Do repetitive botulinum neurotoxin injections induce muscle fibrosis? Sonographic observation of the masseter muscle

In the esthetic field, the masseter muscle is commonly targeted by botulinum neurotoxin for facial contouring. However, multiple botulinum neurotoxin injections have been reported to cause muscle fibrosis. Ultrasonography can be useful for clinical consideration in such cases.

the volume fraction of fibrillar tissue and an increase in nonfibrillar tissue, accompanied by a loss of the linear muscle tissue structure.Furthermore, gene expression analysis showed significant upregulation of several genes in the botulinum neurotoxin-injected legs, whereas only one gene was significantly downregulated.These findings led to the conclusion that high-dose intramuscular botulinum neurotoxin injections can cause microstructural damage to the muscle tissues.
We believe that repetitive injections of botulinum neurotoxin can lead to a reduction in masseter muscle volume, which is equivalent to the effect of high doses of botulinum neurotoxin.In addition, on ultrasonography, the masseter muscle frequently appeared hyperechoic.In this study, we refer to the observed muscle fiber loss with an increase in nonfibrous tissue as "fibrosis." The clinical implications of these findings in patients who have undergone repeated botulinum neurotoxin injections into the masseter muscle should be carefully considered.This study is significant because it provides clinicians with guidance on the use of ultrasonography-guided injection for the masseter muscle, which can help avoid adverse effects such as partial fibrosis.This also indicates that botulinum neurotoxin injections may have no effect on the fully fiberized muscle belly.

| MATERIAL S AND ME THODS
The study was conducted in accordance with the principles outlined Additionally, we were able to identify the deep inferior tendon as a distinct, linear-shaped, and brighter (hyperechoic) structure in the ultrasound image.These identifications were performed by two dermatologists.

| RE SULTS
The results of this study showed that in repeatedly treated patients, muscle fibrosis commonly occurred in the posterior and F I G U R E 1 The schematic images depicting muscle cross sections.The left image (A) demonstrates the control group, while the right image (B) demonstrates the muscle injected with botulinum neurotoxin.The injected muscle appears to have smaller muscle fibers compared to the control muscle, suggesting muscle fibrosis.

V I D E O 1
The video image demonstrates the contraction and relaxation of the masseter muscle with fibrotic tissue (hypoechoic part) between the muscle fibers.
deep portions of the masseter muscle.This fibrosis was observed to be affected by the deep inferior tendon of the masseter muscles.However, in some cases, fibrosis was observed throughout the muscle, including the superficial, deep, or both areas.The repeated injections were administered to address the preferences of Asian individuals who tend to favor a narrower bigonial length, resulting in a narrower jaw in frontal view.Among the 540 female patients, nine patients developed fibrotic tissues, whereas no male patients showed fibrotic images after multiple botulinum neurotoxin injections.Five hundred and eighty-three patients had experienced sonography examinations; however, only nine instances with sonographically identified fibrosis were included in this research.This leads to an incidence rate of 1.5%.

| Case 1
A 33-year-old woman received repetitive botulinum neurotoxin injections every 6 months for 1 year over a period of 5 years.
Ultrasonography was performed to observe the patient's condition (Figure 2A, transverse view).The practitioner advised the patient to receive injections every 6 months.As shown in Figure 2A, fibrosis was observed in the posterior part of the masseter muscle, which may have been caused by multiple deep and superficial injections, including those in the deep inferior part.In many cases, the anterior part did not appear to have been injected as much, resulting in a sharp contrast compared with the posterior part.

| Case 3
A 71-year-old woman received repetitive botulinum neurotoxin injections every 5 months to 1 year for a period of 10 years.
Ultrasonography was performed to assess the patient's condition (Figure 3; transverse view (A) and longitudinal view (B)).In Figure 3A, the transverse view of the masseter muscle reveals fibrosis in both the superficial and deep layers of the muscle.As shown in Figure 3B, the longitudinal view of the masseter muscle showed hyperechoic areas in both the superficial and deep layers.

| Case 4
Ultrasonographic analysis was conducted in a 56-year-old woman who received repetitive botulinum neurotoxin injections for 8 years at intervals of 4 months to 1.6 years.The results, presented in Figure 4A, show a transverse view of partial fibrosis at the middle of the masseter muscle.The deep inferior tendon was visible and divided into the superficial and deep muscles.Additionally, homogeneous echogenicity was observed posteriorly, which could be attributed to a partial parotid gland.

| Case 5
A 60-year-old female patient who received repetitive botulinum neurotoxin injection for a period of 3 years at intervals of 7 months to 1 year was examined using ultrasonography, which revealed partial fibrosis of the middle portion of the masseter muscle with a hyperechoic appearance (Figure 4B).The deep inferior tendon was visible and appeared to divide the superficial and deep muscles.

| Case 6
A female patient, aged 64 years, who received repetitive botulinum neurotoxin injections for 5 years, with an interval of 9 months to 1.2 year, was examined using ultrasonography (Figure 5A, transverse view).Ultrasonographic analysis revealed partial fibrosis with a hyperechoic appearance in the deep belly of the masseter muscle.However, botulinum neurotoxins did not affect the superficial masseter.The deep inferior tendon was identified by dividing the superficial and deep muscle layers.

| Case 7
A female patient, aged 69 years, who received repetitive botulinum neurotoxin injections for 4 years at intervals of 10 months to 1.4 years, was observed using ultrasonography (Figure 5B, transverse view), which revealed complete muscular fibrosis throughout both deep and superficial portions of the masseter muscle, with a uniform hyperechoic appearance.The deep inferior tendon of the masseter muscle could not be identified because of fibrosis.
Muscular fibrosis appears to be more hypoechoic than homogenous parotid gland fibrosis.

| Case 8
A female patient, aged 62 years, who received repetitive botulinum neurotoxin injections at intervals of 6 months to 2 years over a period of 6 years was observed using ultrasonography (Figure 5C, transverse view).The image shows partial muscular fibrosis in the posterior part of the masseter muscle with layered hypoechogenicity.The anterior parts of the muscle fibers appeared normal, and the parotid gland showed homogenous echogenicity.

| Case 9
A female patient, aged 46 years, who received periodic botulinum neurotoxin injections at intervals ranging from 7 months to 1.5 years

| DISCUSS ION
Botulinum neurotoxin is known to be distributed partially and locally in either the superficial or deep muscle belly in a limited manner.The deep inferior tendon separates the superficial and deep bellies of the masseter muscle. 6,7If botulinum neurotoxin consistently affects only one of the bellies, the repeatedly affected masseter muscles may appear fibrotic.In most anatomical studies using cadavers and ultrasound images, the tendinous portion, called the deep inferior tendon, is observed deep in the lower third of the superficial part of the masseter, which divides the superficial and deep bellies of the muscle.Ultrasonography is an effective method for visualizing these structures in clinical practice.
The primary objective of the study conducted by Bae et al.
was to evaluate and compare the effectiveness and safety of two different botulinum neurotoxin injection techniques, the traditional blind injection method and a new method that employed ultrasonographic guidance, when administered to the masseter muscle.After a 1-month follow-up period, the reduction in masseter thickness and facial contour varied significantly between the two groups, with the ultrasonography-guided injection group demonstrating superior results in both measures.The study concluded that the use of ultrasonographic guidance is a more effective and safer approach for injecting botulinum neurotoxins into the masseter muscle. 8 et al. 9 recently reported that botulinum neurotoxin causes muscle fibrosis.However, we observed that muscle fibrosis is due to the atrophy of the muscle; however, the muscle fibers retained their function, as demonstrated in the histological examination. 5sed on our clinical experience, we have observed that conventional blind injection techniques may not always produce the expected level of effectiveness.The masseter muscle is a complex structure with intermingled muscle fibers and compartmentalized tendinous portions.We believe that muscle fiber atrophy resulting from conventional deep injection techniques may occur only in the fibrotic muscle belly, which can make the patient feel that the treatment is ineffective.Our study suggests that practitioners should use ultrasonography to evaluate the masseter muscles and target areas that are not fibrotic in partially fibrotic masseter muscles.For fully fibrotic muscles, practitioners should not administer botulinum neurotoxin injections because they are not effective.

F I G U R E 5
The ultrasonographic analysis reveals partial fibrosis in the deep belly of the masseter muscle (A), with a hyperechoic appearance.The ultrasonography reveals complete muscular fibrosis throughout both the deep and superficial portions of the masseter muscle (B), with a uniform hyperechoic appearance.The image demonstrates a partial muscular fibrosis located at the posterior part of the masseter muscle with layered hypoechogenicity (C).The anterior part of the muscle fibers appears normal, and the parotid gland shows homogenous echogenicity.A transverse view image (D) reveals partial muscular fibrosis located in the posterior section of the masseter muscle, with a hyperechoic needle point evident in the image.
The fibrotic image was mostly observed in the deeper layer of the masseter muscle and this is because many of the literature and the textbooks indicated deeper injection after touching the periosteum has been effected these changes.Also, the females only tend to appear to have fibrotic changes, the result may be resulted because 10 times more specimens were females and the amount of botulinum neurotoxin used for the males use to be the same with the females in most of the cases, while the muscle masses are larger and the higher force of the muscle contraction would have help the toxins to be spread over the muscle bellies than the women.
One limitation of this study is that the sample size of patients with masseteric fibrosis was relatively small, with only nine cases included.While these cases provided valuable insights into the effects of repetitive botulinum neurotoxin injections on the masseter muscle, a larger and more diverse sample size would have strengthened the study's findings and allowed for more robust conclusions.
Additionally, the study's retrospective design may have introduced some inherent biases in data collection and analysis.Further prospective studies with a larger patient cohort and standardized protocols would be beneficial to confirm and generalize the observed fibrotic changes and their clinical implications.Additionally, this study is the uneven distribution of male and female participants.
Among the 583 patients who received multiple botulinum neurotoxin injections into the masseter muscle, there were 540 female participants and only 43 male participants.This gender imbalance may have introduced potential confounding factors and limited the generalizability of the findings, as the effects of botulinum neurotoxin injections and the development of masseteric fibrosis may differ between males and females.
Another potential limitation of this study is the retrospective design, which may have introduced biases in the selection of cases and the data collection process.Prospective studies with well-defined inclusion criteria and data collection methods would enhance the validity of the findings.Additionally, using a control group of patients who did not receive repetitive botulinum neurotoxin injections in the masseter muscle would allow for direct comparisons and better assessment of the specific effects of the injections on fibrosis development.
While this study provides valuable insights into masseter fibrosis caused by repetitive botulinum neurotoxin injections, the small sample size and retrospective design warrant caution in generalizing the results.Further research with larger, more diverse patient cohorts and prospective study designs is necessary to validate and extend these findings, ultimately improving our understanding of the clinical implications of botulinum neurotoxin injections on the masseter muscle.
Given that the primary objective of US observation was for dual plane injections to mitigate the occurrence of paradoxical masseteric bulging, US images were not all documented for immediate intervention.Only the special cases were measured.This has been acknowledged as a limitation in our study, potentially compounded by inherent anatomical variabilities.Nevertheless, our hypothesis is grounded in prior research that explored the effect of high-dose intramuscular botulinum neurotoxin injections on muscle tissues and multiple injection could make these fibrosis as well.

| CON CLUS IONS
in the Declaration of Helsinki, and was approved by the Institutional Review Board (approval no.2-2017-0023; date of approval: June 22, 2017).All patients underwent ultrasonographic observations, and prior to receiving the botulinum neurotoxin treatment, they were requested to provide details of their previous experiences with masseteric botulinum neurotoxin injections.The study included a total of 583 patients (540 females and 43 males) who had previously received multiple botulinum neurotoxin injections into the masseter muscle between February 2017 and May 2020.The study included nine patients with masseteric fibrosis.All the patients provided consent for the study.Ultrasonographic (E-Cube platinum, Inc.Alphinion with an 8-17× linear transducer) analysis was conducted in each patient to observe full and partial masseteric fibrosis after repeated botulinum neurotoxin injections.The ultrasonographic transducer was positioned at the midpoint of the masseter muscle, covering its anterior and posterior borders, insertion, and origin, during both longitudinal and transverse scanning.The fibrotic areas were characterized by a cloudy, grayish appearance in the ultrasonographic image, showing reduced contraction compared to the normal hypoechoic muscle regions.

A
41-year-old woman received repetitive botulinum neurotoxin injections every year for 7 years.Ultrasonography (Figure 2B, transverse view) revealed fibrosis only in the lower posterior part of the masseter muscle.This may have been caused by the injections typically being administered only deep, touching the bony surface, with the deep inferior tendon appearing on the right side of the posterior fibrosus.

F
I G U R E 2 Transverse view ultrasonogram shows the muscle condition after repeated botulinum neurotoxin (A, B).A shows fibrosis in the posterior part of the masseter muscle.B shows fibrosis only in the lower part of the posterior portion.Additionally, the posterior portion displayed partial fibrosis of the parotid gland with homogenous echogenicity.

F I G U R E 3
Ultrasonography reveals the muscle condition in transverse view (A) and longitudinal view (B) after repeated botulinum neurotoxin.A shows fibrosis of the masseter muscle in both the superficial and deep layers.B shows hyperechoic areas of the masseteric muscle in both the superficial and deep layers.F I G U R E 4 Ultrasonographic analysis was conducted in two patients receiving repetitive botulinum neurotoxin injections.A shows a transverse view of partial fibrosis at the middle of the masseter muscle.The deep inferior tendon is visible, dividing the superficial and deep muscle.The ultrasonographic findings reveals partial fibrosis of the masseter muscle at the middle portion with hyperechoic appearance, as shown in B. for a duration of 7 years, was evaluated using ultrasonography.A transverse view (Figure 5D) revealed partial muscular fibrosis in the posterior section of the masseter muscle, with a hyperechoic needle point evident on the image.The patient underwent repeated blind injections, which frequently resulted in inadequate coverage of the front part of the muscle, owing to the usual practice of avoiding injection in front of the masseter while considering the presence of the risorius.
This indicates that although a minority of subjects may exhibit fibrosis, repeated administration of botulinum neurotoxin can contribute to its development.The research underscores the significance of avoiding regions with reduced contractility as targets for botulinum neurotoxin treatment.The hyperechoic appearance of the masseter muscle indicates muscle fibrosis, which can be observed using ultrasonography.Ultrasonography can help predict the efficacy of botulinum neurotoxin injection in cases where it is less effective.As muscle fibrosis can be partial and localized, it is important to determine the degree and location of fibrosis before determining the injection area.In clinical practice, muscle fibrosis may only be present in specific areas where blind injections are administered.