JSV是由欧盟委员会（GAN 603191-PAINCAGE）合作赠款，以支持MM。

涉及动物主题手续已经批准在伦敦大学国王学院的伦理委员会，并按照英国内政部规定（动物科学程序法1986）。 1.膝关节Monoiodoacetate的关节腔内注射<ol><li>众议院8 - 10周龄小鼠中的5组12小时光/暗周期下（上午7:00开灯），食物和水随意让小鼠适应环境1周开始实验前。 </li><li>随机化和5.使用动物数量的组电码笼老鼠实验者盲目治疗。使用体重为随机参数。 </li><li>上注射当天，新鲜在所需浓度制备monoiodoacetate的在无菌盐水（0.9％NaCl）中的溶液中。使用注射无菌生理盐水的对照组小鼠的一类。 MIA的最高推荐剂量为1毫克10微升。 注意:Monoiodoacetate是版本ÿ有毒的。因此，建议在手套和口罩处理粉末和制备溶液时磨损。该溶液应用0.22微米的过滤器进行无菌过滤。 </li><li>通过首先将它们放置在一个腔室中的 O 2的混合物递送2％异氟烷麻醉使用麻醉剂手推车小鼠（流速1.5升/分钟），然后转移小鼠鼻锥部，其中还提供了2％异氟醚的 O 2混合物，正因为如此，在注射期间保持麻醉。放置在眼睛兽医药膏，以免他们晒出来，而在麻醉下。穿手术衣，手套和口罩，同时执行注射过程。 </li><li>通过检查动​​物的缺乏应对的后爪捏刺激确认麻醉。 </li><li>一旦动物的麻醉下，将其放置在它的后面。修剪和擦拭周围用酒精膝关节的区域。碘伏或洗必泰也可被用于˚F或消毒。髌腱（白线波纹管髌骨）会变得可见。 </li><li>为了稳定注射部位，保持膝盖仍然，在弯曲的位置时，通过将食指的膝关节和踝关节的前表面上方的拇指的下方。不需要关节偏好。 </li><li>找到喷射的精确位点，运行连接到一个注射器＆G针沿着膝盖（以便不刺破与尖端的皮肤）水平，直到它找到髌骨下方的间隙。轻轻推压，以纪念该部位，然后抬起针和注射垂直注射器。插入针中所标示的区域中，通过髌腱，垂直于胫骨。无阻力应该可以感觉到。 <ol><li>使用拇指为指导，注入肤浅进入现场。注射后，按摩膝盖以确保均匀的溶液分布。立即丢弃针锐利豳风。 </li><li>将老鼠回到上一个加热垫一个干净的家笼，让他们恢复。保持持续的警惕的动物，直到他们恢复意识合适，这是由他们的测量恢复胸骨斜卧。一旦动物被收回，回到自己的笼子里。 注:建议为染料的使用和立即进行验尸解剖，以确认注射的正确定位的最佳实践和培训。 </li></ol></li></ol> 2.机械过敏的测量（异常性疼痛） 注意:静态机械痛阈值由施加的von Frey毛发后爪的足底表面评估。 <ol><li>带来的小鼠的行为学室和让无节制动物丙烯酸隔间（8厘米×5厘米×10厘米）的线网格之上使其恢复。 <ol><li>火车小鼠处理和2小时习惯的隔间两天之前，冯弗雷头发的应用程序，以冯弗雷头发应用过程中，以限制压力和下地活动。上测试天，habituate动物的隔间长达60分钟前的测试。在所有的行为实验穿戴工作服，手套和口罩。 </li></ol></li><li>应用校准的von Frey毛发（即施加由制造公司的校准，并表示为克数（克限定的力的水平直径增大）的柔性尼龙纤维）的后爪的足底表面，直到光纤弯曲。在测试过程中使用0.008，0.02，0.04，0.07，0.16，0.4，0.6，和1.0克的纤维。 <ol><li>固定在适当位置各发3秒或直到爪子被撤回，后者定义了积极的回应。与0.07克的刺激强度开始，按照"上下法"21小毛适用:标记为X中的戒断反应和O的缺乏反应。适用于力升序，高达1克（切断力），直到检测到响应。 </li><li>通过重复重新测试爪子步骤2.2.1开始，按下面的发挥，产生了撤离的一个力灯丝。 </li><li>然后，依次申请剩余的细丝，通过降力，直到无停药发生。重新申请直到响应观察升序细丝。继续直到六个响应的序列，获得（ 例如 ，OXOXOX），以便通过参照表格值21以获得数k值。 </li><li>表达爪缩回值作为克50％缩爪阈值。使用式（10 [XR + Kδ]）/ 10,000，其中XR =值的序列中使用的（对数单位）最后的von Frey长丝中，k =片状值的，而δ=平均在纤维之间的力差。其中，没有检测到响应，则使用1克21,22的最大应答。 </li></ol></li><li>按照上述（2.2.1-2.2.4）中描述的方法，MIA射液之前评估两个后爪的机械阈值n作为基线值。注射后，评估同侧和对侧足的阈值在MIA后定期天间隔数周，以确定机械异常性疼痛的发展。 注:例如，我们报告测量的阈值0，3，5，7，10，MIA注射后14，21和28天。当它们显示于0.1g或更小的反应的动物被认为是异常性疼痛。正常回应回落0.6内 - 1克范围。 </li></ol> 3.承重赤字的测量注:轴承使用的是重量失能测试仪测量体重的变化。 <ol><li>列车每鼠标走进有机玻璃室的设备和坐在控股框。鼠标放置在保温箱前，提起高考了45°，让鼠标走在和关闭对话框。允许直到他们采取坐姿动物自由移动。这个训练需要至少两天，并保证该动物仍然不升需要整理的腔室的任一侧。 （或根据设备指令）与100g的重量检查使用前校准仪器。 <ol><li>确保每个后爪被放置在适当的记录垫11。每个测量的持续时间为1秒，按照制造商的说明。 </li></ol></li><li>收集承载在从每个记录会话记录垫的每个后爪重量的三次测量，并使用平均值来计算由同侧和对侧足承担的重量差。明示值以克对侧和同侧爪之间的差异。 </li><li>评估MIA注射基线值之前，负重的变化。然后，在经过几周的时间定期重复评估，以确定的门的发展变化。例如，我们报告测量的阈值0，3，5，7，10，MIA注射后14，21和28天。 注:正常重B的50％，抽穗值表示跨同侧和对侧后肢相等的重量分布。动物过敏考虑显示器的负重约45％的变化。机械阈值和负重赤字测量可以在相同的小鼠中进行，作为既不终点影响其他。药理评价，各组动物应该在设定的时间在线路与所用的化合物的药物动力学分布给药后进行测试。 </li></ol>

