Name: a mouse ear model for allergic contact dermatitis evaluation
Uploaded: 2023-03-24T14:55:00
Description: Watch this Scientific Journal Video about A Mouse Ear Model for Allergic Contact Dermatitis Evaluation at JoVE.com

这项工作得到了中国国家自然科学基金（NSFC）对N.-N的支持。Y. （81904212）;江苏省中医药科技项目（YB201995）;以及国家博士后科研人员专项资助项目（2020T130562）。

小鼠的所有护理和治疗均符合扬州大学机构动物护理和使用委员会制定的指导方针，并经机构动物护理和使用委员会批准，项目许可证为SYXK（SU）2022-0044。本研究使用6-8周龄的BALB / c雄性和雌性小鼠。每组由六只小鼠组成（见 材料表）。将笼子放置在温度受控室（22±2°C，12小时光照/黑暗循环）中，自由获取食物和水。实验流程图如图 1所示。
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这里描述的用于诱导小鼠耳朵中ACD样症状的方案可用于研究ACD的病理生理学，并作为开发新药的筛选工具。
建立ACD模型有两个关键步骤：初始致敏和随后的刺激。腹部通常是初始致敏的部位，但随后的刺激部位的选择略有不同。以往的研究表明，大多数学者选择使用DNFB/DNCB或恶唑酮等化学敏化剂在小鼠背部或颈部建立ACD模型，难免要使用刀片或修剪器来挖掘小鼠<sup class="xref"...

过敏性接触性皮炎（ACD）是一种常见的皮肤病，其特征是接触部位出现湿疹样症状，中度病例出现水肿和红斑，严重病例出现丘疹、糜烂、渗出甚至大面积疤痕1。它影响多达 20% 的人口，并且可以影响任何年龄的人2。ACD通常发生在反复接触过敏原的个体中，可能是由个体对家中或工作场所的一种或多种过敏原的免疫反应引起的3。IV型迟发性超敏反应被认为是ACD4的主要免疫反应类型。在反复暴露于过敏原的皮肤区域，循环记忆T细胞大量积累并诱导免疫和炎症反应3，5，6。这项工作的目的是提出一种可靠的实验室技术，用于进一步研究ACD发展中的免疫和炎症反应。
ACD 的发作通常是由于反复接触化学品引起的接触超敏反应。在过去的几十年中，许多研究人员在家鼠7，8，豚鼠9，10和其他动物中开发了各种ACD动物模型，以模拟疾病的发作。大多数实验方法包括两个阶段：腹部致敏（诱导）和在背部或耳垂上提供刺激（刺激）。常用化学物质主要有1-氟-2，4-二硝基苯（DNFB）/1-氯-2，4-二硝基苯（DNCB）8，9，11、噁唑啉酮12、漆酚13等。其中，DNFB和DNCB应用最广泛，首次报道于1958年10月10。镍敏化模型14和光过敏性接触性皮炎模型15也经常使用。
我们提出了一种构建ACD模型的实验方法。该方法在前人研究的基础上，与多个实验进行了对比，对该方法进行了总结和优化。与其他ACD模型相比，该模型具有一些优点，如个体差异小，实验周期短，化学刺激量小等。此外，本研究适用于小鼠，不仅经济，而且在基因敲除或转基因小鼠制备方面有更多的选择16。我们还描述了用于监测实验中ACD进展的各种方法，例如测量耳朵厚度，使用埃文斯蓝染料测量炎症渗出等。该模型不仅可以通过实验室手段分析小鼠耳朵、血液、脾脏等样本，探索ACD的发病机制，而且适用于新治疗方法的临床前评价，具有一定的推广意义。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-Fluoro-2,4-dinitrobenzene (DNFB)</td>
			<td>Merck</td>
			<td>200-734-3</td>
			<td>1-Fluoro-2,4-dinitrobenzene, &#8805;99%</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sinopharm Chemical Reagent Co. LTD</td>
			<td>10000418</td>
			<td>&#8805;99.5%</td>
		</tr>
		<tr>
			<td>Aluminum foil&#160;</td>
			<td>Cleanwrap</td>
			<td>CF-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Evans blue dye</td>
			<td>Solarbio</td>
			<td>314-13-6</td>
			<td>Dye content approx. 80%</td>
		</tr>
		<tr>
			<td>Mouse fixator</td>
			<td>ZHUYANBANG</td>
			<td>GEGD-SM1830</td>
			<td></td>
		</tr>
		<tr>
			<td>Olive oil</td>
			<td>Solarbio</td>
			<td>8001-25-0</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Pipet tip</td>
			<td>Biofount</td>
			<td>FT-200</td>
			<td>10 - 200 &#956;l</td>
		</tr>
		<tr>
			<td>Pipettor</td>
			<td>Eppendorf AG</td>
			<td>3123000250</td>
			<td>20 - 200 &#956;l</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Shanghai Gillette Co. LTD</td>
			<td>74-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Vernier calipers</td>
			<td>Delixi Electric</td>
			<td>DECHOTVCS1200</td>
			<td></td>
		</tr>
	</tbody>
</table>

