Name: intra-arterial delivery of neural stem cells to the rat and mouse brain: application to cerebral ischemia
Uploaded: 2020-06-26T13:30:00
Description: Watch this Scientific Journal Video about Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia at JoVE.com

这项研究得到了以下支持：LC的AHA奖14SDG20480186，山西中医药大学2019-QN07的BZ学科创新团队，以及肯塔基脊髓和头部损伤研究信托基金14-12A的KES和LC奖。

有关动物主题的所有程序都得到肯塔基大学机构动物护理和使用委员会（IACUC）的批准，并采取了适当的照顾，以尽量减少与手术相关的压力或疼痛。
1. 注射导管和手术钩的准备
<ol><li>构造喷射导管（图1）。收集必要的材料，包括：MRE010、MRE025和 MRE050 管材、20 G、26 G 和 27 G 注射针（图2A）、600砂纸、超胶和双组分 5 分钟环氧树?...

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神经系统疾病的干细胞治疗仍处于早期探索阶段。一个主要问题是，没有既定的方法，充分交付SC或NSC到大脑。
虽然静脉注射（IV）、内皮内（IP）或腹内/脑内注射后，在大脑中可以检测到外源性SC/NSC，但每种分娩方法都有缺点。脑内可检测的种群估计在外周注射（IV或IP）时非常低，只占注射或注入细胞的一小部分。脑内注射产生非常焦的分布，并可能直接诱发脑损伤<sup clas...

干细胞（SC）疗法在治疗神经系统疾病方面有着巨大的潜力，包括中风、头部创伤和痴呆症1、2、3、4、5、6。,3,4,5,61,然而，向患病大脑提供外源性SCs的一种有效方法仍然存在问题,2、6、7、8、9、10、11、12、13。6,7,8,9,10,11,12,13通过外周传递途径（包括静脉注射（IV）或内丙酮注射）输送的SC在微循环中受到第一通过滤，特别是在肺、肝、脾和肌肉8、9、13、14,9,13中，14增加了非靶区细胞积累的机会。侵入性脑内注射方法导致局部脑组织损伤和注射,部位,,2、6、8、14、15、16附近的8,1415SCs分布非常有限。6我们最近建立了一种基于导管的动脉内注射方法，以提供外源性神经SC（NSC），本文在焦点缺血性中风的啮齿动物模型中进行了描述。我们诱导瞬态（1小时）缺血-再灌注损伤在一个半球使用硅橡胶涂层灯丝遮挡左中脑动脉（MCA）在小鼠或大鼠17，18，19。17,18,19在这个模型中，我们通过激光多普勒或激光斑点成像17，19，在ipilater半球观察到大约75-85%的脑血流（CBF）抑郁症，19产生一致的神经缺陷17，18，19。17,18,19
为节省时间，视频设置为以两倍于正常速度和常规外科手术，如皮肤准备和伤口闭合缝合，以及使用和设置电动注射器泵不介绍。在啮齿动物实验中风的中脑动脉闭塞（MCAO）模型的背景下，证明了NSC动脉内传递的方法。因此，我们包括瞬态缺血性中风手术，以便稍后演示如何使用同一动物的前手术部位进行第二次手术，即动脉内注射。通过评估外源性NSC的分布和生存，证明了啮齿动物中风模型中动脉内NSC交付的可行性。NSC治疗对降低脑病理和神经功能障碍的疗效将分别报告。

