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J.R. Sabah, H. Davidson, E. McConkey, L. Takemoto; Passage of aqueous humor and vitreous humor protein into the rat lens in vivo . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2629.
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Purpose:The lens is surrounded by the aqueous humor and vitreous humor, which contain soluble proteins. One of the most abundant proteins is albumin. The objective of this study was to assess the possible passage of albumin into the lens, in vivo, which if true could provide nutrients important for metabolism of lens cells. Methods:Rat albumin was covalently labeled with the fluorophore Alexa 488. Bound fluorophore was separated from free fluorophore by gel permeation chromatography. Male CD IGS rats (4–6 weeks of age) were anesthesized, followed by injection of 3 µl containing the protein–Alexa 488 complex into either the anterior chamber or vitreal chamber. At 5 min. postinjection, animals were euthanized, and the lenses removed and immediately fixed in 4% paraformaldehyde in PBS or frozen for further biochemical analysis. Fixed lenses were cut into 200 micron thick sagittal sections using a vibratome, then visualized by confocal microscopy. Frozen lenses were thawed, then homogenized in 1.0 ml of PBS, centrifuged at 14,000 x g for 7 min. Fluorescently labeled albumin in the supernatant fraction was quantitated by gel permeation chromatography, using a TSK 3000 SW column attached to a fluorescence detector. Results:Analysis of fixed, midsagittal sections by confocal microscopy demonstrated that both anterior and vitreal injections resulted in the internalization of fluorescently–labeled albumin at the anterior and posterior sides of the lens. However, fluorescence was more pronounced at the anterior side after injection into the anterior chamber, and more pronounced at the posterior side after injection into the vitreal chamber. Analysis of total soluble lens proteins by gel permeation chromatography using a fluorescence detector confirmed that albumin was internalized into the lens, predominantly in its monomeric form. Conclusions: Albumin, one of the most abundant proteins of the aqueous humor and vitreous humor, is internalized by the lens in vivo, when present in either the aqueous chamber or vitreal chamber.
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