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Effect of duration, and temporal modulation, of monochromatic light on emmetropization in chicks
Vision Research 166 (2020) 12–19
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Effect of duration, and temporal modulation, of monochromatic light on emmetropization in chicks Gregory Lin, Christopher Taylor, Frances Rucker
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New England College of Optometry, Dept. of Biomedical Science, 424 Beacon St., Boston, MA 0211, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Emmetropization Myopia Monochromatic light Temporal Age
Previous experiments disagree on the effect of monochromatic light on emmetropization. Some species respond to wavelength defocus created by longitudinal chromatic aberration and become more myopic in monochromatic red light and more hyperopic in monochromatic blue light, while other species do not. Using the chicken model, we studied the effect of the duration of light exposure, modes of lighting, and circadian interruption on emmetropization in monochromatic light. To achieve this goal, we exposed one-week-old chicks to flickering or steady monochromatic red or blue light for a short (10 days) or long (17 days) duration; other chicks were exposed to white light for 10 days. Refraction and ocular biometry were measured. Activity was measured via a motion detection algorithm and an IR camera. The results showed that in both steady and flickering light, there was a greater increase in axial length and vitreous chamber depth in chicks exposed to red or white light compared to chicks exposed to blue light. With a longer duration of exposure, axial length and vitreous chamber depth differences were no longer observed, except at an intermediate time point. Chicks exposed to red light were more active during the day compared to chicks exposed to blue light. We conclude that our results indicate that with short duration monochromatic light exposure, chicks rely on wavelength defocus to guide emmetropization. With longer exposure from hatching, our results support the notion that responses to wavelength defocus can be transient and that the difference between species may be due to differences in experimental duration and/or interference with circadian activity rhythms.
1. Introduction
(1996), chick (Rucker & Wallman, 2008; Torii, Kurihara et al., 2017; Wang, Schaeffel et al., 2018), guinea pigs (Long, Chen, & Chu, 2009; Liu et al., 2011; Wang, Zhou, Lu, & Chu, 2011; Jiang et al., 2014; Zou et al., 2018) and rhesus monkeys (Liu et al. (2014)). Other experiments have shown unexpected differences in the emmetropization response in monochromatic light. Smith et al. (2015) found that rhesus monkeys who wore long-wavelength filters as lenses for 120 days became more hyperopic than controls that did not wear filtering lenses. Gawne, Siegwart, Ward, and Norton (2017) found that infant tree shrews (22 days old) remained hyperopic after exposure to red light for 13 days, whereas exposure to blue light caused tree shrews to become slightly more myopic. Juvenile and adolescent tree shrews showed similar effects (Gawne, Ward, & Norton, 2017). Thus, both tree shrews and rhesus monkeys respond in an unexplained manner to wavelength defocus. One of the differences between tree shrew and rhesus monkey experiments and others mentioned above was the incorporation of a pseudo-random flickering light that contained a broad range of temporal frequencies (Gawne, Siegwart et al., 2017; Gawne, Ward, &
Evidence suggests that longitudinal chromatic aberration (LCA) provides signals for guiding emmetropization. LCA arises from dispersion, which causes different wavelengths of light to converge at different focal planes. As a result of LCA, longer wavelengths have a longer focal length than shorter wavelengths. Recent experiments have demonstrated species specific emmetropization responses to monochromatic light exposure and the aim of this experiment is to determine whether protocol differences, including flicker and duration of exposure with circadian interruption can explain these differences. In many species the eye can respond appropriately to wavelength defocus induced by LCA. Seidemann and Schaeffel (2002) and Foulds, Barathi, and Luu (2013) observed that chicks reared in short-wavelength, blue light (430 nm), became more hyperopic with decreased axial growth, while chicks reared in long-wavelength, red light (615 nm), became myopic with increased axial growth. A similar hyperopic shift in response to short-wavelength light exposure has been demonstrated in several species (Cichlid fish (Kröger and Wagner
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Corresponding author. E-mail address: [email protected] (F. Rucker).
https://doi.org/10.1016/j.visres.2019.11.002 Received 1 August 2019; Received in revised form 18 October 2019; Accepted 3 November 2019 0042-6989/ © 2019 Elsevier Ltd. All rights reserved.
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2.3. Lighting and flicker conditions
Norton, 2018). In chick, mice and cat, emmetropization has shown a strong temporal frequency dependence. Hyperopic shifts in refraction were seen at high temporal frequencies (Schwahn & Schaeffel, 1997; Crewther & Crewther, 2002; Zhi, Pan et al., 2013; Rucker, Britton et al., 2015; Rucker, Britton et al., 2018; Tian, Zou, Wu, Liu, & Liu, 2019) and myopic shifts at low temporal frequencies (Cremieux, Orban, Duysens, Amblard, & Kennedy, 1989; Crewther & Crewther, 2002; Crewther, Barutchu, Murphy, & Crewther, 2006; Yu, Chen, Tuo, & Zhu, 2011; Rucker, Britton et al., 2015; Rucker, Britton et al., 2018; Murphy, Riddell, Crewther, Simpson, & Crewther, 2019; Tian et al., 2019). Further study is required to clarify the temporal effects of flicker on emmetropization. Another difference between studies was the duration of exposure to monochromatic light and its potential effect on circadian rhythms. Intrinsically photosensitive retinal ganglion cells (iPRGCs), associated with the master circadian clock, respond to light through stimulation of melanopsin in the cell membranes (Berson, Dunn, & Takao, 2002; Hattar, Liao, Takao, Berson, & Yau, 2002) and also to intra-retinal synaptic input from rod and cone driven circuits (Perez-Leon, Warren, Allen, Robinson, & Brown, 2006; Weng, Estevez, & Berson, 2013; Zhao, Stafford, Godin, King, & Wong, 2014). Melanopsin has a peak spectral sensitivity around 480 nm (Panda et al., 2005; Torii, Kojima et al., 2017) and would be minimally stimulated by narrow-band long-wavelength light (626 nm), the wavelength used in the tree shrew experiments (Gawne, Siegwart et al., 2017; Gawne, Ward, et al., 2017) while stimulation would be maintained with short-wavelength light. In this study, we aim to estimate the effect of the duration of exposure to monochromatic light on emmetropization and to examine the effect of monochromatic light exposure on circadian activity rhythms. Lastly, we will use temporally broadband flicker to determine if temporal stimulation interacts with the spectrum of the light to produce variable emmetropization.
Chicks were housed in 48x79x40 cm cages with a maximum of 10 chicks in each cage. Eight strips of RGB LEDS (Superbright NFLSRGBX2) were attached to the roof of the cage. The LED strips were 0.5 m long and were paired together. Each pair was evenly spaced 10 cm from each other. LEDs were controlled via a Raspberry Pi and controlled via custom software written in Python. The strips were set to monochromatic red (628 ± 10 nm) or blue (464 ± 10 nm) light or a broadband white light and produced steady or flickering light depending on the condition. 2.4. Exposure conditions Red and blue lighting conditions were matched to Gawne et al. (2017) conditions both in their spectra and their temporal flicker. In the flicker conditions, lights were set to pseudo-random flicker that contained a broad range of temporal frequencies, flickering over a range of random luminances (4–96% of the maximum luminance) during the day time. Average illuminance was 424 lx for all conditions and was measured with Dr. Meter LX1330B light meter. Mean power (Newport Power Meter) was 266 µW for Blue and 116.2 µW for Red. Luminance (Spectrascan PR670), measured via a reflecting plate placed at the bottom of the cage, was 84 cd/m2 for Blue and 114 cd/m2 for Red. The mean illumination of the flicker conditions differed from Gawne et al. (2017) whose average illuminance varied across time during the wavelength and flicker conditions. In this experiment we aimed to match the mean illuminance across the six conditions (3 color conditions × 2 flicker conditions). The lighting conditions were on a 12 h/12 h light/ dark cycle. 2.5. Short duration condition To compare the effects of monochromatic light and flicker on emmetropization, one-week-old chicks were placed into one of six conditions for a short duration (10 days). The bird numbers in each condition were: (1) Blue Steady (n = 6), (2) Blue Flicker (n = 6), (3) Red Steady (n = 5), (4) Red Flicker (n = 5), (5) White Steady (n = 4), and (6) White Flicker (n = 6). Measurements were made on Day 1, 3, 6, 8, and 10 after initial exposure and the data were collected between 11am and 1 pm. Day 1 in Fig. 1 represents the baseline measurement at 7 days old. Ocular biometry and refractive error were measured again on Day 3, to compare with 10-day-old birds exposed for 10 days in the long duration condition.
