Glutathione attenuates nitric oxide-induced retinal lipid and protein changes.

BACKGROUND: Elevated levels of nitric oxide (NO(•) ), a pro-oxidant that has been associated with numerous retinal diseases, have been implicated in experimental glaucoma models. This study investigated the oxidative effects of sodium nitroprusside (SNP), a nitric oxide donor, on the retinal lipids and proteins and evaluated the potential protective effects of glutathione (GSH). METHODS: Porcine retinal homogenates were incubated with 0, 1, 2, 3, 4 and 5 µm SNP. Malondialdehyde (MDA) levels were assayed spectrophotometrically to quantify lipid peroxidation. Differential protein expressions of 3 µm SNP-treated retinal homogenates were compared with controls after the conduction of two-dimensional gel electrophoresis. Mass spectrometric data was used to identify proteins in NCBInr database. Furthermore, GSH was co-incubated with 3 µm SNP-treated retinal homogenates. MDA levels and protein expressions were compared with SNP-treated controls. RESULTS: SNP significantly increased retinal-MDA levels (p = 0.0002). 2-D gel electrophoresis images displayed a significant change in 13 protein spot expressions (p < 0.05). GSH suppressed SNP-induced MDA elevation (p < 0.0001) and selected protein changes (p < 0.05). SNP down-regulated paraoxonase/arylesterase 2 precursor (PON2), ß-actin and ß-tubulin; however, these effects were prevented by a co-incubation with GSH, as confirmed by Western blots. CONCLUSIONS: Nitric oxide induced lipid and protein changes in retinal tissues. The effects were partially reversed by co-incubation with GSH. Data from this study suggests that nitric oxide-induced retinal oxidative stress induces specific molecular changes. This may enable us to better understand the pathogenesis of glaucoma. Further studies are indicated to explore potential pharmacological applications of GSH in nitric oxide-related retinal diseases.

Clinical device-related article evaluation of morphology and functions of a foldable capsular vitreous body in the rabbit eye.

We previously proposed a new strategy to replace a vitreous body with a novel foldable capsular vitreous body (FCVB). In this study, the FCVB was designed to mimic natural vitreous morphology, and evaluate its physiological functions compared with traditional silicone oil substitutes, in an established rabbit model of proliferative vitreoretinopathy. We found that FCVB was a very good replacement for closely mimicking the morphology and restoring the physiological functions, such as the support, refraction, and cellular barriers, of the rabbit vitreous body. The study has provided us with a novel research and therapy strategy that could effectively mimic the morphology and physiological function of the rabbit vitreous body.

Porcine global flash multifocal electroretinogram: possible mechanisms for the glaucomatous changes in contrast response function.

PURPOSE: The aim of this study was to obtain a better understanding of the cellular contributions to the porcine global flash mfERG by using a pharmacologic dissection method, together with the method using variation of stimulus contrast which has been used to demonstrate mfERG changes in human glaucoma. METHODS: Global flash mfERGs with different stimulus-contrast settings (99%, 65%, 49% or 29%) were recorded from 14 eyes of ten 6-week-old Yorkshire pigs in control conditions and after suppression of inner retinal responses with inhalation of isoflurance (ISO), and injections of tetrodotoxin (TTX) and N-methyl-d-aspartic acid (NMDA). ON- and OFF-pathway responses were isolated by injection of 2-amino-4-phosphonobutyric acid (APB) and cis-2,3-piperidinedicarboylic acid (PDA). RESULTS: The porcine global flash mfERG consisted of an early direct component (DC) and a late induced component (IC). ISO and TTX removed inner retinal contributions to the IC; NMDA application further abolished the oscillatory wavelets in the DC and removed the residual IC waveform. The inner retina contributed regular oscillation-like wavelets (W1, W2 and W3) to the DC and shaped the IC. After removing the inner retinal contributions, the porcine global flash mfERG waveform becomes comparable to that obtained with conventional mfERG stimulation. The remaining waveform (smoothed DC) was mainly contributed by the ON- and OFF-bipolar cells as revealed after APB or PDA injection. Photoreceptors contributed a small signal to the leading edge of N1. The characteristic of contrast response function of DC was demonstrated to be contributed by the inner retinal oscillation-like wavelets. CONCLUSION: We believe that the DC of the porcine global flash mfERG is mainly composed of contributions from photoreceptors, and ON- and OFF-bipolar cells, where inner retinal activity partially shaped the DC with superimposed regular wavelets. However, the IC is dominated by inner retinal activity. The contrast response functions of DC consisted of both outer retinal response and oscillation-like wavelets of the inner retinal response. Both contain different characteristics during contrast modulation of the stimulus, where the changes of W2 of the inner retinal response seem independent of contrast modulation. The DC contrast response feature depends mainly on the relative contribution of inner retinal activities; the loss of inner retinal cells may alter the DC contrast response function, making it tend toward linearity.

