Study Design

This was a prospective case series study performed at Zhongshan Ophthalmic Center, Sun Yat-sen University, China. The patients were prospectively enrolled from January 1, 2011 through October 31, 2011. The study was approved by the ethics committee of Zhongshan Ophthalmic Center, Sun Yat-Sen University. Informed consent for the study was obtained at the time of enrollment. The study adhered to the tenets of the Declaration of Helsinki.

Eligibility

Forty Chinese adults with a diagnosis of benign essential blepharospasm and dry eye disease (Group 3) were consecutively recruited from Zhongshan Ophthalmic Center. The diagnosis of BEB was made according to published standard criteria. Patients with neurologic abnormalities in addition to dystonia and patients with features suggesting dopa-responsive dystonia, myoclonus-dystonia, or secondary dystonias were excluded. Subjects with ocular surface disease, such as keratitis or conjunctivitis, were also excluded. Diagnoses were confirmed by the senior neurologist with extensive experience in movement disorders. Forty subjects with the diagnosis of dry eye disease (Group 2) and 40 unrelated healthy participants (Group 1) were recruited as controls, matched for age and sex.

Further inclusion criteria included the presence of typical symptoms of dry eye disease (dryness, burning, foreign body sensation, ocular pain, photophobia, or easily fatigued eye) and a markedly reduced tear break-up time and Schirmer І test according to the dry eye criteria by Lemp and Pflugfelder and associates. All of the patients diagnosed with DED or DED + BEB were not administered any topical or other DED treatments during the study period.

Assessments

For the diagnosis of dry eye disease and the evaluation of symptoms, each participant’s subjective reports were evaluated after objective observation using a validated Chinese-translated version of the Ocular Surface Disease Index questionnaire (OSDI). The patients in Group 3 were assessed with OSDI again at 1 week after neurotoxin administration. Total OSDI scores were calculated as previously recommended by Schiffman and associates.

Immediately after completing the questionnaire, all subjects underwent a thorough ophthalmologic examination of the anterior segment. Ophthalmologic evaluation of each participant was performed by the same specialist. Ocular examinations consisted of tear break-up time (BUT), the Schirmer І test, and fluorescein staining.

For fluorescein staining, fluorescein strips were wetted with a standardized drop volume of nonpreserved saline solution. The cornea was examined 3 minutes after the last instillation by light passed through a cobalt-blue filter and examined through a biomicroscope containing a Wratten #12 barrier filter. The results were recorded on a cornea diagram as shown in Figure 1 . Punctate staining was recorded using a standardized grading system of 0-3 for each of the 5 areas.

Sketch map showing the division of the corneal surface for measuring fluorescein and standards for clinical scoring of corneal fluorescein staining. A standardized grading system of 0-3 is used for each of the 5 areas on each cornea.

Tear BUT was used to evaluate the stability of the tear film by measuring the time required for the film to break up after complete blinking. BUT was measured by contacting the inferior conjunctiva with a commercially available fluorescein test strip. Subjects were instructed to blink 3 times, followed by gazing straight ahead without blinking. The tear film was observed using a yellow filter under wide lighting. The interval between the last blink and the appearance of the first corneal dry spot was measured. The procedure was repeated 3 times, and the mean value was recorded.

The Schirmer I test (with anesthesia) quantifies tear production. One minute after instillation of the topical anesthetic drops (0.5% proxymetacaine; Alcaine, Alcon, Fort Worth, Texas, USA), a Schirmer strip (filter paper) was placed on the lower conjunctiva between the lateral and the external third. The distance that the tears traveled on the filter paper was measured after 5 minutes. A measured distance less than 5 mm with anesthesia was considered abnormal and indicative of an aqueous-deficient state.

Impression cytology was performed in all subjects. Sheets of cellulose acetate filter paper (type VC, 0.10 μm, VCWP 04700; Millipore Corp, Bedford, Massachusetts, USA) were used to collect cells from the superior and inferior bulbar conjunctiva. The specimens were stained using the procedure previously described by Tseng and examined through a light microscope. Between 0 and 3 points were added from each of the following parameters to calculate the impression cytology (IC) score: the morphology of the epithelial cells; cell-to-cell contact; grade of squamous metaplasia (nucleus-to-cytoplasm ratio); appearance of nuclear chromatin (pyknosis, “snakes,” fragmentation); keratinization; the distribution, number, and morphology of goblet cells; the number and type of inflammatory cells; and mucous aggregation. A total of 0-7 points was typical for normal cell morphology, 8-14 points for slightly pathologic, 15-21 points for moderately pathologic, and 22-30 points for intense pathologic changes.

