Labchart pro v7

Three days after electrode implantation, baseline seizure activity was determined over 3 days. Thereafter, perampanel (2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)benzonitrile, a non-competitive AMPAR antagonist; 8 mg/kg, i.p, Eisai Korea Inc., Seoul, South Korea), GYKI 52,466 (a non-competitive AMPAR inhibitor, 10 mg/kg, i.p.) or saline (vehicle) was daily administered at a certain time of the day (PM 6:00) over a 1-week period [9 (link),54 (link)]. On the basis of previous studies [55 (link),56 (link)], some animals were also given BpV(pic) (2 mg/kg, i.p.) with perampanel or GYKI 52466. EEG was recorded 2 h a day at the same time over a 1-week period. EEG signals were recorded with a DAM 80 differential amplifier (0.1–3000 Hz bandpass; World Precision Instruments, Sarasota, FL, United States) and the data were digitized (1000 Hz) and analyzed using LabChart Pro v7 (ADInstruments, Bella Vista, New South Wales, Australia). Behavioral seizure severity was also evaluated as aforementioned. After recording (18 h after the last treatment), animals were used for Western blot and immunohistochemical study.

Three days after electrode implantation, baseline seizure activity was measured over 3 days. Thereafter, perampanel (2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)benzonitrile; 8 mg/kg, i.p., Eisai Korea Inc., Seoul, Korea), GYKI 52,466 (10 mg/kg, i.p.), or saline (vehicle) was administered daily at PM 6:00 over a 7-day period [31 (link),35 (link),72 (link)]. To select the responders and non-responders, and each dose was chosen as the maximum without adverse effects, based on previous studies [33 (link),34 (link),42 (link),55 (link)]. EEG were detected with a DAM 80 differential amplifier (0.1–3000 Hz bandpass; World Precision Instruments, Sarasota, FL, USA) 2 h a day at the same time over a 7-day period. The data were digitized (1000 Hz) and analyzed using LabChart Pro v7 (ADInstruments, Bella Vista, New South Wales, Australia). Behavioral seizure severity was also evaluated according to Racine’s scale aforementioned. Non-responders were defined as showing no reduction in total seizure occurrence in a 7-day period, as compared with the pre-treatment stage. After recording (18 h after the last treatment), animals were used for Western blot.

Rats were injected intraperitoneally with LiCl (127 mg/kg) 24 h prior to the administration of pilocarpine (30 mg/kg). Twenty minutes before pilocarpine injection, animals were given atropine methylbromide (5 mg/kg) to block the peripheral effect of pilocarpine. SE induction was stopped 2 h after pilocarpine injection by the administration of diazepam (10 mg/kg, i.p.). Diazepam was repeatedly administered as needed. To validate the effect of EGCG or NU335 on seizure susceptibility induced by pilocarpine, electroencephalogram (EEG) signals of electrode-implanted animals were measured with a DAM 80 differential amplifier (0.1–3000 Hz bandpass; World Precision Instruments, Sarasota, FL, USA). EEG activity was measured (2 h recording session), digitized (400 Hz), and analyzed using LabChart Pro v7 (AD Instruments, New South Wales, Australia). The time of seizure onset was defined as the time point showing paroxysmal discharges (4–10 Hz with 2 times higher amplitude than the basal level) that lasted more than 3 seconds. Spectrograms were also automatically calibrated using a Hanning sliding window with 50% overlap [21 (link),22 (link),23 (link)].

SE was induced by a single dose (30 mg/kg) of pilocarpine in rats pretreated (24 h before pilocarpine injection) with 127 mg/kg LiCl, as previously described [8 (link),9 (link),17 (link),18 (link)]. Rats were pretreated with atropine methyl bromide (5 mg/kg i.p.) prior to pilocarpine injection. Two hours after SE, animals were administered diazepam (10 mg/kg, i.p.) to cease behavioral seizures and repeated as needed. As controls, rats were treated with atropine methyl bromide, followed by saline, but not pilocarpine. EEG signals of electrode-implanted animals were recorded with a DAM 80 differential amplifier (0.1–3000 Hz bandpass; World Precision Instruments, Sarasota, FL, USA) during the 2 h recording session for each animal. The EEG signals were digitized and analyzed using LabChart Pro v7 (AD Instruments, New South Wales, Australia). Time of seizure onset was determined when rhythmic paroxysmal depolarizations (4–10 Hz) lasting >3 s with 2 times higher amplitudes than the basal EEG were detected. Two hours after SE onset, diazepam (Valium; Roche, France; 10 mg/kg, i.p.) was administered and repeated as needed.

We applied a modified protocol for the effect of PDI knockdown on spontaneous seizure activity in chronic epileptic rats based on Ko and Kang and Glien et al.53 (link)55 (link). After baseline seizure activity (control siRNA treatment) was determined over 2 days, PDI siRNA was administered over a 7-day period using an osmotic pump (1007D, Alzet, Cupertino, CA, USA). Between trials, the minipump was changed out for another minipump filled with another mixture under isoflurane anesthesia. Every day during the experiment, spontaneous seizure activity was recorded by video-EEG monitoring with 2 h of recording per day at the same time. EEG analysis was performed by LabChart Pro v7 (AD Instruments, NSW, Australia). Behavioral seizure severity was also evaluated according to Racine’s scale56 (link): 1, immobility, eye closure, twitching of vibrissae, sniffing, facial clonus; 2, head nodding associated with more severe facial clonus; 3, clonus of one forelimb; 4, rearing, often accompanied by bilateral forelimb clonus; and 5, rearing with loss of balance and falling accompanied by generalized clonic seizures.

Two days after surgery, rats were given 127 mg/kg LiCl. Twenty-four hours after LiCl treatment, SE was induced by a single dose (30 mg/kg) of pilocarpine. To block the peripheral effect of pilocarpine, atropine methylbromide (5 mg/kg i.p.) was injected into animals 20 min prior to pilocarpine injection. As controls, rats were treated with saline instead of pilocarpine. In electrode-implanted animals, EEG signals were recorded with a DAM 80 differential amplifier (0.1–3000 Hz bandpass, World Precision Instruments, Sarasota, TL, USA), digitized (sampling rates, 1000 Hz), and analyzed using LabChart Pro v7 (AD Instruments, Bella Vista, NSW, Australia). Total EEG power and spectrograms were automatically calculated in 2-hour recording session using a Hanning sliding window with 50% overlap. Two hours after SE, animals received diazepam (Valium; Roche, France; 10 mg/kg, i.p.) to terminate SE.