Mechanisms of emmetropization and what might go wrong in myopia.

Studies in animal models and humans have shown that refractive state is optimized during postnatal development by a closed-loop negative feedback system that uses retinal image defocus as an error signal, a mechanism called emmetropization. The sensor to detect defocus and its sign resides in the retina itself. The retina and/or the retinal pigment epithelium (RPE) presumably releases biochemical messengers to change choroidal thickness and modulate the growth rates of the underlying sclera. A central question arises: if emmetropization operates as a closed-loop system, why does it not stop myopia development? Recent experiments in young human subjects have shown that (1) the emmetropic retina can perfectly distinguish between real positive defocus and simulated defocus, and trigger transient axial eye shortening or elongation, respectively. (2) Strikingly, the myopic retina has reduced ability to inhibit eye growth when positive defocus is imposed. (3) The bi-directional response of the emmetropic retina is elicited with low spatial frequency information below 8 cyc/deg, which makes it unlikely that optical higher-order aberrations play a role. (4) The retinal mechanism for the detection of the sign of defocus involves a comparison of defocus blur in the blue (S-cone) and red end of the spectrum (L + M-cones) but, again, the myopic retina is not responsive, at least not in short-term experiments. This suggests that it cannot fully trigger the inhibitory arm of the emmetropization feedback loop. As a result, with an open feedback loop, myopia development becomes "open-loop".

Effects of short-term exposure to red or near-infrared light on axial length in young human subjects.

PURPOSE: To determine whether visible light is needed to elicit axial eye shortening by exposure to long wavelength light. METHODS: Incoherent narrow-band red (620 ± 10 nm) or near-infrared (NIR, 875 ± 30 nm) light was generated by an array of light-emitting diodes (LEDs) and projected monocularly in 17 myopic and 13 non-myopic subjects for 10 min. The fellow eye was occluded. Light sources were positioned 50 cm from the eye in a dark room. Axial length (AL) was measured before and after the exposure using low-coherence interferometry. RESULTS: Non-myopic subjects responded to red light with significant eye shortening, while NIR light induced minor axial elongation (-13.3 ± 17.3 µm vs. +6.5 ± 11.6 µm, respectively, p = 0.005). Only 41% of the myopic subjects responded to red light exposure with a decrease in AL and changes were therefore, on average, not significantly different from those observed with NIR light (+0.2 ± 12.1 µm vs. +1.1 ± 11.2 µm, respectively, p = 0.83). Interestingly, there was a significant correlation between refractive error and induced changes in AL after exposure to NIR light in myopic eyes (r(15) = -0.52, p = 0.03) and induced changes in AL after exposure to red light in non-myopic eyes (r(11) = 0.62, p = 0.02), with more induced axial elongation with increasing refractive error. CONCLUSIONS: Incoherent narrow-band red light at 620 nm induced axial shortening in 77% of non-myopic and 41% of myopic eyes. NIR light did not induce any significant changes in AL in either refractive group, suggesting that the beneficial effect of red laser light therapy on myopia progression requires visible stimulation and not simply thermal energy.

L-opsin expression in chickens is similarly reduced with diffusers and negative lenses.

Previous studies have shown that the expression of L- and M-opsins was reduced in chicken retina when eyes were covered with diffusers. The purpose of the current study was to find out whether this is a result of altered spatial processing during development of deprivation myopia or merely a consequence of light attenuation by the diffusers. Therefore, retinal luminances were matched by neutral density filters in fellow eyes that served as controls for diffuser-treated eyes. Furthermore, the effects of negative lenses on opsins expression were studied. Chickens wore diffusers or -7D lenses for a period of 7 days and refractive state and ocular biometry were measured at the beginning and at the end of the experiment. Retinal tissue was extracted from both eyes to quantify L-, M- and S-opsins expression by qRT-PCR. It was found that L-opsin expression was significantly lower in eyes wearing diffusers, compared to fellow eyes covered with neutral density filters. Interestingly, L-opsin was also reduced in eyes wearing negative lenses. In summary, this study shows that L-opsin expression is reduced due to the loss of high spatial frequencies and general contrast reduction in the retinal image, rather than by a decline in retinal luminance. Moreover, the fact that L-opsin was similarly reduced in eyes treated with negative lenses and diffusers suggests the existence of a common pathway for emmetropization, but it could also be just a consequence of reduced high spatial frequencies and lower contrast.

