This protocol allows the evaluation of mitochondrial function inside of nerve by parcellation of the isolated mitochondria, a challenging procedure in a small tissue that contains a limited number of mitochondria. The technique provides a possibility to analyze mitochondria functions such as oxygen consumption and ROS production simultaneously in a preserved mitochondria in situ. With multiple substrates, inhibitors, and uncoupler design protocols.
Several diseases share mitochondrial function, in particular neuropathies induced by type two diabetes or chemotherapy. In these diseases, impairments of mitochondrial oxidative phosphorylation is the only sign that dictates disease development. This method provides insights into neurobiology, biochemistry, and pharmacology, with the investigation of mitochondrial disease mechanisms, search for therapies, such as mitochondrial boosters or ameliorated neuropathic pain.
To begin, calibrate the oxygen sensors by pipetting 2.1 milliliters of MR buffer into each chamber. Close it with the stoppers and draw air into the chamber until a bubble is formed. Stir at 37 degrees Celsius for one hour in calibration mode until the oxygen flux per mass stabilizes.
Using the HRR software perform an air calibration of the polarographic oxygen sensors according to the manufacturer's instructions. For 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.
First, transfer the displayed tissue into a small dish containing one milliliter TP buffer for tissue permeabilization. To start permeabilization, transfer the tissue with forceps to another dish containing one milliliter of TP buffer, containing 50 micrograms per milliliter of saponin. Place the plate in a microplate shaker and let it stir gently for 30 minutes.
Then, transfer the tissue with forceps to a fresh dish containing one milliliter MR buffer, and let it stir gently for 10 minutes. Transfer the tissue with forceps to a calibrated HRR chamber. Fill the HRR chambers with 2.1 milliliters of MR buffer.
Add Amplex Red and peroxidase to a final concentration of five micromolar, and two units per milliliter, respectively. Then, add the permeable sciatic nerve. Attach the instrument's fluorescent sensors.
Then, turn off the lights in the control section of the software and click on connect to oxygraph"Go to edit protocols"in the software and insert the tissue weight. Go to layout and choose the specific flux per unit sample"option. Then select plots to simultaneously access the oxygen consumption readout, and if required, the hydrogen peroxide production.
Wait for approximately 10 minutes. Inject two pulses of hydrogen peroxide, each one to a final concentration of 260 micromolar for calibration of the chamber. Inject 20 microliters of succinate, a mitochondrial complex II substrate, to activate the mitochondrial electron transport system.
Add 20 microliters of ADP to activate ATP synthesis. In sequence, add five microliters of cytochrome c as an indicator of membrane integrity. Titrate with aliquots of 0.2 micrograms per milliliter of oligomycin until no further decrease in oxygen consumption is observed.
Titrate with aliquots of 0.5 micromoles per liter FCCP, the mitochondrial uncoupler, until no further increase in oxygen consumption is observed. To end the experiment inject two microliters of antimycin A, do a final concentration of five millimolar and wait for the flow to stabilize. Go to the command bar.
Search for multisensory"in the software. Click on control"then press save file"and disconnect. Open the saved file and select the oxygen flux per mass trays to obtain the experimental oxygen consumption results.
Manually select the window between injections by pressing the shift plus left mouse button. Go to marks, and select statistics to visualize the results for each injection of substrates, inhibitors, and uncoupler protocol. For hydrogen peroxide production perform the same procedure with the amp slope trace.
The decrease in membrane potential promoted by ATP sythase activity accelerated the oxygen consumption. The addition of exogenous cytochrome C promoted only minimal stimulation of respiration, certifying mitochondrial outer membrane integrity for this preparation. Absolute oxygen flows were recorded and only a 6.3%increase was observed in respiration, indicating good quality of the tissue preparation.
The oxygen consumption and ROS production were measured simultaneously in the presence of different substrates providing fuel for the electron transport system. The addition of pyruvate and malate for mitochondrial complex I increased respiration. And further addition of succinate for complex II also increases oxygen consumption.
Therefore other substrates could be tested. The ROS production also increased, indicating the leak of oxygen from the electron transport system. The addition of adenosine diphosphate in saturating concentration increased oxygen consumption driving ATP formation, and decreasing ROS production.
In contrast, oligo titration decreased oxygen consumption and increased ROS production. This suggested that permeablized sciatic nerve could replicate the standard relation in mitochondrial physiology. The addition of FCCP and retinone, aligned with the expected oxygen flux changes, confirm that mitochondrial physiology and bioenergetic profile were preserved in the permeablized sciatic nerve.
The most important step is to gently prepare the tissue section, ensure that all of chamber openings are properly cleaned. Alterations to mitochondria function could be due to dysfunction or a lower mitochondrial number. therefore cited synthase activity and mitochondrial DNA content confirmation by PCR are recommended.
