Stefano Fumagalli is a fellow of the Monzino Foundation.

1。免疫荧光下面的协议是从心脏灌注小鼠（20毫升的PBS，0.1摩尔/升，pH为7.4，随后加入50毫升PBS中冷却的多聚甲醛中4％）中获得冠状脑冷冻切片进行。灌注后，大脑小心移除，并转移到30％蔗糖的PBS中于4℃过夜冷冻保护。 45℃下进行3分钟被密封到小瓶中并储存在-70℃直至使用前 - 大脑，然后迅速通过浸没在冷冻中的异戊烷。冠状脑冷冻切片（20微米）被连续切割，并进行免疫荧光协议11，6。 将所有试剂和样品至室温后方可使用。请注意，抗体和血清中所呈现的工作稀释液造成的，以获得最佳的性能做试验。当不同的抗体和血清的使用，该协议需要被验证。 <p级=“jove_content”&gt;请注意，因为两者的抗CD11b和抗CD68一抗大鼠制成，由抗大鼠的Alexa 546，我们使用了高稀释的抗CD11b的接着用荧光信号放大避免交叉信号全港性系统评估套件（Cy5的酪胺）。我们已经成立了由执行只是步骤1.17所描述的协议，并使用至少7种不同的抗CD11b的稀释液作为记录在表1的最佳工作稀释度的抗CD11b。其结果是，1:30,000已被选定作为唯一稀释实现：1）可见信号用Cy5在激发波长为646纳米; 2）没有用Alexa 546信号在激发波长为532纳米。以这种方式，Alexa的546荧光信号有选择地与CD68表达相关。 最佳稀释度为抗CD11b和抗CD68抗体可能取决于组织的类型而变化，并且必须开始共同的标签协议事先定义。 <ol><li>用PBS洗两次脑冰冻切片。 </li><li>在cubate冷冻切片5分钟，在PBS中含1％H 2 O 2（必需的，因为在荧光扩增需要孵化与辣根过氧化物酶，链霉抗-HRP步骤，参见步骤1.12）。 </li><li>洗涤冷冻切片2倍的PBS。 </li><li>孵育冷冻切片在PBS中含有10％NGS和0.3％的Triton 60分钟。 </li><li>在PBS中含有初级抗体鼠抗CD11b [1:30,000]，10％NGS和0.3％的Triton孵育冷冻切片在4℃下过夜。 </li><li>洗涤冷冻切片2×（5分钟），用PBS。 </li><li>冰冻切片孵育在PBS中含有生物素标记的二抗鼠抗体[1:200]和1％NGS 60分钟。 </li><li>洗涤冷冻切片2倍的PBS。 </li><li>与TNT（：0.1M的Tris-HCl，pH值7.4,0.15 M氯化钠，0.05％吐温20的Tris-HCl，氯化钠，吐温）洗涤冷冻切片。 </li><li>孵育冷冻切片1.5小时在TNB（Tris-盐酸 - 氯化钠 - 封闭缓冲液：0.1M Tris-盐酸，pH值7.4,0.15 M氯化钠，从适当的试剂盒的0.5％封闭试剂（见表试剂））。 </li><li>洗冷冻部3X倍与TNT。 </li><li>孵育含链霉素-HRP [1:100]中冰冻切片TNB。 </li><li>洗3倍冰冻切片与TNT。 </li><li>冰冻切片孵育含有花青5酪胺[1:300]中扩增稀释8分钟。 </li><li>洗涤冷冻切片3倍的PBS。 </li><li>孵育冷冻切片在PBS中含有10％NGS，0.1％的Triton 60分钟。 </li><li>在PBS中含有初级抗体鼠抗CD68 [1:200]，3％NGS和0.3％的Triton孵育冷冻切片在4℃下过夜。 </li><li>洗涤冷冻切片3倍的PBS。 </li><li>冰冻切片孵育在PBS中含有fluorconjugated二抗Alexa的546抗鼠[1:500]和1％NGS 60分钟。 </li><li>洗涤冷冻切片3倍的PBS。 </li><li>孵育冷冻切片含有Hoechst的1微克/毫升的PBS 10分钟。 </li><li>洗涤冷冻切片3倍的PBS。 </li><li>在延长黄金装冰冻切片。 </li></ol> 2。收购三维图像激光共聚焦显微镜<ol><li>选择激励激光器取决于荧光染料被激发的波长（氦 - 氖红为Cy5标记，细胞CD11b;氦氖绿色Alexa546，CD68和紫外二极管为Hoechst公司，核）。 </li><li>选择最佳的分色镜组合的光信号采集。 </li><li>设置图像分辨率最低为800 x 600像素。 </li><li>用落射荧光和逐步增加放大倍率为40倍物镜识别感兴趣的区域。 </li><li>切换到激光扫描显微镜（LSM）的模式。 </li><li>运行重复扫描来调节光电倍增管（PMT）和每个通道的增益。激光器可以逐个打开，以方便套起来。保持增益尽可能低，以避免不必要的非特异性信号。 </li><li>与REPEtitive扫描运行时，将焦点移动控制来定义z轴（总z轴的长度= 10微米）的下部和上部极端。 </li><li>停止重复扫描。 </li><li>定义步长。它应该是尽可能地接近，以像素大小（0.225微米），以获得一个必须是标准1:1尺寸比例在z轴。 </li><li>激活卡尔曼滤波器的至少2倍。 </li><li>启动顺序扫描模式，以避免流血通的效果。 </li><li>去中途沿z轴和运行的xy顺序扫描。检查设置了光电倍增管和增益（良好的信号/噪声比）。如果不理想，请重复步骤2.6。 </li><li>运行XYZ收购。 </li><li>将数据导出为multitiff文件。每个multitiff文件通常包含3个颜色通道（蓝色，绿色和红色）和44的焦平面。 </li></ol> 3。共焦收购和三维渲染的三维视图上传multitiff文件Imaris里的软件，并将它们处理如下： <ol><li>打开软件。 </li><li>选择超越看法。 </li><li>上传multitiff文件。 </li><li>为每个通道选择所需的颜色。 </li><li>通过增加显示调整面板上的最小值从背景中消除噪音。单独调整每个通道。 </li><li>去剖面视图。移动沿z轴寻找的共定位（黄色像素）。 </li><li>点击一个黄色的区域，以查看是否共定位为沿z轴（ 即属于一个固体物体）存在。 Z轴凸起是可见的图中的右侧和底部。 </li><li>拍摄快照。 </li><li>回去超越看法。 </li><li>裁剪三维分离的细胞或细胞群。 </li><li>选择显示调节面板上的一个通道。 </li><li>选择超越/面算法生成器。算法步骤： <ol><li>选择通道。 </li><li>定义阈值（使用相同的最小值在T他显示调节面板）。 </li><li>定义调整大小。 </li><li>定义平滑（最好= 0.200）。 </li><li>定义颜色的外观。 </li><li>结束算法。 </li></ol></li><li>每个通道重复步骤3.12。 </li><li>取消选择荧光通道显示调节面板，并选择所有的三个面 。 </li><li>移动音量，以找到最好的景色。 </li><li>拍摄快照。 </li></ol>

