这项工作得到了上海市临床重症专科项目（批准号：Shslczdzk04001）的支持;上海市科委航海项目（批准号：22YF1444300）;上海中医药大学预算内项目（批准号：2021LK091）.

这项工作是在复旦大学神经生物学研究所疼痛实验室进行的。这些实验已获得批准，并严格遵守国际疼痛研究协会（LASP）为所有外科手术和动物处理制定的实验动物保护指南。本研究使用清洁级 Sprague-Dawley （SD） 大鼠，由 32 只 40-50 日龄的雄性组成，平均体重为 220 ± 1.38 g。这些大鼠购自中国科学院上海生命科学研究院实验动物中心。动物得到适当的照顾，并被安置在一个有独立通风、调节温度（22±1°C）和湿度（40%-50%）的专用房间内。老鼠在笼子里可以获得足够的食物和水。实验动物室遵循12 h的明暗循环，以保持大鼠昼夜节律的规律性，并由指定人员定期更换衬垫。X光片在上海中医药大学附属岳阳中西医结合医院放射科进行。
1. 研究参与者和分组
<ol><li>将32只大鼠分为四组：幼稚（对照），假手术（假手术），CCD（慢性背根神经节压迫）和CCD +推拿（8只大鼠/组）。老鼠可以在动物设施中自由获取食物和水。 注：幼稚大鼠没有干预，而假大鼠组接受了与CCD组大鼠相同的外科手术，但没有在L4和L5椎间孔中留下“L”形不锈钢棒。CCD组大鼠行完全慢性背根神经节压迫模型手术。CCD+推拿组大鼠从CCD手术后第4天开始接受推拿治疗。</li>
</ol>2. 建立动物模型
<ol><li>施用异氟烷麻醉大鼠。一旦大鼠失去知觉（没有甩尾反射或腿部屈曲反射），使用剃须刀剃掉手术区域的毛发。 注：术前15分钟施用镇痛药，大鼠皮下注射曲马多20mg/kg。</li>
	<li>将大鼠固定在泡沫板上（见 材料表），并使用橡皮筋固定其四肢和门牙。用酒精和碘交替的无菌制剂擦拭准备好的区域至少 3 个周期。</li>
	<li>插入“L”形探头（参见 材料表）。<ol><li>用剪刀在皮肤、浅筋膜和深筋膜上逐层做一个2-3厘米的切口。首先，找到髂前上棘，对应于第五腰椎。然后，依次定位第三和第四棘突。</li>
		<li>用齿形镊子夹住棘突并提起，使剪刀靠近棘突的右侧，并剪断附着在棘突右侧的肌肉。</li>
		<li>然后，钝性解剖附着在椎板外表面的肌肉，直到右侧有阻力。突起是颧骨关节。同样，直截了当地解剖颧骨关节上的肌肉和筋膜。 注意：指向大鼠头部方向的横向突将首先在颧骨骺关节的下外前方触摸。横突下方是椎间孔，椎间孔充满神经根和周围的软组织，通常不容易找到。</li>
		<li>首先，使用“L”形探头确定椎间孔的位置，然后使用“L”形不锈钢棒（需要制成直径为0.4毫米，长度为4毫米）将其插入椎间孔。</li>
		<li>如果背根神经节被成功压缩，大鼠将表现出尾巴甩动和腿部屈曲反射。将不锈钢棒插入第四和第五腰椎间孔。然后，逐层缝合（3-0，见 材料表）肌肉、筋膜和皮肤。</li>
	</ol></li>
	<li>将大鼠放入恒温箱中，直到它醒来。醒来后，观察大鼠右后肢功能是否正常。如果有拖拽，则说明手术损伤了运动神经，应丢弃大鼠。如果右后肢功能正常，可以使用大鼠并将其放在笼子中喂食。</li>
</ol>3. 推拿疗法
<ol><li>建立舒适的环境：在开始推拿治疗之前，使大鼠在限制器中适应30分钟，使其适应（图1）。该装置可以完全暴露大鼠的大腿并使其固定，便于推拿操作（见 材料表）。 注意：治疗室的温度应保持在22-26°C之间，湿度应在40%-50%之间。</li>
