Neutralization of the eye and skin irritant benzalkonium chloride using UVC radiation.

PURPOSE: Benzalkonium chloride (BAK) is a widely used disinfectant and preservative which is effective against a wide range of viruses (e.g. SARS-CoV and SARS-CoV-2), bacteria and fungi. However, it is toxic to the eye and skin. This study investigated the neutralization of BAK using ultraviolet C (UVC) radiation as an effort to reduce BAK toxicity potential. METHODS: BAK solutions were irradiated with a germicidal UVC lamp at various doses. Human corneal epithelial cells (HCEC) were then exposed to the UVC-irradiated BAK solutions for 5 minutes. After exposure, the cultures were assessed for metabolic activity using PrestoBlue; for cell viability using confocal microscopy with viability dyes; and for tight junction proteins using immunofluorescence staining for zonula occludens (ZO)-1. RESULTS: UVC radiation reduced BAK toxicity on cell metabolic activity in a dose-dependent manner. When the solution depth of BAK was 1.7 mm, the UVC doses needed to completely neutralize the toxicity of BAK 0.005% and 0.01% were 2.093 J/cm2 and 8.374 J/cm2, respectively. The cultures treated with UVC-neutralized BAK showed similar cell metabolic activity and cell viability to those treated with phosphate buffered saline (PBS) (p = 0.806 ∼ 1.000). The expression of ZO-1 was greatly disturbed by untreated BAK; in contrast, ZO-1 proteins were well maintained after exposure to UVC-neutralized BAK. CONCLUSIONS: Our study demonstrates that the cell toxicity of BAK can be neutralized by UVC radiation, which provides a unique way of detoxifying BAK residues. This finding may be of great value in utilizing the antimicrobial efficacy of BAK (e.g. fighting against SARS-CoV-2) while minimizing its potential hazards to human health and the environment.

Ocular toxicology: synergism of UV radiation and benzalkonium chloride.

PURPOSE: To investigate the combined toxic effect of ultraviolet (UV) radiation and benzalkonium chloride (BAK), a common preservative in ophthalmic eye drops, on human corneal epithelial cells (HCEC). METHODS: Cultured HCEC were exposed to different combined and separate UV (280-400 nm) and BAK solutions at relevant human exposure levels. Human exposure to UV can occur before, during, or after eye drop installation, therefore, three different orders of ocular exposures were investigated: UV and BAK at the same time, UV first followed by BAK, and BAK first followed by UV. Control treatments included testing HCEC exposed to BAK alone and also HCEC exposed to UV alone. In addition, phosphate-buffered saline (PBS) was used as a negative control. After exposure, cell metabolic activity of the cultures was measured with PrestoBlue, and cell viability was determined using confocal microscopy with viability dyes. RESULTS: BAK alone reduced the metabolic activity and cell viability of HCEC in a dose- and time-dependent manner. UV alone at a low dose (0.17 J/cm2) had little toxicity on HCEC and was not significantly different from PBS control. However, UV plus BAK showed combined effects that were either greater than (synergistic) or equal to (additive) the sum of their individual effects. The synergistic effects occurred between low dose UV radiation (0.17 J/cm2) and low concentrations of BAK (0.001%, 0.002%, 0.003%, and 0.004%). CONCLUSIONS: This investigation determined that at relevant human exposure levels, the combination of UV radiation (280-400 nm) and BAK can cause synergistic and additive toxic effects on human corneal epithelial cells. This finding highlights the importance of considering the combined ocular toxicity of BAK and solar radiation in the risk assessment of BAK-preserved ophthalmic solutions.

Effects of Antifungal Soaked Silicone Hydrogel Contact Lenses on Candida albicans in an Agar Eye Model.

PURPOSE: To evaluate the effects of two commercial silicone hydrogel contact lenses (CLs) soaked with natamycin (NA) or fluconazole (FL) on the growth of Candida albicans in an in vitro eye model. METHODS: Three-D printed molds were used as a cast for making eye-shaped models comprising potato dextrose agar. Senofilcon A (SA) and lotrafilcon B (LB) CLs were incubated with either 2 mL of NA or FL at a concentration of 1 mg/mL for 24 hr. To simulate a fungal infection, the eye models were coated with C. albicans. The drug-soaked lenses were placed on top of the eye models. Seven experimental conditions were examined: (1) NA-SA, (2) NA-LB, (3) FL-SA, (4) FL-LB, (5) SA, (6) LB, and (7) control-no lens. At specified time points (t=1, 8, 16, 24, 48 hr), the agar eyes from each experimental condition were removed from the incubator and photographed. The yeast cells from the 24 and 48 hr time point were also analyzed using light microscopy. RESULTS: At 24 and 48 hr, there was considerable growth observed for all conditions except for the NA-SA and NA-LB conditions. When observed under the microscope at 24 and 48 hr, the morphology of the yeast cells in the FL-SA and SA condition were similar to that of the control (oval shaped). There was limited hyphae growth observed for LB and significant visible hyphae growth for the NA-LB group. For NA-SA, NA-LB, and FL-LB groups, the cells were significantly smaller compared with the control. CONCLUSIONS: For NA-SA and NA-LB, there was limited growth of C. albicans observed on the eye models even after 48 hr. Under the microscope, the cell morphology differ noticeably between each testing condition, and is dependent on drug-lens combinations.

Efficacy of antimicrobials against biofilms of Achromobacter and Pseudomonas.

PURPOSE: Achromobacter xylosoxidans and Pseudomonas aeruginosa biofilms can develop in ophthalmic products and accessories such as contact lens cases, leading to the development of ocular infections. This study evaluated the efficacy of the antimicrobials polyaminopropyl biguanide (PAPB) and benzalkonium chloride (BAK) against A. xylosoxidans and P. aeruginosa biofilms. METHODS: Biofilms of A. xylosoxidans and P. aeruginosa used as a comparative control were formed by incubating the bacteria on contact lens cases and on coverslips in phosphate-buffered saline. The biofilms were then exposed to PAPB and BAK for 5 minutes and 4 hours. After exposure, alginate swabs were used to remove the biofilms from the lens cases and the bacteria were plated on tryptic soy agar for determination of survivors. Also, after exposure to these disinfectants, the A. xylosoxidans and P. aeruginosa biofilms were stained with SYTO 9 and propidium iodide. Using a confocal microscope with a 488-nm laser, the number of cells with damaged cell membranes was determined. RESULTS: After 5 minutes of exposure to BAK or PAPB, A. xylosoxidans biofilms were more resistant to the antimicrobial effects of these disinfectants than P. aeruginosa biofilms. After 4 hours, both organisms were reduced by more than 3 logs after exposure to either BAK or PAPB. Confocal microscopy studies revealed that BAK was more effective at damaging A. xylosoxidans and P. aeruginosa cell membranes than PAPB at the concentrations used in ophthalmic products. CONCLUSIONS: Biofilms of the emerging pathogen A. xylosoxidans were more resistant to the disinfectants PAPB and BAK than biofilms of P. aeruginosa. Because of the emergence of A. xylosoxidans and the demonstrated greater resistance to the common ophthalmic preservatives BAK and PAPB than the standard Gram-negative organism P. aeruginosa, A. xylosoxidans biofilms should be assessed in antimicrobial challenge tests to assure the safety of multiuse ophthalmic products.

Application of cyclic biamperometry to viability and cytotoxicity assessment in human corneal epithelial cells.

The application of cyclic biamperometry to viability and cytotoxicity assessments of human corneal epithelial cells has been investigated. Electrochemical measurements have been compared in PBS containing 5.0 mM glucose and minimal essential growth medium. Three different lipophilic mediators including dichlorophenol indophenol, 2-methyl-1,4-naphthoquinone (also called menadione or vitamin K3) and N,N,N',N'-tetramethyl-p-phenylenediamine have been evaluated for shuttling electrons across the cell membrane to the external medium. Transfer of these electrons to ferricyanide in the extra cellular medium results in the accumulation of ferrocyanide. The amount of ferrocyanide is then determined using cyclic biamperometry and is related to the extent of cell metabolic activity and therefore cell viability. To illustrate cytotoxicity assessment of chemicals, hydrogen peroxide, benzalkonium chloride and sodium dodecyl sulfate have been chosen as sample toxins, the cytotoxicities of which have been evaluated and compared to values reported in the literature. Similar values have been reported using colorimetric assays; however, the simplicity of this electrochemical assay can, in principle, open the way to miniaturization onto lab-on-chip devices and its incorporation into tiered-testing approaches for cytotoxicity assessment.

