Blocking neuromuscular junctions to impair muscle overactivity enhances neurological heterotopic ossifications (NHO) development in a mouse model of spinal cord injury

Introduction/Background To ascertain a role for muscle spasticity in the development of NHO, by blocking neuromuscular signalling using botulinum toxin during spinal cord injury-induced NHO formation in a murine model. Material and method Spinal Cord Injury (SCI) was performed on 5–6-week-old C57BL/6 mice. To promote NHO development, cardiotoxin (CDTX) dose injections were administered at the time of the surgery to the right and left hamstring muscles. Botulinum toxin A (BTA) injections were administered at the site of NHO formation in the right hamstrings to block neuromuscular signalling, and mock equivalent volume of PBS was injected in the contralateral site 4 days prior to SCI, and weekly for 3 weeks. Twenty-one days post SCI, quantitative assessment of NHO volume and density within each hamstrings muscle were performed using Micro-computed tomography scan. Results μCTscan analysis revealed the development of NHO in 100% of mice in both hamstrings. NHO volume average was 1.6 mm 3 (± 1.0) in the left hamstrings injected with PBS in contrast to 3.3 mm 3 (± 1.4) in the right hamstrings with BTA. This doubling of NHO volume in BTA-treated hamstrings was statistically significant (Wilcoxon matched-pairs signed rank test, P  = 0.013). Furthermore, NHO were more compact in muscles injected with BTA with a density of 1244.6 mg/cm 3 (± 10.4) and was significantly denser compared to NHO in the PBS-treated contralateral side density of 1231.2 mg/cm 3 (± 20.1) (Wilcoxon matched-pairs signed rank test, P  = 0.027). Conclusion In our murine model of SCI-induced NHO, we demonstrated a doubling of the NHO volume, and increased density burden when treated locally with BTA. These results demonstrate that local administration of BTA exacerbates NHO development leading to an overall increase in bone formation. The outcome suggests that in contrast to risk spasticity, resulting from defective neuromuscular signalling, may be an inhibitor of local NHO development.