This work was supported by grants from the Spanish Ministry of Economy and Competitiveness CSD2010-00045 (MAM), SAF2012-33216 (MAM), SAF2011-23354 (IL) and RENEVAS RD06/0026/0005 (IL), and from the Local Government of Madrid S2010/BMD-2336 (MAM) and S2010/BMD-2349 (IL). IB and MIC are fellows of the Spanish Ministry of Economy and Competitiveness.

注:坚持Complutense大学的动物福利委员会的指导方针所有实验的协议（以下EU指令六百○九分之八十六/ CEE和2003/65 / CE）。 1.脑缺血模型注:本文中的脑缺血模型既包括CCA（颈总动脉）和MCA（大脑中动脉）由结扎永久或暂时性闭塞用尼龙缝合。 <ol><li>手术前的准备<ol><li>通过高压灭菌或用玻璃珠灭菌消毒所有手术器械。消毒用70％乙醇手术区也是如此。 </li><li>麻醉鼠标采用适当的麻醉剂。通过异氟烷2.5％达到诱导并用异氟醚中的80％空气/ 20％氧气的混合物用蒸发器保持1.5％。应用人工泪液的眼睛老鼠，防止干涩而在麻醉下。 </li><li>放置在鼠标上链接到收费加热垫dback设备用在生理水平的外科手术过程中的温度探针放置在直肠和设定温度（36.5±0.5℃）。 </li></ol></li><li>常见的颈内动脉闭塞结扎<ol><li>鼠标放置在背斜卧和固定用胶带。消毒皮肤用70％乙醇或聚维酮碘和切上部腹侧部分的毛发。 </li><li>下一个手术显微镜，使围绕颈部腹面1cm的中线切口。 </li><li>拉开所有软组织（腺组织包括子上颌窦，舌下和腮腺），并确定左颈总动脉（CCA），它是局部横向于气管。缩回肌肉和揭露左CCA。从迷走神经解剖谨慎。 </li><li>一旦切开，通过使用6/0尼龙缝线结扎一个闭塞左CCA。具有永久结或活结结扎（允许再灌注如果短暂性脑缺血模型需要）。 </li><li>把腺体组织到其原来的位置，然后关闭切口颈部皮肤有6/0尼龙线。 注:深水动物进行同样的外科手术如缺血的动物，但CCA不堵塞。 </li></ol></li><li>远端大脑中动脉闭塞结扎<ol><li>将鼠标在其右侧和固定用胶带。消毒头部皮肤用70％乙醇或聚维酮碘和切鼠标头部的毛发（切割之后去除毛发）。 </li><li>下一个手术显微镜，使眼睛和耳朵之间的皮肤切口。将皮肤的路程，用胶带握住。 </li><li>就从向左右侧颞肌水平切口。然后，使右侧颞肌的第二垂直切口（要小心，以避免切颞静脉）。从头骨拉开颞肌，用缝线持有它保持一个干净的颅骨表面。 </li><li>清洁颅骨表面用冷的无菌盐水以检测左大脑中动脉（MCA）的颅骨经由透明性的确切位置。执行一个圆开颅（1毫米直径）配有一个不锈钢的毛刺在颧弓和鳞骨之间的额叶。小心取下的头骨和揭露MCA。 </li><li>适用于少量的冷的无菌盐溶液到大脑的表面上，并通过使用镊子除去脑膜（硬脑膜和蛛网膜）。 </li><li>只是正面和后部MCA分支之间的分歧之前执行留下MCA的远端结扎主干。通过使用一个9/0尼龙缝线结扎一个闭塞左MCA。具有永久结或活结结扎（允许再灌注如果短暂性脑缺血模型需要）。 CCA的和MCA闭塞之间的时间周期是根据运营商的经验，大约10-20分钟。 </li><li>将颞肌到其原来的位置，然后关闭incisio氮对颈部皮肤有6/0尼龙线。 </li><li>返回鼠标笼从麻醉（一般5-10分钟）收回并注入小鼠丁丙诺啡腹腔内（IP）（0.3毫克/千克）。 <ol><li>监控鼠标几个小时来检测任何不适（不要离开鼠标无人看管，直到它恢复足够的意识，保持胸骨斜卧）。因为手术后的重量丢失通常观察，将捣碎食品在培养皿中，以方便进食。 </li></ol></li><li>当动物被完全恢复，返回到其他动物的公司。 </li><li>如果一个短暂缺血模型需要，再次麻醉小鼠并取出CCA和MCA的活结允许再灌注（通常在0.5-2小时（ 图1））。 注:深水动物进行同样的外科手术如缺血动物，但在MCA不堵塞。 </li></ol></li></ol> 2.灌注和Secti脑组织oning <ol><li> 24或缺血后48小时，牺牲鼠标采用了致命剂量的麻醉剂（ 如巴比妥钠，IP 86毫克/千克或异氟醚过量）。 </li><li>灌注小鼠用磷酸盐缓冲液0.1M的随后的4％多聚甲醛（PFA）的磷酸盐缓冲液（pH 7.4）中。 </li><li>取出大脑如下: <ol><li>斩首动物只是脊髓上方。 </li><li>让头部的皮肤后前切口，显露颅骨。使两侧切口在侧壁的交界处及颅底，取出小块骨头。 </li><li>做一个切割，通过沿矢状缝颅骨。 </li><li>取出颅骨使用镊子，露出大脑覆每个半球。用刮刀给大脑从头骨中分离，并将其传输到4％PFA溶液。 </li></ol></li><li>定位后3小时在4％PFA溶液在4ºC。 </li><li>删除PFA解决方案，并incubate为脑48小时在30％的蔗糖溶液来cryoprotect大脑。 </li><li>除去蔗糖溶液和在冷异戊烷（-40℃）快速冷冻大脑。