The authors would like to thank Dr. Jhi-Joung Wang, who is the Vice Superintendent of Education at Chi-Mei Medical Center, and Dr. Chih-Chan Lin from the Laboratory Animal Center, Department of Medical Research, Chi-Mei Medical Center, 901 Zhonghua, Yongkang Dist., Tainan City 701, Taiwan, for providing the shooting venue. They would also like to thank Miss Ling-Yu Tang and Mr. Chung-Ham Wang from the Department of Medical Research, Chi-Mei Medical Center, Tainan, Taiwan, for their valuable assistance in demonstrating the prototype system in real experiments with rats. The author gratefully acknowledges the support provided for this study by the Ministry of Science and Technology (MOST 104- 2218-E-167-001-) of Taiwan.

Ethics Statement: The experimental procedures were approved by the animal ethics committee of Southern Taiwan University of Science and Technology Laboratory Animal Center, National Science Council, Republic of China (Tainan, Taiwan).
1. Constructing the Running Wheel Structure
NOTE: All acrylic should be transparent.&#160;Wash the disassembled wheel with water, then use alcohol to wipe the rubber track and acrylic sheets after each use.&#160;&#160;
<ol>
	<li>Obtain an acrylic running wheel to be 55 cm in diameter and 15 cm in width. 
	NOTE: This wheel is larger than a traditional running wheel (diameter = 35 cm; width = 12 cm) (Figure 1A).</li>
	<li>Using a cutter, cut a quarter-circle opening into one side of the running wheel to act as an entrance and an exit as well for rats (Figure 1B). Place a layer of high-friction rubber track on the inside of the acrylic wheel (Figure 1B).</li>
	<li>Place an iron rod with bearings to connect the running wheel (Figure 1B). Place two acrylic triangular columns on either side of the running wheel to act as the support frame (Figure 1B).</li>
	<li>Attach a 1 mm thick semicircular, transparent acrylic sheet to the external sides of the two triangular columns using screws. Use this sheet to deploy the infrared sensors. Ensure that the acrylic sheets are approximately 3 cm away from each side of the running wheel.</li>
</ol>
2. Deploying the Infrared Sensors and Defining the Effective Exercise Area
NOTE: Take into account the running wheel size and the rat length in the design of an infrared system. A rat only triggers a single sensor at a time. In this experiment, rats are between 20 and 23 cm long.
<ol>
	<li>Drill a hole in the acrylic sheets every 45&#176; (arc interval = 21 cm), with the distance between two holes being roughly equivalent to the length of a test rat. Make the holes the same size as the infrared sensors (Figure 2A). 
	NOTE: For traditional MRWs, drill a hole every 70&#176; (arc interval = 21 cm, Figure 2B).</li>
	<li>During a PRW experiment, maintain the rats at a steady state of running between 0&#176; to 135&#176;. 
	NOTE: Thus define this area as the effective exercise area, while view all the other sections as the ineffective exercise areas. For traditional MRWs, define the effective exercise area as the portion between 0&#176; to 140&#176; (Figure 2B).</li>
</ol>
3. Driving the Running Wheel
<ol>
	<li>Use a brushless DC motor and a motor driver to drive the running wheel.</li>
	<li>Mount a 10 cm diameter rubber disk on the motor&#8217;s central axis (Figure 1B).</li>
	<li>Using the iron frame and springs to support the motor, connect the rubber disk from the motor&#8217;s central axis to the outer side of the running wheel. 
	NOTE: The springs must cooperate with the screws to allow dynamic motor height adjustments and to prevent rubber disk from being disconnected to the running wheel track because of loose springs.</li>
	<li>Operate the motor to drive the 10 cm diameter rubber disk using a microcontroller, and observe the wheel rotate due to the friction between the rubber disk and the runway of wheel, creating a motorized running wheel platform.</li>
	<li>Mount four infrared sensors sequentially between 0&#176; to 135&#176; (Figure 2A). 
	NOTE: For traditional MRWs, mount sensors between 0&#176; to 140&#176; (Figure 2B).</li>
	<li>Connect four pairs of infrared sensors mounted in both acrylic sheets to the microcontroller&#8217;s general pins using single core cables, thereby forming a positioning running wheel system.</li>
</ol>
4. Constructing an Adaptive Acceleration Curve
<ol>
	<li>Three days before the commencement of official 3 week exercise training, train rats by manually operating the running wheel. 
	NOTE: The goal is to allow the rats to become familiar with the running environment, and is to test whether each rat can endure running at 20 m/min.
	<ol>
		<li>During the manually operated training, gradually accelerate the running speed until a rat is unable to keep pace. When this occurs, decrease the speed until the rat regains a steady running pace, and then gradually increase the speed again until the rat reaches 20 m/min (dashed lines in Figure 3). The manual training involves seven rats to construct the training curves.</li>
	</ol>
	</li>
	<li>Using a numerical equation to fit the measured data on Day 3 of the manual testing, calculate the acceleration curves closest to the manual training (the curve with circles, Figure 3). Fit Equation 1 to raw data, where Cini = 8, Cfin = 20 and &#964; = 30 represent the initial velocity, the final velocity and the time constant, respectively). 
	NOTE: This equation adapts to a rat&#8217;s body condition. Therefore, refer to the calculated curve as an adaptive acceleration-training model. 
	<img alt="Equation 1" src="/files/ftp_upload/54354/54354eq1.jpg" />&#160; (1)</li>
	<li>Use Equation 1 for Week 1 of the formal training.</li>
	<li>For Weeks 2 and 3 of the training, adjust the parameters of Equation 1, that is to say, change A from 12 to 22, to allow the speed to reach 30 m/min.</li>
</ol>
5. Controlling the Software Program
NOTE: Exclusively develop a code for the microcontroller-based motor operation and for signal transmission from the infrared sensors to a computer for subsequent data analysis.
<ol>
	<li>Use C programming language to write a software control program containing one main program and two interrupt-service routines for the timer in the microcontroller18.
	<ol>
		<li>Ensure that the main program initializes the microcontroller&#8217;s register and constructs an adaptive acceleration curve model in the microcontroller&#8217;s memory.</li>
		<li>Use the interrupt-service routine of Timer 0 to activate the adaptive acceleration curve and calculate the entire training duration.</li>
		<li>Use the interrupt-service routine of Timer 1 to extract the signal data from the infrared sensors and to transfer the data to the computer.</li>
		<li>Use the main program to record the position of 0o to adjust the speed of running wheel.</li>
	</ol>
	</li>
	<li>Once the receiving IR sensor at 0&#176; is triggered, interpret it as a fall incidence, which is accumulated by the main program. The moment the occurrence times of fall incidences hits the 10% threshold of the number of the rat position detections, deaccelerate the running wheel automatically as a safety measure for trained rats.&#160;NOTE: The speed of running wheel is reduced until the rat can return to the safe zone (0o to 135o) and maintain a stable running state for the safety measure.</li>
