Corneal Viability Research Articles

Nutrient Supplementation Improves Contact Lens-Induced Corneal Cell Damage Based on a SIRC Cellular Model.

The long-term use of contact lenses may damage the structure of the ocular surface and cause metabolic disorders in corneal cells. Vitamins and amino acids help maintain the physiological function of the eye. In the present study, the effects of nutrient (vitamin and amino acid) supplementation on corneal cell repair after contact lens-induced damage was investigated. High-performance liquid chromatography was used to quantify the nutrient contents of minimum essential medium, and the MTT assay was used to measure the viability of corneal cells. A Statens Seruminstitut rabbit cornea cellular model was established to simulate contact lens-induced keratopathy and investigate the effects of vitamin and amino acid supplementations on corneal cell repair. The high water content lens group (78%) has a cell viability as high as 83.3%, whereas the cell viability of the low water content lens group (38%) is only 51.6%. The 32.0% difference between the two groups confirms the correlation between water content of lens and corneal viability. Vitamin B2, vitamin B12, asparagine, and taurine supplementation may help improve contact lens-induced damage.

The Effect of Bioregulators Isolated from Blood Serum and Cornea of the Bovin’s Eye on the Condition of Tissue and Cells of the Rabbit Corneas during Cultivation and Storage

Objective: to study the condition of the cornea, as well as its epithelial and endothelial cells, while maintaining in vitro at various temperature conditions, under the influence of a number of factors, including bioregulators isolated from blood serum and cornea of the bovine, and epidermal growth factor.Methods. The study was carried out on rabbit corneas stored at temperatures of +4, –86 °C, as well as the cultivation of endothelial and epithelial cells isolated from the cornea after storage at these temperatures, followed by histological and immunohistochemical studies.Results. Storage of the cornea at +4 °C for 10 days leads to corneal edema and significantly reduces their transparency, both bioregulators partially prevent a decrease in the transparency of the cornea, while the endothelial layer lyses in groups with the addition of epidermal growth factor and corneal bioregulator; but remains in the cornea with the addition of a serum bioregulator. All three factors contribute to the preservation of the Bowman membrane. In the corneas stored at –86 °C on the 30th day, a preserved endothelial layer was observed, and the epithelium retained its multilayering in all groups with the addition of factors other than the control group. In the control samples, the epithelial layer partially exfoliated, the endothelial layer was almost completely lysed. Both bioregulators stimulated the proliferation of cells isolated from the native cornea and enhanced the action of the epidermal growth factor. Similar results were obtained on cells isolated from stored corneas for 2 weeks at –86 °C. In the case of combined use of the epidermal growth factor and bioregulators on the 30th day, the endothelial layer was mainly preserved, the Descemet’s membrane was not broken. In the control samples, the epithelium was mainly single-layered, partially exfoliated, and the endothelial layer was completely lysed.Conclusion. Storage of cornea during hypothermia (+4 °С) does not provide corneal viability for longer than 10 days. Storage under conditions of cryopreservation (–86 °C) ensures the viability of the cornea for 60 days. Adding bioregulators and an epidermal growth factor to the basic preservation medium allows one to obtain a structurally safe and viable cornea, while all cellular layers of the cornea, including the endothelial layer, are preserved and viable.

Do Donated Corneas Become Transplanted Corneas? The Causes of Discard in Southern Brazil.

To analyze the number of discarded donated corneas and the causes associated with discard in southern Brazil. This retrospective, cross-sectional, and analytic study of donor corneal discards and their associated factors and geospatial distribution was based on a macroregional strategy conducted from 2011 to 2015 in Paraná, southern Brazil. The dataset included all cornea donations from patients who died of cardiac arrest at ages between 3 and 70 years. A total of 9290 donor corneas were identified from 4645 donor patients; of these corneas, 4235 (45.6%) were discarded and 5055 (54.4%) transplanted. Mean age of the donors was 51.13 ± 14.30 years. The main causes of discard were positive serology (49.6%), corneal viability (19.8%), corneal tissue quality (8.5%), and others (16.0%). The discard rate was higher in the 50 to 64 year age group. The corneal discard rate was high in all macroregions studied, with positive serology, viability, and quality of the donated corneas being the main causes of discard. Discards were more prevalent in older age groups (50-64 years and 65 or above age groups). Considering the presented results, the assessment process of potential cornea donors should be changed to reduce losses and costs.

Combined chlorhexidine and PVP-I decontamination of human donor eyes prior to corneal preservation

The aim of this study was to report the efficacy of adding chlorhexidine to the protocol for decontamination of human donor globes prior to excision of corneo-scleral rims for future keratoplasty procedures. In 2005, chlorhexidine was introduced by our eye bank as an additional step in the protocol for decontaminating human donor globes. After 5 years, we prospectively evaluated the number of contaminations. Out of 2,891 globes included in our study, 2,663 globes were processed, of which 36 (1.4%) were considered contaminated. Seventeen contaminations (0.6%) were detected by culturing limbal swabs, directly after decontamination, eight (0.3%) by visible discoloration of the culture medium carrying a corneo-scleral rim, and eleven (0.4%) after inoculation of the culture medium on blood agar plates. Importantly, after 4 weeks of incubation, none of the aerobic and anaerobic cultures taken from the secondary 'transport medium' (dextran containing medium used to transport corneal tissue to the transplantation centre) showed microbiological growth. In conclusion, the combined use of 0.02% chlorhexidine and 0.5% povidone-iodine may allow decontamination of donor globes to a level at which the risk of tissue contamination at the time of transplantation is minimized, while corneal viability is preserved.

A new nondestructive cytometric assay based on resazurin metabolism and an organ culture model for the assessment of corneal viability.

Three-dimensional culture or human corneal equivalents for safety testing are difficult to investigate with classic cytometric or biochemical methods. So a fluorometric method is proposed using resazurin probe. Absorbance and fluorescence spectra of the oxidized and reduced forms of resazurin were performed to determine optimal measurement conditions. More than 100 enucleated porcine eyes were used for this experiment. Twelve corneas were immersed in resazurin solution and fluorescence was measured hourly from 1 to 10 h. Ninety benzalkonium chloride-treated corneas and control corneas were used as toxicity controls, and corneal viability was compared with in vivo rabbit eye irritation. After analysis of spectra, the optimal measurement condition of resazurin metabolism proved to be a fluorescence measurement using 570 nm excitation wavelength and 590 nm emission wavelength. The reduction of resazurin was optimal after 6 h of incubation. Resazurin metabolism by isolated pig corneas varied proportionately with the benzalkonium chloride concentration employed, clearly showing significant differences (P < 0.001) in agreement with the in vivo data. The resazurin metabolism test can be used to evaluate corneal viability and can thus be a potential alternative for toxicological assessments. This assay allows rapid assessment of non destructed samples, with simple equipment and at a reduced cost for continuous monitoring of corneal viability and possibly other three-dimensional cellular models.

Resazurin metabolism assay is a new sensitive alternative test in isolated pig cornea.

The main object of our study was to investigate whether the resazurin metabolism assay is a sensitive surfactant and alcohol toxicity test in isolated pig cornea and to compare this recently developed fluorometric assay with the data collected in the eye irritation reference chemical data bank. Resazurin is a substrate that changes color in response to metabolic activity. Isolated pig corneas were immersed for 10 min in surfactants and alcohol irritant solutions. After incubation, resorufin fluorescence was read and corneal viability was assessed. This corneal viability was compared with the maximal modified average score published in the report of ECETOC. This assay highlighted different concentration-dependent irritation potentials of the three surfactants tested, and the same results were obtained with corneas treated with the alcohols. We observed that the degree of surfactant- and alcohol-induced decrease in corneal viability, using the resazurin reduction test, was correlated with the in vivo irritancy measurements as determined by the Draize test and scored with the Modified Maximum Average Score (MMAS). This assay allowed us to classify the ocular irritancy of the tested surfactants and alcohols in the same ranking order as the Draize classification. Corneal viability measurement can be used as a potential alternative for the toxicological assessment of surfactants and alcohols. The nontoxic, nonradioactive resazurin metabolism assay allows rapid assessment of many samples with simple equipment and at reduced cost for continuous monitoring of corneal viability. This assay seems to be suitable as a toxicological screening test for eye irritation determination.

Importance of donor selection and corneal viability

Importance of donor selection and corneal viability

Measurement of surgically induced corneal deformations using three-dimensional confocal microscopy.

The goal of this study was to develop and apply a new set of experimental techniques for measuring the local deformations induced by partial-thickness corneal incisions in situ. Eight adult cat eyes were enucleated and cannulated, with corneal viability maintained as close to in vivo conditions as possible and intraocular pressure (IOP) carefully controlled. Experimental measurements were made pre/post radial keratotomy (RK) surgery in situ at IOPs of 15, 30, and 45 mm Hg. Incision depth and cross-sectional profiles were measured at the midpoint of selected incisions using three-dimensional (3-D) tandem scanning confocal microscopy (TSCM); central corneal curvature was estimated using a commercial corneal topographical analysis system, and corneal thickness was assessed by both 3-D TSCM and ultrasonic pachymetry. Corneas were then processed for light microscopy and incision depth was measured histologically. Finite element models were developed for comparison with the experimental measurements. There was no significant change in central corneal thickness (-5.3 +/- 3.9%, n = 8) over the course of the experiments, demonstrating that normal endothelial cell function and normal stromal hydration was maintained. The in situ TSCM incision depth measurements were significantly correlated with the histological measurements (slope = 0.95, R = 0.854, p < 0.01, n = 13 incisions). Measured incision gape at the top (anterior) of the stroma was 64.9 +/- 13.4, 87.3 +/- 12.6, and 108.7 +/- 14 microns at IOPs of 15, 30, and 45 mm Hg, respectively. The 3-D incision profiles were nonlinear in shape; comparison with the finite element models suggests that the shape of the wound profile may provide unique information regarding the shear stiffness of the cornea. Overall, the data suggest that TSCM measurements of the cross-sectional profile of the incisions immediately after RK under controlled in situ conditions provide important data regarding the mechanical behavior of the cornea after refractive surgery. These data should provide the foundation for future studies into the relationships between local tissue mechanics and corneal wound healing.

Comparison of pig cornea preservation solutions using quantitative 31P NMR spectroscopy

We compared the abilities of three solutions (bicarbonate-free glucose-phosphate Ringer's solution (EP II), lactated Ringer's solution and physiologic saline solution) to preserve pig cornea by measuring phosphate metabolites with 31P NMR spectroscopy. EP II preserves corneal viability better than others, and preserving extracted cornea also is better than preserving the whole eye.

Dextran Efflux from McCarey-Kaufman-Stored Corneas as Measured by Nuclear Magnetic Resonance

Dextran, a high molecular weight polymer of glucose, is used as an osmotic agent in McCarey-Kaufman (MK) (corneal storage) medium. It has been proposed that Dextran may cause deleterious effects on living cells either by high molecular weight toxicity or by the induction of an immunological reaction against transplanted tissues. Prolonged retention of Dextran in transplanted corneas, therefore, may produce deleterious side effects. The efflux of Dextran from MK-stored human corneas was monitored by transferring corneas to 2 ml of fresh Krebs Ringer bicarbonate solution (BSS) containing 5.5 mM 1-13C-labeled glucose at approximately 1 h intervals for 1-24 h. Chemical shifts of 98.7, 74.5, 72.5, 71.2, 70.6, and 66.5 ppm were found for the naturally abundant 13C NMR spectrum of 40,000 molecular weight Dextran. Peak heights for the 70.6 ppm Dextran chemical shift were measured and plotted against total wash time to determine the slope for Dextran efflux from each cornea. The results of this study suggest that all of the Dextran absorbed into human corneas stored for up to 2 weeks in MK medium is eluted in about 19 h and that the rate of Dextran efflux is unaffected by donor age, time of storage in MK, or corneal viability.

