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Ongoing challenges in diagnosing focal cortical dysplasia (FCD) mandate continuous research and consensus agreement to improve disease definition and classification. An International League Against Epilepsy (ILAE) Task Force (TF) reviewed the FCD classification of 2011 to identify existing gaps and provide a timely update. The following methodology was applied to achieve this goal: a survey of published literature indexed with ((Focal Cortical Dysplasia) AND (epilepsy)) between 01/01/2012 and 06/30/2021 (n = 1349) in PubMed identified the knowledge gained since 2012 and new developments in the field. An online survey consulted the ILAE community about the current use of the FCD classification scheme with 367 people answering. The TF performed an iterative clinico-pathological and genetic agreement study to objectively measure the diagnostic gap in blood/brain samples from 22 patients suspicious for FCD and submitted to epilepsy surgery. The literature confirmed new molecular-genetic characterizations involving the mechanistic Target Of Rapamycin (mTOR) pathway in FCD type II (FCDII), and SLC35A2 in mild malformations of cortical development (mMCDs) with oligodendroglial hyperplasia (MOGHE). The electro-clinical-imaging phenotypes and surgical outcomes were better defined and validated for FCDII. Little new information was acquired on clinical, histopathological, or genetic characteristics of FCD type I (FCDI) and FCD type III (FCDIII). The survey identified mMCDs, FCDI, and genetic characterization as fields for improvement in an updated classification. Our iterative clinico-pathological and genetic agreement study confirmed the importance of immunohistochemical staining, neuroimaging, and genetic tests to improve the diagnostic yield. The TF proposes to include mMCDs, MOGHE, and "no definite FCD on histopathology" as new categories in the updated FCD classification. The histopathological classification can be further augmented by advanced neuroimaging and genetic studies to comprehensively diagnose FCD subtypes; these different levels should then be integrated into a multi-layered diagnostic scheme. This update may help to foster multidisciplinary efforts toward a better understanding of FCD and the development of novel targeted treatment options.

Loss of myelin and altered oligodendrocyte distribution in the cerebral cortex are commonly observed both in postsurgical tissue derived from different focal epilepsies (such as focal cortical dysplasias and tuberous sclerosis) and in animal models of focal epilepsy. Moreover, seizures are a frequent symptom in demyelinating diseases, such as multiple sclerosis, and in animal models of demyelination and oligodendrocyte dysfunction. Finally, the excessive activity reported in demyelinated axons may promote hyperexcitability. We hypothesize that the extracellular potassium rise generated during epileptiform activity may be amplified by the presence of axons without appropriate myelin coating and by alterations in oligodendrocyte function. This process could facilitate the triggering of recurrent spontaneous seizures in areas of altered myelination and could result in further demyelination, thus promoting epileptogenesis.

Epilepsy is a pathologic condition with high prevalence and devastating consequences for the patient and its entourage. Means for accurate diagnosis of type, patient monitoring for predicting seizures and follow up, and efficacious treatment are desperately needed. To improve this adverse outcome, miRNAs and the chaperone system (CS) are promising targets to understand pathogenic mechanisms and for developing theranostics applications. miRNAs implicated in conditions known or suspected to favor seizures such as neuroinflammation, to promote epileptic tolerance and neuronal survival, to regulate seizures, and others showing variations in expression levels related to seizures are promising candidates as useful biomarkers for diagnosis and patient monitoring, and as targets for developing novel therapies. Components of the CS are also promising as biomarkers and as therapeutic targets, since they participate in epileptogenic pathways and in cytoprotective mechanisms in various epileptogenic brain areas, even if what they do and how is not yet clear. The data in this review should help in the identification of molecular targets among the discussed miRNAs and CS components for research aiming at understanding epileptogenic mechanisms and, subsequently, develop means for predicting/preventing seizures and treating the disease.

Summary Objective The contribute of recurring seizures to the progression of epileptogenesis is debated. Seizure-induced brain damage is not conclusively demonstrated both in humans and in animal models of epilepsy. We evaluated the expression of brain injury biomarkers on post-surgical brain tissue obtained from 20 patients with frequent seizures and a long history of drug-resistant focal epilepsy. Methods The expression patterns of specific glial, neuronal and inflammatory molecules was evaluated by immunohistochemistry in the core of type II focal cortical dysplasias (FCD-II), at the FCD boundary (perilesion) and in the adjacent normal-appearing area included in the epileptogenic region. We also analysed surgical specimens from cryptogenic patients not presenting structural alterations at imaging. Results Astroglial and microglial activation, reduced neuronal density, peri-vascular CD3-positive T lymphocytes clustering and fibrinogen extravasation were demonstrated in the core of FCD-II lesions. No pathological immunoreactivity was observed outside the FCD-II and in cryptogenetic specimens, where the occurrence of interictal and ictal epileptiform activity was confirmed by either stereo-EEG or intraoperative electrocorticography. Interpretation Recurrent seizures do not induce the expression of brain damage markers in non-lesional epileptogenic cortex studied on post-surgical tissue from cryptogenic and FCD patients. This evidence argues against the hypothesis that epileptiform activity per se contributes to focal brain injury, at least in the neocortical epilepsies here considered. This article is protected by copyright. All rights reserved.

Objective We evaluated four imaging techniques, i.e. Electroencephalography (EEG)-functional Magnetic Resonance Imaging (MRI) (EEG-fMRI), High-resolution EEG (HR-EEG), Magnetoencephalography (MEG) and 2-[18F]fluoro-2-deoxy-D-glucose positron emission tomography (PET), for the identification of the epileptogenic zone (EZ) in 41 patients with negative MRI, candidate to neurosurgery. Methods For each technique, results were compared to the Stereo-EEG. Diagnostic measures were calculated with respect to the post-surgical outcome, either for all the patients (39/41, two patients excluded) and for the subgroup of patients with the EZ involving more than one lobe (20/41). Results When considered individually, each functional technique showed accuracy values ranging 54,6%–63,2%, having PET, MEG and HR-EEG higher sensitivity, and EEG-fMRI higher specificity. In patients with multilobar epileptogenic zone, functional techniques achieved the best accuracies (up to 80%) when three techniques, including EEG-fMRI, were considered together. Conclusions The study highlights the accuracy of a combination of functional imaging techniques in the identification of EZ in MRI negative focal epilepsy. The best diagnostic yield was obtained if the combination of PET, MEG (or HR-EEG as alternative), EEG-fMRI were considered together. Significance The functional imaging techniques may improve the presurgical workup of MRI negative focal epilepsy, if epileptogenic zone involves more than one lobe.

