CB2 receptor activation causes an ERK1/2-dependent inflammatory response in human RPE cells.

A chronic low-level inflammation contributes to the pathogenesis of age-related macular degeneration (AMD), the most common cause of blindness in the elderly in Western countries. The loss of central vision results from attenuated maintenance of photoreceptors due to the degeneration of retinal pigment epithelium (RPE) cells beneath the photoreceptor layer. It has been proposed that pathologic inflammation initiated in RPE cells could be regulated by the activation of type 2 cannabinoid receptors (CB2). Here, we have analysed the effect of CB2 activation on cellular survival and inflammation in human RPE cells. RPE cells were treated with the selective CB2 agonist JWH-133 in the presence or absence of the oxidative stressor 4-hydroxynonenal. Thereafter, cellular viability as well as the release of pro-inflammatory cytokines and potential underlying signalling pathways were analysed. Our results show that JWH-133 led to increased intracellular Ca2+ levels, suggesting that RPE cells are capable of responding to a CB2 agonist. JWH-133 could not prevent oxidative stress-induced cell death. Instead, 10 µM JWH-133 increased cell death and the release of proinflammatory cytokines in an ERK1/2-dependent manner. In contrast to previous findings, CB2 activation increased, rather than reduced inflammation in RPE cells.

Electroretinography and optical coherence tomography reveal abnormal post-photoreceptoral activity and altered retinal lamination in patients with enhanced S-cone syndrome.

PURPOSE: To compare functional abnormalities of enhanced S-cone syndrome (ESCS), as examined using standard and extended electroretinography (ERG), with structural findings and retinal architecture obtained by spectral domain optical coherence tomography (SD-OCT). METHODS: Four patients with ESCS underwent standard full-field and multifocal ERGs, with extended S-cone and ON/OFF ERG protocols also performed. SD-OCT was also carried out, and longitudinal reflectivity profiles (LRPs) were calculated for the perifoveolar retina. RESULTS: All four patients exhibited pathognomonic full-field ERG findings for ESCS, with delayed responses of similar waveforms to the same intensity flash under both scotopic and photopic conditions. The amplitudes of the full-field ERGs showed considerable variation between patients, which were not related to the extent of the visual field defects. Multifocal ERGs reflected preserved central function in eyes with good visual acuity (Snellen visual acuity >0.7). The ERGs to S-cone-specific stimulation confirmed the expected predominant activity of the S-cone system in all four patients. The ON/OFF ERG recordings revealed abnormal presence of both ON-response and OFF-response activities in three patients; the remaining patient showed only OFF-response activity. SD-OCT showed a significantly thickened outer nuclear layer in all four patients, as obtained by LRP analysis. Furthermore, in the patient with selective preservation of the OFF-response activity, LRP showed reduced numbers of hyper-reflectivity sub-peaks in the inner plexiform layer. CONCLUSION: Patients with ESCS show characteristic full-field ERG waveform abnormality, predominance of S-cone ERG activity, and thickening of the outer nuclear layer on SD-OCT. Moreover, they can also show abnormal post-photoreceptor connectivity through S-cone-related OFF-bipolar cell activity.

Pattern electroretinography of larger stimulus field size and spectral-domain optical coherence tomography in patients with Stargardt disease.

AIMS: To investigate the importance of a larger stimulus field for pattern electroretinography (PERG) in evaluating macular function in Stargardt disease, and to determine the relationship between PERG and spectral-domain optical coherence tomography (SD-OCT). METHODS: In this prospective cross-sectional study, PERG from standard (12 degrees x 16 degrees ) and larger (24 degrees x 32 degrees ) stimulus fields and SD-OCT were recorded in 18 patients with genetically confirmed Stargardt disease, and in 18 control subjects. RESULTS: A PERG P50 response to the larger stimulus field was detectable in 86% of eyes, with a mean P50 amplitude of 2.3 microV, compared with 22% and 1.0 muV for the standard stimulus field. The specificity and sensitivity of PERG to the standard stimulus field were greater than for the larger field. For both PERG P50 and N95, the differences in their amplitudes between the standard and larger stimulus fields correlated significantly with visual acuity and SD-OCT parameters. CONCLUSION: The higher sensitivity and specificity of PERG to the standard stimulus field provide detection of early maculopathy in Stargardt disease, while PERG with the larger stimulus field allows for longer follow-up. The PERG amplitude for the larger stimulus field correlated with severity of transverse photoreceptor loss in SD-OCT. These methods are complementary for evaluation of progression of photoreceptor damage in patients with Stargardt disease.