</ol>
6. Operating the Positioning Running Wheel System
<ol>
	<li>Turn on the microcontroller and wait for an operator to press the button to start each week&#8217;s training model.
	<ol>
		<li>Press the &#8220;Start&#8221; button to begin the training model for Week 1. 
		NOTE: The motor automatically accelerates based on the adaptive acceleration curve until it reaches 20 m/min, and automatically stops after 30 min.</li>
		<li>Press the &#8220;Start&#8221; button to begin the training model for Week 2. 
		NOTE: The motor automatically accelerates based on the adaptive acceleration curve until it reaches 30 m/min, and automatically stops after 30 min.</li>
		<li>Press the &#8220;Start&#8221; button to begin the training model for Week 3. 
		NOTE: The motor automatically accelerates based on the adaptive acceleration curve until it reaches 30 m/min, and automatically stops after 60 min. 
		NOTE: Throughout the entire training process, transmit the signal data received from the infrared sensors to a computer wirelessly.</li>
	</ol>
	</li>
	<li>Using a computer, analyze the location data to obtain an effective exercise measure for the entire exercise process (<img alt="Equation 2" src="/files/ftp_upload/54354/54354eq2.jpg" />). See Equation 2. 
	<img alt="Equation 3" src="/files/ftp_upload/54354/54354eq3.jpg" />&#160; (2) 
	NOTE: EEE, EED and IED represent the effective exercise measure, the effective and ineffective exercise durations, respectively.</li>
</ol>
7. Training the Rats
<ol>
	<li>Randomly divide adult male Sprague-Dawley rats into five groups (n = 9 for each group): the sham, control, treadmill, MRW and PRW groups.</li>
	<li>Conduct a 3 week exercise training for the three exercise groups, i.e. the treadmill, MRW and PRW groups, while do not for the sham and the control groups. 
	NOTE: The 3 week exercise training for each exercise group is 20 m/min for 30 min during Week 1, 30 m/min for 30 min during Week 2 and 30 m/min for 60 min during Week 3.</li>
</ol>
8. Animal and Stroke Model
<ol>
	<li>As stated in Sec. 7.1, randomly divide all the involved adult male Sprague-Dawley rats, weighing between 250-280 g, into 5 groups.</li>
	<li>Weigh all the animals to ensure accurate drug dosage calculations. Anesthetize the rats with sodium pentobarbital (25 mg/kg, intraperitoneally [i.p.]) and a mixture containing ketamine (4.4 mg/ kg, intramuscularly [i.m.]), atropine (0.02633 mg/kg, [i.m.]) and xylazine (6.77 mg/kg, [i.m.]).
	<ol>
		<li>Assess the anesthetic depth by monitoring the breathing rate (normal 70-115 breaths/min), rhythm, depth of respiration, mucous membrane color and regular testing of reflexes, e.g. toe pinch, tail pinch, eyelid/eyelash and palpebral.</li>
	</ol>
	</li>
	<li>Insert temperature probes into the rectum, and maintain the rectal temperatures between 37 to 37.5 &#176;C using separate heating lamps.</li>
	<li>Induce focal ischemia, transient middle cerebral artery occlusion (MCAo) by inserting a filament into the internal carotid artery to occlude the orifice of the middle cerebral artery via external carotid artery approach19.
	<ol>
		<li>Perform the same operating procedures on the sham-operated animals, while do not insert a filament into the internal carotid artery. Maintain focal cerebral ischemia for 1 hr, remove the filament, close the incision, and then leave 1 cm of the nylon suture protruding, which could be withdrawn to allow reperfusion.</li>
	</ol>
	</li>
	<li>Administer subcutaneous (s.c.) injections of an analgesic (buprenorphine (0.05 mg/kg, s.c.)), into the animals for analgesia twice a day for 3 days.</li>
</ol>
9. Assessing Neural Damage
<ol>
	<li>Evaluate neurological and motor functions, respectively, by the neurological severity score (mNSS)20 and an inclined plane test21. 
	NOTE: mNSS is a composite of the motor (muscle status, abnormal movement), sensory (visual, tactile and proprioceptive) and reflex tests. Give one point for failure to perform a task. Rate neurological function on a scale of 0-18 (normal score = 0; maximal deficit score = 18).</li>
	<li>Assess all the rats in terms of behavioral performance the day before and daily over a time span of 7 days after the surgery.</li>
	<li>Measure the hind limb grip strength of the rat using an inclined plane.
	<ol>
		<li>Place rats on the inclined climbing apparatus on a daily basis, and acclimatize the rats to the apparatus and the testing conditions 1 week before testing.</li>
		<li>Place each rat on the apparatus and encourage the rat to climb the platform until the top of the apparatus during the acclimatization period.</li>
		<li>Place the rat at the top of the apparatus with the head downward during testing. Ensure that the body axis of the rat stays along to a 20 x 20 cm2 rubber ribbed surface on the inclined plane starting at an angle of 25&#176;.</li>
		<li>Increase the angle dynamically using a ball screw connected with a stepper motor to determine the maximal angle at which an animal could hold to the plane. Increase the angle of the inclined plane gradually until the mouse failed to hold on the inclined plane, and then detect a sliding down event. The baseline degree of inclined plane is 25&#176; in the beginning.</li>
		<li>Ask two observers (unaware of what treatment the rats had been given), to independently examine and score all the behavioral tests, i.e. the mean of the left- and right-side maximal angles.</li>
	</ol>
	</li>
	<li>Sacri&#64257;ce all the animals on day 7 after MCAo. Perfuse the hearts of the animals under deep anesthesia (sodium pentobarbital 100 mg/kg, i.p.) with saline22. Remove and then immerse the brain tissue in cold saline for 5 min, which is then sliced into 2.0 mm coronal sections using a tissue slicer22.</li>
	<li>Submerge the fresh brain sections into 2, 3, 5-triphenyltetrazolium chloride (TTC) at 37 &#176;C for 30 min, then transfer the slices to 5% formaldehyde solution for &#64257;xation at 4 &#176;C for 24 hr. Place the stained brain slices on a plexiglas holder.</li>
	<li>Photograph the TTC-stained slices with a calibrated scale using a CCD camera linked to a personal computer loaded with image processing software. Use a semi-automated image analysis system as well to estimate the infarcted area (mm2) from each TTC-stained brain slice23.</li>
	<li>Calculate the total infarct volume for each slice by summation of infarcted areas of all brain slices. Mark the unstained area (ischemic brain) separately on each side of 2 mm thick slices, then calculate the infarct volume and the mean value.</li>
	<li>Calculate the corrected infarct volume (CIV) as 
	CIV= {LT- (RT- RI)} <img alt="Equation 4" src="/files/ftp_upload/54354/54354eq4.jpg" /> d (3) 
	NOTE: Where LT and RT denote the areas of the left and right hemispheres in mm2, respectively, RI is the infarcted area in mm2, and d = 2 mm is the slice thickness.</li>
</ol>

