Effects of intraocular irrigation with melatonin on the c-wave of the direct current electroretinogram and on the standing potential of the eye in albino rabbits

The direct current electroretinogram (ERG) and the standing potential (SP) were recorded from both eyes of 14 albino rabbits during intraocular perfusion of one of the eyes, which was vitrectomized, with a recently developed eye irrigation solution (PHS) produced by Pharmacia Ophthalmics. PHS was then replaced by a test solution containing melatonin dissolved in PHS (0.002 M-200 M). The fluids were subsequently alternated (PHS - melatonin - PHS). During uniocular irrigation with melatonin the mean c-wave amplitude and SP level of the intact control eye were increased (c-wave + 24%, p < 0.01; SP + 0.65 mV, p < 0.05) compared with the values during the initial perfusion with PHS. In contrast, the c-wave amplitude of the irrigated eye was markedly decreased in many rabbits during perfusion with melatonin compared with the initial PHS, but the mean reduction was small and not statistically significant. The mean SP level was reduced (-1.54 mV, p < 0.001). This difference between the eyes probably depends on the route by which melatonin reaches the retinal pigment ephithelium and thus whether it primarily affects the apical (as in the irrigated eye) or the basal (as in the control eye) pigment epithelial membrane. A peak in the e-wave amplitude was observed in both eyes during uniocular irrigation with melatonin when compared with the amplitude measured during the initial perfusion with PHS (irrigated eye: + 27%, p < 0.001; control eye + 18%, p < 0.002).     

Nontoxic concentration of antibiotics for intravitreal use — evaluated by human in-vitro ERG

The effects of penicillin G (PC-G) sodium, procaine PC-G, cloxacillin sodium (MCIPC), disodium sulbenicillin (SBPC), cefazolin sodium (CEZ), gentamicin sulfate (GM) and fosfomycin sodium (FOM) on the electroretinogram (ERG) of the human in-vitro eye-cup were studied. The oscillatory potentials (OPs) were selectively and greatly suppressed by 1.0mM PC-G sodium. The OPs and c-wave were suppressed by 0.85mM procaine PC-G. The b-wave and OPs were slightly suppressed by 1.0mM MCIPC. The a-wave, b-wave, OPs and c-wave were not deteriorated by 1.0mM SBPC. The OPs appeared to be selectively suppressed by 1.0mM CEZ. The b-wave was suppressed and the peak latencies of the OPs were delayed by 184g/ml (approximately 0.4mM) GM. The amplitudes of the a-wave and c-wave were slightly enhanced and their peak latencies were slightly delayed by 184g/ml GM. The a-wave, b-wave, OPs and c-wave were not deteriorated by 1.0mM FOM. The results of the present study on the human retina were comparable to those on the albino rabbit retina in our previous studies.     

Effects of low doses of forskolin on the c-wave of the direct current electroretinogram and on the standing potential of the eye

A marked effect of prostaglandins on the b- and c-waves of the direct current electroretinogram was recently reported by our laboratory. The increased b- and c-wave amplitudes in response to prostaglandins may be mediated by cyclic nucleotides acting on fluid and ion transport across the retinal pigment epithelium (RPE). Forskolin is known to increase cyclic adenosine monophosphate in a number of tissues, among them the RPE. To study possible effects of forskolin on the ERG vitrectomy was performed on rabbit eyes, followed by intraocular irrigation with a forskolin solution (10g/ml PHS). Forskolin produced reversible ERG changes with increase in a- (24%, p < 0.001), b- (25%, p < 0.001) and c-wave (53%, p < 0.001) amplitudes and elevation (about 1.0–1.5mV, p < 0.01) of the standing potential of the eye. The increase in c-wave amplitude was significantly greater than that of the a- (p < 0.05) and b- (p < 0.01) wave amplitudes, which seems to imply a primary or at least major effect on the RPE.     

