Our model utilizes female Yucatan miniature pigs typically weighing between 20-25 kg at the time of surgery. Animals are pre-medicated with telazol 4-6 mg/kg intramuscular (IM) injection in conjunction with xylazine 1 mg/kg IM and atropine (0.02-0.04 mg/kg) IM. Animals are induced with either isoflurane (2-3% in O₂) or propofol 2 mg/kg, depending on the study’s anesthetic requirements, and endotracheally intubated. Pigs are given an intravenous (IV) dose of antibiotics (cefazolin, 15 mg/kg), and an IM injection of analgesics (ketoprofen, 3 mg/kg) before surgery. Bupivacaine (1 mg/kg) is injected into the skin and subcutaneous tissue of the incision line as a local anesthetic. A urinary catheter (10 Fr, Jorgensen Laboratories Inc., Loveland, CO, USA) is manually inserted before surgery and is maintained after surgery until the spontaneous urination is restored. Mechanical ventilation is maintained at 10-12 breaths/min and the tidal volume at 12-15 mL/kg (Veterinary Anesthesia Ventilator model 2002, Hallowell EMC, Pittsfield, MA, USA). Standard monitoring is performed for heart rate, respiratory rate, blood pressure, endtidal carbon dioxide, inspired and expired isoflurane levels, and oxygen saturation (pulse oximeter 8600V, Nonin Medical Inc., Markham, ON, USA and Cardell MAX-12HD Veterinary Monitor, Paragon Medical Supply, Inc., Coral Springs, FL, USA). Hydration is maintained with 1.25% dextrose solution and the temperature is measured by a rectal probe and maintained at 37.0-38.0oC with a heating pad (T/Pump, Gaymar Industries, Inc., Orchard Park, NY, USA).

Our most commonly performed injury is at the level of Thoracic 10 (T10) level, but the injury level can be altered depending on the research design. The vertebral levels are easily identified with anatomical landmarks such as the apex of the kyphotic thoracic spine. With the pig in the prone position on the surgical table (Fig. 2), a dorsal midline incision is made between T7 and T14, the length of which is determined by the nature of the experiment. The spinous processes, laminae, and transverse processes of T8-T13 are exposed using monopolar electrocautery. The transverse processes are then removed using a rongeur in order to place pedicle screws at the T6 and T8 or T11 and T13 levels, using 3.5×25 mm poly-axial screws (Vertex screws, Medtronic, Memphis, TN, USA) that are designed for human cervical spine fixation. The location of screw insertion can vary depending on the experimental design. A titanium rod is fixed with the locking caps to mount the weight drop guidance system, to rigidly fix the T9-12 segments during the weight drop, and to provide stability to the spine after SCI. A laminectomy is performed at the T8-T11 level and widened to ensure that a circular window measuring at least 1.2 cm in diameter (impactor diameter, 0.95 cm) is made to expose the dura and spinal cord.

Five minutes before SCI, isof lurane is increased to 3.0% from 0.5% to minimize involuntary movement as a result of the SCI. The weight drop apparatus consists of a guidance rail along which a 50 g impactor falls onto the exposed spinal cord from a defined height (Fig. 3) [5,15]. The guidance system for the impactor is secured rigidly to the rods held by the pedicle screws, and by adjusting the articulating arm, it is positioned directly over the laminectomy defect. The rail guidance system is leveled to ensure that it is aligned vertically in both the coronal and sagittal planes. The laminectomy defect size is checked to ensure that the impactor tip can fall freely on the midline of the dura and spinal cord without any interference. A load cell (LLB215, Futek Advanced Sensor Technology, Irvine, CA, USA) is installed at the end of the impact device to record the force during impact. Finally, the impactor is released and falls along the guidance rail system, and impacts the spinal cord. Immediately after the contusion injury, we placed a 100 g weight on top of the 50 g weight impactor to simulate sustained compression (total 150 g). The duration of compression can be adjusted depending upon the experiment - typically we use 5 minutes of sustained compression. Following 5 minutes of sustained compression, the weight drop apparatus is removed and the wound is closed by layer. However, we could control the degree of cord damage by the impactor height, weight, and the additional compression duration. In the previous study, we analyzed the effect of impactor height on tissue damage by dropping it from various heights - 50, 40, 30, 20, and 10 cm [15]. The height of the impactor drop was very well correlated with the histologic and function outcomes. Also, the additional compression duration and weight after primary impact could be another important factor in SCI, because the contusion from a height of 20 cm with or without sustained compression showed significant metabolic and histologic difference [18]. We ultimately chose 20 cm for the height of the impactor drop, combined with 5 minutes of compression, which produces a lower extremity paralysis that recovers to a Porcine Thoracic Injury Behavioral Scale (PTIBS) of approximately 4 at 12 weeks post-injury (described below).

After the surgery, the animal is maintained on a continuous rate infusion of fentanyl for pain control, which requires close observation. The animals are individually housed in recovery kennels for 7 days. The urinary catheter is removed 7-10 days after SCI, once the animals are able to reflexively empty their bladders. Animals are then housed in pairs on sawdust bedding for 12 weeks, during which behavioral recovery analysis is performed weekly.

