Brain Inflammation, Blood Brain Barrier dysfunction and Neuronal Synaptophysin Decrease after Inhalation Exposure to Titanium Dioxide Nano-aerosol in Aging Rats

Abstract

Notwithstanding potential neurotoxicity of inhaled titanium dioxide nanoparticles (TiO₂ NPs), the toxicokinetics and consequences on blood-brain barrier (BBB) function remain poorly characterized. To improve risk assessment, we need to evaluate the impact on BBB under realistic environmental conditions and take into account vulnerability status such as age. 12-13 week and 19-month-old male rats were exposed by inhalation to 10 mg/m³ of TiO₂ nano-aerosol (6 hrs/day, 5 day/week, for 4 weeks). We showed an age-dependent modulation of BBB integrity parameters suggesting increased BBB permeability in aging rats. This alteration was associated with a significant increase of cytokines/chemokines in the brain, including interleukin-1β, interferon-γ, and fractalkine as well as a decreased expression of synaptophysin, a neuronal activity marker. These observations, in absence of detectable titanium in the brain suggest that CNS-related effects are mediated by systemic-pathway. Moreover, observations in terms of BBB permeability and brain inflammation underline age susceptibility. Even if TiO₂ NPs were not evidenced in the brain, we observed an association between the exposure to TiO₂ NPs and the dysregulation of BBB physiology associated with neuroinflammation and decreased expression of neuronal activity marker, which was further exacerbated in the brain of aged animal's.

Figure 1

Titanium quantification in brains of young adult rats (A) and aging rats (B) of exposed and control animals by inductively coupled plasma mass spectrometry (ICP-MS) at 3 days and 28 days after the end of the inhalation exposure. Results represents the median of n = 5 to 8 animals. Statistical comparison between treated and control groups was by two way ANOVA after Box-Cox transformation on Ti concentration variable and Bonferroni post hoc test.

Figure 2

BBB permeability assessment. The permeability was estimated by the atenolol cerebral concentration and the ratio between atenolol in the brain and plasma (partition coefficient or Kp) at 3 days and 28 days after the end of the inhalation exposure in young adults (A and B) and aging groups (C and D). Each data point represents one animal with median of n = 7 to 9 rats. *P < 0.05; **P < 0.01 compared to corresponding control.

Figure 3

Representative expression of (A,D) von Willebrand factor, green fluorescence; (B,E) claudin-5, red fluorescence; (C,F) vessel-specific claudin-5, yellow overlay in midbrain microvessels (<100 µm in diameter) of (young) 12–13 wk control (A,B,C) or nano-aerosol-exposed (D,E,F) rats at 28 days after the end of exposure. Graph shows quantification of fluorescence shown in panels C and F. A minimum of 3 locations on each section (2 sections per slide), 3 slides and n = 3 per group were analyzed. Scale bar = 100 µm. *p < 0.050 compared to control rat microvessels.

Figure 4

Representative expression of (A,D) von Willebrand factor, green fluorescence; (B,E) claudin-5, red fluorescence; (C,F) vessel-specific claudin-5, yellow overlay in midbrain microvessels (<100 µm in diameter) of (aged) 19 mo control (A,B,C) or nano-aerosol-exposed (D,E,F) rats at 28 days after the end of exposure. Graph shows quantification of fluorescence shown in panels C and F. A minimum of 3 locations on each section (2 sections per slide), 3 slides and n = 3 per group were analyzed. Scale bar = 100 µm. *p < 0.050 compared to control rat microvessels.

Figure 5

Detection of 6 inflammatory markers in young adults and aging rat cerebral tissues (cerebrum + cerebellum) after subacute inhalation exposure to TiO₂ nano-aerosol. Measurement was done in cerebral tissues extracts collected 3 or 28 days after the end of the inhalation exposure by multiplexing approach. Each data point represents one animal with medians of 4 to 7 animals. *P < 0.05; **P < 0.01 compared to corresponding controls.

Figure 6

IL-1β expression (red fluorescence) in the midbrain of (A) 12–13 wk control, (B) 12–13 wk nano-aerosol-exposed; (C) 19 mo control; and (D) 19 mo nano-aerosol-exposed rats at 28 days after the end of exposure. A minimum of 3 locations on each section (2 sections per slide), 3 slides and n = 3 per group were analyzed. Relative fluorescence per unit area is represented in graph shown. *p < 0.050 compared to 12–13 wk control; ^†p < 0.050 compared to 19 mo control. Scale bar = 1000 µm.

Figure 7

IL-1β expression (red fluorescence) in the forebrain of 12–13 wk control (A), 12–13 wk nano-aerosol-exposed (B); 19 mo control (C); and 19 mo nano-aerosol-exposed rats (D) 28 days after the end of exposure. A minimum of 3 locations on each section (2 sections per slide), 3 slides and n = 3 per group were analyzed. Relative fluorescence per unit area is represented in graph shown. *p < 0.050 compared to 12–13 wk control; ^†p < 0.050 compared to 19 mo control. Scale bar = 1000 µm.

Figure 8

mRNA expression of synaptophysin in brain parenchyma at 28 days after the end subacute inhalation exposure to TiO₂ nano-aerosol of young adults and aging rats. Each data point represents one animal with median of n = 4 to 6 animals and RT-qPCR was performed in duplicate for each single preparation of brain tissues. Statistical comparison was performed by Student’s t-test, *P < 0.05.

Figure 9

Synaptophysin expression (red fluorescence) from the midbrain region of (A) 12–13 wk control; (B) 12–13 wk nano-aerosol-exposed; (C) 19 mo control; and (D) 19 mo nano-aerosol-exposed rats at 28 days after the end of exposure. A minimum of 3 locations on each section (2 sections per slide), 2 slides and n = 3 per group were analyzed for total fluorescence. Relative fluorescence per unit area is represented in the graph shown. *P < 0.050 compared to 12–13 wk control; ^†p < 0.050 compared to 19 mo control. Scale bar = 1000 µm.