The Effect of Actinoquinol with Hyaluronic Acid in Eye Drops on the Optical Properties and Oxidative Damage of the Rabbit Cornea Irradiated with UVB Rays

Irradiation of the cornea with UVB rays leads to its oxidative damage, swelling and increased light absorption. We investigated changes in the corneal optics (evaluated by changes of corneal hydration and light absorption) and microscopical disturbances of corneas irradiated with UVB rays as influenced by eye drops containing actinoquinol with hyaluronic acid. Rabbit corneas were irradiated with a daily dose of 0.5 or 1.01 J cm⁻² of UVB rays (312 nm) for 4 days. During irradiation, the eye drops were applied on the right eye and buffered saline (or hyaluronic acid) on the left eye. On day 5 the rabbits were sacrificed and the corneas examined spectrophotometrically for light absorption. The corneal thickness (hydration) was measured using a pachymeter. Corneas of some other rabbits were examined immunohistochemically. After buffered saline treatment UVB rays evoked changes in the corneal optics and induced oxidative damage of the corneas. After actinoquinol-hyaluronic acid application, these changes were diminished. Hyaluronic acid alone was less effective. In conclusion, actinoquinol-hyaluronic acid eye drops decreased changes in corneal optics and suppressed oxidative damage in the UVB-irradiated cornea. However, the effective corneal protection by these eye drops was limited to the lower UVB dose.     

The Reversibility of UV‐B Induced Alterations in Optical Properties of the Rabbit Cornea Depends on Dose of UV Irradiation

Solar UVB radiation evokes photokeratitis, accompanied by increased corneal hydration and changes in corneal transparency, resulting in increased light absorption. Corneal optical properties are disturbed and visual acuity decreased. The aim of this study was to investigate the reversibility of these UVB‐induced changes. Rabbit corneas were irradiated with UVB doses of 0.5 J cm^?2 or 1.01 J cm^?2 during 4 days. Some rabbits were sacrificed after the last irradiation and some 2 months later. Corneas were investigated spectrophotometrically for light absorption, and corneal hydration was evaluated by central corneal thickness with an ultrasonic pachymeter. Corneal impression cytologies were examined immunohistochemically for proinflammatory cytokines and malondialdehyde. The increased corneal light absorption, hydration and the staining of immunohistochemical markers found in corneas after irradiation returned to normal values during 2 months in corneas irradiated with the lower UVB dose. In contrast, in corneas irradiated with the higher UVB dose, a moderate but statistically significant increase in corneal light absorption, hydration and positive immunohistochemical stainings remained as residual changes. This was in contrast to normal corneas, where the staining of proinflammatory cytokines as well as malondialdehyde was negative. In conclusion, the reversibility of UVB‐induced disturbances was dependent on UVB dose.     

Reduced Levels of Tissue Inhibitors of Metalloproteinases in UVB‐Irradiated Corneal Epithelium

Tissue inhibitors of metalloproteinases (TIMPs) are the major endogenous regulators of metalloproteinase activity in tissues. TIMPs are able to inhibit activity of all known matrix metalloproteinases (MMPs) and thus participate in controlling extracellular matrix synthesis and degradation. We showed previously elevated expressions of MMPs in the rabbit corneal epithelium upon UVB exposure and suggested that these enzymes might be involved in corneal destruction caused by excessive proteolysis. The aim of this study was to investigate TIMPs in the corneal epithelium after UV irradiation using immunohistochemical and biochemical methods. We found that as compared to control rabbit corneas where relatively high levels of TIMPs were present in the epithelium, repeated irradiation of the cornea with UVB rays (not with UVA rays of similar doses) significantly decreased TIMPs in corneal epithelial cells. The results of this study point to the suggestion that the decrease in TIMPs in the corneal epithelium after UVB irradiation contributes to increased proteolytic activity of MMPs in UVB‐irradiated corneal epithelium found previously.     

