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The hypoxic environment around the lens is important for maintaining lens transparency. Lens epithelial cells (LECs) play a key role in lens metabolism. We measured oxygen consumption to assess the role of human LECs in maintaining hypoxia around the lens, as well as the impact of systemic and ocular diagnosis on these cells.Baseline cellular respiration was measured in rabbit LECs (NN1003A), canine kidney epithelial cells (MDCK), trabecular meshwork cells (TM-5), and bovine corneal endothelial cells (CCEE) using a XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA), which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro. Following informed written consent, lens capsule epithelial cells were obtained from patients during cataract surgery and were divided into small explants in 96-well plates. Capsules were removed when LECs became confluent. OCR was normalized to the number of cells per well using rabbit LECs as a standard. The effect of patient age, sex, race, and presence of diabetes or glaucoma on oxygen consumption was assessed by using the Mann-Whitney U test and multivariate regression analysis.Primary LECs were obtained from 69 patients. The OCR from donors aged 70 and over was lower than that of those under 70 years (2.21±1.037 vs. 2.86±1.383 fmol/min/cell; p0.05). Diabetic patients had lower OCR than non-diabetic patients (2.02±0.911 vs. 2.79±1.332fmol/min/cell; p0.05), and glaucoma patients had lower OCR than non-glaucoma patients (2.27±1.19 vs. 2.83±1.286 fmol/min/cell; p0.05). Multivariate regression analysis confirmed that donors aged 70 and over (p0.05), diabetic patients (p0.01), and glaucoma patients (p0.05) had significantly lower OCR, independent of other variables. Gender and race had no significant effect on OCR.The lower oxygen consumption rate of human LECs in older donors and patients with diabetes or glaucoma could contribute to cataract development. Diabetes and glaucoma are particularly important factors associated with decreased OCR, independent of age. Ongoing studies are examining pO2 at the anterior surface of the lens in vivo and oxygen consumption in the patient's LECs.

Previous results have shown that Bone Morphogenetic Protein (BMP) signaling is essential for lens specification and differentiation. How BMP signals are regulated in the prospective lens ectoderm is not well defined. To address this issue we have modulated BMP activity in a chicken embryo pre-lens ectoderm explant assay, and also studied transgenic mice, in which the type I BMP receptors, Bmpr1a and Acvr1, are deleted from the prospective lens ectoderm. Our results show that chicken embryo pre-lens ectoderm cells express BMPs and require BMP signaling for lens specification in vitro, and that in vivo inhibition of BMP signals in the mouse prospective lens ectoderm interrupts lens placode formation and prevents lens invagination. Furthermore, our results provide evidence that BMP expression is negatively auto-regulated in the lens-forming ectoderm, decreasing when the tissue is exposed to exogenous BMPs and increasing when BMP signaling is prevented. In addition, eyes lacking BMP receptors in the prospective lens placode develop coloboma in the adjacent wild type optic cup. In these eyes, Bmp7 expression increases in the ventral optic cup and the normal dorsal-ventral gradient of BMP signaling in the optic cup is disrupted. Pax2 becomes undetectable and expression of Sfrp2 increases in the ventral optic cup, suggesting that increased BMP signaling alter their expression, resulting in failure to close the optic fissure. In summary, our results suggest that negative and positive auto-regulation of BMP expression is important to regulate early eye development.

Previous studies of mouse embryos concluded that after the optic vesicle evaginates from the ventral forebrain and contacts the surface ectoderm, signals from the ectoderm specify the distal region of the optic vesicle to become retina and signals from the optic vesicle induce the lens. Germline deletion of Bmp4 resulted in failure of lens formation. We performed conditional deletion of Bmp4 from the optic vesicle to test the function of Bmp4 in murine eye development. The optic vesicle evaginated normally and contacted the surface ectoderm. Lens induction did not occur. The optic cup failed to form and the expression of retina-specific genes decreased markedly in the distal optic vesicle. Instead, cells in the prospective retina expressed genes characteristic of the retinal pigmented epithelium. We conclude that Bmp4 is required for retina specification in mice. In the absence of Bmp4, formation of the retinal pigmented epithelium is the default differentiation pathway of the optic vesicle. Differences in the signaling pathways required for specification of the retina and retinal pigmented epithelium in chicken and mouse embryos suggest major changes in signaling during the evolution of the vertebrate eye.

Purpose The goal of this study was to quantitatively identify the differentially expressed proteins in nuclear cataracts of different ages and normal lens nuclei in humans. Experimental design Forty-eight human lens nucleus samples with hardness grades III, IV were obtained during cataract surgery by extracapsular cataract extraction. Seven normal transparent human lens nuclei were obtained from fresh normal cadaver eyes during corneal transplantation surgery. Lens nuclei were divided into seven groups according to age and optic axis: Group A (average age 80.8 ± 1.2 years), Group B (average age 57.0 ± 4.0 years), Group C average age 80.3 ± 4.5 years), Group D (average age 56.9 ± 4.2 years), Group E (average age 78.1 ± 2.5 years), Group F (average age 57.6 ± 3.3 years) and Group G (seven normal transparent human lenses from normal cadaver eyes, average age 34.7 ± 4.2 years). Water-soluble, water-insoluble, and water-insoluble-urea-soluble protein fractions were extracted from samples. The three-part protein fractions from the individual lenses were combined to form the total proteins of each sample. The proteomic profiles of each group were further analyzed using 8-plex iTRAQ labeling combined with 2D-LC-MS/MS. The data were analyzed with the ProteinPilot software for peptide matching, protein identification, and quantification. Differentially expressed proteins were validated by Western blotting. Results We employed biological and technical replicates and selected the intersection of the two results, which included 80 proteins. Nine proteins were differentially expressed among the 80 proteins identified using proteomic techniques. In age-related nuclear cataracts (ARNC), the expression levels of fatty acid-binding protein and pterin-4-alpha-carbinolamine dehydratase were upregulated, whereas the levels of alpha-crystallin B chain (CRYAB), GSH synthetase, phakinin, gamma-crystallin C, phosphoglycerate kinase 1, betaine-homocysteine S-methyltransferase 1 (BHMT1), and spectrin beta chain were downregulated. These proteins may be associated with abnormal protein aggregation and oxidative stress. GSH synthetase and CRYAB expression levels in the nuclear cataract decreased with age. The mass spectrometric analysis results were consistent with the Western blot validation. Conclusion and clinical relevance The results indicate that CRYAB and GSH synthetase may be involved in ARNC pathogenesis. iTRAQ combined with 2D-LC-MS/MS provides new methods for future studies of pathological mechanisms and protective drug development for ARNC.

