Intrinsic epileptogenicity of dysplastic cortex: Converging data from experimental models and human patients


Address correspondence to Giorgio Battaglia, Molecular Neuroanatomy and Pathogenesis Unit, IRCCS Foundation Neurological Institute “Carlo Besta,” Via Libero Temolo 4, 20126 Milano, Italy.



Focal cortical dysplasia (FCD) is a brain malformation associated with particularly severe drug-resistant epilepsy that often requires surgery for seizure control. The molecular basis for such enhanced propensity to seizure generation in FCD is not as yet elucidated. To investigate cellular and molecular bases of epileptogenic mechanisms and possible effect of severe epilepsy on the malformed cortex we have here performed a parallel analysis of a rat model of acquired cortical dysplasia previously established in our laboratory, i.e., the methylazoxymethanol/pilocarpine (MAM-PILO) rats, and surgical samples from patients with type IIB FCD.

Data from the MAM-PILO rat model and human FCD samples reveal in both conditions: (1) that status epilepticus (SE) and/or seizures can further modify the cellular and molecular settings of the malformed cortex; (2) excitation/inhibition imbalance, and dysregulation of the N-methyl-d-aspartate/ membrane-associated guanylate kinase (NMDA/MAGUK) expression; (3) activation of cell death in neurons and glia. The data therefore highlight the mechanistic relevance of glutamate/NMDA hyperactivation in FCD epileptogenesis and suggest that epilepsy is a pathologic process capable of affecting structure and function of both neurons and glia.

Malformations of cortical development result from pathologic events of a different nature occurring during the process of cortical ontogenesis (Barkovich et al., 2012). They are the neuropathologic substrate in a number of patients with drug-resistant epilepsy. In particular, focal cortical dysplasia (FCD) is the more common brain malformation in patients who are undergoing epilepsy surgery for the relief of intractable seizures (Fauser et al., 2006; Lerner et al., 2009). The classification of FCD has been recently reevaluated by a task force of the International League Against Epilepsy (ILAE) to further define clinical, imaging, and neuropathology features of affected patients (Blümcke et al., 2011). Among the different forms, type IIB FCD, which was first described in a seminal paper by Taylor and colleagues (Taylor et al., 1971), is characterized by distinctive clinical features: early age of onset, electroencephalography (EEG) patterns of continuous interictal spiking activity, severe seizures, and frequent episodes of epileptic status (Palmini et al., 1995; Tassi et al., 2001).

Mechanisms of Hyperexcitability in the Malformed Brain

The mechanisms underlying such severe epilepsy are not completely understood (Schwartzkroin & Wenzel, 2012). However, many data indicate that dysmorphic neurons are strictly related to FCD epileptogenesis. First, stereo-EEG demonstrated that both ictal discharges and interictal rhythmic spiking activity originated from the dysplastic areas where dysmorphic neurons were located (Chassoux et al., 2000). Second, electrocorticographic recordings from patients with FCD demonstrated seizure onset from cortical areas characterized by the presence of dysmorphic neurons and not balloon cells (Marusic et al., 2002; Boonyapisit et al., 2003). Third, electrophysiologic studies in vitro on cortical slices or dissociated neurons from surgery specimens of pediatric FCD patients confirmed that cytomegalic neurons displayed abnormal electrophysiologic properties, thus possibly playing a relevant role in sustaining epileptic discharges in FCD (Cepeda et al., 2003, 2005).

It is also possible that an imbalance between glutamatergic excitatory and γ-aminobutyric acid (GABA)ergic inhibitory inputs may be at the origin of the intrinsic hyperexcitability of the FCD areas. This hypothesis is supported by data indicating N-methyl-d-aspartate (NMDA) receptors as important determinants of FCD-related epilepsy (Ying et al., 1998, 1999; Takase et al., 2008). NR2A/2B NMDA subunits are increased and epileptogenic activities are sensitive to NR2B-specific inhibitors in surgical specimens from epileptic FCD patients (Crino et al., 2001; Möddel et al., 2005). In keeping with these data, we demonstrated a selective increase of NR2B in cortical specimens of FCD human patients (Finardi et al., 2006). Therefore, dysregulation of NMDA-receptor complex expression and function likely plays a role in FCD hyperexcitability.

MAM-PILO Rats: A Model for Human FCD

To better investigate the issue of why the malformed cortex is highly epileptogenic, we have recently developed a rat model of acquired human FCD (Colciaghi et al., 2011) based on prenatal methylazoxymethanol (MAM) treatment (inducing cortical malformations) and postnatal pilocarpine (PILO) treatment (leading to status epilepticus [SE] and epilepsy). This model recapitulates both pathologic conditions of human FCD, that is, abnormal cortical structure and highly recurrent spontaneous seizures. Behavioral and EEG analysis of seizures showed that MAM-PILO rats developed severe epilepsy. The morphologic and molecular analysis of the model demonstrated that SE and epilepsy were able to induce in MAM-PILO rats the presence of abnormally large cortical pyramidal neurons with neurofilament overexpression and recruitment of NMDA regulatory subunits at the postsynaptic membrane. These neurons were similar to the dysmorphic pyramidal neurons observed in human FCD. We have more recently further exploited our MAM-PILO model by analyzing epileptic rats at different time-points after epilepsy onset (18 h after SE onset, as acute SE stage; 3–5 days after the first spontaneous seizure, as early chronic stage; 3 and 6 months after epilepsy onset, as two stages of chronic epilepsy). As appropriate controls, we used MAM rats receiving diazepam (DZP) before PILO, not experiencing either SE or spontaneous seizures (MDP rats). Our analysis showed that (1) epilepsy duration was positively associated with progressive cortical and hippocampal atrophy; (2) dysmorphic cortical neurons overexpressing neurofilaments and NMDA receptors increased in number and became more widespread in the course of epilepsy; (3) recurrent seizures significantly reduced dendritic branching and spine density of dysmorphic pyramidal neurons in both neocortex and hippocampus, as demonstrated by Golgi-Cox analysis; (4) despite the loss of dendritic spines, the glutamatergic input to large cortical and hippocampal pyramidal neurons (soma size ≥400 μm2) was maintained (and that to granule cells even increased), whereas the GABAergic input to the same neurons was decreased, thus creating a statistically significant imbalance between excitatory and inhibitory synapses in both neocortex and hippocampus; and (5) there was a steady activation of the NMDA NR2B regulatory subunits, which at least in the neocortex was progressive during the course of epilepsy. Therefore, data from MAM-PILO rats indicated that the extent of architectural and molecular alterations in both hippocampus and neocortex were related to the duration of epilepsy, thus supporting the intriguing hypothesis of progressive epilepsy-dependent brain abnormalities. Because MAM rats, before pilocarpine treatment, might display different degree of cortical abnormalities, longitudinal magnetic resonance imaging (MRI) analysis during the course of epilepsy should positively confirm the progression of cortical and hippocampal abnormalities in individual MAM-PILO rats.

FCD in Human Patients: The Glutamate Hypothesis

We next extended our investigation in FCD patients to verify the possible effects of severe epilepsy also on the malformed human cortex. To this, we have analyzed surgical samples from eight patients with FCD IIB with different epilepsy duration and nine nondysplastic controls (Finardi et al., 2013). Patients were surgically treated either at the Epilepsy Surgery Center “C. Munari” of the Niguarda General Hospital or at the Neurosurgery Department of the Neurological Institute “C. Besta” in Milano. In the patients with epilepsy, high-resolution MRI and electroclinical analysis including stereo-EEG (SEEG) or video-EEG recordings were used to carefully define the areas of epilepsy onset. In every FCD patient we have compared the dysplastic areas at the origin of epileptic discharges with adjacent areas involved in the epileptic circuitry but not at the origin of seizures. Our results demonstrated that the epileptogenic/dysplastic areas of all patients were characterized by larger dysmorphic neurons, reduced neuronal density, and increased glutamatergic inputs if compared to adjacent areas. These features were likely the result of abnormal cell proliferation and differentiation during brain development of the epileptogenic/dysplastic areas analyzed (Orlova et al., 2010; Blümcke et al., 2011). However, the epileptogenic/dysplastic areas from patients with longer epilepsy history (>10 years) showed dysmorphic neurons with larger soma size, decreased cell density, and increased reactive gliosis with altered astrocytic morphology when compared to those from patients with shorter epilepsy duration (<10 years).

Therefore, in line with what demonstrated in the MAM-PILO model, these data from patients with FCD provide support to the intriguing hypothesis that epilepsy per se can trigger pathologic plasticity of both neurons and glia exacerbating or inducing cytologic abnormalities within the malformed cortex. In addition to the increased glutamatergic input, our Western blot data demonstrated that the NMDA regulatory subunits 2A and 2B and related membrane-associated guanylate kinase (MAGUK) proteins were consistently altered in all patients with FCD that were examined. Therefore, the increased glutamatergic input and altered NMDA/MAGUK expression are likely major determinants in the epileptogenic mechanisms of human FCD, not only contributing to hyperexcitability but also possibly triggering intracellular mechanisms capable of deeply altering brain structure.

Regarding the correlation between epilepsy duration and morphologic changes, it could be argued that epilepsy severity rather than duration is the parameter of choice to be taken into consideration. To quantify epilepsy severity, however, many different clinical features such as seizure frequency, type, and duration, and degree of interictal EEG epileptiform activity should be taken into account, thus making this retrospective evaluation extremely difficult and not reliable. Because the patients with FCD considered were all characterized by severe epilepsy, with seizures recurring many times per week or even per day, we correlated morphometric changes with epilepsy duration, which was a more limited but unequivocal and easily quantifiable parameter in each patient.

Cell Death in the Dysplastic Cortex

The progressive cortical and hippocampal atrophy of MAM-PILO rats and the reduced neuronal cell density observed in the epileptogenic/dysplastic areas of human FCD patients suggested that in both the experimental model and in human patients, SE and/or severe recurrent seizures could activate cell death pathways. We have therefore investigated cell death pathways in MAM-PILO rats at the different epilepsy stages considered (see earlier text). Fluoro Jade B (FJB) staining confirmed that SE was associated with diffuse labeling of degenerating neurons in the neocortex, hippocampus, and thalamus, as already reported by different groups (Narkilahti et al., 2003; Wang et al., 2008). Notably, we found FJB+ degenerating neurons at all epilepsy stages considered and not only after the acute insult, thus suggesting that cell death was a steadily active process, likely correlated to seizure recurrence. In particular, we found FJB+ neurons in the following: (1) superficial and deep neocortical layers at early stages, and deep layers at 3 and 6 months of chronic epilepsy; (2) CA pyramidal neurons at all stages, and dentate gyrus (hilar neurons only) up to 3 months of chronic epilepsy; and (3) ventrolateral/ventrobasal and intralaminar thalamic nuclei at all stages. At chronic epilepsy stages, some neocortical (entorhinal and perirhinal) and thalamic regions were diffusely necrotic. We have also verified the presence of reactive gliosis during the course of epilepsy. As expected, marked increase in glial fibrillary acidic protein (GFAP) immunoreactivity was found in neocortical, hippocampal, and thalamic regions. In all areas considered, astrocyte morphology changed during the course of epilepsy: larger cell bodies, thicker processes, and eccentric nuclei became evident. Hypertrophic astrocytes, with very large opalescent cytoplasm and eccentric nucleus, possibly representing the final morphologic stage of reactive astrogliosis, were evident in the hippocampus of some MAM-PILO rats. We next verified the activation of intracellular death pathways by means of double labeling confocal immunofluorescence (IF) for activated caspase or c-Jun and neuronal and glial markers. At chronic stages, CA pyramidal neurons were positive for both active caspase 3 and p-c-Jun, whereas neocortical pyramidal neurons (including large hypertrophic pyramidal neurons with neurofilament overexpression) were positive for p-c-Jun only. By contrast, GFAP+ astrocytes were frequently labeled for active caspase 3 but not for p-c-Jun, suggesting that different prodeath pathways were preferentially activated in neurons and glia following SE and seizures. Because GFAP+ astrocytes were never FJB+, the presence of caspase 3 could also signify a nonapoptotic role of caspase 3 in cytoskeletal remodeling leading to astrogliosis (Acarin et al., 2007; Aras et al., 2012). In any case, these results clearly indicated that in the dysplastic cortex not only SE but also chronic seizures might induce both neuronal and glial cell death.

Concluding Remarks

Data from the MAM-PILO rat model and the human surgical FCD samples reveal common features shared by both conditions: (1) SE and/or seizures can further modify the cellular and molecular settings of the malformed cortex; (2) there is imbalance between glutamatergic excitation and inhibition, and dysregulation of the NMDA/MAGUK expression; and (3) SE and seizures can induce cell death by activating specific intracellular pathways in neurons and glia.

Even if further experiments should verify what GABAergic interneurons are selectively loss in MAM-PILO rats (Dinocourt et al., 2003), the comparative analysis of the rat model and human patients highlights the mechanistic relevance of glutamate/NMDA hyperactivation in FCD epileptogenesis. In addition, both sets of data suggest that, within the specific context of the epileptogenic malformed brain, epilepsy can be viewed as a maladaptive process affecting the cellular structure and the molecular function of both neurons and glia.


None of the authors have any conflict of interest to declare.

The authors confirm that they have read the Journal's position on issues involved in ethical publication and affirm that this article is consistent with those guidelines.