Background Surgery is a widely accepted treatment option for drug-resistant focal epilepsy. A detailed analysis of longitudinal postoperative seizure outcomes and use of antiepileptic drugs for different brain lesions causing epilepsy is not available. We aimed to analyse the association between histopathology and seizure outcome and drug freedom up to 5 years after epilepsy surgery, to improve presurgical decision making and counselling.Methods In this retrospective, multicentre, longitudinal, cohort study, patients who had epilepsy surgery between Jan 1, 2000, and Dec 31, 2012, at 37 collaborating tertiary referral centres across 18 European countries of the European Epilepsy Brain Bank consortium were assessed. We included patients of all ages with histopathology available after epilepsy surgery. Histopathological diagnoses and a minimal dataset of clinical variables were collected from existing local databases and patient records. The primary outcomes were freedom from disabling seizures (Engel class 1) and drug freedom at 1, 2, and 5 years after surgery. Proportions of individuals who were Engel class 1 and drug-free were reported for the 11 main categories of histopathological diagnosis. We analysed the association between histopathology, duration of epilepsy, and age at surgery, and the primary outcomes using random effects multivariable logistic regression to control for confounding.Findings 9147 patients were included, of whom seizure outcomes were available for 8191 (89.5%) participants at 2 years, and for 5577 (61.0%) at 5 years. The diagnoses of low-grade epilepsy associated neuroepithelial tumour (LEAT), vascular malformation, and hippocampal sclerosis had the best seizure outcome at 2 years after surgery, with 77.5% (1027 of 1325) of patients free from disabling seizures for LEAT, 74.0% (328 of 443) for vascular malformation, and 71.5% (2108 of 2948) for hippocampal sderosis. The worst seizure outcomes at 2 years were seen for patients with focal cortical dysplasia type I or mild malformation of cortical development (50.0%, 213 of 426 free from disabling seizures), those with malformation of cortical development-other (52.3%, 212 of 405 free from disabling seizures), and for those with no histopathological lesion (53.5%, 396 of 740 free from disabling seizures). The proportion of patients being both Engel class 1 and drug-free was 0-14% at 1 year and increased to 14-51% at 5 years. Children were more often drug-free; temporal lobe surgeries had the best seizure outcomes; and a longer duration of epilepsy was associated with reduced chance of favourable seizure outcomes and drug freedom. This effect of duration was evident for all lesions, except for hippocampal sclerosis.Interpretation Histopathological diagnosis, age at surgery, and duration of epilepsy are important prognostic factors for outcomes of epilepsy surgery. In every patient with refractory focal epilepsy presumed to be lesional, evaluation for surgery should be considered. Copyright (C) 2020 Elsevier Ltd. All rights reserved.

Objective Drebrins are crucial for synaptic function and dendritic spine development, remodeling, and maintenance. In temporal lobe epilepsy (TLE) patients, a significant hippocampal synaptic reorganization occurs, and synaptic reorganization has been associated with hippocampal hyperexcitability. This study aimed to evaluate, in TLE patients, the hippocampal expression of drebrin using immunohistochemistry with DAS2 or M2F6 antibodies that recognize adult (drebrin A) or adult and embryonic (pan-drebrin) isoforms, respectively. Methods Hippocampal sections from drug-resistant TLE patients with hippocampal sclerosis (HS; TLE, n = 33), of whom 31 presented with type 1 HS and two with type 2 HS, and autopsy control cases (n = 20) were assayed by immunohistochemistry and evaluated for neuron density, and drebrin A and pan-drebrin expression. Double-labeling immunofluorescences were performed to localize drebrin A-positive spines in dendrites (MAP2), and to evaluate whether drebrin colocalizes with inhibitory (GAD65) and excitatory (VGlut1) presynaptic markers. Results Compared to controls, TLE patients had increased pan-drebrin in all hippocampal subfields and increased drebrin A-immunopositive area in all hippocampal subfields but CA1. Drebrin-positive spine density followed the same pattern as total drebrin quantification. Confocal microscopy indicated juxtaposition of drebrin-positive spines with VGlut1-positive puncta, but not with GAD65-positive puncta. Drebrin expression in the dentate gyrus of TLE cases was associated negatively with seizure frequency and positively with verbal memory. TLE patients with lower drebrin-immunopositive area in inner molecular layer (IML) than in outer molecular layer (OML) had a lower seizure frequency than those with higher or comparable drebrin-immunopositive area in IML compared with OML. Significance Our results suggest that changes in drebrin-positive spines and drebrin expression in the dentate gyrus of TLE patients are associated with lower seizure frequency, more preserved verbal memory, and a better postsurgical outcome.

Objective Activity-dependent changes have been reported in animal models and in human epileptic specimens and could potentially be used as tissue biomarkers to evaluate the propensity of a tissue to generate seizure activity. In this context, cAMP-response element binding protein (CREB) activation was specifically reported in human epileptic foci and related mainly to interictal spike activity. To get further insights into CREB activation in human epilepsy, we analyzed pCREB expression on brain tissue samples from patients who underwent surgery for drug-resistant focal epilepsy, correlating this expression with intracranial stereo-electroencephalography (SEEG) recording in a subgroup. Methods Neocortical specimens from patients with neuropathological diagnosis of no lesion (cryptogenic), malformations of cortical development,mainly type II focal cortical dysplasia (FCD), and hippocampi with and without hippocampal sclerosis have been analyzed by immunohistochemistry. Peritumoral cortex from non-epileptic patients and autoptic samples were used as controls, whereas rat brains were used to test possible loss of pCREB antigenicity due to fixation procedures and postmortem delay. Results pCREB was consistently expressed in layer II neuronal nuclei in regions with normal cortical lamination both in epileptic and non-epileptic surgical tissues. In patients with SEEG recordings, this anatomical pattern was unrelated to the presence of interictal spike activity. Conversely, in the core of type II FCD, as well as in other developmental malformations, pCREB was scattered without any laminar specificity. Furthermore, quantitative data did not reveal significant differences between epileptic and non-epileptic tissues, except for an increased immunoreactivity in the core of type IIB FCD lesion related mainly to reactive glial and balloon cells. Significance The present data argue against the reliability of pCREB immunohistochemistry as a marker of epileptic focus but underscores its layer-related expression, suggesting a potential application in the study of malformations of cortical development, a wide range of diseases arising from perturbations of normal brain development.

