Myopia is one of the most common refractive errors with an increasing prevalence in recent decades.1 Circadian rhythm plays a role in the pathogenesis of myopia. During development of the visual system, a regular circadian rhythm is a pre-requisite for normal ocular growth, axial elongation, and emmetropization.2 A disturbed visual environment would lead to myopic changes in ocular parameters such as optic axis length, choroidal thickness, and vitreous cavity depth.3 Biological neural transmitters such as dopamine and melatonin are involved in myopic development; however, the underlying regulatory mechanism of circadian rhythm is still unclear.4,5
Intrinsically photosensitive retinal ganglion cells (ipRGCs) are the third class of photoreceptor cells of the retina, in addition to rods and cones.6 Studies have found that ipRGCs, although constitute only 0.3 to 0.8% of the total retinal ganglion cells, exert crucial effects in nonimaging visual-forming system, circadian rhythm, and activation of the pupillary light reflex.6
ipRGCs are characterized by the expression of a photoreceptive protein known as melanopsin,7 a photopigment with a distinct action spectrum of maximum absorption at 482 nm.8 Light of 482 nm is a short-wavelength blue light, which influences the diurnal expression of melanopsin and focalizes before the retina, inhibiting ocular growth and resulting in relative hyperopia.9,10 Melanopsin plays a vital role in phototransduction for the circadian pacemaker located in suprachiasmatic nucleolus of the hypothalamus and the maintenance of circadian rhythm.11–13 Diurnal photoentrainment is impaired in melanopsin knockout mice.3 Melanopsin also contributes to the encoding early images in early visual system.14
Melatonin and dopamine are two important neurotransmitters in circadian clock, as well as in refractive error development and visual signaling.15 Melatonin is primarily synthesized by the pineal gland, as well as retinal photoreceptors and ciliary body epithelium cells.16 Melatonin signaling is necessary to circadian rhythm in mice, and deficiency of melatonin would disturb circadian rhythm even at the presence of retinal dopamine.17 Dopamine has widely been investigated as a key factor for myopic development4 and seems to function reciprocally to melanopsin produced by ipRGCs.18,19 These facts indicate that melanopsin may play a role in the refractory development of the eye. Melatonin and dopamine act in a reverse manner. The production and release of dopamine are stimulated by the light, whereas retinal melatonin production is observed in the darkness, and the level of melatonin decreases markedly at the presence of light.20 To our knowledge, the direct effect of melanopsin and its interactions with other myopic regulatory factors on the retinal signaling for refractory development have not been reported. We measured refractive error and the change in two key biometric components in emmetropization, axial length, and corneal radius21 during the development of lens-induced myopia in guinea pigs. Then, we investigated the relations between melanopsin, retinal dopamine, and melatonin, with an aim of exploring the role of melanopsin in the development of myopia.
Eighty 21-day-old tricolored guinea pigs (provided by the Laboratory Animal Department of Central South University) weighing 140 to 170 g and with no sex limitation and of clean grade were randomly divided into four groups: control group, defocus group, defocus + AA92593 (melanopsin antibody) group, and defocus + dimethyl sulfoxide (DMSO) group. Each group comprised 20 guinea pigs. This study was approved by the Medical Ethics Committee of Xiangya Hospital affiliated with Central South University (approval no. 201603242). The use and feeding of the experimental animals fully complied with the “Regulations on Management over Experimental Animals” promulgated by the State Scientific and Technological Commission. During the whole experiment, one guinea pig in the defocus group died, one in the defocus + AA92593 group was infected, and two in the defocus + DMSO group died.