Intermediate uveitis: long-term course and visual outcome.

AIM: In this retrospective cohort study we investigated the long-term course and visual outcomes of intermediate uveitis (IU). METHODS: We performed an institutional study of patients with IU with a follow-up of at least 10 years, followed at a tertiary referral centre. RESULTS: We studied 29 patients with unilateral or bilateral IU. The average age at onset of IU was 31 (range 8-64) years. At onset, three patients (10%) had associated systemic disease (two with sarcoidosis and one with multiple sclerosis) and one patient had granuloma annulare. During the follow-up period, one additional patient was diagnosed with sarcoidosis and one with multiple sclerosis. The percentage of eyes with legal blindness and visual impairment gradually increased over time (from 9/53 (17%) at onset to 15/53 (28%) at 10-year follow-up), with macular oedema, cataract and vitreous opacities being the most common causes of vision loss. The presence of associated anterior uveitis was more frequently noted in patients younger than 20 years at onset. Remissions of intraocular inflammation of at least 1 year were noted in 10/29 (34%) of patients. The mean time-to-remission was 8.6 years; patients who had remissions were younger at the onset of IU than those with ongoing active IU (p = 0.036). Remissions of IU showed borderline association with the absence of systemic disease (p = 0.046). CONCLUSIONS: One-third of IU patients achieved a remission of their intraocular inflammation for longer than 1 year and had a mean time-to-remission of 8.6 years. Patients who were younger at onset of IU were more likely to achieve remission than those who were older at onset.

Mapping of central visual function by microperimetry and autofluorescence in patients with Best's vitelliform dystrophy.

PURPOSE: To evaluate retinal sensitivity and fixation patterns in patients with Best's dystrophy by microperimetry (MP) and to correlate the results with static perimetry and retinal morphology seen by autofluorescence (AF). METHODS: Central 10 degrees visual fields in 11 patients with Best's dystrophy (VA: 0.5+/-0.38) were recorded by the Octopus M2 TOP program and by MP (MP1, Nidek Technologies). AF was recorded by HRA (Heidelberg Engineering). RESULTS: High correlation (R=0.75, -0.76, -0.48) was found between static perimetry (MS, MD and CLV indices) and MP. Based on MP and AF results, three groups of patients were formed. Patients in the first two groups fixated inside the central nonuniform hypo- and hyperfluorescent AF ring area, next to relative (Group 1) or absolute scotoma (Group 2). Inner parts of the retina close to the fovea were most affected, whereas regions closer to the periphery of the 10 degrees visual field showed near normal function. As the disease progressed, there was an evident shift of fixation to preferential retinal locus (PRL) in eight eyes with visual acuity 0.2 or less (Group 3). Fixation shift was superior in four eyes, temporal in two eyes, and nasal in two eyes. CONCLUSION: MP enabled a highly sensitive topographic monitoring of retinal function, showing central or pericentral fixation in the early stages, until loss of central function, in eyes with VA 0.2 or less, caused evident shift of fixation to PRL. PRL was never situated inside the central uniform hypofluorescent area, but corresponded with the hyperfluorescent ring seen with AF imaging.

Genotyping microarray (gene chip) for the ABCR (ABCA4) gene.

