Resolvin E1 improves tear production and decreases inflammation in a dry eye mouse model

Abstract

Purpose: Dry eye (DE) is a common ocular surface disease, particularly among women and the elderly, with chronic symptoms of eye irritation and, in severe cases, blurred vision. Several studies have shown that there is an inflammatory component in DE, although the pathogenesis is not thoroughly understood. Resolvin E1 (RvE1; RX-10001) is an endogenous mediator derived from the omega-3 polyunsaturated fatty acid eicosapentaenoic acid and is involved in inflammation resolution and tissue protection. Here we investigated the role of RvE1 in a DE mouse model.

Methods: Thirteen- to 14-week-old female BALB/C mice were exposed to desiccating conditions. One week after DE exposure, animals were treated topically with drug or vehicle 4 times per day for an additional week. Controls were nontreated animals placed in a normal environment. Schirmer's test was performed before treatment initiation and at days 2 and 4 after treatment. Density of corneal epithelial cells was analyzed in vivo using the Rostock Cornea Module of the Heidelberg Retina Tomograph (HRT-II). Corneas were processed using Western blot analysis and immunofluorescence examination.

Results: Schirmer's test showed a significant decrease in tear production in DE compared with controls. There was no change at 2 and 4 days after treatment with the vehicle, but a significant increase was observed at 2 and 4 days in the RvE1-treated group. The density of the superficial epithelial cells showed a significant decrease after DE compared with controls, which increased after 7 days of RvE1 treatment. Western blot analysis showed that α-smooth muscle actin and cyclooxygenase-2 (COX-2) expression were strongly upregulated after DE and decreased after 7 days of RvE1 treatment. Immunofluorescence confirmed strong positive staining of α-smooth muscle actin and COX-2 in stroma and/or in epithelia after DE, which decreased with RvE1 treatment. The percentage of infiltrating CD⁴+ T cells and CD11b+ cells decreased after RvE1 treatment when compared with DE.

Conclusion: RvE1 promotes tear production, corneal epithelial integrity, and a decrease in inflammatory inducible COX-2. In the stroma, RvE1 inhibits keratocyte transformation to myofibroblasts and lowers the number of monocytes/macrophages in this DE mouse model. These results suggest that RvE1 and similar resolvin analogs have therapeutic potential in the treatment of DE.

FIG. 1.

Schirmer's test in mice treated with resolvin E1 (RvE1). Tear volume was measured after 7 days in dry eye (DE) conditions and then at 2 and 4 days after treatment initiation. Values correspond to mean ± standard error of the mean (SEM). Number of animals tested: control = 7, vehicle = 9, DE = 9, and RvE1 = 13. *Significant differences versus control; **significant differences versus DE.

FIG. 2.

Superficial epithelial cell density in mice treated with RvE1. (A) Representative images of mice cornea epithelial cell surfaces in different groups after 14 days of DE and 7 days of drug/vehicle treatments starting at day 8. (B) Quantification of epithelial cell density; the values correspond to average ± SEM of 3 animals in each group. Six different areas for each animal were quantified. The experiment was repeated 1 time with similar results. *Significant differences versus controls; **significant differences versus DE.

FIG. 3.

Expression of α-smooth muscle actin (α-SMA) in mice treated with RvE1. Animals were in DE conditions for 14 days; drug and vehicle were applied for the last 7 days. (A) Representative images of immunostaining with an α-SMA antibody. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). (B) Western blot analysis shows a band at 47 kDa corresponding to α-SMA after induction of DE; β-actin shows equal loading of the gel. Values are average ± SEM of 3 blots corresponding to 3 different experiments. *Significant difference versus DE. Color images available online at www.liebertonline.com/jop.

FIG. 4.

Expression of cyclooxygenase-2 (COX-2) in mice treated with RvE1. Corneas were obtained after 14 days of DE conditions and treatment with drug and vehicle for the last 7 days. (A) Western blot analysis shows a 78 kDa band corresponding to COX-2. Values correspond to average ± SEM of 3 different experiments. (B) Immunostaining with a COX-2 antibody. The nuclei were counterstained with 4,6-diamidino-2-phenylindole. *Significant differences versus DE. Color images available online at www.liebertonline.com/jop.

FIG. 5.

Effect of RvE1 on CD11b⁺ expression after DE conditions. Mice were in DE conditions for 14 days and treated with drug or vehicle for the last 7 days. (A) Representative images of corneal sections in the different groups. (B) Density of CD11b⁺ cells. Values correspond to average ± SEM of 4 different eyes/group, analyzed as explained in the Methods section. *Significant differences versus control; **significant differences versus DE and vehicle. Color images available online at www.liebertonline.com/jop.

FIG. 6.

Effect of RvE1 on CD4⁺ expression after DE conditions. Mice were in DE conditions for 14 days and treated with drug or vehicle for the last 7 days. (A) Representative images of corneal sections in the different groups. No CD4⁺ cells were found in the normal corneas, whereas DE induced CD4⁺ cell infiltration into cornea. These cells were mostly seen in the anterior stroma close to the limbal area. (B) Density of CD4⁺ cells. Values correspond to average ± SEM of 4 different eyes/group. *p < 0.05. Color images available online at www.liebertonline.com/jop.