这项工作得到了山东省中医药科技计划项目（2021Q080）和齐鲁中医药学院学校传承项目[鲁维信（2022）93]的支持。

该研究获得山东中医药大学附属医院伦理委员会批准（批准文号：2020-29）。
1.实验动物
<ol><li>在标准单笼（12小时光/暗循环，温度20-24°C，空气湿度40%-60%）中饲养18,6个月大的正常级雄性新西兰兔（2.75±0.25kg）。</li>
</ol>2.分组方式
<ol><li>使用随机数法从 18 只新西兰兔子中选择 6 只作为假组，并将剩余的 12 只兔子分配到建模组。</li>
	<li>建模成功后，按随机数法将建模组兔子分为模型组和推拿组，每组6只兔子。</li>
	<li>在推拿组进行推拿干预。在没有推拿的情况下，在假组和模型组中进行相同的抓握和固定。每隔一天运行一次，持续 4 周（图 1）。</li>
</ol>3. KOA模型的建立
<ol><li>在第 1 周对处于标准状态的兔子进行适应性喂养。随意获得水和食物。将兔子右侧放在兔子固定盒中，使它们每天平静15分钟。将他们的头固定在头部固定板上。固定紧固板和螺钉，使兔子无法移动。抓握和固定兔子时戴上防护手套（图1）。</li>
	<li>在第 2 周的第 1、4 和 7 天，将所有 18 只兔子放在兔子固定盒的右侧（图 1）。执行以下操作。</li>
	<li>将3%戊巴比妥钠（1ml / kg）注射到兔子的边缘耳静脉中。用动物剃须刀剃掉兔子的左膝关节，使裸露的皮肤上没有毛发。</li>
	<li>使用医用碘伏和75%酒精从内到外对兔子的左膝关节进行消毒（图2A）。</li>
	<li>将兔子的左膝关节弯曲 60°。插入 Waixiyan 的针头（22G，0.7 mm x 30 mm）。将4%木瓜蛋白酶溶液（0.1mL / kg，2.5kg动物平均0.275mL）注射到建模组的膝关节腔中。将等量的0.9%氯化钠溶液注入假基团中。这种注射剂量对动物的耐受性良好，而不会引起疼痛或痛苦的证据（图2B）。 注：外西燕（EX-LE5）位于髌韧带外侧凹陷，内西燕（EX-LE4）位于髌韧带内侧凹陷20、21、22。</li>
	<li>按压针孔2分钟，以避免溶液溢出。</li>
	<li>将双手放在兔子左膝关节的上方和下方。轻轻被动地弯曲兔子的膝关节，并在生理运动范围（ROM）内伸展10次，使溶液均匀地渗入膝关节腔15。在建模期间每8小时观察一次兔子。当兔子出现隐藏、四肢颤抖、呼吸浅而急促，甚至咬伤和抓挠的迹象时，给予丁丙诺啡 SR （0.18 mg/kg）。</li>
	<li>在第 7 周，观察兔子的左膝屈曲姿势肿胀，膝盖周围肌张力增加，伴有结节和条纹，局部疼痛刺激反应增加，膝关节活动度降低，步态跛行，重心向健康侧转移。这决定了KOA模型的成功（图1，图2C）23,24。</li>
</ol>4. 推拿手法
<ol><li>在推拿操作之前，使用推拿技术参数测定仪进行训练。由同一位专业人员每天训练 1 小时，持续 1 个月。<ol><li>用拇指在推拿操作模拟平台上以5N的力和60次/分钟的频率进行旋转揉捏和按压方法（图3A，C）。</li>
		<li>通过推拿操作参数处理软件分析X、Y、Z轴三个方向的力，并检查屏幕上显示的力的大小、频率和作用时间（图3B、D）。</li>
		<li>评估推拿操作的机械参数，并在训练期间使用软件对推拿操作进行标准化。用拇指保持标准化的旋转揉捏和按压方法，力为5N，频率为60次/分钟，连续操作时间为10分钟。参见图3B，D 25,26,27中操作的标准化定量波形。</li>
	</ol></li>
	<li>将兔子放在兔子固定盒的右侧。轻轻抚摸兔子 10 秒，以舒缓和放松兔子21.然后进行推拿干预。</li>
	<li>用拇指在兔子的左膝周肌肉僵硬，肌腱结和髌骨上进行旋转揉捏方法，以5N的力和60次/ min的频率上下往返操作5分钟。</li>
	<li>用拇指端按杨灵泉（GB 34）、银灵泉（SP 9）、外西岩（EX-LE5）、内西烟（EX-LE4）、鹤鼎（EX-LE2）、雪海（SP 10）、良球（ST 34）、卫中（BL 40）20、21、22，力5N，频率60次/min，各点操作30秒。</li>
	<li>在兔膝关节上执行旋转矫正方法，并分别对组中的每只动物执行3次。<ol><li>用一只手固定股骨。先将另一只手放在膝关节后面，然后分别用拇指和无名指固定胫骨外侧和内侧髁。用食指和中指固定腘窝。施加牵引力和扭转力。</li>
