This study was financed by Instituto Serrapilheira, Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq) and Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel 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.

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
<ol>
	<li>Prepare the Tissue Preservation Buffer (TP Buffer).
	<ol>
		<li>Prepare the following reagents in ultrapure water solution: 10 mM of Ca-EGTA buffer with 0.1 &#956;M 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&#176;C.</li>
	</ol>
	</li>
	<li>Prepare the Mitochondria Respiration Buffer (MR Buffer).
	<ol>
		<li>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 &#176;C.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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 &#176;C in a vial protected from light. 
	&#8203;NOTE: To avoid wearing out the probe by freezing and thawing, make small aliquots for storage no longer than 6 months25.</li>
</ol>
2. Calibration of polarographic oxygen sensors for high-resolution respirometry (HRR)
<ol>
	<li>Clean the HRR chambers of the instrument and syringes (see Table of Materials).
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Apply the following calibration settings in the HRR software (see Table of Materials).
	<ol>
		<li>In HRR software control, add the experimental temperature (37 &#176;C), the parameters for oxygen sensor (gain, 2; polarization voltage, 800 mV), and for amperometric sensor (gain, 1000; polarization voltage, 100 mV).</li>
	</ol>
	</li>
	<li>Calibrate the oxygen sensors.
	<ol>
		<li>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 &#176;C for 1 h in calibration mode until the oxygen flux per mass is stable.</li>
		<li>Perform an air calibration of the polarographic oxygen sensors in the software according to the manufacturer&#39;s protocol24. 
		&#8203;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).</li>
	</ol>
	</li>
</ol>
3. Dissection and permeabilization of the sciatic nerve
<ol>
	<li>Remove the sciatic nerve following the steps below.
	<ol>
		<li>Euthanize the animal by cervical dislocation after removing from its cage and gently restrained to resting on the bench.</li>
		<li>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.</li>
		<li>Weigh the tissue immediately and place it in a vial filled with cold TB Buffer (4 &#176;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.</li>
	</ol>
	</li>
	<li>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).</li>
	<li>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 &#181;L of saponin (from stock solution, step 1.3).
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. Oxygen consumption and ROS production determination
<ol>
	<li>Fill the HRR chambers with 2.1 mL of MR Buffer, add Amplex Red (step 1.4) to a final concentration of 5 &#181;M and peroxidase to 2 U/mL, and add the permeabilized sciatic nerve (step 3).
	<ol>
		<li>Attach the instrument&#39;s fluorescence sensors, turn off the lights in the control section of the software, and press Connect to oxygraph. In &#34;edit protocols&#34; in the software, insert the tissue weight measured in step 3.1.3.</li>
	</ol>
	</li>
	<li>Go to &#34;layout&#34;, choose the &#34;specific flux per unit sample&#34; 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.</li>
	<li>Inject two pulses of H2O2, each one to a final concentration of 260 &#181;M, for later calibration in the chamber.</li>
	<li>Inject 20 &#956;L 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.</li>
	<li>Add 20 &#181;L 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 observed26,27.</li>
	<li>In sequence, add 5 &#181;L 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.</li>
	<li>Titrate with aliquots of 0.2 &#181;g/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 potential26,27.</li>
	<li>Titrate with aliquots of 0.5 &#181;mol/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 &#181;l of antimycin A to a final concentration of 5 &#956;M 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 production26,27.</li>
	<li>Go to the command bar, search for &#34;multisensory&#34; in the software, click on Control &#62; Save file&#160;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&#39;s protocol28.</li>
	<li>Open the saved file and select the &#34;Oxygen Flux per Mass&#34; trace to obtain the experimental oxygen consumption results. Manually select the window between injections by pressing Shift + Left mouse button.
	<ol>
		<li>Go to Marks &#62; Statistics to visualize the results for each injection of substrate/inhibitors/uncoupled protocol. For H2O2 production, perform the same procedure with the &#34;Amp-Slope&#34; 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).</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

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.
<p class="...

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, and they also have machinery for quality control and mitophagy4,5,6.
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&#39;s disease8,9, Parkinson&#39;s disease10,11, amyotrophic lateral sclerosis12,13, and Huntington&#39;s disease14,15. In the peripheral nervous system, loss of mitochondrial function in axons is observed in immune neuropathies, such as Guillain-Barr&#233; syndrome16,17, 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 pathogenesis19,20.
Evaluation of sciatic nerve mitochondrial physiology has emerged as an essential target for investigating neuropathy7,21,22. 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.

