Forty-eight timed pregnant Sprague-Dawley rats (gestational day; GD 8) were purchased from Oriental Yeast Co., Ltd. (Tokyo Japan) and exposed to clean air (control, n = 16), DE (n = 16) and DE-SOA (n = 16) from GD 14 to postnatal day (PND) 21 in the whole body exposure chambers. Food and water were given ad libitum. Date of birth was recorded as PND 0 and the offspring were housed in cages with dam under controlled environmental condition (temperature, 22 ± 0.5 °C; humidity, 50 ± 5%; lights on 07:00–19:00 h). The pups were weaned at PND 21 and 3 ~ 4 pups of same sex were housed in a plastic cage. Sixteen male and female offsprings from the control, DE and DE-SOA groups were used. Experimental design was depicted in the Fig. 1. Social behavioral tests were performed at 10 ~ 13-week-old. Behavioral testing was performed between 09:00 and 13:00 h. Before performing each test, the apparatus to be used was cleaned with 70% ethanol. After completing social behavioral test, the rats (n = 14 from each group) were sacrificed under deep pentobarbital anesthesia and the left and right prefrontal cortex were collected from each group and frozen quickly in liquid nitrogen, then stored at − 80ºC until the extraction of the total RNA. The remaining rats (n = 2 from each group) were sacrificed under deep pentobarbital anesthesia and the whole brain was collected from each group and stored at 10% formalin till histological and immunohistochemical analyses. The experimental protocols were approved by the Ethics Committee of the Animal Care and Experimentation Council of the National Institute for Environmental Studies (NIES), Japan (AE-19-36, AE-20-05). All efforts were made to minimize the number of animals used and their suffering.
Preparation of exposure chambers for clean air, DE or DE-SOA
The whole-body inhalation exposure chambers for clean air, DE or DE-SOA were generated at the National Institute for Environmental Studies, Japan as described previously [25, 29] (Fig. 2). Briefly, an 81-diesel engine (J08C; Hino Motors Ltd., Hino, Japan) was used to generate diesel exhaust. The engine was operated under a steady-state condition for 5 h a day. Our driving condition of diesel engine was not simulated to any special condition as in the real world. The engine operating condition (2000 rpm engine speed and 0 N m engine torque) promotes the generation of high concentrations of nano-size particles. There are three chambers: a control chamber receiving clean air filtered through a HEPA filter and a charcoal filter (referred to as “clean air”), the diluted exhaust (DE which was without mixing O3), DE-SOA which was generated by mixing DE with ozone at 0.6 ppm after secondary dilution. Secondary dilution ratio in DE and DE-SOA chambers were the same which resulted in the same particle and gaseous concentrations when O3 was not mixed. Actually, the concentrations of particles in DE-SOA was higher when O3 was mixed and concentrations of DE and DE-SOA were 101 ± 9 µg/m3 and 118 ± 23 µg/m3, respectively. The increased mass concentration was due to the generation of secondary particles. The temperature and relative humidity inside each chamber were adjusted to approximately 22 ± 0.5 °C and 50 ± 5%, respectively. The particle characteristics inside the exposure chamber were shown in Table 1. In detail, sample air was taken from the inhalation chamber (2.25 m3) using stainless steel tubing. The gas concentrations (CO, CO2, NO, NO2, and SO2) were monitored using a gas analyzer (Horiba, Kyoto, Japan). CO and NOx concentrations in both chambers were similar, but NO and NO2 are different each other because NO was oxidized to NO2 by reacted with O3. The particle size distributions were measured using a scanning mobility particle sizer (SMPS 3034; TSI, MN). The modal sizes of the particles used in the present study were 22.69 ± 1.47 nm for DE and 24.45 ± 1.21 nm for DE-SOA. The particles were collected using a Teflon filter (FP-500; Sumitomo Electric, Osaka, Japan) and a Quartz fiber filter (2500 QAT-UP; Pall, Pine Bush, NY, USA), and the particle mass concentrations were measured using a Teflon filter. The particle weights were measured using an electrical microbalance (UMX 2, Mettler- Toledo, Columbus; OH, USA; readability 0.1 µg) in an air-conditioned chamber (CHAM-1000; Horiba) under constant temperature and relative humidity conditions (21.5 °C, 35%). For the Quartz fiber filter, the quantities of elemental carbon (EC) and organic carbon (OC) were determined using a carbon analyzer (Desert Research Institute, NV, USA). EC to TC ratio in the present study were 0.15 ± 0.06 for the control chamber, 0.36 ± 0.03 for DE chamber and 0.38 ± 0.03 for DE-SOA exposure chamber.
