Glutamatergic Neurons in Rodent Models Respond to Nanoscale Particulate Urban Air Pollutants in Vivo and in Vitro

1In urban environments, exposure to airborne particulate matter (PM) derived from vehicular traffic has been associated with a variety of cardiovascular and respiratory outcomes, for example, increased incidence of asthma, impaired lung growth, and accelerated atherosclerosis (carotid wall thickening).

2Epidemiological evidence suggests that cognitive functions are vulnerable to airborne pollutant exposure across the life span. In older women, low cognitive function was associated with proximity to vehicular traffic, whereas in middle-age adults, lower cognitive performance was associated with exposure to ozone. Cognitive impairments of school-age children have been associated with exposure to black carbon (BC), a marker for vehicle-derived PM. Also, a prospective study of children showed associations of lower IQ with prenatal exposure to vehicle-derived polycyclic aromatic hydrocarbons (PAHs), and autism prevalence was associated with residential proximity at birth to urban freeways. Rodent models also show varying memory impairment after exposure to ozone and vehicle-derived air pollutants.

3Neuropathological findings consistent with these cognitive changes have been reported based on comparisons of samples collected from Mexico City and control cities (Tlaxcala and Veracruz), which represent extremes in ambient PM. Brains of children and young adult accident victims showed a higher prevalence of diffuse amyloid-β peptide deposits, activated glia, and oxidized DNA in residents of highly polluted Mexico City compared with subjects from control cites. Canine brains from Mexico City also showed more extensive deposits of amyloid-β in cerebral arteries and pyramidal neurons, together with markers of inflammation and glial activation, than did the brains of canines from control cities.

4These findings extend to laboratory rodents, which differ importantly from canines as a model for human brain inflammatory responses. The presence of amyloid-β deposits in aging humans and canines adds complexity to environmental responses, because endogenous amyloid itself stimulates local inflammatory responses. The endogenous amyloid-β protein of rodents differs from that of humans and canines in three amino acid residues that render the rodent amyloid-β protein less neurotoxic. Thus, rodent models for brain responses to environmental agents resolve an amyloid-independent phase of inflammatory responses. Inhalation by mice of ambient vehicle-derived PM from an urban freeway for 2–6 weeks induced glial responses with elevations of interleukin-1α (IL-1α), tumor necrosis factor-α (TNFα), nuclear factor κB, and several protein kinases ( 2009; ). These inflammatory changes, together with increased lipid oxidation, suggest that chronic inhalation of vehicle- derived PM can cause inflammation and oxidative stress in the brain.

5Although the bioactivities of airborne PM are incompletely understood, it is clear that the inhaled particle size is critical: the nanosize class (< 200 nm) of PM (nPM) from engineered materials had the greatest toxicity and uptake by vascular and brain cells. Similarly, ambient urban nPM shows enhanced biological effects. In rodent atherosclerosis models, inhaled nPM was more active than are larger particles. Moreover, aqueous suspensions of nPM were cytotoxic to murine macrophages, possibly by generating reactive oxygen species abiotically.

6Inhaled nPM can act on the brain directly and indirectly. Direct entry of inhaled nPM may occur via transport by olfactory neurons from the nasal mucosa, as modeled by the uptake of inhaled 37-nm carbon-13 particles into the brain. Another possible route is through peripheral monocytes, which regularly enter the pool of brain microglia, with enhanced entry during peripheral inflammation. Furthermore, systemic IL-1 and other inflammatory mediators induced by nPM can be transported across the blood–brain barrier (Banks et al. 2002–2003;; ). However, neuronal responses to inhaled nPM have not been reported.

7We therefore studied neuronal cell responses to nPM in vivo after chronic exposure for 10 weeks, using a new technique of reaerosolized nPM collected from urban freeway air for 30 days. We directly exposed in vitro brain cells to aqueous nPM suspensions. Additionally, we tested the possibility that nPM could be neurotoxic through glutamatergic mechanisms, particularly N-methyl-d-aspartic acid (NMDA) receptors, which are implicated in hypoxic-ischemic neuronal damage and excitotoxic cell death. Lastly, we used in vitro models with cultured neurons and glia to evaluate direct and indirect actions of nPM on developing neurons.

8nPM collection and transfer into aqueous suspension. We collected nPM with a high-volume ultrafine particle (HVUP) sampler at 400 L/min flow in Los Angeles City near the CA-110 Freeway. These aerosols represent a mix of fresh ambient PM mostly from vehicular traffic nearby this freeway. The HVUP sampler consists of an ultrafine particle slit impactor, followed by an after-filter holder. The nPM (diameter < 200 nm) was collected on pretreated Teflon filters (20 × 25.4 cm, polytetrafluoroethylene, 2 μm pore; Pall Life Sciences, Covina, CA). We transferred the collected nPM into aqueous suspension by 30 min soaking of nPM-loaded filters in Milli-Q deionized water (resistivity, 18.2 MW; total organic compounds < 10 ppb; particle free; bacteria levels < 1 endotoxin units/mL; endotoxin-free glass vials), followed by vortexing (5 min) and sonication (30 min). As a control for in vitro experiments with resuspended nPM, fresh sterile filters were sham extracted. Aqueous nPM suspensions were pooled and frozen as a stock at –20°C, which retains chemical stability for ≥ 3 months (Li N et al. 2003; Li R et al. 2009). For in vitro experiments, nPM suspensions were diluted in culture medium, vortexed, and added directly to cultures.

9Animals and exposure conditions. The nPM suspensions were reaerosolized by a VORTRAN nebulizer (Vortran Medical Technology 1 Inc., Sacramento, CA) using compressed particle-free filtered air [see Supplemental Material, Figure S1 (doi:10.​1289/ehp.1002973)]. Particles were diffusion dried by passing through silica gel; static charges were removed by passing over polonium-210 neutralizers. Particle sizes and concentrations were continuously monitored during exposure at 0.3 L/min by a scanning mobility particle sizer (SMPS model 3080; TSI Inc., Shoreview, MN). The nPM mass concentration was determined by pre- and postweighing the filters under controlled temperature and relative humidity. Inorganic ions [ammonium (NH4+), nitrate (NO3–), sulfate (SO42–)] were analyzed by ion chromatography. PM-bound metals and trace elements were assayed by magnetic-sector inductively coupled plasma mass spectroscopy. Water-soluble organic carbon was assayed by a GE-Sievers liquid analyzer (GE-Sievers, Boulder, CO). Analytic details for nPM-bound species are given by Li R et al. (2009). Samples of the reaerosolized nPM were collected on parallel Teflon filters for electron paramagnetic resonance (EPR) analysis.

10Mice (C57BL/6J males, 3 months of age) were maintained under standard conditions with ad libitum Purina Lab Chow (Newco Purina, Rancho Cucamonga, CA) and sterile water. Just before nPM exposure, mice were transferred from home cages to exposure chambers that allowed free movement. Temperature and airflow were controlled for adequate ventilation and to minimize buildup of animal-generated contaminants [skin dander, carbon dioxide (CO2), ammonia]. Reaerosolized nPM or ambient air (control) was delivered to the sealed exposure chambers for 5 hr/day, 3 days/week, for 10 weeks. Mice did not lose weight or show signs of respiratory distress. Mice were euthanized after isoflurane anesthesia, and tissue was collected and stored at –80°C. All rodents were treated humanely and with regard for alleviation of suffering; all procedures were approved by the University of Southern California Institutional Animal Care and Use Committee.

11EPR spectroscopy of nPM. The reaerosolized nPM was collected on filters (described above), which were inserted directly in the EPR quartz tube (Bruker EPR spectrometer; Bruker, Rheinstetten, Germany); spectra were measured at 22°C. The g-value was determined following calibration of the EPR instrument using DPPH (2,2-diphenyl-1-picrylhydrazyl) as a standard. The EPR signal for DPPH was measured and the corresponding g-value was calculated. The difference from the known g-value of 2.0036 for DPPH was then used to adjust the observed g-value for the sample.