2. Methods 2.1. Animals Chicks (White leghorn, K strain, Cornell University, Ithaca, NY) were chosen as a model organism because chick eyes grow rapidly and are easily manipulated in experimental conditions (Schaeffel & Feldkaemper, 2015; Wisely et al., 2017). Chicks had access to food and water ad libitum. Use of animals in this study was in compliance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the NECO Institutional Animal Care and Use Committee.
2.6. Long duration condition 16 chicks were hatched in incubators and then transferred immediately to the lighting condition cages. Chicks were placed under a Steady Blue (n = 10) or Steady Red (n = 6) light. In Red, the data from 3 birds was lost as a result of a Lenstar data export error and 1 newly hatched chick died. To examine the possibility of transient duration effects, we compared the measurements from age-matched 10 day-old chicks on Day 3 of the short duration exposure condition (Suppl.
2.2. Hormonal influences In chicks, puberty is generally not reached until 18–21 weeks of age. Given the age of the chicks used, puberty was not a factor in the difference in results between the long and short duration experiments.
Fig. 1. Timeline of the light exposure and measurements for both experiments. The blue colored bar represents when chicks were exposed to the lighting conditions for a short duration. Measurement days are marked on the timeline above. The green colored bar represents when chicks were exposed for a long duration. PREand POST-measurements were made at Days 10 and 17. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 13
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normalized by the number of regions of motion detected. The camera, using night vision mode (using IR), could capture motion during the night. Very little activity was captured during this period (8PM to 8AM) in all the groups. Thus, only the daytime period (8AM to 8PM) was analyzed. 2.10. Circadian activity analysis To equate recording time across days, only the data from the days when no refraction or ocular biometry measurements were collected was analyzed (Days 2, 4, 5, 7, 8, and 9). Our measure of activity is expected to increase with time because the algorithm will detect more pixel changes as the larger birds move within the cage. To statistically control for this effect, analyses was therefore done within each Day. A non-parametric Kruskal Wallis one-way ANOVA of the activity metric was performed. SPSS (version 24, IBM) statistical software was used for analysis.
Fig. 2. Axial length measurements taken after a short duration, or long duration, of exposure to red and blue monochromatic lights and white light. Birds exposed for a short duration in steady light are shown as red, blue and white circles that correspond to the color of the illuminant. Those reared in flicker are shown by diamonds. Birds exposed for a long duration are shown as stars in the corresponding color. Standard error bars are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.11. Component and refraction analysis Refractions and ocular component measurements of both the left and right eyes were averaged. In the short duration condition, a linear model was fit to the data from each measurement day. The primary outcome measure was the slope of the linear regression multiplied by 10 to represent the total change in refraction or the ocular component across 10 days of light exposure. In the long duration condition, the change in the pre- and post-measures were analyzed. In the short duration condition, a 2-way ANOVA with variables of Color and Flicker was computed. Tukey post-hoc t-tests were used to determine if there were differences among individual conditions for each component. Levene’s Test of Equality of Error Variances, based on the median, was run to test for homogeneity of the data and a ShapiroWalk test was run to test for normality. SPSS (version 24, IBM) statistical software was used.
Table 1) and Day 10 of the long duration exposure condition (Suppl. Table 3). The effects of a longer duration of exposure to monochromatic light were assessed via the change in refraction and ocular components between Day 10 and Day 17. No measurements were taken prior to exposure given how fragile the birds are at hatch. The timeline for the measurements can be seen in Fig. 1. 2.7. Eye measurements Refraction and eye measurements were performed on each eye at the same time of day using lid retractors. During measurement, chicks were anesthetized with 1.5% isoflurane gas in 2% oxygen. Refraction was performed using an infrared photorefractometer as described by (Schaeffel, Farkas, & Howland, 1987). Refraction was measured at a distance of about 1 m with a sampling frequency of 62 Hz. The infrared refractometer setup consisted of a camera (IC Imaging Control, Charlotte, NC) with a Pentax 75 mm f/1.4 lens and IR LEDs mounted on the bottom half of the lens. Measurements of the light gradient in the pupil, in the 90 and 180 degree meridians were converted to dioptric values using a predetermined calibration. The mean refraction and standard deviation were calculated from the winsorized mean of 650 samples. Axial length, choroidal thickness, vitreous chamber depth, lens thickness, and anterior chamber depth were measured with a Lenstar LS-900 (Haag-Streit, USA). Five A-scan measurements were collected each measurement day, with each measurement being an average of 16 traces. The peaks in the A-scans corresponding to the ocular biometry were marked via custom software.
3. Results 3.1. Short duration condition Absolute measures of axial length are shown in Fig. 2. The ocular biometry change data for each treatment group is summarized in Fig. 3 and Suppl. Table 2. 3.1.1. Refractive error There were no differences in refractive changes among the light conditions when temporal conditions were pooled (Fig. 3A: Levene’s Test: L = 0.064, p = 0.99; F = 1.74, p = 0.20). Refractive errors were normally distributed with Shapiro-Wilk’s test within each colored condition: Blue (p = 0.43), Red (p = 0.87), White (p = 0.56). Birds exposed to blue light showed a mean change in refractive error of −0.82 ± 0.81 D, while birds in red light showed a mean change of −0.31 ± 0.95 D, and birds in white light showed a change of + 0.44 D ± 0.99 D, over 10 days. Birds in Red and Blue steady light showed no difference in refractive error (p = 0.59). There were no differences in refractive changes between the steady and flicker conditions (Fig. 3B: F = 0.697, p = 0.411). On average, refractive error changed by only −0.08 ± 0.98 D with flicker, while in steady light conditions, refraction changed by only +0.12 ± 0.90 D over 10 days.
2.8. Circadian activity To measure whether circadian rhythms are influenced by the spectral composition, the activity of the chicks was recorded in the short duration Red, Blue and White, flicker conditions. Chick movement was recorded continuously with an infrared video camera throughout the experiment. The monitoring system was paused when the refraction and ocular biometry were measured and during cage cleaning.
3.1.2. Axial length Mean changes in axial length by condition are shown in Fig. 3C and D. Fig. 3C shows the changes in axial length when flicker and steady light conditions are pooled. There was less axial growth when chicks were raised in blue light compared to red and white light (Levene’s Test: L = 0.552, p = 0.55; F = 10.18, p = 0.001). Axial Length
2.9. Circadian activity analysis Activity was recorded via custom motion capture software that computed the area of motion (in pixels/second) and the number of regions in the image via a keyframe algorithm. To equate the changes over each one-minute period, the raw activity in pixels/second was 14
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Fig. 3. Short duration: Mean change in each ocular component calculated over 10 days with treatment starting at 7 days old. Refractive error: A. Pooled and grouped by color. B. Grouped by color and temporal condition. Steady light is represented by diagonally striped bars, flicker by plain bars. Axial length: C. Pooled and grouped by color. D. Grouped by color and temporal condition as in B. Choroid thickness: E. Pooled and grouped by color. F. Grouped by color and temporal condition as in B. Horizontal line in the box indicates the median. Boundaries of the box indicate the 25th and 75th percentile. The whiskers represent the high and low values 1.5x the interquartile range. Dots represent outliers.
Wilks test within each color condition: Blue (p = 0.96), Red (p = 0.67), White (p = 0.20). On average, choroidal thickness increased by only + 0.018 ± 0.039 mm in Blue, +0.012 ± 0.029 mm in Red, and + 0.028 ± 0.036 mm in White light over 10 days. Fig. 3F shows no differences between the flicker conditions for choroidal thickness changes (F = 0.469, p = 0.50). On average, choroidal thickness increased by 0.024 ± 0.033 mm with Flicker and 0.015 ± 0.033 mm with Steady light. There was no interaction between the color and flicker conditions.
measures were normally distributed with Shapiro-Wilks test within each color condition: Blue (p = 0.48), Red (p = 0.57), White (p = 0.68). Tukey HSD post-hoc test showed that axial changes in the Blue and Red conditions differed (p < 0.01) and that Blue and White conditions differed (p = 0.001). On average, the axial length of the chicks raised in Blue increased by 0.945 ± 0.055 mm, less than the axial length increase of 1.089 ± 0.075 mm in Red, and 1.114 ± 0.011 mm in White. As Fig. 3D shows, there was less axial growth in chicks reared in flickering than steady light, irrespective of the light condition (F = 6.176; p < 0.05). In steady light, axial length increased by 1.086 ± 0.108 mm, while with flicker, axial length increased by 1.00 ± 0.120 mm over 10 days. There was no interaction between the light and flicker condition (F = 1.45; p = 0.26).
