Name: a simple and inexpensive running wheel model for progressive resistance training in mice
Uploaded: 2022-04-28T15:00:00
Description: Watch this Scientific Journal Video about A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice at JoVE.com

We would like to thank the Graduate Student Government Association, Office of Student Research, and the Department of Health and Exercise Science at Appalachian State University for providing funding to support this project. Additionally, we would like to thank Monique Eckerd and Therin Williams-Frey for overseeing daily operations of the animal research facility.

This study was approved by Appalachian State University&#39;s Institutional Animal Care and Use Committee (#22-05).
1. Animals
<ol>
	<li>Procure C57BL/6 mice from the in-house mouse colony. 
	NOTE: Male mice 5-8 months of age at the start of the study were used. Daily running activity peaks and plateaus at around 9-10 weeks of age26. Previous studies have demonstrated that old mice (22-24 months) will also perform loaded wheel running<sup class="...

<ol>
	<li>Frontera, W. R., Ochala, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle:+A+brief+review+of+structure+and+function.">Skeletal muscle: A brief review of structure and function.</a> Calcified Tissue International. 96 (3), 183-195 (2015).</li><li>Goldberg, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+synthesis+during+work-induced+growth+of+skeletal+muscle.">Protein synthesis during work-induced growth of skeletal muscle.</a> Journal of Cell Biology. 36 (3), 653-658 (1968).</li><li>Baar, K., Esser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+p70S6k+correlates+with+increased+skeletal+muscle+mass+following+resistance+exercise.">Phosphorylation of p70S6k correlates with increased skeletal muscle mass following resistance exercise.</a> American Journal of Physiology-Cell Physiology. 276 (1), 120-127 (1999).</li><li>Wong, T. S., Booth, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+enlargement+with+weight-lifting+exercise+by+rats.">Skeletal muscle enlargement with weight-lifting exercise by rats.</a> Journal of Applied Physiology. 65 (2), 950-954 (1988).</li><li>Hornberger Jr, T. A., Farrar, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+hypertrophy+of+the+FHL+muscle+following+8+weeks+of+progressive+resistance+exercise+in+the+rat.">Physiological hypertrophy of the FHL muscle following 8 weeks of progressive resistance exercise in the rat.</a> Canadian Journal of Applied Physiology. 29 (1), 16-31 (2004).</li><li>Zhu, W. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Weight+pulling:+A+novel+mouse+model+of+human+progressive+resistance+exercise.">Weight pulling: A novel mouse model of human progressive resistance exercise.</a> Cells. 10 (9), 2459 (2021).</li><li>Tamaki, T., Uchiyama, S., Nakano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+weight-lifting+exercise+model+for+inducing+hypertrophy+in+the+hindlimb+muscles+of+rats.">A weight-lifting exercise model for inducing hypertrophy in the hindlimb muscles of rats.</a> Medicine and Science in Sports and Exercise. 24 (8), 881-886 (1992).</li><li>De Bono, J. P., Adlam, D., Paterson, D. J., Channon, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+quantitative+phenotypes+of+exercise+training+in+mouse+models.">Novel quantitative phenotypes of exercise training in mouse models.</a> American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 290 (4), 926-934 (2006).</li><li>Goh, J., Ladiges, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voluntary+wheel+running+in+mice.">Voluntary wheel running in mice.</a> Current Protocols in Mouse Biology. 5 (4), 283-290 (2015).</li><li>Lightfoot, J. T., Turner, M. J., Daves, M., Vordermark, A., Kleeberger, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+influence+on+daily+wheel+running+activity+level.">Genetic influence on daily wheel running activity level.</a> Physiological Genomics. 19 (3), 270-276 (2004).</li><li>Allen, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+and+skeletal+muscle+adaptations+to+voluntary+wheel+running+in+the+mouse.">Cardiac and skeletal muscle adaptations to voluntary wheel running in the mouse.