S. Metlapally

Ultrastructural analysis of damage to nuclear fiber cell membranes in advanced age-related cataracts from India.

The primary goal was to characterize the structural alterations that occur at the fiber cell interfaces in nuclei of fully opaque cataracts removed by extracapsular cataract surgery in India. The dark yellow to brunescent nuclei, ages 38-78 years, were probably representative of advanced age-related nuclear cataracts. Thick tissue slices were fixed, en bloc stained and embedded for transmission electron microscopy. Stained thin sections contained well-preserved membranes and junctions, although the complex cellular topology often made it necessary to tilt the grid extensively to visualize the membranes. Damage to the fiber cell membranes was noted in all regions of the nucleus. The most important damage occurred within undulating membrane junctions where the loss of membrane segments was common. These membrane breaks were not sites of fusion as membrane edges were detected and cytoplasm appeared to be in contact with extracellular space, which was enlarged in many regions. Dense deposits of protein-like material were frequently observed within the extracellular space and appeared to be similar to protein in the adjacent cytoplasm. The deposits were often 20-50 nm thick, variable in length and located on specific sites on plasma membranes and between clusters of cells or cell processes. In addition, low density regions were seen within the extracellular space, especially within highly undulating membranes where spaces about 100 nm in diameter were observed. The membrane damage was more extensive and extracellular spaces were larger than in aged transparent donor lenses. Because high and low density regions contribute equally to the fluctuations in refractive index, the changes in density due to the observed damage near membranes are likely to produce significant light scattering based on theoretical analysis. The dimensions of the fluctuations in the range 20-100 nm imply that the scattering is probably similar to that of small particles that would increase high-angle scattering visible in the slit lamp. Such damage to membranes would be expected to contribute to the total opacification of the nucleus as the cataract matures. The main sources of the fluctuations appear to be the degradation of membranes and adjacent cytoplasmic proteins, as well as the redistribution of proteins and fragments.

Multilamellar spherical particles as potential sources of excessive light scattering in human age-related nuclear cataracts

The goal of this project was to determine the relative refractive index (RI) of the interior of multilamellar bodies (MLBs) compared to the adjacent cytoplasm within human nuclear fiber cells. MLBs have been characterized previously as 1-4 μm diameter spherical particles covered by multiple lipid bilayers surrounding a cytoplasmic core of variable density. Age-related nuclear cataracts have more MLBs than transparent donor lenses and were predicted to have high forward scattering according to Mie scattering theory, assuming different RIs for the MLB and cytoplasm. In this study quantitative values of relative RI were determined from specific MLBs in electron micrographs of thin sections and used to calculate new Mie scattering plots. Fresh lenses were Vibratome sectioned, immersion fixed and en bloc stained with osmium tetroxide and uranyl acetate, or uranyl acetate alone, prior to dehydration and embedding in epoxy or acrylic resins. Thin sections 70 nm thick were cut on a diamond knife and imaged without grid stains at 60 kV using a CCD camera on a transmission electron microscope (TEM). Integrated intensities in digital electron micrographs were related directly to protein density, which is linearly related to RI for a given substance. The RI of the MLB interior was calculated assuming an RI value of 1.42 for the cytoplasm from the literature. Calculated RI values for MLBs ranged from 1.35 to 1.53. Thus, some MLBs appeared to have interior protein densities similar to or less than the adjacent cytoplasm whereas others had significantly higher densities. The higher density MLBs occurred preferentially in older and more advanced cataracts suggesting a maturation process. The bilayer coats were more often observed in MLBs from transparent donors and early stage cataracts indicating that bilayer loss was part of the MLB maturation, producing large low-density spaces around dense MLB cores. These spaces were frequently observed in advanced cataracts from India as large low-density crescents and annular rings. Predicted scattering from Mie plots using particles with dense cores and low-density rims was higher than reported previously, although the most important factor was the relative RI, not the MLB coat or lack thereof. In conclusion, the measurements confirm the high protein density and RI of some MLB interiors compared to adjacent cytoplasm. This high RI ratio used in the Mie calculations suggests that for 2000 MLBs/mm³, about half that reported for early stage nuclear cataracts from the US, the forward scattering could be more than 30% of the incident light. Therefore, the extent of forward scattering and its influence on macular visual acuity could be important components of ophthalmological evaluations of cataract patients.

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.

Electron tomography of fiber cell cytoplasm and dense cores of multilamellar bodies from human age-related nuclear cataracts.

Human nuclear cataract formation is a multi-factorial disease with contributions to light scattering from many cellular sources that change their scattering properties over decades. The aging process produces aggregation of cytoplasmic crystallin proteins, which alters the protein packing and texture of the cytoplasm. Previous studies of the cytoplasmic texture quantified increases in density fluctuations in protein packing and theoretically predicted the corresponding scattering. Multilamellar bodies (MLBs) are large particles with a core of crystallin cytoplasm that have been suggested to be major sources of scattering in human nuclei. The core has been shown to condense over time such that the refractive index increases compared to the adjacent aged and textured cytoplasm. Electron tomography is used here to visualize the 3D arrangement of protein aggregates in aged and cataractous lens nuclear cytoplasm compared to the dense protein packing in the cores of MLBs. Thin sections, 70 nm thick, were prepared from epoxy-embedded human transparent donor lenses and nuclear cataracts. Tilt series were collected on an FEI T20 transmission electron microscope (TEM) operated at 200 kV using 15 nm gold particles as fiducial markers. Images were aligned and corrected with FEI software and reconstructed with IMOD and other software packages to produce animated tilt series and stereo anaglyphs. The 3D views of protein density showed the relatively uniform packing of proteins in aged transparent lens nuclear cytoplasm and less dense packing of aged cataractous cytoplasm where many low-density regions can be appreciated in the absence of the TEM projection artifacts. In contrast the cores of the MLBs showed a dense packing of protein with minimal density fluctuations. These observations support the conclusion that, during the nuclear cataract formation, alterations in protein packing are extensive and can result in pronounced density fluctuations. Aging causes the MLB cores to become increasingly different in their protein packing from the adjacent cytoplasm. These results support the hypothesis that the MLBs increase their scattering with age and nuclear cataract formation.