The cases included in this study were obtained from the archives of the departments of neuropathology of the Academic Medical Center (University of Amsterdam), the University Medical Center in Utrecht, and the VU University Medical Center (VUMC) in Amsterdam. Twenty-seven brain tissue specimens, removed from patients undergoing surgery for intractable epilepsy, were examined. Informed consent was obtained for the use of brain tissue and for access to medical records for research purposes. All cases were reviewed independently by two neuropathologists, and the diagnosis was confirmed according to the system recently proposed by Palmini et al. for grading the degree of FCD (Palmini et al., 2004). Table S1 summarizes the clinical findings of epilepsy patients and controls. None of the patients with FCD fulfilled the diagnostic criteria for tuberous sclerosis complex (TSC). Table S2 summarizes the neuropathologic findings of FCD specimens and the standard stains used.
All patients underwent presurgical evaluation with phase I investigations consisting of noninvasive tests, history, medical, neurological and neuropsychological assessment, structural neuroimaging, and extensive interictal and ictal electroencephalography (EEG) studies with video monitoring. In phase II, an intracarotid sodium amytal test (Wada test), interictal positron emission tomography (PET), (inter)ictal single photon emission computed tomography (SPECT), and functional magnetic resonance imaging (fMRI) were performed on indication (van Veelen et al., 1990). Patients who underwent implantation of strip and/or grid electrodes for chronic subdural invasive monitoring before resection were excluded from this study. Patients had complex partial seizures (CPS), and all patients had daily seizures, which were resistant to maximum doses of antiepileptic drugs. Seizure duration represents the interval in years from age at seizure onset and age at surgery. The postoperative seizure outcome was classified according to Engel (1993).
Normal-appearing control cortex was obtained at autopsy from six age-matched patients without history of seizures or other neurologic diseases. Autopsied brain tissues from patients with neuroinflammatory pathologies [viral encephalitis and multiple sclerosis (MS)] were also examined (as positive controls). All autopsies were performed within 12 h after death. Histologically normal temporal neocortex (without evidence of significant neuronal loss, gliosis or malformation; epilepsy controls) from four patients undergoing extensive surgical resection of the mesial structures for the treatment of medically intractable complex partial epilepsy was also used for immunocytochemical analysis. This material represents good control tissue, since it is exposed to similar seizure activity, duration of epilepsy, and fixation protocol, and is also useful for investigating whether seizure activity itself triggers the inflammatory response.
Antibody characterization and immunocytochemistry
Antibodies (Abs) specific for glial fibrillary acidic protein (GFAP; polyclonal rabbit, DAKO, Glostrup, Denmark; 1:4,000), vimentin (mouse clone V9; DAKO; 1:1,000), neuronal nuclear protein (NeuN; mouse clone MAB377; Chemi-Con, Temecula, CA, U.S.A.; 1:2,000), neurofilament (SMI311; Sternberger Monoclonals, Lutherville, MD, U.S.A.; 1:1,000), cleaved caspase-3 (rabbit polyclonal, Cell Signaling Technology, Beverly, MA, U.S.A.; 1:100), and phospho-S6 ribosomal protein (Ser235/236; pS6, rabbit polyclonal, Cell Signaling Technology; 1:50) were used in the routine immunocytochemical analysis of FCD cortical specimens to document the presence of a heterogeneous population of cells, apoptosis, and activation of the mTOR pathway (Baybis et al., 2004).
For the detection of the inflammatory cells and proinflammatory pathways the following Abs were used: anti-human leukocyte antigen (HLA)-DP, DQ, DR (HLA-DR; mouse clone CR3/43; DAKO, Glostrup, Denmark; 1:400), anti-CD68 (mouse monoclonal, clone PG-M1; DAKO; 1:200, monocytes, macrophages, microglia), anti-CD3 (mouse monoclonal, clone F7.2.38; DAKO; 1:200, T lymphocytes), anti-CD4 (mouse monoclonal, 4B12; Neomarkers; 1:100, helper/inducer T-lymphocyte subset), anti-CD8 (mouse monoclonal, clone C8/144B; DAKO; 1:100; cytotoxic/suppressor T-lymphocyte subset), anti-CD20 (mouse monoclonal, clone L26; DAKO; 1:400, B lymphocytes), DC-SIGN (CD209; monoclonal mouse, BD Pharmingen, San Diego, CA, U.S.A.), MCP1 (MCP1/CCL2; monoclonal mouse, R&D Systems, Minneapolis, MN, U.S.A.; 1:10), interleukin (IL)-1β [goat polyclonal, sc-1250, Santa Cruz Bio., CA, U.S.A.; 1:70, (Ravizza et al., 2006)], anti-C1q and anti-C3d [rabbit polyclonal, DAKO, Glostrup, Denmark; C1q, 1:100; C3c, C3d, 1:200, (Aronica et al., 2007)].
Immunocytochemistry was carried out as described previously (Aronica et al., 2003). Single-label immunocytochemistry was performed using the Powervision kit (Immunologic, Duiven, The Netherlands) and 3,3-diaminobenzidine as chromogen. Sections were counterstained with hematoxylin. Sections incubated without the primary Ab, with preimmune serum, or with the primary Ab (for IL-1β and MCP1) and an excess of the antigenic peptide were essentially blank. A similar pattern of immunoreactivity was observed in surgical and autopsy control specimens included in this study.
For double-label immunocytochemistry with DC-SIGN (IgG2b) and CD3 (IgG1), we used secondary Ig subtype specific Abs and as chromogens 3-amino-9-ethyl carbazole (AEC, Sigma, St. Louis, MO, U.S.A.) and Fast Blue B salt (Sigma). For double-labeling with HLA-DR and pS6 (as well as for caspase-3 with GFAP, HLA-DR, or NeuN, not shown), we used, as secondary antibodies, Alexa Fluor 568-conjugated anti-rabbit IgG and Alexa Fluor 488 anti-mouse IgG (1:100; Molecular Probes, Leiden, The Netherlands). Sections were mounted with Vectashield containing DAPI (targeting DNA in the cell nucleus; blue emission) and analyzed by means of a laser scanning confocal microscope (Leica TCS SP2; Wetzlar, Germany).