<ol>
	<li>Davalos, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+mediates+rapid+microglial+response+to+local+brain+injury+in+vivo.">ATP mediates rapid microglial response to local brain injury in vivo.</a> Nat. Neurosci. 8, 752-758 (2005).</li><li>Yenari, M. A., Kauppinen, T. M., Swanson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+activation+in+stroke:+therapeutic+targets.">Microglial activation in stroke: therapeutic targets.</a> Neurotherapeutics. 7, 378-391 (2010).</li><li>Iadecola, C., Anrather, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunology+of+stroke:+from+mechanisms+to+translation.">The immunology of stroke: from mechanisms to translation.</a> Nat. Med. 17, 796-808 (2011).</li><li>Jin, R., Yang, G., Li, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+mechanisms+in+ischemic+stroke:+role+of+inflammatory+cells.">Inflammatory mechanisms in ischemic stroke: role of inflammatory cells.</a> J. Leukoc. Biol. 87, 779-789 (2010).</li><li>Schilling, M., Besselmann, M., Muller, M., Strecker, J. K., Ringelstein, E. B., Kiefer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predominant+phagocytic+activity+of+resident+microglia+over+hematogenous+macrophages+following+transient+focal+cerebral+ischemia:+an+investigation+using+green+fluorescent+protein+transgenic+bone+marrow+chimeric+mice.">Predominant phagocytic activity of resident microglia over hematogenous macrophages following transient focal cerebral ischemia: an investigation using green fluorescent protein transgenic bone marrow chimeric mice.</a> Exp. Neurol. 196, 290-297 (2005).</li><li>Perego, C., Fumagalli, S., De Simoni, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+pattern+of+expression+and+colocalization+of+microglia/macrophage+phenotype+markers+following+brain+ischemic+injury+in+mice.">Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice.</a> Journal of Neuroinflammation. 8, 174-193 (2011).</li><li>Zanier, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+umbilical+cord+blood+mesenchymal+stem+cells+protect+mice+brain+after+trauma.">Human umbilical cord blood mesenchymal stem cells protect mice brain after trauma.</a> Crit. CareMed. 39 (11), 2501-2510 (2011).</li><li>Capone, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosphere+derived+cells+exert+a+neuroprotective+action+by+changing+the+ischemic+microenvironment.">Neurosphere derived cells exert a neuroprotective action by changing the ischemic microenvironment.</a> PLoS ONE. 2, e373 (2007).</li><li>Bhatia, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+host+defense+against+Aspergillus+fumigatus+involves+alveolar+macrophages+with+a+predominance+ofalternatively+activated+phenotype.">Rapid host defense against Aspergillus fumigatus involves alveolar macrophages with a predominance ofalternatively activated phenotype.</a> PLoS One. 6, e15943 (2011).</li><li>Raes, G., Noel, W., Beschin, A., Brys, L., de Baetselier, P., Hassanzadeh, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FIZZ1+and+Ym+as+tools+to+discriminate+between+differentially+activated+macrophages.">FIZZ1 and Ym as tools to discriminate between differentially activated macrophages.</a> Dev. Immunol. 9, 151-159 (2002).</li><li>Gesuete, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+C1+inhibitor+in+brain+ischemic+injury.">Recombinant C1 inhibitor in brain ischemic injury.</a> Ann. Neurol. 66, 332-342 (2009).</li><li>Sica, A., Mantovani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+plasticity+and+polarization:+in+vivo+veritas.">Macrophages plasticity and polarization: in vivo veritas.</a> J. Clin. Invest. 122 (3), 787-795 (2012).</li></ol>

作者什么都没有透露。

我们在座的免疫为基础的协议，其次是三维共聚焦分析作为一个强大的方法，调查国产化的M / M型标志物和共表达到缺血区域（更详细的分析见文献6）。此方法结合了M / M活化与三维共焦成像相关的标记物的特异性染色。抗体，血清和fluorconjugated工作稀释度的微调提供了最佳的信号被调查指标的信噪比。图像处理以获得三维渲染允许在细胞群标志物表达的定义，使得所获取的图像表示的M /...

急性脑损伤，小胶质细胞迅速激活并发生剧烈的形态和表型的变化1-3。这种内在的反应相关联招聘血液出生的巨噬细胞迁移到损伤脑实质4,5。小胶质细胞和巨噬细胞的抗原性是无法区分的作用（以下称为M / M）在脑损伤仍有争议。越来越多的研究表明，与什么所述外围巨噬细胞，小胶质细胞和脑招募巨噬细胞可以假定不同的表型，其极端对应于经典的促炎性毒性（M1）或抗炎保护（M2）的表型。在不同的激活状态，包括亲或抗炎因子分泌，神经营养分子和溶酶体活性释放的特征的表型标志物的特定图案，其表达依赖于时间周围环境的演变。这些M / M表型在脑损伤的特征仍然是一知半解。我们使用PMCAO的一套行之有效的小鼠模型来分析的M / M表达和进化中风后。这里提出了一种基于免疫的协议旨在让洞察特定的M / M型标志，其本地化和细胞共同表达在缺血区的外观。我们研究了相关的不同的激活状态，或表型的分子数，即细胞CD11b，白细胞和M / M激活/招聘6-8的一种广泛使用的标记表示的表面标志物CD68溶酶体6,7和的Ym1分泌的标志蛋白替代性活化（M2）巨噬细胞表达和相关的回收和功能恢复9-10。 当两个标记都表达了相同的细胞，但在不同亚细胞区室，独自一人共定位可能不会有太大的INFormative。在这种情况下，可以通过使用单一的平面视图和三维效果进行共表达的分析。在这里，我们描述了一个协议来获取标记共表达的透彻三维分析。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		 </tr>
		 <tr>
			<td>Rat Anti-mouse CD11b</td>
			<td>Kindly provided by Dr. A. Doni, Istituto Clinico Humanitas, Milan, Italy</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rat Anti-mouse CD68 </td>
			<td>AbD Serotec</td>
			<td>MCA 1957</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rabbit Anti-mouse Ym1 </td>
			<td>Stem Cell Technologies</td>
			<td>1404</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Hoechst 33342</td>
			<td>Life technologies</td>
			<td>H21492</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mouse Anti-Neural Nuclei (NeuN)</td>
			<td>CHEMICON</td>
			<td>MAB377</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Biotinilated Goat Anti-Rat antibody</td>
			<td>Jackson Immuno Research</td>
			<td>112-065-143</td>
			<td></td>
		 </tr>
		 <tr>
			<td>TSA Cyanine 5 System </td>
			<td>Perkin Elmer</td>
			<td>NEL705A001KT</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Prolong Gold </td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti-rat alexa 546</td>
			<td>Invitrogen</td>
			<td>A-11081</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti mouse Alexa 488</td>
			<td>Invitrogen</td>
			<td>A-21121</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti-rabbit Alexa 594</td>
			<td>Invitrogen</td>
			<td>A-11037</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Normal Goat Serum</td>
			<td>Vectors Laboratories</td>
			<td>S-1000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Tritin X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma</td>
			<td>P4417-100</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>Cryostat CM1850</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Olympus IX81 confocal microscope </td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>AnalySIS software</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Imaris software 5.0</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Photoshop cs2</td>
			<td>Adobe Systems</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software packages GraphPad Prism version 4.0</td>
			<td>GraphPad Software Inc.</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

three-dimensional confocal analysis of microglia/macrophage markers of polarization in experimental brain injury

