Physiological Effects of Botulinum Neurotoxin A on in vitro Contractile Properties of Skeletal Muscle

Abstract

Clinical treatment with the skeletal muscle relaxant botulinum neurotoxin-A (BoNT-A) reduces excessive muscle tone in a dose-dependent manner in the wrist, fingers, and elbows of patients who experience spasticity after suffering a stroke. However, to date neither in vitro nor in vivo experiments have directly tested effects of mechanical stretch in muscles treated with BoNT-A. PURPOSE: To investigate the potential contribution to muscle damage incurred by repeated active stretches in skeletal muscle following BoNT-A treatment using in vitro METHODS. We hypothesized that, compared to saline-injected controls, BoNT-A treatment of extensor digitorum longus (edL) muscle would both reduce maximal force (Po) and mitigate the extent of mechanical damage (force deficit, Fd) incurred by repeated stretches of the contracting muscle. METHODS: To avoid inducing mechanical damage of the EDL, 10 uL BoNT-A (70 U/kg) was injected into the anterior tibialis (AT) of female CD-1 mice (n=20) weighing at least 28 g. At 24 and 48 hr post-injection, a digit abduction score (DAS) of 4 (total flaccidity) was observed in all mice, indicating diffusion of the toxin from the AT to EDL and digit abductor muscles. At 48 hr post-injection, EDL muscles were surgically removed, placed in a physiologic bath, connected to a force-displacement transducer interfaced with a physiograph, and electrical stimulation was applied. A whole-muscle assay system measured and recorded a number of contractile characteristics, and Fd was calculated. Muscle length and weight were recorded to allow normalization of force to muscle cross-sectional area. RESULTS: Initial data indicated mean normalized force reduction of 20.3% in BoNT-A-exposed EDL muscle at all tested frequencies of stimulation (30, 50, 80, 100, 120, 150 Hz). Force was also reduced an average of 21.9% at each point of the stretch-activation protocol. No significant (p< 0.05) difference was observed for Fd, denned by the equation Fd = (Po before stretch – Po after stretch/ Po before stretch). However variability was significant at all data points. CONCLUSION: BoNT-A treatment may mitigate the extent of tissue damage incurred during vigorous active stretching, which may be important in the management of spasticity in a variety of clinical populations. Because the in vitro assay likely stimulates contraction via direct stimulation of the sarcolemma, thus bypassing the effect of toxin on the nerve, further study using in vivo METHODS is warranted.