Name: micro-computed tomography analysis of the knee in aged dunkin-hartley guinea pigs after intra articular injection
Uploaded: 2024-08-02T13:00:00
Description: The purpose of this protocol is to guide researchers in performing a palpation-guided technique of intra-articular knee injection in guinea pigs and ...

本手稿中概述的研究得到了南卡罗来纳州 SmartState® 药物发现捐赠基金 （PMW）、MUSC 实验动物资源部和 MUSC 药物发现核心的支持。该出版物还得到了美国国立卫生研究院国家转化科学促进中心（National Center for Advancing Translational Sciences of the National Institutes of Health）的支持，资助号为TL1 TR001451和UL1 TR001450，以及美国国立卫生研究院（National Institutes of Health）国家牙科与颅面研究所（National Institute of Dental & Craniofacial Research of the National Institutes of Health），授予号为R01DE029637。

此处描述的所有方法均已获得南卡罗来纳医科大学机构动物护理和使用委员会的批准。该研究遵循3R原则。
1.关节内注射制剂
<ol><li>在开始实验之前，让Dunkin-Hartley豚鼠适应设施至少一周。 注：使用5-（n=2）、9-（n=1）和12-（n=2）月龄雄性豚鼠。男性表现出发育加速和更严重的 OA 表型。</li>
	<li>用电动剃须刀剃除膝盖区域。 注意：小心内侧。</li>
	<li>在异氟?...

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尽管最近在 OA 的对症治疗方面取得了进展，但完全缺乏预防 OA24 发作或延缓进展的治疗药物。目前，严重骨关节炎的唯一治疗方法是关节置换术，这种关节置换术成本高、侵入性强，并可能导致患者发病和死亡25.因此，迫切需要继续对骨关节炎动物模型进行研究，并持续开发新型疗法。有几种动物模型可用于研究 OA 的不同成分，包括自发模型和化学或手术诱?...

