Fig. 4

Neuropathological effects of NP in the brain. After the mice were intranasally instilled with either vehicle (sterile PBS, control group) or NP in PBS (8 mg/kg, NP group) once per day for 81 days, the mice were sacrificed and the brains were harvested. Half of the brain was fixed, sectioning, and used in Niss1 staining or Iba-1 immunofluorescent staining, while the frontal cortex and the hippocampus of the other half were homogenized. a Representative images of Nissl staining in the mPFC and the hippocampus. Zoom-in image demonstrating typical morphology in Nissl staining were presented in the bottom right corner of each image (n = 4 per group for mPFC, n = 3 per group for hippocampus). Scale bar: 50 μm. b Western blot analysis of tau phosphorylation at Serine 396 in the frontal cortex and hippocampus. Image shows four representative protein bands. Band intensity was normalized to that of α-tubulin and analyzed using unpaired Student’s t-tests (n = 8 for each group, except n = 7 for hippocampus control). c Representative images of Iba-1 (red) co-stained with DAPI (blue) in the mPFC and the hippocampus. Typical microglial morphology is highlighted with an arrow. The number and area of the cell bodies with high expression of Iba-1 in each sub-region (area of interest) were measured using Image J and analyzed using an unpaired Mann-Whitney test and an unpaired Student’s t-test, respectively (n = 3 per group). Scale bar: 50 μm. d mRNA expression of cytokines in the frontal cortex and the hippocampus by real time-PCR. Unpaired Student’s t-test was used as statistical analysis in each marker. (n = 6 per group) (e) Western blot analysis of COX-2 expression in the frontal cortex and hippocampus. Image shows four representative protein bands. Band intensity was normalized to that of α-tubulin and analyzed using unpaired Student’s t-tests (n = 8 for each group, except n = 7 for hippocampus control). For all analyses, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared to the controls