所有的实验都普渡大学的动物管理和使用委员会及计划在美国普渡大学实验动物的监督下进行。 博士。杰克·朱迪（jack.judy darpa.mil）的主持下，这项工作是由美国国防高级研究计划局（DARPA）微系统技术办公室（MTO），赞助的可靠神经科技计划的一部分，通过空间和海战系统司令部（SPAWAR）系统中心（SSC）太平洋批准号:N66001-11-1-4013。 的作者想，以感谢米哈伊尔·斯利普琴科，唐就绪，格雷格·里希特，亚伦·泰勒，凯文Eliceiri分享他们的显微镜的专业知识。

解决方案 磷酸盐缓冲液（PBS） -以g / l; 9克NaCl，0.144克KH 2 PO 4，的0.795克Na 2 HPO 4，在pH 7.4 4％甲醛 -毫升/升;202毫升钠磷酸氢二钠溶液（0.4M的Na 2 HPO 4），48毫升磷酸二氢钠溶液（0.4M的NaH 2 PO 4），500毫升8％的甲醛溶液，加入250毫升纯水Q DDI水，在pH = 7.4 HEPES缓冲Hank氏盐渍钠叠氮化物（HBHS） -以g / l，7.5克氯化钠，0.3克氯化钾，0.06克KH 2 PO 4，0.13克的Na 2 HPO 4，2克葡萄糖，2.4克的HEPES，0.05克的MgCl 2 6份H 2 O，0.05克MgSO 4 7份H 2 O，0.165，0.09克NaN的3，在pH 7.4克CaCl 2 洗涤液（WS） - 1％体积/体积，也正常山羊血清，0.3％的Triton X-100，用叠氮化钠（NaN 3）中的HBHS。冷藏WS，在4℃，然后在随后的步骤中使用。 U2 SCA L E解决方案 - 4 M尿素，30％甘油和0.1％的Triton X-100的17 程序所有的实验都普渡大学的动物管理和使用委员会及计划在美国普渡大学实验动物的监督下进行。 1。手术<ol><li>各种无菌外科手术的方法是兼容的组织学方法。立体定位框架的使用和自动插入器建议改善重现性和控制的微电极阵列（MEA）的注入角度相对于立体定向平面。 </li></ol> 注:在这个演示中，我们的手术方法如下:持有的啮齿动物的whIL在立体earbars的主题。e在异氟醚麻醉（1-3％之间）进行的医疗级氧气。缺乏一个脚趾捏试验反映在大鼠或缺乏在鼠标的尾部掐反射确认该动物是完全麻醉。涂抹眼膏，有三个聚维酮碘和乙醇交替洗涤和清洁手术部位。洗净双手，黎明发网，外科手术手套，口罩和隔离衣或防护服。注入静脉推注利多卡因麻醉手术区，创建一个切口，用剪刀或手术刀，明确的骨膜与骨铲土机和棉花喷头，并使用牙钻手机和钻头开颅手术。驱动一个单柄MEA到皮层使用显微操作。有一个手术助手在保持无菌手术条件的援助，并记录了手术的进展。 <ol start="2"><li>植入到大脑的MEA，收集到的图像通过外科手术显微镜通知最终去除的头骨周围的b雨。植入部分的设备将被保留在原位 。 </li></ol> 注:最终收集组织原位设备依赖于密切相匹配的平面与平面的组织切片的设备插入。详细记录设备插入平面相对于大脑是非常重要的。 <ol start="3"><li>装置植入后，应用有机硅弹性体KWIK-SIL（WPI），任何暴露的MEA柄或电缆周围，让治愈。甲灭菌的塑料片，如切断的移液管尖，也可以使用，以帮助固化，同时，包含在一个小的有机硅弹性体以及周围的开颅。 </li><li>应用层由两部分组成的牙科丙烯酸（"喷射液体"和"喷粉"郎牙科的）在KWIK-SIL和任何暴露的头骨。 </li></ol> 注 :在后面的步骤中，headcap将被熔化通过允许以被分离的植入物从头骨。 UV固化丙烯酸在佳银和开颅应避免，因为这非常硬丙烯酸是难以除去购买。视不透明材料植入物周围也应避免明确KWIK-SIL的植入提供了一个在本协议的后续步骤。 <ol start="5"><li>手术后的护理程序，根据当地的动物福利法规，协议的执行，并返回门诊科目单住的笼箱。 </li></ol> 2。灌注和组织收集 注:请参阅Gage等人。在大鼠动物模型19的详细灌注过程演练。 <ol><li>使用麻醉和灌注协议，该机构的动物护理和使用委员会批准。深麻醉后的啮齿类动物（确认缺乏脚趾，捏在大鼠或小鼠的尾巴捏反射的反射）和暴露的心，使的浅剪刀削减左心室和右心房。钝针头插入左心室，并提供室温下PBS。