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.

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.

Effects of Single and Repeated Intravitreal Applications of Atropine on Choroidal Thickness in Alert Chickens.

INTRODUCTION: Atropine, a muscarinic antagonist, is known since the 19th century to inhibit myopia development in children. One of its effects is that it stimulates choroidal thickening. Thicker choroids, in turn, have been linked to myopia inhibition. We used the atropine-stimulated choroidal response in the chicken to learn more about the time courses and amplitudes of the effects of atropine, as well as whether repeated applications lead to accumulation or desensitization. METHODS: Intravitreal injections containing 250 µg atropine sulfate were performed in 1 eye around 10:00 in the morning, the fellow eye received vehicle. Chickens with bilateral vehicle injections served as controls. Choroidal thickness was measured over the day for every 2-3 h in alert animals, using spectral domain optical coherence tomography, with 3-5 independent measurements in each eye. Three experiments were done - (1) single injection and time course measured over 1 day, (2) single injection and time course measured over 4 days, and (3) daily injections and time course measured over 4 days for measuring the effects of atropine on vitreal, retinal, and choroidal dopamine, and 3,4-dihydroxyphenylacetic acid levels by using high-performance liquid chromatography with electrochemical detection. RESULTS: Atropine induced an increase in choroidal thickness by about 60 percent, with a peak amplitude after about 2 h. The effect persisted only for a few hours and had nearly disappeared by evening. Initially, similar amounts of choroidal thickening were observed in vehicle-injected fellow eyes but recovery to baseline was faster. When atropine was injected daily for 4 days, choroids thickened every day with similar amplitudes and time courses, with no signs of either accumulation or desensitization effects. Interestingly, while dopamine release from the retina was stimulated by atropine and followed approximately, the time course of choroidal thickening, its tissue concentration dropped in the choroid. CONCLUSIONS: Even at relatively high intravitreal doses, effects of atropine on choroidal thickness remained transient, similar to its effects on retinal dopamine. With repeated application every day, the diurnal patterns of choroidal thickening could be reproduced for 4 days with similar amplitudes and time courses. The transient nature of the effects of atropine on the choroid may be relevant for application protocols of atropine against myopia.

Human Dopaminergic Neurons Lacking PINK1 Exhibit Disrupted Dopamine Metabolism Related to Vitamin B6 Co-Factors.

PINK1 loss-of-function mutations cause early onset Parkinson disease. PINK1-Parkin mediated mitophagy has been well studied, but the relevance of the endogenous process in the brain is debated. Here, the absence of PINK1 in human dopaminergic neurons inhibits ionophore-induced mitophagy and reduces mitochondrial membrane potential. Compensatory, mitochondrial renewal maintains mitochondrial morphology and protects the respiratory chain. This is paralleled by metabolic changes, including inhibition of the TCA cycle enzyme mAconitase, accumulation of NAD+, and metabolite depletion. Loss of PINK1 disrupts dopamine metabolism by critically affecting its synthesis and uptake. The mechanism involves steering of key amino acids toward energy production rather than neurotransmitter metabolism and involves cofactors related to the vitamin B6 salvage pathway identified using unbiased multi-omics approaches. We propose that reduction of mitochondrial membrane potential that cannot be controlled by PINK1 signaling initiates metabolic compensation that has neurometabolic consequences relevant to Parkinson disease.

Interactions of cone abundancies, opsin expression, and environmental lighting with emmetropization in chickens.

We had previously found that M to L cone abundancy ratios in the chicken retina are correlated with vitreous chamber depth and refractive state in chickens eyes, when they have normal visual exposure but not when they develop deprivation myopia. The finding suggests an interaction between cone abundancies and emmetropization. In the current study, we analyzed how stable this correlation was against changes in environmental variables and strain differences. We found that the correlation was preserved in two chicken strains, as long as they were raised in the laboratory facilities and not in the animal facilities of the institute. To determine the reasons for this difference, spectral and temporal lighting parameters were better adjusted in both places, whereas temperature, humidity, food, diurnal lighting cycles and illuminance were already matched. It was also verified that both strains of chickens had the same cone opsin amino acid sequences. The correlation between M to L cone abundancy and ocular biometry is highly susceptible to changes in environmental variables. Yet undetermined differences in lighting parameters were the most likely reasons. Other striking findings were that green cone opsin mRNA expression was downregulated when deprivation myopia developed. Similarly, red opsin mRNA was downregulated when chicks wore red spectacles, which made them more hyperopic. In summary, our experiments show that photoreceptor abundancies, opsin expression, and the responses to deprivation, and therefore emmetropization, are surprisingly dependent on subtle differences in lighting parameters.

