Doublecortin immunoreactivity in giant cells of tuberous sclerosis and focal cortical dysplasia

Cerebral cortical lesions of tuberous sclerosis (TSC) and focal cortical dysplasia (FCD) show disturbances in laminar architecture and cellular differentiation. We immunohistochemically studied the expression of doublecortin, a fetal neuronal protein that regulates neuronal migration, in the surgical specimens of five TSC and eight FCD patients. In both TSC and FCD, bizarre giant cells showed a variable degree of doublecortin immunoreactivity. Both cytomegalic neurons and balloon cells were positive. The staining tended to be more intense in TSC than in FCD, although there were exceptional cases in both groups. Doublecortin immunoreactivity of normal-sized neural cells was restricted to a small number of astrocytes, and comparable to that in control patients. The persistent expression of doublecortin by giant cells in the postnatal cerebrum is additional evidence of abnormal differentiation, which may be relevant to the pathogenesis of cortical disarray in TSC and FCD.     

Characteristic expression of p57/Kip2 in balloon cells in focal cortical dysplasia

Balloon cells are a pathognomonic cellular feature of various cortical malformations, including focal cortical dysplasia type IIb (FCD IIb), cortical tubers of tuberous sclerosis (TSC) and hemimegalencephaly (HME). In the present study, we investigated the immunohistochemical expression of p57/Kip2, a member of the Cip/Kip family of cyclin‐dependent kinase inhibitory proteins, in balloon cells in surgical specimens taken from 26, 17 and six patients with FCD IIb, TSC and HME, respectively. Characteristic dot‐like reactivity with a faint, intense, reticular and process‐like pattern was confined to the proximal portion of the cytoplasmic processes of the cells. Immunoelectron microscopy revealed the p57/Kip2 reactivity on intermediate filaments in the proximal portion of the processes. The immunohistochemical profile appeared similar to that of CD34; however, a double immunofluorescence study demonstrated that no cells showed reactivity for both p57/Kip2 and CD34. The frequencies of the p57/Kip2‐positive cells in FCD IIb and HME were significantly higher than those in TSC, suggesting that the balloon cells may be heterogeneous. These findings suggest some functional significance of the protein on the cytoplasmic processes of balloon cells and appear consistent with the notion that the cells are abnormally differentiated progenitor cells.     

mTOR-dependent abnormalities in autophagy characterize human malformations of cortical development: evidence from focal cortical dysplasia and tuberous sclerosis

Focal cortical dysplasia (FCD) is a localized malformation of cortical development and is the commonest cause of severe childhood epilepsy in surgical practice. Children with FCD are severely disabled by their epilepsy, presenting with frequent seizures early in life. The commonest form of FCD in children is characterized by the presence of an abnormal population of cells, known as balloon cells. Similar pathological changes are seen in the cortical malformations that characterize patients with tuberous sclerosis complex (TSC). However, the cellular and molecular mechanisms that underlie the malformations of FCD and TSC are not well understood. We provide evidence for a defect in autophagy in FCD and TSC. We have found that balloon cells contain vacuoles that include components of the autophagy pathway. Specifically, we show that balloon cells contain prominent lysosomes by electron microscopy, immunohistochemistry for LAMP1 and LAMP2, LysoTracker labelling and enzyme histochemistry for acid phosphatase. Furthermore, we found that balloon cells contain components of the ATG pathway and that there is cytoplasmic accumulation of the regulator of autophagy, DOR. Most importantly we found that there is abnormal accumulation of the autophagy cargo protein, p62. We show that this defect in autophagy can be, in part, reversed in vitro by inhibition of the mammalian target of rapamycin (mTOR) suggesting that abnormal activation of mTOR may contribute directly to a defect in autophagy in FCD and TSC.     

