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, and they also have machinery for quality control and mitophagy^4,5,6.

Mitochondrial dysfunction is associated with the appearance of several pathological conditions such as cancer, diabetes, and obesity⁷. Disturbances in mitochondrial function are detected in neurodegenerative disorders that affect the central nervous system, as in Alzheimer's disease^8,9, Parkinson's disease^10,11, amyotrophic lateral sclerosis^12,13, and Huntington's disease^14,15. In the peripheral nervous system, loss of mitochondrial function in axons is observed in immune neuropathies, such as Guillain-Barré syndrome^16,17, and in association with high mitochondrial ROS production in axons, these events lead to MAP Kinase activation in Schwann cells¹⁸. This demonstrates that mitochondrial physiology may be essential not only for a site-specific cell, but for an entire tissue. In HIV-associated distal sensory polyneuropathy (HIV-DSP), mitochondria have a role in the mechanism by which the trans-activator of transcription (HIV-TAT) protein allows HIV to replicate efficiently, as well as several other roles in HIV infection pathogenesis^19,20.

Evaluation of sciatic nerve mitochondrial physiology has emerged as an essential target for investigating neuropathy^7,21,22. In diabetic neuropathy, proteomic and metabolomic analyses suggest that most molecular alterations in diabetes affect sciatic nerve mitochondrial oxidative phosphorylation and lipid metabolism⁷. These alterations also seem to be early signs of obesity-induced diabetes²¹. In a mouse model of chemotherapy-induced painful neuropathy, mitochondrial impairment in the sciatic nerve is detected as a decrease in oxidative phosphorylation²², and a reduction of mitochondrial complexes activities, membrane potential, and ATP content²³. However, although several groups have cited mitochondrial dysfunction in neuropathies, these studies are limited to the measurements of activity in mitochondrial complexes with no preservation of the mitochondrial membranes, lacking evaluation of mitochondrial integrity or measurements of ATP content as a parameter for mitochondrial ATP production. In general, a proper assessment of mitochondrial oxygen consumption and ROS production requires the isolation of mitochondria by differential centrifugation in a percoll/sucrose gradient. Isolation of mitochondria can also be a limiting factor for sciatic nerve tissue because of the large amount of tissue needed and mitochondria loss and disruption.

The present study aims to provide a protocol to measure mitochondrial physiology as mitochondrial oxygen consumption and ROS production in the sciatic nerve, preserving mitochondrial membranes and without the need for isolating mitochondria. This protocol is adapted from oxygen consumption measurements in permeabilized muscle fibers²⁴ by high-resolution respirometry (HRR). The advantages of this procedure are the possibility of evaluating mitochondria in small amounts of tissue such as the sciatic nerve and evaluating mitochondrial parameters in situ, thereby preserving the mitochondrial environment, structure, and bioenergetic profile, to obtain a physiologically trustworthy result. The mitochondrial respiratory states were determined with substrates and inhibitors after sciatic nerve permeabilization to properly assess mitochondrial bioenergetics and cytochrome c coefficient for mitochondrial membrane integrity, providing a guide for steps of the mitochondrial electron transport system (ETS) evaluation and calculation of essential parameters. This study can provide tools for answering questions in pathophysiological mechanisms in which sciatic nerve metabolism is implicated, such as peripheral neuropathies.

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 Tissue Preservation Buffer (TP Buffer).
   1. Prepare the following reagents in ultrapure water solution: 10 mM of Ca-EGTA buffer with 0.1 μM of free calcium, 20 mM of imidazole, 20 mM of taurine, 50 mM of K-MES, 0.5 mM of DTT, 6.56 mM of MgCl₂, 5.77 mM of ATP, 15 mM of phosphocreatine (see Table of Materials), pH 7.1. Store at -20°C.
2. Prepare the Mitochondria Respiration Buffer (MR Buffer).
   1. Prepare the following reagents in ultrapure water solution: 0.5 mM of K₂EGTA, 3 mM of MgCl₂, 60 mM of MES, 20 mM of taurine, 10 mM of KH₂PO₄, 20 mM of HEPES, 110 mM of D-sucrose, 1 mg/mL of BSA (fatty acid-free) (see Table of Materials), pH 7.4. Store at -20 °C.
3. Prepare saponin stock solution by dissolving 5 mg of saponin (see Table of Materials) in 1 mL of TP Buffer (step 1.1) and keep it on ice. Saponin is prepared freshly.
4. Prepare Amplex Red by resuspending the powder (see Table of Materials) with DMSO to obtain a stock concentration of 2 mM and store at -20 °C in a vial protected from light.
   ​NOTE: To avoid wearing out the probe by freezing and thawing, make small aliquots for storage no longer than 6 months²⁵.

2. Calibration of polarographic oxygen sensors for high-resolution respirometry (HRR)

1. Clean the HRR chambers of the instrument and syringes (see Table of Materials).
   1. Open HRR chambers, fill with distilled water up to the top and stir for 5 min 3x. Repeat with ethanol and then again with water. Wash stoppers and syringes with water/ethanol/water 3x each.
2. Apply the following calibration settings in the HRR software (see Table of Materials).
   1. In HRR software control, add the experimental temperature (37 °C), the parameters for oxygen sensor (gain, 2; polarization voltage, 800 mV), and for amperometric sensor (gain, 1000; polarization voltage, 100 mV).
3. Calibrate the oxygen sensors.
   1. Pipette 2.1 mL of MR Buffer (step 1.2) into each chamber. Close with the stoppers and draw air into the chamber until a bubble is formed. Stir at 37 °C for 1 h in calibration mode until the oxygen flux per mass is stable.
   2. Perform an air calibration of the polarographic oxygen sensors in the software according to the manufacturer's protocol²⁴.
      ​NOTE: The calibration step is only performed once before an experiment. Additional experiments in the same respiration medium and temperature can be performed after merely washing chambers (step 2.1).

