„PEDIATRIC EPILEPSY SURGERY – predictors of (un)favourable outcome“

DOCTORAL THESIS at the Medical University Vienna

for obtaining the „Doctor of Medical Science“-Degree

by

Dr. Angelika MÜHLEBNER-FAHRNGRUBER n0200413

under the supervision of

ao. Univ. Prof. Dr. Martha Feucht

Division of Neonatology, Pediatric Intensive Care and Neuropediatrics Department of Pediatrics

Medical University Vienna Waehringer Guertel 18-20 A – 1090 Vienna

Vienna, March 18th, 2012

-1- Abstrakt (Deutsch)

Epilepsie ist eine der häufigsten neurologischen Erkrankungen im Kindesalter. Sechzig bis siebzig Prozent der (jungen) Patienten lassen sich medikamentös gut einstellen. Ungefähr ein Drittel aller Kinder mit Epilepsie bleibt jedoch therapierefraktär. Solche Patienten werden gegebenenfalls an einem tertiären Zentrum hinsichtlich eines epilepsie- chirurgischen Eingriffes evaluiert. Alle Patienten erhalten dahingehend ein prolongiertes Video-EEG Monitoring, hochauflösende bildgebende Verfahren sowie eine ausgiebige neuropsychologische Testung. Nach einer temporalen Resektion bleiben ca. 2/3 der Patienten anfallsfrei, bei einer extratemporalen allerdings nur in etwa die Hälfte der Operierten. Zu den häufigsten operierten epileptogenen Läsionen im Kindesalter zählen die kortikalen Entwicklungsstörungen. Ziel dieser Arbeit war eine bessere präoperative und histologische Charakterisierung der Kinder mit Hirnentwicklungsstörungen. Im Zuge dessen konnte eine Kasuistik über einen positiven Outcome nach Temporallappenresektion bei limbischer Enzephalitis veröffentlicht werden (siehe Publikation 1). Weiters erfolgte ein ausgiebiger Review über die neuropathologische Aufarbeitung epilepsie-chirurgischer Resektate (siehe Publikation 2). Zuletzt wurde der Fokus auf histologisch quantifizierbare Kriterien von Hirnentwicklungsstörungen und deren Bedeutung für die prächirurgische Bildgebung gelegt (siehe Publikation 3). Es konnten somit klare Charakteristika (kortikale Dicke, Rinden-Mark Grenze, heterotopes Neuropil in der tiefen weißen Substanz sowie Grad der Myelinisierung) benannt werden, welche in weiterer Folge zu einer Verbesserung in der prächirurgischen Bildgebung führen soll.

-2- Abstract (English)

Among the neurological diseases in childhood, epilepsy has one of the highest frequencies. The most common epileptogenic lesions in children are malformations of the cortex during development. Due to recent progress in diagnostics and treatment, over 60% of newly diagnosed patients may enter remission during treatment with antiepileptic drugs (AEDs). However, as many as 30-40% of patients remain difficult to treat or are drug- resistant, and hence it is important to gain a deeper understanding into their condition so as to ameliorate it. Such patients are usually referred to a tertiary centre for epilepsy- surgery evaluation. This examination includes prolonged video-EEG monitoring, high resolution neuroimaging and extensive neuropsychological evaluation. Following surgery, about 67% of the patients are seizure free after removal of the temporal lobe and about 50% following extratemporal resection. Ideally, anticonvulsive therapy may be stopped pursuant to surgical intervention. The main focus of this thesis was to compare EEG data, neuroimages, and neuropsychological test results from patients before and after surgery. In addition, histological diagnoses were also undertaken. This resulted in the positive surgical outcome in one young female patient. Although it is not common to treat autoimmune-induced limbic encephalitis by resection of the temporal lobe, as a result of analyzing the patient’s data, it became clear that her hippocampus had become atrophied, and this led to persistent epileptic fits. Following the removal of the temporal lobe and hippocampus, the patient recovered and remained seizure free for a year (see publication 1). A further outcome of this work was the publication of an extensive review on neuropathological handling of epilepsy specimens (see publication 2). Importantly, the work helped define clear-cut histological characteristics in brain samples that might positively influence neuroimaging, including: cortical thickness, grey-white matter border, heterotopic neuropil in the deep white matter; and myelination (see publication 3). These findings should help improve presurgical diagnostics, thereby resulting in more a successful outcome of surgical intervention.

-3- Acknowledgements

The thesis was funded by the Jubiläumsfond of the Austrian National Bank (project 12063). Furthermore, I was awarded a scholarship by the Austrian Society of Epileptology to sponsor my first training at the Department of Neuorpathology of the University Erlangen- Nuremberg. First, I want to thank my three supervisors of the Medical University Vienna: Prof. Dr. M. Feucht – Department of Pediatrics and Adolescent Medicine, Prof. Dr. J. Hainfellner – Institute of Neurology and Prof. Dr. D. Prayer – Department of Neuroradiology, who supported me throughout the years of this thesis and were available for critical discussion. Most of the work presented here was carried out at either one of the three departments. Next, Prof. Dr. I. Blümcke of the Department of Neuropathology at the University Erlangen- Nuremberg was invaluable for the conduct of this thesis.

I am very grateful for the support at the clinics and in the lab by all the technicians of the Epilepsy Monitoring Unit for children in Vienna, the Institute of Neurology in Vienna and the Department of Neuropathology in Erlangen.

Katja Kobow, PHD and Roland Coras, MD were very stimulating discussion partners and dear friends throughout my time in Erlangen.

Special thanks to Aner Gurvitz, who helped me a lot with the writing and editing.

Last, I want to thank my husband and my family.

-4- Table of Content

Abstrakt (Deutsch)...... 2 Abstract (English)...... 3 Acknowledgements...... 4 Table of Content...... 5 Glossary...... 7 1 Introduction...... 9 1.1 General background to epilepsy...... 9 1.2 Types of seizures, epilepsies and epilepsy syndromes...... 13 1.2.1 Seizures...... 13 1.2.2 Epilepsies...... 14 1.2.3 Epilepsy syndromes...... 14 Neonatal epileptic seizures and syndromes...... 14 Idiopathic epileptic seizures and syndromes in infancy...... 15 Epileptic encephalopathies of infancy and early childhood...... 15 Severe neocortical epileptic syndromes in infancy and childhood...... 16 Benign childhood focal seizures and related syndromes...... 16 Idiopathic generalized epilepsies...... 16 Structural focal epilepsies...... 16 1.3 Drug management and therapeutic options...... 17 1.4 Surgical intervention in epilepsy...... 17 1.4.1 Presurgical evaluation...... 17 1.4.2 Epilepsy Center Vienna...... 19 1.4.3 Video-EEG Monitoring in Vienna...... 19 1.4.4 Neuroimaging in Vienna...... 19 1.5 Epileptogenic lesions...... 20 1.6 Aims of this thesis...... 22 2 Synopsis...... 23 3 References...... 28 4 Publications...... 31 4. 1 Publication 1: Epilepsy Research 2010 Aug;90(3):295-9 ...... 31 Beneficial effect of epilepsy surgery in a case of childhood non-paraneoplastic limbic

-5- encephalitis ...... 31 4.2 Publication 2: Clinical Neuropathology 2011 Jul-Aug;30(4):164-77 ...... 43 Neuropathological work-up of Focal Cortical Dysplasias using the new ILAE consensus classification system - practical guideline article invited by the Euro-CNS Research Committee...... 43 4.3 Publication 3: Acta Neuropathol. 2011 Nov 27. [Epub ahead of print] ...... 74 Neuropathologic measurements in Focal Cortical Dysplasias: validation of the ILAE 2011 classification system and diagnostic implications for MRI...... 74 5 Curriculum vitae...... 106 6 Publications and Meetings...... 110

-6- Glossary

AED Anti-epileptic drug AD Dr. Anastasia Dressler AM Dr. Angelika Mühlebner BHZ Behandlungszentrum Vogtareuth CA Cornu Ammonis CNPase 2',3'-cyclic nucleotide 3' phosphodiesterase CSF Cerebro-spinal fluid DP Prof. Daniela Prayer EEG Electroencephalography EMU Epilepsy Monitoring Unit for children at the MUVI FCD Focal Cortical Dysplasia FLAIR Fluid attenuated inversion recovery GAD Glutamic acid decarboxylase GFAP Glial fibrillary acidic protein GG Dr. Gudrun Gröppel GK Dr. Gregor Kasprian GTCS Generalized tonic-clonic seizure HA Hippocampal atrophy H&E Hematoxilin & Eosin HHV Human herpes virus HS Hippocampal sclerosis IDH Isocitrate dehydrogenase ILAE International Leaque against Epilepsy IB Prof. Ingmar Blümcke IR Inversion recovery JH Prof. Johannes Hainfellner LE Limbic encephalitis MAP 2 Microtubule-associated protein 2 MCD Malformation of cortical development MD Medical Doctor MF Prof. Martha Feucht

-7- MRI Magnetic Resonance Imaging MS Dr. Maria Schmook MTS Mesial temporal sclerosis NeuN Neuronal nuclei Nissl-LFB Cresyl violet – luxol fast blue NMDA-R N-methyl-D-aspartate receptor NPLE Non-paraneoplastic limbic encephalitis Olig 2 oligodendrocyte lineage transcription factor 2 PBS Phosphate buffered saline PET Positron emission tomography PNLE Paraneoplastic limbic encephalitis SCLC Small cell lung cancer SE Status epilepticus SPECT Single photon emission computer tomography TC Prof. Thomas Czech TSE Turbo spin echo VGKC Voltage-gated potassium channel WHO World Health Organization ZEE Zentrum für Epilepsie Erlangen

-8- 1 Introduction

1.1 General background to epilepsy

With a prevalence of 0.5 -2% amongst the general population, epilepsy is one of the most frequent neurological diseases. In about two thirds of epileptic cases, onset of the disease occurs during childhood and adolescence. Indeed, the maximum rate occurs during the first year of life (100-233/100,000). Thereafter the rate declines in early childhood (60/100,000) and reaches its minimum in juvenile and adults (30-40/100,000) but increases again in the elderly (Panayiotopoulus 2010). According to the World Health Organization (WHO), epilepsy health expenses take up between 0.12 and 1.12% of the total health expenditure, depending on the continent involved, with drug-resistant patients consuming a disproportionate amount of these financial resources. The fiscal magnitude of epilepsy is determined by a combination of factors, including patients’ working activity (indirect costs), cost of pharmacological treatment for the patients and the health system and, to a lesser extent, other health costs e.g. psychological training, electroencephalography (EEG) etc., and the general cost to society (homecare and rehabilitation). Beyond the financial burden of the disease, morbidity, loss of quality of life and well-being of patients and/or their family members should also be considered. Hence, there is an urgent need to gain a deeper understanding into the epilepsy and the brain seizures associated with it (Begley and Beghi 2002). In principle, every brain can seize if exposed to a very strong stimulus, be it either physical or chemical. This phenomenon is not a specific reaction to a very strong impulse, but rather an old phylogenetic mechanism of overreaction. For example, brain seizures are known to occur in great apes and other primates, dogs, cats, mice and rats. In addition, they have been reported in reptiles and amphibians, and additionally in birds (Pitkänen et al. 2005). Seizures refer to abnormal electrical discharges in the brain, these being repetitive and synchronized, whereas the term convulsion (fit) is attributed to ictal semiological clinical presentations or symptoms (see 1.2.2 below); there exist seizures that can be registered on EEG alone, without body convulsions. A single seizure differs from an epileptic event. In the latter, at least two unprovoked convulsions must occur, albeit there is no agreement as to length of the interval involved between the events. Therefore, a single seizure, which might result from a severe impairment of the brain due to intracranial bleeding, infarction, hypoxia, inflammation or even drug/alcohol withdrawal, does not comprise epilepsy. This, however, does not make

-9- single seizures any less life-threatening; on the contrary, epileptic fits may be endured lifelong, whereas acute single seizures often require urgent resuscitation. Although the causes for these disturbances can vary markedly, the fits evoked may be indistinguishable at the level of EEG and other standard measurements (Panayiotopoulus 2010). As mentioned above, epilepsy is defined by the occurrence of two or more unprovoked seizures, which means that the manifestation of an epileptic fit takes place without a preceding brain injury associated with specific elements on EEG (Poeck et Hacke 2001). The refractory period (cf. refractory condition) between two epileptic convulsions yields a different EEG signature to the same period between two acute episodes. In the latter, severe disturbance of brain function can be registered in terms of very slow waves with a low amplitude and repetitive epileptiform discharges (Fig. 1A), whereas in epilepsy, a different pattern occurs. In this case, EEG measurements register mostly sustained background activity representing accurate brain function but additionally show intermittent characteristic spikes or spike-wave complexes, these indicating increased susceptibility of the brain for convulsions (Fig. 1B).

-10- Figure 1. Different EEG patterns between acute (A) and epileptic seizures (B). A: A two year old girl with acute onset of repetitive series of focal seizures due to a malignant brain tumour. B: A 14 year old boy suffering from epilepsy with generalized tonic-clonic seizures.

-11- 1.2 Types of seizures, epilepsies and epilepsy syndromes

Epilepsies are divided into three groups, according to the suspected underlying cause, these being “genetic”, “structural/ metabolic” and “unkown”. In addition to epilepsies, also seizures are typed, and these follow a different classification (Table 1). The Commission on Classification and Terminology of the International League Against Epilepsy (ILAE) has recently (2010) updated the nomenclatures for both epilepsies and seizures, which was last published over a decade earlier (Comission of terminology and classification of the ILAE 1989, Berg et al. 2010).

Table 1. The various types of seizures divided into three major categories

GENERALIZED FOCAL UNKNOWN Tonic-clonic (in any combination) Spasms Absence - typical Absence - atypical Absence – with special features Myoclonic Myoclonic - atonic Myoclonic - tonic Clonic Tonic Atonic Adapted from Berg et al. 2010

1.2.1 Seizures As mentioned before, various types of convulsions exist: generalized; focal; and unknown (spasms). In the interest of brevity, only a short description of the types of seizures is provided for background. Each seizure type shows specific patterns on EEG as well as in clinical presentation. Generalized epileptic fits are thought to originate from a certain point within the brain, whereby bilateral neural networks are recruited to spread rapidly a series of synchronized electrical discharges. Such networks could involve cortical and subcortical structures, e.g. the thalamus, but do not necessarily encompass the whole cortex. Even though separate seizures may seem to be localized, neither the region nor the lateralization stay the same from one seizure to the next. These convulsions can be asymmetric (Berg et al. 2010), i.e. only one of each pair of appendages begins to jerk. Focal seizures are envisioned to be generated within neuronal networks that are restricted to one brain hemisphere. Despite being classified as focal, they can indeed be strictly

-12- localized but may also be more diffuse. The origin of a focal seizure is unchangeable, remaining the same between fits. Nevertheless, the seizure may extend to the other hemisphere (Berg et al. 2010). It has been claimed that some seizure onsets cannot be distinguished with EEG, as they initiate subcortically, where EEG detection is difficult (Harvey and Freeman 2007).

1.2.2 Epilepsies As mentioned earlier, epilepsies are categorised as “genetic”, “structural/ metabolic” or “unknown”. The first class includes all epilepsies where a heredity basis for the fits can be presumed due to known gene alterations or family studies. The second category compiles all epilepsies where a structural defect of the brain is evident, either innate or acquired, but in addition, also defective brain metabolism may lead to seizures. As for the “unknown” grouping, our combined current knowledge still does not allow us to draw conclusions regarding the origin of this type of convulsions (Berg et al. 2010). In the context of epilepsies, while deciding the treatment strategy the treating physician needs to integrate the following points: the types of seizures, the underlying origin, the EEG pattern (ictal = record of a seizure-onset during EEG examination; interictal = in between seizures) and the neurological examination of the patient. By doing so, this could reveal several electro-clinical epilepsy syndromes, which can be age-dependent but may also be due to specific structures of the patient’s brain.

1.2.3 Epilepsy syndromes Although ILAE does not define the difference between epilepsy and epilepsy (or epileptic) syndrome, depending on the researcher involved, the distinction lies within our ability to detect the presence or absence of a clear cause. In those cases were a structural cause can be defined, these are categorized as epilepsy. However, when fits are indeed measurable with EEG and convulsions are presented clinically, albeit there is an absence of a clear brain disorder, these cases tend to be more ambiguous and are hence classified as epilepsy syndrome. At the General Hospital in Vienna, this distinction is not vital for our work. The exact definition of most of the syndromes is beyond the scope of the present thesis and therefore the list of syndromes is not exhaustive.

Neonatal epileptic seizures and syndromes These convulsions occur within the first four weeks of life. Most of them are acute and usually represent a clear sign of imminent neonatal encephalopathy. Furthermore, these episodes are an extreme hazard to the baby's life and may result in neurological

-13- impairment. On the hand, there exist epilepsy syndromes that occur at this age, these being referred to as: benign familial neonatal seizures; benign neonatal convulsions (non- familiar); early myoclonic encephalopathy; and Ohtahara's syndrome. The first and second conditions have a very good prognosis regarding the developmental outcome, whereas the third and fourth diseases have very high rates of morbidity and mortality (Panayiotopoulus 2010).

Idiopathic epileptic seizures and syndromes in infancy These epileptic fits occur exclusively in early childhood, and have an excellent outcome even without treatment with antiepileptic drugs. Febrile seizures represent the most common type of this class, and these usually appear if the body temperature of a child (aged between 6 months and 5 years) rises over 38.5°C due to an infection. They often happen in families with a genetic predisposition. Generalized tonic-clonic convulsions are the most common types among febrile seizures. Simple febrile convulsions (about 70%) occur in neurologically healthy children (a) only once within 24 hours, whereby these are (b) limited to 15 minutes duration, and are (c) generalized rather than focal. The more complex ones, also known as “atypical” have (a) two or more events in a row, (b) last longer, and (c) focal onset may be present. These atypical seizures have a significant increased risk for later development of epilepsy, which ranges from 6-8% in the presence of one characteristic (a-c) up to 49% with all three features visible (Panayiotopoulus 2010).

Epileptic encephalopathies of infancy and early childhood These syndromes are devastating diseases with a high and complex seizure load, which often leads to a severe cognitive decline. For this latter reason, researchers believe that the massive ictal and electrical activities seen with EEG are actually manifestations of constraints applied to the child’s brain maturation processes, thereby leading to neurodevelopmental delay and even regression. Each type of syndrome shows a distinguishing epileptic reaction by the immature brain, depending on its age. The etiologies may be multiple and are certainly not the same for all syndromes. The causes might even be structural, when considering that brain malformations may evoke such syndromes. The following syndromes are included: West syndrome; Dravet syndrome (“severe myoclonic epilepsy in infancy”); Lennox-Gastaut syndrome and “epileptic encephalopathy with continuous spike-and-wave during sleep (CSWS) including “Landau- Kleffner syndrome” (Panayiotopoulus 2010). For a deeper insight into these disorders further reading in specialised textbooks is recommended.

-14- Severe neocortical epileptic syndromes in infancy and childhood All syndromes included below are very rare but grim and no efficacious treatment exists. Kozhevnikov-Rasmussen syndrome is defined as unilateral severe encephalitis of unknown cause associated with hemiatrophy, refractory focal seizures that are mostly motoric (may include epilepsia partialis continua, which is a form of focal motor status epilepticus) and developmental decline as well as paresis. The only treatment option in these children with profound neurological deficits is the removal of the affected brain hemisphere. Furthermore, the phenomenon of partial migrating seizures is also included as a syndrome with a rather poor prognosis (Panayiotopoulus 2010).

Benign childhood focal seizures and related syndromes These are the most common forms of epilepsy in childhood and comprise about 25% of all afebrile seizures. The ILAE acknowledges three syndromes: benign childhood epilepsy with centro-temporal spikes (BCECTS), early onset benign childhood occipital epilepsy (Panayiotopoulus syndrome), late onset childhood occipital epilepsy (Gastaut syndrome). In all three cases, seizures are easy to control with antiepileptic drugs and no impairment of neurological development occurs (Panayiotopoulus 2010).

Idiopathic generalized epilepsies For the sake of being concise, these are only mentioned as they are not part of the present work; the term absence refers to a child’s complete loss of awareness. The following syndromes fall into this category: epilepsy with myoclonic-astatic seizures, epilepsy with myoclonic absences, childhood absence epilepsy, juvenile absence epilepsy, juvenile myoclonic epilepsy and epilepsy with generalized tonic-clonic seizures only. Most of them are a lifelong disease, which can be controlled rather easily with appropriate medication.

Structural focal epilepsies As mentioned in section 1.2.1, focal or partial seizures always originate from mostly a single brain cortex. They are typed according to their anatomical region of origin, as follows: temporal-, frontal-, parietal- and occipital lobe epilepsies. Despite a few hereditary forms, it is presumed that these epilepsies have a structural cause, such as: benign or malignant brain tumours, infectious diseases (viral, bacterial or parasitic, but interestingly not prionic), cerebrovascular disorders, malformations of cortical development, metabolic diseases and traumas. In these forms of epilepsies, therapeutic management might expand from drug treatment to surgical intervention (Panayiotopoulus 2010).

-15- 1.3 Drug management and therapeutic options

The goal of every epilepsy therapy is complete freedom of seizures without medication- induced adverse reactions. In about 50-70% of all epilepsy patients this is possible with only a single adequately chosen antiepileptic drug (AED, Glauser et al 2006). A large variety of drugs exist from which to be chosen, depending on the type of seizure and epilepsy syndrome. Commercially available textbooks on epilepsy therapy are recommended for further reading. In addition to classical AEDs, epilepsy surgery can be taken into account (see below). Furthermore, additional treatment strategies exist such as vagal nerve stimulation and ketogenic diet.

