Name: mouse footpad inoculation model to study viral-induced neuroinflammatory responses
Uploaded: 2020-06-14T14:30:00
Description: Watch this Scientific Journal Video about Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses at JoVE.com

作者承认查尔斯河实验室的出色技术支持，执行组织病理学分析。这项工作由国家神经疾病和中风研究所（RO1 NS033506和RO1 NS060699）资助。资助者在研究设计、数据收集和分析、决定出版或编写手稿方面没有作用。

所有动物实验均按照一项议定书（编号2083-16和2083-19）进行，该议定书由普林斯顿大学动物护理和使用委员会（IACUC）审查和批准。这项工作是严格按照生物安全等级2（BSL-2）的要求进行的，我们拥有一个设备齐全的实验室，得到普林斯顿大学生物安全委员会的批准。包括小鼠脚垫磨损、病毒接种、小鼠解剖和组织收集在内的程序在普林斯顿大学生物防护动物设施室的生物安全柜（BSC）中执行。执...

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此处描述的脚垫接种模型有助于调查在αherpes病毒感染期间神经炎反应的启动和发展。此外，这个体内模型用于建立从PNS到CNS复制和传播的αherpes病毒的动力学。这是其他接种模型的替代，如侧翼皮肤接种模型，它依赖于深皮肤划痕13，或颅内路线，直接将病毒引入CNS 14，15，16。14,15,16因此，使用脚垫模型，可...

这项研究描述了一个脚垫接种模型，以调查病毒从PNS到CNS的复制和传播以及相关的神经炎症反应。脚垫接种模型已被密集用于研究神经元,1、2、32中的αherpes病毒感染。13该模型的主要目标是允许神经热带病毒在到达CNS之前通过PNS到达最大距离。在这里，这个模型被用来获得新的见解，在感染伪狂犬病病毒（PRV）的小鼠中发展一种特定的神经病变（神经病变瘙痒）。
PRV是一种与几种众所周知的病原体（即单纯疱疹1型和2型[HSV1和HSV2]和水痘-佐斯特病毒[VZV]）相关的α疱疹病毒，分别引起冷疮、生殖器病变和水痘4。这些病毒都是泛异性的，能够感染许多不同的细胞类型，而不表现出对特定组织类型的亲和力。然而，它们都表现出一种典型的神经营养，入侵宿主物种的PNS（偶尔还有CNS）。自然宿主是猪，但PRV可以感染大多数哺乳动物。在这些非自然宿主中，PRV感染PNS并诱发一种称为"疯痒"的严重瘙痒，然后是急性死亡55，6。6神经免疫反应在PRV感染的临床结果和发病机制中的作用一直知之甚少。
脚垫接种模型允许PRV在脚垫的表皮细胞中启动感染。然后，感染扩散到感觉和同情神经纤维，内生表皮，汗腺，和真皮。感染通过通过坐骨神经向DRG移动的病毒颗粒在大约60小时内传播。感染通过脊髓传播，当动物垂死（感染后82小时）时，最终到达后脑。在此期间，可以收集、处理和分析组织样本，以进行病毒复制和免疫反应标记。例如，组织学检查和病毒载量定量可以在不同的组织中进行，以确定PRV发病机制中临床、病毒学和神经炎过程的启动和发展之间的相关性。
利用脚垫接种模型，可以研究小鼠PRV诱导瘙痒的细胞和分子机制。此外，该模型可以提供新的见解，在疱疹病毒感染期间病毒引起的神经炎症的启动和发展。更好地了解αherpes病毒引起的神经病变背后的过程，可能导致创新治疗策略的发展。例如，该模型有助于研究疱疹后病变患者（如疱疹、带状疱疹）的神经病变机制，并测试小鼠中针对相应人类疾病的新治疗靶点。

