Associate Professor Sally McFadden

Purpose: The crystalline lens undergoes morphological and functional changes with age and may also play a role in eye emmetropisation. Both the geometry and the gradient index of refraction (GRIN) distribution contribute to the lens optical properties. We studied the lens GRIN in the guinea pig, a common animal model to study myopia. Methods: Lenses were extracted from guinea pigs (Cavia porcellus) at 18¿days of age (n¿=¿4, three monolaterally treated with negative lenses and one untreated) and 39¿days of age (n¿=¿4, all untreated). Treated eyes were myopic (-2.07¿D on average) and untreated eyes hyperopic (+3.3¿D), as revealed using streak retinoscopy in the live and cyclopeged animals. A custom 3D spectral domain optical coherence tomography (OCT) system¿(¿¿=¿840¿nm, ¿¿¿=¿50¿nm) was used to image the enucleated crystalline lens at two orientations. Custom algorithms were used to estimate the lens shape and GRIN was modelled with four variables that were reconstructed using the OCT data and a minimisation algorithm. Ray tracing was used to calculate the optical power and spherical aberration assuming a homogeneous refractive index or the estimated GRIN. Results: Guinea pig lenses exhibited nearly parabolic GRIN profiles. When comparing the two age groups (18- and 39¿day-old) there was a significant increase in the central thickness (from 3.61 to 3.74¿mm), and in the refractive index of the surface (from 1.362 to 1.366) and the nucleus (from 1.443 to 1.454). The presence of GRIN shifted the spherical aberration (-4.1¿µm on average) of the lens towards negative values. Conclusions: The guinea pig lens exhibits a GRIN profile with surface and nucleus refractive indices that increase slightly during the first days of life. GRIN plays a major role in the lens optical properties and should be incorporated into computational guinea pig eye models to study emmetropisation, myopia development and ageing.

SIGNIFICANCE: This study shows that nonvisual mechanism(s) can guide chick eyes to recover from myopia or hyperopia bidirectionally to regain their age-matched length. Because eye growth control is phylogenetically conserved across many species, it is possible that, in general, emmetropization mechanisms are not exclusively based on a local visual feedback system. PURPOSE: Across species, growing eyes compensate for imposed defocus by modifying their growth, showing the visual controls on eye growth and emmetropization. When the spectacle lens is removed, the eyes rapidly recover back to a normal size similar to that in the untreated eyes. We asked whether this recovery process was dependent on visual feedback or whether it might be guided by intrinsic nonvisual mechanisms. METHODS: Chicks wore either a +7 (n = 16) or -7 D (n = 16) lens over one eye for 4 to 7 days; the fellow eye was left untreated. After lens removal, half were recovered in darkness and half in white light. Refractive error and ocular dimensions were measured before and after lens treatment and after recovery with a Hartinger refractometer and A-scan biometer, respectively. RESULTS: Whereas chick eyes completely recovered from prior lens treatment under normal light after 2 days, they also partially recovered from prior hyperopia (by 60%) and myopia (by 69%) after being kept in darkness for 3 days: a +7 and -7 D lens induced a difference between the eyes of +7.08 and -4.69 D, respectively. After recovery in darkness, the eyes recovered by 3.18 and 2.88 D, respectively. CONCLUSIONS: In the absence of visual cues, anisometropic eyes can modify and reverse their growth to regain a similar length to their fellow untreated eye. Because eye growth control is phylogenetically conserved across many species, it is possible that nonvisual mechanisms may contribute more generally to emmetropization and that recovery from anisometropic refractive errors may not be wholly visually controlled.