	<li>推拿标准化：确保治疗师佩戴无线指套，可以监测推拿的压力和频率，并提供实时反馈数据。首先，用指套的反馈数据练习推拿，将力调整到5N，频率调整到2Hz。然后，对大鼠进行相同的操作，在整个过程中保持一致的力量和频率（图2）。 注意：根据我们之前的工作，计算出的最佳压力为 5 N（有关详细信息，请参阅讨论部分）。</li>
	<li>辨识穴位：选择右后肢腓肠肌作为大鼠推拿区，在腓肠肌两头交界处，约在BL5714的位置。</li>
	<li>推拿：确保治疗师面向大鼠大腿的后侧，并用右上肢握住大鼠的右后肢。将拇指垂直放置在 BL57 穴位上，并确保前臂和手指在施加 5 N 压力的同时施加力以执行有节奏的小范围旋转运动（图 3）。</li>
	<li>在治疗过程中，确保操作反馈的力度和频率与预设值相对应。从手术后第四天开始干预，推拿每天进行一次，持续15分钟，持续18天。</li>
</ol>4. 疼痛行为测试
注意：行为测试是在建模前、建模后、干预第 1 天、干预第 3 天、干预第 7 天、干预第 14 天、干预第 17 天和干预第 21 天进行的。
<ol><li>按照以下步骤执行机械刺激响应阈值（爪子退出阈值，PWT）（图 4）。<ol><li>使用von Frey方法测试大鼠脚部机械刺激的反应阈值。将大鼠置于尺寸为20厘米×10厘米×20厘米的透明钢化玻璃隔间中，该玻璃隔间放置在具有10毫米×10毫米孔径的金属丝网格支架上，高度为40厘米。保持室温在23±2°C，周围环境安静。</li>
		<li>用电子 Von Frey 纤维测量机械退出阈值（参见 材料表）。刺激大鼠的脚部中心，直到它明显移动，例如抬起腿或避开它。机器自动记录最大压力值 （N）。</li>
		<li>等待15秒或更长时间，然后再次刺激同一只大鼠。将每次刺激保持在5秒以下，以防止大鼠爪子的触觉过敏。重复测试五次，直到连续三次测量相差小于 10 N。</li>
	</ol></li>
	<li>执行爪子退出潜伏期 （PWL） 以响应热刺激（图 5）。<ol><li>使用 Hargreaves 方法15,16 评估 PWL。将大鼠放入由透明钢化玻璃制成的小腔室中，尺寸为20 cm x 10 cm x 20 cm，带有带通风孔的透明玻璃盖。使用加热板将玻璃盖的中心区域加热至45°C，直至达到稳定温度。</li>
		<li>在行为测试阶段，每天至少2小时使大鼠适应行为实验室，以尽量减少环境因素对测试结果的影响。</li>
		<li>在正式测试之前，将大鼠置于行为实验室30分钟，以使它们适应环境并减少干扰。</li>
		<li>将加热板加热至45°C，并将大鼠的后肢放在加热板上。</li>
		<li>爪子退出潜伏期 （PWL） 定义为从加热开始到爪子退出反射响应热刺激的时间。在每次测试中，连续测试同一后肢三次，并获得该后肢响应潜伏期的平均值。测试后，将大鼠放回笼子喂食。</li>
	</ol></li>
</ol>5. 灌注
<ol><li>制备：事先配制0.9%盐水溶液和4%多聚甲醛溶液。将盐水溶液置于恒温为37°C的烤箱中，并将多聚甲醛溶液储存在4°C的冰箱中以备后用。</li>
	<li>进行麻醉并建立通路。<ol><li>首先将 25% 氨基甲酸乙酯（0.6 mL/100 g，参见 材料表）注射到大鼠腹腔中以诱导深度麻醉。等到未观察到脚趾、角膜或转动反射。将老鼠固定在泡沫板上。</li>
		<li>用剪刀剪开胸骨，逐层打开皮肤和筋膜。切开横膈膜，切掉两侧的肋骨，充分暴露心脏。小心地分离心包。将肺与心脏分开。</li>