Organ Culture System for Assessing the Toxicity of Intraocular Treatment Excipients and Pharmaceuticals.

As the leading cause of blindness, cataracts are a significant burden for the tens of millions of people affected globally by this condition. Chemical exposures, among other environmental factors, are an established cause of cataracts. Ocular toxicity testing can assess whether pharmaceuticals and their components may contribute to lens damage that may lead to cataracts or aid the treatment of cataracts. In vitro studies and in vivo animal testing can be used for assessing the safety of chemicals prior to clinical studies. The Draize test-the current in vivo standard for ocular toxicity and irritancy testing-has been criticized for lack of sensitivity and objective measurements of determining ocular toxicity. In vitro cell-based assays are limited as cell cultures cannot appropriately model an intact functional lens. The method described here is a sensitive in vitro alternative to animal testing, designed to evaluate the response of the intact bovine lens to treatment at both the cellular activity level and for overall refractive performance. The non-toxic reagent resazurin is metabolized in proportion to the level of cell activity. The lens laser-scanner assay measures the ability of the lens to refract incident beams of light to a single point with minimal error, directly relevant to its natural function. The method may be used to determine both acute and delayed changes in the lens, as well as the recovery of the lens from chemical or environmental exposures.

In vitro assessment of the biocompatibility of chemically treated silicone materials with human lens epithelial cells.

Cytotoxicity testing is a regulatory requirement for safety testing of new ocular implants. In vitro toxicity tests determine whether toxic chemicals are present on a material surface or leach out of the material matrix. A method of evaluating the cytotoxicity of ocular implants was developed using fluorescent viability dyes. To assess the assay's sensitivity in detecting toxic substances on biomaterials, zinc diethydithiocarbamate (ZDEC) and benzalkonium chloride (BAK) were deposited on silicone surfaces at different concentrations. Human lens epithelial cells (HLEC) were added to the surface of these treated silicone surfaces and were assessed for viability. The viability of both the adherent and non-adherent cells was determined using confocal microscopy with, annexin V, ethidium homodimer, and calcein. Cell metabolism was also evaluated using resazurin and the release of inflammatory cytokines was quantified using a multiplex Mesoscale Discovery platform. Confocal microscopy was shown to be a sensitive assay for evaluating material toxicity, as significant toxicity (p < 0.05) from ZDEC and BAK-treated surfaces compared to the untreated silicone control was detected. Patterns of cytokine release from cells varied depending on the toxin evaluated and the toxin concentration and did not directly correlate with the reduction in cell metabolic activity measured by alamarBlue.

In vitro ultraviolet-induced damage in human corneal, lens, and retinal pigment epithelial cells.

PURPOSE: The purpose was to develop suitable in vitro methods to detect ocular epithelial cell damage when exposed to UV radiation, in an effort to evaluate UV-absorbing ophthalmic biomaterials. METHODS: Human corneal epithelial cells (HCEC), lens epithelial cells (HLEC), and retinal pigment epithelial cells (ARPE-19) were cultured and Ultraviolet A/Ultraviolet B (UVA/UVB) blocking filters and UVB-only blocking filters were placed between the cells and a UV light source. Cells were irradiated with UV radiations at various energy levels with and without filter protections. Cell viability after exposure was determined using the metabolic dye alamarBlue and by evaluating for changes in the nuclei, mitochondria, membrane permeability, and cell membranes of the cells using the fluorescent dyes Hoechst 33342, rhodamine 123, calcein AM, ethidium homodimer-1, and annexin V. High-resolution images of the cells were taken with a Zeiss 510 confocal laser scanning microscope. RESULTS: The alamarBlue assay results of UV-exposed cells without filters showed energy level-dependent decreases in cellular viability. However, UV treated cells with 400 nm LP filter protection showed the equivalent viability to untreated control cells at all energy levels. Also, UV irradiated cells with 320 nm LP filter showed lower cell viability than the unexposed control cells, yet higher viability than UV-exposed cells without filters in an energy level-dependent manner. The confocal microscopy results also showed that UV radiation can cause significant dose-dependent degradations of nuclei and mitochondria in ocular cells. The annexin V staining also showed an increased number of apoptotic cells after UV irradiation. CONCLUSIONS: The findings suggest that UV-induced HCEC, HLEC, and ARPE-19 cell damage can be evaluated by bioassays that measure changes in the cell nuclei, mitochondria, cell membranes, and cell metabolism, and these assay methods provide a valuable in vitro model for evaluating the effectiveness of UV-absorbing ophthalmic biomaterials, including contact lenses and intraocular lenses.

