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 (17) with respect to gradual progression: (i) rhythmic mouth and facial twitching, (ii) head nodding and wet-dog shakes, (iii) forelimb clonus, (iv) rearing, and (v) loss of balance and falling.

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 (14, 18, 19) were used (Table 1). Tissue sections from the control and NMD brains were collected on probeglass slides (Fisher Scientific, Pittsburg, PA, U.S.A.), and processed for immunohistochemistry by avidin-biotin conjugation method (20). Tissue sections were incubated in 1.5% H₂O₂ in phosphate buffered saline (PBS), treated in 10% normal goat serum in PBS for 30 min in order to block charged sites on the tissue surface, and then incubated with primary antibodies throughout overnight at 4℃. After treatment with 1% avidin-biotinylated horseradish peroxidase (Research Genetics, Huntsville, AL, U.S.A.) for 1 hr at room temperature, the tissue sections were prepared for chromogen reaction with 3-amino-9-ethyl carbazole (AEC, Biomedia, Foster City, CA, U.S.A.). The sections were counter stained with hematoxylin, and examined by a light microscope.

Development of epileptic seizures and EEG abnormalities in NMD and control rats were summarized in Table 2. During the clinical observation and EEG monitoring, only one of 20 rats with NMD presented spontaneous seizures showing staring, mouth and facial twitching, and salivation. However, 17 of 20 rats had seizures after an intraperitoneal KA provocation. During behavioral seizures in NMD rats, the EEG activity was characterized by abnormally amplified rhythmic spike discharges initially developed from the left cortex and then from the left hippocampus (Fig. 1A). In the behavioral manifestations of seizures in NMD rats, 14 rats developed stage 5 seizures (orofacial twitching, wet-dog shakes, forelimb clonus, rearing and falling), and 3 rats developed stage 3 seizures (orofacial twitching, wet-dog shakes and forelimb clonus). Behavioral seizures occurred in NMD rats for 21.3±2.6 min during 5 hr period after the KA provocation, and each seizure lasted 10 to 56 sec (mean 27±6 sec). Two NMD rats showed EEG abnormality without behavioral seizure. In control groups, only one of 5 rats had seizures and the other one showed a mild EEG abnormality after an intraperitoneal KA provocation. The behavioral manifestation of seizures were staring and orofacial twitching (stage 1) in the rat. The seizure occurred for 2.3 min during 2 hr after the KA provocation, and each seizure lasted 5 to 13 sec (mean, 7±2 sec). The seizure spontaneously disappeared at 3 hr after the KA provocation.

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 (Fig. 1B). The affected cortex had nearly one half the thickness of comparable cortex in controls. Cystic changes or hydrocephalus was not noted.

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 (Fig. 1C). Cytomegalic neurons usually have thickened or abnormally aborizing dendrites, and some of them are abnormally oriented (Fig. 1D). Balloon cells found in some human NMD and cortical tubers were not be identified. In the hippocampi, focal disruption of pyramidal neurons in corpus ammonis with neuronal ectopia were seen in six animals. The lesions are developed at area CA1 and CA2 in 4 rats, CA2 and CA3 in 2 rats.

Immunohistochemical expressions of glutamate receptor subunits in the control and NMD cortex are summarized in Table 3. In the control cortex, neurons in layer II/III/IV were diffusely and moderately immunoreactive to NR1 (Fig. 2A) and GluR2, and negative immunoreaction to NR2A/B and GluR2. Neurons in layer V/VI were moderately immunoreactive to NR1 and GluR3 (Fig. 2B), and mildly immunoreactive to NR2A/B and GluR2. NR2A/B presented finely granular or dot-like immunoreactivity in the neuronal cytoplasm. GluR3 immunoreactivity was limited in a few large pyramidal neurons in layer V/VI. Neurons in cortical subplate were mildly immunoreactive to NR1.

In NMD cortex, immunoreactivity for glutamate receptor subunits were demonstrated (Fig. 2C-E). Heterotopic neurons in the layer I and white matter, cytomegalic neurons, and normal-sized dysplastic neurons had strong immunoreactivity to NR1 (Fig. 2C), NR2A/B (Fig. 2D), and GluR2 (Fig. 2E). NR1 and GluR2 were labeled in neuronal cytoplasm and proximal portion of dendrites. NR2A/B showed coarsely granular immunoreactivity in dysplastic neurons. Abnormal neurons in NMD cortex were mildly immunoreactive to GluR3.