Name: modeling stroke in mice: focal cortical lesions by photothrombosis
Uploaded: 2021-05-06T14:00:00
Description: Watch this Scientific Journal Video about Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis at JoVE.com

我们感谢免疫中风联盟（FOR 2879，从免疫细胞到中风恢复）的所有合作伙伴的建议和讨论。这项工作由德国福松斯格梅因沙夫特（DFG，德国研究基金会）在德国系统神经学集群（EXC 2145 SyNergy - ID 390857198）框架内的卓越战略下，以及根据LI-2534/6-1、LI-2534/7-1和LL-112/1-1的赠款资助。

本视频中报道的实验是根据使用实验动物的国家准则进行的，这些议定书得到了德国政府委员会的批准（德国慕尼黑，雷吉龙·冯·奥伯巴耶恩）。这项研究中使用的老鼠是雄性C57Bl/6J小鼠，10-12周大，由德国查尔斯河公司派遣。这些动物被安置在受控温度（22°C±2°C）下，周期为12小时，可获得颗粒食物和水 。
1. 材料和仪器的准备工作
<ol><li>溶解玫瑰孟加拉在0.9%盐水?...

<ol>
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提交的协议描述了光血栓形成的实验性中风模型，用561纳米激光照亮完好的头骨，并进行了玫瑰孟加拉的腹内注射。直到最近，这种模式的使用一直很低，但正在稳步增加。
此模型中中风诱导期间的死亡率不存在。手术过程中，由于麻醉并发症或在符合排除标准后做出牺牲，总死亡率低于5%。为保证该模型的低变异性和可重复性，建议以下排除标准：1） 操作时间超过 30 分钟...

