The authors thank Mr. Masaaki Kaneta, Ms. Hiromi Suzuki, and Ms. Mitsue Kawashima (Department of Regenerative and Infectious Pathology, Hamamatsu University School of Medicine) for their excellent technical assistance. This work was supported by the Japan Society for the Promotion of Science, KAKENHI Grant Number 23590445.

所有实验方案由医学院的滨松大学动物保护委员会的批准。 1.巨细胞病毒（史密斯株）和重组的制备M32增强型绿色荧光蛋白（EGFP）-MCMV <ol><li>根据该方法产生的重组M32-EGFP-MCMV如下（1.2 - 1.9）和如前所述8。 </li><li>使用从野生型MCMV（登录号:U68299）的史密斯菌株的重组病毒。插入EGFP（4361个碱基对; bp）的37089和41450碱基对之间-在通过同源重组MCMV基因组中（M32 M31轨迹）。 </li><li>通过聚合酶链反应扩增的MCMV M32（41450 - 39286碱基对，左侧翼序列和M31（37089 - 39246碱基对，使用下列引物右侧翼序列的基因位点:M32，5&#39;- CTACTAGCTAGCCTTCCGCGAGTCGCTGTATT-3&#39;（正向引物），5 "-CTACTAGCTAGCCTTCCGCGAGTCGCTGTATT-3&#39;（反向引物）; M31，5&#39;- CTAAATTAACTTAAGTCGTCCTCTTCGTCTCACAA-3&#39;（正向引物），5&#39;-AACTAGTCTAGATCGCTCCTGGTTGGTTTTTA-3&#39;（反向引物）。 </li><li>将M32轨迹成使用NheⅠ和BamHⅠ限制性位点的表达EGFP的载体。插入M31轨迹入M32-EGFP用AflII位和XbaI位点-recombinant质粒。切割M32 - EGFP -用NheⅠ和AflII位来自M32 M31序列- EGFP - M31重组质粒，并在H 2 O溶解</li><li>染裂解构建成MCMV（史密斯株）的细胞核使用电穿孔系统以诱导同源重组感染的NIH3T3细胞后24小时感染（HPI）。 </li><li>第3天感染后，共培养以1/1000在6孔板用于感染细胞的表达EGFP的病灶的比率和屏未感染的小鼠胚胎成纤维细胞（MEF）细胞。 </li><li> HarveST从含有绿色荧光灶孔中的含病毒的上清液和十倍稀释。为了纯化，选择单显示绿色荧光灶井。 </li><li>当所有的焦点从限制稀释感染显示EGFP表达产生，病毒制剂是纯粹的，表示没有野生型病毒仍然存在。如前所述17通过空斑测定法量化病毒。 </li><li>在生物安全2级认证的生物安全柜戴手套和屏蔽处理MCMV。 </li><li>通道MCMV（史密斯应变）和重组MCMV在从12天龄的ICR小鼠胚胎制备的MEF先前所描述的17。 </li><li>离心3000×g的16℃，从受感染的MEF培养上清细胞20分钟。 </li><li>超速离心机在70000×g下将上清液40分钟。悬浮含有病毒体颗粒在1ml Tris缓冲盐水和转移到一个预制的线性小号orbitol梯度（25％ - 70％）。 60分钟18超速离心机再次7万×g的。 </li><li>用注射器收获含有病毒体带。由7万×克额外的一步超速离心40分钟沉淀收获病毒颗粒。 </li><li>重悬在1ml磷酸盐缓冲盐水（PBS），并存储的沉淀于-80℃直至感染实验。 </li><li>如前所述19通过空斑测定法量化病毒滴度。 </li><li>可视化（在489纳米，在508纳米的发射激发）通过荧光显微镜（ 图1A）和通过透射电子显微镜（TEM）8（ 图1B）的颗粒结构的重组MCMV颗粒的EGFP表达。 </li></ol> 2.