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Particulate Matter Triggers Depressive-Like Response Associated With Modulation of Inflammatory Cytokine Homeostasis and Brain-Derived Neurotrophic Factor Signaling Pathway in Mice

Particulate Matter Triggers Depressive-Like Response Associated With Modulation of Inflammatory... Abstract Particulate matter (PM) exposure may contribute to depressive-like response in mice. However, few studies have evaluated the adaptive impacts of long-term PM exposure on depressive-like response associated with systemic inflammation and brain-derived neurotrophic factor (BDNF) signaling pathway. We studied the association among depressive-like behaviors, mRNA levels of pro and anti-inflammatory cytokines, and the expression of BDNF signaling pathway in mice by long-term PM exposure. C57BL/6 male mice were exposed to ambient air alongside control mice breathing air filtered through a high-efficiency air PM (HEPA) filter. Depressive-like behaviors were assessed together with proinflammatory, anti-inflammatory cytokine mRNA levels and the modulation of BDNF pathway in hippocampus and olfactory-bulb of mice exposed to PM for 4, 8, and 12 weeks. Exposure to HEPA-filtered air for 4 weeks may exert antidepressant like effects in mice. Proinflammatory cytokines were up-regulated while the expression of BDNF, its high-affinity receptor tropomyosin-related kinase B (TrkB), and the transcription factor (cyclic adenosine monophosphate)-response element-binding protein (CREB) were down-regulated in ambient air mice. However, after 8 weeks, there was no significant difference in the rate of depressive-like behaviors between the 2 groups. After 12 weeks, mice exposed to ambient air again had a higher rate of depressive-like behaviors, significant up-regulation of proinflammatory cytokines, down-regulation of interleukin-10, BDNF, TrkB, and CREB than HEPA mice. Ultrafine PM in brain tissues of mice exposed to ambient air was observed. Our results suggest continuous high-level PM exposure alters the depressive-like response in mice and induces a damage-repair-imbalance reaction. ambient particulate matter, depression, inflammatory cytokines, brain-derived neurotrophic factor (BDNF), hippocampus, olfactory bulb Urban air pollution has become one of the most serious environmental challenges facing humankind in recent decades. Atmospheric particulate matter (PM), which has been classified as a group 1 contaminant by the International Agency for Research on Cancer (IARC) (Feng et al., 2016; Yue et al., 2015) has been extensively implicated in the potentiation of cardiopulmonary morbidity and mortality (Devlin et al., 2014; Wang et al., 2013a,b; Ying et al., 2013). Moreover, according to recent studies, PM exposure contributes to the incidence and development of neuropsychological disorders, including Alzheimer’s disease, Parkinson’s disease, and depression (Block et al., 2009; Dastgheib et al., 2015; Tong et al., 2016). Atmospheric fine particles or ultrafine particles can cross the blood-brain barrier or otherwise enter the brain through the olfactory nerve, facial trigeminal nerve endings, and other routes, causing nerve inflammation, or necrosis directly (Calderon-Garciduenas et al., 2008; Lucchini et al., 2012; Ziemssen et al., 2008). Oxidative stress and systemic inflammation are the central mechanisms by which PM exposure poses a health threat (Carll et al., 2013; Cevallos et al., 2017; Pardo et al., 2015; Wang et al., 2017). PM exposure has potential ability to activate proinflammatory pathways by inducing the expression of inflammatory cytokines in central nervous system (CNS), such as Interleukin-1β (IL-1β), Interleukin-6 (IL-6), antitumor necrosis factor α (TNFα), interferon-γ (IFN-γ) (Block et al., 2009; Cevallos et al., 2017). According to the cytokine hypothesis of depression, proinflammatory cytokines released in excess can lead to neuroinflammation and impaired neurotransmitter and neurotrophin signaling, which may be followed by symptoms typical of depression, such as anorexia, lethargy, lack of pleasure, weight loss, and decreased activity (Raison and Miller, 2003). Conversely, anti-inflammatory cytokines, such as IL-10 and interleukin-1-receptor antagonist (IL-1RA), protect tissues and cells by inhibiting an excessive immune response and can alleviate the depressive-like response in animal models (Van Strien et al., 2010; Watkins et al., 1995). Proinflammatory and anti-inflammatory cytokines interact in a coordinated response to modulate inflammatory processes caused by PM exposure (Delfino et al., 2002; Zhao et al., 2016). Nevertheless, most studies, including those on acute toxicity and in vitro experiments, have failed to evaluate the long-term adaptive, compensatory impacts of PM exposure on systemic inflammation in the CNS. In addition to inflammatory processes, depressive phenotypes are also frequently linked to impairments in neural plasticity, characterized by the altered expression of so-called plasticity genes, such as brain-derived neurotrophic factor (BDNF), its high-affinity receptor tropomyosin-related kinase B (TrkB), and the transcription factor (cyclic adenosine monophosphate)-response-element-binding protein (CREB) (Guo et al., 2014; Młyniec and Nowak, 2015; Vines et al., 2012). Clinical findings have led to the neurotrophin hypothesis of depression, which invokes the suppression of the BDNF-TrkB-CREB pathway (Björkholm and Monteggia, 2016). However, only several studies have focused on acute exposure to PM. Liu et al. (2017) have conducted a 130-min exposure with fine PM inhalation, indicating that no change of BDNF, IL-6, and TNFα in blood were observed in healthy participants. In the study of Bos et al. (2011), serum BDNF levels were not increased in volunteers who had cycled near a major traffic road for 20 min but they were significantly increased when the volunteers had cycled in a room supplied with filtered air. Until now, little information is known about the systematic study concerning the effect of long-term PM exposure on BDNF-TrkB-CREB pathway. Previous studies of the toxicity of long-term ambient particulate air pollution were usually conducted in cities with relatively low PM concentrations, such as a study of neurotoxicity conducted in the United States, where the mean PM2.5 concentration was 16.85 µg/m3 (Fonken et al., 2011) and a study of reproductive toxicity conducted in Canada, where the mean total suspended particle (TSP) concentration was 93.8 ± 17.0 µg/m3 (Yauk et al., 2008). There is limited information on the nervous diseases caused by the much higher ambient PM exposure in China. In this study, C57BL/6 male mice were exposed to either ambient air, and thus to PM, or high-efficiency air PM (HEPA)-filtered air. The aim was to evaluate the impacts of long-term PM exposure on the depressive-like responses of the mice. The study was carried out in Nanjing, a typical mega-city in Eastern China. We examined depressive-like behaviors, the changes in the relative mRNA levels of pro and anti-inflammatory cytokines, and the expression of the BDNF-TrkB-CREB pathway in order to provide new insights into the regulatory mechanism by which long-term, high-level PM exposure contributes to depression in humans. MATERIALS AND METHODS Animals and PM exposure The Comparative Medicine Center of Yangzhou University provided the 6- to 8-week-old healthy C57BL/6 male mice in SPF (Specific Pathogen Free) grade. Once the mice were bought and brought to our laboratory, they were put into the HEPA chamber immediately for at least 1 week to adapt to the basic experimental environment. Then the healthy mice were randomly assigned into 2 experimental chambers for either ambient air exposure or HEPA-filtered air exposure. The chambers (2.0 × 0.6 × 0.8 m) were made of polymethyl methacrylate and placed separately in a vinyl utility shed (2.4 × 2.4 × 1.8 m), such that the mice were not exposed to direct wind or sunlight. The windows (1.7 × 1.0 m) of the shed furnished with nylon mesh (1.0 × 1.0-cm mesh size) could keep the consistency of air quality inside and outside of the shed and provided enough light for the mice. The HEPA chamber was installed a HEPA air purifier (True-HEPA, WINIX model WAC6300, Bemis Manufacturing, Sheboygan Falls, Wisconsin), which was mainly composed of HEPA filter and draught fan, can remove at least 99.97% of particles with a diameter of 0.3 µm, including dust, smoke, pollen, pet dander, and other allergens. The size of HEPA filter is 39 × 27 × 2 cm and replaced every 4 weeks. Two draught fans with the same type and airflow rate were set in front of the 2 chambers to let the air flow in, and then the air was discharged to ambient environment through the pipes connected to the end of the 2 chambers. Two electric heating devices could maintain the temperature of chambers no <18°C ± 1°C. Thus, the only difference between the 2 experimental chambers was that the HEPA filter was not installed for the ambient air exposure group. The mice in both groups were exposed for 4, 8, or 12 weeks, from November 7, 2015 to January 30, 2016. After behavioral testing, the mice were re-exposed to original environment for 3 days before tissue collection. During the exposure period, the mice were fed commercial mouse chow and bottled water from the same sources. All experimental protocols were approved by Model Animal Research Center of Nanjing University. Animals were treated humanely with due consideration to the alleviation of distress and discomfort. PM2.5 sampling and analysis Nanjing (32°03′N, 118°46′E) is an important industrial production area and the main transportation hub in Eastern China. It covers approximately 6600 km2 and had a population of more than 8.1 million. It is in the north subtropical monsoon climate zone. The exposure shed was set up on the