Name: a cell culture model for studying the role of neuron-glia interactions in ischemia
Uploaded: 2020-11-14T14:00:00
Description: Watch this Scientific Journal Video about A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia at JoVE.com

作者感谢通过UIDB/00709/2020项目、POCI-01-0145-FEDER-029311和研究金SFRH/BD/135936/2018向JP提供的资金支持， 通过"2020年中分部"项目 CENTRO-01-0145-FEDER-000013，并通过项目 POCI-01-01-0145-FEDER-022122 向PPBI-葡萄牙生物成像平台提供资金。

所有使用的动物都是按照国家动物研究伦理要求和《欧洲用于实验和其他科学目的的脊椎动物保护公约》（第2010/63/EU号指令）在CICS-UBI健康科学研究中心饲养的。
1. 大鼠胚胎皮层原细胞培养
<ol><li>培养媒介<ol>
<li>通过添加以下补充剂来准备神经基础介质 （NBM）： 2% B27， 0.5 mM 谷氨酰胺， 25 μM 谷氨酸和 120 μg/mL 根霉素.均质化，将pH值调整为7.2，并使用0.22 μm过滤器用真空过?...

<ol>
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这里描述的方法包括从大鼠胚胎皮质组织分离星细胞和神经元，允许建立神经元或星细胞富培养物或神经元胶质培养。它改编自我们小组5的先前研究，其中皮质神经元和神经元丰富的胚胎培养物分离被描述，并描述两种培养物的特点。利用这些培养物，Roque等人发现星形细胞在应对缺血性损伤方面起着关键作用，并表明星形细胞和神经元之间的交流对神经保护5至关...

根据世界卫生组织的数据，每年约有550万人死于缺血性中风。这种情况的特点是血液流向某一大脑区域中断，导致组织氧气和营养物质供应的可逆或不可逆损失，从而改变组织功能，导致线粒体功能障碍、钙失调、谷氨酸兴奋、炎症和细胞损失2，3。,3
除了血管细胞，神经元和胶质细胞也参与缺血性中风的病理生理学。特别是，星形细胞对神经元的维持至关重要，最近被证明对缺血性病变5的反应起着关键作用。这种类型的胶质细胞执行结构支持、抗氧化应激、神经递质合成、细胞细胞通信稳定等功能。与神经元一起，星形细胞在突触传递中起直接作用，调节三磷酸腺苷、伽马-氨基丁酸和谷氨酸7等分子的释放。缺血引起的部分损伤是由谷氨酸的过度释放及其在突触裂口的积累引起的，导致N-甲基-D-阿斯巴酸受体的过度激活，激活下游信号级联，最终导致兴奋8。由于星母细胞能够从突触裂口中去除谷氨酸并将其转化为谷氨酰胺，因此它们对于抵御兴奋性至关重要，从而对缺血产生神经保护作用。这些细胞在缺血引起的神经炎中也起着一定的作用。在缺血性侮辱之后，活性星形细胞发生形态变化（肥大），增殖，并表现出胶质纤维酸蛋白（GFAP）表达的增加。它们可以变成反应性（星体炎），释放亲炎细胞因子，如肿瘤坏死因子-α，白细胞素-1+和白细胞素-1+，并产生自由基，包括一氧化氮和超氧化物，这反过来又可以诱发神经元死亡9，10。9,相比之下，反应性星形细胞也可能起到神经保护作用，因为它们释放抗炎细胞因子，如改变生长因子-β，即中风11后上升调节。此外，它们可以产生胶质疤痕，通过抑制轴芽来限制组织再生;然而，这种胶质疤痕可以分离损伤点与可行的组织，从而防止一个级联波不受控制的组织损伤12，13。1312
因此，必须建立模型，允许研究缺血性损伤下的神经元-胶质相互作用，以找到治疗策略，限制或扭转缺血损伤的影响。与其他用于研究缺血性损伤的模型相比，in vivo,,即体内模型14、15、16、15有机细胞培养17、18、19,18,19和急性脑片1620、21、22，原细胞培养物较不复杂，这使得研究各细胞型在缺血性中风病理生理学中对个体贡献以及每种细胞类型对治疗可能靶点的反应成为可能。20,2122通常，为了研究神经元富集培养物与星细胞富集培养物之间的相互作用，使用产后起源的神经元和胶质细胞23、24,或产后胶质细胞和胚胎神经元25、26。25,26本文提出了一个简单的方法来建立神经元或星细胞富培养物和神经元胶质培养物从同一组织。这些原发性细胞是从大鼠胚胎皮层获得，这个区域经常受到中风27，28,的影响。此外，组织分离只能通过机械程序执行。因此，该协议允许以快速和廉价的方式，以高性能和可重复性的方式，在相同的开发阶段隔离细胞。
可以使用一个共培养系统来探索星母细胞和神经元之间的串扰，在这个系统中，在盖玻片中培养的神经元与多孔板中种子的星细胞的单层保持接触。小石蜡球可用于确保两个细胞培养物的分离。此方法允许在每个单元类型接触之前独立操作它们。例如，可以沉默星形细胞中的特定基因，看看它如何影响神经元脆弱或防止缺血引起的损伤。在体外诱导缺血样条件的方法是氧气和葡萄糖剥夺（OGD）3，它包括3用缺氧大气（95%的空气和5%的CO2）取代常规大气（95%N2和5%CO2），与葡萄糖的遗漏有关。2
所述方法适合以简单、快速、可重复和廉价的方式研究缺血性中风背景下神经元和星形细胞之间的相互作用。