<table><tbody><tr><td>Adenosine 5&#039; triphosphate dissodium salt hydrate</td><td>Sigma-Aldrich</td><td>A26209</td><td></td></tr><tr><td>Adenosine 5&amp;prime;-diphosphate sodium salt</td><td>Sigma-Aldrich</td><td>A2754</td><td></td></tr><tr><td>Amplex Red Reagent</td><td>Thermo Fisher scientific</td><td>A12222</td><td>Amplex Red is prepared in DMSO accordindly with product datasheet</td></tr><tr><td>Antimycin A (from Streptomyces sp.)</td><td>Sigma-Aldrich</td><td>A8674</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A7030</td><td>heat shock fraction, protease free, fatty acid free, essentially globulin free, pH 7, &amp;ge;98%</td></tr><tr><td>Calcium carbonate</td><td>Sigma-Aldrich</td><td>C6763</td><td></td></tr><tr><td>Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)</td><td>Sigma-Aldrich</td><td>C2920</td><td></td></tr><tr><td>Cytochrome c</td><td>Sigma-Aldrich</td><td>C7752</td><td>(from equine heart; small hemeprotein)</td></tr><tr><td>DataLab version 5.1.1.91</td><td>OROBOROS INSTRUMENTS, Austria</td><td></td><td>Copyright (c) 2002 - 13 by Dr. Erich Gnaiger</td></tr><tr><td>Digital orbital microplate shaker 120V</td><td>Thermo Fisher scientific</td><td>88882005</td><td></td></tr><tr><td>DL-Dithiothreitol</td><td>Sigma-Aldrich</td><td>43819</td><td></td></tr><tr><td>EGTA sodium salt</td><td>Sigma-Aldrich</td><td>E8145</td><td></td></tr><tr><td>Hamilton syringe</td><td>Sigma-Aldrich</td><td>HAM80075</td><td>10 uL, 25 uL and 50 uL</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Hydrogen peroxide solution 30% W/W</td><td>Merck</td><td>H1009</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I2399</td><td></td></tr><tr><td>L-(&amp;minus;)-Malic acid</td><td>Sigma-Aldrich</td><td>M7397</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma-Aldrich</td><td>M2393</td><td></td></tr><tr><td>MES sodium salt</td><td>Sigma-Aldrich</td><td>M3885</td><td></td></tr><tr><td>Micro-dissecting forceps, curved</td><td>Sigma-Aldrich</td><td>F4142</td><td></td></tr><tr><td>Micro-dissecting forceps, straight</td><td>Sigma-Aldrich</td><td>F4017</td><td></td></tr><tr><td>O2K - Filter set Amplex Red</td><td>OROBOROS INSTRUMENTS, Austria</td><td>44321-01</td><td>Fasching M, Sumbalova Z, Gnaiger E (2013) O2k-Fluorometry: HRR and H2O2 production in mouse brain mitochondria. Mitochondr Physiol Network 17.17.</td></tr><tr><td>O2K - Fluorescence LED2 - module component Fluorscence-Sensor Green</td><td>OROBOROS INSTRUMENTS, Austria</td><td>44210-01</td><td></td></tr><tr><td>Oligomycin</td><td>Sigma-Aldrich</td><td>O4876</td><td>(from Streptomyces diastatochromogenes; mixture of oligomycins A, B, and C</td></tr><tr><td>OROBOROS Oxygraph-2k</td><td>OROBOROS INSTRUMENTS, Austria</td><td>http://www.oroboros.at</td><td></td></tr><tr><td>Palmitoylcarnitine (Palmitoyl-DL-carnitine-HCl)</td><td>Sigma-Aldrich</td><td>P4509</td><td></td></tr><tr><td>Peroxidase from horseradish</td><td>Sigma-Aldrich</td><td>P8375</td><td></td></tr><tr><td>Petri dishes, polystyrene</td><td>MERCK</td><td>P5606</td><td></td></tr><tr><td>Phosphocreatine disodium salt hydrate</td><td>Sigma-Aldrich</td><td>P7936</td><td></td></tr><tr><td>Potassium dihydrogen phosphate monobasic</td><td>Sigma-Aldrich</td><td>PHR1330</td><td></td></tr><tr><td>Potassium hydroxide</td><td>Sigma-Aldrich</td><td>221473</td><td></td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td></td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>SAE0073</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P5280</td><td></td></tr><tr><td>Sodium succinate dibasic hexahydrate</td><td>Sigma-Aldrich</td><td>S2378</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S9378</td><td></td></tr><tr><td>Taurine</td><td>Sigma-Aldrich</td><td>T0625</td><td></td></tr></tbody></table>

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.

