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D. H. Nguyen, H. Toshida, Q. Ma; Cartilage-Derived Retinoic Acid-Sensitive Protein Expression in Aged Lacrimal Gland. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4249.
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© ARVO (1962-2015); The Authors (2016-present)
Previous gene expression studies in an experimental rat dry eye following chronic loss of parasympathetic activation of secretion identified the cartilage-derived retinoic acid-sensitive protein (CD-RAP) as being significantly upregulated 6-fold in the denervated lacrimal gland (LG). This study examines if expression of CD-RAP is increased as a function of chronological age.
Fischer 344 rats (4, 12, 18, and 24 months, n=3 per age group) were obtained from the National Institute of Aging. LG total RNA (1µg) was used for cDNA synthesis, and the diluted cDNA was used to analyze CD-RAP and M3 muscarinic receptor expression using the iCycler Sybr green and CFX96 real-time PCR detection system. All reactions were performed in triplicate, and the threshold cycle (Ct) for each reaction was determined using the default settings and mean Ct (MCt) was normalized to respective 18S rRNA MCt values. Relative fold change was calculated based on the 2-ΔΔCt method, using the 4 month LG as the baseline expression. Protein lysates (70 µg) were separated by 15% SDS/PAGE, transferred to nitrocellulose membrane, probed with anti-CD-RAP, and visualized using the ECL detection system.
The MCt of the 18S rRNA control gene were similar in all age groups. Compared to expression at 4 month, CD-RAP increased 3.4, 7.0, 4.2-fold at 12, 18, and 24 months, respectively. Western blot analysis of the 4 and 24 month LGs showed higher levels of CD-RAP in the 24 months LG. The expression of M3 muscarinic receptors, when compared to 4 month levels, showed an increase of 1.5, 1.4, and 1.8-fold at 12, 18, and 24 months, respectively. MCts for M3 muscarinic receptors were not different when 12, 18, and 24 months samples were compared.
Although expression of CD-RAP did not increase as a function of chronological age, the persistent and elevated level of CD-RAP suggests pathological changes in matrix remodeling, and as previously observed in a rat dry eye model. Since CD-RAP is a secreted molecule, it may serve as a putative biomarker to assess matrix changes such as tissue fibrosis found in aged and diseased LG.
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