Effects of 7-Methylxanthine on Deprivation Myopia and Retinal Dopamine Release in Chickens.

INTRODUCTION: Intake of 7-methylxanthine (7-MX), an adenosine receptor (AR) antagonist, has been shown to inhibit school myopia in children and deprivation myopia in rhesus monkeys, but the underlying mechanisms are not known. Also retinal dopamine seems to be involved in the control of eye growth, and in the brain, ARs and dopamine receptors interact widely by heteromerization. We have studied whether 7-MX can inhibit deprivation myopia also in chickens and whether inhibition may involve the retinal dopamine system. METHODS: 7-MX was applied by either tube-feeding (100 µg/g body weight, twice a day) or intravitreal injection (12.5 µg, every other day). Forty-eight 2-week-old chicks wore unilateral diffusers and were randomly assigned to either the tube-feeding group (involving 7-MX, vehicle [xanthan gum], or no feeding, for 13 days) or the intravitreal injection group (involving 7-MX, vehicle, or DMSO, for 8 days). Refractions (REs), ocular biometry (AL, VCD), and scleral and choroidal thickness (ChT) were measured before and after treatment. Dopamine and dihydroxyphenylacetic acid (DOPAC) content were determined in retina and vitreous by HPLC at the end of the experiments. RESULTS: No matter how 7-MX was applied, it did not inhibit deprivation myopia in chicks. No significant differences were observed in RE, VCD, AL, and scleral fibrous layer thickness. Feeding 7-MX produced more choroidal thinning in the open contralateral eye compared to control eyes in the vehicle-fed group (-40 ± 14 vs. -1 ± 7 µm, unpaired t test, p < 0.05). DOPAC and dopamine concentration in vitreous and DOPAC concentration in retina did not change with 7-MX. Vitreal dopamine content was significantly decreased in deprived eyes in the groups fed with the vehicle xanthan gum (paired t test, p < 0.01) but not in 7-MX-treated eyes, perhaps indicating a small effect of 7-MX on dopamine. CONCLUSIONS: In our study, 7-MX had no effect on DM in chicks and only minor effects on ChT and retinal dopamine. It remains unclear whether 7-MX inhibits myopia through a retinal mechanism or whether it acts directly on choroid and sclera. In the latter case, the finding that myopia is suppressed in mammals but not birds might be explained by differences in scleral structure.

Studies on retinal mechanisms possibly related to myopia inhibition by atropine in the chicken.

PURPOSE: While low-dose atropine eye drops are currently widely used to inhibit myopia development in children, the underlying mechanisms are poorly understood. Therefore, we studied possible retinal mechanisms and receptors that are potentially involved in myopia inhibition by atropine. METHODS: A total of 250 µg atropine were intravitreally injected into one eye of 19 chickens, while the fellow eyes received saline and served as controls. After 1 h, 1.5 h, 2 h, 3 h, and 4 h, eyes were prepared for vitreal dopamine (DA) measurements, using high-pressure liquid chromatography with electrochemical detection. Twenty-four animals were kept either in bright light (8500 lx) or standard light (500 lx) after atropine injection for 1.5 h before DA was measured. In 10 chickens, the α2A-adrenoreceptor (α2A-ADR) agonists brimonidine and clonidine were intravitreally injected into one eye, the fellow eye served as control, and vitreal DA content was measured after 1.5 h. In 6 chickens, immunohistochemical analyses were performed 1.5 h after atropine injection. RESULTS: Vitreal DA levels increased after a single intravitreal atropine injection, with a peak difference between both eyes after 1.97 h. DA was also enhanced in fellow eyes, suggesting a systemic action of intravitreally administered atropine. Bright light and atropine (which both inhibit myopia) had additive effects on DA release. Quantitative immunolabelling showed that atropine heavily stimulated retinal activity markers ZENK and c-Fos in cells of the inner nuclear layer. Since atropine was recently found to also bind to α2A-ADRs at doses where it can inhibit myopia, their retinal localization was studied. In amacrine cells, α2A-ADRs were colocalized with tyrosine hydroxylase (TH), glucagon, and nitric oxide synthase, peptides known to play a role in myopia development in chickens. Intravitreal atropine injection reduced the number of neurons that were double-labelled for TH and α2A-ADR. α2A-ADR agonists clonidine and brimonidine (which were also found by other authors to inhibit myopia) severely reduced vitreal DA content in both injected and fellow eyes, compared to eyes of untreated chicks. CONCLUSIONS: Merging our results with published data, it can be concluded that both muscarinic and α2A-adrenergic receptors are expressed on dopaminergic neurons and both atropine and α2A-ADR antagonists stimulate DA release whereas α2A-ADR agonists strongly suppress its release. Stimulation of DA by atropine was enhanced by bright light. Results are in line with the hypothesis that inhibition of deprivation myopia is correlated with DA stimulation, as long as no toxicity is involved.

Demyelination and shrinkage of axons in the retinal nerve fiber layer in chickens developing deprivation myopia.

Placing diffusers in front of the eyes induces deprivation myopia in a variety of animal models. As a result of the low pass filtering of the retinal images, less spatial information is available to the retina which should reduce neural activity. Since it has been found that myelination of axons in the central nervous system is modulated by neuronal activity, we have studied whether ganglion cell axons may shrink in response to the restricted visual input. Young chickens were treated for 5 h or 7 days with frosted diffusers to induce deprivation myopia. Nerve fiber layer thickness was measured in vivo, using B-scan OCT. Refractive states were tracked by IR photoretinoscopy, and UV fundus reflectivity by a custom-built device which flashed an LED centered in the camera aperture and recorded pupil brightness after refractive errors were corrected by trial lenses. Moreover, structure and histology of the retinal nerve fibers layer (RNFL) were analyzed ex vivo using transmission electron microscopy and immunohistochemistry. Since chicks have both non-myelinated and myelinated fibers in their RNFL, the thickness of myelin sheaths (G ratio) was measured, as well as the percentage of myelinated axons and the diameters of unmyelinated axons. Short-term deprivation caused an increase in UV fundus reflectivity already after 5 h (measured as pixel grey levels in the pupil: 28 ±â€¯5 vs. 36 ±â€¯10, p < 0.05) and thinning of the myelin sheaths (higher G ratio), compared to untreated control eyes (0.74 ±â€¯0.01 vs. 0.79 ±â€¯0.03, p < 0.05). Neither axon diameters (0.81 ±â€¯0.05 µm vs. 0.82 ±â€¯0.15 µm) nor thickness of the RNFL had changed after only 5 h (42.9 ±â€¯1.3 µm vs. 42.3 ±â€¯2.5 µm). However, after 7 days of diffuser wear, axons had become thinner (0.56 ±â€¯0.14 µm vs. 0.78 ±â€¯0.09 µm vs, p < 0.05), which could explain the thinning of the RNFL (36.3 ±â€¯2.7 µm vs. 42.1 ±â€¯2.4 µm, p < 0.01). Furthermore, myopic eyes had 38% less myelinated axons than untreated eyes as determined by immunohistochemical labelling against myelin basic protein (immunopositive areas in the central retina 1406 ±â€¯341 µm2 vs. 2185 ±â€¯290 µm2 in controls, p < 0.001). Myelin sheaths in the remaining axons remained unchanged (G ratio 0.76 ±â€¯0.02 vs. 0.76 ±â€¯0.03). Our study shows that deprivation myopia is associated with a significant loss of myelinated axons and shrinkage of the axon diameters of certain fibers in the RNFL. Early changes were already detected after 5 h and were accompanied by an increased fundus reflectivity in UV light. These parameters could therefore serve as the biomarkers for myopia development, at least in the chicken.

Changes in fundus reflectivity during myopia development in chickens.

