Electrophysiological consequences of leukotrienes applied on isolated rat retina

Scotopic vision is the result of a cascade of light-dependent biochemical events in rod outer segments (ROS) involving mainly a cGMP-modulation of sodium current. This modification of ionic currents induces changes of membrane potential which generates electroretinographic (ERG) waves. As (i) ERG disturbances are commonly recorded in hypoxic and inflammatory retinal diseases (ii) leukotrienes (LTs), a very potent mediators of inflammation, disturb ionic exchanges in several artificial or natural membrane systems, we undertook the investigation of the effects of LTs on ERG record in mammalian isolated retina. LTB4, LTC4 and LTD4, all induced a dose-dependent marked reduction of the b wave amplitude of ERG. This effect is correlated with a significant decrease in the survival time of the retina. The analysis of the modification of ERG indicates that LTs exhibit a real toxic effect since b wave is mainly affected while P III wave is unchanged. Comparatively with other nervous cells, this phenomenon may be attributed to an increase in Na+ permeability of ROS. It is suggested that LTs may be involved in the development of inflammatory or ischemic retinal diseases.     

Circadian rhythm of active amino acid transport in rat liver

Abstract

The circadian change of the encephalic photosensitivity of quail has been demonstrated. In this study the diurnal variation of the retinal photosensitivity was investigated by the electroretinogram (ERG) to explore the phase relation between the retinal and encephalic systems. The b‐wave, a major component of the ERG, was used as a measure of retinal photosensitivity.

1. In the bird maintained in LD12:12 the b‐wave amplitude of the ERG stayed at a high level for the first 10 h of the light period, and decreased abruptly around the time of light‐off. The decreased level continued until midnight. Thenceforth the b‐wave amplitude recovered progressively to the daytime level before the time of next light on.

2. In the bird maintained in LD16:8, the decrease of the retinal sensitivity in the light phase was initiated prior to the time of expected light‐off, and the onset time of decreasing tendency was advanced by 2 h, with dark adaptation for 30 min given immediately before the ERG measurement. Upon continuous light exposure, the b‐wave amplitude always remained at low level.

3. Periodic changes in retinal sensitivity persisted when the environmental dark phase was temporarily extended for 19 to 30 h.

These observations suggest that the diurnal rhythm of the neural retina in quail might be generated endogenously and appears to control the encephalic photosensitive system.     

Sodium Iodate Selectively Injuries the Posterior Pole of the Retina in a Dose-Dependent Manner: Morphological and Electrophysiological Study

Sequential morphological and functional features of retinal damage in mice exposed to different doses (40 vs. 20 mg/kg) of sodium iodate (NaIO₃) were analyzed. Retinal morphology, apoptosis (TUNEL assay), and function (electroretinography; ERG) were examined at several time points after NaIO₃ administration. The higher dose of NaIO₃ caused progressive degeneration of the whole retinal area and total suppression of scotopic and photopic ERG. In contrast, the lower dose induced much less severe degeneration in peripheral part of retina along with a moderate decline of b- and a-wave amplitudes in ERG, corroborating the presence of regions within retina that retain their function. The peak of photoreceptor apoptosis was found on the 3rd day, but the lower dose induced more intense reaction within the central retina than in its peripheral region. In conclusion, these results indicate that peripheral area of the retina reveals better resistance to NaIO₃ injury than its central part.     

Electrodichroism of Purple Membrane: Ionic Strength Dependence

The dichroism of purple membrane suspension was measured in dc and ac electric fields. From these measurements three parameters can be obtained: the permanent dipole moment, μ, the electrical polarizability, α, and the retinal angle, δ, (relative to the membrane normal). The functional dependence of the dichroism on the electric field is analyzed. There is a small decrease (~2°) in retinal angle going from dark adapted to the light adapted form. No measurable difference in μ, α, and δ was found under the photocycle. The dichroism was measured in two different salt solutions (KCl and CaCl₂) in the range 0-10 mM. The retinal angle increases from 64° to 68° with increasing ionic strength going through a minimum. This is attributed to the changing (decreasing) inner electric field in the membrane. The polarizability, α, consists of two parts. One component is related to the polarization of the purple membrane and the second component to the ionic cloud. The second component decreases with ion concentration approximately as κ^-3 (κ is the Debye parameter) in agreement with a model calculation for the polarization of the ionic cloud. The origin of the slightly ionic strength dependent permanent dipole moment is not well understood.     

