Abstract
Background
Botulinum neurotoxin type A (BoNT-A) is an established minimally invasive treatment for congenital muscular torticollis (CMT); however, current injection strategies rely on empirical anatomical landmarks rather than precise functional targets. Accurate localisation of the sternocleidomastoid muscle (SCM) motor endplate band may improve treatment efficacy by optimising toxin delivery. We aimed to investigate the therapeutic efficacy of BoNT-A injection targeting the motor endplate band of the SCM, as localised by acetylcholinesterase (AChE) whole-muscle staining, in the treatment of CMT.
Methods
Twelve adult New Zealand rabbits underwent AChE whole-muscle staining to reveal the distribution pattern of the SCM motor endplate band. Forty-eight rabbits were randomised into four equal groups: a normal group, a model group (ethanol-induced CMT fibrosis), a conventional injection group (BoNT-A injection at the fibrotic induration), and a motor endplate band injection group (BoNT-A injection at the centre of the motor endplate band). One week after BoNT-A injection, spiral computed tomography (CT) was performed to measure SCM length. Haematoxylin and eosin (H&E) and Masson’s trichrome staining were used to assess muscle fibre cross-sectional area (CSA) and collagen deposition, respectively. Western blotting was employed to detect the protein expression of alpha-smooth muscle actin (α-SMA) and muscle RING-finger protein-1 (MuRF-1).
Results
AChE staining revealed that the motor endplate band was distributed between 20.25%±0.31% and 49.36%±0.41% of the SCM length, with its geometric centre located at 30.07%±0.44% of the muscle length. Two weeks after ethanol-induced modelling, the model group exhibited shortened SCM length, an elevated collagen area ratio, and upregulated α-SMA expression, confirming the successful establishment of the CMT fibrosis model. Following BoNT-A injection at different targets, the motor endplate band injection group demonstrated greater SCM length, lower collagen area ratio, lower α-SMA expression, and higher MuRF-1 expression compared with the conventional injection group; all differences were statistically significant.
Conclusions
The motor endplate band of the SCM is a promising and anatomically defined target for BoNT-A injection in the treatment of CMT. The targeted injection strategy guided by the motor endplate band was associated with significantly greater anti-fibrotic efficacy compared with the conventional injection strategy in suppressing muscle fibrosis and improving muscle function.
Keywords: Congenital muscular torticollis (CMT), botulinum neurotoxin type A (BoNT-A), motor endplate band, acetylcholinesterase staining (AChE staining), sternocleidomastoid muscle (SCM)
Highlight box.
Key findings
• Motor endplate band-targeted botulinum neurotoxin type A (BoNT-A) injection significantly reduced sternocleidomastoid muscle fibrosis, increased muscle length, and produced greater therapeutic efficacy than conventional injection into the fibrotic induration in a rabbit model of congenital muscular torticollis (CMT).
What is known and what is new?
• BoNT-A is an established treatment for CMT, but injection targets are selected empirically because the optimal anatomical target has not been clearly defined.
• This study directly visualised the sternocleidomastoid motor endplate band using whole-muscle acetylcholinesterase staining and demonstrated that targeting this region provides superior anti-fibrotic and functional outcomes compared with conventional injection.
What is the implication, and what should change now?
• These findings support the motor endplate band as the preferred anatomical target for BoNT-A injection in CMT and provide preclinical evidence to guide more precise injection strategies.
• Future clinical studies should evaluate motor endplate band-guided BoNT-A injection to optimise treatment efficacy and facilitate translation from empirical to targeted therapy.
Introduction
Background
Paediatric torticollis is characterised by head and neck deviation with restricted cervical motion and encompasses congenital muscular, congenital osseous, postural, ocular, infectious, neurogenic, and post-traumatic aetiologies (1). Among these, congenital muscular torticollis (CMT) accounts for more than half of all cases (2) and is characterised pathologically by fibrotic induration and persistent contracture of the sternocleidomastoid muscle (SCM) (3).
Current treatments for CMT include orthotic correction, physical rehabilitation, surgical release, and local injection of botulinum neurotoxin type A (BoNT-A) (4). Because of its minimal invasiveness, reversibility, and favourable patient compliance, BoNT-A has become a first-line minimally invasive treatment for mild-to-moderate CMT. BoNT-A acts by blocking acetylcholine release from presynaptic nerve terminals at the neuromuscular junction (motor endplate), thereby inducing chemical denervation to relieve SCM contracture while also inhibiting fibroblast differentiation and proliferation to attenuate muscle fibrosis (5).
Rationale and knowledge gap
Currently, BoNT-A injection targets for CMT are selected primarily using empirical approaches, including multi-point injections distributed along the longitudinal axis of the SCM (30–50 U across 3–4 sites) or a single concentrated injection into the fibrotic induration (30–50 U). Both approaches rely on gross anatomical landmarks rather than precise localisation of intramuscular functional targets, potentially resulting in variable therapeutic efficacy, neutralising antibody formation, and secondary fibrosis at the injection site. A deviation of only 5 mm from the motor endplate can reduce BoNT-A efficacy by approximately 50% (6,7), highlighting the importance of accurately localising the SCM motor endplate band to optimise treatment outcomes.
Visualisation of human motor endplates requires fresh specimens obtained within 3 h of death, making clinical specimen acquisition highly challenging (8). Previous studies have suggested that the intramuscular nerve dense region (INDR) corresponds to the motor endplate band and that its centre (CINDR) may serve as an optimal injection target. Using combined Sihler’s staining and acetylcholinesterase (AChE) staining in a rabbit masseter muscle model, our research group previously demonstrated that the INDR closely corresponds to the motor endplate band and that BoNT-A injection at the CINDR produces significantly greater muscle atrophy than injection into the mid-belly of the muscle (9). This work also showed that whole-muscle AChE staining provides a direct and intuitive method for visualising the motor endplate band in animal models while substantially reducing experimental time compared with Sihler’s staining, thereby offering a convenient and reliable technique for subsequent localisation studies. Accordingly, the present study used whole-muscle AChE staining to directly visualise the motor endplate band of the rabbit SCM.
However, these findings were limited to the normal rabbit masseter muscle and have not been validated in the rabbit SCM, which shares greater similarity with the human SCM. Furthermore, experimental evidence regarding the optimal BoNT-A injection target and its therapeutic efficacy within the fibrotic microenvironment of CMT remains lacking.
Objective
The present study aimed to employ whole-muscle AChE staining to identify the motor endplate band of the rabbit SCM and spiral computed tomography (CT) to determine its corresponding body surface puncture location and injection depth. An ethanol-induced rabbit model of CMT-associated fibrosis was subsequently established. Following BoNT-A injection, SCM length, muscle fibre cross-sectional area (CSA), collagen deposition, and the expression of alpha-smooth muscle actin (α-SMA) and muscle RING-finger protein-1 (MuRF-1) were evaluated in all experimental groups. By integrating morphological, histopathological, and molecular biological analyses, this study systematically evaluated the motor endplate band as the optimal BoNT-A injection target for CMT and preliminarily investigated the molecular mechanisms underlying its therapeutic effects on SCM fibrotic contracture. Together with our group’s previous anatomical localisation of the CINDR in the paediatric SCM (10), these findings provide a theoretical foundation for the clinical translation of targeted BoNT-A therapy for CMT. We present this article in accordance with the ARRIVE reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0659/rc) (11).
Methods
Experimental animals and ethics
Sixty adult New Zealand rabbits (equal numbers of males and females, mean body weight 2.5±0.1 kg) were purchased from the Experimental Animal Centre of Zunyi Medical University and underwent a 1-week adaptive housing period prior to experimentation. The rabbits were housed at a room temperature of 20–24 ℃, with natural lighting and ad libitum access to food and water. All animal experiments were performed under a project license (approval No. ZMU21-2202-116; approval date: 4 January 2022) granted by the Animal Care and Use Committee of Zunyi Medical University, in compliance with national guidelines for the care and use of animals.
Experimental grouping
Of the 60 experimental rabbits, 12 were used for AChE whole-muscle staining to observe the distribution pattern of the SCM motor endplate band. The remaining 48 rabbits were randomised into four groups (n=12 per group, equal numbers of males and females): (I) normal group (no intervention); (II) model group (ethanol-induced CMT fibrosis model, without BoNT-A injection); (III) conventional injection group (CMT model + BoNT-A injection at the fibrotic induration); (IV) motor endplate band injection group (CMT model + BoNT-A injection at the motor endplate band target). The sample size of 12 per group was determined based on our previous study employing the same rabbit model and histological endpoints (9), which demonstrated that n≥10 per group provided >80% statistical power to detect a 20% difference in muscle fibre CSA at a significance level of alpha =0.05.
AChE whole-muscle staining for SCM motor endplate band visualisation
All rabbits were humanely euthanised by overdose injection of pentobarbital sodium (100 mg/kg) via the marginal ear vein. After loss of consciousness and steady cessation of respiration and heartbeat, the bilateral SCM were immediately dissected, and excess fascial tissue on the muscle surface was carefully removed. All tissue harvesting was completed within 3 h post-euthanasia.
The AChE working solution was freshly prepared before use, with a total volume of 200 mL: 400 mg of 2% acetylthiocholine iodide (Darmstadt, Germany) was dissolved in 20 mL of distilled water, and 5.2 mL of 0.1% copper sulphate solution was added. After thorough mixing, the solution was centrifuged at 2,500 r/min for 15 min, and 20 mL of the supernatant was collected as Solution A. Separately, 100 mL of sodium acetate buffer (pH 5.0), 72 mL of distilled water, 4 mL of 3.75% glycine solution, and 4 mL of 0.1% copper sulphate solution were combined to yield Solution B (180 mL). Solutions A and B were mixed to form the AChE working solution.
The processed SCM specimens were fully immersed in the AChE working solution and incubated at 4 ℃ for 48 h. After incubation, the specimens were briefly rinsed with distilled water for 30 s to remove residual working solution. They were then transferred to 10% ammonium sulphide solution and immersed for 60 s for colour development. The developed specimens were fixed and stored in neutral formalin solution (pH 7.0) for subsequent observation and image acquisition.
Localisation of the motor endplate band injection target
SCM specimens with successfully visualised motor endplate bands were photographed and documented. Adobe Photoshop 2025 (Adobe Systems Inc., San Jose, CA, USA) was used for image processing: the most concentrated region of the motor endplate band was selected following the principle of including no more than necessary; the Control + T command was executed, and the resulting geometric centre point was designated as the centre of this region, i.e., the optimal injection target of the motor endplate band. Using the image scale bar as reference, the percentage positions of this target along the SCM length and width were measured to complete the target localisation.
Establishment of the rabbit CMT fibrosis model
Rabbits designated for modelling were anaesthetised by intravenous injection of 10% chloral hydrate (1.2 mL/kg) via the marginal ear vein. After the onset of anaesthesia, the hair from the neck region was removed. A B-mode ultrasound device (LOGIQ 500, 5–10 MHz linear array transducer, GE Healthcare, Chicago, IL, USA) was employed, with the probe positioned perpendicular to the skin surface, to identify the SCM origin and insertion and measure its length. Based on the measurement results, the SCM was divided into upper and lower segments of equal length. The injection segment was randomly determined by coin toss (heads = upper segment, tails = lower segment). Absolute ethanol was injected into the right SCM at a dose of 1 mL/kg, with the left SCM serving as the untreated contralateral control. Fourteen days after modelling, the same physician performed a repeat B-mode ultrasound examination; successful modelling was defined as the presence of a hyperechoic shadow in the right SCM with marked contracture and shortening of the right SCM relative to the left.
B-mode ultrasound-guided BoNT-A injection
Rabbits in the conventional injection and motor endplate band injection groups were anaesthetised and prepared as described. Under real-time guidance of the B-mode ultrasound probe positioned perpendicular to the neck skin, the conventional injection group received BoNT-A injection at the SCM fibrotic induration, whilst the motor endplate band injection group received BoNT-A injection at the centre of the pre-localised motor endplate band region. Both groups received an injection of 2.5 U BoNT-A (Lanzhou Institute of Biological Products Co., Ltd., Lanzhou, China; National Medical Products Administration Approval No. S10970037). This dose was selected to approximate the clinically recommended starting dose for BoNT-A injection in paediatric CMT (approximately 1–2 U/kg), following body surface area-based conversion from human to rabbit. No special postoperative care was administered. All rabbits were individually housed with ad libitum access to food and water under natural lighting.
Spiral CT measurement of SCM length
Because residual villus hair on the neck after skin preparation generated considerable artefact interference under B-mode ultrasound, spiral CT was selected for SCM length measurement. After anaesthesia by the same method, rabbits were placed in the supine position for CT scanning. A 64-slice spiral CT scanner (Erlangen, Germany) was used (tube voltage 120 kV, slice thickness 1 mm, collimation 64×0.75 mm, pitch 1:1). SCM length was measured along the course of the muscle from origin to insertion using the curve measurement tool on the Syngo workstation (Siemens Healthineers, Erlangen, Germany). All measurements were performed by the same radiography technician.
Haematoxylin and eosin (H&E) and Masson’s trichrome staining and histological measurements
One week after BoNT-A injection, rabbits in all groups were humanely euthanised by overdose injection of pentobarbital sodium (100 mg/kg) via the marginal ear vein. A 1 cm × 1 cm × 1 cm muscle tissue block was harvested from the mid-belly of the SCM, fixed in 10% neutral formalin for 24 h, dehydrated through a graded ethanol series, cleared in xylene, embedded in paraffin, and sectioned at 4 μm thickness. Serial sections underwent H&E and Masson’s trichrome staining, respectively. H&E staining was used to observe muscle fibre morphology and measure muscle fibre CSA; Masson’s trichrome staining was used to evaluate collagen fibre deposition. Under a light microscope, five fields (upper, lower, left, right, and centre) were selected per section, with no fewer than 500 muscle fibres counted in total. ImageJ 2022 software (National Institutes of Health, Bethesda, MD, USA) was employed to measure the CSA of individual muscle fibres and to calculate the percentage of collagen fibre area relative to the total field area; the mean value was taken as the muscle fibre CSA and collagen area ratio for each specimen.
Western blot detection of α-SMA and MuRF-1 protein expression in the SCM
Approximately 0.2 cm × 0.2 cm × 0.2 cm of SCM tissue was placed in a glass homogeniser containing Racial and Identity Profiling Act (RIPA) lysis buffer (Beyotime Biotechnology Co., Shanghai, China) and homogenised on ice. The homogenate was centrifuged at 12,000 r/min for 10 min at 4 ℃, and the supernatant was collected. Total protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology Co., Shanghai, China) according to the manufacturer’s instructions. Approximately 2.5 μg of total protein was loaded per lane, separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and wet-transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% skimmed milk at room temperature for 1 h, the membranes were incubated overnight at 4 ℃ with rabbit anti-α-SMA polyclonal antibody (Abcam Trading Co., Shanghai, China), rabbit anti-MuRF-1 polyclonal antibody (Abcam Trading Co.), and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (Abcam Trading Co.), respectively. Following washing with Tris Buffered Saline with Tween 20 (TBST), the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibody (Abcam Trading Co.) for 1 h at room temperature. Enhanced chemiluminescence (ECL) substrate was used for colour development. Band greyscale values were quantified using ImageJ software (version 1.80, National Institutes of Health), and the relative expression levels of α-SMA and MuRF-1 were calculated with GAPDH as the internal reference. The experiment was independently repeated three times.
Statistical analysis
CT image measurements and histological quantitative analyses (CSA and collagen area ratio) were performed by two independent observers who were blinded to group allocation. Western blot band intensities were quantified using ImageJ automated densitometry, which minimises operator-dependent bias. The BoNT-A injection procedure itself could not be fully blinded because the injection targets (motor endplate band versus palpable fibrotic nodule) were anatomically distinct; however, this was mitigated by using a standardised ultrasound-guided protocol for both groups. SPSS 29.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. Quantitative data are expressed as mean ± standard deviation (x̄±s). After normality was confirmed by the Shapiro-Wilk test, paired t-tests were used for comparisons between left and right SCMs, and independent-samples t-tests for comparisons between sexes. One-way analysis of variance (ANOVA) was employed for comparisons of SCM length, muscle fibre CSA, collagen area ratio, and relative expression levels of α-SMA and MuRF-1 among groups, with the least significant difference (LSD) t-test used for post-hoc pairwise comparisons. The significance level was set at α=0.05.
Results
Gross anatomy of the rabbit SCM
Fine dissection of 12 normal adult New Zealand rabbits revealed that the SCM originated from the superior border of the sternum. The sternal portions of the bilateral SCM were fused at the superior sternal border, and no independent clavicular portion was observed; this anatomical pattern was consistent across all 12 specimens. From its sternal origin, the muscle belly coursed posteriorly and superiorly along both sides of the trachea, inserting onto the inferolateral aspect of the mastoid process. The unilateral SCM measured 8.43±0.72 cm in length, 0.85±0.38 cm in width, and 0.21±0.11 cm in thickness. No statistically significant differences in SCM length were observed between the left and right sides or between sexes (P>0.05). The external jugular vein coursed in close apposition to the superficial surface of the middle SCM segment (Figure 1).
Figure 1.
Anatomical dissection of the rabbit sternocleidomastoid muscle. 1= sternum; 2= clavicle; 3= sternocleidomastoid muscle; 4= trachea; 5= external jugular vein; 6= masseter muscle.
Motor endplate band of the SCM
After AChE whole-muscle staining, the SCM exhibited a reddish-brown colouration, with black, serrated, dense bands (the motor endplates) visible on the muscle surface. In the upper-middle portion of the muscle belly, the motor endplates converged to form a dense, rectangular region. This region was distributed between 20.25%±0.31% and 49.36%±0.41% of the muscle length, covering an area of approximately 1.87±0.14 cm2. Its geometric centre was located at 30.07%±0.44% of the muscle length and 51.12%±0.23% of the muscle width (Figure 2). No statistically significant differences in the percentage positions of the centre point were observed between sexes or between left and right sides (P>0.05).
Figure 2.
Motor endplate band of the rabbit sternocleidomastoid muscle. (A) Acetylcholinesterase whole-muscle staining; the red dashed box indicates the motor endplate dense region, and the red dot marks its geometric centre. (B) Magnified view of the red dashed box in (A); red arrows point to the motor endplate band.
CT measurement of SCM length
Two weeks after absolute ethanol modelling, spiral CT scanning showed that the SCM length in the model group, at 6.21±0.28 cm, was significantly shorter than that in the normal group 8.37±0.36 cm; P<0.05 (Figure 3A,3B). One week after BoNT-A injection intervention, repeat CT examination revealed that the SCM length in the motor endplate band injection group (7.54±0.32 cm) was greater than that in the conventional injection group (6.79±0.30 cm; P<0.05; Figure 3C,3D). The SCM length in both injection groups was greater than that in the model group but smaller than that in the normal group, with statistically significant differences among all groups (P<0.05) (Figure 3E).
Figure 3.
CT measurement and intergroup comparison of sternocleidomastoid muscle length. (A) Coronal CT image of the normal group. (B) Coronal CT image of the model group. (C) Coronal CT image of the motor endplate band injection group. (D) Coronal CT image of the conventional injection group. The yellow dashed lines indicate the full SCM length. (E) Bar chart comparing SCM length among the four groups. *, P<0.05. CT, computed tomography; SCM, sternocleidomastoid muscle.
Histological analysis of muscle fibre CSA and collagen fibre deposition
Masson’s trichrome staining revealed only scant blue collagen fibres between muscle fascicles in the normal group, with a collagen deposition area ratio of 8.8%±1.2% (Figure 4A). The model group showed marked collagen fibre hyperplasia with extensive blue-stained deposits; the collagen area ratio was 46.5%±3.8%, significantly elevated compared with the normal group (P<0.05) (Figure 4B). Following BoNT-A injection, the collagen deposition area ratios in the motor endplate band injection group and the conventional injection group decreased to 24.1%±2.6% and 37.7%±3.1%, respectively, both significantly lower than that in the model group but still markedly higher than that in the normal group (P<0.05) (Figure 4C,4D).
Figure 4.
Histological morphology and quantitative analysis of the sternocleidomastoid muscle. (A-D) Masson’s trichrome staining: (A) normal group, showing minimal collagen deposition; (B) model group, showing extensive collagen fibre hyperplasia (blue); (C) motor endplate band injection group, showing markedly reduced collagen deposition compared with the model group; (D) conventional injection group, showing increased collagen deposition compared with the motor endplate band injection group. (E-H) H&E staining: (E) normal group, showing polygonal muscle fibres with compact, orderly arrangement; (F) model group, showing mild morphological distortion of muscle fibres with widened interstitium; (G) motor endplate band injection group, showing pronounced muscle fibre atrophy; (H) conventional injection group, showing milder muscle fibre atrophy compared with the motor endplate band injection group. (I) Bar chart comparing muscle fibre CSA among the four groups. (J) Bar chart comparing collagen fibre deposition area percentage among the four groups. *, P<0.05. CSA, cross-sectional area; H&E, haematoxylin and eosin.
H&E staining showed that muscle fibres in the normal group exhibited a polygonal morphology with clear outlines and compact, orderly arrangement; the CSA was 1,945±152 μm2 (Figure 4E). In the model group, muscle fibres displayed mild morphological distortion with widened interstitium; the CSA was 1,873±168 μm2, slightly reduced compared with the normal group (P<0.05) (Figure 4F). Following BoNT-A injection, muscle fibres in both the motor endplate band injection group and the conventional injection group exhibited pronounced atrophy and morphological disorganisation with widened interstitium; the CSA decreased to 1,056±98 and 1,438±124 μm2, respectively, both significantly lower than those in the normal and model groups (P<0.05) (Figure 4G,4H). Quantitative comparisons of CSA and collagen deposition area ratio among the four groups are shown in Figure 4I,4J.
Western blot detection of α-SMA and MuRF-1 protein expression
Western blot analysis revealed that the relative expression level of α-SMA protein in the model group was elevated compared with the normal group (P<0.05) (Figure 5A,5B). Following BoNT-A injection intervention, the α-SMA expression level in the motor endplate band injection group was lower than that in the conventional injection group; both injection groups exhibited significantly lower levels compared with the normal and model groups (P<0.05) (Figure 5A,5B).
Figure 5.
Western blot detection of α-SMA and MuRF-1 protein expression in the sternocleidomastoid muscle. (A) Representative bands of α-SMA (42 kDa), MuRF-1 (40 kDa), and GAPDH (36 kDa), shown in the order: normal group, model group, motor endplate band injection group, and conventional injection group. (B) Bar chart of greyscale quantitative analysis of α-SMA relative protein expression. (C) Bar chart of greyscale quantitative analysis of MuRF-1 relative protein expression. Data are expressed as mean ± standard deviation; n=12 per group. *, P<0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
The relative expression level of MuRF-1 protein in the model group was mildly elevated compared with the normal group (P<0.05) (Figure 5A,5C). Following BoNT-A injection, MuRF-1 expression in the motor endplate band injection group was higher than that in the conventional injection group; both injection groups showed significantly elevated levels compared with the normal and model groups (P<0.05), with the motor endplate band injection group being higher than the conventional injection group (P<0.05) (Figure 5A,5C).
Discussion
Key findings
The core pathological features of CMT are interstitial fibrosis of the SCM, muscle fibre atrophy, and abnormal collagen deposition (12); however, existing CMT animal models remain relatively limited. Surgical modelling approaches (13), such as carotid artery ligation or balloon compression, can simulate post-ischaemic fibrosis but are associated with considerable trauma, complex procedures, and a focus on vascular injury, which differs to some extent from the pathogenesis of primary myogenic fibrosis in CMT. The present study employed local injection of absolute ethanol to induce an SCM fibrosis model in rabbits (13). Ethanol causes coagulative necrosis of muscle fibres through direct chemical injury, subsequently activating fibroblast proliferation and collagen deposition; this pathological process bears high similarity to the histological changes observed in CMT (14). This model offers the advantages of a short modelling period (2 weeks), simple operation, good reproducibility, and does not involve physical injury to blood vessels or nerves, and has been well validated in studies of this type.
Animals did not exhibit overt head-neck deviation after ethanol modelling in this study; successful model establishment was determined by the detection of intramuscular contractile nodules under B-mode ultrasound. This suggests that the macroscopic phenotype of short-term chemical modelling may be influenced by modelling duration or biomechanical factors (15), warranting further investigation.
From a pathological mechanism perspective, the fibroblast activation-collagen deposition cascade initiated by ethanol-induced chemical myonecrosis exhibits a high degree of histological consistency with the myogenic fibrosis observed in SCM biopsy specimens from patients with CMT (13,14). Although the experimental rabbits in this model did not display prominent head-neck deviation, possibly attributable to compensatory mechanisms of the bilateral cervical muscles during quadrupedal locomotion and the relatively short modelling period (2 weeks), and considering that the gravitational load on the head and neck in the upright human posture is an important biomechanical factor for the manifestation of CMT signs, the detection of intramuscular fibrotic nodules under B-mode ultrasound, the significant collagen deposition on histology, and the upregulation of α-SMA collectively substantiate the successful replication of the core pathological features of CMT at the pathological level. Compared with surgical modelling, chemical modelling has the advantage of not introducing physical injury variables to blood vessels or nerves, thereby more directly simulating the intrinsic nature of primary myogenic fibrosis in CMT.
Strengths and limitations
The present study provides a solution to the clinical dilemma at the animal experimental level: AChE staining confirmed that the motor endplate band is located within a specific interval in the mid-portion of the SCM, and BoNT-A injection centred on this region yielded significantly superior anti-fibrotic effects and functional improvement indices compared with conventional muscle belly injection. Previous human anatomical studies have confirmed that the INDR is highly coincident with the motor endplate band, and the CINDR has been precisely localised, thereby offering an actionable pathway for clinical translation.
Admittedly, the transition from animal experimentation to clinical application still requires human dose exploration and safety assessment (16); however, the present experiment has substantiated, at the proof-of-concept level, the feasibility and superiority of the targeted injection strategy guided by the motor endplate band.
The present study has some limitations. The experimental subjects were adult New Zealand rabbits, whose muscle volume, metabolic characteristics, and fibrosis progression differ from those of humans; extrapolation of the conclusions requires validation through clinical trials (15,16). Furthermore, the ethanol-induced chemical myonecrosis model, although reproducing the key fibrotic features of CMT, does not recapitulate the congenital developmental aetiology of the disease. As the present study was designed primarily to identify the optimal morphological target for BoNT-A injection, this model-level limitation is acceptable for the current proof-of-concept stage; validation in models that better capture the developmental origin of CMT is warranted in subsequent studies. A single BoNT-A dose was employed in the experiment, which only verified the superiority of the target without exploring the optimal dose that can adequately suppress fibrosis without causing excessive atrophy; this requires subsequent dose-gradient investigation. A retrospective power calculation based on the observed effect size for the primary endpoint (muscle fibre CSA, Cohen’s d ≈2.8) confirmed that the study was adequately powered (>99%) with the current sample size.
The observation endpoint was 1-week post-injection, representing a relatively short window; the medium- to long-term effects, including whether fibrosis rebounds, whether atrophy is reversible, and whether repeat injection is necessary, require longer-term follow-up (17). The 1-week time point was selected because peak chemodenervation following BoNT-A injection in rabbit skeletal muscle is documented at approximately 7 days post-injection (6); extending the observation window to 4–8 weeks in future studies would allow assessment of reinnervation dynamics and long-term fibrotic remodelling. The SCM length measured by CT serves as a surrogate indicator of functional improvement and lacks direct behavioural assessments such as cervical range of motion (18); future studies could incorporate functional assessment tools more closely aligned with clinical practice.
Furthermore, the model group did not include a sham injection control (e.g., normal saline injection), which precluded the complete exclusion of potential interference from the puncture procedure itself on the local microenvironment and fibrosis indicators. Subsequent studies should incorporate a sham injection group to differentiate the specific pharmacological effects of BoNT-A from the mechanical effects of needle puncture. Moreover, the present study did not include a combined injection group (motor endplate band plus fibrotic induration), and therefore cannot exclude the possibility that a combined targeting strategy may yield additional benefit.
Comparison with similar research
Two weeks after ethanol modelling, the model group exhibited significant collagen fibre hyperplasia and elevated α-SMA protein expression in the SCM, confirming the successful establishment of the fibrosis model. The mild elevation of MuRF-1 protein in the model group relative to the normal group, with almost non-overlapping data distributions between the two groups, suggests that ethanol-induced myonecrosis may activate the ubiquitin-proteasome pathway, leading to MuRF-1 upregulation (19). This is corroborated by H&E staining, which revealed mildly reduced muscle fibre CSA, widened interstitium, and disorganised arrangement in the model group, indicating that the fibrotic process is accompanied by a certain degree of muscle fibre degenerative change.
BoNT-A injection intervention exhibited clear target-dependent effects: the motor endplate band injection group was superior to the conventional injection group in both reducing α-SMA expression and collagen area ratio, whilst also producing a greater elevation of MuRF-1. This difference is consistent with the mechanism by which BoNT-A blocks acetylcholine release at the neuromuscular junction (5): the closer the injection site to the motor endplate dense region, the stronger the denervation effect and the resultant atrophic effect. Some investigators have expressed concern that BoNT-A may cause irreversible muscle atrophy; however, the results of the present study suggest that, in fibrotic myopathy, the benefit of arresting fibrosis progression may outweigh the risk of transient atrophy. The balance of this double-edged effect requires further dose-response investigation.
In terms of methodology, the innovation of the present study lies in directly visualising the motor endplate band via AChE whole-muscle staining and accurately localising its centre, thereby replacing traditional blind probing or multi-point infiltration injection approaches. The precisely localised motor endplate band was used as the injection target in a CMT pathological model, and the therapeutic superiority of the targeted injection strategy was validated at both tissue morphological and protein molecular levels. However, these findings are derived from a single animal model with a single BoNT-A dose at a single post-injection timepoint and should therefore be interpreted with caution until validated in additional preclinical models.
Explanations of findings
The motor endplate band is the region of highest neuromuscular junction density in skeletal muscle (20,21), and precise identification of its position is a prerequisite for achieving accurate BoNT-A injection. However, the visualisation of human skeletal muscle motor endplate bands is constrained by the requirement for fresh specimens obtained within 3 h of death (8), making direct clinical application challenging. The search for reliable alternative localisation methods has therefore been a key issue in this field.
Previous studies have employed three principal approaches for motor endplate band localisation. Intramuscular nerve anatomical tracing utilises Sihler’s staining or modified Sihler’s staining to reveal the distribution pattern of nerve trunks and their terminal branches (22), thereby indirectly inferring the motor endplate band territory; however, this method is time-consuming and cannot directly display the endplates themselves (23). Histological markers include silver staining of nerve terminals, cholinesterase histochemistry, and immunofluorescence labelling of synapse-associated proteins; these methods, however, may exhibit elevated false-positive rates or background staining interference in certain muscle specimens (24). The INDR-based alternative localisation strategy previously proposed by our research group demonstrated that the INDR is highly consistent with the position of the motor endplate band, and that the CINDR can serve as a reliable alternative target (8), effectively circumventing the technical bottleneck of clinical acquisition of human muscle motor endplate bands.
The Karnovsky-Roots AChE whole-muscle staining method employed in the present study is a modified cholinesterase histochemical technique (25): it can clearly display the motor endplate band as a macroscopically visible tan-coloured transverse band on the whole-muscle specimen, offering the advantages of being both intuitive and straightforward to perform, whilst avoiding the decay of fluorescent signals and interference from autofluorescence (26). Based on this, the present study used the motor endplate band localised by AChE staining as the injection target in the rabbit SCM and confirmed that this target should be the optimal target for BoNT-A injection in the treatment of CMT. Notably, our research group has previously completed the precise localisation of the CINDR in human SCM (9) and provided specific values for puncture position and depth, thereby furnishing direct anatomical evidence for translating AChE-based motor endplate band localisation results into clinical puncture protocols.
With respect to the pathway from animal experimentation to clinical translation, we undertook cross-species validation of the localisation strategy: in the rabbit SCM, AChE whole-muscle staining was used to directly visualise the motor endplate band and determine its percentage position, confirming the therapeutic superiority of targeted injection; on this basis, leveraging the previously completed anatomical localisation data of the CINDR in the human SCM (10), the rabbit motor endplate band percentage positions were converted into body surface-identifiable puncture parameters. The feasibility of this conversion rests on two premises. First, previous studies have confirmed the high positional consistency between the INDR and the motor endplate band (8,9). Second, the rabbit and human SCM share a high degree of anatomical homology in terms of the course from origin to insertion, muscle fibre arrangement orientation, and innervation pattern. This distribution is consistent with the motor endplate band configuration previously described in a rat sternomastoid muscle (27), further supporting the cross-species conservation of the mid-belly motor endplate band pattern. Although the transition from animal percentage-based localisation to human ultrasound-guided puncture still requires resolution of issues such as individual variation correction and real-time image registration, the present study has substantiated the feasibility of this cross-species translational strategy at the proof-of-concept level.
Implications and actions needed
At present, no standardised consensus exists regarding target selection for BoNT-A injection in the clinical treatment of CMT (28). Some practitioners employ manual palpation of the most tense portion of the muscle belly for single-point injection; however, the contractile region does not necessarily overlap with the motor endplate dense region, and target accuracy cannot be guaranteed. Multi-point fractionated injection can increase drug coverage, but the increased number of injection sessions and total drug dosage may exacerbate patient discomfort, particularly in paediatric cases (29). Ultrasound-guided layered injection improves vascular safety; nevertheless, the capability of ultrasound to identify the motor endplate band is limited, and the procedure still relies on the operator’s subjective judgement of muscle belly morphology (30). Nevertheless, the targeted injection strategy proposed herein warrants further validation in larger animal models and, ultimately, in well-designed clinical trials before it can be recommended for routine clinical practice.
Conclusions
The motor endplate band of the SCM is a promising, anatomically defined target for BoNT-A injection in the treatment of CMT. The targeted injection strategy guided by the motor endplate band was associated with significantly greater anti-fibrotic efficacy compared with conventional muscle belly injection. AChE whole-muscle staining accurately localised the percentage position of the optimal target, providing an experimental basis for the clinical translation of CMT treatment from empirical injection to precise targeted intervention.
Supplementary
The article’s supplementary files as
Acknowledgments
We thank the editorial team at Editage for professional language editing of this manuscript, including terminology verification and logical coherence review.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were performed under a project license (approval No. ZMU21-2202-116; approval date: 4 January 2022) granted by the Animal Care and Use Committee of Zunyi Medical University, in compliance with national guidelines for the care and use of animals.
Footnotes
Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0659/rc
Funding: This work was supported by the Health Science and Technology Project of Guizhou Province, China (grant No. gzwkj2025-409), and the Science and Technology Bureau of Zunyi City (grant No. 2023-364). The funding agencies had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation.
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0659/coif). The authors have no conflicts of interest to declare.
Data Sharing Statement
Available at https://tp.amegroups.com/article/view/10.21037/tp-2026-0659/dss
References
- 1. Jianqiang M, Haitian L, Xiaohu F, et al. Disease spectrum of torticollis in children and diagnostic flowchart: A retrospective, single-centre study. J Paediatr Child Health 2024;60:176-82.. 10.1111/jpc.16551 [DOI] [PubMed] [Google Scholar]
- 2. Zhao Z, Deng H, Li Y, et al. Experience with the management of 2599 cases of congenital muscular torticollis and a multicenter epidemiological investigation in 17 hospitals in China. BMC Musculoskelet Disord 2023;24:901.. 10.1186/s12891-023-06983-w [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Płomiński J, Olesińska J, Kamelska-Sadowska AM, et al. Congenital Muscular Torticollis-Current Understanding and Perinatal Risk Factors: A Retrospective Analysis. Healthcare (Basel) 2023;12:13.. 10.3390/healthcare12010013 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Sargent B, Coulter C, Cannoy J, et al. Physical Therapy Management of Congenital Muscular Torticollis: A 2024 Evidence-Based Clinical Practice Guideline From the American Physical Therapy Association Academy of Pediatric Physical Therapy. Pediatr Phys Ther 2024;36:370-421.. 10.1097/PEP.0000000000001114 [DOI] [PubMed] [Google Scholar]
- 5. Pirazzini M, Rossetto O, Eleopra R, et al. Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology. Pharmacol Rev 2017;69:200-35.. 10.1124/pr.116.012658 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Shaari CM, Sanders I. Quantifying how location and dose of botulinum toxin injections affect muscle paralysis. Muscle Nerve 1993;16:964-9.. 10.1002/mus.880160913 [DOI] [PubMed] [Google Scholar]
- 7. Karp BI, Ly A, Alter KE. Localization modalities for botulinum neurotoxin injection. Toxicon 2025;264:108460.. 10.1016/j.toxicon.2025.108460 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Koo HJ, Park HJ, Park GY, et al. Safe Needle Insertion Locations for Motor Point Injection of the Triceps Brachii Muscle: A Pilot Cadaveric and Ultrasonography Study. Ann Rehabil Med 2019;43:635-41.. 10.5535/arm.2019.43.6.635 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Chen R, He L, He X, et al. Experimental study of the center of the intramuscular nerve dense region of the masseter muscle as the optimal botulinum neurotoxin A injection site. Ann Anat 2025;261:152697.. 10.1016/j.aanat.2025.152697 [DOI] [PubMed] [Google Scholar]
- 10. Chen R, Chen H, Zhao J, et al. Target localization for botulinum neurotoxin type A injection in the treatment of pediatric muscular torticollis. Int J Morphol 2026;44:92-8. [Google Scholar]
- 11.National Research Council. Guide for the Care and Use of Laboratory Animals. 8th ed. Washington (DC): National Academies Press (US); 2011. [Google Scholar]
- 12. Mahdy MAA. Skeletal muscle fibrosis: an overview. Cell Tissue Res 2019;375:575-88.. 10.1007/s00441-018-2955-2 [DOI] [PubMed] [Google Scholar]
- 13. Jiang B, Zu W, Xu J, et al. Botulinum toxin type A relieves sternocleidomastoid muscle fibrosis in congenital muscular torticollis. Int J Biol Macromol 2018;112:1014-20.. 10.1016/j.ijbiomac.2018.02.077 [DOI] [PubMed] [Google Scholar]
- 14. Li D, Wang K, Zhang W, et al. Expression of Bax/Bcl-2, TGF-β1, and Type III Collagen Fiber in Congenital Muscular Torticollis. Med Sci Monit 2018;24:7869-74.. 10.12659/MSM.909064 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. McGonigle P, Ruggeri B. Animal models of human disease: challenges in enabling translation. Biochem Pharmacol 2014;87:162-71.. 10.1016/j.bcp.2013.08.006 [DOI] [PubMed] [Google Scholar]
- 16. Delgado MR, Tilton A, Carranza-Del Río J, et al. Efficacy and safety of abobotulinumtoxinA for upper limb spasticity in children with cerebral palsy: a randomized repeat-treatment study. Dev Med Child Neurol 2021;63:592-600.. 10.1111/dmcn.14733 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Multani I, Manji J, Hastings-Ison T, et al. Botulinum Toxin in the Management of Children with Cerebral Palsy. Paediatr Drugs 2019;21:261-81.. 10.1007/s40272-019-00344-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Wijntjes J, van Alfen N. Muscle ultrasound: Present state and future opportunities. Muscle Nerve 2021;63:455-66.. 10.1002/mus.27081 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Peris-Moreno D, Taillandier D, Polge C. MuRF1/TRIM63, Master Regulator of Muscle Mass. Int J Mol Sci 2020;21:6663.. 10.3390/ijms21186663 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20. Yi KH, Lee JH, Lee DK, et al. Anatomical locations of the motor endplates of sartorius muscle for botulinum toxin injections in treatment of muscle spasticity. Surg Radiol Anat 2021;43:2025-30.. 10.1007/s00276-021-02813-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Childers MK. Targeting the neuromuscular junction in skeletal muscles. Am J Phys Med Rehabil 2004;83:S38-44.. 10.1097/01.phm.0000141129.23219.42 [DOI] [PubMed] [Google Scholar]
- 22. Yi KH, Lee HJ, Seo KK, et al. Intramuscular Neural Arborization of the Latissimus Dorsi Muscle: Application of Botulinum Neurotoxin Injection in Flap Reconstruction. Toxins (Basel) 2022;14:107.. 10.3390/toxins14020107 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23. Yi KH, Hu H, Lee JH, et al. Sihler's staining technique: How to and guidance for botulinum neurotoxin injection in human muscles. Clin Anat 2024;37:169-77.. 10.1002/ca.24076 [DOI] [PubMed] [Google Scholar]
- 24. Tse N, Morsch M, Ghazanfari N, et al. The neuromuscular junction: measuring synapse size, fragmentation and changes in synaptic protein density using confocal fluorescence microscopy. J Vis Exp 2014;52220.. 10.3791/52220 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25. KARNOVSKY MJ, ROOTS L. A "DIRECT-COLORING" THIOCHOLINE METHOD FOR CHOLINESTERASES. J Histochem Cytochem 1964;12:219-21.. 10.1177/12.3.219 [DOI] [PubMed] [Google Scholar]
- 26. Davis AS, Richter A, Becker S, et al. Characterizing and Diminishing Autofluorescence in Formalin-fixed Paraffin-embedded Human Respiratory Tissue. J Histochem Cytochem 2014;62:405-23.. 10.1369/0022155414531549 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27. Zhang X, Mu L, Su H, et al. Locations of the motor endplate band and motoneurons innervating the sternomastoid muscle in the rat. Anat Rec (Hoboken) 2011;294:295-304.. 10.1002/ar.21312 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28. Grigoriu AI, Dinomais M, Rémy-Néris O, et al. Impact of Injection-Guiding Techniques on the Effectiveness of Botulinum Toxin for the Treatment of Focal Spasticity and Dystonia: A Systematic Review. Arch Phys Med Rehabil 2015;96:2067-78.e1.. 10.1016/j.apmr.2015.05.002 [DOI] [PubMed] [Google Scholar]
- 29. Limpaphayom N, Kohan E, Huser A, et al. Use of Combined Botulinum Toxin and Physical Therapy for Treatment Resistant Congenital Muscular Torticollis. J Pediatr Orthop 2019;39:e343-8.. 10.1097/BPO.0000000000001302 [DOI] [PubMed] [Google Scholar]
- 30. Kaushik PS, Gowda VK, Shivappa SK, et al. A Randomized Control Trial of Botulinum Toxin A Administration under Ultrasound Guidance against Manual Palpation in Spastic Cerebral Palsy. J Pediatr Neurosci 2018;13:443-7.. 10.4103/JPN.JPN_60_18 [DOI] [PMC free article] [PubMed] [Google Scholar]