Mechanisms of Extraorbital Lacrimal Gland Aging in Mice: An Integrative Analysis of the Temporal Transcriptome.

Purpose: This study used high-throughput RNA sequencing (RNA-Seq) and bioinformatics analysis to investigate the altered transcriptome profile of aging lacrimal glands in mice that occurs over the course of a 24-hour cycle. Methods: Male C57BL/6J mice aged 12 weeks (young) and 20 months (aging) were housed in a pathogen-free setting with a 12-hour light/12-hour dark cycle. Throughout a 24-hour cycle, mouse extraorbital lacrimal glands (ELGs) were collected at eight time points at three-hour intervals. To prepare for the high-throughput RNA-Seq, whole mRNA was extracted. Differentially expressed genes (DEGs) in the young and aging groups were subjected to bioinformatic analysis based on diurnal patterns. Furthermore, the cell populations in which significant DEGs express and signaling pathways occur were validated at the single-cell RNA sequencing (scRNA-seq) level. Results: The total transcriptome composition was significantly altered in aging ELGs compared with that in young mouse ELGs at eight time points during the 24-hour cycle, with 864 upregulated and 228 downregulated DEGs, which were primarily enriched in inflammatory pathways. Further comparative analysis of the point-to-point transcriptome revealed that aging ELGs underwent alterations in the temporal transcriptome profile in several pathways, including the inflammation-related, metabolism-related, mitochondrial bioenergetic function-associated, synaptome neural activity-associated, cell processes-associated, DNA processing-associated and fibrosis-associated pathways. Most of these pathways occurred separately in distinct cell populations. Conclusions: Transcriptome profiles of aging lacrimal glands undergo considerable diurnal time-dependent changes; this finding offers a comprehensive source of information to better understand the pathophysiology of lacrimal gland aging and its underlying mechanisms.