
==== Front
Invest Ophthalmol Vis Sci
Invest Ophthalmol Vis Sci
IOVS
Investigative Ophthalmology & Visual Science
0146-0404
1552-5783
The Association for Research in Vision and Ophthalmology

39292451
10.1167/iovs.65.11.29
IOVS-23-39362
Anatomy and Pathology/Oncology
Anatomy and Pathology/Oncology
Administration of Nicotine Can Inhibit Myopic Growth in Animal Models
Nicotinic Effects on Myopic Growth
Thomson Kate 1
Karouta Cindy 1
Ashby Regan 1 2
1 Centre for Research in Therapeutic Solutions, Faculty of Science and Technology, University of Canberra, Bruce, Canberra, Australia
2 Research School of Biology, Australian National University, Acton, Australia
# Correspondence: Kate Thomson, Faculty of Science and Technology, University of Canberra, University Drive, Bruce, Canberra 2617, Australia; kate.thomson@canberra.edu.au.
* KT and CK contributed equally to this work and should be considered joint first authors.

18 9 2024
9 2024
65 11 2925 8 2024
23 12 2023
Copyright 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Purpose

While previously investigating the mechanism by which atropine inhibits ocular growth, we observed that stimulation of nicotinic receptors can inhibit experimental myopia. This study expands on that preliminary finding and investigates the safety and efficacy of nicotinic stimulation in the inhibition of ocular growth.

Methods

Nicotine's ability to inhibit form-deprivation myopia (FDM), following intravitreal injection (9 chicks per group) or topical application (6 chicks per group), was investigated over three doses. The ability of nicotine to inhibit lens-induced myopia (LIM) was also tested (in 12 chicks). For ocular safety, following 4 weeks of topical treatment with nicotine (n = 10), pupillary reflex, intraocular pressure, corneal curvature/thickness, lens thickness, retinal health (retinal thickness/cell apoptosis), as well as retinal function (electroretinogram recordings) were assessed. We also examined the effects of nicotine on non-ocular autonomic functions in both chicks (n = 5) and mice (n = 5).

Results

Nicotine was observed to significantly inhibit the development of FDM in chicks when administered as an intravitreal injection (P < 0.05) or topical eye drops (P < 0.05), albeit not in a dose-dependent manner. Nicotine also inhibited LIM (P < 0.05) to a similar degree to that seen for FDM. Although ocular health was (for the most part) unaffected by nicotine, the highest topical dose induced a temporary reduction in cardiorespiratory output (P < 0.05).

Conclusions

Nicotine, administered as an intravitreal injection or topical eye drop, significantly inhibits the development of experimental myopia. Although the anti-myopic effects observed presently are interesting, the well-reported side effects (expanded on presently) and addictive properties of nicotine would preclude its clinical use.

myopia
nicotine
acetylcholine
animal models
atropine
==== Body
pmcMyopia (short-sightedness), the leading cause of visual impairment worldwide,1 arises most commonly from the excessive elongation of the eye during development. Over the past 50 years, the prevalence of myopia has increased dramatically, most notably in educationally developed areas of East and Southeast Asia, with estimates predicting that half the world's population may be myopic by 2050.2 Due to its associated sight-threatening pathologies,3 the odds of which increase with the severity of myopia,4 understanding the biochemical processes underlying this condition are critical to facilitate the continual development and optimization of treatments for myopia.

Recently, we reported that modulation of the nicotinic-cholinergic system could inhibit the development of myopia in animal models.5 Specifically, while investigating the role of nicotinic receptors in the pharmacological effects of atropine (the leading drug treatment for human myopia), we observed that administration of nicotine slowed the development of form-deprivation myopia (FDM) in chicks. This finding may help explain the reported association between parental smoking,6–8 or exposure to passive smoke,9 and a lower risk of myopia in humans. However, modulation of nicotinic receptor activity appears to be a complex area, with both stimulation5 and antagonism10 of this receptor class having been associated with anti-myopic effects.

Nicotinic receptors, named for their selective activation by nicotine, are a family of ionotropic receptors permeable to sodium, potassium, and, in some cases, calcium.11,12 Nicotinic receptors are constructed, in the case of neuronal tissues, from a selection of alpha and beta subunits.11,12 These are commonly assembled in α3β4, α4β2, and α7 confirmations.11,12 In the mammalian and avian retina, nicotinic receptors have been localized on ganglion cells, GABAergic and glycinergic amacrine cells, and ON bipolar cells.11,12 In avian and amphibian retinas, nicotinic receptors have also been observed in the outer plexiform layer.11,12 One notable difference between mammalian and avian species is the cholinergic receptor distribution in the ciliary muscle.13 In mammals, the ciliary muscle is innervated by muscarinic cholinergic receptors.13 In contrast, birds possess a striated ciliary muscle, innervated by nicotinic cholinergic receptors.13 With respect to myopia and ocular growth regulation, there are also nicotinic receptors located in the choroid.14 These have primarily been studied with respect to choroidal neovascularization,14 however, given the choroidal responses observed during periods of altered ocular growth (for review see Ref. 15), one may postulate that nicotinic signaling in the choroid may also contribute to nicotinic modulation of growth. The potential expression of nicotinic receptors in the sclera, to the best of our knowledge, has not been assessed.

In this study, we wished to expand on our previous findings, and those of Stone and colleagues,10 and investigate in greater depth the safety and efficacy of nicotinic stimulation in the treatment of experimental myopia to better understand its growth modulatory effects. In this study, efficacy was assessed in chicks by constructing a dose-response curve for nicotine (across 3 log units) with regard to the protection afforded against the development of FDM (as measured by axial length, refraction, and choroidal thickness). Dose-response curves were developed for both intravitreal injection and topical application of nicotine. We also tested the ability of nicotine to inhibit the other major form of experimental myopia (lens-induced myopia [LIM]) at a single intravitreal dose. With regard to safety, when nicotine is administered systemically, a number of significant systemic side effects are observed. These include a well-characterized increase in cardiorespiratory output.16,17 Given the potency of these side effects, we wished to investigate whether nicotine induces any ocular side effects through administration directly to the eye. To this end, we studied the effect of nicotine (after 4 weeks of topical treatment) on the pupillary reflex, intraocular pressure, retinal function (electroretinogram recordings), and retinal health (retinal thickness and tissue apoptosis), as well as investigating the ocular and systemic distribution of this drug. During the above experiments, a transient but significant decrease in cardiorespiratory output was observed following ocular application of nicotine. This response was unexpected based on the previous literature regarding nicotine's systemic effects. Therefore, we also assessed the effects of nicotine on non-ocular autonomic functions in chicks and, additionally, in mice.