		<li>使用镊子将心脏拉向自己，从而拉动并暴露主动脉。将针头、左心室和主动脉对齐在一条直线上，并在同一水平面上。然后将针头从左心室插入主动脉，直到主动脉内可见针头。</li>
		<li>用镊子夹住主动脉和主动脉内的针头，然后用剪刀切开左心房。这时，大量的血液会从左心房喷出。打开盐水溶液的阀门，将盐水注入连接到灌注37°C盐水溶液，总共约150-200mL。</li>
	</ol></li>
	<li>盐水灌注完成后，改用4%多聚甲醛溶液，在4°C下灌注4%多聚甲醛溶液，共约400mL。 开始多聚甲醛灌注时，用一对镊子夹住大鼠的门牙向前拉，同时用一只手握住尾巴向后拉，有利于充分伸展脊柱和增加椎间孔，便于DRG取样。</li>
	<li>在灌注过程中，大鼠的肝脏、肠系膜和大网膜逐渐变白，直到肝脏变硬。然后，减慢流速并将其调整到每秒约 2 滴，直到灌注所有多聚甲醛。</li>
</ol>6.背根神经节集合
注意：灌注后，迅速切断大鼠脊柱的腰部。使用这种定位方法，通过将两侧髂嵴的最高点连接到L5腰椎棘突来定位L5和L4椎间孔，并从椎间孔中取出背根神经节。具体收集方法如下：
<ol><li>从椎管（胸椎管）入口处，将剪刀插入椎管，切断两侧的椎板，直到可以完全去除所有椎板，露出整个椎管。 注意： 切割椎板时注意不要损伤椎管外的脊髓和神经根。</li>
	<li>小心地切除脊髓和后纵韧带。分离附着在椎间孔内孔的硬脑膜。</li>
	<li>用眼科镊子夹住并拉出背根神经节，其形状像珍珠，略带黄色。 注意：由于神经组织的脆弱性和韧性差，在拉出背根神经节时需要掌握力量和拉力方向，不要使用蛮力。</li>
	<li>拔出背根神经节时，确保提前清洁周围的软组织，包括硬脑膜和蛛网膜，以利于背根神经节的顺利拉扯。</li>
	<li>将背根神经节放在吸水纸上，用刀片切断轴突，清洁背根神经节表面的血管。</li>
	<li>修剪后称取背根神经节，浸入浓度为4%、温度为4°C的4%多聚甲醛溶液中足够时间，通常约2-4 h，然后将背根神经节转移到预先配制好的10%、20%、30%PB蔗糖溶液（4°C）中逐步脱水。</li>
</ol>7. 冷冻切片
<ol><li>首先将背根神经节置于0.01M PBS溶液中。摇晃它们10分钟，然后洗掉蔗糖溶液。小心地修剪背根神经节两节的轴突纤维。清洁冷冻切片机的冷冻头，并向其中添加最佳切割温度 （OCT） 化合物（参见 材料表）。</li>
	<li>将冷冻头放在含有液氮的金属外壳表面，等待组织冻结，将其取出，并修剪平整。之后，将冷冻头放在切片机的底座上。背根神经节切片的厚度应为15毫米。将切好的薄片按顺序排列在木箱中，并存放在-20°C冰箱中，避光。</li>
</ol>8.苏木精和曙红染色
<ol><li>将切片置于二甲苯中 2 分钟，并在一系列醇中脱水，包括 100%、95%、80% 和 70%，每次 2 分钟。然后将切片放入蒸馏水中2分钟，在苏木精中放置1分钟，进行自来水冲洗5分钟，并在1%盐醇溶液中进行分化30秒，然后在饱和碳酸锂中进行30秒。</li>
	<li>接下来，将切片分别放入蒸馏水和自来水中 2 分钟，在伊红溶液 （0.5%） 中 5 分钟，在蒸馏水中快速冲洗 1 分钟，在 95% 和 100% 酒精中两轮，每轮 2 分钟，在添加碳酸氢钠的 100% 二甲苯中 30 秒，在二甲苯中三轮，每轮 3 分钟，最后， 用中性香脂密封（见 材料表）。</li>
</ol>

大鼠 DRG 压缩模型的标准化 Tuina 协议

<ol>
	<li>Vos, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=national+incidence,+prevalence,+and+years+lived+with+disability+for+328+diseases+and+injuries+for+195+countries,+1990-2016:+a+systematic+analysis+for+the+Global+Burden+of+Disease+Study+2016.">national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016.</a> The Lancet. 390 (10100), 1211-1259 (2017).</li><li>Amin, R. M., Andrade, N. S., Neuman, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumbar+Disc+Herniation.">Lumbar Disc Herniation.</a> Current Reviews in Musculoskeletal Medicine. 10 (4), 507-516 (2017).</li><li>Stochkendahl, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+Clinical+Guidelines+for+non-surgical+treatment+of+patients+with+recent+onset+low+back+pain+or+lumbar+radiculopathy.">National Clinical Guidelines for non-surgical treatment of patients with recent onset low back pain or lumbar radiculopathy.</a> European Spine Journal. 27 (1), 60-75 (2018).</li><li>Bostelmann, R., Steiger, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comment+on+&amp;#34;An+evidence-based+clinical+guideline+for+the+diagnosis+and+treatment+of+lumbar+disc+herniation+with+radiculopathy&amp;#34.">Comment on &amp;#34;An evidence-based clinical guideline for the diagnosis and treatment of lumbar disc herniation with radiculopathy&amp;#34.</a> The Spine Journal. 14 (9), 2273 (2014).</li><li>Tang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+bone-setting+massage+combined+with+acupuncture+and+moxibustion+on+simple+lumbar+disc+herniation+and+its+effect+on+pain+and+sensory+disorder+of+lower+limbs.">Effect of bone-setting massage combined with acupuncture and moxibustion on simple lumbar disc herniation and its effect on pain and sensory disorder of lower limbs.</a> Chinese Archives of Traditional Chinese Medicine. 38 (10), 244-247 (2020).</li><li>Lin, X. -. Y., Yang, J., Li, H. -. M., Hu, S. -. J., Xing, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+root+ganglion+compression+as+an+animal+model+of+sciatica+and+low+back+pain.">Dorsal root ganglion compression as an animal model of sciatica and low back pain.</a> Neuroscience Bulletin. 28 (5), 618-630 (2012).</li><li>Shi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+studying+the+etiology+and+treatment+of+low+back+pain.">Animal models for studying the etiology and treatment of low back pain.</a> Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society. 36 (5), 1305-1312 (2018).</li><li>Zhao, X. -. y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Tuina+on+the+expression+of+TNF-&#945;,IL-6,TGF-&#946;1+and+CTGF+in+the+degenerative+tissue+of+rabbits&#8242;lumbar+intervertebral+disc.">Effect of Tuina on the expression of TNF-&#945;,IL-6,TGF-&#946;1 and CTGF in the degenerative tissue of rabbits&#8242;lumbar intervertebral disc.</a> Journal of Chengdu Medical College. 14 (6), 741-745 (2019).</li><li>Zhang, L., Li, Z. -. y., Yu, Z. -. y., Yue, X. -. y., Fu, R. -. y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+GABA+and+GABAAR+in+analgesic+loop+of+CCI+rats+by+pressing+manipulation+based+on+&amp;#34;Taking+Tenderness+as+Acupoints&amp;#34;+theory.">Effect of GABA and GABAAR in analgesic loop of CCI rats by pressing manipulation based on &amp;#34;Taking Tenderness as Acupoints&amp;#34; theory.</a> Journal of Shanghai University of Traditional Chinese Medicine. 28 (3), 50-53 (2014).</li><li>Long, B. -. c. a. l., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analgesic+effect+of+massage+on+rats+with+neuropathic+pain+and+its+mechanism.">Analgesic effect of massage on rats with neuropathic pain and its mechanism.</a> Guangxi Medical Journal. 44 (17), 2003-2009 (2022).</li><li>Al-Bedah, A., Ali, G., Aboushanab, T., Qureshi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tui+Na+(or+Tuina)+massage:+A+minireview+of+pertinent+literature,+1970-2017.">Tui Na (or Tuina) massage: A minireview of pertinent literature, 1970-2017.</a> Journal of Complementary and Alternative Medical Research. 3, 1-14 (2017).</li><li>Au, D. W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acupressure+on+anxiety:+a+systematic+review+and+meta-analysis.">Effects of acupressure on anxiety: a systematic review and meta-analysis.</a> Acupuncture in Medicine: Journal of the British Medical Acupuncture Society. 33 (5), 353-359 (2015).</li><li>Hsiung, W. -. T., Chang, Y. -. C., Yeh, M. -. L., Chang, Y. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acupressure+improves+the+postoperative+comfort+of+gastric+cancer+patients:+A+randomised+controlled+trial.">Acupressure improves the postoperative comfort of gastric cancer patients: A randomised controlled trial.</a> Complementary Therapies in Medicine. 23 (3), 339-346 (2015).</li><li>Wu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+interconnection+between+acupoint+&amp;#34;Ch&#233;ngsh&#257;n+(BL57)&amp;#34;+and+sciatic+nerve+in+the+rat.">Neural interconnection between acupoint &amp;#34;Ch&#233;ngsh&#257;n (BL57)&amp;#34; and sciatic nerve in the rat.</a> World Journal of Acupuncture - Moxibustion. 31 (2), 129-135 (2021).</li><li>Banik, R. K., Kabadi, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+Hargreaves+method+for+assessing+threshold+temperatures+for+heat+nociception.">A modified Hargreaves method for assessing threshold temperatures for heat nociception.</a> Journal of neuroscience methods. 219 (1), 41-51 (2013).</li><li>Cheah, M., Fawcett, J. W., Andrews, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Thermal+pain+sensation+in+rats+and+mice+using+the+hargreaves+test.">Assessment of Thermal pain sensation in rats and mice using the hargreaves test.</a> Bio-protocol. 7 (16), e2506 (2017).</li><li>Randall, L. O., Selitto, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+measurement+of+analgesic+activity+on+inflamed+tissue.">A method for measurement of analgesic activity on inflamed tissue.</a> Archives Internationales De Pharmacodynamie Et De Therapie. 111 (4), 409-419 (1957).</li><li>Yan, J. -. t. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Science of Tuina. , (2003).</li><li>Himes, B. T., Tessler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Death+of+some+dorsal+root+ganglion+neurons+and+plasticity+of+others+following+sciatic+nerve+section+in+adult+and+neonatal+rats.">Death of some dorsal root ganglion neurons and plasticity of others following sciatic nerve section in adult and neonatal rats.</a> The Journal of Comparative Neurology. 284 (2), 215-230 (1989).</li></ol>