Sixteen male and female rats were used for social behavior analyses.
Sociability and social novelty preference
Sociability and preference for social novelty test were performed as reported previously . The apparatus used is a rectangular, three-chambered Plexiglas box (100 cm x 100 cm x 35 cm), with equal sizes of the three chambers. The dividing partitions are also made of clear Plexiglas, with small doorways on each (10 cm x 10 cm) that allow free access of the animals among the chambers. Wired cups (diameter 15 cm; height, 30 cm) are placed in each of the side chambers to house unfamiliar animals. For habituation, the subject rats from three different groups (DE or DE-SOA-exposed and the control rats) are placed in the middle chamber and allowed to explore for 5 min. During the habituation phase, the wired cup in each of the side chambers was empty (E). Following habituation, for the sociability test, an unfamiliar rat (stranger 1 (S1), age-matched rat) is placed in the wired cup in one of the side chambers; the subject rats are allowed to explore for 10 min. The location of stranger 1 in the left or right-side chamber is systematically alternated between trials. The social novelty preference test is performed immediately after the sociability test. For this test, another unfamiliar rat (stranger 2 (S2), age-matched rat) is placed in the wired cup on the other side that had been empty during the first 10-minute session, and the subject rat is allowed to explore the two strangers for 10 min. The time spent in exploring the wired cups on either side will be measured. The time that the subject rat spent exploring the wired cup is measured as the time spent with its head facing the cup from a distance of within 1 cm.
Social dominance behavior (Tube test)
Social dominance was tested in a transparent Plexiglas tube measuring 45 cm in length and 4 cm in (inside) diameter, a size just sufficient to permit one rat to pass through without reversing direction . For training, each rat was released at alternating ends of the tube and allows to run through the tube. Each animal was given five training trials on each of two successive days. For the social dominance test, animals were placed at opposite ends of the tube and released simultaneously. An animal was declared the “winner” when its opponent backed out of the tube. The maximum test time was set to 2 min. The tube was cleaned with 70% ethanol before each trial.
Marble burying test
Marble burying test is a useful model of anxiety-like behavior and repetitive behavior. Each rat was placed for 20 min into a clean rat cage (40 cm X 24 cm X 15 cm) with 5 cm deep bedding and 20 glass marbles placed in a regular pattern and evenly spaced. The number of marbles that were buried at least 2/3 of the area by the rat was measured.
Quantification of mRNA expression levels
After completion of behavioral tests, 13-week-old male and femalerats (n = 14 from each group) were sacrificed under deep pentobarbital anesthesia and the left prefrontal cortex was collected from each group for mRNA analyses. Briefly, the total RNA was extracted from the prefrontal cortex samples using the BioRobot EZ-1 and EZ-1 RNA tissue mini kits (Qiagen GmbH, Hilden, Germany). Then, the purity of the total RNA was examined, and the quantity was estimated using the ND-1000 NanoDrop RNA Assay protocol (NanoDrop, Wilmington, DE, USA), as described previously [16, 31]. Next, we performed first-strand cDNA synthesis from the total RNA using SuperScript RNase H−Reverse Transcriptase II (Invitrogen, Carlsbad, CA, USA), according to the Manufacturer’s protocol. We examined the mRNA expression levels using real-time RT-PCR (Light Cycler 96, Roche, Germany). The tissue 18S rRNA level was used as an internal control. The primer sequences used in the present study are shown below. Some primers (5-hydroxytryptamine (serotonin) receptor 5B (5-HT5B), NM_024395; brain-derived neurotrophic factor (BDNF), NM_012513; interleukin (IL)-1β, NM_008361; cyclooxygenase (COX)2, NM_011198; HO-1, NM_010442) were purchased from Qiagen, Sample and Assay Technologies. Other primer was designed in our laboratory as follows: 18S (forward 5’-TACCACATCCAAAAGGCAG-3’, reverse 5’-TGCCCTCCAATGGATCCTC-3’), tumor necrosis factor (TNF) α (forward 5’-GGTTCCTTTGTGGCACTTG-3’, reverse 5’-TTCTCTTGGTGACCGGGAG-3’). Data were analyzed using the comparative threshold cycle method. Then, the relative mRNA expression levels were expressed as mRNA signals per unit of 18S rRNA expression.
