PMID: 23603260 DOI: 10.1016/j.neulet.2013.04.008
2. Materials and methods
All animal procedures were in compliance with the NIH Guide for the Use of Laboratory Animals and approved by the Animal Care and Use Committee of Sun Yat-sen University. Seven-day-old (P7) Sprague-Dawley rat pups (Guangdong Medical Laboratory Animal Co, China) with body weight at 16 ± 3 g were exposed to 1.1% isoflurane (about 0.5 MAC in P7 rats [22]) for 4 h to induce neuronal apoptosis, or to air in a temperature-controlled chamber as we described before [21]. The concentrations of anesthetic gas, oxygen and carbon dioxide (CO2) in the chamber were measured by a gas analyzer (Datex-Ohmeda, Madison, WI).

Four doses of SP600125 (Selleck Chemicals LLC, Houston, TX, USA) (5, 10, 20 or 30 μg) or 12% dimethyl sulfoxide (DMSO) as the vehicle were administered by intracerebroventricular (i.c.v.) injection 15 min before isoflurane exposure. Some rats received 30 μg SP600125 or 12% DMSO only. The injection was performed as described before [6] under isoflurane anesthesia with a 5 μl microsyringe and 0.4 mm external diameter needle. The location of injection was 2.0 mm rostral, 1.5 mm lateral to the lambda and 2.0 mm deep to the skull surface of rats. The injection solution of 5 μl/rat was infused at a constant rate of 2.5 μl/min. The accuracy of i.c.v. injection was verified by methylene blue in our preliminary experiments. All animals were sacrificed 6 h after termination of gas exposure and their hippocampi were used for Western blotting (n = 6) or TdT-mediated dUTP nick end labeling (TUNEL) with fluorescent dye (n = 6).

For Western blotting studies, rat pups were anaesthetized with isoflurane and then sacrificed by decapitation. Hippocampi of rats were isolated immediately on ice and then stored at −80 °C until used. Western blotting was performed as we have described previously [20]. In brief, the protein concentrations of samples were determined using the BCA protein assay (Bio-Rad,Herts, UK). Sixty micrograms of each sample were subjected to Western blot analysis using the following primary antibodies: anti-cleaved caspase-3 at 1:2000 dilution, anti-phospho-JNK at 1:2000 dilution, anti-JNK at 1:2000 dilution, anti-phospho-c-Jun at 1:1000 dilution, anti-phospho-Akt (Ser 473) at 1:2000 dilution, anti-Akt at 1:5000 dilution, anti- phospho-GSK-3β (Ser 9) at 1:2000 dilution, anti-GSK-3β at 1:2000 dilution, anti-Bcl-xL at 1:2000 dilution and anti-β-actin at 1:2000 dilution. All antibodies were purchased from Cell Signaling Technology Company, USA. Images were scanned by an Image Master II scanner (GE Healthcare) and were analyzed using Image Quant TL software (v2003.03, GE Healthcare). The band signals of phospho-JNK, phospho-Akt and phospho-GSK-3β were normalized to their total JNK, Akt and GSK-3β from the same samples. The band signals of other interesting proteins were normalized to those of β-actin and the results in each group were normalized to that of corresponding control group.

For TUNEL studies, rat pups were anaesthetized with isoflurane and perfused transcardially with 4% paraformaldehyde. Their brains were paraffin embedded and sectioned at 6 μm thickness. As we described before [19], four or five sections (200 μm apart) for each animal at the same plane of the hippocampus were chosen for detecting apoptosis using TUNEL fluorescent method (Promega, Madision, WI, USA). The slides were protected from direct light during experiment. Hoechst was used to stain nuclei. The TUNEL positive cells in CA1, CA3 and dentate gyrus (DG) areas of hippocampus were analyzed immediately with NIS-Elements BR imaging processing and analysis software (Nikon Corporation, Japan). The densities of the TUNEL positive cells in CA1, CA3 and DG were calculated by dividing the number of TUNEL positive cells by the area of that brain region.

Data are presented in mean ± SEM. The Graphpad Prism 4.0 software was used to conduct the statistical analyses. A two-tailed P value of less than 0.05 was considered statistically significant. One way ANOVA with Newman–Keuls Multiple Comparison Test was used when data was normally distributed and had equal variances. Otherwise, non-parametric test with Dunn's Multiple Comparisons was used to compare the density of TUNEL positive cells as well as the relative protein abundance data among groups in Western blots.