The authors acknowledge Drs. Patricia Ward, April Herrity and Susan Harkema for their input and guidance, Christine Yarberry for surgical assistance, Yangsheng Chen, Andrea Willhite and Johnny Morehouse for technical assistance and Darlene Burke for assistance with statistics and behavioral assessments. Funding support for this work was provided by the Department of Defense (W81XWH-11-1-0668 and W81XWH-15-1-0656) and the Kentucky Spinal Cord and Head Injury Research Trust (KSCHIRT 14-5).

All methods described have been approved by the University of Louisville Institutional Animal Care and Use Committee (IACUC).
1. Pre-injury Handling and Testing (One Week Before SCI)
<ol>
	<li>Handle each rat for a period of 5 - 10 min once a day for five days. 
	NOTE: Adult male Wistar rats that are ~50 days of age initially and weigh 200 - 225 g are used in this protocol. Rats at this pre-injury time-point are not acclimated to the harness that is used for LT as full use of hindlimbs allows the rat to escape from the jacket.</li>
	<li>Conduct any pre-injury testing that is study-specific (e.g., the authors do metabolic cage assessments for studies that involve the effects of SCI on bladder and bowel function).</li>
</ol>
2. Spinal Cord Contusion25,26,27,28
<ol>
	<li>Anesthetize animals with ketamine (80 mg/kg) and xylazine (10 mg/kg) mixture intraperitoneally according to provided dosage chart (Table 1). Administer supplemental dosing as needed. Test anesthetic depth at least every 10 min by assessing corneal, palpebral, pedal, tail pinch, and pinna reflexes.</li>
	<li>Shave hair from the back of the animal where incision and injury are to occur. Cleanse the surgical area with Dermachlor 4% Surgical scrub. Administer a long-acting general antibiotic (e.g., 0.5 cc Pro-Pen-G subcutaneously).</li>
	<li>Place the anesthetized animal on a heating pad at a low setting to maintain normal body temperature.</li>
	<li>Estimate location of targeted lesion level based upon vertebral protuberances and with a #10 scalpel, make an estimated 5 cm incision on the dorsum of the animal, directly above the midline vertebrae.</li>
	<li>For mid-thoracic contusions, expose the T8/T9 level of spinal cord via removal (with rongeurs) of the overlaying T7 vertebral lamina.</li>
	<li>Using a contusion device such as an infinite horizon impactor29, perform the contusion (for a moderate to severe degree of SCI, use a force of 210 kdyn with no dwell time)18.</li>
	<li>Suture together the muscular layer and fascia over the spinal cord using 4-0 diameter monofilament and close the skin with 9 mm surgical wound clips.</li>
	<li>Administer postoperative drugs, such as gentamicin sulfate (5 mg/kg per day for 5 days; antibiotic to avoid bladder infections) and meloxicam (1 mg/kg subcutaneously, analgesic for first 48 h and then as needed).</li>
	<li>Place animals in a clean cage on a heating pad. Check animal vital signs every 15 minutes until they are fully awake from anesthesia.&#160; During the first post-op day, the animals are encouraged to eat with a sugary treat. For the first 48 h (three times daily at the time of manual crede - see 2.10), rats are monitored for inactivity, vocalization in response to handling, and lack of desire to eat and drink.&#160; If analgesia is found to be inadequate, the veterinary staff is contacted. Throughout the initial two-week recovery phase, the animals are observed for evidence of infection or other complications. Once reflex voiding returns, the animals are tended to twice a day (early morning and late afternoon). Animals with infections or significant weight loss are immediately euthanized. Regarding food and water intake, the cut off point for euthanasia is when the animal has reached anything over 20% weight loss. Normal weight loss after surgery and disuse atrophy of muscles below the level of injury is 15-20%. All animals are weighed at least once per week.</li>
	<li>Perform bladder emptying procedures using the manual Cred&#233; maneuver 3 times a day (8 am, 3 pm, 10 pm) until reflexive bladder function has returned (3 - 6 days on average for contusions)26,30.</li>
</ol>
3. Training Phase
<ol>
	<li>Commence LT no earlier than two weeks post-SCI, as initiating interventions too early may exacerbate secondary injury cascades31.</li>
	<li>Week 1 acclimation to treadmill training: Transport the rats to a quiet room that is dedicated for training.</li>
	<li>On Day 1, randomly and evenly divide the SCI animals into trained and non-trained control groups, to account for potential variability in both the injury itself as well as the degree of spontaneous recovery after contusion. For example, divide rats into 4 separate groups: quadrupedal trained (QT), forelimb-only trained (FT), non-trained control (NT), and non-trained home cage control (HC). A sham group where animals receive a laminectomy but no injury and are otherwise handled the same as the other groups can also be used as an uninjured control group without training.</li>
	<li>Place each animal in the respective harness (Figure 1) and fasten harnesses to the body weight support mechanism above the treadmill via alligator clips which are fastened to weight support springs (Figure 2 and Figure 3). This requires the animal to be fixed in one spot on the treadmill, ensuring that they go in the designated forward direction and speed. 
	NOTE: Due to time and personnel constraints, the authors&#8217; lab conducts daily training in groups of twelve animals, three in each subset group.</li>
	<li>Start the acclimation process following the previously published protocol17. Commence acclimation to LT (start of week 3 post-SCI) with a gradual treadmill exposure regimen, increasing from 10 min on Day 1 to the full target of 58 min over the first week (Table 2). Typically, by Day 4, the animals acclimate well to the training regimen. If an animal does not show progression by the third day of acclimation, the time would be reduced, and extra days added at a more gradual ramp-up (rare occurrence).
	<ol>
		<li>If an animal during the first day or two does not adapt to the confinement of the harness and treadmill, stop the training session, remove it from the harness, place the animal back in its cage, and give it two treats to help reinforce future compliance.&#160;The next day, place the animal in the harness and weight support system again for 10 min. On subsequent days, increase the duration by 20 min initially then continue to increase the training duration daily to achieve full training by Day 10.</li>
	</ol>
	</li>
	<li>Follow the detailed training regimen provided in Table 2.
	<ol>
		<li>Due to limited hind limb use post-injury, rats in the QT group will require manual facilitation for proper paw placement while stepping on the treadmill. Use one finger on each hand (commonly the third digit) to aid in hip/waist support. When the animal requires further assistance in stepping, use this same finger to apply pressure above the knee to initiate stepping. If necessary, use a separate finger (commonly the fifth digit) to aid the foot in stepping. 
		NOTE: The amount of body weight support needed varies from animal to animal and changes as training progresses. The spring support system gives enough assistance to keep the animal positioned for a proper gait. Further support is provided as needed by the trainer per above. Note that a key element of LT is functionally appropriate paw placement for stepping and interlimb coordination that is promoted by the trainer and is independent of the support system.</li>
		<li>For the FT exercise group, adjust the body weight support system to slightly elevate the hind limbs to ensure no sensory stimuli to the paws and no weight bearing is occurring through contact with the treadmill. 
		NOTE: The FT group serves as an exercise and metabolic control, similar to that of a hand-crank exercise in human activity-based training studies.</li>
		<li>Have the NT group harnessed and attached to the body weight support system in a similar fashion as the QT and place the NT group near the QT group on a stationary surface (Figure 2 and Figure 3). 
		NOTE: The NT group receives no activity and controls for any potential effects of being harnessed for an extended time period.</li>
		<li>A home cage group can serve as an additional control. Transport these animals to the training facility as an additional step for this group.</li>
	</ol>
	</li>
	<li>By Day 7 -10 following the start of LT, train each animal once daily, every day until the day of termination of the study. Following each day of training, give each animal a sugary treat to reinforce compliance. Continue daily LT on animals following the 1 h regimen provided in Table 2 for the duration of the study (e.g., 8-12 weeks to mimic the approximate 80 one-hour sessions that are done in clinical studies)9.</li>
</ol>
4. Euthanasia and Tissue Collection
<ol>
	<li>Administer a lethal dose of anesthesia to the animal that adheres to AVMA guidelines on Euthanasia.</li>
	<li>When the heart is just barely beating, immediately begin perfusing the animal in a dedicated fume hood first with cold heparinized saline followed by cold, 4% paraformaldehyde solution.
	<ol>
		<li>Begin by using surgical scissors to make an incision across the diaphragm, exposing the thoracic cavity. Continue to cut through the ribcage rostrally on both sides, removing the ribcage. Insert the perfusion needle into the left ventricle of the heart and clamp needle with hemostats, then clip the right atrium.</li>
		<li>Using a perfusion pump mechanism, allow the cold heparinized saline to flow through the animal&#8217;s blood vessels. Once clear saline flows from the right atrium, switch over to the cold 4% paraformaldehyde solution, until the body has stiffened.</li>
	</ol>
	</li>
	<li>Remove necessary tissue such as kidney, bladder, colon, brain, sensory ganglia, and spinal cord, and store in 4% paraformaldehyde for up to 48 h at 4 &#176;C. After 24 - 48 h, move tissue to 30% sucrose and store at 4 &#176;C.</li>
	<li>Move collected tissue to a 30% sucrose/phosphate buffered cryoprotectant solution until tissue is ready for cutting. To cut tissue, embed in a tissue freezing compound and cut on a cryostat at desired thickness depending on the type of tissue used (e.g., 35 &#181;m for brain and spinal cord tissue, 5 - 7 &#181;m for organ tissues).</li>
</ol>

