This work was supported in part by grants from the National Multiple Sclerosis Society (RG 5001-A-3 to S.J.C.), the National Institutes of Health (NS065808 to E.R.B.; NS078392 to S.J.C.), start-up funds from the UConn Health Center (to SJC) and the Kim Family Fund (UCHC in support of K.I.C.).

所有涉及动物的程序是按照人文关怀和使用实验动物的政策执行提出了实验动物福利办公室（NIH），只有从大学的机构动物照顾及使用委员会（IACUC）批准康涅狄格卫生服务中心。 1，制备混合胶质细胞培养<ol><li>消毒在使用前所有的仪器。加入3毫升含无阳离子的冰冷无菌Hank氏平衡盐溶液（HBSS）（Mg 2 +的或钙离子 ），以保持在冰上，以保持冷3无菌60毫米培养皿。 </li><li>隔离从出生后P0-P2小鼠幼仔的皮质，如前所述17,18。 注意:皮层下结构，如海马，不包括在这些培养物中。 </li><li>小心地取出脑膜，然后分离出皮层转移到新鲜的HBSS和游离组织酶，使用NE根据制造商的协议尤勒尔组织解剖工具包。 </li><li>板中的细胞在无菌的T-175烧瓶中，并在媒体孵育过夜[Dulbecco改良的Eagle培养基（DMEM）中，用10％胎牛血清（FBS）的含有1x青霉素/链霉素。 </li><li>第二天用新鲜培养基更换所有的媒体。继续培育文化，和普通媒体的变化，约3周，直到细胞单层成立。 注意:此单层将被包括星形胶质细胞和小胶质细胞与约10:1的比例19。 </li></ol> 2球细胞诱导混合胶质细胞培养<ol><li>准备ECM包被的盖玻片。 <ol><li>浸泡圆形盖玻片用在无菌培养皿中30分钟，无菌1N的盐酸（HCl）。用无菌水冲洗3次盖玻片，然后浸泡在95％EtOH中至少20分钟。然后让空气干燥。 </li><li>稀释外MATR的地方滴IX（ECM）的蛋白质涂层材料上的封口膜一张（层粘连蛋白10微克/ EG 150-250微升/滴毫升溶解在无菌磷酸盐缓冲液[PBS]）。分配液滴，使盖玻片不会碰的时候放在这些液滴。每个干盖玻片轻轻将上使用镊子液滴。 </li><li>留在液滴在盖玻片约。 4-6小时的培养罩的窗扇内关闭。 </li><li>将ECM包被的盖玻片（ECM涂覆的一面朝上），在每个24孔板的孔中。每次用无菌PBS 3X以及洗净，直到需要保持在4℃。 </li></ol></li><li>准备胶质细胞镀上盖玻片。 <ol><li>从胶质细胞培养物吸媒体，轻轻地用10ml无菌PBS洗胶质细胞单层3倍。接着，加入8-10毫升胰蛋白酶-EDTA溶液与T-175，并孵育5-10分钟，在37℃。 </li><li>当大多数的单层已经分离，加入20 ml完全传媒到该烧瓶中，以停止胰蛋白酶消化。收集含脱落细胞的培养基到50毫升锥形管中并旋转10分钟，在300 XG在室温下。 </li><li>吸出上清液，而不会干扰沉淀，然后轻轻吹打使用P1000吸管的溶液重悬所述细胞于2ml的新鲜完全培养基。 </li><li>确定使用血球细胞的数量，然后重新悬浮的细胞在完全培养基中在所希望的密度（ 例如，10 5个细胞/ ml）。板中的细胞上的细胞外基质包被的盖玻片（ 例如，500微升到24孔板的每孔）。添加额外的新鲜完全培养基中，以每孔3-5天的潜伏期，在37℃，或直至细胞生长至所需的视觉汇合。 </li></ol></li><li>治疗Psychosine。 <ol><li>每瓶psychosine A股浓度（108.3毫米）的二甲基亚砜（DMSO）。稀释此股票在100％乙醇作工作股票可以在n为加入到24孔板各孔中以达到10μM的最终浓度。 </li><li>在加入psychosine的时间，轻轻摇动培养板以混合psychosine到培养基中。通过加入隔日10μM的等效一周补充psychosine。 </li></ol></li><li>经过7天psychosine处理，收集媒体和吸管500微升冷的4％多聚甲醛（PFA）到每口井的。在室温下孵育20分钟，固定细胞。然后，洗涤​​各孔3次，用PBS，然后保持板在4℃下直到处理用于免疫细胞化学（见下文）。 注:细胞培养基可以被收集为酶促或酶联免疫吸附测定法在这一点上，如果需要的。 </li></ol> 3，免疫细胞化学（ICC）的可视化弧面细胞<ol><li>更换PBS以封闭缓冲液[PBS + 0.3％Tween-20的±5％正常山羊血清（NGS），并孵育细胞1小时，于室温下。 </li><li>在cubate固定细胞在初级抗血清伊巴-1 [（1:1,000）稀释在PBS +0.3％Tween-20的±2％NGS]为至少1小时，在室温下，然后用PBS 3×5分钟，洗每个孔/洗。 </li><li>添加适当的次级荧光缀合的抗体来识别第一抗体标记和反染色用4&#39;，6 - 二脒-2 - 苯基吲哚（DAPI，1:1000），以确定细胞核。孵育在二次抗体溶液1小时，在室温下，并用PBS中3×5分钟。 </li><li>挂载每个玻璃罩，以显微镜使用的安装介质的幻灯片。查看幻灯片上的荧光显微镜的视觉和定量分析。 </li></ol> 4，分析和弧面细胞的原代培养的表征<ol><li>使用以下三种形态的标准来确定文化球细胞: <ol><li>确定使用荧光显微镜球状细胞免疫阳性标记的小胶质细胞的标记，IBA-1 +。 </li><li>确定弧面细胞作为polynucleated（ 即含有两种或两种以上的DAPI阳性细胞核）。 </li><li>由一个圆形的形态，从静止小胶质细胞的高度支化的外观独特的识别球状细胞。 注:细胞也可通过流式细胞术从这些培养物中收集到的细胞表面标记物的分析和定量分析。 </li></ol></li><li>测量小胶质细胞和弧面细胞的吞噬活性<ol><li>评估psychosine处理的小胶质细胞的吞噬活性，按照制造商的说明添加FITC标记的胶乳珠的孔中。