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. 2021 Mar 10;9(1):40.
doi: 10.1186/s40478-021-01137-2.

Gut microbial dysbiosis after traumatic brain injury modulates the immune response and impairs neurogenesis

Marta Celorrio  1 , Miguel A Abellanas  1   2   3 , James Rhodes  1 , Victoria Goodwin  1 , Jennie Moritz  1 , Sangeetha Vadivelu  1 , Leran Wang  4 , Rachel Rodgers  4 , Sophia Xiao  1 , Ilakkia Anabayan  1 , Camryn Payne  1 , Alexandra M Perry  1 , Megan T Baldridge  4 , Maria S Aymerich  2   3   5 , Ashley Steed #  1 , Stuart H Friess #  6
Affiliations

Gut microbial dysbiosis after traumatic brain injury modulates the immune response and impairs neurogenesis

Marta Celorrio et al. Acta Neuropathol Commun. .

Abstract

The influence of the gut microbiota on traumatic brain injury (TBI) is presently unknown. This knowledge gap is of paramount clinical significance as TBI patients are highly susceptible to alterations in the gut microbiota by antibiotic exposure. Antibiotic-induced gut microbial dysbiosis established prior to TBI significantly worsened neuronal loss and reduced microglia activation in the injured hippocampus with concomitant changes in fear memory response. Importantly, antibiotic exposure for 1 week after TBI reduced cortical infiltration of Ly6Chigh monocytes, increased microglial pro-inflammatory markers, and decreased T lymphocyte infiltration, which persisted through 1 month post-injury. Moreover, microbial dysbiosis was associated with reduced neurogenesis in the dentate gyrus 1 week after TBI. By 3 months after injury (11 weeks after discontinuation of the antibiotics), we observed increased microglial proliferation, increased hippocampal neuronal loss, and modulation of fear memory response. These data demonstrate that antibiotic-induced gut microbial dysbiosis after TBI impacts neuroinflammation, neurogenesis, and fear memory and implicate gut microbial modulation as a potential therapeutic intervention for TBI.

Keywords: Antibiotics; Fear conditioning; Gut microbial dysbiosis; Microglia; Monocytes; Neurogenesis; T cells; Traumatic brain injury.

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Conflict of interest statement

The authors have no competing interests, and the content of this manuscript has not been published, or submitted for publication elsewhere.

