We thank the Northwestern University Mouse Histology and Phenotyping Laboratoryfor technical support and assistance, and Noboru Mizushima (University of Tokyo) for providing GFP-LC3 transgenic mice. A. R. and C. H. were supported by the startup funds from Northwestern University and the grant from National Institutes of Health (DK094980).

All procedures involving animals were performed according to guidelines approved by the Northwestern University Institutional Animal Care and Use Committee (IACUC).
1. Mouse Models
<ol>
	<li>Use 8-12 week-old mice in the exercise training. To detect exercised-induced autophagy in vivo, use the GFP-LC3 transgenic mice (C57BL/6 background) for imaging studies and C57BL/6 mice for biochemical analyses.</li>
</ol>
2. Exercise-induced Autophagy
<ol>
	<li>Treadmill setup - forced exercise
	<ol>
		<li>Acclimate and train the mice on a 10&#176; uphill open treadmill for 2 days. On day 1, exercise the mice for 5 min at 8 m/min, and on day 2, exercise the mice for 5 min at 8 m/min followed by another 5 min at 10 m/min10. Encourage the mice to run by gentle hand nudge.</li>
		<li>On the 3rd day, let the mice undergo a single bout of running for 90 min on the 10&#176; uphill treadmill, starting at the speed of 10 m/min. After 40 min, increase the treadmill speed at a rate of 1 m/min every 10 min for a total of 30 min, and then increase the speed at the rate of 1 m/min every 5 min until it reaches 17 m/min, for a total of 90 min of exercise time and 1,070 meters of running distance.</li>
		<li>Set the electric stimulus at a low intensity range (1.2 Hz), and encourage the mice to run to avoid repeated foot shock.</li>
		<li>For tissue collection, euthanize the mice by isoflurane inhalation, followed by cervical dislocation immediately after exercise.&#160;Other approved euthanasia methods can also be used, such as CO2 or I.P. injection of other anesthetics. The level of autophagy does not seem to be affected by methods of euthanasia.</li>
	</ol>
	</li>
	<li>Running wheel setup - voluntary exercise
	<ol>
		<li>Place a mouse-running wheel (11.4 cm diameter) inside a cage with a single-housed mouse.</li>
		<li>Verify the running capacity using a bike odometer. Set up the wheel parameter [distance (in mm) per full rotation of the wheel] at 358 (11.4*&#960;). Measure the running distance after 24 hr.</li>
		<li>Let the mouse run voluntarily for 2 weeks with the running wheel. For tissue collection, sacrifice the mouse in the morning after the running period.</li>
	</ol>
	</li>
</ol>
3. Autophagy Flux Evaluation
<ol>
	<li>To measure the autophagic flux under resting and exercise conditions, treat mice with the autophagy inhibitor chloroquine for three days and compare them to mice treated with PBS.
	<ol>
		<li>Dissolve chloroquine in PBS (5 mg/ml), and inject chloroquine into mice intraperitoneally at the dose of 50 mg/kg/day for three consecutive days. Sacrifice mice and collect tissues 3 hr after the last injection.</li>
		<li>For exercised mice, pretreat them with chloroquine for two consecutive days at the dose of 50 mg/kg/day. On the third day, inject the mice with the same concentration of chloroquine, and after 90 min let them run on the treadmill for another 90 min.</li>
		<li>Euthanize the mice by isoflurane inhalation, followed by cervical dislocation. Collect tissues immediately after running, so that the total incubation time of chloroquine is the same as control (resting) mice (3 hr). 
		NOTE: Alternatively, use a single injection of chloroquine to determine the autophagy flux in mouse tissues.</li>
	</ol>
	</li>
</ol>
4. Tissue Harvest and Fixation
<ol>
	<li>Prior to tissue harvest prepare phosphate buffered saline (PBS) and fresh 4% paraformaldehyde (PFA, caution: personal protection equipment required) in PBS at a final pH of 7.4. Store both solutions at 4 &#176;C.</li>
	<li>Fill 30 ml syringe with 15 - 20 ml of 4% PFA (for GFP-LC3 mice). Connect the syringe with a 20 G catheter needle.</li>
	<li>After the exercise, euthanize the mice by isoflurane inhalation, followed by cervical dislocation.</li>
	<li>Place the animal on a clean work surface on its back. Cut and open the thoracic cavity by cutting the diaphragm to expose the heart.</li>
	<li>Make a small incision on the liver to let the blood come out, and inject a total of 15 ml 4% PFA slowly into the right ventricle of the heart via the catheter, until the lung and liver turn completely pale. Perfuse the mouse immediately after the exercise session, using a syringe pump with a constant flow rate of 90 ml/hr.</li>
	<li>After perfusion, remove the needle and collect tissues of interest (e.g. brain11 and skeletal muscle). For skeletal muscle, dissect the vastus lateralis. Briefly, pull the skin of the leg backwards to expose the muscles, localize the quadriceps femoris12, and dissect out vastus lateralis, which is the external muscle attached to the upper portion of the femoral bone.</li>
