本研究由中国浙江省自然科学基金（批准号:LY17H270016），中国国家自然科学基金（批准号:81774331，81873049和81673997）和中国浙江省中医药科学技术项目（批准号:2013ZQ007和2016ZZ011）资助。

涉及动物受试者的程序已获得浙江中医大学医学规范和伦理委员会的批准，并符合中国关于实验动物使用和护理的立法。
1.膝关节内注射单碘乙酸酯
<ol><li>适应一周后，将40只体重180-200g（4-5周龄）的雄性Sprague-Dawley大鼠随机平均分为四组（n = 10只大鼠/组）。 注意:对照组的大鼠将关节内注射50μL盐水，而实验组中的大鼠将分别用溶解在50μL盐水中的0.5，1.5或3mgMIA处理。</li>
	<li>在注射当天，新鲜制备MIA在无菌盐水（0.9%NaCl）中的溶液，浓度为15，30和60mg / mL，并在无菌盐水（0.9%NaCl）中制备10%戊巴比妥溶液。 注意:MIA对粘膜，上呼吸道，眼睛和皮肤和其他组织具有极大的破坏性作用。因此，在制备溶液时，建议戴口罩和手套。</li>
	<li>通过腹膜内注射60mg / kg的氯胺酮和5mg / kg的甲苯噻嗪（均在盐水中制备）麻醉大鼠。用镊子轻轻夹住大鼠的脚趾以确认麻醉。</li>
	<li>将老鼠背朝下放置。刮掉膝盖，用酒精擦拭膝关节周围的区域。</li>
	<li>保持膝盖成90°角，露出髌骨下方的白色髌腱。用指尖按压髌腱，找到髌骨下方的间隙。</li>
	<li>选择间隙和髌腱外侧的交界处作为注射部位。然后，将 26 G 针头垂直插入约 5 mm 的部位。当针头在关节空间时，不应感到阻力。 注意:保持针头垂直于注射部位很重要。</li>
	<li>将 50 μL 盐水或 MIA 溶液注入关节腔。慢慢拔出针头，在注射部位缠上一块纱布，以尽量减少回流和泄漏。麻醉恢复后，取下纱布。</li>
	<li>如第 2 节所述，在注射后 1、7、14、21、28 和 35 天测试疼痛相关行为。</li>
</ol>2. 行为评估
<ol><li>机械撤退阈值 注意:MWT通过冯弗雷测试13 测量，观察者对动物接受的注射不知情。<ol><li>将老鼠放在高架塑料笼子（17 cm x 11 cm x 13 cm）中，金属丝网底座悬挂在桌子上方 50 厘米处。确保测试环境安静，给大鼠30分钟适应环境。</li>
		<li>将冯弗雷针垂直按压在每只大鼠后爪的足底表面上。逐渐（约20 g / s）和线性增加压力，直到发生爪子抬起或舔爪子。</li>
		<li>使用低于上一个阈值的力，以确保阈值是最小撤回力。测试每只大鼠三次以上，至少间隔3-5分钟。</li>
		<li>记录引起爪子撤回反射的最小力。将数据平均为大鼠的MWT。</li>
	</ol></li>
	<li>热抽出延迟 （TWL） 注意:使用足底测试设备（材料表）测量TWL，观察者对动物接受的注射不知情。<ol><li>将一个有机玻璃盒（60厘米x 20厘米x 14厘米，分为6个隔间）放在3毫米厚的玻璃板上，并将老鼠放入盒子中（每个隔间一个）。确保环境安静，室温恒定。</li>
		<li>给大鼠30分钟适应测试环境。</li>
		<li>通过红外辐射计校准热刺激。将所需的红外强度设置为 70 个单位。</li>
		<li>将红外发射器/探测器放在被测爪子中心正下方的容器上。</li>
		<li>按 开始 按钮。计时器将自动启动。一旦爪子发生移动，控制器将自动关闭红外灯并完全停止计时器。</li>
		<li>记录爪子退出和爪子舔舐出现时的反应时间。 注意:测试的阳性反应被视为爪子撤回和爪子舔。如果只发生爪子撤退，则应将其视为大鼠的自愿运动，而不是积极的反应。</li>
		<li>重复测试三次以上。将数据平均为大鼠的TWL。 注意:每次辐射热暴露不应超过 20 秒。同一后爪上的红外刺激应至少间隔 3-5 分钟。</li>