冷冻保存的大脑在-80ºC。 </li><li>获得冠状脑切片（30微米）与冷冻切片。收集10套连续在冷冻保存液的解决方案。每个串行集为300微米的条间的距离能充分品尝小鼠大脑之间1.94和-2.46后部到前囟由周围14-16冠状切片。 </li></ol> 3.尼氏染色及脑梗塞体积估计<ol><li>尼氏染色用甲酚紫<ol><li>摩脑切片成的Superfrost载玻片。对梗塞体积测定由尼氏染色切片的卡瓦列的方法，使用1/20切片采样分数（1/2部分收集在步骤2.7的10串行集​​之一的;约，共7-8段数以600μ的条间的距离; m的使用）。小心以保持适当的前 - 后方向（梗塞区被置于右侧）。 </li><li>如下执行常规尼氏染色: <ol><li>允许滑动安装部分干燥几个小时。 </li><li>再水化载玻片上的切片和染色用5分钟的0.1％甲酚紫溶液。 </li><li>脱水用梯度系列的EtOH（70％，90％和100％;每变化5分钟）。清洁载玻片上的切片用二甲苯，加distrene-80增塑剂二甲苯（DPX）安装媒体并盖上玻璃盖玻片。 </li></ol></li></ol></li><li>利用体视软件来估计梗塞体积在卡瓦列里法尼氏染色连续切片<ol><li>使用表1中所示参数的卡瓦列方法14和一个体视系统，该系统由配有相机，XYZ机动计算机阶段显微镜，和体视软件量化梗塞体积（主下面提供方向都涉及到个人电脑，但方向不同，可以为其它操作系统）。 </li><li>在适当的顺序打开电脑，显微镜，舞台控制器和摄像头。启动体视软件。 </li><li>将10X目标到位，并从客观的下拉菜单中选择10X。 </li><li>走动第一尼氏染色部分，找到一个合适的参考点。点击鼠标来建立参考点（移动至周围，有必要主动喜悦自由按钮）。 </li><li>估计"安装部分厚度"（实际切片厚度考虑到收缩）。按下"焦点位置表"按钮，并计算从底部厚度的部分的顶部。 </li><li>创建轮廓工具的对侧和ipsilesional区和梗死区。 <ol><li>点击菜单中的"显示"，然后选择"显示设置"。这将打开吨他显示设置对话框。 </li><li>选择"等高线"选项卡，然后点击按钮"添加轮廓类型"。 </li><li>创建一个轮廓的每个区域进行量化，并使其轮廓选单中可见。点击OK。 </li></ol></li><li>创建一个标记工具的对侧和ipsilesional半球梗塞面积。 <ol><li>点击菜单中的"显示"，然后选择"显示设置"，打开显示设置对话框。 </li><li>选择"标记"选项卡并更改通过双击在可用的标记命名标记。使他们在标记栏可见。点击OK。 </li></ol></li><li>激活科长。 <ol><li>点击菜单"工具/串行部门经理"上。与"新科"按钮添加新的一节。此按钮打开"串行节设置对话框"。 </li><li>设置"阻止前进"为30微米。设置"安装段厚度"，预计在步骤3.2.5的值。 </li><li>将"评估间隔"为20。使用1/20切片取样率。 </li><li>设置"起始节号"为1。设置为0。单击OK（确定）"为第一部分的顶部Z轴值"。 </li></ol></li><li>画出轮廓勾勒出对侧半球。 <ol><li>选择对侧半球工具，从轮廓下拉菜单（以前在步骤3.2.6创建）。 </li><li>画出轮廓勾勒出对侧半球。 </li><li>要完成跟踪对侧半球，单击鼠标右键，选择"关闭轮廓"。 </li></ol></li><li>估计对侧半球轮廓被卡瓦列里。 <ol><li>点击菜单"探针"，然后选择卡瓦列里估计。设置"栅格间距"为100微米。设置"网格旋转"为0设置"B锁进展"为30微米。点击OK。 </li><li>选择"在对侧半球"从标记栏按钮。右键单击对侧半球轮廓的内侧。 </li><li>选择"画图卡瓦列标记模式"。再次右键单击对侧半球轮廓内，然后选择"画图标记为轮廓"。 </li><li>停用卡瓦列里估计通过点击探头和卡瓦列里估计，并从标记栏取消对侧标记按钮。 </li></ol></li><li>重复同样的步骤对ipsilesional半球梗死区（步骤3.2.7和3.2.8）。每个部分的面积的估计之后保存数据。 </li><li>计算的尼氏染色节的其余部分健侧，ipsilesional和梗死区。 <ol><li>创建一个新的部分，如步骤3.2.6。不要修改的"串行部分设置"对话框中输入的值。单击确定。 </li><html><li>重复步骤3.2.7-3.2.8在新的一节。 </li></ol></li><li>量化的结果导出到一个电子表格文件。 <ol><li>点击"探头"的立体调查菜单。选择"显示探头运行列表"。这将打开"以前的体视运行对话框"。 </li><li>通过点击在他们选择的所有部分。按"查看结果"按钮。这将打开的"卡瓦列里估算结果"估计面积和体积的每个区域都显示在那里。 </li><li>导出结果到电子表格文件，单击"复制所有结果到剪贴板"并粘贴到Excel与每个区域（ 表2）所获得的主要成果文件。 </li></ol></li><li>明示梗死体积为3毫米或为％半球即梗塞（％1H）使用下式15％IH = InfVol / ContrVol * 100，其中的</li></ol></li></ol>ove_content"&gt; InfVol（梗死组织体积）=ΣInfArea 我 ， ContrVol（对侧半球卷）=ΣContrArea 我 中性粒细胞渗脑缺血后的四体视学定量的光学分馏<ol><li>染色的脑切片的: <ol><li>对于由光学精馏塔16推定MCAO后嗜中性粒细胞浸润的总数，使用1/10切片采样级分。