Address correspondence and reprint requests to Dr. R. Spreafico at Dipartimento di Neurofisiologia Sperimentale e Neuroanatomia, Istituto Nazionale Neurologico “C. Besta,” Via Celoria 11, 20133 Milano, Italy. E-mail: email@example.com
Summary: Purpose: We analyzed 26 surgically treated patients operated on for intractable epilepsy associated with type IA (architectural) cortical dysplasia, to investigate neuropathologic and immunocytochemical features, particularly of the γ-aminobutyric acid (GABA)ergic system, and to compare the findings with those observed in normal cortex.
Methods:. Routinely stained slides and serial sections immunostained for neurofilaments (SMI 311), microtubule-associated protein-2 (MAP-2), neuron-specific nuclear protein (NeuN), glial fibrillary acidic protein (GFAP), parvalbumin (PV), calbindin (CB), and calretinin (CR) were processed. Some sections were processed by using single-immunoperoxidase procedures; others were processed for double immunofluorescence labelling and observed by confocal microscopy. The density of inhibitory PV-immunoreactive interneurons was quantitatively assessed in all patients and control cases by using a two-dimensional cell-counting technique on PV immunostained sections.
Results: The density of PV-immunoreactive interneurons was significantly reduced in this group of patients, whereas CB- and CR- positivity appeared similar to those in normal cortex. In five cases, architectural abnormalities, in addition to those that defined type 1A dysplasia, were present and characterized by abnormal clusters of neurons and laminar cellular loss in superficial cortical laminate.
Conclusions: The reduction of PV expression in type IA cortical dysplasia suggests an impairment of the GABAergic system as a possible mechanism for the epileptogenicity; in addition, PV immunoreactivity can be helpful in the neuropathologic characterization of this form of cortical dysplasia.
Focal cortical dysplasias (FCDs) result from a disturbance in cortical development and are characterized by focal anomalies of cortical structure. Such malformations are often associated with intractable epilepsy (1), and some of the affected patients are referred for surgical treatment. Some forms of FCD have been clearly defined (2) and, particularly the type IA (architectural) cortical dysplasia, represent an increasingly recognized cause of temporal lobe epilepsy (3). Neuronal hyperexcitability is attributed to lack of balanced excitatory and inhibitory mechanisms (4). Neuropathologic findings suggest that alterations of the γ-aminobutyric acid (GABA)ergic (inhibitory) cortical neuronal system play a role in focal epilepsies secondary to cortical dysplasia and parvalbumin (PV), calbindin (CB) and calretinin (CR) antibodies are frequently used to study this inhibitory system (5,6). In particular, PV immunoreactivity labels a subpopulation of GABAergic interneurons that include chandelier and basket cells, considered important for controlling pyramidal cell excitability (7).
A reduction of calcium-binding proteins–immunoreactive interneurons has been reported in experimental cortical dysplasia (8) and in human dysplastic cortex (5,6), but in type IA, it was observed in a limited number of cases (9).
On this basis, the aim of this immunocytochemical study was (a) to investigate morphologic changes, particularly involving the inhibitory network, in the temporal cortex of patients operated on for intractable epilepsy associated with type IA (architectural) dysplasia and to compare the findings with those in normal cortex; and (b) to describe architectural abnormalities observed in some cases, in addition to those usually found in this type of dysplasia.
MATERIALS AND METHODS
Temporal lobe specimens were obtained from 26 patients operated on for intractable epilepsy secondary to type IA dysplasia, characterized by abnormal cortical lamination without giant dysmorphic neurons or balloon cells and frequent neuronal heterotopia in the white matter (2,3). Clinical characteristics in these patients are summarized in Table 1. All patients underwent magnetic resonance imaging (MRI) with standardized protocol (3), and tailored temporal lobe and hippocampus resections were performed. Specimens of temporal neocortex from four patients without cortical abnormalities and evidence for seizures, operated on for deep low-grade glial lesions, served as controls. In all cases, the surgery was performed for strictly therapeutic reasons after informed consent.
Table 1. Clinical characteristics of 26 patients operated on for TLE
For routine histologic examination, parts of the surgical specimens were fixed in 10% neutral buffered formalin, embedded in paraffin, and sections (4–10 μm) were stained with hematoxylin and eosin, thionin, luxol fast blue, or Bielschowsky. For the immunocytochemical investigation, part of the cortex from each case was fixed in 4% paraformaldehyde, and vibratome sections (50 μm) were cut and immunostained with different primary antibodies, as reported in Table 2. The immunoperoxidase procedure was described elsewhere (9).
Table 2. Specificity, supplier, and dilution of primary antibodies used
PV-stained sections from pathologic and control cases were quantitatively assessed by using a two-dimensional cell-counting technique. One section per case was viewed with a x10 objective. By using Image Pro-Plus 5.1 software (Media Cybernetics, MD, U.S.A.), a series of contiguous images was captured from the pial surface to the grey–white matter border, forming a single tiled image. A mean area (±SD) of 6.4 ± 1.4 mm2 was considered. All strongly immunopositive neurons within this area were manually counted, and differences in cell density between the two groups were assessed with t test.
In five cases, free-floating sections were incubated in a mixture of the following monoclonal and polyclonal antibodies (MAP2-CB, MAP2-CR, MAP2-PV) and observed with a confocal microscope. Secondary antibodies were from Jackson Immunoresearch (West Grove, PA, U.S.A.) [Cy 2-conjugated goat anti-mouse immunoglobulin G (IgG) diluted 1:200 and Cy3-conjugated goat anti-rabbit IgG diluted 1:600]. The sections were examined under a Radiance 2000 confocal laser scanning microscope (Bio-Rad, Hemel Hempstead, U.K.) equipped with a krypton/argon laser and the images were acquired through separate channels and merged by using Bio-Rad Lasersharp 2000 software.
Control sections were processed without primary antibody; no significant immunostaining was ever observed under these conditions.
Thionin staining showed regular laminar organization, clearly recognizable cortical layers (Fig. 1A), and no cytoarchitectural abnormalities. SMI 311–immunoreactive neurons were present mainly in layers III and V. Scattered immunoreactive cells were also found in layer IV, but never in layer I. SMI 311 immunostaining was particularly intense in cell bodies, basal and apical dendrites of pyramidal neurons, and also in rounded multipolar cells. Anti-NeuN antibody confirmed the normal organization of the cortex. MAP-2 immunoreactivity was present mainly in pyramidal neurons uniformly distributed throughout the cortex and was concentrated in cell bodies and apical dendrites. Glial fibrillary acidic protein (GFAP) antibody labelled only a few cells in the most superficial part of layer I. The three calcium-binding proteins, PV, CB, and CR, are known to be expressed in different, but partially overlapping, subpopulations of GABAergic cortical interneurons. PV immunoreactivity was present in cell bodies, proximal dendrites, and neuropil. PV-positive neurons were present in all layers except layer I. Intensely immunoreactive punctate structures, interpreted as axon terminals or cross-sections of dendrites and axons, were present throughout the cortex but were particularly numerous in layers III ad IV (Fig. 1C). Mean (±SD) PV-positive neuron density was 36.6 ± 1.8 cells/mm2. Small CB-positive neurons were located predominantly in layer II, and some CB-immunoreactive pyramidal cells were observed in layer III. CR-labelled neurons were concentrated in the supragranular layers.
All the samples were characterized, in thionin-stained sections (Fig. 1B), by disruption of cortical layering. Only layer I was clearly discernible. No malformed or giant or undifferentiated cells were observed. SMI 311 positivity appeared less intense in pathologic than in normal tissue, indicating a reduction in neurofilaments. MAP-2– and NeuN-stained sections showed absent cortical lamination and the presence of numerous heterotopic neurons in the underlying white matter. GFAP immunolabeling indicated moderate or intense gray-matter and white-matter gliosis in all the cases.
In all pathologic specimens, CB and CR positivity appeared similar to or slightly less intense than that in normal tissues; PV-positive cells were significantly reduced in different degrees (11.5 ± 8.9 cells/mm2; p < 0.01; Fig. 1D).
In five cases, in addition to the abnormalities used in defining type 1A dysplasia, further alterations were observed. An abnormal band of densely packed neurons below the molecular layer was observed in thionin- and NeuN-stained sections (Fig. 1E). SMI 311 and MAP-2 antibodies also picked out this atypical band of small neurons showing frequently pyramidal morphology and malorientated apical dendrites (Fig. 1F). A reduced neuronal density, observed particularly in NeuN-immunoreactive sections (Fig. 1E), was present just below the band and coupled with gliosis, as revealed by GFAP immunostaining (not shown). In double-labelling experiments, MAP-2–positive cells intermingled with rare CB- and CR-positive interneurons were observed within the band, whereas, in the strip just below it, numerous CB- and CR-labelled cells were present with very few MAP-2–positive neurons (Fig. 1G and H). No PV immunoreactivity was observed.
We found that PV immunoreactivity was significantly reduced in the patients presenting type IA cortical dysplasia (inclusion criterion), while CB and CR immunoreactivity appeared similar to control cases.
Reduced PV immunoreactivity was observed in an animal model of experimental cortical dysplasia (8) and also in human cortical dysplasia, although most of the data have been addressed to type II cortical dysplasia (5,6,9,10). PV represents a preferential marker for the chandelier and basket cells considered important inhibitory interneurons acting on pyramidal cell excitability (7). The low PV immunoreactivity found in our specimens may be due to reduced PV synthesis or loss of PV-immunoreactive cells and terminals. Whatever the cause, reduction of PV immunoreactivity is likely to result in increased cortical excitability. Reduced numbers of PV-containing cells would decrease inhibitory activity overall, whereas decreased PV content within a cell could decrease cell-firing rate. PV appears to buffer transient increases in cytosolic Ca2+ from extracellular and intracellular sources (11) and affects a range of Ca2+-regulated neuronal processes including excitation and synaptic transmission (12).
The presence of an impairment in the GABAergic inhibitory system observed in the present samples could represent a possible mechanism for their excitability and epileptogenicity; these data are in line with the involvement of the GABA-mediated synaptic inhibition reported in patients affected by type II cortical dysplasia (13).
As recently described (2,3), type IA cortical dysplasia is characterized by cortical laminar disorganization frequently associated with white-matter and layer I hypercellularity. These abnormalities are less severe than those observed in other forms of FCD also with cytoskeletal abnormalities and balloon cells (types IB and II). Accordingly, the histopathologic diagnosis of type IA dysplasia could be difficult and could be susceptible to subjective interpretation. The reduced immunoreactivity for PV-reactive cells and terminals reported in this study can be helpful in neuropathologic routine practice to characterize this dysplasia.
In five cases, we found, below layer I, an abnormal band composed of maloriented pyramidal neurons and scattered CB- and CR-positive interneurons, and, just below it, laminar neuronal loss. Similar patterns have been previously noted in surgical samples from patients affected by temporal lobe epilepsy (14,15). Our data suggest that cell loss mainly affect pyramidal neurons, as observed in double-labelled slides. No differences in the clinical data were present in these patients, and we proposed that these abnormalities should be considered a variant of type IA FCD.
Acknowledgment: This work was supported by FIRB RM2, 135-RF2003-RF38, from the Italian Ministry of Health, MIUR-PROT-2003068749-001, and by a grant from the Fondazione Banca del Monte di Lombardia. We thank D. C. Ward for help with the English.