Acute exposure to diesel exhaust impairs adult neurogenesis in mice: prominence in males and protective effect of pioglitazone

Abstract

Adult neurogenesis is the process by which neural stem cells give rise to new functional neurons in specific regions of the adult brain, a process that occurs throughout life. Significantly, neurodegenerative and psychiatric disorders present suppressed neurogenesis, activated microglia, and neuroinflammation. Traffic-related air pollution has been shown to adversely affect the central nervous system. As the cardinal effects of air pollution exposure are microglial activation, and ensuing oxidative stress and neuroinflammation, we investigated whether acute exposures to diesel exhaust (DE) would inhibit adult neurogenesis in mice. Mice were exposed for 6 h to DE at a PM_2.5 concentration of 250-300 μg/m³, followed by assessment of adult neurogenesis in the hippocampal subgranular zone (SGZ), the subventricular zone (SVZ), and olfactory bulb (OB). DE impaired cellular proliferation in the SGZ and SVZ in males, but not females. DE reduced adult neurogenesis, with male mice showing fewer new neurons in the SGZ, SVZ, and OB, and females showing fewer new neurons only in the OB. To assess whether blocking microglial activation protected against DE-induced suppression of adult hippocampal neurogenesis, male mice were pre-treated with pioglitazone (PGZ) prior to DE exposure. The effects of DE exposure on microglia, as well as neuroinflammation and oxidative stress, were reduced by PGZ. PGZ also antagonized DE-induced suppression of neurogenesis in the SGZ. These results suggest that DE exposure impairs adult neurogenesis in a sex-dependent manner, by a mechanism likely to involve microglia activation and neuroinflammation.

Keywords: Adult neurogenesis; Diesel exhaust; Microglia; Neuroinflammation; Pioglitazone.

Fig. 1

Acute DE exposure decreases proliferation in the SGZ (a) and SVZ (b) of male mice. Eight-week-old male and female C57BL/6J mice were exposed to DE (250 μg/m³) or FA for 6 h, and then sacrificed 18 h later. Images shown are representative micrographs of Ki67 immunohistochemistry in female and male mice. Results represent the mean (± SE) with n = 3 per group. Significantly different from FA, **p < 0.01, ***p < 0.001. SGZ subgranular zone of the hippocampus, SVZ subventricular zone

Fig. 2

Effect of acute DE exposure on adult hippocampal neurogenesis in mice. Eight-week-old male and female C57BL/6J mice were given five 100-mg/kg doses of BrdU at 2-h intervals. On the following day they were exposed to DE (250 μg/m³) or FA for 6 h and then sacrificed 21 days later. The images shown are representative micrographs of NeuN/BrdU immunohistochemistry in the SGZ of FA- and DE-exposed mice. Scale bars in detail images represent 10 μm. The number of BrdU-stained cells (a) indicates surviving cells that been born since the beginning of the experiment. Double-stained NeuN/BrdU cells indicate surviving adult-born neurons, which are expressed both as a total number per brain region (b), and as a percentage of all BrdU-positive cells (c). Results represent the mean (± SE) with n = 3 per group. Significantly different from FA, **p < 0.01, ***p < 0.001

Fig. 3

Effect of acute DE exposure on adult neurogenesis in the SVZ of mice. Eight-week-old male and female C57BL/6J mice were given five 100-mg/kg doses of BrdU at 2-h intervals. On the following day, they were exposed to DE (250 μg/m³) or FA for 6 h, and then sacrificed 21 days later. Images shown are representative micrographs of NeuN/BrdU immunohistochemistry in the SVZ of FA- and DE-exposed mice. Scale bars in montage images represent 100 microns, while scale bars in detail images represent 10 μm. The number of BrdU-stained cells (a) indicates surviving cells that been born since the beginning of the experiment. Double-stained NeuN/BrdU cells indicate surviving adult-born neurons, which are expressed both as a total number per brain region (b) and as a percentage of all BrdU-positive cells (c). Data shown represent the mean (± SE) with n = 3 per group. Significantly different from FA, *p < 0.05

Fig. 4

Effect of acute DE exposure on adult neurogenesis in the OB of mice. Eight-week-old male and female C57BL/6J mice were given five 100-mg/kg doses of BrdU at 2-h intervals. On the following day, they were exposed to DE (250 μg/m³) or FA for 6 h and then sacrificed 21 days later. The images shown are representative micrographs of NeuN/BrdU immunohistochemistry in the OB of FA- and DE-exposed mice. Scale bars in detail images represent 10 μm. a Number of BrdU-positive cells indicates surviving cells that been born since the beginning of the experiment. Double-stained NeuN/BrdU cells indicate surviving adult-born neurons, which are expressed both as a total number per brain region (b), and as a percentage of all BrdU-positive cells (c). Data shown represent the mean (± SE) with n = 3 per group. Significantly different from FA, *p < 0.05, **p < 0.01, ***p < 0.001. OB olfactory bulb

Fig. 5

Pioglitazone (PGZ) pre-treatment suppresses microglial activation in the hippocampus of male mice. Eight-week-old male C57BL/6J mice were pre-treated with 12.5 mg/kg/day PGZ or vehicle (VEH) for 4 days up to and including the day of exposure, and were then exposed to DE (250 μg/m³) or FA for 6 h and then sacrificed 18 h later. Representative micrographs (on the right) of Iba1 immunohistochemistry of FA- and DE-exposed mice show microglia from the hippocampus, both as originally captured, and then as defined using tracing tool (inset) to allow for shape descriptor analysis using FIJI. Increases in area of the microglial soma (a) and aspect ratio (d) and decreases in circularity (b) and roundness (c) were indicators of microglial activation. Microglial soma area (a) is measured in μm², circularity (b) is calculated as 4π(area/perimeter²), roundness (c), a measure of inverse aspect ratio, is calculated by R = 4*area/π(major axis)². Aspect ratio (d) is defined as major axis/minor axis. Results represent the mean (± SE) with n = 3 per group. VEH/FA significantly different from VEH/DE; VEH/DE significantly different from PGZ/DE; *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

Effect of pioglitazione on oxidative stress and cytokine expression. Lipid peroxidation (levels of malondialdehyde) and levels of TNF-α mRNA were assessed in the cerebral cortex (a, c), and the hippocampus (b, d) of male mice following 4 days of pre-treatment with either 12. 5-mg/kg/day PGZ or with VEH, followed by a 6-h exposure to DE (250 μg/m³) or FA. Results represent the mean (± SE) with n = 6 per group. Significantly different from FA control, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

Effect of PGZ pre-treatment on DE-induced inhibition of proliferation in the SGZ of male mice. Eight-week-old male C57BL/6J mice were pre-treated with 12.5-mg/kg/day PGZ or VEH for 4 days up to and including the day of exposure, and were then exposed to DE (250 μg/m³) or FA for 6 h and then sacrificed 18 h later. Images shown are representative micrographs of Ki67+ cells in the hippocampus of FA- and DE-exposed mice, without and with PGZ pre-treatment. Significantly different from FA control, *p < 0.05

Fig. 8

Effect of PGZ pre-treatment on DE-induced inhibition of adult hippocampal neurogenesis in male mice. Eight-week-old male C57BL/6J mice were pre-treated with 12.5-mg/kg/day PGZ or VEH for 4 days. On day 3 of pre-treatment, they were given five 100-mg/kg doses of BrdU at 2-h intervals. On day 4, mice were exposed to DE (250 μg/m³) or FA for 6 h, then sacrificed 21 days later. Images shown are representative micrographs of NeuN/BrdU immunohistochemistry in the SGZ of FA- and DE-exposed mice. a BrdU+nuclei indicate surviving cells that been born since the beginning of the experiment. Double-stained NeuN/BrdU cells indicate surviving adult-born neurons, which are expressed both as a total number per brain region (b) and as a percentage of all BrdU-positive cells (c). Results represent the mean (± SE) with n = 3 per group. Significantly different from FA control or between VEH-pre-treatment and PGZ pre-treatment, *p < 0.05, **p < 0.01, ***p < 0.001