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Anstalt \u00f6ffentlichen Rechts"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Cell Commun Signal"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>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.<\/jats:p>","DOI":"10.1186\/s12964-026-02667-9","type":"journal-article","created":{"date-parts":[[2026,1,21]],"date-time":"2026-01-21T00:54:23Z","timestamp":1768956863000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A novel extracellular flux assay workflow uncovers impaired sciatic nerve mitochondrial respiration in diabetic db\/db mice"],"prefix":"10.1186","volume":"24","author":[{"given":"Sebastian","family":"Sill","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"D. 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This study was carried out according to the principles of the 3Rs (Replacement, Reduction and Refinement) to minimize animal suffering and to reduce the number of animals used.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"All authors have approved the manuscript and agree with its submission.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"58"}}