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. 2020 Nov 15:1747:147056.
doi: 10.1016/j.brainres.2020.147056. Epub 2020 Aug 13.

Depletion of gut microbiota is associated with improved neurologic outcome following traumatic brain injury

Dennis W Simon  1 , Matthew B Rogers  2 , Yuan Gao  3 , Garret Vincent  2 , Brian A Firek  2 , Keri Janesko-Feldman  4 , Vincent Vagni  4 , Patrick M Kochanek  5 , John A Ozolek  6 , Kevin P Mollen  2 , Robert S B Clark  7 , Michael J Morowitz  8
Affiliations

Depletion of gut microbiota is associated with improved neurologic outcome following traumatic brain injury

Dennis W Simon et al. Brain Res. .

Abstract

Signaling between intestinal microbiota and the brain influences neurologic outcome in multiple forms of brain injury. The impact of gut microbiota following traumatic brain injury (TBI) has not been well established. Our objective was to compare TBI outcomes in specific pathogen-free mice with or without depletion of intestinal bacteria. Adult male C57BL6/J SPF mice (n = 6/group) were randomized to standard drinking water or ampicillin (1 g/L), metronidazole (1 g/L), neomycin (1 g/L), and vancomycin (0.5 g/L) (AMNV) containing drinking water 14 days prior to controlled cortical impact (CCI) model of TBI. 16S rRNA gene sequencing of fecal pellets was performed and alpha and beta diversity determined. Hippocampal neuronal density and microglial activation was assessed 72 h post-injury by immunohistochemistry. In addition, mice (n = 8-12/group) were randomized to AMNV or no treatment initiated immediately after CCI and memory acquisition (fear conditioning) and lesion volume assessed. Mice receiving AMNV had significantly reduced alpha diversity (p < 0.05) and altered microbiota community composition compared to untreated mice (PERMANOVA: p < 0.01). Mice receiving AMNV prior to TBI had increased CA1 hippocampal neuronal density (15.2 ± 1.4 vs. 8.8 ± 2.1 cells/0.1 mm; p < 0.05) and a 26.6 ± 6.6% reduction in Iba-1 positive cells (p < 0.05) at 72 h. Mice randomized to AMNV immediately after CCI had attenuated associative learning deficit on fear conditioning test (%freeze Cue: 63.7 ± 2.7% vs. 41.0 ± 5.1%, p < 0.05) and decreased lesion volume (27.2 ± 0.8 vs. 24.6 ± 0.7 mm3, p < 0.05). In conclusion, depletion of intestinal microbiota was consistent with a neuroprotective effect whether initiated before or after injury in a murine model of TBI. Further investigations of the role of gut microbiota in TBI are warranted.

Keywords: Antibiotic; Gut-brain axis; Head injury; Inflammation; Microbiome.

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Conflict of interest statement

