Name: sex differences in mouse hippocampal astrocytes after in-vitro ischemia
Uploaded: 2016-10-25T12:30:00.000+00:00
Duration: 8 min 32 s
Description: Watch this Scientific Journal Video about Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia at JoVE.com

Clinical and Translational Science Award program of NCATS UL1 TR0000427 and KL2 TR000428 (Cengiz P), UL1TR000427 to the UW ICTR from NIH/NCATS and funds from Waisman Center (Cengiz P), K08 NS088563-01A1 from NINDS (Cengiz P) and NIH P30 HD03352 (Waisman Center), NIH/NINDS 1K08NS078113 (Ferrazzano P). We would like to thank Albee Messing, PhD, for providing us the GFAP knockout mice.

注:此研究是按照指南的护理的美国国立卫生研究院的实验动物的建议，并进行使用。动物方案经威斯康星大学麦迪逊分校，机构动物护理和使用委员会的大学。即这里提出初级星形胶质细胞培养协议从由张ý19 等和Cengiz第20页等人提出的协议通过。有一些修改。 1.海马解剖和星形胶质细胞文化<ol><li>准备所有必需的试剂和材料，包括在冰上手术剪，光滑细镊子，平头镊子，纸巾，废物袋，70％乙醇和2解剖菜（3.5cm直径）填充用2ml Hank氏平衡盐溶液（HBSS）中的每个。确保手术设备的无菌（釜）前程序和使用所有其他材料（星形胶质细胞电镀液:DMEM + 5％马血清+1％青霉素-链霉素，马血清，HBSS，0.25％胰蛋白酶，盖玻片，培养皿15/50 mL管，T25烧瓶等 ）预先无菌的。 </li><li>轻轻握住和喷雾头和鼠标小狗颈部用70％的乙醇和使用锋利的无菌剪刀斩首[ 图1（1）]。 </li><li>从后执行正中切口沿头皮，露出大脑[ 图1（2-3）]小剪刀把前颅底。 </li><li>从颈部用小剪刀鼻子小心切开头盖骨。然后切头盖骨前方的嗅球和下小脑，断开颅底颅内。 </li><li>作一小切口的颅底和沿中线切开。使用无菌平头镊子剥离的头骨。具有弯曲镊子的帮助下除去脑干。从头骨移除脑并放置到第一解剖盘[ 图1（4-9）] 21。 </li><li>使用弯钳，翻转大脑，使大脑腹面朝上，然后两个半球用锋利的无菌手术刮刀[ 图1（10,11）]分离</li><li>剥开大脑半球并小心地取出膨出脑和丘脑组织用无菌平弯钳揭示海马，在内侧颞叶[ 图1（12-14）小，海马形结构。 </li><li>除去这两个海马裂片[ 图1（15,16）]，并通过与细镊子牵拉小心解剖从裂片脑膜。这一步骤避免了脑膜细胞和成纤维细胞的最终星形胶质细胞培养物污染。 </li><li>转移准备海马瓣进入第二盘填充用HBSS，并将其返回到冰[ 图1（17,18）。从一个单一的鼠标池都海马叶（P0 - P2）准备河马campal星形胶质细胞的文化。这使星形胶质细胞密度适当。肉末用锋利的无菌手术刀片（约4倍）海马叶。 </li><li>抽吸的HBSS从盘使用连接到1毫升吸移管中的无菌尖端和添加0.25％胰蛋白酶的3毫升，混合，转移到15毫升的无菌锥形管中，并在37°C孵育所述组织20分钟轻轻摇动。 </li><li>在300×g离心5分钟，离心管中。吸去上清液小心使用连接到真空管线的无菌玻璃吸管。加入10 mL星形胶质细胞电镀液及磨碎 - 用火抛光的玻璃吸管，直到组织块均匀（20 30倍）。 </li><li>在300×g离心5分钟，离心管中。吸上清液，加入新鲜预热星形胶质细胞电镀媒体2毫升。通过70微米网状过滤器（细胞过滤）细胞进入一个新的50 mL锥形管。 </li><li>在含有3毫升的聚-D-赖氨酸/层粘连蛋白包被的无菌的T25培养瓶中板整个细胞悬浮液星形胶质细胞电镀媒体，然后孵育该烧瓶在37℃在一个5％CO 2培养箱。 </li><li>在按日期抽吸整个媒体I N体外 （DIV）1，DIV 3和DIV 7，并用2毫升的新鲜星形电镀媒体的更换。每一次观察进展，星形细胞融合光镜下而更换星形胶质细胞电镀媒体。 注:在DIV 1中，所有的可行的星形胶质细胞附着在培养瓶的表面，并且包括神经元在上清液浮动死亡或正在死亡的细胞。在格3中，附着的细胞开始分裂，以形成星形细胞层。