PURPOSE: To develop an imaging technique that maps the distribution of free cystine in the rat lens at subcellular resolution and enables cystine accumulation to be compared with the expression of the cystine-glutamate exchanger (Xc-). METHODS: Whole lenses were fixed, cryoprotected, and then cryosectioned in either an equatorial or axial orientation. Sections were double labeled with either a cystine antibody designed to detect free cystine (the oxidized form of cysteine) or an antibody against the light chain of Xc-, and the membrane marker wheat germ agglutinin. Sections were imaged by confocal laser scanning microscopy. Cystine labeling was quantified using image analysis software and an intensity profile plotted as a function of distance from the lens periphery. High performance liquid chromatography (HPLC) was used to determine cyst(e)ine levels in the outer cortex, inner cortex, and core fractions and verify the cystine profiles derived from immunocytochemistry. RESULTS: Qualitative and quantitative imaging approaches both showed that cystine labeling at the lens equator was most intense in the outer cortex, but diminished in the inner cortex before increasing again in the core. HPLC from the outer cortex, inner cortex, and core fractions confirmed high levels of cyst(e)ine in the core relative to the inner cortex. The bimodal distribution of cystine labeling in the outer cortex and core correlated well with the expression of the cystine-glutamate exchanger in these regions, but this was not the case in the inner cortex. A similar bimodal distribution of cystine labeling was observed in axial sections. However, in these sections a track of high-intensity cystine labeling was observed that was associated with the sutures indicating that the sutures act as a delivery pathway to the core. CONCLUSIONS: These imaging approaches have revealed two distinct regions of cystine uptake in the outer cortex and core of the lens. Furthermore, this bimodal distribution of cystine has been shown to be the result of cystine delivery to the core via the sutures. This newly identified pathway opens an opportunity for the delivery of therapeutic antioxidants aimed at preventing age-related nuclear cataract.
PURPOSE: To develop an imaging technique that maps the distribution of free cystine in the rat lens at subcellular resolution and enables cystine accumulation to be compared with the expression of the cystine-glutamate exchanger (Xc-). METHODS: Whole lenses were fixed, cryoprotected, and then cryosectioned in either an equatorial or axial orientation. Sections were double labeled with either a cystine antibody designed to detect free cystine (the oxidized form of cysteine) or an antibody against the light chain of Xc-, and the membrane marker wheat germ agglutinin. Sections were imaged by confocal laser scanning microscopy. Cystine labeling was quantified using image analysis software and an intensity profile plotted as a function of distance from the lens periphery. High performance liquid chromatography (HPLC) was used to determine cyst(e)ine levels in the outer cortex, inner cortex, and core fractions and verify the cystine profiles derived from immunocytochemistry. RESULTS: Qualitative and quantitative imaging approaches both showed that cystine labeling at the lens equator was most intense in the outer cortex, but diminished in the inner cortex before increasing again in the core. HPLC from the outer cortex, inner cortex, and core fractions confirmed high levels of cyst(e)ine in the core relative to the inner cortex. The bimodal distribution of cystine labeling in the outer cortex and core correlated well with the expression of the cystine-glutamate exchanger in these regions, but this was not the case in the inner cortex. A similar bimodal distribution of cystine labeling was observed in axial sections. However, in these sections a track of high-intensity cystine labeling was observed that was associated with the sutures indicating that the sutures act as a delivery pathway to the core. CONCLUSIONS: These imaging approaches have revealed two distinct regions of cystine uptake in the outer cortex and core of the lens. Furthermore, this bimodal distribution of cystine has been shown to be the result of cystine delivery to the core via the sutures. This newly identified pathway opens an opportunity for the delivery of therapeutic antioxidants aimed at preventing age-related nuclear cataract.
Authors: Jan Lewerenz; Sandra J Hewett; Ying Huang; Maria Lambros; Peter W Gout; Peter W Kalivas; Ann Massie; Ilse Smolders; Axel Methner; Mathias Pergande; Sylvia B Smith; Vadivel Ganapathy; Pamela Maher Journal: Antioxid Redox Signal Date: 2012-08-03 Impact factor: 8.401
Authors: Junyuan Gao; Huan Wang; Xiurong Sun; Kulandaiappan Varadaraj; Leping Li; Thomas W White; Richard T Mathias Journal: Invest Ophthalmol Vis Sci Date: 2013-11-01 Impact factor: 4.799
Authors: Ralf Dahm; Jan van Marle; Roy A Quinlan; Alan R Prescott; Gijs F J M Vrensen Journal: Philos Trans R Soc Lond B Biol Sci Date: 2011-04-27 Impact factor: 6.237
Authors: Catherine Cheng; Roberta B Nowak; Junyuan Gao; Xiurong Sun; Sondip K Biswas; Woo-Kuen Lo; Richard T Mathias; Velia M Fowler Journal: Am J Physiol Cell Physiol Date: 2015-03-04 Impact factor: 4.249