The gut microbiome contributes to blood-brain barrier disruption in spontaneously hypertensive stroke prone rats

Abstract

In recent years, it has become apparent that the gut microbiome can influence the functioning and pathological states of organs and systems throughout the body. In this study, we tested the hypothesis that the gut microbiome has a major role in the disruption of the blood-brain barrier (BBB) in the spontaneously hypertensive stroke prone rats (SHRSP), an animal model for hypertensive cerebral small vessel disease (CSVD). Loss of BBB is thought to be an early and initiating component to the full expression of CSVD in animal models and humans. To test this hypothesis, newly born SHRSP pups were placed with foster dams of the SHRSP strain or dams of the WKY strain, the control strain that does not demonstrate BBB dysfunction or develop hypertensive CSVD. Similarly, WKY pups were placed with foster dams of the same or opposite strain. The rationale for cross fostering is that the gut microbiomes are shaped by environmental bacteria of the foster dam and the nesting surroundings. Analysis of the bacterial genera in feces, using 16S rRNA analysis, demonstrated that the gut microbiome in the rat pups was influenced by the foster dam. SHRSP offspring fostered on WKY dams had systolic blood pressures (SBPs) that were significantly decreased by 26 mmHg (P < .001) from 16-20 weeks, compared to SHRSP offspring fostered on SHRSP dams. Similarly WKY offspring fostered on SHRSP dams had significantly increased SBP compared to WKY offspring fostered on WKY dams, although the magnitude of SBP change was not as robust. At ~20 weeks of age, rats fostered on SHRSP dams showed enhanced inflammation in distal ileum regardless of the strain of the offspring. Disruption of BBB integrity, an early marker of CSVD onset, was improved in SHRSPs that were fostered on WKY dams when compared to the SHRSP rats fostered on SHRSP dams. Although SHRSP is a genetic model for CSVD, environmental factors such as the gut microbiota of the foster dam have a major influence in the loss of BBB integrity.

Keywords: cerebral small vessel disease; gut microbiome; hypertension; microbiota-gut-brain axis; vascular cognitive impairment; vascular dementia; white matter disease.

FIGURE 1

Cross fostering protocol to shift the gut microbiome of the fostered offspring to that of the nursing dam. WKYonWKY represents WKY pups fostered on WKY dams, WKYonSHRSP represents WKY pups fostered on SHRSP dams, SHRSPonWKY represents SHRSP pups fostered on WKY dams, and SHRSPonSHRSP represents SHRSP pups fostered on SHRSP dams

FIGURE 2

A, Unweighted Bray-Curtis PCoA plots for fecal microbiota composition from rats in the cross fostering study at ages of 10, 16, and 20 weeks (5-7 per group; P = .001 for each PCoA, PERMANOVA test). B, The abundance ratio of Firmicutes to Bacteroidetes (F : B) in the four groups in the cross fostering protocol. There was a significant main effect of offspring strain (P < .001) and a significant main effect of the strain of the foster dam (P = .044, N = 5-7 per group) using three-way ANOVA. Holm-Sidak post hoc analysis showed that in WKY offspring, there was a significant effect of the foster dam (P = .026) and a significant effect of WKY and SHRSP offspring fostered on SHRSP dam (P < .001)

FIGURE 3

Fold change in message in distal ileum (compared to WKYonWKY) for the four groups of rats in the cross fostering study. Data were analyzed using two-way ANOVA with post hoc Holm-Sidak test where appropriate. Significant main effect (P < .05) for offspring (p), dam (d), and both offspring and dam (pd) (N = 6 per group) demonstrating that the offspring or the fostering dam significantly altered the message independent of other variables. *P < .05 compared to same strain fostered on WKY dam using Holm-Sidak post hoc analysis

FIGURE 4

Fold change in message in brain (compared to WKYonWKY) for the four groups of rats in the cross fostering study. Data were analyzed using two-way ANOVA with post hoc Holm-Sidak test where appropriate. Significant main effect (P < .05) for offspring (p) or fostering dam (d) (N = 6 per group) demonstrating that the offspring alone or the fostering dam alone significantly altered the message independent of other variables.

FIGURE 5

A, Macrophages (Red) as detected by the F4/80 antibody in villi of the distal ileum. Blue = DAPI stain for nuclei. B, Quantitative assessment of macrophages in WKY and SHRSP with different fostering strains. The fluorescence images were quantitated as the number of red pixels (macrophages) normalized to the total number of pixels in the villi. Data were analyzed using two-way ANOVA showing significant effect of the offspring strain P = .026 (two-way ANOVA, N = 4/group); *P = significant effect in SHRSP fostered by SHRSP dam (*P = .009 compared to all other groups, Holm-Sidak post hoc)

FIGURE 6

The effects of cross fostering on systolic blood pressure (SBP). Three-way ANOVA revealed significant differences for offspring strain (P < .001), strain of fostering dam (P < .001), age (P = .033), and a significant interaction between offspring strain and strain of foster dam (P = .021) (N = 6/group). Holm-Sidak post hoc Analysis showed a significant difference between WKYonWKY and WKYonSHRSP (bracket “b,” P = .014), SHRSPonWKY and SHRSPonSHRSP (bracket “a,” P < .001), and individual point differences between SHRSPonSHRSP and SHRSPonWKY (**P = .001 and *P < .05)

FIGURE 7

Plasma zonulin, a marker for the gut barrier integrity, in the four groups of rats in the cross fostering protocol. Data were analyzed using two-way ANOVA with post hoc Holm-Sidak test where appropriate; p = significant effect of pup strain independent of the strain of the fostering dam (P = .008, two-way ANOVA, N = 6 per group); *P < .003 compared to WKYonWKY (post hoc Holm-Sidak method)

FIGURE 8

A, Western blot of IgG in brain parenchymal tissues, a measure of BBB integrity, in the four groups of rats in the cross fostering protocol. GADPH is the loading control. B, The quantitative assessment of Western blots shown in “A” using two-way ANOVA. Two-way ANOVA revealed a significant effect of both the offspring strain (p, P < .001) and the strain of the fostering dam (d, P = .025). **P = .005 and <.001 compared to SHRSPonWKY and WKYonSHRSP, respectively, using post hoc Holm-Sidak (n = 4-5)

FIGURE 9

Lack of relationship between systolic blood pressure (SBP) and Normalized density of IgG in parenchymal tissues, a measures of BBB integrity for SHRSPonWKY A, and SHRSPonSHRSP B, at 20 weeks of age indicating that BBB integrity was not related to SBP. Adjusted R² = 0.000 for both A (P = .755) and B (P = .673), Pearson Product Moment Correlation

FIGURE 10

A, shows immunofluorescent images of microglia/macrophage (IBA-1, green) in the corpus callosum, a white matter structure beneath the cortex. Blue is DAPI staining for the nucleus. The corpus callosum is between the two dashed lines in each of the images. (10B, left panel) The % area of the corpus callosum consisting of microglia in the four groups used in the cross fostering study. Two-way ANOVA revealed a significant main effect for the strain of the pup (P = .008), a significant interaction between the pup strain and the strain of the foster dam (P = .044). *P = .013 compared to SHRSPonWKY and P = .002 compared to WKYonSHRSP. (10B, right panel) The number of microglia/macrophage cell bodies per unit area in the corpus callosum for the four groups used in the cross fostering study. Two-way ANOVA revealed a significant main effect for the strain of the foster dam (P = .022)