Hydrocaffeic and p-coumaric acids, natural phenolic compounds, inhibit UV-B damage in WKD human conjunctival cells in vitro and rabbit eye in vivo

This paper studied the effect on UV-B ocular damage of 10microM hydrocaffeic acid (HCAF) alone and as a mixture (MIX) (5 microM HCAF+5 microM p-coumaric acid). Since ocular UV-B damage is mediated by reactive oxygen species, the aim was to test if HCAF and MIX could reduce oxidation damage in human conjunctival cells (WKD) in vitro and in cornea and sclera of rabbits in vivo. After UVB irradiation (44 J/m(2)) of WKD cells, 8-oxodG levels in DNA were markedly increased and this effect was attenuated by HCAF and MIX. Rabbit eyes were treated by application of HCAF and MIX drops before UV-B exposure (79 J/m(2)). Corneal and scleral DNA oxidation damage, xanthine-oxidase (XO) activity and malondialdehyde levels (MDA) in corneal tissue and prostaglandin E(2) (PGE(2)) in the aqueous humour were reduced by HCAF alone and in combination with p-coumaric acid, showing their potential as a topical treatment against UV-B damage.     

TRAIL: a mechanism of tumor surveillance in an immune privileged site

TRAIL is a recently described member of the TNF superfamily. The ability of TRAIL to induce apoptosis in a large number of tumors has stimulated interest in TRAIL as a tumor therapeutic agent. Although TRAIL mRNA is expressed in a number of tissues, its functional significance to various organs is unknown. Because tumors rarely develop in the eye, we have examined this organ for functional TRAIL expression. Our analysis revealed that TRAIL mRNA and protein are constitutively expressed on numerous ocular structures, including the cornea and retina. More importantly, ocular tissue displays functional TRAIL as determined by in vitro killing of TRAIL-sensitive tumor cell lines. Previous studies have shown that ocular tissue also expresses functional Fas ligand (FasL). To assess the contribution of TRAIL and FasL for tumor cell killing in the eye, cell lines susceptible to both TRAIL and FasL were examined. The results show that ocular tissue kills via either ligand, suggesting a compensatory mechanism between TRAIL and FasL. Collectively, these results provide physiological evidence for ocular TRAIL expression, and suggest a role for this molecule in tumor surveillance in an immune privileged site.     

The role of corneal crystallins in the cellular defense mechanisms against oxidative stress

The refracton hypothesis describes the lens and cornea together as a functional unit that provides the proper ocular transparent and refractive properties for the basis of normal vision. Similarities between the lens and corneal crystallins also suggest that both elements of the refracton may also contribute to the antioxidant defenses of the entire eye. The cornea is the primary physical barrier against environmental assault to the eye and functions as a dominant filter of UV radiation. It is routinely exposed to reactive oxygen species (ROS)-generating UV light and molecular O(2) making it a target vulnerable to UV-induced damage. The cornea is equipped with several defensive mechanisms to counteract the deleterious effects of UV-induced oxidative damage. These comprise both non-enzymatic elements that include proteins and low molecular weight compounds (ferritin, glutathione, NAD(P)H, ascorbate and alpha-tocopherol) as well as various enzymes (catalase, glucose-6-phosphate dehydrogenase, glutathione peroxidase, glutathione reductase, and superoxide dismutase). Several proteins accumulate in the cornea at unusually high concentrations and have been classified as corneal crystallins based on the analogy of these proteins with the abundant taxon-specific lens crystallins. In addition to performing a structural role related to ocular transparency, corneal crystallins may also contribute to the corneal antioxidant systems through a variety of mechanisms including the direct scavenging of free radicals, the production of NAD(P)H, the metabolism and/or detoxification of toxic compounds (i.e. reactive aldehydes), and the direct absorption of UV radiation. In this review, we extend the discussion of the antioxidant defenses of the cornea to include these highly expressed corneal crystallins and address their specific capacities to minimize oxidative damage.     