尼罗河红色荧光微球的制备<ol><li>购买羧基荧光尼罗红微球，并把500微升微珠为根据管径管（0.04- 0.06微米，约3.63×10 12颗粒; 0.1 - 0.3微米，约5.75×10 10颗粒;和1.7 - 2.2微米，约6.85×10 7颗粒）。 </li><li>治疗用500μl0.1M NaOH中的小珠约1天，以除去任何内毒素并在室温重新悬浮在无菌水中的珠。 </li><li>吸附珠粒O / N与来自C57BL / 6小鼠在RT使用之前获得的10％的小鼠血清。 </li><li>为了分离聚集，涡珠和使用前充分超声处理。 </li></ol> 3，巨细胞病毒和荧光微球的ICV注入新生小鼠<ol><li>保持12小时光照/黑暗周期下，在温度控制的设施正常妊娠ICR小鼠。新生儿是在出生的那一天指定为P 0.5。 </li><li>消毒10微升注射器，并与70％的醇的27克针。 </li><li>加载包含MCMV或荧光微注射溶液（5微升）珠成通过小心拉动注射器的柱塞的针头。 </li><li> 4分钟 - 通过将鼠标放在冰上3抑制新生小鼠（P 0.5）。一旦动物是麻醉下，使用脚趾捏反应方法来确定麻醉深度。 </li><li>标记注射部位的位置处约0.7无毒的实验室笔- 1.0毫米外侧到矢状缝和0.7 -从新生儿前囟（ 图2A）1.0毫米尾。 </li><li>插入针头在标记喷射站点2毫米深，在垂直于颅骨表面。作为参考，标记从针与无毒的制造者的尖端2毫米。 </li><li>缓缓注入5μl的MCMV（约5×10 5 PFU）进入侧脑室无需打开头皮（ 图2B）。 </li><li>在另一组老鼠，注入含有荧光微球有5微升的解决方案-通过（0.10.3μm以下，约为5.75×10 8颗粒）同样的方法。 </li><li>停止柱塞运动，以防止回流后20秒 - 慢慢取出针10。 </li><li>要恢复，保持小鼠5 - 10分钟，在一个温暖的容器中，直到运动和一般的响应能力得到恢复。 </li><li>作为一系列时间点（3，12，24，48，和72小时）注射后在第5节收获的大脑。 </li></ol> 4.静脉注射MCMV或荧光微球进入新生小鼠<ol><li> 4分钟 - 通过将鼠标放在冰上3抑制新生小鼠（P 0.5）。 </li><li>用1-ml注射器用35克针P中执行MCMV的静脉注射0.5新生儿。 </li><li>使用透照（静脉取景器），以可视化的颞浅（面）静脉。注射前，确保新生儿使用外科胶带（ 图2C）的透射。 </li><li>虽然戴着放大镜（1.5X），慢慢注入50微升MCMV（约5.45×的; 10 9颗粒）或荧光微珠（0.04 - 0.06微米，约3.63×10 11颗粒; 0.1 - 0.3微米，约5.75×10 9颗粒;和1.7 - 2.2微米，约6.85×10 6个颗粒）到颞浅静脉（ 图2D）。 </li><li>除去针后，使用含有70％乙醇的纱布直至出血停止压力施加到注射部位。 </li><li>给新生儿约5分钟，在一个温暖的容器将其返回到笼子前恢复。 </li><li>作为在一系列的时间点（3，12，24，和72小时）后喷射在部分5中所述收获的大脑。 </li></ol> 5.脑组织样品制备石蜡切片<ol><li>将注入的新生儿在碎冰上一个小塑料盘。 </li><li>一旦动物是麻醉下，使用脚趾捏反应方法来确定anesthes的深度IA。 </li><li>使通过肋骨一个中心或两端横向末端切口打开胸腔。 </li><li>制作与锋利的剪刀中庭削减。注入4％低聚甲醛（PFA）溶液进入心房。在观察到肝脏的自发运动（PFA舞）和减轻色停止灌注。不要抽血。 </li><li>通过首先去除用剪子的头部解剖的新生儿（如前所述20）。 </li><li>使从颈部沿珠中线切开鼻子，露出头骨。 </li><li>一对虹膜剪刀的尖底放入枕骨大孔一侧，沿着头骨的内表面滑动谨慎。 </li><li>使延伸到后颅骨表面的末端边缘的切口，使在对侧的相同切。清除周围的小脑的头骨。 </li><li>小心剥离背面的头骨的一侧，以防止脑损伤。重复此过程对大脑的另一侧。 </li><li>使用刮刀，断绝嗅球和神经连接沿脑的腹侧表面。 </li><li>轻轻大脑从头部分离，修整仍然大脑连接到使用剪刀的头骨和消除大脑硬膜所有。 </li><li>放置脑在含4％PFA的至少10倍，在4℃下的脑部24小时的量的固定剂的小瓶。 </li><li>通过一系列梯度乙醇浴脱水组织以置换水，然后用石蜡渗透，形成一个块。用切片机到4微米厚的片21切蜡块。 </li><li> Deparaffinize和再水合感染脑22的滑动。继续原位杂交石蜡切片。 </li></ol> 6.脑组织样品制备冰冻切片<ol><li>消除大脑以下5.1中列出的步骤 - 5.11。 </li><li>观察荧光微球和M32-EGFP-MCMV，将切除大脑的平底cryomolds。添加包埋介质完全覆盖大脑样本。捕捉冻结预冷正己烷 （-80℃）的cryomolds，把它连接到夹头使用镊子举行cryomolds的边缘前。 </li><li>对于切片，附着在低温恒温预冷盘冷冻的组织块。传送与卡盘的冷冻组织成一个低温恒温器腔和降低温度至-10和-20℃之间，并切割片约8 - 10微米厚的23。 </li><li>通过喷雾固定用cytofixative（异丙醇和聚乙二醇的混合物）的部分。空气干燥在RT 30分钟的部分喷雾后立即，并将它们存储在 - 80ºC直至进一步使用。 </li><li>平衡的部分回至室温，并用PBS洗三次。 </li><li>染色用荧光素异硫氰酸酯（FITC）缀合的G中的各节riffonia simplicifolia凝集素B4以1:1的浓度:100在室温在PBS中的10分钟（ 图5）或用PE缀合的CD31抗体以1:1的浓度:100在室温在PBS 30分钟（ 图6）。 </li><li>用PBS洗三次的章节。 </li><li>洗涤后，染色切片用4&#39;，6-二脒基-2-苯基吲哚（DAPI）来可视化细胞核。安装在防褪色剂和图像DAPI的部分（激发波长345纳米，455纳米发射），EGFP（在489 nm激发，在508 nm发射）和尼罗红（激发波长553纳米，637纳米发射）用荧光显微镜（ 图3，图5，图6）。 </li></ol> 7. 荧光原位杂交（FISH）用于石蜡切片<ol><li>使用包含整个MCMV DNA根细菌人工染色体准备缺口翻译用荧光标记，直接用于DNA 原位杂交FISH探针OME（pSM3fr）19。的FISH探针的浓度为0.1微克/微升。 </li><li> Deparaffinize和再水合感染脑22的滑动。 </li><li>治疗用RNase（PBS中100μg/ ml的）的组织切片以检测病毒DNA。 </li><li> 98℃20分钟 - 用0.05％NP40，在95的0.01M柠檬酸盐缓冲液（pH 6.0）进行抗原修复。冷却该滑动至室温20分钟。 </li><li>在玻璃科普林染色缸，洗涤载玻片在纯水中三次，每次2分钟。执行与0.06％的胃蛋白酶，在37℃下5分钟0.01当量的HCl溶液中的附加抗原修复步骤。 </li><li>漂洗在玻璃科普林染色缸每次滑动在纯水三次2分钟。 </li><li>通过将载玻片从70％的乙醇转印再次脱水组织切片，以85％的乙醇，然后至100％的乙醇。 </li><li>为了制备杂交缓冲液的10毫升，混合1.25毫升原位杂交的盐（3M氯化钠，100毫摩尔Tris-HCl pH 8.0的，100mM磷酸钠pH 6.8的50毫摩尔EDTA）用5毫升去离子甲酰胺，2.5毫升50％硫酸葡聚糖，250μl的50×Denhardt氏溶液，125微升100毫克/毫升的tRNA，和875微升H 2 O </li><li>稀释直接标记与随机识别整个MCMV基因组中的杂交缓冲液中的荧光团的DNA探针。的最终体积为10微升（7微升杂交缓冲液，1微升探针（0.1微克/微升），和2μl蒸馏水）。 </li><li>探针混合物（10微升）添加至每个载玻片，并用盖玻片（15×15mm）的覆盖。密封橡胶水泥盖玻片。变性5分钟的探针混合物在85°C，并完成杂交步骤O / N在42℃。 </li><li>洗用0.3％NP40，0.4倍的SSC载玻片在73℃，2分钟;用0.1％NP40，在73℃1分钟0.4倍的SSC;并用2×SSC两次。 </li><li>染液用DAPI（10毫微克/毫升）和盖上盖玻片幻灯片中的细胞核。 </li><li>安装在防褪色试剂和图像的DAPI（激发在345纳米，在455纳米发射）和EGFP（激发在489纳米，在508纳米发射）的部分用荧光显微镜（ 图4）。 </li></ol>