Expression of tuberin and hamartin in tuberous sclerosis complex-associated and sporadic cortical dysplasia of Taylor's balloon cell type

Focal cortical dysplasia (FCD) type IIB is a malformation of cortical development characterized by presence of balloon cells. These cells share phenotypic features of giant cells found in tuberous sclerosis complex (TSC), but the relationship between FCD type IIB and TSC is not well established. TSC is an autosomal dominant disorder caused by mutation in either of two genes: TSC1, encoding hamartin, and TSC2, encoding tuberin. Both proteins form a complex inhibiting mTOR signalling pathway and thus regulate cell size and proliferation. In this study, tuberin and hamartin expression was evaluated under a confocal microscope in six cases of Taylor's balloon cell type FCD. Three patients met the clinical criteria for TSC. In three other patients, TSC was excluded based on a panel of clinical and radiological examinations. Additionally, two cases of FCD type I and 3 samples of normal brain tissue were used as a reference group. We found loss of tuberin and hamartin expression in FCD type IIB lesions from patients with TSC. In sporadic FCD type IIB cases, only a few tuberin and hamartin positive cells were detected in the white-grey matter junction and in deeper parts of the white matter. Cortical balloon cells showed loss of both tuberin and hamartin. In contrast, the expression of tuberin and hamartin in FCD type I samples was strong, similarly to normal brain tissue. In conclusion, loss of TSC1 and TSC2 products expression in balloon cells of both cortical dysplasia type IIB in TSC-related and sporadic patients suggests that FCD type IIB may represent the focal form of TSC.     

Expression of Bone Morphogenetic Protein-4 in the Cortical Lesions of Focal Cortical Dysplasia IIb and the Tuberous Sclerosis Complex

Focal cortical dysplasia type IIb (FCD IIb) and tuberous sclerosis complex (TSC) are malformations of cortical development (MCDs) and are frequently associated with medically intractable epilepsy. Previous studies have indicated that developmental abnormalities during the early stages of cortical development contribute to the onset of these diseases. Bone morphogenetic protein-4 (BMP-4) is a well-documented key regulator of cortical development. To understand the potential roles of BMP-4 in the cortical lesions associated with MCDs, we investigated the expression pattern of BMP-4 in surgical specimens from patients with FCD IIb (n?=?8) and TSC (cortical tubers; n?=?12), and age-matched normal cortices (CTX) (n?=?8) from autopsy samples were used as controls. The immunohistochemical results demonstrated that the overall immunoreactivity of the BMP-4 staining was diminished in the dysplastic cortices of the FCD IIb and TSC samples compared to the CTX samples. Moderate to strong BMP-4 immunoreactivity, however, was observed in malformed neurons, including dysmorphic neurons, giant neurons, balloon cells, giant cells, and reactive astrocytes. The confocal analysis demonstrated that most malformed neurons expressing BMP-4 were co-labeled with neuronal rather than astrocytic markers, indicating a neuronal lineage. Moreover, the decreased BMP-4 expression within the dysplastic cortex was confirmed by western blot analysis. In conclusion, the downregulation and altered cellular distribution of BMP-4 protein observed in MCDs suggests that BMP-4 may be involved in the pathogenesis of abnormal cortical development.     

Doublecortin‐like (DCL) expression in focal cortical dysplasia and cortical tubers

Purpose: Focal cortical dysplasia type IIB (FCD IIB) and cortical tubers of patients with tuberous sclerosis complex (TSC) are malformations of cortical development that are frequently associated with intractable epilepsy. Their histopathologic and molecular features suggest developmental abnormalities during the early stages of cortical development, which may involve neural progenitor cells. The aim of our study was to define the expression and cell‐specific distribution of doublecortin‐like (DCL), a protein critically involved in neuronal division and radial migration during early corticogenesis, in both FCD and TSC. Methods: DCL was studied in epilepsy surgery cases with FCD IIB (n = 8) and TSC (cortical tubers; n = 8) using immunocytochemistry, confocal analysis, and Western blotting. Results: Autopsy and surgical control neocortical specimens were characterized by modest DCL immunoreactivity (IR) throughout all cortical layers, but DCL IR was not detectable in the white matter. Balloon cells (BCs) in FCD and giant cells (GCs) in TSC expressed strong DCL IR. Most of the large dysplastic neurons (DNs) were positive for DCL in both FCD and TSC. Coexpression of DCL with the neural progenitor or neuroblast markers nestin, GFAPδ, and doublecortin was observed in both FCD and TSC specimens. The increased DCL expression within the dysplastic cortex, compared to control cortex, was confirmed by Western blot analysis. Discussion: The prominent postnatal expression of DCL by BCs/GCs and DNs in FCD and TSC supports an important role for this microtubule associated protein, also during early human cortical development, which could be relevant to the pathogenesis of these developmental glioneuronal malformations.     

