重复电针坐骨神经痛大鼠下丘脑室旁核区神经元的结构

doi:10.3969/j.issn.1673-5374.2013.03.003

摘要/Abstract

摘要：

实验采用去卵巢法制备大鼠学习记忆损伤模型，通过结扎坐骨神经方法制备大鼠慢性神经病理性疼痛模型，观察多次电针大鼠双侧阳陵泉(GB34)-足三里(ST36)穴位引起的累积镇痛效应及下丘脑室旁核区神经细胞突触结构的改变。结果发现，电针2周的大鼠较电针2 d的大鼠热痛阈明显提高，但在去卵巢干预的慢性神经病理性疼痛模型大鼠中，镇痛效果明显减弱。同时，电针2周明显逆转慢性神经病理性疼痛模型大鼠下丘脑室旁核区神经细胞突触后密度变薄和突触间隙变宽。说明多次电针阳陵泉-足三里穴位有累积效应，可通过提高下丘脑室旁核区突触界面结构的可塑性，减轻慢性神经病理性疼痛大鼠的疼痛反应。

关键词: 神经再生, 疼痛, 慢性神经病理性疼痛, 电针, 针刺镇痛, 累积效应, 突触可塑性, 下丘脑, 学习记忆, 基金资助文章, 图片文章

Abstract:

In the present study, a rat model of chronic neuropathic pain was established by ligation of the sciatic nerve and a model of learning and memory impairment was established by ovariectomy to investigate the analgesic effect of repeated electroacupuncture stimulation at bilateral Zusanli (ST36) and Yanglingquan (GB34). In addition, associated synaptic changes in neurons in the paraventricular nucleus of the hypothalamus were examined. Results indicate that the thermal pain threshold (paw withdrawal latency) was significantly increased in rats subjected to 2-week electroacupuncture intervention compared with 2-day electroacupuncture, but the analgesic effect was weakened remarkably in ovariectomized rats with chronic constrictive injury. 2-week electroacupuncture intervention substantially reversed the chronic constrictive injury-induced increase in the synaptic cleft width and thinning of the postsynaptic density. These findings indicate that repeated electroacupuncture at bilateral Zusanli and Yanglingquan has a cumulative analgesic effect and can effectively relieve chronic neuropathic pain by remodeling the synaptic structure of the hypothalamic paraventricular nucleus.

Key words: neural regeneration, acupuncture and moxibustion, chronic neuropathic pain, electroacupuncture, acupuncture analgesia, cumulative effect, synaptic plasticity, hypothalamus, learning and memory, neurobiology, grants-supported paper, photographs-containing paper, neuroregeneration

. 重复电针坐骨神经痛大鼠下丘脑室旁核区神经元的结构 [J]. 中国神经再生研究(英文版), 2013, 8(3): 218-225.

Qiuling Xu, Tao Liu, Shuping Chen, Yonghui Gao, Junying Wang, Lina Qiao, Junling Liu. Correlation between the cumulative analgesic effect of electroacupuncture intervention and synaptic plasticity of hypothalamic paraventricular nucleus neurons in rats with sciatica[J]. Neural Regeneration Research, 2013, 8(3): 218-225.

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引言

材料方法

Design

A randomized, controlled animal study.

Time and setting

The experiments were performed in the Department of Physiology, Institute of Acu-moxibustion, China Academy of Chinese Medical Sciences, China from November 2007 to April 2009.

Materials

A total of 84 clean-grade, female Wistar rats, aged 3–4 months, weighing 240–250 g, were purchased from the Experimental Animal Center of Chinese Academy of Medical Sciences (License No. SCXX-Army 2007-004). Animal care and experimental process were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China^[34].

Methods

Establishment of memory impairment model

After anesthesia, rats in the OVX + CCI, OVX + CCI + EA 2D and OVX + CCI + EA 2W groups underwent OVX. Briefly, bilateral mid-abdominal dorsolateral incisions (about 2 cm long) were made, and both ovaries were removed. Four weeks after OVX, four animals from each group (except the control group) were subjected to a vaginal smear test for verifying OVX success. Forty-five days after OVX, the rats’ learning-memory ability was analyzed by escape latency (place navigation test), swimming distance in the target quadrant and target quadrant crossing times (spatial probe test) in the Morris water maze test for 7 days using a Morris water maze device (DigBehv-MWM Morris Water Maze Video Analysis System, Shanghai Jiliang Co. Ltd., Shanghai, China) as previously described^[35]. Results indicated that the OVX rats had a defect in learning and memory (results not shown).

