A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

Summary

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

Abstract

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.¹ Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.

Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5° (animals rotated at 22.5°), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.

The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

Protocol

1. Set up: Frame and Clamp Design for Holding the Animal

1. The frame and clamp to hold the animal was custom-designed to accommodate the Infinite Horizon (IH) Spinal Cord Injury Impactor.
2. The base of the frame is an aluminum platform cut into the following dimensions (30.2 cm x 20.3 cm x 1.3 cm) in order to fit into the table guide bracket that comes standard with the IH device (Figure 1A).
3. Four Flexaframe support foot plates (Fisher Scientific, Toronto, ON) are attached to the platform and eight Flexaframe support rods (Fisher Scientific, Toronto, ON, 30.5 cm) are assembled using eight Flexaframe support connectors (Figure 1B).
4. Two additional Flexaframe support connectors, attached to the middle two rods, house the custom made holds for the clamp (Figure 1B).
5. The angle by which the animal's spinal cord is rotated with respect to vertical is established by keeping one of the horizontal rod in place and varying the height of the other horizontal rod (Figure 2).
6. The clamp is 35.6 mm long, 25.4 mm high and with a 7.6 mm jaw designed to rigidly grab hold underneath the transverse process from C4 to C6 (Figure 1C). Additional details on the clamp design have been described previously by Choo et al., 2009.

2. Surgery

1. Male Sprague Dawley rats (Charles River Laboratories) weighing 300-350 g were anesthetized by isoflurane (4% for induction and 2% for maintenance) in oxygen (1 L/min).
2. Once the animals are under the plane of anesthesia, the animal is placed in a stereotaxic frame (Kopf, Tujunga, CA).
3. To minimize bleeding during the surgical procedure 0.4 mL of lidocaine (20 mg/mL; Bimeda-MTC Animal Health Inc., Cambridge, Ontario, Canada) with epinephrine is injected intramuscularly around the surgical site in the dorsal neck region.
4. A 4-5 cm dorsal midline incision is made using a sterile scalpel (#15), starting from the base of the skull and extending caudally.
5. Sterile Adson forceps are used to bluntly dissect through the dorsal musculature to reach the spine, and a sterile Alm retractor (Fine Science Tools, North Vancouver, BC) is inserted to keep the muscles spread apart.
6. Using the #15 scalpel, the muscles overlaying the laminae of C4-C7 are scraped off, starting in the midline and sweeping them out laterally.
7. An incision with a sterile scalpel (#15) is made to the muscle attached to the transverse processes on both side of the spine in order to fit the clamp underneath the transverse processes of C4 to C6.
8. With a sterile fine tipped Friedman-Pearson Rongeur (Fine Science Tools, North Vancouver, BC), the left C5 lamina is carefully removed to visualize the dura and the spinal cord.
9. A rod with a diameter of 1.5 mm is slid under the arms to prop the animal up, thereby raising the spine slightly and facilitating clamp insertion.
10. Mount the jaw of the sterile clamp onto lateral transverse processes of C4 to C6 and tighten the screws.
11. Remove the Alm retractor.

3. Spinal Cord Injury

1. After the clamp is mounted on the animal, the animal is moved to the IH impactor.
2. The clamp is inserted into the metal holders on the two middle rods of the frame that have been secured at an angle of 22.5° off of horizontal (Figure 2).
3. The scissor jack that provides stable height adjustments, (VWR, Mississauga, ON) is raised up until the animal lays flat.
4. Ensure that the clamp is horizontal by placing a small cylinder level on top of the clamp and by tightening the screws. It is important that all the screws are tight and the set up is rigid without any movement.
5. The remainder of the procedure is conducted under a microscope (Leica MZ8).
6. Lower and aim the impactor tip (15 mm in diameter, with rounded-off edges ) using the vertical adjustment knob and the two horizontal adjustment knobs on the IH impactor until the center of the impactor tip is hovering above the apex of C6 spinous process.
7. Once the impactor tip is centered, turn the y-axis adjustment knob one and two fifth turn (1.4 mm) to move the tip laterally to the left side and the x axis horizontal adjustment knob to move the impactor tip to the center of C5.
8. Lower the tip until it is just above the dura to verify that the impactor tip is aiming the lateral half of the grey matter.
9. Turn the vertical adjustment knob two turns to raise the tip 4 mm above the dura.
10. Ensure that the impact area is dry by using a cotton swab or stick.
11. Set the desired force to 150 kdyn on the program and click "Start Experiment" to trigger the impactor.
12. After the injury, the wound is closed in layers with 5-0 vicryl sutures. Buprenorphine (0.03 mg/kg SC, Temgesic; Schering-Plough Corporation, Kenilworth, NJ) and Saline (10 mL) is administered subcutaneously before and twice daily for two days after surgery. Animals are closely monitored twice a day for 2 weeks and one a week for 6 weeks post-injury.

4. Representative Results

Twenty Nine male Sprague Dawley rats (Charles River Laboratories) weighing 300-350 g were injured at a force setting of 150 kdyn. The impactor tip was aimed 1.4 mm lateral to midline, at an angle of 22.5° off of vertical. The mean actual force was 155.55 ± 0.73 kdyn. The mean displacement was 1512.72 ± 27.86 μm and the velocity was 120.24 ± 0.52 mm/s (Figure 3).

Behavior Outcome Measures

Functional recovery was assessed using the horizontal ladder test, cylinder rearing test, grooming test, and modified Montoya staircase test¹. Animals were trained before injury and assessed at 2, 4 and 6 weeks post-injury. There were significant forelimb impairments sustained throughout the experimental period.

Horizontal Ladder test. Before the injury, animals made only 4.75 ± 0.73% errors on the ipsilateral forelimb while traversing across the irregularly spaced horizontal ladder. Following the injury, the animals demonstrated marked increases in the percentage of forelimb errors. The ipsilateral forelimb percent errors were 26.97 ± 2.92%, 26.23 ± 2.84% and 22.06 ± 2.05% at 2, 4 and 6 weeks post-injury, respectively (Figure 4A). Importantly, the forelimb impairment on this test was sustained for the 6 weeks.

Cylinder Rearing test. The percentage of ipsilateral forelimb (left + both) usage during exploration decreased significantly after SCI. Prior to the injury, the animals used the ipsilateral forelimb 75.12 ± 2.25%. After the injury the animals used the ipsilateral forelimb 8.59 ± 1.80% at 2 weeks, 14.25 ± 2.65% at 4 weeks and 11.76 ± 2.66% at 6 weeks (Figure 4B).

Modified Montoya Staircase test. The number of pellets retrieved with the ipsilateral forelimb decreased dramatically after the injury. Prior to the injury, the animals collected 84.85 ± 2.88% of the food rewards. However, at 2, 4 and 6 weeks post-injury, the animals retrieved only 30.91 ± 4.03%, 28.94 ± 4.38% and 25.86 ± 3.09% of the pellets (Figure 4C).

Grooming test. There were dramatic decreases in the grooming scores after injury. After SCI, the ipsilateral grooming scores were 2 weeks: 2.00 ± 0.17, 4 weeks: 1.83 ± 0.17 and 6 weeks: 1.79 ± 0.11 (Figure 4D).

Histological Outcomes

White matter and Grey matter sparing. An example of a spinal cord injury at a force of 150 kdyn, angle of 22.5° and lateral aim of 1.4 mm is presented in Figure 5. The injury resulted in substantial damage to the grey and white matter on the ipsilateral side. Both corticospinal and rubrospinal tracts were injured and 23 out of 29 animals had parenchymal damage contained on the ipsilateral side. The longitudinal extent of the damage of the white and grey matter was 2400 rostrally and 2400 caudally (Figure 6). When adding the sections to provide a gross estimation of the "cumulative spread" of white and grey matter sparing (2000 μm rostral and caudal to the epicenter), the ipsilateral side had only 51.8% of the white matter spared and 39.7% of grey matter remaining compared to the contralateral side (Figure 6).

Figure 1. Infinite Horizon spinal cord injury impactor. A. Infinite Horizon impactor and overall set up. B. Frame set up. C. Close-up image of the clamp for holding cervical transverse processes. D. Specification (unit: inch) of the clamp. Suggested tolerance <0.002 in (Choo et al. 2009). Click here to view larger figure.

Figure 2. A. Illustration and corresponding frame set up to achieve spinal cord rotation of 0° (neutral) or B. 22.5° with the lateral aim of 1.4 mm.

Figure 3. Representative force and displacement graphs for Infinite Horizon impactor. The arrow indicates the time that the impactor tip has reached 20 kdyn and the point when the recording of displacement is initiated. The actual force is read from the peak of the force vs. time curve and the corresponding displacement is calculated. The graphs below depict a typical 150 kdyn contusion, A. Displacement vs. time graph, B. Force vs. Time graph. These graphs show that the actual force achieved was 152 kdyn, and the impactor displacement into the cord was measured to be 1287 μm. Click here to view larger figure.

Figure 4. Behavioral assessments for force of 150 kdyn, impactor angulation of 22.5° and aim of 1.4 mm left of midline. A. Horizontal ladder test. B. Cylinder rearing test. C. Modified Montoya staircase test. D. Grooming test. The ipsilateral forelimb resulted in significant and sustained impairments compared to the contralateral forelimb.

Figure 5. Representative images of the injured spinal cord from rostral 1600 μm to caudal 1600 μm.

Figure 6. Histological assessments. A. Percent white and grey matter spared. Matter around 2000 μm of the epicenter of damage was largely spared. B. Cumulative white and grey matter spared within 2000 μm of the epicenter of damage. The ipsilateral side had significantly less spared white and grey matter compared to the contralateral side. Click here to view larger figure.

Discussion

In this paper, we describe a cervical unilateral contusion model using the Infinite Horizon (IH) impactor at a force of 150 kdyn, an angle of 22.5° off of vertical, and a lateral aim of 1.4 mm from the midline. With these settings, we were able to produce sustained behavioral deficits in the ipsilateral forelimb with parenchymal disruption contained largely to the ipsilateral side, where it appeared that considerable damage occurred to the regions where the rubrospinal, reticulospinal, vestibulospinal and corticospin...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Infinite Horizon Impactor	Precision Systems and Instrumentation	IH-0400
Aluminum metal sheet	Metalsupermarlets.com	APT6061/500
Flexaframe support foot plates	Fishers Scientific	1466625Q
Flexaframe support rods	Fishers Scientific	1466610GQ
Flexaframe Support Connectors	Fishers Scientific	1466620Q
Clamp1	Custom made		Choo et al., 2009
Metal holders	Custom made		See above
Impactor tip	Custom made		Diameter: 1.15 mm
Stereotaxic frame	David Kopf Instruments	Model 900
Cylinder Level	YIJIA TOOLS	YJ-SL0620
Microscope	Leica		Model #: MZ8
Laboratory scissor jack	VWR	12620-902