Age-dependent alterations in mouse exorbital lacrimal gland structure, innervation and secretory response

Abstract

Several studies investigated the effect of aging on rat and human lacrimal gland physiology. However, in most of these studies, only two age groups were investigated. Furthermore, those studies did not correlate the age-related histological changes that occur in the lacrimal gland to the functional changes (nerve activity and protein secretion) that might occur with aging. Thus, the purpose of the present study was to investigate the effect of aging on lacrimal gland structure, innervation and function using BALB/c mice at different ages. Exorbital lacrimal glands were removed from 3, 8, 12, 24, and 32-month-old, male BALB/c mice, fixed, embedded and processed for histology and immunohistochemistry. Sections were stained with hematoxylin and eosin to determine morphological changes and lymphocytic infiltration; giemsa to identify mast cells; and Kinyoun's carbol fucsin solution to indicate lipofuscin-like inclusions. Parasympathetic and sympathetic nerves were identified by immunofluorescence techniques. To measure acetylcholine release and protein secretion, lacrimal gland pieces were incubated in Krebs Ringer buffer containing 5 mM KCl (control), 75 mM KCl (depolarizing buffer which activates nerves), carbachol (a cholinergic agonist, 10(-4) M), or phenylephrine (an alpha1-adrenergic agonist, 10(-4) M) for 20 min. The media were collected and analysed for acetylcholine and peroxidase using a spectrofluorometric assay. KCl-, carbachol- and phenylephrine-stimulated peroxidase secretion decreased in lacrimal glands from 8, 12, and 24-month-old mice when compared to 3-month-old animals. Both the density and distribution of parasympathetic and sympathetic nerves surrounding the acini decreased with increasing age. Acetylcholine release from lacrimal gland nerves decreased in 24-month-old mice compared to 3- and 12-month-old animals. Similarly, progressive morphological changes, including increased numbers of lipofuscin-like inclusions, mast cells and lymphocytic infiltration occurred in an age-dependent manner. We conclude that structural alterations of mouse lacrimal gland, including increased accumulation of lipofuscin-like inclusions, chronic inflammation and functional alterations including decreased acetylcholine release and protein secretion occurred with aging.

Fig. 1

Effect of age on KCl-, phenylephrine-, and carbachol-induced peroxidase secretion. Lacrimal glands of 3, 8, 12, and 24-month-old male BALB/c mice were removed and lobules were incubated for 20 min in buffer containing 5 mm KCl (spontaneous secretion), 20 min in 75 mm KCl (evoked secretion), 20 min in the presence of the adrenergic agonist, phenylephrine (10⁻⁴ m) or the muscarinic agonist, carbachol (10⁻⁴ m) and peroxidase secretion was measured. (1A). Peroxidase secretion is expressed as a percentage of the total. (1B). Peroxidase secretion was normalized to the weight of the lacrimal gland. Data are expressed as means±s.e.m. from 3–10 independent experiments. *Indicates significant difference from spontaneously secreted peroxidase. # Indicates significant difference from 8, 12 and 24-month.

Fig. 2

Immunolocalization of nerves containing synaptophysin in the lacrimal gland. Lacrimal glands of 3, 8, 12, 24, and 32-month-old male BALB/c mice were removed and processed for immunohistochemistry with anti-synaptophysin antibody. (2A). Immunofluorescence at 488 nm (green) represents synaptophysin immunoreactivity and autofluorescence of lipofuscin-like inclusions. Fluorescence at 568 nm (red) indicates lipofuscin-like inclusions, which also fluoresce at this wavelength. Overlay of fluorescence observed at 488 and 568 nm distinguishes between the autofluorescence of lipofuscin-like inclusions and the fluorescence of synaptophysin-containing nerves, as synaptophysin-like immunoreactivity is green and whereas lipofuscin-like inclusions are yellow. Arrows indicate areas of nerves. Arrowheads indicate areas of lipofuscin-like inclusions. (2B). Omission of the primary antibody led to a loss of synaptophysin immunoreactivity whereas lipofuscin autofluorescence was still visible in older tissue. Similar results were observed in at least three other experiments. Magnification, ×400.

Fig. 2

Immunolocalization of nerves containing synaptophysin in the lacrimal gland. Lacrimal glands of 3, 8, 12, 24, and 32-month-old male BALB/c mice were removed and processed for immunohistochemistry with anti-synaptophysin antibody. (2A). Immunofluorescence at 488 nm (green) represents synaptophysin immunoreactivity and autofluorescence of lipofuscin-like inclusions. Fluorescence at 568 nm (red) indicates lipofuscin-like inclusions, which also fluoresce at this wavelength. Overlay of fluorescence observed at 488 and 568 nm distinguishes between the autofluorescence of lipofuscin-like inclusions and the fluorescence of synaptophysin-containing nerves, as synaptophysin-like immunoreactivity is green and whereas lipofuscin-like inclusions are yellow. Arrows indicate areas of nerves. Arrowheads indicate areas of lipofuscin-like inclusions. (2B). Omission of the primary antibody led to a loss of synaptophysin immunoreactivity whereas lipofuscin autofluorescence was still visible in older tissue. Similar results were observed in at least three other experiments. Magnification, ×400.

Fig. 3

Immunolocalization of nerves containing vasoactive intestinal peptide (VIP) in the lacrimal gland. Lacrimal glands of 3, 8, 12, 24, and 32-month-old male BALB/c mice were removed and processed for immunohistochemistry with anti-VIP antibody. Immunofluorescence at 488 nm (green) represents VIP-immunoreactivity and autofluorescence of lipofuscin-like inclusions. Fluorescence at 568 nm (red) indicates lipofuscin-like inclusions, which also fluoresce at this wavelength. Overlay of fluorescence observed at 488 and 568 nm distinguishes between the autofluorescence of lipofuscin-like inclusions and the fluorescence of VIP-containing nerves as VIP-like immunoreactivity is green and whereas lipofuscin-like inclusions are yellow. Arrows indicate areas of nerves. Arrowheads indicate areas of lipofuscin-like inclusions. Similar results were observed in at least three other experiments. Magnification, ×400.

Fig. 4

Immunolocalization of nerves containing tyrosine hydroxylase (TH) in the lacrimal gland. Lacrimal glands of 3, 8, 12, 24, and 32-month-old male BALB/c mice were removed and processed for immunohistochemistry with anti-TH antibody. Immunofluorescence at 488 nm (green) represents TH-immunoreactivity and autofluorescence of lipofuscin-like inclusions. Fluorescence at 568 nm (red) indicates lipofuscin-like inclusions, which also fluoresce at this wavelength. Overlay of fluorescence observed at 488 and 568 nm distinguishes between the autofluorescence of lipofuscin-like inclusions and the fluorescence of TH-containing nerves, as TH-like immunoreactivity is green and whereas lipofuscin-like inclusions are yellow. Arrows indicate areas of nerves. Arrowheads indicate areas of lipofuscin-like inclusions. Similar results were observed in at least three other experiments. Magnification, ×400.

Fig. 5

Effect of age on KCl-evoked acetylcholine release. Lacrimal glands of 3, 12, and 24-month-old male BALB/c mice were removed and lobules were incubated for 20 min in buffer containing 5 mm KCl (spontaneous release) or 75 mm KCl (evoked release). (5A). Acetylcholine release is expressed as micromoles of acetylcholine. (5B). The concentration of released acetylcholine was normalized to the weight of the lacrimal gland. Data are expressed as means±s.e.m. from three independent experiments. *Indicates significant difference from spontaneously released acetylcholine.

Fig. 6

Lacrimal glands of 3, 8, 12, 24, and 32-month-old male BALB/c mice were removed and stained with Kinyoun’s carbol fuchsin (Kinyoun’s Carbol). Arrows indicate areas of lipofuscin-like inclusions. Similar results were observed in at least three other experiments. Magnification, ×200.

Fig. 7

Lacrimal gland sections prepared from 3 (n = 7), 8 (n = 4), 12 (n = 6), 24 (n = 6), and 32 (n = 6)-month-old mice were stained with hematoxylin and eosin (for lymphocytic infiltration), giemsa (for mast cell infiltration), or Kinyoun’s carbol fucsin (for lipofuscin-like accumulation). Sections were examined by three investigators and scored using the following criteria: Lymphocytic infiltration was defined as the number of lymphocytic foci present in the lobular and periductal regions and graded as 0 if no foci were present, grade 1 if one to two foci were present, grade 2 if two to four foci were present, and grade 3 if more than four foci were present. Mast cells were graded as 0 if present in blood vessels and connective tissue, 1 if present in interlobular space, 2 if ≤20 mast cells infiltrated the intralobular space, and 3 if more than 20 cells infiltrated the intralobular space. The presence of lipofuscin was grade 0 if not present, grade 1 if present within only few lobules, grade 2 if present in less than 50% of the lobules, and grade 3 if present in more than 50% of the lobules. Data are means±s.e.m. of the individual scores. Data were analysed using the Spearman Rank Correlation test and the P values are shown in the plot.

Fig. 8

Lacrimal glands of 3, 8, 12, 24, and 32-month-old male BALB/c mice were removed and stained with Giemsa. Arrows indicate mast cell infiltration while the arrowhead indicates a degranulating mast cell that is shown at a higher magnification in the insert. Similar results were observed in at least three other experiments. Magnification, ×400.

Fig. 9

Lacrimal glands of 3, 8, 12, 24, and 32-month-old male BALB/c mice were removed and stained with Hematoxylin-eosin (H and E). Arrows indicate areas of lymphocytic infiltration. Similar results were observed in at least three other experiments. Magnification, ×200.