Fecal Microbiota Transplantation Exerts Neuroprotective Effects in a Mouse Spinal Cord Injury Model by Modulating the Microenvironment at the Lesion Site

Abstract

The primary traumatic event that causes spinal cord injury (SCI) is followed by a progressive secondary injury featured by vascular disruption and ischemia, inflammatory responses and the release of cytotoxic debris, which collectively add to the hostile microenvironment of the lesioned cord and inhibit tissue regeneration and functional recovery. In a previous study, we reported that fecal microbiota transplantation (FMT) promotes functional recovery in a contusion SCI mouse model; yet whether and how FMT treatment may impact the microenvironment at the injury site are not well known. In the current study, we examined individual niche components and investigated the effects of FMT on microcirculation, inflammation and trophic factor secretion in the spinal cord of SCI mice. FMT treatment significantly improved spinal cord tissue sparing, vascular perfusion and pericyte coverage and blood-spinal cord-barrier (BSCB) integrity, suppressed the activation of microglia and astrocytes, and enhanced the secretion of neurotrophic factors. Suppression of inflammation and upregulation of trophic factors, jointly, may rebalance the niche homeostasis at the injury site and render it favorable for reparative and regenerative processes, eventually leading to functional recovery. Furthermore, microbiota metabolic profiling revealed that amino acids including β-alanine constituted a major part of the differentially detected metabolites between the groups. Supplementation of β-alanine in SCI mice reduced BSCB permeability and increased the number of surviving neurons, suggesting that β-alanine may be one of the mediators of FMT that participates in the modulation and rebalancing of the microenvironment at the injured spinal cord. IMPORTANCE FMT treatment shows a profound impact on the microenvironment that involves microcirculation, blood-spinal cord-barrier, activation of immune cells, and secretion of neurotrophic factors. Analysis of metabolic profiles reveals around 22 differentially detected metabolites between the groups, and β-alanine was further chosen for functional validation experiments. Supplementation of SCI mice with β-alanine significantly improves neuronal survival, and the integrity of blood-spinal cord-barrier at the lesion site, suggesting that β-alanine might be one of the mediators following FMT that has contributed to the recovery.

Keywords: fecal microbiota transplantation; inflammation; microenvironment; spinal cord injury; vascular repair; β-alanine.

FIG 1

FMT treatment ameliorates pathological features after SCI. (A) Representative images of spinal cord transverse sections after H&E staining. Scale bar = 50 μm. (B) Representative images of spinal cord transverse sections after Nissl staining. Scale bar = 50 μm. (C) The numbers of neurons in the ventral horn of the spinal cord were determined. (D) Representative images of sections spanning from 2 mm rostral to the lesion epicenter to 2 mm caudal after Luxol Fast Blue (LFB) staining. (E) Quantification of spared white matter by LFB staining (n = 4); *, P < 0.05 compared to the SCI group; **, P < 0.01 compared to the SCI group.

FIG 2

FMT treatment restores blood perfusion following SCI. (A to D) 30-s samplings of raw blood flow outputs from the combined probe among different groups. Functional parameters of hemodynamics, including (E) average blood perfusion and (F) relative velocity, were examined by Laser Doppler perfusion monitoring (n = 4). *, P < 0.05 compared to the SCI group; **, P < 0.01 compared to the SCI group (NS: no significant).

FIG 3

FMT treatment alleviates blood vessel damage and increases pericyte coverage of microvessels after SCI. (A) Representative images showing double immunofluorescent staining for CD31 and PDGFRβ at the lesion epicenter. Scale bar = 50 μm. (B) The proportion of CD31-stained area was quantified 4 weeks post-injury on transverse sections. (C) Pericyte coverage was assessed as the ratio of PDGFRβ+ area to CD31⁺ area (n = 4). *, P < 0.05 compared to the SCI group; **, P < 0.01 compared to the SCI group.

FIG 4

FMT treatment improves BSCB integrity 4 weeks after SCI. (A) Representative fluorescent images of Evan’s blue extravasation at the spinal parenchyma. Scale bar = 50 μm. (B) Quantification of extravasated Evan’s blue (n = 4). *, P < 0.05 versus the SCI group; **, P < 0.01 versus the SCI group. (C to D) Expression levels of occludin, ZO-1 and MMP-9 assessed by Western blotting; relative amounts were quantified (n = 4).

FIG 5

FMT treatment alleviates the activation of astrocytes and microglia in the spinal cord after injury. (A) A spinal cord section with outlined gray matter area and a squared inset of ventral horn. (B) Representative images of immunofluorescent staining for Iba-1 (a marker of microglia) and GFAP (a marker of astrocytes) detection in the ventral horn of the injured spinal cord on day 28 (Scale bar = 50 μm). (C) Quantification of the Iba-1-positive area in the ventral horn of spinal cord on day 28 post-SCI (n = 4). (D) Quantification of the GFAP-positive area in the ventral horn of spinal cord on day 28 post-SCI (n = 4). *, P < 0.05 compared to the SCI group; **, P < 0.01 compared to the SCI group.

FIG 6

FMT treatment alters the expression of neurotrophic factors in the spinal cord 4 weeks following injury. (A) Expression of BDNF, GDNF, NT-3 and NGF analyzed by Western blotting. The relative amounts of BDNF (B), GDNF (C), NT-3 (D) and NGF (E) were obtained by semi-quantitative analysis in different groups (n = 4). *, P < 0.05 compared to the SCI group; **, P < 0.01 compared to the SCI group.

FIG 7

Differential amounts of metabolites among the sham, SCI and SCI+FMT groups. (A) PLS-DA score plot based on metabolic profiles in fecal samples from the sham, SCI and SCI+FMT groups. (B) Score scatterplot of OPLS-DA revealing the separation among groups according to metabolic differences. (C) Metabolites significantly altered in sham animals versus SCI group and SCI group versus SCI+FMT group. Those with VIP >1.0 and P < 0.05 (t test) were identified. Venn diagrams demonstrating the amounts of altered metabolites shared among the three groups by the overlap (n = 8). (D) Relative amounts of 22 differentially produced metabolites in the three groups, presented as a heat map.

FIG 8

ALA administration enhances BSCB integrity following SCI. (A) Representative fluorescent images showing Evan’s blue extravasation at the spinal parenchyma. Scale bar = 50 μm. (B) Quantification of extravasated Evan’s blue (n = 4). *, P < 0.05 versus SCI group; **, P < 0.01 versus SCI group. (C to D) Expression of occludin, ZO-1 and MMP-9 4 weeks after SCI examined by Western blotting, and semi-quantitative analysis (n = 4). ALA, β-alanine.

FIG 9

ALA treatment impacts animal behavior and pathological features in SCI mice. (A) Body weight changes during the 4 weeks in different groups. (B) Time course of locomotor function recovery as assessed by the BMS. (C) BMS subscores were assessed on day 28 following SCI (n = 8). (D) Representative images of spinal cord transverse sections after H&E staining and Nissl staining. Scale bar = 50 μm (n = 4). (E) Amounts of neurons in the ventral horn of the spinal cord. *, P < 0.05 compared to the SCI group; **, P < 0.01 compared to the SCI group.