doi: 10.1016/j.bbi.2016.04.003. Epub 2016 Apr 6.
Brain injury induces specific changes in the caecal microbiota of mice via altered autonomic activity and mucoprotein production
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- PMID:27060191
- PMCID: PMC5021180
- DOI: 10.1016/j.bbi.2016.04.003
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Brain injury induces specific changes in the caecal microbiota of mice via altered autonomic activity and mucoprotein production
A Houlden et al. Brain Behav Immun.2016 Oct.
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Abstract
Intestinal microbiota are critical for health with changes associated with diverse human diseases. Research suggests that altered intestinal microbiota can profoundly affect brain function. However, whether altering brain function directly affects the microbiota is unknown. Since it is currently unclear how brain injury induces clinical complications such as infections or paralytic ileus, key contributors to prolonged hospitalization and death post-stroke, we tested in mice the hypothesis that brain damage induced changes in the intestinal microbiota. Experimental stroke altered the composition of caecal microbiota, with specific changes in Peptococcaceae and Prevotellaceae correlating with the extent of injury. These effects are mediated by noradrenaline release from the autonomic nervous system with altered caecal mucoprotein production and goblet cell numbers. Traumatic brain injury also caused changes in the gut microbiota, confirming brain injury effects gut microbiota. Changes in intestinal microbiota after brain injury may affect recovery and treatment of patients should appreciate such changes.
Keywords: Cerebral ischemia; Gut; Inflammation; Microbiota; Mucoprotein; Noradrenaline; Stroke.
Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.
Figures
Neurological deficit and bacterial community profiling of brain injury mice and controls by DGGE: (A) Brain injury is shown after 60 min MCAo and 72 h reperfusion as identified by cresyl violet staining, dotted line indicates infarct boarder. (B) Neurological deficit score for mice post surgery significant effect of treatment (ANOVA: F4,20 = 33.88, p = <0.01) post hoc TukeyHSD confirmed stroke increase neurological deficit (p < 0.001), with no difference between 4 h or 72 h post brain injury (p = 0.58) Error bars are standard error of the mean, n = 5. (C) NMDS analysis of caecal bacterial communities assessed by DGGE of the 16S rRNA gene, profile of Naïve, Sham and Brain injury mice caecal bacterial communities at t = 4 h and 72 h post operation. Samples are as follows: ○ = t 0; △ = t 4 h; □ = t 72 h post treatment; white = naïve; grey = sham; black = brain injury. Axis represent scale for simularity distance scores between sample centered to (0,0). PERMANOVA: Naïve vs. Sham and Brain injury at 4 h post operation (adonis: F.Model2,12 = 1.34, p = 0.159), Naïve vs. Sham 72 h (adonis: F.Model1,8 = 3.59, p = 0.016); Naïve vs. Brain Injury 72 h, (adonis: F.Model1,8 = 5.90, p = 0.008); Sham 72 h vs. Brain Injury 72 h, (adonis: F.Model1,8 = 1.51, pr = 0.17). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
16S amplicon pyrosequencing analysis of mouse caecum from niave, sham and brain injury mice. Bacterial species relative abundances from rarefied OTU data (1866 sequences) were used for NMDS analysis Samples are as follows: ○ = t 0; □ = t 72 h post treatment; white = naïve; grey = sham; black = brain injury. Axis represent scale for simularity distance scores between sample centered to (0,0). Pair-wise comparisons were undertaken to identify significant differences using PERMANOVA. Naïve vs. Sham (adonis: F.Model1,8 = 2.12, p = 0.008); Naïve vs. Brain Injury (adonis: F.Model1,8 = 2.24, p = 0.008); Sham vs. Brain injury, (adonis: F.Model1,8 = 1.02, p = 0.484).
Proportions of bacterial taxa that were identified to change significantly as a result of experimental stroke or sham surgery. ANOVA was undertaken on all taxa identified with FDR correction of P-values. (A)Peptococcaceae proportions of community between naïve, sham, and brain injury mice were significantly different (p-adjust = 0.026). with labels a and b denoting treatments significantly different (p < 0.05) identified using TukeyHSD posthoc test, (B)Prevotellaceae proportions of community between naïve, sham, and brain injury mice were significantly different (p-adjust = 0.019) with labels a and b denoting treatments significantly different (p ⩽ 0.05) identified using TukeyHSD posthoc test. Upper and lower limits of box represent 75th and 25th percentile, solid line in median, dotted line mean. Dots represent actual values for each mouse.