Surgical pathology of epilepsy‐associated non‐neoplastic cerebral lesions: A brief introduction with special reference to hippocampal sclerosis and focal cortical dysplasia

Among epilepsy‐associated non‐neoplastic lesions, mesial temporal lobe epilepsy with hippocampal sclerosis (mTLE‐HS) and malformation of cortical development (MCD), including focal cortical dysplasia (FCD), are the two most frequent causes of drug‐resistant focal epilepsies, constituting about 50% of all surgical pathology of epilepsy. Several distinct histological patterns have been historically recognized in both HS and FCD, and several studies have tried to perform clinicopathological correlations. However, results have been controversial, particularly in terms of post‐surgical seizure outcome. Recently, the International League Against Epilepsy constituted a Task Forces of Neuropathology and FCD within the Commission on Diagnostic Methods, to establish an international consensus of histological classification of HS and FCD, respectively, based on agreement with the recognition of the importance of defining a histopathological classification system that reliably has some clinicopathological correlation. Such consensus classifications are likely to facilitate future clinicopathological studies. Meanwhile, we reviewed the neuropathology of 41 surgical cases of mTLE, and confirmed three type/patterns of HS along with no HS, based on the qualitative evaluation of the distribution and severity of neuronal loss and gliosis within hippocampal formation, that is, HS type 1 (61%) equivalent to “classical” Ammon's horn sclerosis, HS type 2 (2%) representing CA1 sclerosis, HS type 3 (17%) equivalent to end folium sclerosis, and no HS (19%). Furthermore, we performed a neuropathological comparative study on mTLE‐HS and dementia‐associated HS (d‐HS) in the elderly, and confirmed that neuropathological features differ between mTLE‐HS and d‐HS in the distribution of hippocampal neuronal loss and gliosis, morphology of reactive astrocytes and their protein expression, and presence of concomitant neurodegenerative changes, particularly Alzheimer type and TDP‐43 pathologies. These differences may account, at least in part, for the difference in pathogenesis and epileptogenicity of HS in mTLE and senile dementia. However, the etiology and pathogenesis of most epileptogenic lesions are yet to be elucidated.     

CD34-immunoreactive balloon cells in cortical malformations

Balloon cells are histopathological hallmarks of various cortical malformations, i.e., focal cortical dysplasia (Taylors type, FCD IIb), hemimegalencephaly (HME) or cortical tubers (tuberous sclerosis, TSC). Whether this intriguing cell type results from similar pathogenetic pathways remains to be shown. Here, we analyzed the immunohistochemical distribution pattern of the CD34 epitope in surgical specimens from 34 patients with FCD IIb, compared to that of 6 patients with TSC and 3 patients with HME. In normal brain, CD34 occurs only transiently during neurulation, but cannot be detected in mature neuroectodermal cell progenies. In contrast, 58% of our patients showed CD34 immunoreactivity within a subpopulation of balloon cells. Interestingly, CD34-positive balloon cells were confined to the white matter, but never observed in neocortical layers. Furthermore, balloon cells expressing neurofilament protein were also restricted to white matter, whereas GFAP-positive balloon cells were observed either in white or gray matter location. Clinical characteristics did not significantly differ between patients with CD34-positive versus CD34-negative lesions. No significant correlation was found between CD34 expression and genetic alterations of the TSC1 gene, which is affected in many FCD and TSC patients and which plays a role in the regulation of cell size. Further studies are warranted to clarify the restricted expression of CD34 in balloon cells of the white matter.     