脑卒中小胶质细胞/巨噬细胞后（M / M）进行快速活化，包括新颖的表面抗原和生产的建立和维持炎症反应介质的表达显着形态学和表型的改变。新的证据表明，M / M是高塑性细胞急性脑损伤后，可以假定经典的促炎症（M1）或替代消炎（M2）的激活。但是M / M型标志物表达，他们的共存和时间演化的脑损伤的完整的特征是人仍下落不明。 免疫荧光协议专门染色的M / M激活相关标志物可在缺血性脑进行。在这里，我们提出了免疫为基础的协议，其次是三维共聚焦分析，作为一个强大的方法，调查本地化和共表达M / M型标志，如图案细胞CD11b，CD68，的Ym1，在小鼠局灶性脑缺血引起的大脑中动脉（PMCAO）的永久性闭塞模型。污渍区域的二维分析表明，每个标记是关联到一个定义的M / M的形态，并具有一个给定的定位中的缺血性损伤。 M / M的表型标记物的共表达模式可以通过在局部缺血区域的三维共聚焦成像来评估。图像可以在一个确定的容积被收购（10μm的z轴和一个0.23微米的步长大小，对应于180×135×10微米的体积）与连续扫描模式，以尽量减少渗色的效果，并避免波长重叠。图像，然后处理由软件Imaris里的手段来获得三维渲染。三维效果图实视图允许在集群细胞标志物表达的定义。我们表明，M / M具有分化向多个表型的能力，这取决于在病变部位和Ti的位置我受伤后。

当标记协议和共焦的采集进行入缺血区得到的结果的一个例子示于图1A和1B。获得的图像的二维视图显示，在缺血后24小时（A），溶酶体标记CD68（绿色）表示肥厚ameboid CD11b的细胞（红色）出现在缺血中心。在边境区（B）的CD11b阳性细胞显示圆形胞体和积极的CD68分枝过程。 Z轴投影（A和B的右侧和底部）允许的记录是否在?...

Watch this Scientific Journal Video about Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury at JoVE.com

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

一种方式来获得新的见解的大脑炎症反应的复杂性呈现。我们描述了免疫为基础的协议，其次是三维共聚焦分析，以探讨局灶性脑缺血小鼠模型共表达小胶质细胞/巨噬细胞表型标志物的图案。

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 ...

three-dimensional-confocal-analysis-microgliamacrophage-markers

Research

JoVE Journal

Neuroscience

11.5K 次观看.  IRCCS - Istituto di Ricerche Farmacologiche Mario Negri. 一种方式来获得新的见解的大脑炎症反应的复杂性呈现。我们描述了免疫为基础的协议，其次是三维共聚焦分析，以探讨局灶性脑缺血小鼠模型共表达小胶质细胞/巨噬细胞表型标志物的图案。 

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

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

科研

神经科学

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

协议星形胶质细胞的三维共聚焦形态分析

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

人 Intraepidermal 神经纤维内线粒体三维成像及分析

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

慢性压迫性损伤小鼠步态的自动分析

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

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 ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

常见马莫塞的全皮质电皮质阵列的慢性植入

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

脑结构三维形状建模与分析

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional deficits. Numerous studies by our group and others have shown that mTBI stimulates cytokine expression and activates microglia, decreases cerebral blood flow and metabolism, and impairs cerebrovascular reactivity. Moreover, several works have reported an association between derangements in these neuroinflammatory and hemodynamic markers and cognitive impairments. Herein we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI in mice. Specifically, we describe how to perform a weight-drop model of mTBI, how to longitudinally measure cerebral blood flow using a non-invasive optical technique called diffuse correlation spectroscopy, and how to perform a Luminex multiplexed immunoassay on brain tissue samples to quantify cytokines and immunomodulatory phospho-proteins (e.g., within the MAPK and NF&#954;B pathways) that respond to and regulate activity of microglia and other neural immune cells. Finally, we detail how to integrate these data using a multivariate systems analysis approach to understand the relationships between all of these variables. Understanding the relationships between these physiologic and molecular variables will ultimately enable us to identify mechanisms responsible for mTBI.

This protocol presents methods to characterize the neuroinflammatory and hemodynamic response to mild traumatic brain injury and to integrate these data as part of a multivariate systems analysis using partial least squares regression.

创伤性脑损伤的神经炎症和血流动力学反应的系统分析

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional ...

Three-Dimensional Motor Nerve Organoid Generation

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

三维运动神经器官生成

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and ...