Objective The microscopic review of hematoxylin-eosin-stained images of focal cortical dysplasia type IIb and cortical tuber of tuberous sclerosis complex remains challenging. Both entities are distinct subtypes of human malformations of cortical development that share histopathological features consisting of neuronal dyslamination with dysmorphic neurons and balloon cells. We trained a convolutional neural network (CNN) to classify both entities and visualize the results. Additionally, we propose a new Web-based deep learning application as proof of concept of how deep learning could enter the pathologic routine. Methods A digital processing pipeline was developed for a series of 56 cases of focal cortical dysplasia type IIb and cortical tuber of tuberous sclerosis complex to obtain 4000 regions of interest and 200 000 subsamples with different zoom and rotation angles to train a neural network. Guided gradient-weighted class activation maps (Guided Grad-CAMs) were generated to visualize morphological features used by the CNN to distinguish both entities. Results Our best-performing network achieved 91% accuracy and 0.88 area under the receiver operating characteristic curve at the tile level for an unseen test set. Novel histopathologic patterns were found through the visualized Guided Grad-CAMs. These patterns were assembled into a classification score to augment decision-making in routine histopathology workup. This score was successfully validated by 11 expert neuropathologists and 12 nonexperts, boosting nonexperts to expert level performance. Significance Our newly developed Web application combines the visualization of whole slide images with the possibility of deep learning-aided classification between focal cortical dysplasia IIb and tuberous sclerosis complex. This approach will help to introduce deep learning applications and visualization for the histopathologic diagnosis of rare and difficult-to-classify brain lesions.

Summary Objective Several studies have reported that inhibitory networks are altered in dysplastic tissue obtained from epilepsy surgery specimens. A consistent decrease in the number of inhibitory interneuronal subpopulation that expresses parvalbumin (PV) was reported in postsurgical tissue from patients with focal cortical dysplasia (FCD). We tested if the decrease in PV protein expression observed in epileptic tissue corresponds to a parallel impairment in the γ-aminobutyric acid (GABA)ergic compartment. Methods We analyzed postsurgical tissue from 30 surgically treated patients who underwent surgery for intractable epilepsy including 26 patients with FCD (types I, II, and III) and 4 patients without any microscopic visible lesion (cryptogenic) as controls. Serial sections were processed using in situ hybridization with GAD-65 and GAD-67 probes and immunocytochemistry with antibody against PV. The density of inhibitory PV-immunoreactive interneurons in relation to GABAergic cells was estimated in controls and in all different pathologic groups by using a two- and three-dimensional (2D and 3D) cell-counting technique. Field fraction and line profile analyses were added to estimate immunostaining proportion and distribution of PV signal generated in gray matter. Results A reduction of PV-positive cells and PV-immunoreactivity was observed exclusively in FCD type I/III specimens compared with cryptogenic tissue from control patients with a poor postsurgical outcome. In FCD type II, a profound rearrangement in the cortical distribution of PV immunoreactivity was observed, without a quantitative reduction of the number of neurons and terminals. In situ hybridization did not reveal significant variations of GAD expression in any FCD subtype. Significance Our study suggests a preservation of inhibitory networks in FCD postsurgical tissue, demonstrated by a substantial normal count of GABAergic neurons. A selective PV expression impairment is demonstrated in FCD type I and III and an abnormal, but not reduced, distribution of PV cells and terminals is confirmed in type II FCD. Possible functional consequences are discussed.

BACKGROUND AND PURPOSE: Conventional MR imaging has limitations in detecting focal cortical dysplasia. We assessed the added value of 7T in patients with histologically proved focal cortical dysplasia to highlight correlations between neuropathology and ultra-high-field imaging. MATERIALS AND METHODS: Between 2013 and 2019, we performed a standardized 7T MR imaging protocol in patients with drug-resistant focal epilepsy. We focused on 12 patients in whom postsurgical histopathology revealed focal cortical dysplasia and explored the diagnostic yield of preoperative 7T versus 1.5/3T MR imaging and the correlations of imaging findings with histopathology. We also assessed the relationship between epilepsy surgery outcome and the completeness of surgical removal of the MR imaging–visible structural abnormality. RESULTS: We observed clear abnormalities in 10/12 patients using 7T versus 9/12 revealed by 1.5/3T MR imaging. In patients with focal cortical dysplasia I, 7T MR imaging did not disclose morphologic abnormalities (n = 0/2). In patients with focal cortical dysplasia II, 7T uncovered morphologic signs that were not visible on clinical imaging in 1 patient with focal cortical dysplasia IIa (n = 1/4) and in all those with focal cortical dysplasia IIb (n = 6/6). T2*WI provided the highest added value, disclosing a peculiar intracortical hypointense band (black line) in 5/6 patients with focal cortical dysplasia IIb. The complete removal of the black line was associated with good postsurgical outcome (n = 4/5), while its incomplete removal yielded unsatisfactory results (n = 1/5). CONCLUSIONS: The high sensitivity of 7T T2*-weighted images provides an additional tool in defining potential morphologic markers of high epileptogenicity within the dysplastic tissue of focal cortical dysplasia IIb and will likely help to more precisely plan epilepsy surgery and explain surgical failures.

Immune changes occur in experimental and clinical epilepsy. Here, we tested the hypothesis that during epileptogenesis and spontaneous recurrent seizures (SRS) an impairment of the endogenous anti-inflammatory pathway glucocorticoid receptor (GR)-annexin A1 (ANXA1) occurs. By administrating exogenous ANXA1, we studied whether pharmacological potentiation of the anti-inflammatory response modifies seizure activity and pathophysiology. We used an in vivo model of temporal lobe epilepsy based on intrahippocampal kainic acid (KA) injection. Video-electroencephalography, molecular biology analyses on brain and peripheral blood samples, and pharmacological investigations were performed in this model. Human epileptic cortices presenting type II focal cortical dysplasia (IIa and b), hippocampi with or without hippocampal sclerosis (HS), and available controls were used to study ANXA1 expression. A decrease of phosphorylated (phospho-) GR and phospho-GR/tot-GR protein expression occurred in the hippocampus during epileptogenesis. Downstream to GR, the anti-inflammatory protein ANXA1 remained at baseline levels while inflammation installed and endured. In peripheral blood, ANXA1 and corticosterone levels showed no significant modifications during disease progression except for an early and transient increase poststatus epilepticus. These results indicate inadequate ANXA1 engagement over time and in these experimental conditions. By analyzing human brain specimens, we found that where significant inflammation exists, the pattern of ANXA1 immunoreactivity was abnormal because the typical perivascular ANXA1 immunoreactivity was reduced. We next asked whether potentiation of the endogenous anti-inflammatory mechanism by ANXA1 administration modifies the disease pathophysiology. Although with varying efficacy, administration of exogenous ANXA1 somewhat reduced the time spent in seizure activity as compared to saline. These results indicate that the anti-inflammatory GR-ANXA1 pathway is defective during experimental seizure progression. The prospect of pharmacologically restoring or potentiating this endogenous anti-inflammatory mechanism as an add-on therapeutic strategy for specific forms of epilepsy is proposed.

Summary Objective Hippocampal sclerosis (HS) is the most frequent neuropathologic finding in patients undergoing surgery for intractable temporal lobe epilepsy (TLE). The International League Against Epilepsy (ILAE) has recently proposed a new classification of HS based on specific patterns of cell loss. The aim of this study was to investigate the relationships between HS types, their etiologic factors, and the short- and long-term postsurgical outcomes of patients undergoing surgery because of drug-resistant TLE with HS. Methods Two hundred thirteen patients with a neuropathologic diagnosis of HS and a minimum follow-up of 2 years were divided on the basis of their ILAE HS type and further classified into: (1) isolated HS, (2) HS associated with focal cortical dysplasia (FCD IIIa), or (3) HS associated with other lesions. Their clinical and neuropathologic data were correlated with their Engel class postsurgical outcomes. Results The main findings were the following: (1) HS type 1 was associated with a longer duration of epilepsy; (2) >80% of the patients had an Engel class I short- and long-term outcomes, regardless of HS type and associated pathology; (3) short- and long-term postsurgical outcomes were less satisfactory in the patients who were completely seizure-free (Engel class Ia), and patients with HS type 2 had better long-term seizure outcomes than those with type 1; (4) the concomitant presence of FCD contributed to a worse outcome, regardless of HS type; and (5) a shorter duration of epilepsy significantly correlated with an Engel class Ia outcome. Significance These data suggest that HS type and associated pathologies may predict the risk of recurrence, but other variables such as the duration of epilepsy need to be considered. A common neuropathologic classification system may help to identify preoperative predictive factors and improve the selection of patients who may benefit from epilepsy surgery.

The contribution of recurring seizures to the progression of epileptogenesis is debated. Seizure-induced brain damage is not conclusively demonstrated either in humans or in animal models of epilepsy. We evaluated the expression of brain injury biomarkers on postsurgical brain tissue obtained from 20 patients with frequent seizures and a long history of drug-resistant focal epilepsy.The expression patterns of specific glial, neuronal, and inflammatory molecules were evaluated by immunohistochemistry in the core of type II focal cortical dysplasias (FCD-II), at the FCD boundary (perilesion), and in the adjacent normal-appearing area included in the epileptogenic region. We also analyzed surgical specimens from cryptogenic patients not presenting structural alterations at imaging.Astroglial and microglial activation, reduced neuronal density, perivascular CD3-positive T-lymphocyte clustering, and fibrinogen extravasation were demonstrated in the core of FCD-II lesions. No pathological immunoreactivity was observed outside the FCD-II or in cryptogenetic specimens, where the occurrence of interictal and ictal epileptiform activity was confirmed by either stereo-electroencephalography or intraoperative electrocorticography.Recurrent seizures do not induce the expression of brain damage markers in nonlesional epileptogenic cortex studied in postsurgical tissue from cryptogenic and FCD patients. This evidence argues against the hypothesis that epileptiform activity per se contributes to focal brain injury, at least in the neocortical epilepsies considered here. Ann Neurol 2017;82:331-341.

Type II focal cortical dysplasia (FCD II) is a malformation of cortical development, frequently associated with intractable epilepsy, characterised by cortical dyslamination, dysmorphic neurons (DNs) and balloon cells (BCs). We investigated the expression of pS6 (downstream target) and pPDK1-pAkt (upstream targets) as evidence for mTOR pathway activation and their co-expression with Interleukin-1β in FCD II surgical specimens and compared the findings with control non-epileptic tissue, non-malformed epileptic tissue or acquired epilepsy-Rasmussen's Encephalitis (RE) occasionally presenting pS6 and Interleukin-1β positive abnormal neurons. Downstream mTOR activation was demonstrated in almost all abnormal cells in both FCD II and RE. Conversely, upstream activation in FCD II was observed in the majority of BCs, in a proportion of DNs, not presenting Interleukin-1β expression, but not at all in RE scattered abnormal neurons. Based on these findings we suggest that the presence of BCs and DNs in FCD II could be due to a first upstream mTOR pathway PI3K-Akt-mediate event occurring very early during cortical development in the large proportion of abnormal cells; followed by the appearance of additional pS6 positive DNs promoted by the presence of a later inflammatory processes.

Aim. The extracellular matrix glycoprotein reelin plays a crucial role in the control of neuronal migration and during development is expressed by Cajal-Retzius cells in the marginal zone. The purpose of this study was to investigate the possible involvement of reelin in the pathogenesis of human nodular heterotopia, a malformation of cortical development frequently associated with focal drug-resistant epilepsy. Methods. Five patients presenting with subcortical nodular heterotopia and referred for epilepsy surgery, after a comprehensive presurgical investigation, were considered. The surgical specimens were studied by combining immunohistochemistry, double immunofluorescence, and in situ hybridisation procedures. Results. The selected cases were characterised by the presence of multiple nodules presenting in the core cell-free zones, reminiscent of the cortical molecular layer. In all cases, small reelin-positive cells, without typical Cajal-Retzius cell features, were distributed inside the nodules and localised in these cell body-sparse regions. Conclusion. The presented data corroborate the hypothesis that reelin might be involved in human heterotopic nodular formation.