		<li>用一只手固定股骨。用另一只手的拇指和小指固定髌骨的内侧和外侧边缘。用食指、中指和无名指固定髌骨根部。施加扭转力。</li>
		<li>保持牵引力方向与胫骨长轴平行，扭转力方向与下西岩方向一致。用手指将皮肤固定到位，以避免皮肤和手指之间发生摩擦。</li>
	</ol></li>
</ol>5. 膝关节ROM的测量
注意： 测量前，让兔子平静下来。测量统计员和操作员彼此不同。
<ol><li>在实验开始和每周结束时测量每组兔子左膝关节的活动度。</li>
	<li>将兔子放在兔子固定盒的右侧，用一只手固定其左股骨。</li>
	<li>将医用关节镜的圆圈中心与兔子左膝关节的外侧中心对齐。延长固定臂，使其平行于连接圆中心和大转子的线延伸。伸展活动臂，使其平行于胫骨的纵轴。</li>
	<li>将另一只手放在胫骨的纵轴上，距离膝关节约 9 厘米。以 750°/s 的角速度手动施加大约 850-3 g 的扭矩28.</li>
	<li>执行此操作直到兔子的膝关节不再移动。记录测角仪在关节停止移动时显示的度数;这是膝关节ROM。阅读时，请确保视线垂直于尺子表面。</li>
	<li>测量每个膝盖的 ROM 3 次并取平均值28。</li>
</ol>6.苏木精-伊红（HE）染色
<ol><li>样品采集<ol><li>在干预结束后1周（图1），将兔子右侧放在兔子固定盒中（兔子躺在右侧时更有可能保持放松）。将戊巴比妥（100mg / kg）注射到兔子的耳边静脉中，以进行人道安乐死29,30。</li>
		<li>用手术刀、剪刀和止血钳快速打开左膝腔，去除附着在股骨远端软骨周围的软组织。</li>
		<li>用咬合镊子收集股骨远端约 1 cm x 1 cm 的软骨标本，并将其放入生理盐水中进行清洁。</li>
	</ol></li>
	<li>固定和脱钙<ol><li>将软骨置于4%多聚甲醛溶液中并固定72小时。</li>
		<li>用流水冲洗12小时。在乙二胺四乙酸（EDTA）脱钙溶液中脱钙6周。每 3 天更换一次 EDTA 脱钙溶液。当骨组织变得柔软有弹性，可以很容易地弯曲，并用针头顺利刺穿时，确定脱钙的终点31。</li>
	</ol></li>
	<li>包埋部分脱水<ol><li>将试样放入自动脱水机中进行脱水。</li>
		<li>将打蜡和修剪的组织放在装有溶解石蜡的方形容器的底部1小时。将它们放入冷却炉中，直到冷却并凝固成硬块。在切片机中将石蜡包埋的组织块切成4μm的厚度。</li>
		<li>在漂白机中展开部分，然后将它们放在粘合剂载玻片上，编号，然后用切片烘烤机和烤箱烘干。</li>
	</ol></li>
	<li>脱蜡和补水<ol><li>将切片在65°C烘烤60分钟。</li>
		<li>将切片浸泡在二甲苯中 7 分钟，然后在新鲜二甲苯中再浸泡 2 轮，每次浸泡 7 分钟。</li>
		<li>将切片浸泡在无水乙醇中5分钟，然后在95%乙醇，85%乙醇和75%乙醇中分别浸泡2分钟。</li>
		<li>将切片浸泡在蒸馏水中2分钟。</li>
	</ol></li>
	<li>苏木精染色：用苏木精染色切片 20 秒。用流水冲洗部分。将切片浸泡在盐酸乙醇分馏中3秒。用自来水冲洗切片 5 分钟。</li>
	<li>曙红复染：用曙红染色切片 30 秒。用自来水冲洗部分。</li>
	<li>脱水以确保样品的透明度<ol><li>将切片放入95%乙醇中两次，每次3秒，然后放入无水乙醇中3秒。</li>
		<li>再次，将切片置于无水乙醇中1分钟，然后进行2轮二甲苯洗涤，每次1分钟。</li>
	</ol></li>
	<li>切片密封：取出切片，滴上中性口香糖密封剂，盖上盖玻片，让切片在通风橱中晾干，直到无味。</li>
	<li>拍摄样品：在光学显微镜的视场下以 100 倍的镜头观察和拍摄。</li>
	<li>评估：通过每组32 的 Mankin 评分评估软骨组织。</li>
</ol>7. 数据分析
<ol><li>使用分析软件对实验数据进行统计分析。当数据采用正态分布时，通过t检验比较两组样本，通过单因素方差分析比较多组样本。</li>
	<li>将结果表示为平均值±标准差 （SD）。使用商业软件将结果表示为统计图。差异在 p < 0.05 时具有统计学意义。</li>
</ol>

