*Shin-ichi Yoshioka *Division of Neuropsychiatry, Faculty of Medicine, Tottori University, Yonago, Japan .
Purpose: In humans, prenatal or postnatal brain insults such as hyperthermia, x-ray irradiation, malnutrition, or exposure to toxic substances may produce brain defects. These brain defects result in an increased postnatal seizure susceptibility. Methotrexate (MTX), a folic acid antagonist, interferes with DNA synthesis and is teratogenic or embryotoxic in humans as well as in experimental animals. In addition, MTX exposure during pregnancy is known to result in brain malformations. The aim of this study was to determine whether in utero exposure to MTX in rats modifies the development of amygdaloid kindling at immature ages and the persistence of seizure susceptibility during development.
Methods: Pregnant Wistar rats received a single intraperitoneal injection of MTX (5 mg/kg) dissolved in saline or 1 ml/kg of vehicle at gestational day 15 (day 15 of embryonic development). On day 12 after birth (postnatal day 12; PN12), pup rats were stereotaxically implanted with a twisted bipolar electrode into the right amygdala under pentobarbital (PTB) anesthesia. A reference electrode was placed in the frontal sinus. On PN 14, after the determination of the afterdischarge threshold (ADT), the rats received electrical stimulation at an ADT level of 60 Hz sine wave for 1 s once every 30 min until the appearance of five stage 5 seizures. Subsequently, the electrodes were removed, and the rats were allowed to grow. On PN 63, bipolar electrodes were again implanted into the ipsilateral amygdala under PTB anesthesia. A reference electrode was placed in the frontal bone. According to whether 70-day-old rats were given MTX treatment and/or initial kindling on PN 14, four groups were designated as vehicle-treatment plus kindling group (CC), MTX-treatment plus kindling group (MC), vehicle-treatment plus rekindling group (CK), and MTX-treatment plus rekindling group (MK). After the determination of the ADT on PN 70, the rats received the same electrical stimulation at the ADT level for 1 s once daily until the appearance of five stage 5 seizures. The behavioral seizure stages of amygdaloid kindling were assessed by using the classification proposed by Moshé for immature rats and by Racine for adult rats. Electrographic recording in the right amygdala and cortex also was performed before and during kindling stimulation and the duration of the afterdischarge (AD) elicited at each stimulation was noted.
Results: In the 14-day-old pup rats, prenatal MTX treatment decreased significantly the number of stimulations required to reach the first stage 5 seizure compared with vehicle-treated rats. In addition, the cumulative AD durations elicited up to the tenth kindling stimulation after the initial stimulation (cumulative × 10 ADDs) were significantly shorter in MTX-treated rats than in control rats. When the rats were kindled on PN 70, no significant difference was found in the number of stimulations needed to induce the first stage 5 seizure between the CC and the CK groups. However, the number of stimulations required to induce the first stage 5 seizure was significantly lower in the MK group than in the CC and CK groups. Additionally, the cumulative ×10 ADDs was significantly longer in the MK group than in the CC group.
Conclusions: These results suggest that the susceptibility to kindling-induced seizure in immature rats is enhanced by in utero exposure to MTX, and that the enhanced seizure susceptibility acquired during the immature age persists into maturity.