<table>
	<tbody>
		<tr>
			<td>20 G needle</td>
			<td>Becton &#38; Dickinson</td>
			<td>BD PrecisionGlide 305175</td>
			<td>preparation of injection catheter</td>
		</tr>
		<tr>
			<td>26 G needle</td>
			<td>Becton &#38; Dickinson</td>
			<td>BD PrecisionGlide 305111</td>
			<td>preparation of injection catheter</td>
		</tr>
		<tr>
			<td>27 G needle</td>
			<td>Becton &#38; Dickinson</td>
			<td>BD PrecisionGlide 305136</td>
			<td>preparation of injection catheter</td>
		</tr>
		<tr>
			<td>4-0 NFS-2 suture with needle</td>
			<td>Henry Schein Animal Health</td>
			<td>56905</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>6-0 nylon suture</td>
			<td>Teleflex/Braintree Scientific</td>
			<td>104-s</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>STEMCELL Technologies</td>
			<td>7922</td>
			<td>cell detachment solution</td>
		</tr>
		<tr>
			<td>blade</td>
			<td>Bard-Parker</td>
			<td>10</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>Buprenorphine-SR Lab</td>
			<td>ZooPharm</td>
			<td>Buprenorphine-SR Lab&#174;</td>
			<td>analgesia (0.6-1 mg/kg over 3 d)</td>
		</tr>
		<tr>
			<td>Calcium/magnisum free PBS</td>
			<td>VWR</td>
			<td>02-0119-0500</td>
			<td>NSC dissociation</td>
		</tr>
		<tr>
			<td>DCX antibody</td>
			<td>Millipore</td>
			<td>AB2253</td>
			<td>immunostaining</td>
		</tr>
		<tr>
			<td>GFAP antibody</td>
			<td>Invitrogen</td>
			<td>180063</td>
			<td>immunostaining</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein Animal Health</td>
			<td>50562-1</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>MCAO filament for mouse</td>
			<td>Doccol</td>
			<td>702223PK5Re</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>MCAO filament for rat</td>
			<td>Doccol</td>
			<td>503334PK5Re</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>MRE010 catheter</td>
			<td>Braintree Scientific</td>
			<td>MRE010</td>
			<td>preparation of injection catheter</td>
		</tr>
		<tr>
			<td>MRE025 catheter</td>
			<td>Braintree Scientific</td>
			<td>MRE025</td>
			<td>preparation of injection catheter</td>
		</tr>
		<tr>
			<td>MRE050 catheter</td>
			<td>Braintree Scientific</td>
			<td>MRE050</td>
			<td>preparation of injection catheter</td>
		</tr>
		<tr>
			<td>Nu-Tears Ointment</td>
			<td>NuLife Pharmaceuticals</td>
			<td>Nu-Tears Ointment</td>
			<td>eye care during surgery</td>
		</tr>
		<tr>
			<td>S&#38;T Forceps - SuperGrip Tips JF-5TC Angled</td>
			<td>Fine Science Tools</td>
			<td>00649-11</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>S&#38;T Forceps - SuperGrip Tips JF-5TC Straight</td>
			<td>Fine Science Tools</td>
			<td>00632-11</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>Superglue</td>
			<td>Pacer Technology</td>
			<td>15187</td>
			<td>preparation of injection catheter</td>
		</tr>
		<tr>
			<td>syringe pump</td>
			<td>Kent Scientific</td>
			<td>GenieTouch</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>Tuj1 antibody</td>
			<td>Millipore</td>
			<td>MAb1637</td>
			<td>immunostaining</td>
		</tr>
		<tr>
			<td>two-component 5 minute epoxy</td>
			<td>Devcon</td>
			<td>20445</td>
			<td>preparation of injection catheter</td>
		</tr>
		<tr>
			<td>Vannas spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>vascular clamps</td>
			<td>Fine Science Tools</td>
			<td>00400-03</td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>Zeiss microscope</td>
			<td>Zeiss</td>
			<td>Axio Imager 2</td>
			<td>microscopy</td>
		</tr>
	</tbody>
</table>

intra-arterial delivery of neural stem cells to the rat and mouse brain: application to cerebral ischemia

神经干细胞（NSC）疗法是中风、创伤性脑损伤和神经退行性疾病的新兴创新疗法。与颅内分娩相比，NSC的动脉内给法侵入性较小，在脑腹皮瘤中产生更分散的NSC分布。此外，动脉内分娩允许大脑循环中的第一通效应，减少细胞在周围器官（如肝脏和脾脏）中诱捕的可能性，这是与周围体外注射相关的并发症。在这里，我们详细介绍了小鼠和大鼠通过普通胡萝卜动脉（小鼠）或外体胡萝卜动脉（大鼠）在缺血性中风后将NSC传递到缺血性中风的异化半球的方法。使用标有 GFP 的 NSC，我们说明了在缺血损伤发生后 1d、1 周和 4 周内在整个啮齿动物异半球中实现的广泛分布，在缺血损伤场址内或附近具有较高的密度。除了长期存活，我们还显示G进P标记细胞在4周内分化的证据。此处为 NSC 描述的动脉内输送方法也可用于治疗化合物的施用，因此对多个物种的各种 CNS 损伤和疾病模型具有广泛的适用性。

在缺血性大脑中很容易检测到标有GPC的NSC，主要是在缺血性半球，特别是在五角细胞和沿着损伤边缘（图6）。在成像和分析过程中，检查员是单盲的。
例如，在注射后 1 d 时，在小鼠海马体内检测到 NSC。即使在这个早期点（图6A），NSC的子集也显示了凹陷陀螺中未成熟的神经元标记DCX的共表达（图6A）。
在中?...