骨关节炎 （OA） 影响了 3250 万美国成年人。它是由关节软骨的进行性损失，关节内和关节周围的组织轻度炎症以及骨赘和骨囊肿的形成引起的1,2。症状通常在疾病的后期阶段表现出来，目前的治疗方法只能提供姑息性缓解，并且具有全身性副作用。缺乏改善疾病的药物源于对疾病潜在机制的了解不足3.因此，对治疗 OA 的改进药物存在着关键且持续的医疗需求。
有几种 OA 动物模型可用于检查疾病过程的不同组成部分4.虽然存在几种手术模型，包括前交叉韧带的横断和内侧半月板的不稳定，但这些都是侵入性的，需要高水平的技术技能5。化学诱导模型是侵入性相对较小的程序，通常用于研究 OA 疼痛机制6.一种广泛使用的小鼠模型涉及通过关节内膝部注射碘乙酸钠 （MIA） 诱导 OA。该模型产生可重复、稳健和快速的疼痛样表型，可以通过改变 MIA 剂量7 进行分级。诱导该模型的技术细节在前面已经描述过 7.由于它们的解剖学差异，将这种技术应用于较大的啮齿动物（如豚鼠）很困难。一些差异包括与小鼠8 中看到的远端融合相比，豚鼠相邻骨骼和关节空间周围的肌肉组织增加，以及铂骨和胫骨的关节融合。Dunkin-Hartley 豚鼠是一种广泛使用的豚鼠品系，是一种已建立的 OA 动物模型，因为它们会自然发展为这种疾病，从而为研究通过关节内注射给药的新型疗法对疾病进展的影响提供了一个强大的模型9。Dunkin-Hartley 豚鼠在三个月大时开始发展 OA，雄性表现出发育加速和更严重的表型10。在豚鼠中，OA 随着年龄的增长而进展，在 12 个月时，影像学检查显示相关病理学明显11。自发性 OA 模型，如 Dunkin-Hartley 模型，不需要任何干预来诱导 OA，从而概括了人类疾病表型的发展和进展，从而提供了一个强大的转化模型10。此外，当在给定动物的单膝关节中单侧施用新型疗法时，骨关节炎的自发发展允许内部控制。在分析数据时，这种内部控制可以最大限度地减少动物间变异的影响，并可能有助于减少动物总数。 
X 射线显微计算机断层扫描 （μCT） 分析是一种强大的工具，可以对12 级 OA 严重程度进行定量评估。μCT涉及扫描从旋转样品或旋转X射线源和检测器13获得的多个高分辨率X射线图像。然后，将三维（3D）体积数据以堆叠图像切片14的形式重建。由于矿化骨在 μCT 上具有出色的对比度，因此该模式可用于评估 3D 特征并对与 OA 15,16,17 相关的变化进行定量分析。与更广泛使用的工具相比，μCT 具有多项优势，包括组织病理学和步态分析。 与对一个或几个组织切片的组织学评估相比，μCT 扫描整个关节，并对 OA 病变提供更全面的评估18。虽然步态分析可以辨别关节功能随时间推移的症状性变化，但关节变化早在与 OA 相关的功能变化之前就已经发生。μCT 可以在跛行开始之前提供更灵敏的 OA 发展测量。两个特别相关的定量测量包括骨矿物质密度和小梁厚度，因为两者都在 OA19,20 的进展过程中增加。将分析分为软骨下板和小梁骨会有所帮助，因为它们具有不同的特征，以实现更可靠的测量和比较。
该方法的总体目标是帮助研究人员成功地对豚鼠进行关节内注射。所提出的方案使用5-（n=2）、9-（n=1）和12-（n=2）月龄的完整雄性Dunkin-Hartley豚鼠;手术可以外推到其他需要关节内膝关节注射的豚鼠品系和年龄。在自发的 OA 模型中，如 Dunkin-Hartley 模型，通常需要长时间监测疾病进展和对连续治疗的反应，持续数周至数月9。这种扩展的方案导致多次关节内注射，因此拥有适当的注射技术以防止不良事件非常重要，包括疼痛、跛行或感染，所有这些都会影响动物福利并混淆研究结果，同时需要额外的动物进行研究。所提出的方案描述了豚鼠关节内注射的方法和随后的μCT数据分析。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>200 Proof Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2701</td>
			<td>sterilizing agent</td>
		</tr>
		<tr>
			<td>3D.SUITE software</td>
			<td>Bruker</td>
			<td></td>
			<td>&#956;-CT analyzing software</td>
		</tr>
		<tr>
			<td>Betadine Surgical Scrub</td>
			<td>Avrio Health</td>
			<td>67618-151-16</td>
			<td>sterilizing agent</td>
		</tr>
		<tr>
			<td>Insulin syringe with needle</td>
			<td>Ulticare</td>
			<td>91008</td>
			<td>to perform injections</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal</td>
			<td>803249</td>
			<td>anesthesize animal</td>
		</tr>
		<tr>
			<td>Neutral Buffered Formalin</td>
			<td>Fisher Scientific</td>
			<td>23-427098</td>
			<td>Fix tissue</td>
		</tr>
		<tr>
			<td>Nrecon Software</td>
			<td>Bruker</td>
			<td></td>
			<td>&#956;-CT reconstruction software</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Cytiva</td>
			<td>SH30258.01</td>
			<td>control and diluting agent</td>
		</tr>
		<tr>
			<td>SkyScan 1176</td>
			<td>Bruker</td>
			<td></td>
			<td>to scan samples&#160;</td>
		</tr>
	</tbody>
</table>

micro-computed tomography analysis of the knee in aged dunkin-hartley guinea pigs after intra articular injection