Methods

Animals and Housing

One-day-old male White Leghorn chicks (Gallus gallus) were obtained from Barter and Sons Hatchery (Horsley Park, New South Wales, Australia) and housed in temperature-controlled rooms under fluorescent white (cool white, correlated color temperature 4000 K) laboratory lighting (approximately 500 lux) on a 12:12 hour light:dark cycle, with lights on at 9 AM and off at 9 PM. Chicks 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).

For assessment of pupillary reflex and cardiorespiratory function in the presence of nicotine, C56BL/6J mice (Mus musculus, obtained from the University of Canberra's breeding colony) were kept in individually ventilated cages, with experiments commencing at 8 weeks of age. Mice were housed at 23°C on a 12:12 hour light:dark cycle (8 AM:8 PM; 500 lux fluorescent lights [cool white, correlated color temperature 4000 K]) and had access to unlimited amounts of food and water.

Authorization to conduct experiments using animals was given by the University of Canberra Animal Ethics Committee under the ACT Animal Welfare Act 1992 (Project Numbers: Chicken CEAE 20-98, Mouse CEAE 80-89) and conformed to the ARVO Resolution for the Use of Animals in Ophthalmic and Vision Research.

Experiment 1: Effectiveness of Nicotine Against the Development of Experimental Myopia (Intravitreal Injections)

To expand on our previous work,5 and to investigate if nicotine inhibits the development of experimental myopia in a dose-dependent manner, three different doses of nicotine were administered as a daily intravitreal injection to chicks developing FDM (Table 1). As no dose-response was observed during FDM treatment, and to examine whether nicotine's effects were conserved during LIM, nicotine was also administered at a single dose to chicks wearing negative lenses (see Table 1).

Table 1. Drug Administration, Dosage, and Number of Chicks Allocated Across Experiments 1 and 2

Treatment	Dose (nmol)	Dose (µg)	Concentration (mM)	Number of Animals	
Experiment 1 - dose-response curve (intravitreal injections; 6 days of treatment)	
 Age-matched untreated controls	—	—	—	12	
 FDM only	—	—	—	12	
 FDM/placebo (PBS)	—	—	—	12	
 LIM only	—	—	—	12	
 FDM/nicotine	1.50	0.24	0.15	9	
 FDM/nicotine	15.00	2.43	1.50	11	
 FDM/nicotine	150.00	24.33	15.00	12	
 LIM/nicotine	150.00	24.33	15.00	12	
 Nicotine (no optical treatment)	150.00	24.33	15.00	12	
Experiment 2 - dose-response curve (topical eye drops; 3 days of treatment)	
 FDM only	—	—	—	12	
 FDM/nicotine	1.20	0.20	0.015	6	
 FDM/nicotine	12.00	1.95	0.15	12	
 FDM/nicotine	120.00	19.46	1.50	6	
 FDM/nicotine	1200.00	194.64	15.00	6	
FDM, form-deprivation myopia; LIM, lens-induced myopia.

Volumes administered: intravitreal injection = 10 µL; and topical eye drops = 80 µL (two 40 µL drops).

Myopia Induction, Drug Treatment, and Measurement of Ocular Parameters

Experimental myopia was induced monocularly by fitting a translucent diffuser (FDM) or –10 diopter (D) negative lens (LIM) over the left eyes of the chicks, with the right eyes left untreated to serve as an internal contralateral control. The procedures for fitting diffusers and negative lenses were undertaken as previously described.18

For all preparations, nicotine (N3876; Sigma-Aldrich) was freshly prepared (pH 7.5) in 1× phosphate buffered saline (PBS) with the tested doses (see Table 1) chosen based on ED50 data from cell culture19 and nicotine’s previously observed effectiveness in our laboratory.5 For 6 consecutive days, the chicks were given a daily 10 µL intravitreal injection of nicotine (or placebo) to the left eyes at lights on (9 AM) using a 30-gauge needle (Terumo) fitted to a Hamilton syringe (100 µL capacity) under light anesthesia (5% in 1L 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).

To assess the development of myopia, refraction and ultrasound measures were taken from the treated (left) and contralateral control (right) eyes on the day prior to the commencement of treatment (day 0, baseline) and on the day following treatment cessation (day 7 of diffuser- or negative lens-wear). In short, refraction and ocular biometry were measured using automated infrared photoretinoscopy (the system was provided courtesy of Professor Frank Schaeffel, University of Tuebingen, Germany) and A-scan ultrasonography (Biometer AL-100; Tomey Corporation, Nagoya, Japan). Biometry measures were performed under light isoflurane anesthesia, as described above. The thickness of the anterior chamber, the lens and vitreal chamber, as well as overall axial length (cornea to retina), was measured by A-scan ultrasonography.20 Retinal and choroidal thickness were measured by optical coherence tomography (OCT; Spectralis, Heidelberg Engineering, Heidelberg, Germany) for the 1.5 mM intravitreal injection treatment group and the 1.5 mM, 0.15 mM, and 0.015 mM topical eye drop treatment groups. For OCT measures, the instrument was aligned perpendicular to the eye and focused through the pupil, the pecten oculi was aligned in the bottom right quadrant of the fundus image and seven parallel line scans were performed, with the top of the pecten level to the bottom line of the scan. These seven-line scans were taken to ensure the area centralis, which is located superior and nasal to the pectin,21,22 was captured. Retinal and choroidal thickness measures were averaged across the seven- line scans (resolution mode = high resolution, scan angle = 20 degrees, scan type = B-scan, 1024 × 496 pixels, line scan, eye tracking not engaged, scan rate of live image = 85 kHz, and measurement wavelength = 820 nm). For all OCT measures, the chicks were anaesthetized (described above) and their eyes opened using calipers, with moisturizing drops continuously added to ensure no drying of the corneal surface. To ensure that the OCT was aligned perpendicular to the eye of the animal, once anesthetized, the head of the animal was placed in a custom mount that ensured a consistent angle for measurement.

