Combination of Defined CatWalk Gait Parameters for Predictive Locomotion Recovery in Experimental Spinal Cord Injury Rat Models

Abstract

In many preclinical spinal cord injury (SCI) studies, assessment of locomotion recovery is key to understanding the effectiveness of the experimental intervention. In such rat SCI studies, the most basic locomotor recovery scoring system is a subjective observation of animals freely roaming in an open field, the Basso Beattie Bresnahan (BBB) score. In comparison, CatWalk is an automated gait analysis system, providing further parameter specifications. Although together the CatWalk parameters encompass gait, studies consistently report single parameters, which differ in significance from other behavioral assessments. Therefore, we believe no single parameter produced by the CatWalk can represent the fully-coordinated motion of gait. Typically, other locomotor assessments, such as the BBB score, combine several locomotor characteristics into a representative score. For this reason, we ranked the most distinctive CatWalk parameters between uninjured and SC injured rats. Subsequently, we combined nine of the topmost parameters into an SCI gait index score based on linear discriminant analysis (LDA). The resulting combination was applied to assess gait recovery in SCI experiments comprising of three thoracic contusions, a thoracic dorsal hemisection, and a cervical dorsal column lesion model. For thoracic lesions, our unbiased machine learning model revealed gait differences in lesion type and severity. In some instances, our LDA was found to be more sensitive in differentiating recovery than the BBB score alone. We believe the newly developed gait parameter combination presented here should be used in CatWalk gait recovery work with preclinical thoracic rat SCI models.

Keywords: CatWalk; gait parameter; linear discriminant analysis; locomotion recovery; preclinical development; spinal cord injury.

Graphical abstract

Figure 1.

An example of CatWalk image captured from below an injured rat. The paws in contact with the glass walkway are indicated with the colored boxes (magenta: right hindpaw; yellow: left forepaw).

Figure 2.

Scatter plot of several gait parameters obtained from the individual run data from Study 1. The injured (red) and uninjured (dark gray) animals are denoted by color here. The different shapes in each group represent the different individuals. a, Forepaw swing time and forepaw stride length. b, Forepaw duty cycle and hindpaw BOS. c, Forepaw BOS and number of paw support = [Support_One + 2 × Support_Diagonal + 2 × Support_Girdle + 2 × Support_Lateral + 3 × Support_Three + 4 × Support_Four]/100.

Figure 3.

(a, b) Combinations of gait parameters (p_LDA) and BBB scores for each group in Study 1 without treatment (mean ± SEM). (c, d) Treated and untreated pLDA and BBB scores for moderate contusions. (e, f) Treated and untreated pLDA and BBB scores for moderate-severe contusions. UI: uninjured; Veh.Mod.: vehicle moderate SCI; Veh.Mod-Sev.: vehicle moderate-severe SCI; Exp.Mod.: experimental moderate SCI; Exp.Mod-Sev.: experimental moderate severe SCI; Exp.Mod-Sev.: experimental moderate severe SCI; *p < 0.05; **p < 0.01; ####p_{compared with UI} < 0.0001.

Figure 4.

(a, b) Combinations of gait parameters (p_LDA) and BBB scores for vehicle and young groups in Studies 2 and 3, respectively (mean ± SEM). (c-d) The Two-way ANOVA in Study 2 shows significant effects on pLDA of both groups (p  = 0.037) and time (p < 0.00001), without significant interaction. In the RI-controlled BBB score (Study 2), significant effects of both groups (p  = 0.003) and time (p < 0.00001) were observed, as well as significant interaction (p  = 0.049). (e-f) The Two-way ANOVA in Study 3 shows significant effect on pLDA of both group (p < 0.00001) and time (p < 0.00001), with significant interaction (p < 0.001). In the BBB score (Study 3), significant effect of time (p < 0.00001) was observed, but no significant effect of group and interaction; *p < 0.05, ***p < 0.001, ****p < 0.0001 Prog.: progesterone; Veh.: vehicle.

Figure 5.

a, Combinations of gait parameters (p_LDA) of uninjured rats and vehicle SCI rats in Study 2 (S2: Th8 contusion), Study 4 (S4: Th8/9 dorsal hemisection), and Study 5 (S5: C4 bilateral dorsal column lesion). b, Combinations of gait parameters (p_LDA) of vehicle SCI rats in Studies 2 and 4. c, BBB scores of vehicle SCI rats in Studies 2 and 4; mean ± SEM; ***p < 0.001, ****p < 0.0001, ###p_{compared with UI} < 0.001, ####p_{compared with UI} < 0.0001; UI: uninjured; Veh.: vehicle; Cont.: contusion; DH.: dorsal hemisection.

Figure 6.

Schematic representation of the p_LDA scores for varying lesion types and severities.