Hydroxyproline concentrations in ocular tissues of Arabian camel (Camelus dromedarius Linn.)

Hydroxyproline (Hyp) concentrations (total, free, peptide-bound and protein-bound) in camel eye tissues were determined. Total Hyp concentration was highest in iris, followed by ciliary body, sclera, cornea, lens and retina; the difference between total Hyp concentration of iris and sclera (P < 0.05) and cornea, lens and retina (P < 0.001) was statistically significant. Cornea had the highest concentration of free Hyp, followed by ciliary body, retina, iris, sclera and lens (P < 0.001). Peptide-bound Hyp concentration was highest in iris, followed by lens, cornea, ciliary body, retina and sclera (P < 0.001). Iris also had the highest concentration of protein-bound Hyp, followed by ciliary body, sclera, cornea, retina and lens; the difference in the protein-bound Hyp concentration between iris and sclera (P < 0.05) and cornea, retina and lens (P < 0.001) was statistically significant. Iris was also found to have the highest concentration of collagen, followed by ciliary body, sclera, cornea, lens and retina; the difference between the collagen concentration of iris and sclera (P < 0.05) and cornea, lens and retina (P < 0.001) was statistically significant. These variations may result from differences in the collagen structure and/or composition in these tissues.     

Developmental analysis of ocular morphogenesis in alpha A-crystallin/diphtheria toxin transgenic mice undergoing ablation of the lens.

The role of the lens in early eye development was examined in transgenic mice carrying the cytotoxic diphtheria toxin A gene driven by hamster alpha A-crystallin promoter sequences. Mice hemizygous for this construct are microphthalmic and contain a vacuolated and highly disorganized lens, whereas adult homozygous mice are completely ablated of the lens and lack a pupil, aqueous and posterior chamber, vitreous humor, iris, and ciliary body and show extensive convolution of the sensory retina. Developmental analysis of animals homozygous for the transgene revealed that the optic cup and lens vesicle form normally and that ablation of the lens occurs as a gradual degenerative process beginning between Days 12 and 13 of gestation. Degeneration of the lens vesicle coincides with retarded growth and development of the neuroretina, sclera, and cornea. The anterior lip of the optic cup fails to differentiate into the normal epithelium of the iris and ciliary body and the vitreous body does not develop. Although the retinal layers apparently form normally, retinal folding becomes prominent following lens degeneration. These results suggest that development of a functional lens from Embryonic Day 12.5 onward is critical for formation of the ciliary epithelium, iris, and vitreous body, as well as for appropriate growth, development, and maintenance of morphology of the retina, cornea, sclera, and optic nerve. Our results also provide information on the time course of DT-A-mediated cell destruction in vivo and are discussed in context with previous lens ablation studies and the importance of developmental analysis for interpretation of the extent to which morphogenetic aberrations are concurrent with or secondary to genetic ablation of the target tissue.     

Nucleofection of whole murine retinas

The mouse retina constitutes an important research model for studies aiming to unravel the cellular and molecular mechanisms underlying ocular diseases. The accessibility of this tissue and its feasibility to directly obtain neurons from it has increased the number of studies culturing mouse retina, mainly retinal cell suspensions. However, to address many questions concerning retinal diseases and protein function, the organotypic structure must be maintained, so it becomes important to devise methods to transfect and culture whole retinas without disturbing their cellular structure. Moreover, the postmitotic stage of retinal neurons makes them reluctant to commonly used transfection techniques. For this purpose some published methods employ in vivo virus-based transfection techniques or biolistics, methods that present some constraints. Here we report for the first time a method to transfect P15-P20 whole murine retinas via nucleofection, where nucleic acids are directly delivered to the cell nuclei, allowing in vitro transfection of postmitotic cells. A detailed protocol for successful retina extraction, organotypic culture, nucleofection, histological procedures and imaging is described. In our hands the A-33 nucleofector program shows the highest transfection efficiency. Whole flat-mount retinas and cryosections from transfected retinas were imaged by epifluorescence and confocal microscopy, showing that not only cells located in the outermost retinal layers, but also those in inner retinal layers are transfected. In conclusion, we present a novel method to successfully transfect postnatal whole murine retina via nucleofection, showing that retina can be successfully nucleofected after some optimization steps.     

Analysis of lens cell fate and eye morphogenesis in transgenic mice ablated for cells of the lens lineage

Transgenic mice carrying the diphtheria toxin A gene driven by mouse gamma 2-crystallin promoter sequences manifest microphthalmia due to ablation of fiber cells in the ocular lens. Here we map ablation events in the lens by crossing animals hemizygous for the ablation construct with transgenic mice homozygous for the in situ lacZ reporter gene driven by identical gamma 2-crystallin promoter sequences. By comparing the spatial distribution of lacZ-expressing cells and the profile of gamma-crystallin gene expression in the lenses of normal and microphthalmic offspring, the contributions of specific cell types to lens development were examined. The results suggest that phenotypically and developmentally distinct populations of lens fiber cells are able to contribute to the lens nucleus during organogenesis. We also show that dosage of the transgene and its site of integration influence the extent of ablation. In those mice homozygous for the transgene and completely lacking cells of the lens lineage, we show that the sclera, cornea, and ciliary epithelium are reduced in size but, otherwise, reasonably well formed. In contrast, the anterior chamber, iris, and vitreous body are not discernible while the sensory retina is highly convoluted and extensively fills the vitreous chamber.     

Structure,histochemistry, ontogenetic development,and regeneration of the ocellus of Cladonema radiatum dujardin (cnidaria,hydrozoa, anthomedusae)

The ectodermal eyes, 45–55 μm in diameter, of the cnidarian hydrozoan Cladonema radiatum Dujardin possess a lens approximately 15 μm in diameter enveloped by an eyecup (retina). An overlying layer of intensely vacuolated distal process of the adjoining epithelial cells forms a transparent cornea. The eyecup is composed of three cell types: basal cells, melanin-containing pigment cells, and photoreceptor cells. The last two cell types occur in the ratio of approximately 2:1. Histogenesis of the eye both during ontogeny and regeneration is described from light and electron microscopic investigations. During ontogeny the cell types forming the retina are derived from a compact group of morphologically undifferentiated cells, but during regeneration a primordium is formed by regeneration cells. In both cases the lens is built from distal nonnucleated cytoplasmic portions pinched off from the pigment cells. The cornea is formed by distal lamellar processes of the ocellus adjoining the epithelial cells. Through EM-histochemical methods (silver impregnation and DOPA-oxidase reaction) the pigment of the chromatophores of the retina was identified as melanin.     

Preparation and square wave electroporation of retinal explant cultures

This protocol details organotypic cultures of developing mouse, monkey and human retinas, which can be maintained for up to 2 weeks. Intact retinas are placed on polycarbonate filters floating on explant culture medium and fed every day with previously prepared retinal conditioned medium. Developing mouse retinas from E12.5 to P12 have been successfully cultured using this protocol as well as retinas from the equivalent stages of human and monkey development. Although this protocol does not require any special equipment, it provides a relatively high throughput. Retinal explant cultures lend themselves to complex pharmacological and genetic manipulations that are currently not feasible in vivo. A detailed procedure for square wave electroporation of retinal explants is also included to provide a high-throughput means to alter gene expression in the developing retina. This protocol for the preparation of retinal conditioned explant medium requires 4 d. Other steps of this protocol can be completed in 2 h.     

Whole-mount immunohistochemistry: a high-throughput screen for patterning defects in the mouse cerebellum.

Large-scale mouse mutagenesis experiments now under way require appropriate screening methods. An important class of potential mutants comprises those with defects in the development of normal cerebellar patterning. Cerebellar defects are likely to be identified often because they typically result in ataxia. Immunohistochemistry (IHC) is commonly used to reveal cerebellar organization. In particular, the antigen zebrin II (=aldolase C), expressed by stripes of Purkinje cells, has been valuable in revealing cerebellar pattern abnormalities. The development of whole-mount procedures in Drosophila, chick, and Xenopus embryos allows complex patterns to be studied in situ while preserving the integrity of the structure. By combining procedures originally designed for embryonic and early postnatal tissue analyses, we have developed a whole-mount IHC protocol using anti-zebrin II, which reveals the complex topography of Purkinje cells in the adult mouse cerebellum. Furthermore, the procedure is effective with a number of other antigens and works well on both perfusion-fixed and immersion-fixed tissue. By use of this approach, normal adult murine cerebellar topography and patterning defects caused by mutation can be studied without the need for three-dimensional reconstruction.     

Transdifferentiation of ocular tissues in larval Xenopus laevis

Transdifferentiation phenomena offer a useful opportunity to study experimentally the mechanisms on which cell phenotypic stability depends. The capacities of vertebrate eye tissues to reprogram cell differentiation are well known in avian and mammalian embryos, and in larval and adult newt. From research into the capacity of anuran eye tissues to reprogram differentiation into a new pathway, considerable data have accumulated concerning the transdifferentiative capacities of eye tissues in larval Xenopus laevis. This work reviews the data concerning the transdifferentiative phenomena of eye tissues in that species and, based on these, aims to establish the extent of our knowledge about the mechanism controlling these processes. In larval Xenopus laevis the outer cornea can regenerate a lens by a lens-transdifferentiation process triggered and substained by a factor(s), probably of a protein nature, produced by the neural retina. In a normal eye phenotypic stability of the outer cornea is guaranteed by the presence of the inner cornea and lens, which prevent the spread of retinal factor(s). The stimulus for lens transdifferentiation of the outer cornea can be supplied by other tissues as well, but this capacity is not widely distributed. The iris and retinal pigmented epithelium can transdifferentiate into neural retina if isolated from the surrounding tissues and implanted in the vitreous chamber. As for lens transdifferentiation of the outer cornea, retinal transdifferentiation of the iris can be stimulated by certain nonocular tissues as well.     

The distribution of pressure behind a soft contact lens

We determine the pressure distribution behind a soft contact lens that is necessary to keep the lens in conformity with an axisymmetric substrate. The substrate consists of two regions: a central portion, the cornea, supposed to be an ellipsoid; and a peripheral region, the sclera, taken to be a sphere. The pressure is obtained as part of a numerical solution of the axisymmetric equilibrium equations for an initially curved, linearly elastic membrane. The relaxed shape of the lens is assumed to be an axisymmetric ellipsoid with a central curvature and a shape factor different from those of the cornea. The variation in the thickness of the lens from its center to edge is approximated by a polynomial. Pressure distributions are obtained for several typical soft contact lens fittings.     

A lattice model for computing the transmissivity of the cornea and sclera.

The method of photonic band structure is used to calculate the frequencies of light that propagate in lattice models of the cornea and sclera of the mammalian eye, providing an explanation for transparency in the cornea that first properly accounts for multiple scattering of light. Each eye tissue is modeled as an ordered array of collagen rods, and photonic band structure methods are used to solve Maxwell's equations exactly for these models, a procedure that automatically effectively includes all orders of multiple scattering. These calculations show that the dispersion relation for the cornea is linear in the visible range, implying that the cornea is transparent. We show that the transmissivity is approximately 97% by using an effective medium approximation derived from the photonic band structure results and applicable in the visible region. In contrast, the dispersion relation for the model in the sclera is not linear in the visible region, and there are band gaps in this region that could play an important role in the transmission of light in the sclera.     

Genomic characterization and embryonic expression of the mouse Bigh3 (Tgfbi) gene

Mutations in human BIGH3 (TGFB1), a gene identified after treatment of an adenocarcinoma cell line with TGF-beta, have been observed in patients with granular Groenouw type I, Reis-Bücklers, Thiel-Behnke, Avellino, and Lattice type I and IIIa, six autosomal dominant corneal dystrophies linked to chromosome 5q. In order to gain insight into the physiological role of this gene, we characterized the genomic structure of the mouse Bigh3 and its expression in murine embryos. The gene spans 30 kb on mouse chromosome 13 and has 17 exons. Embryonic expression of Bigh3 is observed in the mesenchyme of the first and second branchial arches as early as dpc 11.5 and is particularly strong in the mesenchyme of numerous tissues throughout all the development stages. In fetal eye, the expression is first seen at 11.5 dpc in the mesenchyme surrounding the optic stalk, extends toward the sclera and choroid by 14.3 dpc and reaches the cornea by 17.5 dpc. Because the physiological role of BIGH3/Bigh3 is still largely unknown, embryonic expression in organs like heart, vessels, and intestine may help to identify new functions which could be searched for in patients and in knock-out animal models. The characterization of the murine structure is a prerequisite for the making of such models.     

The transparent lens and cornea in the mouse and zebra fish eye

The lens and cornea combine to form a single optical element in which transparency and refraction are the fundamental biophysical characteristics required for a functional visual system. Although lens and cornea have different cellular and extracellular specializations that contribute to transparency and refraction, their development is closely related. In the embryonic mouse, the developing cornea and lens separate early. In contrast, zebra fish lens and cornea remain connected during early development and the optical properties of the cornea and lens observed by slit lamp and quasielastic laser light scattering spectroscopy (QLS) are more similar in the zebra fish eye than in the mouse eye. Optical similarities between cornea and lens of zebra fish may be the result of similarities in the cellular development of the cornea and lens.     

Material properties of the cornea and sclera: a modelling approach to test experimental analysis

Material properties of cornea and sclera are important for maintaining the shape of the eye and the requisite surface curvatures for optics. They also need to withstand the forces of external and internal musculature and fluctuations in intraocular pressure (IOP). These properties are difficult to measure and variable results have been reported. A previously published experimental procedure, from which the material properties of the eyeball coats were obtained, has been modelled in this paper using Finite Element Analysis, in order to test the accuracy of the experiment. Material parameters were calculated from the model and the resulting relationships between stress and strain for the cornea and sclera compared to their experimentally obtained counterparts. The comparison between model and experiment was close for the sclera but more varied for the cornea. The pressure vessel model can be applied for measuring the material properties of the sclera but is less accurate for the cornea.     

Targeted ablation of alpha-crystallin-synthesizing cells produces lens-deficient eyes in transgenic mice

Genetic ablation techniques were used to study the role of the lens in mammalian eye development. Ablation was accomplished by microinjecting murine eggs with chimeric DNA constructs in which the alpha A-crystallin gene regulatory sequence (-366 to +46) was fused to the highly cytotoxic diphtheria toxin gene coding sequence. For genetic ablation to be successful the promoter regulating expression should be specific and completely silent in cells necessary for normal mouse development. In this report, we describe the generation and analysis of transgenic mice with this readily discernible phenotype: aphakia or eyes without lens. Of the 109 live-born pups, eight carried the transgene and could be grouped according to the apparent severity of eye malformations. Lines 4, 5 and 6 founder (F0) mice had the most severe phenotype. Histological analysis revealed: marked reduction in eye size, total absence of lens, increased retinal cell density and extensive whorling of the retinal fibre layers. The line 1 F0 mouse displayed a distinct lens opacity and lines 2, 3 and 8 F0 mice were mosaics with a relatively mild, but most unusual phenotype. Their eyes contained a small, highly vacuolated lens. The progeny of these mosaics that inherited the transgene, however, again exhibited the severe phenotype. The aberrant structures of the eyes in which complete genetic ablation of the lens has been achieved suggest that the lens plays a pivotal role in the development of multiple components of the murine eye.