Methylphenidate Inhibits the Development of Myopia by Altering Dopamine and Norepinephrine Reuptake

Abstract

Purpose

Dopaminergic dysregulation plays a critical role in myopia development in animal models. Although its relevance to human myopia remains uncertain, the observation that methylphenidate hydrochloride (MPH)—a dopamine (DA) and norepinephrine (NE) uptake inhibitor—slows myopia progression in children suggests a possible link. This study aimed to investigate whether MPH can inhibit myopic growth and elucidate the underlying mechanisms using an animal model.

Methods

MPH was administered via oral, topical, or intravitreal routes for 7 days (minimum 5 per group) to chicks undergoing form-deprivation myopia (FDM). Myopia was assessed by refraction and axial length. Retinal DA and NE dynamics—including synthesis, release, uptake, breakdown (DA only), extracellular levels, and receptor sensitivity—were evaluated using mass spectrometry and chronoamperometry (minimum 5 per group). DA and NE receptors were pharmacologically blocked (DA = spiperone, SCH-23390; and NE = yohimbine) to determine their role in MPH's anti-myopic effects.

Results

MPH inhibited FDM via all administration routes (oral = 55%, P < 0.05, topical = 45%, P < 0.05, and intravitreal = 87%, P < 0.05 protection against myopic growth). It enhanced DA and NE synthesis while blocking their uptake, resulting in elevated extracellular levels. MPH's anti-myopic effects were abolished when DA or NE receptors were pharmacologically blocked. Additionally, NE receptor stimulation alone inhibited FDM (P < 0.05).

Conclusions

MPH suppresses experimental myopia, with its effects linked to increased extracellular levels of DA and NE. These findings align with the anti-myopic effects observed in clinical studies, supporting a role for DA in human myopia and suggesting that NE may also contribute to the regulation of ocular growth.

The refractive disorder myopia (short-sightedness) poses a significant global health risk due to its high prevalence¹ and the sight-threatening pathologies associated with progression toward high myopia (<−6 diopters [D]).^2,3 Studies in animal models, including nonhuman primates, have suggested that the dopaminergic system plays a central role in the development of myopia (for a review, see Ref. 4). Specifically, a reduction in extracellular dopamine (DA) within the retina is one of the earliest biochemical changes observed in animal models of myopia. Further, pharmacological stimulation of the dopaminergic system inhibits the development of experimental myopia in all species investigated.^5–32 Light-induced DA release has also been postulated to underlie the epidemiological finding that time spent outdoors is protective against the onset of myopia,³³ a hypothesis supported by animal studies.^34–37

Although DA has been extensively studied in animal models, investigating its role in humans remains challenging. Consequently, its involvement in human myopia development—including its hypothesized role in the protective effects associated with time spent outdoors—has not yet been definitively established. In an effort to explore this, a recent clinical study by Gurlevik et al.³⁸ examined whether lower rates of myopia progression were seen in children (aged 8–18 years of age) prescribed the DA modulator methylphenidate hydrochloride (MPH) for the treatment of attention deficit hyperactivity disorder (ADHD). ADHD, which is characterized by a persistent pattern of developmentally inappropriate inattention, hyperactivity, or impulsivity, is one of the most common behavioral disorders in children and adolescents, affecting 6% to 10% of children and adolescents worldwide.³⁹ MPH, commercially known as Ritalin, treats the symptoms of ADHD by blocking the reuptake of DA and norepinephrine (NE) through their respective cellular transporters.^40,41 This facilitates the accumulation of DA and NE in the extracellular space, promoting extended binding and stimulation of target receptors.^40,41

The small study by Gurlevik and colleagues³⁸ assessed the rate of myopia development, over a period of 12 months, in children aged 8 to 18 years old (MPH treatment for ADHD = 19; and untreated age-matched control group = 20). It should be noted that MPH also inhibits the reuptake of NE, a molecule not heavily studied in the context of myopia (a knowledge gap this manuscript helps to address).

By removing the confounding factor of an ADHD diagnosis, this current study looked to understand whether MPH can inhibit the development of myopia in an animal model,⁴² although not with the intention of proposing MPH as a clinical treatment for myopia. To replicate its use in humans, MPH was administered to chicks via ingestion at a dosage scaled for body size, with the caveat that pharmacokinetic differences between species may still influence drug efficacy. Following this, to examine whether MPH can inhibit ocular growth when targeted directly to the eye (without the confounding effects of systemic distribution), MPH was administered through two additional means. First, MPH was administered directly to the presumed target tissue (the retina) via intravitreal injection. This ensures a known amount of the drug directly strikes the retina. However, due to the invasive nature of this procedure, which can introduce confounding factors (for a review, see Ref. 43), this study also investigated the effects of applying MPH as topical eye drops. For all 3 avenues of treatment, ocular and systemic distribution of MPH was assessed 30 minutes post-application to understand penetration levels of the retina. Additionally, blood levels of MPH were monitored over a 6-hour period post-intramuscular administration to analyze pharmacokinetics. Given that MPH is a behavioral modifier (e.g. increases in sustained attention and memory consolidation, euphoria, nervousness, wakefulness/insomnia, and appetite suppression^44,45), the behavior of treated chicks was also examined.

To better understand how MPH may inhibit myopia development, this study investigated if (and how) MPH may alter DA and NE retinal activity (i.e. synthesis, release, uptake, breakdown [DA only], and total extracellular levels, as well as receptor sensitivity). Furthermore, to investigate a potential causal link, we studied if pharmacological manipulation of DA and NE receptor activity altered the anti-myopic effects of MPH.

Methods

Animals and Housing

One-day-old male White Leghorn chickens (Gallus gallus) were obtained from Aviagen Hatchery (Goulburn, New South Wales, Australia) and housed in temperature-controlled rooms under normal laboratory lighting (approximately 500 lux, Phillips TLD36W840ALTO [correlated color temperature 4000 K, see previous work for full spectral details³⁵]) on a 12:12 hour light:dark cycle, with lights on at 9 AM and off at 9 PM. The chickens had access to unlimited amounts of food and water and were given 4 days to become accustomed to their environment before the initiation of experiments on post-hatching day 5 (P5). Authorization to conduct experiments using animals was given by the University of Canberra Animal Ethics Committee under the ACT Animal Welfare Act 1992 (Project Number: CEAE 11943) and conformed to the ARVO Resolution for the Use of Animals in Ophthalmic and Vision Research.

Drug Administration and Preparation

The dosage of MPH administered to animals was equivalent to that prescribed to children aged 6 to 10 years (the primary age range for myopia onset in areas of the world with high prevalence rates).⁴⁶ According to the Australian Medicines Handbook,⁴⁷ the recommended MPH dose for children aged between 6 and 10 years old is 25 mg/day. This falls within the dosage range recommended by the US Food and Drug Administration and the UK National Health Service.^48,49 The World Health Organization's Child Growth Standards⁵⁰ lists the median weight of a child between the ages of 6 and 10 as 21 to 31 kg. This would equate to a dose range of between 1.2 mg/kg/day to 0.8 mg/kg/day. This dosage also concurs with a recent meta-analysis of 11 randomized clinical trials and 38 cohort studies, which found the maximum dose of MPH to range from 0.8 to 1.8 mg/kg/day.⁵¹

Based on these data, we administered the median dosage of 1 mg/kg/day (equivalent to the dosage received at the median weight of a child aged 8 years old). Due to the smaller size of the chick (50 grams; average body weight at P5), this equated to 0.05 mg of MPH per day (irrespective of the method of delivery).

