Quantitative assessment of botulinum toxin injection on blink rate in blepharospasm

ABSTRACT Purpose To objectively measure the blink rate in patients with blepharospasm managed by botulinum toxin type-A injections. Methods In this prospective, non-interventional case series, the complete blink rates of subjects were measured before incobotulinumtoxina injection and at follow-up within 4 weeks using slow-motion video-taping. Additionally, subjects graded the frequency of blinking, the severity of light-sensitivity, and the severity and frequency of dry eye symptoms on a categorical visual analog scale. The results are reported as median (range). Results Ten subjects were enrolled, with nine females. The total duration of treatment was 70 (5–116) months with total of 27.5 (2–51) injections. The subjects were grouped as short-time (<52w) or long-time (>52w) treatments. The median age, follow-up time, and injected doses were 73.5 (49–81) years, 21 (14–28) days, and 38 (8–47) units, respectively, with no significant difference between groups. The total complete blinks per minute before incobotulinumtoxina injection was 39 (23–64) which decreased to 18.5 (1–60) at follow-up (p = 0.004). The average change in complete blink rate was −67.4 ± 23.7% in long-time and −45.2 ± 31.2% in short-time groups (mean ± SD, p = 0.01). The total self-graded frequency of blinking and light-sensitivity decreased significantly at follow-up (p = 0.004, p = 0.047, respectively). Similar patterns of subject reported grades were seen in both groups. Conclusion Videotaping is a low-cost method for objective measurement of blink rate in blepharospasm patients after incobotulinumtoxina injection. There was a significant reduction in blink rate after incobotulinumtoxina injections with higher percentage of change in the long-time treatment group. Incobotulinumtoxina injection also significantly improves subjective photophobia.


Introduction
Benign essential blepharospasm (BEB) is a focal dystonia manifested by involuntary eyelid protractor and periorbital muscle spasms leading to uncontrolled closure of the eyelids, repetitive facial and eyelid movements and twitching, and increased frequency of spontaneous blinking. 1BEB is often a function-altering condition which interferes with daily activities such as reading, driving, and working. 2 Moreover, photooculodynia and light-sensitivity are debilitating characteristics of BEB in the majority of cases, which may not resolve after improvement of involuntary motor movements by surgical and medical treatments. 3lepharospasm can also have a significant relation with ocular surface diseases.Eye discomfort associated with ocular surface diseases may cause secondary (reflex) blepharospasm with eyelid contractions and increased rate of blinking.Secondary blepharospasm should be distinguished from essential blepharospasm and is usually managed by treating the underlying ocular surface condition in addition to adjunct botulinum toxin injection. 4,5It is also important to recognize that dry eye diseases are very commonly observed concurrently in cases of benign essential blepharospasm. 6he most common treatment of BEB is injection of botulinum toxin type-A into the eyelid protractors to reduce involuntary eyelid contractions and spasms. 7otulinum toxin injections have also been reported to be a useful adjunct in treating secondary blepharospasm. 5The treatment dose, pattern, and interval between injections varies for each individual patient and should be customized based on clinical findings and therapeutic response. 8The designs of most studies evaluating the effectiveness of peri-ocular and eyelid muscle botulinum toxin injections on blepharospasm signs and symptoms (such as blink rate) are mainly based on using subjective self-graded questionnaires completed by the subjects. 9In this study, the effect of botulinum toxin injection on the blink rate is objectively measured.Also, subject-reported effects of botulinum toxin injections on the frequency of blinking, the severity of light sensitivity, severity and frequency of dry eye symptoms are assessed using a visual analog scale.

