作者要感谢 Paola 洛佩斯 Pieraldi 的技术援助。A.H.M. 感谢8G12MD007600 和 U54-NS083924 支持这一出版物的赠款。我们感谢 NIH-NIMHD-G12-MD007583 赠款的设施支持。d.e.r. 对 NIHNIGMS-R25GM110513 提供的奖学金表示感谢。我们感谢使用通用仪器区和 Dr. Priscila 加西亚萨纳布里亚的援助, 以利用 RCMI 方案的光学成像设施的赠款 G12MD007583。此外, 我们要感谢何塞帕迪拉在拍摄和编辑视觉协议方面的杰出作用。

产后大鼠 (大) 1-4 天后被用来分离皮质。安乐死的方法是斩首, 由 NIH 的指南批准. 
 1. 准备手术用的仪器和材料 <ol> <li> 灭菌釜中的仪器 (温度: 121 和 #186; C、压力:15 psi, 时间:30 分钟) 使用钢盒或即时密封灭菌袋。请参阅 材料表 中的材料. </li>
</ol> 2。完整的 DMEM 准备 <ol> <li> 在包含700毫升蒸压水的一公升烧杯中, 在室温下添加 Dulbecco 和 #39 的改性鹰和 #39;
	<li> 添加3.7 克/升碳酸氢钠, 而溶液用磁力搅拌器搅拌. </li>
	<li> 调整 pH 值为 7.4, 用蒸压水将容积调到1升, 然后过滤。为了培养目的, 用 10% FBS 和1% 青霉素/链霉素来补充所需的培养基容量, 并在37和 #730 加热;请参阅 材料表 中的材料. 
	 注意: ph 值被调整到7.4 通过插入 ph 计电极入媒介。媒体必须搅拌, 然后慢慢添加1M 氢氧化钠使用滴管. </li>
</ol> 3。原星形星形胶质细胞培养 注意: 播种后, 必须每三天更改一次单元格, 并且单元格可以长到 70-100 (11-13 天)。在第三天, 轻敲烧瓶几次, 以解除胶质细胞和胶质祖细胞的文化, 删除所有的和 #39; 老 #39; 媒体, 洗两次使用10毫升的 pbs, 吸 pbs, 然后添加新鲜的新媒体。请参阅 材料表 中的材料. 
<ol> <li> 新生鼠脑解剖 <ol> <li> 在组织培养罩中进行解剖。获得1-4 天的新生大鼠幼崽 (2 只老鼠大脑使用每 75 cm 2 烧瓶). </li>
		<li> 准备三 60 mm 的培养皿和吸管5毫升的完整 DMEM. 
		 注意: 他们可以划分如下: #1 为每只老鼠的大脑, #2 所有大脑皮层的大脑, 和 #3 所有的去皮皮质 (没有脑膜). </li>
		<li> 抓住小狗和喷雾70% 乙醇。斩首它, 并丢弃在一个小的生物危害废物容器的身体. </li>
		<li> 使用微解剖镊子, 按住头部并将头部的皮肤切开以露出颅骨. </li>
		<li> 用另一把剪刀, 使 #34; #34; 从头骨后部向鼻子的切口开始。用一副镊子轻轻取下颅骨. </li>
		<li> 用镊子将裸露的大脑取出, 并将大脑放在以前充满培养基的培养皿中. </li>
		<li> 为后续步骤使用另一组无菌仪器. </li>
		<li> 使用微解剖钳将一个大脑轻轻地放在一个 60 mm 培养皿的倒盖上。当培养皿放在无菌纱布上时, 可以清晰地观察大脑, 更容易解剖. </li>
		<li> 用微解剖钳将大脑稳定下来, 用微解剖钳的锋利的一端轻轻地沿着中线裂, 将脑半球分开. </li>
		<li> 偏转和剥离皮层, 留下白质, 并转移到培养皿, 以前标记 #2。重复所有的大脑的过程, 结合所有的解剖皮层的培养皿标记 #2. </li>
		<li> 从每个皮层下方取出海马体并丢弃它. </li>
		<li> 使用微解剖镊子和 a 和 #160; 60 mm 培养皿从单个皮质叶轻轻剥离脑膜, 并将其放入标记为 #3 的培养皿中. </li>
	</ol> </li> <li> 组织分离: 均质搅拌器方法 <ol> <li> 将组织/中型悬浮液 (大脑的去皮皮层) 倒入无菌的搅拌机袋中, 并添加足够的介质以使袋子中的总容积为5毫升. </li>
		<li> 将包放入均质搅拌机中, 将大约2厘米的袋子放在关闭的门上方。细胞离解是做在2.5 分钟的高速. 
		 注意: 或者, 这可以通过关闭袋子和轻轻地敲打它与一个烧杯在引擎盖。确保在袋和烧杯之间使用布, 以避免摩擦. </li>
		<li> 将电池悬浮液倒入数字60和 #160; 网筛, 然后将过滤过的流倒入100筛, 使其能够按重力进行过滤. </li>
		<li> 使用15毫升管, 离心机的细胞悬浮在 200 x g 在临床离心机 (优选摆动斗转子) 5 分钟. </li>
		<li> 将上清液倒入烧杯中, 然后使用血清吸管, re-suspend 并将细胞颗粒与5毫升的培养基分离. </li>
	</ol> </li> <li> 单元计数 <ol> 与微, 收集100和 #181; 1.5 毫升离心管中的细胞悬浮, 并添加100和 #181;
		<li> 插入10和 #181; L 在例中收集到的悬浮物, 并计算在正方形的四角中所有可行的细胞. </li>
		<li> 在准备烧瓶前计算细胞密度的公式为: 
		<img alt="Equation 1" src="/files/ftp_upload/55108/55108eq1.jpg"/> <img alt="Equation 2" src="/files/ftp_upload/55108/55108eq2.jpg"/> </li> <li> 每 25 cm 2 烧瓶中添加至少50万单元格。每瓶 DMEM 5 毫升的吸管。混合物不应到达烧瓶的颈部。把它们放在37和 #176; C 孵化器在 5% CO 2, 为3天, 然后更改媒体. </li>
	</ol> </li> <li> 星形胶质细胞纯化技术: 介质变化, 机械和化学策略 注: 胶质细胞的代谢率比其他类型的高, 因此, 在营养缺乏条件, 这些细胞在几天后显示出低的生存率。与星形胶质细胞不同的是, 在这种环境中, 小胶质物质不受营养缺乏和增殖的影响 18 。为了保持胶质细胞数量的减少, 作者每3天改变形细胞培养介质。这种在细胞介质中的不断变化也会减少胶质的种群, 在星细胞单层的顶部可以生长在 19 烧瓶中。
	<ol> <li> 使用巴斯德吸管将每个烧瓶中的换药胶质细胞吸入介质。用滴的方式 (5 毫升/烧瓶) 清洗残留在每个烧瓶中的碎片. </li>
		<li> 在每个烧瓶 (5 毫升/烧瓶) 中添加无菌过滤 DMEM (带有抗生素和胎儿牛血清) 滴。在圆周运动中轻轻地搅动以覆盖整个表面. </li>
		<li> 将单元格放置在37#176;C 孵化器在 5% CO 2 为8-10 天, 直到烧瓶达到 70-100 (和 #62; 95% 板覆盖). 
		 注意: 几项协议表明, 当星形胶质细胞到达 70-100, 机械方法, 例如摇晃文化从2到 24 h, 导致细胞的分离在这些星形胶质细胞膜顶端, 主要小胶质和前体细胞 19 , 20 . </li>
		<li> 非星细胞在培养中通过在 180 rpm 30 分钟的轨道晃动而减少。在清液中去除细胞后, 新鲜的培养基被 pipetted 到烧瓶中 (~ 15 毫升). </li>
		<li> 消除胶质祖细胞, 增加震动速度为 200 rpm 为 6 h 21 , 22 , 23 , 吸气上清, 和吸管新鲜媒体. 
		 注意: 为了减少小胶质细胞在培养中的存在, 两种化学方法在原代培养方法中可以连续 (不同时) 使用: 糖胞嘧啶 (胞苷-C) 和 l-亮氨酸甲酯 (LME) 24 . </li>
		<li> 立即当细胞到达 70-100 (100%), 3-4 天后, 机械去除小胶质细胞, 这些电池可以处理8和 #181; M 的有丝分裂化合物糖胞嘧啶 (胞苷-c) 在 DMEM 5 天 (变化到新鲜的糖胞苷-c 在 DMEM 日报). 
		 注意: 添加阿糖胞苷的时间点是至关重要的, 因为这种化合物是一种有丝分裂药物, 它可以减少快速分裂的细胞的数量, 如小胶质物质 19 , 25 和成纤维细胞 26.与小胶质细胞相反, 当汇合时, 星形胶质停止通过接触抑制增殖 27 . </li>
		<li> 允许2天的细胞从胞苷-C 处理恢复. </li>
		<li> 为了进一步减少胶质的污染, 在细胞到达70-100 后, 使用50毫米的 l-亮氨酸甲酯 (LME) 在 DMEM 固定在 pH 7.4, 1 h at 37 和 #730; C. 
		 注意: LME 穿过细胞膜和细胞器, 经过水解, l-亮氨酸在这些细胞的溶酶体中积累。这种氨基酸的积累导致渗透肿胀和溶酶体的破裂 28 , 29 。LME 在消除小胶质细胞方面是非常有效的, 与其他单元相比 20 , 30 。
	</ol> </li> </ol> 4。6井板中培养星形胶质细胞的研究 <ol> <li> 涂层片与多聚-d-赖氨酸 <ol> <li> 在每个 35 mm 的 petri 盘中放入一个片, 然后添加100和 #181; 稀释的聚 d-赖氨酸溶液对每个片和等待 1 h. </li>
		<li> 取出聚 d-赖氨酸, 用2毫升无菌水冲洗两次. </li>
		<li> 移除水并等待片在引擎盖内晾干。将片存储在-20 和 #176; C 最多两周. 
		 注意: 冷冻稀释的聚-d-赖氨酸溶液;它可以存储长达两周. </li>
	</ol> </li> <li> Trypsinization <ol> <li> 使用巴斯德吸管, 从烧瓶中吸取培养基。添加5毫升/瓶消毒过滤 PBS 轻轻冲洗细胞. </li>
		<li> 从 PBS 中吸出, 然后添加5毫升/烧瓶的无菌过滤0.25% 胰蛋白酶和0.5 毫米 EDTA 溶液。将烧瓶放置在37和 #730; C, 5% CO 2 孵化器为 10 min. 
		 注意: 当细胞孵化, 温暖5毫升/烧瓶新鲜完整的 DMEM 在37和 #176; C. </li>
		<li> 经过10分钟的孵化, 鼓动, 以确保所有的细胞都解除了烧瓶。快速添加5毫升的温暖完整的 DMEM 每瓶, 以中和的胰蛋白酶. </li>
		<li> 轻轻地将单元格向上和向下移几次以分解任何 #34; 丛 #34;将悬浮细胞从两个烧瓶转移到15毫升圆锥离心管. </li>
		<li> 离心5分钟, 在 200 x g 收集细胞颗粒。在500和 #181 中重新悬浮颗粒, 然后将细胞放入烧瓶中. 
		 注意: 用细胞进行分析的例子: 1) 反转录聚合酶链反应 (rt-pcr) 分析基因表达;2) 免疫印迹评价蛋白质表达;3) 流式细胞仪分析, 定量的神经祖细胞和感兴趣的蛋白质的数量;4) 免疫荧光分析蛋白质的表达和定位. </li>
	</ol> </li> <li> 播种星形胶质细胞 <ol> 在处理主星形胶质细胞与胰蛋白酶 (参见 <li> 4.2 ) 后, 收集100和 #956; 1.5 毫升离心管中的细胞悬液, 并添加100和 #956; 0.5% 台台盼蓝. 
		<li> 添加10和 #956; 我收集的悬浮在台盼蓝例和计数所有的细胞是可行的. </li>
		<li> 在准备烧瓶前, 使用步骤3.3 中的公式计算单元格的密度. </li>
		<li> 在每个井中加入一个无菌、多聚 d-赖氨酸涂层片, 准备井的菜肴。吸管5万细胞和填充其余的体积在每个井菜与新鲜培养基 (约2毫升). </li>
		<li> 将井盘放入37和 #730; C 恒温箱 5% CO 2 , 在5-7 天后, 星形胶质细胞可能生长并覆盖片的整个表面. </li>