Experiment 2: Feasibility of Topical Administration for Nicotine Treatment

Given the complications associated with frequent intravitreal injections (for review see Ref. 23), a preferable avenue for longer-term pharmacological experiments using nicotine would be through administration as topical eye drops. However, there are several significant biological impediments that preclude many drugs from penetrating the eye to be therapeutically effective when applied as an eye drop.24–27 Therefore, nicotine was administered to chicks as topical eye drops to, first, establish how well nicotine penetrates the eye and, second, to examine how topical nicotine affects the development of FDM.

Topical doses were chosen to be of equivalency to the amount striking the retina following intravitreal injections. This was based on the prediction that a maximum of 4% of the topical solution may penetrate to the retina. Thus, for the lower topical dose (12 nmoles), it is assumed that 0.48 nmoles may reach the retina. This is in the equivalent range to the expected dose that would reach the retina following intravitreal injection at the lowest concentration, assuming a maximum retinal penetration of 80% (1.2 nmoles). Predictions of retinal penetration were based on the literature,24–27 and on previous penetration seen in our laboratory for other small polar molecules.5,28–31

Drug Distribution

To assess how topical nicotine is distributed throughout the eye, the middle concentration investigated above (1.5 mM) was applied to otherwise untreated eyes as 80 µL eye drops (2 × 40 µL drops, 120 nmoles). This dose was chosen as it represents the highest effective concentration before which changes in autonomic functions are observed (see below for further details on autonomic changes). Drops were applied to both eyes at the above concentration.

Blood and ocular samples (tears, cornea, aqueous humour, iris/lens/ciliary body, vitreous humour, retina, choroid/retinal pigment epithelium [RPE], and sclera) were collected 30 minutes post-application of the eye drops and stored at –80°C until use. For each timepoint, samples were collected from both nicotine treated animals and from age-matched untreated controls (4 chicks per timepoint, per condition). Samples were analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a previously developed method32 adapted to measure nicotine levels (see appendix for full methods). Nicotine levels in all collected samples were assessed and compared between nicotine treated and untreated age-matched control animals.

Effectiveness of Nicotine Eye Drops Against the Development of FDM

To investigate if topical nicotine affects the development of FDM, nicotine (2 × 40 µL eye drops) was administered daily to the left eye, at one of four doses (see Table 1), at lights on (9 AM) for 3 consecutive days (see the allocation of chicks that is outlined in Table 1).

During initial visual observations, it was noted that the highest dose of nicotine tested (15 mM) induced a temporary and short-lived autonomic response (described further below). While chicks recovered from this within 5 minutes, the nicotine treatment period was reduced from 6 days (in the intravitreal injection experiment) to 3 days (the shortest timeframe to observe a robust growth response in our laboratory) to avoid potential adverse effects associated with longer term treatment.

For all eye drop experiments, diffusers were fitted and FDM development was measured (minus OCT analysis) as described above for intravitreal injections. Due to the reduction in the experimental timeline, ocular parameters were measured on day 4 of diffuser-wear instead of day 7.

Experiment 3: The Effects of Nicotine on the Autonomic Response

As noted, during experiment 2, the initial visual observations detected a temporary and short-lived alteration in animal alertness and respiratory rates after administration with the highest dose of topical nicotine (15 mM [1200 nmoles]). These effects, as well as their relation to previously reported effects of systemic nicotine treatment, are discussed in more detail later. However, it should be noted that the direction of this autonomic response (toward a reduction in cardiorespiratory output) was opposite to that previously observed in systemic nicotine studies. Therefore, we investigated more closely the effects of topically applied nicotine on autonomic functions. For this analysis, body temperature, heart rate, and breathing rate of the chicks were measured for 5 minutes following topical treatment with nicotine (Table 2). Temperature measurements were recorded using a feedback system linked to a T-type thermocouple rectal probe (AD Instruments, Dunedin, New Zealand; built courtesy of RSPE, ANU), whereas breathing rate and heart rate were recorded using a 26T PowerLab with LabChart software (AD Instruments, Dunedin, New Zealand). Positive and negative needle electrodes were placed in the right and left thighs of each animal, respectively, while a reference electrode was placed subcutaneously in their back. To investigate if nicotine-induced changes in autonomic functions were also seen in mammals, or whether this phenomenon was isolated to birds, the mice were assessed in the same manner as that stated for the chicks. For all autonomic tests, both chicks (n = 5) and mice (n = 5) were anaesthetized with isoflurane (as outlined above) and measures compared to baseline and placebo values.

Table 2. Drug Administration and Dosage to Investigate the Effect of Nicotine on the Autonomic Response in Chicks and Mice

Treatment	Dose (nmol)	
Group 1 - The effect of nicotine (topical eye drops, day 1)	
 Baseline	—	
 Placebo (PBS)	—	
 Nicotine	120	
 Nicotine	1200	
Group 2 – Co-administration of nicotine with a nicotinic antagonist (topical eye drops, day 2)	
 Baseline	—	
 Vecuronium bromide (nicotinic antagonist)	126	
 Nicotine/vecuronium bromide (nicotinic antagonist)	1200/126	
Group 3 - Co-administration of nicotine with a serotonergic antagonist (topical eye drops, day 3)	
 Baseline	—	
 Mianserin (serotonergic antagonist)	40	
 Nicotine/mianserin (serotonergic antagonist)	1200/40	

Tests of autonomic function were broken down into three groups: group 1 = the effect of nicotine, group 2 = co-administration of nicotine with a nicotinic antagonist, and group 3 = co-administration of nicotine with a serotonergic antagonist (see Table 2). Each group of tests was performed on a separate day to allow the animals to recover from drug treatment as the same set of five animals was used for each experimental grouping. The first group of tests studied the effects of two doses of nicotine eye drops on autonomic functions. The highest of these two doses (1200 nmoles) had been observed to induce a visible change in behavior/respiration during experiment 2. In contrast, the lower dose tested (120 nmoles) did not appear to induce any visible changes in the animals.

