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

Abstract

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 Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript 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 2 000 MLBs/ mm 3...

Structural and Functional Properties, Chaperone Activity and Posttranslational Modifications of Alpha-Crystallin and its Related Subunits in the Crystalline Lens: N-acetylcarnosine, Carnosine and Carcinine act as Alpha- Crystallin/Small Heat Shock Protein Enhancers in Prevention and Dissolution of Cataract in Ocular Drug Delivery Formulations of Novel Therapeutic Agents

“…During cataractogenesis, formation of high molecular mass (HMM) aggregates cause fluctuations in protein density, resulting in light scattering and a concomitant decrease in transparency. Other factors such as multilamellar bodies and morphological changes could also contribute substantially to lens opacity [15][16][17][18] and not compaction [19]. The organellefree 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 published work to Metlapally et al [19] has provided analysis of the cytoplasmic texture to transparent and advanced cataractous lenses and related the extent of texturing to the nuclear scattering observed using the Debye-Bueche theory for inhomogeneous materials. The extent of forward scattering and its influence on macular visual acuity could be important components of ophthalmological evaluations of cataract patients [16]. Filipin cytochemistry in combination with freeze fracture revealed that the amount of cholesterol in the normal fibre membranes was fully comparable with the amount of cholesterol in the deviating membranes of the focal opacities and the multilamellar bodies [17].…”

Effect of Light Scattering due to Multilamellar Bodies in the Human Eye

“…1). Hence, resulting m 1 = n l /n m = n i /n lb = 1.49/1.5 and m 2 = n l /n m = n lb /n c = 1.5/1.42 (relative refractive indices of the first and the second layer, respectively) [4,6]. The characteristics of scattering are essentially controlled by simple factors (r, λ , m l ).…”

“…When photons pass through MLBs formed by layers with a refractive index different from the core of the MLBs, the scattering angle range increases even more and hence the forward scattering decreases. In research done by Costello [4,6,7], the MBLs are considered to be potential sources for excess light scattering in age-related nuclear cataracts and described likely candidates for large scattering particles. Thus, to be considered as large particles suggested that their major contributions would be low-angle scattering owing to their lipid-rich layer and fluctuations in the cytoplasm might be responsible for key fluctuations in the local refraction index essential to produce significant light scattering.…”

“…Thus, opacity of this kind of cataracts is the consequence of the formation of lipid bilayers in the particles that are inside the lens. MLBs contain a core of cytoplasmic proteins surrounded by multiple lipid-rich bilayers forming a membrane, given rise to one of the major sources of scattering in the human nuclei [4][5][6][7]. Those coatings come to have 3 to 10 thin bilayers forming the coat of the MLBs and the thickness of each bilayer is about 5 nm, thus the thickness range of the coat is from 15 nm to 50 nm [7].…”