	</ol> </li> </ol> 5。主星形胶质细胞 OGD 协议 <ol> <li> OGD 培养基准备 <ol> <li> 补充 Dulbecco 和 #39; 改良的鹰和 #39 的无培养基葡萄糖, 3.7 克/升碳酸氢钠, 1% 青霉素/链霉素。请参阅 表 4 中的材料. </li>
		<li> 用 95% N 2 /5% CO 2 在10分钟的流量中清除20毫升的葡萄糖自由介质 (用流量计表示), 方法是将血清吸管插入介质中的气体系统. 
		 注意: 在清除氧气介质后, 将 pH 值调整为 7.4 (请参阅步骤 2.1 中的说明). </li>
		<li> 使用50毫升管过滤系统过滤清除无糖培养基. 
		 注意: 为每项实验制作新鲜的 OGD 培养基, 在使用前冒泡 95% N 2 /5% CO 2 , 以确保细胞暴露在氧气清除的环境中. </li>
	</ol> </li> <li> 实验过程 <ol> 在组织培养罩内, 从先前准备好的片中删除星形胶质细胞 (参考步骤 4.3.5), 并用蜂窝 PBS 清洗两次, 以去除电池上的葡萄糖. <li>
		<li> 添加2毫升的 OGD 介质对每个井并且安置井盘在缺氧室里面与他们的盖子. </li>
		<li> 将气体混合物连接到入口阀门, 并使出口阀门保持打开状态。用 95% N 2 /5% CO 2 的气体混合物冲洗腔室, 在15升/分为 5 min. </li>
		<li> 停止气体流量, 首先关闭出口阀门上的夹具, 然后关闭气体入口阀门导管上的夹具. 
		 注意: 通过用肥皂水覆盖密封室, 确保不漏气。如果密封牢固, 则不应形成气泡. </li>
		<li> 将整个房间置于37和 #176 的细胞培养箱中; C 为 OGD 1 小时或6小时. </li>
		<li> 孵育时间结束后, 吸气 OGD 介质, 并仔细吸管2毫升的完整 DMEM, 每4.67 和 #160; cm 2 常进程 24 h. </li>
	</ol> </li> </ol> 6。原星形星形胶质细胞免疫荧光制剂的协议 注意: 要评估星形胶质星的纯度, 不同的脑神经元, 如神经细胞, 小胶质, 和突可以被检测使用不同的细胞标记进行免疫荧光。增殖标志物、PCNA、碘碘 (PI) 可用于 OGD 后常条件下的原发性星形胶质细胞。请参阅 材料表 中的材料. 
<ol> <li> 免疫荧光制剂 注: 这是一种通用的免疫荧光协议, 可以进行较小的修改以达到最佳的信号 ( 如 , 更长的潜伏期,孵化温度)。 表 1 中的每个标记都可以根据制造商和 #39 的协议使用。
	<ol> <li> 要评估细胞的生存能力, 细胞可以孵育5分钟, 在37和 #176; C 与 PI (5 和 #181; M 在完全 DMEM) 和洗涤两次与2毫升 PBS 为 5 min. 显示 PI 染色的细胞是不可行的. 
		 注意: 使用步骤5.2.6 中处理的单元格. </li>
		<li> 在 PI 染色后固定细胞, 每4.67 和 #160 增加2毫升4% 福尔马林; cm 2 好, 20 分钟4和 #730; C。对于标有 PCNA 的样品, 优选的固定溶液为甲醇10分钟, 在4和 #730; C. 
		 警告: 福尔马林和甲醇是有毒的. 
		 注意: 始终检查每个公司的特定抗体协议. </li>
		<li> 用2毫升 PBS 清洗细胞5分钟, 重复此步骤3次. </li>
		<li> 在室温下孵育细胞 (0.5% 非离子表面活性剂, 10% FBS 在 PBS 中), 温度为30分钟, 温和摇动. </li>
		<li> 用2毫升 PBS 清洗细胞5分钟, 重复洗涤3次. </li>
		<li> 在 PBS 的 1% FBS 的溶液中孵育4和 #176; C, 过夜 (16 小时), 与原抗体 (PCNA, 胶质蛋白). </li>
		<li> 删除主抗体溶液, 用2毫升 PBS 洗涤细胞5分钟, 并重复此步骤3次. </li>
		<li> 在 PBS 中与荧光结合的二次抗体孵育, 在 1% FBS 的溶液中, 在室温下为 1 h. </li>
		<li> 用2毫升 PBS 清洗细胞5分钟, 重复此步骤3次。添加1和 #181; DAPI (4 和 #39;, 6 和 #39;-diamidino-2-phenylindole) 为5分钟, 染色细胞核. </li>
		<li> 2 毫升 pbs 5 分钟清洗细胞, 重复此步骤两次, 并保持 pbs. </li>
		<li> 使用安装介质在显微镜幻灯片上准备片. </li>
	</ol> </li> <li> 共焦显微镜 <ol> 设置显微镜后, 将片侧放在显微镜台上。
		<li> 使用该软件, 选择 543nm GFAP-Cy3 共轭抗体的激光束. </li>
	</ol> </li> </ol> 7。细胞活力测定方法 <ol> <li> 处理和量化星形胶质细胞, 按照步骤4.3 和 4.4. </li>
	<li> 添加 1x10 4 单元格/好到 96-井板, 并将它们长到 70-100. </li>
	<li> 使用步骤5中的方法, 将培养的96井板暴露为常 1 h OGD 或 6 h OGD. </li>
	<li> 在 OGD 治疗后, 常培养基被添加到所有的细胞中 24 h, 并使用 MTT 试剂测定法测定其生存能力 (作为制造商和 #39 的指示). </li>
	<li> 从5毫克/毫升的 mtt 溶液, 增加10% 的总体积每井和孵化3小时, 在37和 #730; C 与 MTT 试剂. </li>
	<li> 使用200和 #181 去除介质和重晶体; L 二甲基亚砜。在 570 nm 的分光光度计上阅读该板. 
	 注意: 相对于控制细胞而言, 减少吸光度将与生存能力降低相关. </li>
</ol>