Electroretinographic detection of human brain dopamine response to oral food stimulation

Objective:

The activity of dopamine‐dependent retinal signaling can be assessed using electroretinography. Response of this system to oral food stimulation might provide accessible insight into the brain dopamine response to oral stimuli as retinal dopamine concentration is dependent upon mid brain dopamine concentration was postulated.

Design and Methods:

Nine individuals had cone ERG (b wave) response to oral food stimulation and oral methylphenidate (MPH) administration measured on separate days, and completed self reported eating behavior questionnaires.

Results:

Significant and similar increases in b wave response to both stimuli (P = 0.012 and P = 0.042, MPH and food, respectively) and significant correlations of the food stimulated b wave amplitude with binge eating related behavior as measured by the Gormally Binge Eating Scale (r = 0.68, P = 0.044) and self‐reported trait hunger as measured by the Stunkard and Messick Three Factor Eating Questionnaire (r = 0.67, P = 0.048) were found.

Conclusion:

The significant increase in food stimulated dopamine dependent b wave amplitude and correlation with methylphenidate stimulated b wave amplitude suggest that ERG may offer a relatively inexpensive and accessible methodology for potentially assess dopaminergic responses to food and other externally applied stimuli that have been implicated in the pathogenesis of a number of human diseases.     

P2X<Subscript>7</Subscript>R modulation of visually evoked synaptic responses in the retina

P2X₇Rs are distributed throughout all layers of the retina, and thus, their localisation on various cell types puts into question their specific site(s) of action. Using a dark-adapted, ex vivo mouse retinal whole mount preparation, the present study aimed to characterise the effect of P2X₇R activation on light-evoked, excitatory RGC ON-field excitatory post-synaptic potentials (fEPSPs) and on outer retinal electroretinogram (ERG) responses under comparable conditions. The pharmacologically isolated NMDA receptor-mediated RGC ON-fEPSP was reduced in the presence of BzATP, an effect which was significantly attenuated by A438079 and other selective P2X₇R antagonists A804598 or AF27139. In physiological Krebs medium, BzATP induced a significant potentiation of the ERG a-wave, with a concomitant reduction in the b-wave and the power of the oscillatory potentials. Conversely, in the pharmacologically modified Mg²⁺-free perfusate, BzATP reduced both the a-wave and b-wave. The effects of BzATP on the ERG components were suppressed by A438079. A role for P2X₇R function in visual processing in both the inner and outer retina under physiological conditions remains controversial. The ON-fEPSP was significantly reduced in the presence of A804598 but not by A438079 or AF27139. Furthermore, A438079 did not have any effect on the ERG components in physiological Krebs but potentiated and reduced the a-wave and b-wave, respectively, when applied to the pharmacologically modified medium. Therefore, activation of P2X₇Rs affects the function in the retinal ON pathway. The presence of a high concentration of extracellular ATP would most likely contribute to the modulation of visual transmission in the retina in the pathophysiological microenvironment.     

Naringenin protects RPE cells from NaIO3-induced oxidative damage in vivo and in vitro through up-regulation of SIRT1

BackgroundDry age-related macular degeneration (dAMD) leads to serious burden of visual impairment and there is no definitive treatment. Previous studies have showed that naringenin (NAR) significantly increased electroretinography (ERG) c-wave in sodium iodate (NaIO₃)-treated rats and viability of NaIO₃-treated ARPE-19 cells. But the underlying mechanism is still unknown.PurposeWe tested the hypothesis that anti-oxidation mediated by Sirtuin 1 (SIRT1) was important to the protective effect of NAR on dAMD.Study design/MethodsNaIO₃-induced mice retinopathy and ARPE-19 cells injury models were established. In vivo, the protective effect of NAR eye drops on retina was evaluated by flash ERG (FERG) recording and histopathological examination. In vitro, viability of ARPE-19 cells, and the levels of lactic dehydrogenase (LDH), reactive oxygen species (ROS) and carbonyl protein were detected. Protein expression of SIRT1 was analyzed by immunochemical staining, immunofluorescence and western blotting.ResultsNAR eye drops improved retinal function and morphology and normalized the protein expression of SIRT1 in mice exposed to NaIO₃. NAR promoted the survival of ARPE-19 cells in a concentration-dependent manner. NAR up-regulated SIRT1 protein expression, and decreased levels of ROS and carbonyl protein. Moreover, EX527, a selective inhibitor of SIRT1, abolished the effects of NAR on the cell viability and ROS. In addition, SRT1720, a selective agonist of SIRT1, improved the viability of cells and suppressed the production of ROS.ConclusionOur findings indicate that SIRT1-mediated anti-oxidation contributes to the protective effect of NAR eye drops on dAMD.     

Visual cells and visual pigments of the lamprey,Lampetra fluviatilis

Summary The long and short photoreceptors in the lamprey retina possess similar cone-like outer segments where many disks are infoldings of the outer plasmic membrane. Following the treatment by the Hartwig's (1967) method, outer segments of the long receptors are stained red, and those of the short receptor are stained blue, like the cones and rods in higher vertebrates, resp. (Fig. 1). Microspectrophotometry has shown that the short cells contain P517₁ whereas the long receptors possess P555₁ (Fig. 3). Spectral sensitivity of the dark-adapted retina measured by electroretinographic b-wave and aspartate-isolated receptor potential, corresponds to P517 (Figs. 5, 8). Judging from the receptor potential, the short receptors do not saturate at high illuminances and contribute to the retinal function in photopic conditions as well (Fig. 7). Photopic ERG is of a typical cone-dominant shape (Fig. 4).It is concluded that the long photoreceptors of the lamprey retina are cones whereas the short cells should be regarded as a peculiar kind of rods which possess cone ultrastructure and can operate in scotopic as well as in photopic conditions.Abbreviation LRP late receptor potential     

Production of leukotrienes and prostaglandins in the rat uterus during periimplantation period

We have measured by radioimmunoassay the production of leukotrienes (LTC₄ and LTB₄) and prostaglandins (PGE₂ and PGF_2α) in the rat uterus on Days 1 through 6 of pregnancy. The production is defined as the synthesis minus the degradation for a defined period. The production of LTC₄ or LTB₄ remained unaltered on days 1–3, but exhibited a marked increase on Day 4 showing a peak at noon. This was then followed by a sharp decline on Day-5 morning. A small but consistent peak in uterine LT production was also noticed on Day-5 noon prior to implantation and this was followed by a decline on Day-6 morning i.e. after initiation of implantation. The production profile of PGE₂ and PGE_2α showed a striking resemblance to that of LTs; one exception being that maximal PG production was noticed on Day-4 morning and preceded the peak production of LTs. These vasoactive arachidonate derivatives reached their peak production rates at around the time when a surge in estrogen level is noticed in the uterus on Day 4. Implantation is a local proinflammatory type of reaction that is associated with increased uterine vascular permeability. Vascular changes in inflammatory reactions are provoked by two kinds of chemical mediators: (1) vasodilators and (2) agents that increase vascular permeability. PGs (especially of the E series) are known as vasodilators, while LTs and histamine mediate increases in vascular permeability. Therefore, an interaction between LTs, PGs, and histamine could be important for uterine preparation for implantation and/or implantation .     

Processing of retinal signals in normal and HCN deficient mice

This study investigates the role of two different HCN channel isoforms in the light response of the outer retina. Taking advantage of HCN-deficient mice models and of in vitro (patch-clamp) and in vivo (ERG) recordings of retinal activity we show that HCN1 and HCN2 channels are expressed at distinct retinal sites and serve different functions. Specifically, HCN1 operate mainly at the level of the photoreceptor inner segment from where, together with other voltage sensitive channels, they control the time course of the response to bright light. Conversely, HCN2 channels are mainly expressed on the dendrites of bipolar cells and affect the response to dim lights. Single cell recordings in HCN1^−/− mice or during a pharmacological blockade of I_h show that, contrary to previous reports, I_kx alone is able to generate the fast initial transient in the rod bright flash response. Here we demonstrate that the relative contribution of I_h and I_kx to the rods'' temporal tuning depends on the membrane potential. This is the first instance in which the light response of normal and HCN1- or HCN2-deficient mice is analyzed in single cells in retinal slice preparations and in integrated full field ERG responses from intact animals. This comparison reveals a high degree of correlation between single cell current clamp data and ERG measurements. A novel picture emerges showing that the temporal profile of the visual response to dim and bright luminance changes is separately determined by the coordinated gating of distinct voltage dependent conductances in photoreceptors and bipolar cells.     

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.     

Correspondences in the Behavior of the Electroretinogram and of the Potentials Evoked at the Visual Cortex

Electrical potentials from the eye (ERG) and from the contralateral visual cortex were recorded in response to flashes of white and of colored light of various intensities and durations. The evoked potentials were found to parallel the behavior of the ERG in several significant respects. Selective changes in the ERG brought about by increasing the light intensity and by light adaptation led to parallel selective changes in the cortical responses. The dual waves (b₁, b₂) of the ERG were found to have counterparts in two cortical waves (c₁, c₂) which, in respect to changes in light intensity and to light adaptation, behaved analogously to the two retinal components. The responses evoked at high intensity showed only the diphasic c₁-potential. As stimulus intensity was lowered the c₁-wave decreased in magnitude and a delayed c₂-component appeared. The c₂-potential increased in amplitude as light intensity of the flash was further reduced. Eventually the c₂-wave, too, decreased as stimulus reduction continued. There was no wave length specificity in regard to either the duplex b-waves or duplex cortical waves. Both appeared at all wave lengths from 454 mµ to 630 mµ. The two cortical waves evoked by brief flashes of colored light showed all the behavior to changes in stimulus intensity and to light adaptation that occurred with white light.     

In the Early Stages of Diabetes,Rat Retinal Mitochondria Undergo Mild Uncoupling due to UCP2 Activity

In order to maintain high transmembrane ionic gradients, retinal tissues require a large amount of energy probably provided by a high rate of both, glycolysis and oxidative phosphorylation. However, little information exists on retinal mitochondrial efficiency. We analyzed the retinal mitochondrial activity in ex vivo retinas and in isolated mitochondria from normal rat retina and from short-term streptozotocin-diabetic rats. In normal ex vivo retinas, increasing glucose concentrations from 5.6mM to 30mM caused a four-fold increase in glucose accumulation and CO₂ production. Retina from diabetic rats accumulated similar amounts of glucose. However, CO₂ production was not as high. Isolated mitochondria from normal rat retina exhibited a resting rate of oxygen consumption of 14.6 ± 1.1 natgO (min.mg prot)^-1 and a respiratory control of 4.0. Mitochondria from 7, 20 and 45 days diabetic rats increased the resting rate of oxygen consumption and the activity of the electron transport complexes; under these conditions the mitochondrial transmembrane potential decreased. In spite of this, the ATP synthesis was not modified. GDP, an UCP2 inhibitor, increased mitochondrial membrane potential and superoxide production in controls and at 45 days of diabetes. The role of UCP2 is discussed. The results suggest that at the early stage of diabetes we studied, retinal mitochondria undergo adaptations leading to maintain energetic requirements and prevent oxidative stress.     

Electroretinographic study of the magnetic compass in European robins

Migratory birds are known to be sensitive to external magnetic field (MF). Much indirect evidence suggests that the avian magnetic compass is localized in the retina. Previously, we showed that changes in the MF direction could modulate retinal responses in pigeons. In the present study, we performed similar experiments using the traditional model animal to study the magnetic compass, European robins. The photoresponses of isolated retina were recorded using ex vivo electroretinography (ERG). Blue- and red-light stimuli were applied under an MF with the natural intensity and two MF directions, when the angle between the plane of the retina and the field lines was 0° and 90°, respectively. The results were separately analysed for four quadrants of the retina. A comparison of the amplitudes of the a- and b-waves of the ERG responses to blue stimuli under the two MF directions revealed a small but significant difference in a- but not b-waves, and in only one (nasal) quadrant of the retina. The amplitudes of both the a- and b-waves of the ERG responses to red stimuli did not show significant effects of the MF direction. Thus, changes in the external MF modulate the European robin retinal responses to blue flashes, but not to red flashes. This result is in a good agreement with behavioural data showing the successful orientation of birds in an MF under blue, but not under red illumination.     

Human Retinal Light Sensitivity and Melatonin Rhythms Following Four Days in Near Darkness

The rods in the retina are responsible for night vision, whereas the cone system enables day vision. We studied whether rod function in humans exhibits an endogenous circadian rhythm and if changes occur in conditions of prolonged darkness. Seven healthy subjects (mean age±SD: 25.6±12.3 yr) completed a 4.5‐day protocol during which they were kept in complete darkness (days 1 and 4) and near darkness (<0.1 lux red light, days 2 and 3). Electroretinography (ERG) and saliva collections were done at intervals of at least 3 h for 27 h on days 1 and 4. Full‐field ERGs were recorded over 10 low‐intensity green light flashes known to test predominantly rod function. As a circadian marker, salivary melatonin concentration was measured by radioimmunoassay. The ERG data showed that rod responsiveness to light progressively diminished in darkness (significantly lower a‐ and b‐wave amplitudes, longer b‐wave implicit time). The decrease in amplitude (b‐wave) from day 1 to day 4 averaged 22±14%. After correction for the darkness‐related linear trend, the circadian variations in ERG indices were weak and usually non‐significant, with slightly higher responsiveness to light during the day than night. Rod sensitivity (by K index) tended to decrease. Strikingly, the overall amount of melatonin secretion (area under 24 h curve) also decreased from day 1 to day 4 by 33.1±18.9% (p=.017). The drift of the melatonin rhythm phase was within the normal range, less than 56 min over three days. There was no significant correlation between the changes in ERG responses and melatonin. In conclusion, scotopic retinal response to (low‐intensity) light and the amount of melatonin secreted are diminished when humans are kept in continuous darkness. Both processes may have a common underlying mechanism implicating a variety of neurochemicals known to be involved in the regulation of both photoreceptor and pineal gland function.     

Treatment with 670 nm Light Up Regulates Cytochrome C Oxidase Expression and Reduces Inflammation in an Age-Related Macular Degeneration Model

Inflammation is an umbrella feature of ageing. It is present in the aged retina and many retinal diseases including age-related macular degeneration (AMD). In ageing and in AMD mitochondrial function declines. In normal ageing this can be manipulated by brief exposure to 670 nm light on the retina, which increases mitochondrial membrane potential and reduces inflammation. Here we ask if 670 nm exposure has the same ability in an aged mouse model of AMD, the complement factor H knockout (CFH^−/−) where inflammation is a key feature. Further, we ask whether this occurs when 670 nm is delivered briefly in environmental lighting rather than directly focussed on the retina. Mice were exposed to 670 nm for 6 minutes twice a day for 14 days in the form of supplemented environmental light. Exposed animals had significant increase in cytochrome c oxidase (COX), which is a mitochondrial enzyme regulating oxidative phosphorylation.There was a significant reduction in complement component C3, an inflammatory marker in the outer retina. Vimetin and glial fibrillary acidic protein (GFAP) expression, which reflect retinal stress in Muller glia, were also significantly down regulated. There were also significant changes in outer retinal macrophage morphology. However, amyloid beta (Aβ) load, which also increases with age in the outer retina and is pro-inflammatory, did not change. Hence, 670 nm is effective in reducing inflammation probably via COX activation in mice with a genotype similar to that in 50% of AMD patients even when brief exposures are delivered via environmental lighting. Further, inflammation can be reduced independent of Aβ. The efficacy revealed here supports current early stage clinical trials of 670 nm in AMD patients.     

Components of the ERG of the moth, Galleria mellonella

Origin of the components of the electroretinogram of the moth Galleria mellonella have been investigated. Successive recordings taken at different depths through the retina and optic ganglion indicate that the positive and maintained negative phases of the ERG originate in the retina while a phasic negative potential occurs in the ganglion, but under certain conditions may invade the retina too. There appears to be no correlation between amplitude and adaptation for the positive and slow negative phases nor will agents applied to the ganglion obliterate the positive phase. If either NaCl or Ringer solution is applied to the retina the positive phase is abolished. Isotonic KCl does not have this effect. Ringer's and NaCl applied to the ganglion do not affect the ERG. Damage to the retinal basement membrane also appears to abolish the positive phase. We interpret these effects to indicate the ion-selective permeability of the basement membrane.     

Caffeic acid phenethyl ester and its amide analogue are potent inhibitors of leukotriene biosynthesis in human polymorphonuclear leukocytes

Background

5-lipoxygenase (5-LO) catalyses the transformation of arachidonic acid (AA) into leukotrienes (LTs), which are important lipid mediators of inflammation. LTs have been directly implicated in inflammatory diseases like asthma, atherosclerosis and rheumatoid arthritis; therefore inhibition of LT biosynthesis is a strategy for the treatment of these chronic diseases.

Methodology/Principal Findings

Analogues of caffeic acid, including the naturally-occurring caffeic acid phenethyl ester (CAPE), were synthesized and evaluated for their capacity to inhibit 5-LO and LTs biosynthesis in human polymorphonuclear leukocytes (PMNL) and whole blood. Anti-free radical and anti-oxidant activities of the compounds were also measured. Caffeic acid did not inhibit 5-LO activity or LT biosynthesis at concentrations up to 10 µM. CAPE inhibited 5-LO activity (IC₅₀ 0.13 µM, 95% CI 0.08–0.23 µM) more effectively than the clinically-approved 5-LO inhibitor zileuton (IC₅₀ 3.5 µM, 95% CI 2.3–5.4 µM). CAPE was also more effective than zileuton for the inhibition of LT biosynthesis in PMNL but the compounds were equipotent in whole blood. The activity of the amide analogue of CAPE was similar to that of zileuton. Inhibition of LT biosynthesis by CAPE was the result of the inhibition of 5-LO and of AA release. Caffeic acid, CAPE and its amide analog were free radical scavengers and antioxidants with IC₅₀ values in the low µM range; however, the phenethyl moiety of CAPE was required for effective inhibition of 5-LO and LT biosynthesis.

Conclusions

CAPE is a potent LT biosynthesis inhibitor that blocks 5-LO activity and AA release. The CAPE structure can be used as a framework for the rational design of stable and potent inhibitors of LT biosynthesis.     

Inhibition of glycolysis in the retina by oxidative stress: prevention by pyruvate

Intraocular generation of reactive oxygen species (ROS) with consequent oxidative stress has been shown to be a significant factor in the pathogenesis of many vision-impairing diseases such as cataracts and retinal degenerations. Previous studies have shown that pyruvate can inhibit such oxidative stress. This is attributable to its property of scavenging various ROS and consequently inhibiting many of the apparent toxic reactions such as lipid peroxidation and loss of tissue thiols. It is hence expected that ROS will have an adverse effect on tissue metabolism also. The present investigations were hence undertaken to study the possibility that while scavenging ROS, the compound could be effective also in preventing the inhibition of tissue metabolism as well. Since glycolysis constitutes the major bioenergetic source of the retina, the objective of the present studies was to ascertain if the effects of pyruvate are indeed reflected in the maintenance of this pathway even when the tissue is exposed to ROS. This hypothesis was examined by incubating retinal explants in ROS-generating medium in the absence and presence of pyruvate and measuring ³H₂O generated from 5-³H glucose. In addition, the lactate generated was also measured. As hypothesized, ROS-induced inhibition of glycolysis indexed by the decrease in ³H₂O as well as lactate formation was significantly prevented by pyruvate. This effect was also reflected by the elevation of NAD/NADH ratio, a major pacemaker of glycolysis.