Figure 6.

Effect of exogenous TGF-β1 treatment on TGF-β/ALK5 signaling and on BBB permeability. (A) Western blot analysis of microvessels isolated from rats treated with saline or λ-carrageenan in the presence and absence of SB431542, a selective ALK5 inhibitor. Cytoplasmic and nuclear extracts from rat brain microvessels (20 µg) were resolved on a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to a PVDF membrane. Samples were analyzed for expression of p-Smad2 and p-Smad3. P-Smad2 was detected using the polyclonal antibody Ser465/467 (1:500 dilution) and p-Smad3 was detected using the polyclonal antibody Ser423/425 (1:500 dilution). Relative levels of p-Smad2 and p-Smad3 were determined by densitometric analysis and the nucleus/cytoplasm ratio was calculated. Results are expressed as mean ± s.d. of three separate experiments. Asterisks represent data points that are significantly different from control. (B) Changes in paracellular permeability to ¹⁴C-sucrose during peripheral inflammatory pain in the presence and absence of TGF-β1. Graph shows the percent of radioactivity detected in the brain as compared with that in the perfusate (% RBr) for the four treatment groups 3 h after injection of 3% λ-carrageenan or 0.9% saline into the plantar surface of the right hind paw. Transforming growth factor-β1 (12.5 ng/kg) was injected i.p. 30 mins before footpad injection. Results are expressed as mean ± s.d. of six animals per treatment group.