Systematic Analysis of Transcriptomic Profile of the Effects of Low Dose Atropine Treatment on Scleral Fibroblasts using Next-Generation Sequencing and Bioinformatics.

This study has two novel findings: it is not only the first to deduct potential genes involved in scleral growth repression upon atropine instillation from a prevention point of view, but also the first to demonstrate that only slight changes in scleral gene expression were found after atropine treatment as side effects and safety reasons of the eye drops are of concern. The sclera determines the final ocular shape and size, constituting of scleral fibroblasts as the principal cell type and the major regulator of extracellular matrix. The aim of our study was to identify differentially expressed genes and microRNA regulations in atropine-treated scleral fibroblasts that are potentially involved in preventing the onset of excessive ocular growth using next-generation sequencing and bioinformatics approaches. Differentially expressed genes were functionally enriched in anti-remodeling effects, comprising of structural changes of extracellular matrix and metabolic pathways involving cell differentiation. Significant canonical pathways were correlated to inhibition of melatonin degradation, which was compatible with our clinical practice as atropine eye drops are instilled at night. Validation of the dysregulated genes with previous eye growth-related arrays and through microRNA-mRNA interaction predictions revealed the association of hsa-miR-2682-5p-KCNJ5 and hsa-miR-2682-5p-PRLR with scleral anti-remodeling and circadian rhythmicity. Our findings present new insights into understanding the anti-myopic effects of atropine, which may assist in prevention of myopia development.

On Intraspecific and Interspecific Variation in Teleost Scleral Ossification.

Scleral ossicles are bony elements found along the eyes of many fishes, amphibians, and reptiles. These bones provide a superficial layer of support to the eye and may facilitate visual acuity. Previous research has shown that scleral ossicle diversity is generally limited among teleosts, but that scleral ossicles have been lost numerous times among teleosts inhabiting benthopelagic habitats (Franz-Odendaal. Anat Rec 291 (2008) 161-168). In this study, we further investigate these patterns of intraspecific and interspecific variation by examining eyes from multiple individuals of 10 riverine teleosts native to Kentucky as well as one population of the Mexican blind cavefish, Astyanax mexicanus, and by re-analyzing a quantitative database of scleral ossicle number and depth preference from over 100 teleosts using newly resolved teleost phylogenies. Consistent with the limited diversity of most teleost families, we find that intraspecific variation in scleral ossicle number and size is virtually nonexistent among the species sampled, although we do find evidence of additional interspecific variation among the Cyprinodontiformes, as well as dramatic intrapopulation variation among cavefish from Chica Cave. Although our data replicates the negative relationship between scleral ossicle number and the depth preference previously found among teleosts (Franz-Odendaal. Anat Rec 291 (2008) 161-168), even when accounting for phylogenetic relationships, our results further reveal that this relationship is relatively weak. We conclude that further sampling may reveal additional interspecific and even intraspecific variation among some groups of teleosts, and that depth could serve as a proxy for other life history traits that more directly influence teleost scleral ossicle diversity such as prey-capture strategies. Anat Rec, 302:1238-1249, 2019. © 2019 Wiley Periodicals, Inc.

The pilocarpine-induced ciliary body contraction affects the elastic modulus and collagen of cornea and sclera in early development.

The aim of this study was to investigate the effects of pilocarpine-induced ciliary body constant contraction for a long time period on the elastic modulus and collagen in corneal and scleral tissues in the early developmental stage. Twelve one-month-old New Zealand white rabbits were randomly monocularly treated with pilocarpine to cause ciliary body constant contraction. After 1- and 2-months, the sclera tissues and the eyeballs were obtained to assess the cornea and three regions of the sclera-anterior, equatorial and posterior. The corneal tissues and the three regions of the scleral tissues were cut into strips for elastic modulus measurement using an Instron 5544, the size distribution of collagen fibrils was examined using electron microscopy, the samples were homogenized, and the concentration of hydroxyproline was measured to determine the collagen content. For corneal tissues, long-term pilocarpine-induced ciliary body constant contraction did not affect the elastic modulus and collagen. For scleral tissues, long-term pilocarpine-induced ciliary body constant contraction affected the elastic modulus, diameter of collagen fibrils and collagen content in the equatorial and posterior sclera, however, the anterior sclera were unaffected. These results suggested that pilocarpine-induced ciliary body contraction could affect the scleral structure and lead to deformation of the sclera and eyeball, thereby affecting visual functions function in the process of ocular emmetropization.