Figures

Fig. 1
Fig. 1
Microbial dysbiosis prior to and during TBI exacerbates neurodegeneration and fear memory alterations. a Experimental design. Graphs depict b Richness index and c Shannon diversity index. Genera with a frequency > 3% annotated, mean values are plotted ± SEM, unpaired t-test, **** p < 0.0001, n = 9–25 per group. d Representative image of NeuN + cells in CA3 region of injured hippocampi (indicated by the dotted line) and e stereological quantification. f Representative images of Iba1 + cells in the CA3 region of ipsilateral hippocampi (indicated by the dotted line) and g stereological quantification. Scale bars are 250 µm. Mean values are plotted ± SEM, unpaired t-test **p < 0.01, n = 8–9 per group. h Fear conditioning scheme. Quantification of % total freezing time i conditioning, j contextual memory and k cued memory. Mean values are plotted ± SEM, unpaired t-test, *p < 0.05, n = 4–5 per group. Abbreviations: VNAM, vancomycin, neomycin-sulfate, ampicillin and metronidazole; CCI, controlled cortical impact
Fig. 2
Fig. 2
Local immune alterations in the brain 3 days after TBI and antibiotic induced gut microbial dysbiosis. a Experimental design. b–j Brain leucocyte profile characterization by flow cytometry. b Gating strategy of Ly6Chigh cells. Quantification of cell absolute numbers in the hippocampi, amygdalae, and cortices for c leukocytes (CD45), d CD3 T cells (CD11b−CD3+), e CD4 T cells (CD11b−CD3+CD4+), f CD8 T cells (CD11b−CD3+CD8+), g DN T cells (CD11b−CD3+CD4−CD8−), h T reg cells (CD11b−CD4+CD25+), i monocytes (CD45highCD11b+Ly6C+) and j microglia (CD45lowCD11b+). Mean values are plotted ± SEM, unpaired t test, **p < 0.01. k Representatives 20X images of ipsilateral hippocampi of sham, control-injured and antibiotic-injured mice. Scale bars are 100 µm. l Quantification of fluorescent intensity from (k), two-way ANOVA, antibiotic F(1,14) = 0.015, p = 0.9, injury F(1,14) = 29.2, p < 0.0001. m Protein cytokine and chemokine quantification from whole ipsilateral hippocampi. Mean values are plotted ± SEM, unpaired t-test, *p < 0.05. Abbreviations: VNAM, vancomycin, neomycin-sulfate, ampicillin and metronidazole; CCI, controlled cortical impact. Hip: hippocampus; Amy, amygdala; Cx, cortex
Fig. 3
Fig. 3
Local immune alterations in the brain 7 days after TBI and antibiotic induced gut microbial dysbiosis. a Experimental design. Graphs depict b Richness index and c Shannon diversity index from injured mice. Genera with a frequency > 3% annotated, mean values are plotted ± SEM, unpaired t-test, *** p < 0.001, n = 6 per group. d Gating strategy of CD3+ cells in the brain. e–j Quantification of cell absolute numbers in the hippocampi, amygdalae, and cortices. Mean values are plotted ± SEM, two-way ANOVA followed by Tukey's multiple comparisons. F statistic for antibiotic*injury presented unless otherwise noted (*p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001), n = 7–8 per group. e leukocytes (CD45), F(1,24) = 7.28, p = 0.013; f CD3 T cells (CD11b−CD3+), F(1,24) = 12.02, p = 0.002; g CD4 T cells (CD11b−CD3+CD4+), F(1,24) = 19.51, p = 0.0002; h CD8 T cells (CD11b−CD3+CD8+), F(1,24) = 9.541, p = 0.0053; i DN T cells (CD11b−CD3+CD4−CD8−), Finjury (1,24) = 12.11, p = 0.0019; and, j T reg cells (CD11b−CD4+CD25+), Finjury (1,22) = 6.725, p = 0.017, Fantibiotic (1,22) = 5.228, p = 0.032. k Representative 10X images (top panel, scale bars are 100 µm) and 40X (bottom panel, scale bars are 20 µm) for CD3 + staining in the ipsilateral hippocampi (as indicated by black arrows). Abbreviations: VNAM, vancomycin, neomycin-sulfate, ampicillin and metronidazole; CCI, controlled cortical impact. Hip: hippocampus; Amy, amygdala; Cx, cortex
Fig. 4
Fig. 4
Microglia morphology changes 7 days after TBI and antibiotic induced gut microbial dysbiosis. a Experimental design. b Gating strategy of Ly6Chigh in the hippocampus. c–f Quantification of absolute cell numbers in the brain. Mean values are plotted ± SEM, two-way ANOVA followed by Tukey's multiple comparisons. F statistic for antibiotic*injury presented unless otherwise noted (*p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001). c microglia (CD45lowCD11b+), n = 6–8 per group, Finjury (1,23) = 7.45, p = 0.012. MFI of d TLR4, hippocampus Finjury (1,13) = 6.575, p = 0.024, Fantibiotic (1,13) = 9.9, p = 0.008, and cortex Fantibiotic (1,13) = 14.58, p = 0.0021; e MHCII, hippocampus Fantibiotic (1,13) = 6.32, p = 0.026, cortex Finjury (1,13) = 14.93, p = 0.002, Fantibiotic (1,13) = 9.48, p = 0.009, on microglia (CD45lowCD11b+) gated populations. f monocytes (CD45highCD11b+Ly6C+), F(1,24) = 6.24, p = 0.02. g Quantitative measurement of Il-1α, Il-1β, Il-2, Il-10, Il-17, Tnf-α, Ifn-γ and Ccxl13 transcripts, unpaired t test, *p < 0.05. h