Patients and methods
In this prospective non-interventional case series, subjects with the diagnosis of benign essential blepharospasm (BEB) or secondary blepharospasm managed with peri-ocular and eyelid protractor muscle injection of incobotulinumtoxina (IBTX-A) (Xeomin, Merz Therapeutics, Frankfort, Germany) were enrolled.The subjects visited from July to November 2021 at the Alkek Eye Center, Baylor College of Medicine, Houston, TX.This study was approved by the Institutional Review Board (IRB) of Baylor College of Medicine, and all subjects were recruited after oral and written informed consent.All injections were performed by the same oculofacial surgeon (MTY) using the technique as previously described. 10The decision for the dose of IBTX-A to inject was made based on the severity of the symptoms and prior responses to the treatment.All previous injections of the enrolled subjects were performed by the same surgeon.
The subjects were evaluated immediately before injection of IBTX-A (at least 3 months after their last injection), and within 4 weeks after injection for followup.Subjects were instructed not to change their dry eye therapy during this period.The demographic data and medical histories were recorded by chart review.The gathered data included age, gender, duration, and numbers of IBTX-A injections, and the management of dry eye symptoms.

Self -graded questionnaire with visual analog scale
At each visit, a self-graded questionnaire (Supplementary Material S1) was completed by subjects including four questions to grade the frequency of blinking, severity of light-sensitivity, severity of dry eye symptoms and frequency of dry eye symptoms on a 100 mm (equally divided into 5 increments) visual analog scale (0, normal, and 5, severe).The other two questions were whether the IBTX-A injection (1) decreases the frequency of their blinking and (2) reduces the severity of their light-sensitivity, which the subjects were choosing as one of the options of "Yes," "No," or "Maybe."

Measurement of blink rate
To measure the number of complete blinks, the subjects' faces were video recorded while watching a threeminute movie clip (all video recordings were performed by an iPhone 12 pro with the same camera zoom and 1080p HD slow-motion at 240 fps).The same setup in a single examination room was used for all visits.The same movie clip was played, the distance to the display was 1 m at the level of patients' eyes, the display was 17" in size using the same screen brightness, and room illumination during all exams.The face video recordings were used to count the number of complete blinks over 3 minutes.All videos were watched by the same two individuals (GY and SCP), and the counted blinks were averaged.Only complete blinks (obvious touching of eyelid margins in slow-motion videos) were counted.The number of blinks per minute was calculated by dividing the total number of blinks in 3 minutes by 3. The blink rate change was calculated using the following formula:

Statistical analysis
The data are presented as median (range) or mean ± standard deviation (SD).Non-parametric paired comparisons were performed using Wilcoxon matched-pairs signed rank test for before-after comparisons.Mann-Whitney test was used for non-parametric comparisons between the groups.Student's T-test was performed for parametric comparisons.P-value lower than 0.05 is considered significant.All statistical analyses were performed using GraphPad Prism version 9.3.0 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com.

Demographic features
Ten subjects with benign essential blepharospasm or secondary blepharospasm being treated with botulinum toxin injections were enrolled in this prospective interventional case series.In total, nine subjects were female, and one subject was male.The median age of all subjects was 73.5 (range, 49-81) years.The duration between the IBTX-A injection and follow-up assessment was between 14 and 28 days (median, 21 days).The median historical total duration of treatment with IBTX-A injections was 70 (range, 5-116) months, and the median of the historical total number of injections was 27.5 (range, 2-51).Since the duration of treatment with IBTX-A for controlling the symptoms of BEB have a significant effect on the results, the subjects were divided into two groups of subjects under treatment for short-time (less than 50 months) and subjects under treatment for longtime (more than 50 months).The median duration of BTX-injection treatments and number of injections for long-time subjects were 113 (91-116) and 38 (32-51), respectively; while these values were 27 (5-49) and 6 (2-23), respectively, for short-time treatment subjects (p < 0.05 for both comparisons, Table 1).There was no significant difference in median age and follow-up time between short-time and long-time treatment groups (Table 1).