Previous studies have shown that changes in functional activity in the retina can be visualized as changes in fundus reflectivity. When the image projected on the retina is low pass filtered or defocused by covering the eye with a frosted diffuser or a negative lens, it starts growing longer and develops myopia. We have tested the hypothesis that the resulting altered retinal activity may show up as changes in fundus reflectivity. Fundus reflectivity was measured in chickens in vivo, both in visible (400-800 nm, white) and near ultraviolet (UV) light (315-380 nm). Two CCD cameras were used; a RGB camera and a camera sensitive in near UV light (peak sensitivity at 360 nm). White and UV LEDs, respectively, placed in the center of the camera lens aperture, served as light sources. Software was written to flash the LEDs and record the average brightness of the pupil that was illuminated by light reflected from the fundus. The average pixel grey level (px) in the pupil was taken as a measure of the amount of reflected light while refractive errors were corrected by trial lenses after pupil brightness was corrected for pupil size. It was found that myopic eyes had brighter pupils in UV light, compared to eyes with normal vision, no matter whether myopia was induced by diffusers or negative lenses (48 ± 9 vs. 28 ± 3, p<0.001 and 47 ± 7 vs. 27 ± 2, respectively). Using SD-OCT in alert chickens it was found that the retinal nerve fiber layer (RNFL) and the retinal ganglion cell layer (RGCL) in the central retina became thinner already at early stages of myopia development, compared to controls (31.2 ± 5.8 µm vs. 43.9 ± 2.6 µm, p<0.001 and 36.9 ± 1.2 µm vs. 44 ± 0.5 µm, respectively). While the decrease in RNFL thickness occurred concomitantly with the increase in UV reflectivity, it remains unclear whether these changes were causally linked. Thinning of the RNFL could be due to reduced neural activity in retinal ganglion cells but also due to metabolic changes in the retina during myopia development.

Effects of Light of Different Spectral Composition on Refractive Development and Retinal Dopamine in Chicks.

Purpose: There is ample evidence that retinal dopamine (DA) is involved in the biochemical signaling cascade that controls emmetropization, but it is unknown how its release depends on the spectral composition of ambient light. We have studied DA release, refractive development, and growth in chicken eyes that were exposed to light of different spectral bands, and had either normal vision or were covered by frosted diffusers to induce myopia. Methods: Experiment 1: After spending the night in the dark, chicks were exposed to white room light (spectral range, 430-630 nm) or kept in the dark. Additional chicks were unilaterally exposed to blue (peak at 470 nm), red (620 nm), or UV lighting (375 nm) for 30 minutes and their fellow eyes covered with black occluders to minimize light exposure. Experiment 2: In the second experiment, chicks wore diffusers over one eye to induce deprivation myopia and were raised for 5 days in either white room light or in lighting supplied by UV, blue, or red light-emitting diodes (LEDs). Refractive states were recorded daily with infrared photoretinoscopy, and ocular dimensions at the start and end of the experiment with A-scan ultrasonography. DA and its metabolites were measured in retina and vitreous by high pressure liquid chromatography-electrochemical detection (HPLC-ED) in all cases. Results: Compared to chicks kept in the dark, retinal DA and vitreal 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations were clearly elevated after 30 minutes in white light. Vitreal DOPAC was also increased in red, blue, and UV lighting, compared to the fellow eyes covered with black occluders (black occluder versus blue light: 1.31 ± 0.32 vs. 1.70 ± 0.37; red: 1.26 ± 0.33 vs. 1.64 ± 0.38; UV: 1.13 ± 0.19 vs. 1.63 ± 0.21 ng/0.1 g wet weight). Chickens developed significantly less deprivation myopia, with shorter eyes, when raised under UV and blue lighting for 5 days, compared to under red and white light. Eyes with normal vision became more hyperopic in blue and UV lighting. Vitreal DOPAC levels were lowest after 5 days of exposure to UV lighting. Conclusions: Red, blue, and UV lighting all stimulated the release of retinal DA, but there were wavelength-dependent differences in DA release and metabolism. Less deprivation myopia developed in UV and blue lighting, compared to white and red light. The application of these findings to humans is limited by the fact that, different from chicks, humans have very low sensitivity in the near-UV region of the spectrum.

