Electrophysiological correlates of rod outer segment disc shedding in rabbit retina

We hypothesize that the b-wave of the electroreginogram (ERG) can provide a precise, noninvasive measure of the morning rod disc shedding event. ERGs were recorded in the dark from photoentrained rabbits using a 500 nm test light. Histological examination of the pigment epithelium confirmed that an increase in large phagosome content occurred shortly after light onset. In albino rabbits the b-wave decreased in amplitude at about 30 min after the time of normal light onset. The corresponding decrease in sensitivity required to produce this change in amplitude was 0.11 log unit. No ERG effect was seen in pigmented rabbits.     

The effects of prolonged dark exposure on visual thresholds in young and adult rats.

A comparison of electroretinogram (ERG) thresholds measured on rats reared in continuous darkness and under cyclic lighting conditions shows that by 30 days of age the dark-reared animals have achieved significantly lower thresholds than the animals reared under cyclic illumination. Ten days of continuous dark exposure produces this same increase in sensitivity in adult rats reared in cyclic lighting. These changes in sensitivity appear to reflect structural changes occurring within the rod outer segments.     

Rod and cone disc shedding in the rhesus monkey retina: A quantitative study

Four Rhesus monkeys were maintained for several months on a daily lighting schedule of 12 hr light and 12 hr darkness (12L:12D). The animals were then fixed by intracardiac perfusion at 1 and 5 hr following the onset of light, and at 1 and 5 hr after the offset of light. The number of phagosomes/mm of retinal pigment epithelium (RPE) was counted in the light microscope for both foveal and extrafoveal locations. At extrafoveal sites, phagosomes within the RPE cell bodies and those within the ensheathing processes bordering cone outer segments were counted separately. Counts at foveal and extrafoveal locations suggest that the rate of disc shedding, and therefore the rate of membrane replacement, is probably less for foveal cones than it is for rods. The number of phagosomes above the rod tips 1 hr following light onset and their diminution several hours later suggests that an increase in rod shedding, similar to the “burst” of rod shedding reported for other species, also exists in the monkey retina. The number and location of phagosomes 5 hr after the offset of light indicates that there could be another phase of increased rod shedding somewhere around the middle of the dark period. Phagosome counts in the foveal RPE were highest 5 hr after light offset, however the counts at 1 and 5 hr following light onset were nearly as high. The number of RPE phagosomes at both foveal and extrafoveal locations was lowest 1 hr after light offset. Counts of phagosomes within the sheaths of extrafoveal cones showed that about 20% contained one or more phagosomes within their sheaths. Of those sheaths that did contain phagosomes, approximately 25% had multiple phagosomes. There appears to be a considerable period of time during which extrafoveal cone phagosomes remain within the ensheathing processes, moving from the cone tips toward the apical RPE surface. Rod and cone disc shedding in the monkey retina are probably cyclic processes—although the temporal pattern of shedding may be different than that reported for other species.     

Light damage in the developing retina of the albino rat: an electroretinographic study

The albino rat retina is severely damaged by exposure to bright light. The degree of damage depends upon the intensity of the exposure and its duration. In the present study, electroretinographic (ERG) responses were measured in rats exposed at different ages during the period of retinal development to 24 hr of bright light and then transferred to darkness for about 2 months. The ERG data indicated that if the bright-light exposure was done prior to 20 days of age, the retina was resistant to the light damage, and the dark-adapted ERG responses measured later were normal. In rats older than 20 days, light damage increased with age until, in rats exposed to light at age 30 days, the ERG was unrecordable. Further experiments showed that light exposure did damage the 15-day-old rat retina; however, complete recovery was attained within 15 days postexposure.     

原发性视网膜色素变性光感受器细胞损害的临床分析

目的：用视网膜电图(ERG)与暗适应(DA)检测评价视网膜色素变性(RP)患者视杆细胞和视锥细胞的功能改变。 方法：100例RP患者(198眼)和正常人30例(60眼)对照研究，应用Nicolet compact Ⅳ电生理仪和Fridmann视野计(FVFA)MK₂程序分析分别作ERG和暗适应(DA)检查。 结果：93例RP患者184眼ERG正常者4眼(2.1%)，b波近消失者120眼(65.2%)，b波降低者60眼(32.6%)．后者多数呈杆-锥型损害，少数呈锥杆型攒害。89例170眼DA检查发现84.7%光敏感阈值升高，68.8%严重升高. 结论：大多数RP患者杆体细胞最先受损，部分病例锥体细胞似乎活性损害在前，其次是锥，杆细胞广泛损害。 （中华眼底病杂志，1995，11：147-150）     

Effect of early and late light exposure on the inherited retinal degeneration in rats

The inherited retinal dystrophy of RCS rats is slowed by maintaining the animals from birth in total darkness. Adult dystrophic rats raised in darkness from birth show nearly four times the ERG amplitude of animals kept in cyclic light conditions. Light deprivation, however, need not begin at birth for the full benefit of the treatment to occur. Rats reared in cyclic light and then transferred to darkness at 15–29 days of age show ERGs in adult-hood equal to animals raised in darkness from birth.Early dark rearing for as long as 45 days does not protect the retina from effects of subsequent light exposure as measured by ERG amplitude.Thus, early rearing conditions neither permanently damage nor permanently protect the dystrophic retina of the RCS rat.     

Encapsulated cell-based intraocular delivery of ciliary neurotrophic factor in normal rabbit: dose-dependent effects on ERG and retinal histology

PURPOSE: ERG and histologic changes were investigated in normal rabbits after intravitreal implantation of encapsulated cell technology (ECT) devices releasing ciliary neurotrophic factor (CNTF). METHODS: Fifteen adult New Zealand White albino rabbits had ECT devices secreting CNTF at 22, 5, or 0 ng/d implanted in the superior temporal quadrant of the left eye. The low dose has been shown to produce substantial rescue of photoreceptors in the rcd1 canine model of retinal degeneration. Right eyes were untreated. Ganzfeld dark- and light-adapted ERGs and clinical observations were performed at 5, 15, and 25 days after implantation. Rod a-waves and rod and cone b-waves and outer nuclear layer (ONL) morphology were evaluated at 25 days. RESULTS: Clinical examination showed minimal changes in a few CNTF-treated eyes, including vitreous membranes and engorgement of iris vessels at day 25. Retinas appeared normal. CNTF did not significantly affect the rod a- or b-waves, although the b-wave amplitude tended to be larger in CNTF-treated retinas at low flash intensities. The cone b-wave amplitude was significantly reduced in high-dose eyes at some flash intensities. The ONL area in high-dose eyes was significantly greater because of increased thickness than in fellow retinas. ONL cell size was significantly increased, and staining density decreased in CNTF-treated retinas. CONCLUSIONS: CNTF, given by intravitreal ECT device at doses that protect photoreceptors in a canine model of retinal degeneration (5 ng/d), did not adversely affect either rod or cone ERG function of normal rabbit retina. The cone ERG was more sensitive to suppression being reduced, at low flash intensities, by 22 ng/d. Dose-related changes in the ONL and photoreceptor cell nuclei did not represent a toxic effect, because they were not associated with deficits in the rod ERG over a broad range of intensities.     

Dark adaptation in locally detached retina

Nonrhegmatogenous retinal detachments were formed in the eyes of Dutch rabbits by subretinal injection of Hanks' balanced salt solution. The electroretinogram (ERG) was recorded locally from the acutely detached retina, and simultaneously from the surrounding attached retina (vitreal ERG [VERG]), before and after exposure to diffuse intense irradiation. Light adaptation elevated b-wave threshold for both the local ERG (LERG) and VERG by about 3 log units; thresholds for both responses recovered fully within 60-90 min after the irradiation. The normal time course of dark adaptation of the LERG suggests the occurrence of substantial rhodopsin regeneration in the rod photoreceptors of nonrhegmatogenously detached retina. These results differ from reports that visual pigment regeneration is slow in central serous chorioretinopathy, possibly because our detachments were studied within hours of formation, whereas some photoreceptor degeneration may be present in older clinical detachments.     

Retinal function and histopathology in rabbits treated with Topiramate

Purpose To evaluate retinal function and histopathology in rabbits treated orally with the anti-epileptic drug topiramate.Methods Six rabbits were treated with a daily oral dose of topiramate during a period of eight months. Six rabbits receiving water served as controls. Blood samples were analyzed for determination of topiramate serum levels in order to ensure successful drug exposition. Standardized full-field electroretinograms (ERGs) were performed before treatment and then at 2, 3 and 8 months during the treatment period. After terminating treatment the rabbits were sacrificed and the morphology of the sectioned retina was studied.Results After eight months of treatment the full-field ERG demonstrated normal rod function in treated and control rabbits, but the light adapted 30 Hz flicker b-wave amplitude was significantly reduced in the treated rabbits. This was the case for both the light adapted (Wilcoxon signed ranks test, P = 0.046) and the dark adapted (Wilcoxon signed ranks test, P = 0.028) 30 Hz flicker response from the treated rabbits. Retinal immunohistology revealed a severe accumulation of GABA in amacrine cells and in the inner plexiform layer in 4 of 6 treated rabbits compared to the controls.Conclusions Topiramate, orally administrated to rabbits, may cause a significant reduction of the retinal function demonstrated by the reduced b-wave amplitude in the full-field ERG, as well as changes in immunohistology characterized by a severe accumulation of GABA in the inner retina. The retinal dysfunction and the morphological changes indicate that topiramat may damage the retina, similarly to vigabatrin (another anti-epileptic drug).     

