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.

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.

Conserved characteristics of ocular refractive development - Did the eye evolve once?

It has been speculated that the unitary eyes of vertebrates and molluscs, and the compound eyes of insects and crustaceans, evolved separately. On the other hand, the common use of rhodopsin as a photoreceptor molecule, and the conservation of Pax6 as a master control gene for eye development, suggest instead that the eye evolved once. Yet, recently the molecular genetics that had seemed to suggest a definitive answer to this evolutionary point has once again become cloudy. Here we propose an alternative approach to addressing the question of eye evolution through comparative analyses of physiological optics. Serendipitous discoveries involving form deprivation and defocusing with young monkeys and chicks demonstrated the conserved importance of visual experience on eye development. Similar results have been demonstrated in teleosts, although differences exist in eye anatomy, physiology and optics. In particular, since fish grow throughout life, these effects can also be demonstrated in adults. In comparison, the cephalopod eye is an often-cited example of convergent evolution with the vertebrate eye, although considerable developmental differences exist. Nevertheless, squid eyes from animals raised under alternative lighting exhibit anatomical and refractive changes that agree with those found in vertebrates. Together, these observations provide functional and structural support for the view that the eye evolved once. Because of their very compressed lifespans (only one to two years) cephalopods may be ideal animal models for the study of ocular refractive development.

Refractive plasticity of the developing chick eye: a summary and update.

PURPOSE: To summarize the OPO 1992 Classic Paper: Refractive plasticity of the developing chick eye (12: 448-452) and discuss recent findings in refractive development. SUMMARY AND RECENT FINDINGS: The classic paper shows that when lightweight plastic goggles with rigid contact lens inserts are applied to the eyes of newly hatched chicks, the eye responds accurately to defocus between -10 and +20 D, although hyperopia develops more rapidly. While the changes largely are due to change in axial length, high levels of hyperopia are associated with corneal flattening. Also, newly hatched chicks are better able to compensate for the induced defocus than chicks that are 9 days old. In addition, astigmatism of 2-6 D can be produced by applying 9 D toric inducing lenses on the day of hatching, and the most myopic meridian coincides with the power meridian of the inducing lens. This astigmatism appears to be primarily due to corneal toricity. Furthermore, the greatest magnitude was produced when the plano meridian of the inducing lens was placed 45° from the line of the palpebral fissure. Since our publication in 1992, it has been shown that similar results can be produced in a variety of species, including; tree shrews, marmosets, monkeys and fish. Considerable effort has been spent in trying to determine what the eye uses, if not the brain, as the signal to the sign of the defocus. Accommodation, chromatic aberration, diurnal variation, astigmatism and higher order monochromatic aberrations have all been considered. Choroidal thinning and thickening play a role in myopia and hyperopia development, respectively, in chicks. High light levels (15,000 lux) increase the rate at which chicks compensate for positive lenses and decrease the compensation rate for negative lenses. However these light levels do not prevent the eye from fully compensating for either type of lens. It has also been shown that brief periods of normal vision prevent the development of form deprivation myopia. Finally, the importance of the peripheral retina in refractive development has been explored and lenses designed to reduce relative peripheral hyperopia have resulted in variable effects as far as myopia control is concerned. CONCLUSIONS: A growing body of evidence, from both animal models and human clinical trials indicates that the development of myopia is related both to genetics and environment / lifestyle. Nevertheless, we are far from understanding how this interaction takes place.

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.

Blood flow dynamics in the snake spectacle.

The eyes of snakes are shielded beneath a layer of transparent integument referred to as the 'reptilian spectacle'. Well adapted to vision by virtue of its optical transparency, it nevertheless retains one characteristic of the integument that would otherwise prove detrimental to vision: its vascularity. Given the potential consequence of spectacle blood vessels on visual clarity, one might expect adaptations to have evolved that mitigate their negative impact. Earlier research demonstrated an adaptation to their spatial layout in only one species to reduce the vessels' density in the region serving the foveal and binocular visual fields. Here, we present a study of spectacle blood flow dynamics and provide evidence of a mechanism to mitigate the spectacle blood vessels' deleterious effect on vision by regulation of blood flow through them. It was found that when snakes are at rest and undisturbed, spectacle vessels undergo cycles of dilation and constriction, such that the majority of the time the vessels are fully constricted, effectively removing them from the visual field. When snakes are presented with a visual threat, spectacle vessels constrict and remain constricted for longer periods than occur during the resting cycles, thus guaranteeing the best possible visual capabilities in times of need. Finally, during the snakes' renewal phase when they are generating a new stratum corneum, the resting cycle is abolished, spectacle vessels remain dilated and blood flow remains strong and continuous. The significance of these findings in terms of the visual capabilities and physiology of snakes is discussed.

