The authors have no financial interest in the subject matter of this poster. The Effect of Topical Epigallocatechin Gallate (EGCG) Treatment on Murine Dry Eye Hyun Soo Lee, Andre Okanobo, Nambi Nallasamy, Sunil K. Chauhan, M. Reza Dana Schepens Eye Research Institute Boston, Massachusetts Department of Ophalmology, Harvard Medical School and Massachusetts Eye and Ear Infirmary, Boston, Massachusetts

Introduction Dry eye is the most common cause for clinical disease in ophthalmology and it is widely recognized that dry eye disease (DED) is associated with inflammation of the ocular surface. Epigallocatechin gallate (EGCG), one of main extracts in green tea, has an inhibitory effect on inflammation by reducing the expression of IL-1, IL-6, MCP-1 and TNF-α through inhibition of NF-kB activation.

Hypothesis Topical EGCG administration may have a therapeutic effect for the treatment of DED by inhibiting proinflammatory cytokines through the modulation of NF-kB. Here, we aimed to investigate the efficacy of topical EGCG on a murine model of dry eye.

Materials and Methods Female 7-8 week old C57BL/6 mice were housed in the controlled environment chamber (relative humidity < 30%, constant temperature of 21°C to 23°C, and airflow of 15 L/min for all day) to induce DED. Subcutaneous administration of scopolamine (tid) was used to maximize ocular dryness. Forty-eight hours after induction of dry eye, mice were randomly divided into the following 4 different groups: 1) untreated group, 2) topical vehicle treatment, 3) topical 0.01% EGCG, and 4) topical 0.1% EGCG.

Materials and Methods Corneal fluorescein staining (using the National Eye Institute grading scheme) at baseline (day 0), 48 hrs, and day 4 and day 9 after induction of DED Immunohistochemical staining (day 9) 1) corneal CD11b+ cells, 2) NF-kB p65 Real-time PCR (day 9) : corneal cytokines IL-1β, TNF-α, monocyte chemotactic protein (MCP)-1 Corneal TUNEL assay (day 9)

Results: corneal fluorescein staining score Topical 0.1% EGCG treatment produced a significant decrease in the corneal fluorescein staining score compared with the vehicle and untreated groups at days 9. Asterisk indicates P<.0001 vs untreated, P=.001 vs vehicle, P=.006 vs 1.0% EGCG. But there was no significant difference between 0.01% EGCG and vehicle. Data are presented as mean and SEM (error bars).

Results: CD11b + cells in dry eye cornea Center Periphery ‡ ‡ † ‡ † † * Both 0.01% and 0.1% EGCG treatment decrease the number of CD11b + cells in the center and periphery of dry eye corneas, compared with the untreated and vehicle groups. Asterisk indicates P <.05 ; dagger, P<.01 ; and double dagger, P<.001. Data are presented as mean and SEM (error bars).

Results: real-time PCR Real-time polymerase chain reaction results showing that corneas treated with 0.1% and 0.01% EGCG have significantly decreased relative expression of IL-1β (A) and monocyte chemoattractant protein (MCP)-1(C) transcripts in dry eye cornea. Data were normalized to GAPDH mRNA and values are expressed as fold change over normal control corneas. Data from three independent experiments are presented as mean and SEM (error bars) A BC * IL-1bTNF-α MCP-1 * †

Results : Immunohistochemistry of NF-kB p65 The p65 protein was localized in the cytoplasm of the corneal epithelial cells of normal controls (A). Significant nuclear translocation for p65 protein was observed in the epithelium of the untreated DED cornea (C). In contrast, 0.1% EGCG (G) decreased the translocation of NF-kB p65 compated to the vehicle- treated group (E). Left column: Immunohistochemistry staining of NF-kB p65. Right column: DAPI (4’, 6 diamidino-2-phenylindole) staining of nucleus. ) Normal control ) Untreated ) Vehicle ) 0.1% EGCG

Results : Tunel assay ‡ * † * The number of apoptotic cells was significantly increased in dry eye corneal epithelial layer compared with normal controls (P<.0001 vs normal control). Although 0.01% and 0.1% EGCG showed decrease in the number of apoptotic cells compared to the untreated group (P=.009 and P=.003, respectively), But topical 1.0% EGCG significantly increased the corneal epithelial apoptosis compared with 0.01% and 0.1% EGCG groups (P=.013 and P=.007, respectively)

Conclusions Topical EGCG treatment is able to reduce the clinical signs and inflammatory changes in experimental dry eyes through suppressing the inflammatory cytokine expression and infiltration of CD11b+ cells in the cornea. Our study suggests that EGCG, a potential pharmacologically active agent available for topical administration, could be used therapeutically for the treatment of dry eye.