交付<em>在体内</em>急性间歇性缺氧新生儿鼠害总理下区源性神经祖细胞培养
Summary
本文介绍了管理间歇性缺氧的短期产后1-8天的小鼠或大鼠幼仔的方法。这种方法有效地引起上都在低氧暴露30分钟收获培养的神经祖细胞的一个强大的组织水平“启动效应”。
Abstract
Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to adequate numbers prior to transplantation. Identifying approaches to refine this process, to augment the production of all CNS cell types (i.e., neurons), and to possibly contribute to therapeutic cell therapy protocols is a high research priority. This report describes an easily applied in vivo “pre-conditioning” stimulus which can be delivered to awake, non-anesthetized animals. Thus, it is a non-invasive and non-stressful procedure. Specifically described are the procedures for exposing mouse or rat pups (aged postnatal day 1-8) to a brief (40-80 min) period of intermittent hypoxia (AIH). The procedures included in this video protocol include calibration of the whole-body plethysmography chamber in which pups are placed during AIH and the technical details of AIH exposure. The efficacy of this approach to elicit tissue-level changes in the awake animal is demonstrated through the enhancement of subsequent in vitro expansion and neuronal differentiation in cells harvested from the subventricular zone (SVZ). These results support the notion that tissue level changes across multiple systems could be observed following AIH, and support the continued optimization and establishment of AIH as a priming or conditioning modality for therapeutic cell populations.
Introduction
该方法的目标是可重复地提供全身降低环境氧的间歇发作新生儿啮齿动物。使用间歇性缺氧（1H）操纵干细胞生物学的基本原理源于体外细胞培养实验，其中的 O 2含量的生长培养基被改变。具体地，当相比于20％O 2，干/祖细胞群的细胞的扩展培养在3％ 的 O 2的结果增加的增殖的“标准”的条件下，降低的细胞凋亡和增加的产量神经1,2。
本组有全身IH的管理显著的经验，并已在呼吸可塑性3-7进行了IH的作用，广泛的研究。这项工作，最近发现，即慢性IH在啮齿动物的CNS 8-10增加神经发生，形成基础急性在体内的勘探缺氧作为预处理刺激（ 即 ，之前的组织收获）与神经干/祖细胞（NPC的）11的随后的培养。值得注意的是，当小鼠幼仔暴露于一个简短（<1小时）期间的急性间歇性缺氧（AIH），从室下区（SVZ）收获该细胞已经显著增加了对扩张为神经球或贴壁单层细胞的能力。该AIH协议也与一个“神经元命运的”转录因子（Pax6的）表达增加有关。
因此 ，在体内 AIH协议可能以“素”之前筹备文化提供了一种方法。例如，对于这种方法的应用可包括在移植之前扩大细胞群体进损伤的中枢神经系统，或者简单地增加培养细胞的神经元分化之前在体外实验 。另外，由于这是一种全身递送，任何器官，组织或细胞是类似的研究的候选者。因此，书面协议是可能适用于范围广泛的间歇氧操纵小型哺乳动物的研究。
有一定的优势，以这种方式。在其他发表的作品，新生儿被视为与大坝低压室 ，其中在治疗之前允许长期给药，少搬运垃圾，并维持治疗9时产妇接触。目前的方法绕过重复治疗，以繁殖雌性，或使用不同的坝每个实验。该协议还允许精确垫料匹配和年龄匹配的新生儿研究。有代表性的数据证明这种协议的另一个关键力量，即迅速与AIH在交付时，引发神经干细胞生物学的强大和一致的生物反应。这将建立一个先例，该协议引起组织特异性和细胞水平biologi从而改变细胞生物学CAL变化。
本报告将勾勒出详细的程序用于揭露啮齿动物幼仔AIH和SVZ细胞群体分析生长为神经球。
Protocol
Representative Results
Discussion
This work reports the development of a protocol to expose neonatal rodents to AIH. The parameters described here effectively alter in situ neural stem cell biology, which is observable over several rounds of cell passage. Specifically, AIH increases the number of non-adherent neurospheres, the expansion of cells within each neurosphere (refected by sphere diameter), the expansion of adherent NPC populations, and the presence of neuroblasts in both non-adherent and adherent populations. It should be emph…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors acknowledge funding sources responsible for this work: 5K12HD055929 (HHR), 5R01NS080180-02 (DDF).
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Mouse plethysmography chambers | Buxco | PLY4211 | |
| Flow meter | Porter | F150 | |
| Bias flow unit | AFPS | ||
| Baseline Gas Mix | Airgas | AIZ300 | Compressed Air |
| Hypoxic Gas Mix | Airgas | X03NI72C2000189 | 10% Oxygen, balance nitrogen |
| Oxygen Meter | Teledyne | AX-300 |
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