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
<ol><li>对于定时怀孕交配, 请考虑在交配后的一天中午 E0.5。在一天结束时对老鼠进行配对, 并在清晨检查插头, 以减少交配时间的变异性。</li>
	<li>在手术前准备玻璃针。拉针和削减1/3 它的长度, 然后锐化在45°角使用微磨床与水滴, 直到毛孔大小为 50-70 µm. 检查针尖在立体镜的质量和破损。</li>
	<li>把病毒整除在冰上。就在手术前, 将 ZIKV 注射剂装入准备好的玻璃注射针中。组装气密注射注射器 (50 µL 容积), 鲁尔锁附件和油管, 并用矿物油将组装好的注射器与油管浸透。一旦饱和, 连接针, 并绘制〜 6-7 µL ZIKV 入针 (1 µL 是 1.7 x 106 TCID50/mL)。</li>
	<li>注射妊娠 C57BL/6J 或 129S1/SvImJ 小鼠胚胎天 E14.5 胚胎 (颅内注射) 或 E10.5 胚胎 (用于宫内注射) 盐酸氯胺酮 (80-100 毫克/千克) 和甲苯噻嗪 (5-10 毫克/千克) 稀释的无菌盐水溶液腹腔在母亲身上引起麻醉。另外, 麻醉母亲与监测的异氟醚异氟醚提供的吸入, 并在废气清除, 如果适当的。注射丁丙诺啡-SR (0.5-1.0mg/千克), 以诱发镇痛。</li>
	<li>捏试验麻醉母亲的脚趾头或尾部, 然后躺在动物认可的加热垫 (热垫 100 x 200 2.5 毫米) 仰卧。脚趾夹钳是不育的, 不用于处理组织在手术中。或者, 戴手套的手可以用来测试脚趾夹戒的反射。 注: 请参考您的机构兽医人员的首选围手术期加热方法。在这个加热垫和动物之间放置了一个盖子。</li>
	<li>在眼睛上加眼膏。</li>
	<li>剃掉腹部的表面, 用碘和酒精消毒三次。交替碘和酒精湿巾;从手术部位抹去圆形的运动。</li>
	<li>用无菌布将手术部位周围的皮肤披上, 以避免切口和器械的污染。 注: 悬垂开口不应大于准备好的手术部位;在褶皱的开口上不应该看到头发。</li>
	<li>用镊子将母亲的皮肤从腹部掐开, 用不育的手术剪刀在内侧矢状线的 1-1.5 厘米线上切开下腹部。这会把皮肤切开, 进一步进入腹腔, 这样胚胎就会暴露出来了。 注: 手术剪刀用于切割皮肤和腹膜层。每项手术都使用无菌器械和手套。</li>
	<li>在一个小的不育纱布中间切开一条狭缝, 并应用在手术开口的顶端。水合无菌生理盐水。通过切口将胚胎拉到无菌纱布上, 注意不要将超过 4-5 的胚胎一次聚焦在一个子宫角上。</li>
	<li>在接种前和整个注射过程中, 用无菌生理盐水水合胚胎, 以确保它们不干燥。跟踪哪些胚胎被注射, 以及它们在子宫角内的位置。因此, 单个胚胎被视为单独的实验, 因为胚胎在子宫内发育时不会改变位置。(继续步骤1.15 为宫内注射)。</li>
	<li>轻轻地将刮刀放在胚头下。用一盏灯照亮胚胎, 以可视化头部和头骨缝合线。 注: 采用无菌技术, 包括无菌手术手套和器械。在无菌物品 ( 如灯 ) 纵后 , 手套应更换为新的无菌手套之前 , 处理无菌仪器和领域。</li>
	<li>通过操纵胚胎与灯 (在头后面为可见性) 和自由手指来定位头部, 直到胚胎的头部被直接推到子宫壁上, 并与非显性手一起举行。 注意: 定位胚胎是一个关键的部分和技术, 采取了最大的实践。过多的手指压力会破坏导致致命的胚胎膜, 没有足够的压力会使注射变得困难。</li>
	<li>使用沿头骨缝合的血管作为指导。注射 ZIKV 病毒 (〜1µL, 1.7 x 106 TCID50/mL 墨西哥隔离 MEX1-44) 到 E14.5 胚胎大脑的侧脑室与组装的注射器和针。使用控制介质作为假注入。</li>
	<li>为了改善妊娠的保留, 避免病毒注射的两个胚胎旁边的卵巢和两个胚胎旁边的上阴道 (图 1A)。总的来说, 每只垃圾注射的胚胎不超过 6, 以减少手术时间, 防止胚胎丢失。</li>
	<li>将注入的胚胎放回怀孕的水坝, 用0.5 毫升无菌生理盐水填满腹腔。要关闭, 首先缝合腹部腹膜肌, 然后第二缝合外部皮肤层与4.0 无菌缝线。最好使用间断缝线;如果老鼠咀嚼缝线, 就有可能裂开伤口。</li>
	<li>将鼠标放在一个放在加热垫上的笼子里, 如果需要, 可以让鼠标逃到一个不加热的区域。监测的母亲, 而他们恢复 (1-2 小时) 从麻醉。根据个别实验, 在手术后进行不同时间的胚胎发育。</li>
	<li>
宫内注射
	<ol>
<li>与一个子宫喇叭和胚胎暴露, 使用1毫升注射器和27G 针注射 100 ul Zika 病毒 (106 TCID50单位暂停在 100 ul DMEM), 或控制媒介, 进入宫内空间或胎盘组织。注意哪些胚胎已经注射用于胚胎分期解剖。有关更多详细信息, 请参见以前发布的工作18。</li>