<table>
	<tbody>
		<tr>
			<td>24 -well culture plates</td>
			<td>Thermo Fischer Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>95% N2/5% CO2 gas cylinder</td>
			<td>ArL&#237;quido</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse conjugated to Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td>1/1000 dilution; incubation period - 1 h at room temperature</td>
		</tr>
		<tr>
			<td>Anti-rabbit conjugated to Alexa Fluor 546</td>
			<td>Invitrogen</td>
			<td>A11010</td>
			<td>1/1000 dilution; incubation period - 1 h at room temperature</td>
		</tr>
		<tr>
			<td>B27 supplement (50x)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako Fluorescence Mounting Medium</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose anhydrous</td>
			<td>Fisher Scientific</td>
			<td>G/0450/60</td>
			<td>3.4 g/L</td>
		</tr>
		<tr>
			<td>Epifluorescence microscope</td>
			<td>Zeiss</td>
			<td>AxioObserver Z1x</td>
			<td>63x objective</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Biochrom</td>
			<td>S0615</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G1272</td>
			<td>120 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G8415</td>
			<td>25&#181;M</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G3126</td>
			<td>0.5 mM</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H1399</td>
			<td>2 &#181;M; incubation period - 10 min at room temperature</td>
		</tr>
		<tr>
			<td>Hypoxia incubation chamber</td>
			<td>Stemcell Technologies</td>
			<td>27310</td>
			<td>Chamber used for OGD induction</td>
		</tr>
		<tr>
			<td>Insulin from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>I5500</td>
			<td>5 mg/L</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Sigma-Aldrich</td>
			<td>K-002</td>
			<td>87.5 mg/Kg</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium Eagle medium</td>
			<td>Sigma-Aldrich</td>
			<td>M0268</td>
			<td>warm up to 37 &#176;C before use</td>
		</tr>
		<tr>
			<td>Mouse Anti-MAP2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>Sc-74421</td>
			<td>1/500 dilution; incubation period overnight at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>warm up to 37 &#176;C before use</td>
		</tr>
		<tr>
			<td>Paraffin pastilles for histology</td>
			<td>Sigma-Aldrich</td>
			<td>1.07164</td>
			<td>Solidification point 56-58&#176;C</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma -Aldrich</td>
			<td>P6148</td>
			<td>4% in PBS</td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin</td>
			<td>Biochrom</td>
			<td>A 2213</td>
			<td>penicillin (12U/mL) /streptomycin (12&#181;g/mL)</td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P1024</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti-GFAP</td>
			<td>DAKO</td>
			<td>Z0334</td>
			<td>1/2000 dilution; incubation period overnight at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate</td>
			<td>Fisher Scientific</td>
			<td>S/4240/60</td>
			<td>2.2g/L</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Sigma-Aldrich</td>
			<td>X1126</td>
			<td>12 mg/Kg</td>
		</tr>
	</tbody>
</table>

a cell culture model for studying the role of neuron-glia interactions in ischemia