兔模型中用推拿治疗膝关节骨关节炎

作者声明没有潜在的利益冲突。

实验方案的设计对于研究推拿治疗KOA的机制尤为重要。通过在Waixiyan注射木瓜蛋白酶对兔子进行KOA建模。外显妍位于髌韧带外隐窝内，易于定位，膝关节屈曲时股骨与胫骨的关节间隙较大，便于注入膝关节腔，防止损伤周围组织，因此易于建立KOA模型34。在KOA兔推拿干预期间，模型兔被适当定位以利于推拿给药。兔子被放在兔子固定盒中，身体健康，头部固定，情绪得到安抚，全身放松，不挣扎。为了规范推拿的强度和频率，提高推拿干预的均匀性，推拿由经过严格推拿技术参数测定仪推拿操作培训的操作人员进行推拿。
该协议的关键步骤是使用推拿结合膝关节旋转矫正方法来治疗KOA。术前，从髋部到脚踝触诊兔子患下肢，重点观察膝关节周围区域，探查肌腱结和肌肉僵硬，然后屈伸膝关节观察内西燕和外西妍的身高，进行精准推拿操作。旋转揉捏按压法可缓解肌肉紧张和痉挛，改善膝关节血液循环，促进炎症物质代谢，减轻肿胀和疼痛35,36。针灸穴位是疾病的反应和治疗点，GB 34和EX-LE2是治疗KOA的高敏感点，通过刺激它们可以作用于肥大细胞，从而影响5-羟色胺、类胰蛋白酶和组胺37的释放。数据挖掘显示，GB 34、SP 9、EX-LE5、EX-LE4、EX-LE2、SP 10、ST 34、BL 40均为临床常用穴位38,39。这些穴位具有缓解肌腱和打结、活化血液循环和缓解疼痛的作用38,39.研究表明，刺激上述穴位可降低血清肿瘤坏死因子-α和白细胞介素-1β等炎症因子水平，抑制软骨细胞骨架的破坏，从而治疗KOA 39,40,41。
在主动阻力运动下进行的膝关节旋转矫正方法，与屈曲和压缩等被动运动技术相比，易于执行，并避免了调节膝关节结构的潜在损伤风险。角度影响扭矩，膝关节内部闭合扭矩和外展扭矩之间的不平衡会增加异常关节负荷，从而触发 KOA42,43,44,45。一些研究表明，推拿干预与定期阻力运动可以提高骨密度，缓解肌肉痉挛，恢复肌肉质量和力量，减轻疼痛，并有效治疗KOA 46,47,48,49,50。由于兔膝比人膝小，不能进行自主阻力运动，将原来的两人推拿改为单人双手推拿，以保证膝关节旋转矫正法在阻力12下更好的功效和易操作性。现在，通过一只手固定兔子的股骨来模拟阻力运动，另一只手通过对下西岩施加扭转和牵引力来进行膝关节旋转矫正方法，使外西岩和内西燕处于同一高度，并调整胫骨内侧和外侧平台的相对高度。膝关节还可以向内和向外调节，以调节其向内和向外的扭矩，促进轴向对准，调整股胫关节和股髌关节的对齐，恢复膝关节的正常受力结构，减轻膝关节负荷，增加膝关节稳定性，恢复膝关节的正常活动度和生理功能42， 43、44、45.