作者没有什么可透露的。

课题组对早期推拿操作的参数进行了相关研究。首先，设置推拿操作的力强度很重要。在临床推拿中，从业者根据自己的经验和患者的主观感受调整用力强度，通过沟通达到最佳的推拿效果。然而，这在动物实验中是不可行的。在动物实验中，“反应阈值”用于定义推拿操作的强度。这些根据动物的本能反应来判断施加的推拿力是否合适。Randall17 使用这种方法来研究大鼠炎症模...

在临床环境中，由于各种原因，观察到神经根受压引起的神经系统病理性疼痛是很常见的。这种神经性疼痛最典型的形式是腰椎间盘突出症 （LDH），它通常是持续的、反复发作的且难以治愈。全球约有 9% 的人口受到乳酸脱氢酶的影响，导致严重的社会和经济负担1.由于人类生产和生活方式的改变，这种类型的神经性疼痛的发病率逐年增加，患者有年轻化的趋势2.尽管使用非甾体类止痛药，但患者的症状仍不能完全缓解。因此，用于治疗乳酸脱氢酶引起的疼痛的替代疗法（如推拿）越来越受到关注。
推拿疗法是乳酸脱氢酶的一种保守治疗形式，在世界范围内的各种临床实践指南中被广泛推荐用于预防和治疗腰痛 3,4。研究表明，推拿可显著降低乳酸脱氢酶患者的血清IL-6和肿瘤坏死因子-α（TNF-α）等炎症因子，同时改善患者的疼痛和腰椎功能损害5。然而，推拿疗法缓解疼痛效果背后的具体机制尚不清楚。
动物模型是研究 LDH6 引起的神经性疼痛的宝贵工具。它们允许进行行为测量以评估推拿疗法的有效性，并提供乳酸脱氢酶的病理生理学样本。例如，可以从大腿背根神经节采集样本来验证背根神经节细胞的变化。背根神经节 （CCD） 模型的慢性压迫通常用于评估 LDH 的病理生理学，因为它对背根神经节细胞的形态造成损害，这与椎间盘突出引起的神经压迫临床病例中观察到的病理变化一致7.
许多学者对穴位按压镇痛进行了几次动物实验8,9,10。然而，在动物模型上实施穴位按摩操作时，他们经常模仿人类穴位按摩。穴位按摩的治疗效果受大小、频率和施加力的方向等因素的影响11,12,13。如果实验缺乏统一的穴位按摩标准，如手术的力度、频率、持续时间等，可能会导致实验结果出现一定偏差。本文介绍了一套基于CCD大鼠特点的穴位按压治疗方案，并促进了动物模型中标准化穴位按压操作的发展。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#34;L&#34; stainless steel rod (4 mm long and 0.4 mm in diameter)</td>
			<td>hand-made</td>
			<td>/</td>
			<td>For CCD models making</td>
		</tr>
		<tr>
			<td>ALMEMO admeasuring apparatus</td>
			<td>ahlborn</td>
			<td>2450-1</td>
			<td>Mechanical Withdrawal Threshold test</td>
		</tr>
		<tr>
			<td>Constant temperature slicer CM-1900</td>
			<td>Leica</td>
			<td>1491950C1US</td>
			<td>For specimen production</td>
		</tr>
		<tr>
			<td>Disinfectant (iodine) 100 mL/bottle</td>
			<td>LIRCON/Shandong Lilkang</td>
			<td>/</td>
			<td>For disinfection</td>
		</tr>
		<tr>
			<td>Disposable sterile syringe 5 mL</td>
			<td>Shanghai Misha Wa Medical Industry</td>
			<td>/</td>
			<td>For injection</td>
		</tr>
		<tr>
			<td>Electron microscope CX-31</td>
			<td>Olympus, Japan</td>
			<td>BJ002318</td>
			<td>For specimen observation</td>
		</tr>
		<tr>
			<td>Finger pressure recordings</td>
			<td>Suzhou Changxian Optoelectronic Technology</td>
			<td>CX1003w</td>
			<td>For Tuina manipulation</td>
		</tr>
		<tr>
			<td>Foam board (35 cm x 20 cm)</td>
			<td>hand-made</td>
			<td>/</td>
			<td>It is our homemade apparatus for fixing rats</td>