Multiplex Cytokine Immunobead Assay

Tears were collected before any other test and at a minimum of 10 minutes after the patient had completed the questionnaire. Unstimulated tear samples were collected nontraumatically from the external canthus of open eyes in each participant, avoiding an additional tear reflex as much as possible. Glass capillary micropipettes (Microcaps 2 μL; Drummond Scientific, Broomall, Pennsylvania, USA) were used to collect 10 μL of tears. Each sample was then collected in a sterile collection tube at 4 C and stored at −80 C until assayed. The samples were obtained from the right eye and left eye of each individual and were not pooled.

Cytokine levels in tear samples were determined by a Human Multi Analyte Profiling Base Kit A (Fluorokine MAP; LUH000; R&D Systems, Wiesbaden, Germany) using a Luminex 200 detector (Luminex, Austin, Texas, USA) according to the manufacturer’s instructions. Briefly, tear samples were added to wells containing an appropriate cytokine bead mixture that included human monoclonal antibodies specific for interleukin (IL)-1β, IL-2, IL-6, IL-8/CXCL8, IL-10, IL-17, tumor necrosis factor (TNF)-α, and vascular endothelial growth factor (VEGF) and incubated overnight at 4°. Washed beads were further incubated with biotin-labeled anti-human cytokine antibodies for 1 h at room temperature followed by incubation with streptavidin-phycoerythrin for 30 minutes. Samples were analyzed using Luminex 200 and Statlia software (Brendan Technologies Inc, Carlsbad, California, USA). Standard curves of known concentrations of recombinant human cytokines were used to convert median fluorescence intensity (MFI) to cytokine concentration in pg/mL. Only the linear portions of the standard curves were used to quantify cytokine concentrations.

Botulinum Neurotoxin Type A Injection for Treatment of Benign Essential Blepharospasm and Dry Eye Disease Patients

The group of 40 Chinese patients with DED accompanied by BEB received treatment with botulinum neurotoxin type A injections. By diluting 100 U of the botulinum neurotoxin type A (Allergan, Inc, Irvine, California, USA) in 2 mL of saline that included no preservatives (0.9% NaCl) before use, a solution with a working concentration of 5 U botulinum neurotoxin type A/0.1 mL was obtained. The dose of botulinum neurotoxin type A per injection site was 2.5 U. The injection locations are presented in Figure 2 . To prevent drug interactions, no other drugs were used during the study.

Sketch map showing the botulinum neurotoxin A injection sites for patients with dry eye disease accompanied by benign essential blepharospasm.

Examinations After Treatment

According to the procedures above, the posttreatment examinations, including the OSDI questionnaire, tear film BUT, Schirmer I test (with anesthesia), fluorescein staining, and impression cytology, were conducted 1 week after the botulinum neurotoxin type A injections. In addition, the severity of the contractions in the eyebrow and eyelid were recorded before botulinum neurotoxin type A injection and 1 week later. The classification of severity was based on Cohen’s blepharospasm evaluation scale from 0-4: 0 for none; 1 for increased blinking caused by external stimuli; 2 for mild, noticeable fluttering that is not incapacitating; 3 for moderate, very noticeable spasm that is mildly incapacitating; 4 for severely incapacitating spasm (unable to drive, read, etc). Moreover, tears of 6 patients after treatment were collected for multiplex cytokine immunobead assay.

Statistical Analysis

The data are presented as the arithmetic mean ± standard deviation. Comparisons of sex and symptoms were performed with the χ ² test. Student t test or analysis of variance (ANOVA) with Tukey post hoc test was used for other statistical comparisons. P ≤ .05 was considered statistically significant. All statistical tests were performed using GraphPad Prism 5.0 software (Graph-Pad Prism, Inc, San Diego, California, USA; http://www.graphpad.com ).