ON and OFF receptive field processing in the presence of optical scattering.

The balance of ON/OFF pathway activation in the retina plays a role in emmetropization. A new myopia control lens design uses contrast reduction to down-regulate a hypothesized enhanced ON contrast sensitivity in myopes. The study thus examined ON/OFF receptive field processing in myopes and non-myopes and the impact of contrast reduction. A psychophysical approach was used to measure the combined retinal-cortical output in the form of low-level ON and OFF contrast sensitivity with and without contrast reduction in 22 participants. ON responses were lower than OFF responses (ON 1.25 ± 0.03 vs. OFF 1.39 ± 0.03 log(CS); p < 0.0001) and myopes showed generally reduced sensitivities (myopes 1.25 ± 0.05 vs. non-myopes 1.39 ± 0.05 log(CS); p = 0.05). These findings remained unaffected by contrast reduction (p > 0.05). The study suggests that perceptual differences in ON and OFF signal processing between myopes and non-myopes exist but cannot explain how contrast reduction can inhibit myopia development.

GABAB Receptor Activation Affects Eye Growth in Chickens with Visually Induced Refractive Errors.

This study aims to explore the role of GABAB receptors in the development of deprivation myopia (DM), lens-induced myopia (LIM) and lens-induced hyperopia (LIH). Chicks were intravitreally injected with 25 µg baclofen (GABABR agonist) in one eye and saline into the fellow eye. Choroidal thickness (ChT) was measured via OCT before and 2, 4, 6, 8, 24 h after injection. ChT decreased strongly at 6 and 8 h after baclofen injection and returned back to baseline level after 24 h. Moreover, chicks were monocularly treated with translucent diffusers, -7D or +7D lenses and randomly assigned to baclofen or saline treatment. DM chicks were injected daily into both eyes, while LIM and LIH chicks were monocularly injected into the lens-wearing eyes, for 4 days. Refractive error, axial length and ChT were measured before and after treatment. Dopamine and its metabolites were analyzed via HPLC. Baclofen significantly reduced the myopic shift and eye growth in DM and LIM eyes. However, it did not change ChT compared to respective saline-injected eyes. On the other hand, baclofen inhibited the hyperopic shift and choroidal thickening in LIH eyes. All the baclofen-injected eyes showed significantly lower vitreal DOPAC content. Since GABA is an inhibitory ubiquitous neurotransmitter, interfering with its signaling affects spatial retinal processing and therefore refractive error development with both diffusers and lenses.

Imposed positive defocus changes choroidal blood flow in young human subjects.

PURPOSE: It has previously been found that imposing positive defocus changes axial length and choroidal thickness after only 30 min. In the present study, we investigated whether these changes may result from an altered choroidal blood flow. METHODS: Eighteen young adult subjects watched a movie from a large screen (65 in.) in a dark room at 2 m distance. A 15-min wash-out period was followed by 30 min of watching the movie with a monocular positive defocus (+ 2.5D). Changes in axial length and ocular blood flow were measured before and after the defocus, by using low-coherent interferometer (LS 900, Haag-Streit, Switzerland) and a laser speckle flowgraphy (LSFG) RetFlow unit (Nidek Co., LTD, Japan), respectively. Three regions were analyzed: (1) the macular area, where choroidal blood flow can be measured, (2) the optic nerve head (ONH), and (3) retinal vessel segments. RESULTS: Changes in choroidal blood flow were significantly and negatively correlated with changes in axial length that followed positive defocus in exposed eyes (R = - 0.67, p < 0.01). The absolute values of changes in choroidal blood flow in the defocused eyes were significantly larger than in the fellow control eyes (2.35 ± 2.16 AU vs. 1.37 ± 1.44 AU, respectively, p < 0.05). ONH and retinal blood flow were not associated with the induced changes in axial length. CONCLUSIONS: Positive defocus selectively alters choroidal, but not retinal or ONH blood flow in young human subjects after short-term visual exposure. The results suggest that blood flow modulation is involved in the mechanism of choroidal responses to optical defocus.

