PURPOSE

Loss of function of the lacrimal gland (LG), which produces the aqueous tear film, is implicated in age-related dry eye. To better understand this deterioration, we evaluated changes in lipid metabolism and inflammation in LGs from an aging model.

METHODS

LG sections from female C57BL/6J mice of different ages (young, 2-3 months; intermediate, 10-14 months; old,  ≥24 months) were stained with Oil Red-O or Toluidine blue to detect lipids. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and western blotting of LG lysates determined differences in the expression of genes and proteins related to lipid metabolism. A photobleaching protocol to quench age-related autofluorescence was used in LG sections to evaluate changes in immunofluorescence associated with NPC1, NPC2, CTSL, and macrophages (F4/80, CD11b) with age using confocal fluorescence microscopy.

RESULTS

Old LGs showed increased lipids prominent in basal aggregates in acinar cells and in extra-acinar sites. LG gene expression of Npc1, Npc2, Lipa, and Mcoln2, encoding proteins involved in lipid metabolism, was increased with age. NPC1 was also significantly increased in old LGs by western blotting. In photobleached LG sections, confocal fluorescence microscopy imaging of NPC1, NPC2, and CTSL immunofluorescence showed age-associated enrichment in macrophages labeled to detect F4/80. Although mononuclear macrophages were detectable in LG at all ages, this novel multinucleate macrophage population containing NPC1, NPC2, and CTSL and enriched in F4/80 and some CD11b was increased with age at extra-acinar sites.

CONCLUSIONS

Lipid-metabolizing proteins enriched in F4/80-positive multinucleated macrophages are increased in old LGs adjacent to sites of lipid deposition in acini.