Lymphocyte infiltration of neocortex and hippocampus after a single brief seizure in mice J. Silverberg a,e,g, * , D. Ginsburg a,f , R. Orman b , V. Amassian a,b,c , H.G. Durkin a,d,e,g , M. Stewart a,b,c a School of Graduate Studies, State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States b Department of Physiology & Pharmacology, State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States c Department of Neurology, State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States d Department of Pathology, State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States e Department of Medicine, State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States f Department of Anesthesiology, State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States g Center for Allergy and Asthma Research, State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States article info Article history: Received 9 July 2009 Received in revised form 16 September 2009 Accepted 3 October 2009 Available online 12 October 2009 Keywords: Epilepsy Seizure Blood–brain barrier Maximal electric stimulation MES Neuroimmunology Microglia CD4 CD8 T cells B cells abstract Various immune responses have been described in epileptic patients and animal models of epilepsy, but immune responses in brain after a single seizure are poorly understood. We studied immune responses in brain after a single brief generalized tonic–clonic seizure in mice. C57bl/6 mice, either unanesthetized or anesthetized (pentobarbital, ethyl chloride) received either electrical (15–30 mA, 100 Hz, 1 s) or sham stimulation (subcutaneous electrodes over frontal lobe, no current). Electrical stimulation of unanesthe- tized mice resulted in tonic–clonic convulsions with hind-limb extension (maximal seizure), tonic–clonic convulsions without hind-limb extension (submaximal seizure), or no seizure. In contrast, such stimula- tion of anesthetized mice did not result in seizure. Mice were killed at 1 h–7 days after seizure. Brains or regions dissected from brain (neocortex, hippocampus, midbrain, cerebellum) of each group were pooled, single cell suspensions prepared, and cells separated according to density. CD4 + (CD3 + CD45 Hi ) and CD8 + (CD3 + CD45 Hi ) T cell and CD45R + (CD45 Hi ) B cell numbers were determined by flow cytometry. At 24 h after a maximal seizure, CD4 + and CD8 + T cells and CD45R + B cells appeared in brain, reaching peak num- bers at 48 h, but were no longer detected at 7 days. CD4 + T cells and CD45R + B cells were preferentially found in neocortex compared with hippocampus, whereas CD8 + T cells were preferentially found in hip- pocampus at 24 h after a maximal seizure. In contrast, virtually no lymphocytes were detected in brains of unstimulated or sham stimulated mice, unanesthetized stimulated mice after submaximal or no sei- zure, and anesthetized stimulated mice at 1 h–7 day. Neither Ly6-G+ neutrophils nor erythrocytes were detected in brains of any animals, nor was there any detectable increase of blood–brain barrier perme- ability by uptake of Evans Blue dye. The results indicate that lymphocyte entry into brain after a single brief seizure is due to a selective process of recruitment into cortical regions. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction A wide array of brain insults, including trauma and infection, are associated with inflammatory responses in brain and develop- ment of seizure activity (Vezzani and Granata, 2005; Vezzani et al., 2008). Studies in animal models and patients have largely aimed at defining the cascade of events from brain injury to epilepsy. Severe, prolonged seizures in animals lead to specific patterns of neuronal death in cortical and subcortical areas of brain, followed by specific patterns of neuronal circuit reorganization that favor occurrences of future seizures (Engel et al., 2001). Various inflammatory re- sponses in brain, to both seizures per se and secondary cellular damage, have been identified (Biagas et al., 1992; Borges et al., 2003, 2004; Hirschberg et al., 1998; Sheibani et al., 2004; Stevens et al., 2002; Taniwaki, 2001; Vezzani et al., 1999). A portion of the brain inflammatory responses to a seizure involves resident brain cells, e.g., microglia and astrocytes (Borges et al., 2003; Tan- iwaki, 2001; Vezzani et al., 1999), whereas other aspects of the brain immune responses may involve infiltration of circulating leu- kocytes (Biagas et al., 1992; Borges et al., 2004; Hirschberg et al., 1998; Sheibani et al., 2004; Stevens et al., 2002). In both animal models of status epilepticus and patients with epi- lepsy, increased numbers of lymphocytes have been described in brain. In mice with pilocarpine-induced status epilepticus, lympho- cytes were detected in hippocampus and thalamus (Borges et al., 0889-1591/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbi.2009.10.006 * Corresponding author. Address: SUNY Downstate Medical Ctr., Department of Pathology, Box 25, 450 Clarkson Ave., Brooklyn, NY 11203, United States. Fax: +1 718 270 3289. E-mail address: JonathanISilverberg@gmail.com (J. Silverberg). Brain, Behavior, and Immunity 24 (2010) 263–272 Contents lists available at ScienceDirect Brain, Behavior, and Immunity journal homepage: www.elsevier.com/locate/ybrbi