In Vivo Corneal Nerve Imaging and Quantification

Patients underwent examination with the Heidelberg retina tomograph III in vivo corneal confocal microscope. The subject's eyes were anesthetized using a drop of 0.4% benoxinate hydrochloride, and Viscotears were applied on the front of the eye for lubrication. A drop of viscoelastic gel was placed on the tip of the objective lens, and a sterile disposable Perspex cap was placed over the lens allowing optical coupling of the objective lens to the cornea. The patient was instructed to fixate on a target with the eye not being examined. Several scans of the entire depth of the cornea were recorded by turning the fine focus of the objective lens backward and forward for ~2 min using the section mode, which enables manual acquisition and storage of single images of all corneal layers. This provides en face two-dimensional images with a lateral resolution of ~2 µm/pixel and final image size of 400 × 400 pixels of the subbasal nerve plexus of the cornea from each patient and control subject. This layer is of particular relevance for defining neuropathic changes since it is the location of the main nerve plexus that supplies the overlying corneal epithelium. Each nerve fiber bundle contains unmyelinated fibers, which run parallel to Bowman’s layer before dividing and terminating as individual axons underneath the surface epithelium (26 (link)). Five images per patient from the center of the cornea were selected and examined in a masked and randomized fashion (27 (link)). Three corneal nerve parameters were quantified: 1) CNFD, the total number of major nerves per square millimeter of corneal tissue; 2) corneal nerve branch density (CNBD), the number of branches emanating from all major nerve trunks per square millimeter of corneal tissue; and 3) corneal nerve fiber length (CNFL), the total length of all nerve fibers and branches (mm/mm²) within the area of corneal tissue. CNFD and CNFL are considered to reflect overall nerve fiber degeneration, whereas CNBD reflects nerve fiber regeneration, which is partially also captured by CNFL.