Abstract Epidemiological studies have revealed that ambient fine particulate matter (PM2.5) exposure is closely associated with autism spectrum disorder (ASD). However, there is a relative paucity of laboratory data to support this epidemic finding. In order to assess the relationship between PM2.5 exposure and ASD, neonatal male Sprague–Dawley rats were chosen and exposed to PM2.5 (2 or 20 mg/kg body weight, once a day) by intranasal instillation from postnatal day 8 to 22. It was found that when exposed to PM2.5 in the early neonatal period for two weeks, both groups of the exposure rats manifested typical behavioral features of autism, including communication deficits, poor social interaction, and novelty avoidance. And, we further found, among five ASD candidate genes we chose, both the mRNA level and protein expression of SH3 and multiple ankyrin repeat domains 3 (Shank3) decreased significantly in the rat hippocampus after high dose of PM2.5 exposure. Moreover, results showed that PM2.5-exposure significantly increased the levels of proinflammatory cytokines, interleukin 1β, interleukin 6, and tumor necrosis factor alpha in the hippocampus and prefrontal cortex. The expression of glial fibrillary acidic protein and ionized calcium-binding adapter molecule, markers of astrocytes and microglial cell activation, respectively, also increased in the exposed animals. Our work provides new data on the link between postnatal exposure to ambient PM2.5 and the onset of ASD-like symptoms in human beings, and the increased inflammatory response and abnormalities in Shank3 expression in the brain may contribute to the mechanisms of PM2.5 exposure-induced ASD. air pollution, fine particulate matter, autism spectrum disorder, behavioral assessment, Shank3 gene Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder which presents two core symptoms: impaired social communication and interaction, and restricted or repetitive behaviors (Association, 2013). The prevalence of ASD is increasing worldwide (Naigles et al., 2016), and is four to five times greater in males than in females (Blumberg et al., 2013). Although genetics plays an important role in these conditions, environmental exposures, particularly during the in utero or early postnatal stages, are increasingly being recognized as potential risk factors for ASD (Kim and Leventhal, 2015; Reich-Erkelenz et al., 2015). Although considerable efforts have been made to clean up the environment, air pollution remains a major problem and poses continuing risks to health in general. In recent decades, several epidemiological studies have linked exposure to air pollution during development to the occurrence of ASD. Two recent case–control studies (Kalkbrenner et al., 2015; Raz et al., 2015) showed that higher maternal exposure to air pollution particulate matter (PM2.5) during pregnancy, particularly during the third trimester, was associated with a greater risk of the child developing ASD. Talbott et al. (2015) reported that both prenatal and postnatal exposures to PM2.5 may be associated with an increased risk of ASD, consistent with previous studies that indicated that exposure to traffic-related PM2.5 air pollution during pregnancy and during the first year of life was associated with ASD (Volk et al., 2013). Furthermore, mice exposed to air pollution during the early postnatal period developed features of ASD (Allen et al., 2014, 2017). The latest epidemiological studies have posed a hypothesis that pre- or postnatal exposure to ambient pollutants, particularly PM2.5, contributes to the risk of ASD in children (Suades-Gonzalez et al., 2015). The precise etiology of ASD remains poorly understood. Evidence indicating that immune dysfunction/inflammation plays a role in ASD has accumulated (Theoharides et al., 2013). Hence, we hypothesized that early-postnatal PM2.5 exposure is a potential contributor to ASD, and that neuroinflammation may serve as a mechanism linking PM2.5 and ASD-like abnormalities. In the present study, we investigated the effects of early postnatal PM2.5 exposure on a battery of behavioral dimensions and gene expression in the brain, in order to establish whether such exposure can lead to ASD-like phenotypes in rats. MATERIALS AND METHODS PM2.5 Sampling and Processing Ambient PM2.5 samples were collected at the research building (fifth floor) of the Institute of Health and Environmental Medicine, which is located in an urban area of Tianjin, China, by using an impactor sampler (Laoying 2030, Qingdao, China), placed at the intersection of two very busy streets, between December 2014 and March 2015. Cumulative samples were collected onto quartz fiber (diameter: 80 mm) at an air-flow rate of 100 L/min over 24 h. PM2.5 mass concentrations were measured using a DustMate particulate detector (Turnkey Instruments, Northwich, UK). The particles were extracted from the filters via sonication, followed by lyophilization (ALPHA2-4 LD, Martin Christ, Osterode am Harz, Germany), and were then weighed and stored at −80 °C. PM2.5 was collected on a Teflon filter in the same condition above for the analysis of chemical composition. Sixteen polycyclic aromatic hydrocarbons (PAHs) were analyzed by an Agilent 7890A-5975C gas chromatography–mass spectrometry network system (Agilent Technologies, Wilmington, DE, USA) according to the National Environmental Protection Standard of PR China (HJ646-2013), and nine elements were measured by inductively coupled plasma optical emission spectrometer (ICP Optima 8300, Perkin Elmer, USA). As previously reported, the per-minute ventilation of rats is about 0.16 L (Crosfill and Widdicombe, 1961), and the daily respiratory volume reaches 0.23 m3. We detected an average PM2.5 concentration of 187 µg/m3 in the sampling period. Therefore, the equivalent PM2.5 exposure dose for rats would be about 43 µg/day. Given the body weight (bw) of early postnatal rats, we set a PM2.5 exposure dose of 2 µg/g bw to simulate actual exposure level and a 20 µg/g bw dose to explore the acute neurodevelopmental toxicological effects of PM2.5 on ASD, and the mechanism underlying those effects. Animal Groups and Treatment Research and animal care procedures were approved by the Animal and Human Use in Research Committee of the Tianjin Institute of Health and Environmental Medicine, and all animal experiments were performed in accordance with relevant guidelines and regulations. The experimental design of the animal study is shown in Supplementary Figure 1. Sprague–Dawley rat litters were obtained from the Lab Animal Center, Institute of Health and Environmental Medicine, Tianjin, China. Considering the prevalence of ASD in males, and to avoid uncertain sex-dependent differences, we analyzed only male rats in this study. The litters were adjusted to six male pups per dam at postnatal day (PND) 3, to preclude litter-specific effects, every single pup in litters was from the breeding cohort with different dams, and litter size was counterbalanced when assigning litters to a treatment group. Pups were randomly divided into four groups: blank control (Control); vehicle exposed (Saline); 2 µg/g bw PM2.5 exposures (Low), and 20 µg/g bw PM2.5 exposure (High). From PND 8, pups were administered saline vehicle or two dose of PM2.5 suspension via intranasal instillation, through both nostrils in a supine position, once per day, for 2 weeks. Pups in the control group were only taken out without instillation. All animals were housed in a room with a 12-h light/dark cycle and temperature of 20–24 °C and humidity of 50–60%, with food and water given ad libitum. Rats were allowed to habituate to the laboratory environment for 5 days prior to the start of the experiment. Behavioral Assessment Ultrasonic vocalizations analysis Ultrasonic vocalization (USV) analysis was performed on PND 16. Rat pups were separated from their mother and littermates in the home cage, and were placed in the center of a sound-proofed test chamber. The isolation calls, or USVs, were recorded using a 1/4-in. Brüel & Kjær model 4938 microphone (Brüel & Kjær, B&K, Nærum, Denmark) connected to a B&K 2669 preamplifier and a B&K 2690 conditioning amplifier. The microphone was placed 10 cm above the test subject. The USVs were recorded and analyzed using B&K 3560-C PULSE hardware with PULSE LabShop Version 13.5.0 software for 5 min. Three-chambered social test This test was conducted as previously described (Crawley, 2004) with some modifications in three sessions within a three-chambered box (60 cm × 40 cm × 22 cm) that was equipped with retractable doorways that permitted access to each chamber. The sessions were as follows. Session 1—Habituation: Pups were first placed in the middle chamber; the doorways were opened and pups were allowed to explore the other two side chambers freely. Session 2—Sociability: At the end of the period of habituation to the empty box, pups were made to interact with a never-before-met and age-matched rat enclosed in a wire cup placed in a side chamber and an empty wire cup placed in the other side chamber. Session 3—Social novelty: After the sociability test was completed, a new unfamiliar rat was placed in the wire cup that had been empty during the previous session. Each session lasted 5 min. The time spent sniffing each cup and the time spent in each chamber were recorded using the SuperMaze video analysis system (Xinruan, Shanghai, China). Marble burying test Pups were individually placed in a Plexiglas cage (42 cm × 24 cm × 17 cm) filled with approximately 5-cm-deep bedding, with a flat, even surface. A total of 20 glass marbles (1.4-cm diameter) were arranged on the bedding surface, and were evenly spaced in 5 rows of 4. The test pup was allowed to explore the testing cage and marbles for 30 min. The total number of marbles buried (to two-thirds of their depth) was recorded. Novel object recognition test The novel object recognition (NOR) task was performed as previously described (Leger et al., 2013) with some modifications. The test consisted of three trials: acclimation, object familiarization, and object recognition. The subject pups were first allowed to habituate to the test cage (the three-chambered apparatus mentioned earlier, but without the partition) for 5 min. During the familiarization session, the pups were exposed to two identical objects (Greiner® cell-culture flask filled