Name: a rat model of ecohiv brain infection
Uploaded: 2021-01-21T14:30:00
Description: Watch this Scientific Journal Video about A Rat Model of EcoHIV Brain Infection at JoVE.com

这项工作由NIH赠款HD043680、MH106392、DA013137和NS100624资助。

所有动物协议均由南卡罗来纳大学动物护理和使用委员会审查和批准（联邦保证号：D16-00028）。六只成年雄性F344/N大鼠被配对安置在12/12光下的受控环境中：暗循环，可获得食物和水。所有动物都受到照料，使用国家卫生研究院在《实验室动物护理和使用指南》中制定的准则。
1. 293 个 FT 细胞中的病毒包装
<ol><li>培养293 FT细胞（5×105/mL）在明胶涂层75厘米2 烧...

<ol>
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在本议定书中，我们建立了大鼠感染EcoHIV的艾滋病毒模式。具体来说，我们描述了EcoHIV的双边立体毒性注射到皮层，在注射后7天成功地诱导了大鼠大脑中的活性HIV感染。此外，我们证明，大鼠的EcoHIV感染可能是研究HAND关键方面的一个很好的生物系统。在EcoHIV感染后八周，大鼠表现出严重的神经认知障碍，其中包括在NAc的MSN中时间处理和突触功能障碍的改变。鉴于动物模型对神经HIV和<sup class="xref"...

生物系统增强了我们对HIV-1相关神经认知障碍（HAND）及其基础神经机制2的理解。确定哪个生物系统最适合任何特定的研究，往往取决于利益问题2。宿主动物模型范围的限制对艾滋病毒-1疾病发展的研究提出了挑战。为了研究HIV-1病毒的复制和发病机理，Potash等人3日创建了活性HIV-1感染的小鼠模型，用生态MLV gp80取代了HIV表面包络糖蛋白gp120的编码区域，从而成功地在4号小鼠身上进行了病毒复制。在有幻想的HIV（EcoHIV）小鼠注射尾静脉后，观察到许多特征与HIV-1血清阳性个体（如受感染的淋巴细胞和巨噬细胞，针对抗病毒免疫反应，以及炎症3，5，6）相似。
虽然老鼠和老鼠都是Muridae的成员，但基本物种的差异可能会影响它们适合特定的实验问题7。因此，将EcoHIV感染模型推广到大鼠（通常用于药物滥用和神经认知障碍的研究）将有利于神经HIV的研究。例如，其较大的尺寸使朱拉导管植入药物自我管理程序更实用8。大鼠的药物自我管理技术被用来评估艾滋病毒-19的动机。此外，许多神经认知/行为任务最初是为10号大鼠设计的。在这里，我们报告利用大鼠EcoHIV的立体毒性注射来扩展EcoHIV感染模式，并提供了一个关键的机会来解决与神经HIV和HAND相关的新问题。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>293FT cells</td>
			<td>ThermoFisher Scientific</td>
			<td>R70007</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic solution</td>
			<td>Cellgro</td>
			<td>30004CI</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Corning BioCoatGelatin 75cm&#178; Rectangular Canted Neck Cell Culture Flask with Vented Cap</td>
			<td>Life Technologies</td>
			<td>354488</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning DMEM with L-Glutamine, 4.5 g/L Glucose and Sodium Pyruvate</td>
			<td>Life Technologies</td>
			<td>10013CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>637-137</td>
			<td></td>
		</tr>
		<tr>
			<td>drill</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>World Precision Instruments</td>
			<td>14095</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #7 Forceps</td>
			<td>World Precision Instruments</td>
			<td>14097</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Snap-Cap Microcentrifuge Biopur Safe-Lock Tubes</td>
			<td>Life Technologies</td>
			<td>22600028</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethicon Vicryl Plus Antibacterial, 4-0 Polyglactin 910 Suture, 27in. FS-2</td>
			<td>Med Vet International</td>
			<td>VCP422H</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton</td>
			<td>1701</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen Lipofectamine 3000 Transfection Reagent</td>
			<td>Life Technologies</td>
			<td>L3000015</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Forceps</td>
			<td>World Precision Instruments</td>
			<td>15914</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>World Precision Instruments</td>
			<td>500216</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentivirus-Associated p24 ELISA Kit</td>
			<td>Cell Biolabs, inc.</td>
			<td>VPK-107-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Lenti-X Concentrator</td>
			<td>Takara</td>
			<td>PT4421-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td>11058021</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold</td>
			<td>Fisher Scientific</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Sevoflurane</td>
			<td>Merritt Veterinary Supply</td>
			<td>347075</td>
			<td></td>
		</tr>
		<tr>
			<td>stereotaxic apparatus</td>
			<td>Kopf Instruments</td>
			<td>Model 900</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost Plus Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-154%</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Scissors</td>
			<td>World Precision Instruments</td>
			<td>500086</td>
			<td></td>
		</tr>
	</tbody>
</table>