12Cell culture and nPM exposure. Hippocampal slices from postnatal day 10–12 rats were cultured 2 weeks in a humidified incubator (35°C/5% CO2) with nPM suspensions added for 24–72 hr of exposure. Primary neurons from embryonic day 18 rat cerebral cortex were plated at 20,000 neurons/cm2 on cover slips coated with poly-d-lysine/laminin and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with B27, at 37°C in 5% CO2 atmosphere. Primary glial cultures from cerebral cortex of neonatal day 3 rats (F344) were plated at 200,000 cells/cm2 in DMEM/F12 medium supplemented with 10% fetal bovine serum and 1% l-glutamine and incubated as described above. For conditioned medium experiments, glial cultures were treated with 10 mg nPM/mL; after 24 hr, media were transferred by pipette to neuron cultures.

13Neurite outgrowth and toxicity assays. After treatments, neurons were fixed in 4% paraformaldehyde and immunostained with anti–β-III-tubulin (1:1,000, rabbit; Sigma Chemical Co., St. Louis, MO); F-actin was stained by rhodamine phalloidin (1:40; Molecular Probes, Carlsbad, CA). A neurite was defined as a process extending from the cell soma of the neuron that was immunopositive for both β-III-tubulin (green) and F-actin (red). The length of neurites was measured using NeuronJ software. Growth cones were defined by the presence of actin-rich filopodia and lamellipodia. Collapsed growth cones were defined as actin-rich neuritic endings in which filopodia and lamellipodia were indistinguishable. In neurite outgrowth and growth cone collapse assays, individual neurons were selected from two cover slips per condition; n is the total number of neurons analyzed per treatment. Cytotoxicity in slice cultures was assayed by lactate dehydrogenase (LDH) release to media and by cellular uptake of propidium iodide (PI). Neuronal viability was assayed by Live/Dead Cytotoxicity Kit (Invitrogen, Carlsbad, CA) by computer-assisted image analysis of fluorescent images. Mitochondrial reductase was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 585 nm in undifferentiated PC12 cells. For viability assays, n is the total number of hippocampal slices analyzed (LDH release and PI uptake) or the total number of cell culture wells analyzed per condition.

14Immunoblotting. Mouse hippocampi were homogenized using a glass homogenizer in cold lysis buffer as described by. After sample preparation, 20 μg protein was electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gels, followed by transfer to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% bovine serum albumin for 1 hr and probed with primary antibodies overnight at 4°C: anti-GluA1 (glutamate receptor subunit 1; 1:3,000, rabbit; Abcam, Cambridge, MA), anti-GluA2 (1:2,000, rabbit; Millipore, Billerica, MA), anti-PSD95 (1:1,000, mouse; Abcam), anti-synaptophysin (1:5,000, mouse; Stressgene; Enzo, Plymouth Meeting, PA), and anti-β-III tubulin (loading control; 1:15,000, rabbit; Sigma), followed by incubation with secondary antibodies (1:10,000) conjugated with IRDye 680 (rabbit, LI-COR Biosciences, Lincoln, NE) and IRDye 800 (mouse, LI-COR). Immunofluorescence was detected by infrared imaging (Odyssey, LI-COR).

15Quantitative polymerase chain reaction (qPCR). Total cellular RNA was extracted from cerebral cortex of nPM-exposed mice and rat primary glia (Tri Reagent; Sigma), and cDNA (2 μg RNA; Superscript III kit; Invitrogen) was analyzed by qPCR, with primers appropriate for mouse (in vivo) or rat (in vitro). Genes examined by qPCR were CD14, CD68, CD11b, CD11c, GFAP (glial fibrillary acidic protein), IFN-γ (interferon-γ), IL-1α, IL-1, IL-6, and TNFα. Data were normalized to β-actin.

16Statistical analysis. Data are expressed as mean ± SE. The numbers of individual measurements (n) are described above and listed in the figure legends. Single and multiple comparisons used Student’s t-test (unpaired) and one-way analysis of variance (ANOVA)/Tukey’s honestly significant difference, with statistical significance defined as p < 0.05.

17nPM collection and characterization. Urban ambient nPM was collected on filters for 30 days, transferred to sterile aqueous suspension, and reaerosolized for inhalation [Supplemental Material, Figure S1 (doi:10.1289/ehp.1002973)]. The reaerosolized nPM particle number and mass concentrations were 2.54 ± 0.47 × 105 particles/cm3 (mean ± SE) and 468 ± 25 μg/m3, respectively; most (> 95%) particles were < 180 nm in diameter (Figure 1). The size distribution of reaerosolized nPM (Figure 1A) was comparable to typical ambient aerosols of fractioned nPM at peak traffic times at this location (Figure 1B). The chemical composition was also similar for NH4+, NO3–, SO42–, water-soluble organics (Figure 1C), and water-soluble redox active metals (Figure 1D). However, transfer of BC (Figure 1C) and of the most hydrophobic organic components (PAHs, organic acids) was incomplete (see Supplemental Material, Figure S2).

18We observed definitive free radical signals on EPR spectroscopy in a sample of reaerosolized nPM collected during mouse exposure (Figure 1E). The g-value of 2.0029 with a peak-to-peak difference (ΔHp–p) of 7.5 Gauss is characteristic of stable carbon-based free radicals. These long-lived free radicals had persisted in frozen aqueous suspension for at least 30 days since the last day of collection from ambient air.

19Chronic nPM mouse exposure. Male mice exposed to reaerosolized nPM for 10 weeks (3 days/week, 5 hr/day; 150 hr total), showed neuronal responses in the hippocampus, a brain region that mediates learning and memory. Neuronal glutamate receptor subunits in hippocampus responded selectively to nPM inhalation: GluA1 protein levels were decreased by 35% (Figure 2A), whereas GluA2 was not significantly altered (Figure 2B). We also observed no significant change in levels of the major synaptic proteins synaptophysin (presynaptic) and PSD95 (postsynaptic) (Figure 2C,D), consistent with the normal histological appearance of hippocampal neuron layers (data not shown).

20Glial activation was indicated by increased mRNA of the microglial markers CD14 (innate immune receptor) (Figure 3A) and CD68 (macrosialin) (Figure 3B), and of astrocytic GFAP (Figure 3C). Two proinflammatory cytokine mRNAs increased, IL-1α (Figure 3D) and TNFα (Figure 3E), confirming previous immunohistochemical observations in mice exposed to ambient nPM from the same urban site [see Supplemental Material, Table S1 (doi:10.1289/ehp.1002973)]. IL-1β mRNA was also increased, whereas IL-6, IFN-γ, CD11b, and CD11c mRNAs were not significantly changed (data not shown).

21In vitro nPM brain cell exposure. We evaluated direct effects of nPM suspensions on developing rat hippocampal slice and embryonic neuron cultures. Initial studies calibrated the range of nPM effects using the neuronlike PC12 cell line. By the MTT assay, nPM induced oxidative stress with dose responses from 1 to 20 μg/mL and EC50 (50% effective concentration) of 10 μg/mL [see Supplemental Material, Figure S3 (doi:10.1289/ehp.​1002973)]. Because of neuronal changes in glutamate receptors in vivo (Figure 2A), we examined hippocampal slice cultures from neonatal rats for nPM cytotoxicity in combination with glutamatergic drugs. Release of LDH was increased by nPM at 1 μg/mL during a 48-hr exposure and was blocked by (2R)-amino-5-phosphonopentanoate (AP5; NMDA receptor antagonist) (Figure 4A). In the presence of NMDA (a glutamatergic agonist), nPM was also neurotoxic (based on PI uptake in CA1 pyramidal neurons) (Figure 4B). We chose this NMDA dose to target CA1 neurons (effects on CA3 and dentate granule neurons are not shown).

