UVB-irradiated Cornea Research Articles

The Healing of Oxidative Injuries with Trehalose in UVB-Irradiated Rabbit Corneas.

Our previous research revealed that trehalose, a nonreducing disaccharide of glucose and an important stress responsive factor, proved to have anti-inflammatory, antiapoptotic, and particularly antioxidant properties in UVB-irradiated corneas. Trehalose reduced oxidative stress in corneas induced by UVB irradiation, by means of a decrease in the antioxidant/prooxidant imbalance in the corneal epithelium. In this study, we demonstrate that trehalose of 3% or 6% concentration in eye drops directly decreases oxidative stress in UVB-irradiated corneas, by removing the excessive amount of reactive oxygen species (ROS). Trehalose drops applied on corneas during UVB irradiation once daily for four days resulted in a reduction or even absence of the oxidative stress, DNA damage, and peroxynitrite formation (detected by nitrotyrosine residues), seen in buffer-treated corneas. Furthermore, trehalose treatment applied curatively after repeated irradiation for the subsequent fourteen days led to the renewal of corneal transparency and significant suppression or even absence of neovascularization. This was in contrast to buffer-treated irradiated corneas, where the intracorneal inflammation was developed and the untransparent corneas were vascularized. In conclusion, the treatment of UVB-irradiated corneas with trehalose eye drops removed the excessive amount of ROS in the corneal epithelium, leading to the suppression of oxidative stress and favorable corneal healing. The 6% trehalose showed a higher intensive antioxidant effect.

Trehalose in ophthalmology.

Trehalose, a disaccharide of glucose, is a naturally occurring nontoxic and nonreducing bioactive sugar. Trehalose is synthetized by many organisms when cells are exposed to stressful conditions, including dehydration, heat, oxidation, hypoxia or even anoxia. Although trehalose is not synthesized by mammalian cells, it has recently been demonstrated to have a number of important properties that indicate its utility in humans. Trehalose enables wound healing by protecting cells, especially cell membranes, from oxidative injury and dessication. When the injured cornea is treated with trehalose, corneal inflammation, scar formation and corneal neovascularization are suppressed. In dry eye disease, trehalose decreased cell apoptosis and reduced oxidative, inflammatory and proteolytic activity at the ocular surface. In UVB irradiated cornea, trehalose suppressed photodamage evoked by UVB rays. It decreased the intracorneal inflammation and reduced corneal neovascularization. Trehalose prevented postoperative fibrous scar formation after ocular surgery, such as glaucoma filtration surgery. The non-toxicity of trehalose allows its administration in humans for extended periods and enables its use in various disease states.

RETRACTED ARTICLE: Therapeutic effect of molecular hydrogen in corneal UVB-induced oxidative stress and corneal photodamage

The aim of this study is to examine whether molecular hydrogen (H2) is able to reduce oxidative stress after corneal damage induced by UVB irradiation. We previously found that UVB irradiation of the cornea caused the imbalance between the antioxidant and prooxidant enzymes in the corneal epithelium, followed by the imbalance between metalloproteinases and their physiological inhibitors (imbalances in favour of prooxidants and metalloproteinases) contributing to oxidative stress and development of the intracorneal inflammation. Here we investigate the effect of H2 dissolved in PBS in the concentration 0.5 ppm wt/vol, applied on rabbit corneas during UVB irradiation and healing (UVB doses 1.01 J/cm2 once daily for four days). Some irradiated corneas remained untreated or buffer treated. In these corneas the oxidative stress appeared, followed by the excessive inflammation. Malondiladehyde and peroxynitrite expressions were present. The corneas healed with scar formation and neovascularization. In contrast, in H2 treated irradiated corneas oxidative stress was suppressed and malondiladehyde and peroxynitrite expressions were absent. The corneas healed with the restoration of transparency. The study provides the first evidence of the role of H2 in prevention of oxidative and nitrosative stress in UVB irradiated corneas, which may represent a novel prophylactic approach to corneal photodamage.

Trehalose treatment accelerates the healing of UVB-irradiated corneas. Comparative immunohistochemical studies on corneal cryostat sections and corneal impression cytology.

