A baseline human corneal subbasal nerve density has been determined by laser-scanning IVCM using rigorous methods. The methods and results could aid in the future assessment of corneal nerves in various patient populations.

IMPORTANCE It is unknown whether a neurotrophic deficit or pathologic nerve morphology persists in keratoconus in the long term after corneal collagen cross-linking (CXL) treatment. Nerve pathology could impact long-term corneal status in patients with keratoconus. OBJECTIVE To determine whether CXL treatment of keratoconus results in normalization of subbasal nerve density and architecture up to 5 years after treatment. DESIGN, SETTING, AND PARTICIPANTS Observational study of 19 patients with early-stage keratoconus indicated for a first CXL treatment with longitudinal follow-up to 5 years postoperatively (examinations were performed from 2009 to 2015; analysis was performed from February to May 2015) and 19 age-matched healthy volunteers at a primary care center and a university hospital ophthalmology department. EXPOSURE The patients with keratoconus underwent standard epithelial-off UV-A/riboflavin CXL treatment with 30-minute UV-A exposure at 3 mW/cm 2 irradiance. MAIN OUTCOMES AND MEASURES Central corneal subbasal nerve density and subbasal nerve architecture by use of laser-scanning in vivo confocal microscopy; subbasal nerve analysis by 2 masked observers and by use of a fully automated method; wide-field mosaics of subbasal nerve architecture by use of an automated method; and ocular surface touch sensitivity by use of contact esthesiometry. RESULTS Mean (SD) age of the 19 patients with keratoconus was 27.5 (7.1) years (range, 19-44 years), and minimal corneal thickness was 428 (36) μm (range, 372-497 μm). Compared with the mean (SD) preoperative subbasal nerve density of 21.0 (4.2) mm/mm 2 in healthy corneas, the mean (SD) preoperative subbasal nerve density of 10.3 (5.6) mm/mm 2 in the corneas of patients with stage 1 or 2 keratoconus was reduced 51% (mean difference, 10.7 mm/mm 2 [95% CI, 6.8-14.6 mm/mm 2 ]; P < .001). After CXL, nerves continued to regenerate for up to 5 years, but nerve density remained reduced relative to healthy corneas at final follow-up (mean reduction, 8.5 mm/mm 2 [95% CI, 4.7-12.4 mm/mm 2 ]; P < .001) despite recovery of touch sensitivity to normal levels by 6 months. Preoperatively, more frequent nerve loops, crossings, and greater crossing angles were observed in the corneas of patients with keratoconus compared with healthy corneas. Postoperatively, the frequency of nerve looping increased, crossings were more frequent, and nerve tortuosity increased. Wide-field mosaics indicated persistent disrupted orientation of the regenerating subbasal nerves 5 years after CXL. CONCLUSIONS AND RELEVANCE Keratoconus is characterized by a neurotrophic deficit and altered nerve morphology that CXL treatment does not address, despite providing a positive biomechanical effect in the stroma. Given the widespread use of CXL in the management of patients with keratoconus, the progression of abnormal innervation after CXL should be recognized.