Mitoapocynin Attenuates Organic Dust Exposure-Induced Neuroinflammation and Sensory-Motor Deficits in a Mouse Model

Abstract

Increased incidences of neuro-inflammatory diseases in the mid-western United States of America (USA) have been linked to exposure to agriculture contaminants. Organic dust (OD) is a major contaminant in the animal production industry and is central to the respiratory symptoms in the exposed individuals. However, the exposure effects on the brain remain largely unknown. OD exposure is known to induce a pro-inflammatory phenotype in microglial cells. Further, blocking cytoplasmic NOX-2 using mitoapocynin (MA) partially curtail the OD exposure effects. Therefore, using a mouse model, we tested a hypothesis that inhaled OD induces neuroinflammation and sensory-motor deficits. Mice were administered with either saline, fluorescent lipopolysaccharides (LPSs), or OD extract intranasally daily for 5 days a week for 5 weeks. The saline or OD extract-exposed mice received either a vehicle or MA (3 mg/kg) orally for 3 days/week for 5 weeks. We quantified inflammatory changes in the upper respiratory tract and brain, assessed sensory-motor changes using rotarod, open-field, and olfactory test, and quantified neurochemicals in the brain. Inhaled fluorescent LPS (FL-LPS) was detected in the nasal turbinates and olfactory bulbs. OD extract exposure induced atrophy of the olfactory epithelium with reduction in the number of nerve bundles in the nasopharyngeal meatus, loss of cilia in the upper respiratory epithelium with an increase in the number of goblet cells, and increase in the thickness of the nasal epithelium. Interestingly, OD exposure increased the expression of HMGB1, 3- nitrotyrosine (NT), IBA1, glial fibrillary acidic protein (GFAP), hyperphosphorylated Tau (p-Tau), and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL)-positive cells in the brain. Further, OD exposure decreased time to fall (rotarod), total distance traveled (open-field test), and olfactory ability (novel scent test). Oral MA partially rescued olfactory epithelial changes and gross congestion of the brain tissue. MA treatment also decreased the expression of HMGB1, 3-NT, IBA1, GFAP, and p-Tau, and significantly reversed exposure induced sensory-motor deficits. Neurochemical analysis provided an early indication of depressive behavior. Collectively, our results demonstrate that inhalation exposure to OD can cause sustained neuroinflammation and behavior deficits through lung-brain axis and that MA treatment can dampen the OD-induced inflammatory response at the level of lung and brain.

Keywords: inflammation; microglia; mitoapocynin; neurodegeneration; organic dust.

Figure 1

Organic dust extract (ODE) induces gross and histological inflammatory changes in nasal passage and fluorescent lipopolysaccharide (LPS) travel to the olfactory bulb after inhalation. Mice were exposed to various treatments outlined in the Supplementary Table 1. Paraffin embedded sections from perfused (4 paraformaldehyde) mice brain and decalcified upper respiratory tract are shown (A–C). Compared to control, Fluorescent LPS (white arrowhead) was observed in nasal turbinate and the olfactory bulb of the brain (A: i–iv, micrometer bar = 100 μm, 20x). When compared to control, mice exposed to ODE showed atrophic changes in the olfactory epithelium (double sided arrow) and fewer nerve bundles (black arrowhead) in nasopharyngeal meatus. Mitoapocynin (MA) treatment partially rescued olfactory epithelium atrophy and nerve bundle loss (B: i–iii, micrometer bar = 100 μm, 20x). Immunofluorescent staining of the nasopharyngeal meatus (coronal sections) with acetylated α-tubulin showed a significant loss of cilia (white arrow) in mice treated with ODE (C: i–iii, micrometer bar = 100 μm, 20x), and MA treatment had no effect on cilia loss (C: vii). Goblet cell numbers and nasal epithelium thickness (two-sided arrow) were analyzed by a blinded investigator (C: iv–vi, micrometer bar = 50 μm, 40x). Compared to control, ODE exposure induced goblet cell hyperplasia and thickening of the nasal epithelium, and MA treatment had no effect (C: viii, ix). Upon visual gross examination of freshly dissected non-perfused brains, ODE-exposed mice showed signs of congestion when compared to mice administered with normal saline and MA (C11), which appears to reduce congestion in brain (D: i–iii).

Figure 2

ODE induces HMGB1 expression in various regions of mice brain. Paraffin embedded sections from perfused mice brain were stained with anti-HMGB1 (Cy3, red) antibody and nuclei were identified with 4', 6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, blue) (A). HMGB1 expression in immunohistochemistry (IHC) was quantified by a blinded investigator. Compared to control, ODE-exposed mice showed higher amounts of HMGB1 staining in the corpus callosum, cerebellum, frontal cortex, and olfactory bulb of the brain. MA (C11) treatment significantly reduced HMGB1 expression (B). Whole cell lysate from freshly dissected mice brain were processed for western blot analysis. HMGB1 and β-actin antibodies (house-keeping protein) detected 25 and 42 kD bands, respectively. Densitometry of normalized bands showed that, compared to controls, ODE exposure increased the HMGB1 protein level in mice brain and that MA treatment significantly reduced HMGB1 protein levels (C,D) (n = 5, *exposure effect, ** indicates a significant change with respect to control, # MA treatment effect, ## indicates significant change with respect to MA (mitoapocynin). p < 0.05, micrometer bar = 50 μm).

Figure 3

ODE induces 3-Nitrotyrosine generation in various regions of mice brain. Paraffin-embedded sections from perfused mice brain were stained with anti-3-nitrotyrosine (NT; white arrow; Fluorescein isothiocyanate, FITC; green) antibody and nuclei were identified with DAPI (blue) (A). 3-NT generation in IHC was quantified by a blinded investigator. Compared to control, ODE-exposed mice showed higher amounts of 3-NT staining in the cerebellum, frontal cortex, and olfactory bulb of the brain. MA (C11) treatment significantly reduced 3-NT generation only in olfactory bulb area of the brain (B) (n = 5, *exposure effect, **indicates a significant change with respect to control, # MA treatment effect, ## indicates significant change with respect to MA (mitoapocynin), p < 0.05, micrometer bar = 50 μm).

Figure 4

ODE induces IBA1 expression in various regions of mice brain. Paraffin-embedded sections from perfused mice brain were stained with anti-IBA1 (white arrow, Cy3, red) antibody, and nuclei were identified with DAPI (blue). (A) IBA1 expression in IHC was quantified by a blinded investigator. Compared to control, ODE-exposed mice showed higher amounts of IBA1 staining in the corpus callosum, cerebellum, frontal cortex, hippocampus, and olfactory bulb of the brain. MA (C11) treatment significantly reduced IBA1 expression in the corpus callosum, cerebellum frontal cortex, and olfactory bulb regions of brain (B). Whole cell lysate from freshly dissected mice brain were processed for western blot analysis. IBA1 and β-actin antibodies (house-keeping protein) detected 13 and 42 kD bands, respectively. Densitometry of normalized bands showed that, compared to controls, ODE exposure increased the IBA1 protein level in mice brain, and that MA treatment significantly reduced IBA1 protein level (C,D) (n = 5, *exposure effect, # MA treatment effect, p < 0.05, micrometer bar = 50 μm).

Figure 5

ODE induces glial fibrillary acidic protein (GFAP) expression in various regions of mice brain. Paraffin-embedded sections from perfused mice brain were stained with anti-GFAP (white arrow, FITC, green) antibody and nuclei were identified with DAPI (blue) (A). GFAP expression in IHC was quantified by a blinded investigator. Compared to control, ODE-exposed mice showed higher amounts of GFAP staining in the corpus callosum, frontal cortex, and olfactory bulb of the brain. MA (C11) treatment significantly reduced GFAP expression in the corpus callosum, frontal cortex, and olfactory bulb regions of brain (B) (n = 5, *exposure effect, # MA treatment effect, p < 0.05, micrometer bar = 50 μm).

Figure 6

ODE induces hyperphosphorylated Tau (p-Tau) expression in various regions of mice brain. Paraffin-embedded sections from perfused mice brain were stained with anti p-Tau (white arrow, FITC, green) and anti-NeuN (neuronal marker; Cy3, red) antibodies. Also, nuclei were identified with DAPI (blue) (A). p-Tau expression in IHC was quantified by a blinded investigator. Compared to control, ODE-exposed mice showed higher amounts of p-Tau staining in the cerebellum and olfactory bulb of the brain. MA (C11) treatment significantly reduced p-Tau expression in the cerebellum region of brain (B). Whole cell lysate from freshly dissected mice brain were processed for western blot analysis. p-Tau and β-actin antibodies (house-keeping protein) detected 79 and 42 kD bands, respectively. Densitometry of normalized bands showed that, compared to controls, ODE exposure increased the p-Tau protein level in mice brain and MA treatment significantly reduced p-Tau protein level (C,D) (n = 5, *exposure effect, # MA treatment effect, p < 0.05, micrometer bar = 50 μm).

Figure 7

ODE exposure induces neurodegenerative changes in various regions of mice brain. Paraffin-embedded sections from perfused mice brain were labeled with deoxyuridine triphosphate (dUTP)-FITC (white arrow; apoptosis marker, FITC, green), and nucleus was stained with DAPI (blue) (A). The total number of cells (DAPI, blue) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells (FITC, green) per field (20X) were counted in a total of five random fields by a blinded investigator. Compared to control, ODE-exposed mice showed a higher number of TUNEL positive cells in the corpus callosum, cerebellum, frontal cortex, and olfactory bulb of the brain. MA (C11) significantly reduced the number of TUNEL positive cells in the corpus callosum, cerebellum, frontal cortex, and olfactory bulb regions of the mice brain (B) [n = 5, *exposure effect, # MA (C11) treatment effect, p < 0.05, micrometer bar = 50 μm].

Figure 8

ODE exposure induces neuro-motor and neuro-sensory changes in mice. As compared to control, ODE-exposed mice displayed a significant decrease in the time to fall from the rotarod in an accelerated profile, whereas mice administered with oral dosages of MA (C11) showed a significant higher time to fall from the rotarod when compared to ODE-exposed group (A). As compared to control, ODE-exposed mice showed significantly lower exploratory locomotor activity. Whereas, mice administered with oral dosages of MA(C11) showed a significant higher exploratory locomotor activity when compared to ODE-exposed (B). As compared to control, ODE exposed mice showed a significantly lower ability of olfactory sensation and mice administered with MA (C11) showed a significant higher sense of olfactory ability when compared to mice exposed to only ODE (C).