Magnetic resonance imaging-positive temporal lobe atrophy with temporo-polar grey/white matter abnormalities (usually called 'blurring') has been frequently reported in patients with temporal lobe epilepsy associated with hippocampal sclerosis. The poor distinction of grey and white matter has been attributed to various causes, including developmental cortical abnormalities, gliosis, myelin alterations, a non-specific increase in temporal lobe water content and metabolic/perfusion alterations. However, there is still no consensus regarding the genesis of these abnormalities and no histopathological proof for a structural nature of magnetic resonance imaging changes. The aim of this study was to investigate the pathological substrate of temporo-polar blurring using different methodological approaches and evaluate the possible clinical significance of the abnormalities. The study involved 32 consecutive patients with medically intractable temporal lobe epilepsy and hippocampal sclerosis who underwent surgery after a comprehensive electroclinical and imaging evaluation. They were divided into two groups on the basis of the presence/absence of temporo-polar blurring. Surgical specimens were examined neuropathologically, and selected samples from both groups underwent high-field 7 T magnetic resonance imaging and ultrastructural studies. At the clinical level, the two groups were significantly different in terms of age at epilepsy onset (earlier in the patients with blurring) and epilepsy duration (longer in the patients with blurring). Blurring was also associated with lower neuropsychological test scores, with a significant relationship to abstract reasoning. On 7 T magnetic resonance image examination, the borders between the grey and white matter were clear in all of the samples, but only those with blurring showed a dishomogeneous signal in the white matter, with patchy areas of hyperintensity mainly in the depth of the white matter. Sections from the patients with blurring that were processed for myelin staining revealed dishomogeneous staining of the white matter, which was confirmed by analyses of the corresponding semi-thin sections. Ultrastructural examinations revealed the presence of axonal degeneration and a significant reduction in the number of axons in the patients with blurring; there were no vascular alterations in either group. These data obtained using different methodological approaches provide robust evidence that temporo-polar blurring is caused by the degeneration of fibre bundles and suggest slowly evolving chronic degeneration with the redistribution of the remaining fibres. The article also discusses the correlations between the morphological findings and clinical data.

Summary Purpose: Neuropathologic investigations frequently reveal the presence of architectural cortical dysplasia in patients with temporal lobe epilepsy (TLE), sometimes as an isolated finding but more commonly associated with hippocampal sclerosis (HS) and white matter abnormalities. The histologic pattern and the developmental origin of these alterations are not clear, and their diagnostic criteria are poorly defined. The aim of this study was to investigate the expression patterns of layer-specific genes in cortical specimens from patients with TLE presenting different subtypes of cortical malformations in order to elucidate the disorganization of the laminar architecture of such epileptogenic abnormalities and provide evidence to enable a more objective neuropathologic diagnosis. Methods: We analyzed the expression patterns of CUX2, RORBETA, ER81, NURR1, and CTGF genes, respectively specific markers of layers II–III, IV, V, VI, and VIb, in surgical samples by means of in situ hybridization and compared them with those observed in control cortices. The pathologic samples included typical architectural dysplasia (group 1); temporal lobe sclerosis, a variant of architectural dysplasia (group 2); and white matter heterotopic neuronal aggregates, namely small lentiform nodules (group 3). These abnormalities may have been associated or not with HS. Key Findings: All of the genes had a laminar expression pattern in normal cortices, whereas groups 1 and 2 showed alterations mainly involving layers V and VI, and highlighted by the altered distribution of ER81- and NURR1-positive cells. The expression of ER81 and NURR1 genes was different among the groups, and atypical coexpression of NURR1 and CUX2 mRNA was detected in the neurons making up the small lentiform nodules. Significance: These findings indicate that defects in cortical organization involving the deeper cortical neurons may be a common etiopathogenic mechanism in group 1 and 2 cortical dysplasia, whether isolated or associated with HS, and that developmental disorders may also be present in the white matter (group 3). They also provide evidence that the layer-specific genes can be usefully used to investigate the neuropathology of human cortical dysplasia.

Background: Gap junctions are specialized channels composed of several connexins, membrane proteins that mediate electrical and metabolic coupling between cells. Previous data have suggested that changes in the expression of Cx43, the main astrocytic Cx isoform, may be involved in seizure activity in human epileptic tissue. However, Cx43 has never been examined in focal cortical dysplasia (FCD) and in other human refractory epilepsies. Methods: We analyzed Cx43 protein localization and Cx43 mRNA levels in surgical specimens of cortex from a cohort of patients with intractable epilepsy vs control nonepileptic tissue. Samples had neuropathologically defined diagnosis of cryptogenic epilepsy or epilepsy secondary to FCD. Results: Cx43 immunoreactivity, which labeled punctate elements, did not reveal distinctive features in cryptogenic epilepsy and FCD type IA and IIA. A peculiar pattern of immunolabeling was instead observed in FCD type IIB, in which large aggregates of Cx43-immunopositive puncta were clustered around subsets of balloon cells and astrocytes. Further characterization revealed that these balloon cells do not express markers of precursor cells, such as CD34. Quantitative real-time reverse transcriptase PCR showed elevated levels of Cx43 transcript in a subgroup (25%) of cryptogenic epilepsy specimens compared to control and FCD ones. Conclusions: Our study points out that a rearrangement of Cx43-positive elements is part of abnormal tissue organization in FCD type IIB, and that cryptogenic epilepsies include forms with increased Cx43 mRNA expression. The data implicate functional consequences of altered Cx43 expression, and therefore of altered gap junctional coupling, in abnormal network properties of subtypes of human refractory epilepsies.