该协议的目的是指导研究人员在豚鼠中执行触诊引导的关节内膝关节注射技术并使用显微计算机断层扫描进行评估。Dunkin-Hartley豚鼠是骨关节炎研究的稳健模型，因为它们会自发地在膝盖上发展骨关节炎。关节内给药是研究研究药物 在体内效果的常用方法。在人类中，通过关节内注射给药的治疗剂可以缓解疼痛并延缓骨关节炎的进一步进展。与任何物种一样，将针头引入关节空间可能会导致受伤，从而导致疼痛、跛行或感染。这些不良事件会损害动物福利，混淆研究结果，并需要额外的动物来实现研究目标。因此，当务之急是开发适当的注射技术来预防并发症，特别是在需要多次、重复关节内注射的纵向研究中。使用所提出的方法，五只豚鼠在全身麻醉下接受了双侧膝关节注射。注射后七天，对动物进行人道安乐死，以分析骨关节炎的严重程度。麻醉或膝关节注射后未发生不良事件，包括跛行、疼痛或感染。膝关节的 X 射线显微计算机断层扫描分析可以检测与骨关节炎相关的病理变化。显微计算机断层扫描数据表明，骨关节炎在老年动物中更为严重，表现为骨密度和小梁厚度随年龄增长而增加。这些结果与组织学变化和改良 Mankin 评分一致，这是一种已建立且广泛使用的评分系统，用于评估这些相同动物的关节炎严重程度。该方案可用于改进豚鼠的关节内注射。

在对活体动物进行关节内注射之前，在三具大鼠尸体上实施上述方案，以确保正确的注射位置。在练习过程中，使用上述方法将 50 μL 70% 新的亚甲蓝染料注射到双侧膝关节中。这相当于六次练习注射。注射后，通过切开关节空间的颅方面、髌骨远端和髶韧带来解剖膝关节，以可视化关节空间并验证染料沉积的位置。由于新的亚甲蓝染料是一种亮蓝色溶液，因此可以粗略地看到染料沉积的位置。尸?...

Dunkin-Hartley豚鼠是用于骨关节炎研究的既定动物模型。由于各种原因，此类研究可能受益于关节内注射，包括研究新型药物或治疗疾病。我们描述了一种在豚鼠中进行关节内膝关节注射的方法，以及随后评估关节炎相关膝关节变化的显微计算机断层扫描分析。

老年Dunkin-Hartley豚鼠关节内注射后膝关节的显微计算机断层扫描分析

The purpose of this protocol is to guide researchers in performing a palpation-guided technique of intra-articular knee injection in guinea pigs and ...

