Short- and long-term effects of botulinum toxin A injection into the masseter muscle of rats: immunohistochemical and ultrastructural study

Abstract

Introduction: In the head and neck area of human, botulinum toxin A (BTX) injections into the masticatory muscle are used for several indications such as trismus, bruxism, clenching, migraine, temporomandibular joint disorders or masseter hypertrophy. For more evidence-based applications of BTX injection into the masseter muscle, well-designed randomized clinical trials and basic studies about the responses and the recovery of muscle tissues for a long time after BTX injection are more needed. The purpose of this animal model study was to investigate the atrophy and recovery of masseter muscle following bilateral BTX injection using two different dose of BTX in the short and long term. Materials and Methods: Mature male rats (n=30) were randomized into 3 groups. Rats of each group received saline (control group), 5 units of BTX (5 U BTX group) or 10 units of BTX (10 U BTX group) into the each masseter muscle bilaterally. The amount of daily food intake and body weight was measured until 12 weeks after the injection. The thickness of the masseter muscle was also measured using ultrasonography weekly. A half of animals were sacrificed at 2 weeks after the injection. The other animals were sacrificed at 12 weeks after injection. Specimens of the injected masseter muscle were processed for immunohistochemical determination of myosin heavy chain (MyHC) composition and ultrastructural analysis. Results: There was no statistically significant difference among groups in the body weight and the amount of food consumption after 2 weeks. The maximal atrophy of masseter muscle after BTX injection was found at 6 weeks. A longer time than 12 weeks was required for BTX injected masseter muscle to recover completely. There was no significant difference in muscle thickness according to the injected dose of BTX. The expression level of MyHC type I and type IIa in the BTX treated masseter muscles was significantly increased at 2 weeks. However, the expression level of MyHC type I and type IIa was not different from that of the control group after 12 weeks regardless of the injected dose of BTX. Abnormalities of myofilaments were observed in both BTX treatment groups at 12 weeks. The mitochondrial swelling and alteration of cristae were prominent in the 10 U BTX group after 12 weeks. Conclusions: BTX injection led to changes in the MyHC composition in the short term, but the MyHC composition was almost recovered after 12 weeks. BTX injection to the masseter muscle of rats with a high dosage might be associated with increased mitochondrial susceptibility to apoptosis as a delayed phenomenon.