Estimating ON and OFF contributions to the photopic hill: normative data and clinical applications.

BACKGROUND: With progressively brighter stimuli, the amplitude of the b-wave of the human photopic electroretinogram (ERG) first increases to a maximal value (Vmax) and then decreases to finally reach a plateau, a phenomenon known as the photopic hill (PH). A mathematical model combining a Gaussian (G) and a logistic (L) growth function was previously proposed to fit this unusual luminance-response curve, where the G and L functions were suggested to represent, respectively, the OFF and ON retinal pathway contributions to the building of the PH. METHOD: The PHs of patients presenting stationary diseases affecting specifically the ON (3 CSNB-1) or OFF (4 CPCPA) retinal pathways as well as patients affected with retinitis pigmentosa (14 RP) of different stages or etiology were analyzed using this mathematical model and compared to the PHs of a group of 28 normal subjects. RESULTS: The PH of the CSNB-1 patients had a much larger contribution from the G function compared to normal subjects, whereas the opposite was observed for the CPCPA patients. On the other hand, analysis of data from RP patients revealed variable G-L contributions to the building of their PH. CONCLUSION: In this study, we confirm the previous claim that the luminance-response function of the photopic ERG b-wave can be decomposed into a Gaussian function and a logistic growth function representing, respectively, the OFF and ON retinal pathways. Furthermore, our findings suggest that this mathematical decomposition could be useful to further segregate and potentially follow the progression of retinopathies such as RP.

Postnatal hyperoxia and the developing rat retina: beyond the obvious vasculopathy.

Although a great deal of emphasis has been placed on the vasculopathy that is associated with oxygen-induced retinopathy (OIR), our studies also revealed significant and irreversible structural (retinal histology) and functional (scotopic and photopic electroretinograms) impairments that were significantly more severe in pigmented Long-Evans rats compared to the more commonly used albino Sprague Dawley rats. In the following pages, we will highlight what we have learned about the retinal pathophysiological processes of OIR taking place in strains of both rats with the hope that this will trigger investigations into new therapeutic strategies to complement those geared at preventing the vasculopathy.

The effect of oxygen and light on the structure and function of the neonatal rat retina.

The neonatal rat is born with its eyes closed and an immature visual system, that some say is equivalent to that of a human fetus at 26 weeks of gestation. From birth, the visual system of the newborn rat will gradually mature, the first manifestation of that being the opening of the eye which usually take place at postnatal day 14. Complete maturation of the retina and visual pathways is normally reached at the end of the first month of life. The neonatal rat model thus represents a unique paradigm to study the normal and abnormal maturation of the primary visual pathways that normally occurs in utero in human subjects. Our laboratory has, over the past decade, developed two animal models of postnatally induced retinopathy, namely the Oxygen-Induced Retinopathy (OIR) that share several common features with the human Retinopathy of Prematurity (ROP) and the Light-Induced Retinopathy that is viewed by some as a valid model of some forms of Retinitis Pigmentosa (RP). The following pages review what is known of the pathophysiological processes taking place and suggest possible therapeutic avenues that could be explored in order to halt the degenerative process.

Comparing the retinal structures and functions in two species of gulls (Larus delawarensis and Larus modestus) with significant nocturnal behaviours.

Ring-billed gulls (Larus delawarensis) and gray gulls (Larus modestus) are two species active both by day and night. We have investigated the retinal adaptations that allow the diurnal and nocturnal behaviours of these two species. Electroretinograms and histological analyses show that both species have a duplex retina in which cones outnumber rods, but the number of rods appears sufficient to provide vision at night. Their retinas respond over the same scotopic dynamic range of 3.4logcdm(-2), which encompasses all of the light levels occurring at night in their photic environment. The amplitudes of the scotopic saturated a- and b-wave responses as well as the photopic saturated b-wave response and the photopic sensitivity parameter S are however higher in ring-billed gulls than in gray gulls. Moreover, the process of dark adaptation is about 30min faster in gray gulls than in ring-billed gulls. Our results suggest that both species have acquired in the course of their evolution functional adaptations that can be related to their specific photic environment.

Cyclooxygenase-2 in human and experimental ischemic proliferative retinopathy.