a rat model of ecohiv brain infection

据充分研究，EcoHIV感染小鼠模型在研究艾滋病毒相关神经并发症方面具有重大效用。建立EcoHIV感染大鼠模型，用于研究药物滥用和神经认知障碍，将有利于神经HIV和HIV-1相关神经认知障碍（HAND）的研究。在本研究中，我们演示了使用有幻想的艾滋病毒（EcoHIV）成功创建了活性艾滋病毒感染的老鼠模型。首先，EcoHIV 的扁桃体结构在培养的 293 FT 单元中包装了 48 小时。然后，有条件的介质被集中和滴答作响。接下来，我们将EcoHIV-EGFP的双边立体毒性注射到F344/N大鼠脑组织中。感染一周后，在受感染的脑组织中检测到EGFP荧光信号，表明EcoHIV成功地诱发大鼠的艾滋病毒感染。此外，还对微胶质细胞标记物Ibba1进行了免疫染色。结果表明，微胶质是窝藏EcoHIV的主要细胞类型。此外，EcoHIV大鼠在时间处理方面表现出改变，这是HAND潜在的神经行为机制，感染后8周出现突触功能障碍。本研究将EcoHIV艾滋病毒-1感染模型共同扩展至大鼠，为研究大脑中的HIV-1病毒库以及手部和药物滥用等相关合并症提供了宝贵的生物系统。

条件介质是从感染了293FT细胞的EcoHIV-EGFP的扁豆病毒中收集的。接下来，它被浓缩和滴答作响，然后立体地注入F344/N大鼠的大脑（皮质区域）。注射后七天，老鼠被牺牲，图像从冠状脑切片从胸围5.64毫米到布雷格玛-4.68毫米不等。在 图1A中，整个大脑都有显著的EcoHIV-EGFP信号，特别是在皮层和海马凹痕陀螺中。此外，与Iba1和EcoHIV-EGFP探头的双重标记提供了有力的证据，证明微?...

Watch this Scientific Journal Video about A Rat Model of EcoHIV Brain Infection at JoVE.com

A Rat Model of EcoHIV Brain Infection

在这里，我们提出了一个协议，建立一个新的大鼠模式，使用有幻想的艾滋病毒（EcoHIV），这是关键，以提高我们对HIV-1病毒库在大脑中的理解，并提供一个系统，以研究艾滋病毒相关的神经认知障碍和相关的合并症（即药物滥用）。