Studies on the interactions of retinal dopamine with choroidal thickness in the chicken.

PURPOSE: Recently, an increasing number of studies relied on the assumption that visually induced changes in choroidal thickness can serve as a proxy to predict future axial eye growth. The retinal signals controlling choroidal thickness are, however, not well defined. We have studied the potential roles of dopamine, released from the retina, in the choroidal response in the chicken. METHODS: Changes in retinal dopamine release and choroidal thickness changes were induced by intravitreal injections of either atropine (250 µg or 360 nMol), atropine combined with a dopamine antagonist, spiperone (500 µMol), or spiperone alone and were tracked by optical coherence tomography (OCT). To visually stimulate dopamine release, other chicks were exposed to flicker light of 1, 10, or 400 Hz (duty cycle 0.2) and choroidal thickness was tracked. In all experiments, dopamine and 3,4-Dihydroxyphenylacetic acid (DOPAC) were measured in vitreous, retina, and choroid by high-performance liquid chromatography with electrochemical detection (HLPC-ED). The distribution of the rate-limiting enzyme of dopamine synthesis, tyrosine hydroxylase (TH), neuronal nitric oxide synthase (nNOS), vascular endothelial growth factor (VEGF), and alpha2A adrenoreceptors (alpha2A-ADR) was studied in the choroid by immunofluorescence. RESULTS: The choroid thickened strongly in atropine-injected eyes, less so in atropine + spiperone-injected eyes and became thinner over the day in spiperone alone-, vehicle-, or non-injected eyes. Flickering light at 20 lx, both 1 and 10 Hz, prevented diurnal choroidal thinning, compared to 400 Hz, and stimulated retinal dopamine release. Correlation analysis showed that the higher retinal dopamine levels or release, the thicker became the choroid. TH-, nNOS-, VEGF-, and alpha2A adrenoreceptor-positive nerve fibers were localized in the choroid around lacunae and in the walls of blood vessels with colocalization of TH and nNOS, and TH and VEGF. CONCLUSIONS: Retinal DOPAC and dopamine levels were positively correlated with choroidal thickness. TH-positive nerve fibers in the choroid were closely associated with peptides known to play a role in myopia development. Findings are in line with the hypothesis that dopamine is related to retinal signals controlling choroidal thickness.

Myopia: why the retina stops inhibiting eye growth.

In myopia, the eye grows too long, and the image projected on the retina is poorly focused when subjects look at a distance. While the retina normally controls eye growth by visual processing, it seems to give up during myopia development. But what has changed? To determine whether the sharp image is in front or behind the retinal plane, a comparison of image sharpness in red and blue would provide a reliable cue because focal planes are about 1.3 D apart due to longitudinal chromatic aberration (LCA). However, up to now, it could not be demonstrated that the retina does, in fact, such a comparison. We used a new approach: movies were digitally filtered in real time to present either the blue channel of the RGB color format unfiltered while green and red were blurred ("blue in focus"), or the red channel was unfiltered while green and blue were blurred ("red in focus") accordingly to the human LCA function. Here we show that, even though filtered movies looked similar, eyes became significantly shorter when the movie was sharp in the red plane but became longer when it was presented sharp in the blue plane. Strikingly, the eyes of young subjects who were already myopic did not respond at all-showing that their retina could no longer decode the sign of defocus based on LCA. Our findings resolve a long-standing question as to how the human retina detects the sign of defocus. It also suggests a new non-invasive strategy to inhibit early myopia development: keeping the red image plane on a computer screen sharp but low pass filtering the blue.

Impact of cone abundancy ratios and light spectra on emmetropization in chickens.

