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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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?…
Representative Results
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?…
Discussion
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 20 G needle | Becton & Dickinson | BD PrecisionGlide 305175 | preparation of injection catheter |
| 26 G needle | Becton & Dickinson | BD PrecisionGlide 305111 | preparation of injection catheter |
| 27 G needle | Becton & Dickinson | BD PrecisionGlide 305136 | preparation of injection catheter |
| 4-0 NFS-2 suture with needle | Henry Schein Animal Health | 56905 | surgery |
| 6-0 nylon suture | Teleflex/Braintree Scientific | 104-s | surgery |
| Accutase | STEMCELL Technologies | 7922 | cell detachment solution |
| blade | Bard-Parker | 10 | surgery |
| Buprenorphine-SR Lab | ZooPharm | Buprenorphine-SR Lab® | analgesia (0.6-1 mg/kg over 3 d) |
| Calcium/magnisum free PBS | VWR | 02-0119-0500 | NSC dissociation |
| DCX antibody | Millipore | AB2253 | immunostaining |
| GFAP antibody | Invitrogen | 180063 | immunostaining |
| Isoflurane | Henry Schein Animal Health | 50562-1 | surgery |
| MCAO filament for mouse | Doccol | 702223PK5Re | surgery |
| MCAO filament for rat | Doccol | 503334PK5Re | surgery |
| MRE010 catheter | Braintree Scientific | MRE010 | preparation of injection catheter |
| MRE025 catheter | Braintree Scientific | MRE025 | preparation of injection catheter |
| MRE050 catheter | Braintree Scientific | MRE050 | preparation of injection catheter |
| Nu-Tears Ointment | NuLife Pharmaceuticals | Nu-Tears Ointment | eye care during surgery |
| S&T Forceps – SuperGrip Tips JF-5TC Angled | Fine Science Tools | 00649-11 | surgery |
| S&T Forceps – SuperGrip Tips JF-5TC Straight | Fine Science Tools | 00632-11 | surgery |
| Superglue | Pacer Technology | 15187 | preparation of injection catheter |
| syringe pump | Kent Scientific | GenieTouch | surgery |
| Tuj1 antibody | Millipore | MAb1637 | immunostaining |
| two-component 5 minute epoxy | Devcon | 20445 | preparation of injection catheter |
| Vannas spring scissors | Fine Science Tools | 15000-08 | surgery |
| vascular clamps | Fine Science Tools | 00400-03 | surgery |
| Zeiss microscope | Zeiss | Axio Imager 2 | microscopy |
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Citar este artigo
Zhang, B., Joseph, B., Saatman, K. E., Chen, L. Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia. J. Vis. Exp. (160), e61119, doi:10.3791/61119 (2020).