The remaining two groups of autonomic tests examined whether the effects observed during treatment with the higher of the two doses of nicotine (1200 nmoles) could be blocked by co-administration with an opposing nicotinic receptor antagonist (vecuronium bromide, which has been reported not to affect compensation to negative lenses33) or, as nicotine may cross react with the serotonergic 5HT3 receptor,34 a serotonergic receptor antagonist (mianserin).

Experiment 4: The Effects of Nicotine on Ocular Safety

To assess the safety of nicotine on ocular tissue, topical drops were given to the chicks for a period of 4 weeks at the highest effective dose (120 nmoles), at which the cardiorespiratory system was unaffected (as assessed in the experiment 3). Drops were given daily at lights on (9 AM) for 4 weeks (n = 10 per group). Measures of ocular safety were compared to age-matched untreated control chicks (n = 10 per group).

Following 4 weeks of treatment, corneal thickness (DGH Pachmate 2 pachymeter; DGH Technology Inc., USA), intraocular pressure (Icare TA01i tonometer, Icare, Finland), and corneal curvature were measured as previously described29 to determine any effects on the refractive surface. The effect of nicotine on pupil size was also assessed in both the chicks (n = 5) and the mice (n = 5). Mice were included in this analysis, as a negative control, as the iris in mice is innervated by muscarinic, not nicotinic-cholinergic receptors. The pupillary response was first measured after dark-adaption (1 hour, <1 lux). Following this, and while still dark adapted, the pupillary response to a light flash (10 seconds, 100 lux) was assessed. For the chicks, nicotine was compared with animals that received no drug treatment. In mice, nicotine was compared to pilocarpine (510 nmoles, muscarinic-cholinergic agonist, induces pupil constriction), atropine (150 nmoles, muscarinic-cholinergic antagonist, induces pupil dilation), as well as animals that received no drug treatment. Retinal function was assessed by electroretinogram (ERG), after which the chicks were euthanized and retinal samples collected and tested for signs of cell damage or death (apoptosis) using TUNEL analysis. Both ERG and TUNEL analysis were performed using previously outlined protocols.29

Data and Statistical Analysis

A power calculation was undertaken to determine the group sizes required to achieve 80% power in observing a minimum of a 1 D treatment effect (relative to control values) in refraction when the standard deviation (SD) is approximately 0.5 D: n1=σ12+σ22/Kz1-α/2+z1-β2Δ2n1=0.52+0.52/11.96+0.84212n1=4

For experiment 1, to account for potential removal of diffusers (at which point, the chicks were removed from the experiment and were not reported) over a longer treatment period (7 days), group sizes were increased to 12 chicks. For experiment 2 (topical eye drops), group sizes were increased to a minimum of 6 chicks. A total of four chicks (all in experiment 1) were not included in the analyses due to diffuser loss (see Table 1 for final group numbers).

Refraction and ocular biometry measurements are presented as means ± standard error of the means, with the data analyses representing the difference between the treated and the contralateral control eye following treatment. Figures represent the percent protection elicited by a treatment against the development of FDM or LIM, calculated using the differences between treated and contralateral control eyes. Percent protection was calculated as follows: %Protection=100-DifferencetocontralateralcontrolsindrugtreatedgroupDifferencetocontralateralcontrolsinFDMorLIMonlygroup×100

LC-MS/MS measurements are presented as the average peak area ratios (PARs) of analyte to internal standard (peak area of nicotine:peak area of deuterated serotonin) with pairwise comparisons listed in the tables within the results.

Measures of ERG recordings, pupil dilation, and autonomic function are presented as the means ± standard error of the means of each variable (e.g. respiratory rate is presented in beats/minute). All figures are presented as box plots, to demonstrate the interquartile range, overlayed with dots depicting the values measured from individual animals.

Prior to statistical analysis, all data were first tested for normality and homogeneity of variance (Shapiro-Wilk test). Once normality and homogeneity were confirmed, statistical analysis for treatment effect was undertaken using a 1-way univariate analysis of variance (ANOVA) for refraction, ocular biometry, LC-MS/MS, and autonomic measures, whereas a multivariate analysis of variance (MANOVA) was used to analyze ERG data. Analyses were undertaken in the statistical software program SPSS (IBM, Armonk, NY, USA) with a cutoff of 0.05 for statistical significance.

Results

Intravitreal Injections of Nicotine Inhibit the Development of Experimental Myopia, but not in a Dose-Dependent Manner

Following 6 days, the development of FDM had been significantly inhibited by daily administration of nicotine (intravitreal injection), as measured by axial length (ANOVA = F (3, 41) = 41.78, P < 0.05; Figs. 1A, 1B, Supplementary Table S1) and refraction (ANOVA = F (3, 41) = 27.96, P < 0.05; Figs. 1C, 1D, Supplementary Table S1). Interestingly, this protection was not dose-dependent, with each of the three tested concentrations (15, 1.5, and 0.15 mM) providing a similar effect with respect to axial elongation (approximately 75% protection; ANOVA = F (2, 30) = 0.014, P = 0.987) and myopic refractive shifts (approximately 60% protection; ANOVA = F (2, 30) = 1.282, P = 0.293). Daily intravitreal injections of nicotine (150 nmoles) did not induce any significant changes in retinal thickness in otherwise untreated eyes after 3 days of treatment but did induce significant choroidal thickening (Fig. 2). FDM was unaffected by the daily administration of a vehicle alone, suggesting that the protection observed was due to the presence of nicotine, not an injection effect (axial length = P = 0.451, refraction = P = 0.635; see Supplementary Table S1).

Figure 1. Intravitreal injections of nicotine inhibit the development of form-deprivation myopia (FDM). Over a 6-day period, FDM was inhibited in chicks (A, B) axial length and (C, D) refraction by daily administration of nicotine (minimum of 9 per group). The degree of protection was similar for all doses tested. Statistics represent a significant difference to form-deprivation only (no drug treatment) animals (*P < 0.05). For panels A and C, box plots represent the interquartile ranges of the percent protection produced by each pharmacological agent relative to the form-deprivation only group. For panels B and D, box plots represent the interquartile ranges for the difference between the treated eye and contralateral control eye in each animal. Black dots represent individual values. Data are summarized in further detail in Supplementary Table S1.