with sand, 9.5 cm × 2.5 cm × 4.2 cm) that were placed at an equal distance from the wall for 5 min. Object recognition was performed after 30 min. During that stage, the two familiar objects were replaced, one with an identical copy (to ensure that there are no residual olfactory cues on the previously used object) and the other with a novel object (Abcam® Lego brick, 7.6 cm ×1.9 cm ×5.5 cm). The old and new two objects have the approximate size and no odor with differ in shape and texture. Animals were left in the cage for 5 min. Investigative behaviors, such as sniffing the object or touching the object while looking at it, were considered as exploration of the objects. The time spent exploring the old or the novel object during the recognition phase was recorded. The box was cleaned with 75% ethanol once a subject completed the test. Olfactory habituation/dishabituation test The olfactory habituation/dishabituation (OHD) test was conducted as previously described (Laugeray et al., 2014). Cotton swabs were used as the medium for each odor stimulus. Each pup was habituated to a clean cage equipped with a clean dry cotton swab for 30 min. The test consisted of the presentation of several odors in a sequence of 15× 2 min: 3 water, 3 almond odor, 3 banana odor, 3 social odor 1, and 3 social odor 2. The almond and banana odors were made using food flavoring. All the nonsocial odors were prepared by dipping the swab in the respective solution for 2 s. The two social odors were obtained by swabbing two cages of unfamiliar male rats. Exploration was defined as a distance of <1 cm between the subject’s nose and the swab. Tissue Collection Some rats in each group were euthanized by rapid decapitation on PND 22 to avoid the known effects of anesthetics on neurochemistry, allowing us to assess the immediate and persistent effects of PM2.5 exposure on the developing rats CNS. The hippocampus and prefrontal cortex were isolated, on ice, and were immediately frozen in liquid nitrogen. The tissues were then stored at –80 °C until use. Proinflammatory Cytokine Evaluation With Enzyme-linked Immunosorbent Assay The frozen rat hippocampus and prefrontal cortex were homogenized in phosphate-buffered saline. Total protein was measured using the bicinchoninic acid assay (Boster, Wuhan, China). The interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (BlueGene Biotech, Shanghai, China) following the manufacturer’s instructions. Determination of Gene Expression by Quantitative Real-Time PCR Total RNA was extracted from homogenized hippocampi ipsilateral using TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa Bio, Dalian, China) according to the manufacturer’s protocol. Reverse transcription was conducted using a Transcriptor First Strand cDNA Synthesis Kit (TaKaRa Bio, Dalian, China). Quantitative real-time PCR (qPCR) was finally performed to specifically amplify contactin associated protein like-2 (Cntnap2), SH3 and multiple ankyrin repeat domains 3 (Shank3), neurexin-1 (Nrxn1), methyl CpG-binding protein 2 (Mecp2), fragile X mental retardation 1 (Fmr1), glial fibrillary acidic protein (Gfap), and ionized calcium-binding adapter molecule 1 (Iba1). β-Actin gene was used as an internal control. Primer sequences are shown in Supplementary Table 1. The qPCR was performed under the following thermal cycling conditions: one cycle of 3 min at 95 °C, followed by 39 cycles at 95 °C for 5 s, 58 °C for 10 s, and 72 °C for 20 s, using Takara PCR Thermal Cycler Dice Real Time system (TaKaRa Bio, Dalian, China). For relative quantification, gene expression was normalized to expression of β-actin housekeeping gene and compared by 2−ΔΔCt method. Western Blot Analysis Hippocampal tissues were homogenized in RIPA lysis buffer (Solarbio, China). Proteins were separated in SDS-PAGE gel and then transferred to PVDF membrane (Millipore). Following incubation in blocking buffer (Tris-buffered saline with 5% nonfat dry milk and 0.1% Tween 20) and probed with primary antibodies directed to GFAP, IBA1, and β-actin (Proteintech), SHANK3 (Abcam). The peroxidase conjugated Affinipure goat anti-mouse and goat anti-rabbit IgG (Proteintech) were used as special secondary antibodies. The proteins were visualized using an enhanced chemiluminescence solution (Millipore). Densitometric analysis for quantification was performed by using Gel-Pro Analyzer software (Gel-Pro Analyzer v6.0, Media Cibernetics, USA). Statistical Analysis All data were analyzed using GraphPad software (GraphPad Prism v6.0, GraphPad Software, San Diego, CA). Two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test was used for analysis of the three-chambered social test and NOR test results. A repeated-measures three-way ANOVA (treatment × odor × presentation) followed by Tukey’s test was used for analysis of olfactory habituation/dishabituation test results, whereas an one-way ANOVA followed by Tukey’s test was used for the analysis of the other test results. Multiple comparisons correction was not done. The data are presented as means ± standard error of the mean. p < .05 was considered to indicate statistical significance. RESULTS Analysis of PM2.5 The various chemical components of PM2.5 are presented in Supplementary Table 2. Benzo(b)fluoranthene was the prevalent PAH, whereas zinc and lead represented the most abundant metals. Naphthalene, acenaphthylene, acenaphthene, aluorene, aibenzo(a,h)anthracene, and arsenic were not detected. Rats Exposed to PM2.5 Exhibit Behavioral Abnormalities Related to ASD The main objective of this study was to assess whether postnatal air pollution exposure could induce childhood autistic traits in a PM2.5-exposed rat model. Behavior is easily affected by the previous encounters. There are many behavioral tests in this study, in order to eliminate possible confounds, we set a blank control group to indicate the impact of intranasal instillation treatment on the behavioral outcomes of pups. There was no significant change between the control and saline groups in any of the analyses performed in this study, which suggests that instillation of saline did not affect rat behavior. Hence, the data of the control group were not included in the between-group comparisons. Communication deficit is a hallmark of ASD. USVs emitted by pups separated from their mother and littermates are used to assess social communication between mice/rats (Servadio et al., 2015). In order to test communication, USVs were recorded for 5 min from PM2.5-exposed or control rat pups. The results showed a significantly fewer number of USVs in PM2.5-exposed pups (Figure 1A and Supplementary Figure 2, F(3, 37) = 76.69, p < .001), and revealed a dose–response relationship. These data suggest that the communication development of the exposed pups lagged behind that of control pups. Figure 1. View largeDownload slide Particulate matter (PM2.5)-exposed rats exhibit communicative deficits and abnormal marble-burying behaviors. A, The total number of ultrasonic vocalizations (USVs) made by pups after intranasal instillation of 2 µg/g (Low) or 20 µg/g (High) PM2.5 solution or vehicle (Saline). B, Quantification of total number of marbles buried during the marble-burying test. C, Amount of time spent sniffing water and odor-soaked swabs in olfactory habituation/dishabituation test. Error bars represent the standard error of the mean (SEM). *p < .05, **p < .01, ***p < .001 (n = 9–11 per group). Figure 1. View largeDownload slide Particulate matter (PM2.5)-exposed rats exhibit communicative deficits and abnormal marble-burying behaviors. A, The total number of ultrasonic vocalizations (USVs) made by pups after intranasal instillation of 2 µg/g (Low) or 20 µg/g (High) PM2.5 solution or vehicle (Saline). B, Quantification of total number of marbles buried during the marble-burying test. C, Amount of time spent sniffing water and odor-soaked swabs in olfactory habituation/dishabituation test. Error bars represent the standard error of the mean (SEM). *p < .05, **p < .01, ***p < .001 (n = 9–11 per group). The OHD test measures olfaction, memory, and social communication (Servadio et al., 2015). Significant habituation and dishabituation to nonsocial and social odors were observed in all group animals in this test (Figure 1C, main effect of treatment: F(3, 35) = 7.049, p = .0008; main effect of odor: F(14, 490) = 102.5, p < .0001; main effect of presentation: F(35, 490) = 0.7995, p < .0001; treatment × odor interaction: F(42, 490) = 0.7157, p = .9095; treatment × presentation interaction: F(42, 490) = 0.6584, p = .6243; presentation ×odor interaction: F(42, 490) = .9852, p = .8465). The trend of sniffing during the triple presentation of each odor was consistent. In the nonsocial odor phase, moderate sniffing behavior was elicited by the first presentation of an odor-wet swab, which declined across the second and third exposure (habituation). The first presentation of a social odor elicited significantly more sniffing (dishabituation), which then declined across the second and third presentations. The sniffing time of rats exposed to high-dose PM2.5 was significantly less than that in the unexposed rats during the first presentation of social odor 1 and social odor 2, whereas no difference was noted for nonsocial cues and for the second and third presentations of social odor cues. Social interaction deficits are one of the core characteristic features of ASD. The three-chambered sociability test has been commonly used to assess the social behavior of rodent models of ASD (Crawley, 2007). In the habituation session of this test, only the low dose animals spent less time in the left chamber than in the center, suggested that subjects almost had no a priori preference for either chamber before the introduction of a social target (Figure 2A, main effect of treatment: F(3, 105) = 0.1374, p = .9375). In the sociability session, all group animals showed a preference for the social target; however, the interaction time of the high-dose exposure group was significantly shorter than that of the other groups (Figure 2B, main effect of treatment: F(1, 70) =200.2, p < .0001). Furthermore, in the social novelty session, rats from the high-dose exposure group spent an equal amount of time interacting with novel and familiar rats, thus indicating no preference for social novelty, whereas rats from other groups spent more time