Effect of incobotulinumtoxina injection on blink rate: objective measurements
The rates of complete blinks for subjects were measured before IBTX-A injection and at follow-up while watching a video by slow-motion face videotaping.The total units of injected IBTX-A, and the measured blink rates for each subject are summarized in Table 1.The total median (range) of the injected IBTX-A dose was 38 (8-47) units.The dose of injected IBTX-A was not significantly different between long-time and short-time treatment group subjects (Table 1).The total (all subjects) median (range) blink rate before IBTX-A injection was 39 (23-64) blinks per minute, while the blinking rate was significantly decreased to 18.5 (1-60) blinks per minute (p = 0.004, Table 1) at the post injection follow-up.Although there was no difference in blink rate between the long-time and short-time treatment groups before injection of IBTX-A (27 (23-64) and 52 (28-59), respectively, p = 0.25), the blink rate at followup was significantly different between the two groups (13 (1-30) and 33 (13-60), respectively, p = 0.032).Moreover, on average, the long-time treatment group had a significantly higher percentage of change (decrease) in blink rate (−67.4 ± 23.7%) compared to short-time treatment group (−23.1 ± 20.1%, p = 0.01, Table 1).The total average change in blink rate was −45.2 ± 31.2% (Table 1).

Effect of incobotuilnumtoxina injection on subject reported blinking frequency and severity of light-sensitivity
Table 2 presents the subject reported grade of blinking frequency and light-sensitivity before IBTX-A injection and at follow-up, respectively.The total median (range) of blinking (0, normal and 5, constant blinking) reported by subjects was 4.5 (2-5) before IBTX-A injection, which significantly decreased to 1.75 (0-4.5) at followup (p = 0.004).The significant difference in the grade of frequency of blinking before injection (4 (2-5)) and at follow-up (2 (1-4.5) was found in short-time treatment subjects (p = 0.031, Table 2).There was also a significant difference in the grade of frequency of blinking in longtime treatment subjects (p = 0.048).As summarized in Table 2, all subjects believed that the IBTX-A injections were decreasing the frequency of their blinking.
Although in total, four subjects believed that the treatment had no effect on their light-sensitivity, and six subjects believed that their light-sensitivity might be (four subjects), or definitely (two subjects) improved by the IBTX-A injection (Table 1), the total median (range) of subject reported light-sensitivity (0, none, and 5, severe/constant) significantly decreased from 3.5 (0-5) before IBTX-A injection to 2.75 (0-4) at follow-up (p = 0.023).While the grade of light-sensitivity was not significantly different in each treatment time group (Table 2).

Incobotulinumtoxina injection and subject reported severity and frequency of dry eye symptoms
All the enrolled subjects in this study have been under treatment for dry eye disease.Artificial tears and selective immunosuppressives (cyclosporine or lifitegrast) were the main treatment regiments of the subjects (Supplementary Table S1).The subject reported grade of dry eye symptoms including dry eye severity and dry eye frequency are summarized in Supplementary Table S1.In total, the median (range) grade of severity of dry eye symptoms changed from 2.5 (0-5) before IBTX-A injection to 2 (0-5) at follow-up (p = 0.476).The grade of frequency of dry eye symptoms changed from 3 (0-4) before injection to 1.5 (0-4) at follow-up (p = 0.148).The grade of frequency of dry eye was decreased from 3 (0-3) to 1 (0-2), and the grade of severity of dry eye was decreased from 3 (1-4) to 2 (0-2.5) in long-time treatment group.These grades were changed from 3 (1-4) to 2 (0-2.5) and from 2 (0-5) to 3.5 (0.5-5) in shorttime treatment group, respectively.No significant difference was found in the subjects who reported dry eye frequency and severity grades between before injection and at follow-up in both short-time and long-time treatment groups (Supplementary Table S1).