Xianlin Campus of Nanjing University, where is near the northern industrial zones of the city (Supplementary Figure 1). PM concentrations in the HEPA-filtered air and ambient air were monitored by 2 DUSTMATE monitors (Turnkey Instruments Ltd., UK). The 24-h sampling of PM2.5 in ambient air was carried out by 2 high-volume PM samplers (model TE-6070VFC, Tisch Environmental, Inc. Cleves, Ohio) operating at a flow rate of 1.13 m3/min on a quartz fiber filter (Whatman QM-A) filter. The samplers were set up outside, approximately 3 m from the experimental shed. The main constituents of PM2.5, including water-soluble inorganic ions, carbonaceous fractions, and trace elements (TEs), were analyzed. A chemical mass closure was introduced to specifically obtain information on the chemical composition of PM2.5 collected in the study area. Details of chemical analysis and chemical mass closure can be found in our previous report (Li et al., 2016). Behavioral testing Behavioral tests were carried out by 2 trained observers blinded to the groups. At least 12 mice per group were used for each behavioral test after each exposure period to ensure experimental accuracy. The tail suspension test (TST) is a widely used test of depressive-like behavior in which depression is inferred from the increased duration of immobility. Mice both acoustically and visually isolated were suspended 50 cm above the floor using adhesive tape placed approximately 1 cm from the tip of their tails. A mouse was judged to be immobile when it hung down passively/ceased moving its limbs and body, making only those movements necessary to breathe. The total duration of immobility was recorded during the last 4 min of the 6-min long testing period (Wang et al., 2013c). To further assess depressogenic-like effect of ambient air exposure, the mice were also evaluated in the forced swim test (FST). Briefly, the mice were individually forced to swim in an open cylindrical container (diameter 10 cm, height 25 cm), containing water at a depth of 19 cm and a temperature of 25°C ± 1°C. The mice were considered to be immobile when they ceased struggling and remained floating motionless in the water, making only the small movements necessary to keep their head above water. An increase in the duration of immobility is indicative of a depressant-like effect (Kaster et al., 2007). The behaviors were assessed in a 6-min period; the duration of immobility within the last 4 min of the test was recorded. Brain tissue dissection The whole brain of mice was removed from the cut head and placed on an ice-cooled dissection slab. The selected brain areas were immediately snap frozen in liquid nitrogen and stored at −80°C until use. Briefly, the olfactory bulb was in the forepart of the brain and can be removed first. The hippocampus was located in the bottom of cerebral cortex and above the thalamus, which is easily identified by its difference in color from the occipital cortex. To dissect out the hippocampus, we placed the brain on its ventral side, peeled off cortical layer and then removed all the cortex connected to hippocampus. Analysis of gene transcripts Fresh tissues of the hippocampus and olfactory bulb were collected, flash frozen and stored at −80°C. Total RNA was isolated from tissues using Trizol reagent (Invitrogen, USA) according to manufacturer’s instructions. cDNA was synthesized from mRNA using a first-stand cDNA synthesis kit (Fermentas, Lithuania), which was used as a template to perform real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR was carried out using Applied Biosystems StepOnePlus real-time PCR System (LifeTechnologies, USA) according to the manufacturer’s instructions. The qRT-PCR procedure was as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Data were collected and analyzed by using PCR system software based on 2−ΔΔCT threshold cycle method (Livak and Schmittgen, 2001). The GAPDH transcript served as a housekeeping gene. The primers for the amplification of the genes were designed using Primer 5.0 and listed in Supplementary Table 1. qRT-PCR assays were conducted in triplicate for each sample to ensure experimental accuracy. Morphological analyses of the hippocampus and olfactory bulb The hippocampus and olfactory bulb were dissected out from the freshly isolated mouse brains on an ice tray and then fixed for 24 h in a centrifuge tube containing 4% formaldehyde. After routine paraffin embedding and hematoxylin-eosin staining, the 0.5-μm-thick tissue sections were examined by optical microscopy (Nikon Eclipse E100, Japan). Details of this method can be found elsewhere (Ying et al., 2017; Zhang et al., 2010). High-resolution transmission electron microscopy Ambient PM in tissues of hippocampus and olfactory bulb was investigated by high-resolution transmission electron microscopy (HR-TEM) using a FEI Tecnai F20 TEM instrument operated at 200 kV, and equipped with an energy-dispersive X-ray spectroscopy (EDS). When compared with the conventional method for TEM Samples (Maher et al., 2016), we omitted steps of uranium lead double staining to avoid disturbances to EDS analysis. All reagents were prepared from ultrapure Milli-Q water and pre-filtered (<0.1-μm PTFE membrane filter) to preclude any particulate contamination. Bright-, dark-field, and HR-TEM imaging together with elemental analysis with EDS were used to investigate PM in the mouse brain tissues. Statistical analysis The data are presented as the mean ± SEM. All statistical analyses were performed using SPSS software, version 23.0. Data obtained from the behavioral testing of mice exposed to ambient air versus HEPA-filtered air at 4, 8, and 12 weeks were analyzed using a repeated measures 2-way ANOVA, in which the 2 factors were the group and the exposure period. The groups were analyzed individually in a 1-way ANOVA followed by Tukey’s post hoc test. The mean values of the 2 groups were compared using a paired t test. A p value ≤ .05 was considered to indicate statistical significance. RESULTS PM Concentration and Components The PM10, PM2.5, and PM1 levels in the ambient air and HEPA-filtered air during the exposure time are presented in Figure 1. The average concentrations of PM10, PM2.5, and PM1 were 92.65 µg/m3 (range 44.90–188.9 µg/m3), 68.26 µg/m3 (30.70–142.3 µg/m3), and 58.93 µg/m3 (range 25.56–137.7 µg/m3), respectively. About 7.14% and 34.52% of the daily PM10 and PM2.5 concentrations exceeded the 24-h limit of 150 and 75 µg/m3 specified by the Chinese National Ambient Air Quality Standard, respectively. The ratios of PM2.5/PM10 and PM1/PM2.5 were 0.744 and 0.854, respectively, indicating that fine PM, and especially PM1, should be given high priority in assessments of PM pollution. In contrast, in the HEPA-filtered air the average concentrations of TSP, PM10, PM2.5, and PM1 monitored were 9.31 µg/m3 (range 1.64–18.86 µg/m3), 8.26 µg/m3 (1.35–16.86 µg/m3), 6.41 µg/m3 (1.08–14.29 µg/m3), and 2.35 µg/m3 (0.40–5.46 µg/m3). There were considerable differences in the PM concentrations between ambient air and HEPA-filtered air, confirming the rationale and reliability of our experimental model to compare the effects of PM exposure. Figure 1. View largeDownload slide Concentrations of PM10, PM2.5, and PM1 from November 7, 2015 to January 30, 2016 in the ambient air of study area of Nanjing, China (A) and in the HEPA-filtered air (B). Figure 1. View largeDownload slide Concentrations of PM10, PM2.5, and PM1 from November 7, 2015 to January 30, 2016 in the ambient air of study area of Nanjing, China (A) and in the HEPA-filtered air (B). The relative contributions of various compounds to the PM2.5 mass were evaluated by considering secondary inorganic aerosols (SIA, defined as the sum of SO42-, NO3-, and NH4+), organic matter (OM), elemental carbon (EC), mineral dust (MD), sea salt (SS), and TEs. The results are shown in Supplementary Figure 2. The average SIA, OM, EC, SS, MD, and TE levels accounted for 27.41% (range 16.37%–33.95%), 37.91% (30.51%–48.01%), 7.78% (4.98%–11.95%), 2.20% (1.18%–3.73%), 8.25% (3.89%–15.32%), and 2.00% (range 1.09%–3.92%) of the PM2.5 mass, respectively. The unaccounted mass of 14.44% (8.47%–19.86%) was probably due to the presence of water or inadequate estimation of crustal components. The analysis clearly showed that OM and SIA were the main components of PM2.5 during the exposure periods. Depressive-Like Behavior Analyses Depressive-like responses were evaluated by subjecting the mice in both groups to the TST and FST (Figure 2). Depressogenic-like effects of ambient air exposure were determined in 2-way repeated measures ANOVA (p < .001). The effect of the exposure period (p = .001 and .031 for the TST and FST, respectively) and of the interaction between the exposure period and ambient air exposure (p < .001) were also significant, both for the TST and the FST. Figure 2. View largeDownload slide Effects of PM exposure on the depressive-like response in mice subjected to the TST (A) and FST (B). In both, an increased immobility time indicates a depressive-like state. Bars indicate the SEM (n ≥ 12). *p < .05; **p < .01, 2-tailed t test. Figure 2. View largeDownload slide Effects of PM exposure on the depressive-like response in mice subjected to the TST (A) and FST (B). In both, an increased immobility time indicates a depressive-like state. Bars indicate the SEM (n ≥ 12). *p < .05; **p < .01, 2-tailed t test. Mice exposed to ambient air for 4 weeks exhibited a longer immobility time than mice exposed to HEPA-filtered air (p < .01 for both tests). However, there were no significant differences at 8 weeks (p > .05 for both tests), although by 12 weeks the immobility time had again increased significantly in mice exposed to ambient air (p < .01 for both tests). The immobility time of mice exposed to ambient air followed a significant U-shaped fluctuation