在缺乏支持性的条件下，文化是几乎没有任何神经细胞生长的。在DIV 7，星形胶质细胞层为约80 - 90％汇合和几个小胶质细胞以及少突胶质细胞前体细胞是存在于星形细胞层的顶部。 </li><li>吸从烧瓶电镀介质，加入新鲜预热的星形胶质细胞电镀液2毫升，漂洗在第二DIV 11再次删除，当星形胶质细胞汇合，准备子培养。 </li><li>加入2毫升0.25％的胰蛋白酶，轻轻转动该烧瓶几次并用无菌玻璃吸管吸出胰蛋白酶。 </li><li>加入2毫升0.25％胰蛋白酶，让瓶中坐4的组织培养罩 - 在室温5分钟。 </li><li>除去胰蛋白酶，并保持该烧瓶在37℃在一个5％CO 2培养箱10分钟。加入5毫升预热的星形胶质细胞电镀液，并通过点击烧瓶对你的手掌分离星形细胞层（3 - 4次），其次是温和的研磨实现完全分离和收集在一个15毫升的锥形管的星形胶质细胞。 </li><li>离心管中，在300×g离心5分钟，吸出上清液，并添加1毫升新鲜星形胶质电镀介质。 </li><li>添加细胞悬浮液10μL的对血球和使用倒置相差显微镜（10X物镜）计数在中央大网格面积（1mm 2中）的细胞。 MULtiply 10 4来估算每毫升的细胞数。一个T25培养瓶中将产生〜1×10 6个总解离细胞。种子〜1×10 5细胞中在24孔培养皿的孔12毫米直径的聚-D-赖氨酸预包被的玻璃盖玻片2mL的星形胶质细胞电镀介质。 </li><li>在一个5％CO 2培养箱在37℃。 14（第2节） - 在DIV 12进行OGD /再氧化。 </li><li>治疗星形细胞培养在DIV 3用5mM L-亮氨酸甲基酯（LME）的盐酸直到DIV 11如果缺乏的小胶质细胞的海马培养是期望的。 LME孵育后，受到文化晃动（1小时300转），以去除污染的小胶质细胞。 注:LME是已被广泛地用作一种方法来消除增殖胶质22上的小胶质细胞毒性剂。 </li></ol> 2. OGD /再氧化处理<ol><li>从盖玻片含胶质细胞贴壁吸文化传媒（DIV 12 - 14）和冲洗3次轻轻旋转24孔培养皿保持盖玻片用含有（以mM计）1毫升等渗OGD溶液（pH 7.4）几次:0葡萄糖，21的NaHCO 3，120氯化钠，5.36氯化钾，0.33 的 Na 2 HPO 4， 0.44 KH 2 PO 4，1.27 氯化钙 ，和0.81 硫酸镁 。 </li><li>添加0.2mL的OGD溶液很好覆盖盖玻片并在含有94％的N 2，1％O 2缺氧培养箱孵育2小时，和5％ 的 CO 2。与缺氧的前30分钟，以促进气体交换轨道摇床（50rpm下）轻轻混匀。 </li><li>孵育常氧对照细胞在5％CO 2和大气中相同，只是在加入5.5毫葡萄糖的OGD溶液的缓冲液2小时。 </li><li>对于再氧化，吸液OGD并添加星形胶质细胞的2毫升电镀液。孵育在5％CO 2和大气中以37℃进行5小时。 </li></ol> 3.免疫细胞化学染色&lt;/ P&gt; <ol><li>使用连接到真空管线中的无菌玻璃吸管吸出培养基，并通过添加的0.1M Tris缓冲盐水（TBS）的1mL中迅速漂洗盖玻片一次（154毫摩尔NaCl，16毫Trizma碱，84毫摩尔Tris盐酸，pH 7.4）中到孔中。吸并在1X磷酸盐缓冲液（PBS）加入2毫升4％多聚甲醛（PFA）的。在室温下孵育15分钟。 </li><li>抽吸并添加1毫升的0.1M的TBS，然后放置在摇床上2分钟。重复3次。添加1毫升封闭液（10％山羊血清，10毫克/毫升BSA，0.025％的Triton X-100的0.1M的TBS），在37℃的摇床上孵育30分钟。 </li><li>抽吸阻断溶液并添加初级小鼠单克隆抗GFAP抗体（0.2毫升1:500稀释在封闭溶液中）和兔多克隆抗S100B（1:500），或兔多克隆抗HIF1α（1:200），60分钟在上一个摇床上37℃。 </li><li>或者，一些星形细胞孵育用兔多克隆抗IBA1（1:200）和鼠问oclonal抗MAP2访问培养纯度。 </li><li>吸一次抗体，并通过添加1毫升的0.1M的TBS并放置在摇床上进行2分钟，然后吸出冲洗盖玻片。重复两次。 </li><li>在60分钟，在37℃的摇床上封闭溶液二次山羊抗兔488缀合的抗小鼠568缀合的抗体的1:200稀释:添加1 0.2毫升。 </li><li>抽吸的第二抗体，并通过添加1毫升的0.1M的TBS和地点上的摇床上进行2分钟，然后吸出冲洗盖玻片。重复两次。 </li><li>通过放置在位于上滑盖干燥幻灯片从井和干删除盖玻片。通过对反转的的Vectashield单滴一个新的幻灯片盖玻片山安装hardset培养基DAPI（1.5纳克/μL）。 </li><li>使用两种20X干燥或60X油浸物镜共聚焦显微镜图像盖玻片。获取图像（512×512）DAPI（405纳米EX / 450纳米EM）和荧光标记的488 GFAP抗体（488纳米EX / 515纳米EM）。