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...

Watch this Scientific Journal Video about Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry at JoVE.com

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

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.

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple ...

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Research

JoVE Journal

Biochemistry

2.3K Views.  Universidade Federal do Rio de Janeiro. 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. 

Video: Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Developmental Biology

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Medicine

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Biology

Determining Respiration in Human Sperm Cells by High-Resolution Respirometry

High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm mitochondrial activity, so the measurement of sperm mitochondrial function is an indicator of sperm quality. High-resolution respirometry is a method of measuring the oxygen consumption of cells or tissues in a closed-chamber system. This technique can be implemented to measure respiration in human sperm and provides information about the quality and integrity of the sperm mitochondria. High-resolution respirometry allows the cells to move freely, which is an a priori advantage in the case of sperm. This technique can be applied with intact or permeabilized spermatozoa and allows for the study of intact sperm mitochondrial function and the activity of individual respiratory chain complexes. The high-resolution oxygraph instrument uses sensors to measure the oxygen concentration coupled with sensitive software to calculate the oxygen consumption. The data are used to calculate respiratory indices based on the oxygen consumption ratios. Consequently, the indices are the proportions of two oxygen consumption rates and are internally normalized to the cell number or protein mass. The respiratory indices are an indicator of sperm mitochondrial function and dysfunction.

Author Spotlight: Advancing Male Infertility Research by Unraveling Sperm Metabolism and Mitochondrial Function 

The analysis of sperm mitochondrial function by high-resolution respirometry permits the measurement of the oxygen consumption of freely moving spermatozoa in a closed-chamber system. The technique can be applied to measure respiration in human spermatozoa, which provides information on sperm mitochondrial characteristics and integrity.

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm ...

High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and assessment of respiratory complexes using diversified substrate-uncoupler-inhibitor titration (SUIT) protocols. Here, the usage of two high-resolution respirometry devices is demonstrated, and a basic collection of protocols applicable for the analysis of cultured cells, skeletal and heart muscle fibers, and soft tissues such as the brain and liver are presented. Protocols for cultured cells and tissues are provided for a chamber-based respirometer and cultured cells for a microplate-based respirometer, both encompassing standard respiration protocols. For comparative purposes, CRISPR-engineered HEK293 cells deficient in mitochondrial translation causing multiple respiratory system deficiency are used with both devices to demonstrate cellular defects in respiration. Both respirometers allow for comprehensive measurement of cellular respiration with their respective technical merits and suitability dependent on the research question and model under study.

Assessing oxidative phosphorylation using high-resolution respirometers has become an integral part of the functional analysis of mitochondria and cellular energy metabolism. Here, we present protocols for the analysis of cellular energy metabolism using chamber and microplate-based high-resolution respirometers and discuss the key benefits of each device.

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and ...

High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring mitochondrial function is fundamental in biomedical research. Skeletal muscle is a robust source of mitochondria, particularly in animals with a very high aerobic capacity, such as horses, making them ideal subjects for studying mitochondrial physiology. This article demonstrates the use of high-resolution respirometry with concurrent fluorometry, with freshly harvested skeletal muscle mitochondria, to quantify the capacity to oxidize substrates under different mitochondrial states and determine the relative capacities of distinct elements of mitochondrial respiration. Tetramethylrhodamine methylester is used to demonstrate the production of mitochondrial membrane potential resulting from substrate oxidation, including calculation of the relative efficiency of the mitochondria by calculating the relative membrane potential generated per unit of concurrent oxygen flux. The conversion of ADP to ATP results in a change in the concentration of magnesium in the reaction chamber, due to differing affinities of the adenylates for magnesium. Therefore, magnesium green can be used to measure the rate of ATP synthesis, allowing the further calculation of the oxidative phosphorylation efficiency (ratio of phosphorylation to oxidation [P/O]). Finally, the use of Amplex UltraRed, which produces a fluorescent product (resorufin) when combined with hydrogen peroxide, allows the quantification of reactive oxygen species production during mitochondrial respiration, as well as the relationship between ROS production and concurrent respiration. These techniques allow the robust quantification of mitochondrial physiology under a variety of different simulated conditions, thus shedding light on the contribution of this critical cellular component to both health and disease.