<ol>
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S., Kosugi, I., Tsutsui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclosporine+inhibits+mouse+cytomegalovirus+infection+via+a+cyclophilin-dependent+pathway+specifically+in+neural+stem/progenitor+cells.">Cyclosporine inhibits mouse cytomegalovirus infection via a cyclophilin-dependent pathway specifically in neural stem/progenitor cells.</a> J Virol. 81 (17), 9013-9023 (2007).</li><li>Britt, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cytomegalovirus:+propagation,+quantification,+and+storage.">Human cytomegalovirus: propagation, quantification, and storage.</a> Curr Protoc Microbiol. Chapter 14, Unit 14E 13 (2010).</li><li>Kawasaki, H., Kosugi, I., Arai, Y., Iwashita, T., Tsutsui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+embryonic+stem+cells+inhibit+murine+cytomegalovirus+infection+through+a+multi-step+process.">Mouse embryonic stem cells inhibit murine cytomegalovirus infection through a multi-step process.</a> PLoS One. 6 (3), e17492 (2011).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J Vis Exp. (65), (2012).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+sections+of+paraffin-embedded+tissues.">Cutting sections of paraffin-embedded tissues.</a> CSH Protoc. , (2008).</li><li>Chi, V., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry:+paraffin+sections+using+the+Vectastain+ABC+kit+from+vector+labs.">Immunohistochemistry: paraffin sections using the Vectastain ABC kit from vector labs.</a> J Vis Exp. (8), e308 (2007).</li><li>Wilsbacher, L. D., Coughlin, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cardiomyocyte+development+using+immunofluorescence+in+embryonic+mouse+heart.">Analysis of cardiomyocyte development using immunofluorescence in embryonic mouse heart.</a> J Vis Exp. (97), (2015).</li><li>Ohshima, 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=Intraperitoneal+and+intravenous+deliveries+are+not+comparable+in+terms+of+drug+efficacy+and+cell+distribution+in+neonatal+mice+with+hypoxia-ischemia.">Intraperitoneal and intravenous deliveries are not comparable in terms of drug efficacy and cell distribution in neonatal mice with hypoxia-ischemia.</a> Brain Dev. 37 (4), 376-386 (2015).</li><li>Kim, J. Y., Grunke, S. D., Levites, Y., Golde, T. E., Jankowsky, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebroventricular+viral+injection+of+the+neonatal+mouse+brain+for+persistent+and+widespread+neuronal+transduction.">Intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction.</a> J Vis Exp. (91), e51863 (2014).</li><li>Glascock, J. 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=Delivery+of+therapeutic+agents+through+intracerebroventricular+(ICV)+and+intravenous+(IV)+injection+in+mice.">Delivery of therapeutic agents through intracerebroventricular (ICV) and intravenous (IV) injection in mice.</a> J Vis Exp. (56), (2011).</li></ol>

