Study and Patients
In this prospective study, patients with DED were examined and screened for enrolment at a tertiary referral center (Department of Ophthalmology, University of Magna Grecia, Catanzaro, Italy) between March 2022 and December 2022.
The study was approved by the local Ethics Committee (Comitato Etico Regione Calabria – Sezione Area Centro, protocol n. 239–2022). A detailed informed consent for participation in the study was signed by all patients, in accordance with the 1964 Declaration of Helsinki.
Inclusion criterion was the diagnosis of DED not successfully controlled with conventional tear substitutes. Patients were excluded if one or more of the following conditions were present: recent (within 3 months) ocular surgery, systemic disease or therapies affecting tear secretion, concomitant ocular diseases or use of other topical medications (e.g., corticosteroids, NSAIDs). Baseline demographic and clinical characteristics of enrolled patients are summarized in Table 1.
Table 1
Baseline demographic and clinical patient’s characteristics
Age, mean value (SD), years | 67.00 (8.00) |
Sex (M/F) | 23/37 |
Caucasian race, N (%) | 60 (100) |
TMH, median value (IQR), mm | 0.28 (0.21–0.39) |
NIKBUT first, median value (IQR), s | 4.01 (2.87–5.88) |
NIKBUT average, mean value (SD), s | 9.63 (5.03) |
SANDE score, median value (IQR) | 60.60 (52.21–68.90) |
Bulbar redness score, median value (IQR) | 1.35 (1.02–1.60) |
MGL scale, median value (IQR) | 1.50 (1.00–2.00) |
Abbreviations: SD, standard deviation; IQR, interquartile range; TMH, tear meniscus height; NIKBUT, non-invasive breakup time; SANDE, Symptom Assessment in Dry Eye; MGL, meibomian glands loss; mm, millimeters; s, seconds. |
Patients who satisfied study criteria were enrolled and treated with Lacricomplex® according to the following therapeutic regimen: 2 drops 4 times daily for 60 days in both eyes.
Ocular Surface Workup
All patients underwent non-invasive examination of the ocular surface using Keratograph 5M (Oculus, Wetzlar, Germany), before starting treatment (T0) and 30 ± 2 days (T1) and 60 ± 4 days (T2) after, for the evaluation of: i) tear meniscus height (TMH); ii) non-invasive break-up time (NIBUT) a) first, b) average, c) class (0:>10 seconds [s]; I: 6–10 s; II 3–6 s; III < 3 s); iii) bulbar redness; iv) infrared meibography for evaluating meibomian glands loss (MGL). Symptoms of ocular discomfort were assessed using the Symptom Assessment in Dry Eye (SANDE) questionnaire.
Tear Analysis
In a subgroup of patients with adequate amount of tears (TMH value ≥ 0.25 millimetres [mm] at T0), a sample of 10 µL of tears was collected to evaluate the concentration of Proenkephalin and Met/Leu-enkephalinproenkephalin. Briefly, tears collected by capillary tube were blown into microcentrifuge tubes, stored at − 80°C, and used within 2 months. Total protein amount in the tears were determined by performing a Bradford Assay according to the manufacturer's recommendations (Protein assay Dye reagent concentrate, Bio-Rad, Hercules, US-CA). The absorbance was measured at 595 nm with a spectrophotometer (NanoDrop One, Thermo Scientific™) and the protein concentrations were de-rived using a bovine serum albumin (BSA) calibration curve. Thirty-five micrograms of tear proteins were used for Western Blot analyses and 10 micrograms for Coomassie Blue staining. Separation by SDS-PAGE, blotting, and incubation with primary and secondary antibodies were performed as de-scribed elsewhere.22 Chemiluminescence reaction was carried out with Clarity Western ECL Substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the signal acquisition was performed through the ChemiDoc Xrs + by the Image Lab software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Antisera and monoclonal antibodies used in the present work are the following: HRP-conjugated anti-mouse (Sigma Aldrich, Saint Louis, MO, USA) and anti-human Met/Leu-enkephalin (cat. No. sc-47705; Santa Cruz Biotechnology, Santa Cruz, CA, USA). In the Coomassie Blue staining following SDS-PAGE the polyacrylamide gel was stained with a staining solution (0.4% Coomassie Blue, 50% methanol, 10% acetic acid) for 30 min at room temperature. The gel was sequentially soaked into a destaining solution-I (50% methanol, 10% acetic acid) for 30–60 min until the protein bands were dis-cretely observable. Signals were analyzed by ImageJ software.
Outcomes
The primary outcome was the changes of objective signs and subjective symptoms occurring after the study treatment. The secondary outcome was the change of tears content (Proenkephalin and Met/Leu-enkephalinproenkephalin) registered after treatment in a subgroup of patients with adequate amount of tears.
Statistical Analysis
Statistical analyses were performed Prism version 9.4.0 (GraphPad Software Inc., San Diego, CA, USA). Normally distributed data were expressed as mean ± standard deviation (SD), otherwise as median values with interquartile range (IQR). Parametric and nonparametric tests were chosen on the basis of data normality. The Anderson-Darling test and Kolmogorov-Smirnov tests were applied to assess if data were normally distributed. Student t test, Mann-Whitney U test, Dunnett’s multiple comparison test and Friedman test were used to compare variables, when appropriate. A p-value less than 0.05 was considered statistically significant. To determine the sample size of the study, a priori power analysis was performed based on the data of the study of Lambiase et al. In total, 19 patients were required to detect a mean change of SANDE from baseline of 16.1 points, with a power of 0.95 and a P value of 0.05.23