		</tr>
		<tr>
			<td>MERSILK W2512</td>
			<td>Johnson &#38; Johnson</td>
			<td>/</td>
			<td>For tissue suture</td>
		</tr>
		<tr>
			<td>Neutral balsam</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10004160</td>
			<td>For specimen production</td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>China National Chemical Reagent</td>
			<td>/</td>
			<td>For specimen production</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Sigma-Aldrich</td>
			<td>P3761</td>
			<td>For anesthesia of rat</td>
		</tr>
		<tr>
			<td>Plantar Test Apparatus (Hargreaves Method) for Mice and Rats</td>
			<td>IITC Life Science</td>
			<td>/</td>
			<td>Paw Withdrawal Latency</td>
		</tr>
		<tr>
			<td>Precision electronic scale for experiment JY3002</td>
			<td>Shanghai Precision Scientific Instrument</td>
			<td>/</td>
			<td>Weighing of rat</td>
		</tr>
		<tr>
			<td>Rat hair clipper</td>
			<td>Philips</td>
			<td>HP6341/00</td>
			<td>Shaving of rat fur</td>
		</tr>
		<tr>
			<td>Restrainer for rats</td>
			<td>Tongji University (self-made)</td>
			<td>/</td>
			<td>It is a homemade apparatus made by Tongji University, which can effectively immobilize the rats and fully expose their hind limbs.</td>
		</tr>
		<tr>
			<td>Tissue-Tek&#160;O.C.T. Compound</td>
			<td>SAKURA</td>
			<td>4583</td>
			<td>For specimen production</td>
		</tr>
		<tr>
			<td>Uratan</td>
			<td>China National Chemical Reagent</td>
			<td>/</td>
			<td>For anesthesia of rat</td>
		</tr>
		<tr>
			<td>X-ray detector XR-600</td>
			<td>Dongguan Kaso Electronic Technology</td>
			<td>/</td>
			<td>Examination of CCD models</td>
		</tr>
		<tr>
			<td>xylene</td>
			<td>Shanghai Sinopharm Group</td>
			<td>100092</td>
			<td>For specimen production</td>
		</tr>
	</tbody>
</table>

analgesic effect of tuina on rat models with compression of the dorsal root ganglion pain

神经性疼痛是一种普遍存在的疾病，影响 6.9%-10% 的人口，由各种病因引起的神经损伤引起，例如腰椎间盘突出症、椎管狭窄和椎间孔狭窄。虽然推拿作为一种传统的中国手法疗法，在临床上显示出治疗神经性疼痛的镇痛作用，但其潜在的神经生物学机制尚不清楚。动物模型对于阐明推拿的基本原理至关重要。在这项研究中，我们提出了一种标准化的推拿方案，用于压迫背根神经节 （DRG） 的大鼠，其中包括通过将不锈钢棒插入椎间孔来诱导 DRG 压迫，在受控环境中以特定的位置、强度和频率参数进行推拿操作，并评估推拿治疗的行为和组织病理学结果。本文还讨论了该研究的潜在临床意义和局限性，并提出了未来推拿研究的方向。

In clinical settings, it is common to observe neurological pathological pain caused by nerve root compression due to various reasons. The most typical form of this neuropathic pain is lumbar disc herniation (LDH), which is often persistent, recurrent, and difficult to cure. Approximately 9% of the global population is affected by LDH, leading to significant social and economic burdens1. The incidence of this type of neuropathic pain is increasing yearly, with a trend toward younger patients, due to changes in human production and lifestyle2. Despite the use of non-steroidal painkillers, patients&#39; symptoms cannot be completely alleviated. As a result, alternative therapies, such as Tuina, for treating pain caused by LDH have gained increasing attention.
Tuina therapy, a form of conservative treatment for LDH, is widely recommended in various clinical practice guidelines worldwide for preventing and treating lower back pain3,4. Research has shown that Tuina can significantly lower inflammatory factors such as serum IL-6 and tumor necrosis factor-alpha (TNF-&#945;) levels in LDH patients while improving patients&#39; pain and lumbar function impairment5. However, the specific mechanism behind Tuina therapy&#39;s pain-relieving effects remains unclear.
Animal models are a valuable tool for studying neuropathic pain caused by LDH6. They allow for behavioral measurements to evaluate the effectiveness of Tuina therapy and provide samples of the pathological physiology of LDH. For example, samples from the dorsal root ganglia in the thigh can be taken to verify changes in dorsal root ganglion cells. The chronic compression of the Dorsal Root Ganglion (CCD) model is commonly used to evaluate the pathological physiology of LDH, as it causes damage to the morphology of dorsal root ganglion cells that are consistent with the pathological changes seen in clinical cases of nerve compression caused by disc herniation7.
Many scholars have conducted several animal experiments on acupressure analgesia8,9,10. However, when implementing acupressure operations on animal models, they often imitate human acupressure. The therapeutic effect of acupressure is affected by factors such as the size, frequency, and the direction of the applied force11,12,13. If the experiment lacks a unified acupressure standard, such as the force, frequency, and duration of the operation, this may cause some deviation in the experimental results. This article introduces a set of acupressure treatment plans based on the characteristics of CCD rats, and promotes the development of standardized acupressure operations in animal models.

作者聚焦： 推拿对压迫背根神经节疼痛的大鼠模型的镇痛作用  

推拿对大鼠模型的镇痛作用，压迫背根神经节疼痛

Tuina analgesia is widely used in clinical practice. Our research focuses on the mechanism of action of Tuina analgesia, and investigate how Tuina achieve its analgesic effect. Achieving standardization in Tuina is currently the most significant obstacle.The effectiveness of Tuina is influenced by several factors such as treatment, area type, intensity, and frequency. The current protocol is optimized in two aspect based on the earlier research. Firstly, the force and frequency of pushing and holding operations were standardized and controlled by force-measuring gloves.Secondly, the results of imaging and animal behavior test were integrated to assess the results of modeling.

推拿疗法可以帮助降低CCD建模引起的大鼠机械和热刺激阈值 推拿治疗17天后，接受推拿治疗的CCD大鼠与未治疗的CCD组之间的PWT阈值存在显着差异（P = 0.021，<0.05）（图6 和 表1）。
CCD组接受推拿治疗的大鼠从治疗开始疼痛阈值就有所改善，取模后第14天CCD组与接受推拿治疗组的热痛阈值差异有统计学意义（P = 0.0047?...

Watch this Scientific Journal Video about Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain at JoVE.com

本文介绍了一种使用推拿疗法治疗大鼠背根神经节慢性压迫的操作方法，以及一种基于疼痛行为和组织病理学结果评估其有效性的方法。

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar ...

推拿镇痛在临床上应用广泛。我们的研究重点是推拿镇痛的作用机制，并探讨推拿如何达到镇痛效果。实现推拿标准化是目前最大的障碍。推拿的有效性受多种因素影响，例如治疗、区域类型、强度和频率。在前期研究的基础上，从两个方面对当前协议进行了优化。首先，通过测力手套对推力和保持操作的力和频率进行标准化和控制;其次，将影像学结果与动物行为试验结果相结合，对建模结果进行评价;

analgesic-effect-tuina-on-rat-models-with-compression-dorsal-root

Research

JoVE Journal

Medicine

Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain

922 次观看.  Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Shanghai University of Traditional  Chinese Medicine. 推拿镇痛在临床上应用广泛。我们的研究重点是推拿镇痛的作用机制，并探讨推拿如何达到镇痛效果。实现推拿标准化是目前最大的障碍。推拿的有效性受多种因素影响，例如治疗、区域类型、强度和频率。在前期研究的基础上，从两个方面对当前协议进行了优化。首先，通过测力手套对推力和保持操作的力和频率进行标准化和控制;其次，将影像学结果与动物行?...

