Carbon black nanoparticle preparation and characterization
Carbon black (CB) powder (Printex 90®, a gift from Evonik, Frankfurt, Germany) is composed of 99.9% carbon. CB aerosols were generated using a high-pressure acoustical generator (HPAG, IEStechno, Morgantown, WV). The output of the generator was fed into a Venturi pump (JS-60 M, Vaccon, Medway, MA) to further de-agglomerate the particles. The nano-CB aerosol/air mix was sampled in real-time with a light scattering device (PDR-1500, Thermo Environmental Instruments Inc., Franklin, MA) to estimate the aerosol mass concentration within the exposure chamber. Stable mass concentrations were maintained in real-time via software feedback loops. 37 mm PTFE filters were used for gravimetric measures concurrent with the PDR-1500 measures to obtain a calibration factor; gravimetric measures were also performed during exposures to calculate true mass concentrations. The particle count size distribution was measured using a high resolution electrical low-pressure impactor (ELPI, Dekati, Tampere, Finland). Particle mass size distribution of the CB aerosol was measured from the exposure chamber with a cascade impactor (Nano-MOUDI, 115R, MSP Corp, Shoreview, MN).
Aerosol characterization was verified throughout a given exposure by collecting CB particle samples on filters, and making hourly gravimetric measurements with a microbalance. This approach was also used to collect samples for transmission electron microscopy (TEM) and scanning electron microscopy (SEM).
Male Sprague Dawley rats (8 weeks, 250–275 g) were obtained from Hilltop Laboratories (Scottdale, PA) and housed in an AAALAC approved facility at WVU. All procedures were approved by the WVU Institutional Animal Care and Use Committee (protocol 1602000621) and conformed to the most current National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals. Housing conditions included 12:12 h light:dark cycle, 20–26 °C, 30–70% relative humidity, acclimatization for 48–72 h prior to any procedure and had ad libitum access to food and water.
Whole body inhalation exposure
Rats (sham-control group: age 53 ± 1 days; mass 288 ± 3 g. CB group age 53 ± 1 days; mass 303 ± 4 g.) were exposed to a target concentration of CB for 6 h: 6 mg/m3 (n = 10/group). Rats were randomly assigned to control or experimental groups and were individually housed in cages within a stainless steel chamber during exposure. The exposure chamber was 22″ × 20″ × 20″ (wdh) with an approximate volume of 144 L. The airflow through the chamber was approximately 28 LPM during exposures. Bedding material soaked with water is used in the exposure chamber to maintain comfortable humidity (30–70%) and temperature (20–26 °C) during the exposure. Sham-control animals were exposed to HEPA filtered air only.
Tissue collection and processing
Tissue harvesting were performed 24 h after exposure. Euthanasia was performed via exsanguination under deep anesthesia (5% induction, 2% maintenance with isoflurane gas) followed by organ removal. Plasma aliquots and whole tissues (arteries, brain, lung) were snap frozen in liquid nitrogen and stored at −80 °C. Solid tissues were pulverized on dry ice to granular powder. Tissue was homogenized with RIPA buffer (Thermo Fisher Scientific, Waltham, MA) or MILLIPLEX MAP buffer (MilliporeSigma, Burlington, MA). Total protein was determined by Direct Detect Spectrometer (MilliporeSigma).
The Meso Scale Discovery (MSD, Rockville, MD, USA) V-PLEX Rat Pro-inflammatory Panel 2 Kit (K15059G) was used to quantify tissue (50 μg) and plasma (25 µl) concentrations of IFN-γ, IL-1β, IL-4, IL-5 IL-6, IL-10, IL-13, KC/GRO, and TNF-α. All samples were run in duplicate. Plates were processed according to the manufacturer’s instructions using the MSD MESO Sector 600. Data were analyzed using MSD Discovery Workbench 4.0 software .
Vascular injury panels
Vascular injury was measured in plasma using the Rat Vascular Injury Panels 1 and 2 (RV1MAG-26 K & RV2MAG-26 K, MilliporeSigma). Samples were processed according to manufacturer recommendations, and data captured using Luminex Magpix (MilliporeSigma).
Biomarker data calculations were performed using GraphPad Prism 9. Differences between sham-control and CB exposed groups were identified by using unpaired t-test or Mann–Whitney tests according to data distribution (n ≥ 8 for all biomarkers per experimental group). Significance was established as p ≤ 0.05. Descriptive statistics are provided for CB aerosol characterization.