# A novel extracellular flux assay workflow uncovers impaired sciatic nerve mitochondrial respiration in diabetic db/db mice

## TL;DR

A new method for measuring mitochondrial function in sciatic nerves reveals impaired respiration in diabetic mice, offering a tool for studying and treating diabetic neuropathy.

## Contribution

An optimized ex vivo workflow for measuring sciatic nerve mitochondrial respiration with novel normalization and computational techniques.

## Key findings

- Diabetic db/db mice show reduced basal and maximal mitochondrial respiration in sciatic nerves.
- ATP-linked respiration and proton leak are also decreased in diabetic sciatic nerves.
- The new workflow enables stable and reproducible assessment of mitochondrial bioenergetics in peripheral nerves.

## Abstract

Abnormal mitochondrial function contributes to the development of diabetic neuropathy by inducing oxidative stress and altering energy metabolism, ultimately leading to neuronal damage. However, direct and reproducible assessment of mitochondrial bioenergetics in peripheral nerves remains technically challenging. Here, we describe an optimized ex vivo approach for measuring mitochondrial respiration in isolated murine sciatic nerve segments using extracellular flux analysis. Although extracellular flux-based assays have previously been applied to nerve tissue, we introduce key methodological refinements, including optimized tissue preparation, assay conditions, and injection parameters, to achieve stable and reproducible mitochondrial responses. The innovations of our work include the use of mass spectrometry-acquired mitochondrial protein abundances (MitoCarta 3.0) for normalization of the extracellular flux data as well as the application of new computational algorithms that enable straightforward evaluation of mitochondrial toxicity, uncoupling, and alterations in oxidative phosphorylation. Using this workflow, we show that sciatic nerves from diabetic db/db mice exhibit impaired mitochondrial respiration compared to lean C57BLKS/J controls, characterized by reduced basal and maximal respiration, lower ATP-linked respiration, and decreased proton leak. Our approach provides a robust platform for studying mitochondrial bioenergetics in peripheral nerves and offers a scalable tool for evaluating therapeutic interventions in diabetic neuropathy and related disorders.

The online version contains supplementary material available at 10.1186/s12964-026-02667-9.