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

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)

1. 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.
2. 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).

2. Spinal Cord Contusion^25,26,27,28

1. 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.
2. 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).
3. Place the anesthetized animal on a heating pad at a low setting to maintain normal body temperature.
4. 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.
5. For mid-thoracic contusions, expose the T8/T9 level of spinal cord via removal (with rongeurs) of the overlaying T7 vertebral lamina.
6. 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.
7. 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.
8. 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).
9. Place animals in a clean cage on a heating pad. Check animal vital signs every 15 minutes until they are fully awake from anesthesia.  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.  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.
10. Perform bladder emptying procedures using the manual Credé 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.

3. Training Phase

1. Commence LT no earlier than two weeks post-SCI, as initiating interventions too early may exacerbate secondary injury cascades³¹.
2. Week 1 acclimation to treadmill training: Transport the rats to a quiet room that is dedicated for training.
3. 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.
4. 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’ lab conducts daily training in groups of twelve animals, three in each subset group.
5. 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).
   1. 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. 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.
6. Follow the detailed training regimen provided in Table 2.
   1. 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.
   2. 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.
   3. 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.
   4. A home cage group can serve as an additional control. Transport these animals to the training facility as an additional step for this group.
7. 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.

4. Euthanasia and Tissue Collection

1. Administer a lethal dose of anesthesia to the animal that adheres to AVMA guidelines on Euthanasia.
2. 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.
   1. 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.
   2. Using a perfusion pump mechanism, allow the cold heparinized saline to flow through the animal’s blood vessels. Once clear saline flows from the right atrium, switch over to the cold 4% paraformaldehyde solution, until the body has stiffened.
3. 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 °C. After 24 – 48 h, move tissue to 30% sucrose and store at 4 °C.
4. 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 µm for brain and spinal cord tissue, 5 – 7 µm for organ tissues).