骨关节炎（OA）是一种退行性关节疾病，影响着3250万美国成年人。通常，OA症状仅在疾病变得严重后才会出现。缺乏改善疾病的药物源于对引发和驱动疾病的机制知之甚少。因此，对用于治疗 OA 的改进药物存在着关键且持续的医疗需求。Dunkin-Hartley 豚鼠是一种成熟的 OA 动物模型，因为它们会自然发展这种疾病，为研究通过关节内注射给药的新型疗法对疾病进展的影响提供了一个强大的模型。由于矿化骨在显微 CT 上具有出色的对比度，因此该模式可用于评估 3D 特征并对与 OA 相关的变化进行定量分析。两个特别相关的定量测量包括骨矿物质密度和小梁厚度，因为两者在疾病进展过程中都会增加。所描述的豚鼠关节内注射方法和随后的显微CT数据分析将有助于减少研究变异性并提高数据分析的效率。用电动剃须刀剃掉膝盖。对动物进行麻醉，并通过缺乏脚趾捏合反应来确认足够的麻醉深度。将无菌眼部润滑剂放在双眼中，以防止干燥和受伤。使用圆周运动清洁注射部位三次，并使用稀释的磨砂膏和酒精溶液交替进行。应使用无菌手套。该视频包括使用高压灭菌器硝基手套。在整个手术过程中应使用无菌技术。将膝盖弯曲 90 度，将手指向髌骨远端移动，通过弯曲和伸展后肢来定位关节空间远端的凹槽。胫骨可以感觉到髌骨远端的骨骼结构。一旦确定了胫骨和髌骨的位置，关节就在它们之间。小心地将胰岛素针插入关节间隙内髌骨远端的中线。针头应插入皮肤下方一到两毫米。慢慢注入体积。通过弯曲和伸展关节几次来按摩膝盖，以确保药物的正确分布。将含有福尔马林的样品放入兼容的容器中。根据制造商的建议校准 micro-CT 机器的暗场和明场暴露。使用 18 微米的铝铜滤光片扫描样品。使用偏移相机使用 0.7 度旋转步长进行 360 度。从显微 CT 图像中选择一个切片。检查错位补偿。在设置下，应用平滑、光束硬化、CS 旋转和环形伪影。选择“开始”以开始重建。选择 VOI 并将样品定向为垂直对齐，以便以后进行分析。将编辑后的 VOI 另存为新文件夹。选择要分析的图像范围，从软骨下板开始。建议对小梁中的软骨下板进行拆分分析，因为它们具有不同的骨骼特征。为每张图像选择感兴趣的区域，以确保它包含骨骼。选择二元选择。调整直方图，使骨骼中的背景完全分离。选择“骨矿物质密度”选项卡。将该数据保存到新的分析数据文件夹中。选择自定义处理。首先，执行阈值并选择“自动”，然后运行。然后选择“去斑点”，然后选择“删除黑色斑点”。重复 Despeckle，然后选择 Remove white speckles。选择“3D 分析”，然后选择基本值和附加值。A 显示一个被解剖的膝关节，关节腔内存在亚甲蓝染料，显示注射正确。B 是浅注射，导致皮下空间形成水泡。C表示关节空间内缺乏亚甲蓝染料，表明注射不正确。将软骨下板（如 A 所示）和小梁骨（如 B 所示）的分析分开可能会有所帮助，因为有时它们对不同的骨分析具有不同的参数。对于骨矿物质密度，我们看到 12 个月大的豚鼠与 5 个月大的豚鼠相比，12 个月大的豚鼠的平均数有所增加，这在软骨下板和小梁骨中都是如此，这表明骨矿物质密度随着时间的推移而增加。此外，对于小梁厚度，我们看到 12 个月大的豚鼠和 9 个月大的豚鼠和 5 个月大的豚鼠之间的厚度（以毫米为单位）有所增加，这表明小梁厚度随着时间的推移而增加。改良的Mankin评分随着豚鼠年龄的增长而增加，如图A所示，图B至D显示甲苯胺蓝染色。B是5个月大的豚鼠，C是9个月大的豚鼠，D是12个月大的珍珠猪。图C中的黑色星号表示蛋白多糖丢失，而图D中的黑色星号表示裂隙，图D中的白色星号表示细胞不足。目前，严重骨关节炎的唯一治疗方法是置换治疗，这种疗法成本高昂、侵入性强，并可能导致患者发病和死亡。因此，迫切需要继续对OA的动物模型进行研究，并持续开发新型疗法。尽管关节内注射是豚鼠骨关节炎研究不可或缺的一部分，但该程序尚未得到系统的详细说明。正确的注射程序对于确保动物福利和减少动物总数至关重要，特别是对于需要在数月内多次注射的研究。为了系统地评估OA相关的变化，我们描述了micro-CT分析。Micro-CT 对 OA 变化的评估比对整个样本（而不是一个或几个组织切片）的扫描进行组织学检查更深入。虽然Dunkin-Hartley豚鼠为研究OA进展和新型疗法的效果提供了一个稳健的模型，但该模型存在局限性。与手术或化学诱导的 OA 后快速发展的变化相比，自发模型通常需要更长的研究期。此外，研究人员可以通过对每只动物的一个肢体进行治疗来减少动物间变异性的影响，使对侧肢体可以作为内部控制。

Research

JoVE Journal

Medicine

Micro-Computed Tomography Analysis of the Knee in Aged Dunkin-Hartley Guinea Pigs after Intra Articular Injection

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

微型计算机断层扫描成像制备小鼠冠状动脉血管逆行灌注和灌装

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

科研

医学

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

药物输送到微型计算机断层扫描成像缺血后肢血管灌注与微滤渗透

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

评估人体脂肪注射生存能力的裸鼠与微型计算机断层扫描

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

采用微计算机断层扫描对肿瘤发展的评估和后续反应来治疗肺癌的小鼠模型

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

结合近红外荧光成像和微计算机断层扫描直接可视化脑血栓

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Cochlear Implantation in the Guinea Pig

豚鼠耳蜗植入术

Cochlear implants are highly efficient devices that can restore hearing in subjects with profound hearing loss. Due to improved speech perception ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

油酸注射液在猪急性呼吸窘迫综合征模型中的研究

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

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

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 ...

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

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 ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

用于慢性病大型哺乳动物心脏模型对比增强显影显微计算机断层扫描的组织制备技术

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...