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.

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.

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.

Identification of myopia-related marker proteins in tilapia retinal, RPE, and choroidal tissue following induced form deprivation.

PURPOSE: Experimentally induced myopia is characterized by axial elongation of the eye. The molecular pathways leading to this condition are largely unknown, even though many candidate proteins have been proposed to be involved in this process. This study has identified proteins that were differentially expressed in myopic and control combined retina, retinal pigment epithelium (RPE), and choroidal tissue in tilapia (Oreochromis niloticus). METHODS: Form deprivation was used to induce myopia in tilapia (n = 3). In this initial study on tilapia retina, RPE and choroid, 2-D differential in gel electrophoresis (DIGE) and mass spectrometry were used to identify differentially expressed proteins. Homology-based gene cloning was used to obtain full sequence data for one of the identified proteins. RESULTS: A total of 18 protein spots separated by 2-D electrophoresis exhibited statistically significant differences in expression between the myopic and contralateral control combined retinal, RPE, and choroidal tissue. Three proteins were identified at a significance level of p < 0.05, as annexin A5 (down-regulated 47%), Gelsolin (down-regulated 27%), and TCP-1 (CCT) (down-regulated 54%). DNA sequencing of tilapia annexin A5 shows an amino acid sequence identity of 84.5% with the homologous Japanese ricefish annexin max2. CONCLUSIONS: A proteomics approach has been used to identify differentially expressed proteins in form-deprived combined retinal, RPE, and choroidal tissue from myopic versus normal eyes. The identified proteins may be components of pathways involved in myopia pathogenesis.

Effects of 400 nm, 420 nm, and 435.8 nm radiations on cultured human retinal pigment epithelial cells.

The present study demonstrates narrowband short-wavelengths radiation- (400, 420, and 435.8 nm) induced cellular damage of cultured human retinal pigment epithelial cells using in vitro biological assays to determine wavelengths that are responsible for photochemical lesions of the retina. This work involved the exposure of retinal pigment epithelial (RPE) cells (ARPE-19) to narrowband light of three different wavelengths (400, 420, and 435.8 nm) using a xenon arc lamp and interference filters. Cellular viability, mitochondrial distribution, and nucleic acid (both DNA and RNA) damage were quantified after various energy levels of exposure, using the Alamar blue assay, and confocal laser scanning microscopy with two fluorescent stains (Rhodamine 123 and Acridine Orange). The results clearly show that 400 nm light radiation can cause significant dose-dependent decreases in RPE cell viability as well as degradations of DNA/RNA and mitochondria in RPE cells, while 420 and 435.8 nm light radiation cause no cellular damage. While further evaluations may be needed to assess specificity and confounding factors of these assessment tools, the results may be a matter for consideration in future IOL design efforts.

Contributions of mouse genetic background and age on anterior lens capsule thickness.

Accurate lens capsule thickness measurements are necessary for studies investigating mechanical characteristics of the capsule. Confocal Z-axis imaging was used to measure the anterior lens capsule thickness of living intact lenses with minimal tissue manipulation. Measurements of the anterior capsule thickness is reported for the first time in young and old mice from four inbred strains, BALB/c, FVB/N, C57BL/6, and 129X1, and the outbred strain ICR. Our data demonstrates that the mouse anterior lens capsule continues to grow postnatally similar to that described in other mammals. It is also shown there is a significant difference in anterior lens capsule thickness between unrelated mouse strains, suggesting that capsule thickness is a quantitative trait shared by strains with common ancestry. Measurements, taken from other regions of FVB/N capsules revealed the anterior pole to be the thickest, followed by the equatorial region and posterior pole. In addition to mouse, anterior capsule measurements taken from intact cattle, rabbit, rat lenses, and human capsulotomy specimens correlated with the overall size of the animal.

Regional variation in the refractive-index of the bovine and human cornea.

