Yang Dian-Ping, Zhang Ying, Lin Pei-Min, Mao An-Qiong, Liu Qing
School of Integrated Traditional Chinese and Western Medicine, Southwest Medical University, Luzhou 646000, Sichuan Province, China.
Department of Anesthesiology, Affiliated Hospital of TCM of Southwest Medical University, Luzhou 646000, Sichuan Province.
Zhongguo Zhen Jiu. 2022 Sep 12;42(9):1029-36. doi: 10.13703/j.0255-2930.20210617-0004.
To observe the effect of electroacupuncture (EA) at "Huantiao" (GB 30) and "Weizhong" (BL 40) on the activation of glial cells, the expression of brain-derived neurotrophic factor (BDNF), excitability and the number of dendritic spines of neurons in the spinal dorsal horn in rats with spared nerve injury (SNI) of sciatic nerve, and to explore the analgesic mechanism of EA on SNI.
PartⅠ: Sixty SD rats were randomly divided into a sham operation group, a model group, an EA group and a sham EA group, 15 rats in each group. Except the sham operation group, the SNI rat model was established in the remaining groups. The rats in the sham operation group were only treated with incision without damaging the nerve. The rats in the EA group were treated with EA at "Huantiao" (GB 30) and "Weizhong" (BL 40) on the affected side, continuous wave, frequency of 2 Hz, current intensity of 1 mA, 30 minutes each time, once a day, for 14 days. The rats in the sham EA group were treated with EA at points 0.5 cm next to "Huantiao" (GB 30) and "Weizhong" (BL 40) on the affected side; the manipulation, EA parameters and treatment course were the same as the EA group. The latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex were detected 1 day before modeling and 3, 7 and 14 days after modeling. Fourteen days after modeling, Western blot was used to detect the protein expressions of ionized binding adapter junction protein 1 (Iba-1), glial fibrillary acidic protein (GFAP), BDNF and c-Fos in the spinal dorsal horn; the expressions of Iba-1 and c-Fos proteins in the spinal dorsal horn were detected by immunofluorescence staining; immunohistochemical method was used to detect the expression of GFAP protein in the spinal dorsal horn; Golgi staining was used to detect the number of dendritic spines in spinal dorsal horn neurons. PartⅡ: Thirty SD rats were randomly divided into a control group, a BDNF group and a BDNF+anti-TrkB group, 10 rats in each group. The control group was treated with intrathecal injection of 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and dimethyl sulfoxide (DMSO); the BDNF group was treated with intrathecal injection of 10 μg rat recombinant BDNF dissolved in 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and DMSO; the BDNF+anti-TrkB group was treated with intrathecal injection of 10 μg rat recombinant BDNF and 30 μg tyrosine kinase receptor B (TrkB) antibody dissolved in 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and DMSO. The threshold of mechanical foot retraction reflex was detected 1 day before intrathecal injection and 1, 3 and 7 days after injection. Seven days after injection, the expression of c-Fos protein in the spinal dorsal horn was detected by Western blot and immunofluorescence staining.
PartⅠ: Compared with the sham operation group, 3, 7 and 14 days after modeling, the latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex in the model group were decreased (<0.05); 7 and 14 days after modeling, compared with the model group, the latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex in the EA group were increased (<0.05). The expressions of Iba-1, GFAP, BDNF, c-Fos proteins and the number of neuronal dendritic spines in the spinal dorsal horn in the model group were higher than those in the sham operation group (<0.05); the expressions of Iba-1, BDNF, c-Fos proteins and the number of neuronal dendritic spines in the EA group were lower than those in the model group (<0.05). PartⅡ: 3 and 7 days after intrathecal injection, the threshold of mechanical foot retraction reflex in the BDNF group was lower than that in the control group (<0.05); the threshold of mechanical foot retraction reflex in the BDNF+anti-TrkB group was higher than that in the BDNF group (<0.05). The expression of c-Fos protein in spinal dorsal horn in the BDNF group was higher than that in the control group (<0.05); the expression of c-Fos protein in spinal dorsal horn in the BDNF+anti-TrkB group was lower than that in the BDNF group (<0.05).
The analgesic effect of EA at "Huantiao" (GB 30) and "Weizhong" (BL 40) on SNI rats may be related to inhibiting the activation of microglia in the dorsal horn of the spinal cord, thereby blocking the signal of microglia-BDNF-neuron, and finally reducing the excitability of neurons.
观察电针“环跳”（GB 30）、“委中”（BL 40）对坐骨神经 spared nerve injury（SNI）大鼠脊髓背角胶质细胞激活、脑源性神经营养因子（BDNF）表达、神经元兴奋性及树突棘数量的影响，探讨电针对 SNI 的镇痛机制。
第一部分：60 只 SD 大鼠随机分为假手术组、模型组、电针组和假电针组，每组 15 只。除假手术组外，其余组建立 SNI 大鼠模型。假手术组仅行切口处理，不损伤神经。电针组于患侧“环跳”（GB 30）、“委中”（BL 40）行电针治疗，连续波，频率 2 Hz，电流强度 1 mA，每次 30 分钟，每日 1 次，共 14 天。假电针组于患侧“环跳”（GB 30）、“委中”（BL 40）旁开 0.5 cm 处行电针治疗；操作、电针参数及疗程同电针组。于造模前 1 天及造模后 3、7、14 天检测热足缩反射潜伏期及机械性足缩反射阈值。造模 14 天后，采用 Western blot 检测脊髓背角离子化结合衔接蛋白 1（Iba-1）、胶质纤维酸性蛋白（GFAP）、BDNF 和 c-Fos 的蛋白表达；采用免疫荧光染色检测脊髓背角 Iba-1 和 c-Fos 蛋白表达；采用免疫组化方法检测脊髓背角 GFAP 蛋白表达；采用 Golgi 染色检测脊髓背角神经元树突棘数量。第二部分：30 只 SD 大鼠随机分为对照组、BDNF 组和 BDNF+抗 TrkB 组，每组 10 只。对照组鞘内注射 10 μL 含 1︰1 的 0.9%氯化钠溶液和二甲基亚砜（DMSO）的混合液；BDNF 组鞘内注射 10 μg 溶解于 10 μL 含 1︰1 的 0.9%氯化钠溶液和 DMSO 混合液中的大鼠重组 BDNF；BDNF+抗 TrkB 组鞘内注射 10 μg 大鼠重组 BDNF 和 30 μg 酪氨酸激酶受体 B（TrkB）抗体溶解于 10 μL 含 1︰1 的 0.9%氯化钠溶液和 DMSO 的混合液。于鞘内注射前 1 天及注射后 1、3、7 天检测机械性足退缩反射阈值。注射 7 天后，采用 Western blot 和免疫荧光染色检测脊髓背角 c-Fos 蛋白表达。
第一部分：与假手术组比较，造模后 3、7、14 天，模型组热足缩反射潜伏期及机械性足缩反射阈值降低（<0.05）；造模后 7、14 天，与模型组比较，电针组热足缩反射潜伏期及机械性足缩反射阈值升高（<0.05）。模型组脊髓背角 Iba-1、GFAP、BDNF、c-Fos 蛋白表达及神经元树突棘数量高于假手术组（<0.05）；电针组脊髓背角 Iba-1、BDNF、c-Fos 蛋白表达及神经元树突棘数量低于模型组（<0.05）。第二部分：鞘内注射后 3、7 天，BDNF 组机械性足退缩反射阈值低于对照组（<0.05）；BDNF+抗 TrkB 组机械性足退缩反射阈值高于 BDNF 组（<0.05）。BDNF 组脊髓背角 c-Fos 蛋白表达高于对照组（<0.05）；BDNF+抗 TrkB 组脊髓背角 c-Fos 蛋白表达低于 BDNF 组（<0.05）。
电针“环跳”（GB 30）、“委中”（BL 40）对 SNI 大鼠的镇痛作用可能与抑制脊髓背角小胶质细胞激活，阻断小胶质细胞-BDNF-神经元信号，最终降低神经元兴奋性有关。
