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Abstract
Previous studies have shown that changes in functional activity in the retina can be visualized as changes in fundus reflectivity. When the image projected on the retina is low pass filtered or defocused by covering the eye with a frosted diffuser or a negative lens, it starts growing longer and develops myopia. We have tested the hypothesis that the resulting altered retinal activity may show up as changes in fundus reflectivity. Fundus reflectivity was measured in chickens in vivo, both in visible (400-800 nm, white) and near ultraviolet (UV) light (315-380 nm). Two CCD cameras were used; a RGB camera and a camera sensitive in near UV light (peak sensitivity at 360 nm). White and UV LEDs, respectively, placed in the center of the camera lens aperture, served as light sources. Software was written to flash the LEDs and record the average brightness of the pupil that was illuminated by light reflected from the fundus. The average pixel grey level (px) in the pupil was taken as a measure of the amount of reflected light while refractive errors were corrected by trial lenses after pupil brightness was corrected for pupil size. It was found that myopic eyes had brighter pupils in UV light, compared to eyes with normal vision, no matter whether myopia was induced by diffusers or negative lenses (48 ± 9 vs. 28 ± 3, p<0.001 and 47 ± 7 vs. 27 ± 2, respectively). Using SD-OCT in alert chickens it was found that the retinal nerve fiber layer (RNFL) and the retinal ganglion cell layer (RGCL) in the central retina became thinner already at early stages of myopia development, compared to controls (31.2 ± 5.8 µm vs. 43.9 ± 2.6 µm, p<0.001 and 36.9 ± 1.2 µm vs. 44 ± 0.5 µm, respectively). While the decrease in RNFL thickness occurred concomitantly with the increase in UV reflectivity, it remains unclear whether these changes were causally linked. Thinning of the RNFL could be due to reduced neural activity in retinal ganglion cells but also due to metabolic changes in the retina during myopia development.
© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Myopia (near-sightedness) is the most common ocular disorder in young people, where distant objects are focused in front of the retina. Its prevalence is currently still rising, especially in Southeast Asia, but also in Europe and the United States. It has been extrapolated that about half of the world population will be myopic by 2050 [1]. Given that only a small percentage of variance of refractive errors (<10 percent) can be explained by genetic variants that were detected in Genome-Wide Association Studies [2–4], the major stimulus for myopia development must reside in the visual environment [5–9]. A large number of studies have focused on the biological mechanisms of myopia development and the visual cues that might trigger the enhanced growth of the eyeball (i.e [10].). However, there is currently no biomarker available that could be used to follow potential myopia-related changes in the retina in vivo. In this study, we have analyzed changes in fundal reflectivity and retinal layer thickness that might be associated with early stages of myopia development in the chicken model.
1.1 Previous attempts to measure reflectivity of the fundus
Already in 1952, Rushton introduced quantitative densitometry to measure photopigment density based on the spectral characteristics of reflected light in the living human eye [11–14]. Van Norren and van de Kraats used continuous recordings of fundus reflectivity in white light to analyze the time kinetics of visual pigment bleaching and regeneration [15]. Optical density and topography of the macular pigment were also measured by fundus reflectometry [16]. Orientation and directionality of the photoreceptors (the Stiles-Crawford effect) were explored in vivo by measuring the amount of light reflected by the retina in the pupil plane [17–20].
1.2 Previous attempts to measure the state of retinal activity
The amount of light and spectral distribution of light reflected in a tissue depends not only on pigment densities and distributions but also on the distribution of structures with different refractive indices. Refractive indices in the intracellular space are related to the metabolic state of a cell [21]. Furthermore, biochemical changes may induce structural modifications in the cell that can also change reflectivity [22–26]. In retinal glia cells it was shown that enhanced neural activity increases light transmittance by causing cellular swelling, related to higher extracellular potassium levels. In the rat optic nerve, swelling of astrocytes was elicited by stimulation with flicker light [27]. With increasing amplitudes of the flicker electroretinogram (ERG), blood flow also increases to deliver nutrients and oxygen to the cells with higher metabolic demands. Both in monkeys and humans it was found that a spot of flickering green light projected on the retina reduces fundus reflectivity in the near-infrared range selectively in the stimulated area [26,28,29]. The reasons for these changes have not been fully understood [30–32], despite extensive studies by optical coherence tomography (OCT). OCT images are based on differences in refractive index between the different retinal layers. Based on OCT B-scans, Hillman et al [29] proposed that visual stimulation may affect optical path lengths in photoreceptors outer segments. Another explanation for reduced reflectivity was that increased neural blood flow induced higher light absorption by hemoglobin [33–35]. Buerk et al. stimulated a large area of the retina (30 degrees) and found that blood flow in the optic nerve head increased already after a few seconds, as did potassium ion concentrations [36]. In summary, these findings support the idea that visual experience, shaping neural activity as well as retinal and choroidal blood flow, can show up as changes in fundus reflectivity.