PURPOSE: Given the refractive importance of the human cornea, surprisingly little attention has been directed to the study of local variation in corneal refractive-index. This in vitro and in vivo study measures the refractive-index of different portions of the bovine and human cornea. METHODS: Fifty fresh bovine corneas (obtained from an abattoir) and 10 human subjects were used for the study. The refractive-index of the central, nasal, and temporal corneal epithelium was measured with a bench-top Abbe refractometer in the case of bovine corneas and with a hand-held refractometer with humans. RESULTS: The mean (+/-standard deviation) refractive-indices of the central, nasal, and temporal bovine corneal epithelium were 1.3760 (+/-0.003), 1.3757 (+/-0.002), and 1.3746 (+/-0.002), respectively. Refractive-indices of the anterior and posterior bovine corneal stroma were 1.3731 (+/-0.002) and 1.3708 (+/-0.004), respectively. The mean (+/-standard deviation) refractive-index in the central, nasal, and temporal periphery of the human cornea epithelium were 1.3970 (+/-0.001), 1.3946 (+/-0.001), and 1.3940 (+/-0.001), respectively. CONCLUSION: There are small local differences in the refractive-index of the bovine and human corneal epithelium and the refractive-index of the epithelium is higher than that of the anterior and posterior stroma of the bovine cornea.

The role of the lens in refractive development of the eye: animal models of ametropia.

Research with young mammals and chicks has shown that the visual environment can affect the refractive development of the eye by enhancing or slowing axial eye growth, but the effect on the refractive components of the eye, the lens and cornea, are less clear. A review of the literature indicates that the lens is minimally affected, if at all, and results vary depending on whether the lens is studied in an isolated state or with the accommodative apparatus intact. Research has shown that the development of myopia or hyperopia in young chicks alters lens focal length and magnitude of the accommodative response. However, the result may be indirect or passive due to the effect of the change in size and shape of the globe on the articulation between the ciliary body and lens. Recent research has also investigated the role of the lens in induced refractive error development in a fish, tilapia (Oreochromis niloticus). Translucent goggles were sutured over one eye for 4 weeks to induce form deprivation myopia while the untreated eye served as an untreated contralateral control. In addition to measuring refractive state and intraocular dimensions, a scanning laser system was used to determine the optical quality of excised lenses. All the deprived fish eyes developed significant amounts of myopia and the vitreous and anterior chambers of the treated eye were significantly longer axially than those of the untreated contralateral eyes. No significant change in optical quality was found between lenses of the myopic and non-myopic eyes and the fish recovered completely from the myopia five days after the goggle was removed. The results show that although fish, unlike higher vertebrates, are capable of lifelong growth, the visual environment is an important factor controlling ocular development in this group as well, and eye development is not strictly genetically determined. This review indicates that lens growth and optical development is independent from the refractive development of the whole eye.

Effect of age on ocular irritancy as measured with in vitro bovine lenses.

PURPOSE: This project studied the effect of age on optical quality of cultured bovine lenses exposed to a number of common surfactants and alcohol. METHODS: Lenses from calves (8-18 months) and cows (2-3 years) were isolated aseptically and studied optically for 96 h after treatment with various commercial surfactants and an alcohol. Potential eye irritancy was evaluated using a scanning laser in vitro assay system which records the change in focal characteristics (back vertex distance variability or BVDV) of the bovine lenses. Lenses were divided into a total of 14 groups. Both calf and cow lenses (a total of 257 lenses were used) were arranged into control, 0.01% BAK, 1% SDS, 1.0% Triton X-100, 100% ethanol, 10% Tween-20 and 1.0% Tween-20 treatment groups. RESULTS: The cationic surfactant BAK caused the most amount of optical change to the bovine lenses, followed by SDS, Triton X-100, ethanol and then Tween-20. There was also a significant difference in BVDV between the cow and calf groups for all the treated groups, except for Tween-20, with the calf lenses showing greater optical damage. In the case of 10% Tween-20, both cow and calf lenses show equal optical damage while at 1.0% both groups show no effect and are no different from the untreated control lenses. CONCLUSION: Younger bovine lenses are more sensitive to the surfactants and alcohol tested when compared to their older counterparts, indicating that younger eyes may be more sensitive to these chemicals. The results further indicate that age is a factor that should be taken into account in assessing ocular risk.

The role of Hsp70 and Hsp90 in TGF-beta-induced epithelial-to-mesenchymal transition in rat lens epithelial explants.