Effect of Two Different UVA Doses on the Rabbit Cornea and Lens

The aim of the present paper was to examine the irradiation effect of two doses of UVA rays (365 nm) on the rabbit cornea and lens. Corneas of anesthetized adult albino rabbits were irradiated with UVA rays for 5 days (daily dose 1.01 J cm⁻² in one group of rabbits and daily dose 2.02 J cm⁻² in the second group of animals). The third day after the last irradiation, the rabbits were killed, and their eyes were employed for spectrophotometrical, biochemical and immunohistochemical investigations. Normal eyes served as controls. Absorption spectra of the whole corneal centers were recorded over the UV–VIS (visible) spectral range. Levels of antioxidant and prooxidant enzymes, nitric oxide synthases and nitric oxide (indirectly measured as nitrate concentration) were investigated in the cornea. Malondialdehyde, a byproduct of lipid peroxidation, was examined in the cornea and lens. The results show that the staining for endothelial nitric oxide synthase was more pronounced in corneas irradiated with the higher UVA dose. Otherwise, UVA rays at either dose did not significantly change corneal light absorption properties and did not cause statistically significant metabolic changes in the cornea or lens. In conclusion, UVA rays at the employed doses did not evoke harmful effects in the cornea or lens.     

Ultraviolet Light‐Induced Cyclobutane Pyrimidine Dimers in Rabbit Eyes

Sunlight exposure of the eye leads to pathologies including photokeratitis, cortical cataracts, pterygium, actinic conjunctivitis and age‐related macular degeneration. It is well established that exposure to ultraviolet (UV) radiations leads to DNA damage, mainly cyclobutane pyrimidine dimers (CPDs). CPD formation is the principal factor involved in skin cancer. However, the exact mechanism by which sunlight induces ocular pathologies is not well understood. To shed light on this issue, we quantified the CPD formation onto DNA of rabbit ocular cells following UVB exposure. We found that CPDs were induced only in the structures of the ocular anterior chamber (cornea, iris and lens) and were more concentrated in the corneal epithelium. Residual UVB that pass through the cornea are completely absorbed by the anterior layers of the iris. CPDs were also detected in the central portion of the lens that is not protected by the iris (pupil). By determining the UV‐induced DNA damage formation in eyes, we showed that anterior ocular structures are a reliable physical barrier that protects the subjacent structures from the toxic effects of UV. Although the corneal epithelium is the structure where most of the CPDs were detected, no cancer is related to solar exposure.     

Antioxidant protection in cultured corneal cells and whole corneas submitted to UV-B exposure

Several corneal pathologies are characterized by the presence of reactive oxygen species (ROS); therefore, we evaluated the protection afforded by pirenoxine and melatonin to corneal cell culture and whole rabbit cornea from ultraviolet exposure and other oxidant systems. Rabbit cornea cell (SIRC) plates and whole corneas were exposed to UV-B (80 or 800 mJ/cm2) or incubated with fMLP-stimulated autologous macrophages, in the presence or absence of pirenoxine or melatonin (10(-5) M). The protective activity of compounds was assessed by measuring superoxide anion formation, inhibition of oxidation and mitochondrial viability. Moreover the ex vivo protective effect of pirenoxine and melatonin was verified in the whole cornea submitted to UV-B exposure in vitro. Our experimental data demonstrate that pirenoxine and melatonin were able to inhibit the superoxide formation and oxidative effect in cell culture and whole rabbit corneas submitted to UV-B exposure or to incubation with fMLP-stimulated autologous macrophages. Mitochondrial viability was restored in epithelial cells of rabbit cornea but not in SIRCs. Moreover, both compounds are also able to increase ex vivo epithelial corneal cell defences against the in vitro UV-B induced lipid peroxidation.     

Inhibition of corneal neovascularization by rapamycin

The purpose of this study was to determine whether rapamycin could inhibit corneal angiogenesis induced by basic fibroblast growth factor (bFGF). Using human dermal microvascular endothelial cells (HDMECs), we examined the effect of rapamycin on cell proliferation and migration, and the expression of vascular endothelial growth factor (VEGF). The rabbit's eye was implanted intrastromally into the superior cornea with pellet containing bFGF for the control group and pellet containing bFGF and rapamycin for the rapamycin group. Biomicrographically, corneal angiogenesis was evaluated for 10 days after pellet implantation. The neovascularized cornea also was examined histologically. bFGF induced corneal neovascularization was significantly reduced by treatment with rapamycin. Using in vitro model, rapamycin strongly inhibited bFGF induced proliferation, migration, and VEGF secretion of HDMECs. We could observe that the bFGF induced corneal angiogenesis was inhibited by rapamycin in a micropocket rabbit model. The score of neovascularization was significantly decreased in the rapamycin group than in the control group at 10 days after pellet implantation. Histologically, the cornea of rapamycin group also showed much less new vessels than that of control group. Collectively, rapamycin appears to inhibit bFGF induced angiogenesis in a rabbit corneal micropocket assay and may have therapeutic potential as an antiangiogenic agent.     

