The Transcription Factor Bhlhb4 Is Required for Rod Bipolar Cell Maturation

AbstractRetinal bipolar cells are essential to the transmission of light information. Although bipolar cell dysfunction can result in blindness, little is known about the factors required for bipolar cell development and functional maturation. The basic helix-loop-helix (bHLH) transcription factor Bhlhb4 was found to be expressed in rod bipolar cells (RB). Electroretinograms (ERGs) in the adult Bhlhb4 knockout (Bhlhb4−/−) showed that the loss of Bhlhb4 resulted in disrupted rod signaling and profound retinal dysfunction resembling human congenital stationary night blindness (CSNB), characterized by the loss of the scotopic ERG b-wave. A depletion of inner nuclear layer (INL) cells in the adult Bhlhb4 knockout has been ascribed to the abolishment of the RB cell population during postnatal development. Other retinal cell populations including photoreceptors were unaltered. The timing of RB cell depletion in the Bhlhb4−/− mouse suggests that Bhlhb4 is essential for RB cell maturation

Assessing Photoreceptor Structure Associated with Ellipsoid Zone Disruptions Visualized with Optical Coherence Tomography

Purpose: To compare images of photoreceptor layer disruptions obtained with optical coherence tomography (OCT) and adaptive optics scanning light ophthalmoscopy (AOSLO) in a variety of pathologic states.Methods: Five subjects with photoreceptor ellipsoid zone disruption as per OCT and clinical diagnoses of closed-globe blunt ocular trauma (n = 2), macular telangiectasia type 2 (n = 1), blue-cone monochromacy (n = 1), or cone-rod dystrophy (n = 1) were included. Images were acquired within and around photoreceptor lesions using spectral domain OCT, confocal AOSLO, and split-detector AOSLO.Results: There were substantial differences in the extent and appearance of the photoreceptor mosaic as revealed by confocal AOSLO, split-detector AOSLO, and spectral domain OCT en face view of the ellipsoid zone.Conclusion: Clinically available spectral domain OCT, viewed en face or as B-scan, may lead to misinterpretation of photoreceptor anatomy in a variety of diseases and injuries. This was demonstrated using split-detector AOSLO to reveal substantial populations of photoreceptors in areas of no, low, or ambiguous ellipsoid zone reflectivity with en face OCT and confocal AOSLO. Although it is unclear if these photoreceptors are functional, their presence offers hope for therapeutic strategies aimed at preserving or restoring photoreceptor function

The Reliability of Parafoveal Cone Density Measurements

Background Adaptive optics scanning light ophthalmoscopy (AOSLO) enables direct visualisation of the cone mosaic, with metrics such as cone density and cell spacing used to assess the integrity or health of the mosaic. Here we examined the interobserver and inter-instrument reliability of cone density measurements. Methods For the interobserver reliability study, 30 subjects with no vision-limiting pathology were imaged. Three image sequences were acquired at a single parafoveal location and aligned to ensure that the three images were from the same retinal location. Ten observers used a semiautomated algorithm to identify the cones in each image, and this was repeated three times for each image. To assess inter-instrument reliability, 20 subjects were imaged at eight parafoveal locations on one AOSLO, followed by the same set of locations on the second AOSLO. A single observer manually aligned the pairs of images and used the semiautomated algorithm to identify the cones in each image. Results Based on a factorial study design model and a variance components model, the interobserver study\u27s largest contribution to variability was the subject (95.72%) while the observer\u27s contribution was only 1.03%. For the inter-instrument study, an average cone density intraclass correlation coefficient (ICC) of between 0.931 and 0.975 was calculated. Conclusions With the AOSLOs used here, reliable cone density measurements can be obtained between observers and between instruments. Additional work is needed to determine how these results vary with differences in image quality

Foveal Cone Structure in Patients With Blue Cone Monochromacy

Purpose: Blue cone monochromacy (BCM) is a rare inherited cone disorder in which both long- (L-) and middle- (M-) wavelength sensitive cone classes are either impaired or nonfunctional. Assessing genotype-phenotype relationships in BCM can improve our understanding of retinal development in the absence of functional L- and M-cones. Here we examined foveal cone structure in patients with genetically-confirmed BCM, using adaptive optics scanning light ophthalmoscopy (AOSLO). / Methods: Twenty-three male patients (aged 6-75 years) with genetically-confirmed BCM were recruited for high-resolution imaging. Eight patients had a deletion of the locus control region (LCR), and 15 had a missense mutation-Cys203Arg-affecting the first two genes in the opsin gene array. Foveal cone structure was assessed using confocal and non-confocal split-detection AOSLO across a 300 × 300 µm area, centered on the location of peak cell density. / Results: Only one of eight patients with LCR deletions and 10 of 15 patients with Cys203Arg mutations had analyzable images. Mean total cone density for Cys203Arg patients was 16,664 ± 11,513 cones/mm2 (n = 10), which is, on average, around 40% of normal. Waveguiding cone density was 2073 ± 963 cones/mm2 (n = 9), which was consistent with published histological estimates of S-cone density in the normal eye. The one patient with an LCR deletion had a total cone density of 10,246 cones/mm2 and waveguiding density of 1535 cones/mm2. / Conclusions: Our results show that BCM patients with LCR deletions and Cys203Arg mutations have a population of non-waveguiding photoreceptors, although the spectral identity and level of function remain unknown