Discussion
We present the objective measurement of blink rate and subjective assessment of symptoms in blepharospasm patients before and after eyelid and peri-ocular  1).This objective measurement can be valuable in not only assessing the efficacy of the treatment but also in counselling patients regarding treatment options.
From the subjects' perspective, IBTX-A injection significantly attenuated the frequency of blinking, and the severity of light-sensitivity (Table 2).However, although dry eye frequency and severity scores decreased, they were not significantly changed following IBTX-A injection (Supplementary Table S1).][13][14] Here, we are introducing the objective measurement of blink rate as an indicator of clinical response to IBTX-A injection, which allows performing correlational studies.Objective measurement of the blink rate showed an average of 45.2% decrease in blink rate and a median number of 18.5 blinks per minute following peri-ocular injection of IBTX-A.Considering 10-20 blinks per minute as normal, the IBTX-A injection decreased the blink rate of subjects to within a normal range.Subject 1 is an example of the importance of objective measurement of blink rate in BEB cases in addition to subjective measurement.Although this case had significant objective reduction in the blink rate (before injection, 50, follow-up, 2, blinks/min), the subjective score of blinking was not dramatically reduced (before injection, 5, follow-up, 3).This could be due to self-sense of the subject's blinking which considers subtle incomplete blinks as complete blinks.In accordance with our results, Ferrazzano et al. reported the decrease in blink rate in BEB patients 1 month after botulinum toxin injection using a video recording protocol (blinks/min (mean ± SE), 67.6 ± 9 pre-injection, 41.8 ± 9 at follow-up, P < 0.001). 15In a similar study, a high-speed camera and a custommade software were used to measure the blinking parameters in BEB patients and evaluate the effect of botulinum toxin.Significant reduction in blinking frequency, velocity and amplitude was observed in BEB subjects treated with botulinum toxin injections. 16Jang et al. also evaluated the blink profile in blepharospasm patients using an ocular surface interferometer, suggesting patients with higher ages, shorter closure time, and faster closing speed responded better to botulinum toxin-A injection. 17wever, the method using an ocular surface interferometer for evaluation of the blinking might not necessarily assess the usual blinking since the patients need to hold their head in the device rather than having no contact with the device.
In this study, we included both patients with BEB and secondary blepharospasm from ocular surface disease since both groups were presenting for botulinum toxin injections for their eyelid spasms.However, we would not expect significant differences in treatment effect between the two groups of patients.Since the effect of IBTX injections on the orbicularis oculi muscle should be agnostic to the underlying condition, we believe that our objective measurements of blink rate are still valid in considering the effect of botulinum toxin injections in blepharospasm.
Although measuring the blink rate in blepharospasm patients has benefits for clinical management and research purposes, counting the blink rate in the clinic requires a sophisticated and reliable method.Our team recently reported the use of an infrared sensor mounted on a headset to measure the blink rate. 18However, the infrared sensor technology is not capable of distinguishing between incomplete and complete blinks.Therefore, in this study, we used slow-motion facial videotaping to measure the rate of complete blinks.Furthermore, there are several parameters in blinking dynamics, other than blink rate, such as blink velocity, blink amplitude, blink apraxia and blink force that are altered in blepharospasm patients and would impact the visual function and quality of life.6][17] The facial videotaping approach has the potential of evaluating several parameters of blinking dynamics and should be considered in future studies.Further technological and software advancements will help automatize the blink dynamics' measurement.
To better present the obtained data, we divided the enrolled subjects into short-time and long-time treatment groups based on the duration of IBTX-A injection treatments.Higher decrease in the blink rate was found in long-term treatment group compared to short-term treatment group (Table 1).Previous studies reported that there is no requirement to increase the doses of injected IBTX-A 8 ; however, the real-world clinical practice scenarios are different.The injected doses of IBTX-A are individualized for each subject based on clinical judgment of the severity of blepharospasm immediately after diagnosis.The goal is to manage the patients with lowest possible IBTX-A dose, especially in the early phases of the treatment to find the optimal maintenance dose.In the clinical course of BEB as a lifelong disease, the condition progresses and worsens in the first few years and then it stabilizes.This could be the reason that a higher decrease was seen in long-time treatment subjects compared to short-time treatment group.Patients in the long-time group have already been optimized for their treatment dose.However, this does not downgrad the efficiency of IBTX-A injection in reduction of blink rate in short-term treatment group (Table 1).This is again emphasizing the importance of objective measurement of blink rate for efficient management and followup of the cases.The underlying recovery mechanism leading to return of muscle function following injection of IBTX-A is terminal nerve sprouting results in formation of new neuromuscular junctions in affected muscle. 19Moreover, long-term injection of IBTX-A may lead to muscle atrophy. 20Alteration in muscle recovery or muscle atrophy could also be involved mechanisms in higher decrease of blink rate in longtime treatment group.