3.2. Intermediate Duration 3.2.1. Refractive error Overall, in 10-day old, age-matched chicks, the refractions were more hyperopic in Blue than in Red (Fig. 4A: F = 47.08; p = 0.01). The difference in refractive error in Red and Blue Steady lighting was greater after a shorter duration of exposure (Blue-Red = 2.84D) than a longer duration of exposure (Blue-Red = 1.42D) (Levene’s L = 2.06, p = 0.14; F = 5.21: p < 0.05). This result indicates a transient response to wavelength defocus.
3.1.3. Choroid Choroidal changes in each illumination and flicker condition are shown in Fig. 3E and F. The lighting condition did not affect choroidal thickness (Levene’s Test: L = 0.587, p = 0.71: F = 0.266, p = 0.77). Choroidal measurements were normally distributed with the Shapiro15
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Fig. 4. Absolute refractive and axial measures for age-matched, 10 day-old chicks, in the short and long duration conditions after exposure to steady colored illuminants for 3 or 10 days, respectively. A. Overall, refractions were more hyperopic in Blue than in Red. There was a greater difference in refractive error in red and blue light in the short duration condition as shown by the bar extending between the duration conditions. B. Axial lengths were longer in the long duration condition. In the long duration condition, the axial length in red light was longer than in blue light. C. There was no difference in choroidal thickness. A p-value of p < 0.05 is represent by *, a p-value of p < 0.01 by **. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
refractive errors were normally distributed in Blue (p = 0.80) and Red (p = 0.22) conditions.
3.2.2. Axial length At 10 days old, with pooled color data, chicks had shorter axial lengths (9.105 ± 0.114 mm) after a short duration of exposure than a long duration (9.260 ± 0.210 mm) exposure (Fig. 4B: Levene’s L = 0.684, p = 0.57; F = 12.9, P = 0.002). Examining each condition separately, at 10-days old there was no difference in axial length between Blue and Red after a short exposure (F = 3.031, p = 0.12), but axial length changed more in Red than in Blue after a long exposure (F = 14.334, p = 0.003). At this intermediate time point, a longer duration of exposure to monochromatic light increases eye growth, particularly in Red.
3.3.2. Axial length Between 10 and 17 days of exposure, there were no further differences in axial length changes between the two illumination conditions (Fig. 5B: Levene’s Test: L = 0.011, p = 0.92; t = 0.259, p = 0.62). In Blue, the change in axial length was + 0.898 ± 0.089 mm from Day 10 to Day 17, while in Red, the average change in axial length was + 0.871 ± 0.103 mm. Shapiro-Wilks test shows that changes in axial length measures were normally distributed in Blue (p = 0.15) and Red (p = 0.45) conditions.
3.2.3. Choroid At 10 days-old, there was no difference in choroidal thickness in Red and Blue (Fig. 4C: Levene’s L = 1.03p = 0.40; F < 0.001, p = 0.99) and no difference with exposure duration (F = 0.787, p = 0.39).
3.3.3. Choroid As Fig. 5C shows, after a total exposure duration of 17 days, choroidal thickness increased in Red compared to Blue (Levene’s Test: L = 0.312, p = 0.59; t = 13.62, p < 0.01). In Blue, choroids thickened + 0.003 ± 0.268 mm, and in Red, +0.054 ± 0.021 mm. Shapiro-Wilks test shows that choroid measures were normally distributed in Blue (p = 0.31) and Red (p = 0.26) conditions.
3.3. Long duration condition Increasing the exposure duration from 10 to 17 days did not reproduce or enhance the changes in ocular biometry observed after a short duration exposure. Change values can be seen in Fig. 5 and Suppl. Table 4 and can be calculated from pre-and post-measurement values in Suppl. Table 3.
3.3.4. Weight There was no difference in weight gain in the chicks between the lighting conditions (t = 0.224, p = 0.64). Chicks raised in Blue weighed on average 51.92 ± 2.6 g on Day 10 and 132.0 ± 9.2 g on Day 17. Chicks raised in Red weighed on average 53.6 ± 1.96 g on Day 10 and 135.6 ± 7.3 g on Day 17.
3.3.1. Refraction Fig. 5A shows that after an additional 7 days of exposure when chicks were 17 days-old, there were no further differences in refractive error changes between the two illumination conditions (Levene’s Test: L = 3.042, p = 0.11; t = 2.48, p = 0.14). In Blue, the change in refractive error was −0.69 ± 1.10 D from Day 10 to Day 17 and in Red it was + 0.12 ± 0.39 D. Shapiro-Wilks test shows that the changes in
3.4. Activity There was very minimal activity at night with no significant differences between the groups (F = 1.91; p = 0.57). Fig. 6 shows values 16
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Fig. 5. Long duration: Changes in ocular measurements after 17 days of exposure to Blue or Red steady light from hatching. A. Changes in refractive error. B. Changes in axial length. C. Changes in choroidal thickness. Red exposure resulted in thicker choroids than Blue. Asterisks represent a p-value < 0.01. Graphical design as in Fig. 3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
White, but increased activity in Red Flicker versus Blue and White Flicker (all comparisons, p < 0.01). For all days, activity was measured to be 482.8 ± 170 Δpixels/min for chicks raised in Blue Flicker, 622.4 ± 165 Δpixels/min for Red Flicker, and 426.8 ± 135 Δpixels/ min for White Flicker. There was no difference in activity between the morning and afternoon Blue Flicker (F = 0.63, p = 0.44), Red Flicker (F = 0.263, p = 0.61), or White Flicker (F = 2.95, p = 0.1).
4. Discussion This experiment examined the effects of exposure duration and flicker on emmetropization when chicks were exposed to monochromatic light. We also examined the possibility that monochromatic light interferes with circadian activity rhythms. With a short duration exposure, we found that monochromatic red or broadband white light led to more axial growth than monochromatic blue light, yet there was no refractive difference. It is possible the lack of refractive effect may be due to differences in corneal changes in red and blue light (Rucker, Britton et al., 2015) but these were not measured. The axial results concurred with the anticipated response to wavelength defocus for emmetropization. A longer duration of exposure did not reproduce the difference in axial growth seen with the short duration of exposure, but the eye was already longer in Red than Blue by Day 10, and further changes in refraction and axial length were small. Prolonged exposure to monochromatic light had the effect of increasing growth stimulation, particularly in red light, as seen at the Day 10 intermediate time point. We speculate that this large increase in axial growth is due to circadian interference, as suggested by the increase in activity patterns in Red compared to Blue. In support of this hypothesis, Wang, Schaeffel et al. (2018) demonstrated a minimal change in retinal dopamine levels in chick with exposure to 620 nm red light, with a trend towards higher retinal dopamine release in UV light compared to white light. Presuming these changes in retinal dopamine are related to stimulation of the circadian system, it is notable that
Fig. 6. Box plot of Activity. Horizontal line in the box indicates the median. Boundaries of the box indicate the 25th and 75th percentile. The whiskers represent the high and low values 1.5x the interquartile range. Dots represent outliers. The box plot represents activity measured during day time only.
measured during lights on. Overall, the data for the activity measure were not normally distributed, so the non-parametric Kruskal-Wallis test was used for analysis. On Day 2, a difference among the lighting conditions was observed (Levene’s L = 3.753, p = 0.12; χ2(2) = 22.04, p < 0.01). Tukey posthoc tests showed that increased activity was seen in chicks reared in Red (p < 0.001) and Blue Flicker (p < 0.001) compared to White Flicker. Differences among the conditions were observed on Day 4 (Levene’s L = 3.518, p = 0.04; χ2(2) = 33.282, p < 0.01), Day 5 (Levene’s L = 0.074, p = 0.93; χ2(2) = 12.75, p = 0.002), Day 7 (Levene’s L = 1.907, p = 0.16; χ2(2) = 17.79, p < 0.01), Day 8 (Levene’s L = 1.538, p = 0.22; χ2(2) = 16.85p < 0.01), and Day 9 (Levene’s L = 1.335, p = 0.27, χ2(2) = 12.57p = 0.002). Unlike Day 2, these subsequent days did not show increased activity in Blue versus 17
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affect the flicker response. In comparison, Gawne et al. (2017) also found a greater myopic refractive shift in broad-band flickering blue light than in steady blue light, while Gawne et al. (2018) found similar refractive changes in both steady and flickering blue light. When shortwavelength-sensitive cones are stimulated with sinusoidal, rather than broad-band, flicker at a low temporal frequency they are more sensitive than when stimulated with higher temporal frequencies (Smithson & Mollon, 2004). It was thought that the presence of higher temporal frequencies in the pseudo-random noise used in this experiment and Gawne et al. (2017) may have reduced the S-cone response and driven the emmetropization system toward the development of myopia (Rucker, Britton et al., 2015; Rucker, Britton et al., 2018). The color independent reduction in eye growth with broad-band flicker is in agreement with the finding that a color insensitive reduction in eye growth occurs with high frequency luminance flicker (Rucker, Britton et al., 2015). Previous studies have suggested that the use of continuous wear narrow band, binocular, red filters, for prolonged periods may reduce the magnitude of myopic refractions in monkeys. However, if this effect is due to circadian interference then the optical benefits may be overwhelmed by the deleterious effects of circadian rhythm disruption on health, including increased risk of cancer (review: (Feillet, van der Horst, Levi, Rand, & Delaunay, 2015) metabolic changes leading to obesity, mood disorders and cardiovascular disease (Chepesiuk, 2009; Anisimov, Vinogradova, Panchenko, Popovich, & Zabezhinski, 2012, Stevens, Brainard, Blask, Lockley, & Motta, 2013).