Retinal Neuroprotective Effects of Flibanserin, an FDA-Approved Dual Serotonin Receptor Agonist-Antagonist

Purpose: To assess the neuroprotective effects of flibanserin (formerly BIMT-17), a dual 5-HT1A agonist and 5-HT2A antagonist, in a light-induced retinopathy model. Methods: Albino BALB/c mice were injected intraperitoneally with either vehicle or increasing doses of flibanserin ranging from 0.75 to 15 mg/kg flibanserin. To assess 5-HT1A-mediated effects, BALB/c mice were injected with 10 mg/kg WAY 100635, a 5-HT1A antagonist, prior to 6 mg/kg flibanserin and 5-HT1A knockout mice were injected with 6 mg/kg flibanserin. Injections were administered once immediately prior to light exposure or over the course of five days. Light exposure lasted for one hour at an intensity of 10,000 lux. Retinal structure was assessed using spectral domain optical coherence tomography and retinal function was assessed using electroretinography. To investigate the mechanisms of flibanserin-mediated neuroprotection, gene expression, measured by RT-qPCR, was assessed following five days of daily 15 mg/kg flibanserin injections. Results: A five-day treatment regimen of 3 to 15 mg/kg of flibanserin significantly preserved outer retinal structure and function in a dose-dependent manner. Additionally, a single-day treatment regimen of 6 to 15 mg/kg of flibanserin still provided significant protection. The action of flibanserin was hindered by the 5-HT1A antagonist, WAY 100635, and was not effective in 5-HT1A knockout mice. Creb, c-Jun, c-Fos, Bcl-2, Cast1, Nqo1, Sod1, and Cat were significantly increased in flibanserin-injected mice versus vehicle-injected mice. Conclusions: Intraperitoneal delivery of flibanserin in a light-induced retinopathy mouse model provides retinal neuroprotection. Mechanistic data suggests that this effect is mediated through 5-HT1A receptors and that flibanserin augments the expression of genes capable of reducing mitochondrial dysfunction and oxidative stress. Since flibanserin is already FDA-approved for other indications, the potential to repurpose this drug for treating retinal degenerations merits further investigation

Quantitative optical coherence tomography angiography of vascular abnormalities in the living human eye

Retinal vascular diseases are important causes of vision loss. A detailed evaluation of the vascular abnormalities facilitates diagnosis and treatment in these diseases. Optical coherence tomography (OCT) angiography using the highly efficient split-spectrum amplitude decorrelation angiography algorithm offers an alternative to conventional dye-based retinal angiography. OCT angiography has several advantages, including 3D visualization of retinal and choroidal circulations (including the choriocapillaris) and avoidance of dye injection-related complications. Results from six illustrative cases are reported. In diabetic retinopathy, OCT angiography can detect neovascularization and quantify ischemia. In age-related macular degeneration, choroidal neovascularization can be observed without the obscuration of details caused by dye leakage in conventional angiography. Choriocapillaris dysfunction can be detected in the nonneovascular form of the disease, furthering our understanding of pathogenesis. In choroideremia, OCT's ability to show choroidal and retinal vascular dysfunction separately may be valuable in predicting progression and assessing treatment response. OCT angiography shows promise as a noninvasive alternative to dye-based angiography for highly detailed, in vivo, 3D, quantitative evaluation of retinal vascular abnormalities.National Institutes of Health (U.S.) (Grant R01-EY023285)National Institutes of Health (U.S.) (Grant R01-EY024544)National Institutes of Health (U.S.) (Grant DP3 DK104397)National Institutes of Health (U.S.) (Grant R01-EY11289)National Institutes of Health (U.S.) (Grant K08-EY021186)National Institutes of Health (U.S.) (Grant T32-EY23211)National Institutes of Health (U.S.) (Grant P30-EY010572)Clinical and Translational Science Award Grant UL1TR000128Research to Prevent Blindness, Inc. (United States) (Grant and Career Development Award CD-NMT-0914-0659-OHSU)United States. Air Force Office of Scientific Research (Foundation Fighting Blindness Career Development Award FA9550-10-1-0551)German Research Foundation (Grant DFG-HO-1791/11-1)German Research Foundation (Grant DFG-GSC80-SAOT

Intravitreal Moxifloxacin: Retinal Safety Study with Electroretinography and Histopathology in Animal Models