		<li>将胚胎注入到怀孕的水坝中, 用0.5 毫升无菌生理盐水填满腹腔。要关闭, 首先缝合腹部腹膜肌, 然后第二缝合外部皮肤层与4.0 无菌缝线。最好使用间断缝线;如果老鼠咀嚼缝线, 就有可能裂开伤口。</li>
		<li>将鼠标放在一个放在加热垫上的笼子里, 如果需要, 可以让鼠标逃到一个不加热的区域。监测的母亲, 而他们恢复 (1-2 小时) 从麻醉。根据个别实验, 在手术后进行不同时间的胚胎发育。</li>
	</ol>
</li>
</ol>2. Zika 病毒新生儿脑内接种: P0/P1
<ol><li>确保气密注射注射器 (10 µL 量) 和针头不堵塞。通过装载三20毫升一次性注射器和26口径针头进行清洁和测试: 一份含盐水, 一份含70% 乙醇, 一个用空气清除溶液。使用10% 漂白剂消毒, 如果以前用于病毒注射。</li>
	<li>把病毒放在冰上。用盐水填充注射器以减少注射器中的死容积, 并绘制0.75 µL 的空气, 将盐水溶液与注射材料分离。</li>
	<li>为注射的幼崽设置一个温暖的湿润恢复室。使用加热块, 放置一个 closeable 容器 (如空尖盒) 与无菌纱布和 humidify 生理盐水。在开始注射前预热。</li>
	<li>设置 microinjector, 包括泵和控制器。确定特定注射器的设备类型代码 (即, 对于10µL 注射器, 设备类型为 "D")。确定注射速度。</li>
	<li>收集的产后一天0或1幼崽 (P0/P1) 附近的手术设置。用病毒装载注射注射器。用70% 乙醇消毒幼头坐骑。</li>
	<li>将幼崽裹在薄薄的纱布里, 将它们完全掩埋在冰层中, 以达到麻醉状态。幼崽的 cryoanesthetized 为5分钟. 确保幼崽有足够的麻醉注射通过执行尾巴/脚趾捏没有反应。这大约产生15分钟的麻醉状态。</li>
	<li>将幼崽放在头上, 用碘和乙醇 (三倍) 消毒头部表面, 然后用无菌擦拭 (异丙醇或70% 乙醇) 擦拭乾燥。</li>
	<li>用黑色标记标记 lambda, 并使用立体定向仪器记录 lambda 上的坐标。测量从 lambda 到眼睛边缘的距离, 从 lambda 的立体定位坐标开始计算2/5 的距离, 从 lambda 到眼睛 (两个半球, 如果你注射两个)。</li>
	<li>在为注射部位创建孔之前重新检查麻醉深度。使用26口径针仔细和表面刺穿头皮和头骨在注射位置 (头骨是非常软的新生儿), 以创建一个开放的注射针。</li>
	<li>将注射针头降低到穿刺部位, 一旦针头刚刚突破皮肤, 用立体定向仪计算注射部位 (侧脑室) 的1毫米深度。</li>
	<li>一旦针被降低到注射深度, 程序 microinjector 注射: 1 µL 病毒, 以 10 nL/秒的速度, 注射大约1µL 1.5 分钟 (~ 1 ul 3.4 x 105 TCID50/mL ZIKV)。</li>
	<li>当注射完成后, 等待三十年代, 并通过旋转螺钉 (背), 在每增加后等待三十年代, 以0.5 毫米的增量收回针。 注意: 这减少了注射病毒的渗漏。</li>
	<li>一旦删除, 要么快速加载针头和注射器与更多的病毒 (不同的病毒或模拟控制应加载到单独的注射器/针头) 或删除幼崽从头部安装到预热的湿室。每隔几分钟监视幼犬以测量恢复。用它的垃圾代替幼崽。</li>
</ol>3. Zika 病毒成人脑内接种
<ol><li>用盐酸氯胺酮 (80-100 毫克/千克) 和甲苯噻嗪 (5-10 毫克/千克) 稀释在无菌生理盐水中注射成年小鼠腹腔麻醉。注射丁丙诺啡-SR (0.5-1.0mg/千克), 以诱发镇痛。脚趾/尾部捏老鼠, 以确保其麻醉状态, 然后将其安装在立体定向仪器 (带加热垫), 以固定头部在手术期间。</li>
	<li>在眼睛上添加眼膏, 防止老鼠的眼睛在手术过程中干燥。把头皮从眼睛后面刮到耳朵的开始。用碘和70% 乙醇三次消毒暴露的皮肤。交替碘和酒精湿巾;从手术部位抹去圆形的运动。</li>
	<li>在切割皮肤前重新检查麻醉深度。使用手术刀, 使一个0.5 厘米的切口沿内侧矢状线头部在消毒的位置, 暴露 bregma。使用带有棉尖涂抹器的滚动运动, 清除脑膜组织颅骨的表面, 同时将皮肤从注射部位推开。</li>
	<li>从 bregma 开始, 将外科钻头对准, 并使用以下坐标识别注射部位: AP-0.5 毫米, ML: +/-1.5 毫米. 使用控制脚踏踏板, 钻入颅骨缓慢, 只钻骨, 直到一个洞已经清除针。</li>
	<li>用 microinjector 泵和气密注射器 (10 µL 容积) 在立体定向上更换钻头。在盐水溶液和病毒之间留下一个小气泡, µL 4-5 的病毒。确定特定注射器容积的设备类型代码 (即, D 为 10 ul 容积注射器)。将针降低到 DV: 从脑表面-1.5 毫米。注射1µL 的病毒, 程序的速度 10 nL/秒, 注射约1µL 超过1.5 分钟。</li>