	<li>For further fixation and dehydration, place the tissues of GFP-LC3 mice in 4% PFA for 24 hr at 4 &#176;C, and then transfer them to 15% sucrose-PBS at 4 &#176;C overnight or until the tissues have settled, and then to 30% sucrose-PBS at 4 &#176;C overnight or for longer storage. Keep the samples in the dark as much as possible as they may be sensitive to strong light.</li>
	<li>For western blot analysis snap freeze the tissues from C57BL/6 mice directly in liquid nitrogen, and store them at -80 &#176;C.</li>
</ol>
5. Imaging Analysis of GFP-LC3 Puncta
<ol>
	<li>Tissue process
	<ol>
		<li>Pre-treat the tissues (previously fixed in PFA and stored in 30% sucrose) in embedding medium such as &#34;Optimal Cutting Temperature compound&#34; (OCT) for a few min in a labeled small petri dish.</li>
		<li>Transfer the tissue in a labeled cryomold containing enough fresh OCT to cover the tissue. Orient the sectioning surface toward the bottom of the cryomold (cross section for the vastus lateralis muscle and sagittal section for the hemi-brain). Avoid formation of bubbles.</li>
		<li>Freeze the samples by placing the cryomold for a few min in a covered foam cooler filled with dry ice, until the OCT turns white. Wrap individual samples in labeled foils, seal them in a plastic bag, and store them at -80 &#176;C overnight or longer.</li>
		<li>Use a cryostat to cut sample sections at a thickness of 10 &#181;m, and mount the sections on the slides. Store the slides at -20 &#176;C, and always keep the slides away from light whenever possible.</li>
	</ol>
	</li>
	<li>Slide preparation
	<ol>
		<li>Remove slides from the freezer and thaw them at room temperature. Cover the tissue section with a drop of mounting media with DAPI. Place a coverslip of appropriate size on top and avoid bubble formation.</li>
		<li>Use nail polish to seal the edges of the coverslip, allow nail polish to dry in the dark at room temperature, and then store the slides in a light-tight container at 4 &#176;C, or proceed with imaging capture of GFP-LC3 puncta.</li>
	</ol>
	</li>
	<li>Quantification of GFP - LC3 puncta by fluorescence microscopy
	<ol>
		<li>Perform epifluorescence microscopy, capturing pictures in the same condition (exposure and settings) for all the samples. For GFP, use an emission wavelength of 525 nm; for DAPI, use 490 nm. Acquire at least ten images per sample using a 60X objective for vastus lateralis and the frontal cortex region of the brain.</li>
		<li>Quantify the number of GFP-LC3 puncta in a tissue area of 2,500 &#181;m2 in each image for statistical analysis, blinded for experimental groups. Calculate the average number from 10 images of each sample, using the automatic measurement function of &#34;Object Count&#34;. Settings for the vastus lateralis muscle are: Threshold L = 10, H = 200; 2X smooth; 1X clean, and settings for the brain frontal cortex are: Threshold L = 11, H = 200; 2X smooth; 1X clean.</li>
	</ol>
	</li>
</ol>
6. Western Blot Analysis on Autophagy Markers
<ol>
	<li>Tissue process
	<ol>
		<li>Prepare the lysis buffer: 50 mM Tris-HCl; 150 mM NaCl; 1 mM EDTA; 1% Triton X-100; protease inhibitor and phosphatase inhibitor (freshly added).</li>
		<li>Remove the tissue of C57BL/6 mice from -80 &#176;C storage. Cut the muscle tissue into small pieces with a razor blade prior to the following process. Add 800 &#181;l (for hemi-brain) or 500 &#181;l (for vastus lateralis) of cold lysis buffer to the sample. Homogenize the sample at 4 &#176;C with a homogenizer at a medium speed.</li>
		<li>Fully lyse the homogenized mixture for another 30 min to 1 hr at 4 &#176;C with rotation.</li>
		<li>Spin down the homogenate at 12,000 x g for 10 min at 4 &#176;C, discard the pellet, and collect the supernatant in a new tube.</li>
	</ol>
	</li>
	<li>Autophagy analysis
	<ol>
		<li>Determine the protein concentration of tissue lysates using the BCA Protein assay kit according to the manufacturer&#39;s specifications. Normalize each sample to the same concentration.</li>
		<li>Combine the sample with the 2x Laemmli sample buffer at a 1:1 ratio, boil it at 95 &#176;C for 5 - 10 min, and proceed with western blot analysis on autophagy markers13,14, such as p62 (anti-p62 antibody, 1:500 dilution) and LC3 (anti-LC3 antibody, 1:500 dilution). Boil the samples immediately after addition of the sample buffer, as longer incubation on ice may generate multiple degraded bands of LC3.</li>
		<li>Quantify p62 and LC3 bands by densitometry analysis using ImageJ, and normalize the value to the corresponding actin band.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Autophagy is a catabolic process that provides energy and reduces cytotoxicity by lysosomal degradation of cytoplasm components or damaged organelles. Studying autophagy is important to understand the regulation of cellular homeostasis and the mechanisms of stress response. New models and methodologies are emerging in the research field15, to study how impaired autophagy contributes to numerous pathological processes16,17.
