Inulin supplementation prior to mild traumatic brain injury mitigates gut dysbiosis, and brain vascular and white matter deficits in mice

Abstract

Introduction: Mild traumatic brain injury (mTBI) has been shown to negatively alter bacterial diversity and composition within the gut, known as dysbiosis, in rodents and humans. These changes cause secondary consequences systemically through decreased bacterial metabolites such as short chain fatty acids (SCFAs) which play a role in inflammation and metabolism. The goal of the study was to identify if giving prebiotic inulin prior to closed head injury (CHI) could mitigate gut dysbiosis, increase SCFAs, and improve recovery outcomes, including protecting cerebral blood flow (CBF) and white matter integrity (WMI) in young mice.

Methods: We fed mice at 2 months of age with either inulin or control diet (with cellulose as fiber source) for two months before the CHI and continued till the end of the study. We analyzed gut microbiome composition and diversity, determined SCFAs levels, and measured CBF and WMI using MRI. We compared the results with Naïve and Sham-injury mice at 24 hours, 1.5 months, and 3-4 months post-injury.

Results: We found that both CHI and Sham mice had time-dependent changes in gut composition and diversity after surgery. Inulin significantly reduced the abundance of pathobiont bacteria, such as E. coli, Desulfovibrio spp and Pseudomonas aeruginosa, in Sham and CHI mice compared to mice fed with control diet. On the other hand, inulin increased SCFAs-producing bacteria, such as Bifidobacterium spp and Lactobacillus spp, increased levels of SCFAs, including butyrate and propionate, and significantly altered beta diversity as early as 24 hours post-injury, which lasted up to 3-4 months post-injury. The mitigation of dysbiosis is associated with protection of WMI in fimbria, internal and external capsule, and CBF in the right hippocampus of CHI mice, suggesting protection of memory and cognitive functions.

Discussion: The results indicate that giving inulin prior to CHI could promote recovery outcome through gut microbiome modulation. As inulin, microbiome analysis, and MRI are readily to be used in humans, the findings from the study may pave a way for a cost-effective, accessible intervention for those at risk of sustaining a head injury, such as military personnel or athletes in contact sports.

Keywords: MRI; cerebral blood flow; inulin; microbiome; prebiotics; short chain fatty acids; traumatic brain injury; white matter integrity.

FIGURE 1

Study design. Mice started diet (control or inulin) 2 months prior to injury at 2 months of age. Fecal samples were taken periodically throughout the study at (1) 2 months of age (prior to diet administration), (2) 24 hours prior to injury, (3) 24 hours post-injury, (4) 1.5 months post-injury, and (5) 3 months post-injury. Diffusion tensor imaging was taken at 1.5 months post-injury and 3-4 months post-injury to determine white matter integrity. Arterial spin labeling was taken 3-4 months post-injury to determine cerebral blood flow.

FIGURE 2

Alpha diversity and beta diversity analysis 24 hours, 1.5 months, and 3 months post-injury. Shannon index is used for all alpha diversity analyses, and Bray-Curtis index is used for all beta diversity analyses. Alpha diversity at (A) 24 hours, (B) 1.5 months, and (C) 3 months post-injury. Beta diversity at (D) 24 hours, (E) 1.5 months, and (F) 3 months post-injury. *p < 0.05.

FIGURE 3

Analysis of pathobiont bacteria and beneficial bacteria at 24 hours, 1.5 months and 3 months post-injury. (A) At 24 hours post-injury, control-fed mice show higher levels of (E) coli and Shigella spp than inulin-fed mice in Sham and CHI groups. (B) At 1.5 months post-injury, control-fed mice show higher levels than inulin-fed mice of L. bacterium 28-4 and Desulfovibrio spp in Naïve, Sham, and CHI mice and higher levels of P. aeruginosa in Naïve and CHI mice. (C) At 3 months post-injury, there are higher levels in control-fed mice than inulin-fed mice of L. bacteria 28-4 and Streptococcus spp in Naïve, Sham, and CHI mice, higher levels of Desulfovibrio spp in Sham and CHI mice, and higher levels of (E) coli, Shigella spp, and P. aeruginosa in CHI mice. (D) At 24 hours post-injury, inulin-fed mice show higher levels than control-fed mice of Bifidobacterium spp in Naïve, Sham, and CHI groups and higher levels of Lactobacillus spp in CHI mice. (E) At 1.5 months post-injury, inulin-fed mice show higher levels than control-fed mice of Bifidobacterium spp in Naïve, Sham, and CHI groups and higher levels of Lactobacillus spp. in Sham and CHI groups. (F) At 3 months post-injury, inulin-fed mice show higher levels than control-fed mice of Bifidobacterium spp. in Naïve, Sham, and CHI groups and higher levels of Lactobacillus spp in Sham and CHI groups.

FIGURE 4

SCFAs analysis 3-4 months post-injury. (A) Acetate significantly increased in the control-fed mice compared to the inulin-fed mice in the Naïve and Sham groups. (B) Butyrate significantly increased in inulin-fed mice compared to control-fed mice in Naïve, Sham, and CHI groups. (C) Propionate increased in inulin-fed mice compared to control-fed mice in Sham and CHI groups. Data are mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

FIGURE 5

White matter integrity analysis and cerebral blood flow 3-4 months post-injury. Sham-inulin mice had higher FA than the sham-control in (A) right and (B) left corpus callosum, and (C) left external capsule. (D) CHI-inulin mice had higher FA than the CHI-control mice in the right internal capsule. (E) CHI-inulin mice had higher CBF in the right hippocampus than the CHI-control mice. Data are mean ± SD. *p < 0.05.