In vitro analysis of the interaction of tear film inflammatory markers with contemporary contact lens materials.

PURPOSE: Several clinical studies have suggested that reusable silicone hydrogel contact lens materials exhibit a two-times increased rate of corneal infiltrative events compared to reusable hydrogels. One potential factor contributing to this complication relates to the differential uptake of tear film-based pro-inflammatory cytokines. The purpose of this study was to use an in vitro assay to investigate whether four pro-inflammatory cytokines differed in their uptake onto six contemporary contact lens materials. METHODS: Conventional hydrogel (etafilcon A, omafilcon A) and silicone hydrogel (balafilcon A, comfilcon A, senofilcon A, somofilcon A) contact lens materials were soaked in solutions containing pro-inflammatory cytokines IL-1ß, IL-6, IL-8 and TNF-α. Samples of the soaking solutions were collected over various time points and analyzed using the Meso Scale Discovery system, which served as a measurement of cytokine uptake onto the contact lens materials. RESULTS: Both conventional hydrogels (etafilcon A, omafilcon A) and two of the four silicone hydrogels tested (balafilcon A, comfilcon A), exhibited some uptake of IL-1ß, IL-8 or TNF-α (p < 0.05). Senofilcon A and somofilcon A did not exhibit uptake of any of these cytokines (p > 0.05). There was no uptake of IL-6 onto any of the contact lens materials investigated (p > 0.05). CONCLUSION: The contact lens materials tested did not exhibit any uptake of IL-6 and furthermore, did not exhibit more than 10 ± 3 % to 25 ± 12 % uptake of IL-1ß, IL-8 or TNF-α. Numerous factors could contribute to the reported increase in corneal infiltrative events with reusable silicone hydrogel materials, however, based on these results, it appears that uptake of these four cytokines are unlikely to contribute to this finding.

The Effect of Antimicrobial Peptides on the Viability of Human Corneal Epithelial Cells.

Antimicrobial peptides are polypeptides composed of less than 100 amino acids and are a class of antibiotics with strong activity against some infectious bacteria. This study examined the safety of four chosen antimicrobial peptides using primary human corneal epithelial cells (HCEC) and explored their potential therapeutic use. The efficacy of the peptides was also studied by evaluating the minimum inhibitory concentrations (MIC) against Gram-negative and Gram-positive bacteria. One of the peptides (polymyxin E) was found to have antibacterial efficacy against a common Gram-negative bacterium (MIC 1.56 µg/mL for Pseudomonas aeruginosa), and another one (nisin) was found to have antibacterial efficacy against a common Gram-positive bacterium (MIC 125 µg/mL for Staphylococcus aureus). Metabolic activity and live/dead/apoptotic effects were measured with fluorescent dyes after HCEC were exposed to the peptides for 30 min. Three of the peptides exhibited lower toxicity against HCEC than a currently marketed eye drop product. Regarding both efficacy and safety, two of the peptides (polymyxin E and nisin) were found to have potential use for treating ocular infections.

The efficacy of povidone-iodine, hydrogen peroxide and a chemical multipurpose contact lens care system against Pseudomonas aeruginosa on various lens case surfaces.

