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. 2024 Feb 5;14(1):2990.
doi: 10.1038/s41598-024-53430-4.

Acute gastrointestinal permeability after traumatic brain injury in mice precedes a bloom in Akkermansia muciniphila supported by intestinal hypoxia

Anthony J DeSana  1   2 , Steven Estus  1   3 , Terrence A Barrett  4   5 , Kathryn E Saatman  6   7
Affiliations

Acute gastrointestinal permeability after traumatic brain injury in mice precedes a bloom in Akkermansia muciniphila supported by intestinal hypoxia

Anthony J DeSana et al. Sci Rep. .

Abstract

Traumatic brain injury (TBI) increases gastrointestinal morbidity and associated mortality. Clinical and preclinical studies implicate gut dysbiosis as a consequence of TBI and an amplifier of brain damage. However, little is known about the association of gut dysbiosis with structural and functional changes of the gastrointestinal tract after an isolated TBI. To assess gastrointestinal dysfunction, mice received a controlled cortical impact or sham brain injury and intestinal permeability was assessed at 4 h, 8 h, 1 d, and 3 d after injury by oral administration of 4 kDa FITC Dextran prior to euthanasia. Quantification of serum fluorescence revealed an acute, short-lived increase in permeability 4 h after TBI. Despite transient intestinal dysfunction, no overt morphological changes were evident in the ileum or colon across timepoints from 4 h to 4 wks post-injury. To elucidate the timeline of microbiome changes after TBI, 16 s gene sequencing was performed on DNA extracted from fecal samples collected prior to and over the first month after TBI. Differential abundance analysis revealed that the phylum Verrucomicrobiota was increased at 1, 2, and 3 d after TBI. The Verrucomicrobiota species was identified by qPCR as Akkermansia muciniphila, an obligate anaerobe that resides in the intestinal mucus bilayer and produces short chain fatty acids (e.g. butyrate) utilized by intestinal epithelial cells. We postulated that TBI promotes intestinal changes favorable for the bloom of A. muciniphila. Consistent with this premise, the relative area of mucus-producing goblet cells in the medial colon was significantly increased at 1 d after injury, while colon hypoxia was significantly increased at 3 d. Our findings reveal acute gastrointestinal functional changes coupled with an increase of beneficial bacteria suggesting a potential compensatory response to systemic stress after TBI.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Study timeline of microbiome normalization, TBI and fecal collection. (A) Prior to fecal collection, the microbiome was normalized by performing a whole bowel irrigation with polyethylene glycol (PEG) followed by repeated inoculations with a cecal slurry (green triangles) collected from age and sex-matched mice. Fecal samples were collected (light blue arrow) prior to injury and at several timepoints out to 4 wks post-injury. Examples of Nissl stain from (B) a sham control and (C) controlled cortical impact (CCI) mice at early and chronic timepoints illustrate injury progression from a hemorrhagic contusion to cortical cavitation.
Figure 2
Figure 2
CCI results in a transient increase in intestinal permeability. Intestinal permeability was increased at 4 h following CCI when compared to sham animals binned from all timepoints. Serum concentration of FITC dextran returned to sham levels beginning at 8 h and remained there at 1 and 3 d post-injury. Whiskers represent minimum and maximum, box represents 25th-75th percentile, and line represents median value (**p < 0.01; nsham = 2–3/timepoint, nCCI = 6–9/timepoint). Data for individual sham groups can be found in Supplemental Fig. 4.
Figure 3
Figure 3
CCI does not alter morphology of the ileum or colon. Representative images of the crypt-villi structure from (A) sham and (B) CCI mice. Swiss-rolled cross sections are shown at low magnification with a box denoting the area shown at high magnification. Green lines depict the distance measurements acquired. (C) Quantification of crypt-villi depth for each timepoint shows no alteration in the ileum after CCI. Representative images illustrating crypt depth measurements in the colon (green lines) from (D) sham and (E) CCI mice. Quantification of the crypt depth suggests that CCI did not alter crypt morphology in the (F) medial or (G) distal colon. In the distal colon, a main effect of time after injury was present. Whiskers represent minimum and maximum, box represents 25th–75th percentile, and line represents median value (nsham = 3–6/timepoint, nCCI = 6–10/timepoint).