PURPOSE: This study investigates the effects of heat shock treatment and the role of Hsp70 and Hsp90 on tranforming growth factor beta 2 (TGF-beta2)-induced epithelial-to-mesenchymal transition (EMT) in rat lens epithelial explants. METHODS: Rat lens epithelial explants from 7 to 10 day-old Wistar rats were dissected and incubated for 24 h before treatment. The explants were divided into eight treatment groups: control (culture medium), fibroblast growth factor-2 (FGF-2), TGF-beta2, and TGF-beta2+FGF-2 under normal culture conditions and heat shocked conditions. The explants were heat shocked at 45 degrees C before treatment with the respective media. H&E staining was performed on whole-mount epithelial explants from each group. Immunofluorescence staining for alpha-smooth muscle actin (alpha-SMA), F-actin, and E-cadherin was also used to determine EMT and fibrotic plaque formation in the lens epithelial explants. Apoptotic cell death was determined using the TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) assay. Confocal microscopy was used to visualize immunoreactivity in the whole-mount epithelial explants. Western blot analysis of alpha-SMA, E-cadherin, Hsp70, and Hsp90 were also performed. RESULTS: TGF-beta2-induced EMT and plaque formation in the lens epithelial explants. The simultaneous treatment of epithelial explants with TGF-beta2+FGF-2-induced the most significant morphological changes and EMT. Heat shock treatment of lens epithelial explants before TGF-beta2 treatment did not inhibit plaque formation, but there was significant reduction of alpha-SMA expression and greater E-cadherin expression when compared to the non-heat shocked TGF-beta2-treated explants. Interestingly, TGF-beta-induced apoptotic cell death was significantly lower in the heat shocked explants compared to the non-heat shock lens explants. Heat-induced accumulation of Hsp70 and Hsp90 expression was reduced in the heat shocked groups at day 4 of treatment. CONCLUSIONS: TGF-beta2-induced EMT was significantly reduced in the heat shocked TGF-beta2 lens epithelial explants. After four days of culture, there is a reduction in expression of Hsp70 and Hsp90 in the heat-shocked groups, indicating that the lens epithelial cells are under a less stressful condition than the non-heat shocked groups. In conclusion, molecular chaperones can play a protective role against TGF-beta2-induced EMT and enhance cell survival.

Biocompatibility of contact lens solutions using confocal laser scanning microscopy and the in vitro bovine cornea.

PURPOSE: This study used high-resolution research confocal laser scanning microscopy to study the epithelium of fresh bovine corneas treated with two contact lens solutions. METHODS: Fresh bovine corneal buttons were stained with a combination of the cell-permeable mitochondrial- and nuclear-specific dyes rhodamine 123 and Hoechst 33342 or acridine orange and imaged with a confocal laser scanning microscope. High-resolution X,Y images of the corneal epithelium were taken with a Zeiss 510 confocal laser scanning microscope at a 0.44-062 -microm Z step, and three-dimensional images of the entire thickness of the corneal epithelium were reconstructed. Semiautomated quantitative MatLab software analysis was applied to count the mitochondria in each optical slice. The effects of treatment for 30 minutes with OPTI-FREE Express and ReNu MultiPlus No Rub were investigated and compared to Hanks' balanced salt solution controls. RESULTS: This study showed the feasibility of using the confocal laser scanning microscope with fluorescent dyes to study organelles throughout the epithelial layers of fresh unfixed bovine corneas. The mitochondria in the intermediate and basal epithelium are abundant and range in length from less than 1 microm to threads greater than 15 microm, with a tendency to accumulate around the nuclei. Image analysis indicated significantly fewer mitochondria only in the superficial epithelium after treatment with ReNu MultiPlus No Rub treatment. OPTI-FREE Express treatment produced significantly fewer mitochondria not only in the superficial but also in the intermediate epithelial layers. CONCLUSIONS: OPTI-FREE Express has the potential for deeper mitochondrial effects compared to ReNu MultiPlus No Rub and Hanks' balanced salt solution controls.

Eyes of a lower vertebrate are susceptible to the visual environment.

