Repeatability and reproducibility of corneal biomechanical parameters derived from Corvis ST.

INTRODUCTION: The aim of this study was to evaluate repeatability and reproducibility of newly calculated biomechanical parameters of the cornea, developed by our research group. METHODS: One eye from each of the 23 healthy subjects was measured three times consecutively, three times at different daytimes and on three different days. The within-subject standard deviation and coefficient of variation, as well as the intraclass correlation coefficient, were calculated for every parameter in each group. RESULTS: Excellent repeatability and reproducibility (coefficient of variation < 5%, intraclass correlation coefficient > 0.75) was found for corrected values measured at A1, HC, and A2 time points (2nd A2 Time, 2nd A1 Time, 2nd HC Time, 2nd HC Def Amp and 2nd A1 Def Amp). Corneal-specific stiffness parameters, which showed good repeatability and reliability, were DA_cor (coefficient of variation = 4.02%, intraclass correlation coefficient = 0.919), KcLinear (coefficient of variation = 4.03%, intraclass correlation coefficient = 0.895), areaForceCornea (coefficient of variation = 3.34%, intraclass correlation coefficient = 0.853) and E2 (coefficient of variation = 4.1%, intraclass correlation coefficient = 0.78). Overall, most parameters fell into the category of good reliability (high intraclass correlation coefficient) and poor reproducibility (low coefficient of variation), including all the parameters describing extraocular deformation (DA_ext, AEPvED, AUC EDef, areaForceExtra, Kg and µg). Comparing the coefficient of variation values for intrasession, intersession and daytime measurements, there were no indices for diurnal changes. CONCLUSION: Most parameters showed good repeatability and reliability. The extraocular stiffness parameters showed poor reproducibility. KcLinear can serve as a very reliable and repeatable indicator of corneal stiffness.

CROP - The Clinico-Radiologico-Ophthalmological Paradox in Multiple Sclerosis: Are Patterns of Retinal and MRI Changes Heterogeneous and Thus Not Predictable?

BACKGROUND: To date, no direct scientific evidence has been found linking tissue changes in multiple sclerosis (MS) patients, such as demyelination, axonal destruction or gliosis, with either steady progression and/or stepwise accumulation of focal CNS lesions. Tissue changes such as reduction of the retinal nerve fiber layer (RNFL) and the total macular volume (TMV), or brain- and spinal cord atrophy indicates an irreversible stage of tissue destruction. Whether these changes are found in all MS patients, and if there is a correlation with clinical disease state, remains controversial. The objective of our study was to determine, whether there was any correlation between the RNFL or TMV of patients with MS, and: (1) the lesion load along the visual pathways, (2) the ratios and absolute concentrations of metabolites in the normal-appearing white matter (NAWM), (3) standard brain atrophy indices, (4) disease activity or (5) disease duration. METHODS: 28 MS patients (RRMS, n = 23; secondary progressive MS (SPMS), n = 5) with moderately-high disease activity or long disease course were included in the study. We utilised: (1) magnetic resonance imaging (MRI) and (2) -spectroscopy (MRS), both operating at 3 Tesla, and (3) high-resolution spectral domain-OCT with locked reference images and eye tracking mode) to undertake the study. RESULTS: There was no consistency in the pattern of CNS metabolites, brain atrophy indices and the RNFL/TMV between individuals, which ranged from normal to markedly-reduced levels. Furthermore, there was no strict correlation between CNS metabolites, lesions along the visual pathways, atrophy indices, RNFL, TMV, disease duration or disability. CONCLUSIONS: Based on the findings of this study, we recommend that the concept of 'clinico-radiologico paradox' in multiple sclerosis be extended to CROP-'clinico-radiologico-ophthalmological paradox'. Furthermore, OCT data of MS patients should be interpreted with caution.

Corrigendum: "High-Resolution Spectral Domain-Optical Coherence Tomography in Multiple Sclerosis, Part II - The Total Macular Volume. The First Follow-Up Study Over 2 Years".

[This corrects the article on p. 20 in vol. 5, PMID: 24605107.].

Detection of Retinal Nerve Fiber Layer Defects in Alzheimer's Disease Using SD-OCT.

INTRODUCTION: Our aim is to examine the clinical value of spectral-domain optical coherence tomography (Spectralis OCT) to detect retinal nerve fibre layer defects in patients with clinically defined Alzheimer's disease (AD). MATERIAL AND METHODS: This cross-sectional study included 22 patients with AD (mean age: 75.9 ± 6.1 years) and 22 healthy age- and sex-matched controls. Neuro-ophthalmologic examinations and a series of high-resolution OCT examinations of the peripapillary retinal nerve fiber layer (RNFL) thickness using the Spectralis 3.5-mm circle scan protocol with ART-Modus and eye tracking were obtained, and compared to age- and sex-matched healthy control subjects. RESULTS: Patients with AD showed a significant decrease in RNFL thickness in the nasal superior sector compared to the control group (101.0 ± 18.18 µm versus 122.8 ± 28.08 µm; P < 0.0001). In all other sectors, independently of disease duration, no significant difference in RNFL thickness compared to controls was detected. Using the advanced age- and gender-matched measurement model, 32 out of 42 eyes (76.19%) as pathologic with 67 abnormal sectors were detected. DISCUSSION: As examined by spectral-domain OCT, patients with mild to moderate stages of AD showed a significant reduction of RNFL thickness in the nasal superior sector. Nevertheless, successive studies are needed.

High-Resolution Spectral Domain-Optical Coherence Tomography in Multiple Sclerosis, Part II - the Total Macular Volume. The First Follow-Up Study over 2 Years.

BACKGROUND: Recent studies investigating the use of optical coherence tomography (OCT) in multiple sclerosis (MS) patients have resulted in wide-ranging and often contradictory outcomes. This is mainly due to the complex etiology and heterogeneity of MS, physiological variations in the retinal nerve fiber layer (RNFL) and/or total macular volume (TMV), and limitations in methodology. It remains to be discovered whether any retinal changes in MS develop continuously or in a stepwise fashion, and whether these changes occur in all or a subset of patients. High-resolution spectral domain-OCT devices (SD-OCT) would be required to detect subtle retinal changes and longitudinal studies would have to be carried out to investigate retinal changes over time. In addition, if the hypothesis is correct, then retinal and global brain tissue changes should be detected in a substantial majority of MS patients and detection should be possible with a high degree of disease activity and/or long disease course. METHODOLOGY: In order to address the factors above, 37 MS patients (relapsing-remitting, n = 27; secondary progressive, n = 10) were examined prospectively on two occasions with a median interval of 22.4 ± 0.5 months [range 19-27]. SD-OCT was utilized with the Spectralis 3.5 mm circle scan protocol (with locked reference images and eye-tracking mode). None of the patients had optic neuritis 12 months prior to study entry or during the observation period. PRINCIPAL FINDINGS: The initial TMV pattern differed between study participants, but remained relatively unchanged over the 2-year observation period despite high disease activity or long disease course. The TMV correlated well with the RNFL. CONCLUSION: The significance of differences in TMV (and RNFL) between study participants remains unclear. Until these differences have been explored further, OCT data in MS patients should be interpreted with caution.

Comparison of VEP with contrast sensitivity and other measurements of central visual function.

PURPOSE: In order to evaluate alternative visual acuity testing techniques, especially to discriminate between small changes and for high visual acuity, we conducted a study covering several state-of-the-art techniques. METHODS: In this cross-sectional study, a homogeneous cohort of healthy and young patients (n = 33; 66 eyes) underwent ETDRS vision acuity (VA) testing, testing for contrast sensitivity (CS), VA determination with spatial frequency sweep visual evoked potentials (VEP) and a series of examinations of perifoveal retinal nerve fibre layer thickness (RNFLT) using Spectralis SD-OCT. To simulate the effect of artificial media opacity, CS, and VEP were repeated with Bangerter foils. RESULTS: We found that Bangerter foils can be used to reduce VA effectively measured by VA testing and VEP VA. CS correlated significantly with VA (correlation coefficients ranging from 0.54 to 0.77). VEP may be used to estimate VA; nevertheless, we found no significant correlation. RNFLT did not correlate significantly with VA. CONCLUSION: CS seems to correlate well with VA when used for high VA. All other used examinations seem to have difficulties distinguishing between small differences in VA or when the VA is high.

Comparison of visual evoked potentials and retinal nerve fiber layer thickness in Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) is a long term progressive neurodegenerative disease and might affect the retinal nerve fiber layer thickness (RNFLT) of the eye. There is increasing evidence that visual evoked potentials (VEP), which are an objective way to indicate visual field loss, might be affected by the disease as well. MATERIALS AND METHODS: About 22 patients (mean age: 75.9 ± 6.1 years; 14 women) with mild-to-moderate AD and 22 sex-matched healthy patients were examined. We compared the use of VEP and RNFLT using the latest high-resolution spectral domain optical coherence tomography with eye-tracking capabilities for optimized peripapillary scan centering for the first time in AD patients. RESULTS: The mean MMSE score was 22.59 ± 5.47 in the AD group, and did not significantly correlate with the VEP latencies. We found no significant difference between the VEP latencies of the AD patients and those of the control patients. No peripapillary sector of the retina had a RNFLT significantly correlated with the VEP latencies. DISCUSSION: We demonstrated that pattern VEP did not show any significant correlation despite subtle loss in RNFLT. It remains open whether additional flash VEP combined with RNFLT analysis may be useful in diagnosing AD, particularly for mild-to-moderate stages of the disease.

Interpretation of RNFLT values in multiple sclerosis-associated acute optic neuritis using high-resolution SD-OCT device.

PURPOSE: Optical coherence tomography (OCT) has emerged as the technique of choice in measuring the retinal nerve fibre layer (RNFL) quantitatively. It is suggested that RNFL reduction may correlate with lesion burden and diffuse axonal degeneration in the whole CNS of patients with multiple sclerosis (MS). However, RNFL changes because of optic neuritis (ON) must be taken into account. METHODS: Twenty-three patients with acute ON (46 eyes) associated with clinical definite MS (23 ON eyes, 23 fellow eyes) and 23 sex- and age-matched healthy controls were studied. Retinal nerve fibre layer thickness (RNFLT) was measured at baseline, using a high-resolution spectral domain OCT (SD-OCT) applying circular, peripapillary OCT scans with a novel eye-tracking mechanism. RESULTS: The internal OCT software was able to identify RNFL atrophy in three out of five of the acute ON eyes and one out of four of the fellow eyes with previous ON episodes. Retinal nerve fibre layer thickness of two ON (8.7%) and five fellow eyes (21.7%) was overestimated, thus located within the 95% and 5% confidence interval of the company standard values (not marked pathologic). In contrast, our comparison with age- and sex-matched controls revealed RNFL atrophy suggestive of prior, clinically silent RNFL loss in ON and fellow eyes (30.4%). CONCLUSION: Retinal nerve fibre layer thickness measurements at a single time-point seem to have a limited role in detecting prior clinically silent optic nerve injury. Our data suggest that affected eyes should be compared with the fellow eyes and a sufficient number of age- and sex-matched controls to allow the detection of even subtle RNFL changes at baseline. The role of OCT for disease monitoring of MS must be evaluated in detail, as ON is often the initial symptom of MS.

Retinal blood flow velocity measured by retinal function imaging in retinitis pigmentosa.

BACKGROUND: To measure the retinal blood flow velocity in patients with retinitis pigmentosa using the retinal function imaging technique. METHODS: The clinical observational investigation included a study group of five eyes of five patients (age: 55.7 ± 8.6 years) with retinitis pigmentosa (RP) and a control group of five eyes of five healthy subjects. We used a randomly chosen eye of the RP patients, and compared its results to the normal subjects using a mixed linear model, correcting for heart rate, age, and gender. RESULTS: The mean blood velocity in the narrow retinal veins (1.7 ± 0.35 cm/s versus 3.0 ± 0.35 cm/s; P < 0.001) and wide retinal veins (1.5 ± 0.35 cm/s versus 3.1 ± 0.30 cm/s; P < 0.001) was significantly lower in the study group than in the control group not correcting for heart rate, age or gender. Correspondingly, the arterial blood flow velocity was significantly lower in the study group than in the control group for the narrow arterial vessels (2.3 ± 0.55 versus 4.2 ± 0.5; P = 0.006) and for the wide retinal arteries (2.5 ± 1.05 cm/s versus 4.8 ± 1.0 cm/s; P < 0.001). CONCLUSIONS: Using the retinal function imaging technology revealed significantly lower retinal blood flow velocities in the small and large retinal vessels in patients with retinitis pigmentosa than in healthy subjects. This corresponds with the known decrease in the retinal vessel diameters as observed upon ophthalmoscopy in patients with retinitis pigmentosa. Retinal function imaging technology may hold promise for measurements of retinal blood flow parameters.

High resolution spectral domain optical coherence tomography (SD-OCT) in multiple sclerosis: the first follow up study over two years.

BACKGROUND: "Non-invasive, faster and less expensive than MRI" and "the eye is a window to the brain" are recent slogans promoting optical coherence tomography (OCT) as a new surrogate marker in multiple sclerosis (MS). Indeed, OCT allows for the first time a non-invasive visualization of axons of the central nervous system (CNS). Reduction of retina nerve fibre layer (RNFL) thickness was suggested to correlate with disease activity and duration. However, several issues are unclear: Do a few million axons, which build up both optic nerves, really resemble billions of CNS neurons? Does global CNS damage really result in global RNFL reduction? And if so, does global RNFL reduction really exist in all MS patients, and follow a slowly but steadily ongoing pattern? How can these (hypothesized) subtle global RNFL changes be reliably measured and separated from the rather gross RNFL changes caused by optic neuritis? Before generally being accepted, this interpretation needs further critical and objective validation. METHODOLOGY: We prospectively studied 37 MS patients with relapsing remitting (n = 27) and secondary progressive (n = 10) course on two occasions with a median interval of 22.4±0.5 months [range 19-27]. We used the high resolution spectral domain (SD-)OCT with the Spectralis 3.5 mm circle scan protocol with locked reference images and eye tracking mode. Patients with an attack of optic neuritis within 12 months prior to the onset of the study were excluded. PRINCIPAL FINDINGS: Although the disease was highly active over the observation period in more than half of the included relapsing remitting MS patients (19 patients/32 relapses) and the initial RNFL pattern showed a broad range, from normal to markedly reduced thickness, no significant changes between baseline and follow-up examinations could be detected. CONCLUSIONS: These results show that caution is required when using OCT for monitoring disease activity and global axonal injury in MS.

RNFL thickness in MS-associated acute optic neuritis using SD-OCT: critical interpretation and limitations.

PURPOSE: Axonal loss is considered a key prognostic factor in diagnosing and monitoring the progress of multiple sclerosis (MS). The purpose of our research was to determine whether the measurement of retinal nerve fibre layer thickness (RNFLT) as measured with high-resolution spectral-domain optical coherence tomography (SD-OCT) differs between optic nerve injury following acute optic neuritis (ON) or following unregistered subclinical axonal damage in patients with MS. METHODS: High-resolution SD-OCT measurements of RNFLT were initially carried out in the acute phase of ON and again after 3 months, in 25 patients with clinical definite MS and 25 sex- and age-matched healthy controls, all at the University Eye Hospital, Vienna. RESULTS: Conventional OCT-based RNFLT analysis correctly identified all three patients with initial RNFL swelling. However, only two of three acute ON eyes with a history of ON were registered with RNFLT decrease in seven peripapillary sectors (PPs). The remaining have only been revealed using RNFLT symmetry comparison. Two of 22 (9%) first-episode ON eyes were labelled as pathologic. The number and metric RNFL values of pathologically labelled PPs remained unchanged after 3 months. Our age- and sex-match-based measurement model, with patients with MS being plotted individually and towards the fellow eye, identified all acute ON eyes (with a history of prior ON) with RNFLT reduction in 11 PPs. A global RNFL loss was registered in 36.4% (eight of 22 eyes). However, in 72%, or 16 of 22 ON eyes presenting with first episode of acute ON, a segmental RNFL loss was initially registered in 39 PPs upon baseline examination. The number of PPs with identified axonal decrease increased to a total of 48 PPs within the observational period. CONCLUSIONS: Spectral-domain optical coherence tomography imaging of identical scanning locations, combined with an optimized scan centring around the optic disc, offers the technological potential of detecting prior, subtle, clinically unregistered optic nerve injury within MS individuals. Significant discrepancy in RNFLT to the potential ON eye may be achieved by comparing OCT metrics with the fellow eye and a sufficient number of age and sex-matched controls.

Central serous chorioretinopathy shows reduced retinal flow circulation in retinal function imaging (RFI).

PURPOSE: To assess retinal blood flow in patients with central serous chorioretinopathy (CSR). METHODS: The hospital-based observational comparative study included a study group with 12 patients (age: 45 ± 13 years) with an acute onset of CSR and a control group of 12 subjects matched for age and gender with the study group. The diagnosis was substantiated by fluorescein angiography and optical coherence tomography. All study participants underwent measurement of retinal blood perfusion using the retinal function imager (RFI). RESULTS: The retinal blood flow velocity in the retinal veins was significantly lower in the study group than in the control group (2.76 ± 0.53 mm/s versus 3.33 ± 0.76 mm/s; p = 0.03).The difference between the study group and control group was more marked for the larger retinal veins (2.87 ± 0.51 mm/s versus 3.47 ± 0.48 mm/s; p = 0.001) than for the smaller veins (2.69 ± 0.53 mm/s versus 3.42 ± 1.05 mm/s; p = 0.04). Both groups did not differ significantly in the data of the retinal arterial flow velocities. CONCLUSIONS: The data suggest an abnormal retinal venous blood flow regulation in patients with active CSR.

Assessment of central corneal thickness using OCT, ultrasound, optical low coherence reflectometry and Scheimpflug pachymetry.

PURPOSE: Accurate measurement of central corneal thickness (CCT) is essential in refractive surgery and advanced glaucoma diagnostics. The gold standard for pachymetry is full-contact ultrasound-based pachymetry. As this method is associated with potential sources of error, noncontact methods have been introduced. The aim of this study was to compare CCT results measured using 4 different techniques. METHODS: In this analysis of 20 patients (40 eyes) at the University Eye Hospital Heidelberg, Germany, we compared a slit-lamp-mounted optical coherence tomography (OCT) system (SL-OCT, Heidelberg Engineering, Heidelberg, Germany), conventional ultrasound pachymetry (IOPac, Heidelberg Engineering), optical low coherence reflectometry (OLCR, Haag-Streit, Germany), and scanning-slit pachymetry (Orbscan). RESULTS: Comparison among the 4 groups did not show significant differences, except the comparison of OLCR to Orbscan; the mean was significantly different (p=0.0247) and the Orbscan detected slightly thicker values than the other methods. CONCLUSIONS: Orbscan, SL-OCT, and OLCR provide non-touch technology, without the need for local anesthesia, and limiting the risk of infection or artifacts. Extreme care must be used interpreting the results obtained from Orbscan, as this technique may overestimate the CCT significantly.

Heterogeneous pattern of retinal nerve fiber layer in multiple sclerosis. High resolution optical coherence tomography: potential and limitations.

BACKGROUND: Recently the reduction of the retinal nerve fibre layer (RNFL) was suggested to be associated with diffuse axonal damage in the whole CNS of multiple sclerosis (MS) patients. However, several points are still under discussion. (1) Is high resolution optical coherence tomography (OCT) required to detect the partly very subtle RNFL changes seen in MS patients? (2) Can a reduction of RNFL be detected in all MS patients, even in early disease courses and in all MS subtypes? (3) Does an optic neuritis (ON) or focal lesions along the visual pathways, which are both very common in MS, limit the predication of diffuse axonal degeneration in the whole CNS? The purpose of our study was to determine the baseline characteristics of clinical definite relapsing-remitting (RRMS) and secondary progressive (SPMS) MS patients with high resolution OCT technique. METHODOLOGY: Forty-two RRMS and 17 SPMS patients with and without history of uni- or bilateral ON, and 59 age- and sex-matched healthy controls were analysed prospectively with the high resolution spectral-domain OCT device (SD-OCT) using the Spectralis 3.5mm circle scan protocol with locked reference images and eye tracking mode. Furthermore we performed tests for visual and contrast acuity and sensitivity (ETDRS, Sloan and Pelli-Robson-charts), for color vision (Lanthony D-15), the Humphrey visual field and visual evoked potential testing (VEP). PRINCIPAL FINDINGS: All 4 groups (RRMS and SPMS with or without ON) showed significantly reduced RNFL globally, or at least in one of the peripapillary sectors compared to age-/sex-matched healthy controls. In patients with previous ON additional RNFL reduction was found. However, in many RRMS patients the RNFL was found within normal range. We found no correlation between RNFL reduction and disease duration (range 9-540 months). CONCLUSIONS: RNFL baseline characteristics of RRMS and SPMS are heterogeneous (range from normal to markedly reduced levels).

Function of the tryptophan metabolite, L-kynurenine, in human corneal endothelial cells.

PURPOSE: Penetrating keratoplasty has been the mainstay for the treatment of blindness and is the most common form of tissue transplantation worldwide. Due to significant rates of rejection, treatment of immunological transplant reactions is of wide interest. Recently in a mouse model, the overexpression of indoeleamine 2,3 dioxigenase (IDO) was led to an extension in corneal allograft survival. L-kynurenine is a tryptophan metabolite, which may render activated T-cells apoptotic and therefore might modulate an allogenous transplant reaction. The function of L-kynurenine in the human cornea remains unclear. We analyzed the expression levels of IDO in human corneal endothelial cells (HCECs) and downstream tryptophan/kynurenine mechanisms in cell culture. METHODS: An immunological activation profile was determined in proliferation assays of monocytes from healthy donors. Reversed-phase high pressure liquid chromatography (HPLC), western blot, real time polymerase chain reaction (PCR), and microarray analyses were used. The expression of IDO and immunological infiltration of rejected human corneal allografts (n=12) were analyzed by immunohistochemistry. RESULTS: We found IDO and an associated tryptophan/kynurenine transporter protein exchange mechanism upregulated by inflammatory cytokines in HCECs. The inhibition of T-cell proliferation might depend on rapid delivery of the tryptophan metabolite, L-kynurenine, to the local corneal environment. Microarray analysis gives evidence that the large amino acid transporter 1 (LAT1) transporter protein is responsible for this mechanism. CONCLUSIONS: Our data support that adequate levels of functional L-kynurenine might contribute to the maintenance of a relative immune privilege in the ocular anterior chamber, thereby contributing to the preservation of corneal allogeneic cells.

Paroxysmal nocturnal hemoglobinuria may cause retinal vascular occlusions.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the classic triad of haemolytic anaemia, thrombophilia and cytopenia with the majority of cases occurring in adulthood. PNH constitutes a nonmalignant clonal disease of hematopoietic stem cells harboring somatic mutations in the X-linked phosphatidyl inositol glycan complementation group-A (PIG-A) gene. METHODS: We report for the first time retinal venous vascular occlusion as the primary manifestation of PNH. A patient of untypical age for retinal vascular occlusions presented with a history of 4 weeks of progressive reduction in visual acuity. RESULTS: The screening tests for thrombophilia were not successful. However, elevated LDH was detected, leading to the diagnosis of PNH. CONCLUSIONS: To date, no report shows retinal vascular occlusion as the primary symptom leading to the diagnosis PNH. This article describes, for the first time, that this rare disease needs to be considered in the differential diagnosis of retinal vascular occlusions.

Indoleamine 2,3-dioxygenase protects corneal endothelial cells from UV mediated damage.

Indoleamine-2,3-dioxygenase (IDO) is an intracellular enzyme present in dendritic cells and macrophages. It is a known modulator of T-cell response and contributes to the UV protection of the lens. There yet is no information on IDO activity in the corneal endothelium, protecting the endothelial cells from light mediated damage. We exposed murine corneal endothelial cells (MCEC) with different doses of UV-B light 280-320 nm, probed for IDO mRNA (real-time PCR) and assessed apoptosis rate (flow cytometry) and caspase-3-activity in the cells. The metabolites of the IDO catalysed reaction, l-kynurenine, was also measured. Malondialdehyde was detected for quantification of UV-B-induced oxidative stress. To investigate specificity, IDO effects were blocked by 1-methyl-tryptophan. The effects of IDO overexpression in the MCEC were assessed by transfection of an expression vector. MCEC consistently express IDO at low levels. Exposure to UV-B light led to a dose-responding upregulation of IDO; IDO was found competent converting l-tryptophan into l-kynurenine. Irradiation led to increased apoptosis and caspase-3-activity of MCEC. Supplementation of l-kynurenine or overexpression of IDO in the MCEC could reduce apoptosis significantly following UV-B irradiation. Inhibition of IDO by 1-MT was potent to reverse this effect. IDO and its metabolite l-kynurenine can protect corneal endothelial cells from UV-B-induced oxidative stress and apoptosis. It may be an active protection mechanism against corneal endothelial damage.

Vitamins inhibit oxidant-induced apoptosis of corneal endothelial cells.

PURPOSE: To determine the effects of vitamins A, C, and E supplementation on lipid peroxidation and apoptosis in corneal endothelial cells. METHODS: Murine corneal endothelial cells were maintained in tissue culture medium supplemented with free iron ions, known to lead to increased lipid peroxidation. The concentration of antioxidative vitamins (ascorbic acid, tocopherol, and retinoic acid) in the cells and supernatant was determined using reversed-phase high-performance liquid chromatography. Apoptosis was assessed by quantification of caspase-3-like activity, using annexin-V/propidium iodide stains for flow cytometry. Lipid peroxidation was assessed using the malondialdehyde method. Supplementation of antioxidative vitamins was tested in the setting of apoptosis. RESULTS: Increasing levels of free iron led to a rapid loss of antioxidative vitamins in the supernatant and corneal endothelial cells. This was correlated with rising levels of malondialdehyde and increased apoptosis. Supplementation with ascorbic acid or alpha-tocopherol alone was not sufficient to prevent lipid peroxidation in the cells, whereas a combination of vitamins C and E was able to do so. In contrast, supplementation with vitamin A alone significantly reduced oxidative stress and apoptosis. CONCLUSIONS: We present an in vitro model to test the direct influence of vitamin supplementation on corneal endothelial cells with regard to lipid peroxidation and apoptosis. We show that supplementation with antioxidative vitamins of corneal endothelial cells significantly prevents the generation of free-radical injury, lipid peroxidation, and consequent apoptosis.