Introduction 58 81 10 16 19 10 22 25 53 46 81 1 36 29 63 68 69 70 50 81 81 10 13 45 VEGFB 56 73 Materials and methods Subjects The cases included in this study were obtained from the databases of the Departments of Neuropathology of the Academic Medical Center (University of Amsterdam; UVA) in Amsterdam and the University Medical Center in Utrecht (UMCU). We examined surgically resected tissue from nine patients undergoing epilepsy surgery for focal cortical dysplasia. Informed consent was obtained for the use of brain tissue and for access to medical records for research purposes. The tissue was obtained and used in a manner compliant with the Declaration of Helsinki. 48 1 74 17 Table 1 Summary of clinical findings of patients with focal cortical dysplasia Patient/sex/age (years) Diagnosis Duration of epilepsy (years) Seizure type Engel class 1/M/11 FCD IIB 11 CPS I 2/M/31 FCD IIB 20 CPS I 3/F/25 FCD IIB 9 CPS I 4/F/22 FCD IIB 21 CPS/SGS I 5/M/18 FCD IIB 14 CPS I 6/M/17 FCD IIB 10 CPS I 7/F/16 FCD IIB 11 CPS I 8/M/29 FCD IIB 21 CPS I 9/M/28 FCD IIB 21 CPS I FCD CPS SGS Normal-appearing control cortex/white matter from temporal region was obtained at autopsy from five adult control patients (male/female: 2/3; mean age 42, range 17–55) without history of neurological diseases. All autopsies were performed within 12 h after death (post mortem delay: 11, 11.5, 9, 8.5, 6). The cause of death was represented by acute myocardial infarction. In addition, four of the nine FCD cases contained sufficient amount of perilesional zone (normal-appearing cortex/white matter adjacent to the lesion), for comparison with the autopsy specimens. This material represents good disease control tissue, since it is exposed to the same seizure activity, drugs, fixation time, and the age and gender are also the same. Tissue preparation Tissue was fixed in 10% buffered formalin and embedded in paraffin. Two representative paraffin blocks per case (containing the complete lesion or the largest part of the lesion resected at surgery) were sectioned, stained, and assessed. Paraffin-embedded tissue was sectioned at 6 μm, mounted on organosilane-coated slides (Sigma, St Louis, MO) and used for histological and immunocytochemical reactions as described below. Frozen tissue from control cortex and FCD tissue, stored at −80°C, was used for western blot analysis. Antibody characterization To document the presence of a heterogeneous population of cells, we used the following antibodies: glial fibrillary acidic protein (GFAP; polyclonal rabbit, DAKO, Glostrup, Denmark; 1:4,000; monoclonal mouse, DAKO; 1:50), vimentin (mouse clone V9, DAKO; 1:1,000), MAP2 (polyclonal rabbit; Chemicon; 1:500), neuronal nuclear protein (NeuN; mouse clone MAB377, Chemicon, Temecula, CA, USA; 1:2,000), non-phosphorylated neurofilament (SMI311; Sternberger monoclonals, Lutherville, MD; 1:1,000), human leukocyte antigen (HLA)-DP, -DQ, -DR (CR3/43; monoclonal mouse, DAKO; 1:400), CD68 (mouse clone PG-M1, DAKO; 1:200) and CD31 (mouse clone JC70A, DAKO; 1:100). 51 n n 49 64 80 1 56 Fig. 1 Representative immunoblot of VEGFA, VEGFB, VEGFR-1, and VEGFR-2 in total homogenates from control cortex and FCD tissue. Expression of β-actin (as reference protein) is shown in the same protein extracts  n n 65 8 Immunocytochemistry 2 2  ® ® Evaluation of immunostaining Semi-quantitative evaluation of immunoreactivity 3 54 2 2 2 3 54 Table 2 VEGFA, VEGFB, VEGFR-1, and VEGFR-2 distribution in different cellular types in cases of FCD (% of cases with immunoreactive cells)  n Neurons Astrocytes Balloon cells − + ++ − + ++ − + ++ VEGFA 0 22% 78% 0 11% 89% 0 11% 89% VEGFB 0 33% 67% 78% 22% 0 78% 22% 0 VEGFR-1 0 55% 45% 0 22% 78% 0 33% 67% VEGFR-2 0 11% 89% 55% 45%  0 22%  45% 33% FCD − + ++ Frequency of cell staining 4 23 54 P Results Human material and histological features 1 48 6 Expression of VEGF and VEGFR in normal temporal cortex and FCD Cellular distribution of VEGFA 2 2 Fig. 2 a b inset c d arrows e f g h i arrows i j l green j red k yellow l m o green m red n yellow o p green red Scale bar a  2 2 6 2 2 6 2 Cellular distribution of VEGFB 3 Fig. 3 a inset aI inset aII b c arrows d arrows e g green e red f g h j green h red i yellow j k green red Scale bar a  3 2 6 3 3 2 6 3 3 Cellular distribution of VEGFR-1 4 4 Fig. 4 a b a b a b arrowheads c d c arrows d arrows Inset c green red Inset d green red e g arrows arrowheads e h j green h red i j k m green k red l m Scale bar a  4 2 6 4 4 4 4 Cellular distribution of VEGFR-2 5 5 Fig. 5 a b a b a b c d arrows c arrowheads d e f arrow e arrow f arrowheads e f Inset e green red g i green g red h i Scale bar a  5 2 6 5 2 2 5 Fig. 6 Materials and methods a e b f c g d h a d e h P  Discussion 13 44 56 13 26 33 69 76 Expression of VEGFA and VEGFB in normal temporal cortex 39 41 42 72 8 78 24 36 42 42 8 36 78 Differential cellular distribution of VEGFA and VEGFB in FCD 8 27 59 78 79 5 7 48 12 13 44 56 56 56 21 38 61 44 31 HIF1α VEGFA 2 3 15 54 77 14 35 76 77 71 11 42 52 60 55 56 75 13 47 9 28 32 67 18 43 62 36 68 69 This is an observational study and we were, therefore, not able to investigate the spatio-temporal regulation of the VEGF system. Further research in animal models of cortical dysplasia is clearly needed to elucidate the role of VEGFs and their signaling pathways in the histogenesis or epileptogenesis of developmental disorders. Expression of VEGF receptors in normal temporal cortex 8 11 78 79 Differential cellular distribution of VEGFR-1 and VEGFR-2 in FCD 8 11 37 39 66 79 56 56 8 30 52 8 34 57 40 45 6 11 20 43 67 43 13 44 56 Conclusions Our observed cellular distribution of VEGFA, VEGFB, and their signaling receptors indicate that different cellular components of FCD are involved in VEGF-signaling. In this context, future studies, using both in vivo and in vitro models, will be important to achieve a better understanding of the role of the VEGF-mediated pathways in the histogenesis and epileptogenesis of developmental lesions associated with intractable chronic epilepsy. Presently, signaling via VEGF receptors is not targeted by existing therapies in epileptic patients, but it can be potentially useful in view of its involvement in the regulation of neurogenesis, inflammation, and BBB integrity. However, an effective therapeutic intervention based on modulation of the VEGF system has to take into consideration the specific role of VEGFA and VEGFB and the multiple effects (protective and/or detrimental) reported for VEGFA.