Introduction: In vivo ¹³C NMR measurements have indicated neurotransmitter cycling is supported by neuronal glucose oxidation¹. Anesthetics are routinely used during surgery, and for the measurements of energy metabolism in different neurological and neuropsychiatric disorders. However, the impact of anesthetics on GABAergic and glutamatergic, the major inhibitory and excitatory neurons, neuronal metabolic activity is not clear. Isoflurane and urethane are the most commonly used anesthetics in rodent studies. Isoflurane is believed to decrease the g-aminobutyric acid type A receptors (GABA_AR) activity³, while urethane (ethyl carbamate) affects excitatory and inhibitory signaling along with the ion channel activity⁴. This study aims to evaluate the impacts of isoflurane and urethane on glutamatergic and GABAergic neurometabolic activity across the brain.
Methods: All animal experiments were approved by the Animal Ethics Committee of CCMB, Hyderabad. Three months old male C57BL6 mice (n=24) were divided into the following groups: Group A: Awake (n=8); Group B: Isoflurane (n=8); Group C. Urethane (n=8). Mice were fasted for 4 hours prior to study. The anesthesia in Group B animals was maintained by 1.5% isoflurane mixed in air while in Group C intraperitoneal administration of urethane (1.5 g/kg). Mice were infused [1,6-¹³C₂]glucose post 45 min of anesthetics by a bolus variable infusion protocol⁴. The blood was withdrawn from the retro-orbital sinus at 8 min, and the cerebral metabolism was arrested using a Focused Beam Microwave Irradiation system (4 kW, 1 s), (MMW-05, Muromachi Kikai Co., Ltd. Japan) at 10 min after the [1,6-¹³C₂]glucose administration. The ¹³C labeling of brain metabolites was measured in extracts of different brain regions by ¹H-[¹³C]-NMR spectroscopy at 600 MHz NMR spectrometer (Bruker Biospin, Germany)⁵. The cerebral metabolic rates of glucose oxidations in glutamatergic and GABAergic neurons were evaluated by accumulation of ¹³C label into different neurometabolites⁶. One-way ANOVA analysis was carried out using GraphPad Prism to find the statistical significance of differences among groups. Values are presented as mean±SD. A P value less than 0.05 was considered significant.

Results: ¹H-[¹³C]-NMR spectra from the cerebral cortex under isoflurane and urethane are depicted in Fig. 1. The decreased ¹³C labeling of neurometabolites under isoflurane and urethane is clearly visible in ¹³C edited NMR spectrum (lower panel). The levels of neurometabolites were estimated from. The One-way ANOVA analysis of neurometabolites levels estimated from the un-edited (upper panel) spectrum indicated that isoflurane increased the level of alanine (p<0.001), and decreased the levels of aspartate (p<0.01) and succinate (p<0.01) in forebrain (Table 1). Urethane did not perturb neurometabolites homeostasis across the brain. The concentrations of ¹³C labeled Asp_C3 (p≤0.001), Glu_C4 (p≤0.01), Glu_C3 (p<0.01), GABA_C2 (p≤0.05), and Gln_C4 (p<0.01) were reduced significantly across the brain in mice maintained under isoflurane and urethane anesthetics when compared with awake (Table 2). Consequently, the estimated rates of glucose oxidation in glutamatergic neurons (CMR_Glc(Glu)) were reduced across the brain in mice maintained under isoflurane (p≤0.001) and urethane (p≤0.001) (Fig. 2). Similarly, inhibitory neuronal metabolic activity (CMR_Glc(GABA)) was also reduced severely under isoflurane (p≤0.001) and urethane (p≤0.001). Correlation analysis between inhibitory and excitatory neurometabolic rates indicates that CMR_Glc(GABA) correlates positively (slope 0.299, R² 0.992) with CMR_Glc(Glu) in the cerebral cortex across the brain (Fig. 3).
Discussion: Both isoflurane and urethane suppressed the excitatory and inhibitory activity associated with glutamatergic and GABAergic neurons across the brain. Additionally, Ala_C3 was decreased in urethane-treated mice (0.12±0.02 µmol/g, p<0.001) when compared with awake (0.21±0.04 µmol/g). In contrast, the labeling of Ala_C3 was increased (p<0.001) increase in the isoflurane group of mice (0.34±0.05 µmol/g) suggesting an uncoupling between glycolysis and oxidative phosphorylation under isoflurane. Most importantly, there was a positive correlation between CMR_Glc(GABA) and CMR_Glc(Glu) suggesting that the inhibitory and excitatory neurotransmission are in direct proportion with the brain activity.

Acknowledgements

This work was supported by the Council for Scientific and Industrial Research (CSIR), Government of India (Health Care Theme NCP/MLP0139).

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