保留采集参数的20X 60X和组内保持不变。 </li></ol> 4.性别决定采用PCR <ol><li>热小狗脚趾或在50mM的NaOH 95℃45分钟手指剪辑和中和等体积的1M Tris，pH值6.8的。 </li><li>添加1μL的提取的DNA溶液的19微升以下混合物的:5皮摩尔引物为生成素和Sry基因的基因，1×反应缓冲液，各0.2mM的脱氧核苷酸和8有用Taq聚合酶。 </li><li>使用的引物序列; Sry基因5&#39;TCATGAGACTGCCAACCACAG3&#39;，5&#39;CATGACCACCACCACCACCAA3&#39;和生成素5&#39;TTACGTCCATCGTGGACAGC3"，5&#39;TGGGCTGGGTGTTAGTCTTA3"23。 </li><li>在95℃95℃3分钟，然后30℃变性个循环15秒，退火58℃15秒，和延伸率在72℃1分钟:执行以下PCR方案。在此之后30个循环，将反应结束以最终72℃延伸1分钟。 </li><li>单独的PCR产品在紫外光照射下电泳在含溴化乙锭的2％琼脂糖凝胶和可视化。 （ 图2A） 注意！溴化乙锭是一种潜在的致癌物质，必须小心处理和妥善处置按机构的规定。 </li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+DNA+extraction+and+PCR-sexing+of+mouse+embryos.">Rapid DNA extraction and PCR-sexing of mouse embryos.</a> Molecular reproduction and development. 60, 225-226 (2001).</li><li>Wolterink-Donselaar, I. G., Meerding, J. M., Fernandes, 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+method+for+gender+determination+in+newborn+dark+pigmented+mice.">A method for gender determination in newborn dark pigmented mice.</a> Lab Anim (NY). 38, 35-38 (2009).</li><li>Uliasz, T. F., Hamby, M. E., Jackman, N. A., Hewett, J. A., Hewett, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+primary+astrocyte+cultures+devoid+of+contaminating+microglia.">Generation of primary astrocyte cultures devoid of contaminating microglia.</a> Methods Mol Biol. 814, 61-79 (2012).</li><li>Raponi, E., 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=S100B+expression+defines+a+state+in+which+GFAP-expressing+cells+lose+their+neural+stem+cell+potential+and+acquire+a+more+mature+developmental+stage.">S100B expression defines a state in which GFAP-expressing cells lose their neural stem cell potential and acquire a more mature developmental stage.</a> Glia. 55, 165-177 (2007).</li><li>Souza, D. G., Bellaver, B., Souza, D. O., Quincozes-Santos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+adult+rat+astrocyte+cultures.">Characterization of adult rat astrocyte cultures.</a> PloS one. 8, e60282 (2013).</li><li>Puschmann, T. B., Dixon, K. J., Turnley, A. 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的作者都没有竞争或利益冲突。

