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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve.

Abstract

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.

Introduction

Mitochondria are essential for maintaining cell viability and perform numerous cell functions such as energy metabolism (glucose, amino acid, lipid, and nucleotide metabolism pathways). As the primary site of reactive oxygen species (ROS) production, mitochondria are central in several cell signaling processes such as apoptosis and participate in the synthesis of iron-sulfur (Fe-S) clusters, mitochondrial protein import and maturation, and maintenance of their genome and ribosomes1,2,3. The mitochondrial membrane dynamics network is controlled by fusion and fission processes,....

Protocol

The present protocol is approved by the Ethics Committee on the Use of Animals in Research, CCS/UFRJ (CEUA-101/19), and National Institutes of Health guidelines for the care and use of experimental animals. The sciatic nerve is isolated from four-month-old male C57BL/6 mice, euthanized by cervical dislocation as per the institutional guidelines. The protocol steps are optimized to avoid mitochondrial deterioration. Therefore, in this protocol, calibration of polarographic oxygen sensors was performed prior to mouse sciat.......

Representative Results

The mitochondrial oxygen consumption by the permeabilized sciatic nerve is represented in Figure 2. The red trace represents the O2 flux per unit mass in pmol/s.mg. After recording a basal oxygen consumption with endogenous substrates (routine respiration), succinate (SUCC) is injected to record complex II (succinate dehydrogenase)-driven respiration, resulting in an increase in the oxygen consumption rate. In sequence, a saturating concentration of ADP is added, activating ATP sy.......

Discussion

Several diseases or conditions accompanying neuropathies have mitochondrial dysfunction as a risk factor. The evaluation of mitochondrial function in peripheral nerves is essential to elucidate how the mitochondria act in these neurodegenerative conditions. The assessment of mitochondrial function is laborious due to the difficulty of the isolation method and the scarcity of material. Thus, the development of tissue permeabilization techniques that do not require the isolation of mitochondria is essential.

Acknowledgements

This study was financed by Instituto Serrapilheira, Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES). We are grateful to Dr. Antonio Galina Filho, Dr. Monica Montero Lomeli and Dr. Claudio Masuda for the support with laboratory facilities, and Dr. Martha Sorenson for kind and valuable comments in improving the article.

....

Materials

NameCompanyCatalog NumberComments
Adenosine 5' triphosphate dissodium salt hydrateSigma-AldrichA26209
Adenosine 5′-diphosphate sodium saltSigma-AldrichA2754
Amplex Red ReagentThermo Fisher scientificA12222Amplex Red is prepared in DMSO accordindly with product datasheet
Antimycin A (from Streptomyces sp.)Sigma-AldrichA8674
Bovine Serum AlbuminSigma-AldrichA7030heat shock fraction, protease free, fatty acid free, essentially globulin free, pH 7, ≥98%
Calcium carbonateSigma-AldrichC6763
Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)Sigma-AldrichC2920
Cytochrome cSigma-AldrichC7752(from equine heart; small hemeprotein)
DataLab version 5.1.1.91OROBOROS INSTRUMENTS, AustriaCopyright (c) 2002 - 13 by Dr. Erich Gnaiger
Digital orbital microplate shaker 120VThermo Fisher scientific88882005
DL-DithiothreitolSigma-Aldrich43819
EGTA sodium saltSigma-AldrichE8145
Hamilton syringeSigma-AldrichHAM8007510 uL, 25 uL and 50 uL
HEPESSigma-AldrichH3375
Hydrogen peroxide solution 30% W/WMerckH1009
ImidazoleSigma-AldrichI2399
L-(−)-Malic acidSigma-AldrichM7397
Magnesium chloride hexahydrateSigma-AldrichM2393
MES sodium saltSigma-AldrichM3885
Micro-dissecting forceps, curvedSigma-AldrichF4142
Micro-dissecting forceps, straightSigma-AldrichF4017
O2K - Filter set Amplex RedOROBOROS INSTRUMENTS, Austria44321-01Fasching M, Sumbalova Z, Gnaiger E (2013) O2k-Fluorometry: HRR and H2O2 production in mouse brain mitochondria. Mitochondr Physiol Network 17.17.
O2K - Fluorescence LED2 - module component Fluorscence-Sensor GreenOROBOROS INSTRUMENTS, Austria44210-01
OligomycinSigma-AldrichO4876(from Streptomyces diastatochromogenes; mixture of oligomycins A, B, and C
OROBOROS Oxygraph-2kOROBOROS INSTRUMENTS, Austriahttp://www.oroboros.at
Palmitoylcarnitine (Palmitoyl-DL-carnitine-HCl)Sigma-AldrichP4509
Peroxidase from horseradishSigma-AldrichP8375
Petri dishes, polystyreneMERCKP5606
Phosphocreatine disodium salt hydrateSigma-AldrichP7936
Potassium dihydrogen phosphate monobasicSigma-AldrichPHR1330
Potassium hydroxideSigma-Aldrich221473
RotenoneSigma-AldrichR8875
SaponinSigma-AldrichSAE0073
Sodium pyruvateSigma-AldrichP5280
Sodium succinate dibasic hexahydrateSigma-AldrichS2378
SucroseSigma-AldrichS9378
TaurineSigma-AldrichT0625

References

  1. Pfanner, N., Warscheid, B., Wiedemann, N. Mitochondrial protein organization: from biogenesis to networks and function. Nature Reviews Molecular Cell Biology. 20 (5), 267-284 (2019).
  2. Sena, L. A., Chandel, N. S. Phys....

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