Collagen IV in the developing lens capsule.

The lens capsule is a specialized thickened basement membrane that completely surrounds the lens and provides anchoring sites for zonules, the filamentous bodies that suspend the lens. Like other basement membranes, the lens capsule contains collagen IV, which is a family of six polypeptides, subunits alpha1(IV)-alpha6(IV), each of which is encoded by a distinct gene. We have investigated the presence of collagen IV subunits in the developing lens capsule by using confocal immunohistochemistry and antibodies against each of the six collagen IV subunits. In murine embryos, subunits alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV) were detected in the basement membrane surrounding the lens vesicle, and they persisted in the capsule until adulthood. In contrast, neither collagen alpha3(IV) nor alpha4(IV) was detected in the lens capsule until 2 weeks postnatal. Similarly, we detected no collagen alpha3(IV) or alpha4(IV) in lens capsules of 54-day human embryos, while collagen alpha3(IV) and alpha4(IV) were detected in adult humans. Thus, in the lens capsule, there is a developmental shift in detectable collagen IV subunits; early in development we observed subunits alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV), which is consistent with the presence of fibrillar [alpha1alpha1alpha2] and elastic [alpha5alpha5alpha6] protomers, but later in development components of the more cross-linked [alpha3alpha4alpha5] protomer appear. An elastic lens capsule may be necessary in order to accommodate rapid lens growth in early development, whereas later in development a stronger, more cross-linked capsule may be necessary in order to tolerate the stress caused by postnatal accommodation and disaccommodation of the lens.     

Calculating the diffusion coefficient in the eye lens

An experimental investigation of the appearance of a cataract under intensive irradiation is carried out. On the basis of the results obtained a model is proposed of calculating the diffusion coefficient which can model the crystalline lens, its individual peculiarities being taken into account.     

Macromolecular organization of bovine lens capsule

Rabbit antisera to type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin were used to localize these proteins in cross-sections of bovine anterior lens capsule. The antisera were exposed to (a) 10-micron frozen-thawed sections of formaldehyde-fixed tissue for examination in the light microscope by the indirect immunofluorescence method and (b) formaldehyde-fixed and L. R. White plastic-embedded thin sections for electron microscopic examination by the protein A-gold technique. The intensity of immunofluorescence was both uniform and strong throughout for type IV collagen, laminin and entactin, but patchy and weak for fibronectin. Electron microscopic immunolabeling with protein A-gold showed that all five components were distributed throughout the full thickness of the membrane, albeit the density of gold particles was not identical for all basement membrane proteins. In general, the number of particles per micron2 was greatest for type IV collagen and entactin, moderate for laminin and heparan sulfate proteoglycan and low for fibronectin. The ultrastructure of the lens capsule as examined by the electron microscope revealed a relatively uniform parallel alignment of filaments, thought to be collagenous. Since the distribution of the filaments corresponds well with the observed immunocytochemical pattern it is concluded that type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin co-localize throughout the cross-section of the anterior lens capsule.     

MALDI-MS-imaging of whole human lens capsule

The ocular lens capsule is a smooth, transparent basement membrane that encapsulates the lens and is composed of a rigid network of interacting structural proteins and glycosaminoglycans. During cataract surgery, the anterior lens capsule is routinely removed in the form of a circular disk. We considered that the excised capsule could be easily prepared for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-MSI) analysis. MALDI-MSI is a powerful tool to elucidate the spatial distribution of small molecules, peptides, and proteins within tissues. Here, we apply this molecular imaging technique to analyze the freshly excised human lens capsule en face. We demonstrate that novel information about the distribution of proteins by MALDI-MSI can be obtained from this highly compact connective tissue, having no evident histo-morphological characteristics. Trypsin digestion carried out on-tissue is shown to improve MALDI-MSI analysis of human lens capsules and affords high repeatability. Most importantly, MALDI-MSI analysis reveals a concentric distribution pattern of proteins such as apolipoprotein E (ApoE) and collagen IV alpha-1 on the anterior surface of surgically removed lens capsule, which may indicate direct or indirect effects of environmental and mechanical stresses on the human ocular lens.     

Multiaxial mechanical behavior of the porcine anterior lens capsule

The biomechanics of the lens capsule of the eye is important both in physiologic processes such as accommodation and clinical treatments such as cataract surgery. Although the lens capsule experiences multiaxial stresses in vivo, there have been no measurements of its multiaxial properties or possible regional heterogeneities. Rather all prior mechanical data have come from 1-D pressure–volume or uniaxial force-length tests. Here, we report a new experimental approach to study in situ the regional, multiaxial mechanical behavior of the lens capsule. Moreover, we report multiaxial data suggesting that the porcine anterior lens capsule exhibits a typical nonlinear pseudoelastic behavior over finite strains, that the in situ state is pre-stressed multiaxially, and that the meridional and circumferential directions are principal directions of strain, which is nearly equibiaxial at the pole but less so towards the equator. Such data are fundamental to much needed constitutive formulations.     

A structural constitutive model for the human lens capsule

Published data on the mechanical performance of the human lens capsule when tested under uniaxial and biaxial conditions are reviewed. It is concluded that two simple phenomenological constitutive models (namely a linear elastic model and a Fung-type hyperelastic model) are unable to provide satisfactory representations of the mechanical behaviour of the capsule for both of these loading conditions. The possibility of resolving these difficulties using a structural constitutive model for the capsule, of a form that is inspired by the network of collagen IV filaments that exist within the lens capsule, is explored. The model is implemented within a rectangular periodic cell. Prescribed stretches are imposed on the periodic cell and the network is allowed to deform in a non-affine manner. The performance of the constitutive model correlates well with previously published test data. One possible application of the model is in the development of a multi-scale analysis of the mechanics of the human lens capsule.     

Synthesis of lens capsule and plasma membrane glycoproteins by lens epithelial cells and fibers in the rat

The lens of the eye possesses a capsule which is a greatly hypertrophied basement membrane. To investigate the synthesis of glycoproteins destined for this capsule, 3H-fucose was injected into the vitreous body of intact rats weighing approximately 200 gm. The animals were killed from 10 min to 14.5 months later, and their lenses were processed for electron microscope radioautography. At 10 min after injection, more than 58% of the silver grains were localized to the Golgi apparatus of the lens epithelial cells. By day 1, the heaviest sites of reaction were the plasma membrane (more than 50% of total label), the basal cytoplasm, and the adjacent lens capsule, where a heavy band of reaction was seen. The remainder of the capsule exhibited a lighter diffuse reaction. In the lens fibers, the label was at first localized to clusters of vesicles but then migrated to the plasma membrane and to the region of the capsule adjacent to the basal surface of these fibers. Light microscope radioautographs of the lens capsule at later time intervals revealed that by 1 month after injection the diffuse reaction had disappeared, and only the strongly labeled band remained. By 14.5 months after injection, this band had migrated partially across the lens capsule, but the capsule itself had increased considerably in thickness. On the other hand, the distance between the labeled band and the free edge of the capsule had decreased from that seen at the time of injection.     

FGF-2 release from the lens capsule by MMP-2 maintains lens epithelial cell viability

The lens is an avascular tissue, separated from the aqueous and vitreous humors by its own extracellular matrix, the lens capsule. Here we demonstrate that the lens capsule is a source of essential survival factors for lens epithelial cells. Primary and immortalized lens epithelial cells survive in low levels of serum and are resistant to staurosporine-induced apoptosis when they remain in contact with the lens capsule. Physical contact with the capsule is required for maximal resistance to stress. The lens capsule is also a source of soluble factors including fibroblast growth factor 2 (FGF-2) and perlecan, an extracellular matrix component that enhances FGF-2 activity. Matrix metalloproteinase 2 (MMP-2) inhibition as well as MMP-2 pretreatment of lens capsules greatly reduced the protective effect of the lens capsule, although this could be largely reversed by the addition of either conditioned medium or recombinant FGF-2. These data suggest that FGF-2 release from the lens capsule by MMP-2 is essential to lens epithelial cell viability and survival.     

Regional multiaxial mechanical properties of the porcine anterior lens capsule

The lens capsule of the eye plays fundamental biomechanical roles in both normal physiological processes and clinical interventions. There has been modest attention given to the mechanical properties of this important membrane, however, and prior studies have focused on 1-D analyses of the data. We present results that suggest that the porcine anterior lens capsule has a complex, regionally dependent, nonlinear, anisotropic behavior. Specifically, using a subdomain inverse finite element method to analyze data collected via a new biplane video-based test system, we found that the lens capsule is nearly isotropic (in-plane) near the pole but progressively stiffer in the circumferential compared to the meridional direction as one approaches the equator. Because the porcine capsule is a good model of the young human capsule, there is strong motivation to determine if similar regional variations exist in the human lens capsule for knowledge of such complexities may allow us to improve the design of surgical procedures and implants.     

Three-dimensional structure of type IV collagen in the mammalian lens capsule.

