Name: excitotoxic stimulation of brain microslices as an in vitro model of stroke
Uploaded: 2014-02-04T14:45:00
Description: Watch this Scientific Journal Video about Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke at JoVE.com

这项工作是由国家健康和医学研究委员会澳大利亚，猎人医学研究所，以及纽卡斯尔大学支持的研究经费。

所有的程序都来自纽卡斯尔大学动物护理和伦理委员会，并按照相关指引及法规，包括新南威尔士州动物研究法，新南威尔士州动物研究法规和实务的澳大利亚守则进行审批保养和科学用途的动物。 1。脑组织解剖<ol><li>断头牺牲12周岁雄性大鼠。老鼠可以是由惊人的和断头处死，或者可以先异氟醚麻醉（5％感应，1.5-2％维护）在70％的N 2和30％O 2，然后斩首?...

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本文中， 体外技术microslices可用于检查涉及兴奋性中毒和缺血介导的细胞死亡在完整成熟的脑组织的分子机制的生成进行说明。该技术生产的活组织（ 图1-3），即代谢类似于较大的器官切片15。此外，这种microslice模式紧密对应观察下面的体内 10介导的兴奋性毒性脑损伤的反应。 90秒脑 microslices与AMPA，NMDA或谷氨酸体外刺激诱 ​​导多种分子...

中风的最常见的形式是缺血性中风，当脑血管变得闭塞而发生。该组织缺血导致从停止血流引起膜的去极化普遍，兴奋性神经递质的释放，细胞内钙，从而导致细胞死亡的活性的持续升高的途径1。这个过程已经被称为“兴奋性中毒”，并且是参与神经元死亡的一个常见的途径由多种病症，包括中风2制备。参与兴奋性毒性及其他神经细胞死亡的级联信号通路的抑制是一个有吸引力的方式来限制中风后神经元损伤。 确定在兴奋性毒性和缺血性中风的确切分子机制可以用整体动物系统时会很困难。因此，初级胚胎和“神经元样”（ 例如 neuroblasto毫安和腺癌永生化线）细胞培养系统经常被使用。这些模型的主要优点是它们易于操作的，相对低成本，和细胞死亡可容易地测量和定量。然而，信号通路可以通过使用3,4的培养条件，以及未成熟的神经元改变和永生线可以表达不同的受体和较成熟的大脑5-8信号分子。此外，培养的神经元只允许一个小区类型的检测（或2，如果一个共存系统使用的），而完整的脑组织是异质的，含有多种与彼此交互的细胞类型。器官切片培养系统（薄植脑组织）也被使用，并且这些模型使细胞的异质群体的研究，因为他们被发现在体内 。但是，可以从每个动物使用这种技术时获得的组织只有数量有限，切片可未进行培养，只要永生化细胞系，并且介质给长期培养可导致改变的信号转导途径和受体的切片。尽管成熟的大脑可以被用来生成器官切片，从不成熟的脑切片更适于培养，而且更普遍使用。因此，有必要开发一种模仿或代表完整成熟的大脑，是易于使用，其中神经信号传导通路可以检查模型。 本文中， 在体外 ，涉及可用于阐明参与细胞死亡的分子机制下列的兴奋毒性或缺血性损伤完好的神经组织的技术进行说明。这种技术减少了要执行的实验所需的动物数量，是可重现的，并产生其行为在代谢上类似的方式来放大器官切片活组织。此外，神经回路，细胞相互作用，和突触后共同mpartment部分仍然完好。用生理缓冲使细胞膜“重新封装”，并且使细胞恢复其原来的膜电阻9。这种脑片模型能够忠实地模仿观察到下列介导的兴奋性毒性脑损伤10的反应，并且可以用来检查涉及中风的分子机制。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
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		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Guillotine</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Used to decapitate animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical equipment</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Forceps, scissors, tweezers, etc, for brain removal and dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">McIlwain chopper</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Used to generate 100-400 &#956;m brain sections.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>McIlwain choppers are manufactured/distributed by a range of companies including Mickle Engineering, Harvard Apparatus, Campden Instruments and Ted Pella.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Round bottom plastic tubes</td>
			<td style="width:71px;">Greiner</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water bath</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>For keeping tissue at 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aerating apparatus</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Used to keep microslices in a humidified, oxygenated environment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Flat bottomed polystyrene tubes</td>
			<td style="width:71px;">Nunc</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dounce Homogenizer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

excitotoxic stimulation of brain microslices as an in vitro model of stroke

检查涉及神经病理症状的分子机制，如缺血性中风，可以使用整个动物系统时是困难的。因此，伯或“神经元样”细胞培养系统通常利用。虽然这些系统相对容易的工作，并且是有用的模型系统中各种功能的结果（如细胞死亡）可以很容易量化的研究成果和途径培养未成熟的神经元（如兴奋毒性介导的细胞死亡途径）不一定是相同的成熟大脑观察到，或者在完整的组织。因此，有必要开发模型，其中在成熟的神经组织的细胞机制可以被检查。我们已经开发出一种体外技术，可以用于研究各种在完好的神经组织分子途径。本文描述的技术利用大鼠皮质组织，但该技术可以适用于使用组织从多种物种（如小鼠，兔，豚鼠，鸡）或脑区域（例如，海马，纹状体， 等等 ）。此外，各种刺激/治疗方法可以被使用（例如，兴奋毒性，抑制剂的施用等 ）。总之，本文所述的脑切片模型可用于研究多种参与兴奋性毒性介导的脑损伤的分子机制。

使用此过程产生Microslices是可行的，并有多种物种（例如大鼠，小鼠和鸡），可用于产生microslices。已被利用的可行性的三个独立的措施：呼吸速率（ 图1），腺嘌呤核苷酸的比例（ 图2），和组织的钾含量（ 图3）。采用这些措施，它已经证明，microslices保持活力至少2小时后生成。 脑microslices可以用来检查各种信号转导分子，下列各种刺?...

Watch this Scientific Journal Video about Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke at JoVE.com

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

我们已开发了可用于检查涉及兴奋性毒性介导的脑损伤的分子机制的脑切片的模型。这种技术产生可行的成熟的脑组织，并减少所需的实验动物数量，同时保持神经回路，细胞相互作用，和突触后车厢部分完好无损。

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