Daily Injection But Not Continuous Infusion of Apomorphine Inhibits Form-Deprivation Myopia in Mice

Abstract

Purpose.: To compare the effects of daily injection versus continuous infusion of a nonspecific dopamine agonist, apomorphine (APO), on refraction and ocular growth in normal postnatal mice and mice with form-deprivation myopia (FDM).

Methods.: The C57BL/6 mice were subjected (or not) to monocular FD by covering the left eye with a frosted goggle and leaving the right (fellow) eye uncovered. During postnatal days 28 to 56, both groups received APO (5 mg/kg/d) or vehicle either as daily intraperitoneal injection or by continuous subcutaneous infusion with mini-pumps. After these treatments, binocular refractions were measured by photoretinoscopy and binocular ocular dimensions were measured by optical coherence tomography. Monocular photopic flash electroretinograms were recorded from non-FD mice.

Results.: In normal mice, daily injection or continuous infusion of APO did not affect normal postnatal development of refraction. However, in the FD group, daily APO-injection attenuated ocular growth and also myopia development, as reflected in the interocular differences for APO-injected mice compared with vehicle-injected mice: (1) refraction, −1.04 ± 0.37 diopter (D) (APO-injection) compared with −4.14 ± 0.77 D (vehicle-injection) (P < 0.05); (2) vitreous chamber depth: −0.002 ± 0.005 mm compared with 0.032 ± 0.009 mm (P < 0.05); and (3) axial length: 0.000 ± 0.005 mm compared with 0.057 ± 0.007 mm (P < 0.05). By contrast, continuous APO-infusion failed to affect these biometric parameters. Furthermore, daily APO-injection decreased the ERG a- and b-wave amplitudes, whereas continuous APO-infusion increased these responses.

Conclusions.: In monocularly FD mice, daily APO-injection, but not continuous infusion, attenuated myopia development. Therefore, evaluating different dopamine agonist administration paradigms is important for identifying effective dopamine-based treatment for myopia.

Myopia is increasingly becoming a significant public health issue. In advanced cases, it can lead to severe visual impairment and even blindness. Approximately 80% to 90% of students graduating from high school in East Asia have this condition,¹ with similar trends in North America and Europe as well.¹ Recently, several reports demonstrated that outdoor activity reduces the risk of myopia development in young individuals,^2–4 and this benefit is postulated to be associated with light-induced increases in retinal dopamine (DA) release, a key neurotransmitter.^5,6 

Dopamine is thought to play a role in the regulation of eye growth. One indication of its importance is that in chickens, experimental myopia development, such as form-deprivation myopia (FDM), is associated with reduced retinal DA levels in the FDM eye.^7–9 In addition, after recovery from the FDM condition, DA levels return to normal.⁹ Furthermore, DA receptor activation inhibited FDM development in chickens,¹⁰ guinea pigs,^11,12 and rhesus monkeys¹³ treated with levodopa (a precursor of DA) or apomorphine (APO, a nonselective DA agonist). However, there is also contradictory evidence for an association between myopia development and a decline in DA levels, because depletion of retinal DA by 6-hydroxydopamine^14,15 or reserpine¹⁵ also inhibits FDM development in chickens. The reason for this apparently paradoxical effect is still unclear. 

Retinal DA is released exclusively from amacrine and interplexiform cells. This neurotransmitter interacts with two different DA receptor subtypes designated D1-like receptors (D1Rs) and D2-like receptors (D2Rs). The D1Rs are coupled to the Gα_s/olf protein and their activation by DA increases the rate of cAMP synthesis, whereas activation of D2Rs is coupled to the Gα_i/o protein and instead decreases cAMP level.¹⁶ The D2Rs have a relatively higher affinity for DA than D1Rs.¹⁷ In chickens, the role of D1R in FDM development is currently controversial, because several studies suggest that only D2R takes part in the regulation of ocular growth.^18,19 The findings of the latter studies are also consistent with the finding of an earlier study showing that APO-induced inhibition of FDM was completely blocked by simultaneous administration of spiperone, a D2R selective antagonist, but not by SCH23390, a D1R selective antagonist.¹⁰ However, others found that both relatively selective D1R and D2R agonists mimicked the inhibitory effect of APO on FDM,²⁰ whereas both D1R and D2R antagonists enhanced this FDM response.¹⁵ 

