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

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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型中的疼痛行为。不同的是，Randall在局部炎症区域进行手术，而这个实验在非损伤区域进行手术。重度推拿操作会使老鼠产生尖叫和逃避反射。此重力强度的设置决定了正式的推拿力强度小于此重力强度。基于150只大鼠在5-25 N之间最大尖叫和逃逸反射压力值的分布，平均值和标准差分别为9.93和3.018（图11），我们之前的工作计算出最佳压力值为5 N。其次，推拿法的工作频率在《推拿科学》中描述为120-160次/分18。因此，本实验中的推拿频率设置为2 Hz。
CCD 模型是一种可能具有创伤性的手术模型，因为它需要暴露于关节突和椎间孔。因此，大鼠在手术后需要1-3天才能恢复。根据文献和初步实验数据，大鼠在术后第4天疼痛行为趋于稳定。这为观察推拿干预的镇痛效果提供了有利时机。然而，长期推拿可能不利于大鼠的镇痛作用，并可能导致组织损伤。本研究比较了推拿与揉捏5、15、30 min的镇痛效果，最终选择15 min作为干预的最佳持续时间。
这种方法有局限性，因为只有一部分临床操作用于选择操作和身体部位。BL57（成山）位于大鼠的肱三头肌中，由L4和L5的背根神经节支配19。由于肱三头肌较厚，适合推拿，按摩该区域可对用于实验取样的大鼠腰椎L4和L5背根神经节产生直接影响。
在成型过程中，需要通过X射线验证来确保“L”形不锈钢棒准确插入CCD疼痛模型中L4和L5的椎间孔。在拍摄 X 射线之前，我们给予腹膜内 25% 持续时间的麻醉 （0.6 mL/100 g） 以麻醉大鼠。X射线证实“L”形不锈钢棒成功插入L4和L5的椎间孔（图12）。
本研究主要通过观察推拿治疗操作，评价推拿对CCD大鼠的治疗效果。该团队进行了动物实验，探索如何设置推拿参数，选择合适的部位，并确定合适的治疗时间。这为未来推拿镇痛效果的研究提供了标准化、可重复的动物模型干预示范。

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><tbody><tr><td>&quot;L&quot; 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 &amp;amp; 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&amp;nbsp;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。

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、0.0056、0.0049、<0.01）（图7 和 表2）。
推拿疗法的应用并未改善CCD模型引起的细胞坏死 基于背根神经节的HE染色，CCD组大鼠用“L”形不锈钢棒进行物理压缩，发生细胞膜损伤和细胞凋亡（图8）。相比之下，对照组大鼠具有整齐的边缘，完整的神经元轮廓和完整的细胞体（图9）。在CCD+推拿组中，大鼠背根神经节中的一些神经元轮廓不完整（图10）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65535/65535fig01.jpg"> 图1：大鼠限制器。 它是同济大学自制的仪器，可以有效地固定老鼠，充分暴露其后肢。银色螺丝控制着固定老鼠尾巴的挡板并约束老鼠。黑色螺钉将设备调整到大鼠的长度。 <a href="https://www.jove.com/files/ftp_upload/65535/65535fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65535/65535fig02.jpg"> 图 2：佩戴无线触觉力测量指套。 测量和显示手指按压力和频率的设备，在推拿操作过程中提供有关强度和频率的实时反馈。（a） 压力传感器和传动设备。（b） 手指压力测量。（c） 推拿过程中测得的力。 <a href="https://www.jove.com/files/ftp_upload/65535/65535fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65535/65535fig03.jpg"> 图3：大鼠推拿的身体位置和穴位。 大鼠约束器可以完全暴露BL57的位置。四肢与拇指一起抓住大鼠的下肢，将其固定到位，以便大鼠在治疗过程中可以安静地配合。 <a href="https://www.jove.com/files/ftp_upload/65535/65535fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65535/65535fig04.jpg"> 图4.爪子退出阈值测试。 这是一项测量大鼠疼痛行为的测试。它涉及机械刺激他们的脚并测量他们的爪子退出潜伏期。（a）显示了该装置，（b）显示了在实验过程中如何在小鼠身上操作该装置。 <a href="https://www.jove.com/files/ftp_upload/65535/65535fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65535/65535fig05.jpg"> 图 5：爪子拔出延迟测试。 这种设置通过大鼠对聚光灯产生的热量的敏感性来测量大鼠的疼痛阈值。 <a href="https://www.jove.com/files/ftp_upload/65535/65535fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65535/65535fig06.jpg"> 图 6：爪子退出阈值测试的结果。测量值使用平均值正负标准误差 （）<img alt="Equation 1" src="/files/ftp_upload/65535/65535eq01.jpg" style="margin: auto;"> 表示。机械刺激诱导的各组大鼠不同阶段后肢戒断反射阈值试验结果。从第17天开始，CCD+推拿组与CCD模型组<差异有统计学意义（P 0.05）。 <a href="https://www.jove.com/files/ftp_upload/65535/65535fig06large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65535/65535fig07.jpg"> 图 7：爪子拔出延迟测试的结果。 测量值使用平均值正负标准误差 （）<img alt="Equation 1" src="/files/ftp_upload/65535/65535eq01.jpg" style="margin: auto;"> 表示。各组大鼠不同阶段热刺激诱导的下肢退缩反射阈值检测结果。CCD+推拿组与CCD模型组从第7天开始比较，差异有统计学意义（P < 0.05）。 <a href="https://www.jove.com/files/ftp_upload/65535/65535fig07large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65535/65535fig08.jpg"> 图8：CCD组背根神经节神经元的显微镜观察（纵向切片）。 该图像是通过扫描背根神经节的 HE 染色样本获得的。图中的红色椭圆表示一些神经元正在经历坏死。这表明CCD模型对背根神经节中的神经元造成了损害。比例尺 = 50 μm. <a href="https://www.jove.com/files/ftp_upload/65535/65535fig08large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/65535/65535fig09.jpg"> 图 9.幼稚组背根神经节神经元的显微镜观察（纵切面和横截面）。 如上图所示，（a）为纵截面，（b）为横截面。大鼠背根神经节空白群聚集区无CCD组空缺现象。神经元是连续和紧凑的，细胞体被填充。卫星神经胶质细胞位于神经元之间。比例尺：（a），50 μm;（b）， 100 μm. <a href="https://www.jove.com/files/ftp_upload/65535/65535fig09large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/65535/65535fig10.jpg"> 图10：CCD+推拿组背根神经节神经元的显微观察（横截面）。 神经元是连续的，并且存在细胞溶解现象。这表明，在短期内，按摩疗法无法改善CCD模型引起的细胞坏死（红色椭圆）。比例尺 = 100 μm。 <a href="https://www.jove.com/files/ftp_upload/65535/65535fig10large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/65535/65535fig11.jpg"> 图11：推拿压力测试。 具有最小嘶嘶声逃逸反射压缩值的大鼠大小各不相同，范围从5N到25N。横坐标代表压力程度，纵坐标代表老鼠的数量。 <a href="https://www.jove.com/files/ftp_upload/65535/65535fig11large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/65535/65535fig12.jpg"> 图12：CCD大鼠的X射线图像。 使用大鼠模型的 X 射线成像验证 CCD 制造。（a） 显示在垂直平面上拍摄的 X 射线，以及 （b） 显示在矢状面上拍摄的 X 射线。两种不同的横截面 X 射线可以更清楚地看到“L”形不锈钢棒的位置。 <a href="https://www.jove.com/files/ftp_upload/65535/65535fig12large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td>
			<td>N</td>
			<td>建模前</td>
			<td>第1天</td>
			<td>第3天</td>
			<td>第7天</td>
			<td>第14天</td>
			<td>第17天</td>
			<td>第21天</td>
		</tr><tr><td>幼稚</td>
			<td>8</td>
			<td>18.1±1.4</td>
			<td>16.8+1.5</td>
			<td>16,8±1,5</td>
			<td>18.6±1.7</td>
			<td>16,8±1,5</td>
			<td>15,6±1,8</td>
			<td>16,8±1,5</td>
		</tr><tr><td>假</td>
			<td>8</td>
			<td>18.0±1.7</td>
			<td>18.6±1.7</td>
			<td>16.0±1.3</td>
			<td>17,6±1,7</td>
			<td>16,8±1,5</td>
			<td>18.9±1.7</td>
			<td>16,8±1,5</td>
		</tr><tr><td>CCD系列</td>
			<td>8</td>
			<td>17.7±1.1</td>
			<td>10.7±2.8#</td>
			<td>10.0±1.5</td>
			<td>4.4±2.2</td>
			<td>3.8±1.7</td>
			<td>4.1±2.4</td>
			<td>3.8±2.5</td>
		</tr><tr><td>CCD+推拿</td>
			<td>8</td>
			<td>18.9±1.7</td>
			<td>9.8±2.3#</td>
			<td>8.3±1.4</td>
			<td>4.8±1.2</td>
			<td>5.8±2.0</td>
			<td>7.2±1.8*</td>
			<td>7.5±1.8*</td>
		</tr><tr><td colspan="9">#建模前和 D1 建模前的比较，第 <0.05 页。</td>
		</tr><tr><td colspan="9">*CCD组与CCD+推拿组的比较，第<0.05页。</td>
		</tr></tbody></table>表 1：爪子退出阈值，PWT （<img alt="Equation 1" src="/files/ftp_upload/65535/65535eq01.jpg" style="margin: auto;">， s）。建模前和 D1 建模的比较， #p < 0.05。CCD组与CCD+推拿组比较，*p < 0.05。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td>
			<td>N</td>
			<td>建模前</td>
			<td>第1天</td>
			<td>第3天</td>
			<td>第7天</td>
			<td>第14天</td>
			<td>第17天</td>
			<td>第21天</td>
		</tr><tr><td>幼稚</td>
			<td>8</td>
			<td>11.9±1.2</td>
			<td>12.0±1.6</td>
			<td>12.2±1.9</td>
			<td>12.4±1.1</td>
			<td>12.2±1.9</td>
			<td>12.0±1.4</td>
			<td>12.0±1.4</td>
		</tr><tr><td>假</td>
			<td>8</td>
			<td>11.9±1.2</td>
			<td>11.6±1.5</td>
			<td>12.2±1.9</td>
			<td>11.6±1.5</td>
			<td>12.2±0.9</td>
			<td>11.6±1.5</td>
			<td>11.6±1.5</td>
		</tr><tr><td>CCD系列</td>
			<td>8</td>
			<td>10.8±1.1</td>
			<td>8.9±0.7#</td>
			<td>7.9±0.8</td>
			<td>7.7±0.5</td>
			<td>7.8±1.0</td>
			<td>7.7±0.8</td>
			<td>7.7±0.8</td>
		</tr><tr><td>CCD+推拿</td>
			<td>8</td>
			<td>11.3±1.5</td>
			<td>9.1±0.6#</td>
			<td>8.0±0.7</td>
			<td>8.3±0.7*</td>
			<td>8.9±0.6*</td>
			<td>9.1±0.7*</td>
			<td>9.2±0.9*</td>
		</tr><tr><td colspan="9">#建模前和 D1 建模前的比较，p < 0.05。</td>
		</tr><tr><td colspan="9">CCD组与CCD+推拿组的比较，*p < 0.05。</td>
		</tr></tbody></table>表 2：爪子退出延迟，PWL （<img alt="Equation 1" src="/files/ftp_upload/65535/65535eq01.jpg" style="margin: auto;">， s）。建模前和 D1 建模的比较，#p < 0.05。CCD组与CCD+推拿组的比较，*p < 0.05。

