Address correspondence to Roberto Spreafico, IRCCS Foundation Neurological Institute “C. Besta,” Via Celoria 11, Milano, Italy. E-mail: email@example.com
Purpose: Despite the availability of detailed electroclinical and imaging data, only a few neuropathological studies of nodular heterotopia have been published. The aim of this study was to describe the neuropathological features of various types of nodular heterotopia obtained from patients undergoing surgery for intractable epilepsy.
Methods: Specimens of heterotopic nodules taken from 24 patients were neuropathologically investigated using routine and immunocytochemical procedures, and the data were compared with magnetic resonance imaging (MRI), electroclinical findings, and surgical outcomes.
Results: The neuropathological data distinguished two groups. Group 1 (14 patients, 78% in Engel class 1) had similar characteristics regardless of the size, number, or location of the nodules, with both projecting and local circuit neurons in the nodules intermingled with glial cells. Thirteen patients had focal cortical dysplasia. The nodules were identified by MRI in all cases. In group 2 (10 patients, 90% in Engel class 1), all of the nodules were within the temporal lobe and associated with hippocampal sclerosis or gangliogliomas. They were very small (undetected by MRI) and mainly formed by projecting neurons with no evidence of glial cells. All of the patients had cortical dysplasia.
Discussion: The distinctive neuropathological features of the nodules in the two groups suggest different etiopathogenetic mechanisms. In group 2, the presence of nodular formations in association with cortical dysplasia and either hippocampal sclerosis or ganglioglioma raises a question concerning so-called dual pathology in the temporal lobe.
Heterotopia are malformations of cortical development (MCD) characterized by the presence of apparently normal brain cells in abnormal positions. They are divided into three broad categories: (1) individual misplaced neurons, also called excessive single neurons in the white matter (neuronal heterotopia); (2) heterotopic nodules of grey matter (nodular heterotopia); and (3) continuous bands of grey matter between the cortical mantle and the ventricles (laminar heterotopia).
Before the introduction of MRI, grey matter heterotopia were usually only identified at autopsy, but modern high-resolution imaging techniques, such as thin-section volumetric MRI, allow the macroscopic morphology and extent of NH to be precisely defined. They can therefore now be diagnosed in vivo, and it is possible to investigate their association with neurological deficits and epilepsy. Despite the large amount of electroclinical, genetic, and MRI data, there is still no exhaustive description of the neuropathology and intrinsic organization of the nodules in the different forms of NH, partially because only a few autoptic cases have been published, and surgical specimens are still rare (Dubeau et al., 1995; Eksioglu et al., 1996; Hannan et al., 1999; Santi & Golden, 2001; Kakita et al., 2002; Thom et al., 2004; Tassi et al., 2005; Wieck et al., 2005).
The aim of this study was to describe the histopathological and immunocytochemical features of NH (MRI detectable or not) and the organization of the adjacent epileptogenic cortical areas, on the basis of surgical specimens taken from 24 patients operated on for intractable epilepsy at the “C. Munari” Epilepsy Surgery Center. The surgical outcomes related to the different types of NH are also described.
Materials and Methods
Twenty-four (4%) out of 583 patients, operated on for intractable epilepsy from June 1996 to May 2006 at the “C. Munari” Epilepsy Surgery Center at Niguarda Hospital, Milan, Italy, were retrospectively selected for the presence of NH by neuropathological reviewing of permanent slides. Preoperative high-resolution MRI of each patient was retrospectively revised independently by two neuroradiologists, at the light of the neuropathological findings.
Clinical and presurgical workup
All of the 24 patients had undergone an extensive presurgical evaluation involving their clinical history (including age at seizure onset, mode of presentation, and the characteristics and frequency of the seizures), a neuropsychological work-up, and MRI and video electroencephalography (vEEG) recordings as previously described (Tassi et al., 2005). All of the patients underwent long-term vEEG recordings, and 14 (58%) also underwent invasive presurgical stereo EEG (sEEG) as the epileptogenic zone was not identified with sufficient precision (Cossu et al., 2005). In addition to the anatomoelectroclinical data, the final surgical approach was also based on the patients' neurological condition and the likelihood of postsurgical neurological deficits. Surgery was performed only after informed consent had been given by the patient (or the parents of intellectually impaired individuals), and its outcome was determined by means of Engel's classification (Engel, 1987) after at least 1 year of follow-up.
Histology and immunolocalization
After resection, the tissue specimens were fixed in 10% neutral-buffered formalin, and paraffin-embedded sections (4–10 μm thick) were stained with hematoxylin-eosin (H&E), Luxol Fast Blue (LFB), and thionin for routine neuropathology testing, or processed for immunolocalization.
The specimens taken from 21 patients were large enough to be able to process some of the tissue for further immunohistochemical studies. These tissue samples were fixed in paraformaldehyde 4% in 0.1 M phosphate buffer, pH 7.2, vibratome cut into 50-μm serial sections, and processed for immunohistochemistry using antibodies against glial fibrillary acidic protein (GFAP), intermediate filament protein vimentin (VIM), neuronal nuclear protein (NeuN), nonphosphorylated neurofilaments (SMI 311), microtubule-associated proteins 2a and 2b (MAP2), and the calcium-binding proteins parvalbumin (PV), calbindin (CB), and calretinin (CR). The characteristics and working dilutions of the primary antibodies are shown in Table 1.
Table 1. Source, type, specificity, and working dilutions of the primary antibodies
Intermediate filament glial fibrillary acidic protein
1:500 (P) 1:15.000 (V)
Intermediate filament vimentin
Radial glia and reactive astrocytes
Neuronal nuclear protein
Microtubule associated proteins 2a and b
Subpopulations of GABAergic interneurons
Subpopulations of GABAergic interneurons
Subpopulations of GABAergic interneurons
The details of the immunohistochemical procedures have been previously described (Spreafico et al., 1998; Tassi et al., 2005). Briefly, after incubation in 10% (vol/vol) normal serum in order to mask nonspecific adsorption sites, the sections were incubated for 24 h in primary antibody, rinsed in 0.01% phosphate-buffered saline (PBS), and incubated for 1 h in the appropriate biotinylated secondary antibody (goat anti-rabbit or horse anti-mouse immunoglobulins; Vector, Burlingame, CA, U.S.A.) diluted 1:200 in PBS, and then in avidin-biotinylated complex (ABC kit; Vector), diluted 1:100 in PBS. The immunoreaction was revealed using 0.075% 3-3′-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO, U.S.A.) as the chromogen substrate with 0.002% H2O2 in 0.05 M Tris-HCl buffer. The sections were then washed in Tris-HCl, mounted on gelatin-coated slides, dehydrated, and coverslipped. One of nine sections was stained with 0.1% thionin as a structural control.
At the time of surgery, the 24 patients (Tables 2 and 3) had a mean age of 30 years (sd 8, range 17–42). Their mean age at the time of epilepsy onset was 11 years (sd 6, range 0–25), the mean duration of epilepsy was 19 years (sd 9, range 6–37), and mean seizure frequency was 21 per month (sd 24, range 1–100).
Table 2. Anatomical and neuropathological characteristics
Two distinct groups emerged on the basis of the neuropathological data, the clinical, neurophysiological, and neuroradiological findings, and the presurgical approaches. One group consisted of 14 patients (58%) with similar histopathological findings, characteristic MRI features of PNH, and frequent multilobar involvement during seizures requiring invasive investigations in all but one. The other group consisted of 10 patients (42%) whose small NH (only revealed upon neuropathological investigation), history, clinical seizure characteristics, vEEG monitoring and radiological studies allowed the definition of temporal lobe epilepsy. Only one of these patients underwent sEEG because of atypical ictal semiology suggesting possible primary extratemporal involvement.
Group 1 (patients 1–14)
PNH was diagnosed in these patients (seven females and seven males) on the basis of presurgical MRI. Their mean age at the time of epilepsy onset was 11 years (sd 6, range 1–17), and the mean duration of epilepsy was 20 years (sd 9, range 8–37). At the time of surgery, they had a mean age of 31 years (sd 8, range 21–42) and a mean seizure frequency of 20 per month (sd 18, range 2–60). All the males with a unilateral PNH, were born preterm at the seventh month of pregnancy. One patient with unilateral PNH and three with bilateral PNH were mentally retarded (IQ < 80).
MRI led to a diagnosis of subependymal nodular heterotopia (SEH) in four patients (unilateral in three and bilateral symmetric in one) and subcortical nodular heterotopia (SCH) in the remaining 10 patients (unilateral in eight and bilateral asymmetric in two), with the nodules stretching from the periventricular area toward the subcortical regions. The nodules were confined to the temporal lobe in only four patients.
Thirteen patients underwent sEEG. In 10 cases (71%), the epileptogenic zone (EZ) involved multiple lobes; in the remaining four cases (29%), it was restricted to the temporal lobe. The clinical and anatomical data are summarized in Tables 2 and 3.
All of the patients were followed up for at least 1 year. Ten patients (71%) with unilateral subcortical or subependymal PNH are now in class I (six, or 43%, are in class Ia); three patients are in class III, and one is in class IV. Antiepileptic drugs have been completely withdrawn in one case and are being reduced in a further nine (Table 3).
Histology and immunohistochemistry
All of the nodules in the sections routinely processed for neuropathology had similar morphological features and, regardless of their size and location, appeared as clearly delimited islands of grey matter enveloped by dense white matter bundles from which some myelinated fibers penetrate the nodules (Fig. 1A and 1B). Moderate gliosis was observed with GFAP-positive cells scattered inside the nodules, frequently around blood vessels; the intensity of the gliosis paralleled that found in the overlaying cortex.
Specimens from 12 patients were available for further immunohistochemistry studies. NeuN-positive neurons were uniformly distributed inside the nodules, without any clear clustering (Fig. 1C), whereas MAP2 immunoreactivity revealed clusters of small and medium-sized neurons of different morphology with haphazardly oriented apical dendrites, intermingled with areas of fewer positive neurons (Fig. 1E). In general, as observed in the overlaying cortex, SMI 311 immunoreactivity was less than that of MAP2, revealing only a few cell bodies and numerous dendritic processes (Fig. 1D). The edges of the nodules showed bipolar and pyramidal neurons highly stained by MAP2 or SMI 311 antibodies, with their major axis and dendritic processes parallel to the bordering fibers (Fig. 1, D and E).
Subpopulations of interneurons (identified by their expression of the calcium-binding proteins PV, CB, and CR) were scattered within the nodules without any apparent laminar organization. Some patients showed PV-positive cells intermingled with unlabeled areas (Fig. 1F) which, in adjacent sections, resulted in filled MAP2-positive cells (Fig. 1E).
There were numerous scattered or clustered NeuN-positive neurons in the white matter, and one patient (Patient 4) showed a thin ribbon of heterotopic neurons extending from the cortex to the nodules. Vimentin immunostaining did not reveal any specific labeling suggesting remnants of radial glial fibers.
The cortex of two patients (Patients 11 and 13) was disrupted by heterotopic nodules involving the cortical mantle. In four patients (Patients 2, 3, 11, and 12), small and packed gyri were observed without any cytological alterations, suggesting polymicrogyric cortex.
Thirteen of the fourteen patients had a dysplastic cortex overlaying the nodules. This was diagnosed as type IA focal cortical dysplasia (FCD), characterized by disrupted cortical lamination in 11 cases [two had no layer IV (Patients 3 and 5), and a third showed a clustered layer II (Patient 6)]; the remaining two patients, who had bilateral heterotopia (Patients 13 and 14), were diagnosed as having type IB FCD because, in addition to the laminar disorganization, they also had large, nondysmorphic pyramidal neurons scattered throughout the cortex (Palmini et al., 2004). Reduced PV immunolabeling in all of these patients confirmed the diagnosis of type I FCD (Garbelli et al., 2006).
Hippocampal structures were resected in 10 patients, but the ablated tissue was sufficiently preserved for a precise histopathological diagnosis in only eight, none of whom showed any signs of hippocampal sclerosis (HS).
Group 2 (patients 15–24)
In these 10 patients (42%), small heterotopic nodules were only recognized upon histopathological investigation, as neither the presurgical MRI evaluation nor the retrospective imaging review guided by the neuropathological findings revealed any grey or white matter abnormality suggesting the presence of heterotopia.
The patients' clinical and radiological characteristics are summarized in Tables 2 and 3.
Three of the patients were female (30%), and seven were male (70%). Their mean age at the time of epilepsy onset was 10 years (sd 7, range 0–25), and the mean duration of epilepsy was 19 years (sd 9, range 6–33). The mean age at surgery was 29 years (sd 7, range 17–37), and the mean seizure frequency was 23 per month (sd 32, range 1–100). Five patients (50%) had febrile seizures.
MRI led to a diagnosis of double pathology (FCD plus HS) in four patients because of hypoplasia and blurring of the temporal lobe coupled with a reduction in the size of the hippocampus and signal alterations; HS alone was diagnosed in a further four patients; and a low-grade tumor was suggested in the remaining two.
Only one patient underwent sEEG to identify the epileptogenic zone precisely. Surgery was limited to the temporal lobe in all cases.
Nine patients (90%) are currently in class Ia and Ic, and one patient is in class III. Antiepileptic drugs have been completely withdrawn in three patients and are being reduced in six patients (Table 3).
Histology and immunohistochemistry
In addition to routine histopathological investigations, immunohistochemistry was used to investigate the samples taken from nine of the 10 patients.
The nodular formations consisted of a few scattered small and round nodules, whose major axis was generally oriented in the direction of the main course of the white matter fiber bundles (Fig. 2A–2C), which were detected close to grey matter immediately below layer VI of the temporal neocortex (Patients 16, 18, 19, and 21–24) or mesial temporal structures (the amygdala in Patient 15 and the hippocampus in Patients 17 and 20).
In the LFB-stained sections, the nodules consisted of densely packed round shaped neurons with large nuclei surrounded by a thin rim of cytoplasm (Fig. 2C and insert) and were bounded by a broad mesh of myelinated fibers (Fig. 2E). MAP2 immunoreactivity revealed that almost all the neurons were labeled and had short and poorly branching proximal dendrites without any preferential orientation; there was also a very intense MAP2-positive neuropil (Fig. 2C). Immunostaining with SMI 311 showed only a few labeled dendrites on the borders of the nodules, and CB-, CR-, and PV-positive interneurons were rarely detected within them. In two cases (Patients 16 and 18), irregularly shaped nodules were closely aligned or intermingled with columns of medium-sized heterotopic neurons (Fig. 2D).
Despite the presence of intense gliosis in the surrounding white matter, no GFAP-positive cells were observed within the nodules (Fig. 2F).
FCD was diagnosed in all of the patients (Table 3): type IA in nine patients and type IB in one. In six cases (Patients 15, 16, 17, 18, 21, and 24), anti-NeuN and anti-MAP2 antibodies showed that layer II was abnormally packed with neurons and intense gliosis as has been previously described (Garbelli et al., 2006).
Hippocampal structures were resected in all of the patients, and HS was histopathologically confirmed in seven patients (the specimen from one patient was not sufficiently preserved to make a correct diagnosis). The remaining two patients showed a different degree of hippocampal gliosis without neuronal loss, and the diagnosis of ganglioglioma suspected on the basis of the MRI findings was histopathologically confirmed.
Our previous neuropathological findings in nine patients with NH identified by MRI allowed us to describe the morphological and immunocytochemical characteristics of heterotopic nodules and the overlying cortex (Tassi et al., 2005). The present study extended our analysis to a larger group of 24 patients undergoing surgery for intractable epilepsy, and the results suggest that two homogeneous subgroups can be distinguished on the basis of their neuropathological, electroclinical, and neuroimaging characteristics.
The first group was characterized by two major observations. Despite their broad clinical and imaging spectra, which allow different anatomoelectroclinical classifications (see Battaglia et al., 2006 for references), all of the nodules seemed to have similar morphological and immunocytochemical characteristics, regardless of their size and number (single or multiple), lobe, and depth of location (subependymal and/or subcortical). Similarly organized nodules have also been found in patients with the filamin 1 gene mutation (Kakita et al., 2002; Thom et al., 2004). Thom et al. (2004) found that the nodules contain all of the subtypes of inhibitory interneurons and that their morphology seemed to be normal. In five cases, they also observed the presence of cell-free zones with small vessels reminiscent of the molecular layer and glial cells with radiating fibers similar to radial pattern of superficial gliosis suggesting a rudimentary laminar pattern.
We observed cell-free regions in our PV-stained sections, but the adjacent areas seemed to be filled with NeuN- and MAP2-positive neurons. Moreover, GFAP immunoreactivity inside the nodules was very scarce and did not reveal any radial pattern suggesting Chaslin's gliosis. However, the fact that we did not detect any pattern reminiscent of the molecular layer in our cases may have been due to the different types of material (autoptic versus surgical specimens).
The second observation concerned the neocortex overlying the nodules. Although MRI suggested abnormal gyrations with normal signal intensities in some patients, there were no histopathological abnormalities consistent with typical polymicrogyria, although all but one patient had cortical dysplasia. Kakita et al. (2002) also found a dysplastic cortex in their autoptic case, but Thom et al. (2004) observed a less pronounced PV-immunoreactive plexus in an apparently normal cortex. In our patients, laminar disruption of the overlaying cortex was associated with a considerable reduction in PV immunoreactivity, thus further supporting the diagnosis of FCD (Garbelli et al., 2006).
Surgical outcome was particularly good in this group of patients (71% class I), and it is worth noting that this was also true in the patients with unilateral heterotopia regardless of the size and location of the nodules. This confirms that surgery should be considered in such cases after a careful presurgical work-up.
The presurgical MRI investigations of the 10 patients in the second group revealed HS plus FCD in four patients, HS alone in four, and tumors in two. The neuropathological diagnosis confirmed the imaging findings, but also revealed the presence of FCD in all of the patients, as well as small nodules in the white matter that were not detected by MRI. The histopathological and immunocytochemical characteristics of these nodules were completely different from those of the first group, as they consisted of densely packed, round MAP2-positive cells whose morphological features resembled those of immature neurons; they also contained rare calcium-binding protein-immunopositive interneurons and no GFAP-positive cells. The morphological characteristics of the cells, together with their positions and relationships with the surrounding white matter fibers, give an impression of clusters of incorrectly migrated neurons entrapped by bundles of fibers, and it is these histopathological characteristics that allow us to classify them as nodular heterotopia rather than glioneuronal hamartomas. A glioneuronal hamartoma is in fact a small intracortical aggregate of randomly oriented neurons, astrocytes, and oligodendroglia-like cells usually observed in the grey matter of the neocortex, hippocampus, or amygdala (Gòmez-Ansòn et al., 2000), whereas a heterotopia is an accumulation of neurons whose migration to the cerebral cortex has been prematurely arrested (Barkovich & Kuzniecky, 2000).
Interestingly, we found that the nodules in the temporal lobe associated with FCD coupled with HS or gangliogliomas. There have been increasing reports of an association between HS and FCD or low-grade glioneuronal tumors (Lévesque et al., 1991; Raymond et al., 1994; Li et al., 1997; Fauser et al., 2004), and the concomitance of apparently multiple temporal lobe pathologies raises the question of a possibly common malformative process.
In any case, regardless of their etiology, defective neuronal migration in early development seems to be involved in the formation of various NH, thus suggesting that these mechanisms were impaired in all of our cases in both groups.
The nodules observed in our group 2 patients were smaller than those observed in group 1, were always detected just below the grey matter, and were associated with cortical alterations that were particularly evident in the granular and supragranular layers. This suggests that the impaired neuronal migration occurred during the late phases of corticogenesis, in accordance with the inside-out mechanisms of cortical development. Conversely, the large number of neurons forming the white matter nodules in the group 1 patients suggests that other mechanisms such as neuroblast overproliferation may also have been involved (Dobyns et al., 1996). Our histological findings indicate that, although dysplastic, the cortex overlying these large nodules was not abnormally thin, thus suggesting that the neurons are overproduced during corticogenesis.
In brief, the two groups identified on the basis of their anatomoelectroclinical correlations and neuropathologyical data seem to be completely distinct. The first included different subtypes of MRI-detectable PNH with similar histopathological characteristics features and the electroclinical pictures of the patients were so complex that invasive presurgical sEEG recordings were necessary to be able to identify the epileptogenic zone precisely. In the second, the NH were only detected neuropathologically and were associated with HS or gangliogliomas; furthermore, all of the patients had typical temporal lobe epilepsy, and most did not not require the use of invasive recordings.
This study was supported by the Italian Ministry of Health, EU grant Functional Genomics and Neurobiology of Epilepsy (EPICURE), contract no. LSHM-CT-2006-0373315, Associazione P. Zorzi, and Fondazione del Banco di Lombardia (FBML).
Conflict of interest: We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. The authors have no personal, commercial, academic, or financial conflicts of interest to disclose.