1.3 Evidence for changes in “retinal activity” during myopia induction
To induce deprivation myopia in an animal model, it is sufficient to spatially low pass filter the retinal image [37]. In a low pass filtered image, it can be expected that ON/OFF antagonistic ganglion cells are scarcely stimulated when their receptive field sizes are smaller than the wavelength at the highest transmitted spatial frequency. However, in the presence of high frequency Ganzfeld flicker, rapid temporal luminance modulations are provided which may restore some of the activity of these cells even in the presence of spatially low pass filtered images [38]. In chickens, myopia can be suppressed by stroboscopic light (i.e [39].). The inhibition of myopia in chicks by flicker light with various duty cycles was correlated with the amplitude of Ganzfeld flicker electroretinogram [40]. While it is expected that the amplitude in the electroretinogram depends on the length of the eye [41] and is inversely correlated with its axial length [42], it is more interesting to look into functional changes in the retina when myopia just starts to develop. In fact, changes in the induced component of the mfERG were found in children already when they just started to develop myopia and had still less than 0.5D [43]. Chen et al showed that the delayed mfERG response in myopic subjects was not due to longer eyes but due to changes in retinal processing [44]. It was proposed that reduced and delayed ERG responses in myopic eyes may result from changes in synaptic transmission between photoreceptors and bipolar cells or biochemical changes in the inner retina [44,45]. Given that functional changes occur in the retina at early stages of myopia development in humans, we hypothesize that there should also be early changes in fundal reflectivity. However, to our knowledge this has never been tested. If present, such changes could serve as an in-vivo biosensor for early changes in retinal processing during myopia development.
2. Methods
2.1 Animals
Fifteen male white leghorn chickens were obtained from a local hatchery in Kirchberg, Germany. Chickens were kept under a 12/12 hour light/dark cycle. Illuminance was 500 lux during the light phase from 8AM to 8PM. Food and water were provided ad libitum. All measurements were taken in alert, hand-held chickens. Experiments were approved by the local University Commission of Animal Welfare and were in accordance with the ARVO Statement for care and use of Animals in Ophthalmic and Vision Research.
2.2 Treatment
Experiment 1. At the age of 10 days, one eye of 10 chicks was covered with a frosted diffuser to induce deprivation myopia (DM) [46]. In the other 5 chickens, a 7D negative lens was placed in front of one eye to induce lens-induced myopia (LIM). Fellow eyes had normal vision. Treatment lasted 7 days in all cases. Baseline measurements of refractions, thickness of retinal layers and fundus reflectivity were taken before diffusers or lenses were attached, as well as at three time points during the treatment period (Fig. 1). The data of all 15 chicks were included in the correlation analyses.
Fig. 1 Time points of data collection. Treatment with negative lenses (n = 5) or diffusers (n = 10) started at day 0, when chicks were 10 days old and was continued for 7 days. After 40 hours, chicks had refractive error shifts of less than 1.5D (except for one), on the fifth day they were not yet fully myopic (refractive error shift less than 3D; referred to “pre-myopic” stage), and on the last day of the experiment, 9 chicks developed myopia (2 treated with negative lenses and 7 treated with diffusers).
Chicks were clustered into three categories for the analyses shown in Fig. 7(A)-(F): (1) early changes (2) “pre-myopic” stage, (3) myopia (negative refraction). To detect early changes in fundus reflectivity and retinal thickness, the first sampling point was already after 40 hours of treatment when the induced relative myopia was still lower than 1.5D. One chick which had already developed more than 1.5D of relative myopia was excluded from the analysis at this time point (diffuser n = 9, lens n = 5; Fig. 7(A), 7(D)). The next measurement was done on day 5 of treatment when chicks had less than 3D of refractive error shift. At this time point all lens-treated chicks felt into this category but two chicks, which did not respond to the deprivation at all, were excluded from the analysis (diffuser n = 8, lens n = 5; below referred to as “pre-myopic stage”). The last measurement was taken on the last day of treatment and included all myopic chickens. Two lens treated chicks and 7 diffuser treated chicks fulfilled the inclusion criteria.