Nutrient deprivation (starvation) and pharmacological induction are commonly used approaches to induce autophagy in vitro and in vivo. These methods, especially for animal models, may show adverse effects that can influence the overall results. For example, since it requires at least 48 hr of starvation to induce a detectable level of autophagy, the animals may not have energy needed for regular motor activity, which affects the outcome of many subsequent behavioral studies. Yet pharmacological inducers may also lead to side effects due to their lack of specificity to the autophagy pathway. The major autophagy inducer rapamycin and its derivatives suppress mTOR activity and cause metabolic dysfunction and immunosuppression18,19,20, which should be taken into consideration in the experimental design for long-term treatment. Thus, we have been working on more physiological and robust ways for autophagy activation in animal models.
Recently we and others have identified exercise as an effective, faster and safer autophagy inducer in vivo7,8,9. Both forced exercise by treadmill and voluntary exercise by running wheel have been used to analyze the effects of exercise on autophagy activation, with variations in exercise duration and intensity21,22. For example, an hour of treadmill running (starting at speed of 10 m/min to a maximum of 40 m/min) induces abundant LC3 lipidation in skeletal muscle21, and longer-term voluntary wheel running for 3 months or 4 - 5 weeks also increases basal autophagy and enhances the expression of autophagy proteins in skeletal muscle21,22.
Here we described and compared the two methods (treadmill and running wheel) to exercise mice, and presented optimized shorter protocols with high efficiency in autophagy induction. Each approach has pros and cons. A single bout of 90 min of forced exercise on treadmill is sufficient to induce autophagy in skeletal muscle and brain. We have done a time course of treadmill exercise7, and found that the exercise conditions described here induce the maximal level of autophagy in mouse skeletal muscle, which is also validated by others23. Under these conditions, mice undergo aerobic exercise; as previously reported, aerobic exercise is induced by prolonged running with gradual increases in speeds on a treadmill 24,25,26, whereas the anaerobic condition is obtained by a short duration of high intensity exercise, which increases muscle mass but does not necessarily enhance exercise endurance27,28. Furthermore, the running speed reported here in this protocol positively correlates with oxygen consumption capacity, by indirect methods to assess the aerobic capacity29. Therefore, aerobic exercise on a treadmill is a fast and effective way to induce autophagy.
However, forced running in an enclosed space may exert stress to mice. Thus, to avoid giving additional stress to animals, we use finger nudges or wire tassels as a method to keep mice running, instead of the built-in electric shock. Compared to the treadmill, the use of running wheel has many advantages. It is less stressful, does not require researchers&#39; observation time, and is convenient to study long-term effects of autophagy activation. Yet exercise on a running wheel is voluntary and thus generates variability in terms of running distance and speed among different mice or the same mouse on different days. Therefore, it is necessary to run mice on a wheel for an extended period of time (e.g., several weeks) to minimize the individual variability. Importantly, the approach also requires using an odometer at night to verify that the mouse is actually running. We measured the average running distance of wild-type C57BL/6 mice over a period of 2 weeks, and found that they run approximately 1 km/night at the beginning of training (day 1) and can run up to 8 - 10 km/night at day 14. Overall, the running wheel is an effective method to stimulate autophagy in most organs, although, it is harder to capture autophagosome accumulation in the skeletal muscle compared to using the treadmill. The reason is that skeletal muscle has a high autophagic flux and a fast autophagosome degradation rate, which requires immediate tissue harvesting after running to detect the autophagy induction by GFP-LC3 puncta measurement. In either protocol, rapid PFA perfusion and fixation of the tissues after animal euthanasia is a critical step of the procedure to preserve the autophagosome structures that are otherwise easily degraded during dissection.
This protocol demonstrates how to use aerobic exercise to stimulate autophagy in mouse tissues, including brain and skeletal muscle. These methods can be used to study the mechanisms of the autophagy pathway in the maintenance of tissue function, such as mitochondrial maintenance23, and to study the long-term effects of autophagy induction on the regulation of behavior and health of animal disease models, such as enhancing the analgesic effects of cannabinoids9. Both the treadmill and running wheel methods induce a good level of autophagy; yet it is important to consider their differences when choosing the best approach to meet different research goals.

