Stroke and the gut microbiome: chronic gut dysbiosis in experimental stroke

Written by Allison L Brichacek & Candice M Brown (West Virginia University)

This is an image depicting the gut microbiome in the gastrointestinal tract.

Stroke is a leading cause of mortality and disability worldwide, which results in a significant socioeconomic burden for stroke survivors and their families. The global burden continues to increase, with a rapid rise among developing nations. Ischemic strokes comprise ~85% of all strokes, while the remainder are hemorrhagic strokes [1,2]. Ischemic stroke results in a substantial systemic proinflammatory response that may promote the onset or exacerbate pre-existing inflammation in local tissues [3]. Pre-existing systemic inflammation plays an important role in stroke outcome and functional recovery; conversely, stroke also generates systemic inflammatory responses that impact multiple peripheral organ systems.

One of the emerging areas in stroke research is how the gut microbiome contributes to brain and systemic inflammatory responses post-stroke. While previous studies have addressed the short-term effects of stroke (over several days) on the gut microbiome, much less is known about the long-term effects of stroke (over several weeks) on the gut microbiota [4–6]. The goal of our current work is to identify longitudinal changes in the gut microbiota post-stroke and how these changes may impact functional recovery, neuroinflammation and gut dysbiosis.


Chronic brain inflammation and functional outcomes following ischemic stroke

Our lab uses a common model of experimental stroke called transient middle cerebral artery occlusion to study ischemic stroke. An experimental stroke is achieved by placing an intraluminal filament through the internal carotid artery to occlude the middle cerebral artery, which results in restricted blood flow to the cortex and striatum [7,8].

We have observed that experimental stroke in male mice results in the persistent elevation of neurological severity scores accompanied by deficits in sensorimotor function for up to 4 weeks post-stroke. Histological analysis of brain tissue from these mice showed the inflammatory status of brain immune cells such as microglia and astrocytes, which remained elevated up to 1-month post-stroke.

These findings are important because our mouse model may mimic the functional deficits and chronic proinflammatory phenotype in stroke patients who suffer from long-term loss of motor function after a stroke. The ability to model chronic human clinical deficits in an experimental stroke model may allow researchers to identify pathways and potential therapeutic targets that can be tested in clinical trials for human stroke patients.

Does stroke cause similar chronic changes that alter the gut microbiome?

The microbiome facilitates several important functions related to health such as: facilitating food digestion, boosting the immune system and influencing mental health. Poor diet has been shown to be one of the greatest risk factors for stroke in the USA, so it is likely that stroke patients already have a distinct microbiome composition prior to stroke onset compared with healthy controls [9]. However, our studies have focused on the direct effects of ischemic stroke on the gut microbiome, as these experiments will provide insight on specific changes in the gut microbiota and how they may lead to chronic gut dysbiosis in stroke patients.

Our laboratory employed 16S rRNA gene sequencing of the V3-V4 bacterial 16S hypervariable region. We observed the overall upregulation of the phylum Firmicutes and decline in phylum Bacteroidetes, resulting in an elevated Firmicutes:Bacteroidetes (F/B) ratio between 2–4 weeks post-stroke. The F/B ratio is commonly used as an indicator for gut dysbiosis in several disease models, including metabolic disease, irritable bowel syndrome and social behaviors [10–12]. We also observed that the elevated F/B ratio was accompanied by leukocyte infiltration and morphological changes associated with pathology in the small intestine. Based on our results, we suggest that prebiotic or probiotic supplementation of the gut microbiota may be a viable therapeutic target to treat gut dysbiosis and improve long-term outcomes in stroke patients.

What are the future directions and challenges for stroke microbiome work?

Identifying changes in the abundance of specific gut bacteria taxa is an important first step in understanding how stroke impacts the gut microbiome. One of the next critical steps is to determine how these changes affect the host, or the stroke patient. This can be assessed through the identification of metabolites produced by gut bacteria, such as short-chain fatty acids (SCFA), and how these metabolites impact signaling pathways that alter brain function, behavior and inflammation. Sadler et al. recently showed that supplementation with a SCFA combination of sodium propionate, sodium butyrate and sodium acetate for several weeks prior to stroke improved post-stroke recovery through a mechanism that likely includes the modulation of both systemic and local immune responses [13].