<ol>
	<li>Mayo, N. E., Wood-Dauphinee, S., Cote, R., Durcan, L., Carlton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity,+participation,+and+quality+of+life+6+months+poststroke.">Activity, participation, and quality of life 6 months poststroke.</a> Arch Phys Med Rehabil. 83 (8), 1035-1042 (2002).</li><li>Duncan, P. W., Goldstein, L. B., Horner, R. D., Landsman, P. B., Samsa, G. P., Matchar, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similar+motor+recovery+of+upper+and+lower-extremities+after+stroke.">Similar motor recovery of upper and lower-extremities after stroke.</a> Stroke. 25 (6), 1181-1188 (1994).</li><li>Raichlen, D. A., Gordon, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+exercise+capacity+and+brain+size+in+mammals.">Relationship between exercise capacity and brain size in mammals.</a> PLoS One. 6 (6), (2011).</li><li>Trejo, J. L., Carro, E., Torres-Aleman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+insulin-like+growth+factor+I+mediates+exercise-induced+increases+in+the+number+of+new+neurons+in+the+adult+hippocampus.">Circulating insulin-like growth factor I mediates exercise-induced increases in the number of new neurons in the adult hippocampus.</a> J Neurosci. 21 (5), 1628-1634 (2001).</li><li>Zhang, F., Wu, Y., Jia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+preconditioning+and+brain+ischemic+tolerance.">Exercise preconditioning and brain ischemic tolerance.</a> Neuroscience. 177, 170-176 (2011).</li><li>Wang, R. Y., Yang, Y. R., Yu, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+treadmill+training+on+infarction+in+rats.">Protective effects of treadmill training on infarction in rats.</a> Brain Res. 922 (1), 140-143 (2001).</li><li>Ding, 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=Exercise+pre-conditioning+reduces+brain+damage+in+ischemic+rats+that+may+be+associated+with+regional+angiogenesis+and+cellular+overexpression+of+neurotrophin.">Exercise pre-conditioning reduces brain damage in ischemic rats that may be associated with regional angiogenesis and cellular overexpression of neurotrophin.</a> Neuroscience. 124 (3), 583-591 (2004).</li><li>Li, J., Luan, X. D., Clark, J. C., Rafols, J. A., Ding, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotection+against+transient+cerebral+ischemia+by+exercise+pre-conditioning+in+rats.">Neuroprotection against transient cerebral ischemia by exercise pre-conditioning in rats.</a> Brain Res. 26 (4), 404-408 (2004).</li><li>Leasure, J. L., Jones, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forced+and+voluntary+exercise+differentially+affect+brain+and+behavior.">Forced and voluntary exercise differentially affect brain and behavior.</a> Neuroscience. 156 (3), 456-465 (2008).</li><li>Chen, C. C., 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=A+Forced+running+wheel+system+with+a+microcontroller+that+provides+high-intensity+exercise+training+in+an+animal+ischemic+stroke+model.">A Forced running wheel system with a microcontroller that provides high-intensity exercise training in an animal ischemic stroke model.</a> Braz J Med Biol Res. 47 (10), 858-868 (2014).</li><li>Chen, C. -. C., 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=Improved+infrared-sensing+running+wheel+systems+with+an+effective+exercise+activity+indicator.">Improved infrared-sensing running wheel systems with an effective exercise activity indicator.</a> PLoS One. 10 (4), (2015).</li><li>Fantegrossi, W. E., Xiao, W. R., Zimmerman, S. 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+technology+for+modulating+locomotor+activity+as+an+operant+response+in+the+mouse:+Implications+for+neuroscience+studies+involving+&#34;exercise&#34;+in+rodents.">Novel technology for modulating locomotor activity as an operant response in the mouse: Implications for neuroscience studies involving &#34;exercise&#34; in rodents.</a> J Neurosci Methods. 212 (2), 338-343 (2013).</li><li>Hayes, K., 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=Forced,+not+voluntary,+exercise+effectively+induces+neuroprotection+in+stroke.">Forced, not voluntary, exercise effectively induces neuroprotection in stroke.</a> Acta Neuropathol. 115 (3), 289-296 (2008).