Watch this Scientific Journal Video about Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia at JoVE.com

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

报告缺血性中风后，通过普通胡萝卜动脉（小鼠）或外体皮动脉（大鼠）提供神经干细胞的方法，可适应注射溶液或悬浮液。注射的细胞广泛分布于整个脑帕伦奇玛，在分娩后可检测到长达30天。

神经干细胞向大鼠和小鼠大脑的动脉内传递：对脑缺血的应用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

中风是全世界死亡和残疾的主要原因。虽然脑血管闭塞的血栓解和手术切除可以在部分患者中进行，但仍迫切需要有效的治疗中风。在这里，我们演示了一种基于导管的动脉内干细胞到缺血半球的传递，作为中风治疗的潜在方案。首先，我们介绍如何准备注射导管和手术钩。我们为动脉内注射设计了两种类型的注射导管。设计一用于注射不需要冲洗管的溶液或悬浮液。设计二允许注射神经干细胞，然后冲洗死量，以提供完整的体积。要构建一组喷射导管，您需要 26 个仪表和 20 个仪表指针、10 到 15 厘米长度的 MRE 25 导管和 3 厘米长的 MRE 50 和 MRE 10 导管。切断针尖，抛光砂纸上的金属端，重新打开管子并平滑边缘，然后用蒸馏水冲洗，以清理孔并清除碎屑。该图显示了剪下针尖后 20 量针的闭端，在抛光后用平滑孔重新打开。在显微镜下，将 20 测量针插入 MRE 50 管的端。弯曲 26 表针，然后穿透 20 表针尖的末尾下方的管子。用超级胶水固定 26 表针。然后嵌入两个针轮毂和管和环氧树脂，以建立一个坚实的块，以提高强度。你还需要三个手术钩，以保持组织的位置，以更好地暴露手术位。从 27 个测量针中切开一个半到两厘米长的针轴，并在砂纸上抛光两端，直到变暗。使用小型止血夹将轴弯曲成一端的挂钩，另一端为环形。插入 10 至 15 厘米长的 MRE 25 导管穿过环，用清晰的手术胶带固定。使用同一方法制作两个挂钩。钩和导管系统应浸泡在70%乙醇中过夜进行灭菌。涉及动物主体的所有程序都得到肯塔基大学机构动物护理和使用委员会的批准。我们使用ENT胚胎小鼠大脑培养GP，正神经干细胞。GFP阳性胚胎可以在荧光显微镜上的 FITC 通道中轻松可视化。而野生型胚胎没有显示荧光使用相同的暴露。只有当暴露时间增加100倍时，野生型胚胎的自荧光才可见。胚胎皮层被收集，并用于建立神经干细胞培养遵循制造商的协议。这些神经干细胞可以形成神经球，可以超过10代，他们可以区分成胶质是神经元。胚胎干细胞标记组，如纳米，SSEA1，Oct4和Sox3可用于识别干细胞属性，而神经球。神经球在介质中培养，遵循制造商的协议，并在第三和五段之间用于神经干细胞的传递。在小鼠和大鼠的左半球诱导缺血性中风，使用基于灯丝的中脑动脉闭塞，MCAO根据物种进行一些修改。在小鼠中，灯丝通过常见的胡萝卜状动脉CCA插入，并推进到内胡萝卜状动脉，ICA。而在大鼠中，由于工作空间较大，丝丝通过外皮动脉ECA引入国际癌症中心。在这里，我们演示小鼠手术。沿着颈部做中线切口后，使用钝解剖分离底层组织。使用由 27 量针轴制作的三个显微手术钩，实现更好的视场。黑色方块表示下一步的手术区域。左侧 CCA 上方的肌肉层小心打开，以暴露左侧 CCA 和迷走神经。按照 CCA 进行，直到您到达并识别分叉。应谨慎行事，不要伸展、取代或挤压此 CCA 或迷走神经。小心地打开清晰的结缔组织，覆盖CCA和迷走神经。释放此 CCA 下方的空间，并尽可能隔离 CCA 茎。螺纹双链六O编织尼龙手术缝合线下CCA。剪切中间点以留下 CCA 基础的缝合线。在 CCA 下放置另一个尼龙缝合线。在上缝合上系上一个滑点，保持松动。分离两个下部缝合线。用中间缝合线打滑结，保持松动。用下缝合线系上外科医生的结，并尽可能将结移至近端。向下修剪缝合端。进一步公开除分叉之外的 CCA。将上部松动滑到分叉上，拧紧，以防止血液回流。将硅橡胶涂层 70 尼龙 MCAO 缝合线放在 CCA 旁边。