22Neurite outgrowth of primary E18 neuronal cultures was inhibited by nPM (2 μg/mL) and rescued by AP5 (Figure 4C), consistent with neurotoxicity assays of cultured slices (Figure 4A). The presence of nPM decreased the number of intact growth cones by 25% and decreased neuron viability by 20% (Figure 4D).

23Glia also responded to nPM but at ≥ 5-fold higher concentrations than neuronal responses. In mixed glial cultures (astrocytes and microglia, 3:1), 12 μg/mL nPM increased IL-1α and TNFα mRNA (Figure 4E). IL-1β and IL-6 mRNAs were also increased, whereas IFN-γ mRNA was not significantly changed (data not shown). Indirect effects of nPM on neurons through glial secretions were examined by measuring neurite outgrowth in conditioned medium from nPM-treated glia, which showed an inhibition of neurite outgrowth by 30% and a reduction of neurite number by 25% (Figure 4F).

24Neuronal glutamatergic functions responded to urban nPM in vivo after chronic inhalation by adult mice for 10 weeks and in vitro after 48-hr exposures of neonatal hippocampal slice or embryonic neuron cultures. Inflammatory and oxidative mechanisms were also implicated. The adverse effects of nPM on embryonic neurite outgrowth may be a model for developmental impairments.

25We used a novel technology for experimental exposure to airborne PM. Ambient freeway nPM was collected on filters for 1 month and then transferred into an aqueous suspension for reaerosolization at the time of rodent exposure. This approach eliminates the expensive, space-limited, on-site collection facilities for exposing animals to ambient air and has the advantage of collecting nPM over long periods, which averages daily variations. The frozen nPM aqueous suspension can be stored for reaerosolization or direct addition to cultured brain cells. The composition of reaerosolized nPM versus ambient samples was similar except for its lower content of BC and of the most hydrophobic PAHs and other organic components. Nonetheless, despite the reduced nPM content of some of these components, the glial responses to reaerosolized nPM closely approximated those in prior studies of inhaled ambient nPM from this freeway site [see Supplemental Material, Table S1 (doi:10.1289/ehp.1002973)].

26We observed two new cellular reactions to nPM in rodent microglia and neurons: chronic nPM inhalation induced the microglial-monocytic markers CD14 (innate immune receptor) and CD68/macrosialin (lysosomal-associated membrane protein) and reduced neuronal GluA1 receptor protein levels. Thus, the microglial activation observed in Mexico City brains from humans and canines might have resulted from the nPM present in the complex mixture of urban air pollutants. Although the GluA1 receptor is associated with excitotoxic pathways (e.g., ), we found no evidence for gross neurodegeneration by the normal appearance of hippocampal neuron layers or by the unchanged levels of two synaptic proteins, synaptophysin and PSD95.

27In vitro models with developing brain cells indicate neurotoxicity of aqueous suspensions of nPM. In cultured neonatal hippocampal slices exposed to nPM for 48 hr, CA1 neurons showed increased uptake of PI, an indicator of cell death. We examined CA1 neurons because of their vulnerability to damage from cerebral ischemia and Alzheimer disease. Because of likely glia–neuron interactions in cultured slices, we also examined the effects of nPM on cultured embryonic neurons. Direct neuronal effects of nPM included decreased neurite extension, which again was attenuated by AP5. These responses are consistent with the role of glutamatergic NMDA receptor signaling during neurite outgrowth in developing neurons.

28These in vitro models suggest that nPM has both direct neurotoxicity through glutamatergic mechanisms and indirect neurotoxicity through glial secretions. Indirect effects on neurons from nPM-induced glial secretions are indicated by the inhibition of neurite outgrowth in conditioned medium from nPM-treated glia. TNFα, which is induced in nPM-treated glia (Figure 4E) and can inhibit neurite outgrowth, is a candidate for further study in glia–neuron synergies of nPM neurotoxicity. Another mechanism is suggested by the rapid induction of hydrogen peroxide (H2O2) production by nPM in macrophages within 1 hr. Glutamatergic NMDA receptors are sensitive to H2O2. It will be difficult to evaluate the relative roles of indirect (systemic) influences from cytokines and circulating macrophages from direct effects of nPM transported into the brain by olfactory neurons (see introductory remarks). Nonetheless, further in vivo studies are needed to identify the range of neuronal systems responsive to inhaled nPM.

29The biochemical basis for nPM neurotoxicity could involve free radicals, including reactive oxygen species. Filter-collected nPM from reaerosolized nPM aqueous suspensions in the present studies has an EPR signal characteristic of stable carbon-based free radicals. This EPR signal is provisionally generated from the nPM-associated graphitelike BC assayed in the reaerosolized nPM sample (Figure 1C). We detected these signals in nPM collected at least 30 days before measurement. Stable free radicals in nPM are consistent with the generation of singlet oxygen and other ROS in vitro. In fact, nPM suspensions from this Los Angeles site generated ROS under abiotic conditions.

30The evident neurotoxicity of nPM suggests links between urban air pollution and brain health across the life span. The impairment of neurite outgrowth by nPM is consistent with epidemiological evidence of developmental effects of air pollution in children, which include an association with autism. The interaction of nPM with glutamatergic (NMDA) excitotoxicity suggests that cerebral ischemia, which also involves glutamatergic excitotoxicity, could be exacerbated by nPM. The increased risk of cardiovascular events during surges of air pollution (“inhaling a heart attack”) can reduce cerebral blood flow, with additional risks to the brain. For example, nPM effects could compound neuronal damage in CA1 neurons from cerebral ischemia during stroke or transient ischemia. Moreover, the association of PM with accelerated carotid atherosclerosis (increased carotid artery intima-media thickness) has direct relevance to cognitive aging because of the links between cardiovascular disease and age-related cognitive decline. Urban airborne pollutants may also be a risk factor in Alzheimer disease, as suggested by, who observed that brain amyloid-β peptide deposits in postmortem samples from young adult humans exposed to urban pollution were more prevalent in carriers of apolipoprotein E4, a genotype at high risk for Alzheimer disease.

31An important unknown is the actual brain accumulation of inhaled PM, which may be transported inside the brain by olfactory neurons from the nasal mucosa. Additionally, because of its small size, nPM is not efficiently cleared by mucosal surfaces or alveolar macrophages and is distributed throughout the body, including the brain.

32Particle size distributions are important for public health. Notably, the smallest PM size for which the U.S. Environmental Protection Agency (EPA) provides quantitative risk assessment (U.S. EPA 2009) is the “fine” PM, having and aerodynamic diameter < 2.5 μm (PM2.5). Although the term “ultrafine” particle has been used for nPM, these designations do not define a specific size range and do not reflect their similarity in size or properties to spherical engineered nanoparticles or other types of nanosized particles.

33Lastly, we note the health problems associated with the global growth of airborne pollutants and the continuing increase of urban vehicular traffic and power generation from fossil fuel. The National Research Council report America’s Climate Choices: Panel on Advancing the Science of Climate Change briefly identified increasing air pollution in urban health but did not discuss endangerment of the brain. Despite the apparent greater toxicity of nPM relative to larger PM, its overall health effects are not well defined by the U.S. EPA (2009). The present findings with portable nPM should facilitate studies on the neurotoxicity and associated risks of nPM to brain development and aging.

34Responses of rodent neurons to airborne nanosized particles from vehicular traffic provide a new model to study associations of urban air pollution to cognitive impairment and neuropathology. Our findings support glutamatergic mechanisms of neuronal response to nPM that may be relevant to interactions of air pollution with cerebral ischemia.