Alterations of Phosphatidylinositol 3-Kinase Pathway Components in Epilepsy-associated Glioneuronal Lesions

Summary:  Low-grade glioneuronal lesions involving tumors such as gangliogliomas and focal cortical dysplasias (FCD) predispose individuals to pharmacoresistant epilepsy. A frequent variant of FCD is composed of dysplastic cytomegalic neurons and Taylor-type balloon cells (FCD_IIb). Those are similar to cellular elements, which are present in cortical tubers in the autosomal dominant inherited tuberous sclerosis complex (TSC). This phacomatosis is caused by mutations in the TSC1 or TSC2 genes. Recent data have indicated accumulation of distinct allelic variants of TSC1 also in FCD_IIb. TSC1 represents a key factor in the phosphatidylinositol 3-kinase (PI3K) pathway. A variety of alterations in the PI3K-pathway have been recently reported in epilepsy-associated glioneuronal malformations. Here, we discuss pathogenetic similarities and differences between cortical dysplasias as well epilepsy-associated glioneuronal tumors and TSC-associated cortical tubers with a focus on PI3K-pathway components including ezrin, radixin and moesin (ERM), which represent downstream effectors involved in cytoskeleton-membrane interference. No evidence has been found for mutational events of ERM genes to play a major pathogenetic role in epilepsy-associated glioneuronal malformations. In contrast, aberrant expression of ERM proteins in FCDs and gangliogliomas was observed. These alterations may relate to compromised interactions of dysplastic cellular components in epilepsy-associated glioneuronal lesions and be involved in aberrant PI3K-pathway signaling in epilepsy-associated malformations. However, the underlying cause of PI3K-pathway activation and the functional relationship of PI3K-pathway activity to generation of seizures in epilepsy-associated glioneuronal lesions will need to be determined in the future.     

Isolated cortical tuber in an infant with genetically confirmed tuberous sclerosis complex 1 presenting with symptomatic West syndrome

Tuberous sclerosis complex (TSC) is an autosomal dominant hereditary disorder caused by mutations in either TSC1 on chromosome 16 or TSC2 on chromosome 9, clinically characterized mainly by facial angiofibroma, epilepsy, and intellectual disability. Cortical dysplasias, subependymal nodules, and subependymal giant cell astrocytoma are characteristic central nervous system lesions among 11 major features in the current clinical diagnostic criteria for TSC. We encountered an unusual case of genetically confirmed TSC1 presenting with symptomatic West syndrome due to an isolated cortical dysplasia in the left occipital lobe of a six‐month‐old male infant who did not meet the clinical diagnostic criteria for TSC. The patient underwent left occipital lesionectomy at age 11 months and has been seizure‐free for nearly six years since then. Histological examination of the resection specimen revealed cortical neuronal dyslamination with abundant dysmorphic neurons and ballooned cells, consistent with focal cortical dysplasia (FCD) type IIb. However, the lesion was also accompanied by unusual features, including marked calcifications, dense fibrillary gliosis containing abundant Rosenthal fibers, CD34‐positive glial cells with abundant long processes confined to the dysplastic cortex, and multiple nodular lesions occupying the underlying white matter, consisting exclusively of ballooned cell and/or balloon‐like astrocytes with focal calcifications. Genetic testing for TSC1 and TSC2 using the patient's peripheral blood revealed a germline heterozygous mutation in exon 7 (NM_000368.5: c.526dupT, p.Tyr176fs) in TSC1. Isolated FCD with unusual features such as calcification, dense fibrillary gliosis, Rosenthal fibers and/or subependymal nodule‐like lesions in the white matter may indicate the possibility of a cortical tuber even without a clinical diagnosis of TSC. Identification of such histopathological findings has significant implications for early and accurate diagnosis and treatment of TSC, and is likely to serve as an important supplementary feature for the current clinical diagnostic criteria for TSC.     

