Exposure to Environmental Toxins: Potential Implications for Stroke Risk via the Gut- and Lung-Brain Axis.

Recent evidence indicates that exposure to environmental toxins, both short-term and long-term, can increase the risk of developing neurological disorders, including neurodegenerative diseases (i.e., Alzheimer's disease and other dementias) and acute brain injury (i.e., stroke). For stroke, the latest systematic analysis revealed that exposure to ambient particulate matter is the second most frequent stroke risk after high blood pressure. However, preclinical and clinical stroke investigations on the deleterious consequences of environmental pollutants are scarce. This review examines recent evidence of how environmental toxins, absorbed along the digestive tract or inhaled through the lungs, affect the host cellular response. We particularly address the consequences of environmental toxins on the immune response and the microbiome at the gut and lung barrier sites. Additionally, this review highlights findings showing the potential contribution of environmental toxins to an increased risk of stroke. A better understanding of the biological mechanisms underlying exposure to environmental toxins has the potential to mitigate stroke risk and other neurological disorders.

Post-ischemic ubiquitination at the postsynaptic density reversibly influences the activity of ischemia-relevant kinases.

Ubiquitin modifications alter protein function and stability, thereby regulating cell homeostasis and viability, particularly under stress. Ischemic stroke induces protein ubiquitination at the ischemic periphery, wherein cells remain viable, however the identity of ubiquitinated proteins is unknown. Here, we employed a proteomics approach to identify these proteins in mice undergoing ischemic stroke. The data are available in a searchable web interface ( https://hochrainerlab.shinyapps.io/StrokeUbiOmics/ ). We detected increased ubiquitination of 198 proteins, many of which localize to the postsynaptic density (PSD) of glutamatergic neurons. Among these were proteins essential for maintaining PSD architecture, such as PSD95, as well as NMDA and AMPA receptor subunits. The largest enzymatic group at the PSD with elevated post-ischemic ubiquitination were kinases, such as CaMKII, PKC, Cdk5, and Pyk2, whose aberrant activities are well-known to contribute to post-ischemic neuronal death. Concurrent phospho-proteomics revealed altered PSD-associated phosphorylation patterns, indicative of modified kinase activities following stroke. PSD-located CaMKII, PKC, and Cdk5 activities were decreased while Pyk2 activity was increased after stroke. Removal of ubiquitin restored kinase activities to pre-stroke levels, identifying ubiquitination as the responsible molecular mechanism for post-ischemic kinase regulation. These findings unveil a previously unrecognized role of ubiquitination in the regulation of essential kinases involved in ischemic injury.

Post-ischemic ubiquitination at the postsynaptic density reversibly influences the activity of ischemia-relevant kinases.

Ubiquitin modifications alter protein function and stability, thereby regulating cell homeostasis and viability, particularly under stress. Ischemic stroke induces protein ubiquitination at the ischemic periphery, wherein cells remain viable, however the identity of ubiquitinated proteins is unknown. Here, we employed a proteomics approach to identify these proteins in mice undergoing ischemic stroke. The data are available in a searchable web interface ( https://hochrainerlab.shinyapps.io/StrokeUbiOmics/ ). We detected increased ubiquitination of 198 proteins, many of which localize to the postsynaptic density (PSD) of glutamatergic neurons. Among these were proteins essential for maintaining PSD architecture, such as PSD95, as well as NMDA and AMPA receptor subunits. The largest enzymatic group at the PSD with elevated post-ischemic ubiquitination were kinases, such as CaMKII, PKC, Cdk5, and Pyk2, whose aberrant activities are well-known to contribute to post-ischemic neuronal death. Concurrent phospho-proteomics revealed altered PSD-associated phosphorylation patterns, indicative of modified kinase activities following stroke. PSD-located CaMKII, PKC, and Cdk5 activities were decreased while Pyk2 activity was increased after stroke. Removal of ubiquitin restored kinase activities to pre-stroke levels, identifying ubiquitination as the responsible molecular mechanism for post-ischemic kinase regulation. These findings unveil a previously unrecognized role of ubiquitination in the regulation of essential kinases involved in ischemic injury.

Protocol for microbiota analysis of a murine stroke model.