We had previously found that chicken eyes with normal visual experience grow larger when they have more L cones, relative to M cones. It is not known whether also S cone abundancies may affect eye size, whether cone abundancy ratios can also affect the amount of deprivation myopia that is induced by diffusers in front of the eyes, and whether broadband white light with added energy in the blue may reduce the development of deprivation myopia. Therefore, chickens were monocularly treated with diffusers and raised under three different light conditions with increasing amounts of energy in the blue but with matched total illuminance. L, M and S cones were counted in fresh retinal tissues after the experiments. It was found that adding energy in the blue did not significantly inhibit deprivation myopia, nor did it make uncovered eyes more hyperopic. However, more S cones, relative to L cones, were correlated with more hyperopic refractions in eyes with normal vision. M to L, L to S and M to S cone ratios were also correlated with the amount of induced deprivation myopia. Interestingly, in deprivation myopia, the correlations between cone abundancy ratios with refractive states had reverted signs: eyes with more S cones developed more myopia. Since cone abundancy ratios remained correlated in both eyes, no matter whether eyes had normal vision, were deprived or were exposed to different light spectra, they appear genetically determined. We conclude that, among other factors, inherited cone abundancy ratios determine both normal refractive development and deprivation myopia in the chicken while adding more blue light to a broadband light spectrum had no effect.

Transient Eye Shortening During Reading Text With Inverted Contrast: Effects of Refractive Error and Letter Size.

Purpose: Myopes have a reduced ability to elicit transient axial eye shortening after imposed positive defocus, which may be due to changes in the biochemical signaling cascade controlling choroidal thickness. We have investigated whether reading with inverted text contrast can still elicit transient axial eye shortening in myopes, like it has been shown in emmetropes. Methods: Changes in axial length before and after reading were measured with the Lenstar LS-900. Text with inverted contrast was read from a large screen at 2 m distance (angular subtense 35.9°, screen luminance matched in all conditions to 86 ± 7 cd/m²) for 30 minutes. Moreover, we investigated the effects of letter sizes. Two text sizes were tested: "small" text (letter height 13.75 arcmin) and "large" text (letter height 34.39 arcmin). Results: Reading text with inverted contrast induced eye shortening (-10.2 ± 9.5 µm) in myopic eyes (n = 11; refraction -3.5 ± 1.9 diopters [D]), showing that an inhibitory signal was still generated by the retina as in emmetropes. In 15 subjects (refraction +1.7 to -4.2 D) we found that small text does not elicit significant differences in axial length (P = 0.09). However, with large text, changes in axial length were clearly different for the both contrast polarities (standard contrast, +1.7 ± 9.0 µm; inverted contrast, -9.7 ± 8.9 µm; P = 0.0017). Conclusions: Although positive defocus may not be an effective intervention to inhibit further eye growth in myopes, other visual stimuli can still trigger choroidal thickening and possibly generate signals to decrease myopia progression. Translational Relevance: Our results have shown the optimized text features, which may have a positive impact on myopia control.

Positions of the horizontal and vertical centre of rotation in eyes with different refractive errors.

PURPOSE: To determine how the position of the centre of rotation of the eyeball is related to axial length and refractive error when horizontal and vertical eye movements are performed. METHODS: A custom-built eye tracker was used that determined the centre of rotation of the eye (COR) from lateral displacements of the pupil centre. Horizontal and vertical eye movements were studied in the right eyes, and each measurement performed five times in 59 subjects (32 females) with an average age of 36.6 ± 9.1 years. Spherical equivalent refractive errors ranged from -9.7 to +6.8 D with an average error of -1.5 ± 2.9 D. Axial lengths were measured with the ZEISS IOL Master 500. RESULTS: The mean horizontal centre of rotation (COR) of the right eye for a saccade from 0° to ±11.9° was 15.3 ± 1.5 mm behind the corneal apex, while the average vertical COR for the same angle of eccentricity was 12.5 ± 1.4 mm, indicating that the horizontal COR was 2.8 ± 1.7 mm behind the vertical COR. In right eyes, horizontal COR was significantly correlated with axial length (r = 0.28, p = 0.02) but not with the spherical equivalent refractive error (r = 0.39, p = 0.90). Similarly, vertical COR was significantly correlated with axial length (r = 0.25, p = 0.03) but not with the spherical equivalent refractive error (r = 0.17, p = 0.90). CONCLUSIONS: While it might be expected that the COR is dependent on axial length, the correlation was not strong. Interestingly, the location of the COR was substantially different for horizontal and vertical eye movements which may relate to the flatter curvature of the eyeball in the vertical meridian, compared to the horizontal, as described in previous studies.

Mice Lacking Gpr179 with Complete Congenital Stationary Night Blindness Are a Good Model for Myopia.