Nestin expression in ganglioglioma

It has been suggested that gangliogliomas represent a neoplastic transformation of a dysplastic focus or heterotopia. Other theories propose that gangliogliomas arise from multipotent stem cells with the ability to differentiate along glial and neuronal cell lines. Our goal was to characterize the expression of nestin, a neuroepithelial precursor/stem cell antigen, in gangliogliomas along with other pathological and clinical features of this entity. The clinical and operative features of 18 recent cases meeting the histological criteria for ganglioglioma were reviewed. The expression of nestin, microtubule-associated protein 2 (MAP2), neurofilament, and glial fibrillary acidic protein (GFAP) was assessed by immunohistochemistry and confocal scanning laser microscopy. Abundant MAP2- and nestin-positive neuronal cells were found by immunohistochemistry in all 18 gangliogliomas. GFAP staining was found in reactive and lesional astrocytes but not in cells of neuronal morphology. Confocal microscopy demonstrated colocalization of nestin and MAP2 in select neuronal cells. The true lineage of gangliogliomas remains controversial. Our findings confirm the presence of cells within these lesions that harbor a persistent stem cell cytoskeletal protein (nestin). Further insight into the cytoskeletal derangement of nestin-positive neuronal cells may shed further light on the pathogenesis of gangliogliomas and its associated epilepsy.     

Activation of leukocyte immunoglobulin-like receptor B2 signaling pathway in cortical lesions of pediatric patients with focal cortical dysplasia type IIb and tuberous sclerosis complex

BackgroundsFocal cortical dysplasia type IIb (FCD IIb) and tuberous sclerosis complex (TSC) are very frequently associated with epilepsy in pediatric patients. Human leukocyte immunoglobulin-like receptor B2 (LILRB2) participates in the process of neurite growth, synaptic plasticity, and inflammatory reaction, suggesting a potential role of LILRB2 in epilepsy. However, little is known about the distribution and expression of LILRB2 in cortical lesions of FCD IIb and cortical tubers of TSC.MethodsIn this study, we have described the distribution and expression of LILRB2 signaling pathway in cortical lesions of pediatric patients with FCD IIb (n = 15) and TSC (n = 12) relative to age-matched autopsy control samples (CTX, n = 10), respectively. The protein levels of LILRB2 pathway molecules were assessed by western blotting and immunohistochemistry. The expression pattern was investigated by immunohistochemistry and double labeling experiment. Spearman correlation analysis to explore the correlation between LILRB2 protein level and seizure frequency.ResultsThe protein levels of LILRB2 and its downstream molecules POSH, SHROOM3, ROCK1, ROCK2 were increased in cortices of patients compared to CTX. Protein levels of LILRB2 negatively correlated with the frequency of seizures in FCD IIb and TSC patients, respectively. Moreover, all LILRB2 pathway molecules were strongly expressed in dysmorphic neurons, balloon cells, and giant cells, LILRB2 co-localized with neuron marker and astrocyte marker.ConclusionTaken together, the special expression patterns of LILRB2 signaling pathway in cortical lesions of FCD IIb and TSC implies that it may be involved in the process of epilepsy.     