Glucose concentration profiles of normal and ultraviolet radiation-exposed rabbit corneas

The passage of glucose within the cornea has been thought to occur by passive diffusion processes. However, corneal glucose concentration profiles have not been established to support this notion. While microfluorometric methods of metabolite assay typically have been used as a means of assessing regional brain metabolism, this unique methodology of tissue isolation and metabolite determination has not previously been applied to the cornea. Since this technique permits metabolite quantification on microgram-sized tissue samples, a co-ordinated corneal glucose concentration profile can be obtained. Tissue preparation consisted of liquid nitrogen freezing, cryo-sectioning, and freeze-drying, with storage at −20°C. The sections were thawed under vacuum pump, subsectioned, weighed, and assayed for glucose concentration (by dry weight). This study established a glucose concentration profile of the epithelium, anterior stroma, midstroma, posterior stroma, and endothelium for the normal pigmented rabbit cornea. A glucose concentration profile for UV radiation-exposed rabbit corneas also was documented. The UV radiation glucose profile data indicate the presence of an active transport mechanism capable of delivering glucose into the corneal epithelium against a concentration gradient. The presence of a transport system that ‘pulls’ glucose through the deeper corneal layers thus would make epithelial integrity important for the maintenance of overall corneal viability.

Extending Corneal Storage With 2.5% Chondroitin Sulfate (K-Sol)

ABSTRACT K-Sol ™ corneal storage medium maintains corneal viability for at least ten days, compared with McCareyKauf man (M-K) medium, which does so for a reported maximum of three days. Since many eye banks still use M-K medium exclusively, stored corneas must be used without undue delay, and shipping time further hastens the planned surgery time. Transferring the shipped M-K medium-stored cornea to K-Sol offers a simple way of increasing storage time in our eye bank. Ninety-five out of 100 transferred corneas (95%) remained clear 3 to 18 months postoperatively. There were no primary graft failures, and transferred corneal mates were clear. A comparable series of 100 corneas stored in M-K medium alone, or in K-SoI alone, were clear in 93% and 97% of the transplants. Analysis of donor rims after keratoplasty indicated statistically significantly more endothelial cell loss during storage in corneas transferred to K-SoI compared with 18 corneas preserved only in M-K medium for a similar length of time. There was no statistical significance between the transferred group and the group preserved in only K-Sol. Transferring M-K medium stored corneas to K-SoI medium may be a useful means of extending corneal storage time, resulting in improved tissue utilization.

Extending corneal storage with 2.5% chondroitin sulfate (K-Sol).

K-Sol corneal storage medium maintains corneal viability for at least ten days, compared with McCarey-Kaufman (M-K) medium, which does so for a reported maximum of three days. Since many eye banks still use M-K medium exclusively, stored corneas must be used without undue delay, and shipping time further hastens the planned surgery time. Transferring the shipped M-K medium-stored cornea to K-Sol offers a simple way of increasing storage time in our eye bank. Ninety-five out of 100 transferred corneas (95%) remained clear 3 to 18 months postoperatively. There were no primary graft failures, and transferred corneal mates were clear. A comparable series of 100 corneas stored in M-K medium alone, or in K-Sol alone, were clear in 93% and 97% of the transplants. Analysis of donor rims after keratoplasty indicated statistically significantly more endothelial cell loss during storage in corneas transferred to K-Sol compared with 18 corneas preserved only in M-K medium for a similar length of time. There was no statistical significance between the transferred group and the group preserved in only K-Sol. Transferring M-K medium stored corneas to K-Sol medium may be a useful means of extending corneal storage time, resulting in improved tissue utilization.

Corneal alanine metabolism demonstrated by NMR spectroscopy.

This study confirms the feasibility of monitoring corneal metabolism of an amino acid using high resolution NMR spectroscopy. Alanine metabolism to lactate was detected in intact, freshly isolated rabbit corneas. Progressively decreasing alanine resonances were noted after 4-5 hours of incubation while increasing amounts of lactate were detected. No similar signal was noted in de-epithelialized tissue. This study demonstrates the ability of corneal epithelium to utilize alanine as a metabolic substrate. This technique may be useful to determine the essential amino acids and their optimal concentrations necessary to maintain corneal viability during storage.