Protection of Retinal Ganglion Cells and Retinal Vasculature by Lycium Barbarum Polysaccharides in a Mouse Model of Acute Ocular Hypertension

Acute ocular hypertension (AOH) is a condition found in acute glaucoma. The purpose of this study is to investigate the protective effect of Lycium barbarum polysaccharides (LBP) and its protective mechanisms in the AOH insult. LBP has been shown to exhibit neuroprotective effect in the chronic ocular hypertension (COH) experiments. AOH mouse model was induced in unilateral eye for one hour by introducing 90 mmHg ocular pressure. The animal was fed with LBP solution (1 mg/kg) or vehicle daily from 7 days before the AOH insult till sacrifice at either day 4 or day 7 post insult. The neuroprotective effects of LBP on retinal ganglion cells (RGCs) and blood-retinal-barrier (BRB) were evaluated. In control AOH retina, loss of RGCs, thinning of IRL thickness, increased IgG leakage, broken tight junctions, and decreased density of retinal blood vessels were observed. However, in LBP-treated AOH retina, there was less loss of RGCs with thinning of IRL thickness, IgG leakage, more continued structure of tight junctions associated with higher level of occludin protein and the recovery of the blood vessel density when compared with vehicle-treated AOH retina. Moreover, we found that LBP provides neuroprotection by down-regulating RAGE, ET-1, Aβ and AGE in the retina, as well as their related signaling pathways, which was related to inhibiting vascular damages and the neuronal degeneration in AOH insults. The present study suggests that LBP could prevent damage to RGCs from AOH-induced ischemic injury; furthermore, through its effects on blood vessel protection, LBP would also be a potential treatment for vascular-related retinopathy.     

Genotoxicity of pesticides: a review of human biomonitoring studies

Pesticides constitute a heterogeneous category of chemicals specifically designed for the control of pests, weeds or plant diseases. Pesticides have been considered potential chemical mutagens: experimental data revealed that various agrochemical ingredients possess mutagenic properties inducing mutations, chromosomal alterations or DNA damage. Biological monitoring provides a useful tool to estimate the genetic risk deriving from an integrated exposure to a complex mixture of chemicals. Studies available in scientific literature have essentially focused on cytogenetic end-points to evaluate the potential genotoxicity of pesticides in occupationally exposed populations, including pesticide manufacturing workers, pesticide applicators, floriculturists and farm workers. A positive association between occupational exposure to complex pesticide mixtures and the presence of chromosomal aberrations (CA), sister-chromatid exchanges (SCE) and micronuclei (MN) has been detected in the majority of the studies, although a number of these failed to detect cytogenetic damage. Conflicting results from cytogenetic studies reflect the heterogeneity of the groups studied with regard to chemicals used and exposure conditions. Genetic damage associated with pesticides occurs in human populations subject to high exposure levels due to intensive use, misuse or failure of control measures. The majority of studies on cytogenetic biomarkers in pesticide-exposed workers have indicated some dose-dependent effects, with increasing duration or intensity of exposure.Chromosomal damage induced by pesticides appears to have been transient in acute or discontinuous exposure, but cumulative in continuous exposure to complex agrochemical mixtures.Data available at present on the effect of genetic polymorphism on susceptibility to pesticides does not allow any conclusion.     

Central corneal thickness considered an index of corneal hydration of the UVB irradiated rabbit cornea as influenced by UVB absorber

UVB radiation from sunlight induces an acute corneal inflammation, photokeratitis, accompanied by changes in corneal hydration. We employed a method of ultrasonic pachymetry for daily examination of central corneal thickness as an index of corneal hydration of the rabbit cornea repeatedly irradiated by UVB radiation (312 nm, daily dose of 0.25 J/cm(2) during three or four days) as influenced by UVB absorber (actinoquinol combined with hyaluronic acid) dropped on the ocular surface during irradiation. One day after the third irradiation procedure the animals were sacrificed and corneas examined immuno-histochemically for peroxynitrite formation, a marker of oxidative damage, the antioxidant aldehyde dehydrogenase 3A1 and endothelial nitric oxide synthase, an enzyme generated nitric oxide. Results show that UV absorber combined with hyaluronic acid protected the cornea against UVB-induced changes in corneal thickness and microscopical disturbances to the cornea (both seen after buffered saline application) until the fourth experimental day. These UVB doses are equivalent to a daily exposure of 2.5 hrs of the human cornea to solar UVB radiation for three consecutive days. It is suggested that actinoquinol/ hyaluronic acid drops might be helpful for the human eye in the defence against photooxidative and other oxidative processes.     