使用微型剪刀，在CCA上切一个小切口，紧放在下结旁边。通过此切口将 MCAO 缝合线尖插入 CCA，并将其推进到上结。调整中间结的位置并拧紧它，直到它能够将 MCAO 缝合线保持到位。不要过度拧紧结。松开上结，但在同一位置再打一个松滑结。将 MCAO 缝合线推进到 ICA 中。确认 MCAO 缝合线将分叉传递到 ICA 中。缝合的路径应该是相对直的，进入更深的组织，因为它的进展。检查缝合端的银色标记。确保它达到分叉。再次检查 MCAO 缝合线的方向。调整上部和中结，确保无血泄漏。进一步推进 MCAO 缝合线，拧紧两个上结，确保 MCAO 缝合到位。开始记录一小时的缺血时间。一个小时的缺血后，我们麻醉动物，并重新打开皮肤伤口。暴露手术区域。找到上部和中滑点。轻轻松开上结。轻轻拉出 MCAO 缝合线，直到白色硅橡胶部分达到中间结。拧紧上结，将 MCAO 缝合线保持到位，以避免出血。轻轻松开中间滑点。拉出 MCAO 缝合线，直到其端部通过上结。拧紧上结。将中间滑点放在 CCA 的切口上方。拧紧中间滑点。拆下上结。中间结的修剪缝合端。关闭皮肤伤口，让动物恢复。根据我们的经验，神经干细胞在中风后一到三天之间通过这条动脉途径安全注射。同样，小鼠和大鼠手术在神经干细胞注射方面也有细微的变异。在这里，我们演示小鼠手术。在中风后的第三天，麻醉动物，重新打开伤口，并重新暴露CCA。在 CCA 下铺设双绞线手术缝合线，并在中点切割，以获得两个缝合线。正确放置两个缝合线。用上缝合做一个滑点。放在分叉的下面。拧紧上滑口。用中间缝合做另一个滑点。在中风手术的剩余修剪结上方切一个小切口。修剪 MRE 10 导管的尖端，角度约为 45 度。使用此锋利的端在 CCA 上输入切口。小心正确调整入口角度和用于插入导管的力。否则，导管可能无法进入 CCA 或可能刺穿 CCA。插入导管并前进，直到尖端达到上结。拧紧中间滑点，将导管保持到位，并确保无血连杆。松开上结。用上缝合打滑结。调整 CCA 内导管的角度和两个结的位置，直到血液快速回流可见，表示导管和 CCA 流量之间的成功通信。启动注射器泵。导管中的血将被冲洗回CCA。五分钟后终止注射。把上结拉上。松开中间结并松开导管。把中间结拉上。修剪所有结。关闭伤口，让动物恢复。在动脉注射后，标有GP的神经干细胞大多存在于气管半球。在这里，我们显示注射后一天海马凹陷陀螺的分布。在ipilateral半球，一些GP阳性神经干细胞在皮层和线状体中局部，表达双皮质，一个不成熟神经元的标记。这些是来自假和中风动物的代表性图像，有或没有神经干细胞注射在注射后七天。在分娩后30天，只有轻微的自动荧光可以通过感染车辆注射的动物的 FITC 通道检测到。在注射的干细胞中，在受伤的大脑中可以发现标有GP神经干细胞，并表达各种细胞标记，如格洛标记GP或神经元标记图胡安。每个图像中的白色箭头分别指示表达 GFP 或 tujuan 标记的神经干细胞。总之，动脉内注射是一种可行的方法，用于中风治疗的神经干细胞的输送。神经干细胞可以存活下来，并分化成受伤大脑中不同的细胞类型。最后，这里介绍的动脉注射方法也可用于提供溶液和悬浮液。

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

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

基因传递到产后大鼠脑非心室质粒注射和电穿孔

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

从成年鼠脑的常规和非常规地区日益增长的神经干细胞

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

神经干细胞和祖细胞在小鼠脑开发基因毒性应激后的评估细胞周期进程

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

脑血流动力学和的光散射特性的同步评估<em&gt;体内</em&gt;大鼠脑使用多光谱漫反射成像

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...