The anterior lens capsule provides a thick, easily handled model system for the study of the organization of type IV collagen, the main component of basement membranes. We have used the technique of rapid freezing, deep-etch, and rotary replication to study the three-dimensional organization of the collagen skeleton in mammalian lens capsule after a variety of extraction procedures. In all cases the collagen appeared as a densely packed three-dimensional branching network of fine microfibrils. The organization of the microfibrils appears to show some regularity, with branch points approximately 40 nm apart. Most junctions are three-way and the network forms predominantly five-sided figures. This closely resembles the collagenous network described by Yurchenco and Ruben (1987, 1988) in human amniotic basement membrane and EHS tumor matrix, but extends their findings to another system for which X-ray diffraction data are available. The three-dimensional network is discussed in terms of molecular packing of type IV collagen in light of the information available from the diffraction data.     

Studies on the assembly of the rat lens capsule. Biosynthesis of a cross-linked collagenous component of high molecular weight.

1. Intact rat lenses in tissue culture synthesize hydroxy[3H]proline-containing polypeptides of apparent mol.wt. approx. 180000, which become assembled into aggregates of higher molecular weight with time. 2. Both the 180000-mol.wt. species and the aggregates are components of the deoxycholate-insoluble base-membrane matrix. 3. Formation of the high-molecular-weight aggregate is accompanied by the biosynthesis of the reducible hydroxylysine-derived cross-link hydroxylysino-5-oxo-norleucine. 4. Hydroxylysino-5-oxonorleucine and dehydrohydroxylysinonorleucine are the major reducible cross-links present in intact foetal and 1-month-old calf lens capsules.     

Finite element model of stresses in the anterior lens capsule of the eye

Continuum biomechanics continues to aid in the design of clinical procedures and biomedical devices. Such design requires detailed information on the biomechanical properties of the tissues of interest as well as appropriate methods of analysis. In this paper, we use a fully nonlinear, virtual work based finite element method to study the normal stress field in the anterior lens capsule of the porcine eye. The analysis shows that recently measured regional variations in material symmetry may combine with regional variations in membrane thickness to yield a nearly uniform and equibiaxial stress field in normalcy. These findings are discussed in terms of potential implications to the underlying mechanobiology and in particular, in terms of perturbations in stress that result from cataract surgery, the most commonly performed surgical procedure in North America.     

Finite element model of stresses in the anterior lens capsule of the eye

Continuum biomechanics continues to aid in the design of clinical procedures and biomedical devices. Such design requires detailed information on the biomechanical properties of the tissues of interest as well as appropriate methods of analysis. In this paper, we use a fully nonlinear, virtual work based finite element method to study the normal stress field in the anterior lens capsule of the porcine eye. The analysis shows that recently measured regional variations in material symmetry may combine with regional variations in membrane thickness to yield a nearly uniform and equibiaxial stress field in normalcy. These findings are discussed in terms of potential implications to the underlying mechanobiology and in particular, in terms of perturbations in stress that result from cataract surgery, the most commonly performed surgical procedure in North America.     

Experimental lens capsular bag model for posterior capsule opacification

An in vitro culture model enabling posterior capsule opacification (PCO) to be investigated was developed and established by using low-melting-point (LMP)-agarose gel to support the capsular bag. After removal of the cornea from rodent and porcine eyeballs, the lens zonules were dissected. Whole lens explants were embedded into 2 % (37 °C) LMP-agarose gel solution. As performed routinely in cataract surgery, capsulotomy and lens fiber removal were carried out in the solidified LMP-agarose gel as sham cataract surgery. The LMP-agarose-gel-supported capsular bag/lens epithelial cell (CB-LEC) complexes were maintained in Dulbecco’s modified Eagle medium supplemented with 10 % fetal bovine serum in an anterior face-down position. The proliferation and migration of LECs into the posterior capsule were observed every 12 h by phase-contrast microscopy. Epithelial cells were observed at the central portion of the CB-LEC complexes after 56.57?±?16.56 h (n?=?7) and 106?±?14.03 h (n?=?6) of culture, for rodent and porcine lenses, respectively. The solidified gel allowed clear microscopic observations and whole-mount immunostaining evaluations of the whole area of the capsular bag. Histological examinations revealed the proliferation, migration, and transdifferentiation of LECs related to posterior capsule opacification. This new in vitro culture model provides experimental benefits by maintaining the natural contour of the capsule without implants inside or outside of the capsule. In addition, this model system allows pharmacological and histological evaluations of the cultured CB-LEC complexes without additional manipulations.