Figure 2. Effects of intravitreal injections of 1.5 mM nicotine on retinal and choroidal thickness. (A) Representative fundus and optical coherence tomography (OCT) image with the retinal nerve fiber layer (RNFL), retinal pigment epithelium (RPE), and choroidal and scleral boundaries marked. Daily intravitreal injections of nicotine (150 nmoles) did not induce any significant changes in (B) retinal thickness in otherwise untreated eyes after 3 days of treatment, but did induce significant (C) choroidal thickening. Statistics represent a significant difference to otherwise untreated chicks (*P < 0.05). Box plots represent the interquartile ranges of retinal and choroidal thickness (µm), black dots represent individual values.

To confirm that nicotine can also inhibit the other main paradigm of experimental myopia, LIM, nicotine was administered daily (at a single dose = 150 nmoles), for 6 days, to chicks wearing –10 D lenses. Nicotine induced a similar level of protection against the axial elongation (approximately 70%, P < 0.05; see Supplementary Table S1) and refractive shift (approximately 65%, P < 0.05; see Supplementary Table S1) associated with LIM to that observed in the chicks developing FDM.

When administered to eyes receiving no diffuser or negative-lens treatment, intravitreal injections of nicotine (150 nmoles) did not affect normal ocular development, with respect to either axial growth (P = 0.872; see Supplementary Table S1) or refractive development (P = 0.633; see Supplementary Table S1).

Following Topical Application, Nicotine was Distributed to All Layers of the Eye Where It Inhibited the Development of FDM

When analyzed using LC-MS/MS, nicotine was detectable in all ocular layers 30 minutes after topical administration (Fig. 3, Supplementary Table S2). The concentration of nicotine was highest in the combined iris/lens/ciliary body samples, followed by the aqueous humor and retina, with low levels of nicotine detected in the tears, cornea, vitreous, RPE, and choroid. There was also notable absorption of nicotine into the bloodstream, with these levels being second only to the combined iris/lens/ciliary body samples. Nicotine was not detected in any untreated control samples.

Figure 3. Distribution of nicotine 30 minutes following topical treatment. Box plots represent the interquartile ranges of the peak area ratio (ratio of nicotine's peak area to that of the internal standard [serotonin-d4]) per milligram of sample. Black dots represent individual values. Nicotine was detectable and quantifiable in all samples (n = 4 chicks per group). Data are summarized in further detail in Supplementary Table S2.

Following topical application of the highest dose of nicotine (1200 nmoles), the chicks were observed to become lethargic, with their respiratory rate becoming slow. The effects were rapid (within seconds) and short-lived, with the animals returning to normal behavior/respiration within 2 to 5 minutes. These adverse changes were not seen at lower topical doses, nor were they seen in response to intravitreal injections. However, to avoid potential harm, topical experiments were reduced from 7 days (as occurred for intravitreal injections) to 3 days (the minimal time needed to see a robust treatment effect).

Like that observed for intravitreal injections, topical administration of nicotine significantly inhibited experimental myopia relative to FDM alone (axial elongation = ANOVA = F (4, 37) = 4.46, P < 0.05; Figs. 4A, 4B, Supplementary Table S3; refraction: ANOVA = F (4, 37) = 5.94, P < 0.05; Figs. 4C, 4D, see Supplementary Table S3). This protection was also not significantly dose-dependent, with each of the four concentrations (15, 1.5, 0.15, and 0.015 mM) providing a similar level of protection against both axial elongation (approximately 60% protection; ANOVA = F (3, 26) = 0.61, P = 0.61) and myopic refractive shifts (approximately 50% protection; ANOVA = F (3, 26) = 0.71, P = 0.55). Like that observed for intravitreal injections, topical nicotine induced significant choroidal thickening (Supplementary Fig. S1). Although the degree of effectiveness against axial elongation was similar to that observed for intravitreal injections, there was a 10% drop in protection against the myopic refractive shift. As with many other drugs in the field, the protection afforded by nicotine was more variable with drops than injections.

Figure 4. Topical application of nicotine eye drops inhibits the development of form-deprivation myopia (FDM). FDM was inhibited in chicks A, B) axial length and (C, D) refraction by topical eye drops of 4 ascending doses of nicotine over a 3-day period (n = 6 per group). Statistics represent a significant difference to form-deprivation only (no drug treatment) animals (*P < 0.05). For panels A and C, box plots represent the interquartile ranges of the percent protection produced by each pharmacological agent relative to the form-deprivation only group. For panels B and D, box plots represent the interquartile ranges for the difference between the treated eye and contralateral control eye in each animal. Data are summarized in further detail in Supplementary Table S3.

When administered as either intravitreal injections or topical eye drops, nicotine did not induce changes in corneal curvature/thickness (P = 0.819; Supplementary Fig. S2), anterior chamber depth (P = 0.104; see Supplementary Tables S1, S3), or lens thickness (P = 0.900; see Supplementary Tables S1, S3). Rather, its protection was elicited by slowing vitreal chamber elongation and inducing choroidal thickening (P < 0.05; see Fig. 2).

Autonomic Responses to Nicotine Treatment

As noted, the highest dose (1200 nmoles) of nicotine eye drops induced a significant change in respiration rate and rhythm. Therefore, we wished to further examine the effects of topical nicotine treatment on non-ocular autonomic responses. This was examined by studying the effects of topical nicotine on internal body temperature, heart rate, and respiratory rate in chicks and, to study these effects in a mammalian model, mice (see Fig. 5 for a representative recording). Respiratory and heart rate recordings were performed continuously for 5 minutes prior to treatment (baseline) and for 5 minutes following treatment, whereas temperature was recorded every 30 seconds. We studied the effect of nicotine on autonomic responses at both a dose where the drug was (1200 nmoles) and was not (120 nmoles) found to visually affect respiration.

Figure 5. Representative autonomic responses to nicotine treatment. Representative traces for (A) respiration and (B) heart rate from a single chick undergoing (i) baseline recordings, and recordings immediately following (ii) placebo treatment, (iii) 1.5 mM nicotine treatment, and (iv) 15 mM nicotine treatment. Respiratory measures are represented over a 20 second recording interval, whereas heart rate measures are represented over a 3 second recording interval commencing 10 seconds after treatment.