Experiment 2. At the age of 13 days, 7 chickens were unilaterally treated with diffusers. Fundus reflectivity in UV light was measured in the beginning and after 3 and 5 hours of treatment. Ocular biometry and retinal thickness were measured before and after 5 hours of imposed vision blur.
2.3 Measurements of refractive state and ocular dimensions
Refractions were determined by automated eccentric infrared photorefraction [47]. Ocular dimensions were determined by A-scan ultrasonography [48] under local corneal anesthesia (2% xylocaine solution) on the last day of the experiment.
2.4 Fundus reflectivity
A monochrome camera sensitive in the near UV range (300-420nm; SONY XC-EU50-CE, ALRAD Imaging, Camberley, England) with a peak sensitivity at 365 nm combined with a UV LED with a peak emission at 375 nm (LED375L, 5 mm; ThorLabs) centered in the camera lens aperture was used to measure fundus UV reflectivity. In chicks, the ocular media transmit light to more than 90 percent down to at least 350 nm [49]. A special UV transmitting camera optics was used (UV-Lens f/4, 60 mm, Carl Zeiss, Jena). In the visible range, fundus reflectivity was measured using a RGB CCD camera equipped with a 50 mm lens (DFK21 AU04, The Imaging Source, Bremen, Germany) with high sensitivity between 400 and 780 nm, and peak sensitivity at 500 nm. For measurements in visible light, a high power white LED (Nichia NSPW500CS, 5 mm; Conrad Electronics, Germany) with an emission peak at 460 nm served as light source. Software was developed under Visual C + + 8.0 to flash the LEDs and to analyze the brightness of the pupil, after back-illumination by light reflected from the fundus. Because automated detection of the pupil margins by the software was not always reliable, the operator could manually mark 4 positions at the pupil border and the software performed automatically a circle fit (Fig. 2(A)). Pixel grey levels in the pupil were averaged and displayed on the screen, together with their standard deviation. The software corrected for the effects of pupil sizes, assuming that pupil brightness increases linearly with pupil area when a point source is used as described by Choi [50]. In the case of UV light, pixels were monochrome (Y800). In the case of the RGB camera, a simultaneous analysis was possible for the RGB channels (Fig. 2(A)). In the case of the RGB camera it was verified that there was a linear relationship between luminance (in cd/m2) and pixel grey level over the range of measurements in the current study (between 40 to 100; R2 = 0.99, pixel values = 0.34*luminance + 38).
Fig. 2 (A) Screenshot of the software output. The software grabbed a frame while the LED in the center of the camera aperture was flashed for 25 msec. The user could mark the pupil margins by mouse click. The software determined the average pixel grey levels in the image of the pupil, together with its standard deviation. It also automatically corrected for differences in pupil diameters. With the color camera, RGB channels were separately analyzed (four pictures on the right). (B) Because the set-up matched the optics of isotropic photorefraction, refractive errors had a strong impact on pupil brightness. Light returning from the fundus is focused in space at distances that depend on the refractive error of the eye. Accordingly, light distributions and pupil brightness vary in the camera plane. To minimize the impact of this factor, refractive error of each eye was corrected by trial lenses during the measurements.
Chickens were cooperative and the operator could tilt their head in any direction until the eyes were aligned with the camera. After an image was grabbed, the alignment of the eye could be verified by the position of the first Purkinje image in the pupil (see example in Fig. 6). Since many measurements could be done in a short time, enough images were available to select those with the eyes properly aligned. A complicating factor was the varying amount of refractive error. The optical configuration used for the measurements of fundus reflectivity was the same as for isotopic photorefraction [51,52], meaning that light returning from a bright point in the fundus is focused in space at different distances, depending on the refractive state of the eye. Accordingly, light distributions in the camera plane vary with refractive state, causing also differently bright pupils. To overcome this problem, refractive errors had to be corrected by trial lenses, placed in front of the eye (Fig. 2(B)).
