Current Eye Research, Early Online, 1–10, 2014 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2014.914540 ORIGINAL ARTICLE Effect of Melatonin and Analogues on Corneal Wound Healing: Involvement of Mt 2 Melatonin Receptor Almudena Crooke 1 , Ana Guzman-Aranguez 1 , Aranzazu Mediero 1 , Pilar Alarma-Estrany 1 , Gonzalo Carracedo 2 , Teresa Pelaez 1 , Assumpta Peral 2 and Jesu ´ s Pintor 1 1 Departamento de Bioquı ´mica y Biologı ´a Molecular IV, Facultad de O ´ ptica y Optometrı ´a, Universidad Complutense de Madrid, Madrid, Spain, and , 2 Departamento de O ´ ptica II, Facultad de O ´ ptica y Optometrı ´a, Universidad Complutense de Madrid, Madrid, Spain ABSTRACT Purpose: We have investigated the effect of melatonin and its analogues on rabbit corneal epithelial wound healing. Methods: New Zealand rabbits were anaesthetised and wounds were made by placing Whatman paper discs soaked in n-heptanol on the cornea. Melatonin and analogues (all 10 nmol) were instilled. Wound diameter was measured every 2 hours by means of fluorescein application with a Topcon SL-8Z slit lamp. Melatonin antagonists (all 10 nmol) were applied 2 hours before the application of the n-heptanol-soaked disc and then every 6 hours together with melatonin. To confirm the presence of MT 2 receptors in corneal epithelial cells immunohistochemistry, Western blot and RT-PCR assays in native tissue and in rabbit corneal epithelial cells were performed. The tear components were extracted then processed by HPLC to quantify melatonin in tears. Results: Migration assays revealed that melatonin and particularly the treatment with the MT 2 agonist IIK7, accelerated the rate of healing (p50.001). The application of the non-selective melatonin receptor antagonist luzindole and the MT 2 antagonist DH97 (but not prazosin), prevented the effect of melatonin on wound healing (both p50.001). Immunohistochemistry, Western blot and RT-PCR assays showed the presence of MT 2 melatonin receptor in corneal epithelial cells. In addition, we have identified melatonin in tears and determined its daily variations. Conclusions: These data suggest that MT 2 receptors are implicated in the effect of melatonin on corneal wound healing regulating migration rate. This suggests the potential use of melatonin and its analogues to enhance epithelial wound healing in ocular surface disease. Keywords: Corneal wound healing, melatonin, melatonin agonists, melatonin antagonists, melatonin receptors INTRODUCTION Melatonin is an indolamine produced by the pineal gland; it participates in many functions including the coordination of circadian rhythms. 1–5 Some functions of melatonin are mediated by stimulating MT 1 and MT 2 receptors as well as the putative MT 3 receptor 6–8 , while others may involve nuclear binding sites 9,10 or being receptor independent. 11,12 Although melatonin is most widely known as a product of the pineal gland 13 , melatonin synthesis also occurs in extra- pineal sites such as the eye. 14,15 Melatonin is synthe- sized by retinal photoreceptors and ciliary epithelial cells 16,17 and it modulates a variety of physiological processes in the eye. Particularly, in retina, melatonin modulates the photoreceptor outer segment shedding rate 18 , horizontal cell sensitivity to light 19 , and dopa- mine release. 20 In the anterior chamber, melatonin Correspondence: Jesu ´s Pintor, Departamento de Bioquı ´mica y Biologı ´a Molecular IV, Facultad de O ´ ptica y Optometrı ´a, Universidad Complutense de Madrid, C/Arcos de Jalo ´n 118, 28037 Madrid, Spain. Tel: +34-91-3946859. Fax:+34-91-3946885. E-mail: jpintor@vet.ucm.es Received 2 December 2013; revised 13 March 2014; accepted 02 April 2014; published online 14 May 2014 1 Curr Eye Res Downloaded from informahealthcare.com by 83.53.32.37 on 06/03/14 For personal use only.