该团队之前的临床研究已经证明了这种方法在治疗KOA方面的疗效，动物研究表明，它在治疗KOA12方面比硫酸氨基葡萄糖更有效。推拿可介导白细胞介素1β（IL-1β）和细胞外信号调节激酶1/2（ERK1/2）-核转录因子κB（NF-κB）信号通路，降低兔外周血清和关节液中IL-1β的浓度，增加B细胞淋巴瘤-2（Bcl-2）的表达水平，降低ERK1/2、Bcl-2相关x蛋白、NF-κB p65和半胱氨酸天冬氨酸蛋白酶3的表达水平。这有助于调节软骨细胞的凋亡和增殖，平衡无序的软骨细胞内部环境，从而改善软骨的病理变化12。
这种方法的局限性在于推拿的干预是由人而不是机器进行的，操作者很难在推拿的强度和频率上达到完全的同质性。
综上所述，推拿可有效减轻膝关节炎症，抑制膝关节软骨退化，逐步恢复正常生理活动能力，本研究可为推拿治疗膝关节疾病的机制提供科学可行的研究方案。

膝骨关节炎（KOA）是一种膝关节退行性疾病，主要表现为膝关节疼痛、肿胀、变形、活动受限等，致残率高，女性患病率较高，2019年全球骨关节炎患者5.2781亿，其全球患病率占OA全球总患病率的60.6%1.临床上，KOA的治疗通常分为非手术治疗和手术治疗。非手术治疗包括物理治疗、药物治疗和富血小板血浆注射治疗 2,3。推拿是中医常用、安全、可靠、有效的治疗方法。本研究采用推拿结合膝关节旋转矫正法治疗KOA。推拿技术，如旋转揉捏和按压法，可以平衡肌肉组织，减轻疼痛，调节炎症因子水平，改善组织代谢，抑制关节软骨退化4,5。膝关节旋转矫正法可以调整下肢骨骼和关节的对齐方式，改善膝关节间隙，恢复正常受力线，平衡下肢生物力学6,7,8,9。抗阻运动可以增强肌肉质量和力量，促进软骨组织更新10,11。一项初步研究发现，这种推拿方案在治疗 KOA 方面明显比口服硫酸氨基葡萄糖胶囊更有效，起效更快，并显着抑制软骨细胞变性和受损软骨组织的修复12。在KOA的治疗中，与推拿治疗相比，非甾体抗炎药有不良反应和不理想的长期疗效，手术风险和费用相对较高，并且需要一定的适应证才能进行手术治疗，并伴有术后问题和假体周围并发症13,14,15.与药物治疗和手术相比，推拿治疗 KOA 具有多种优势，包括减少副作用、降低风险、增强安全性、成本效益和更持久的疗效。此外，它还可以有效缓解膝关节疼痛、肿胀、爆裂和活动受限 6,13,16,17。
然而，推拿治疗KOA的机制有待明确，这限制了KOA治疗方案的改进和完善。因此，通过动物实验研究推拿干预KOA的机制是一种有效的方法。与老鼠相比，兔子具有温顺的气质和较大的膝关节。其解剖结构和软骨生化指标与人类相似，是推拿18研究膝关节病机制的合适课题。将木瓜蛋白酶注射到兔膝关节腔中建立的KOA模型具有建模时间短、创伤小、成功率高、存活率高、病理机制与KOA19相似等优点。本研究旨在建立推拿干预KOA的科学可行的动物实验方案，并探讨推拿的机制。

