Stroke: A significant unmet clinical need

Stroke presents as the fifth leading cause of death in the United States. Minimal treatment options are at the disposal of acute and chronic stroke patients. Lack of oxygen and nutrients result in primary cell death, however inflammation-induced secondary cell death is responsible for debilitating neurological deficits prevalent in stroke patients. Targeting the inflammation associated with secondary cell death serves as a promising therapeutic mechanism to decrease neurologic deficits post-stroke.

     Initially, the central nervous system (CNS) was believed to solely play an integral role in stroke progression, however accumulating evidence indicates a major role of the peripheral nervous system in stroke-associated secondary cell death. Furthermore, the abnormal increase in inflammation in the gut and brain post-stroke indicates that the gut-brain axis may mediate stroke progression (Venkat et al., 2018; Singh et al., 2018; Arumugam et al., 2017). Microorganisms and bacteria maintain normal gut functions as well as anti-inflammatory microbiomes. Peripheral microbiomes have been identified in the skin, oral cavity, vagina, and the gut (Sharma et al., 2018). Altered metabolism and dysfunction of the immune system often result in gut dysbiosis, an imbalance of microbiota populations, which may influence the brain during the onset and progression of stroke via the gut-brain axis.

     Although the enteric nervous system, or gastrointestinal (GI) tract, independently functions from the CNS, the GI tract can connect to the CNS through digestive activities that involve both parasympathetic and sympathetic controls. The CNS regulates functions of the GI via neural fibers that transmit sensory information. The gut-brain axis has been implicated in the pathology of stroke and other neurological disorders. Interrogating the processes involved in the homeostasis and dysfunctional gut-brain axis may uncover novel insights into the disease and treatment of the disease. Dysregulation in the gut microbiota, resulting from a stroke, may trigger a similar pathological brain microbiome. Conversely, transplanting stem cells to the gut may restore its healthy microbiome, thus protecting the brain from the damaging effects seen from a deviation in the gut microbiota (Figure 1). Investigations designed to unravel gut dysbiosis with a focus on inflammatory microbiota may serve as a fertile ground for deciphering innovative pathways of secondary cell death, as well as novel therapeutic targets for stroke.

Stroke-induced cognitive impairments and inflammation

Approximately 25-30% of patients develop cognitive impairment immediately or after a delay post-stroke (Kalaria et al., 2016). Both cognitive impairment and dementia occur about three months after a stroke episode. Advanced age, family history, genetic variants, vascular comorbidities and having had a previous or recurrent ischemic stroke are the main risk factors for developing dementia. Additional comorbidities such as hypertension, diabetes, obesity, and dyslipidemia may increase the risk of presenting this cognitive deficit after stroke, so it is essential to understand the mechanisms that accompany a stroke injury (Kalaria et al.,2016). Hippocampal injuries and white matter injuries caused by stroke and cerebral microbleeds contribute to the pathophysiology of cognitive decline after a stroke (Lo Coco et al., 2016; Sun et al., 2014).

     Ischemic stroke-induced dementia is accompanied by neuroanatomic lesions. Infarctions in specific areas of the brain, such as the entorhinal cortex and the hippocampus, are key factors mediating cognitive impairment and are associated with the severity of dementia (Tomlinson et al., 1970). White matter lesions are common features of brain parenchymal damage from small cerebrovascular disease (Sun et al., 2014). Stroke survivors exhibit hyperintensity volumes of white matter coincident with the onset of dementia and damage to the blood-brain barrier (BBB) (Kalaria et al., 2016). Furthermore, small vessel disease closely accompanies the pathophysiology of cerebrovascular accidents, suggesting its contribution to cognitive impairment and functional loss, mainly in elderly patients (Lo Coco et al., 2016). Understanding the pathology of stroke, with an emphasis on cognitive behaviors, can help us develop new therapeutic approaches for patients with this pathology.