a mouse ear model for allergic contact dermatitis evaluation

皮肤是人体的第一道防线，也是最容易受到环境化学物质影响的器官之一。过敏性接触性皮炎（ACD）是一种常见的皮肤病，表现为局部皮疹、发红和皮肤病变。ACD的发生和发展受遗传和环境因素的影响。虽然近年来许多学者构建了一系列ACD模型，但这些模型的实验方案各不相同，这使得读者很难很好地建立它们。因此，稳定高效的动物模型对进一步研究特应性皮炎的发病机制具有重要意义。在这项研究中，我们详细介绍了一种使用1-氟-2，4-二硝基苯（DNFB）在小鼠耳朵中诱导ACD样症状的建模方法，并描述了几种在建模过程中评估皮炎严重程度的方法。该实验方案已在部分实验中成功应用，在ACD研究领域具有一定的促进作用。

在反复的DNFB刺激下，DNFB组小鼠耳朵表现出与ACD相当的明显临床症状，敏感区域表现出发红、干燥甚至侵蚀渗出的典型症状。然而，耳部给予纯水（对照组）或溶剂对照（载体组）没有产生类似的症状（图4）。
同时，在DNFB组中，与未经治疗的右耳相比，DNFB刺激后左耳的厚度显着增加（图5A），而对照组和载体组没有显着差异（<str...

Watch this Scientific Journal Video about A Mouse Ear Model for Allergic Contact Dermatitis Evaluation at JoVE.com

A Mouse Ear Model for Allergic Contact Dermatitis Evaluation

在这里，我们描述了通过1-氟-2，4-二硝基苯（DNFB）诱导小鼠耳朵过敏性接触性皮炎的方法以及如何评估过敏性接触性皮炎的严重程度。

用于过敏性接触性皮炎评估的小鼠耳模型

Skin is the human body&#39;s first line of defense and one of the most exposed organs to environmental chemicals. Allergic contact dermatitis (ACD) is a ...

