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>

<ol>
	<li>Dreher, A. 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=Spectrum+of+Disease+and+Outcome+in+Children+with+Symptomatic+Congenital+Cytomegalovirus+Infection.">Spectrum of Disease and Outcome in Children with Symptomatic Congenital Cytomegalovirus Infection.</a> J of Pediatr. 164 (4), 855-859 (2014).</li><li>Lanzieri, T. 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=Long-term+outcomes+of+children+with+symptomatic+congenital+cytomegalovirus+disease.">Long-term outcomes of children with symptomatic congenital cytomegalovirus disease.</a> J of Perinatol. 37 (7), 875-880 (2017).</li><li>Naseer, M. I., 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=A+novel+WDR62+mutation+causes+primary+microcephaly+in+a+large+consanguineous+Saudi+family.">A novel WDR62 mutation causes primary microcephaly in a large consanguineous Saudi family.</a> Ann Saudi Med. 37 (2), 148-153 (2017).</li><li>Abuelo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcephaly+Syndromes.">Microcephaly Syndromes.</a> Semin Pediatr Neurol. 14 (3), 118-127 (2007).</li><li>Nicholas, A. K., 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=WDR62+is+associated+with+the+spindle+pole+and+is+mutated+in+human+microcephaly.">WDR62 is associated with the spindle pole and is mutated in human microcephaly.</a> Nat Genet. 42 (11), 1010-1014 (2010).</li><li>Pulvers, J. N., 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=Mutations+in+mouse+Aspm+(abnormal+spindle-like+microcephaly+associated)+cause+not+only+microcephaly+but+also+major+defects+in+the+germline.">Mutations in mouse Aspm (abnormal spindle-like microcephaly associated) cause not only microcephaly but also major defects in the germline.</a> Proc Natl Acad Sci U S A. 107 (38), 16595-16600 (2010).</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=Zika+virus+infection+disrupts+neurovascular+development+and+results+in+postnatal+microcephaly+with+brain+damage.">Zika virus infection disrupts neurovascular development and results in postnatal microcephaly with brain damage.</a> Development. 143 (22), 4127-4136 (2016).</li><li>Li, C., 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+Disrupts+Neural+Progenitor+Development+and+Leads+to+Microcephaly+in+Mice.">Zika Virus Disrupts Neural Progenitor Development and Leads to Microcephaly in Mice.</a> Cell Stem Cell. 19 (5), 672 (2016).</li><li>Miki, T., Fukui, Y., Takeuchi, Y., Itoh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+study+of+the+effects+of+prenatal+X-irradiation+on+the+development+of+cerebral+cortex+in+rats.">A quantitative study of the effects of prenatal X-irradiation on the development of cerebral cortex in rats.</a> Neurosci Res. 23, 241-247 (1995).</li><li>Cragan, J. D., 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=Population-based+microcephaly+surveillance+in+the+United+States,+2009+to+2013:+An+analysis+of+potential+sources+of+variation.">Population-based microcephaly surveillance in the United States, 2009 to 2013: An analysis of potential sources of variation.</a> Birth Defects Res Part A Clin Mol Teratol. 106 (11), 972-982 (2016).</li><li>Cragan, J. D., 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=Baseline+Prevalence+of+Birth+Defects+Associated+with+Congenital+Zika+Virus.">Baseline Prevalence of Birth Defects Associated with Congenital Zika Virus.</a> MMWR Morb Mortal Wkly Rep. 66 (8), 219-220 (2017).</li><li>Mlakar, J., 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+Associated+with+Microcephaly.">Zika Virus Associated with Microcephaly.</a> N Engl J Med. 374 (10), 951-958 (2016).</li><li>Jaenisch, T., Rosenberger, D., Brito, C., Brady, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+of+microcephaly+after+Zika+virus+infection+in+Brazil,+2015+to+2016.">Risk of microcephaly after Zika virus infection in Brazil, 2015 to 2016.</a> Bull World Health Organ. 95 (3), 191-198 (2017).</li><li>Clancy, B., Darlington, R. B., Finlay, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+developmental+time+across+mammalian+species.">Translating developmental time across mammalian species.</a> Neuroscience. 105 (1), 7-17 (2001).</li><li>Driggers, R. W., 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+Infection+with+Prolonged+Maternal+Viremia+and+Fetal+Brain+Abnormalities.">Zika Virus Infection with Prolonged Maternal Viremia and Fetal Brain Abnormalities.</a> N Engl J Med. 374 (22), 2142-2151 (2016).</li><li>Semple, B. D., Blomgren, K., Gimlin, K., Ferriero, D. M., Noble-Haeusslein, L. 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 &amp;micro;L, Model 1701 LT SYR</td><td>Hamilton</td><td>80001</td><td>for embryonic inoculation</td></tr><tr><td>10 &amp;micro;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>&lt;strong&gt;Antibodies&lt;/strong&gt;</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 (&lt;strong class=&quot;xfig&quot;&gt;Figure 2&lt;/strong&gt;, &lt;strong class=&quot;xfig&quot;&gt;Figure 3&lt;/strong&gt;)</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

Universidad de Cartagena UDEC

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

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

人胎脑神经干细胞 Zika 病毒感染的免疫细胞化学分析

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

科研

发育生物学

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 ...

神经科学

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

免疫与感染

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

利用干细胞衍生脑 Organoids体外培养人脑组织 Zika 病毒感染的模拟研究

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.

登革热病毒诱发急性病毒性脑炎样疾病的鼠害模型

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

从小鼠多个器官分离和定量寨卡病毒

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

脑室微注射脂多糖到斑马鱼幼虫中以评估神经炎症和神经毒性

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...

Developing an In Vitro Model of Zika Virus Infection in Cerebral Organoids

Source: Salick, M. R., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/56404">Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids.</a> J. Vis. Exp. (2017).
This video demonstrates the establishment of an in vitro model of Zika virus infection in a developing brain. Human cerebral organoids containing rosettes of stem cells, neural progenitor cells (NPCs), and neurons are infected with the Zika virus. The virus infects NPCs and triggers apoptosis, disrupting organoid development.

在脑类器官中开发寨卡病毒感染的体外模型

1. Creating Cerebral Organoids from iPSC / hESC 2D Culture (Day 0)
Note: This protocol assumes that stem cell (SC) maintenance is conducted in xeno-free ...

JoVE Encyclopedia of Experiments

Neuropathology

Infectious Diseases

Generation of a Murine Model with a Central Nervous System Infection

Source: Shen, T., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59132">A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease.</a> J. Vis. Exp. (2019)
This video demonstrates the generation of a mouse model with a CNS infection. A suckling pup is injected with the virus both intracranially and intraperitoneally. The virus spreads, infecting the CNS causing neuronal cell death and inflammation. The pup is assessed for symptoms of infection as the disease progresses.

生成具有中枢神经系统感染的小鼠模型

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Establishing a Model of Zika Virus-Induced Neurodegeneration in an Adult Mouse

Source: Herrlinger, S. A. et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/56486">Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult.</a> J. Vis. Exp. (2018)
The video demonstrates intracerebral injection of Zika virus suspension in an adult mouse to induce neurodegeneration. An anesthetized mouse is secured in a stereotaxic frame, and a hole is drilled into the exposed skull for injection. A suspension of the Zika virus is injected into the brain tissue. The viral infection causes inflammation, leading to neurodegeneration.

建立寨卡病毒诱导的成年小鼠神经退行性变模型

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Adult ...