A previous study model (21–23) on miRNA data acquisition, processing and statistical analysis was used. A flow chart of the experimental protocol is shown in Fig. 1.

Animal protocols, including the method for euthanasia, were approved by the Animal Experimental Ethics Review Committee of Nippon Medical School (Tokyo, Japan; approval no.: 24-005). All procedures followed the guidelines of the International Association for the Study of Pain (24). Specific pathogen-free male Wistar rats aged six weeks old, (Saitama Experimental Animal Supply Co., Saitama, Japan) were purchased and acclimated under temperature-controlled conditions on a 12-h light/dark cycle, starting at 06:00 and 18:00, respectively, for a week prior to the commencement of experiments. All rats received food and water ad libitum, and the average starting weight of the rats was 250±10 g.

During the experiment, each rat was housed in a plastic box (45×32×23 cm), which was large enough for the rat to move and breathe fresh air. The rats were minimally handled in order to reduce stress. The tails of the rats were placed through a small hole in the wall of the box, a catheter was inserted into the tail vein without anesthesia and normal saline was infused continuously at a rate of 1.0 ml/h. The rats were allowed to breathe spontaneously and were supplied with an air-oxygen mixture [fraction of inspired oxygen (FiO₂) = 0.40]. Body temperature was maintained at 37.0°C with a heat lamp.

The rats were randomized into three groups (n=7 in each group) and each group received either sevoflurane, propofol or no anesthetic (control group). In the anesthesia groups, the rats were anesthetized with sevoflurane or propofol for 6 h (between 9:00 and 15:00). Rats undergoing inhalational anesthesia (sevoflurane) were placed in plastic boxes supplied with sevoflurane (Sevofrane^®; Maruishi Pharmaceutical Co., Ltd., Osaka, Japan; 2.4% gas-air mixture) (25) and 40% oxygen (6 l/min). Normal saline was administered via a venous catheter at a rate of 1.0 ml/h. Rats undergoing intravenous anesthesia (propofol) were housed in plastic boxes supplied with 40% oxygen at a rate of 6 l/min and were administered 1% propofol (Diprivan^®; AstraZeneca, Osaka, Japan; 600 μg/kg/min) (26) via a venous catheter (rate of 9.0 ml/h for rats weighing 250 g). In the control group, the rats were not administered anesthetics; however, they were similarly housed in plastic boxes supplied with 40% oxygen at a rate of 6 l/min and normal saline was administered via a venous catheter at a rate of 1.0 ml/h. Rats in the control group were not allowed access to food and water. All rats were allowed to breathe spontaneously. Animals were sacrificed immediately following completion of anesthesia at 15:00. Within 3 mins after decapitation, the whole brain was rapidly removed and the bilateral hippocampus was directly removed from the brain, as described previously (27,28).

Physiological variables were measured in three separate groups of rats not used for hippocampal miRNA evaluation (n=7 each), which were treated with sevoflurane, propofol or no anesthetic (control group). For all rats, a catheter was inserted into the tail vein without anesthesia and normal saline was infused continuously at a rate of 1.0 ml/h. During the experiment, each rat was placed in a plastic cage (45×32×23 cm) supplied with an air-oxygen mixture [(FiO₂) = 0.40] and allowed to breathe spontaneously. The body temperatures of the rats were maintained at 37.0°C with a heat lamp and their rectal temperatures were measured. As a surgical procedure, the left femoral artery was cannulated to obtain blood samples for measuring the arterial PO₂, arterial PCO₂, arterial blood pH, heart rate and arterial blood pressure (Table I). Anesthetics were administered as described for the miRNA analysis groups. In the sevoflurane and propofol physiological groups, anesthesia was maintained for an additional 3 or 6 h after surgery. In the control group, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg) for the surgery and it was maintained for 3 h. The rats were placed in a rat tunnel in the plastic cages and allowed to recover from the anesthesia. Blood samples were obtained from all five groups.

Hippocampal samples were washed twice with cold phosphate-buffered saline, immediately placed in RNAlater solution (Applied Biosystems, Inc., Foster City, CA, USA) and stored at −80°C for one day. Following storage, the samples were defrosted and the RNAlater solution was rapidly separated from the samples by centrifugation (10,000 × g, 4°C, 5 min). Total RNA was extracted from each sample using a mirVana™ miRNA Isolation kit (Applied Biosystems, Inc.) according to the manufacturer’s instructions. RNA quantity and quality were assessed by measuring absorbance using a NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA). Any RNA samples with a 260/280 nm absorbance ratio >1.8 were subjected to quantitative analysis. Total RNA samples containing miRNA were used for quantitative reverse transcription-polymerase chain reaction (qRT-PCR).

The miRNA expression profiles in the hippocampal samples were analyzed using TLDA Rodent miRNA cards v.3.0 A and B (Applied Biosystems, Inc.), a set of miRNA low-density arrays which enables simultaneous qRT-PCR for 373 preloaded rat miRNA targets and three endogenous controls (Mamm U6, U87 and Y1). All miRNA targets are cataloged in the miRBase database (http://www.mirbase.org/). All procedures were based on those of a previous study (29). Briefly, TLDA was performed as a two-step process. During the first step, 1,200 ng of total RNA per sample was reverse-transcribed using Megaplex RT Primer Pools A and B (Applied Biosystems, Inc.), up to 381 stem-looped primers per pool and a TaqMan miRNA Reverse Transcription kit (Applied Biosystems, Inc.). In the second step, each of the resulting complementary DNA pools was diluted, mixed with TaqMan Universal PCR Master mix (Applied Biosystems, Inc.) and deionized distilled water (Wako Pure Chemical Industries, Ltd., Tokyo, Japan), and loaded into one of the eight fill ports on the TLDA microfluidic card. The cards were briefly centrifuged for 1 min at 315 × g to distribute samples to the multiple wells connected to the fill ports and were then sealed to prevent well-to-well contamination. The cards were processed and analyzed using a 7900HT Fast Real-Time PCR system (Applied Biosystems, Inc.).

