Optimization of mitochondrial isolation techniques for intraspinal transplantation procedures
Optimization of mitochondrial isolation techniques for intraspinal transplantation procedures
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DOI：
10.1016/j.jneumeth.2017.05.023
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发表时间：
2017-08-01
影响因子：
3
通讯作者：
Rabchevsky, Alexander G.
中科院分区：
文献类型：
--
作者：
Gollihue, Jenna L.;Patel, Samir P.;Rabchevsky, Alexander G.
背景：适当的线粒体功能对于维持正常的细胞生物能量和离子稳态至关重要。在严重组织损伤的情况下，如创伤性脑和脊髓损伤，线粒体受损和不受调节，导致细胞死亡。神经损伤后的线粒体移植是一个相对未开发的领域，其理论基础是用外源性呼吸能力线粒体代替受损的线粒体可以恢复整个组织的生物能量。新方法：优化了体外制备离体线粒体悬液的工艺。用涡轮绿色荧光蛋白（tGFP）标记从细胞培养中分离的线粒体，用于体外和体内成像和跟踪目的。结果：我们使用延时共聚焦成像技术发现外源性荧光标记线粒体在短暂共孵育后进入PC-12细胞。此外，我们发现线粒体可以注射到脊髓中，免疫组织化学证据表明宿主细胞在24小时内摄取线粒体。与现有方法相比，我们的方法利用转基因荧光标记线粒体，这是一种无毒且光稳定的替代方法。向分离的线粒体添加底物有助于移植前室温下恢复III态呼吸。这些实验描述了使用转基因细胞系分离耦合良好的线粒体的精细方法，这些线粒体具有永久荧光标记，可以实时跟踪体外移植的线粒体，以及原位成像。结论：这些技术为检测线粒体移植对脊髓损伤及其他神经损伤动物模型的潜在治疗作用奠定了基础。(C) 2017 Elsevier B.V.版权所有
Background: Proper mitochondrial function is essential to maintain normal cellular bioenergetics and ionic homeostasis. In instances of severe tissue damage, such as traumatic brain and spinal cord injury, mitochondria become damaged and unregulated leading to cell death. The relatively unexplored field of mitochondrial transplantation following neurotrauma is based on the theory that replacing damaged mitochondria with exogenous respiratory-competent mitochondria can restore overall tissue bioenergetics.New method: We optimized techniques in vitro to prepare suspensions of isolated mitochondria for transplantation in vivo. Mitochondria isolated from cell culture were genetically labeled with turbo-green fluorescent protein (tGFP) for imaging and tracking purposes in vitro and in vivo.Results: We used time-lapse confocal imaging to reveal the incorporation of exogenous fluorescently-tagged mitochondria into PC-12 cells after brief co-incubation. Further, we show that mitochondria can be injected into the spinal cord with immunohistochemical evidence of host cellular uptake within 24 h.Comparison to existing methods: Our methods utilize transgenic fluorescent labeling of mitochondria for a nontoxic and photostable alternative to other labeling methods. Substrate addition to isolated mitochondria helped to restore state III respiration at room temperature prior to transplantation. These experiments delineate refined methods to use transgenic cell lines for the purpose of isolating well coupled mitochondria that have a permanent fluorescent label that allows real time tracking of transplanted mitochondria in vitro, as well as imaging in situ.Conclusions: These techniques lay the foundation for testing the potential therapeutic effects of mitochondrial transplantation following spinal cord injury and other animal models of neurotrauma. (C) 2017 Elsevier B.V. All rights reserved.