Sustained anxiolytic and antidepressant effects of botulinum toxin A in blepharospasm patients beyond motor symptom control

Abstract

BACKGROUND

The previous studies have primarily focused on the influence of botulinum toxin A (BoNT-A) injection on emotions during the period of peak motor symptom improvement in blepharospasm patients, based on facial feedback hypothesis.

AIM

To evaluate the sustained anxiolytic and antidepressant effects of BoNT-A in blepharospasm patients beyond motor symptom control.

METHODS

We recruited benign essential blepharospasm patients with BoNT-A treatment and collected their data to compare scale scores of Jankovic Rating Scale, Blepharospasm Disability Index, Self-rating Anxiety Scale (SAS), Self-rating Depression Scale (SDS), Hamilton Anxiety Scale and Hamilton Depression Scale between pre-treatment (baseline) and pre-reinjection (treatment), to further assess the effects of repeated treatments with BoNT by using sub-group analyses in the certain special states.

RESULTS

A total of 21 eligible blepharospasm patients were with the mean age of 58.4 years and a male-to-female ratio of 1:6. Significantly decreases in the subscale scores of SDS and SAS, including SDS well-being index, decreased capacity and hard to decide, SAS inability to sit still and headache were showed at post-a single BoNT-A injection when scale scores of Jankovic Rating Scale and Blepharospasm Disability Index were matched between baseline and post-treatment. With each additional BoNT-A injection, the odds ratio of patients with the moderate depressive symptoms decreased by 92.6%. Moreover, BoNT treatment remained a decrease in the subscale scores of SDS and SAS in patients with repeated injections.

CONCLUSION

This study is to demonstrate that repeated BoNT-A injection have a long-lasting relief for anxiety and depressive symptoms in blepharospasm even after its motor symptom-modulating effects have diminished.

Key Words: Botulinum toxin A; Repeated injection; Neuromuscular junction; Anxiety; Depression; Blepharospasm

Core Tip: Botulinum toxin A (BoNT-A) injection significantly decreases subscale scores of Self-rating Anxiety Scale and Self-rating Depression Scale in blepharospasm even after its effects on facial muscle activity have subside. BoNT-A treatment is an independent factor for the depressive symptom improvement, especially, well-being index in the certain state. BoNT-A might alleviate somatic anxiety but not mental anxiety at a relative long follow-up. Repeated BoNT-A treatment may improve the anxiety and depressive symptoms.

INTRODUCTION

Blepharospasm is a chronic, idiopathic, recurrent and progressive disease, characterized by motor symptoms, i.e., bilateral involuntary spasms of the orbicularis oculi muscle[1,2]. Moreover, its clinical manifestations include non-motor symptoms[3] such as psychiatric disorders, sleep disturbances, cognitive impairment, and ocular symptoms.

Botulinum toxin (BoNT) is a severe neurotoxicity secreted by the bacterium Clostridium botulinum[4]. BoNT-A is used as the first-line treatment for blepharospasm to achieve a short-term relief of motor symptoms[5-7], based on the principle of smooth muscle relaxation around the eyes through the transient inhibition of neuromuscular nerve signals[8]. Recently, its effects on relieving depression and anxiety in blepharospasm patients have been reported[9-12]. For example, Dong et al[12] injected BoNT-A into the bilateral orbicularis oculi, eyebrow-lowering muscle, frontal muscle, and frown muscle of patients with benign essential blepharospasm patients at a dose of 50-100 U. They found that the mean scores of the Self-rating Anxiety Scale (SAS) and the Self-rating Depression Scale (SDS) decreased, significantly. Zhang et al[9] applied BoNT-A to the orbicularis oculi and glabellar complex in patients with blepharospasm, and observed that the mean scores for Blepharospasm Disability Index (BSDI), Personal Health Questionnaire Depression Scale 8, and the Generalized Anxiety Disorder 7-item scale improved significantly at follow-up compared to the initial visit. These findings suggested that BoNT can improve the symptoms of anxiety and depression in blepharospasm patients.