curve (p < .01 for both tests). It is probably that exposure to HEPA-filtered air exerts antidepressant like effects in mice for TST and FST at 4 and 12 weeks. Thus, we presumed the initial depressive-like response may have been followed by a transient recovery by 8 weeks, after which it was further aggravated. Relative mRNA Expression of Proinflammatory Cytokines Supplementary Tables 2 and 3 showed the relative mRNA expression levels of proinflammatory (IL-1β, TNFα, IL-6, and IFN-γ), anti-inflammatory (IL-10 and IL-1RA), as well as BDNF, TrkB, and CREB in hippocampus and olfactory bulb of mice exposed to ambient air and HEPA-filtered air after 4, 8, and 12 weeks. To better compare the results obtained with the 2 groups, the gene expression levels of the mice exposed to ambient air are presented as percent of the control (HEPA-filtered air) group (Figs. 3–5), and the mRNA expressions of HEPA mice across weeks were shown in Supplementary Figure 6. Figure 3. View largeDownload slide Proinflammatory cytokine expression in mice exposed to PM for 4, 8, and 12 weeks. The data are expressed as the percent in mRNA expression of mice exposed to ambient of HEPA-filtered (control) air. IL-1β, TNFα, IL-6, and IFN-γ expression in the hippocampus (A, C, E, and G, respectively), and in the olfactory bulb (B, D, F, and H, respectively). Bars indicate the SEM (n ≥ 3). *p < .05, **p < .01, 1-tailed t test. Figure 3. View largeDownload slide Proinflammatory cytokine expression in mice exposed to PM for 4, 8, and 12 weeks. The data are expressed as the percent in mRNA expression of mice exposed to ambient of HEPA-filtered (control) air. IL-1β, TNFα, IL-6, and IFN-γ expression in the hippocampus (A, C, E, and G, respectively), and in the olfactory bulb (B, D, F, and H, respectively). Bars indicate the SEM (n ≥ 3). *p < .05, **p < .01, 1-tailed t test. Figure 4. View largeDownload slide Anti-inflammatory cytokine expression in mice exposed to PM for 4, 8, and 12 weeks. The data are expressed as percent in mRNA expression in mice exposed to ambient of HEPA-filtered (control) air. IL-10 and IL-1RA expression in the hippocampus (A and C, respectively), and in the olfactory bulb (B and D, respectively). Bars indicate the SEM (n ≥ 3). *p < .05, **p < 0.01, #p = .0598, 2-tailed t test. Figure 4. View largeDownload slide Anti-inflammatory cytokine expression in mice exposed to PM for 4, 8, and 12 weeks. The data are expressed as percent in mRNA expression in mice exposed to ambient of HEPA-filtered (control) air. IL-10 and IL-1RA expression in the hippocampus (A and C, respectively), and in the olfactory bulb (B and D, respectively). Bars indicate the SEM (n ≥ 3). *p < .05, **p < 0.01, #p = .0598, 2-tailed t test. Figure 5. View largeDownload slide BDNF, TrkB, and CREB mRNA expression in the hippocampus and olfactory bulb of mice exposed to PM for 4, 8, and 12 weeks. The data are expressed as percent in mRNA expression in mice exposed to ambient of HEPA-filtered (control) air. BDNF, CREB, and TrkB expression in the hippocampus (A, C, and E, respectively) and in the olfactory bulb (B, D, and F, respectively). Bars indicate the SEM (n ≥ 3). *p < .05, **p < .01, 1-tailed t test. Figure 5. View largeDownload slide BDNF, TrkB, and CREB mRNA expression in the hippocampus and olfactory bulb of mice exposed to PM for 4, 8, and 12 weeks. The data are expressed as percent in mRNA expression in mice exposed to ambient of HEPA-filtered (control) air. BDNF, CREB, and TrkB expression in the hippocampus (A, C, and E, respectively) and in the olfactory bulb (B, D, and F, respectively). Bars indicate the SEM (n ≥ 3). *p < .05, **p < .01, 1-tailed t test. As seen in Figures 3A, 3C, and 3E, the mRNA expression levels of most of the proinflammatory cytokines were significantly up-regulated in the hippocampus of mice exposed to ambient air: IL-1β (p = .029, .013, and .009 for 4, 8, and 12 weeks, respectively), TNFα (p = .016 and .007 for 4 and 12 weeks, respectively), IL-6 (p = .012 and .001 for 8 and 12 weeks, respectively). PM exposure had no effect on IFN-γ mRNA expression in the hippocampus (p > .05, Figure 3G). For IL-1β, TNFα, and IL-6, their mRNA expression levels were strongly elevated in mice exposed to ambient air for 12 weeks (232.6%, 309.8%, and 418.4% of control for IL-1β, TNFα, and IL-6, respectively; Figure 3) compared with those exposed for 4 and 8 weeks. In contrast to the hippocampus, there were only 4 significant up-regulations of proinflammatory cytokines in the olfactory bulb: IL-1β at 12 weeks (p = .048; Figure 3B), TNFα at 4 and 12 weeks (p = .043 and .011, respectively; Figure 3D), and IL-6 at 12 weeks (p = .035; Figure 3F). PM exposure did not lead to the up-regulation of IFN-γ expression; instead, the levels of this cytokine decreased in mice exposed to ambient air for 4 weeks (p = .016; Figure 3H), indicating differences in the actions and/or various sources of proinflammatory cytokines (Dieme et al., 2012). Similar to mRNA expression in the hippocampus, the largest increases in proinflammatory cytokine mRNA expression in the olfactory bulb of mice exposed to ambient air mainly occurred at 12 weeks (181.6%, 193.3%, and 201.9% of control for IL-1β, TNFα, and IL-6, respectively; Figure 3). Relative mRNA Expression of Anti-inflammatory Cytokines As shown in Figure 4, the expression of IL-10 and IL-1RA mRNA in the hippocampus and olfactory bulb at 4 weeks did not differ significantly between the 2 groups (p > .05), whereas at 8 and 12 weeks the expression of these cytokines changed, albeit in opposite directions. In mice exposed to ambient air, IL-10 expression in hippocampus was strongly up-regulated at 8 weeks (p = .016, Figure 4A) and marginal significantly down-regulated in the hippocampus at 12 weeks (p = .060; Figure 4A). In contrast, IL-1RA mRNA expression in the olfactory bulb of these mice decreased at 8 weeks (p = .026; Figure 4D) but increased at 12 weeks, both in the hippocampus and the olfactory bulb (p = .002 and .044, respectively; Figs. 4C and 4D). The asynchronous activation of different anti-inflammatory cytokines demonstrates the complexity of the immune regulatory network response to PM exposure (Delfino et al., 2002; Nikasinovic et al., 2006). The largest increases in IL-10 and IL-1RA mRNA expression occurred at 8 and 12 weeks, respectively (Figure 4, Supplementary Tables 1 and 2). At 8 weeks, IL-10 expression in the hippocampus and olfactory bulb was 242.8% and 188.6% of control, respectively, in ambient air than in HEPA-filtered air. At 12 weeks, IL-1RA expression in the hippocampus and olfactory bulb was 210.8% and 131.9% of control, respectively. Ratios of Pro/Anti-inflammatory Cytokines The increased ratios of pro/anti-inflammatory cytokines indicated an imbalance of the immune system (Parissis et al., 2004) similar to that previously documented in patients with depression (Dhabhar et al., 2009). As shown in Supplementary Figures 3A and 3B, the ratios of TNFα/IL-10, IL-6/IL-10, and IFN-γ/IL-10, but not of IL-1ß/IL-1RA, were significantly higher after 4 and 12 weeks than after 8 weeks both in hippocampus and olfactory bulb of mice exposed to ambient air. In particular, sharp increase was found in hippocampus in the expression of TNFα/IL-10 (12.034) and IL-6/IL-10 (11.894) (Supplementary Figure 3A). In mice exposed to HEPA-filtered air, in contrast, these ratios did significantly differ at any of the 3 time points (Supplementary Figs. 3C and 3D). Relative mRNA Expression of BDNF, TrkB, and CREB As shown in Figure 5 and in Supplementary Tables 1 and 2, exposure to ambient air induced a general down-regulation of BDNF, TrkB, and CREB mRNA expression in the hippocampus and olfactory bulb, especially at 4 and 12 weeks (p < .05). These results indicated the inhibition of the BDNF-TrkB-CREB signaling pathway by PM exposure. In contrast to the down-regulation at 4 and 12 weeks, at 8 weeks only BDNF mRNA expression in the olfactory bulb and TrkB mRNA expression in the hippocampus differed significantly versus the control (p = .012 and .045, respectively; Figs. 5B and 5C). The differences in the expression levels of the other markers in the 2 groups were not significant (p > .05; Figs. 5A, 5D–5F). Plotting the relatively higher expression levels of BDNF, TrkB, and CREB resulted in approximately inverted U-shaped curves, which revealed the close associations between BDNF-TrkB-CREB pathway and the depressive-like responses of the mice exposed to PM. Morphological Changes in the Hippocampus and Olfactory Bulb The morphological changes in the hippocampus in mice exposed to PM in ambient air are shown in Supplementary Figure 4. At 4 weeks, these changes included pyramidal cell swelling, fainter staining of the cytoplasm, and nuclear displacement (Supplementary Figure 4D), as shown by yellow arrows. At 8 weeks, pyramidal cell atrophy, narrowing of the cell body, intense cytoplasmic staining, and initial signs of necrosis were seen (Supplementary Figure 4E), as shown by black arrows, whereas by 12 weeks the amount of pyramidal cell atrophy and necrosis had increased substantially (Supplementary Figure 4F). The cells from mice exposed to HEPA-filtered air were morphologically intact, with only few atrophic pyramidal cells seen in the examined sections (Supplementary Figs. 4A–C). As shown by the yellow arrow in Supplementary Figure 5D, the observed changes in olfactory bulb of mice exposed to ambient air for 4 weeks consisted of granulosa cell swelling, cytoplasm vacuoles, and nuclear displacement. At 8 weeks, slight granular cell necrosis, nucleus pyknosis, intense cytoplasmic staining were apparent (Supplementary Figure 5E), as shown by black arrow. At 12 weeks, granular cell necrosis was the most prominent feature (Supplementary Figure 5F). In contrast, there was no significant inflammatory response or signs of apoptosis in olfactory bulb tissue sections from mice exposed to HEPA-filtered air (Supplementary Figs. 5A–C). Generally, the tissue damage in the hippocampus and olfactory bulb demonstrated their gradual deterioration during 12 weeks of PM exposure. Although after 4 weeks, the symptoms were mainly those of inflammation, more serious tissue damage was apparent at 8 weeks and a considerable worsening at 12 weeks, when extensive nerve cell apoptosis was seen. The latter was consistent with the changes in the mRNA expression levels of proinflammatory cytokines. HR-TEM of the Hippocampus and Olfactory Bulb The abundant presence of PM (20–200-nm diameter) in the hippocampus and olfactory bulb of mice exposed to ambient air for 12 weeks was demonstrated by HR-TEM (Figure 6). The elemental analysis with EDS for PM was shown in Supplementary Figure 7. The sizes of these particles were similar to those of the magnetite nanoparticles observed in human brain (Maher et al., 2016). The particles were well dispersed in these brain structures and readily distinguished. In contrast, no extraneous particles were observed in the hippocampus and olfactory bulb of mice exposed to HEPA-filtered air. The combined HR-TEM and EDS analyses identified 3 categories of PM: (1) polygonal, angular particles containing high levels of silicon (Si), aluminum (Al), magnesium (Mg), etc. (Supplementary Figure 7A), consistent with their MD origin (Li et al., 2016), (2) roughly spherical particles containing heavy metals such as iron (Fe), chromium (Cr), cadmium (Cd), lead (Pb) (Supplementary Figure 7B), suggesting the high-temperature formation of nanospheres via combustion and/or friction (Maher et al., 2016) and (3) roughly spherical, single or aggregate carbon particles, containing high contents of carbon (C) and oxygen (O) (Supplementary Figs. 7C and 7D), identified the presence of fly ash from industrial coal combustion (Li et al., 2016). Figure 6. View largeDownload slide HR-TEMs of the olfactory bulb (A, B, G, H) and hippocampus (C, D, E, F) reveals 3 distinct types of PM, polygonal aluminosilicate particles (A and B), metal-bearing particles (C and D), single or aggregate carbon particles (E, F, G, and H). Osmic acid staining. Figure 6. View largeDownload slide HR-TEMs of the olfactory bulb (A, B, G, H) and hippocampus (C, D, E, F) reveals 3 distinct types of PM, polygonal aluminosilicate particles (A and B), metal-bearing particles (C and D), single or aggregate carbon particles (E, F, G, and H). Osmic acid staining. DISCUSSION Our study showed that long-term PM exposure triggers a depressive-like response in mice that is associated with changes in inflammatory cytokines and modulation of the BDNF-TrkB-CREB pathway in the hippocampus and olfactory bulb. Generally, the toxicity of PM depends on the composition, concentration and size of the particles (Fukagawa et al., 2013; Pieters et al., 2015; Wang et al., 2013a,b). The specific surface area of PM facilitates itself to be bound heavy metals, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, viruses, and other toxic substances, which could lead to the generation of reactive oxygen species and the release of inflammatory cytokines (Pardo et al., 2015; Yue et al., 2015). The mean concentrations of PM2.5 in the ambient air from our study area during 4, 8, and 12 weeks were 54.20, 73.15, and 77.42 µg/m3, respectively, which were much higher than the 24-h concentration proposed by the World Health Organization (25 µg/m3). PM1 accounted for 85.4% of the PM2.5 mass. Ultrafine particles were observed in the hippocampus and olfactory bulb tissues of mice exposed to ambient air (Figure 6), where they may further damage the CNS. According to the cytokine hypothesis of depression, the release of proinflammatory cytokines induces depression via neuroendocrine as well as neural biochemical changes (Block et al., 2009; Elder et al., 2006; Ying et al., 2013). In our study, the immobility time in TST and FST increased in mice exposed to ambient air for 4 and 12 weeks and which were accompanied by the up-regulation of mRNAs encoding the proinflammatory cytokines IL-1β, TNFα, and IL-6. These results are in good agreement with the cytokine hypothesis. The up-regulation of anti-inflammatory cytokines, in contrast, can retard the development of depressive symptoms (Arimoto et al., 2007; Qian et al., 2006) consistent with the observed up-regulation of IL-10 in hippocampus and the weakened depressive status in mice exposed to PM for 8 weeks. In addition, the higher expression of pro and anti-inflammatory cytokines in the hippocampus than in the olfactory bulb in response to PM exposure (Figs. 3 and 4) may reflect its greater vulnerability to injury and inflammation and its abundance of inflammatory cytokine receptors, including those for IL-1β, TNFα, and IL-6 (Fonken et al., 2011; Maier and Watkins, 1998). The depressogenic-like effect in mice exposed to ambient air for 4 and 12 weeks was accompanied by the down-regulation of the nutritional factor BDNF. At 8 weeks, its relative up-regulation was coincided with the relief of depressive-like response. This result suggests a close relationship between nutritional factors such as BDNF and depression (Fang et al., 2013; Vines et al., 2012). Based on the observed changes in the trends of inflammatory cytokines, it may be that the expression of inflammatory cytokines induces changes in the BDNF-TrkB-CREB pathway. Support for this hypothesis comes from studies showing that proinflammatory cytokines decrease serotonin (5-HT) neurotransmission by lowering the concentration of this neurotransmitter or changing the sensitivity of the postsynaptic serotonin 1A (5-HT1A) and serotonin 2A (5-HT2A) receptors, which leads to a deactivation of cAMP/protein kinase A and CREB, thus reducing the expression and secretion of BDNF (Fang et al., 2013; Regmi et al., 2014). An alternative explanation relies on a possible blockade of the mitogen-activated protein kinases signaling pathway caused by inflammatory cytokines, which is also associated with the regulation of BDNF-TrkB-CREB expession (De Vry et al., 2016; Zhao et al., 2017). Interestingly, after the initial depressive-like response at 4 weeks, mice exposed to ambient air showed an improvement in their depressive status at 8 weeks, consistent with the observed up-regulation of IL-10. As an important anti-inflammatory cytokine, IL-10 inhibits microglial activation and limits the secretion of proinflammatory cytokines (Pan et al., 2013). It is reported that systemic inflammatory response syndrome is a proinflammatory response responsible for killing infectious organisms through activation of the immune system (Bone, 1996), whereas the compensatory anti-inflammatory response syndrome is a global deactivation of the immune system responsible for restoring homeostasis (Ward et al., 2008; Berry et al., 2010). Thus, the up-regulation of IL-10 may help to restore immune system balance and suppress the CNS damage to some extent at 8 weeks. The positive impact can be further confirmed by the up-regulation of BDNF, TrkB, and CREB mRNA expression at 8 weeks (Figure 5). Then, it may be concluded that IL-10 inhibited the excessive release of proinflammatory cytokines, which then restored BDNF transduction pathways, at least partially. This activation of immune compensation may have slowed down the development of the depressive-like response initially induced by PM exposure at 8 weeks. However, at 12 weeks the depressive-like response was again observed in mice exposed to ambient air and it was accompanied by the further up-regulation of proinflammatory cytokines (IL-1β, TNFα, and IL-6) and the down-regulation of IL-10, BDNF, TrkB, and CREB mRNA. These changes occurred in parallel with neuronal damage, seen as widespread pyramidal cell apoptosis (see Supplementary Figs. 4 and 5). Increased TNFα/IL-10, IL-6/IL-10, and IFN-γ/IL-10 ratios also suggested immune dysfunction in mice (Supplementary Figure 3). Although the initial depressive-like response was probably subsequently weakened by an immune compensation mechanism, a continued high concentration of PM (Figure 1) and/or a prolonged exposure time may have ultimately caused an imbalance of the immune system, manifested as excessive proinflammatory cytokine release and neural cell apoptosis. In summary, inflammatory cytokines and BDNF-TrkB-CREB pathway were involved in the depressive-like response of PM-exposed mice. The respective changes were consistent with a damage-repair imbalance model after long-term, high-level PM exposure. Our study also confirmed the importance of the hippocampus and olfactory bulb in the development of the depressive-like response caused by PM exposure in mice. Further efforts are required to identify the systemic response mechanism that relates the PM-induced changes in inflammatory cytokine levels and BDNF-TrkB-CREB pathway modulation. This will contribute to new approaches to preventing depression and other CNS diseases provoked by exposure to air pollution. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. FUNDING This study was supported by the National Natural Science Foundation of China (grant nos. 41501549 and 41771533), the Natural Science Foundation of Jiangsu Province, China (grant no. BK20171339). ACKNOWLEDGMENTS We would like to thank Prof James S. Quinn from McMaster University for his kind help on the design of the exposure shed in this study. REFERENCES Arimoto T. , Choi D. , Lu X. , Liu M. , Nguyen X. V. , Zheng N. , Stewart C. A. , Kim H. , Bing G. ( 2007 ). Interleukin-10 protects against inflammation-mediated degeneration of dopaminergic neurons in substantia nigra . Neurobiol. Aging 28 , 894 – 906 . Google Scholar CrossRef Search ADS PubMed Berry P. A. , Antoniades C. G. , Wendon J. ( 2010 ). 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Particulate Matter Triggers Depressive-Like Response Associated With Modulation of Inflammatory Cytokine Homeostasis and Brain-Derived Neurotrophic Factor Signaling Pathway in Mice

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Abstract

Abstract Particulate matter (PM) exposure may contribute to depressive-like response in mice. However, few studies have evaluated the adaptive impacts of long-term PM exposure on depressive-like response associated with systemic inflammation and brain-derived neurotrophic factor (BDNF) signaling pathway. We studied the association among depressive-like behaviors, mRNA levels of pro and anti-inflammatory cytokines, and the expression of BDNF signaling pathway in mice by long-term PM exposure. C57BL/6 male mice were exposed to ambient air alongside control mice breathing air filtered through a high-efficiency air PM (HEPA) filter. Depressive-like behaviors were assessed together with proinflammatory, anti-inflammatory cytokine mRNA levels and the modulation of BDNF pathway in hippocampus and olfactory-bulb of mice exposed to PM for 4, 8, and 12 weeks. Exposure to HEPA-filtered air for 4 weeks may exert antidepressant like effects in mice. Proinflammatory cytokines were up-regulated while the expression of BDNF, its high-affinity receptor tropomyosin-related kinase B (TrkB), and the transcription factor (cyclic adenosine monophosphate)-response element-binding protein (CREB) were down-regulated in ambient air mice. However, after 8 weeks, there was no significant difference in the rate of depressive-like behaviors between the 2 groups. After 12 weeks, mice exposed to ambient air again had a higher rate of depressive-like behaviors, significant up-regulation of proinflammatory cytokines, down-regulation of interleukin-10, BDNF, TrkB, and CREB than HEPA mice. Ultrafine PM in brain tissues of mice exposed to ambient air was observed. Our results suggest continuous high-level PM exposure alters the depressive-like response in mice and induces a damage-repair-imbalance reaction. ambient particulate matter, depression, inflammatory cytokines, brain-derived neurotrophic factor (BDNF), hippocampus, olfactory bulb Urban air pollution has become one of the most serious environmental challenges facing humankind in recent decades. Atmospheric particulate matter (PM), which has been classified as a group 1 contaminant by the International Agency for Research on Cancer (IARC) (Feng et al., 2016; Yue et al., 2015) has been extensively implicated in the potentiation of cardiopulmonary morbidity and mortality (Devlin et al., 2014; Wang et al., 2013a,b; Ying et al., 2013). Moreover, according to recent studies, PM exposure contributes to the incidence and development of neuropsychological disorders, including Alzheimer’s disease, Parkinson’s disease, and depression (Block et al., 2009; Dastgheib et al., 2015; Tong et al., 2016). Atmospheric fine particles or ultrafine particles can cross the blood-brain barrier or otherwise enter the brain through the olfactory nerve, facial trigeminal nerve endings, and other routes, causing nerve inflammation, or necrosis directly (Calderon-Garciduenas et al., 2008; Lucchini et al., 2012; Ziemssen et al., 2008). Oxidative stress and systemic inflammation are the central mechanisms by which PM exposure poses a health threat (Carll et al., 2013; Cevallos et al., 2017; Pardo et al., 2015; Wang et al., 2017). PM exposure has potential ability to activate proinflammatory pathways by inducing the expression of inflammatory cytokines in central nervous system (CNS), such as Interleukin-1β (IL-1β), Interleukin-6 (IL-6), antitumor necrosis factor α (TNFα), interferon-γ (IFN-γ) (Block et al., 2009; Cevallos et al., 2017). According to the cytokine hypothesis of depression, proinflammatory cytokines released in excess can lead to neuroinflammation and impaired neurotransmitter and neurotrophin signaling, which may be followed by symptoms typical of depression, such as anorexia, lethargy, lack of pleasure, weight loss, and decreased activity (Raison and Miller, 2003). Conversely, anti-inflammatory cytokines, such as IL-10 and interleukin-1-receptor antagonist (IL-1RA), protect tissues and cells by inhibiting an excessive immune response and can alleviate the depressive-like response in animal models (Van Strien et al., 2010; Watkins et al., 1995). Proinflammatory and anti-inflammatory cytokines interact in a coordinated response to modulate inflammatory processes caused by PM exposure (Delfino et al., 2002; Zhao et al., 2016). Nevertheless, most studies, including those on acute toxicity and in vitro experiments, have failed to evaluate the long-term adaptive, compensatory impacts of PM exposure on systemic inflammation in the CNS. In addition to inflammatory processes, depressive phenotypes are also frequently linked to impairments in neural plasticity, characterized by the altered expression of so-called plasticity genes, such as brain-derived neurotrophic factor (BDNF), its high-affinity receptor tropomyosin-related kinase B (TrkB), and the transcription factor (cyclic adenosine monophosphate)-response-element-binding protein (CREB) (Guo et al., 2014; Młyniec and Nowak, 2015; Vines et al., 2012). Clinical findings have led to the neurotrophin hypothesis of depression, which invokes the suppression of the BDNF-TrkB-CREB pathway (Björkholm and Monteggia, 2016). However, only several studies have focused on acute exposure to PM. Liu et al. (2017) have conducted a 130-min exposure with fine PM inhalation, indicating that no change of BDNF, IL-6, and TNFα in blood were observed in healthy participants. In the study of Bos et al. (2011), serum BDNF levels were not increased in volunteers who had cycled near a major traffic road for 20 min but they were significantly increased when the volunteers had cycled in a room supplied with filtered air. Until now, little information is known about the systematic study concerning the effect of long-term PM exposure on BDNF-TrkB-CREB pathway. Previous studies of the toxicity of long-term ambient particulate air pollution were usually conducted in cities with relatively low PM concentrations, such as a study of neurotoxicity conducted in the United States, where the mean PM2.5 concentration was 16.85 µg/m3 (Fonken et al., 2011) and a study of reproductive toxicity conducted in Canada, where the mean total suspended particle (TSP) concentration was 93.8 ± 17.0 µg/m3 (Yauk et al., 2008). There is limited information on the nervous diseases caused by the much higher ambient PM exposure in China. In this study, C57BL/6 male mice were exposed to either ambient air, and thus to PM, or high-efficiency air PM (HEPA)-filtered air. The aim was to evaluate the impacts of long-term PM exposure on the depressive-like responses of the mice. The study was carried out in Nanjing, a typical mega-city in Eastern China. We examined depressive-like behaviors, the changes in the relative mRNA levels of pro and anti-inflammatory cytokines, and the expression of the BDNF-TrkB-CREB pathway in order to provide new insights into the regulatory mechanism by which long-term, high-level PM exposure contributes to depression in humans. MATERIALS AND METHODS Animals and PM exposure The Comparative Medicine Center of Yangzhou University provided the 6- to 8-week-old healthy C57BL/6 male mice in SPF (Specific Pathogen Free) grade. Once the mice were bought and brought to our laboratory, they were put into the HEPA chamber immediately for at least 1 week to adapt to the basic experimental environment. Then the healthy mice were randomly assigned into 2 experimental chambers for either ambient air exposure or HEPA-filtered air exposure. The chambers (2.0 × 0.6 × 0.8 m) were made of polymethyl methacrylate and placed separately in a vinyl utility shed (2.4 × 2.4 × 1.8 m), such that the mice were not exposed to direct wind or sunlight. The windows (1.7 × 1.0 m) of the shed furnished with nylon mesh (1.0 × 1.0-cm mesh size) could keep the consistency of air quality inside and outside of the shed and provided enough light for the mice. The HEPA chamber was installed a HEPA air purifier (True-HEPA, WINIX model WAC6300, Bemis Manufacturing, Sheboygan Falls, Wisconsin), which was mainly composed of HEPA filter and draught fan, can remove at least 99.97% of particles with a diameter of 0.3 µm, including dust, smoke, pollen, pet dander, and other allergens. The size of HEPA filter is 39 × 27 × 2 cm and replaced every 4 weeks. Two draught fans with the same type and airflow rate were set in front of the 2 chambers to let the air flow in, and then the air was discharged to ambient environment through the pipes connected to the end of the 2 chambers. Two electric heating devices could maintain the temperature of chambers no <18°C ± 1°C. Thus, the only difference between the 2 experimental chambers was that the HEPA filter was not installed for the ambient air exposure group. The mice in both groups were exposed for 4, 8, or 12 weeks, from November 7, 2015 to January 30, 2016. After behavioral testing, the mice were re-exposed to original environment for 3 days before tissue collection. During the exposure period, the mice were fed commercial mouse chow and bottled water from the same sources. All experimental protocols were approved by Model Animal Research Center of Nanjing University. Animals were treated humanely with due consideration to the alleviation of distress and discomfort. PM2.5 sampling and analysis Nanjing (32°03′N, 118°46′E) is an important industrial production area and the main transportation hub in Eastern China. It covers approximately 6600 km2 and had a population of more than 8.1 million. It is in the north subtropical monsoon climate zone. The exposure shed was set up on the Xianlin Campus of Nanjing University, where is near the northern industrial zones of the city (Supplementary