For all preparations, MPH (M325880, Toronto Research Chemicals, molecular weight 269.77 g/mol) was freshly dissolved in nuclease-free water. For MPH administration, the chicks were treated via 1 of 3 avenues: (1) 20 µL syringe fed, (2) 2 × 40 µL topical eye drops to the left (treated) eye, or (3) 10 µL intravitreal injection into the left (treated) eye. MPH was administered each day at 10 AM, which was 1 hour after the onset of the light phase. Intravitreal injections (using a 30-gauge needle [Terumo] fitted to a Hamilton syringe) were performed under light isoflurane anesthesia (5% isoflurane in 1 L of medical grade oxygen per minute; Veterinary Companies of Australia, Kings Park, New South Wales, Australia) using a vaporizer gas system (Stinger Research Anesthetic Gas Machine [2848], Advanced Anesthesia Specialists, Payson, AZ, USA).

For all 3 avenues of MPH administration, the chicks were treated for a period of 7 days (Fig. 1A; see Table 1 for experimental groupings and numbers), with an additional control group receiving vehicle solution alone (nuclease-free water, pH 7.4). Given the greater control over drug delivery to the retina, intravitreal injections were used to examine potential dose-dependent effects of MPH on myopia development. To assess this, two additional doses (1 and 2 log units below the “human equivalent” dose) were tested (see Table 1 for details).

Figure 1.

Experimental timeline. Chicks were housed under a 12:12 hour light:dark cycle (lights-on at 9 AM) and given 4 days post-hatching (P1–P4) to become accustomed to their surroundings (white bars) prior to the commencement of experiments on P5. (A) Ocular parameters (refraction and axial length) were measured following 7 days of treatment (P12) with methylphenidate hydrochloride (MPH). Chicks were fitted with translucent diffusers on P5, with MPH administered daily (at 10 AM) from P5 to P12 through one of three routes (ingestion, topical eye drops, or intravitreal injections). Behavioral monitoring was undertaken each day over two 15-minute periods (15 minutes and 5 hours following treatment). (B) MPH distribution was measured in otherwise untreated chicks administered with MPH via ingestion, topical eye drops, or intravitreal injections. Blood and ocular tissue were collected (dashed arrow) 30 minutes following treatment on P5. (C) The effects of diffuser-wear and MPH (administered via ingestion) on dopaminergic and noradrenergic functions (i.e. analyte levels, receptor sensitivity, as well as the rate of synthesis, release, uptake, and catabolism) were evaluated in retinal and vitreal tissue collected (dashed arrow) 2 hours following treatment on P5. (D) Ocular parameters (refraction and axial length) were measured following 3 days of treatment with MPH alone or in conjunction with a dopaminergic and/or noradrenergic receptor antagonist. Chicks were fitted with translucent diffusers on P5, with drugs administered daily (at 10 AM) from P5 to P7. MPH was administered via ingestion, whereas antagonists were administered via an intravitreal injection.

Table 1.

Allocation of Animals Across the Experimental Paradigms Investigated

Treatment	Dose, mg	Dose, µmoles	Number of Animals
Effects of MPH on the development of myopia
Ingestion (20 µL)
• Age-matched untreated controls	–	–	4
• No ocular treatment + MPH	0.05	0.185	5
• FDM only	–	–	10
• FDM + placebo	–	–	5
• FDM + MPH	0.05	0.185	9
Topical eye drops, 2 × 40 µL drops
• FDM only	–	–	7
• FDM + placebo	–	–	6
• FDM + MPH	0.05	0.185	7
Intravitreal injection, 10 µL
• FDM only	–	–	6
• FDM + placebo	–	–	5
• FDM + MPH	0.05	0.185	6
• FDM + MPH	0.005	0.019	5
• FDM + MPH	0.0005	0.002	6
MPH distribution and pharmacokinetics
• MPH, ingestion	0.05	0.185	2*
• MPH, topical eye drops	0.05	0.185	2*
• MPH, intravitreal injection	0.05	0.185	2*
• MPH, intramuscular injection	0.05	0.185	2
Effects of MPH on dopaminergic and noradrenergic functions
Vitreal NE and retinal DA and NE levels
• Age-matched untreated controls	–	–	5
• Control + MPH, intravitreal injection	0.05	0.185	5
• FDM only	–	–	5
• FDM + MPH, intravitreal injection	0.05	0.185	5
The effect of MPH treatment route on extracellular DA levels
• Age-matched untreated controls	–	–	6
• FDM only	–	–	7
• FDM + placebo, intravitreal injection	–	–	7
• FDM + MPH, ingestion	0.05	0.185	6
• FDM + MPH, topical eye drops	0.05	0.185	6
• FDM + MPH, intravitreal injection	0.05	0.185	7
DA and NE synthesisa/ releaseb/ uptakec/ breakdownd/ receptor sensitivitye
• Age-matched untreated controls	–	–	10a,b,c/5d,e
• No ocular treatment + MPH	0.05	0.185	10a,b,c/5d,e
DA release – positive controls
• Age-matched untreated controls	–	–	5
• Mercaptosuccinate	15	0.010	5
• Tetrabenazine	0.2	0.0006	5
• Glibenclamide	0.1	0.0003	5
• Tolbutamide	0.8	0.003	5
• Dark-adaptation†	–	–	5
• Light†	–	–	5
Effects of dopaminergic and noradrenergic antagonists on the anti-myopic effects of MPH
• FDM only	–	–	5
• FDM + MPH	0.05	0.185	5
• FDM + MPH + placebo	0.05	0.185	5
• FDM + MPH + DA-Ant	0.002f,g	0.005f,g	5
• FDM + MPH + NE-Ant	0.0004h	0.001h	5
• FDM + MPH + DA-Ant + NE-Ant	0.002f,g/0.0004h	0.005f,g/0.001h	5

DA, dopamine; DA-Ant, dopaminergic antagonists (spiperone^f and SCH-23390^g); DA-Ant/NE-Ant, combination of spiperone, SCH-23390 and yohimbine; FDM, form-deprivation myopia; MPH, methylphenidate hydrochloride; NE, norepinephrine; NE-Ant, noradrenergic antagonist (yohimbine^h).

^*Both eyes from the same animal were treated and analyzed, meaning a total of four eyes were measured.

^†Animals were dark adapted overnight (dark-adaptation) before being exposed to 500 lux fluorescent light for 30 minutes (light) to induce an increase in dopamine release.

Superscript letters indicate the numbers used in each experiment (see section headings). Placebo solutions were comprised of nuclease free water (pH 7.4).

Myopia Induction and Measurement

Form-deprivation myopia (FDM) was induced monocularly by fitting a translucent diffuser over the left eyes of the chickens, with the right eyes left untreated and serving as an internal contralateral control. The fitting of diffusers was undertaken as previously described.⁵²

To assess the degree of myopia development, refraction and ultrasonography measures were taken for both the treated (left) and contralateral control (right) eyes of each animal prior to the commencement of experiments and within 2 to 5 hours following treatment on the final day of the experimental period (see Fig. 1A). Refraction and biometric measures were undertaken as previously described.⁵²

Behavioral Monitoring

To assess whether MPH induced behavioral changes that might underlie its anti-myopic effects, several well-established behavioral tests were conducted, as detailed in Supplementary Tables S1 and S2. Animals were observed by a treatment-blind assessor for two 15-minute periods daily—once 15 minutes after MPH administration and again 5 hours post-treatment. During each session, behaviors were recorded based on established criteria for “normal”⁵³ and “stressed”^54,55 chick behavior, as well as MPH-associated behaviors previously described in other animal models.^56–59 Behavioral data were quantified as the number of chicks (“events”) exhibiting each behavior per experimental group.

