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 running27.</li>
	<li>House the mice individually in a standard rodent cage with a wire lid and keep the cage in a controlled environment (20-24 &#176;C with a 12:12 h light:dark cycle).</li>
	<li>Provide standard rodent chow and water ad libitum.</li>
</ol>
2. Running wheel apparatus
<ol>
	<li>Running wheel setup: 
	NOTE: Running wheels are assembled/set up similarly for all running protocols, except for adding 1 g or 2.5 g load magnets.
	<ol>
		<li>Glue a single 1 g sensor magnet to the outer middle circumference of the running wheel (Figure 1).</li>
		<li>Use this wheel with a single 1 g sensor magnet for only the first week of wheel acclimation.</li>
		<li>Loaded wheel running (LWR; identical loading protocol to PoWeR17): Follow steps 2.1.4-2.1.6.</li>
		<li>Week 2 for LWR requires 2 g of load (see Table 1) .</li>
		<li>Glue two 1 g magnets side-by-side onto the outer circumference of the wheel (Figure 2A). 
		NOTE: Here, it is helpful to use tape to hold the magnets in place until the glue firmly dries; otherwise, they may be attracted to the sensor magnet and become dislodged.</li>
		<li>Apply additional load in weeks 3, 4, and 6 by placing another 1 g magnet on top of either of the magnets already present. 
		NOTE: No glue is necessary as the magnets firmly adhere to one another. For example, with 6 g of load in week 6, the magnets will each be stacked three-high (Figure 2B).</li>
		<li>High loaded wheel running (HLWR): Follow steps 2.1.8-2.1.11. 
		NOTE: The HLWR protocol requires three sets of wheels. Assembling different sets of wheels allows the researcher to reuse wheel setups for other mice once the wheel is thoroughly cleaned and sanitized (numbers of each set should be determined by the researcher based on cohort/group size).</li>
		<li>The first set of wheels (required for week 2 only) will have a single 2.5 g magnet; glue (refer to the NOTE below Step 2.1.5) one 2.5 g magnet onto the outer circumference of the wheel (Figure 3A).</li>
		<li>The second set of wheels (required for week 3 only) will have two 2.5 g magnets; glue (refer to the NOTE below Step 2.1.5) two 2.5 g magnets side-by-side onto the outer circumference of the wheel (Figure 3B).</li>
		<li>The third set of wheels (required for week 4 and beyond) will have three 2.5 g magnets side-by-side; glue (refer to the NOTE below Step 2.1.5) three 2.5 g magnets side-by-side onto the outer circumference of the wheel (Figure 3C).</li>
		<li>Apply additional load for weeks 6 and 8 by placing another 2.5 g magnet on top of either of the magnets already present (Figures 3D, E).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63933/63933fig01.jpg" /> 
Figure 1: Basic running wheel with single 1 g sensor magnet glued to the middle outer circumference of the wheel. <a href="https://www.jove.com/files/ftp_upload/63933/63933fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63933/63933fig02.jpg" /> 
Figure 2: Loaded running wheel (LWR) with sensor magnet and 1 g loading magnets. (A) Example of 2 g of load, two 1 g magnets glued side-by-side to the outer edge of wheel; (B) example of 6 g of load, two 1 g magnets glued side-by-side to the outer edge of wheel with an additional 4 g of load applied. <a href="https://www.jove.com/files/ftp_upload/63933/63933fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63933/63933fig03.jpg" /> 
Figure 3: High loaded running wheel (HLWR) with sensor magnet and 2.5 g loading magnets. (A) example of 2.5 g of load, one 2.5 g magnet glued to the outer edge of wheel; (B) example of 5 g of load, two 2.5 g magnets glued side-by-side to the outer edge of wheel; (C) example of 7.5 g of load, three 2.5 g magnets glued side-by-side to the outer edge of wheel; (D) example of 10 g of load, three 2.5 g magnets glued side-by-side to the outer edge of wheel, with an additional 2.5 g of load applied; (E) example of 12.5 g of load, three 2.5 g magnets glued side-by-side to the outer edge of wheel, with an additional 5 g of load applied. <a href="https://www.jove.com/files/ftp_upload/63933/63933fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Cage assembly
<ol>
	<li>Assemble running wheels using a cage equipped with a digital bike computer to monitor time exercising (h) and distance traveled (km). Average speed (km/h) is derived arithmetically.
	<ol>
		<li>Ensure that a fresh battery is inserted into the bike computer prior to assembly.</li>
