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

Figures

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  Figure 1. Representative images of 8-hydroxy-2′-deoxyguanosine (8-OHdG, red) in the hippocampus CA1 region in either (A) apolipoprotein E-null (ApoE−/−) mice exposed to filtered air (FA); (B) ApoE−/− mice exposed to FA+captopril (FA+ACEi); (C) ApoE−/− mice exposed to mixed vehicle exhaust (MVE: 200 µg PM/m3 mixed gasoline and diesel exhaust, 6 h/day, 7 days/week, 30 days); or (D) ApoE−/− mice exposed to MVE+ACEi. (E) Fluorescence was quantified and represented as mean fluorescence for the CA1 region in each animal. n = 3 animals per group, four sections per animal. Blue fluorescence = nuclear Hoechst stain. Scale bar = 100 µm, 40× magnification (CA1). Statistical analyses were conducted using a two-way pairwise ANOVA. * = p ≤ 0.05, ** = p ≤ 0.01.

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  Figure 2. Representative images of NADPH oxidase (NOX2, red) in the CA1 region of the hippocampus in either (A) ApoE−/− mice exposed to filtered air (FA); (B) ApoE−/− mice exposed to FA + captopril (FA+ACEi); (C) ApoE−/− mice exposed to mixed vehicle exhaust (MVE: 200 µg PM/m3 mixed gasoline and diesel exhaust, 6 h/day, 7 days/week, 30 days); or (D) ApoE−/− mice exposed to MVE+ACEi. (E) Fluorescence was quantified and represented as mean fluorescence for the CA1 region in each animal. n = 3 animals per group, four sections per animal. Blue fluorescence = nuclear Hoechststain. Scale bar = 100 µm, 40× magnification (CA1). (F) Real-time RT-qPCR analysis of cerebral mRNA expression of NADPH oxidase subunit gp91phox, n = 4–7 animals per group. Statistical analyses were conducted using a two-way pairwise ANOVA. Data is expressed as mean normalized gene expression using ∆∆CT values, with GAPDH used as the housekeeping gene. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001.

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  Figure 3. Representative images of interleukin-1 beta (IL-1ß, red) and Von Willebrand Factor (vWF, green) in the parenchyma surrounding the cerebral vasculature in either (A) ApoE−/− mice exposed to filtered air (FA); (B) ApoE−/− mice exposed to FA + captopril (FA+ACEi); (C) ApoE−/− mice exposed to mixed vehicle exhaust (MVE: 200 µg PM/m3 mixed gasoline and diesel exhaust, 6 h/day, 7 days/week, 30 days); or (D) ApoE−/− mice exposed to MVE + ACEi. (E) Fluorescence was quantified and represented as mean fluorescence for standardized areas in surrounding cerebral vessels (vWF positive) in each animal. n = 3 animals per group, two slides per animal, two sections per slide, 8–10 vessels on each section. Blue fluorescence = nuclear Hoechst stain; 20× magnification (scale bar = 200 µm). (F) Mean normalized gene expression of cerebral IL-1β was quantified using RT-qPCR, n = 4–6 animals per group. Statistical analyses were conducted using a two-way pairwise ANOVA. * = p ≤ 0.05, ** = p ≤ 0.01.

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  Figure 4. Plasma angiotensin (Ang) II levels, as determined by ELISA, in ApoE−/− mice exposed to either filtered air (FA) or mixed gasoline and diesel vehicle emissions (MVE: 200 µg/m3 PM for 6 h/day, 5 days/week, 30 days) with or without captopril treatment (ACEi; 4 mg/kg/day) in the drinking water. n = 6 per group. Statistical analyses were conducted using a two-way pairwise ANOVA. * = p ≤ 0.05, **** = p ≤ 0.0001.

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  Figure 5. Representative images of angiotensin type 1 receptor (AT1, red) in the CA1 region of the hippocampus in (A) ApoE−/− mice exposed to water + filtered air (FA); (B) ApoE−/− mice exposed to captopril + FA (FA+ACEi); (C) ApoE−/− mice exposed to water + mixed vehicle exhaust (MVE: 200 µg PM/m3 mixed gasoline and diesel exhaust, 6 h/day, 7 days/week, 30 days); or (D) ApoE−/− mice exposed to MVE + captopril treatment (MVE+ACEi). (E) Fluorescence was quantified and represented as mean fluorescence for the CA1 region in each animal. n = 3 animals per group, four sections per animal. Blue fluorescence = nuclear Hoechst stain. Scale bar = 100 µm, 40x magnification (CA1 region). (F) Mean normalized gene expression of the AT1 receptor was quantified in the cerebrum using RT-qPCR, n = 5 animals per group. Statistical analyses were conducted using a two-way pairwise ANOVA. * = p ≤ 0.05.

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  Figure 6. Real time RT-qPCR analysis of cerebral mRNA expression of (A) amyloid beta precursor protein (APP), (B) beta-secretase 1 (ΒACE1), (C) Aph-1 homolog B gamma-secretase subunit (APH1B) mRNA expression in cerebral tissue from ApoE−/− mice exposed to either filtered air (FA), FA with concurrent treatment of captopril (FA+ACEi) or mixed vehicle exhaust (MVE) (200 µg PM/m3 mixed gasoline and diesel exhaust, 6 h/day, 7 days/week, 30 days), or MVE with concurrent treatment of captopril (MVE+ACEi). Data is expressed as mean normalized gene expression using ∆∆CT values, with GAPDH used as the housekeeping gene, n = 6–8 animals per group. (D–G) Representative images of β-secretase (ΒACE1, red) in the hippocampus CA1 region of ApoE−/− mice exposed to (D) FA, (E) FA+ACEi, (F) MVE, or (G) MVE + ACEi. (H) Fluorescence was quantified and represented as mean fluorescence for the CA1 region in each animal. n = 3 animals per group, four sections per animal. Blue fluorescence = nuclear Hoechst stain. Scale bar = 100 µm, 40× magnification (CA1). Statistical analyses were conducted using a two-way pairwise ANOVA * = p ≤ 0.05, ** = p ≤ 0.01.