Autophagy is an evolutionarily conserved degradation pathway, which is induced in response to various stress conditions such as starvation and hypoxia1,2. During autophagy, double-membrane vesicles, called autophagosomes, incorporate unnecessary or damaged subcellular components and transport them into lysosomes for degradation3. Basal autophagy is essential for cellular function and organism development, and impaired basal autophagy is implicated in many disorders, including neurodegeneration, tumorigenesis and type 2 diabetes4,5,6.
The best-known physiological autophagy inducer is starvation. However, it has two major limitations. First, starvation takes a long period to effectively induce autophagy in animals, e.g., 48 hr of food restriction in mice in most organs. Second, starvation barely induces brain autophagy, due to a relatively stable nutrient supply in the brain. In fact, it is also difficult to detect autophagy induction by small-molecule inducers, as many drugs cannot pass the blood brain barrier. Thus, to better analyze the function of autophagy activation in disease pathogenesis, we recently discovered that exercise is a more potent physiological method to induce autophagy in a short period of time7,8,9. Compared with starvation, autophagy is effectively induced by treadmill running as fast as 30 min. Thus, exercise is a convenient and potent physiological approach to study the mechanism of autophagy in mediating health benefits and preventing diseases.
There are several protein markers for the detection of autophagy activity, including LC3 and p62. LC3 (microtubule-associated protein 1A/1B-light chain 3) is a cytosolic protein (LC3-I form) that is conjugated to PE (phosphatidylethanolamine) upon autophagy induction. PE-lipidated LC3 (LC3-II form) is recruited onto autophagosomal membranes and can be used to visualize autophagosomes when labeled with GFP. Its translocation from the cytosol to punctate structures of autophagosomes under microscopy is an indication of autophagy induction. p62 is a cargo receptor for autophagy substrates (such as ubiquitination proteins), and is incorporated into autophagosomes as well. Since the protein is degraded in lysosomes along with autophagosomes, its levels can be used to measure the autophagy flux. Here we show how to use these markers to quantify autophagy in different mouse tissues induced by aerobic exercise, including forced exercise (treadmill) and voluntary exercise (running wheels). The same procedures can also be applied to in vivo measurement of autophagy after treatment of other inducers.