	</ol></li>
	<li>步态模式分析 注意:使用成像系统（材料表）测量步态模式分析，观察者对动物接受的注射不知情。<ol><li>通过使用步行舱两端的旋钮将步行舱的长度调整为适合大鼠的长度（例如，61厘米）。</li>
		<li>在正式实验之前，将一只大鼠放入步行舱并训练大鼠以 18 厘米/秒的速度不间断地运行至少 5 个步程循环。 注意:当老鼠第一次被放置在步行舱中时，速度可以设置为大约 20 厘米/秒，并在奔跑约 2 秒时关闭。然后将速度设置为 18 厘米/秒。如果老鼠在行走过程中停下来或后退，用隔板轻轻拍打老鼠的背部。尝试将速度从 18 厘米/秒逐渐降低到 10 厘米/秒。如果大鼠最终未能进行测试，则可以选择另一只大鼠进行测试。</li>
		<li>慢慢增加跑步机的速度，直到达到目标速度（18 厘米/秒）。使用安装在透明跑步机皮带下方的高速数字摄像机捕获至少5秒的大鼠连续运动的视频。</li>
		<li>测试每只大鼠至少相隔5分钟以获得至少三次不间断的运行。</li>
		<li>在成像软件中绘制一个"边界框"来定义动物行走图像的边界，并输入每个视频的运行速度。选择视频作为一个组，并通过软件自动处理视频。处理视频后，软件将输出多个电子表格来报告步态指数，包括站姿、摆动、制动、推进、踏频、步数顺序等。</li>
		<li>计算总爪面积 （cm 2）、平均步幅 （cm） 和统一步幅。 注意:总爪面积是每组大鼠四只爪子总面积的平均值，爪面积定义为在步道周期的站立阶段与跑步机接触的最大爪面积。步幅是同一爪子在一个完整步幅中的初始接触之间的距离。单位步幅 = 平均步幅 （cm）/体长 （cm）。</li>
	</ol></li>
</ol>3. 组织病理学和免疫组织化学分析
<ol><li>通过腹膜内注射150mg / kg的戊巴比妥钠（在盐水中制备）对大鼠实施安乐死。立即切除膝关节进行组织学分析。</li>
	<li>在10%福尔马林中固定关节24小时，在磷酸盐缓冲盐水（PBS）中用10%EDTA脱钙8周，然后将关节嵌入石蜡中。 注意:福尔马林会引起眼睛、皮肤和呼吸道刺激。它应该在引擎盖中处理。</li>
	<li>用切片机将石蜡嵌入接头切成3mm厚的接头，并漂浮在含有蒸馏水的40°C水浴中。</li>
	<li>将切片转移到载玻片上。将载玻片干燥过夜，并将载玻片储存在室温（RT）下以继续以下染色。</li>
	<li>将载玻片放入60°C烘箱中4小时以脱蜡。</li>
	<li>将载玻片分别浸入二甲苯，二甲苯，100%乙醇，100%乙醇，95%乙醇，80%乙醇和75%乙醇中，在室温下5分钟。</li>
	<li>用苏木精和伊红（H&E），safranin-O（SO）和阿尔新蓝苏木精（ABH）以及针对大鼠II型胶原蛋白（Col2），X型胶原蛋白（Col10）和基质金属蛋白酶13（MMP13）的抗体染色切片，如步骤3.8-3.12所述。</li>
	<li>H&E 染色<ol><li>用去离子H2O冲洗载玻片3分钟。用苏木精染色载玻片3-5分钟。</li>
		<li>用去离子H 2O 3x 冲洗载玻片，每次 1 分钟，直到表面上没有苏木精残留。</li>
		<li>用伊红染色载玻片30秒。然后，用去离子H2O 3x冲洗载玻片，每次1分钟，直到表面上没有曙红残留。</li>
		<li>将载玻片浸入0.1%氨中10-20秒。然后，用去离子H 2O 3x 冲洗载玻片，每次 1 分钟。</li>
		<li>分别将幻灯片依次浸入95%乙醇，100%乙醇，二甲苯，二甲苯和二甲苯中1分钟。</li>
		<li>用中性树脂盖玻片。</li>
	</ol></li>
	<li>SO 染色<ol><li>用去离子H2O冲洗载玻片3分钟。用苏木精染色载玻片3-5分钟。</li>