Mutations in GPR179 are one of the most common causes of autosomal recessive complete congenital stationary night blindness (cCSNB). This retinal disease is characterized in patients by impaired dim and night vision, associated with other ocular symptoms, including high myopia. cCSNB is caused by a complete loss of signal transmission from photoreceptors to ON-bipolar cells. In this study, we hypothesized that the lack of Gpr179 and the subsequent impaired ON-pathway could lead to myopic features in a mouse model of cCSNB. Using ultra performance liquid chromatography, we show that adult Gpr179-/- mice have a significant decrease in both retinal dopamine and 3,4-dihydroxyphenylacetic acid, compared to Gpr179+/+ mice. This alteration of the dopaminergic system is thought to be correlated with an increased susceptibility to lens-induced myopia but does not affect the natural refractive development. Altogether, our data added a novel myopia model, which could be used to identify therapeutic interventions.

"Emmetropic, but not myopic human eyes distinguish positive defocus from calculated defocus in monochromatic red light".

Studies in animal models have provided evidence that broadband light and chromatic cues are necessary for successful emmetropization. We have studied this question in young human subjects by measuring short-term changes in axial length when they watched movies with calculated defocus (2.5D) or optically defocused movies (+2.5D) with red interference filters (620 ± 10 nm). Since filters cut luminance down by a factor of 10, a control experiment with neutral density filters (ND 1.0) was done. Ten myopes and 10 emmetropes were studied. Four experimental conditions were tested on two separate days. On the first day, movies with calculated defocus, and defocused by positive lenses were watched with ND filters. On the second day, movies with the same defocus patterns were watched with the red filters. Movies were presented on a large TV screen (LG OLED65C9, 65″) in a dark room at 2 m distance for 30 min. Changes in axial length before and after each stimulation were measured with the Lenstar (LS 900, with autopositioning system; Haag-Streit). Interestingly, the effects of calculated defocus or optical positive defocus on axial length were suppressed by 1.0 ND filters in myopes and emmetropes, with no clear trend. In contrast, narrow-band red light suppressed eye elongation with calculated defocus but not eye shortening with positive defocus in emmetropes. In myopes, as previously found in white light, there was a trend of axial eye elongation with positive lenses. In conclusion, the effect of positive lenses on eye growth did not require chromatic cues.

Functional integration of eye tissues and refractive eye development: Mechanisms and pathways.

Refractive eye development is a tightly coordinated developmental process. The general layout of the eye and its various components are established during embryonic development, which involves a complex cross-tissue signaling. The eye then undergoes a refinement process during the postnatal emmetropization process, which relies heavily on the integration of environmental and genetic factors and is controlled by an elaborate genetic network. This genetic network encodes a multilayered signaling cascade, which converts visual stimuli into molecular signals that guide the postnatal growth of the eye. The signaling cascade underlying refractive eye development spans across all ocular tissues and comprises multiple signaling pathways. Notably, tissue-tissue interaction plays a key role in both embryonic eye development and postnatal eye emmetropization. Recent advances in eye biometry, physiological optics and systems genetics of refractive error have significantly advanced our understanding of the biological processes involved in refractive eye development and provided a framework for the development of new treatment options for myopia. In this review, we summarize the recent data on the mechanisms and signaling pathways underlying refractive eye development and discuss new evidence suggesting a wide-spread signal integration across different tissues and ocular components involved in visually guided eye growth.

A low-cost, high-performance video-based binocular eye tracker for psychophysical research.

We describe a high-performance, pupil-based binocular eye tracker that approaches the performance of a well-established commercial system, but at a fraction of the cost. The eye tracker is built from standard hardware components, and its software (written in Visual C++) can be easily implemented. Because of its fast and simple linear calibration scheme, the eye tracker performs best in the central 10 degrees of the visual field. The eye tracker possesses a number of useful features: (1) automated calibration simultaneously in both eyes while subjects fixate four fixation points sequentially on a computer screen, (2) automated realtime continuous analysis of measurement noise, (3) automated blink detection, (4) and realtime analysis of pupil centration artifacts. This last feature is critical because it is known that pupil diameter changes can be erroneously registered by pupil-based trackers as a change in eye position. We evaluated the performance of our system against that of a wellestablished commercial system using simultaneous measurements in 10 participants. We propose our low-cost eye tracker as a promising resource for studies of binocular eye movements.

A retrospective analysis of the therapeutic effects of 0.01% atropine on axial length growth in children in a real-life clinical setting.

BACKGROUND: Several randomized controlled studies have demonstrated the beneficial effects of 0.01% atropine eye drops on myopia progression in children. However, treatment effects may be different in a routine clinical setting. We performed a retrospective analysis of our clinical data from children to investigate the effect of 0.01% atropine eye drops on myopia progression in a routine clinical setting. METHODS: Atropine-treated children were asked to instill one drop of 0.01% atropine in each eye every evening at 5 days a week. Myopic children who did not undergo atropine treatment served as controls. Objective refraction and ocular biometry of 80 atropine-treated and 103 untreated children at initial visit and 1 year later were retrospectively analyzed. RESULTS: Myopic refractions in the treated and untreated children at initial visit ranged from -0.625 to -15.25 D (-4.21 ± 2.90 D) and from -0.125 to -9.375 D (-2.92 ± 1.77 D), respectively. Ages at initial visit ranged from 3.2 to 15.5 years (10.1 ± 2.7 years) in the treated and from 3.4 to 15.5 years (11.2 ± 3.0 years) in untreated children. Two-factor ANOVA for age and atropine effects on axial length growth confirmed that axial length growth rates declined with age (p<0.0001) and revealed a significant inhibitory effect of atropine on axial length growth (p<0.0015). The atropine effect on axial length growth averaged to 0.08 mm (28%) inhibition per year. Effects on refraction were not statistically significant. CONCLUSION: The observed atropine effects were not very distinctive: Statistical analysis confirmed that atropine reduced axial length growth, but to an extent of minor clinical relevance. It was also shown that beneficial effects of 0.01% atropine may not be obvious in each single case, which should be communicated with parents and resident ophthalmologists.

Loss of Gap Junction Delta-2 (GJD2) gene orthologs leads to refractive error in zebrafish.

Myopia is the most common developmental disorder of juvenile eyes, and it has become an increasing cause of severe visual impairment. The GJD2 locus has been consistently associated with myopia in multiple independent genome-wide association studies. However, despite the strong genetic evidence, little is known about the functional role of GJD2 in refractive error development. Here, we find that depletion of gjd2a (Cx35.5) or gjd2b (Cx35.1) orthologs in zebrafish, cause changes in the biometry and refractive status of the eye. Our immunohistological and scRNA sequencing studies show that Cx35.5 (gjd2a) is a retinal connexin and its depletion leads to hyperopia and electrophysiological changes in the retina. These findings support a role for Cx35.5 (gjd2a) in the regulation of ocular biometry. Cx35.1 (gjd2b) has previously been identified in the retina, however, we found an additional lenticular role. Lack of Cx35.1 (gjd2b) led to a nuclear cataract that triggered axial elongation. Our results provide functional evidence of a link between gjd2 and refractive error.

Natural environment statistics in the upper and lower visual field are reflected in mouse retinal specializations.

Pressures for survival make sensory circuits adapted to a species' natural habitat and its behavioral challenges. Thus, to advance our understanding of the visual system, it is essential to consider an animal's specific visual environment by capturing natural scenes, characterizing their statistical regularities, and using them to probe visual computations. Mice, a prominent visual system model, have salient visual specializations, being dichromatic with enhanced sensitivity to green and UV in the dorsal and ventral retina, respectively. However, the characteristics of their visual environment that likely have driven these adaptations are rarely considered. Here, we built a UV-green-sensitive camera to record footage from mouse habitats. This footage is publicly available as a resource for mouse vision research. We found chromatic contrast to greatly diverge in the upper, but not the lower, visual field. Moreover, training a convolutional autoencoder on upper, but not lower, visual field scenes was sufficient for the emergence of color-opponent filters, suggesting that this environmental difference might have driven superior chromatic opponency in the ventral mouse retina, supporting color discrimination in the upper visual field. Furthermore, the upper visual field was biased toward dark UV contrasts, paralleled by more light-offset-sensitive ganglion cells in the ventral retina. Finally, footage recorded at twilight suggests that UV promotes aerial predator detection. Our findings support that natural scene statistics shaped early visual processing in evolution.