Effect of dopamine and haloperidol on the c-wave and light peak of light-induced retinal responses in chick eye

The relation between dopaminergic cells (and centrifugal fibers), the electroretinogram (ERG) c-wave, and the light peak were electrophysiologically investigated by observing the effects of a retrobulbar conduction block and intravitreal injection of either dopamine or haloperidol on these retinal responses. The retrobulbar conduction block (1% lidocaine) caused a decrease in the amplitude of the c-wave and the light peak in newly hatched chicks. Injections (2–20 l) containing dopamine (0.1–10 mM) or haloperidol (1.3–13 mM) were given intravitreously while the responses were recorded. Although intravitreous injection of saline for control resulted in no observable change in the responses, dopamine selectively augmented the c-wave of ERGs and the light peak, but not the a-, b-, and d-waves. Haloperidol decreased first the light peak and later the c-wave. The augmentation of the retinal responses by dopamine and their reduction by haloperidol was statistically significant. The estimated threshold concentration of dopamine in the vitreous cavity was 1–3.5 M.Since in many species the interplexiform cells have been found to contain dopamine, we hypothesize that the modulatory effects on the c-wave and the light peak in this preparation may be due to a centrifugal feed-back loop which includes the interplexiform cells to the horizontal and bipolar cells and the horizontal cells to the cones.     

Adrenergic effects on the corneal and intraretinal direct-current electroretinogram and on the standing potential of albino rabbit eyes

This study was undertaken to investigate further the responsiveness of the albino rabbit retinal pigment epithelium and the inner retina to adrenergic agents as reflected in changes of the direct-current electroretinogram and of the standing potential of the eye. After unilateral vitrectomy on albino rabbits, a continuous intraocular perfusion with a reference solution was established. The reference solution was then alternated with the test solution. The direct-current electroretinogram and the standing potential were recorded from both eyes with a scleral contact lens and a reference electrode connected to matched calomel half-cells. Anin vivo experimental technique that allows intraocular perfusion of a test substance and simultaneous intraretinal microelectrode measurements was also used. The-adrenergic agonist phenylephrine (0.04µM, n = 8) produced a reversible increase in c-wave amplitude (48%, p < 0.001) and also a small increase in b-wave amplitude (12%, p < 0.002). There was no significant influence on the a-wave amplitude. The standing potential was elevated at 1694 ± 362µV (mean ± SEM) (p < 0.002). The ₂-adrenergic agonist clonidine caused similar effects on the electroretinogram, although at a higher concentration (40µM, n = 5), with an elevation of the c-wave (25%, p < 0.001) and a small b-wave increase (12% p < 0.002). No significant influence on the a-wave or on the standing potential was found. Intraretinal direct-current electroretinogram-recordings during intraocular perfusion with phenylephrine showed an increase in transepithelial potential (p < 0.004; n = 6), accompanied by a reduction of the slow PIII (p < 0.0035; n = 6). The c-wave increase resulting from-adrenergic stimulation seems to be generated partly across the retinal pigment epithelium, with an increase in transepithelial potential, combined with a reduction of the slow PIII. The elevation of the b-wave amplitude, together with the influence on the slow PIII, suggests-adrenergic effects also on the inner retina. The experimental technique used in this study with intraocular perfusion after vitrectomy and simultaneous intraretinal direct-current recordings seems to be a practicable method for studies of the influence of pharmacologic agents on the retina and the retinal pigment epithelium.Abbreviations DC direct current - RPE retinal pigment epithelium - SP standing potential - TEP transepithelial potential     

Effects of intraocular perfusion with two alternating irrigation solutions on the simultaneously recorded electroretinogram of albino rabbits

The direct current electroretinogram (DC-ERG) of 15 unilaterally vitrectomized albino rabbits was studied during continuous intraocular irrigation with a recently developed solution (PHS) produced by Pharmacia Ophthalmics. The mean amplitudes of the b- and c-waves were somewhat lower than corresponding values of previously investigated reference subjects, but not statistically different. In 11 of the 15 rabbits PHS was then replaced by the commonly used balanced salt solution (BSS). The fluids were subsequently repeatedly alternated (PHS, BSS, PHS, BSS, PHS). During irrigation with BSS the average amplitude of the b-wave was reduced to 87% (p < 0.02) and that of the c-wave to 63% (p < 0.001) of the levels obtained during perfusion with PHS. In two rabbits the ERG was studied about one month after vitrectomy, where PHS-irrigation was used both during surgery and for 30 min after its completion. No marked differences were observed between the treated and the untreated eyes as to the b- and c-wave amplitudes at stable stages of the recording. Thus, judged by the ERG, retinal and pigment epithelial functions were influenced unfavorably by BSS, whereas they seemed to be well preserved with PHS. From this point of view PHS appears to be an excellent irrigation solution for vitreoretinal surgery that ought to be clinically tested.     

