Passive Mechanical Properties of Skeletal Muscle Following Botulinum Toxin Type A Injection Demonstrate Sexual Dimorphisms

The purpose of this study was to investigate passive mechanical of skeletal muscle following Botulinum Toxin Type A (BoNT-A) injection. We hypothesize that we will observe higher passive stresses within injected muscle that increase with time. 20 male and 20 female C57BL6 mice aged 6 weeks were injected with 1 unit/kg of BoNT-A in a single lateral gastrocnemius (LG), contralateral non-injected limb was the control. At 2-, 4-, 8-, & 12-weeks (2W, 4W, 8W, 12W) post-injection (n= 5m, 5f at each time point) bilateral LG muscles were dissected and mechanically tested. Muscle passive forces were normalized to physiological cross-sectional area (PCSA) calculated from muscle mass.[3] The normalized stress-strain curves and mass were compared across both injected and non-injected samples. To check for sexual dimorphisms, we compared male to female samples. All data is given in average ± SD. Stress vs. strain curves were compared using non-linear least squares regression comprising of a sum of squares F test. Muscles injected with BoNT-A demonstrated lower mass across all timepoints. A two-way ANOVA (sex and time points) found no significant difference between the sexes but a significance in time since injection (p = 0.006). Muscle mass decreased until 4W where the muscle mass averaged 81%± 8 of the non-injected limbs mass. Mass began to recover between 4W and 8W and at 12W recovered to 92%± 7. Indicating muscle mass does not fully recover in the first twelve weeks following a single BoNT-A injection, though active effects are thought to wear off around 8W. Comparing the stress-strain profiles male and female groups to one another we observed similar trends up until 8W within male and female, with non-significant differences between the sexes and control vs. BoNT-A injected muscle. However at 8W and 12W, we see a divergence that showed the stresses of the females samples being greater than their male cohorts. By 12W both the female contralateral control and BoNT-A groups stresses were larger than the male counterparts (34.6% & 48.1% respectively). But within each sex, only at 12 weeks was the BoNT-A injected muscle stiffer than the control. In summary, we observed significant muscle atrophy that did not recover by 12W post BoNT-A injection. We interestingly observed significant differences between male and female muscle properties in both the contralateral control and BoNT-A injected samples starting at 8W post-injection, with BoNT-A muscles being stiffer in both sexes at 12W. The sources of these sexual dimorphisms are unclear and should be the focus of future studies in addition to exploring the longer-term effects of BoNT-A (6 months to 1 year). References: [1] Binder-Markey et al. (2021), Front Neurol 12; [2] Binder-Markey et al. (2021), J Biomech 129; [3] Ward et al. (2020), Front Physiol 11; [4] Dutta et al. (2016); Funding for this work was provided by an Individual Researcher Biomedical Award from The Hartwell Foundation and Foundation for Physical Therapy Research This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.