Development of the New Zealand White Rabbit Eye: I. Pre- and Postnatal Development of Eye Tunics.

The New Zealand white (NZW) rabbit has been and is right now regularly utilized in ophthalmic surgery evaluation. Inside NZW rabbit eye, the visibility of ocular structures throughout surgical procedure is fantastic. Younger rabbits are used in different ages for the evaluation of ophthalmic surgery. Complete studies of ocular development in the NZW rabbits have not been reported previously. The aim of the present investigation was to describe the major landmarks and the time course of the pre- and post-natal development of the complete eye tunics of the NZW rabbit to give a superb model as well as a fruitful area for further ophthalmological investigations. Serial histological sections of NZW rabbit prenatal (E13-E28) and post-natal (P1-P14) stages were examined, respectively. The eye of the NZW rabbit developed in a similar manner to that of the human and domestic animals eyes; the principal differences were at the time of occurrence of certain developmental events, absence of pigmentation which represent an exploited benefit for ophthalmic surgery, remarkable Bowman's membrane at E25, poor developed ciliary stroma and juvenile retinal layer until P9. In human, the basic morphogenetic processes of the development of eye tunics are completed towards the end of the first half of gestation period. However, the latter represents the beginning stage of the development of eye tunics in the rabbit. Thus, allowing various extensive ophthalmic researches to be performed.

A wide temporal window for conjunctival papillae development ensures the formation of a complete sclerotic ring.

BACKGROUND: The conjunctival papillae are epithelial thickenings of the conjunctiva that are required for the induction of underlying bones (the scleral ossicles). These transient papillae develop and become inductively active over an extended temporal period (HH 30-36, 6.5-10 dpf). While their inductive capacity was discovered in the mid-1900s, little is known about their development. RESULTS: Through a series of timed surgical ablations followed by in situ hybridization for Bmp2, we show that the ring of conjunctival papillae is not altered if the conjunctival epithelium is ablated either prior to or shortly after papillae induction (i.e., HH 29-30, 6.5-7 dpf). A conjunctival papilla ablated at or prior to HH 34 (8 dpf), when the complete ring is present, regenerates and quickly becomes inductively active, inducing an underlying scleral condensation with only a slight delay. This regenerative capacity extends until HH 35.5, a full 36 hours beyond the normal timeline of papillae induction. As such, the period of epithelial competency for papilla induction is longer than previously identified. CONCLUSIONS: Papilla regeneration is a mechanism that ensures the formation of a complete sclerotic ring and provides another level of redundancy for the induction of a complete sclerotic ring during the normal inductive period. Developmental Dynamics 246:381-391, 2017. © 2017 Wiley Periodicals, Inc.

Anatomical differences of the protein profile in the rabbit sclera during growth.

We aimed to investigate the proteome changes in anatomical regions of sclera during growth and development of the rabbit. Sclera from New Zealand white rabbits of three ages (1 month, 2 months and 6 months) was dissected into three segments - anterior, equatorial, and posterior. A total of 36 samples were divided into groups by age and anatomical region. Tryptic digests of total proteins were analyzed by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). Label-free quantification based on spectral counts was used to determine the relative protein abundance and identify proteins with statistically significant differences between age groups or anatomical regions of the sclera. Western blotting was performed to validate some of the differentially expressed proteins. A total of 840 non-redundant proteins was identified in the sclera at different ages and regions with protein and peptide false discovery rate (FDR) at ≤1.0% and ≤0.1%, respectively. Differentially expressed proteins were identified by comparing age or anatomical region. Among these, periostin showed decreasing abundance with age, while myocilin, latent-transforming growth factor beta-binding protein 2, hyaluronan, proteoglycan link protein 1 and selenbp1 showed increasing abundance with age. In mature rabbits, alcohol dehydrogenase showed region-related differences in the sclera. Periostin showed an age-related decrease while selenbp1 showed an age-related increase in abundance in the anterior region. Vitronectin and extracellular superoxide dismutase had greater expression with age in the equatorial and posterior regions, respectively. The age related differential expression of periostin and selenbp1 was confirmed by western blotting. In conclusion, the protein profile of sclera showed age- and region-related differences. The differential protein profiles provide a baseline for understanding changes in the protein expression in the young and mature rabbit that appears to show regional changes. The changes observed in the present study add to the existing knowledge about regional alterations in biomechanical properties of sclera during growth.