A cyclic adenosine monophosphate agonist elevates the b- and c-waves of the rabbit direct-current electroretinogram

The effects of the stable cyclic adenosine monophosphate analogue adenosine 3, 5-cyclic monophosphorothioate Sp-isomer (Sp-cAMPS) on the direct-current electroretinogram and the standing potential of the eye were studied. Corneal recordings were obtained from unilaterally vitrectomized albino rabbit eyes during alternating intravitreal perfusions with Sp-cAMPS and a control solution (Pharmacia eye irrigating solution). The contralateral eye was used as a control. To evaluate further the effects on the c-wave,in vivo intraretinal microelectrode measurements were made during simultaneous intravitreal perfusion of Sp-cAMPS and irrigating solution, respectively. Sp-cAMPS in concentrations of 1, 10 and 100µM was tested by corneal direct-current electroretinography. There was no significant effect on the a-wave amplitude. The b-wave amplitude was reversibly elevated at an Sp-cAMPS concentration of 100µM (p<0.01, n=7). The c-wave amplitude was reversibly elevated at a concentration of 10µM (p<0.001, n=8), and this effect was more pronounced at 100µM (p<0.001, n=7). The SP increased reversibly at a concentration of 100µM (p<0.001, n=7). Microelectrode recordings were performed with Sp-cAMPS at a concentration of 100µM. The recordings showed significant increases in both the transepithelial potential (p<0.01, n=3) and the slow PIII (p<0.01, n=3). The effects of Sp-cAMPS on the b-wave as well as on the two components of the c-wave suggest influences on both the inner retina and the retinal pigment epithelium of the rabbit eye.Abbreviations PHS Pharmacia eye irrigating solution - AMP adenosine monophosphate - Sp-cAMPS adenosine 3, 5 - cyclic monophosphorothioate Sp-isomer     

Nontoxic concentration of kanamycin and gentamicin for intravitreal use — evaluated by in vitro ERG

The effects of kanamycin (KM) and gentamicin (GM) on the in-vitro electroretinogram of the albino rabbit were studied. The b-wave and oscillatory potentials (OPs) were unchanged by 0.1 mM KM. The photopic b-wave and OPs were slightly suppressed by 0.4 mM. The b-wave and OPs were not deteriorated by 23 µg/ml (approximately 0.05 mM) GM. The photopic b-wave and OPs were slightly suppressed by 46 µg/ml (approximately 0.1 mM) GM. The minimum concentration affecting the ERG was tentatively defined as the mean of the minimum concentration needed to change the ERG and the maximum concentration which induced no discernible changes in the ERG. The minimum concentration of KM and GM affecting the ERG were 0.25mM (approximately 150 g/ml) and 35 g/ml (approximately 0.075 mM) respectively. The minimum concentration of KM affecting the ERG was higher than its minimum inhibitory concentration against Staphylococcus aureus and Streptococcus pneumoniae. The minimum concentration of GM affecting the ERG was higher than its mimimum inhibitory concentration against Staphylococcus aureus, Streptococcus pneumoniae and Pseudomonas aeruginosa.     

Influence of isobutylmethylxanthine on the direct current electroretinogram of albino rabbit eyes

The direct current electroretinogram and the standing potential of the eye were recorded from both eyes of unilaterally vitrectomized albino rabbits. The effect of intraocular irrigation with the nonselective phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) was compared with that of a balanced salt control solution. IBMX (0.5mM) produced a reversible increase in the amplitudes of the a-wave (19%, p < 0.02), b-wave(21%, p < 0.001) and c-wave (12%, p < 0.01) of the electroretinogram. A small elevation of the standing potential was found (0.4 ± 0.2mV), but this increase was not statistically significant (p > 0.05). The results indicate a primary and principal influence of IBMX on the photoreceptors.     