At the lower dose tested, a small (but not significant) decrease was observed in the breathing rate for mice. Other than this, no effect on autonomic responses were observed in either the chicks or mice. In contrast, when the higher dose of topical nicotine was applied (1200 nmoles), a significant decrease in respiratory rate, as well as a loss of rhythmicity, was observed in both chicks (Fig. 6) and mice (Fig. 7) compared to baseline (P < 0.05 for both chicks and mice) and placebo levels (P < 0.05 for both chicks and mice). There was also a small (but not significant) decrease in heart rate observed in the chicks (see Fig. 6), but not the mice (see Fig. 7). Animals recovered from these effects, returning to baseline levels, 5 minutes post-treatment. Co-administration of nicotine with an agent intended to block its action at nicotinic-cholinergic receptors (vecuronium bromide) or serotonergic receptors (mianserin34) abolished the cardiorespiratory changes observed (see Figs. 6, 7). This supports the hypothesis that, at high concentrations, nicotine is activating a local autonomic reflex (i.e. the oculorespiratory reflex). No significant effects on temperature were observed with any treatment.

Figure 6. Autonomic responses of chicks to topical nicotine treatment. (i) Temperature, (ii) heart rate, and (iii) breathing rate were measured in chicks undergoing treatment with nicotine (n = 5 per group). Chicks (n = 5 per group) were treated with (A) two different doses of nicotine (120 and 1200 nmoles), (B) nicotine in the presence of a nicotinic receptor antagonist (vecuronium bromide, 126 nmoles), and (C) nicotine in the presence of a serotonergic receptor antagonist (mianserin, 40 nmoles). Each test (A, B, and C) was performed on a separate day. Statistics represent a significant difference to baseline (*P < 0.05) and placebo treated values (^P < 0.05). Box plots represent the interquartile ranges of each autonomic response being tested, the black dots represent individual values. Recordings were undertaken for 5 minutes following treatment.

Figure 7. Autonomic responses of mice to topical nicotine treatment. (i) Temperature, (ii) heart rate, and (iii) breathing rate were measured in mice undergoing treatment with nicotine (n = 5 per group). Mice (n = 5 per group) were treated with (A) two different doses of nicotine (120 and 1200 nmoles), (B) nicotine in the presence of a nicotinic receptor antagonist (vecuronium bromide, 126 nmoles), and (C) nicotine in the presence of a serotonergic receptor antagonist (mianserin, 40 nmoles). Each test (A, B, and C) was performed on a separate day. Statistics represent a significant difference to baseline (*P < 0.05) and placebo treated values (^P < 0.05). Box plots represent the interquartile ranges of each autonomic response being tested, black dots represent individual values. Recordings were undertaken for 5 minutes following treatment.

Effects of Nicotine Treatment on Ocular Safety

As noted, topical application of a 1.5 mM (120 nmoles) solution of nicotine was the highest effective dose at which cardiorespiratory functions were unaffected. To expand on this, we investigated the ocular safety of nicotine topically applied at this concentration over a 4-week period.

With regard to the anterior segment of the eye, 4 weeks of topical nicotine did not affect intraocular pressure (P = 0.481; see Supplementary Fig. S2) corneal curvature (P = 0.779; see Supplementary Fig. S2), or lens power (P = 0.401; see Supplementary Fig. S2) when calculated using Bennet's equation (adjusted for chicks35). As expected, due to the presence of nicotinic receptors in the chick iris, nicotine eye drops induced a short-term constriction of the pupil in dark adapted chicks (P < 0.05; Fig. 8A). The pupillary light reflex was also removed in the chicks treated with nicotine, with these effects persisting for 30 minutes. Dark adapted mice (which, like humans, have an iris that is innervated by muscarinic-cholinergic receptors) had dilated pupils when left untreated or administered with topical nicotine (Fig. 8B). In contrast, pupils in mice treated with a muscarinic-cholinergic agonist (pilocarpine) were constricted even during dark adaptation (P < 0.05; see Fig. 8B). Those mice receiving topical nicotine showed a pupillary light response after dark adaption (P < 0.05; see Fig. 8B), whereas neither atropine (P = 0.347) nor pilocarpine (P = 0.076) treated eyes showed a light response due to complete dilation or constriction of the pupil, respectively (see Fig. 8B).

Figure 8. Pupil diameter following topical nicotine treatment. Pupil diameter was measured in (A, B) chicks and (C, D) mice (n = 5 per animal model) before and 5 minutes following treatment with nicotine eye drops (120 nmoles). Pupil measurements were performed in (A, C) dark adapted animals (kept for 60 minutes at <1 lux) and (B, D) in response to a 100-lux flash (10 seconds) in previously dark-adapted animals. For chicks, nicotine treatment induced pupil constriction in (A) dark adapted animals (relative to controls), and thus abolished the (B) pupillary light reflex. For mice, (C) dark-adapted animals showed pupil dilation in both untreated and nicotine treated animals. In contrast, the administration of pilocarpine (muscarinic receptor agonist, 510 nmoles), caused the pupil to constrict and remain constricted during (C) dark adaptation. Following dark adaptation, untreated and nicotine treated mice showed a (D) pupillary light response to a 100-lux flash. In contrast, no change in pupil size was seen in atropine (muscarinic receptor antagonist) or pilocarpine treated mice in response to a (D) light flash due to prior dilation or constriction of the pupil, respectively. Statistics represent a significant difference to untreated animals (*P < 0.05). Box plots represent the interquartile ranges of each group, dots represent individual values.

With regard to the posterior eye, no change in retinal thickness (discussed earlier, P = 0.878; see Fig. 2) or retinal TUNEL labeling (a measure of cell damage/death; Fig. 9) was observed in nicotine treated eyes when compared to age-matched untreated chicks. With regard to retinal function, ERG measures showed a small increase in the light adapted b-wave amplitude compared to otherwise untreated chicks (P < 0.05; Figs. 10A, 10B). Interestingly, this increase in amplitude was not observed following a 4-day washout period (P = 0.311; Figs. 10C, 10D).

Figure 9. Fluorescent visualization of TUNEL staining following topical nicotine treatment. Chick retinae are visualized using 20× magnification. (A) Positive control (DNase I treatment was undertaken to simulate the DNA strand breaks characteristic of apoptosis); (B) negative control (no enzyme); (C) nicotine treated; (D) placebo treated; and (E) age-matched untreated control. In panels B to E, fluorescence was only observed in the oil droplets located in the photoreceptor layer. Within the same panels (B to E), the exposure settings used to aquire (i) the left half of the image represent those used to visualize the positive control (A). The (ii) right side of the image represents the overexposed version of the same retinal section to allow the tissue to be visible.