<ol>
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作者没有什么可透露的。

本协议描述了从大鼠皮层星形胶质细胞的分离。在这种方法, 这是至关重要的, 以减少污染与其他细胞类型, 如小胶质, 突, 和成纤维细胞。为了减少小胶质细胞的数量, 可以采取几个步骤: 改变介质, 轨道震动和化学处理。一旦通过选择性细胞标记或最突出的细胞污染物, 通过免疫荧光证实了培养纯度, 就可以进行实验。例如, 抗电离钙结合剂分子 1 (Iba-1) 抗体可以用来检测小胶质细胞。神经元死亡在培养早期开始, 这些细胞与胶质前体细胞 (OPCs) 一起在烧瓶攻丝和媒体改变3天31。Oligodendrocytic 前体细胞可以用 olig2 或 NG2 等抗体来鉴定。GalC 可以用来识别不成熟或成熟的突, 但在这个文化阶段, 这些不太可能被发现。另外, 可以量化非胶质蛋白阳性细胞, 并估计污染物。这最后的方法不区分特定的细胞, 但将是一个更快的和更经济的方法来确定 non-astrocytic 细胞在文化中的百分比。
一旦形成了星形胶质细胞的富集培养, 就可以进行 OGD 实验来研究星形胶质细胞的反应性。通过冒泡代替氧气来清除溶液中的氮气是常用的协议32。在我们的方法论中, 我们使用冒泡法, 用氮气 (95% N2, 5% CO2) 将氧气清除到无糖介质中, 流速为15升/分, 为10分钟, 总容积为0.025 升。为了确认氧气被清除, 我们用氧气计测量液体和空气中残留的氧气浓度。清除后介质中的氧浓度为10分钟, 从7.9 毫克/升降至0.3 毫克/升。为了确定必须清除腔室以替换氧气的时间, 使用 N2进行清除的方法不低于商会制造商的建议 (20 升/分, 2-5 分钟)。在5分钟的清除15升/分钟, 氧气浓度保持在会议厅是0%。使用类似腔室的其他方法已以8-20 升/分钟的流速清除, 5-10 分钟的时间以氮气替代空气氧量33,34。
与其他细胞模型一样, OGD 有局限性, 结果应该仔细解释。OGD 可能对神经元有害, 但是, 星形胶质细胞可以耐受这些条件达 24 h35。另一个限制是, 星形胶质细胞在这个系统中是孤立的, 缺乏来自其他电池的信号。克服这种限制的一种方法是在相同的缺血样条件下使用来自其他细胞的条件介质。或者, 在 OGD36之后, 可以将细胞因子或其他因素添加到介质中。
另一种方法来研究能量缺乏的影响, 细胞, 是增加哇的媒体。 这种化合物主要抑制钠/钾泵, 它耗尽了 ATP 的胞内生产, 类似于 OGD37,38的影响。然而, 这种方法的缺点是, 它的所有机制都没有得到充分的理解, 并导致不相干的影响, 其中引入变量, 构成一个不切实际的缺血性中风的情况。
总之, OGD 方法是一个孤立的细胞系统, 使我们可以直接测试的影响, 不同的星药物靶向的体外, 可以进一步促进神经保护。它还提供了一个研究基本星形胶质细胞生物学的工具。这个模型可以建立一个强有力的证据基础, 以证明使用脑卒中的体内模型开发药物的实验条件。

