ABSTRACT

Introduction: Botulinum neurotoxin A (BoNTA) has long been used as a therapeutic agent and has been widely accepted as a cosmetic agent in recent years. It can inhibit function and induce structural changes in skeletal muscle. Methods: Specimens of fresh dissected human masseter muscle were used to observe the ultrastructural changes that occurred at 6 and 12 months following BoNTA injection. Results: The findings observed were muscle fiber distortion, sarcomere shortening, mitochondrial vacuolar degeneration, glycogen accumulation, and H and M band disruption in the triad of tubules. At 12 months after injection, there was still evidence of degenerative changes in muscle ultrastructure, whereas most organelles exhibited a normal structure. Discussion: Profound ultrastructural and organelle disfiguring changes were observed after BoNTA injection into human masseter muscle. Most changes were transient, however, and were resolved by 12 months after injection.

Keywords

Botulinum toxin A, human masseter muscle, ultrastructural changes, electron microscope,  mitochondria, sarcomere

Botulinum neurotoxin A (BoNTA) was first used therapeutically by local injection in 1980.1 The mechanism of action of BoNTA is inhibition of the synaptic release of acetylcholine, thereby blocking neuromuscular transmission.2 A study of biochemical changes in neuromuscular junctions after BoNTA injection illustrated its mechanism of action.3 General histologic and ultrastructural changes in muscle tissue after treatment have also been studied.4鈥�6 Because of difficulties with human tissue sampling, ultrastructural changes in human striated muscles after BoNTA injection have not been studied. This study examines specimens of human masseter muscle by electron microscopy (EM) to assess their ultrastructural changes at different times after BoNTA injection.

MATERIALS AND METHODS

This study was approved by the ethics committee of Weifang Medical University. After informed consent was obtained, 9 Asian women with bilateral masseter muscle hypertrophy (who planned to undergo partial removal of the masseter muscle to narrow the lower face at the Plastic Surgery Hospital of Weifang Medical University) were selected as study participants. The ages of patients ranged from 20 to 30 years. Three patients received local injections of BoNTA in the masseter muscles bilaterally 6 months prior to surgery, and 3 patients received injections 12 months prior to the surgery. Three patients who did not receive local injections of BoNTA before masseter muscle removal were included as controls. The masseter muscles of all 9 patients were partially removed bilaterally, and the specimens were immediately pretreated in the operating room. In total, 18 specimens were processed for EM. The BoNTA injected in the patients was Hengli BoNTA (Lanzhou Institute of Biological Products, Lanzhou Gansu Province, People鈥檚 Republic of China); a dose of 50 units was used for the muscle on each side.

An appropriate amount of masseter muscle, as judged by the patients鈥� surgeons, was removed from the patients via an intraoral incision under general anesthesia. A 1-mm2 fragment of muscle tissue was extracted from the fiber-rich parts of the severed masseter muscle. The tissue fragment was immediately submerged in 2.5% glutaraldehyde fixation fluid and preserved at 4 8C. The specimens were processed for EM by standard methods.7

An HL 7500 transmission electron microscope (Hitachi, Chiyoda, Japan) was used. Analysis of all specimens was performed by the same researcher.

RESULTS

Facial Contour

The width of the lower face started to decrease 2鈥�3 weeks after injection, and, by 4鈥�6 months postinjection, the width had decreased by approximately 10%, as measured by CT scan. The volume of the muscle then began slowly to increase, and, by 12 months postinjection, the width was 5% less than that before injection.

Muscle Fibers and Cellular Structure

The sarcomeres in the patients without BoNTA injection were aligned well and were of consistent length. In contrast, the arrangement of masseter muscle fibers at 6 months after BoNTA injection was very distorted, the sarcomeres were shortened, and the change in ultrastructure was unequivocal. The length of the sarcomeres at 12 months after injection was restored to that of the control group, but the arrangement was still relatively distorted, and the ultrastructure was obviously different from that in the control group (Fig. 1).

FIGURE 1. Changes in the muscle fibers of the masseter muscle and the cellular structure at different time points. (A) Muscle fiber arrangement without BoNTA injection. (B) At 6 months after BoNTA injection, the arrangement was disordered, the sarcomeres were shortened, and the change in ultrastructure was obvious. (C) At 12 months after injection, the length of the sarcomeres was restored to the level of the control group, but the arrangement was still relatively disordered. BoNTA, botulinum toxin A.

Mitochondria

It was difficult to observe normal mitochondrial structure in human masseter muscles at 6 months after BoNTA injection because of extensive vacuolar degeneration, and some vacuoles exhibited glycogen accumulation. Myelin sheath-like changes and cristae breakage were evident in many mitochondria. (Figs. 2, 3). At 12 months after BoNTA injection, many mitochondria had normal morphology, clear internal structure, and larger than normal volume. Many mitochondria still exhibited vacuolar degeneration, although the size of the vacuoles was smaller than that at 6 months after injection. Mitochondria with myelin sheath-like changes were still present, but there were fewer of these than at 6 months after injection (Fig. 4).

FIGURE 2. Changes in typical mitochondrial morphology at different time points after intramuscular injection of BoNTA. (A) Mitochondria without BoNTA injection. (B) At 6 months after BoNTA injection, extensive vacuolar degeneration was present (white arrow), and in some vacuoles there was glycogen accumulation (black arrow). (C) At 12 months after BoNTA injection, the most mitochondria were larger than normal in size and demonstrated normal internal structure (arrow). BoNTA, botulinum toxin A.

FIGURE 3. At 6 months postinjection, all the observed mitochondria had abnormal morphology. Shown here are myelin sheath-like changes (A; black arrow), vacuolar degeneration and glycogen accumulation (B), and cristae breakage (C; arrow). G, glycogen accumulation; V, vacuolar degeneration.

FIGURE 4. (A) Many mitochondria with normal morphology, clear internal structure, and larger than normal volume were observed at 12 months after injection (arrow). Some mitochondria still exhibited myelin sheath-like changes (B; arrow) and vacuolar degeneration (C), but the number of mitochondria with abnormal morphology was significantly reduced compared with that at 6 months after injection. V, vacuolar degeneration.

Triad of Tubules

The structure of the triad of tubules of masseter muscles without BoNTA injection was clear, with the H band and the M band in full alignment. At 6 months after BoNTA injection, the triad had lost its normal morphology and the structure was abnormal, with the H and M bands arranged in a disorderly manner. At 12 months after BoNTA injection, the triad structure was largely restored to the preinjection morphology, although the H and M bands were still not completely aligned; the Z-line was mostly normal, and its basic structure could be identified (Fig. 5).

FIGURE 5. Changes in the triad of tubules at different times after BoNTA injection. (A) The structure of the triad of tubules without BoNTA injection was clear (arrow). (B) At 6 months after BoNTA injection, the triad had lost its normal morphology (arrow). (C) At 12 months after injection, the triad structure was restored to the preinjection morphology (arrow). BoNTA, botulinum toxin A.

DISCUSSION

We investigated a total of 18 specimens from 9 patients to observe the effect of BoNTA on the ultrastructure of human masseter muscles and found that the ultrastructure of this muscle was substantially changed after BoNTA injection. These changes consisted of rearrangement of the sequence of muscle fibers (similar to the changes following denervation by nerve section)8,9 and changes to the structures of the nuclei and mitochondria. Vacuolar degeneration of mitochondria was consistent with the mitochondrial ultrastructural changes in patients with mitochondrial encephalomyopathies,10 and increased glycogen accumulation was consistent with the mitochondrial ultrastructural changes in patients with mitochondrial myopathy.11 We also observed the phenomenon of glycogen accumulation that we had seen in patients with myasthenia gravis in our other unpublished studies.

 

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