Focal length variability and protein leakage as tools for measuring photooxidative damage to the lens

Hypericin is the ingredient used to standardize the popular over-the-counter antidepressant medication St. John's Wort. Because hypericin readily produces singlet oxygen and other excited state intermediates, it is a very efficient phototoxic agent in the eye that can potentially induce the development of the cataract photooxidative mechanism. Hypericin absorbs in the UV and visible ranges, binds to the lens crystallins (alpha, beta and gamma) and damages these proteins through a photooxidative mechanism. Effects were measured previously using fluorescence, UV and mass spectrometry. We report here two additional methods to monitor lens damage: (1) measuring focal length variability using a ScanTox instrument and (2) measuring protein leakage from the damaged lens. Because nonenzymic glycation results in free radical production, we chose to use elevated glucose concentrations as a convenient model for studying oxidative stress. To compare and contrast photooxidative damage against oxidative damage to the lens, we also measured the focal length variability and protein leakage induced by the presence of elevated glucose concentrations. We found that the total accumulated protein leakage was positively correlated (r = 0.9) with variability in focal length. Lenses treated with hypericin and irradiated with UVB had an increase in focal length variability as compared with the lenses that were only UVB-irradiated. Lenses without UVB irradiation had much lower focal length variability than irradiated lenses. For non-hypericin-treated lenses, UVB-irradiated lenses had a larger variability (4.58 mm) than the unirradiated lenses (1.78 mm). The lenses incubated in elevated glucose concentrations had a focal length variability (3.23 mm) equivalent to that of the unirradiated hypericin-treated lenses (3.54 mm). We conclude that photooxidative damage by hypericin results in changes in the optical properties of the lens, protein leakage and finally cataract formation. In contrast to this, high concentrations of glucose induced protein leakage but not changes in optical properties or the opacity associated with a cataract. This work provides further evidence that people should protect their eyes from intense sunlight when taking St. John's Wort.     

The Role of Calcium in UVB-lnduced Damage in Irradiated Ocular Lenses

Abstract— The purpose of this study was to evaluate the role of altered calcium homeostasis in the development of irreversible membrane damage in the UVB-irradiated ocular lens. In particular, experiments were designed to determine whether restricting calcium influx could prevent membrane damage that typically leads to ion imbalances and lens opacification following short-term exposure to ultraviolet light (UVB). The influx of calcium was reduced by culturing lenses in a low-calcium culture medium containing 0.3 mM Ca²+ rather than physiological concentrations of 1.6 mM. This low-calcium protocol retarded calcium accumulation in UVB-irradiated lenses for 2 days of culture, and opacification was delayed by 24 h. Loss of transparency did occur during the second day of culture, but more slowly than in irradiated lenses cultured in normal-calcium medium. Membrane damage was assessed by evaluating loss in cation transport activity, assessed by measuring ⁸⁶Rb uptake into cultured lenses. Uptake was markedly inhibited in UVB-irradiated lenses and low-calcium culture did not prevent this inhibition of cation transport, a finding that explains why low-calcium protocol did not help maintain sodium homeostasis in irradiated lenses. Inhibition of cation transport and sodium accumulation eventually caused lens hydration and light scattering during extended culture in the absence of significant calcium elevation. Additional experiments were done to establish whether initial damage sustained by membranes could be repaired through the biosynthesis of new membrane proteins. Incorporation of ¹⁴C-histidine in membranes of the UVB-exposed lens was measured to assess membrane synthesis essential for repairing membrane damage. The rate of membrane protein synthesis, assessed by measuring incorporation of labeled amino acids, declined in UVB cataract, despite the prevention of calcium accumulation. These results suggest that one explanation for irreversible gain in sodium and calcium content accompanying opacification is the inability of lenses to replenish damaged membrane proteins comprising ion channels or transporters.     