免疫染色嗜中性粒细胞通过使用与大鼠抗Ly6G抗体（1A8），并在自由浮动部分的右边的第二抗体的常规免疫荧光技术。此外，与像DAPI或TOPRO核复染机，尽管它不是必须的嗜中性粒细胞的识别，可以使更容易检测梗塞面积。 </li><li>摩脑切片与放置在右侧的梗塞面积的Superfrost显微镜载玻片。 </li></ol></li><li> Quantific在缺血性脑渗透白细胞由光学精馏塔ATION <ol><li> 表3中所示的使用参数通过用体视装置中的光学分馏方法来量化在缺血性脑浸润白细胞。从第3节重复步骤3.2.1-3.2.5。 </li><li>创建一个轮廓的工具，为梗塞区，因为它已经在第3条的步骤3.2.6进行了描述。 </li><li>创建的标记工具，嗜中性粒细胞，因为它已在第3条的步骤3.2.7进行了描述。 </li><li>激活科长，并创建一个新的部分作为步骤3.2.8。在这种情况下，设置"评估间隔"作为10（1/10切片采样馏分用于估计嗜中性粒细胞数）。 </li><li>选择"梗塞区"轮廓刀具从轮廓下拉菜单（以前在步骤4.2.1创建）。画出一个轮廓，在10X目标，勾勒出梗死区。改变目标100X执行neutroph金正日量化（不要忘了，从客观下拉菜单中选择100X）。 </li><li>点击菜单中的"探头"，然后选择"光分馏塔"。设置为230 X和Y网格大小的"计算框架的XY放置"。设置"网格旋转"为0设置"阻止前进"为30微米。点击OK。 </li><li>请从标记栏的"中性粒细胞"按钮。马克在梗死区染色中性粒细胞。一旦完成，点击"探头"和"光学分馏"取消光学分馏探头。停用从标记栏的中性粒细胞按钮。 </li><li>重复同样的步骤，对每个部分（4.2.4-4.2.9）。 </li><li>量化的结果导出到一个电子表格文件。 <ol><li>点击"探头"的立体调查菜单。选择"显示探头运行列表"，打开"以前的体视奔跑"对话框。 </li><li>通过点击在他们选择的所有部分。按"查看结果"按钮，打开"光学分馏结果"，其中显示中性粒细胞的数量估计。 </li><li>通过点击"复制所有结果到剪贴板"并粘贴到一个电子表格文件将结果导出为电子表格文件。 </li></ol></li></ol></li></ol> 5.脑解离和流式细胞仪分析注:通过流式细胞术在新鲜的脑组织髓细胞亚群的表征可以被用作一个替代的固定和免疫染色的脑切片进行先前的嗜中性粒细胞的表征。 <ol><li>脑解离和单细胞悬浮液制备<ol><li>使以10ml /一个的RPMI（罗斯威尔公园纪念研究所培养基）的动物装有8毫升的RPMI中，加入1ml 30％的Percoll和1ml 10倍PBS（磷酸盐缓冲盐水）的-Percoll溶液。 </li><li> ř如在步骤2.3中所述MCAO后emove小鼠脑。 </li><li>在显微镜下，解剖了脑缺血小鼠同侧皮层（或假和天真的动物类似的区域）从大脑的核心和梗死周围区。重量的脑组织，并将其放置到5ml冰冷的RPMI-珀（可以为实验组之间正常化进行重量步骤）。 </li><li>离解在用Potter-Elvehjem型组织研磨机用特氟隆杵单细胞悬浮液的组织。 </li><li>转移细胞悬液至50ml离心管并加入5更毫升的RPMI-的Percoll溶液的样品。 </li><li>离心细胞悬浮液在7800×g离心30分钟，在25ºC。离心后，确保相应于髓鞘的白色色层出现在溶液的顶部。 </li><li>小心地取出髓鞘层和过滤器的全细胞悬浮液，包括沉淀的细胞，通过40-μm的尼龙筛straine河</li><li>加入10 mL的RPMI对过滤器，以确保所有细胞被过滤，并放置在50毫升的管中的溶液中。添加30毫升的RPMI和离心机50 ml细胞悬浮液在600×g离心在室温10分钟。 </li><li>弃上清，悬浮颗粒白细胞用1毫升的PBS。加入4毫升裂解缓冲液（150mM的氯化铵 ，10mM的碳酸氢钠和0.1mM EDTA）中，并培育的细胞在室温下2-3分钟，以从细胞悬浮液溶解红细胞。离心该溶液，在600×g离心10分钟，在4ºC。 </li></ol></li><li>细胞染色和流式细胞仪<ol><li>制备标记鸡尾酒流式细胞 ，含有200微升的PBS-BSA（1％）1:100的Fc-座1时40分的抗CD45-PercP大鼠，1点40抗Ly6G-APC大鼠，一点40的反的CD11b-FITC大鼠和一点40反-LY-6C-PE每个样品鼠。 </li><li>重悬的细胞中的标记鸡尾酒孵育的样品在冰中45分钟。 </li><li>洗标签鸡尾酒用1ml冷PBS中，并离心将细胞在600×g离心10分钟，在4ºC。悬浮用100μl的FACS流动的粒料，并获得在整个细胞悬液通过流式细胞术。 </li><li>使用流式细胞仪分析软件来表征和量化的细胞群。 <ol><li> CD45 LO的CD11b +:对应于在这个协议中制备的标记的鸡尾酒主白细胞亚群可以如下表征驻地小神经胶质细胞; 喜 CD45 +细胞CD11b阮文黎-6G +:中性粒细胞; 喜 CD45 +细胞CD11b阮文黎-6G -阮文黎-6C 喜 :促炎单核细胞。 </li></ol></li></ol></li></ol>