		<li>在1%酸性酒精（约3秒）中快速分化。然后，用去离子H2O 3x冲洗载玻片，每次1分钟，直到表面上没有苏木精残留。</li>
		<li>用快速绿色（FCF）溶液染色载玻片5分钟。然后，用去离子H 2O 3x 冲洗载玻片，每次 1 分钟。</li>
		<li>用SO染色载玻片1-2分钟。然后，用去离子H 2O 3x 冲洗载玻片，每次 1 分钟。</li>
		<li>用1%乙酸冲洗载玻片1-2分钟以去除残留的FCF。用去离子H2O冲洗载玻片1分钟。</li>
		<li>分别将幻灯片依次浸入95%乙醇，100%乙醇，二甲苯，二甲苯和二甲苯中1分钟。</li>
		<li>用中性树脂盖玻片。</li>
	</ol></li>
	<li>ABH 染色<ol><li>用去离子H2O冲洗载玻片3分钟。将载玻片浸入1%酸性酒精中30秒，然后在纸巾上短暂沥干（不要冲洗）。</li>
		<li>将载玻片浸入ABH中1小时。然后，用去离子H 2O 3x 冲洗载玻片，每次 1 分钟，直到表面上没有 ABH 残留。</li>
		<li>在1%酸性酒精（约3秒）中快速分化。用去离子H2O 3x冲洗载玻片，每次1分钟。</li>
		<li>将载玻片浸入0.5%铵水中15秒。然后，用去离子H 2O 3x 冲洗载玻片，每次 1 分钟。</li>
		<li>将载玻片浸入95%乙醇中1分钟。将载玻片浸入伊红/橙色G溶液中1.5分钟。</li>
		<li>分别将幻灯片依次浸入95%乙醇，100%乙醇，二甲苯，二甲苯和二甲苯中1分钟。</li>
		<li>用中性树脂盖玻片。</li>
	</ol></li>
	<li>根据曼金的评分系统14，在显微镜下检查所有载玻片，并通过双盲观察以0-13的等级进行统计评分。</li>
	<li>免疫组化<ol><li>用去离子H2O冲洗载玻片3分钟。</li>
		<li>将载玻片浸入0.1M柠檬酸钠中，并将载玻片置于60°C烘箱中4小时以取出抗原。</li>
		<li>将载玻片浸入PBS中的0.3%Triton X-100中10分钟。然后，用PBS 2x冲洗载玻片，每次3分钟。</li>
		<li>在室温下在3%H2O2 甲醇溶液中孵育切片10分钟以阻断内源性过氧化物酶活性。用PBS 2x冲洗载玻片，每次3分钟。</li>
		<li>在室温下用 5% 山羊血清在 PBS 中孵育切片 30 分钟以阻断任何非特异性结合。用PBS 2x冲洗载玻片，每次3分钟。</li>
		<li>将切片在4°C下孵育过夜，与100μLPBS稀释（1:1，000）抗大鼠II型胶原蛋白（Col2），X型胶原蛋白（Col10）和基质金属蛋白酶13（MMP13）的一抗。用PBS 2x冲洗载玻片，每次3分钟。</li>
		<li>用 100 μL PBS 稀释的 （1:1，000） 二抗（山羊抗小鼠二抗或山羊抗小鼠二抗）在室温下孵育切片 20 分钟。 用 PBS 冲洗载玻片 2x，每次 3 分钟。</li>
		<li>用 100 μL 3，3′-二氨基联苯胺 （DAB） 工作溶液孵育切片。当显色反应使表位位点变成棕色时监测反应。 注意:DAB是一种致癌物质。使用 DAB 工作时，请务必戴手套并在头罩中工作。 注意:显色时间可能从几秒钟到10分钟不等。</li>
		<li>一旦切片上出现棕色，用去离子 H2O 2x 冲洗载玻片，每次 3 分钟。</li>
		<li>将载玻片浸入苏木精中1-2分钟以复染载玻片。然后，用去离子H 2O 3x 冲洗载玻片，每次 1 分钟。</li>
		<li>分别将幻灯片依次浸入95%乙醇，100%乙醇，二甲苯，二甲苯和二甲苯中1分钟。</li>
		<li>用中性树脂盖玻片。</li>
		<li>在显微镜下观察切片中抗体染色的颜色。</li>
	</ol></li>
</ol>

