An NINDS Grant NS05033 (JCC) supported this work. The University of Connecticut RAC, SURF and OUR programs provided additional support.

注：动物的程序批准了康涅狄格IACUC大学和符合美国国立卫生研究院的指导方针。人体组织和​​数据分析及程序均符合并经IRB康涅狄格州的大学，并符合美国国立卫生研究院的指导方针。 1.鼠标：侧脑室脑室周围白质细胞的完整性和三维建模分析 1.1）准备鼠标侧脑室墙全挂载的<ol><li>准备鼠标侧脑室整个坐骑免疫组织化学（IHC）前面所描述的16,17。 </li></ol> 1.2）免疫组化侧脑室分析<ol><li>修复和部分老鼠的脑组织如前所述18,19。 <ol><li>简言之，将冲洗小鼠transcardially与正常，室温盐水，直至器官已清除（由浅着色表示），随后室温下4％多聚甲醛（PFA）的0.1M磷酸盐缓冲盐水（PBS），直到组织已加强。 </li><li>从头骨中取出大脑。使用虹膜切除剪刀沿矢状缝颅底削减。 </li><li>暴露的头骨和镊子取出大脑。沉浸大脑在4％PFA固定剂过夜，在4℃。然后洗3次，每次20分钟，用PBS。 </li></ol></li><li>准备免疫组化和体积分析连续切片。 <ol><li>采用显微手术刀，做一个小切口沿纵向左半球的皮层，让左半球的鉴定右半球时，浮动部分的免疫染色。 </li><li> vibratome切片过程中包住固定在3％琼脂糖以增加结构支撑的大脑。在蒸馏水温暖3％琼脂糖（重量/体积），直到完全使用一个微波或热板溶解。 </li><li>用刀片，在与小脑取出大脑的尾部冠状切，留下了平坦和均匀的表面。适用超级胶水到组织阶段和朝上嗅球的新切割表面定位大脑。 </li><li>当液体琼脂糖溶液冷却到刚刚温暖触摸的温度，均匀涂抹在大脑完全包住整个大脑。允许琼脂糖固化。 </li><li>部琼脂糖包裹大脑冠上的vibratome到50微米的部分，与串联置于含有PBS中，以保持连续的顺序一个24孔板部分。 注：一般为12井前看到，让A1开始侧脑室的出现，数字移动通过对B6和骑自行车回至A1用于一个大脑，以收集组织开​​始5段。这使得从相同的大脑的多个不同的部分可以在同一个井处理。对于侧脑室量分析，组织收集停止时侧脑室秒和第三脑室合并，导致每孔约3段或36段总数。 </li><li>使用贴有20-200微升枪头移液管，以确保浮动部分不意外吸入吸出PBS。块和透化在10％血清的浮动部分，0.1％的Triton X-100（TX）的PBS（PBS-TX）处理1小时，在室温下进行。使用300微升的溶液，每孔以覆盖自由浮动的组织切片在24孔板中并在摇板执行部分的所有的孵育。 </li><li>吸出封闭溶液，并在4℃孵育在PBS-TX过夜（至少12小时），初级抗体部分。对于使用冠状切片心室容积痕迹，使用抗S100β抗体标记室管膜细胞。 </li><li>吸去第一抗体溶液和洗涤切片3次，每次10分钟，用PBS-TX。 </li><li>在孵育的暗部1小时在室温彩画TURE用荧光二级抗体，以1浓度：1000，在10％血清，PBS-TX。保持在暗部的所有剩余的孵育步骤。 </li><li>吸去二级抗体溶液孵育切片用4&#39;，6-二脒基-2-苯基吲哚在PBS（DAPI，10微克/ ml）处理5分钟至染液细胞核。吸出DAPI溶液和冲洗切片3次，每次10分钟，用PBS。 </li><li>山路段上连续使用皮质标志幻灯片保存左与右半球的方向。空气干燥切片在黑暗中，然后盖玻片通过施加一薄层安装媒体向滑动，并轻轻放置在上面的玻璃盖玻片。允许盖片载玻片干燥过夜，在室温下进行。 </li></ol></li></ol> 1.3）侧脑室分割的三维重建注：执行侧脑室跟踪使用绘图软件上直立落射荧光microscoPE与自动阶段和一个数字CCD照相机进行荧光检测。 <ol><li>打开荧光显微镜设备，并启动绘图软件在荧光模式。打开“显示选项”加载需要跟踪的正确工具栏和停靠工具面板。选项​​&gt;显示选项&gt;附件，然后选择“主”和“标记”工具栏。在同一个菜单中，选择“相机直方图”，“多通道控制”，“相机设置”，然后在“图像采集&#39;停靠工具面板。” </li><li>观察基于强大的S-100β的荧光标记室管膜细胞该行侧脑室脑室的开端。识别含有侧脑室第一块组织。 </li><li>创建一个新的数据文件，并通过点击图像窗口上方左侧脑室指定为阶段运动的参考点。寻找小切口从东方右半球离开了。保持横跨跟踪文件导入轮廓串行重建时，以帮助在相对位置侧脑室一个一致的参考点。 </li><li>从轮廓相应的预设轮廓式下拉菜单， 如 “左侧侧脑室。”使用独特的颜色为每个左和右侧脑室描记的。如果改变轮廓名称和颜色都需要，进入选项&gt;显示首选项&gt;轮廓，然后修改的轮廓颜色的名称，或选择“添加轮廓类型”。 </li><li>在选择了“左侧侧脑室&#39;轮廓，单击沿室管膜衬的顶面，开始跟踪轮廓。通过继续单击先后沿顶面追踪心室。 <ol><li>对于不规则区域需要更多的技巧，请单击并按住鼠标左键，以便追查放开手脚。按Ctrl + Z，或去牛逼O选项&gt;撤消撤消之前的轮廓点。移动使用箭头键跟踪窗口或通过一个轮廓点关闭屏幕并允许窗口自动中心本身。要完成心室跟踪点击右键，选择“关闭轮廓”。 </li></ol></li><li>选择新的轮廓色（轮廓型）使用下拉菜单右侧脑室。跟踪右心室如步骤1.3.4左侧。 </li><li>如果改变轮廓名称和颜色都需要，右键单击轮廓上并选择更改轮廓类型来选择合适的颜色。 </li><li>添加标记沿中线协助对准串行轮廓的三维重建。要添加标记，选择适当的标记（用&#39;X&#39;）从左侧的工具栏标记，然后点击删除它们跟踪屏幕上。导入标记以及轮廓的每个组织切片痕迹。 </li><li>要保存组织的痕迹，选择File&gt; Save数据文件作为，并创建一个新的文件夹和文件名。数描记[幻灯片＃] - [＃组织]为了便于识别，从连续切片痕迹。例如，在第二滑动的第三组织部分有目录的每个大脑内的文件名&#39;2-3&#39;。 </li><li>脑组织中每个串行部分在整个大脑追查心室，排除脑室壁上的附着力地区重复步骤1.3.4至1.3.9。 </li><li>如果心室有附着力，子区段的前部区域的从那些背侧和腹侧的粘附位点的区域。每个心室（ 例如 ，&#39;背左心室“和”腹左心室&#39;）创建两个新的轮廓类型。在第一组织切片用粘合性，痕量侧脑室与原始侧脑室轮廓和新背部和腹部的轮廓，以确保一个完整心室重构可以被创建。 </li><li>在第后路一个ventriclË壁粘附，创建一组新的轮廓颜色为每个心室（ 例如 ，&#39;后路左心室&#39;）的后部。双跟踪同时与当前的附着力和新轮廓后这类型的第一重接合部。 </li></ol> 1.4）侧脑室三维重建<ol><li>对齐和编译鼠标侧脑室生成三维重建和容积数据的串行轮廓描记。 </li><li>开启3D重建方案。单击文件&gt;打开并选择第一个跟踪串行部分的轮廓文件。导入左和右心室的轮廓描以及任何添加的标记。 </li><li>要选择轮廓，单击“选择对象”按钮（光标的图标），并通过举办“Ctrl键”选择两个心室和标记。要建立轮廓的Z位置，点击右键，选择“修改Z位置”。第一组织切片设置为0微米。 </li> &lt;li&gt;要保存的重建中，选择文件&gt;另存数据文件作为。节约每一文件导入后，如果做错了复苏的重新加载一个文件一样简单。 </li><li>下一组织切片添加到重建，单击文件&gt;附加到显示屏。选择要追加的.dat文件，并检查“合并”对话框。 </li><li>按照步骤1.4.3再次调整新导入的跟踪文件的Z位置。选择在主窗口中只有新添加的左侧和右侧的轮廓，以避免移动的其他痕迹。从以前的轮廓为50微米的部分Z位置设置每个连续的跟踪文件到50微米。 （第一个轮廓：Z = 0，第二个轮廓：Z = 50，第三个轮廓：Z = 100等） </li><li>要调整X，选择轮廓和标记Y位置，单击“启用翻译与鼠标”左键并拖动的轮廓，使其与以往的组织轮廓。保留两个当前跟踪的左右轮廓选允许同步运动。 </li><li>要旋转的痕迹顺时针或逆时针排队中线标志，选择“Z轴旋转”按钮。要在整个翻转Y轴轮廓（如果左轮廓右）同时选择进口的轮廓和标记，单击右键，选择“设置旋转角度”，然后输入“180”到“Y旋转”。 </li><li>确保脑室和中线标记保存重建文件，如步骤1.4.4之前最好对齐。 </li><li>直到所有已导入和正确对齐重复步骤1.4.5至1.4.9，其后每组织切片。 </li><li>要查看序列重建的容积数据，单击分析&gt;“标记和区域分析”，然后选择“3D轮廓摘要”。选择在左侧面板中的所有轮廓，然后点击“三维可视化”按钮显示最终的3D重建。 </li></ol>2.人类：侧脑室脑室周围白质细胞的完整性和三维建模分析 2.1）人力MRI数据分析注：协议中列出创建3D图像重建和侧脑室的体积量化和评估随时间变化的体积使用纵向叠加分析。要注意在磁共振数据采集（ 例如 ，机器和磁铁强度，切片厚度，取向和分辨率）和采集后处理的一致性是非常重要的标准列入数据集20是很重要的。 <ol><li>心室分割 注：ITK /快从高分辨率的T1加权像用来分割心室。可替代地，可以使用其他的免费软件如Freesurfer。 <ol><li>打开ITK /快，文件&gt;打开灰度图像。选择需要的文件并打开一个文件.nii（NITFI文件）。 </li><li>通过滚动ANAT每omical飞机，并指出心室异常（狭窄的区域，或大或小的时间牛角等 ）。在主工具箱单击“蛇ROI工具”。点击“段3D”。选择“强区”选项。 </li><li>点击“图像预处理”。选择&#39;上面&#39;，包括低于一定强度阈值的区域，以突出的投资回报率在白色。使用滑动条记录最大强度。设置阈值，以最大强度（记录此值）的15％。平滑参数设置为10。点击“确定”，然后“下一步”。 </li><li>添加10-12小（2号）“气泡”每个心室：两个前额叶角，七沿侧脑室体，一个在枕角，一个在的颞角的上表面均匀间隔侧脑室。 “参数选择”，并设置曲率力0.4。点击播放符号，开始主动轮廓演化。 </li><li>点击“更新网”可视化表面;选中“自动更新”。停止分割时侧脑室的所有领域都包括在感兴趣区域（ROI）的区域。避免列入第三脑室。点击“完成”。 </li><li>手动编辑图像，如果有必要通过以下的方法以除去不需要的区域（通常需要擦除第三脑室泄漏）。手动使用“3D手术刀”工具来去除多余的区域或手动使用“画笔”工具“清除标签”为活动图纸标签删除任何包含超出投资回报率。 </li><li>保存分割结果作为.nii图像（&#39;NIFTI&#39;文件格式）。 </li><li>要获得总成交量心室，选择“分割&gt;卷”与统计;获得使用设置的颜色标签心室的总体积。 </li></ol></li><li>纵向表示侧脑室从MRIScans 注：使用芒果软件，纵向的ROI重叠定性和定量表明随着时间的推移受试者内变化心室体积。 <ol><li>打开芒果。单击第一个投资回报率的时间点（基线）绿色的文件&gt;加载的投资回报率。文件&gt;第二次投资回报率的时间点为红色的负荷的投资回报率。保存纵向叠加通过选择文件&gt;另存为;分配每个图像作为“文件名_ [第一时间点时代] - [第二个时间点的年龄] .nii”。 </li><li>打开ITK-SNAP。重新组合的投资回报为“灰度图像”，通过选择细分&gt;负荷图像&gt;组合的投资回报率的文件。为了获得纵向数据量运行以下命令：分割&gt;音量统计&gt;标签1（红色）=膨胀体积;标签2（绿色）=狭窄量;标签3（蓝色）=基本卷。 </li></ol></li></ol> 2.2）人力脑室周围组织准备和分析<ol><li>与人体组织工作安全须知<ol><li>获得appropriate安全培训（ 例如 ，血源性病原体课程）。 </li><li>观察标准（通用）的安全防范措施。戴手套。接触任何普通设备或表面不属于一次​​性之前更换手套。常规标签与“人体组织使用”指定的人体组织中工作时处理的任何项目。 </li><li>遵循接触人体组织的所有项目去污做法。漂白所有污染的液体以处置前至少24小时，治疗污染表面与漂白浸毛巾（ 例如 ，台式），或通过直接在漂白放置的物体（ 例如镊子）。 </li><li>制定应急计划，与人体组织直接在皮肤上，其中可能包括浸泡纸巾漂白和保持患处（5-10分钟）接触。 </li><li>使用适当的废物处置的光临与人体组织接触的所有物品。 </li></ol></li><li>侧脑室整装准备<ol><li> B具有完整半球eginning（固定在10％福尔马林和用0.1M PBS中彻底漂洗），切穿整个半球使用大刀厚1.5厘米冠状切片。 注意：所有固定协议需要事先抗体验证。 </li><li>标签部分前后开始用含有侧脑室第一部分。使用字母数字标记系统，用字母（从前到后），并款用数字（顶端向下）标记该切片。避免破坏室管膜的表面。 </li><li>采用显微外科手术刀，解剖左心室壁1厘米深，保持一个连续的部分，整个侧壁。根据需要进行染色以及创建适当大小的部分细分墙。对室壁另一侧缺口部分，以确定上/下方向。 </li><li>通过将组织切片在10mM柠檬酸钠缓冲液（pH 6.0）在100℃下10-20分钟使用双boile执行抗原修复r（下最佳孵育时间根据经验确定）。冲洗3次在PBS / 0.1％的Triton X-100（PBS-TX）。 </li><li>使用PBS / PBS-TX 1小时，在室温下，在10％马血清的块。 </li><li>孵育初级抗体为48小时，4℃。以可视化心室墙衬，整个免疫荧光染色与坐骑以下抗体组合：鼠抗β-catenin的（1：250）;山羊抗GFAP（1：250）;兔抗水通道蛋白（1：400）。 </li><li>请在黑暗中所有的后续步骤。漂洗组织3次在PBS中孵育第二抗体（1：500），24小时，在4℃或2小时，在室温下，和漂洗另外3次在PBS中。在DAPI孵育组织在室温下7分钟，随后与另一漂洗3次在PBS中。 </li><li>通过覆盖蜡底菜用封口膜组织准备进行最后的清扫。填写菜用PBS，用细镊子把组织中的菜，避免与制作室管膜壁接触。 </li><li>根据解剖显微镜，坚决SECU再在其一侧用针组织。有22.5°显微捅刀，创建统一的薄片（约300微米厚）侧脑室壁。对于大部份或难以弯曲部分，切成段最后安装及注意事项位置之前，切成小方块。 </li><li>仔细用细镊子，注意到区域的位置和方向上的幻灯片处切开部分室管膜的一面向上滑上。组织覆盖薄层aquapolymount的，照顾，以避免气泡。将盖玻片组织，增加额外的aquapolymount必要填补盖玻片下的任何间隙。 </li><li>放置在黑暗及干燥装部分为2-3天，在室温下进行。店slidebox在4℃。 </li></ol></li><li>免疫组化和组织学分析<ol><li>使用共焦显微镜，以查看荧光免疫组织化学，设置在成像焦平面在心室表面，如通过使用Ó确定˚Fβ连环蛋白（标记为室管膜细胞的心尖粘着连接蛋白）。划定室管膜细胞覆盖和地表星形胶质细胞增生（GFAP染色在心室表面）的区域。 </li><li>记录和蒙太奇整个心室的表面，使用重叠的图像，覆盖整个表面。要创建序列图像的蒙太奇，打开Adobe公司的Photoshop。使用File&gt;自动化&gt; Photomerge的&gt;交互式布局选择图像叠加，必要时作出手动调整。 </li><li>创建新图层跟踪蒙太奇生成完整的室管膜细胞覆盖或心室表面胶质增生的卡通代表。重复每个幻灯片或投资回报率。编译所有部分重建地图室壁，并链接到MRI上重建相应的区域。 </li></ol></li></ol>

<ol>
	<li>Del Bigio, M. 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The authors have nothing to disclose.

我们提出的工具和可用于在小鼠和人类中评估大脑脑室系统的完整性协议。这些工具，然而，也可以应用到进行，或在老化14,21,22的过程中，由于受伤，疾病改变其它脑结构或器官系统。策略提出的软件趁其允许剖面和纵向的MRI序列的比对，以产生特定区域或感兴趣结构的3D体积表示。纵向MRI序列允许的发生随着时间的推移，并且可以扩展到包括大脑总体积，为侧脑室总脑体积比，和/或其...

一个室管膜细胞单层线大脑提供的脑脊髓液（CSF）和间质液（ISF）的1-3之间的双向屏障和传输功能的脑室系统。这些功能有助于保持大脑毒物和无生理平衡2,3中。在人类中损失此衬里通过损伤或疾病的部分不会出现导致再生替换为在其他上皮衬里找到;而室管膜细胞覆盖的损失似乎导致脑室周围星形胶质细胞增生与星形胶质细胞覆盖在心室表面溶蚀室管膜细胞的区域的小梁。严重的影响到重要的CSF / ISF交换和通关机制，将预期从这个皮层1,2,4-7损失导致。 人类衰老的共同特征被放大侧脑室（脑室）和相关的脑室周围水肿作为observ由MRI和液体衰减反转恢复MRI（磁共振成像/ FLAIR）8-14编调查脑室和心室衬里的细胞组织之间的关系，死后人MRI序列进行匹配与侧脑室脑室周围组织的组织学制备。在脑室病例，胶质增生的重要方面开展了沿侧脑室壁室管膜代替小区覆盖。如果不是由基于MRI容积分析发现心室扩张，室管膜细胞衬里是完整，胶质细胞增生并没有沿着心室衬里6检测。这种组合的方式表示使用部分wholemount筹备或整个侧脑室壁和心室体积6三维建模侧脑室衬里细胞完整性的第一个全面的文档，详细的变化。几种疾病（阿尔茨海默氏症，精神分裂症）和损伤（创伤性脑损伤）显示脑室扩大作为早期神经病理学特征。的室管膜细胞衬里从而领域剥蚀将预期可干扰正常室管膜细胞功能和妥协CSF / ISF流体和溶质交换之间的稳态平衡。因此，改变的脑室系统，其细胞组合物，以及所产生的结果底层或邻近脑结构进行更彻底的检查将最终开始更多地揭示与脑室扩大相关的神经病理学。 缺乏多模成像数据的，尤其是纵向的数据序列，用有限的访问相应的组织学组织样品一起使人类脑病理学的分析困难。在人体衰老或疾病发现的表型造型往往可以达到的小鼠模型和动物模型成为我们的最佳手段之一，以探讨有关人类疾病的发生和发展的问题。在一些研究中健康年轻小鼠所描述的侧脑室壁的细胞结构和底层干细胞小生境4,7-15。这些研究已经通过延长老化6,15包括脑室壁上的三维建模和细胞分析。无论是脑室周围胶质细胞增生，也不脑室老年小鼠中观察到的，而老鼠显示一个相对强劲的subventicular区（SVZ）干细胞小生境下层为一个完整的室管膜细胞衬里6,15。因此，在这两种一般的保养和侧脑室衬里的完整性存在老化6,15的过程中撞击物种特异性差异。因此，最好使用小鼠来询问在人类中发现的条件，需要进行表征，而在任何建模范例适当考虑这两个物种之间的差异。这里，我们目前的程序，以评估在人类和M纵向变化侧脑室和相关组织脑室周围乌斯河。我们的程序包括3D渲染和小鼠和人脑室体积测量，并利用脑室周围组织整装准备免疫组化分析，既细胞组织和结构进行了表征。连同这些程序提供表征在脑室系统的变化和相关的脑室周围组织的一种手段。

<table border="0" cellpadding="0" cellspacing="0" width="881">
	<tbody>
		<tr height="44">
			<td height="44" style="height:44px;width:227px;">Name of the Materal/Equipment</td>
			<td style="width:363px;">Company</td>
			<td style="width:131px;">Catalog Number</td>
			<td style="width:160px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>21600-069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy Sciences</td>
			<td>19210</td>
			<td>Use at 4% in PBS, 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Horse Serum</td>
			<td>Life Technologies</td>
			<td>16050</td>
			<td>10% in PBS-TX (v/v)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Goat Serum</td>
			<td>Life Technologies</td>
			<td>16210</td>
			<td>10% in PBS-TX (v/v)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100 (TX)</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>0.1% in PBS (v/v)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome</td>
			<td>Leica</td>
			<td>VT1000S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence Microscope</td>
			<td>Zeiss</td>
			<td>Imager.M2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Camera</td>
			<td>Hamamatsu</td>
			<td>ORCA R2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope Stage Controller</td>
			<td>Ludl Electronic Products</td>
			<td>MAC 6000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereology software</td>
			<td>MBF Bioscience</td>
			<td colspan="2">Stereo Investigator 11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereology software</td>
			<td>ImageJ/NIH</td>
			<td>NIH freeware</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3D Reconstruction software</td>
			<td>MBF Bioscience</td>
			<td colspan="2">Neurolucida Explorer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscope</td>
			<td>Leica</td>
			<td>TCS SP2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MRI Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Freesurfer</td>
			<td colspan="2">https://surfer.nmr.mgh.harvard.edu/fswiki/DownloadAndInstall</td>
			<td>Segmentation and Volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ITK-Snap</td>
			<td>http://www.itksnap.org/pmwiki/pmwiki.php</td>
			<td></td>
			<td>Segmentation and Volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-image Analysis GUI (Mango)</td>
			<td>http://ric.uthscsa.edu/mango/</td>
			<td></td>
			<td>Longitudinal overlay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Whole Mount Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">22.5&#176; microsurgical straight stab knife</td>
			<td>Fisher Scientific</td>
			<td>NC9854830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">parafilm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">wax bottom dissecting dish&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fine forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">aquapolymount</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Microscope</td>
			<td>Leica</td>
			<td>MZ95</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Whole Mount Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti-b-catenin</td>
			<td>BD Bioschiences, San Jose, CA, USA</td>
			<td></td>
			<td>1:250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-GFAP</td>
			<td>Santa Cruz Biotechnology</td>
			<td></td>
			<td>1:250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit anti-AQP4 (aquaporin-4)</td>
			<td>&#160;Sigma-Aldrich</td>
			<td></td>
			<td>1:400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coronal Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-S100&#946; antibody</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8217;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Life Technologies</td>
			<td>D-1306</td>
			<td>10 &#181;g/mL in PBS</td>
		</tr>
	</tbody>
</table>

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.

根据免疫染色为50μm冠状切片和三维重建（ 图3）的小鼠侧脑室的轮廓跟踪允许量数据被收集在使用小鼠作为疾病或损伤的模型系统不同实验范式。临界此过程是区，其中侧脑室壁彼此粘附的排斥。由subsegmenting心室的区域并指定一个不同的颜色为每个区域（ 图3C），邻接部分可以遵从与区域和总体积可以从编译的子段被计算。 类似的研究可以使用用?...

Watch this Scientific Journal Video about 3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse at JoVE.com

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

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 ...

3d-modeling-lateral-ventricles-histological-characterization

Research

JoVE Journal

Neuroscience

13.9K 次观看.  University of Connecticut. Using MRI scans (human), 3D imaging software, and immunohistological analysis, we document changes to the brain’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. 

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

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

脑室周围白质软化的小鼠模型

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

科研

神经科学

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

使用扩频萧条的情节和慢性偏头痛的神经免疫信号建模<em在体外</em

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

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

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

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

成年鼠脑铸造三维成像和形态分析

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

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

鼠标感觉神经支配的皮肤和四肢骨骼纤维组织的制备及免疫染色

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Manual Drainage of the Zebrafish Embryonic Brain Ventricles

Cerebrospinal fluid (CSF) is a protein rich fluid contained within the brain ventricles. It is present during early vertebrate embryonic development and persists throughout life. Adult CSF is thought to cushion the brain, remove waste, and carry secreted molecules1,2. In the adult and older embryo, the majority of CSF is made by the choroid plexus, a series of highly vascularized secretory regions located adjacent to the brain ventricles3-5. In zebrafish, the choroid plexus is fully formed at 144 hours post fertilization (hpf)6. Prior to this, in both zebrafish and other vertebrate embryos including mouse, a significant amount of embryonic CSF (eCSF) is present . These data and studies in chick suggest that the neuroepithelium is secretory early in development and may be the major source of eCSF prior to choroid plexus development7.
eCSF contains about three times more protein than adult CSF, suggesting that it may have an important role during development8,9. Studies in chick and mouse demonstrate that secreted factors in the eCSF, fluid pressure, or a combination of these, are important for neurogenesis, gene expression, cell proliferation, and cell survival in the neuroepithelium10-20. Proteomic analyses of human, rat, mouse, and chick eCSF have identified many proteins that may be necessary for CSF function. These include extracellular matrix components, apolipoproteins, osmotic pressure regulating proteins, and proteins involved in cell death and proliferation21-24. However, the complex functions of the eCSF are largely unknown.
We have developed a method for removing eCSF from zebrafish brain ventricles, thus allowing for identification of eCSF components and for analysis of the eCSF requirement during development. Although more eCSF can be collected from other vertebrate systems with larger embryos, eCSF can be collected from the earliest stages of zebrafish development, and under genetic or environmental conditions that lead to abnormal brain ventricle volume or morphology. Removal and collection of eCSF allows for mass spectrometric analysis, investigation of eCSF function, and reintroduction of select factors into the ventricles to assay their function. Thus the accessibility of the early zebrafish embryo allows for detailed analysis of eCSF function during development.

We present a method to collect cerebrospinal fluid (CSF) and to create a system which lacks CSF within the embryonic zebrafish brain ventricular system. This allows for further examination of CSF composition and its requirement during embryonic brain development.

手动排水的斑马鱼胚胎脑室

Cerebrospinal fluid  (CSF) is a protein rich fluid contained within the brain ventricles. It is  present during early vertebrate embryonic development and ...

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.
Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.

Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.

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 ...

Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans

The corticospinal pathway is the major pathway connecting the brain with the muscles and is therefore highly relevant for movement control and motor learning. There exists a number of noninvasive electrophysiological methods investigating the excitability and plasticity of this pathway. However, most methods are based on quantification of compound potentials and neglect that the corticospinal pathway consists of many different connections that are more or less direct. Here, we present a method that allows testing excitability of different fractions of the corticospinal transmission. This so called H-reflex conditioning technique allows one to assess excitability of the fastest (monosynaptic) and also polysynaptic corticospinal pathways. Furthermore, by using two different stimulation sites, the motor cortex and the cervicomedullary junction, it allows not only differentiation between cortical and spinal effects but also assessment of transmission at the corticomotoneural synapse. In this manuscript, we describe how this method can be used to assess corticomotoneural transmission after low-frequency repetitive transcranial magnetic stimulation, a method that was previously shown to reduce excitability of cortical cells. Here we demonstrate that not only cortical cells are affected by this repetitive stimulation but also transmission at the corticomotoneuronal synapse at the spinal level. This finding is important for the understanding of basic mechanisms and sites of neuroplasticity. Besides investigation of basic mechanisms, the H-reflex conditioning technique may be applied to test changes in corticospinal transmission following behavioral (e.g., training) or therapeutic interventions, pathology or aging and therefore allows a better understanding of neural processes that underlie movement control and motor learning.

The aim of the present study was to assess changes in transmission at the corticomotoneuronal synapses in humans after repetitive transcranial magnetic stimulation. For this purpose, an electrophysiological method is introduced that allows assessment of pathway specific corticospinal transmission, i.e. differentiation of fast, direct corticospinal pathways from polysynaptic connections.

非侵入性评估人类皮质神经元传播的变化

The corticospinal pathway is the major pathway connecting the brain with the muscles and is therefore highly relevant for movement control and motor ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

室管膜纤毛在小鼠脑侧脑室实时成像

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Cortex-，Hippocampus-，Thalamus-，下丘脑 - ，横向隔核 - 纹状体专用<em&gt;在子宫内</em&gt;电穿孔的C57BL / 6小鼠

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...