THYMULIN REVERSES INFLAMMATORY HYPERALGESIA AND MODULATES THE INCREASED CONCENTRATION OF PROINFLAMMATORY CYTOKINES INDUCED BY I.C.V. ENDOTOXIN INJECTION B. SAFIEH-GARABEDIAN, a * C. I. OCHOA-CHAAR, b S. POOLE, c C. A. MASSAAD, b S. F. ATWEH, b S. J. JABBUR b AND N. E. SAADE ´ b a Department of Biology, Faculty of Arts and Sciences, American Uni- versity of Beirut, Riad El Solh, Beirut 1107-2020, Lebanon b Neuroscience Program, Faculty of Medicine, American University of Beirut, Beirut, Lebanon c Division of Endocrinology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK Abstract—The immunomodulatory thymic hormone thymulin has been shown previously to possess anti-inflammatory actions in the periphery. In this study, we have examined the effect of i.c.v. injections of either endotoxin (ET) or thymulin, in separate groups of conscious rats, on pain-related behav- ior and cytokine levels in different areas of the brain. Further- more, we investigated the effect of pretreatment with either i.c.v. or i.p. injections of thymulin on endotoxin-induced hy- peralgesia and the effect of pretreatment with i.c.v. thymulin on endotoxin-induced up-regulation of cytokine levels. Our results demonstrate that i.c.v. injection of endotoxin (1 g in 5 l saline) resulted in a significant decrease in the nocicep- tive thresholds as assessed by different pain tests, with peak hyperalgesia at 3h. However, thymulin at different doses, when injected (i.c.v.), had no significant effect on pain related behavior. Pretreatment (i.c.v.) with thymulin (0.1, 0.5 and 1 g in 5 l saline) 20 min before endotoxin (i.c.v.) injection (1 g in 5 l saline) reduced, in a dose dependent manner, the endotoxin-induced hyperalgesia and exerted differential ef- fects on the up-regulated levels of cytokines in different ar- eas of the brain. The results provide behavioral and immu- nochemical characterization of a rat model for intracerebral inflammation and indicates a neuroprotective role for thymu- lin in the CNS. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: brain, neuroinflammation, hyperalgesia, cytokines, endotoxin. Ample evidence implicates inflammatory processes in the development of a number of neurodegenerative diseases and demonstrates that neurons and microglia can serve as targets and/or sources for various cytokines, which are believed to be involved in the neuropathology (Spranger and Fontana, 1996; Lemke et al., 1999; Schoneboom et al., 2000). An important aspect of brain injury is its asso- ciation with increased production of cytokines (Selmaj and Raine, 1988; Balasingam et al., 1994; Rothwell and Lu- heshi, 2000). Moreover, it is now established that many of these inflammatory molecules, commonly associated with the peripheral immune system, are also produced within the CNS (Quan et al., 1998; Konsman et al., 2002). The pro-inflammatory cytokines, including interleukin 1 (IL- 1), tumor necrosis factor  (TNF-) and IL-6, have been shown to play important roles in the development of sick- ness behavior and the ensuing hyperalgesia (Dantzer, 2001; Poole et al., 2000). As illustration, endotoxin-in- duced hyperalgesia is associated with increases in the levels of central and peripheral IL-1, TNF- and IL-6 (Safieh-Garabedian et al., 1997; Nadeau and Rivest, 2000). Furthermore, i.c.v. administration of these mole- cules has been found to elicit hyperalgesia (Oka et al., 1993; and for review, see Hori et al., 2000). Despite diffi- culties in defining precisely the physiological and behav- ioral signs of CNS inflammation, increasing evidence indi- cates that many pathological states are associated with pain-related behavior (Tasker et al., 1991; Boivie, 1994). Considerable research is currently directed on targeting proinflammatory mediators like cytokines and the tran- scription factors responsible for their expression, as a pos- sible novel therapeutic approach for inflammation-associ- ated neurodegeneration (Dinarello et al., 1993; Huber et al., 2000). Thymulin is a peptide hormone derived from thymic epithelial cells and is mainly involved in various immune functions (Safieh-Garabedian et al., 1992). Besides its immunomodulating role, several recent reports have indi- cated that thymulin is capable of interacting directly and/or indirectly with the nervous system (for review see Safieh- Garabedian et al., 1999). Systemic injection of this hor- mone has been shown, in high concentrations, to reduce the hyperalgesia and the up-regulated cytokines following endotoxin (ET) injection (Safieh-Garabedian et al., 1998), whereas in low concentrations, injected on its own, it in- creases the levels of these molecules and results in hy- peralgesia (Safieh-Garabedian et al., 2000). This study has two aims: first, to characterize the effect of i.c.v. injection of ET in awake rats by assessing its nociceptive effect using the paw withdrawal (PW) and the hotplate (HP) tests and to determine the effects of pretreat- ment with thymulin on the observed hyperalgesia; second, *Corresponding author. Tel: +96-1-1-350-000x3896; fax: +96-1-1- 744-461. E-mail address: bared@aub.edu.lb (B. Safieh-Garabedian). Abbreviations: CSF, cerebrospinal fluid; EAE, experimental allergic encephalomyelitis; ET, endotoxin; HP, hot plate; IL, interleukin; PW, paw withdrawal; TNF, tumor necrosis factor. Neuroscience 121 (2003) 865– 873 0306-4522/03$30.00+0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00500-1 865