning of eIectroacupuncture intervention, there was statistical difference in the number of proliferative spinal cord ceils under the optical microscope （ x 200） between the 3 groups （43.67 ＋/- 8.19,18.77 ＋/- 3.55,6.30 ＋/- 2.32, F = 382. 413, P = 0. 000） ; the number of proliferative spinal cord cells was more in electroacupuneture group compared to model group（ q = 20. 889 ,P = 0. 000 ） and sham - operated group（ q = 31. 348, P = 0. 000 ） , and the number of proliferative spinal cord cells was more in model group compared to sham -operated group （q = 10. 459,P = 0.000）. At 7 days after the beginning of intervention, there was statistical difference in the number of Nestin positive expres- sion cells of spinal cord under the optical microscope （ × 200）between the 3 groups （23.37 ＋/-5.10,10.77 ＋/-3.26,4.43 ＋/-1.33, F = 218.084, P = 0. 000 ） ; the number of Nestin positive expression cells was more in electroacupuncture group compared to model group （ q = 15. 770,P = 0.000 ） and sham - operated group （ q = 23. 696, P = 0.000 ） , and the number of Nestin positive expression ceils was more in model group compared to sham - operated group （ q = 7. 926, P = 0. 000 ）. At 7 days after the beginning of intervention, there was statistical difference in the number of red nucleus neuronal cells under the optical microscope （ ~ 200 ） between the 3 groups （513.40 ＋/- 46.79, 417.90 ＋/-43.03,578.40 ＋/-43.91, F = 32. 760, P = 0. 000 ） ;the number of red nucleus neuronal ceils was more in electroacupuncture group compared to model group （ q = 6. 771, P = 0. 000 ） and was less compared to sham - operated group （ q = 4. 608, P = 0. 000 ）, and the number of red nucleus neuronal cells was less in model group compared to sham - operated group （q = 11. 379, P = 0. 000 ）. Conclusion ： Electroacupuncture at Point JIAJI（ EX - B2 ） and Point governor vessel can obviously improve the neural function of hindlimbs in rabbits with acute spinal cord injury. It can promote the differentiation and proliferation of neural stem cells and inhibit the apoptosis of red nucleus neuronal cells, which may be the mechanisms of action for repairing the nervous tissues at the injuried segments and recirculating the neural pathway.