The lid wiper and muco-cutaneous junction anatomy of the human eyelid margins: an in vivo confocal and histological study.

The inner border of the eyelid margin is critically important for ocular surface integrity because it guarantees the thin spread of the tear film. Its exact morphology in the human is still insufficiently known. The histology in serial sections of upper and lower lid margins in whole-mount specimens from 10 human body donors was compared to in vivo confocal microscopy of eight eyes with a Heidelberg retina-tomograph (HRT II) and attached Rostock cornea module. Behind the posterior margin of the Meibomian orifices, the cornified epidermis stopped abruptly and was replaced by a continuous layer of para-keratinized (pk) cells followed by discontinuous pk cells. The pk cells covered the muco-cutaneous junction (MCJ), the surface of which corresponded to the line of Marx (0.2-0.3 mm wide). Then a stratified epithelium with a conjunctival structure of cuboidal cells, some pk cells, and goblet cells formed an epithelial elevation of typically about 100 µm initial thickness (lid wiper). This continued for 0.3-1.5 mm and formed a slope. The MCJ and lid wiper extended all along the lid margin from nasal to temporal positions in the upper and lower lids. Details of the epithelium and connective tissue were also detectable using the Rostock cornea module. The human inner lid border has distinct zones. Due to its location and morphology, the epithelial lip of the lid wiper appears a suitable structure to spread the tear film and is distinct from the MCJ/line of Marx. Better knowledge of the lid margin appears important for understanding dry eye disease and its morphology can be analysed clinically by in vivo confocal microscopy.

Influence of benzalkonium chloride on langerhans cells in corneal epithelium and development of dry eye in healthy volunteers.

PURPOSE: To investigate the influence of benzalkonium chloride (BAC) on corneal Langerhans cells (LCs) and on the development of dry eye. METHODS: A randomized double-blind clinical trial was performed in 20 healthy volunteers. One eye of each subject was treated with a 0.01% BAC solution (and the fellow eye with a placebo solution) three times daily for 12 weeks. The distribution and density of LCs in the central and peripheral corneal epithelium were evaluated by in-vivo confocal laser-scanning microscopy (CLSM). The subjects were monitored for dry eye (subjective discomfort, slit-lamp biomicroscopy, tear film break-up time, Schirmer's test). RESULTS: In the BAC group, compared with placebo, a marked increase in LC density was found in the central cornea at Week 6 and in the central and peripheral cornea at Week 12. LC density then decreased again in both zones after the end of treatment, falling toward (or even below) baseline levels. Significant changes in the LC count relative to baseline were found at Week 12 in the central and peripheral cornea in the BAC group as well as in the peripheral cornea in the placebo group. LC density in the BAC group increased more rapidly in the central than in the peripheral cornea. During therapy the BAC group showed no signs of dry eye. CONCLUSION: 12-week application of a 0.01% BAC solution in healthy volunteers induces a significant increase in LCs in the central cornea at Week 12 without dry-eye changes. The return of LC counts toward (or even below) baseline levels just four weeks after the end of BAC administration demonstrates the rapid normalization of the inflammatory environment.

Structural-functional correlations of corneal innervation after LASIK and penetrating keratoplasty.

PURPOSE: To compare corneal subbasal nerve fiber distribution and corneal sensation in healthy humans with findings obtained in regenerated subbasal nerves after LASIK and penetrating keratoplasty (PK). METHODS: In a comparative case series study, in vivo confocal laser-scanning microscopy was used to investigate subbasal nerve fiber bundles in healthy individuals and at various time points after surgery in patients who had undergone LASIK and corneal grafting. Corneal sensation was measured (Cochet-Bonnet esthesiometer). RESULTS: In normal corneas investigated, image superimposition revealed the consistent appearance of curved nerve fibers showing a whorl-like pattern with clockwise orientation. Nerve fibers parallel to Bowman's layer originating peripherally traveled radially inwards to a point located at the lower nasal quadrant. This pattern was not seen in any of the patients after LASIK or PK. Regenerated nerve fibers were thinner, more curved, and showed abnormal branching in nearly all patients. Normal corneal neuro-anatomical architecture remained absent even months after total restoration of corneal sensation. After LASIK, normal sensation was regained independently of normal subbasal nerve anatomy. Corneal grafts have shown some recovery of subbasal nerve morphology, at least in the graft periphery, but not complete recovery of function. CONCLUSIONS: It would appear that normal corneal sensation after LASIK or PK does not always depend on normal subbasal nerve anatomy but on the collateral organization of subbasal nerve fibers.

The microstructure of cornea verticillata in Fabry disease and amiodarone-induced keratopathy: a confocal laser-scanning microscopy study.

PURPOSE: The purpose of this study is to describe cornea verticillata in Fabry disease and in amiodarone-induced keratopathy and to compare the corneal microstructure of both types. PATIENTS AND METHODS: Ten eyes from ten normal subjects, 28 eyes from 22 patients with Fabry disease confirmed by molecular genetic studies, and 16 eyes from 11 patients receiving amiodarone were examined by slit-lamp microscopy and in-vivo confocal laser-scanning microscopy (CLSM) with following three-dimensional reconstruction of the individual corneal layers. Five patients with Fabry disease were monitored during the course of enzyme replacement therapy (ERT). RESULTS: Evidence of cornea verticillata was found by slit-lamp microscopy both in patients with Fabry disease and in those with amiodarone-induced keratopathy; CLSM revealed the same pattern of hyper-reflective deposits in the basal cell layer of corneal epithelium in both sets of patients. Microdot changes in the anterior stroma were more prevalent in patients receiving amiodarone but do not presuppose the simultaneous presence of cornea verticillata. The bulbar conjunctiva was found to be normal in all patients. The tarsal conjunctival epithelium contained round hyper-reflective structures, which are also encountered physiologically, but these were more common in patients with Fabry disease. In one out of the five patients examined, monitoring of corneal changes over time during ERT disclosed a regressive tendency of the deposits in the epithelial basal cell layer documented by CLSM. CONCLUSIONS: The microstructural corneal changes typically seen in cornea verticillata in both Fabry disease and in amiodarone-induced keratopathy can be successfully visualized by confocal in-vivo microscopy at the level of the basal cell layer. By analogy, with the grading system for cornea verticillata based on slit-lamp microscopy, staging of these deposits in the basal cell layer can also be performed following in-vivo CLSM. The microdots in the anterior stroma as well as the changes observed in the tarsal conjunctiva should be regarded as having less diagnostic value because such changes may also occur in normal subjects. The utility of CLSM as a tool for monitoring ERT in Fabry disease over time needs to be confirmed in studies with larger sample sizes conducted over a longer period.

Two cases of keratitis and corneal ulcers caused by Burkholderia gladioli.

We report two cases of protracted keratitis complicated by corneal ulcer. Burkholderia gladioli, primarily known as a plant pathogen, was cultured from corneal swabs. The epithelial defects healed after extended adequate antibiotic therapy. Despite identical patterns of both strains in restriction fragment length polymorphism analysis, a common infection source was not detectable.

In vivo three-dimensional confocal laser scanning microscopy of the epithelial nerve structure in the human cornea.

PURPOSE: Evaluation of a new method for in vivo visualization of the distribution and morphology of human anterior corneal nerves. METHOD: The anterior cornea was examined to a depth of 100 microm in four human volunteers with a confocal laser scanning microscope (CLSM) using a Rostock Cornea Module (developed in house) attached to a Heidelberg Retina Tomograph II (Heidelberg Engineering, Germany). Optical sections were digitally reconstructed in 3D using AMIRA (TGS Inc., USA). The scanned volumes had a greatest size of 300 x 300 x 40 microm and voxel size of 0.78 x 0.78 x 0.95 microm. RESULTS: The spatial arrangement of the epithelium, nerves and keratocytes was visualized by in vivo 3D-CLSM. The 3D-reconstruction of the volunteers' corneas in combination with the oblique sections gave a picture of the nerves in the central human cornea. Thin nerves run in the subepithelial plexus aligned parallel to Bowman's layer and are partially interconnected. The diameter of these fibres varied between 1.0 and 5 microm. Thick fibres rose out of the deeper stroma. The diameter of the main nerve trunks was 12+/-2 microm. Branches penetrating the anterior epithelial cell layer could not be visualized. CONCLUSIONS: 3D-CLSM allows analysis of the spatial arrangement of the anterior corneal nerves and visualization of the epithelium and keratocytes in the living human cornea. The developed method provides a basis for further studies of alterations of the cellular arrangement and epithelial innervation in corneal disease. This may help to clarify alterations of nerve fibre patterns under various clinical and experimental conditions.

In vivo confocal laser scanning microscopy of the cornea in dry eye.

BACKGROUND: We carried out an investigation into the morphological and quantitative corneal properties in dry eye with various underlying pathologies. METHODS: Ten patients with aqueous tear deficiency, 8 with dysthyroid ophthalmopathy, 8 with chronic lagophthalmos and 10 normal participants were examined. Confocal microscope images were taken at the centre and at the lower and upper periphery of the cornea. Quantitative and morphological assessments of the epithelium, of the sub-basal nerves, of the stroma and the endothelium were made. The epithelial and corneal thicknesses were measured. RESULTS: The mean superficial and intermediate epithelial cell densities in the central cornea in the patient groups were significantly lower than in normal participants (p<0.01). The peripheral epithelial thickness was smaller (p<0.01); it was smallest in the lagophthalmos group. The cornea was thinner in the patient groups (p<0.01). For sub-basal nerves, the density had decreased (p<0.05), and in lagophthalmos the number of beadlike formations had increased (p<0.001); in some patients we found irregular branching patterns. CONCLUSIONS: Dry eye patients showed significant alterations in the cornea, presumably due to increased desquamation of the superficial cell layer. This was most pronounced at the lower periphery of the cornea in patients with exposure keratopathy.

In vivo confocal microscopy of the ocular surface.

Over the past two decades, the applications of in vivo confocal microscopy to the investigation of ocular surface diseases in the living eye have been greatly extended. Confocal microscopy enables detailed investigation of tarsal and palpebral conjunctiva, central and peripheral cornea, tear film, and lids, and it allows evaluation of the ocular surface at the cellular level. High-quality imaging in both contact and noncontact modes has allowed new understanding of the functions of the ocular surface system, and in the coming years, such knowledge will become increasingly comprehensive and specific. Confocal microscopy may provide a link between well-established ex vivo histology and in vivo study of ocular pathology, not only in clinical science but also in clinical practice. The purpose of this review is to summarize the current knowledge about in vivo confocal microscopy of the ocular surface.