The overall goal of this experiment is to physiologically activate autophagy in mice by forced or voluntary running, and then measure the levels of autophagy in specific tissues. This method can help answer key questions about autophagy, in particular, how autophagy and action contributes to the beneficial effects mediated by aerobic exercise. The main advantage of this technique is that physical exercise is a fast aerobic sway between autophagy in vivo using mice.
For this experiment, start with eight to twelve week old C57 black six mice, harboring a trans gene to detect exercise induced autophagy in vivo, known as GFP-LC3. For details on the use of mice treadmills, see the following JoVE publication. Set the electrical stimulus of the treadmill to low intensity.
On their first two sessions, acclimate the mice to a ten degree uphill open treadmill. On the first day, exercise the mice for five minutes with the treadmill running at eight meters per minute. On the second day, run the mice for ten minutes, initially at eight meters per minute, and after five minutes, ramp up the speed to ten meters per minute.
On the third day, run the mice for a full 90 minutes. Start the treadmill at ten meters per minute and encourage the mice to run using gentle nudges to avoid repeated foot shocks. It is critical to observe and manipulate the mice so they don't receive too many electric shocks during the 90 minute run.
Throughout the run, use your fingers to encourage the mice to stay on the treadmill. After 40 minutes, 6increase the speed by one meter per minute. After 50 minutes, increase the speed by one meter per minute again.
After 60 minutes, increase the speed by another meter per minute. After 70 minutes, start increasing the speed every five minutes so that the last five minutes of the 90 minute run are 17 meters per minute. After the end of the trial, immediately euthanize the mice for tissue collection if desired.
For this test, house the mice individually. Use the same strain as previously described. Prepare an 11.4 centimeter diameter mouse running wheel with an attached bike odometer.
Each rotation should measure 358 millimeters of travel. Move the mouse into the cage containing the wheel and let the mouse use it freely for two weeks. Every 24 hours, record the distance run by the mouse from the odometer.
After the two weeks, harvest tissues for analysis as needed. To measure the autophagic flux, treat the mice with the autophagy inhibitor, chloroquine, for three days. Dissolve the chloroquine in PBS at five milligrams per milliliter and inject it into mice intraperitoneally at the dose of 50 micrograms per gram.
Give the mice one injection daily for three consecutive days and harvest tissues three hours after the last injection. For the treadmill exercised mice, start the 90 minute run 90 minutes after the injection on the third day so the run ends three hours after the injection. Then euthanize the mice and immediately harvest their tissues.
Prepare to profuse an animal within 90 minutes of being exercised by preparing the fixative. Load a 30 milliliter syringe with 15 to 20 milliliters of freshly made four percent paraformaldehyde and connect the syringe with a 20 gauge catheter needle. Attach the loaded syringe pump and set it to 90 milliliters per hour of continuous flow.
With the animal on a clean work surface, in a supine position, cut open the thoracic cavity through the diaphragm to expose the heart. Now, insert the catheter needle into the right ventricle. Next, make a small incision on the liver to drain the blood and start the pump.
Inject the fixative until the lung and liver turn completely pale. Usually, 15 milliliters of fixative will suffice. After the profusion, remove the needle and collect tissues of interest.
To collect skeletal muscle, dissect the vastus lateralis. Briefly pull the skin of the leg backwards to expose the muscles and localize the quadriceps femoris. Then, dissect out the vastus lateralis, which is the external muscle attached to the upper portion of the femoral bone.
For further fixation and dehydration, place the harvested tissues in four percent paraformaldehyde for 24 hours at four degrees celsius. The next day, transfer the tissues to 15 percent sucrose PBS and let them soak at four degrees celsius overnight or until they have settled in the solution. Then, transfer the tissues to 30 percent sucrose PBS at four degrees celsius for overnight or longer.
Keep the tissues in the dark as much as possible during these baths. Next, place the tissues in an embedding medium such as OCT and cut them into smaller pieces if necessary. Then, allow them to acclimate for a few minutes in a small petri dish.
Afterwards, transfer them to cryomolds with enough OCT to cover. In the molds, orient the sectioning surfaces toward the bottom and avoid forming bubbles. Then, freeze the samples in a covered foam cooler filled with dry ice until the OCT turns white.
Now, wrap individual samples in labeled foils, seal them in a plastic bag and store them at negative 80 degrees celsius until they can be processed. Using GFP tagged LC3 as a reporter system, autophagy induction was observed in mice exercised as described. After autophagy stimulation, LC3 translocated from the cytosol to the autophagosome in punctuate structures.
Exercised mice had many more GFP-LC3 puncta in both skeletal muscle and the cerebral cortex compared to resting condition mice. Western blot analysis using an LC3 antibody showed that exercise significantly induced the generation of lipid conjugative LC3-II, suggesting that there was increased conversion of cytosolic LC3-I into LC3-II. Next, the degradation of the autophagic cargo receptor, p62, was measured.
90 minutes of treadmill activity caused a higher degradation of p62 in skeletal muscle than the resting condition. This effect was rescued by an injection with chloroquine prior to exercise. As chloroquine is an inhibitor of lysosomal degradation, these data indicate that the observed phenomena is due to an elevated autophogagic flux rather than a block in autophagosome degradation.
After its development, this technique had precursors in the field of autophagy to explore the effects on the mechanism of exercise in the degradation of metabolism and animal behaviors. While attempting this procedure, it's important to monitor the mice while they are running. Following this procedure, are the methods like electric microscopy.
Animal microscopy can be performed in order to answer additional questions like exercise in select autophagy pathways, such as mitophagy and lipophagy.
