Effects of morning and evening exposures to blue light of varying illuminance on ocular growth rates and ocular rhythms in chicks.

Recent evidence indicates that moderate levels of blue light are sufficient to suppress the nighttime rise in serum melatonin in humans, suggesting that luminous screens may be deleterious to sleep cycles and to other functions. Little is known however, about the effects of exposures to blue light on ocular physiology. We tested the effects of transient blue light exposures of various illuminances on ocular growth rates and ocular rhythms in chicks. 10-day old chicks were exposed to narrow band blue light (460 nm) of specific illuminance for 4 h in the evening (ZT8-ZT12) or the morning (ZT0-ZT4) for 9 days; for the remainder of the day they were in white light (588 lux). For the evening, four illuminances were tested: 0.15 lux (n = 15), 200 lux (radiometrically matched to white controls; n = 16), 600 lux (photometrically matched to white controls; n = 15) or 1000 lux (n = 8). The 600 lux condition was also tested using a 2-h duration (n = 8). The 200 and 600 lux conditions were extended to 14 and 21 days (n = 8 each). For morning exposures, 200 lux (n = 9), 600 lux (n = 9) and 1000 lux (n = 8) were tested. Controls remained in white light (n = 23). Ocular dimensions were measured by A-scan ultrasonography on days 1 and 9 to assess growth rates. On day 8 or 9, measurements were made at 6-h intervals over 24 h starting at noon to assess rhythm parameters. Evening exposure to blue light stimulated ocular growth rates relative to controls for all except the bright condition (0.15 lux, 200 lux, 600 lux vs bright and white respectively: 845 µm, 838 µm, 898 µm vs 733 µm and 766 µm; p < 0.05 for all comparisons). 2 h exposures to 600 lux were similarly effective (915 µm vs 766 µm; p < 0.05). Morning exposures only resulted in growth stimulation for the 200 lux condition (200 lux vs white: 884 µm vs 766 µm; p < 0.05). Furthermore, for this group only, growth of the anterior chamber had a significant contribution to the overall effect (vs white: p < 0.05), and choroids showed significant thickening. For evening exposures to 200 and 600 lux, the growth stimulatory effect lasted for 14 days (p = 0.01); by 21 days only the 600 lux group still differed (p < 0.0001). Evening exposures caused circadian disruptions in the choroidal thickness rhythms, and morning exposures disrupted both axial and choroidal rhythms. Exposure to 4 h of blue light at lower illuminances (less than 1000 lux) at transition times of lights-on and lights-off stimulates ocular growth rates and affects ocular rhythms in chicks, suggesting that such exposures may be deleterious to emmetropization in children.

Visual Image Quality Impacts Circadian Rhythm-Related Gene Expression in Retina and in Choroid: A Potential Mechanism for Ametropias.

Purpose: Stimulated by evidence implicating diurnal/circadian rhythms and light in refractive development, we studied the expression over 24 hours of selected clock and circadian rhythm-related genes in retina/retinal pigment epithelium (RPE) and choroid of experimental ametropias in chicks. Methods: Newly hatched chicks, entrained to a 12-hour light/dark cycle for 12 to 14 days, either experienced nonrestricted vision OU (i.e., in both eyes) or received an image-blurring diffuser or a minus 10-diopter (D) or a plus 10-D defocusing lens over one eye. Starting 1 day later and at 4-hour intervals for 24 hours, the retina/RPE and choroid were separately dissected. Without pooling, total RNA was extracted, converted to cDNA, and assayed by quantitative PCR for the expression of the following genes: Opn4m, Clock, Npas2, Per3, Cry1, Arntl, and Mtnr1a. Results: The expression of each gene in retina/RPE and in choroid of eyes with nonrestricted vision OU varied over 24 hours, with equal levels OU for most genes and times. Altered visual input influenced gene expression in complex patterns that varied by gene, visual input, time, and eye, affecting experimental eyes with altered vision and also contralateral eyes with nonrestricted vision. Discussion: Altering visual input in ways known to induce ametropias alters the retinal/RPE and choroidal expression of circadian rhythm-related genes, further linking circadian biology with eye growth regulation. While further investigations are needed, studying circadian processes may help understand refractive mechanisms and the increasing myopia prevalence in contemporary societies where lighting patterns can desynchronize endogenous rhythms from the natural environmental light/dark cycle.

The effects of brief high intensity light on ocular growth in chicks developing myopia vary with time of day.