changes in circadian rhythms in proteoglycan synthesis in chick have been associated with changes in axial length (Nickla, Rada, & Wallman, 1999). This finding of an increase in growth in red light may partially explain the observation by Foulds et al. (2013) who observed an unusually large difference in vitreous chamber depth between red and blue lighting conditions after 14 days of light exposure from hatch. The difference in choroidal effects with a longer duration of exposure to red light could be attributed to a lack of stimulation of the iPRGCs in red monochromatic light and a shift in the diurnal choroidal rhythms as found in chick, monkey and humans (Nickla, 1996; Nickla, Wildsoet, & Wallman, 1998; Nickla, Wildsoet, & Troilo, 2002; Nickla, 2006; Burfield, Patel, & Ostrin, 2018). In tree shrew, choroidal thickening contributed to the hyperopic refractive effects observed in red monochromatic light in the longer duration experiments (Gawne, Siegwart et al., 2017; Gawne, Ward et al., 2017). In binocularly exposed monkey and tree shrew, reared in red light, hyperopic changes were associated with corresponding changes in vitreous chamber depth (Smith et al., 2013; Smith et al., 2015; Gawne, Siegwart et al., 2017; Gawne, Ward et al., 2017), though axial changes were not noted at any age. Another interesting finding was that monocularly reared monkeys wearing red filters did not show vitreous changes. Monocular rearing would have allowed sustained circadian stimulation. However, measurements of circadian rhythms in the choroid and in retinal and vitreal dopamine and/or DOPAC levels would be required to confirm this hypothesis. Previous experiments have demonstrated that disruption of the circadian rhythms affects choroidal thickness in chicks (Nickla, Wildsoet et al., 1998; Nickla, Rada, et al., 1999) and there are differences in ocular circadian rhythms between emmetropic and myopic humans (Burfield et al., 2018; Burfield, Carkeet, & Ostrin, 2019). There was evidence of a transient effect of monochromatic light on emmetropization, in that when the refractive changes were examined at the Day 10 intermediate time point, there was a greater difference in refractive error between red and blue light conditions after 3 days of exposure compared to 10 days of exposure. This result suggests that the refractive effects of wavelength defocus diminish over time and that there is a critical period where cone stimulation with monochromatic light can alter emmetropization in a manner predicted by wavelength defocus. This is consistent with the transient refractive effects observed in tree shrew. Tree shrews exposed to blue light became hyperopic during the early phase at around 27 days of visual experience before becoming myopic in both steady and flickering light during the late phase (Gawne et al., 2018). The authors suggested that the tree shrew loses its ability to detect the sign of defocus via feedback mechanisms in narrowband blue light. Thus, it is clear that depriving an animal of certain wavelengths for prolonged periods interferes with the emmetropization process. The duration of the exposure to monochromatic light in this experiment differed from that in other experiments (Foulds et al., 2013; Smith et al., 2015; Hung, Arumugam, Sankaridurg, & Smith, 2016; Gawne, Siegwart et al., 2017; Gawne, Ward et al., 2017; Gawne et al., 2018). Foulds et al. (2013) used 1-day old chicks and exposed them to the lighting conditions for up to 28 days. Gawne & Siegwart et al. (2017) exposed tree shrews at 1 month of age. The eyes of the tree shew typically open 3 weeks after birth. Thus, the animals had several days of visual experience (DVE) at the time of exposure, followed by 13 days in the experiment. Rhesus monkey were first exposed to lighting conditions at 25 ± 2 days of age and exposure was continued up to 146 ± 7 days of age (Smith et al., 2013; Smith et al., 2015). Given the different rates of visual and ocular development among species, matching exposure time does not equate the experience required for development. To address this concern, we examined the effect of two different durations of exposure in chick. The primary aim was to use the methods of Gawne, Ward et al. (2017) and Gawne et al. (2017) while studying tree shrew, in the chick model. Our results indicate that emmetropization is sensitive to the broad-band flicker presented, but the color of the light source did not
5. Summary In this study we showed that the duration of exposure to different monochromatic lighting conditions affects refractive changes. Differences in refraction and eye growth found with shorter duration exposures to red and blue monochromatic light, were not observed with longer duration exposures probably because of transient changes in ocular growth in response to wavelength defocus and a general increase in eye growth in monochromatic light. This instability may be due to the influence of monochromatic light on circadian activity rhythms. These results may help to explain the species differences observed in monochromatic light experiments via differences in the experimental protocols or differences in the underlying biology of circadian rhythms, though species differences cannot be excluded. Acknowledgements Research reported in this publication was supported by the National Eye Institute of the National Institutes of Health under Award Number R01EY023281 and by the NIH T35 Program. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.visres.2019.11.002. References Anisimov, V., Vinogradova, I., Panchenko, A., Popovich, I., & Zabezhinski, M. A. (2012). Light-at-night-induced circadian disruption, cancer and aging. Current Aging Science, 5, 170–177. Berson, D. M., Dunn, F. A., & Takao, M. (2002). Phototransduction by retinal ganglion cells that set the circadian clock. Science, 295(5557), 1070–1073. Burfield, H. J., Carkeet, A., & Ostrin, L. A. (2019). Ocular and systemic diurnal rhythms in emmetropic and myopic adults. Investigative Ophthalmology & Visual Science, 60(6), 2237–2247. Burfield, H. J., Patel, N. B., & Ostrin, L. A. (2018). Ocular biometric diurnal rhythms in emmetropic and myopic adults. Investigative Ophthalmology and Visual Science, 59(12), 5176–5187.
