Acute effects of dietary retinoic acid on ocular components in the growing chick

Transcription

1 Experimental Eye Research 83 (2006) 949e961 Acute effects of dietary retinoic acid on ocular components in the growing chick Sally A. McFadden a, *, Marc H.C. Howlett a, James R. Mertz b, Josh Wallman c a Psychology, School of Behavioural Sciences, Faculty of Science and IT, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia b New England College of Optometry, Boston, MD, USA c Department of Biology, City College of the City University of New York, NY 10013, USA Received 3 March 2006; accepted in revised form 8 May 2006 Available online 22 June 2006 Abstract When the eyes of chicks are induced to grow toward myopia or hyperopia by having them wear spectacle lenses or diffusers, opposite changes take place in the retina and choroid in the synthesis and levels of all-trans Retinoic Acid (RA). To explore whether RA plays a causal role in the regulation of eye growth, we fed young chicks RA (doses 0. to 24 mg/kg) either twice a day or on alternate days or only once. Refractive error was measured with a Hartinger refractometer; ocular length, lens-thickness and choroidal thickness were measured by A-scan ultrasound. The amount of RA present in ocular tissues was determined using HPLC. Oral delivery of RA effectively increased RA in ocular tissues within 8 h. During the first day after feeding RA at levels above 8 mg/kg, the rate of ocular elongation tripled, the choroid thickened and lens thickening was inhibited. The day following a dose of RA, the rate of ocular elongation was inhibited and the lens thickened more than normal. Nonetheless, the cumulative effect of repeated doses was that the eye became longer and the lens became thinner than normal, with no net change in refractive error. The rate of elongation was also increased by feeding 13-cis RA, and was reduced by citral, an inhibitor of RA synthesis. Surprisingly, birds fed RA while being kept in darkness also had normal refractive errors despite increased ocular elongation, and birds wearing either þ6 D or 6 D spectacle lenses compensated normally for the lenses despite the enhanced ocular elongation caused by the RA. These results suggest that RA may act at the level of a coordinated non-visual regulatory system which controls the growth of the various ocular components, arguing that emmetropization does not depend entirely on vision. Crown Copyright Ó 2006 Published by Elsevier Ltd. All rights reserved. Keywords: retinoic acid; myopia; hyperopia; emmetropization; form deprivation; chick 1. Introduction The eye of a neonate grows towards a state in which the optical properties and physical dimensions are precisely matched (emmetropia). Visual factors partially regulate this process, since perturbations of vision disrupt the growth towards emmetropia. Depriving the neonate eye of form vision results in eyes with longer axial lengths and myopic refractive errors (Howlett * Corresponding author. Tel.: þ ; fax: þ addresses: sally.mcfadden@newcastle.edu.au (S.A. McFadden), psmch@alinga.newcastle.edu.au (M.H.C. Howlett), mertzj@neco.edu (J.R. Mertz), wallman@sci.ccny.cuny.edu (J. Wallman). and McFadden, 2006; Lodge et al., 1994; Norton, 1990; Troilo and Judge, 1993; Wallman and Adams, 1987; Wallman et al., 1978; Wiesel and Raviola, 1977). Changing the eye s effective focal length with spectacle lenses induces compensatory changes in ocular length and refractive error which are sensitive to both the sign and magnitude of the imposed defocus (e.g. chicks, Schaeffel et al., 1988; monkeys, Hung et al., 199; tree shrews, Shaikh et al., 1999; Venkataraman et al., 200; guinea pigs, McFadden et al., 2004; and marmosets, Whatham and Judge, 2001). It is unknown what visual cues guide ocular growth and how these cues translate into chemical and physical changes. Evidence supports the possible involvement of dopamine (Guo et al., 199; Stone et al., 1989), vasoactive intestinal /$ - see front matter Crown Copyright Ó 2006 Published by Elsevier Ltd. All rights reserved. doi: /j.exer

2 90 S.A. McFadden et al. / Experimental Eye Research 83 (2006) 949e961 polypeptide (Seltner and Stell, 199; Stone et al., 1988), nitric oxide (Fujikado et al., 1997; Nickla and Wildsoet, 2004), enkephalin (Seltner et al., 1997), basic fibroblast growth factor (Rohrer and Stell, 1994), transforming growth factor b (Honda et al., 1996; Rohrer and Stell, 1994), glucagon (Buck et al., 2004; Feldkaemper and Schaeffel, 2002) and retinoic acid (Bitzer et al., 2000; McFadden et al., 2004; Mertz and Wallman, 2000; Seko et al., 1998) in the control of eye growth. Of these, only glucagon and retinoic acid show evidence of changing in opposite directions when positive and negative spectacle lenses are worn. Strong evidence supports an important role for a vitamin-a derivative, all-trans retinoic acid (RA), in the control of eye growth. In many cell types RA acts by binding to retinoid receptors which activate gene transcription of target genes whose function is to limit growth (Blaner and Olson, 1994). RA and its associated molecules are present in, and synthesized by, the retina and choroid (Fischer et al., 1999; Mertz and Wallman, 2000; Mey et al., 1997; Saari et al., 1982) and have visual functions in the retina (Weiler et al., 1998, 2000; Zhang and McMahon, 2000). The involvement of RA in emmetropization is suggested by the finding that the synthesis of both retinal and choroidal RA is modified by visual conditions which change eye growth. In chicks and guinea pigs, the level of RA is elevated in the retina in eyes with enhanced ocular growth (after form deprivation or negative lens-wear and reduced in eyes with inhibited ocular growth (after positive lens-wear) (McFadden et al., 2004; Seko et al., 1998). Furthermore, intravitreal injection of inhibitors of RA synthesis, reduce the degree of myopia produced by form deprivation (Bitzer et al., 2000). The level of RA in the chick choroid is also correlated with the direction of eye growth, but in the opposite direction to that seen with retinal