2.5 Spectral domain optical coherence tomography (SD-OCT)
SD-OCT measurements were taken in alert, hand-held chickens. The thickness of the retinal layers was measured in B-scans with the Spectralis HRA + OCT 2016 (Heidelberg Engineering GmbH) using the software provided by the manufacturer (version 6.5). We focused our analysis on the inner retinal layers because previous studies in human subjects had shown reduced thickness of the middle and inner retina in moderate and high myopia [53,54]. Since chickens have no fovea but only a diffuse “area centralis”, the alignment of the eye is demanding. The root of the pecten was used as a landmark (the pecten is a heavily perfused structure protruding into the vitreous body of avian eyes) [55,56]. OCT examinations took place in the area centralis, located superior and nasally from the root of the pecten. In this region, visual acuity reaches a maximum of about 7 cyc/deg [57] and RNFL and retinal ganglion cell layer (RGCL) have a similar thickness, as previously described based on histological studies [58,59], and also shown in Fig. 3. Thickness of these layers was directly measured in the OCT scans, as well as the thickness of the layers between the inner plexiform layer (IPL) and the retinal pigment epithelium (RPE) (Fig. 3). Three B-scans, selected based on their best quality, were analyzed for each eye. The thickness of the RNFL and the RGCL in the area centralis could be measured in OCT B-scans with small standard deviations in repeated measurements (about 2.2µm). Inter-individual variance was also low (about 3.6µm).
Fig. 3 OCT B-scan of the retinal layers in an alert chicken. The dashed box includes the region of the area centralis. The segments of retinal thickness were analyzed (1) retinal nerve fiber layer (RNFL) plus the retinal ganglion cell layer (GC), marked in red, (2) the sum of inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptors (PR) and retinal pigment epithelium (RPE) thickness (marked in blue). For comparison, a conventional histological section is shown on the right. Note that all retinal layers, the choroid (CH) and the two layers of the sclera (SC), fibrous and cartilaginous layer, are clearly visible in both, OCT and histological images.
2.6 Statistics
Statistical analyses were performed with the commercially available software package R (R 3.3.3, R Core Team, R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria). Averages of repeated measurements were taken for statistical analysis. Differences between treated and contralateral control eyes at each time point were analyzed using paired Student’s t-tests. Repeated-measures MANOVA was used to analyze the effect of diffuser and lens treatment on fundus reflectivity and RNFL thickness over time. For comparison between diffuser-treated, lens-treated and control eyes at one treatment point an un-paired Student`s t-test with Bonferroni correction was used. The effects of diffuser or lens wear over increasing refractive error were evaluated by Pearson’s correlation coefficient.
3. Results
3.1 Development of myopia
Seven of 10 chickens treated with diffusers and two of the 5 chickens treated with negative lenses developed relative myopia between −0.25 to −6D (average −1.6 ± 1.8D, with a relative myopic shift of maximally 7D, Fig. 4(A)). Two chickens treated with diffusers were poor responders with only a minor myopic shift of only about 1D, and a difference in axial length between treated and control eye of less than 50 µm after 7 days of treatment.
Fig. 4 (A) Refractive development in individual chickens treated with either diffusers (black lines) or negative lenses (grey lines). The solid thick line denotes the average refraction of the control eyes. Nine out of 15 chickens developed myopia within 7 days, with an average myopic shift of 3.6D. Four eyes did not develop myopia although they were growing more than their untreated fellow eyes. Two chickens were “non-responders” with less than 1D of relative myopic shift (dashed lines). (B) Average refractions of each group, excluding the two “non-responders” (diffuser n = 8, lens n = 5, control n = 13) during the treatment period. Refraction at day 7 was −1.3 ± 2.1D in the group treated with diffusers and −0.3 ± 1.3D in the group treated with negative lenses. Control eyes remained slightly hyperopic during the whole experiment with an average refraction of 2.3 ± 0.2D. Error bars represent standard deviations.
The average refraction in the deprived eyes of all responding chicks was −0.3 ± 1.6D (n = 8) after 5 days of treatment and reached −1.3 ± 2.1D after 7 days of treatment. Refraction in the lens treated eyes was 0.9 ± 0.6D (n = 5) after 5 days and −0.3 ± 1.3D after 1 week of lens wear (Fig. 4(B)). Control eyes were measured slightly hyperopic during the whole experiment with an average refraction of 2.3 ± 0.2D, similar to typical refractions of other control chickens in the lab. Four chicks had longer eyes after the treatment period, but did not display more myopic refractive errors. Possible reasons include flattening of the anterior segment of the eye with associated longer focal lengths of the optics. In fact, anterior chamber depths were less in the treated compared to the control eyes by 160 ± 10µm. Nevertheless, there was a significant correlation between axial elongation, relative to the untreated fellow eye, and change in refractive state in eyes treated with diffusers (R = 0.70, p = 0.02). In the group treated with negative lenses, this correlation did not achieve significance (R = 0.62, p = 0.2), also because of the low number of myopic animals in this group (Fig. 5). The average change in axial length in all chickens (n = 15) between treated and control eyes was 385 ± 232µm.
Fig. 5 Correlation between changes in refractive errors and axial length. There was a large inter-individual variability in myopia development in the 15 chickens in the two groups, treated with either diffusers (open symbols) or negative lenses (filled symbols). Measurements were taken at the end of the treatment period after 7 days. Each data point denotes one chicken. Differences between both eyes in axial lengths between treated and control eye (Δ axial length) were correlated with differences in refractive errors.
3.2 Changes in fundus reflectivity in white and UV light
During all measurements of fundus reflectivity, individual refractive error was corrected by trial lenses, placed in front of the eye. A typical measurement of pupil brightness in myopic and control eyes after white and UV light flashes is shown in Fig. 6. Pupil brightness increased in UV light when myopia was induced. In contrast, fundus reflectivity in white light did not change during the diffuser or lens treatment period (baseline vs. day 7: 67 ± 2 px vs. 70 ± 4 px and 66 ± 2 px vs. 68 ± 3 px, respectively).
Fig. 6 Pupil brightness during a flash of (A) near-UV and (B) visible broadband white light in untreated control eyes and after induction of myopia.
Fundus reflectivity in UV light remained unchanged in control eyes over the treatment period (first day: 30 ± 5 px, last day: 27 ± 3 px, Fig. 7(A)-(C)), whereas pupils of myopic due to the treatment with diffusers or negative lenses became brighter in UV light than pupils in their fellow eyes with unobstructed vision (diffusers: 48 ± 9 px vs. 28 ± 3 px, p<0.001; lenses: 47 ± 7 px vs. 27 ± 2 px; Fig. 7(C), 6(A)). Interestingly, eyes treated with diffusers displayed increased UV reflectivity already after 40 hours, compared to control eyes, and also compared to minus lens-treated eyes (diffuser vs. contralateral control eye vs. lens: 34 ± 3 px vs. 28 ± 1 px vs. 27 ± 2 px, un-paired t-test with Bonferroni correction, p<0.01, Fig. 7(A)). For the lens-treated eyes, it took two more days for a significant increase in UV reflectivity (day 5: 37 ± 4 px vs. 29 ± 1 px; p<0.05; Fig. 7(B)). In the current study, inter-individual variability of induced myopia was high and the amount of myopia was generally lower than in previous studies from the lab [8]. This might be due to the fact that there was a change in chicken strain by the supplier. Looking at the time course of changes in all chickens which responded to the treatment with diffusers (n = 8, Fig. 7(G)), a significant increased UV fundus reflectivity was measured (Fig. 7(G)). MANOVA analysis for repeated measurements showed a highly significant effect of treatment, time and a time and treatment interaction on fundus reflectivity (MANOVA, p<0.001 for each of these variables). Also in lens treated chicks (n = 5) a significantly increasing UV fundus reflectivity (MANOVA, p<0.001 for treatment, p<0.01 for time, p<0.05 for time and treatment interaction) was found.
Fig. 7 Differences in fundus reflectivity in UV light (A, B, C) and RNFL + RGCL thickness (D, E, F) between treated and control eyes at the three time points of measurement (40 hours, “pre-myopic state”, myopia). Dots represent data of individual eyes. Line plots (G, H) denote changes in fundus reflectivity in UV light (G) and RNFL + RGCL thickness (H) over time, including baseline measurements in all animals (dotted lines). After exclusion of data of two non-responders, there were 8 chicks in the diffuser group and 5 in the lens-treated group. (A, B) UV reflectivity started to increase from the first days of treatment with diffusers. (C) Fundus reflectivity was about 40% higher in the myopic eyes than in controls after 7 days of treatment. (D, E) Significant thinning of the RNFL + RGCL was detected between the third and fifth day of the experiment. (F) The effect was larger in eyes that were treated with diffusers. (paired Student’s t-test for treated vs. control eye, un-paired Student’s t-test with Bonferroni correction for diffuser vs. lens vs. control eye). (G) In both, diffuser (black line) and negative lens (grey line) treated eyes, UV fundus reflectivity increased significantly over time. (H) In treated eyes, a decrease in RNFL + RGCL thickness was measured. Control eyes (dashed lines) remained unchanged over 7 days of experiment. Error bars represent standard error. Significance levels * p< 0.05; ** p<0.01; ***p<0.001.
