VP is a participant in the Charit&#233; Clinical Scientist Program, funded by the Charit&#233; - Universit&#228;tsmedizin Berlin and the Berlin Institute of Health. TB is an SNSF PostDoc Mobility fellow. The authors receive grant support from EinsteinStiftung/A-2012-153 to PV.

伦理声明:涉及动物受试者实验是根据由Landesamt附耳GESUNDHEIT UND Soziales，柏林，德国（G0298 / 13）和ARRIVE标准规定的准则和规定执行，如适用。在这项研究中，10至12周龄的雄性C57BL / 6J小鼠。 1.横向慢性颅窗口准备<ol><li>用皮下注射氯胺酮（90毫克/千克）和赛拉嗪（10毫克/千克）的执行麻醉。测试与痛觉刺激足够的镇静作用。 </li><li>消毒手术器械和手术区域用70％的乙醇。 </li><li>从颈部用啮齿动物剃须刀的眼睛中取出头皮。 </li><li>在立体定位框架固定的头部。 </li><li>使用上双眼泛醇眼膏，以避免脱水。 </li><li>清洁手术区，以去除所有毛发和74.1％乙醇的3层和10％的2-心得消毒它。 </li><li>执行中线从颈部到用手术刀眼睛切口。 </li><li>跨度皮瓣4隆起缝线。 </li><li>通过用解剖刀到颞肌开始点刮它关闭在左半球小心取出骨膜。 注意:此制剂也用于创建牙科胶，其固定在盖玻璃良好的粘合面积。 </li><li>通过在使用microdrill骨瓣的周缘变薄颅骨执行与直径4mm的额顶叶开颅。应用生理盐水，同时钻探，以避免热损伤。 </li><li>提升骨瓣与插管并使用microforceps将其删除。 </li><li>用生理盐水仔细冲洗广泛。 </li><li>混合所述牙科胶，直到它具有适当的稠度和不流体（ 即，胶不应产生线程了）。将其放在周围的开颅骨头。 </li><li>放置一个玻璃盖的直径在准备好的胶水6毫米，次修复牙胶电子休息。可以肯定，这是滴水不漏。等到胶水是用钳子测试它干又硬。 注:另外一个灌溉加速固化过程。 </li><li>取出隆起缝线和关闭与皮肤缝线伤口。 </li><li>拉起鼠标的侧翼的皮肤上。插入皮下针，轻轻地替代0.5毫升无菌生理盐水皮下的体液平衡。 </li><li>手术后，保持动物的热回收笼90分钟。等到动物是让他们无人之前完全清醒。等待，直到动物它们返回到与其他动物的笼子前完全恢复。 </li><li>重复皮下盐水体积替代约12小时后，如在步骤1.16中描述。 </li><li>通过管饲法或直接手术（ 例如，对乙酰氨基酚（10毫克/毫升）或其他非甾体抗炎药）后总是适用镇痛到口腔。 </li><li>检查<html>动物手术后，每天的状况，始终在培养皿使饮食简单，避免了手术后的关键减肥提供泥地板上动物性食物。 注:活体显微镜可以在颅窗口制备后的第一天进行。 </li><li>应用异氟醚麻醉并固定在头支架的动物。打开皮肤缝合，并用干净棉球和无菌生理盐水的窗口。 24小时后，颅窗口应该由该时间点，这使得成像充满脑脊液。通过建立显微镜协议执行18成像。 </li></ol> 2.远端缺血注:缺血过程应CW准备后进行大约5天。这最小化从所引起的CW制备与中风引起的免疫反应的免疫反应的干扰。 "SRC ="/文件/ ftp_upload / 54701 / 54701fig1.jpg"/&gt; 图1.概述远端缺血的。答:这是关于运前的船只很好的概述。后第一次接触双极B的船只。之后第二双极接触C.的船只。 D.有关船只，目前已完全关闭的概述。 E.最后概述与较低的放大倍率。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54701/54701fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol><li>麻醉使用麻醉面罩和适当麻醉政权的老鼠，在咨询的兽医人员（ 例如，诱导1.5 - 通过 1.5％异氟醚2/3 N 2 O和1/3 O 2 - 2％异氟醚和1.0保养蒸发器）。 </li><li>刮胡子，除去毛和消毒的皮肤和周围的皮毛与相应的代理（例如，70％乙醇），并随后干燥。 </li><li>保持体温的小鼠在36.5℃，在手术过程中±0.5°C。反馈控制的加热垫，根据鼠标的直肠温度下加热，强烈建议。 </li><li>放置动物中的横向位置。固定在麻醉面罩的鼻子和调整异氟烷浓度为1.0 - 1.5％。 </li><li>应用湿药膏（泛醇），以两只眼睛。 </li><li>用于CW准备做皮肤切口。 </li><li>轻轻分开皮肤与下方识别颞肌。 </li><li>调整高频发生器的能量（5 - 7 W）和使用双极性模式。 </li><li>使用电镊子和小心从头骨移除颞肌，创造了肌瓣，无需完全除去肌肉。 </li><li>确定马华在颞区的喙部分透明的头骨下方，背侧复古眼眶升窦。如果马华分叉不能被识别，试图找出最船只喙。 </li><li>薄MCA分支与microdrill同时不断冲洗，以避免热损伤上面的头骨。 </li><li>提起与插管骨​​和microforceps删除它。 </li><li>减少高频发生器的能量到3 - 5 W. </li><li>从上面的方法动脉，轻轻两侧双极钳触碰它无需提起容器。 </li><li>近端和远端凝结动脉至脉管分叉。 </li><li>等待30秒，然后轻轻触摸动脉，以确保血液流动被永久中断。如果观察到一个再通重复电。 </li><li>固定用1或2个针颞肌的肌肉插入覆盖骨缺损，如果可能的话。 </li><li>缝合伤口和动物放入加热的回收箱。一般情况下，挥发性ANEST后迅速恢复的动物hesia。 </li><li>对于体积替代，应用0.5毫升无菌盐水皮下的，如在步骤1.16中描述。 </li><li>手术后，让动物们留在加热回收笼90分钟。等到动物是让他们无人之前自觉。其中只有返回与其他动物的笼子时，他们完全康复。 </li><li>重复体积的取代，如在步骤1.16所解释的，经过12小时。 </li><li>申请通过饮水（ 例如，对乙酰氨基酚（10毫克/毫升）或其他非甾体抗炎药）术后镇痛。 </li><li>每天检查手术后的动物的医疗条件。在地板上的培养皿提供捣碎动物食品，以简化的饮食，并尽量减少术后体重减轻。 </li></ol> 3.假治疗<ol><li>相同执行所有程序，步骤1和2，上面包括CW准备 - 除了不凝结暴露大脑中描述tery。 </li></ol>

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The authors have nothing to disclose.

中风是长期的伤残和死亡全世界1的主要原因之一。除了急性期治疗，研究新的方法和机制，加快和提高中风后恢复仍然是一个主要的医疗目标7。试验行程研究者经常使用局灶性脑缺血啮齿动物模型。实际上，模型诱导瞬时或永久MCAO模仿最常见的类型患者4局灶性脑缺血中的一个。除了MCA的近端闭塞，为远侧MCAO手术闭塞灯丝模型可能是在实验性卒中研究5,19中最常用的模型。这里，我们描述永久远侧MCAO的基本技术有横向连续组合，将提供对在老鼠纵向活体显微镜的机会。高病灶体积的可重复性，以及非常低死亡率，特别是对于研究的长期结果，ARE本小鼠模型的主要优点。在此皮层中风模型，血管中的笔划区和梗死周围区域可以通过慢性CW被可视化。使用多荧光萤光videomicroscopic系统，血流动力学和循环细胞的动态招募可以可视化。血管是通过使用荧光标记的大分子的可视化，如葡聚糖或白蛋白。细胞可以通过荧光染料或通过遗传模型，如与GFP阳性的动物的骨髓嵌合体进行标记。另外，为了研究细胞 - 细胞相互作用和血管外细胞的动态，双光子激光扫描共焦显微镜可以应用。可以进行成像到低于皮层表面250微米。再次，血管使用荧光标记的大分子染色，细胞是遗传标记（ 例如，通过使用转基因GFP的巢蛋白小鼠）。 颅窗口手术是在不打开硬脑膜经由开颅进行。一个主要缺陷是意外伤害与microdrill打开颅骨下面时硬脑膜层和皮质。因此，这种技术要求，以避免到硬脑膜和皮质，其诱导免疫反应和影响的显微镜结果损坏了一些技术技能。可替代地，一个潜在的稀疏颅骨模型由因为剩余颅骨不太可靠显微镜质量的限制，尤其是在长期的。频繁，重复的颅骨变薄是必要的，而在连续波模式，窗口质量持续数月，直到颅骨再生或硬脑膜增厚影响成像质量14,20。这种模式有稀疏的颅骨制剂的调制是可能的。硬脑膜层应留在脑，以避免皮质任何损伤或激发。仅当直接施用于缺血区中的实验模型期望可以硬膜被重新感动，仔细，不伤害任何桥静脉。 在对比模型组合CW和通过循环光敏剂的辐射诱导靶向血管闭塞，导致只有非常小的缺血性病变，远侧MCAO模型模拟的大多数都位于皮质MCA区13人类笔划。为了避免与一个短暂的炎症反应的干扰，由于CW准备，窗口应几天远端缺血手术前准备。 一些测试，以评估在啮齿类动物中的功能，以及行为方面是可用的（ 例如，步态分析，旋转试验，极测试，粘合剂去除试验，楼梯测试，旷场试验和Morris水迷宫）21。在所有这些测试中，遭受缺血小鼠比对照动物相对于短期和中期成果进行成功少。然而，关于评估长期结果，必须承认，功能测试的敏感性是远端MCAO方面非常有限，以及温和近端MCAO 19,21-23。 远端MCAO，由训练有素的医生所进行的，可以少于20分钟内被诱导，并且能够产生高重现性的缺血性病变。但是，重复性要求混杂因素的彻底控制。在手术技术的差异可能导致梗塞面积24的差异。不同的小鼠品系会显示不同的中风结果由于菌株间脑血管解剖差异。此外，由于体温影响的神经元损伤和易感性缺血，与低温导致较小的病灶和热疗到更严重的缺陷25,26，温度控制和维护是在该模型中其它缺血模型27高度相关，以及。在一般情况下，生理参数，如血液pressu重和血液气体，是结果的重要的混杂因素和必须控制为28。此外，麻醉剂的选择是非常重要的，因为某些试剂可以施加直接的神经保护作用，或通过血管活性属性29间接地作用。因此，暴露于麻醉应当标准化，并保持尽可能短。最后，住房条件，如使用浓缩的，可能会影响中风结果为好，因此应该标准化和研究报告中列出30。以生产新的治疗方法，标准化，质量控制和报告发展的相关临床前结果是极为重要的31。

Stroke is among the principal causes of long-term disability and death worldwide1, coming second after coronary heart disease. In addition, stroke is the primary cause of long-term disability, underscoring its tremendous socioeconomic impact6-8. Beyond acute treatment, investigating new approaches and mechanisms to accelerate and enhance recovery after stroke remains a prime medical goal7. 
In the last few decades, data from experimental stroke research has contributed substantially to understanding the complex pathophysiological cascades triggered by ischemia9,10. Excitotoxicity, apoptosis, peri-infarct depolarization, and inflammation have been identified as the most relevant mediators of cell death following focal cerebral ischemia. Moreover, using animal models of cerebral ischemia, important concepts, diagnostic modalities, and therapeutic approaches have been developed and validated (e.g., "penumbra" and thrombolysis)11.