Development of FDM in mice resembles that of other animal models,^21,22 although its progression rate is slower. The effects of APO, a nonselective DA agonist, on FDM development has been characterized in chickens,¹⁰ guinea pigs,¹¹ and rhesus monkey models,¹³ but not in mice. As APO has a relatively short half-life,²³ it is commonly administered for clinical²⁴ and experimental purposes²⁵ other than myopia research by either repeated injection or by continuous infusion to prolong its effects. In studies on Parkinson's disease,²⁶ nerve injury,²⁷ and striatal dopaminergic terminals,²⁸ the effects of APO differ according to whether it is administered by repeated injection or continuous infusion. It is postulated that this difference is attributable to differences in the duration of DA receptor stimulation. Injection of APO induces pulsatile DA receptor stimulation, whereas continuous infusion elicits steady-state activation.²⁹ These two different activation patterns are associated with distinct biological responses. For instance, continuous infusion with a DA receptor agonist likely produces DA receptor desensitization,³⁰ whereas daily injection of DA receptor agonist can produce pulsatile DA receptor activation, leading to behavioral supersensitization, such as levodopa-induced dyskinesia.³¹ 

The effect of routes and timing of APO administration on the FDM development in mice is unknown. In this study, we determined the effects of differences in APO routes of administration on FDM development in mice. This was done by comparing the effects of APO administered by daily injection and by continuous infusion on changes in refraction (RE) and ocular dimensions of FDM mice. 

Methods

Animals

The study was approved by the Animal Care and Ethics Committee at Wenzhou Medical College (Wenzhou, China). All experiments were conducted according to the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research. Four-week-old C57BL/6, wild-type, male mice (n = 98) were obtained from the Animal Breeding Unit at Wenzhou Medical College. All animals were raised in standard transparent mouse cages (24 × 18 × 13 cm) with a 12-hour light/12-hour dark cycle (light from 8 AM to 8 PM) for 4 weeks. Light provided by incandescent bulbs produced an ambient luminance of approximately 500 lux on the cage floor. The rooms were kept at 22 ± 2°C, and mice received food and water ad libitum. All of the conditions were maintained during the 4 weeks of treatment. 

Experimental Design

The C57BL/6 mice were administered APO daily by intraperitoneal injection (n = 62) or by continuous subcutaneous infusion via implanted mini-pumps (n = 36). Half of each group were monocularly form-deprived (FD, with only left eyes wearing goggles as described below); the other half were not FD and served as controls. The control mice were subjected to one of two different treatments: (1) daily injection of vehicle (VEH-injection, n = 16); continuous infusion of vehicle (VEH-infusion, n = 6); (2) daily injection of APO (APO-injection, n = 14); continuous infusion of APO (APO-infusion, n = 11). Similarly, the FD groups were also subjected to one of two treatments: (1) daily injection of vehicle (FD-VEH-injection, n = 16); continuous infusion of vehicle (FD-VEH-infusion, n = 8); (2) daily injection of APO (FD-APO-injection, n = 16); and continuous infusion of APO (FD-APO-infusion, n = 11). Based on our previous studies, we chose 4-week-old mice because the spatial vision in the mice appears to reach a stable, mature level at this developmental stage,³² and they survive after mini-pump implant surgery³³ and are susceptible to FD.²² 

Form deprivation was achieved by gluing a frosted, hemispherical, thin plastic shell (goggle) over the left eye of each animal.^22,34 The shell acted as a light diffuser and remained in place for 4 weeks. Collars made from thin plastic were fitted around the neck to prevent the mice from removing the diffusers. The daily injection or continuous infusion of APO and vehicle were begun at the same time as the FD treatments. 

