Early molecular response and microanatomical changes in the masseter muscle and mandibular head after botulinum toxin intervention in adult mice

Publication date: Available online 28 December 2017
Source:Annals of Anatomy - Anatomischer Anzeiger
Author(s): Julián Balanta-Melo, Viviana Toro-Ibacache, Maria Angélica Torres-Quintana, Kornelius Kupczik, Carolina Vega, Camilo Morales, Nadia Hernández-Moya, Manuel Arias-Calderón, Carolina Beato, Sonja Buvinic
BackgroundMasseter muscle paralysis induced by botulinum toxin type A (BoNTA) evokes subchondral bone loss in mandibular heads of adult rats and growing mice after 4 weeks. However, the primary cellular and molecular events leading to altered bone remodeling remain unexplored. Thus, the aim of the current work has been to assess the molecular response that precedes the early microanatomical changes in the masseter muscle and subchondral bone of the mandibular head in adult mice after BoNTA intervention.MethodsA pre-clinical in vivo study was performed by a single intramuscular injection of 0.2U BoNTA in the right masseter (experimental) of adult BALB/c mice. The contralateral masseter was injected with vehicle (control). Changes in mRNA levels of molecular markers of bone loss or muscle atrophy/regeneration were addressed by qPCR at day 2 or 7, respectively. mRNA levels of receptor activator of nuclear factor-κB ligand (RANKL) was assessed in mandibular heads, whilst mRNA levels of Atrogin-1/MAFbx, MuRF-1 and Myogenin were addressed in masseter muscles. In order to identify the early microanatomical changes at day 14, fiber diameters in transversal sections of masseter muscles were quantified, and histomorphometric analysis was used to determine the bone per tissue area and the trabecular thickness of subchondral bone of the mandibular heads.ResultsAn increase of up to 4-fold in RANKL mRNA levels were detected in mandibular heads of the BoNTA-injected sides as early as 2 d after intervention. Moreover, a 4-6 fold increase in Atrogin-1/MAFbx and MuRF-1 and an up to 25 fold increase in myogenin mRNA level were detected in masseter muscles 7 d after BoNTA injections. Masseter muscle mass, as well as individual muscle fiber diameter, were significantly reduced in BoNTA-injected side after 14 d post-intervention. At the same time, in the mandibular heads from the treated side, the subchondral bone loss was evinced by a significant reduction in bone per tissue area (- 40%) and trabecular thickness (- 55%).ConclusionsOur results show that masseter muscle paralysis induced by BoNTA leads to significant microanatomical changes by day 14, preceded by molecular changes as early as 2 d in bone, and 7 d in muscle. Therefore, masseter muscle atrophy and subchondral bone loss detected at 14 d are preceded by molecular responses that occur during the first week after BoNTA intervention.



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