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>

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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://www-jove-com.remotexs.ntu.edu.sg/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://www-jove-com.remotexs.ntu.edu.sg/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://www-jove-com.remotexs.ntu.edu.sg/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://www-jove-com.remotexs.ntu.edu.sg/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://www-jove-com.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Experimental Strategies to Bridge Large Tissue Gaps in the Injured Spinal Cord after Acute and Chronic Lesion

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the spinal cord, tumor resections, or tissue defects resulting from traumatic accidents can aid in facilitating general tissue repair as well as regenerative growth of nerve fibers into and beyond the affected area. Two experimental treatment strategies are presented: (1) implantation of a novel microconnector device into an acutely and completely transected thoracic rat spinal cord to readapt severed spinal cord tissue stumps, and (2) polyethylene glycol filling of the SCI site in chronically lesioned rats after scar resection. The chronic spinal cord lesion in this model is a complete spinal cord transection which was inflicted 5 weeks before treatment. Both methods have recently achieved very promising outcomes and promoted axonal regrowth, beneficial cellular invasion and functional improvements in rodent models of spinal cord injury.
The mechanical microconnector system (mMS) is a multi-channel system composed of polymethylmethacrylate (PMMA) with an outlet tubing system to apply negative pressure to the mMS lumen thus pulling the spinal cord stumps into the honeycomb-structured holes. After its implantation into the 1 mm tissue gap the tissue is sucked into the device. Furthermore, the inner walls of the mMS are microstructured for better tissue adhesion.
In the case of the chronic spinal cord injury approach, spinal cord tissue - including the scar-filled lesion area - is resected over an area of 4 mm in length. After the microsurgical scar resection the resulting cavity is filled with polyethylene glycol (PEG 600) which was found to provide an excellent substratum for cellular invasion, revascularization, axonal regeneration and even compact remyelination in vivo.

Severe spinal cord injuries often result in tissue defects. Two possibilities are described to successfully bridge such gaps to promote tissue adaptation, regenerative responses and functional improvement in rats via implantation of a mechanical microconnector system after acute injury and five weeks after complete spinal cord transection.

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

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 ...

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

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Chicken Embryo Spinal Cord Slice Culture Protocol

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Experimental Strategies to Bridge Large Tissue Gaps in the Injured Spinal Cord after Acute and Chronic Lesion

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Laminectomy and Spinal Cord Window Implantation in the Mouse

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

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