Horses have an exceptional aerobic exercise capacity, making equine skeletal muscle an important tissue for both the study of equine exercise physiology as well as mammalian mitochondrial physiology. This article describes techniques for the comprehensive assessment of mitochondrial function in equine skeletal muscle.

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring ...

High-Resolution Analysis of Mitochondrial Function in Human PBMCs

High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these changes&#160;do not always reflect what occurs in other tissues, such as skeletal muscle, these cells are an accessible and valuable source of viable mitochondria from human subjects. PBMCs are exposed to systemic signals that impact their bioenergetic state. Thus, expanding our tools to interrogate mitochondrial metabolism in this population will elucidate mechanisms related to disease progression. Functional assays of mitochondria are often limited to using respiratory outputs following maximal substrate, inhibitor, and uncoupler concentrations to determine the full range of respiratory capacity, which may not be achievable in vivo. The conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by ATP-synthase results in a decrease in mitochondrial membrane potential (mMP) and an increase in oxygen consumption. To provide a more integrated analysis of mitochondrial dynamics, this article describes the use of high-resolution fluorespirometry to measure the simultaneous response of oxygen consumption and mitochondrial membrane potential (mMP) to physiologically relevant concentrations of ADP. This technique uses tetramethylrhodamine methylester (TMRM) to measure mMP polarization in response to ADP titrations following maximal hyperpolarization with complex I and II substrates. This technique can be used to quantify how changes in health status, such as aging and metabolic disease, affect the sensitivity of mitochondrial response to energy demand in PBMCs, T-cells, and monocytes from human subjects.

Author Spotlight: New Insights into PBMC Mitochondrial Responses Using Fluorespirometry

Methods for studying mitochondrial bioenergetics under physiologically relevant substrate concentrations in immune cells are limited. We provide a detailed protocol that uses high-resolution fluorespirometry to assess changes in the response of the mitochondrial membrane potential to energy demand in human T-cells, monocytes, and peripheral mononuclear cells.

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these ...

Real-Time Quantification of Mitochondrial Respiration in Muscle Fibers

Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle is linked to prevalent metabolic disorders (e.g., diabetes and obesity), muscular dystrophies, and sarcopenia. While there are many techniques to evaluate mitochondrial content and morphology, the hallmark method to assess mitochondrial function is the measurement of mitochondrial oxidative phosphorylation (OXPHOS) by respirometry. Quantification of mitochondrial OXPHOS provides insight into the efficiency of mitochondrial oxidative energy production and cellular bioenergetics. A high-resolution respirometer provides highly sensitive, robust measurements of mitochondrial OXPHOS in permeabilized muscle fibers by measuring real-time changes in mitochondrial oxygen consumption rate. The use of permeabilized muscle fibers, as opposed to isolated mitochondria, preserves mitochondrial networks, maintains mitochondrial membrane integrity, and ultimately allows for more physiologically relevant measurements. This system also allows for the measurement of fuel preference and metabolic flexibility - dynamic aspects of muscle energy metabolism. Here, we provide a comprehensive guide for mitochondrial OXPHOS measurements in human and mouse skeletal muscle fibers using a high-resolution respirometer. Skeletal muscle groups are composed of different fiber types that vary in their mitochondrial fuel preference and bioenergetics. Using a high-resolution respirometer, we describe methods for evaluating both aerobic glycolytic and fatty acid substrates to assess fuel preference and metabolic flexibility in a fiber-type-dependent manner. The protocol is versatile and applicable to both human and rodent muscle fibers. The goal is to enhance the reproducibility and accuracy of mitochondrial function assessments, which will improve our understanding of an organelle important to muscle health.

Author Spotlight: Unveiling Mitochondrial Function and Cellular Metabolic Adaptation in Metabolic Diseases

Here, we describe a comprehensive method for measuring mitochondrial oxidative phosphorylation in fresh permeabilized skeletal muscle fibers from either human or mouse muscle. This method allows for the real-time quantification of mitochondrial respiration and the assessment of fuel preference and metabolic flexibility while preserving existing mitochondrial networks and membrane integrity.

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle ...

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Summary

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