Changes of Ocular Surface and the Inflammatory Response in a Rabbit Model of Short-Term Exposure Keratopathy

Purpose

To evaluate the ocular surface change and the inflammatory response in a rabbit model of short-term exposure keratopathy.

Methods

Short term exposure keratopathy by continuous eyelid opening was induced in New Zealand white rabbits for up to 4 hours. Ultrasound pachymetry was used to detect central total corneal thickness. In vivo confocal microscopy and impression cytology were performed to evaluate the morphology of ocular surface epithelium and the infiltration of inflammatory cells. Immunohistochemistry for macrophage,neutrophil, CD4(+) T cells, and CD8(+) T cells were performed to classify the inflammatory cells. Scanning electron microscopy(SEM) was performed to detect ocular surface change.The concentrations of IL-8, IL-17, Line and TNF-αwere analyzed by multiplex immunobead assay. TUNEL staining was performed to detect cellular apoptosis.

Results

Significant decrease ofcentral total cornealthickness were found within the first 5 minutes and remained stable thereafter, while there were no changes of corneal epithelial thickness.No significant change of corneal, limbal and conjunctival epithelial morphology was found by in vivo confocal microscopy except the time dependent increase of superficial cellular defects in the central cornea. Impression cytology also demonstrated time dependent increase of sloughing superficial cells of the central cornea. Aggregations ofinflammatory cells were found at 1 hour in the limbal epithelium, 2 hours in the perilimbal conjunctival epithelium, and 3 hours in the peripheral corneal epithelium.In eyes receiving exposure for 4 hours, the infiltration of the inflammatory cells can still be detected at 8 hours after closing eyes.Immunohistochemical study demonstrated the cells to be macrophages, neutrophils, CD4-T cells and CD-8 T cells.SEM demonstrated time-depending increase of intercellular border and sloughing of superficial epithelial cells in corneal surface. Time dependent increase of IL-8, IL-17 and TNF-α in tear was found.TUNEL staining revealed some apoptotic cells in the corneal epithelium and superficial stroma at 3 hours after exposure.

Conclusions

Short term exposure keratopathy can cause significant changes to the ocular surface and inflammatory response. Decrease of central total corneal thickness, aggregation of inflammatory cells, and cornea epithelial cell and superficial keratocyte apoptosis were found no less than 4 hours following the insult.     

Genotoxic evaluation of workers employed in pesticide production

Pesticides are widely used throughout the world in agriculture to protect crops and in public health to control diseases. Nevertheless exposure to pesticides can represent a potential risk to humans. Pesticide manufacturing unit workers are prone to possible occupational pesticide exposure. Therefore, this study was performed to evaluate the genotoxic effect of pesticide exposure in these workers. In the present investigation 54 pesticide workers and an equal number of control subjects were assessed for genome damage in blood lymphocytes utilizing the chromosomal aberration analysis and the buccal epithelial cell by adopting the micronucleus test. The results suggested that pesticide workers had a significantly increased frequency of chromosomal aberrations when compared with controls (mean+/-S.D., 8.43+/-2.36 versus 3.32+/-1.26; P<0.05). Similarly, the pesticides exposed workers showed a significant increase in micronucleated cells compared with controls (1.24+/-0.72 versus 0.32+/-0.26; P<0.05). Analysis of variance revealed that occupational exposure to pesticides had a significant effect on frequency of micronuclei (P<0.05), whereas smoking, age, gender and alcohol consumption had no significant effect on genetic damage (P>0.05). However, no association was found between years of exposure, smoking, age, gender, alcohol consumption and higher levels of genetic damage as assessed by the chromosomal aberration assay (P>0.05). Our findings indicate that occupational exposure to pesticides could cause genome damage in somatic cells.     

Transmission of 308 nm excimer laser radiation for ophthalmic microsurgery--medical, technical and safety aspects

A surgical system using 308 nm excimer laser radiation transmitted by quartz fibers is described. In-vitro surgery has been performed on the cornea, in the anterior chamber angle, on the lens and in the vitreous with a special application tip for each procedure. Possible damage of ocular structures by 308 nm radiation is discussed. In-vitro experiments showed that UV exposure of intraocular structures sensitive to 308 nm radiation can be reduced by appropriate design of the application tips and by the application of UV absorbing drugs.     