Currently, the mechanism by which BoNT-A alleviates anxiety and depression remains unclear. The prevailing hypothesis is the facial feedback hypothesis[13], which proposes that the musculature of the face responds to emotional changes with a unique speed and sensitivity and the facial expressions associated with happiness, sadness, anger, fear, surprise, and disgust. Muscular manipulations that BoNT causes paralysis of negative emotional muscles may result in more positive facial expressions and more positive emotional states in affected individuals[14-17]. In other words, behavioral and psychophysiological symptoms in blepharospasm with BoNT treatment should be synchronous due to crosstalk between temporary paralysis of facial muscles and facial feedback action inducted by BoNT. Accordingly, previous studies, including evaluation of the short- and long-term effects of BoNT-A therapy, have primarily focused on the influence of BoNT-A injection on emotions during the period of peak motor symptom improvement in blepharospasm patients[9-12,18,19]. However, in certain special states, there is no correlation between motor symptoms and emotions. For example, inhibition of neuromuscular nerve signals by a single BoNT injection typically wears off after 3-4 months[20], whereas anti-depression of BoNT continued over 24 weeks in patients with major depression[21]. Thus, not all of emotional changes are associated with facial muscle expressions. We hypothesized that the attenuation of the effects on facial features may not lead to in an abatement of emotional responses during BoNT therapy.

However, there is no data on whether these beneficial mood effects persist independently once the muscle-paralyzing effects of BoNT-A have worn off and motor symptoms have returned. Therefore, this study specifically aimed to investigate this dissociation. We evaluate the sustained effects of BoNT-A on anxiety and depression in blepharospasm patients using the disappearance of temporary muscle paralysis induced by BoNT as a marker. The aim is to assess whether BoNT-A exerts a sustained effect on anxiety and depressive symptoms in blepharospasm after its effects on abnormal facial muscle activity have subsided, to identify the specific symptom of responsive to BoNT-A, and to further assess the effects of repeated BoNT treatments in the certain special states.

MATERIALS AND METHODS

Study design and eligibility

We recruited outpatients with facial movement disorders undergoing ongoing BoNT-A treatment at Nanjing Brain Hospital between September 2022 and June 2024. Inclusion criteria were that the patients were: (1) Diagnosed with benign essential blepharospasm by two experienced clinicians; and (2) Received BoNT-A injection therapy. Exclusion criteria were: (1) Use of medications that may affect disease symptoms, including anticholinergics, benzodiazepines, anti-Parkinson’s drugs, and antipsychotics; (2) Patients with additional psychiatric diagnoses (e.g., major depression or anxiety disorder); and (3) Contraindications for BoNT-A injection therapy, such as infection or skin lesions at the injection site or any medical conditions that may increase the risk of BoNT-A injection.

Because without data on the sustained anxiolytic and antidepressant effects of BoNT-A in blepharospasm patients beyond motor symptom control, we powered the study only for the effect of BoNT-A on depressive and anxious symptoms during the period of peak motor symptom improvement. The assessment scales included SDS and Hamilton Depression Scale (HAMD) for depressive symptoms and SAS and Hamilton Anxiety Scale (HAMA) for anxious symptoms. Based on outcomes of previous literatures, we used PASS software (version 11) and set α of 0.05 and power of 0.9 to calculate the sample size of the study. When the sample size reached 21, the power value was 1.00000.

Data collection and follow-up

Demographic information of eligible patients, including age, sex, education, age at disease onset, and frequency of BoNT-A injections, was collected. Patients received BoNT-A injections (BoNT-A, BOTOX^®, United States) under ultrasound guidance. Furthermore, the total dose of each injection was about 50 units, diluted to 2 mL of physiological saline at a concentration of 0.9%. The injection sites were determined according to the regions of the facial muscle spasm, mainly including orbicularis oculi, corrugator, and procerus, etc. (Figure 1). Importantly, the follow-up time point was set before reinjection. That is, the BoNT-A injection had lost its effect on controlling abnormal facial muscle activity and patients’ motor symptoms were sufficiently severe to require reinjection. The interval from initial BoNT-A to reinjection was recorded. The data before the initial treatment were considered baseline data, and data collected before BoNT-A reinjection were considered post-treatment data.

Figure 1 Schematic representation of botulinum toxin A injection muscle. ①: Orbicularis oculi; ②: Procerus; ③: Corrugator.

Outcomes

The primary outcome was the evaluation of motor symptoms, anxiety and depression at pre-treatment (baseline) and pre-reinjection (treatment) stages. The assessment scales included Jankovic Rating Scale (JRS) and BSDI for motor symptoms[22,23], SAS[24] and HAMA[25] for anxious symptoms, SDS[26] and HAMD[27] for depressive symptoms. JRS, comprising two indices, severity and frequency, is a standard rating scale for quantifying blepharospasm symptoms. The BSDI assesses the impact on quality of life and the degree of disability in blepharospasm patients. SAS, SDS, HAMA, and HAMD, are rating scales that are designed to quantify anxiety and depressive symptoms. SAS and SDS are self-rated scales, whereas HAMA and HAMD are rated by interviewers.

The secondary outcome was evaluated using SAS and SDS subscale scores at baseline vs treatment. The version of SAS contains 20 items, divided into four subscale scores: Anxiety (anxiety, fear, panic, feeling crazy and flushed face), somatic control (panic, premonition of misfortune, inability to sit still, dyspnea, hyperhidrosis and sleep disorder), vestibular sensations (trembling hands and feet, palpitation, dizziness, syncope and tingling of hands and feet), and gastrointestinal/muscular sensations (headache, fatigue, stomach pain and indigestion, frequent urination and nightmare)[28]. The version of SDS also contains 20 items, divided into five subscale scores: Well-being index (decreased capacity, hard to decide, feeling of uselessness, empty-minded and loss of interest), depressed mood index (dejection, weepiness, sense of worthlessness), somatic symptoms index (palpitation and fatigue), optimism index (thought disorder and despair), and items not clustered into an index (heavy in the morning and light at night, sleep disorder, loss of appetite, hyposexual interest, weight loss, constipation, insecurity and irritability)[29]. The SAS four-factor model and the SDS five-factor model were intended to enhance the stability and the comprehensiveness of the scales. All assessments were conducted by two experienced clinicians who were blinded to this study.

Statistical analysis

The Statistical Package for the Social Sciences (SPSS, version 27.0; IBM, Armonk, NY, United States) was used for all statistical analyses. Continuous variables were tested for normality using the Shapiro-Wilk test. Normally distributed data were expressed as the mean ± SD, whereas non-normally distributed data were expressed as the median and interquartile range. Categorical variables were expressed as frequencies or percentages, as appropriate. For group comparisons, the independent sample T test was used for normally distributed data; and the Mann-Whitney U test was used for non-normally distributed data. Spearman correlation analysis was performed to assess potential associations between BoNT-A treatment and anxiety and depressive symptoms, with motor symptoms taken into consideration. These analyses were conducted for the total group, as well as across subgroups categorized by BoNT-A treatment history. Multivariate logistic regression analyses were used to further explore the relationship between BoNT-A treatment (independent variable) and anxiety and depressive symptoms (categorical dependent variables) when controlling for potential confounders, such as age, sex, education, age at disease onset, and frequency of BoNT-A injections. The scale scores and subscale scores from assessments were treated as the categorical dependent variables. Based on each scale score range, we categorized the SDS total scale into three groups: Moderate (≥ 32), mild (30-31) and normal (25-29). Similarly, we grouped SDS well-being index into three categories: Moderate (> 6), mild (6) and normal (5). BoNT-A treatment [no (1), yes (2)] was treated as the independent variables and dichotomized, with the first category serving as the reference. Subgroup analyses stratified by BoNT-A treatment history (BoNT-naïve and repeated injection) were performed to assess the effects of multiple BoNT-A treatments on anxiety and depressive symptoms in blepharospasm patients. All P values were two-tailed, and the significance level was set at 0.05.

RESULTS

A total of 21 patients with benign essential blepharospasm were recruited from Nanjing Brain Hospital between September 1, 2022 and June 30, 2024. Flow diagram of inclusion and exclusion are shown in Figure 2. The patients’ demographic and clinical characteristics at baseline are presented in Table 1. The mean age was 58.4 ± 9.5 years with a male-to-female ratio of 1:6. The mean age at onset of motor symptom was 50.8 ± 9.5 years, and the patients had received BoNT-A injection an average of 9.3 ± 7.6 times. The mean evaluation endpoint was 23.4 ± 6.1 weeks post-BoNT-A injection. Among these patients, three were BoNT-naïve, while the remaining eighteen received ongoing treatment with repeated injections.

Figure 2 Flow diagram describing the study population, number included and excluded. BoNT-A: Botulinum toxin A; BEB: Benign essential blepharospasm.

Table 1 Demographic and clinical characteristics of eligible patients at baseline, n (%)/mean ± SD.

	Patients (n = 21)
Age, years	58.4 ± 9.5
Sex (female)	18 (85.7)
Education
Less than high school	14 (66.7)
High school	4 (19.1)
Bachelor or higher	3 (14.3)
Age at onset (years)	50.8 ± 9.5
Frequency of BoNT-A injection	9.3 (7.6)
Endpoint of evaluation (weeks)	23.4 ± 6.1
Assessment of anxious symptoms
SAS total score (median, IQR)	30.0 (25.5, 35.5)
Anxiety and panic	5.0 (5.0, 7.5)
Somatic control	6.0 (5.0, 8.0)
Vestibular sensations	5.0 (5.0, 5.5)
Gastrointestinal/muscular sensations	6.0 (5.0, 8.0)
HAMA (median, IQR)	2.0 (0, 3.5)
Assessment of depressive symptoms
SDS total score (median, IQR)	30.0 (25.0, 34.0)
Well-being index	5.0 (5.0, 8.0)
Depressed mood index	3.0 (3.0, 3.0)
Somatic symptoms index	2.0 (2.0, 3.0)
Optimism index	2.0 (2.0, 3.0)
Items not clustered into an index	10.0 (8.0, 12.0)
HAMD (median, IQR)	2.0 (0, 3.0)
Assessment of motor symptoms
JRS (median, IQR)	6.0 (5.0, 7.0)
JRS-severity	3.0 (2.0, 3.5)
JRS-frequency	3.0 (3.0, 3.0)
BSDI (median, IQR)	1.0 (0.7, 1.9)

BoNT-A: Botulinum toxin A; SAS: Self-rating Anxiety Scale; IQR: Interquartile range; HAMA: Hamilton Anxiety Scale; SDS: Self-rating Depression Scale; HAMD: Hamilton Depression Scale; JRS: Jankovic Rating Scale; BSDI: Blepharospasm Disability Index.

As showed in Table 2, the comparisons of indices related to motor symptoms, anxiety and depression revealed a decrease in SAS the item scores for inability to sit still (P = 0.019) and headache (P = 0.039), as well as in the SDS well-being index subscale (P = 0.029) including the items of decreased capacity (P = 0.019) and hard to decide (P = 0.019) following BoNT-A treatment. Marginal differences between baseline and treatment were found in the scores of HAMA (P = 0.094), the SAS gastrointestinal/muscular sensations subscale including the items of stomach pain and indigestion (P = 0.076) and frequent urination (P = 0.075), and the scores of the SDS optimism index subscale (P = 0.091) including the items of thought disorder (P = 0.081) and despair (P = 0.081), and SDS items of feeling of uselessness (P = 0.052) and emptiness (P = 0.094). In parallel, no significant differences between baseline and treatment were observed in BSDI or JRS scores or their subscales.

Table 2 Comparison of motor symptom, anxious and depression scales and their subscale scores in blepharospasm between baseline and treatment, median (interquartile range).