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The authors declare that they have no competing financial interests.

这里介绍的脑缺血模型给出高重现性梗死体积测定24-48小时，MCA结扎用不同的方法8,15,17后7天。此MCAO模型具有低死亡率（小于1％）相比，给他人，最小化在研究中使用的动物数量。获得死亡率率如此之低的一个关键步骤是保持适当的无菌条件，以避免感染诱导中风后可能损害生存。这个MCAO模型不仅可以用作永久性MCAO模型，这被认为是对转化研究18临床相关的模式，但也可作为由CCA和MCA的瞬态结扎一个过渡模型与在期望的时间的活结和后再灌注19。此方法已被成功地用于在小鼠和大鼠17,20。所有这些证据表明，缺血通过结扎脑缺血与多个应用程序的高通用的型号。一个criti这种技术的校准步骤是，它需要在立体显微镜下微创手术;开颅应执行非常小心，以不损坏颧骨以及MCA。然而，（不进行它们经受外科手术，但CCA和MCA结扎）利用假动物提供了有用的工具，以鉴别手术过程依赖效应。脑损伤的以下这种技术的范围内可通过几种方法来量化。我们的脑切片，Nissl染色和由卡瓦列体积的随后估计的协议允许对受损区域的精确量化，最大限度地减少在这种类型的研究中所使用的串行自脑切片也可以用于不同的免疫组织化学分析的小鼠的数目。对于这种方法的一个更好的性能，这是至关重要的，以选择在体视软件中使用的适当的参数（ 表1），这将是必要的，用于估计第Ë体积的不同区域的由式:V = 1 / SSF * A F * T *ΣPI（SSF是片抽样比，t为各部分的平均厚度，f是网格间距的面积和ΣP点撞击结构的数量）。 远端MCAO通过连接可以是有用的表征渗透白细胞和免疫细胞亚群8,13参与脑损伤后1-4的炎症过程。在这里，我们提出了两种不同的方法对脑免疫细胞特性，精确的体视学方法和流式细胞仪分析表征好多个白细胞亚群。 趁着串行脑切片，嗜中性粒细胞的总数的定量可通过光学分馏方法16，其估计的细胞的总数量从细胞取样机智的数量来实现HA系统随机抽样（SRS）一套公正的虚拟计算空间覆盖感兴趣的整个区域，在我们的例子中梗死区域，不带偏见的虚拟计算空间之间的距离均匀的方向的X，Y和Z.该方法提供了一个准确的工具结扎后估计在不同的时间在局部缺血性脑总嗜中性粒细胞数。虽然它没有被在本研究中所示，该协议也可以被局部缺血21之后以及在像其他浸润白细胞的局部缺血性脑发现的任何其他细胞群的准确定量用于估计不同的中性粒细胞亚群（单核细胞/巨噬细胞）而且估计存活的神经元，甚至中风后神经量化。为准确估计在所期望的区域中的最重要的步骤是适当的参数的选择，如图所示的那些表3中用于在缺血区的中性粒细胞的定量。这些参数将被用于在体视软件通过使用等式N =ΣQ-×1 / SSF×1 / ASF×1 / TSF（ΣQ-是细胞计数的总数来计算总阳性细胞数目（N）的分馏，社保基金的部分采样率，ASF是采样率区域，TSF是抽样比厚度）5。虽然这种方法比其它量化方法要慢（例如，中性粒细胞标记物通过组织，代表图像或每场的中性粒细胞的数量的密度的毫克分析），它具有的优点是无偏和固体技术，该技术提供了精确的定量细胞数。 大脑白细胞隔离方法允许同时识别和几种免疫细胞亚型的定量，而不需要偏压系统通过体内染色或遗传操作。随后的细胞从其特征髓P分拣opulations或它们的免疫磁性分离，可用于多种下游应用，如基因或蛋白质表达的进一步研究。的嗜中性粒细胞，单核细胞和小胶质细胞用这种方法获得的精确表征提供高特异性相对于现有的方法，如免疫组织化学研究中，一个优点是，允许分配特定的功能，以不同的骨髓细胞介导脑先天免疫应答。此外，它可以进一步扩展到使用相应的标签表征其他脑种群，如血液出生的巨噬细胞（细胞CD11b + CD45 您好 CD68 +），并且其可以应用于用于其它CNS病症或损伤的研究。因此，该技术提供了一种重要的工具，以探索在脑炎症反应的异质性。这种技术的主要局限性在于从新鲜的脑组织的白细胞悬浮液的制备方法，它可以改变所述细胞或它们的抗原变更激活状态。尽管该技术允许一个更详细的定性表征相比免疫组化研究，它提供了基于细胞的分离较不准确的定量。尽管这样，我们的细胞分离协议的效率类似于其他发表的方法9，它可以有效地用于检测甚至经受不同处理8 MCAO组之间的控制和缺血组之间或脑的免疫细胞的数目的差异。 关键步骤这个协议是组织解剖，组织破坏过程，以及髓磷脂的去除。关于组织采集，归一化步骤可以包括（通过称重收集的组织），以避免变性，由于不同解剖表演。此外，还可发生梗死体积不同的缺血组关系正常化（此前磁R所确定esonance）。另一种方式来解决这个问题的方法是使用两个缺血组和对照组的整个同侧半球，或者甚至使用缺血小鼠的对侧半球作为对照，以尽量减少使用的动物的数量。虽然这种方法避免了每个夹层之间的差别，它有一个主要的缺点;稀释因子​​由增加的细胞的总数，但尚未髓细胞是专门设在同侧皮层的芯和梗死周围区域的数量增加。关于组织破坏，该协议说明的步骤对脑细胞的机械破碎，避免酶处理和防止表面抗原蚀变9,22，对于炎性细胞亚群的进一步定性和定量分析的重要问题。另外所关注的细胞悬浮液的制备，从脑样品髓鞘去除是高度推荐的步骤，以避免与downst干扰令的应用，如免疫细胞分离或流式细胞术23,24。这可以用不同的方法，如蔗糖或珀梯度，或抗髓鞘珠来实现。在这里，并根据该比较用的Percoll使用不同的方法对脑细胞悬浮液分离，机械破碎组合以除去髓鞘用于提高细胞产量和存活率25以前的研究。

