Repeated gestational exposure to diesel engine exhaust affects the fetal olfactory system and alters olfactory-based behavior in rabbit offspring

Abstract

Background: Airborne pollution, especially from diesel exhaust (DE), is known to have a negative effect on the central nervous system in exposed human populations. However, the consequences of gestational exposure to DE on the fetal brain remain poorly explored, with various effects depending on the conditions of exposure, as well as little information on early developmental stages. We investigated the short-term effects of indirect DE exposure throughout gestation on the developing brain using a rabbit model. We analyzed fetal olfactory tissues at the end of gestation and tested behaviors relevant to pups' survival at birth. Pregnant dams were exposed by nose-only inhalation to either clean air or DE with a content of particles (DEP) adjusted to 1 mg/m³ by diluting engine exhaust, for 2 h/day, 5 days/week, from gestational day 3 (GD3) to day 27 (GD27). At GD28, fetal olfactory mucosa, olfactory bulbs and whole brains were collected for anatomical and neurochemical measurements. At postnatal day 2 (PND2), pups born from another group of exposed or control female were examined for their odor-guided behavior in response to the presentation of the rabbit mammary pheromone 2-methyl-3-butyn-2-ol (2MB2).

Results: At GD28, nano-sized particles were observed in cilia and cytoplasm of the olfactory sensory neurons in the olfactory mucosa and in the cytoplasm of periglomerular cells in the olfactory bulbs of exposed fetuses. Moreover, cellular and axonal hypertrophies were observed throughout olfactory tissues. Concomitantly, fetal serotoninergic and dopaminergic systems were affected in the olfactory bulbs. Moreover, the neuromodulatory homeostasis was disturbed in a sex-dependent manner in olfactory tissues. At birth, the olfactory sensitivity to 2MB2 was reduced in exposed PND2 pups.

Conclusion: Gestational exposure to DE alters olfactory tissues and affects monoaminergic neurotransmission in fetuses' olfactory bulbs, resulting in an alteration of olfactory-based behaviors at birth. Considering the anatomical and functional continuum between the olfactory system and other brain structures, and due to the importance of monoamine neurotransmission in the plasticity of neural circuits, such alterations could participate to disturbances in higher integrative structures, with possible long-term neurobehavioral consequences.

Keywords: Airborne pollution; Bulbar neurotransmitter disturbances; Diesel exhaust; Gestational exposure; Nano-particulate matter; Olfactory dysfunction; Olfactory toxicity; Olfactory-based behavior; Pheromonal response.

Fig. 1

Effects of DE exposure on the olfactory mucosa. a to d. Micrograph of an olfactory mucosa from control (a, c) and exposed (b, d) GD28 fetuses. The histological coloration reveals the anatomical alterations of the olfactory mucosa (b) and sub-mucosa (d) in exposed GD28 fetuses, compared to control GD28 fetuses (a and c, respectively). Scale bar = 25 μm. e to h. Electron micrograph of dendritic knobs of an olfactory sensory neuron and of an olfactory axon bundle from control (e and g) or exposed (f and h) GD28 fetuses. Note the decrease of cell organelles in the dendrite knobs of the OSN in exposed GD28 fetuses (f), compared to control GD28 fetuses (e). The white arrows indicate axonal hypertrophy of the olfactory axons of exposed fetuses (h), not observed in control fetuses (g). Note the condensation and marginalization of the chromatin of the ensheating cells of exposed fetuses when compared to controls. Scale bar = 5 μm. bv = blood vessels; nc: nasal cavity; dk = dendritic knok; oc = olfactory cilia; nu = nucleus, is = intercellular space

Fig. 2

Effects of DE exposure on the olfactory bulb. a and b. Micrograph of an olfactory bulb section from control (a) and exposed (b) GD28 fetuses. The histological coloration reveals the anatomical alterations of the different layers of the olfactory bulbs of exposed GD28 fetuses (b), compared to control ones (a) and the decrease in axonal ends and the presence of areas empty of biological material around glomeruli of exposed fetuses (b and b1) when compared to controls (a and a1). Granule cells clusters seem disrupted on exposed fetuses (b3) when compared to controls (a3). No anatomical differences in the mitral (Mt) cell layer of exposed fetuses (b2) and control (a2) fetuses were observed. Scale bar: 50 μm. c and d. Electron micrograph of the glomerular layer of olfactory bulbs from control (c) and exposed (d) GD28 fetuses. Note a significant decrease in axonal endings and the presence of empty areas (black stars) around the glomerulus from exposed fetuses (d), compared to the glomerulus from control fetuses (c). The PG cells of exposed fetuses exhibit a hypertrophy and a condensation and marginalization of their chromatin (white arrows) compared to PG cells from control fetuses. Scale bar: 20 μm. e and f. Electron micrograph of the granular layer of olfactory bulbs from control and exposed GD28 fetuses. Note the disruption of granular cells clusters, along with a condensation and marginalization of their chromatin, in exposed fetuses (f), compared to the granular clusters from control fetuses (e). Scale bar: 5 μm. nu = nucleus, is = intercellular space

Fig. 3

Electron micrograph showing the presence of particle-like structures in exposed GD28 fetuses’ olfactory tissues. a and b. Electron micrograph of the apical zone of the olfactory epithelium from exposed GD28 fetuses. a. Olfactory sensory neurons cilia with the structure in the framework enlarged at higher magnification (a1). The white arrows point out isolated particle-like structures (15–20 nm in diameter), which appeared inside olfactory cilia or in contact with olfactory cilia. b. Olfactory sensory neurons cytoplasm, with the structure in the white framework at higher magnification (b1). The white arrows point out lysosome-like vesicles containing NP-like particles (15–20 nm in diameter). Scale bars: a and b = 5 μm; a1 and b1 = 200 nm. c. Electron micrograph of the olfactory glomerulus from an exposed fetus showing the presence of NP-like particles in the cytoplasm of a PG cell. c1, c2 and c3 are enlargements at higher magnification to point out lysosome-like vesicles containing NP-like particles (5 nm in diameter). Scale bars: c. = 10 μm; c1 = 5 μm; c2 = 1 μm; c3 = 50 nm. nc = nasal cavity; dk = dendritic knok; oc = olfactory cilia; nu = nucleus, mi = mitochondrion

Fig. 4

Quantification of dopaminergic and cholinergic inputs into GD28 olfactory tubercle. a Relative mean density of TH+ labeled fibers in the olfactory tubercle. The quantitative estimation did not show any significant difference between the two groups. b Relative means density of AChE activity in the olfactory tubercle. There was no significant difference between the two groups regarding the DE exposure. However, we observed a sex-dependent difference between the two groups, which interacted significantly with DE exposure

Fig. 5

Percentages of positive behavioral responses following to the presentation of the rabbit mammary pheromone 2MB2. Results are expressed as mean ± SEM for each group. # p < 0.01 represented a significant difference between the groups and the expected percentage (60%). The percentage of positive responses in exposed fetuses (41%) was significantly lower, whereas that of control fetuses (66%) was not significantly different. **p < 0.01 represented a significant difference between the two groups. The comparison of the percentages showed a significant difference between the two groups

Fig. 6

Experimental protocol. Pregnant New-Zealand white female rabbits were exposed by nose-only inhalation to either clean air (control group; N = 14) or DE containing diluted DEP (1 mg/m³) (exposed group; N = 14) for 2 h/day, 5 days/week, from gestational day 3 (GD3) to day 27. At GD28, 12 dams (N = 5 controls; N = 7 exposed) were euthanized and their fetuses sacrificed by decapitation. Fetal olfactory mucosa (OM), hemi-olfactory bulbs (OB) and whole brains were dissected. Random samples of the latter structures were chosen for structural and chemical measures Eighteen exposed or control dams (N = 9 each) gave birth to F1 offspring. The day of birth was settled as postnatal day 0 (PND0). At PND2, offspring were examined for their odor-guided behavior in response to the presentation of the rabbit mammary pheromone 2-Methyl-3-butyn-2-ol (2MB2). They were then raised in control conditions in order to study long-term and intergenerational effects of this gestational DE exposure.