Influence of Mild Chronic Stress and Social Isolation on Acute Ozone-Induced Alterations in Stress Biomarkers and Brain-Region-Specific Gene Expression in Male Wistar-Kyoto Rats

Abstract

Individuals with psychosocial stress often experience an exaggerated response to air pollutants. Ozone (O₃) exposure has been associated with the activation of the neuroendocrine stress-response system. We hypothesized that preexistent mild chronic stress plus social isolation (CS), or social isolation (SI) alone, would exacerbate the acute effects of O₃ exposure on the circulating adrenal-derived stress hormones, and the expression of the genes regulating glucocorticoid stress signaling via an altered stress adaptation in a brain-region-specific manner. Male Wistar-Kyoto rats (5 weeks old) were socially isolated, plus were subjected to either CS (noise, confinement, fear, uncomfortable living, hectic activity, and single housing), SI (single housing only, restricted handling and no enrichment) or no stress (NS; double housing, frequent handling and enrichment provided) for 8 weeks. The rats were then exposed to either air or O₃ (0.8 ppm for 4 h), and the samples were collected immediately after. The indicators of sympathetic and hypothalamic-pituitary axis (HPA) activation (i.e., epinephrine, corticosterone, and lymphopenia) increased with O₃ exposure, but there were no effects from CS or SI, except for the depletion of serum BDNF. CS and SI revealed small changes in brain-region-specific glucocorticoid-signaling-associated markers of gene expression in the air-exposed rats (hypothalamic Nr3c1, Nr3c2 Hsp90aa1, Hspa4 and Cnr1 inhibition in SI; hippocampal HSP90aa1 increase in SI; and inhibition of the bed nucleus of the stria terminalis (BNST) Cnr1 in CS). Gene expression across all brain regions was altered by O₃, reflective of glucocorticoid signaling effects, such as Fkbp5 in NS, CS and SI. The SI effects on Fkbp5 were greatest for SI in BNST. O₃ increased Cnr2 expression in the hypothalamus and olfactory bulbs of the NS and SI groups. O_3, in all stress conditions, generally inhibited the expression of Nr3c1 in all brain regions, Nr3c2 in the hippocampus and hypothalamus and Bdnf in the hippocampus. SI, in general, showed slightly greater O₃-induced changes when compared to NS and CS. Serum metabolomics revealed increased sphingomyelins in the air-exposed SI and O₃-exposed NS, with underlying SI dampening some of the O₃-induced changes. These results suggest a potential link between preexistent SI and acute O₃-induced increases in the circulating adrenal-derived stress hormones and brain-region-specific gene expression changes in glucocorticoid signaling, which may partly underlie the stress dynamic in those with long-term SI.

Keywords: Fkbp5; bed nucleus of the stria terminalis; chronic stress; gene expression; glucocorticoids; hippocampus; hypothalamus; metabolomics; neuroendocrine; olfactory bulbs; ozone; social isolation.

Figure 1

Effect of stressors on physiological responses. (A): Regardless of stress conditions, O₃ decreased blood lymphocytes across all stress groups. (B): Corticosterone (CORT) was increased across all stress groups concomitant with the decrease in lymphocytes. (C): ACTH levels at the selected time point were unaltered across all stress and exposure groups. (D,E): Plasma epinephrine was significantly elevated by exposure to O₃ with no effect on plasma norepinephrine levels. (F): BDNF plasma levels were significantly decreased by chronic stress and social isolation regardless of exposures. NS = no stress controls; CS = chronic stress group; SI = social isolation group. Data are represented by boxplots with a dot plot overlay to express the distribution of each data point where n = 5–12. @ represents a statistical difference (p < 0.05; see details in the text) between exposure groups within the same stress group. # represents a statistical difference (p < 0.05; see details in the text) between stress groups within the same exposure group.

Figure 2

Effect of stressors on genes related to stress receptors. (A): Across all brain regions and stress groups, there was no change in Crhr1 gene expression. (B): Crhr2 expression was altered by O₃ exposure in the no stress (NS) and social isolation (SI) group for hypothalamus (HYP) and NS group for olfactory bulb (OB). Also, in the OB, both chronic stress (CS) and SI had diminished responses to O₃ exposure. (C): Nr3C1 expression was significantly decreased by O₃ exposure across all stress groups in the hippocampus (HIP) and OB. Nr3C1 expression was also decreased in the NS and SI groups in the bed nucleus of the stria terminalis (BNST) and the NS and CS groups in the HYP. In the HYP, expression in the SI group was lower than that in the NS group. (D): Nr3c2 expression was decreased by O₃ for all three stress groups in the HIP. A similar decrease was observed in the HYP and OB, but only the CS groups were significantly decreased. In the BNST, O₃ exposure in the SI group increased expression of Nr3c2. In the BNST and HYP, CS and S groups had diminished expression compared to NS, respectively. Data are represented by boxplots with a dot plot overlay to express the distribution of each data point where n = 5–12. @ represents a statistical difference (p < 0.05; see details in the text) between exposure groups within the same stress group. # represents a statistical difference (p < 0.05; see details in the text) between stress groups within the same exposure group.

Figure 3

Effect of stressors on genes related to glucocorticoid-associated chaperones. (A): Fkbp4 was significantly altered by exposure to O₃ in the hippocampus (HIP) for the no stress (NS) and chronic stress (CS) groups and in the hypothalamus (HYP) for the NS group. (B): Fkbp5 was significantly increased by O₃ exposure in all brain regions. Also, the O₃-induced increase in Fkbp5 was higher than that in the NS group in the bed nucleus of stria terminalis (BNST) while it was lower in the olfactory bulb (OB). (C): Hsp90aa1 expression was significantly higher in the BNST when exposed to O₃ compared to the NS group. In the HYP, the social isolation (SI) group had higher baseline Hsp90aa1 expression in the air group compared to NS. In the HYP, both exposure groups were significantly altered by SI compared to NS. (D): Hspa4 was decreased by O₃ exposure in the OB for all three stressor groups. In the HYP, the air-exposed group in SI had lower expression than that of the NS. Data are represented by boxplots with a dot plot overlay to express the distribution of each data point where n = 5–12. @ represents a statistical difference (p < 0.05; see details in the text) between exposure groups within the same stress group. # represents a statistical difference (p < 0.05; see details in the text) between stress groups within the same exposure group.

Figure 4

Effect of stressors on genes related to neurotrophic, neurotransmitter and cannabinoid factors. (A): O₃ exposure significantly decreased Bdnf expression in the hippocampus (HIP) region for all three stress groups (NS = no stress controls; CS = chronic stress group; SI = social isolation group) and in the SI group in the olfactory bulb (OB). (B): Th expression was increased across all stress groups in response to O₃ exposure in the hippocampus (HIP) but decreased in the SI within the bed nucleus of the stria terminalis (BNST). (C): Cnr1 expression was increased by O₃ exposure in the SI group within the BNST and hypothalamus (HYP) and decreased by O₃ in the NS group within the OB. In the BNST, the baseline air-exposed group in the CS was lower than that of the NS. A lower baseline was also found in the SI of the HYP. Data are represented by boxplots with a dot plot overlay to express the distribution of each datapoint where n = 5–12. @ represents a statistical difference (p < 0.05; see details in the text) between exposure groups within the same stress group. # represents a statistical difference (p < 0.05; see details in the text) between stress groups within the same exposure.