3.3 Changes in RNFL and RGCL thickness during myopia development
Thickness of retinal layers was analyzed using SD-OCT. In eyes treated with negative lenses and diffusers, thinning of RNFL + RGCL was not yet detectable after 40 hours (Fig. 7(D)) but significant thinning of the RNFL + RGCL occurred between day 3 to 5 of treatment in both diffuser- and lens-treated chicks (34.9 ± 5.3 µm vs. 42.9 ± 5.1 µm and 39.3 ± 3.5 µm vs. 44.7 ± 1.5 µm, respectively; both p<0.05; Fig. 7(E)). Thinning was even more pronounced at the end of the treatment in all nine myopic eyes (7 treated with diffusers 31.2 ± 5.8 µm vs. 43.9 ± 2.6 µm, p<0.001 and two eyes treated with negative lenses 36.9 ± 1.2 µm vs. 44 ± 0.5 µm; Fig. 7(F), n.s.). Looking at the time course of changes in all chickens which responded to the treatment with diffusers (n = 8), a decreased RNFL + RGCL thickness was measured (Fig. 7(H)). MANOVA analysis for repeated measurements showed a highly significant effect of treatment, time and a time and treatment interaction on RNFL + RGCL thickness (MANOVA, p<0.01 for treatment, p<0.001 for time and time and treatment interaction). Also in lens treated chicks (n = 5) a significantly decreasing RNFL + RGCL thickness (MANOVA, p<0.05 for treatment) was found. It was striking that, except for the RNFL + RGCL, there was no significant thinning of retinal layers, even though the posterior globe expanded (194 ± 18 µm vs. 220 ± 19 µm).
The differences in fundus reflectivity between treated and control eyes were significantly correlated with the amount of induced myopia, both in diffuser and negative lens-treated chickens (R = 0.66; p<0.0001 and R = 0.77; p<0.001, respectively; Fig. 8(A)). Fundus reflectivity in UV light was not only correlated with induced myopia but also with ocular axial length (diffuser (n = 8): R2 = 0.45, lens (n = 5): R2 = 0.7). RNFL + RGCL thickness was significantly negatively correlated with the amount of induced myopia in chicks treated with diffusers (Fig. 8(B), R = 0.69; p<0.001). In chicks treated with lenses, such a correlation was not observed (Fig. 8(B); R = 0.19; p = 0.49). Instead, RNFL + RGCL thickness were significantly negatively associated with axial length in lens treated eyes (diffuser (n = 8): R2 = 0.28, lens (n = 5): R2 = 0.89).
Fig. 8 (A) Correlations between changes in refraction and fundus reflectivity in UV light. Fundus reflectivity in UV light increased with increasing myopia (open symbols: diffusers, R = 0.82, p<0.0001; filled symbols: lenses, R = 0.77, p<0.001). (B) Correlations between changes in refraction and thickness of RNFL + RGCL. Thickness declined with increasing myopia in eyes with diffusers (R = 0.82, p<0.001), but not in eyes with negative lenses (R = 0.19, p = 0.49). Each data point represents one chicken. Data are pooled from the three time points of measurement. Data of the two non-responders were excluded.
3.4 Correlations between changes in fundal reflectivity and RNFL and RGCL thickness during myopia development
A significant correlation between UV reflectivity and thickness of the RNFL + RGCL was found in eyes treated with diffusers (R = 0.64; p<0.001). In eyes treated with negative lenses, there was only a trend (R = 0.46; p = 0.08) which is likely due to the smaller range of changes in RNFL + RGCL thickness in eyes treated with lenses (Fig. 9). Even though a correlation by itself does not prove a causal link, it is possible that enhanced UV reflectivity resulted from thinning of the RNFL + RGCL.
