ITF2357 regulates NF-κB signaling pathway to protect barrier integrity in retinal pigment epithelial cells.

The robust integrity of the retinal pigment epithelium (RPE), which contributes to the outer brain retina barrier (oBRB), is compromised in several retinal degenerative and vascular disorders, including diabetic macular edema (DME). This study evaluates the role of a new generation of histone deacetylase inhibitor (HDACi), ITF2357, in regulating outer blood-retinal barrier function and investigates the underlying mechanism of action in inhibiting TNFα-induced damage to RPE integrity. Using the immortalized RPE cell line (ARPE-19), ITF2357 was found to be non-toxic between 50 nM and 5 µM concentrations. When applied as a pre-treatment in conjunction with an inflammatory cytokine, TNFα, the HDACi was safe and effective in preventing epithelial permeability by fortifying tight junction (ZO-1, -2, -3, occludin, claudin-1, -2, -3, -5, -19) and adherens junction (E-cadherin, Nectin-1) protein expression post-TNFα stress. Mechanistically, ITF2357 depicted a late action at 24 h via attenuating IKK, IκBα, and p65 phosphorylation and ameliorated the expression of IL-1ß, IL-6, and MCP-1. Also, ITF2357 delayed IκBα synthesis and turnover. The use of Bay 11-7082 and MG132 further uncovered a possible role for ITF2357 in non-canonical NF-κB activation. Overall, this study revealed the protection effects of ITF2357 by regulating the turnover of tight and adherens junction proteins and modulating NF-κB signaling pathway in the presence of an inflammatory stressor, making it a potential therapeutic application for retinal vascular diseases such as DME with compromised outer blood-retinal barrier.

Application of Neuron-Selective Fluorescent Probe, NeuA, To Identify Mouse Retinal Degeneration.

The retina is part of the central nerve system (CNS) and has various interneurons and sensory neurons such as photoreceptor cells. Retinitis pigmentosa (RP) is an inherited condition that is characterized by photoreceptor degeneration. Herein, we developed a fluorescent probe-NeuA-for detecting retinal neuronal cells and applied NeuA to discriminate between healthy and RP retinas. The staining pattern of NeuA in the retinas of healthy and RP mouse models was examined in vitro, ex vivo and in vivo using confocal microscopy, the fluorescent fundus microscopy and optical coherent tomography (OCT). NeuA strongly stained the outer segment layer of photoreceptor cells and some bipolar cells in the healthy retina, but there was only weak staining in the photoreceptor degenerated retinas. Therefore, NeuA probe can be used as the detecting RP tools in the preclinical conditions.

The Role of Autophagy in Chemical Proteasome Inhibition Model of Retinal Degeneration.

We recently demonstrated that chemical proteasome inhibition induced inner retinal degeneration, supporting the pivotal roles of the ubiquitin-proteasome system in retinal structural integrity maintenance. In this study, using beclin1-heterozygous (Becn1-Het) mice with autophagic dysfunction, we tested our hypothesis that autophagy could be a compensatory retinal protective mechanism for proteasomal impairment. Despite the reduced number of autophagosome, the ocular tissue morphology and intraocular pressure were normal. Surprisingly, Becn1-Het mice experienced the same extent of retinal degeneration as was observed in wild-type mice, following an intravitreal injection of a chemical proteasome inhibitor. Similarly, these mice equally responded to other chemical insults, including endoplasmic reticulum stress inducer, N-methyl-D-aspartate, and lipopolysaccharide. Interestingly, in cultured neuroblastoma cells, we found that the mammalian target of rapamycin-independent autophagy activators, lithium chloride and rilmenidine, rescued these cells against proteasome inhibition-induced death. These results suggest that Becn1-mediated autophagy is not an effective intrinsic protective mechanism for retinal damage induced by insults, including impaired proteasomal activity; furthermore, autophagic activation beyond normal levels is required to alleviate the cytotoxic effect of proteasomal inhibition. Further studies are underway to delineate the precise roles of different forms of autophagy, and investigate the effects of their activation in rescuing retinal neurons under various pathological conditions.

Potential of subconjunctival aflibercept in treating choroidal neovascularization.

The study aimed to evaluate the intraocular pharmacokinetics and efficacy of aflibercept after subconjunctival injection in animal models for treating choroidal neovascularization (CNV) associated with Age-Related Macular Degeneration (AMD). New Zealand albino rabbits received aflibercept (2000 µg/50 µl) in one eye, and the other eye was used as control. At 7, 14, 21 and 28 days, the animals were sacrificed to dissect the ocular tissues, and serum was collected at 1hr, 3 h, 1, 7, 14, 21 and 28 days. The concentration of aflibercept in various ocular tissues and serum were measured using the immunoassay technique. The concentration maximum (Cmax) at the Retinal Pigment Epithelium (RPE)-choroid complex and retina in treated eyes was 261.55 and 33.83 ng/gm, respectively. The area under the curve (AUC0-last) for RPE-Choroid and retina were 2094.02 and 290.33 days. ng/gm respectively. The time maximum (Tmax) for the ocular tissues was reached on day 7. In the vitreous humour, a lower level of aflibercept was retrieved. The Cmax (1766.84 ng/mL) in the serum was reached on day 1, followed by a decline in the concentration till the end of the study period. In treated eyes, the levels of aflibercept in most of the ocular tissues were maintained for at least 21 days above the invitro IC50 concentration. The results of the efficacy study show that subconjunctival aflibercept could reach the therapeutic target to inhibit CNV. The subconjunctival aflibercept could be a less invasive route for treating CNV with AMD.

NOD-like Receptors in the Eye: Uncovering Its Role in Diabetic Retinopathy.

Diabetic retinopathy (DR) is an ocular complication of diabetes mellitus (DM). International Diabetic Federations (IDF) estimates up to 629 million people with DM by the year 2045 worldwide. Nearly 50% of DM patients will show evidence of diabetic-related eye problems. Therapeutic interventions for DR are limited and mostly involve surgical intervention at the late-stages of the disease. The lack of early-stage diagnostic tools and therapies, especially in DR, demands a better understanding of the biological processes involved in the etiology of disease progression. The recent surge in literature associated with NOD-like receptors (NLRs) has gained massive attraction due to their involvement in mediating the innate immune response and perpetuating inflammatory pathways, a central phenomenon found in the pathogenesis of ocular diseases including DR. The NLR family of receptors are expressed in different eye tissues during pathological conditions suggesting their potential roles in dry eye, ocular infection, retinal ischemia, cataract, glaucoma, age-related macular degeneration (AMD), diabetic macular edema (DME) and DR. Our group is interested in studying the critical early components involved in the immune cell infiltration and inflammatory pathways involved in the progression of DR. Recently, we reported that NLRP3 inflammasome might play a pivotal role in the pathogenesis of DR. This comprehensive review summarizes the findings of NLRs expression in the ocular tissues with special emphasis on its presence in the retinal microglia and DR pathogenesis.

Surgical Approaches for Cell Therapeutics Delivery to the Retinal Pigment Epithelium and Retina.

Developing successful surgical strategies to deliver cell therapeutics to the back of the eye is an essential pillar to success for stem cell-based applications in blinding retinal diseases. Within this chapter, we have attempted to gather all key considerations during preclinical animal trials.Guidance is provided for choices on animal models, options for immunosuppression, as well as anesthesia. Subsequently we cover surgical strategies for RPE graft delivery, both as suspension as well as in monolayers in small rodents, rabbits, pigs, and nonhuman primate. A detailed account is given in particular on animal variations in vitrectomy and subretinal surgery, which requires a considerable learning curve, when transiting from human to animal. In turn, however, many essential subretinal implantation techniques in large-eyed animals are directly transferrable to human clinical trial protocols.A dedicated subchapter on photoreceptor replacement provides insights on preparation of suspension as well as sheet grafts, to subsequently outline the basics of subretinal delivery via both the transscleral and transvitreal route. In closing, a future outlook on vision restoration through retinal cell-based therapeutics is presented.