为了研究生理和病理条件下，在属性的性别差异和功能星形胶质细胞，在细胞培养物中制备性控初级星形胶质细胞是利用一个重要工具。在本研究中，我们报道了高效和可再现的方法来培养新生性控海马星形胶质细胞的高度富集的同质群体（P0-P2）的C57BL / 6（野生型）或K19F（GFAP空）小鼠幼仔体外 。建立这种方法可以帮助研究者了解性控海马星形胶质细胞的生理和病理功能体外缺血?...

星形胶质细胞是中枢神经系统（CNS）最重要的关键球员之一。越来越多的证据表明，星形胶质细胞的作用是比提供支持神经元多。事实上，在生理条件下的星形胶质细胞的作用可以是非常复杂的，如引导显影轴突1的迁移，调节CNS血流2中，并保持突触组织液3的pH稳态，和参与血脑屏障4和突触传递5。在病理条件下，星形细胞损伤响应与被叫其中形态，数量，位置，地形（相对于从损伤的距离）的星形胶质细胞和功能可在异构方式6,7-改变反应性星形胶质细胞增生的过程。看到下面的星形胶质细胞增生新生儿缺氧缺血性脑病，也许有利于新生儿的发病率和死亡率<s达&gt; 8。 最近的临床和实验研究表明，大脑损伤的严重程度似乎是性别依赖性和，作为相对于男性新生儿对缺氧/缺血（HI）相关的脑损伤的影响更加敏感，导致更严重的神经成果女性具有可比性脑损伤9-11。虽然损伤的定位取决于胎龄和持续时间和损伤的严重程度，海马是足月HI后在CNS中最常见的影响区域中的一个，并增加海马星形胶质细胞增生被上调的确认胶质纤维酸性蛋白（GFAP）新生儿HI 7,10,12,13后3天。星形胶质细胞功能的性别差异被证明在这两个新生儿和成人啮齿动物脑缺血后14,15。另外，男性星形细胞易感性的体外缺血通过增加CEL所示升死亡相比，在文化16女性皮层星形胶质细胞。 性别差异在子宫内开始并持续直至死亡17。在过去十年中，包括在细胞培养和体内研究，在实验条件下男女的重要性一直医学和NIH研究所的重点寻求基础知识，在生理和病理条件17,18见过的性别差异。可靠的方法开发分离和维护绝育手术海马星形胶质细胞的人口有必要了解的新生儿HI的病理后果的性别差异的细胞学基础。本研究的目的是提供的技术准备，以评估以下氧气/葡萄糖剥夺（OGD）和复氧（再氧化），诱导GFAP免疫反应阳性星形胶质细胞的作用，从新生小鼠丰富的性别特异性海马星形胶质细胞培养HI在细胞培养环境。这种技术可以用来测试有关含氧量正常和缺血性的条件下在新生儿男性和女性海马星形胶质细胞的任何假设。

