Figure 5

PSLs affect splenic cognate antigen specific proliferation and expression of TNFα, iNOS and ARG-1. (a) Cognate antigen specific proliferation (10 dpi) of splenic cultures was assessed by culturing splenic cells from vehicle, PCL and PSL treated animals with MOG. Proliferation was assessed by [3H]thymidine incorporation. Non-stimulated cultures were used as control (dotted line). Data represent the mean ± SEM of four experiments. (b) The size of the splenic white pulp was determined using ImageJ software. Three cryosections per animal were stained with CD200R, a marker for myeloid cells, after which the surface area surrounded by the marginal metallophilic macrophages was determined. Five images (4× magnification) per section were taken to calculate the mean white pulp size. Data represent the mean ± SEM of four animals. (c,d) Comparison of fold changes between vehicle, PCL and PSL-treated spleens 10 dpi. Relative quantification of iNOS (c) and TNFα (d) was accomplished by using the comparative C_t method. Data were normalized to the most stable reference genes, determined by Genorm (Pgk1 and Rpl13a). Data represent the mean ± SEM of 4 experiments. (e) Spleen cryosections were stained with ARG-1 after which the total corrected fluorescence was determined using ImageJ software, as described previously [65]. Three cryosections were stained and 6 images (10× magnification) were taken per section. Data represent the mean ± SEM of four animals.