Fig. 9 Correlations between the thickness of the RNFL + RGCL and fundus reflectivity in UV light. In eyes treated with diffusers, the two variables were clearly correlated (R = 0.64, p<0.001) but there was only a trend in lens-treated eyes. As in Fig. 8, data are pooled from all time points of measurement and do not include data from two non-responders.
3.5 Short-term effects of diffusers on fundus UV reflectivity
Seven chickens wore diffusers for 5 hours. The treatment time was too short to induce measurable changes in ocular biometry (axial length: diffuser vs. control: 9.32 ± 0.29mm vs. 9.30 ± 0.29mm; vitreous chamber depth: 5.34 ± 0.10mm vs. 5.32 ± 0.10mm, RNFL thickness: 42.8 ± 2.0µm vs. 43.7 ± 2.8µm, and retinal thickness: 265 ± 15µm vs. 275 ± 12µm). Nevertheless, fundus reflectivity in UV light increased, with a trend after 3 hours and a significant elevation after 5 hours (diffusers 39 ± 6.3px vs. control 30 ± 2.1px, paired Student’s t-test, p<0.01, Fig. 10).
Fig. 10 Changes in fundus reflectivity in UV light during the first 5 hours of diffuser wear. Eyes with diffusers developed higher reflectivity compared with fellow eyes with unobstructed vision (39 ± 6.3px vs. 30 ± 2.1px, paired Student’s t-test p<0.01). Each dot represents data from an individual animal. Error bars represent standard errors. Significance levels * p< 0.05; ** p<0.01; ***p<0.001.
4. Discussion
In an attempt to identify potential biomarkers of myopia development that could be used in vivo, we found that fundus reflectivity in near UV light increased during induction of myopia in the chicken model. We also found that the increase in UV reflectivity was correlated with the amount of induced deprivation and lens induced myopia. As a possible morphological correlate we found that the retinal nerve fiber layer (RNFL) and the retinal ganglion cell layer (RGCL) were thinning in correlation with the increase of the UV reflectivity. However, a short term experiment showed that fundus reflectivity was already increased after 5 hours of diffuser wear, clearly too early to generate measurable changes in ocular biometry and retinal layer thickness. These findings suggest that the changes in UV reflectivity were not (only) due to the thinning of the RNFL.
In eyes in which myopia was induced by negative lenses, such changes were also observed but they were less pronounced and also delayed in time. However the amount of myopia induced by the lenses was also lower and there were less chickens available in the lens-treated group. Myopia in animal models is inherently variable. In general, imposing defocus by lenses represents a closed-loop trigger of the emmetropization feedback loop and generates therefore less variable refractions than retinal image degradation by diffusers which represent an open-loop condition as the eye has no chance to improve retinal image by any kind of growth [48,60]. The amount of induced deprivation myopia is therefore largely determined by genetic factors. In fact, Chen et al. has shown that chickens can be selectively bred to develop either high or low amounts of deprivation myopia in only two generations [61]. In the current study, it was helpful that the induced deprivation myopia was highly variable because the variability permitted correlations with RNFLs and RGCLs, as well as with fundus reflectivity.
4.1 Why might there be selective thinning of the RNFL and the RGCL?
A striking finding was that the inner retinal layers (RNFL + RGCL) were thinning during development of myopia but that these changes could not simply be attributed to stretching since the other retinal layers did not change significantly, even at the end of the experiment when the eyes were clearly longer and myopic (thickness between IPL and RPE - myopic eyes: 194 ± 18 µm vs. controls: 220 ± 19 µm). A possible explanation is that, with diffusers in front of the eyes, the retinal image is low pass-filtered and also has low contrast, both factors reducing spatial information. It is likely that retinal ganglion cells, sampling the retinal image with their circular ON or OFF antagonistic field structures, are less stimulated when diffusers are worn. We hypothesize that less information is transmitted by the ganglion cell axons which could induce smaller axon diameters. Recently, it was shown in the auditory system that axon diameters and myelination are experience dependent [62]. Mice that were partially deprived from acoustic stimulation by wearing ear plugs developed reduced myelination and axons diameters in the trapezoid body. The decline in myelin sheet thickness amounted to up to about 50 percent. In our study, the thinning of the RNFL + RGCL reached about 30 percent (Fig. 8(B)). Lazari et al. [63], discussing the study by Sinclair et al. [62], emphasized that “experience-related reductions of myelin and axons diameters persist also in adulthood”. It is known that information flux in an axon is dependent on its diameter. In fact, there might be a dynamic adaptation of “cable volume” to its “information capacity”, as has been described by Sterling for the axons of rods and cones in the retina [64]. We propose that the thickness of the RNFL and the RGCL might be related to the average amount of captured visual information. Extrapolated to humans, it could be that reduced RNFL + RGCL thickness might represent a biomarker for a previous history of low pass filtering and defocus. However, it is clear that a direct demonstration is necessary that thinning of the RNFL + RGCL is due to thinning of the ganglion cell axons.
