Comparative functional analysis of mice after local injection with botulinum neurotoxin A1, A2, A6, and B1 by catwalk analysis.

Botulinum neurotoxins (BoNTs) are potent neurotoxins and are the causative agent of botulism, as well as valuable pharmaceuticals. BoNTs are divided into seven serotypes that comprise over 40 reported subtypes. BoNT/A1 and BoNT/B1 are currently the only subtypes approved for pharmaceutical use in the USA. While several other BoNT subtypes including BoNT/A2 and/A6 have been proposed as promising pharmaceuticals, detailed characterization using in vivo assays are essential to determine their pharmaceutical characteristics compared to the currently used BoNT/A1 and/B1. Several methods for studying BoNTs in mice are being used, but no objective and quantitative assay for assessment of functional outcomes after injection has been described. Here we describe the use of CatWalk XT as a new analytical tool for the objective and quantitative analysis of the paralytic effect after local intramuscular injection of BoNT subtypes A1, A2, A6, and B1. Catwalk is a sophisticated gait and locomotion analysis system that quantitatively analyzes a rodent's paw print dimensions and footfall patterns while traversing a glass plate during unforced walk. Significant changes were observed in several gait parameters in mice after local intramuscular injection of all tested BoNT subtypes, however, no changes were observed in mice injected intraperitoneally with the same BoNTs. While a clear difference in time to peak paralysis was observed between BoNT/A1 and/B1, injection of all four toxins resulted in a deficit in the injected limb with the other limbs functionally compensating and with no qualitative differences between the four BoNT subtypes. The presented data demonstrate the utility of CatWalk as a tool for functional outcomes after local BoNT injection through its ability to collect large amounts of quantitative data and objectively analyze sensitive changes in static and dynamic gait parameters.