Glutamate-induced retinal lipid and protein damage: the protective effects of catechin.

Glutamate toxicity has been implicated in various retinal diseases. Green tea leaf extract catechin has protective effects against cellular toxicity. This study investigated the effects of catechin on the glutamate-treated retina. Porcine retinal homogenates were incubated with glutamate (20 nmol) at 37 degrees C for 60 min. Catechin was co-incubated with the glutamate-treated retina in the same condition. The malondialdehyde (MDA) levels were determined as an index of lipid peroxidation (LPO). Differential protein expressions were derived from two-dimensional gel electrophoresis. Mass spectrometry was conducted to identify the proteins. Glutamate increased the retinal MDA (p<0.0001) and catechin reversed the effect (p<0.0001). There were significant changes in seven proteins after the glutamate treatment (p<0.05), namely, heterogeneous ribonucleoprotein, thioredoxin peroxidase, 5-hydroxytryptamine receptor, pyruvate dehydrogenase, ARHA protein, peroxiredoxin 6 and proteasome. Catechin significantly reversed the changes in thioredoxin peroxidase, 5-hydroxytryptamine receptor, peroxiredoxin 6 and pyruvate dehydrogenase (p<0.05). Our study shows that (a) retinal glutamate toxicity is mediated by LPO and protein modification, and (b) catechin ameliorates the toxicity.

Pharmacologically defined components of the normal porcine multifocal ERG.

Multifocal electroretinograms (mfERG) from isoflurane anesthetized pigs were recorded and sequential application of TTX, NMDA, APB and PDA were used to identify contributions to the mfERG from inner retinal neurons, ON-pathway, OFF-pathway and photoreceptors. The cellular origins of the first-order kernel (K1) and the first slice of the second-order kernel (K2.1) porcine mfERG are contributed from both inner and outer retina. For the K1 waveform, the n1 involved responses of cone photoreceptors and OFF-bipolar cells. The leading edge of p1 is dominated by ON-bipolar cell depolarization. The rear edge of p1, n2 and p2 are dominated by ON-bipolar activities and shaped by the activities of OFF-bipolar cells and retinal cells with NMDAr and voltage-gated sodium channels other than ganglion cells. The p3 is mainly inner retinal activities. For the K2.1 waveform, the p1 and n1 are the summation of activities of ON-, OFF-bipolar cells and retinal cells rich in NMDAr and voltage-gated sodium channels other than ganglion cells. The p2 seems to be related to the ganglion cells. Better understanding of the cellular origins of the normal porcine mfERG will be useful for comparing and defining the functional changes that may occur in diseased retinas.

Pinoline and N-acetylserotonin reduce glutamate-induced lipid peroxidation in retinal homogenates.

Glutamate is a neurotransmitter associated with oxidative retinal disorders. Pinoline (PIN) and N-acetylserotonin (NAS) are newly identified neural protectors. We investigated the glutamate-induced lipid peroxidation (LPO) and the protective effects of PIN and NAS in the retina. Porcine retinal homogenates were treated with different concentrations of glutamate. The malondialdehyde (MDA) level per unit weight of protein was quantified spectro-photometrically as an index of LPO. The glutamate concentration that induced a significant increase in retinal MDA was determined. The glutamate-treated retinal homogenate was then co-incubated with 5 different concentrations (0, 35.7, 71.5, 143 and 286 microM) of PIN, NAS or their combinations (concentration corresponding to 25, 50 and 75% of protection). Glutamate induced a significant dose-dependent increase in retinal MDA (p<0.0001). Co-incubation with PIN or NAS significantly suppressed the glutamate-induced MDA (p<0.01) in a dose-dependent manner (p<0.0001). The concentrations to inhibit 50% of LPO were 132.8 and 98.6 microM for PIN and NAS, respectively. In summary, elevated glutamate induced retinal LPO. Both PIN and NAS suppressed the glutamate-induced LPO and a synergic protection was evident after incubation in PIN/NAS mixtures.

Melatonin and lipid uptake by murine fibroblasts: clinical implications.

