Recovery after stroke is a multicellular process encompassing neurons, resident immune cells, and brain-invading cells. Stroke alters the gut microbiome, which in turn has considerable impact on stroke outcome. However, the mechanisms underlying gut–brain interaction and implications for long-term recovery are largely elusive. Here, we tested the hypothesis that short-chain fatty acids (SCFAs), key bioactive microbial metabolites, are the missing link along the gut–brain axis and might be able to modulate recovery after experimental stroke. SCFA supplementation in the drinking water of male mice significantly improved recovery of affected limb motor function. Using in vivo wide-field calcium imaging, we observed that SCFAs induced altered contralesional cortex connectivity. This was associated with SCFA-dependent changes in spine and synapse densities. RNA sequencing of the forebrain cortex indicated a potential involvement of microglial cells in contributing to the structural and functional remodeling. Further analyses confirmed a substantial impact of SCFAs on microglial activation, which depended on the recruitment of T cells to the infarcted brain. Our findings identified that microbiota-derived SCFAs modulate poststroke recovery via effects on systemic and brain resident immune cells.
SIGNIFICANCE STATEMENT Previous studies have shown a bidirectional communication along the gut–brain axis after stroke. Stroke alters the gut microbiota composition, and in turn, microbiota dysbiosis has a substantial impact on stroke outcome by modulating the immune response. However, until now, the mediators derived from the gut microbiome affecting the gut-immune-brain axis and the molecular mechanisms involved in this process were unknown. Here, we demonstrate that short-chain fatty acids, fermentation products of the gut microbiome, are potent and proregenerative modulators of poststroke neuronal plasticity at various structural levels. We identified that this effect was mediated via circulating lymphocytes on microglial activation. These results identify short-chain fatty acids as a missing link along the gut–brain axis and as a potential therapeutic to improve recovery after stroke.
Digital Object Identifier (DOI)
This work was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, LI2534/2-1), the European Research Council (ERC-StG 802305), the Vascular Dementia Research Foundation, and the Munich Cluster for Systems Neurology (EXC 2145 SyNergy) ID 390857198 to A.L., the Texas Institute for Brain Injury and Repair to M.P.G. and A.M.S., and National Institutes of Health/National Institute of Neurological Disorders and Stroke (NS088555) to A.M.S.
Extended Data (available for download as the additional files listed at the end of this record)
SCFA concentrations are reduced after stroke. (A) Plasma concentration (μM) showing a decrease of total SCFA (sum of acetate, propionate and butyrate) in mice 3 days after fMCAo stroke surgery (grey bars) compared to sham (open bars). N=4 per group (Mann-Whitney U test). (B) No difference of body weight in grams at baseline (BL) until D42 post PT in mice supplemented with SCFA (grey line) or control (black line). N=14/15 per group (2-way ANOVA with Holm-Sidak correction for multiple testing). (C) Lesion size as measured in pixels did not differ between SCFA (grey bar) and control mice (open bar) at any time points. N=11/10 and statistical test with multiple T tests per time point and Holm-Sidak method correction for multiple testing.
SCFA supplementation does not alter tissue injury in 3 focal stroke models. Infarct volume from Nissl staining in mice supplemented with control mix (open bar) or SCFA (grey bar). (A) 24 h after PT, N=10 per group; (B) 14 d after PT, N=10/9 per group; (C) 5 d after dMCAo, N=6/9 per group; (D) 14 d after dMCAo, N=9/8 per group; and (E) 14 d after fMCAo, N=5/7 per group. Statistical analyses were performed using the Mann-Whitney U test. (F) As in Figure 2E, histogram of the relative frequency (fraction) of spines found at different lengths 14 d after PT in the contralateral cortex (bin width = 0.2 μm). (G) Quantification of short (0.2 μm) and long (1.4 μm) spines in control (open bars) and SCFA (grey bars) treated mice. N=4/5 per group, Mann-Whitney U test. (H) The size in pixels of synapses stained with VGlut1 (left) and Homer1 (right). Quantification revealed larger synapse areas in the peri-lesional cortex which was significantly ameliorated with SCFA supplementation. Contra: contralateral hemisphere; ipsi: ipsilateral hemisphere, N=3/4 mice (3 sections per mouse), statistical analysis performed with Kruskal-Wallis test with Dunn's multiple comparison correction.
SCFA supplementation increases plasma concentrations. Mice were pre-treated with antibiotic mix for 4 weeks, and then additionally given control (ABX, open bars) or SCFA supplementation (ABX+SCFA, grey bars). Total plasma concentration (μM) of total SCFA (acetate, propionate and butyrate) showing significant increase after SCFA supplementation compared to control treated mice, N=4/5 per group (Mann-Whitney U test).
SCFA supplementation does not affect cerebral endothelial expression. Relative mRNA expression (RE) for the tight-junction and adhesion molecules Claudin-5 (left), ICAM-1 (middle) and VCAM-1 (right) from the peri-lesional cortex in control (open bars) and SCFA (grey bars) treated mice (Mann-Whitney U test).
Sadler, Rebecca; Cramer, Julia V; Heindl, Steffanie; Kostidis, Sarantos; Betz, Dene; Zuurbier, Kielen R; Northoff, Bernd H.; Heijink, Marieke; Goldberg, Mark P.; Plautz, Erik J.; Roth, Stefan; Malik, Rainer; Dichgans, Martin; Holdt, Lesca M.; Benakis, Corinne; Giera, Martin; Stowe, Ann M.; and Liesz, Arthur, "Short-Chain Fatty Acids Improve Poststroke Recovery via Immunological Mechanisms" (2020). Neurology Faculty Publications. 53.
Figure 1-1: SCFA concentrations are reduced after stroke
inline-supplementary-material-2.pdf (272 kB)
Figure 2-1: SCFA supplementation does not alter tissue injury in 3 focal stroke model
inline-supplementary-material-3.pdf (134 kB)
Figure 3-1: SCFA supplementation increases plasma concentrations
inline-supplementary-material-4.pdf (143 kB)
Figure 4-1: SCFA supplementation does not affect cerebral endothelial expression