Genetic variation in the ABCR (ABCA4) gene has been associated with five distinct retinal phenotypes, including Stargardt disease/fundus flavimaculatus (STGD/FFM), cone-rod dystrophy (CRD), and age-related macular degeneration (AMD). Comparative genetic analyses of ABCR variation and diagnostics have been complicated by substantial allelic heterogeneity and by differences in screening methods. To overcome these limitations, we designed a genotyping microarray (gene chip) for ABCR that includes all approximately 400 disease-associated and other variants currently described, enabling simultaneous detection of all known ABCR variants. The ABCR genotyping microarray (the ABCR400 chip) was constructed by the arrayed primer extension (APEX) technology. Each sequence change in ABCR was included on the chip by synthesis and application of sequence-specific oligonucleotides. We validated the chip by screening 136 confirmed STGD patients and 96 healthy controls, each of whom we had analyzed previously by single strand conformation polymorphism (SSCP) technology and/or heteroduplex analysis. The microarray was >98% effective in determining the existing genetic variation and was comparable to direct sequencing in that it yielded many sequence changes undetected by SSCP. In STGD patient cohorts, the efficiency of the array to detect disease-associated alleles was between 54% and 78%, depending on the ethnic composition and degree of clinical and molecular characterization of a cohort. In addition, chip analysis suggested a high carrier frequency (up to 1:10) of ABCR variants in the general population. The ABCR genotyping microarray is a robust, cost-effective, and comprehensive screening tool for variation in one gene in which mutations are responsible for a substantial fraction of retinal disease. The ABCR chip is a prototype for the next generation of screening and diagnostic tools in ophthalmic genetics, bridging clinical and scientific research.

Pattern ERG and psychophysical functions in Best's disease.

The aim of the study was to asses the neurosensory retinal function in 12 patients (24 eyes) with different stages of Best's disease, by determining how pattern and full field flash ERG responses were related to visual acuity, stage of disease and extent of visual field loss. All patients had typically abnormal EOG responses and normal full field-flash ERG responses. Patients were stratified in two groups according to visual acuity. In the first group 12 eyes with visual acuity better than 0.5, all amplitudes and latencies of PERG P50 and N95 responses were in the normal range. Small central scotoma was detected by static perimetry in four of these eyes. In the second group of 12 eyes with visual acuity 0.5 or less, PERG showed reduced both P50 and N95 amplitudes in five eyes, and N95 solely, in two eyes. All patients had central scotomas detected by static perimetry. Progression of the disease, seen in deterioration of visual acuity and progression of central visual field defects, corresponded well with reduction of both PERG P50 and N95 amplitudes. There was no correlation found between visual acuity and EOG responses. Our results show that in Best's distrophy, pattern ERG is getting abnormal with progression of the disease, indicating relative preservation of neurosensory retina in initial stages of the disease. In contrast to EOG - being abnormal in all the patients regardless of the stage of disease - and full field-flash ERG - being normal in most of the patients - PERG gives opportunity for electrophysiological determination of the progression of the disease.

Standard for pattern electroretinography. International Society for Clinical Electrophysiology of Vision.

The pattern electroretinogram (PERG) is a retinal response evoked by viewing an alternating checkerboard or grating. It receives clinical and research attention because it can provide information about inner retinal cells and the macula. However, clinicians may have trouble choosing between different techniques for recording the PERG that have been described in the literature. The International Society for Clinical Electrophysiology of Vision has prepared a standard for a basic PERG recording procedure to aid new users in obtaining reliable responses and to encourage more uniformity among existing users.

Light-emitting diodes and half-cell electrodes in experimental recording of electroretinogram c-wave.

A method of electroretinogram c-wave recording in the rat was developed that uses a contact lens electrode connected through a saline bridge with a silver-silver chloride half-cell. A cluster of light-emitting diodes, regulated by an electronic light-emitting diode stimulator, was used as a light source. The method enables recordings of c-waves of 0.4-1.6 mV amplitude as well as other electroretinogram components in narrow limits of variation. The two main sources of response variability are voltage shunting and eyeball protrusion.

New noncorneal HK-loop electrode for clinical electroretinography.