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Correlation of morphology with biochemical and metabolic data.</a> Journal of Bone and Joint Surgery. 53 (3), 523-537 (1971).</li><li>Yan, 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=Chondroprotective+effects+of+platelet+lysate+towards+monoiodoacetate-induced+arthritis+by+suppression+of+TNF-&#945;-induced+activation+of+NF-&#312;B+pathway+in+chondrocytes.">Chondroprotective effects of platelet lysate towards monoiodoacetate-induced arthritis by suppression of TNF-&#945;-induced activation of NF-&#312;B pathway in chondrocytes.</a> Aging. 11 (9), 2797-2811 (2019).</li><li>Yan, B., 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=Intra-Articular+Injection+of+Extract+Attenuates+Pain+Behavior+and+Cartilage+Degeneration+in+Mono-Iodoacetate+Induced+Osteoarthritic+Rats.">Intra-Articular Injection of Extract Attenuates Pain Behavior and Cartilage Degeneration in Mono-Iodoacetate Induced Osteoarthritic Rats.</a> Frontiers in Pharmacology. 9, 1360 (2018).</li><li>Wang, 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=Agkistrodon+ameliorates+pain+response+and+prevents+cartilage+degradation+in+monosodium+iodoacetate-induced+osteoarthritic+rats+by+inhibiting+chondrocyte+hypertrophy+and+apoptosis.">Agkistrodon ameliorates pain response and prevents cartilage degradation in monosodium iodoacetate-induced osteoarthritic rats by inhibiting chondrocyte hypertrophy and apoptosis.</a> Journal of Ethnopharmacology. 231, 545-554 (2019).</li><li>Yamada, E. F., 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=Evaluation+of+monosodium+iodoacetate+dosage+to+induce+knee+osteoarthritis:+Relation+with+oxidative+stress+and+pain.">Evaluation of monosodium iodoacetate dosage to induce knee osteoarthritis: Relation with oxidative stress and pain.</a> International Journal of Rheumatic Diseases. 22 (3), 399-410 (2019).</li><li>Schuelert, N., McDougall, 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=Electrophysiological+evidence+that+the+vasoactive+intestinal+peptide+receptor+antagonist+VIP6-28+reduces+nociception+in+an+animal+model+of+osteoarthritis.">Electrophysiological evidence that the vasoactive intestinal peptide receptor antagonist VIP6-28 reduces nociception in an animal model of osteoarthritis.</a> Osteoarthritis and Cartilage. 14 (11), 1155-1162 (2006).</li><li>Lee, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choline,+an+alpha7+nicotinic+acetylcholine+receptor+agonist,+alleviates+hyperalgesia+in+a+rat+osteoarthritis+model.">Choline, an alpha7 nicotinic acetylcholine receptor agonist, alleviates hyperalgesia in a rat osteoarthritis model.</a> Neuroscience Letters. 548, 291-295 (2013).</li><li>Piesla, 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=Abnormal+gait,+due+to+inflammation+but+not+nerve+injury,+reflects+enhanced+nociception+in+preclinical+pain+models.">Abnormal gait, due to inflammation but not nerve injury, reflects enhanced nociception in preclinical pain models.</a> Brain Research. 1295, 89-98 (2009).</li><li>Udo, M., 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=Monoiodoacetic+acid+induces+arthritis+and+synovitis+in+rats+in+a+dose-+and+time-dependent+manner:+proposed+model-specific+scoring+systems.">Monoiodoacetic acid induces arthritis and synovitis in rats in a dose- and time-dependent manner: proposed model-specific scoring systems.</a> Osteoarthritis and Cartilage. 24 (7), 1284-1291 (2016).</li><li>Guingamp, 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=Mono-iodoacetate-induced+experimental+osteoarthritis:+a+dose-response+study+of+loss+of+mobility,+morphology,+and+biochemistry.">Mono-iodoacetate-induced experimental osteoarthritis: a dose-response study of loss of mobility, morphology, and biochemistry.</a> Arthritis &amp; Rheumatism. 40 (9), 1670-1679 (1997).</li><li>Jeong, J. 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=Eupatilin+Exerts+Antinociceptive+and+Chondroprotective+Properties+in+a+Rat+Model+of+Osteoarthritis+by+Downregulating+Oxidative+Damage+and+Catabolic+Activity+in+Chondrocytes.">Eupatilin Exerts Antinociceptive and Chondroprotective Properties in a Rat Model of Osteoarthritis by Downregulating Oxidative Damage and Catabolic Activity in Chondrocytes.</a> PLoS ONE. 10 (6), 0130882 (2015).</li><li>Cook, J. 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=Animal+models+of+cartilage+repair.">Animal models of cartilage repair.</a> Bone &amp; Joint Research. 3 (4), 89-94 (2014).</li><li>Little, C. B., Zaki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+constitutes+an+"animal+model+of+osteoarthritis"--the+need+for+consensus.">What constitutes an "animal model of osteoarthritis"--the need for consensus.</a> Osteoarthritis and Cartilage. 20 (4), 261-267 (2012).</li></ol>