<ol>
	<li>Ahuja, C. S., 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=Traumatic+spinal+cord+injury.">Traumatic spinal cord injury.</a> Nature Reviews Disease Primers. 3, 17018 (2017).</li><li>Behrman, A. L., Harkema, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locomotor+training+after+human+spinal+cord+injury:+a+series+of+case+studies.">Locomotor training after human spinal cord injury: a series of case studies.</a> Physical Therapy. 80 (7), 688-700 (2000).</li><li>Anderson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+recovery:+priorities+of+the+spinal+cord-injured+population.">Targeting recovery: priorities of the spinal cord-injured population.</a> Journal of Neurotrauma. 21 (10), 1371-1383 (2004).</li><li>Steadman, C. J., Hubscher, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+function+after+spinal+cord+injury:+innervation,+assessment,+and+treatment.">Sexual function after spinal cord injury: innervation, assessment, and treatment.</a> Current Sexual Health Reports. 8 (2), 106-115 (2016).</li><li>Behrman, A. 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=Locomotor+training+progression+and+outcomes+after+incomplete+spinal+cord+injury.">Locomotor training progression and outcomes after incomplete spinal cord injury.</a> Physical Therapy. 85 (12), 1356-1371 (2005).</li><li>Dietz, V., Harkema, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locomotor+activity+in+spinal+cord-injured+persons.">Locomotor activity in spinal cord-injured persons.</a> Journal of Applied Physiology. 96 (5), 1954-1960 (2004).</li><li>Harkema, S., 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=Effect+of+epidural+stimulation+of+the+lumbosacral+spinal+cord+on+voluntary+movement,+standing,+and+assisted+stepping+after+motor+complete+paraplegia:+a+case+study.">Effect of epidural stimulation of the lumbosacral spinal cord on voluntary movement, standing, and assisted stepping after motor complete paraplegia: a case study.</a> The Lancet. 377 (9781), 1938-1947 (2011).</li><li>Harkema, 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=Locomotor+training:+as+a+treatment+of+spinal+cord+injury+and+in+the+progression+of+neurologic+rehabilitation.">Locomotor training: as a treatment of spinal cord injury and in the progression of neurologic rehabilitation.</a> Archives of physical medicine and rehabilitation. 93 (9), 1588-1597 (2012).</li><li>Jayaraman, 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=Locomotor+training+and+muscle+function+after+incomplete+spinal+cord+injury:+case+series.">Locomotor training and muscle function after incomplete spinal cord injury: case series.</a> The Journal of Spinal Cord Medicine. 31 (2), 185-193 (2008).</li><li>Behrman, A. L., Bowden, M. G., Nair, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroplasticity+after+spinal+cord+injury+and+training:+an+emerging+paradigm+shift+in+rehabilitation+and+walking+recovery.">Neuroplasticity after spinal cord injury and training: an emerging paradigm shift in rehabilitation and walking recovery.</a> Physical Therapy. 86 (10), 1406-1425 (2006).</li><li>Edgerton, V. R., Tillakaratne, N. J., Bigbee, A. J., de Leon, R. D., Roy, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+the+spinal+neural+circuitry+after+injury.">Plasticity of the spinal neural circuitry after injury.</a> Annual Review of Neuroscience. 27, 145-167 (2004).</li><li>Barbeau, H., Rossignol, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+locomotion+after+chronic+spinalization+in+the+adult+cat.">Recovery of locomotion after chronic spinalization in the adult cat.</a> Brain Research. 412 (1), 84-95 (1987).</li><li>Lovely, R. G., Gregor, R., Roy, R., 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=Effects+of+training+on+the+recovery+of+full-weight-bearing+stepping+in+the+adult+spinal+cat.">Effects of training on the recovery of full-weight-bearing stepping in the adult spinal cat.</a> Experimental Neurology. 92 (2), 421-435 (1986).</li><li>Multon, S., Franzen, R., Poirrier, A. -. L., Scholtes, F., Schoenen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+treadmill+training+on+motor+recovery+after+a+partial+spinal+cord+compression-injury+in+the+adult+rat.">The effect of treadmill training on motor recovery after a partial spinal cord compression-injury in the adult rat.</a> Journal of Neurotrauma. 20 (8), 699-706 (2003).</li><li>Moraud, E. 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=Closed-loop+control+of+trunk+posture+improves+locomotion+through+the+regulation+of+leg+proprioceptive+feedback+after+spinal+cord+injury.">Closed-loop control of trunk posture improves locomotion through the regulation of leg proprioceptive feedback after spinal cord injury.</a> Scientific Reports. 8 (1), 76 (2018).</li><li>Hubscher, C. H., 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+exercise+training+on+urinary+tract+function+after+spinal+cord+injury.">Effects of exercise training on urinary tract function after spinal cord injury.</a> American Journal of Physiology-Renal Physiology. 310 (11), F1258-F1268 (2016).</li><li>Ward, P. 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=Novel+multi-system+functional+gains+via+task+specific+training+in+spinal+cord+injured+male+rats.">Novel multi-system functional gains via task specific training in spinal cord injured male rats.</a> Journal of Neurotrauma. 31 (9), 819-833 (2014).</li><li>Ward, P. 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=Optically-induced+neuronal+activity+is+sufficient+to+promote+functional+motor+axon+regeneration+in+vivo.">Optically-induced neuronal activity is sufficient to promote functional motor axon regeneration in vivo.</a> PloS One. 11 (5), e0154243 (2016).</li><li>Edgerton, V. R., 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=Retraining+the+injured+spinal+cord.">Retraining the injured spinal cord.</a> The Journal of physiology. 533 (1), 15-22 (2001).</li><li>Angeli, C. A., Edgerton, V. R., Gerasimenko, Y. P., Harkema, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altering+spinal+cord+excitability+enables+voluntary+movements+after+chronic+complete+paralysis+in+humans.">Altering spinal cord excitability enables voluntary movements after chronic complete paralysis in humans.</a> Brain. 137 (5), 1394-1409 (2014).</li><li>Behrman, A. L., Ardolino, E. M., Harkema, S. 