Figure 1). PM concentrations in the HEPA-filtered air and ambient air were monitored by 2 DUSTMATE monitors (Turnkey Instruments Ltd., UK). The 24-h sampling of PM2.5 in ambient air was carried out by 2 high-volume PM samplers (model TE-6070VFC, Tisch Environmental, Inc. Cleves, Ohio) operating at a flow rate of 1.13 m3/min on a quartz fiber filter (Whatman QM-A) filter. The samplers were set up outside, approximately 3 m from the experimental shed. The main constituents of PM2.5, including water-soluble inorganic ions, carbonaceous fractions, and trace elements (TEs), were analyzed. A chemical mass closure was introduced to specifically obtain information on the chemical composition of PM2.5 collected in the study area. Details of chemical analysis and chemical mass closure can be found in our previous report (Li et al., 2016). Behavioral testing Behavioral tests were carried out by 2 trained observers blinded to the groups. At least 12 mice per group were used for each behavioral test after each exposure period to ensure experimental accuracy. The tail suspension test (TST) is a widely used test of depressive-like behavior in which depression is inferred from the increased duration of immobility. Mice both acoustically and visually isolated were suspended 50 cm above the floor using adhesive tape placed approximately 1 cm from the tip of their tails. A mouse was judged to be immobile when it hung down passively/ceased moving its limbs and body, making only those movements necessary to breathe. The total duration of immobility was recorded during the last 4 min of the 6-min long testing period (Wang et al., 2013c). To further assess depressogenic-like effect of ambient air exposure, the mice were also evaluated in the forced swim test (FST). Briefly, the mice were individually forced to swim in an open cylindrical container (diameter 10 cm, height 25 cm), containing water at a depth of 19 cm and a temperature of 25°C ± 1°C. The mice were considered to be immobile when they ceased struggling and remained floating motionless in the water, making only the small movements necessary to keep their head above water. An increase in the duration of immobility is indicative of a depressant-like effect (Kaster et al., 2007). The behaviors were assessed in a 6-min period; the duration of immobility within the last 4 min of the test was recorded. Brain tissue dissection The whole brain of mice was removed from the cut head and placed on an ice-cooled dissection slab. The selected brain areas were immediately snap frozen in liquid nitrogen and stored at −80°C until use. Briefly, the olfactory bulb was in the forepart of the brain and can be removed first. The hippocampus was located in the bottom of cerebral cortex and above the thalamus, which is easily identified by its difference in color from the occipital cortex. To dissect out the hippocampus, we placed the brain on its ventral side, peeled off cortical layer and then removed all the cortex connected to hippocampus. Analysis of gene transcripts Fresh tissues of the hippocampus and olfactory bulb were collected, flash frozen and stored at −80°C. Total RNA was isolated from tissues using Trizol reagent (Invitrogen, USA) according to manufacturer’s instructions. cDNA was synthesized from mRNA using a first-stand cDNA synthesis kit (Fermentas, Lithuania), which was used as a template to perform real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR was carried out using Applied Biosystems StepOnePlus real-time PCR System (LifeTechnologies, USA) according to the manufacturer’s instructions. The qRT-PCR procedure was as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Data were collected and analyzed by using PCR system software based on 2−ΔΔCT threshold cycle method (Livak and Schmittgen, 2001). The GAPDH transcript served as a housekeeping gene. The primers for the amplification of the genes were designed using Primer 5.0 and listed in Supplementary Table 1. qRT-PCR assays were conducted in triplicate for each sample to ensure experimental accuracy. Morphological analyses of the hippocampus and olfactory bulb The hippocampus and olfactory bulb were dissected out from the freshly isolated mouse brains on an ice tray and then fixed for 24 h in a centrifuge tube containing 4% formaldehyde. After routine paraffin embedding and hematoxylin-eosin staining, the 0.5-μm-thick tissue sections were examined by optical microscopy (Nikon Eclipse E100, Japan). Details of this method can be found elsewhere (Ying et al., 2017; Zhang et al., 2010). High-resolution transmission electron microscopy Ambient PM in tissues of hippocampus and olfactory bulb was investigated by high-resolution transmission electron microscopy (HR-TEM) using a FEI Tecnai F20 TEM instrument operated at 200 kV, and equipped with an energy-dispersive X-ray spectroscopy (EDS). When compared with the conventional method for TEM Samples (Maher et al., 2016), we omitted steps of uranium lead double staining to avoid disturbances to EDS analysis. All reagents were prepared from ultrapure Milli-Q water and pre-filtered (<0.1-μm PTFE membrane filter) to preclude any particulate contamination. Bright-, dark-field, and HR-TEM imaging together with elemental analysis with EDS were used to investigate PM in the mouse brain tissues. Statistical analysis The data are presented as the mean ± SEM. All statistical analyses were performed using SPSS software, version 23.0. Data obtained from the behavioral testing of mice exposed to ambient air versus HEPA-filtered air at 4, 8, and 12 weeks were analyzed using a repeated measures 2-way ANOVA, in which the 2 factors were the group and the exposure period. The groups were analyzed individually in a 1-way ANOVA followed by Tukey’s post hoc test. The mean values of the 2 groups were compared using a paired t test. A p value ≤ .05 was considered to indicate statistical significance. RESULTS PM Concentration and Components The PM10, PM2.5, and PM1 levels in the ambient air and HEPA-filtered air during the exposure time are presented in Figure 1. The average concentrations of PM10, PM2.5, and PM1 were 92.65 µg/m3 (range 44.90–188.9 µg/m3), 68.26 µg/m3 (30.70–142.3 µg/m3), and 58.93 µg/m3 (range 25.56–137.7 µg/m3), respectively. About 7.14% and 34.52% of the daily PM10 and PM2.5 concentrations exceeded the 24-h limit of 150 and 75 µg/m3 specified by the Chinese National Ambient Air Quality Standard, respectively. The ratios of PM2.5/PM10 and PM1/PM2.5 were 0.744 and 0.854, respectively, indicating that fine PM, and especially PM1, should be given high priority in assessments of PM pollution. In contrast, in the HEPA-filtered air the average concentrations of TSP, PM10, PM2.5, and PM1 monitored were 9.31 µg/m3 (range 1.64–18.86 µg/m3), 8.26 µg/m3 (1.35–16.86 µg/m3), 6.41 µg/m3 (1.08–14.29 µg/m3), and 2.35 µg/m3 (0.40–5.46 µg/m3). There were considerable differences in the PM concentrations between ambient air and HEPA-filtered air, confirming the rationale and reliability of our experimental model to compare the effects of PM exposure. Figure 1. View largeDownload slide Concentrations of PM10, PM2.5, and PM1 from November 7, 2015 to January 30, 2016 in the ambient air of study area of Nanjing, China (A) and in the HEPA-filtered air (B). Figure 1. View largeDownload slide Concentrations of PM10, PM2.5, and PM1 from November 7, 2015 to January 30, 2016 in the ambient air of study area of Nanjing, China (A) and in the HEPA-filtered air (B). The relative contributions of various compounds to the PM2.5 mass were evaluated by considering secondary inorganic aerosols (SIA, defined as the sum of SO42-, NO3-, and NH4+), organic matter (OM), elemental carbon (EC), mineral dust (MD), sea salt (SS), and TEs. The results are shown in Supplementary Figure 2. The average SIA, OM, EC, SS, MD, and TE levels accounted for 27.41% (range 16.37%–33.95%), 37.91% (30.51%–48.01%), 7.78% (4.98%–11.95%), 2.20% (1.18%–3.73%), 8.25% (3.89%–15.32%), and 2.00% (range 1.09%–3.92%) of the PM2.5 mass, respectively. The unaccounted mass of 14.44% (8.47%–19.86%) was probably due to the presence of water or inadequate estimation of crustal components. The analysis clearly showed that OM and SIA were the main components of PM2.5 during the exposure periods. Depressive-Like Behavior Analyses Depressive-like responses were evaluated by subjecting the mice in both groups to the TST and FST (Figure 2). Depressogenic-like effects of ambient air exposure were determined in 2-way repeated measures ANOVA (p < .001). The effect of the exposure period (p = .001 and .031 for the TST and FST, respectively) and of the interaction between the exposure period and ambient air exposure (p < .001) were also significant, both for the TST and the FST. Figure 2. View largeDownload slide Effects of PM exposure on the depressive-like response in mice subjected to the TST (A) and FST (B). In both, an increased immobility time indicates a depressive-like state. Bars indicate the SEM (n ≥ 12). *p < .05; **p < .01, 2-tailed t test. Figure 2. View largeDownload slide Effects of PM exposure on the depressive-like response in mice subjected to the TST (A) and FST (B). In both, an increased immobility time indicates a depressive-like state. Bars indicate the SEM (n ≥ 12). *p < .05; **p < .01, 2-tailed t test. Mice exposed to ambient air for 4 weeks exhibited a longer immobility time than mice exposed to HEPA-filtered air (p < .01 for both tests). However, there were no significant differences at 8 weeks (p > .05 for both tests), although by 12 weeks the immobility time had again increased significantly in mice exposed to ambient air (p < .01 for both tests). The immobility time of mice exposed to ambient air followed a significant U-shaped fluctuation curve (p < .01 for both tests). It is probably that exposure to HEPA-filtered air exerts antidepressant