BACKGROUND: Intravitreal neovascular diseases, as in ischemic retinopathies, are a major cause of blindness. Because inflammatory mechanisms influence vitreal neovascularization and cyclooxygenase (COX)-2 promotes tumor angiogenesis, we investigated the role of COX-2 in ischemic proliferative retinopathy. METHODS AND RESULTS: We describe here that COX-2 is induced in retinal astrocytes in human diabetic retinopathy, in the murine and rat model of ischemic proliferative retinopathy in vivo, and in hypoxic astrocytes in vitro. Specific COX-2 but not COX-1 inhibitors prevented intravitreal neovascularization, whereas prostaglandin E2, mainly via its prostaglandin E receptor 3 (EP3), exacerbated neovascularization. COX-2 inhibition induced an upregulation of thrombospondin-1 and its CD36 receptor, consistent with the observed antiangiogenic effects of COX-2 inhibition; EP3 stimulation reversed effects of COX-2 inhibitors on thrombospondin-1 and CD36. CONCLUSIONS: These findings point to an important role for COX-2 in ischemic proliferative retinopathy, as in diabetes.

Effects of the intravitreal administration of dopaminergic ligands on the b-wave amplitude of the rabbit electroretinogram.

In the retina of mammals, dopamine (DA) is generally released by amacrine cells and is known to alter the physiology of most retinal cells. It is well known that DA reduces the amplitude of the b-wave of the electroretinogram (ERG) in rabbit. However, the specific receptor subtypes that mediate this action have not yet been elucidated. To do this, we recorded flash ERGs before and after the intravitreal injection of D1-like DA receptor agonists (SKF38393, A77693) and antagonist (SCH23390), and of D2-like agonist (R(-)-propylnorapomorphine hydrochloride; NPA) and antagonist ((S)-(-)-sulpiride). Contralateral control eyes were injected with the vehicle only. Both D1 agonists provoked a reduction of the ERG b-wave amplitude (34.0% and 59.2% of the pre-injection level, respectively). The D2-like agonist NPA had no significant effects on ERG components. Unexpectedly, both D1- and D2-like antagonists also reduced the b-wave amplitude (28.9% and 59.8%). Overall, these data suggest that the previously described effect of DA on the rabbit ERG b-wave came from activation of D1-like receptors. On the basis of the effects observed with D2-like antagonist, a subtle contribution of D2-like presynaptic receptors cannot be ruled out.

Oxidants, nitric oxide and prostanoids in the developing ocular vasculature: a basis for ischemic retinopathy.

The choroid is the main source of oxygen to the retina. In contrast to the adult, the absence of autoregulation of choroidal blood flow in the newborn leads to hyperoxygenation of the retina. In the immature retina which contains relatively low levels of antioxidants this hyperoxygenation favors peroxidation including the generation of biologically active isoprostanes, and results in vasoconstriction and vascular cytotoxicity leading to ischemia, which predisposes to the development of a vasoproliferative retinopathy, commonly termed retinopathy of prematurity. During frequently encountered oxidative stress to the perinate, the combined absence of vascular autoregulation and excessive oxygen delivery to the eyes of the developing subject is largely the result of a complex epigenetic and genetic interplay between prostanoids and nitric oxide (NO) systems on vasomotor regulation. The effects of certain prostaglandins are NO-dependent; conversely, those of NO have also been found to be largely prostaglandin I(2)-mediated in the eye; and NO synthase expression seems to be significantly regulated by other prostaglandins apparently through activation of functional perinuclear prostanoid receptors which affect gene transcription. The increased production of both prostaglandins and NO in the perinate augment ocular blood flow and as a result oxygen delivery to an immature retina partly devoid of antioxidant defenses. The ensuing peroxidation results in impaired circulation (partly thromboxane A(2)-dependent) and vascular integrity, leading to ischemia which predisposes to abnormal preretinal neovascularization, a major feature of ischemic retinopathy. Because tissue oxygenation is largely dependent upon circulation and critical in the generation of reactive oxygen species, and since the latter exert a major contribution in the pathogenesis of retinopathy of prematurity, it is important to understand the mechanisms that govern ocular blood flow. In this review we focus on the important and complex interaction between prostanoid, NO and peroxidation products on circulatory control of the immature retina.