As described below, RE and ocular biometric parameters, including corneal radius of curvature (CRC) and the axial dimensions of ocular components, were measured in both eyes of each animal before (4 weeks old) and at the end of the 4 weeks of treatment. Photopic flash ERGs were recorded at 3 weeks from the right eyes of non-FD, control mice in both the VEH and APO treatment groups, to assess the effects on retinal function of the two APO treatments. 

Pharmacological Treatment

Solution of APO (Tocris Bioscience, Bristol, UK) was prepared in distilled H₂O containing 1 mg/mL ascorbic acid (ICN Biomedicals, Inc., Irvine, CA, USA) to retard oxidation. Based on previous studies,^35,36 we chose daily APO (5 mg/kg body weight), for both daily injection intraperitoneally and continuous infusion subcutaneously. Solutions of 1 mg/mL ascorbic acid were used for the VEH groups. 

In the experiment involving daily injection of APO, the solution was injected at approximately 9 AM in the peritoneal cavity using a syringe attached to a 29-gauge needle. The injection site was the lower right or left quadrant of the abdomen on alternate days. The APO solution was freshly prepared every day before each injection. 

In the continuous infusion of APO experiments, the solution was administered by ALZET osmotic mini-pumps (DURECT Corporation, Cupertino, CA, USA) that were implanted subcutaneously to deliver their content at continuous and controlled rates. After taking into account the animal weight (15 g body weight on average), APO solubility, and desired delivery rate, the ALZET osmotic mini-pump Model 1002 was chosen. According to the manufacturer, the nominal pumping rate was 0.25 μL/h for 14 days. The mini-pumps were filled with APO, 12.5 μg/μL in solution, to achieve delivery of 5 mg/kg/d APO or solvent alone for VEH control mice. In the latter protocol, the dose of 5 mg/kg APO was distributed across each day in contrast to the single bolus delivery with the injection protocol. Thus, the mice receiving daily injections of APO would have been exposed to higher doses of APO, if transiently. 

To implant the mini-pumps, each mouse was anesthetized with a subcutaneous injection of 15% ketamine hydrochloride (70 mg/kg) and 2% xylazine hydrochloride (10 mg/kg). The implantation site in the right scapular region of the back was shaved and disinfected with alcohol. A 1-cm incision was made, followed by implantation of the mini-pump under the skin, which was then closed with vicryl 4.0 sutures. The total operation duration was less than 5 minutes. Verification of delivery from the mini-pump was achieved by measurement of the residual volume in the mini-pump reservoir after explant. As the Model 1002 osmotic mini-pump is designed for continuous delivery for only 14 days of delivery, initially implanted pumps were replaced after 2 weeks to cover the remaining 2 weeks of the experiments. 

Biometric Measurements

Refraction.

The refractive state was measured in a darkened room with a custom-built eccentric infrared photoretinoscope calibrated according to a published procedure.^22,37 Briefly, each unanesthetized animal was gently restrained, and its position adjusted until the first Purkinje image in the photoretinoscope was in the center of the pupil, indicating an on-axis measurement. The data were recorded using software designed by Schaeffel et al.,²² and each measurement was repeated at least three times. 

Corneal Radius of Curvature and Axial Components.

The CRC, anterior chamber depth (ACD, from the posterior corneal surface to the anterior lens surface), lens thickness (LT, from the anterior lens surface to the posterior lens surface), vitreous chamber depth (VCD, from the posterior lens surface to the retinal nerve fiber layer), and axial length (AL, from the anterior corneal surface to the retinal nerve fiber layer) were measured with a custom-made, ultra-long depth, high-resolution spectral-domain optical coherence tomography (SD-OCT) system.^38,39 After being anesthetized, the mice were placed in a cylindrical holder and mounted on the positioning stage in front of the modified slit lamp. The operator achieved on-axis alignment by adjusting the position of the mouse eye until both the x- and y-scans were oriented horizontally on the iris and a vertical specular reflex was present. The raw OCT data were exported and analyzed by custom-designed software to obtain the CRC and axial components.^38,39 Each measurement was determined based on the mean of three OCT-recorded images. 