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-Based+Therapy:+From+basic+science+to+clinical+application+for+recovery+after+spinal+cord+injury.">Activity-Based Therapy: From basic science to clinical application for recovery after spinal cord injury.</a> Journal of Neurologic Physical Therapy. 41, S39-S45 (2017).</li><li>Hubscher, C. H., 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=Improvements+in+bladder,+bowel+and+sexual+outcomes+following+task-specific+locomotor+training+in+human+spinal+cord+injury.">Improvements in bladder, bowel and sexual outcomes following task-specific locomotor training in human spinal cord injury.</a> PloS One. 13 (1), e0190998 (2018).</li><li>Rejc, E., Angeli, C. A., Bryant, N., Harkema, S. J. <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+stand+and+step+training+with+epidural+stimulation+on+motor+function+for+standing+in+chronic+complete+paraplegics.">Effects of stand and step training with epidural stimulation on motor function for standing in chronic complete paraplegics.</a> Journal of Neurotrauma. 34 (9), 1787-1802 (2017).</li><li>Hall, B. 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=Spinal+cord+injuries+containing+asymmetrical+damage+in+the+ventrolateral+funiculus+is+associated+with+a+higher+incidence+of+at-level+allodynia.">Spinal cord injuries containing asymmetrical damage in the ventrolateral funiculus is associated with a higher incidence of at-level allodynia.</a> The Journal of Pain. 11 (9), 864-875 (2010).</li><li>Hubscher, C. H., Johnson, R. D. <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+acute+and+chronic+midthoracic+spinal+cord+injury+on+neural+circuits+for+male+sexual+function.+II.+Descending+pathways.">Effects of acute and chronic midthoracic spinal cord injury on neural circuits for male sexual function. II. Descending pathways.</a> Journal of Neurophysiology. 83 (5), 2508-2518 (2000).</li><li>Hubscher, C. H., Johnson, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+spinal+cord+injury+induced+changes+in+the+responses+of+thalamic+neurons.">Chronic spinal cord injury induced changes in the responses of thalamic neurons.</a> Experimental Neurology. 197 (1), 177-188 (2006).</li><li>Ward, P. J., Hubscher, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+polyuria+in+a+rat+spinal+contusion+model.">Persistent polyuria in a rat spinal contusion model.</a> Journal of Neurotrauma. 29 (15), 2490-2498 (2012).</li><li>Scheff, S. W., Rabchevsky, A. G., Fugaccia, I., Main, J. A., Lumpp, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+modeling+of+spinal+cord+injury:+characterization+of+a+force-defined+injury+device.">Experimental modeling of spinal cord injury: characterization of a force-defined injury device.</a> Journal of Neurotrauma. 20 (2), 179-193 (2003).</li><li>Ferrero, S. 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=Effects+of+lateral+funiculus+sparing,+spinal+lesion+level,+and+gender+on+recovery+of+bladder+voiding+reflexes+and+hematuria+in+rats.">Effects of lateral funiculus sparing, spinal lesion level, and gender on recovery of bladder voiding reflexes and hematuria in rats.</a> Journal of Neurotrauma. 32 (3), 200-208 (2015).</li><li>Smith, R. R., 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=Swim+training+initiated+acutely+after+spinal+cord+injury+is+ineffective+and+induces+extravasation+in+and+around+the+epicenter.">Swim training initiated acutely after spinal cord injury is ineffective and induces extravasation in and around the epicenter.</a> Journal of Neurotrauma. 26 (7), 1017-1027 (2009).</li><li>Oelke, 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=A+practical+approach+to+the+management+of+nocturia.">A practical approach to the management of nocturia.</a> International Journal of Clinical Practice. 71 (11), e13027 (2017).</li><li>Claydon, V., Steeves, J., Krassioukov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthostatic+hypotension+following+spinal+cord+injury:+understanding+clinical+pathophysiology.">Orthostatic hypotension following spinal cord injury: understanding clinical pathophysiology.</a> Spinal Cord. 44 (6), 341 (2006).</li><li>Antunes-Rodrigues, J., De Castro, M., Elias, L. L., Valenca, M. M., McCANN, 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=Neuroendocrine+control+of+body+fluid+metabolism.">Neuroendocrine control of body fluid metabolism.</a> Physiological Reviews. 84 (1), 169-208 (2004).</li><li>C&#244;t&#233;, M. -. P., Azzam, G. A., Lemay, M. A., Zhukareva, V., Houl&#233;, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+increase+in+neurotrophic+factors+is+associated+with+an+enhanced+modulation+of+spinal+reflexes+after+spinal+cord+injury.">Activity-dependent increase in neurotrophic factors is associated with an enhanced modulation of spinal reflexes after spinal cord injury.</a> Journal of Neurotrauma. 28 (2), 299-309 (2011).</li><li>Dupont-Versteegden, E. E., 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-induced+gene+expression+in+soleus+muscle+is+dependent+on+time+after+spinal+cord+injury+in+rats.">Exercise-induced gene expression in soleus muscle is dependent on time after spinal cord injury in rats.</a> Muscle &amp; Nerve: Official Journal of the American Association of Electrodiagnostic Medicine. 29 (1), 73-81 (2004).</li><li>De Leon, R., Hodgson, J., Roy, R., 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=Full+weight-bearing+hindlimb+standing+following+stand+training+in+the+adult+spinal+cat.">Full weight-bearing hindlimb standing following stand training in the adult spinal cat.</a> Journal of Neurophysiology. 80 (1), 83-91 (1998).</li><li>Pearson, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Progress in brain research. 143, 123-129 (2004).</li><li>Gerasimenko, 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=Feed-forwardness+of+spinal+networks+in+posture+and+locomotion.">Feed-forwardness of spinal networks in posture and locomotion.</a> The Neuroscientist. 23 (5), 441-453 (2017).</li><li>Courtine, 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=Transformation+of+nonfunctional+spinal+circuits+into+functional+states+after+the+loss+of+brain+input.">Transformation of nonfunctional spinal circuits into functional states after the loss of brain input.</a> Nature Neuroscience. 12 (10), 1333 (2009).</li></ol>

