Regulation of scleral cell contraction by transforming growth factor-β and stress competing roles in myopic growth

Reduced extracellular matrix accumulation in the sclera of myopic eyes leads to increased ocular extensibility and is related to reduced levels of scleral transforming growth factor-β (TGF-β). The current study investigated the impact of this extracellular environment on scleral cell phenotype and cellular biomechanical characteristics. Scleral cell phenotype was investigated in vivo in a mammalian model of myopia using the myofibroblast marker, α-smooth muscle actin (α-SMA). In eyes developing myopia α-SMA levels were increased, suggesting increased numbers of contractile myofibroblasts, and decreased in eyes recovering from myopia. To understand the factors regulating this change in scleral phenotype, the competing roles of TGF-β and mechanical stress were investigated in scleral cells cultured in three-dimensional collagen gels. All three mammalian isoforms of TGF-β altered scleral cell phenotype to produce highly contractile, α-SMA-expressing myofibroblasts (TGF-β3 > TGF-β2 > TGF-β1). Exposure of cells to the reduced levels of TGF-β found in the sclera in myopia produced decreased cell-mediated contraction and reduced α-SMA expression. These findings are contrary to the in vivo gene expression data. However, when cells were exposed to both the increased stress and the reduced levels of TGF-β found in myopia, increased α-SMA expression was observed, replicating in vivo findings. These results show that although reduced scleral TGF-β is a major contributor to the extracellular matrix remodeling in the myopic eye, it is the resulting increase in scleral stress that dominates the competing TGF-β effect, inducing increased α-SMA expression and, hence, producing a larger population of contractile cells in the myopic eye

Mie light scattering calculations for an Indian age-related nuclear cataract with a high density of multilamellar bodies

Purpose: Multilamellar bodies (MLBs) are lipid-coated spheres (1–4 µm in diameter) found with greater frequency in the nuclear region of human age-related cataracts compared with human transparent lenses. Mie light scattering calculations have demonstrated that MLBs are potential sources of forward light scattering in human age-related nuclear cataracts due to their shape, size, frequency, and cytoplasmic contents, which often differ in refractive index from their surroundings. Previous studies have used data from several non-serial tissue sections viewed by light microscopy to extrapolate a volume and have assumed that MLBs are random in distribution. Currently, confocal microscopy is being used to examine actual tissue volumes from age-related nuclear cataracts and transparent lenses collected in India to confirm MLB shape, size, frequency, and randomness. These data allow Mie scattering calculations to be done with directly observed MLBs in intact tissue. Methods: Whole Indian donor lenses and Indian lens nuclei after extracapsular cataract extraction were immersion-fixed in 10% formalin for 24 h and in 4% paraformaldehyde for 24 h before sectioning with a Vibratome. The 160 µm thick sections were stained for 24 h in the lipid dye DiI (1,1’-dilinoleyl-3,3,3′,3′ tetramethylindocarbocyanine, 4-chlorobenzenesulfonate), washed, stabilized in Permount under coverslips and examined with a Zeiss LSM 510 confocal microscope. Individual volumes of tissue (each typically 500,000 µm3) were examined using a plan-apochromat 63X oil (NA=1.4) lens. Other lenses were prepared for electron microscopy and histological examination using previously described procedures. Results: Analysis of tissue volumes within Indian age-related nuclear cataracts and transparent lenses has confirmed that most MLBs are 1–4 µm in diameter and typically spherical with some occurring as doublets or in clusters. Most Indian cataracts and transparent lenses are similar to samples obtained in the United States. One cataract contained as many as 400,000 MLBs per mm3 –100 times more than in cataracts collected in the United States. Pairwise distribution analysis has revealed that MLBs even in this exceptional case are found with a distribution that appears to be random. Mie calculations indicate that more than 90% of the incident light could be scattered by the high density of MLBs. Conclusions: An important finding was that one advanced Indian cataract contained many more MLBs than cataracts examined from India and previously from the United States. This indicates that specific conditions or susceptibilities may exist that promote the formation of excessive MLBs. Based on the extremely high frequency, as well as their spherical shape, large size, and apparent random distribution, the MLBs are predicted according to Mie light scattering calculations to cause high amounts of forward scattering sufficient to produce nuclear opacity

Simple fixation and storage protocol for preserving the internal structure of intact human donor lenses and extracted human nuclear cataract specimens

Purpose: Increased use of phacoemulsification procedures for cataract surgeries has resulted in a dramatic decrease in the availability of cataractous nuclear specimens for basic research into the mechanism of human cataract formation. To overcome such difficulties, a fixation protocol was developed to provide good initial fixation of human donor lenses and extracted nuclei, when available, and is suitable for storing or shipping cataracts to laboratories where structural studies could be completed. Methods: Cataractous lens nuclei (n=19, ages 12 to 74 years) were obtained from operating suites after extracapsular extraction. Transparent human donor lenses (n=27, ages 22 to 92 years) were obtained from the Ramayamma International Eye Bank. After the dimensions were measured with a digital caliper, samples were preserved in 10% formalin (neutral buffered) for 24 h and followed by fixation in 4% paraformaldehyde (pH 7.2) for 48 h. Samples were stored cold (4 °C) in buffer until shipped. Samples were photographed and measured before further processing for transmission electron microscopy. Results: The dimensions of the samples varied slightly after short fixation followed by 1 to 5 months’ storage before transmission electron microscopy processing. The mean change in the axial thickness of the donor lenses was 0.15±0.21 mm or 3.0±5.4%, while that of the extracted nuclei was 0.05±0.24 mm or 1.8±7.6%. Because the initial concern was whether the nuclear core was preserved, thin sections were examined from the embryonic and fetal nuclear regions. All cellular structures were preserved, including the cytoplasm, complex edge processes, membranes, and junctions. The preservation quality was excellent and nearly equivalent to preservation of fresh lenses even for the lens cortex. Cell damage characteristic of specific nuclear cataract types was easily recognized. Conclusions: The novel fixation protocol appears effective in preserving whole donor lenses and cataractous nuclei over a wide age range. Dimensions varied only 2%–3%, and fiber cell damage correlated well with standard fixation. These methods enable researchers and clinicians in remote settings to preserve donor lenses and rare examples of extracapsular extractions for detailed examination at later times

Dynamic accommodation without feedback does not respond to isolated blur cues.

The aim of this study was to determine whether dynamic accommodation responds to isolated blur cues without feedback, and without changes in the distance of the object. Nine healthy subjects aged 21-40years were recruited. Four different aberration patterns were used as stimuli to induce blur with (1) the eye's natural, uncorrected, optical aberrations, (2) all aberrations corrected, (3) spherical aberration only, or (4) astigmatism only. The stimulus was a video animation based on computer-generated images of a monochromatic Maltese cross. Each individual video was generated for each subject off-line, after measuring individual aberrations at different accommodation levels. The video simulated sinusoidal changes in defocus at 0.2Hz. Dynamic images were observed through a 0.8mm pinhole placed at a plane conjugated with the eye's pupil, thus effectively removing potential feedback stemming from accommodation changes. Accommodation responses were measured with a Hartmann-Shack aberrometer for the four different aberration patterns. The results showed that seven out of nine subjects did not respond to any stimuli, whereas the response of the other two subjects was erratic and they seemed to be searching rather than following the stimulus. A significant reduction in average accommodative gain (from 0.52 to 0.11) was obtained when the dioptric demand cue was removed. No statistically significant differences were found among the experimental conditions used. We conclude that aberration related blur does not drive the accommodation response in the absence of feedback from accommodation

Analysis of nuclear fiber cell cytoplasmic texture in advanced cataractous lenses from Indian subjects using Debye–Bueche theory

Alterations in ultrastructural features of the lens fiber cells lead to scattering and opacity typical of cataracts. The organelle-free cytoplasm of the lens nuclear fiber cell is one such component that contains vital information about the packing and organization of crystallins critical to lens transparency. The current work has extended analysis of the cytoplasmic texture to transparent and advanced cataractous lenses from India and related the extent of texturing to the nuclear scattering observed using the Debye-Bueche theory for inhomogeneous materials. Advanced age-related nuclear cataracts (age-range 38–75 years) and transparent lenses (age-range 48–78 years) were obtained following extracapsular cataract removal or from the eye bank, at the L. V. Prasad Eye Institute. Lens nuclei were Vibratome-sectioned, fixed and prepared for transmission electron microscopy using established techniques. Electron micrographs of the unstained thin sections of the cytoplasm were acquired at 6500X and percent scattering for wavelengths 400–700 nm was calculated using the Debye-Bueche theory. Electron micrographs from comparable areas in an oxidative-damage sensitive (OXYS) rat model and normal rat lenses preserved from an earlier study were used, as they have extremely textured and smooth cytoplasms, respectively. The Debye-Bueche theoretical approach produces plots that vary smoothly with wavelength and are sensitive to spatial fluctuations in density. The central lens fiber cells from advanced cataractous lenses from India and the OXYS rat, representing opaque lens nuclei, produced the greatest texture and scattering. The transparent human lenses from India had a smoother texture and less predicted scattering, similar to early cataracts from previous studies. The normal rat lens had a homogeneous cytoplasm and little scattering. The data indicate that this method allowed easy comparison of small variations in cytoplasmic texture and robustly detected differences between transparent and advanced cataractous human lenses. This may relate directly to the proportion of opacification contributed by the packing of crystallins. The percent scattering calculated using this method may thus be used to generate a range of curves with which to compare and quantify the relative contribution of the packing of crystallins to the loss of transparency and scattering observed

Pediatric cataract, myopic astigmatism, familial exudative vitreoretinopathy and primary open-angle glaucoma co-segregating in a family

Purpose: To describe an Australian pedigree of European descent with a variable autosomal dominant phenotype of: pediatric cortical cataract (CC), asymmetric myopia with astigmatism, familial exudative vitreoretinopathy (FEVR), and primary open-angle glaucoma (POAG). Methods: Probands with CC, FEVR, and POAG were enrolled in three independent genetic eye studies in Tasmania. Genealogy confirmed these individuals were closely related and subsequent examination revealed 11 other family members with some or all of the associated disorders. Results: Twelve individuals had CC thought to be of childhood onset, with one child demonstrating progressive lenticular opacification. One individual had severe retinal detachment while five others had dragged retinal vessels. Seven individuals had POAG. Seven individuals had myopia in at least one eye ≤-3 Diopters. DNA testing excluded mutations in myocilin, trabecular meshwork inducible glucocorticoid response (MYOC) and tetraspanin 12 (TSPAN12). Haplotype analysis excluded frizzled family receptor 4 (FZD4) and low density lipoprotein receptor-related protein 5 (LRP5), but only partly excluded EVR3. Multipoint linkage analysis revealed multiple chromosomal single-nucleotide polymorphisms (SNPs) of interest, but no statistically significant focal localization. Conclusions: This unusual clustering of ophthalmic diseases suggests a possible single genetic cause for an apparently new cataract syndrome. This family’s clinical ocular features may reflect the interplay between retinal disease with lenticular changes and axial length in the development of myopia and glaucoma

Characterizing New-Onset Exudation in the Randomized Phase 2 FILLY Trial of Complement Inhibitor Pegcetacoplan for Geographic Atrophy.

To evaluate clinical characteristics of eyes in which investigator-determined new-onset exudative age-related macular degeneration (eAMD) developed during the FILLY trial.Post hoc analysis of the phase 2 study of intravitreal pegcetacoplan in geographic atrophy (GA).Patients with GA secondary to age-related macular degeneration (AMD), n = 246.Either 15 mg intravitreal pegcetacoplan or sham given monthly or every other month for 12 months followed by a 6-month off-treatment period.Time of new eAMD onset in the study eye, history of eAMD in the fellow eye, presence of double-layer sign (DLS) on structural OCT in the study eye, changes in retinal anatomic features by structural OCT and fluorescein angiography (FA), and changes in visual acuity.Exudation was reported in 26 study eyes across treatment groups over 18 months. Mean time to eAMD diagnosis was 256 days (range, 31-555 days). Overall, a higher proportion of patients with a baseline history of eAMD in the fellow eye (P = 0.016) and a DLS in the study eye (P = 0.0001) demonstrated eAMD. Among study eyes in which eAMD developed, 18 of 26 (69%) had history of fellow-eye eAMD and 19 of 26 (73.1%) had DLS at baseline, compared with 76 of 217 study eyes (35%; P = 0.0007) and 70 of 215 study eyes (32.5%; P0.0001), respectively, in which eAMD did not develop. All 21 patients with structural OCT imaging at the time of eAMD diagnosis demonstrated subretinal fluid, intraretinal cysts, or both consistent with exudation. Among 17 patients who underwent FA at eAMD diagnosis, 10 showed detectable macular neovascularization (MNV), all occult lesions. Development of eAMD did not have an appreciable impact on visual acuity, and all patients responded to anti-vascular endothelial growth factor (VEGF) therapy.Intravitreal pegcetacoplan slowed the rate of GA growth and was associated with an unexpected dose-dependent increased incidence of eAMD with no temporal clustering of onset. Exudative AMD seemed to be associated with baseline eAMD in the contralateral eye and a DLS, suggestive of nonexudative MNV, in the study eye. The safety profile of pegcetacoplan was acceptable to proceed to phase 3 studies without adjustments to enrollment criteria