Main Reagents and Instruments
AA92593 obtained was from Sigma-Aldrich (St. Louis, MO; SML0865). Rabbit antimelanopsin polyclonal antibody was from Abcam, Cambridge, United Kingdom (ab19383), and rabbit anti-GAPDH polyclonal antibody was from Bioss Biotechnology Co., Ltd., Beijing, China (bs-2188R). Standard dopamine and melatonin substances were obtained from Sigma-Aldrich (BP468 and M5250). The −6 D lenses were from Hong Kong Optical Lens Co., Ltd., Hong Kong, China. The computerized optometry unit was from OCULUS, Arlington, Germany (PARK 1). The keratometer was from Nidek, Aichi, Japan (ARK-510A). The ophthalmic A/B ultrasound was from Tianjin Meda Co., Ltd., Tianjin, China. Aicevoos digital illuminometer (V8) was from Zhongce Hongtu Measuring Instrument Co., Ltd., Wuhan, China. Aosvi binocular continuous zoom stereoscope (XTL-2600) was from Aosvi Optical Instrument Co., Ltd., Shenzhen, China.
Myopia Modeling in Guinea Pigs
Animal experiments in this study were adherent to the ethical principles stated in the Association for Research in Vision and Ophthalmology guidelines. The myopia modeling in guinea pigs used in this study has previously been reported.4 Briefly, the left eye was used for myopia modeling. A self-made frame with a −6.00 D lens was sutured and fixed to the soft tissue around the orbit, and the lens was wiped daily to keep it clean. The −6.00 D optical resin lens had a diameter of 12 mm and inner arc curvature of 9.61 mm. The guinea pigs were kept in separate cages with a daily light and dark cycle of 12 hours each, with the light period from 7:00 am to 7:00 pm. Indoor fluorescent lamps were used for lighting. The eye illuminance of the guinea pigs in the cages was measured daily with a digital illuminance meter, which was controlled between 480 and 520 lux.
After the defocus eyes wore concave lenses for 2 weeks, the corneal curvature radius was measured with a keratometer, the refractive error (in diopters) was measured using a computerized optometry unit after instillation of compounded tropicamide to induce mydriasis, and axial length (accurate to 0.01 mm) was measured with A-scan ultrasound. Each measurement was repeated three times, and the average values were taken.
AA92593 was dissolved in 5% DMSO. On days 0 and 8 of wearing lenses, a volume of 5 μL of AA92593 was injected into the vitreous cavities of the defocus + AA92593 group with a microinjector. In the defocus + DMSO group, 5 μL of 5% DMSO was injected into the vitreous cavities as the control.
The Expression of Melanopsin Protein Was Measured with Retina Immunofluorescence Staining
Three retina specimens from each group were used for retinal immunofluorescence staining to detect the melanopsin protein. Excess pentobarbital sodium (Nembutal; AKORN, Lake Forest, IL) was injected intraperitoneally to kill the guinea pigs; their eyeballs were then removed. Each anterior segment and each vitreous cavity was removed under a stereomicroscope, and the optic disc and nerve were cut off. The retinal neuroepithelium layer was separated in the phosphate-buffered saline (PBS) solution, and the peripheral part of the retina was symmetrically cut, resulting in a four-leaf clover shape. Each specimen was rinsed with PBS and fixed with 4% paraformaldehyde solution. The membranes were permeated with 0.5% Triton X-100 (AR-0341, Dingguo, Beijing, China), and normal goat serum was used for blocking. Three hundred microliters of the first antibody (rabbit antimelanopsin polyclonal antibody, 1:100) was added dropwise and kept at 4°C overnight. Then the fluorescein-labeled second antibody (Alexa Fluor-555, A32732, Invitrogen, Carlsbad, CA) was added dropwise, and 4’,6-diamindino-2-phenylindole (DAPI) was used to stain the nucleus. A laser confocal scanning microscope was used to observe the tissue samples and take photographs after the slide was sealed.