The discovery of cytosolic RNA granule (RG) component proteins associated with human cataract has initiated investigations on post-transcriptional mechanisms of gene expression control in the lens. Application of established mouse lens epithelial cell lines (LECs) can provide rapid insights on RG function in lens cells, especially because mouse mutants in several RG components are not available. However, although these LECs represent potential reagents for such analyses, they are uncharacterized for lens gene expression or RG formation. Therefore, a detailed molecular and cellular characterization of three permanent mouse LECs 17EM15, 21EM15 and αTN4 is performed in this study. Comparative analysis between microarray gene expression datasets on LEC 21EM15 and iSyTE lens tissue demonstrates that 30% of top 200 iSyTE identified lens-enriched genes are expressed in these cells. Majority of these candidates are independently validated to either have lens expression, function or linkage to cataract. Moreover, analysis of microarray data with genes described in Cat-Map, an online database of cataract associated genes and loci, demonstrates that 131 genes linked to cataract loci are expressed in 21EM15 cells. Furthermore, gene expression in LECs is compared to isolated lens epithelium or fiber cells by qRT-PCR and by comparative analyses with publically available epithelium or fiber-specific microarray and RNA-seq (sequencing) datasets. Expression of select candidate genes was validated by regular and real-time quantitative RT-PCR. Expression of lens epithelium-enriched genes Foxe3, Pax6, Anxa4 and Mcm4 is up-regulated in LEC lines, compared to isolated lens fiber cells. Moreover, similar to isolated lens epithelium, all three LECs exhibit down-regulation of fiber cell-expressed genes Crybb1, Mip and Prox1 when compared to fiber cells. These data indicate that the LEC lines exhibit greater similarity to lens epithelium than to fiber cells. Compared to non-lens cell line NIH3T3, LECs exhibit significantly enriched expression of transcription factors with important function in the lens, namely Pax6, Foxe3 and Prox1. In addition to these genes, all three LECs also express key lens- and cataract-associated genes, namely Dkk3, Epha2, Hsf4, Jag1, Mab21l1, Meis1, Pknox1, Pou2f1, Sfrp1, Sparc, Tdrd7 and Trpm3. Additionally, 21EM15 microarrays indicate expression of Chmp4b, Cryab and Tcfap2a among others important genes. Immunostaining with makers for Processing bodies (P-bodies) and Stress granules (SGs) demonstrates that these classes of RGs are robustly expressed in all three LECs. Moreover, under conditions of stress, 17EM15 and αTN4 exhibit significantly higher numbers of P-bodies and SGs compared to NIH3T3 cells. In sum, these data indicate that mouse LECs 21EM15, 17EM15 and αTN4 express key lens or cataract genes, are similar to lens epithelium than fiber cells, and exhibit high levels of P-bodies and SGs, indicating their suitability for investigating gene expression control and RG function in lens-derived cells.

Precise shaping of the eye is crucial for proper vision. Here, we use experiments on chick embryos along with computational models to examine the mechanical factors involved in the formation of the optic vesicles (OVs), which grow outward from the forebrain of the early embryo. First, mechanical dissections were used to remove the surface ectoderm (SE), a membrane that contacts the outer surfaces of the OVs. Principal components analysis of OV shapes suggests that the SE exerts asymmetric loads that cause the OVs to flatten and shear caudally during the earliest stages of eye development and later to bend in the caudal and dorsal directions. These deformations cause the initially spherical OVs to become pear-shaped. Exposure to the myosin II inhibitor blebbistatin reduced these effects, suggesting that cytoskeletal contraction controls OV shape by regulating tension in the SE. To test the physical plausibility of these interpretations, we developed 2-D finite-element models for frontal and transverse cross-sections of the forebrain, including frictionless contact between the SE and OVs. With geometric data used to specify differential growth in the OVs, these models were used to simulate each experiment (control, SE removed, no contraction). For each case, the predicted shape of the OV agrees reasonably well with experiments. The results of this study indicate that differential growth in the OV and external pressure exerted by the SE are sufficient to cause the global changes in OV shape observed during the earliest stages of eye development.

PurposeVitreous liquefaction and subsequent posterior vitreous detachment can lead to several sight-threatening diseases, including retinal detachment, macular hole and macular traction syndrome, nuclear cataracts, and possibly, open-angle glaucoma. In this study, we tested the ability of three novel synthetic chondroitin sulfate proteoglycan mimics to preserve the structure and physical properties of enzymatically-degraded bovine vitreous.MethodsChondroitin sulfate proteoglycan mimics, designed to bind to type II collagen, hyaluronic acid, or both, were applied to trypsin- or collagenase-treated bovine vitreous in situ and in vitro. Rheology and liquefaction tests were performed to determine the physical properties of the vitreous, while Western blots were used to detect the presence and degradation of soluble collagen II (α1). Deep-etch electron microscopy (DEEM) identified the ultrastructure of mimic-treated and untreated enzyme-degraded bovine vitreous.ResultsProteoglycan mimics preserved the physical properties of trypsin-degraded bovine vitreous and protected against vitreous liquefaction. Although the collagen-binding mimic maintained the physical properties of collagenase-treated vitreous, liquefaction still occurred. Western blots indicated that the mimic provided only marginal protective ability against soluble collagen degradation. Deep-etch electron microscopy, however, showed increased density and isotropy of microstructural components in mimic-treated vitreous, supporting the initial result that vitreous structure was preserved.ConclusionsProteoglycan mimics preserved bovine vitreous physical properties after enzymatic degradation. These compounds may be useful in delaying or preventing the pathological effects of age-related, or enzymatically-induced, degradation of the vitreous body.

Murine lacrimal, harderian and meibomian glands develop from the prospective conjunctival and eyelid epithelia and produce secretions that lubricate and protect the ocular surface. Sox9 expression localizes to the presumptive conjunctival epithelium as early as E11.5 and is detected in the lacrimal and harderian glands as they form. Conditional deletion showed that Sox9 is required for the development of the lacrimal and harderian glands and contributes to the formation of the meibomian glands. Sox9 regulates the expression of Sox10 to promote the formation of secretory acinar lobes in the lacrimal gland. Sox9 and FGF signaling were required for the expression of cartilage-associated extracellular matrix components during early stage lacrimal gland development. Fgfr2 deletion in the ocular surface epithelium reduced Sox9 and eliminated Sox10 expression. Sox9 deletion from the ectoderm did not affect Fgf10 expression in the adjacent mesenchyme or Fgfr2 expression in the epithelium, but appeared to reduce FGF signaling. Sox9 heterozygotes showed a haploinsufficient phenotype, in which the exorbital branch of the lacrimal gland was absent in most cases. However, enhancement of epithelial FGF signaling by expression of a constitutively active FGF receptor only partially rescued the lacrimal gland defects in Sox9 heterozygotes, suggesting a crucial role of Sox9, downstream of FGF signaling, in regulating lacrimal gland branching and differentiation.

