Requirement for Crk and CrkL during postnatal lens development.

The Crk and CrkL adaptor proteins have SH2 and SH3 domains and play essential overlapping, as well as distinct, roles in many biological processes, ranging from cell structure and motility to proliferation. Conditional ablation of both Crk and CrkL in neuronal progenitor cells, using a Nestin-Cre transgene, resulted in severe defects in postnatal eye development, including progressive eye closure, lens rupture, and retinal malformation. These phenotypes were not observed in the presence of a single wild-type allele of either Crk or CrkL. We found that the lens in knockout mice started to rupture and disintegrate between postnatal days 7 and 12, although the structure of the retina was relatively well maintained. As the lens deteriorated further, the outer nuclear layer in the posterior of the retina enlarged and developed ruffles. Cre recombination occurred in the lens and retina of the knockout mice. Furthermore, the posterior lens capsule of the knockout mouse was thinner at postnatal days 0.5 and 3, suggesting that the defective lens capsule caused rupturing of the lens near the posterior pole. These results indicate that Crk and CrkL play essential overlapping roles in postnatal lens development.

Contributions of mouse genetic background and age on anterior lens capsule thickness.

Accurate lens capsule thickness measurements are necessary for studies investigating mechanical characteristics of the capsule. Confocal Z-axis imaging was used to measure the anterior lens capsule thickness of living intact lenses with minimal tissue manipulation. Measurements of the anterior capsule thickness is reported for the first time in young and old mice from four inbred strains, BALB/c, FVB/N, C57BL/6, and 129X1, and the outbred strain ICR. Our data demonstrates that the mouse anterior lens capsule continues to grow postnatally similar to that described in other mammals. It is also shown there is a significant difference in anterior lens capsule thickness between unrelated mouse strains, suggesting that capsule thickness is a quantitative trait shared by strains with common ancestry. Measurements, taken from other regions of FVB/N capsules revealed the anterior pole to be the thickest, followed by the equatorial region and posterior pole. In addition to mouse, anterior capsule measurements taken from intact cattle, rabbit, rat lenses, and human capsulotomy specimens correlated with the overall size of the animal.

Estudio experimental sobre la repercusión anatómica de la zónula de Zinn tras la introducción de un anillo endocapsular en ojos de cerdo / Anatomical repercusion on the zonular apparatus after a capsular tension ring insertion in swine eyes

Objetivos: Valorar experimentalmente la integridad morfológica de los ligamentos suspensorios de la zónula de Zinn (LSZZ) tras la introducción de un anillo de distensión capsular (ADC) mediante un estudio descriptivo. Métodos: Se utilizaron siete ojos de cerdo a los cuales se realizó una extracción extracapsular de cristalino transparente. A cuatro se introdujo un ADC con pinzas y al quinto con inyector. Los otros dos se utilizaron como control. Se examinó la integridad anatómica y la nueva configuración de los LSZZ con un microscopio electrónico de barrido. Resultados: No se observaron alteraciones en la integridad de los LSZZ. Los LSZZ adoptan tras la introducción de un ADC una nueva trayectoria de repliegue consecuencia de la nueva disposición del saco capsular. Conclusiones: En un modelo experimental conojos de cerdo no se encontraron lesiones en los LSZZ por lo que parece que, en cápsulas elásticas y de gran diámetro, la inserción de un ADC no produce roturas en los LSZZ

Purpose: To carry out an experimental and descriptive study that exhibits the anatomical repercussions on the zonular apparatus after a capsular tensionring (CTR) is inserted. Methods: CTRs were inserted in five swine eyes (four with forceps, one with an injector). Two additional eyes were left untouched for control purposes.The integrity of the suspensory ligament was examined by scanning electron microscopy. Results: We did not observe alterations in the integrity of the zonular apparatus. The suspensory ligaments adopted a new fold configuration. Conclusions: CTR insertion is a safe therapeutic tool to maintain the integrity of the lens zonules inelastic, large diameter capsular bags

Vitronectin is present in epithelial cells of the intact lens and promotes epithelial mesenchymal transition in lens epithelial explants.