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None of the authors have competing interests or conflicting interests.

用这种方法，我们描述用于通过关节内注射MIA的在膝关节和敏感性的评估，以在后肢非伤害性和伤害性刺激的小鼠诱导关节炎样疼痛的优选方法。 MIA注入与持续性疼痛的行为，即改变后肢负重，并提到机械超敏反应（异常性疼痛）的发展有关。这种静态测量可以通过步态分析在跑步机上或通过走秀分析自由移动的动物来补充。 MIA模型响应常规的止痛治疗24，这表明它们可能是雪亮的治疗方法是有用的。而MIA的注射是在技术上没有困难，关节囊可以在注射期间刺穿，从而在胶囊外MIA的泄漏，和随后的故障以诱导软骨细胞的毒性。事实上，MIA的全身注射能在啮齿类动物的致命第二对组织和软骨细胞比其他细胞MIA可能造成的影响可能混淆的结果，除了是不可取的。因此，它必须强调的是非常小心需要给予MIA的注射，因为它是该模型的一个重要组成部分，并需要被考虑到喷射时进入关节空间的信心。该协议有助于实现这一目标。 这里描述的方案的目标是确保动物提供整个试验期间一致疼痛样的反应。另外，它们允许通过改变用于诱导病理学15,23 MIA的剂量疾病严重程度的调整。既疾病状态和疼痛样行为的快速诱导允许疼痛调节化合物的及时评估。这是在现有的手术和自发OA开发模式，这可能需要更长的时间内开发出过敏有利。此外，特别是对于自发模型，所述疾病病理学术不表现在所有动物中（约20％ - 80％7），而在MIA模型与应答器显著发病相关联。此外，自发模型不适于在负重的变化的测量，OA在两个膝盖开发。当考虑行为的测量，动物需要评估过程中要保持冷静和放松。做到这一点，作为记录的测量之前和由反复处理，其允许动物熟悉实验者在协议所详述，由早期训练。一个关键点，以减轻压力是始终使用同一实验者的行为测试，因为不断变化将引起前面提到的问题。像任何模式，OA的MIA模式还带有局限性，如关节破坏的快速性，它并不像OA病变发展缓慢的患者。解决这个问题的一个办法是用su命令切换到补充这一模式OA的rgical模式。使用中，化合物的开发在MIA化学模型允许对的OA样疼痛的开发和维护的使用预防性和治疗方案。最后，MIA模型将补充敲除小鼠表型的性状的研究，有助于进一步了解OA疾病。

在临床上，骨关节炎（OA）或退行性关节病，是一种痛苦的和衰弱状况，其特征关节软骨，在和关节周围的组织的轻度炎症，有时形成骨赘骨囊肿的渐进性丧失。 OA患者报告持续性疼痛1和显示在关节炎关节2-4的敏感性增加的压力和伤害性刺激。目前，没有治愈与现有治疗方法和镇痛药处方来缓解这种状况带来的痛苦，有某种程度的成功5 OA。但是，OA疼痛仍正在开发的OA的临床问题和动物模型来改善我们的OA相关的疼痛机制的理解和披露的新的治疗靶点。 有可用的具有不同特点的OA 6几种动物模型。手术的方法，如前交叉韧带切断，可以使用。然而，它们涉及熟练手术干预和在大鼠主要执行，而内侧半月板（DMM）的不稳定在小鼠被使用。 OA的自发发展发生在豚鼠和自发的关节退变已在3至16个月年龄7,8 C57黑鼠的报道。自发的OA模型不涉及任何干预来诱导条件，但它们有内在的可变性，因此，招致较大的号码和成本9,10。化学诱导的模型，另一方面，需要比外科模型更侵入性操作，因此，更容易实现，并允许OA病变的研究在不同的阶段。这些模型包括在抗炎剂，免疫毒素，胶原酶，木瓜蛋白酶，或monoiodoacetate的膝盖，如果它们逃离关节空间，可能是有毒的单一注射。 OA的所有化学车型，MIA是最常使用的，PArticularly测试药物制剂的功效，以治疗疼痛，因为这个模型产生可再现的，健壮和快速疼痛样表型，可以通过改变MIA剂量11-15进行分级。 在啮齿类动物中MIA的关节内注射再现OA样病变和可以分析和量化的功能障碍。 MIA是甘油醛-3-磷酸酶的抑制剂，扰乱细胞的糖酵解并最终导致细胞死亡16,17。 MIA的关节内注射会导致软骨细胞死亡，从而导致软骨退变和随后的软骨下骨改建如骨的外观骨赘18,19。 作为MIA在大鼠该实用程序前20名进行了说明，在本文中，我们专注于MIA诱发骨关节炎的小鼠的方法，因为这种模式是与基因敲除小鼠的可用性正在越来越多地使用。我们描述了一个程序为injec极少量到膝盖和测量灵敏度在后肢有毒和非伤害性刺激方法和灰。 该方法的击穿将有助于减少可变性，因此，细化模型并减少所需的研究的动物数量。

<table><tbody><tr><td>Monoiodoacetate</td><td>Sigma-Aldrich</td><td>I-2512-25G</td><td>&amp;#8805; 98% purity</td></tr><tr><td>0.9% Saline</td><td>Mini-Plasco basic</td><td>365 4840</td><td></td></tr><tr><td>Isoflurane</td><td>Merial</td><td>DNI 4090/1</td><td></td></tr><tr><td>26 G Needle</td><td>Fisher Scientific</td><td>12947606</td><td></td></tr><tr><td>50 &amp;#956;l Hamilton Syringe</td><td>Sigma-Aldrich</td><td>20701</td><td></td></tr><tr><td>Von Frey Hairs</td><td>Linton Instruments</td><td>NC 122775-99</td><td></td></tr><tr><td>Incapacitance tester</td><td>Linton Instruments</td><td></td><td>Delivery on Request</td></tr><tr><td>Testing Cage Rack&amp;nbsp;</td><td>Ugo Basile</td><td>37450</td><td></td></tr><tr><td>Compact Anesthetic system</td><td>Vet-Tech</td><td>AN001B</td><td></td></tr><tr><td>Medical O&lt;sub&gt;2&lt;/sub&gt;</td><td>BOC</td><td>101-F</td><td></td></tr><tr><td>Aldasorbers</td><td></td><td>Vet -Tech</td><td>AN006A</td></tr></tbody></table>

the monoiodoacetate model of osteoarthritis pain in the mouse

患者的主要症状有骨关节炎（OA）是受痛途径中外围以及中心的变化所引发的疼痛。目前治疗方法的OA疼痛如NSAIDS或鸦片剂既不充分有效也不缺乏有害的副作用。 OA的动物模型正在开发，以提高我们的OA相关的疼痛机制的理解和定义的治疗药物的新靶点。目前在啮齿类动物OA的可用型号包括手术和化学干预到一个膝关节。该monoiodoacetate（MIA）模型已成为大鼠和小鼠在OA模型关节破坏的标准。的模式，这是更容易在大鼠来执行的，涉及MIA的注入诱导在同侧肢快速疼痛样反应，这可以通过不同剂量的注射来控制电平的膝关节。 MIA的关节腔内注射抑制glycer破坏软骨细胞糖酵解醛-3-磷酸脱氢酶和软骨细胞死亡，新血管形成，软骨下骨坏死和崩溃的结果，以及炎症。关节软骨和骨破坏的形态变化是反射的患者病理的某些方面。随着关节损伤，MIA注射诱导同侧后爪和负重赤字是衡量和量化简称机械感。这些行为的改变类似的一些由患者人口报告，从而确认在膝盖在MIA注射作为OA中疼痛的有用和相关的临床前模型中的症状。 这篇文章的目的是描述MIA关节内注射和过敏相关的开发行为记录的方法，用一记强调必要步骤，提供一致和可靠的记录。

我们最近已报道，0.5的注射- 1毫克MIA在小鼠膝关节诱导称为在同侧后爪的机械超敏性（异常性疼痛）和负重赤字长达4周，尽管起始点是剂量依赖性23。 在图1中所报告的数据构成以下的范围内的膝注射剂量在同侧后爪MIA引起的机械超敏性的时间过程的一个例子。具体地，MIA（0.5毫克/小鼠）的最低剂量诱导的阈值的50％相比，减少食盐的第10天的注入，和阈值注入后减小到那些盐水对照的70％的28的一天。为0.75mg MIA的中间剂量导致的阈值逐渐减少即分别降低了80％，比第10天生理盐水对照阈值和低仍高达28天1毫克MIA的最高剂量与一个符号相关联的在阈值量占用滴在第5天，并在第10天进一步降低，这是持续高达28一天。 在图2中报告的数据提供的是与MIA注射膝关节关联负重的变化的例子。在本组实验中，而0.5毫克MIA中的剂量没有引起在负重整个研究的28天时间显著变化，0.75毫克MIA剂量导致在重量的显著减少第10天由同侧爪承担起。值得注意的是，负重不对称0.75毫克MIA的关联机构可能会产生研究23之间的可变和不一致的结果。相反，1毫克MIA的剂量通常导致再现的承重不对称和图2中的数据表明承载在同侧后爪从第3天直到观察期末显著轻量化。正如预期的那样，萨利NE处理的动物没有表现出负重的变化。 <img alt="图1" src="/files/ftp_upload/53746/53746fig1.jpg" /> 图1 机械的发展异常性疼痛后MIA注入。同侧和对侧的后爪的爪退缩阈值之前和MIA（0.5，0.75，和1毫克/小鼠）和盐水（0.9％NaCl）中的注射后评估中，n = 8 - 10只/组。 * P &lt;0.05，** P &lt;0.01，*** P &lt;0.001相对于盐水处理组;双向重复测量ANOVA接着学生纽曼两比较事后检验。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53746/53746fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53746/53746fig2.jpg" /> 图2. 承重赤字的发展对后MIA注射剂的变化在两后肢之间的身体重量分布计算为[（生于生于同侧和对侧足重的同侧足/总重）×100]前和MIA的注射液（后进行了评估0.5，0.75，和1mg /小鼠）和盐水（0.9％氯化钠）中，n = 8 - 8 10只小鼠/组。 * P &lt;0.05，** P &lt;0.01，*** P &lt;0.001相对于盐水处理组。双向重复测量ANOVA接着学生纽曼两比较事后检验。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53746/53746fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse at JoVE.com

