The authors have nothing to disclose.

这项研究进行了按照批准的医学科学，伊朗大学（德黑兰，伊朗）伦理委员会提出的指南护理和使用实验动物（NIH）的政策。 1.动物，手术和标本准备注：准备脑组织的三维共聚焦显微镜分析。 <ol><li>分割动物随机分为两组：Aβ1-40 -注射（N = 8），和Aβ1-40 -注射用染料木黄酮处理（N = 8）;手术前施用染料木素（1​​0mg / kg的稀释在车辆中，如聚乙氧基化蓖麻油）灌胃1小时。 </li><li>麻醉动物用氯胺酮（100毫克/千克）和赛拉嗪（10毫克/千克）。捏脚趾后，确认正确的麻醉缺乏撤退反射的。在手术过程中使用软膏的眼睛，以防止干燥。 </li><li>将动物立体定位仪和剃的头。应用IODINE溶液清洗切口前的头皮和手术过程中保持操作现场无菌，以减少感染的风险。海马注射Aβ1-40立体定向（4微升）在-3.5毫米后到前囟，±2毫米横向中线和-2.8毫米以下硬脑膜，根据大鼠脑图谱8。对于立体定位方法请参见上一公布9。 </li><li>提供术后观察/护理，直至动物充分意识到，以确保动物的舒适性。保持动物温暖的恢复过程中，不把它们返回到与其他动物的笼子，直到完全康复。手术后，在动物的注意任何感染的迹象。 </li><li>氯胺酮（150毫克/千克）手术三周后深麻醉动物。通过灌注用0.9％生理盐水动物其次是4％多聚甲醛的0.1M PBS（pH值= 7.4）执行穿心固定，然后取出并在10所描述修复大脑&lt;/ SUP&gt;。 </li><li>在4℃后固定在4％多聚甲醛的大脑2-3天。 </li><li>嵌入石蜡块组织通过使用组织灌封设备，以及避免下填充或过度填充盒，因为它可能与正确对准或切片干扰。注意在石蜡块中的组织的方向。 注：大鼠海马，例如，靠近大脑半球的后部。因此，组织应该嵌入的方式，切片开始于脑的后部。使用Paxinos地图集8为指导，以达到所期望的解剖结构。 </li><li>将切片机远离空气气流或门道，因为空气流动做出艰难的部分的处理。 </li><li>使用部分的厚度≥20微米，因为这与深度是必要的，以产生完整的星形胶质细胞的Z-堆栈的图像。不要使用第一对夫妇的部分，因为他们可能有不想要的厚度由于热ēxpansion。 </li><li>放置部分温水（5±2 C，低于熔化温度为石蜡）刚好足够长的平坦化部分的表面上。 注：在膨胀部分的会干扰结构的形态。用微小的画笔仔细地转移部分。收集二点五十八每张幻灯片上的部分。 </li><li>在直立位置的齿条商店滑动并让它们在37℃干燥几个小时或O / N。 </li></ol> 2，免疫组化<ol><li> Deparaffinize切片在二甲苯中，2×10分钟，和再水合通过醇梯度（99.8％，95％，70％，每10分钟）和PBS。 </li><li>孵育抗胶质纤维酸性蛋白（GFAP）稀释1：1500于PBS含0.25％牛血清白蛋白（BSA），0.25％的Triton X-100和3.5％正常血清（O / N，在4℃）。 </li><li>在PBS中进行3×5分钟洗涤部分。 </li><li>孵育次级碱性磷酸酶共轭antibodieS表示1小时（1：100，21 C，在PBS中稀释）。 </li><li>在PBS洗的章节3×5分钟。 注：为二级抗体后，用正确方法洗手的部分，以减少背景染色是很重要的。 </li><li>孵育在黑暗的部分与液体永久红显色（15-20分钟）。注意：使用前准备的解决方案不超过30分钟。 </li><li>在PBS中进行3×5分钟洗涤部分。 </li><li>最后，染色4-6二脒基-2-苯基吲哚的核（DAPI; 1：500，在PBS中稀释）之前以覆盖打滑。镜前存放在冰箱24-48小时的幻灯片。 </li></ol> 3.共聚焦显微镜注：对于定量评价（见下文），选择一个星形胶质细胞具有清晰可见DAPI染色细胞核以最小的重叠枝，以减少错误的风险。生产的Z-Stack图像非常耗时。要有耐心，并且在加工过程中不停止。立式激光共聚焦显微镜为used将星形胶质细胞创建3D图像。 <ol><li>放置在显微镜阶段滑动，并选择63X物镜。 </li><li>打开成像软件后点击Start系统。点击定位标签。去分配，并选择GFP来调整合适的光。 </li><li>打开快门，以反射光。找到反射左轮手枪右侧的快门，并选择应该由星形胶质细胞（绿色，红色或紫色）被反射的颜色;红色（FSet20 WF）在这里被使用。 注意：不要忘记在显微镜的目镜直接聚焦的画面。 </li><li>为了找到最佳的焦点，点击全封闭，放在屏幕模式显微镜针，然后单击采集卡上。 </li><li>点击采集卡在智能安装程序;打开一个窗口。从列表中选择合适的荧光团（ 即 DAPI和Alexa Fluor 555，在当前项目中）。颜色是自动选择。 </li><li>点击最佳的信号，并应用，然后点击设置曝光;计算机将自动设置曝光参数。 </li><li>为了优化图像，进入通道窗口。取消标记TRACK2，突出Track1和点击直播。如果需要的话聚焦影像。 </li><li>在通道窗口调整下面的键;点击1AU自动优化针孔。这是很重要的是要做到这一步，否则共焦图象将是最优的。调整增益有最好的强度（保持低于800此设置，以减少额外的噪声）。最后，设置数字增益2和3之间。 </li><li>取消标记TRACK1，点击TRACK2并重复步骤3.8 TRACK2。然后停止直播。 </li><li>进入采集模式窗口。提高图像通过优化帧大小和平均。为了改变框的大小去X * Y，并选择1024×1024。选择一个缓慢的速度来创造一个更好的形象。 </li><li>去平均，然后选择一个数≥4。这个平均值表示将被平均，以全国生产的扫描数Ë获得的图像。现在点击捕捉按钮，保存拍摄的2D图像。 </li><li>对于3D影像，在智能设置标记的Z-Stack项目造成的Z-Stack窗口中打开。 </li><li>设置的Z堆叠的第一和最后位置，即细胞的深度。首先点击直播。然后用显微镜的聚焦驱动集中星形胶质细胞在组织中的最上位置，其中的Z-Stack是开始。点击组第一。然后向下集中在组织，其中Z-Stack的扫描，应停止星形胶质细胞的最低位置。单击设置最后。然后停止直播。 </li><li>选择时间间隔; 1.01微米用在目前的研究;选择的时间间隔可以通过点击最优设置的片数来进行。 </li><li>点击开始实验。保存图像，一旦扫描完成。这个图像将被用于下列参数的测量：表面积和星形胶质细胞领土体积，整个胶质细胞包括胸罩nches，细胞体，和细胞核。 </li><li>获得使用10X或20X物镜按照步骤3.6-3.11所述2D图像。这个图像可以被用于测量抗体的荧光免疫反应性，和星形胶质细胞密度（个/ 1000微米2）的强度。 </li></ol> 4，星形胶质细胞密度和胶质纤维酸性蛋白+荧光强度<ol><li>打开20X 2D图像。在图像窗口左侧点击组织相容（Hisogram）。 </li><li>为了计算星形胶质细胞的密度，选择在20X目标三连冠的视野。算显示出一个清晰的体躯具有最小，重叠在田里分支的星形胶质细胞的数量。 </li><li>量化荧光强度中，选择使图像窗口的页脚一个合适的工具（矩形或圆形），并选择的区域进行测定。注意该图像下的平均值和标准偏差自动出现。在目前的研究中，在0.7毫米2的STR之间的矩形区域iatum和叶片内侧齿状回用。 </li></ol> 5.星形胶质细胞分行<ol><li>量化分支的长度和总数目，使用中的图像解析软件3D图像（具有20X获得）。沿着选定的星形胶质细胞的每个分支的长度手工画一条线。然后选择测量工具专门用于测量线的长度。为了量化分支的数量，选择计数工具计数标注的项目数。 </li></ol> 6.体积和表面积注：在3D图像分析软件手动标记的结构，需要重点和培训。在开始实验前练习多次。 <ol><li>量化星形细胞细胞核，躯体，单元主体和领土的体积和表面积，3D图像（在步骤3.1-3.16收购）转移到一个3D图像分析软件如Volocity 6.1。 </li><li>打开软件，并创建一个新的图书馆。将所有图像向左灰色列中的新库。然后，选择一个3D图像。在右边，有不同的信道用不同的颜色。打开一个感兴趣信道;例如测量细胞核时，蓝色通道的DAPI。手动更改亮度，以获得最佳的对比度。 </li><li>选择工具菜单，去进行音量，并产生一个单一的3D图像了Z堆栈图像。点击属性，在编辑菜单，可以肯定的是X，Y和Z属性显示;否则是不可能进行三维分析。 </li><li>点击工具栏上的手绘RIO工具图标。为了测量星形胶质细胞核的体积和表面积周围画DAPI标记的核的线。 </li><li>为了测量星形胶质细胞体的体积和表面积周围绘制胞体不含分支的线。 </li><li>为了测量整个星形胶质细胞（细胞体含分支机构）的体积和表面积周围画苏摩下面的一行各分公司的表面。 </li><li>为了评估星形胶质细胞领土的体积和表面积画树枝的顶端之间的一条线。 注：按键盘上的Delete删除不必要的图纸。 </li><li>点击在图像窗口创建测量协议的顶部测量菜单上;打开包含不同的工具的窗口。 </li><li>拖动查找对象到窗口（窗格）的顶部。举一个描述性的名称，人口1，选择相关的颜色，然后点击测量。打开另一个窗口。 </li><li>选择一个参数，即 ，体积或表面积。单击确定。该数据将显示在表格中。 </li><li>保存测量菜单上的数据单击并选择使测量项目。 </li><li>从打开的窗口中被称为一个新的测量项目。然后给一个名称的数据，然后点击确定。 </li><li>最后，出口数据统计软件如图形垫或Excel程序。使用t检验来分析数据，然后绘制2D克APHS。 </li></ol>

<ol>
	<li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60 (3), 430-440 (2008).</li><li>Pellerin, L., 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=Activity-dependent+regulation+of+energy+metabolism+by+astrocytes:+an+update.">Activity-dependent regulation of energy metabolism by astrocytes: an update.</a> Glia. 55 (12), 1251-1262 (2007).</li><li>Sofroniew, M. V., Vinters, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes:+biology+and+pathology.">Astrocytes: biology and pathology.</a> Acta Neuropathol. 119 (1), 7-35 (2010).</li><li>Pekny, M., Nilsson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+activation+and+reactive+gliosis.">Astrocyte activation and reactive gliosis.</a> Glia. 50 (4), 427-434 (2005).</li><li>Sofroniew, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+dissection+of+reactive+astrogliosis+and+glial+scar+formation.">Molecular dissection of reactive astrogliosis and glial scar formation.</a> Trends Neurosci. 32 (12), 638-647 (2009).</li><li>Anderova, M., 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=Cell+death/proliferation+and+alterations+in+glial+morphology+contribute+to+changes+in+diffusivity+in+the+rat+hippocampus+after+hypoxia-ischemia.">Cell death/proliferation and alterations in glial morphology contribute to changes in diffusivity in the rat hippocampus after hypoxia-ischemia.</a> J Cereb Blood Flow Metab. 31 (3), 894-907 (2011).</li><li>Hatten, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+regulation+of+astroglial+morphology+and+proliferation+in+vitro.">Neuronal regulation of astroglial morphology and proliferation in vitro.</a> J Cell Biol. 100 (2), 384-396 (1985).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A stereotaxic atlas of the rat brain. , (1998).</li><li>Kirby, E. D., Jensen, K., Goosens, K. A., Kaufer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+surgery+for+excitotoxic+lesion+of+specific+brain+areas+in+the+adult+rat.">Stereotaxic surgery for excitotoxic lesion of specific brain areas in the adult rat.</a> J Vis Exp. (65), e4079 (2012).</li><li>Bagheri, M., 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=Amyloid+beta(1-40)-induced+astrogliosis+and+the+effect+of+genistein+treatment+in+rat:+a+three-dimensional+confocal+morphometric+and+proteomic+study.">Amyloid beta(1-40)-induced astrogliosis and the effect of genistein treatment in rat: a three-dimensional confocal morphometric and proteomic study.</a> PloS One. 8 (10), e76526 (2013).</li><li>Chvatal, A., Anderova, M., Kirchhoff, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+confocal+morphometry+-+a+new+approach+for+studying+dynamic+changes+in+cell+morphology+in+brain+slices.">Three-dimensional confocal morphometry - a new approach for studying dynamic changes in cell morphology in brain slices.</a> J Anat. 210 (6), 671-683 (2007).</li><li>Kulkarni, P. M., 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=Quantitative+3-D+analysis+of+GFAP+labeled+astrocytes+from+fluorescence+confocal+images.">Quantitative 3-D analysis of GFAP labeled astrocytes from fluorescence confocal images.</a> J Neuroscie Methods. 15 (246), 38-51 (2015).</li><li>Wagner, D. 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=Object-based+analysis+of+astroglial+reaction+and+astrocyte+subtype+morphology+after+ischemic+brain+injury.">Object-based analysis of astroglial reaction and astrocyte subtype morphology after ischemic brain injury.</a> Acta Neurobiol Exp. 73 (1), 79-87 (2013).</li></ol>

这项工作是由郡议会东约特兰省的，（瑞典）和细胞分子研究中心授予在医学大学伊朗（德黑兰，伊朗）的支持。

在目前的协议，我们采用三维激光共聚焦形态来评估与星形胶质细胞形态进行了相关的12个不​​同的参数。为了这个目的，大鼠Aβ1-40的海马组织-诱导的星形胶质细胞增生，有或没有染料木素预处理作为抗炎剂被使用。通过使用3D图像和形态的软件，我们能够表明染料木黄酮对星形胶质细胞增生，即星形胶质细胞形态的影响。 变化的脑组织的细胞内和?...

在健康的中枢神经系统（CNS），星形胶质细胞发挥 ​​血流量，能量代谢，突触功能和可塑性，和细胞外离子的调节中起重要作用的神经递质和动态平衡1-3。此外，星形胶质细胞，以不同的有害刺激和异常时，例如通过反应性星形胶质细胞增生其特点是肥大，增殖和星形胶质细胞4,5-功能重塑外伤，感染，局部缺血或神经变性响应。 反应性星形胶质可以设计在组织中的炎症反应和修复过程，因此，可能会影响治疗性干预的临床结果。因此，星形细胞已经在过去几十年，作为治疗性干预的各种影响CNS疾病的潜在目标接收到的关注神经学家。 星形胶质细胞通常有一个星状的形状，以及-Defined分支中传遍了SOMA 6。在大脑中的疾病状况，星形分支成为旋绕和显示肿胀的端部7中，例如在淀粉样蛋白β（Aβ）的存在。 本文介绍了协议分析由共聚焦显微镜获得的星形胶质细胞的三维图像。测量了每个星形细胞十二个不同的量化参数：所述表面区域和星形胶质细胞的领土（覆盖有星形细胞的组织），整个细胞（包括分支），细胞体，和细胞核的体积;的总长度和分支的数目;用于星形细胞抗体检测的荧光强度;和星形胶质细胞的密度（个/ 1000微米2）。为了这个目的，我们使用脑切片从大鼠暴露于海马内注射Aβ1-40的含或不含染料木黄酮处理作为抗炎物质。所描述的协议可以用于形态的alysis 在体外或体内在不同条件下的不同细胞类型。

<table border="0">
	<tbody>
		<tr>
			<td>Amyloid beta 1-40</td>
			<td>Sigma Aldrich</td>
			<td>79793</td>
			<td>Keep in -70 &#176;C</td>
		</tr>
		<tr>
			<td>Genistein</td>
			<td>Sigma Aldrich</td>
			<td>446-72-0</td>
			<td>keep in -20 &#176;C</td>
		</tr>
		<tr>
			<td>polycolonal rabbit antibodies against glial fibrillary acidic protein</td>
			<td>DAKO</td>
			<td>Z0334</td>
			<td></td>
		</tr>
		<tr>
			<td>alkaline phosphate-conjugated swine anti-rabbit IgG antibodies</td>
			<td>DAKO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Permanent Chromogen</td>
			<td>DAKO</td>
			<td>K0640</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid permanent Red Substrate Buffer</td>
			<td>DAKO</td>
			<td>K0640</td>
			<td></td>
		</tr>
		<tr>
			<td>Cremophor EL</td>
			<td>Sigma Aldrich</td>
			<td>27963</td>
			<td>Polyethoxylated castor oil - Step 1.1</td>
		</tr>
		<tr>
			<td>LSM 700 Confocal Laser Scanning Microscopy</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Volocity 6.3</td>
			<td>Perkin Elmer Inc.,</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image Analysis 2000</td>
			<td>Tekno Optic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Streotaxic apparatus</td>
			<td>Stoelting</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Graph pad Prism 5</td>
			<td>Graph pad software Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

protocol for three-dimensional confocal morphometric analysis of astrocytes

如神经胶质细胞在大脑中，星形胶质细胞在中枢神经系统中多样化的功能角色。中的有害刺激的存在下，星形细胞修改他们的功能和结构性质，一个称为反应性星形胶质细胞增生的条件。这里，一个协议的星形胶质细胞的形态性质的评估呈现。这个协议包括12个不同参数，即表面积和所覆盖的星形胶质细胞（星形胶质细胞境）的组织，在整个星形胶质细胞，包括分支，细胞体，和细胞核，体积定量分支以及总长度和数量，强度用于星形胶质细胞的检测抗体的荧光免疫反应性，和星形胶质细胞密度（个/ 1000微米2）。为了这个目的所创建的三维（3D）的共焦显微镜图像和3D图像分析软件如Volocity 6.3用于测量。大鼠脑组织暴露于淀粉样蛋白1-40 </子&gt;（Aβ1-40）具有或不具有治疗性干预是用于呈现方法。该协议也可以被用于其它细胞在体内或体外条件的三维形态测定分析。

本节介绍由三维形态分析产生的定性和定量的观测的一些例子。对于前面所提到的全部12个参数的完整结果，请参考我们以前出版10。 定性观察星形胶质细胞表现出薄或厚的分支中通常是长期的Aβ1-40注射的老鼠（ 图1）。几个小星芒状星形细胞与短支链，观察在大脑皮质，和星形胶质细胞的一种紧凑网络发生在胼胝体。 <p c...

Watch this Scientific Journal Video about Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes at JoVE.com

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

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 ...

protocol-for-three-dimensional-confocal-morphometric-analysis

Research

JoVE Journal

Neuroscience

8.7K 次观看.  Linköping University. 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.

视频: Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

视网膜节的形态分析

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

科研

神经科学

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

小鼠多巴胺和三维胶原基质检测纹状体外植体的解剖和文化

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

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 ...

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 ...

Denver Papillae Protocol for Objective Analysis of Fungiform Papillae

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure consistent scoring between scorers. This protocol builds off of a need that has arisen from the last two decades of taste research using FP as a proxy for taste pore density. FP density has historically been analyzed using Miller &#38; Reedy&#8217;s 1990 characterizations of their morphology: round, stained lighter, large, and elevated. In this work, the authors forewarned that stricter definitions of FP morphology needed to be outlined. Despite this call to action, follow up literature has been scarce, with most studies continuing to cite Miller &#38; Reedy&#8217;s original work. Consequently, FP density reports have been highly variable and, combined with small sample sizes, may contribute to the discrepant conclusions on the role of FP in taste sensitivity. The Genetics of Taste Lab explored this apparent inconsistency in counting and found that scorers were individually prioritizing the importance of these characteristics differently and had no guidance for when a papilla had some, but not all, of the reported qualities of FP. The result of this subjectivity is highly variable FP counts of the same tongue image. The Denver Papillae Protocol has been developed to remedy this consequence through use of a dichotomous key that further defines and prioritizes the importance of the characteristics put forth by Miller &#38; Reedy. The proposed method could help create a standard way to quantify FP for researchers in the field of taste and nutritional studies.

Here we present a protocol to measure fungiform papilla density from digital photographs. This method builds prioritization and objective characteristic metrics into the original descriptive work on fungiform papillae by Miller &#38; Reedy (1990).

为菌状乳头的客观分析丹佛乳头协议

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure ...

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 ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

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 ...

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. ...

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

We describe procedures for processing newborn rat brain tissue to obtain high-resolution electron micrographs for morphometric analysis of synaptic vesicle distribution at nerve terminals. The micrographs obtained with these methods can also be used to study the morphology of a number of other cellular components and their dimensional structural relationships.