<table><tbody><tr><td>Treadmill</td><td>Columbus Instruments</td><td>150-RM Exer 3/6</td><td></td></tr><tr><td>Mouse running wheel</td><td>Super Pet</td><td>100079365</td><td>diameter 11.4 cm</td></tr><tr><td>Odometer</td><td>Bell</td><td>DASHBOARD 100</td><td></td></tr><tr><td>Syringe pump</td><td>KD Scientific</td><td>KDS100</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Nikon</td><td>Model: inverted microscope ECLIPSE</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM 1850UV</td><td></td></tr><tr><td>Homogenizer</td><td>IKA</td><td>003737001 / Model: T10 Basic S1</td><td></td></tr><tr><td>Chloroquine</td><td>CAYMAN CHEMICAL COMPANY</td><td>14194</td><td></td></tr><tr><td>Parafolmaldehye</td><td>SIGMA-ALDRICH</td><td>P6148</td><td>Personal protection equipment required. This product may release formaldehyde gas, a chemical known to cause cancer.</td></tr><tr><td>Mounting media</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>p62 antibody</td><td>BD Biosciences</td><td>610833</td><td></td></tr><tr><td>LC3 antibody</td><td>Novus Biologicals</td><td>NB100-2220</td><td></td></tr><tr><td>2x Laemmli Sample Buffer</td><td>Bio-Rad Laboratories</td><td>161-0737</td><td></td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td></td></tr></tbody></table>