Daphnia pulex Fed UVB-lrradiated Chlamydomonas reinhardtii Show Decreased Survival and Fecundity†

Abstract The ability to avoid or tolerate UVB radiation (290–320 nm) probably reduces the quality of phytoplankton as food for zooplankton. Ultraviolet avoidance forces motile algae lower in the water column, reducing net primary productivity. Production of UV-absorbing compounds as a tolerance mechanism takes carbon and energy away from cell growth; these compounds are also less useful as food for herbivores. We assessed the food quality of UVB-irradiated Chlamydomonas reinhardtii Dang, by analysis of cohort life tables for the herbivore Daphnia pulex Leydig. Algal cultures were grown under fluorescent lights interspersed with UV bulbs (20°C, 16 h light: 8 h dark) and covered with either cellulose acetate (+UVB) or Mylar D (-UVB). After a 7 day UVB exposure, carotenoid levels of C. reinhardtii were significantly greater than in unexposed cultures; there were no significant differences in chlorophyll, lipid, protein or nonstructural carbohydrate levels. After an initial decrease in growth rate, UVB-exposed algae recovered and final growth rates of both cultures were similar after 7 days. Daphnia neonates were raised under optimal conditions (20°C, 16 h light:8 h dark) and fed either UVB-irradiated or control algae. Daphnia fed UVB-irradiated algae had significantly increased mortality, decreased growth rate at the time of first clutch production, delayed release of the first clutch and decreased overall fecundity (P < 0.05). Daphnia were also less efficient at clearing UVB-irradiated algae from the water column (17% removal in 18 h) when compared with Daphnia that grazed upon nonirradiated algae (85% removal in 18 h). These data suggest that even though UVB-irradiated algal populations can recover from their initial decline, their predators may not be able to utilize them as an effective food source. Therefore, the impacts of UVB radiation within     

Reconstruction of Rabbit Corneal Layer Composed of Corneal Fibroblasts and Corneal Epithelium on the Lyophilized Amniotic Membrane

Introduction Thehumancornea,animportantopticalcompo nentthatfocusesthelightontheiris,isatransparent avasculartissuethatalsoplaysaprotectiveroleforthe eye.Therefore,anyinjury,diseaseorcelldamagethat canleadtoopacificationofthecornea,canimpairthe vision,and…     

EARLY CELL KINETIC EFECTS OF A SINGLE DOSE OF NARROW-BANDED ULTRAVIOLET B IRRADIATION ON THE RAT CORNEAL EPITHELUM

Abstract— The right eyes of 40 rats were exposed to a signal erythemogenic dose fo ultraviolet B irradiation (UVB) at 297nm. The irradiation was directed perpenddicualr to the center of the cornea. The left eyes served as controls. The animals were randomly assigned into 10 groups. The labelling index (LI) after pluse labeling the tritiated thymidine and the mitotic rate (MR) after Colcemid administration were registered in the corneal epithelium at predetermined intervals up to 96 h after the irradiation. A mathematical method was used to corealted corresponding corneal areas from the different animals. In the central the LI was considerably reduced up to 36h after the irradiation. The LI increased toward the peripheral cornea and reached normal values at the limbal area. The MR was also reduced up to 36h. However, this reduction was over the entire epithelium. The block in cell proliferation was followed by increased proliferation.     

UV-mediated DNA strand breaks in corneal epithelial cells assessed using the comet assay procedure

Ultraviolet (UV)-mediated DNA damage in various tissues has been well documented. However, research on the damaging effect of UV irradiation on the DNA of corneal epithelium is scarce, even though this is of interest because the cornea is directly exposed to damaging solar (UV) radiation. In this study, we developed a corneal epithelium Comet assay model to assess the background DNA damage (as strand breaks) in cells retrieved from different layers of the porcine corneal epithelium, and to investigate the effect of UV irradiation on DNA damage in corneal epithelial cells. Results show that the background DNA strand breaks decreased significantly (P < 0.001) toward deeper layers of the epithelium. Exposure to the same intensity (0.216 J/cm2) of UVA, UVB and UVC caused a significant (P < 0.001) increase in DNA strand breaks of deeper-layer cells: mean +/- SD %DNA scores (10 gels per treatment, with 100 irradiated cells scored per gel) were 10.2% +/- 1.4% for UVA, 27.4% +/- 4.6% for UVB, and 14.7% +/- 1.8% for UVC compared with 4.2% +/- 0.5% for controls (ambient room light). This study has shown for the first time that the Comet assay for DNA strand breaks can be used successfully with corneal epithelial cells. This report will support future studies investigating environmental influences on corneal health and the assessment of possible protective strategies, and in applying DNA lesion-specific versions of the Comet assay in this corneal epithelial cell model.     