<table>
	<tbody>
		<tr>
			<td>Antibody anti-PRV gB</td>
			<td>Made by the lab</td>
			<td></td>
			<td>1/500 dilution</td>
		</tr>
		<tr>
			<td>Aqua-hold2 pap pen red</td>
			<td>Fisher scientific</td>
			<td>2886909</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact emery boards-24 count (100/180 grit nail files)</td>
			<td>Revlon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Complete EDTA-free Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice (5-7 weeks)</td>
			<td>The Jackson Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI solution (1mg/ml)</td>
			<td>Fisher scientific</td>
			<td>62248</td>
			<td>1/1000 dilution</td>
		</tr>
		<tr>
			<td>Disposable sterile polystyrene petri dish 100 x 15 mm</td>
			<td>Sigma-Aldrich</td>
			<td>P5731500</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Hyclone, GE Healthcare life Sciences</td>
			<td>SH30022</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phophate Buffer Saline (PBS) solution</td>
			<td>Hyclone, GE Healthcare life Sciences</td>
			<td>SH30028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Hyclone, GE Healthcare life Sciences</td>
			<td>SH30088</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine curved scissors stainless steel</td>
			<td>FST</td>
			<td>14095-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G mounting media</td>
			<td>Fisher scientific</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution, neutral buffered 10%</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Isothesia Isoflurane</td>
			<td>Henry Schein</td>
			<td>NDC 11695-6776-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube 2ml</td>
			<td>Denville Scientific</td>
			<td>1000945</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtube 1.5ml</td>
			<td>SARSTEDT</td>
			<td>72692005</td>
			<td></td>
		</tr>
		<tr>
			<td>Negative goat serum</td>
			<td>Vector</td>
			<td>S-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>154022</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Glide needle 18G</td>
			<td>BD</td>
			<td>305196</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades steel back</td>
			<td>Personna</td>
			<td>9412071</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA lysis buffer (RLT)</td>
			<td>Qiagen</td>
			<td>79216</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Beads, 5 mm</td>
			<td>Qiagen</td>
			<td>69989</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost/plus microscopic slides</td>
			<td>Fisher scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue lyser LT</td>
			<td>Qiagen</td>
			<td>69980</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek OCT</td>
			<td>Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>488 (goat anti-mouse)</td>
			<td>Life Technologies</td>
			<td>A11029</td>
			<td>1/2000 dilution</td>
		</tr>
	</tbody>
</table>

mouse footpad inoculation model to study viral-induced neuroinflammatory responses

该协议描述了一种脚垫接种模型，用于研究小鼠α疱疹病毒感染期间神经炎症反应的启动和发展。由于αherpes病毒是周围神经系统（PNS）的主要入侵者，该模型适用于病毒复制的动力学、从PNS到CNS的传播以及相关的神经炎症反应。脚垫接种模型允许病毒颗粒从脚垫表皮的主要感染部位传播到内层、汗腺和真皮的感官和同情神经纤维。感染通过坐骨神经传播到背根神经（DRG），并最终通过脊髓传播到大脑。在这里，鼠标脚垫接种了伪狂犬病病毒（PRV），一种与单纯疱疹病毒（HSV）和水痘-佐斯特病毒（VZV）密切相关的α疱疹病毒。该模型表明，PRV感染诱发严重炎症，其特点是中性粒细胞渗透和DRG。ELISA随后在同质组织中检测到高浓度的炎症细胞因子。此外，观察到DRG中的PRV基因和蛋白质表达（通过qPCR和IF染色）与亲炎细胞因子的产生之间有很强的相关性。因此，脚垫接种模型提供了更好地了解αherpes病毒引起的神经病变背后的过程，并可能导致创新治疗策略的发展。此外，该模型可以指导对周围神经病变的研究，如多发性硬化症和相关的病毒引起的对PNS的损害。最终，它可以作为药物开发具有成本效益的体内工具。

小鼠脚垫接种模型允许对体内αherpes病毒感染的免疫发病机制进行表征，包括将感染从接种的脚垫复制和扩散到神经系统，以及诱导特定的神经炎症反应。
在这项研究中，我们首先对鼠标后脚垫进行了磨蚀，或者用PRV（PRV-Becker）的毒性菌株对磨损区域进行模拟接种或接种。在控制脚垫中可以看到磨损部位。作为愈合过程的一部分，在磨损部位形成地壳（图1，<...

Watch this Scientific Journal Video about Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses at JoVE.com

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

脚垫接种模型是描述体内病毒引起的神经炎反应的宝贵工具。特别是，它提供了病毒动力学和相关免疫病理过程在周围神经系统启动的明确评估。

小鼠脚垫接种模型，研究病毒引起的神经炎反应

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus ...