	Baseline (n = 21)	Treatment (n = 21)	P value
Anxious and subscale scores
SAS	30 (25.5, 35.5)	27 (25, 30)	0.211
Anxiety and panic	5 (5, 7.5)	5 (5, 6)	0.580
Anxiety	1 (1, 1)	1 (1, 1)	0.712
Fear	1 (1, 1)	1 (1, 1)	1.000
Panic	1 (1, 2)	1 (1, 1.5)	0.429
Feeling crazy	1 (1, 1)	1 (1, 1)	0.299
Flushed face	1 (1, 1)	1 (1, 1)	0.288
Somatic control	6 (5, 8)	5 (5, 7.5)	0.441
Premonition of misfortune	1 (1, 2)	1 (1, 1.5)	0.755
Inability to sit still	1 (1, 1.5)	1 (1, 1)	0.019a
Dyspnea	1 (1, 1)	1 (1, 1)	0.317
Hyperhidrosis	1 (1, 1.5)	1 (1, 1)	0.738
Sleep disorder	1 (1, 2)	1 (1, 2)	0.808
Vestibular sensations	5 (5, 5.5)	5 (5, 6)	0.601
Trembling hands and feet	1 (1, 1)	1 (1, 1)	0.554
Palpitation	1 (1, 1)	1 (1, 1)	0.973
Dizziness	1 (1, 1)	1 (1, 1.5)	0.244
Syncope	1 (1, 1)	1 (1, 1)	0.317
Tingling of hands and feet	1 (1, 1)	1 (1, 1)	0.592
Gastrointestinal/muscular sensations	6 (5, 8)	5 (5, 6)	0.134
Headache	1 (1, 2)	1 (1, 1)	0.039a
Fatigue	1 (1, 1.5)	1 (1, 2)	0.407
Stomach pain and indigestion	1 (1, 1)	1 (1, 1)	0.076
Frequent urination	1 (1, 1.5)	1 (1, 1)	0.075
Nightmare	1 (1, 1)	1 (1, 1)	0.408
HAMA	2 (0, 3.5)	0 (0, 1.5)	0.094
Depression and subscale scores
SDS	30 (25, 34)	26 (25, 28.5)	0.096
Well-being index	5 (5, 8)	5 (5, 5)	0.029a
Decreased capacity	1 (1, 1.5)	1 (1, 1)	0.019a
Hard to decide	1 (1, 1.5)	1 (1, 1)	0.019a
Feeling of uselessness	1 (1, 2)	1 (1, 1)	0.052
Empty-minded	1 (1, 1.5)	1 (1, 1)	0.094
Loss of interest	1 (1, 1)	1 (1, 1)	0.158
Depressed mood index	3 (3, 3)	3 (3, 3)	0.607
Dejection	1 (1, 1)	1 (1, 1)	0.639
Weepiness	1 (1, 1)	1 (1, 1)	0.317
Sense of worthlessness	1 (1, 1)	1 (1, 1)	0.317
Somatic symptoms index	2 (2, 3)	2 (2, 3)	0.924
Palpitation	1 (1, 1)	1 (1, 1)	0.152
Fatigue	1 (1, 1)	1 (1, 2)	0.435
Optimism index	2 (2, 3)	2 (2, 2)	0.091
Thought disorder	1 (1, 1.5)	1 (1, 1)	0.081
Despair	1 (1, 1.5)	1 (1, 1)	0.081
Items not clustered into an index	10 (8, 12)	9 (8, 10.5)	0.327
Heavy in the morning and light at night	1 (1, 2)	1 (1, 1)	0.334
Sleep disorder	1 (1, 3)	1 (1, 2.5)	0.820
Loss of appetite	1 (1, 1)	1 (1, 1)	1.000
Hyposexual interest	1 (1, 1)	1 (1, 1)	0.592
Weight loss	1 (1, 1)	1 (1, 1)	0.317
Constipation	1 (1, 1)	1 (1, 1)	0.576
Insecurity	1 (1, 1)	1 (1, 1)	0.311
Irritability	1 (1, 1.5)	1 (1, 1)	0.419
HAMD	2 (0, 3)	1 (0, 3)	0.395
Motor symptom and subscale scores
JRS	6 (5, 7)	6 (4, 6.5)	0.393
JRS-severity	3 (2, 3.5)	3 (2, 3)	0.685
JRS-frequency	3 (3, 3)	3 (2, 3)	0.190
BSDI	1 (0.71, 1.9)	0.83 (0.35, 1.415)	0.255

^aP < 0.05.

SAS: Self-rating Anxiety Scale; HAMA: Hamilton Anxiety Scale; SDS: Self-rating Depression Scale; HAMD: Hamilton Depression Scale; JRS: Jankovic Rating Scale; BSDI: Blepharospasm Disability Index.

Spearman correlation analysis results are shown in Figure 3 and Supplementary Table 1. BoNT-A treatment was significantly and negatively associated with the scores of the SDS well-being index (r = -0.342, P = 0.027) subscale, including the items of decreased capacity (r = -0.368, P = 0.017), and difficulty deciding (r = -0.367, P = 0.017), and the item scores of the SAS inability to sit still (r = -0.367, P = 0.017) and headache (r = -0.322, P = 0.038). No correlations between BSDI or JRS scores and SAS, SDS, HAMA or HAMD scores were observed in the total sample.