Figure 10. Light adapted electroretinogram (ERG) responses following topical nicotine treatment. (A) A-wave amplitude; (B) A-wave latency; (C) B-wave amplitude; and (D) B-wave latency (n = 5 chicks per group). Statistics represent a significant difference to untreated animals measured at the same timepoint (*P < 0.05). Box plots represent the interquartile ranges of each group, dots represent individual values.

Discussion

When administered as either intravitreal injections or topical eye drops, nicotine was observed to significantly inhibit the development of FDM in chicks, albeit not in a dose-dependent manner. Nicotine was also capable of inhibiting LIM to a similar degree to that seen for FDM. These findings may help explain the epidemiological observation that exposure to smoking is associated with a lower risk of myopia,6–8,36–38 although this association is not always found.39,40

Like many other drugs investigated in the field,20,28,29,41–46 nicotine had no effect on normal ocular development, suggesting the pathways underlying normal and myopic eye growth are different in some fundamental way. Although nicotine did not induce any significant changes in ocular health, at the highest dose tested, nicotine eye drops induced a temporary autonomic reflex that led to a significant reduction in cardiorespiratory output. Therefore, although the anti-myopic effects observed presently are interesting, the well-reported side effects16,17 (as well as those observed in this study) and addictive properties of nicotine (for review see Ref. 47) would preclude its clinical use.

Administration of Nicotine has Significant Anti-Myopic Effects

When administered as an intravitreal injection, all doses of nicotine tested in this study significantly inhibited the development of FDM and LIM, indicating that nicotinic stimulation can slow ocular growth. Relative to intravitreal injection, topically applied nicotine showed similar anti-myopic effects when ocular penetration rates are accounted for. For both injections and drops, nicotine blocked myopic growth by inhibiting vitreal chamber elongation, while inducing choroidal thickening.

Interestingly, Stone and colleagues10 have previously reported that nicotinic-cholinergic antagonists also inhibit the development of experimental myopia in chicks. Specifically, the daily intravitreal administration of dihydro-b-erythroidine hydrobromide, mecamylamine, methyllycaconitine citrate, or administration of chlorisondamine diiodide every second day, over a period of 1 week inhibited the development of FDM by 50% to 100% (as measured by refraction and axial length).10 Interestingly, not all nicotinic antagonist have been observed to affect myopic growth, with vecuronium bromide ineffective at inhibiting LIM in chicks.33 The anti-myopic effects of both nicotinic agonists and antagonists is somewhat surprising, as these agents would be expected to have opposing actions. It should be noted, that a similar complexity has been reported for the muscarinic-cholinergic system, with administration of both agonists and antagonists reported to inhibit FDM (for review see Ref. 5).

What could explain both stimulation and inhibition of nicotinic receptors showing anti-myopic effects? First, it could be that any deviation in nicotinic-cholinergic receptor activity away from homeostatic norms (i.e. stimulation or inhibition) can inhibit ocular growth. Second, agonists and antagonists may have different sites of action (e.g. agonists functioning within the retina and antagonists functioning within the choroid). Third, this complexity may be due to a number of these cholinergic agonists and/or antagonists not functioning through their expected mode of action. For example, due to cross-reactivity with other receptor families, several of these compounds could act through non-cholinergic pathways, such as serotonergic, GABAergic, glutamatergic, melatonin, etc. Finally, nicotine exposure can lead to receptor desensitization.48 This may temporarily reduce nicotinic signaling, thus producing an antagonist-like response. However, as described previously,48–61 several compensatory mechanisms exist to ensure the body remains sensitive to acetylcholine (or nicotine) stimulation even following receptor desensitization.

Without further work, it is difficult to ascertain which of the above explanations, if any, are correct. This highlights a difficulty in any pharmacological investigation, in which interpretation of the results are complicated by a number of factors, including the role of receptor subtypes, receptor distribution (the potential for multiple target sites), receptor specificity (cross-reactivity), drug pharmacokinetics (e.g. distribution and duration of effect), genetic variability (differences between individuals with regard to receptor structure/function), interactions with the disease state (i.e. myopia induction), and compensatory mechanisms in response to drug treatment (e.g. receptor desensitization and/or downregulation). The use of genetic tools to manipulate tissue-specific expression of potential target receptors would be required to increase confidence in our understanding of the mode-of-action; although such tools come with their own complications with regard to how well they represent normal physiological conditions.

Distribution of Nicotine and its Potential Site of Action

Thirty minutes following topical application, nicotine had been distributed to all layers of the eye. The highest concentration was found within the combined tissue sample of the iris, lens, and ciliary body, which accounted for 81% of the total nicotine detected across samples. Within the other ocular samples, the aqueous humour contained 1.4%, the retina 1.2%, the choroid 0.9%, tears 0.4%, cornea 0.2%, and the vitreous body 0.1%. A significant amount of nicotine was also detected in the blood, accounting for 14.8% of the total drug detected.

Based on the extensive distribution of nicotine, it is unclear (from pharmacological data alone) the “site of action” at which nicotine induces its anti-myopic effects. The presumed target had been that of the retina, the source of initial growth signals. However, a number of factors make it difficult to determine the location and subtype of receptor that may be involved in nicotine's anti-myopic effects. First, nicotinic receptors, to which nicotine can bind and modulate all subtypes (discussed previously), are ubiquitously expressed throughout the eyes.11,12 Second, nicotine was found to penetrate, at biologically relevant levels, all potential target tissues within the eyes. Finally, nicotine treatment induced biochemical or physiological changes in all major target tissues: the retina (changes in electrophysiology and, previously, dopamine levels5), the choroid (thickening), and the sclera (reduced scleral remodeling/axial elongation). To elucidate the location and receptor subtype of nicotine's anti-myopic effects, future studies would need to make use of genetic tools (e.g. gene knockdown/knockout/overexpression) to demonstrate a causal effect.