The authors have nothing to disclose.

Our methods of ABT on rats after SCI is a novel therapeutic intervention. While other methods of exercise and step training in animal models may exist35,36,37, this method mimics LT carried out clinically in the SCI human population, where we have seen promising results23. With the combination of our setup, regimen, and use of control animals, the results obtained from utilizing our training paradigm will help to understand the benefits of ABT after SCI. Future applications of this protocol include observing the described outcomes of ABT at different training timeframes as well as the effect that ABT has on recovery from different levels and extents of injury.
One limitation of this design is the length of time for such experiments. Given that our training regimen for each animal requires 1 hour per day, every day for 10 weeks, substantial personnel time and an organized schedule is a necessity. An important aspect that requires special attention involves the FT group, which has unique harnesses with hook-and-loop material straps to secure the hind limbs above the treadmill for the elimination of weight support. It is important to ensure that the animal does not receive weight support, which is why a platform is not positioned under the rat's hind paws. In addition, as previous studies have indicated that the sensory input is a principal driver of locomotor system plasticity in the spinal cord38,39,40, there is a constant need of handling the QT group to assist with stepping much the same as physical therapists in the clinical setting. 
An important modification made to the commercially available treadmill system used for the animals was reversing the polarity. After exposing the motor, the positive and negative wires were switched which reverses the direction the treadmill moves. This allows for more space and easier access to reach and help train the animals (the system comes with a shock grid at one end that is designed to prevent non-harnessed, spinally intact animals from stepping off the treadmill belt).

Globally, between 250,000 - 500,000 new spinal cord injury (SCI) cases arise either due to degeneration, diseases, or most commonly (up to 90%) trauma1. After traumatic SCI, a series of physiologic events take place that result in neurological deficits that affect a multitude of bodily functions. Due to the chronic deficits that follow SCI, the development and testing of effective treatment modalities is crucial. Until recently, rehabilitation strategies have most commonly focused on recovery of mobility2,3. Following SCI, patients rank bladder/urinary, bowel, and sexual functions among the highest quality of life complications in need of better management1,4,5. Therefore, targeting bladder, bowel, and sexual function is of utmost importance from a rehabilitation standpoint1,4,5.
Exercise and locomotor training (LT) are commonly utilized rehabilitative therapies in the SCI patient population with many benefits such as cardiovascular function, bladder/urinary function, and mobility6,7,8,9,10. It is for this reason we utilize a similar modality in our pre-clinical rat SCI model. It is our goal to determine what effects LT has on SCI Wistar rats, specifically regarding both upper (kidney) and lower (bladder, external urethral sphincter) urinary tract function, bowel function, and sexual function. Further, LT has been shown to be sufficient in activating neuromuscular systems below the level of injury which may influence the amount of plasticity within the central nervous system (CNS)11,12.
The success of LT in pre-clinical studies is well documented in both large13,14 and small15,16,17,18,19 SCI animal models. Evidence suggests that afferent sensory input provided by LT is sufficient to stimulate spinal reflex pathways that result in plasticity and improvements to sensory-motor function9,20. LT benefits regarding autonomic functions have not been well characterized. For this reason, we implement our training paradigm with a focus on autonomic outcome measures, using four distinct groups that include two non-trained controls and a metabolic/exercise non- weight bearing group alongside an LT group that mimics the timing, session duration, manual assistance and weight support that are used in clinical studies19,21,22,23,24.

