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Neuronal migration disorders (NMDs) constitute the main pathologic substrate of medically intractable epilepsy in human. This study is designed to investigate the changes in expression of glutamate receptor subtypes on radiation-induced NMD in rats. The lesion was produced by intrauterine irradiation (240 cGy) on E17 rats, and then 10 weeks old rats were used for the study. The pathologic and immunohistochemical findings for glutamate receptor subunit proteins on NMD cortex were correlated with development of behavioral seizures and EEG abnormality. Spontaneous seizures uncommonly occurred in NMD rats (5%); however, clinical stages of seizures were significantly increased in NMD rats by an administration of kainic acid. Brains taken from irradiated rats revealed gross and histopathologic features of NMD. Focal cortical dysplasia was identified by histopathology and immunohistochemistry with neurofilament protein (NF-M/H). Significantly strong NR1 and NR2A/B immunoreactivities were demonstrated in cytomegalic and heterotopic neurons of NMD rats. The results of the present study indicate that epileptogenesis of NMD might be caused by upregulation of glutamate receptor expression in dysplastic neurons of the rat cerebral cortex with NMDs.
Previous studies have reported that the main pathological features in temporal lobe epilepsy (TLE) are Ammon's horn sclerosis (AHS) or hippocampal sclerosis (HS), neoplasia and glioneuronal malformations (
NMD lesions, including cortical dysplasia (CD) or microdysgenesis, are caused by impeded normal neurogenesis and migration of neurons during the fetal brain development (
Animal models of NMD have been generated in rodents by intrauterine exposure to methylazoxymethanol acetate (MAM), ionizing radiation, or ethanol, and prenatal cortical freezing (
A recent study has demonstrated that NMDAR2 subunit protein is selectively expressed in the dysplastic neurons of the human epileptic neocortex (
Wistar rats were housed and maintained according to the animal care guide lines of the Research Institute of Medical Science, Chonnam National University. Age-matched pregnant rats, 20 weeks old, were exposed to 240 cGy of external radiography irradiation from a linear accelerator (Mevatron 7600, Siemens, U.S.A.) on gestational day 17 (E17). Gestational day 1 (E1) was counted when insemination was detected by vaginal smear. Newborn rats were obtained from the irradiated mother rats, and they were maintained carefully. Ten weeks old rats were used in this study. There were four experimental groups consisting of control (n=10) and NMD rats (n=30) for clinical, histopathological and immunohistochemical study.
For the clinical observation of seizure development and EEG monitoring, a stereotaxic operation was carried out from control (n=5) and NMD rats (n=20). Nine weeks old rats were anesthetized with intraperitoneal injection of sodium phenobarbital (50 mg/kg), and fixed in an animal stereotaxic frame (David Kopf, Tujunga, CA, U.S.A.). Bipolar electrodes were placed in the left frontal cortex and hippocampus. Electrodes were fixed on scalp with dental cement and connected to an electrode socket. The rats were let free for a week to recover from the operation. For standard EEG recording, the electrode socket were connected via freely moving interface cables to an EEG monitor. Development of spontaneous seizure or seizure induced by an intraperitoneal injection (1 mg/kg) of kainic acid (KA, Nacalitesque, Kyoto, Japan) was monitored continuously for 5 hr in each rat. The clinical seizures were rated on 5 stages (
Ten weeks old rats, control (n=5) and NMD (n=10) groups, were anesthetized with intraperitoneal injection of sodium phenobarbital (50 mg/kg), and then they were perfused intracardially with phosphate-buffered saline (PBS, pH 7.40). The brains were removed, and fixed in buffered formalin for 48 hr. Serial coronal sections, 3 mm thick, were made from the formalin-fixed brains, and gross abnormalites were described. Then, the brain sections were embedded in paraffin, and cut with a microtome into 6 m thick tissue sections.
