Fifteen adult patients (seven men, eight women; mean age, 59.9 ± 19.4 years) with a diagnosis of Mooren's ulcer who were willing to comply with the protocol and who provided informed consent were enrolled in the study. Examination procedures were board reviewed. The study complied with the principles of the Declaration of Helsinki for research involving human subjects. Ten eyes of seven patients who had active peripheral corneal ulcers in the peripheral cornea, with pain and the adjacent inflamed and hyperemic conjunctiva and sclera, were diagnosed as patients with active Mooren's ulcer. Twelve eyes of nine patients with a history of Mooren's ulcer, whose corneal ulcers had not relapsed for the past 12 months, were diagnosed as Mooren's ulcers in remission. One patient had bilateral Mooren's ulcers, whose right eye was in remission and whose left eye was active. Patients who had rheumatoid arthritis or other collagen vascular diseases, staphylococcal marginal keratitis accompanied by blepharitis, infectious ulcers, giant cell arteritis, local infectious causes, including herpes simplex and herpes zoster, Terrien's degeneration, pellucid marginal degeneration, senile furrow degeneration, ocular rosacea, or leukemia, were excluded.
6 To exclude rheumatoid arthritis and other collagen diseases, blood tests of RA factor, antinuclear antibody, anti-Ro/SSA antibody, anti-La/SSB antibody, CRP, and CBC were performed. To exclude staphylococcal marginal keratitis or other infectious diseases, we performed bacterial cultures from conjunctival sac swabs, eye discharge, or margin of ulcers. To exclude herpes simplex and herpes zoster infections, we requested SRL Laboratory (Tokyo, Japan) to perform herpes antigen test by the fluorescein antibody (FA) method using scratch samples from the ulcer beds. Other diseases meeting the exclusion criteria were checked by clinical record and anamnesis.
Patients with active Mooren's ulcers underwent in vivo laser confocal microscopy and a complete ophthalmic examination, including measurement of visual acuity and intraocular pressure, slit lamp microscopic examination, and dilated fundus examination at the enrollment visit, day 0. At the study follow-up visits (scheduled on weeks 1, 4, and 8 after enrollment), in vivo laser confocal microscopy and ophthalmic examination were performed, consisting of visual acuity testing, intraocular pressure, and slit lamp microscopic examination. At the enrollment visit, patients with Mooren's ulcers in remission underwent in vivo laser confocal microscopy and ophthalmic examination, including slit lamp microscopic examination.
All patients at day 0 had anterior segment photographs and slit lamp microscopic examination, including fluorescein staining of the ocular surface. The extent of limbal involvement with ulcers and adjacent gray-white infiltration was expressed in degrees (0–360°). To measure it, the corneal circumference of a slit lamp photograph was divided in 24 sections of 15° each. The count of sections of limbal involvement along with ulcers was then recalculated in degrees. The extent of limbal involvement in eyes with Mooren's ulcer in remission was regarded as 0°, according to the protocol of the study.
In vivo laser confocal scanning laser microscopy (CSLM) was performed on all subjects with a new-generation confocal microscope (the Rostock Corneal Software, ver. 1.2, Heidelberg Retina Tomograph II [RCM/HRT II]; Heidelberg Engineering GmbH). After the administration of topical anesthesia with 0.4% oxybuprocaine, the subject's chin was placed in the chin rest. The objective of the microscope was an immersion lens (magnification 63×; Carl Zeiss, Oberkochen, Germany) covered by a polymethylmethacrylate (PMMA) cap (Tomo-cap; Heidelberg Engineering GmbH). Comfort gel (Bausch & Lomb, GmbH, Berlin, Germany) was used as a coupling agent between the applanating lens cap and the conjunctiva. By adjusting the controller, the center of the cap was applanated onto the cornea, and in vivo digital images of the cornea were visualized directly on the computer screen. When the first superficial cells were seen, the digital micrometer gauge was set at 0; then, when the operator pressed on the foot pedal, sequence images were recorded by a charge-coupled device (CCD) color camera (maximum 30 frames/s) while the focal plane was gradually moved forward into the corneal stroma. Corneal and adjacent limbal conjunctival lesions were scanned while the applanating lens was moved through the entire length of the ulcer with minute vertical or horizontal movements. Four nonoverlapping areas with images of inflammatory cells were selected from all vertical scan areas, and inflammatory cell density (ICD) was counted at the basal cell layer level of the corneal epithelium in each 400 × 400-μm frame, with the help of the accompanying software. To avoid overlap, the scans were performed along the entire length of the ulcers on the limbal side, central corneal side, and superior/inferior edges, at all four sites with minute movements of the PMMA cap, and three nonoverlapping scans per site with best resolution quality were selected and underwent ICD calculations. The depth chosen for ICD calculations in the ulcers corresponded to the level of the healthy basal epithelium along the edge of the ulcer. White round cells of 5- to 15-μm diameter were chosen for counting ICD, and images with many dendritic cells were not used, because it is hard to count dendritic cells accurately. The mean ICD of each (limbal side, central side, superior edge, and inferior edge) site and the mean of ICDs of the four sites was calculated. Confocal microscopy scans were performed by one examiner who was masked to the diagnosis of the subjects. Analysis of the scans was performed by three different investigators who were also masked to the diagnosis in each case. For intraobserver variability testing, the same investigator performed the same ICD analysis on three occasions. For evaluating intraobserver variability, these investigators carried out ICD calculations, and the differences between the investigator calculations were sought.
The laser source used in the Retina Tomograph II/Rostock Corneal Module (Heidelberg Engineering) is a diode laser with a wavelength of 670 nm. Two-dimensional images consisted of 384 × 384 picture elements, covering an area of 400 × 400-μm field of the view (FOV). The transverse field of view was captured with the 400 FOV lens. Digital resolution was quoted as 1 μm/pixel transversely and 2 μm/pixel longitudinally by the manufacturer.
Eyes with active Mooren's ulcer were treated with topical corticosteroids and additional therapy, including systemic corticosteroids, topical cyclosporine, systemic cyclosporine, conjunctival excision, and/or keratoepithelioplasty in a severity-based stepladder approach.
1 Eyes with Mooren's ulcer in remission continued to receive the previous treatment protocols.