提供一个共〜10毫升PBS〜5毫升/分钟在小鼠和〜200毫升的PBS在〜100 ml / min的大鼠。看的肝脏从血液清除。 </li><li>注入组织学相关的化学品标签transcardially，如果需要的话，用注射器交付小心谨慎以避免引入气泡。 </li></ol> 注:血管标签（DII）和核酸染液的染料（ 如赫斯特33342）交付灌注过程中的化学品标签的例子。如果管理得当，这些组织的标记应标注在整个动物20。 <ol start="3"><li>提供缓冲，室温，4％的甲醛transcardially固定动物。避免鼻中隔穿孔整个的心脏，它可以证明的期间肺鼻子排水灌注。固定在大肌肉震颤显示完整的灌注。提供一个共〜10毫升固定液〜5毫升/分钟在小鼠和〜200毫升固定剂在〜100 ml / min的大鼠。 </li><li>灌注后，杀头的动物，用咬骨钳或剪刀，头放入4％甲醛溶液中过夜，脑干或小脑的一部分暴露在4°C </li><li>一至四个小时洗涤液之间的间隔，在三个转变的PBS洗净头部。 </li><li>将固定磁头HBHS叠氮化钠在4°C为几天到几个月。 </li></ol> 3。脑去除原位设备 注:执行本节在通风橱中，而穿着适当的个人防护装备。在返回固定头，定期HBHS解决方案避免组织干燥。 <ol><li>各地丙烯酸头盖骨使用钳子和小剪刀，小心地剖析了皮肤和其他组织。 </li><li>虽然穿着热我nsensitive手套，使用电烙铁，清除牙丙烯酸和公开区基本明确KWIK-SIL（ 图2a）。 </li></ol> 注意:此步骤的目的是允许微剪刀访问植入设备的位置处进入大脑的一个适当的角度，从而使脑植入的元件可能是分离的组件固定在颅骨上。 <ol start="3"><li>在手术显微镜下，有机硅弹性体具有弯曲的微剪刀切成，用镊子除去小块佳实向下的位置时，器件进入大脑。 </li><li>继续沿着弯曲的微型剪刀单独的设备组件，多晶硅和颅骨植入大脑中的组件开颅手术的表面切割。通过公开的设备组件再次使用显微镜的放大倍率和切割时非常谨慎，小心，以避免推动或拖动小鬼lants在固定的组织。 </li><li>去除骨周围的headcap小心用咬骨钳。用铲子分离的大脑植入设备的头骨。店的解决方案在HBHS的大脑，直到准备好切。 </li><li>将大脑充满HBHS在玻璃培养皿中，并用剃刀刀片删除多余的组织，如脊髓，小脑，或嗅球，这取决于它们是否是相关的位置的装置植入。使用的脑块（泰德培拉）和刀片部大脑和密切匹配的角度植入设备建立一个平面，这是通过在一个大小合适的脑块放置大脑，大脑这样，任何取向可见部分的植入物是平行刀片指南，和放置在一个刀片导向从植入物的至少2毫米的剃刀刀片。按刀片穿过组织。安装这面到vibratome平台。或者为剃刀刀片安装在显微操作作为大脑块创建密切匹配的植入物的方向的平面内，可以用在类似的控制的方式。 </li><li>将冰下vibratome平台中，使组织表面简单的纸巾上干燥后，坚持到vibratome使用超级胶水的大脑。胶在上一步中创建的扁平组织平面的舞台。具有非对称的组织尺寸，取向等的样品被粘下来的vibratome刀片的移动方向，以帮助避免可能敲组织超过刀片平行的最宽尺寸。胶水集后（约1-2分钟），添加冷藏（〜4℃）的PBS组织周围的vibratome菜。 </li><li>收藏报错厚200-400微米之间，包括控制组织的组织切片，用刀片从浩角为10°，使用的最大振动速度（〜100赫兹）和一个缓慢的叶片进展率（&lt;0.2毫米每秒）rizontal。小心地用刷子或铲子铲和商店在HBHS在24孔板收集切片在4°C。 </li><li>一旦原位的移动设备的肉眼或手术显微镜可见，收集较薄的部分接近100微米或更小的增量，在分片的移动设备。 </li></ol> 注:典型的硅微植入物是在甲醛固定的啮齿动物大脑皮层组织300和500微米之间的从设备表面用肉眼可见。 <ol start="10"><li>随着原位设备，现在已经接近表面的组织，收集&gt; 250μm的组织切片内的设备。必要的厚度估计可以辅助通过引用以前收集的切片。 </li></ol> 注:大型设备，multishank设备和角度的设备可能需要更厚的组织切片（&gt; 500微米）捕捉到的设备在一个组织块重新成员应用标签的渗透和显微成像深度将是有限的，在最后的数据收集这些很厚的片。