Animals and ethosuximide treatment
All procedures were in full compliance with approved institutional animal care and use protocols. We used female WAG/Rij rats bred at our institution, which originated from the Radiobiological Institute, TNO, in Rijswijk (Reinhold, 1966
), and age-matched nonepileptic control female Wistar rats from Charles River Laboratories (Wilmington, MA). These two strains were chosen for the experiments since they are genetically similar, both being Wistar substrains, but the WAG/Rij rats have a much higher rate of SWD than ordinary Wistars. Because even ordinary (unselected) Wistar rats can occasionally show a SWD phenotype (Coenen et al., 1992
; Coenen & Van Luijtelaar, 2003
), the nonepileptic control rats were screened by EEG and if significant SWD occurred, they were eliminated from the experiment. No selection procedure was used for the WAG/Rij rats. Animals were housed in groups of 2 or 3, and kept on a 12-h light/dark cycle (lights on at 07:00 h) with unlimited access to food and water. Following implants of recording electrodes, animals were single housed to prevent injuries.
Ethosuximide (ESX) was administered orally at a dose of 300 mg/kg/day by adding it to the drinking water beginning at the time of weaning (p21–p23). This dosage and route were selected based on prior work, and on initial pilot experiments. In previous rat studies, EEG recordings showed suppression of SWD shortly after single oral or IP doses of ethosuximide ranging from 12.5 to 200 mg/kg (Micheletti et al., 1985
; Vergnes et al., 1985
; Peeters et al., 1988
; Wahle & Frey, 1990
; van Rijn et al., 2004
). However, prior work showed that SWD returned within a few hours after single doses (Micheletti et al., 1985
; Peeters et al., 1988
), and the half-life of ethosuximide is considerably shorter in rats than in humans (Faingold & Browning, 1987
; Mifsud et al., 2001
). Therefore, we added ethosuximide to the drinking water, which was freely available to the rats, in the hope that this would produce a more stable or nearly continuous delivery of the medication. Based on pilot measurements, we determined that the rats drink approximately 120 cc/kg/day, and drink slightly more (~200 cc/kg/day) in the first 1.5 months. Therefore, ethosuximide 300 mg/kg/day was given using 250 mg/5 ml syrup (Pharmaceutical Associates, Inc. Greenville, SC) by adding 3 cc of syrup per 100 cc H2
O for p21 through p45, and 5 cc per 100 cc H2
O for p45 onward. In initial pilot experiments, we tried lower doses of ethosuximide (200 mg/kg/day in three animals) but this did not consistently block SWD. Higher doses (400 mg/kg/day in three animals) caused significant lethargy, hair loss and gait ataxia. The dose of 300 mg/kg/day was chosen because it was effective in completely blocking SWD, and was also well tolerated without any side effects of lethargy, ataxia, hair loss, or reduced food intake. All animals were evaluated for these adverse effects three times weekly throughout the experiments, and none were detected with the 300 mg/kg/day dose.
Water bottles were coated on the outside with black paint since the medication is light-sensitive. Medication in the bottles was replaced at least weekly to ensure that therapeutic doses were continuously available.
There were four groups of animals in the first experiment, and three groups of animals in the second experiment (). The four groups in Experiment 1 () were: nonepileptic (NE) control rats on water (n = 8), NE control rats on ethosuximide 300 mg/kg/day (n = 8), WAG/Rij rats on water (n = 7), and WAG/Rij rats on ethosuximide 300 mg/kg/day (n = 8). In Experiment 1, animals on ethosuximide were treated from weaning (p21) until they were sacrificed at 5 months. The three groups in Experiment 2 () were: WAG/Rij rats on water (n = 13), WAG/Rij rats on ethosuximide 300 mg/kg/day for p21 through age 5 months (ESX 4 months group) (n = 11), and WAG/Rij rats on ethosuximide for p21 through age 8 months (ESX continuous group) (n = 13).
Figure 1 Experimental design for measuring effects of early ethosuximide treatment on ion channel expression (A) and epilepsy (B). (A) Experiment 1: Epileptic WAG/Rij rats and nonepileptic Wistar control rats (NE) were given either normal drinking water (H2O), (more ...)
Serum ethosuximide levels (National Medical Services; Willow Grove, PA) were measured at the time that animals were sacrificed. Oral ethosuximide 300 mg/kg/day produced therapeutic blood levels (88 ± 10 µg/dl; mean ± S.E.M.), and effective seizure blockade without toxic side effects (see above).
Surgery and recordings
At age 4.75 months all animals were implanted with electrodes for EEG recordings. Under ketamine (100 mg/kg), xylazine (10 mg/kg), and acepromazine (1 mg/kg) anesthesia, we implanted tripolar electrodes (Part # MS333/3-A, Tripolar electrode uncut untwisted 0.005, Pedestal Height: 8 mm, Internal control # 8LMS3333XXXE; Plastics One, Inc., Roanoke, VA, U.S.A.) using a stereotactic frame (David Kopf Instruments, Tujunga, CA, U.S.A.). To provide good electrical contact before wrapping around skull screws, the ends of the recording electrodes were prepared by scraping off all the polyimide insulation and exposing stainless steel wire up to 10 mm from the tip, leaving insulation intact proximally, as verified under the microscope. Level of anesthesia was monitored by respiration, heart rate, glabrous skin perfusion, and response to foot pinch. Small burr holes (using Micro Drill Steel Burrs, 2.3 mm shaft diameter, 44 mm overall length; Item # 19007-14, Fine Science Tools, Foster City, CA, U.S.A.) were made in the skull without disturbing the dura and electrodes were secured to the skull using stainless steel screws (Part # 0-80X1/16, Internal control # 8L010121201F, with shaft length = 1.60 mm, head diameter = 2.50 mm, shaft diameter = 1.57 mm; Plastics One, Inc.). EEG recording electrodes were placed at frontal cortex (AP +2.0, ML +2.0 mm), and parietal cortex (AP −6.0, ML +2.0 mm) and a ground electrode was placed in the midline over the cerebellum. An additional anchoring screw, without electrode, was placed at ML −2.5 mm, equal distance between the coronal suture and lamdoidal suture. Dental acrylic (Cat # 1255710; Henry Schein, Inc., Indianapolis, IN, U.S.A.; Lang Jet Denture Repair Acylic) was used to fix the electrode pedestal in place.
Animals were given a 1 week recovery period after surgery. EEG signals were recorded via commutator (Plastics One, Inc.) using a Grass CP 511 amplifier (Grass-Telefactor, Astro Med, Inc., West Warwick, RI). Band pass frequency filter settings were 1–300 Hz. Signals were digitized at a sampling rate of 1 kHz with an NI USB-6008 A/D converter and LabView 7.1 software (National Instruments, Austin, TX), and analyzed using Spike 2 (Cambridge Electronic Design, Cambridge, UK).
Continuous EEG data were recorded from awake-behaving rats between 10:00 a.m. and 4:00 p.m.. For Experiment 1 (), recordings were obtained from each animal for 2 h per day over a 3-day period (6 h total per animal), and animals were then sacrificed for histology. For Experiment 2 (), recordings were obtained from each animal for 3 h per day at the following time intervals in the ESX 4 months group, and age-matched animals in the other two groups: 1 day before stopping ESX; and 1, 14, 30, 60, and 90 days after stopping ESX.
Rats were anesthetized with ketamine/xylazine (80/5 mg/kg i.p.) and then underwent intracardiac perfusion with 0.01 M phosphate buffer solution (PBS) followed by a 4% solution of cold-buffered paraformaldehyde. Brains were postfixed and cryoprotected in 30% sucrose in PBS, and coronal sections (10 µm) of the cerebral hemispheres were cut. Separate serially consecutive slices with identical preparations were used for Nav1.1, Nav1.6, and HCN1 immunostaining. Slices were mounted onto slides and incubated in blocking solution (5% normal goat serum and 1% bovine serum albumin in PBS) containing 0.1% Triton X-100 and 0.02% sodium azide at room temperature for 30 min. Slides were then incubated with subtype-specific antibodies to either sodium channel α-subunits Nav1.1 (residues 465–481, 1:100 dilution, Alomone, Jerusalem, Israel), Nav1.6 (residues 1,042–1,061, 1:100, Alomone), or HCN1 antibody (rabbit anti-rat, 1:100 dilution, Chemicon, Temecula, CA, U.S.A.) overnight at 4 °C. Slides were washed in PBS and then incubated with goat anti-rabbit IgG-Cy3 (1:2,000, Amersham, Piscataway, NJ, U.S.A.). Immunofluorescence signal was detected using fluorescein illumination (emission wavelength 516–565 nm).
Analysis of immunocytochemistry data
Semiquantitative densitometry of immunostaining signals was performed with a Nikon Eclipse TE300 microscope using IPLab v3.0 Image Processing software (Scanalytics, Inc., Fairfax, VA, U.S.A.). The quantification process was done with the experimenter blinded to the identity of the experimental group. Signal intensities were determined by outlining individual cortical pyramidal neurons, and IPLab integrated densitometry functions were used to calculate mean signal intensities for each cell. Results from identical regions and layers of cortex in WAG/Rij (epileptic) rats were compared to nonepileptic Wistar (control) rats processed in parallel. Immunopositivity was quantified by averaging multiple counts within a defined area (1.9 × 104
) within layers II–III of the S1BF somatosensory cortex, where changes in channel expression have been demonstrated previously in epileptic WAG/Rij rats (Klein et al., 2004
). Only neurons with distinct borders whose nuclei fell within the plane of section were analyzed. Approximately 50 neurons were analyzed for each antibody (Nav1.1, Nav1.6, and HCN1) per animal (150 cells total per animal). Analysis of neurons from left versus right somatosensory cortex showed no significant difference in level of expression within each group of rats, so these data were combined. Mean immunofluorescence of neurons with each antibody from each set of animals was compared using one-way ANOVA with post hoc Bonferroni adjustment for multiple comparisons. All statistical analyses were run in SPSS (v. 14, SPSS, Inc., Chicago, IL, U.S.A.). An alpha level of 0.05 was used as a threshold for statistical significance. All data are presented as mean ± S.E.M.
Analysis of EEG data
SWDs were defined as large-amplitude (>2× the background EEG peak-to-peak amplitude) rhythmic 7–8 Hz discharges with typical spike-wave morphology lasting >1.0 s. Intervals containing artifact or slow wave sleep were excluded from the analysis. Start and end times for all SWDs were marked. Number of seizures, and seizure durations were then calculated. Percent time in SWD was determined as (sum of SWD interval durations/ total usable recording time) × 100%. The readers who analyzed the EEG files were blinded to the type of rat and treatment (water, ESX 4 months, or ESX continuous).
Power spectral analysis was performed for marked SWD intervals, as well as for all EEG data included in the above analysis. Power spectra were calculated for all animals and groups using Spike2 software, with scripts provided by Cambridge Electronic Design (Cambridge, U.K.), as well as in house programming in MATLAB 7.3 (MathWorks, Natick, MA, U.S.A.). Bin size for the FFT was 1.024 s.
Statistical analyses were run in SPSS (v. 14, SPSS, Inc.) using one-way ANOVA or MANOVA for repeated measures with Wilks’ Lambda multivariate analysis, followed by post hoc analyses if appropriate. As with the immunocytochemistry data, an alpha level of 0.05 was used as a threshold for statistical significance.