Photokeratitis from 193 nm argon-fluoride laser radiation

The threshold for photokeratitis at 193 nm was obtained for the rabbit cornea using an ArF excimer laser. Because ablation occurs at a level below that for photokeratitis, it was necessary to expose the cornea to a lengthy series of low-energy exposures. It is concluded that the 193 nm photons have such a shallow penetration depth, being limited to the outermost epithelial cells, that classical photokeratitis occurs from the fluorescence emitted at the corneal epithelial absorption site. An intact tear film may help to protect the cornea from low-level, scattered 193 nm laser radiation.     

Phototoxicity in human lens epithelial cells promoted by St. John's Wort

St. John's Wort (SJW), an over-the-counter antidepressant, contains hypericin, which absorbs light in the UV and visible ranges and is phototoxic to skin. To determine if it also could be phototoxic to the eye, we exposed human lens epithelial cells to 0.1-10 microM hypericin and irradiated them with 4 J/cm2 UV-A or 0.9 J/cm2 visible light. Neither hypericin exposure alone nor light exposure alone reduced cell viability. In contrast, cells exposed to hypericin in combination with UV-A or visible light underwent necrosis and apoptosis. The ocular antioxidants lutein and N-acetyl cysteine did not prevent damage. Thus, ingested SJW is potentially phototoxic to the eye and could contribute to early cataractogenesis. Precautions should be taken to protect the eye from intense sunlight while taking SJW.     

Transmission spectra of light to the mammalian retina

A simple method has been developed to determine the optical properties of the anterior segment of the intact eye. This consists of a probe that is inserted into the posterior sclera and detects light passing through the anterior segment. The probe is connected to a charge-coupled device spectrophotometer via a fiber optic bundle. It was determined that the young rat anterior segment transmits light down to 300 nm, whereas calf and rabbit eyes transmit no UVB and only part of the UVA to the posterior segment. The absorbing species in these animals is most likely NAD(P)H, which has an absorption maximum at approximately 345 nm and is associated with zeta-crystallin. A young primate anterior segment transmits almost no UV with a steep increase in transmission at CA 400 nm. Because some experiments employed a light tube that is used to illuminate the retina during vitrectomies, this method can be used to determine the transmission spectra of the anterior segment of humans in vivo.     

A novel corneal adhesive based on functionally coupled PEG-lysozyme hydrogel for wound closure after surgical eye surgery

Corneal wound closure for surgical eye surgeries or accidents is typically performed to prevent pathogens from the sterile intraocular environment and avoid potential postoperative complications. Tissue adhesives are increasingly employed for corneal wound closure with superior treatment efficiency and less adverse effects. In this study, we successfully develop a novel corneal adhesive based on functionally coupled PEG-lysozyme (PEG-LZ) hydrogels for wound closure after surgical eye surgeries. PEG-LZ hydrogels have plenty of micropores and gradually decreased pore size with increasing concentration from 10%, 15% to 20% (w/v), in which PEG-LZ (15%) represents the suitable pH value, gelation time and elastic modulus. PEG-LZ hydrogels have no in vitro cytotoxicity and excellent ex vivo wound closure effectiveness in porcine eyes. The in vivo wound sealant in rabbit eyes by PEG-LZ hydrogels presents a superior therapeutic effect compared with the conventional methods of stromal hydration and suture, in terms of the wound closure percent, mean corneal thickness, percent of wound gaping, and the Descemet membrane detachment. PEG-LZ hydrogels do not induce obvious histological pathology changes. The PEG-LZ corneal adhesive is considered as a tissue adhesive alternative for wound closure after surgical eye surgeries.     