</a> Journal of Applied Physiology. 90 (5), 1900-1908 (2001).</li><li>Ishihara, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+running+exercise+with+increasing+loads+on+tibialis+anterior+muscle+fibres+in+mice.">Effects of running exercise with increasing loads on tibialis anterior muscle fibres in mice.</a> Experimental Physiology. 87 (2), 113-116 (2002).</li><li>Kurosaka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+voluntary+wheel+running+on+satellite+cells+in+the+rat+plantaris+muscle.">Effects of voluntary wheel running on satellite cells in the rat plantaris muscle.</a> Journal of Sports Science and Medicine. 8 (1), 51-57 (2009).</li><li>Lambert, M. I., Noakes, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+running+increases+VO2max+and+running+performance+in+rats.">Spontaneous running increases VO2max and running performance in rats.</a> Journal of Applied Physiology. 68 (1), 400-403 (1990).</li><li>Rodnick, K. J., Reaven, G. M., Haskell, W. L., Sims, C. R., Mondon, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variations+in+running+activity+and+enzymatic+adaptations+in+voluntary+running+rats.">Variations in running activity and enzymatic adaptations in voluntary running rats.</a> Journal of Applied Physiology. 66 (3), 1250-1257 (1989).</li><li>Sexton, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+adaptations+in+rat+hindlimb+skeletal+muscle+after+voluntary+running-wheel+exercise.">Vascular adaptations in rat hindlimb skeletal muscle after voluntary running-wheel exercise.</a> Journal of Applied Physiology. 79 (1), 287-296 (1995).</li><li>Dungan, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+myonuclear+density+during+skeletal+muscle+hypertrophy+in+response+to+training+is+reversed+during+detraining.">Elevated myonuclear density during skeletal muscle hypertrophy in response to training is reversed during detraining.</a> American Journal of Physiology-Cell Physiology. 316 (5), 649-654 (2019).</li><li>Ishihara, A., Roy, R. R., Ohira, Y., Ibata, Y., Edgerton, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertrophy+of+rat+plantaris+muscle+fibers+after+voluntary+running+with+increasing+loads.">Hypertrophy of rat plantaris muscle fibers after voluntary running with increasing loads.</a> Journal of Applied Physiology. 84 (6), 2183-2189 (1998).</li><li>Konhilas, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loaded+wheel+running+and+muscle+adaptation+in+the+mouse.">Loaded wheel running and muscle adaptation in the mouse.</a> American Journal of Physiology-Heart and Circulatory Physiology. 289 (1), 455-465 (2005).</li><li>Legerlotz, K., Elliott, B., Guillemin, B., Smith, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voluntary+resistance+running+wheel+activity+pattern+and+skeletal+muscle+growth+in+rats:+Wheel+running+activity+pattern+and+muscle+growth.">Voluntary resistance running wheel activity pattern and skeletal muscle growth in rats: Wheel running activity pattern and muscle growth.</a> Experimental Physiology. 93 (6), 754-762 (2008).</li><li>Mobley, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+resistance-loaded+voluntary+wheel+running+increases+hypertrophy+and+differentially+affects+muscle+protein+synthesis,+ribosome+biogenesis,+and+proteolytic+markers+in+rat+muscle.">Progressive resistance-loaded voluntary wheel running increases hypertrophy and differentially affects muscle protein synthesis, ribosome biogenesis, and proteolytic markers in rat muscle.</a> Journal of Animal Physiology and Animal Nutrition. 102 (1), 317-329 (2018).</li><li>D'Hulst, G., Palmer, A. S., Masschelein, E., Bar-Nur, O., De Bock, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voluntary+resistance+running+as+a+model+to+induce+mTOR+activation+in+mouse+skeletal+muscle.">Voluntary resistance running as a model to induce mTOR activation in mouse skeletal muscle.</a> Frontiers in Physiology. 10, 1271 (2019).