Electroretinograms

Photopic flash ERGs were recorded with a custom-built Ganzfeld dome connected to a computer-based system (Q450SC UV; Roland Consult, Wiesbaden, Germany).⁴⁰ White LED stimuli of two intensities, 6.3 cd·s/m² and 20 cd·s/m² were used to generate and record cone responses under photopic conditions, with a background white light at 25 cd/m². The interstimulus interval was 0.4 second at all intensities. Ganzfeld illumination with white light was applied for 2.4 ms. Fifty signals were averaged for photopic measurements. The signal was amplified 1000-fold and bandpass filtered between 1 and 100 Hz. 

In the APO-injection experiments, recordings were started 20 minutes after APO or VEH-injection. General anesthesia was achieved as mentioned above, and the pupils were dilated with 1% tropicamide and 2.5% phenylephrine hydrochloride. A small amount of 2.5% methylcellulose gel was applied to the eye, and a custom Ag/AgCl wire loop electrode was placed over the cornea as an active electrode. Needle reference and ground electrodes were inserted into the cheek and tail, respectively. Recordings were started from the lower light intensity and progressed to higher level. Body temperature was maintained by placing the animals on a 37°C warming pad during the experiment. 

Statistics

Descriptive statistics and data analysis were performed using the Statistical Package for the Social Sciences version 19 (SPSS, Inc., Chicago, IL, USA). Data were expressed as means ± SEMs. The distributions of measured data were tested for normality. Baseline biometric parameters recorded from the various groups were analyzed by one-way ANOVA. After 4 weeks of the treatment, intergroup differences (APO-injection/infusion versus VEH-injection/infusion groups) on biometric parameters were compared by two-way ANOVA. Repeated measures ANOVA was used to analyze the changes in ERGs. Differences were defined as significant at P less than 0.05 and highly significant at P less than 0.001. 

Results

Health-Related Effects of Surgery, APO, and FD Treatments

Mini-pump implantation did not result in any case of mortality or infection and all mice remained in good health throughout the 4-week experimental period. There were also no significant differences in the weights of the various treatment groups, either at the beginning or end of the 4-week treatment period (data not shown). 

Effects of Daily Injection of APO on RE, Ocular Dimensions, and ERGs

Before the FD or APO manipulations, there were no significant differences between experimental groups in relation to RE and biometric parameters (P > 0.05, one-way ANOVA). 

Neither the FD manipulation (the FD groups versus the non-FD groups) nor the APO treatment (APO-injection versus VEH-injection) had any effect on the fellow non-FD eyes (P > 0.05, two-way ANOVA, Table 1). Thus, the non-FD eyes of the FD groups were used as references against which to assess biometric changes in deprived eyes, with all treatment effects being expressed as interocular differences, that is, the differences between FD (left eyes) and fellow eyes (right eyes). 

Table 1

 

Effects of Monocular FD and Daily Injection of APO for 4 Weeks on RE and Ocular Dimensions

 

At 4 weeks of treatment, there was a significant interaction between FD treatment and APO treatment (F_1,58 = 12.307, P = 0.001 for RE, F_1,58 = 8.491, P = 0.005 for VCD, F_1,58 = 21.983, P < 0.001 for AL, two-way ANOVA). The interocular RE difference for the FD-APO-injection group was reduced to 25% of that of the FD-VEH-injection group (F_1,58 = 20.258, P < 0.001, two-way ANOVA, post hoc simple effect analysis, Fig. 1A). In parallel with the refractive changes, the interocular VCD and AL differences were larger in the FD-VEH-injection group than in the FD-APO-injection group (F_1,58 = 11.855, P = 0.001 for VCD, F_1,58 = 43.867, P < 0.001 for AL, two-way ANOVA, post hoc simple effect analysis, Figs. 1B, 1C). Thus, APO-injection attenuated the FD-induced changes in RE, VCD, and AL (Fig. 1). 

