Anatomical and functional outcomes following a precise, graded, dorsal laceration spinal cord injury in C57BL/6 mice

Abstract

To study the pathophysiology of spinal cord injury (SCI), we used the LISA-Vibraknife to generate a precise and reproducible dorsal laceration SCI in the mouse. The surgical procedure involved a T9 laminectomy, dural resection, and a spinal cord laceration to a precisely controlled depth. Four dorsal hemisection injuries with lesion depths of 0.5, 0.8, 1.1, and 1.4 mm, as well as normal, sham (laminectomy and dural removal only), and transection controls were examined. Assessments including the Basso Mouse Scale (BMS), footprint analysis, beam walk, toe spread reflex, Hargreaves' test, and transcranial magnetic motor-evoked potential (tcMMEP) analysis were performed to assess motor, sensorimotor, and sensory function. These outcome measures demonstrated significant increases in functional deficits as the depth of the lesion increased, and significant behavioral recovery was observed in the groups over time. Quantitative histological examination showed significant differences between the injury groups and insignificant lesion depth variance within each of the groups. Statistically significant differences were additionally found in the amount of ventral spared tissue at the lesion site between the injury groups. This novel, graded, reproducible laceration SCI model can be used in future studies to look more closely at underlying mechanisms that lead to functional deficits following SCI, as well as to determine the efficacy of therapeutic intervention strategies in the injury and recovery processes following SCI.

FIG. 1.

Mouse stabilizer set-up for LISA-Vibraknife lesion. (A) Bilateral spinal column stabilizers; lateral view (top panel), ventral view (bottom panel). (B) The mouse is placed in a U-shaped metal channel (arrows on the right and left), and the blade (arrow in the middle) is positioned over the spinal cord surface under a dissecting microscope.

FIG. 2.

Acute anatomical qualitative assessment of the lesion. (A) The dorsal (D), ventral (V), and lateral (L) surfaces of the spinal cord at 20 min following a laceration injury of 0.5 mm (top row), 0.8 mm (second row), 1.1 mm (third row), and 1.6 mm (Tx; last row). Arrows point to corresponding histological images of each lesion (last column on the right); small black arrows pinpoint the lesion depth. Note the absence of dorsal surface deformation or contusion. (B) Demonstrates the difference in the lesion depths when comparing the LISA-Vibraknife lesions to those generated by another surgeon using microscissors. The microscissor injury was supposed to generate a 0.8-mm-deep lesion; however, the microscissor lesion depth is less than the 0.5-mm laceration injury shown in the left panel. Arrows point to images of the spinal cord following an acute microscissor injury (D and L panels on the left), compared to a 0.8-mm laceration injury (D and L panels on the right).

FIG. 3.

BMS and beam walk scores over time. (A) BMS scores demonstrate significant improvement in over-ground locomotion behavior over time within each of the groups (*p < 0.05; **p < 0.01), excluding the normal and sham groups. In the 0.5-mm group: scores at 14 and 42 dpi were significantly higher than the 1-dpi scores (p < 0.05; n = 8–13). In the 0.8-mm group: scores at 7, 14, 28, and 42 dpi were significantly greater than their 1-dpi scores (p < 0.01; n = 9–14); the 3-dpi scores were also significantly higher than the 1-dpi scores (p < 0.05). In the 1.1-mm group: scores at 7, 14, 28, and 42 dpi were significantly higher than the 1- and 3-dpi scores (p < .001); the 28- and 42-dpi scores were also significantly higher than the 7-dpi scores (p  < 0.05; n = 11–14). For the 1.4-mm group, the scores at 42, 28, and 14 dpi were significantly higher than the 1-, 3-, and 7-dpi scores (p < 0.01; n = 9–15); the 14-dpi scores were significantly higher than the 7- (p < 0.05), 3-, and 1-dpi scores (p < 0.01). For the 1.6-mm (Tx) group, the scores at 42, 28, 14, and 7 dpi were significantly higher than the 1- and 3-dpi scores (p < 0.01; n = 8–10); the 42-dpi scores were also significantly higher than the 7-dpi scores (p < 0.01). (B) Beam walk. The normal/sham group scored significantly higher than the 0.5-, 0.8-, and 1.1-mm groups throughout the course of the study (p < 0.01). The 0.5-mm group (*) was significantly lower than the normal/sham group and significantly higher than the 0.8- and 1.1-mm groups at all time points. The 0.8- and 1.1-mm groups were significantly lower (**) than the normal/sham and 0.5-mm group at all time points, but were never significantly different from each other (p < 0.01; n = 8–13).

FIG. 4.

Footprint analysis. (A) Stride length. The 0.8-mm group's stride lengths were significantly shorter (**) than the normal, sham (p < 0.01), and 0.5-mm groups (p < 0.05; n = 5–6). The 1.1-mm group's stride lengths were significantly shorter than those of all other groups (*p < 0.05). (B) Stance length. The 0.5-, 0.8-, and 1.1-mm groups' stances were significantly shorter compared to the normal group (#p < 0.05; n = 5–6). (C) Base of support (BOS). The 0.5-, 0.8- (#p < 0.05), and 1.1-mm (*p < 0.01) groups' BOS were significantly smaller compared to the normal and sham groups. (D) Regularity index (RI). The 0.8-mm group had significantly lower stepping regularity (%) than the normal/sham and 0.5-mm groups (#p < 0.05). The 1.1-mm group's average was significantly lower than that of the normal/sham group (***p < 0.001) and the 0.5-mm group (#p < 0.05). (E) Toe drags/step cycle. At 14, 28, and 42 dpi, the 0.5-mm group showed a significant reduction in the number of toe drags/cycle than at 1 dpi (**p < 0.01; n = 7–13). The 0.8-mm group at 7 (#p < 0.05), 14, 28, and 42 dpi (**p < 0.01) showed significant reductions in the average numbers of toe drags/cycle compared to 1 dpi (n = 7–11). The 28- and 42-dpi averages were also significantly lower than the number of toe drags/cycle at 7 dpi (*p < 0.05). The 1.1-mm group showed no significant change in the number of toe drags/cycle over the course of the study.

FIG. 5.

Hargreaves' noxious thermal stimulus. The average forepaw responses of the 1.4-mm group were significantly faster than those of the other groups (***p < 0.001; n = 31–48). The hindpaw response times of the 0.8-mm group were significantly less than the response times of the normal and 0.5-mm groups (*p < 0.05). The 1.1- and 1.4-mm groups' average response times were significantly less than those of the normal and 0.5-mm groups (***p < 0.001). The 1.4-mm group also responded significantly slower than the 0.8-mm group (#p = 0.003).

FIG. 6.

Transcranial magnetic motor-evoked potentials. The amplitudes of the tcMMEP recordings from the normal, 0.5-, and 0.8-mm groups were significantly greater than those of the 1.1- and 1.4-mm groups (**p < 0.01; n = 7–30). Representative average amplitude waveforms are shown for each injury level. The normal, 0.5, and 0.8 mm groups' amplitudes were not significantly different from each other.

FIG. 7.

Axonal tracing. The top panel shows a 0.5-mm lesioned mouse spinal cord; note the disruption of the CST fibers (BDA-labeled fibers are indicated by the arrows) at the rostral border of the lesion. No BDA labeling can be seen crossing the lesion. The bottom panel shows a 0.5-mm lesioned cord; note how the CTB labeling stops at the caudal edge of the lesion (arrows). Top to bottom: dorsal to ventral; left to right: rostral to caudal.