Evidence suggests that the relevant variable in the anti-myopigenic effect of increased time spent outdoors is the increase in light intensity. Because light is the strongest Zeitgeber, it is plausible that the effects of bright light exposure depend on time of day, and may impact circadian rhythms. In these studies, we asked whether the effects on eye growth rates and ocular rhythms of brief daily exposures to bright light differed depending on time of day in eyes developing myopia in response to form deprivation (FD) or negative lens-induced hyperopic defocus (LENS). We also studied the effects of concurrent exposures to brief hyperopic defocus and bright light. Exp. 1: Starting at 12d, chicks wearing monocular diffusers or -10 D lenses were exposed to 3 daily hours (h) of bright light (30K lux) in the morning (FD: n = 12; LENS: n = 7) or evening (FD: n = 21; LENS: n = 7) for a total of 6 exposures. Controls wore diffusers or lenses but weren't exposed to bright light ("not bright" FD: n = 14; LENS: n = 9). Exp. 2: Untreated chicks were exposed to 3 h bright light in the morning (n = 12) or evening (n = 14) for a total of 6 exposures. Controls were not exposed to bright light (n = 11). Exp. 3: Chicks were exposed to 2 h simultaneous monocular hyperopic defocus and bright light in the morning (n = 11), mid-day (n = 7) or evening (n = 8) for 5 exposures. "Not bright" lens-wearing controls were data from published work (Nickla et al., 2017). High frequency A-scan ultrasonography was done at the start and end to measure growth rates. The FD group in Exp. 1 and the morning and evening groups in Exp. 3 were measured at 6-h intervals over the final 24 h to determine parameters for the rhythms in axial length and choroidal thickness. 1. Brief bright light in the evening inhibited eye growth in eyes wearing diffusers or lenses relative to "not bright" controls(interocular differences: FD: 316 vs 468 µm, p = 0.026; LENS: 233 vs 438 µm, p = 0.03); morning bright light had no effect. There was no differential effect of time of day of exposure on the rhythm in axial length; for choroid thickness, "time" accounted for the variance between groups (2-way ANOVA interaction p = 0.027). 2. In binocularly untreated chicks, bright light in the morning had a small but significant growth stimulatory effect relative to evening exposures (803  vs 679 µm/7d; post-hoc p = 0.048). 3. Eyes exposed to simultaneous hyperopic defocus and bright light were significantly more inhibited relative to "not bright" controls for morning exposures (interocular differences: -207 vs 69 µm; p < 0.01). In conclusion, the effects of brief periods of bright light on the growth controller depended on the time of day of exposure and on the contemporaneous state ofocular growth .

Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration.

Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the Abca4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in Abca4-/- mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness. No effective treatment currently exists for STGD1. Here we show by several approaches that ABCA4 is additionally expressed in RPE cells. (i) By in situ hybridization analysis and by RNA-sequencing analysis, we show the Abca4 mRNA is expressed in human and mouse RPE cells. (ii) By quantitative immunoblotting, we show that the level of ABCA4 protein in homogenates of wild-type mouse RPE is about 1% of the level in neural retina homogenates. (iii) ABCA4 immunofluorescence is present in RPE cells of wild-type and Mertk-/- but not Abca4-/- mouse retina sections, where it colocalizes with endolysosomal proteins. To elucidate the role of ABCA4 in RPE cells, we generated a line of genetically modified mice that express ABCA4 in RPE cells but not in photoreceptors. Mice from this line on the Abca4-/- background showed partial rescue of photoreceptor degeneration and decreased lipofuscin accumulation compared with nontransgenic Abca4-/- mice. We propose that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis of distal photoreceptor OS. ABCA4 deficiency in the RPE may play a role in the pathogenesis of STGD1.

Complement modulation in the retinal pigment epithelium rescues photoreceptor degeneration in a mouse model of Stargardt disease.

Recessive Stargardt macular degeneration (STGD1) is caused by mutations in the gene for the ABCA4 transporter in photoreceptor outer segments. STGD1 patients and Abca4-/- (STGD1) mice exhibit buildup of bisretinoid-containing lipofuscin pigments in the retinal pigment epithelium (RPE), increased oxidative stress, augmented complement activation and slow degeneration of photoreceptors. A reduction in complement negative regulatory proteins (CRPs), possibly owing to bisretinoid accumulation, may be responsible for the increased complement activation seen on the RPE of STGD1 mice. CRPs prevent attack on host cells by the complement system, and complement receptor 1-like protein y (CRRY) is an important CRP in mice. Here we attempted to rescue the phenotype in STGD1 mice by increasing expression of CRRY in the RPE using a gene therapy approach. We injected recombinant adeno-associated virus containing the CRRY coding sequence (AAV-CRRY) into the subretinal space of 4-wk-old Abca4-/- mice. This resulted in sustained, several-fold increased expression of CRRY in the RPE, which significantly reduced the complement factors C3/C3b in the RPE. Unexpectedly, AAV-CRRY-treated STGD1 mice also showed reduced accumulation of bisretinoids compared with sham-injected STGD1 control mice. Furthermore, we observed slower photoreceptor degeneration and increased visual chromophore in 1-y-old AAV-CRRY-treated STGD1 mice. Rescue of the STGD1 phenotype by AAV-CRRY gene therapy suggests that complement attack on the RPE is an important etiologic factor in STGD1. Modulation of the complement system by locally increasing CRP expression using targeted gene therapy represents a potential treatment strategy for STGD1 and other retinopathies associated with complement dysregulation.

Biocompatibility of a Synthetic Biopolymer for the Treatment of Rhegmatogenous Retinal Detachment.

OBJECTIVE: The aim of this study is to evaluate the retinal safety and toxicity of a novel synthetic biopolymer to be used as a patch to treat rhegmatogenous retinal detachment. METHODS: Thirty one adult wild type albino mice were divided in 2 groups. In Group A (n=9) 0.2 µl balanced salt solution (BSS) and in Group B (n=22), 0.2 µl biopolymer was injected in the subretinal space. Trans-scleral subretinal injection was performed in one eye and the fellow eye was used as control. In both groups, in vivo color fundus photography, electroretinogram (ERG), spectral domain optical coherence tomography (SD-OCT) were performed before injection and at days 7 and 14 post-intervention. Histological analysis was performed following euthanization at days 1, 7 and 21 post-injection. RESULTS: The biopolymer was visualized in the subretinal space in vivo by SD-OCT and post-life by histology up to 1 week after the injection. There were no significant differences in ERG parameters between the two groups at 1 and 2 weeks post-injection. Minimal inflammatory response and loss of photoreceptor cells was only observed in the immediate proximity of the site of scleral perforation, which was similar in both groups. Overall integrity of the outer, inner retina and retinal pigment epithelial (RPE) layers was unaffected by the presence of the biopolymer in the subretinal space. CONCLUSIONS: Functional and histological evaluation suggests that the synthetic biopolymer is non-inflammatory and non-toxic to the eye. It may represent a safe therapeutic agent in the future, for the treatment of rhegmatogenous retinal detachment.

Overexpression of rod photoreceptor glutamic acid rich protein 2 (GARP2) increases gain and slows recovery in mouse retina.

BACKGROUND: The rod photoreceptor cGMP-gated cation channel, consisting of three α- and one ß subunit, controls ion flow into the rod outer segment (ROS). In addition to the ß-subunit, the Cngb1 locus encodes an abundant soluble protein, GARP2 that binds stoichiometrically to rod photoreceptor cGMP phosphodiesterase type 6 (PDE6). To examine the in vivo functional role of GARP2 we generated opsin promoter-driven transgenic mice overexpressing GARP2 three-fold specifically in rod photoreceptors. RESULTS: In the GARP2 overexpressing transgenic mice (tg), the endogenous channel ß-subunit, cGMP phosphodiesterase α-subunit, peripherin2/RDS and guanylate cyclase I were present at WT levels and were properly localized within the ROS. While localized properly within ROS, two proteins cGMP phosphodiesterase α-subunit (1.4-fold) and cGMP-gated cation channel α-subunit (1.2-fold) were moderately, but significantly elevated. Normal stratification of all retinal layers was observed, and ROS were stable in numbers but were 19% shorter than WT. Analysis of the photoresponse using electroretinography (ERG) showed that tg mice exhibit no change in sensitivity indicating overall normal rod function, however two parameters of the photoresponse significantly differed from WT responses. Fitting of the rising phase of the ERG a-wave to an accepted model of phototransduction showed a two-fold increase in phototransduction gain in the tg mice. The increase in gain was confirmed in isolated retinal tissue and by suction electrode recordings of individual rod photoreceptor cells. A measure of response recovery, the dominant time constant (τD) was elevated 69% in isolated retina compared to WT, indicating slower shutoff of the photoresponse. CONCLUSIONS: GARP2 may participate in regulating visual signal transduction through a previously unappreciated role in regulating phototransduction gain and recovery.

Identification of DES1 as a vitamin A isomerase in Müller glial cells of the retina.

Absorption of a light particle by an opsin-pigment causes photoisomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the resulting apo-opsin requires chemical re-isomerization of the photobleached chromophore. This is carried out by a multistep enzyme pathway called the visual cycle. Accumulating evidence suggests the existence of an alternative visual cycle for regenerating opsins in daylight. Here we identified dihydroceramide desaturase-1 (DES1) as a retinol isomerase and an excellent candidate for isomerase-2 in this alternative pathway. DES1 is expressed in retinal Müller cells, where it coimmunoprecipitates with cellular retinaldehyde binding protein (CRALBP). Adenoviral gene therapy with DES1 partially rescued the biochemical and physiological phenotypes in Rpe65(-/-) mice lacking isomerohydrolase (isomerase-1). Knockdown of DES1 expression by RNA interference concordantly reduced isomerase-2 activity in cultured Müller cells. Purified DES1 had very high isomerase-2 activity in the presence of appropriate cofactors, suggesting that DES1 by itself is sufficient for isomerase activity.

Knockout of GARPs and the ß-subunit of the rod cGMP-gated channel disrupts disk morphogenesis and rod outer segment structural integrity.

Ion flow into the rod photoreceptor outer segment (ROS) is regulated by a member of the cyclic-nucleotide-gated cation-channel family; this channel consists of two subunit types, alpha and beta. In the rod cells, the Cngb1 locus encodes the channel beta-subunit and two related glutamic-acid-rich proteins (GARPs). Despite intensive research, it is still unclear why the beta-subunit and GARPs are coexpressed and what function these proteins serve. We hypothesized a role for the proteins in the maintenance of ROS structural integrity. To test this hypothesis, we created a Cngb1 5'-knockout photoreceptor null (Cngb1-X1). Morphologically, ROSs were shorter and, in most rods that were examined, some disks were misaligned, misshapen and abnormally elongated at periods when stratification was still apparent and degeneration was limited. Additionally, a marked reduction in the level of channel alpha-subunit, guanylate cyclase I (GC1) and ATP-binding cassette transporter (ABCA4) was observed without affecting levels of other ROS proteins, consistent with a requirement for the beta-subunit in channel assembly or targeting of select proteins to ROS. Remarkably, phototransduction still occurred when only trace levels of homomeric alpha-subunit channels were present, although rod sensitivity and response amplitude were both substantially reduced. Our results demonstrate that the beta-subunit and GARPs are necessary not only to maintain ROS structural integrity but also for normal disk morphogenesis, and that the beta-subunit is required for normal light sensitivity of the rods.

The ups and downs of peer review.

This article traces the history of peer review of scientific publications, plotting the development of the process from its inception to its present-day application. We discuss the merits of peer review and its weaknesses, both perceived and real, as well as the practicalities of several major proposed changes to the system. It is our hope that readers will gain a better appreciation of the complexities of the process and, when serving as reviewers themselves, will do so in a manner that will enhance the utility of the exercise. We also propose the development of an international on-line training program for accreditation of potential referees.

The Drosophila ninaG oxidoreductase acts in visual pigment chromophore production.

The Drosophila ninaG mutant is characterized by low levels of Rh1 rhodopsin, because of the inability to transport this rhodopsin from the endoplasmic reticulum to the rhabdomere. ninaG mutants do not affect the biogenesis of the minor opsins Rh4 and Rh6. A genetic analysis placed the ninaG gene within the 86E4-86E6 chromosomal region. A sequence analysis of the 15 open reading frames within this region from the ninaG(P330) mutant allele identified a stop codon in the CG6728 gene. Germ-line transformation of the CG6728 genomic region rescued the ninaG mutant phenotypes, confirming that CG6728 corresponds to the ninaG gene. The NinaG protein belongs to the glucose-methanol-choline oxidoreductase family of flavin adenine dinucleotide-binding enzymes catalyzing hydroxylation and oxidation of a variety of small organic molecules. High performance liquid chromatography analysis of retinoids was used to gain insight into the in vivo role of the NinaG oxidoreductase. The results show that when Rh1 is expressed as the major rhodopsin, ninaG flies fail to accumulate 3-hydroxyretinal. Further, in transgenic flies expressing Rh4 as the major rhodopsin, 3-hydroxyretinal is the major retinoid in ninaG+, but a different retinoid profile is observed in ninaG(P330). These results indicate that the ninaG oxidoreductase acts in the biochemical pathway responsible for conversion of retinal to the rhodopsin chromophore, 3-hydroxyretinal.