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Julie Elizabeth Whitcomb, Susan S Lee, Michael Robinson, Jie Shen, Elizabeth Spencer, Meagan Krueger, Harvey Pollack, Rex Moats, Yang Tang, Mayssa Attar; Methods for an Enhanced 3D Interpretation of Autoradiographic Ocular Drug Distribution Data. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4120.
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© ARVO (1962-2015); The Authors (2016-present)
Understanding drug distribution in the eye following various routes of administration is important for optimizing drug delivery. The physiologic and anatomic barriers that affect drug distribution to the site of action can be studied with autoradiography (ARL), which detects radiolabeled drug compounds on two dimensional film, but lacks anatomical landmarks for spatial reference. Herein, we describe a method to enhance ARL images for 3D interpretation.
A model radiolabeled compound (MW: 430 g/mol) was dosed unilaterally via daily repeated topical dose and a single intravitreal injection (n = 2 monkeys). The frozen heads were sectioned into 43 transverse cross sections 20 µm thick through the eye. Sections were mounted on a lexan plate, photographically imaged (Figure 1a), then exposed on phosphor imaging screens (Figure 1b) with blood standards for radioactivity quantitation. A novel algorithm was designed to align the anatomical photographic images to the ARL slides. Radioactivity concentration as a function of image density was generated and the individual images stacked to render a 3D volume.
The 3D reconstruction provided an anatomically accurate and quantitative representation of the 2D phosphor images. The blood standards were used to translate the log-linear concentration of the radioactivity to a quantitative heat map (Figure 1c) to interpretat the dimensional gradient. Interpolation between the individual slices was done to visually illustrate the regional drug distribution between the two routes of administration (Figure 2). Overall the topical dosing resulted in localized distribution in the anterior region; whereas, the intravitreal dose was more dispersed throughout the entire eye.
Traditional ARL films are generally difficult to interpret in a 3D space and the drug distribution is difficult to interpret without any anatomic references. Quantitative 3D renderings of drug distribution from 2D digital autoradiographic images is a useful tool for understanding local drug delivery in relation to the anatomical structures and can be applied to other labeling techniques to better understand drug distribution.
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