</li><li>Soffe, Z., Radley-Crabb, H. G., McMahon, C., Grounds, M. D., Shavlakadze, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+loaded+voluntary+wheel+exercise+on+performance+and+muscle+hypertrophy+in+young+and+old+male+C57Bl/6J+mice:+Exercise+and+muscle+hypertrophy+in+old+mice.">Effects of loaded voluntary wheel exercise on performance and muscle hypertrophy in young and old male C57Bl/6J mice: Exercise and muscle hypertrophy in old mice.</a> Scandinavian Journal of Medicine and Science in Sports. 26 (2), 172-188 (2016).</li><li>White, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voluntary+resistance+wheel+exercise+from+mid-life+prevents+sarcopenia+and+increases+markers+of+mitochondrial+function+and+autophagy+in+muscles+of+old+male+and+female+C57BL/6J+mice.">Voluntary resistance wheel exercise from mid-life prevents sarcopenia and increases markers of mitochondrial function and autophagy in muscles of old male and female C57BL/6J mice.</a> Skeletal Muscle. 6 (1), 45 (2016).</li><li>Murach, K. A., McCarthy, J. J., Peterson, C. A., Dungan, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+mice+mighty:+Recent+advances+in+translational+models+of+load-induced+muscle+hypertrophy.">Making mice mighty: Recent advances in translational models of load-induced muscle hypertrophy.</a> Journal of Applied Physiology. 129 (3), 516-521 (2020).</li><li>Swallow, J. G., Garland, T., Carter, P. A., Zhan, W. -. Z., Sieck, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+voluntary+activity+and+genetic+selection+on+aerobic+capacity+in+house+mice+(Mus+domesticus).">Effects of voluntary activity and genetic selection on aerobic capacity in house mice (Mus domesticus).</a> Journal of Applied Physiology. 84 (1), 69-76 (1998).</li><li>Murach, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late-life+exercise+mitigates+skeletal+muscle+epigenetic+aging.">Late-life exercise mitigates skeletal muscle epigenetic aging.</a> Aging Cell. 21 (1), 13527 (2022).</li><li>Mackay, A. D., Marchant, E. D., Louw, M., Thomson, D. M., Hancock, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise,+but+not+metformin+prevents+loss+of+muscle+function+due+to+doxorubicin+in+mice+using+an+in+situ+method.">Exercise, but not metformin prevents loss of muscle function due to doxorubicin in mice using an in situ method.</a> International Journal of Molecular Sciences. 22 (17), 9163 (2021).</li><li>Godwin, J. S., Hodgman, C. F., Needle, A. R., Zwetsloot, K. A., Andrew, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+heat+shock+accelerates+recovery+from+impact-+induced+skeletal+muscle+damage+in+mice.">Whole-body heat shock accelerates recovery from impact- induced skeletal muscle damage in mice.</a> Conditioning Medicine. 2 (4), 184-191 (2020).</li><li>Wen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MyoVision:+Software+for+automated+high-content+analysis+of+skeletal+muscle+immunohistochemistry.">MyoVision: Software for automated high-content analysis of skeletal muscle immunohistochemistry.</a> Journal of Applied Physiology. 124 (1), 40-51 (2018).</li><li>Manzanares, G., Brito-da-Silva, G., Gandra, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voluntary+wheel+running:+Patterns+and+physiological+effects+in+mice.">Voluntary wheel running: Patterns and physiological effects in mice.</a> Brazilian Journal of Medical and Biological Research. 52 (1), 7830 (2019).</li><li>Bartling, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-related+differences+in+the+wheel-running+activity+of+mice+decline+with+increasing+age.">Sex-related differences in the wheel-running activity of mice decline with increasing age.</a> Experimental Gerontology. 87, 139-147 (2017).</li><li>Zwetsloot, K. A., Westerkamp, L. M., Holmes, B. F., Gavin, T. 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Existing resistance exercise models in rodents have proven invaluable for skeletal muscle research; however, many of these models are invasive, involuntary, and/or time- and labor-intensive. LWR is an excellent model that not only induces similar muscular adaptations as those observed in other well-accepted resistance exercise training models, but also provides a chronic, low-stress exercise stimulus for the animal with minimal time/labor commitment by the researcher. Additionally, since LWR models require minimal direct...