For histopathologic evaluation, the tissue sections were stained with hematoxylin and eosin (H&E). Bielschowsky silver stain and immunohistochemical stains for glial fibrillary acidic protein (GFAP, Dako, Glostrup, Denmark), neurofilament protein (NF-M/M, Sternberger-Mannheim, Luthervill, MD, U.S.A.), microtubule-associated protein 2 (MAP2, Boehringer-Mannheim, Germany), vimentin (Dako, Glostrup, Denmark) were performed in selected cases. For the investigation of glutamate receptor changes in human and experimental rat NMDs, a series of antibodies for glutamate receptor subunits (
Development of epileptic seizures and EEG abnormalities in NMD and control rats were summarized in
Coronal sections of the brain in control group showed well defined gray and white matter of the cerebral cortex, and intact corpus callosum. NMD group that had been irradiated on E17 consistently showed microcephaly, agenesis of corpus callosum, thinning of the gray matter of cerebral cortex, and ill-defined border of the gray and white matter (
Histologically, control brains showed 4 layers of the gray matter consisting of I, II/III/IV, V/VI layers of human-equivalent, and cortical subplate layer. The laminations were relatively well identified on ×40 magnification field by light microscopic examinations. In irradiated brains, histopathologic features of NMD were noted. NMD in the experimental animals was characterized by loss of normal lamination of the gray matter, neuronal heterotopia in the layer I and white matter, and presence of cytomegalic neurons. Nodular clusters of normal-sized cytomegalic neurons were seen in the layer I and subpial surface. Neurons were dyslaminated randomly throughout the gray matter of the neocotex. Nodular clusters of cytomegalic neurons in the cortex were noted in seven animals, and the lesion was more clearly identified by immunohistochemical stain for neurofilament protein (
Immunohistochemical expressions of glutamate receptor subunits in the control and NMD cortex are summarized in
In NMD cortex, immunoreactivity for glutamate receptor subunits were demonstrated (
The experimental cerebral cortical malformation produced by an intrauterine irradiation was initially studied in an attempt to understand cortical development (
In the present study, spontaneous clinical seizures associated with epileptiform EEG activity was observed in only one out of 20 NMD rats (5%). However, most of NMD rats (90%) developed clinical seizures after small dose of KA provocation. Clinical stage, duration, and frequency of seizures were significantly increased in NMD rats than controls. These clinical data suggested that NMD might cause epileptic seizures by way of an intrinsic epileptogenecity or decreasing the threshold to excitatory neurotransmitter-induced seizures. Experimental and clinical studies suggest that NMD could increase seizure susceptibility (
Brains from adult animals that have been irradiated on E16 or E17 show five major gross and microscopic abnormalities consisting of microcephaly, diffuse cortical dysplasia, neuronal heterotopia, focal areas of ectopic neurons in the hippocampus, and agenesis or severe hypoplasia of the corpus callosum (
In the present study, brains from 10 weeks old rats with irradiation on E17 also show gross and microscopic abnormalities. Nodular clusters of neurons in layer I up to the pial surface and the periventricular white matter are commonly seen. Focal cortical dysplasia, which is similar to grade III of NMD in human, is consistently observed in the neocortex. The focal lesion is hardly identified by gross examination, but relatively well defined by light microscopic examinations of routine H&E, or silver-stained tissue slides. Immunohistochemical stains for neurofilament proteins and MAP2 are helpful to find abnormally dense stained lesion of focal cortical dysplasia. The focal lesion is easily identified on ×2-×40 magnification field of light microscope. Cytomegalic or normal-sized neurons bearing abnormal neurofilament proteins in their cytoplasm and dendrites are frequently disoriented. Balloon cells are not noted in these animals.
Recent studies have demonstrated that NMDAR plays an important role in hyperexcitability associated with human or some animal models of human epilepsy (
In the present study, cerebral cortex associated NMD showed significantly increased expression of NR2A/B subunit proteins by immunohistochemistry, while control cortex showed positive reaction in a few large pyramidal neurons in the deep gray matter (layer V/VI of human cortex). NR1, GluR2, and GluR3 showed slightly increased immunoreactivities in NMD cortex than control. These results strongly suggest that NMDA receptors composed of both NR1 and NR2A/B are also increased in the NMD lesions of experimental rats. The intracellular mechanisms by which NR2A/B increases in the abnormal neurons of NMD are still unknown. NMD results from disturbances of normal development of cerebral cortex, which consists of neuroblast proliferation, neuronal migration and differentiation, and manifests in an aberrant synaptic connection, and an altered regulation of intracytoplasmic organelles (
This study was supported by a grant of Korea Health 21 R&D project (02-PJ1-PG3-21305-0001), Ministry of Health & Welfare, Republic of Korea.
Pathophysiologic features in a rat with neuronal migration disorder (NMD). EEG from an NMD rat with kainic acid provocation (1 mg/kg, ip) shows abnormal rhythmic spikes initially developed from the cortex, and then from the hippocampus (
Immunohistochemistry for glutamate receptor subunit proteins in the control and NMD cortex. Control cortex shows diffuse and moderate expression of NR1 in the neurons throughout the gray matter (
Primary antibodies for glutamate receptor subunit proteins
Clinical features of control and radiation-induced NMD rats
*Clinical stage of seizure (see reference 21). †intraperitoneal injection of kainic acid (1 mg/kg). ‡Duration of total seizures and each seizure were checked for 5 hr after kainic acid provocation.
Immunohistochemical findings of glutamate receptor subunits in the control and NMD rats
-; negative, +; mild, ++; moderate, +++; strong immunoreactivity, CxS; cortical subplate, NMD; neuronal migration disorder.
*; granular or dot-like immunoreactivity, †; limited in a few large pyramidal neurons.