In addition to behavioral observations, daily food and water intake was measured per cage and normalized by the number of animals housed, as individual housing was not feasible. For clarity, repeated treatment conditions across batches (e.g. age-matched untreated controls, form-deprivation, and form-deprivation + placebo) were consolidated into a single data point for each of these conditions. To complement these assessments, fecal output was also measured 45 minutes post MPH treatment.

Ocular Distribution of MPH

To assess the biodistribution of 0.05 mg MPH across 3 administration routes, biological samples (blood, tears, cornea, aqueous humor, iris/lens/ciliary body, vitreous humor, retina, and choroid) were collected from 2 animals (4 eyes per treatment avenue) 30 minutes post-administration (Fig. 1B; Table 1). Further, to investigate the pharmacokinetics of 0.05 mg MPH in the systemic circulation of chicks, blood samples from a prior experiment were analyzed 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 6 hours post intramuscular administration (2 per timepoint; see Table 1). It is important to note that most commercially available forms of MPH, including the formulation used in this study, consist of a racemic mixture containing both the L- and D-enantiomers. Our analysis did not differentiate between the pharmacologically active D-enantiomer and the less active L-enantiomer.

For sample collection, chickens were heavily anesthetized using isoflurane and euthanized by decapitation. Immediately prior to euthanization, tear samples were collected using Shirmer strips. Following euthanization, blood (100 µL) was rapidly collected from the exposed carotid artery by syringe. Each eye was rapidly removed, and the aqueous humor was collected using needle aspiration. Eyes were then hemisected equatorially to float both the anterior and posterior eye cup in 1x phosphate buffered saline (PBS; pH 7.4; Sigma-Aldrich). This facilitated the collection of the remaining ocular samples listed above. Samples were snap frozen and stored at –80°C until analysis. Samples were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS; Agilent 1260 Infinity HPLC interfaced with an Agilent 6410 quadrupole mass spectrometer) following a method adapted from that previously described by our group¹¹ (see Supplementary Methods, section 1.1 for full LC-MS/MS methods).

Effects of MPH on Retinal DA and NE Dynamics

Although the central effects of MPH are well-documented across a range of species, this study investigated its impact in chicks and on the retinal DA and NE systems—the two primary targets for MPH. Specifically, total levels of DA and NE were measured in vitreal and/or retinal samples 2 hours after one of the following treatments: (1) FDM alone, (2) FDM combined with intravitreal MPH administration (0.05 mg), (3) intravitreal MPH administration (0.05 mg) in otherwise untreated animals, or (4) aged-matched untreated control animals. The full methodology is listed in the Supplementary Materials (section 1.1). To evaluate potential differences in DA release across treatment routes, vitreal concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC)—used as a proxy for DA release—were measured 2 hours following oral, topical, and intravitreal administration of MPH (0.05 mg). DA and NE levels were measured using LC-MS/MS (Fig. 1C; Table 1), with the full methodology listed in the Supplementary Materials (section 1.1).

We subsequently examined the effects of oral MPH treatment (0.05 mg) on DA and NE dynamics in eyes that had not undergone myopia induction, by comparing these neurotransmitter profiles to those in naïve animals that had not been exposed to MPH. Assessments were conducted 2 hours post-administration and included measurements of DA and NE synthesis, release, uptake rates, and receptor sensitivity. Additionally, DA breakdown was evaluated. For detailed methodology, adapted from published works,^60–69 refer to the Supplementary Materials (sections 1.3–1.7).

Investigating Whether the Increased Bioavailability of DA and NE at Their Respective Receptors Drives the Anti-Myopic Effects of MPH

To investigate a potential causal link between retinal DA and NE activity and the anti-myopic effects of MPH, we examined whether blocking DA and NE receptor activity altered MPH efficacy. Form-deprived animals receiving daily MPH were divided into four groups, each receiving a daily intravitreal injection of one of the following:

• 1.A cocktail of D1 (SCH-23390, D054; Sigma-Aldrich; 0.002 mg per day) and D2 (spiperone, S7395; Sigma-Aldrich; 0.002 mg per day) receptor antagonists;
• 2.A nonspecific NE receptor antagonist (yohimbine, Y3125; Sigma-Aldrich; 0.0004 mg per day);
• 3.A cocktail containing a D1, D2, and nonspecific NE receptor antagonist (SCH-23390 [0.002 mg per day], spiperone [0.002 mg per day], and yohimbine [0.0004 mg per day]); or
• 4.A vehicle solution (water, pH 7.4).

Drugs were administered daily for a period of 3 days, with axial length and refraction measured before and after the treatment period (Fig. 1D). As the specific DA receptor subtype mediating MPH's anti-myopic effects remains unknown, both D1 and D2 receptors were blocked. Thus, due to the use of a nonspecific antagonist for NE and the simultaneous blockade of both D1 and D2 receptors for DA, the specific receptor subtypes involved in MPH's anti-myopic effects cannot be determined from the current results.

For administration of the D1/D2 cocktail, spiperone and SCH-23390 were freshly dissolved in 0.1% ascorbic acid (in water, pH 7.4), whereas the nonspecific NE receptor antagonist yohimbine was freshly dissolved in water (pH 7.4; see Table 1 for experimental groupings, numbers, and drug dosage). For MPH administration, chicks were treated through a 20 µL syringe feed (containing 0.05 mg of MPH), whereas all other drugs were given through a 10 µL intravitreal injection into the left (treated) eye of anesthetized animals (described above).

The Effects of NE on the Development of Experimental Myopia

Although the ability of DA agonists to inhibit myopia is well-established, the role of NE receptor stimulation remains less understood. To evaluate whether NE could suppress myopic growth, it was administered daily (at lights-on [9 AM]) to chicks undergoing form-deprivation over a 3-day period (see Table 1). Axial length and refraction were measured before and after the treatment period. Each day, a 1 mM NE solution was made fresh in 1 × PBS (pH 7.4) and administered to anesthetized chickens as a 10 µL intravitreal injection into the left (treated) eye (described above).

Data and Statistical Analysis

As previously noted,^7,70 a power analysis indicated that a minimum group size of 4 animals was required to achieve 80% power to detect a 1 D change in refraction, assuming a standard deviation (SD) of approximately 0.5 D.

For measurements of ocular DA and NE dynamics, a power calculation determined that the minimum group size of 5 was required to achieve 80% power in observing a 25% reduction in DOPAC levels when the SD is 0.03, as previously observed in our laboratory.⁷¹

Refractive and axial length measurements are presented as means ± standard error of the means, with the data representing the difference between the treated and the contralateral control eye at the end of treatment. Behavioral data are presented as the number of each behavioral “event” observed per group. LC-MS/MS data are presented as the peak area ratio (PAR) ± standard error of the means. Chronoamperometry data are presented as the number of events, concentration of DA/NE detected, or uptake rate of DA/NE ± standard error of the means. All figures, with the exception of behavior and receptor sensitivity, are presented as box plots representing the interquartile ranges, with dots representing individual data points.

Prior to statistical analysis, all data were first tested for normality and homogeneity of variance (Shapiro-Wilk test). All statistical analyses for the effect of treatment, with the exception of behavior, were undertaken using a 1-way univariate analysis of variance (ANOVA) followed by post hoc Student's t-test with Bonferroni correction for multiple testing. Behavior was analyzed using a chi-square analysis. All analyses were undertaken in the statistical software program SPSS (IBM, Armonk, NY, USA) with a cutoff of 0.05 for statistical significance.