中风被定义为 "急性神经功能障碍的血管来源, 无论是突然或快速发展的症状和体征, 对应于大脑中的重点领域的参与"1,2。有两种类型的中风: 出血和缺血性。当血管功能障碍是由动脉瘤或动静脉故障引起的, 伴有动脉后破裂的减弱, 这称为出血性中风3 , 在大多数情况下, 导致死亡。当血栓或栓子阻碍血流, 导致暂时剥夺氧气和葡萄糖的大脑区域, 它被称为缺血性中风4。不滋养细胞周围的受影响地区或缺血核心导致了自我平衡和代谢失衡, 精力充沛的功能障碍, 神经元死亡和炎症5, 这可能导致 life-long 残疾患者的6。
缺血性中风是一种多因素损伤, 涉及多种类型的细胞, 在不同的时间点反应和发挥作用。许多相互作用为研究单个细胞的行为创造了一个困难的环境。那么, 我们如何研究一个特定的细胞类型在如此复杂的环境下的贡献呢？一个接受的体外模型的缺血包括暴露细胞到氧和葡萄糖剥夺 (OGD), 在一定时期内, 然后恢复细胞到常环境。该系统模拟缺血性中风后再灌注血。在这种方法中, 细胞或组织被暴露在一个无糖的介质中, 在一个净化氧气的环境中, 使用一个专门的缺氧室。OGD 潜伏期可以从几分钟到24小时不等, 这取决于要测试的假说。研究表明, 根据 OGD 和常环境的时间, 可以实现中风的特定表型 (即、急性或亚慢性)。主要孤立星形胶质细胞, 暴露于 OGD 后恢复到常条件, 是一个研究细胞模型模拟中风体外7。利用 OGD 可以在脑卒中的环境下揭示孤立细胞的独立分子机制。
随着我们对星形胶质细胞生物学知识的增加, 它已经变得明显, 它们对于维持突触和维持神经修复、发育和可塑性是至关重要的8。在正常情况下, 星形胶质细胞释放和反应的因子, 因子, 生长因子和 gliotransmitters, 保持代谢平衡和内稳态的突触5,9。在急性 neuroinflammation, 如缺血性中风, 这些细胞可以成为反应, 显示长期过度表达的胶质纤维酸性蛋白 (胶质), 并显示肥大在其形态学5,10,11,12. 随着缺血性梗塞的发展, 星形胶质细胞所提供的稳态对正常的谷氨酸摄取、能量代谢、活性分子的交换和抗氧化活性的影响, 如13。
重新激活的星形胶质细胞在梗塞组织周围增殖, 而白细胞则向损伤区域14迁移。星增殖可以用标记物如增殖细胞核抗原 (PCNA)、Ki67 和尿 (BrdU)15来测量。这种增殖反应是以时间为根据的方式产生的, 它有助于形成胶质瘢痕, 一组不可逆转的反应性星形胶质细胞沿受损部位的软组织损伤后9。这个疤痕的初始功能之一是限制免疫细胞外渗从这个地区。然而, 研究表明, 疤痕成为一个物理障碍的轴突延长, 因为他们释放分子抑制轴突生长, 并创建一个物理屏障, 防止轴突在受伤地区的延伸16。然而, 有科学证据表明, 脊髓损伤后, 完全防止胶质瘢痕形成会损害轴突的再生17。因此, 必须在所研究的伤害的框架内考虑具体的星反应的背景。
所提出的方法可用于研究星形胶质细胞在缺氧性葡萄糖剥夺后的个体化功能, 并可根据研究者想要回答的问题进行修正。例如, 除了形态学变化和在不同 OGD 时间表达的标记, 清星形胶质细胞暴露于 OGD 可以进一步分析, 以确定这些细胞释放出的可溶性因子, 或作为条件介质来评估其在其他脑细胞中的作用。这种方法可以研究星形胶质细胞的反应性, 这可能导致解释的因素, 控制和调节他们的反应在缺血性中风的情况。