The availability of experimental stroke models, combined with non-invasive imaging modalities (e.g., magnetic resonance imaging (MRI), computed tomography, or laser speckle contrast analysis), enables the researcher to investigate hyperacute and chronic pathophysiological changes induced by the ischemic insult in a longitudinal manner12. Along with studying the spatiotemporal profile of the evolving lesion, changes resembling neuronal plasticity can be investigated and correlated to functional outcomes and histological findings. Within the last few years, further methodological advances have been made using the combination of cerebral ischemia models and in vivo microscopy via cranial windows13. These new techniques allow investigators to analyze the neurovascular unit at the cellular and molecular level, with great analytic power in the acute, subacute, and chronic phases following focal cerebral ischemia14. Moreover, in vivo microscopy imaging of microcirculatory dynamics has revealed novel aspects of cerebral microvasculature function and angioarchitecture, with significant pathophysiological relevance15-17. 
In this protocol, we present how to perform a chronic CW preparation lateral to the sagittal sinus and how to surgically induce a distal stroke underneath the window. This mouse model can be applied to sequential imaging of acute, subacute, and chronic changes following focal cerebral ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Binocular surgical microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000 C</td>
			<td></td>
		</tr>
		<tr>
			<td>Light source for microscope</td>
			<td>Zeiss</td>
			<td>CL 6000 LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad with rectal probe</td>
			<td>FST</td>
			<td>21061-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic frame</td>
			<td>Kopf</td>
			<td>Model 930</td>
			<td></td>
		</tr>
		<tr>
			<td>Anaethesia system for isoflurane</td>
			<td>Draeger</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abott</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont forceps #5</td>
			<td>FST</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont forceps #7</td>
			<td>FST</td>
			<td>11271-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Bipolar Forceps</td>
			<td>Erbe</td>
			<td>20195-501</td>
			<td></td>
		</tr>
		<tr>
			<td>Bipolar Forceps&#160;</td>
			<td colspan="2">Erbe&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 20195-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdrill</td>
			<td colspan="2">FST&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 18000-17&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>FST</td>
			<td>12010-14</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 silk suture</td>
			<td>Feuerstein, Suprama</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7-0 silk suture</td>
			<td>Feuerstein,Suprama</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 silk suture</td>
			<td>Feuerstein, Suprama</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Veterinary Recovery Chamber</td>
			<td>Peco Services</td>
			<td>V1200</td>
			<td></td>
		</tr>
	</tbody>
</table>

lateral chronic cranial window preparation enables in vivo observation following distal middle cerebral artery occlusion in mice

局灶性脑缺血（即缺血性中风）可能会导致重大的脑损伤，导致神经功能严重受损并因此导致运动和认知障碍的主机。它的高患病率带来了严重的健康负担，中风是长期的伤残和死亡全世界1的主要原因之一。神经功能的恢复是，在大多数情况下，只有部分。迄今为止，治疗选择是非常有限的，尤其是由于对溶栓2,3窄时间窗。确定的方法来加速从中风复苏仍尖端医疗的目标;然而，这已经阻碍了机械见解不足到恢复过程。试验行程研究者经常使用局灶性脑缺血啮齿动物模型。除了急性期，中风的研究越来越集中于亚急性期和慢性期脑缺血。大多数研究者中风申请永久或TRAN在小鼠或大鼠大脑中动脉闭塞过性。在患者中，MCA的闭塞是缺血性中风4的最常见的原因之一。除了使用灯丝模型MCA的近端闭塞，远端MCA的外科闭塞可能是在实验性卒中研究5中最常用的模型。远端（到lenticulo-纹动脉的分支）马华支闭塞通常免去了纹状体和主要影响大脑皮层。血管闭塞可以是永久的或暂时的。相对于长期预后病变体积和非常低的死亡率高重复性是这种模式的主要优点。在这里，我们将演示如何进行慢性颅窗口（CW）准备外侧为矢状窦，事后如何手术诱导采用开颅手术方法的窗口下方的远端行程。这种方法可应用于以下通过缺血的急性和慢性的变化序列成像外延照射，共焦激光扫描，和双光子活体显微镜。

时间轴和有代表性的结果示于图2和3。颅窗口制备，用小颅窗口外侧上矢状窦（ 图2 B，C，D）的结果在由有经验的外科医生进行的时非常低的死亡率和发病率。所有10只动物存活，所有慢性的CW可用于高质量成像，甚至手术后28天。有一个与伤口感染或其他并发症没有问题。 由于短曝光挥发性麻醉并只有轻微的脑损伤，约10 - 远侧MCAO后15分钟，并转移至加热的恢复笼中，所有的动物都清醒，在回收笼子自由移动，并与同窝相互作用。在一般情况下，远端MCAO模型的死亡率小于5％，主要发生血管我的结果njury和缺血手术过程中出血以后。在下面远端缺血28天的观察期内死亡率只发生非常罕见。形态，病变可使用组织学或MRI（ 图3的A，B）进行评估。此外，MRI测量提供了机会，以评估纵向的方式病变体积和进展。一种MRI进行24小时后缺血清楚描绘位于慢性CW下方的缺血性损伤，而下面的假手术，无损伤皮层组织中发现（ 图3A）。在MRI结果清楚地显示病变部位的严格限制到皮质，从而不放过纹状体，在对比的是丝状MCAO模型（ 图3B）。横向慢性CW制备使皮质脉管和微循环的长期可视通过落射荧光显微镜（ 图3C中 ，上半部分），并通过双光子显微镜皮质下区域（Figu重新3C，下部）。此外，也可以将图像的分子途径和细胞 - 细胞相互作用，用荧光标记的细胞或自体荧光测量，如荧光寿命成像。 如图3D中所示，远侧MCAO模型导致高度重现缺血性损害。关于梗死体积，我们预期在一组手术低于15％的标准偏差。如上所述，在对比近端MCAO模型，死亡率是相当低的远端模型5。使用顺序MRI，病变，体积和水肿进展局灶性脑缺血进行评估。在24小时和永久远端缺血后96小时MRI显示T2高信号的无显著进展。 <img alt="图2" src="/files/ftp_upload/54701/54701fig2.jpg" /> 图2:慢性颅窗口准备。 （A）代表时间表。 &lt;强&gt;（B）的标记，其中开颅发生，横向于颞肌的面积，内侧上矢状窦，和背侧前囟。 （C）开颅手术后，脑表面，具有完整的硬脑膜层和到位的玻璃盖;圆圈表示远端MCAO后缺血区的位置。 （D）的具有固定护罩玻璃成品慢性颅窗口，准备了几个星期（B =前囟，TM =颞肌，SSS =矢状窦，局部缺血的AI =区域）重复成像。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54701/54701fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54701/54701fig3.jpg" /> 图 3: 横向慢性CW和远端缺血或假手术相结合。 （一）MRI SH之后进行24小时上午手术不显示任何损伤皮层组织。 （B）中的MRI进行24小时后缺血清楚描绘位于慢性CW下方缺血损伤（*）。 （C）的活体落射荧光成像（上部）和皮质脉管的双光子成像（下部）。 （D）脑梗死体积通过MRI在24小时评估和缺血后96小时显示平均病变13.162.3毫米3在24小时和96小时体积12.21.9毫米3。每个点代表一个单独的动物（每组n = 10只动物，平均值±SEM）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54701/54701fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice at JoVE.com

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

远端大脑中动脉分支（缺血）的手术闭塞是实验性卒中研究常用的模式。该原稿描述永久MCAO的基本技术，有横向颅窗口的插入，这提供了在小鼠中纵向活体显微镜的机会相结合。

横向慢性颅窗口准备启用<em&gt;在体内</em&gt;观察继末节小鼠大脑中动脉闭塞

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

lateral-chronic-cranial-window-preparation-enables-vivo-observation

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

11.5K 次观看.  Charité-Universitätsmedizin. 远端大脑中动脉分支（缺血）的手术闭塞是实验性卒中研究常用的模式。该原稿描述永久MCAO的基本技术，有横向颅窗口的插入，这提供了在小鼠中纵向活体显微镜的机会相结合。 

视频: Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

焦距脑缺血模型大鼠大脑中动脉的血管内缝合闭塞

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

科研

神经科学

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

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

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 ...

Piezo High Accuracy Surgical Osteal Removal (PHASOR): A Technique for Improved Cranial Window Surgery in Mice

Multiphoton microscopy has been widely adapted for imaging neurons in vivo. Repeated imaging requires implantation of a cranial window or repeated thinning of the skull. Cranial window surgery is typically performed with a high speed rotary drill, and many investigators find it challenging to prevent the drill from damaging the delicate dura and blood vessels. Extensive training and practice is required to remove the bone without damage to underlying tissue and thus cranial window surgery can be difficult, time consuming, and produce tissue damage. Piezoelectric surgery, which is extensively used for maxillofacial and dental surgery, utilizes ultrasonic vibrations to remove bone without damaging soft tissues. We have developed a method applying piezoelectric surgery to improve cranial window surgery in mice in preparation for multiphoton imaging. Comparisons within our lab find that the method requires less surgery time and has a lower average rate of complications due to dural bleeding than cranial window surgery with a rotary drill.

Piezo High Accuracy Surgical Osteal Removal PHASOR: A Technique for Improved Cranial Window Surgery in Mice

Piezoelectric surgery has led to improvements in human maxillofacial and dental surgery. We have developed a protocol to optimize piezoelectric surgery for cranial window surgery in mice.

压电高精度手术骨切除 (相量): 改进小鼠颅骨窗手术的技术

Multiphoton microscopy has been widely adapted for imaging neurons in vivo. Repeated imaging requires implantation of a cranial window or repeated ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

模拟小鼠中风:通过颈外动脉的短暂性脑中动脉闭塞

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

植入颅窗以在清醒小鼠 中重复体内 成像

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. Development of these tools is crucial for understanding complex behaviors and cognition and for advancing clinical applications. However, it remains a challenge to densely record from cell populations stably and continuously over long time periods. Many popular electrodes, such as tetrodes and silicon arrays, feature large cross-diameters that produce damage upon insertion and elicit chronic reactive tissue responses associated with neuronal death, hindering the recording of stable, continuous neural activity. In addition, most wire bundles exhibit broad spacing between channels, precluding simultaneous recording from a large number of cells clustered in a small area. The carbon fiber microelectrode arrays described in this protocol offer an accessible solution to these concerns. The study provides a detailed method for fabricating carbon fiber microelectrode arrays that can be used for both acute and chronic recordings in vivo. The physical properties of these electrodes make them ideal for stable and continuous long-term recordings at high cell densities, enabling the researcher to make robust, unambiguous recordings from single units across months.

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

This protocol describes a procedure for constructing carbon fiber microelectrode arrays for chronic and acute in vivo electrophysiological recordings in mouse (Mus musculus) and ferret (Mustela putorius furo) from multiple brain regions. Each step, following the purchase of raw carbon fibers to microelectrode array implantation, is described in detail, with emphasis on microelectrode array construction.

用于慢性和急性体内记录的碳纤维微电极阵列 的 构建和实现

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. ...

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

小鼠脑中动脉闭塞诱导脑卒中后脑肌粘连血管病治疗模型

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The ...

Assessment of Thermal Damage from Robot-Drilled Craniotomy for Cranial Window Surgery in Mice

Cranial window surgery allows for the imaging of brain tissue in live mice with the use of multiphoton or other intravital imaging techniques. However, when performing any craniotomy by hand, there is often thermal damage to brain tissue, which is inherently variable surgery-to-surgery and may be dependent on individual surgeon technique. Implementing a surgical robot can standardize surgery and lead to a decrease in thermal damage associated with surgery. In this study, three methods of robotic drilling were tested to evaluate thermal damage: horizontal, point-by-point, and pulsed point-by-point. Horizontal drilling utilizes a continuous drilling schematic, while point-by-point drills several holes encompassing the cranial window. Pulsed point-by-point adds a &#34;2 s on, 2 s off&#34; drilling scheme to allow for cooling in between drilling. Fluorescent imaging of Evans Blue (EB) dye injected intravenously measures damage to brain tissue, while a thermocouple placed under the drilling site measures thermal damage. Thermocouple results indicate a significant decrease in temperature change in the pulsed point-by-point (6.90 &#176;C &#177; 1.35 &#176;C) group compared to the horizontal (16.66 &#176;C &#177; 2.08 &#176;C) and point-by-point (18.69 &#176;C &#177; 1.75 &#176;C) groups. Similarly, the pulsed point-by-point group also showed significantly less EB presence after cranial window drilling compared to the horizontal method, indicating less damage to blood vessels in the brain. Thus, a pulsed point-by-point drilling method appears to be the optimal scheme for reducing thermal damage. A robotic drill is a useful tool to help minimize training, variability, and reduce thermal damage. With the expanding use of multiphoton imaging across research labs, it is important to improve the rigor and reproducibility of results. The methods addressed here will help inform others of how to better use these surgical robots to further advance the field.

Cranial windows have become a ubiquitously implemented surgical technique to allow for intravital imaging in transgenic mice. This protocol describes the use of a surgical robot that performs semi-automated bone drilling of cranial windows and can help reduce surgeon-to-surgeon variability and partially mitigate thermal blood-brain barrier damage.

机器人钻孔开颅术对小鼠颅窗手术热损伤的评估

Cranial window surgery allows for the imaging of brain tissue in live mice with the use of multiphoton or other intravital imaging techniques. However, ...

建立经颅大脑中动脉闭塞小鼠模型

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

作者聚焦:中风研究的创新技术和未来方向 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes.

改进的经颅大脑中动脉闭塞模型研究老年小鼠脑卒中结局

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. ...