Figure 3 Heat map of correlation between botulinum toxin A therapy and Jankovic Rating Scale, Blepharospasm Disability Index, Self-rating Anxiety Scale, Hamilton Anxiety Scale, Self-rating Depression Scale and Hamilton Depression Scale total and their subscale scores in a whole group and subgroups. ^aP < 0.05, N: No significance. BoNT-A: Botulinum toxin A; SAS: Self-rating Anxiety Scale; HAMA: Hamilton Anxiety Scale; SDS: Self-rating Depression Scale; HAMD: Hamilton Depression Scale; JRS: Jankovic Rating Scale; BSDI: Blepharospasm Disability Index.

After controlling for other variables, stepwise multiple logistic regressions showed (Table 3) that BoNT-A treatment was an independent predictor of the SDS total score and the SDS well-being index in blepharospasm patients, i.e., with each additional BoNT-A injection, the odds ratio (OR) for patients having an SDS ≥ 32 decreased by 92.6% [OR = 0.074, 95% confidence interval (CI): 0.008-0.678, P = 0.021] and the OR for having an SDS well-being index > 6 decreased by 90.2% (OR = 0.098, 95%CI: 0.011-0.892, P = 0.039).

Table 3 Stepwise multiple logistic regression about Self-rating Depression Scale and Self-rating Anxiety Scale total and their subscale scores with botulinum toxin A treatment.

Index1	Category1	OR2	P value2	95%CI2
SDS total score	25-29	Reference
	30-31	0.588	0.559	0.099-3.491
	≥ 32	0.074	0.021a	0.008-0.678
SDS well-being index	5	Reference
	6	0.684	0.795	0.039-11.949
	> 6	0.098	0.039a	0.011-0.892

^aP < 0.05.

¹Dependent variables.

²Independent variable (botulinum toxin A).

SDS: Self-rating Depression Scale; OR: Odds ratio; CI: Confidence interval.

To explore the effect of repeated BoNT-A injections on anxiety and depression, the subgroup analyses were conducted. In the repeated BoNT injection subgroup, significant differences between baseline and treatment were observed for the SAS items inability to sit still (P = 0.018), the SDS well-being index subscale including the items of decreased capacity (P = 0.036) and hard to decide (P = 0.036), and the SDS item thought disorder (P = 0.036) (Supplementary Table 2). In this sub-group, the correlation analysis revealed that the SAS inability to sit still scores (r = -0.401, P = 0.015), and the SDS items decreased capacity (r = -0.354, P = 0.034), difficulty deciding (r = -0.354, P = 0.034) and thought disorder (r = -0.354, P = 0.034) were negatively correlated with BoNT-A injection (Figure 3 and Supplementary Table 1). Furthermore, subgroup stepwise multiple logistic regressions confirmed that the incidence of SDS ≥ 32 (OR = 0.092, 95%CI: 0.010-0.877, P = 0.038) significantly decreased in patients who received repeated BoNT injections. Moreover, no significant differences were found in the BoNT-naïve sub-group. Due to the exceedingly small sample size, formal statistical comparison for the BoNT-naïve subgroup was not feasible; results are presented descriptively only.

DISCUSSION

This study demonstrates that repeated treatment with BoNT-A maintains anti-anxiolytic and antidepressant effects even after its effects on facial muscle activity have subsided in blepharospasm. We found that a single injection of BoNT-A influences anxious and depressive symptoms, with improvement observed in the SDS well-being index, specifically, decreased capacity and difficulty deciding when control of motor symptoms had already diminished in the overall blepharospasm cohort. BoNT treatment was identified as an independent intervention factor for the SDS total score, particularly, the well-being index in blepharospasm patients. Notably, BoNT treatment relieved both anxiety and depressive symptoms in patients with repeated injections, whereas a single BoNT-A injection did not improve the anxiety or depressive symptoms in BoNT-naïve patients.