activating autophagy by aerobic exercise in mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

This protocol describes two different methods to induce autophagy in mouse tissues by aerobic exercise: a total of 90 min of forced exercise on a multi-lane treadmill proceeded by two days of acclimation; or two weeks of voluntary exercise on a running wheel used by single-housed mice. In each exercise protocol, we can measure the autophagy flux by fluorescence microscopy and western blot analysis in various organs.
We used a transgenic mouse line expressing GFP-tagged LC3 as a reporter system to monitor autophagy by exercise1. Upon autophagy induction, LC3 translocates from the cytosol to the autophagosome in punctate structures. After sectioning, formation of GFP-LC3 puncta can be directly visualized by fluorescence microscopy (Figure 1A). Alternatively, autophagosome structures can also be immunostained by an LC3 antibody for imaging. Either 90 min of treadmill exercise or 2 weeks of voluntary running increased the number of GFP-LC3 puncta in both skeletal muscle (vastus lateralis) and cerebral cortex, compared to the resting condition (Figure 1B). It should be noted that the frontal cortex region has been the major region in the brain where autophagy is clearly induced by either method so far. Exercise also induced the conversion of LC3 from the cytosolic form (LC3-I) to the lipid-conjugated form (LC3-II), which can be detected by western blot analysis (Figure 1C).
Exercise-induced increment of autophagosomes (represented by LC3-II and GFP-LC3 puncta) is due to an elevated autophagic flux, rather than a block in autophagosome degradation, assessed by the use of inhibitors of lysosomal degradation, such as bafilomycin A1 or chloroquine. Here we measured the degradation of the autophagic cargo receptor p62 as an example. Exercise (90 min of treadmill) caused a higher degradation of p62 in skeletal muscle than the resting condition, which was rescued by injection with chloroquine prior to exercise (Figure 2). The similar results are also observed with voluntary exercise by running wheels. Thus, aerobic exercise by treadmill or running wheels induces autophagy in vivo, measured by the steady-state level of LC3 and the degradation of p62.
<img alt="Figure 1" src="/files/ftp_upload/55099/55099fig1.jpg" /> 
Figure 1. Aerobic Exercise Induces Autophagy in Mouse Tissues. Representative images (A) and quantification (B) of GFP-LC3 puncta in skeletal muscle (vastus lateralis) and brain (frontal cortex) of GFP-LC3 transgenic mice under the control condition (resting), after 90 min of forced exercise (treadmill) or after 2 weeks of voluntary exercise (wheel). Results represent mean &#177; s.e.m of 10 pictures per mouse. N = 5 mice. The following emission wavelengths were used: GFP - 525 nm; DAPI - 490 nm. (C) Western blot detection of LC3 in skeletal muscle (vastus lateralis) from rested and exercised (by treadmill) mice. Quantification data represent the level of LC3-II normalized to actin (left) and the ratio of LC3-II to LC3-I (right). N = 3 mice. Statistic is comparing each value to the control sample. Results represent mean &#177; s.e.m. *, P &#60; 0.05; **, P &#60; 0.01; ***, P &#60; 0.001, t-test. Scale bar, 25 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/55099/55099fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55099/55099fig2.jpg" /> 
Figure 2. Exercise Increases Autophagy Flux in Mouse Skeletal Muscle. (A) Western blot detection of p62 in vastus lateralis from rested and exercised mice in the presence or absence of the lysosomal inhibitors chloroquine. (B) (Left) Quantification analysis of p62 normalized to the corresponding actin band. (Right) The p62 flux is determined by subtracting the normalized densitometric value of PBS-treated p62 from that of chloroquine-treated p62. Results represent mean &#177; s.e.m. N = 3 mice. *, P &#60; 0.05, t-test. <a href="http://cloudflare.jove.com/files/ftp_upload/55099/55099fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Activating Autophagy by Aerobic Exercise in Mice

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

activating-autophagy-by-aerobic-exercise-in-mice

Nanyang Technological University

Research

JoVE Journal

Biology

11.7K Views.  Northwestern University. Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

Video: Activating Autophagy by Aerobic Exercise in Mice

Assessment of Murine Exercise Endurance Without the Use of a Shock Grid: An Alternative to Forced Exercise

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most common method for assessing exercise endurance in mice utilizes forced running on a motorized treadmill equipped with a shock grid. Animals who quit running are pushed by the moving treadmill belt onto a grid that delivers an electric foot shock; to escape the negative stimulus, the mice return to running on the belt. However, avoidance behavior and psychological stress due to use of a shock apparatus can interfere with quantitation of running endurance, as well as confound measurements of postexercise serum hormone and cytokine levels. Here, we demonstrate and validate a refined method to measure running endurance in na&#239;ve C57BL/6 laboratory mice on a motorized treadmill without utilizing a shock grid. When mice are preacclimated to the treadmill, they run voluntarily with gait speeds specific to each mouse. Use of the shock grid is replaced by gentle encouragement by a human operator using a tongue depressor, coupled with sensitivity to the voluntary willingness to run on the part of the mouse. Clear endpoints for quantifying running time-to-exhaustion for each mouse are defined and reflected in behavioral signs of exhaustion such as splayed posture and labored breathing. This method is a humane refinement which also decreases the confounding effects of stress on experimental parameters.

