Corneal Nerve Fiber Imaging by CCM

Subjects were examined using a Heidelberg Retina Tomograph 3 equipped with a Rostock Cornea Module (Heidelberg Engineering, Heidelberg, Germany). The laser source used in the module was a diode laser with a wavelength of 670 nm. The two‐dimensional images acquired had a definition of 384 × 384 pixels over an area of 0.16 mm² with a lateral digital resolution of 1 μm/pixel and a depth resolution of 2 μm/pixel. Each eye examined was anesthetized with one drop of 0.4% benoxinate hydrochloride (Santen Pharmaceutical, Osaka, Japan). The objective lens of the cornea module was disinfected with 70% isopropyl alcohol swabs. A drop of Comfort Gel (Dr Mann Pharma, Berlin, Germany) was applied to the tip of the lens and a disposable sterilized Tomocap was mounted on the holder to cover the objective lens. After applying Comfort Gel to the Tomocap, the lens was slowly advanced forward until the gel touched the cornea, allowing optical contact between the objective lens and the corneal epithelium during the examination. Correct alignment and contact with the cornea were monitored by magnified images captured by a camera tangential to the eye. After focusing on the corneal epithelium, the nerve fiber layer (sub‐basal layer) was recorded by fine turning the focus. It took 3–5 min to acquire 10–20 satisfactory images of the corneal nerve fibers. Four to five high‐quality images of the sub‐basal layer were used to analyze morphological parameters of the corneal nerve fibers. The images collected were used to quantify the following parameters to define corneal nerve fiber changes: (i) corneal nerve fiber density (CNFD; /mm²); (ii) corneal nerve fiber length (CNFL; mm/mm²); (iii) corneal nerve fiber branch density (CNBD; /mm²); (iv) length of the corneal nerve branch emanating from the major nerve trunk (CNBL; mm/mm²); (v) tortuosity; and (vi) frequency of beading (/0.1 mm). All measurements, except for tortuosity, were made using Image J (Texelcraft, Tokyo, Japan); tortuosity was determined according to the grading system proposed by Oliveira‐Soto and Efron¹⁵. The reproducibility of the assessment of morphological parameters by CCM was evaluated in 14 healthy volunteers by repeating the CCM examination with different examiners and calculating the coefficient of variation (CV) for individual parameters.