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

14.0K 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

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

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). ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

修改一个Colliculo  - 丘脑鼠脑切片，收纳室部件的3-D打印和多尺度光学成像

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume three-dimensionally. While MRI is an extremely sensitive tool for detecting tissue abnormalities, association of signal changes with an underlying pathological process is usually not straightforward. In the central nervous system, for example, inflammation, demyelination, axonal damage, gliosis, and neuronal death may all induce similar findings on MRI. As such, interpretation of MRI scans depends on the context, and radiological-histopathological correlation is therefore of the utmost importance. Unfortunately, traditional pathological sectioning of brain tissue is often imprecise and inconsistent, thus complicating the comparison between histology sections and MRI. This article presents novel methodology for accurately sectioning primate brain tissues and thus allowing precise matching between histology and MRI. The detailed protocol described in this article will assist investigators in applying this method, which relies on the creation of 3D printed brain slicers. Slightly modified, it can be easily implemented for brains of other species, including humans.

The overall goal of this protocol is to accurately align magnetic resonance imaging (MRI) image volumes with histology sections via the creation of customized 3D-printed brain holders and slicer boxes.

利用3D打印技术来合并MRI与组织学:一个大脑切片协议

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

探索深空-揭开人脑侧脑室的脑室结构解剖

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have been established by tamoxifen induced deletion of Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. An accurate and quantitative method to assess lesion burden and progression is essential to harness the full value of these animal models. Here, we demonstrate the induction of CCM disease in a mouse model and the use of the contrast enhanced X-ray micro computed tomography (micro-CT) method to measure CCM lesion burden in mouse brains. At postnatal day 1 (P1), we used 4-hydroxytamoxifen (4HT) to activate Cre recombinase activity from the Cdh5-CreErt2 transgene to cleave the floxed allele of Ccm2. CCM lesions in mouse brains were analyzed at P8. For micro-CT, iodine based Lugol&#39;s solution was used to enhance contrast in brain tissue. We have optimized the scan parameters and utilized a voxel dimension of 9.5 &#181;m, which lead to a minimum feature size of approximately 25 &#181;m. This resolution is sufficient to measure CCM lesion volume and number globally and accurately, and provide high-quality 3-D mapping of CCM lesions in mouse brains. This method enhances the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

小鼠脑海绵体畸形的诱导及显微 CT 成像

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

医学

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

小鼠大脑皮层细胞组织的大尺度三维成像

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12&#8211;15 and 5&#8211;6 &#181;m&#160;per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies.

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

一种基于微 ct 的小动物脑损伤和定位电极表征方法

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that ...

A Needle Model for Vascular Cognitive Impairment Studies in Mice

A Mouse Model for Vascular Cognitive Impairment and Dementia Based on Needle-guided Asymmetric Bilateral Common Carotid Artery Stenosis

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue rising as the population ages, it is critical to establish a robust animal model for the disease. This paper presents a novel method of creating a mouse model of VCID that is based on asymmetric bilateral common carotid artery stenosis, which mimics human chronic cerebral hypoperfusion caused by carotid atherosclerosis.
Briefly, common carotid arteries (CCAs) are ligated to different gauge needles (32 G for the right CCA and 34 G for the left CCA) using 7-0 silk sutures followed by immediate needle removal. The remaining suture rings cause persistent blood flow reduction and long-term cognitive impairment associated with white matter injury, microinfarcts, and reactive gliosis, thus closely mimicking the pathogenesis of VCID. Importantly, in this needle model, the clinical representations do not revert with time, providing reliable long-term cognitive impairment. Moreover, the survival rate 24 weeks post surgery was 81.6%, which is higher compared to the other established models of VCID with a similar level of blood flow reduction.
Additional advantages include low material cost and compatibility with MRI to monitor brain injury in live animals since no metal is implanted. The main challenge in employing the needle model of VCID is the requirement for developing advanced surgical skills since mouse CCAs are less than 0.6 mm in diameter and are very fragile. High-quality visual representation of the surgery will thus help researchers to master this technique and advance our understanding of VCID, potentially leading to the development of novel therapeutic modalities to decrease the devastating cognitive decline associated with VCID.

The needle method of asymmetric bilateral common carotid artery stenosis is proposed to create a mouse model for vascular cognitive impairment and dementia. It results in longer-term outcomes compared to previously established models and is compatible with live MRI. Visual representation demonstrating the procedure provides guidance for mastering the surgery.

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue ...