Traffic-Related Emissions Induce Angiotensin II-Dependent Oxidative Stress in the Hippocampus of ApoE-Null Male Mice

Description

This study tested the hypothesis that exposure to traffic-generated air pollution (MVE) in apolipoprotein null (ApoE-/-) mice leads to increased oxidative stress and amyloid processing, associated with increased renin-angiotensin system (RAS) signaling. Methods: Male ApoE-/- mice (6-8 weeks old) on a high-fat diet were treated with either an ACE inhibitor (captopril, 4 mg/kg/day) or water and exposed to filtered air (FA) or MVE (200µg PM/m³) for 30 days. Real-time qPCR was used to analyze cerebral endpoints, double immunofluorescence to analyze protein expression in the CA1 region of the hippocampus, and an ELISA to quantify plasma angiotensin II levels.

Steps to reproduce

Immunofluorescence: Brains were fixed in Histochoice (VWR, Irving, TX) at 4°C overnight, embedded in Tissue Freezing Medium (TBS, IMEB Inc., San Marcos, CA) and frozen at -80°C until sectioning. Frozen brain sections were cut (coronal plane) 10 µm thickness between Bregma 0-Bregma 2.12 mm, and were prepared for either single or double immunofluorescence, as previously described by our laboratory 50, using the following primary antibodies: rabbit anti-AGTR-1 pAb (1:1000, Novus, Centennial, CO, NBP1-77078AF555), mouse anti-beta amyloid mAb (1:500, Novus, NBP2-13075AF488), mouse anti-BACE1 mAb (1:1000, Santa Cruz, Dallas, TX, sc-33711), mouse anti-8-OHdG mAb (1:1000, Santa Cruz, sc-66036), sheep anti-Von Willebrand Factor pAb (1:1000, Abcam, Waltham, MA, Ab11713), and rabbit anti-IL-1β pAb (1:1000, Abcam, ab9722). Primary antibodies were conjugated with appropriate secondary antibodies at a 1:2 (primary: secondary) concentration, using Alexa Fluor 555 donkey anti-rabbit (Invitrogen, A31572), Alexa Fluor 555 goat anti-mouse (Invitrogen, A21422), or Alexa Fluor 488 donkey anti-sheep (Invitrogen, A11015). Negative controls were processed using only secondary antibodies to ensure there was no non-specific binding (supplemental Figure S1). Two slides with two brain sections per slide were used for analysis, with an n=3 per group. Slides were imaged under fluorescent microscopy at 40x with the appropriate excitation/emission filter, digitally recorded, and analyzed with image densitometry with ImageJ software (NIH). The fluorescence in the CA1 area of the hippocampus (3-6 sections) was analyzed using beacons to quantify the specific region for consistency across sections and slides. Real time RT-qPCR: Gene expression of cerebral APP, BACE1, Aph1B, gp47phox, gp91phox, ACE1, AT1 receptor was analyzed using the appropriate forward and reverse primers, as previously described. Briefly, RNA was isolated from midbrain cerebral tissue (~20 mg of tissue) using a Tissue Lyser system and AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Germantown, MD), following the manufacturer protocol. RNA quality and concentration were measured on a Cytation 3, using a Take3 plate (BioTek, Winooski, VT). Real-time RT-qPCR was performed on a BIORAD CFX96 Touch Real-Time PCR system (Hercules, CA) and analyzed using ΔΔCT. Ang II plasma ELISA: Angiotensin II was measured in plasma using an ELISA kit (CUSABIO Life Sciences, Houston, TX, murine Ang II kit, #ABIN366511), following the manufacturer’s protocol. Sample values were derived from a standard curve (generated from serial dilution of known standards), and results are expressed as pg/ml/mg tissue. Statistics: A 2-way ANOVA with post hoc Holm Sidak’s test was used to analyze statistical significance between treatment and exposure, and treatment x exposure interactions for each endpoint. Statistical analyses were conducted using GraphPad Prism v10.0.

Institutions

• University of North Texas

  TX, Denton

Categories

Funders

• National Institute of Environmental Health Sciences

  North Carolina, United States

  Grant ID: R00ES016586
• National Institute of Environmental Health Sciences

  North Carolina, United States

  Grant ID: 2R15ES026795-02

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