Fig. 7
From: The pathogenic role of c-Kit+ mast cells in the spinal motor neuron-vascular niche in ALS
Post-paralysis masitinib treatment prevents mast cell accumulation and trafficking into the spinal cord of ALS mice. SOD1G93A 140d old symptomatic mice were orally (gavage) daily treated with 50 mg/Kg of masitinib (or vehicle) for 10 days and immunohistochemical analysis was performed to characterize the presence of mast cells in the lumbar spinal cord. A Representative confocal images show the analysis of c-Kit+ cells (red – white arrows) accumulated in the surroundings of motor neurons (white dotted lines) in the lumbar spinal cord of SOD1G93A mice after vehicle or masitinib treatment. Magenta squares show high magnification 3D images of typical c-Kit+ mast cells in both conditions. The graph shows that masitinib treatment significantly prevents the accumulation of c-Kit+ cells in the parenchyma of the spinal cord when compared with vehicle-treated mice. B Representative confocal images that show the analysis of chymase+ mast cells (green—white arrows) associated with motor neurons (white dotted lines) in the spinal cord of vehicle- and masitinib-treated SOD1G93A mice. Magenta squares show high magnification 3D images of typically degranulating mast cells in proximity to motor neurons. Quantitative analysis in the graph shows that masitinib treatment significantly prevents the accumulation of degranulating chymase+ cells in the spinal cord. C Confocal microscopy analysis of the microvasculature pathology in SOD1G93A treated with vehicle or masitinib. The number of vessels strings and vessels sprouts were quantified between groups in Collagen-I stained spinal cords. The graph shows that after 10 days of treatment with masitinib, there is a significant reduction of microvasculature alterations. D The scheme shows the experimental protocol for the i.v. delivery of c-Kit+/CFSE+ cells, after 10 days of the vehicle or masitinib treatment. E After 10 days of vehicle (upper panels) or masitinib (lower panels) treatment, c-Kit+ mast cells precursors (red) stained with CFSE dye (green) were i.v. injected in symptomatic 150d old SOD1G93A mice. c-Kit+/CFSE+ precursors (magenta arrows) were analyzed in the periphery of blood vessels stained with EB dye systemically perfused after euthanasia (white). Magenta squares show high magnification 3D images of typical c-Kit+/CFSE+ cells infiltrating the spinal cord. Masitinib treatment significantly reduced the number of c-Kit+ cells infiltrating the spinal cord parenchyma of SOD1G93A mice. The graph shows the quantitative analysis of c-Kit+ /CFSE+ cells in the surroundings of motor neurons. F Non-Tg, and vehicle- and masitinib-treated SOD1G93A mice were systemically perfused after euthanasia with EB to measure the perivascular dye extravasation in the parenchyma of the spinal cord. Note the significant perivascular EB extravasation in SOD1G93A vehicle-treated mice when compared to Non-Tg littermates. Masitinib significantly reduced perivascular extravasation after 10 days of treatment. The graph shows the quantitative analysis of perivascular EB extravasation among groups. Quantitative data are expressed as mean ± s.e.m.; data were analyzed by a two-tailed Mann–Whitney test (A–C, E) and one-way ANOVA (F) with *p < 0,05, ***p < 0.001 and ****p < 0.0001 considered significant. n = 4 animals/condition. Scale bars: 10 μm (low magnification panels) and 5 μm (insets)