Results

MPH Treatment Slows the Rate of Myopia Development

Control Paradigms

Prior to the commencement of experiments, there were no significant differences between animals allocated to any of the treatment groups with respect to refraction (Wilks’ Lambda = 0.543, F(3, 79) = 2.186, P = 0.119; see Fig. 2), or axial length (Wilks’ Lambda = 0.557, F(3, 79) = 2.064, P = 0.136; see Fig. 2). For all FDM only groups (with one group run alongside each mode of MPH administration), diffuser-wear induced a significant myopic shift in refraction (Wilks’ Lambda = 0.075, F(2, 21) = 29.563, P < 0.001) and axial elongation (Wilks’ Lambda = 0.013, F(2, 21) = 186.614, P < 0.001) when compared with untreated contralateral control values (Table 2). The axial length changes seen (see Fig. 2) represented elongation of the vitreous chamber (Wilks’ Lambda = 0.022, F(2, 21) = 75.824, P < 0.001), with no significant differences observed in anterior chamber depth (ACD) and lens thickness between FDM only and contralateral control eyes (Supplementary Table S4; ACD - Wilks’ Lambda = 0.228, F(2, 21) = 10.713, P = 0.079, Lens - Wilks’ Lambda = 0.363, F(2, 21) = 7.558, P = 0.104). As there was no significant difference in the degree of myopia development among the three FDM only groups tested (refraction - ANOVA F(2, 21) = 2.417, P = 0.064; axial length - ANOVA F(2, 21) = 0.787, P = 0.541), these data are combined as a single group for ease of visualization in Figure 2. As previously observed,⁷² administration of a vehicle solution (water) through ingestion, topical eye drops, or intravitreal injection did not affect the myopic shift or axial elongation associated with FDM (see Fig. 2; Table 2). In untreated controls, FDM only chicks, and groups undergoing treatment via ingestion or topical eye drops, the refractive changes observed correlated strongly with the changes seen in axial length (R² = 0.71; Supplementary Fig. S1). For chicks undergoing treatment via intravitreal injections, although the same trend was observed in the two measures, the correlation between refraction and axial length was not as strong (R² = 0.49).

Figure 2.

The effect of methylphenidate hydrochloride (MPH) on refraction and axial length following 7 days of treatment. Differences in (A) refraction and (B) axial length between treated and contralateral control eyes during form-deprivation (FDM) and MPH treatment. Form-deprivation induced a significant myopic shift in refraction and axial elongation, which was inhibited by all three routes of MPH administration. Box plots represent the interquartile ranges of each group, whereas dots represent individual data points (each group consisted of a minimum of 5 animals). Statistics represent a significant difference (P < 0.05) between treated and contralateral control eyes (*) or to FDM only values (#). Placebo = water.

Table 2.

Raw Data and Pairwise Comparisons Analyzing the Effect of Methylphenidate Hydrochloride (MPH) on the Development of Experimental Myopia

	Refraction, Diopters			Axial Length, mm
Condition	Left Eye	Right Eye	Difference	Left Eye	Right Eye	Difference
FDM only	−3.12 ± 0.19*	2.94 ± 0.06	−6.06 ± 0.19*	9.41 ± 0.06*	8.73 ± 0.03	0.68 ± 0.04*
Ingestion
FDM + MPH	−1.34 ± 0.32*,†	3.03 ± 0.05	−4.38 ± 0.34†	8.91 ± 0.08*,†	8.61 ± 0.05	0.30 ± 0.06*,†
FDM + Placebo	−2.89 ± 0.43*	3.69 ± 0.22	−6.59 ± 0.40*	9.54 ± 0.09*	9.01 ± 0.07	0.53 ± 0.06*
Control	2.68 ± 0.19†	2.85 ± 0.19	−0.18 ± 0.02†	8.60 ± 0.03†	8.61 ± 0.03	−0.02 ± 0.01†
Control + MPH	2.94 ± 0.08†	3.02 ± 0.10	−0.08 ± 0.05†	8.65 ± 0.03†	8.65 ± 0.04	0.00 ± 0.03†
Topical eye drops
FDM + MPH	−1.06 ± 0.22*,†	2.87 ± 0.17	−3.93 ± 0.25*,†	9.15 ± 0.09*,†	8.78 ± 0.03	0.37 ± 0.06*,†
FDM + placebo	−3.51 ± 0.51*	2.71 ± 0.15	−6.23 ± 0.46*	9.44 ± 0.08*	8.83 ± 0.04	0.61 ± 0.06*
Intravitreal injections
FDM + MPH (lowest dose)	−1.33 ± 0.32*,†	2.90 ± 0.13	−4.23 ± 0.35*,†	8.85 ± 0.10†	8.73 ± 0.04	0.12 ± 0.09†
FDM + MPH (low dose)	−1.22 ± 0.52*,†	3.08 ± 0.10	−4.30 ± 0.56*,†	8.84 ± 0.12†	8.72 ± 0.07	0.12 ± 0.10†
FDM + MPH (“human equivalent” dose)	−0.43 ± 0.25*,†	3.00 ± 0.05	−3.43 ± 0.29*,†	8.92 ± 0.07†	8.82 ± 0.04	0.09 ± 0.03†
FDM + placebo	−3.64 ± 0.65*	2.82 ± 0.08	−6.46 ± 0.69*	9.22 ± 0.05*	8.59 ± 0.03	0.64 ± 0.02*

FDM, form-deprivation myopia; MPH, methylphenidate hydrochloride; Placebo = water.

Values presented are means ± standard error.

^*P < 0.05, significantly different from untreated contralateral control values.

^†P < 0.05, significantly different from FDM only.

MPH Can Prevent the Development of Experimental Myopia in Chickens

As contralateral control values (axial length and refraction) were no different between groups (refraction – ANOVA F(9, 69) = 0.808, P = 0.610; axial length – ANOVA F(9, 69) = 1.778, P = 0.098), or relative to age-matched untreated control values (refraction – ANOVA F(10, 72) = 0.723, P = 0.700; axial length – ANOVA F(10, 72) = 2.262, P = 0.058), MPH data are plotted as differences between the treated (left) eye and contralateral (right) eye (see Fig. 2).

When MPH was administered through ingestion for a 7-day period, it significantly inhibited both the myopic shift in refraction (approximately 30% protection; P < 0.05) and the excessive axial elongation of the eye (approximately 55% protection; P < 0.05) associated with diffuser-wear (see Fig. 2; Table 2). When fed to animals undergoing no optical treatment, MPH had no effect on refraction (P = 0.21) or axial length (P = 0.69) when compared with age-matched untreated controls (see Fig. 2; Table 2).

As with ingestion, topically administered MPH significantly inhibited FDM following 7 days of treatment (approximately 35% protection against refractive changes [P < 0.05], and approximately 45% protection against axial length changes [P < 0.05]); see Fig. 2; Table 2). Similarly, when administered through intravitreal injections, all three doses of MPH significantly suppressed the development of FDM (refraction - ANOVA F (3, 20) = 7.55, P < 0.05; axial length - ANOVA F (3, 20) = 11.20, P < 0.05; see Fig. 2; Table 2). MPH did not show a dose-dependent effect (refraction - ANOVA F (2, 17) = 1.23, P = 0.32; axial length - ANOVA F (2, 17) = 0.04, P = 0.96), instead, all three doses inhibited the development of FDM to a similar degree (refraction = 30.2%, 29%, and 43.4% across the 3 doses; axial length = 82.4%, 82.4%, and 86.8% across the 3 doses).