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Chepesiuk, R. (2009). Missing the dark: Health effects of light pollution. Environmental Health Perspectives, 117, 20–27. Cremieux, J., Orban, G. A., Duysens, J., Amblard, B., & Kennedy, H. (1989). Experimental myopia in cats reared in stroboscopic illumination. Vision Research, 29(8), 1033–1036. Crewther, S. G., Barutchu, A., Murphy, M. J., & Crewther, D. P. (2006). Low frequency temporal modulation of light promotes a myopic shift in refractive compensation to all spectacle lenses. Experimental Eye Research, 83(2), 322–328. Crewther, D. P., & Crewther, S. G. (2002). Refractive compensation to optical defocus depends on the temporal profile of luminance modulation of the environment. NeuroReport, 13(8), 1029–1032. Feillet, C., van der Horst, G., Levi, F., Rand, D., & Delaunay, F. (2015). Coupling between the circadian clock and dell cycle oscillators: Implication for healthy cells and malignant growth. Frontiers in Neurology, 6, 1–7. Foulds, W. S., Barathi, V. A., & Luu, C. D. (2013). Progressive myopia or hyperopia can be induced in chicks and reversed by manipulation of the chromaticity of ambient light. Investigative Ophthalmology and Visual Science, 54(13), 8004–8012. Gawne, T. J., Siegwart, J. T., Jr., Ward, A. H., & Norton, T. T. (2017). The wavelength composition and temporal modulation of ambient lighting strongly affect refractive development in young tree shrews. Experimental Eye Research, 155, 75–84. Gawne, T. J., Ward, A. H., & Norton, T. T. (2017). Long-wavelength (red) light produces hyperopia in juvenile and adolescent tree shrews. Vision Research, 140, 55–65. Gawne, T. J., Ward, A. H., & Norton, T. T. (2018). Juvenile Tree Shrews Do Not Maintain Emmetropia in Narrow-band Blue Light. Optometry and Vision Science, 95(10), 911–920. Hattar, S., Liao, H. W., Takao, M., Berson, D. M., & Yau, K. W. (2002). Melanopsincontaining retinal ganglion cells: Architecture, projections, and intrinsic photosensitivity. Science, 295(5557), 1065–1070. Hung, L. F., Arumugam, B., Sankaridurg, P., & Smith, E. L. (2016). Effects of Long-wavelength Lighting Combined with Positive Lens Wear on Emmetropization in Young Rhesus Monkeys. IOVS, 57(7), 5526. Jiang, L., Zhang, S., Schaeffel, F., Xiong, S., Zheng, Y., Zhou, X., ... Qu, J. (2014). Interactions of chromatic and lens-induced defocus during visual control of eye growth in guinea pigs (Cavia porcellus). Vision Research, 94, 24–32. Kröger, R. H., & Wagner, H. J. (1996). The eye of the blue acara (Aequidens pulcher, Cichlidae) grows to compensate for defocus due to chromatic aberration. Journal of Comparative Physiology A, 179(6), 837–842. Liu, R., Hu, M., He, J. C., Zhou, X. T., Dai, J. H., Qu, X. M., ... Chu, R. Y. (2014). The effects of monochromatic illumination on early eye development in rhesus monkeys. Investigative Ophthalmology and Visual Science, 55(3), 1901–1909. Liu, R., Qian, Y. F., He, J. C., Hu, M., Zhou, X. T., Dai, J. H., ... Chu, R. Y. (2011). Effects of different monochromatic lights on refractive development and eye growth in guinea pigs. Experimental Eye Research, 92(6), 447–453. Long, Q., Chen, D., & Chu, R. (2009). Illumination with monochromatic long-wavelength light promotes myopic shift and ocular elongation in newborn pigmented guinea pigs. Cutaneous and Ocular Toxicology, 28(4), 176–180. Murphy, M. J., Riddell, N., Crewther, D. P., Simpson, D., & Crewther, S. G. (2019). Temporal whole field sawtooth flicker without a spatial component elicits a myopic shift following optical defocus irrespective of waveform direction in chicks. PeerJournal, 7, e6277. Nickla, D. L. (1996). Diurnal rhythms and eye growth in chicks. City University of New York. Nickla, D. L. (2006). The phase relationships between the diurnal rhythms in axial length and choroidal thickness and the association with ocular growth rate in chicks. Journal of Comparative Physiology A: Sensory, Neural, and Behavioral Physiology, 192(4), 399–407. Nickla, D. L., Rada, J. A., & Wallman, J. (1999). Isolated chick sclera shows a circadian rhythm in proteoglycan synthesis perhaps associated with the rhythm in ocular elongation. Journal of Comparative Physiology A, 185(1), 81–90. Nickla, D. L., Wildsoet, C. F., & Troilo, D. (2002). Diurnal rhythms in intraocular pressure, axial length, and choroidal thickness in a primate model of eye growth, the common marmoset. Investigative Ophthalmology and Visual Science, 43(8), 2519–2528. Nickla, D. L., Wildsoet, C., & Wallman, J. (1998). Visual influences on diurnal rhythms in ocular length and choroidal thickness in chick eyes. Experimental Eye Research, 66(2), 163–181.
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Effect of duration, and temporal modulation, of monochromatic light on emmetropization in chicks Gregory Lin, Christopher Taylor, Frances Rucker
T
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New England College of Optometry, Dept. of Biomedical Science, 424 Beacon St., Boston, MA 0211, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Emmetropization Myopia Monochromatic light Temporal Age
Previous experiments disagree on the effect of monochromatic light on emmetropization. Some species respond to wavelength defocus created by longitudinal chromatic aberration and become more myopic in monochromatic red light and more hyperopic in monochromatic blue light, while other species do not. Using the chicken model, we studied the effect of the duration of light exposure, modes of lighting, and circadian interruption on emmetropization in monochromatic light. To achieve this goal, we exposed one-week-old chicks to flickering or steady monochromatic red or blue light for a short (10 days) or long (17 days) duration; other chicks were exposed to white light for 10 days. Refraction and ocular biometry were measured. Activity was measured via a motion detection algorithm and an IR camera. The results showed that in both steady and flickering light, there was a greater increase in axial length and vitreous chamber depth in chicks exposed to red or white light compared to chicks exposed to blue light. With a longer duration of exposure, axial length and vitreous chamber depth differences were no longer observed, except at an intermediate time point. Chicks exposed to red light were more active during the day compared to chicks exposed to blue light. We conclude that our results indicate that with short duration monochromatic light exposure, chicks rely on wavelength defocus to guide emmetropization. With longer exposure from hatching, our results support the notion that responses to wavelength defocus can be transient and that the difference between species may be due to differences in experimental duration and/or interference with circadian activity rhythms.
1. Introduction
(1996), chick (Rucker & Wallman, 2008; Torii, Kurihara et al., 2017; Wang, Schaeffel et al., 2018), guinea pigs (Long, Chen, & Chu, 2009; Liu et al., 2011; Wang, Zhou, Lu, & Chu, 2011; Jiang et al., 2014; Zou et al., 2018) and rhesus monkeys (Liu et al. (2014)). Other experiments have shown unexpected differences in the emmetropization response in monochromatic light. Smith et al. (2015) found that rhesus monkeys who wore long-wavelength filters as lenses for 120 days became more hyperopic than controls that did not wear filtering lenses. Gawne, Siegwart, Ward, and Norton (2017) found that infant tree shrews (22 days old) remained hyperopic after exposure to red light for 13 days, whereas exposure to blue light caused tree shrews to become slightly more myopic. Juvenile and adolescent tree shrews showed similar effects (Gawne, Ward, & Norton, 2017). Thus, both tree shrews and rhesus monkeys respond in an unexplained manner to wavelength defocus. One of the differences between tree shrew and rhesus monkey experiments and others mentioned above was the incorporation of a pseudo-random flickering light that contained a broad range of temporal frequencies (Gawne, Siegwart et al., 2017; Gawne, Ward, &
Evidence suggests that longitudinal chromatic aberration (LCA) provides signals for guiding emmetropization. LCA arises from dispersion, which causes different wavelengths of light to converge at different focal planes. As a result of LCA, longer wavelengths have a longer focal length than shorter wavelengths. Recent experiments have demonstrated species specific emmetropization responses to monochromatic light exposure and the aim of this experiment is to determine whether protocol differences, including flicker and duration of exposure with circadian interruption can explain these differences. In many species the eye can respond appropriately to wavelength defocus induced by LCA. Seidemann and Schaeffel (2002) and Foulds, Barathi, and Luu (2013) observed that chicks reared in short-wavelength, blue light (430 nm), became more hyperopic with decreased axial growth, while chicks reared in long-wavelength, red light (615 nm), became myopic with increased axial growth. A similar hyperopic shift in response to short-wavelength light exposure has been demonstrated in several species (Cichlid fish (Kröger and Wagner
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Corresponding author. E-mail address: [email protected] (F. Rucker).
https://doi.org/10.1016/j.visres.2019.11.002 Received 1 August 2019; Received in revised form 18 October 2019; Accepted 3 November 2019 0042-6989/ © 2019 Elsevier Ltd. All rights reserved.
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2.3. Lighting and flicker conditions
Norton, 2018). In chick, mice and cat, emmetropization has shown a strong temporal frequency dependence. Hyperopic shifts in refraction were seen at high temporal frequencies (Schwahn & Schaeffel, 1997; Crewther & Crewther, 2002; Zhi, Pan et al., 2013; Rucker, Britton et al., 2015; Rucker, Britton et al., 2018; Tian, Zou, Wu, Liu, & Liu, 2019) and myopic shifts at low temporal frequencies (Cremieux, Orban, Duysens, Amblard, & Kennedy, 1989; Crewther & Crewther, 2002; Crewther, Barutchu, Murphy, & Crewther, 2006; Yu, Chen, Tuo, & Zhu, 2011; Rucker, Britton et al., 2015; Rucker, Britton et al., 2018; Murphy, Riddell, Crewther, Simpson, & Crewther, 2019; Tian et al., 2019). Further study is required to clarify the temporal effects of flicker on emmetropization. Another difference between studies was the duration of exposure to monochromatic light and its potential effect on circadian rhythms. Intrinsically photosensitive retinal ganglion cells (iPRGCs), associated with the master circadian clock, respond to light through stimulation of melanopsin in the cell membranes (Berson, Dunn, & Takao, 2002; Hattar, Liao, Takao, Berson, & Yau, 2002) and also to intra-retinal synaptic input from rod and cone driven circuits (Perez-Leon, Warren, Allen, Robinson, & Brown, 2006; Weng, Estevez, & Berson, 2013; Zhao, Stafford, Godin, King, & Wong, 2014). Melanopsin has a peak spectral sensitivity around 480 nm (Panda et al., 2005; Torii, Kojima et al., 2017) and would be minimally stimulated by narrow-band long-wavelength light (626 nm), the wavelength used in the tree shrew experiments (Gawne, Siegwart et al., 2017; Gawne, Ward, et al., 2017) while stimulation would be maintained with short-wavelength light. In this study, we aim to estimate the effect of the duration of exposure to monochromatic light on emmetropization and to examine the effect of monochromatic light exposure on circadian activity rhythms. Lastly, we will use temporally broadband flicker to determine if temporal stimulation interacts with the spectrum of the light to produce variable emmetropization.