<table><tbody><tr><td>Exer-3R treadmill</td><td>Columbus Instruments</td><td></td><td>reversed polarity of the motor</td></tr><tr><td>Body weight support system</td><td>N/A</td><td>N/A</td><td>modified spring scales with alligator clips</td></tr><tr><td>Rat harness</td><td>N/A</td><td>N/A</td><td>Our harnesses are custom made; please refer to Figure 1 for visual.</td></tr><tr><td>Infinite Horizon (IH) impactor device</td><td>Precision Systems and Instrumentation</td><td>Model 0400</td><td></td></tr><tr><td>Ketamine HCl</td><td>Hospira</td><td>NDC 0409-2053-10</td><td></td></tr><tr><td>Xylazine (AnaSed Injection)</td><td>Akorn Animal Health</td><td>NDC 59399-110-20</td><td></td></tr><tr><td>Meloxicam (Eloxiject)</td><td>Henry Schein Animal Health</td><td>NDC 116695-6925-2</td><td></td></tr><tr><td>Gentamicin Sulfate (GentaFuse)</td><td>Henry Schein Animal Health</td><td>NDC 11695-4146-1</td><td></td></tr><tr><td>urethane, 97%</td><td>Argos Organics</td><td>CAS 51-79-6</td><td></td></tr><tr><td>4-0 monofilament suture kit (4-0 Ethilon Nylon Suture)</td><td>Ethicon, LLC</td><td>205016</td><td></td></tr><tr><td>Michel suture clips (9mm Auto Clips)</td><td>MikRon Precision, Inc.</td><td>1629</td><td></td></tr><tr><td>Heating pad</td><td>Mastex Industries, Inc</td><td>Model 500</td><td></td></tr><tr><td>Tootie Fruitys cereal</td><td>Malt O Meal</td><td></td><td>For training reward</td></tr><tr><td>Male Wistar rats</td><td>Envigo</td><td></td><td></td></tr><tr><td>Size 10 surgical scalpel blades</td><td>Miltex</td><td>SKU: 4-110</td><td></td></tr></tbody></table>

activity-based training on a treadmill with spinal cord injured wistar rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