该模型不仅可用于探索ACD的发病机制，而且适用于新治疗方法的临床前进化，具有重要意义。这是一种稳定高效的动物模型，具有实验周期短、操作方便、症状明显、成功率高等诸多优点。我们的实验方案非常详细且易于操作。确保正确遵循协议的每个步骤。在开始建模之前，让小鼠适应环境一周，自由获得食物和水。在建模当天，使用紫外线灯和75%酒精，清洁和消毒环境和台面。使用棉签将肥皂水涂抹在小鼠的腹部，并用刀片或剃须刀沿毛发生长的方向剃除该区域。称量鼠标并比较每组之间的重量变化。在进行腹部致敏刺激之前，确保剃须引起的小鼠腹部皮肤的任何轻微损伤完全恢复。准备0.5%DNFB溶液，并用丙酮与橄榄油混合物以4：1的比例稀释。使用移液器枪吹气并混合20次，以彻底混合DNFB溶液。用移液器将25微升0.5%DNFB溶液涂抹在腹部剃须区域的中间，并用移液器尖端的光滑侧轻轻涂抹以使其均匀分散。DNFB刺激30秒后，将鼠标放在没有垫料的空笼子中，以防止小鼠擦掉DNFB溶液。当溶液完全干燥时，将鼠标放回原来的笼子，自由获取食物和水。对于耳敏化刺激，定向小鼠身体并使耳廓的外缘朝下，以防止溶液在DNFB刺激期间进入耳道。在第4天和第10天，使用移液器将20微升载体或0.2%DNFB溶液缓慢均匀地涂抹到左耳廓的内表面上，并使用移液器的光滑侧轻轻地分配溶液。右耳不经治疗。等到DNFB溶液干燥，然后将鼠标放回笼子中。腹部和耳朵致敏刺激后，从第一天开始每两天称重一次小鼠。将每只小鼠的体重与零日进行比较，并评估ACD对体重变化时小鼠体重的影响。从第一天开始，每两天拍摄一次小鼠耳朵的高分辨率照片以记录ACD临床症状。使用游标卡尺，每天在同一时间测量耳廓厚度以获得准确的结果。当有轻微堵塞时，停止游标卡尺继续向内夹紧，以防止组织对小鼠耳朵造成损害。保持位置固定并记录双耳的数据。记录三个数据的平均值，并以微米为单位评估耳朵肿胀。为了评估第11天的炎症肿胀程度，准备0.5%伊文思蓝染料溶液并用PBS稀释。要用固定器固定鼠标，请打开盖子，握住鼠标尾巴，使鼠标的头部面对固定器，让鼠标本能地爬进固定器。盖上盖子。使鼠标尾巴从盖子上的孔中出来，并调整固定器的长度以露出整个鼠标尾巴。用酒精棉球反复擦拭尾巴，或用温水浸泡30秒，轻轻捏住尾巴根部填充，扩张两侧静脉。在冷光源的照射下，使用1毫米胰岛素针将埃文斯蓝染料溶液缓慢注射到小鼠尾静脉中。等待 15 分钟拍摄鼠标耳朵的照片。DNFB组小鼠耳朵表现出与ACD相当的明显临床症状，敏感区域表现出发红，干燥，侵蚀和渗出的典型症状。然而，耳部给予纯水或溶剂控制没有产生任何类似于DNFB组的症状。在DNFB组中，与未经治疗的右耳相比，DNFB刺激后左耳的厚度显着增加。对照组和载体组无显著差异。DNFB组小鼠的左耳在注射埃文斯蓝染料后变成深蓝色，在视觉上与右耳不同。对照组和载体组中小鼠的左耳和右耳颜色大致相同。DNFB或简单的载体刺激使小鼠体重增加略有减慢，但没有导致显着的体重减轻。小鼠耳朵中反复的DNFB刺激导致脾脏肿大和脾脏指数升高。相比之下，载体组小鼠的脾脏指数没有显着变化。确保DINFB溶液完全混合并均匀地涂在鼠标耳朵上，而不会流入耳道。这是建模成功的关键。

Research

JoVE Journal

Immunology and Infection

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

科研

免疫与感染

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

鼠标型号病原体引起的慢性炎症局部及全身站点

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

甲醛暴露对大鼠新生辣椒素致过敏性皮炎模型的影响

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

红外测温仪在被动系统过敏反应和食品过敏评价中的应用

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

新生儿多药脓毒症的对照小鼠模型

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

小鼠特拉切斯刷细胞的分离与定量评价

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. ...

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

用于研究呼吸道病毒感染期间先天免疫细胞活化的非接触式共培养模型

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The ...

Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted ...

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

双室渗透支持系统中的犬肠类器官

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral ...