Institute of Metabolic Disease, Baylor Scott and White Research Institute, Dallas, TX, United States.
Institute of Metabolic Disease, Baylor Scott and White Research Institute, Dallas, TX, United States.
J Neurosci Methods. 2018 Sep 1;307:188-193. doi: 10.1016/j.jneumeth.2018.05.016. Epub 2018 May 23.
Assessing neurotransmitter metabolism in the brain is essential in studying the effects of drugs, dietary modification and characterizing transgenic mouse models of human neurodegenerative diseases. Regional brain concentrations of parent neurotransmitters and related metabolites are informative and provide a snap shot of the steady-state levels. The choice in method of sacrificing mice may differ from one laboratory to another, and the technique in removal of brain may have limitations depending on speed in which tissue can be dissected and frozen to prevent post-mortem changes.
In order to better assess neurotransmitter metabolism in an effective and standardized manner we evaluated microwave irradiation as a method of sacrificing mice. Mice were sacrificed by CO asphyxiation followed by cervical dislocation or microwave irradiation at 4 Kw for 1.1 s. Brain tissue was harvested into five regions and stored at -80 °C until analysis by either LC-MS/MS for acetylcholine, choline and GABA, or HPLC-EC for dopamine, serotonin and norepinephrine and related metabolites.
The results of our study showed considerable differences in the levels of neurotransmitters between the two methods of sacrifice. Overall, the concentrations of neurotransmitters were higher in mice sacrificed by microwave irradiation, except for GABA, which was lower.
COMPARISON WITH EXISTING METHOD(S): Previous microwave irradiation studies employed presently outdated equipment and neurotransmitter analysis methods, and were not as comprehensive.
The combination of microwave irradiation with LC-MS/MS and HPLC-EC detection allows accurate and sensitive measurement of several neurotransmitter systems in discrete mouse brain regions.
评估大脑中的神经递质代谢对于研究药物、饮食改变的影响以及表征人类神经退行性疾病的转基因小鼠模型至关重要。亲神经递质和相关代谢物的脑区浓度具有信息性，可提供稳态水平的快照。从一个实验室到另一个实验室，牺牲小鼠的方法选择可能会有所不同，并且由于组织可以被解剖和冷冻以防止死后变化的速度限制，因此去除大脑的技术可能存在局限性。
为了更有效地评估神经递质代谢并使其标准化，我们评估了微波辐射作为一种牺牲小鼠的方法。小鼠通过 CO 窒息然后颈椎脱位或 4 Kw 微波辐射 1.1 s 来牺牲。脑组织被收获到五个区域中，并储存在-80°C 下，直到通过 LC-MS/MS 分析乙酰胆碱、胆碱和 GABA，或通过 HPLC-EC 分析多巴胺、血清素和去甲肾上腺素及其相关代谢物。
我们的研究结果表明，两种牺牲方法之间的神经递质水平存在相当大的差异。总体而言，除 GABA 外，用微波辐射牺牲的小鼠中的神经递质浓度较高。
以前的微波辐射研究使用的是过时的设备和神经递质分析方法，并且不那么全面。
微波辐射与 LC-MS/MS 和 HPLC-EC 检测的结合允许在离散的小鼠脑区中准确和灵敏地测量几种神经递质系统。