S-cone ERGs elicited by a simple technique in normals and in tritanopes.

PURPOSE: To measure changes in the relative spectral sensitivities of the dark adapted and light adapted ERG and thus to establish the possible contribution of rods to the 'blue cone' ERG elicited by flashes of blue light. BACKGROUND: Short wavelength stimuli in the light-adapted eye evoke small rounded b-waves which have been considered to be S-cone responses. We have recorded such responses from tritanopes, which called the assumptions into question. METHODS: Small ERGs were recorded to blue and green flashes. The stimulus was a Ganzfeld which employed light emitting diodes. ERGs were obtained in both the dark-adapted eye and after light adaptation to intense orange light (peak wavelength 610 nm). The change in sensitivity with light adaptation and the relative spectral sensitivity was determined from the voltage/log light intensity functions, using a 10 microV criterion. RESULTS: (1) peak times and changes in sensitivity did not help distinguish light-adapted rod from possible S-cone responses; (2) analysis of the change in the ratio of blue:green sensitivity from darkness to 4.4 log Td. 610 nm background suggests that in seven normal subjects, 90% or more of the ERG evoked by 440 nm flashes is generated by S-cones; (3) three tritanopes have insignificantly reduced S-cone responses. CONCLUSIONS: (1) clinical techniques used to isolate S-cone ERGs are appropriate; (2) there are at least two types of tritanope and in those we investigated, functional S-cones are probably displaced into the retinal periphery.     

Development and degeneration of retina in rds mutant mice: altered disc shedding pattern in the albino heterozygotes and its relation to light exposure

The number of phagosomes in the retinal pigment epithelium (RPE) of normal albino mice, maintained in cyclic light, is highest at the time of onset of light. The number declines to the lowest level around the start of the dark period. If the dark period is prolonged, the pattern of shedding remains cyclic but the phagosome count is higher than normal. If the light period is prolonged the phagosome count remains low and the rise to the peak is delayed. The number of phagosomes in the RPE of albino rds/+ mice, maintained in cyclic light, peaks to higher than normal level and the peak is recorded near the end of the light period. If the dark period is prolonged the phagosome count in the RPE of rds/+ mice remains lower than in the similarly treated normal mice. The phagosome count in the RPE of rds/+ mice, exposed to a prolonged light period, remains higher than in the similarly treated normal mice. In both normal and rds/+ mice, born and reared in total darkness, the pattern of disc shedding is very different but the rds/+ mice show a relatively higher frequency of phagosomes in the RPE than the normal mice. The phagosomes in the rds/+ RPE are larger than normal. An increase in size appears to correspond with increased rate of shedding in the rds/+ mice and to a lesser extent also in normal mice. Thus, the pattern of disc shedding in the albino rds/+ mice, which is different from the normal albino mice, also shows a different reaction to changes in the environmental light.     

Photoreceptor disc shedding in eye cups. Inhibition by deletion of extracellular divalent cations

To further define the medium requirements for in vitro rod disc shedding and phagocytosis in eye cups of Xenopus laevis, the effect of deletion of divalent cations was examined. Calcium-free medium completely eliminated both normal diurnal disc shedding (initiated by light onset) and dark-primed disc shedding (initiated by a period of darkness followed by additional darkness or light). The effect was reversible. Furthermore, the events that occurred during the initial dark-priming period did not require extracellular millimolar calcium, since the addition of calcium (1.8 mM) after an initial hour of darkness in calcium-free medium resulted in a marked increase in disc shedding, regardless of the subsequent lighting condition. Magnesium-free medium did not inhibit light-evoked shedding. However, magnesium-free medium partially inhibited disc shedding in one of the two lighting paradigms used to elicit dark-primed disc shedding. This suggests that the extracellular divalent cation requirement varies for different lighting paradigms that promote shedding. The inhibition of disc shedding by magnesium-free medium was morphologically distinct from calcium-free medium; the inhibition in magnesium-free medium was correlated uniquely with a reduction in the interdigitation between the photoreceptor and the retinal pigment epithelium.     

