Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

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 ribosomes^1,2,3. 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 sciatic nerve tissue dissection and permeabilization.

1. Preparation of reagents

1. Prepare the Tissu....

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.

Materials

Name	Company	Catalog Number	Comments
Adenosine 5' triphosphate dissodium salt hydrate	Sigma-Aldrich	A26209
Adenosine 5′-diphosphate sodium salt	Sigma-Aldrich	A2754
Amplex Red Reagent	Thermo Fisher scientific	A12222	Amplex Red is prepared in DMSO accordindly with product datasheet
Antimycin A (from Streptomyces sp.)	Sigma-Aldrich	A8674
Bovine Serum Albumin	Sigma-Aldrich	A7030	heat shock fraction, protease free, fatty acid free, essentially globulin free, pH 7, ≥98%
Calcium carbonate	Sigma-Aldrich	C6763
Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)	Sigma-Aldrich	C2920
Cytochrome c	Sigma-Aldrich	C7752	(from equine heart; small hemeprotein)
DataLab version 5.1.1.91	OROBOROS INSTRUMENTS, Austria		Copyright (c) 2002 - 13 by Dr. Erich Gnaiger
Digital orbital microplate shaker 120V	Thermo Fisher scientific	88882005
DL-Dithiothreitol	Sigma-Aldrich	43819
EGTA sodium salt	Sigma-Aldrich	E8145
Hamilton syringe	Sigma-Aldrich	HAM80075	10 uL, 25 uL and 50 uL
HEPES	Sigma-Aldrich	H3375
Hydrogen peroxide solution 30% W/W	Merck	H1009
Imidazole	Sigma-Aldrich	I2399
L-(−)-Malic acid	Sigma-Aldrich	M7397
Magnesium chloride hexahydrate	Sigma-Aldrich	M2393
MES sodium salt	Sigma-Aldrich	M3885
Micro-dissecting forceps, curved	Sigma-Aldrich	F4142
Micro-dissecting forceps, straight	Sigma-Aldrich	F4017
O2K - Filter set Amplex Red	OROBOROS INSTRUMENTS, Austria	44321-01	Fasching 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 Green	OROBOROS INSTRUMENTS, Austria	44210-01
Oligomycin	Sigma-Aldrich	O4876	(from Streptomyces diastatochromogenes; mixture of oligomycins A, B, and C
OROBOROS Oxygraph-2k	OROBOROS INSTRUMENTS, Austria	http://www.oroboros.at
Palmitoylcarnitine (Palmitoyl-DL-carnitine-HCl)	Sigma-Aldrich	P4509
Peroxidase from horseradish	Sigma-Aldrich	P8375
Petri dishes, polystyrene	MERCK	P5606
Phosphocreatine disodium salt hydrate	Sigma-Aldrich	P7936
Potassium dihydrogen phosphate monobasic	Sigma-Aldrich	PHR1330
Potassium hydroxide	Sigma-Aldrich	221473
Rotenone	Sigma-Aldrich	R8875
Saponin	Sigma-Aldrich	SAE0073
Sodium pyruvate	Sigma-Aldrich	P5280
Sodium succinate dibasic hexahydrate	Sigma-Aldrich	S2378
Sucrose	Sigma-Aldrich	S9378
Taurine	Sigma-Aldrich	T0625