A new noncorneal electrode for clinical electroretinography was developed. It consists of a thin wire forming a loop modeled to fit into the lower conjunctival sac. Electrical contact is made with the scleral conjunctiva through an exposed portion of otherwise insulated wire. The recorded pattern electroretinograms are in the same amplitude range as if recorded by the gold foil electrode, while the flash electroretinograms with the new electrode are of about two-thirds the amplitude of corneal electrodes. The new electrode is more durable and hence less expensive than gold foil electrodes and can likewise be used without topical anesthetic. Cleaning is easy and effective. The electrode rarely causes discomfort and produces stable responses for at least 2 hours. The electrode aims to match stability of skin electrodes with sensitivity of fragile foil and fiber electrodes.

Diurnal variations in the electroretinographic c-wave and retinal melatonin content in rats with inherited retinal dystrophy.

The inability of retinal pigment epithelium to phagocytose shed photoreceptor disks is a cause of retinal degeneration in the Royal College of Surgeons rat; retinal pigment epithelial phagocytosis and disk shedding are regulated by the diurnal rhythm of retinal melatonin level. The diurnal rhythms of the electroretinogram (particularly that of the retinal pigment epithelial potential, the electroretinographic c-wave) and retinal melatonin content were thus investigated in Royal College of Surgeons rats from postnatal day 17 to 24, the period preceding retinal degeneration. The amplitudes of both the b- and c-waves of the electroretinogram fell significantly during the peak time of rod disk shedding and rose after the time of expected light off in the control and dystrophic rats. While the b-wave rhythms did not differ between the two strains, diurnal changes in the c-wave were significantly less distinct in the dystrophic rats than in controls. This difference may reflect lack of phagocytosis in dystrophic rats. Furthermore, the ERG c-wave was significantly larger and prolonged, and the retinal melatonin content higher, in dystrophic rats of this age group than in controls. It appears that retinal melatonin metabolism may play an important role in the maintenance of retinal pigment epithelial and photoreceptor function.

Central fiber contribution to W-shaped visual evoked potentials in patients with optic neuritis.

We studied W-shaped waveforms that occurred in full-field responses to pattern large-field stimulation in patients who had optic neuritis. Affected eyes showed no absolute scotomata; visual acuity was normal at the time of recording. To evaluate the contribution of macular- and paramacular-derived components to the development of the W-shaped waveforms in the patients, half-field and central full-field stimulation was used. The responses were compared with those obtained with the use of experimental scotomata in healthy subjects. The W-shaped waveforms recorded in the patients closely resembled the responses observed in healthy subjects after the introduction of experimental scotomata. In all affected eyes, half-field stimulation showed absence of the ipsilateral P100 component or its interaction with the P135 component. Enhanced paramacular N105 and P135 components were seen over the contralateral hemisphere. Responses to central full-field stimulation were an attenuated and prolonged P100 in the majority of affected eyes. Results of our study showed that W-shaped waveforms in response to large full-field stimulation may reflect impaired function of macular fibers. These electrophysiologic findings, however, were not always associated with evidence of a central field defect demonstrated by Friedmann perimetry.

Pattern electroretinogram recorded by skin electrodes in early ocular hypertension and glaucoma.

Diagnostic value of transient pattern electroretinogram (PERG), recorded by skin electrodes, was compared with Goldmann perimetry in cases of ocular hypertension and glaucoma. According to the assumption that the PERG mostly reflects activity of the retinal ganglion cells, and histological evidence that 30-50% atrophy of the retinal ganglion cells is necessary to cause defects in visual field, we wanted to assess if i) this method could be more sensitive in detecting early glaucomatous damage than routine Goldmann perimetry in eyes with normal or only borderline elevated intraocular pressure in the time of PERG recording (first group of patients), and ii) how the PERG amplitude corresponds to ganglion cell loss, expected in the eyes with already detectable initial glaucomatous visual field defects, according to Goldmann II/2 isopter, with normal or borderline elevated intraocular pressure in the time of PERG recording (second group). In the group with no visual field defects subnormal amplitude of the major positive component of the PERG, N1-P1, was detected in three of 30 eyes (10%), while in the group with initial visual field defects N1-P1 amplitude was subnormal in 6 of 11 eyes (54%). The amplitude of the major negative PERG component, P1-N2, was found normal in all eyes of the first group and subnormal in 5 eyes (45%) of the second group.