interacting with the novel animal (Figure 2C, main effect of treatment: F(1, 70) = 72.93, p < .0001); these findings indicate that high-dose PM2.5-exposure may affect the sociability of rats. Figure 2. View largeDownload slide Neonatal PM2.5-exposed rats show social abnormalities. A–C, Three-chambered social test. A, Time spent in each chambers during habituation. B, Time spent sniffing the stranger rat and the empty cup during the sociability test. C, Time spent sniffing the familiar and novel rat during the social novelty test. D–F, Novel object recognition test. D, Mean total time spent sniffing two similar objects during 5 min. E, Mean total time exploring the novel and familiar object during 5 min. F, The percentage of object discrimination ([Novel object interaction/total interaction with both objects] × 100). Error bars represent the SEM. *p < .05, **p < .01, ***p < .001 (n = 9–11 per group). Figure 2. View largeDownload slide Neonatal PM2.5-exposed rats show social abnormalities. A–C, Three-chambered social test. A, Time spent in each chambers during habituation. B, Time spent sniffing the stranger rat and the empty cup during the sociability test. C, Time spent sniffing the familiar and novel rat during the social novelty test. D–F, Novel object recognition test. D, Mean total time spent sniffing two similar objects during 5 min. E, Mean total time exploring the novel and familiar object during 5 min. F, The percentage of object discrimination ([Novel object interaction/total interaction with both objects] × 100). Error bars represent the SEM. *p < .05, **p < .01, ***p < .001 (n = 9–11 per group). The marble burying test is commonly used to detect repetitive digging behavior (Servadio et al., 2015). In the current study, significant differences in marble-burying behavior were observed between PM2.5-exposed and control rats (Figure 1B and Supplementary Figure 3, F(3, 35) = 37.56, p < .001). PM2.5-exposed rats showed little interest in burying marbles. In many cases, it appeared that the PM2.5-exposed rats did not even exhibit digging behavior, and that the marbles moved only because the rats walked over them. The NOR test is used to evaluate cognition, particularly recognition memory, in rodents (Leger et al., 2013). During the object familiarization session of this test, all four groups spent an equal amount of time exploring the two identical objects, indicating that rats had no preference for any particular side (Figure 2D, main effect of treatment: F(1, 70) = 0.09545, p = .7583). During the object recognition session, all groups, except for the high-dose exposure group, showed a preference for the novel object; in fact, PM2.5-exposed rats spent significantly less time exploring the novel object than did control animals (Figure 2E, main effect of treatment: F(1, 70) = 92.43, p < .0001). Moreover, the discrimination index in exposed rats was reduced, relative to the control animals (Figure 2F, F(3, 34) = 12.96, p < .0001). Taken the results of marble burying test together, suggesting that high-dose animals might exhibit novelty avoidance compared with the unexposed ones. PM2.5-Exposed Rats Exhibit Abnormal Inflammatory Changes Many autistic individuals display immune abnormalities. In order to evaluate the effect of PM2.5 exposure on the production of proinflammatory cytokine, ELISA assays were used to measure the levels of IL-1β, IL-6, and TNF-α in the hippocampus and prefrontal cortex. After 2 weeks of treatment, the IL-1β and TNF-α levels significantly increased in the hippocampus and prefrontal cortex in both the 2 and 20 µg/g PM2.5-exposed rats. In particular, the hippocampal IL-1β levels in the 2 and 20 µg/g PM2.5-exposed rats were 1.6- and 2.7-fold higher than those in the vehicle-treated rats (Figure 3A, F(3, 28) = 68.19, p < .0001), whereas the cortical IL-1β levels were 2.2- and 5.1-fold higher, respectively (Figure 3D, F(3, 28) = 166.6, p < .0001). Furthermore, the hippocampal TNF-α levels increased by 1.9- and 3.8-fold, whereas the cortical TNF-α levels were 2.2- and 4.6-fold higher in the 2 and 20 µg/g PM2.5-exposed rats when compared with the vehicle-treated rats, respectively (Figure 3C, F(3, 28) = 116.8, p < .0001; and Figure 3F, F(3, 28) = 245.9, p < .0001). Only 20 µg/g PM2.5-exposure led to an increase in IL-6 levels in the hippocampus (2.0-fold) and prefrontal cortex (3.5-fold) (Figure 3B, F(3, 28) = 85.65, p < .0001; and Figure 3E, F(3, 28) = 33.95, p < .0001). Figure 3. View largeDownload slide Proinflammatory cytokine levels after neonatal exposure to PM2.5. A–F, IL-1β, IL-6, and TNF-α levels in the hippocampus (A, B, and C, respectively) and cortex (D, E, and F, respectively) of rats in each group were measured using the ELISA assay. Cytokine levels are presented as pg/mg protein. Error bars represent the SEM. *p < .05, **p < .01, ***p < .001 (n = 6 per group). Figure 3. View largeDownload slide Proinflammatory cytokine levels after neonatal exposure to PM2.5. A–F, IL-1β, IL-6, and TNF-α levels in the hippocampus (A, B, and C, respectively) and cortex (D, E, and F, respectively) of rats in each group were measured using the ELISA assay. Cytokine levels are presented as pg/mg protein. Error bars represent the SEM. *p < .05, **p < .01, ***p < .001 (n = 6 per group). GFAP, an astrocyte marker, and IBA1, a microglia marker, can be used to detect glial activation during neuroinflammation. The qPCR results revealed that Gfap (F(3, 32) = 57.89, p < .0001) and Iba1 (F(3, 32) = 27.35, p < .0001) mRNA expression were significantly increased in both the treatment groups, although the high-dose group displayed a greater elevation (Figs. 4F and 4G). Furthermore, enhanced expression of GFAP (F(3, 28) = 36.07, p < .0001) and IBA1 (F(3, 28) = 28.4, p < .0001) protein was also observed by immunoblotting analysis in the 20 µg/g PM2.5-exposed group (Figs. 4J–L). These data indicate that PM2.5-exposure may lead to hippocampal glial activation, and may thus play an important role in the initiation of neuroinflammation. Figure 4. View large Download slide Expression levels of autism spectrum disorder candidate genes or protein in the hippocampus after PM2.5 exposure. Hippocampal Cntnap2 (A), Fmr1 (B), Mecp2 (C), Nrxn1 (D), Shank3 (E), Gfap (F), and Iba1 (G) mRNA expression levels of rats in each group measured by quantitative real-time PCR. H, Expression of SHANK3 protein in the hippocampus on western blot analysis. I, The densities of the immunoreactive bands of SHANK3 were quantified and presented as the percent change relative to the control samples. K, Western blot analysis of GFAP and IBA1 in the hippocampus in the control and exposure group. Quantification of the immunoreactive band densities of GFAP (J) and IBA1 (L). Full-length blots are presented in Supplementary Figure 4. Error bars represent the SEM. **p < .01 and ***p < .001 (n = 6 per group). Figure 4. View large Download slide Expression levels of autism spectrum disorder candidate genes or protein in the hippocampus after PM2.5 exposure. Hippocampal Cntnap2 (A), Fmr1 (B), Mecp2 (C), Nrxn1 (D), Shank3 (E), Gfap (F), and Iba1 (G) mRNA expression levels of rats in each group measured by quantitative real-time PCR. H, Expression of SHANK3 protein in the hippocampus on western blot analysis. I, The densities of the immunoreactive bands of SHANK3 were quantified and presented as the percent change relative to the control samples. K, Western blot analysis of GFAP and IBA1 in the hippocampus in the control and exposure group. Quantification of the immunoreactive band densities of GFAP (J) and IBA1 (L). Full-length blots are presented in Supplementary Figure 4. Error bars represent the SEM. **p < .01 and ***p < .001 (n = 6 per group). Exposure to PM2.5 Induces Altered Gene Expression Involved in ASD It has long been suspected that genetics and environmental exposures can interact to increase ASD risk in certain individuals. qPCR was conducted to measure the hippocampal mRNA levels of five ASD candidate genes at PND 22 in male controls and PM2.5-exposed rats. Unexpectedly, we found that Shank3 was significantly downregulated in high-dose PM2.5-exposed rats (Figure 4E, F(3, 32) = 16.81, p < .0001), whereas no effect of PM2.5-exposure on Cntnap2 (F(3, 32) = 2.103, p = .1194), Nrxn1 (F(3, 32) = 2.269, p = .0994), Mecp2 (F(3, 32) = 0.9495, p = .4284), or Fmr1 (F(3, 32) = 2.25, p = .1015) was detected (Figs. 4A–D). We verified the change in SHANK3 protein expression by western blot analysis, and found a similar trend to that of the Shank3 mRNA levels (Figs. 4H and 4I, F(3, 28) = 14.09, p < .0001). These data suggest that postnatal exposure to PM2.5 alters the expression of the genes related to ASD. DISCUSSION The brain growth spurt (BGS) is an important period of brain development which is characterized by a number of rapid processes in the brain, such as neural connections establishment, synaptogenesis and proliferation of glia cells accompany with myelination (Rice and Barone, 2000). In mammals, the period of BGS in terms of onset and duration varies from species to species. In humans, it begins during the third trimester of pregnancy and continues throughout the first 2 years of life, whereas in rodents, such as mice and rats, the BGS is neonatal, spanning the first 3–4 weeks of life (Viberg et al., 2008). Most of the different transmitter systems undergo qualitative and quantitative changes during this developmental period. And this is also the period when brain is vulnerable to environmental toxicants. PM2.5 is an air pollutant that elicits major concerns regarding public health, and contains toxic substances, such as PAHs, metals, organic matter, and elemental carbon. In the present study, concentrations of PAHs and metals in PM2.5, particularly benzo[b]fluoranthene, benzo[a]anthracene, benzo[k]fluoranthene, benzo[a]pyrene, pyrene, lead, zinc, and manganese, are considered of potential neurotoxic risk, in accordance with the reported developmental neurotoxicity because these toxicants has been associated with the triggering of inflammation, generation of ROS, and lipid peroxidation (Fortoul et al., 2015; Jedrychowski et al., 2015; Peterson et al., 2015). Previous studies have suggested that PM2.5-exposure during pregnancy or childhood is related to neurodevelopmental delays (Basagana et al., 2016). A growing number of epidemiological studies have suggested that