Western Blot Measurement of Retinal Melanopsin Protein Expression
The retinal neuroepithelium layer was used for Western blot testing to detect the expression of retinal melanopsin protein in the control group (n = 8), the defocus group (n = 7), the defocus + AA92593 group (n = 7), and the defocus + DMSO group (n = 6). The retinal neuroepithelium layer was removed and weighed, lysed with cell lysis buffer (1:10), and followed by centrifugation at 9000g at 4°C for 15 minutes. The supernatant was collected and saved at −20°C. A bicinchoninic acid protein quantification kit was used for protein quantification. A 25-μg sample of protein was separated by 10% discontinuous sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Protein was electrotransferred to nitrocellulose membrane, which then was blocked with 5% skimmed milk. Rabbit antimelanopsin polyclonal antibody was added (1:500) and was kept at 4°C overnight. Horseradish peroxidase–labeled goat antirabbit IgG was incubated for 90 minutes after elution. Chemiluminescent reagent was added to react for 5 minutes after the membrane was washed, after which it underwent x-ray exposure, image displaying, and photographic fixing. Bandscan 5.0 image analysis software was used to analyze the gray level of the target strip, and GAPDH was used as the internal control to calculate the relative expression of melanopsin protein (relative expression of melanopsin = gray level of melanopsin strip/gray level of GAPDH strip).
High-performance Liquid Chromatography Electrochemical Measurement of Dopamine and Melatonin
The retinal neuroepithelium layer was used for the high-performance liquid chromatography electrochemical (HPLC-EC) to measure the content of dopamine and melatonin. Nine samples per group were removed, weighed, and stored in liquid nitrogen. Chromatographic conditions were as follows: Waters μBondapak C18 reverse-phase column (150 × 3.9 mm, 5 μm). The mobile phase comprised the aqueous phase (containing 2.8 g of B8, 6.8 g of KH2PQ4, 18 mg of ethylenediamine tetraacetic acid [EDTA] per 500 mL, pH 3.3), methanol, and acetonitrile (78:19:3), with a flow rate of 0.75 mL/min, a working voltage of 0.7 V, and an injection volume of 10 μL. The preservation times of dopamine and melatonin were 6.2 and 8.6 minutes, respectively. The content (in nanograms) of dopamine and melatonin per milligram of nerve retina was calculated.
All data were represented by means ± standard deviations and processed using the SPSS 22.0 analysis package (IBM Corp., Armonk, NY). The guinea pigs' corneal radius of curvature, refractive error, axial length, relative expression of melanopsin protein and dopamine, and melatonin content were tested using one-way ANOVA to evaluate the differences among groups. Student unpaired t test with Bonferroni correction was applied for multiple comparisons between groups. The refractive error and axial length of the guinea pig eyes in the defocus group and defocus + AA92593 group, which were used for HPLC detection, underwent a correlation analysis with the melatonin content to calculate the Pearson correlation coefficient. P < .05 indicates statistical significance.
Change in Refractive Error in Guinea Pig Eyes
In the control group, the eyeballs of the 35-day-old guinea pigs demonstrated mild hyperopia (+0.95 ± 0.53 D). After 2 weeks of wearing the −6.00 D lens over the left eye, the defocus group showed an increased axial length and development of myopia (Fig. 1). The degree of myopia (−2.59 ± 0.97 D) was greater, and axial length was longer (8.15 ± 0.11 mm), in the defocus eyes in the defocus + AA92593 group compared with those of the defocus group (−1.98 ± 0.82 D [P = .05] and 8.05 ± 0.09 mm [P = .008]). The defocus eye receiving intravitreal injection of DMSO showed no effect on the axial length or myopia development when compared with the defocus group. There was no statistically significant difference in the corneal radius of curvature between the groups (F = 2.239, P = .09, one-way ANOVA).