<table><tbody><tr><td>&lt;strong&gt;Instruments for Surgery - Step 1&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Operating scissor 5.5&amp;rdquo;</td><td>Roboz Company</td><td>RS-6812</td><td>Tools used to decapitate the rats.</td></tr><tr><td>Curved forceps 7&amp;rdquo;&amp;nbsp;</td><td>Roboz Company</td><td>RS-5271</td><td>Holds the skin of the rat while the skull is removed.</td></tr><tr><td>Micro-dissecting scissors 4&amp;rdquo;&amp;nbsp;</td><td>Roboz Company</td><td>RS-5882</td><td>Cuts both the skin and skull of the rat.</td></tr><tr><td>Micro-dissecting forceps 4&amp;rdquo; angled, fine sharp&amp;nbsp;</td><td>Roboz Company</td><td>RS-5095</td><td>Holds the skin of the rat while the skull is removed.</td></tr><tr><td>Micro-dissecting forceps 4&amp;rdquo; slightly curved 0.8</td><td>Roboz Company</td><td>RS-5135</td><td>Tool used to separate cortices.</td></tr><tr><td>Micro-dissecting tweezers</td><td>Roboz Company</td><td>RS-4972</td><td>Peels brain meninges.</td></tr><tr><td>Dissection microscope</td><td>Olympus</td><td>SZX16</td><td>Important for removing the meninge from the cortices.</td></tr><tr><td>&lt;strong&gt;DMEM Preparation - step 2&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>GibCo. Company</td><td>11995-065</td><td>Supports the growth of cells.</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich Company</td><td>S7277</td><td>Supplement for the cell culture media.</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>GibCo. Company</td><td>10437-010</td><td>Serum-supplement for the cell culture.</td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>GibCo. Company</td><td>15140-148</td><td>Inhibits the growth of bacterias in the cell culture.</td></tr><tr><td>Filter System 1L with 0.22um pore</td><td>Corning</td><td>431098</td><td></td></tr><tr><td>&lt;strong&gt;Astrocyte culture - step 3&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Serological pipets 5mL</td><td>VWR</td><td>89130-896</td><td>To pipette DMEM to containers with cells.</td></tr><tr><td>Serological pipets 10mL</td><td>VWR</td><td>89130-898</td><td>To pipette DMEM to containers with cells.</td></tr><tr><td>Serological pipets 25mL</td><td>VWR</td><td>89130-900</td><td>To pipette DMEM to containers with cells.</td></tr><tr><td>Centrifuge conical tube 15mL</td><td>Santa Cruz Biotechnology</td><td>sc-200250</td><td></td></tr><tr><td>Safe-lock tube 1.5mL</td><td>Eppendorf</td><td>022363204</td><td></td></tr><tr><td>Barrier Tips 200 uL</td><td>Santa Cruz Biotechnology</td><td>sc-201725</td><td></td></tr><tr><td>Barrier Tips 1 mL</td><td>Santa Cruz Biotechnology</td><td>sc-201727</td><td></td></tr><tr><td>Biohazard Orange Bag 14 x 19&quot;</td><td>VWR</td><td>14220-048</td><td></td></tr><tr><td>60mm petri dishes</td><td>Falcon</td><td>351007</td><td></td></tr><tr><td>Sterile gauze pads</td><td>Honeywell Safety</td><td>89133-086</td><td></td></tr><tr><td>Stomacher 80 Biomaster</td><td>Sewar Lab System</td><td>030010019</td><td>Triturate the brain tissue.</td></tr><tr><td>Stomacher 80 Blender Sterile Bags</td><td>Sewar Lab System</td><td>BA6040</td><td>Sterile bag for the stomacher cell homogenizer.</td></tr><tr><td>Beaker 400mL</td><td>Pyrex</td><td>1000</td><td></td></tr><tr><td>Sterile cell dissociation sieve, mesh #60&amp;nbsp;</td><td>Sigma-Aldrich Company</td><td>S1020</td><td>To obtain a uniform single cell suspension.</td></tr><tr><td>Sterile cell dissociation sieve, mesh #100</td><td>Sigma-Aldrich Company</td><td>S3895</td><td>To obtain a uniform single cell suspension.</td></tr><tr><td>Invert phase microscope</td><td>Nikon</td><td>Eclypse Ti-S</td><td>Verify cells for contamination or abnormal cell growth.</td></tr><tr><td>75cm2 sterile flasks</td><td>Falcon</td><td>353136</td><td></td></tr><tr><td>Multi-well plate</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>Micro cover glasses (coverslips), 18mm, round</td><td>VWR</td><td>48380-046</td><td></td></tr><tr><td>Bright-Line hemacytometer</td><td>Sigma-Aldrich Company</td><td>Z359629</td><td></td></tr><tr><td>Pasteur pipettes</td><td>Fisher Scientific</td><td>13-678-20D</td><td></td></tr><tr><td>Ethyl alcohol&amp;nbsp;</td><td>Sigma-Aldrich Company</td><td>E7023</td><td></td></tr><tr><td>L-leucine methyl ester hydrochloride 98% (LME)</td><td>Sigma-Aldrich Company</td><td>L1002</td><td>Promotes the elimination of microglia cells in the primary cortical astrocyte cultutre.</td></tr><tr><td>Cytosine &amp;beta;-D-arabinofuranoside (Ara-C)</td><td>Sigma-Aldrich Company</td><td>C1768</td><td></td></tr><tr><td>Poly-D-Lysine Hydrobromide, mol wt 70,000-150,000</td><td>Sigma-Aldrich Company</td><td>P0899</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>GibCo. Company</td><td>15400-054</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma-Aldrich Company</td><td>T8154</td><td></td></tr><tr><td>Phosphate buffer saline (PBS) tablets</td><td>Calbiochem</td><td>524650</td><td></td></tr><tr><td>Sterile Water</td><td>Sigma-Aldrich Company</td><td>W3500</td><td></td></tr><tr><td>&lt;strong&gt;&amp;nbsp;OGD Medium Preparation - step 5&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge conical tube 50 mL</td><td>VWR</td><td>89039-658</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium-free glucose</td><td>Sigma-Aldrich Company</td><td>D5030</td><td>Supports the growth of cells.</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich Company</td><td>S7277</td><td>Supplement for the cell culture media.</td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>GibCo. Company</td><td>15140-148</td><td>Inhibits the growth of bacterias in the cell culture.</td></tr><tr><td>200mM&amp;nbsp; L-glutamine&amp;nbsp;</td><td>GibCo. Company</td><td>25030-081</td><td>Amino acid that supplements the growth of cells.</td></tr><tr><td>Phospahet buffer saline (PBS) tablets</td><td>Calbiochem</td><td>524650</td><td></td></tr><tr><td>Filter System 50mL with 0.22um pore</td><td>Corning</td><td>430320</td><td></td></tr><tr><td>Centrifuge conical tube 50 mL</td><td>VWR</td><td>89039-658</td><td></td></tr><tr><td>Single Flow Meter&amp;nbsp;</td><td>Billups-Rothenberg</td><td>SMF3001</td><td>Measure gas flow in oxygen purge.</td></tr><tr><td>Hypoxia Incubator Chamber&amp;nbsp;</td><td>StemCell</td><td>27310</td><td>Generates a hypoxic environment for the cell culture.</td></tr><tr><td>Traceable Dissolved Oxygen Meter</td><td>VWR</td><td>21800-022</td><td></td></tr><tr><td>95% N2/ 5% CO2 Gas Mixture</td><td>Linde</td><td></td><td>Purges the environment of oxygen.</td></tr><tr><td>&lt;strong&gt;primary astrocyte immunofluorescence - step 6&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Phosphate buffer saline (PBS) tablets</td><td>Calbiochem</td><td>524650</td><td></td></tr><tr><td>Formaline Solution Neutral Buffer 10%</td><td>Sigma-Aldrich</td><td>HT501128</td><td>Solution used to fix cells.</td></tr><tr><td>Methanol&amp;nbsp;</td><td>Fisher</td><td>A4544</td><td>Solution used to fix cells.</td></tr><tr><td>Non-ionic surfactant (Triton X-100)</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>GibCo. Company</td><td>10437-010</td><td>Serum-supplement for the cell culture.</td></tr><tr><td>Anti-NeuN</td><td>Cell Signaling</td><td>24307</td><td>Detects mature neurons, serves to validate the astrocytic culture.</td></tr><tr><td>Anti-PCNA</td><td>Cell Signaling</td><td>2586</td><td>Detects proliferating cells.</td></tr><tr><td>Propidium Iodide (PI)</td><td>Sigma-Aldrich Company</td><td>P4170</td><td>Apoptosis staining.</td></tr><tr><td>Anti-Olig1</td><td>Abcam</td><td>AB68105</td><td>Detects mature oligodendrocytes.</td></tr><tr><td>Anti-Iba1+</td><td>Wako</td><td>016-20001</td><td>Detects microglial cells.</td></tr><tr><td>Anti-GFAP Conjugated with Cy3&amp;nbsp;</td><td>Sigma-Aldrich Company</td><td>C9205</td><td>Detects reactive astrocytes in the treated cells.</td></tr><tr><td>Alexa Fluor 488</td><td>Molecular Probe Life Technology</td><td>A1101</td><td>Anti-Mouse Secondary Antibody.</td></tr><tr><td>Alexa Fluor 555</td><td>Molecular Probe Life Technology</td><td>A21428</td><td>Anti-Rabbit Secondary Antibody.</td></tr><tr><td>4&amp;rsquo;,6&amp;rsquo;-diamidino-2-phenylindole (DAPI)</td><td>Sigma-Aldrich Company</td><td>D9542</td><td>Nuclear staining.</td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td></td><td></td></tr></tbody></table>

monitoring astrocyte reactivity and proliferation in vitro under ischemic-like conditions

缺血性中风是一种复杂的脑损伤, 血栓或栓子阻碍血液流向大脑的部分。这导致了缺氧和葡萄糖的匮乏, 导致能量衰竭和神经元死亡。在缺血性中风的侮辱, 星形胶质细胞成为反应和增殖周围的伤害现场, 因为它的发展。在这种情况下, 很难研究星形胶质细胞对缺血脑区的特定贡献。因此, 本文介绍了一种方法, 研究原星形胶质细胞的反应性和增殖在一个体外模型的缺血样环境, 称为氧葡萄糖剥夺 (OGD)。星形胶质细胞是从1-4 岁的新生大鼠中分离出来的, 并通过胶质纤维酸性蛋白 (胶质细胞) 和细胞核染色对非特异性星的数量进行评估。星形胶质细胞受 OGD 条件的时间可以定制, 以及他们所接触到的氧气的百分比。这种灵活性允许科学家在不同的细胞组体外描述缺血样状态的持续时间。本文讨论了诱导星形胶质细胞反应性、肥大形态学和增殖细胞核抗原 (PCNA) 免疫荧光法测定 OGD 的时间范围。星形胶质细胞除增殖外, 还具有能量和氧化应激, 并通过释放可溶性因子而对 OGD 反应。这种培养基可以收集并用于分析星形胶质细胞释放的分子在没有细胞相互作用的原代神经细胞中的作用。总之, 这种原细胞培养模型可以有效地用于了解孤立星形胶质细胞在损伤中的作用。