		<li>Set wheel size during initial bike computer programming (see manufacturer&#39;s instructions); calculate per revolution distance by measuring the outer circumference of the running wheel (e.g., 3,580 mm for the wheel type used herein).</li>
	</ol>
	</li>
	<li>Place the bike computer sensor within a solid surface on the outside of the cage lid, directly above where the sensor magnet of the wheel is located. Ensure that all computer and sensor components are contained within a solid barrier outside the cage to prevent mice from chewing on components.
	<ol>
		<li>Utilize the lid of an empty pipette tip box with a small rectangle cut out for the magnetic bike sensor to reside, and the main part of the box (with the tip rack grid removed) to hold the bike computer and wire (Figure 4A).</li>
		<li>Drill two holes through the corners of the solid surface to secure the magnetic bike sensor and running wheel stand in place on the outside of the cage (Figure 4A).</li>
	</ol>
	</li>
	<li>Insert the running wheel base, upside down, through the gaps in the cage lid but on top of the solid surface described in step 3.2 (Figure 4B).
	<ol>
		<li>Secure the wheel base and computer sensor to the top of the cage with hardware (Figure 4C, D).</li>
	</ol>
	</li>
	<li>Ensure that the sensor magnet and computer sensor are spaced no more than 1 cm apart to allow for proper recording of wheel movement (most standard bike computer sensors are bi-directional and will record positive wheel movement in either direction of rotation).</li>
	<li>Attach the appropriate running wheel (as described above) to the wheel base from the inside of the cage lid, and securely place the lid onto the cage (Figure 4E, F).</li>
	<li>With the wheel hanging from the cage lid, ensure at least 2.5 cm of clearance from the cage floor. 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.</li>
	<li>During experimentation, record data from the bike computer at a consistent interval schedule to ensure accurate activity monitoring.
	<ol>
		<li>Recognize that mice are a nocturnal species; therefore, most of their natural cage activity (including wheel running) will be performed during the dark hours of the light cycle.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63933/63933fig04.jpg" /> 
Figure 4: Running wheel cage assembly. (A) Bike computer and magnetic sensor placed in solid surface/tray; (B) inverted wheel base placed on top of solid surface/tray and sensor (top view; note the two holes in sensor surface/tray for securing base to cage lid with hardware), (C) inverted wheel base with hardware assembled (bottom view); (D) inverted wheel base with hardware assembled (top view); (E) full cage assembly (top view); and (F) full cage assembly (side view). <a href="https://www.jove.com/files/ftp_upload/63933/63933fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Exercise training loading protocols
<ol>
	<li>Individually house sedentary (SED) mice for 9 weeks in a cage containing a locked running wheel to prevent any running. 
	NOTE: Table 1 provides the loading schedule for the LWR (PoWeR) and HLWR protocols used in the experimental design.</li>
	<li>Reduce load for the LWR and HLWR groups, if necessary, to ensure that mice continue to exercise for the entire 9 week protocol.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td style="min-width: 80px;"></td>
			<td style="min-width: 80px;"></td>
			<td colspan="9" style="min-width: 80px;">Week</td>
		</tr>
		<tr>
			<td style="min-width: 80px;"></td>
			<td style="min-width: 80px;"></td>
			<td style="min-width: 80px;">1</td>
			<td style="min-width: 80px;">2</td>
			<td style="min-width: 80px;">3</td>
			<td style="min-width: 80px;">4</td>
			<td style="min-width: 80px;">5</td>
			<td style="min-width: 80px;">6</td>
			<td style="min-width: 80px;">7</td>
			<td style="min-width: 80px;">8</td>
			<td style="min-width: 80px;">9</td>
		</tr>
		<tr>
			<td rowspan="2" style="min-width: 80px;">LWR (n = 4)</td>
			<td style="min-width: 80px;">Load (g)</td>
			<td style="min-width: 80px;">0.0</td>
			<td style="min-width: 80px;">2.0</td>
			<td style="min-width: 80px;">3.0</td>
			<td style="min-width: 80px;">4.0</td>
			<td style="min-width: 80px;">5.0</td>
			<td style="min-width: 80px;">5.0</td>
			<td style="min-width: 80px;">6.0</td>
			<td style="min-width: 80px;">6.0</td>
			<td style="min-width: 80px;">6.0</td>
		</tr>
		<tr>