PURPOSE: To determine the antimicrobial efficacy of a povidone-iodine system (PVP-I; cleadew, OPHTECS Corporation, Kobe, Japan), a peroxide system (AOSEPT Plus with HydraGlyde, Alcon, Fort Worth, TX), and a chemical multipurpose system (renu fresh, Bausch & Lomb, Rochester, NY) on contact lens case surfaces that are both in contact and not in contact with the solutions during lens disinfection. METHODS: The surfaces of the inner walls, underside of the lid, and lens holder (if applicable) of the cases were inoculated with P. aeruginosa ATCC 27853. The cases were disinfected with the solutions as per their manufacturer instructions. After disinfection, the inoculated surfaces were swabbed and the amount of surviving P. aeruginosa was determined. Following this experiment, separate cases were inoculated and disinfected as before. This time the cases were agitated after recommended disinfection time and the amount of P. aeruginosa in the disinfecting solution was quantified immediately, and again after resting for 7 days. Experiments were conducted in triplicate (n = 3). RESULTS: Units are expressed in log CFU. All three solutions significantly reduced P. aeruginosa on direct-contact surfaces (all p < 0.039). On non-contact surfaces, the reduction of P. aeruginosa in the PVP-I system (pre-disinfection: 6.8 ± 0.5, post-disinfection: 1.0 ± 0.0; p < 0.001) was significant, but not for the hydrogen peroxide system (pre-disinfection: 6.3 ± 0.6, post: 5.5 ± 0.5; p = 0.194) and the chemical multipurpose system (pre-disinfection: 6.6 ± 0.1, post-disinfection: 5.6 ± 0.8; p = 0.336). After 7 days post-disinfection, no P. aeruginosa regrowth was observed in the PVP-I system (Day 1: 1.0 ± 0.0, Day 7: 1.0 ± 0.0; p = 1) and the chemical multipurpose system (Day 1: 4.2 ± 0.2, Day 7: 1.8 ± 0.9; p = 0.012), however regrowth was observed in the hydrogen peroxide system (Day 1: 3.4 ± 0.6, Day 7: 6.1 ± 0.4; p = 0.003). CONCLUSION: The PVP-I system was more effective against P. aeruginosa on non-contact surfaces than the hydrogen peroxide system or the chemical multipurpose system and is capable of inhibiting regrowth of P. aeruginosa for at least 7 days post-disinfection.

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells.

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H2O2), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-α) assay, and cells were evaluated for viability using fluorescent dyes. The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H2O2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation.

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 °C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

Cytotoxic and inflammatory effects of contact lens solutions on human corneal epithelial cells in vitro.

PURPOSE: To ascertain the effect that four contact lens (CL) multipurpose solutions (MPS) have on the viability and release of pro-inflammatory cytokines from human corneal epithelial cells (HCEC). METHODS: HCEC were exposed to four different MPS at various concentrations for 18 hours. The cells were also exposed to phosphate buffer, borate buffer, and PHMB. The cell viability was evaluated using the alamarBlue assay. The release of pro-inflammatory cytokines was measured using a Multiplex electrochemiluminescent assay. RESULTS: MPS-A, MPS-B and MPS-C all reduced cell metabolic activity p < 0.05 from control with MPS-A showing the greatest cytotoxic effect (maximum reduction, 90.6%). In contrast, MPS-D showed no significant reductions in cytotoxicity except at the highest concentration tested (19% reduction at 20% MPS concentration). Of the four cytokines evaluated MPS-C showed a substantial increase in the release of IL-1ß, IL-6, IL-8, and TNF-α at higher concentrations when compared to control p < 0.05. At the 20% concentration of MPS-A and MPS-B the release of IL-1 ß increased p < 0.05 but the release of IL-6, IL-8, and TNF-α decreased. MPS-D did not cause a change in the release of cytokines IL-1ß, IL-6, IL-8 and TNF-α p > 0.05. Exposing the cells to borate buffer and PHMB caused an increase in the release of TNF-α p < 0.05. CONCLUSIONS: This investigation demonstrates that at different concentration levels, several of the MPS tested showed a decrease in viability and an increase in the release of inflammatory cytokines from HCEC. The borate buffer component as well as PHMB appears to contribute to this pro-inflammatory reaction.

The Effect of Denatured Lysozyme on Human Corneal Epithelial Cells.

