Method Article

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

19.5K views

⸱

DOI:

10.3791/51846

⸱

August 14th, 2014

 ,  , 
1Research Service, VA Puget Sound Health Care System, 2Seattle Institute for Biomedical and Clinical Research, 3Division of Gerontology and Geriatric Medicine, Department of Medicine, University of Washington, 4Geriatric Research, Education, and Clinical Center, VA Puget Sound Health Care System

In This Article

Summary

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.

Abstract

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 naïve 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.

Introduction

Obesity, insulin resistance, and type 2 diabetes are interrelated metabolic disorders that exert profound effects on the health of both the U.S. and worldwide populations 1-4. Endurance exercise can prevent, as well as treat, these conditions5,6. Moreover, assessment of gait speed and endurance are utilized clinically as diagnostic tests for frailty, sarcopenia, and the consequences of other disorders such as chronic obstructive pulmonary disease, in human subjects 7.

The biochemical pathways underlying the beneficial effects of endurance exercise on body composition and insulin sensitivity are beginning to be elucidated, using genetically or pharmacologically modified mice that display enhanced or reduced exercise capacity 8-11. However, many such studies have utilized motorized treadmills equipped with shock grids to force mice to run 8-11. 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. Such procedures may introduce psychological stress and avoidance behavior as confounding factors affecting experimental parameters12. Other methods of measuring endurance, such as quantitation of ambulatory activity using a beam-break apparatus or quantitation of in-cage wheel running, may be confounded by modulation of circadian cycles, anxiety, inadvertent training, or food seeking behavior12-16. Moreover, these procedures require single housing, another source of psychological stress for mice. Therefore, a direct measurement of exercise endurance not confounded by stress is needed.

To obviate these concerns, our laboratory has developed and validated a method to assess maximal running capacity and sustained running speed in untrained, naïve mice using a motorized treadmill not equipped with a shock grid. The space at the end of the running belt, where the shock grid usually resides, then becomes a platform for mice to rest, and provides a place for mice that refuse to run to sit until removed from the apparatus. Mice are encouraged to run by a human observer using gentle tapping or touching with a tongue depressor, coupled with sensitivity to the voluntary willingness to run on the part of the mouse. This method has been used to quantify differences in exercise endurance between genetically-modified and control C57BL/6 mice that differ in expression of the muscle derived cytokine interleukin-15 (IL-15)16,17. This method is a humane refinement that decreases the confounding effects of negative reinforcement caused by use of a shock grid.

Access restricted. Please log in or start a trial to view this content.

Protocol

The procedure described here was approved by the VA Puget Sound Institutional Animal Care and Use Committee, and complies with the ILAR Guide for the Care and Use of Laboratory Animals.

1. Experimental Preparation

  1. Decide beforehand if blood or tissue collection soon after exercise is necessary, and if so, determine the post-exercise interval for conduction of such procedures. Examples include determination of exercise induced hormone or cytokine release into the circulation (necessitating blood collection), determination of exercise induced expression of mRNA species of interest (necessitating euthanasia followed by tissue collection). If immediate post exercise blood or tissue collection is indicated, have materials for these procedures on hand in the procedure room. Receive institutional review board approval and perform according to regulatory guidelines. Not all experiments require postexercise blood or tissue collection; a simple comparison of running endurance may be all that is needed.
  2. Decide in advance on experimental groups and postexercise procedures for mice (see description above).
  3. Place the treadmill on a sturdy table in a quiet room dedicated during the running trials to this procedure.
  4. Set the treadmill for mouse-sized lanes and at an inclination appropriate to the protocol. The inclination of +5o is used here to obviate muscle damage due to eccentric exercise (lengthening the muscle at the same time as contraction) that occurs during downhill running.
  5. Place an absorbent pad below the treadmill belt to catch feces.

2. Acclimation of Mice to the Treadmill

Decide if a “sedentary” (no exercise) group of mice is necessary, and expose sedentary mice to the treadmill without running as outlined in Steps 2.1 - 2.5. Determine if 2 or 4 experimental groups are required, e.g., Sedentary/Exercise (2 groups); Exercised Controls/Exercised Treatment (2 groups, no sedentary animals); or, Sedentary/Exercised x Control/Treatment (4 groups). The “treatment” may be a transgenic or knockout genotype, a pharmacological regimen, special diet, or other experimental intervention.