MPH is Distributed to all Layers of the Eye Following Treatment, Irrespective of the Route of Administration

To measure the ocular distribution of MPH following treatment, biological samples (blood, tears, cornea, aqueous humor, iris/lens/ciliary body, vitreous humor, retina, and choroid) were collected 30 minutes post-administration of 0.05 mg MPH (via ingestion, topical eye drops, or intravitreal injection). When measured via LC-MS/MS, MPH was detectable in all layers of the eye (Fig. 3; Table 3). As expected, the highest levels of MPH within ocular tissues were observed following intravitreal injections (peaking in the vitreous), followed by ingestion and topical eye drops (both peaking in the choroid). With regard to systemic distribution of MPH, the highest blood levels were observed following ingestion, followed by topical eye drops and intravitreal injections.

Figure 3.

The distribution of methylphenidate hydrochloride (MPH) in ocular tissue and blood. Liquid chromatography-tandem mass spectrometry was used to measure MPH levels 30 minutes following treatment with 0.05 mg of MPH, administered via (A) ingestion, (B) topical eye drops, or (C) intravitreal injection. Data are presented as pie charts showing the relative distribution of MPH between the different tissues measured.

Table 3.

The Distribution of Methylphenidate Hydrochloride (MPH) in Ocular Tissue and Blood

Tissue	Ingestion	Topical Eye Drops	Intravitreal Injections
Cornea	0.0006 ± 0.0001	0.0015 ± 0.0003	0.0082 ± 0.0021
Aqueous humor	0.0015 ± 0.0004	0.0093 ± 0.0069	0.1665 ± 0.0521
Iris/ lens/ ciliary body	0.0531 ± 0.0116	0.0126 ± 0.0011	0.9420 ± 0.3287
Vitreous humor	0.0144 ± 0.0030	0.0221 ± 0.0063	4.0598 ± 1.6288
Retina	0.0038 ± 0.0008	0.0151 ± 0.0044	0.1994 ± 0.0635
Choroid	0.0560 ± 0.0222	0.0274 ± 0.0089	0.1242 ± 0.0623
Blood	0.3322 ± 0.0573	0.0346 ± 0.0087	0.0170 ± 0.0064

Liquid chromatography-tandem mass spectrometry was used to measure MPH levels 30 minutes following treatment (0.05 mg MPH was administered for each mode of administration). Data are presented as the mean peak area ratio (MPH:internal standard) ± standard error.

To assess the pharmacokinetics of MPH in chicks, Figure 4 illustrates blood concentrations over a 6-hour period following intramuscular administration. Notably, peak levels were observed at 30 minutes (T_max) rather than at the first measured timepoint (15 minutes), suggesting a brief lag phase during which the drug transitioned from the injection site (upper thigh) into the systemic circulation. MPH remained detectable throughout the 6-hour monitoring period, although concentrations approached the limits of detection beyond 2 hours (see Table 4). Based on these data, the estimated half-life of MPH in the chick's systemic circulation is approximately 30 minutes.

Figure 4.

Pharmacokinetics of methylphenidate hydrochloride (MPH) in the systemic circulation of chicks. Liquid chromatography-tandem mass spectrometry was used to measure MPH levels 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 6 hours following intramuscular administration of 0.05 mg of MPH. MPH levels are expressed as the peak area ratio of MPH to the internal standard. Data are presented as mean ± standard error.

Table 4.

The Pharmacokinetics of Methylphenidate Hydrochloride (MPH)

Time, Hours	Peak Area Ratio
0.25	0.0166 ± 0.0105
0.50	0.0343 ± 0.0091
1	0.0083 ± 0.0049
2	0.0015 ± 0.0005
4	0.0006 ± 0.0003
6	0.0005 ± 0.0001

Liquid chromatography-tandem mass spectrometry was used to measure MPH levels in blood following 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 6 hours after intramuscular administration of MPH (0.05 mg). Data are presented as the mean peak area ratio (MPH:internal standard) ± standard error.

Dopaminergic and Noradrenergic Dynamics are Altered During MPH Treatment

Effects of MPH on DA and NE Levels

Consistent with previous reports,^11,71 dopaminergic output was reduced 2 hours post fitting of diffusers. Specifically, there was a significant decrease in retinal and vitreal levels of DA's primary metabolite (DOPAC) when compared with age-matched untreated eyes and contralateral controls (P < 0.05; Fig. 5; Supplementary Table S5, Supplementary Fig. S2), suggestive of a decrease in extracellular DA levels. This led to a significant reduction in the retinal DOPAC:DA ratio during form-deprivation (P < 0.05). MPH injections, either into form-deprived or otherwise untreated eyes, induced a significant increase in DA and DOPAC levels in both retinal and vitreal samples (P < 0.05).

Figure 5.

The effect of methylphenidate hydrochloride (MPH) on dopamine (DA) and norepinephrine (NE) levels. Liquid chromatography-tandem mass spectrometry was used to measure (A) vitreal 3,4-dihyroxyphenylacetic acid (DOPAC) levels (a proxy for extracellular retinal DA levels), (B) retinal DA levels, (C) vitreal NE levels, and (D) retinal NE levels. Analyte levels were measured in age-matched untreated control eyes (untreated), diffuser treated eyes (form-deprivation myopia [FDM]) and contralateral control eyes (Contra) of animals treated with methylphenidate hydrochloride. For analysis of vitreal DOPAC levels, MPH was administered through three different routes: ingestion, topical eye drops, and intravitreal injection (I.I.). For analysis of retinal DA levels and NE levels, MPH was administered via intravitreal injections. Box plots represent the interquartile ranges of each group, whereas dots represent individual data points (each group consisted of a minimum of 5 animals). Data are presented as the peak area ratio (peak area of the analyte:peak area of the internal standard). Dotted lines represent the average values observed in age-matched untreated control eyes, whereas dashed lines represent the average values observed in FDM only eyes. Statistics represent a significant difference (P < 0.05) to untreated control eyes (U lane: *), FDM only eyes (M lane: ^#), or contralateral control eyes (C lane: ^). Placebo = water.

For NE, form-deprivation did not alter vitreal (P = 0.678) or retinal levels (P = 0.722) of this catecholamine 2 hours post fitment of diffusers (see Fig. 5; Supplementary Table S5). However, like that seen for DA, MPH injections led to a significant increase in retinal NE levels 2 hours post-treatment in both form-deprived (P < 0.05) or otherwise untreated animals (P < 0.05).

To assess how different modes of MPH administration influence extracellular DA levels, a subset analysis measured vitreal DOPAC levels 2 hours post-treatment via ingestion, eye drops, and intravitreal injection (see Fig. 5; Supplementary Table S5). DOPAC levels were significantly elevated in both form-deprived and untreated eyes following all three delivery methods (P < 0.05 for each). However, the magnitude of effect followed a consistent rank order: intravitreal injection > eye drops > ingestion.

DA Dynamics During MPH Treatment

To study the effects of MPH on DA synthesis rates, the conversion of tyrosine-d₄ to dopamine-d₄ was measured using LC-MS/MS (Fig. 6Ai, Supplementary Table S6). When administered to chicks via ingestion, MPH treatment induced a significant increase in the rate of DA synthesis in retinal tissue 2 hours post-treatment (P < 0.05). Similarly, when spiked directly into the assay mix, MPH also induced a significant increase in the rate of DA synthesis (P < 0.05; see Supplementary Table S6).

Figure 6.