Purpose: During contact lens wear, the amount of lysozyme deposited on contact lenses varies depending on the lens material. The binding of lysozyme to some contact lens materials may result in a conformational change that denatures the protein to an inactive form. This investigation evaluated the effect that denatured lysozyme has on human corneal epithelial cells (HCECs) by measuring cell viability and the release of inflammatory cytokines. Methods: HCECs were exposed to lysozyme that was denatured to various activity levels. After 24-hour exposure to the lysozyme (1.9 mg/mL) in growth media, the cells were evaluated for cell viability using confocal microscopy. The metabolic activity of the cells was determined using an alamarBlue assay. Cell supernatants were analyzed for inflammatory cytokines. Results: Using confocal microscopy, there was no detectable change in the viability of the HCECs after exposure to the denatured lysozyme. However, using alamarBlue, a decrease in the metabolic activity of the HCECs exposed to denatured lysozyme was detected. HCECs exposed to lysozyme that was 67%, 47%, and 22% active showed a reduction in metabolic activity when compared with native (100% active) lysozyme and the media controls (P < 0.05). Exposure to the denatured lysozyme also caused an increase in the release of inflammatory cytokines (P < 0.05) from the HCECs. Conclusions: The results of this study show that denatured lysozyme can have a detrimental effect on HCECs. Both a reduction in metabolic activity and an increase in the release of inflammatory cytokines occurred after HCEC exposure to denatured lysozyme.

Assessment of biofilm formation of E. meningoseptica, D. acidovorans, and S. maltophilia in lens cases and their growth on recovery media.

PURPOSE: Bacterial biofilm formation in contact lens cases is a risk factor in the development of both microbial and infiltrative keratitis. This investigation evaluated three emerging pathogens: Stenotrophomonas maltophilia, Elizabethkingia meningoseptica, and Delftia acidovorans for biofilm formation and metabolic activity in lens cases. Also, growth of these bacteria on different media was assessed to optimize recovery conditions. METHODS: The three bacteria were incubated in lens cases with different concentrations of tryptic soy broth. Biofilm formation was evaluated by measuring metabolic activity using MTT and enumerating the number of viable bacteria. To determine the optimal recovery media, dilutions of these microorganisms were plated on six different media. The number of colony forming units (CFU) was recorded after 48, 72, and 96 h of incubation at 32°C and 37°C for S. maltophilia, and at 37°C for E. meningoseptica and D. acidovorans. RESULTS: All three microorganisms established biofilms in the lens cases, with significant numbers of CFU recovered. Biofilms of S. maltophilia and E. meningoseptica were metabolically active. Significant reduction in metabolic activity and number of viable S. maltophilia occurred when the incubation temperature was raised from 32°C to 37°C (p<0.05). The metabolic activity of the biofilms increased with greater organic load present. The highest percent recovery for all three organisms was given by Columbia blood agar, followed by chocolate. CONCLUSION: Based on the results, the presence of the three emerging pathogens present in lens cases and from corneal isolates can be accurately determined if proper growth media and incubation temperatures are utilized.

Use of the viability reagent PrestoBlue in comparison with alamarBlue and MTT to assess the viability of human corneal epithelial cells.

INTRODUCTION: PrestoBlue is a new resazurin based reagent to assess cell viability and cytotoxicity. It is claimed to be a fast and highly sensitive assay. Here, we compared PrestoBlue, alamarBlue, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazonium bromide (MTT) in assessing cell viability of human corneal epithelial cells (HCEC), and investigated the effect of plate color, reading mode, and plate storage on the performance of PrestoBlue assay. METHODS: The viability of different numbers of healthy HCEC and the toxicity of various chemicals on HCEC were evaluated using PrestoBlue (fluorescence), alamarBlue (fluorescence), and MTT (absorbance). The sensitivities of the three assays were compared. In the PrestoBlue assay, three plate colors and two reading modes were used and compared in assessing the toxic effect of sodium dodecyl sulfate (SDS). The PrestoBlue solutions after reaction were stored and measured on day 1, 2, 3, 5, and 7. The fluorescence readings obtained on different days were then compared. RESULTS: Both PrestoBlue and alamarBlue were able to detect 5000 healthy cells after 30min incubation and 1000 cells after 1h, 2h, and 4h incubation; while MTT was able to detect 5000 cells after 3h incubation. In the assessment of the toxicity of various chemicals, PrestoBlue and alamarBlue performed similarly. There was no significant difference between the results obtained by these two reagents. All the three plate colors and two reading modes showed similar results in the PrestoBlue assay in assessing the toxicity of SDS. Plate storage up to 7days did not affect the result of the PrestoBlue assay. CONCLUSION: Our study suggests that in evaluating the viability of HCEC, PrestoBlue is more sensitive than MTT, but similar to alamarBlue. The plate color, reading mode and plate storage up to 7days did not affect the performance of the PrestoBlue assay.

Comparison of the effects of ophthalmic solutions on human corneal epithelial cells using fluorescent dyes.

