Increased Substance P Expression in the Ocular Surface in Murine Dry Eye Disease

Abstract

Purpose: Substance P is a key mediator of pain and neurogenic inflammation. In the present study, we analyzed protein and mRNA expression levels of substance P in the ocular surface to evaluate their association with the early induction phase of dry eye disease (DED).

Methods: DED was induced in 6-week-old female C57BL/6 mice by bilateral extra-orbital lacrimal gland excision followed by their placement in a controlled environmental chamber. Corneal fluorescent staining (CFS) was evaluated at days -1, 3, 6, and 13, and corneas and conjunctivae were harvested at days 4, 7, and 14 to analyze protein and mRNA expression levels of substance P. Substance P protein expression levels were measured with a competitive enzyme immunoassay kit (R&D Systems, Minneapolis, MN) and then adjusted to the total protein amount. Expression of substance P mRNA was analyzed by real-time PCR. One way ANOVA test was used for comparison of the expression levels and Pearson correlation was used for correlation analysis.

Results: Substance P protein expression levels were increased in both the cornea and conjunctiva on days 4 and 7 compared to the sham operated control group (Cornea: 1.9 fold on day 4, 2.0 fold on day 7, P<0.05; Conjunctiva: 2.8 fold on day 4, 2.2 fold on day 7, P<0.05). By day 14, levels of substance P decreased to baseline levels. We observed similar kinetics for the mRNA expression of substance P. Increased protein expression of substance P in the cornea correlated with increased CFS scores until day 7 (r = 0.900, P<0.001).

Conclusions: Protein and mRNA expression levels of substance P are increased in the early induction phase of DED and correlate with increased CFS scores.

Keywords: 480 cornea: basic science • 614 neuropeptides • 533 gene/expression