Cellular and molecular assessment of rose bengal photodynamic antimicrobial therapy on keratocytes, corneal endothelium and limbal stem cell niche.

Rose Bengal Photodynamic Antimicrobial Therapy (RB-PDAT) is a novel potential treatment for progressive infectious keratitis. The principle behind this therapy is using Rose Bengal as a photosensitizer that can be activated by green light and results in the production of oxygen free radicals which in turn eradicate the microorganism. Given RB-PDAT's mechanism of action and the potential cytotoxic effects, concerns regarding the safety of this technique have arisen. The purpose of this study was to evaluate the effect of RB-PDAT on keratocytes, while focusing on the safety profile that the photo-chemical reaction has on the limbal stem cell (LSC) niche and endothelial cell layer of the treated cornea. To perform RB-PDAT, Rose Bengal solution (0.1% RB in BSS) was applied to the right cornea of rabbits for 30 min and then irradiated by a custom-made green LED light source (525 nm, 6 mW/cm2) for 15 min (5.4 J/cm2). Three rabbits were sacrificed and enucleated after 24 h for evaluation. TUNEL assay and immunohistochemistry for endothelium and limbal stem cell viability were performed on whole mounts and frozen sections in treated and control eyes. LSC of both eyes were isolated and cultured to perform MTT viability and proliferation, and scratch wound healing assays under time-lapse microscopy. Interestingly, while Rose Bengal dye penetration was superficial, yet associated cellular apoptosis was evidenced in up to 1/3 of the stromal thickness on frozen sections. TUNEL assay on whole mounts showed no endothelial cell death following treatment. Immunohistochemistry on frozen sections of LSC displayed no structural difference between treated and non-treated eyes. There was no difference in LSC proliferation rates and scratch wound healing assay demonstrated adequate cell migration from treated and non-treated eyes. The current study suggests that even though penetration of the RB dye has been shown to be limited, oxidative stress produced by RB-PDAT can reach deeper into the corneal stroma. Nevertheless, our results show that performing RB-PDAT is safe on the corneal endothelium and has no effect on LSC viability or function.

Regional assessment of energy-producing metabolic activity in the endothelium of donor corneas.

PURPOSE: We characterized mitochondrial respiration and glycolysis activity of human corneal endothelium, and compared metabolic activity between central and peripheral regions. METHODS: Endothelial keratoplasty-suitable corneas were obtained from donors aged 50 to 75 years. The endothelium-Descemet membrane complex (EDM) was isolated, and 3-mm punches were obtained from central and peripheral regions. Endothelium-Descemet membrane punches were assayed for mitochondrial respiration (oxygen consumption) and glycolysis (extracellular acidification) using an extracellular flux analyzer. Enzymatic (citrate synthase, glucose hexokinase) and mitochondrial density (MitoTracker) assays also were performed. RESULTS: Ten corneas were analyzed per assay. Metabolic activity for mitochondrial respiration and glycolysis showed expected changes to assay compounds (P < 0.01, all pairwise comparisons). Basal mitochondrial respiration and glycolysis activity did not differ between regions (P > 0.99). Similarly, central versus peripheral activity after assay compound treatment showed no significant differences (P > 0.99, all time points). The intracorneal coefficient of variation for basal readings between two and four peripheral punches was 18.5% of the mean. Although peripheral samples displayed greater enzymatic activity than central samples (P < 0.05), similar to extracellular flux results, mitochondrial density did not differ between regions (P = 0.78). CONCLUSIONS: Extracellular flux analysis of oxygen and pH is a valid technique for characterizing metabolic activity of human corneal endothelium. This technique demonstrates high reproducibility, allows quantification of metabolic parameters using small quantities of live cells, and permits estimation of overall metabolic output. Neither oxygen consumption nor extracellular acidification differed between central and peripheral regions of transplant suitable corneas in this series. Our results show that endothelial cell health can be quantified biochemically in transplant suitable corneas.

Quality assessment of corneal storage media and their components.

BACKGROUND: To keep the loss of endothelial cell density in donor corneas to a minimum, a storage medium which is adjusted to their nutritional needs is necessary. Different media, used either serum-supplemented or serum-free, are available. The quality of medium- and serum-batches as well as support of endothelial cell viability by the medium are to be tested with a quality assured screening system that allows routine examination. METHODS: A screening system was developed which is based on cell-culture tests with the well-established human corneal endothelial cell line HCEC-12, and therefore can be performed without the need for donor corneas. The cells are plated at a defined density in cell-culture dishes, and are cultured for a defined period of time in the test media. Evaluation is carried out by assaying cell count, activity of cell metabolism (resazurin conversion), and determining the number of apoptotic and necrotic cells (combined vital staining with YO-PRO®-1/propidium iodide and subsequent flow cytometry). RESULTS: Human corneal endothelial cells that are cultured in a medium which is adjusted to their nutritional needs achieve higher cell numbers and show a higher metabolic rate. Simultaneously, the percentage of apoptotic and necrotic cells is lower. The screening system developed in this study allows for easy and reliable detection of slightest differences between different media, different processing steps for same media, and different supplements, as well as different serum batches. CONCLUSIONS: The differentiated results show that the screening system is sensitive enough to show even minor quality differences. Therefore, it is more suitable than the hitherto commonly used growth assay with primary, mostly porcine, corneal endothelial cells.