The authors have nothing to disclose.

在动物模型中，ICV，腹膜内，直接胎盘和IV感染已被用于研究病毒性脑炎的发病机制。我们专注于新生小鼠的IC​​V和静脉注射模型的程序简单和直接喷射粒子的利益为目标区域。虽然腹腔内感染是一个简单的方法，病毒颗粒通过间接方法5,24全身蔓延。胎盘直接感染是一个很好的方法来研究胚胎全身感染。然而，该方法需要特别的训练，以产生稳定的结果，并具有低的成功率。感染的方?...

当研究病毒性脑炎，病毒颗粒的初始分布是非常重要的，以了解疾病的发病机制和大脑识别病毒靶。大多数病毒范围从20至300纳米的尺寸，虽然潘多拉病毒是大小为1超过700nm。在感染的急性期的病毒颗粒的分布可取决于颗粒的尺寸，细胞受体的分布，或所述细胞受体的病毒的亲和性。在动物模型中，脑室内（ICV），腹膜内，直接胎盘和静脉内（IV）的感染已被用于研究病毒性脑炎的发病机制。 ICV接种病毒经常被用来建立在小鼠的中枢神经系统（CNS）的感染。使用这种技术的研究报告广泛感染，尤其是在脑室周围区域的细胞，并在与所述脑脊液（CSF），simila直接接触的大脑区域R以病毒ventriculoencephalitis的效果。腺相关病毒（AAV）的颗粒尺寸小（20 -直径为25纳米）有利于在整个ICV感染2-4大脑及其传播。 5腹腔内，胎盘直接6和静脉注射7代表造血全身用药。通过血-脑屏障（BBB）的病毒颗粒的渗透允许它们到达新生儿脑的实质，表示漫小胶质结节8,9。 巨细胞病毒（CMV）是属于疱疹病毒科的共同病毒。在美国，50％ - 在80％的人有过巨细胞病毒感染的40岁CMV感染是很少有害的，但可能会导致免疫功能低下患者和胎儿疾病。所有交付的，为0.2％ - 2％是天生的巨细胞病毒10，产生严重的症状，如小头畸形，脑室周围钙化，小脑发育不全，MICR眼炎，视神经萎缩11,12。此外，智力低下，耳聋，视觉缺陷，癫痫发作和癫痫发生非致命感染巨细胞病毒的婴儿13,14 10％左右。的CNS功能障碍是CMV先天性异常的最常见的典型症状。更多的孩子被永久每年由先天性巨细胞病毒不是唐氏综合症，胎儿酒精综合征或脊柱裂15禁用。有对抗CMV没有接种疫苗可在目前，要求需要一种安全有效的疫苗。研究在感染的最早阶段与它们的受体的CMV颗粒的相互作用是重要的是了解疫苗接种的效果。 Ventriculoencephalitis和小胶质细胞弥漫性结节是CMV的两个主要病理特征脑炎16。 （ - 300纳米150）在感染的急性期传播通过大脑的CMV粒子如何它一直不明朗第二如何细胞受体的分布以及它们的病毒的亲和性向病毒传播。川崎等人已经评估ICV和从颗粒以及它们在病毒感染的早期阶段受体（β1整合）的分布的透视IV感染。我们已发现，巨细胞病毒颗粒和β1整合素的表达的传播感染的两个侧脑室和IV感染8的最早阶段是公相关。 ICV感染是ventriculoencephalitis模型和IV感染是弥漫性结节小胶质细胞的典范。研究病毒或荧光颗粒的动力学将使颗粒尺寸，与细胞受体的病毒的相互作用，和BBB渗透的大脑中的机构的作用的有用信息。以下方案可用于调查在CNS任何病毒感染和病毒载体。