Functional organization of the brain with malformations of cortical development

We examined the localization of cerebral functions in 28 patients with focal epilepsy and malformations of cortical development (MCDs). Polymicrogyria occurred in nine, hemimegalencephaly in four, heterotopia in eight, and focal cortical dysplasia (FCD) in nine cases. We used simple (sensomotor, visual) or complex (language, memory) functional magnetic resonance imaging (fMRI) paradigms. Two thirds of MCDs were activated by simple fMRI paradigms, whereas they less frequently showed activity during complex cognitive fMRI paradigms. During simple paradigms, all disturbances of cortical organization (polymicrogyria, schizencephaly, and mild-type FCD) showed activity, whereas other MCDs (disturbances of earlier steps of cortical development: hemimegalencephaly, Taylor-type FCD, and heterotopia) showed activity in only 44% (p < 0.01). The association between the pathophysiology and morphology of MCDs confirms the recently proposed classification system. Both focal neurological signs (p < 0.05) and focal electroencephalogram slowing (p < 0.05) independently correlated with MCD inactivity, confirming that fMRI showed neuronal functions of MCDs. Conclusively, fMRI visualizes the MCD functions and their relationship to the eloquent cortex, providing useful information before epilepsy surgery. Surgery of cortical organization disturbances should be cautiously performed because these malformations may participate to some degree in brain functions.     

Expression and cell distribution of group I and group II metabotropic glutamate receptor subtypes in taylor-type focal cortical dysplasia

PURPOSE: Focal cortical dysplasia (FCD) is known to be a major cause of intractable epilepsy. The cellular mechanism(s) underlying the epileptogenicity of FCD remain largely unknown. Because recent studies indicate that metabotropic glutamate receptor subtypes (mGluRs) play a role in epileptogenesis, we investigated the expression and cellular distribution pattern of mGluRs in FCD specimens. METHODS: Immunocytochemical expression of group I and group II mGluR subtypes was investigated in 15 specimens of human FCD obtained during epilepsy surgery. RESULTS: Strong mGluR1alpha and mGluR5 (group I mGluRs) immunoreactivity (IR) was observed in the majority of FCD specimens in dysplastic as well as in heterotopic neurons. mGluR1alpha was expressed in a subpopulation of neurons (mainly large dysplastic cells), whereas mGluR5 was represented in a higher percentage of dysplastic neuronal cells. Group II mGluRs (mGluR2/3) IR was observed less frequently than that in group I mGluRs and generally appeared in <10% of the dysplastic neurons. IR for all three mGluR subtypes was observed in balloon cells. mGluR2/3 appeared to be most frequently expressed in glial fibrillary acidic protein (GFAP)-positive balloon cells (glial type), and mGluR1alpha, in microtubule-associated protein (MAP)2-positive cells (neuronal type). mGluR5 was present in the majority of balloon cells. Occasionally glial mGluR1alpha IR was observed in bizarre glial cells with di- or multinuclei. Reactive astrocytes were intensively stained, mainly with mGluR5 and mGluR2/3. CONCLUSIONS: The cellular distribution of mGluR subtypes, with high expression of mGluR1alpha and mGluR5 in dysplastic neurons, suggests a possible contribution of group I mGluRs to the intrinsic and high epileptogenicity of dysplastic cortical regions.     

Focal cortical dysplasia of Taylor's balloon cell type: mutational analysis of the TSC1 gene indicates a pathogenic relationship to tuberous sclerosis

Focal cortical dysplasia (FCD) is characterized by a localized malformation of the neocortex and underlying white matter. Balloon cells, similar to those observed in tuberous sclerosis, are present in many cases (FCD(bc)). In these patients, a hyperintense funnel-shaped subcortical lesion tapering toward the lateral ventricle was the characteristic finding on fluid-attenuated inversion recovery magnetic resonance imaging scans. Surgical lesionectomy results in complete seizure relief. Although the pathogenesis of FCD(bc) remains uncertain, histopathological similarities indicate that FCD(bc) may be related pathogenetically to tuberous sclerosis. Here, we studied alterations of the TSC1 and TSC2 genes in a cohort of patients with chronic, focal epilepsy and histologically documented FCD(bc) (n = 48). DNA was obtained after microdissection and laser-assisted isolation of balloon cells, dysplastic neurons, and nonlesional cells from adjacent normal brain tissue. Sequence alterations resulting in amino acid exchange of the TSC1 gene product affecting exons 5 and 17 and silent base exchanges in exons 14 and 22 were increased in patients with FCD(bc) compared with 200 control individuals (exon 5, 2.3% FCD(bc) vs 0% C; exon 17, 35% FCD(bc) vs 1.0% C; exon 14, 37.8% FCD(bc) vs 15% C; exon 22, 45% FCD(bc) vs 23.8% C). Sequence alterations could be detected in FCD(bc) and in adjacent normal cells. In 24 patients, DNA was suitable to study loss of heterozygosity at the TSC1 gene locus in microdissected FCD(bc) samples compared with control tissue. Eleven FCD(bc) cases exhibited loss of heterozygosity. In the TSC2 gene, only silent polymorphisms were detected at similar frequencies as in controls. Our findings indicate that FCD(bc) constitutes a clinicopathological entity with distinct neuroradiological, neuropathological, and molecular genetic features. These data also suggest a role of the TSC1 gene in the development of FCD(bc) and point toward a pathogenic relationship between FCD(bc) and the tuberous sclerosis complex.     

A comparison of cell phenotypes in hemimegalencephaly and tuberous sclerosis

Hemimegalencephaly, an uncommon sporadic nonfamilial congenital dysplastic abnormality of the central nervous system, constitutes a pathological spectrum of neuronal migration disorders, but consistently includes abnormal large neurons similar to those in the cortical tubers of tuberous sclerosis. Microscopically, there are also cells with homogeneous and weakly eosinophilic cytoplasm with a single eccentric nucleus, sometimes called balloon cells (likewise prominent in tuberous sclerosis). We looked for immunohistochemical and ultrastructural differences in the large neurons and balloon cells between hemimegalencephaly and tuberous sclerosis. Microtubule-associated protein 1B and 2, phosphorylated and non-phosphorylated neurofilament and synaptophysin identify the large neurons and distinguish them from balloon cells in both entities. Balloon cells in hemimegalencephaly showed no immunoreactivity for TSC2 gene product, tuberin, and vimentin, but similar cells in tuber tissue showed consistent immunoreactivity. Balloon cells in hemimegalencephaly showed no immunoreactivity for glial fibrillary acidic protein, but some cells in tubers showed such immunoreactivity. Ultrastructurally, balloon cells in hemimegalencephaly contained very few lysosomes, microfilaments, and microtubules, but abundant lipofuscin granules. Similar cells in tubers had prominent lysosomes, more microfilaments and microtubules, and very few lipofuscin granules. The resemblance between abnormal cells in hemimegalencephaly and tuberous sclerosis is superficial; their immunohistochemistry and electron microscopic profiles show distinct differences. Received: 4 January 1999 / Revised: 1 March 1999 / Accepted: 15 March 1999     

Analysis of chromosomal instability in focal cortical dysplasia of Taylor’s balloon cell type

Focal cortical dysplasias (FCD) represent a frequent finding in patients with chronic intractable epilepsy. Neuropathological hallmarks include localized dyslamination of the neocortex and neuronal heterotopias in white matter. Balloon cells, similar to those occurring in cortical tubers of patients with tuberous sclerosis (TSC) are observed in numerous patients. These lesions were classified as FCD type IIb (FCD IIb). Recent findings indicate an accumulation of TSC1 polymorphisms as well as loss of heterozygosity (LOH) and/or microsatellite instability (MSI) at the TSC1 locus on chromosome 9q in FCD IIb. Here, we tested the hypothesis of whether chromosomal instability constitutes a genome-wide phenomenon in this patient cohort. Seven microsatellite markers based on a reference panel recommended by the international workshop on microsatellite instability were analyzed in 14 surgical FCD IIb specimens. DNA from single laser-microdissected cells, i.e., balloon cells versus control neurons obtained from adjacent cortex was harvested for PCR amplification and subsequent fluorescent fragment length gel electrophoresis. Our analysis revealed only rare instances of LOH and MSI at genomic loci on 2p and 17q, whereas no alterations were found at informative markers on chromosomes 1p, 5q and 18q. In addition, no loss of repair protein expression (MSH2 or MLH1) has been identified in balloon cell nuclei of FCD IIb specimens. The present data suggest solitary LOH and MSI events at genomic localizations others than the TSC1 locus to occur in FCD IIb. Our findings lend further support to the hypothesis that the molecular pathogenesis of FCD IIb is associated with TSC1.     

Surgical pathologic features of cerebral cortical lesions taken from 600 patients with intractable epilepsy

Objectives: To determine the scope of histopathological variation in lesions responsible for epileptogenesis, I retrospectively reviewed the features of surgical specimens taken consecutively from 600 patients with intractable epilepsy. Methods: The patients were divided into three groups on the basis of age at seizure onset: 94 patients with infantile onset (before 1 year of age), 307 patients with juvenile onset (between 1 and 12 years of age), and 199 patients with adolescent/adult onset (at 13 years of age or beyond). Histological and immunohistochemical evaluations of the surgical specimens were performed. Results: In the infant group, seizure duration was significantly shorter than in the other groups, and malformations caused by abnormalities of cortical development, including focal cortical dysplasia (FCD) type IIa/b, tuberous sclerosis, hemimegalencephaly, and polymicrogyria were predominant, whereas in the juvenile and adolescent/adult groups, other lesions such as hippocampal sclerosis (HS), tumors, FCD type I, and vascular lesions were frequently observed. For patients with HS, seizure duration in the juvenile group was significantly longer than in the adolescent/adult group. FCD type IIIa was noted in nearly half of patient with HS in both juvenile and adolescent/adult groups. The causative tumors included dysembryoplastic neuroepithelial tumors, gangliogliomas, astrocytomas, and other glioneuronal and glial tumors. Conclusion: Various histopathological entities and types, showing clear predominance depending on the age at seizure onset, were observed in patients with epilepsy. These features appear to provide information on the pathomechanisms of the lesions and their clinical relevance in affected patients.     

Immunohistochemical expression of fibroblast growth factor (FGF)‐2 in epilepsy‐associated malformations of cortical development (MCDs)

To elucidate the biological significance of dysplastic cells in malformations of cortical development, an immunohistochemical study was performed to investigate fibroblast growth factor‐2 (FGF‐2) expression in corticectomy specimens from epilepsy patients, including focal cortical dysplasia (FCD) with balloon cells (BCs) (n = 4; age/sex = 2M, 14F, 24M, 45M), tubers of tuberous sclerosis complex (TSC‐tubers) (n = 2; 1F, 3F), FCD without BCs (n = 3; 23F, 23M, 25M), and gliotic lesions (n = 3; 12M, 25M, 29M). The nucleus and/or cytoplasm of astrocytes in all cases examined were positive for FGF‐2; however, FGF‐2 immunoreactivity was not detected in oligodendroglial cells. In all dysplastic lesions, FGF‐2 was detected in the astrocytic nuclei, and cytoplasm and/or nuclei of BCs. Dysplastic neurons (DNs) in FCD with BCs and TSC‐tubers were variably positive for FGF‐2 in the cytoplasm, but FGF‐2 was not detected in the neurons of FCD without BCs. The number of FGF‐2 immunoreactive cells (FGF‐2‐IR%) in FCD with BCs (46.0 ± 4.1%) was higher than that in FCD without BCs (19.8 ± 3.1%) and gliotic lesions (19.5 ± 3.3%) with statistical significance (P < 0.001). These results, together with previous reports showing FGF‐2 expression in neuroblasts and glioblasts in human fetal brain, and mainly in astrocytes in adult brain, suggest that FGF‐2 expression in MCDs reflects incomplete differentiation and maturation of dysplastic cells, and that FGF‐2‐IR% is associated with histological subtypes of MCD, reflecting the timing of insults underlying the pathogenesis of each disorder.