  1. Allow the mouse to acclimate to the procedure room in its transported home cage, with cage mates, for 1 - 2 hr.
  2. Select a single mouse and record tag number; weigh mouse and record weight.
  3. Place mouse in the treadmill without the motor turned on for acclimation, about 5 min. Turn power on, without the treadmill belt running, and let the mouse acclimate to the machine noise for another 5 min.
  4. Remove mice in the “sedentary” groups (if used; see Note 1) from the treadmill at this point and return them to their cage or conduct post-exercise procedures as per the experimental design (see Note 2).

3. Starting the Run-to-Exhaustion test

  1. After acclimation, turn the belt to a low speed (10 m/min) when the animal is exploring the belt and not on the platform.
  2. Gently tap or lift the hindquarters of the mouse with a tongue depressor, one to three times, to encourage reluctant animals to stay on the treadmill and run.
    NOTE: Most mice will run readily, but they sometimes stop for short periods. When this occurs, use the tongue depressor to gently tap or lift the rear of the animal to encourage resumption of running. Often, the mice do not need to be touched with the tongue depressor; noticing the hand with the tongue depressor coming towards them causes the mice to run some more.
  3. Start a laboratory timer once the mouse is running at the initial speed of 10 m/min.
  4. Turn the speed up slowly when the mouse is at the top (uphill end) of the belt, at increments of 1 m/min, about every 2 min.
  5. Turn the speed down to the previous setting if the mouse repeatedly runs in short bursts and not at a steady pace.
  6. Determine the maximal sustained running speed of each mouse (usually 14 - 17 m/min for C57BL/6 mice) by adjusting the speed and watching the mouse. Record this speed.
    NOTE: The location of the animal on the treadmill cues the investigator as to the willingness of the animal to run at a higher speed. If the mouse is at the far (uphill) end and about to run into the wall, it is ready to run at a higher speed. If the mouse is running at a steady pace in the middle of the treadmill it may be at its maximal speed, or may need further adjustment, depending on the quality of the run.

4. Endpoints and Mice that Stop

  1. Use the tongue depressor to gently tap or lift the hindquarters of the animals that stop, to encourage resumption of running (see Note 3.2). Do not stop the timer for temporary interruptions in running.
  2. Identify and remove mice that refuse to run . Note this on the data sheet. Depending on your experimental design, mice that refuse can be retested on a different day
    NOTE: Some mice will completely refuse to run. Such mice will run in short bursts, then stop to groom or “pedal”, that is, sit on the platform and use their front legs on the belt. The posture of these mice is hunched, with all four feet planted under the animal to keep from being pushed back onto the treadmill by the tongue depressor. Occasionally a mouse becomes aggressive towards the tongue depressor when unwilling to run, biting the tongue depressor or climbing up it as a way of escape. It is clear within the first 5 min of the trial whether the mouse will, or will not, run. Remove mice that refuse to run from the analysis, but do not count them as “sedentary” mice.
  3. Identify exhausted mice and stop the timer.
    NOTE: Exhaustion is defined by three successive stops and refusal to resume running despite gentle encouragement, plus physical signs of exhaustion such as labored breathing and splayed posture. Mice recover from exhaustion within 30 to 60 min.
  4. Record the total amount of time spent running at all speeds.
  5. Depending on the experimental design, either return the mouse is to its cage, or conduct immediate postexercise procedures such as blood or tissue collection as per institutional guidelines.
  6. Wipe the belt clean with a germicidal towelette after testing each individual mouse so that the scent of the previous mouse does not influence behavior of subsequent mice tested.
  7. Test the next mouse. Use a fresh tongue depressor for each mouse. Mice that reside in the same cage can be tested sequentially on the same day without additional acclimation to the procedure room.

Access restricted. Please log in or start a trial to view this content.