<table><tbody><tr><td>Tris; tris(hydroxymethyl)- aminomethane</td><td>Sigma-Aldrich</td><td>T-6791</td><td></td></tr><tr><td>HCl</td><td>Sigma-Aldrich</td><td>H-1758</td><td></td></tr><tr><td>pEGFP-N1 vector&amp;nbsp;</td><td>Clontech</td><td>#6085-1</td><td></td></tr><tr><td>D-sorbitol</td><td>Sigma-Aldrich</td><td>S-1876</td><td></td></tr><tr><td>SPHERO TM Fluorescent Polystyrene Nile Red 0.04 - 0.06</td><td>Spherotech, Inc.</td><td>FP-00556-2</td><td></td></tr><tr><td>SPHERO TM Fluorescent Polystyrene Nile Red 0.1 - 0.3</td><td>Spherotech, Inc.</td><td>FP-0256-2</td><td></td></tr><tr><td>SPHERO TM Fluorescent Polystyrene Nile Red 1.7 - 2.2</td><td>Spherotech, inc.&amp;nbsp;</td><td>FP-2056-2</td><td></td></tr><tr><td>10% mouse serum</td><td>DAKO&amp;nbsp;</td><td>X0910</td><td></td></tr><tr><td>C57BL/6 mouse</td><td>SLC, Inc.</td><td></td><td></td></tr><tr><td>ICR mouse</td><td>SLC, Inc.</td><td></td><td></td></tr><tr><td>Modified Microliter Syringes (7000 Series)</td><td>Hamilton company</td><td></td><td></td></tr><tr><td>35-gauge needle</td><td>Saito Medical</td><td></td><td></td></tr><tr><td>A Wee Sight Transilluminator</td><td>Phillips Healthcare</td><td>1017920</td><td></td></tr><tr><td>O.C.T.Compound</td><td>Sakura Finetek</td><td>4583</td><td></td></tr><tr><td>RNase A</td><td>Sigma-Aldrich</td><td>R4642</td><td></td></tr><tr><td>Nonidet(R) P-40</td><td>Nacalai</td><td>25223-04</td><td></td></tr><tr><td>citrate buffer (pH 6) x 10</td><td>Sigma-Aldrich</td><td>C9999-100ml</td><td></td></tr><tr><td>pepsin</td><td>Sigma-Aldrich</td><td>P6887</td><td></td></tr><tr><td>EDTA</td><td>dojindo</td><td>N001</td><td></td></tr><tr><td>Formamide</td><td>TCI</td><td>F0045</td><td></td></tr><tr><td>Dextran sulfate sodium salt</td><td>Sigma-Aldrich</td><td>42867-5G</td><td></td></tr><tr><td>Denhardt&#039;s Solution (50x)</td><td>ThermoFishcer sceintific</td><td>750018</td><td></td></tr><tr><td>Yeast tRNA (10 mg/ml)</td><td>ThermoFishcer sceintific</td><td>AM7119</td><td></td></tr><tr><td>SSC 20x</td><td>Sigma-Aldrich</td><td>S6639</td><td></td></tr><tr><td>DAPI</td><td>ThermoFishcer sceintific</td><td>D1306</td><td></td></tr><tr><td>n-Hexane</td><td>Sigma-Aldrich</td><td>296090</td><td></td></tr><tr><td>superfrost plus glass</td><td>ThermoFishcer sceintific</td><td>12-55-18</td><td></td></tr><tr><td>Cytokeep II</td><td>Nippon Shoji Co.</td><td></td><td></td></tr><tr><td>FITC-conjugated Griffonia simplicifolia isolectin B4</td><td>Vector laboratories, Inc.</td><td>L1104</td><td></td></tr><tr><td>Anti-Mouse CD31 (PECAM-1) PE</td><td>ebioscience</td><td>12-0311</td><td></td></tr><tr><td>ProLong&amp;nbsp; Gold</td><td>ThermoFishcer sceintific</td><td>P36934</td><td></td></tr><tr><td>BIOREVO</td><td>KEYENCE</td><td>BZ-9000E</td><td></td></tr></tbody></table>

intracerebroventricular and intravascular injection of viral particles and fluorescent microbeads into the neonatal brain

在病毒性脑炎的发病机制的研究中，感染的方法是至关重要的。第一的两个主要感染途径到大脑的是血源性途径，其中涉及内皮细胞和脑的周细胞的感染。第二个是脑室内（ICV）的路线。一旦中枢神经系统（CNS）内，病毒可能会传播到经由脑脊液蛛网膜下腔，脑膜，和脉络丛。在实验模型中，中枢神经系统的病毒式分发的最初阶段不充分表征，并且目前还不清楚只有某些细胞是否最初感染。这里，我们已经分析了在感染的急性期巨细胞病毒（CMV）颗粒的分布，被称为主血症，ICV或血管内（IV）注射以下到新生小鼠的大脑。在ICV注射模型，5微升小鼠CMV（MCMV）或荧光微珠在midpoi分别注入侧脑室NT使用一个27克针10微升注射器耳朵和眼睛之间。在IV注射模型中，使用1-ml注射器用35克针。一个透射被用于可视化颞浅（面）新生小鼠的静脉中。我们注入50微升MCMV或荧光微球进入颞浅静脉。脑收集在不同时间点后喷射。 原位杂交方法，使用该检测MCMV基因组。通过荧光显微镜观察荧光微球或绿色荧光蛋白表达重组MCMV颗粒。这些技术可以应用到许多其它病原体调查脑炎的发病机制。

