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Pathology type Number of Gender Mean age Localization Mean duration of cases (M/F) (years/range) epilepsy (years/
range)
mMCD 6 5/1 21.5 (19-27) 3 fr/3t 17.3 (11-32)
FCD IIa 5 3/2 34.2 (18-45) 4 fr/1t 22.4 (14-26)
FCD IIb 6 2/4 33 (21 –45) 4 fr/2t 24 (15-40)
Cortical Tubers (TSC) 6 3/3 7.1 (3-16) 4 fr/2t 5.8 (0.8 –13)
Controls/autopsy 14 8/6 27.0 (2-48) 6 fr/8t -
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obtained and used in accordance with the Declaration of Helsinki and the AMC Research Code provided by the Medical Ethics Committee and approved by the committee of the UMCU Biobank. This study was also approved by the Ethical Committee of the Medical University of Vienna. All cases were reviewed independently by two neuropathologists and the diagnosis of FCD was confirmed according to the international consensus clas- sification system recently proposed for grading FCD 21. All patients with cortical tubers fulfilled the diagnostic criteria for TSC 22. None of the FCD patients fulfilled the diag- nostic criteria for TSC. Table 1 summarizes the clinical findings of patients with MCD and epilepsy (6 mMCD, 5 FCD IIa, 6 FCD IIb, 6 TSC-tubers, 4 TSC2/2 TSC1; pre-operative seizure frequency/month, mean/SEM: mMCD: 19.8 ± 6.7; FCD II: 149 ± 68.7; TSC 114.8 ± 24.2); seizure frequencies were recorded (video-electroencephalographic monitoring) at the time of the preoperative evaluation. One tuber specimen was obtained post-mor- tem (age 32 years; male; TSC2). Hippocampal specimens from patients with Alzheimer’s disease (AD; n = 4; 3 females and 1 males; Braak stage V and VI, age: 81.7 ± 2.8) were also examined as positive controls. In addition, normal-appearing control cortex and white matter was obtained at autopsy from 6 young adult control patients (Table 1), without a history of seizures or other neurological diseases. All autopsies were performed within 24 hours after death.
Tissue preparation and immunohistochemistry
Brain tissue from control and MCD patients was fixed in 10% buffered formalin and embedded in paraffin. Paraffin-embedded tissue was sectioned at 5 μm, mounted on pre-coated glass slides (Star Frost, Waldemar Knittel GmbH, Braunschweig, Germany) and used for histology and immunohistochemistry. One representative paraffin block per case was sectioned, stained and assessed. Sections were processed for haematoxylin eosin stainings, as well as for immunohistochemical stainings for a number of neuronal and glial markers and antibodies against the constitutive (β1, β5) and immunoprotea- some (β1i, β5i) subunits (Table 2). These antibodies have been extensively tested on human liver and brain tissues 23, 24, including surgical brain specimens from patients with mesial temporal lobe epilepsy revealing bands at the expected molecular weight (17; sup- plementary Fig. 1). To detect differences in labeling related to technical variables such as tissue fixation, we also tested the antibodies in specimens of selected regions (tempo- ral cortex/hippocampus) collected at autopsy and immediately fixed in formalin for 24
Table 1 Summary of clinical findings of epilepsy patients and controls.
FCD: Focal Cortical Dysplasia; TSC: Tuberous Sclerosis Complex; mMCD: mild malformations of cortical development; M = male; F = female. fr: frontal; t: temporal.