Evaluation of toxic potential of captan: Induction of hsp70 and tissue damage in transgenic Drosophila melanogaster (hsp70-lacZ) Bg9

The study investigated the working hypothesis that a widely used fungicide captan exerts toxic effects on nontarget organisms. Transgenic Drosophila melanogaster (hsp70-lacZ) was used as a model by assaying stress gene expression as an endpoint for cytotoxicity and also to evaluate whether stress gene expression is sufficient enough to protect and to prevent tissue damage against toxic insult of the chemical. The study was further extended to understand the effect of the pesticide on development, life cycle, and reproduction of the organism and finally to evaluate a concentration of the chemical to be nontoxic to the organism. The study showed that (i) captan causes cytotoxicity at and above 0.015 ppm; (ii) at 0.0015 ppm captan, absence of hsp70 expression in the exposed organism was evaluated as the concentration referred to as no observed adverse effect level (NOAEL) for Drosophila; (iii) emergence pattern of flies was affected only at the highest concentration of captan by 4 days, while hatching and survivorship were unaffected even at this concentration; (iv) reproductive performance was significantly affected only at 125.0 and 1250.0 ppm captan, while in the lower dietary concentrations no such deleterious effects were observed; (v) at 1250.0 ppm, hsp70 failed to protect the cells from toxicant assault after 48 h exposure, thus leading to tissue damage as revealed by Trypan Blue staining. The present study shows the cytotoxic potential of captan and further reveals the application of stress genes in determining NOAEL and its expression as bioindicator of exposure to environmental contaminants.     

Absence of ocular effects after either single or repeated exposure to 10 mW/cm(2) from a 60 GHz CW source

This study was designed to examine ocular effects associated with exposure to millimeter waves (60 GHz). Rabbits served as the primary experimental subjects. To confirm the results of the rabbit experiments in a higher species, the second phase of the study used nonhuman primates (Macaca mulatta). First, this study used time-resolved infrared radiometry to assess the field distribution patterns produced by different antennas operating at 60 GHz. These results allowed us to select an antenna that produced a uniform energy distribution and the best distance at which to expose our experimental subjects. The study then examined ocular changes after exposure at an incident power density of 10 mW/cm(2). Acute exposure of both rabbits and nonhuman primates consisted of a single 8 h exposure, and the repeated exposure protocol consisted of five separate 4 h exposures on consecutive days. One eye in each animal was exposed and the contralateral eye served as the sham-exposed control. After postexposure diagnostic examinations, animals were euthanized and the eyes were removed. Ocular tissue was examined by both light and transmission electron microscopy. Neither microscopic examinations nor the diagnostic procedures performed on the eyes of acute and repeatedly exposed rabbits found any ocular changes that could be attributed to millimeter-wave exposure at 10 mW/cm(2). Examination of the primates after comparable exposures also failed to detect any ocular changes due to exposure. On the basis of our results, we conclude that single or repeated exposure to 60 GHz CW radiation at 10 mW/cm(2) does not result in any detectable ocular damage.     

Environmental light and endogenous antioxidants as the main determinants of non-cancer ocular diseases

The human eye is constantly exposed to sunlight and artificial lighting. Exogenous sources of reactive oxygen species (ROS) such as UV light, visible light, ionizing radiation, chemotherapeutics, and environmental toxins contribute to oxidative damage in ocular tissues. Long-term exposure to these insults places the aging eye at considerable risk for pathological consequences of oxidative stress. Furthermore, in eye tissues, mitochondria are an important endogenous source of ROS. Over time, all ocular structures, from the tear film to the retina, undergo oxidative stress, and therefore, the antioxidant defenses of each tissue assume the role of a safeguard against degenerative ocular pathologies. The ocular surface and cornea protect the other ocular tissues and are significantly exposed to oxidative stress of environmental origin. Overwhelming of antioxidant defenses in these tissues clinically manifests as pathologies including pterygium, corneal dystrophies, and endothelial Fuch's dystrophy. The crystalline lens is highly susceptible to oxidative damage in aging because its cells and their intracellular proteins are not turned over or replaced, thus providing the basis for cataractogenesis. The trabecular meshwork, which is the anterior chamber tissue devoted to aqueous humor drainage, has a particular susceptibility to mitochondrial oxidative injury that affects its endothelium and leads to an intraocular pressure increase that marks the beginning of glaucoma. Photo-oxidative stress can cause acute or chronic retinal damage. The pathogenesis of age-related macular degeneration involves oxidative stress and death of the retinal pigment epithelium followed by death of the overlying photoreceptors. Accordingly, converging evidence indicates that mutagenic mechanisms of environmental and endogenous sources play a fundamental pathogenic role in degenerative eye diseases.     