Following this training protocol, it has been documented that only the QT animals demonstrate superior locomotor function when compared to the other groups18. However, due to the nature of our lab, our primary focus is to investigate non-locomotor benefits of activity-based task-specific training (ABT), including bladder, bowel, and sexual function. For instance, we have previously published data that shows LT results in an exercise-induced reduction of polyuria in both QT and FT groups of SCI rats (Figure 4)17. Also, an injury-induced decrease in transforming growth factor-&#946; (TGF-&#946;) expression in the kidney&#39;s, indicative of an altered immune response, was not seen in QT and FT groups, which had TGF-&#946; levels similar to sham (no injury) animals. In the same study17, awake cystometry was performed before euthanasia and tissue collection. The maximum amplitude of bladder contractions during void cycles was not significantly different across sham, QT, and FT groups, while NT groups remained significantly altered. Together, these data indicate a positive exercise outcome on kidney health and bladder function, thus improving urinary function after SCI.
The mechanisms underlying polyuria within the SCI population is currently not clear but is likely multi-factorial32. Some have hypothesized, for example, that pooling of fluid in the lower limbs while SCI individuals are in a wheelchair can lead to fluid overload and increased fluid elimination during postural shifts (such as moving from sitting to lying)33. Such an explanation does not hold for the pre-clinical model, which has led us to focus initially on arginine vasopressin (AVP), the hormone which controls fluid homeostasis in the body and can be modulated with exercise. AVP controls fluid homeostasis through activation of the V2 receptor in the kidneys which facilitates water resorption from the renal collecting ducts34. Preliminary evidence from a pilot experiment (chronic time-point with one lesion severity - 210 kdyn impact force) indicate a beneficial effect of exercise (LT and FT) on V2 receptor levels in the rat kidney (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58983/58983fig1.jpg" /> 
Figure 1: Custom-made harnesses sized for male Wistar rats. Both QT and NT animals are placed in the same type of jacket (A) allowing for the use of hind limbs in the case of QT animals. There are additional straps sewed onto the harness used for FT animals (B) to raise up the hind limbs, assuring no body weight support. The large hook-and-loop material portions of the harness allow for easy adjustments to different sized animals and to any changes in the size of an individual animal over time. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58983/58983fig1large.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/58983/58983fig2.jpg" /> 
Figure 2: Training station setup. Body weight support mechanism surrounding the treadmill for either NT (far left), QT (middle), or FT (right) groups. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58983/58983fig2large.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/58983/58983fig3.jpg" /> 
Figure 3: Training station with animals. Top (A) and (B) side views showing body weight support mechanism and location of attachment support clips to the harnesses. Note that the hind limbs of the FT animal (B) is raised and off the treadmill belt. Inset (C) portrays a closer view of the clip fastened to the harness. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58983/58983fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58983/58983fig4.jpg" /> 
Figure 4: ABT effects on rat polyuria after SCI. The total volume of urine output (A) increased after SCI (*; p &#60; 0.05) and returned closer to baseline after 9 weeks of LT training in both QT and FT groups but remained increased in the NT group relative to the trained groups (#; p &#60; 0.05). All groups demonstrated increased urine output compared to baseline at 9 weeks and increased void volume (B). It is important to note that the number of voids (C) and the amount of water intake (D) remained the same across all groups. Values are means &#177; standard error. This figure is republished with author permission17. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58983/58983fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58983/58983fig5.jpg" /> 
Figure 5: ABT effects on rat kidney. Western blot results for rat kidney levels of V2 receptors in 5 groups of 4 rats each (20 total), showing expression levels for the protein bands provided in panel A and group mean densitometry analysis results of the bands (using ImageJ; OD = optical density) in panel B, indicating a significant (*; p &#60; 0.05) decrease in receptors at a chronic time-point (12 weeks) post-SCI and no decrease relative to baseline (sham surgical controls) for groups receiving 10 weeks of one-hour daily ABT. Error bars represent standard error. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58983/58983fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Ketamine/Xylazine Dose Chart</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Effective dose:</td>
			<td colspan="4">***Using 100 mg/mL ketamine stock and 20 mg/mL xylazine stock***</td>
		</tr>
		<tr>
			<td>80 mg/kg ketamine</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 mg/kg xylazine</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="5">1.0 mL mixture Injection = 0.62 mL ketamine stock (100 mg/mL) + 0.38 mL xylazine stock (20 mg/mL)</td>
		</tr>
		<tr>
			<td>Animal Weight</td>
			<td colspan="2">Mixture Injection</td>
			<td>Animal Weight</td>
			<td>Mixture Injection</td>
		</tr>
		<tr>
			<td>(g)</td>
			<td>(mL)</td>
			<td></td>
			<td>(g)</td>
			<td>(mL)</td>
		</tr>
		<tr>
			<td>100</td>
			<td>0.13</td>
			<td></td>
			<td>275</td>
			<td>0.36</td>
		</tr>
		<tr>
			<td>105</td>
			<td>0.14</td>
			<td></td>
			<td>285</td>
			<td>0.37</td>
		</tr>
		<tr>
			<td>110</td>
			<td>0.14</td>
			<td></td>
			<td>290</td>
			<td>0.38</td>
		</tr>
		<tr>
			<td>115</td>
			<td>0.15</td>
			<td></td>
			<td>300</td>
			<td>0.39</td>
		</tr>
		<tr>
			<td>120</td>
			<td>0.16</td>
			<td></td>
			<td>305</td>
			<td>0.4</td>
		</tr>
		<tr>
			<td>125</td>
			<td>0.16</td>
			<td></td>
			<td>310</td>
			<td>0.4</td>
		</tr>
		<tr>
			<td>130</td>
			<td>0.17</td>
			<td></td>
			<td>315</td>
			<td>0.41</td>
		</tr>
		<tr>
			<td>135</td>
			<td>0.18</td>
			<td></td>
			<td>320</td>
			<td>0.42</td>
		</tr>
		<tr>
			<td>140</td>
			<td>0.18</td>
			<td></td>
			<td>325</td>
			<td>0.42</td>
		</tr>
		<tr>
			<td>145</td>
			<td>0.19</td>
			<td></td>
			<td>330</td>
			<td>0.43</td>
		</tr>
		<tr>
			<td>150</td>
			<td>0.2</td>
			<td></td>
			<td>335</td>
			<td>0.44</td>
		</tr>
		<tr>
			<td>155</td>
			<td>0.2</td>
			<td></td>
			<td>340</td>
			<td>0.44</td>
		</tr>
		<tr>
			<td>160</td>
			<td>0.21</td>
			<td></td>
			<td>345</td>
			<td>0.45</td>
		</tr>
		<tr>
			<td>165</td>
			<td>0.21</td>
			<td></td>
			<td>350</td>
			<td>0.46</td>
		</tr>
		<tr>
			<td>170</td>
			<td>0.22</td>
			<td></td>
			<td>355</td>
			<td>0.46</td>
		</tr>
		<tr>
			<td>175</td>
			<td>0.23</td>
			<td></td>
			<td>360</td>
			<td>0.47</td>
		</tr>
		<tr>
			<td>180</td>
			<td>0.23</td>
			<td></td>
			<td>365</td>
			<td>0.47</td>
		</tr>
		<tr>
			<td>185</td>
			<td>0.24</td>
			<td></td>
			<td>370</td>
			<td>0.48</td>
		</tr>
		<tr>
			<td>190</td>
			<td>0.25</td>
			<td></td>
			<td>375</td>
			<td>0.49</td>
		</tr>
		<tr>
			<td>195</td>
			<td>0.25</td>
			<td></td>
			<td>380</td>
			<td>0.49</td>
		</tr>
		<tr>
			<td>200</td>
			<td>0.26</td>
			<td></td>
			<td>385</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>205</td>
			<td>0.27</td>
			<td></td>
			<td>390</td>
			<td>0.51</td>
		</tr>
		<tr>
			<td>210</td>
			<td>0.27</td>
			<td></td>
			<td>395</td>
			<td>0.51</td>
		</tr>
		<tr>
			<td>215</td>
			<td>0.28</td>
			<td></td>
			<td>400</td>
			<td>0.52</td>
		</tr>
		<tr>
			<td>220</td>
			<td>0.29</td>
			<td></td>
			<td>410</td>
			<td>0.53</td>
		</tr>
		<tr>
			<td>225</td>
			<td>0.29</td>
			<td></td>
			<td>420</td>
			<td>0.55</td>
		</tr>
		<tr>
			<td>230</td>
			<td>0.3</td>
			<td></td>
			<td>430</td>
			<td>0.56</td>
		</tr>
		<tr>
			<td>235</td>
			<td>0.31</td>
			<td></td>
			<td>440</td>
			<td>0.57</td>
		</tr>
		<tr>
			<td>240</td>
			<td>0.31</td>
			<td></td>
			<td>450</td>
			<td>0.59</td>
		</tr>
		<tr>
			<td>245</td>
			<td>0.32</td>
			<td></td>
			<td>460</td>
			<td>0.6</td>
		</tr>
		<tr>
			<td>250</td>
			<td>0.33</td>
			<td></td>
			<td>470</td>
			<td>0.61</td>
		</tr>
		<tr>
			<td>255</td>
			<td>0.33</td>
			<td></td>
			<td>480</td>
			<td>0.62</td>
		</tr>
		<tr>
			<td>260</td>
			<td>0.34</td>
			<td></td>
			<td>490</td>
			<td>0.64</td>
		</tr>
		<tr>
			<td>265</td>
			<td>0.34</td>
			<td></td>
			<td>500</td>
			<td>0.65</td>
		</tr>
		<tr>
			<td>270</td>
			<td>0.35</td>
			<td></td>
			<td>510</td>
			<td>0.66</td>
		</tr>
	</tbody>
</table>
Table 1: Anesthesia dosage chart based upon individual animal&#39;s weight.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Training Time 
			(min)</td>
			<td>Speed (cm/s)</td>
			<td>Duration (min)</td>
		</tr>
		<tr>
			<td>0-1</td>
			<td>6</td>
			<td>1</td>
		</tr>
		<tr>
			<td>1-2</td>
			<td>8.4</td>
			<td>1</td>
		</tr>
		<tr>
			<td>2-3</td>
			<td>10.8</td>
			<td>1</td>
		</tr>
		<tr>
			<td>3-8</td>
			<td>13.2</td>
			<td>5</td>
		</tr>
		<tr>
			<td>8-13</td>
			<td>10.8</td>
			<td>5</td>
		</tr>
		<tr>
			<td>13-28</td>
			<td>13.2</td>
			<td>15</td>
		</tr>
		<tr>
			<td>28-33</td>
			<td>10.8</td>
			<td>5</td>
		</tr>
		<tr>
			<td>33-38</td>
			<td>6</td>
			<td>5</td>
		</tr>
		<tr>
			<td>38-43</td>
			<td>8.4</td>
			<td>5</td>
		</tr>
		<tr>
			<td>43-58</td>
			<td>13.2</td>
			<td>15</td>
		</tr>
	</tbody>
</table>
Table 2: Training regimen of speed settings the treadmill should be on corresponding to the time spent at each speed.