Measurement of glutamate concentration
After completing social behavioral test, the rats (n = 14 from each group) were sacrificed under deep pentobarbital anesthesia and the right prefrontal cortex was collected from six male and female rats of each group and frozen quickly in liquid nitrogen, then stored at − 80ºC until protein analysis. The right prefrontal cortex from 6 rats of each male and female groups were homogenized in a falcon tube containing 10 ml of cool sterile saline and centrifuged at 3000 rpm/min for 5 min at 4˚C. The supernatant was used for subsequent glutamate detection by glutamate research ELISA assay kit (Ref: BA E-2300, Neuroscience. Inc., Tokyo, Japan) according to the manufacturer's instructions.
Histology and mast cell analyses
Two male and female rats from each group were sacrificed under deep pentobarbital anesthesia and the whole brain was collected and stored at 10% formalin for histology and mast cell analyses. General morphology was examined by H & E stain. Mast cell was examined by toluidine blue staining. Briefly, a 1% stock solution of toluidine blue in 70% ethanol was dissolved in 0.5% NaCl (pH 2.2–2.3). The slides were immersed in solution for 30 min, then washed twice with distilled water and dehydrated using a series of increasing concentrations of ethanol, and finally immersed in butyl acetate ester. Cover slips were applied, and then the slides were allowed to dry overnight. Photomicrographic digital images of the prefrontal cortex regions were taken using a charged coupled device (CCD) camera connected to a light microscope.
Two male and female rats from each group were used for histology and mast cell analyses. Microglial activation in the prefrontal cortex was examined. The tissue sections were stained with microglial marker Iba1 as described previously . Briefly, the brain sections were immersed in absolute ethanol, followed by 10% H2O2 for 10 min each at room temperature. After rinsing in 0.01-M phosphate buffer saline, the sections were blocked with 2% normal swine serum in PBS for 30 min at room temperature and then reacted with goat polyclonal anti-Iba1 (diluted 1:100; abcam: ab5076; Tokyo, Japan) in PBS for 1 h at 37 °C. Thereafter, the sections were reacted with biotinylated donkey anti- goat IgG (1:300 Histofine; Nichirei Bioscience, Tokyo, Japan) in PBS for 1 h at 37 °C. The sections were then incubated with peroxidase-tagged streptavidin (1:300, ABC KIT) containing PBS for 1 h at room temperature. After a further rinse in PBS, Iba1 immunoreactivity was detected using a Dako DAB Plus Liquid System (Dako Corp., Carpinteria, CA, USA). To detect the immunoreactivity of Iba1 in the prefrontal cortex, photomicrographic digital images (150 dpi, 256 scales) of the prefrontal cortex regions were taken using a charged coupled device (CCD) camera connected to a light microscope.
2.1 Statistical analysis
All the data were expressed as the mean ± standard error (S.E.). The statistical analysis was performed using the StatMate II statistical analysis system for Microsoft Excel, Version 5.0 (Nankodo Inc., Tokyo, Japan). The data were analyzed using a one-way analysis of variance with a post-hoc analysis using the Bonferroni/Dunn method. Differences were considered significant at P < 0.05.