Representative images of Iba1 + cells in the CA3 region of ipsilateral hippocampi (indicated by the dotted line) and i stereological quantification, Finjury (1,22) = 74.8, p < 0.0001, Fantibiotic (1,22) = 5.94 p = 0.023. Scale bars are 100 µm. j Representative three-dimensional reconstruction images of microglia cells. Scale bars are 15 um. k–o Quantification of microglia morphology. k Dendrite length, Finjury (1,20) = 124.3, p < 0.0001, Fantibiotic (1,20) = 30.9, p < 0.0001; l number of segments, Finjury (1,20) = 94.3, p < 0.0001, Fantibiotic (1,20) = 21.7, p < 0.0002; m number of branch points, Finjury (1,20) = 104.4, p < 0.0001, Fantibiotic (1,20) = 24.4, p < 0.0001; n number of terminal points, Finjury (1,20) = 88.0, p < 0.0001, Fantibiotic (1,20) = 18.78 p < 0.0004; and o volume, Fantibiotic (1,20) = 22.63 p < 0.0002. Abbreviations: VNAM, vancomycin, neomycin-sulfate, ampicillin and metronidazole; CCI, controlled cortical impact; MFI, median fluorescence intensity; TLR4, toll-like receptor 4; MHCII, major histocompatibility complex II
Fig. 5
Fig. 5
Impact of TBI and antibiotic induced gut microbial dysbiosis on neurogenesis. a Representative images of NeuN + cells in the ipsilateral hippocampi. Mean values are plotted ± SEM, two-way ANOVA followed by Tukey's multiple comparisons. F statistic for antibiotic*injury presented unless otherwise noted (*p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001). b Stereological quantification, Finjury (1,19) = 80.2, p < 0.0001. c Experimental design. d Representative fluoroscopy of the SGZ in the DG of the hippocampus labeled with BrdU (magenta), NeuN (red), and DCX (green) (1cell expressing BrdU/DCX/NeuN, 2cell expressing BrdU/DCX and and 3cell expressing BrdU only). e Quantification of stained cells per area per condition. f Summation of total neuronal lineage cells per area of hippocampi, F(1,14) = 20.8, p < 0.0005. Abbreviations: VNAM, vancomycin, neomycin-sulfate, ampicillin and metronidazole; CCI, controlled cortical impact; SGZ, subgranular zone; DG, dentate gyrus; DCX, doublecortin; BrdU, 5-Bromo-2´-Deoxyuridine
Fig. 6
Fig. 6
Long term alterations of local immunity after microbiota dysbiosis at the time of TBI. a Experimental design. Graphs depict b Richness index and c Shannon diversity index. Genera with a frequency > 3% annotated, mean values are plotted ± SEM, unpaired t-test, *** p < 0.001, n = 3 per group. d Gating strategy of CD3+ cells and CD25+ cells in the ipsilateral hippocampi. e–l Quantification of the cell absolute numbers in the brain. Mean values are plotted ± SEM, two-way ANOVA followed by Tukey's multiple comparisons. F statistic for antibiotic*injury presented unless otherwise noted (*p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001). n = 7–8 mice per group. e leukocytes (CD45), f CD3 T cells (CD11b−CD3+), Finjury (1,30) = 15.52, p = 0.0005; g CD4 T cells (CD11b−CD3+CD4+), F(1,30) = 5.704, p = 0.023; h CD8 T cells (CD11b−CD3+CD8+), i DN T cells (CD11b−CD3+CD4−CD8−), Finjury (1,30) = 18.91, p = 0.0001; j Treg cells (CD11b−CD4+CD25+), hippocampus, F(1,30) = 9.011, p = 0.0054, cortex, F(1,30) = 5.443, p = 0.0265; k monocytes (CD45highCD11b+Ly6C+), and l microglia (CD45lowCD11b+). m MFI of TLR4 in the microglia population, Finjury (1,11) = 19.85, p = 0.0010. Abbreviations: VNAM, vancomycin, neomycin-sulfate, ampicillin and metronidazole; CCI, controlled cortical impact. Hip: hippocampus; Amy, amygdala; Cx, cortex
Fig. 7
Fig. 7
Alterations in fear memory response and subcellular populations three months after TBI in the setting of gut microbial dysbiosis. a Experimental design. Graphs depict b Richness index and c Shannon diversity index. Mean values are plotted ± SEM, unpaired t-test, *** p < 0.001, n = 3 per group. Fear conditioning 3-day paradigm and quantification of % total freezing time of d conditioning learning, e contextual memory and f cued memory. g Representative images of NeuN + cells in the pyramidal layer of CA3 (indicated by the dotted line) with h stereological quantification and i hippocampi volumes. j Representative images of Iba1 + cells in the injured ipsilateral hippocampi. Scale bars are 250 µm. k Stereological analysis of microglia density as shown in (h). Mean values are plotted ± SEM, unpaired t-test, *p < 0.05, n = 8–9 mice per group. l Representative three-dimensional reconstructive images of microglia (scale bars are 15 um). m-q Quantification of microglia morphology in hippocampi. m Dendrite length, n number of segments, o number of branch points, p number of terminal points and q volume. Abbreviations: VNAM, vancomycin, neomycin-sulfate, ampicillin and metronidazole; CCI, controlled cortical impact. Hip: hippocampus; Amy, amygdala; Cx, cortex
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