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. 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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 border="0" cellpadding="0" cellspacing="0" width="690">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Tris; tris(hydroxymethyl)- aminomethane</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:80px;">T-6791</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl</td>
			<td>Sigma-Aldrich</td>
			<td>H-1758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pEGFP-N1 vector&#160;</td>
			<td>Clontech</td>
			<td>#6085-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">D-sorbitol</td>
			<td>Sigma-Aldrich</td>
			<td>S-1876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPHERO TM Fluorescent Polystyrene Nile Red 0.04-0.06</td>
			<td>Spherotech, Inc.</td>
			<td>FP-00556-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPHERO TM Fluorescent Polystyrene Nile Red 0.1-0.3</td>
			<td>Spherotech, Inc.</td>
			<td>FP-0256-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPHERO TM Fluorescent Polystyrene Nile Red 1.7-2.2</td>
			<td>Spherotech, inc.&#160;</td>
			<td>FP-2056-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% mouse serum</td>
			<td>DAKO&#160;</td>
			<td>X0910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6 mouse</td>
			<td>SLC, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ICR mouse</td>
			<td>SLC, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Modified Microliter Syringes (7000 Series)</td>
			<td>Hamilton company</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35-gauge needle</td>
			<td>Saito Medical</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A Wee Sight Transilluminator</td>
			<td>Phillips Healthcare</td>
			<td>1017920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O.C.T.Compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase A</td>
			<td>Sigma-Aldrich</td>
			<td>R4642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nonidet(R) P-40</td>
			<td>Nacalai</td>
			<td>25223-04</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">citrate buffer (pH6) x10</td>
			<td>Sigma-Aldrich</td>
			<td colspan="2">C9999-100ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pepsin</td>
			<td>Sigma-Aldrich</td>
			<td>P6887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>dojindo</td>
			<td>N001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formamide</td>
			<td>TCI</td>
			<td>F0045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran sulfate sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>42867-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Denhardt&#39;s Solution (50X)</td>
			<td>ThermoFishcer sceintific</td>
			<td>750018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast tRNA (10 mg/mL)</td>
			<td>ThermoFishcer sceintific</td>
			<td>AM7119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SSC x20</td>
			<td>Sigma-Aldrich</td>
			<td>S6639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>ThermoFishcer sceintific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-Hexane</td>
			<td>Sigma-Aldrich</td>
			<td>296090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">superfrost plus glass</td>
			<td>ThermoFishcer sceintific</td>
			<td>12-55-18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytokeep II</td>
			<td>Nippon Shoji Co.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC-conjugated Griffonia simplicifolia isolectin B4</td>
			<td>Vector laboratories, Inc.</td>
			<td>L1104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse CD31 (PECAM-1) PE</td>
			<td>ebioscience</td>
			<td>12-0311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong&#160; Gold</td>
			<td>ThermoFishcer sceintific</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BIOREVO</td>
			<td>KEYENCE</td>
			<td style="width:80px;">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

19.2K 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

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

可伸缩流控喷油器阵列病毒完整的3 - D的大脑回路为目标

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

科研

神经科学

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 ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

立体定向注射病毒载体的基因操作在小鼠脊髓条件

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

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 ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.

Demyelinating diseases can be modeled in animals by focal application of lysolecithin into the CNS. A single injection of lysolecithin into mouse spinal cord produces a lesion that spontaneously repairs over time. The goal is to study factors involved in de- and remyelination, and to test agents for enhancing repair.

实验脱髓鞘和小鼠脊髓髓鞘再生由溶血卵磷脂的焦点注射

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

低聚淀粉样蛋白-β的立体灌注到小鼠海马

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

正常小鼠β淀粉样肽侧脑室注射敏锐地诱导老年痴呆样认知缺陷

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In addition to its peripheral function, its involvement in brain functions has also been suggested. Resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, has been known to induce long-term desensitization of TRPV1, and this desensitization has been an alternative approach for investigating the physiological relevance of TRPV1-expressing cells. Here we describe a protocol for intracerebroventricular (i.c.v.) treatment with RTX in mice. Procedures are described for testing nociception to peripheral TRPV1 stimulation (RTX test) and mechanical stimulation (tail pressure test) then follow. Although the nociceptive responses of mice that had been administered RTX i.c.v. were comparable to those of the control groups, RTX-i.c.v.-administered mice were insensitive to the analgesic effect of acetaminophen, suggesting that i.c.v. RTX treatment can induce supraspinal-selective TRPV1 desensitization. This mouse model can be used as a convenient experimental system for studying the role of TRPV1 in brain/supraspinal function. These techniques can also be applied to studies of the central actions of other drugs.

The transient receptor potential vanilloid type 1 (TRPV1) in the supraspinal region has been suggested to play some roles in the brain function. Described here is a protocol for intracerebroventricular injection of resiniferatoxin for supraspinal TRPV1 desensitization in mice. Procedures for some pain tests are also presented.

小鼠用树脂叶毒素和疼痛测试进行脑血管内治疗

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

在Vivo直接重新编程居民胶质细胞到内神经通过病毒载体的脑内注射

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps.&#160;It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.
Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant&#160;brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

将小鼠大脑显微切割成功能和解剖学上不同的区域

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain ...