Fig. 7

Representative images of neocortex sections from naïve (a), EAE-affected (b; cs 2.0) and EAE-affected MSC-treated (c; cs 1.0) mice, sacrificed 24 h after MSC treatment, and processed according to the dual RNAscope IHC/ISH method applied to concomitant detection of GFAP protein and Ccl2 mRNA; morphometric analyses of GFAP/Ccl2 cell populations and Ccl2 fluorescence intensity (d, e). a Few GFAP⁺ astrocytes show a barely detectable expression of Ccl2 (arrows) in naïve mice. b In EAE-affected mice, GFAP⁺ astrocytes express high levels of Ccl2 (arrows), the Ccl2 probes also marking a large GFAP⁻ cell population (arrowheads). c A few Ccl2 expressing cells, both GFAP⁺ (arrows) and GFAP⁻ (arrowheads), are recognizable in MSC-treated mice. d Morphometric quantitation of GFAP⁻/Ccl2⁺ cells shows a significant increase in this cell population in EAE-affected mice compared with naïve controls; this increase is also detected, although to a significantly lesser extent, in EAE-affected MSC-treated mice; in both treated and not treated EAE-affected mice, GFAP⁺/Ccl2⁺ astrocytes are significantly more numerous compared to naïve mice, whereas the neocortex of EAE-affected MSC-treated mice is characterized by the prevalence of the GFAP⁺/Ccl2⁻ astrocyte population. e The fluorescence intensity corresponding to Ccl2 mRNA expression was increased in EAE-affected mice but was reverted to levels observed in naïve mice upon MSC treatment. Data are reported as means ± SD (n = 3, n = 4, n = 4; 3 sections each), and the Bonferroni post-test was used to compare all groups after two-way ANOVA (d) or one-way ANOVA (e). Clinical score of EAE-affected mice (mean cs 2.1) and MSC-treated mice (mean cs 2.0). TOPRO-3 nuclear counterstaining. Scale bar: a-c 20 µm