形态，表型和基因表达的小胶质细胞的改变，以及外渗和激活血源性巨噬细胞和嗜中性粒细胞被认为脑损伤后1-4中的病理生理级联中发挥关键作用。这个协议提供了两种方法来估计局部缺血大脑的不同髓样细胞亚群的数量和执行他们的表型表征。此外，它也提供了脑缺血的小鼠实验模型，它由两个远侧大脑中动脉的暂时或永久的结扎（MCA）和颈总动脉（CCA）的说明，其中包括如何评估梗塞通过使用体视软件精确卡瓦列利方法。 表征缺血脑的骨髓细胞亚群的第一种方法是基于光学分馏方法5一个视学方法。这是最常用的体视探头在生命科学，并提供一个高的精确度与误差5-7的低系数。这是为小区量化的最佳选择，当组织被切段，因为它避免了由于组织成段的碎片对细胞估计偏置。这种方法是一种非常强大的方式来描述数字，动态和缺血组织8的中性粒细胞浸润的亚表型的改变。 第二表征方法是基于从康帕内拉和合作者9的改性协议，它提供了一个简单的方法来隔离脑白细胞悬浮液并通过流式细胞术来表征它们。与传统的免疫组织化学技术，流式细胞术表征允许小胶质细胞之间进行区分（CD45 LO的CD11b +）和渗透基于它们的diff髓样细胞（CD45 您好的CD11b +）CD45 9-13 erent表达水平。另外，促炎性单核细胞（CD45 您好的CD11b +莱曼6G -莱曼6C 喜 ），嗜中性粒细胞（CD45 您好的CD11b +莱曼6G +）和缺血组织的其它白细胞子集能够被区分。这种方法提供了一个可靠和快速检测，以评估脑缺血实验模型神经炎症。然而，组织处理可以影响的激活状态，并在局部缺血组织提供半定量表征发现不同细胞群的编号。

<table><tbody><tr><td>Photonic Led F1</td><td>WPI</td><td>571329-8</td><td>Equipment</td></tr><tr><td>Temp Controller&amp;nbsp;</td><td>Panlab&amp;nbsp;</td><td>HB 101/2</td><td>Equipment</td></tr><tr><td>Volvere GX</td><td>NSK</td><td>Ne22L</td><td>Equipment</td></tr><tr><td>Microscope</td><td>WPI</td><td>PZMIII-BS</td><td>Equipment</td></tr><tr><td>Stainless Steel Burrs</td><td>FST</td><td>19007-14</td><td>Surgical material</td></tr><tr><td>Forceps Dumont #5/45&amp;nbsp;</td><td>FST</td><td>11251-35</td><td>Surgical material</td></tr><tr><td>Forceps Dumont #5SF</td><td>FST</td><td>11252-30</td><td>Surgical material</td></tr><tr><td>Suture 6/0</td><td>LorcaMarin</td><td>55108</td><td>Surgical material</td></tr><tr><td>Suture 9/0</td><td>LorcaMarin</td><td>61966</td><td>Surgical material</td></tr><tr><td>Nikon Eclipse&amp;nbsp;</td><td>Nikon</td><td>TE300</td><td>Equipment</td></tr><tr><td>Isoflurane</td><td>Esteve</td><td>571329-8</td><td>Chemical</td></tr><tr><td>Sodium Pentobarbital</td><td>Vetoquinol</td><td>570681</td><td>Chemical</td></tr><tr><td>Freezing microtome</td><td>Leica Microsystems GmbH</td><td>SM2000R</td><td>Equipment</td></tr><tr><td>Superfrost slides</td><td>Thermo Scientific&amp;nbsp;</td><td>2014-07</td><td>Lab material</td></tr><tr><td>Cresyl violet acetate</td><td>Sigma</td><td>C5042</td><td>Chemical</td></tr><tr><td>Microscope&amp;nbsp;</td><td>Nikon</td><td>Nikon Eclipse TE300</td><td>Equipment</td></tr><tr><td>XYZ motorized computer stage and controller</td><td>Ludl electronics Products</td><td></td><td>Equipment</td></tr><tr><td>Stereo Investigator System</td><td>Microbrightfield</td><td>Version 7.003 software</td><td>Software</td></tr><tr><td>5 ml Tissue Grinder, Potter-Elv with teflon pestle</td><td>Thomas Scientific</td><td>0913X70</td><td>Lab Material</td></tr><tr><td>Polypropylene 50 ml Oak Ridge Centrifuge Tube</td><td>Nalgene</td><td>3119-0050&amp;nbsp;</td><td>Lab material</td></tr><tr><td>Percoll</td><td>Sigma</td><td>p1644-100</td><td>Chemical</td></tr><tr><td>RPMI 1640</td><td>Lonza</td><td>BE12-702F</td><td>Chemical</td></tr><tr><td>Beckman Ultracentrifugue</td><td>Beckman Coulter</td><td></td><td>Equipment</td></tr><tr><td>Beckman Coulter ultracentrifuge rotor 45 Ti</td><td>Beckman Coulter</td><td></td><td>Equipment</td></tr><tr><td>BD Sterile Cell Strainer, 40 Micron</td><td>BD</td><td>BD 352340</td><td>Lab Material</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A3733-500G</td><td>Reagment</td></tr><tr><td>FcR Blocking Reagent, mouse</td><td>Miltenyi</td><td>130-092-575</td><td>Antibody</td></tr><tr><td>CD11b-FITC, human and mouse</td><td>Miltenyi</td><td>130-098-085</td><td>Antibody</td></tr><tr><td>CD45-PE, mouse</td><td>Miltenyi</td><td>130-102-596</td><td>Antibody</td></tr><tr><td>Anti-Ly-6G-APC, mouse</td><td>Miltenyi</td><td>130-102-936</td><td>Antibody</td></tr><tr><td>PerCP/Cy5.5 anti-mouse Ly-6C</td><td>Biolegend</td><td>128012</td><td>Antibody</td></tr><tr><td>BD FACS Flow</td><td>BD</td><td>342003</td><td>Reagment</td></tr><tr><td>BD FACSCalibur; 4-color</td><td>BD</td><td>342975</td><td>Equipment</td></tr><tr><td>BD Cell Quest Pro Software</td><td>BD</td><td></td><td>Software</td></tr><tr><td>FlowJo software</td><td>Treestar inc.</td><td></td><td>Software</td></tr></tbody></table>

stereological and flow cytometry characterization of leukocyte subpopulations in models of transient or permanent cerebral ischemia