<table><tbody><tr><td>0.9 % sodium chloride injection</td><td>Sichuan Keren Pharmaceutical Co.</td><td>Z22121903</td><td></td></tr><tr><td>-20&amp;deg;C refrigerator</td><td>Haier</td><td>BD-328WL</td><td></td></tr><tr><td>4 % fixative solution</td><td>Solarbio</td><td>P1110</td><td></td></tr><tr><td>4&amp;deg;C refrigerator</td><td>Haier</td><td>SC-315DS</td><td></td></tr><tr><td>Anhydrous ethanol</td><td>Sinopharm</td><td></td><td></td></tr><tr><td>Automatic tissue dewatering machine</td><td>Dakowei (Shenzhen) Medical Equipment Co.</td><td>HP30</td><td></td></tr><tr><td>Blast drying oven</td><td>Shanghai Yiheng Scientific Instruments Co.</td><td>DHG-9070A</td><td></td></tr><tr><td>Coverslip</td><td>Biyuntian</td><td>FCGF50</td><td></td></tr><tr><td>Electric thermostat water bath</td><td>Shanghai Yiheng Scientific Instruments Co.</td><td>HWS-26</td><td></td></tr><tr><td>Embedding freezing table</td><td>Changzhou Paishijie Medical Equipment Co.</td><td>BM450</td><td></td></tr><tr><td>Embedding machine</td><td>Changzhou Paishijie Medical Equipment Co.</td><td>BM450A</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid decalcification solution</td><td>Servicebio</td><td>G1105-500ML</td><td></td></tr><tr><td>Fluorescent inverted microscope</td><td>Leica</td><td>Leica DM IL LED</td><td></td></tr><tr><td>Hematoxylin-eosin staining kit</td><td>Cisco Jet</td><td>EE0012</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Laiyang Economic and Technological Development Zone Fine Chemical Plant</td><td></td><td></td></tr><tr><td>Medical joint goniometer</td><td>KOSLO</td><td></td><td></td></tr><tr><td>Neutral gum</td><td>Cisco Jet</td><td>EE0013</td><td></td></tr><tr><td>Normal-grade male New Zealand rabbit</td><td>Jinan Xilingjiao Breeding and Breeding Center</td><td>SCXK (Lu) 2020 0004</td><td></td></tr><tr><td>Papain(3000 U/mg)</td><td>Bioss</td><td>D10366</td><td></td></tr><tr><td>Pathological tissue bleaching and drying instrument</td><td>Changzhou Paishijie Medical Equipment Co.</td><td>PH60</td><td></td></tr><tr><td>Pet electric clippers</td><td>Codos</td><td>CP-3180</td><td></td></tr><tr><td>Rabbit fixing box</td><td>any brand</td><td></td><td></td></tr><tr><td>Rotating Slicer</td><td>Leica</td><td>531CM-Y43</td><td></td></tr><tr><td>Tuina technique parameter determination instrument</td><td>Shanghai DuKang Instrument Equipment Co. Ltd.</td><td>ZTC-&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/65763/65763eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;</td><td></td></tr><tr><td>Ventilator</td><td>TALY ELECTRIC</td><td>C32</td><td></td></tr><tr><td>Xylene</td><td>Fuyu Reagent</td><td></td><td></td></tr></tbody></table>