Establishment of CCI models

Chronic neuropathic pain model was established by ligation of the unilateral sciatic nerve according to a modification of previously published methods^[36]. Under intraperitoneal anesthesia with a mixed solution of 28 mg/100 g urethane (Beijing Chemistry Reagent, Beijing, China) and 3.3 mg/100 g chloralose (Sigma-Aldrich, St. Louis, MO, USA) and routine sterilization, the left sciatic nerve was exposed at the mid-thigh level by blunt dissection through the biceps femoris muscle. Four constrictive ligatures (4-0 surgical suture) were tied around the nerve at the distal end close to the nerve bifurcation at spaces about 1.0 mm apart. The ligature was considered to be suitable if local and moderate muscular contraction of the leg was clearly observed. Following local application of antibiotics (sodium penicillin, 9 000–10 000 U/rat), the muscle and skin tissues were sutured in layers. To reduce experimental variability, all surgeries were performed by the same operator. For OVX + CCI rats, CCI surgery was performed 4–5 days after the Morris water maze test.

EA intervention

Bilateral Zusanli (5 mm beneath the capitulum fibulae and lateral-posterior to the knee-joint) and Yanglingquan (about 5 mm superior-lateral to Zusanli ) were punctured with filiform needles (Hua Tuo acupuncture needle, Suzhou Medical Appliance Factory, Suzhou, China; Gauge 28, 0.5 cun) to a depth of 2–3 mm and electrically stimulated using a HAN’s EA Apparatus (LH202, Beijing Huawei Industrial Developing Company, Beijing, China). EA (2/15 Hz, 1 mA) was administered for 30 minutes, once daily beginning on the 4^th day after CCI surgery for 2 weeks for rats in the CCI + EA 2W and OVX + CCI + EA 2W groups, and on the 16^th day after CCI surgery for 2 days for rats in the CCI + EA 2D and OVX + CCI + EA 2D groups.

Thermal pain threshold detection

Each rat was placed into a black cloth bag with the hindlimbs and tail exposed to move freely. A mobile radiant heat source (high-intensity light beam with radiant heat dolorimeter; Thermotron, Holland, Michigan, USA) was focused on the plantar surface of the hind paw. Paw withdrawal latency (i.e., pain threshold) of the bilateral footplates was measured three times with an interval of 3–5 minutes between detections. To avoid potential tissue damage, the cutoff time of the radiant heat radiation was set to 20 seconds. Thermal pain threshold detection was conducted prior to as well as 4, 8, 12, 16 and 18 days after CCI. Pain thresholds were measured in rats from the CCI + EA and OVX + CCI + EA groups on the following morning for observing the post-effects of EA.

Transmission electron microscopy observation of hypothalamic ultrastructure

At the end of each experiment and under deep anesthesia, the rat was perfused transcardially by using a solution of 2% paraformaldehyde + 2% glutaraldehyde. Then, the brain tissue containing the PVN of the hypothalamus^[37] was cut into small cubic pieces (about 1 mm³), fixed with 3% glutaraldehyde (EMS Company, Shanghai, China) and 1% osmium tetroxide, sequentially, for 2 hours, dehydrated with ethanol, submerged in acetone, embedded in 812 Epon-Araldite, sectioned at 250-nm thickness, and stained using uranyl acetate and lead citrate. The brain sections were then examined under a JEM-1230 transmission electron microscope (JEOL Ltd, Tokyo, Japan) and areas of interest that contained synapses were imaged using a NIS-Elements BR2.30 (Nikon, Tokyo, Japan) operated at 200 keV and a Gatan 2K × 2K CCD camera at a magnification of 50 000 ×. The average synaptic cleft width, the thickness of the postsynaptic density and the length of the active zone in 10 optional visual fields of sections of each brain (four rats/group) were assessed. The values for the synaptic active zone (the length of the postsynaptic density, the thickness of the postsynaptic density and the synaptic cleft width) were measured separately according to previously published methods^[38] (Figure 6). The curvature of the synaptic interface was also measured^[39](Figure 7).

Statistical analysis

Data were expressed as mean ± SD. Differences in paw withdrawal latency were assessed using one-way analysis of variance with repeated measures when appropriate. Least significant difference, t-test was used to compare data between two groups. A value of P < 0.05 was considered statistically significant.

讨论

文章快阅

延伸阅读

1实验设计构思介绍：
边缘系统（海马、下丘脑等）是针刺镇痛的重要部位，也是神经记忆、内分泌等机体功能活动调节的关键部位，尤其是下丘脑。针刺的信息可影响这些部位的神经元的活动，对抗慢性痛的信号，从而启动一系列复杂有序的与针刺镇痛累积效应有关的神经元及其细胞内蛋白质分子的编码、信息储存和译码的过程。

2 国内外同类研究水平的介绍：
针刺疗法，是通过穴位刺激调动和促进人体自我愈合能力的一种自然疗法。临床经验证明：治疗一种病，一般1-2次或3-4次治疗后，患者会觉得症状有改善，逐渐，发现效应累积。美国Megan Long 和Dipl AC在他们的文章：“针刺和东方医学”（Acupuncture and Oriental Medicine，2003）指出：针刺是一种累积治疗模式，需要多次的重复。有关针刺累积效应的临床和实验的系统研究，国外尚未见有这方面的报道。国内，陈美仁等治疗90例坐骨神经痛患者，随机分为温针灸组、西药（口服尼美舒利）组和穴位注射（6542注射液）组（各30例），温针灸肾俞、环跳等，每日1次，10天为1个疗程，共3个疗程。治疗后，有效率分别为90.00%、73.33%和63.33%。随疗程的增加各组患者的痛阈逐渐提高，与治疗前比，第1，2个疗程有增加，但并不显著； 3个疗程后痛阈的增加均有显著性(P<0.01); 其中，温针灸的疗效明显优于其余两组 (P<0.01)。王宁华报告，重复经皮神经电刺激正常老年人受试者膝关节周围外膝眼、足三里等,每日2次、每次60 min。 6次刺激后，重复经皮神经电刺激组显示热痛阈显著增加，并对疼痛具有累加性抑制作用。说明，针灸治疗可明显减轻疼痛并具有累积效应。

动物实验证明：在白陶土-鹿角菜胶造成的大鼠慢性关节炎疼痛的模型上，反复电针“足三里／三阴交”等可加强镇痛效应，并可促进炎症的缓解过程、加速肢体功能的恢复。针刺大鼠“百会”等1，3， 5，10天，随着针刺时间的延长，脑缺血大鼠海马CA3区血管内皮素、细胞间黏附分子的表达渐明显降低，血管内皮VIII因子相关抗原的表达则明显增加，呈现出累加效应。韩济生院士在福氏佐剂引发大鼠关节炎的模型上，观察到：第一次电针后即时已呈现镇痛效应，停针后反而发生痛敏反应；若隔日一次电针，连续９次，痛敏不再出现，针效积累，肿胀消失，体质量增加。在正常大鼠重复经皮神经电刺激“足三里／三阴交”，发现每两天一次电刺激的镇痛效应能累加，但后效应减弱；如果７天一次刺激，其镇痛效应可逐渐累加。董志强等报告，在结扎大鼠造成慢性压迫性损伤疼痛的模型上，电针大鼠“环跳”-“阳陵泉”1-3周后，痛敏反应逐渐减弱，脊髓背根神经节内脑源性神经营养因子mRNA的表达也呈累积性加强。陈丁生等的研究结果表明，电针大鼠“三阴交”等每周3次，共5周，可抑制链脲佐菌素（造成实验性糖尿病）引起的Na+-K+/ATPase活性的下降，停止治疗后4周，针刺的这种作用仍能维持一定的水平。我们的工作证明，在实验性动脉粥样硬化的家兔模型上采用“心兪－厥阴兪”穴位贴敷法，观察到：贴敷2.5－3.5月后，主动脉弓、腹主动脉和心肌血管内膜增厚的程度呈不同程度抑制的趋势；在2.5月时受到一定程度的抑制，而3.5月时受到明显的抑制，效果累积；同时，穴位贴敷对减轻血总胆固醇、低密度脂蛋白－胆固醇含量的升高3.5月时有明显效果。韩济生实验室还报告，在正常大鼠上，用100Hz每两天电针一次，共5次，其镇痛效应逐步降低，即出现明显的针刺耐受现象；用2Hz 每2天一次给“足三里－三阴交” 重复经皮神经电刺激时, 镇痛效应可累加，但后效应减弱。在慢性疼痛模型上，2/5Hz电针30分钟，间隔1小时，镇痛效应可逐渐增强，而间隔4小时多次电针，则针刺效果不能逐步加强。

