Matthias Böhnke

Confocal microscopy of the cornea

This paper provides the clinician and the researcher with an in-depth manual on the use of a scanning-slit confocal light microscope for the clinical examination and investigation of the living human cornea in vivo. The scope of the paper includes a thorough explanation of the principles of various types of confocal microscopes as well as their limitations, a comprehensive review of the development of biomicroscopy of the eye, and a comparison of confocal microscopy and other optical techniques such as optical coherence tomography. The early work of Ridley, Goldmann and others on point illumination in early confocal instruments is described. The main part of the paper describes and illustrates the clinical examination of the living human cornea with the confocal microscope. Figures (many in color) from the normal cornea, the cornea with known parthologies, and the postsurgical cornea are selected for their educational value. Photographs of standard light microscopy of fixed, human corneal sections are compared with confocal maicroscopic images. Where appropriate, slit lamp color photographs are compared with confocal microscopic images. The clinical scanning-slit confocal microscope is an evolving instrument for biomicroscopy of the living eye.

Corneal thickness and endothelial density before and after cataract surgery

BACKGROUND/AIMS Deturgescence of the corneal stroma is controlled by the pumping action of the endothelial layer and can be monitored by measurement of central corneal thickness (pachymetry). Loss or damage of endothelial cells leads to an increase in corneal thickness, which may ultimately induce corneal decompensation and loss of vision. Little is known about the effect of moderate reductions in endothelial cell number on the thickness of the corneal stroma. This study aimed to investigate this matter further using patients who had incurred moderate decreases in their endothelial cell counts as a result of cataract surgery. METHODS Central corneal thickness was measured 1 day before surgery, 1 day after surgery, and again at 3 months or 1 year. Endothelial cell counts were also performed 1 day before surgery and thereafter at 3 months or 1 year after surgery. The relationship between these two parameters was assessed statistically. Precise measurements of central corneal thickness were made by optical low coherence reflectometry. For comparative purposes, this parameter was also determined by ultrasonic pachymetry. Central corneal endothelial cell numerical density was estimated on photomicrographs taken with a specular microscope. RESULTS All patients had significant postoperative corneal swelling on the day after surgery; preoperative values were restored by 3 and 12 months, even though significant endothelial cell losses had occurred. No correlation existed between central corneal thickness and central corneal endothelial cell numerical density. Measurements estimated by ultrasonic pachymetry were more variable and significantly higher than those determined by optical low coherence reflectometry. CONCLUSION As long as the numerical density of the corneal endothelial cells does not fall below the physiological threshold, a moderate decrease in this parameter does not compromise the pumping activity of the layer as a whole.

Central corneal thickness measurements in patients with normal tension glaucoma, primary open angle glaucoma, pseudoexfoliation glaucoma, or ocular hypertension

BACKGROUND/AIMS Recent studies have revealed patients with ocular hypertension to have thicker than normal central corneas and those with normal tension glaucoma to have thinner than normal ones, as determined by ultrasonic pachymetry. Since corneal thickness measurements and applanation tonometric estimates of intraocular pressure (IOP) correlate positively, monitoring of the former parameter have served as the basis for adjusting readings pertaining to the latter, with the consequence that many patients have had to be reclassified. With a view to validating these pachymetric studies, the central corneal thickness was determined in patients with normal tension glaucoma, primary open angle glaucoma, pseudoexfoliation glaucoma, or ocular hypertension, as well as that of normal subjects, using optical low coherence reflectometry, which is a new and more precise method than ultrasonic pachymetry. METHODS 34 patients with normal tension glaucoma, 20 with primary open angle glaucoma, 13 with pseudoexfoliation glaucoma, and 12 with ocular hypertension, together with 21 control subjects, were included in this observational, concurrent case-control study. One eye per individual was randomly selected for investigation. IOP was measured by Goldmann applanation tonometry and central corneal thickness by optical low coherence reflectometry. RESULTS Central corneal thickness was significantly higher (p ⩽0.001) in patients with ocular hypertension than in normal individuals or in subjects with either normal tension glaucoma, primary open angle glaucoma, or pseudoexfoliation glaucoma, there being no significant differences between the latter four groups. Patients with ocular hypertension were also significantly younger (p ⩽0.003) than those within any of the three glaucomatous groups. CONCLUSION This study confirms that a significant number of patients with ocular hypertension have normal IOPs after the appropriate adjustments have been made for deviations from normal in their central corneal thickness. The accurate measurement of this latter parameter is important not only for individual patient care, in permitting more precise estimations of IOP, but also for clinical studies, in assuring a more reliable classification of subjects.

Primary open-angle glaucoma is associated with sleep apnea syndrome.

Introduction: The etiology of primary open-angle glaucoma remains unclear. Various risk factors, including vascular abnormalities, have been associated with this disease. Sleep-associated diseases, like sleep apnea syndrome, might also represent a risk factor. Sleep apnea syndrome is characterized by repetitive upper airway obstructions during sleep, inducing hypoxia and sleep disruption with the risk of cardiovascular and neurological sequelae. In this study, we determined the prevalence of sleep apnea syndrome in primary open-angle glaucoma patients. Methods: Overnight transcutaneous finger oximetry was performed in 30 consecutive patients having primary open-angle glaucoma. We assessed the oximetry disturbance index during night sleep, a parameter used to diagnose sleep apnea syndrome and to grade its severity. Results: Sleep apnea syndrome was more prevalent among primary open-angle glaucoma patients compared to normal historic controls of the same age and sex distribution (χ2 = 9.35, d.f. = 3, p < 0.025). The oximetry disturbance index grade was significantly larger in the primary open-angle glaucoma group compared to normal controls (U = 3,352, p = 0.01). According to the oximetry disturbance index, 20% (6/30) of primary open-angle glaucoma patients had sleep apnea syndrome. Conclusion: Primary open-angle glaucoma is associated with sleep apnea syndrome. Early recognition and treatment of sleep apnea syndrome are important to avoid cardiovascular and neurological complications.

Long-term Contact Lens Wear Induces a Corneal Degeneration with Microdot Deposits in the Corneal Stroma

OBJECTIVE Confocal in vivo real-time microscopy was applied to study the corneal morphology in long-term contact lens wearers. DESIGN In a cross-sectional study, patients with a history of long-term contact lens wear underwent corneal confocal microscopy. The authors investigated 13 patients with a history of up to 26 years of soft contact lens wear, 11 patients with a history of up to 25 years of rigid gas permeable contact lens wear, and a control group of 29 normal subjects without a history of contact lens wear. INTERVENTION Scanning slit-confocal microscopy was performed with a 50x/1.0 NA water immersion objective. Corneal optical sections were recorded in real time without further digital processing and reviewed frame by frame. MAIN OUTCOME MEASURES Video frames selected from all corneal layers were evaluated qualitatively. The new finding of panstromal microdot deposits was quantitated in a scoring system ranging from 0 to 4+. Corneal endothelial cell densities were counted with the fixed frame technique. RESULTS Epithelial microcystic changes and alterations of endothelial cell morphology were found to a variable extent as described previously. A new finding was there were highly reflective panstromal microdot deposits in the corneal stroma. For this new disease, a scoring system ranging from 0 to 4+ was established. In the control group, 0 of 29 patients had stromal microdot deposits. In the soft contact lens group, 13 of 13 patients had panstromal microdot deposits with a mean score of 3.1 (range, 1-4), and in the hard contact lens group, 11 of 11 had a mean score of 1.9 (range, 1-4) for corneal microdot deposits. CONCLUSIONS With confocal microscopy, a new type of chronic stromal change has been observed in all subjects with long-term contact lens wear. Because subjects with soft contact lens wear had a more pronounced corneal degeneration than did subjects with gas permeable lenses, the authors assume the deposits to be induced by chronic hypoxia. The condition of stromal microdot degeneration as observed with confocal microscopy may be the early stage of a significant corneal disease, which eventually may affect large numbers of patients after decades of contact lens wear.

Confocal microscopy after implantation of intrastromal corneal ring segments

OBJECTIVE Confocal in vivo real-time microscopy was used to study the corneal morphologic features in eyes after Intrastromal Corneal Ring Segments (ICRS; now called KeraVision INTACS, KeraVision, Inc., Fremont, CA) implantation. DESIGN Noncomparative, interventional case series. PARTICIPANTS The authors performed confocal real-time microscopy on a total of 21 eyes from 11 patients. Seventeen eyes from 10 patients (five female, five male; mean age 32.3 years; range 22-42 years) underwent uncomplicated ICRS surgery to correct myopia and were examined after surgery (average 8.6 months; range 2-15 months). Three patients had the ICRS implanted into only one eye, and those eyes were compared with the untreated fellow eyes. One eye of another patient was examined 1 and 6 months after ICRS removal. INTERVENTION Flying slit-confocal microscopy was performed with water immersion objectives in the corneal center and near the nasal or temporal ICRS. Corneal optical sections were recorded in real time without further digital processing and were reviewed frame by frame. MAIN OUTCOME MEASURES Video frames selected from all corneal layers were evaluated qualitatively and quantitatively. RESULTS In the central cornea, we found normal morphologic features at all layers. In peripheral sections, epithelial cells with highly reflective nuclei in the basal cell layer were observed in six of 17 eyes (35%) implanted with ICRS. We found an intact corneal nerve plexus and undisturbed corneal endothelium immediately underneath the ICRS. Around the ICRS, moderate fibrosis was seen. In one eye, linear structures in bamboo-like orientation were detected after ICRS removal in the last keratocyte layer underneath the collapsed tunnel. CONCLUSIONS Whereas the central corneal zone appears unchanged, the corneal stroma adjacent to the ICRS displays a slight, but distinct, activation of wound healing. Epithelial cells with highly reflective nuclei in this region may be an indicator for an increased biologic stress caused by the device.

Confocal microscopy reveals persisting stromal changes after myopic photorefractive keratectomy in zero haze corneas.

AIMS Micromorphological examination of the central cornea in myopic patients 8–43 months after excimer laser photorefractive keratectomy (PRK), using the slit scanning confocal microscope. METHODS Patients were selected from a larger cohort of individuals on the basis of full corneal clarity (haze grading 0 to +1; mean 0.3) and their willingness to participate in the study. 15 eyes of 10 patients with myopic PRK (−4 to −11 D; mean 6.7) and an uneventful postoperative interval of 8–43 months (mean 26) were examined. Contact lenses had been worn by eight of the 10 patients for 4–11 years (mean 6.7) before surgery. Controls included the five untreated fellow eyes of PRK patients, 10 healthy, age matched volunteers without a history of ocular inflammation or contact lens wear, and 20 patients who had worn rigid gas permeable (n=10) or soft contact lenses (n=10) for 2–11 years. Subjects were examined with a real time flying slit, scanning confocal microscope using ×25 and ×50 objectives. RESULTS In PRK treated patients and contact lens wearers, basal layer epithelial cells sporadically displayed enhanced reflectivity. The subepithelial nerve plexus was observed in all individuals, but was usually less well contrasted in the PRK group, owing to the presence of a very discrete layer of subepithelial scar tissue, which patchily enhanced background reflectivity. Within all layers of the stroma, two distinct types of abnormal reflective bodies were observed in all PRK treated eyes, but in none of the controls. One had the appearance of long (>= 50 μm), slender (2–8 μm in diameter) dimly reflective rods, which sometimes contained bright, punctate, crystal-like inclusions, arranged linearly and at irregular intervals. The other was shorter (<25 μm), more slender in form (<1 μm in diameter), and highly reflective; these so called needles were composed of crystal-like granules in linear array, with an individual appearance similar to the bright punctate inclusions seen in rods, but densely packed. Both of these unusual structures were confined, laterally, to the ablated area, but were otherwise distributed throughout all stromal layers, with a clear predominance in the anterior ones. These rods and needles were observed in all PRK treated corneas, irrespective of previous contact lens wear. On the basis of qualitative inspection, the incidence of rods and needles did not appear to correlate with either the volume of tissue ablated or the length of the postoperative interval. In contact lens wearing controls, highly reflective granules, reminiscent of those from which the needles were composed, were found scattered as isolated entities throughout the entire depth and lateral extent of the corneal stroma, but rods and needles were never encountered. The corneal endothelium exhibited no obvious abnormalities. CONCLUSION Confocal microscopy 8–43 months after PRK revealed belated changes in the corneal stroma. These were manifested as two distinct types of abnormal reflective bodies, which had persisted beyond the stage when acute wound healing would have been expected to be complete. The clinical significance of these findings in the context of contrast visual acuity and long term status of the cornea is, as yet, unknown.

Continuous Non-contact Corneal Pachymetry with a High Speed Reflectometer

PURPOSE We developed an instrument that permits non-contact, continuous, high speed and high precision monitoring of corneal thickness and tested the stability and reproducibility of measurements made over extended time periods and under various conditions of low surface reflectivity encountered during protracted exposure of the unmoistened corneal surface to ambient air. METHODS The optical pachymeter (basic component of a broad-band, all-fiber Michelson interferometer) was used to monitor changes in the central corneal thickness of enucleated porcine eyes. Measurements were performed on three groups of eight eyes, each with different surface characteristics: intact epithelium, mechanically abraded epithelium, and 90 microm excimer laser keratectomy. Corneal thickness was monitored continuously with values recorded every 2 to 3 minutes for periods up to 1 hour in the absence of surface rinsing. RESULTS The thicknesses of all unmoistened corneas could be monitored with a precision of 1 microm (ascertained using a calibration glass plate and a living human cornea) over the entire observation period. Under ambient air conditions, deturgescence occurred in each case, and ranged from 1 to 5 microm/min. The rate of corneal thinning was fairly constant during the first 15 minutes of monitoring, but was nonlinear thereafter. Corneas with an intact epithelium had the lowest thinning rate with only 10% of the original thickness lost during the course of 1 hour. Deturgescence increased to 25% in corneas that had mechanical removal of the epithelium and to 28.5% in those that had an anterior excimer laser keratectomy, during a similar time-period. CONCLUSION With this new interferometric method, continuous, non-contact measurement of corneal thickness is possible to within a precision of 1 microm for periods up to 1 hour, even under the modified surface conditions after photoablative keratectomy. This device may be useful for on-line monitoring of ablation depths during such procedures.

Incidence of bacterial and fungal contamination of donor corneas preserved by organ culture.

We reviewed the results of sterility testing from culture media of 1,134 donor corneas preserved by organ culture at 37°C in our eye bank. All corneas were stored in minimal essential medium containing 2% fetal calf serum, 0.1 mg/ml penicillin G, 0.1 mg/ml streptomycin, and 2.5 u.g/ ml amphotericin B. After removal of ocular adnexal tissue, donor globes were rinsed with sterile saline solution, incubated in 3% polyvinylpyrrolidone-iodine solution for 3-5 min, and subsequently rinsed again with sterile saline solution. Samples for microbiological evaluation were obtained from the initial evaluation medium, at every medium change (every 10 days), and from the medium used for deswelling of the individual cornea 1 day before transplantation. Incidence of contamination was 0.53% (6 of 1,134 corneas). Three corneas were contaminated by Micrococcus species, three by fungi. We conclude from our study that a combination of rinsing donor globes with sterile saline solution, the initial use of a disinfectant, and the employment of penicillin/streptomycin and amphotericin B in the organ culture medium, which have been commonly used in cell culture for decades, result in a low incidence of bacterial and fungal contamination of corneas preserved by organ culture.

Corneal grafting of donor tissue preserved for longer than 4 weeks in organ-culture medium.

In vitro viability of the endothelium of corneas stored in organ-culture medium is longer than in other storage techniques. This article reports on the clinical results of 20 penetrating keratoplasties performed using corneas stored for >4 weeks in organ culture. Seventeen of the procedures were elective and three were emergencies. Mean preservation time was 35.6 ± 7 days (mean ± SD) and mean follow-up was 16.2 months. During preservation, an endothelial cell loss of 10% was noted. Seventeen grafts remained transparent, and three failed between 3 and 24 months postoperatively. Mean endothelial cell density was 2,153 ± 372/mm2 at 4 months and 1,854 ± 390/mm2 at 12 months. Central pachymetry was 548 ± 53 μm at 4 months and 528 ± 57 μm at 12 months after surgery. Corneas stored for up to 48 days in organ culture can be used in elective as well as in emergency procedures.