Epilepsia, 48(4):743–751, 2007 Blackwell Publishing, Inc. C  2007 International League Against Epilepsy Expression of Adhesion Factors Induced by Epileptiform Activity in the Endothelium of the Isolated Guinea Pig Brain In Vitro ∗ Laura Librizzi, ∗ Maria Cristina Regondi, ∗ ‡Chiara Pastori, †Simona Frigerio, ∗ Carolina Frassoni, and ∗ Marco de Curtis ∗ Department of Clinical Epileptology and Experimental Neurophysiology, †Laboratory of Neurobiology and Neurodegenerative Therapies, Istituto Nazionale Neurologico, Milan, Italy; ‡Neurology Residency School University of Milano-Bicocca, Monza, Italy Summary: Purpose: Brain inflammation has been recently considered in the pathogenesis of focal epilepsies. Synthesis of pro-inflammatory mediators in the brain was described both in experimental models of seizures and in human postsurgical tissue. Inflammatory mediators may up-regulate endothelial ad- hesion molecules, therefore promoting adhesion and homing of leucocytes into the brain. In the present study, expression of inducible adhesion factors in brain endothelium was verified af- ter pharmacological induction of seizure-like activity in specific brain areas of the in vitro isolated guinea pig brain. Methods: Experiments were performed in isolated guinea-pig brains maintained in vitro by arterial perfusion. In this prepara- tion, brief application of the GABAa receptor–antagonist, bicu- culline, consistently induced focal ictal discharges in the limbic region that secondarily diffuse to the neocortex, as verified by simultaneous electrophysiological recording of extracellular ac- tivity. At the end of the electrophysiological experiment (after 5 h in vitro), brains were fixed and immunostaining for adhe- sion molecules P-selectin and ICAM-1 and for Fos protein was evaluated. Results: Immunohistochemical analysis of isolated brains in which seizure-like activity was induced revealed expression of inducible adhesion factors P-selectin and ICAM-1 in the en- dothelium of small–medium size brain vessels. In particular, the expression of these molecules was consistently observed in all areas involved in epileptic seizure-like ictal activity (limbic cor- tices and neocortex), and was infrequently found in regions that generated interictal spiking (piriform cortex), suggesting a trig- ger role played by seizures for endothelial activation. An increase in Fos protein expression was evident in all analyzed limbic ar- eas and in the neocortex, indicating a correlation between the areas of neuronal and endothelial activation. In control brains maintained in vitro for comparable times without induction of epileptiform activity, no immunoreactivity for Fos and adhesion molecules was observed. Conclusions: Seizure-like activity in an in vitro isolated brain preparation induces the expression of adhesion molecules in the cerebral endothelium. These observations indicate that local en- dothelial activation may represent a crucial step for the devel- opment of an inflammatory response induced by seizures, and suggest a possible novel pathogenic mechanism during the pro- cess of epileptogenesis. Key Words: Fos—ICAM-1—Isolated guinea pig brain in vitro—P-selectin—Seizures. Increasing evidence strongly suggests that an inflam- matory reaction occurs in the brain during the estab- lishment of an epileptic condition. Synthesis of pro- inflammatory cytokines interleukin-1β (IL-1β ) and tumor necrosis factor α (TNFα) (Minami et al., 1991; Vezzani et al., 2002; Vezzani and Granata, 2005) alterations in gene expression (Lynch et al., 1996) and blood–brain bar- rier (BBB) dysfunctions (Zucker et al., 1983; Cornford, 1999) can be triggered in the central nervous system by seizures (Vezzani and Granata, 2005). Cytokine synthesis Accepted January 4, 2007. Address correspondence and reprint requests to Marco de Curtis, De- partment of Clinical Epileptology and Experimental Neurophysiology, Istituto Nazionale Neurologico, via Celoria 11, 20133 Milano, Italy, E- mail: decurtis@istituto-besta.it doi: 10.1111/j.1528-1167.2007.01047.x in the brain was detected mainly in microglia and astro- cytes (Vezzani et al, 1999), although it was shown that a subset of hippocampal neurons could also express IL-1 beta in epileptic rats (De Simoni et al., 2000). More- over, high IL-6 levels were found in plasma and in the cerebrospinal fluid of epileptic patients after tonic–clonic seizures (Peltola et al.,1998, 2002), and increased neu- ronal and glial expression of pro-inflammatory mediators was demonstrated in brain specimens obtained from pa- tients surgically treated for drug-resistant epilepsy (Cre- spel et al., 2002; Maldonado et al., 2003). Synthesis and release of cytokines both contribute to cause transient changes in the functional and struc- tural features of the BBB, that lead to an impairment of its integrity (Zucker et al., 1983). In different ex- perimental models of BBB and of cerebral diseases, 743