Skeletal muscle mass comprises approximately 40% of body mass in adult humans; thus, maintaining skeletal muscle mass throughout life is critical. Skeletal muscle mass plays an integral role in energy metabolism, maintaining core body temperature, and glucose homeostasis1. The maintenance of skeletal muscle is a balance between protein synthesis and protein degradation, but many gaps still exist in the understanding of the intricate molecular mechanisms that drive these processes. To study the molecular mechanisms that regulate the maintenance and growth of muscle mass, human subjects&#39; research models often employ resistance exercise-based interventions, since mechanical stimuli play an integral role in the regulation of skeletal muscle mass. While human subjects research has been successful, the time necessary to exhibit adaptations and ethical concerns regarding invasive procedures (i.e., muscle biopsies) limit the quantity of data that can be obtained. While the adaptations to resistance exercise are fairly ubiquitous across mammalian species, animal models provide the benefit of being able to precisely control the diet and exercise regimen while also allowing for the collection of whole tissues throughout the body, such as the brain, liver, heart, and skeletal muscle.
Many resistance training models have been developed for use in rodents: synergistic ablation2, electrical stimulation3,4, weighted ladder climbing5, weighted sled pulling6, and canvassed squatting7. It is evident that all of these models, if done correctly, can be effective models to induce skeletal muscle adaptations, such as hypertrophy. However, the downfalls of these models are that they are mostly involuntary, not part of normal rodent behavior, time-/labor-intensive, and invasive.
Fortunately, many mouse and rat strains voluntarily run long distances when given access to a running wheel. Moreover, free-running wheel (FWR) exercise models do not rely on extensive conditioning, positive/negative reinforcement, or anesthesia to force movement or muscle activity8,9. Running activity depends greatly on mouse strain, sex, age, and an individual basis. Lightfoot et al. compared the running activity of 15 different mouse strains and found that daily running distance ranges from 2.93 km to 7.93 km, with C57BL/6 mice running the farthest, regardless of sex10. FWR is commonly accepted as an excellent model for inducing endurance adaptations in skeletal and cardiac muscles11,12,13,14,15,16; however, utilizing wheel running in resistance training models is less commonly investigated.
As one could suspect, the hypertrophic effect of wheel running might be augmented by adding resistance to the running wheel, termed loaded wheel running (LWR), thus requiring greater efforts to run on the wheel to more closely mimic resistance training. Using varied methods of load application, previous studies have demonstrated that the LWR model utilizing rats and mice routinely displayed increases in limb muscle mass of 5%-30% in a matter of 6-8 weeks17,18,19,20,21. Furthermore, D&#39;hulst et al. demonstrated that a single bout of LWR led to a 50% greater increase&#160;in activation of the protein synthesis signaling pathway compared to FWR22. Wheel resistance has been most commonly applied by a friction-based, constant loading method, whereby a magnetic brake or tension bolt is utilized to apply wheel resistance12,19,23,24. One caveat of the friction-based, constant load method is that when moderate to high resistance is applied, the animal cannot overcome the high resistance to initiate movement of the wheel, effectively ceasing training. Most importantly, many of the cage and wheel systems used for rodent running wheel models are quite costly and require specialized equipment.