			<td style="min-width: 80px;">%BM</td>
			<td style="min-width: 80px;">--</td>
			<td style="min-width: 80px;">8%</td>
			<td style="min-width: 80px;">11%</td>
			<td style="min-width: 80px;">15%</td>
			<td style="min-width: 80px;">19%</td>
			<td style="min-width: 80px;">19%</td>
			<td style="min-width: 80px;">23%</td>
			<td style="min-width: 80px;">23%</td>
			<td style="min-width: 80px;">23%</td>
		</tr>
		<tr>
			<td rowspan="2" style="min-width: 80px;">HLWR (n =7)</td>
			<td style="min-width: 80px;">Load (g)</td>
			<td style="min-width: 80px;">0.0</td>
			<td style="min-width: 80px;">2.5</td>
			<td style="min-width: 80px;">5.0</td>
			<td style="min-width: 80px;">7.5</td>
			<td style="min-width: 80px;">7.5</td>
			<td style="min-width: 80px;">10.0</td>
			<td style="min-width: 80px;">10.0</td>
			<td style="min-width: 80px;">12.5</td>
			<td style="min-width: 80px;">12.5</td>
		</tr>
		<tr>
			<td style="min-width: 80px;">%BM</td>
			<td style="min-width: 80px;">--</td>
			<td style="min-width: 80px;">10%</td>
			<td style="min-width: 80px;">19%</td>
			<td style="min-width: 80px;">28%</td>
			<td style="min-width: 80px;">28%</td>
			<td style="min-width: 80px;">38%</td>
			<td style="min-width: 80px;">38%</td>
			<td style="min-width: 80px;">48%</td>
			<td style="min-width: 80px;">48%</td>
		</tr>
	</tbody>
</table>
Table 1. Loaded wheel running protocols
5. In situ muscle function testing, tissue harvesting, and tissue analysis
<ol>
	<li>Following the 9 week training intervention, anesthetize mice using inhaled isoflurane (4% induction; 2% maintenance) with supplemental oxygen, and ensure proper anesthetic plane monitoring throughout the procedure.</li>
	<li>Perform an in situ muscle function test on the gastrocnemius, plantaris, soleus (GPS) complex to test isometric muscular strength28. Establish a force-frequency curve by directly stimulating the sciatic nerve with 27 G electrode needles at 11 ascending frequencies between 1-300 Hz, with tetanic contractions occurring around 100-150 hz29.</li>
	<li>Immediately following the muscle function test, euthanize the mice via cervical dislocation and confirm euthanasia by removing the heart. Carefully excise the plantaris and soleus muscles and record the wet tissue mass.</li>
	<li>Coat each muscle sample in an embedding medium (OCT) and mount it on a cork. Freeze it in liquid nitrogen-cooled isopentane, and store it at -80 &#176;C until further immunohistochemical (IHC) analysis is performed on sections of muscle tissue (10 &#181;m thick).</li>
	<li>Analyze muscle fiber CSA using immunofluorescence for laminin. Measure fiber CSA using an automatic image quantification platform30.</li>
</ol>
6. Statistical analysis
<ol>
	<li>Express all data as mean &#177; SD.</li>
	<li>Perform statistical analyses using statistical analysis software with significance set at p &#8804; 0.05.</li>
	<li>Compare wheel running and training volume data with repeated measures two-way ANOVA.</li>
	<li>Compare body mass, tissue mass, CSA, and muscle function with a one-way ANOVA. If significant F-ratios are found, compare within-group differences using Fisher LSD post hoc analyses.</li>
	<li>Calculate effect sizes, then interpret them as 0.01, 0.06, and 0.14 for small, medium, and large effect sizes, respectively.</li>
</ol>

<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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPK+regulates+basal+skeletal+muscle+capillarization+and+VEGF+expression,+but+is+not+necessary+for+the+angiogenic+response+to+exercise:+AMPK+and+the+skeletal+muscle+angiogenic+response+to+exercise.">AMPK regulates basal skeletal muscle capillarization and VEGF expression, but is not necessary for the angiogenic response to exercise: AMPK and the skeletal muscle angiogenic response to exercise.</a> The Journal of Physiology. 586 (24), 6021-6035 (2008).</li></ol>

The authors have no conflicts of interest to disclose.

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 ...

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3.3K Views.  Appalachian State University. 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. 

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

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. ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...

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

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