Abstract
Purpose::
To determine the potential mechanisms that contribute to the decline in lacrimal gland function with age.
Methods::
Lacrimal gland pieces were prepared from 3-, 8-,12- and 24-month-old male C57BL/6 mice for histochemical analysis. Changes in indicators of oxidative stress were assessed by quantifying the accumulation and/or activity of lipofuscin-like inclusions, lipid peroxidation (LPO), reduced glutathione (GSH), superoxide dismutases (SOD), and inducible nitric oxide synthase (iNOS). Levels of LPO and GSH were measured by biochemical assays. Levels of the SOD isoforms, and iNOS were measured by Western blotting or immunofluorescence microscopy. For measurement of secretion, pieces of lacrimal gland were incubated for 20 min. intervals in Krebs Ringer buffer containing 5mM KCl (control), 75mM KCl (depolarizing buffer to activate nerves, evoked), carbachol (a cholinergic agonist, 10-4 M), phenylephrine (an α1-adrenergic agonist, 10-4 M) or the phorbol ester PMA (a protein kinase C activator, 10-7M). The latter three compounds cause agonist-induced secretion. After incubation, the media were collected and the total amount of peroxidase released into the media was analyzed using a spectrofluorometric assay.
Results::
With increasing age, progressive morphological changes occurred in the lacrimal gland including accumulation of large cytoplasmic lipofuscin-like granules, increased number of mast cells, increased lymphocytic infiltration, and lobular and acinar atrophy. The level of LPO increased in an age-dependent manner in lacrimal gland homogenates. The level of GSH decreased in lacrimal gland homogenates of 24- month-old mice compared to 3-month-old ones. iNOS was differentially expressed (increase followed by a decrease) in 12- and 24-month-old glands as compared to 3-month-old glands. Expression of SOD did not change with increasing animal age. Evoked and agonist-induced peroxidase secretion decreased in an age-dependent manner.
Conclusions::
In the lacrimal gland considerable structural alteration, increased in indicators of oxidative stress, and decreased agonist induced protein secretion occurs with increasing age. These changes appear to result in the impairment of neurotransmitter release and lacrimal acinar cell intracellular signaling pathways that stimulate protein secretion.
Keywords: aging • oxidation/oxidative or free radical damage • cornea: tears/tear film/dry eye