Non-invasive assessment of corneal endothelial permeability by means of electrical impedance measurements.

The permeability of the corneal endothelial layer has an important role in the correct function of the cornea. Since ionic permeability has a fundamental impact on the passive electrical properties of living tissues, here it is hypothesized that impedance methods can be employed for assessing the permeability of the endothelial layer in a minimally invasive fashion. Precisely, the main objective of the present study is to develop and to analyze a minimally invasive method for assessing the electrical properties of the corneal endothelium, as a possible diagnostic tool for the evaluation of patients with endothelial dysfunction. A bidimensional model consisting of the main corneal layers and a four-electrode impedance measurement setup placed on the epithelium has been implemented and analyzed by means of the finite elements method (FEM). In order to obtain a robust indicator of the permeability of the endothelium layer, the effect of the endothelium electrical properties on the measured impedance has been studied together with reasonable variations of the other model layers. Simulation results show that the impedance measurements by means of external electrodes are indeed sufficiently sensitive to the changes in the electrical properties of the endothelial layer. It is concluded that the method presented here can be employed as non-invasive method for assessing endothelial layer function.

Gas stress test for assessment of corneal endothelial function.

PURPOSE: This study was undertaken to evaluate the endothelial pump function by monitoring both corneal swelling response under hypoxia and dehydration response following hypoxia in vivo. METHODS: Humidified nitrogen gas was used to obtain corneal swelling, and humidified gas mixed with oxygen and nitrogen was used for corneal dehydration. First, in 6 young volunteers, we investigated the most suitable oxygen level for evaluating pump function by changing oxygen levels. Then, with the optimal oxygen level, we attempted to evaluate pump function in 53 normal subjects, 5 Fuchs' dystrophy patients, and 3 iridocorneal endothelial syndrome (ICE) patients. RESULTS: Swelling rate showed similar values regardless of age, but both dehydration rate and swelling rate plus dehydration rate decreased with aging. The swelling rate of 5 guttata corneas was significantly higher than that of age-matched control corneas. In contrast, dehydration rate markedly decreased in guttata corneas, while the swelling rate plus dehydration rate of guttata corneas was comparable to that of age-matched corneas. In the 3 ICE corneas, however, swelling rate, dehydration rate, and swelling rate plus dehydration rate were markedly lower than those of both the fellow corneas and the age-matched control corneas. CONCLUSION: These observations lead us to conclude that in order to evaluate pump function, it is necessary to monitor not only dehydration response following hypoxia but also swelling response under hypoxia.

Assessment of swelling-activated Cl- channels using the halide-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl)quinolinium.

This study describes a quantitative analysis of the enhancement in anion permeability through swelling-activated Cl- channels, using the halide-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Cultured bovine corneal endothelial monolayers perfused with NO3- Ringer's were exposed to I- pulses under isosmotic and, subsequently, hyposmotic conditions. Changes in SPQ fluorescence due to I- influx were significantly faster under hyposmotic than under isosmotic conditions. Plasma membrane potential (Em) was -58 and -32 mV under isosmotic and hyposmotic conditions, respectively. An expression for the ratio of I- permeability under hyposmotic condition to that under isosmotic condition (termed enhancement ratio or ER) was derived by combining the Stern-Volmer equation (for modeling SPQ fluorescence quenching by I-) and the Goldman flux equation (for modeling the electrodiffusive unidirectional I- influx). The fluorescence values and slopes at the inflection points of the SPQ fluorescence profile during I- influx, together with Em under isosmotic and hyposmotic conditions, were used to calculate ER. Based on this approach, endothelial cells were shown to express swelling-activated Cl- channels with ER = 4.9 when the hyposmotic shock was 110 +/- 10 mosM. These results illustrate the application of the SPQ-based method for quantitative characterization of swelling-activated Cl- channels in monolayers.

Non-invasive assessment of the donor corneal endothelium using ocular redox fluorometry.

AIMS: To investigate the usefulness of ocular redox fluorometry for evaluating donor corneal endothelial viability. METHODS: Corneas from 42 recipients of penetrating keratoplasty and four donor corneas were examined by ocular redox fluorometry. Autofluorescence from reduced pyridine nucleotides (PN) and oxidised flavoproteins (Fp) of the human corneal endothelium were measured non-invasively, and the PN/Fp ratio was used as a tissue metabolic indicator. Specular microscopy and electron microscopy were also performed. RESULTS: Both the quality of specular microscopic image and the PN/Fp ratio were significantly correlated with the degree of corneal endothelial damage determined by histological examination. Corneas with poor specular microscopic image showed significantly decreased PN/Fp ratio compared with corneas with good or fair specular images (p = 0.041 and 0.027, respectively). The PN/Fp ratio increased in corneas with mildly damaged endothelium but decreased in corneas with severely damaged endothelium determined by histological examination. Evaluation of corneal endothelium by combination of specular microscopy and ocular redox fluorometry showed excellent association with that of histopathological examination (p < 0.0001). CONCLUSION: Ocular redox fluorometry is useful for assessing donor corneal endothelial viability. Combination of ocular redox fluorometry and specular microscopy may increase the ability of donor cornea selection.