The effect of methylphenidate hydrochloride (MPH) on dopamine (DA) and norepinephrine (NE) dynamics. For (A) DA and (B) NE, the effects of MPH on synthesis, release, uptake and (in the case of DA) breakdown were measured. Analyte synthesis (i) was measured for both molecules using liquid chromatography-tandem mass spectrometry, determining the synthesis of (A) d₄-dopamine following the application of d₅-tyrosine, and (B) NE following the application of DA in retinal protein extracts. Chronoamperometry was used to measure (ii) the number of release events over a 10 second period and (iii) the uptake rate of each analyte in retinal biopsy punches mounted on screen-printed electrodes. Finally, (iv) the effect of MPH on the activity of one of the critical enzymes involved in DA breakdown, monoamine oxidase (MAO), was measured using a commercial fluorometric assay. MPH treatment was associated with a significant increase in the synthesis rate of each analyte and a significant decrease in the uptake rate of each analyte. Box plots represent the interquartile ranges of each group, whereas dots represent individual data points (each group consisted of 5 animals). Statistics represent a significant difference to control eyes (* P < 0.05).

The effect of MPH on retinal DA release and uptake rates was assessed using chronoamperometry. To validate the sensitivity and reliability of this technique in detecting changes in neurotransmitter dynamics, a series of positive control experiments were conducted. These included pharmacological and lighting manipulations known to elicit predictable responses in DA signaling (Supplementary Fig. S3, Supplementary Table S7), confirming that chronoamperometry effectively captured relevant changes in DA activity.

MPH ingestion did not alter DA release characteristics (see Fig. 6Aii, Supplementary Fig. S4, Supplementary Table S7). Specifically, there were no significant changes in the number of DA transient events (ANOVA, F(2, 28) = 2.015, P = 0.169), the average DA released per transient event (ANOVA, F(2, 28) = 0.441, P = 0.513), or the amount of DA released in response to potassium chloride stimulation (ANOVA, F(2, 28) = 0.654, P = 0.427). In contrast, MPH treatment was associated with a significant reduction in DA uptake rate (ANOVA, F(2, 36) = 12.772, P < 0.05; Fig. 6Aiii, Supplementary Fig. S5, see Supplementary Table S7).

To evaluate whether the changes in extracellular DA levels seen during MPH treatment are due to changes in DA breakdown activity, a fluorometric activity assay was undertaken for monoamine oxidase (MAO), one of the primary enzymes involved in DA breakdown (Fig. 6Aiv; see Supplementary Table S6). MPH treatment did not have any significant effects on DA catabolism, with no change in MAO activity observed (P = 0.952).

To evaluate the effects of MPH on D2-like receptor sensitivity, binding of the D2-like antagonist spiperone over a range of concentrations was quantified using LC-MS/MS (Fig. 7; Supplementary Table S8). Two-way ANOVA analysis found that MPH treatment, assessed 2 hours post-ingestion, was not associated with a significant change in D2-like receptor sensitivity (F(1, 49) = 1.7422, P = 0.201) across any concentration of spiperone (F(4, 46) = 3.358, P = 0.081). Instead, this analysis only observed a statistically significant effect on binding with regard to the concentration of spiperone applied (F(4, 46) = 131.891, P < 0.05).

Figure 7.

The effect of methylphenidate hydrochloride (MPH) treatment on dopaminergic and adrenergic receptor sensitivity. Liquid chromatography-tandem mass spectrometry was used to measure the binding of (A) the D2-like dopaminergic receptor antagonist spiperone and (B) the adrenergic receptor antagonist yohimbine over a range of concentrations in animals treated with methylphenidate hydrochloride (MPH). No significant effects of treatment were observed between MPH treated animals and untreated controls. Each group consisted of five animals.

NE Dynamics During MPH Treatment

Like that observed for DA, 2 hours post-ingestion of MPH, there was an increase in the rate of NE synthesis (studied by looking at the conversion of DA to NE using LC-MS/MS (Fig. 6Bi, see Supplementary Table S6, P < 0.05). A similar increase in NE synthesis was observed when MPH was spiked directly into the assay mix (P < 0.05; see Supplementary Table S6). Ingestion of MPH also significantly reduced the rate of NE uptake (P < 0.05; see Fig. 6B, Supplementary Table S7), but did not affect the release of NE. Specifically, MPH did not induce any significant changes in the number of NE release events (P = 0.817) or the amount of NE released per event (P = 0.795) over a 10-second recording period (see Fig. 6B, Supplementary Table S7).

The effects of MPH on adrenergic receptor sensitivity were evaluated by measuring the binding of yohimbine over a range of concentrations (see Fig. 7B, Supplementary Table S8). MPH treatment was not associated with a significant change in adrenergic receptor sensitivity at any of the concentrations of yohimbine tested. Rather, like that observed for DA receptor binding, a significant effect on binding was only associated with the different concentrations of yohimbine itself.

The Anti-Myopic Effects of MPH are Attenuated by Co-Treatment With Dopaminergic or Noradrenergic Receptor Antagonists

To investigate whether the changes in DA and NE dynamics are likely to underly the protective effects of MPH against FDM, animals fitted with diffusers were co-treated with MPH and (1) dopaminergic (spiperone and SCH-23390), (2) noradrenergic (yohimbine), or (3) a combination of dopaminergic and noradrenergic receptor antagonists (Fig. 8; Table 5; Supplementary Table S9). Intravitreal injection of a placebo solution (water) did not alter the anti-myopic effects of MPH (refraction – P = 0.393; axial length – P = 0.978). In contrast, the protective effects of MPH were significantly attenuated by co-treatment with a D1-like (SCH-23390)/D2-like (spiperone) receptor antagonist cocktail (refraction – P < 0.05; axial elongation, P < 0.05), or the noradrenergic receptor antagonist yohimbine (refraction – P < 0.05; axial elongation, P < 0.05) compared with animals given MPH alone. When the dopaminergic and noradrenergic antagonists were combined, a significant reduction in MPH's anti-myopic effects were observed in refractive measurements (P < 0.05), with animals no different to the FDM only group (P = 0.344). Although the axial length data would indicate a similar trend, due to the spread of observations, these chicks were no different in axial length to those values seen in both the FDM only (P = 0.094) and the MPH treated groups (P = 0.078). Worth noting, the combined administration of a DA and NE receptor antagonist was no more effective at blocking the actions of MPH than either a DA (refraction – P = 0.773; axial length – P = 0.952) or NE antagonist alone (refraction – P = 0.999; axial length – P = 0.748).

Figure 8.

The effect of dopaminergic and noradrenergic antagonists on the anti-myopic effects of methylphenidate hydrochloride (MPH). Differences in (A) refraction and (B) axial length between treated and contralateral control eyes during form-deprivation (FDM; Myopia) and systemic MPH treatment either alone or alongside intravitreal injections of dopaminergic antagonists (DA-Ant; spiperone and SCH-23390), noradrenergic antagonists (NE-Ant; yohimbine), or a combination of both (DA-Ant/NE-Ant). Box plots represent the interquartile ranges of each group, whereas dots represent individual data points (each group consisted of 5 animals). Statistics represent a significant difference (P < 0.05) to FDM only values (#) or to FDM + MPH values (^). Placebo = water.

Table 5.