RA. Thus the rate of choroidal synthesis of RA decreases in response to form deprivation or 1 D spectacle lens-wear and increases in eyes recovering from form deprivation or wearing þ1 D lenses (Mertz and Wallman, 2000). Because the visual manipulations just described cause changes both in ocular growth and in the level of RA, we artificially elevated levels of ocular RA in chicks through feeding and examined their ocular elongation rates. There is evidence that RA given orally to guinea pigs results in increased ocular elongation (McFadden et al., 2004). To study the interaction between the effects of RA on eye elongation and eye growth arising from visual manipulations, we also fed chicks RA while they were either raised in the dark or wore spectacle lenses. Since serum albumin delivers RA to tissues in normal animals (Blaner and Olson, 1994), RA absorbed from the gut would be likely to be bound to serum albumin and distributed throughout the eye. To confirm this, we measured the levels of RA in both the retina and choroid. Some of these results have been published as an abstract (Mertz et al., 1999). 2. Materials and methods 2.1. Animals Day-old white leghorn chicks (Gallus gallus domesticus, Truslow Farms, MD, USA) were group-housed in temperature-controlled (32 C) metal brooders under a 14 L/ 10 D lighting cycle (lights on at 8am) with water and food (Purina chick food) freely available. Care and use of animals were in compliance with the World Medical Association statement on Animal Use in Biomedical Research and in accordance with the NIH guidelines for the care and use of animals in research Preparation and administration of RA and Citral In all experiments, systemic doses of all-trans retinoic acid (RA), 13-cis retinoic acid (13-cis RA) or citral (a competitive inhibitor of the dehydrogenases responsible for the generation of retinoic acid; 3,7-dimethyl-2,6-octadiena, Sigma, St Louis, MO) were mixed with 0.2 ml of peanut oil (Vehicle). Reference to RA, 13-cis RA and citral throughout the text should be interpreted as RA & peanut oil, 13-cis RA & peanut oil and citral & peanut oil, Vehicle refers to those chicks administered 0.2 mls of peanut oil alone. RA and 13-cis RA were dissolved in diethyl ether before being added to the peanut oil. The diethyl ether was then gently blown off with nitrogen gas. Dosage levels were calculated from a mean chick weight of 100 g. Awake chicks were dosed by gavage into the crop region. When dosage occurred after ultrasound, chicks were allowed to recover from the anesthetic prior to the gavage treatment A-scan and refractive error measurements Axial ocular dimensions were measured by A-scan ultrasonography using a 30 MHz transducer (Panametrics Model 17699), with a Sonix 8100 a/d board sampling at 100 MHz, as described previously (Nickla et al., 1998). The length of the eye from the anterior surface of the cornea to the back of the sclera is referred to here as ocular length, to avoid confusion with the term axial length, which usually refers to the distance from cornea to retina, and therefore is influenced by changes in the choroidal thickness. Refractive errors were measured using a modified Hartinger refractometer (Wallman and Adams, 1987). Both refractive error (RE) and ultrasound measurements were undertaken while chicks were lightly anaesthetized with 1% halothane in oxygen. Although no cycloplegic agent was used, halothane causes pupillary dilation and hence probably some degree of cycloplegia (Nickla et al., 1998) Experimental paradigms We conducted five experiments to ascertain whether systemic administration of RA might induce changes in ocular elongation (Table 1). The drug doses listed in Table 1 were given at each of the time points indicated Experiment 1. Effect of feeding RA on the level of RA in the retina and choroid (A) To determine the time-course of changes in tissue levels of RA, birds were fed RA once, and retinal and

3 S.A. McFadden et al. / Experimental Eye Research 83 (2006) 949e Table 1 Experimental paradigms Experiment Substance fed Timing of drug gavage Measurement details No. of (mg/kg/dose) Times of day Age (days) chicks Exp. 1: Measurement of ocular levels of RA 1A: Time course RA: 12 0, 2, 8 or 24 h after RA at 14 days of age am 14 RA: 24 8 or 12 h after RA at 14 days of age. 8 1B: Dose response Vehicle RA: 2 6 RA: 8 10 am 14 8 h after RA at 14 days of age. 4 RA: 12 3 RA: 24 4 Exp. 2: Effect of RA, an RA agonist and an RA inhibitor 2A RA: am, 10 pm 6e9 US: daily at 10 am from 6e10 days of age RE: 10 am on day 6 and day 10. Vehicle 2B 13-cis RA: am, 10 pm 6e9 US: Daily at 10 am for 4 days from 6e10 days of age. citral: 44 9 am, 1 pm, 6 pm 10 am, 10 pm 6 7e9 Vehicle 9 am, 1 pm, 6 pm 10 am, 10 pm Exp. 3: Time-course 3A: Single dose RA: am 7 US: 10 am and 4 pm on day 7 and daily at 10 am from 8e11 days of age. Vehicle 10 am 7 3B: Circadian RA: am, 10 pm 6e9 US: every 6 h from 6e10 days of age. 3C: Alternate RA: 24 (in 0.4 ml peanut oil) 10 am every alternate day 6 7e9 6e16 US: daily at 10 am from 6e17 days of age. 3 Exp. 4: Dose-response 4 Vehicle RA: 0. US: daily at 10 am for 4 days from RA: 2 10 am, 10 pm 6e9 6e10 days of age. RA: 8 RA: 24 Exp. : Visual manipulations A: Darkness RA: am, 10 pm 8e10 Raised in dark for 3 days (age 8e11). RE and US at 10 am at 8 and 11 days of age. Vehicle B: þ6 D Lens RA: 18 (day 8), RA: 36 (day 9), RA: 4 (day 10) 10 am 8e10 Lenses were worn for 3 days (age 8e11). RE and US: 10 am at 8 and 11 days of age. Vehicle 6 C: 6 D Lens RA: 18 (day 8), 7 RA: 36 (day 9), RA: 4 (day 10) 10 am 8e10 Vehicle US, ultrasound measurements of ocular length and other internal ocular parameters; RE, refractive error. Procedures were done in the following order: RE, US, drug gavage. Vehicle was 0.2 ml of peanut oil. RA was mixed with 0.2 ml of peanut oil except in Experiment 3C. As an example of how to read the table, in Experiment 2A, five chicks were fed 24 mg/kg of RA twice each day for 3 days (i.e., a total of 48 mg/kg/day) between 6e9 days of age; and five were fed Vehicle over the same period. US was done every day at 10 am between 6e10 days of age, and RE was measured at 10 am on day 6 (before any dosage) and on day 10 (24 h after the last dose). 6 choroidal levels of RA were measured at various times (see Table 1). (B) To determine the effect of different doses of RA on the tissue levels, eyes were removed 8 h after various RA dosages (Table 1). In both cases, central 8 mm punches were dissected to separate the retinal and choroidal layers, and the RPE was removed by gentle manipulation with a camel hair brush. Levels of RA were obtained in each tissue by normal phase HPLC using an identical procedure to

4 92 S.A. McFadden et al. / Experimental Eye Research 83 (2006) 949e961 that previously described for scleral tissue (Mertz and Wallman, 2000) Experiment 2. Effects of repeated Ingestion of RA, an RA agonist and an RA inhibitor To determine the effect of feeding RA on ocular elongation, chicks were fed either Vehicle or RA and their ocular parameters and RE measured (see Table 1 for dosage and timing). We also fed chicks an agonist of all-trans RA (13-cis RA) and an inhibitor of RA formation, citral Experiment 3. Time-course of effects of RA To study the time-course of RA-induced ocular changes, we did three experiments (Table 1): (a) chicks were fed RA or Vehicle once and measured 6 h later, and then daily for the next 4 days; (b) chicks were fed RA twice a day and measured every 6 h; and (c) chicks were fed RA on alternate days and measured daily Experiment 4. Dose-response of effects of RA We varied the dose of RA in different groups of chicks as shown in Table Experiment. Effect of visual manipulations (a) Light deprivation. To determine the influence of vision on the RA-induced ocular elongation, chicks were fed either RA or Vehicle, while being raised in darkness from 8e 11 days of age after having been on a normal light cycle prior to dark rearing. Weight gain was normal in the dark. The first drug administration occurred after 30 min of darkness. Birds were exposed to very dim red light during the daily 30 s gavage procedure. Measurements and timing are shown in Table 1. (b) Lens wear. Chicks wore a spectacle lens on one eye for 3 days from 8 to 11 days of age while receiving either RA or Vehicle. In lens-wear experiments the changes in ocular elongation begin a day or two after the lenses are fitted, just when we find that the effects of constant daily doses of RA diminish. Thus RA was given in increasing amounts once each day (Table 1), as pilot experiments showed that such a dose regime was able to sustain the accelerated rate of RA-induced ocular elongation, and it ensured that RA was active during the same period in which lens-wear increases eye growth (Kee et al., 2001). Lenses (PMMA) were attached at their edges to a Velcro Ò ring and the mating ring was fitted around one eye using collodion adhesive (Fisher Scientific, Fairlawn, NJ). Half the birds in each group were fitted with a þ6 D lens and the other half with a 6 D lens. The fellow eyes served as controls for the RA-induced ocular elongation alone. Lenses were cleaned twice daily. Chicks were measured as indicated in Table Data presentation and analysis There were no significant differences between the left and right eyes within an animal in all cases, so the left eye data were averaged with the right eye data (except in Experiment B and C in which one eye wore a lens). Statistical comparisons between drug groups were performed after normalizing the data by subtracting the average value prior to treatment. In all experiments, the amount of daily change was the raw value for each day minus the value for the preceding day. Statistical analyses used repeated measures (RM) or one- or two-way ANOVA (SPSS 9.0) as specified. Post-hoc analyses used Tukey s Least Significant Difference (LSD) or Dunnett s pairwise multiple comparisons. The data given on total change refers to the accumulated change over the entire course of the experiment (e.g., day 6 to 10 in Experiment 1). When the total change in the eyes of the experimental animals was compared to that in Vehicle-fed chicks, the p value refers to a two-tailed t-test. 3. Results 3.1. Effect of feeding RA on ocular levels of RA (Experiment 1) Feeding chicks a single dose of RA (12 or 24 mg/kg) raised the levels of RA in both the retina and choroid (Experiment 1; Fig. 1A): at 2 h the levels in the choroid and retina were approximately ten times the endogenous levels (shown as time zero); these levels peaked at 8 h to over 100 times the endogenous levels and fell at 24 h to 70 times the endogenous levels in the choroid and to 2½ times the endogenous levels in the retina. Twenty-four h after a dose of 12 mg/kg, the level of RA was more elevated in the choroid than that in the retina (Fig. 1A). At 8 h after feeding RA, the levels of RA in the retina and choroid increased monotonically from 8e24 mg/kg (Fig. 1B, One way ANOVA, F ¼ 29. and F ¼ 31.0 respectively, p < in both cases). Relative to baseline, a dose of 2 mg/kg of RA nearly doubled the amount of RA, although this change was not statistically significant (retina: 0.8 vs ng; Choroid: 0.23 vs. 0.6 ng respectively) Effects of repeated ingestion of RA, an RA agonist and an RA inhibitor (Experiment 2) RA fed over several days (Experiment 2A) led to a sustained increase in ocular length (Fig. 2A, total change: RA, þ mm; Vehicle, þ mm, p < 0.00). The immediate effect was a dramatic acceleration in the rate of ocular elongation, on the first day being four times that of birds fed Vehicle (Fig. 2B, RM ANOVA, F ¼ 46.12, p < 0.001). The amount of additional elongation each day declined over the course of the experiment (RM ANOVA, F ¼ 10.6, p < 0.01), despite the twice-daily feedings, so that by the fourth day, the rate of additional daily elongation (in a substantially elongated eye) was no longer significantly different from birds fed Vehicle (Fig. 2B). Nevertheless, over the 4 days of the experiment, the eye had elongated twice as much as normal on average (RA, 138 mm/day; Vehicle, 7 mm/day like untreated chicks, 72 mm/day (Nickla et al., 1998).