1.4 Surgical intervention in epilepsy

Thirty percent of all epilepsy patients are drug resistant. Drug resistance in this context is usually defined as failure of the patient to improve following treatment using two regimens of appropriate AEDs (mono- or polytherapy) that are well tolerated (Kwan and Brodie 2010, Kwan et al., 2010). Some of these drug-resistant patients could be helped with surgery, with a 74-84% chance of becoming seizure free (Cole and Wiebe 2008, de Tisi et al. 2011). Patients with proven drug resistance should be referred to tertiary epilepsy centers for prompt evaluation. Surgery has become an important option in epilepsy treatment over the last 30 years. Since this thesis is concerned with surgery in young epilepsy patients, descriptions of presurgical assessments is focused on children. It transpires that morbidity and mortality rates following surgery in the young are not higher compared to the situation in adults. Therefore, drug-resistant children should be evaluated for surgery early so as to avoid later developmental problems (Skirrow et al. 2011, Dunkley et al. 2011, Maton et al. 2008).

1.4.1 Presurgical evaluation Surgery may only be considered in cases where seizures are focal in nature. Diagnostics ahead of surgery consist of the following techniques: long-term video-EEG monitoring; high-resolution magnetic resonance imaging (MRI); neuropsychological and developmental evaluation; positron emission tomography (PET); and where necessary ictal single photon emission computer tomography (SPECT). The dysfunctional areas of the cortex need to be defined before surgical removal of brain structures can be considered. The establishment of such areas with the right diagnostic tools is the fundamental basis of epilepsy surgery at dedicated centres, such as the one in Vienna.

-16- By convention, the following regions are considered in presurgical evaluation (Rosenow and Lüders 2001):

The seizure-onset zone This refers to the portion of the cortex where the inception of seizures is visualised. Usually, the determination is possible with scalp EEG, albeit some patients require invasive EEG recordings for data gathering. This latter technique includes the implantation of electrodes directly onto the brain surface or into brain structures. In addition, ictal SPECT can be of help.

The epileptogenic zone This term is more a theoretical concept than a physical structure. In addition to the aforementioned seizure-onset zone it also includes the cortical areas which have the potential to generate epileptic fits on their own should the seizure-onset zone be removed. To date, there is no diagnostic option to examine this zone.

The epileptogenic lesion This refers to the only radiologically visible structural lesion assumed to cause epilepsy. Nowadays, high-resolution MRI is used.

The symptomatogenic zone It is that part of the brain, which, when impacted by epileptic discharges, generates clinical seizure symptoms, referred to as ictal semiology. Nevertheless, this region does not necessarily overlap with the seizure-onset zone.

The irritative zone This region refers to that part of the brain producing interictal epileptiform discharges as seen with scalp EEG.

The functional deficit zone This defines the area of the cortex which shows deficits in the neurological examination during the interictal period. In general, PET and interictal SPECT are done to delineate this region.

-17- 1.4.2 Epilepsy Center Vienna The epilepsy centre of Vienna was established in 1995 as a consortium of the Departments of Neurology, Neurosurgery, Pediatrics and Adolescent Medicine, Neuroradiology, Nuclear Medicine and the Institute of Neurology. To date, over 500 patients with chronic drug-resistant regional/structural epilepsy, among them 200 children and adolescents below the age of 18 years have been evaluated and had undergone surgery at our centre. Unlike the situation in other life-threatening events requiring surgery, the one involved here is a protracted process, beginning with skull removal and often electrode implantation, followed by extensive EEG, and finally resection and remounting of the skull and suturing of the scalp. Post-operative follow-up visits of the pediatric patients are performed three months after surgery, then once per year until the age of 18 years (further follow-up, if needed is performed at the Department of Neurology).

1.4.3 Video-EEG Monitoring in Vienna At the Epilepsy Monitoring Unit (EMU) for children at the Department of Pediatrics there are four beds available for simultaneous video-EEG recordings. The collected material is reviewed for either diagnostic evaluation of the epilepsy syndrome or for detailed determination of the various regions prior to surgical intervention by experienced childhood epileptologists (M.F., A.M., A.D. and G.G.), see section 1.4.1 above. The findings are then discussed with specialists in epileptology, neuroradiology and neurosurgery (M.F., D.P. and T.C.) to determine the surgical procedure.

1.4.4 Neuroimaging in Vienna At our centre a standardized MRI protocol is established to detect epileptogenic lesions, where only a slight modification to this protocol is applied when dealing with adults. All scans take place at the Department of Radiology on a Siemens 3Tesla machine. The diagnostics are undertaken by experienced neuroradiologists (D.P., G.K. and M.S.). Children are scanned with the following sequences: axial fluid attenuated inversion recovery (FLAIR) T2* with 3-4mm slice thickness, paracoronal T1 Volume (1-2mm) of the whole head and T2 turbo spin echo (TSE) and inversion recovery (IR) (2mm). If pathology is suspected within the temporal lobe, all sequences are perpendicular to the hippocampal angle.

-18- Figure 2: Focal cortical dysplasia. Right frontal FCD on an axial FLAIR weighted image.

1.5 Epileptogenic lesions

A variety of neuropathological changes can be found in surgical specimens obtained from epilepsy patients: hippocampal sclerosis, glioneuronal tumors, vascular malformations, ischemia, intracerebral hemorrhage, glial scars, inflammation, and malformations of cortical development (MCDs). MCDs are increasingly recognised in children and adolescents with drug-resistant epilepsies, however, their histopathological characterisation remains a challenge. The problem lies within existing classification schemes (Taylor et al 1971; Palmini et al 2004; Blümcke et al 2011), which do not cover the full range of changes observed in the histopathological specimens of these patients. The same problems exist with respect to MRI classification (Barkovich et al, 2001; Tassi et al, 2002; Colombo et al, 2003). In 1971, Taylor and colleagues defined “Focal Cortical Dysplasia (FCD)” for circumscribed MCDs in ten patients after surgical intervention (Taylor et al 1971). Thereafter, the ranges of FCD widened to comprise also disturbances in cortical architecture without abnormal neurons. In 2004, Palmini and co-workers came up with the most widely accepted classification system (Palmini et al, 2004).

-19- In addition, also the ILAE published a broadened scheme for FCD (Blümcke et al 2011). FCD type I covers singular FCDs involving defective cortical architecture in one or more parts of the brain. FCD type II is equal to what was previously described by the two groups of Taylor and Palmini (Taylor et al 1971, Palmini et al 2004, Blümcke et al 2011). This category is divided by the absence (type IIa) and the presence of balloon cells (type IIb). The new FCD type III subgroup contains those variants of cortical dyslamination combined with- or next to other principal lesions. These include hippocampal sclerosis (FCD type IIIa), glio-neuronal tumors (IIIb), vascular malformations (IIIc) or brain injuries aquired during early life (IIId). Previously defined seizure outcome varies tremendously between FCD subtypes (Fauser et al. 2004, Krsek et al. 2008; 2009). According to our own two groups of 43 children with histologically proven FCD and 34 with hippocampal sclerosis, the outcome lies within the published range. The average number of seizure-free children among all our epilepsy surgery patients is already about 60%.

-20- 1.6 Aims of this thesis

In summary, children represent a major group of patients with epilepsy. About 35% of them do not respond to traditional antiepileptic drug treatment at all (Fusco and Vigevano 2004). Furthermore, in 70-80% of the children and young adults disorders of cognitive development can be observed (Bast et al 2006). Consequently, these patients should be referred to epilepsy surgery evaluation early. Despite other epileptogenic lesions (e.g. hippocampal sclerosis) little is known about the ideal candidacy for epilepsy surgery in patients with FCD. Therefore, the aims of this thesis were the following:

• To create a database determining and characterizing all patients with FCDs through careful evaluation of patients' histories; • Thus, leading to the report of positive surgical outcome in one girl; • To type all neuropatholoical specimens from the Vienna Pediatric epilepsy surgery program according to the new ILAE classification system for FCD (Blümcke et al 2011) and introduction of all data in the European Epilepsy Brain Bank; • To define clear-cut histological characteristics in brain samples of FCDs taking the following parameters into account: cortical thickness, grey-white matter border, heterotopic neuropil in the deep white matter; and myelination; • To find clinical implications that might increase the predictive value of presurgical MRI.

-21- 2 Synopsis

In a first step, all clinical data available on pediatric patients at the Vienna epilepsy surgery program were carefully reviewed. A database was created to characterise all children and young adults. In the course of this work we detected a highly interesting case of childhood-onset limbic encephalitis (publication 1: Muehlebner et al, 2010). The acute onset with series of temporal lobe seizures and initial hyperintensity of one hippocampus on MRI lead to an eager exploration to detect an immunological cause e.g. specific anti-neuronal antibodies. However, rapid progression to unilateral hippocampal atrophy and resistance of the seizures to antiepileptic drug treatment made the girl a candidate for epilepsy surgery. The search for specific antibodies continued on the resected hippocampus which showed neuronal cell loss in the CA1 sub-field of the cornu ammonis and distinct inflammation. No auto-antibodies could be found. In the follow-up she was seizure-free for more than a year. However, seizures returned after 18 months and she was therefore readmitted to our department for implantation of subdural grids and depth electrodes. After invasive recordings she could undergo surgical intervention again (additional resection of a more posterior part of the affected hippocampus) and up to date she is seizure-free. Next, we focused on the population of patients with FCDs. To get better insight into the spectrum of neuropathological changes, brain tissue gained from all pediatric epilepsy patients who had undergone epilepsy surgery at the Vienna University Hospital since 2000 were evaluated and reclassified retrospectively. All material available from our patients (n= 77) was carefully reviewed and systematically analyzed. In seven children brain tissue was either unavailable or of poor quality. During the histological analysis, classification of malformations of cortical development – as in other centres - became a major issue of debate. In addition, a European Brain Bank was initiated at the Department of Neuropathology of the University Erlangen-Nuremberg. Therefore, all FCD specimens were sent to Erlangen for referential review and reclassification according to the new ILAE classification system as well as inclusion into the brain bank. During a nine months stay at the University of Erlangen (which was partially financed by the Austrian Chapter of the ILAE), consecutive analysis of all epilepsy specimens included in the European Epilepsy Brain Bank subsuming the Viennese samples focused on FCD was performed under the supervision of Prof. Dr. I. Blümcke. The work lead to a review paper regarding the detailed neuropathological work-up of epilepsy surgery specimens. The main focus of this paper lies accordingly on the newly introduced ILAE classification.

-22- Each FCD variant was carefully presented and additional graphical material introduced to extend the original publication (publication 2: Blümcke & Mühlebner, 2011). Further, we identified 52 epilepsy patients with very well preserved neocortical samples and histological evidence for FCD from the epilepsy centres Erlangen, Vogtareuth and Vienna among the European Epilepsy Brain Bank. These were studied and reclassified according to the 2011 classification. Histopathologic parameters suspected to be relevant also for presurgical neuroimaging were included. These consisted of quantitative measurements of cellular profiles, cortical thickness, heterotopic neurons in white matter, and myelination. All findings were correlated between FCD subtypes and controls (age- and localisation matched, n=36) using SPSS for statistical analysis. As a result of the testing neuronal body and nucleic diameters of dysmorphic neurons in both FCD Type II variants were significantly increased. In addition, the width of the neocortex was significantly broader in both types. Only the clear loss of myelin content in the presence of balloon cells could distinguish FCD Type IIb from IIa. Further, our analysis showed, that deficiency in myelination in FCD Type IIb may be due to an impaired oligodendroglial lineage differentiation. In this context, the loss of myelin might contribute to the so-called “transmantle sign” - a characteristic feature for FCD in presurgical MRI. In contrast, a thinner cortical ribbon and higher numbers of neuronal cells could be identified in FCD Type Ia. However, based on the inconsistency of cortical thickness within the control group these criteria did not reach significance. The grey-white matter border and the deep white matter showed increased amount of neuropil in all FCD subtypes. Consequently, many patients with clinically suspected FCD remain MRI-negative. Thus, development of MRI protocols based on histopathologic criteria may improve visual detection (publication 3: Mühlebner et al 2011).

Finally, we focused on clinico-pathological variants of childhood drug-resistant temporal lobe epilepsy (TLE): pre- and postsurgical data of all 34 patients with hippocampal sclerosis, who underwent epilepsy surgery at our centre, were reviewed. Due to either inadequate brain tissue or MRI scans of poor quality only 17 could be included into the study (14 females; 9 left TLE). All children had presurgical evaluation and annual postoperative follow-up investigations including video-EEG monitoring, neuropsychological and developmental examination as well as modern neuroimaging (MRI, PET, SPECT). Candidacy for - and type - of surgery were decided by an experienced pediatric epilepsy surgery board (MF, DP and TC). Outcome was defined according to the ILAE proposal for classification of outcome (Wieser et al, 2001). Video-EEG monitoring including sphenoidal

-23- electrodes was analyzed by experienced epileptologists (MF, GG, AD, AM) with regard to clinical lateralizing signs defining the clinical symptomatogenic zone, the seizure onset zone and the irritative zone; Interictal EEG findings were grouped into three categories:

a “temporo-mesial EEG field” (spikes mainly localised in sphenoidal electrodes and very little spread) an “extended EEG field” (marked additional involvement of lateral temporal, i.e. temporo-frontal and temporo-occipital electrodes) a “generalized EEG field” (seizure onset exclusively temporal, but generalised interictal epileptiform changes)

MRI examinations were performed on a 1,5 Tesla system with sequences as follows: axial FLAIR sequence of the entire brain (5mm), paracoronal (perpendicular to the course of the hippocampus) turbo SE T2w sequence (2mm), Inversion-recovery and FLAIR sequence (3mm). MRI was reviewed with respect to presence of hippocampal atrophy/sclerosis, small temporal lobe, blurring of the grey-white matter junction, signal changes of the subcortical white matter, and thickening of the adjacent cortex. Images were initially reviewed by GK and DP and afterwards re-evaluated by MS blinded to the previous results and histological evaluation. Histopathological specimens were carefully orientated, trimmed and sectioned in the plane and according to its axis. The specimens were routinely processed for histopathology. In addition to hematoxylin and eosin (HE) stains, immunohistochemistry was performed. We stained tissue sections with antibodies against neuronal nuclei (NeuN, Chemicon; 1:1000), microtubule associated protein 2 (MAP2, Chemicon, 1:500), glial fibrillary acid protein (GFAP, Dako, 1:3000), non-phosphorylated neurofilament (SMI32, Sternberger, 1:200). Histopathological analysis and diagnosis done by JH and AM was blinded to MRI changes. Formol-fixed paraffin-embedded tissue was sent to the Department of Neuropathology at the University Hospital Erlangen for reference pathological classification according to the 2011 ILAE classification system, with special attention to the novel category of FCD IIIa. Patients were grouped according to type of surgery (anterior temporo-polar resection vs. selective amygdalohippocampectomy) and presence/absence of FCD IIIa within the temporal lobe. All 17 children showed hippocampal sclerosis (HS).

-24- Group 1: Six children (5 females) had anterior temporo-polar resection. All six children displayed additional FCD IIIa. The age of epilepsy onset in this group was 7 (+/- 2,7) years. All six patients had a history of febrile seizures. Five patients showed an extended EEG field, one a generalized EEG field, not one displayed a temporo-mesial field. In five the typical MRI changes of architectural dysplasia were found. One child was mentally impaired. All six of them were seizure free after surgery (Wieser 1a).

Group 2: Five children (5 females) who underwent anterior temporo-polar resection showed HS without FCD IIIa. The average age of epilepsy onset in this group was 4 (+/- 5,5) years. Two patients had febrile seizures. Four showed an extended EEG field, one showed generalized epileptiform discharges. Only one displayed the typical MRI changes of architectural dysplasia. One child was mentally impaired. Postsurgical outcome was Wieser 1a in two children, Wieser 2 was found in one and Wieser 3 in two patients.

Group 3: Six patients (4 females) underwent selective amygdalohippocampectomy and therefore temporal cortex was not available for tissue analysis. The average age of epilepsy onset in this group was 5 (+/- 1,5) years. four patients had febrile seizures. Four children presented the temporo-mesial EEG field, whereas two showed a generalized EEG pathology. None of them had changes in the temporal lobe on MRI scan or was mentally impaired. Five were seizure free after surgery (Wieser 1a), one had outcome Wieser 3.

If histopathological diagnosis did not match (1 case) decision was made in favour of IB. In case of MRI changes a consensus was made between DP, GK and MS to overcome different diagnosis (2 cases).

-25- anterior temporo-polar anterior temporo- selective amygdalo- resection polar resection hippocampectomy + FCD IIIa Cases 6 5 6 architectural dysplasia on MRI scan 5 1 0 Sex 5w, 1m 5w 4w, 2m age at epilepsy onset 7 (+/- 2,7) 4 (+/- 5,5) 5 (+/- 1,5) Febrile seizure 6 2 4 EEG field 1. extended 5 4 0 2. temporo-mesial 0 0 4 3. generalized 1 1 2 encephalopathy 1 1 0 Outcome Wieser 1a 6 2 5 Outcome Wieser 2 0 1 0 Outcome Wieser 3 0 2 1

The 2011 ILAE classification for focal cortical dysplasia includes recent work on temporal lobe sclerosis in order to form a new subgroup of MCDs (Thom et al, 2007). The FCD type IIIa is defined through cortical dysplasia within the temporal lobe plus hippocampal sclerosis. In our series, six children showed typical histopathological features of this new entity. As shown above, this subgroup showed distinct clinical features: onset of epilepsy was later (7 vs 4 years), history of febrile seizures was reported more frequently (6 vs 2) and outcome was more favourable (Wieser 1a in 6 vs 2). In summary, the combination of certain clinical characteristics, clear-cut signal alterations on MRI scan, sufficient surgical resection as well as certain histopathological diagnosis is associated with a favourable outcome in childhood temporal lobe epilepsy. On the other hand, missing morphological features of malformation of cortical development seems to influence outcome negatively (Mühlebner et al, 2012 in prep.). Our findings of outcome after surgery are consistent with the literature (Krsek et al, 2009; Lerner et al, 2009; Téllez-Zenteno JF et al, 2010).

-26- 3 References

Barkovich AJ, Kuzniecky RI, Jackson GD, Guerrini R and Dobyns WB (2001). Classification system for malformations of cortical development: update 2001. Neurology. 57:2168-78. Bast T, Ramantani G, Seitz A and Rating D (2006). Focal cortical dysplasia: prevalence, clinical presentation and epilepsy in children and adults. Acta Neurol Scand. 113:72-81. Begley CE and Beghi E (2002). The economic cost of epilepsy: a review of the literature. Epilepsia. 43 Suppl 4:3-9. Berg AT, Berkovic SF, Brodie MJ, Buchhalter J, Cross JH, van Emde Boas W, Engel J, French J, Glauser TA, Mathern GW, Moshé SL, Nordli D, Plouin P and Scheffer IE (2010). Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005-2009. Epilepsia. 51:676-85. Blumcke I, Thom M, Aronica E, Armstrong DD, Vinters HV, Palmini A, Jacques TS, Avanzini G, Barkovich AJ, Battaglia G, Becker A, Cepeda C, Cendes F, Colombo N, Crino P, Cross JH, Delalande O, Dubeau F, Duncan JS, Guerrini R, Kahane P, Mathern GW, Najm I, Özkara C, Raybaud C, Represa A, Roper SN, Salamon N, Schulze- Bonhage A, Tassi L, Vezzani A and Spreafico R (2011) The clinico-pathological spectrum of Focal Cortical Dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission. Epilepsia. 52:158-174. Blumcke I and Muhlebner A (2011). Neuropathological work-up of focal cortical dysplasias using the new ILAE consensus classification system - practical guideline article invited by the Euro-CNS Research Committee. Clin Neuropathol. 30:164-77. Cole A and Wiebe S (2008). Debate: Should antiepileptic drugs be stopped after successful epilepsy surgery? Epilepsia. 49 (Suppl 9):29-34. Colombo N, Tassi L, Galli C, Citterio A, Lo Russo G, Scialfa G and Spreafico R (2003). Focal cortical dysplasias: MR imaging, histopathologic, and clinical correlations in surgically treated patients with epilepsy. AJNR. 24:724-33. Commission on Classification and Terminology of the International League Against Epilepsy. (1989) Proposal for revised classification of epilepsies and epileptic syndromes. Epilepsia 30:389–399. Dunkley C, Kung J, Scott RC, Nicolaides P, Neville B, Aylett SE, Harkness W and Cross JH (2011). Epilepsy surgery in children under 3 years. Epilepsy Res. 93:96-106.