Hypothermia Protects and Prolongs the Tolerance Time of Retinal Ganglion Cells against Ischemia.

PURPOSE: Hypothermia has been shown to be neuroprotective in the therapy of ischemic stroke in the brain. To date no studies exist on the level of the inner retina and it is unclear if hypothermia would prolong the ischemic tolerance time of retinal ganglion cells, which are decisive in many ischemic retinopathies. METHODS: Bovine eyes were enucleated and stored either at 21°C or 37°C for 100 or 340 minutes, respectively. Afterwards the globes were dissected, the retina was prepared and either the spontaneous ganglion cell responses were measured or the retina was incubated as an organotypic culture for additional 24 hours. After incubation the retina was either processed for histology (H&E and DAPI staining) or real-time PCR (Thy-1 expression) was performed. RESULTS: Hypothermia prolonged ganglion cell survival up to 340 minutes under ischemic conditions. In contrast to eyes kept at 37°C the eyes stored at 21°C still showed spontaneous ganglion cell spiking (56.8% versus 0%), a 5.8 fold higher Thy-1 mRNA expression (not significant, but a trend) and a preserved retinal structure after 340 minutes of ischemia. CONCLUSION: Hypothermia protects retinal ganglion cells against ischemia and prolongs their ischemic tolerance time.

Changes in dopamine and ZENK during suppression of myopia in chicks by intense illuminance.

High ambient illuminances have been found to slow the development of deprivation myopia in several animal models. Almost complete inhibition of myopia was observed in chickens when intermittent episodes of high illuminance were alternated with standard office illuminance (50% duty cycle, alternate periods of 1 min 15,000 lux and 1 min 500 lux, continued for 10 h per day), or when illuminances were increased to 40,000 lux. Since the mechanisms by which bright light suppresses myopia are poorly understood, we have studied the roles of two well-established signaling molecules in myopia, dopamine and ZENK, in the chicken. In line with previous studies, we found that retinal dopamine release (as reflected by vitreal DOPAC content) was severely reduced during development of deprivation myopia. We found that illuminance of 15,000 lux, provided by quartz-halogen lamps, partially rescued the drop in retinal dopamine release. The finding is in line with the assumption that dopamine is involved in the light-induced inhibition of myopia. No differences in vitreal DOPAC were found when bright light was provided continuously or with 1:1 min alternating exposure with 500 lux. As previously described by others, wearing diffusers suppressed the expression of ZENK protein in glucagonergic amacrine cells (GACs) but neither continuous nor 1:1 min alternating bright to normal light could rescue the suppression of ZENK in GACs. While it is well known that light increases global retinal ZENK mRNA and protein levels, the changes of ZENK protein induced specifically in GACs by diffuser wear appear independent of light levels.

Animal models in myopia research.

Our current understanding of the development of refractive errors, in particular myopia, would be substantially limited had Wiesel and Raviola not discovered by accident that monkeys develop axial myopia as a result of deprivation of form vision. Similarly, if Josh Wallman and colleagues had not found that simple plastic goggles attached to the chicken eye generate large amounts of myopia, the chicken model would perhaps not have become such an important animal model. Contrary to previous assumptions about the mechanisms of myopia, these animal models suggested that eye growth is visually controlled locally by the retina, that an afferent connection to the brain is not essential and that emmetropisation uses more sophisticated cues than just the magnitude of retinal blur. While animal models have shown that the retina can determine the sign of defocus, the underlying mechanism is still not entirely clear. Animal models have also provided knowledge about the biochemical nature of the signal cascade converting the output of retinal image processing to changes in choroidal thickness and scleral growth; however, a critical question was, and still is, can the results from animal models be applied to myopia in children? While the basic findings from chickens appear applicable to monkeys, some fundamental questions remain. If eye growth is guided by visual feedback, why is myopic development not self-limiting? Why does undercorrection not arrest myopic progression even though positive lenses induce myopic defocus, which leads to the development of hyperopia in emmetropic animals? Why do some spectacle or contact lens designs reduce myopic progression and others not? It appears that some major differences exist between animals reared with imposed defocus and children treated with various optical corrections, although without the basic knowledge obtained from animal models, we would be lost in an abundance of untestable hypotheses concerning human myopia.