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1.
Figure 1.. Adult C57BL/6 mice were randomized to standard drinking water or AMNV-containing drinking water and subjected to CCI.
Experiment 1 was pre-treatment design with a 72h sacrifice for histology (n=6/group). CCI was performed with 5m/s velocity, 50msec dwell, 1.2mm depth. Experiment 2 was post-treatment design with behavior and lesion volume as endpoints (n=8–12/group). CCI was performed with 6m/s velocity, 50msec dwell, 2.0mm depth. AMNV - ampicillin, metronidazole, neomycin, vancomycin
Figure 2.
Figure 2.. 16S analysis of gut microbiome.
A) Plot of number of observed species and Shannon entropy (alpha diversity) between samples prior to randomization, at the time of CCI, and at 72h after CCI. Mice given AMNV antibiotics had significantly reduced alpha diversity (p<0.05) at the time of CCI. B) NMDS plot of weighted UniFrac distances between samples. Samples from pre-randomization, pre- and post-CCI with standard drinking water, and pre- and post-CCI+AMNV cluster separately (PERMANOVA p<0.05). C) Relative abundance of microbial taxa showing reduction in microbial diversity in AMNV-treated mice and enrichment of Verrucomicrobiaceae sp. in mice on basal and high-fiber diet. CCI, controlled cortical impact; AMNV, ampicillin neomycin metronidazole vancomycin
Figure 3.
Figure 3.. AMNV-treated water effectively depletes bacteria in the gut lumen.
In situ labeling for bacterial 16S rRNA with EUB338 (red) and DAPI nuclear stain (blue) within the intestinal lumen of cecum of mice (n=6/group). Images taken at 72h after CCI demonstrate depletion of bacteria in mice given AMNV antibiotics in drinking water. AMNV, ampicillin neomycin metronidazole vancomycin
Figure 4.
Figure 4.. Histologic evaluation of hippocampal neuronal density and microglial activation at 72h after CCI.
A) Shown are representative 5μm sections through dorsal hippocampus. Scale bar = 500μm. B) Quantification (n=6/group) of surviving CA1 region hippocampal neurons per 0.1mm demonstrates a significant injury effect (naïve vs. CCI: 23.7 ± 1.4 vs. 8.8 ± 2.1 cells/0.1mm, p<0.05) and significantly increased CA1 neuronal density in AMNV antibiotic treated mice (CCI vs. CCI+AMNV: 8.8 ± 2.1 vs. 15.2 ± 1.4 cells/0.1mm, p<0.05). No effect on CA3 neuronal survival was seen. C) Quantification of Iba-1 positive cells in the hippocampus was performed and normalized to CCI. In AMNV treated mice, there was a 26.6±6.6% reduction in Ibs-1 positive cells in the dorsal hippocampus (n=6/group; p<0.05). CCI, controlled cortical impact; AMNV, ampicillin neomycin metronidazole vancomycin
Figure 5.
Figure 5.. Histologic evaluation of cecum for change in gut morphology or barrier protein expression at 72h after CCI.
Sacrifice (n=6/group) was performed at 72h after CCI and cecum embedded for histologic analysis. Shown are 5μm sections stained with H&E, DAPI nuclear stain (blue), and ZO-1 (red). No significant blunting or necrosis of villi or inflammatory cell infiltrate was observed in any group. The bowel of AMNV antibiotic treated mice was edematous. ZO-1 expression was significantly decreased after CCI (Naïve vs. CCI mean fluorescence intensity: 23.3±2.4 vs. 12.9±0.8; p<0.01). This decrease was attenuated in mice that received AMNV antibiotics (CCI vs. CCI+AMNV: 12.9±0.8 vs. 31.2±2.6; p<0.001). Scale bar = 100μm. CCI, controlled cortical impact; AMNV, ampicillin neomycin metronidazole vancomycin
Figure 6.
Figure 6.. AMNV antibiotic treated mice have attenuated cued learning deficit and lesion volume after TBI.
On days 21–22 after injury the fear conditioning task was performed to assess cued memory acquisition (n=12/group). A) On fear conditioning task, percent time frozen during cue was significantly decreased in mice after CCI (Naïve vs. CCI: 63.7 ± 2.7% vs. 41.0 ± 5.1%; p<0.01). Cued learning deficit was attenuated in CCI+AMNV mice which showed no learning deficit relative to naïve mice (Naïve vs. CCI+AMNV: 63.7 ± 2.7% vs. 54.8 ± 5.0%, p=NS) B) Recovery was determined as the % change in activity during the 60 secs after cue relative to the 60 secs during cue. A significant recovery deficit was observed after CCI (Naïve vs. CCI: 43.7 ± 6.1% vs. 21.7 ± 9.4%, p<0.05). No difference was seen in % recovery between naïve and CCI+AMNV groups. C) At completion of behavioral testing, mice were sacrificed and serial coronal sections from frozen tissue were taken, stained with H&E, and analyzed to quantify lesion volume. Mice given AMNV after CCI had significantly reduced lesion volume (CCI vs. CCI+AMNV: 26.1 ± 0.8mm2 vs. 24.6 ± 0.7mm2, p<0.05). Data analyzed by one-way ANOVA or Student t test as appropriate. D) Representative 10μm coronal sections taken from mice sacrificed on day 22 after CCI and used for quantification of lesion volume. The brain was cryopreserved and stored at −80°C. 10μm sections were stained with H&E. Shaded regions indicate lesion area. CCI, controlled cortical impact; AMNV, ampicillin neomycin metronidazole vancomycin
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