Another method that researchers are utilizing to prove that the microbiome becomes more pathogenic post-stroke is to employ fecal transplants from stroke-injured animals into healthy animals [6]. Taken together, these findings are important because they demonstrate that the altered microbiota profile is both a result of the stroke and a driver of post-stroke systemic inflammation, which ultimately impacts stroke recovery.

In spite of numerous clinical trials conducted over the past 25 years only one drug, tissue plasminogen activator (r-tPA or alteplase), has received approval by the US FDA to treat ischemic stroke. Several anti-inflammatory drugs have shown great promise in preclinical studies, but did not achieve therapeutic efficacy in clinical studies [14]. The findings from our laboratory and others suggest that modulation of the gut microbiome by administration of prebiotics and/or probiotics may be a potential therapeutic strategy to target inflammation. We propose that targeted prebiotic and/or probiotic formulations can be administered to individuals as a strategy to either prevent the occurrence of stroke or to improve post-stroke recovery and quality of life.

Nevertheless, there are multiple issues that must be addressed before any results can be translated into potential therapies. For example, species differences between mice and humans may limit some of the translational impact of preclinical findings. Rapidly evolving improvements in 16S rRNA sequencing technology have allowed investigators to identify taxonomic differences in bacterial abundance at the species and strain levels. Armed with this information, investigators must determine the biological relevance of their finding as it is unlikely that targeting a single bacterium will have a substantial impact within a microbiota community that typically includes thousands of commensal bacteria species. In spite of these challenges, the research in this field has the ability to advance our understanding of the complex and dynamic interrelationship among the gut microbiota, the brain and systemic inflammation in ischemic stroke.

Find out more about this work from the Brown Lab here.

References

[1] Benjamin EJ, Muntner P, Alonso A et al. Heart disease and stroke statistics—2019 update: a report from the American Heart Association. Circulation 139(10), doi:10.1161/CIR.0000000000000659 (2019).

[2] Johnson CO, Nguyen M, Roth GA et al. Global, regional, and national burden of stroke, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Neurol. 18(5), 439–458 (2019).

[3] Anrather J, Iadecola C. Inflammation and stroke: an overview. Neurotherapeutics 13(4), 661–670 (2016).

[4] Stanley D, Moore RJ, Wong CHY. An insight into intestinal mucosal microbiota disruption after stroke. Sci. Rep. 8, 568 (2018).

[5] Singh V, Roth S, Llovera G et al. Microbiota dysbiosis controls the neuroinflammatory response after stroke. J. Neurosci. 36(28), 7428–7440 (2016).

[6] Spychala MS, Venna VR, Jandzinski M et al. Age-related changes in the gut microbiota influence systemic inflammation and stroke outcome. Annals Neurol. 84, 23–36 (2018).

[7] Macrae IM. Preclinical stroke research–advantages and disadvantages of the most common rodent models of focal ischaemia. Br. J. Pharmacol. 164(4), 1062–1078 (2011).

[8] Liu F, McCullough LD. The middle cerebral artery occlusion model of transient focal cerebral ischemia. Methods Mol. Biol.1135, 81–93 (2014).

[9] Spence J. Nutrition and risk of stroke. Nutrients 11(3), 647 (2019).

[10] Duan R, Zhu S, Wang B, Duan L. Alterations of gut microbiota in patients with irritable bowel syndrome based on 16S rRNA-targeted sequencing: a systematic review. Clin. Transl. Gastroenterol. 10(2), e00012 (2019).

[11] Robertson RC, Seira Oriach C, Murphy K et al. Omega-3 polyunsaturated fatty acids critically regulate behaviour and gut microbiota development in adolescence and adulthood. Brain Behav. Immun. 59, 21–37 (2017).

[12] Palmnäs MSA, Cowan TE, Bomhof MR et al. Low-dose aspartame consumption differentially affects gut microbiota–host metabolic interactions in the diet-induced obese rat. PLoS ONE 9(10), e109841 (2014).

[13] Sadler R, Cramer JV, Heindl S et al. Short-chain fatty acids improve poststroke recovery via immunological mechanisms. J. Neurosci. 40(5), 1162–1173 (2020).

[14] Veltkamp R, Gill D. Clinical trials of immunomodulation in ischemic stroke. Neurotherapeutics 13(4), 791–800 (2016).

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