</li><li>Arida, R. M., Scorza, C. A., da Silva, A. V., Scorza, F. A., Cavalheiro, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+spontaneous+versus+forced+exercise+in+rats+on+the+staining+of+parvalbumin-positive+neurons+in+the+hippocampal+formation.">Differential effects of spontaneous versus forced exercise in rats on the staining of parvalbumin-positive neurons in the hippocampal formation.</a> Neurosci Lett. 364 (3), 135-138 (2004).</li><li>Waters, R. 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=Selection+for+aerobic+capacity+affects+corticosterone,+monoamines+and+wheel-running+activity.">Selection for aerobic capacity affects corticosterone, monoamines and wheel-running activity.</a> Physiol Behav. (4-5), 1044-1054 (2008).</li><li>Ke, Z., Yip, S. P., Li, L., Zheng, X. -. X., Tong, K. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+voluntary,+involuntary,+and+forced+exercises+on+brain-derived+neurotrophic+factor+and+motor+function+recovery:+A+rat+brain+ischemia+model.">The effects of voluntary, involuntary, and forced exercises on brain-derived neurotrophic factor and motor function recovery: A rat brain ischemia model.</a> PLoS One. 6 (2), (2011).</li><li>Caton, S. J., 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=Low-carbohydrate+high-fat+diets+in+combination+with+daily+exercise+in+rats:+Effects+on+body+weight+regulation,+body+composition+and+exercise+capacity.">Low-carbohydrate high-fat diets in combination with daily exercise in rats: Effects on body weight regulation, body composition and exercise capacity.</a> Physiol Behav. 106 (2), 185-192 (2012).</li><li>. C8051F330/1/2/3/4/5 datasheet Available from: <a target="_blank" href="https://www.silabs.com/Support%20Documents/TechnicalDocs/C8051F33x.pdf">https://www.silabs.com/Support%20Documents/TechnicalDocs/C8051F33x.pdf</a> (2006)</li><li>Longa, E. Z., Weinstein, P. R., Carlson, S., Cummins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke. 20 (1), 84-91 (1989).</li><li>Chen, J. 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=Therapeutic+benefit+of+intravenous+administration+of+bone+marrow+stromal+cells+after+cerebral+ischemia+in+rats.">Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats.</a> Stroke. 32 (4), 1005-1011 (2001).</li><li>Chang, M. -. W., Young, M. -. S., Lin, M. -. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inclined+plane+system+with+microcontroller+to+determine+limb+motor+function+of+laboratory+animals.">An inclined plane system with microcontroller to determine limb motor function of laboratory animals.</a> J Neurosci Methods. 168 (1), 186-194 (2008).</li><li>Gartshore, G., Patterson, J., Macrae, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+ischemia+and+reperfusion+on+the+course+of+brain+tissue+swelling+and+blood-brain+barrier+permeability+in+a+rodent+model+of+transient+focal+cerebral+ischemia.">Influence of ischemia and reperfusion on the course of brain tissue swelling and blood-brain barrier permeability in a rodent model of transient focal cerebral ischemia.</a> Exp Neurol. 147 (2), 353-360 (1997).</li><li>Chen, F., 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=Rodent+stroke+induced+by+photochemical+occlusion+of+proximal+middle+cerebral+artery:+Evolution+monitored+with+MR+imaging+and+histopathology.">Rodent stroke induced by photochemical occlusion of proximal middle cerebral artery: Evolution monitored with MR imaging and histopathology.</a> Eur J Radiol. 63 (1), 68-75 (2007).</li><li>Almenning, I., Rieber-Mohn, A., Lundgren, K. M., Lovvik, T. S., Garnaes, K. K., Moholdt, 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+high+intensity+interval+training+and+strength+training+on+metabolic,+cardiovascular+and+hormonal+outcomes+in+women+with+polycystic+ovary+syndrome:+a+pilot+study.">Effects of high intensity interval training and strength training on metabolic, cardiovascular and hormonal outcomes in women with polycystic ovary syndrome: a pilot study.</a> PLoS One. 10 (9), (2015).</li><li>Costigan, S. A., Eather, N., Plotnikoff, R. C., Taaffe, D. R., Lubans, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-intensity+interval+training+for+improving+health-related+fitness+in+adolescents:+a+systematic+review+and+meta-analysis.">High-intensity interval training for improving health-related fitness in adolescents: a systematic review and meta-analysis.</a> Br J Sports Med. 49 (19), (2015).</li></ol>