Chicks were housed in 48x79x40 cm cages with a maximum of 10 chicks in each cage. Eight strips of RGB LEDS (Superbright NFLSRGBX2) were attached to the roof of the cage. The LED strips were 0.5 m long and were paired together. Each pair was evenly spaced 10 cm from each other. LEDs were controlled via a Raspberry Pi and controlled via custom software written in Python. The strips were set to monochromatic red (628 ± 10 nm) or blue (464 ± 10 nm) light or a broadband white light and produced steady or flickering light depending on the condition. 2.4. Exposure conditions Red and blue lighting conditions were matched to Gawne et al. (2017) conditions both in their spectra and their temporal flicker. In the flicker conditions, lights were set to pseudo-random flicker that contained a broad range of temporal frequencies, flickering over a range of random luminances (4–96% of the maximum luminance) during the day time. Average illuminance was 424 lx for all conditions and was measured with Dr. Meter LX1330B light meter. Mean power (Newport Power Meter) was 266 µW for Blue and 116.2 µW for Red. Luminance (Spectrascan PR670), measured via a reflecting plate placed at the bottom of the cage, was 84 cd/m2 for Blue and 114 cd/m2 for Red. The mean illumination of the flicker conditions differed from Gawne et al. (2017) whose average illuminance varied across time during the wavelength and flicker conditions. In this experiment we aimed to match the mean illuminance across the six conditions (3 color conditions × 2 flicker conditions). The lighting conditions were on a 12 h/12 h light/ dark cycle. 2.5. Short duration condition To compare the effects of monochromatic light and flicker on emmetropization, one-week-old chicks were placed into one of six conditions for a short duration (10 days). The bird numbers in each condition were: (1) Blue Steady (n = 6), (2) Blue Flicker (n = 6), (3) Red Steady (n = 5), (4) Red Flicker (n = 5), (5) White Steady (n = 4), and (6) White Flicker (n = 6). Measurements were made on Day 1, 3, 6, 8, and 10 after initial exposure and the data were collected between 11am and 1 pm. Day 1 in Fig. 1 represents the baseline measurement at 7 days old. Ocular biometry and refractive error were measured again on Day 3, to compare with 10-day-old birds exposed for 10 days in the long duration condition.
2. Methods 2.1. Animals Chicks (White leghorn, K strain, Cornell University, Ithaca, NY) were chosen as a model organism because chick eyes grow rapidly and are easily manipulated in experimental conditions (Schaeffel & Feldkaemper, 2015; Wisely et al., 2017). Chicks had access to food and water ad libitum. Use of animals in this study was in compliance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the NECO Institutional Animal Care and Use Committee.
2.6. Long duration condition 16 chicks were hatched in incubators and then transferred immediately to the lighting condition cages. Chicks were placed under a Steady Blue (n = 10) or Steady Red (n = 6) light. In Red, the data from 3 birds was lost as a result of a Lenstar data export error and 1 newly hatched chick died. To examine the possibility of transient duration effects, we compared the measurements from age-matched 10 day-old chicks on Day 3 of the short duration exposure condition (Suppl.
2.2. Hormonal influences In chicks, puberty is generally not reached until 18–21 weeks of age. Given the age of the chicks used, puberty was not a factor in the difference in results between the long and short duration experiments.
Fig. 1. Timeline of the light exposure and measurements for both experiments. The blue colored bar represents when chicks were exposed to the lighting conditions for a short duration. Measurement days are marked on the timeline above. The green colored bar represents when chicks were exposed for a long duration. PREand POST-measurements were made at Days 10 and 17. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 13
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normalized by the number of regions of motion detected. The camera, using night vision mode (using IR), could capture motion during the night. Very little activity was captured during this period (8PM to 8AM) in all the groups. Thus, only the daytime period (8AM to 8PM) was analyzed. 2.10. Circadian activity analysis To equate recording time across days, only the data from the days when no refraction or ocular biometry measurements were collected was analyzed (Days 2, 4, 5, 7, 8, and 9). Our measure of activity is expected to increase with time because the algorithm will detect more pixel changes as the larger birds move within the cage. To statistically control for this effect, analyses was therefore done within each Day. A non-parametric Kruskal Wallis one-way ANOVA of the activity metric was performed. SPSS (version 24, IBM) statistical software was used for analysis.
Fig. 2. Axial length measurements taken after a short duration, or long duration, of exposure to red and blue monochromatic lights and white light. Birds exposed for a short duration in steady light are shown as red, blue and white circles that correspond to the color of the illuminant. Those reared in flicker are shown by diamonds. Birds exposed for a long duration are shown as stars in the corresponding color. Standard error bars are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.11. Component and refraction analysis Refractions and ocular component measurements of both the left and right eyes were averaged. In the short duration condition, a linear model was fit to the data from each measurement day. The primary outcome measure was the slope of the linear regression multiplied by 10 to represent the total change in refraction or the ocular component across 10 days of light exposure. In the long duration condition, the change in the pre- and post-measures were analyzed. In the short duration condition, a 2-way ANOVA with variables of Color and Flicker was computed. Tukey post-hoc t-tests were used to determine if there were differences among individual conditions for each component. Levene’s Test of Equality of Error Variances, based on the median, was run to test for homogeneity of the data and a ShapiroWalk test was run to test for normality. SPSS (version 24, IBM) statistical software was used.
Table 1) and Day 10 of the long duration exposure condition (Suppl. Table 3). The effects of a longer duration of exposure to monochromatic light were assessed via the change in refraction and ocular components between Day 10 and Day 17. No measurements were taken prior to exposure given how fragile the birds are at hatch. The timeline for the measurements can be seen in Fig. 1. 2.7. Eye measurements Refraction and eye measurements were performed on each eye at the same time of day using lid retractors. During measurement, chicks were anesthetized with 1.5% isoflurane gas in 2% oxygen. Refraction was performed using an infrared photorefractometer as described by (Schaeffel, Farkas, & Howland, 1987). Refraction was measured at a distance of about 1 m with a sampling frequency of 62 Hz. The infrared refractometer setup consisted of a camera (IC Imaging Control, Charlotte, NC) with a Pentax 75 mm f/1.4 lens and IR LEDs mounted on the bottom half of the lens. Measurements of the light gradient in the pupil, in the 90 and 180 degree meridians were converted to dioptric values using a predetermined calibration. The mean refraction and standard deviation were calculated from the winsorized mean of 650 samples. Axial length, choroidal thickness, vitreous chamber depth, lens thickness, and anterior chamber depth were measured with a Lenstar LS-900 (Haag-Streit, USA). Five A-scan measurements were collected each measurement day, with each measurement being an average of 16 traces. The peaks in the A-scans corresponding to the ocular biometry were marked via custom software.
3. Results 3.1. Short duration condition Absolute measures of axial length are shown in Fig. 2. The ocular biometry change data for each treatment group is summarized in Fig. 3 and Suppl. Table 2. 3.1.1. Refractive error There were no differences in refractive changes among the light conditions when temporal conditions were pooled (Fig. 3A: Levene’s Test: L = 0.064, p = 0.99; F = 1.74, p = 0.20). Refractive errors were normally distributed with Shapiro-Wilk’s test within each colored condition: Blue (p = 0.43), Red (p = 0.87), White (p = 0.56). Birds exposed to blue light showed a mean change in refractive error of −0.82 ± 0.81 D, while birds in red light showed a mean change of −0.31 ± 0.95 D, and birds in white light showed a change of + 0.44 D ± 0.99 D, over 10 days. Birds in Red and Blue steady light showed no difference in refractive error (p = 0.59). There were no differences in refractive changes between the steady and flicker conditions (Fig. 3B: F = 0.697, p = 0.411). On average, refractive error changed by only −0.08 ± 0.98 D with flicker, while in steady light conditions, refraction changed by only +0.12 ± 0.90 D over 10 days.
2.8. Circadian activity To measure whether circadian rhythms are influenced by the spectral composition, the activity of the chicks was recorded in the short duration Red, Blue and White, flicker conditions. Chick movement was recorded continuously with an infrared video camera throughout the experiment. The monitoring system was paused when the refraction and ocular biometry were measured and during cage cleaning.