5 S.A. McFadden et al. / Experimental Eye Research 83 (2006) 949e in Experiments 2A and 2B (þ mm vs. þ mm respectively, p > 0.). In contrast, citral administration nearly halved the total amount of ocular elongation over the course of the experiment when compared to that in the chicks fed Vehicle on the same schedule (total change, þ mm vs. þ mm respectively, p < 0.00). Unlike the immediate effects of RA, this inhibition in ocular elongation was significant only after 2 to 3 days (Fig. 2C). Like RA, the effects were transient and the rate of elongation returned to normal thereafter (Fig. 2D) Time course of the effects of RA on ocular elongation (Experiment 3) Fig. 1. Level of RA in the retina (filled symbols) and choroid (unfilled symbols): A) after chicks were dosed once with 12 or 24 mg/kg of RA (0 time point shows baseline endogenous levels of RA); and B) 8 h after chicks were fed different doses of RA (0 mg/kg ¼ Vehicle only). *p < 0.0, ***p < 0.001, error bars are the SEM. Feeding the RA agonist, 13-cis RA, also led to sustained increases in eye size compared to Vehicle controls (total change, þ mm vs. þ mm respectively, p < 0.0), and these changes were also rapid. Twenty four hours after the dosage started, the eyes of birds fed 13-cis RA elongated significantly more than did those of Vehiclefed controls, and these differences were sustained for the 4 days of the experiment (Fig. 2C, RM ANOVA, F ¼ 28.48, p < 0.00). As with the all-trans RA, the amount of daily change in eye elongation declined with repeated applications, with the rate of ocular elongation not different to Vehicle by the third day (Fig. 2D). Compared to all-trans RA, 13-cis RA elicited slightly smaller increases in ocular elongation over the duration of the experiment (total change, þ vs. þ mm respectively, p < 0.01), while the total change in ocular length for birds fed Vehicle were similar In two experiments, we found an extraordinary increase in the ocular length starting 6 h after the first dose of RA: In Experiment 3A a change of þ81 mm (Fig. 3A) being double that in the chicks fed Vehicle (þ37 mm, LSD p < 0.001) and in Experiment 3B a change of þ133 mm (Fig. 3C). The greatest amount of eye elongation occurred during the light part of the cycle (Fig. 3C), as occurs in untreated chicks of the same strain (Nickla et al., 1998). By 24 h, the eye had elongated by 116 mm after a single dose of RA (Experiment 3A, Fig. 3B) and by 289 mm after two doses (Experiment 3B, Fig. 3C) or an average of 240 mm when fed on alternate days (Experiment 3C, Fig. 3D). This immediate stimulation of ocular elongation by RA was followed by a reversal. This was shown by the rate of elongation being significantly below that of Vehicle-fed birds (Fig. 3B, 24e48 h, Experiment 3A). Furthermore, when RA was fed only on alternate days, the rate of ocular elongation was slower than normal (untreated chicks ¼þ70 mm/day, chicks fed RA ¼ as low as 38 mm/day, Fig. 3D, days 14 and 16). This reversal was not enough to offset the stimulation phase, so that over the 11 days of Experiment 3C, the eye progressively elongated by an average of 13 mm/day, twice that of untreated chicks (Nickla et al., 1998) Effects of RA ingestion on other ocular parameters (Experiments 2 and 3) Choroidal response The choroid thickened in birds treated with RA, but unlike ocular elongation, the maximum response was sometimes delayed and transient. In Experiment 2A, maximum thickness occurred 2 days after the first dose of RA, and then declined to normal over the following two days (Fig. 4A). In birds treated only every other day, choroidal thickening rose and fell together with the rate of ocular elongation (Fig. 3D). The choroidal increases were stronger than the reversals which followed, so that the net effect was that the choroid thickness increased from 186 to 30 mm over the 11 days (RM ANOVA, F ¼ 8.1, p < 0.001). The thickening of the choroid seems not to have been a delayed reaction to ocular elongation because there was no correlation between an individual bird s ocular length at 7 days of age, and the choroid thickness at either

6 94 S.A. McFadden et al. / Experimental Eye Research 83 (2006) 949e961 Fig. 2. Ocular length (A and C) and daily change in ocular length (B and D) after repeated doses (upward arrows) of retinoic acid, 13-cis retinoic acid or citral (Experiment 2). Values in A and C are normalised to the mean length prior to treatment at 6 days of age. The additional symbols on day 6 show the raw values for each group. *p < 0.0, **p < 0.01, ***p < 0.001, p values are the difference relative to Vehicle (RM ANOVA, LSD) unless otherwise indicated, error bars ¼ SEM. 7 or 8 days of age (Experiment 2A; r 2 ¼ 0.00 and respectively). In normal chicks, ocular elongation peaks during the day while choroidal thickness peaks at night (Nickla et al., 1998). Feeding RA changed this rhythm so