上述结果显示，针刺等穴位刺激对疼痛的缓解和对某些疾病的治疗作用确有明显的累积效应，这种作用与刺激的量、时间间隔、疾病的性质等有密切的关系。

有关针刺累积作用机制的研究目前报道极为少见。Kendall 1989年，曾将针刺效应的机制归纳为：神经学，生物化学，和生物电学3个方面。针刺体表穴位后，可激活局部组织的Aδ和C类纤维，将信号通过脊髓背角上传到脊髓和脑内高位中枢；缓激肽、组织胺、Leukotrin等生物活性物质是启动针刺穴位效应的重要介质；在中枢神经系统中，5-羟色胺、P物质、脑啡肽、内啡肽等神经递质介导针刺效应。此外，针刺局部穴位的机械刺激可产生电能的变化，加强针刺诱发的神经冲动的传导。下丘脑等边缘系统在针刺镇痛和对机体活动的调控中发挥着极为重要作用。我们过去的工作证明，电针家兔“内关”可激活下丘脑后区神经元电活动，75.0%产生兴奋反应，而“足三里”主要产生抑制性效应（62.5%）。c-fos是一种主要的基因蛋白，也是一个神经元代谢活动增强的标志物。郭惠夫等人报告，2Hz 和100Hz电针诱发下丘脑视前区、视上核、杏仁核、中脑中央中核、延脑孤束核等部位细胞c-fos基因表达没有明显差异；2Hz 电针使前脑啡肽原mRNA表达大大增高；100Hz电针能使前强啡肽原基因转录加速，而2Hz电针没有这一作用。用c-fos/c-jun反义寡核苷酸阻断的方法证明：fos/jun是电针引起前强啡肽原基因表达的转录因子。用原位杂交技术观察到，电针24h后前脑啡肽原mRNA合成显著增加，提示该类表达为临床针刺累加效应的原因之一。给予佐剂性关节炎大鼠每周两次电针，于每周末测定脊髓灌流液中P物质、胆囊收缩素和甲硫氨酸脑啡肽，反复电针（3-4周）可使至炎后脊髓释放增加的P物质释放显著减少，至炎后减少的胆囊收缩素恢复正常，释放增加的甲硫氨酸脑啡肽提前出现。反复的穴位电刺激使阿片肽及其受体的功能发生的可塑性变化，可能是其累加治疗作用的机制之一。

上述结果表明：针刺体表穴位的信号可抵达下丘脑等脑内各有关结构，激活或抑制中枢神经元的活动，这是其“平衡阴阳”、动员机体体内的抗病和愈合功能紊乱的能力，实现“除百病”效果的基础；中枢神经系统是人体和动物体调控其各种功能活动的指挥中心。多次的针刺刺激穴位所产生的累积效应，与神经递质的释放与合成、神经元之间联系的加强、神经元内信号的转导通路的激活、基因表达等过程密切相关；下丘脑是长时程记忆的关键部位，也是体内植物性神经系统、神经内分系统的调节中枢。因此，我们有理由设想：反复针刺之所以能够产生累加疗效，很可能是与中枢神经系统内有关脑区、核团，特别是边缘系统（海马、下丘脑）神经元的记忆、编码、解码过程，突触的可塑性、神经内分泌等功能活动的改变密不可分。

3与其他同类研究的比较：
实验以神经记忆为切入点，从动物的整体行为反应、神经结构的可塑性改变、细胞内信号转导通路的活动、等一系列变化结合起来，分析针刺的累积效应，具有创新性，目前尚未见有如此系统研究的报道。

4 文章创新性：
检索数据库：MEDLINE数据库（1965-2009）、U.S.PATENT DATABASE（1979-2009）国外医学系列（1984-2009）及中国专利数据库（1985-2009），中国中医药期刊文献数据库（1984-2007）、中医药新闻数据库（1984-2009）、中国生物医学文献数据库（1978-2009）、CNKI期刊文献全文数据库（1994-2009）；检索时间：2010年11月17日，检索关键词：针刺、电针、慢性压迫性神经损伤、累积效应；检索出相关文章数量：一般相关文献2篇，本课题组发表的密切相关文献3篇及验证说明：国外文献未查到相关报道，国内文献未查到以海马-下丘脑-垂体-肾上腺为轴，从相关行为学改变，蛋白激酶活性、基因表达结合起来探讨针刺镇痛累积效应机理的探讨。