Pilocarpine Model of Epilepsy (Dingledine and Coulter): Sprague-Dawley rats (180-200g) were purchased from Charles-River (Raleigh, NC). All animals were given an injection of methylscopolamine (1mg/kg, i.p., Sigma) to minimize cholinergic peripheral effects of pilocarpine. After 30 minutes, animals received pilocarpine hydrochloride (300-350 mg/kg, s.c., Sigma) or saline. Seizures were classified according to Racine (1972) and Schauwecker and Steward (1997) with slight modifications (Borges et al., 2003). SE was defined by continuous seizure activity consisting of at least stage 3 seizures and one stage 5 or 6 seizure or several stage 4 seizures. SE was terminated with pentobarbital (25 mg/kg, i.p., Sigma) after 90 min of SE activity. Animals were monitored daily and given dextrose in lactate Ringer solution (~1mL, i.p.) when needed. Animals were euthanized by decapitation. The brains were rapidly removed, bisected down the midline. One half of the brain was placed in 4% paraformaldehyde and stored at 5°C until shipped. The other half was frozen on dry ice and stored at -70°C to -80°C until shipped. Kainate Model of Epilepsy (Wadman and Nadler): Sprague Dawley rats (175-250g) were purchased from Charles-River, Raleigh (Nadler group) or Harlan, Netherlands (Wadman group). All animals were injected with kainic acid (5mg/kg, i.p.) or saline hourly until status epilepticus was achieved. The Wadman group used an initial intraperitoneal injection of 7.5 mg/kg, followed by hourly 5mg/kg until SE induction. All animals were given 5-6 injections before SE onset. The Nadler group allowed SE to self-terminate after 6-8 hours, while the Wadman group stopped seizure activity with pentobarbital 90 min after SE onset. SE onset was defined by continuous stage 4 and stage 5 seizure activity for 3 hours. Animals were euthanized by decapitation. The brains were rapidly removed and bisected down the midline. One half of the brain was placed in 4% paraformaldehyde and stored at 5°C until shipped. The other half was frozen on dry ice and stored at -70°C to -80°C until shipped. Self-Sustained Status Epilepticus Model of Epilepsy (Wasterlain): Male Sprague-Dawley rats were purchased from Charles River, Raleigh at 280-300g. The animals were implanted with a stimulating electrode in the angular bundle in reference to lambda (AP +0.5mm, ML +4.5mm--left side, DV -4.0mm from surface of brain). Electrodes were positioned under isoflurane anesthesia (4% in O2), and animals were treated for post-operative pain with buprenorphine for three days following surgery. They were allowed to recover for 14 days before initiating SE. EEG was recorded from skull screws located over the dorsal hippocampi. SE was initiated by 30 minutes of perforant path stimulation. The stimulation wave form consisted of 10 second trains of 20hz biphasic square waves delivered at 1 train per minute superimposed on continuous 2 hz monophasic square waves. All square waves were 1 millisecond in duration. Animals received no treatment following stimulation of the perforant path and were included in the study only if they remained in SE for at least 10 minutes after the end of perforant path stimulation. Control animals were implanted but not stimulated. The experimental animals were euthanized at 1 day, 3 days, and 10 days after initiating SE. Animals were euthanized by isoflurane anesthesia followed by decapitation. The brains were rapidly removed, bisected down the midline. One half of the brain was placed in 4% paraformaldehyde and stored at 5°C until shipped. The other half was frozen on dry ice and stored at -70°C until shipped. All samples were shipped to the Dingledine laboratory. The fixed hemispheres were sent to Robert Slovitor for Nissl histology, and the frozen hemispheres of the pilocarpine and kainate models were sent to Duke University (Pate Skene) for laser capture microscopy of dentate granule cells. Granule cells from the SSSE model were captured at Emory University.
Growth protocol
Male Sprague-Dawley rats were used. Individually housed animals were handled and allowed to acclimate for 1 week prior to experiments with food and water available ad libitum. All animals weighed 175-250g at the time of experimentation.
Extracted molecule
total RNA
Extraction protocol
LCM and RNA Isolation: Ten micron coronal sections were taken through the hippocampus and collected onto uncoated microscope slides, refrozen on dry ice, and stored in a -80°C freezer until staining for LCM. For staining, sections were fixed in ice-cold 70% ethanol for 2 min, rinsed in water, dipped in cresyl violet stain, then rinsed and dehydrated to xylene. Sections were dried in a fume hood and LCM was performed within 1 hour. LCM was performed using an Arcturus Pixcell IIe system with transmission illumination (Arcturus, CA) and the following parameters: spot size = 25-30μm; power = 50-65mW; and duration = 1700-2500μs per hit. The dentate gyrus was harvested from 10-16 sections per subject in order to get at least 10ng total RNA for amplification. LCM HS Caps (Arcturus) were used to collect dentate granule cells with 2-5 sections per cap, resulting in 2-8 LCM Caps used per subject. Total RNA was immediately extracted from each LCM cap using the PicoPure Isolation Kit protocol (Arcturus) with DNase digestion (Quiagen Rnase-free DNase Set), and stored at -80°C until RNA quality verification and quantification using Agilent RNA 6000 PicoChips. Pico Kit instructions were followed to analyze the RNA isolations from each subject, and the PicoChip was run on the Agilent 2100 Bioanalyzer. The results of the PicoChip determined whether the RNA was of high enough quality to keep for amplification and later hybridization. Only total RNA with a RNA Integrity Number (RIN) > 7.0 was kept to include in a pool of total RNA from each LCM cap per subject in order to obtain one sample with a minimum of 10ng total RNA. Once a total RNA pool was complete for each subject, the sample was run through the BioAnalyzer, quantity and quality recorded, and samples were frozen at -80°C until shipment to TGEN (Phoenix, AZ, USA) for RNA amplification and microarray hybridization.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol
Hybridization protocol
10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Rat Genome 230 2.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Scan protocol
GeneChips were scanned using the GeneArray Scanner G2500A.
Data processing
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The mean target intensity of each array was set to 150.