主要的关注之一星文化是存在的其他细胞, 如神经元, 突, 成纤维细胞, 和小胶质。在图 1中, 从大鼠皮层分离出来的细胞每3天就有一次改变, 或者未经治疗或在 1 h. 24 小时后被添加 LME, 细胞被 immunostained 为胶质蛋白和 1-4 DAPI。未经处理的细胞显示平均39% 非胶质细胞阳性, 而 LME 治疗的细胞显示8%。这些结果表明, LME 的治疗和媒介的变化有效地减少了非胶质细胞的4.75 倍, 产生了丰富的星形星的文化。为了确定1小时或6小时 OGD 影响星形胶质细胞的生存能力, 在这种方法之后,图 2显示了一种 mtt 法测定星形胶质细胞的培养, 24 h 后的侮辱。在任何条件下均无明显差异, 表明两次可用于研究星形胶质细胞的反应性。细胞增殖是 OGD 在星形细胞中引起的作用之一。增殖细胞核抗原 (PCNA) 在图 3中从10% 在常细胞中增加, 到30% 后 1 h OGD, 显示预期的体内星响应15。最后, 星形胶质细胞暴露在6小时 OGD, 其次为 24 h 常条件。经过这段时间后, 对胶质纤维进行免疫荧光检查。在常条件下, OGD 暴露的细胞被比较。没有 OGD 的星形胶质细胞显示了传统的星状形态学 (图 4A), 而接受 OGD 的单元格明显呈现出反应性星形胶质细胞的肥大 (图 4B)。这种肥大可以在常和 OGD 条件下的皮层大鼠星形胶质细胞的相应的差分干涉造影 (DIC) 图像中可视化 (图 5)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55108/55108fig1.jpg"> 
图 1.用胶质蛋白和 DAPI 对原代皮质大鼠星形胶质细胞培养纯度进行免疫荧光验证.(A) 左面板表示胶质蛋白阳性细胞 (红色), 中间板显示细胞核 (DAPI, 蓝色), 右面板显示非 lme 处理 (上部面板) 和 lme 处理 (下部面板) 单元的合并图像。白色箭头显示非胶质细胞阳性染色。(B) 在 lme 和非 lme 处理的细胞中对非胶质蛋白阳性细胞核进行定量。进行了未配对 t 检验 (p 和 #60; 0.0001), 误差线用扫描电镜表示平均值. 缩放条形图代表50微米, 图片以20x 放大倍数显示。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55108/55108fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55108/55108fig2.jpg"> 
图 2.24 h OGD 星形胶质细胞的活性测定.星形胶质细胞被培养在96井板和暴露于常, 1 h OGD, 或6小时 OGD 条件。在治疗后, 细胞被添加到常培养基 24 h 和生存能力用 MTT 法测定。570 nm 的吸光度显示 OGD 细胞在培养中的细胞活力与常条件下的细胞比较。用 one-way 方差分析 (p = 0.9669) 对数据进行了解析, 并给出了用扫描电镜表示平均值的误差条.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55108/55108fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55108/55108fig3.jpg"> 
图 3。大鼠星形胶质细胞的增殖在暴露于缺氧和葡萄糖缺乏时增加, 并可通过 PCNA 作为标记物进行检测.(A) 左面板代表 PCNA 阳性细胞 (绿色), 中间的面板显示细胞核 (DAPI, 蓝色), 右面板显示合并图像的常细胞 (上部面板) 和1小时 OGD 处理的细胞 (下部面板)。(B) 常与 OGD 治疗细胞的 PCNA 阳性细胞百分率。对未配对的测试进行了 (p 和 #60; 0.0001) 和误差线代表的平均数与 SEM. 缩放条形图代表50微米, 图片以20x 放大倍数显示。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55108/55108fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55108/55108fig4.jpg"> 
图 4.胶质纤维酸性蛋白在原代大鼠皮层星形胶质细胞培养中的 Cy3 表达.(a) 星形胶质细胞在常环境中表现出特征的星状形态。中间丝蛋白, 胶质, 填充细胞体, 并延伸到薄细胞质过程。(B) 6 h OGD 暴露的星形胶质细胞采用肥大形态学。鳞片代表20微米, 照片在共聚焦显微镜下以40x 倍的倍率拍摄。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55108/55108fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55108/55108fig5.jpg"> 
图 5.常和缺氧的皮层星形胶质细胞.星形胶质细胞在常条件下 (左上面板) 或 6 h OGD (左下面板), 染色使用 Cy3-conjugated 抗体对胶质纤维。相应的差分干涉对比度 (DIC) 图像显示在右侧面板上。缩放条代表20微米, 照片以20x 倍的倍率拍摄。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55108/55108fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>目的</td>
			<td>抗体/标记</td>
			<td>描述</td>
		</tr>
		<tr>
			<td rowspan="5">星形胶质细胞培养验证</td>
			<td>Iba-1</td>
			<td>检测胶质单元格</td>
		</tr>
		<tr>
			<td>NeuN</td>
			<td>识别成熟神经元</td>
		</tr>
		<tr>
			<td>Olig1</td>
			<td>检测成熟突</td>
		</tr>
		<tr>
			<td>Olig2, NG2</td>
			<td>检测胶质前体细胞</td>
		</tr>
		<tr>
			<td>galc</td>
			<td>识别未成熟或成熟的突</td>
		</tr>
		<tr>
			<td>扩散</td>
			<td>pcna</td>
			<td>结合 p36 蛋白, 表达在高浓度的增殖细胞</td>
		</tr>
		<tr>
			<td rowspan="2">细胞活力</td>
			<td>碘碘化物 (PI)</td>
			<td>绑定到 DNA, 这个标记表明缺乏细胞活力</td>
		</tr></tbody></table>tr ><tr>
			<td>mtt</td>
			<td>测量线粒体功能</td>
		</tr>
		<tr>
			<td>反应</td>
			<td>gfap</td>
			<td>星形胶质细胞反应性指标</td>
		</tr>
	

表1。用于染色各类细胞和细胞过程的试剂清单

Watch this Scientific Journal Video about Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions at JoVE.com

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

缺血性中风是一个复杂的事件, 其中星形胶质细胞对受氧葡萄糖剥夺 (OGD) 影响的脑区的具体贡献是很难研究的。本文介绍了一种获取孤立星形胶质细胞的方法, 并研究了其在 OGD 条件下的反应性和增殖。

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 ...

monitoring-astrocyte-reactivity-proliferation-vitro-under-ischemic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

9.8K 次观看.  Universidad Central del Caribe. 缺血性中风是一个复杂的事件, 其中星形胶质细胞对受氧葡萄糖剥夺 (OGD) 影响的脑区的具体贡献是很难研究的。本文介绍了一种获取孤立星形胶质细胞的方法, 并研究了其在 OGD 条件下的反应性和增殖。

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

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

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 ...

神经科学

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

生物工程

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

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

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

协议星形胶质细胞的三维共聚焦形态分析

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

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately.

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

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 ...

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 ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

评估血脑屏障网状血管形成和破坏的体外测定

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

医学

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 ...

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

大鼠星形胶质细胞和小胶质细胞的原代培养及其在肌萎缩侧索硬化症研究中的应用

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro&#160;experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

小鼠星形胶质细胞的分离和直接神经元重编程

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...