视频: 作者聚焦： 推拿对压迫背根神经节疼痛的大鼠模型的镇痛作用  

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar disc herniation, spinal canal stenosis, and intervertebral foramen stenosis. Although Tuina, a traditional Chinese manual therapy, has shown analgesic effects in clinical practice for the treatment of neuropathic pain, its underlying neurobiological mechanisms remain unclear. Animal models are essential for elucidating the basic principles of Tuina. In this study, we propose a standardized Tuina protocol for rats with compression of the dorsal root ganglion (DRG), which involves inducing DRG compression by inserting a stainless steel rod into the intervertebral foramen, performing Tuina manipulation with specific parameters of location, intensity, and frequency in a controlled environment, and assessing the behavioral and histopathological outcomes of Tuina treatment. This article also discusses the potential clinical implications and limitations of the study and suggests directions for future research on Tuina.

This article presents a manipulation for treating chronic compression of the dorsal root ganglion in rats using Tuina therapy, along with a method for evaluating its effectiveness based on pain behavior and histopathological results.

科研

医学

Establishment of Murine Chronic Root Ganglion Compression Model

To establish the animal model, start by securing an anesthetized rat on a foam board, fastening its limbs and incisors using rubber bands. Wipe the prepared area with a 75%alcohol swab. Next, use scissors to make a two to three centimeter incision through the skin, superficial fascia, and deep fascia layer by layer.Locate the anterior superior iliac spine, followed by the third and fourth spinous processes. Clamp the spinous process with toothed forceps and lift it to allow the scissors to be close to the right side of the spinous process. Cut the muscle attached to the right side of the spinous process.Then bluntly dissect the muscle attached to the outer surface of the vertebral plate until resistance is felt. Similarly, bluntly dissect the muscle and fascia on the zygapophysial joint. Use an L-shaped probe to locate the intervertebral foramen.Next, insert an L-shaped stainless steel rod into the fourth and fifth lumbar intervertebral foramen. Proceed to suture the muscle, fascia, and skin layer by layer using 3-0 sutures. Place the rat in a thermostatic box until it wakes up and observe the function of the rat's right hind limb to ensure normalcy.

小鼠慢性根神经节压迫模型的建立

Tuina Therapy Administration to Murine Root Ganglion Compression Model

To standardize the tuina therapy, first, practice tuina using the finger sleeves feedback data, adjusting force to 5 newtons and frequency to 2 hertz. Next, perform tuina on immobilized rats, maintaining consistent force and frequency. Identify the acupoint by selecting the gastrocnemius muscle of the right hind limb at the junction of its two heads, approximately at BL 57.Perform the tuina by positioning the thumb vertically on the BL 57 acupoint and moving in rhythmic, small-range rotary motions exerting 5 newtons of pressure. After 17 days of tuina therapy, a significant difference was observed in paw withdrawal thresholds between the CCD rats receiving tuina therapy and the untreated CCD group. The rats in the CCD group receiving tuina therapy showed improvement in pain threshold from the beginning of treatment.Thermal pain thresholds differed significantly between the CCD group and the tuina group from day 14.

推拿治疗小鼠根神经节压迫模型

Murine Pain Behavioral Testing to Study the Effect of Tuina Therapy

To conduct the Von Fray test, begin by placing the rats in a transparent tempered glass compartment on a metal wire grid stand at a height of 40 centimeters. Next, measure the mechanical withdrawal threshold with Von Frey electronic fibers. Stimulate the rat's foot until it reacts noticeably, such as by lifting the leg or avoiding it.To assess the paw withdrawal latency, or PWL using the Hargreaves method, focus the spotlight on the center of the rat's foot and press the start button. Monitor the rat for behaviors such as foot retraction or paw licking, and press the start button again to record the time. Perform the test on the same hind limb three consecutive times.After 17 days of Tuina therapy, a significant difference was observed in paw withdrawal thresholds between the CCD rats receiving Tuina therapy and the untreated CCD group. The rats in the CCD group receiving Tuina therapy showed improvement in pain threshold from the beginning of treatment. Thermal pain thresholds differed significantly between the CCD group and the Tuina group from day 14.

作者聚焦： 推拿对压迫背根神经节疼痛的大鼠模型的镇痛作用

作者聚焦：推拿对压迫背根神经节疼痛的大鼠模型的镇痛作用