The study adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from all enrolled patients. We examined 126 eyes of 126 consecutive Caucasian subjects—30 eyes affected with untreated OH and 96 eyes affected with POAG in medical therapy—referred to the Ophthalmic Clinic of University Chieti-Pescara, Italy. Fifteen consecutive healthy eyes were used as control. The demographic characteristics of patients are reported in
Table 1 .
None of the normal subjects had a history of any topical or systemic therapy, nor had they been affected by any ocular or systemic diseases in the past 6 months. All eyes showed a best corrected visual acuity ≥8/10, a refractive error ≤4 D (spherical equivalent), an intraocular pressure (IOP) lower than 20 mm Hg, corneal pachymetry ranging from 530 to 560 μm, an absence of signs of glaucomatous optic neuropathy, and normal results in a visual field examination (Humphrey 30-2 full-threshold; Carl Zeiss Meditec, Oberkochen Germany). All hypertensive eyes were completely normal except for the IOPs, which ranged from 22 to 27 mm Hg (mean of three measurements at 9 AM, 12 noon, and 4 PM). Conversely, glaucomatous eyes had IOPs at the time of diagnosis ranging from 22 to 34 mm Hg (mean of three measurements at 9 AM, 12 noon, and 4 PM), a full-threshold visual field test (Humphrey 30-2; Carl Zeiss Meditec) showing at least three contiguous points on the total deviation probability plot at the less than 2% level, Glaucoma Hemifield Test results outside normal limits, and classic ophthalmoscopic signs of glaucomatous optic neuropathy (cupping, neural rim notching, and saucerization). Only patients with glaucoma who were receiving topical hypotensive therapy that had started at least 6 months before the day of IVCM examination and had remained unmodified during the past 6 months were enrolled in the study. Moreover, none of the patients had a history of any topical or systemic diseases or therapies in the past 6 months, to avoid any significant interaction that could have occurred with ocular surface tissues. A history of using contact lenses was considered an exclusion criterion in all cases. Among patients with glaucoma, 28 were in monotherapy with topical β-blockers (group 1; 17 eyes with preserved timolol maleate 0.5% and 11 eyes with preserved levobunolol hydrochloride 0.5%), 46 were in monotherapy with prostaglandin analogues (group 2; 16 with latanoprost 0.005%, 15 with travoprost 0.004% and 15 with bimatoprost 0.03%), and 22 patients were in associative unfixed combination therapy (group 3; eight with latanoprost-timolol, six with travoprost-timolol, and eight with bimatoprost-timolol).
Each subject enrolled in the study, after a complete ophthalmic examination performed from 10 to 12 AM, including visual acuity evaluation, applanation tonometry, slit lamp biomicroscopy, gonioscopy, and funduscopy, was carefully examined with a digital confocal laser-scanning microscope (LSM; HRT II Cornea Module; Heidelberg Engineering GmbH, Heidelberg, Germany).
The technical characteristics of this instrument and the details of conjunctival examination have been described.
16 The confocal assessment of bulbar conjunctiva in this case series was performed by placing the seated patient in front of the microscope, the head was set steady with a headrest, and the eye was properly aligned to obtain tangential optical sections of the superior and temporal bulbar conjunctiva, with a dedicated target mobile bright red light provided with the instrument that the patient had to fix with the fellow eye. A digital camera was used to acquire images of the lateral view of the eye and objective lens to check the position of the objective lens on the surface of the eye during each scan. The LSM objective was gently placed in contact with the ocular surface separated by a PMMA (polymethyl methacrylate) contact cap and a drop of 0.2% polyacrylic gel (Viscotirs Gel; CIBA Vision Ophthalmics, Marcon, Venezia, Italy) served as a coupling medium. Sequential images derived from automatic scans and manual frames were acquired throughout upper and temporal bulbar conjunctiva at the intermediate layer (10–20 μm) of each examined eye.
For the IVCM assessment, we microscopically evaluated the bulbar conjunctiva on identifying epithelial microcysts (defined as intraepithelial and extracellular optically empty spaces), such as those reported in the conjunctival wall of functioning filtering blebs
(Fig. 1) .
12 13 14 15 The main parameters considered in the IVCM examination were the mean microcyst density (cysts per square millimeters) and the total microcyst area (in square micrometers). The results were reported as the average of measures performed in the superior and temporal conjunctiva (from 3 to 6 mm from the limbus), analyzing six images (three in the superior conjunctiva and three in the temporal conjunctiva; 300 × 300 μm), selected by the IVCM operator (MN) in a masked fashion. The surface area of epithelial microcysts was calculated by using ImageJ, an open source software (http://rsb.info.nih.gov/ij/ available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD), as described elsewhere.
14
In addition, to verify the findings obtained in the sampled fields, we extended the analysis area to the whole conjunctival superior region in three OH eyes not included in the sample analysis, finally obtaining a planar reconstruction that showed the actual distribution of epithelial microcysts
(Fig. 2) .
The mean time of the IVCM examination from the onset of the disease was 27.1 ± 10.9 months (range, 6–42) and 41.3 ± 7.3 months (range, 12–64) in patients affected with OH and POAG, respectively.