Watch this Scientific Journal Video about Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats at JoVE.com

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

activity-based-training-on-treadmill-with-spinal-cord-injured-wistar

Research

JoVE Journal

Neuroscience

8.0K Views.  University of Louisville. This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

Video: Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Getting to Compliance in Forced Exercise in Rodents: A Critical Standard to Evaluate Exercise Impact in Aging-related Disorders and Disease

There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these include improving cognitive function and physical performance. As a result, there is an increase in the number of animal studies employing exercise. It is argued that one intrinsic value of forced exercise is that the investigator has control over the factors that can influence the impact of exercise on behavioral outcomes, notably exercise frequency, duration, and intensity of the exercise regimen. However, compliance in forced exercise regimens may be an issue, particularly if potential confounds of employing foot-shock are to be avoided. It is also important to consider that since most cognitive and locomotor impairments strike in the aged individual, determining impact of exercise on these impairments should consider using aged rodents with a highest possible level of compliance to ensure minimal need for test subjects. Here, the pertinent steps and considerations necessary to achieve nearly 100% compliance to treadmill exercise in an aged rodent model will be presented and discussed. Notwithstanding the particular exercise regimen being employed by the investigator, our protocol should be of use to investigators that are particularly interested in the potential impact of forced exercise on aging-related impairments, including aging-related Parkinsonism and Parkinson&#8217;s disease.

Exercise may alleviate behavioral impairments associated with neurodegenerative diseases. Studies of the mechanisms underlying exercise-mediated effects have increased in disease models, but the neurobiological background accompanying these diseases, specifically aging, is likely not considered. Here, we illustrate measures to increase the likelihood of a successful exercise paradigm in aged rats.

There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these ...

Behavior

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 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 ...

Training Persons with Spinal Cord Injury to Ambulate Using a Powered Exoskeleton

Powered exoskeletons have become available for overground ambulation in persons with paralyses due to spinal cord injury (SCI) who have intact upper extremity function and are able to maintain upright balance using forearm crutches. To ambulate in an exoskeleton, the user must acquire the ability to maintain balance while standing, sitting and appropriate weight shifting with each step. This can be a challenging task for those with deficits in sensation and proprioception in their lower extremities. This manuscript describes screening criteria and a training program developed at the James J. Peters VA Medical Center, Bronx, NY to teach users the skills needed to utilize these devices in institutional, home or community environments. Before training can begin, potential users are screened for appropriate range of motion of the hip, knee and ankle joints. Persons with SCI are at an increased risk of sustaining lower extremity fractures, even with minimal strain or trauma, therefore a bone mineral density assessment is performed to reduce the risk of fracture. Also, as part of screening, a physical examination is performed in order to identify additional health-related contraindications. 
Once the person has successfully passed all screening requirements, they are cleared to begin the training program. The device is properly adjusted to fit the user. A series of static and dynamic balance tasks are taught and performed by the user before learning to walk. The person is taught to ambulate in various environments ranging from indoor level surfaces to outdoors over uneven or changing surfaces. Once skilled enough to be a candidate for home use with the exoskeleton, the user is then required to designate a companion-walker who will train alongside them. Together, the pair must demonstrate the ability to perform various advanced tasks in order to be permitted to use the exoskeleton in their home/community environment.

Training a person with paralysis to ambulate using a powered exoskeleton may present challenges. The goals are to present the candidate selection criteria and the training procedures for exoskeletal-assisted walking and other mobility skills that can be progressed as the participant's skill level improves.

Powered exoskeletons have become available for overground ambulation in persons with paralyses due to spinal cord injury (SCI) who have intact upper ...

Bioengineering

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human Brown-S&#233;quard syndrome&#160;is a common type of incomplete SCI caused by a lesion to one half of the spinal cord which results in paralysis and loss of&#160;proprioception&#160;on the same (or ipsilesional) side as the injury, and loss of pain and temperature sensation on the opposite (or contralesional) side. Adequate methodologies for producing a spinal cord lateral hemisection (HX) and assessing neurological impairments are essential to establish a reliable animal model of Brown-S&#233;quard syndrome. Although lateral hemisection model plays a pivotal role in basic and translational research, standardized protocols for creating such a hemisection and assessing unilateralized function are lacking. The goal of this study is to describe step-by-step procedures to produce a rat spinal lateral HX at the 9th thoracic (T9) vertebral level. We, then, describe a combined behavior scale for HX (CBS-HX) that provides a simple and sensitive assessment of asymmetric neurological performance for unilateral SCI. The CBS-HX, ranging from 0 to 18, is composed of 4 individual assessments which include unilateral hindlimb stepping (UHS), coupling, contact placing, and grid walking. For CBS-HX, the ipsilateral and contralateral hindlimbs are assessed separately. We found that, after a T9 HX, the ipsilateral hindlimb showed impaired behavior function whereas the contralateral hindlimb showed substantial recovery. The CBS-HX effectively discriminated behavioral functions between ipsilateral and contralateral hindlimbs and detected temporal progression of recovery of the ipsilateral hindlimb. The CBS-HX components can be analyzed separately or in combination with other measures when needed. Although we only provided visual descriptions of the surgical procedures and behavioral assessments of a thoracic HX, the principle may be applied to other incomplete SCIs and at other levels of the injury.