tuina intervention in rabbit model of knee osteoarthritis

膝骨关节炎（KOA）的主要特征是膝关节软骨和周围软组织的退行性变化。推拿治疗KOA的疗效已得到证实，但其潜在机制有待研究。本研究旨在建立推拿治疗的KOA兔模型，以揭示其潜在机制。为此，将 18 只 6 个月大的正常级雄性新西兰兔随机分为假手术组、模型组和推拿组，每组 6 只兔子。将4%木瓜蛋白酶溶液注入膝关节腔中，建立KOA模型。推拿组采用推拿联合膝关节旋转矫正法干预4周。在假手术组和模型组中仅进行标准抓握和固定。在1周的干预结束时，观察膝关节关节活动度（ROM），并进行软骨苏木精-伊红（HE）染色。研究表明，推拿可以抑制软骨细胞凋亡，修复软骨组织，恢复膝关节活动度。综上所述，本研究证明了推拿治疗KOA模型兔的科学可行性，凸显了其在KOA和类似膝关节相关疾病研究中的潜在应用。

膝关节活动受限和软骨组织损伤的程度反映了 KOA 的严重程度。膝关节的 ROM 反映了膝关节运动的限制程度。膝关节活动度越小，膝关节活动受限越严重。相反，膝关节ROM越大，膝关节运动程度越正常。HE染色观察软骨组织的形态和结构，反映软骨组织损伤程度。软骨组织表面越不规则，裂缝和缺损的存在越多，软骨细胞数量越少，软骨层厚度越薄，细胞排列越无序，细胞分布越不均匀，层越不清晰，潮线越不清晰完整， Mankin评分越高，膝关节软骨组织损伤越严重，反之，软骨组织越正常，为32。在建立KOA模型时，可以通过观察兔膝关节运动的限制程度来判断建模的成功23,24。推拿的疗效可以通过观察推拿12干预兔膝关节时膝关节运动受限程度和软骨组织损伤程度的改善来确定。
第7周后，比较两组兔子的左膝关节，发现建模组的肌肉更僵硬，运动受限，ROM为74.67°±1.21°，低于假手术组的140.17°±1.33°，表明建模成功（图2C， 图4）。
经第12周测量分析，假手术组、模型组和推拿组的膝关节活动度分别为140.33°±1.37°、76.33°±1.37°、134.33°±1.51°，推拿组膝关节活动度显著高于模型组（p < 0.01），说明推拿可以改善KOA兔的膝关节功能（图5）。
各组家兔左膝关节软骨HE染色显示，假手术组软骨组织表面光滑完整，软骨细胞数量为331.67±13.98，软骨层厚度为259.42±41.97 μm，细胞排列良好，分布均匀，水平清晰， 潮汐线清晰、连续、完整，曼金得分为0.33±0.52。与假手术组相比，模型组软骨组织表面不规则，有缺损和裂缝，软骨细胞数量为29.50±8.04，软骨层厚度为103.15±24.64 μm，细胞无序，分布不均匀，层不清晰，潮汐线不清晰不完整，Mankin评分为9.33±1.03。与模型组相比，推拿组软骨表面规则，缺损和裂隙较少，软骨细胞数量为291.83±8.18，软骨层厚度为183.58±15.34 μm，细胞排列更整齐，分布略显不均匀，层更清晰，潮汐线相对清晰完整， Mankin的得分为3.00±0.63（图6）15,23,33。推拿组的细胞数、软骨层厚度、Mankin评分均明显优于模型组（p < 0.001），提示推拿可以修复受损的软骨组织。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65763/65763fig01.jpg"> 图1：KOA模型兔的建立和推拿方案。 在兔子适应性喂养1周后，在兔子的左膝关节上建立KOA模型6周，在建模开始的第1,4和7天注射4%木瓜蛋白酶溶液。推拿干预兔子左膝关节4周，隔天1次。喂食1周后，测量兔子左膝关节的ROM并取样。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65763/65763fig01large.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/65763/65763fig02.jpg"> 图2：KOA模型的建立 。 （A）准备并消毒兔子的左膝关节。（B）从外西燕插入针头，分别将4%木瓜蛋白酶溶液和0.9%氯化钠溶液注射到模型组和假手术组的膝关节腔中。（C）成功成型的左膝关节活动受限的KOA模型兔。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65763/65763fig02large.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/65763/65763fig03.jpg"> 图 3：使用推拿技术参数测定仪进行推拿技术训练。 （A） 用拇指训练旋转揉捏方法。（B）用拇指旋转揉捏方法的曲线。（C） 用拇指端训练按压方法。（D）拇指端按压方式的曲线。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65763/65763fig03large.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/65763/65763fig04.jpg"> 图 4：建模前后兔子的左膝关节 ROM。对假手术组和建模组的数据进行t检验处理，结果用平均值表示±SD。 ns p > 0.05，***p < 0.001。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65763/65763fig04large.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/65763/65763fig05.jpg"> 图 5：兔子的左膝关节 ROM。 假手术组、模型组和推拿组采用方差分析处理数据，结果表示为平均值±标准差。在第 1 周，所有三组的 ROM 均略有增加。与假手术组相比，模型组和推拿组在建模时的ROM逐渐降低（p < 0.001）。与模型组相比，推拿组干预后ROM逐渐升高（p < 0.001）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65763/65763fig05large.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/65763/65763fig06.jpg"> 图6：兔膝关节软骨HE染色 。 （A）假组的HE染色。（B）模型组的HE染色。（C）推拿组HE染色。（D）组间软骨细胞数的比较。（E）组间软骨层厚度的比较。（F） 组间 Mankin 评分的比较。数据采用方差分析处理，结果表示为平均值±标准差。12周后，HE染色可见，该组软骨组织结构完整，细胞数量和排列正常;模型组软骨组织结构破坏，细胞数量少，排列无序;推拿组软骨组织完整，细胞数量和排列相对正常。 ***p < 0.001。比例尺 = 100 μm，N =6。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65763/65763fig06large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Using a Rabbit Model to Explore the Efficacy of Tuina in Treating Knee Osteoarthritis at JoVE.com

作者聚焦：使用兔子模型探索推拿治疗膝骨关节炎的疗效 

该协议描述了一种在膝骨关节炎的兔子模型中进行推拿干预的方法。

推拿干预膝骨关节炎兔模型

Knee osteoarthritis (KOA) is mainly characterized by degenerative changes in the knee joint's cartilage and surrounding soft tissues. The efficacy of ...

在比较两种类型的治疗和手术时，推拿治疗全膝骨关节炎具有多种优势，包括减少副作用、降低风险、增强安全性、成本效益和更持久的疗效。此外，它还可以有效消除膝关节疼痛、肿胀、爆裂和活动受限。这项研究提供了一个科学合理、负担得起的研究细节，将有助于探索推拿治疗膝关节疾病有效性的潜在机制。本研究证明了推拿治疗膝骨关节炎模型疣的科学可行性，突出了其在膝骨关节炎和类似膝关节相关疾病研究中的潜在应用。

tuina-intervention-in-rabbit-model-of-knee-osteoarthritis

Universidad San Sebastian

Research

JoVE Journal

Medicine

Tuina Intervention in Rabbit Model of Knee Osteoarthritis

1.4K 次观看.  Shandong University of Traditional Chinese Medicine. 在比较两种类型的治疗和手术时，推拿治疗全膝骨关节炎具有多种优势，包括减少副作用、降低风险、增强安全性、成本效益和更持久的疗效。此外，它还可以有效消除膝关节疼痛、肿胀、爆裂和活动受限。这项研究提供了一个科学合理、负担得起的研究细节，将有助于探索推拿治疗膝关节疾病有效性的潜在机制。本研究证明了推拿治疗膝骨关节炎模型疣的科学可行性?...

视频: 作者聚焦：使用兔子模型探索推拿治疗膝骨关节炎的疗效 

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 ...