that both measures were in phase, peaking during the day over the 4 days of Experiment 3B (first two cycles shown in Fig. 3C) Vitreous chamber In untreated birds, the vitreous elongates by approximately 44 mm/day (Nickla et al., 1998). In chicks fed RA, the vitreous chamber deepened on average by mm/day, 2½ times the normal rate, while that in chicks fed Vehicle, increased by only mm/day perhaps due to the repeated anesthesia. Over the 4 days of dosing, the vitreous chamber in RA-fed birds increased five times greater than that in the Vehicle-fed birds (total change: RA, þ ; Vehicle, þ mm; p < 0.00; Experiment 2A; Fig. 4B) Lens thickness In contrast to the stimulatory effects of RA on ocular elongation, RA caused the lens to thicken half as much as in birds fed Vehicle (total change: RA, þ mm; Vehicle, þ mm; p < 0.01; Experiment 2A, Fig. 4C). This decline in the rate of lens thickening relative to that in birds fed Vehicle occurred rapidly e within the first 24 h (RA: þ16 9 mm, Vehicle: þ 7 mm, RM ANOVA F ¼ 24.13, LSD, p < 0.01). When measured every 6 h, the normal lens thickening ( mm/day) was inhibited from the first time-point after RA was fed (at time 0 the lens was mm, 6 h later it was mm, and at 24 h was mm; Experiment 3B). When RA was given to chicks on alternate days, the rapid inhibition of lens thickening was followed by an increase in lens thickness the following day (Experiment 3C, paired t-test on averages, p < 0.00). The day after RA was fed to chicks, the average daily increase in lens thickness was only 7 mm (SD ¼ 20 mm), but it thickened a normal amount each following day (2 1 mm) Refractive error Although chicks fed RA twice-daily for 4 days (Experiment 2A) had vitreous chambers 0.4 mm deeper than those of birds fed Vehicle, their refractive errors did not change between 6 and 10 days of age (RA: day 6, þ D; day 10, þ D; Vehicle: day 6, D; day 10, þ D, RM ANOVA, F ¼ 0.4, p ¼ 0.) and did not differ significantly from the Vehicle-fed birds (total change: RA, D; Vehicle, þ D, p ¼ 0.33) Anterior chamber, retina and sclera There were no significant effects of feeding RA in Experiment 2A on the depth of the anterior chamber or the thickness of the sclera, although the retina thinned slightly in proportion to the vitreous expansion (10 mm thinner for 0.4 mm expansion, r 2 ¼ 0.42, p < 0.001), possibly a passive result of the increased eye size.

7 S.A. McFadden et al. / Experimental Eye Research 83 (2006) 949e961 9 Fig. 3. Time course of changes in ocular length and choroid thickness after RA administration (upward arrows). A) Normalised ocular length (to that at 7 days of age) after a single dose of RA (filled symbols) or Vehicle (open symbols) in Experiment 3A. The additional symbols at day 7 show the raw values. p values are RA compared to Vehicle (RM ANOVA, LSD). B) Corresponding differences between each successive measure in Experiment 3A show a doubling in ocular expansion rate 6 h after RA was given, followed by a subsequent transient inhibition in ocular elongation rate (down arrowhead). C) Rates of ocular elongation and choroidal expansion change in parallel when RA is fed twice daily (Experiment 3B). Both peak during the daylight hours. The night is shown by the filled bars. D) RA given every second day resulted in ocular elongation and choroidal thickening 24 h after each dose, followed by increasing amounts of rebound inhibition the following day (Experiment 3C). *p < 0.0, **p < 0.01, ***p < 0.001, error bars ¼ SEM. 3.. Dose-response of RA (Experiment 4) Stimulation of ocular elongation by RA showed a steep dose-response function. Neither 0. nor 2 mg/kg had any significant overall effect, whereas 8 and 24 mg/kg had maximal effects of over three times the normal daily rate of ocular elongation when measured 24 h after dosing (2, 114, 91, 217 and 21 mm for 0, 0., 2, 8, 24 mg/kg RA respectively, one-way ANOVA, F ¼ 14., LSD, p < for 8 and 24 mg/kg). These effects were maintained over the 4 days of dosing (Fig. A, one-way ANOVA, F ¼ 9.9, p < 0.001). Although the two highest dose groups were similar in terms of overall ocular elongation, vitreous chamber elongation and inhibition in lens thickening were significantly greater for 24 mg/kg of RA compared to 8 mg/kg (Fig. B,C). Both the thickening in the lens (Fig. C) and choroid (Fig. D) were sensitive to the smaller doses of RA. Thickening of the lens was inhibited within 24 h by small doses of RA (0 mg/kg: þ39 mm, Fig. 4. Time course of changes in: A) choroidal thickness; B) vitreous chamber depth; and C) lens thickness for chicks fed 24 mg/kg of RA (filled symbols) or Vehicle (open symbols) twice daily (arrows) in Experiment 2A. Data is normalised to the average of both groups at 6 days of age. Raw values at 6 days of age are also shown. *p < 0.0, **p < 0.01, ***p < 0.001, p values are RA compared to Vehicle (RM ANOVA, LSD), error bars ¼ SEM.