like effects in mice for TST and FST at 4 and 12 weeks. Thus, we presumed the initial depressive-like response may have been followed by a transient recovery by 8 weeks, after which it was further aggravated. Relative mRNA Expression of Proinflammatory Cytokines Supplementary Tables 2 and 3 showed the relative mRNA expression levels of proinflammatory (IL-1β, TNFα, IL-6, and IFN-γ), anti-inflammatory (IL-10 and IL-1RA), as well as BDNF, TrkB, and CREB in hippocampus and olfactory bulb of mice exposed to ambient air and HEPA-filtered air after 4, 8, and 12 weeks. To better compare the results obtained with the 2 groups, the gene expression levels of the mice exposed to ambient air are presented as percent of the control (HEPA-filtered air) group (Figs. 3–5), and the mRNA expressions of HEPA mice across weeks were shown in Supplementary Figure 6. Figure 3. View largeDownload slide Proinflammatory cytokine expression in mice exposed to PM for 4, 8, and 12 weeks. The data are expressed as the percent in mRNA expression of mice exposed to ambient of HEPA-filtered (control) air. IL-1β, TNFα, IL-6, and IFN-γ expression in the hippocampus (A, C, E, and G, respectively), and in the olfactory bulb (B, D, F, and H, respectively). Bars indicate the SEM (n ≥ 3). *p < .05, **p < .01, 1-tailed t test. Figure 3. View largeDownload slide Proinflammatory cytokine expression in mice exposed to PM for 4, 8, and 12 weeks. The data are expressed as the percent in mRNA expression of mice exposed to ambient of HEPA-filtered (control) air. IL-1β, TNFα, IL-6, and IFN-γ expression in the hippocampus (A, C, E, and G, respectively), and in the olfactory bulb (B, D, F, and H, respectively). Bars indicate the SEM (n ≥ 3). *p < .05, **p < .01, 1-tailed t test. Figure 4. View largeDownload slide Anti-inflammatory cytokine expression in mice exposed to PM for 4, 8, and 12 weeks. The data are expressed as percent in mRNA expression in mice exposed to ambient of HEPA-filtered (control) air. IL-10 and IL-1RA expression in the hippocampus (A and C, respectively), and in the olfactory bulb (B and D, respectively). Bars indicate the SEM (n ≥ 3). *p < .05, **p < 0.01, #p = .0598, 2-tailed t test. Figure 4. View largeDownload slide Anti-inflammatory cytokine expression in mice exposed to PM for 4, 8, and 12 weeks. The data are expressed as percent in mRNA expression in mice exposed to ambient of HEPA-filtered (control) air. IL-10 and IL-1RA expression in the hippocampus (A and C, respectively), and in the olfactory bulb (B and D, respectively). Bars indicate the SEM (n ≥ 3). *p < .05, **p < 0.01, #p = .0598, 2-tailed t test. Figure 5. View largeDownload slide BDNF, TrkB, and CREB mRNA expression in the hippocampus and olfactory bulb of mice exposed to PM for 4, 8, and 12 weeks. The data are expressed as percent in mRNA expression in mice exposed to ambient of HEPA-filtered (control) air. BDNF, CREB, and TrkB expression in the hippocampus (A, C, and E, respectively) and in the olfactory bulb (B, D, and F, respectively). Bars indicate the SEM (n ≥ 3). *p < .05, **p < .01, 1-tailed t test. Figure 5. View largeDownload slide BDNF, TrkB, and CREB mRNA expression in the hippocampus and olfactory bulb of mice exposed to PM for 4, 8, and 12 weeks. The data are expressed as percent in mRNA expression in mice exposed to ambient of HEPA-filtered (control) air. BDNF, CREB, and TrkB expression in the hippocampus (A, C, and E, respectively) and in the olfactory bulb (B, D, and F, respectively). Bars indicate the SEM (n ≥ 3). *p < .05, **p < .01, 1-tailed t test. As seen in Figures 3A, 3C, and 3E, the mRNA expression levels of most of the proinflammatory cytokines were significantly up-regulated in the hippocampus of mice exposed to ambient air: IL-1β (p = .029, .013, and .009 for 4, 8, and 12 weeks, respectively), TNFα (p = .016 and .007 for 4 and 12 weeks, respectively), IL-6 (p = .012 and .001 for 8 and 12 weeks, respectively). PM exposure had no effect on IFN-γ mRNA expression in the hippocampus (p > .05, Figure 3G). For IL-1β, TNFα, and IL-6, their mRNA expression levels were strongly elevated in mice exposed to ambient air for 12 weeks (232.6%, 309.8%, and 418.4% of control for IL-1β, TNFα, and IL-6, respectively; Figure 3) compared with those exposed for 4 and 8 weeks. In contrast to the hippocampus, there were only 4 significant up-regulations of proinflammatory cytokines in the olfactory bulb: IL-1β at 12 weeks (p = .048; Figure 3B), TNFα at 4 and 12 weeks (p = .043 and .011, respectively; Figure 3D), and IL-6 at 12 weeks (p = .035; Figure 3F). PM exposure did not lead to the up-regulation of IFN-γ expression; instead, the levels of this cytokine decreased in mice exposed to ambient air for 4 weeks (p = .016; Figure 3H), indicating differences in the actions and/or various sources of proinflammatory cytokines (Dieme et al., 2012). Similar to mRNA expression in the hippocampus, the largest increases in proinflammatory cytokine mRNA expression in the olfactory bulb of mice exposed to ambient air mainly occurred at 12 weeks (181.6%, 193.3%, and 201.9% of control for IL-1β, TNFα, and IL-6, respectively; Figure 3). Relative mRNA Expression of Anti-inflammatory Cytokines As shown in Figure 4, the expression of IL-10 and IL-1RA mRNA in the hippocampus and olfactory bulb at 4 weeks did not differ significantly between the 2 groups (p > .05), whereas at 8 and 12 weeks the expression of these cytokines changed, albeit in opposite directions. In mice exposed to ambient air, IL-10 expression in hippocampus was strongly up-regulated at 8 weeks (p = .016, Figure 4A) and marginal significantly down-regulated in the hippocampus at 12 weeks (p = .060; Figure 4A). In contrast, IL-1RA mRNA expression in the olfactory bulb of these mice decreased at 8 weeks (p = .026; Figure 4D) but increased at 12 weeks, both in the hippocampus and the olfactory bulb (p = .002 and .044, respectively; Figs. 4C and 4D). The asynchronous activation of different anti-inflammatory cytokines demonstrates the complexity of the immune regulatory network response to PM exposure (Delfino et al., 2002; Nikasinovic et al., 2006). The largest increases in IL-10 and IL-1RA mRNA expression occurred at 8 and 12 weeks, respectively (Figure 4, Supplementary Tables 1 and 2). At 8 weeks, IL-10 expression in the hippocampus and olfactory bulb was 242.8% and 188.6% of control, respectively, in ambient air than in HEPA-filtered air. At 12 weeks, IL-1RA expression in the hippocampus and olfactory bulb was 210.8% and 131.9% of control, respectively. Ratios of Pro/Anti-inflammatory Cytokines The increased ratios of pro/anti-inflammatory cytokines indicated an imbalance of the immune system (Parissis et al., 2004) similar to that previously documented in patients with depression (Dhabhar et al., 2009). As shown in Supplementary Figures 3A and 3B, the ratios of TNFα/IL-10, IL-6/IL-10, and IFN-γ/IL-10, but not of IL-1ß/IL-1RA, were significantly higher after 4 and 12 weeks than after 8 weeks both in hippocampus and olfactory bulb of mice exposed to ambient air. In particular, sharp increase was found in hippocampus in the expression of TNFα/IL-10 (12.034) and IL-6/IL-10 (11.894) (Supplementary Figure 3A). In mice exposed to HEPA-filtered air, in contrast, these ratios did significantly differ at any of the 3 time points (Supplementary Figs. 3C and 3D). Relative mRNA Expression of BDNF, TrkB, and CREB As shown in Figure 5 and in Supplementary Tables 1 and 2, exposure to ambient air induced a general down-regulation of BDNF, TrkB, and CREB mRNA expression in the hippocampus and olfactory bulb, especially at 4 and 12 weeks (p < .05). These results indicated the inhibition of the BDNF-TrkB-CREB signaling pathway by PM exposure. In contrast to the down-regulation at 4 and 12 weeks, at 8 weeks only BDNF mRNA expression in the olfactory bulb and TrkB mRNA expression in the hippocampus differed significantly versus the control (p = .012 and .045, respectively; Figs. 5B and 5C). The differences in the expression levels of the other markers in the 2 groups were not significant (p > .05; Figs. 5A, 5D–5F). Plotting the relatively higher expression levels of BDNF, TrkB, and CREB resulted in approximately inverted U-shaped curves, which revealed the close associations between BDNF-TrkB-CREB pathway and the depressive-like responses of the mice exposed to PM. Morphological Changes in the Hippocampus and Olfactory Bulb The morphological changes in the hippocampus in mice exposed to PM in ambient air are shown in Supplementary Figure 4. At 4 weeks, these changes included pyramidal cell swelling, fainter staining of the cytoplasm, and nuclear displacement (Supplementary Figure 4D), as shown by yellow arrows. At 8 weeks, pyramidal cell atrophy, narrowing of the cell body, intense cytoplasmic staining, and initial signs of necrosis were seen (Supplementary Figure 4E), as shown by black arrows, whereas by 12 weeks the amount of pyramidal cell atrophy and necrosis had increased substantially (Supplementary Figure 4F). The cells from mice exposed to HEPA-filtered air were morphologically intact, with only few atrophic pyramidal cells seen in the examined sections (Supplementary Figs. 4A–C). As shown by the yellow arrow in Supplementary Figure 5D, the observed changes in olfactory bulb of mice exposed to ambient air for 4 weeks consisted of granulosa cell swelling, cytoplasm vacuoles, and nuclear displacement. At 8 weeks, slight granular cell necrosis, nucleus pyknosis, intense cytoplasmic staining were apparent (Supplementary Figure 5E), as shown by black arrow. At 12 weeks, granular cell necrosis was the most prominent feature (Supplementary Figure 5F). In contrast, there was no significant inflammatory response or signs of apoptosis in olfactory bulb tissue sections from mice exposed to HEPA-filtered air (Supplementary Figs. 5A–C). Generally, the