ARTICLE Evaluation of Effect of Topical Tacrolimus Treatment on Herpetic Stromal Keratitis in a Rat Model Erdem Eris¸, M.D., Nurs¸en Yüksel, M.D., Dilara Pirhan, M.D., Aynur Karadenizli, M.D., Mehmet Aslan, M.D., Gülçin Gacar, Ph.D., Gülay Erman, B.S., Cansu Subas¸ ı, M.Sc., Hüseyin Uzuner, Ph.D., Demir Kürs¸at Yıldız, M.D., and Erdal Karaöz, Ph.D. Objectives: To investigate the effectiveness of topical tacrolimus treatment on herpetic stromal keratitis (HSK) in a rat model. Methods: The development of HSK was monitored for 14 days after the inoculation of rats with herpes simplex type 1 virus. Rats that developed HSK were divided into four groups as follows: (1) topical antiviral treatment (control), (2) topical antiviral and 1% prednisolone acetate, (3) topical antiviral and 0.03% tacrolimus ointment, and (4) topical antiviral plus 0.1% tacrolimus ointment. After 14 days of treatment, the severity levels of HSK were scored and compared with the levels before the treatment. The expression of CD3, CD4, and CD8 was evaluated by flow cytometry. The development of the disease was evaluated clinically and histologically. Results: Significant improvement in vascularization was observed in the groups with the drug treatment in addition to the antiviral agent (P,0.05), but there was no obvious difference within groups 2, 3, and 4 in the vascularization severity. The regression of corneal edema was 8.05%6 6% in group 1, 25.17%614.55% in group 2 (P¼0.01), 36.40%621.69% in group 3 (P¼0.03), and 46.39%614.96% in group 4 (P¼0.00). A signif- icant decrease in the number of inflammatory cells in the groups with the drug treatment was evaluated by immunohistochemical staining and con- firmed by flow cytometry analysis. Conclusions: Topical tacrolimus treatment caused a significant decrease in corneal vascularization accompanied by a lower number of inflammatory cells in the experimental HSK corneal edema model. Therefore, topical tacrolimus has the potential to be used in the treatment of HSK. Key Words: Cornea—Herpetic stromal keratitis—Inflammation— Neovascularization—Rat. (Eye & Contact Lens 2016;42: 163–170) H erpetic stromal keratitis (HSK) is a chronic recurrent inflam- matory disease occurring due to infection by the herpes simplex type 1 virus (HSV-1). 1 Because of recurrences, the disease may cause visual morbidity associated with a wide range of sequelae because of corneal vascularization, irreversible stromal scarring, and lipid keratopathy. Herpetic stromal keratitis is believed to represent a T-cell–mediated immunopathologic basis 2 that primarily involves CD4 + and CD8 + cells. 2,3 Vessels of neovascularization are more susceptible to leakage and contribute to blurred vision by allowing the escape of inflammatory cells. 4 After corneal vascularization is evident, it is difficult to manage HSK lesions, and patients need to be treated with topical steroids for a long period. If steroid treatment is kept short because of the side effects, then HSK recurrences occur, causing an increase in the overall HSK recurrence rate. 5 Thus, new alternative therapies are needed for the treatment of HSK. Currently, much research reveals that alternatives to the steroid treatment of HSK, the use of nonsteroid anti-inflammatory drugs, implantation of amniotic membrane, and cyclosporine A were tested. 6–11 Topical cyclosporine application was also shown to be effective in HSK treatment, and its effect is concentration dependent. 10 Tacrolimus, a potent immunosuppressive macrolide isolated from the soil fungus Streptomyces tsukubaensi, acts primarily by suppressing both B-cell and T-cell activation, T helper cell– mediated responses, and the production of interleukin (IL)-2, such as cyclosporine A. As a result, both drugs inhibit the acti- vation and proliferation of inflammatory cells. 12,13 Both topical cyclosporine A and tacrolimus have effectively been used in treatments of ocular surface diseases, such as superior limbic keratoconjunctivitis, atopic keratoconjunctivitis, and penetrating keratoplasty. 14–18 Although it is 100 times more effective than cyclosporine, no significant systemic side effect was reported for the treatment by topical tacrolimus. 19 To the best of our knowledge, this is the first prospective study on the use of tacrolimus on HSK. In this study, we evaluated the effectiveness of topical tacrolimus on HSK in a rat model. We used immunohistochemistry (IHC), flow cytometry, and clinical approaches to evaluate the effects of different doses of topical tacrolimus. MATERIALS AND METHODS Animals Seventy-two Wistar albino rats weighing 250 to 350 g and aging 6 to 8 weeks were used in this study. The animal studies were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the research was approved by the Ethics Committee of Kocaeli University Medical Faculty. Virus Production Herpes simplex virus type 1 human strain stock was kindly provided by Ayhan Kubar, Professor of Medical Microbiology at Gulhane Military Medical Academy. From the Departments of Ophthalmology (E.E., N.Y., D.P., M.A.), Medical Microbiology (A.K., H.U.), Stem Cell (G.G., G.E., C.S., E.K.), and Pathology (D.K.Y.), Faculty of Medicine, Kocaeli University, Kocaeli, Turkey. The authors have no funding or conflicts of interest to disclose. Address correspondence to Dilara Pirhan, M.D., Department of Oph- thalmology, Faculty of Medicine, Kocaeli University, Kocaeli 41380, Turkey; e-mail: dpirhan@yahoo.com.tr Accepted March 29, 2015. DOI: 10.1097/ICL.0000000000000162 Eye & Contact Lens  Volume 42, Number 3, May 2016 163 Copyright @ Contact Lens Association of Opthalmologists, Inc. Unauthorized reproduction of this article is prohibited.