Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple causes, including autoimmune diseases, diabetes, infections, inherited disorders, and tumors. Assessing mitochondrial function in mouse peripheral nerves can be challenging due to the small sample size, a limited number of mitochondria present in the tissue, and the presence of a myelin sheath. The technique described in this work minimizes these challenges by using a unique permeabilization protocol adapted from one used for muscle fibers, to assess sciatic nerve mitochondrial function instead of isolating the mitochondria from the tissue. By measuring fluorimetric reactive species production with Amplex Red/Peroxidase and comparing different mitochondrial substrates and inhibitors in saponin-permeabilized nerves, it was possible to detect mitochondrial respiratory states, reactive oxygen species (ROS), and the activity of mitochondrial complexes simultaneously. Therefore, the method presented here offers advantages compared to the assessment of mitochondrial function by other techniques.

Automated summary

Mitochondrial dysfunction in peripheral nerves is associated with various diseases, and assessing mitochondrial function in mouse peripheral nerves can be challenging. This study presents a unique permeabilization protocol that allows for the evaluation of mitochondrial function in sciatic nerves, overcoming the limitations of small sample size, limited mitochondrial quantity, and presence of myelin sheath. By measuring reactive species production and comparing different mitochondrial substrates and inhibitors, this method enables the simultaneous detection of mitochondrial respiratory states, ROS, and mitochondrial complex activity.