Dependence of the b-wave on the potassium concentration in the isolated superfused rabbit retina

The amplitude of the b-wave of the isolated superfused rabbit retina is drastically reduced with increasing potassium concentration (10 and 20 mM respectively) in the perfusate like in frog retina. These results are in agreement with the idea of the glial origin of the b-wave, but an influence of potassium on synaptic transmission remains a possibility. For these results the conditions for tissue survival are imperative. When the retina was superfused with a plasma saline mixture kept at 35°C, b-wave amplitudes for different preparations varied between 300 V and 900 V and loss of sensitivity was tolerated till 15% in one preparation. The temperature quotient for the amplitude of b-wave was 4–6 between 35° and 25°C, for the peak time about two.     

Electrooculographic and electroretinographic study in the chicken after dopamine and haloperidol

The implication of dopamine in the modulation of the standing potential of the eye was tested in the chicken through an indirect electrooculographic method and direct current electroretinogram (ERG) recording after haloperidol, a mixed D1-D2 antagonist. The standing potential of the eye was reduced within 15 min after intravitreal injection of the antagonist (150 g). This effect is rapidly reversed by an application of dopamine. The fast oscillation was preserved but the light peak was either strongly reduced or abolished. The dark trough showed an apparently normal time course. The intensity-voltage function was studied for the various ERG components. After haloperidol the b-wave and the c-wave were strongly reduced, whereas the a-wave was little affected. Together with previous data obtained with intraocular injections of dopamine, our data suggest the involvement of dopamine in the modulation of the standing potential. They also support the hypothesis that the light peak, which is generated by a photoreceptor-pigment epithelium interaction, is influenced by dopamine or by a related substance. The modulatory effect could also be due to a balance between several neurotransmitter systems.     

Effects of β-agonists on b- and c-waves implicit for adrenergic mechanisms in cat retina

Nylidrin (buphenine) is a -adrenergic agonist known to dilate peripheral vessels and used therapeutically in retinal degeneration and glaucoma. We studied retinal function under -agonists in arterially perfused cat eyes and observed a dose-dependent, reversible increase in b-wave amplitude and a decrease in c-wave amplitude in concentrations from 4.5 to 120M. A half maximal response was obtained at 40 to 50M. The optic nerve response to light showed dose-dependent reversible changes under nylidrin. Standing potential, light peak, intraocular pressure, vascular resistance, and diameter of or retinal vessels showed no consistent changes under nylidrin.The effects were inhibited by each of the -blocking agents propranolol, ICI 118, and oxprenolol (in sequence of decreasing potency).Another potent ₂-agonist, clenbuterol, was used to determine the extent to which the responses to nylidrin were due to -receptor-mediated action. Clenbuterol had similar effects on the b-wave and optic nerve response at slightly higher concentrations (30–200 M) but more variable effects on the c-wave.The data are interpreted as functional evidence that -adrenergic mechanisms are involved in retinal signal processing. This concept is corroborated by identification of -adrenergic binding sites in cat retina (Bruinink et al., 1986).     

The negative response of the flash electroretinogram in glaucoma

The existence of a negative ERG component following the b-wave has been known for a long time. Recently, in unilateral macaque experimental glaucoma, a negative response in flash electroretinograms under scotopic as well as photopic conditions has been shown to be greatly reduced or absent compared to the healthy fellow eye. The aim of this pilot study was to test whether a late negative electroretinogram component is reduced also in human glaucoma patients under different stimulus conditions. Dark-adapted ganzfeld flash electroretinograms were recorded after 30 min of dark using two test conditions, obtained as optimal in pilot studies on controls. Under the scotopic condition I white Xenon-flashes of intensity 0.53 Log photopic Td s were presented on a low white background of 1.38 Log scotopic Td. Under the more photopic condition II orange flashes of intensity –0.37 Log photopic Td s were presented on a blue-adapting background of 2.5 Log scotopic Td. Nine controls and 18 patients with advanced glaucoma were analyzed. The amplitude of the negative response was not significantly reduced in glaucoma patients (condition I: –28.5±23.7 V; condition II: –25.2±19.7 V) compared to controls (condition I: –41.4±36.6 V; condition II: –31.3±26.2 V). The peak latency of the responses under condition I and II did not differ significantly between patients and controls. Thus, the late negative electroretinogram component in ganzfeld flash electroretinograms obtained under scotopic and more photopic conditions does not seem to distinguish as easy between human controls and glaucoma patients as animal experiments suggest.     

