Ultra fast in vivo microwave irradiation for enhanced metabolic stability of brain biopsy samples during HRMAS NMR analysis

Abstract

High resolution magic-angle spinning (HRMAS) NMR spectroscopy is a well established technique for ex vivo metabolite investigations but experimental factors such as ischemic delay or mechanical stress due to continuous spinning deserve further investigations. Cortical brain samples from rats that underwent ultrafast in vivo microwave irradiation (MWp group) were compared to similar samples that underwent standard nitrogen freezing with and without exposure to domestic microwaves (FN and FN+MWd groups). One dimensional 1H HRMAS NMR spectra were acquired and 16 metabolites of interest were quantified. Within each group 3 samples underwent long lasting acquisition (up to 15h). Statistically significant differences in metabolite concentrations were observed between groups for metabolites associated to post mortem biochemical changes and/or anaerobic glycolysis including several neurotransmitters. Spectral assessment over time showed a drastic reduction of biochemical variations in both MW groups. Only 2/16 metabolites exhibited significant signal variations after 15h of continuous spinning and acquisition in the MWp group. This number increased to 10 in the FN group. We confirmed limited anaerobic metabolism and post mortem degradation after ultra fast in vivo MW irradiation. Furthermore, spectra obtained after MWp and MWd irradiation exhibited an extremely stable spectral pattern over extended periods of continuous acquisition.