如果可能的话，应避免撞击装置的vibratome刀片，因为这可能会导致拖动设备通过组织和形态的变形。 4。组织处理及结算所<ol><li>洗净切片，3倍于每洗5分钟HBHS </li><li>孵育在硼氢化钠（5毫克加入NaBH 4/1毫升HBHS）总共为30分钟（15分钟的每一侧的切片）。翻转片，采用不锈钢或聚四氟乙烯涂层的微勺子，小画笔（特德·佩拉）。动作速度慢和周到的提高，每片翻转的成功。避免接触的任何区域周围的植入装置用刷子。在可能的情况下，将越来越多的解决方案需要（2毫升），允许一个组织切片更容易操作和翻转。 </li></ol>注意:此步骤，减少内源性自发荧光;如果采用荧光标记灌注过程中，或XFP转基因标记是跳过此步骤。 <ol start="3"><li>在洗涤液:在室温下洗涤​​5分钟，每片3倍洗净。 </li></ol> 注:搅拌过程中清洗和培养步骤是可选的。研究人员推测，微妙的生硬植入物与周围的脑组织，可能会出现不和谐的搅拌。然而，在温和的速率（&lt;60转）移动一个轨道混合器可避免这种情况的解决方案，同时增加的移动。 <ol start="4"><li>座冲洗缓冲液中2小时，在室温下（1小时后）倒装片。 </li><li>初级抗体（冲洗缓冲液中稀释）孵育约48小时（24小时，每一侧上的切片），在4℃下</li><li>执行6快3分钟的清洗液清洗。 </li><li>执行6 1小时，洗涤（3洗涤上课冲洗缓冲液中的每一侧上的组织切片）（储存于4℃下如果洗衣机过夜）。 </li><li>在4°C的二抗（用清洗液稀释）孵育约48小时（24小时每边一个） </li><li>执行6快3分钟的清洗液清洗。 </li><li>执行，1小时洗（每边洗3次），清洗液储存于4°C如果洗涤隔夜。 </li><li>删除洗涤液，并添加甘油为基础的"U2 - SCA L E"结算解决方案。允许对于非常厚的组织切片（&gt; 500微米）厚的片（250-500微米）或2-3周清除约1周，在4℃下</li><li>覆盖板带箔和存储在4°C。 </li><li>安装在幻灯片容纳厚的组织切片（ 图2b，2c）的结算解决方案安装介质和密封透明指甲油。在4°C避光保存。 </li></ol> 5。全景成像的全钛ssue和设备接口<ol><li>使用较长的工作距离物镜（&gt; 2毫米），开始用激光扫描共聚焦显微镜或双光子显微镜成像。我们将展示一个蔡司LSM 710，卡尔·蔡司"禅"软件（2010），和一个计算机控制的XY转换阶段植入物周围的3D全景图像。 </li></ol> 注意:正确结算解决方案，无论是在软件，通过纠正领上的目标的折射率，或在采集后的处理的数据。在这个演示中，我们进入了一个"U2"结算解决方案，徕卡的禅显微镜软件到1.4折射率值，此设定巧妙地调整Z-步的时间间隔折射率不匹配。 <ol start="2"><li>在装置附近的组织，大致评估相应的z轴的窗口，并在z维度上的数据收集设置为每个荧光通道使用低激光功率（〜00.5〜5％）和较大的z步骤大小（&gt; 25微米）。 </li></ol> 注:包括额外的空间的上方和下方，当设定z轴时，设立为全景数据收集，作为组织可能不会完全位于平面上的载玻片（〜20μm的每个方向）。 <ol start="3"><li>在XY中心最终的全景;使用Zen全景软件所设定的目标，确定收集需要为每个轴（X和Y），以支付您感兴趣的区域的位置。 </li><li>重新评估和完成的z轴的采集窗口。 </li><li>手动设置的检测器的灵敏度和激光功率斜坡适当增加深度的目的通常是维持大致相同的图像强度无论成像深度进入组织切片，但也避免高背景噪声。 </li><li>收集每个荧光图像数据系列。如果有必要，以避免重叠信号，运行激光线小号顺序。 </li></ol> 注:如果可用，设置软件自动将数据保存到硬盘驱动器在收集过程中。 <ol start="7"><li>更改软件设置，以收集的"反射"和"透射比"的数据，并使用较长的，更渗透波长的激光（ 例如 633纳米）捕捉到这些渠道。 "反射"和"透射比"收集和确定合适的激光功率检测器的灵敏度，周围的植入装置重复同样的收藏系列。 </li><li>导出的数据后处理，量化和分析。 </li></ol>