Stratum corneum lipid abnormalities in UVB-irradiated skin

Stratum corneum (SC) lipids are of particular importance in maintaining the permeability barrier function. Although many studies have demonstrated that UVB irradiation of mammalian skin reduces barrier function, the responsible alterations in SC lipid profiles are not known. In this study, we investigated both compositional and morphological alterations in SC lipids with the development of barrier abnormalities caused by daily UVB irradiation in hairless rat skin. The UVB irradiation of suberythemal doses (0.5 minimal erythema dose) significantly increased transepidermal water loss (TEWL) relative to nonirradiated control, indicating a diminished barrier function. Under these conditions, the total amounts of major SC lipid species (ceramides, cholesterol, free fatty acids) in UVB-irradiated SC did not differ from those in nonirradiated SC. However, electron microscopic observations revealed marked abnormalities in the intercellular domains of UVB-irradiated SC, where naturally occurring intercellular multilamellar structures were often absent and leaving the area with the appearance of an empty space. Moreover, in UVB-irradiated SC, individual corneocytes often showed small amounts of intercellular deposition product with abnormal lamellar structure, where lamellar body sphingomyelinase activity was present. These observations demonstrated a partial failure of lamellar body secretion in UVB-irradiated SC and suggested that a defect in the secretion of lamellar body-derived lipids and enzymes to SC intercellular space is, at least in part, responsible for the observed abnormal intercellular structure and barrier disruption.     

Susceptibility to Effects of UVB Irradiation on Induction of Contact Sensitivity, Relevance of Number and Function of Langerhans Cells and Epidermal Macrophages

Sensitization on skin exposed to acute low-dose UVB irradiation separates normal humans into two phenotypically distinct groups: One group, following sensitization on UVB-irradiated skin, develops contact sensitivity, designated UVB resistant (UVB-R) and the second group, following sensitization on UVB-irradiated skin, fails to develop contact sensitivity, designated UVB susceptible (UVB-S). To investigate whether UVB susceptibility in humans is related to antigen-presenting activity in the skin we studied the effect of UVB irradiation on the number and function of the epidermal antigen-presenting cells in volunteers identified as UVB-R and UVB-S. Single cell suspensions of epidermal cells from control skin and skin exposed to 3 minimal erythema doses (MED) of UVB 3 days previously were stained for Langerhans cells (CD1a+HLA-DR+) and epidermal macrophages (CD1a-HLA-DR+). The UVB exposure of the skin significantly decreased the percentage of Langerhans cells (UVB-R: n = 7, P < 0.02, UVB-S: n = 6, P < 0.03) and increased the percentage of epidermal macrophages (UVB-R: n = 7, P < 0.03, UVB-S: n = 6, P < 0.03) however to the same degree in both the UVBR and the UVB-S group. To study the effect on Langerhans cell alloreactivity, epidermal cells were harvested immediately after UVB irradiation. However, in both UVB-R and UVB-S subjects the Langerhans cell alloreactivity was blocked to the same degree immediately after UVB irradiation compared to nonirradiated epidermal cells. To determine the effect of UVB irradiation on epidermal macrophages, epidermal cells were harvested 3 days after UVB irradiation. Irradiated epidermal cells from both UVB-R and UVB-S subjects demonstrated a strong antigen-presenting capacity compared to epidermal cells from control skin leading to activation of T cells that mainly secrete interferon (1FN)-γ and not interleukin (IL)-4. In conclusion we found that UVB susceptibility was not correlated with the number of Langerhans cells or epidermal macrophages in the skin at the same time of sensitization. Neither was it correlated with the capacity of Langerhans cells nor UVB-induced epidermal macrophages to activate T cells in vitro.     

The photophysics and photobiology of the eye

The eye consists of three major segments: the cornea, lens and retina. The main function of the anterior ocular tissue, the cornea and the lens is to transmit and focus light on the retina without distortion. They also filter out UV light (less than 400 nm) and prevent it from reaching the retina. Much of the light reaching the retina is used for sight. However, light can have numerous other effects on the constituents of the eye, both beneficial and deleterious. This article reviews the interaction of light with the eye, various protective mechanisms, the possible role of light in aging and disease states and the role of light in biological processes other than sight such as mood, hormonal secretions and the cyclic growth and phagocytosis of the rods and cones.