The authors have nothing to disclose.

This protocol describes a highly effective running wheel system for&#160;reducing the severity of the effects of the stroke&#160;in animals. As a rat-friendly testbed, this platform is designed as well in such a way that a stable running speed can be maintained by rats throughout a running process by means of a predetermined adaptive acceleration curve. In typical training systems, preset training speeds and durations are set manually. Once an exercise commences, a preset speed is reached very shortly. In this context, i...

Strokes exist continuously as a financial burden to countries globally, leaving countless patients physically and mentally disabled1,2. There is clinical evidence to suggest that regular exercise can improve nerve regeneration and strengthen neural connections3,4, and it is also shown that exercise can decrease the risk of suffering ischemic strokes5. With either a treadmill or a running wheel as an exercise training system, rodents, such as rats, serve as a proxy for humans for testing the effectiveness of exercises in a vast majority of clinical experiments6-8. A training system normally involves training a rat for a certain period of time, during which a rat runs at a certain speed. Therefore, the training intensity is generally calculated according to the exercise speed and duration6-8. The same approach is applied to estimate the amount of exercise required for neurophysiological protection. However, the experimental exercises are sometimes found to be ineffective, such as when a rat stumbles, falls, or grabs the rails once they are unable to catch up with the running wheel speed9-11. Needless to say, incidents of ineffective exercise significantly reduce the exercise benefit. Even though there is no any universally accepted approach currently to quantify the effective exercises for reducing brain damage, the level of effective exercises still stands as an objective appraisal for clinical researchers to illustrate the benefits of exercise in the discipline of neurophysiology.
There exist a number of limitations on commercially available animal mobility systems used in today&#39;s brain damage reduction&#160;experiments12. In a treadmill case, rats are forced to run by means of electric shocks, inducing tremendous psychological stress on the animals and thereby interference in the final neurophysiological test results8,13,14. Running wheels can be categorized into two types, namely voluntary and forced. Voluntary running wheels allow rats to run naturally, creating excessive variability due of the differences in the rats&#39; physical traits and abilities15, while motorized running wheels (MRWs) employ a motor to turn the wheel, forcing rats to run. Despite also being a form of forced training, MRWs imposes less psychological stress on rats than treadmills13,16,17. However, experiments using MRWs have reported that rats sometimes interrupt the exercise by grabbing the rails on the wheel track and refusing to run at speeds exceeding 20 m/min9. These examples show that animal mobility systems currently available have an inherent disadvantage that inhibits effective exercising. For objective rat training purposes, the development of a highly effective training system but with low interference is therefore viewed as an urgent issue for neurophysiological exercise experiments.
This study presents a highly effective running wheel system for experiments on&#160;reducing the severity of the effects of the stroke11. In addition to a reduced number of interference factors during a training process, this system detects the running position of a rat using infrared sensors embedded in the wheel, thereby achieving a more reliable estimate of effective exercise activity. The psychological stress imposed by traditional treadmills and the frequent exercise interruptions in MRWs both skew the objectivity of the resulting exercise estimates. A positioning running wheel (PRW) system presented in this study is developed in an attempt to minimize the unwanted interference while providing a reliable training model for quantifying effective exercise.

<table><tbody><tr><td>Brushless DC motor</td><td>Oriental Motor</td><td>BLEM512-GFS</td><td></td></tr><tr><td>Motor driver</td><td>Oriental Motor</td><td>BLED12A</td><td></td></tr><tr><td>Motor reducer</td><td>Oriental Motor</td><td>GFS5G20</td><td></td></tr><tr><td>Speedometer</td><td>Oriental Motor</td><td>OPX-2A</td><td></td></tr><tr><td>Treadmill</td><td>Columbus Instruments</td><td>Exer-6M</td><td></td></tr><tr><td>Infrared transmitter&amp;nbsp;</td><td>Seeed Studio</td><td>TSAL6200</td><td></td></tr><tr><td>Infrared Receiver</td><td>Seeed Studio</td><td>TSOP382</td><td></td></tr><tr><td>Microcontroller</td><td>Silicon Labs</td><td>C8051F330</td><td></td></tr><tr><td>CCD camera</td><td>Canon Inc.</td><td>EOS 450D</td><td></td></tr><tr><td>Image processing software</td><td>Adobe Systems Incorporated</td><td>ADOBE Photoshop CS5 12.0</td><td></td></tr><tr><td>Image analysis</td><td>Media Cybernetics</td><td>Pro Plus 4.50.29</td><td></td></tr><tr><td>Sodium pentobarbital</td><td>Sigma-Aldrich (Saint Louis, MO, USA)</td><td>SIGMA P-3761</td><td></td></tr><tr><td>Ketamine</td><td>Pfizer (Kent, UK)</td><td>&amp;nbsp;1867-66-9</td><td></td></tr><tr><td>Atropine</td><td>Taiwan Biotech Co., Ltd. (Taoyuan, Taiwan)</td><td>A03BA01</td><td></td></tr><tr><td>Xylazine</td><td>Sigma-Aldrich (Saint Louis, MO, USA)</td><td>SIGMA X1126</td><td></td></tr><tr><td>Buprenorphine</td><td>Sigma-Aldrich (Saint Louis, MO, USA)</td><td>B9275</td><td></td></tr></tbody></table>

an innovative running wheel-based mechanism for improved rat training performance

