Age-dependent changes in rat lacrimal gland anti-oxidant and vesicular related protein expression profiles

Abstract

Purpose: Anti-oxidation and exocytosis are important for maintaining exocrine tissue homeostasis. During aging, functional and structural alterations occur in the lacrimal gland (LG), including oxidative damage to proteins, lipids, and DNA. The aims of the present study were to determine in the aging LG: a) the effects of aging on LG structure and secretory activity and b) changes in the expression of oxidative stress markers.

Methods: To address these goals, tear secretion composition and corneal impression cytology were compared between male Wistar rats of 2 (control) and 24 (aged) months. LG morphology and the expression levels of vitamin E and malonaldehyde (MDA) were evaluated to determine the anti-oxidant activity and lipid peroxidation, respectively. RT-PCR and western blot analysis were used for the analysis of Ras related in brain GTPase protein (Rab) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of the secretory machinery (i.e.; Rab 3d, Rab 27, vesicle-associated membrane protein-2 (Vamp-2), and syntaxin).

Results: Histological analysis of aged rats revealed a higher frequency of corneal epithelia metaplasia. In the acinar cells, organelles underwent degeneration, and lipofucsin-like material accumulated in the cytoplasm along with declines in the anti-oxidant marker vitamin E. Rab3d and Rab27b mRNA levels fell along with Rab3d protein expression, whereas syntaxin levels increased.

Conclusions: These findings indicate that exocytotic and anti-oxidant mechanisms become impaired with age in the rat LG. In parallel with these structural alterations, functional declines may contribute to the pathophysiology caused by tear film modification in dry eye disease.

Figure 1

Impression cytology of the corneas of control (A) and aged (B) rats. Grades from 0 to 3 were given for each sample, based on size, nucleus, and the presence of mucus (Scale bar=25 µm).

Figure 2

LG histology in the control and aged groups. Control (A) and aged (B) samples stained with H&E (Scale bar=100 µm). Unstained samples of control (C) and aged (D), to demonstrate autofluorescence of LG (white arrows: lipofucsin-like deposits; Scale bar=100 µm). TEM (Scale bar=2.5 µm) of control (E) and aged (F) samples revealing details of LG acinar cells.

Figure 3

Effect of the aging on mRNA expression in LG. The ratio between Rab 3d/Actb (A), Rab 27/Actb (B), and Vamp-2/Actb (C) mRNAs are expressed as the mean±standard error of densitometric arbitrary units (*p<0.05, Mann–Whitney U test). Results are representative of three independent experiments.

Figure 4

Effect of aging on the expression of the ratio of (A) Rab 3d/GAPDH, (B) Syntaxin 1A/GAPDH, and (C) Vamp 2/GAPDH in LG. Tissues from both groups were excised and homogenates were analyzed by western blot (*p<0.05, Mann–Whitney U test). Results are representative of three independent experiments.