Changes in expression of inflammatory–associated genes in the rat lacrimal gland after loss of muscarinic, parasympathetic activation.

Abstract

Abstract: : Purpose: Previous microarray and real–time PCR results suggest an elevated expression of inflammation–associated genes in the rat lacrimal gland (LG) following chronic loss of parasympathetic activation. In this study, real–time PCR was used to verify expression of several proinflammatory genes in the rat LG. Methods: Male Long–Evans rats underwent unilateral pre–ganglionic parasympathetic sectioning of the greater superficial petrosal nerve and were sacrificed after 7 days. cDNA was synthesized from 1 µg of total RNA from contralateral (Ctla) and experimental LG (N=3). Semi–quantitative real–time PCR was carried out using Sybr Green. All reactions were done either in duplicate or triplicate using the iCycler IQ real–time detection system. The 18S rRNA internal control was run in separate reactions. Mean cycle threshold (MCt) for the genes in the Ctla and experimental RLG were normalized to 18S rRNA MCt values. Relative fold change was calculated for each gene using the 2^–ΔΔCT method (Livak and Schmittgen, 2001). Confirmation was defined as a change greater than or equal to a two–fold change by both real–time PCR and DNA microarray analyses or when fold change from real–time PCR exceeded that of the microarray analysis. Results: The differential expression of nuclear factor kappa B p105 subunit (NFKB p105), allograft inflammatory factor–1 (AIF–1), and leukocyte antigen MRC–OX44 was confirmed in the experimental LG by real–time PCR. Expression of NFKB p105, AIF–1, and leukocyte antigen MRC–OX44 increased 2.71, 4.69, and 4.63 fold in the experimental LG, respectively. However, caspase 6 with a fold change of 1.34 calculated by real–time PCR did not qualify for our definition of confirmation. Lastly, MCt of the 18S rRNA (mean + SEM) was not significantly different between Ctla (16.08 + 0.55) and experimental LGs (15.87 + 0.30). Conclusions: Microarray analysis and real–time PCR have shown major changes in genes involved in immune regulation in the lacrimal gland after removal of parasympathetic control. These changes occur in the absence of an increase in the M3 muscarinic receptor.

Keywords: cornea: tears/tear film/dry eye • lacrimal gland • inflammation