Our findings have provided new insights into the effects of BoNT-A therapy on depressive and anxiety symptoms in blepharospasm patients. First, we observed that a single BoNT-A injection decreased SDS total scores in blepharospasm patients even after its effect on motor symptoms had subsided. Multiple logistic regression analysis confirmed that BoNT-A treatment was an independent factor in the improvement of depressive symptoms in blepharospasm patients, particularly, in those who received multiple BoNT-A injections. This suggests that after attenuation of its motor symptom effect, BoNT-A treatment continues to alleviate depression in blepharospasm patients. Moreover, as BoNT-A treatment is an independent factor for depressive symptom improvement, these antidepressant effects are unlikely to be secondary to the restoration of regular facial muscle activity in the specific state.

Second, to identify the important symptom dimensions and evaluate the relationship between BoNT-A therapy and specific symptom domains, we analyzed subscale scores. Initially, no significant difference was observed between pre- and post-BoNT injection in SAS total scores for anxiety. However, contrasted groups with similar total scores may present different patterns of item endorsement. By examining subscale scores, we identify specific areas of change; after BoNT-A had lost its control over motor symptoms, it appeared to improve somatic anxiety symptoms, including headaches, neck and back pain and inability to sit still. Somatic anxiety is an important component of overall anxiety symptomatology. A previous study with a relatively short follow-up duration found that BoNT-A therapy significantly improved both somatic and mental anxiety factor scores at 12 weeks post-treatment[30]. In contrast, our findings indicate that BoNT-A is prone to alleviate somatic anxiety but not mental anxiety at a relatively long follow-up.

BoNT holds excellent potential for pain treatments. It has been used for several chronic pain diseases, including, headaches and neuropathic pain[31,32]. Blepharospasm patients frequently complain of facial pain or headache. It was reported that BoNT-A and onabotulinumtoxinA significantly improved scores of pain-related disability and depressive and somatic symptoms after 6 months[33]. Initially, the analgesic effect of BoNT-A has been considered secondary to its action for muscle relaxation. Recently, BoNT-A has also been shown to affects the release of neurotransmitters involved in pain perception[8]. BoNT-A interferes with sensory transmission through its interaction with the heterotrimeric soluble N-ethylmaleimide-sensitive factor-attachment protein receptor complex[34], which inhibits the release of neurotransmitters, such as glutamate, substance P, and calcitonin gene-related peptide[35]. Our findings support evidence that BoNT-A may improve anxiety originating from headaches neck and back pain.

Additionally, it is interesting that our injection sites for blepharospasm treatment are in the local glabella region, where the inability to sit still symptoms are considered a systemic somatic symptom. In theory, peripheral BoNT-A injection at the local site is unable to improve systemic symptoms. However, there are reports that BoNT injections have been reported to be helpful in some patients with tics, tardive and levodopa-induced dyskinesia, and restless legs syndrome[36]. It is speculated that the results of BoNT-A in the amelioration of inability to sit still would be expected to be similar to those of levodopa-induced dyskinesia or restless legs syndrome. Overall, we suggest that BoNT-A treatment may relieve anxious symptoms by cessation of cholinergic transmission in the somatic nervous systems.

Third, it was found that BoNT-A therapy focused on improving the SDS well-being index subscale through alleviation of decreased capacity, difficulty in decision, feelings of uselessness and empty-minded symptoms, according to the assessment of SDS subscale scores. Evidence that BoNT-A therapy with compound positive impact on social health and psychological well-being were supported in aesthetic procedures[37]. However, unlike evaluation scales of well-being used in cosmetology or other fields, the well-being subscale in the SDS contains items whose literal phrasing suggests a positive state, but whose content is equivalent to symptoms such as psychomotor retardation, confusion, and hopelessness, and underlines the possibility that the respondent may be reacting to the positive element in the items rather than to the specific symptom (or lack of it) that the item is supposed to represent[29]. Although, BoNT-A therapy is known to have beneficial effects on headaches, suppressing excessive sweating, and tremors, which could plausibly influence well-being index in blepharospasm[38-40], our results, unexpectedly, showed that BoNT-A therapy was also an independent influencing factor in decreasing the well-being index in blepharospasm patients. We speculate that the effect of BoNT-A on the well-being index is not secondary to motor symptom improvement, but may represent another independent therapeutic pathway.