PURPOSE: Recently, it has been found that form deprivation myopia can be induced in fish (tilapia). This study examined the sensitivity of the tilapia eye to positive and negative lenses. It further investigated the sensitivity of the fish eye to form deprivation by examining the effect of fish weight. METHODS: Twenty-five Nile tilapia (Oreochromis niloticus; group 1) were weighed (range, 26-101 g) and killed, and their eyes were measured to provide normative data regarding fish eye size, body weight, and refractive state. Goggles with lenses of refractive powers in water of either +15 D (group 2, n = 7) or -12 D (group 3, n = 7) were sutured over the right eye of for 2 weeks to induce hyperopia or myopia. The untreated contralateral eye served as a control. An additional six fish (group 4), each wearing a goggle with an open central area, were used to evaluate the effect of the goggle itself. Refractive measurements for these 20 fish were made before and after treatment, after which the fish were killed, the eyes were removed, and axial lengths were measured from frozen sections. Another 21 fish were treated with goggles with lenses for 2 weeks, after which the goggle was removed and the refractive states of both eyes were measured every day for 6 days (day 19) and then after 28 days. These fish were placed in one group (group 5) wearing negative (-12 D) lenses (n = 8; average weight, 25.5 g) and two groups (groups 6, 7) of different size (average weights, 13.9 g [n = 5] and 26.9 g [n = 8], respectively) wearing positive (+15 D) lenses during the treatment period. In addition, translucent goggles were applied for 2 weeks to induce form deprivation myopia in three groups of fish (groups 8, 9, 10) of different weights, averaging 16.0 g (n = 7), 57.4 g, (n = 8), and 98.4 g, (n = 7), to provide an evaluation of the effect of weight on the development of form deprivation myopia. RESULTS: In untreated fish (group 1), the axial length of the eye, ranging from 5.86 mm to 7.16 mm, was proportional to weight (26.5-101 g), whereas refractive state shifted from hyperopia (+15D for 10-g fish) toward emmetropia. The +15D lens-treated fish (group 2) became hyperopic relative to the contralateral eye (+7.7 +/- 1.6 D; mean +/- SD), whereas the -12 D lenses (group 3) induced myopia relative to the control eye (-8.4 +/- 0.8 D) within 2 weeks. Hyperopic eyes were shorter (4.16 +/- 0.11 mm vs. 4.28 +/- 0.06 mm) and myopic were eyes longer (3.96 +/- 0.36 mm vs. 3.84 +/- 0.27 mm) than their contralateral control eyes. There were no significant differences in eye size or refractive state between treated and untreated eyes of fish wearing open goggles. In the groups that were allowed to recover (groups 5, 6, 7), the fish treated with minus lenses developed an average of -9.8 +/- 1.9 D myopia, whereas +15 D lenses induced average hyperopia amounts of +8.1 +/- 1.4 D (group 6) and +6.2 5 +/- 2.87 D (group 7). All these fish recovered completely within 2 weeks once the goggles with lenses were removed. Pretreatment and posttreatment refractive results indicated that the contralateral control eyes were affected by the positive and negative lens treatments, though to a lesser extent. Form deprivation myopia was induced in all three different weight groups, averaging -11.9 +/- 2.9 D for group 8, 6.3 +/- 2.5 D for group 9, and -2.3 +/- 1.0 D for group 10. All form-deprived eyes and those treated with positive and negative lenses recovered-i.e., little or no difference resulted in refractive state or dimensions between the treated and untreated eyes-to pretreatment levels within 1 week of goggle removal. CONCLUSIONS: Tilapia, a lower vertebrate species, exhibits positive and negative lens-induced refractive change, as is the case for higher vertebrates. In addition, the level of sensitivity to form deprivation is weight dependent.

In vitro assays for evaluating the ultraviolet B-induced damage in cultured human retinal pigment epithelial cells.

The present study demonstrates broadband UV-B-induced damage of cultured human retinal pigment epithelial cells as an effort to develop an in vitro model that can be used, along with in vivo research and other in vitro efforts, to evaluate the need for retinal UV protection in humans after cataract removal. The human retinal pigment epithelial cell line, ARPE-19, was cultured in two groups: control and treated. Treated cells were irradiated with three broadband UVB radiations at energy levels of 0.05, 0.1 and 0.2J/cm(2). After irradiation, cells were incubated for 48h while cellular viability, morphology, and phagocytotic activity were analyzed using the Alamar blue assay, confocal microscopy, and fluorescent microspheres. Confocal analysis concentrated on the study of the cell nuclei and mitochondria. The Alamar blue assay of UV-B-exposed cells showed dose and time-dependent decreases in cellular viability in comparison to control cells. Loss of cell viability was measured at the two higher energy levels (0.2 and 0.1J/cm(2)), but the cell group exposed to 0.05J/cm(2) showed no significant viability change at 1-h time point. Morphological evaluation also showed dose and time-dependent degradation of mitochondria and nucleic acids. Cells exposed with 0.05J/cm(2) UVB did not show significant degradation of mitochondria and nucleic acids during the entire culture period. Phagocytotic activity assay data for UVB-exposed cells showed dose-dependent decreases in phagocytotic activity in comparison with the control cells. The control cells have significantly greater capacities for uptake than the 0.1 and 0.2J/cm(2) UV-B-exposed cells, while the 0.05J/cm(2) UV-B-exposed cell group showed no significant difference from the control cell group. The findings suggest that UVB radiation-induced cultured RPE cell damage can be evaluated by assays that probe cellular viability, morphological change, and phagocytotic activity, and that these assay methods together provide a valuable in vitro model for ultraviolet radiation-induced retinal toxicology research.