Effects of Glucose Administration on Development of Sclera in Chick Embryos.

OBJECTIVE: To determine the effect of glucose administration on the development of sclera in the chick embryo Gallus domesticus. STUDY DESIGN: Experimental study. PLACE AND DURATION OF STUDY: Anatomy Department, CPSPRegional Centre, Islamabad, from January 2013 to January 2014. METHODOLOGY: The study was carried out in two main groups, control Aand experimental B, which were subdivided into three subgroups comprising 30 eggs each. The group Awas injected with normal saline (0.3 ml) in the egg albumen. The group B was injected with 0.3 ml of 5% w/v solution of glucose equivalent to 15 mg of glucose. Subgroups A1 and B1 were opened on day 10 of incubation. Subgroups A2 and B2 were sacrificed on day 12 of incubation. Eggs from subgroups A3 and B3 were opened on day 15 of incubation. Experimental subgroups were compared with matched control subgroups and quantitative data was analysed statistically. RESULTS: Administration of glucose resulted in changes in thickness of sclera. The mean thickness (µm) of sclera at day 10 of incubation was 43.54 ±2.45 in control subgroup and 43.03 ±5.86 in the experimental subgroup (p=0.673). The mean thickness (µm) of sclera at day 15 of incubation 77.48 ±8.32 in control subgroup and 73.99 ±8.62 in experimental subgroup (p=0.145). The mean number of chondrocytes/unit area of hyaline cartilage of sclera in day 10 was 17.40 ±1.44 control subgroup and 14.57 ±1.87 in the experimental subgroup (p < 0.001). The mean number of chondrocytes/unit area of hyaline cartilage of sclera on day 15 was 10.02 ±0.86 in the control subgroup and 9.54 ±0.59 in the experimental subgroup (p=0.025). There was disrupted ossicular formation indicating adverse effects on the development of bony sclera as well. CONCLUSION: Administration of glucose caused alteration in the histology of sclera in developing chick embryos.

Scleral Mechanisms Underlying Ocular Growth and Myopia.

In the regulation of ocular growth, scleral events critically determine eye size and thus the refractive status of the eye. Increased scleral matrix remodeling can lead to exaggerated eye growth causing myopia and additionally increased risk of ocular pathological complications. Thus, therapies targeting these changes in sclera hold potential to limit such complications since sclera represents a relatively safe and accessible drug target. Understanding the scleral molecular mechanisms underlying ocular growth is essential to identifying plausible therapeutic targets in the sclera. This section provides a brief update on molecular studies that pertain to the sclera in the context of ocular growth regulation and myopia.

The dynamic sclera: extracellular matrix remodeling in normal ocular growth and myopia development.

Myopia is a common ocular condition, characterized by excessive elongation of the ocular globe. The prevalence of myopia continues to increase, particularly among highly educated groups, now exceeding 80% in some groups. In parallel with the increased prevalence of myopia, are increases in associated blinding ocular conditions including glaucoma, retinal detachment and macular degeneration, making myopia a significant global health concern. The elongation of the eye is closely related to the biomechanical properties of the sclera, which in turn are largely dependent on the composition of the scleral extracellular matrix. Therefore an understanding of the cellular and extracellular events involved in the regulation of scleral growth and remodeling during childhood and young adulthood will provide future avenues for the treatment of myopia and its associated ocular complications.