8 96 S.A. McFadden et al. / Experimental Eye Research 83 (2006) 949e961 Fig.. Dose-response functions of RA. Total change (from day 6 to 10) in A) ocular length; B) vitreous chamber depth; and C) lens thickness. D) Initial change in choroidal thickness over the first 24 h after various doses of RA and Vehicle alone (dose ¼ 0) in Experiment 4. *p < 0.0, **p < 0.01, ***p < 0.001, p values from LSD after one-way ANOVA, error bars ¼ SEM. 0. mg/kg: þ4 mm, F ¼ 16.7, LSD, p < 0.01), with higher doses causing the lens to initially shrink (8 mg/kg: 30 mm, 24 mg/kg: 27 mm). Over the 4 days of treatment, the degree of lens thickening was inversely proportional to the dose of RA given (Fig. C). The choroidal thickening was transient, being affected at 24 h (Fig. D) and 48 h, but returning to normal thereafter Effect of RA on chicks with visual manipulations (Experiment ) To see whether the maintenance of emmetropia despite the RA-induced ocular elongation was due to visual compensatory mechanisms, we fed chicks RA and kept them in darkness or had them wear positive or negative lenses RA in darkness Darkness had no influence on the RA-induced ocular changes. First, as in chicks reared under a normal photoperiod, the eyes of RA-treated chicks elongated approximately twice as much as the Vehicle-fed chicks over the 4 day course of this experiment (Fig. 6A). This elongation was primarily from an increased depth of the vitreous chamber (total change: RA, þ0.064 mm, Vehicle, þ0.034 mm; day 11: RA, þ mm, Vehicle, þ mm, t-test, p < 0.01). Second, the crystalline lens of the RA-fed chicks was thinner than that in chicks fed Vehicle (Fig. 6B), as was the case in chicks raised in normal lighting (Fig. 4C). Third, the refractive errors were not statistically different from those of the Vehicle-fed birds (day 11, LSD, p ¼ 0.1; total change: RA, þ0.3 D, Vehicle, 1.1 D, p ¼ 0.09), and if anything, were relatively hyperopic (Fig. 6C) RA and spectacle lenses We asked whether the RA-induced elongation changes interact with those that occur as a result of spectacle lenswear. We found that these two effects were simply added together, and that the refractive error was determined solely by the lens power worn. To make this comparison, we needed the RA and lens-wear to be active at the same time. We found that the progressive doses of RA were effective in increasing ocular length on each of the 3 days of lens wear. Specifically, the eye which did not wear a lens (fellow eye) of birds fed RA elongated significantly more than in those fed Vehicle (Dunnett comparison after two-way RM ANOVA: þ6 D, p < 0.001; 6 D, p < 0.01). The average rate of elongation

9 S.A. McFadden et al. / Experimental Eye Research 83 (2006) 949e Fig. 6. Effect of darkness on response to feeding RA twice daily on A) ocular length; B) lens thickness; and C) refractive errors of chicks fed either 24 mg/kg RA or Vehicle twice daily and raised in total darkness in Experiment A. Measures were taken before (d 8) and after 4 days of RA or Vehicle administration (d 11). *p < 0.0, **p < 0.01, only p values for Day 11 indicated (LSD after RM ANOVA on raw data), error bars ¼ SEM. in the fellow-eyes of the Vehicle-fed birds was normal (77 mm/ day, Nickla et al., 1998) while those fed RA more than doubled their elongation rate on each of the second and third days of progressive dosing (by 86 and 83 mm more than the Vehicle controls respectively). (a) Refractive error (RE). All eyes were approximately emmetropic before lenses were worn at 8 days of age ( D, n ¼ 48). The fellow eyes of chicks fed Vehicle did not change over the 3 days of lens-wear (shift of þ0.3 D), similar to that in untreated lens-wearing birds (Winawer and Wallman, 2002). The non-lens-wearing eyes of birds fed RA also remained emmetropic (Fig. 7A). Eyes which wore lenses compensated for the power of the lens worn, whether they were fed RA or Vehicle. There was no significant difference in RE between these two groups either for the change over the 3 days of lens-wear (Fig. 7A) or for the RE at the end of the lens-wear period (þ6 D lens: RA, þ D vs. Vehicle, þ D; 6 D lens: RA, D vs. Vehicle, D; p > 0.0 in both cases). The effect of the lenses was slightly stronger in birds also fed RA, but not significantly so (RE difference between the lens-wearing eye relative to its fellow eye: þ6 D lens; RA, þ D vs. Vehicle, þ. 1.8 D; 6 D lens; RA, D vs. Vehicle, D; p > 0.0 in both cases). Thus, eyes which wore þ6 D spectacle lenses became hyperopic by approximately 6 D, while those that wore 6 D lenses became approximately 6 D myopic. (b) Ocular elongation. The effects of RA and lens-wear were independent. Wearing 6 D lenses increased the ocular elongation over 3 days by 87 mm (Fig. 7B, Vehicle eyes), Fig. 7. Effect of feeding RA combined with wearing either þ6 D (top row) or 6 D spectacle lenses (bottom row). Changes over 3 days in A) refractive error; B) ocular length; C) vitreous chamber depth; and D) crystalline lens thickness of chicks fed RA (left panels) or Vehicle (right panels). The fellow eye did not wear a lens. The additive effects of RA and lens-wear are demonstrated by the arrows in C, bottom panel, in which the filled arrow shows the effect of RA alone and unfilled arrows show the effect of the lens alone). *p < 0.0, **p < 0.01, ***p < 0.001, p values compare groups joined by solid lines (LSD after RM ANOVA), Error bars ¼ SD.