Melanopsin Expression in Retinal Neuroepithelium
Immunofluorescence staining of the retinal tissue revealed that melanopsin protein was mainly expressed on the cell surface and dendrites of ipRGCs and partially expressed in the cytoplasm. In the defocus + AA92593 group, the percentage of ipRGCs with positive expression of melanopsin protein of the defocus eyes was reduced (Fig. 2C). The Western blotting analysis showed the relative expression of retinal melanopsin protein in the defocus group (0.67 ± 0.11) and the control group (0.73 ± 0.09), and the difference between the two groups was not statistically significant (P > .99). The relative expression of retinal melanopsin protein in the defocus + AA92593 group (0.20 ± 0.06) decreased, and the difference was statistically significant compared with the control group (0.73 ± 0.09, P < .0001), the defocus group (0.67 ± 0.11, P < .0001), and the defocus + DMSO group (0.75 ± 0.10, P < .0001; Fig. 3B).
Changes in Retinal Neuroepithelium Dopamine and Melatonin Content
The contents of dopamine and melatonin in the retinal neural epithelium of the guinea pigs in the control group were 1.02 ± 0.30 and 0.36 ± 0.09 ng/mg, respectively. The content of dopamine in the defocus group decreased to 0.64 ± 0.18 ng/mg (P = .008), whereas the melatonin content was 0.38 ± 0.09 ng/mg (P = .58). The intravitreal injection of AA92593 into the defocus eyes resulted in a marked increase in melatonin content in retinal neuroepithelium (0.55 ± 0.13 ng/mg; P = .01) compared with the defocus group, but had no marked effect on the dopamine content (0.61 ± 0.17 ng/mg; P > .99; Figs. 4A, B). The intravitreal injection of DMSO had no effect on either the dopamine or melatonin content in the defocus eyes. In the defocus + AA92593 group, the melatonin content of the retinal neuroepithelium was correlated with the refractive error (Pearson correlation coefficient = −0.68, P = .006) and axial length (Pearson correlation coefficient = 0.74, P = .02; Figs. 4C, D).
ipRGCs play an important role in circadian rhythm and pupillary response to light, and ipRGCs account for 0.3 to 0.8% of the total number of retinal ganglion cells.7 Approximately 60% of the cell bodies are distributed in the ganglion cell layer, and 40% of the cell bodies are distributed in the inner nuclear layer.6 ipRGCs not only can directly sense light signal stimulation through melanopsin but also can receive signal input from retinal cone and rod cells through bipolar cells.22 In addition, ipRGCs have synaptic connections with dopamine amacrine cells and Müller cells.23 These features constitute the anatomical and physiological basis for ipRGCs to participate in the retinal neural signal network and also suggest that ipRGCs may affect the myopia development by regulating retinal myopia signal factors.23 In this study, we used the mature model of guinea pig lens-induced myopia to study the relationship between ipRGCs and myopia development. It was found that melanopsin protein was mainly expressed on the cell body and dendrites of guinea pig ipRGCs. Wearing a −6.00 D lens induced myopia development in the defocus eyes, with a relative myopia degree of −2.96 D, but had no notable effect on the expression of retinal melanopsin protein. Intravitreal injection of melanopsin antagonist AA92593 not only reduced the expression of retinal melanopsin protein but also increased the degree of myopia in the defocus eyes. However, the magnitude of increased myopia is small in the melanopsin inhibition group, and no significant difference is shown compared with the defocus group. We suspect that the reason may lie in the incomplete block of melanopsin expression. These results show that retinal melanopsin has a protective effect against the development of myopia, and ipRGCs are involved in the occurrence and development of myopia. Ostrin24 found that there is no correlation between the ipRGCs-mediated pupillary response to light and the development of refractive error, so ipRGCs may participate in the development process of myopia by regulating the circadian rhythm of the eyes.
Melanopsin is a G protein–coupled receptor, participating in phototransduction for the maintenance of circadian rhythm and contributing to early image forming.12,14 As a photosensitive protein produced by ipRGCs, its sensitive wavelength is 482 nm, and its expression is regulated by light and circadian rhythm, being the highest in the morning and lowest in the evening.8 Light with a length of 482 nm is blue light, which not only can regulate circadian rhythm changes by affecting the secretion of melanopsin but also can play an important role in the development of the eye refractory state.16 Studies of the relationship between monochromatic light and myopia have found that long-wavelength light (red) focusing after the retina can induce relative myopia development, whereas short-wavelength light (blue) focusing before the retina can inhibit eyeball growth and develop relative hyperopia.9,10 Qian et al.10 also confirmed that, in a blue light environment, the lens-induced myopia axial length extension of guinea pigs slowed down, and the myopia degree was reduced, whereas putting guinea pigs in a white light environment could partially reverse this effect. The study demonstrated that blue light can inhibit the development of lens-induced myopia. In this study, the competitive inhibitor of melanopsin was used to block the effect of melanopsin, which increased the sensitivity of the eyes to optical defocus signals. It also accelerated and increased the axial length extension and myopia development of the defocus eyes. This result not only supports the view that blue light has a protective effect against myopia, but also is an explanation of its protective mechanism.
Circadian rhythm is the most common biological rhythm, involving sleep, food and water intake, body temperature, and endocrine system feedback loops.25–27 Circadian rhythm is mainly regulated by suprachiasmatic nucleus of hypothalamus,28 and the light signal is its most sensitive zeitgeber.29 In the process of eyeball growth and development, regular circadian rhythm is an indispensable condition for its emmetropization.3 In the normal visual environment, the vertebrate axial extension has pronounced circadian rhythm changes, with the peak occurring around midday.3 In animal models and human myopia, the normal rhythm of this axial length change is broken, and the axial length extension speeds up at night, causing the axial length to exceed the rate of emmetropization, resulting in myopia.30 In addition, choroidal thickness, vitreous cavity depth, IOP, and anterior chamber depth all have observed circadian rhythms, and these factors are all related to biological rhythms of axial length extension.31–33
Dopamine and melatonin are important neural signal factors in the retina, and their synthesis and release have pronounced circadian rhythms. Dopamine levels are higher in the daytime and lower at night, whereas melatonin is the opposite. In animal models of myopia, it has been found that both dopamine and melatonin are involved in the occurrence and development of myopia, especially form-deprivation myopia.4,16 It is generally believed that dopamine participates in retinal light adaptation and inhibits melatonin synthesis and secretion through the D2/D4 receptor.34 Melatonin participates in the dark adaptation process and can inhibit dopamine synthesis and secretion through the MT2 receptor.35 The balance between the two plays a crucial role in the regulation of the circadian rhythm of the eye.36 In this study, we found that optical defocus can induce a decrease of the retinal dopamine content in guinea pigs, but it had no effect on the melatonin content in the retina. Intravitreal injection of AA92593 antagonized the effect of melanopsin, accelerating lens-induced myopia development and upregulating the melatonin content in the retina, but had no effect on the dopamine content in the retina. It is previously reported that the melanopsin participated in regulation of melatonin based on an evidence that blue light of 460 to 480 nm absorbed by melanopsin is the most potent wavelength for acute suppression of melatonin.37,38 In our study, the melatonin content in the retina was significantly correlated with refractive error and axial length, which indicated that the protective effect of melanopsin against myopia was related to the change of the melatonin content in the retina.
However, there are limitations to this study. First, the research was conducted among guinea pigs because guinea pigs develop myopia in a reasonably short period and respond well to lens-induced refraction; however, because human eyes grow slower and lens accommodation behaves differently compared with animal myopic models,39 the retinal circuitry and refractive status development may be different when applying to human. Second, our biometric parameters and samples were collected during daytime; some reported the refraction status of the eyes tends to be more myopic in the morning than in the evening.40 The data collecting time may have influenced the results.
In conclusion, retinal melanopsin inhibits the development of lens-induced myopia, and this effect may be related to the expression of retinal melatonin. Levels of dopamine may not affect the function of melanopsin in myopia development. Retinal melanopsin may be a potential target in the prevention of myopia development, although the underlying mechanism for this effect needs further study.
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