Figure 1

 

Effects of daily injection of APO on refraction and ocular dimensions. Biometric measurements in VEH-injection, APO-injection, FD-VEH-injection, and FD-APO-injection groups before and after 4 weeks of treatment. (A) RE; (B) VCD; (C) AL. *P < 0.05, **P < 0.001, differences between FD-APO-injection and FD-VEH-injection group, two-way ANOVA.

After 4 weeks of treatment, neither FD nor daily APO-injection, applied alone or in combination, had any effect on CRC, ACD, or LT, even after 4 weeks of treatment (P > 0.05, two-way ANOVA). 

For ERGs, daily injection of APO significantly reduced a-wave amplitudes (F_1,12 = 3.887, P = 0.072 for interaction effect, F_1,12 = 5.485, P = 0.037 for main effect, two-way repeated ANOVA, Fig. 2D) and b-wave amplitudes for the higher, 20 cd·s/m² stimulus only (F_1,12 = 11.420, P = 0.005 for interaction effect, F_1,12 = 10.092, P = 0.008 for post hoc simple effect analysis, two-way repeated ANOVA, Fig. 2E). 

Figure 2

 

Effects of daily injection of APO on ERGs. (A) Sample of original ERG traces for daily injection treatments. Left column: VEH-injection. Right column: APO-injection. (B) Mean a-wave implicit times of daily APO-injection administration. (C) Mean b-wave implicit times of daily APO-injection administration. (D) Mean a-wave amplitudes of daily APO-injection administration. (E) Mean b-wave amplitudes of daily APO-injection administration. For VEH-injection group: n = 8; APO-injection group: n = 6. Only right eyes were tested. *P < 0.05 compared with VEH-injection group, two-way repeated ANOVA. The origin of the y-axis is offset from zero to make the difference easier to visualize.

Effects of Continuous Infusion of APO on RE, Ocular Dimensions, and ERGs

There were no significant differences between any of the groups, for any of the baseline measurements (P > 0.05, one-way ANOVA). Also, neither the FD nor the APO-infusion treatment had any effect on the fellow eyes in respective treatment groups (P > 0.05, two-way ANOVA, Table 2). 

Table 2

 

Effects of Monocular FD and Continuous Infusion of APO for 4 Weeks on RE and Ocular Dimensions

 

There was no significant interaction between FD treatment and continuous infusion of APO on interocular differences of RE (F_1,32 = 0.042, P = 0.840), VCD (F_1,32 = 0.089, P = 0.767), or AL (F_1,32 = 0.896, P = 0.351) (two-way ANOVA, Fig. 3). The FD significantly increased the interocular differences in RE (F_1,32 = 61.258, P < 0.001), VCD (F_1,32 = 7.222, P = 0.011), and AL (F_1,32 = 13.330, P = 0.001) (two-way ANOVA, Fig. 3) after 4 weeks of treatment. However, unlike daily injection of APO, its continuous infusion had no effect on the FD-induced changes in RE (F_1,32 = 1.401, P = 0.245), VCD (F_1,32 = 0.089, P = 0.767), or AL induced by FD (F_1,32 = 0.758, P = 0.391) (two-way ANOVA, Fig. 3). 

Figure 3

 

Effects of continuous infusion of APO on refraction and ocular dimensions. Biometric measurements in VEH-infusion, APO-infusion, FD-VEH-infusion, and FD-APO-infusion groups before and after 4 weeks of treatment. (A) RE; (B) VCD; (C) AL.

Once again, after 4 weeks of treatment, FD had no effect on CRC, ACD, or LT, nor did continuous infusion of APO (P > 0.05 two-way ANOVA). 

