Briefly, the following surgery and instrumentation was completed with sterile technique: 1) Precordial silver wire electrocardiogram (ECG) electrodes were implanted subcutaneously. 2) An aortic catheter (28-gauge Teflon) was inserted via the left carotid artery to record arterial blood pressure. 3) A 0.9-mm Renathane catheter was threaded into the left jugular vein to administer parental solutions. 4) Three subcutaneous EEG needle electrodes were placed into the skull above the right and left temporal lobes, and a third reference electrode was placed in the neck region. 5) Temperature was measured by a vaginal thermistor and servo-regulated at 37°C. 6) A silicone cannula was inserted in the urethra to continuously record urine output. During surgery or any possible irritating manipulation, the anesthetic isoflurane level was at >1.5%, which maintained the following states: 1) the electroencephalogram (EEG) was synchronized and dominated by high-voltage slow-wave activity; 2) mean arterial pressure was 100 mmHg, the heart rate, 420 beats/min, and 3) there were no evident EEG, blood pressure or heart rate responses to surgical manipulation. Isoflurane was delivered into the inspiratory gas stream by a precision mass-flow controller. After the initial surgery, Isoflurane was gradually lowered (over 1-2 days) and maintained at <0.5% during the remaining experimental period. Rats were ventilated through a per os coaxial tracheal cannula at 72 breaths/min with an inspiratory and expiratory ratio of 1:2 and a minute volume of 180–200 ml and gas concentrations of 50% O2, 47% N2, and 3% CO2, delivered by a precision volumetric respirator. Intermittent respiratory hyperinflations (6 per hour at 15 cmH2O), positive end-expiratory pressure (1.5 cmH2O), and expiratory CO2 monitoring were continuous. Neuromuscular blockade (NMBA) was induced on the first day (100 µg iv. alpha-cobrotoxin) and maintained by continuous infusion (250 µg/day, iv). Mechanical ventilation was initiated immediately after the NMBA induction.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted according to the standard Affymetrix protocol including Trizol extraction and Rneasy column cleanup
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 3 g total RNA (Expression Analysis Technical Manual, 2007, Affymetrix).
Hybridization protocol
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on Affymetrix Rat Gene 1.0 ST Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the GeneChip Scanner 3000.
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 trimmed mean target intensity of each array was arbitrarily set to 800. The RMA algorithm was then used to generate the Sample data table values.