Raw Data and Pairwise Comparisons Analyzing the Effect of Dopamine (DA) and Norepinephrine (NE) Binding in the Anti-Myopic Effect of Methylphenidate Hydrochloride (MPH) on the Development of Experimental Myopia

	Refraction, Diopters			Axial Length, mm
Condition	Left Eye	Right Eye	Difference	Left Eye	Right Eye	Difference
FDM	−1.88 ± 0.26*	2.24 ± 0.08	−4.12 ± 0.22‡	9.54 ± 0.09*	9.17 ± 0.09	0.37 ± 0.02‡
FDM + MPH	−0.46 ± 0.27*,†	2.28 ± 0.08	−2.74 ± 0.22†	9.30 ± 0.05*,†	9.11 ± 0.05	0.19 ± 0.02†
FDM + MPH + placebo	−0.94 ± 0.17*,†	2.10 ± 0.10	−3.04 ± 0.20†	9.37 ± 0.10*	9.18 ± 0.09	0.19 ± 0.02†
FDM + MPH + DA-Ant	−1.74 ± 0.32*,‡	2.58 ± 0.27	−4.32 ± 0.26‡	9.29 ± 0.01*,†	9.01 ± 0.03	0.28 ± 0.02†,‡
FDM + MPH + NE-Ant	−2.24 ± 0.34*,‡	2.18 ± 0.07	−4.42 ± 0.34‡	9.35 ± 0.02*	9.08 ± 0.03	0.27 ± 0.02†,‡
FDM + MPH + DA-Ant + NE-Ant	−2.18 ± 0.14*,‡	2.24 ± 0.09	−4.42 ± 0.15‡	9.24 ± 0.10	8.96 ± 0.08	0.29 ± 0.04

DA-Ant, dopaminergic antagonists (spiperone and SCH-23390); DA-Ant/NE-Ant, combination of spiperone, SCH-23390, and yohimbine; FDM, form-deprivation myopia; MPH, methylphenidate hydrochloride; NE-Ant, noradrenergic antagonist (yohimbine); Placebo = water.

Values presented are means ± SEM.

^*P < 0.05, significantly different from untreated contralateral control values.

^†P < 0.05, significantly different from FDM only.

^‡P < 0.05, significantly different from FDM + MPH.

MPH Does Not Affect Normal Chicken Behavior

MPH administration (whether by ingestion, topical eye drops, or intravitreal injection) did not significantly affect animal behavior, food or water intake, fecal output, or final body weight in either form-deprived or untreated control animals (Wilks’ Lambda = 0.512, F(1, 80) = 1.909, P = 0.202; Supplementary Table S10). Importantly, no stressed or abnormal animal behaviors were observed over the 7-day treatment period.

Pharmacological Administration of NE Inhibits the Development of FDM

Following the finding that antagonism of NE receptors can block the anti-myopic effects of MPH, we investigated further the role of NE in myopia. Although total NE levels in the retina were unchanged during myopia development (as outlined earlier; see Fig. 5), daily intravitreal administration of NE itself, significantly inhibited the development of FDM over a 3-day period of treatment (refraction – 61% protection, P < 0.05; axial length – 89% protection, P < 0.05; Fig. 9; Supplementary Tables S11, S12). These results indicate that stimulation of the NE system yields outcomes comparable to those well-documented for activation of the DA system.^5–32

Figure 9.

The effect of norepinephrine (NE) administration on the development of experimental myopia. Differences in (A) refraction and (B) axial length between treated and contralateral control eyes during form-deprivation (FDM; Myopia) and NE treatment. After 3 days of treatment, form-deprivation induced a significant myopic shift in refraction and axial elongation, which was inhibited by a daily 10 µL intravitreal injection of 1 mM NE. Box plots represent the interquartile ranges of each group, whereas dots represent individual data points (n = 7 per group). Statistics represent a significant difference (P < 0.05) between treated and contralateral control eyes (*) or to FDM only values (#).

Discussion

Prompted by recent clinical observations,³⁸ this study aimed to clarify the extent to which MPH can inhibit myopic growth and to explore the underlying mechanisms. Consistent with human data, we found that daily administration of 0.01 to 1 mg/kg MPH over a 7-day period significantly suppressed experimental myopia in chicks, regardless of the delivery method.

In line with previously reported central nervous system effects,^73,74 MPH enhanced retinal synthesis of DA and NE, while concurrently reducing their uptake. This led to elevated extracellular levels of both neurotransmitters, increasing their bioavailability at target receptors. Importantly, blocking either DA or NE receptor activity significantly attenuated the anti-myopic effects of MPH.

Our data support clinical observations that MPH can inhibit myopic progression and, in doing so, support the hypothesis that DA, as in animal models, plays a critical role in human refractive development and myopia. Additionally, the data reveal a novel and potentially important role for NE in regulating ocular growth. However, these results should not be interpreted as an endorsement for the clinical use of MPH in the treatment of myopia.

Although the mechanistic effects of MPH were seen in both myopic and control eyes, ocular growth was only affected in those eyes experiencing visual manipulation (diffuser-wear). With the exception of insulin,^75,76 this is a common finding within the field, with a large array of compounds found to alter the rate of myopia development, yet have no effect on normal ocular growth.^{7,11,18,20,23,24,77–79} As previously noted,^30,72 this suggests that the pathways involved in “normal” and “abnormal” (experimental myopia) eye growth are fundamentally different, particularly in their sensitivity to manipulations of DA and NE pathway activity. Such a fundamental change in the sensitivity of the retina to growth modulatory cues also appears to occur within the human condition (for a review, see Ref. 80). For example, compared with emmetropes, myopes have a weaker/altered response to positive defocus and chromatic cues,^81–86 reduced ON-pathway responses (which become larger than values seen in emmetropes in response to inverted text contrast),^87–89 deficits in flicker^90–97 and contrast^98–100 sensitivity, and greater accommodation errors.^101,102

MPH Treatment Inhibits the Development of Experimental Myopia

Experimental myopia was inhibited by MPH treatment regardless of the administrative route. The highest level of protection was observed in response to intravitreal injection (approximately 85% against axial elongation), followed by systemic administration (approximately 55% against axial elongation) and topical application (45% against axial elongation). The degree of protection observed was, for the most part, correlated with the amount of MPH that penetrated the retina.

The current findings support the anti-myopic effects of MPH observed in a pilot study conducted by Gurlevik and colleagues in a patient population,³⁸ as well as those reported in a recent retrospective population-based cohort analysis from Taiwan.¹⁰³ However, further work is still needed, with several studies having previously reported mixed results with regard to the effects of MPH on refractive development.^104–107 These studies, however, were (for the most part) not directly assessing the effects of MPH on myopia, and, in some cases, the lack of a control/placebo group makes it difficult to assess changes in refractive development.

It should be noted that the effect size of MPH treatment observed presently was weaker than that seen by Gurlevik and colleagues³⁸ who found that MPH treatment abolished axial elongation and refractive progression over their 12-month trial period in children/teenagers aged 8 to 18 years. However, effect sizes are difficult to compare due to inherent differences between experimentally induced myopia in animal models, and the development of myopia in children. Also worth noting is that the anti-myopic properties of MPH are smaller than those observed for other pharmacological agents that directly target the DA or NE systems (i.e. administration of dopaminergic agonists, the DA precursor levodopa, DA itself [for a review, see Ref. 4], or NE itself; Fig. 9).

