Localization of Initial Uptake of Macromolecules into Whole Rat Lens

Abstract

Abstract: : Purpose: Recent studies have demonstrated that large molecular weight macromolecules such as proteins, plasmids and carbohydrates can translocate across the capsule and into lens epithelial cells and fiber cells. Our approach to understanding this process is first to determine if the translocation of macromolecules across the capsule and into superficial lens cells occurs equally along all regions of the lens (central epithelium, germative zone epithelium, differentiating epithelium or posterior capsule)or is preferential to specific regions of the lens. Methods: Whole rat lenses were obtained and cultured as previously described (Boyle et al., 2002). Fluorescently-labeled protein (alpha crystallin) or carbohydrate (dextran) were added to the medium and incubated for given periods of time. At different time periods, lenses were fixed, vibratome sectioned, and the translocation of fluorescent macromolecules was visualized by confocal microscopy. Results: At early time periods (0, 1 hours), there was very little, if any, uptake of fluorescently-labeled macromolecule into superficial lens cells in contact with the capsule in any region of the lens. After 2 hrs of incubation, fluorescent macromolecules were observed in the cytoplasm of central epithelial cells and concentrated at the superficial capsule in the germative to central epithelial regions. In addition, the first indications of uptake into the lens involved epithelial cells and superficial fiber cells located in the central anterior and anterior germative regions. A single layer of fluorescently-labeled macromolecules, 2-3 cell layers deep from the epithelium-fiber cell interface, was also observed in the equatorial region. There was very little, if any, uptake of fluorescently-labeled macromolecule into posterior cells of the lens. At later time periods after 4-6 hours of incubation, macromolecules were detected in all superficial cells of the lens. Conclusions: The initial uptake of protein (alpha crystallin) and carbohydrate (dextran) involves specific regions of the lens. There is little, if any, initial uptake of macromolecules by posterior cells of the lens. Preferentially uptake of macromolecules into lens cells involves the central and germative regions, suggesting that these parts of the epithelium may play an important role in uptake of aqueous humor components that are thought to be necessary for the metabolic maintenance of cells throughout the lens.

Keywords: anatomy • microscopy: confocal/tunneling • crystallins