作者没有什么可透露的。

MIA诱导的OA大鼠模型是一种成熟且广泛使用的模型。关节内注射MIA最初引起严重和急性炎症，这导致OA的更长和退行性阶段17，18。在这项研究中，我们通过MWT和TWL测量了伤害敏感性，并使用成像系统评估了步态改变。先前的报告发现，注射MIA可以提高传入膝关节纤维的敏感性，导致伤害感受，这反映在热痛觉过敏和降低的机械阈值19，20上。已经证明步态改变与伤害感受增强有关，这表明步态模式可用于评估疼痛模型21。因此，MIA诱导的模型主要用于评估OA相关的疼痛和筛选口服药物以及关节注射药物3，6。
虽然与手术诱导OA模型相比，将MIA注入关节腔更简单、更快捷，但在建模中仍然存在关键点。首先，大鼠的关节腔很小，注射前应确认其位置。其次，MIA是有毒的，因此MIA的剂量应仔细选择。据报道，MIA可以以剂量和时间依赖性的方式诱导关节软骨损伤（通过OARSI组织学评分和Mankin评分评估），表明可以通过调节MIA22，23的浓度来调节关节病变的进展和严重程度。发现MIA在高剂量10，18，24下诱导疼痛和氧化应激标志物。以前的报告表明，在大鼠中注射1.5毫克剂量的MIA会产生类似于人膝关节OA18，25的炎症过程。此外，重要的是在整个行为测试中使用相同的实验者，并提前让大鼠熟悉环境，以减少焦虑并避免影响实验结果。
如上所述，OA动物模型通常分为自发模型和诱导模型。关节内注射MIA由于几个优点而被广泛使用:1）操作简单;2）易于感应和重现性;3）剂量和严重程度可控;4）建模时间短;5）适合小动物和大型动物。然而，与其他动物模型一样，MIA诱导的OA模型也有几个缺点。注射MIA后广泛的细胞死亡和快速的关节破坏与人类的自发或创伤后OA不一致26。此外，关节腔内残留的MIA可能会影响后续关节内治疗的效果，导致使用MIA模型的结果不确定。在该模型中，是否在治疗性注射前清洗关节腔仍然是一个悬而未决的问题。总的来说，没有一个单一的动物模型可以完美地概括人类OA的各个方面，但是可用的模型种类繁多，可以将多个模型综合应用于最相关的问题。