Regarding photopic ERGs, continuous infusion of APO significantly increased both b-wave implicit times (F_1,6 = 0.818, P = 0.401, for interaction effect, F_1,6 = 8.727, P = 0.025 for main effect, two-way repeated ANOVA, Fig. 4C) and a-wave amplitudes (F_1,6 = 0.039, P = 0.849, for interaction effect, F_1,6 = 6.181, P = 0.047, for main effect, two-way repeated ANOVA, Fig. 4D) for both intensities. 

Figure 4

 

Effect of continuous infusion of APO on ERGs. (A) Sample of original ERG traces for infusion treatment. Left column: VEH-infusion. Right column: APO-infusion. (B) Mean a-wave implicit times of continuous APO-infusion administration. (C) Mean b-wave implicit times of continuous APO-infusion administration. (D) Mean a-wave amplitudes of continuous APO-infusion administration. (E) Mean b-wave amplitudes of continuous APO-infusion administration. For VEH-infusion group: n = 4; APO-infusion group: n = 4. Only right eyes were tested. *P < 0.05 compared with VEH-infusion group, two-way repeated ANOVA. The origin of the y-axis is offset from zero to make the difference easier to visualize.

Differences Between Daily Injection or Continuous Infusion of APO on RE, Ocular Dimensions, and ERGs

For the 4-week treatment period used in this study, daily injection of APO completely inhibited FD-induced RE changes and almost completely inhibited its effect on VCD and AL. In contrast, continuous infusion of APO had no significant effect on the FD-induced changes in RE, VCD, and AL. We analyzed biometric changes in the FD-APO-injection, FD-VEH-injection, FD-APO-infusion, and FD-VEH-infusion groups by using two-way ANOVA with factors of APO treatment (APO versus vehicle) and administration route (injection versus infusion). The effect of APO treatment on myopia was dependent on the APO treatment paradigms (daily injection versus continuous infusion) (Table 3; Fig. 5). Neither daily injection nor continuous infusion of APO had any significant effect on the FD-induced changes in ACD, LT, or CRC (Table 3). 

Table 3

 

Two-Way ANOVA for Treatment and Administration Route

 

Figure 5

 

Differences between APO-injection and APO-infusion administration on RE, VCD, and AL. Biometric measurements in FD-VEH-injection, FD-APO-injection, FD-VEH-infusion, and FD-APO-infusion groups after 4 weeks of treatment. *P < 0.05, difference between FD-APO-injection and FD-APO-infusion group, two-way ANOVA.

After 3 weeks of daily injection or continuous infusion of APO or vehicle, we analyzed ERG changes by using three-way repeated ANOVA with factors of APO treatment (APO versus vehicle), administration route (injection versus infusion), and stimulus intensity. The effects of APO on ERGs were dependent on the administration paradigm: Daily injection of APO significantly decreased a- and b-wave amplitudes, whereas continuous infusion of APO significantly increased a- and b-wave amplitudes. The a- and b-wave implicit times were differentially affected by APO treatment paradigms. Thus, the daily injection and continuous infusion of APO produced opposite effects on ERG measurements (Table 4; Fig. 6). 

Table 4

 

Statistical Analysis of the Effect of Treatment, Administration Route, and Interactions on ERGs

 

Figure 6

 

Differences between APO-injection and APO-infusion administration on photopic ERGs. Electroretinogram measurements in VEH-injection, APO-injection, VEH-infusion, and APO-infusion groups. (A) Mean a-wave implicit times. (B) Mean b-wave implicit times. (C) Mean a-wave amplitudes. (D) Mean b-wave amplitudes. Only right eyes were tested. *P < 0.05, **P < 0.001, differences between APO-injection and APO-infusion group, three-way repeated ANOVA. The origin of the y-axis is offset from zero to make the difference easier to visualize.