Cavin-2 regulates the activity and stability of endothelial nitric-oxide synthase (eNOS) in angiogenesis.

Angiogenesis is a highly regulated process for formation of new blood vessels from pre-existing ones. Angiogenesis is dysregulated in various pathologies, including age-related macular degeneration, arthritis, and cancer. Inhibiting pathological angiogenesis therefore represents a promising therapeutic strategy for treating these disorders, highlighting the need to study angiogenesis in more detail. To this end, identifying the genes essential for blood vessel formation and elucidating their function are crucial for a complete understanding of angiogenesis. Here, focusing on potential candidate genes for angiogenesis, we performed a morpholino-based genetic screen in zebrafish and identified Cavin-2, a membrane-bound phosphatidylserine-binding protein and critical organizer of caveolae (small microdomains in the plasma membrane), as a regulator of angiogenesis. Using endothelial cells, we show that Cavin-2 is required for in vitro angiogenesis and also for endothelial cell proliferation, migration, and invasion. We noted a high level of Cavin-2 expression in the neovascular tufts in the mouse model of oxygen-induced retinopathy, suggesting a role for Cavin-2 in pathogenic angiogenesis. Interestingly, we also found that Cavin-2 regulates the production of nitric oxide (NO) in endothelial cells by controlling the stability and activity of the endothelial nitric-oxide synthase (eNOS) and that Cavin-2 knockdown cells produce much less NO than WT cells. Also, mass spectrometry, flow cytometry, and electron microscopy analyses indicated that Cavin-2 is secreted in endothelial microparticles (EMPs) and is required for EMP biogenesis. Taken together, our results indicate that in addition to its function in caveolae biogenesis, Cavin-2 plays a critical role in endothelial cell maintenance and function by regulating eNOS activity.

AAV-mediated gene delivery of the calreticulin anti-angiogenic domain inhibits ocular neovascularization.

Ocular neovascularization is a common pathological feature in diabetic retinopathy and neovascular age-related macular degeneration that can lead to severe vision loss. We evaluated the therapeutic efficacy of a novel endogenous inhibitor of angiogenesis, the calreticulin anti-angiogenic domain (CAD180), and its functional 112-residue fragment, CAD-like peptide 112 (CAD112), delivered using a self-complementary adeno-associated virus serotype 2 (scAAV2) in rodent models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. The expression of CAD180 and CAD112 was elevated in human umbilical vein endothelial cells transduced with scAAV2-CAD180 or scAAV2-CAD112, respectively, and both inhibited angiogenic activity in vitro. Intravitreal gene delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly inhibited ischemia-induced retinal neovascularization in rat eyes (CAD180: 52.7% reduction; CAD112: 49.2% reduction) compared to scAAV2-mCherry, as measured in retinal flatmounts stained with isolectin B4. Moreover, the retinal structure and function were unaffected by scAAV2-CAD180 or scAAV2-CAD112, as measured by optical coherence tomography and electroretinography. Moreover, subretinal delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly attenuated laser-induced choroidal neovascularization in mouse eyes compared to scAAV2-mCherry, as measured by fundus fluorescein angiography (CAD180: 62.4% reduction; CAD112: 57.5% reduction) and choroidal flatmounts (CAD180: 40.21% reduction; CAD112: 43.03% reduction). Gene delivery using scAAV2-CAD180 or scAAV2-CAD112 has significant potential as a therapeutic option for the management of ocular neovascularization.

Mfsd2a Is a Transporter for the Essential ω-3 Fatty Acid Docosahexaenoic Acid (DHA) in Eye and Is Important for Photoreceptor Cell Development.