缺血性中风是一种临床疾病，其特征是脑组织灌注过低，导致缺氧和葡萄糖剥夺，并随之导致神经元丧失。许多证据表明，胶质细胞和神经元细胞之间的相互作用在缺血事件后产生有益的作用。因此，要探索潜在的保护机制，开发模型，允许在缺血环境中研究神经元-胶质相互作用是重要的。在这里，我们提出了一个简单的方法，从大鼠胚胎皮层分离星形细胞和神经元，通过使用特定的培养媒体，允许建立神经元或星细胞富培养物或神经元胶质培养物具有高产量和可重复性。
为了研究星核细胞和神经元之间的串扰，我们提出了一种基于共培养系统的方法，其中在盖玻片中培养的神经元与多孔板中镀在一体的单层星细胞保持接触。这两种文化由小石蜡球保持分离。这种方法允许对每种细胞类型进行独立操作和应用特定的治疗，这在许多研究中代表着优势。
为了模拟缺血性中风期间发生的情况，培养物受到缺氧和葡萄糖剥夺协议。该协议是研究神经元-胶质相互作用在缺血性中风中的作用的有用工具。

为了描述培养物的特征，免疫细胞化学评估表达GFAP或 MAP2的细胞数量，广泛使用的星形细胞和神经元的标记物（图2），在每种类型的皮质培养物中执行。这一分析显示，富含星细胞的培养物呈现了97%的表达GFAP的细胞（图2A）。关于神经元富集培养，78%的细胞表示 MAP2，4% 的细胞表示 GFAP，18% 的细胞同时表示 GFAP 和 MAP2 阴性（图 2B?...

Watch this Scientific Journal Video about A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia at JoVE.com

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

在这里，我们提出了一个简单的方法，使用特定的培养媒体，允许建立神经元和星细胞丰富的区域性，或神经元胶质培养物从胚胎皮层，具有高产量和可重复性。

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 ...

缺血性中风是一种临床疾病，其特征是脑组织灌注过低，导致神经元丧失。大量证据表明，胶质和小便池细胞之间的相互作用在缺血事件后产生压力效应。为了探索潜在的保护机制，开发允许研究缺血环境中神经元-胶质相互作用的模型非常重要。在这里，我们提出了一个简单的方法，从大鼠胚胎皮层分离星形细胞和神经元，通过使用特定的文化媒介，允许建立神经元或星细胞丰富的区域性或新胶质文化具有高产量和可重复性。为了研究星核细胞和神经元之间的串扰，我们提出了一种基于共培养系统的方法，即在盖滑中培养的神经元与多孔板中镀在一体的单层星细胞保持接触。这两种文化由小石蜡球保持一部分。这种方法允许独立操作和将特定处理应用到每种细胞类型，这在许多研究中代表了优势。为了模拟缺血性中风期间发生的情况，培养物受到缺氧和葡萄糖剥夺协议。该协议是研究神经元-胶质相互作用在缺血性中风中的作用的有用工具。从大鼠胚胎皮层绝缘开始。胚胎是在妊娠15天后从一组大鼠身上获得的，并被放置在一个含有PBS的无菌猎鹰管中。还在蛋黄袋胚胎内被放置在含有冷 PBS 的培养皿中。在剪刀和钳子的帮助下，蛋黄囊被打破，胚胎被取出，放在另一个含有冷PBS的培养皿中，放在冰袋上。打破蛋黄囊时必须非常小心，不要损坏胚胎。将胚胎置于解剖显微镜下，用扭动器轻轻固定胚胎。初始切口应与皮层平行。小心不要斩首动物。头皮和小心删除不损害皮质脑组织。下一个切口将皮层分开。去除组织中的血管。最后，使用移液器，将皮质组织转移到带PBS的猎鹰管中。单细胞悬浮液是通过使用直径减小的尖端机械消化皮质脑组织获得的。确保组织在消化后均匀化，在400G下将材料离心三分钟。丢弃上流水，在培养中重新悬浮沉积物，以前加热到37度。使用 Neubauer 腔室计算悬浮液中存在的细胞总数，并准备稀释以产生足够的细胞密度。最后，在多井中播种细胞，并在37度孵育。为了为共培养系统准备材料，在加热块中加热石蜡，直到其变成液体。然后，借助立体玻璃巴斯德移液器，在以前放置在多井的盖滑上准备小球体，并涂上聚-D-Lysine。在两种培养物接触前24小时，改变神经元和星细胞的培养培养，以补充其NBM与或不带热灭活的FBS。当两种培养物准备好使用时，将位于盖上、带石蜡球的神经元转移到含有星形细胞的井中，使用以前浸入70%乙醇的钳子。将两种细胞类型置于接触中后，等待8至12小时，然后开始不同的刺激和过程。氧气和葡萄糖的剥夺是在生长七天的培养中进行的。去除培养培养素，用HSSS培养剂洗两次，无需葡萄糖补充。确保所有含有葡萄糖的介质都洗净。密封缺氧室，将含有95%氮气和5%二氧化碳的气体混合物加入4分钟，流量为20升，以更换室内的氧气。然后，停止流动，将缺氧室放在37度的孵化器中。在缺氧和葡萄糖剥夺期后，用适当的培养库代替无葡萄糖补充的培养素，并在37度下孵育细胞。为了描述皮质培养的类型，我们在三种皮质培养物中进行了免疫组织化学，以评估表达GFPA或 MAP2的细胞数量，这些细胞分别是分别广泛用于天体细胞和神经元细胞的标记。这些分析表明，当这个协议，我们能够获得一个纯丰富的富集的星细胞与约97%的细胞表达GFAP。关于神经元丰富的培养，我们验证了约78%的细胞表达 MAP2。然而，我们识别约18%的GFAP和 MAP2阴性细胞。对于神经元-胶质皮质培养，观察到约49%的细胞为 MAP2 阳性。31% 为 GFAP 阳性。20% 他们不对 Gfap 或 MAP2 负责。皮质培养建立7天后，神经元-胶质培养和神经元富集培养接受OGD手术4小时和6小时。在此过程之后，细胞被标记为 MAP2，然后使用荧光显微镜量化 MAP2 阳性细胞的数量。在神经元-胶质培养中，当培养被提交到4小时的OGD周期时，神经元的数量减少了约30%。经过6小时的OGD期后，病变的延长增加，达到神经元损失的60%左右。关于暴露于OGD的神经元的丰富培养，观察到 MAP2 细胞的数量减少约 41% 和 64%。此外，据观察，在神经元丰富的培养文化中，与在4小时内也接触OGD的神经元胶质培养相比，损伤延长略有增加。总之，这里代表一个体外模型，研究以简单、快速、昂贵和可重复的方式建立的缺血性中风。此外，所述方法还允许实现神经元和富含星细胞的初级培养，但也允许神经元-胶质培养。因此，提供一个伟大的体外模型，建模几个脑部疾病与更高的复杂程度，比不朽的细胞系和纯粹的神经元或胶质培养。

Research

JoVE Journal

Neuroscience

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats

在实验室老鼠研究求可卡因电击诱导恢复原状的程序

The most insidious aspect of drug addiction is the high propensity for relapse.  Animal models of relapse, known as reinstatement procedures, have been ...

科研

神经科学

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

方法研究抗精神病药物的作用机制在<em&gt;秀丽隐杆线虫</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

一<em&gt;在体外</em&gt;成年小鼠肌肉，神经准备留学肌肉传入放电的放电特性

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

尼古丁诱导的钙信号和神经递质的释放了沿着腹侧海马轴突实时成像

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

间接神经元的星形胶质细胞共培养试验：一个<em&gt;体外</em&gt;设置 - 为神经元的神经胶质细胞相互作用的详细调查

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy

用于估计电惊厥治疗大鼠模型中细胞数的光学分级技术

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells ...