PMID: 25663300 DOI: 10.1007/s11064-015-1529-x
Materials and Methods
Chemicals and Reagents for Metabolomic Analysis
Methanol (pesticide residue grade), bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) plus 1 % trimethylchlorosilane (TMCS) (REGIS Technologies Inc. Morton Grove, IL, USA), and amino acid standard solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). L-2-chlorophenylalanine (internal standard) was obtained from Shanghai Hengbai Biotech Co. Ltd. (Shanghai, China). All other chemicals and reagents were purchased from Anpel Company (Shanghai, China). Distilled water was prepared using the Milli-Q Reagent-Water System (Millipore, MA, USA).

Animals and Anesthesia
Sprague–Dawley (SD) rats used in the present study were obtained from the Animal Care Center of Fudan University. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee, Fudan University. According to the flow chart of the experimental protocol (Fig. 1), the rat pups (body weight: 12.1 ± 0.1 g) at postnatal day 7 (P7) were divided into two groups: control (Group C) and sevoflurane-treated (Group S).
P7 rats in group S were placed in a sealed chamber ventilated with 3 % sevoflurane in 100 % oxygen for 6 h and sevoflurane concentration was continuously measured through a gas sample line by using a monitor (Datex Ohmeda S/5, Helsinki, Finland) whereas those in group C were placed in a similar chamber for 6 h under identical experimental conditions without sevoflurane exposure. The temperature in the sealed chamber was maintained at 33–35 °C with a heating pad. The total survival percentage of P7 rats in group S after 6-h anesthesia was 90 %. After treatment, the rat pups were returned to their dams for lactation. The rats in the same litter were used for each experiment and they were sacrificed by rapid decapitation at 12 h after sevoflurane exposure. The frontal cortex was harvested and stored at −80 °C until use. The preparations of the brain samples and the number of animals used were described in their methods, respectively.

Blood Gas Analysis
P7 rats (n = 4 each group) were used to assess the effect of sevoflurane treatment on arterial blood gases. Arterial blood sampling from the left cardiac ventricle was performed immediately after the end of sevoflurane anesthesia according to the previous described method [16].We measured partial pressures of carbon dioxide (PaCO2) and oxygen (PaO2), pH, and blood lactate and glucose levels with a Radiometer ABL 800 blood gas analyzer (Radiometer, Copenhagen, Denmark).

GC–MS Analysis
Prior to metabolic profiling, frontal cortex (n = 6/group) were homogenized with 50 μL L-2-chlorophenylalanine in a 2-mL centrifuge tube. Then, 0.4 mL methanol-chloroform (3:1, V:V) as extraction liquid was added to each homogenate. After 2 min of vortex-mixing, the samples were centrifuged at 12,000 rpm for 10 min at 4 °C and 400 μL of supernatant from each sample was transferred into a new 2-mL glass tube. The supernatants of cortical samples were concentrated to complete dryness at a temperature of 50 °C for approximately 30 min using the TurboVap nitrogen evaporator (Caliper Life Science, Hopkinton, MA). Afterward, 100 µL of anhydrous toluene (stored with sodium sulfate) was added to each of the dried tissues. Following 1 min of vortex-mixing, the samples were evaporated to dryness using the evaporator to ensure the complete elimination of any traces of water which might interfere with the subsequent GC–MS analysis. Then, 80 μL MOX reagent was added to the dried samples, vortex-mixed for 2 min, and incubated at 37 °C for at least 2 h as a methoximation step. Derivatization reaction aimed to increase the volatility of polar metabolites was then initiated by adding 100 μL of BSTFA (with 1 % TMCS) to each sample, vortex-mixed for 2 min, and incubated at 70 °C for 60 min. Following the incubation, each sample was vortex-mixed for 2 min and carefully transferred to the autosampler vials for subsequent GC–MS analysis [17].

GC–MS analysis was performed on an Agilent 7890A gas chromatography system coupled with an Agilent 5975C mass spectrometer (Agilent, USA). The system utilized a DB-5MS capillary column coated with 5 % diphenyl cross-linked with 95 % dimethylpolysiloxane (30 μm × 250-μm inner diameter, 0.25-μm film thickness; J&W Scientific, Folsom, CA, USA). A 1-μL aliquot of the analyte was injected in splitless mode. Helium was used as the carrier gas, the front inlet purge flow was 3 mL/min, and the gas flow rate through the column was 1 mL/min. The initial temperature was kept at 80 °C for 2 min, then raised to 240 °C at a rate of 5 °C/min, and finally to 290 °C at a rate of 10 °C/min for 11 min. The injection, transfer line, and ion source temperatures were 280, 270, and 220 °C, respectively. The energy was −70 eV in electron impact mode. The mass spectrometry data were acquired in full-scan mode with the m/z range of 20–600 at a rate of 100 spectra per second after a solvent delay of 492 s. Chroma TOF4.3X software of LECO Corporation were used to acquire mass spectrometric data [18]. Mass spectra of all detected compounds were compared with spectra in the National Institute of Standards and Technology (NIST, http://www.nist.gov/index.html) and Fiehn databases. The peaks with similarity index of more than 70 % were selected and named the putative metabolite identities.

Multivariate Data Analysis
The resulting GC–MS data were first processed by normalizing peak area of each analyte based on total integral area calculation performed using an in-house script (Microsoft Office Excel). All processed data were then mean-centered and unit-variance scaled before they were subjected to principal component analysis (PCA) (version 11.5, SIMCA-P software, Umetrics, Umea, Sweden) to identify clustering trend, as well as detect and exclude outliers. Quality control (QC) samples for cortical tissues were prepared by randomly pooling 5 μL from each of the five samples belonging to the test groups. QC samples were analyzed at constant intervals to ensure that the data acquisition for GC/MS metabolic profiling was reproducible for all samples. Variable importance in the projection (VIP) cutoff value was defined as 1.00.

Western Blot Analysis
The frontal cortical tissues were homogenized in RIPA buffer (Millipore, Temecula, CA, USA) containing complete protease inhibitor cocktail and 2 mM phenylmethylsulfonyl fluoride. The lysates were collected and centrifuged at 12,000 rpm for 30 min at 4 °C. After the protein samples were quantified using a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA), the cleaved caspase-3 expression was detected by western blot analysis according to our previous method [4]. Data were expressed as mean ± SD. The changes were presented as a percentage of those of the control group. One-hundred-percent of caspase-3 activation refers to control level for the purpose of comparison to that in Group S.

Measurement of ROS Levels
The frontal cortex was cleaned in PBS and dissociated in trypsin solution, and stopped using DMEM solution. A single-cell suspension was obtained by using a 70-μm mesh. The chemiluminescent probe with flow cytometry technique was used for detection of intracellular ROS level according to a previously described method [19]. Briefly, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) probe was added to the cell suspensions at a final concentration of 10 μmol/L and incubated at 37 °C, protected from light for 1 h, followed by flow cytometry (FACSCanto, BD Biosciences, San Jose, CA, USA) measurement. The formation of the oxidized fluorescent derivative 2′,7′-dichlorofluorescein (DCF) was monitored with excitation light at 488 nm and emission light at 525 nm, and normalized by protein concentration. By quantifying fluorescence intensity of DCF, the ROS levels in both groups were calculated.

Mitochondrial Cardiolipin Assay
Extraction of mitochondria was performed using the mitochondria isolation kit (Shanghai Genmed Scientifics Inc., China). Briefly, the frontal cortex was lysed in precooled centrifuge tubes and disrupted by 80 passes in the homogenizer with a tight fitting Dounce homogenizer. The homogenate was then centrifuged for 10 min at 1,500g at 4 °C. The mitochondria-rich supernatant was then collected and centrifuged for 10 min at 10,000g at 4 °C. The mitochondrial pellets were then washed with 2 mL of preservation medium (25 mmol/L potassium phosphate; 5 mmol/L MgCl2, pH 7.2) and centrifuged for 5 min at 10,000g at 4 °C. Purified mitochondrial samples were freeze-thawed three times and suspended to 5.5 mg/mL in PBS before use. The mitochondrial cardilopin contents were quantified by the microplate reader method using the high affinity 10-N-nonyl acridine orange (NAO) for cardiolipin of freshly isolated mitochondria [20]. Briefly, reagents (90 μL) from cardilopin assay kits (Shanghai GenMed Scientifics Inc., Shanghai, China) was added into mitochondrial sample (10 μL) on the microplate. The microplate was gently shaken and incubated in a dark room for 20 min at room temperature. Then fluorescence intensity was measured with excitation light at 580 nm and emission light at 630 nm. The cardilopin contents were expressed as relative fluorescence unit (RFU) and normalized by protein concentration.

Electron Microscopy
The rat brain was perfused with normal saline solution followed by phosphate-buffered 2.5 % glutaraldehyde and 4 % paraformaldehyde 12 h after sevoflurane treatment, then the frontal cortex was sliced into sections of approximately 1 mm2, and kept in the same glutaraldehyde solution for 12 h at room temperature. Samples were postfixed in 1 % osmium tetroxide for 2 h, dehydrated in a series of alcohol solutions at 4 °C, immersed in propylene oxide, and embedded in Araldite 502 resin at 60 °C. Ultrathin (0.5 μm) sections were placed on grids and stained with uranyl acetate and lead citrate before examination with a transmission electron microscope (Philips CM-120, Eindhoven, The Netherlands). The organelles of neuronal cells were observed and imaged at 10,000× magnification.

Statistical Analysis
We performed one-way ANOVA to determine differences in caspase-3 activation and cardiolipin contents, and independent Student’s t test to compare the difference in arterial blood gas analysis and ROS levels. Independent t tests with Welch’s correction were then used for statistical comparison of discriminant metabolite levels between Group C and Group S, which determined for sevoflurane-induced alteration of metabolic profiling in neonatal rat model. The significance level was set at p < 0.05.