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

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  

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。

推拿对大鼠模型的镇痛作用，压迫背根神经节疼痛

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

Nanyang Technological University

Research

JoVE Journal

Medicine

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

1.3K 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  

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

胃食管反流与小鼠手术模型

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

科研

医学

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

骨关节炎疼痛的鼠标Monoiodoacetate模式

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.

We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

比浊法对人洗涤血小板:所述Pannexin1抑制剂的影响亮蓝FCF对胶原诱导的聚集

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

A basic protocol to evaluate the toxicity of chemicals in a model animal, Caenorhabditis elegans, is described. The method is convenient and useful for the development of pharmaceuticals as well as for the risk assessment of various environmental pollutants.

测定化学物质对线虫线虫生长和繁殖的影响

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

人体滋养层模型研究母体肥胖炎症对胎盘自噬的调节作用

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

Spontaneous and Evoked Measures of Pain in Murine Models of Monoarticular Knee Pain

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are necessary and useful for studying the mechanisms of pain and for evaluating efficacy of arthritis therapies. Measuring pain in animal models of arthritis is challenging. We have developed methods for measuring evoked and spontaneous pain in three models of murine arthritis. We quantitate the evoked pain responses of mice subjected to firm palpation of a painful knee. We also evaluate spontaneous pain by the proportion of weight and the amount of time placed on each of their 4 limbs after induction of arthritis pain in one knee. Joint pain in these mouse models produces a significant increase in evoked pain responses and an alteration in weight bearing. Since mice are quadrupeds, they offload the painful limb to the contralateral limb, to the forelimbs, or some combination. These methods are simple, require minimal equipment, and are reproducible and sensitive for detecting pain. They are useful for studying both disease-modifying arthritis treatments and analgesics in mice.

We have developed an evoked measure of arthritis pain and coupled it with a standardized method for measuring spontaneous pain in different murine models of chemically induced arthritis. These measures are sensitive and reproducible for different types of joint pain.

单关节膝关节疼痛小鼠模型疼痛的自发性和诱发性测量

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are ...

Three-Dimensional Finger Motion Tracking during Needling: A Solution for the Kinematic Analysis of Acupuncture Manipulation

Three-dimensional (3D) motion tracking has been used in many fields, such as the researches of sport and medical skills. This experiment aimed to use 3D motion tracking technology to measure the kinematic parameters of the joints of fingers during acupuncture manipulation (AM) and establish three technical indicators "amplitude, velocity and time". This method can reflect the operation characteristics of AM and provide quantitative parameters along three axes of multiple finger joints. The current evidence shows that the method has great potential for future applications such as the study of the dose-effect relationship of acupuncture, teaching, and learning of AM, and the measurement and preservation of famous acupuncturists' AM.

This experimental method describes a solution for the kinematic analysis of acupuncture manipulation with three-dimensional finger motion tracking technology.

针刺过程中的三维手指运动跟踪:针灸操作运动学分析的解决方案

Three-dimensional (3D) motion tracking has been used in many fields, such as the researches of sport and medical skills. This experiment aimed to use 3D ...

<meta charset="utf8"></head><body>评估推拿疗法对慢性缩窄大鼠的疼痛缓解效果

Characteristics of Pain Changes in Rats with Nerve Injury Within 24 hours After One-Time Tuina Intervention

Tuina, as an external treatment method of traditional Chinese medicine, has been proven to have an analgesic effect on peripheral neuropathic pain (pNP) in clinical and basic research. However, the optimal time point for the analgesic effect of tuina may vary according to different injury sensations, affecting the exploration of the initiation mechanism of tuina analgesia. 
The research used minor chronic constriction injury (minor CCI) model rats to simulate pNP and used the intelligent tuina manipulation simulator to simulate the three methods (point-pressing, plucking, and kneading) and three acupoints (Yinmen BL37, Chengshan BL57, and Yanglingquan GB34) for performing tuina therapy. The study evaluated the changes in pain within 24 h and the optimal time point for the efficacy of tuina analgesia in rats with minor CCI models by testing cold sensitivity threshold (CST), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL). Furthermore, the study evaluated IL-10 and TNF-&#945; expression changes through Elisa detection. The results show that tuina has both immediate and sustained analgesic effects. For the three different injury sensitivity thresholds of CST, MWT, TWL, and two cytokines of IL-10 and TNF-&#945;, the analgesic efficacy of tuina within 24 h after intervention is significantly different at different time points.

<meta charset="utf8"></head><body>作者聚焦：探索神经损伤大鼠“三法三穴”推拿镇痛的启动机制

We present a protocol using the tuina manipulation simulator to perform the "Three-Manipulation and Three-Acupoint" tuina therapy for minor chronic constriction injury rats and evaluate the effective analgesic time points of tuina within 24 h by testing pain changes through behavioral analysis and changes in inflammatory factor expression using enzyme-linked immunosorbent assay.

神经损伤大鼠1次推拿干预后24 h内疼痛变化特征

Tuina, as an external treatment method of traditional Chinese medicine, has been proven to have an analgesic effect on peripheral neuropathic pain (pNP) ...

推拿对大鼠模型的镇痛作用，压迫背根神经节疼痛

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此视频中的章节

胃食管反流与小鼠手术模型

骨关节炎疼痛的鼠标Monoiodoacetate模式

比浊法对人洗涤血小板:所述Pannexin1抑制剂的影响亮蓝FCF对胶原诱导的聚集

测定化学物质对线虫线虫生长和繁殖的影响

人体滋养层模型研究母体肥胖炎症对胎盘自噬的调节作用

单关节膝关节疼痛小鼠模型疼痛的自发性和诱发性测量

针刺过程中的三维手指运动跟踪:针灸操作运动学分析的解决方案

神经损伤大鼠1次推拿干预后24 h内疼痛变化特征

神经损伤大鼠1次推拿干预后24 h内疼痛变化特征

神经损伤大鼠1次推拿干预后24 h内疼痛变化特征