Discussion

This study is an extension of previous reports in which it was shown that daily injection of APO reduced FDM-induced AL elongation in chickens,¹⁰ guinea pigs,¹¹ and monkeys.¹³ Our findings clearly indicate that in mice, daily APO-injection also offered protection against FDM development. Although endogenous DA in retinas of non-FD mouse eyes can tonically activate DA receptors under physiological conditions, systemic daily administration of APO may preferentially affect the FDM eyes with depleted retinal DA, normalizing DA receptor activity. Thus, consistent with findings in other species, our analysis showed that VCDs and ALs were increased in FDM eyes compared with fellow eyes, and daily injection of APO inhibited these FD-induced changes. 

Although the mode of action of APO-injection-mediated inhibition of FDM is not clear, it is possible that retinal DA receptors take part in this process. It is reasonable to assume that APO affects other receptors besides DA receptors, because it is reported to interact with serotonin and adrenergic receptors, although it has lower affinity for the latter receptors.²⁶ The biological function of DA-induced signaling is mainly dictated by extracellular DA levels, which are modulated by changes in DA release, DA reuptake, and DA metabolism.⁴¹ However, because of the technical challenges associated with working with the very small size of mouse retinas, we were not able to determine retinal DA levels in our study and thus to assess the effects of FD and APO treatments. 

As an alternative to measuring extracellular DA levels, we evaluated instead the effect of our APO treatments on retinal function by measuring their effects on ERGs. It is generally accepted that the ERG a-wave reflects the activity of retinal photoreceptors,⁴² whereas the ERG b-wave reflects bipolar cell activity.⁴³ Changes in ERGs after systemic administration of dopaminergic drugs can be taken as an indication of retinal DA receptor activation. For example, APO-injection may decrease the amplitude of the ERG b-wave in rabbits by activating retinal DA receptors.^44,45 We also found that photopic ERG b-wave amplitudes were significantly decreased by daily injection of APO. Therefore, daily injection of APO in our study could have affected ocular growth and refractive development through interacting with retinal DA receptors. 

Although the stability of APO in the mini-pumps was not directly assessed, that APO remained chemically stable and biologically active over the 14 days of implantation is consistent with results from previous related studies involving mini-pumps and similar experimental procedures,^27,28,35,46 as well as from an in vitro experiment in which the biological activity of APO was tested after storage in a mini-pump at 37°C for 2 weeks (data not shown). 

We did not attempt to measure retinal APO content, but it is likely that its steady-state circulating concentration during continuous infusion was lower than the peak concentration reached with once-a-day injections. It has been reported that 5 mg/kg/d APO-infusion was sufficient to stimulate the DA receptors, reversing the behavioral changes resulting from chronic functional impairment of dopaminergic neurons of the substantia nigra produced by 1-methyl-4-phenyl-1,2,3,6–tetrahydropyridine.³⁶ This result suggests that APO-infusion can be delivered to the brain and stimulate the DA receptors there. It is likely that in our study, APO-infusion delivery was sufficient to reach the retina because the blood–retinal barrier is more permeable to lipophilic compounds than the blood–brain barrier.⁴⁷ Thus, systemic administration (injection or infusion) of APO would probably have stimulated both extraretinal and intraretinal DA receptors, especially because the levels of DA receptor expression in the retina and brain are reported to be related to one another.^48,49 Although there is reasonable evidence that systemic administration of APO acts on retinal DA receptors to exert its control of myopia development, extraretinal DA receptors could also play a role in FDM. Additional studies using focal deletion of retinal and extraretinal DA receptors are required to clarify the role of the latter receptors. One possible source of error in our study is that we used different photoretinoscopes, which may confound the difference we observed in RE between daily injection and continuous infusion of APO. However, the detection of the interocular difference between FDM and fellow eyes and the parallel difference in axial changes seen after the daily injection (but not continuous infusion) of APO treatment suggests that the RE difference is likely attributable to the different APO treatment paradigms, rather than the use of different photoretinoscopes. 

Dopamine concentrations vary in association with normal day/night rhythms. Dopamine production and release increase during daytime hours and decrease at night.⁵⁰ It has been proposed that DA acts through D2Rs to inhibit melatonin production, and modulate responses to this rhythm.⁵¹ Given the timing of the APO injections in our study, animals would have experienced transiently high levels of APO during the day and low levels at night, similar to the natural (circadian) variations in retinal DA. It is thus possible that the timing of our injections was a contributing factor in observed inhibitory effect of daily APO injections on FDM. 

Other possibilities for why myopia development is dependent on the APO delivery protocol stems from differences in APO affinity for the two DA receptor subtypes. The APO affinity for rat D2Rs is greater than that for D1Rs by up to 22-fold,⁵² and the APO dose-response curve is biphasic. Apomorphine decreases D2R-dependent protein kinase C activity at low concentrations (0.1 μM), but increases the D1R-dependent protein kinase C activity at higher concentrations (10 μM).⁵³ It is possible that low circulating levels of APO, as achieved with APO-infusion, were insufficient to stimulate D1Rs, whereas the high peak levels obtained with APO injections were adequate to activate the latter receptors. 

If the above assumption is correct, then the difference between the effects of daily injection versus infusion of APO on ERG responses may reflect whether or not D1R receptors were activated. The D1 agonists reduce b-wave amplitude in rabbits⁵⁴ and monkeys,⁵⁵ whereas D2 agonists increase b-wave amplitudes in iguanas.⁵⁶ Although other conflicting results have been reported for the effects of dopaminergic drugs on ERGs,⁵⁷ in general, retinal D1Rs and D2Rs elicit physiological responses that are usually opposite to one another, when selectively activated. However, it is not clear whether APO-injection and APO-infusion had different effects on FDM by working through different DA receptor subtypes. Further studies are warranted to evaluate the effects of more selective D1R and D2R agents than APO on FDM. Such pharmacological studies could be combined with appropriately genetically altered mice to provide additional insights into the role of these two types of DA receptors.⁵⁸ 

The finding of opposite effects on ERGs by two different treatment paradigms can be taken as an indication that APO was delivered at a concentration adequate for stimulating retinal DA receptors. This also suggests that the lack of an effect of continuous APO-infusion on myopia development is unlikely due to the inaccessibility of APO to retinal DA receptors. Instead, this likely reflects functional differences, such as different DA kinetics (i.e., pulse versus continuous activation) and different levels of DA receptor activation (i.e., upregulation versus physiological activation). 

In the present study, we found that daily systemic APO-injection in mice had effects on FDM eye growth similar to those described in other species in which APO was instead delivered locally rather than systemically. Thus, the mouse is not an outlier compared with other animal models with respect to FDM development and responses to DA agonists. In contrast, continuous APO-infusion administration, like constant light stimulation, did not block FDM development. We propose that normal diurnal DA rhythms affect the development of myopia in FD eyes. In turn, both the exact timing and magnitude of the concentration maxima may result in different pharmacological responses. Additional studies in FDM mice using different modes of drug administration, including further control over drug delivery rate, and in which different receptor subtypes are examined, will provide greater insight into designing effective protocols for pharmacological treatment of FDM development. 

Acknowledgments

The authors thank Frank Schaeffel for providing support with our eccentric infrared photoretinoscope, Peter Reinach from Wenzhou Medical University, and Tim Corson from the Volunteer Editor Program of IOVS for editorial support that improved the manuscript. 

Supported by National Basic Research Program of China (973 project) number 2011CB504602, National Natural Science Foundation of China (81422007, 81371047, and 30973278), Natural Science Foundation of Zhejiang (LZ14H120001), Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents, Program for New Century Excellent Talents in the University, National Ministry of Education Grant NCET-10–0977, and National Young Excellent Talents Support Program. The authors alone are responsible for the content and writing of the paper. 

Disclosure: T. Yan, None; W. Xiong, None; F. Huang, None; F. Zheng, None; H. Ying, None; J.-F. Chen, None; J. Qu, None; X. Zhou, None