Apoptotic action of botulinum toxin on masseter muscle in rats: early and late changes in the expression of molecular markers.

The purpose of this study was to compare the early or late expression levels of p65, Bcl-2, and type II myosin and the frequency of TUNEL-positive nuclei in the rat masseter muscle after injection of different concentrations of botulinum toxin-A (BTX-A). We injected either 5 U or 10 U of BTX-A into both masseter muscles of the rat. As a control group, the same volume of saline was injected. After 2 or 12 weeks, the animals were sacrificed. Subsequently, a biopsy and immunohistochemical staining of the samples were performed using a p65, Bcl-2, or type II myosin antibody. Additionally, a TUNEL assay and transmission electron microscopic analysis were performed. The expression of p65, Bcl-2, and type II myosin increased significantly with increasing concentrations of BTX-A at 2 weeks after BTX-A injection (P < 0.05). The number of TUNEL-positive nuclei was also significantly increased in the BTX-A-treated groups in comparison to the saline-treated control at 2 weeks after BTX-A injection (P < 0.05). Elevated expression of Bcl-2 was also observed in 10-unit BTX-A-treated group at 12 weeks after injection (P < 0.05). At 12 weeks after injection, the number of enlarged mitochondria was increased, and many mitochondria displayed aberrations in cristae morphology after BTX-A injection. In conclusion, BTX-A injection into the masseter muscle increased the expression level of p65, Bcl-2, and type II myosin at an early stage. The morphological changes of mitochondria were more evident at 12 weeks after injection.