Molecular Imaging Laboratory, Department of Radiology, Howard University, Washington, DC, USA.
Department of Pharmacotherapy and Outcomes Science, School of Pharmacy, Virginia Commonwealth University, Richmond, VA, USA.
NMR Biomed. 2021 Feb;34(2):e4451. doi: 10.1002/nbm.4451. Epub 2020 Nov 30.
The study of cerebral metabolites relies heavily on detection methods and sample preparation. Animal experiments in vivo require anesthetic agents that can alter brain metabolism, whereas ex vivo experiments demand appropriate fixation methods to preserve the tissue from rapid postmortem degradation. In this study, the metabolic profiles of mouse hippocampi using proton magnetic resonance spectroscopy ( H-MRS) were compared in vivo and in situ with or without focused beam microwave irradiation (FBMI) fixation. Ten major brain metabolites, including lactate (Lac), N-acetylaspartate (NAA), total choline (tCho), myo-inositol (mIns), glutamine (Gln), glutamate (Glu), aminobutyric acid (GABA), glutathione (GSH), total creatine (tCr) and taurine (Tau), were analyzed using LCModel. After FBMI fixation, the concentrations of Lac, tCho and mIns were comparable with those obtained in vivo under isoflurane, whereas other metabolites were significantly lower. Except for a decrease in NAA and an increase in Tau, all the other metabolites remained stable over 41 hours in FBMI-fixed brains. Without FBMI, the concentrations of mIns (before 2 hours), tCho and GABA were close to those measured in vivo. However, higher Lac (P < .01) and lower NAA, Gln, Glu, GSH, tCr and Tau were observed (P < .01). NAA, Gln, Glu, GSH, tCr and Tau exhibited good temporal stability for at least 20 hours in the unfixed brain, whereas a linear increase of tCho, mIns and GABA was observed. Possible mechanisms of postmortem degradation are discussed. Our results indicate that a proper fixation method is required for in situ detection depending on the targeted metabolites of specific interests in the brain.
本研究主要采用基于质子磁共振波谱（1H-MRS）的方法，对比分析了在体和原位条件下，经聚焦束微波辐射（FBMI）固定和未经固定两种方式下，小鼠海马代谢物的轮廓图谱。利用 LCModel 软件对 10 种主要的脑代谢物（乳酸盐（Lac）、N-乙酰天冬氨酸（NAA）、总胆碱（tCho）、肌醇（mIns）、谷氨酰胺（Gln）、谷氨酸（Glu）、γ-氨基丁酸（GABA）、谷胱甘肽（GSH）、总肌酸（tCr）和牛磺酸（Tau））进行了分析。经 FBMI 固定后，Lac、tCho 和 mIns 的浓度与异氟烷麻醉状态下在体实验所测值相当，而其他代谢物的浓度显著降低。除 NAA 减少和 Tau 增加外，FBMI 固定的脑组织在 41 小时内，其余代谢物均保持稳定。未经 FBMI 固定时，mIns（前 2 小时）、tCho 和 GABA 的浓度与在体实验所测值相近，但 Lac 水平较高（P <.01），NAA、Gln、Glu、GSH、tCr 和 Tau 水平较低（P <.01）。在未固定的脑组织中，NAA、Gln、Glu、GSH、tCr 和 Tau 至少在 20 小时内具有良好的时间稳定性，而 tCho、mIns 和 GABA 呈线性增加。讨论了死后降解的可能机制。本研究结果表明，针对特定脑区感兴趣的代谢物，在原位检测时需要采用适当的固定方法。