生态HIV脑感染的老鼠模型

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

使用大鼠，该协议可用于建立使用幻想艾滋病毒的活性艾滋病毒感染的新模式。与大鼠，建立EcoHIV感染模型，用于研究药物滥用和神经认知障碍，可能有利于研究神经HIV和HIV-1相关的神经认知障碍。演示程序将是李海龙，我实验室的一名研究员。对于将病毒包装成293个T细胞，种子每毫升DMEM的5倍10至5293T细胞，在两个明胶涂层的75平方厘米烧瓶中各补充10%FBS，并在37摄氏度下孵育细胞，直到培养物达到50%的共融度。在转染当天，在 1.5 毫升微中福管中稀释 750 微升每瓶中等试剂中的 22.5 微升转染试剂，并涡流溶液三秒钟。将20微克的奇美EcoHIV质粒DNA稀释到750微升的介质中，在第二个1.5毫升微中微流管中每瓶与彻底混合。将稀释后的DNA与稀释的输血试剂与温和混合相结合，在室温下孵育15分钟。在孵化结束时，将每个病毒悬浮添加到 10 毫升的预热 DMEM 介质中，并将每个病毒补充溶液添加到 293 个 T 细胞的一个烧瓶中。经过两天的细胞培养后，将烧瓶中的超自然分子集中到一个50毫升的圆锥管中进行离心，并将澄清的超自然分子转移到一个新的50毫升管中。在澄清的超自然剂中加入八毫升 Lenti-X 浓缩器，并与温和的反转混合。在摄氏四度下进行两天的孵化后，将混合物离心，小心地去除超母体，然后用100毫升PBS的100微升轻轻补充颗粒，并使用P24 ELISA试剂盒来滴定病毒浓度。对于 EcoHIV-EGFP 注射，在确认对踏板反射缺乏反应后，剃掉大脑区域的头发，用 70% 乙醇和氯西丁磨砂对暴露的皮肤进行两次消毒。将大鼠固定在立体氧化装置的易发位置，并通过头皮中线的皮肤切口五到六厘米。在 0.8 毫米横向标记一个钻孔位置，在每个头骨位置标记一个直径为 0.4 毫米的孔，然后标记一个 1.2 毫米的轮盘孔。将每毫升滴定 EcoHIV Lentivirus 解决方案的第六个转导单元加载 1.04 倍 10 到 10 微升注射注射器中，并将注射器固定到立体氧化装置中。将针头移近一个钻孔的表面，并将针头 2.5 毫升插入孔中。以每分钟 0.2 微升病毒的速度注入一微升病毒溶液。当所有的病毒都注射后，将针头留在注射区域内五分钟，然后慢慢收回针头，直到针头在老鼠头骨之外。用 4-0 丝线缝合关闭皮肤切口，用 70% 乙醇对溶液进行消毒，然后将大鼠放在带加热垫的恢复室中，并附有监控，直到重新入职。病毒输液一到八周后，在确认对踏板反射缺乏反应后，将大鼠固定在烟罩内的超平位置，沿着胸腔中线将皮肤固定。切开隔膜以打开胸腔，并将 20 个仪表乘 25 毫米针插入左心室。立即用剪刀打开右中庭，以每分钟 5 毫升的流速将 50 毫升预冷 100 毫升 PBS 灌注。当所有的PBS都交付后，用100毫升的冷4%副甲醛香水。当所有的固定剂都交付后，从头骨中取出大脑，用新鲜的4%副甲醛在一夜之间修复组织。第二天早上，将样品放在40毫升的100毫升PBS中，在50毫升的管子里放置大约三天，直到大脑稳定到管子底部，然后在零下80摄氏度下将甲基丁醇中的脑组织冷冻两分钟。冷冻后，使用零下20摄氏度的低温喷涂和细画笔获得50微米厚的日冕部分。获得所有部分后，用 300 微升防褪色介质覆盖样品，并安装 22 个 50 毫升盖片。当介质干燥时，通过共聚焦显微镜对部分进行成像。伦蒂病毒注射七天后，大鼠冠状脑切片图像显示整个脑组织中存在大量 EcoHIV-EGFP 信号，尤其是在皮层和海马凹痕陀螺内。与Iba1和EcoHIV-EGFP探头的双重标记提供了强有力的证据，证明微胶质是大脑中含有EcoHIV表达的主要细胞类型。感染后八周，EcoHIV注射的动物对控制盐水的间隔表现出相对的麻木不仁，与盐水控制相比，间隔功能相对平坦。此外，EcoHIV注射的老鼠在树突状脊柱形态上表现出深刻的变化，短树突状脊柱相对频率增加，头部和颈部直径相对于对照动物增加就证明了这一点。立体氧化注射前条件介质的浓度和滴定对于确保整个实验的一致性和可复制结果至关重要。EcoHIV 的这种特定区域立体毒性注射在大脑中提供了有效的 HIV 感染，并在大鼠中产生时间处理缺陷。利用大鼠的立体毒性艾滋病毒注射来扩展EcoHIV感染模式，为解决与手头神经艾滋病毒有关的新问题提供了关键机会。

Research

JoVE Journal

Neuroscience

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

基因传递到产后大鼠脑非心室质粒注射和电穿孔

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

科研

神经科学

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

特定脑区的兴奋毒性损伤的成年大鼠立体定向手术治疗

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

可卡因使用障碍大鼠模型中提示反应性测定的一种协议

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

轻度宫内灌注圈狭窄的大鼠模型

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

一种体内评价大鼠缺血性中风模型中血脑屏障的破坏

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

基于改进多平台法的大鼠中心疲劳模型

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

利用体外大鼠脑切片研究变性主要事件的一种替代方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

A Novel In Vitro Model of Blast Traumatic Brain Injury

一种新的脑外伤体损伤模型

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

在鼠模型中使用聚焦超声波打开位置特定血脑屏障开口的台面方法

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

鼠脑组织疏水组织清除方法

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...