PMID: 25099925 PMCID: PMC4169313 DOI: 10.1213/ANE.0000000000000380
Methods
Animals
The experimental procedures and protocols used in this study were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania. All efforts were made to minimize the number of animals used and their suffering. Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were housed with a 12-hour light-dark cycle at 22°C, with food and water provided ad libitum. Thirty-eight postnatal day 7 (P7) rats were used for the ELISA and Western blots and 11 for immunohistochemistry, with approximately equal numbers of male and female rat pups randomly assigned to each condition.

Anesthesia exposure
The groups of rats were exposed to treatments in parallel. The minimum number of rats in each group was determined by a power analysis. We anticipated a large effect size would be clinically significant and chose an effect size of 1.3, and using the desired statistical level of 0.8 and probability level of 0.05, determined a minimum sample size per group (2-tailed hypothesis) of 11 animals. P7 rats were placed in plexiglass chambers resting in a 37°C water bath to maintain a constant environmental temperature. The rat pups were exposed in these chambers to carrier gas (30% oxygen balanced in nitrogen) for 30 min and then 1.5% ISO for 6 h the following day (1.5% ISO), or preconditioned (PC) with a 30 min 1.5% ISO exposure and then exposed to 1.5% ISO for 6 h the following day (PC + 1.5% ISO). The control animals were exposed to carrier gas (30% oxygen balanced in nitrogen) for 30 min and then carrier gas again for 6 h the following day in the plexiglass chambers but not in the water bath. Exposure to ISO for 30 min alone at P7 has been shown not to be detrimental12 and thus this control group was not included. In order to maintain a steady state of anesthetic gas and to prevent accumulation of expired carbon dioxide within the chamber, we used 6 liters of total gas flow throughout the experiments. The ISO, oxygen and carbon dioxide levels in the chamber were monitored using IR absorbance (Ohmeda 5330, Datex-Ohmeda, Louisville, CO) as described in our previous studies.4,15,24 Two rats died during exposure to 1.5% ISO for 6 hrs, 1 from the ISO alone group and the other from the PC plus ISO group.

Determination of plasma S100β
Two hours after the completion of the anesthetic treatment, P7 rats from the control, 1.5% ISO and PC+1.5%ISO groups were deeply anesthetized with 2–3% ISO. Blood (0.1 ml) was collected from the left ventricle and centrifuged to separate the plasma. We measured levels of S100β, a neuronal injury marker, using Sangtec 100 ELISA kits (DiaSorinInc, Stillwater, MN) following the manufacturer’s protocol and as we described previously.25 Briefly, 50 µl of plasma from each rat was placed in each well of a 96-well-plate and mixed with 150 µl of tracer from the kit, and incubated for 2 hours. Afterwards, 3,3’,5,5’tetramethylbenzidine substrate and stop solution were added to each well. The optical density was read at 450 nm. The sensitivity was determined by plotting the standard curve and then measuring concentrations of the samples from the standard curve.

Western Blot Assays
Western blots were performed as we described previously.15,24 Two hours after the ISO exposure, after the mice were anesthetized and blood samples collected from the heart (see above), the mice were perfused with ice-cold saline through the heart and the parietal cortex dissected, frozen in liquid nitrogen and stored at −80. At the time of the assay, the brain tissue from the P7 rat cortical tissue was thawed and homogenized and the total protein concentrations were quantified. The proteins were then separated by 12% gel electrophoresis and were transferred to a nitrocellulose membrane. The blots were incubated with an antibody against cleaved caspase-3 (Cell Signaling #9664), caspase-12 (Cell Signaling #2202), or Beclin-1 (Cell Signaling #3495). The density was measured by Quantity One software (BIO-RAD version 4.5.0) and GS-800 Densitometer (BIO-RAD, Hercules, CA) and the data are expressed as the percent of control of the means from 1 blot per animal per group.

Immunohistochemistry
Immunohistochemical localization of caspase-3 was performed in a separate group of P7 rats, as previously described.15 Briefly, 2 hours after the ISO exposure, P7 pups were deeply anesthetized with ISO and transcardially perfused with ice cold saline before the brains were removed, fixed with 4% paraformaldehyde, cryprototected in 30% sucrose, frozen in isopentane and stored at −80°C. Coronal cryosections (10µm) were incubated in 3% hydrogen peroxide, 10% normal goat serum and cleaved caspase-3 antibody (1:400; Cell Signaling Technology, #9664) overnight at room temperature. The next day, the sections were incubated with Alexa Fluor® 594 goat anti-rabbit IgG and coverslipped using ProLong® Gold Antifade Reagent containing the nuclear stain, DAPI (Invitrogen). Quantitative imaging was conducted on an Olympus IX70 microscope equipped with a Cooke SensiCam camera (Applied Scientific Instrumentation, Eugene, OR) and IP lab 4.0 software (Biovision Technologies, Exton, PA). Caspase-positive and total number of cells were counted in the CA1 region of the hippocampus and the adjacent parietal cortex at 20× magnification. The brain sampled and analyzed in parietal cortex was the same region used in the Western blot from the opposite brain hemisphere. The mean number of cells was calculated from 3 sections per animal and the data expressed as the percentage of caspase-3 positive cells in each region.

Statistical analysis
All data were analyzed using the Mann-Whitney U test to determine between-group differences and exact p-values using STATA statistical software. Differences were considered statistically significant at p<0.01.