This study presents an animal mobility system, equipped with a positioning running wheel (PRW), as a way to quantify the efficacy of an exercise activity for reducing the severity of the effects of the stroke&#160;in rats. This system provides more effective animal exercise training than commercially available systems such as treadmills and motorized running wheels (MRWs). In contrast to an MRW that can only achieve speeds below 20 m/min, rats are permitted to run at a stable speed of 30 m/min on a more spacious and high-density rubber running track supported by a 15 cm wide acrylic wheel with a diameter of 55 cm in this work. Using a predefined adaptive acceleration curve, the system not only reduces the operator error but also trains the rats to run persistently until a specified intensity is reached. As a way to evaluate the exercise effectiveness, real-time position of a rat is detected by four pairs of infrared sensors deployed on the running wheel. Once an adaptive acceleration curve is initiated using a microcontroller, the data obtained by the infrared sensors are automatically recorded and analyzed in a computer. For comparison purposes, 3 week training is conducted on rats using a treadmill, an MRW and a PRW. After surgically inducing middle cerebral artery occlusion (MCAo), modified neurological severity scores (mNSS) and an inclined plane test were conducted to assess the neurological damages to the rats. PRW is experimentally validated as the most effective among such animal mobility systems. Furthermore, an exercise effectiveness measure, based on rat position analysis, showed that there is a high negative correlation between the effective exercise and the infarct volume, and can be employed to quantify a rat training in any type of brain damage reduction&#160;experiments.

This section is devoted to comparisons, made 1 week after surgery, on the mNSS scores, incline plane test results and brain infarct volumes among five groups. Figure 4A and 4B present the average mNSS scores and the average of incline plane test results, respectively. The PRW group appears as the best in terms of mNSS improvement. The significant differences between PRW and MRW and between treadmill and PRW clearly suggest that the PRW protects against st...

Watch this Scientific Journal Video about An Innovative Running Wheel-based Mechanism for Improved Rat Training Performance at JoVE.com

An Innovative Running Wheel-based Mechanism for Improved Rat Training Performance

This study presents an innovative running wheel-based animal mobility system to quantify an effective exercise activity in rats. A rat-friendly testbed is built, using a predefined adaptive acceleration curve, and a high correlation between the effective exercise rate and the infarct volume suggests the protocol&#39;s potential for stroke prevention experiments.

This study presents an animal mobility system, equipped with a positioning running wheel (PRW), as a way to quantify the efficacy of an exercise activity ...

an-innovative-running-wheel-based-mechanism-for-improved-rat-training

Universidad de Cartagena UDEC

Research

JoVE Journal

Behavior

8.8K Views.  National Chin-Yi University of Technology. This study presents an innovative running wheel-based animal mobility system to quantify an effective exercise activity in rats. A rat-friendly testbed is built, using a predefined adaptive acceleration curve, and a high correlation between the effective exercise rate and the infarct volume suggests the protocol's potential for stroke prevention experiments.

Video: An Innovative Running Wheel-based Mechanism for Improved Rat Training Performance

A Fully Automated Rodent Conditioning Protocol for Sensorimotor Integration and Cognitive Control Experiments

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. Higher cognitive functions that require precise control over sensorimotor responses such as decision-making and attentional modulation, however, are typically assessed in nonhuman primates. Despite the richness of primate behavior that allows multiple variants of these functions to be studied, the rodent model remains an attractive, cost-effective alternative to primate models. Furthermore, the ability to fully automate operant conditioning in rodents adds unique advantages over the labor intensive training of nonhuman primates while studying a broad range of these complex functions.
Here, we introduce a protocol for operantly conditioning rats on performing working memory tasks. During critical epochs of the task, the protocol ensures that the animal's overt movement is minimized by requiring the animal to 'fixate' until a Go cue is delivered, akin to nonhuman primate experimental design. A simple two alternative forced choice task is implemented to demonstrate the performance. We discuss the application of this paradigm to other tasks.

A fully automated protocol for rodent operant conditioning is proposed. The protocol relies on precise temporal control of behavioral events to investigate the extent to which this control influences neural activity underlying sensorimotor integration and cognitive control experiments.

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. ...

Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes. As such, all behaviors manifest through the motor system. In the fields of neuroscience and orthopedics, locomotion is a commonly evaluated behavior for a variety of disease models. For example, locomotor recovery after traumatic injury to the nervous system is one of the most commonly evaluated behaviors 1-3. Though locomotion can be evaluated using a variety of endpoint measurements (e.g. time taken to complete a locomotor task, etc), semiquantitative kinematic measures (e.g. ordinal rating scales (e.g. Basso Beattie and Bresnahan locomotor (BBB) rating scale, etc)) and surrogate measures of behaviour (e.g. muscle force, nerve conduction velocity, etc), only kinetics (force measurements) and kinematics (measurements of body segments in space) provide a detailed description of the strategy by which an animal is able to locomote 1. Though not new, kinematic and kinetic measurements of locomoting rodents is now more readily accessible due to the availability of commercially available equipment designed for this purpose. Importantly, however, experimenters need to be very familiar with theory of biomechanical analyses and understand the benefits and limitations of these forms of analyses prior to embarking on what will become a relatively labor-intensive study. The present paper aims to describe a method for collecting kinematic and ground reaction force data using commercially available equipment. Details of equipment and apparatus set-up, pre-training of animals, inclusion and exclusion criteria of acceptable runs, and methods for collecting the data are described. We illustrate the utility of this behavioral analysis technique by describing the kinematics and kinetics of strain-matched young adult, middle-aged, and geriatric rats.