A prominent debilitating symptom of patients with BEB is photophobia (or light-sensitivity), which significantly alters lifestyle by avoiding outdoor or even indoor activities in well-lighted areas.Among current questionnaires utilized for evaluating the severity of BEB symptoms and signs, there is no direct question about the severity of light-sensitivity.Only in the Blepharospasm Disability Scale (BDS) questionnaire is the presence of photophobia evaluated by asking whether the subject needs to wear sunglasses outdoor and/or indoor. 12owever, this question did not contribute strongly to the reliability of the BDS questionnaire and showed a weak correlation with other items, and it was removed in modified versions of this questionnaire, now called the Blepharospasm Disability Index (BSDI). 1,12,13oreover, peri-ocular IBTX-A injection improved frequency and severity of photophobia in cases with neuropathic ocular pain with symptoms of dryness, burning, and photophobia out of proportion of ocular surface findings. 21In this current study, the subjects were asked to score their severity of light-sensitivity on a visual analog scale before and after IBTX-A injection.A significant improvement in severity of light-sensitivity has been observed following injection of IBTX-A.This could serve as evidence to conduct initiatives for modifying current or designing new questionnaires.
There is also a strong co-incidence between BEB and dry eye diseases (DED). 6It is still unclear whether DED triggers BEB, or BEB results in DED. 22In this study, the enrolled subjects had somewhat similar management for their dry eye condition (Supplementary Table S1).We recorded the subject reported severity and frequency of dry eye before IBTX-A injection and at follow-up using a visual analog scale.No significant difference was found between the previous IBTX-A injection and follow-up grades for both frequency and severity of dry eye.Because of the trend toward improvement, it is possible the improvement might reach significance with a greater sample size.Various results have been reported in previous studies regarding the effect of IBTX-A injection on improvement of dry eye signs and symptoms.Some studies showed subjective and objective improvements in DED following IBTX-A injection, 21,[23][24][25] while other studies have not found significant benefit of IBTX-A injection on DED. 22,26Girard et al. reported that the tear osmolarity and Schirmer's I-test results are not changing after IBTX-A injection, and low production of tears is present in BEB patients before starting the treatment with IBTX-A injection. 22However, Kocabeyoglu et al. reported that the results of tear break-up time (TBUT), lissamine green (LG) staining, and Schirmer's I-test improved temporarily following injection of IBTX-A in BEB patients and returned to baseline 6 months after injection. 27On the other hand, a significant improvement in ocular surface disease index (OSDI) was found compared to the baseline which lasted for 6 months following injection. 27here are limitations to this study that should be addressed in future studies.Higher number of enrolled subjects with comprehensive grading of blepharospasm symptoms, light-sensitivity, and dry eye symptoms would be very helpful for better understanding of the role of IBTX-A injection in management of blepharospasm.Also, identical injection to follow-up duration would eliminate any potential bias in data interpretation.Developing future technologies to measure velocity, and strength of blinking is necessary for inclusion of a broad number of elements and outcomes in the analysis.

Conclusion
This study objectively measured and evaluated the blink rate of blepahrospasm patients before and after incobotulinumtoxina injection.There was a significant reduction in blink rate after incobotulinumtoxina injections.Moreover, generated data indicate the effectiveness of incobotulinumtoxina injection on reducing patients' symptoms of photophobia.Although no significant effect of incobotulinumtoxina injection on patientreported dry eye symptoms was found, future studies are required to objectively evaluate the dry eye disease conditions during the management of blepharospasm patients.

Table 1 .
Demography of included subjects in the prospective interventional case series study and collected/measured parameters.

Table 2 .
Summary of the results of the visual-analog scale questionnaire on frequency of blinking and light-sensitivity.