tissue damage in the hippocampus and olfactory bulb demonstrated their gradual deterioration during 12 weeks of PM exposure. Although after 4 weeks, the symptoms were mainly those of inflammation, more serious tissue damage was apparent at 8 weeks and a considerable worsening at 12 weeks, when extensive nerve cell apoptosis was seen. The latter was consistent with the changes in the mRNA expression levels of proinflammatory cytokines. HR-TEM of the Hippocampus and Olfactory Bulb The abundant presence of PM (20–200-nm diameter) in the hippocampus and olfactory bulb of mice exposed to ambient air for 12 weeks was demonstrated by HR-TEM (Figure 6). The elemental analysis with EDS for PM was shown in Supplementary Figure 7. The sizes of these particles were similar to those of the magnetite nanoparticles observed in human brain (Maher et al., 2016). The particles were well dispersed in these brain structures and readily distinguished. In contrast, no extraneous particles were observed in the hippocampus and olfactory bulb of mice exposed to HEPA-filtered air. The combined HR-TEM and EDS analyses identified 3 categories of PM: (1) polygonal, angular particles containing high levels of silicon (Si), aluminum (Al), magnesium (Mg), etc. (Supplementary Figure 7A), consistent with their MD origin (Li et al., 2016), (2) roughly spherical particles containing heavy metals such as iron (Fe), chromium (Cr), cadmium (Cd), lead (Pb) (Supplementary Figure 7B), suggesting the high-temperature formation of nanospheres via combustion and/or friction (Maher et al., 2016) and (3) roughly spherical, single or aggregate carbon particles, containing high contents of carbon (C) and oxygen (O) (Supplementary Figs. 7C and 7D), identified the presence of fly ash from industrial coal combustion (Li et al., 2016). Figure 6. View largeDownload slide HR-TEMs of the olfactory bulb (A, B, G, H) and hippocampus (C, D, E, F) reveals 3 distinct types of PM, polygonal aluminosilicate particles (A and B), metal-bearing particles (C and D), single or aggregate carbon particles (E, F, G, and H). Osmic acid staining. Figure 6. View largeDownload slide HR-TEMs of the olfactory bulb (A, B, G, H) and hippocampus (C, D, E, F) reveals 3 distinct types of PM, polygonal aluminosilicate particles (A and B), metal-bearing particles (C and D), single or aggregate carbon particles (E, F, G, and H). Osmic acid staining. DISCUSSION Our study showed that long-term PM exposure triggers a depressive-like response in mice that is associated with changes in inflammatory cytokines and modulation of the BDNF-TrkB-CREB pathway in the hippocampus and olfactory bulb. Generally, the toxicity of PM depends on the composition, concentration and size of the particles (Fukagawa et al., 2013; Pieters et al., 2015; Wang et al., 2013a,b). The specific surface area of PM facilitates itself to be bound heavy metals, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, viruses, and other toxic substances, which could lead to the generation of reactive oxygen species and the release of inflammatory cytokines (Pardo et al., 2015; Yue et al., 2015). The mean concentrations of PM2.5 in the ambient air from our study area during 4, 8, and 12 weeks were 54.20, 73.15, and 77.42 µg/m3, respectively, which were much higher than the 24-h concentration proposed by the World Health Organization (25 µg/m3). PM1 accounted for 85.4% of the PM2.5 mass. Ultrafine particles were observed in the hippocampus and olfactory bulb tissues of mice exposed to ambient air (Figure 6), where they may further damage the CNS. According to the cytokine hypothesis of depression, the release of proinflammatory cytokines induces depression via neuroendocrine as well as neural biochemical changes (Block et al., 2009; Elder et al., 2006; Ying et al., 2013). In our study, the immobility time in TST and FST increased in mice exposed to ambient air for 4 and 12 weeks and which were accompanied by the up-regulation of mRNAs encoding the proinflammatory cytokines IL-1β, TNFα, and IL-6. These results are in good agreement with the cytokine hypothesis. The up-regulation of anti-inflammatory cytokines, in contrast, can retard the development of depressive symptoms (Arimoto et al., 2007; Qian et al., 2006) consistent with the observed up-regulation of IL-10 in hippocampus and the weakened depressive status in mice exposed to PM for 8 weeks. In addition, the higher expression of pro and anti-inflammatory cytokines in the hippocampus than in the olfactory bulb in response to PM exposure (Figs. 3 and 4) may reflect its greater vulnerability to injury and inflammation and its abundance of inflammatory cytokine receptors, including those for IL-1β, TNFα, and IL-6 (Fonken et al., 2011; Maier and Watkins, 1998). The depressogenic-like effect in mice exposed to ambient air for 4 and 12 weeks was accompanied by the down-regulation of the nutritional factor BDNF. At 8 weeks, its relative up-regulation was coincided with the relief of depressive-like response. This result suggests a close relationship between nutritional factors such as BDNF and depression (Fang et al., 2013; Vines et al., 2012). Based on the observed changes in the trends of inflammatory cytokines, it may be that the expression of inflammatory cytokines induces changes in the BDNF-TrkB-CREB pathway. Support for this hypothesis comes from studies showing that proinflammatory cytokines decrease serotonin (5-HT) neurotransmission by lowering the concentration of this neurotransmitter or changing the sensitivity of the postsynaptic serotonin 1A (5-HT1A) and serotonin 2A (5-HT2A) receptors, which leads to a deactivation of cAMP/protein kinase A and CREB, thus reducing the expression and secretion of BDNF (Fang et al., 2013; Regmi et al., 2014). An alternative explanation relies on a possible blockade of the mitogen-activated protein kinases signaling pathway caused by inflammatory cytokines, which is also associated with the regulation of BDNF-TrkB-CREB expession (De Vry et al., 2016; Zhao et al., 2017). Interestingly, after the initial depressive-like response at 4 weeks, mice exposed to ambient air showed an improvement in their depressive status at 8 weeks, consistent with the observed up-regulation of IL-10. As an important anti-inflammatory cytokine, IL-10 inhibits microglial activation and limits the secretion of proinflammatory cytokines (Pan et al., 2013). It is reported that systemic inflammatory response syndrome is a proinflammatory response responsible for killing infectious organisms through activation of the immune system (Bone, 1996), whereas the compensatory anti-inflammatory response syndrome is a global deactivation of the immune system responsible for restoring homeostasis (Ward et al., 2008; Berry et al., 2010). Thus, the up-regulation of IL-10 may help to restore immune system balance and suppress the CNS damage to some extent at 8 weeks. The positive impact can be further confirmed by the up-regulation of BDNF, TrkB, and CREB mRNA expression at 8 weeks (Figure 5). Then, it may be concluded that IL-10 inhibited the excessive release of proinflammatory cytokines, which then restored BDNF transduction pathways, at least partially. This activation of immune compensation may have slowed down the development of the depressive-like response initially induced by PM exposure at 8 weeks. However, at 12 weeks the depressive-like response was again observed in mice exposed to ambient air and it was accompanied by the further up-regulation of proinflammatory cytokines (IL-1β, TNFα, and IL-6) and the down-regulation of IL-10, BDNF, TrkB, and CREB mRNA. These changes occurred in parallel with neuronal damage, seen as widespread pyramidal cell apoptosis (see Supplementary Figs. 4 and 5). Increased TNFα/IL-10, IL-6/IL-10, and IFN-γ/IL-10 ratios also suggested immune dysfunction in mice (Supplementary Figure 3). Although the initial depressive-like response was probably subsequently weakened by an immune compensation mechanism, a continued high concentration of PM (Figure 1) and/or a prolonged exposure time may have ultimately caused an imbalance of the immune system, manifested as excessive proinflammatory cytokine release and neural cell apoptosis. In summary, inflammatory cytokines and BDNF-TrkB-CREB pathway were involved in the depressive-like response of PM-exposed mice. The respective changes were consistent with a damage-repair imbalance model after long-term, high-level PM exposure. Our study also confirmed the importance of the hippocampus and olfactory bulb in the development of the depressive-like response caused by PM exposure in mice. Further efforts are required to identify the systemic response mechanism that relates the PM-induced changes in inflammatory cytokine levels and BDNF-TrkB-CREB pathway modulation. This will contribute to new approaches to preventing depression and other CNS diseases provoked by exposure to air pollution. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. FUNDING This study was supported by the National Natural Science Foundation of China (grant nos. 41501549 and 41771533), the Natural Science Foundation of Jiangsu Province, China (grant no. BK20171339). ACKNOWLEDGMENTS We would like to thank Prof James S. Quinn from McMaster University for his kind help on the design of the exposure shed in this study. REFERENCES Arimoto T. , Choi D. , Lu X. , Liu M. , Nguyen X. V. , Zheng N. , Stewart C. A. , Kim H. , Bing G. ( 2007 ). Interleukin-10 protects against inflammation-mediated degeneration of dopaminergic neurons in substantia nigra . Neurobiol. Aging 28 , 894 – 906 . Google Scholar CrossRef Search ADS PubMed Berry P. A. , Antoniades C. G. , Wendon J. ( 2010 ). 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Toxicological Sciences – Oxford University Press

Published: Apr 23, 2018

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