在病毒性脑炎的发病机理的研究，感染的方法是很重要的。血行路线表示内皮细胞和脑的周细胞的急性感染，而ICV路线表示急性感染通过蛛网膜下腔经由脑脊液蔓延，延伸到脑膜和脉络丛。为了分析在急性脑炎颗粒的第一分配，原位杂交检测MCMV基因组和M32-EGFP-MCMV颗粒或荧光微珠直接观察被使用。 MCMV重组生成（M3...

Watch this Scientific Journal Video about Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain at JoVE.com

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

病毒颗粒和荧光微球侧脑室和血管内注入新生儿脑

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

intracerebroventricular-intravascular-injection-viral-particles

Research

JoVE Journal

Neuroscience

19.3K 次观看.  Hamamatsu University School of Medicine. Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization. 

视频: Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

颅内注射腺相关病毒载体

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

科研

神经科学

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

交付的治疗药物通过侧脑室（ICV）和静脉（IV）注射液在小鼠

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

医学

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

工程神经前体宫内移植,用于Vivo神经结构研究

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

遗传学

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

使用<em&gt;在体内</em&gt;影像监测进展的实验小鼠巨细胞病毒感染的新生儿

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

免疫与感染

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 ...

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 ...

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

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 and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

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

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 ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

在新生小鼠静脉注射

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

生物学

在新生小鼠中进行鞘内注射的综合方案

Intrathecal Injection of Newborn Mouse for Genome Editing and Drug Delivery

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and treatments. By directly delivering medications and treatments into the cerebrospinal fluid of the central nervous system, this method achieves higher localized drug concentrations while reducing systemic side-effects compared to other routes such as intravenous, subcutaneous, or intramuscular injections. Its importance extends beyond clinical settings, as intrathecal injection plays a vital role in preclinical studies focused on treating neurogenetic disorders in rodents and other large animals, including non-human primates. However, despite its widespread application, intrathecal injection in young, particularly neonatal pups, poses significant technical challenges due to their small size and fragile nature. Successful and reliable administration of intrathecal injections in newborn mice requires meticulous attention to detail and careful consideration of various factors. Thus, there is a crucial need for a standardized protocol that not only provides instructions but also highlights key technical considerations and good laboratory practices to ensure procedural consistency, as well as the safety and welfare of the animals.
To address this unmet need, we present a detailed and comprehensive protocol for performing intrathecal injections specifically in newborn pups on postnatal day 1 (P1). By following the step-by-step instructions, researchers can confidently perform intrathecal injections in neonatal pups, enabling the accurate delivery of drugs, antisense oligos, and viruses for gene replacement or genome editing-based treatments. Furthermore, the importance of adhering to good laboratory practices is emphasized to maintain the well-being of animals and ensure reliable experimental outcomes. This protocol aims to address the technical challenges associated with intrathecal injections in neonatal mice, ultimately facilitating advances in the field of neurogenetic research that aims to develop potential therapeutic interventions.

作者聚焦: 鞘内注射 – 一种高效可靠的递送方法，用于测试新生小鼠大脑中基因编辑的功效

The present protocol outlines step-by-step instructions for performing intrathecal injections in neonatal mice for gene editing and drug delivery.

新生小鼠鞘内注射用于基因组编辑和药物递送

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and ...

病毒颗粒和荧光微球侧脑室和血管内注入新生儿脑

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颅内注射腺相关病毒载体

交付的治疗药物通过侧脑室（ICV）和静脉（IV）注射液在小鼠

工程神经前体宫内移植,用于Vivo神经结构研究

使用

扩展胚胎及成体神经干细胞

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

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

在新生小鼠静脉注射

新生小鼠鞘内注射用于基因组编辑和药物递送

新生小鼠鞘内注射用于基因组编辑和药物递送