A Laser-Guided Spinal Cord Displacement Injury in Adult Mice

Abstract

Mouse models are unique for studying molecular mechanisms of neurotrauma because of the availability of various genetic modified mouse lines. For spinal cord injury (SCI) research, producing an accurate injury is essential, but it is challenging because of the small size of the mouse cord and the inconsistency of injury production. The Louisville Injury System Apparatus (LISA) impactor has been shown to produce precise contusive SCI in adult rats. Here, we examined whether the LISA impactor could be used to create accurate and graded contusive SCIs in mice. Adult C57BL/6 mice received a T10 laminectomy followed by 0.2, 0.5, and 0.8 mm displacement injuries, guided by a laser, from the dorsal surface of the spinal cord using the LISA impactor. Basso Mouse Scale (BMS), grid-walking, TreadScan, and Hargreaves analyses were performed for up to 6 weeks post-injury. All mice were euthanized at the 7th week, and the spinal cords were collected for histological analysis. Our results showed that the LISA impactor produced accurate and consistent contusive SCIs corresponding to mild, moderate, and severe injuries to the cord. The degree of injury severities could be readily determined by the BMS locomotor, grid-walking, and TreadScan gait assessments. The cutaneous hyperalgesia threshold was also significantly increased as the injury severity increased. The terminal lesion area and the spared white matter of the injury epicenter were strongly correlated with the injury severities. We conclude that the LISA device, guided by a laser, can produce reliable graded contusive SCIs in mice, resulting in severity-dependent behavioral and histopathological deficits.

Keywords: SCI; behavioral test; contusion; mouse; tissue displacement.

FIG. 1.

The parameters of injury produced by the Louisville Injury System Apparatus (LISA) impactor. (A) Tissue displacement. (B) Impact velocity. (C) Injury duration time. All values are mean ± SEM (0.2 mm, n = 10; 0.5 mm, n = 10; 0.8 mm, n = 10. ***p < 0.0001.)

FIG. 2.

Spontaneous locomotor recovery up to 6 weeks post-injury. (A) Basso Mouse Scale (BMS) locomotor scores showed a significant difference between the 0.2 mm and 0.5 mm (***p < 0.001), 0.2 mm and 0.8 mm (&&&p < 0.001), and 0.5 mm and 0.8 mm (###p < 0.001) injury groups. (B) The grid-walking test showed hindlimb foot drop errors among different groups. The foot drop errors were slightly increased in the 0.2 mm group and significantly increased in the 0.5 mm group compared with sham (###p < 0.001). There was a significant difference between the 0.5 mm and the 0.2 mm injury groups (***p < 0.001). Sham, n = 6; 0.2 mm, n = 10; 0.5 mm, n = 8; 0.8 mm, n = 4. All values are mean ± SEM.

FIG. 3.

Gait analysis using TreadScan. (A) Representative mouse image illustrating the selected gait parameters. (B) The average stance time decreased by 20% (p < 0.05) in the 0.2 mm and 48% (p < 0.001) in the 0.5 mm injury groups compared with sham. In the 0.5 mm group, the average stance time significantly decreased by 35% compared with the 0.2 mm group (p < 0.01). (C, D) The average rear-track width and average print length showed no difference in the 0.2 mm group, but significantly decreased by 47% and 46% in the 0.5 mm group as compared with sham group, respectively (p < 0.001). Compared with the 0.2 mm group, the average rear-track width and print length were significantly reduced by 43% (p < 0.001) and 35% (p < 0.01), respectively, in the 0.5 mm group. (E) The average toe spread was significantly decreased in the 0.2 mm group by 27% (p < 0.05) and in the 0.5 mm group by 59% (p < 0.001) compared with sham. In the 0.5 mm group, the average toe spread significantly decreased by 44% compared with the 0.2 mm group (p < 0.01). Sham, n = 6; 0.2 mm, n = 10; 0.5 mm, n = 8. Values are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Color image is available online at www.liebertpub.com/neu

FIG. 4.

Interlimb coordination using TreadScan. (A) Schematic representation of mouse interlimb coordination. (B) Average maximum lateral deviation (LatD max) showed no difference in the 0.2 mm group and significantly decreased by 29% (p < 0.001) in the 0.5 mm injury group compared with sham. The LatD max showed a significant decrease in the 0.5 mm group compared with the 0.2 mm group (25% decrease, p < 0.001). (C) The average minimum lateral deviation (LatD min) was significantly decreased in the 0.2 mm (25%) and 0.5 mm groups (63%) as compared with sham (p < 0.001). Compared with the 0.2 mm group, the LatD min significantly decreased by 51% in the 0.5 mm group (p < 0.001). (D) The minimum longitudinal deviation (LongD min) significantly increased in the 0.5 mm group by 76% (p < 0.001) and 66% (p < 0.01) compared with the 0.2 mm and sham groups, respectively. There was no statistically significant difference between the 0.2 mm and sham groups. Sham, n = 6; 0.2 mm, n = 10; 0.5 mm, n = 8. Values are mean ± SEM. **p < 0.01, ***p < 0.001.

FIG. 5.

Hargreaves test on the 3rd week post-SCI. The withdrawal latencies of the hindpaws were significantly increased in both the 0.2 and 0.5 mm injury groups compared with sham (p < 0.05, p < 0.01, respectively). However, there was no significant difference between the 0.2 mm and 0.5 mm injury groups. Sham, n = 6; 0.2 mm, n = 10; 0.5 mm, n = 8. Values are mean ± SEM. *p < 0.05, **p < 0.01.

FIG. 6.

Lesion area and spared white matter measured at the injury epicenter. (A) Cresyl violet eosin staining showed increased lesion areas as the injury severity increased (upper row). Luxol Fast Blue staining demonstrated decreased spared myelin areas as the injury severity increased (lower row). (B) Comparison of percent lesion area among different groups. (C) Comparison of percent spared white matter (WM) area among different groups. Sham, n = 6; 0.2 mm, n = 10; 0.5 mm, n = 8; 0.8 mm, n = 4. All values are mean ± SEM. ***p < 0.001.

FIG. 7.

Correlations of displacement with behavioral and histological parameters at 6 weeks post-spinal cord injury (SCI). Correlation between displacement and (A) Basso Mouse Scale (BMS) score, (B) foot drop errors, (C) average stance time, (D) average minimum lateral deviation (latD min), (E) percentage of lesion area, and (F) spared white matter area. 0.2 mm, n = 10; 0.5 mm, n = 8; 0.8 mm, n = 4.