Figure 4. Downregulation of pSMAD after CA treatment.
(A) In vivo bioluminescence of diseased SBE-Luc mice depicts pSMAD signaling. Mice were imaged on the indicated days after immunization. The highly increased level of pSMAD signaling in the vehicle control group (top row) is almost completely abrogated by CA treatment (bottom row). One representative mouse of each group is followed up. sr, steradian. (B and C) Statistical evaluation of the experiment shown in A. Mean intensity of bioluminescence in (B) brains and (C) spinal cords of immunized SBE-Luc mice. Mean ± SEM of each group is shown (n = 5 per group). *P < 0.05 (Student’s t test). (D) Cerebellar slices are stained for pSMAD, confirming its upregulation during EAE and its downregulation due to CA treatment. One representative slide of each group is shown. Scale bars: 100 μm. (E) Statistical evaluation of the experiment shown in D. pSMAD staining intensities were evaluated with MetaMorph software and normalized on the healthy control group. Statistical analysis of 6 cerebellar slices per mouse is shown (n = 5 mice per group; mean ± SEM). *P < 0.02 (Student’s t test). (F–I) Fluorescence double staining of inflamed spinal cords for pSMAD (red) and the following cell markers (blue): (F) astrocytes, (G) microglial cells, (H) neurons, and (I) CD4+ T cells. T cells show high levels of nuclear staining for pSMAD, and therefore are responsive to TGF-β, as do microglia and neurons but not astrocytes. Spinal cords are from untreated diseased mice. Scale bars: 50 μm.