PMID: 30959098 DOI: 10.1016/j.ijdevneu.2019.04.002
2 Material and methods
All animal protocols were approved by the animal research committee of Akita University, Japan (Approval number: a-1-2625). Seven-day-old (P7) Wistar rats (male and female) rat pups (body weight, 12–15 g) were used in this study. Animals were housed under standard conditions (12 h light/12 h dark cycle at 22 °C) in the Animal Research Laboratory at Akita University. All efforts to reduce the number of animals and their suffering were made. The animals were randomly divided into 6 groups (n = 10 per group) as follows: no anesthesia and no injection (sham), no anesthesia and intraperitoneal 25 μg/kg DEX (control), intraperitoneal saline (DEX 0), intraperitoneal 6.6 μg/kg DEX (DEX 6.6), intraperitoneal 12.5 μg/kg DEX (DEX 12.5), and intraperitoneal 25 μg/kg DEX (DEX 25).

After 30 min intraperitoneal injection on P7, the pups were put into a plastic chamber, exposed to 3% sevoflurane with 2 L/min of 21% oxygen for 4 h, and returned to their mother's cage. The oxygen and sevoflurane concentration were measured using a gas analysis system (GE Healthcare BioSciences, Pittsburgh, PA). The chamber was maintained at 30 ± 1 °C using an infrared heat lamp during the exposure.

Cognitive tests
2.1.1 Morris water maze
Spatial memory retention was examined using the Morris water maze by blinded observer as described previously (Goyagi, 2018). At P27 – P29, acquisition trials were executed 4 times per day for 3 successive days. The latency and the swimming path length to reach the hidden platform were measured using a video image motion analyzer (DVTrack DVT-11; Muromachi Kikai Co. Ltd, Tokyo, Japan). If the rat could not reach the hidden platform within 90 s, it was placed on the platform for 30 s during an acquisition trial. At P47 – P49, retention trials were executed 4 times per day. If the rats failed to find the platform within 90 s, the latency was regarded as 90 s. In this study, a probe trial was not done during the acquisition trials.

2.1.2 Fear conditioning test
Fear conditioning was performed to evaluate contextual memory retention using the fear conditioning system (MK-450RSQ; Muromachi Kikai Co., Ltd, Tokyo, Japan) as described previously (Goyagi, 2018). The apparatus consisted of a clear rectangular Plexiglas box with a floor of for the delivery of electric currents. At P42, the rats were placed on the cleaned parallel metallic rods to be accustomed to new environment for 1 min, before they were presented with a 70-dB white noise for 30 s A mild foot shock (0.4-mA) was administered through the metallic rods during the last 1 s of the tone presentation. The tone-shock pairing was repeated once per minute for the next 2 min. The rats were left in the cage for an additional 60 s before returned to their cage. At P49, cued fear memory was tested by placing rats into an unrelated environment for 90 s without any tone and presenting the auditory cue for a further 60 s used for conditioning. Freezing time was measured by the percent of time during the tone presentation using a video image motion analyzer (DVTrack DVT-11; Muromachi Kikai Co. Ltd, Tokyo, Japan).

Histological analyses
2.2.1 Neuronal nuclei staining
After finished the water maze task and fear conditioning test at P49, the rats’ brains were removed and embedded in paraffin following the perfusion of heparinized saline then 150 mL of 4% paraformaldehyde in phosphate buffer (pH 7.4) to use further neuronal nuclei (NeuN) stain, as described previously (Goyagi, 2018). In brief, 3-μm-thick serial transverse sections were incubated with a mouse monoclonal antibody to NeuN antigen (NeuN; 1:100 diluted in blocking solution; Millipore Corporation, Temecula, CA) for 10 min at 37 °C. Immunodetection was performed using avidin-horse radish peroxidase complexes with biotinylated antibodies to rabbit and mouse IgG (MILLIPORE IHC SelectR Immunoperoxidase Secondary Detection System; Millipore Corporation), with diaminobenzidine. Then we counterstained those with hematoxylin. The NeuN-positive cells express as mature typical neurons after growth. We counted the number of NeuN-positive cells in bilateral 500 μm × 300 μm areas in the CA1 hippocampus, amygdala, and cerebral cortical layer 3, as described previously (Goyagi, 2018).

2.2.2 Positive cell density map (PCDM)
The PCDM was made as described previously (Goyagi, 2018; Wada et al., 2006). In brief, the composite image was FFT- bandpass-filtered using the Image J program (National Institute of Health, Bethesda, MD) to eliminate low-frequency drifts (>20 pixels [50 μm]) and high-frequency noises (<1 pixel [2.5 μm]). The PDCM was made with a custom-made program using MATLAB (MathWorks INC., Natick, MA) (Wada et al., 2006), then adjusted for each section automatically and enumeration of NeuN-positive cells in each 100 μm × 100 μm square section. Finally, the normalized PCDMs were seen as averaged for each group (Fig. 6 A). As mentioned our previous study (Goyagi, 2018), the PCDMs were analyzed whether the DEX-treated groups showed increased NeuN cell density compared with the DEX 0 group. The areas were mapped as colored to indicate significantly increased normal neurons in blocks where the P value was less than 0.05 (Fig. 6B).

Statistical analysis
The escape latency, the swimming speed, the swimming path length, the freezing time, and the number of NeuN-positive cells are expressed as means ± standard deviation (SD). Comparisons of these variables among the groups were performed using a one-way or two-way analysis of variance (ANOVA) for multiple comparisons followed by Bonferroni post hoc tests. Each PCDM using a Gaussian filter of the block size (SD = 100 μm) was analyzed using t-tests for each block. Differences with p-values less than 0.05 were considered statistically significant. We performed all analyses using GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA).