Figure 1.
Albumin as a marker of BBB dysfunction.
In control medium-injected animals, very little BBB disruption was evident at 2 weeks (A), 3 weeks (B) or 4 weeks (C) following injection. Conversely, in tumor-inoculated animals an increase in albumin staining was evident at 2 weeks (D) and extremely pronounced at 3 weeks (E) and 4 weeks (F). This was confirmed using color deconvolution, which revealed a highly significant increase in BBB disruption, as assessed by albumin staining at both 3 and 4 weeks. Scale bars = 2 mm. Image are representative of n = 6 per group. Color deconvolution analysis n = 6 per group/time point (** p<0.01, ***p<0.001 compared to controls).
Figure 2.
SP immunolabelled sections of peritumoral vessels.
At 2 weeks (A), 3 weeks (B) and 4 weeks (C) following in culture medium injected animals, minimal SP immunoreactivity was observed in the vasculature. Following tumor cell inoculation at 2 weeks (D) a marked increase in perivascular SP staining was evident, this intensity of staining was sustained at both the 3 weeks (E) and 4 weeks (F) time points. This was confirmed by color deconvolution analysis of perivascular SP staining, which revealed a significant increase in SP immunoreactivity in tumor inoculated animals when compared to controls at 3 weeks (p = 0.039) (G). Images are representative of n = 6 per group. Scale bar = 50 µm. Color deconvolution analysis n = 6 per group (*p<0.05 compared to controls).
Figure 3.
NK1 receptor immunoreactivity.
A marked increase in NK1 receptor staining was observed in the area surrounding the tumor at 3 weeks (E) and 4 weeks (F) following tumor inoculation at low power when compared to the controls of the same time points (A–C). At 2 (G), 3 (H) and 4 (I) weeks following culture medium injection, there was minimal NK1 staining around the vasculature, especially when compared to the tumor inoculated animals where a moderate increase was evident at 2 (J) and 3 (K) weeks, but clearly increased at 4 weeks (L). The qualitative assessment was confirmed by color deconvolution, revealing a significant increase in overall NK1 receptor immunoreactivity when compared to controls (p = 0.0327) (M) as well as a significant increase in NK1 receptor staining in the tumor inoculated hemisphere when compared to the contralateral hemisphere of the same animals (p = 0.0086) (N). A–F Scale bars = 2 mm, G–L Scale bars = 50 µm. Images are representative of n = 6 per group. Color deconvolution analysis n = 6 per group (*p<0.05 compared to controls).
Figure 4.
Effects of NK1 antagonist treatment on BBB permeability to Evans blue and edema formation.
Using Evan's blue (EB) (A) as a marker of BBB disruption treatment with both the NK1 antagonist Emend and dexamethasone resulted in a reduction of BBB permeability back to control levels (p = 0.0237). Similarly, NK1 antagonist and dexamethasone treatment resulted in a significant reduction of brain water content (B) compared to tumor inoculated vehicle treated mice (p = 0.0188). Brain water content n = 5 per group, EB n = 6 per group (*p<0.05 compared to shams for EB, *p<0.05 compared to vehicle for WWDW).