	<li>当注射完成后, 等待三十年代, 并删除针在0.5 毫米增量, 等待三十年代后每回合。这减少了注射病毒的回流和渗漏。</li>
	<li>注射完毕后, 使用镊子松开皮肤, 恢复外露颅骨。用4.0 缝线缝合头皮, 从立体定向仪器中取出鼠标。将鼠标放在一个固定在加热垫上的笼子里, 让鼠标在需要的地方逃逸到一个不加热的区域, 并在他们恢复 (1-2 小时) 麻醉时进行监测。</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+development+in+rodents+and+humans:+Identifying+benchmarks+of+maturation+and+vulnerability+to+injury+across+species.">Brain development in rodents and humans: Identifying benchmarks of maturation and vulnerability to injury across species.</a> Prog Neurobiol. 106, 1-16 (2013).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+Infects+Neural+Progenitors+in+the+Adult+Mouse+Brain+and+Alters+Proliferation.">Zika Virus Infects Neural Progenitors in the Adult Mouse Brain and Alters Proliferation.</a> Cell Stem Cell. 19 (5), 593-598 (2016).</li><li>Vermillion, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrauterine+Zika+virus+infection+of+pregnant+immunocompetent+mice+models+transplacental+transmission+and+adverse+perinatal+outcomes.">Intrauterine Zika virus infection of pregnant immunocompetent mice models transplacental transmission and adverse perinatal outcomes.</a> Nat. Commun. 8, 14575 (2017).</li><li>Goodfellow, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+Induced+Mortality+and+Microcephaly+in+Chicken+Embryos.">Zika Virus Induced Mortality and Microcephaly in Chicken Embryos.</a> Stem Cells Dev. 25 (22), 1-27 (2016).</li><li>Dick, G. W. A., Kitchen, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+(I).+Isolations+and+serological+specificity.">Zika Virus (I). Isolations and serological specificity.</a> Trans R Soc Trop Med Hyg. 46 (5), 509-520 (1952).</li><li>Shao, Q., Herrlinger, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+African+Zika+virus+MR-766+is+more+virulent+and+causes+more+severe+brain+damage+than+current+Asian+lineage+and+Dengue+virus.">The African Zika virus MR-766 is more virulent and causes more severe brain damage than current Asian lineage and Dengue virus.</a> Development. , (2017).</li></ol>