     Multivariate lesion-mapping can be used on ischemic stroke patients in order to identify strategic regions of the brain for post-stroke cognitive impairment. There are specific neuronal structures, such as the left angular gyrus, left basal ganglia, and the white matter around the left basal ganglia that display pathological alterations associated with cognitive impairment (Zhao et al., 2018). With this information on brain organ-specific behavioral outcomes, comprehensive models are warranted to recapitulate post-stroke cognitive impairment towards a better understanding of the brain pathology and its treatment.

     Mechanistic consequences of stroke-induced cognitive dysfunction may involve cellular damage to specific regions of the brain (Kalaria et al., 2016). Brain insult to the hippocampus causes stroke survivors to be at a higher risk for cognitive impairment (Allan et al., 2011; Kokmen et al., 1996). In tandem, neuroinflammation and immune system dysregulation can lead to cell death, which promotes further detrimental effects on the physiological structure and cognitive functions of the brain (McColl et al., 2007; Meisel and Meisel, 2011; Gold et al., 2011; Swardfager et al., 2013; Brown et al., 2020). Pathophysiological pathways leading to dementia may be influenced by these proinflammatory mechanisms (Kinney et al., 2018). Specifically, microglia and astrocytes exhibit a dampened cytokine response caused by cerebral atrophy in stroke-induced dementia patients (Chen et al., 2016; Li et al., 2020; Wen et al., 2020).

     Primary stroke lesions induce secondary cell death via neuroinflammatory pathways and dysregulation of the immune response, leading to worsened cognitive deficits and physiological damage within the brain. A possible link exists between post-stroke neuroinflammation and the associated cognitive deficits with GI microbiota via bidirectional communication between the gut and the brain. Such gut-brain axis crosstalk manifests in germ-free mice colonized with microbiota acquired from stroke mice. After the recipient mice experienced cortical stroke injury, they displayed greater infarct volumes when compared to the nonrecipient mice. The recipient mice also expressed elevated levels of the inflammatory T-cells Th1 and Th17; these specific T-cells may be involved in the pathogenesis of stroke (Yamashiro et al., 2017). The elevated levels of Th1 and Th17 suggest not only a relationship between stroke-induced neuroinflammation and gut microbiota, but more specifically that altered microbiota may worsen post-stroke inflammation. This gut dysbiosis exacerbates physiological and cognitive dysfunction. In parallel, evaluation of the anti-inflammatory neuroprotective effects of administering antibiotics prior to stroke revealed a therapeutic link between neuroinflammation and microbiota modifications. The mice that received antibiotic treatment prior to stroke injury, demonstrated a 60% decreased infarct volume post-stroke, when compared to the control group (Benakis et al., 2016). Furthermore, the promotion of immune and neuroinflammation modulation was demonstrated by increased levels of regulatory T-cells and decreased levels of cells inhibiting effector T-cells interleukin-17⁺ γδ (Benakis et al., 2016). Approaches that restore GI microbiota to normal levels, while also enhancing anti-inflammatory cytokines, should be further explored due to the possible beneficial effects this would have in preventing cognitive dysfunction related to stroke and other neurological diseases.

Cell therapy-mediated sequestration of inflammation in gut and brain

Therapeutic techniques targeting the gut microbiome show major promise in their abilities to treat the damaging effects of stroke. Essential bacteria within the gut may assist in mitigating neuroinflammation, thus slowing neurodegeneration and the resulting psychological deficits fundamental in stroke patients. Microbiota-based treatments, such as prebiotics and probiotics, have demonstrated positive outcomes in decreasing modifications of the hippocampus and enhancing cognition in mouse models (Romo-Araiza et al., 2018; Abraham et al., 2019; Chen et al., 2017; Zhou et al., 2019; MahmoudianDehkordi et al., 2019). These therapeutic strategies hold great potential in their application to stroke models. Various probiotic regimens, such as Clostridium butyricum, Lactobacillus Plantarum ZDY2013, certain beneficial bacterial mixtures, and bacterial metabolites have all appeared to alleviate the symptoms of stroke-related disorders (Xie et al., 2016; Sun et al., 2016). Lactobacillus is a highly influential type of probiotic bacteria found within the host gut that may enhance cognitive function and mood, as well as to mitigate inflammation associated with aging (Pluta et al., 2021). After a brain infarction in monkeys, there was a relative decrease in the level of Lactobacillus, further suggesting a connection between these beneficial bacterial metabolites in the gut that appears to be associated with brain health (Pluta et al., 2021).

     The overproduction of free radicals and pro-inflammatory cytokines plays a critical role in the advancement of ischemic-related damage post-stroke. Accordingly, the suppression of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), and free radicals may help to attenuate neuronal damage after ischemic injury. A significant reduction in TNF-α and malondialdehyde levels in the brains of stroke animals that received two weeks of probiotic treatment prior to middle cerebral artery occlusion (Akhoundzadeh et al., 2018). These findings demonstrate the antioxidant activity and pro-inflammatory cytokine suppression capabilities of probiotic treatment. For some cases involving acute stroke-caused deficiencies, the GI tract might necessitate a mass alteration or an entire repopulation of gut microbiota through fecal transplantation (Spychala et al., 2018).

     Stem cell-based regeneration has recently become a popular target for stroke treatment. Stem cell transplantation has broadened the range of therapeutic stroke targets from central to peripheral organs (Otero-Ortega et al., 2018; Sun et al., 2018). Peripherally administered stem cells after a neurovascular insult tend to migrate heavily to the spleen (Crowley et al., 2017). This preferential migration of stem cells towards the spleen further advances the concept that stem cells are inflammation-homing biologics that may exert neuroprotection via sequestration of systemic inflammatory response (Crowley et al., 2017). Further research regarding cell-based regeneration may reveal optimal cell-based treatment for stroke and other neurological disorders (Otero-Ortega et al., 2018). Profiling of different microbiomes in the brain and gut may provide novel insights in the pathology of PD (Thomson-Luque et al., 2018; Cheemalapati et al., 2016). The intestinal tract of PD patients is decorated by pro-inflammatory microbiota that stimulate the pathology known as “leaky gut”, a condition characterized by inflammatory mediators and bacteria moving from the gut mucosa into the hosts blood (Castelli et al., 2020). Additionally, the GI tract of PD patients contains a dissimilar gut microbiota when compared to healthy individuals (Castelli et al., 2020). Our studies on PD demonstrate the crucial function of the microbiome as a biomarker and a potential therapeutic target (Lee et al., 2019; Lee et al., 2019). PD treatment and diagnosis primarily focus on the depletion of nigrostriatal dopamine in the brain, however, abnormal GI function precedes the onset of common symptoms seen in the brain of PD patients (Lee et al., 2019; Lee et al., 2019). These findings suggest that the gut microbiome could reveal further insights into PD pathology and its treatment. Our most recent studies revealed upregulation of three specific gut microbes: LAB158, BAC303, and EREC482 in PD animal models. These specific microbes play a pivotal role in gut-to-brain pro-inflammation in response to injury. Moreover, these same microbiota were downregulated after stem cell treatment (Lee et al., 2019; Lee et al., 2019). Based on these findings, gut inflammatory microbiota may serve as biomarkers for neurodegeneration, as well as potent therapeutic indices of cell in PD.

Conclusion

The gut microbiome plays a key role in stroke pathology. Targeting the gut and its inflammatory microbiome may serve as a robust stroke therapeutic. Cognizant that gut dysbiosis closely accompanies stroke provides the impetus to probe the gut and its microbiome in an effort to develop innovative concepts on stroke pathology and its treatment.

Acknowledgments

The authors thank the entire staff of Dr. Borlongan’s Neural Transplant Laboratory for critical discussions of this manuscript. C.V.B. is funded by National Institutes of Health (NIH) R01NS090962, NIH R01NS102395, and NIH R21NS109575.

Declaration of competing interests

The authors declare no competing interests.

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