Figure 4
Figure 4
CCI results in decreased alpha diversity as measured by Shannon diversity index and altered beta diversity as measured by Bray–Curtis dissimilarity and weighted UniFrac distance. Alpha diversity: (A) the Shannon diversity index was significantly reduced in mice with CCI as compared to sham controls, but this injury effect did not depend on collection timepoint. (B) Faith’s phylogenetic diversity was not altered by injury or time point. Statistical comparison by mixed-effects analysis (whiskers represent minimum and maximum, box represents 25th–75th percentile, and line represents median value). Beta diversity: Principal coordinate analysis visualizations for (C) Bray–Curtis Dissimilarity (p = 0.002), (D) UniFrac distance (p = 0.053), and (E) weighted UniFrac distance (p = 0.009). Statistical comparison of Beta Diversity by PERMANOVA (nsham = 6, nCCI = 7).
Figure 5
Figure 5
Early increase in abundance of taxa Verrucomicrobiota following CCI. (A) The phylum Verrucomicrobiota is differentially abundant in CCI animals compared to time-matched sham animals at 1, 2, and 3 d post-injury using ANCOM-BC. A log-fold change value (y-axis) of 0 indicates equivalent taxa abundance while above 0 indicates increased abundance in sham mice, and a log-fold change value below 0 indicates that a taxa is more abundant in CCI animals. (B) This taxa is differentially abundant down to an order level (Verrucomicrobiales) in CCI animals compared to time-matched sham animals at 2 and 3 d post-injury. Data are represented as log-fold change and standard error (## q < 0.01). (C) The Verrucomicrobiota species Akkermansia muciniphila is increased at 2 and 3 d post-injury as determined by qPCR. Data are represented as mean and standard deviation (*p < 0.05; nsham = 6, nCCI = 7).
Figure 6
Figure 6
Goblet cells are altered in the medial colon but not in the ileum or distal colon. Intestinal tissue was stained with Alcian Blue to label mucins within goblet cells and counterstained with nuclear fast red. Representative images of the distal third of the small intestine, the ileum, from (A) 1 d sham and (B) 1 d CCI mice. (C) Counts of goblet cells per villi were equivalent for sham and CCI groups but varied across timepoints. Representative images of colon tissue from both the (D, E) medial and (G, H) distal colon of 1 d sham and 1 d CCI mice, respectively. (F) CCI increased goblet cell density in the medial colon at 1 d, (G) but no effect of injury was observed in the distal colon. A main effect of time post-injury was present in both regions of the colon. Whiskers represent minimum and maximum, box represents 25th–75th percentile, and line represents median value (*p < 0.05; nsham = 3–6/timepoint; nCCI = 6–10/timepoint).
Figure 7
Figure 7
CCI induces colon hypoxia at 3 d post-injury. Representative images of hypoxyprobe-1 staining (HP1, red) in the colon of (A) sham and (B) 3 d post-CCI mice. (C) Traces of hypoxia intensity as a function of distance from the lumen, averaged from three crypts within three randomly selected fields of view from sham (blue), 1 d CCI (red), and 3 d CCI (black). (D) Area under the hypoxia intensity curve is increased at 3 d post-CCI compared to sham mice. (E) Peak hypoxia is also increased at 3 d post-CCI. Whiskers represent minimum and maximum, box represents 25th–75th percentile, and line represents median value (*p < 0.05; nsham = 11, nCCI = 6–7/timepoint).
Figure 8
Figure 8
Graphical summary of findings. We hypothesize that TBI leads to a transient disruption of the intestinal barrier that initiates a colonic response characterized by increased goblet cell density and hypoxia, facilitating an increase in A. muciniphila as a beneficial compensatory response to limit damage to the GI tract. Figure created with BioRender.com.
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