PURPOSE. Posterior scleral remodeling accompanies myopia. In guinea pigs developing myopia, the region around the optic nerve (peripapillary zone, PPZ) rapidly expands followed by inhibition in eye size in the periphery. We studied the differential gene expression in the sclera that accompanies these changes. METHODS. Guinea pigs were form-deprived (FD) for 2 weeks to induce myopia, while the fellow eye served as a control. After 2 weeks, the PPZ and the peripheral temporal sclera were isolated in representative animals to extract the RNA. RNA sequencing was undertaken using an Illumina HiSeq 2000, with differential expression analyzed using Voom and pathways analyzed using the Ingenuity Pathway Analysis tool. RNA from additional PPZ and peripheral temporal sclera in FD and fellow eyes was used for validation of gene expression using quantitative real-time PCR (qRT-PCR). RESULTS. In myopic sclera, 348 genes were differentially expressed between PPZ and the peripheral temporal region (corrected P < 0.05), of which 61 were differentially expressed in the PPZ between myopic and control eyes. Pathway analyses of these gene sets showed the involvement of Gai signaling along with previously reported gamma-aminobutyric acid (GABA) and glutamate receptors among numerous novel pathways. The expression pattern of three novel genes and two myopia-related genes was validated using qRT-PCR. CONCLUSIONS. Gene expression changes are associated with the rapid elongation that occurs around the optic nerve region during the development of myopia. A prominent change in Gai signaling, which affects cAMP synthesis and thus collagen levels, may be critical in mediating the regional changes in myopic sclera.

Myopia is generally regarded as a failure of normal emmetropization process, however, its underlying molecular mechanisms are unclear. Retinal protein profile changes using integrated SWATH and MRM-HR MS were studied in guinea pigs at 3- and 21-days of age, where the axial elongation was significantly detected. Differential proteins expressions were identified, and related to pathways which are important in postnatal development in retina, proliferation, breakdown of glycogen-energy and visual phototransduction. These results are significant as key retinal protein players and pathways that underlying emmetropization can be discovered. All raw data generated from IDA and SWATH acquisitions were accepted and published in the Peptide Atlas public repository (http://www.peptideatlas.org/) for general release (Data ID PASS00746). A more comprehensive analysis of this data can be obtained in the article ¿Integrated SWATH-based and targeted-based proteomics provide insights into the retinal emmetropization process in guinea pig¿ in Journal of Proteomics (Shan et al., 2018) [1].

The current study aimed to investigate the differential protein expression in Guinea pig retinas in response to lens-induced myopia (LIM) before fully compensated eye growth. Four days old Guinea pigs (n=5) were subjected to -4D LIM for 8 days. Refractive errors were measured before and at the end of the lens wear period. Ocular dimensions were also recorded using high-frequency A-scan ultrasonography. After the LIM treatment, retinas of both eyes were harvested and soluble proteins were extracted. Paired retinal protein expressions in each animal were profiled and compared using a sensitive fluorescence difference two-dimensional gel electrophoresis. The quantitative retinal proteomes of myopic and control eye were analysed using computerised DeCyder software. Those proteins that were consistently changed with at least 1.2-fold difference (P<0.05) in the same direction in all five animals were extracted, trypsin digested and identified by tandem mass spectrometry. Significant myopia was induced in Guinea pigs after 8 days of lens wear. The vitreous chamber depth in lens-treated eyes was found to be significantly elongated. Typically, more than 1,000 protein spots could be detected from each retina. Thirty-two of them showed differential expression between myopic and untreated retina. Among these proteins, 21 spots were upregulated and 11 were downregulated. Eight protein spots could be successfully identified which included ß-actin, enolase 1, cytosolic malate dehydrogenase, Ras-related protein Rab-11B, protein-L-isoaspartate (D-aspartate) O-methyltransferase, PKM2 protein, X-linked eukaryotic translation initiation factor 1A and ACP1 protein. The present study serves as the first report to uncover the retinal 2D proteome expressions in mammalian Guinea pig myopia model using a top-down fluorescent dyes labelling gel approach. The results showed a downregulation in glycolytic enzymes that may suggest a significant alteration of glycolysis during myopia development. Other protein candidates also suggested multiple pathways which could provide new insights for further study of the myopic eye growth.

Myopia is generally regarded as a failure of normal emmetropization process, however, its underlying molecular mechanisms are unclear. To investigate the retinal protein profile changes during emmetropization, we studied differential protein expressions of ocular growth in young guinea pigs at 3 and 21 days old respectively, when significant axial elongation was detected (P < 0.001, n = 10). Independent pooled retinal samples of both eyes were subjected to SWATH mass spectrometry (MS) followed by bioinformatics analysis using cloud-based platforms. A comprehensive retina SWATH ion-library consisting of 3138 (22,871) unique proteins (peptides) at 1% FDR was constructed. 40 proteins were found to be significantly up-regulated and 8 proteins down-regulated during emmetropization (=log2 of 0.43 with =2 peptides matched per protein; P < 0.05). Using pathway analysis, the most significant pathway identifiable was ¿phototransduction¿ (P = 1.412e-4). Expression patterns of 7 proteins identified in this pathway were further validated and confirmed (P < 0.05) with high-resolution Multiple Reaction Monitoring (MRM-HR) MS. Combining discovery and targeted proteomics approaches, this study for the first time comprehensively profiled protein changes in the guinea pig retina during normal emmetropization-associated eye growth. The findings of this study are also relevant to the myopia development, which is the result of failed emmetropization. Significance: Myopia is considered as a failure of emmetropization. However, the underlying biochemical mechanism of emmetropization, a visually guided process in which eye grows towards the optimal optical state of clear vision during early development, is not well understood. Retina is known as the key tissue to regulate this active eye growth. we studied eye growth of young guinea pigs and harvested their retinal tissues. A comprehensive SWATH ion library with identification of a total 3138 unique proteins were established, in which 48 proteins exhibited significant differential expressions between 3 and 21 days old. After MRM-HR confirmation, ¿phototransduction¿ were found as the most active pathway during emmetropic eye growth. This study is the first in discovering key retinal protein players and pathways which are presumably orchestrated by biological mechanism(s) underlying emmetropization.

PURPOSE. It has been proposed that the peripheral retina, responding to local optical defocus, contributes to myopia and associated altered eye growth in humans. To test this hypothesis, we measured the changes in central (on-axis) and peripheral ocular dimensions in guinea pigs wearing a concentric bifocal spectacle lens design with power restricted to the periphery. METHODS. Five groups of guinea pigs (n = 83) wore either a unifocal (UF) spectacle lens (-4, 0, or +4 Diopters [D]), or a peripheral defocus (PF) spectacle lens that had a plano center (diameter of 5 mm) with either -4 or +4 D in the surround (-4/0 or +4/0 D). The overall optical diameter of all lenses was 12 mm. Lenses were worn over one eye from 8 to 18 days of age for negative and plano lenses, or from 8 to 22 days of age for positive lenses. Refractive error was measured centrally and 30° off-axis in the temporal and nasal retina. The shape of the eye was analyzed from images of sectioned eyes. RESULTS. Lenses of -4 D UF induced myopia, reflecting enhanced ocular elongation, which was centered on the optic nerve head and included the surrounding peripapillary zone (PPZ, 188 in diameter). Some ocular expansion, including within the PPZ, also was recorded with -4/0 and +4/0 D PF lenses while the +4 D UF lens inhibited rather than enhanced elongation, centrally and peripherally. CONCLUSIONS. Peripheral defocus-induced ocular expansion encompasses the PPZ, irrespective of the sign of the inducing defocus. Understanding the underlying mechanism potentially has important implications for designing multifocal lenses for controlling myopia in humans and also potentially for understanding the link between myopia and glaucoma.

Custom Spectral Optical Coherence Tomography (SOCT) provided with automatic quantification and distortion correction algorithms was used to measure the 3-D morphology in guinea pig eyes (n = 8, 30 days; n = 5, 40 days). Animals were measured awake in vivo under cyclopegia. Measurements showed low intraocular variability (<4% in corneal and anterior lens radii and <8% in the posterior lens radii, <1% interocular distances). The repeatability of the surface elevation was less than 2 µm. Surface astigmatism was the individual dominant term in all surfaces. Higher-order RMS surface elevation was largest in the posterior lens. Individual surface elevation Zernike terms correlated significantly across corneal and anterior lens surfaces. Higher-order-aberrations (except spherical aberration) were comparable with those predicted by OCT-based eye models.

METHODS. To induce accelerated growth and myopia, guinea pigs wore a -5 diopter (D) lens over one eye from 4 to 11 days of age. To induce inhibited growth, the lens was removed after 7 days of -5 D lens wear, and the eye allowed to recover from myopia for 3 days. Ocular parameters and Egr-1 mRNA levels were subsequently assessed, and compared to untreated fellow eyes and eyes from untreated littermates. Possible circadian changes in Egr-1 mRNA levels were also determined in 18 additional animals by taking measures every 4 hours during a 24-hour cycle.

Many lateral eyed birds are bifoveate or have dual retinal specialisations corresponding with their lateral and frontal viewing modes. For example, the eye design in the pigeon, which is typical of many granivorous birds, incorporates an optic axis for lateral distant viewing and a frontal area in the temporal retina associated with close binocular viewing. In this paper we ask how these two separate visual axis are used to focus on objects, not only at different eccentricities, but also at different absolute distances. Using simultaneous infrared (IR) photoretinoscopy and IR keratometry, we find that the pigeons optical system can indeed simultaneously support differential refractive states on the lateral and frontal axis. Maps of the refractive error (RE) and corneal power in cyclopleged and anaesthetized pigeons, reveal small but antagonistic gradients in different sectors of the visual field. In particular, we find that at rest, the frontal field is relatively myopic and yet the cornea has less power in this sector. We studied how these RE and corneal power maps actively vary during a reaching behaviour. We found that during the saccadic like peck response, the optic axis is not focussed on the grain and has only 30% of the required accommodation power. In contrast, we calculate that the frontal axis does have adequate power to focus on the grain. Furthermore, at the last stationary head fixation position (which occurs at an eye-grain distance of 67mm, SD=3.9mm) before the final descent when the eyes begin to close, accommodation on the frontal axis can be supported wholly by the cornea, which allows dramatic RE changes (-13D, SD=7.7) without unduly effecting the refractive error on the optic axis (-2D, SD=1.7). We propose that the shape of the cornea facilitates this local accommodation mechanism and that this dual visual system can be simultaneously focussed at disparate distances in space.

The amplitude of saccadic eye movements is adjusted by the oculomotor system. If a target is surreptitiously moved during saccades so that the eye lands beyond the target, a gradual decrease in the saccadic gain (saccade-size divided by target-displacement) ensues. What error signals guide this adaptation? We propose that visual attention helps keep track of the target across saccades, so that saccade accuracy can be determined by comparing the location of the fovea with the locus of attention. If attention plays this role, saccadic errors might be more apparent when the size of the attentional field is small than when it is large. We tested this by a conventional saccade adaptation paradigm in which the target stepped 8Ã0 and then stepped back by 30% during each saccade, while the subject did one of two psychophysical tasks demanding either a small or large attentional field. Specifically, the saccade target consisted of a large outer (8.5°) and a small inner (0.8°) ring, each with several breaks, rotating in opposite directions. After each saccade, the number of breaks transiently changed. Subjects reported the number of breaks present during this period in the attended ring. Saccadic gain was assessed both during adaptation and by comparing blocks of saccades before and after 250 adaptation trials using a spot target that disappeared upon saccade onset. After adaptation, saccadic gain had decreased four times as much if subjects attended to the small ring than to the large (small ring: -0.11, SD, 0.031; large ring: -0.027, SD, 0.038, paired t-test, p<0.01). Also, 4 of the 5 subjects had significantly negative gain slopes during the adaptation when attending to the small ring but none did when attending to the large ring. These findings argue that attention plays an important role in the adjustment of saccadic gain and suggest that the spatial scale of attention influences whether saccades are deemed to be accurate or in need of correction.

© OSA 2016.We quantified anterior-segment geometry in both control and lens-treated eyes of a guinea pig model in vivo, using custom-developed optical coherence tomography. Myopic eyes showed longer axial-lengths, thinner corneas, longer anterior-chamber-depth and steeper anterior lens.

© OSA 2016.Myopia is induced when growing eyes are exposed to hyperopic defocus or reduced vision. The associated changes and factors that influence the development of myopia in the guinea pig eye are described.