-27- Fauser S, Schulze-Bonhage A, Honegger J, Carmona H, Huppertz HJ, Pantazis G, Rona S, Bast T, Strobl K, Steinhoff BJ, Korinthenberg R, Rating D, Volk B, Zentner J (2004) Focal cortical dysplasias: surgical outcome in 67 patients in relation to histological subtypes and dual pathology. Brain. 127:2406-2418. Fusco L and Vigevano F (2004). Indications for surgical treatment of epilepsy in childhood: a clinical and neurophysiological approach. Acta Paediatr Suppl. 445:28-31.µ§ Glauser T, Ben-Menachem E, Bourgeois B, Cnaan A, Chadwick D, Guerreiro C, Kalviainen R, Mattson R, Perucca E and Tomson T (2006). ILAE treatment guidelines: evidence- based analysis of antiepileptic drug efficacy and effectiveness as initial monotherapy for epileptic seizures and syndromes. Epilepsia. 47:1094-120. Harvey AS and Freeman JL (2007). Epilepsy in hypothalamic hamartoma: clinical and EEG features. Semin Pediatr Neurol.14:60-4.Kwan P, Arzimanoglou A, Berg AT, Brodie MJ, Hauser WA, Mathern G, Moshé, Perucca E, Wiebe S and French J (2010). Definition of drug-resistant epilepsy: Consensus proposal by the ad hoc Task Force of the ILAE Commission on Therapeutic Strategies. Epilepsia. 51:1069-1077. Kwan P and Brodie MJ (2010). Definition of refractory epilepsy: defining the indefinable? Lancet Neurol. 9:27-29. Krsek P, Maton B, Korman B, Pacheco-Jacome E, Jayakar P, Dunoyer C, Rey G, Morrison G, Ragheb J, Vinters HV, Resnick T, Duchowny M (2008). Different features of histopathological subtypes of pediatric focal cortical dysplasia. Ann Neurol. 63:758-769. Krsek P, Pieper T, Karlmeier A, Hildebrandt M, Kolodziejczyk D, Winkler P, Pauli E, Blumcke I, Holthausen H (2009). Different presurgical characteristics and seizure outcomes in children with focal cortical dysplasia type I or II. Epilepsia. 50:125-137. Lerner JT, Salamon N, Hauptman JS, Velasco TR, Hemb M, Wu JY, Sankar R, Donald Shields W, Engel J Jr, Fried I, Cepeda C, Andre VM, Levine MS, Miyata H, Yong WH, Vinters HV, Mathern GW (2009). Assessment and surgical outcomes for mild type I and severe type II cortical dysplasia: a critical review and the UCLA experience. Epilepsia. 50:1310-35. Maton B, Jayakar P, Resnick T, Morrison G, Ragheb J and Duchowny M (2008). Surgery for medically intractable temporal lobe epilepsy during early life. Epilepsia. 49:80-7. Muehlebner A, Groeppel G, Pahs G, Hainfellner JA, Prayer D, Czech T and Feucht M (2010). Beneficial effect of epilepsy surgery in a case of childhood non-paraneoplastic limbic encephalitis. Epilepsy Res. 90:295-9.

-28- Mühlebner A, Coras R, Kobow K, Feucht M, Czech T, Stefan H, Weigel D, Buchfelder M, Holthausen H, Pieper T, Kudernatsch M and Blümcke I (2011). Neuropathologic measurements in focal cortical dysplasias: validation of the ILAE 2011 classification system and diagnostic implications for MRI. Acta Neuropathol. [Epub ahead of print]. Palmini A, Najm I, Avanzini G, Babb T, Guerrini R., Foldvary-Schaefer N., Jackson G., Luders H O, Prayson R, Spreafico R and Vinters HV (2004). Terminology and classification of the cortical dysplasias. Neurology. 6:S2-8. Panyiotopoulos CP (author, 2010). A clinical guide to epileptic syndromes and their treatment. Revised 2nd edition. Springer. Pitkänen A, Schwartzkroin PA and Moshè SL (editors, 2005). Models of seizures and epilepsy. 1st edition. Elsevier. Poeck and Hacke (authors, 2001). Neurologie. 11th edition. Springer. Rosenow F and Lüders H (2001). Presurgical evaluation in epilepsy. Brain. 124:1683-700. Skirrow C, Cross JH, Cormack F, Harkness W, Vargha-Khadem F and Baldeweg T (2011). Long-term intellectual outcome after temporal lobe surgery in childhood. Neurology. 276:1330-7. Tassi L, Colombo N, Garbelli R, Francione S, Lo Russo G, Mai R, Cardinale F, Cossu M, Ferrario A, Galli C, Bramerio M, Citterio A and Spreafico R (2002). Focal cortical dysplasia: neuropathological subtypes, EEG, neuroimaging and surgical outcome. Brain. 125:1719-32. Taylor DC, Falconer MA, Bruton CJ and Corsellis JA (1971). Focal Dysplasia of the cerebral cortex in epilepsy. J Neurol Neurosurg Psych. 34:369-87. Téllez-Zenteno JF, Hernández Ronquillo L, Moien-Afshari F and Wiebe S (2010). Surgical outcomes in lesional and non-lesional epilepsy: a systematic review and meta-analysis. Epilepsy Res. 89:310-8. Thom M, Eriksson S, Martinian L, Caboclo LO, McEvoy AW, Duncan JS and Sisodiya SM 2009). Temporal lobe sclerosis associated with hippocampal sclerosis in temporal lobe epilepsy: neuropathological features. J Neuropathol Exp Neurol. 68:928-38. de Tisi J, Bell GS, Peacock JL, McEvoy AW, Harkness W, Sander JW and Duncan JS (2011). The long-term outcome of adult epilepsy surgery, patterns of seizure remission, and relapse: a cohort study. Lancet. 378:1388-95. Wieser HG, Blume WT, Fish D, Goldensohn E, Hufnagel A, King D, Sperling MR, Luders H Pedley TA (2001). ILAE Commission Report. Proposal for a new classification of outcome with respect to epileptic seizures following epilepsy surgery. Epilepsia. 42:282- 6.

-29- 4 Publications

4. 1 Publication 1: Epilepsy Research 2010 Aug;90(3):295-9

Beneficial effect of epilepsy surgery in a case of childhood non- paraneoplastic limbic encephalitis

Angelika Muehlebner, MD1,2; Gudrun Groeppel, MD1; Gerald Pahs, MD1, Johannes A. Hainfellner, MD2; Daniela Prayer, MD3; Thomas Czech, MD4; and Martha Feucht, MD1.

Affiliations: 1 Department of Pediatrics 2 Institute of Neurology 3 Department of Radiology, Division of Neuroradiology 4 Department of Neurosurgery Medical University Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria

Contact information for the corresponding author: Univ. Prof. Dr. Martha Feucht Department of Pediatrics Medical University Vienna Waehringer Guertel 18-20; 1090 Vienna Austria Tel: 0043- 1-40400-3385; FAX 00431 40400 2277 E-Mail: [email protected]

-30- Summary

This 15-year-old girl had subacute onset of secondary generalized seizures, confusion, and subsequent memory decline. MRI showed bilateral hippocampal swelling progressing to unilateral mesial temporal sclerosis (MTS) within 12 months. Epilepsy surgery was performed, and laboratory data were consistent with non-paraneoplastic limbic encephalitis. 18 months after epilepsy surgery, the patient is seizure-free with stable cognitive functions.

Keywords: Mesial temporal lobe epilepsy, drug-resistance, unilateral hippocampal sclerosis, epilepsy surgery, limbic encephalitis

-31- Introduction

Autoimmune limbic encephalitis (LE) is an inflammatory disease primarily restricted to the hippocampus. Clinical characteristics include subacute onset, profound cognitive impair­ ment (especially memory decline), psychiatric disorders, and epileptic (usually complex partial) seizures (Bien et al., 2007a). Serial brain MRIs have shown uni- or bilateral swollen temporomesial structures at disease onset and progressive temporomesial sclerosis/at­ rophy (Urbach et al., 2006). Histopathology shows medial temporal lobe inflammation, i.e. infiltrating round cells (T lymphocytes) and activated microglial cells, which sometimes form nodules. LE can occur as a paraneoplastic phenomenon (PNLE) (Gultekin et al., 2000) associ­ ated with onconeuronal antibodies to intracellular antigens, mainly Hu, CRMP5/CV2, Ma2, amphiphysin in patients with small cell lung cancer (SCLC), neuronal surface antibodies to voltage-gated potassium channel (VGKC) in and glutamic acid decarboxylase (GAD) in combination with SCLC or thymoma and antibodies to N-methyl-D-aspartate receptor (NM­ DA-R) together with ovarian teratoma (Dalmau et al., 2008a). Removal of the tumour seems to improve neurological outcome (Dalmau et al., 2008a). Other causes of LE may be direct invasion of a virus, usually human herpesvirus (HHV) – 6 (Theodore et al., 2008) and autoimmune processes without tumour association. Antibodies to specific neuronal cell-surface membrane proteins have been discovered in different forms of non-paraneo­ plastic limbic encephalitis (NPLE), in 18% of 53 patients anti-VGKC (Buckley et al., 2001, Malter et al., in press), in 17% of 53 patients anti-GAD (Mata et al., 2008, Malter et al., in press), in 7,5% of 53 patients anti-NMDA-R (Dalmau et al., 2008b, Malter et al., in press) antibodies. However, in about 20-55% of NPLE, no such auto-antibodies have been found (Graus et al., 2008; Malter et al., in press). NPLE do not show a good response to conven­ tional antiepileptic drugs (AEDs), but respond to immunotherapies. Immunosuppression is therefore widely considered as first line treatment in these patients (Bataller et al., 2007). NPLE has been identified almost exclusively in adults, whereas most paediatric-onset LE seem to be paraneoplastic (Florance et al., 2009). We report a fifteen year old girl with NPLE and favourable outcome after epilepsy surgery.

-32- Case

This healthy 15-year-old girl presented with memory and concentration problems, followed by episodes of nausea, epigastric sensations, confusion and blurred language since January 2007. The patient was otherwise healthy, with no medical problems in the past. Development had been normal. She attended high school with recent decline in academic performance. Family history was unremarkable. In July 2007, she was hospitalized because of two secondary generalized tonic-clonic seizures (GTCS). Clinical examinations at admission were normal, as were routine blood tests. Initial evaluation did not suggest neoplastic, toxic or metabolic aetiology. Virus-PCR (including adeno-, coxsackie-, enteroviruses, measles, mumps, rubella, influenza A + B, JC virus, HSV 1 + 2, EBV, CMV, VZV and HHV 6 + 7)and diagnostic work-up for bacterial or fungal cause of infection were negative. Immunological parameters including ASLO, anti- DNA, streptococcal DNAse, ANA, ANCA and Lupus serology were normal. Cerebrospinal fluid (CSF) showed 6 cells/µl (primarily lymphocytes) and normal protein content. Neuropsychological testing revealed normal full-IQ, but deficits in verbal and non-verbal episodic memory. Magnetic resonance imaging (MRI) displayed bilateral abnormal hyperintense signals in the mesial temporal areas on T2/ FLAIR images, more prominent on the left side (fig 1A - B). FDG-Positron-Emission-Tomography (PET) showed reduced tracer-uptake of the left mesiotemporal region. Diagnosis of LE was based on the features of (1) “limbic” signs and symptoms for ≤ 5years (impaired recent memory, temporal lobe seizures and affective abnormalities) and (2) prototypical MRI evidence of mediotemporal encephalitis. Paraneoplastic aetiologies were excluded following the Bonn protocol for tumour search in limbic encephalitis (Malter et al, in press) including abdominal ultrasound examination and computer tomography scan with contrast enhancement of the chest and abdominal region, gynaecological examination, and serological tests for both cancer-specific (CEA, CA125 and CA 19-9) and tissue-specific tumour markers (AFP, beta-HCG, Thyroglobulin and neuron-specific Enolase). All results were negative and therefore the LE was classified as non-paraneoplastic. For autoantibody screening serum was analyzed for anti- Yo, Hu. Ri and Ma antibodies (immunoblot and indirect immunoflorescence), anti-GAD antibodies at the Medical University of Vienna and sent to Vincent A (University of Oxford, Weatherall Institute of Molecular Medicine) for antibodies against VGKC and NMDA-R. Results were negative. NPLE was diagnosed and treatment with both 1g of methylprednisolone/day for 3 days and levetirazetame was initiated. However, a few month later complex-partial

-33- seizures occurred with increasing frequency (1-2/d) despite high doses of levetiracetame, carbamazepine, oxcarbazepine, topiramate and clobazame. School performance declined further and neuropsychological re-evaluation in March 2008 showed cognitive deterioration. MRI scans three and eight months after disease-onset (October 2007 and March 2008), demonstrated development of left hippocampal atrophy (HA) and T2/FLAIR signal increase indicating hippocampal sclerosis (HS) (fig 1 C and D). EEG-video-monitoring from May 2008 showed interictal spikes and seizure onset in the left sphenoidal electrode (fig. 2 A). Left anterior 2/3 temporal-lobectomy was performed on July 25th 2008. The resected specimen, divided into several parts (anterior, middle and posterior part of the hippocampus as well as the amygdala) were carefully orientated, trimmed and sectioned in the plane perpendicular to its longitudinal axis. The specimens were routinely processed for histopathology. In addition to hematoxylin and eosin (HE) stains, immunohistochemistry was performed. We stained tissue sections with antibodies against CD 3 (Neomarkers, Thermo Fisher Scientific Inc, Fremont, CA, USA, 1:100), CD 4 (Novocastra, Leica Microsystem, Germany, 1:10), CD 8 (Dako, Glostrup, Denmark, 1:100), CD 20 (Dako, Glostrup, Denmark, 1:200), CD 45 RO (OPD 4, Dako, Glostrup, Denmark, 1:100), CD 68 (Dako, Glostrup, Denmark, 1:1000), Caspase 3 (Cell Signalling Technology Inc, Danvers, MA, USA, 1:100) and Caspase 9 (Chemicon, 1:20). For antigen retrieval (except CD 4), the slides were pretreated in a microwave oven for 10 min in 10mM citric acid buffer at pH 6. For IgG-staining 4µm thick sections were deparaffinized in xylene. Endogenous peroxidase was blocked by incubation in 0.03% hydrogen peroxide for 30 min. The sections were rehydrated through a descending ethanol series and rinsed in distilled water. The slides were heated in 10 mM citrate buffer. Slides were then exposed to 3% milk powder in phosphate buffered saline (PBS) for 30 min at room temperature to reduce non- specific background. The slides were washed with PBS several times and incubated with patient’s serum 1:1000 in 1.5% milk powder in PBS at 4°C overnight. The sections were then washed in PBS and incubated with biotinylated rabbit anti-human IgG secondary antibody 1:1000 in 1.5% milk powder in PBS for 2 h at room temperature. All the sections were counterstained with Meyer’s hematoxylin, dehydrated, and coverslipped. Negative controls were performed by omitting the primary antibody, by using irrelevant isotype control antibodies, and by using age-matched control cases from patients without recurring seizures.

-34- For immunological studies routine screening procedures for identification of serum autoantibodies with indirect immunofluorescence were used. Rat cerebellum/brainstem frozen sections served as substrate, FITC-conjugated rabbit anti human IgG as conjugate. For visualization of antibody binding a rabbit anti human secondary antibody, which was conjugated with horse raddish peroxidase, was used. All signals were detected with the EnVision kit (Dakocytomation K5007, Glostrup, Denmark).

The low-power microscopic findings of the hippocampus showed a typical pattern of HA/HS (fig. 2 B), characterized by severe loss of neurons and gliosis of the CA 1 sector of the pyramidal cell layer. The other subfields showed fewer changes. The granular neurons of the dentate gyrus were slightly dispersed. The high-power view showed chronic inflammation and infiltration with macrophages (fig. 2 C). Especially in the CA1 subfield, the infiltrates were composed of CD 3+ CD 8+ T-lymphocytes (more than 8 cells/ high power field) and CD 68+ macrophages (fig 2 D, E). There was also microglial activation detectable. Positive signals with Caspase 3 and 9 were not retrieved. Results of immunohistochemical staining of patient’s brain for IgG were not different from age matched controls.

18 months after surgery, the patient is still seizure-free on stable AED treatment with lacosamide. MRI three and twelve months after surgery showed a normal post-surgical situation without signal alteration of the right mesial structures. Neuropsychological testing in November 2008 and July 2009 revealed significant improvement of general intellectual performance, long-term memory, serial reproduction and visual-motoric coordination. Follow-up tumour screening remained negative.

-35- Discussion

Clinical outcome data on non-paraneoplastic limbic encephalitis in paediatric patients are rare. We report a case of paediatric-onset NPLE, subsequent unilateral HA/HS and drug- resistant TLE as previously described in adult patients (Bien et al., 2007b). Rapid evolution of hippocampal swelling into atrophy and sclerosis of the mesial structures 3 to 8 months after disease-onset is demonstrated by serial MRI. As the girl never experienced convulsive status epilepticus (SE) during the disease course, it seems unlikely that these findings were the consequence of acute seizure activity (Nohria et al., 1994, Tien et al., 1995, Wieshmann et al., 1997). Further (and in contrast to our case where histopathology showed chronic inflammation consistent with LE), significant inflammation was not reported in surgically resected hippocampal specimen of SE patients and brain biopsies performed in seven children with febrile infection-related epilepsy syndrome (FIRES) showed gliosis but no inflammation (van Baalen et al., 2010). Despite intensive antibody search and detailed tissue analysis, the underlying cause of LE in our patient remains unclear. However, antibodies may have been present earlier and already disappeared at the time of testing (Jarius et al., 2008).

Long-term immunosuppression, which is widely considered the first-line treatmet of LE (independent of clinical symptomatology and autoantibody findings), was not administered in our case, because response is variable and patients with antibodies to cell-membrane antigens usually respond better (Bataller et al., 2007). Further, we found only a few case reports and one study addressing immunotherapy in paediatric NPLE (Kröll-Seger et al., 2009, Akman et al., 2009, Florance et al., 2009). Finally, our patient rapidly developed unilateral AHS/TLE, which made the girl an ideal candidate for epilepsy surgery. Although long-term prognosis still remains uncertain excellent seizure control and favourable cognitive outcome have been achieved for now 18 months. This so far favourable outcome indicates that – despite the presumed progressive nature of LE - epilepsy surgery may be a treatment option in some of these patients.

-36- Acknowledgment

Financial support for this project exclusively comes from the Anniversary Fund of the Central Bank of the Republic of Austria (awarded to Dr Feucht), Grant Number ÖNB- 12036. This study is part of the doctoral thesis project “Pediatric Epilepsy Surgery – Predictors of (un)favourable Outcome”, see also www.meduniwien.ac.at/clins. We thank Dr. Bien (University of Bonn, Medical Centre) for critical discussion, the technical staff of the Vienna Paediatric Epilepsy Monitoring Unit for their support and Dr. Höftberger as well as I. Leisser for perfect technical assistance.

Disclosure

The authors declare no conflicts of interest associated with this manuscript. Full consent for publication of material relating to them in a peer reviewed journal was obtained from the patient and her mother (legal representative). We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

-37- References

Akman, C.I., Patterson, M.C., Rubinstein, A., Herzog, R., 2009. Limbic encephalitis associated with anti-GAD antibody and common variable immune deficiency. Dev Med Child Neurol 51, 563-7. Bataller, L., Kleopa, K.A., Wu, G.F., Rossi, J.E., Rosenfeld, M.R., Dalmau, J., 2007. Autoimmune limbic encephalitis in 39 patients: immunophenotypes and outcomes. J Neurol Neurosurg Psychiatry 78, 381-5. Bien, C.G., Elger, C.E., 2007a. Limbic encephalitis: a cause of temporal lobe epilepsy with onset in adult life. Epilepsy Behav. 10, 529-38. Bien, C.G., Urbach, H., Schramm, J., Soeder, B.M., Becker, A.J., Voltz, R., Vincent, A., Elger, C.E., 2007b. Limbic encephalitis as a precipitating event in adult-onset temporal lobe epilepsy. Neurology 69, 1236-44. Buckley, C., Oger, J., Clover, L., Tuzun, E., Carpenter, K., Jackson, M., Vincent, A., 2001. Potassium channel antibodies in two patients with reversible limbic encephalitis. Ann Neurol 50, 73-8. Dalmau, J., Rosenfeld, M.R., 2008a. Paraneoplastic Syndromes of the CNS. Lancet Neurol 7, 327-40. Dalmau, J., Gleichman, A.J., Hughes, E.G., Rossi, J.E., Peng, X., Lai, M., Dessain, S.K., Rosenfeld, M.R., Balice-Gordon, R., Lynch, D.R., 2008b. Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies. Lancet Neurol 7, 1091-8. Florance, N.R., Davis, R.L., Lam, C., Szperka, C., Zhou, L., Ahmad, S., Campen, C.J., Moss, H., Peter, N., Gleichman A.,J., Glaser C.A., Lynch, D.R., Rosenfeld, M.R., Damlau, J., 2009. Anti-N-Methyl-D-Aspartate receptor (NMDAR) encephalitis in children and adolescents. Ann Neurol 66, 11-18. Graus, F., Saiz, A., Lai, M., Bruna, J., Lopez, F., Sabater, L., Blanco, Y., Rey, M.J., Ribalta, T., Dalmau, J., 2008. Neuronal surface antigen antibodies in limbic encephalitis: clinical- immunologic associations. Neurology 71, 930-6. Gultekin, S.H., Rosenfeld, M.R., Voltz, R., Eichen, J., Posner, J.B., Dalmau, J., 2000. Paraneoplastic limbic encephalitis: neurological symptoms, immunological findings and tumour association in 50 patients. Brain 123, 1481-94. Jarius, S., Hoffmann, L.A., Stich, O., Clover, L., Rauer, S., Vincent, A., Voltz, R., 2008. Relative frequency of VGKC and “classical” paraneoplastic antibodies in patients with limbic encephalitis. J Neurol 255, 1100-1.

-38- Kröll-Seger, J., Bien, C.G., Huppertz, H.J., 2009. Non-paraneoplastic limbic encephalitis associated with antibodies to potassium channels leading to bilateral hippocampal sclerosis in a pre-pubertal girl. Epileptic Disord 11, 54-9. Malter, M., Urbach, H., Vincent, A., Bien, C.G., 2010. Antibodies to glutamic acid decarboxylase define a form of limbic encephalitis. Ann Neurol in press. Mata, S., Muscas, G.C., Naldi, I., Rosati, E., Paladini, S., Cruciatti, B., Bisulli, F., Paganini, M., Mazzi, G., Sorbi, S., Tinuper, P., 2008. Non-paraneoplastic limbic encephalitis associated with anti-glutamic acid decarboxylase antibodies. J Neuroimmunol 199, 155-9. Theodore, W.H., Epstein, L., Gaillard, W.D., Shinnar, S., Wainwright, M.S., Jacobson, S., 2007. Human herpes virus 6B: a possible role in epilepsy? Epilepsia 49, 1828-37. Nohria, V., Lee, N., Tien, R.D., Heinz, E.R., Smith, J.S., DeLong, G.R., Skeen, M.B., Resnick, T.J., Crain, B., Lewis, D.V., 1994. Magnetic resonance imaging evidence of hippocampal sclerosis in progression: a case report. Epilepsia 35, 1332-36. Urbach, H., Soeder, B.M., Jeub, M., Klockgether, T., Meyer, B., Bien, C.G., 2006. Serial MRI of limbic encephalitis. Neuroradiology 48, 380-6. Tien, R.D., Felsberg, G.J., 1995. The hippocampus in status epilepticus: demonstration of signal intensity and morphologic changes with sequential fast spin-echo MR imaging. Radiology 194, 249-56. Van Baalen, A., Häusler, M., Boor, R., Rohr, A., Sperner, J., Kurlemann, G., Panzer, A., Stephani, U., Kluger, G., 2010. Febrile infection-related epilepsy syndrome (FIRES): A nonencephalitic encephalopathy in childhood. Epilepsia, epub ahead of print. Wieshmann, U.C., Woermann, F.G., Lemieux, L., Free, S.L., Bartlett, P.A., Smith, S.J.M., Duncan, J.S., Stevens, J.M., Shorvon, S.D., 1997. Development of hippocampal atrophy: a serial magnetic resonance imaging study in a patient who developed epilepsy after generalized status epilepticus. Epilepsia 38, 1238-41.

