Femtosecond laser cutting of human corneas for the subbasal nerve plexus evaluation.

Assessment of various morphological parameters of the corneal subbasal nerve plexus is a valuable method of documenting the structural and presumably functional integrity of the corneal innervation in health and disease. The aim of this work is to establish a rapid, reliable and reproducible method for visualization of the human corneal SBP using femtosecond laser cut corneal tissue sections. Trephined healthy corneal buttons were fixed and processed using TissueSurgeon-a femtosecond laser based microtome, to obtain thick tissue sections of the corneal epithelium and anterior stroma cut parallel to the ocular surface within approximately 15 min. A near infrared femtosecond laser was focused on to the cornea approximately 70-90 µm from the anterior surface to induce material separation using TissueSurgeon. The obtained corneal sections were stained following standard immunohistochemical procedures with anti-neuronal ß-III tubulin antibody for visualization of the corneal nerves. Sections that contained the epithelium and approximately 20-30 µm of anterior stroma yielded excellent visualisation of the SBP with minimal optical interference from underlying stromal nerves. In conclusion, the results of this study have demonstrated that femtosecond laser cutting of the human cornea offers greater speed, ease and reliability than standard tissue preparation methods for obtaining high quality thick sections of the anterior cornea cut parallel to the ocular surface.

Comparative quantitative assessment of the human corneal sub-basal nerve plexus by in vivo confocal microscopy and histological staining.

PurposeThis study was designed to compare and contrast quantitative data of the human corneal sub-basal nerve plexus (SBP) evaluated by two different methods: in vivo confocal microscopy (IVCM), and immunohistochemical staining of ex vivo donor corneas.MethodsSeven parameters of the SBP in large-scale IVCM mosaicking images from healthy subjects were compared with the identical parameters in ex vivo donor corneas stained by ß-III-tubulin immunohistochemistry. Corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), average weighted corneal nerve fiber tortuosity (CNFTo), corneal nerve connection points (CNCP), average corneal nerve single-fiber length (CNSFL), and average weighted corneal nerve fiber thickness (CNFTh) were calculated using a dedicated, published algorithm and compared.ResultsOur experiments showed significantly higher values for CNFL (50.2 vs 21.4 mm/mm2), CNFD (1358.8 vs 277.3 nerve fibers/mm2), CNBD (847.6 vs 163.5 branches/mm2), CNFTo (0.095 vs 0.081 µm-1), and CNCP (49.4 vs 21.6 connections/mm2) in histologically staining specimens compared with IVCM images. In contrast, CNSFL values were higher in IVCM images than in histological specimens (32.1 vs 74.1 µm). No significant difference was observed in CNFTh (2.22 vs 2.20 µm) between the two groups.ConclusionsThe results of this study have shown that IVCM has an inherently lower resolution compared with ex vivo immunohistochemical staining of the corneal SBP and that this limitation leads to a systematic underestimation of several SBP parameters. Despite this shortcoming, IVCM is a vital clinical tool for in vivo characterization, quantitative clinical imaging, and evaluation of the human corneal SBP.

Quantitative assessment of central and limbal epithelium after long-term wear of soft contact lenses and in patients with dry eyes: a pilot study.

PurposeAnalysis of microstructural alterations of corneal and limbal epithelial cells in healthy human corneas and in other ocular conditions.Patients and methodsUnilateral eyes of three groups of subjects include healthy volunteers (G1, n=5), contact lens wearers (G2, n=5), and patients with dry eyes (G3, n=5) were studied. Imaging of basal (BC) and intermediate (IC) epithelial cells from central cornea (CC), corneal limbus (CL) and scleral limbus (SL) was obtained by in vivo confocal microscopy (IVCM). An appropriate image analysis algorithm was used to quantify morphometric parameters including mean cell area, compactness, solidity, major and minor diameter, and maximum boundary distance.ResultsThe morphometric parameters of BC and IC demonstrated no significant differences (P>0.05) between groups. Comparison between three corneal locations (CC, CL, and SL) within the groups showed significant differences (P<0.05) with mean values of cell area, compactness, solidity, and major and minor diameter of BC that increase from CC to limbus. The BC were round and regular in the central cornea (P<0.05) compared with CL and SL.ConclusionsIVCM enables high-quality confocal images from central corneal and limbal epithelium. This quantitative study demonstrated morphological differences in the basal and intermediate epithelium between limbus and central cornea, and found no differences between contact lens wearers, dry eyes, and normal subjects.