The current study was undertaken to uncover the role of melatonin in lipid metabolism in the murine fibroblasts. The results show melatonin in vitro enhances lipid accumulation and lipid droplet formation in this cell line. Using oil red O staining, it was found that when oleic acid was present in the culture media, melatonin at doses of 0.1-2mM, significantly increased the lipid concentrations in the cells. However, low levels of melatonin, with or without oleic acid, did not influence lipid metabolism in the cultured fibroblasts. When a non-specific melatonin receptor antagonist, luzindole 10 microM was co-incubated with 1mM melatonin, the stimulatory effects of melatonin on lipid accumulation in these cells was significantly reduced. It appears that the effects of melatonin on lipid metabolism in murine fibroblasts is mediated by melatonin membrane receptors.

Protective effects of melatonin in experimental free radical-related ocular diseases.

Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine with a range of antioxidative properties. Melatonin is endogenously produced in the eye and in other organs. Current evidence suggests that melatonin may act as a protective agent in ocular conditions such as photo-keratitis, cataract, glaucoma, retinopathy of prematurity and ischemia/reperfusion injury. These diseases are sight-threatening and they currently remain, for the most part, untreatable. The pathogenesis of these conditions is not entirely clear but oxidative stress has been proposed as one of the causative factors. Elevated levels of various reactive oxygen and nitrogen species have been identified in diseased ocular structures. These reactants damage the structure and deplete the eye of natural defense systems, such as the antioxidant, reduced glutathione, and the antioxidant enzyme superoxide dismutase. Oxidative damage in the eye leads to apoptotic degeneration of retinal neurons and fluid accumulation. Retinal degeneration decreases visual sensitivity and even a small change in the fluid content of the cornea and crystalline lens is sufficient to disrupt ocular transparency. In the eye, melatonin is produced in the retina and in the ciliary body. Continuous regeneration of melatonin in the eye offers a frontier antioxidative defense for both the anterior and posterior eye. However, melatonin production is minimal in newborns and its production gradually wanes in aging individuals as indicated by the large drop in circulating blood concentrations of the indoleamine. These individuals are possibly at risk of contracting degenerative eye diseases that are free radical-based. Supplementation with melatonin, a potent antioxidant, in especially the aged population should be considered as a prophylaxis to preserve visual functions. It may benefit many individuals worldwide, especially in countries where access to medical facilities is limited.

Effects of vitamin E and pinoline on retinal lipid peroxidation.

BACKGROUND: Pinoline is a pineal indoleamine naturally found in the retina. This study compared the effects of pinoline and vitamin E on the copper (I)-induced retinal lipid peroxidation (LPO). METHODS: Porcine retinal homogenates were mixed with 120 micro M copper (I) solution. The mixtures were co-incubated with various concentrations of pinoline or trolox (water-soluble vitamin E analogue) at 37 degrees Centigrade for 60 minutes. The amounts of malondialdehyde (MDA) and protein were assayed to quantify the LPO. RESULTS: Copper (I) ions significantly increased the MDA concentration in the retinal homogenates (p < 0.0007). Both pinoline and trolox significantly suppressed MDA in a dose-dependent manner (p < 0.0001) and their effects were significantly different (p = 0.004). The concentrations that inhibited 50 per cent of LPO were 0.24 mM and 0.68 mM for pinoline and trolox, respectively. DISCUSSION: Pinoline suppressed the LPO at a potency of 2.8 times compared with trolox. The results support an anti-oxidative role for pinoline in the retina. Further study is required to characterise the pharmacological potency of pinoline in vivo.

Production of hypoxia-induced corneal edema in aged eyes.

PURPOSE: Corneal thickness assessment is a common clinical procedure applied in corneal and contact lens care. This study aims to investigate the effect of age on hypoxia-induced corneal swelling. METHODS: Eighteen male subjects were equally divided into the younger [(23.7 +/- 0.8) years old] and older [(74.4 +/- 2.5) years old] groups. Each subject wore a thick soft contact lens (uniform thickness of 0.3 mm) on the left cornea. With the contact lens in place, the baseline central corneal thickness was measured using a specially designed photo-pachometer. The lens was then patched behind the closed eyelids, producing an extremely hypoxic stress to the cornea. The change in central corneal thickness was monitored every 20 minutes with momentary disruptions to the hypoxic stress over the next 2 hours. The increase in thickness was taken as an index of corneal edema. The rate of change in corneal thickness, as derived from a non-linear mathematical model, was compared between groups. RESULTS: The corneal thickness of both age groups increased significantly with time (P < 0.0001). The mean corneal swelling constant for the older subjects was 16.5 x 10(-3) (S.E.M. = 2.65 x 10(-3)) and the value for the younger subjects was 46.5 x 10(-3) (S.E.M. = 3.25 x 10(-3)). The difference was statistically significant (P < 0.0001). CONCLUSION: Aging cornea has a slower hypoxia-induced edema response compared with the younger group. Whether it is caused by a decreased corneal lactate production or an increased resistance to physical expansion deserves further investigation.