A method to assess exercise endurance in laboratory mice without the use of a shock grid is demonstrated. This method is a humane refinement that can decrease the confounding effects of stress on experimental parameters.

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most ...

Behavior

Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by international guidelines for patients suffering from cardiovascular diseases, more specifically, lower extremity artery diseases, where the patients' walking capacity is considerably altered, affecting their quality of life. 
Traditionally, both low continuous exercise and interval training have been used. Recently, supramaximal training has also been shown to improve athletes' performances via vascular adaptations, amongst other mechanisms. The combination of this type of training with hypoxia could bring an additional and/or synergic effect, which could be of interest for certain pathologies. Here, we describe how to perform supramaximal intensity training sessions in hypoxia on healthy mice at 150% of their maximal speed, using a motorized treadmill and a hypoxic box. We also show how to dissect the mouse in order to retrieve organs of interest, particularly the pulmonary artery, the abdominal aorta, and the iliac artery. Finally, we show how to perform ex vivo vascular function assessment on the retrieved vessels, using isometric tension studies.

High-intensity training in hypoxia is a protocol that has been proven to induce vascular adaptations potentially beneficial in some patients and to improve athletes&#39; repeated sprint ability. Here, we test the feasibility of training mice using that protocol and identify those vascular adaptations using ex vivo vascular function assessment.

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Developmental Biology

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

Immunology and Infection

Short Session High Intensity Interval Training and Treadmill Assessment in Aged Mice

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, with regimens less than or equal to 10 min is of particular interest as several human studies feature routines as short as a few minutes a couple times a week. However, there is a paucity of animal studies that model the impacts of short session HIIT. Here, we describe a methodology for an individually tailored and progressive short session HIIT regimen of 10 min given 3 days a week for aged mice using an inclined treadmill. Our methodology also includes protocols for treadmill assessment. Mice are initially acclimatized to the treadmill and then given baseline flat and uphill treadmill assessments. Exercise sessions begin with a 3 min warm-up, then three intervals of 1 min at a fast pace, followed by 1 min at an active recovery pace. Following these intervals, the mice are given a final segment that starts at the fast pace and accelerates for 1 min. The HIIT protocol is individually tailored as the speed and intensity for each mouse are determined based upon initial anaerobic assessment scores. Additionally, we detail the conditions for increasing or decreasing the intensity for individual mice depending on performance. Finally, intensity is increased for all mice every two weeks. We previously reported in this protocol enhanced physical performance in aged male mice and here show it also increases treadmill performance in aged female mice. Advantages of our protocol include low administration time (about 15 min per 6 mice, 3 days a week), strategy for individualizing for mice to better model prescribed exercise, and a modular design that allows for the addition or removal of the number and length of intervals to titrate exercise benefits.

Short session (&#8804;10 min) high intensity interval training (HIIT) is emerging as an alternative to longer exercise modalities, yet the shorter variants are rarely modeled in animal studies. Here, we describe a 10 min, 3 day a week, uphill treadmill HIIT protocol that enhances physical performance in male and female aged mice.

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and further explore whether its mechanism is related to the impact of mitophagy. This study also aimed to demonstrate the therapeutic effect of safflower acid on oxidative stress in cardiomyocytes and explore whether its mechanism is related to the effect of mitophagy. Here, an H2O2-based oxidative stress model was constructed and assessed the degree of oxidative stress injury of cardiomyocytes by detecting the levels of lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH Px). Reactive oxygen species (ROS)-detecting fluorescent dye DCFH-DA, JC-1 dye, and TUNEL dye were employed to assess mitochondrial damage and apoptosis. Autophagic flux was measured by transfecting Ad-mCherry-GFP-LC3B adenovirus. Mitophagy-related proteins were then detected via western blotting and immunofluorescence. However, crocetin (0.1-10 &#181;M) could significantly improve cell viability and reduce apoptosis and oxidative stress damage caused by H2O2. In cells with excessive autophagic activation, crocetin could also reduce autophagy flow and the expression of mitophagy-related proteins PINK1 and Parkin, and reverse the transfer of Parkin to mitochondria. Crocetin could reduce H2O2-mediated oxidative stress damage and the apoptosis of H9c2 cells, and its mechanism was closely related to mitophagy.

