Name: establishing mouse models for zika virus-induced neurological disorders using intracerebral injection strategies: embryonic, neonatal, and adult
Uploaded: 2018-04-26T13:00:00
Description: Watch this Scientific Journal Video about Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult at JoVE.com

作者想感谢罗切斯特大学的 Abdellatif Benraiss 博士, 他的导师和讨论与学习成人外科和新生儿技术有关。作者还要感谢 UGA 博士在他的立体定向设备的使用和与建立这种技术的方法有关的讨论, 以及研究学院科学家 (弧形) 基金会的进步为他们的支持和我们的 NIH 支持 (NINDS 赠款 R01NS096176-02, R01NS097231-01, & F99NS105187-01)。

所有动物使用协议遵循南加州大学和佐治亚大学的动物保育指南。妊娠大坝和成人的安乐死方法按照批准的协议进行: 二氧化碳窒息, 其次是颈椎脱位, 以确保安乐死。新生幼崽通过斩首被安乐死。
警告: 以下协议涉及处理致病病毒。在处理病毒时应采取适当的预防措施。所有议定书必须在使用前由适当的机构委员会批准。
1. Zika 病毒的胚胎脑内接种
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<ol>
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这里描述的是一种在胚胎、新生儿和成人阶段 ZIKV 脑内接种的方法, 用于研究 ZIKV 诱发的脑发育损伤。虽然直截了当, 调查人员应该采取一些考虑, 以确保研究的质量和有关人员的安全。
DENV 与黄属 ZIKV 密切相关。DENV 并没有与人类的儿童脑功能紊乱有因果关系。DENV2 可以通过胚胎接种方法 (1 µL 3.4 x 105 TCID50/mL) 成功地感染和复制发育中的大脑 (图 2D</stron...

小头是由于脑发育缺陷而导致的一种情况, 其特点是新生儿的头部大小小于平均水平。小头儿童表现出一系列的症状, 其中包括发育迟缓、癫痫、智力残疾、听力丧失、视力问题以及运动和平衡等问题, 这取决于疾病的严重性和原因1,2,3。这种情况是多因素的性质, 与遗传, 传染性的代理和环境因素, 导致小头4,5,6,7,8, 9。在 2015-2016 ZIKV 爆发之前, 根据 CDC10, 在美国诊断出1万出生的8儿童患有小头。在 2月1日st 2016年世界卫生组织宣布了 Zika 病毒一个国际关注的公共卫生紧急状态由于母亲的 ZIKV 传染的小头诊断的惊人的增量11, 12。疾病控制中心最近对美国 ZIKV 病例的一项研究表明, 与未感染的个人相比, 产妇 ZIKV 感染导致儿童发展小头的风险增加了20倍, 而来自美国的 4% ZIKV 受感染的母亲导致具有小头11的子级。据报道, 巴西 ZIKV 感染妊娠期间小头相关出生缺陷的发生率已影响到受感染母亲中多达17% 的婴儿, 这表明拉丁美洲的其他因素可能导致增加的风险13. 虽然我们知道 ZIKV 可能导致小头和发病机制在神经祖细胞 (NPC) 人口7,8,14, ZIKV 的完全发病机制在开发的脑子里仍然难以捉摸。重要的是发展动物模型, 以进一步调查的疾病机制的基础上的大脑异常与 ZIKV 感染有关。
为了直接研究 ZIKV 对脑发育的影响, 我们首先开发了用 ZIKV7的胚胎 14.5 (E14.5) 脑接种的小鼠模型。这个阶段被选择了, 因为它被认为是在人类妊娠的第一个月的结尾的代表14。幼崽可以存活到产后5天 (P5) 与这种胚胎脑注射方法 (~ 1 µL 1.7 x 106组织培养感染剂量 (TCID50/mL))。这些产后幼崽在受感染的人类婴儿中表现出相似观察到的一系列表型, 包括心室增大、神经元丢失、轴突稀疏、astrogliosis 和胶质激活12、15。新生老鼠的大脑相对不成熟, 类似于怀孕中期的人脑发育阶段16, 老鼠大脑发育包括一个主要的产后部分。为了研究晚期妊娠期感染, 还介绍了一种产后感染的方法。新生儿感染 ZIKV 在 P1 可以存活13天后注射。在老鼠之前的17中已经描述了血液产生的成人阶段感染, 但需要使用干扰素 (干扰素) 调节因子 (IRF-3) 转录因子,-5,-7 三次击倒菌株。该协议描述了一种接种 ZIKV intraventricularly 的方法, 以规避在成人中禁用小鼠模型的抗病毒反应。虽然这绕过了小鼠的免疫系统, 这种注射途径并不直接模仿典型的感染途径。为了直接解决这种差异, 实验者可以在宫内感染 ZIKV 而不是颅内通路。通过以前的工作18, 我们在这个胚胎感染协议中简要描述了这一技术。
使用此技术实现的 Zika 病毒菌株包括墨西哥隔离 MEX1-447、19和 1947年20隔离的非洲隔离 MR-766。Zika MEX1-44 于 1月2016年在墨西哥恰帕斯被感染的白纹蚊蚊子隔离。我们通过在加尔维斯顿 (UTMB) 的德克萨斯大学医学处获得了这种病毒。此外, 登革热病毒2型 (DENV2) 接种了这种技术的比较研究。DENV2, 应变 S16803 (序列基因库 GU289914), 被隔离从一个病人样本从泰国1974年和传代在 C6/36 细胞。病毒是传代两次在维罗细胞被世界文献中心的新兴病毒和虫媒病毒 (WRCEVA) 之前, 老鼠注射。这表明, 这项技术同样适用于各种 ZIKV 和其他 flaviviruses 可能对大脑发育有影响的菌株。

