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Cryopreservation and functional analysis of cardiac autonomic neurons.
Journal of Neuroscience Methods ( IF 3 ) Pub Date : 2020-05-11 , DOI: 10.1016/j.jneumeth.2020.108724
Tess Torregrosa 1 , Sophie Webster 1 , Chiamaka Aghaizu 1 , Jonathan R Soucy 1 , Christopher Bertucci 1 , Leigh Plant 2 , Abigail N Koppes 3 , Ryan A Koppes 1
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BACKGROUND Generally, primary neurons are isolated and seeded within hours of isolation, but cryopreservation, documented for a small number of central and peripheral neuronal subtypes, can contribute to improved utility and reduce the cost of developing new in vitro models. The preservation of cells of the autonomic nervous system (ANS), specifically sympathetic and parasympathetic neurons, has not been explored. NEW METHOD In this work, we establish a method for preserving cardiac ANS neurons as well as evaluating the phenotypical changes of dissociated superior cervical ganglia (sympathetic neurons) and intracardiac ganglia (parasympathetic neurons) for up to a month of storage in liquid nitrogen. RESULTS Neuron populations maintained a viability of at least 35%, and the extent of neurite outgrowth was not different from fresh cells, regardless of the storage duration studied. Expression of tyrosine hydroxylase and choline acetyl transferase were maintained over one month of cryopreservation in sympathetic and parasympathetic populations, respectively. Electrophysiological recordings for both neuron types indicate sustained characteristic resting potentials, excitability, and action potentials after more than one month in liquid nitrogen. COMPARISON WITH EXISTING METHODS Primary cultures of the autonomic nervous system have been previously established for in vitro investigations. This is the first example of preserving primary ANS neuron cultures for long-term on-demand use. CONCLUSIONS This report describes a readily implemented method for cryopreserving sympathetic and parasympathetic neurons that does not alter neither morphological nor electrophysiological characteristics. This methodology expands the utility of ANS cultures for use in morphological and functional assays.

中文翻译:

心脏自主神经元的冷冻保存和功能分析。

背景技术通常,原代神经元是在分离后的几个小时内分离并接种的,但是低温保存已被证明具有少量的中枢和周围神经元亚型,可有助于提高实用性并降低开发新的体外模型的成本。尚未探索自主神经系统(ANS)的细胞,特别是交感神经和副交感神经元的保存。新方法在这项工作中,我们建立了一种保存心脏ANS神经元并评估解离的上颈神经节(交感神经元)和心内神经节(副交感神经元)的表型变化的方法,该方法最多可在液氮中存储一个月。结果神经元种群的生存力至少为35%,神经突向外生长的程度与新鲜细胞没有不同,不管所研究的存储期限如何。在交感神经和副交感神经群体中,冷冻保存一个月后,酪氨酸羟化酶和胆碱乙酰基转移酶的表达得以维持。两种神经元类型的电生理记录都表明,在液氮中放置一个多月后,它们具有持续的特征性静息电位,兴奋性和动作电位。与现有方法的比较先前已经建立了自主神经系统的原代培养物用于体外研究。这是为长期按需使用而保存主要ANS神经元文化的第一个示例。结论本报告描述了一种易于实施的冷冻保存交感神经和副交感神经元的方法,该方法不会改变形态或电生理特性。这种方法扩展了ANS培养物在形态学和功能分析中的用途。
更新日期:2020-05-11
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