Intermittent episodes of bright light suppress myopia in the chicken more than continuous bright light.

PURPOSE: Bright light has been shown a powerful inhibitor of myopia development in animal models. We studied which temporal patterns of bright light are the most potent in suppressing deprivation myopia in chickens. METHODS: Eight-day-old chickens wore diffusers over one eye to induce deprivation myopia. A reference group (n = 8) was kept under office-like illuminance (500 lux) at a 10:14 light:dark cycle. Episodes of bright light (15 000 lux) were super-imposed on this background as follows. Paradigm I: exposure to constant bright light for either 1 hour (n = 5), 2 hours (n = 5), 5 hours (n = 4) or 10 hours (n = 4). Paradigm II: exposure to repeated cycles of bright light with 50% duty cycle and either 60 minutes (n = 7), 30 minutes (n = 8), 15 minutes (n = 6), 7 minutes (n = 7) or 1 minute (n = 7) periods, provided for 10 hours. Refraction and axial length were measured prior to and immediately after the 5-day experiment. Relative changes were analyzed by paired t-tests, and differences among groups were tested by one-way ANOVA. RESULTS: Compared with the reference group, exposure to continuous bright light for 1 or 2 hours every day had no significant protective effect against deprivation myopia. Inhibition of myopia became significant after 5 hours of bright light exposure but extending the duration to 10 hours did not offer an additional benefit. In comparison, repeated cycles of 1:1 or 7:7 minutes of bright light enhanced the protective effect against myopia and could fully suppress its development. CONCLUSIONS: The protective effect of bright light depends on the exposure duration and, to the intermittent form, the frequency cycle. Compared to the saturation effect of continuous bright light, low frequency cycles of bright light (1:1 min) provided the strongest inhibition effect. However, our quantitative results probably might not be directly translated into humans, but rather need further amendments in clinical studies.

Bright light induces choroidal thickening in chickens.

PURPOSE: Bright light is a potent inhibitor of myopia development in animal models. Because development of refractive errors has been linked to changes in choroidal thickness, we have studied in chickens whether bright light may exert its effects on myopia also through changes in choroidal thickness. METHODS: Three-day-old chickens were exposed to "bright light" (15,000 lux; n = 14) from 10 AM to 4 PM but kept under "normal light" (500 lux) during the remaining time of the light phase for 5 days (total duration of light phase 8 AM to 6 PM). A control group (n = 14) was kept under normal light during the entire light phase. Choroidal thickness was measured in alert, hand-held animals with optical coherence tomography at 10 AM, 4 PM, and 8 PM every day. RESULTS: Complete data sets were available for 12 chicks in bright light group and nine in normal light group. The striking inter-individual variability in choroidal thickness (coefficient of variance: 23%) made it necessary to normalize changes to the individual baseline thickness of the choroid. During the 6 hours of exposure to bright light, choroidal thickness decreased by -5.2 ± 4.0% (mean ± SEM). By contrast, in the group kept under normal light, choroidal thickness increased by +15.4 ± 4.7% (difference between both groups p = 0.003). After an additional 4 hours, choroidal thickness increased also in the "bright light group" by +17.8 ± 3.5%, while there was little further change (+0.6 ± 4.0%) in the "normal light group" (difference p = 0.004). Finally, the choroid was thicker in the "bright light group" (+7.6 ± 26.0%) than in the "normal light group" (day 5: -18.6 ± 26.9%; difference p = 0.036). CONCLUSIONS: Bright light stimulates choroidal thickening in chickens, although the response is smaller than with experimentally imposed myopic defocus, and it occurs with some time delay. It nevertheless suggests that choroidal thickening is also involved in myopia inhibition by bright light.

An updated view on the role of dopamine in myopia.

