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A versatile EPR toolbox for the simultaneous measurement of oxygen consumption and superoxide production.

ABSTRACT: In this paper, we describe an assay to analyze simultaneously the oxygen consumption rate (OCR) and superoxide production in a biological system. The analytical set-up uses electron paramagnetic resonance (EPR) spectroscopy with two different isotopically-labelled sensors: ¹⁵N-PDT (4-oxo-2,2,6,6-tetramethylpiperidine-d₁₆-¹⁵N-1-oxyl) as oxygen-sensing probe and ¹⁴N-CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine, a cyclic hydroxylamine, as sensor of reactive oxygen species (ROS). The superoxide contribution to CMH oxidation is assessed using SOD or PEGSOD as controls. Because the EPR spectra are not superimposable, the variation of EPR linewidth of ¹⁵N-PDT (linked to OCR) and the formation of the nitroxide from ¹⁴N-CMH (linked to superoxide production) can be recorded simultaneously over time on a single preparation. The EPR toolbox was qualified in biological systems of increasing complexity. First, we used an enzymatic assay based on the hypoxanthine (HX)/xanthine oxidase (XO) which is a well described model of oxygen consumption and superoxide production. Second, we used a cellular model of superoxide production using macrophages exposed to phorbol 12-myristate 13-acetate (PMA) which stimulates the NADPH oxidase (NOX) to consume oxygen and produce superoxide. Finally, we exposed isolated mitochondria to established inhibitors of the electron transport chain (rotenone and metformin) in order to assess their impact on OCR and superoxide production. This EPR toolbox has the potential to screen the effect of intoxicants or drugs targeting the mitochondrial function.

SUBMITTER: Donatienne d'Hose

PROVIDER: S-EPMC7804984 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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A versatile EPR toolbox for the simultaneous measurement of oxygen consumption and superoxide production.

Donatienne d'Hose Danhier Pierre P Northshield Heidi H Isenborghs Pauline P Jordan Bénédicte F BF Gallez Bernard B

Redox biology 20201228

In this paper, we describe an assay to analyze simultaneously the oxygen consumption rate (OCR) and superoxide production in a biological system. The analytical set-up uses electron paramagnetic resonance (EPR) spectroscopy with two different isotopically-labelled sensors: 15N-PDT (4-oxo-2,2,6,6-tetramethylpiperidine-d16-15N-1-oxyl) as oxygen-sensing probe and 14N-CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine, a cyclic hydroxylamine, ...[more]

PMID: 33418140

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Project description:Lesioning and neurophysiological studies have facilitated the elucidation of cortical functions and mechanisms of functional recovery following injury. Clinical translation of such studies is contingent on their employment in non-human primates (NHPs), yet tools for monitoring and modulating cortical physiology are incompatible with conventional lesioning techniques. To address these challenges, we developed a toolbox validated in seven macaques. We introduce the photothrombotic method for inducing focal cortical lesions, a quantitative model for designing experiment-specific lesion profiles and optical coherence tomography angiography (OCTA) for large-scale (~5 cm2) monitoring of vascular dynamics. We integrate these tools with our electrocorticographic array for large-scale monitoring of neural dynamics and testing stimulation-based interventions. Advantageously, this versatile toolbox can be incorporated into established chronic cranial windows. By combining optical and electrophysiological techniques in the NHP cortex, we can enhance our understanding of cortical functions, investigate functional recovery mechanisms, integrate physiological and behavioral findings, and develop neurorehabilitative treatments. MOTIVATION The primate neocortex encodes for complex functions and behaviors, the physiologies of which are yet to be fully understood. Such an understanding in both healthy and diseased states can be crucial for the development of effective neurorehabilitative strategies. However, there is a lack of a comprehensive and adaptable set of tools that enables the study of multiple physiological phenomena in healthy and injured brains. Therefore, we developed a toolbox with the capability to induce targeted cortical lesions, monitor dynamics of underlying cortical microvasculature, and record and stimulate neural activity. With this toolbox, we can enhance our understanding of cortical functions, investigate functional recovery mechanisms, test stimulation-based interventions, and integrate physiological and behavioral findings.

Dataset Information

A versatile EPR toolbox for the simultaneous measurement of oxygen consumption and superoxide production.

Publications

A versatile EPR toolbox for the simultaneous measurement of oxygen consumption and superoxide production.

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