3. Dissection and permeabilization of the sciatic nerve

1. Remove the sciatic nerve following the steps below.
   1. Euthanize the animal by cervical dislocation after removing from its cage and gently restrained to resting on the bench.
   2. In the euthanized animal, make an incision with scissors in the lower back, starting near the spine and proceeding down the thigh toward the foot. Remove skin and muscle attached to the nerve, and then cut and remove the entire sciatic nerve.
   3. Weigh the tissue immediately and place it in a vial filled with cold TB Buffer (4 °C). Perform steps 3.2-3.3 on ice.
      NOTE: The wet tissue weight is used to normalize oxygen consumption and ROS production flow in the following steps. If the tissue cannot be weighed immediately, conserve it in cold TB Buffer. The procedure is performed in fresh tissue and must not be frozen to avoid mitochondrial damage.
2. For the tissue preparation, place the sciatic nerve in a Petri dish with enough TP Buffer to cover it. Hold one end of the nerve with forceps, and with another pair of forceps, pull out the nerve bundles horizontally.
   NOTE: This procedure needs to be done in less than 10 min to avoid tissue deterioration. The tissue will be ready when it can be visualized as transparent foggy layers, opposite the previous white opaque tissue (Figure 1).
3. First, transfer the splayed tissue into a small dish containing 1 mL of TP Buffer for tissue permeabilization. To start permeabilization, transfer the tissue with forceps to a dish containing 1 mL of TP Buffer and 10 µL of saponin (from stock solution, step 1.3).
   1. Shake in a microplate shaker gently for 30 min, then transfer the tissue with forceps to a fresh dish containing MR Buffer (1 mL) and shake gently for 10 min. Transfer the tissue with forceps to a calibrated HRR chamber.

4. Oxygen consumption and ROS production determination

1. Fill the HRR chambers with 2.1 mL of MR Buffer, add Amplex Red (step 1.4) to a final concentration of 5 µM and peroxidase to 2 U/mL, and add the permeabilized sciatic nerve (step 3).
   1. Attach the instrument's fluorescence sensors, turn off the lights in the control section of the software, and press Connect to oxygraph. In "edit protocols" in the software, insert the tissue weight measured in step 3.1.3.
2. Go to "layout", choose the "specific flux per unit sample" option, and select Plots to simultaneously access the oxygen consumption readout and, if desired, the H₂O₂ production. Wait ~10 min.
   NOTE: This time is required to stabilize the basal flow of oxygen consumption with no added substrate (basal). Before further injections, ensure that the oxygen flow is stabilized.
3. Inject two pulses of H₂O₂, each one to a final concentration of 260 µM, for later calibration in the chamber.
4. Inject 20 μL of succinate (see Table of Materials), a mitochondrial complex II substrate, to activate the mitochondrial electron transport system.
   NOTE: Different mitochondrial substrates can be added at this point to evaluate mitochondrial function by different complexes. Representative results with varying substrates for mitochondrial complexes I and II are shown in Figure 2 and Figure 3. At this point, an increase in O₂ consumption and H₂O₂ production, simultaneously, is observed in Figure 3.
5. Add 20 µL of adenosine diphosphate (ADP) to activate adenosine triphosphate (ATP) synthesis.
   NOTE: ADP stimulates ATP synthesis and reduces the membrane potential. An increase in O₂ consumption and a decrease in the production of H₂O₂ are expected to be observed^26,27.
6. In sequence, add 5 µL of cytochrome c (see Table of Materials) as an indicator of membrane integrity.
   NOTE: If the tissue is well prepared and permeabilized, cytochrome c should not increase oxygen consumption by more than 15%. If it occurs, check the troubleshooting section for recommendations.
7. Titrate with aliquots of 0.2 µg/mL of oligomycin (see Table of Materials) until no further decrease in O₂ consumption is observed.
   NOTE: Oligomycin acts by inhibiting ATP synthesis leading to a decrease in O₂ flow and an enhancement in H₂O₂ formation favored by the high membrane potential^26,27.
8. Titrate with aliquots of 0.5 µmol/L of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (see Table of Materials), the mitochondrial uncoupler, until it is possible to observe no further increase in O₂ consumption. To finish the experiment, inject 2 µl of antimycin A to a final concentration of 5 μM and wait for flow stabilization.
   NOTE: A decrease in H₂O₂ production is observed due to the membrane potential dissipation after injecting FCCP. Antimycin A inhibits complex III, thereby preventing the flow of electrons. Therefore, mitochondria-dependent O₂ consumption is impaired, decreasing oxygen flux per mass and stimulating electron leakage, increasing H₂O₂ production^26,27.
9. Go to the command bar, search for "multisensory" in the software, click on Control > Save file and Disconnect.
   NOTE: The addition of other substrates and inhibitors can be performed according to the question under study. An example is described in the representative results. H₂O₂ calibration is performed after finishing the experiment, according to the manufacturer's protocol²⁸.
10. Open the saved file and select the "Oxygen Flux per Mass" trace to obtain the experimental oxygen consumption results. Manually select the window between injections by pressing Shift + Left mouse button.
    1. Go to Marks > Statistics to visualize the results for each injection of substrate/inhibitors/uncoupled protocol. For H₂O₂ production, perform the same procedure with the "Amp-Slope" trace.
       NOTE: When selecting the window, avoid artifacts of volume injections by choosing a window where oxygen (or H₂O₂) is more stable and constant. Examples of selected windows are represented by black braces in the representative results (Figure 2 and Figure 3).

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