Animals
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 pregnant mice were exposed in the exposure chamber for 5 h per day (from 10:00 pm to 3:00 am), 5 days a week excluding weekends from gestational day 14 to postnatal day 21 with their pups.
The number of pups born were 128 (60 male and 68 female) in the control, 130 (68 male and 62 female) in the DE and 135 (63 male and 72 female) in the DE-SOA groups. We used 3 male and 3 female pups from each dam (total 48 male and 48 female pups). Among them, 16 male and 16 female rats were used for 3 chamber social behavior test and marble burying test, among them 14 were used for neurochemical analyses and 2 were used for immunohistochemical analyses. Another 16 male and 16 female rats were used for test animals for social dominance test and other 16 male and 16 female rats were used for pair match. Furthermore, 4 male and 4 female rats from each group were used for aged match stranger 1 and stranger 2.
The pups were weaned at PND 21 and 3~4 pups of same sex were housed in a plastic cage. 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 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 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 [22, 28] (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 Nm 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 ± 23μg/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.
Behavioral Assessment
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 [22]. 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 [29]. 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 [13, 30]. 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), {"type":"entrez-nucleotide","attrs":{"text":"NM_024395","term_id":"281306772"}}NM_024395; brain-derived neurotrophic factor (BDNF), {"type":"entrez-nucleotide","attrs":{"text":"NM_012513","term_id":"397174765"}}NM_012513; interleukin (IL)-1β, {"type":"entrez-nucleotide","attrs":{"text":"NM_008361","term_id":"921274059","term_text":"NM_008361"}}NM_008361; cyclooxygenase (COX)2, {"type":"entrez-nucleotide","attrs":{"text":"NM_011198","term_id":"922959878"}}NM_011198; HO-1, NM_010442) and neuroligin3 (Nlgn3), {"type":"entrez-nucleotide","attrs":{"text":"NM_008361","term_id":"921274059","term_text":"NM_008361"}}NM_134336 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) a (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.
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.