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Wangfei Wang, Tara Nguyen, Dingcai Cao, David R Pepperberg; Guanidine hydrochloride treatment of rat isolated eye tissues for quantification of amyloid-beta. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3574a. doi: https://doi.org/.
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Amyloid-beta (Aβ), a group of aggregation-prone peptides (lengths: 38-43 amino acids), is found in tissues of eye and brain. In brain, dysregulated Aβ metabolism is thought to promote Alzheimer’s disease progression. Recent studies suggest that Aβ dysregulation may also accelerate the progression of age-related macular degeneration (AMD), raising interest in developing approaches that efficiently recover Aβ from eye tissues. Aβ-containing brain tissues possess soluble (i.e., extractable by phosphate-buffered saline; PBS) and insoluble (aggregated) Aβ; the denaturing agent guanidine hydrochloride (GuHCl) has been used in brain studies to facilitate recovery of insoluble forms (ref. 1). We have investigated the use of GuHCl extraction to recover Aβ40 (length: 40 amino acids), a principal Aβ form, from eye tissues.
Sprague-Dawley rats (ages: 9-12 months) were used. Following euthanasia, lens/vitreous, retina and RPE/choroid were separately recovered from the enucleated eye (2). Tissues from one eye of each animal were homogenized (as single or pooled samples) in PBS supplemented with 5 M GuHCl (pH: 7.4), incubated overnight, then centrifuged. The supernatant was de-salted (PBS-equilibrated column), and the filtrate analyzed for Aβ40 (ELISA) and protein (Bradford). Tissues of the fellow eye were homogenized in PBS lacking GuHCl (“PBS-only”) (pH 7.4) and centrifuged, for similar analysis of the supernatant. Both the GuHCl/PBS and the PBS-only media contained 2% (v/v) EDTA-free protease inhibitor.
Data were obtained from the eyes of 8 male and 4 female rats. For treatment with PBS-only, Aβ40 in pmol per g protein (pmol/g) was 1.72 ± 1.52 (mean ± SEM) for RPE/choroid, 0.61 ± 0.34 for retina, and 0.11 ± 0.03 for lens/vitreous. For overnight treatment with GuHCl/PBS, Aβ40 in pmol/g was 9.90 ± 2.27 for RPE/choroid, 6.88 ± 1.80 for retina, and 1.70 ± 0.67 for lens/vitreous.
The substantially higher recovery of Aβ40 obtained with GuHCl treatment encourages the use of GuHCl-supplemented extraction medium in studies that investigate how eye tissue abnormalities (induced genetically or pharmacologically) that model AMD-associated features correlate with changes in Aβ metabolism.References: (1) Youmans et al. (2011) J. Neurosci. Meth. 196:51-59. (2) Parthasarathy et al. (2015) Exper. Eye Res. 138:134-144.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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