Prevention of postasphyxia electroretinal dysfunction with a pyridoxal hydrazone.

The newborn retina is particularly sensitive and frequently subjected to peroxidative stresses that result in visual sequelae. We compared two iron chelators, deferoxamine and a newer compound, pyridoxal isonicotinoyl hydrazone (PIH), in protecting the retina of newborn pigs (1-3 d old) from asphyxia-reoxygenation insults. Animals were treated IV with either saline, deferoxamine 15.2 mumol/kg (10 mg/kg) or PIH 34.8 mumol/kg (10 mg/kg); n = 10 in each treatment group. Scotopic and photopic electroretinograms (ERG) were recorded before and 40 min after drug treatment as well as 45 min following a 5-min period of asphyxia by interrupting ventilation. In separate animals the indices of peroxidation, malondialdehyde (MDA: TBARS) and hydroperoxides, were measured in retina at the same times. In saline-treated animals, there was a marked increase in MDA and hydroperoxide concentrations in the retina following the asphyxia-reoxygenation period. This was associated with a decrease in the a- (photoreceptor generated) and b-wave (generated by Müller and bipolar cells) amplitudes measured under photopic (cone-mediated response) and scotopic (rod-mediated response) conditions, and an increase in their implicit times. PIH and deferoxamine prevented the postasphyxial increase in MDA and hydroperoxides. However, only PIH prevented the postasphyxial changes in a- and b-wave amplitudes and implicit times, whereas deferoxamine markedly altered the preasphyxial ERG and provided only partial postasphyxial protection simply to the retinal outer segment. Our findings indicate that the iron chelator PIH effectively inhibits peroxidation and retinal electrophysiological alterations secondary to asphyxia-reoxygenation-induced oxidative stresses to newborn animals, whereas deferoxamine adversely affects retinal function; hence, PIH may be a preferred alternative to deferoxamine.

Light induces peroxidation in retina by activating prostaglandin G/H synthase.

Prostaglandin G/H synthase (PGHS) has been shown to generate peroxides to a significant extent in the retina and absorbs light at the lower end of the visible spectrum. We postulated that PGHS could be an important initial source of peroxidation in the retina exposed to light, which would in turn alter retinal function. Exposure of pig eyes (in vivo) to light (350 fc/3770 lx) caused after 3 h a 50% increase and by 5 h a 30% decrease in a- and b-wave amplitudes of the electroretinogram (ERG) which were comparable at 380-650 nm and 380-440 nm but were not observed at wavelengths > 450 nm. These effects of light were prevented by free radical scavengers (dimethylthiourea and high-dose allopurinol) and PGHS inhibitors (naproxen and diclofenac), but stable analogs of prostaglandins did not affect the ERG. Both increases and subsequent decreases in ERG wave amplitudes following light exposure in vivo were associated with increases in retinal prostaglandin and malondialdehyde (peroxidation product) levels, which were inhibited by the nonselective PGHS blockers, naproxen and diclofenac. Similar observations were made in vitro on isolated porcine eyecups as well as on retinal membranes exposed to light (250 fc/ 2700 lx) 380-650 nm and 380-440 nm but not at > 500 nm. Both PGHS-1 and PGHS-2 contributed equivalently to light-induced prostaglandin synthesis, as shown after selective PGHS-2 blockers, but mRNA expression of PGHS-1 and 2 was not affected by light. Finally, light stimulated activities of pure PGHS-1 and PGHS-2 isozymes, and these were also shown to produce superoxide radical (detected with fluorogenic spin trap, proxyl fluorescamine). Taken together, data suggest that PGHS- (1 and 2) is activated by short wavelength visible light, and in the retina is an important source of reactive oxygen species which in turn alter retinal electrophysiological function. PGHS thus seems a likely chromophore in setting forth photic-induced retinal injury. Findings provide an explanation for increased sensitivity of the retina to visible light predominantly at the far blue range of its spectrum.

The photopic electroretinogram in congenital stationary night blindness with myopia.