Nicotine did not Induce a Dose-Dependent Effect

Surprisingly, there was no dose-dependent protection afforded by nicotine, irrespective of the mode of delivery. Rather, all doses exhibited roughly 75% to 80% protection against axial elongation and 50% to 60% protection against the myopic shift in refraction. This was somewhat unexpected, as in other systems, nicotine has been frequently observed to produce a dose-based response over the concentration range tested presently.62–68 Furthermore, the majority of compounds tested in the field of myopia, including other cholinergic agents, exhibit dose-dependent protection against myopia, although the shape of this response can vary (e.g. logarithmic, biphasic, sinusoidal, multiphasic, etc.).20,28–32,69–72 For example, for nicotinic receptor antagonists, Stone and colleagues10 observed a multiphasic dose-response for the majority of the compounds investigated. The lack of dose-response observed presently may therefore suggest that even at the lowest concentration applied, nicotinic receptors were saturated and producing their maximal effect.

Nicotine does not Induce Notable Changes in Ocular Health

Chronic treatment with nicotine, as topical drops, induced no remarkable ocular changes (i.e. corneal thickness/curvature, lens thickness, IOP, and retinal thickness/cell viability) over a 4-week period. The only notable changes associated with nicotine were in light-adapted ERG recordings, as well as dark-adapted pupil size. Specifically, there was a small (but significant) increase in b-wave amplitude, which has been reported previously.73 Variations in b-wave amplitude represent changes in the activity of ON and OFF bipolar cells. What functional or clinical significance such a small amplitude change would have is unclear, with no other aspect of the ERG recording showing any drug-induced effects. Furthermore, the changes observed in b-wave activity appear to be temporary, as following drug washout (4 days) b-wave amplitude had returned to normal. With regard to pupil size, there was an approximately 25% decrease in diameter in dark-adapted chicks. This is consistent with previous reports,74 with the chick iris being innovated by nicotinic rather than muscarinic (mammals) receptors. What role modulation of the iris may play in the anti-myopic effects of nicotine is unclear and would require further investigation to understand.

High Doses of Topical Nicotine Appear to Induce the Oculorespiratory Autonomic Reflex

As noted, at the highest topical dose, nicotine was observed to induce lethargy and a decreased respiration rate in chicks. This response persisted for 2 to 5 minutes before full recovery. A more detailed analysis of autonomic output observed that, at a topical concentration of 15 mM, nicotine induced a 45% reduction in respiration rate, a loss of breathing rhythmicity, and a 13% reduction in heart rate (although this did not reach statistical significance). These autonomic changes were seen in both chicks and mice, suggesting a conserved response across birds and mammals.

Three potential mechanisms could explain these observed effects. First, following systemic absorption, nicotine may bind its receptors within the autonomic centers of the brain or directly within the affected organs. Nicotine is a sympathomimetic drug and, when administered systemically, has been reported to increase the heart rate and cardiac contractility, constrict cutaneous and coronary blood vessels, and transiently increase blood pressure.16,17 High levels of nicotine were detected in the blood 30 minutes following topical treatment in this study, so it is feasible that nicotine may have induced autonomic changes through this mechanism. However, the autonomic responses observed in this study are opposite to that previously reported during systemic nicotine treatment,16,17 suggestive that these cardiorespiratory changes are driven by a different pathway.

Second, nicotine may bind to 5HT3 serotonin receptors in the brain, inducing serotonin syndrome, due to the structural homology of 5HT3 with nicotinic receptors.34 This syndrome (also known as serotonin toxicity), which occurs in response to inappropriate increases in synaptic levels of serotonin within the brain, is associated with an increase in heart rate, blood pressure, and body temperature.75 However, this serotonergic mechanism also appears an unlikely candidate for the symptoms observed in this study due to the direction of the response and the speed at which it was observed.

Finally (and most likely), following topical application, nicotine may induce an oculorespiratory reflex. This reflex is brought about by activation of parasympathetic pathways within the extraocular muscles (this commonly occurs through physical deformation of the tissue).76 The signal is rapidly carried through the ophthalmic division of the trigeminal nerve to the pneumotaxic respiratory center in the pons which stimulates the medullary respiratory area.76 By acting through the phrenic and other respiratory nerves, this causes a reduction in respiratory rates, irregular respiratory movements, and, in severe cases, respiratory arrest.76 A similar reflex, the oculocardiac reflex, is induced through the same pathway and leads to a reduction in heart rate, arrhythmia, and syncope (for review see Ref. 77). The rapid speed at which the oculorespiratory and oculocardiac reflex can manifest and dissipate fits strongly with the current findings. Importantly, the cardiorespiratory changes induced by these pathways are in the correct direction for what was presently observed.

As one might expect, co-administration of a nicotinic antagonist blocked the effects of nicotine on cardiorespiratory functions. Interestingly, co-administration with a serotonergic receptor antagonist was also able to reduce nicotine's effects. As serotonergic stimulation can affect ocular muscle activity,78 it is therefore possible that the oculorespiratory reflex was induced through nicotine binding to serotonergic and/or nicotinic receptors. Alternatively, due to the similarities in receptor structure discussed above, the serotonergic antagonist mianserin may be blocking nicotine's activity at nicotinic receptors.

Nicotine is known to have numerous adverse events when systemically administered. For example, systemic nicotine treatment increases the heart rate and cardiac contractility, constricts cutaneous and coronary blood vessels, and transiently increases blood pressure.16,17 Thus, although producing a significant anti-myopic effect, the known adverse events associated with systemic distribution of nicotine combined with the currently observed oculorespiratory changes, would heavily impede any use of nicotine as a clinical intervention for myopia or other visual disorders.

Conclusions

Intravitreal administration of nicotine was able to significantly inhibit the development of both FDM and LIM. Nicotine remained effective when administered as topical eye drops, but interestingly did not induce a dose-dependent protection through either route of administration. Although nicotine did not induce any notable effects on ocular safety, the highest dose tested for topical eye drops was associated with a short-lived alteration in cardiorespiratory autonomic output. This adds to the known safety and dependency concerns associated with nicotine use. Therefore, although effective at inhibiting ocular growth in animal models, nicotine would not be an appropriate clinical treatment for myopia.

Supplementary Material

Supplement 1

Acknowledgments

Funded by ANU Connect Ventures through a Discovery Translation Fund grant (Project ID: DTF311).

Disclosure: K. Thomson, None; C. Karouta, None; R. Ashby, None
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