Figure 8. SCFA restore microglia malformation and immaturity in GF mice.

(a) Relative cecal weight of six SPF (SPF contr.), six GF mice fed with sodium-matched water (GF contr.) or nine GF mice treated with SCFAs (GF SCFA) for 4 weeks. Symbols represent data from individual mice. Data are presented as mean ± s.e.m. Significant differences were determined by an unpaired t test (***P < 0.001). P values: SPF (contr.) versus GF (contr.), 0.0001; SPF (contr.) versus GF (SCFA.), <0.0001. (b) CNS histology of the cerebral cortex that was subjected to immunohistochemistry for Iba-1 to detect microglia (left) and quantification (right). Scale bars represent 50 μm. Representative pictures of six examined control mice and nine SCFA-treated mice are displayed, respectively. Each symbol represents one mouse and three to four sections per mouse were examined. Data are presented as mean ± s.e.m. Significant differences were determined by an unpaired t test (*P < 0.05). P values: SPF (contr.) versus GF (contr.), 0.0456; GF (contr.) versus GF (SCFA.), 0.0118. (c) Expression of Ddit4 mRNA measured by qRT-PCR in microglia isolated from SPF (contr.) (black bar), GF (contr.) (white bar) or GF mice treated with SCFA (gray bar). Data are presented as mean ± s.e.m. with six control samples and nine SCFA-treated samples. Significant differences were determined by an unpaired t test (*P < 0.05, **P < 0.01). P values: SPF (contr.) versus GF (contr.), 0.0064; GF (contr.) versus GF (SCFA.), 0.0306; SPF (contr.) versus GF (SCFA.), 0.0056. (d) Morphology of representative cortical microglia (scale bars represent 15 μm, d) and Imaris-based quantification of cellular parameters (e). Each symbol displays one mouse with three measured cells per animal. Six SPF and GF control animals and nine SCFA-treated GF animals were investigated. Data are presented as mean ± s.e.m. Significant differences were determined by an unpaired t test (*P < 0.05, **P < 0.01, ***P < 0.001). P values: dendrite length SPF (contr.) versus GF (contr.), 0.0001; GF (contr.) versus GF (SCFA), 0.0008; SPF (contr.) versus GF (SCFA), 0.0247; number of segments SPF (contr.) versus GF (contr.), 0.0001; GF (contr.) versus GF (SCFA), 0.0021; number of branch points SPF (contr.) versus GF (contr.), 0.0001; GF (contr.) versus GF (SCFA), 0.0027; number of terminal points SPF (contr.) versus GF (contr.), 0.0001; GF (contr.) versus GF (SCFA), 0.0018; volume SPF (contr.) versus GF (contr.), 0.0001; GF (contr.) versus GF (SCFA), 0.0004. (f) Quantifications of the percentages of positively labeled cells and MFIs for CSF1R, F4/80 and CD31 on microglia from six SPF (contr.), six GF (contr.) and nine GF (SCFA) mice. Each symbol represents one mouse. Data are presented as mean ± s.e.m. Significant differences were determined by an unpaired t test (*P < 0.05, **P < 0.01, ***P < 0.001). P values: CSF1R (percentage of positive cells) SPF (contr.) versus GF (contr.), 0.0094; GF (contr.) versus GF (SCFA), 0.0156; CSF1R (MFI) SPF (contr.) versus GF (contr.), 0.0076; GF (contr.) versus GF (SCFA), 0.0427; F4/80 (percentage of positive cells) SPF (contr.) versus GF (contr.), 0.0204; GF (contr.) versus GF (SCFA), 0.0367; SPF (contr.) versus GF (SCFA), 0.0002; F4/80 (MFI) SPF (contr.) versus GF (SCFA), 0.0194; CD31 (percentage of positive cells) SPF (contr.) versus GF (contr.), 0.0009; SPF (contr.) versus GF (SCFA), 0.0023. (g) Iba-1 immunhistochemistry (left) and quantification (right) of cortical sections from four FFAR2-deficient (SPF-FFAR2^ko) or five competent (SPF^wt) mice. Scale bars represent 50 μm. Representative pictures are displayed. Each symbol represents one mouse. Three to four sections per mouse were examined. Data are presented as mean ± s.e.m. (h,i) Imaris-based three-dimensional reconstruction of representative microglia from SPF-FFAR2^ko and (SPF^wt) mice (scale bars represent 15 μm) and quantification of the cellular parameters (i). Each symbol shows one mouse with at least three measured cells per animal. Four FFAR2-deficient (SPF-FFAR2^ko) and five competent (SPF^wt) mice were analyzed. Data are presented as mean ± s.e.m. Significant differences were determined by an unpaired t test (***P < 0.001). P values: dendrite length, 0.0007; number of segments, 0.0007; number of branch points, 0.0006; number of terminal points, 0.0008; volume, 0.0008. (j) Ffar2 mRNAs levels measured by qRT-PCR for the indicated cells and tissues, respectively. Data are expressed as ΔCT ratio of Ffar2 mRNA expression versus endogenous Actb and exhibited as mean ± s.e.m. Bar represents means ± s.e.m. with at least three samples in each group. n.d., not detectable. (k) Immunofluorescence images of FFAR2 (aqua green) on Iba-1+ (red) microglia or macrophages of wild-type mice. Asterisks indicate double-positive cells in the spleen (upper row) and FFAR2-negative microglia in the cortex (lower row) of wild-type mice. Arrows highlight FFAR2-negative endothelial cells. Nuclei were stained with DAPI (blue). Scale bars represent 50 μm.