Studies in human subjects have shown that changes in neural retina are not uncommon among myopic subjects [41,65]. Most of the studies measuring RNFL abnormalities were carried out on glaucomatous eyes [66–69]. Nevertheless, thinning of the RNFL has also previously been reported in myopic human subjects [70–74]. While most studies in humans showed that RNFL thickness is reduced only in high myopia, recent work showed that such changes can already be detected in low and intermediate levels of myopia although only in specific subfields. It was concluded that a specific pattern exists for myopia influencing RNFL thickness [70].
4.2 What could cause the changes in UV reflectivity when myopia develops?
The increased UV reflectivity after 40 hours of treatment could be related to a change in layer thickness and /or a re-arrangement inside axons in the nerve fiber layer of the retina. However, in the current study, a wide range of baseline thicknesses was found for the RNFL and RGCL (35 – 53 µm) already in untreated eyes. Despite the variance in thickness, fundus reflectivity in UV light was always low in untreated eyes. A thin RNFL and RGCL can therefore not be the only reason for the high fundus reflectivity in eyes with diffusers. In line with our findings, previous studies have shown that changes in RNFL reflectivity occur mainly in the short wavelengths range (<560nm) [75–78]. In chicks, changes in reflectivity were restricted to the near UV range; they can therefore not be measured in humans since the ocular media scarcely transmit UV light. In addition to changes in thickness, other optical properties may also change such as biochemical parameters in the axons which depend on retinal activity. Huang and colleagues proposed a biophysical model of RNFL reflectivity speckles. They considered axon bundles as cylindrical structures, which actively transport molecules between cells, producing a characteristic speckle pattern. Each change in the dynamics in this mechanism could change the speckle texture. In-vitro studies have shown changes of RNFL reflectivity over time when retinal activity was modulated [79,80]. In other studies it was also found that differences in reflectivity can occur e.g. during axonal degeneration in rat model of glaucoma [81]. In our experiment we used incoherent light. An advantage of our method is that changes in RNFL and RGCL in the chicken can be easily detected by measuring UV reflectivity of the fundus in vivo. Therefore, UV reflectivity can be considered a biomarker for myopia development in the chicken (Fig. 11). Another possible explanation for the changes in fundus reflectivity could be changes in biochemical messengers during the development of DM and LIM, for instance decreased dopamine levels or downregulation of the transcription factor Erg-1 which should change retinal metabolism and ultrastructure, also affecting reflectivity [82,83].
Fig. 11 Proposed biomarker of myopia development. Compared to control eyes with normal visual exposure (open circles in the bottom) eyes developing myopia had thinner RNFL + RGCL and higher UV reflectivity (encircled in red). Data from the last day of experiment which included only animals which developed myopia after 7 days of treatment (DM: n = 7 and LIM: n = 2).
5. Summary and conclusions
In chickens, fundus reflectivity in near UV light increases concomitantly with development of myopia. At the same time, the thickness of the retinal nerve fiber layer (RNFL) and the retinal ganglion cell layer (RGCL) decreases. If myopia was induced by negative lenses, similar changes were detected but with some time delay. We hypothesize that thinning of the nerve fiber layer may be due to the reduced visual information that is transmitted through the optic nerve when the eye is covered by a diffuser or a lens. If this would be true, previously experienced poor retinal image quality should be reflected in a thinner RNFL and RGCL. While such changes can be measured in humans and chicks by OCT, they can also be studied in chickens by simply measuring fundus UV reflectivity.
Funding
European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie Research Training Network MyFun Grant (MSCA-ITN-2015-675137).
Acknowledgments
We acknowledge the support by Open Access Publishing Fund of University of Tübingen.
Disclosures
The authors declare that there are no conflicts of interest related to this article.
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