<table><tbody><tr><td>&lt;strong&gt;&lt;em&gt;Astrocyte culture media&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM, high glucose</td><td>cellgro</td><td>10-013-CV</td><td></td></tr><tr><td>Horse Serum</td><td>Gibco</td><td>26050-070</td><td>Final Concentration: 10%</td></tr><tr><td>Penicillin-Streptomycin</td><td>Cellgro&amp;nbsp;</td><td>30-002-CI</td><td>Final Concentration: 1%</td></tr><tr><td>L-Leucine methyl ester hydrochloride</td><td>Aldrich</td><td>L1002-25G</td><td>Final Concentration: 5 mM</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Solution for brain tissue digestion&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HBSS</td><td>Life Technologies</td><td>14170-088</td><td></td></tr><tr><td>Trypsin</td><td>cellgro</td><td>25-050-CI</td><td>Final Concentration: 0.25%</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Other&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>70% (vol/vol) ethanol</td><td>Roth</td><td>9065.2</td><td></td></tr><tr><td>Poly-D-Lysine (PDL) 12 mm round coverslips&amp;nbsp;</td><td>Corning</td><td>354087</td><td></td></tr><tr><td>Water</td><td>Sigma</td><td>W3500</td><td>Cell-culture grade</td></tr><tr><td>PBS</td><td>cellgro</td><td>21-040-CV</td><td>Cell-culture grade</td></tr><tr><td>0.05% Trypsin-EDTA&amp;nbsp;</td><td>Life Technologies</td><td>25300-062</td><td></td></tr><tr><td>70 &amp;mu;m Sterile cell strainer&amp;nbsp;</td><td>Fisher scientific</td><td>22363548</td><td></td></tr><tr><td>3.5 cm Petri dish</td><td>BD Falcon</td><td>353001</td><td></td></tr><tr><td>15 mL Falcon tube</td><td>BD Falcon</td><td>352096</td><td></td></tr><tr><td>50 mL Falcon tube</td><td>BD Falcon</td><td>352070</td><td></td></tr><tr><td>Forceps, fine&amp;nbsp;</td><td>Dumont</td><td>2-1032; 2-1033</td><td># 3c; # 5</td></tr><tr><td>Forceps, flat tip</td><td>KLS Martin</td><td>12-120-11</td><td></td></tr><tr><td>13 cm surgical scissors</td><td>Aesculap</td><td>BC-140-R</td><td></td></tr><tr><td>Confocal Microscope</td><td>Nikon</td><td>A1RSi&amp;nbsp;</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5805000.017</td><td>5804R</td></tr><tr><td>Orbital Shaker</td><td>Thermo Scientific</td><td>SHKE 4450-1CE</td><td>MaxQ 4450&amp;nbsp;</td></tr><tr><td>Anti-IBA1</td><td>Wako</td><td>019-19741</td><td>Rabbit monoclonal</td></tr><tr><td>Anti-MAP2</td><td>Sigma</td><td>M2320</td><td>Mouse monoclonal</td></tr><tr><td>Anti-HIF1alpha</td><td>abcam</td><td>ab179483</td><td>Rabbit monoclonal</td></tr><tr><td>Anti-S100B</td><td>Sigma</td><td>HPA015768</td><td>Rabbit polyclonal</td></tr><tr><td>Anti-GFAP (cocktail)</td><td>Biolegend</td><td>837602</td><td></td></tr><tr><td>VECTASTAIN Elite ABC Kit (Rabbit IgG)</td><td>Vector Labs</td><td>PK-6101</td><td>Contains 4 Reagents&amp;nbsp;</td></tr><tr><td>Goat Anti Rabbit Alexa-Fluor 488</td><td>Invitrogen</td><td>A11070</td><td></td></tr><tr><td>Goat Anti Mouse Alexa-Fluor 568</td><td>Invitrogen</td><td>A11004</td><td></td></tr></tbody></table>

sex differences in mouse hippocampal astrocytes after in-vitro ischemia

星形胶质细胞增生缺氧/缺血（HI）相关的脑损伤起着新生儿增加发病率和死亡率的作用。最近的临床研究表明，大脑损伤的严重程度似乎是性别依赖性的，并且该男性新生儿是HI有关的脑损伤的影响更加敏感，导致更严重的神经系统的结果相比，女性具有可比的脑损伤。可靠的方法来隔离和维护绝育手术海马星形胶质细胞的高度浓缩的人口的发展是至关重要的了解新生儿HI的病理后果的性别差异细胞基础。在这项研究中，我们描述了创建经受体外缺血，缺氧缺糖模型，随后复氧性具体海马星形细胞培养的方法。随后的反应性星形胶质被immunostai检查宁为胶质纤维酸性蛋白（GFAP）和S100B。这种方法提供了一个有用的工具来研究男性和女性的海马星形胶质细胞的新生之后HI的作用，分别。

理解的生理或病理生理条件下性控星形细胞机能的作用已经通过体外的条件下培养这些细胞被极大地阐明。执行性控培养的重要方面是确定鼠标小狗的性别其使用前。我们的基因通过PCR和通过视觉评价（ 图2）16所确定的小鼠的性别。采用PCR性别鉴定的方法与McClive和辛克莱修改获得通过，快速，简便和高重现性的方法23。 图2A</stron...