Flavio Villani *, Veronica Pelliccia , Giuseppe Didato , Giovanni Tringali , Francesco Deleo , Rita Garbelli , Laura Rossini , Roberto Spreafico a Division of Clinical Epileptology and Experimental Neurophysiology, ‘‘Carlo Besta’’ Neurological Institute Foundation, Milan, Italy b Epilepsy Surgery Center ‘‘C. Munari’’, Niguarda Hospital, Milan, Italy Division of Neurosurgery III, ‘‘Carlo Besta’’ Neurological Institute Foundation, Milan, Italy

The present study included analysis of data from 243 patients surgically treated for Temporal Lobe Epilepsy (TLE). Resection was confined to the temporal lobe, with at least two years of follow-up, and specimens sufficiently preserved to allow a precise evaluation of both lateral neocortex and hippocampus. The frequency of different types of lesion and hippocampal sclerosis (HS), isolated or associated with neocortical lesions, risk factors and surgical outcomes in relation to neuropathological findings were evaluated. We found tumours in 33% of patients, malformations of cortical development (MCD) in 45%, isolated HS in 14%, no lesion in 5% and less common lesions in 3%. HS was present in 8% of tumour cases and 70% of MCD. Statistical analysis of antecedents was significantly associated only with febrile seizures (FS). In MCD patients with no history of FS, a strong association between HS and duration of epilepsy was revealed. A Class I outcome was identified in 87% of cases with tumours and 79% in cases with MCD. In 93 patients the antiepileptic drug therapy was withdrawn. Our findings show that MCD, which is significantly associated with HS, is the most common lesion in TLE and support the concept that an optimal outcome is obtained when mesial and neocortical structures are removed. FS are particularly relevant in patients with focal cortical dysplasia and HS.

In the present report, the correlations between ex vivo high-resolution imaging and specific histological and ultrastructural patterns in type II focal cortical dysplasia (FCD) have been studied to explain the differences in the magnetic resonance imaging (MRI) detection of dysplasia and to contribute to the presurgical imaging evaluation of this pathology.Surgical specimens from 13 patients with FCD IIa/b were submitted to 7T MRI scanning, and then analyzed histologically and ultrastructurally to compare the results with the MRI findings. Region of interest (ROI)-based measures on T2-weighted images (T2wi) were quantitatively evaluated in the lesion and in adjacent perilesional gray and white matter.Matched histological sections and 7T T2wi showed that the core of the lesion was characterized by patchy aggregates of abnormal cells and fiber disorganization related to inhomogeneity of intracortical signal intensity. The quantitative approach on T2wi can help to distinguish the lesions and perilesional areas even in a clinical MRI-negative case. The ultrastructural study showed that the strong signal hyperintensity in the white matter of FCD IIb was related to a dysmyelination process associated with severe fiber loss and abnormal cells. Less severe histopathological features were found in FCD IIa, thus reflecting their less evident MRI alterations.We suggest that white matter abnormalities in type IIb FCD are due to defects of the myelination processes and maturation, impaired by the presence of balloon cells. To reveal the presence and the border of type II cortical dysplasia on MRI, a quantitative ROI-based analysis (coefficient of variation) is also proposed.

Introduction: Neuro-vascular rearrangement occurs in brain disorders, including epilepsy. Platelet-derived growth factor receptor beta (PDGFRβ) is used as a marker of perivascular pericytes. Whether PDGFRβ + cell reorganization occurs in regions of neuro-vascular dysplasia associated with seizures is unknown. Methods : We used brain specimens derived from epileptic subjects affected by intractable seizures associated with focal cortical dysplasia (FCD) or temporal lobe epilepsy with hippocampal sclerosis (TLE-HS). Tissues from cryptogenic epilepsy, non-sclerotic hippocampi or peritumoral were used for comparison. An in vivo rat model of neuro-vascular dysplasia was obtained by pre-natal exposure to methyl-axozy methanoic acid (MAM). Status epilepticus (SE) was induced in adult MAM rats by intraperitoneal pilocarpine. MAM tissues were also used to establish organotypic hippocampal cultures (OHC) to further assess pericytes positioning at the dysplastic microvasculature. PDGFRβ and its colocalization with RECA-1 or CD34 were used to segregate perivascular pericytes. PDGFRβ and NG2 or IBA1 colocalization were performed. Rat cortices and hippocampi were used for PDGFRβ western blot analysis. Results: Human FCD displayed the highest perivascular PDGFRβ immunoreactivity, indicating pericytes, and presence of ramified PDGFRβ + cells in the parenchyma and proximal to microvessels. Tissues deriving from human cryptogenic epilepsy displayed a similar pattern of immunoreactivity, although to a lesser extent compared to FCD. In TLE-HS, CD34 vascular proliferation was paralleled by increased perivascular PDGFRβ + pericytes, as compared to non-HS. Parenchymal PDGFRβ immunoreactivity co-localized with NG2 but was distinct from IBA1 + microglia. In MAM rats, we found pericyte-vascular changes in regions characterized by neuronal heterotopias. PDGFRβ immunoreactivity was differentially distributed in the heterotopic and adjacent normal CA1 region. The use of MAM OHC revealed microvascular-pericyte dysplasia at the capillary tree lining the dentate gyrus (DG) molecular layer as compared to control OHC. Severe SE induced PDGFRβ + immunoreactivity mostly in the CA1 region of MAM rats. Conclusion: Our descriptive study points to microvascular-pericyte changes in the epileptic pathology. The possible link between PDGFRβ + cells, neuro-vascular dysplasia and remodeling during seizures is discussed.

We present the results of a retrospective study on 10 patients operated on for intractable epilepsy associated with nodular heterotopia as identified by high resolution MRI. Seven patients had unilateral heterotopia, one patient had symmetric bilateral heterotopia and two patients had asymmetric bilateral heterotopia. By stereo-electroencephalogram (SEEG) (nine patients) interictal activity within nodules was similar in all cases, and ictal activity never started from nodules alone but from the overlying cortex or simultaneously in nodules and cortex. Excellent outcomes ([Engel class Ia, 1987][1]) were achieved in the seven patients with unilateral heterotopia, showing that surgery can be highly beneficial in such cases when the epileptogenic zone is carefully located prior to surgery by MRI and particularly SEEG. For the bilateral cases surgical outcomes were Engel IIa (one patient) or Engel IIIa (two patients). Histological/immunohistochemical studies of resected specimens showed that all nodules had similar microscopic organization, even though their extent and location varied markedly. The overlying cortex was dysplastic in nine patients, but of normal thickness. We suggest that nodule formation may be the result of a dual mechanism: (i) failure of a stop signal in the germinal periventricular region leading to cell overproduction; and (ii) early transformation of radial glial cells into astrocytes resulting in defective neuronal migration. The intrinsic interictal epileptiform activity of nodules may be due to an impaired intranodular GABAergic system. [1]: #ref-5

Purpose: Periventricular nodular heterotopia (PNH) is the most common human brain dysgenesis, very frequently characterized by focal drug-resistant epilepsy. To understand the cellular mechanisms underlying its intrinsic hyperexcitability, we investigated the expression of glutamate-receptor subunits and related proteins in four human patients affected by PNH. Methods: PNH was diagnosed by means of magnetic resonance imaging. The epileptogenic area was revealed by depth electrode recordings and removed during epilepsy surgery. Sections from the removed cerebral tissue were analyzed by means of immunocytochemistry (ICC), with antibodies directed against N-methyl-D-aspartate (NMDA)-receptor subunits, the a subunit of the Ca 2+ /calmodulin-dependent kinase II (αCaMKII), and its active phosphorylated form. Results: The ICC data demonstrated that the subcortical heterotopic nodules were consistently characterized by lower expression of aCaMKII and its activated form. In more pronounced cases (i.e., when the extension of the nodules to the neocortex determined clear layering abnormalities), the heterotopic tissue also was characterized by a decreased expression of NMDA-receptor subunits, which was particularly evident in the dendritic compartment. Conclusions: These data suggest the existence of an alteration of αCaMKII and the NMDA-receptor complex in the epileptogenic brain tissue of human PNH, which may play a role in the basic mechanisms of hyperexcitability associated with this brain dysgenesis.

Item does not contain fulltext Background: Detailed neuropathological information on the structural brain lesions underlying seizures is valuable for understanding drug-resistant focal epilepsy. Methods: We report the diagnoses made on the basis of resected brain specimens from 9523 patients who underwent epilepsy surgery for drug-resistant seizures in 36 centers from 12 European countries over 25 years. Histopathological diagnoses were determined through examination of the specimens in local hospitals (41%) or at the German Neuropathology Reference Center for Epilepsy Surgery (59%). Results: The onset of seizures occurred before 18 years of age in 75.9% of patients overall, and 72.5% of the patients underwent surgery as adults. The mean duration of epilepsy before surgical resection was 20.1 years among adults and 5.3 years among children. The temporal lobe was involved in 71.9% of operations. There were 36 histopathological diagnoses in seven major disease categories. The most common categories were hippocampal sclerosis, found in 36.4% of the patients (88.7% of cases were in adults), tumors (mainly ganglioglioma) in 23.6%, and malformations of cortical development in 19.8% (focal cortical dysplasia was the most common type, 52.7% of cases of which were in children). No histopathological diagnosis could be established for 7.7% of the patients. Conclusions: In patients with drug-resistant focal epilepsy requiring surgery, hippocampal sclerosis was the most common histopathological diagnosis among adults, and focal cortical dysplasia was the most common diagnosis among children. Tumors were the second most common lesion in both groups. (Funded by the European Union and others.) 9 p.

In the present report we describe the neuropathological characteristics of tissue surgically resected from three patients affected by intractable epilepsy secondary to cortical dysplasia. Common features, suggestive of a focal cortical dysplasia of Taylor, were observed in all specimens. Immunocytochemical procedures were performed using neuronal and glial markers and the sections were observed at light traditional and confocal microscopes. This part of the investigation pointed out: 1. cortical laminar disruption; 2. very large neurons displaying a pyramidal or round shape; 3. ballooned cells; 4. decrease of calcium binding proteins immunoreactivity; 5. abnormal nets of parvalbumin- and glutamic acid decarboxylase-positive puncta around giant neurons but not around ballooned cells. Ultrastructural investigation on the same material provided evidence of a high concentration of neurofilaments in giant neurons and of glial intermediate filaments in ballooned cells. In addition, immunolabeled GABAergic terminals clustered around giant neurons were not found to establish synapses on their cell bodies. The present data, derived from a limited sample of patients but showing very consistent features, suggest that in Taylor's type of cortical dysplasia a disturbance of migratory events could be paralleled by a disruption of cell differentiation and maturation and by an impairment of synaptogenesis. This latter mechanism seemed to affect especially the inhibitory elements, and could account for the hyperexcitability of this tissue and thus for the high epileptogenicity of Taylor's dysplasia.

Rasmussen encephalitis (RE) is a progressive inflammatory disorder characterized by brain hemiatrophy, unilateral focal deficits, and drug-refractory focal epilepsy. Epilepsia partialis continua (EPC) is a hallmark of the disease. Several immunomodulatory treatments may slow but not halt the disease progression. The treatment of choice still relies on surgical hemispheric disconnection, which is burdened by heavy neurologic morbidity. More limited cortical resections, although more tolerable, are usually considered to be, at best, only transiently effective in RE. Hemispheric disconnections may be not feasible when neurologic functions are preserved and the dominant hemisphere is affected. Adult patients with a milder RE course that preserves neurologic function for a long period are particularly at risk of developing severe deficits after surgery. In this study we present the histories of two patients with adult-onset RE who have undergone selective cortical resections to control EPC, avoiding, at the same time, the severe postsurgical deficits that may be induced by hemispheric disconnective surgery. The good result obtained on EPC has been stable over a prolonged period; however, this result was not paralleled by the stop of neurologic progression in one of the two cases. A PowerPoint slide summarizing this article is available for download in the Supporting Information section http://dx.doi.org/10.1111/epi.12596/supinfo.

Aquaporin-4 (AQP4), the prominent water channel in the brain, is particularly concentrated in astrocytic endfeet membranes lining the capillaries and the pia. This localisation pattern makes it well suited to regulate the flow of water and the homeostasis of the interstitial fluid surrounding the neurons. Using immunocytochemical and Western blot techniques, we investigated the expression of AQP4 and some related proteins (i.e. glial fibrillary acidic protein, glial glutamate transporter 1, the endothelial marker CD34, and dystrophin) in tissue taken from epileptic patients. To this end, we used surgical samples containing focal cortical dysplasia (FCD) Type IIB, samples of normal-appearing (cryptogenic) cortex, and samples from non-epileptic patients as controls. AQP4 expression and distribution in the cryptogenic patients were similar to those observed in the control cases, mainly concentrated around blood vessels. In the patients with FCD type IIB, severe malformation in cortical development, the protein was more expressed and the distribution pattern of AQP4 immunoreactivity was different, being strong in the neuropil and around several dysplastic neurons, whereas the vessels appeared to be less intensely stained by AQP4 and dystrophin. As the efficiency of AQP4 in regulating water and ion homeostasis in extracellular space depends on its spatial distribution in astrocytes, the different distribution of AQP4 protein in the FCD type IIB samples may modify fluid homeostasis control and the regular functioning of neuronal cells.

Although the term ‘neuropathology’ tends to refer to an ‘old-fashioned’ discipline, the development of new methodologies such as immunocytochemistry and in situ hybridization, applied to the surgical specimens obtained from patients affected by partial refractory seizures, has renewed interest in the field. Neuropathology of epilepsy has moved from a static concept to a dynamic interpretation of different forms of epilepsy – i.e., from a mere descriptive anatomical view to a morphofunctional interpretation of the disease. Therefore, the current goal of neuropathological investigation of epilepsy is not only to make a correct diagnosis of the pathological process underlying seizures, but also to gain more insights on the pathogenesis and physiopathology of epilepsies. For these reasons, the neuropathologist must be considered as a fundamental partner in a team involving epileptologists, neuropsychologists, neuroradiologists, and neurosurgeons – to provide a comprehensive approach to cure and care of epileptic patients. The present paper illustrates some of the major new methodological aspects of neuropathology of epilepsy, and highlights recent exciting results and future challenges.

Summary Purpose: Despite the availability of detailed electroclinical and imaging data, only a few neuropathological studies of nodular heterotopia have been published. The aim of this study was to describe the neuropathological features of various types of nodular heterotopia obtained from patients undergoing surgery for intractable epilepsy. Methods: Specimens of heterotopic nodules taken from 24 patients were neuropathologically investigated using routine and immunocytochemical procedures, and the data were compared with magnetic resonance imaging (MRI), electroclinical findings, and surgical outcomes. Results: The neuropathological data distinguished two groups. Group 1 (14 patients, 78% in Engel class 1) had similar characteristics regardless of the size, number, or location of the nodules, with both projecting and local circuit neurons in the nodules intermingled with glial cells. Thirteen patients had focal cortical dysplasia. The nodules were identified by MRI in all cases. In group 2 (10 patients, 90% in Engel class 1), all of the nodules were within the temporal lobe and associated with hippocampal sclerosis or gangliogliomas. They were very small (undetected by MRI) and mainly formed by projecting neurons with no evidence of glial cells. All of the patients had cortical dysplasia. Discussion: The distinctive neuropathological features of the nodules in the two groups suggest different etiopathogenetic mechanisms. In group 2, the presence of nodular formations in association with cortical dysplasia and either hippocampal sclerosis or ganglioglioma raises a question concerning so-called dual pathology in the temporal lobe.

Focal cortical dysplasia (FCD) is a pathology that is characterized by the abnormal development of the neocortex. Indeed, a wide range of abnormalities in the cortical mantle have been associated with this pathology, including cytoarchitectonic alterations and the presence of dysmorphic neurons, balloon cells and ectopic neurons in the white matter. FCD is commonly associated with epilepsy, and hence we have studied the ultrastructure of cortical tissue resected from three subjects with intractable epilepsy secondary to cortical dysplasia to identify possible alterations in synaptic circuitry, using correlative light and electron microscopic methods. While the balloon cells found in this tissue do not appear to receive synaptic contacts, the ectopic neurons in the white matter were abnormally large and were surrounded by hypertrophic basket formations immunoreactive for the calcium-binding protein parvalbumin. Furthermore, these basket formations formed symmetrical (inhibitory) synapses with both the somata and the proximal portion of the dendrites of these giant ectopic neurons. A quantitative analysis revealed that in the dysplastic tissue, the density of excitatory and inhibitory synapses was different from that of the normal adjacent cortex. Both increases and decreases in synaptic density were observed, as well as changes in the proportion of excitatory and inhibitory synapses. However, we could not establish a common pattern of changes, either in the same patients or between different patients. These results suggest that cortical dysplasia leads to multiple changes in excitatory and inhibitory synaptic circuits. We discuss the possible relationship between these alterations and epilepsy, bearing in mind the possible limitations that preclude the extrapolation of the results to the whole population of epileptic patients with dysplastic neocortex.

Summary: Purpose: The aim of this study was to correlate the electroclinical and radiologic data with the neuropathologic findings and surgical outcome in epileptic patients with epilepsy and Taylor's focal cortical dysplasia (TFCD) and to characterize further the abnormal intermediate filaments expression in the balloon cell present in the peculiar dysplasia. Methods: We retrospectively selected 13 TFCD patients who underwent surgery for intractable epilepsy with the aim of removing the magnetic resonance (MR)-detectable lesion and/or the epileptogenic zone defined by stereoelectroencephalographic recordings. The surgical specimens were analyzed by means of routine neuropathologic and immunocytochemical studies. Antisera against different intermediate filaments also were used in serial adjacent sections to evaluate their coexpression in balloon cells. Results: Histopathologic abnormalities typical of TFCD were found not only within the MR-visible lesions but also in most of the epileptogenic zones with no MR signal alterations. Furthermore, the MR-visible lesions contained a high proportion of cells with an abnormal expression of intermediate filament proteins. After a long follow-up, 10 of the patients are now seizure free. Conclusions: Our findings indicate that highly epileptogenic zones may correspond to tissue alterations not revealed by neuroimaging. Furthermore, the immunocytochemical data show that the dysplastic tissue detected by MR contained high concentrations of cells filled with abnormal intermediate filaments. The detected colocalization of neuronal and glial markers in balloon cells indicates a failure of cellular commitment during development.