ambient PM2.5 pollution has adverse effects on maternal and fetal development. Exposure to elevated levels of PM2.5 in early life has been correlated with increased frequency of ASD. Because the diagnosis of ASD is mainly based on behavioral criteria, many behavioral approaches have been used to detect ASD-like symptoms in rodent models, with consideration of known clinical manifestations in humans (Pasciuto et al., 2015; Servadio et al., 2015). Here, we performed a battery of behavioral tests; we demonstrated that an early postnatal PM2.5-exposed male rats exhibit communication deficits, poor social interaction, and novelty avoidance, which are all autistic-like behaviors. The deficits in communication reported here in PM2.5-exposed rats were assessed by both an analysis of USVs emitted by pups isolated from their dam and by the OHD. Isolation-induced USVs are regarded as important communication signals eliciting maternal care behaviors (Branchi et al., 2001; Scattoni et al., 2009). Abnormal isolation-induced USVs have been found in several genetic and environmental rodent models of ASDs (Kogan et al., 2015; Nakatani et al., 2009). USV follows a fairly predictable developmental pattern in infant rats; USVs emerge a day or two after birth, peak during the first week at approximately 100/min, and then gradually decline by 17–20 days after birth (Winslow, 2009). Therefore, we calculated the number of USVs emitted by maternally isolated pups on PND 16 before they declined completely, and found that PM2.5-exposed pups vocalized significantly less than unexposed pups, indicating that PM2.5-exposure affected the social communication of rats to some degree. This observation could easily translate to the atypical cry shown in infants at risk of ASD (Esposito and Venuti, 2010; Sullivan et al., 2013). Olfaction can also be used to understand social interaction, which is considered as another level of rodent communication (Silverman et al., 2010). Novel odors initially elicit exploratory sniffing, but the sniffing time reduces with repeated exposure to the same odor. Similarly, social odors elicit considerably higher levels of sniffing than nonsocial odors (Pasciuto et al., 2015). In the present study, normal olfactory habituation/dishabituation was observed in the all four groups. However, rats exposed to high-dose PM2.5 showed significantly less sniffing time during the first presentation of social odors. This deficit might contribute to the poor social interaction in three-chambered social test and be attributed to impairments in the main olfactory system rather than the accessory olfactory system because many studies have now shown that the brain often processes social cues differently than non-social ones (Laugeray et al., 2014). The marble burying in rodents is thought to reflect repetitive and perseverative behavior, and most ASD rodent models display high levels of marble burying (Amodeo et al., 2012; Santini et al., 2013). We intended to assess the repetitive behavior after PM2.5-exposure using this test; however, unexpectedly, we found that PM2.5-exposed rats barely buried the marbles. Moreover, the exposed rats showed little interest in exploring unfamiliar objects in the NOR test. These abnormal behaviors can perhaps be interpreted as an avoidance of novel inanimate objects, which is also found in children with ASD (Anckarsater et al., 2006; Kootz et al., 1982), and this neophobic tendency might be responsible to the social deficit observed in the three-chambered social interaction task. Actually, the OHD test and NOR test were also designed as control experiments in order to eliminate possible confounds to the specific tests, including olfactory deficit, general memory impairment, and lack of novelty preference. And what we found in these tests led to the discovery of lack of novelty preference was a social memory deficit (social novelty test) or a social attention deficit (OHD). Further evidence for the link between neonatal exposure to PM2.5 and ASD-like deficits can be found in the changes in certain ASD susceptibility genes in exposed rats. By predicting the association of gene expression with environmental affects and behaviors from the online database, we targeted and evaluated five ASD candidate genes, in the brain regions of the male rats by qPCR. We found that PM2.5-exposure significantly downregulated the expression of Shank3, a similar trend was also observed for the corresponding protein level. SHANK3 is a synaptic scaffolding protein that is predominantly found in the postsynaptic region of excitatory synapses, and plays important roles in the formation, maturation, and maintenance of synapses (Uchino and Waga, 2013). Mutations in SHANK3 have been identified in ASD patients (Durand et al., 2007; Nemirovsky et al., 2015; Uchino and Waga, 2013). Furthermore, rodent models based on a Shank3 mutation exhibit phenotypes related to ASD. Compared with wild-type animals, Shank3-mutant mice/rats show abnormal social interactions (Wang et al., 2011; Yang et al., 2012) and altered USVs (Bozdagi et al., 2010), consistent with the results in the present study. Shank3ΔC/ΔC mice failed to show a preference for the