Department of Anatomy, Faculty of Medicine, University of Gazi, Ankara, Turkey Dose-Dependent Immunohistochemical Changes in Rat Cornea and Retina after Oral Methylphenidate Administration E. Tunc 1  , D. Erdogan 2 , R. Gozil 3  , M. Bahcelioglu 3*  , E. Calguner 3 , G. Take 2 , G. Caglar 2 and H. Oktem 3 Addresses of authors: 1 Department of Anatomy, Faculty of Medicine, University of Ufuk, Balgat, 06520, Ankara, Turkey; 2 Department of Histology and Embryology; 3 Department of Anatomy, Faculty of Medicine, University of Gazi, Besevler, 06500, Ankara, Turkey; *Corresponding author: Tel.: 90 312 202 4618; fax: 90 312 212 4647; e-mail: meltemb@gazi.edu.tr With 2 figures and 3 tables Received March 2008; accepted for publication June 2008 Summary Methylphenidate hydrochloride (MPH), more commonly known as Ritalin, is a piperidine derivative and is the drug most often used to treat attention deficit/hyperactivity dis- order, one of the most common behavioural disorders of children and young adults. The aim of this study was to investigate dose-dependent immunohistochemical Dopamine 2 receptor (D2) expression and apoptosis in the rat cornea and cornea. In this study, 27 female pre-pubertal Wistar albino rats, divided into three different dose groups (5, 10 and 20 mg/kg) and their control groups, were used. They were treated orally with methylphenidate dissolved in saline solution for 5 days per week during 3 months. At the end of the third month, after perfusion fixation, eye tissue was removed. Paraffin sections were collected for immunohisto- chemical and terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labelling assay studies. In our study, we observed that the cornea D2 receptor reactivity showed a dose-related increase after MPH treatment, especially in basal cells of the epithelium and a dose-dependent decrease in the retinal ganglion cell which was statistically meaning- ful. Analysis of the cornea thickness results showed no meaningful difference between groups. Apoptotic cell num- ber showed a meaningful increase in the high dose treated group compared to the other groups of the study. The data suggest that Ritalin has degenerative effect on the important functional part of the eye, such as cornea and retina and its activating dopaminergic mechanism via similar neuronal paths, functionally and structurally, to induce morpholo- gical changes. As a result, we believe that this morphological changes negatively effecting functional organization of the affected cornea and retina. Introduction Attention-deficit/hyperactivity disorder (ADHD), the most common behavioural disorder of childhood, is heterogeneous: symptoms of impulsivity, hyperactivity and inattention are expressed with various degrees of severity (American Psychi- atric Association, 1994). Methylphenidate hydrochloride (Ritalin) (MPH) is a psychostimulant that is widely used in the treatment of ADHD in adolescents and adults and is also diverted for recreational purposes (Kollins et al., 2001; Swanson and Volkow, 2003). This psychostimulant binds and inhibits the dopamine transporter (Schweri et al., 1985; Gatley et al., 1996) and norepinephrine reuptake (Kuczenski and Segal, 1997). Monoamine receptors are membrane-bound receptors located throughout the body in neuronal and non-neuronal tissues where they mediate a diverse range of responses to the endogenous catecholamines epinephrine, norepinephrine, dopamine and serotonin (Grueb et al., 2006). Dopaminergic receptors are members of the same superfamily which can be divided into two subfamilies (D1-like receptors –D1, D5-, D2-like receptors D2, D3, D4) the properties of which are defined pharmacologically and biochemically (Strange, 1992). Cavallotti et al., 1999 showed D1-like and D2-like dopamine receptors in sections of the rabbit cornea suggesting their possible role in the control of corneal functions, whereas other researchers investigated their role on retina (Reis et al., 2007). The use of the Ritalin for the treatment of attention deficit/ hyperactivity disorder has increased dramatically in recent years since its first use in 1950s (El-Zein et al., 2005). Since then, only few studies have been conducted on the potential for serious side effects, such as mutagenicity and carcinogenicity, in animals or in humans (El-Zein et al., 2005). The expanded use of methylphenidate stimulates the need to expend research studies on the potential toxicity of this drug. Our aim was to investigate dose-dependent immunohisto- chemical D2 expression and apoptosis in the rat cornea and retina, to demonstrate possible toxicity of the long-term and high-dose use of the methylphenidate. Materials and Methods Animals and treatment The experimental protocol was approved by the local Ethical Committee for animal studies and conducted at Gazi University Faculty of Medicine. In the experimental protocol, twenty-seven female Wistar albino rats with a weight of 110 g (±20), divided into three different dose groups (5, 10 and 20 mg/kg) and their control groups, were used. Pre-pubertal (35 day-old) rats, as indicated in the literature, were treated orally with MPH dissolved in saline solution for 5 days per week for 3 months. The animals were synchronized to a light-dark cycle (lights on from 08:00 h to 20:00 h) beginning   The authors equally contributed to this work Ó 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag GmbH Anat. Histol. Embryol. 38, 128–132 (2009) doi: 10.1111/j.1439-0264.2008.00909.x ISSN 0340–2096