骨关节炎（OA）是世界上最常见的关节疾病，估计影响成人10-12%的人口1。最普遍受累的关节是膝关节，OA在老年人中发病率较高，尤其是女性2。作为一种慢性疾病，OA在几十年内逐渐发展为关节衰竭，症状包括软骨丢失，滑膜炎症，骨癣菌病，功能下降和慢性疼痛3。根据世界卫生组织（WHO）的数据，OA是女性中第四大最普遍的疾病，也是男性中第八大最流行的疾病。到2020年，OA可能成为人类第四大致残性疾病4。然而，目前可用的OA疗法仅解决症状，并延长关节置换手术的时间5。
人类患者的自发性OA往往需要很长时间才能产生关节相关疼痛等临床症状6。在OA的早期阶段，疼痛通常是间歇性的，并且随着疾病的进展变得更加频繁和严重，使其成为患者的主要主诉7。因此，在过去的半个世纪中，已经开发了广泛的OA疼痛动物模型来促进疼痛缓解疗法。OA模型通常分为自发模型和诱导模型。自发模型包括自然发生的模型和转基因模型，它们可以更密切地模拟人类原发性OA的过程8。诱导模型一般可分为两类:1）手术或其他创伤诱导的创伤后OA;或2）关节内注射软骨毒性或促炎物质3。这些模型为OA的病理生理学研究奠定了基础，并为开发减轻疼痛和增加功能的药物做出了巨大贡献。
最近，OA建模最广泛使用的诱导剂是单碘乙酸酯（MIA）。MIA是甘油醛-3-磷酸脱氢酶的抑制剂，可引起软骨基质的变化、降解、软骨流失、滑膜炎等变化，与人类骨关节炎的病理变化相似9.已经注意到，关节内注射MIA在MIA给药后28天引起持续疼痛，表明MIA模型可能有助于研究慢性伤害性疼痛10，11，12。在这项研究中，雄性Sprague-Dawley大鼠接受关节内注射，膝关节中含有0.5、1.5或3mg的MIA。通过注射后1、7、14、21、28和35天的机械和热敏感性评估来测量MIA诱导的关节疼痛的严重程度。在此基础上，选择1.5mg的MIA作为最终浓度，以评估注射后28天的步态模式和组织学变化。

<table border="0" cellpadding="0" cellspacing="0" style="width:783px;" width="781">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Anti-Collagen II antibody</td>
			<td style="width:255px;">Abcam(UK)</td>
			<td style="width:115px;">34712</td>
			<td style="width:191px;">Primary antibody for immunohistochemistry (IHC)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Anti-Collagen X (Col10) antibody</td>
			<td style="width:255px;">Abcam(UK)</td>
			<td style="width:115px;">49945</td>
			<td style="width:191px;">Primary antibody for IHC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">DigiGait Imaging System</td>
			<td style="width:255px;">Mouse Specifics (Boston, MA, USA)</td>
			<td style="width:115px;"></td>
			<td style="width:191px;">Equipment for gait patterns analyses</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Eosin</td>
			<td style="width:255px;">Sigma-Aldrich</td>
			<td style="width:115px;">861006</td>
			<td style="width:191px;">The dye for HE staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Fast Green FCF</td>
			<td style="width:255px;">Sigma-Aldrich</td>
			<td style="width:115px;">F7252</td>
			<td style="width:191px;">The dye for SO staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Goat anti-mouse antibody</td>
			<td style="width:255px;">ZSGQ-BIO (Beijing, China)</td>
			<td style="width:115px;">PV-9002</td>
			<td style="width:191px;">Secondary antibody for IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Goat anti-rabbit antibody</td>
			<td style="width:255px;">ZSGQ-BIO (Beijing, China)</td>
			<td style="width:115px;">PV-9001</td>
			<td style="width:191px;">Secondary antibody for IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Hematoxylin</td>
			<td style="width:255px;">Sigma-Aldrich</td>
			<td style="width:115px;">H3163</td>
			<td style="width:191px;">The dye for HE staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">MIA</td>
			<td style="width:255px;">Sigma-Aldrich</td>
			<td style="width:115px;">I4386-10G</td>
			<td style="width:191px;">powder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">MMP13</td>
			<td style="width:255px;">Cell Signaling Technology, Inc. (Danvers, MA, USA)</td>
			<td style="width:115px;">69926</td>
			<td style="width:191px;">Primary antibody for IHC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Modular tissue embedding center</td>
			<td style="width:255px;">Thermo Fisher Scientific (USA)</td>
			<td style="width:115px;">EC 350</td>
			<td style="width:191px;">Produce paraffin blocks.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Plantar Test apparatus</td>
			<td style="width:255px;">UgoBasile (Italy)</td>
			<td style="width:115px;">37370</td>
			<td style="width:191px;">Equipment for TWL assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">PrimeScript RT reagent Kit (Perfect Real Time)</td>
			<td style="width:255px;">TaKaRa Biotechnology Co. Ltd. (Dalian, China)</td>
			<td style="width:115px;">RR037A</td>
			<td style="width:191px;">Extracte total RNA from cultured cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Rotary and Sliding Microtomes</td>
			<td style="width:255px;">Thermo Fisher Scientific (USA)</td>
			<td style="width:115px;">HM325</td>
			<td style="width:191px;">Precise paraffin sections.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Safranin-O</td>
			<td style="width:255px;">Sigma-Aldrich</td>
			<td style="width:115px;">S2255</td>
			<td style="width:191px;">The dye for SO staining</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Tissue-Tek VIP 5 Jr</td>
			<td style="width:255px;">Sakura (Japan)</td>
			<td style="width:115px;"></td>
			<td style="width:191px;">Vacuum Infiltration Processor</td>
		</tr>
	</tbody>
</table>

