Özet

生态HIV脑感染的老鼠模型

Published: January 21, 2021
doi:

Özet

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)。

Abstract

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。

Introduction

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。

Protocol

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 培养293 FT细胞(5×105/mL)在明胶涂层75厘米2 烧…

Representative Results

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?…

Discussion

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"…

Açıklamalar

The authors have nothing to disclose.

Acknowledgements

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

Materials

293FT cells ThermoFisher Scientific R70007
Antibiotic-Antimycotic solution Cellgro 30004CI 100X
Corning BioCoatGelatin 75cm² Rectangular Canted Neck Cell Culture Flask with Vented Cap Life Technologies 354488
Corning DMEM with L-Glutamine, 4.5 g/L Glucose and Sodium Pyruvate Life Technologies 10013CV
Cover glass VWR 637-137
drill
Dumont #5 Forceps World Precision Instruments 14095
Dumont #7 Forceps World Precision Instruments 14097
Eppendorf Snap-Cap Microcentrifuge Biopur Safe-Lock Tubes Life Technologies 22600028
Ethicon Vicryl Plus Antibacterial, 4-0 Polyglactin 910 Suture, 27in. FS-2 Med Vet International VCP422H
Hamilton syringe Hamilton 1701
Invitrogen Lipofectamine 3000 Transfection Reagent Life Technologies L3000015
Iris Forceps World Precision Instruments 15914
Iris Scissors World Precision Instruments 500216
Lentivirus-Associated p24 ELISA Kit Cell Biolabs, inc. VPK-107-5
Lenti-X Concentrator Takara PT4421-2
Opti-MEM I Reduced Serum Medium Life Technologies 11058021
Paraformaldehyde Sigma-Aldrich 158127-500G
Paraformaldehyde Sigma P6148
ProLong Gold Fisher Scientific P36930
Sevoflurane Merritt Veterinary Supply 347075
stereotaxic apparatus Kopf Instruments Model 900
SuperFrost Plus Slides Fisher Scientific 12-550-154%
Vannas Scissors World Precision Instruments 500086

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Bu Makaleden Alıntı Yapın
Li, H., McLaurin, K. A., Mactutus, C. F., Booze, R. M. A Rat Model of EcoHIV Brain Infection. J. Vis. Exp. (167), e62137, doi:10.3791/62137 (2021).

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