Data analysis was performed using DataAssist software v2.0 (Applied Biosystems, Inc.). The resulting data were expressed as threshold cycle (Ct) values, where Ct represents a unitless value defined as the fractional cycle number at which the sample fluorescence signal passes a fixed threshold above baseline. The relative quantities of all the miRNAs were calculated by the comparative Ct method. ΔCt is the difference in Ct values derived from the experimental samples and the controls, and ΔΔCt represents the difference between paired samples, as calculated by the following formula: ΔΔCt = ΔCt of sample following anesthesia − ΔCt of the control group. The expression ratio shows the relative quantity of the target gene (Xtarget) to the control gene (Xcontrol). The fold change was computed by the following formula: Xtarget/Xcontrol = 2^−ΔΔCt. The 2^−ΔΔCt method is a convenient method for analyzing the relative changes in gene expression from qRT-PCR experiments. Relative quantification (RQ) values (relative levels of miRNA expression) were calculated by this comparative Ct method using RQ Manager version 1.2.1 (Applied Biosystems, Inc.) (30). Graphic displays were visualized as heat map results of hierarchical clustering. Distances between samples and assays were calculated for hierarchical clustering as determined by ΔCt values, using Euclidean distance.

Values are expressed as the mean ± standard deviation. Analysis of variance (ANOVA) followed by Tukey-Kramer test was used to compare the physiological data and miRNA expression levels in the sevoflurane anesthesia, propofol anesthesia and control groups. P<0.05 was considered to indicate a statistically significant difference in all tests. Tukey-Kramer test was applied to identify genes and miRNAs that were significantly differentially expressed on exposure to anesthetic agents. ANOVA followed by Tukey-Kramer test was performed using Kyplot software, version 5.0 (KyensLab Incorporated, Tokyo, Japan).

Following induction of sevoflurane or propofol anesthesia, all rats lost consciousness within 5 min, with loss of corneal reflexes and the withdrawal response to pain. No rats moved spontaneously or died until the end of anesthesia. Data for all animals were used.

The possible effects of anesthesia on a number of physiological parameters were investigated in three separate groups of rats. Fig. 1 shows a flow chart of the experimental protocol. The physiological data for the treatment groups during anesthesia were compared with those of the control group, in which no anesthesia was administered following the termination of anesthesia for the cannulation of the femoral artery. Low mean blood pressure and high PCO₂ were observed following the administration of anesthetic drugs. However, no significant differences in the physiological parameters among the administration groups and control group were identified (Table I). Hypoxia, hyper/hypocapnia, hypotension or hypothermia were not observed in any of the three groups.

In order to identify suitable endogenous controls, three different candidate miRNAs were analyzed for variance in gene expression with DataAssist software v2.0. The statistical method ranked the candidate endogenous control genes with an excellent correlation of raw stability values. The most stably expressed Y1 was selected as the endogenous control and the relative miRNA expression levels were normalized against Y1. Ct values >35 indicated that their expression levels were too low for accurate analysis. The miRNA expression Ct value was therefore standardized at ≤35.

The results of the TLDA analysis of the hippocampi are shown in Tables II–IV (n=7 for each group; significance level, P<0.05). There were 750 miRNAs on the TLDA plate; 373 miRNAs were associated with rats and the rest with mice. TLDA analysis revealed 279 expressed miRNAs (74.8%), which were expressed in the sevoflurane, propofol and control groups. Significant differences were observed in the levels of 33 of the 279 expressed miRNAs (11.8%) among the three groups in response to the anesthetic agents, and when compared with the control group, significant differences were found in 26 of the 279 expressed miRNAs (9.3%). Following sevoflurane anesthesia, the levels of four miRNAs were significantly increased and those of 12 were significantly reduced compared with those of the control group. By contrast, following propofol anesthesia, the levels of 11 miRNAs were significantly reduced but no miRNAs exhibited significantly elevated levels. Of the total 33 significantly differentially expressed miRNAs, one miRNA was common to both anesthetics, whereas 14 miRNAs were significantly differentially expressed (Tables II–IV).

Cluster analysis from TLDA data is shown in Fig. 2. Significantly differentially expressed miRNAs were converted into a heat map in order to improve the understanding of the miRNA expression patterns in each group. Heat maps for the calculated clusters are commonly used gene-expression analyses and are constructed for visualization of gene expression levels. In the present study, heat maps illustrated the gene expression levels from the hippocampal samples following treatment with anesthetics using ΔCT values. The color intensity on the heat map corresponds to the absolute intensity of the miRNA expression. Each column represents a sample and each row shows miRNA in the heat map. By individually clustering columns and rows, the heat map simultaneously presents the separate samples and miRNA clustering in one graphic. The dendrograms of clustering analysis for the samples and miRNAs are shown to the top and left of the heat map. This indicated the relatedness of the samples as determined by the overall miRNA expression values. The dendrogram separates into three main branches: The sevoflurane anesthesia, the propofol anesthesia and the control groups.