Neuropathology of Developmental Disorders Associated with Epilepsy

Chapter 14

Harry V. Vinters

Noriko Salamon

Hajime Miyata

Negar Khanlou

Gary W. Mathern

Introduction

Structural lesions associated with epilepsy can be categorized into the following groups: (a) malformative, (b) neoplastic, (c) familial/metabolic, (d) vascular/traumatic, (e) infectious/inflammatory, and (f) associated with Ammon’s horn (hippocampal, mesial temporal) sclerosis.⁴⁴^,¹⁸⁴ Several authors have suggested that some of these pathologic processes may overlap in epilepsy patients,¹²^,¹⁶ and dual pathology (e.g., a corticectomy showing features of both a malformative and destructive lesion) may occasionally be encountered.¹¹⁶ Some of the causal lesions are amenable to definitive surgical treatment (e.g., low-grade neoplasms), whereas others (e.g., metabolic diseases) frequently undergo diagnostic brain biopsy, sometimes unintentionally. This chapter focuses on developmental malformations of the neocortex that account for the majority of structural lesions seen in infants and children with intractable epilepsy, especially those with infantile spasms (IS). (The other categories of epileptogenic structural lesions are discussed elsewhere.) More subtle malformations are also recognized with increasing frequency in adults with epilepsy.

Neocortical malformations have usually been considered to represent developmental disorders of neuronal migration (or vascular formation), and have variously been classified with regard to morphology or putative etiology.⁸^,¹³⁵^,¹³⁸ The categorization of these heterogeneous lesions is increasingly being modified by (a) the availability of high-resolution multimodality neuroimaging techniques that can be used to predict pathologic abnormalities in a given patient²⁴^,²⁶^,¹⁵⁸ and (b) molecular genetic clues to pathogenesis based on techniques such as gene expression profiling following laser capture microdissection of surgical specimens.³² The genetic basis of some types of lissencephaly (e.g., Miller-Dieker syndrome) is now quite well understood.³²^,¹⁴⁷ They can be roughly categorized as follows: (a) cortical dysplasia (CD), which accounts for the majority of malformations associated with pediatric epilepsy and encompasses the full spectrum of neuronal migration disorders (NMDs), sometimes also described as malformations of cortical development (MCDs), ranging from the most subtle to the most severe; (b) structural lesions associated with tuberous sclerosis complex (TSC); (c) Sturge-Weber-Dimitri syndrome (SWDS), also known as encephalotrigeminal angiomatosis; (d) neurofibromatosis type 2, which may be associated with meningio-angiomatosis; and (e) vascular malformations.

This chapter discusses clinicopathologic associations between the recognized pediatric epilepsy syndromes and their neuroradiologic and neuropathologic substrates. We review the terminology used to characterize these complex malformative lesions in the light of modern neuroimaging data. We give a brief overview of development of the cerebral cortex and identify the points of susceptibility at which the error or errors (genetic or environmental) resulting in cortical malformations may occur.

Clinicopathologic Features

Epilepsy has traditionally been classified into syndromes based on clinical presentation and electroencephalographic (EEG) findings. However, there is often a marked discrepancy between the clinical phenomenology of a seizure disorder and its neuropathologic substrate(s). Infantile spasms (IS, West syndrome) and Lennox-Gastaut syndrome can be seen with a wide range of cerebral lesions,⁶⁹^,¹⁰⁶^,¹⁵³ indicating the nonspecific nature of these seizure disorders. The clinical form of epilepsy seen in a given patient appears to depend more on when during cerebral development the lesion occurred than on the specific type or topographic distribution of lesions.⁶⁹^,¹⁰⁸ It has been suggested that the central nervous system (CNS) lesions associated with IS can be functionally categorized into three groups: (a) diffuse, (b) focal or multifocal cerebral lesions, and (c) cases with minimal neuropathologic change.¹⁵³ Diffuse hemispheric lesions include hemimegalencephaly (HME), agyria/pachygyria-lissencephaly, and Aicardi syndrome.¹ Focal and multifocal lesions include CD and destructive lesions (vascular and infectious), as well as cortical tubers seen in patients with TSC.

Developmental malformations of the neocortex (malformations of cortical development, MCDs) can be considered a spectrum of CD resulting from derangement of the normal process of cortical development.¹¹⁷^,¹²⁰^,¹³⁰^,¹³¹^,¹⁴² This spectrum consists of a range of morphologic features associated with multiple putative etiologic factors, including genetic and environmental (e.g., destructive) influences. CD encompasses the full spectrum of neocortical malformations, ranging from the most subtle (microdysgenesis) to the most severe (HME), and includes such conditions as agyria/pachygyria-lissencephaly, polymicrogyria (PMG), and focal CD. CD, therefore, comprises a spectrum of derangements in neocortical development that are associated with a range of morphologic features and with multiple putative etiologic factors, including genetic and environmental influences. The resultant neuropathologic features may reflect abnormalities that probably occur within discrete time windows during brain development. Clinically there is an inverse correlation between the size and severity of CD and the age at clinical presentation,²⁶^,¹¹⁷ supporting the notion that there is a predominance of pathologically severe CD in neonates and infants with seizures, including those

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with IS.⁷³ Although CD accounts for the majority of malformations associated with pediatric epilepsy, other malformative lesions are also capable of producing these epilepsy syndromes, and they frequently show neuropathologic changes that overlap significantly with those seen in patients with CD.

Jellinger⁶⁹ noted that the clinical severity of a given seizure disorder appears to be more closely related to the timing of the insult (whether genetic or environmental) and its effect on the processes of development than to the nature of the lesion itself. This may explain some of the heterogeneity of neuropathologic lesions seen in children with epilepsy because the clinical phenotype results not only from the lesion or putative insult to the developing CNS, but also from the developmental processes it affects.

A puzzling feature noted in pediatric epilepsy is the finding in cases of IS of diffuse and transient suppression of cortical activity in the presence of focal or multifocal lesions.¹⁵³ This diffuse cortical suppression has not been linked to particular distributions or types of neuropathologic change. Rather, Robain and Vinters suggested that this feature of IS is associated with neocortical immaturity.¹⁵³ The pattern of evolution of IS from transient and diffuse suppression of cortical discharge to ultimately focal or generalized seizures may represent a maturation of the malformed cortex.¹⁵³

Nosology

The nosology and understanding of the full spectrum of CD are evolving and reflect a progressive elucidation of the basis of these extremely complex lesions. Initially, cortical malformations were referred to largely by their gross characteristics (i.e., agyria/pachygyria-lissencephaly, HME, microgyria). As investigators discovered the range of microscopic cortical malformations that produce epilepsy but show no (or relatively mild) gross abnormalities, additional terms such as microdysgenesis,⁵⁷^,¹⁰⁷^,¹⁰⁸ dysplastic cortical architecture (not otherwise specified),¹⁵⁷ focal cortical dysplasia,¹¹⁹^,¹⁷⁴ and generalized or diffuse cortical dysplasia⁷⁸^,⁸³^,⁹⁶ were added to the literature. The nomenclature of CD has evolved through several schema of classification, each intended to provide correlations with morphology of seizures and reflect etiologic mechanisms.⁸^,⁹^,¹¹⁷ Focal cortical dysplasia was initially used to specify lesions in which cytomegalic neurons were present,¹⁷⁴ although its frequent use to refer to a localized region of CD renders the term somewhat ambiguous. Some investigators prefer to use a traditional classification of migrational disorders into four main groups: (a) agyria/pachygyria-lissencephaly, (b) microgyria-polymicrogyria, (c) dysplastic cortical architecture, and (d) heterotopias.¹⁵⁷ Others, to denote the belief that these lesions are related and reflect developmental abnormalities along a continuous spectrum, have chosen to refer to this group of lesions as neuronal migration disorders or NMDs,⁸^,¹³⁰^,¹³¹ cerebral dysgenesis,¹⁵⁹ or synaptic dysgenesis.¹³ The nosology of developmental neocortical malformations will continue to evolve, especially in the era of high-resolution magnetic resonance imaging (MRI), and reflect a progressive understanding of the underlying biology of these complex lesions (see Chapter 259). A recent consensus conference resulted in a proposal to subclassify CD or grade its severity (in surgical resection specimens) using simple morphologic criteria identifiable by any experienced neuropathologist, for example, presence or absence of cortical disorganization, enlarged or dysmorphic neuronal cell bodies, and “balloon cells” (see later discussion).¹³² This classification scheme has been adopted widely enough that surgical pathology reports on corticectomy specimens now frequently contain the terminology “Cortical dysplasia, Palmini type….”

Development of the Neocortex

Overview of Neocortical Development

The neuropathologic changes seen in children with epilepsy frequently represent the end results of insults to a rapidly developing brain. This section briefly summarizes the normal process of neocortical development and identifies the points of susceptibility at which the error or errors (genetic, environmental, or both) result in cases of pediatric epilepsy—usually due to CD. We also briefly introduce some recent advances in the genetic and molecular mechanisms that regulate cortical development. Many excellent reviews have summarized the historical evolution in our understanding of the complex neurobiologic, cellular, and molecular processes that work in concert to create a normally functioning cerebral cortex.³⁰^,⁵²^,⁶⁷^,¹⁴¹^,¹⁶⁰ Neocortical development after neural tube formation can roughly be considered to be the result of a series of overlapping processes: (a) cell proliferation in the ventricular zone and subventricular zone (VZ/SVZ), (b) early differentiation of neuroblasts and glioblasts, (c) programmed cell death of neuronal precursors and neurons, (d) migration of neuroblasts to form the cortical plate, (e) late neuronal migration, (f) organization and maturation of the cortex, and (g) synaptogenesis.¹^,⁸^,¹⁵⁷^,¹⁷⁵ Abnormalities of these processes result in abnormalities of cortical architecture and, by inference, its electrophysiologic properties. Most developmental disorders of the brain commonly associated with epilepsy are believed to originate from the perturbation of developmental events after the embryonic period, i.e., after 6 weeks’ gestation, when cell proliferation starts along the wall of the neural tube. This generates a collection of matrix cells,⁴⁵ or precursor cells for all neuroblasts and glioblasts, forming ventricular and subventricular zones (VZ/SVZ) in the pallium, as well as the ganglionic eminence in the subpallium (Table 1).

Table 1 Major stages of human central nervous system development and associated epileptic disorders

Stages	Time of occurrence (wk)	Morphologic changes and events	Corresponding disorders commonly associated with intractable epilepsy
Embryonic period
Formation and separation of germ layers	2	Neural plate	Enterogenous cysts and fistulas Split notochord syndrome
Dorsal induction: primary neurulation	3–4	Neural tube, neural crest and derivatives	Anencephaly, encephalocele
		Closure of rostral and caudal neuropores	Chiari malformation
Ventral induction: telencephalization	4–6	Development of forebrain and face	Holoprosencephaly
		Formation of cerebral vesicles Optic and olfactory placodes	Dandy-Walker malformation
Fetal period
Neuronal and glial proliferation	6–16	Cell proliferation in ventricular and subventricular zones	Microcephaly Hemimegalencephaly
		Early differentiation of neuroblasts and glioblasts	Cortical tuber of tuberous sclerosis Cortical dysplasia with balloon cells
		Programmmed cell death	Dysembryoplastic neuroepithelial tumor ganglioglioma, gangliocytoma
Migration	12–24	Migration of cortical neurons	Type I lissencephaly
		Formation of corpus callosum	Type II lissencephaly (cobblestone) Heterotopia
Perinatal period
Organization	24 to postnatal	Late neuronal migration	Polymicrogyria, schizencephaly
		Organization and maturation of cerebral cortex Synaptogenesis	Cortical dysplasia without balloon cells Mild cortical dysplasia “Microdysgenesis”
Myelination	24 to 2 yr postnatally		Destructive lesions Myelination disorders
Modified from Aicardi (1992), Barkovich et al. (2005), and ten Donkelaar et al. (2006).

Programmed cell death (PCD) is an essential mechanism for normal brain development, determining the size and shape of the nervous system. In normal brain development, there is a 25% to 50% overproduction of neuroblasts; these undergo physiologic PCD.¹⁵⁹ Neuroblasts and glia undergo this process as well. PCD appears to be under tight genetic control, as demonstrated in the Caenorhabditis elegans model.³⁹ It is an active process that can be blocked by inhibitors of protein synthesis and RNA transcription.⁷⁰ Failure of PCD may lead to mechanical barriers to migration and abnormal numbers of neurons. Supplemental to PCD, there is a conspicuous elimination of synapses occurring during development that is essential to remodeling of the cortex.¹⁴⁰ This may be accomplished by different mechanisms, and competition for trophic substances has been suggested as one. Synapse elimination is highly intertwined with the remodeling of cortical connections and is a highly dynamic process¹⁴⁰ demonstrated both in vivo and in vitro.¹⁸⁰

Terminal differentiation of a neuroblast appears to be a multistep process.²²^,¹⁰²^,¹²⁸ Cell surface properties and extracellular matrix molecules play a crucial role in influencing migration and terminal differentiation of neuroblasts. Neural adhesion and migration are governed by a series of morphoregulatory molecules, cell-adhesion molecules (CAMs), and substrate-adhesion molecules (SAMs). CAMs

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are transmembrane molecules bearing extracellular domains that are homologous to domains of the immunoglobulin superfamily³⁸ and are engaged in homotypic binding. The binding functions interact with the cytoskeleton because CAMs are transmembrane molecules. These molecules are involved in cell adhesion, axon binding, growth cone interactions, and other migrational mechanisms. SAMs, extracellular matrix molecules, are involved in the regulation of cell shape, motion, and process extension.³⁶ These molecules are expressed in a spatial-temporal fashion and are under the control of homeobox-containing genes that are known to govern place-dependent morphology.⁷⁴^,⁷⁵ The time-dependent levels of expression of the molecules are characteristic of a given anatomic area but are dynamically regulated and subject to local influences. The activities of these molecules are further modulated by neural activity itself.³⁷ These morphoregulatory molecules can mediate neuron–neuron interactions (NCAMs), neuron–glia interactions (Ng-CAMs), or neuron–extracellular matrix interactions.³⁰^,⁶⁰^,⁶¹ Extracellular matrix molecules also appear to be important in cell motion, attraction, repulsion, and growth cone migration; soluble trophic factors have an important role in these processes.³⁴

Radial Migration of Neuroblasts from the Subventricular Zone

Radial migration refers to the process by which neuroblasts from the VZ/SVZ migrate along the processes of radial glia to reach the (neo)cortex. Radial glia initially have cell bodies in the ventricular zone and end-feet on the pial surface; with time, they detach themselves from the ventricular lining and migrate toward the cortex.^32a^,⁴¹ They function as a permissive scaffold on which neuroblasts may migrate from the ventricular zone to the cortical plate.⁶⁰ At approximately 4 weeks’ gestation, the neural tube forms with a simple pseudostratified neuroepithelium, component cells of which then proliferate around the developing ventricular system, eventually becoming the ventricular zone. The ventricular zone can be described as a mosaic of precursors that will give rise to neurons, astrocytes, and oligodendroglia, as demonstrated using retroviral markers.¹³⁹ The first postmitotic cells form the preplate or primordial plexiform layer⁹⁸^,¹¹⁰^,¹¹²^,¹⁶⁸^,¹⁹⁴ above the ventricular zone and beneath the pia. Toward the end of the embryonic period, cells from the ventricular zone migrate to form the cortical plate within the preplate; that is, the cortical plate is formed dividing

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the preplate (preplate splitting) into (a) a thin superficial component, the marginal zone, and (b) a thick, deeper component, the subplate. The intermediate zone or future subcortical white matter also appears by this stage,¹⁵⁷ and this cortical plate formation continues until 16 weeks’ gestation.⁹ The neocortex is formed in an “inside-out” fashion: Neuroblasts born first, destined for the deepest cortical layers, migrate first, whereas neuroblasts born later, destined for the more superficial cortical layers, migrate past the already present cortical neuroblasts,⁶ eventually forming a six-layered structure.¹⁶³ Even after this point, however, neuroblasts continue to migrate through the intermediate zone (future white matter) to the cortex, a process that can continue up to a few months after birth.¹⁶³ Abnormalities of radial glia may occur through various molecular mechanisms either focally or diffusely in malformations of cortical development (MCDs), including Fukuyama congenital muscular dystrophy (FCMD)¹⁶⁹ and tuberous sclerosis complex (TSC).¹³³ Alterations of signaling pathways that regulate microtubule dynamics either directly or indirectly may also result in a derangement of radial glial fibers, as has been demonstrated in reeler mice⁶⁴ and Lis1 mutant mice.¹⁹ Radial glia have long been known to serve as guides for migrating neuroblasts and finally give rise to cortical astrocytes. However, recent evidence in mice indicates that radial glial cells also generate neurons in the developing cerebral cortex. Three distinct subsets are thus identified, including subpopulations important in gliogenesis, neurogenesis, and both.⁷^,⁵⁸

Tangential Migration of GABAergic Interneurons From Ganglionic Eminence

Tangential migration refers to the process by which neuroblasts from the ganglionic eminence migrate in a nonradial, “neurophilic” fashion, possibly along neuronal processes rather than radial glial fibers, moving tangential to the pial surface to form complex three-dimensional neural structures.¹⁴³ Ganglionic eminence, primarily considered to be a source of basal ganglia neurons, consists of three parts. The medial ganglionic eminence, derived from the diencephalon, gives rise to globus pallidus. The lateral ganglionic eminence, derived from the telencephalon, gives rise to the caudate nucleus and putamen. The caudal ganglionic eminence primarily gives rise to amygdala. Both lateral and median ganglionic eminences, however, are also involved in the formation of cerebral cortex. In fact, mechanically separating the ganglionic eminence from the cortex results in a loss of calbindin and γ-aminobutyric acid (GABA)–positive neurons in the cortex,² and 35% of cortical GABAergic interneurons arise form both ganglionic eminences by way of tangential migration.³^,⁸⁷ This contrasts with the origin of pyramidal neurons of the cerebral cortex, which arise from the ventricular zone of the pallium by way of radial migration. Although 65% of cortical GABAergic interneurons arise from the ventricular zone of the pallium, they are considered to migrate in nonradial, neurophilic fashion at least within the VZ/SVZ.⁸⁷ Migration of GABAergic interneurons seems to be much more complex, because “ventricle-directed migration” has also been demonstrated in mice.¹²² GABAergic interneurons are immunoreactive for calcium-binding proteins such as calbindin. Studies of macaque monkey brain have suggested that calcium-binding-protein–containing interneurons make up 90% of all GABAergic neurons in the cerebral cortex.⁹¹ The number and distribution of calcium-binding-protein–containing neurons in the neocortex can be reorganized in early postnatal life¹⁴⁹; by 28 weeks after birth, the laminar distribution of calbindin-immunoreactive cells shifts from layer IV to layer II. It has been suggested that doublecortin (DCX) is important for nonradial migration because it is rather selectively expressed in tangentially oriented postmitotic neurons in the subventricular and intermediate zones.¹¹³

Origin of Cajal-Retzius Cells and Their Biologic Significance

The marginal zone, future molecular layer or cortical layer I, is composed largely of Cajal-Retzius cells. They secrete the extracellular glycoprotein reelin that is required for the normal inside-out positioning of neurons as they migrate from the ventricular zone along radial glia. Human Cajal-Retzius cells, characterized by the combined expression of reelin and p73, are transient cells, are present from the preplate stage at 8 weeks’ gestation, and gradually increase in number (by tangential migration) until they disappear by the end of gestation.¹¹⁰^,¹¹³ One possible origin of Cajal-Retzius cells is considered to be the boundary between prospective hippocampus and choroid plexus epithelium or “cortical hem” in the dorsal telencephalon.¹¹³ In mice carrying mutations in RELN (reeler mice) and in disabled-1 (Dab1) as well as in mice carrying double mutations of both very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), normal neuroblast migration with an inside-out fashion is inverted.¹⁷⁸ This suggests a role for these genes in the control of cell positioning in the developing central nervous system and even predicts a pattern of cytoarchitectural alteration in patients carrying alterations in the reelin/lipoprotein receptor/Dab1 pathway, as well as RELN mutations causing lissencephaly.⁶³ LIS1 also appears to have important effects on neuronal migration, and significant interactions with Cajal-Retzius cells: LIS1 deficiency negatively affects the migration and differentiation of both doublecortin- and reelin-positive neurons in the developing human brain.¹¹⁴

FIGURE 1. Hemimegalencephaly (HME) in a 6-month-old child. Axial (A) and coronal (B) T2-weighted magnetic resonance imaging (MRI) images show markedly enlarged left cerebral hemisphere with thickened gyri and smooth surface, consistent with HME. The left lateral ventricle and caudate head are deformed. White matter is diffusely hypointense compared with the unaffected right cerebral hemisphere, suggesting accentuated myelination. C: Intraoperative photograph of the region of HME oriented with the frontal lobe (F) in the top left and the temporal lobe (T) in the lower middle portions of the image. Notice the diffuse cortical disorganization of all gyri. This child had a monozygotic twin who was normal. (Panel B is from Salamon N, Andres M, Chute DJ, et al. Contralateral hemimicrencephaly and clinical-pathological correlations in children with hemimegalencephaly. Brain. 2006;129:352–365; with permission of the editor of Brain and Oxford University Press.)

Origin of Superficial Granular Layer and Its Biologic Significance

The superficial or subpial granular layer (SGL) is a transient cell layer and appears beneath the pial surface between 13 and 24 weeks’ gestation.¹⁸^,⁴⁴ Cells in the SGL originate from the basal periolfactory subventricular zone¹⁸^,⁴⁶^,¹¹¹ and migrate tangentially beneath the pia to cover the neocortical marginal zone. Cells in this layer express interneuron markers such as calretinin, calbindin, and GABA,¹⁴⁵ suggesting that they are equivalent to GABAergic interneurons. The biologic significance of the SGL, however, remains to be elucidated. Programmed cell death may, at least in part, contribute to elimination of the SGL.¹⁶⁶ There is controversy in this research area because the SGL is also suggested to be an additional source of cortical interneurons; it disappears probably as the result of inward or ventricle-directed migration of its component cells.¹²²^,¹⁴⁴^,¹⁴⁵

Subplate Neurons as Pioneer Cells to Form Early Thalamocortical Projections

The human subplate contains large multipolar neurons. Subplate neurons in the developing cerebrum, although they are transient and most disappear in early postnatal life, are believed to be important in organizing cortical connections in the developing cerebrum. They are believed to act as pioneer corticofugal axons.²⁹^,⁴⁹^,⁵⁰^,¹⁰¹

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FIGURE 2. Representative micrographs of the resection specimen obtained from the child with magnetic resonance imaging (MRI) and intraoperative appearances illustrated in Fig. 1. A: Low-magnification view of a representative region of cortex shows a modest degree of neuronal disorganization. B: A focus of polymicrogyria, one of many seen in the resection specimen. Also note a “rind” of disorganized glial tissue covering the pia, with apparent extension into the subarachnoid space (arrows). (Hematoxylin and eosin stain.) (See the color insert.)

FIGURE 3. Focal cortical dysplasia extensively involving the left parietal lobe. T2-weighted axial (A) and coronal (B) images show thickened gyri and hyperintense signal in the white matter of the left parietal lobe. The left lateral ventricle is enlarged (arrow).

FIGURE 4. Axial FLAIR (fluid attenuated inversion recovery) (A) and T2-weighted axial (B) images demonstrate hyperintense signal area in the subcortical white matter of the right frontal operculum in a 4-year-old girl. The gray matter in this region is thickened, but there is no mass effect. The findings are suggestive of cortical dysplasia. C: View of the lesion intraoperatively (arrow). Histopathology of this lesion showed classic features of Palmini grade IIB cortical dysplasia. F, frontal lobe.

Neuropathology and Pathophysiologic Significance of Cortical Dysplasia

The Pediatric Epilepsy Surgery program at UCLA Center For the Health Sciences, active since 1986, has enabled us to examine over 500 surgically resected specimens from infants and children with intractable seizures, ranging from partial lobectomies to complete and partial (functional) hemispherectomies. The most common morphologic substrate for this was CD, this being of etiologic importance as a cause of intractable pediatric seizures in >80% of children <3 years of age. The extent of CD neuropathology can be predicted by high-resolution neuroimaging studies. Such neuroimaging allows for stratification of CD cases into those that show hemimegalencephaly (HME), with diffuse enlargement of the gray and white matter, including thickening of the cortical ribbon, within an entire cerebral hemisphere (Figs. 1 and 2); hemispheric CD, with multifocal CD affecting one cerebral hemisphere (although not causing enlargement of that hemisphere); and multilobar, lobar, or focal CD (Figs. 3 and 4), the latter affecting as few as one or two adjacent gyri. HME is easily recognized by neuroimaging. The MRI findings include an enlarged cerebral hemisphere and markedly thickened gyri, with loss of sulcation. There is deformity and enlargement of the ipsilateral ventricle. Palmini type IIB CD is also easily identified by MRI, with focal thickness of the gyrus (gyri) and associated hyperintense T2-weighted signal changes in the adjacent white matter. It is difficult to visualize Palmini type I CD by MRI; however, on combining this with other modalities, such as positron emission tomography (PET) and magnetic source imaging (MSI), the detectability of this

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lesion increases and foci of subtle gray–white matter blurring may be visualized intraoperatively.

Macroscopic heterotopia and polymicrogyria (PMG) are occasionally seen in resection specimens from such patients. Loss of the normal cortex–white matter junction, best appreciated with a Klüver-Barrera or other myelin stain, is a frequent accompaniment and excellent predictor of severe microscopic CD. Many specimens, however, exhibit no striking gross cortical abnormalities. CD can be further characterized with regard to specific and easily identifiable microscopic abnormalities,

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which include cortical laminar disorganization (Fig. 5), single heterotopic white matter neurons, excess neurons in the neocortical molecular layer, marginal glioneuronal heterotopia, white matter neuronal heterotopia, neuronal cytomegaly with or without associated dysmorphic features of the cytoplasm (the latter almost invariably accompanied by cytoskeletal abnormalities) (Fig. 6), and balloon cell change (Fig. 7).¹¹⁷ These microscopic features can be used as the basis for a grading system for CD; one widely used schema is that presented recently by Palmini et al.¹³²

FIGURE 5. A: Relatively normally organized cerebral cortex from a surgical resection specimen. B: Region with severe cortical dysplasia shows pronounced cortical disorganization, with abnormal crowding of neurons and abnormal orientation of many cells. Both panels are from micrographs photographed at the same magnification from sections stained with routine hematoxylin and eosin stains. (See the color insert.)

FIGURE 6. Neuronal disorganization and dysmorphism in cortical dysplasia/malformations of cortical development. A: Intermediate-magnification micrograph shows crowded and abnormally oriented neuronal cell bodies. B: Same features shown at a higher magnification. C: Variably enlarged and abnormally distributed neurons near the cortex–white matter junction. D: Magnified view of a dysmorphic neuron (arrow) with clumping of Nissl substance around the nucleus and clearing of the peripheral cytoplasm. Gemistocytic astrocytes are seen distributed around this neuron. (All panels are from hematoxylin and eosin–stained sections.) (See the color insert.)

Cortical laminar disorganization is the most ubiquitous microscopic finding, obviously because it represents a defining histopathologic feature of CD. Because neocortical architecture is the end result of the developmental processes of proliferation of neuronal precursors, migration, terminal differentiation, PCD, and cortical remodeling (see earlier discussion), abnormalities in any of these processes may result in abnormal cortical architecture. Cortical disorganization (as well as cytologic abnormalities in individual neurons, especially when fairly subtle) can be highlighted using immunohistochemistry that incorporates primary antibodies to neurofilament epitopes (Fig. 8). Although many neurons still reside in the intermediate zone/white matter in the last trimester of pregnancy and even into postnatal life,⁸⁰^,¹⁶³ the phenomenon of single heterotopic neurons in the white matter is accentuated in CD.¹⁰⁸ It is present in the majority of our patients with CD, and has been demonstrated in other series using morphometric techniques.¹⁰⁵ It has been suggested that injury to the radial glial fibers leads to a stranding of the migrating neuroblasts within the white matter, where they further differentiate into

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mature neurons.¹⁵⁹ Alternatively, overproduction of neurons late in neurogenesis may lead to crowding of migrating neurons toward the cortical surface.⁵ Morphometric analysis has also demonstrated a statistically significant increase in the number of neurons within the molecular layer of the cortex in epileptic patients versus controls,¹⁰⁷ and this is considered to be evidence of a slight maldevelopment of the neocortex, sometimes described as microdysgenesis, although this term is now felt to be imprecise and best avoided.¹³² Persistence of the superficial granular layer (SGL) has been seen in association with many cortical malformations.¹¹⁷ Marginal glioneuronal heterotopia consists of excrescences of disorganized neuroglial tissue extending from the pial surface into the subarachnoid space. They are often found in association with persistent SGL and tend to occur in the same brain region as the other malformations. These lesions may be associated with a failure of the glia limitans.¹¹⁷ White matter neuronal heterotopia consists of disorganized masses of neurons in the white matter that usually occur in a periventricular position with a nodular morphology, although rare instances of laminar subcortical bands of heterotopic gray matter have been known to produce the appearance of a double cortex. It has been suggested that these are associated with injury to a group of radial glia, leading to failure of a group of neuroblasts to migrate.¹⁵⁹ Alternatively, a defect in genes controlling neuroglial interactions, neuroblast proliferation, and PCD has been suggested as being causal.¹⁵⁷

PMG denotes small meandering gyri, often with bridging of the sulci by fusion of the molecular layers. It consists of two histologic types: Four-layered PMG is most frequently considered to result from a destructive lesion occurring approximately at 20 to 24 weeks’ gestation, whereas an unlayered form is believed to result from an insult earlier in development (at approximately 13–16 weeks).⁵ Whether PMG represents a destructive lesion with secondary malformation or a primary malformative lesion continues to be debated.¹²⁴^,¹²⁶^,¹⁵⁸

Neuronal cytomegaly denotes enlarged neurons, some of which may also be dysmorphic (Fig. 9); these were first described by Taylor et al.¹⁷⁴ in association with seizure-producing focal cortical malformations or regions of dysplasia. Nerve cell hypertrophy was convincingly shown by Bignami et al.¹⁵ using quantitative histochemistry in a case of HME. The differentiation of cytomegalic versus dysmorphic neurons is of importance in assigning a Palmini grade to a given lesion. Neurons that show enlargement of their cell bodies only are, in association with cortical architectural disorganization, typical of Palmini type IB lesions, whereas corticectomies that also contain dysmorphic neurons (defined as showing abnormalities of shape with abnormal orientation, cytoskeletal structure, and atypical dendritic arborizations) are characteristic of Palmini type II CD; when balloon cells are absent, the lesion is described as Palmini type IIA, and when they are present, it becomes Palmini type IIB. (Palmini IIB CD corresponds to what has been described as Taylor-type focal cortical dysplasia [T-FCD].) Dysmorphic neurons bear an extremely complex dendritic arborization as well as an abundance of perisomatic synapses and a paucity of axosomatic synapses.²⁸^,¹⁵⁴ Increased neuronal size has been associated with an increased DNA and RNA content, as well as increased nuclear and nucleolar volume suggestive of heteroploidy.¹³^,⁹⁵ Many cytomegalic and dysmorphic neurons contain cytoskeletal abnormalities. Argyrophilic, neurofibrillary-like tangles, and cytoplasmic vacuoles have been demonstrated within many such neurons,¹⁸⁶ as has the existence of paracrystalline intracytoplasmic structures

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visible on ultrastructural examination.³³ These neurofibrillary-like cytoplasmic inclusions are, like the neurofibrillary tangles seen in Alzheimer disease, strongly immunoreactive with antibodies to high- and medium-molecular-weight neurofilament (including phosphorylated and nonphosphorylated proteins), ubiquitin, and tau. However, they differ from the neurofibrillary tangles of Alzheimer disease in that they do not harbor paired helical filaments.³⁵ It is of interest that many cells within a focus of CD can coexpress neuronal and astrocytic epitopes, a phenomenon that can be easily demonstrated using confocal laser microscopy or immunohistochemistry on serial sections (Fig. 10).³²

Balloon cells, showing considerable similarity to gemistocytic astrocytes, have eccentric nuclei and ballooned, opalescent eosinophilic cytoplasm (Fig. 7). They often demonstrate binucleation or dysmorphic nuclei, sometimes showing bridges of nucleoplasm between two separate islands of nuclear material within a cell. They are noted to cluster at the cortex–white matter junction or may be abundant within subcortical white matter.³²^,¹⁸⁴ Frequently, they are admixed with dysmorphic and enlarged neuronal cell bodies. Ultrastructurally, they are packed with filaments ranging in size from 400 to 600 nm in length and 30 nm in thickness, interspersed with non–membrane-bound, electron-dense, helical structures.⁴² Vinters et al.¹⁸⁶ and others have demonstrated dual staining of many cells in dysplastic cortex (including some balloon cells) with antibodies to both neuronal and glial markers (synaptophysin and glial fibrillary acidic protein), implying either a failure of the cells to commit to a specific phenotype or a dedifferentiation. The resemblance of balloon cells to cells found within the cortical tubers of TSC has suggested the possibility that cases of CD harboring balloon cell change may represent a forme fruste of TSC,³²^,⁴²^,¹³⁰^,¹³¹^,¹⁵⁵ as is discussed later. At least one group¹⁷⁹ has suggested that T-FCD (Palmini type IIB cortical dysplasia), when resected, has good postsurgical outcome.

FIGURE 7. Typical balloon cells, with variably pronounced nuclear atypia. Hematoxylin and eosin stains. A: Balloon cells often show binucleation (arrow). Paired balloon cells are also seen (arrowheads). B: A balloon cell with a nuclear bridge connecting two nuclei (arrow). C: Balloon cell with marked nuclear atypia and/or nuclear invagination with nuclear budding (arrow). Also note the Marinesco body-like intranuclear inclusion. D: Balloon cell with multinucleation and/or micronucleation (arrow). Bars = 50 μm. (From Crino PB, Miyata H, Vinters HV. Neurodevelopmental disorders as a cause of seizures: neuropathologic, genetic, and mechanistic considerations. Brain Pathol. 2002;12:212–233; with permission.) (See the color insert.)

FIGURE 8. Neurofilament (N52, high-molecular-weight neurofilament antibody) immunohistochemistry of surgically resected corticectomies. A: Relatively normal cortex from a patient with temporal lobe epilepsy. Some, although not all, of the pyramidal neurons in layers III and V as well as multipolar interneurons (arrow) are positive for N52. B: Focal cortical dysplasia. Neuronal dyslamination and dysmorphic, multipolar, cytomegalic neurons are evident. C: Relatively normal cortex adjacent to a region of cortical dysplasia. More pyramidal neurons (in layers III and V) are more strongly immunoreactive for N52 than in control cortex (A), and an abnormally oriented pyramidal neuron (arrow) is also easily identified. The pial surface is at the top in each panel. Bars = 100 μm. (From Crino PB, Miyata H, Vinters HV. Neurodevelopmental disorders as a cause of seizures: neuropathologic, genetic, and mechanistic considerations. Brain Pathol. 2002;12:212–233; with permission.) (See the color insert.)

FIGURE 9. Histologic features of severe cortical dysplasia. A: Dysmorphic and enlarged neuronal cell bodies. Arrow indicates a cell that shows a “neuronal” nucleus but pale, glassy amphophilic cytoplasm (lacking Nissl substance) of the type more commonly seen in gemistocytic astrocytes. B, C: Dysmorphic, enlarged neurons with coarseness of the cytoplasm, suggestive of neurofibrillary change. (All panels are micrographs from hematoxylin and eosin–stained sections.). (See the color insert.)

FIGURE 10. Balloon cells show various patterns of neuroectodermal differentiation. Some show only glial (glial fibrillary acidic protein [GFAP]) or neuronal (neurofilament) differentiation, whereas others show both GFAP and neurofilament immunoreactivity. Only rare balloon cells are immunoreactive for phosphorylated neurofilament (arrowhead, panel A). However, most are immunoreactive with nonphosphorylated neurofilament (panel B) with varying intensities. Some are immunoreactive for both GFAP and nonphosphorylated neurofilament (double arrows, panels B and C). Symbols and arrows in all panels indicate the same cell identified in serial sections. Arrowhead indicates a balloon cell that is phosphorylated neurofilament (p-NF)-positive, NF-positive, and GFAP-negative. Arrows indicate balloon cells that are p-NF-negative, NF-positive, and GFAP-negative. Asterisks indicate balloon cells that are p-NF and NF-negative but GFAP-positive. Double arrow indicates a balloon cell that is p-NF-negative, NF-positive, and GFAP-positive. Bars = 50 μm. (Panel A, section stained with primary antibody to phospho-NF, panel B with primary antibody to NF, panel C with primary antibody to GFAP). (From Crino PB, Miyata H, Vinters HV. Neurodevelopmental disorders as a cause of seizures: neuropathologic, genetic, and mechanistic considerations. Brain Pathol. 2002;12:212–233; with permission.) (See the color insert.)

FIGURE 11. Hemimegalencephaly (HME) in a 13-month-old child with tuberous sclerosis complex (TSC). A: Axial T2-weighted image shows thickened gyri of the left frontal lobe, with an enlarged left cerebral hemisphere, suggesting HME. Most of the white matter in the left cerebral hemisphere is poorly myelinated. A well-defined subcortical hyperintense T2-weighted signal is noted in the left anterior cingulate gyrus, consistent with a tuber (white arrow). B: Postcontrast T1-weighted coronal image shows patchy foci of enhancement in the left cerebral hemispheric white matter, suggesting locations of tubers. A small, round focus of enhancement in the inferior aspect of the right frontal horn of the lateral ventricle (white arrowhead) represents a subependymal nodule, also characteristic of TSC. C: Intraoperative view of the left cerebral hemisphere showing multiple areas of abnormal cortical organization. The frontal lobe is in the upper left and the parietal lobe is in the upper right of the image.

Pathogenesis of Cortical Dysplasia

Although it is accepted that cortical dysplasia involves abnormal cerebral cortical development, it is unclear when it occurs

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and how it produces seizures. Most classification systems of MCDs are based on speculating when the first development steps involving cell proliferation, neuronal migration, and cortical organization abnormality occurs to produce the malformed cortex. Previously, the presumed mechanisms of surgically treated CD pathogenesis have been believed to be defects in neuronal migration to explain subcortical heterotopic neurons, and altered periventricular neuroglial differentiation to account for the abnormal cytomegalic and dysmorphic neurons and balloon cells.¹⁴⁶^,¹⁶¹^,¹⁷⁹ Although such mechanisms appear to be operant in genetic forms of malformations of cortical development, clinicopathologic investigations have challenged this singular interpretation in surgically treated cortical dysplasia.⁸^,²³^,²⁴^,²⁵^,²⁶

In recent studies, the UCLA group has found evidence that surgically treated cortical dysplasia seems to involve retention of cells of the human pre- and subplate along with overproliferation of cortical neurons. Prenatal human subplate cells have morphologic features similar to those of dysmorphic neurons found in postnatal CD tissue.²³ The normal human subplate contains large multipolar neurons similar to cytomegalic neurons, and polymorphous and fusiform neurons with thick primary dendrites along with inverted pyramidal-shaped neurons, a feature seen in dysmorphic CD neurons.⁸⁰ Most human subplate cells degenerate in the 4 to 6 weeks prior to birth,²⁹^,⁴³ which coincides with increasing definition of the gray–white matter junction and secondary gyral folding.²⁷ Furthermore, toward the end of normal neurogenesis, periventricular radial glial cells attach themselves to the tailing processes of the last-produced cortical pyramidal neurons and migrate toward the cortex, where they detach and eventually transform into protoplasmic astrocytes.⁸²^,¹²¹^,¹²³ This may explain why balloon cells in CD tissue have morphologic and other characteristics similar to those of radial glia.⁴⁰^,¹⁷⁶^,¹⁹³

The UCLA group has proposed that CD pathogenesis probably involves partial failure of later phases of corticogenesis. As a consequence, subplate and radial glial degeneration and transformation would be reduced or prevented, giving the appearance of abnormal dysmorphic cells in postnatal CD tissue. In addition, failure of late cortical maturation could explain the

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presence of abnormally thickened gyri with indistinct cortical gray–white matter junctions in MRI scans of CD patients.²⁴ The timing of these events during cortical development would explain the different forms of CD identified by MRI and severity of CD by histopathology. Developmental alterations during the late second or early third trimester would account for severe CD, like hemimegalencephaly, whereas events occurring closer to birth (after the subplate has nearly degenerated) would explain milder forms of CD. In addition, it appears that there is an overproduction of neurons in later phases of cortical development. MRI cerebral hemisphere volumes were normal or increased in the case of hemimegalencephaly, and cortical thickness was the same or slightly increased.⁵^,¹⁵⁸ Furthermore, neuronal densities were increased in the upper gray matter, molecular layer, and subcortical white matter. The location of excess neurons would be consistent with the idea that this process occurred in later periventricular cell cycles (i.e., the ones toward the end of neurogenesis). Thus, heterotopic subcortical white matter neurons are likely the result of excessive late generated pyramidal neurons trying to migrate toward the already overly crowded cortical ribbon, in combination with residual prenatal subplate neurons that failed to degenerate prior to birth.

Cortical Tubers of Tuberous Sclerosis Complex (TSC)

Tuberous sclerosis complex (TSC, or Bourneville disease) is an autosomal-dominant, multisystem disorder in which the CNS, eyes, kidneys, skin, and heart are most commonly

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affected by malformative, hamartomatous, or neoplastic lesions.³¹^,⁵¹^,⁸⁴^,¹⁶²^,¹⁸⁷ It has an incidence of 1 in 9,400 to 10,000 births³¹^,⁵¹; however, accurate estimates are difficult to ascertain because of the markedly variable penetrance of the disorder; it may go unrecognized for many years, and a high rate of spontaneous TSC gene mutations has been described.³¹^,¹⁸⁸ The clinical presentation of an individual with TSC may be with infantile spasms, autism, or mental retardation. Approximately 85% of TSC patients who come to medical attention have experienced an epileptic seizure at some time. Because genetic analysis to confirm the diagnosis of TSC remains unavailable to most physicians, diagnostic criteria for TSC have been enunciated.¹⁵¹^,¹⁵² In the 1992 iteration, these included primary features of the disease (facial angiofibromas, multiple subungual fibromas, a [histologically confirmed] cortical tuber, [histologically confirmed] subependymal nodule (SEN) or subependymal giant cell astrocytoma (SEGA), [radiographically confirmed] multiple calcified SENs protruding into the ventricular cavity, and multiple retinal astrocytomas); secondary features (an affected first-degree relative, cardiac rhabdomyoma [confirmed by histopathology or radiographically], retinal hamartoma or achromic patch, cerebral tubers [radiographically confirmed], a Shagreen patch, forehead plaque, [histopathologically confirmed] pulmonary lymphangiomyomatosis or renal cysts); and tertiary features (hypomelanotic macules, [radiographically confirmed] renal cysts, etc.). In the 1998 revised criteria, some clinical features previously thought to be pathognomonic for TSC were considered less specific, whereas clinical/radiographic features of the disease were subdivided into major and minor categories based on their apparent degree of specificity for TSC. Based on these newer revised criteria, a definitive diagnosis of TSC is made by the confirmation of two or more distinct types of lesion in a patient, rather than multiple lesions of the same type (e.g., tubers) in the same organ system. In other words, no single lesion is one that defines the disease.

Neuropathology of Cortical Tubers as a Cause of Seizures

Individuals with TSC may rarely present with HME (Fig. 11). However, the characteristic brain abnormalities of TSC include neocortical tubers (Figs. 12 and 13), SENs, and SEGAs. Cortical tubers are very often associated with infantile spasms and intractable epilepsy in children. The lesions manifest as enlarged gyri in which the cortex/white matter junction has become blurred, resembling sporadic, severe cortical dysplasia.³²^,¹¹⁷ Histologically, these lesions show disorganized neocortex, with a variety of dysmorphic, markedly enlarged neurons and bizarre gemistocytic astrocyte-like “balloon cells” having eccentric nuclei containing relatively coarse chromatin and glassy eosinophilic cytoplasm (Fig. 13). Balloon cells seen in TSC strongly resemble those seen in severe cortical dysplasia or focal cortical dysplasia of Taylor type. This observation has frequently raised the possibility that cases of CD with

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balloon cell change represent a forme fruste of TSC.⁴²^,¹³⁰^,¹³¹^,¹⁵⁵ Balloon cells, like those seen in FCD, show morphologic and immunohistochemical features of both neurons and astrocytes, suggesting a failure of commitment in neuroglial differentiation (Fig. 14), that is, they are variably immunoreactive, even among cells within a tuber, for glial fibrillary acidic protein (GFAP) and neuronal markers. Balloon cells may cluster together, particularly in the subpial region or subcortical white matter, or be scattered among dysmorphic neurons. Tubers may show prominent punctate calcification. Whereas cortical tubers demonstrate reduced immunoreactivity for the synaptic protein synapsin 1,⁸⁸ giant cells within TSC tubers show dramatic halos of synaptophysin immunoreactivity resembling those noted in gangliogliomas, as well as strong immunostaining with antibodies to the microtubule-associated protein 2 (MAP-2).¹⁹² Alpha B-crystallin, a member of the heat-shock-protein family of peptides, is found in abundance within dysgenetic cells of tubers and within both SEGAs and SENs.⁶⁶ Dysmorphic cytomegalic neurons express high levels of tuberin, as do individual cells within SEGAs and SENs. In a developmental time frame, tuberin appears to be present in most neuronal populations of the CNS from at least 20 weeks of gestation, with an apparent upregulation of its expression after 40 weeks of gestation.¹⁸⁷ Hamartin was found, albeit with a weaker signal, in the same cell types during CNS development.

Whereas architectural disarray is a defining feature of a tuber, cellularity of a lesion may be extremely variable, although when high cell density is noted, a tuber may resemble a ganglioglioma.¹⁵⁰^,¹⁸⁷ Although the proliferative potential of these lesions appears to be low, as judged by immunohistochemistry for Ki-67, other markers of cellular proliferation (e.g., collapsin response mediator protein 4 [CRMP4], doublecortin) are expressed within giant cells of cortical tubers and SEGAs (human-derived material) and SENs of Eker rats, suggesting that they may represent newly generated cells that have migrated into tubers from the subventricular zone.⁸⁶

Genetic and Molecular Biologic Aspects of Tuberous Sclerosis Complex Pathogenesis

Our understanding of the molecular pathogenesis of TSC represents a triumph of multidisciplinary, multicenter (often multicontinent) collaborations through the early 1990s focused on characterizing the complex gene defects that cause this disorder.⁴¹^,⁶⁸^,¹³⁷^,¹⁸¹ TSC is caused by mutations in one of two nonhomologous tumor suppressor genes: TSC1¹⁸¹ on chromosome 9 (9q34) encoding a 130-kDa protein, hamartin, and TSC2⁴¹ on chromosome 16 (16p13.3) encoding a 200-kDa protein, tuberin. About half of TSC families show linkage to each of the two identified genes. TSC1 mutations, accounting for a minority of mutations identified, are slightly less common in sporadic TSC patients and are more common in familial cases (13%–50%).⁹⁴ The putative functions of both TSC1 and TSC2 gene products have been intensively studied using

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mostly Drosophila and rodent models of TSC, but also using human tissues and loss-of-heterozygosity analyses of TSC hamartomas. These initially suggested that both TSC1 and TSC2 gene products, even before the details of their encoded proteins were known, had growth suppressor properties.²⁰^,⁵³^,⁵⁴ Significant interactions of TSC genes with intracellular signaling pathways have been implicated, as shown in FIGURE 15. Hamartin possesses a coiled-coil domain in its carboxy region, suggesting the possibility of a functional protein–protein interaction with tuberin¹⁸¹ to regulate cell proliferation and cell cycle progression.²¹^,⁶²

FIGURE 12. Tuber of tuberous sclerosis complex. A: Axial FLAIR (fluid attenuated inversion recovery) image shows a focus of hyperintensity in the left parietal lobe subcortical white matter (arrow). This patient also has multiple similar foci in the cerebrum. B: Axial T1-weighted inversion recovery image shows foci of subependymal nodules (arrow) along the ventricular wall.

FIGURE 13. Cortical tuber in a child with tuberous sclerosis complex (TSC). A: Low-magnification view shows a disorganized collection of neuroglial cells, some enlarged. B: Magnified view of the lesion showing “balloon cells” with eccentric nuclei and glassy eosinophilic cytoplasm (arrows), admixed with dysmorphic, markedly enlarged neurons containing Nissl substance in their cytoplasm (arrowheads). C: A cluster of balloon cells within the tuber. D: An abnormal neuroglial cell showing morphologic features of both dysplastic neuron and balloon cell (arrow). Note the eccentric nucleolated nucleus and the enlarged pale but slightly basophilic cytoplasm, raising the possibility that the cell represents a transitional form of neuroglial cell. E: Various degrees of punctate calcification (arrows) and calcifications along vessel walls (arrowheads) are often seen in TSC tubers. F: Morphologically normal cerebral cortex (for comparison with features of the tuber). (All panels are micrographs from sections stained with hematoxylin and eosin.) (See the color insert.)

Hamartin may also interact with other proteins, including the ERM (ezrin, radixin, and moesin) family of actin-binding proteins, to activate small GTPases of the Rho subfamily (Rho GTPases).⁸⁵ Rho GTPases are important regulators of the actin cytoskeleton and are thought to be involved in neuronal developmental processes including neuronal migration, establishment of polarity, axon growth and guidance, dendrite elaboration and plasticity, and synapse formation.⁹² ERM proteins belong to the band-4.1 superfamily of membrane-cytoskeleton linking proteins.¹⁷³ These proteins are believed to function in multiple different fashions according to their interaction with various membrane proteins, Ras superfamily GTPases, and the actin cytoskeleton, and appear to be involved in the formation of microvilli, cell–cell adhesion, maintenance of cell shape, cell motility, and membrane trafficking.⁹⁰ The fact that hamartin binds to ezrin in vivo and can modulate the activity of RhoA (Ras homologous member A)⁸⁵ suggests that tuberin and hamartin may be attached to the membrane-cytoskeletal cortex through activated ERM proteins.⁷² Evidence from several reports suggests that ERM proteins function at a position upstream and downstream of Rho GTPases to regulate cellular adhesion and motility.¹⁰³^,¹⁷⁰^,¹⁷¹ ERM proteins (ezrin and moesin) are expressed in germinal matrix cells, migrating cells, and radial glial fibers in the developing human brain,⁷² correlating with RhoA expression in proliferating and migrating cells in the developing rat brain.¹²⁹ Dysfunction of tuberin and hamartin may perturb communication between ERM proteins and Rho GTPase to cause abnormal neuronal migration, polarity, and morphology, resulting in the formation of dysplastic

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cortex. Hamartin and tuberin are coexpressed within a population of abnormal neuroglial cells,⁷¹ and both TSC gene products and ERM proteins are also coexpressed within a subpopulation of abnormal neuroglial cells in TSC tubers,⁷² suggesting the upregulation of ERM proteins within these cells in response to a TSC gene mutation. Abnormalities of radial glia have also been implicated in the pathogenesis of brain lesions of TSC.¹³³

Tuberin contains a conserved 163-amino acid carboxy-terminal region that exhibits sequence homology to the catalytic domain of a GTPase-activating protein (GAP) for the low-molecular-weight GTPase Rap1¹⁸⁹ and for Rab5.¹⁹¹ Based on in situ hybridization with a digoxigenin-labeled cDNA probe, TSC2 mRNA was found to be widely expressed in various cell types throughout the body, including epithelia, lymphocytes, and endocrine organs; within the CNS, it was prominently and selectively expressed within neurons, especially motor neurons, including cortical, brainstem, and spinal cord neurons.¹⁰⁹ Widespread expression of the TSC2 gene within developing and adult nervous system was noted in another study using reverse transcription-polymerase chain reaction (RT-PCR), Northern blot, and in situ hybridization analysis.⁴⁸ The results of a study in mice showed that tuberin localized to the perinuclear region of cerebellar Purkinje cells, whereas hamartin was noted to distribute along neuronal or astrocytic processes.⁵⁶ Bases on human autopsy and biopsy material, TSC2 mRNA and tuberin were found in abundance in many CNS cell types, including neurons and ependymal cells.⁷⁹ TSC2-negative fibroblasts show inactivation of the cyclin-dependent kinase inhibitor p27.¹⁶⁵ Mutations in TSC2 may result in constitutive activation of Rap1, leading to enhanced proliferation or incomplete cellular differentiation.¹⁶⁴

TSC1 and TSC2 gene products colocalize within tubers (and sometimes within individual dysmorphic cells) of patients with TSC.⁷¹ Tissue culture experiments in various cell types, using both confocal laser microscopy and coimmunoprecipitation, show that both hamartin and tuberin interact with the G2/M cyclin-dependent kinase CDK1.²¹ It has further been suggested that hamartin and tuberin have separable functions in mammalian cell cycle regulation,¹¹⁵ that is, that hamartin itself has the ability to modulate cell proliferation independent of the presence of functional tuberin, and binding to hamartin is not always essential for tuberin to affect cell proliferation. Tsc1 and Tsc2, Drosophila homologs of TSC1 and TSC2, function together in vivo to negatively regulate cell size, cell proliferation, and organ size in the insulin signaling pathway (PI3Kinase-Akt/PKB-mTOR-S6K-S6) at a position downstream of dAkt (Drosophila Akt) and upstream of dS6k (Drosophila S6 kinase).¹³⁶ This has been clearly confirmed in surgically resected TSC tubers by means of quantitative immunohistochemical evaluation using tissue microarray methodology,¹¹⁸ and constitutive activation of S6K has been observed in TSC tubers but not in focal cortical dysplasia of Taylor type (T-FCD or CD Palmini type IIB), suggesting one difference between these MCDs (Fig. 16).¹¹^,¹¹⁸ Recent studies have also revealed Rheb (Ras homolog enriched in brain) GAP activity of tuberin playing a role in regulation of S6K and 4E-BP1,⁶⁵^,¹⁶⁷^,¹⁹⁵ indicating indirect regulation of the insulin signaling pathway by TSC genes through the inhibition of Rheb activity.

FIGURE 14. Immunohistochemical findings in a tuberous sclerosis complex (TSC) tuber. Double-label immunohistochemistry confirms colocalization of glial fibrillary acidic protein (GFAP) (brown color) and nonphosphorylated neurofilament (purplish-blue) in a subpopulation of balloon cells (arrowheads) in a TSC tuber, suggesting a failure of commitment in neuroglial differentiation. Note that some abnormal neuroglial cells are immunoreactive for GFAP (arrow) or nonphosphorylated neurofilament (double arrows). Scale bar = 100 μm. (See the color insert.)

FIGURE 15. Schematic summary of the functions of tuberous sclerosis complex (TSC) genes. The functions of both TSC1 and TSC2 gene products have been intensively studied using mostly Drosophila, tissue culture, and rodent models. This diagram briefly summarizes significant interactions of TSC genes with intracellular signaling pathways. CDK, cyclin-dependent kinase; MAP, microtubule-associated protein.

Sturge-Weber-Dimitri Syndrome/Encephalo-Trigeminal Angiomatosis

This is a rare, nonfamilial, neurocutaneous syndrome of unknown etiology¹²⁴^,¹²⁶^,¹⁸² encountered in surgical specimens from infants and children with intractable epilepsy, although much less commonly than destructive and malformative/hamartomatous lesions. The frequency of Sturge-Weber-Dimitri (SWD) syndrome is estimated to be 1 per 50,000 live births,⁵⁹ and 75% to 90% of children with SWD syndrome

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develop partial seizures by 3 years of age.⁹⁷ Clinicopathologic reports describe the association of the cerebral lesion, usually localized to the occipital cortex, with facial capillary hemangioma (“port-wine stain”) in the distribution of the ophthalmic division of the trigeminal nerve, and provide excellent accounts of the natural history of the disorder.¹²⁷^,¹⁸²^,¹⁹⁰ Visceral angiomas may be encountered in some patients.¹⁴ Neuroimaging features are highly characteristic (Fig. 17).

Neuropathologic abnormalities in cortical resection specimens (Fig. 18) are easily appreciated at low magnification, and soft tissue radiographs of the sliced specimen may show the characteristic “tram-track” pattern of neocortical calcification. The leptomeningeal angiomatosis is a key diagnostic feature of SWD syndrome,¹⁷⁷ characterized by some authors as a venous angioma,¹⁹⁰ consisting of dilated and tortuous thin-walled blood vessels within the subarachnoid space and pia, which may extend into the underlying cerebral cortex and even subcortical white matter. The cortex itself shows calcifications centered on microvessels, with associated neuronal loss, astrocytic gliosis, and extensive cortical atrophy¹²⁴^,¹²⁶ that is assumed to result from ischemic phenomena secondary, at least in part, to the meningeal angiomatosis. Associated malformations such as polymicrogyria, agyria/pachygyria, heterotopias, and cortical disorganization can also be seen.¹⁸² Ultrastructural studies of the parenchymal calcifications in Sturge-Weber brain have suggested that the earliest calcium deposits occur within perithelial cells of small blood vessels, and that the underlying cause of the calcification may be anoxic injury to endothelial, perithelial, and glial mitochondria due to stasis and abnormally increased vascular permeability of vessels in the hemangioma.⁵⁵^,¹²⁵

Neurofibromatosis, Meningio-angiomatosis, and Other Neurocutaneous Syndromes

Central neurofibromatosis (NF-2) is a genetic disorder characterized by neoplastic and dysplastic lesions of Schwann cells, meningeal cells, and glia.⁸⁹^,¹⁰⁴ It is associated with (a) Schwannomas, both central and peripheral, including bilateral acoustic Schwannomas; (b) meningiomas; (c) gliomas; and (d) glial hamartomas. It is inherited in an autosomal-dominant fashion, with a high rate of sporadic mutations (up to 50%).⁸⁹^,¹⁰⁴ The NF-2 gene, postulated to be a tumor-suppressor gene, has been localized to chromosome 22q12 and encodes a widely expressed protein, merlin (moesin, ezrin, radixin–like protein) or schwannomin, which is a new member of the protein 4.1 family of cytoskeleton-associated proteins.⁸⁹^,⁹³ Although seizures can develop in patients with NF-2, these are usually caused by a primary neoplasm rather than a primary malformative lesion. The most frequent malformations seen in association with NF-2 are meningio-angiomatosis and glial hamartomas. Meningio-angiomatosis⁷⁷^,¹³⁴ is a rare malformation of the cerebral cortex of unknown etiology, described in more detail in Chapter 12, where it is placed in the context of other neoplasms that are associated with seizures. Rare neurocutaneous syndromes, such as epidermal nevus syndrome and hypomelanosis of Ito,⁸¹ have also been associated with pediatric epilepsy, serving to emphasize the interrelated development of the CNS and overlying mesenchyme. The pathologic changes seen within the cerebral cortex of these patients are virtually identical to those seen in patients with CD.

FIGURE 16. Tissue microarray (TMA) analysis of insulin signaling pathways in tuberous sclerosis complex (TSC) tubers, focal cortical dysplasia, and control tissues. The left column represents sample cores from a TSC tuber (A, D, G, J, M); the middle column represents focal cortical dysplasia (FCD) with balloon cells (Palmini type IIB) (B, E, H, K, N); and the right column represents histologically normal cerebral cortex (C, F, I, L, O). Stained with hematoxylin and eosin (H&E) (A–C), p-mTOR (D–F), p-p70S6K (G–I), p-S6 (J–L), and p-eIF4G (M–O). Immunostains were performed on consecutive serial sections, and the same cells can be easily identified in different stains (e.g., arrowheads in D and G). Expression of p-p70S6K appears to be specific to the TSC tuber (panel G). Note the population of abnormal neuroglial cells in the TSC tuber expressing p-S6 and/or p-eIF4G (e.g., central area of each core shown in panels J and M), despite negative expression of p-mTOR and p-p70S6K (the same areas indicated by asterisks in panels D and G). Each core has a 0.6-mm diameter. Bar = 100 μm. eIF4G, eukaryotic translation initiation factor 4G; mTOR, mammalian target of rapamycin; p-, phospho-; p70S6K, 70-kDa ribosomal protein S6 kinase; S6, 40S ribosomal protein S6. For details of methodology, see Miyata et al. (2004). (From Miyata H, Chiang ACY, Vinters HV. Insulin signaling pathways in cortical dysplasia and TSC-tubers: tissue microarray analysis. Ann Neurol. 2004;56:510–519; with permission.) (See the color insert.)

Vascular Malformations

Cerebral vascular malformations are a group of “congenital” developmental abnormalities of the cerebral blood vessels, which can be divided, based on pathologic features, into five main groups: (a) arteriovenous malformations, (b) cavernous hemangiomas, (c) venous angiomas, (d) capillary telangiectases, and (e) varix or aneurysm of the great vein of Galen (which is actually an arteriovenous malformation or fistula).⁷⁶^,¹⁰⁰^,¹⁸³ The term “congenital” is placed in quotation marks because neuropathologists almost never encounter these lesions (with the exception of vein of Galen aneurysms) in infants or young children. Despite this, there is a widespread belief that the nidus of a future vascular malformation is present early in brain development. Arteriovenous malformations

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(AVMs) consist of a tangle of tightly interwoven blood vessels in the leptomeninges and underlying cerebral cortex and white matter. Brain tissue fragments among and adjacent to the abnormal vascular channels of a hemangioma (AVM) often show disorganization and reactive gliotic change, some of which may suggest the propensity of the tissue to generate seizures. These vascular lesions contain arteries, veins, and “arterialized veins,” muscularized vessels intermediate between an arterial and venous channel (Fig. 19). Cavernous hemangiomas are composed of a cluster of dilated, ectatic, and hyalinized vessels with no normal intervening brain parenchyma, and there is almost always abundant surrounding evidence of old hemorrhage. Venous angiomas contain dilated ectatic and venous channels with normal intervening brain parenchyma. Capillary telangiectases consist of dilated capillaries separated by normal brain parenchyma. The varix of vein of Galen (vein of Galen aneurysm) is a large, dilated, ectatic vein of Galen, which is usually in direct connection to a branch of one of the major arterial blood vessels at the base of the brain; varix of vein of Galen can therefore be considered a variant of AVM.⁴⁷^,⁷⁶^,¹⁰⁰

Clinically, AVMs are the vascular malformations most likely to cause symptoms, although cavernous hemangiomas and occasionally venous angiomas can be symptomatic.⁹⁹ Mechanisms of seizure genesis by these lesions include subclinical hemorrhage, compression and scarring of brain tissue around the vascular malformation, and a “steal” of blood from normal brain, rendering it at risk for seizure activity. Capillary telangiectases usually represent an incidental finding at autopsy and are almost never found within cortex or subcortical white matter.⁴⁷^,⁷⁶ Although hemorrhage is the most common and severe manifestation of AVMs, seizures occur at some time in up to 70% of affected patients.⁹⁹^,¹⁰⁰ Seizures are also frequently

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seen (in addition to congestive heart failure) in infants and neonates with varix of the vein of Galen.⁷⁶^,⁹⁹^,¹⁰⁰^,¹⁷²

FIGURE 17. Sturge-Weber-Dimitri syndrome (SWDS). A: Postcontrast T1-weighted axial image shows global right cerebral hemispheric atrophy with smooth leptomeningeal enhancement along the right parieto-occipital region. There is faint meningeal enhancement in the right frontal operculum. Enlarged deep white matter veins are also seen in the right frontal region. The right frontal calvarium is thicker than the left. These features are consistent with SWDS. B: Intraoperative photograph illustrating marked angiomatosis of the arachnoidal surface in the frontal region over the sensorimotor cortex.

FIGURE 18. Sturge-Weber-Dimitri syndrome, microsco-pic features. A: Dense angiomatosis of meningeal vessels in the occipital region (arrow). B, C: Punctate calcifications (arrows) in the underlying brain parenchyma. (See the color insert.)

FIGURE 19. Massive hemispheric vascular malformation, best categorized as an arteriovenous malformation, from a 9-year-old boy who underwent hemispherectomy. A: Operative photograph of the vascular malformation in situ. B: Representative histologic section showing two complete (and one partial) vascular channels with ectasia and striking variability in wall thickness. C: Higher magnification of the region shown by the arrow in panel B, highlighting the degree of fibromuscular thickening of this segment of the vessel (many of the cells immunostained with antibodies to smooth muscle actin, not shown). Brain parenchyma adjacent to the vascular channels showed dystrophic changes, with focally pronounced calcification. Hematoxylin and eosin (panels B and C), ×45 (panel B), ×175 (panel C). (Panel A courtesy of Dr. Warwick Peacock and Mr. Eric Behnke.)

Summary and Conclusions

The neuropathologist plays a crucial role in the evaluation of the complex lesions described in this chapter, which produce seizures as a major clinical manifestation. She or he has a major role in giving a definitive diagnosis for the structural abnormality/abnormalities present within a corticectomy specimen and at least suggesting how it may have contributed to intractable seizures (the latter assignment often more challenging than the former). Gross and microscopic examinations remain the gold standard for the diagnosis of CD as well as for other nonmalformative structural lesions of the neocortex.

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Correlation between pathologic features and clinical presentation is essential for understanding biologic behavior.¹⁸⁵ Furthermore, neuropathologic examination of resected tissue can yield important clues regarding the timing of a putative developmental aberration that led to the development of a lesion, which can aid in understanding the causes and course of CD and related developmental abnormalities. Because the resected specimens represent a valuable resource for furthering our understanding of cortical development and maldevelopment, the neuropathologist plays a role as a conduit for the resected tissue, ensuring maximal effective use for diagnosis and research that utilizes the specimen. Research questions can only be addressed (in human brain tissue specimens) using optimally preserved resection specimens that are preserved for electrophysiologic, neurochemical, and molecular investigations.⁴^,¹⁷ However, when such tissue is available, it is as valuable as—and arguably more valuable than—tissues obtained from experimental animals. For example, laser capture microscopy coupled with molecular analytic techniques has facilitated the study of neurotransmitter receptor subunit analysis in HME.¹⁰

Malformative lesions of the neocortex are responsible for the majority of structural pathologic features seen in infants and children with seizure disorders, especially in those with IS. CD, which represents a spectrum of neuropathologic changes associated with disruption of development of the normal neocortex, accounts for most of these malformations, although structural lesions associated with TSC, SWDS, NF-2, and meningio-angiomatosis and vascular malformations are also seen. Multiple etiologic factors have been associated with these developmental disorders, including genetic mutations, prenatal vascular insults,¹⁴⁸ and toxic and environmental exposures.

Although advances have been made in understanding these complex developmental lesions, especially from neuroimaging and molecular perspectives, much remains to be learned. Promising animal models have been developed¹⁵⁶ in which cortical malformations have been induced in rats. These experimental lesions, which conferred an increased propensity for seizures, demonstrated some changes identical to those seen in patients with CD, including cortical laminar disorganization, neurons in the cortical molecular layer, and periventricular and laminar heterotopia.¹⁵⁶ Experimental models such as this one provide an excellent opportunity for examining the basic biologic derangements that occur in the evolution of epileptogenic cortical malformations. Continued morphologic, immunohistochemical, clinicopathologic, and molecular research is necessary to characterize better the time course and associations of these lesions. Investigations seeking to understand the molecular events involved in the basic processes of cell proliferation, migration, terminal differentiation, PCD, and cortical remodeling will help to elucidate the fundamental mechanisms of brain development and significantly aid in the understanding of cortical malformations responsible for seizure disorders.

Acknowledgments

We thank Alex Brooks, Nelly Vehabedian, and Matthew Lynch for ongoing expert technical assistance and help with manuscript preparation and Carol Appleton for preparation of many of the figures. We also thank Drs. Michael Menchine, Jessica Emelin, Michael DeRosa, Michael W. Johnson, and Michael A. Farrell for past contributions to this work. H.V.V. is supported in part by the Daljit S. & Elaine Sarkaria Chair in Diagnostic Medicine. G.W.M. is supported by PHS/NIH grants R01 NS 38992 and P01 NS 02808. H.M. is supported in part by the Grant-in-Aid for Young Scientists (A) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (#17689040) and by the Japan Epilepsy Research Foundation (H16–009).

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	Epilepsy: A Comprehensive Textbook 2nd Edition
	© 2008 Lippincott Williams & Wilkins

Epilepsy: A Comprehensive Textbook2nd Edition

Epilepsy: A Comprehensive Textbook
2nd Edition