Fig 2: Mast cells interact with astrocytes and motor neurons expressing Stem Cell Factor in ALS mice. A Representative immunohistochemical confocal images showing the characterization of the c-Kit ligand expression, SCF, in the lumbar spinal cord of Non-Tg and symptomatic SOD1G93A mice. Note the significant upregulation of SCF in reactive GFAP+ astrocytes (white arrows) in symptomatic ALS mice as compared with Non-Tg littermates. Motor neurons (white asterisks) express similar levels of SCF in both Non-Tg and SOD1G93A mice. The magenta square shows a high magnification analysis of a SCF+ motor neuron surrounded by reactive astrocytes co-expressing high levels of GFAP and SCF (colocalization in yellow). The graph to the right shows the quantitative analysis of the co-localization of SCF and GFAP in spinal cord sections of SOD1G93A as compared with Non-Tg mice. B High magnification confocal analysis of c-Kit+cells lacking granules that spatially associate with SFC + /GFAP+ reactive astrocytes. Squares show higher magnification 3D images of c-Kit+ cells (red) that surround reactive astrocytes. The graph shows the quantitative analysis of c-Kit+ cells surrounding SCF+ astrocytes. C High magnification confocal analysis of c-Kit + cells that closely interact with SFC+ motor neurons. Squares show higher magnification 3D images of c-Kit+ cells (red). The graph shows the quantitative analysis of c-Kit+ cells surrounding SCF+ motor neurons. Data are expressed as mean ± s.e.m. For statistical analysis two-tailed Mann–Whitney test was used, with ***p < 0.0001 considered significant. n = 4 animals/condition. Scale bars: 20 µm (A) and 10 µm and 5 µm (B, C)

Fig 3: Schematic hypothesis about the pathogenic role of mast cells in ALS. c-Kit+ MC precursors infiltrate from the blood through altered microvascular elements that surround the degenerating/damaged motor neurons (MTN) and accumulate in the ALS spinal cord. The c-Kit ligand SCF expressed in reactive astrocytes and motor neurons promotes chemoattraction of c-Kit+ precursors that differentiate into MC bearing granular morphology and expressing typical MC markers exerting a potentially toxic effect on the microvasculature, glial cells, and motor neurons. Pharmacological inhibition of c-Kit reduces the MC number and the microvasculature pathology

Fig 4: Ex vivo generation of c-Kit+ mast cells from the spinal cord of ALS rats. A The scheme shows the procedure followed to obtain primary cultures of mast cells from the adult spinal cord of symptomatic SOD1G93A rats and Non-Tg littermates. Spinal cords were maintained in the presence of IL-3 (20 ng/mL) and SCF (20 ng/mL) for 2, 7, and 14 days in vitro (DIV), and then mast cell markers, c-Kit, chymase, Cox-2, and CD45 were analyzed by flow cytometry and immunocytochemistry. B Flow cytometry analysis of c-Kit+ cultured mast cells after 2 and 7 DIV. Representative density plots show the expression of c-Kit at 2 and 7 days. The graph to the right shows the quantitative analysis of c-Kit expression in cells cultured from Non-Tg and SOD1G93A rats. C Representative cytological and confocal immunohistochemical images of mast cells isolated from symptomatic SOD1G93A spinal cord. Left panels show representative bright-field images of non-adherent cells with showing typical granular mast cells (upper panel) and metachromatic granular mast cells stained with toluidine blue (lower panel). Panels to the right show immunohistochemical phenotyping by confocal microscopy of cultured mast cells expressing typical markers such as c-Kit, CD45, chymase, and Cox-2 after 14 days in culture. Quantitative data are expressed as mean ± s.e.m. Data were analyzed by the Mann–Whitney test with *p < 0,05 considered significant. n = 4 animals/condition. Scale bars: 5 µm