Eye photoreceptor membrane discs in outer rod segments are highly enriched in the visual pigment rhodopsin and the ω-3 fatty acid docosahexaenoic acid (DHA). The eye acquires DHA from blood, but transporters for DHA uptake across the blood-retinal barrier or retinal pigment epithelium have not been identified. Mfsd2a is a newly described sodium-dependent lysophosphatidylcholine (LPC) symporter expressed at the blood-brain barrier that transports LPCs containing DHA and other long-chain fatty acids. LPC transport via Mfsd2a has been shown to be necessary for human brain growth. Here we demonstrate that Mfsd2a is highly expressed in retinal pigment epithelium in embryonic eye, before the development of photoreceptors, and is the primary site of Mfsd2a expression in the eye. Eyes from whole body Mfsd2a-deficient (KO) mice, but not endothelium-specific Mfsd2a-deficient mice, were DHA-deficient and had significantly reduced LPC/DHA transport in vivo Fluorescein angiography indicated normal blood-retinal barrier function. Histological and electron microscopic analysis indicated that Mfsd2a KO mice exhibited a specific reduction in outer rod segment length, disorganized outer rod segment discs, and mislocalization of and reduction in rhodopsin early in postnatal development without loss of photoreceptors. Minor photoreceptor cell loss occurred in adult Mfsd2a KO mice, but electroretinography indicated visual function was normal. The developing eyes of Mfsd2a KO mice had activated microglia and up-regulation of lipogenic and cholesterogenic genes, likely adaptations to loss of LPC transport. These findings identify LPC transport via Mfsd2a as an important pathway for DHA uptake in eye and for development of photoreceptor membrane discs.

Meta-analysis of genome-wide association studies in multiethnic Asians identifies two loci for age-related nuclear cataract.

Age-related cataract is a leading cause of blindness worldwide, especially in developing countries where access to cataract surgery remains limited. Previous linkage and candidate gene studies suggested genetic influences on age-related nuclear cataract but few genetic markers have been identified thus far. We conducted genome-wide association studies on 4569 Asians (including 2369 Malays and 2200 Indians), and replicated our analysis in 2481 Chinese from two independent cohorts (1768 Chinese in Singapore and 803 Chinese in Beijing). We confirmed two genome-wide significant loci for nuclear cataract in the combined meta-analysis of four cohorts (n = 7140). The first locus was at chromosome 3q25.31 in KCNAB1 (rs7615568, fixed-effect Pmeta = 2.30 × 10(-8); random-effect Pmeta = 1.08 × 10(-8)). The second locus was at chromosome 21 in the proximity of CRYAA (rs11911275, fixed-effect Pmeta = 2.77 × 10(-8); random-effect Pmeta = 1.98 × 10(-9)), a major protein component of eye lens. The findings were further supported by up-regulation and down-regulation of KCNAB1 and CRYAA in human lens capsule, respectively, as the severity of nuclear cataract increases. The results offer additional insights into the pathogenesis of nuclear cataract in Asians.

Mutations in SCO2 are associated with autosomal-dominant high-grade myopia.

Myopia, or near-sightedness, is an ocular refractive error of unfocused image quality in front of the retinal plane. Individuals with high-grade myopia (dioptric power greater than -6.00) are predisposed to ocular morbidities such as glaucoma, retinal detachment, and myopic maculopathy. Nonsyndromic, high-grade myopia is highly heritable, and to date multiple gene loci have been reported. We performed exome sequencing in 4 individuals from an 11-member family of European descent from the United States. Affected individuals had a mean dioptric spherical equivalent of -22.00 sphere. A premature stop codon mutation c.157C>T (p.Gln53*) cosegregating with disease was discovered within SCO2 that maps to chromosome 22q13.33. Subsequent analyses identified three additional mutations in three highly myopic unrelated individuals (c.341G>A, c.418G>A, and c.776C>T). To determine differential gene expression in a developmental mouse model, we induced myopia by applying a -15.00D lens over one eye. Messenger RNA levels of SCO2 were significantly downregulated in myopic mouse retinae. Immunohistochemistry in mouse eyes confirmed SCO2 protein localization in retina, retinal pigment epithelium, and sclera. SCO2 encodes for a copper homeostasis protein influential in mitochondrial cytochrome c oxidase activity. Copper deficiencies have been linked with photoreceptor loss and myopia with increased scleral wall elasticity. Retinal thinning has been reported with an SC02 variant. Human mutation identification with support from an induced myopic animal provides biological insights of myopic development.