该协议允许在病毒感染期间研究小鼠周围神经系统神经刺激反应的启动和发展。有了这个模型，感染从外围到周围和中枢神经系统的距离更大，从而可以最大限度地评估神经刺激反应的动力学。将五到七周大麻醉的 C57BL/6 小鼠置于手术垫上的 supine 位置后，确认对踏板反射缺乏反应。并使用 100 到 180 砂砾磨板，用大约 20 次板的通行证轻轻擦擦脚跟和行走垫之间一个后脚垫的厚大皮肤。然后使用细钳慢慢剥下被磨损分离的地层角膜，以暴露地层巴萨勒。轻轻混合后，在磨损的脚垫上涂抹20微升的合适滴度病毒。仅在控制动物的磨损脚垫中同时进行模拟接种。每次应用后，用针头轴轻轻擦脚垫10次，便于每10分钟吸附一30分钟。当磨擦脚垫干燥时，让小鼠通过监测恢复，直到胸骨卧放，然后将动物放入单独的笼子进行临床随从和取样。在适当的实验时间点，收集病毒暴露的器官，包括心脏，肺，脾脏，胰腺，肝脏，肾脏和膀胱到单独的1.5毫升微管的冰上。然后收获脚跟和行走垫之间的磨光脚垫，将样品放入单独的1.5毫升微管的冰上。接下来，将鼠标放在易发位置，用70%乙醇将毛皮淋湿。要露出椎柱，在骨盆区域做一个小切口，将皮肤从后肢剥到头部。要切除前肢和后肢，请使用剪刀与两侧的脊柱平行并靠近脊柱。并移除头骨底部的头部。用剪刀把头骨从前体巨子打开到前骨。并使用钳子向横向拉开头骨。然后用钳子轻轻地挖出大脑，把大脑放入冰上1.5毫升的微管中。使用弯曲的剪刀清洁肌肉、脂肪和软组织的脊柱，并切除尾巴。然后将完整的脊柱放入15毫升的无菌冰冷PBS管中，在冰上不超过3小时。解剖后，将脊髓转移到组织培养盘中，以便去除剩余的软组织。使用剃须刀刀片在 S2、L1 和 T1 椎骨的脊柱上进行三次横向切口。将碎片转移到含有无菌冰冷 PBS 的单个 15 毫米餐具中。跟踪和标记段的原点，供以后分析。接下来，使用钳子将一个段的侧向上固定，并使用剃须刀刀片将单个纵向切口穿过中线，将柱分成两半。将两半放在一个新的培养皿中，在原始方向上含有新鲜、无菌的冰冷 PBS，以便能够区分脊柱的左右两侧。在解剖显微镜下，使用细钳将脊髓从脊椎的两半中轻轻剥到骨管方向。然后将两个脊髓的两半放在单独的1.5毫升微管中，在冰上含有500微升的无菌冰冷PBS。要露出背根结节，请轻轻地将脑膜从脊柱的一侧取出到另一侧。将裸露的白色脊柱神经从脊柱中拉出。从脊椎部分收获圆形、透明的后根结节，成为15毫米的冰冷PBS培养皿。并收集同性与反面的后根结节，正如刚刚证明的。从另外两个脊柱段采集脊髓神经和后根结节后，以高速离心所有组织管。然后吸进超自然剂，将样品在液氮中闪冻，储存在零下80摄氏度，直到均质。对于组织均质化，在称出每个样品的100毫克之前，先将冷冻组织管解冻在冰上。转移到含有无菌珠子和500微升放射性免疫沉淀测定缓冲液的两毫升微离心管中。接下来，以20个周期/秒的室温在室温下将组织以20个周期/秒中断两分钟，然后是1分钟的等待期，20个周期/秒，再中断两分钟。在第二轮均质化后，以高速离心组织，然后将每个上经剂的500微升转移到一个新的适当标记的管中。储存在零下20摄氏度，直到下游分析。在控制脚垫中可以看到磨损的站点。作为愈合过程的一部分，地壳在磨损地点形成是典型的。相比之下，接种假狂犬病的小鼠在人道终点处表现出严重炎症，其特点是肿胀和发红。对接种的脚垫和后根结节进行组织病理学检查，发现伪狂犬病足部部分内表皮坏死和严重皮肤炎症。在受感染的 DRG 部分也观察到嗜中性粒细胞的渗透。与感染后7小时和82小时的对子相比，在感染的小鼠的脚垫和后根结节中测量了G-CSF蛋白表达显著增加。与对子相比，在感染伪狂犬病小鼠的脊髓、大脑、心脏和肝脏组织感染后82小时也观察到显著的G-CSF水平。此外，从感染后24小时开始，在所有被检测的伪狂犬病小鼠的组织中检测到相当水平的IL-6。在垂死状态下，在脚垫、后根结节、脊髓和大脑中检测到伪狂犬病。在伪狂犬病感染小鼠的后根结节神经元中，用高细胞质表达的psuedorabies糖蛋白gB。为了确保成功感染，重要的是，一个列中的病毒液滴不会从磨损的足垫下降。这种动物模型可以起到平台作用，测试抗炎和抗病毒药物的疗效，以防止病毒引起的周围神经病变。

Research

JoVE Journal

Immunology and Infection

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

实验小鼠模型阴道接种和样品采集的协议念珠菌阴道炎

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of ...

科研

免疫与感染

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

鼻咽癌相关淋巴组织（NALT）在鼠标的响应中的作用研究疫苗

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

人源化的小鼠模型来研究细菌感染瞄准微血管

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

增加了恢复时间，并减少LPS管理研究迷走神经刺激机制的有限炎症反应

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

IL-8瞬时转化的小鼠模型<em&gt;体内</em&gt;长期监测炎症反应

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

一种研究人体 T 细胞召回反应的吸水泡协议

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

一种研究食细胞诱导免疫应答的 mellonella 口服液模型

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

活体细胞毒性试验研究肿瘤特异性 CD8+ t 细胞反应中的免疫调节

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...