小胶质细胞的活化，以及血源性巨噬细胞和嗜中性粒细胞的外渗，被认为中风后发挥脑损伤的关键作用。这些骨髓细胞亚群可显示不同的表型和功能，需要加以区别和特点，研究他们的监管和贡献的组织损伤。该协议提供了两种不同的方法对脑免疫细胞特性:精确的体视学方法和流式细胞仪分析。的体视的方法是基于在光学分馏方法，其计算在感兴趣（梗塞脑）的区域的细胞的总数量由系统随机抽样估计。第二表征方法提供了一种简单的方法来隔离脑白细胞悬浮液并通过流式细胞术来表征它们，允许小胶质细胞的表征，渗入的单核细胞和缺血组织的嗜中性粒细胞。此外，它也可为details在小鼠脑缺血模型，只影响大脑皮质，产生高度可重复的梗塞与死亡率的低速率，并且过程进行组织学大脑处理由卡瓦列方法来表征梗死体积。

此处示出的脑缺血模型生成只在皮质梗塞看出在不影响纹状体组织自MCA的lenticulostriatal分支灌溉纹状体不遮挡（ 图1）。通过Nissl染色，受损区域可以被确定为一个低色素皮层区域（ 图2）。这个模型是在24小时MCAO（％IH 15.89±0.28）由卡瓦列方法在尼氏染色切片估计（ 图2）后其特征在于，高度可再现的梗死体积。梗塞体积由所述卡瓦列方法的估计是具有低误差被反映在误差（CE）的贡诺森在对侧和ipsilesional半球的系数，以及在梗死区（ 表2）的准确的方法。受损组织体积可以表示为mm 3，但也可作为使用式中的部分3.2.13％IH。此外，估计所述ipsilesional和对侧区域的总体积的允许水肿指数的计算来校正梗死体积，并避免损伤组织（ 表2）的过高估计。 给定的脑组织的连续切片加工，这是可以被利用来执行细胞亚群的总数目的准确估计，如渗透中性粒细胞，在缺血区域中，使用光学分馏方法（图3和表4）与表3所示的参数，该协议估计嗜中性粒细胞（Ly6G阳性细胞）中的23328±3623梗塞面积的总数在24小时和82856±8143在48小时的小鼠pMCAO（ 图3D）之后。在与先前的研究一致，中性粒细胞浸润的直接相关的梗死面积（ 图3E）。吨估算他由光学分馏方法号嗜中性粒细胞的是具有低的错误是受贡诺森（ 表4）的CE反射的准确的方法。 白细胞分离和流式细胞术表征协议允许47922±23174骨髓细胞的缺血的小鼠的ipsilesional半球的皮质的隔离。这包括在细胞悬浮液中发现的总的事件的10-30％。绝大多数使用此协议本低的FSC参数，相关联的细胞碎片（ 图4）捕捉到的事件。的CD11b染色表明的CD11b +细胞具有更高的FSC值（ 图4），这表明细胞碎片不标记该标记物，如前面所指出的，这可以通过设置阈值的FSC在200 9被排除在进一步分析。细胞碎片用这种方法得到的可变的量表明的差异上样过程ING（定时，组织保护，样品的温度，高效髓鞘去除等 ）可占它。此外，使用细胞过滤的，还需要避免在样品中的细胞夹具的存在;此步骤需要事先对细胞染色来完成。使用在图5中这是基于CD11b和CD45的表达中所示的门控策略，我们可以驻留和浸润骨髓细胞之间在缺血组织区别。当与幼稚和与假手术组，其中，这些细胞大多关联到的CD45的低表达指示小胶质细胞是缺血后增殖（ 图4，图5和表5）相比，此细胞CD11b +群体的增加在缺血半球。这种差异可能是由于从外周细胞浸润，这证明在缺血脑一个的CD11b + CD45 您好细胞亚群的出现（ 图4图5和表5），这是天真和虚伪的大脑低不少。浸润到的CD11b +细胞群脑缺血的贡献是一个非常动态的过程4。在MCAO模型由结扎，它可以有所不同，从30％到视时的表征已经完成时的病灶的尺寸和总的CD11b +细胞的60％。嗜中性粒细胞，其特点为阮文黎-6G +细胞是在24小时发现缺血后用脑缺血的这种模式的缺血性小鼠大脑中最多的渗入细胞群体，因为他们构成的CD11b + CD45的70-80％ 喜 。细胞其余大多是细胞CD11b + CD45 喜阮文黎-6G亚群-阮文黎-6C 喜促炎单核细胞。在这种模式下，该群体将在数在缺血脑区增加从24至MCAO之后48小时。 图1:手术过程的MCA结扎（A）颞肌的收缩后，一个小开颅在鼠标头骨进行。 MCA是一个结，或用9/0缝线活结结扎。（B）由缺血模型生成的皮质病变的代表形象。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52031/52031fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52031/52031fig2.jpg" /> 图2:梗死体积由卡瓦列里的定量。 （A）的尼氏染色脑切片代表图像永久性MCA结扎后24小时。高倍率显示尼氏染色后hipocromatic面积标识受损区域（核心）MCAO后。梗死周围（PI）的区域也被示出。（B）中通过使用卡瓦列方法在串行尼氏染色切片，24小时MCAO后（N = 50只小鼠表示为％梗死半球（％1H）梗死体积的定量）。 <img alt="图3" src="/files/ftp_upload/52031/52031fig3.jpg" /> 图3:在梗死区24中性粒细胞和结扎后48小时的体视学定量分析，使用光学分馏方法。在缺血区中性粒细胞浸润（Ly6G阳性细胞）在缺血后24小时（A）代表的形象。划界显示梗塞区（B，C）的光学解剖为嗜中性粒细胞的定量由光学分馏的实例。（D）的总嗜中性粒细胞在梗塞区域的定量在24和​​48小时（N = 4只小鼠）。（E）的嗜中性粒细胞的梗死面积在24和​​MCA结扎后48小时数的相关性。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52031/52031fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52031/52031fig4.jpg" /> 图4:脑白细胞，天真，假缺血（MCAO 24小时后）半球的物理参数（SSC和FSC）的基础上获得的代表点图散布分析事件表达的CD11b（红色）一个群体被确定在所有组。这一人群根据CD45的表达门控和特征。细胞表达CD45（绿色）的低水平存在于缺血性和非缺血性脑皮质和对应于小胶质细胞。与此相反，细胞表达高水平的CD45（黄色）只在缺血半球和假手术组程度较轻发现。 CD11b表达CD45 +喜人口的进一步分析表明，嗜中性粒细胞（LY-6G +细胞，蓝色）和促炎单核细胞（LY-6C 喜细胞，橙）在大脑中发现的主要细胞亚群浸润中风后24小时， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52031/52031fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52031/52031fig5.jpg" /> 图5:门控策略来区分居民从浸润骨髓细胞在缺血组织中的CD11b +细胞首先根据同种型荧光强度（A）的门控。缺血脑的细胞悬浮液的代表性点图显示在面板的右上方角落。在另外，使用这种技术获得的事件的总数和为细胞CD11b +事件的数量的典型值是示出（无细胞的/局部缺血性脑半球;（A）中的CD11b +细胞的CD45的荧光强度分析显示在面板B.一个代表性的点图分析的门控的CD11b +亚群的CD45和CD11b表达的显示在面板的向上右上角。另外，使用这种技术的每缺血性脑半球获得的CD45 LO CD45 您好细胞的典型数示出（B）中 。 <table border="0" cellpadding="0" cellspacing="0"><colgroup><col span="2" /></colgroup><tbody><tr><td colspan="2"> 用于卡瓦列方法参数 </td></tr><tr><td>部分厚度（T） </td><td> 30微米</td></tr><tr><td>目标</td><td> 10X </td></tr><tr><td>切片取样率（SSF） </td><td> 1/ 20 </td></tr><tr><td>部分之间的距离</td><td> 600微米</td></tr><tr><td>栅格间距</td><td> 100微米</td></tr></tbody></table> 表1:用于梗死组织的体视学定量参数。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 结果 </td><td> 对侧 </td><td> Ipsilesional </td><td> 梗塞 </td></tr><tr><td>面积（平方微米） </td><td> 151460000 </td><td> 155060000 </td><td> 22600000 </td></tr><tr><td>体积（μm³） </td><td> 908.76亿</td><td> 930.36亿</td><td> 135.6亿</td></tr><tr><td>量的修正 在投影（μm³） </td><td> 89957100000 </td><td> 92046300000 </td><td> &lt;STRONG&gt; 13416600000 </td></tr><tr><td>误差系数（冈德森） m = 0的</td><td> 0.068 </td><td> 0.077 </td><td> 0.067 </td></tr><tr><td>误差系数（冈德森） 米= 1 </td><td> 0.015 </td><td> 0.017 </td><td> 0.015 </td></tr><tr><td>误差系数（冈德森） α（Q） </td><td> 0.068 </td><td> 0.077 </td><td> 0.067 </td></tr><tr><td colspan="2"> ％半球梗死 </td><td colspan="2"> 14.90 </td></tr><tr><td colspan="2"> 脑水肿（IPS卷/续卷） </td><td colspan="2"> 1.02 </td></tr></tbody></table> 表2:对侧，ipsilesional和梗死体积通过使用立体声研究者软件的卡瓦列方法的代表性实例。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td col跨度="2"&gt; 用于光学分馏参数 </td></tr><tr><td>部分厚度（TSF） </td><td> 30微米</td></tr><tr><td>目标</td><td> 100X </td></tr><tr><td>切片取样率（SSF） </td><td> 1/10 </td></tr><tr><td>算盘高度</td><td> 40微米</td></tr><tr><td>算盘宽度</td><td> 40微米</td></tr><tr><td> X网格尺寸</td><td> 230微米</td></tr><tr><td> X网格尺寸</td><td> 230微米</td></tr><tr><td>安全防范</td><td> 2微米</td></tr><tr><td>光学Disector高度</td><td> 14微米</td></tr></tbody></table> 表3:通过使用立体声研究者软件用于渗透的中性粒细胞的脑缺血与光学精馏塔探测后的体视量化参数。 <table border="0" cellpadding="0" cellspaCING ="0"&gt; <tbody><tr><td colspan="2"> 中性粒细胞的光学分馏估算 </td></tr><tr><td>采样站点数量</td><td> 430 </td></tr><tr><td>形状因子</td><td> 6.24 </td></tr><tr><td>总标记计数</td><td> 166 </td></tr><tr><td>预计中性粒细胞通过光学分馏</td><td> 117,608.03 </td></tr><tr><td>误差的系数（龚德森），m = 0的</td><td> 0.22 </td></tr><tr><td>误差的系数（龚德森）时，m = 1 </td><td> 0.09 </td></tr></tbody></table> 表4:脑缺血与光学分馏探测后估计渗透的中性粒细胞通过使用立体声研究者软件代表例子。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> 的CD11b + </td><td> 嗜中性粒细胞 </td><td> 单核细胞 </td><td> 小胶质细胞 </td></tr><tr><td> 天真 </td><td> 25863±4,575.8 </td><td> 473±75.8 </td><td> 525±191.4 </td><td> 19012±1523 </td></tr><tr><td> 深水24小时 </td><td> 24563±5263 </td><td> 873±192.5 </td><td> 1124±391.5 </td><td> 23734±2,910 </td></tr><tr><td> pMCAO 24小时 </td><td> 47922±23174 </td><td> 4874±748.7 </td><td> 4826±1345 </td><td> 35395±10833 </td></tr></tbody></table> 表5:估计骨髓细胞的脑缺血术方法的流程后，代表性的结果。

Watch this Scientific Journal Video about Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia at JoVE.com

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

脑的骨髓细胞的表征下列行程可以通过体视用光学分馏方法来进行，或者通过大脑的流式细胞仪分析白细胞悬浮液。两者都是有用的技术来执行，在缺血性脑中发现的主要的骨髓细胞亚群的准确的表型区分。

白细胞亚群在暂时或永久的脑缺血模型体视学和流式细胞仪鉴定

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

stereological-flow-cytometry-characterization-leukocyte

Nanyang Technological University

Research

JoVE Journal

Medicine

15.5K 次观看.  Universidad Complutense de Madrid y Instituto de Investigación Hospital 12 de Octubre, Madrid. 脑的骨髓细胞的表征下列行程可以通过体视用光学分馏方法来进行，或者通过大脑的流式细胞仪分析白细胞悬浮液。两者都是有用的技术来执行，在缺血性脑中发现的主要的骨髓细胞亚群的准确的表型区分。 

视频: Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

After brain stroke microglia/macrophages (M/M) undergo rapid activation with dramatic morphological and phenotypic changes that include expression of novel surface antigens and production of mediators that build up and maintain the inflammatory response. Emerging evidence indicates that M/M are highly plastic cells that can assume classic pro-inflammatory (M1) or alternative anti-inflammatory (M2) activation after acute brain injury. However a complete characterization of M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is still missing.	
	Immunofluorescence protocols specifically staining relevant markers of M/M activation can be performed in the ischemic brain. Here we present immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate the pattern of localization and co-expression of M/M phenotype markers such as CD11b, CD68, Ym1, in mouse model of focal ischemia induced by permanent occlusion of the middle cerebral artery (pMCAO). Two-dimensional analysis of the stained area reveals that each marker is associated to a defined M/M morphology and has a given localization in the ischemic lesion. Patterns of M/M phenotype marker co-expression can be assessed by three-dimensional confocal imaging in the ischemic area. Images can be acquired over a defined volume (10 &mu;m z-axis and a 0.23 &mu;m step size, corresponding to a 180 x 135 x 10 &mu;m volume) with a sequential scanning mode to minimize bleed-through effects and avoid wavelength overlapping. Images are then processed to obtain three-dimensional renderings by means of Imaris software. Solid view of three dimensional renderings allows the definition of marker expression in clusters of cells. We show that M/M have the ability to differentiate towards a multitude of phenotypes, depending on the location in the lesion site and time after injury.

A way to gain new insights into the complexity of the brain inflammatory response is presented. We describe immunofluorescence-based protocols followed by three-dimensional confocal analysis to investigate the pattern of co-expression of microglia/macrophage phenotype markers in a mouse model of focal ischemia.

偏振的实验性脑损伤小胶质细胞/巨噬细胞标志物的三维共聚焦分析

After brain stroke microglia/macrophages (M/M) undergo  rapid activation with dramatic morphological and phenotypic changes that  include expression of ...

科研

神经科学

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

免疫与感染

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

医学

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

基于短暂性脑缺氧缺血性血栓性中风模型

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

通过流式细胞术分析来自中枢神经系统的小胶质细胞和单核细胞衍生的巨噬细胞

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

横向慢性颅窗口准备启用<em&gt;在体内</em&gt;观察继末节小鼠大脑中动脉闭塞

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

对大脑中动脉闭塞再灌注损伤诱发中风小鼠的手术方法

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be altered by severe brain injury. This is evidenced by the fact that pneumonia is a major cause of mortality in patients who have suffered from ischemic stroke. The goal of this protocol is to describe the use of multicolor flow cytometric analysis to identify 13 types of immune cells in the lungs of mice, including alveolar macrophages, interstitial macrophages, CD103+ or CD11b+ dendritic cells (DCs), plasmacytoid DCs, eosinophils, monocytes/monocyte-derived cells, neutrophils, lymphoid-derived&#160; T and B cells, NK cells, and NKT cells, following ischemic stroke induction by transient middle cerebral artery occlusion. Moreover, we describe the preparation of lung homogenates using a bead homogenization method, to determine the expression levels of 13 different cytokines or chemokines simultaneously by multiplex bead arrays coupled with flow cytometric analysis. This protocol can also be used to investigate the pulmonary immune response in other disease settings, such as infectious lung disease or allergic disease.

This protocol describes the use of flow cytometry to identify the changes in immune cell composition, cytokine profile, and chemokine profile in the pulmonary environment following transient middle cerebral artery occlusion, a murine model of ischemic stroke.

肺环境中免疫细胞和促炎介质的表征

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be ...

从小鼠脑和颅骨骨髓中获得单细胞悬液

Isolating Immune Cells from Mouse Brain and Skull

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only in the brain, but also in the skull. A key step toward analyzing changes in immune cell populations in both the brain and skull bone marrow after brain damage (e.g., stroke) is to obtain sufficient numbers of high-quality immune cells for downstream analyses. Here, two optimized protocols are provided for isolating immune cells from the brain and skull bone marrow. The advantages of both protocols are reflected in their simplicity, speed, and efficacy in yielding a large quantity of viable immune cells. These cells may be suitable for a range of downstream applications, such as cell sorting, flow cytometry, and transcriptomic analysis. To demonstrate the effectiveness of the protocols, immunophenotyping experiments were performed on stroke brains and normal brain skull bone marrow using flow cytometry analysis, and the results aligned with findings from published studies.

作者聚焦：研究脑缺血后的复杂免疫反应

To investigate the immune response to brain disorders, one common approach is to analyze changes in immune cells. Here, two simple and effective protocols are provided for isolating immune cells from murine brain tissue and skull bone marrow.

从小鼠大脑和颅骨中分离免疫细胞

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only ...