The data acquired from the impactor tip during the actual contusive injury is collected using a custom Labview (V8.6, National Instruments, Austin, TX, USA) program at 100 kHz, followed by post-filtering and then customized with Matlab (V2008b, The Mathworks, Matick, MA, USA) using a bi-directional 4-way filter with 5 kHz low-pass cutoff frequency. Injury parameters such as impact force, displacement, velocity, and impulse are measured, providing relevant information on injury severity and variability.

To characterize hindlimb locomotor recovery, we created the Porcine Thoracic Injury Behavior Scale (PTIBS) [15]. The PTIBS stratifies hind limb locomotor function into ten categories, ranging from absent hind limb movement (score 1) to normal walking (score 10). A PTIBS score of 1-3 ref lects “hindlimb dragging”, scores of 4-6 reflect varying degrees of “stepping” ability, and scores of 7-10 reflect varying degrees of “walking” ability. A detailed description and functional components of each PTIBS score are listed in Tables 1 and 2, respectively. Hindlimb function is recorded using three highdefinition camcorders located behind the animal, so that the observer can visualize the movement of the hindlimbs and hips as the animal moves away from the camera. One week before the injury, videos are recorded of the animals to establish a “baseline” locomotor behavior score. If an animal fails to achieve a score of 10 at baseline, the animal will be removed from the study. Post-injury, videos are recorded once per week for 12 weeks and subsequently scored by two independent, blinded observers.

The reliability of PTIBS is relatively high under conditions of proper training. In our previous study [15], 154 video clips were scored by three observers (two trained technicians and an undergraduate student) on two occasions, 3 weeks apart. The intra-observer correlation coefficients were 0.954, 0.992, and 0.972 for the three observers and the inter-observer correlation coefficients were 0.961, 0.927, and 0.918. In addition, we analyzed the relationship between the histologic damage of the spinal cord and PTIBS. The PTIBS scores are positively correlated with white and gray matter sparing (see below in the “Histologic analysis”) 12 weeks post-injury. The correlation coefficient between the PTIBS score and the spared white matter was R=0.888 (p <0.001) and gray matter was q=0.748 (p <0.001). Also, the PTIBS scores are highly correlated with total (white and gray matter) spared tissue with a coefficient of q=0.881 (p <0.001). Also, the neurofilament immunoreactivity showed a positive correlation with the PTIBS score as a coefficient of q=0.663 (p =0.002).

In a recent study [26], we analyzed the correlation between the impact degree and PTIBS. The impact degree falls into three groups; mild, moderate, and severe. A mild impact degree represents an injury from a 10 cm drop height, moderate represents 20 cm, and severe represents 40 cm. One week after SCI, functional outcomes were most severely impaired. The average PTIBS score for the mild, moderate, and severe injury groups, were 4.0, 3.2, and 2.0, respectively. At 12 weeks after injury, the average PTIBS scores were 6.5, 4.25, and 3.0, respectively. Impact height had a significant effect on the PTIBS scores (p<0.0001) between all injury groups from 6 to 12 weeks after SCI. This data suggests that the impact force is strongly correlated with behavioral recovery.

For histological evaluation of damage induced by injury, a 10 cm segment of the thoracic spinal cord centered around the lesion is collected, post-fixed in 4% paraformaldehyde for 86 hours at 4oC, and cryopreserved by transferring the tissue through a series of solutions with gradually increasing sucrose concentrations (12%, 22%, and 30%). Spinal cord segments are divided into 1 cm blocks, one of them centering the impact site, kept frozen at -80oC, and cryo-sectioned with a thickness of 20 μm.

For the measurement of spared white and gray matter, a series of cross-sections are stained with Eriochrome Cyanine R as described in Rabchevsky et al. [19] and counterstained with Neutral Red (Fig. 4). The images are then captured with an optical microscope (Leica DM5000B, Leica, Concord, Canada) using a 2.5× or 5.0× objective lens. In the uninjured tissue, the staining allows clear differentiation between the white matter, gray matter and the pia by the color of the stain and the shape. In the injured tissue, the spared white matter is defined as a region showing dense blue staining. We defined uninjured gray matter as tissue containing a normal gray matter cytoarchitecture and keeping the residual blue color with visible neutral red staining speck. The cumulative preserved white and gray matter is calculated by summing the percentage of spared white and gray matter in a segment around epicenter, for example from 1.52 cm rostral to 1.52 cm caudal to the epicenter [16]. This provides a general volumetric representation of the extent of the injury.

In a recent study [26], we analyzed the correlation between the impact degree (sham, mild, moderate, severe) and histology. The height of injury had a significant effect on the amount of spared tissue, calculated with the area under the curve of the total percent spared tissue from 13.6 mm rostral and 13.6 mm caudal of the epicenter (p <0.0001). Cumulative spared tissue was greatest in the SHAM group (3380±16.0 cumulative%) compared to the mild (2720±98.0 cumulative%, p =0.0022), moderate (1990±140 cumulative%, p <0.0001) and severe (1650±88.0 cumulative%, p <0.0001) groups. The cumulative spared tissue was significantly greater in the mild SCI group as compared to the moderate SCI group (p=0.0008) and the severe SCI group (p <0.0001). The spared tissue was significantly higher between the 3.2 to 11.2 mm sections caudal to the epicenter in the moderate SCI group comparing with the severe SCI group, but there were no statistical differences between the cumulative areas (p =0.12). This data suggests that the impact force is correlated with tissue damage.