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D. H. Nguyen, S. Ma, G. K. Kousoulas; Aging of the Rat Lacrimal Gland Correlates With Increased Expression of Genes Involved in Protein Processing and Modification. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1904. doi: https://doi.org/.
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Previous microarray and real-time PCR analysis confirmed increased expression of several genes associated with protein processing and modification: macrophage metalloelastase (MME), lysosomal acid lipase (LAL), and cathepsin E in the lacrimal gland (LG) of a rat denervated dry eye model. This study examines whether the expression of these genes is increased with age.
Total RNA was isolated from young (4 mo and 12 mo) and aged (18 mo and 24 mo) LGs of F344 rats (n=4 per age group). RNA integrity (RI) was assessed using the RNA LabChipTM, and all RNA samples had a RI number (RIN) of greater than 9.2. DNA-free RNA (1µg) was used for cDNA synthesis, and PCR reactions were performed in triplicate using the iCyclerTM Sybr Green and IQTM real-time detection system. Melt curve analysis was used to confirm the authenticity of the amplified product. The threshold cycle (Ct) for each reaction was determined using the default settings, and mean Ct was normalized to respective 18S control Ct values. Relative fold change was calculated using the 2-ΔΔCt method. Western blot and immunohistochemical analyses were carried out using anti-mouse MME (1:1000) and the ECL and ABC detection system. A secondary antibody alone was used as a control.
Expression of MME increased to 5.7-fold at 12 mo and to 10- and 13.3-fold at 18 mo and 24 mo, respectively, when compared to the level at 4 mo. Expression of LAL increased to almost 2-fold at 18 mo and to 4.3-fold at 24 mo. Cathepsin E increased to 4-, 7.1-, and 124.4-fold at 12, 18, and 24 mo, respectively. Western blot showed increased levels of MME with age. Immunohistochemistry showed sparse and moderate MME staining in the 12 mo, and more intense staining in the 18 mo LG that are mostly localized to membrane of acini; in the 24 mo LG, intense staining was found on acini membrane and cytoplasm.
Aging in the LG is associated with persistent increase in expression of genes involved in protein processing and modification that may suggest aberrant morphologic changes. These changes were previously observed after parasympathetic denervation, indicating that parasympathetic control of LG function is decreased with age.
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