Recently, Dungan et al. developed a progressive weighted-wheel-running (PoWeR) model, which applies a load to the wheel asymmetrically via external masses adhered to a single side of the wheel. The unbalanced wheel loading and variable resistance of the PoWeR model are thought to encourage continued running activity and promote shorter bursts of loaded wheel running in mice, more closely imitating the sets and repetitions performed with resistance training17. Despite the average running distance being 10-12 km per day, the PoWeR model yielded a 16% and 17% increase in plantaris muscle wet mass and fiber cross-sectional area (CSA), respectively. Despite many practical advantages, the PoWeR model of LWR does have some limitations. As recognized by the authors, the PoWeR model is a high-volume &#34;hybrid&#34; stimulus that is reflective of a blended endurance/resistance exercise model (i.e., concurrent training in humans), as opposed to a more strictly resistance exercise-based model, potentially introducing an interference effect and contributing to the less pronounced hypertrophy or different mechanisms by which hypertrophy is induced25. Ensuring that a concurrent training phenomenon does not occur in what is intended to be a resistance exercise training model is imperative. Therefore, the PoWeR model was modified to develop a LWR model that utilizes higher loads than previously used to more closely resemble a resistance training model. Herein, details are provided for a simple and inexpensive 9 week progressive resistance training LWR model in C57BL/6 mice.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 g disc neodymium magnets</td>
			<td>Applied Magnets</td>
			<td>ND018-6</td>
			<td>Used for all sensor magnets and 1 g increments of wheel loading</td>
		</tr>
		<tr>
			<td>2.5 g disc neodymium magnets</td>
			<td>Applied Magnets</td>
			<td>ND022</td>
			<td>Used for 2.5 g increments of wheel loading</td>
		</tr>
		<tr>
			<td>8-32 x 1&#34; stainless steel screws</td>
			<td>Amazon</td>
			<td></td>
			<td>https://www.amazon.com/gp/product/B07939RS23/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&#38;psc=1</td>
		</tr>
		<tr>
			<td>8-32 Wing Nuts</td>
			<td>Amazon</td>
			<td></td>
			<td>https://www.amazon.com/gp/product/B07YYWW2SB/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&#38;th=1</td>
		</tr>
		<tr>
			<td>10 &#181;L pipette tip box (empty)</td>
			<td>Thermo Scientific</td>
			<td>2140</td>
			<td>We used empty ART Pipette tip boxes, but any similar sized boxes/trays would suffice</td>
		</tr>
		<tr>
			<td>Extreme Liquid Glue</td>
			<td>Loctite</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin primary antibody</td>
			<td>Novus Biologicals</td>
			<td>NB300-144AF647</td>
			<td>primary antibody conjugated with AF657; 1:200 in PBS containing 10% normal goat serum</td>
		</tr>
		<tr>
			<td>Lithium 3 V battery</td>
			<td>n/a</td>
			<td>CR2032</td>
			<td></td>
		</tr>
		<tr>
			<td>M10 (3/16&#34; x 1 1/4&#34;) stainless steel fender washers</td>
			<td>Amazon</td>
			<td></td>
			<td>https://www.amazon.com/gp/product/B00OHUHEU8/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&#38;th=1</td>
		</tr>
		<tr>
			<td>MyoVision: Automated Image Quantification Platform&#160;</td>
			<td>Wen et al. (2017)</td>
			<td>v1.0</td>
			<td>https://www.uky.edu/chs/center-for-muscle-biology/myovision</td>
		</tr>
		<tr>
			<td>Polycarbonate rodent cage (430 mm L x 290 mm W x 201 mm H), with narrow width stainless steel wired bar lid</td>
			<td>Orchid Scientific</td>
			<td>Polycarbonate Rat Cage Type II</td>
			<td>https://orchidscientific.com/product/rat-cage/ - 1519974600758-c29bc1c5-6dfa</td>
		</tr>
		<tr>
			<td>Sigma Sport 509 Bike Computer</td>
			<td>Sigma Sport</td>
			<td></td>
			<td>Does not need to be this model in particular, but must have distance and time monitoring capabilities</td>
		</tr>
		<tr>
			<td>Silent Spinner Running Wheel (mini 11.4 cm)</td>
			<td>Kaytee</td>
			<td>SKU# 100079369</td>
			<td>https://www.kaytee.com/all-products/small-animal/silent-spinner-wheel</td>
		</tr>
	</tbody>
</table>