缺血性中风仍然是21世纪 发达国家的主要死因和成人残疾，2017年全球约有270万人死于缺血性中风。即使科学界作出巨大努力，也很少有治疗方法。此外，由于排除标准如此之高，许多患者无法获得这些已经有限的选择，因此迫切需要新的治疗方法来改善中风后的功能恢复。
考虑到 体外 模型无法复制大脑的复杂相互作用，动物模型对于临床前中风研究至关重要。老鼠是中风研究领域最常用的动物模型。这些小鼠模型大多旨在通过阻断中脑动脉（MCA）内的血流来诱导梗塞，因为大部分人类中风病变位于MCU区域2。虽然这些模型可以更好地回顾人类中风病变，但它们涉及高梗塞体积变异性的抽搐手术。
自从罗森布卢姆和艾尔-萨班于1977年提出光血栓模型3，后来该模型应用于老鼠沃森等人，它已成为广泛应用于缺血性中风研究5，6。光血栓中风模型诱导局部和定义的皮质梗塞，由于以前注入血液中的光敏染料的光活化。这会导致暴露在光线下的船只局部血栓。简言之，在接触注射光敏染料的光线后，诱发内皮细胞膜局部氧化损伤，导致血小板聚集和血栓形成，随后局部扰乱脑血流7。
这种技术的主要优点在于其执行的简单性和将病变引向所需区域的可能性。与其他实验性中风模型不同，执行光血栓中风模型需要少量的手术专业知识，因为病变是通过对完整头骨的照明诱导的。此外，划定良好的边界（图2A和图5B）和灵活性诱导病变到特定的大脑区域，可以促进研究细胞反应内缺血或完整的皮质区域8。由于这些原因，这种方法适用于皮质可塑性细胞和分子机制的研究。
在过去几十年中，人们越来越担心研究小组之间缺乏可复制性，这引发了所谓的复制危机。在2015年10月协调了第一个临床前随机对照多中心试验研究后，一个改进临床前研究的拟议工具11、12、13被证实，独立实验室临床前研究不重复的一个原因是实验性中风模型和结果参数缺乏足够的标准化。因此，当免疫中风联合体成立（https://immunostroke.de/）时，一个旨在了解中风恢复机械原理背后的大脑免疫相互作用的合作，每个研究小组中所有实验性中风模型的标准化至关重要。
这里描述的是上述研究联合体中使用的光血栓模型诱导的标准化程序。简言之，一只动物接受了麻醉，在腹内接受了玫瑰孟加拉注射（10μL/g），从布雷格玛留下的3毫米完整的头骨立即被561纳米激光照亮20分钟（图1）。此外，还报告了分析此模型中风结果的相关理论和行为方法。所有方法都基于实验室制定和使用的标准操作程序。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>561 nm wavelenght laser</td>
			<td>Solna</td>
			<td>Cobolt HS-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma Life Science</td>
			<td>695092</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia system for isoflurane</td>
			<td>Drager</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ApopTag Peroxidase In Situ Apoptosis Detection Kit</td>
			<td>Millipore</td>
			<td>S7100</td>
			<td></td>
		</tr>
		<tr>
			<td>Bepanthen pomade</td>
			<td>Bayer</td>
			<td>1578681</td>
			<td></td>
		</tr>
		<tr>
			<td>C57Bl/6J mice</td>
			<td>Charles River</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Collimeter</td>
			<td>Thorlabs</td>
			<td>F240APC-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotons</td>
			<td>NOBA Verbondmitel Danz</td>
			<td>974116</td>
			<td></td>
		</tr>
		<tr>
			<td>Cresyl violet</td>
			<td>Sigma Life Science</td>
			<td>C5042-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Thermo Scientific CryoStarNX70</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>CLN Chemikalien Laborbedorf</td>
			<td>521005</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>CLN Chemikalien Laborbedorf</td>
			<td>522078</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 99%</td>
			<td>CLN Chemikalien Laborbedorf</td>
			<td>ETO-5000-99-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Macherey-Nagel</td>
			<td>432018</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>FST</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FST</td>
			<td>11616-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating blanket</td>
			<td>FHC DC Temperature Controller</td>
			<td>&#160;40-90-8D</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbot</td>
			<td>B506</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopentane</td>
			<td>Fluka</td>
			<td>59070</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Inresa Arzneimittel GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Speckle</td>
			<td>Perimed</td>
			<td>PeriCam PSI HR</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayor Scissors</td>
			<td>FST</td>
			<td>1410-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline PH: 7.4</td>
			<td>Apotheke Innestadt Uni Munchen</td>
			<td>P32799</td>
			<td></td>
		</tr>
		<tr>
			<td>Protective glasses</td>
			<td>Laser 2000</td>
			<td>NIR-ZS2-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Rose Bengal</td>
			<td>Sigma Aldrich</td>
			<td>198250-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Roti-Histokit mounting medium</td>
			<td>Roth</td>
			<td>6638.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline solution</td>
			<td>Braun</td>
			<td>131321</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomikroskop</td>
			<td>Zeiss</td>
			<td>Stemi DV4</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic frame</td>
			<td>Stoelting</td>
			<td>51500U</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Slides</td>
			<td>Thermo Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylacine</td>
			<td>Albrecht</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling stroke in mice: focal cortical lesions by photothrombosis

在发达国家，中风是导致死亡和成人残疾的主要原因。尽管对新的治疗策略进行了广泛的研究，但中风患者的治疗选择仍然有限。因此，需要对病理生理途径进行更多的研究，如中风后炎症、血管生成、神经元可塑性和再生。鉴于 体外 模型无法重现大脑的复杂性，实验性中风模型对于分析和随后评估这些机制的新药靶点至关重要。此外，迫切需要为克服所谓的复制危机而为所有程序制定详细的标准化模型。作为免疫冲程研究财团的一项努力，描述了一个标准化的光血栓小鼠模型，该模型使用玫瑰孟加拉的腹内注射和561纳米激光照亮完好的头骨。该模型允许在没有侵入性手术的情况下将中风的效果分配给大脑的任何皮质区域：因此，使中风的研究在大脑的各个区域。在本视频中，演示了光血栓模型中中风感应的手术方法以及理论分析。

此处描述的模型是玫瑰孟加拉注射的光血栓冲程模型，完整的头骨照明为 20 分钟，在光纤上恒定的 561 nm 波长和 25 mW 输出功率。虽然完整的光栓手术持续30分钟，但动物保持在低麻醉下，脑损伤适中。大约10分钟后，转移到他们的笼子，所有的动物都醒了，在笼子里自由移动，并与垃圾伙伴互动。
在中风诱导后24小时使用青紫罗兰色染色连续冠脑部分（图2A）</...

