Anti-synaptophysin

nPM collection and transfer into aqueous suspension. We collected nPM with a high-volume ultrafine particle (HVUP) sampler (Misra et al. 2002 ) 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 (Ning et al. 2007 (link)). 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.
Animals 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 (SO₄^2–)] 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.
Mice (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.
EPR 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.
Cell culture and nPM exposure. Hippocampal slices from postnatal day 10–12 rats were cultured 2 weeks in a humidified incubator (35°C/5% CO₂) (Jourdi et al. 2005 (link)) 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 (Rozovsky et al. 2005 (link)). 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 (Rozovsky et al. 1998 (link)). For conditioned medium experiments, glial cultures were treated with 10 mg nPM/mL; after 24 hr, media were transferred by pipette to neuron cultures.
Neurite 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 (Meijering et al. 2004 (link)). Growth cones were defined by the presence of actin-rich filopodia and lamellipodia (Kapfhammer et al. 2007 ). 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) (Jourdi et al. 2005 (link)). 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 (Mosmann 1983 (link)). 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.
Immunoblotting. Mouse hippocampi were homogenized using a glass homogenizer in cold lysis buffer as described by Jourdi et al. (2005) (link). 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).
Quantitative 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.
Statistical 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.

The brain sections containing the hippocampus were incubated in 3% hydrogen peroxide overnight and then were rinsed in 0.1 PB 3 times for 10 min. After blocking in 10% normal goat serum (NGS), 0.5% Triton X-100, and 0.1 M phosphate-buffered saline (PBS) for 2 h at room temperature, the brain sections were incubated in a 1:1000 dilution of anti-synaptophysin made in rabbits (Sigma, Lot no. 310333) diluted into 1% NGS in PBS at 4 °C overnight, followed by 1:100 goat anti-rabbit antibody-conjugated Rhodamine diluted in PBS (Millipore lot no. 2775066, Burlington, MA, USA) for 1 h. The sections were counter-stained with 1 μg/mL of DAPI and then washed in PB. The synaptophysin-labeled presynaptic vesicle proteins in the CA1 of the hippocampus were observed under a fluorescent microscope (Zeiss Axio Scope. A1, Tokyo, Japan) and were photographed using the 20× objective. The density of the synaptophysin was analyzed using the ImageJ program (https://imagej.net/ij/). The grid in which 1000 µm² square areas were laid over the synaptophysin labeling area, and 10 of these square areas were systematically selected for counting and analyzing the synaptophysin density. These data were compared to those of the five animal groups.

In this study, IHC, ICC and Western blots assays utilized the following primary antibodies: Mouse anti-β-actin antibody (Abcam, #ab8226, Cambridge, UK), Mouse anti-GFAP antibody (Cell Signaling Technology, #3670S, Boston, MA, USA), Goat anti-PSD95 antibody (Abcam, #ab12093), Rabbit anti-synaptophysin antibody (Abcam, #ab32127), Rabbit anti-LAMP1 antibody (Abcam, #ab24170), Rabbit anti-MEGF10 antibody (Sigma-Aldrich, #ABC10, St. Louis, MO, USA).