作者没有什么可透露的。

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 ...

establishing-mouse-models-for-zika-virus-induced-neurological

Research

JoVE Journal

Neuroscience

8.5K 次观看.  University of Georgia. 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。

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

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.

Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.

建立颅内脑肿瘤异种移植肿瘤的生长和对治疗的反应与后续利用生物发光成像分析

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

科研

神经科学

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

成年鼠脑铸造三维成像和形态分析

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear. Since there is no cell line that satisfactorily represents mammalian hair cells, research on hair cells relies on primary organ cultures. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice (Figure 1) 1-6. The utricle is a vestibular organ, and the hair cells of the utricle are similar in both structure and function to the hair cells in the auditory organ, the organ of Corti. The adult mouse utricle preparation represents a mature sensory epithelium for studies of the molecular signals that regulate the survival, homeostasis, and death of these cells.
Mammalian cochlear hair cells are terminally differentiated and are not regenerated when they are lost. In non-mammalian vertebrates, auditory or vestibular hair cell death is followed by robust regeneration which restores hearing and balance functions 7, 8. Hair cell regeneration is mediated by glia-like supporting cells, which contact the basolateral surfaces of hair cells in the sensory epithelium 9, 10. Supporting cells are also important mediators of hair cell survival and death 11. We have recently developed a technique for infection of supporting cells in cultured utricles using adenovirus. Using adenovirus type 5 (dE1/E3) to deliver a transgene containing GFP under the control of the CMV promoter, we find that adenovirus specifically and efficiently infects supporting cells. Supporting cell infection efficiency is approximately 25-50%, and hair cells are not infected (Figure 2). Importantly, we find that adenoviral infection of supporting cells does not result in toxicity to hair cells or supporting cells, as cell counts in Ad-GFP infected utricles are equivalent to those in non-infected utricles (Figure 3). Thus adenovirus-mediated gene expression in supporting cells of cultured utricles provides a powerful tool to study the roles of supporting cells as mediators of hair cell survival, death, and regeneration.

Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles.

解剖成年小鼠的椭圆囊和腺病毒介导的支撑细胞的感染

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear.  Since there ...

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

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. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

逆行神经，大港，并用荧光染料的神经根装载

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

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

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

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

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

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

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

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

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

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

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

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

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 ...