Scleral gene expression during recovery from myopia compared with expression during myopia development in tree shrew.

PURPOSE: During postnatal refractive development, the sclera receives retinally generated signals that regulate its biochemical properties. Hyperopic refractive error causes the retina to produce "GO" signals that, through the direct emmetropization pathway, cause scleral remodeling that increases the axial elongation rate of the eye, reducing the hyperopia. Myopia causes the retina to generate "STOP" signals that produce scleral remodeling, slowing the axial elongation rate and reducing the myopia. Our aim was to compare the pattern of gene expression produced in the sclera by the STOP signals with the GO gene expression signature we described previously. METHODS: The GO gene expression signature was produced by monocular -5 diopter (D) lens wear for 2 days (ML-2) or 4 days (ML-4); an additional "STAY" condition was examined after eyes had fully compensated for a -5 D lens after 11 days of lens wear (ML-11). After 11 days of -5 D lens wear had produced full refractive compensation, gene expression in the STOP condition was examined during recovery (without the lens) for 2 days (REC-2) or 4 days (REC-4). The untreated contralateral eyes served as a control in all groups. Two age-matched normal groups provided a comparison with the treated groups. Quantitative real-time PCR was used to measure mRNA levels for 55 candidate genes. RESULTS: The STAY group compensated fully for the lens (treated eye versus control eye, -5.1±0.2 D). Wearing the lens, the hyperopic signal for elongation had dissipated (-0.3±0.3 D). In the STOP groups, the refraction in the recovering eyes became less myopic relative to the control eyes (REC-2, +1.3±0.3 D; REC-4, +2.6±0.4 D). In the STAY group, three genes showed significant downregulation. However, many genes that were significantly altered in GO showed smaller, nonsignificant, expression differences in the same direction in STAY, suggesting the gene expression signature in STAY is a greatly weakened form of the GO signature. In the STOP groups, a different gene expression pattern was observed, characterized by mostly upregulation with larger fold differences after 4 days than after 2 days of recovery. Eleven of the 55 genes examined showed significant bidirectional GO/STOP regulation in the ML-2 and REC-2 groups, and 13 genes showed bidirectional regulation in the ML-4 and REC-4 groups. Eight of these genes (NPR3, CAPNS1, NGEF, TGFB1, CTGF, NOV, TIMP1, and HS6ST1) were bidirectionally regulated at both time points in the GO and STOP conditions. An additional 15 genes showed significant regulation in either GO or STOP conditions but not in both. CONCLUSIONS: Many genes are involved in scleral remodeling and the control of axial length. The STOP (recovery) gene expression signature in the sclera involves some of the same genes, bidirectionally regulated, as the GO signature. However, other genes, regulated in GO, are not differentially regulated in STOP, and others show differential regulation only in STOP.

Scleral micro-RNA signatures in adult and fetal eyes.

INTRODUCTION: In human eyes, ocular enlargement/growth reflects active extracellular matrix remodeling of the outer scleral shell. Micro-RNAs are small non-coding RNAs that regulate gene expression by base pairing with target sequences. They serve as nodes of signaling networks. We hypothesized that the sclera, like most tissues, expresses micro-RNAs, some of which modulate genes regulating ocular growth. In this study, the scleral micro-RNA expression profile of rapidly growing human fetal eyes was compared with that of stable adult donor eyes using high-throughput microarray and quantitative PCR analyses. METHODS: Scleral samples from normal human fetal (24 wk) and normal adult donor eyes were obtained (n=4 to 6, each group), and RNA extracted. Genome-wide micro-RNA profiling was performed using the Agilent micro-RNA microarray platform. Micro-RNA target predictions were obtained using Microcosm, TargetScan and PicTar algorithms. TaqMan® micro-RNA assays targeting micro-RNAs showing either highest significance, detection, or fold differences, and collagen specificity, were applied to scleral samples from posterior and peripheral ocular regions (n=7, each group). Microarray data were analyzed using R, and quantitative PCR data with 2^-deltaCt methods. RESULTS: Human sclera was found to express micro-RNAs, and comparison of microarray results for adult and fetal samples revealed many to be differentially expressed (p<0.01, min p= 6.5x10(11)). Specifically, fetal sclera showed increased expression of mir-214, let-7c, let-7e, mir-103, mir-107, and mir-98 (1.5 to 4 fold changes, p<0.01). However, no significant regionally specific differences .i.e., posterior vs. peripheral sclera, were observed for either adult or fetal samples. CONCLUSION: For the first time, micro-RNA expression has been catalogued in human sclera. Some micro-RNAs show age-related differential regulation, higher in the sclera of rapidly growing fetal eyes, consistent with a role in ocular growth regulation. Thus micro-RNAs represent potential targets for ocular growth manipulation, related to myopia and/or other disorders such as scleral ectasia.