Watch this Scientific Journal Video about Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis at JoVE.com

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

这里描述的是光血栓中风模型，其中中风通过完整的头骨产生，在施用光敏染料后，使用激光照明诱导永久微血管闭塞。

小鼠模型中风：光血栓形成的焦皮质病变

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

光血栓中风模型方法可能适合细胞和分子研究皮质可塑性，因为边界有限，在皮层的不同区域诱导损伤的灵活性有限。光血栓形成模型与其他中风模型有三大优势，即将病变引向所需区域的可能性、高可重复性和低死亡率。适当的玫瑰孟加拉剂量是关键的成功和模型的稳定性。结果取决于技术规范，如激光功率、交叉应用正时和未受影响皮层的光学先令。首先溶解玫瑰孟加拉在0.9%盐溶液的最终浓度为每毫升10毫克。使用热铅消毒器对所有仪器进行细致消毒，并在手术前后使用微生物消毒喷雾对所有表面进行消毒。准备一个装满盐水溶液的注射器，以保持操作区域水分和准备麻醉气体。测量小鼠的体重，以调整要注射的玫瑰孟加拉的剂量。设置相关反馈控制加热垫，以保持鼠标体温。一旦小鼠完全麻醉并固定在立体定向框架中，轻轻插入直肠探头，在整个外科手术过程中监测小鼠的体温。将 Dex 泛酚眼药膏涂抹在眼睛上，用消毒剂清洁皮肤和周围毛皮。用剪刀做一个两到两厘米半厘米的纵向切口，并缩回暴露头骨。使用棉花轻轻取出腹膜并定位冠状缝合线。打开 561 纳米激光时戴上防护眼镜，在左侧标记加号 3 毫米，然后关闭激光，在标记的坐标上挂上直径为 4 毫米的孔的贴纸。将鼠标内注射玫瑰孟加拉。将激光束置于距离头骨四到五厘米的地方。打开激光并照亮头骨 20 分钟。通过涂抹两滴0.9%的盐水来补充头骨水分。在缝注伤口后，将鼠标置于37摄氏度的恢复室中，以便从麻醉中恢复过来。一小时后，将鼠标放回笼子里，放在温度控制室。连续冠心病脑部段的晶体紫罗兰色在中风诱导24小时后用于梗塞体积进入分析。这种中风模型的变异性较低，平均梗塞量为29.3立方毫米，占一个脑半球的23%。光粒体病变导致复合神经评分所示的中度和长期感觉运动损伤。手术后24小时，中风动物的神经评分发生了显著变化。尽管差异仍然存在，但中风小鼠会随着时间而改善。在受玫瑰孟加拉加照明的老鼠中，手术后24小时体重和体温下降。然而，在手术后的三天内，观察到了恢复情况。利用激光成像证实了缺血变化。结果表明，仅由玫瑰孟加拉或激光照明不会产生病变，而两者同时应用则产生一个圆形低沉灌水区，周围环绕着狭窄的寡头区域。手术后24小时评估梗塞体积的水晶紫罗兰色和隧道染色表明，玫瑰孟加拉或激光照明手术均未造成组织损伤。另一方面，玫瑰孟加拉加激光照明产生了一个划好分界病变。重要的是要测量动物的体重来调整玫瑰孟加拉注射。验尸官切换器的正确状态对于诱导动物之间一致的病变非常重要。由于此协议使头骨完好无损，甚至可以通过颅窗执行，因此可以与宽场或多光子在体内成像相结合。

Research

JoVE Journal

Neuroscience

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

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

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

总钙在神经元的电子探针X射线显微分析测量

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

建模星形细胞瘤发病机制<em&gt;在体外</em&gt;和<em&gt;体内</em&gt;使用星形胶质细胞或条件，遗传工程小鼠神经干细胞

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

实验脱髓鞘和小鼠脊髓髓鞘再生由溶血卵磷脂的焦点注射

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

大鼠皮层神经元丝状肌动蛋白（F-肌动蛋白）泪点的量化

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

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

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

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 ...

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

小鼠原代多元体的体外模拟？

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

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

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 ...