novel social target in the three-chambered social interaction test (Kouser et al., 2013). Reduced social interaction and abnormal social novelty recognition was also found in Shank3B–/– mice (Peca et al., 2011). Most importantly, we are confused to find a reduced marble burying in PM2.5-exposed rats until reduction of Shank3 was determined. In contrast to most other ASD models, marble burying behavior is significantly reduced in Shank3 mutant model, which was also be interpreted as novelty avoidance (Jaramillo et al., 2016; Kouser et al., 2013; Speed et al., 2015). Furthermore, a recent study (Wei et al., 2016) reported that PM2.5 and its extracts interfered with gene-specific DNA methylation and mRNA expression of ASD candidate genes in neuron cells, and that Shank3 was downregulated due to upregulation of promoter DNA methylation. Increasing evidence has shown widespread immune alterations in the brains and periphery of ASD individuals, and early life immune disruption/inflammation is associated with ASD (Noriega and Savelkoul, 2014; Theoharides et al., 2013). In the present study, we showed that PM2.5-exposure significantly increased the expression of IL-1β, IL-6, and TNF-α in the brains of neonatal rats. Elevated expression of GFAP and IBA1, which are markers of astrocyte and microglial activation, was also noted in the exposed animals. Consistent with these findings, neuroinflammatory responses, reflected by increased CNS cytokine levels and glial activation across multiple brain regions, were observed in male mice exposed to ultrafine concentrated ambient particles (Allen et al., 2014). Neuroglial cells contribute in a series ways to the regulation of immune responses in the CNS and play important roles in neuronal function and homeostasis. Astrocytes can secrete proinflammatory cytokines (ie, IL-1β, IL-6, and TNF-α), chemokines, and metalloproteinases that can magnify immune reactions within the CNS after activated by environmental triggers. Similarly, the activation of microglial may result in dysfunction of synaptic stripping, cortical plasticity, and immune surveillance (Pardo et al., 2005). TNF-α is produced by various of cells during inflammatory events (Gruys et al., 2005) and it can regulate neuronal cell proliferation or cell death, and play an important role in synaptic pruning (Cacci et al., 2005; Stellwagen and Malenka, 2006). IL-1β and IL-6 can also influence neuronal survival, proliferation, synapse formation, migration, and differentiation (Onore et al., 2012). Collectively these findings suggest that cytokines are both necessary for normal neurodevelopment and behavior and that any perturbation in the cytokine network can impact neurodevelopment and may result in behavioral abnormalities. Because astrocytes and microglia are not altered in Shank3 transgenic autism model (Cope et al., 2016), the neuroinflammatory response and Shank3 abnormalities may be the parallel consequences of PM2.5 exposure, and perhaps cause neurological impairments, particularly synaptic deficit, may be responsible for the pathogenesis and ASD-like symptoms in male rats. A limitation in our study is only the effects of postnatal PM2.5 exposure of male rats were elevated. And in this study, a series of biological and behavioral measures were taken from a single experiment on a single cohort of rats whereas multiple comparisons correction was not done. Another limitation is the exposure dose of the rat pups which estimated from the respiratory rate of adult rats made the dose higher than actual exposure level. In addition, instillation exposure was used rather than a more physiologically relevant inhalation exposure. Nasal instillation has been shown to be an effective delivery route for acute exposures of PM2.5 (Bai et al., 2015; Riva et al., 2011). However, the differences in the relative size of the olfactory mucosa and olfactory bulb between rats and humans (Dorman et al., 2002), and the different dose rate and deposition pattern between a single bolus dose of PM2.5 and real conditions, made it might not be indicative of human exposure level. This model of administration may not necessarily reflect the actual PM2.5 exposure in human. Nevertheless, it is very useful for comparative studies of particle toxicity. In summary, our combined results suggest that neonatal PM2.5-exposure may cause communication deficits, poor social interaction, and novelty avoidance, and may also induce downregulation of Shank3 expression, and increased neuroinflammatory responses in male rats. To our knowledge, this is the first report to show PM2.5-induced phenotype related to the core symptoms of ASD. Our findings may provide novel experimental evidence supporting the hypothesis that an etiological association exists between particulate air pollution exposure and the pathophysiology of ASD. To confirm the contribution of air pollution to ASD risk, additional laboratory studies should be performed using a prenatal exposure model, and by ensuring whole-body inhalation exposure, which is more akin to the human context. 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Published: Mar 1, 2018
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