Locomotion is often examined as a behavioural outcome in various models of disease in fields such as neuroscience and orthopedics. This video paper intends to describe a method for collecting ground reaction forces and kinematics from rats during unrestrained locomotion.

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes.  As such, all behaviors manifest through the ...

Neuroscience

Acquisition of a High-precision Skilled Forelimb Reaching Task in Rats

Movements are the main measurable output of central nervous system function. Developing behavioral paradigms that allow detailed analysis of motor learning and execution is of critical importance in order to understand the principles and processes that underlie motor function. Here we present a paradigm to study movement acquisition within a daily session of training (within-session) representing the fast learning component and primary acquisition as well as skilled motor learning over several training sessions (between-session) representing the slow learning component and consolidation of the learned task. This behavioral paradigm increases the degree of difficulty and complexity of the motor skill task due to two features: First, the animal realigns its body prior to each pellet retrieval forcing renewed orientation and preventing movement execution from the same angle. Second, pellets are grasped from a vertical post that matches the diameter of the pellet and is placed in front of the cage. This requires a precise grasp for successful pellet retrieval and thus prevents simple pulling of the pellet towards the animal. In combination with novel genetics, imaging and electrophysiological technologies, this behavioral method will aid to understand the morphological, anatomical and molecular underpinnings of motor learning and memory.

A paradigm is presented to analyze the acquisition of a high-precision skilled forelimb reaching task in rats.

Movements are the main measurable output of central nervous system function. Developing behavioral paradigms that allow detailed analysis of motor ...

Investigating Motor Skill Learning Processes with a Robotic Manipulandum

Skilled reaching tasks are commonly used in studies of motor skill learning and motor function under healthy and pathological conditions, but can be time-intensive and ambiguous to quantify beyond simple success rates. Here, we describe the training procedure for reach-and-pull tasks with ETH Pattus, a robotic platform for automated forelimb reaching training that records pulling and hand rotation movements in rats. Kinematic quantification of the performed pulling attempts reveals the presence of distinct temporal profiles of movement parameters such as pulling velocity, spatial variability of the pulling trajectory, deviation from midline, as well as pulling success. We show how minor adjustments in the training paradigm result in alterations in these parameters, revealing their relation to task difficulty, general motor function or skilled task execution. Combined with electrophysiological, pharmacological and optogenetic techniques, this paradigm can be used to explore the mechanisms underlying motor learning and memory formation, as well as loss and recovery of function (e.g. after stroke).

A paradigm is presented for training and analysis of an automated skilled reaching task in rats. Analysis of pulling attempts reveals distinct subprocesses of motor learning.

Skilled reaching tasks are commonly used in studies of motor skill learning and motor function under healthy and pathological conditions, but can be ...

The Knob Supination Task: A Semi-automated Method for Assessing Forelimb Function in Rats

Tasks that accurately measure dexterity in animal models are critical to understand hand function. Current rat behavioral tasks that measure dexterity largely use video analysis of reaching or food manipulation. While these tasks are easy to implement and are robust across disease models, they are subjective and laborious for the experimenter. Automating traditional tasks or creating new automated tasks can make the tasks more efficient, objective, and quantitative. Since rats are less dexterous than primates, central nervous system (CNS) injury produces more subtle deficits in dexterity, however, supination is highly affected in rodents and crucial to hand function in primates. Therefore, we designed a semi-automated task that measures forelimb supination in rats. Rats are trained to reach and grasp a knob-shaped manipulandum and turn the manipulandum in supination to receive a reward. Rats can acquire the skill within 20 &plusmn; 5 days. While the early part of training is highly supervised, much of the training is done without direct supervision. The task reliably and reproducibly captures subtle deficits after injury and shows functional recovery that accurately reflects clinical recovery curves. Analysis of data is performed by specialized software through a graphical user interface that is designed to be intuitive. We also give solutions to common problems encountered during training, and show that minor corrections to behavior early in training produce reliable acquisition of supination. Thus, the knob supination task provides efficient and quantitative evaluation of a critical movement for dexterity in rats.

This manuscript describes a semi-automated task that quantifies supination in rats. Rats reach, grasp, and supinate a spherical manipulandum. The rat is rewarded with a pellet if the turn angle exceeds a criterion set by the user. This task increases throughput, sensitivity to injury, and objectivity compared to traditional tasks.

Tasks that accurately measure dexterity in animal models are critical to understand hand function. Current rat behavioral tasks that measure dexterity ...

Automated Rat Single-Pellet Reaching with 3-Dimensional Reconstruction of Paw and Digit Trajectories

Rodent skilled reaching is commonly used to study dexterous skills, but requires significant time and effort to implement the task and analyze the behavior. Several automated versions of skilled reaching have been developed recently. Here, we describe a version that automatically presents pellets to rats while recording high-definition video from multiple angles at high frame rates (300 fps). The paw and individual digits are tracked with DeepLabCut, a machine learning algorithm for markerless pose estimation. This system can also be synchronized with physiological recordings, or be used to trigger physiologic interventions (e.g., electrical or optical stimulation).

Rodent skilled reaching is commonly used to study dexterous skills, but requires significant time and effort to implement the task and analyze the behavior. We describe an automated version of skilled reaching with motion tracking and three-dimensional reconstruction of reach trajectories.

Rodent skilled reaching is commonly used to study dexterous skills, but requires significant time and effort to implement the task and analyze the ...

Utilizing a Reconfigurable Maze System to Enhance the Reproducibility of Spatial Navigation Tests in Rodents

Several maze shapes are used to test spatial navigation performance and behavioral phenotypes. Traditionally, each experiment requires a unique maze shape, thus requiring several separate mazes in different configurations. The maze geometry cannot be reconfigured in a single environment to accommodate scalability and reproducibility. The reconfigurable maze is a unique approach to address the limitations, allowing quick and flexible configurations of maze pathways in a repeatable manner. It consists of interlocking pathways and includes feeders, treadmills, movable walls, and shut-off sensors. The current protocol describes how the reconfigurable maze can replicate existing mazes, including the T-shaped, plus-shaped, W-shaped, and figure-eight mazes. Initially, the T-shaped maze was constructed inside a single experimental room, followed by modifications. The rapid and scalable protocol outlined herein demonstrates the flexibility of the reconfigurable maze, achieved through the addition of components and behavioral training phases in a stepwise manner. The reconfigurable maze systematically and precisely assesses the performance of multiple aspects of spatial navigation behavior.

The present protocol describes a reconfigurable maze, a unique system for testing spatial navigation and behavioral phenotypes in rodents. The adaptability of this maze system enables the execution of various experiments in a single physical environment. The ease of structural rearrangement generates reliable and reproducible experimental results.

Several maze shapes are used to test spatial navigation performance and behavioral phenotypes. Traditionally, each experiment requires a unique maze ...

Tracking Individual Running Metrics in Mice Using a Voluntary Wheel Running Protocol that Minimizes Social Isolation

Voluntary wheel running (VWR) in rodents is used to evaluate performance and endurance and to simulate exercise. In contrast to other exercise paradigms that require aversive stimulus to induce active movement, wheel running is voluntary. VWR generally consists of placing a running wheel inside the mouse home cage. However, analyzing exercise parameters can typically only be conducted when mice are individually housed, while overall cage effects can be reported when mice are group-housed. Therefore, the current models force experimenters to choose between measuring individual exercise parameters in single-housed mice or measuring group effects in group-housed mice; depending on the research question and model, this could be limiting. 
Therefore, the goal of this method is to demonstrate a protocol that assesses VWR in a mouse model by quantifying running parameters in individual mice, with minimal isolation. We report on device design, preoperative preparations, and experiment setup. Results of a 6-week training protocol using this VWR model suggest it provides sufficient stimulus to increase spatial object recognition memory, comparable to the clinical effect of exercise. This simple and inexpensive model facilitates investigation into the effect of VWR on multiple measurements of wheel activity, minimizing factors that could limit or affect experimental outcomes.

This protocol describes an effective voluntary wheel-running model that can be implemented in various research contexts. Unlike other voluntary running models, this method avoids isolation housing while tracking running metrics in individual mice.

Voluntary wheel running (VWR) in rodents is used to evaluate performance and endurance and to simulate exercise. In contrast to other exercise paradigms ...

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

Automated Assessment of Fine Motor Performance in Mice Using the Erasmus Ladder

Monitoring Fine and Associative Motor Learning in Mice Using the Erasmus Ladder

Behavior is shaped by actions, and actions necessitate motor skills such as strength, coordination, and learning. None of the behaviors essential for sustaining life would be possible without the ability to transition from one position to another. Unfortunately, motor skills can be compromised in a wide array of diseases. Therefore, investigating the mechanisms of motor functions at the cellular, molecular, and circuit levels, as well as understanding the symptoms, causes, and progression of motor disorders, is crucial for developing effective treatments. Mouse models are frequently employed for this purpose.
This article describes a protocol that allows the monitoring of various aspects of motor performance and learning in mice using an automated tool called the Erasmus Ladder. The assay involves two phases: an initial phase where mice are trained to navigate a horizontal ladder built of irregular rungs (&#34;fine motor learning&#34;), and a second phase where an obstacle is presented in the path of the moving animal. The perturbation can be unexpected (&#34;challenged motor learning&#34;) or preceded by an auditory tone (&#34;associative motor learning&#34;). The task is easy to conduct and is fully supported by automated software.
This report shows how different readouts from the test, when analyzed with sensitive statistical methods, allow fine monitoring of mouse motor skills using a small cohort of mice. We propose that the method will be highly sensitive to evaluate motor adaptations driven by environmental modifications as well as early-stage subtle motor deficits in mutant mice with compromised motor functions.

Author Spotlight: Unveiling Neural Mechanisms Through Automated Evaluation of Motor Learning and Myelin Plasticity Studies Using the Erasmus Ladder 

This article presents a protocol that allows a non-invasive and automated assessment of fine motor performance, as well as adaptive and associative motor learning upon challenges, using a device called the Erasmus Ladder. Task difficulty can be titrated to detect motor impairment ranging from major to subtle degrees.

Behavior is shaped by actions, and actions necessitate motor skills such as strength, coordination, and learning. None of the behaviors essential for ...