Oxygen levels in the eye are generally low and tightly regulated. Oxygen enters the eye largely by diffusion from retinal arterioles and through the cornea. In intact eyes, oxygen from the retinal arterioles diffuses into the vitreous body. There is a decreasing oxygen gradient from the retina to the lens, established by oxygen consumption by ascorbate in the vitreous fluid and lens metabolism. Age-related degeneration of the vitreous body or removal during vitrectomy exposes the posterior of the lens to increased oxygen, causing nuclear sclerotic cataracts. Lowering oxygen in the vitreous, as occurs in patients with ischemic diabetic retinopathy, protects against cataracts after vitrectomy. Vitrectomy and cataract surgery increase oxygen levels at the trabecular meshwork and with it the risk of open angle glaucoma. Two additional risk factors for glaucoma, African heritage and having a thinner cornea, are also associated with increased oxygen in the anterior chamber angle. Preservation of the vitreous body and the lens, two important oxygen consumers, would protect against nuclear sclerotic cataracts and open angle glaucoma. Delaying removal of the lens for as long as possible after vitrectomy would be an important step in delaying ocular hypertension and glaucoma progression.

Lens induction is a classical embryologic model to study cell fate determination. It has been proposed earlier that specific changes in core histone modifications accompany the process of cell fate specification and determination. The lysine acetyltransferases CBP and p300 function as principal enzymes that modify core histones to facilitate specific gene expression. Herein, we performed conditional inactivation of both CBP and p300 in the ectodermal cells that give rise to the lens placode. Inactivation of both CBP and p300 resulted in the dramatic discontinuation of all aspects of lens specification and organogenesis, resulting in aphakia. The CBP/p300(-/-) ectodermal cells are viable and not prone to apoptosis. These cells showed reduced expression of Six3 and Sox2, while expression of Pax6 was not upregulated, indicating discontinuation of lens induction. Consequently, expression of αB- and αA-crystallins was not initiated. Mutant ectoderm exhibited markedly reduced levels of histone H3 K18 and K27 acetylation, subtly increased H3 K27me3 and unaltered overall levels of H3 K9ac and H3 K4me3. Our data demonstrate that CBP and p300 are required to establish lens cell-type identity during lens induction, and suggest that posttranslational histone modifications are integral to normal cell fate determination in the mammalian lens.

This unit describes a protocol for embedding, sectioning, and immunocytochemical analysis of pluripotent stem cell-derived 3-D organoids. Specifically, we describe a method to embed iPSC-derived retinal cups in low-melt agarose, acquire thick sections using a vibratome tissue slicer, and perform immunohistochemical analysis. This method includes an approach for antibody labeling that minimizes the amount of antibody needed for individual experiments and that utilizes large-volume washing to increase the signal-to-noise ratio, allowing for clean, high-resolution imaging of developing cell types. The universal methods described can be employed regardless of the type of pluripotent stem cell used and 3-D organoid generated. © 2016 by John Wiley & Sons, Inc.

In the early embryo, the eyes form initially as relatively spherical optic vesicles (OVs) that protrude from both sides of the brain tube. Each OV grows until it contacts and adheres to the overlying surface ectoderm (SE) via an extracellular matrix (ECM) that is secreted by the SE and OV. The OV and SE then thicken and bend inward (invaginate) to create the optic cup (OC) and lens vesicle, respectively. While constriction of cell apices likely plays a role in SE invagination, the mechanisms that drive OV invagination are poorly understood. Here, we used experiments and computational modeling to explore the hypothesis that the ECM locally constrains the growing OV, forcing it to invaginate. In chick embryos, we examined the need for the ECM by (1) removing SE at different developmental stages and (2) exposing the embryo to collagenase. At relatively early stages of invagination (Hamburger–Hamilton stage HH14 $$-$$ ), removing the SE caused the curvature of the OV to reverse as it ‘popped out’ and became convex, but the OV remained concave at later stages (HH15) and invaginated further during subsequent culture. Disrupting the ECM had a similar effect, with the OV popping out at early to mid-stages of invagination (HH14 $$-$$ to HH14 $$+$$ ). These results suggest that the ECM is required for the early stages but not the late stages of OV invagination. Microindentation tests indicate that the matrix is considerably stiffer than the cellular OV, and a finite-element model consisting of a growing spherical OV attached to a relatively stiff layer of ECM reproduced the observed behavior, as well as measured temporal changes in OV curvature, wall thickness, and invagination depth reasonably well. Results from our study also suggest that the OV grows relatively uniformly, while the ECM is stiffer toward the center of the optic vesicle. These results are consistent with our matrix-constraint hypothesis, providing new insight into the mechanics of OC (early retina) morphogenesis.

SUMMARYWe previously found that lenses lacking the Acvr1 gene, which encodes a bone morphogenetic protein (BMP) receptor, had abnormal proliferation and cell death in epithelial and cortical fiber cells. We tested whether the tumor suppressor protein p53 (encoded by Trp53) affected this phenotype. Acvr1 conditional knockout (Acvr1CKO) mouse fiber cells had increased numbers of nuclei that stained for p53 phosphorylated on serine 15, an indicator of p53 stabilization and activation. Deletion of Trp53 rescued the Acvr1CKO cell death phenotype in embryos and reduced Acvr1-dependent apoptosis in postnatal lenses. However, deletion of Trp53 alone increased the number of fiber cells that failed to withdraw from the cell cycle. Trp53CKO and Acvr1;Trp53DCKO (double conditional knockout), but not Acvr1CKO, lenses developed abnormal collections of cells at the posterior of the lens that resembled posterior subcapsular cataracts. Cells from human posterior subcapsular cataracts had morphological and molecular characteristics similar to the cells at the posterior of mouse lenses lacking Trp53. In Trp53CKO lenses, cells in the posterior plaques did not proliferate but, in Acvr1;Trp53DCKO lenses, many cells in the posterior plaques continued to proliferate, eventually forming vascularized tumor-like masses at the posterior of the lens. We conclude that p53 protects the lens against posterior subcapsular cataract formation by suppressing the proliferation of fiber cells and promoting the death of any fiber cells that enter the cell cycle. Acvr1 acts as a tumor suppressor in the lens. Enhancing p53 function in the lens could contribute to the prevention of steroid- and radiation-induced posterior subcapsular cataracts.

The lens is composed of a thin metabolically active outer layer, consisting of epithelial and superficial fibre cells. Lying within this outer shell are terminally differentiated, metabolically inactive fibre cells, which are divided into an outer cortex and central nucleus. Mature fibre cells contain a very high protein concentration, which is important for the transparency and refractive power of the lens. These proteins are protected from oxidation by reducing substances, like glutathione, and by the low-oxygen environment around the lens. Glutathione reaches the mature fibre cells by diffusing from the metabolically active cells at the lens surface. With age, the cytoplasm of the nucleus becomes stiffer, reducing the rate of diffusion and making nuclear proteins more susceptible to oxidation. Low pO2is maintained at the posterior surface of the lens by the physical and physiological properties of the vitreous body, the gel filling the space between the lens and the retina. Destruction or degeneration of the vitreous body increases exposure of the lens to oxygen from the retina. Oxygen reaches the lens nucleus, increasing protein oxidation and aggregation and leading to nuclear cataract. We suggest that maintaining low pO2around the lens should prevent the formation of nuclear cataracts.

There are conflicting reports about whether BMP signaling is required for eyelid closure during fetal development. This question was addressed using mice deficient in BMP or TGFβ signaling in prospective eyelid and conjunctival epithelial cells. Genes encoding two type I BMP receptors, the type II TGFβ receptor, two BMP- or two TGFβ-activated R-Smads or the co-Smad Smad4 were deleted from the ocular surface ectoderm using Cre recombinase. Only mice with deletion of components of the BMP pathway had an `eyelid open at birth' phenotype. Mice lacking Fgf10 or Fgfr2 also have open eyelids at birth. To better understand the pathways that regulate BMP expression and function during eyelid development, we localized BMPs and BMP signaling intermediates in Fgfr2 and Smad4 conditional knockout (CKO) mice. We found that Fgfr2 was required for the expression of Bmp4, the normal distribution of Shh signaling and for preserving the differentiation of the conjunctival epithelium. FGF signaling also promoted the expression of the Wnt antagonist Sfrp1 and suppressed Wnt signaling in the prospective eyelid epithelial cells, independently of BMP function. Transcripts encoding Foxc1 and Foxc2, which were previously shown to be necessary for eyelid closure, were not detectable in Smad4CKO animals. c-Jun, another key regulator of eyelid closure, was present and phosphorylated in eyelid periderm cells at the time of fusion, but failed to translocate to the nucleus in the absence of BMP function. Smad4CKO mice also showed premature differentiation of the conjunctival epithelium, conjunctival hyperplasia and the acquisition of epidermal characteristics, including formation of an ectopic row of hair follicles in place of the Meibomian glands. A second row of eyelashes is a feature of human lymphedema-distichiasis syndrome, which is associated with mutations in FOXC2.

A network of capillaries branches from the hyaloid vascular system and surrounds the mammalian lens throughout much of its embryonic development. These vessels are presumed to be important for the growth and maturation of the lens, although the lenses of non-mammalian vertebrates have no comparable vessels. Over expression of VEGF-A in the lens increases the extent of these capillaries, but it is not known whether VEGF-A from the lens is necessary for their formation or survival. To address this question, we deleted Vegfa in the lens. This prevented the formation of the capillary networks adjacent to the lens capsule, but did not alter nearby hyaloid vessels at the surface of the retina. Postnatal lenses lacking Vegfa were smaller than wild type and, by 1 month of age, many had mild nuclear opacities. These opacities regressed with age. The lens is hypoxic throughout most of life and VEGF-A expression is often regulated by the transcription factor, hypoxia inducible factor-1. Lenses lacking Hif1a were of apparently normal size, had markedly reduced levels of mRNA for VEGF-A and glyceraldehyde-3-phosphate dehydrogenase, but had normal-appearing capillaries covering their surface. We conclude that VEGF-A from the lens is necessary for the formation of the normal hyaloid vascular system and that lack of these capillaries was the most likely cause of growth retardation during fetal and early postnatal lens development. In the absence of HIF-1 function, sufficient VEGF-A is produced by the lens to promote capillary formation. Further study is needed to explain the formation of the mild opacities seen in some lenses lacking Vegfa and their regression later in life.

The vertebrate lens provides an excellent model to study the mechanisms that regulate terminal differentiation. Although fibroblast growth factors (FGFs) are thought to be important for lens cell differentiation, it is unclear which FGF receptors mediate these processes during different stages of lens development. Deletion of three FGF receptors (Fgfr1-3) early in lens development demonstrated that expression of only a single allele of Fgfr2 or Fgfr3 was sufficient for grossly normal lens development, while mice possessing only a single Fgfr1 allele developed cataracts and microphthalmia. Profound defects were observed in lenses lacking all three Fgfrs. These included lack of fiber cell elongation, abnormal proliferation in prospective lens fiber cells, reduced expression of the cell cycle inhibitors p27(kip1) and p57(kip2), increased apoptosis and aberrant or reduced expression of Prox1, Pax6, c-Maf, E-cadherin and alpha-, beta- and gamma-crystallins. Therefore, while signaling by FGF receptors is essential for lens fiber differentiation, different FGF receptors function redundantly.

Although linked to several vitreoretinal pathologies including traumatic retinal tears, breaks, and symptomatic vitreomacular traction, the dynamic material behavior of the vitreous body in response to mechanical loads is not well understood. The purpose of this study was to evaluate spatiotemporal patterns of collagen fiber reorganization and vitreous deformation (strain) in response to tensile and compressive forces. Using thick slabs of bovine eyes we examined collagen fiber reorganization following tensile and compressive step-loading with quantitative polarized light imaging. Strains were measured from sparse marker arrays and temporal collagen behavior was estimated from creep compliance rheological tests. Results showed that under applied loads (1) collagen fibers became significantly more aligned at the vitreous base (near the pars plana and the ciliary body), (2) vitreous located directly behind the lens deformed significantly more than surrounding regions, and (3) changes in collagen fiber alignment occurred on a short (

It was recently demonstrated that deficiency of a conserved RNA binding protein (RBP) and RNA granule (RG) component Tdrd7 causes ocular defects including cataracts in human, mouse and chicken, indicating the importance of posttranscriptional regulation in eye development. Here we investigated the function of a second conserved RBP/RG component Caprin2 that is identified by the eye gene discovery tool iSyTE.In situ hybridization, Western blotting and immunostaining confirmed highly enriched expression of Caprin2 mRNA and protein in mouse embryonic and postnatal lens. To gain insight into its function, lens-specific Caprin2 conditional knockout (cKO) mouse mutants were generated using a lens-Cre deleter line Pax6GFPCre. Phenotypic analysis of Caprin2(cKO/cKO) mutants revealed distinct eye defects at variable penetrance. Wheat germ agglutinin staining and scanning electron microscopy demonstrated that Caprin2(cKO/cKO) mutants have an abnormally compact lens nucleus, which is the core of the lens comprised of centrally located terminally differentiated fiber cells. Additionally, Caprin2(cKO/cKO) mutants also exhibited at 8% penetrance a developmental defect that resembles a human condition called Peters anomaly, wherein the lens and the cornea remain attached by a persistent stalk.These data suggest that a conserved RBP Caprin2 functions in distinct morphological events in mammalian eye development.

To explore how limbal niche cells (LNCs) may control quiescence, self-renewal, and corneal epithelial lineage commitment/differentiation of limbal epithelial progenitor/stem cells (LEPCs), we have established an in vitro sphere assay by reunion between the two cell types in three-dimensional Matrigel. The resultant sphere exhibits inhibition of corneal epithelial lineage commitment/differentiation and marked clonal growth of LEPCs, of which the latter is correlated with activation of canonical Wnt signaling. Herein, we have created a similar reunion assay in immobilized heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3), which is purified from amniotic membrane (AM) and consists of a complex formed by hyaluronic covalently linked to heavy chain 1 of inter-α-inhibitor and noncovalently linked to pentraxin 3. The resultant spheres exhibited similar suppression of corneal epithelial lineage commitment/differentiation but upregulation of quiescence markers including nuclear translocation of Bmi-1, and negligible clonal growth of LEPCs. This outcome was correlated with the suppression of canonical Wnt but activation of noncanonical (Planar cell polarity) Wnt signaling as well as BMP signaling in both LEPCs and LNCs. The activation of BMP signaling in LNCs was pivotal because nuclear translocation of pSmad1/5/8 was prohibited in hLEPCs when reunioned with mLNCs of conditionally deleted Bmpr1a;Acvr1DCKO mice. Furthermore, ablation of BMP signaling in LEPCs led to upregulation of cell cycle genes, downregulation of Bmi-1, nuclear exclusion of phosphorylated Bmi-1, and marked promotion of the clonal growth of LEPCs. Hence, HC-HA/PTX3 uniquely upregulates BMP signaling in LNCs which leads to BMP signaling in LEPCs to achieve quiescence, helping explain how AM transplantation is clinically useful to be used as a matrix for ex vivo expansion of LEPCs and to treat corneal blindness caused by limbal stem cells deficiency. Stem Cells 2015;33:3341–3355

Previous studies reported that characteristic lens opacities were present in Alzheimer Disease (AD) patients postmortem. We therefore determined whether cataract grade or lens opacity is related to the risk of Alzheimer dementia in participants who have biomarkers that predict a high risk of developing the disease. AD biomarker status was determined by positron emission tomography-Pittsburgh compound B (PET-PiB) imaging and cerebrospinal fluid (CSF) levels of Aβ42. Cognitively normal participants with a clinical dementia rating of zero (CDR=0; N=40) or with slight evidence of dementia (CDR=0.5; N=2) were recruited from longitudinal studies of memory and aging at the Washington University Knight Alzheimer's Disease Research Center. The age, sex, race, cataract type and cataract grade of all participants were recorded and an objective measure of lens light scattering was obtained for each eye using a Scheimpflug camera. Twenty-seven participants had no biomarkers of Alzheimer dementia and were CDR=0. Fifteen participants had biomarkers indicating increased risk of AD, two of which were CDR=0.5. Participants who were biomarker positive were older than those who were biomarker negative. Biomarker positive participants had more advanced cataracts and increased cortical light scattering, none of which reached statistical significance after adjustment for age. We conclude that cataract grade or lens opacity is unlikely to provide a non-invasive measure of the risk of developing Alzheimer dementia. Key Words: Lens opacity, cataract, Scheimpflug imaging, Alzheimer disease biomarkers, preclinical Alzheimer disease

Retraction of: “Sclera-Choroid-RPE Transport of Eight β-Blockers in Human, Bovine, Porcine, Rabbit, and Rat Models” by Rajendra S. Kadam, Narayan P. S. Cheruvu, Henry F. Edelhauser, and Uday B. Kompella (Invest Ophthalmol Vis Sci. 2011;52:5387–5399) doi:10.1167/iovs.10-6233 A scientific misconduct investigation by University of Colorado Denver (Anschutz Medical Campus) concluded that this paper contains falsified and/or fabricated data. Specifically, Figures 1 and 3: Some LC-MS peak areas in the β-blocker transport data reported in these figures were falsified to create smooth monotonic transport curves with smaller error bars. Tables 2, 3; Figures 2, 4, 5, 7, 8, and 10: All use the primary transport data from curves in Figures 1 and 3, hence the analyses reported will all be contaminated by falsified data from Figure 1 and/or Figure 3. The university recommended the paper be retracted from publication and the Editor-in-Chief, David C. Beebe, agreed. The paper is therefore being retracted by ARVO from IOVS.

We study the molecular mechanisms that are essential for development and maintenance of transparency in the eye lens. Toward this goal, we have applied the bioinformatics-based gene discovery tool ...

To report intraocular oxygen tension in eyes of diabetic and nondiabetic patients.A prospective, interventional consecutive case series.Oxygen was measured with an optical oxygen sensor in patients who were undergoing vitrectomy. Before turning on the infusion fluid, intraocular oxygen tension was measured in two locations: adjacent to the lens and in the mid vitreous cavity.Fifty eyes from 50 patients were included in the study. Twenty-one eyes were from diabetic patients and 29 eyes were from nondiabetic patients. The mean oxygen tension adjacent to the lens was significantly lower in diabetic than in nondiabetic patients (8.4 +/- 0.7 mm Hg vs 10.7 +/- 0.8 mm Hg; P.05). Similarly, the mean oxygen tension in the center of the vitreous cavity was lower in diabetic than in nondiabetic patients (5.7 +/- 0.7 mm Hg vs 8.5 +/- 0.6 mm Hg; P.001). In subgroup analyses, previous panretinal photocoagulation or cataract surgery did not affect oxygen levels significantly in the vitreous of diabetic or nondiabetic patients.Eyes from diabetic patients have significantly lower intraocular oxygen tension than in eyes from nondiabetic patients. Because oxidative damage to the lens nucleus and increased intraocular oxygen tension have been associated with nuclear sclerotic cataract, these findings may help explain recent reports of an apparent protective effect of diabetes mellitus against nuclear sclerotic cataract.

Fibroblast growth factors (FGFs) play important roles in many aspects of development, including lens development. The lens is derived from the surface ectoderm and consists of an anterior layer of epithelial cells and elongated, terminally differentiated fiber cells that form the bulk of the tissue. FGF signaling has been implicated in lens induction, proliferation, and differentiation. To address the role of FGFs in lens development, we inactivated FGF receptor-2 (Fgfr2) using a Cre transgene that is expressed in all prospective lens cells from embryonic day 9.0. Inactivation of Fgfr2 shows that signaling through this receptor is not required for lens induction or for the proliferation of lens epithelial cells. However, Fgfr2 signaling is needed to drive lens fiber cells out of the cell cycle during their terminal differentiation. It also contributes to the normal elongation of primary lens fiber cells and to the survival of lens epithelial cells.

We isolated the chicken homologue of opticin (cOptc), a member of the small leucine-rich repeat protein (SLRP) family. cOptc is expressed in the brain and the neural tube from stage 9 onward. At later stages, cOptc is expressed in the ciliary epithelium of the eye, optic stalk, Rathke's pouch, pharyngeal pouches, nasal pit and otic vesicle.

This study evaluated in vivo imaging capabilities and safety of qualitative monitoring of oxygen saturation of hemoglobin (sO_2) of rabbit ciliary body tissues obtained with acoustic resolution (AR) photoacoustic tomography (PAT). AR PAT was used to collect trans-scleral images from ciliary body vasculature of seven New Zealand White rabbits. The PAT sO_2 measurements were obtained under the following conditions: when systemic sO_2 as measured by pulse oximetry was between 100% and 99% (level 1); systemic sO_2 as measured by pulse oximetry was between 98% and 90% (level 2); and systemic sO_2 as measured by pulse oximetry was less than 90% (level 3). Following imaging, histological analysis of ocular tissue was conducted to evaluate for possible structural damage caused by the AR PAT imaging. AR PAT was able to resolve anatomical structures of the anterior segment of the eye, viewed through the cornea or anterior sclera. Histological studies revealed no ocular damage. On average, sO_2 values (%) obtained with AR PAT were lower than sO_2 values obtained with pulse oximetry (all p < 0.001): 86.28 ± 4.16 versus 99.25 ± 0.28, 84.09 ± 1.81 vs. 95.3 ± 2.6, and 64.49 ± 7.27 vs. 71.15 ± 10.21 for levels 1, 2 and 3 respectively. AR PAT imaging modality is capable of qualitative monitoring for deep tissue sO_2 in rabbits. Further studies are needed to validate and modify the AR PAT modality specifically for use in human eyes. Having a safe, non-invasive method of in vivo imaging of sO_2 in the anterior segment is important to studies evaluating the role of oxidative damage, hypoxia and ischemia in pathogenesis of ocular diseases.

PURPOSE Collagen fiber remodeling in the vitreous body has been implicated in cases of vitreomacular traction, macular hole, and retinal detachment, and also may occur during pharmacologic vitreolysis. The purpose of this study was to evaluate quantitative polarized light imaging (QPLI) as a tool for studying fiber organization in the vitreous and near the vitreoretinal interface in control and enzymatically perturbed conditions. METHODS Fiber alignment was measured in anterior-posterior sections of bovine and porcine vitreous. Additional tests were performed on bovine lenses and nasal-temporal vitreous sections. Effects of proteoglycan degradation on collagen fiber alignment using trypsin and plasmin were assessed at the microstructural level using electron microscopy and at the global level using QPLI. RESULTS Control vitreous showed fiber organization patterns consistent with the literature across multiple-length scales, including the global anterior-posterior coursing of vitreous fibers, as well as local fibers parallel to the equatorial vitreoretinal interface and transverse to the posterior interface. Proteoglycan digestion with trypsin or plasmin significantly increased fiber alignment throughout the vitreous (P < 0.01). The largest changes (3×) occurred in the posterior vitreous where fibers are aligned transverse to the posterior vitreoretinal interface (P < 0.01). CONCLUSIONS Proteoglycan loss due to enzymatic vitreolysis differentially increases fiber alignment at locations where tractions are most common. We hypothesize that a similar mechanism leads to retinal complications during age-related vitreous degeneration. Structural changes to the entire vitreous body (as opposed to the vitreoretinal interface alone) should be evaluated during preclinical testing of pharmacological vitreolysis candidates.

Purpose To measure oxygen (pO 2 ) in eyes of patients undergoing intraocular surgery and identify correlations with central corneal thickness (CCT). Design Prospective, cross-sectional study. Methods setting: Institutional. patient population: 124 patients undergoing cataract and/or glaucoma surgery. observation procedure: Prior to surgery, an oxygen sensor was introduced into the anterior chamber (AC) via peripheral corneal paracentesis. The tip of the flexible fiberoptic probe was positioned for 3 measurements in all patients: (1) near central corneal endothelium; (2) in mid-AC; and (3) in AC angle. In patients undergoing cataract extraction, additional measurements were taken (4) at the anterior lens surface and (5) in the posterior chamber. main outcome measures: pO 2 measurements at 5 locations within the eye were compared to central corneal thickness measurements by multivariate regression analyses. Results There was a statistically significant inverse correlation between CCT and pO 2 in the anterior chamber angle ( P = .048). pO 2 was not significantly related to CCT at any other location, including beneath the central cornea. Regression analysis relating CCT to age, race, and oxygen levels in all 5 locations in the anterior segment revealed an association of a thinner cornea with increasing age ( P = .007). Conclusions Physiologic correlations with central corneal thickness may provide clues to understanding why a thinner cornea increases the risk of open glaucoma. Associations between glaucoma risk, CCT, and pO 2 in the AC angle suggest that exposure of the outflow system to increased oxygen or oxygen metabolites may increase oxidative damage to the trabecular meshwork cells, resulting in elevation of intraocular pressure.

As recently as two decades ago, textbooks on vitreous taught students of ophthalmology that the vitreous gel had no real function in the adult eye other than to serve as a space-occupying, optically clear structure. In fact, one such textbook published in 1994 stated, “Apart from its role in oculogenesis, the vitreous has no well substantiated function so that an eye devoid of gel is not adversely affected” [1]. However, beginning in 2005 with published reports of oxygen tension measurements in the human vitreous gel before and after vitrectomy surgery [2] and culminating with reports in 2009 that the human vitreous gel consumes oxygen in an ascorbate-dependent manner [3], a new understanding of the vitreous gel and its role in vitreoretinal physiology and pathology has emerged.

During the development of the anterior segment of the eye, neural crest mesenchyme cells migrate between the lens and the corneal epithelium. These cells contribute to the structures lining the anterior chamber: the corneal endothelium and stroma, iris stroma, and trabecular meshwork. In the present study, removal of the lens or replacement of the lens with a cellulose bead led to the formation a disorganized aggregate of mesenchymal cells beneath the corneal epithelium. No recognizable corneal endothelium, corneal stroma, iris stroma, or anterior chamber was found in these eyes. When the lens was replaced immediately after removal, a disorganized mass of mesenchymal cells again formed beneath the corneal epithelium. However, 2 days after surgery, the corneal endothelium and the anterior chamber formed adjacent to the lens. When the lens was removed and replaced such that only a portion of its anterior epithelial cells faced the cornea, mesenchyme cells adjacent to the lens epithelium differentiated into corneal endothelium. Mesenchyme cells adjacent to lens fibers did not form an endothelial layer. The cell adhesion molecule, N-cadherin, is expressed by corneal endothelial cells. When the lens was removed the mesenchyme cells that accumulated beneath the corneal epithelium did not express N-cadherin. Replacement of the lens immediately after removal led to the formation of an endothelial layer that expressed N-cadherin. Implantation of lens epithelia from older embryos showed that the lens epithelium maintained the ability to support the expression of N-cadherin and the formation of the corneal endothelium until E15. This ability was lost by E18. These studies provide evidence that N-cadherin expression and the formation of the corneal endothelium are regulated by signals from the lens. N-cadherin may be important for the mesenchymal-to-epithelial transformation that accompanies the formation of the corneal endothelium.

A critical event in avian corneal development occurs when the acellular primary stroma swells and becomes populated by mesen- chymal cells that migrate from the periphery. These cells then deposit the mature stromal ma- trix that exhibits the unique features necessary for corneal function. Our previous work corre- lated the disappearance of collagen type IX immu- noreactivity at stage 27 (51 ⁄2-6 days) with matrix swelling and invasion. To investigate further the mechanism of this disappearance, we employed immunohistochemistry after tissue fixation with Histochoice, a non-crosslinking fixative, immuno- blot analysis of protein extracts, and gel sub- strate chromatography (zymography) to detect endogenous proteolytic activity. We found that corneas fixed in Histochoice retain immunoreac- tivity for type IX collagen for 1-2 days after corneal swelling. This immunoreactivity, how- ever, becomes extractable from tissue sections of unfixed corneas at the time of initiation of stro- mal swelling and mesenchymal cell invasion. Im- munoblot analysis confirmed that, following swelling, immunoreactivity for collagen IX de- creased substantially in corneas, but not in the vitreous body, which served as a comparison. Analysis of ammonium sulfate (AS) fractions of such extracts indicated that, at the time of swell- ing, much of the immunoreactivity for type IX collagen in cornea shifted from the AS precipitate (containing high molecular weight molecules) to the AS supernatant (containing smaller frag- ments). In contrast, collagen IX immunoreactiv- ity from the vitreous was precipitated by ammo- nium sulfate throughout the period of study. Collagen type II, a major fibrillar collagen in both the corneal stroma and vitreous, remained in the high molecular weight fraction at all times exam- ined. Zymography detected the presence of the latent (proenzyme) form of gelatinase A (MMP-2) before corneal swelling and invasion (4 days), and both the latent and active forms of the en- zyme after corneal swelling. This suggests tissue- specific, developmentally regulated proteolysis of collagen IX as a trigger for corneal matrix swelling. Dev. Dyn. 1998;212:27-37.

Purpose Previous studies that measured liquefaction and oxygen content in human vitreous suggested that exposure of the lens to excess oxygen causes nuclear cataracts. Here, we developed a computational model that reproduced available experimental oxygen distributions for intact and degraded human vitreous in physiologic and environmentally perturbed conditions. After validation, the model was used to estimate how age-related changes in vitreous physiology and structure alter oxygen levels at the lens. Methods A finite-element model for oxygen transport and consumption in the human vitreous was created. Major inputs included ascorbate-mediated oxygen consumption in the vitreous, consumption at the posterior lens surface, and inflow from the retinal vasculature. Concentration-dependent relations were determined from experimental human data or estimated from animal studies, with the impact of all assumptions explored via parameter studies. Results The model reproduced experimental data in humans, including oxygen partial pressure (Po2) gradients (≈15 mm Hg) across the anterior-posterior extent of the vitreous body, higher oxygen levels at the pars plana relative to the vitreous core, increases in Po2 near the lens after cataract surgery, and equilibration in the vitreous chamber following vitrectomy. Loss of the antioxidative capacity of ascorbate increases oxygen levels 3-fold at the lens surface. Homogeneous vitreous degeneration (liquefaction), but not partial posterior vitreous detachment, greatly increases oxygen exposure to the lens. Conclusions Ascorbate content and the structure of the vitreous gel are critical determinants of lens oxygen exposure. Minimally invasive surgery and restoration of vitreous structure warrant further attention as strategies for preventing nuclear cataracts.

The aim of the study was to investigate the protective effects of intact vitreous gel on the lens after pharmacologic vitreolysis and hyperoxia exposure in rats in vivo.Eyes of Sprague-Dawley rats were induced to posterior vitreous detachment (PVD) by pharmacologic vitreolysis, and the rats with and without PVD were treated with hyperoxia 3 h per day for 5 months. Lens transparency was monitored by a slit-lamp biomicroscope. A series of biochemical measurements were made in extracts of the lens cortex and nucleus. Ascorbate levels were measured in the aqueous and vitreous humors.No significant differences in lens transparency or morphology were observed in all groups, and no significant biochemical changes were observed in the cortex or nucleus of lenses of the PVD group. In the lens nucleus, the values of water-soluble protein concentration in PVD + hyperoxia group were lower than that of the PVD group. The levels of water-soluble proteins, glutathione (GSH) and ascorbate decreased in the hyperoxia group with an intact vitreous body. Vitreolysis enhanced the effect of hyperoxia, decreasing soluble protein, GSH and ascorbate below the levels seen in eyes with vitreolysis alone. The levels of antioxidants and soluble proteins were lower in the lens nucleus, and the effects of vitreolysis plus hyperoxia were more significant in the nucleus. Hyperoxia and hyperoxia plus vitreolysis reduced catalase activity and increased oxidized GSH to a greater extent in the lens cortex, although these treatments increased protein-GSH mixed disulfides in both regions. Long-term hyperoxia also lowered ascorbate levels in the vitreous and aqueous humors, an effect that was enhanced by vitreolysis.Exposure to excess molecular oxygen produces significant oxidative damage to the lens, especially the lens nucleus. These effects were enhanced by pharmacologic vitreolysis, indicating that intact vitreous gel protects the lens from oxidative damage.

Pax6 encodes a specific DNA-binding transcription factor that regulates the development of multiple organs, including the eye, brain and pancreas. Previous studies have shown that Pax6 regulates the entire process of ocular lens development. In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific populations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification in telencephalon. In the pancreas, Pax6 is essential for the differentiation of alpha-, beta- and delta-islet cells. To elucidate molecular roles of Pax6, chromatin immunoprecipitation experiments combined with high-density oligonucleotide array hybridizations (ChIP-chip) were performed using three distinct sources of chromatin (lens, forebrain and beta-cells). ChIP-chip studies, performed as biological triplicates, identified a total of 5,260 promoters occupied by Pax6. 1,001 (133) of these promoter regions were shared between at least two (three) distinct chromatin sources, respectively. In lens chromatin, 2,335 promoters were bound by Pax6. RNA expression profiling from Pax6(+/-) lenses combined with in vivo Pax6-binding data yielded 76 putative Pax6-direct targets, including the Gaa, Isl1, Kif1b, Mtmr2, Pcsk1n, and Snca genes. RNA and ChIP data were validated for all these genes. In lens cells, reporter assays established Kib1b and Snca as Pax6 activated and repressed genes, respectively. In situ hybridization revealed reduced expression of these genes in E14 cerebral cortex. Moreover, we examined differentially expressed transcripts between E9.5 wild type and Pax6(-/-) lens placodes that suggested Efnb2, Fat4, Has2, Nav1, and Trpm3 as novel Pax6-direct targets. Collectively, the present studies, through the identification of Pax6-direct target genes, provide novel insights into the molecular mechanisms of Pax6 gene control during mouse embryonic development. In addition, the present data demonstrate that Pax6 interacts preferentially with promoter regions in a tissue-specific fashion. Nevertheless, nearly 20% of the regions identified are accessible to Pax6 in multiple tissues.

During their maturation, lens cells lose all membrane bound organelles, including mitochondria. In chicken embryos this process begins in the central lens fibers beginning around embryonic day 12 (E12). Transforming growth factor beta (TGF beta) is a multipotent growth modulator thought to play a role in numerous developmental processes. TGF beta 1 has been localized to mitochondria in rat liver cells and muscle cells. In the present study, we examined the expression of TGF beta isoform mRNAs and proteins during chicken embryonic lens development. PCR analysis demonstrated TGF beta 2 and TGF beta 3 transcripts in the lens epithelium and fibers throughout pre- and post-hatching development. TGF beta isoforms were detected throughout the lens epithelium and fibers early in development (E6). However by E19, the distribution of TGF beta 2 and TGF beta 3 transcripts and proteins coincided with regions of the lens that contained mitochondria. In addition, intense TGF beta staining was observed in the basal portions of the equatorial epithelial cells, a region with abundant mitochondria. Transcripts for TGF beta 1 and TGF beta 4 were not detected in any tissue or time frame examined. Similarly, no immunostaining for TGF beta 1 was observed.

Purpose Ascorbic acid (AsA) is an important antioxidant in the eye. Ascorbic acid is usually transported by sodium-dependent AsA transporters (SVCTs), and dehydroascorbic acid (DHA) by glucose transporters (GLUTs). This study investigates these AsA-related transporters in human compared with mouse eyes. Methods Five pairs of human donor eyes and 15 pairs of mouse eyes were collected. Immunofluorescence and in situ hybridization were performed to detect SVCTs and GLUTs expression in the ciliary epithelium, retina, and lens epithelial cells (LECs). These tissues were isolated with laser microdissection followed by extraction of total RNA. Quantitative PCR (qPCR) was performed to examine the mRNA level of SVCTs and GLUTs in human and mouse ocular tissues. Results Immunofluorescence and in situ hybridization showed SVCT2 and GLUT1 expression in human ciliary epithelium with varied distributions. Sodium-dependent AsA transporter 2 is expressed only in the pigmented epithelium (PE), and GLUT1 is predominately expressed in the nonpigmented epithelium (NPE). However, SVCT2 was not identified in mouse ciliary epithelium, whereas GLUT1 expressed in both PE and NPE. Laser microdissection and qPCR revealed high levels of SVCT2 mRNA in human RPE cells and murine neural retina. Sodium-dependent AsA transporter 1 mRNA could be detected only in human and murine LECs. Glucose transporter 3 and GLUT4 mRNA could not be detected in either the human or mouse ciliary processes or in the lens epithelium. Conclusions These fundamental findings indicate AsA transporter expression in eyes of humans is significantly different compared with mice. This may explain why human aqueous and vitreous humors contain higher AsA levels compared with other animals.

Invagination of epithelia is an essential morphogenetic process that occurs in early eye development. The mechanics of the tissue forces necessary for eye invagination are not yet understood [1]. The eyes begin as two optic vesicles that grow outwards from the forebrain and adhere to the surface ectoderm. At this point of contact, both the surface ectoderm and optic vesicle thicken, forming the lens placode and retinal placode, respectively. The two placodes then bend inward to create the lens vesicle and bilayered optic cup (OC) [1, 2].Copyright © 2012 by ASME

Differences in brain structure between species have long fascinated evolutionary biologists. Understanding how these differences arise requires knowing how they are generated in the embryo. Growing evidence in the field of evolutionary developmental biology (evo-devo) suggests that morphological differences between species result largely from changes in the spatiotemporal regulation of gene expression during development. Corresponding changes in functional cellular behaviors (morphogenetic mechanisms) are only beginning to be explored, however. Here we show that spatiotemporal patterns of tissue contractility are sufficient to explain differences in morphology of the early embryonic brain between disparate species. We found that enhancing cytoskeletal contraction in the embryonic chick brain with calyculin A alters the distribution of contractile proteins on the apical side of the neuroepithelium and changes relatively round cross-sections of the tubular brain into shapes resembling triangles, diamonds, and narrow slits. These perturbed shapes, as well as overall brain morphology, are remarkably similar to those of corresponding sections normally found in species such as zebrafish and Xenopus laevis (frog). Tissue staining revealed relatively strong concentration of F-actin at vertices of hyper-contracted cross-sections, and a finite element model shows that local contraction in these regions can convert circular sections into the observed shapes. Another model suggests that these variations in contractility depend on the initial geometry of the brain tube, as localized contraction may be needed to open the initially closed lumen in normal zebrafish and Xenopus brains, whereas this contractile machinery is not necessary in chick brains, which are already open when first created. We conclude that interspecies differences in cytoskeletal contraction may play a larger role in generating differences in morphology, and at much earlier developmental stages, in the brain than previously appreciated. This study is a step toward uncovering the underlying morphomechanical mechanisms that regulate how neural phenotypic differences arise between species.

PURPOSE To determine whether the gauge of vitrectomy instrumentation is associated with the progression of nuclear sclerotic cataract. METHODS A prospective interventional and observational study of patients undergoing vitrectomy surgery for various retinal conditions. Patients had Scheimpflug lens photography in the operated and fellow eye at baseline and at 6 months and 12 months after vitrectomy surgery. RESULTS Of 42 eyes included in the analysis, 11 had 20-gauge surgery, 22 had 23-gauge surgery, and 9 had 25-gauge surgery. In all operated eyes, vitrectomy surgery led to the significant progression of nuclear sclerotic cataract, compared with the fellow, unoperated eye. This small study was unable to detect a difference in nuclear sclerotic progression when comparing small-gauge surgery (23 and 25 gauge) with standard 20-gauge surgery. CONCLUSION Removal of the vitreous gel using any-gauge vitrectomy surgery leads to significant progression of nuclear sclerotic cataract at 6 months and 12 months. The findings are consistent with the hypothesis that the vitreous gel is important in protecting the lens from increased exposure to oxygen that leads to the formation of nuclear sclerotic cataract. This increased exposure to oxygen occurs as a result of removing the vitreous gel and is independent of the gauge of vitrectomy instrumentation.

Results of electrical, dye-coupling and morphological studies have previously suggested that gap junctions mediate communication between the anterior epithelium of the lens and the underlying lens fiber cells. This connection is believed to permit ‘metabolic cooperation’ between these dissimilar cell types and may be of particular importance to the fiber cells, which are thought incapable of autonomous ionic homeostasis. We reinvestigated the nature of the connection between epithelial and fiber cells of the embryonic chicken lens using fluorescence confocal microscopy and freeze-fracture analysis. In contrast to earlier studies, our data provided no support for gap-junction-mediated transport from the lens epithelium to the fibers. Fluorescent dyes loaded biochemically into the lens epithelium were retained there for more than one hour. There was a decrease in epithelial fluorescence over this period, but this was not accompanied by an increase in fiber cell fluorescence. Diffusional modeling suggested that these data were inconsistent with the presence of extensive epithelium-fiber cell coupling, even if the observed decrease in epithelial fluorescence was attributed exclusively to the diffusion of dye into the fiber mass via gap junctions. Furthermore, the rate of loss of fluorescence from isolated epithelia was indistinguishable from that measured in whole lenses, suggesting that decreased epithelial fluorescence resulted from photobleaching and leakage of dye rather than diffusion, via gap junctions, into the fibers. Analysis of freeze-fracture replicas of plasma membranes at the epithelial-fiber cell interface failed to reveal evidence of gap-junction plaques, although evidence of endocytosis was abundant. These studies were done under conditions where the location of the fracture plane was unambiguous and where gap junctions could be observed in the lateral membranes of neighboring epithelial and fiber cells. Paradoxically, tracer molecules injected into the fiber mass were able to pass into the epithelium via a pathway that was not blocked by incubation at 4 degrees C or by treatment with octanol and which excluded large (approximately 10 kDa) molecular mass tracers. Together with previous measurements of electrical coupling between fiber cells and epithelial cells, these data indicate the presence of a low-resistance pathway connecting these cell types that is not mediated by classical gap junctions.