This article details a method for assessing mitochondrial function in brain slices from healthy and neurodegenerative mice. The protocol involves measuring the oxygen consumption rate (OCR) using an extracellular flux analyzer to evaluate mitochondrial impairment.
Place brain slice punches from healthy and neurodegenerative mice on sample-holding screens.
Flip the screens and insert them into a tissue plate.
Take a pre-calibrated sensor cartridge with sensor probes. Load the compounds—pyruvate, oligomycin, FCCP, and antimycin A—into the injection ports.
Place the cartridge on the tissue plate. Initiate the run in an extracellular flux analyzer.
The probes measure the slice’s oxygen consumption rate or (OCR).
With the first injection, pyruvate enters the mitochondria, generating electron carriers.
These carriers donate electrons to Complexes I and II of the electron transport chain or (ETC), initiating an electron transfer reaction that increases oxygen consumption and OCR.
Oligomycin inhibits Complex V, halting ATP synthesis and decreasing OCR.
FCCP, a protonophore, disrupts the proton gradient, increasing oxygen consumption and OCR.
Antimycin A binds to Complex III and stops the ETC, minimizing OCR.
Compare the OCR profiles.
A decreased OCR in the neurodegenerative slices indicates mitochondrial impairment.
Suction one piece of punched brain tissue, and carefully place the tissue onto the mesh side of the capture screen. A circular piece of plastic with the mesh attached to one side. Using a paper tissue, gently dry the capture screen and remove the moisture, which allows the tissue to become sticky and attached to the center of the mesh.
Next, hold the capture screen, slice-side down with tweezers, and place it into one of the wells of the incubating islet plate. Then, incubate the islet plate at 37 degrees Celsius in an incubator for at least 30 minutes, to allow temperature and pH equilibration before running the assay. Dilute the desired compounds and modified artificial cerebrospinal fluid without BSA to the final stock concentration of the working concentration for ports A to D, respectively.
Gently preload 75 microliters of the diluted compounds into the appropriate injection ports of the sensor cartridge by placing the tips halfway into the injection ports at a 45-degree angle, with the tip against the wall of the injection port, as complete insertion may cause compound leakage through the port. Afterward, withdraw the tips from the ports carefully without creating air bubbles, and do not tap any portion of the cartridge to avoid alleviating air bubbles. Then, visually inspect the injection ports for even loading, ensuring all liquid is in the port and no residual drops are present on top of the cartridge.
After placing the sensor cartridge onto the utility plate, put it into an incubator for 30 minutes to allow it to heat up to 37 degrees Celsius while handling carefully by only holding onto the utility plate and moving as little as possible. First, load the assay template in the software, and then press the green Start button. Then load the sensor cartridge on the utility plate into the instrument tray, ensuring that the plate sits correctly and is flat, loading the drug-filled sensor cartridge into the analyzer for calibration.
Afterward, follow the instructions on the screen in order to calibrate and equilibrate the sensors. Once the equilibration step is done, remove the calibration plate, and replace it with the islet plate containing the mesh and tissue slices. Then measure the oxygen consumption rate in each well of the plate, using the assay protocols as described in the manuscript. Following, analyze the oxygen consumption rate, measurement data, and the coupling efficiency data.
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