Fourth, we observed that repeated BoNT treatment ameliorates anxious or depressive symptoms, whereas the single BoNT-A injection did not improve the anxiety and depressive symptoms in BoNT-naïve patients. Typically, the duration of BoNT action in movement disorder treatment spans 3-4 months[20]. During this period, motor axon sprouts form at the presynaptic nerve terminals, and the original nerve terminals regain exocytic function, leading to the restoration of neuromuscular transmission[20], so repeated injections are required. The experiments of Rogozhin et al[40] in mice reported that neuromuscular junction (NMJ) remodeling often results in NMJs with abnormal structure but substantially normal function, moreover, and that recovery of NMJs in the mouse epitrochleoanconeus muscle is slower after multiple exposures to BoNT-A, due to the accumulation of structurally abnormal NMJs after the initial exposure[41]. Hence, their findings may explain that BoNT-A’s sustainable effectiveness is possible after multiple exposures.

Lastly, our results presented that the anxiolytic and antidepressant effects of BoNT-A outlast its motor effects in blepharospasm patients. Generally, BoNT-A injections in the glabellar region have long been proposed to improve mood by disrupting the feedback loops that reinforce negative emotions and by enhancing positive social interactions in the correct context[15]. With a deeper understanding of the mechanisms of BoNT action, recent evidence indicates that beyond its well-known local effects at the NMJ, BoNT can affect central circuits[42]. Some studies have revealed a novel neuronal trafficking pathway in which BoNT-A is retrogradely transported and trans-synaptically migrated. For example, cleavage of the BoNT-A substrate SNAP-25 was detected in the rat facial nucleus following delivery of BoNT-A to the whisker pad[43]. BoNT is taken up by peripheral afferents and transported to central terminals, where it inhibits neurotransmitter release, thereby decreasing the activation of second-order neurons[44]. As a results, BoNT-A may remotely act on the central nervous system following peripheral injection[45,46]. We speculate that the transmembrane synapse hypothesis-whereby BoNT-A triggers central nervous system and systemic effects-may lead to the relief of depressive and anxious symptom in the absence of motor symptom improvement.

Limitation

First, the small sample size of BoNT-A-naïve patients may cause false negatives, although the subgroup findings are just preliminary. Preliminary observations in our small cohort indicate no effect in BoNT-naïve patients, but this requires validation in a larger sample. Second, the subscales of HAMD and HAMA were not assessed, because the patients in our study did not present severe depressive and anxious symptoms. Finally, the influence of BoNT-A injections on central nervous system mechanisms related to alleviating depressive symptoms in blepharospasm patients were not contained in this study. For subsequent studies, it is feasible to consider adding functional magnetic resonance imaging examinations to observe activity changes in brain regions related to emotion (such as the amygdala and prefrontal cortex), or detecting the concentrations of neurotransmitters related to anxiety and depression (e.g., serotonin, glutamate) in serum/cerebrospinal fluid. Meanwhile, electromyography can be used to quantify the degree of delayed recovery of NMJs and correlate it with the improvement of emotional symptoms, so as to verify the hypothetical mechanisms.

CONCLUSION

This study demonstrates that repeated treatment with BoNT-A maintains anti-anxiolytic and antidepressant effects when its effect on facial muscle activity is over a course of treatment in blepharospasm. It is found that a single injection of BoNT-A influences anxiety and depressive symptoms when it has been out of the control for motor symptoms. BoNT treatment is an independent intervention factor of SDS total score, especially, well-being index. These phenomena of depression relief are possible not secondary to an establishment of a normal to facial muscle activity in the certain state, but be another independent approach. The transmembrane synapse hypothesis triggering central nervous system and systemic effects may the reason of the effect of BoNT-A on depressive and anxious symptom relief in the absence of motor symptoms. Moreover, it is interesting that BoNT treatment may relieve anxiety and depressive symptoms in patients with repeated treatment, while the single BoNT-A injection does not improve emotions in BoNT-naïve patients. Multiple exposures to BoNT-A prolonging recovery of the NMJ may be likely to sustainable effectiveness.

ACKNOWLEDGEMENTS

We would like to thank the Department of Geriatrics of Nanjing Brain Hospital for the cooperation and medical support to our study. And we are thankful to the patients and their caregivers for providing information. We thank for Jing Wu from School of Biomedical Engineering and Informatics, Nanjing Medical University (Nanjing, China) reviews the statistical methods of this study.