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

骨关节炎疼痛的鼠标Monoiodoacetate模式

骨关节炎（OA）或退行性关节病，是与该保持仅部分地由可用镇痛药控制疼痛有关的衰弱状态。动物模型正在开发以改善我们的OA相关的疼痛机制的理解。在这里，我们描述了用于办公自动化疼痛鼠标monoiodoacetate模型的方法。

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 ...

the-monoiodoacetate-model-of-osteoarthritis-pain-in-the-mouse

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

35.0K 次观看.  King's College London. 骨关节炎（OA）或退行性关节病，是与该保持仅部分地由可用镇痛药控制疼痛有关的衰弱状态。动物模型正在开发以改善我们的OA相关的疼痛机制的理解。在这里，我们描述了用于办公自动化疼痛鼠标monoiodoacetate模型的方法。 

视频: The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

在的鼠标Aortocaval瘘的技术方面

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

科研

医学

Inflammatory TMJ Pain Mouse Model: Bite Force and Von Frey Filament Assessments

Temporomandibular Joint Pain Measurement by Bite Force and Von Frey Filament Assays in Mice

Temporomandibular joint (TMJ) pain and osteoarthritis (OA) are common and debilitating disorders that impair patients' quality of life. The mechanisms driving diseases-related pain are poorly understood, and current treatments fail to provide effective and long-term therapeutic effects. Additionally, pain assessment in research, particularly orofacial pain, poses several challenges that complicate studies in both clinical and basic science settings. Therefore, we have established an inflammatory TMJ pain mouse model via intra-articular injection of CFA (Complete Freund's Adjuvant) and evaluated pain behaviors by bite force measurement and the von Frey filament test. Mice with CFA injection exhibited orofacial pain behaviors compared to PBS injection, including reduced bite force and head withdrawal threshold in the von Frey filament test. These methods are relatively easy to execute to have reproducible results and can be potentially extended to pain studies for other disease models related to TMJ disorders. Together, bite force, and the von Frey filament tests are reliable in measuring orofacial pain, as demonstrated in CFA injection-induced painful TMJOA mouse models.

This protocol describes how measuring bite force and head withdrawal threshold can capture pain behavior in Complete Freund's Adjuvant (CFA)-induced inflammatory temporomandibular joint (TMJ) pain.

Temporomandibular joint (TMJ) pain and osteoarthritis (OA) are common and debilitating disorders that impair patients' quality of life. The mechanisms ...

行为学

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 ...

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, ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

骨关节炎模型中骨髓、骨髓、淋巴结和合成组织内免疫细胞子集的流动细胞测量分析

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness

Subchondral bone thickening and sclerosis are the major hallmarks of osteoarthritis (OA), both in animal models and in humans. Currently, the severity of the histologic subchondral bone thickening is mostly determined by visual estimation based semi-quantitative grading systems. This article presents a reproducible and easily executed protocol to quantitatively measure subchondral bone thickness in a mouse model of knee OA induced by destabilization of the medial meniscus (DMM). This protocol utilized ImageJ software to quantify subchondral bone thickness on histologic images after defining a region of interest in the medial femoral condyle and the medical tibial plateau where subchondral bone thickening usually occurs in DMM-induced knee OA. Histologic images from knee joints with a sham procedure were used as controls. Statistical analysis indicated that the newly developed quantitative subchondral bone measurement system was highly reproducible with low intra- and inter-observer variabilities. The results suggest that the new protocol is more sensitive to subtle or mild subchondral bone thickening than the widely used visual grading systems. This protocol is suitable for detecting both early and progressing osteoarthritic subchondral bone changes and for assessing in vivo efficacy of OA treatments in concert with OA cartilage grading.

This methodology article presents a software-assisted quantitative measurement protocol to quantify histologic subchondral bone thickness in murine osteoarthritic knee joints and normal knee joints as controls. This protocol is highly sensitive to subtle thickening and is suitable for detecting early osteoarthritic subchondral bone changes.

软件辅助骨关节炎软骨下骨厚度的定量测量

Subchondral bone thickening and sclerosis are the major hallmarks of osteoarthritis (OA), both in animal models and in humans. Currently, the severity of ...

Destabilization of the Medial Meniscus and Cartilage Scratch Murine Model of Accelerated Osteoarthritis

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are used to mimic many aspects of the disease. The present protocol describes the destabilization and cartilage scratch model (DCS) of post-traumatic osteoarthritis. Based on the widely used destabilization of the medial meniscus (DMM) model, DCS introduces three scratches on the cartilage surface. The current article highlights the steps to destabilize the knee by transecting the medial meniscotibial ligament followed by three intentional superficial scratches on the articular cartilage. The possible analysis methods by dynamic weight-bearing, microcomputed tomography, and histology are also demonstrated. While the DCS model is not recommended for studies that focus on the effect of osteoarthritis on the cartilage, it enables the study of osteoarthritis development in a shorter time window, with special focus on (1) osteophyte formation, (2) osteoarthritic and injury pain, and (3) the effect of cartilage damage in the whole joint.

The present protocol describes the controlled microblade scratches on the surface of the articular cartilage after destabilizing the mouse knee by cutting the medial miniscotibial ligament. This animal model presents an accelerated form of osteoarthritis (OA) suitable for studying osteophyte formation, osteosclerosis, and early-stage pain.

加速性骨关节炎内侧半月板和软骨划痕鼠模型的不稳定

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are ...

<meta charset="utf8"></head><body>双后爪注射增强镇痛测试

A Modified Inflammatory Pain Model to Study the Analgesic Effect in Mice

The hot plate test is widely used to evaluate analgesic effects on inflammatory pain in mice. A commonly used model of inflammatory pain was induced with an intraplantar injection of carrageenan in one hind paw. However, the findings from our laboratory showed that mice with a single-hind-paw injection of carrageenan lifted their paws to avoid thermal nociception during the hot plate test. Because of this response,&#160;previous injection method cannot accurately reflect the thermal pain threshold. Thus, we investigated a new method to avoid this issue. In the present study, we modified the previous method by injecting carrageenan into both hind paws to establish the model of inflammatory pain. The results demonstrated that both-hind-paw injection with carrageenan was sensitive and a better method to induce inflammatory pain when using the hot plate test than single-hind-paw injection. On the basis of these findings, we designed further experiments in which mice with either both-hind-paw or single-hind-paw injection of carrageenan were treated intragastrically with celecoxib (30 mg/kg). The results of the hot plate test showed that celecoxib augmented the thermal pain threshold in mice with both-hind-paw injection of carrageenan but not in mice with single-hind-paw injection of carrageenan. In summary, we developed a superior method to induce a model of inflammatory pain to evaluate analgesic effect.

<meta charset="utf8"></head><body>作者聚焦:评估镇痛效果的增强方法 — 小鼠双后爪角叉菜胶注射液

Here, we present a modified inflammatory pain model with the both-hind-paw carrageenan injection to evaluate the analgesic effect.

研究小鼠镇痛作用的改良炎症性疼痛模型

The hot plate test is widely used to evaluate analgesic effects on inflammatory pain in mice. A commonly used model of inflammatory pain was induced with ...

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