10 98 S.A. McFadden et al. / Experimental Eye Research 83 (2006) 949e961 RA by itself increased it by 187 mm (RA and Vehicle fellow eyes compared), and RA with 6 D lenses increased it by 286 mm, nearly a perfect addition. Similarly, with the þ6 D lenses, the inhibition of the lens effect was subtracted from the RA stimulation of elongation (Fig. 7B). In this case, after one day, the lenses had not yet exerted their effect so that the eyes of chicks fed RA were growing 100 mm/day faster than those fed Vehicle. However, by the next day, the effect of wearing positive lenses had reduced ocular elongation by similar amounts in chicks fed RA or Vehicle (RA, 60 mm/day; Vehicle, 44 mm/day; data not shown). (c) Vitreous chamber depth. Simultaneous RA and 6 D lens-wear increased vitreous depth by 183 mm/day, while that in the fellow eyes of the birds fed Vehicle increased by 32 mm/day, slightly less than that found in normal chicks of the same strain (44 mm/day) (Nickla et al., 1998). Eyes treated with both RA and 6 D lenses increased their vitreous chamber by 467 mm more than in fellow eyes of chicks fed Vehicle over the 3 days (Fig. 7C, bottom panel), being the sum of the effect of the lens alone (10 mm, dashed arrow) and RA alone (32 mm, solid arrow). Similarly, the reduction in elongation caused by wearing a þ6 D lens subtracted from the RAinduced increase in vitreous depth (Fig. 7C, top panel). (d) Crystalline lens. Feeding RA caused the crystalline lens to thicken less, whether or not the eye was also wearing a spectacle lens (Fig. 7D). Wearing a þ6 D lens without RA significantly reduced lens thickness (Fig. 7D, top panel, p < 0.01), whereas wearing a 6 D spectacle lens did not. Thus positive lens wear alone also induces inhibition in crystalline lens thickening. The effects of combined RA and lens-wear on crystalline lens thickness were additive. (e) Choroid. The response of the choroid to RA combined with a positive spectacle lens was also additive, but transient and restricted to the first 24 h (lens alone, þ172 mm increase; RA alone, þ8 mm; both combined, þ234 mm). In contrast, the response to wearing a negative lens was similar, with or without RA (with, 9 mm; without 104 mm). 4. Discussion Because a number of studies have shown that RA synthesis is altered when eyes are induced to grow towards myopia or hyperopia, one might expect that feeding RA would either do nothing, if RA were simply a correlate, but not a cause, of the altered refractive errors, or else that it would cause growth toward myopia (increased RA in the retina is associated with growth toward myopia) or growth toward hyperopia (increased RA in the choroid is associated with growth toward hyperopia). Instead, we report a puzzling trio of results. First, feeding RA causes large increases in eye length without changes in the refractive status of the eye even if the animals were given the RA while kept in darkness. Furthermore, the acceleration of ocular elongation does not interfere with normal refractive compensation to positive or negative lenses. Thus, whatever the action of RA (whether specific or toxic), it acts at a level where changes in the eye size are integrated with changes in the components to permit the eye to become larger without changes in refractive status. Second, when RA was fed, eyes elongated within six hours, and the normal thickening of the lens was inhibited. The rapidity of the elongation is surprising, given that ocular growth usually takes several days (Kee et al., 2001). Nevertheless, RA normally acts by modulating transcription and transcriptional regulation is rapid. Third, after a day, the rate of elongation fell to less than normal and the choroid thinned. This could have occurred either as a result of a fundamentally biphasic action of RA or as a result of a compensation for the increases in eye-length that occurred during the first day Phase 1: Immediate stimulation of ocular elongation The acceleration of ocular elongation after feeding RA differs from the ocular growth caused by form-deprivation or wearing negative lenses in that it is more rapid and greater in magnitude, and that it is accompanied by three changes (a thickening of the choroid, a flattening of the cornea and a reduced thickening of the lens) that are usually produced by visual conditions that slow ocular elongation, for example, wearing positive lenses. The rate of elongation also increases substantially and rapidly in guinea pigs after feeding RA (McFadden et al., 2004). The extreme rapidity of the onset of accelerated elongation gives one pause. We report here a substantial increase in ocular elongation 6 h after feeding RA, and significant increases at 2 h have also been reported (Sheng and Wallman, 200). Most changes in eye growth produced by wearing spectacle lenses generally take days (Kee et al., 2001), presumably because remodeling of the extracellular matrix and addition of new material must take place. Although the magnitude of the ocular elongation we find after feeding RA is greater than that associated with wearing negative lenses or after form deprivation, the doses of RA that we used were quite high. In our experiments, the smallest effective elongation induced in the first day was more than 4 times control rates, and this occurred with a dose of 8 mg/kg of RA twice daily (Fig. A). A single dose of 8 mg/kg increased the level of RA in retinal tissue more than 66 times 8 h later (Fig. 1B). This is much higher than reported levels of RA in the chick retina after visual manipulations (Mertz et al., 1999; McFadden et al., 2004; Seko et al., 1998), although in these studies, RA levels were determined after several days of visual manipulation had elapsed. The steep RA dose-response function suggests that a threshold may exist for affecting ocular elongation. Cultured bovine RPE cells increase their RA metabolism as a function of concentration up to a maximum level (Doyle et al., 199). Perhaps in the chick similar processes may prevent accumulation of RA in the RPE/retina when fed below a threshold dose Phase 2: Inhibition of ocular elongation When chicks were fed RA only once, the acceleration of ocular elongation was followed by an inhibition the following day (Fig. 3B); when RA was fed repeatedly only on alternate

11 S.A. McFadden et al. / Experimental Eye Research 83 (2006) 949e days, the strong immediate stimulation of ocular elongation was followed a day later by an inhibition of ocular elongation (Fig. 3D). When normal chicks wear positive lenses, ocular elongation is also inhibited after a delay of a day or two (Kee et al., 2001), perhaps in part because of elevated levels of RA in the choroid, which directly inhibits the synthesis of extracellular matrix in the sclera (Mertz and Wallman, 2000) as well as other growth factors secreted by the choroid (Marzani and Wallman, 1997; Rada et al., 2001). We speculate that feeding chicks RA may impose a myopic defocus phenotype on the choroid (the level of RA in the choroid remains over 70 times the endogenous levels for a day after feeding), which in turn results in the inhibition of scleral growth much as though positive lenses were worn. In both the case of the chicks wearing lenses and the chicks fed RA, the delay might be intrinsic to the sclera. We find that even when sclera is treated with RA directly in tissue culture, the inhibition of proteoglycan synthesis is delayed by 10 h (unpublished data). Alternatively, the inhibition might represent an intrinsically biphasic action of RA, either because there are multiple RA receptors whose actions have different time courses or because the retinoic acid receptors are desensitized by the continuous presence of high levels of