PURPOSE: Extracellular matrix (ECM) accumulates during the development of posterior capsule opacification (PCO). Vitronectin, an ECM component that is generally prominent in wound healing, has been detected in PCO specimens. Here we set out to investigate the distribution of vitronectin in the lens and determine how it, and other ECM components, influence the lens epithelial phenotype. METHODS: Rat lens epithelial explants were cultured on vitronectin, fibronectin, and laminin substrata. Explants were monitored for cell migration and the appearance of markers for epithelial mesenchymal transition (EMT), using phase contrast microscopy and immunohistochemistry, respectively. Explants were also monitored for evidence of Smad signaling. Vitronectin expression was analyzed in embryonic and postnatal rodent lens development by immunohistochemistry, western blotting, and in situ hybridization. RESULTS: Vitronectin, like fibronectin and laminin, provided a good substratum for cellular attachment and migration. However, in the case of vitronectin and fibronectin, this was accompanied by a major phenotypic change. On either vitronectin or fibronectin, but not laminin, most of the cells became elongated, spindle-shaped and were strongly reactive for filamentous alpha-smooth muscle actin. In these respects this transition was typical of the well known TGFbeta-induced EMT. In explants cultured on vitronectin and fibronectin, but not laminin, cell nuclei showed prominent reactivity for Smad 2/3. Vitronectin was also shown to be expressed during embryonic and postnatal development. Initially mRNA and protein were detected in all lens cells, however as development progressed, expression became restricted to cells of the epithelium and transition zone. CONCLUSIONS: The results clearly show that lens cell engagement with a vitronectin or a fibronectin, but not laminin, substratum has a potent EMT promoting effect and that Smad 2/3 signaling is involved. Thus when considering strategies to slow or prevent PCO, these results highlight the need to take into account ECM molecules such as vitronectin that have the capacity to promote EMT.

Lens major intrinsic protein (MIP)/aquaporin 0 expression in rat lens epithelia explants requires fibroblast growth factor-induced ERK and JNK signaling.

Lens major intrinsic protein (MIP), exclusive to the vertebrate lens, otherwise known as MIP26 and Aquaporin 0, is abundantly expressed as a lens fiber membrane protein. Although relatively less efficient compared with other aquaporins, MIP is suggested to function as a water channel, as an adhesion molecule, and is required for lens transparency. Because MIP is specifically expressed in lens fiber cells, we investigated in this study the activation of Mip expression after triggering differentiation of rat lens epithelia explants by fibroblast growth factor (FGF)-2. Here, we show that Mip expression in the lens cells is regulated by FGF-2. Using Real time PCR we demonstrate that endogenous Mip levels in the explants were up-regulated upon FGF-2 stimulation, in a concentration-dependent manner. Up-regulation of Mip at the transcriptional level was simultaneous with the activation of the FGF down-stream signaling components, ERK1/2 and JNK. Specific inhibitors, UO126 for ERK1/2 and SP600125 for JNK, abrogated Mip expression in response to FGF-2 in the explants. This inhibition pattern was recapitulated in reporter assays for transfection of the rat lens epithelia explants, driven by the Mip promoter (-1648/+44). Our studies show that ERK1/2 and JNK signaling pathways are required for Mip expression in lens epithelia explants induced to differentiate by FGF-2.

Expression of the matricellular protein SPARC in murine lens: SPARC is necessary for the structural integrity of the capsular basement membrane.

SPARC (Secreted Protein, Acidic and Rich in Cysteine) is a matricellular glycoprotein that modulates cell proliferation, adhesion, migration, and extracellular matrix (ECM) production. Although SPARC is generally abundant in embryonic tissues and is diminished in adults, we have found that the expression of SPARC in murine lens persists throughout embryogenesis and adulthood. Our previous studies showed that targeted ablation of the SPARC gene in mice results in cataract formation, a pathology attributed partially to an abnormal lens capsule. Here we provide evidence that SPARC is not a structural component of the lens capsule. In contrast, SPARC is abundant in lens epithelial cells, and newly differentiated fiber cells, with stable expression in wild-type mice up to 2 years of age. Pertubation of the lens capsule in animals lacking SPARC appears to be a consequence of the invasion of the lens cells situated beneath the capsule. Immunoreactivity for SPARC in the lens cells was uneven, with minimal reactivity in the epithelial cells immediately anterior to the equator. These epithelial cells appeared essentially noninvasive in SPARC-null mice, in comparison to the centrally located anterior epithelial cells, in which strong labeling by anti-SPARC IgG was observed. The posterior lens fibers exhibited cytoplasmic extensions into the posterior lens capsule, which was severely damaged in SPARC-null lenses. The expression of SPARC in wild-type lens cells, together with the abnormal lens capsule in SPARC-null mice, indicated that the structural integrity of the lens capsule is dependent on the matricellular protein SPARC. The effects of SPARC in the lens appear to involve regulation of lens epithelial and fiber cell morphology and functions rather than deposition as a structural component of the lens capsule.

Morphologic alterations in the anterior lens capsule of canine eyes with cataracts.