Previous studies have reported that subjects affected with congenital stationary night blindness and myopia demonstrated some photopic (cone) abnormalities in their electroretinogram (ERG). By comparing the photopic ERG elicited with a threshold and a suprathreshold stimulus it was found that, at threshold, no significant differences were noted both in the peak time and in the amplitude of ERGs evoked from CSNB and normal subjects. However, a more powerful stimulus (16 times the threshold) yields a significant difference in the ERGs recorded from the two groups. ERGs recorded from CSNB patients are decreased in amplitude with a b-wave peak time that remains normal. First derivative analysis of the ERG wave along with a selective recording of the oscillatory components of the ERG suggest that the only visible anomaly in the suprathreshold photopic ERG of CSNB patients is an absence of the two oscillations normally seen on the ascending portion of the b-wave. Data obtained on normal subjects are also reported that try to explain the functional significance of these two oscillatory potentials.

Graded contribution of retinal maturation to the development of oxygen-induced retinopathy in rats.

PURPOSE: Newborn rats exposed to hyperoxia during the first days of life have been shown to exhibit not only vasculopathy but also permanent changes in the structure and function of the retina. Given that the rat retina is immature at birth and that the maturation process continues until the opening of the eyes at 14 days of life, this study was conducted to investigate the susceptibility of the retina to oxygen toxicity as a function of the degree of retinal maturity reached at the time of oxygen exposure. METHODS: Newborn rats were exposed to hyperoxia during selected postnatal day intervals. Scotopic electroretinograms were recorded at 30 and 60 days of age, and retinal histology was obtained at the end of the study. RESULTS: There was a strong correlation between the duration of the hyperoxic event and the structural and functional consequences in the retina. However, the repercussions were significantly more profound when the exposure to oxygen occurred within the second week of life (6-14 days), compared with earlier (0-6 days) or later periods (14-28 days). CONCLUSIONS: The results strongly suggest that the structural and functional retinal changes secondary to postnatal hyperoxia are not only the direct consequence of exposure to high levels of oxygen (i.e., free radicals), but also are determined by the level of retinal maturity reached at the time of oxygen exposure. The results also indicate that the structural anomalies precede the functional impairments.

Preservation of neural function in the perinate by high PGE(2) levels acting via EP(2) receptors.

Despite increasingly frequent and longer lasting hypoxic episodes during progressive labor, the neonate is alert and vigorous at birth. We investigated whether high levels of PGs during the perinatal period assist in preserving neural function after such "stressful" hypoxic events. Visual evoked potentials (VEPs) and electroretinograms (ERGs) were recorded before and 45 min after mild moderate asphyxic hypoxia (two 4-min asphyxic-hypoxic periods induced by interrupting ventilation at 8-min intervals) in newborn piglets <12 h old treated or not treated with inhibitors of PG synthase (ibuprofen or diclofenac) with or without PG analogs. At 45 min after the hypoxic episode, P2 and b-wave amplitudes were slightly decreased and latencies were delayed. These changes in the VEP and ERG returned to near normal by 120 min. Ibuprofen and diclofenac decreased brain and retinal PG levels and markedly intensified 45 min after hypoxia-induced changes in VEP and ERG, but cerebral and retinal blood flows improved. Combined treatment with PG synthase inhibitor in combination with 16,16-dimethyl-PGE(2) (a PGE(2) analog), but not with PGI(2) and PGF(2alpha) analogs, and in combination with the EP(2) receptor agonist butaprost (but not EP(1) or EP(3) agonists), prevented ibuprofen- and diclofenac-aggravated postasphyxia electrophysiological changes. In conclusion, high levels of PGE(2) in nervous tissue, via actions on EP(2) receptors, seem to contribute to preservation of neural function in the perinate subjected to frequent hypoxic events.

Role of thromboxane in retinal microvascular degeneration in oxygen-induced retinopathy.

Microvascular degeneration is an important event in oxygen-induced retinopathy (OIR), a model of retinopathy of prematurity. Because oxidant stress abundantly generates thromboxane A2 (TxA2), we tested whether TxA2 plays a role in retinal vasoobliteration of OIR and contributes to such vascular degeneration by direct endothelial cytotoxicity. Hyperoxia-induced retinal vasoobliteration in rat pups (80% O2 exposure from postnatal days 5-14) was associated with increased TxB2 generation and was significantly prevented by TxA2 synthase inhibitor CGS-12970 (10 mg x kg(-1) x day(-1)) or TxA2-receptor antagonist CGS-22652 (10 mg x kg(-1) x day(-1)). TxA2 mimetics U-46619 (EC50 50 nM) and I-BOP (EC50 5 nM) caused a time- and concentration-dependent cell death of neuroretinovascular endothelial cells from rats as well as newborn pigs but not of smooth muscle and astroglial cells; other prostanoids did not cause cell death. The peroxidation product 8-iso-PGF2, which is generated in OIR, stimulated TxA2 formation by endothelial cells and triggered cell death; these effects were markedly diminished by CGS-12970. TxA2-dependent neuroretinovascular endothelial cell death was mostly by necrosis and to a lesser extent by apoptosis. The data identify an important role for TxA2 in vasoobliteration of OIR and unveil a so far unknown function for TxA2 in directly triggering neuroretinal microvascular endothelial cell death. These effects of TxA2 might participate in other ischemic neurovascular injuries.

Modulations of collicular visual responses by acoustic stimuli in rabbits.

This study analyzes the influences of an acoustic stimulus upon neuronal light responses of superficial layers of the superior colliculus in anesthetized and paralyzed rabbits. The results have revealed that even if visually-responsive cells fail to be excited by sound, the latter is still capable of modifying light-evoked discharge. The influence may be "short-term" (the discharge rate recovers within 500 ms) or it may be "long-term" (the firing rate remains modified for several seconds). This audio-visual interaction depends upon several factors: the time of occurrence of both stimuli, the physical aspects of the visual target, the relative positions of the speaker and the visual receptive field, and finally, the sensitivity of the unit to movement direction. Data indicates that cells of the most dorsal (hence visual) layers of the superior colliculus are influenced by sound. It is concluded that the colliculus may use the sound as an additional cue to orientate the animal. Also, collicular cells could "memorize" for several seconds various features present in the environment.

A new speculum electrode for electroretinography.

A Storz model E4110 infant speculum was modified to record electroretinograms (ERGs) from anesthetized rabbits. The electrode combines the advantage of a corneal contact lens electrode (with blepharostat) without its major disadvantage, namely: corneal abrasion. Addition of i.v. tubings glued to both arms of the speculum allows for constant wetting of the eyeball with 0.9% NaCl. The latter not only prevents the eye (and cornea) from drying, but also favors the recording of reproducible ERGs with a noise level comparable to ERGs recorded with a corneal contact lens electrode.

Human strabismus: evaluation of the interhemispheric transmission time and hemiretinal differences using a reaction time task.

Experimentally induced strabismus in visually immature cats leads to abnormal development of the posterior corpus callosum. This, in turn, should lead to abnormal interhemispheric integration of unilaterally presented visual information. To test whether strabismus produces deficits in the human commissural visual system, the interhemispheric transmission time (ITT) was compared in strabismic and normal subjects. Simple unimanual reaction times (RT) were tested in 30 subjects in response to a lateralized target presented monocularly at 4 degrees and 35 degrees nasally and temporally from the fovea along the horizontal meridian. This method was also used to examine the effect of strabismus on the central and peripheral portions of each hemiretina. The results showed that in strabismic subjects with or without amblyopia, the ITT did not differ significantly from normals at both eccentricities. In non-amblyopic strabismic patients, RTs in the central and peripheral portions of hemiretina were comparable to normals. However, a reduced speed of response was found in the central visual field (4 degrees) in the amblyopic eye. Our results suggest that the ITT is normal in strabismic subjects and that the longer RTs in the central portion of the nasal and temporal hemiretina of the amblyopic eye may be associated with the severe amblyopic condition.

Lateral geniculate cell responses to electrical stimulation of the retina.

Electrical stimulation of the retina evokes at the optic tract level rhythmic bursts of activity whose temporal structure is predictable from the polarity of the stimulation and the receptive field type. The reaction of lateral geniculate units to this input was studied in fast and slow relay cells as well as in interneurons. The results revealed that fast relaty cells presented a response whose temporal structure remained essentially unmodified in comparison to that observed at the optic tract level: that both anodal and cathodal polarities produced rythmic pattern of excitation the latency of which depended upon receptive field type and polarity applied. In slow relay cells and interneurons responses with equal latencies could be evoked for both polarities. Following cortical depression with 3 M KCl the latency of first bursts was unaffected in relay cells, while about one third of interneurons showed a temporal pattern which was similar to that recorded at the optic tract level after the treatment. This suggests that both ON and OFF retinal networks converge upon one geniculate slow P cell and interneuron, whereas fast relay cells are mostly driven by one of the two systems. Furthermore this convergence may be achieved through visual cortex in some units.

Influence of the visual cortex upon receptive field organization of lateral geniculate cells in rabbits.

In anesthetized rabbits, the receptive fields of lateral geniculate cells were mapped prior to and following the interruption of the corticogeniculate feed-back. Visual cortex (V.C.) was depressed by a focal application of 3 M KCl. The responsiveness of the V.C. was verified by monitoring the visually evoked potentials. In off- and on-center cells, the surround excitatory responses were remarkably reduced and even fully abolished in most units. In contrast, the center excitation remained unmodified. These effects were reversible. In some on-center units the center response had also decreased, and was replaced by an evoked inhibitory response. Relay cells and interneurons which yielded on and off responses over the entire area of the receptive field exhibited a loss of only one of the evoked discharges. It is concluded that the V.C. exerts mostly a specific desinhibitory action upon the geniculate network. This action affects either the center or the surround responses differentially. The results are compared with those obtained from cats.

Influence of a local inactivation in the superior colliculus on lateral geniculate responses in rabbits.

The influence of the superficial layers of the superior colliculus (SC) on responses of the lateral geniculate nucleus was studied in anesthetized and paralyzed rabbits. Fifty to one hundred nanoliters of inactivating drugs were injected through a micropipette positioned in the SC in register with the geniculate cells. The latter were tested with discrete moving and stationary targets presented in random sequences. Results revealed that the SC exerted a dual facilitative (n = 19) and inhibitory (n = 25) influence. The latter segment of the response pattern was modified while the initial portion remained unchanged. In some cases differential effects were observed, that is, the responses to one particular stimulus were more affected than responses to other modalities. However, in the majority of cells it was the on-off responses which were most affected. These findings point toward complex influences of the SC upon geniculate responses in rabbits. This complexity is also supported by findings that in cats the colliculo-geniculate synapses are of various types.

Local excitability in the superior colliculus influences evoked responses of lateral geniculate cells in rabbits.

In anesthetized, immobilized rabbits recordings were made simultaneously from cells in the Lateral Geniculate Nucleus (CGL) and Superior Colliculus (CS), in order to study how the CS influences the CGL. The experimental protocol consisted of three steps. In the initial step (first control) the light stimulus was triggered electronically. In the second step (Test), the same stimulus was triggered by a spontaneous spike arising from a collicular cell. Thus the stimulus presentation was time-locked to collicular endogenous activity. The third step was the same as the first and constituted a second control. The frequencies of stimulus application were gated to be approximately the same. The results indicated that the CS exerts two separate effects on CGL units. In 37 pairs (26%), conditioning the stimulus presentation to collicular firing produced a significant enhancement of geniculate responses. In 24 pairs (17%), the geniculate responses declined. In 82 pairs (57%), no significant influence was noted. The colliculo-geniculate influence is transient. The effects peaked between 100 to 200 msec after the collicular spike and returned to their control levels within 300 msec. Collicular cells producing a decline were encountered mostly in the ventral part of the stratum griseum superficiale, and the stratum opticum, whereas collicular cells that were related to an increased geniculate response were more frequently found dorsally. Increments were more pronounced if the distance (D) between receptive fields was short (0 degrees less than D less than 40 degrees) or if the collicular and geniculate fields were far apart (120 degrees less than D less than 180 degrees). The decrement effect was attenuated as the distance separating the two receptive fields. This study suggests that the superior colliculus is capable of generating an internal signal powerful enough to modulate at the geniculate nucleus the visual message conveyed toward the visual cortex. A possible role of the CS in the initiation of the corollary discharge is briefly discussed.