Results

This procedure for measuring exercise endurance accurately reflects the molecular and metabolic profile of different strains of C57BL/6 mice that differ in expression of the cytokine IL-15 16,17. Transgenic mice that overexpress IL-15 (IL-15 TG mice) exhibit significantly increased run-to-exhaustion times compared to littermate controls, while mice that lack IL-15 (IL-15 KO mice) exhibit significantly reduced run times prior to reaching exhaustion (Figure 1A). Published studies have shown that...

Access restricted. Please log in or start a trial to view this content.

Discussion

Described here is a method to assess voluntary running endurance in laboratory mice using a motorized treadmill without the use of a shock grid. This method can reveal differences among sub-lines of C57BL/6 mice that differ in expression of the cytokine IL-15, which in turn causes differences in expression of factors that underlie exercise endurance16,17. In keeping with the principles of the “Three R’s” in laboratory animal science, this method can be used as a more humane alternative, or re...

Access restricted. Please log in or start a trial to view this content.

Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

Supported by Merit Review #BX001026 from the Department of Veterans Affairs (LSQ), and use of resources and facilities at VA Puget Sound Health Care System, the Transgenic Resource Core at the University of Washington Nathan Shock Center of Excellence in the Basic Biology of Aging (NIA #5P30AG-013280), and the University of Washington Diabetes Endocrinology Research Center (NIH #P30 DK-17047). We thank Cynthia Pekow DVM and Kari L. Koszdin DVM, VA Puget Sound, provided helpful comments on the manuscript.

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Open Rodent Treadmill Exer-3/6Columbus Instruments, Columbus OH1050RMThis catalog number is for models without shock grid. Shock grids can be removed manually from older models (Eco 3/6)
Sani Cloth Germicidal TowlettesPDI- Professional Disposables, Inc., Orangeburg, NYusually ordered through local facilityContains 10% isopropyl alcohol; any equivalent product can be used.
Tongue depressorsAny local supplier
Laboratory timerAny supplier
IL-15 TG mice*JAX, Bar Harbor, ME011002Murine IL-15 transgene expressed from modified human alpha-skeletal actin promotor with altered signal sequence to facilitate secretion.
C57BL/6J mice (wild-type)*JAX, Bar Harbor, ME000664Control mice for IL-15 TG
IL-15 KO mice*Taconic Farms, Germantown, NY4269-MHomozygous IL-15 "knockout" mice on C57BL/6 background
C57BL/6NTac (wild-type)*Taconic Farms, Germantown, NYB6-MControl mice for IL-15 KO
*Optional; mouse lines and treatments can be specific to the experimental protocol.