Nine loci for ocular axial length identified through genome-wide association studies, including shared loci with refractive error.

Refractive errors are common eye disorders of public health importance worldwide. Ocular axial length (AL) is the major determinant of refraction and thus of myopia and hyperopia. We conducted a meta-analysis of genome-wide association studies for AL, combining 12,531 Europeans and 8,216 Asians. We identified eight genome-wide significant loci for AL (RSPO1, C3orf26, LAMA2, GJD2, ZNRF3, CD55, MIP, and ALPPL2) and confirmed one previously reported AL locus (ZC3H11B). Of the nine loci, five (LAMA2, GJD2, CD55, ALPPL2, and ZC3H11B) were associated with refraction in 18 independent cohorts (n = 23,591). Differential gene expression was observed for these loci in minus-lens-induced myopia mouse experiments and human ocular tissues. Two of the AL genes, RSPO1 and ZNRF3, are involved in Wnt signaling, a pathway playing a major role in the regulation of eyeball size. This study provides evidence of shared genes between AL and refraction, but importantly also suggests that these traits may have unique pathways.

Genome-wide association study identifies ZFHX1B as a susceptibility locus for severe myopia.

Severe myopia (defined as spherical equivalent < -6.0 D) is a predominant problem in Asian countries, resulting in substantial morbidity. We performed a meta-analysis of four genome-wide association studies (GWAS), all of East Asian descent totaling 1603 cases and 3427 controls. Two single nucleotide polymorphisms (SNPs) (rs13382811 from ZFHX1B [encoding for ZEB2] and rs6469937 from SNTB1) showed highly suggestive evidence of association with disease (P < 1 × 10(-7)) and were brought forward for replication analysis in a further 1241 severe myopia cases and 3559 controls from a further three independent sample collections. Significant evidence of replication was observed, and both SNP markers surpassed the formal threshold for genome-wide significance upon meta-analysis of both discovery and replication stages (P = 5.79 × 10(-10), per-allele odds ratio (OR) = 1.26 for rs13382811 and P = 2.01 × 10(-9), per-allele OR = 0.79 for rs6469937). The observation at SNTB1 is confirmatory of a very recent GWAS on severe myopia. Both genes were expressed in the human retina, sclera, as well as the retinal pigmented epithelium. In an experimental mouse model for myopia, we observed significant alterations to gene and protein expression in the retina and sclera of the unilateral induced myopic eyes for Zfhx1b and Sntb1. These new data advance our understanding of the molecular pathogenesis of severe myopia.

Genetic variants on chromosome 1q41 influence ocular axial length and high myopia.

As one of the leading causes of visual impairment and blindness, myopia poses a significant public health burden in Asia. The primary determinant of myopia is an elongated ocular axial length (AL). Here we report a meta-analysis of three genome-wide association studies on AL conducted in 1,860 Chinese adults, 929 Chinese children, and 2,155 Malay adults. We identified a genetic locus on chromosome 1q41 harboring the zinc-finger 11B pseudogene ZC3H11B showing genome-wide significant association with AL variation (rs4373767, ß = -0.16 mm per minor allele, P(meta) =2.69 × 10(-10)). The minor C allele of rs4373767 was also observed to significantly associate with decreased susceptibility to high myopia (per-allele odds ratio (OR) =0.75, 95% CI: 0.68-0.84, P(meta) =4.38 × 10(-7)) in 1,118 highly myopic cases and 5,433 controls. ZC3H11B and two neighboring genes SLC30A10 and LYPLAL1 were expressed in the human neural retina, retinal pigment epithelium, and sclera. In an experimental myopia mouse model, we observed significant alterations to gene and protein expression in the retina and sclera of the unilateral induced myopic eyes for the murine genes ZC3H11A, SLC30A10, and LYPLAL1. This supports the likely role of genetic variants at chromosome 1q41 in influencing AL variation and high myopia.

Involvement of GABA transporters in atropine-treated myopic retina as revealed by iTRAQ quantitative proteomics.

Atropine, a muscarinic antagonist, is known to inhibit myopia progression in several animal models and humans. However, the mode of action is not established yet. In this study, we compared quantitative iTRAQ proteomic analysis in the retinas collected from control and lens-induced myopic (LIM) mouse eyes treated with atropine. The myopic group received a (-15D) spectacle lens over the right eye on postnatal day 10 with or without atropine eye drops starting on postnatal day 24. Axial length was measured by optical low coherence interferometry (OLCI), AC-Master, and refraction was measured by automated infrared photorefractor at postnatal 24, 38, and 52 days. Retinal tissue samples were pooled from six eyes for each group. The experiments were repeated twice, and technical replicates were also performed for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. MetaCore was used to perform protein profiling for pathway analysis. We identified a total of 3882 unique proteins with <1% FDR by analyzing the samples in replicates for two independent experiments. This is the largest number of mouse retina proteome reported to date. Thirty proteins were found to be up-regulated (ratio for myopia/control > global mean ratio + 1 standard deviation), and 28 proteins were down-regulated (ratio for myopia/control < global mean ratio - 1 standard deviation) in myopic eyes as compared with control retinas. Pathway analysis using MetaCore revealed regulation of γ-aminobutyric acid (GABA) levels in the myopic eyes. Detailed analysis of the quantitative proteomics data showed that the levels of GABA transporter 1 (GAT-1) were elevated in myopic retina and significantly reduced after atropine treatment. These results were further validated with immunohistochemistry and Western blot analysis. In conclusion, this study provides a comprehensive quantitative proteomic analysis of atropine-treated mouse retina and suggests the involvement of GABAergic signaling in the antimyopic effects of atropine in mouse eyes. The GABAergic transmission in the neural retina plays a pivotal role in the maintenance of axial eye growth in mammals.

Intravitreal Neuroglobin Mitigates Primate Experimental Glaucomatous Structural Damage in Association with Reduced Optic Nerve Microglial and Complement 3-Astrocyte Activation.

Current management of glaucomatous optic neuropathy is limited to intraocular pressure control. Neuroglobin (Ngb) is an endogenous neuroprotectant expressed in neurons and astrocytes. We recently showed that exogenous intravitreal Ngb reduced inflammatory cytokines and microglial activation in a rodent model of hypoxia. We thus hypothesised that IVT-Ngb may also be neuroprotective in experimental glaucoma (EG) by mitigating optic nerve (ON) astrogliosis and microgliosis as well as structural damage. In this study using a microbead-induced model of EG in six Cynomolgus primates, optical coherence imaging showed that Ngb-treated EG eyes had significantly less thinning of the peripapillary minimum rim width, retinal nerve fibre layer thickness, and ON head cupping than untreated EG eyes. Immunohistochemistry confirmed that ON astrocytes overexpressed Ngb following Ngb treatment. A reduction in complement 3 and cleaved-caspase 3 activated microglia and astrocytes was also noted. Our findings in higher-order primates recapitulate the effects of neuroprotection by Ngb treatment in rodent EG studies and suggest that Ngb may be a potential candidate for glaucoma neuroprotection in humans.

A Duration-Dependent Interaction Between High-Intensity Light and Unrestricted Vision in the Drive for Myopia Control.

Purpose: To evaluate the duration-dependent and synergetic impact of high-intensity light (HL) and unrestricted vision (UnV) on lens-induced myopia (LIM) development in chickens. Methods: Myopia was induced in one eye in chicks (10 groups, n = 126) from day 1 posthatching (D1) until day 8 (D8) using -10 diopter (D) lenses. Fellow eyes remained uncovered as controls. Nine groups were exposed daily to 2, 4, or 6 hours of HL (15,000 lux), UnV (removal of -10 D lens), or both (HL + UnV). One group served as the LIM group without any interventions. Ocular axial length (AL), refractive error, and choroidal thickness were measured on D1, D4, and D8. Outcome measures are expressed as interocular difference (IOD = experimental eye - control eye) ± SEM. Results: By D8, LIM increased AL (0.36 ± 0.04 mm), myopic refraction (-9.02 ± 0.37 D), and choroidal thinning (-90.27 ± 16.44 µm) in the LIM group (all, P < 0.001). Compared to the LIM group, exposure to 2, 4, or 6 hours of HL, UnV, or HL + UnV reduced myopic refraction in a duration-dependent manner, with UnV being more effective than HL (P < 0.05). Only 6 hours of HL + UnV (not 2 or 4 hours) prevented LIM and was more effective than UnV (P = 0.004) or HL (P < 0.001) in reducing myopic refraction and more effective than HL (P < 0.001) in reducing axial elongation. Conclusions: Daily exposure to 2, 4, or 6 hours of HL, UnV, or HL + UnV reduced lens-induced myopic refraction in a duration-dependent manner in chickens. Only 6 hours of HL + UnV completely stopped LIM development. The synergetic effect of HL and UnV is dependent on the duration of the interventions.

Posterior scleral birefringence measured by triple-input polarization-sensitive imaging as a biomarker of myopia progression.

In myopic eyes, pathological remodelling of collagen in the posterior sclera has mostly been observed ex vivo. Here we report the development of triple-input polarization-sensitive optical coherence tomography (OCT) for measuring posterior scleral birefringence. In guinea pigs and humans, the technique offers superior imaging sensitivities and accuracies than dual-input polarization-sensitive OCT. In 8-week-long studies with young guinea pigs, scleral birefringence was positively correlated with spherical equivalent refractive errors and predicted the onset of myopia. In a cross-sectional study involving adult individuals, scleral birefringence was associated with myopia status and negatively correlated with refractive errors. Triple-input polarization-sensitive OCT may help establish posterior scleral birefringence as a non-invasive biomarker for assessing the progression of myopia.

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.

Recovery From Form-Deprivation Myopia in Chicks Is Dependent Upon the Fullness and Correlated Color Temperature of the Light Spectrum.

Purpose: The purpose of this study was to evaluate the impact of full-spectrum light-emitting diodes mimicking sunlight (Sunlike LEDs) on ocular growth and refractive error development in a chicken model of myopia. Methods: One-day old chicks (n = 39) were distributed into 3 groups and raised for 28 days in isoluminant (approximately 285 lux) fluorescent (n = 18, [FL-4000], correlated color temperature [CCT] = 4000 K) or Sunlike LED (n = 12, [SL-4000], CCT = 4000 K; n = 9, [SL-6500], CCT = 6500 K) white lighting environments. Form-deprivation myopia was induced monocularly from day 1 post-hatching (D1) until D14. On D14, form deprivation was halted and the recovery of form-deprived (FD) eyes was monitored until D28. Axial length (AL), refraction, choroidal thickness, and anterior chamber depth were measured in vivo on D1, D7, D14, D22, and D28. Differences in outcome measures between eyes and groups were compared using 2-way repeated-measures ANOVA. Results: AL and myopic refraction of FD eyes increased similarly among groups during form-deprivation. FD eyes of animals raised under SL-4000 (D22: P < 0.001 and D28: P < 0.001) and SL-6500 (D22: P = 0.006 and D28: P < 0.001) recovered faster from axial elongation compared with animals raised under FL-4000. The refractive status of FD eyes reared under SL-6500, not under FL-4000 or SL-4000, was similar to control eyes on D28 (P > 0.05). However, SL-4000 and SL-6500 exhibited similar refraction on D28 than FL-4000 (P > 0.05). Choroidal thickness was significantly greater in FD eyes of chickens raised under SL-6500 than in animals raised under FL-4000 (P = 0.03). Conclusions: Compared to fluorescent light, moderate intensities of full-spectrum Sunlike LEDs can accelerate recovery from form-deprivation myopia in chickens, potentially through a change in the choroid-mediated pathway.