固定之前用PFA添加荧光标记的乳胶珠48小时。修复珠处理（如上文第2.4节）细胞和流程免疫细胞（如第3节所述）。 </li><li>选择荧光基团标记的第二抗体，使它们不重叠的荧​​光团标记的珠粒的荧光发射光谱。 </li><li> Analyzë伊巴-1 +小胶质细胞通过识别那些细胞的吞噬型材共定位与荧光标记的珠子。 注:Psychosine将激活小胶质细胞。吞噬的小珠将单核和多核伊巴-1 +细胞在此培养环境内识别。 </li></ol></li></ol> 5，感应距离纯化小胶质文化弧面细胞注:另一种方法来破译星形胶质细胞和小胶质细胞之间的细胞间信号是在体外球细胞模型的另一个优势。初级小胶质细胞为replating或共同培养的纯化可以通过降低粘附性的培养来实现，如下面的步骤（参见第5.2节）中描述。 <ol><li>收集从集流混合胶质细胞培养介质到50ml锥形管中，并于37℃培养箱中保持它。 </li><li>小胶质细胞的分离，从初级混合胶质细胞培养。<ol><li>从汇合混合胶质细胞培养T175烧瓶中除去培养基后，加入温水摇动媒体[组成的完全培养基+的25mM 4-（2 - 羟乙基）-1 - 哌嗪乙磺酸（HEPES）+的25mM碳酸氢钠（Sigma公司）]。摇动烧瓶3-4小时，在轨道摇床上以100rpm（37℃）。收集这些摇瓶媒体到50ml锥形管。 注:该媒体将包括从烧瓶少贴小胶质细胞。 </li><li>旋转介质，在300×g离心10分钟，在室温下进行。吸出上清液，然后重悬浮细胞于2ml的星形胶质细胞条件培养基（在步骤5.1收集）。 </li><li>建立使用血细胞计数器，然后板的小神经胶质细胞上衬底涂覆的盖玻片上获得的细胞数量（参见第2.1节），用于免疫细胞化学，或镀到超低粘附孔板弧面细胞共培养与其他细胞类型的将来采集，如少突胶质细胞（如递减在下面的部分进项税）。 注意:一旦镀金的小胶质细胞可以与如上所述（见2.3节）psychosine（10微米）来治疗。 </li></ol></li><li>共培养基层少突胶质细胞的细胞毒性分析。 注:收集从低粘附片用于随后的共培养到少突胶质细胞的原代培养物，或者少突胶质细胞祖细胞psychosine处理的小胶质细胞，可用于检查这些弧面状细胞在体外的潜在细胞毒性作用。 <ol><li>开发利用已建立的方法18,20初级少突胶质细胞的培养。收集psychosine处理的小胶质细胞，轻轻地吸取到从孔的底部分离的松散的贴壁细胞。收集细胞，离心分离机，在300×g离心10分钟，然后小心地重新悬浮在少突胶质细胞分化培养基[的Neurobasal介质组成，L-谷氨酰胺（1倍），B-27补充剂（1×），和三碘甲状腺氨酸（T3中，10纳克/毫升）]。 </li><li>计数使用血球细胞的数量，然后播种的小胶质细胞上的少突胶质细胞培养在1×10 5个细胞/ ml，从而以近似1:2的小胶质细胞/少突胶质细胞的比例。孵育作为共培养3天。 </li><li>固定细胞的免疫细胞化学（见2.4节）。收集的细胞培养基用于ELISA或根据需要进行化学分析。 </li></ol></li></ol>

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The authors declare no conflicts of interest.

这里所描述的协议提供了一种新的模式系统，其中研究开发和激活小胶质细胞和球状细胞的功能特性。通过QQ，MSN 等前期工作。使用HEK293细胞系提供本协议的发展为球细胞形成21学习的模板。同样重要的是要指出，来自在该模型中的球状细胞来自原弧面细胞识别在GLD不同。举例来说，我们经常观察到小神经胶质细胞在我们的鼠文化quadranucleation，而球状细胞（GLD）经常被发现含有超?...

球细胞脑白质营养不良（GLD），也被称为克拉伯病，是一种致命的脱髓鞘疾病的功能突变的galatocerebrosidase（GALC）1基因造成的损失。政府物流服务署的最普遍的形式是由发生在儿童早期代表，往往前五年2,3岁特点是运动和认知能力下降，导致过早死亡的一个积极的临床过程中，婴儿的变体。基因检测是用来验证GLD 4的诊断。政府物流服务署的神经病理学发现广泛脱髓鞘，神经萎缩，星形胶质细胞增生和肿胀多有核小胶质细胞的存在被称为球状细胞5-7。弧面细胞的鉴定，常含在细胞质筒状包裹体，一直是政府物流服务署的定义特征，在过去97年虽然这些显眼细胞的特异功能一直难以实现。 非MYEL的参与GLD中的发病inating神经胶质细胞（小胶质细胞和星形胶质细胞），一直被认为是本病8到深刻的脱髓鞘二级响应。有趣的是，这种疾病的最初描述，在1916年5由克努兹克拉贝，包含已命名的脂质碎片多核巨噬细胞的报道形成"球状细胞&#39;并且是本病的决定性特征。 球状​​细胞病理学（GLD）的共同特征，虽然他们在政府物流服务署的角色长期以来被忽视了。有趣的是，这些细胞中，GLD的CNS组织中的最早的特性的变化。这种知识的缺乏可能是由于该多核巨噬细胞，称为巨核细胞中的其他疾病的形成中，通常认为是病理学的结果，而不是一个初始致病驱动力9的假设。因此，很少有研究调查了WHI机制CH弧面细胞是从吞噬细胞形成，特别是在政府物流服务署的中枢神经系统。本报告中所述的程序集中于球细胞的形成在CNS中的重要性和我们先前示范体外小胶质细胞以及这些细胞的那psychosine诱导的多核化表现出较高水平的细胞吞噬活性的。根据这些意见，球状细胞鸟类观察者的大脑经常含有PAS阳性的碎片，暗示高水平的吞噬活性。球状细胞也发现免疫阳性铁蛋白（一小胶质细胞标记物）10，KP-1 / CD68（单核细胞标记物），有的还积极为波形蛋白（一种中间丝蛋白，星形胶质细胞的标志物和激活的小胶质细胞），11， HLA-DRA（一个MHCII表面受体），和TNF-α7和IBA-112（用于识别的小胶质细胞钙结合蛋白）。基于标记这个集合，球状细胞从培养小胶质细胞起源独特的表型。 尽管他们的独特性，具体功能和选区于GLD发病的贡献已经在很大程度上被忽视。球状​​细胞已被认为是慢性脱髓鞘的次要结果。然而，过去的研究探讨球状细胞的时序关联到政府物流服务署的脑白质病变​​已经确定球状细胞在胚胎后期存在于出生后早期阶段;时间少突胶质细胞凋亡和公开脱髓鞘13前。因此，在GLD的神经病理学发展的时间顺序显示，球状细胞形成预先脱髓鞘在此疾病14。这导致我们在GLD早期形成球状的细胞可能代表一个决定性致病事件，而不是次要的，反应要少突胶质细胞受损15回应假设。此外，在政府物流服务署小胶质细胞活性的失调也被认为是事实上的R限流的hematopeotic干细胞疗法的远期疗效治疗本病16。因此，调查的细胞功能和小胶质细胞的调节，球状的细胞，以应对psychosine预计将提供在政府物流服务署的发病机制的新见解。 直到最近，缺乏在其中，研究球状细胞形成一个合适的模型已经限定的确切功能及这些细胞GLD的病理作用的了解。在最近的研究中，确定该弧面状细胞可被形成在直接响应psychosine，积聚在GLD病原脂质毒素。我们发现，小胶质细胞，但不巨噬细胞，被激活并转化成球状的细胞在初级胶质细胞培养响应于psychosine 15。该转化成球状的细胞被发现的胞外蛋白酶，基质金属蛋白酶（MMP）-3 15介导。最近，我们扩展了这些调查结果，并决定在此体外模型系统，开发出psychosine激活的小胶质细胞和球状细胞强效有毒的少突胶质细胞和少突胶质祖细胞。因此，当在GLD，psychosine和地层之前脱髓鞘将支持在该疾病的小胶质细胞的新兴初级和可能的致病作用弧面细胞的早期积累的上下文中考虑。 我们建议的球细胞形成的研究将揭示政府物流服务署的发病机制，这将有助于我们对这个疾病的认识新的信息。此外，政府物流服务署对这种新的细胞模型可提供一种新的形式从中新的治疗方法，以解决这种疾病的病理变化可以进行测试。因此，在本报告中，我们提供了体外发育非髓鞘神经胶质细胞的原代培养psychosine诱发球状细胞的详细协议。

<table><tbody><tr><td>Hank&amp;rsquo;s balanced salt solution (HBSS) containing no cations (Mg&lt;sup&gt;2+&lt;/sup&gt; and Ca&lt;sup&gt;2+&lt;/sup&gt;).</td><td>Life technologies</td><td>14175-095</td><td></td></tr><tr><td>Neural Tissue Dissociation Kit</td><td>Miltenyi</td><td>130-092-628</td><td></td></tr><tr><td>40 &amp;#956;m cell-strainer</td><td>Fisherbrand</td><td>22363547</td><td></td></tr><tr><td>Hank&amp;rsquo;s balanced salt solution (HBSS) containing cations (Mg&lt;sup&gt;2+&lt;/sup&gt; and Ca&lt;sup&gt;2+&lt;/sup&gt;).</td><td>Gibco</td><td>14025-092</td><td></td></tr><tr><td>Dulbecco&#039;s modified eagle medium (DMEM)</td><td>Gibco</td><td>11995-065</td><td></td></tr><tr><td>fetal bovine serum (FBS)</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>Penicilin/Streptomycin</td><td>Life technologies</td><td>15070-063</td><td></td></tr><tr><td>Laminin</td><td>Sigma</td><td>L2020</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Life technologies</td><td>25299-056</td><td></td></tr><tr><td>Psychosine</td><td>Sigma</td><td>P9256</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Electron Microscopy Science</td><td>19208</td><td></td></tr><tr><td>Normal Goat Serum (NGS)</td><td>Invitrogen</td><td>PCN5000</td><td></td></tr><tr><td>Iba-1</td><td>WAKO</td><td>019-19741</td><td></td></tr><tr><td>Alexa Fluor conjugated antisera</td><td>Life Technologies</td><td>Various</td><td></td></tr><tr><td>Mounting Media</td><td>Southern Biotech</td><td>OB100-01</td><td></td></tr><tr><td>Phagocytic Assay Kit</td><td>Cayman Chemicals</td><td>500290</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>BP310-500</td><td></td></tr></tbody></table>

an in vitro model for the study of cellular pathophysiology in globoid cell leukodystrophy

多成核的小神经胶质细胞的确切功能，称为球状的细胞，即是在球状细胞脑白质营养不良（GLD）的中枢神经系统中唯一地丰富目前还不清楚。在认识这个差距已经阻碍了缺乏适当的体外研究模型。在这里，我们描述了一个主要的鼠神经胶质培养体系中，治疗psychosine结果在小神经胶质细胞的多核类似GLD中发现的特征球状细胞。使用这个新系统，我们定义的条件和分析模式弧面细胞的研究。可能利用此模型系统在我们以前的研究，其中确定了基质金属蛋白酶（MMP）的潜在作用进行了验证-3 GLD。这种新颖的体外系统可以是其中以研究这些独特的细胞的形成和功能，而且还潜在治疗操纵，一个有用的模型。

这个协议，因为写的，预计需时约36天，从开始到结束（参见图1:实验工作流程计划）来完成。一直以来，我们经历了&#39;弧面状"细胞的这种原代培养体系的发展是既可靠和可重复性:多核细胞的响应psychosine的形成始终7天的治疗观察。 使用IBA-1在用核反染色结合将使识别的大小胶质细胞的免疫细胞化学染色，圆形多核细胞（ 图1b）。在某些情况下在这?...

Watch this Scientific Journal Video about An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy at JoVE.com

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

Globoid cells are a defining pathological feature of Krabbe disease, a leukodystrophy currently lacking an effective long-term therapy. We have developed a cell culture model to study the innate biology and pathogenic potential of activated microglia and their transformation into globoid cells.

一<em&gt;在体外</em&gt;模型细胞病理生理学的球细胞脑​​白质营养不良的研究

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell ...

an-vitro-model-for-study-cellular-pathophysiology-globoid-cell

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

7.8K 次观看.  University of Connecticut Health Center. Globoid cells are a defining pathological feature of Krabbe disease, a leukodystrophy currently lacking an effective long-term therapy. We have developed a cell culture model to study the innate biology and pathogenic potential of activated microglia and their transformation into globoid cells. 

视频: An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

生产和使用慢病毒的有选择性地转导原少突胶质前体细胞<em&gt;在体外</em&gt;髓鞘测定

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

科研

发育生物学

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

大鼠少突胶质祖文化的制备和使用双红外荧光扫描Oligodendrogenesis的量化

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

Derivation of Glial Restricted Precursors from E13 mice

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

从E13小鼠胶质限制前体的推导

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

神经科学

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

一个<em&gt;体外</em&gt;急性脑片的少突胶质细胞定向T细胞攻击模式

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

免疫与感染

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases.

In this protocol, we present the procedures in establishing myotonic dystrophy 1 myoblast models, including optimized C2C12 cell maintenance, gene transfection/transduction, and myocyte differentiation.

在C2C12肌细胞造型强直性肌营养不良1

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

医学

Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory neurons and glial satellite cells. These are useful and versatile models to investigate a variety of biological responses associated with physiological and pathological conditions of the peripheral nervous system (PNS) ranging from neuron-glial interaction, neuroplasticity, neuroinflammation, and viral infection. The usage of DRG explant is scientifically advantageous compared to simplistic single cells models for multiple reasons. For instance, as an organotypic culture, the DRG explant allows ex vivo transfer of an entire neuronal network including the extracellular microenvironment that play a significant role in all the neuronal and glial functions. Further, DRG explants can also be maintained ex vivo for several days and the culture conditions can be perturbed as desired. In addition, the harvested DRG can be further dissociated into an in vitro co-culture of primary sensory neurons and satellite glial cells to investigate neuronal-glial interaction, neuritogenesis, axonal cone interaction with the extracellular microenvironment, and more general, any aspect associated with the neuronal metabolism. Therefore, the DRG-explant system offers a great deal of flexibility to study a wide array of events related to biological, physiological, and pathological conditions in a cost-effective manner.

In this report, the advantages of organotypic cultures and dissociated primary cultures of mouse-derived dorsal root ganglia are highlighted to investigate a wide range of mechanisms associated with neuron-glial interaction, neuroplasticity, neuroinflammation, and response to viral infection.

成年小鼠背根节外植体和分离细胞模型调查可塑性和对包括病毒感染在内的环境侮辱的反应

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

在体内病态的人类基因组建模使用斑马鱼

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

生物学

Ex Vivo Myelination and Remyelination in Cerebellar Slice Cultures as a Quantitative Model for Developmental and Disease-Relevant Manipulations

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in vitro, although scalable,&#160;does not recapitulate axonal myelination. OPC-neuron cocultures and OPC-fiber cultures allow for the examination of in vitro myelination, but they lack additional cell types that are present in vivo, such as astrocytes and microglia. In vivo&#160;mouse models, however, are less amenable to chemical, environmental, and genetic manipulation and are much more labor intensive. Here, we describe&#160;an&#160;ex vivo mouse cerebellar slice culture (CSC) quantitative system that is useful for: 1) studying developmental myelination, 2) modeling demyelination and remyelination, and 3) conducting translational research. Sagittal sections of the cerebellum and hindbrain are isolated from postnatal day (P) 0&#8211;2 mice, after which they myelinate&#160;ex vivo&#160;for 12 days. During this period, slices can be manipulated in various ways, including the addition of compounds to test for an effect on developmental myelination. In addition, tissue can be fixed for electron microscopy to assess myelin ultrastructure and compaction. To model disease, CSC can be subjected to acute hypoxia to induce hypomyelination. Demyelination in these explants can also be induced by lysolecithin, which allows for the identification of factors that promote remyelination. Aside from chemical and environmental modifications, CSC can be isolated from transgenic mice and are responsive to genetic manipulation induced with Ad-Cre adenoviruses and tamoxifen. Thus, cerebellar slice cultures are a fast, reproducible, and quantifiable model for recapitulating myelination.

Presented is a protocol for an ex vivo quantitative model of demyelination and remyelination using mouse cerebellar slice cultures. This method closely recapitulates an in vivo model with its full complement of CNS cell types in intact tissue, while maintaining the chemical, genetic, and environmental amenability of an in vitro system.

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

从多个中枢神经系统区域解剖和分离小鼠神经胶质细胞

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

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生产和使用慢病毒的有选择性地转导原少突胶质前体细胞