Effect of optical defocus on multifocal ERG responses.

BACKGROUND: Multifocal electroretinography (mfERG) is a sensitive technique to probe retinal function topographically. Various conditions such as macular degeneration decrease the first-order kernel (K1) response magnitude of mfERG. Previous studies have given inconsistent results on the effect of optical defocus due to poor controls. This study investigated the effect of optical defocus on the K1 response of the central retina using a well-controlled method. METHODS: Twenty subjects were recruited to undergo mfERG measurement using the VERIS 4.0 system. A four millimetre artificial pupil was fitted before each fully-dilated right eye, optically corrected for the viewing distance. The implicit times and response amplitudes of n1 (first negative trough) and p1 (first positive peak) under three different optical defocus conditions (zero dioptres, +1.00 D and +3.00 D) were measured. RESULTS: The implicit times of n1 and p1 did not demonstrate any significant variation from the central macula to para-macula under different optical defocus conditions. The response amplitude of n1 did not show any changes from the central macula to para-macula but the response amplitude of the central macular p1 showed a significant reduction by 12 per cent under +1.00 D defocus and +3.00 D defocus. CONCLUSIONS: Optical defocus causes mild reduction in mfERG at the central macula but there are no significant changes in the periphery. A full optical correction is recommended for mfERG measurement to minimise the reduction of the macular response due to optical defocus.

Effects of pupil diameter on light detection and temporal modulation.

PURPOSE: This study compared the effects of pupil variation on light detection and temporal modulation across the central visual field. METHODS: Light detection sensitivity (LDS) and low flickering frequency (6 Hz) temporal modulation sensitivity (TMS) of 20 young subjects were measured from the central visual field of the right eyes using an autumated perimeter (Medmont M600). The measurements were taken under 3 artificial pupils, i.e. 3 mm, 4.3 mm and 6 mm diameters. The sensitivities were grouped and averaged for different retinal eccentricities (3 degrees, 6 degrees, 10 degrees, and 15 degrees). RESULTS: TMS and LDS were reduced with increasing retinal eccentricities (P < 0.001) and decreasing pupil diameters (P < 0.001). TMS collected from all pupil diameters were significantly different from each other (P < 0.001). Similarly, LDS under 3 mm pupil was statistically different from those of 4.3 mm and 6 mm (P < 0.003). Comparison of the hills of vision showed that pupil variation resulted in significantly different slopes (P = 0.001). The slopes were also found to be significantly different between TMS and LDS (P = 0.012). CONCLUSIONS: The data showed that dilated pupil resulted in significantly higher sensitivities than those of smaller pupil for both visual functions. The difference in the slopes of hills of vision also suggested that the variation in retinal illumination affected the visual responses differently at various retinal eccentricities for TMS and LDS.

The performance of color deficient individuals on airfield color tasks.

BACKGROUND: The pseudo-isochromatic plate (PIP) test (e.g., Ishihara test) is the clinical test commonly used to assess color vision. Upon failure of this test, candidates are typically reassessed using the Farnsworth Lantern (FALANT) test to determine their fitness for occupations which require normal color vision. We were interested in determining to what extent clinical tests can predict real life color naming performance, particularly in the context of "airside drivers" (any airport vehicle operators who drive on the airfield). METHODS: There were 24 male subjects with a color vision deficiency, as defined by the Ishihara test, who participated in this study. They were further assessed using the D-15 and lantern color vision tests. All subjects then participated in two separate color naming tasks. These tasks consisted of naming surface colors and colored-lights of the type used on the airfield of the Hong Kong International Airport (HKIA). RESULTS: Of the 24 subjects, 15 failed both D-15 and the FALANT tests. Out of these 15 subjects, 8 also failed the naming tasks. The FALANT test showed very good agreement (87.5%) with the Ishihara test. Similar to the Ishihara, FALANT tests had 100% sensitivity in identifying the subjects who failed the naming tasks. The agreement between the Ishihara and D-15 tests was 62.5%. DISCUSSION: In common with previous studies, our results show that clinical tests cannot predict accurately who will fail color naming tasks of the type normally encountered in the real-life work environment. The high false positive values of the clinical tests in relation to color naming tasks suggest that people with color deficiency may not be given a fair opportunity to demonstrate their true ability in performing the task.

Nitric oxide and hydroxyl radical-induced retinal lipid peroxidation in vitro.

BACKGROUND: Free radicals can cause cellular oxidation and consequent membrane lipid peroxidation (LPO). The significance of these effects on retinal tissue is unclear. This study compared the retinal LPO products, after the incubation with nitric oxide or hydroxyl radical, with those of two commonly studied tissues-kidney and liver. METHODS: Retina, liver and kidney were obtained from Sprague-Dawley rats. These samples were homogenised and incubated with variously 2, 20 or 200 mM iron (II) or sodium nitroprusside (SNP) solution for 60 minutes at 37 degrees C. The amounts of malondialdehyde (MDA) were measured and the concentrations per unit weight protein provided an index of LPO. RESULTS: After the iron (II) and SNP treatments, MDA levels were significantly different among the three tissues (p < 0.0001) in a dose-dependent manner (p < 0.0001). The retinal MDA level was significantly higher than those of the kidney (p < 0.001) and the liver (p < 0.001). For the iron (II)-treated homogenates, the differences in MDA were statistically significant in the retina (p = 0.0001) and the liver (p = 0.0004). For the SNP-treated homogenates, the differences in MDA were all statistically significant in the retina (p = 0.002), the liver (p < 0.0001) and the kidney (p < 0.0001). There was no statistical difference in retinal MDA concentrations between iron (II) and SNP treatments (p = 0.41). DISCUSSION: Retinal tissue is several times more susceptible to free radical-induced LPO than liver and kidney tissue. The retinal responses to SNP and iron (II) treatments were comparable, suggesting that NO(*)- and (*)OH-induced LPO damage shared similar mechanism in vitro. Future work is required to identify the protective system in living retina.

Total retinal nitric oxide production is increased in intraocular pressure-elevated rats.

Nitric oxide (NO) is a well-known vaso-dilator but its regulation in the retina is unclear. This study was conducted to quantify total NO production and retinal ganglion cell (RGC) loss in an experimental glaucoma model. Three quarters of the peri-limbal/episcleral drainage vessels and anterior angle (right eyes) of Sprague-Dawley rats were thermally blocked using laser irradiation, while the left eyes served as controls. We measured the intraocular pressure (IOP) of both eyes using a digital tonometer (Tonopen) 21, 28 and 35 days after the laser treatment. After 35 days, we determined the total NO level in retinas and remaining ocular tissues for the laser-treated and control eyes using a spectro-photometric assay. The viable RGC numbers were also determined by counting the cell bodies stained retrogradely by fluoro-gold. The laser treatment significantly increased the IOP 2.0-2.6x throughout the whole periodof measurements (P<0.0001). The mean total RGC number decreased significantly from 98725+/-5383 (+/-S.E. (M.)) to 69276+/-5592, or 29.8% reduction, in the laser-treated eyes after 35 days (P=0.008). The mean total NO level in the laser-treated retina was significantly increased by 2.4x compared with controls (P=0.016), but no significant difference was found in the eyecups (P>0.05). Laser treatment resulted in significant IOP elevation and RGC loss, suggesting that thermal coagulation of the perilimbal region may provide an alternate protocol for glaucoma study. NO level was increased by two-fold in the retina but not in other ocular tissues. Since NO is capable of producing powerful peroxynitrite anions and hydroxyl radicals, elevated level of NO has a potential role in glaucoma.

Addition lens alleviates reading-induced ocular stress.

BACKGROUND: Near tasks have been associated with binocular stress to induce myopia. The aim of this study was to investigate the effects of accommodation on reading-induced near heterophoria. METHODS: We measured the near heterophoria of 22 young adults before and after 30 minutes of reading. The reading task comprised a column of local English newsletter studied monocularly at 33 cm. One of three addition lenses (that is, 0.00 D, +1.50 D and +3.00 D) was randomly incorporated into the optical prescription. The difference in near heterophoria between the pre- and post-reading task was recorded. The experiment was completed on separate days for the other lens powers. RESULTS: Reading for 30 minutes with a plano lens addition (control) increased the near heterophoria by 3.81 +/- 0.95 prism dioptres (SEM) toward exo-deviation (p < 0.002). Addition of a +3.00 D lens significantly decreased the reading-induced exophoric shift to 1.36 +/- 0.55 prism dioptres (SEM). Similarly, a +1.50 D lens reduced the exophoric shift to 3.14 +/- 0.85 prism dioptres (SEM) but the difference was not statistically significant when compared with the control. CONCLUSIONS: The results showed that close work might cause eye strain via the extraocular muscles. Incorporation of plus lens into the optical correction caused a power-dependent reduction in the stress, that is, smaller exophoric shift. Whether binocular stress contributes to myopia and its response to addition lens therapy deserve further investigation.