PURPOSE: To investigate the effect of differently preserved ophthalmic solutions on the viability and barrier function of human corneal epithelial cells (HCEC) using fluorescent dyes. METHODS: HCEC monolayers were exposed to the ophthalmic solutions containing benzalkonium chloride (BAK), edetate disodium, polyquad, stabilized oxychloro complex (Purite), sodium perborate, or sorbic acid for 5 min, 15 min, and 1 h. At 24 h after exposure, the cultures were assessed for metabolic activity using alamarBlue. The enzyme activity, membrane integrity, and apoptosis were evaluated using confocal microscopy. Barrier function was assessed using sodium fluorescein. RESULTS: The metabolic assay showed that the BAK-preserved ophthalmic solutions significantly reduced cell viability after a 5-min exposure compared to the phosphate buffered saline treated control (P<0.05). Using confocal microscopy, the micrographs showed that BAK caused a reduction in the enzyme activity, increased membrane permeability, and decreased the number of viable cells. Ophthalmic solutions with new preservatives had varying time-dependent adverse effects on cell viability, and the preservative-free solution had the least effect on HCEC. Sodium fluorescein permeability showed that HCEC monolayers treated with BAK-preserved solutions were more permeable to sodium fluorescein than those treated by the other ophthalmic solutions (P<0.05). CONCLUSIONS: BAK-preserved solutions had greater adverse effects on metabolic activity, enzyme activity, membrane integrity, cell viability, and barrier function than the solutions that were not preserved with BAK. Our study suggests that BAK-free especially, preservative-free ophthalmic solutions are safer alternatives to BAK-preserved ones.

Utilization of in vitro methods to determine the biocompatibility of intraocular lens materials.

In vitro methods for measuring the adhesion and viability of lens epithelial cells on implant devices are needed to assess material biocompatibility. We investigated whether the use of confocal microscopy and spectrophotometric methods could determine the viability and adhesion of cells on a silicone biomaterial. Human lens epithelial cells adhered to silicone were treated with 0.01% benzalkonium chloride (cationic surfactant), 0.1% sodium dodecyl sulfate (anionic surfactant), and 10% Tween 20 (nonionic surfactant). Cell viability was then assessed using two fluorescent dyes (calcein and ethidium homodimer-1). Adhesion was determined directly by measuring the number of attached cells after surfactant treatment and by an indirect method that utilized the colorimetric agent crystal violet. The number of viable cells remaining on the biomaterial was determined both immediately after exposure and after the cells were allowed to grow for 1 day following surfactant exposure. The measurements for adhesion showed that the anionic surfactant weakened cell surface binding more than the cationic or nonionic surfactant. This study demonstrated that confocal microscopy in conjunction with crystal violet as an indirect colorimetric indicator can quantify the viability and adhesion of human lens epithelial cells attached to a material surface.

Rabbit models of contact lens--associated corneal hypoxia: a review of the literature.

PURPOSE: To provide a review of the literature on rabbit models of contact lens-associated corneal hypoxia. METHODS: The literature was searched for articles describing corneal hypoxia in the rabbit coincident with contact lens wear. The articles were identified by key word searches for rabbit models, contact lens, hypoxia, and cornea. RESULTS: Designing a rabbit model of contact lens-associated corneal hypoxia requires identification of the key determinants of the physiologic response. The retention of soft contact lenses, particularly high-water content lenses, is problematic in the rabbit eye, in which blink frequency is on the order of minutes. An intact nictitating membrane may be beneficial in the hydration and, therefore, retention of soft contact lenses. Although tarsorrhaphy improves lens retention, lid closure also enhances the hypoxic response and may obscure the benefits of the greater oxygen transmissibility of soft contact lenses. The duration of lens wear in most reviewed studies was 24 hours or less, with only a few studies evaluating the hypoxic effect of lens wear of 1 week or more. Methods commonly used to characterize corneal hypoxia in rabbits include pachymetry, tear film lactate dehydrogenase measurements, clinical observations of limbal hyperemia, and more invasive methods of assessing cell proliferation and apoptosis. In summary, the ideal rabbit model of contact lens-associated corneal hypoxia depends on the question to be answered. CONCLUSIONS: This review tabulates the known studies, highlights key considerations in study design, and describes useful methods in characterization of the hypoxic response.