Andrew W. Siu

The efficacy of vitamin E and melatonin as antioxidants against lipid peroxidation in rat retinal homogenates.

Abstract: Free radical‐induced oxidation can cause severe cell damage in biological systems. Melatonin, a pineal secretory product, is a recently identified antioxidant that protects cells from the damaging effects of free radicals. We compared the effect of melatonin and vitamin E, another antioxidant, against lipid peroxidation (LPO) in rat retinal homogenates. The aim was to characterize the antioxidative efficacy of melatonin in retina, a tissue highly susceptible to oxidative damage. The LPO product, malondialdehyde (MDA), was determined to provide an index of cell damage in vitro. After the incubation with iron(II) ions, the free radical scavenging effectiveness of four different concentrations (i.e., 0.5, 1.0, 2.0, and 4.0 mM) of vitamin E and melatonin were determined by comparing the final levels of MDA. Lipid peroxidation product levels were significantly reduced in a dose‐response manner by all concentrations of vitamin E. Melatonin, in concentrations of either 2.0 or 4.0 mM, also significantly reduced LPO. Statistical analysis of the data showed that vitamin E treatment always yielded a lower level of LPO products than did the same concentration of melatonin. The concentrations of each agent required to inhibit 50% of the lipid damage (IC50) were 0.69 mM and 4.98 mM for vitamin E and melatonin, respectively. Both vitamin E and melatonin protect the retina against LPO in a dose‐dependent manner. Although the IC50 value for melatonin is about 7.2 times higher than that of vitamin E, melatonins pharmacological and physiological role in the treatment and/or prevention of certain retinal diseases in vivo should be further investigated.

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.

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.

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.

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.

Effects of melatonin on the nitric oxide treated retina

Aims: Nitric oxide (NO) is a free radical which reportedly causes damage to living cells. This study evaluated the damaging effect of NO and the protection of melatonin on the retina in vivo. Methods: Female Wistar rats (230–250 g) received two intraperitoneal injections of either melatonin (5 mg/kg) or vehicle alone. After general anaesthesia, the animals received 1 μl intravitreal injections of 0.9% saline and 1 mM sodium nitroprusside (SNP) into the right eye and the left eye, respectively. The animals were divided into two groups and then sacrificed after 24 hours (day 1) and 96 hours (day 4). The mean inner retinal layer thickness (mIRLT), the number of retinas expressing hyperchromatic (HC) nuclei in the inner nuclear layer (INL) and the apoptotic ganglion cell detection were compared. Results: After 1 day, SNP significantly increased the mIRLT by 45% (p = 0.004), initiated more INL nuclear HC expression (p = 0.01) and apoptotic nuclei (p<0.05) compared with the control eyes. Injection of melatonin ameliorated these changes. On day 4, SNP demonstrated similar effects in all parameters on the retina. After the injection of melatonin, both INL HC expression and apoptotic ganglion nuclei in the SNP treated eyes were similar to the controls but the mIRLT was significantly greater than in controls (p = 0.006). Conclusion: Uncontrolled NO elevation caused morphological and nuclear changes in the retina. Melatonin significantly suppressed the NO induced increase in mIRLT, INL HC expression, and apoptotic ganglion cells on day 1, but not after day 4. Melatonin may have a protective role in the NO elevated retina.

The effect of light scattering on multifocal electroretinography

Purpose: Unclear ocular media is a very common condition of older eyes characterized by significant light scattering and image degradation. The multifocal electroretinography (MERG) is a useful objective technique to measure retinal activity but its validity in the presence of cloudy ocular media remains unclear. We tested the MERG under controlled light scattering conditions using a liquid crystal diffuser (LCD) that simulated different degrees of image degradation.

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 rnacular 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 wellcontrolled method.

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.

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.