3.1.2. Axial length Mean changes in axial length by condition are shown in Fig. 3C and D. Fig. 3C shows the changes in axial length when flicker and steady light conditions are pooled. There was less axial growth when chicks were raised in blue light compared to red and white light (Levene’s Test: L = 0.552, p = 0.55; F = 10.18, p = 0.001). Axial Length
2.9. Circadian activity analysis Activity was recorded via custom motion capture software that computed the area of motion (in pixels/second) and the number of regions in the image via a keyframe algorithm. To equate the changes over each one-minute period, the raw activity in pixels/second was 14
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Fig. 3. Short duration: Mean change in each ocular component calculated over 10 days with treatment starting at 7 days old. Refractive error: A. Pooled and grouped by color. B. Grouped by color and temporal condition. Steady light is represented by diagonally striped bars, flicker by plain bars. Axial length: C. Pooled and grouped by color. D. Grouped by color and temporal condition as in B. Choroid thickness: E. Pooled and grouped by color. F. Grouped by color and temporal condition as in B. Horizontal line in the box indicates the median. Boundaries of the box indicate the 25th and 75th percentile. The whiskers represent the high and low values 1.5x the interquartile range. Dots represent outliers.
Wilks test within each color condition: Blue (p = 0.96), Red (p = 0.67), White (p = 0.20). On average, choroidal thickness increased by only + 0.018 ± 0.039 mm in Blue, +0.012 ± 0.029 mm in Red, and + 0.028 ± 0.036 mm in White light over 10 days. Fig. 3F shows no differences between the flicker conditions for choroidal thickness changes (F = 0.469, p = 0.50). On average, choroidal thickness increased by 0.024 ± 0.033 mm with Flicker and 0.015 ± 0.033 mm with Steady light. There was no interaction between the color and flicker conditions.
measures were normally distributed with Shapiro-Wilks test within each color condition: Blue (p = 0.48), Red (p = 0.57), White (p = 0.68). Tukey HSD post-hoc test showed that axial changes in the Blue and Red conditions differed (p < 0.01) and that Blue and White conditions differed (p = 0.001). On average, the axial length of the chicks raised in Blue increased by 0.945 ± 0.055 mm, less than the axial length increase of 1.089 ± 0.075 mm in Red, and 1.114 ± 0.011 mm in White. As Fig. 3D shows, there was less axial growth in chicks reared in flickering than steady light, irrespective of the light condition (F = 6.176; p < 0.05). In steady light, axial length increased by 1.086 ± 0.108 mm, while with flicker, axial length increased by 1.00 ± 0.120 mm over 10 days. There was no interaction between the light and flicker condition (F = 1.45; p = 0.26).
3.2. Intermediate Duration 3.2.1. Refractive error Overall, in 10-day old, age-matched chicks, the refractions were more hyperopic in Blue than in Red (Fig. 4A: F = 47.08; p = 0.01). The difference in refractive error in Red and Blue Steady lighting was greater after a shorter duration of exposure (Blue-Red = 2.84D) than a longer duration of exposure (Blue-Red = 1.42D) (Levene’s L = 2.06, p = 0.14; F = 5.21: p < 0.05). This result indicates a transient response to wavelength defocus.
3.1.3. Choroid Choroidal changes in each illumination and flicker condition are shown in Fig. 3E and F. The lighting condition did not affect choroidal thickness (Levene’s Test: L = 0.587, p = 0.71: F = 0.266, p = 0.77). Choroidal measurements were normally distributed with the Shapiro15
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Fig. 4. Absolute refractive and axial measures for age-matched, 10 day-old chicks, in the short and long duration conditions after exposure to steady colored illuminants for 3 or 10 days, respectively. A. Overall, refractions were more hyperopic in Blue than in Red. There was a greater difference in refractive error in red and blue light in the short duration condition as shown by the bar extending between the duration conditions. B. Axial lengths were longer in the long duration condition. In the long duration condition, the axial length in red light was longer than in blue light. C. There was no difference in choroidal thickness. A p-value of p < 0.05 is represent by *, a p-value of p < 0.01 by **. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
refractive errors were normally distributed in Blue (p = 0.80) and Red (p = 0.22) conditions.
3.2.2. Axial length At 10 days old, with pooled color data, chicks had shorter axial lengths (9.105 ± 0.114 mm) after a short duration of exposure than a long duration (9.260 ± 0.210 mm) exposure (Fig. 4B: Levene’s L = 0.684, p = 0.57; F = 12.9, P = 0.002). Examining each condition separately, at 10-days old there was no difference in axial length between Blue and Red after a short exposure (F = 3.031, p = 0.12), but axial length changed more in Red than in Blue after a long exposure (F = 14.334, p = 0.003). At this intermediate time point, a longer duration of exposure to monochromatic light increases eye growth, particularly in Red.
3.3.2. Axial length Between 10 and 17 days of exposure, there were no further differences in axial length changes between the two illumination conditions (Fig. 5B: Levene’s Test: L = 0.011, p = 0.92; t = 0.259, p = 0.62). In Blue, the change in axial length was + 0.898 ± 0.089 mm from Day 10 to Day 17, while in Red, the average change in axial length was + 0.871 ± 0.103 mm. Shapiro-Wilks test shows that changes in axial length measures were normally distributed in Blue (p = 0.15) and Red (p = 0.45) conditions.
3.2.3. Choroid At 10 days-old, there was no difference in choroidal thickness in Red and Blue (Fig. 4C: Levene’s L = 1.03p = 0.40; F < 0.001, p = 0.99) and no difference with exposure duration (F = 0.787, p = 0.39).
3.3.3. Choroid As Fig. 5C shows, after a total exposure duration of 17 days, choroidal thickness increased in Red compared to Blue (Levene’s Test: L = 0.312, p = 0.59; t = 13.62, p < 0.01). In Blue, choroids thickened + 0.003 ± 0.268 mm, and in Red, +0.054 ± 0.021 mm. Shapiro-Wilks test shows that choroid measures were normally distributed in Blue (p = 0.31) and Red (p = 0.26) conditions.
3.3. Long duration condition Increasing the exposure duration from 10 to 17 days did not reproduce or enhance the changes in ocular biometry observed after a short duration exposure. Change values can be seen in Fig. 5 and Suppl. Table 4 and can be calculated from pre-and post-measurement values in Suppl. Table 3.
3.3.4. Weight There was no difference in weight gain in the chicks between the lighting conditions (t = 0.224, p = 0.64). Chicks raised in Blue weighed on average 51.92 ± 2.6 g on Day 10 and 132.0 ± 9.2 g on Day 17. Chicks raised in Red weighed on average 53.6 ± 1.96 g on Day 10 and 135.6 ± 7.3 g on Day 17.
3.3.1. Refraction Fig. 5A shows that after an additional 7 days of exposure when chicks were 17 days-old, there were no further differences in refractive error changes between the two illumination conditions (Levene’s Test: L = 3.042, p = 0.11; t = 2.48, p = 0.14). In Blue, the change in refractive error was −0.69 ± 1.10 D from Day 10 to Day 17 and in Red it was + 0.12 ± 0.39 D. Shapiro-Wilks test shows that the changes in
3.4. Activity There was very minimal activity at night with no significant differences between the groups (F = 1.91; p = 0.57). Fig. 6 shows values 16
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Fig. 5. Long duration: Changes in ocular measurements after 17 days of exposure to Blue or Red steady light from hatching. A. Changes in refractive error. B. Changes in axial length. C. Changes in choroidal thickness. Red exposure resulted in thicker choroids than Blue. Asterisks represent a p-value < 0.01. Graphical design as in Fig. 3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
White, but increased activity in Red Flicker versus Blue and White Flicker (all comparisons, p < 0.01). For all days, activity was measured to be 482.8 ± 170 Δpixels/min for chicks raised in Blue Flicker, 622.4 ± 165 Δpixels/min for Red Flicker, and 426.8 ± 135 Δpixels/ min for White Flicker. There was no difference in activity between the morning and afternoon Blue Flicker (F = 0.63, p = 0.44), Red Flicker (F = 0.263, p = 0.61), or White Flicker (F = 2.95, p = 0.1).