Here we describe surgical procedures to produce a reliable spinal cord lateral hemisection (HX) at the 9th thoracic level in adult rats and neurobehavioral assessments designed for detecting asymmetric deficits after such a unilateral injury.

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human ...

Medicine

Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are valuable tools to understand the neural mechanisms involved in locomotor recovery after SCI and to design therapies for clinical populations. There are several experimental SCI models including contusion, compression, and transection injuries that are used in a wide variety of species. A hemisection involves the unilateral transection of the spinal cord and disrupts all ascending and descending tracts on one side only. Spinal hemisection produces a highly selective and reproducible injury in comparison to contusion or compression techniques that is useful for investigating neural plasticity in spared and damaged pathways associated with functional recovery. We present a detailed step-by-step protocol for performing a thoracic hemisection at the T8 vertebral level in the rat that results in an initial paralysis of the hindlimb on the side of the lesion with graded spontaneous recovery of locomotor function over several weeks. We also provide a locomotor scoring protocol to assess functional recovery in the open-field. The locomotor assessment provides a linear recovery profile and can be performed both early and repeatedly after injury in order to accurately screen animals for appropriate time points in which to conduct more specialized behavioral testing. The hemisection technique presented can be readily adapted to other transection models and species, and the locomotor assessment can be used in a variety of SCI and other injury models to score locomotor function.

The rat thoracic spinal hemisection is a valuable and reproducible model of unilateral spinal cord injury to investigate the neural mechanisms of locomotor recovery and treatment efficacy. This article includes a detailed step-by-step guide to perform the hemisection procedure and to assess locomotor performance in an open-field arena.

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are ...

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats&#8217; paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method&#8217;s limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Automated gait analysis is a feasible tool to evaluate functional recovery in rodent models of peripheral nerve injury and spinal cord contusion injury. While it requires only one setup to assess locomotor function in various experimental models, meticulous hard- and soft-ware adjustment and training of the animals is highly important.

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current limitations in immunosuppression and functional neuromotor recovery. Many animal models have been developed for studying unique features of VCA, but here we present a robust reproducible model of orthotopic hind limb transplant in rats designed to simultaneously investigate both aspects of current VCA limitation: immunosuppression strategies and functional neuromotor recovery. At the core of the model rests a commitment to meticulous, time-tested microsurgical techniques such as hand sewn vascular anastomoses and hand sewn neural coaptation of the femoral nerve and the sciatic nerve. This approach yields durable limb reconstructions that allow for longer lived animals capable of rehabilitation, resumption of daily activities, and functional testing. With short-term treatment of conventional immunosuppressive agents, allotransplanted animals survived up to 70 days post-transplant, and isotransplanted animals provide long lived controls beyond 200 days post-operatively. Evidence of neurologic functional recovery is present by 30 days post operatively. This model not only provides a useful platform for interrogating immunological questions unique to VCA and nerve regeneration, but also allows for in vivo testing of new therapeutic strategies specifically tailored for VCA.

This protocol presents a robust, reproducible model of vascularized composite allotransplant (VCA) geared toward simultaneous study of immunology and functional recovery. The time invested in meticulous technique in a right mid-thigh hind limb orthotopic transplant with hand sewn vascular anastomoses and neural coaptation yields the ability to study functional recovery.

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current ...

A Minimally Invasive, Fast Spinal Cord Lateral Hemisection Technique for Modeling Open Spinal Cord Injuries in Rats

Open spinal cord injury techniques modeling laceration-like injuries are time-consuming and invasive because they involve laminectomy. This new technique eliminates laminectomy by removing two spinous processes and lifting, then tilting the caudal vertebral arch. The surgical area opens up without the need for laminectomy. Lateral hemisection is then performed with direct visible control under a microscope. The trauma is minimized, requiring only a small bone wound. 
This technique has several advantages: it is faster and, therefore, less of a burden for the animal, and the bone wound is smaller. Because the laminectomy is eliminated, there is less chance for unwanted injury to the spinal cord, and there are no bone splinters that can cause problems (bone splinters embedded in the spinal cord can cause swelling and secondary damage). The vertebral canal remains intact. The main limitation is that the hemisection can only be performed in the intervertebral spaces. 
The results show that this technique can be performed much faster than the traditional surgical approach, using laminectomy (11 min vs. 35 min). This technique can be useful for researchers working with animal models of open spinal cord injury as it is widely adaptable and does not require any additional specialized instrumentation.

Here, we describe a new, fast technique modeling open spinal cord injury in rats that eliminates laminectomy. Lateral hemisection is performed while viewing through a microscope. The technique is versatile and can also be used in the cervical, thoracic, and lumbar regions of the spinal cord of other animals.

Open spinal cord injury techniques modeling laceration-like injuries are time-consuming and invasive because they involve laminectomy. This new technique ...

Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics is still unclear. The present study describes a motion analysis approach that utilizes three-dimensional (3D) reconstruction from video captures. Using this technology, disuse-evoked alterations of walking performances were observed in rodents exposed to a simulated microgravity environment by unloading their hindlimb by their tail. After 2 weeks of unloading, the rats walked on a treadmill, and their gait motions were captured with four charge-coupled device (CCD) cameras. 3D motion profiles were reconstructed and compared to those of control subjects using the image processing software. The reconstructed outcome measures successfully portrayed distinct aspects of distorted gait motion: hyperextension of the knee and ankle joints and higher position of the hip joints during the stance phase. Motion analysis is useful for several reasons. First, it enables quantitative behavioral evaluations instead of subjective observations (e.g., pass/fail in certain tasks). Second, multiple parameters can be extracted to fit specific needs once the fundamental datasets are obtained. Despite hurdles for broader application, the disadvantages of this method, including labor intensity and cost, may be alleviated by determining comprehensive measurements and experimental procedures.

The present protocol describes three-dimensional motion tracking/evaluation to depict gait motion alteration of rats after exposure to a simulated disuse environment.

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics ...