Alimohammadi1; M. Andersson

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Correlation of botulinum toxin dose with neurophysiological parameters of efficacy and safety in the glabellar muscles: a double blind, placebo controlled randomized study

Mohammad Alimohammadi1; M. Andersson2, A. R. Punga3*

1 Department of Medical Sciences, Uppsala University, 2 Q-MED, a Galderma Division, Uppsala, Sweden, 3 Department of Clinical Neurophysiology, Uppsala University
Running title: Neurophysiological parameters and botulinum toxin in glabella

Background: Despite the extensive use of Botulinum toxin type A (BoNT-A) in cosmetic treatments for glabellar frown lines; the neurophysiological dose-response effect in the glabellar muscles remains unknown.

Objectives: To characterize neurophysiological parameters that correlate with the effect of BoNT-A in the glabellar muscles and diffusion to surrounding ocular muscles.

Methods: A randomized, double blind, placebo-controlled study of BoNT-A, on glabellar frown lines was conducted in 16 healthy women (31-64 years) with four different dose groups: placebo (0.9% NaCl), 5, 10 or 20 units Vistabel. Time points: baseline and 2, 4, 12 and 24 weeks following BoNT-A injection. Neurophysiological efficacy measurement of the corrugator supercilii muscles included: compound motor action potential (CMAP) and electromyography (EMG). Assessment of BoNT-A diffusion effects included use of CMAP of the nasalis muscle, EMG and single-fiber EMG (SFEMG) of the orbicularis oculi muscle. Photography graded glabellar frown lines (sentence needs completion).

Results: CMAP reduction correlated well with intramuscular BoNT-A dose and separated the dose groups. Muscle paralysis, measured by EMG, started from 2 weeks and was not entirely reversed in the 10 or 20 unit groups at 24 weeks. Aesthetically, all subjects receiving BoNT-A were satisfied with the reduced glabellar frown lines. Limited diffusion of effects to orbicularis oculi was detected with SFEMG.

Conclusions: We developed a novel neurophysiologic strategy for effect evaluation of BoNT-A in glabellar muscles. CMAP and EMG correlated with given BoNT-A dose and are more defined neurophysiological parameters than clinical glabellar photo scales. This study provides better understanding of the glabellar dose-response and safety effect of BoNT-A. Keywords: Botulinum toxin, glabellar muscles, CMAP, EMG
Mohammad Alimohammadi; MD, PhD, Department of Medical Sciences, Uppsala University, 75185 Uppsala, Sweden. E-mail:


Botulinum toxin type A (BoNT-A), a very potent neurotoxin, prevents the release of acetylcholine (ACh) from the nerve terminal into the neuromuscular junction (NMJ)1. The reduced ACh release results in neuromuscular blockade and subsequent phamacological denervation. This effect remains until the reinnervation process has been completed(note--innervation is reestablished but reinnervation is not complete. It is a lengthy process); which results in recovery of the original (or new???—there is literature that shows new NMJs in lid muscle specimens after botulinum –Alderson, Holds et al.) NMJs within approximately 12 weeks2, usually with limited temporary formation of nerve sprouting during the recovery process. BoNT-A is used in a wide range of medical treatments of muscular hyperactivity/tension, extending from spasticity and dystonia to aesthetic treatment of wrinkles caused by underling muscle tension, primarily in the area between the eyes (the glabellar complex). Glabellar frown lines are caused by tension in the corrugator supercilii and procerus muscles and BoNT injection into the glabellar muscles results in paralysis and hence diminished glabellar frown lines

BoNT-A exerts its predominant action in the peripheral nervous system. Diffusion has been reported to contralateral facial muscles upon unilateral BoNT-A application3 as well as remote spread from the site of injection, even to arm muscles upon facial muscle injection 4. These documented possibilities of still unknown pharmacologic BoNT-A effects emphasize the importance of correct administration of the toxin in terms of injection points, dose selection, as well as appropriate effect surveillance.

Visual clinical rating scales that focus on wrinkle depth and maximal muscle contraction are commonly used as efficacy measures of BoNT in facial muscles . Nevertheless, these scales are not sensitive enough to detect subtle difference in effect between various BoNT-A formulations or doses 7-9 or even simple parameters, such as details about the exact onset of action. Neurophysiological studies comparing different BoNT products have mainly analyzed the compound motor action potential (CMAP), i.e. the collective muscle response upon motor nerve stimulation, and then almost exclusively in the small foot extensor muscle 10-12. Few studies have addressed neurophysiology in facial muscles in a limited fashion, however such measures would enable evaluation of time to onset and duration of treatment effect upon BoNT-A injection in the tiny glabellar muscles.

The aim of this study was to evaluate whether neurophysiological measures correlated with the local effect of BoNT-A in the glabellar muscles as well as with the effect of spread to surrounding facial muscles. We found that neurophysiological measures are highly sensitive for assessment of dose-response effect upon BoNT-A injection in the glabellar muscles.



16 healthy women, aged 31-64 years, were enrolled from January until March 2012. Inclusion criteria were visible glabellar frown lines at rest or prominent glabellar muscles. Exclusion criteria were a diagnosis/symptoms of a neuromuscular disorder (e.g. Myasthenia Gravis), allergy to BoNT-A, previous BoNT-A injection, plastic surgery/implants in the facial area, ongoing infection, systemic disease or inflammation surrounding injection areas, earlier unspecific severe allergic reactions, coagulation disorder or medication with anticoagulants or muscle relaxants, pregnancy or lactation.


The study was approved by the Regional Ethical Review Board of Uppsala (Dnr: 2011/247)) and the Medical Products Agency (EudraCT nr: 2011-004636-66). All subjects were enrolled after written informed consent.

Study design:

The study was designed as randomized double blind, placebo-controlled where the evaluating clinical neurophysiologist (ARP) and the study subjects were blinded to the given substance and dose until after the study was finalized(was the neurophysiologic still blinded when data were analyzed?). All examinations were conducted at the Department of Clinical Neurophysiology, Uppsala University Hospital from January until September 2012. The neurophysiological and photographical baseline examinations were performed before the intramuscular injection of Vistabel® or placebo (Table 1). The study subjects were randomized into four groups, with four individuals in each group. The same injection volume, 0.1 ml, was given by a dermatologist (MOA) to all subjects in 5 standardized injection points using EMG-guided technique with disposable hypodermic needle electrodes (Bo-ject DHN 37,37 mm x 0.46 mm; 26G, Alpine Biomed, Skovlunde, Denmark) to ensure central muscle injection. Group I received 1 unit/injection (total 5 units); group II received 2 units/injection (total 10 units); Group III received 4 units/injection, (total 20 units); and group IV received 0.9% NaCl/injection. The recommended dose for treatment of glabellar frown lines is 20 units total (4 units/injection).

Neurophysiological examinations and parameters

All neurophysiological examinations were made on Keypoint equipment (Medtronic). The primary hypothesis (neurophysiological effects of BoNT-A), was evaluated by the following examinations bilaterally (Table 1):

  1. Motor neurography (CMAP): stimulation of the temporal branch of the facial nerve with surface electrodes, 4 cm lateral to the eye, and surface recording over the corrugator supercilii.


  1. Electromyography (EMG): a concentric facial EMG electrode (30G; 25 mm x 0.33 mm; Alpine Biomed, Skovlunde, Denmark) was placed in the corrugator supercilii and analyzed spontaneous activity (indicating denervation), quantitative motor unit potentials (MUPs) at slight voluntary activation and maximal muscle contraction.

To evaluate the secondary hypothesis (effects of spread to injection in adjacent muscles), we performed:

  1. Motor neurography (CMAP): The zygomatic branch of the facial nerve was stimulated 1.5 cm anterior to the tragus of the ear. Surface electrodes were placed over the muscle belly of the nasalis muscle.

  2. EMG in the ocular orbicularis oculi muscle. (What method is used for quantification of the signal. What are the reference values?

  3. Single-fiber EMG (SFEMG) (SFEMG requires a SF needle electrode—see comments attached): a disposable concentric facial EMG needle electrode15 (30G; 25 mm x 0.33 mm; Alpine Biomed, Skovlunde, Denmark) was inserted in the lateral lower part of the orbicularis oculi muscle under slight voluntary contraction. The jitter (s) was expressed as the mean consecutive difference (MCD) of 20 analyzed potential pairs16. Filter settings were set to 1000 Hz - 10 kHz.

Photodocumentation of glabellar frown lines and subject’s diary

The study subjects were placed between two fluorescent strip lamps, fixed in the ceiling, at all follow-up visits. A Nikon D5000 with 105 mm Macro objective was used for photography of the glabellar area in three different modes: 1) relaxed mode 2) frontalis muscle contraction 3) maximal glabellar muscle contraction. Glabellar frown lines in the relaxed state and at maximal contraction were graded on a four-point clinical severity scale from minimum 0 points to maximal 3 points 6. Additionally, study subjects were equipped with a diary to document any treatment-related adverse events.

Statistical methods

For the endpoints where a measurement was available from left and right sides, the average for each subject (N=16) was used in the calculations. Both absolute and percentage change were calculated for each subject, relating post-treatment values to baseline values. Statistical evaluations were performed based on these within-subject changes where the hypothesis was that the true mean of change was zero (1-sample t-test). Comparison of within-subject changes between dose groups was performed using a 2-sample t-test where the hypothesis to test was that the true mean of change was equal. A two-sided p-value  0.05 was considered significant.


Clinical characteristics and aesthetic treatment results

All 16 enrolled women (age range 31-64 years, mean 44 years) were naive to previous BoNT-A treatment. None of them met any exclusion criteria, confirmed by neurological examination and SFEMG analysis of neuromuscular transmission. All subjects were examined at 0, 2, 4, 12 and 24 weeks. Fifteen subjects experienced no adverse events. One of the study subjects, in the 20-unit dose group, experienced dry skin, seborrheic eczema, in the glabellar region. Glabellar frown lines in the relaxed state were graded from 0 to 2 and after BoNT-A treatment, the glabellar frown score did not necessarily change (Figure 1; Table 2). The mean baseline score at maximum frown was 2.5 (range: 1 - 4). At 4 weeks, the number of subjects per group that had improved at least one point on the photo scale was 0, 2, 2 and 4 for the 0-, 5-, 10- and 20-unit group respectively. The photos were able to separate subjects between placebo and BoNT-A, however separation between the active doses was not possible. Although blinded, all subjects who received the BoNT-A felt that they had received the active substance and 11 of 12 persons, regardless of BoNT-A dose, were satisfied with the aesthetic outcome. In addition, one of the persons who received placebo experienced aesthetic improvement and positive impact on her self-confidence, indicating a well-known placebo effect 17.

Reduction of CMAP in the glabellar muscles correlates with given dose of BoNT-A

Initial CMAP ranged from amplitude of 0.8-1.6 mV for the left and 0.9-1.7 mV for the right corrugator supercilii muscles. CMAP amplitude and area were substantially reduced after 2 weeks (Figure 2A) in all subjects who received BoNT-A. In a few cases CMAP area and amplitude further dropped from 2 to 4 weeks. The placebo group was easily identified, with CMAP around 100% (10%) of baseline at all follow-up examinations (Figure 2B).

In the standard dose group (20-unit), CMAP amplitude was reduced to 28% of baseline at 2 weeks, with a slight increase from 2 to 4 weeks. CMAP amplitude in the 20-unit group increased to 49% at 12 weeks and further to 66% at 24 weeks, indicating a large residual treatment effect (Figure 2B). Considering the half (10-unit) and quarter (5-unit) doses, the relationship between results obtained with the three doses was preserved throughout the study with 20 units consistently showing the largest reduction in amplitude and 5 units the smallest (Figure 2B). All doses differed significantly to placebo, except the 5-unit dose at week 24. The smaller differences that existed between doses were only detected statistically between 5 units and 20 units at week 2. However, even with only 4 subjects per group, CMAP amplitude appears a useful tool in discriminating between doses (Figure 3). As a control, CMAP from the nasalis muscle showed no signs of reduction (data not shown).

Change in EMG pattern following BoNT-A

The EMG pattern in the placebo group was similar at baseline and all follow-up visits, without abnormal spontaneous activity at rest and maximal voluntary glabellar muscle contraction. In the 20-unit BoNT-A group, abnormal spontaneous activity, including positive sharp waves and fibrillations, occurred from 2 weeks post-injection and became more pronounced at 4 weeks, suggesting pharmacological denervation of the corrugator supercilii muscles. The denervation activity was followed by low amplitude, unstable MUPs or total block of voluntary contraction, i.e. no MUPs were detected by EMG (When exactly did this occur? How could a CMAP or EMG amplitude be recorded when no MUPS were present?). At 12 weeks, denervation activity was absent and ongoing reinnervation was observed with the maximal EMG activity (discuss how this was quantified) increasing to approximately 25%, whereas maximal contraction reached only approximately 40% at 24 weeks in this group (Figure 5). The EMG picture was quite similar in the 10-unit dose groups and neither of these dose groups obtained maximal voluntary contraction at 24 weeks (Figure 4 and 5). One person in the 10-unit dose group had as much denervation activity(how was denervation activity quantified to make this comparison?) as in the 20-unit dose groups, but otherwise less denervation activity was noted in the 10- and 5-unit dose groups. Inter-individual variability was therefore important. The EMG picture was generally less affected in the 5-unit dose group with earlier appearance of polyphasic MUPs, indicating a more rapid reinnervation process, with maximal contraction capacity comparable to placebo at 24 weeks.

BoNT-A injection in the glabellar area caused disturbed neuromuscular transmission in the ocular muscles

SFEMG in the orbicularis oculi showed a mean MCD of 28 s (range: 23-32 s) in the whole group at baseline. In the 12 subjects who received BoNT-A, mean MCD at 2 weeks increased significantly to 35 s (range 30-39 s; p0.001), whereas mean MCD in the placebo group was unchanged at 29 s. When studying individual dose groups, there was a trend of increasing jitter values at 2 week with higher doses of BoNT-A (Table 2). The change (mean  SD) in jitter values between week 0 and 2 was +1.9% (11.6%) in the placebo group (p=0.761), +32.8% (23.8%) in the 5-unit group (p=0.070), +24.9% (10.6%) in the 10-unit group (p=0.018) and +26.1(16.8%) in the 20-unit group (p=0.053). Was blocking seen in any of the groups? No subjects indicated clinical eye closure weakness at week 2 and thus, the detected disturbance of neuromuscular transmission was defined as subclinical. There were no signs of pathological EMG pattern in the orbicularis oculi muscle at 2 weeks, indicating that the subclinical neuromuscular transmission failure did not cause substantial pharmacological denervation.


We report on neurophysiological parameters in the glabellar muscles that correlate with given doses of BoNT-A. Hitherto, the only existing measure for the muscle paralyzing effect of BoNT-A in the glabellar muscles is the validated clinical glabellar photo scale . Since the pharmacological effect of BoNT-A is indeed muscle fiber paralysis, it is important to focus on real neurophysiological parameters for quantification of the functional effect, including onset, duration of action and side effects. The photo scale was able to separate placebo and active dose groups, however not between the different BoNT-A doses. This study was not specifically designed to evaluate the effect photographically and hence the photographic procedure was not fully standardized, making subtle dose differences difficult to detect. Furthermore, the wide range of wrinkle scores at baseline could have an impact on the photographic evaluation for this small sample-size. Nevertheless, our report indicates that photographs alone do have limitations in grading the objective effect of different BoNT-A doses.

One previous report, using EMG, has implied that BoNT-A reduces EMG amplitude in the frontalis muscle upon voluntary contraction14. After an initial reduction of amplitude at 2 and 4 weeks, reinnervation and/or recovery of the original NMJs starts and the amplitude of certain MUPs increase, resulting in false high or low values depending on the reinnervation/recovery process. What is meant by a false high value?? And, value of what? EMG or CMAP? Even in the hands of an experienced neurophysiologist, we did not find a correlation between EMG amplitude and BoNT-A dose. This study instead demonstrated that the combination of CMAP and EMG allows for grading of different doses of BoNT-A in the glabellar muscles. These methods enable objective measurement of BoNT-A effect, even in cases where initial glabellar frown lines are almost absent and where the photographic scale fails to shown an objective effect at rest.

In clinical neurophysiology, presence of denervation activity is often used to assess the efficacy of BoNT-A in injected muscles, to determine resistance due to BoNT-A antibodies. From this study, it is evident that with a standard dose injection, denervation activity appeared at 2 weeks and was maximal up to 4 weeks. In the cases of half/quarter dose, however, the amount of denervation activity was modest, except in cases of a small initial glabellar muscle mass. Was the amount of denervation quantified? And if so, what method was used?? Thus, the muscle size and dose of BoNT-A are important factors to consider, since the between-subject variability was most probably caused by differences in these variables.

The maximum effect on CMAP and EMG was obtained within 4 weeks, with indication of a more delayed maximal effect for 5 units and 10 units, when compared to 20 units. Further, the duration of effect of BoNT-A for cosmetic indications is usually reported to be 3-4 months18. However, these data are mainly derived from clinical studies where the observation period was terminated at specific time points. Only a few studies have carried on to, for example, 6 months: a longer-lasting effect was identified in about 30% of patients in examples of these prolonged studies 17. Nevertheless, we confirm that the “real” neurophysiological effect actually lasts longer, since no subject recovered their function even at 24 weeks in the 10- or 20-unit dose groups. There was also a clear dose-response effect on recovery, with completed reinnervation/recovery in the 5-unit group already at 12 weeks and incomplete/ongoing reinnervation/recovery in the 10- and 20-unit groups at the same time. Hence, our data support that muscle recovery depends on formation and maturation of new NMJs and/or recovery of old existing NMJs following pharmacological denervation of BoNT-A19.

Intriguingly, there was a positive aesthetic effect on the glabellar frown lines with only 5 units of BoNT-A. A parallel reduction of CMAP was seen also in this quarter dose group, although the ability to maximally contract the corrugator muscles returned earlier. The major aesthetic difference between the quarter versus standard dose groups was an accompanying paralysis of the adjacent medial part of the frontalis muscle in the 20-unit group, resulting in lateral eyebrow lift known as “Dr Spock”. Some patients prefer this effect, however, less frontalis muscle involvement (as in the 5-unit dose group) looks more “natural”. Since 20 units is regarded as the recommended dose of the specific BoNT-A for treatment of glabellar frown lines, this study suggests individual tailoring of dose is beneficial, based on desired visual appearance.

Adverse effects of BoNT-A after injection in the glabellar area include a risk of blepharoptosis ranging from as little as 2.5 to as much as 9.5% of cases . We did not observe blepharoptosis and, with the exception of one case with seborrhoic eczema; no adverse events were reported. The EMG guided injection technique, ensuring that the BoNT-A was indeed injected intramuscularly, could have been a significant contribution to the absence of adverse events.

The disturbed neuromuscular transmission in the ocular muscle, which we observed, could have been caused by regional effect of BoNT-A. Since the orbicularis oculi is very close to the glabellar injection sites, this could be explained by a physical spread of toxin or through small intramuscular vessels. All subjects who received BoNT-A developed increased jitter in the ocular muscle and therefore this effect might be related to volume of liquid injected and not dose. Minimizing the risk of BoNT-A diffusion is probably most dependent on correct injection in the target muscle, which is especially important in the facial area, with proximity to the vital bulbar muscles.

In conclusion, we successfully characterized novel neurophysiological parameters for detailed evaluation of the dose/response effect of BoNT-A injection in the glabellar muscles. Reduction of CMAP amplitude and maximal EMG activity in the corrugator supercilii muscles correlate well with given dose of BoNT-A and are sensitive objective measures, for example in the development of new BoNT-A formulations. Additionally, 25% of the standard dose BoNT-A used in cosmetic dermatology is sufficient to paralyze the glabellar muscles and result in a more natural relaxed aesthetic outcome effect than standard doses which can result in “frozen forehead “ look.


The authors are grateful to Jan-Erik Anheller for monitoring the study and to Andy Pickett for reviewing the manuscript. The authors thank professor Erik Stålberg for valuable scientific discussion.

Conflict of interest

The study was an investigator-initiated study and the manuscript was prepared solely by the authors. MOA and ARP have received consulting fees from Q-MED AB. MAA is an employee of Q-MED AB, a Galderma Division.

Author contribution

MOA and ARP designed the study, enrolled, treated and examined the study subjects, collected data and wrote the manuscript. MAA performed the statistical analysis on the outcome data from the study.


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14 Karsai S, Adrian R, Hammes S et al. A randomized double-blind study of the effect of Botox and Dysport/Reloxin on forehead wrinkles and electromyographic activity. Archives of dermatology 2007; 143: 1447-9.

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Table 1. Study design with time points. The examination at 0 weeks was done before injection of botulinum toxin A. All other time points were follow-up examinations to study the neurophysiological effect of BoNT-A in the glabellar muscles. notably corrugator supercilii and the surrounding facial muscles. CMAP, compound motor action potential; EMG, electromyography; SFEMG, single fiber EMG.

0 weeks

2 weeks

4 weeks

12 weeks

24 weeks

Neurological examination























Table 2. Comparison between glabellar frown line scores and neurophysiological parameters of the glabellar muscles. CMAP; compound motor action potential.

Change in glabellar score at rest (mean)

Change in glabellar score at contraction (mean)

CMAP % reduction

(mean left/right corrugator)





5 units




10 units




20 units




Table 3. Comparison of neuromuscular jitter (µs) as measured by SFEMG at baseline (week 0) and 2 weeks after injection of placebo or different dose of BoNT-A. There is a trend of increasing jitter between increasing BoNT doses at week 2. p = test of hypothesis that the true mean of within-subject change in SFEMG jitter (µs) between week 0 and week 2 is zero (1-sample t-test). *p  0.05, significant.










(week 0)





























Week 2





























Figure 1. Representative clinical photographs from individuals in different randomised group. Panel A represents Placebo, Panel B 5 units, Panel C 10 units and Panel D 20 units. The visual appearance of the glabellar region at rest, voluntary contraction of the frontal muscles and glabellar muscles respectively. Glabellar frown scale scores (right column) was assessed at different time points at rest. The figure shows that differences in glabellar frown lines as well as ability to contract the glabellar muscles can be seen upon treatment with botulinum toxin, however, the accuracy of the glabellar frown scale is not optimal.

Figure 2. CMAP amplitude as percent of baseline value over time for each subject (A) and the mean value ± SEM in each dose group (B). The different dose groups can be separated based on mean CMAP amplitude.

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