osteoarthritis pain model induced by intra-articular injection of mono-iodoacetate in rats

目前的骨关节炎（OA）动物模型可分为自发模型和诱导模型，两者都旨在模拟人类OA的病理生理变化。但是，疼痛作为OA晚期的主要症状，影响患者的日常生活，可用的模型并不多。单碘乙酸（MIA）诱导模型是应用最广泛的OA疼痛模型，主要用于啮齿动物。MIA是甘油醛-3-磷酸脱氢酶的抑制剂，可导致软骨细胞死亡，软骨变性，骨赘和动物行为的可测量变化。此外，在MIA诱导的模型中可以检测到基质金属蛋白酶（MMP）和促炎细胞因子（IL1 β和TNF α）的表达变化。这些变化与人类OA病理生理状况一致，表明MIA可以诱导可测量和成功的OA疼痛模型。本研究旨在描述大鼠关节内注射MIA的方法，并讨论由此产生的疼痛相关行为和组织病理学变化。

通过这种方法，我们在大鼠中建立了OA疼痛模型并检测了由此产生的变化。MWT和TWL分别反映机械性异常性疼痛和热痛觉过敏。如图 1所示，MIA诱导的机械性异常性疼痛和热痛觉过敏以剂量依赖性方式存在。值得注意的是，MWT的下降从21 d达到峰值至28 d，然后反弹，表明现阶段可能发生关节修复，但3 mg MIA组的MWT仍处于较低水平。TWL的变化与MWT大致一致（图2）。
在此基础上，我们选择1.5mg的MIA作为最终剂量，并评估注射后28天的步态模式和组织学变化。步态参数（总爪面积和单位步幅）反映了与疼痛相关的行为。28天后，MIA组的步态参数水平（包括总爪面积（图3A）和单位步幅（图3B））显着降低，表明MIA诱导大鼠骨关节炎相关的关节疼痛。随着组织病理学载玻片上Mankin评分的增加，MIA组明显可见软骨变性、胶原蛋白破坏和基质紊乱（图4）。如图5所示，1.5 mg MIA引起MMP13和Col10的显着上调，以及Col2的显着下调。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60649/60649fig1.jpg"> 图1:注射MIA后MTW的发展。 注射MIA（0.5，1.5或3mg /大鼠）和盐水（0.9%NaCl）后评估后爪的机械撤退阈值，n = 10只大鼠/组。值表示为标准偏差±平均值。 **P < 0.01 与盐水处理组;单因素方差分析，然后是费舍尔最小显著差异（LSD）比较。 <a href="https://www.jove.com/files/ftp_upload/60649/60649fig1large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60649/60649fig2.jpg"> 图2:注射MIA后TWL的发展。 注射MIA（0.5，1.5或3mg /大鼠）和盐水（0.9%NaCl）后评估后爪的热撤回潜伏期，n = 10只大鼠/组。值表示为标准偏差±平均值。 **P < 0.01 与盐水处理组 （NC）;单因素方差分析，然后是费舍尔的LSD比较。 <a href="https://www.jove.com/files/ftp_upload/60649/60649fig2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60649/60649fig3.jpg"> 图 3:MIA 注射后 28 天的步态分析。 （A） 爪子总面积（厘米2）。总爪面积:每组大鼠四只爪子总面积的平均值。（B） 单位步幅。单位步幅 = 平均步幅 （厘米）/体长 （厘米）。n = 10只大鼠/组。数值表示为第28天±平均值SD. ##P <0.01与盐水处理组（NC）相比。这个数字是从Yan等人15修改而来的。 <a href="https://www.jove.com/files/ftp_upload/60649/60649fig3large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60649/60649fig4.jpg"> 图 4:MIA 治疗后第 28 天大鼠膝关节的组织病理学观察（HE、SO 和 AHB 染色）和 Mankin's 评分。 n = 10只大鼠/组。比例尺 = 40 μm。数值表示为平均值±SD.##P < 0.01与盐水处理组（NC）。单因素方差分析，然后是费舍尔的LSD比较。这个数字是从Yan等人16修改而来的。 <a href="https://www.jove.com/files/ftp_upload/60649/60649fig4large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60649/60649fig5.jpg"> 图 5:第 28 天大鼠软骨中 MMP13、Col2 和 Col10 表达的免疫组织化学观察。 比例尺= 50μm.N = 10只大鼠/组。这个数字是从Yan等人16修改而来的。 <a href="https://www.jove.com/files/ftp_upload/60649/60649fig5large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about Osteoarthritis Pain Model Induced by Intra-Articular Injection of Mono-Iodoacetate in Rats at JoVE.com