4. Discussion This experiment examined the effects of exposure duration and flicker on emmetropization when chicks were exposed to monochromatic light. We also examined the possibility that monochromatic light interferes with circadian activity rhythms. With a short duration exposure, we found that monochromatic red or broadband white light led to more axial growth than monochromatic blue light, yet there was no refractive difference. It is possible the lack of refractive effect may be due to differences in corneal changes in red and blue light (Rucker, Britton et al., 2015) but these were not measured. The axial results concurred with the anticipated response to wavelength defocus for emmetropization. A longer duration of exposure did not reproduce the difference in axial growth seen with the short duration of exposure, but the eye was already longer in Red than Blue by Day 10, and further changes in refraction and axial length were small. Prolonged exposure to monochromatic light had the effect of increasing growth stimulation, particularly in red light, as seen at the Day 10 intermediate time point. We speculate that this large increase in axial growth is due to circadian interference, as suggested by the increase in activity patterns in Red compared to Blue. In support of this hypothesis, Wang, Schaeffel et al. (2018) demonstrated a minimal change in retinal dopamine levels in chick with exposure to 620 nm red light, with a trend towards higher retinal dopamine release in UV light compared to white light. Presuming these changes in retinal dopamine are related to stimulation of the circadian system, it is notable that
Fig. 6. Box plot of Activity. Horizontal line in the box indicates the median. Boundaries of the box indicate the 25th and 75th percentile. The whiskers represent the high and low values 1.5x the interquartile range. Dots represent outliers. The box plot represents activity measured during day time only.
measured during lights on. Overall, the data for the activity measure were not normally distributed, so the non-parametric Kruskal-Wallis test was used for analysis. On Day 2, a difference among the lighting conditions was observed (Levene’s L = 3.753, p = 0.12; χ2(2) = 22.04, p < 0.01). Tukey posthoc tests showed that increased activity was seen in chicks reared in Red (p < 0.001) and Blue Flicker (p < 0.001) compared to White Flicker. Differences among the conditions were observed on Day 4 (Levene’s L = 3.518, p = 0.04; χ2(2) = 33.282, p < 0.01), Day 5 (Levene’s L = 0.074, p = 0.93; χ2(2) = 12.75, p = 0.002), Day 7 (Levene’s L = 1.907, p = 0.16; χ2(2) = 17.79, p < 0.01), Day 8 (Levene’s L = 1.538, p = 0.22; χ2(2) = 16.85p < 0.01), and Day 9 (Levene’s L = 1.335, p = 0.27, χ2(2) = 12.57p = 0.002). Unlike Day 2, these subsequent days did not show increased activity in Blue versus 17
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affect the flicker response. In comparison, Gawne et al. (2017) also found a greater myopic refractive shift in broad-band flickering blue light than in steady blue light, while Gawne et al. (2018) found similar refractive changes in both steady and flickering blue light. When shortwavelength-sensitive cones are stimulated with sinusoidal, rather than broad-band, flicker at a low temporal frequency they are more sensitive than when stimulated with higher temporal frequencies (Smithson & Mollon, 2004). It was thought that the presence of higher temporal frequencies in the pseudo-random noise used in this experiment and Gawne et al. (2017) may have reduced the S-cone response and driven the emmetropization system toward the development of myopia (Rucker, Britton et al., 2015; Rucker, Britton et al., 2018). The color independent reduction in eye growth with broad-band flicker is in agreement with the finding that a color insensitive reduction in eye growth occurs with high frequency luminance flicker (Rucker, Britton et al., 2015). Previous studies have suggested that the use of continuous wear narrow band, binocular, red filters, for prolonged periods may reduce the magnitude of myopic refractions in monkeys. However, if this effect is due to circadian interference then the optical benefits may be overwhelmed by the deleterious effects of circadian rhythm disruption on health, including increased risk of cancer (review: (Feillet, van der Horst, Levi, Rand, & Delaunay, 2015) metabolic changes leading to obesity, mood disorders and cardiovascular disease (Chepesiuk, 2009; Anisimov, Vinogradova, Panchenko, Popovich, & Zabezhinski, 2012, Stevens, Brainard, Blask, Lockley, & Motta, 2013).
changes in circadian rhythms in proteoglycan synthesis in chick have been associated with changes in axial length (Nickla, Rada, & Wallman, 1999). This finding of an increase in growth in red light may partially explain the observation by Foulds et al. (2013) who observed an unusually large difference in vitreous chamber depth between red and blue lighting conditions after 14 days of light exposure from hatch. The difference in choroidal effects with a longer duration of exposure to red light could be attributed to a lack of stimulation of the iPRGCs in red monochromatic light and a shift in the diurnal choroidal rhythms as found in chick, monkey and humans (Nickla, 1996; Nickla, Wildsoet, & Wallman, 1998; Nickla, Wildsoet, & Troilo, 2002; Nickla, 2006; Burfield, Patel, & Ostrin, 2018). In tree shrew, choroidal thickening contributed to the hyperopic refractive effects observed in red monochromatic light in the longer duration experiments (Gawne, Siegwart et al., 2017; Gawne, Ward et al., 2017). In binocularly exposed monkey and tree shrew, reared in red light, hyperopic changes were associated with corresponding changes in vitreous chamber depth (Smith et al., 2013; Smith et al., 2015; Gawne, Siegwart et al., 2017; Gawne, Ward et al., 2017), though axial changes were not noted at any age. Another interesting finding was that monocularly reared monkeys wearing red filters did not show vitreous changes. Monocular rearing would have allowed sustained circadian stimulation. However, measurements of circadian rhythms in the choroid and in retinal and vitreal dopamine and/or DOPAC levels would be required to confirm this hypothesis. Previous experiments have demonstrated that disruption of the circadian rhythms affects choroidal thickness in chicks (Nickla, Wildsoet et al., 1998; Nickla, Rada, et al., 1999) and there are differences in ocular circadian rhythms between emmetropic and myopic humans (Burfield et al., 2018; Burfield, Carkeet, & Ostrin, 2019). There was evidence of a transient effect of monochromatic light on emmetropization, in that when the refractive changes were examined at the Day 10 intermediate time point, there was a greater difference in refractive error between red and blue light conditions after 3 days of exposure compared to 10 days of exposure. This result suggests that the refractive effects of wavelength defocus diminish over time and that there is a critical period where cone stimulation with monochromatic light can alter emmetropization in a manner predicted by wavelength defocus. This is consistent with the transient refractive effects observed in tree shrew. Tree shrews exposed to blue light became hyperopic during the early phase at around 27 days of visual experience before becoming myopic in both steady and flickering light during the late phase (Gawne et al., 2018). The authors suggested that the tree shrew loses its ability to detect the sign of defocus via feedback mechanisms in narrowband blue light. Thus, it is clear that depriving an animal of certain wavelengths for prolonged periods interferes with the emmetropization process. The duration of the exposure to monochromatic light in this experiment differed from that in other experiments (Foulds et al., 2013; Smith et al., 2015; Hung, Arumugam, Sankaridurg, & Smith, 2016; Gawne, Siegwart et al., 2017; Gawne, Ward et al., 2017; Gawne et al., 2018). Foulds et al. (2013) used 1-day old chicks and exposed them to the lighting conditions for up to 28 days. Gawne & Siegwart et al. (2017) exposed tree shrews at 1 month of age. The eyes of the tree shew typically open 3 weeks after birth. Thus, the animals had several days of visual experience (DVE) at the time of exposure, followed by 13 days in the experiment. Rhesus monkey were first exposed to lighting conditions at 25 ± 2 days of age and exposure was continued up to 146 ± 7 days of age (Smith et al., 2013; Smith et al., 2015). Given the different rates of visual and ocular development among species, matching exposure time does not equate the experience required for development. To address this concern, we examined the effect of two different durations of exposure in chick. The primary aim was to use the methods of Gawne, Ward et al. (2017) and Gawne et al. (2017) while studying tree shrew, in the chick model. Our results indicate that emmetropization is sensitive to the broad-band flicker presented, but the color of the light source did not
5. Summary In this study we showed that the duration of exposure to different monochromatic lighting conditions affects refractive changes. Differences in refraction and eye growth found with shorter duration exposures to red and blue monochromatic light, were not observed with longer duration exposures probably because of transient changes in ocular growth in response to wavelength defocus and a general increase in eye growth in monochromatic light. This instability may be due to the influence of monochromatic light on circadian activity rhythms. These results may help to explain the species differences observed in monochromatic light experiments via differences in the experimental protocols or differences in the underlying biology of circadian rhythms, though species differences cannot be excluded. Acknowledgements Research reported in this publication was supported by the National Eye Institute of the National Institutes of Health under Award Number R01EY023281 and by the NIH T35 Program. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.visres.2019.11.002. References Anisimov, V., Vinogradova, I., Panchenko, A., Popovich, I., & Zabezhinski, M. A. (2012). Light-at-night-induced circadian disruption, cancer and aging. Current Aging Science, 5, 170–177. Berson, D. M., Dunn, F. A., & Takao, M. (2002). Phototransduction by retinal ganglion cells that set the circadian clock. Science, 295(5557), 1070–1073. Burfield, H. J., Carkeet, A., & Ostrin, L. A. (2019). Ocular and systemic diurnal rhythms in emmetropic and myopic adults. Investigative Ophthalmology & Visual Science, 60(6), 2237–2247. Burfield, H. J., Patel, N. B., & Ostrin, L. A. (2018). Ocular biometric diurnal rhythms in emmetropic and myopic adults. Investigative Ophthalmology and Visual Science, 59(12), 5176–5187.
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