Hydrocaffeic and p-coumaric acids,natural phenolic compounds,inhibit UV-B damage in WKD human conjunctival cells in vitro and rabbit eye in vivo

This paper studied the effect on UV-B ocular damage of 10µm hydrocaffeic acid (HCAF) alone and as a mixture (MIX) (5µm HCAF+5µm p-coumaric acid). Since ocular UV-B damage is mediated by reactive oxygen species, the aim was to test if HCAF and MIX could reduce oxidation damage in human conjunctival cells (WKD) in vitro and in cornea and sclera of rabbits in vivo. After UVB irradiation (44 J/m²) of WKD cells, 8-oxodG levels in DNA were markedly increased and this effect was attenuated by HCAF and MIX. Rabbit eyes were treated by application of HCAF and MIX drops before UV-B exposure (79 J/m²). Corneal and scleral DNA oxidation damage, xanthine-oxidase (XO) activity and malondialdehyde levels (MDA) in corneal tissue and prostaglandin E₂ (PGE₂) in the aqueous humour were reduced by HCAF alone and in combination with p-coumaric acid, showing their potential as a topical treatment against UV-B damage.     

HPM长期辐照的眼生物效应研究

目的：研究高功率微波（HPM）在不同平均功率和不同重复频率条件下长期多次照射对眼组织结构的生物效应,为我国HPM安全防护提供生物学依据。方法：采用自行研制的HPM效应模拟源远场平面波（峰值功率密度50W／cm^2）分3个不同平均功率水平,每天6min持续照射1个月,并在照后5个时间点通过眼底镜、裂隙灯观察、组织病理学方法等研究HPM长期照射对动物眼重要部位结构的生物效应。结果：HPM照射后眼角膜、晶状体、眼底等组织结构都出现了不同程度的病理变化,并呈现出一定的时效和量效关系;其中角膜病变依剂量不同可分别于照后2月到照后6月恢复正常,而晶状体病变在观察期内（照后6月）仍未见恢复,照射后动物眼底动静脉和毛细血管稍有扩张充血,但未见瘢痕、裂隙、出血等表现。结论：实验所用剂量范围内的HPM重复照射可以对动物角膜、晶状体、玻璃体等部位造成一定程度的生物效应,并呈现出一定的量效关系;在实验的观察期内,角膜和眼底依照射剂量不同可分别于照后不同期间恢复正常,但晶状体混浊在观察期末仍未见恢复,能否发展成微波白内障尚需观察。     

Retinal dysplasia and progressive atrophy in dogs irradiated during ocular development

Beagle dogs were given a single, whole-body gamma-radiation exposure at various stages during ocular development and were evaluated for the presence of ocular lesions. Dogs were exposed during middle or late pregnancy at 28 or 55 days postcoitus (dpc) or as neonates at 2 days postpartum (dpp). Mean whole-body and ocular doses ranged from 1.0 to 3.8 Gy. Dogs were sacrificed and ocular lesions were evaluated at 70 days, 2 years, or 4 years of age. Retinal dysplasias and atrophy were the most striking lesions related to radiation exposure. These lesions were bilateral and focal to diffuse in nature, and they increased in severity with increasing radiation dose. The stage of development at irradiation had a marked effect on the distribution of retinal lesions, with the most severe changes being present in that portion of the retina undergoing differentiation at the time of the insult. In dogs sacrificed at 70 days of age the lesions were primarily dysplasias consisting of ectopic nuclear aggregates in the photoreceptor layer, retinal folds, and retinal rosettes. With increasing age (up to 4 years), there appeared to be progression of the extent of the clinically evident lesions, and there was a change in the nature of the lesions from dysplasia to atrophy. This was accompanied by marked attenuation of the retinal vasculature.     

Homeostatic expansion of CD4(+) T cells upregulates VLA-4 and exacerbates HSV-induced corneal immunopathology

Using a viral-induced immunopathology model, we showed that when CD4(+) T cells were allowed to undergo homeostatic expansion prior to ocular herpes simplex virus infection, mice developed more severe inflammatory lesions with the increased severity associated with enhanced effector function of ocular CD4(+) T cells, and blocking their functional activity reduced the lesion severity. Additionally, homeostatically expanded CD4(+) T cells upregulated VLA-4, and in vivo administration of anti-VLA-4 mAb significantly decreased the homeostatic proliferation. Furthermore, blocking of VLA-4 interaction also diminished the infiltration of CD4(+) T cells into the cornea and decreased lesion severity. Our results imply that homeostatic expansion of T cells, as could occur in a virus-induced lymphopenia, may generate cells with enhanced effector function that can contribute to tissue damage.     

Uncovering the beginning of diabetes: the cellular redox status and oxidative stress as starting players in hyperglycemic damage

Early hyperglycemic insult can lead to permanent, cumulative damage that might be one of the earliest causes for a pre-diabetic situation. Despite this, the early phases of hyperglycemic exposure have been poorly studied. We have previously demonstrated that mitochondrial injury takes place early on upon hyperglycemic exposure. In this work, we demonstrate that just 1 h of hyperglycemic exposure is sufficient to induce increased mitochondrial membrane potential and generation. This is accompanied (and probably caused) by a decrease in the cells’ NAD⁺/NADH ratio. Furthermore, we show that the modulation of the activity of parallel pathways to glycolysis can alter the effects of hyperglycemic exposure. Activation of the pentose phosphate pathway leads to diminished effects of glucose on the above parameters, either by removing glucose from glycolysis or by NADPH generation. We also demonstrate that the hexosamine pathway inhibition also leads to a decreased effect of excess glucose. So, this work demonstrates the need for increased focus of study on the reductive status of the cell as one of the most important hallmarks of initial hyperglycemic damage.     

Ocular Signs Correlate Well with Disease Severity and Genotype in Fabry Disease

Ocular signs in Fabry disease have generally been regarded to be primarily of diagnostic value. We explored whether ocular findings, alone or in particular in combination with the α-galactosidase A gene mutation, have predictive value for disease severity. Data from the Fabry Outcome Survey (FOS), a large, global database sponsored by Shire, were selected for adult patients who had undergone ophthalmological examination. Three ocular signs were assessed: cornea verticillata, tortuous conjunctival and/or retinal vessels, and cataract. Fabry disease severity was measured using FOS Mainz Severity Score Index and modifications thereof. Ophthalmological data were available for 1203 (699 female, 504 male) adult patients with eye findings characteristic of Fabry disease in 55.1%. Cornea verticillata had a similar distribution in women (51.1%) and men (50.8%), whereas tortuous vessels and Fabry cataract were somewhat more frequent in men than in women. Patients with cornea verticillata, selected as the principal ocular sign for this study, had more severe disease (median score, 20.0) versus those without ocular signs (11.0; P<0.001). This finding could be confirmed by applying age adjusted severity scores. Moreover, the prevalence of cornea verticillata was significantly higher in patients with null (male, 76.9%; female, 64.5%) and missense (male, 79.2%; female, 67.4%) mutations versus mild missense (male, 17.1%; female, 23.1%) and the p.N215S (male, 15.0%; female, 15.6%) mutations (P<0.01). Our analyses show a correlation between the prevalence of ocular changes in Fabry disease and disease severity. Consequently, information on ocular findings and α-galactosidase A gene mutation may help assess the risk for more severe Fabry disease. These observed findings are of notable clinical importance, as Fabry disease is characterized by high clinical course variability and only weak genotype-phenotype correlation at the individual patient level. Further confirmatory studies are needed.