A large body of data is available to support the hypothesis that dopamine (DA) is one of the retinal neurotransmitters involved in the signaling cascade that controls eye growth by vision. Initially, reduced retinal DA levels were observed in eyes deprived of sharp vision by either diffusers ("deprivation myopia", DM) or negative lenses ("lens induced myopia", LIM). Simulating high retinal DA levels by intravitreal application of a DA agonist can suppress the development of both DM and LIM. Also more recent studies using knock-out mouse models of DA receptors support the idea of an association between decreased DA levels and DM. There seem to be differences in the magnitude of the effects of DA on DM and LIM, with larger changes in DM but the degrees of image degradation by both treatments need to be matched to support this conclusion. Although a number of studies have shown that the inhibitory effects of dopamine agonists on DM and LIM are mediated through stimulation of the D2-receptor, there is also recent evidence that the balance of D2- and D1-receptor activation is important. Inhibition of D2-receptors can also slow the development of spontaneous myopia in albino guinea pigs. Retinal DA content displays a distinct endogenous diurnal, and partially circadian rhythm. In addition, retinal DA is regulated by a number of visual stimuli like retinal illuminance, spatial frequency content of the image, temporal contrast and, in chicks, by the light input from the pineal organ. A close interaction was found between muscarinergic and dopaminergic systems, and between nitric oxide and dopaminergic pathways, and there is evidence for crosstalk between the different pathways, perhaps multiple binding of the ligands to different receptors. It was shown that DA agonists interact with the immediate early signaling molecule ZENK which triggers the first steps in eye growth regulation. However, since long treatment periods were often needed to induce significant changes in retinal dopamine synthesis and release, the role of dopamine in the early steps is unclear. The wide spatial distribution of dopaminergic amacrine cells in the retina and the observation that changes in dopamine levels can be locally induced by local retinal deprivation is in line with the assumption that dopaminergic mechanisms control both central and peripheral eye growth. The protective effect of outdoor activity on myopia development in children seems to be partly mediated by the stimulatory effect of light on retinal dopamine production and release. However, the dose-response function linking light exposure to dopamine and to the suppression of myopia is not known and requires further studies.

Effects of intravitreal insulin and insulin signaling cascade inhibitors on emmetropization in the chick.

PURPOSE: Intravitreal insulin has been shown to be a powerful stimulator of myopia in chickens, in particular if the retinal image is degraded or defocused. In most tissues, the insulin receptor activates two main signaling pathways: a) the mitogen-activated protein kinase (MAPK) cascade (e.g., mitogen-activated protein kinasem kinase [MEK] and extracellular regulated kinase [ERK]) and b) the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. In the current study, insulin was injected, and these pathways were separately inhibited to determine which is activated when the retinal image is defocused by spectacle lenses. METHODS: Chicks were treated with either +7 D, -7 D, or no lenses. They were intravitreally injected with insulin, the MEK inhibitor U0126, the PI3K inhibitor Ly294002, or a combination of insulin and one of the inhibitors. Refractions and ocular dimension were measured at the beginning and after four days of treatment. The retinal proteins of the chicks were measured with western blots after 2 h and four days of treatment. Incubation occurred with anti-Akt1, anti-Erk1/2, anti-phospho-Akt(Thr308), and anti-phospho-Erk1/2((Thr202/Tyr204)) antibodies, and the ratio between the relative intensity of the phospho-form and the total-form was calculated. RESULTS: Chicks wearing positive lenses and injected with saline and with PI3K inhibitor compensated for the imposed defocus and became hyperopic. Insulin injections and insulin plus PI3K inhibitor injections prevented lens-induced hyperopia, whereas the MEK inhibitor alone and insulin plus MEK inhibitor had no effect. Obviously, the MEK inhibitor suppressed the effect of insulin on eye growth in the plus lens-treated animals. Chicks treated with negative lenses and injected with insulin, or with insulin plus MEK inhibitor, overcompensated for the imposed defocus. This effect of insulin was not detected in eyes injected with PI3K inhibitor plus insulin, suggesting that the PI3K inhibitor suppressed the effects of insulin in minus lens-treated animals. Insulin increased the ratio of phospho-Akt/total-Akt in animals with normal visual exposure but even more so in chicks wearing plus or minus lenses. The increase was blocked by simultaneous PI3K inhibitor injections in control eyes but not in lens-treated eyes. Insulin also increased the ratio of phospho-ERK/total-ERK in animals with normal visual exposure and in animals wearing positive lenses, compared to U0126- and Ly294002-injected eyes. In contrast, no significant activation of the MEK/ERK pathway was observed in the negative lens-treated animals. CONCLUSIONS: Intravitreal insulin promoted axial eye growth and stimulated both signaling pathways. The PI3K/Akt pathway was activated in control and plus and minus lens-treated eyes, but the MEK/ERK pathway was activated only with positive lenses or no lenses. With negative lenses, insulin did not stimulate the MEK/ERK signaling cascade. Independent of the pathway stimulated after insulin binding, the effect on insulin was always the same: an increase in eye growth.

Ultrasonography and optical low-coherence interferometry compared in the chicken eye.

PURPOSE: To compare ocular biometry [anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD), and axial length (AL)] using A-scan ultrasonography and optical low-coherence interferometry (OLCI) in the chicken eye. METHODS: Two-week-old chicks (n = 42) were measured. Bland-Altman plots and repeatability and correlation analyses were calculated for both methods. RESULTS: There was a high correlation between both methods for ACD (r = 0.6144, p < 0.0001), VCD (r = 0.9595, p < 0.0001), and AL (r = 0.9290, p < 0.0001) but not for LT (r = 0.1604, p = 0.144). Measurements by OLCI were more consistent (smaller coefficients of variation and higher intraclass correlation). Bland-Altman plots showed that ultrasound provided larger values for LT, VCD, and AL but not for ACD [differences between ultrasound and OLCI (mean ± SD): ACD = -0.11 ± 0.12 mm; LT = 0.10 ± 0.09 mm; VCD = 0.25 ± 0.08 mm; AL = 0.50 ± 0.16 mm]. CONCLUSIONS: A high correlation between both techniques was found for three of the four parameters (ACD, VCD, and AL). However, as the absolute values were different, both techniques cannot replace each other mainly because (1) one is non-contact and the other contact and can induce a minor indentation of the cornea and (2) each device uses different types of waves that cross the ocular interfaces differently. While consistency and repeatability were better by OLCI, a disadvantage is that, different from humans, it can only be used in anesthetized chicks.

Insulin, insulin-like growth factor-1, insulin receptor, and insulin-like growth factor-1 receptor expression in the chick eye and their regulation with imposed myopic or hyperopic defocus.

PURPOSE: Insulin stimulates eye growth in chicks and this effect is greatly enhanced if the retinal image is degraded by the defocus of either sign. However, it is unclear whether the insulin receptor (IR) is expressed at all in the chicken retina in animals 1-2 weeks post-hatching. We have investigated IR expression and whether IR transcript abundance varies in the fundal layers. To elucidate the possible role of insulin and insulin-like growth factor (IGF)-1 signaling in eye growth regulation, mRNA (mRNA) levels were measured for insulin, IGF-1, IR, and IGF-1 receptor (IGF-1R) during imposed negative or positive defocus. METHODS: Chicks were treated binocularly with positive or negative spectacle lenses for 4 or 24 h, or they remained untreated (n=6, for each treatment group). Northern blot analyses were performed to screen for transcription variants in the different fundal layers of untreated animals. Real-time PCR was used to quantify IR, IGF-1R, IGF-1, and insulin mRNA levels in the different fundal layers of the chick eye in the three treatment groups. RESULTS: IR mRNA was found in all the studied tissues, although there is evidence of tissue-specific transcript variations. Three major transcripts were detected for IR. The brain, retina, and choroid showed the longest transcript (4.3 kb), which was not present in the liver. Nevertheless, the liver and brain showed a second transcript (2.6 kb) not present in the retina and choroid. A short transcript (1.3 kb) was the predominant form in the liver and choroid, and it seems to be present in the retinal pigment epithelium (RPE) and sclera as well. In the retina, no significant gene expression changes were found when defocus was imposed. Interestingly, in the RPE, both IR and IGF-1R were already downregulated after short periods (4 h) of positive lens wear. In contrast, IR and IGF-1R were upregulated in the choroid and fibrous sclera during treatment with negative, but not positive, lenses. CONCLUSIONS: Differences observed in the IR transcript length in different tissues suggest possibly different functions. The differential regulation of IR and IGF-1R in the RPE, choroid, and fibrous sclera is consistent with their involvement in a signaling cascade for emmetropization.