科研

医学

Preparation, Procedures and Evaluation of Platelet-Rich Plasma Injection in the Treatment of Knee Osteoarthritis

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited effect on the repair of cartilage and on bone regeneration. Platelet-rich plasma (PRP) is an autologous bioactive substance that can repair cartilage injury and accelerate bone regeneration effectively. However, reporting of PRP preparation protocols in clinical studies is highly inconsistent, with the majority of studies providing insufficient information to allow the protocol to be reproduced. We describe a repeatable method of preparing PRP visually, the treatment of KOA using PRP intra-articular injection, and methods of evaluating the outcome. PRP was prepared using manual double centrifugation. The PRP layer was extracted from peripheral blood and used for knee joint cavity injection. Evaluations included assessments of blood platelet concentrations and clinical outcomes. Preparation of PRP by manual centrifugation requires less apparatus and is less costly than plasma filtration or centrifugation using equipment. The centrifugation time of our double centrifugation method was 6 and 5 minutes for the respective centrifugations at forces of 800 and 1400 x g, respectively, to allow for the consistent preparation of standardized PRP. However, a manual method is susceptible to operator error, and PRP batch preparation is not available. Intra-articular injection of PRP proved to be an effective treatment for knee osteoarthritis. The entire treatment procedure took less than 30 minutes, the blood platelet concentration of PRP could be standardized, and treatment was proven to be effective when evaluated by follow-up.

Knee osteoarthritis is frequently seen in the orthopedic department. We introduce in detail the entire knee osteoarthritis treatment process with platelet-rich plasma injection, including preparation, procedures, and evaluation.

富血小板血浆注射液治疗膝关节骨关节炎的制备、程序及评价

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Transthoracic Echocardiographic Examination in the Rabbit Model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

兔子模型中的跨胸超声心动图检查

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...

The Lower Body Positive Pressure Treadmill for Knee Osteoarthritis Rehabilitation

Here, based on a clinician&rsquo;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

Here, based on a clinician&#8217;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

下体正压跑步机用于膝关节骨关节炎康复

Here, based on a clinician&rsquo;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in ...

Osteoarthritis Pain Model Induced by Intra-Articular Injection of Mono-Iodoacetate in Rats

The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the pathophysiological changes of human OA. However, as the main symptom in the late stage of OA, pain affects the patients&#39; daily life, and there are not many available models. The mono-iodoacetate (MIA)-induced model is the most widely used OA pain model, mainly used in rodents. MIA is an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, which causes chondrocyte death, cartilage degeneration, osteophyte, and measurable changes in animal behavior. Besides, expression changes of matrix metalloproteinase (MMP) and pro-inflammatory cytokines (IL1 &#946; and TNF &#945;) can be detected in the MIA-induced model. Those changes are consistent with OA pathophysiological conditions in humans, indicating that MIA can induce a measurable and successful OA pain model. This study aims to describe the methodology of intra-articular injection of MIA in rats and discuss the resulting pain-related behaviors and histopathological changes.

This study describes the method of intra-articular injection of mono-iodoacetate in rats and discusses the resulting pain-related behaviors and histopathological changes, which provide references for future applications.

大鼠关节内注射单碘乙酸酯诱导的骨关节炎疼痛模型

The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

手术小鼠模型中骨关节炎的标准化形态学评价

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

大鼠膝骨关节炎的标准化推拿手法

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

作者聚焦:了解大鼠推拿操作的机制 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

推拿操作以减少膝关节骨关节炎大鼠的炎症和软骨损失

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...

推拿干预膝骨关节炎兔模型

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此视频中的章节

骨关节炎疼痛的鼠标Monoiodoacetate模式

富血小板血浆注射液治疗膝关节骨关节炎的制备、程序及评价

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

兔子模型中的跨胸超声心动图检查

下体正压跑步机用于膝关节骨关节炎康复

大鼠关节内注射单碘乙酸酯诱导的骨关节炎疼痛模型

手术小鼠模型中骨关节炎的标准化形态学评价

推拿操作以减少膝关节骨关节炎大鼠的炎症和软骨损失

推拿操作以减少膝关节骨关节炎大鼠的炎症和软骨损失

推拿操作以减少膝关节骨关节炎大鼠的炎症和软骨损失