The effects of ozone exposure and sedentary lifestyle on neuronal microglia and mitochondrial bioenergetics of female Long-Evans rats

Abstract

Ozone (O₃) is a widespread air pollutant that produces cardiovascular and pulmonary dysfunction possibly mediated by activation of central stress centers. Epidemiological data suggest that sedentary lifestyles may exacerbate responses to air pollutants such as O₃. We sought to assess neurological changes in response to O₃ exposure and an active lifestyle. We developed an animal model in which female Long-Evans rats were either sedentary or active with continuous access to running wheels starting at postnatal day (PND) 22 until the age of PND 100 and then exposed to O₃ (0, 0.25, 0.5 or 1.0 ppm) 5 h/day for two consecutive days. We found significantly more reactive microglia within the hippocampus (HIP) in animals exposed to O₃ in both sedentary and active rats. No changes were detected in astrocytic coverage. We next analyzed mitochondrial bioenergetic parameters (complex I, complex II and complex IV). Complex I activity was significantly affected by exercise in hypothalamus (HYP). Complex II activity was significantly affected by both exercise and O₃ exposure in the HIP. Concomitant with the changes in enzymatic activity, there were also effects on expression of genes related to mitochondrial bioenergetics and antioxidant production. These results demonstrate that O₃ induces microglia reactivity within stress centers of the brain and that mitochondrial bioenergetics are altered. Some of these effects may be augmented by exercise, suggesting a role for lifestyle in O₃ effects on brain mitochondrial bioenergetics parameters in agreement with our previous reports on other endpoints.

Keywords: Astrocytes; Bioenergetics; Exercise; GFAP; Iba1; Microglia; Mitochondria; Oxidative Stress Genes; Ozone.

Fig. 1.. Experimental design to understand the interactive effects of lifestyle and O₃ exposure.

Female Long Evans rats were obtained at PND22 and were assigned as sedentary or active (running wheel) until PND100. After PND96, both groups were exposed to O₃ (0, 0.25, 0.5 or 1 ppm) for 5 hours/day on two consecutive days. Immediately after O₃ exposure, rats were euthanized, brains removed, half brain was fixed in 4% formaldehyde for immunohistochemistry and the other half brain was dissected for hippocampus and hypothalamus. Brain regions were quick frozen for analysis of mitochondrial bioenergetics and oxidative stress gene expression. *Indicates that parameters that were analyzed by other team members and some results were published already.

Fig. 2.

Changes in GFAP immunoreactivity in the hippocampus and hypothalamus in response to O₃ and exercise. Female rats with access to a running wheel and those without access (sedentary) were exposed to 0 ppm and 1 ppm O₃ for 5 h/day for 2 consecutive days. Slices of 50 μm were taken from corresponding bregma levels across all animals for dorsal hippocampus and hypothalamus and were stained with a combination of astrocytic and microglia markers with immunofluorescence. Representative images of the astrocytic marker, GFAP, are depicted here by inverting the greyscale image of the corresponding color channel for GFAP from images taken at 20X (A). GFAP immunoreactivity was traced via NIS Elements software and area (μm2) data was analyzed within each brain region (B). Images have been scaled 2X for better visualization in print and therefore do not contain the full area of analysis. Scale bar represents 10 μm. Data plotted as means ± SEM, n = 3 animals.

Fig. 3.

Changes in Iba1 immunoreactivity in the hippocampus and hypothalamus in response to O₃ and exercise. Female rats with access to a running wheel and those without access (sedentary) were exposed to 0 ppm and 1 ppm O₃ for 5 h/day for 2 consecutive days. Slices of 50 m were taken from corresponding bregma levels across all animals for dorsal hippocampus and hypothalamus and were stained with a combination of astrocytic and microglia markers with immunofluorescence. Representative images of the microglial marker, Iba1, are depicted here by inverting the greyscale image of the corresponding color channel for Iba1from images taken at 20X (A). Iba1 containing cells were counted and classified, based on their morphology, as either “resting” or “reactive.” The percent of “reactive” cells divided by the total Iba1 positive cells for each image were analyzed within each brain region (B). Images have been scaled 2X for better visualization in print and therefore do not contain the full area of analysis. Scale bar represents 10 m. Red arrows indicate microglia with representative reactive morphology. Data plotted as means ± SEM, n = 3 animals. * p < 0.05 indicate significant differences between O₃ levels within exercise groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.

Mitochondrial Complex Enzyme Activities in The Hippocampus of Sedentary and Active Rats Following O₃ Exposure. Data plotted as means ± SEM, n = 5 animals. Asterisks indicate significant differences between activity levels within the same O₃ doses (* p < 0.05; *** p < 0.001).

Fig. 5.

Mitochondrial Gene Expression In The Hippocampus Of Sedentary And Active Rats Following O₃ Exposure. Data plotted as means ± SEM, n = 5 animals. Asterisks indicate significant differences between activity levels within the same O₃ doses (* p < 0.05; ** p < 0.01). Hashtags indicate significant differences between different O₃ doses within the same activity group (# p < 0.05).

Fig. 6.

Mitochondrial Complex Enzyme Activities in The Hypothalamus of Sedentary and Active Rats Following O₃ Exposure. Data plotted as means ± SEM, n = 5 animals. Asterisks indicate significant differences between activity levels within the same O₃ doses (* p < 0.05).

Fig. 7.

Mitochondrial Gene Expression in The Hypothalamus of Sedentary and Active Rats Following O₃ Exposure. Data plotted as means ± SEM, n = 5 animals. Asterisks indicate significant differences between activity levels within the same O₃ doses (* p < 0.05). Hashtags indicate significant differences between different O₃ doses within the same activity group (# p < 0.05).

Fig. 8.

Oxidative Stress Related Gene Expression in Hypothalamus of Sedentary and Active Rats Following O₃ Exposure. Data plotted as means ± SEM, n = 5 animals. Asterisks indicate significant differences between activity levels within the same O₃ doses (* p < 0.05; ** p < 0.01). Hashtags indicate significant differences between different O₃ doses within the same activity group (# p < 0.05; ## p < 0.01).

Fig. 9.

Oxidative Stress Related Gene Expression in The Hippocampus of Sedentary and Active Rats Following O₃ Exposure. Data plotted as means ± SEM, n = 5 animals. Asterisks indicate significant differences between activity levels within the same O₃ doses (* p < 0.05). Hashtags indicate significant differences between different O₃ doses within the same activity group (# p < 0.05).