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

本研究描述了大鼠关节内注射单碘乙酸酯的方法，并讨论了由此产生的疼痛相关行为和组织病理学变化，为今后的应用提供参考。

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 ...

osteoarthritis-pain-model-induced-intra-articular-injection-mono

Universidad San Sebastian

Research

JoVE Journal

Medicine

7.9K 次观看.  Zhejiang Chinese Medical University. 本研究描述了大鼠关节内注射单碘乙酸酯的方法，并讨论了由此产生的疼痛相关行为和组织病理学变化，为今后的应用提供参考。

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

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

科研

医学

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

实验诱发青光眼通过磁性微球注射眼

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

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 ...

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

Circumlimbal 缝合法诱导大鼠和小鼠青光眼模型的建立

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

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 ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

一种生成周期负荷诱导的大鼠膝关节内软骨病变模型的非侵入性方法

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

开发Wistar白化大鼠股骨截骨模型的协议

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...

FSN 疗法治疗膝骨关节炎疼痛

Fu's Subcutaneous Needling for Knee Osteoarthritis Pain

Fu's subcutaneous needling (FSN) is a new acupuncture and dry needling technique based on traditional Chinese medicine. It rapidly produces long-lasting effects in soft tissue injuries, particularly in painful musculoskeletal conditions, by providing stimulation primarily in the subcutaneous area. Osteoarthritis (OA) is the most common joint disease in adults worldwide and is often accompanied by a painful syndrome of structural changes in the peripheral joints of the knee. However, the etiology of OA pain is not fully understood, though myofascial trigger points (MTrPs) are commonly found in the lower limb muscles (so-called "tightened muscles") of patients with knee OA. 
FSN has been used in many fields for the treatment of acute pain problems and can relieve muscle contraction from MTrPs, thereby improving the local circulation. This study recruited patients with pain from knee OA into an FSN group or a transcutaneous electrical nerve stimulation (TENS) group with three treatment sessions and a follow-up over the course of 2 weeks. The results showed that FSN was effective in treating soft tissue pain around the knee with OA. This study aimed to establish and visualize three key technical indicators during FSN therapy, including the FSN needle insertion point and layer; the frequency and duration of the swaying movement; and the manipulation of the reperfusion approach. These findings have great potential for future applications in myofascial pain treatment, especially for pain management. Following this protocol could enhance FSN skills.

作者聚焦： Fu 的皮下针刺治疗膝骨关节炎疼痛  

We present a protocol for using Fu's subcutaneous needling for knee osteoarthritis pain, which combines swaying movement and reperfusion approach techniques. This protocol has great potential for future applications in myofascial pain treatment and could enhance Fu's subcutaneous needling (FSN) manipulation skills.

傅氏皮下针刺治疗膝关节骨关节炎疼痛

Fu's subcutaneous needling (FSN) is a new acupuncture and dry needling technique based on traditional Chinese medicine. It rapidly produces long-lasting ...