Quantitative Analysis of Corneal Sub-basal Nerve Plexus and Inflammatory Cells in Large-area Mosaics Obtained by In Vivo Confocal Microscopy

Abstract

Purpose: To quantify corneal sub-basal nerve and inflammatory cell density from large-area corneal mosaics in order to evaluate the possible differences between right and left eye and to compare the results of manual versus automated analysis.

Methods: Using laser-scanning in vivo confocal microscopy, the central cornea of 83 subjects (166 eyes) was imaged by an experienced examiner. Images of the sub-basal plexus were used to assemble the best possible mosaic. Mosaics from both eyes of each patient were thereafter subjected to both manual and automated tracing of nerves and manual counting of inflammatory cells. ImageJ/NeuronJ was used for manual analysis while a custom program was used for automated analysis. The results of both manual and automated density analysis were compared.

Results: Large-area mosaics could be assembled in all eyes, with an average mosaic size of 6.3 mm², representing an average of 40 single confocal fields of view. A single mosaic took an average of 106 minutes to assemble from an average of 522 individual input image frames by a fully automated procedure. Mosaic tracing by the manual Neuron-J method took on average 35 minutes per mosaic, and yielded sub-basal nerve density values that differed from the fully automated method, which took less than one minute per mosaic to fully trace and analyze. Left and right eyes were often found to have differing nerve patterns and nerve and cell densities, while the mosaics also revealed local variations in nerve and inflammatory cell density.

Conclusions: High quality, large-area mosaics of the corneal sub-basal nerve plexus can be obtained and assembled with an automated approach. Nerve and cell density, however, can vary with eye, analysis method, and microscopic region within the plexus. This brings into question the validity of multiple single-image and single-eye analyses of these parameters.