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. 2021 Nov 27;10(12):3332. doi: 10.3390/cells10123332

Figure 6.

Figure 6

Microglia upregulate OSMR expression in spinal cord neurosphere cells. (A): QPCR for indicated cytokines in RNA extracted in sham-operated and injured spinal cord (3 days after SCI). n = 3 independent experiments. Numbers indicate the fold change compared to sham-operated spinal cords. Statistical tests were performed with Bootstratio (Clèries et al., 2012) compared to control spinal cords. (B): Immunofluorescences for GFP performed in CX3CR1+/GFP mice to reveal microglia (green) associated with ependymal cells. White arrowheads and arrows show microglia somas and processes respectively, close or within the ependymal cell layer. Scale bars = 10 µm. (C): Left-hand panel: detection of the OSM cytokine in the supernatant of BV-2 microglial cell culture by ELISA (n = 4 independent experiments). Right-hand panel: detection of indicated cytokines in BV-2 microglia by QPCR (n = 3 independent experiments). (D): Influence of BV-2 cells on OSMR expression in spinal cord neurosphere cells. Left-hand panel: QPCR for OSMR. RNA were extracted from GFP+ spinal cord neurosphere cells cultured without (control) and with BV-2 cells. Values represent fold change compared to control neurospheres. Statistical test was performed with Bootstratio (Clèries et al., 2012) compared to control neurospheres. n = 3 independent experiments. Lower panel: Immunofluorescences for OSMR in GFP+ spinal cord neurosphere cells cultured without (control) or with BV-2 microglia. Scale bars = 10 µm. Right-hand panel: Quantification of immunofluorescences (n = 6 fields). test= two tailed t-test. (E): Influence of BV-2 cells on GFAP expression in spinal cord neurosphere cells. Left-hand panel: QPCR for GFAP. RNA were extracted from spinal cord neurosphere cells cultured without (control) and with BV-2 cells. Values represent fold change compared to control neurospheres. Statistical test was performed with Bootstratio (Clèries et al., 2012) compared to control neurospheres. n = 3 independent experiments. Lower panel: Immunofluorescences for GFAP in GFP+ spinal cord neurosphere cells cultured without (control) or with BV-2 microglia. Scale bars = 10 µm. Right-hand panel: Quantification of immunofluorescences (n = 10 fields). test= two tailed t-test. ***, p < 0.001; **, p < 0.01; *, p ≤ 0.05. n.s. = not significant. IF = immunofluorescence.