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作者申报，财务或其他有没有相关的披露。

的"设备采集组织学"（DCHist）这里演示的方法，使关闭的形态保存下来的脑组织和组织之间的相互作用植入的组织学评价。 DCHist组织收集需要仔细分离的骷髅装设备组件从组件在大脑中植入。 DCHist还需要收集了厚厚的病理组织切片（&gt; 250微米）。这些组织切片，一次标记，清除，安装和成像，可以提供新的见解植入损伤或后续长期留置设备。使用先进的显微镜工具，组织和移动设备之间的接口可以被成像和高细节分析，如在图3-5中示出。 在其目前的形式依赖于技术的能力，慢慢地由两部分组成的牙科水泥和KWIK-SIL点的装置植入的挖掘了headcap的。利用牙科水泥，可以烧掉或以其他方式拆除和清晰的硅弹性体，通过它可以引导小剪刀，在视觉上极大的帮助成功地分离的植入装置从它的头骨安装的组件。的头骨和下上面的大脑试图削减的设备，以收集在原位不推荐，因为骷髅式的植入物被拉出的大脑有显着的量。 更薄，更小的植入物，如单柄多边环境从NeuroNexus技术，虽然是最适合DCHist，单个设备柄或微电极阵列的原则并不限于。采集和图像处理的策略是广泛适用于多柄的设备和更大的植入物，如套管的要求，他们必须从任何颅骨安装的分离和收集在厚的组织切片。虽然作者主要集中在组织分析皮质植入物周围，驱动进入大脑更深的设备也可以被收集和成像。植入物插入的深度应不影响植入物的捕获切片中的提供该设备不很大，从已知的插入角度偏离。 存在限制的有用的应用程序的DCHist方法。是具有挑战性的脑组织切片的图像，通过数百微米，尤其是在白质区域，虽然光透明的解决方案可以大大提高在各种组织中的成像深度21。为了进一步改善成像深度，可以采用双光子激发显微镜可沿与所述的光透明。 另一个潜在的局限性所描述的方法可以是特殊的荧光免疫组织化学方法和抗体的研究人员。被动扩散典型的驱动器将这些标记通过固定的组织和抗原结合位点上。标签渗透最大化，同时避免了高层次的背景标记的情况下，基础的情况下，最终的抗体浓度必须确定工作的研究人员。抗体标记不应该是可变的，处理相同的片之间，但不同的抗体，可能有很大的不同，其穿透能力和标记抗原，轻松地标记抗原抗体的许多数百微米深和其他标记抗原只有几十微米深。我们描述的，通过施加高于典型浓度的抗体标签的应用程序的多个天改善这种扩散，并在溶液中含有稀洗涤剂​​和封闭血清。定期翻转片，甚至也促进了标签。抗原修复步骤和替代固定过程（ 例如，戊二醛，微波等），可能是适当的特异性抗原。二次抗体 - fluoroch的罗马的结合物可能也各不相同性能标签厚的部分，虽然这并没有被观察作者使用Invitrogen公司的Alexa Fluor标签的。或者，在感兴趣的细胞类型的表达荧光蛋白的转基因动物受试者可能被利用，避免与抗体标签渗透的问题，尽可能多的荧光蛋白质，如绿色荧光蛋白，甲醛处理后保留它们的荧光，并在组织切片中可能会立即对可视化。 DCHist是一套功能强大的技术来捕获和分析的影响对脑组织植入的微型器件。耦合与在体内评估的电质量和电极阻抗数据22这个组织的协议可以大大提高我们的理解生理变异和退化的生物来源。领域的植入神经义肢装置，特别是可能受益的详细DCHist IMA晋完整的设备/组织接口的，告知进一步发展生物中性MEA设备。

神经义肢的研究领域，旨在协助患有各种残疾和疾病，绕过病变或受损的身体结构，通过CNS接口设备1,2个人。脑植入的微型器件，如微电极阵列（MEAs）中，可用于记录或刺激大脑结构，从而允许建立的长期的电子设备之间的接口和中枢神经系统组织3-5。穿透多边环境协定所带动的设备进入脑组织，具有特别的承诺，由于靠近内，他们提出电极附近的神经元6一个相对较小的双向接口。 但是，复杂的组织反应导致从长期植入穿透多边环境协定，往往造成在变量和逐渐降解的电生理在数天至数月的信号 - 噪声比，和在电气阻抗增加ANCE电极位置和地面7,8。这些变化包括在假定的起源激活的小胶质细胞，星形胶质细胞反应沿微型器件，以及从周围组织植入设备9-11神经元的损失或迁移。了解这些组织周围的变化慢性，渗透多边环境协定的主要挑战是在捕捉周围长期植入装置12的完整的组织界面的组织学数据的难度。组织与组织学分析设备/组织界面仍然存在于当前的设备移除组织的协议将提高。一个未受干扰的装置，残留在该组织，生物的影响相对微妙的相互作用，如生物相容性涂层13,14或在电极表面15,16的电结算的利用率，可以相对于植入物的成像和分析。 ĤERE我们展示了一个方法来收集，过程，和图像的完整的微型器件接口的详细周围脑组织的显微镜为基础的分析。在该方法中，移动设备和周围的脑组织厚（&gt; 250微米）的组织使用vibratome部分内收集。为了提高标签渗透到这些巨厚的切片组织学，荧光组织化学和免疫组化的标签，适用于高浓度的解决方案，包含多天的封闭血清和洗涤剂。一种光学结算溶液提高显微成像深度，并且组织被安装在随后的激光扫描共聚焦显微镜17的 2 -双面腔室。要捕捉完整的病理接口，在成像过程中使用计算机控制的翻译阶段收集的Z-Stack全景沿着植入物的长度。除了影像应用组织标签，收集激光反射回来植入通过组织帮助和透射光定位设备接口，与周围组织。组织准备使用此"移动设备捕获组织学"（DCHist）协议提供成像获得的形态保存的组织/设备的相互作用，从而提高了在先前的移动设备去除的组织学协议18。

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td>赫斯特33342 </td><td> Invitrogen公司</td><td> 14533 </td><td> DNA标记</td></tr><tr><td>兔抗Iba1 </td><td>和光（日本） </td><td> 019-19741 </td><td>单核细胞抗体</td></tr><tr><td>牙科丙烯酸</td><td>郎牙科（各分销商） </td><td>喷气义齿修复的粉末和液体</td><td> Headcap建设</td></tr><tr><td> KWIK-SIL </td><td>世界精密仪器</td><td> KWIK-SIL </td><td>覆盖暴露颅骨植入物</td></tr><tr><td>正常山羊血清</td><td>杰克逊immunoresearch公司</td><td> 005-000-121 </td><td>组织处理</td></tr><tr><td>的Triton X-100的</td><td> Sigma-Aldrich公司</td><td> X100-500ML </td><td>组织处理</td></tr><tr><td>尿素</td><td> Sigma-Aldrich公司</td><td> U4883 </td><td>清除解决方案</td></tr><tr><td>甘油</td><td> Sigma-Aldrich公司</td><td> G20225 </td><td>清除解决方案</td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>弯曲的微型剪刀</td><td>世界精密仪器</td><td> 14208 </td><td>切割植入前除去脑</td></tr><tr><td>剃刀片</td><td>特德·佩拉</td><td> （各种尺寸） </td><td>用于脑块</td></tr><tr><td>丙烯酸脑矩阵的</td><td>特德·佩拉</td><td> 15054 </td><td>在组织中创建初始平面脑块</td></tr><tr><td>塑料滑梯</td><td>特德·佩拉</td><td> 260225 </td><td>安装组织</td></tr><tr><td>盖玻片</td><td>特德·佩拉</td><td> （各种尺寸） </td><td>安装组织</td></tr><tr><td>电极阵列</td><td> NeuroNexus技术</td><td> （各种图案） </td><td>例如多边环境协定</td></tr><tr><td> Vibratome </td><td>徕卡</td><td> VT1000小号</td><td>提出的方法中使用的特定的系统</td></tr><tr><td> Vibratome刀片</td><td> （不同的供应商） </td><td></td><td>采集片</td></tr><tr><td> 24孔培养板</td><td> （不同的供应商） </td><td></td><td>存储和处理组织切片</td></tr><tr><td>共聚焦显微镜电动XY扫描台</td><td>卡尔·蔡司显微镜</td><td>蔡司LSM 710和"禅宗2010&#39;软件</td><td>提出的方法中使用的特定的系统</td></tr><tr><td>烙铁</td><td> （不同的供应商） </td><td></td><td>挖掘headcap </td></tr></tbody></table>

intact histological characterization of brain-implanted microdevices and surrounding tissue

脑植入的微型器件，如微电极阵列的设计和利用，研究的目的是长期与周围脑组织的临床相关设备的接口。这些植入物周围的组织被认为反应的存在下，随着时间的推移的设备，其中包括绝缘"周围的设备的"胶质瘢痕形成。然而，这些组织的变化的组织学分析的典型后进行explanting设备，在一个过程中，可以破坏感兴趣的组织的形态。 在这里，我们展示了一个协议，其中皮质植入设备周围的啮齿动物脑组织中收集完整。我们描述了如何，一旦灌注固定液，大脑除去，切片在这样一种方式，以避免explanting设备。我们概述了荧光抗体标记和有用的信息，而厚的组织的光透明的方法一节。最后，我们证明了这些组织切片为了研究大脑植入设备周围的生物界面的安装和成像。

脑植入多边环境可以在组织切片收集从脑嵌入式组件通过第一分离任何颅骨安装组件。 图2a示出的结果中除去1侧的牙科水泥headcap和佳实的MEA电缆周围的一部分的在大鼠颅骨。甲烙铁是用来去除在通风橱中的牙科用粘固剂和佳实。慢慢挖掘通过佳实向下固定脑表面的电缆和任何非注入的MEA结构接下来微剪刀切断。 切片后，标签和清算组织的，简单的自定义制作的幻灯片是有用的安装厚的组织切片（ 图2b）。植入的微型器件包含一个安装的大脑切片如图2c所示。成像可以让你通过任何一方的幻灯片评估周围的设备的接口，主要表现在图3 </st荣&gt;，小胶质细胞的MEA沿着硅的支持，从一个侧面可见（ 图3a）和小胶质细胞沿电极位置和走线的另一端是可见的（ 图3b）。 一旦被安装，可将组织成像的使用的XY翻译阶段。整个组织切片的荧光标记的概述可以在所需的分辨率（ 图4）产生的。植入的微型器件周围的形态保存的组织界面的详细检查可以收集在高倍率下的局部组织的响应（ 图5）分析。 <img src="/files/ftp_upload/50126/50126fig1.jpg" alt="图1" fo:content-width="6.5in" fo:src="/files/ftp_upload/50126/50126fig1highres.jpg"/> 图1。步骤概述（AC）的脑微植入手术是国际泳联汉化与佳柏实紧接在器件周围的聚合物的层，其次由丙烯酸类水泥覆盖。安乐死后，（D，E）的丙烯酸系层烧掉，通过有机硅弹性体通过切割从植入的元件和安装在颅骨上的移动设备的组件分离。 （f和g）脑然后除去从颅骨和被切割并安装到vibratome阶段。 （ 香港 ）收集组织切片，包括含有该装置的组织切片。 （L）组织处理，并安装在自定义的详细显微镜室。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50126/50126fig1large.jpg" target="_blank">点击此处查看大图</a> 。 <img src="/files/ftp_upload/50126/50126fig2.jpg" alt="图2"/> 图2（a）明确KWIK-SIL硅弹性体苏rrounding电极阵列电缆（箭头）已公开的燃烧掉一个窗口，在使用电烙铁在灌注啮齿动物头的牙科水泥盖。 （b）为容纳到两侧的厚组织切片成像，简单的"滑动室"，可通过切割的塑料载玻片和附着盖玻片周围的组织的任一侧和安装溶液。 （c）在大鼠脑组织切片显示与的完整植入物（红色箭头）后安装。任一侧的组织的快速访问与此安装程序的显微镜成像。规模，面板（*）为25毫米，在底部的对面，而面板（ 乙，丙）是80毫米在底部的对面。 <img src="/files/ftp_upload/50126/50126fig3.jpg" alt="图3" fo:content-width="3.5in" fo:src="/files/ftp_upload/50126/50126fig3highres.jpg"/> 图3最大诠释。ensity的z突起的40微米厚的图像堆栈，收集周围的背面侧（a）和一个植入的微电极阵列的前面的（b）。小胶质细胞（标记的抗-Iba1和Alexa Fluor 633，白色）和激光的光反射率（红），从装置表面收集，依次从一个400微米厚的组织切片的两侧成像。由于大脑植入物通常是不透明的，安装光纤接入到双方的标记的组织结构"的背后，"植入显微镜物镜提供了一个明确的说法。比例尺为50μm。 <img src="/files/ftp_upload/50126/50126fig4.jpg" alt="图4"/> 图4。标记DCHist切片成像，激光扫描共聚焦显微镜使用电脑控制的舞台上。 （ 一 ）细胞核（用Hoechst 33342染色）和（b）的单核细胞/小胶质细胞（标记的抗-Iba1）同时成像在分开的信道。 （c）发送的光，反射率也被收集，表示4周植入的位置相对于给Hoechst和Iba1数据。 （四）覆盖的所有通道，但传输也同时显示。出现在右图像中的白色矩形面积扩大。比例尺1毫米。 <img src="/files/ftp_upload/50126/50126fig5.jpg" alt="图5" fo:content-width="4.5in" fo:src="/files/ftp_upload/50126/50126fig5highres.jpg"/> 图5，使用计算机控制的显微镜载物台和适当的软件，全景成像数据可以被收集，植入物周围。反射率（a）和透射率（b）允许设备的国产化，免疫荧光或化学品标签（C，抗Iba1标记的小胶质细胞和Macrophages）的组织成分以及完整的组织界面进行详细的成像。比例尺条为200μm。

Watch this Scientific Journal Video about Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue at JoVE.com

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

在这里，我们提出了一个组织学方法进行拍摄，标签，光清理和完整的脑组织界面成像在啮齿类动物的脑组织周围长期植入的微型器件。对于理解各种穿透脑植入物在其周围组织的影响，结果，从包括该方法的技术是有用的。

脑植入的微型器件及周围组织的完整的组织学特性研究

Research  into the design and utilization of brain-implanted microdevices, such as  microelectrode arrays, aims to produce clinically relevant devices ...

intact-histological-characterization-brain-implanted-microdevices

Nanyang Technological University

Research

JoVE Journal

Neuroscience

16.8K 次观看.  Purdue University. 在这里，我们提出了一个组织学方法进行拍摄，标签，光清理和完整的脑组织界面成像在啮齿类动物的脑组织周围长期植入的微型器件。对于理解各种穿透脑植入物在其周围组织的影响，结果，从包括该方法的技术是有用的。

视频: Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

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

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

由运动神经元调制生理，形态学和神经化学的表征

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

在完整的视网膜电生理特性表达GFP的细胞群

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

在完整的成年斑马鱼大脑电生理记录

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional units of the ventricles are ciliated epithelial cells termed ependymal cells, which line the ventricles and through ciliary action are capable of generating laminar flow of CSF at the ventricle surface. This monolayer of ependymal cells also provides barrier and filtration functions that promote exchange between brain interstitial fluids (ISF) and circulating CSF. Biochemical changes in the brain are thereby reflected in the composition of the CSF and destruction of the ependyma can disrupt the delicate balance of CSF and ISF exchange. In humans there is a strong correlation between lateral ventricle expansion and aging. Age-associated ventriculomegaly can occur even in the absence of dementia or obstruction of CSF flow. The exact cause and progression of ventriculomegaly is often unknown; however, enlarged ventricles can show regional and, often, extensive loss of ependymal cell coverage with ventricle surface astrogliosis and associated periventricular edema replacing the functional ependymal cell monolayer. Using MRI scans together with postmortem human brain tissue, we describe how to prepare, image and compile 3D renderings of lateral ventricle volumes, calculate lateral ventricle volumes, and characterize periventricular tissue through immunohistochemical analysis of en face lateral ventricle wall tissue preparations. Corresponding analyses of mouse brain tissue are also presented supporting the use of mouse models as a means to evaluate changes to the lateral ventricles and periventricular tissue found in human aging and disease. Together, these protocols allow investigations into the cause and effect of ventriculomegaly and highlight techniques to study ventricular system health and its important barrier and filtration functions within the brain.

Using MRI scans (human), 3D imaging software, and immunohistological analysis, we document changes to the brain&#8217;s lateral ventricles. Longitudinal 3D mapping of lateral ventricle volume changes and characterization of periventricular cellular changes that occur in the human brain due to aging or disease are then modeled in mice.

脑室周围组织的侧脑室及组织学特性对人类和鼠标3D建模

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional ...

Electroencephalographic, Heart Rate, and Galvanic Skin Response Assessment for an Advertising Perception Study: Application to Antismoking Public Service Announcements

The evaluation of advertising, products, and packaging is traditionally performed through methods based on self-reports and focus groups, but these approaches often appear poorly accurate in scientific terms. Neuroscience is increasingly applied to the investigation of the neurophysiological bases of the perception of and reaction to commercial stimuli to support traditional marketing methods. In this context, a particular sector or marketing is represented by public service announcements (PSAs). The objective of this protocol is to apply electroencephalography (EEG) and autonomic signal analysis to study responses to selected antismoking PSAs. Two EEG indices were employed: the frontal alpha band EEG asymmetry (the Approach Withdrawal (AW) index) and the frontal theta (effort index). Furthermore, the autonomic Emotional Index (EI) was calculated, as derived from the Galvanic Skin Response (GSR) and Heart Rate (HR) signals. The present protocol describes a series of operational and computational steps required to properly estimate, through the aforementioned indices, the emotional and cerebral reaction of a group of subjects towards a selected number of antismoking PSAs. In particular, a campaign characterized by a symbolic communication style (classified as "awarded" on the basis of the prizes received by specialized committees) obtained the highest approach values, as estimated by the AW index. A spot and an image belonging to the same PSA campaign based on the "fear arousing appeal" and with a narrative/experiential communication style (classified as "effective" on the basis of the economical/health-related improvements promoted) reported the lowest and highest effort values, respectively. This is probably due to the complexity of the storytelling (spot) and to the immediateness of the image (a lady who underwent a tracheotomy). Finally, the same "effective" campaign showed the highest EI values, possibly because of the empathy induced by the testimonial and the explicitness of the message.

The following protocol describes a series of operational and computational steps required to properly estimate the emotional and cerebral reaction of a group of subjects towards a selected number of Public Service Announcements (PSAs) against smoking, aired in the USA and Europe during the period between 1998 and 2015.

脑电图, 心率和电皮肤反应评估为广告知觉研究: 应用到禁烟公共服务公告

The evaluation of advertising, products, and packaging is traditionally performed through methods based on self-reports and focus groups, but these ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

轻度宫内灌注圈狭窄的大鼠模型

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

密度梯度离心法分离小鼠原发胶质细胞的研究

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...