References

  1. Hill, J. A., Wyatt, H. R., Reed, G. W., Peters, J. C. Obesity and the environment: where do we go from here? Science. 299 (5608), 853-855 (2003).
  2. Kahn, S. E., Hull, R. L., Utzschneider, K. M. Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature. 444 (7121), 840-846 (2006).
  3. Van Gaal, L. F., Mertens, I. L., De Block, C. E. Mechanisms linking obesity with cardiovascular disease. Nature. 444 (7121), 875-880 (2006).
  4. Pischon, T., Nothlings, U., Boeing, H. Obesity and cancer. Proc Natl Acad Sci USA. 67 (2), 128-145 (2008).
  5. Benton, C. R., Wright, D. C., Bonen, A. PGC-1α-mediated regulation of gene expression and metabolism: Implications for nutrition and exercise prescriptions. Appl. Physiol. Nutr. Metab. 33 (5), 843-862 (2008).
  6. Handschin, C., Spiegelman, B. M. The role of exercise and PGC1alpha in inflammation and chronic disease. Nature. 454 (7203), 463-469 (2008).
  7. Vasunilashorn, S., et al. Use of the Short Physical Performance Battery score to predict loss of ability to walk 400 meters: analysis from the InCHIANTI study. J. Gerontol. A Biol. Med. Sci. 64 (2), 223-229 (2009).
  8. Wang, Y. X., et al. Regulation of muscle fiber type and running endurance by PPARδ. PLoS Bio. 2 (10), e294(2004).
  9. LeBrasseur, N. K., et al. Myostatin inhibition enhances the effects of exercise on performance and metabolic outcomes in aged mice. J. Gerontol. A Biol. Med. Sci. 64 (9), 940-948 (2009).
  10. Burch, N., et al. Electric pulse stimulation of cultured murine muscle cells reproduces gene expression changes of trained mouse muscle. PLoS ONE. 5 (6), e10970(2010).
  11. Li, L., et al. Mitochondrial biogenesis and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) deacetylation by physical activity. Diabetes. 60 (1), 157-167 (2011).
  12. Knab, A. M., et al. Repeatability of exercise behaviors in mice. Physiol. & Behavior. 98 (4), 433-440 (2009).
  13. He, Y., et al. IL-15 Receptor deletion results in circadian changes of locomotor and metabolic behavior. J. Mol. Neurosci. 41 (2), 315-321 (2010).
  14. Wu, X., Hsuchou, H., Kastin, A. J., Rood, J. C., Pan, W. Essential role of interleukin-15 receptor in normal anxiety behavior. Brain Behav. Immun. 24 (8), 1340-1346 (2010).
  15. Pistilli, E. E., et al. Loss of IL-15 receptor α alters the endurance, fatigability, and metabolic characteristics of mouse fast skeletal muscles. J. Clin. Invest. 121 (8), 3120-3132 (2011).
  16. Quinn, L. S., Anderson, B. G., Conner, J. D., Wolden-Hanson, T. W. IL-15 overexpression promotes endurance, oxidative energy metabolism, and muscle PPARδ, SIRT1, PGC-1α, and PGC-1β expression in male mice. Endocrinology. 154 (1), 232-245 (2013).
  17. Quinn, L. S., Anderson, B. G., Conner, J. D., Wolden-Hanson, T., Marcell, T. J. IL-15 is required for post-exercise induction of the pro-oxidative mediators PPARdelta and SIRT1. Endocrinology. , (2013).
  18. Ward, J. M., Anver, M. R., Mahler, J. F., Devor-Henneman, D. E. Chapter 13, Pathology of mice commonly used in genetic engineering (C57BL/6; 129; B6,129; and FVB/N). The Pathology of Genetically Engineered Mice. Ward, J. M., Mahler, J. F., Maronpot, R. R., Sundberg, J. P. , Iowa State University Press. Ames, IA, Chapter. 161-179 (2000).
  19. Lightfoot, J. T., Turner, M. J., Debate, K. A., Kleeberger, S. R. Interstrain variation in murine aerobic capacity. Med. Sci. Sports Exerc. 33 (12), 2053-2057 (2001).
  20. Turner, M. J., Kleeberger, S. R., Lightfoot, J. T. Influence of genetic background on daily running-wheel activity differs with aging. Physiol. Genomics. 22 (1), 76-85 (2005).
  21. Haight, T. J., van der Laan, M. J., Manini, T., Tager, I. B. Direct effects of leisure-time physical activity on walking speed. J. Nutr. Health Aging. 17 (8), 666-673 (2013).
  22. Studenski, S. Gait speed and survival in older adults. JAMA. 305 (1), 50-58 (2011).
  23. Jacobsen, K. R., Kalliokoski, O., Teilmann, A. C., Hau, J., Abelson, K. S. The effect of isoflurane anaesthesia and vasectomy on circulating corticosterone and ACTH in BALB/c mice. Gen. Comp. Endocrinol. 179 (3), 406-413 (2012).
  24. Sellers, T. L., Jaussi, A. W., Yang, H. T., Heninger, R. W., Winder, W. W. Effect of the exercise-induced increase in glucocorticoids on endurance in the rat. J. Appl. Physiol. 65 (1), 173-178 (1988).
  25. Cook, M. D., et al. Forced treadmill exercise training exacerbates inflammation and causes mortality while voluntary wheel training is protective in a mouse model of colitis. Brain Behav. Immun. 33, 46-56 (2013).

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

Mouse TreadmillVoluntary RunningShock Grid AlternativeTreadmill AcclimationExhaustion EndpointsIL 15 TransgenicMaximal Sustained SpeedGentle EncouragementTime to Exhaustion

Related Articles