MPH Inhibits Myopic Growth by Increasing the Bioavailability of DA and NE

MPH increased the rate of DA and NE retinal synthesis. This occurred irrespective of whether animals were pre-treated with MPH, or if MPH was directly added to the synthesis assay mix. This would indicate that the increase in synthesis rates observed was due to the direct presence and/or binding of MPH to tyrosine hydroxylase (DA synthesis), dopa-decarboxylase (DA synthesis), and DA β-hydroxylase (NE synthesis), rather than altering the system at an upstream point. Whereas not heavily investigated, MPH has previously been observed to alter DA synthesis dynamics through direct interaction with tyrosine hydroxylase in vitro.¹⁰⁸ Although MPH enhanced synthesis rates, it did not affect the release dynamics of either DA or NE. Thus, the increase in extracellular levels of DA and NE observed presently appears to be driven primarily by the effects of MPH on uptake (discussed next).

As is well established in the brain,^73,109–111 the primary outcome of MPH administration was a reduction in the cellular uptake for both DA and NE. MPH achieves this by binding to the active sites of the dopamine transporter (DAT) and norepinephrine transporter (NET).^112–114 This binding prevents the conformational changes required for these transporters to reabsorb DA and NE into the presynaptic neuron.^112–115

The resulting reduction in uptake led to a significant increase in the extracellular levels of both neurotransmitters, and with it an increase in their bioavailability at target receptors. This increased bioavailability is presumed to be critical for MPH's anti-myopic effects, as pharmacological blockade of either DA or NE receptors abolished its efficacy. Importantly, MPH does not directly alter receptor structure,¹¹⁶ activity (current results), or expression levels¹¹⁷; rather, it enhances the availability of the endogenous ligands (see Fig. 10).

Figure 10.

Diagrammatic representation of methylphenidate hydrochloride (MPH) induced changes in dopamine (DA) and norepinephrine (NE) activity. MPH administration induces a similar set of changes in (A) DA, and (B) NE activity in the retina. Specifically, MPH was able to: (1) enhance the rate of synthesis of both DA and NE through modulating the activity of their respective rate limiting enzymes (tyrosine hydroxylase [TH] and DA β-hydroxylase [DBH]); (2) inhibit the uptake of DA (through DAT) and NE (through NET), increasing the extracellular levels of both biomolecules; and (3) increase the bioavailability of DA and NE at their respective target receptors. The anti-myopic effects of MPH were lost by coadministration of pharmacological agents that inhibit the activity of DA and NE receptors. This would suggest that the increased bioavailability of DA and NE at their target receptors drives the anti-myopic effects of MPH. MAO (monoamine oxidase), DOPAC (3,4-dihydroxyphenylacetic acid), COMT (catechol-O-methyltransferase), NMN (normetanephrine).

Unexpectedly, the anti-myopic effects of MPH appear to be mediated through two concurrent catecholaminergic pathways (DA and NE), or specifically the activation of their target receptors. Whereas the role of DA in experimental myopia is well established, the involvement of NE is less explored. Although some evidence suggests that α-adrenergic agonists can inhibit experimental myopia, only a limited number of studies have investigated NE's role in refractive development.^118,119 The current findings indicate that total retinal NE levels remain unchanged during myopic growth. However, exogenous administration of NE was found to inhibit the development of experimental myopia.

Pharmacokinetics of MPH in Chickens and its Translation to Humans

MPH has previously been shown to be pharmacologically active in birds (pigeon and Japanese quail^120,121). Building on this, the current findings demonstrate that MPH is also functionally active in chickens and is capable of modulating ocular growth through changes in extracellular levels of DA and NE. If the chick model accurately reflects human physiology, these results support the hypothesis that MPH can influence refractive development. Moreover, they offer a mechanistic explanation for the anti-myopic effects observed in clinical studies.^38,103

However, the pharmacokinetics of MPH (including absorption, distribution, metabolism, receptor subtype interactions, binding affinity, and elimination) can differ between chickens and humans, an important consideration when interpreting findings in a translational context. Interspecies similarities in drug pharmacokinetics are often compound-specific, with differences largely attributed to variations in hepatic enzyme activity (particularly cytochrome P450 isoenzymes), as well as gastrointestinal anatomy and physiology.¹²² These interspecies differences may affect drug efficacy, duration of action, and therapeutic outcomes. Given that chickens have a relatively larger liver-to-body mass and liver-to-blood volume ratio compared with humans,^123,124 it is plausible that this contributes to more rapid hepatic metabolism and clearance of MPH.

To our knowledge, no studies have directly compared the pharmacokinetics of MPH in chickens to those in humans. Our data show that following intramuscular administration in chicks, blood levels of MPH peak over the first hour and remain detectable for up to 6 hours, although concentrations approach the limits of detection beyond the initial peak. This corresponds to an estimated half-life of approximately 30 minutes in chicks—a markedly shorter duration than the 2 to 4 hour half-life commonly reported in humans for the same MPH formulation.^{109,125–131} Consequently, the therapeutic window in chicks is significantly narrower than that observed in humans. This makes the anti-myopic effects of MPH observed in our study even more noteworthy, as they were achieved despite the shorter systemic exposure in chicks compared to that which would be observed in a clinical population.

MPH did not Significantly Affect Behavior

No significant changes in behavior were observed in MPH treated chicks over the experimental period. This would indicate that the anti-myopic properties of MPH are driven by its localized binding within the eye rather than being a secondary outcome to changes in behavior. This concurs with previous reports regarding the effects of MPH in birds, in which behavioral changes are rarely observed at the doses used in this study.^120,121,132 This lack of effect on behavior does differ from several reports in rodents examining similar doses of MPH,^133–137 although these behavioral changes are not consistently observed.^135,138,139 The effects of MPH on animal behavior have also been reported to be affected by a wide range of additional variables, such as age, pharmacokinetics and metabolism, sex, interval and duration of treatment, genetic background, and behavioral testing methods.^{137,138,140,141} In the context of this study, smaller MPH-induced changes may also have gone unnoticed, as the current study focused on large behavioral changes.

Potential Interactions Between Mental and Behavioral Disorders and Myopia

The rising diagnosis and prevalence of mental and behavioral disorders, including ADHD, increases the likelihood of significant overlap with children also diagnosed with myopia. There are a growing number of studies examining the overlap in prevalence between these conditions, with correlations reported between myopia and ADHD^103,142 (although this is not always observed^{106,143–145}). This potential correlation raises two important questions: (1) does ADHD predispose children to myopia (this may be due to genetic, neurochemical, or behavior changes)?; and (2) does MPH treatment, which modulates a pathway suggested to be critical for myopia (that of DA), alter the onset and/or progression of this visual disorder? A recent analysis of retrospective population-based cohort data in Taiwan found that children diagnosed with ADHD, but who did not receive treatment, had a higher risk of myopia (1.22-fold) compared to children without ADHD.¹⁰³ In contrast, and in agreement with previous studies,³⁸ children treated for ADHD had the lowest risk (0.61-fold) of myopia.¹⁰³ When taken together with the current findings, this emphasizes a potential role of the DA and NE system in human myopia. This also raises a further question: do other compounds used to treat mental and behavioral disorders, which target the dopaminergic or other neurotransmitter systems implicated in myopia, influence ocular growth?¹⁴⁶ We do not currently have an answer to this question.

Conclusions

This study demonstrates that MPH, administered via ingestion, topical eye drops, or intravitreal injection, can inhibit the development of experimental myopia. The anti-myopic effects of MPH were associated with increased extracellular accumulation of DA and NE in the retina, enhancing their bioavailability at respective receptor families. This increased bioavailability appears to be critical for MPH's efficacy, as pharmacological blockade of either DA or NE receptors abolished its anti-myopic effects.

Taken together, the current findings support the clinical observation that MPH can inhibit myopic progression and support a role for DA in human refractive development. Additionally, the data suggest a novel and potentially important role for NE in the regulation of ocular growth.