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

Based on in vitro experiments, this study revealed the mechanism of crocetin in repairing oxidative stress damage of cardiomyocytes by influencing mitophagy, in which the PINK1/Parkin signaling pathway plays an important role.

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and ...

Medicine

Exploring T2DM Exercise Interventions: A Static Training Device for Mice

Static Strength Training Method for Type 2 Diabetic Mice

The treatment of type 2 diabetes mellitus (T2DM) is a major difficulty in improving patient health. Exercise is one of the main interventions for T2DM. Static strength training is one of the key forms of traditional sports in China. Research shows that static strength training is an effective clinical method for T2DM intervention, but there is no experimental device suitable for static training in mice. One of the difficulties in moving from clinical to basic research is to design appropriate experimental devices. In order to further study the mechanism of static training intervention in T2DM, a simple method for making a static training device for mice is introduced in this paper. This device has the advantages of simple operation, cheap material, and high feasibility. Previous studies conducted under this protocol have shown that static training can effectively reduce blood glucose levels and improve the mitochondrial function of skeletal muscle cells in T2DM mice. The purpose of introducing this device is to promote research on the mechanism of traditional exercise in the intervention of T2DM and to lay a foundation for the quantitative intervention of exercise.

Author Spotlight: Advancing Diabetes Research with Static Exercise Training in Mice

This protocol provides a simple method of making static training equipment for mice. The device maintains the muscle isometric contraction of the limbs of mice so as to verify the intervention effect of traditional exercise on type 2 diabetes (T2DM) and provides new exercise therapy for the clinical treatment of T2DM.

The treatment of type 2 diabetes mellitus (T2DM) is a major difficulty in improving patient health. Exercise is one of the main interventions for T2DM. ...

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most recently, weighted-sled pulling, are highly effective at providing a hypertrophic stimulus to induce skeletal muscle adaptations. While these models have proven invaluable for skeletal muscle research, they are either invasive or involuntary and labor-intensive. Fortunately, many rodent strains voluntarily run long distances when given access to a running wheel. Loaded wheel running (LWR) models in rodents are capable of inducing adaptations commonly observed with resistance training in humans, such as increased muscle mass and fiber hypertrophy, as well as stimulation of muscle protein synthesis. However, the addition of moderate wheel load either fails to deter mice from running great distances, which is more reflective of an endurance/resistance training model, or the mice discontinue running nearly entirely due to the method of load application. Therefore, a novel high-load wheel running model (HLWR) has been developed for mice where external resistance is applied and progressively increased, enabling mice to continue running with much higher loads than previously utilized. Preliminary results from this novel HLWR model suggest it provides sufficient stimulus to induce hypertrophic adaptations over the 9 week training protocol. Herein, the specific procedures to execute this simple yet inexpensive progressive resistance-based exercise training model in mice are described.

This procedure describes a translatable progressive loaded running wheel resistance training model in mice. The primary advantage of this resistance training model is that it is entirely voluntary, thus reducing stress for the animals and the burden on the researcher.

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most ...

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans.&#160;The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis.

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

The present protocol describes the induction of autophagy in the Drosophila melanogaster larval fat body via nutrient depletion and analyzes changes in autophagy using transgenic fly strains.

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. ...

Activating Autophagy by Aerobic Exercise in Mice

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Chapters in this video

Assessment of Murine Exercise Endurance Without the Use of a Shock Grid: An Alternative to Forced Exercise

Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Short Session High Intensity Interval Training and Treadmill Assessment in Aged Mice

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

Static Strength Training Method for Type 2 Diabetic Mice

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body