Four subjects with an established diagnosis of keratoconus were recruited for the study. Exclusion criteria were previous contact lens wear, history of ocular trauma or surgery, ocular disease other than keratoconus, and systemic disease that may affect the cornea. Two subjects were men and two were women (mean age, 44 ± 9 years).
All research protocols adhered to the tenets of the Declaration of Helsinki. Before enrollment, informed, written consent was obtained from all subjects after an explanation of the nature and possible consequences of the study. The protocol used was approved by the Auckland ethics committee.
Slit lamp biomicroscopy was performed on all eyes, and each subject exhibited one or more of the following clinical signs: central corneal stromal thinning, Fleisher’s ring, Vogt’s striae or Munson’s sign. No eyes had signs of corneal hydrops or scarring. In all cases, slit-scanning elevation topography (Orbscan II; Bausch & Lomb Surgical, Rochester, NY) was performed to confirm the clinical diagnosis and to classify further the severity of keratoconus. The modified Rabinowitz-McDonnell test was used to confirm the diagnosis of keratoconus,
6 and the severity of keratoconus was classified according to the steepest reading on the keratometric map (mild, <45 D; moderate, 45–52 D; and severe, >52 D).
Laser scanning in vivo confocal microscopy was subsequently performed on all subjects (Heidelberg Retina Tomograph II, Rostock Corneal Module [RCM]; Heidelberg Engineering GmbH, Heidelberg, Germany). This microscope utilizes a 670-nm red wavelength diode laser source. It is a class 1 laser system and so, by definition, does not pose any ocular safety hazard. However, to guarantee the safety of the operator and subjects, the manufacturers have imposed a limit on the maximum period of exposure for patient and operator of 3000 seconds (50 minutes) in any single examination period. A 60× objective water-immersion lens with a numerical aperture of 0.9 (Olympus, Tokyo, Japan) and a working distance, relative to the applanating cap, of 0.0 to 3.0 mm was used. The dimensions of each image produced using this lens are 400 μm × 400 μm, and the manufacturers quote transverse resolution and optical section thickness as 2 and 4 μm, respectively. The RCM uses an entirely digital image capture system.
Eyes were anesthetized with a drop of 0.4% benoxinate hydrochloride (Chauvin Pharmaceuticals, Romford, UK). Viscotears (Carbomer 980, 0.2%; Novartis, Basel, Switzerland) was used as a coupling agent between the applanating lens cap and the cornea. During the examination, all subjects were asked to fixate on distance targets arranged in a grid pattern, as described previously.
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The cornea was scanned, using the device’s “section mode” to obtain high-quality images of the sub-basal nerve plexus in each position. The section mode enables instantaneous imaging of a single area of the cornea at a desired depth. The overall examination took approximately 40 minutes to perform for each subject, including breaks every few minutes and a total in vivo confocal exposure time of less than 20 minutes. None of the subjects experienced any visual symptoms or corneal complications as a result of the examinations.
A software program (Freehand 10; Macromedia, San Francisco, CA) was used to arrange images from each eye into wide-field montages of the sub-basal nerve plexus. Sub-basal nerve density measurements were performed on the montage using a caliper tool (analySIS 3.1; Soft Imaging System, Münster, Germany). In all cases, a standard frame size of 0.8 × 0.8 mm (area, 0.64 mm2) was selected. Nerve density was assessed in the region of the central cornea by measuring the total length of nerves per defined frame.