may be key targets in the regulation of this miRNA in response to inflammatory molecules [19,20]. In the present study we evaluated the role of miR-146a in human astrocytes. We first assessed the expression levels of miR- 146a in epilepsy-associated glioneuronal lesions (ganglioglioma, GG; focal cortical dysplasia, FCD type IIb) characterized by prominent activation of the innate immune response in astroglial cells [3,23–25] and analyzed the cellular distribution in FCD type IIb. Furthermore, we investigated the regulation of miR-146a in response to inflammatory molecules in cultured human glial cells (U373 glioblastoma cell line and human astrocytes cell cultures) and further evaluated its role in regulating TLR/IL-1R-in- terleukin-1 receptor-associated kinase (IRAK)-NF-kB signaling, particularly in astroglia. Materials and Methods Human Material The cases included in this study were obtained from the archives of the departments of neuropathology of the Academic Medical Center (University of Amsterdam) and the University Medical Center in Utrecht. A total of 6 brain tissue specimens, removed from patients undergoing surgery for intractable epilepsy, were examined. Informed written consent was obtained for the use of brain tissue and for access to medical records for research purposes. Tissue was obtained in accordance with the Declaration of Helsinki and the AMC Research Code provided by the Medical Ethics Committee of the AMC. All cases were reviewed independently by two neuropathologists and the diagnosis was confirmed according to the international consensus classification system recently proposed for grading FCD [23]. For the GG we used the revised WHO classification of tumors of the central nervous system [26]. Table S1 summarizes the clinical findings of epilepsy patients and controls. None of the FCD patients fulfilled the diagnostic criteria for tuberous sclerosis complex (TSC). All patients underwent presurgical evaluation with investigations consisting of non-invasive tests, including history, medical, neurological and neuropsychological assessment, structural neu- roimaging and extensive interictal and ictal electroencephalo- graphic (EEG) studies with video monitoring. 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 maximal doses of anti-epileptic drugs. Seizure duration represents the interval in years from age at seizure onset and age at surgery. Normal- appearing control cortex was obtained at autopsy from 6 age- matched patients without history of seizures or other neurological diseases. Autopsied brain tissues from patients with neuro- inflammatory pathologies (viral encephalitis; herpes simplex encephalitis and [27] and rabies encephalitis [28]) were also examined as positive controls). All autopsies were performed within 12 hours after death. Furthermore, we also used histologically normal temporal neocortex from patients undergo- ing extensive surgical resection of the mesial structures for the treatment of medically intractable complex partial epilepsy to control for a possible effect of recurrent seizures, antiepileptic drugs or post mortem interval on the expression of miR146a. Tissue Preparation Brain tissue from control patients and surgical tissue block from patients with epilepsy was snap frozen in liquid nitrogen and stored at 280uC until further use (RNA isolation for RT-PCR). Additional tissue was fixed in 10% buffered formalin and miR-146a and Astrocyte Inflammatory Response embedded in paraffin. Paraffin-embedded tissue was sectioned at 6 mm, mounted on pre-coated glass slides (Star Frost, Waldemar Knittel GmbH, Brunschweig, Germany) and two sections were used for in situ hybridizations and immunocytochemistry. Two additional sections were used for the double staining, combining in situ hybridization with immunocytochemistry (in the same sections) with different antibodies, as described below. One representative paraffin block per case (containing the complete lesion or the largest part of the lesion resected at surgery) were sectioned, stained and assessed. Sections of all specimens were processed for haematoxylin eosin (HE), luxol fast blue (LFB) and Nissl stains, as well as for immunocytochemical stainings for a number of neuronal and glial markers to confirm the diagnosis of FCD IIb. In situ Hybridization In situ hybridization for miR-146a was performed using a 59 fluorescein labeled 19 mer antisense oligonucleotide containing Locked Nucleic Acid (LNA) and 29OME RNA moieties (FAM - AacCcaTggAauTcaGuuCucA, capitals indicate LNA, lower case indicates 29OME RNA). The oligo’s were synthesized by Ribotask ApS, Odense, Denmark. The hybridizations were done on 6 mm sections of paraffin embedded materials described previously [29]. The hybridization signal was detected using a rabbit polyclonal anti-fluorescein/Oregon green antibody (A21253, Molecular probes, Invitrogen) and a horse radish peroxidase labeled goat anti-rabbit polyclonal antibody (P0448 Dako, Glostrup Denmark) as secondary antibody. Signal was detected with chromogens 3- amino-9-ethyl carbazole (St. Louis, MO, USA) or Vector NovaRed (Vector Laboratories, Burlingame, CA, USA) and the nuclei were stained with haematoxylin. Slides were sealed with glycerol-gelatin (St. Louis, MO, USA). Sections without probe or with scramble probes were blank. For the double staining, combining immunocytochemistry with in situ hybridization, sections were first processed for immunocytochemistry as pre- viously described [24] with GFAP (glial fibrillary acidic protein; polyclonal rabbit, DAKO, Glostrup, Denmark; 1:4000) or HLA- DR (anti-human leukocyte antigen (HLA)-DP, DQ, DR (mouse clone CR3/43; DAKO, Glostrup, Denmark; 1:400) using Fast Blue B salt (St. Louis, MO, USA) or Vector Blue substrate (Vector Laboratories, Burlingame, CA, USA) as chromogen. After washing, sections were processed for in situ hybridization as described above. Images were captured with an Olympus microscope (BX41, Tokyo, Japan) equipped with a digital camera (DFC500, Leica Microsystems-Switzerland Ltd., Heerbrugg, Switzerland). RNA Isolation For RNA isolation, 800 ml Trizol LS Reagent (Invitrogen, Carlsbad, CA, USA) was added to 0.1–0.56106 cells. After addition of 200 mg glycogen and 200 ml chloroform, the aqueous phase was isolated using Phase Lock tubes (5 Prime GmBH, Hamburg, Germany). RNA was precipitated with isopropyl alcohol, washed with 75% ethanol and dissolved in water. The concentration and purity of RNA were determined at 260/ 280 nm using a nanodrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Real-time Quantitative PCR Analysis (qPCR) Five micrograms of total RNA were reverse-transcribed into cDNA using oligo dT primers. Five nmol oligo dT primers were annealed to 5 mg total RNA in a total volume of 25 ml, by incubation at 72uC for 10 min, and cooled to 4uC. Reverse transcription was performed by the addition of 25 ml RT-mix, PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e44789