-39- Figures

Figure 1

Legend to Figure 1: A – D: Coronary T2 and axial FLAIR MRI sequences showing bilateral signal enhancement and swelling of the mesial temporal lobe, whereas hyperintensity was more distinct on the left side (A, July 2007; B, August 2007). Decrease of swelling, beginning of hippocampal atrophy (C, October 2007). Left hippocampal atrophy with signal increase on FLAIR sequence indicating hippocampal sclerosis (D, March 2008).

-40- Figure 2

Legend to Figure 2: A Interictal EEG with sharp slow waves left temporal. B – E Microscopic findings. Ammon’s horn sclerosis with lesioning of the CA1 subfield (B, NeuN, 12,5x). Perivascular infiltrates composed of lymphocytes and macrophages (arrowheads) (C, H-E, 200x). Immunohistochemical labelling of macrophages in the CA 1 subfield (D, CD 68, 200x, brown label). CD 8+ T-lymphocytes infiltrating the lesional zone (more than 8 cells/ high power field) in the CA 1 subfield (E, CD 8, arrows, 200x, brown label).

-41- 4.2 Publication 2: Clinical Neuropathology 2011 Jul-Aug;30(4):164-77

Neuropathological work-up of Focal Cortical Dysplasias using the new ILAE consensus classification system - practical guideline article invited by the Euro-CNS Research Committee

Ingmar Blümcke and Angelika Mühlebner

Department of Neuropathology, University Hospital Erlangen, Germany Schwabachanlage 6 DE – 91054 Erlangen, Germany Telephone: +49-9131-8526031 Fax: +49-9131-8526033

-42- Abstract

FCDs are increasingly recognized in patients with drug-resistant epilepsies, and many patients benefit from tailored resection strategies. Yet, postsurgical seizure control cannot be sufficiently predicted and specification of FCD variants remains difficult during presurgical monitoring. The International League against Epilepsy (ILAE) has published a new consensus classification system for Focal Cortical Dysplasias (FCDs). Based on a review of imaging data, electro-clinical features and postsurgical seizure control correlation with neuropathological findings specify three clinico-pathological FCD subtypes: FCD Type I is characterized by aberrant radial (FCD Type Ia) or tangential lamination of the neocortex (FCD Type Ib) affecting one or multiple lobes. FCD Type II is characterized by cortical dyslamination and dysmorphic neurons without (Type IIa) or with balloon cells (Type IIb). It is important to note, that these types should not associate with any other structural brain lesion (isolated FCD). In contrast, a new FCD Type III is introduced, which occur in combination with hippocampal sclerosis (FCD Type IIIa), or with epilepsy- associated tumors (FCD Type IIIb). FCD Type IIIc is found adjacent to vascular malformations, whereas FCD Type IIId can be diagnosed in association with other epileptogenic lesions obtained in early life (i.e., traumatic injury, ischemic injury or encephalitis). Histopathological features are very similar to those observed in FCD Type I, but likely present postnatal development and maturation failures acquired by the principal lesion. This first international consensus classification may encourage neuropathologists to focus their attention onto this important histopathological group. Addressing more precisely defined clinico-pathological entities will also help to clarify underlying pathomechanisms and, thereby, improve treatment strategies for patients with difficult-to-treat epilepsies.

Keywords: Epilepsy, Seizures, Hippocampal Sclerosis, Tumors, Cortical Dysplasia, Dysmorphic

-43- Introduction

In 1971, David Taylor and colleagues coined the term “Focal Cortical Dysplasia (FCD)” to describe localized malformative brain lesions in a series of ten patients with drug resistant epilepsy [1]. Surgical resection offered a successful strategy to achieve seizure control in these difficult-to-treat patients, and histopathological examination revealed dysmorphic neurons in all, as well as balloon cells in half of this series. Since then, the spectrum of “Focal Cortical Dysplasias” increased and include nowadays also architectural disturbances without significant cellular abnormalities. Hence, the histopathological, clinical and neuroradiological classification of these lesions remains difficult or even controversial. The most widely recognized classification system was published by Andre Palmini in 2004 [2]. It was the report of an international working group, hence only little contribution and recognition of neuropathology data was included. Unfortunately, this classification has not yielded a reliable prediction of proposed FCD variants and postsurgical seizure control [3]. This applies in particular to so-called Type I FCDs. A recently published neuropathological study showed that inter- and intraobserver reproducibility of FCD Type I cases were in the range of only 50% when using the Palmini system [4]. A Task Force of the international League against Epilepsy was addressing this clinically important issue and re-defined clinico-pathological FCD subtypes [5, 6]. 30 colleagues* from different medical and scientific disciplines agreed to use histopathological hallmarks as most reliable classifying parameters. Another effort was to recognize neurodevelopmental concepts for plasticity and regeneration in the postnatal brain as helpful to understand variant patterns of cortical dyslamination in FCD [7]. On the other hand, this new consensus classification does not define each lesion’s epileptogenicity nor does it predict postsurgical seizure control. These issues need to be addressed in prospective studies, when modern electrophysiological characteristics, imaging data and surgical resection strategies become comparable and accessible for meta-analysis of FCD variants. The Task Force has agreed, therefore, on a classification scheme extending the Palmini system. There are three FCD subtypes to distinguish (Table 1). FCD Type I refers to isolated focal cortical dysplasias affecting cortical architecture in one or multiple lobes. Although we have not achieved consensus about the etiology of this subgroup, FCD Type I is likely congenital and most frequent in young patients with severe, drug-resistant seizures and psycho-motor retardation [8, 9]. In addition, MRI recognition may be quite difficult to obtain and likely to reflect only hemispheric hypoplasia [9]. The second FCD

-44- variant is identical to that described by Taylor and previously termed FCD Type II by Palmini et al [4]. The new FCD Type III subgroup will now include those variants of cortical dyslamination associated with or adjacent to other principal lesions. Prominent examples include hippocampal sclerosis (FCD Type IIIa), glio-neuronal tumors (FCD Type IIIb), vascular malformations (FCD Type IIIc) or brain injuries aquired during early life (FCD Type IIId). Associated FCD variants are likely to be more frequent than isolated subtypes in surgical series of epilepsy patients [10]. However, these FCD variants may well occur during postnatal development and maturation periods [11]. The new “FCD classification system” also takes into account insights from experimental neurodevelopmental studies [7], i.e., sustained plasticity and neurogenesis in the postnatal brain, which is compromised by various pathogenic conditions. Similarily, the “dysmature cerebral developmental hypothesis” suggested that there is partial failure in later phases of cortical development that might explain the distinctive histopathology of CD and that local interactions of dysmature cells with normal postnatal neurons promote seizures [12].

Footnote * Members of the ILAE Task Force: Ingmar Blümcke (chair), Maria Thom, Eleonora Aronica, Dawna Armstrong, Harry V. Vinters, Andre Palmini, Thomas S Jacques, Giuliano Avanzini, James Barkovich, Giorgio Battaglia, Albert Becker, Carlos Cepeda, Fernando Cendes, Peter Crino, J. Helen Cross, Nadia Colombo, Francois Dubeau, John Duncan, Renzo Guerrini, Philippe Kahane, Gary Mathern, Imad Najm, Çiğdem Özkara, Charles Raybaud, Alfonso Represa, Steven Roper, Noriko Salamon, Andreas Schulze- Bonhage, Laura Tassi, and Roberto Spreafico

-45- Table 1: ILAE consensus classification system for FCD

FCD Type I FCD Ia : abnormal radial FCD Ib: abnormal FCD Ic: abnormal radial (isolated) cortical lamination tangential cortical and tangential cortical lamination lamination

FCD Type II FCD IIa: with dysmorphic neurons FCD IIb: dysmorphic neurons and (isolated) balloon cells

FCD Type III FCD IIIa: Cortical FCD IIIb: Cortical FCD IIIc: Cortical FCD IIId: Cortical (associated lamination lamination lamination lamination with principal abnormalities in abnormalities abnormalities abnormalities lesion) the temporal lobe adjacent to a glial adjacent to adjacent to any other associated with or glio-neuronal vascular lesion acquired hippocampal tumor malformation during early life, e.g., sclerosis trauma, ischemic injury, encephalitis

Legend: The three-tiered ILAE classification system for Focal Cortical Dysplasias (FCD) distinguishes isolated forms (FCD Type I and II) from those cortical layer abnormalities associated with another principal lesion (FCD Type III).

-46- Neuropathological Work-up and Histopathological Protocols

The new ILAE classification system requires surgical brain specimen processed with standardized histopathological procedures. Tissue should always be fixed in formalin (10%). On bloc resections should be carefully orientated and cut perpendicular to the cortical surface (3 – 5 mm section thickness, Figure 1). Tangential gating should be avoided. We suggest to further process every second or third slice (if the resected specimen is very large) and always include those areas with macroscopically visible changes, such as blurred grey-white matter border or increased cortical thickness (Figure 1). Remaining unfixed tissue slices should be snap frozen in liquid nitrogen and long-term stored at -80°C to allow molecular-biological or genetic analysis. Overnight fixation is recommended for larger specimens. Standardized paraffin embedding allows fine sectioning for subsequent staining procedures. Four to seven µm paraffin sections are most appropriate for histochemical and immunohistochemical stains. Thicker sections (<20 µm) may yield a better visualization of cortical layers, but penetration of antibodies needs longer time periods. However, different standardized laboratory methods are available and will yield specific patterns, which should be adapted and confirmed if different from that recommended below (Table 2). Hematoxylin & Eosin (H & E) staining should be performed on every slice, whereas additional stainings should be conducted only after preselection. If H & E staining is conclusive, sections with anatomically well preserved cortical orientation including representative white matter areas should be considered. To evaluate cortical architecture and myelination the following stainings or antibodies are suggested: H & E, cresyl violet, luxol-fast blue, Klüver-Barrera, NeuN. In order to assess cytological features we recommend MAP2, NeuN, phosphorylated and non- phosphorylated neurofilament protein. Balloon cells can be best studied using Vimentin which also stains reactive astrocytes. To exclude tumor cell infiltration into neocortex suspicious for FCD Type IIIb (tumor infiltration would almost always exclude the diagnosis of associated FCD) proliferation index (Ki67), mutation specific isocitrate dehydrogenase (IDH1) and CD34 should be conducted. IDH1 detects individual tumor cells in up to 75% of low grade astrocytoma and oligodendroglioma [13]. A principal pathology should always be determined in order to distinguish between isolated and associated FCD variants (Figure 2).

-47- Table 2: Histochemical and immunohistochemical stains recommended for the histopathological work-up of surgical FCD specimens

Stain/ Variants/ Type I Type II Type III Antibody Clones H & E + + + CV Nissl + + + Myelin Luxol –FB + + + Klüver - B EVG - - +/- NeuN A60 + + + MAP2 Clone C + + + p-NFP 2F11 + + + np-NFP SMI32 + + + GFAP 6F2 +/- +/- +/- Vimentin 3B4 +/- + - CD34 QBend10 - - + IDH1 H09 - - + Ki67 Mib1 - - +/-

Legend: H & E = Haematoxylin and Eosin stain; CV = cresyl violet (syn. Nissl staining); Myelin can be visualized using Luxol-Fast Blue or Klüver-Barrera stainings; EVG = elastic - Van Gieson, p-NFP – phosphorylated neurofilament protein, np-NFP – non phosphorylated neurofilament protein, GFAP – glial fibrillary acidic protein; IDH1: mutation specific isocitrate dehydrogenase 1; Ki67 – proliferation marker, + = recommended to confirm diagnosis; +/- = may be helpful; - = not required.

-48- Focal Cortical Dysplasia Type I

Focal Cortical Dysplasia Type I is a malformation affecting one or multiple lobes. No other gross (principal) pathology can be detected (during presurgical MRI and by microscopic analysis). Recent MRI data point towards hemispheric hypoplasia [9]. Neuropathological hallmarks include abnormal cortical layering affecting both radial migration and maturation of neurons (FCD Type Ia) or the six-layered horizontal composition of the neocortex (FCD Type Ib). The combination of both variants should be classified as FCD Type Ic. These malformations are likely congenital but also compromise postnatal development. Young children with catastrophic epilepsies and psycho-motor retardation are usually affected [14]. Genetic councelling should be always envisaged to exclude any symptomatic epileptic encephalopathy (i.e. Dravet Syndrome). Surgical resection strategies are often regarded as palliative treatment option. Indeed, complete seizure control was reported in only 50% of patients [8], but attenuated seizure burden ameliorate psycho-motor development in many children. The following terminology and definitions were adopted from [6].

Histopathological features in FCD Type I

Microcolumns in FCD Type Ia. Microcolumns resemble ontogenetic columns described during normal cortical development [15] (Figure 3). They can be also seen at lower frequency and with fewer neurons in non-epileptic brain samples as well as in the vicinity of other principal lesions (see below). The border towards white matter is usually less sharply demarcated due to increased numbers of heterotopic neurons. Cellular abnormalities can be encountered in this variant, and include (1) immature small diameter neurons [16] or (2) hypertrophic pyramidal neurons outside Layer 5. The diagnosis of FCD Type I variants will need particular attention, however, when studying agranular or dysgranular areas of the temporo-polar lobe [17].

Horizontal layer abnormalities in FCD Type Ib. Failure to establish a 6-layered tangential composition of the isocortex is a hallmark of this variant (and should, therefore, always be used with caution in non-six layered allo- or proisocortical areas). The entire neocortical architecture may be affected without any recognizable layering (with the exception of Layer 1). Other subtypes are restricted to abnormal layering of Layer 2, Layer 4 or both. Layer 2 can be either missing or is significantly depleted of the characteristic population of small pyramidal neurons. This pattern results in a blurred demarcation

-49- between Layers 1 and 2, as well as between Layers 2 and 3, whose boundaries are very well defined in non-epileptic controls. Layer 4 can also be missing or is obscured and less distinguishable from Layers 3 and 5 (Figure 2). The border with white matter is usually less sharply demarcated due to increased neuronal cells. Cellular abnormalities can be encountered in this variant, and include (1) immature neurons with a small diameter or (2) hypertrophic pyramidal neurons outside Layer 5 or (3) normal neurons with disoriented dendrites (Figure 4). These observations and other cellular alterations requiring sophisticated neuroanatomical techniques are not mandatory, however, to establish the diagnosis of FCD Type I variants.

Histopathological findings in FCD Type Ic. This variant refers to those isolated lesions, in which histopathological inspection reveals both, abnormal radial and tangential cortical lamination. Histopathological hallmarks are identical to those specified above. This FCD variant is diagnosed only as an isolated lesion and not in combination with any other pathology. It has to be clarified in the future, however, whether such lesions occur within patients with more widespread abnormalities linked to mental retardation and/or multiple congenital abnormality syndromes.

Immunohistochemistry in FCD Type I

Immunohistochemical analysis should be mandatory for the differential diagnosis of FCD Type I subtypes. Cortical layer abnormalities can be visualized using antibodies directed against NeuN. Densities of heterotopic neurons (cells per mm²) vary among different lobes and reference values must be adopted from one’s own laboratory experience/counting protocols. As a rough estimate, more than 30 MAP2-immunoreactive neurons/mm² within deep white matter location (measured > 500µm from cortical ribbon) may be considered as a significant excess of heterotopic neurons. Immunohistochemical reactions are very helpful for the differential diagnosis from FCD Type II or III variants. Dysmorphic neurons in FCD Type II are readily identified in routine H & E staining and accumulate phosphorylated or non phosphorylated neurofilament proteins (Figure 4). Balloon cells always react with antibodies against vimentin and nestin. To distinguish a FCD adjacent from cortex infiltrated by a neoplasm, CD34 and IDH1 immunoreactivity should be applied.

-50- Focal Cortical Dysplasia Type II

Focal Cortical Dysplasia Type II is a malformation presenting with disrupted cortical lamination and specific cytological abnormalities, which differentiates FCD Type IIa (dysmorphic neurons without balloon cells) from FCD Type IIb (dysmorphic neurons and balloon cells). However, histopathologically similar lesions are observed in cortical tubers and other gross MCDs, i.e. hemimegalencephaly or schizencephaly. FCD Type IIb can often be detected by presurgical MRI and present with a “transmantle sign” in FLAIR sequences [18]. In contrast, FCD Type IIa is less clearly visible by structural MRI but may be detectable using specialized post-processing protocols [6]. Children and adolescents frequently develop drug-resistance and surgical resection offers complete seizure control in up to 80% of all patients [19]. The following terminology and definitions were adopted from [6].

Histopathological findings in FCD Type II

Cortical dyslamination is always present. In FCD Type II, there is no identifiable cortical layering except Layer 1 (Figure 3). Whether cortical thickness is normal or increased remains to be clarified but is likely not be changed significantly [20, 21]. This is a discriminating feature from FCD Type I, in which only individual cortical layers are obscured or cortical thickness may be decreased.

Junction at gray/white matter is usually blurred with increased heterotopic neurons and neuropil in white matter. These neurons may also be dysmorphic (see below). The precise border between cortex and white matter is usually difficult to delineate.

Altered myelin content in white matter. In FCD Type IIb, there is usually a reduction of myelin staining in the underlying white matter, which can be histochemically verified using Luxol-Fast-Blue or similar appropriate staining protocols during routine neuropathological work-up of surgical specimens. However, there is yet no published data available clarifying neither the origin of reduced myelin content nor suggesting significant differences between FCD subgroups.

Dysmorphic neurons were first described by Crome [22] and Taylor [1]. Dysmorphic neurons are visible only in FCD Type II lesions and are characterized by severe cytological abnormalities: (a) Neuronal cell diameters and cell nucleus diameter are significantly

-51- enlarged; (b) Nissl substance is aggregated and displaced towards the cell membrane; (c) Phosphorylated and non-phosphorylated neurofilament isoforms accumulate in their cytoplasm (Figure 4). Dysmorphic neurons are similar in Type IIa and Type IIb. In the center of lesion, dysmorphic neurons cluster in large aggregates. They are distributed throughout the entire cortical thickness and dislocate also into the white matter. However, distant from the core of the main lesion isolated dysmorphic neurons can also be observed. In addition, multiple FCD Type II lesions have been recognized and individually contribute to seizure generation [23].

Balloon Cells are the distinguishing hallmark of FCD Type IIb. They present with a large cell body and opalescent glassy eosinophilic cytoplasm (using H & E stain), which lacks Nissl substance. Multiple nuclei are often present. Balloon cells can occur at any cortical location (including Layer 1) and are often found in the underlying white matter. Balloon cells may gather in small aggregates but can also be found displaced within adjacent “normal” brain tissue. Balloon cells commonly accumulate intermediate filaments Vimentin and Nestin [18, 24]. They have variable GFAP and neurofilament staining patterns. In rare examples, co-expression of both markers was reported suggesting glial and neuronal lineage determination, i.e. intermediate cells [25]. Balloon cells have gross histomorphological similarities with giant cells, and can be observed in cortical tubers from patients with Tuberous Sclerosis Complex (Figure 4).

Immunohistochemistry in Focal Cortical Dysplasia Type II

Dysmorphic neurons can often be identified already in routine H&E staining. As dysmorphic neurons always accumulate either phosphorylated or non-phosphorylated neurofilament proteins, antibodies directed against these variants can aid in the assessment of abnormal shape, size, orientation, dendritic profile and cortical positioning. The differentiation between FCD Type IIa and IIb depends on the presence of balloon cells, which can be confirmed/excluded using vimentin and nestin labeling. This staining procedure should be applied if balloon cells cannot be identified in H & E stains or if gemistocytic astrocytes are difficult to exclude (e.g. presence of glial scarring due to intracerebral depth recordings or along previous surgical resection borders). Balloon cells also express the GFAP-delta variant or other stem cell markers, which may be helpful for histopathological diagnosis [26, 27].

-52- Focal Cortical Dysplasia Type III

Focal Cortical Dysplasia Type III refers to cortical lamination abnormalities associated with a principal lesion, usually adjacent to or affecting the same cortical area/lobe. They may be similar to those observed for FCD Type I, although specific patterns can be identified. Four subtypes have been proposed: FCD Type IIIa, when associated with hippocampal sclerosis; FCD Type IIIb, when associated with tumors; FCD Type IIIc, when associated with vascular malformations and FCD Type IIId when associated with any other principal lesion acquired during early life. This new category has not yet been clinically well described, although many patients were included in cohorts of previously published Palmini FCD Type I series [3]. A controversial (although less likely) issue remains the possible correlation between FCD Type IIIa and MRI signal enhancement in an atrophic temporal pole [28]. Other correlations may point to variant epileptogenic networks in the temporal lobe [29] or patterns of hippocampal cell loss [30]. Epileptogenicity has been, however, frequently documented within architecturally abnormal neocortex in HS patients [31]. In general, seizure control is similar if compared to the principal lesion itself [8]. The following terminology and definitions were adopted from [6].

Histopathological Findings in FCD Type IIIa

There are two distinct findings that specifically match the associated FCD Type IIIa variant (Figure 5):

Temporal Lobe Sclerosis (TLS): In approx 10% of temporal lobe surgical specimens obtained from HS patients, an abnormal band of small and clustered “granular” neurons can be observed in the outer part of Layer 2, designated also as Temporal Lobe Sclerosis [32, 33]. TLS is likely to present severe neuronal cell loss in Layers 2 and 3 with associated laminar gliosis (GFAP-positive astrogliosis) and cortical re-organisation. Horizontal bundles of myelinated axons can be observed to a variable degree in these cases using H & E - Luxol-Fast-Blue stainings. In 40% of HS/TLS cases more severe involvement of the temporal pole is seen, whereas extensive involvement throughout the temporal lobe occurs in 20%. There is no correlation between this FCD variant and MRI findings in these patients.

-53- Small “lentiform” heterotopias or heterotopic neurons in white matter: In HS patients, small “lentiform” nodular heterotopias can be identified within the temporal lobe. They usually remain undetected by MRI [34]. Radial orientation along the gray/white matter junction is characteristic and cellular composition is usually formed by projecting neurons. These small “lentiform” heterotopias are distinct from the larger nodular heterotopias, which are readily identified by MRI, may be present in any location of the white matter and are histologically characterized by projecting and local circuit neurons [34]. A diagnostic pitfall results from a similar normal anatomical structure located within the depth of the temporal lobe close to the claustrum.

The following patterns should not be included as FCD Type IIIa variants:

(1) Neuronal cell loss within the hippocampus, amygdala and entorhinal cortex. This is a characteristic pattern in many (if not in all) HS patients and usually termed mesial temporal sclerosis (MTS)

(2) HS with single heterotopic neurons in the deep white matter of temporal lobe, but no other architectural alteration. These neuronal heterotopias include also blurring of gray/white matter junction. The pathogenic and epileptogenic significance of this frequent finding has yet to be clarified.

(3) HS and any other principal lesion in the temporal lobe, i.e. tumors, FCD Type IIa/IIb, vascular malformations, glial scars or MCDs (other than FCD Type IIIa) should be classified as “Dual Pathology”. The term “Dual Pathology” is, therefore, not appropriate to describe HS with associated FCD Type IIIa lesions.

-54- Histopathological Findings in FCD Type IIIb

The histopathological hallmark of this new FCD variant is an altered architectural (cortical dyslamination, hypoplasia without six-layered structure) and/or cytoarchitectural composition (hypertrophic neurons) of the neocortex, which occur adjacent to tumors (Ganglioglioma, Dysembryoplastic Neuroepithelial Tumor (DNT, syn. DNET) or other epilepsy-associated neoplasms (for review see [10]). It is important to exclude tumor infiltration in areas of cortical abnormalities before establishing the diagnosis of FCD. The etiology and pathogenesis of FCD Type IIIb remains to be determined, but is likely an acquired process. It should not be considered, therefore, as “Double Pathology” (see glossary, Figure 6).

Histopathological Findings in FCD Type IIIc

Alterations in architectural (cortical dyslamination, hypoplasia) or cytoarchitectural composition of the neocortex (hypertrophic neurons), which occur adjacent to vascular malformations (cavernomas, arteriovenous malformations, leptomeningeal vascular malformations, telangiectasias, meningioangiomatosis). The etiology and pathogenesis of FCD Type IIIc remains to be determined, but is likely an acquired process (Figure 7). It should not be considered, therefore, as “Double Pathology”.

The histopathologic pattern is similar to that described for other FCD Type III variants, and can be identified adjacent to the principal lesion. Cortical architecture may be severely disturbed. However, we cannot exclude the possibility, that the compromised cortical architecture is acquired secondary to the development of the principal lesion, but seizure activity may arise from altered networks in this affected cortical area [35].

Histopathological Findings in FCD Type IIId

The histopathological hallmark of this new FCD variant is an altered architectural (cortical dyslamination, hypoplasia without six-layered structure) or cytoarchitectural composition (hypertrophic neurons) of the neocortex, which occur adjacent to other lesions acquired during early life (not included into FCD Type IIIa-c). These lesions comprise a large spectrum including traumatic brain injury [11, 36], glial scarring after prenatal or perinatal ischemic injury or bleeding and inflammatory or infectious diseases, i.e. Rasmussen encephalitis, limbic encephalitis, bacterial or viral infections (Figure 8).

-55- Focal Cortical Dysplasia associated with clinically suspected principal lesion, but lesion not available for histopathological examination (FCD Type III, not otherwise specified, NOS)

If FCD Type I patterns are histopathologically detected in a patient with a clinically suspected principal lesion, but (1) the principal lesion is not available for microscopic inspection (entire sample should be embedded and sectioned for microscopic inspection), or (2) tissue may not be available for microscopic analysis after the surgical procedure, the neuropathological diagnosis of FCD Type III (NOS) should be considered.

As a consequence, the signing neuropathologist should always have access to clinical information and imaging findings (Figure 2). This information should not bias the histopathological diagnosis but allow to judge whether appropriate and representative tissue was received from the operation theatre. If no specific diagnosis can be achieved, a descriptive formulation should be given. “Probable or suspect FCD” was not recommended by the ILAE Task Force as diagnostic terms.

Immunohistochemistry in Focal Cortical Dysplasia Type III

There is yet no specific immunohistochemical marker available to characterize FCD Type III variants, but NeuN, Map2 and neurofilament proteins should always be looked for in FCD Type III specimens. Cortical infiltration of neoplastic cells should always be excluded before making the diagnosis of FCD Type IIIb. We suggest using a panel of antibodies including CD34 (ganglioglioma; [37]), MAP2 [38], p53, IDH1 and Mib1 (the latter are often enhanced in diffuse gliomas). NeuN immunoreactivity is always helpful to identify 6-layered cortical organization in 4µm thin paraffin embedded histological preparations.

-56- Glossary

There is considerable debate regarding the terminology used for abnormal cell types, which has been inconsistently used in previous classification systems. The following definitions were based on microscopic inspection of 4-7µm thin sectioned, formalin-fixed and paraffin embedded surgical specimens. Terminology and definitions were adapted from [5].

Balloon cells have a large cell body with opaque eosinophilic cytoplasm which lacks Nissl substance on Haematoxylin & Eosin stains. They rarely express cytoplasmic/ immunohistochemical differentiation with glial (GFAP) or neuronal markers (NFP). Multiple nuclei can be seen.

Double Pathology refers to two independent lesions affecting one or multiple lobes, but not including hippocampal sclerosis. This definition assumes that both lesions evolve from an independent pathogenesis, i.e. a cavernoma in one cerebral hemisphere and a ganglioglioma in the other. Electrophysiology will be necessary to characterize the “most likely” epileptogenic lesion.

Dual Pathology is not yet comprehensively defined [39], and is still ambiguously used in clinical and histopathological practice. We propose the following definition: Dual Pathology refers only to patients with hippocampal sclerosis, who have a second principal lesion affecting the brain (which may be located also outside the ipsilateral temporal lobe), i.e. tumor, vascular malformation, glial scar, limbic/Rasmussen encephalitis, or MCD (including FCD Type IIa/IIb). Ipsilateral temporo-polar atrophy with increased T2 signal changes on MRI is not included as its histopathological correlate has yet to be specified. Of note, histopathologically confirmed architectural abnormalities in the temporal lobe associated with HS should not be diagnosed as FCD Type I or “Dual Pathology” but FCD Type IIIa.

Dyslamination is a compromized tangential or radial organization of cortical architecture. It may be observed in any of the proposed FCD subtypes.

-57- Dysmorphic neurons are essential component of FCD Types IIa and IIb. Their soma and nuclei are abnormally large. They are disoriented in the cortex with abnormal aggregates of Nissl substance and phosphorylated or non-phosphorylated neurofilament accumulation in cytoplasm. They mostly represent altered pyramidal neurons but can also show features consistent with those of interneurons.

Dysplasia (syn. Dysgenesis, Malformation) is a general term referring to any tissue that is imperfectly developed in embryonic or fetal life. However, Dysplasia is a diagnostic term used here to identify specific malformations of the cortex, the so-called Focal Cortical Dysplasias (FCDs) irrespective of their diverse histological appearances which are addressed by this classification system.

Dysplastic neurons are the neuronal components of glio-neuronal tumors, i.e. Gangliogliomas and Dysembryoplastic Neuroepithelial Tumor.

Ectopia is a normally formed organ or tissue in an abnormal site within the body. We do not refer to this definition in our classification system.

Hamartoma is a tumor-like non-neoplastic mass (> 1mm) of malformed tissue [40], composed of normal cells in their normal site which exhibit disorganized architecture. Hamartia is a small glio-neuronal lesion which is not grossly visible (< 1mm).

Heterotopia misplaced tissue or cells within their normal organ of origin.

Hypertrophic neurons resemble large pyramidal cells of Layer 5 abnormally located in Layers 1, 2, 3 or 4. Dendrites’ orientation and arborisation may be altered, but there is no obvious intracellular pathology affecting the nucleus or Nissl substance.

Immature neurons develop from neuroblasts and have a small diameter and cell size (< 250 mm²). They do not accumulate non-phosphorylated neurofilaments. They are observed in large numbers in vertically oriented microcolumns (FCD Type Ia).

Principal Lesions comprise any anatomical lesion with etiologically defined pathogenesis of either neoplastic, genetic, infectious, traumatic or metabolic origin. This includes the spectrum of epilepsy-associated tumors, vascular malformations, MCDs, encephalitis, traumatic scars, bleeding, vascular infarction, mitochondrial/metabolic dysfunction and genetic syndromes.

Conflict of interest:

The authors declare no conflict of interest.

-58- Summary

Table 3

Type Ia Type Ib Type Ic Type IIa Type IIb Type IIIa Type IIIb Type IIIc Type IIId

Cortical abnormalities MC LL MC/LL Dis Dis TLS/LL MC/LL MC/LL MC/LL

Dysmorphic Neurons 0 0 0 + + 0 0 0 0

Balloon cells 0 0 0 0 + 0 0 0 0

Immature Neurons + ± ± ± ± ± ± ± ±

Hypertrophic neurons ± ± ± ± ± ± ± ± ±

WM Changes Het Het Het Het/no ML Het/ML Het/LH Het Het Het

Legend: MC – microcolumns; LL – Loss of individual layers; Dis – entire cortical lamination disrupted (with exception of Layer 1); TLS –temporal lobe sclerosis; 0 – not present; + = present; ± = variable; Het – increased heterotopic neurons in gray/white matter junction and deep white matter location; ML – myelin loss; LH – lentiform heterotopia; WM – white matter.

-59- Figures

Figure 1: Macroscopic processing of surgical specimen – a guide

Legend to Figure 1: A: Right temporal pole of a 56 year old patient with therapy refractory temporal lobe epilepsy. The specimen should be adjusted according to gyral patterns and then cut from right to left (arrow). Scale bar on top in mm. B: There is no macroscopic evidence for structural abnormalities. We suggest, therefore, embedding every second slice into paraffin for further microscopic evaluation.

-60- Figure 2: Guidelines for a protocol in the neuropathological assessment of FCD in epilepsy resection specimens

Legend to Figure 2: As adapted from [5]. Vascular = vascular malformation, MTS = mesial temporal sclerosis, nd = not determined. Section thickness for: H & E (haematoxylin and eosin) = 4-7µm ; cresyl violet (CV) or Nissl = 12-14µm; immunohistochemistry = 4-7µm. Suggested special stains and immunohistochemistry also referenced in Table 3. NFPs=neurofilament proteins (both non-phosphorylated NFP and phosphorylated NFP should be included). GFAP – glial fibrillary acidic protein. CD34, IDH1 (mutation specific isocitrate dehydrogenase 1) and Mib1 recommended markers to specify tumor biology (when FCD Type IIIb suspected). Myelin stains include Klüver-Barrera or Luxol Fast Blue. EVG – elastic - Van Gieson to examine vascular malformation. Note cytoarchitectural variation in superior temporal gyrus, parahippocampal gyrus, peri-amygdala cortex, subiculum and entorhinal cortex. DN = dysmorphic neurons, BC = balloon cells.

-61- Figure 3: Architectural abnormalities in FCD variants

Legend to Figure 3: A, B, G, H Normal appearing neocortex (Nissl-LFB and NeuN). C, D: Distinct microcolumnar arrangements of small diameter neurons can be detected in FCD Type Ia, when surgical specimen is cut perfectly perpendicular to the pial surface and paraffin embedded sections were used. (Nissl-LFB, NeuN). E, F: Tangential altered neocortical architecture in FCD Type Ib (Nissl-LFB, NeuN). I - L: Dyslamination with dysmorphic neurons in FCD Type IIa (I, J) oder IIb (K, L). Scale bar in L indicates 500µm and applies to all images.

-62- Figure 4: Cytological abnormalities in FCD variants

Legend to Figure 4: A, B: Balloon cells in FCD Type IIb (HE, vimentin). C, D: Dysmorphic neurons in FCD Type IIa and IIb (HE, SMI32). E, F: Hypertrophic neurons in FCD Ib (SMI 32). Scale bar in A - D and F indicates 50µm. Scale bar in E indicates 100µm.

-63- Figure 5: FCD Type IIIa

Legend to Figure 5: A: Abnormal layer 2 (TLS, arrow, NeuN). Scale bar indicates 100µm. C: Heterotopic “lentiforme” neuronal islands in the subcortical white matter (MAP2). Scale bar indicates 100µm.

-64- Figure 6: FCD Type IIIb

Legend to Figure 6: A: Macroscopic appearance of a glio-neuronal tumor with nodular appearance resembling DNT (arrow). Scale bar indicates 1000µm. Courtesy from Dr. Macaulay (Halifax, Canada). B: The specific glio-neuronal element in DNT (HE). Scale bar indicates 200µm. C: Floating neurons (HE). Scale bar indicates 20µm. D, E: Microcolumnar appearance of the adjacent neocortex (Nissl-LFB, NeuN). Scale bar indicates 200µm.

-65- Figure 7: FCD Type IIIc

Legend to Figure 7: A, B: Patient with Sturge-Weber syndrome: Abnormal cortical architecture beneath a meningoangiomatous lesion (asterisk, HE, NeuN). Scale bar in B indicates 500µm and applies also to A.

-66- Figure 8: FCD Type IIId

Legend to Figure 8: A, B: Child with perinatal hemorrhage and glio-mesodermal scarring in the temporal lobe (HE, Prussian blue stain). Scale bar indicates 50µm. C: Abnormal cortical architecture (arrow) adjacent to scar with nodular heterotopias (asterisk, NeuN). Scale bar in C indicates 1000µm D: Small nodular heterotopias (arrow, MAP2). Scale bar indicates 200µm.

-67- References

1. Taylor DC, Falconer MA, Bruton CJ, Corsellis JA. Focal dysplasia of the cerebral cortex in epilepsy. J Neurol Neurosurg Psychiatry. 1971; 34: 369-387.

2. Palmini A, Najm I, Avanzini G, Babb T, Guerrini R, Foldvary-Schaefer N, Jackson G, Luders HO, Prayson R, Spreafico R, Vinters HV. Terminology and classification of the cortical dysplasias. Neurology. 2004; 62: S2-8.

3. Blumcke I, Vinters HV, Armstrong D, Aronica E, Thom M, Spreafico R. Malformations of cortical development and epilepsies. Epileptic Disord. 2009; 11: 181-193.

4. Chamberlain WA, Cohen ML, Gyure KA, Kleinschmidt-DeMasters BK, Perry A, Powell SZ, Qian J, Staugaitis SM, Prayson RA. Interobserver and intraobserver reproducibility in focal cortical dysplasia (malformations of cortical development). Epilepsia. 2009; 50: 2593-2598.

5. Blumcke I, Spreafico R. An international consensus classification for focal cortical dysplasias. Lancet Neurol. 2011; 10: 26-27.

6. Blümcke I, Thom M, Aronica E, Armstrong DD, Vinters HV, Palmini A, Jacques TS, Avanzini G, Barkovich AJ, Battaglia G, Becker A, Cepeda C, Cendes F, Colombo N, Crino P, Cross JH, Delalande O, Dubeau F, Duncan JS, Guerrini R, Kahane P, Mathern GW, Najm I, Özkara C, Raybaud C, Represa A, Roper SN, Salamon N, Schulze- Bonhage A, Tassi L, Vezzani A, Spreafico R. The clinico-pathological spectrum of Focal Cortical Dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission. Epilepsia. 2011; 52: 158-174.

7 Battaglia G, Becker AJ, Loturco J, Represa A, Baraban SC, Roper SN, Vezzani A. Basic mechanisms of MCD in animal models. Epileptic Disord. 2009; 11: 206-214.

8. Tassi L, Garbelli R, Colombo N, Bramerio M, Lo Russo G, Deleo F, Milesi G, Spreafico R. Type I focal cortical dysplasia: surgical outcome is related to histopathology. Epileptic Disord. 2010; 12: 181-191.

9. Blumcke I, Pieper T, Pauli E, Hildebrandt M, Kudernatsch M, Winkler P, Karlmeier A, Holthausen H. A distinct variant of focal cortical dysplasia type I characterised by magnetic resonance imaging and neuropathological examination in children with severe epilepsies. Epileptic Disord. 2010; 12: 172-180. Neuropathology work-up of the ILAE Classification System of FCD page 26.

-68- 10. Blumcke I. Neuropathology of focal epilepsies: a critical review. Epilepsy Behav. 2009; 15: 34-39.

11. Marin-Padilla M, Parisi JE, Armstrong DL, Sargent SK, Kaplan JA. Shaken infant syndrome: developmental neuropathology, progressive cortical dysplasia, and epilepsy. Acta Neuropathol (Berl). 2002; 103: 321-332.

12. Cepeda C, Andre VM, Levine MS, Salanion N, Miyata H, Vinters HV, Mathern GW. Epileptogenesis in pediatric cortical dysplasia: The dysmature cerebral developmental hypothesis. Epilepsy & Behavior. 2006; 9: 219-235.

13. Cendes F, Cook MJ, Watson C, Andermann F, Fish DR, Shorvon SD, Bergin P, Free S, Dubeau F, Arnold DL. Frequency and characteristics of dual pathology in patients with lesional epilepsy. Neurology. 1995; 45: 2058-2064.

14. Wolf HK, Wiestler OD. Surgical pathology of chronic epileptic seizure disorders. Brain Pathol. 1993; 3: 371-380.

15. Krsek P, Pieper T, Karlmeier A, Hildebrandt M, Kolodziejczyk D, Winkler P, Pauli E, Blumcke I, Holthausen H. Different presurgical characteristics and seizure outcomes in children with focal cortical dysplasia type I or II. Epilepsia. 2009; 50: 125-137.

16. Rakic P. Specification of cerebral cortical areas. Science. 1988; 241: 170-176.

17. Hildebrandt M, Pieper T, Winkler P, Kolodziejczyk D, Holthausen H, Blumcke I. Neuropathological spectrum of cortical dysplasia in children with severe focal epilepsies. Acta Neuropathol. 2005; 110: 1-11.

18. Ding SL, Van Hoesen GW, Cassell MD, Poremba A. Parcellation of human temporal polar cortex: a combined analysis of multiple cytoarchitectonic, chemoarchitectonic, and pathological markers. J Comp Neurol. 2009; 514: 595-623.

19. Urbach H, Scheffler B, Heinrichsmeier T, von Oertzen J, Kral T, Wellmer J, Schramm J, Wiestler OD, Blumcke I. Focal cortical dysplasia of Taylor's balloon cell type: a clinicopathological entity with characteristic neuroimaging and histopathological features, and favorable postsurgical outcome. Epilepsia. 2002; 43: 33-40.

20. Krsek P, Maton B, Korman B, Pacheco-Jacome E, Jayakar P, Dunoyer C, Rey G, Morrison G, Ragheb J, Vinters HV, Resnick T, Duchowny M. Different features of histopathological subtypes of pediatric focal cortical dysplasia. Ann Neurol. 2008; 63: 758-769.

21. Andres M, Andre VM, Nguyen S, Salamon N, Cepeda C, Levine MS, Leite JP, Neder L,

-69- Vinters HV, Mathern GW. Human Cortical Dysplasia and Epilepsy: An Ontogenetic Neuropathology work-up of the ILAE Classification System of FCD page 27 Hypothesis Based on Volumetric MRI and NeuN Neuronal Density and Size Measurements. Cereb Cortex. 2005; 15: 194-210.

22. Chandra PS, Salamon N, Nguyen ST, Chang JW, Huynh MN, Cepeda C, Leite JP, Neder L, Koh S, Vinters HV, Mathern GW. Infantile spasm-associated microencephaly in tuberous sclerosis complex and cortical dysplasia. Neurology. 2007; 68: 438-445.

23. Crome L. Infantile cerebral gliosis with giant nerve cells. J Neurol Neurosurg Psychiatry. 1957; 20: 117-124.

24. Fauser S, Sisodiya SM, Martinian L, Thom M, Gumbinger C, Huppertz HJ, Hader C, Strobl K, Steinhoff BJ, Prinz M, Zentner J, Schulze-Bonhage A. Multi-focal occurrence of cortical dysplasia in epilepsy patients. Brain. 2009; 132: 2079-2090.

25. Garbelli R, Munari C, De Biasi S, Vitellaro-Zuccarello L, Galli C, Bramerio M, Mai R, Battaglia G, Spreafico R. Taylor's cortical dysplasia: a confocal and ultrastructural immunohistochemical study. Brain Pathol. 1999; 9: 445-461.

26. Talos DM, Kwiatkowski DJ, Cordero K, Black PM, Jensen FE. Cell-specific alterations of glutamate receptor expression in tuberous sclerosis complex cortical tubers. Ann Neurol. 2008; 63: 454-465.

27. Martinian L, Boer K, Middeldorp J, Hol EM, Sisodiya SM, Squier W, Aronica E, Thom M. Expression patterns of glial fibrillary acidic protein (GFAP)-delta in epilepsyassociated lesional pathologies. Neuropathol Appl Neurobiol. 2009; 35: 394- 405.

28. Yasin SA, Latak K, Becherini F, Ganapathi A, Miller K, Campos O, Picker SR, Bier N, Smith M, Thom M, Anderson G, Helen Cross J, Harkness W, Harding B, Jacques TS. Balloon cells in human cortical dysplasia and tuberous sclerosis: isolation of a pathological progenitor-like cell. Acta Neuropathol. 2010; 120: 85-96.

29. Colombo N, Salamon N, Raybaud C, Ozkara C, Barkovich AJ. Imaging of malformations of cortical development. Epileptic Disord. 2009; 11: 194-205.

30. Kahane P, Bartolomei F. Temporal lobe epilepsy and hippocampal sclerosis: lessons from depth EEG recordings. Epilepsia. 2010; 51 Suppl 1: 59-62.

-70- 31. Blumcke I, Pauli E, Clusmann H, Schramm J, Becker A, Elger C, Merschhemke M, Meencke HJ, Lehmann T, von Deimling A, Scheiwe C, Zentner J, Volk B, Romstock J, Stefan H, Hildebrandt M. A new clinico-pathological classification system for mesial temporal sclerosis. Acta Neuropathol. 2007; 113: 235-244. Neuropathology work-up of the ILAE Classification System of FCD page 28

32. Fauser S, Schulze-Bonhage A. Epileptogenicity of cortical dysplasia in temporal lobe dual pathology: an electrophysiological study with invasive recordings. Brain. 2006; 129: 82-95.

33. Thom M, Eriksson S, Martinian L, Caboclo LO, McEvoy AW, Duncan JS, Sisodiya SM. Temporal Lobe Sclerosis Associated With Hippocampal Sclerosis in Temporal Lobe Epilepsy: Neuropathological Features. J Neuropathol Exp Neurol. 2009; 68: 928-938.

34. Garbelli R, Meroni A, Magnaghi G, Beolchi MS, Ferrario A, Tassi L, Bramerio M, Spreafico R. Architectural (Type IA) focal cortical dysplasia and parvalbumin immunostaining in temporal lobe epilepsy. Epilepsia. 2006; 47: 1074-1078.

35. Meroni A, Galli C, Bramerio M, Tassi L, Colombo N, Cossu M, Lo Russo G, Garbelli R, Spreafico R. Nodular heterotopia: a neuropathological study of 24 patients undergoing surgery for drug-resistant epilepsy. Epilepsia. 2009; 50: 116-124.

36. Ferrier CH, Aronica E, Leijten FS, Spliet WG, Boer K, van Rijen PC, van Huffelen AC. Electrocorticography discharge patterns in patients with a cavernous hemangioma and pharmacoresistent epilepsy. J Neurosurg. 2007; 107: 495-503.

37. Lombroso CT. Can early postnatal closed head injury induce cortical dysplasia. Epilepsia. 2000; 41: 245-253.

38. Blumcke I, Giencke K, Wardelmann E, Beyenburg S, Kral T, Sarioglu N, Pietsch T, Wolf HK, Schramm J, Elger CE, Wiestler OD. The CD34 epitope is expressed in neoplastic and malformative lesions associated with chronic, focal epilepsies. Acta Neuropathol. 1999; 97: 481-490.

39. Blumcke I, Müller S, Buslei R, Riederer BM, Wiestler OD. Microtubule-associated protein-2 immunoreactivity: a useful tool in the differential diagnosis of low-grade neuroepithelial tumors. Acta Neuropathol (Berl). 2004; 108: 89-96.

-71- 40. Blumcke I, Pieper T, Pauli E, Hildebrandt M, Kudernatsch M, Winkler P, Karlmeier A, Holthausen H. Magnetic resonance imaging and neuropathological examination characterize a distinct variant of Focal Cortical Dysplasia Type I in children with severe epilepsies. Epileptic Dis. 2010; (epub ahead of print).

41. Rojiani AM, Emery JA, Anderson KJ, Massey JK. Distribution of heterotopic neurons in normal hemispheric white matter: a morphometric analysis. J Neuropathol Exp Neurol. 1996; 55: 178-183. Neuropathology work-up of the ILAE Classification System of FCD page 29

42. Emery JA, Roper SN, Rojiani AM. White matter neuronal heterotopia in temporal lobe epilepsy: a morphometric and immunohistochemical study. J Neuropathol Exp Neurol. 1997; 56: 1276-1282.

-72- 4.3 Publication 3: Acta Neuropathol. 2011 Nov 27. [Epub ahead of print]

Neuropathologic measurements in Focal Cortical Dysplasias: validation of the ILAE 2011 classification system and diagnostic implications for MRI

Angelika Mühlebner 1,2, Roland Coras 1, Katja Kobow 1, Martha Feucht 2, Thomas Czech 3, Hermann Stefan 4, Daniel Weigel 5, Hans Holthausen 6, Tom Pieper 6, Manfred Kudernatsch 6 and Ingmar Blümcke 1

1Department of Neuropathology, University Hospital Erlangen, 91054 Erlangen, Germany 2Department of Pediatrics, Medical University Vienna, 1090 Vienna, Austria 3Department of Neurosurgery, Medical University Vienna, 1090 Vienna, Austria 4Epilepsy Center, Department of Neurology, University Hospital Erlangen, 91054 Erlangen, Germany 5Department of Neurosurgery, University Hospital Erlangen, 91054 Erlangen, Germany 6Neuropediatric Clinic and Clinic for Neurorehabilitation, Epilepsy Center for Children and Adolescents, Schoen-Klinik Vogtareuth, 83569 Vogtareuth, Germany

Correspondence to: Prof. Dr. Ingmar Blümcke Department of Neuropathology University Hospital Erlangen Schwabachanlage 6 91054 Erlangen Germany fon: 0049 9131 85 26031 fax: 0049 9131 85 26033 [email protected]

-73- Abstract

Focal Cortical Dysplasias (FCD) represent a composite group of cortical malformations and are increasingly recognised as morphological substrate for severe therapy-refractory epilepsy in children and young adults. However, presurgical evaluation remains challenging as not all FCD variants can be reliably detected by high-resolution magnetic resonance imaging (MRI). Here, we studied a cohort of 52 epilepsy patients with neuropathological evidence for FCD using the 2011 classification of the International League against Epilepsy (ILAE) and systematically analyzed those histopathologic features applicable also for MRI diagnostics. Histopathologic parameters included quantitative measurements of cellular profiles, cortical thickness, heterotopic neurons in white matter, and myelination and were compared between FCD subtypes and age- /localization matched controls (n=36) using multivariate analysis. Dysmorphic neurons in both FCD Type II variants showed significantly increased diameters of their cell bodies and nuclei. In addition, cortical thickness was significantly increased with a distinct loss of myelin content specifying FCD Type IIb from IIa. The data further suggested, that myelination deficits in FCD Type IIb result from compromised oligodendroglial lineage differentiation and we concluded that the “transmantle sign” is a unique finding in FCD Type IIb. In contrast, FCD Type Ia was characterized by a smaller cortical ribbon and higher neuronal densities, but these parameters failed to reach statistical significance (considering age- and location-dependent variability in controls). All FCD variants showed abnormal grey-white matter boundaries with increased numbers of heterotopic neurons. Similar results were obtained also at deep white matter location. Thus, many FCD variants may indeed escape visual MRI inspection, but suspicious areas with increased or decreased cortical thickness as well as grey-white matter blurring may be uncovered using post-processing protocols of neuroimaging data. The systematic analysis of well-specified histopathological features could be helpful to improve sensitivity and specificity in MRI detection during pre-surgical work-up of patients with drug-resistant focal epilepsies.

Key words: malformations, neocortex, MRI, seizures, epilepsy, neuropathology

-74- Introduction

Focal Cortical Dysplasias (FCD) are increasingly recognized as underlying neuropathological condition in therapy refractory epilepsy patients. Since their first description by Taylor et al. in 1971, several FCD classification schemes have been introduced [2, 26, 34, 36]. Most recently, a three-tired international consensus classification system for FCDs was proposed by the ILAE, which included clinical, imaging and neuropathological findings [5]. Focal Cortical Dysplasia Type I is, thereby, recognized as an isolated malformation with abnormal cortical layering, either showing vertical persistence of developmental microcolumns (FCD Type Ia) or loss of the horizontal hexalaminar structure (FCD Type Ib). Focal Cortical Dysplasia Type II presents with altered cortical layering and specific cytological abnormalities including FCD Type IIa (dysmorphic neurons without balloon cells) and FCD Type IIb (dysmorphic neurons and balloon cells). Focal Cortical Dysplasia Type III comprises architectural abnormalities associated with either hippocampal sclerosis (FCD Type IIIa), tumours (FCD Type IIIb), vascular malformations (FCD Type IIIc) or other principal lesions acquired during early life (FCD Type IIId) [6]. However, neuropathological diagnosis remains often difficult and will require, therefore, standardized morphometric parameters, i.e., quantitative measurements of (i) dysmorphic neuronal cell body and nucleus diameters, (ii) neuronal cell densities, (iii) cortical thickness, or (iv) heterotopic neurons in white matter. Indeed, a recent study demonstrated that the diagnosis of neuropathologically ill-defined FCD subtypes will reach only moderate inter- and intraobserver agreement [7]. In our present study, we systematically evaluated a panel of neuropathologic features in FCD subtypes as classified by the ILAE 2011 scheme. These figures may present a basis for the reliable histopathologic diagnosis of epilepsy surgery brain specimens. These neuropathologic parameters may be also recognizable in presurgical high-field MRI and, thereby, help to interpret different presurgical imaging protocols and to classify FCD variants [9]. This is an important clinical issue, as the underlying cause in patients with focal epilepsies has a predictive value for long-term postsurgical seizure control [35]. As an example, excellent seizure control is reported in 70-100 % of patients with FCD Type IIb [20, 21, 40]. On the contrary, postsurgical outcome is less beneficial in Palmini’s FCD Type I with a range of complete seizure control between 21 to 67 % [13, 20, 21]. For this reason, neuroimaging strategies, such as positron emission tomography, ictal single photon emission tomography (SPECT) as well as high resolution structural and functional magnetic resonance imaging (MRI) were continuously improved to identify FCD subtypes as well as

-75- additional epileptogenic pathologies. A successful approach to increase sensitivity of structural MRI is the implementation of postacquisition digital processing methods such as texture analysis, statistic parametrical mapping (SPM) and voxel based morphometry (VBM) [3, 29, 41, 42]. VBM was reported to increase detection of an epileptogenic lesion in 59 % of patients previously diagnosed as MRI-negative [8]. SPM showed also focally increased T2 signal in 23 out of 45 patients with otherwise MRI-negative therapy-refractory seizures [29]. Here we applied VBM to 40 samples and measured this and that using. In addition the results were validated using these techniques. The results are discussed in reference to disease brains in children.

-76- Material and Methods

Subjects

A total of 105 patients with drug-resistant epilepsy and Focal Cortical Dysplasia underwent epilepsy surgery between 2003 and 2011 at the Zentrum für Epilepsie Erlangen (ZEE), Behandlungszentrum Vogtareuth (BHZ) and Epilepsie Monitoring Unit für Kinder Wien (EMU). Extensive presurgical evaluation including video-EEG monitoring, high-resolution MR imaging and neuropsychological testing [19] was performed in each patient to characterize the epileptogenic area, and to achieve seizure control by tailored surgical resection strategies. Only patients from which we received anatomically well preserved, en bloc resected neocortical tissue were included into the present study (n = 52). Informed consent was given from all patients included into our study. The age- and localization-matched control group consisted of 24 autopsy cases (age: mean = 31.79 ± 14.95; range = 2 – 54 years; localization: 19 frontal, 2 parietal, 10 temporal, 4 occipital; gender: 12 males, 12 females) and 12 cortical specimens from brain biopsies (age: mean = 52.42 ± 13.54; range = 36 – 77 years; localization: 6 frontal, 6 temporal; 7 males, 5 females). None of these patients reported about a clinical seizure history.

Tissue preparation and staining protocols

Surgical specimens were immediately submitted to the Neuropathology Dept. for further processing. The tissue was carefully orientated, cut perpendicular to the pial surface, fixed overnight in 4 % formaldehyde and routinely processed into liquid paraffin. Sections were cut at 4 µm with a microtome (Microm, Heidelberg, Germany), and mounted on positively charged slides (Superfrost + Menzel, Germany). Each specimen was histopathologically classified according to the recently published classification scheme on Focal Cortical Dysplasia using haematoxylin & eosin (H & E) as well as Cresyl violet – Luxol fast blue (Nissl - LFB). An immunohistochemical examination of all surgical specimens was performed using the following panel of antibodies and respective dilutions: MAP2 (microtubule-associated protein 2, 1:100, clone c, courtesy of Dr. Riederer), CNPase (2',3'- cyclic nucleotide 3' phosphodiesterase, 1 : 200, clone 11-5B, Millipore, Temecula, Canada), Olig2 (oligodendrocyte lineage transcription factor 2, 1 : 100, IBL, Minneapolis, USA),

-77- NeuN (neuronal nuclei, 1:1,000, clone A-60, Millipore), non-phosphorylated neurofilament H (1:100, clone SMI-32, Covance, Emeryville, USA), GFAP (glial fibrillary acidic protein, 1:800, clone 6F2, Dako, Glostrup, Denmark) and vimentin (1:500, clone 3B4, Dako). Autopsy cases were pre-treated with Triton X (1 % for 1 h) prior to incubation with anti- Olig2 antibody. The slides were air dried overnight at 37°C using an incubator. All immunohistochemical stainings were performed with a Ventana semiautomated staining machine (Nexus; Ventana, Illkirch, France) and the Ventana DAB staining system according to the manufacturer’s protocol. Image analysis was performed on a microcomputing imaging system (ColorView II CCD camera, AnalySIS imaging software, Soft imaging system, Münster, Germany) equipped to a BX51 microscope (Olympus, Tokyo, Japan). Images for evaluation of cortical thickness, heterotopic neurons at the grey-white matter border, neuropil intensity of the deep white matter and myelination were obtained at 1.25x magnification (feed size: 5.763 x 4.323 mm) representing 24.913 mm² (Figure 1 a - d). Olig2-postive cells were counted on images at 10x magnification (feed size: 721.100 x 540.800 µm). Nine images for neuronal measurements were taken at 20x magnification within deep cortical layers (layer 4-6) and aligned (feed size: 936.900 x 702.000 µm, Figure 1 e). Qualitative and semi-quantitative image analysis was performed with Cell^F imaging software (Olympus, Hamburg, Germany). The mean and standard deviation was calculated for every parameter and used for statistical analysis.

Semi-quantitative measurements

Cortical thickness

Cortical thickness was determined only in cortical regions cut perpendicular to the pial surface (see Figures 1 and 2), and which always included the maximum degree of pathology: MAP2 stainings were used to delineate the border between grey and white matter (Figure 1 a). In each microscopic section the anatomically well-preserved cortical ribbon was classified into “bottom of sulcus”, “intermediate zone” and “crown of gyrus” (Figure 1 a) and separately analyzed. In all three cortical areas the distance between pial surface and grey-white matter border was assessed. If the grey-white matter border was difficult to delineate, the suspected border was approved through serial sections, stained with CNPase, LFB or NeuN. All measurements were performed in triplicates. “Deep white matter” was considered at a distance > 500 µm apart from the grey-white matter boundary.

-78- Heterotopic neurons in white matter, myelination and oligodendrocytes

Quantification of heterotopic neuropil at grey-white matter borders is a difficult task as it shows large variability already in human control post-mortem tissue. We propose a new measurement protocol that calculates the ratio between optical density fields (250 x 500 µm, representing 0.625 mm²) obtained from deep grey matter and adjacent white matter (Figure 1 b). The computer program calculated MAP2 signal intensity according to HSI colour space (hue= 182.8/ 37.03°; saturation= 0/ 92 %; intensity= 61/ 214 %). Five individual measurements were performed per sample, ratios calculated and statistically compared between groups and all other morphological as well as clinical variables (age, localization). We assume that this technique is less susceptible for any observer bias, which may occur when studying only one single ROI at the white matter border of interest. We assessed heterotopic neuropil from the “deep white matter” in three randomly placed visual fields (500 x 500 µm, representing 0.750 mm²) by calculating MAP2 signal intensities as described above (Figure 1c). Intensity of CNPase signal was calculated accordingly using HSI colour space (hue= 60/ 31.41°; saturation= 9/ 96 %; intensity= 52/ 240 %). In addition, we performed Olig2-positive cell counts in 3 randomly placed 500 x 500 µm frames (representing 0.750 mm²) and all numbers refer to mm2.

Cellular densities and profiles

Neuronal density measurements were performed using Nissl-LFB stained sections (Figure 1e). Neurons were counted in a 500 x 500 µm frame (representing 0.250 mm²) within deep cortical layers (layer 4 - 6) and all numbers refer to mm². Areas of neuronal cell bodies were obtained from the 25 largest nerve cells within the frame by adjusting a smoothed polygon around each neuronal cell body (Figure 1 f). Neuronal cell body diameters were taken along the main neuronal axis (longest diameter) and then perpendicular through the level of the nucleus (shortest diameter; see Figure 1 g). The same procedure was conducted for the measurement of neuronal nuclei (Figure 1 g), as well as for balloon cells in FCD Type IIb.

-79- FCD classification and anatomical specification

Tissue specimens were categorized according to the following histopathological diagnoses [6]: “FCD Type Ia”, “FCD Type IIa”, “FCD Type IIb” and “FCD Type III” (comprising FCD Type IIIa/IIIb/IIIc/IIId). No specimens contained multiple FCD variants. Semi-quantitative measurements were performed from microscopic images in 11 patients with histopathological features of FCD Type Ia (age: mean = 5.36 ± 3.11; range = 1 – 11 years; gender: 4 males, 7 females). Only two cases of FCD Type Ib were encountered in our series. However, anatomical preservation in one surgical specimen was not adequate to be included into the present analysis. To the best of our knowledge, no patient with FCD Type Ic has been ever reported yet. Localization of cortical resection was temporal in three and occipital in eight patients. The following cortical regions showed histopathologic abnormalities and were separately studied: “bottom of sulcus” in a total of 16, “intermediate zone” in 15, “crown of gyrus” in 6 surgical specimens. Ten patients showed histopathological features of FCD Type IIa (age: mean = 10.90 ± 8.97; range = 1 – 28 years; gender: 6 males, 4 females). Absence of balloon cells was confirmed immunohistochemically in all cases using antibodies against vimentin [40]. Localization of cortical resection was frontal in five, parietal in two, temporal in three and occipital in one patient. Dysmorphic neurons were prominent at the “bottom of sulcus” in 11, at the “intermediate zone” in 10, and “crown of gyrus” in 5 specimens. Seventeen patients with FCD Type IIb were included (age: mean = 9.38 ± 7.55; range = 2 – 33 years; gender: 9 males, 8 females). Localization of cortical resection was frontal in five, parietal in two, temporal in three and occipital in two patients. The following cortical subregions were included into our analysis: 21 “bottom of sulcus”, 21 “intermediate zone”, and 18 “crown of gyrus”. Fifteen patients with FCD Type III (5 FCD Type IIIa and hippocampal sclerosis, two FCD Type IIIb and glio-neuronal tumours, four FCD Type IIIc and vascular malformations, four FCD Type IIId and prenatal infarction; mean age at surgery = 23.53 ± 18.22 years; range = 6 – 51 years; gender: 8 males, 7 females). Localization of cortical resection was frontal in one, parietal in one, temporal in eight and occipital in five patients; region of interest: 1 “bottom of sulcus”, 15 “intermediate zone”, 7 “crown of gyrus”. In total, 54 “bottom of sulcus”, 61 “intermediate zone” and 42 “crown of gyrus” were available in the control group.

-80- Statistical analysis

Statistical analysis was performed on SPSS 18 (IBM, PASW Statistics, USA). Uni- and multivariate ANOVA, student’s t-test, Pearson’s correlation as well as univariate and multivariate linear regression were used to analyse the data. Dunnet’s post hoc testing for multiple comparisons was performed if subgroups where compared to the control group. Tukey’s post hoc testing for multiple comparisons was used for analysis of neuronal measurements and subgroup analysis of the control group. P-values were considered significant if < 0.05. P-values are rounded to next 2 decimal digits. Data was tested for normal distribution with Shapiro-Wilk’s method and “heterotopic neurons in the grey - white matter border”, “neuropil intensity of the deep white matter” and “myelination of the deep white matter” was left skewed and therefore logarithmized.

-81- Results

Cortices are thicker in diseased brain cells

Cortical thickness is an important anatomical hallmark, which can be assessed by microscopy as well as MRI analysis (Figure 2). Microscopic measurements have to be performed always at a plane of section perpendicular to the pial surface (penetrating blood vessels should serve as control). When examining regions with maximum FCD pathology, our analysis revealed significantly increased cortical thickness in both FCD Type IIa (p < 0.01; mean = 3,098.42 µm; SD = 904.02 µm) and FCD Type IIb subgroups (univariate ANOVA with Dunnet’s post hoc testing for multiple comparisons; p < 0.01, mean = 2,958.07 µm; SD = 641.13 µm). In addition, FCD Type IIId showed a tendency for decreased cortical thickness (p = 0.07). The results were most robust at the bottom of sulcus region including 157 control values, as well as 42 FCD Type Ia, 33 FCD Type IIa, 60 FCD Type IIb and 33 FCD Type IIIa/IIIb/IIIc/IIId values (Table 1). Univariate linear regression confirmed the impact of histopathological diagnosis on cortical thickness (p < 0.01), which remained significant, when all data were adjusted for age and localization (p = 0.02). However, multivariate linear regression confirmed also that cortical thickness differed with localization (p < 0.01) and by trend with age (p = 0.06). Next, we studied the “intermediate cortical zone”, in which abnormal radial cortical layering was most often recognized, i.e. microcolumns in FCD Type Ia. The analysis was based on 120 controls, as well as 24 FCD Type Ia, 21 FCD Type IIa, 45 FCD Type IIb and 20 FCD Type IIIa/IIIb/IIIc/IIId values. Significant differences were observed between FCD subgroups using univariate ANOVA (p < 0.01), but Dunnet’s post hoc testing for multiple comparisons did only reveal a tendency when compared to controls (p = 0.10, data not shown). There were no differences at the “crown of gyrus” between our FCD cases. However, we also observed large variance of the grey - white matter border in controls, and have excluded this particular area from any further statistical analysis. Subgroup analysis in the “bottom of sulcus” region of our control group included 75 frontal, 9 parietal, 54 temporal, 18 occipital measurements and revealed a tendency towards decreased cortical thickness in elderly patients (Pearson’s correlation: p = 0.08; R² linear = 0.06), whereas parietal cortex was significantly thicker than frontal or temporal lobes (one-way univariate ANOVA with Tukey’s post hoc testing for multiple comparisons: p < 0.01, mean = 3037.13 µm; SD = 582.82 µm).

-82- Abnormal neuronal cell densities

The 2011 ILAE FCD classification system did not specify abnormal neuronal phenotypes with histopathological measures. Although MRI resolution is far too coarse to detect individual neuronal profiles, cellular densities are likely to alter T2 and FLAIR signals in high resolution MRI. Therefore, we have studied neuronal cell densities in cortical areas with maximum degree of FCD subtype pathology compared to age – and location matched controls. We also included measurements of maximum/minimum diameters for neuronal cell size and nuclei to allow a reliable neuropathologic assessment of abnormal cellular profiles. Interestingly, our present analysis revealed significantly reduced neuronal cell densities (neurons/ mm²) in FCD Type IIb (multivariate ANOVA with Dunnet’s post hoc testing for multiple comparison: p < 0.00, mean = 94.00, SD = 29.76; Table 1). In contrast, neuronal cell numbers were increased in FCD Type Ia (multivariate ANOVA with Dunnet’s post hoc testing for multiple comparison: p < 0.01, mean = 324.57, SD = 128.86) and FCD Type IIIc (multivariate ANOVA with Dunnet's post hoc testing for multiple comparison: p < 0.01, mean = 374.00, SD = 91.07). The analysis was based on neuronal density measurements within deep cortical layers (layers 4 – 6) in 29 controls, 7 FCD Type Ia, 7 FCD Type IIa, 9 FCD Type IIb and 15 FCD Type IIIa/IIIb/IIIc/IIId patients. Subgroup analysis for localization (using 8 frontal, 6 parietal, 9 temporal and 6 occipital specimens) and age dependency was also performed in the control group. Indeed, higher neuronal cell densities were encountered in the occipital (when studying deep cortical layers) compared to all other lobes (multivariate ANOVA with Tukey’s post hoc testing for multiple comparisons: p < 0.05, mean = 304.47, SD = 47.13). In order to discriminate whether FCD Type Ia or FCD Type IIIc is different from controls (these lesions commonly occurred in the temporal and occipital lobe) a two-tailed student’s t test was performed and revealed no differences, but a higher cell density could be confirmed in FCD Type IIIc (p < 0.00 ). Neuronal densities were not age dependent.

Abnormal cell body and nuclear diameter

We also measured the diameter of neuronal cell bodies and their nuclei within deep cortical layers. Both parameter were highly significant increased in all FCD Type IIa (multivariate ANOVA with Dunnet’s post hoc testing for multiple comparison: p = 0.00, mean = 25.76 (size) / 15.97 (nucleus) µm, SD = 4.01/ 2.31 µm²) and IIb variants

-83- (multivariate ANOVA with Tukey’s post hoc testing for multiple comparison: p = 0.00, mean = 26.33/ 16.41 µm, SD = 16.41/ 3.36 µm; Table 1). In FCD Type IIb, balloon cells revealed a long diameter of 38.98 ± 6.61, and short diameter of 32.72 ± 4.50. Their largest nuclear diameter was calculated as 14.87 ± 1.39. There was no size difference between our cases. In FCD Type Ia, increased neuronal cell body diameters were encountered (multivariate ANOVA with Tukey’s post hoc testing for multiple comparison: p = 0.03, mean = 21.64 µm, SD = 3.75). These values were different from our previous study [17], which result from measurements of infragranular cell layers (compared to supragranular layers) in the present analysis. Subgroup analysis in the control group showed no differences regarding age and localization. Similar results were obtained using the best fitted area of neuronal bodies and nucleus (data not shown). The results are summarized in Table 1.

Heterotopic neurons in the grey-white matter border

In the third part of our semi-quantitative morphometric analysis, we studied white matter changes focussing on heterotopic neurons. In contrast to previous reports [11, 28, 39], we specifically addressed the boundary between grey and white matter, as this interface was often shown to be compromised in MRI studies [9, 31]. We used the ratio between MAP2 signal intensities (detecting neuronal cell bodies and dendritic ramification) obtained from (1) very deep grey matter (as reference value for overall staining intensity) and (2) adjacent white matter measured at the bottom of sulcus only (Figure 1 b). Indeed, this ratio was significantly increased in all epilepsy patients using univariate ANOVA with Dunnet’s post hoc testing for multiple comparisons (p < 0.01), indicating abnormal grey-white matter blurring at all lobar regions studied (Figure 3 a – d). Statistical analysis was based on 195 values from the control group, 65 values from FCD Type Ia, 40 from FCD Type IIa, 90 from FCD Type IIb and 80 from FCD Type IIIa/IIIb/IIIc/IIId. A multivariate linear regression model also showed major impact of the diagnosis on blurred grey-white matter border when adjusted for age and localization (p < 0.01). In contrast, neither age nor localization revealed significant results. Separate subgroup analysis was also performed in controls regarding localization and age dependency (including 105 frontal, 15 parietal, 50 temporal and 25 occipital values). There were always higher MAP2 signal intensities in the temporal lobe compared to frontal samples (univariate ANOVA with Tukey’s post hoc testing for multiple comparisons: p = 0.02; mean = 0.2; SD = 0.12). No correlation could be found between age and blurred grey-white matter boundaries.

-84- Neuropil intensity in the deep white matter

MAP2 signal intensity revealed an increased number of heterotopic neurons in the “deep white matter” of all epilepsy specimens when compared to controls (univariate ANOVA with Dunnet’s post hoc testing for multiple comparisons, p = 0.00, Figure 3 e - h). Ninety control values, 39 FCD Type Ia, 21 FCD Type IIa, 54 FCD Type IIb and 27 FCD Type IIIa/IIIb/IIIc/IIId were included. When age and localization were included into the multivariate linear regression model, the histopathologic diagnosis associated most significantly with increased heterotopic neuropil intensities (p < 0.01). However, localization reached also significant levels (p < 0.01) whereas age did not. Localization and age dependency were separately analyzed in controls (including 48 frontal, 9 parietal, 21 temporal and 12 occipital values). No differences between regions and no correlation between age and heterotopic neuropil could be detected (data not shown).

Myelination and oligodendroglia

Our last morphometric paradigm addressed myelin content as well as semi-quantitative densities of oligodendroglia. Analysis of CNPase immunoreactivity revealed significantly altered myelination patterns only in FCD Type IIb (ANOVA with Dunnet’s post hoc testing for multiple comparison, p < 0.01, mean = 140824.37 µm²/ 24.21 %, SD = 196510.96 µm²/ 28.15 %, Figure 4 g and h). Twenty-one control values, 18 FCD Type Ia, 18 FCD Type IIa, 30 FCD Type IIb and 16 FCD Type IIIa/IIIb/IIIc/IIId values were available for statistical analysis. When including all these figures into a univariate linear regression analysis, the histopathologic diagnosis associated most significantly with CNPase signal alterations (p = 0.03; data not shown), as well as the lesions’ localization (p = 0.01), whereas age did not. The number of Olig2-positive cells within the white matter was dramatically reduced in the presence of balloon cells in two/thirds of our 17 FCD Type IIb cases. There was no correlation between the loss of myelin and increased neuropil intensity of the deep white matter. Intriguingly, oligodendroglial nuclei were abnormally configurated in these FCD Type IIb specimens (Figure 4 k) compared to the characteristic round shaped oligodendroglial nuclei in normal white matter (Figure 4 j / 5 g). We also observed a highly significant correlation between reduced myelin content and oligodendroglial cell numbers (Pearson’s correlation: p = 0.00, R² linear = 0.50, Figure 4 i). Despite pre-treatment, anti- Olig2 staining achieved no reliable immunohistochemical reaction patterns in our autopsy controls precluding further subgroup analysis.

-85- Discussion

The 2011 ILAE consensus classification of Focal Cortical Dysplasias qualitatively specified neuropathological criteria for each subgroup but did not establish quantitative data to achieve better sensitivity and specificity in routine microscopic diagnosis. Our present data fill this gap. It also highlights specific morphological parameter for FCD subgroups which might improve sensitivity and specificity in MRI detection during pre-surgical work-up of patients with drug-resistant focal epilepsies.

Focal Cortical Dysplasia Type I

The 2011 ILAE classification introduced this new FCD subtype (FCD Type Ia), because it likely represents a specific clinico-pathologic entity [6, 20]. Patients are young and characterized by early seizure onset, severe psycho-motor retardation and mild hemispheric hypoplasia without MR visibility of any other lesion [4, 17, 21, 35]. Drug resistance is frequent and surgical resection procedures achieved seizure control in only 21 % of children in an initial series addressing this isolated FCD subtype [21]. With increasing diagnostic and neurosurgical experience, however, the perspective for this peculiar group of young patients to become seizure free has dramatically improved to almost 50 % of cases [18]. Indeed, all FCD Type I patients from our present study belong to this clinico-pathologic variant. Neuropathological hallmarks included abnormal cortical layering effecting both radial migration and maturation of neurons (Figure 2 c and d). This aberrant pattern was most clearly visible in the “intermediate zone” of either the temporal or the occipital lobes, resembling those neurodevelopmental minicolumns described by the “radial unit lineage model” [27]. Furthermore, cortical thickness was decreased in our series of patients but this parameter failed to prove statistical confidence. The broad variation of cortical thickness in our age- and location matched control group most likely accounts for this result, as recent MRI data pointed to a significantly smaller hemisphere (in FCD Type Ia patients) ipsilateral to the ictal onset [4]. In addition, neuronal cell density tended to be increased which might point towards immaturity of the neuronal cells of these lesions. These findings are consistent with the recently introduced concept of post- migrational FCD [33]. However, to date, there are no molecular-biological nor genetic data available to clarify or even suggest the underlying pathomechanism in this difficult-to-treat- epileptic disorder.

-86- Focal Cortical Dysplasia Type II

This subtype was originally described by Taylor and colleagues in 1971, and comprised already two histopathological variants: FCDs with or without balloon cells [10, 36]. Barkovic’ MRI classification system assigned both variants to different pathomechanisms, i.e. FCD with balloon cells resulting from abnormal proliferation, whereas FCD without balloon cells as abnormal cortical organisation [2]. Current classification systems continued to separate both entities as FCD Type IIa and IIb. Yet, there is no distinguishing evidence for the clinical course, neither the aetiology nor for separate molecular-genetic or biological pathomechanisms [26]. Indeed, dysmorphic neurons are very similar between both variants and cannot be distinguished by our morphometric analysis (see Table 1). It was the presence of balloon cells, accompanied by lack of myelin and oligodendrocytes that makes the difference (Figures 4 and 5). When the centre of the lesion was well represented in our en bloc resected surgical specimens, lack of myelination revealed a funnel – like shape from the bottom of sulcus into the deep white matter in two thirds of our 17 cases with FCD Type IIb. Increased neuropil intensity was missing in the same area. Indeed, this pattern very much resembled that described by MRI studies as “transmantle sign” and we concluded that the abnormal MRI signal derives from the abnormally low myelin content (Figure 4). Another intriguing finding was the obvious lack of oligodendroglia in the same subgroup of specimens. This was always accompanied by increased numbers of balloon cells suggesting that (besides abnormal neuronal differentiation, i.e. dysmorphic neurons) oligodendroglia lineage differentiation is also compromised in patients suffering from FCD Type IIb. A detailed molecular genetic or cell- biological characterization of oligodendroglial lineage differentiation and balloon cells was beyond the scope of this present work [12, 22, 43]. However, the presence of “mild” FCD Type IIb variants (those showing little myelin deficiency, higher oligodendroglia numbers and fewer balloon cells; see Figure 4 i) may be compatible with a continuous lesional spectrum of FCD Type II subtypes: one end of the spectrum including lesions with only dysmorphic neurons and an almost normal white matter organisation (FCD Type IIa), whereas “full-blown” FCD Type IIb presents with many balloon cells, lack of oligodendroglial differentiation and a very large dysmyelination area, easily visible on structural MRI. The same may apply for the pathogenesis of FCD Type II variants, as most reports identified an altered mTOR signalling pathway in either FCD Type IIa or Type IIb [25, 30]. Molecular targets may be different and could separate both variants: TSC1 alterations have been so far demonstrated exclusively in FCD Type IIb lesions (balloon

-87- cells), whereas FCD Type IIa predominately associates with TSC2 alterations [24]. Our findings will have clinical impact for patient counselling and neuroimaging analysis. Indeed, complete resection of FCD Type IIb can be visually guided and allow seizure control in up to 100 % of patients, which is the best record for epilepsy surgery in all different pathologies encountered so far. In contrast, FCD Type IIa is less frequent and more likely to escape visually guided MRI reading, even in highly experienced centres. Our data point to abnormally increased cortical thickness in FCD Type IIa lesions (as well as abnormal grey-white matter boundary do to increased neuronal heterotopia), which should be detectable by systematic use of modern post-processing protocols, i.e. VBM or SPM [1]. However, cortical thickness can be difficult to address on MRI unless very thin sections (1 mm or below) are acquired as a volumetric data set and reformatted on a plane perpendicular to the gyrus or displayed as a 2-dimensional “flattened” cortex.

Focal Cortical Dysplasia Type III

This entity is newly introduced into the ILAE 2011 classification system and refers to a compilation of cortical abnormalities associated with and adjacent to another principal lesion. A most prominent example refers to FCD Type IIIa encountered in MTLE patients with hippocampal sclerosis (formerly classified as FCD Type Ia in Palmini’s system). This diagnosis was probably very much fostered by abnormal anterior lobe white matter signal changes detectable by MRI as well as from ictogenic EEG patterns evolving independent from hippocampal location. Yet, not all patients showed histomorphologically confirmed and approved FCDs. Thom et al. suggested, that one easy to recognize FCD Type IIIa variant should be termed “Temporal Lobe Sclerosis” (TLS), as its pathogenesis is rather reactive than malformative [37]. This notion is supported by recent findings from Garbelli et al. using high-resolution 7T MRI in the temporal lobe in HS patients [14]. They suggest the origin of abnormal white matter intensities (accompanied by anterior temporal lobe atrophy) to derive from punctuate myelin loss being of reactive/degenerative nature rather than malformative. The very similar post-operative seizure control in patients with only a principal lesion compared to those with an associated FCD further argue against an independent origin. In our study, we focused on those FCD Type III variants, which showed a macroscopically otherwise normal neocortex, i.e. FCD Type IIIa and IIIb. In contrast, FCD IIIc and IIId comprise a broad spectrum of heavily distorted cortical ribbons due to adjacent glio-mesodermal scarring or vascular malformations which may be represented by the

-88- finding of smaller cortical ribbons in FCD Type IIId and higher neuronal cells within the cortex in FCD Type IIIc. These lesions are usually easy to detect at microscopic levels as well as by MRI. In contrast, FCD IIIa and IIIb variants showed no gross abnormalities, and neither cortical thickness nor neuronal profiles were distinguishable from controls. These features make them more likely to escape visually guided MRI detection. On the other hand, we consistently observed neuronal heterotopias (measured as increased MAP2- immunoreactive neuropil intensities) at the grey-white matter border as well as in deep white matters areas. These abnormalities could be recognizable by high resolution MRI and may point to an associated FCD. Of note, our histopathologic analysis was not attempting to clarify the nature nor developmental stage of these neurons, as nicely addressed in previous studies [11, 16, 38, 39]. Another issue of continuous discussion relates to the specificity of neuronal heterotopias in different epileptogenic conditions. Some authors suggested that increased heterotopic neuropil will be recognized in most long-term epilepsy specimens. Our data does not contradict this hypothesis. Others proposed neuronal heterotopia even as a specific diagnosis, i.e. as mild form of cortical malformation in Palmini’s classification system (mMCD Type II) [26]. Yet, we can only speculate about the origin of heterotopic neuronal profiles in our FCD samples, occurring either in deep areas or at the grey-white matter interface. They may derive from resting adult stem/precursor cells, as recent neurodevelopmental studies provide evidence for neurogenic radial glia in the outer subventricular zone of human neocortex [15], a region that will turn into white matter at later maturation stages. In rat models as well as young children, increased hippocampal neurogenesis was shown following repetitive seizures [32]. This may apply also to cortical epilepsies but remains to be shown. The functional impact of aberrantly located white matter neurons to seizure susceptible neuronal networks is another controversial issue, as seizure initiation from white matter location is not very well documented [23].

-89- Acknowledgment

We kindly thank B. Rings for her expert technical assistance. This work was supported by the EU FP6 EPICURE project (LSH-CT-2006-037315) and the funds of the Österreichische Nationalbank (Anniversary Fund, project number: 12063)”. This study will be part of the doctoral thesis (AM), entitled “Pediatric Epilepsy Surgery: Predictors of (un)favourable Outcome” (www.meduniwien.ac.at/clins).

We declare no conflict of interest.

-90- References

1. Andres M, Andre VM, Nguyen S, Salamon N, Cepeda C, Levine MS, Leite JP, Neder L, Vinters HV, Mathern GW (2005) Human Cortical Dysplasia and Epilepsy: An Ontogenetic Hypothesis Based on Volumetric MRI and NeuN Neuronal Density and Size Measurements. Cereb Cortex 15:194-210.

2. Barkovich AJ, Kuzniecky RI, Jackson GD, Guerrini R, Dobyns WB (2005) A developmental and genetic classification for malformations of cortical development. Neurology 65:1873-1887.

3. Bernasconi N, Duchesne S, Janke A, Lerch J, Collins DL, Bernasconi A (2004) Whole- brain voxel-based statistical analysis of gray matter and white matter in temporal lobe epilepsy. Neuroimage 23:717-723.

4. Blumcke I, Pieper T, Pauli E, Hildebrandt M, Kudernatsch M, Winkler P, Karlmeier A, Holthausen H (2010) A distinct variant of focal cortical dysplasia type I characterised by magnetic resonance imaging and neuropathological examination in children with severe epilepsies. Epileptic Disord 12:172-180.

5. Blumcke I, Spreafico R (2011) An international consensus classification for focal cortical dysplasias. Lancet Neurol 10:26-27.

6. Blumcke I, Thom M, Aronica E, Armstrong DD, Vinters HV, Palmini A, Jacques TS, Avanzini G, Barkovich AJ, Battaglia G, Becker A, Cepeda C, Cendes F, Colombo N, Crino P, Cross JH, Delalande O, Dubeau F, Duncan JS, Guerrini R, Kahane P, Mathern GW, Najm I, Özkara C, Raybaud C, Represa A, Roper SN, Salamon N, Schulze- Bonhage A, Tassi L, Vezzani A, Spreafico R (2011) The clinico-pathological spectrum of Focal Cortical Dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission. Epilepsia 52:158-174.

7. Chamberlain WA, Cohen ML, Gyure KA, Kleinschmidt-DeMasters BK, Perry A, Powell SZ, Qian J, Staugaitis SM, Prayson RA (2009) Interobserver and intraobserver reproducibility in focal cortical dysplasia (malformations of cortical development). Epilepsia 50:2593-2598.

8. Colliot O, Antel SB, Naessens VB, Bernasconi N, Bernasconi A (2006) In vivo profiling of focal cortical dysplasia on high-resolution MRI with computational models. Epilepsia 47:134-142.

-91- 9. Colombo N, Salamon N, Raybaud C, Ozkara C, Barkovich AJ (2009) Imaging of malformations of cortical development. Epileptic Disord 11:194-205.

10. Crome L (1957) Infantile cerebral gliosis with giant nerve cells. J Neurol Neurosurg Psychiatry 20:117-124.

11. Emery JA, Roper SN, Rojiani AM (1997) White matter neuronal heterotopia in temporal lobe epilepsy: a morphometric and immunohistochemical study. J Neuropathol Exp Neurol 56:1276-1282.

12. Englund C, Folkerth RD, Born D, Lacy JM, Hevner RF (2005) Aberrant neuronal-glial differentiation in Taylor-type focal cortical dysplasia (type IIA/B). Acta Neuropathol (Berl) 109:519-533.

13. Fauser S, Schulze-Bonhage A, Honegger J, Carmona H, Huppertz HJ, Pantazis G, Rona S, Bast T, Strobl K, Steinhoff BJ, Korinthenberg R, Rating D, Volk B, Zentner J (2004) Focal cortical dysplasias: surgical outcome in 67 patients in relation to histological subtypes and dual pathology. Brain 127:2406-2418.

14. Garbelli R, Zucca I, Milesi G, Mastropietro A, D'Incerti L, Tassi L, Colombo N, Marras C, Villani F, Minati L, Spreafico R (2011) Combined 7-T MRI and histopathologic study of normal and dysplastic samples from patients with TLE. Neurology 76:1177-1185.

15. Hansen DV, Lui JH, Parker PR, Kriegstein AR (2010) Neurogenic radial glia in the outer subventricular zone of human neocortex. Nature 464:554-561.

16. Hardiman O, Burke T, Phillips J, Murphy S, O'Moore B, Staunton H, Farrell MA (1988) Microdysgenesis in resected temporal neocortex: incidence and clinical significance in focal epilepsy. Neurology 38:1041-1047.

17. Hildebrandt M, Pieper T, Winkler P, Kolodziejczyk D, Holthausen H, Blumcke I (2005) Neuropathological spectrum of cortical dysplasia in children with severe focal epilepsies. Acta Neuropathol 110:1-11.

18. Kessler-Uberti S, Pieper T, Eitel H, Pascher B, Hartlieb T, Getzinger T, Karlmeier A, Winkler PA, Kudernatsch M, Kolodziejczyk D, Blumcke I, Staudt M, Holthausen H (2011) 12 years of pediatric epilepsy surgery - the Vogtareuth experience. Neuropediatrics 42:32-33.

19. Kral T, Clusmann H, Urbach J, Schramm J, Elger CE, Kurthen M, Grunwald T (2002) Preoperative evaluation for epilepsy surgery (Bonn Algorithm). Zentralbl Neurochir 63:106-110.

-92- 20. Krsek P, Maton B, Korman B, Pacheco-Jacome E, Jayakar P, Dunoyer C, Rey G, Morrison G, Ragheb J, Vinters HV, Resnick T, Duchowny M (2008) Different features of histopathological subtypes of pediatric focal cortical dysplasia. Ann Neurol 63:758-769.

21. Krsek P, Pieper T, Karlmeier A, Hildebrandt M, Kolodziejczyk D, Winkler P, Pauli E, Blumcke I, Holthausen H (2009) Different presurgical characteristics and seizure outcomes in children with focal cortical dysplasia type I or II. Epilepsia 50:125-137.

22. Lamparello P, Baybis M, Pollard J, Hol EM, Eisenstat DD, Aronica E, Crino PB (2007) Developmental lineage of cell types in cortical dysplasia with balloon cells. Brain 130:2267-2276.

23. Loup F, Picard F, Yonekawa Y, Wieser HG, Fritschy JM (2009) Selective changes in GABAA receptor subtypes in white matter neurons of patients with focal epilepsy. Brain 132:2449-2463.

24. Majores M, Blumcke I, Urbach H, Meroni A, Hans V, Holthausen H, Elger CE, Schramm J, Galli C, Spreafico R, Wiestler OD, Becker AJ (2005) Distinct allelic variants of TSC1 and TSC2 in epilepsy-associated cortical malformations without balloon cells. J Neuropathol Exp Neurol 64:629-637.

25. Orlova KA, Tsai V, Baybis M, Heuer GG, Sisodiya S, Thom M, Strauss K, Aronica E, Storm PB, Crino PB (2010) Early progenitor cell marker expression distinguishes type II from type I focal cortical dysplasias. J Neuropathol Exp Neurol 69:850-863.

26. Palmini A, Najm I, Avanzini G, Babb T, Guerrini R, Foldvary-Schaefer N, Jackson G, Luders HO, Prayson R, Spreafico R, Vinters HV (2004) Terminology and classification of the cortical dysplasias. Neurology 62:S2-8.

27. Rakic P (2009) Evolution of the neocortex: a perspective from developmental biology. Nat Rev Neurosci 10:724-735.

28. Rojiani AM, Emery JA, Anderson KJ, Massey JK (1996) Distribution of heterotopic neurons in normal hemispheric white matter: a morphometric analysis. J Neuropathol Exp Neurol 55:178-183.

29. Rugg-Gunn FJ, Boulby PA, Symms MR, Barker GJ, Duncan JS (2005) Whole-brain T2 mapping demonstrates occult abnormalities in focal epilepsy. Neurology 64:318-325.

30. Schick V, Majores M, Koch A, Elger CE, Schramm J, Urbach H, Becker AJ (2007) Alterations of phosphatidylinositol 3-kinase pathway components in epilepsy- associated glioneuronal lesions. Epilepsia 48 Suppl 5:65-73.

-93- 31. Schijns OE, Bien CG, Majores M, von Lehe M, Urbach H, Becker A, Schramm J, Elger CE, Clusmann H (2011) Presence of temporal gray-white matter abnormalities does not influence epilepsy surgery outcome in temporal lobe epilepsy with hippocampal sclerosis. Neurosurgery 68:98-106; discussion 107.

32. Siebzehnrubl F, Blumcke I (2008) Neurogenesis in the Human Hippocampus and its Relevance to Temporal Lobe Epilepsies. Epilepsia 49 55-65.

33. Spreafico R (2010) Are some focal cortical dysplasias post-migratory cortical malformaitons ? Epileptic Disord 12:169-171.

34. Tassi L, Colombo N, Garbelli R, Francione S, Lo Russo G, Mai R, Cardinale F, Cossu M, Ferrario A, Galli C, Bramerio M, Citterio A, Spreafico R (2002) Focal cortical dysplasia: neuropathological subtypes, EEG, neuroimaging and surgical outcome. Brain 125:1719-1732.

35. Tassi L, Garbelli R, Colombo N, Bramerio M, Lo Russo G, Deleo F, Milesi G, Spreafico R (2010) Type I focal cortical dysplasia: surgical outcome is related to histopathology. Epileptic Disord 12:181-191.

36. Taylor DC, Falconer MA, Bruton CJ, Corsellis JA (1971) Focal dysplasia of the cerebral cortex in epilepsy. J Neurol Neurosurg Psychiatry 34:369-387.

37. Thom M, Eriksson S, Martinian L, Caboclo LO, McEvoy AW, Duncan JS, Sisodiya SM (2009) Temporal Lobe Sclerosis Associated With Hippocampal Sclerosis in Temporal Lobe Epilepsy: Neuropathological Features. J Neuropathol Exp Neurol 68:928-938.

38. Thom M, Martinian L, Parnavelas JG, Sisodiya SM (2004) Distribution of cortical interneurons in grey matter heterotopia in patients with epilepsy. Epilepsia 45:916-923.

39. Thom M, Sisodiya S, Harkness W, Scaravilli F (2001) Microdysgenesis in temporal lobe epilepsy. A quantitative and immunohistochemical study of white matter neurones. Brain 124:2299-2309.

40. Urbach H, Scheffler B, Heinrichsmeier T, von Oertzen J, Kral T, Wellmer J, Schramm J, Wiestler OD, Blumcke I (2002) Focal cortical dysplasia of Taylor's balloon cell type: a clinicopathological entity with characteristic neuroimaging and histopathological features, and favorable postsurgical outcome. Epilepsia 43:33-40.

41. Wagner J, Weber B, Urbach H, Elger CE, Huppertz HJ (2011) Morphometric MRI analysis improves detection of focal cortical dysplasia type II. Brain (epub ahead of print).

-94- 42. Wehner T, Luders H (2008) Role of neuroimaging in the presurgical evaluation of epilepsy. J Clin Neurol 4:1-16.

43. Yasin SA, Latak K, Becherini F, Ganapathi A, Miller K, Campos O, Picker SR, Bier N, Smith M, Thom M, Anderson G, Helen Cross J, Harkness W, Harding B, Jacques TS (2010) Balloon cells in human cortical dysplasia and tuberous sclerosis: isolation of a pathological progenitor-like cell. Acta Neuropathol 120:85-96.

-95- Tables

Table 1: Neuropathologic measurements in FCDs

cortical neuronal shortest diameter of thickness density neuronal neuronal diagnosis n [µm] p-value [count/ mm²] p-value diameter [µm] p-value nucleus [µm] p-value bottom of intermediate intermediate intermediate slucus zone zone zone 2033.10 214.50 18.83 13.11 frontal 8 > 0.05 > 0.05 > 0.05 > 0.05 (± 373.12) (± 54.21) (± 2.76) (± 1.73) 3037.13 207.33 17.86 13.22 parietal 6 < 0.01* > 0.05 > 0.05 > 0.05 (± 582.82) (± 49.99) (± 2.05) (± 1.45) controls 1997.53 200.44 17.07 12.22 temporal 9 > 0.05 > 0.05 > 0.05 > 0.05 (± 432.61) (± 33.07) (± 2.72) (± 2.21) 2440.68 304.00 17.45 11.53 occipital 6 > 0.05 < 0.01* > 0.05 > 0.05 (± 489.52) (± 47.13) (± 1.19) (± 1.19) 1925.13 324.57 21.64 14.85 FCD Type Ia 7 > 0.05 0.04* 0.03* > 0.05 (± 312.53) (± 128.86) (± 3.75) (± 2.15) 3098.42 193.71 25.76 15.97 FCD Type IIa 7 < 0.01* > 0.05 < 0.01* < 0.01* (± 904.02) (± 94.00) (± 4.01) (± 2.31) 2958.07 92.44 26.33 16.41 FCD Type IIb 9 < 0.01* < 0.01* < 0.01* < 0.01* (± 641.13) (± 29.76) (± 4.01) (± 3.36) 2287.38 251.20 16.68 12.37 FCD Type IIIa 5 > 0.05 > 0.05 > 0.05 > 0.05 (± 927.63) (± 136.39) (± 0.94) (± 1.05) 2287.38 251.20 16.68 12.37 FCD Type IIIa 5 > 0.05 > 0.05 > 0.05 > 0.05 (± 927.63) (± 136.39) (± 0.94) (± 1.05) 2188.51 116.00§ 18.1§ 12.15§ FCD Type IIIb 3 > 0.05 - - - (± 919.92) (± 0.00) (± 0.00) (± 0.00) 1541.82 374.00 18.48 12.97 FCD Type IIIc 5 > 0.05 < 0.01* > 0.05 > 0.05 (± 261.15) (± 91.07) (± 1.92) (± 0.98) 1695.68 331.00 19.18 12.89 FCD Type IIId 4 0.07 > 0.05 > 0.05 > 0.05 (± 296.38) (± 93.72) (± 2.69) (± 1.17)

Legend to Table 1: FCD Type IIa and IIb showed increased cortical thickness and neuronal densities (multivariate ANOVA with Tukey’s post hoc testing for multiple comparisons). Cortical thickness was also increased in the parietal cortex of controls (compared to all other lobes; multivariate ANOVA with Tukey’s post hoc testing). Neuronal cell body and nucleus diameters were significantly increased in both FCD Type IIa and IIb variants (multivariate ANOVA with Tukey’s post hoc testing). Asterisks indicate level of significance (p < 0.05) when compared to entire control group. All data presented in mean (± standard deviation). Neuronal / nuclear diameters were obtained at infragranular layers.

-96- Figures

Figure 1: Applied methodology

Legend to Figure 1: a: Anti-MAP2 staining of the frontal cortex in a 42 year old male autopsy control. Rectangles referred to three regions of interest: I - „bottom of sulcus“; II - „crown of gyrus“; III - „intermediate zone“. b: Semi-automated assessment of the grey - white matter border. The ratio between optical density fields (250 x 500 µm) obtained from

-97- deep grey and adjacent white matter was calculated (see Material and Methods). c: Anti- MAP2 staining measured in “deep white matter”, 500µm apart from the grey matter border. d: Anti-CNPase staining of the frontal cortex in a 49 year old surgical control. Scale bars in a – d = 1000 µm. No counterstaining was performed in a – d. e: Nissl - LFB staining of the frontal cortex in a 42 year old male autopsy control. Rectangle referred to deep cortical layers 4-6, from which neuronal measurements were obtained. Scale bar = 2000 µm. f: The size of neuronal cell bodies was measured using a smoothed polygon (see Material and Methods). g: On the left: largest diameter of a neuronal cell body was estimated at the level of the main cellular axis. The smallest diameter was obtained perpendicular to the main axis at the level of the nucleolus. On the right: same procedure was applied to measure the diameter of each cell nucleus. Scale bar = 20 µm (applies also to f).

-98- Figure 2: Cortical abnormalities in FCD subtypes

Legend to Figure 2: a and b: Neocortex of a control case. L1 – L6 = cortical layers. Nissl- LFB, NeuN. c and d: Vertical, microcolumnar arrangements in FCD Type Ia. Nissl-LFB, NeuN. e and f: Architectural dyslamination with abundant dysmorphic neurons in FCD Type IIa. Nissl-LFB, NeuN. g and h: Architectural dyslamination with abundant

-99- dysmorphic neurons and balloon cells in FCD Type IIb. At this magnification, balloon cells were not specifically visible using Nissl-LFB. i and j: “Temporal Lobe Sclerosis” with horizontally organized neuronal clusters in Layer 2. Patients suffered also from hippocampal sclerosis. This lesion classifies, therefore, as FCD ILAE Type IIIa. Nissl-LFB, NeuN. k and l: Architectural abnormalities associated with glio-neuronal tumours. This lesion classifies as FCD ILAE Type IIIb. Nissl-LFB, NeuN. Scale bar in F = 500µm and applies to all images.

-100- Figure 3: Heterotopic neuropil in white matter

Legend to Figure 3: a: Increased MAP2 immunoreactivity in white matter (as %) was detected in all epilepsy patients (FCD I, IIa, IIb, III) compared to controls (C). p < 0.05. b – d: Grey-white matter boundary (as shown by anti-MAP2 staining) was normal in controls (b), but severely blurred in FCD Type Ia (c) or FCD Type IIb (d). e: Abundant heterotopic neuropil was detected in deep white matter of all epilepsy patients (FCD I, IIa, IIb, III) compared to controls (C). p < 0.05. f – h: Deep white matter neurons are rarely detectable at a distance > 500 µm apart from the grey matter border in controls (f), but frequently encountered in FCD Type Ia (g) or FCD Type IIb (h). Scale bar in H = 100 µm and applies also to b – d and f – g.

-101- Figure 4: Comprehensive neuropathology and imaging findings in FCD Type IIb

Legend to Figure 4: a: T2-weighted FLAIR MRI of a 14 year old girl with therapy- refractory epilepsy and a right frontal lesion suspicious for FCD with transmantle sign (arrowhead). b: Nissl-LFB staining revealed increased cortical thickness and myelin loss at

-102- the “bottom of sulcus”. This pattern corresponds to the transmantle MRI sign. c: Cortical thickness was significantly increased (p < 0.01) in FCD Type IIb when measured at the “bottom of sulcus” region. C = control group; IIb = FCD Type IIb. p < 0.05. Error bars ± standard deviation. d and e: H - E stainings revealed histopathologic hallmarks of FCD ILAE Type IIb. d = balloon cell. e = dysmorphic neuron. f: Balloon cells in the white matter at the level of the “transmantle sign”. Anti-vimentin immunohistochenistry. g: Loss of anti- CNPase staining at same level of transmantle sign shown in b (serial sections). h: Myelin intensities were significantly reduced in FCD Type IIb. C = control group; IIb = FCD Type IIb. p < 0.05. Error bar ± standard deviation. i: Significant correlation between myelin content and oligodendroglial cell numbers. p < 0.01. Dashed line indicates regression and dotted lines 95% confidence intervals. cross = controls, star = FCD Type Ia, circle = FCD Type IIa, square = FCD Type IIb, diamond = FCD Type III. j and k: Olig2 immunoreactive cells were depicted from boxed areas with high vs. low CNPase content in Figure g. Note the reduced number of oligodendroglia in areas with abundant balloon cells (arrowheads in k). In addition, oligodendroglial cell nuclei were abnormal (k) compared to areas of intact myelin (j). Scale bar in b = 500 µm and applies also to g. Scale bar in d = 10 µm and applies also to e, j and k. Scale bar in f = 100 µm.

-103- Figure 5: Comprehensive neuropathology and imaging findings in FCD Type IIa

Legend to Figure 5: a: T2-weighted FLAIR of a 21 year old woman with therapy-refractory focal epilepsy. There was no lesion detectable at presurgical MRI. Arrow points to the resected epileptogenic zone as identified by subdural EEG recordings. b: Nissl-LFB staining revealed increased cortical thickness and almost normal myelination beneath the lesional cortex. c: Mean cortical thickness was significantly increased in our FCD Type IIa cohort (measured always at the “bottom of sulcus”). C = controls; IIa = FCD Type IIa. p < 0.01. Error bar ± standard deviation. d and e: Dysmorphic neurons in FCD Type IIa (d: Nissl - LFB, e: anti - SMI32 staining). f: Anti-CNPase staining showed normal CNPase activity in deep white matter/ serial section to b). g: Myelin staining patterns (anti-CNPase) were not different between FCD Type IIa and controls. Error bar = standard deviation.

-104- 5 Curriculum vitae

Dr. Angelika Mühlebner-Fahrngruber

Department of Pediatrics Medical University of Vienna Waehringer Guertel 18-20 1090 Vienna Austria Phone: +43 1 40400 3805 Fax: +43 1 40400 2277 mail: [email protected]

Personal data

Date of birth May 18th, 1984

Citizenship Austria

-105- Education

Medical University Vienna: doctoral program „Clinical Neurosciences“ 2008 - 2011 Title: “Pediatric Epilepsy Surgery - Predictors for (un)favourable Outcome“ Supervisor: Dr. Feucht

Medical University Vienna: 2002 – 2008 Medicine Graduation on July 24th, 2008

College: 1994 – 2002 Marchettigasse, Vienna VI first class honour degree

1990 – 1994 Primary school St. Marien, Vienna VI

Abroad

Resident at the Department of Neuropathology, University January 2011 – June 2011 Erlangen-Nuremberg

Fellowship at the Department of Neuropathology, University March 2010 – May 2010 Erlangen-Nuremberg

University Copenhagen Winter term 2006/07 Medicine (ERASMUS program)

-106- Scientific experience

Resident at the Department of Pediatrics Since August 2011 (Medical University Vienna)

Resident at the Department of Pediatrics July 2010 – December 2010 (Medical University Vienna), Neonatology third party funding

Resident at the Department of Pediatrics March 2009 – February 2010 (Medical University Vienna), Epilepsy Monitoring Unit third party funding

Resident for Neuropathology at the Institute of Neurology August 2008 – February 2009 (Medical University Vienna) third party funding

Diploma work Max F. Perutz Laboratories Vienna April – September 2007 Supervisor: Dr. Skern Novel binding partners of the poxviral proteins A46R and A52R

Department of Surgery: October 2005 – July 2006 Supervisor: Dr. Spittler Phosphorylation of the MAP kinases p38 and ERK1/2

Techniques

DNA methods cloning, gel electrophoresis, PCR

Cell culture transfection, expression, extractpreparation

Protein methods SDS-PAGE, Western Blot, silver staining

Flow cytometry ex-vivo measurement of extra-/ intracellular phosphorylation Beckman-Coulter Cytomics signalling molecules of PBMCs FC 500

Histology Immunohistochemistry

Electroencephalography Certified on Feb. 17th, 2011

-107- Clinical studies

SP954: An Open-Label Multicenter, Multinational Study of 2010, SI Lacosamide as First Add-On Antiepileptic Drug (AED) Treatment in Subjects with Partial-Onset Seizures

22.10.2008 ICH/ GCP training

-108- 6 Publications and Meetings

Publications

Mühlebner A, Gröppel G, Pahs G, Kasprian G, Schmook M, Prayer D, Czech T, Feucht M. Childhood temporal lobe epilepsy: beyond hippocampal sclerosis. In preparation.

Mühlebner A, Coras R, Kobow K, Feucht M, Czech T, Hermann Stefan, Daniel Weigel, Michael Buchfelder, Hans Holthausen, Tom Pieper, Manfred Kudernatsch, Blümcke I. Neuropathologic measurements in Focal Cortical Dysplasias: validation of the ILAE 2011 classification system and diagnostic implications for MRI. Acta Neuropathologica. 2011 Nov 27. [Epub ahead of print] .

Blümcke I and Mühlebner A. Neuropathological work-up of focal cortical dysplasias using the new ILAE consensus classification system - practical guideline article invited by the Euro-CNS Research Committee. Clin Neuropathol. 2011 Jul-Aug;30(4):164-77.

Dorfer C, Kasprian G, Mühlebner A, Czech T. Giant Solid-Cystic Hypothalamic Hamartoma: A Case Report. 2011 Feb;30(2):E7.

Muehlebner A, Groeppel G, Pahs G, Hainfellner JA, Prayer D, Czech T, Feucht M. Beneficial effect of epilepsy surgery in a case of childhood non-paraneoplastic limbic encephalitis. Epilepsy Res. 2010 Aug;90(3):295-9. Epub 2010 Jun 12.

Meetings

Meeting of the German, Swiss and Austrian section of the May 2011 ILAE, Graz, invited talk EU taskforce for Pediatric Epilepsy Surgery, Vienna, December 2010 invited talk International Congress of Neuropathology, Salzburg September 2010 invited workshop

-109- European Epilepsy Congress, Rhodos July 2010 scientific poster

September 2009 Meeting of the European Epilepsy Registry

Meeting of the American Society of Neuroradiology, May 2009 Vancouver scientific poster

2nd Colloqium on Epilepsy Surgery - Mai 2009 Pediatric Epilepsy Surgery, Lyon scientific poster

November 2008 ÖGNP Meeting in St. Pölten

Verein für Psychiatrie und Neurologie; Imaging in limbic December 2010 encephalitis, D. Prayer

Symposium Neuroradiologicum, MRI Imaging in October 2010 nonneoplastic Limbic Encephalitis, M. Scharitzer

Scholarships

November 2011 Ernst Niedermeyer Prize 2011

Scholarship for the attendance of the 4th Eilat International September 2011 Educational Course: PHARMACOLOGICAL TREATMENT OF EPILEPSY

Scholarship of the Austrian Section of the International March 2010 – May 2010 League Against Epilepsy

2006 Merit scholarship of the Medical University Vienna

-110-