Reconstitution of the photoreceptor-pigment epithelium interface: L-glutamate stimulation of adhesive interactions and rod disc shedding after recombination of dissociated Xenopus laevis eyecups.

In order to investigate adhesive interactions between photoreceptor and pigment epithelial cells, we have mechanically separated neural retinas from Xenopus laevis eyecups and then recombined the tissues in vitro. When tissue pairs are incubated in a defined medium, cell-cell contact is achieved within 3 hr. However, the average proportion of reassembled eyecups in which photoreceptor outer segments interdigitate with epithelial microvilli is limited. Furthermore, rod disc shedding does not take place in these cultures, even following a dark to light transition. When recombined tissues are placed in medium supplemented with 12 mM L-glutamate, retinal reattachment is enhanced and there is a four-fold increase in epithelial phagosome content. The positive effect of excitatory amino acid exposure on shedding, however, is restricted to regions where visual and epithelial cells interdigitate. These results indicate that re-establishment of cell contact may be necessary for shedding of apical disc membranes prior to their engulfment by the epithelium. While reattachment is not affected by pre-incubation of separated tissues in normal medium, rod photoreceptors fail to undergo membrane turnover in response to L-glutamate if a delay of 1 hr or more is interposed between isolation of the retina and its recombination with the pigment epithelium. This is probably due to a decline in retinal function in culture, since a similar preincubation of the pigment epithelium prior to reassembly with a freshly isolated retina does not inhibit the shedding response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diurnal rhythm in the electroretinogram of the Royal College of Surgeons (RCS) pigmented rat

RCS pigmented rats, born and raised in cyclic light for 22-23 days and then placed in darkness for up to 24 hr, showed a diurnal rhythm in the rod electroretinogram (ERG) that was comparable with that seen in normal pigmented rats. a-Wave and b-wave sensitivities were 21- and 34% lower, respectively, 1.5 hr after expected light onset compared with those at other times of day. In contrast to findings in normal rats, however, these sensitivity decreases in the RCS rats were not associated with an increase in the frequency of large phagosomes in the pigment epithelium; phagosome frequency was subnormal and relatively constant over the day. These findings suggest that depressed ERG sensitivities 1.5 hr after expected light onset in both normal and RCS pigmented rats reflect altered rod photoreceptor function associated with the process of outer-segment disc shedding and not with engulfment of shed discs into phagosomes by the pigment epithelium.     

Retinal toxicity of intravitreal triamcinolone acetonide at high doses in the rabbit

In order to study acute retinal toxicity of intravitreal triamcinolone acetonide (TA) at high doses in an animal model, thirty New Zealand albino rabbits were injected with intravitreal TA. The animals were divided in five groups: Group 1 received an intravitreal injection of 0.1 mL balanced salt solution; Group 2, 0.1 mL of the solvent (0.99 mg of benzyl alcohol); Group 3, received 4 mg/0.1 mL TA; Group 4, 20mg/0.1 mL TA; and Group 5, 30 mg/0.1 mL TA. A standard light and dark adapted electroretinogram (ERG) was obtained prior and 28 days after the injection. The animals were sacrificed 28 days after the injection and the eyes were enucleated and examined by electron (EM) and light microscopy (LM) using hematoxylin-eosin, Nissl fluorescent, and immunohistochemistry (glial fibrillary acidic protein). No statistically significant differences in ERG before and 28 days after the injection were found. LM and EM did not show retinal damage in any animal. One eye developed bacterial endophthalmitis 14 days after the injection. Intravitreal TA up to 30 mg does not seem to have acute toxic effects on the function (ERG) or the structure (LM, EM) of the retina of albino rabbits.     

Postnatal development of flicker sensitivity in guinea pigs

Background: The retinal response to flickering stimuli (steady state ERG) recruits many retinal elements and is a sensitive indicator of early retinal dysfunction. This study reports the post‐natal maturation of the steady state ERG response in guinea pigs. Methods: The steady state ERG response to flickering stimuli (0.6 to 20 Hz) was recorded from dark adapted (more than 12 hrs) English Shorthair guinea pigs (n = 7) using flashes that produced rod and cone dominated responses. Temporal sensitivity functions and critical fusion frequencies (GFF) were derived over a range of ages from postnatal day (PND) 1 to 45. Results: Guinea pig rod and cone temporal sensitivity functions show shape characteristics and CFF similar to humans. Furthermore, the post‐natal development of the guinea pig temporal characteristics is also similar to that of humans—they are present at birth and mature rapidly post‐natally. The time‐course of CFF maturation is similar for rod and cone mediated responses. Conclusions: These data show that the temporal response and its maturation in the guinea pig retina is similar to that in humans. Therefore, we propose that the guinea pig is a particularly useful animal model to study retinal disease in early childhood.     

Excitatory amino acids and rod photoreceptor disc shedding: analysis using specific agonists

L-Glutamate and L-aspartate stimulate photoreceptor disc shedding. In order to evaluate the possible involvement of a receptor, we examined the effects of specific excitatory amino acid agonists on rod photoreceptor disc shedding and neural retinal toxicity. Using eyecups from both Xenopus laevis and Rana pipiens, we found that kainate, quisqualate, and N-methyl-D-aspartate (NMDA) were all neurotoxic, but that kainate caused a more extensive inner retinal lesion. Kainate also caused disc shedding at concentrations as low as 10 microM; dihydrokainate, a structural analogue, was at least 100-fold less potent. In contrast, quisqualate induced disc shedding only at concentrations above 5.0 mM, and NMDA had no effect on disc shedding at any concentration examined. Our results suggest that excitatory amino acids act via a receptor of the kainate type to effect disc shedding. The mechanism in the retina or photoreceptor-pigment epithelial complex by which an excitatory amino acid receptor system influences disc shedding remains to be identified.     

Study on choroidal blood flow at dark and light adaptation. 1. Choroidal blood flow at dark adaptation

The effect of dark adaptation on choroidal blood flow (CBF) was studied in albino rabbits. CBF was measured by the hydrogen clearance method. There was no significant change of CBF at dark adaptation in both short term dark adaptation, in which CBF had been expected to increase due to increased oxygen demand of the outer retina, and long term dark adaptation, in which CBF had been expected to decrease due to depressed function of visual cells and retinal pigment epithelium. The results show that dark adaptation does not influence CBF, in spite of the close relationship between the outer retina and the choroidal circulation. The large blood flow of the choroidal circulation may not need the reaction mechanism for the functional changes of the outer retina.     

Retinal function in Bothnia dystrophy. An electrophysiological study

Using prolonged dark adaptometry, standard dark adaptation (DA) and prolonged DA full-field electroretinograms (ERGs), we analysed the retinal function in patients with Bothnia dystrophy (BD), a variant of recessive retinitis punctata albescens (RPA). A compromised rod and cone function, a likely dysfunction of the Müller cells, and indications of disturbed neuronal function of the inner retina, were found. With prolonged DA, a gradual increase in retinal sensitivity to light and an improvement of the ERG components occurred. The findings indicate a prolonged synthesis of photopigments, retardation of the visual process in the retinal pigment epithelium (RPE), and a loss of retinal cells, probably starting at a relatively early age in BD.     

Retinal light damage in rats exposed to intermittent light. Comparison with continuous light exposure

Visible light-induced photoreceptor cell damage resulting from exposure to multiple intermittent light-dark periods was compared with damage resulting from continuous light in albino rats maintained in a weak cyclic-light environment or in darkness before light treatment. The time course of retinal damage was determined by correlative measurements of rhodopsin and visual cell DNA at various times after light exposure, and by histopathological evaluation. The effect of intense light exposures on rhodopsin regeneration and on the level of rod outer segment docosahexaenoic acid was also determined. For rats previously maintained in weak cyclic light, 50% visual cell loss was measured 2 weeks after 12 1 hr light/2 hr dark periods, or following 24 hr of continuous light. A comparable 50% loss of visual cells was found in dark-reared rats after only 5 hr of continuous illumination or 2-3 hr of intermittent light. As judged by histology, cyclic-light-reared rats incurred less retinal pigment epithelial cell damage than dark-reared animals. In both experimental rat models intermittent light exposure resulted in greater visual cell damage than continuous exposure. Visual cell damage from intermittent light was found to depend on the duration of light exposure and on the number of light doses administered. Measurements of rhodopsin and DNA 2 hr and 2 weeks after light exposure of up to 8 hr duration revealed that visual cell loss occurs largely during the 2 week dark period following light treatment. The loss of docosahexaenoic acid from rod outer segments was also greater in rats exposed to intermittent light than in animals treated with continuous light. It is concluded that intermittent light exposure exacerbates Type I light damage in rats (involving the retina and retinal pigment epithelium) and the schedule of intense light exposure is a determinant of visual cell death.