Introduction Botulinum toxin is frequently administered serially to maintain therapeutic muscle paralysis, but repeated dose effects on muscle function are largely unknown. This study characterized the muscle response to 2 onabotulinum toxin (BoNT) injections separated by 3 months. Methods Animal subjects received a single toxin injection (n=8), 2 BoNT injections separated by 3 months (n= 14), or 1 BoNT and 1 saline injection separated by 3 months (n=8). Results The functional effect of 2 serial injections was exponentially greater than the effect of a single injection. While both groups treated with a single BoNT injection had decreased torque in the injected leg by about 50% relative to contralateral legs, the double BoNT injected group had decreased torque by over 95% relative to the pre-injection level. Both single and double BoNT injections produced clear signs of fiber-type grouping. Discussion These experiments demonstrate a disproportionately increased effect of a second BoNT injection.

The effect of physical manipulation on the outcome of neurotoxin (NT) injection was studied in a rat tibialis anterior (TA) model system where dorsiflexion torque could be measured precisely. After determination of initial torque, all rats received a one-time botulinum toxin A (BTX-A) injection (dose 6.0 units/kg in a volume of 100μL) into the TA midbelly. Four experimental groups were studied: one group was subjected to BTX-A injection alone (BTX-A only, n=8), one was subjected to BTX-A injection followed immediately by 10 isometric contractions (ISO; n=9), and the third was subjected to BTX-A followed immediately by 10 muscle passive stretch/release cycles (PS; n=10). After 1 month, maximum dorsiflexion torque of the injected and contralateral legs was determined followed by quantification of TA fiber area. Post-injection torque was significantly reduced by around 80% in all NT-treated extremities 1 month after injection (p<0.05). While all NT-treated extremities demonstrated a significant torque decrease relative to their pre-injection levels, ISO and PS groups demonstrated significantly lower torques compared with the BTX-A only group which received no physical manipulation (p<0.05) indicating greater efficacy. Perhaps even more surprising was that the ISO and PS groups both demonstrated a significantly smaller contralateral effect compared with the BTX-A only group that received no manipulation (p<0.05) indicating a decreased systemic-effect. Muscle fiber size generally correlated with dorsiflexion torque. These data demonstrate that both neuromuscular activity (seen in the ISO group) and muscle movement (seen in the PS group) increased the efficacy of BTX-A and decreased the systemic side effects.Neurotoxins (NT) that block neuromuscular transmission are used to treat muscular spasticity secondary to cerebral palsy, stroke, and head injury. 1-3 Therapeutic effects of NT treatment on spasticity include improved muscle function, facilitation of the effects of physical therapy, and delayed surgery. However, there are immediate and long-term negative side effects related to NT treatments, including systemic weakness and resistance to the toxin. [4][5][6]

The data demonstrate that, after a single BT-A injection, although gross muscle morphology recovered over a 12-month time period, loss of contractile function did not recover. Muscle Nerve 57: 435-441, 2018.

Introduction In this study we provide global transcriptomic profiling and analysis of botulinum toxin A (BoNT-A)–treated muscle over a 1-year period. Methods Microarray analysis was performed on rat tibialis anterior muscles from 4 groups (n =4/group) at 1, 4, 12, and 52 weeks after BoNT-A injection compared with saline-injected rats at 12 weeks. Results Dramatic transcriptional adaptation occurred at 1 week with a paradoxical increase in expression of slow and immature isoforms, activation of genes in competing pathways of repair and atrophy, impaired mitochondrial biogenesis, and increased metal ion imbalance. Adaptations of the basal lamina and fibrillar extracellular matrix (ECM) occurred by 4 weeks. The muscle transcriptome returned to its unperturbed state 12 weeks after injection. Conclusion Acute transcriptional adaptations resemble denervated muscle with some subtle differences, but resolved more quickly compared with denervation. Overall, gene expression, across time, correlates with the generally accepted BoNT-A time course and suggests that the direct action of BoNT-A in skeletal muscle is relatively rapid.

In the present study we evaluated the morphological aspect and changes in the area and incidence of muscle fiber types of long-term regenerated rat tibialis anterior (TA) muscle previously submitted to periodic contusions. Animals received eight consecutive traumas: one trauma per week, for eight weeks, and were evaluated one (N = 8) and four (N = 9) months after the last contusion. Serial cross-sections were evaluated by toluidine blue staining, acid phosphatase and myosin ATPase reactions. The weight of injured muscles was decreased compared to the contralateral intact one (one month: 0.77 ± 0.15 vs 0.91 ± 0.09 g, P = 0.03; four months: 0.79 ± 0.14 vs 1.02 ± 0.07 g, P = 0.0007, respectively) and showed abundant presence of split fibers and fibers with centralized nuclei, mainly in the deep portion. Damaged muscles presented a higher incidence of undifferentiated fibers when compared to the intact one (one month: 3.4 ± 2.1 vs 0.5 ± 0.3%, P = 0.006; four months: 2.3 ± 1.6 vs 0.3 ± 0.3%, P = 0.007, respectively). Injured TA evaluated one month later showed a decreased area of muscle fibers when compared to the intact one (P = 0.003). Thus, we conclude that: a) muscle fibers were damaged mainly in the deep portion, probably because they were compressed against the tibia; b) periodic contusions in the TA muscle did not change the percentage of type I and II muscle fibers; c) periodically injured TA muscles took four months to reach a muscle fiber area similar to that of the intact muscle.