RA, or because catabolic enzymes that degrade RA are induced at the same time as the RA effects. Similarly, the delay in the inhibitory effect of citral, a competitive inhibitor of the dehydrogenases that convert retinol to RA, might reflect the time required to reduce the activity of the cytosolic dehydrogenases, plentiful in the RPE and neural retina (Drager et al., 1998). Such a mechanism would be consistent with our being able to prevent the inhibition of ocular elongation by progressively increasing the RA dose. Humans chronically treated with RA for acute promyelocytic leukemia rapidly develop increased rates of plasma clearance of RA apparently because of RA inducing its own oxidative catabolism (Muindi et al., 1992). Indeed, there is evidence that the transcription of a principal RA catabolic enzyme, cyp26, is under the control of an RAR receptor (Ozpolat et al., 2002) Differences between the effects of RA and of visual manipulations The changes we see after feeding RA are rather different from those observed after spectacle lens wear. In our experiments, we have increased the level of RA simultaneously in the retina and choroid, while normally after lens wear, changes in the level of RA in the chick are in opposite directions in these two tissues. Perhaps because of this, immediately after the feeding of RA commenced, we saw three changes that would normally never occur together: An increase in the rate of ocular elongation (normally associated with wearing negative lenses), a thickening of the choroid and a cessation of the normal lenticular thickening (both associated with wearing positive lenses). In contrast, when chicks wear negative or positive spectacle lenses, the choroid thins or thickens immediately, followed by changes in the rate of ocular elongation a day or two later (Kee et al., 2001). The delay in these visually evoked changes in ocular elongation are probably not due to a delayed increase in retinal RA. Only six hours of þ7 D lens-wear is required to up-regulate retinal mrna of the RA receptor RAR-b (Bitzer et al., 2000). Furthermore, when chicks wear positive lenses for ten minutes and then are placed in darkness, both the retinal and choroidal RA are changed (in opposite directions) at 6 h (Richiert et al., 2004) Specificity of RA effect Because of the dramatic immediate effects of feeding RA on ocular elongation and because of the high doses used and the steep dose/response curve, one can question whether these are physiological effects or reflect toxicity of the RA. The reasons for not viewing the effects as toxic are: (a) When we fed the RA on alternate days, ocular elongation increased and decreased on alternate days throughout the 11-day regimen; (b) when we fed citral, which inhibits synthesis of RA, we see changes opposite to those caused by RA e it seems unlikely that both are toxicity effects; and (c) the visually mediated compensation for positive and negative spectacle lenses occurs despite the feeding of RA. Therefore, it seems unlikely that the effects we observe are substantially the manifestations of toxic reactions. However, even were it the case that the RA effects were due to toxicity, it would still be quite interesting that one can make a chemical lesion that causes such tightly coordinated changes in the ocular components such that the eyes maintain their emmetropia. 4.. Is the coordination of ocular components a secondary effect? An alternate explanation for the maintenance of emmetropia despite the enormous acceleration of ocular elongation caused by feeding RA is that RA caused only a change in ocular elongation, with the other changes being visually mediated homeostatic responses to maintain emmetropia. The evidence does not support this view either. Both the choroidal and lenticular changes were present even if chicks were raised in darkness, implying that visually guided homeostasis is not necessary. In fact, whether chicks were fed RA in the light or the dark, the refractive status of the eyes showed very little change over several days, remaining approximately emmetropic despite their substantially increased ocular length. If the RA-fed chicks had only increased their ocular length, the refractive error would have changed by approximately 2 D/ day (Schaeffel and Howland, 1988; Wallman et al., 199). Furthermore, when negative lenses were worn by chicks fed RA, the eye elongated even more, as though the visual response is independent of the increased ocular elongation caused by the exogenous RA. Alternatively, the choroidal and lenticular changes might arise in response to the increased ocular elongation from homeostatic mechanisms that are non-visual (Wallman and Winawer, 2004). However, even doses of RA too low to cause changes in vitreous chamber or ocular length cause changes in both the lens and choroid (Fig., 2 mg/kg).

12 960 S.A. McFadden et al. / Experimental Eye Research 83 (2006) 949e961 Therefore, we tentatively conclude that, surprising as it may seem, the fed RA results in coordinated changes in ocular length and in the eye s focal length thereby maintaining emmetropia despite the enlarged eye. This may be accomplished by the RA causing a decrease in the curvature of the cornea (Napier and Mertz, 2003) and causing the lens to be thinner than normal (Fig. 4C), thus compensating for the increased ocular elongation. It has been suggested that the aqueous humor may supply retinoic acid to the lens epithelial cells in human eyes (Wakabayashi et al., 1994). If the same is true for the chick, then RA could be exerting a direct effect upon the crystalline lens and cornea. Although such an explanation is difficult to refute, it strains credulity to imagine that such direct effects on separate tissues would cause an exactly balanced effect on the refractive status so that emmetropia is maintained. Even when chicks wore defocusing lenses while being fed RA, the lens-wearing eyes compensated for the imposed changes to their effective focal length, despite the greatly accelerated ocular elongation upon which they were superimposed. We prefer to argue that there exists a stage at which the growth of the eye is intrinsically coordinated, maintaining the balance between increases in the focal length and increases in the physical length. RA is well known to play an important role within other growth contexts, for example, during early CNS development, RA acts together with Hedgehog signalling and fibroblast growth factor, to determine the patterning of the ventral axis of the eye (Brent, 200), and in mesoderm segmentation, RA is essential for the establishment of the left-right symmetry of somite formation (Giuseppe et al., 2006). Our results suggest that during ocular development, RA also acts upon some intrinsic homeostatic mechanism which coordinates the growth of different ocular structures independent of visual input. In summary, feeding chicks RA resulted in a dramatic increase in ocular length caused by a surprisingly rapid transient increase in the rate of ocular elongation, which was accompanied by a thickening of the choroid and reduced thickening of the lens. This was followed by a rebound inhibition in ocular elongation, as if RA had imposed a myopic defocus phenotype on the choroid. Because the abnormal increases in ocular length were accompanied by changes in the eye s optics resulting in no change in RE, even in darkness, we conclude that the growth of the various ocular components is tightly coordinated and RA may act at the level of a non-visual mechanism which regulates ocular growth. Acknowledgements This work was supported by NIH EY and RR03060 and by the Australian Research Council. 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