OBJECTIVE: To examine the morphologic changes in the anterior lens capsule and lens epithelium of canine eyes with cataracts. SAMPLE POPULATION: Anterior lens capsules from the eyes of 25 dogs with cataracts and from an additional 10 canine globes with lenses subjectively assessed to be normal. PROCEDURE: Thickness of each anterior lens capsule was measured by use of a digital microscopic camera and imaging software. All 25 capsules from eyes with cataracts were submitted for light microscopy; 4 were also submitted for electron microscopy. RESULTS: Thickness of the anterior lens capsule increased with age for the normal lenses and the lenses with cataracts; the change with age was similar for both groups. Light microscopy revealed fibrous metaplasia of lens epithelial cells in 7 of 25 anterior lens capsules with focal thickenings of the posterior aspect of the capsule. Electron microscopy revealed deposition of collagen and basement membrane-like material by fibroblast-like cells. CONCLUSIONS: Results indicate that thickness of the anterior lens capsule in dogs increases with age and that this increase in thickness is not significantly different between normal lenses and lenses with cataracts. In addition, epithelial cells from lenses with cataracts may undergo metaplasia to form plaques composed of fibrous tissue and ectopic basement membrane produced by epithelial cells.

Ex vivo canine lens capsular sac explants.

BACKGROUND: Lens capsular sac explants from human cadaver eyes were used to investigate posterior capsular opacification (PCO). The purpose of this study was to characterize a similar model using canine tissue and to determine whether transferrin (Tf), transforming growth factor beta-2 (TGF-beta2), and insulin-like growth factor-1 (IGF-1) are secreted by lens epithelial cells (LEC) of these ex vivo sacs. METHODS: The lens from canine eyes was removed by extracapsular cataract extraction, the lens sac dissected free, pinned to a petri dish, and cultured in either serum-supplemented or serum-free medium. Morphologic characteristics and growth rate to confluence on the posterior capsule were studied by phase-contrast microscopy. Vimentin, alpha smooth muscle actin, and panTGF-beta expression by LEC were determined by immunohistochemistry. Tf, TGF-beta2, and IGF-1 levels were measured by ELISA in the supernatant of sacs cultured in serum-free medium. RESULTS: The mean time to confluence of LEC onto the posterior capsule was 5.4+/-1.1 days (n=22) and 14.7+/-3.7 days (n=14) for sacs in serum-supplemented and serum-free medium, respectively. Following development of confluence, explants displayed opacification and light scatter from cellular proliferation and capsular contraction. Confluent LEC expressed vimentin, alpha smooth muscle actin, and TGF-beta2, and both Tf and TGF-beta2 were secreted into the culture supernatant. CONCLUSION: Canine lens sac explants have characteristics virtually identical to those of human origin, and appear to be a useful alternative tissue source for this model when human cadaver eyes are unavailable. Tf and TGFbeta-2, but not IGF-1, are secreted by LEC in explanted lens sacs and may influence the proliferation and metaplasia of LEC during the development of PCO.

Intraocular lenses for pediatric implantation: biomaterials, designs, and sizing.

Posterior chamber intraocular lenses (IOLs) are being implanted in children with increasing frequency. However, with rare exceptions, only IOLs designed for adults are currently available. These lenses may be difficult to insert into small eyes. Since the pediatric crystalline lens is smaller than that of adults and because the capsular bag does not continue to grow after lensectomy, it is worthwhile to determine the biomaterials, designs, and sizes that may be appropriate for pediatric implantation. In a study of 50 pediatric eyes obtained postmortem, we have documented an estimated growth curve for the developing crystalline lens between birth and 16 years of age. Ninety percent of crystalline lens growth occurs during the first two years of life. Based on these data and this study using the Miyake posterior view analysis of implanted standard and prototype IOLs, we recommend the following: Clinical trials of capsular IOLs, downsized to approximately 10.0 mm diameter, are appropriate for children under two years of age. Capsular IOLs are defined as flexible open-loop, one-piece, all poly(methyl methacrylate), modified C-loop designs made specifically for in-the-bag placement. Because the rapid growth phase of the lens is complete by the age of two, we believe that downsizing the IOL is not necessary after this age unless axial length measurements indicate an unusually small eye. Standard flexible 12.0 mm to 12.5 mm diameter capsular IOLs can be safely implanted. Such lenses could be tolerated throughout life, obviating the need for later IOL exchange.

Acidic and basic FGF in ocular media and lens: implications for lens polarity and growth patterns.

We have shown previously that FGF induces lens epithelial cells in explant culture to proliferate, migrate and differentiate into fibre cells in a progressive concentration-dependent manner. In situ, these processes occur in a distinct anterior-posterior pattern in clearly defined regions of the lens. Thus anterior-posterior differences in the bio-availability of FGF in the lens environment may play a role in determining lens polarity and growth patterns. In this study, using heparin chromatography and western blotting (or ELISA), we established that both acidic and basic FGF are present in the aqueous and vitreous (the ocular media that bathe the anterior and posterior compartments of the lens, respectively). In addition, substantially more FGF was recovered from vitreous than from aqueous. Both forms of FGF were also detected in lens fibre cells and capsule. A truncated form of basic FGF (less than 20 x 10(3) M(r)) predominated in every case with traces of higher M(r) forms in lens cells. For acidic FGF, the classical full-length form (about 20 x 10(3) M(r)) predominated in lens cells and a truncated form was found in vitreous. The capsule contained a higher M(r) form. Using our explant system, we also tested the biological activity of ocular media and FGF fractions obtained from vitreous and lens cells. Vitreous but not aqueous contained fibre-differentiating activity. Furthermore, virtually all the fibre-differentiating activity of vitreous was shown to be FGF-associated, as follows: (a) this activity remained associated with FGF during fractionation of vitreous by heparin and Mono-S chromatography and (b) the activity of the major FGF-containing fraction was blocked by antibodies to acidic and basic FGF. Posterior, but not anterior, capsule was shown to have mitogenic activity, which was neutralised by FGF antibodies and associated only with the cellular surface. These results support our hypothesis that FGF is involved in determining the behaviour of lens cells in situ. In particular, a key role for FGF in determining lens polarity and growth patterns is suggested by the anterior-posterior differences in the bio-availability of FGF in the ocular media and capsule.

The roles of laminin and fibronectin in the development of the lens capsule.

This study examines the distribution of laminin and fibronectin in the rat lens capsule during development. Both these extracellular matrix glycoproteins are localised in the interspace between presumptive lens and presumptive retina as well as in their basal laminae. The lens capsule arises from multilayering of the basal lamina of the lens cells. Immunofluorescence localises both laminin and fibronectin in the capsule at 16 days of embryonic development, although reactivity for fibronectin is much weaker than for laminin. In the 19 day embryo only laminin is detected. This indicates that during embryonic development fibronectin becomes a minor component of lens cell ECM and is not accumulated in the developing capsule. The roles of laminin and fibronectin in promoting cell migration during development were analysed in explant cultures. Lens epithelial explants from 16, 17 and 19 day old embryos and neonatal rats were grown on a laminin or fibronectin substratum. Lens cells from all ages of rats migrated on the laminin substratum, whereas lens cells progressively lost the ability to migrate on a fibronectin substratum as the age of the donor increased. This developmental loss of ability to migrate on fibronectin in vitro coincides with the developmental loss of fibronectin from the lens capsule in vivo. Therefore, we propose that whilst both laminin and fibronectin may be important for promoting migration of lens cells on their substratum at early stages of lens morphogenesis, during development laminin takes over as the key molecule that promotes migration on the capsule.

A morphometric analysis of the development of the rat lens capsule.

This study analysed patterns of growth of the lens capsule by measuring capsule thickness at 13, 16 and 19 days of embryonic development and 2, 21, 140 and 600 days of post-natal development. The major findings were that, at early stages of embryonic development, the posterior capsule was thicker than the anterior capsule. However, at later stages, the posterior capsule did not increase in thickness whereas the anterior capsule continued to thicken so that, by 2 days of post-natal development, the situation was reversed and the anterior capsule was significantly thicker than the posterior capsule. This trend continued and by 600 days post-natal development, the anterior capsule was 7.5 times thicker than the posterior capsule. In these older lenses the capsule tapered sharply in thickness from the anterior to the posterior equatorial region. These regional differences in thickness of the lens capsule, and the changes reported during development, may reflect changes in capsule production by epithelial and fibre cells as they differentiate.

Influence of undernutrition on the lenticular epithelium in neonatal and weanling albino rats.

Protein malnutrition, a morbid nutritional disorder of the developing countries, is known to interfere with cell replication. The influence of undernutrition on the lens which represents a unique combination of static, conditional renewal and constant cell renewal systems is not completely understood. In the present investigation it has been shown that undernutrition results in poor synthetic and proliferative activities of lenticular epithelium in neonatal and post-weaning rats. It is supposed that protein deficiency per se may not produce clinical diseases, but may increase the susceptibility of lenses to exogenous and/or endogenous insults.