Nanoparticulate matter exposure results in neuroinflammatory changes in the corpus callosum

Abstract

Epidemiological studies have established an association between air pollution particulate matter exposure (PM2.5) and neurocognitive decline. Experimental data suggest that microglia play an essential role in air pollution PM-induced neuroinflammation and oxidative stress. This study examined the effect of nano-sized particulate matter (nPM) on complement C5 deposition and microglial activation in the corpus callosum of mice (C57BL/6J males). nPM was collected in an urban Los Angeles region impacted by traffic emissions. Mice were exposed to 10 weeks of re-aerosolized nPM or filtered air for a cumulative 150 hours. nPM-exposed mice exhibited reactive microglia and 2-fold increased local deposition of complement C5/ C5α proteins and complement component C5a receptor 1 (CD88) in the corpus callosum. However, serum C5 levels did not differ between nPM and filtered air cohorts. These findings demonstrate white matter C5 deposition and microglial activation secondary to nPM exposure. The C5 upregulation appears to be localized to the brain.

Fig 1. IBA-1 and GFAP staining for reactive glial cells in the medial corpus callosum of mice exposed to filtered air or nanoparticulate matter (nPM).

(A) IBA-1 positive cell counts in each experimental group. The nPM group showed significantly greater microglial cell count (n = 8) compared to the filtered air group (n = 8, p = 0.047). * signifies p < 0.05. (B) Below; low magnification representation of region analyzed. Above; filtered air and nPM exposed mice stained for IBA-1 in the corpus callosum (40x). The upper left hand corner is a high magnification representation of a single cell. (C) IBA-1 cell body to dendritic process ratio in each experimental group. The nPM cohort had a significantly increased ratio (n = 8) compared to the filter group (n = 8, p < 0.001). ** signifies p < 0.001. (D) GFAP positive cell counts in each experimental group. There was no significant difference in astrocyte cell count between groups (p = 0.983) (E) Below; low magnification representation of region analyzed. Above; filtered air and nPM exposed mice stained for GFAP in the corpus callosum (40x). The upper left hand corner is a high magnification representation of a single cell. Error bars represent standard deviation. Scale bars are presented on the lower right corner of the images.

Fig 2. Immunohistochemical analysis of C5, C5α, and C5a receptor (CD88) in the corpus callosum of animals exposed to filtered air or nanoparticulate matter (nPM).

(A) Filtered air (n = 8) or nPM (n = 8) exposed mice stained for C5 (red) in the corpus callosum. Nuclei (DAPI) are stained in blue (400x). (B) Low magnification representation of region analyzed. (C) C5 immunostaining in the corpus callosum was significantly higher in nPM exposed animals compared to the filtered air group (p = 0.001). (D) Filtered air (n = 8) and nPM (n = 8) exposed mice stained for C5α (red) in the corpus callosum. Nuclei (DAPI) are stained in blue (400x). (E) Low magnification representation of region analyzed. (F) C5α immunostaining in the corpus callosum of nPM exposed animals was significantly greater than in the filtered air group (p = 0.02). (G) Filtered air (n = 18) and nPM (n = 18) exposed mice stained for CD88 (red) in the corpus callosum. Nuclei (DAPI) are stained in blue (200x). (H) Low magnification representation of region analyzed. (I) CD88 immunostaining in the corpus callosum was significantly higher in nPM exposed animals compared to the filtered air group (p = 0.04). * signifies p< 0.05, ** signifies p ≤ 0.001. Error bars represent standard deviation. Scale bars indicate 50 μm.

Fig 3. Double immunofluorescence staining of C5 with IBA-1, GFAP, or ChAT in the corpus callosum of animals exposed to nanoparticulate matter (nPM).

(A) Co-staining of IBA-1 (red) with C5 (green) in the corpus callosum of a mouse exposed to nPM. Nuclei (DAPI) are stained in blue (200x). (B) Co-staining of GFAP (red) with C5 (green) in the corpus callosum of a mouse exposed to nPM. Nuclei (DAPI) are stained in blue (200x). (C) Co-staining of ChAT (red) with C5 (green) in the corpus callosum of a mouse exposed to nPM. Nuclei (DAPI) are stained in blue (200x). Scale bars are presented on the lower right corner of the images.

Fig 4. ELISA of serum C5 and TNF-alpha in animals exposed to filtered air or nanoparticulate matter (nPM).

Protein concentrations expressed as log values. (A) Serum C5 levels did not differ between the nPM and filtered air groups (p = 0.785). (B) TNF-alpha levels were significantly elevated in the nPM group compared to the filtered air group (p = 0.0419). * signifies p< 0.05. Error bars represent standard deviation.