In vitro effects of insulin and RPE on choroidal and scleral components of eye growth in chicks.

In chick eyes, exogenous insulin prevents the choroidal thickening caused by wearing positive lenses and increases ocular elongation and scleral glycosaminoglycan (GAG) synthesis, an indicator of eye growth. Using in vitro eye-cups, a novel experimental system, we examined the role of the retinal pigment epithelium (RPE) and insulin on choroidal thickness and scleral GAG synthesis. Specifically, we asked whether insulin causes the release of diffusible factors from the RPE that affect the choroid. We studied the effect of insulin on choroidal thickness and scleral GAG synthesis by making eye-cups consisting of RPE, choroid, and sclera (RCS), choroid and sclera (CS), or just sclera from pairs of eyes. One eye-cup was cultured in 0.037, 0.37, 3.7 or 37 µM insulin dissolved in L-15 medium, and its pair was cultured in L-15 medium without insulin. Choroidal thickness in eye-cups was measured by A-scan ultrasonography before and after 20 h of incubation. Sulfate incorporation into GAGs (scleral GAG synthesis) was measured after 44 h of incubation. To further study the effect of RPE and insulin on the choroids, we prepared pairs of CS eye-cups cultured with vs. without RPE transplanted from donor eyes, in the presence or absence of 37 µM insulin. To study if insulin caused the RPE to produce diffusible factors that affected the choroid, we prepared medium conditioned by the RPE in the presence (experimental conditioned medium) or absence (control conditioned medium) of 37 µM insulin for 20 h. Experimental and control conditioned media were pooled separately, and an equal volume of medium containing 37 µM insulin was added to both experimental and control media. Pairs of CS eye-cups were cultured in conditioned medium (experimental vs. control). Choroidal thickness was measured before and after 20 h of incubation. Choroids in all eye-cups thickened after 20 h of incubation. Insulin reduced this natural choroidal thickening seen in culture significantly, but only if the RPE was present. This effect was dose-dependent and strongest at 37 µM. Insulin increased scleral GAG synthesis in both RCS and CS eye-cups, having a greater effect in the CS eye-cups. Insulin had no effect on scleral GAG synthesis in scleral eye-cups. Choroids of CS eye-cups cultured with transplanted RPE plus insulin thickened significantly less than choroids of eye-cups cultured with insulin but without the RPE. The reduction in choroidal thickening was similar to that seen in eye-cups with intact RPE (RCS). Choroidal thickening of CS eye-cups cultured with experimental conditioned medium was significantly reduced compared with their pairs cultured with control conditioned medium. In vitro, as in vivo, insulin prevents choroidal thickening and increases scleral GAG synthesis. Insulin causes the RPE to synthesize diffusible molecules that inhibit choroidal thickening. Insulin might also cause the choroid to produce secondary signals that affect scleral GAG synthesis.

The choroid as a sclera growth regulator.

Emmetropization is a vision dependent mechanism that attempts to minimize refractive error through coordinated growth of the cornea, lens and sclera such that the axial length matches the focal length of the eye. It is generally accepted that this visually guided eye growth is controlled via a cascade of locally generated chemical events that are initiated in the retina and ultimately cause changes in scleral extracellular matrix (ECM) remodeling which lead to changes in eye size and refraction. Of much interest, therefore, are the molecular mechanisms that underpin emmetropization and visually guided ocular growth. The choroid, a highly vascularized layer located between the retina and the sclera is uniquely situated to relay retina-derived signals to the sclera to effect changes in ECM synthesis and ocular size. Studies initiated by Josh Wallman clearly demonstrate that the choroid plays an active role in emmetropization, both by modulation of its thickness to adjust the retina to the focal plane of the eye (choroidal accommodation), and well as through the release of growth factors that have the potential to regulate scleral extracellular matrix remodeling. His discoveries prompted numerous investigations on the molecular composition of the choroid and changes in gene expression associated with visually guided ocular growth. This article will review molecular and functional studies of the choroid to provide support for the hypothesis that the choroid is a source of sclera growth regulators that effect changes in ocular growth in response to visual stimuli.

Scleral changes induced by atropine in chicks as an experimental model of myopia.

PURPOSE: To determine the effects of intravitreal atropine on scleral growth in the form-deprived chick as an experimental model of myopia. METHODS: Five groups of five chicks were studied from day 0-12 post-hatching. One group remained untreated (C), and four were form-deprived by monocular light diffusers to induce myopia. Two groups (RL and A) wore diffusers for 9 days, and the other two groups (D and D + A) wore diffusers throughout the study. Group D received no further treatment (myopia positive control). Groups A and D + A received intravitreal injections of atropine for days 9-12. Measurements of refractive error and axial length were performed on days 0, 9, and 12. Sclera changes were assessed in cartilaginous and fibrous layers by histological analysis. RESULTS: All form-deprived eyes had a myopic refractive error on day 9. All atropine-treated groups were hyperopic on day 12. The effect of atropine was most evident in Group D + A in which diffusers were maintained throughout treatment and changes in refractive error were statistically significant. The observed changes in axial length were in line with the changes in refractive error. The scleral fibrous layer thickness increased, and the sceral cartilaginous layer underwent a slight thinning compared to Group D, the myopia positive control. CONCLUSIONS: If the signals that induce growth remain during atropine treatment, morphological changes in sclera are produced: the scleral fibrous layer thickened, and the sceral cartilaginous layer thinned. These changes resulted in refractive error recovery, and the ocular growth was stopped. The data suggested the atropine was acting throughout the scleral fibrous layer.

Chicks use changes in luminance and chromatic contrast as indicators of the sign of defocus.

As the eye changes focus, the resulting changes in cone contrast are associated with changes in color and luminance. Color fluctuations should simulate the eye being hyperopic and make the eye grow in the myopic direction, while luminance fluctuations should simulate myopia and make the eye grow in the hyperopic direction. Chicks without lenses were exposed daily (9 a.m. to 5 p.m.) for three days on two consecutive weeks to 2 Hz sinusoidally modulated illumination (mean illuminance of 680 lux) to one of the following: in-phase modulated luminance flicker (LUM), counterphase-modulated red/green (R/G Color) or blue/yellow flicker (B/Y Color), combined color and luminance flicker (Color + LUM), reduced amplitude luminance flicker (Low LUM), or no flicker. After the three-day exposure to flicker, chicks were kept in a brooder under normal diurnal lighting for four days. Changes in the ocular components were measured with ultrasound and with a Hartinger Coincidence Refractometer (aus Jena, Jena, East Germany. After the first three-day exposure, luminance flicker produced more hyperopic refractions (LUM: 2.27 D) than did color flicker (R/G Color: 0.09 D; B/Y Color: -0.25 D). Changes in refraction were mainly due to changes in eye length, with color flicker producing much greater changes in eye length than luminance flicker (R/G Color: 102 µm; B/Y Color: 98 µm; LUM: 66 µm). Our results support the hypothesis that the eye can differentiate between hyperopic and myopic defocus on the basis of the effects of change in luminance or color contrast.

Evaluation of gene expression profiles and pathways underlying postnatal development in mouse sclera.

PURPOSE: The aim of this study was to identify the genes and pathways underlying the growth of the mouse sclera during postnatal development. METHODS: Total RNA was isolated from each of 30 single mouse sclera (n=30, 6 sclera each from 1-, 2-, 3-, 6-, and 8-week-old mice) and reverse-transcribed into cDNA using a T7-N(6) primer. The resulting cDNA was fragmented, labeled with biotin, and hybridized to a Mouse Gene 1.0 ST Array. ANOVA analysis was then performed using Partek Genomic Suite 6.5 beta and differentially expressed transcript clusters were filtered based on a selection criterion of ≥ 2 relative fold change at a false discovery rate of ≤ 5%. Genes identified as involved in the main biologic processes during postnatal scleral development were further confirmed using qPCR. A possible pathway that contributes to the postnatal development of the sclera was investigated using Ingenuity Pathway Analysis software. RESULTS: The hierarchical clustering of all time points showed that they did not cluster according to age. The highest number of differentially expressed transcript clusters was found when week 1 and week 2 old scleral tissues were compared. The peroxisome proliferator- activated receptor gamma coactivator 1-alpha (Ppargc1a) gene was found to be involved in the networks generated using Ingenuity Pathway Studio (IPA) from the differentially expressed transcript cluster lists of week 2 versus 1, week 3 versus 2, week 6 versus 3, and week 8 versus 6. The gene expression of Ppargc1a varied during scleral growth from week 1 to 2, week 2 to 3, week 3 to 6, and week 6 to 8 and was found to interact with a different set of genes at different scleral growth stages. Therefore, this indicated that Ppargc1a might play a role in scleral growth during postnatal weeks 1 to 8. CONCLUSIONS: Gene expression of eye diseases should be studied as early as postnatal weeks 1-2 to ensure that any changes in gene expression pattern during disease development are detected. In addition, we propose that Ppargc1a might play a role in regulating postnatal scleral development by interacting with a different set of genes at different scleral growth stages.

Muller glia, vision-guided ocular growth, retinal stem cells, and a little serendipity: the Cogan lecture.

Hypothesis-driven science is expected to result in a continuum of studies and findings along a discrete path. By comparison, serendipity can lead to new directions that branch into different paths. Herein, I describe a diverse series of findings that were motivated by hypotheses, but driven by serendipity. I summarize how investigations into vision-guided ocular growth in the chick eye led to the identification of glucagonergic amacrine cells as key regulators of ocular elongation. Studies designed to assess the impact of the ablation of different types of neurons on vision-guided ocular growth led to the finding of numerous proliferating cells within damaged retinas. These proliferating cells were Müller glia-derived retinal progenitors with a capacity to produce new neurons. Studies designed to investigate Müller glia-derived progenitors led to the identification of a domain of neural stem cells that form a circumferential marginal zone (CMZ) that lines the periphery of the retina. Accelerated ocular growth, caused by visual deprivation, stimulated the proliferation of CMZ progenitors. We formulated a hypothesis that growth-regulating glucagonergic cells may regulate both overall eye size (scleral growth) and the growth of the retina (proliferation of CMZ cells). Subsequent studies identified unusual types of glucagonergic neurons with terminals that ramify within the CMZ; these cells use visual cues to control equatorial ocular growth and the proliferation of CMZ cells. Finally, while studying the signaling pathways that stimulate CMZ and Müller glia-derived progenitors, serendipity led to the discovery of a novel type of glial cell that is scattered across the inner retinal layers.

Genetic deletion of the adenosine A2A receptor confers postnatal development of relative myopia in mice.

PURPOSE: To critically evaluate whether the adenosine A2A receptor (A2AR) plays a role in postnatal refractive development in mice. METHODS: Custom-built biometric systems specifically designed for mice were used to assess the development of relative myopia by examining refraction and biometrics in A2AR knockout (KO) mice and wild-type (WT) littermates between postnatal days (P)28 and P56. Ocular dimensions were measured by customized optical coherence tomography (OCT), refractive state by eccentric infrared photorefraction (EIR), and corneal radius of curvature by modified keratometry. Scleral collagen diameter and density were examined by electron microscopy on P35. The effect of A2AR activation on collagen mRNA expression and on soluble collagen production was examined in cultured human scleral fibroblasts by real-time RT-PCR and a collagen assay kit. RESULTS: Compared with WT littermates, the A2AR KO mice displayed relative myopia (average difference, 5.1 D between P28 and P35) and associated increases in VC depth and axial length from P28 to P56. Furthermore, the myopic shift in A2AR KO mice was associated with ultrastructural changes in the sclera: Electron microscopy revealed denser collagen fibrils with reduced diameter in A2AR KO compared with WT. Last, A2AR activation induced expression of mRNAs for collagens I, III, and V and increased production of soluble collagen in cultured human scleral fibroblasts. CONCLUSIONS: Genetic deletion of the A2AR promotes development of relative myopia with increased axial length and altered scleral collagen fiber structure during postnatal development in mice. Thus, the A2AR may be important in normal refractive development.

Effects of poly(2-hydroxyethyl methacrylate) and poly(vinyl-pyrrolidone) hydrogel implants on myopic and normal chick sclera.

There has been generally little attention paid to the utilization of biomaterials as an anti-myopia treatment. The purpose of this study was to investigate whether polymeric hydrogels, either implanted or injected adjacent to the outer scleral surface, slow ocular elongation. White Leghorn (Gallus gallus domesticus) chicks were used at 2 weeks of age. Chicks had either (1) a strip of poly(2-hydroxyethyl methacrylate) (pHEMA) implanted monocularly against the outer sclera at the posterior pole, or (2) an in situ polymerizing gel [main ingredient: poly(vinyl-pyrrolidone) (PVP)] injected monocularly at the same location. Some of the eyes injected with the polymer were fitted with a diffuser or a -10D lens. In each experiment, ocular lengths were measured at regular intervals by high frequency A-scan ultrasonography, and chicks were sacrificed for histology at staged intervals. No in vivo signs of either orbital or ocular inflammation were observed. The pHEMA implant significantly increased scleral thickness by the third week, and the implant became encapsulated with fibrous tissue. The PVP-injected eyes left otherwise untreated, showed a significant increase in scleral thickness, due to increased chondrocyte proliferation and extracellular matrix deposition. However, there was no effect of the PVP injection on ocular elongation. In eyes wearing optical devices, there was no effect on either scleral thickness or ocular elongation. These results represent "proof of principle" that scleral growth can be manipulated without adverse inflammatory responses. However, since neither approach slowed ocular elongation, additional factors must influence scleral surface area expansion in the avian eye.

Human transscleral albumin permeability and the effect of topographical location and donor age.

PURPOSE: To quantify the permeability coefficient of albumin across human sclera and to assess topographical and age-related variation. METHODS: Equatorial superotemporal scleral tissue from 15 donor eyes (mean age 60 years; range 39-84) was mounted in a modified Ussing chamber. Additional tissue was taken from the anterior and posterior superotemporal regions of six eyes, and equatorial superonasal, and inferotemporal regions of a further six eyes. Fluorescein isothiocyanate (FITC)-labeled, 0.412 mM, bovine albumin was placed in one hemichamber facing the internal scleral surface, and the rate of transscleral flux was determined over 24 hours, at 25 degrees C, with a spectrophotometer. RESULTS: Permeability coefficient for equatorial superotemporal scleral tissue at 25 degrees C (+/-SD) was 0.83 +/- 0.50 x 10(-6) cm . s(-1). The permeability coefficient adjusted for 37 degrees C (+/-SD) was 1.43 +/- 0.86 x 10(-6) cm . s(-1). The effect of donor age was assessed for the 15 equatorial superotemporal samples. Regression analysis showed a significant decline in scleral diffusion of albumin with increasing donor age (P = 0.0166). There was no significant difference in diffusion over the different topographical regions tested. The partition coefficient of permeability to albumin also showed a decline with increasing donor age (P = 0.001). CONCLUSIONS: The permeability and partition coefficients of human sclera both significantly decline with increasing donor age. Permeability coefficient shows no significant variation over the different topographical regions tested. The decrease in albumin permeability with increasing donor age may have pharmacokinetic implications when considering transscleral diffusion of high-molecular-weight compounds.