PURPOSE. To determine whether moxifloxacin can be used safely as an intraocular antibiotic, retinal safety of intravitreal moxifloxacin was studied with electroretinography (ERG) and histopathology in animal models. METHODS. Moxifloxacin was injected into mouse eyes at intravitreal concentrations of 5 to 500 g/mL and into rabbit eyes at 150 g/mL. As the control, the vehicle was injected into the fellow eyes of each animal. Four weeks after injection, ERG recordings were performed, and animal eyes were processed for histologic examination. RESULTS. ERG studies showed no significant difference between control and moxifloxacin-injected eyes at any dose in either the mouse or rabbit model. Histologic examination revealed no retinal abnormality in mice at 5 to 100 g/mL or in rabbits at 150 g/mL intravitreal moxifloxacin. In mice at 500 g/mL, occasional focal retinal necroses were observed, suggesting isolated retinal toxicity at this concentration of moxifloxacin. CONCLUSIONS. Intravitreal moxifloxacin, up to 100 g/mL in mice or 150 g/mL in rabbits, caused no ERG or retinal histologic abnormality. These results indicate that moxifloxacin is a safe intravitreal antibiotic in mouse and rabbit animal models. If proven safe and efficacious by further study in humans, intravitreal injection of moxifloxacin could be considered as an alternative to currently used antibiotics in selected patients with resistance or allergy to the more traditional antibiotics

Results at 2 Years after Gene Therapy for RPE65-Deficient Leber Congenital Amaurosis and Severe Early-Childhood–Onset Retinal Dystrophy

PurposeTo provide an initial assessment of the safety of a recombinant adeno-associated virus vector expressing RPE65 (rAAV2-CB-hRPE65) in adults and children with retinal degeneration caused by RPE65 mutations.DesignNonrandomized, multicenter clinical trial.ParticipantsEight adults and 4 children, 6 to 39 years of age, with Leber congenital amaurosis (LCA) or severe early-childhood–onset retinal degeneration (SECORD).MethodsPatients received a subretinal injection of rAAV2-CB-hRPE65 in the poorer-seeing eye, at either of 2 dose levels, and were followed up for 2 years after treatment.Main Outcome MeasuresThe primary safety measures were ocular and nonocular adverse events. Exploratory efficacy measures included changes in best-corrected visual acuity (BCVA), static perimetry central 30° visual field hill of vision (V30) and total visual field hill of vision (VTOT), kinetic perimetry visual field area, and responses to a quality-of-life questionnaire.ResultsAll patients tolerated subretinal injections and there were no treatment-related serious adverse events. Common adverse events were those associated with the surgical procedure and included subconjunctival hemorrhage in 8 patients and ocular hyperemia in 5 patients. In the treated eye, BCVA increased in 5 patients, V30 increased in 6 patients, VTOT increased in 5 patients, and kinetic visual field area improved in 3 patients. One subject showed a decrease in BCVA and 2 patients showed a decrease in kinetic visual field area.ConclusionsTreatment with rAAV2-CB-hRPE65 was not associated with serious adverse events, and improvement in 1 or more measures of visual function was observed in 9 of 12 patients. The greatest improvements in visual acuity were observed in younger patients with better baseline visual acuity. Evaluation of more patients and a longer duration of follow-up will be needed to determine the rate of uncommon or rare side effects or safety concerns

Measurement and Reproducibility of Preserved Ellipsoid Zone Area and Preserved Retinal Pigment Epithelium Area in Eyes With Choroideremia

PURPOSE: To identify valid and reproducible methods for quantifying anatomic outcome measures for eyes with choroideremia (CHM) in clinical trials. DESIGN: Reliability analysis study. METHODS: In this multicenter study, patients with confirmed genetic diagnosis of CHM were enrolled. All cases underwent spectral-domain optical coherence tomography (SDOCT) and fundus autofluorescence (FAF) imaging. Two graders independently delineated boundaries of preserved autofluorescence (PAF) and pre-served ellipsoid zone (EZ) on FAF and OCT images, respectively. The results of the 2 independent gradings of both FAF and OCT images were compared to assess the reproducibility of the grading methods. RESULTS: A total of 148 eyes from 75 cases were included. In 21% of eyes PAF and in 43% of eyes preserved EZ had extended beyond the image capture area. After exclusion of these eyes and low-quality images, 114 FAF and 77 OCT images were graded. The mean PAF areas from 2 independent gradings were 3.720 +/- 3.340 mm(2) and 3.692 +/- 3.253 mm2, respectively. Intraclass correlation coefficient (ICC) for these gradings was 0.996. The mean preserved EZ areas from 2 independent gradings were 2.746 +/- 2.319 mm2 and 2.858 2.446 mm2, respectively. ICC for these gradings was 0.991. CONCLUSIONS: Quantifying preserved retinal pigment epithelium and EZ areas on FAF and OCT images, respectively, in CHM patients is highly reproducible. These variables would be potential anatomic outcome measures for CHM clinical trials and could be studied and tracked longitudinally in choroideremia. (C) 2017 Elsevier Inc. All rights reserved.Peer reviewe