Paradoxical enhancement of the ERG c-wave by a small dose of sodium iodate

The early effects of sodium iodate (NaIO3) on the ERG c- and b-waves were studied in rabbits. When 30 mg NaIO3/kg body weight was administered intravenously, the c-wave amplitude decreased and was replaced by a potential of opposite polarity. However, the administration of 20 mg/kg of NaIO3 enhanced the c-wave remarkably. Twenty min after administration, the mean c-wave amplitude significantly increased (P less than 0.001) to 211 +/- 6.2% of the pre-administration amplitude, whereas that of the control was 108 +/- 13.0%. The b-wave amplitude showed no significant changes. When 10 mg/kg of NaIO3 was administered, the enhancement of the c-wave was not remarkable. These effects were similar in both pigmented and albino rabbits. A lower blood concentration of NaIO3 applied to the basal side of the retinal pigment epithelium (RPE) may initially affect only the basal membrane of the RPE resulting in the decrease in the basal membrane resistance (Rba). This would explain the transient increase in the c-wave amplitude with 20 mg NaIO3/kg.     

Absence of indoleamine-accumulating neurons in the retina of humans and cynomolgus monkeys

Summary Indoleamine-accumulating neurons have previously been detected in cats, rabbits, goldfish, chicken, pigeons, and Cebus monkeys, and were therefore also looked for in humans and an Old World monkey, Cynomolgus irus. The monkey eyes were injected intravitreally with 50 g 5,6-dihydroxytryptamine, 5 g -methylnoradrenaline, or both drugs simultaneously. The human retinas were incubated in either drug.Previous observations on the distribution of dopaminergic neurons were confirmed in the two species, but no indoleamine-accumulating neurons were detected. The result emphasizes the similarity between human and Old World monkey retinas and contrasts them with the retina of the New World monkeys.     

Effects of quisqualic acid on the corneal and intraretinal direct-current electroretinogram and on the standing potential of the rabbit eye

Quisqualic acid, an excitatory amino acid agonist, has been shown to stimulate inositol phosphate production in the rabbit retina. Inositol trisphosphate serves as a second messenger and increases intracellular calcium. We investigated the influence of quisqualic acid on the direct-current electroretinogram and on the standing potential of the rabbit eye. After unilateral vitrectomy, the corneal direct-current electroretinogram and the standing potential were recorded from both eyes of albino rabbits during simultaneous unilateral intravitreal perfusion with quisqualic acid alternating with control solution. The contralateral eye was used as a control. Intravitreal perfusion with 100-µM and 200-µM quisqualic acid elevated the standing potential significantly. This elevation was accompanied by a significant increase in c-wave amplitude and a significant decrease in b-wave amplitude. Quisqalic acid at 200-µM concentration decreased the a-wave amplitude also.In vivo intraretinal recordings showed that intravitreal perfusion with quisqualic acid at 200-µM concentration significantly increased the retinal pigment epithelial component of the c-wave. We conclude that quisqualic acid influences the direct-current electroretinogram and the standing potential apparently through its action on the retinal pigment epithelium. A possible mode of action is increased production of inositol trisphosphate, followed by an increase in intracellular release of calcium ions and an increase in basal chloride conductance. The decrease in a- and b-wave amplitudes indicates direct effects of quisqualic acid also on the neural retina.Abbreviations EAA excitatory amino acid - IP inositol phosphate - NMDA N-methyl-Daspartate - PPI phosphoinositide - QA quisqualic acid     

Toxicity of 1-(β-d-arabinofuranosyl)cytosine after intravitreal injection in the rabbit eye

The retinal toxicity of intravitreally injected 1-(-d-arabinofuranosyl)cytosine (cytarabine) was examined in 7 chinchilla rabbits to determine if cytarabine can be used as local therapy for vitreoretinal non-Hodgkin's lymphoma. Fractionated dose of 600 g, 1500 g, and 2700 g cytarabine in stabilized saline were given intravitreally in one eye (2 × 300 g, 5 × 300 g, and 3 × 900 g, respectively, with an interval time of 24 h) and stabilized saline in the other eye as control. Toxic effects were evaluated with biomicroscopy, direct ophthalmoscopy, fluorophotometry, electroretinography, light, and electron microscopy. Toxic effects were found with the 1500 g and 2700 g doses only. They consisted of a temporary impairment of the blood retina barrier function for fluorescein as measured by fluorophotometry and an irreversible change of the b-wave in the electroretinograms. No histopathologic changes were seen under the light microscope. Electron microscopic examination showed aberrations in the synaptic pedicles of the photoreceptor cells at a dose of 1500 g cytarabine. The results suggest that the cytarabine dose that is expected to be therapeutic for vitreoretinal non-Hodgkin's lymphoma (about 90 g given in three doses of 30 g) is non-toxic for ocular structures.Correspondence to: J.A. van Best     