Intestinal microbiota changes correlate with gut noradrenaline (NE) levels (A) Mice that had undergone 60 min MCAo and 72 h reperfusion show significantly increased NE levels in gut tissue homogenates (ANOVA: F2,12 = 10.02, p = 0.003; Posthoc TukeyHSD significance <0.001, with the labels a and b denoting samples significantly different, error bars are standard error of the mean, n = 5. (B) Peptococcaceae relative abundance correlate with intestinal NE levels (Regression: R2 = 0.370, F1,13 = 7.56, p = 0.02), Equation of line y = 0.322 * x + 0.114. (C) Prevotellaceae decrease in the intestinal proportionally to increasing NE levels after injury (Regression: R2 = 0.512, F1,13 = 13.58, p = 0.003), Equation of line y = −3.299 * x + 3.480. Samples are as follows: ○ = Naïve; △ = Sham 72 h post treatment; □ = Brain injury 72 h post treatment.
Brain injury results in altered noradrenaline (NE) release and mucoprotein production in the caecum. (A) Tissue uptake and stimulated release of3H-NE was significantly increased in the caecum 72 h after experimental stroke (**P < 0.01,***P < 0.0001, unpairedt test), error bars are standard error of the mean, n = 6–8). (B) Tyrosine hydroxilase immunopositive nerve fibers (arrowheads) are most abundant at the basis of the intestinal epithelium found often in the proximity of goblet cells in mice 72 h after brain injury (cresyl violet counterstain). (C) Haematoxylin and eosin (H&E), Azan andperiodic acid-Schiff-alcian blue (PAS) staining of paraffin-embedded caecum sections is shown, 72 h after sham surgery or experimental stroke. PAS staining identifies mucoprotein-containing cells (magenta – neutral mucins, blue – acidic mucins, magenta/blue –mixed mucins) and indicates less mucoproteins associated with the apical part of intestinal epithelial cells (shown by arrowheads on insert). (D) Quantification of PAS staining (C). Brain injury is associated with less mucin-containing cells in the caecum compared to sham animals (*P < 0.05,***P < 0.0001, one-way ANOVA followed by TukeyHSD posthoc test), error bars are standard error of the mean, n = 4. Scale bars: B – 50 μm; C – 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Impact of manipulations of the sympathetic nervous system. (A) Impact on community microbiota assessed by NMDS analysis of 16S amplicon illumina sequencing relative abundances from rarefied OTU data (36,904 sequences). Controls = white, 6-OHDA treated = Light grey, Increased systemic sympathetic autonomic tone = dark grey. 6-OHDA samples were significantly different to controls (adonis F.model1,12 = 2.90, p = 0.0012), whereas Increased systemic sympathetic autonomic tone had no impact. Significant correlations were identified with the Phyla of Firmicutes (R2 = 0.60, p = 0.008), Bacteroidetes (R2 = 0.58, p = 0.01) and the Bacteroidetes family S24-7 (R2 = 0.78, p = 0.017). (B)Periodic acid-Schiff-alcian blue (PAS) staining of paraffin-embedded caecum sections is shown in control mice and after 6-OHDA administration or atomoxetine/yohimbine treatment that leads to mild increase in sympathetic tone (Inc. symp). Number of PAS-positive goblet cells is significantly reduced 72 h after 6-OHDA compared to Control (p < 0.001) and Inc. symp. (&p < 0.05) mice. Scale bar: 100 μm. Error bars are standard error of the mean, n = 5–6. (C) Significant shifts (∗) in the proportions of Prevotellaceae as a result of 6-OHDA treatment p < 0.05. Upper and lower limits of box represent 75th and 25th percentile, solid line in median, dotted line mean. Dots represent actual values for each mouse.
Impact of traumatic brain injury (TBI) on microbiota. (A) Cresyl violet staining indicates brain injury induced by TBI in the cerebral cortex. Scale bar: 500 μm. (B) Impact on community microbiota as a result of TBI assessed by NMDS analysis of 16S amplicon Ilumina sequencing. Rarefied OTU data (36,904 sequences) was used with samples as follows: Controls = white, TBI = dark grey. A strong correlation between neurological deficit and community compositions was seen label N (R2 = 0.6797 p = 0.0013). Other significant correlations explaining the shifts in NMDS were identified and marked with the letters as follows (A) Clostridiales (R2 = 0.76 p = 0.015), (B) Bacteroidetes (R2 = 0.63 p = 0.02), (C) α-proteobacteria (R2 = 0.59 p = 0.039), (D) Proteobacteria (R2 = 0.73 p = 0.008), (E) Cyanobacteria (R2 = 0.76 p = 0.002), (F) Porphyromonadaceae (R2 = 0.66 p = 0.027). (C)Periodic acid-Schiff-alcian blue (PAS) staining of paraffin-embedded caecum sections is shown 72 h after sham surgery or traumatic brain injury (TBI). Scale bar: 100 μm. Error bars are standard error of the mean, n = 7–8. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Comment in
- Brief commentary for brain, behavior, and immunity.Auvinen P.Auvinen P.Brain Behav Immun. 2016 Oct;57:8-9. doi: 10.1016/j.bbi.2016.06.003. Epub 2016 Jun 4.Brain Behav Immun. 2016.PMID:27266392No abstract available.
References
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- Benjamini Y., Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Ser. B. 1995;57:289–300.
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