<table>
	<tbody>
		<tr>
			<td>Flexible Drive Shaft Drill Hanging Motor</td>
			<td>Leica</td>
			<td>39416001</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Stereotax</td>
			<td>Kopf</td>
			<td>04557R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro4 Microsyringe Pump Controller</td>
			<td>WPI</td>
			<td>SYS-MICRO4</td>
			<td></td>
		</tr>
		<tr>
			<td>UMP3 UltraMicroPump</td>
			<td>WPI</td>
			<td>UMP3</td>
			<td></td>
		</tr>
		<tr>
			<td>Modulamp</td>
			<td>Schott</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer-lock tubing (19-gauge)</td>
			<td>Hamilton</td>
			<td>90619</td>
			<td></td>
		</tr>
		<tr>
			<td>Melting Point Capillary</td>
			<td>Kimble</td>
			<td>34500-99</td>
			<td>Glass needle</td>
		</tr>
		<tr>
			<td>Fluoro-Max: Red Fluorescent Microspheres</td>
			<td>Thermo Scientific</td>
			<td>R25</td>
			<td>No dilution; Use for practice injections</td>
		</tr>
		<tr>
			<td>10 &#181;L, Model 1701 LT SYR</td>
			<td>Hamilton</td>
			<td>80001</td>
			<td>for embryonic inoculation</td>
		</tr>
		<tr>
			<td>10 &#181;L, Model 1701 RN SYR, Small Removable NDL, 26s ga, 2 in, point style 2</td>
			<td>Hamilton</td>
			<td>80030</td>
			<td>for neonate/adult</td>
		</tr>
		<tr>
			<td>4-0 Ethilon Nylon Sutures</td>
			<td>Ethicon</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>VWR</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Grinder</td>
			<td>Narishige</td>
			<td>EG-44</td>
			<td></td>
		</tr>
		<tr>
			<td>Fastgreen FCF Dye</td>
			<td>Sigma</td>
			<td>F7252</td>
			<td>inject with 0.5% Dye</td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flavivirus group antigen antibody</td>
			<td>Millipore</td>
			<td>MAB10216</td>
			<td>ms IgG2a 1:400 (Figure 2, Figure 3)</td>
		</tr>
		<tr>
			<td>Pax6</td>
			<td>DBHB</td>
			<td>Pax6-s</td>
			<td>ms IgG1 1:20</td>
		</tr>
	</tbody>
</table>

establishing mouse models for zika virus-induced neurological disorders using intracerebral injection strategies: embryonic, neonatal, and adult

Zika 病毒 (ZIKV) 是目前在北部, 中美洲和南美流行的黄。现在建立的 ZIKV 可能导致小头和额外的大脑异常。然而, 在发育中的大脑中 ZIKV 的发病机制仍然不清楚。脑外科方法经常用于神经科学研究, 以解决正常和异常的大脑发育和大脑功能的问题。该协议利用经典的外科技术, 并描述了一种方法, 使一个模型 ZIKV 相关的人类神经系统疾病的小鼠神经系统。虽然直接脑接种没有模型的正常模式的病毒传播, 该方法允许调查人员提出有针对性的问题的后果后, ZIKV 感染的大脑发育。本协议描述 ZIKV 脑室内接种的胚胎、新生儿和成人阶段。一旦掌握, 这种方法就可以成为一种简单而又可重复的技术, 只需几个小时即可完成。

我们对胚胎脑 ZIKV 接种方法的代表性图像显示在描述脑内注射 (图 1A) 和宫内和 intraplacental 注射 (图 1B) 的图示中, 说明方法对妊娠期大坝和胚胎进行手术治疗 (胚胎接种协议)。图 2A展示了 ZIKV (MEX1-44) 感染 (immunostained 与抗体抗黄组抗原, 绿色) 在 E18.5 大脑皮层。Pax6 (红色) 标签的 npc 在发展中的皮层。ZIKV 被?...

Watch this Scientific Journal Video about Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult at JoVE.com

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

应用脑注射策略建立小鼠 Zika 病毒诱发神经系统疾病模型: 胚胎、新生儿和成人

本文介绍了一种在小鼠中建立 Zika 病毒诱导小头模型的方法。该协议包括胚胎、新生儿和成人期 Zika 病毒的脑内接种方法。

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly ...

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Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

扩展胚胎及成体神经干细胞<em&gt;在子宫内</em电穿孔或病毒立体定向注射

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

来自祖，人脑少突胶质细胞培养系统

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

星形细胞转运电流派生谷氨酸间隙的时间进程与反卷积分析

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...