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.

In this study, 24 C57BL/6 mice (6.3 &#177; 0.7 months at the start of this study) were randomly assigned to one of three treatment groups: sedentary (SED), loaded wheel running (LWR; same as PoWeR described by Dungan et al.17), or high LWR (HLWR), and then completed their respective 9 week protocol. After the acclimation week (week 1), there were no group or group x time differences in running distance or training volume (Figure 5).
<p class="jove_content biglegen...

Watch this Scientific Journal Video about A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice at JoVE.com

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

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 ...

This protocol allows researchers to resistance train large cohorts of mice at a much lower cost compared to models that use commercially available running wheel equipment. The primary advantage of this resistance training model is that it is entirely voluntary, which reduces stress for the animals and time commitment for the researcher. This model can help better understand the cellular and molecular mechanisms that regulate muscle mass in response to exercise training.By design, this loaded wheel running model is relatively simple to execute. However, it is still recommended that researchers perform pilot testing in the unique laboratory setting to estimate the running performance of mice prior to experimentation. Demonstrating this procedure will be PJ Koopmans, a graduate research assistant in my laboratory.To set up the running wheel apparatus, glue a single one gram sensor magnet to the outer middle circumference of the running wheel and use this wheel for only the first week of wheel acclimation. Loaded wheel running requires two grams of load, hence glue two magnets of one gram side by side onto the outer circumference of the wheel. A tape can be used to hold the magnets in place until the glue firmly dries.As the weeks pass, apply additional load in weeks 3, 4, 5, and 7 by placing another one gram magnet on top of either magnet already present. As these magnets firmly adhere to one another, no glue will be required. High loaded wheel running setup requires three sets of wheels.The first set of wheels, which is required for week two only has a single 2.5 gram magnet glued onto the outer circumference of the wheel. The second set of wheels, which is required for week three only, has two 2.5 gram magnets glued side by side onto the outer circumference of the wheel. The third set of wheels required for four weeks and beyond has three 2.5 gram magnets glued side by side onto the outer circumference of the wheel.Apply additional load for week six and eight by placing another 2.5 gram magnet on top of either of the magnets already present. Ensure that a fresh battery is inserted into the bike computer before the assembly. Then assemble the running wheels using a cage equipped with a digital bike computer to monitor time and distance traveled during exercise.The average speed in kilometers per hour is derived arithmetically. Set the wheel size during initial bike computer programming and calculate per revolution distance by measuring the outer circumference of the running wheel. To ensure that all computer and sensor components are contained within a solid barrier outside the cage to prevent mice from chewing on the components, utilize the lid of an empty pipette tip box with a small rectangle cutout for the magnetic bike sensor and the main part of the box to hold the bike computer, and wire.Drill two holes through the corners of the pipette tip box lid to secure the magnetic bike sensor and running wheel stand in place on the outside of the cage and insert the running wheel base upside down through the gaps in the cage lid but on top of the solid surface. Secure the wheel base and computer sensor to the top of the cage with hardware. Ensure that the bike computer sensor and the pipette tip box lid is directly above where the sensor magnet of the wheel is located and that the sensor magnet and computer sensor are spaced no more than one centimeter apart to allow the proper recording of wheel movement.Attach the appropriate running wheel to the wheel base before placing the lid onto the cage, then securely placing the lid onto the cage. With the wheel hanging from the cage lid, ensure at least 2.5 centimeters of clearance from the cage floor. Then place a minimal amount of bedding material in the cage to ensure that the wheel spins freely but does not become impeded by the buildup of bedding.As mice are an nocturnal species, most of their natural cage activity, including wheel running, will be performed during the dark hours of the light cycle. During the experiment, record this data from the bike computer at a consistent interval scheduled to ensure accurate activity monitoring. Individually house sedentary mice for nine weeks in a cage containing a locked running wheel to prevent any running.Reduce load for the loaded wheel running and high loaded wheel running groups if necessary to ensure that mice continue to exercise for the entire nine a week protocol as per the loading schedule. During the study, mice were randomly assigned to one of three treatment groups, namely sedentary, loaded wheel running, or high loaded wheel running, and then completed their respective nine week protocol. After the one week of acclimation, there were no group or group by time differences in running distance or training volume.Normalized soleus mass was 21.4%larger in the high loaded wheel running group than in the sedentary group despite no difference in fiber cross-sectional area. Although plantaris muscle mass and average fiber cross-sectional area did not displace statistically significant differences, there appears to be a shift in the the proportion of fibers with a larger cross-sectional area in the plantaris of high loaded wheel running compared to sedentary and loaded wheel running. There were no significant differences in twitch or peak force of the GPS complex between groups as measured by an NC2 muscle function test.It is important to ensure the wheel spins freely within the cage and that the wheel sensor magnet is close to the bike computer sensor so wheel rotation is unimpeded and the bike computer accurately records running data. Following this procedure, researchers can perform subsequent analyses such as contractile function or immunohistochemical techniques to examine the various physiological responses further to exercise training.