Effects of buphenine (nylidrin) on the perfused mammalian eye

Buphenine (nylidrin), a -adrenergic agonist, is used therapeutically for its vasodilating effect on the peripheral circulation and possibly on the cerebral circulation as well. In spite of its ophthalmic usage in degenerative retinal disease and glaucoma, buphenine's ocular effects and their mechanisms are not sufficiently established. Consquently, we studied the action of 4.5–120 M buphenine in isolated, arterially perfused cat eyes, and then used light-evoked electrical signals, perfusion flow rates and the diameters of the retinal vessels as parameters for the drug's effect. Our findings showed that buphenine induced a marked, dose-related, reversible increase in the amplitude of the electroretinogram (ERG) b-wave, a decrease in the c-wave, but no significant changes in the standing potential and light peak of the DC-ERG. The compound action potential of the optic nerve revealed dose-dependent and reversible changes in configuration. However, the flow of perfusate was not affected by the drug, and the diameter of retinal vessels did not change significantly. Our studies suggest that the interaction of buphenine with retinal adrenergic receptors is not related to the vasculature present but to elements involved in information processing. It is likely that these receptors are linked to neurons, since the -agonist affected the ERG b-waves as well as the compound action potential of the optic nerve.Presented in part at the 78th Congress of the Swiss Ophthalmological Society, St. Moritz, September 1985     

Concentration-dependent effects of dopamine on the direct current electroretinogram of pigmented rabbits during prolonged intermittent recording

The direct-current (DC) electroretinogram (ERG) was studied in 24 pigmented rabbits. Four experiments were performed, each including six animals. One eye was injected intravitreally with 0.1 ml dopamine (DA) with an estimated concentration in the vitreous body of 0.0025 mM, 0.025 mM, 0.25 mM and 2.5 mM, respectively. The contralateral eye was injected with the same amount of saline. Following the injection the animals were dark adapted for 30 min and then exposed to repeated light stimuli of low intensity for almost 3 hours (series I: 1 stimulus per 3 min, 10 s duration, light intensity 6.8 × 10² lux). After another 30 min period of dark adaptation repeated light stimuli of high intensity were presented to the eyes (series II: 1 stimulus per 70s, 10 s duration, light intensity 6.8 × 10⁴ lux) for 33 min. In the control eyes, a slow increase with time of the a-, b-and c-wave amplitudes was observed during series I. During series II, the a- b-and c-wave amplitudes were markedly reduced between the first and the second light stimulus, but subsequently grew to a peak. The behavior of the ERG in the eyes injected with dopamine was not different from that observed in the control eyes at the lowest concentration of the drug. At higher concentrations the b-and c-wave amplitudes were reduced compared with the control eyes, and did not show the slow increase with time observed in these eyes during series I. Peak responses observed during series II in the control eyes was increasingly suppressed in the eyes treated with dopamine. Results of ERG recordings suggest that dopamine influences retinal adaptation in rabbits in a dose dependent manner.     

Nontoxic concentration of amphotericin B for intravitreal use — evaluated by in vitro ERG

Amphotericin B (AMPH)-induced changes of the electroretinogram (ERG) were studied in the in vitro eye-cup of the albino rabbit. The a-wave, b-wave, oscillatory potentials and c-wave were not changed by 1 M AMPH, and these all were slightly suppressed by 5 M AMPH. These changes were mostly reversible. The b-wave and oscillatory potentials were greatly suppressed by 50 M AMPH. These changes were only partially reversible 75 minutes after re-perfusion without AMPH. Address for offprints Kazuo Kawasaki, M.D., Department of Ophthalmology Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa, 920 Japan.