Watch this Scientific Journal Video about Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia at JoVE.com

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

星形胶质细胞是中枢神经系统（CNS）最重要的关键球员之一。在这里，我们报告，以研究男性和女性新生儿幼崽的星形胶质细胞功能的体外缺血后背后的机制性控海马星形胶质细胞培养协议的实用方法。

性别差异在小鼠海马星形胶质细胞<em&gt;在体外</em&gt;缺血

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

sex-differences-mouse-hippocampal-astrocytes-after-vitro

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

13.3K 次观看.  University of Wisconsin. 星形胶质细胞是中枢神经系统（CNS）最重要的关键球员之一。在这里，我们报告，以研究男性和女性新生儿幼崽的星形胶质细胞功能的体外缺血后背后的机制性控海马星形胶质细胞培养协议的实用方法。 

视频: Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

小鼠脑皮层星形胶质细胞的分离和培养

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

科研

神经科学

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

树突分支中的海马区对小鼠的齿状回评

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

在缺血样条件下监测星形胶质细胞的反应性和增殖体外

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

小鼠新生儿缺氧缺血性脑损伤模型的神经行为评估

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and cardiac arrest are remarkably sexually dimorphic diseases, and both induce cerebral ischemia. However, progress in understanding the vascular cognitive impairment, and then developing sex-specific treatments, has been partly limited by challenges in investigating the brain microcirculation from mouse models in functional studies. Here, we present an approach to examine the capillary-to-arteriole signaling in an ex vivo hippocampal capillary-parenchymal arteriole (HiCaPA) preparation from mouse brain. We describe how to isolate, cannulate, and pressurize the microcirculation to measure arteriolar diameter in response to capillary stimulation. We show which appropriate functional controls can be used to validate the HiCaPA preparation integrity and display typical results, including testing potassium as a neurovascular coupling agent and the effect of the recently characterized inhibitor of the Kir2 inward rectifying potassium channel family, ML133. Further, we compare the responses in preparations obtained from male and female mice. While these data reflect functional investigations, our approach can also be used in molecular biology, immunochemistry, and electrophysiology studies.

The present manuscript details how to isolate hippocampal arterioles and capillaries from the mouse brain and how to pressurize them for pressure myography, immunofluorescence, biochemistry, and molecular studies.

Ex Vivo 加压希马卡毛细管- 功能研究准备

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

研究神经-胶质相互作用在缺血中的作用的细胞培养模型

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections

Astrocytes possess an astounding degree of morphological complexity that enables them to interact with nearly every type of cell and structure within the brain. Through these interactions, astrocytes actively regulate many critical brain functions, including synapse formation, neurotransmission, and ion homeostasis. In the rodent brain, astrocytes grow in size and complexity during the first three postnatal weeks and establish distinct, non-overlapping territories to tile the brain. This protocol provides an established method for analyzing astrocyte territory volume and astrocyte tiling using free-floating tissue sections from the mouse brain. First, this protocol describes the steps for tissue collection, cryosectioning, and immunostaining of free-floating tissue sections. Second, this protocol describes image acquisition and analysis of astrocyte territory volume and territory overlap volume, using commercially available image analysis software. Lastly, this manuscript discusses the advantages, important considerations, common pitfalls, and limitations of these methods. This protocol requires brain tissue with sparse or mosaic fluorescent labeling of astrocytes, and is designed to be used with common lab equipment, confocal microscopy, and commercially available image analysis software.

This protocol describes methods for sectioning, staining, and imaging free-floating tissue sections of the mouse brain, followed by a detailed description of the analysis of astrocyte territory volume and astrocyte territory overlap or tiling.

厚自由漂浮组织切片中星形胶质细胞区域体积和瓷砖的分析

Astrocytes possess an astounding degree of morphological complexity that enables them to interact with nearly every type of cell and structure within the ...

远端中动脉闭塞开发缺血性卒中小鼠模型

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

作者聚焦：在小鼠中建立可靠的远端 MCA 闭塞模型用于中风研究

Here, we present a protocol to establish a distal middle cerebral artery occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6J mice and evaluate the subsequent neurological behavior and histopathological features.

使用远端中动脉闭塞技术诱导小鼠急性缺血性卒中

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic ...

性别差异在小鼠海马星形胶质细胞

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此视频中的章节

小鼠脑皮层星形胶质细胞的分离和培养

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

树突分支中的海马区对小鼠的齿状回评

在缺血样条件下监测星形胶质细胞的反应性和增殖体外

小鼠新生儿缺氧缺血性脑损伤模型的神经行为评估

Ex Vivo 加压希马卡毛细管- 功能研究准备

研究神经-胶质相互作用在缺血中的作用的细胞培养模型

厚自由漂浮组织切片中星形胶质细胞区域体积和瓷砖的分析

使用远端中动脉闭塞技术诱导小鼠急性缺血性卒中

使用远端中动脉闭塞技术诱导小鼠急性缺血性卒中