a-simple-inexpensive-running-wheel-model-for-progressive-resistance

Research

JoVE Journal

Biology

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model

Preclinical models of restenosis are essential to unravel the pathophysiological processes that lead to in-stent restenosis and to optimize existing and future drug-eluting stents.
A variety of antibodies and transgenic and knockout strains are available in rats. Consequently, a model for in-stent restenosis in the rat would be convenient for pathobiological and pathophysiological studies. 
In this video, we present the full procedure and pit-falls of a rat stent model suitable for high throughput stent research. We will show the surgical procedure of stent deployment, and the assessment of in-stent restenosis using the most elegant technique of OCT (Optical Coherence Tomography). This technique provides high accuracy in assessing plaque CSAs (cross section areas) and correlates well with histological sections, which require special and time consuming embedding and sectioning techniques. OCT imaging further allows longitudinal monitoring of the development of in-stent restenosis within the same animal compared to one-time snapshots using histology.

This video demonstrates how to use a preclinical inexpensive and reliable model to study pathobiological and pathophysiological processes of in-stent restenosis development. Longitudinal in vivo monitoring using OCT (Optical Coherence Tomography) and analysis of OCT images are also demonstrated.

Preclinical models of restenosis are essential to unravel the pathophysiological processes that lead to in-stent restenosis and to optimize existing and ...

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

In vivo Measurement of Knee Extensor Muscle Function in Mice

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the ...

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most animal laboratories.&#160;Contrary to the conventional approach for double ligation of the femoral artery (DLFA), a smaller incision on the right inguinal region was made to expose the proximal femoral artery (FA) to perform DLFA. Then, using a 7-0 suture, the incision was dragged to the knee region to expose the distal FA. Magnetic resonance imaging (MRI) on bilateral hind limbs was used to detect FA occlusion after the surgery. At 0, 1, 3, 5, and 7 days after the surgery, functional recovery of the hind limbs was visually assessed and graded using the Tarlov scale. Histologic evaluation was performed after euthanizing the animals 7 days after DLFA. The procedures were successfully performed on the right leg in ten ApoE-/- mice, and no mice died during subsequent observation. The incision sizes in all 10 mice were less than 5 mm (4.2 &#177; 0.63 mm). MRI results showed that FA blood flow in the ischemic side was clearly blocked. The Tarlov scale results demonstrated that hind limb function significantly decreased after the procedure and slowly recovered over the following 7 days. Histologic evaluation showed a significant inflammatory response on the ischemic side and reduced microvascular density in the ischemic hind limb. In conclusion, this study introduces a modified technique using a miniature incision to perform hind limb ischemia (HLI) using DLFA.

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades.

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most ...