The UVB-irradiated cornea is damaged by oxidative stress. Toxic oxygen products induced by UVB radiation in the cornea are insufficiently removed by antioxidants, whose numbers decrease with increasing UVB irradiation. In addition, the UVB-irradiated cornea suffers from hypoxic conditions because damaged corneal cells cannot utilize oxygen normally, although the supply of oxygen to the cornea is unchanged (normal). This contributes to attenuated re-epithelialization, corneal neovascularization and apoptotic cell death. Our previous publications reported that trehalose applied on the corneal surface during irradiation significantly suppressed UVB-induced corneal oxidative damage. The results of this study provide for the first time important evidence that trehalose applied on the surface of corneas for two weeks following repeated UVB irradiation (312 nm, daily dose 0.5 J/cm2) accelerated corneal healing, restored corneal transparency and suppressed corneal neovascularization. Compared to buffered saline treatment, following which caspase-3, nitrotyrosine, malondialdehyde and urokinase-type plasminogen activator were still strongly expressed in the corneal epithelium two weeks after irradiation and corneal neovascularization was evident, apoptotic cell death was already significantly reduced after one week of trehalose application. The expression of other markers of injury returned to normal levels during two weeks of trehalose treatment. In conclusion, our results show that trehalose accelerated healing of the UVB irradiated cornea, very probably via suppression of hypoxia-response injury. In addition, immunohistochemical results on corneal cryostat sections corresponded with those obtained using corneal impression cytologies, thus confirming that corneal impression cytologies are useful for diagnostic purposes.

Favorable effects of trehalose on the development of UVB-mediated antioxidant/pro-oxidant imbalance in the corneal epithelium, proinflammatory cytokine and matrix metalloproteinase induction, and heat shock protein 70 expression

Trehalose, a nonreducing disaccharide of glucose, is synthesized as a stress response factor when cells are exposed to stressful conditions. In the cornea, oxidative stress plays the key role in the development of acute corneal inflammatory response to UVB rays, photokeratitis. We found previously that trehalose reduced UVB-induced oxidative effects on the formation of cytotoxic peroxynitrite, apoptotic corneal epithelial cell death and changes in corneal optics. The aim of the present study was to examine whether trehalose might inhibit UVB-mediated proinflammatory cytokine and matrix metalloproteinase induction and the development of an antioxidant/pro-oxidant imbalance in the corneal epithelium, changes found previously to be strongly involved in the acute corneal UVB-induced inflammation. The expression of heat shock protein 70 as a potential biomarker for corneal UVB-induced damage was also examined. The corneas of New Zealand white rabbits were irradiated with UVB rays, 312 nm, daily dose of 0.5 J/cm(2) for 4 days. During the irradiation, trehalose drops were applied on the right eye and buffered saline on the left eye. One day after the end of irradiations, the animals were killed and the corneas examined immunohistochemically for the expression of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase), pro-oxidant xanthine oxidoreductase/xanthine oxidase, proinflammatory cytokines (interleukin-6, interleukin-8), matrix metalloproteinase-9 and heat shock protein 70. After buffered saline treatment during UVB irradiation, an antioxidant/pro-oxidant imbalance appeared in the corneal epithelium: The expression of antioxidant enzymes was highly reduced, whereas the expression of pro-oxidant xanthine oxidase was increased. The pronounced expression of pro-inflammatory cytokines, matrix metalloproteinase and heat shock protein 70 was found in the UVB-irradiated corneal epithelium. Trehalose application significantly suppressed all the above-mentioned UVB-induced corneal disturbances. Trehalose favorably influenced the oxidative damage of the cornea caused by UVB rays. Trehalose suppressed proinflammatory cytokine induction. It is suggested that suppression of proinflammatory cytokines contributed strongly to reduced matrix metalloproteinase and xanthine oxidase expression in the UVB-irradiated corneal epithelium and to the decreased development of an antioxidant/pro-oxidant imbalance. The overexpression of heat shock protein 70 found in UVB-irradiated cornea after buffered saline treatment was reduced after trehalose application.

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.

Expression of tissue inhibitors of matrix metalloproteinases 1 and 2 in corneal epithelium after UV irradiation

Abstract Purpose Tissue inhibitors of metalloproteinases (TIMPs) are capable of inhibiting the activities of all known matrix metalloproteinases (MMPs) and therefore they play a key role in maintaining the balance between synthesis and degradation of basic extracellular matrix components (collagens, glycosaminoglycans) in the normal cornea. However, under pathological conditions, disruption of this balance may appear due to the increased expression of MMPs or decreased expression of TIMPs. Purpose of this study was to investigate whether changes in expression of TIMPs might contribute to increased proteolytic activity of MMPs in corneas irradiated by UV rays. Methods In the first group of rabbits the corneas were irradiated with UVA lamp (365 nm, once a day during 4 days, a dose per day 1.01 J/cm2), in the second group with UVB lamp (312 nm, once a day during 4 days, a dose per day 1.01 J/cm2). Normal corneas served as controls. TIMP‐1 and TIMP‐2 were examined in cryostat sections immunohistochemically using mouse monoclonal anti‐TIMP‐1 and anti‐TIMP‐2 antibodies. Results Immunohistochemical examination showed that UVA rays did not change the expression of TIMPs studied in the corneal epithelium. In contrast, UVB rays induced the decreased expression of TIMPs in corneal epithelial cells. From both enzymes investigated in UVB irradiated corneas the decrease of TIMP‐1 expression was more significant than expression of TIMP‐2. Conclusion Even if further studies are necessary, our results point to the suggestions that insufficient inhibition of MMPs due to the decreased presence of TIMPs in corneas irradiated by UVB rays may lead to corneal damage from excessive proteolytic degradation of corneal proteins.

Light Absorption Properties of the Rabbit Cornea Repeatedly Irradiated with UVB Rays

Under normal conditions, the cornea absorbs the majority of UVB (ultraviolet B, 280-320 nm) rays, which is very important for the protection of the inner eye against their damaging effect. Our previous studies have shown that repeated irradiation of the rabbit cornea with UVB rays for 5 days (daily dose of 1.01 J cm(- 2)) caused photokeratitis accompanied by swelling (hydration) of the corneal stroma, thinning of the corneal epithelium and decrease in antioxidants. The purpose of this study was to examine the light absorption properties of such damaged rabbit cornea. Results of both spectrophotometry of the whole corneal buttons and corneal tissue dissolved in sodium hydroxide show that because of above mentioned disturbances, UVB-irradiated cornea absorbs more light throughout the whole measurable UV-VIS spectral range than the normal cornea. Increased corneal thickness (result of hydration), changes of corneal transparency (the cornea becomes grayish) and some increase in protein content all contribute to the increased light absorption of UVB irradiated corneas. We suggest that the UVB-irradiated cornea, although damaged and nearly without antioxidants, might actually through its higher UV absorbance protect the inner eye against further damage from UVB rays.

Effects of inhibition of urokinase -type plasminogen activator (u-PA) by amiloride in the cornea and tear fluid of eyes irradiated with UVB

The purpose of the present study was to test our hypothesis that amiloride, a specific u-PA inhibitor, effectively decreases u-PA activity in cornea as well as in tear fluid and favourably affects corneal healing. Therefore, comparative histochemical and biochemical studies of u-PA and the effects of amiloride were performed on rabbit corneas and tear fluid using the sensitive fluorogenic substrate Z-Gly–Gly–Arg-7-amino-4-trifluoromethylcoumarin. Rabbit eyes were repeatedly irradiated with UVB for 9 days and during the irradiation topically treated with amiloride (1 mg/ml saline) or placebo (saline) (dropwise, 5 times daily). Results show that in placebo-treated eyes, UVB evoked the appearance of u-PA activity in cornea and tear fluid in early stages of irradiation, and u-PA levels increased during irradiation. Corneal epithelium was gradually lost and remnants of the epithelium as well as keratocytes in the upper part of corneal stroma showed high u-PA activity. Finally, corneas lost their epithelium completely. In corneal stroma, numerous u-PA-containing inflammatory cells were present. Corneas were vascularized. When amiloride was dropped on the eye surface on the first day of irradiation and subsequently daily until the end of the experiment, u-PA activity in both cornea and tear fluid was strongly inhibited. Corneas were covered with a continuous epithelium until the end of the experiment. The number of inflammatory cells was significantly decreased. Corneal vascularization was reduced by 50%. In conclusion, early application of amiloride inhibited u-PA activity in UVB-irradiated corneas as well as in tear fluid and diminished the development of corneal pathology.

Long-term effects of a single dose of ultraviolet-B on albino rabbit cornea--I. in vivo analyses.

Both eyes of female albino rabbits (1.9 kg) were exposed to a single dose of UV-B (300 +/- 9 nm; 0.125 J/cm2 total dose) between 13.30 and 15.00 h. The average irradiance was 209 +/- 4 microW/cm2 delivered over 612 +/- 13 s. At various time periods thereafter (every 12 h for 3 days, 6, 7, 14, 28, 42, 56, 112, 224 and 336 days post-irradiation), the animals were subjected to a full slit lamp examination to evaluate the status of the cornea and the anterior segment along with optical or ultrasonic pachometry of central corneal thickness. The results were compared with studies on age-matched rabbits over the same time period. In response to the UV-B irradiation, the corneas showed a modest edema (20% increase in central corneal thickness) that peaked at 48 h. Nearly normal central corneal thickness returned in 6 days and followed by a secondary very slight swelling (less than 5%) that resolved by 14 days. The edema was accompanied by keratitis over the same period. Thereafter, both control and UV-B irradiated corneas progressively increased in thickness with age. Biomicroscopy also revealed the appearance of granular opacities in the corneal epithelium that peaked at 72-96 h and resolved over 28 days. In addition, very small microdot opacities of the corneal epithelium were present in the UV-B irradiated corneas that reached maximum at 72 h but persisted to some degree throughout the evaluation period. Biomicroscopy also revealed a progressive disruption of the homogeneous nature of the corneal stroma by the appearance of large 'bread crumb'-like opacities that started at 72 h and was still present at the end of the evaluation period. These results suggest that long-term evaluation of the cornea is important after acute UV-B exposure and indicate that acute exposure to UV-R can produce corneal changes resembling those reported following chronic exposure to UV-R-rich environments.