Investigations on the microbiota in neurological diseases such as stroke are increasingly common; however, stroke researchers may have limited experience with designing such studies. Here, we describe a protocol to conduct a stroke microbiota study in mice, from experimental stroke surgery and sample collection to data analysis. We provide details on sample processing and sequencing and provide a reproducible data analysis pipeline. In doing so, we hope to enable researchers to conduct robust studies and facilitate identification of stroke-associated microbial signatures. For complete details on the use and execution of this protocol, please refer to Sorbie et al. (2022).1.

T cells modulate the microglial response to brain ischemia.

Neuroinflammation after stroke is characterized by the activation of resident microglia and the invasion of circulating leukocytes into the brain. Although lymphocytes infiltrate the brain in small number, they have been consistently demonstrated to be the most potent leukocyte subpopulation contributing to secondary inflammatory brain injury. However, the exact mechanism of how this minimal number of lymphocytes can profoundly affect stroke outcome is still largely elusive. Here, using a mouse model for ischemic stroke, we demonstrated that early activation of microglia in response to stroke is differentially regulated by distinct T cell subpopulations - with TH1 cells inducing a type I INF signaling in microglia and regulatory T cells (TREG) cells promoting microglial genes associated with chemotaxis. Acute treatment with engineered T cells overexpressing IL-10 administered into the cisterna magna after stroke induces a switch of microglial gene expression to a profile associated with pro-regenerative functions. Whereas microglia polarization by T cell subsets did not affect the acute development of the infarct volume, these findings substantiate the role of T cells in stroke by polarizing the microglial phenotype. Targeting T cell-microglia interactions can have direct translational relevance for further development of immune-targeted therapies for stroke and other neuroinflammatory conditions.

The gut-brain axis in ischemic stroke: its relevance in pathology and as a therapeutic target.

The gut contains the largest reservoir of microorganisms of the human body, termed as the gut microbiota which emerges as a key pathophysiological factor in health and disease. The gut microbiota has been demonstrated to influence various brain functions along the "gut-brain axis". Stroke leads to intestinal dysmotility and leakiness of the intestinal barrier which are associated with change of the gut microbiota composition and its interaction with the human host. Growing evidence over the past decade has demonstrated an important role of these post-stroke changes along the gut-brain axis to contribute to stroke pathology and be potentially druggable targets for future therapies. The impact of the gut microbiota on brain health and repair after stroke might be attributed to the diverse functions of gut bacteria in producing neuroactive compounds, modulating the host's metabolism and immune status. Therefore, a better understanding on the gut-brain axis after stroke and its integration in a broader concept of stroke pathology could open up new avenues for stroke therapy. Here, we discuss current concepts from preclinical models and human studies on the bi-directional communication along the microbiota-gut-brain axis in stroke.

Increasing transparency and reproducibility in stroke-microbiota research: A toolbox for microbiota analysis.

Homeostasis of gut microbiota is crucial in maintaining human health. Alterations, or "dysbiosis," are increasingly implicated in human diseases, such as cancer, inflammatory bowel diseases, and, more recently, neurological disorders. In ischemic stroke patients, gut microbial profiles are markedly different compared to healthy controls, whereas manipulation of microbiota in animal models of stroke modulates outcome, further implicating microbiota in stroke pathobiology. Despite this, evidence for the involvement of specific microbes or microbial products and microbial signatures have yet to be identified, likely owing to differences in methodology, data analysis, and confounding variables between different studies. Here, we provide a set of guidelines to enable researchers to conduct high-quality, reproducible, and transparent microbiota studies, focusing on 16S rRNA sequencing in the emerging subfield of the stroke-microbiota. In doing so, we aim to facilitate novel and reproducible associations between the microbiota and brain diseases, including stroke, and translation into clinical practice.

Stroke affects intestinal immune cell trafficking to the central nervous system.

Stroke is an acute neurological disease with a strong inflammatory component that can be regulated by the intestinal microbiota and intestinal immune cells. Although stroke has been shown to alter immune cell populations in the gut, the dynamics of cell trafficking have not been elucidated. To study the trafficking of gut-derived immune cells after stroke, we used mice expressing the photoconvertible protein Kikume Green-Red, which turns form green to red when exposed to violet light. Mice underwent laparotomy and the small intestine was exposed to violet laser light. Immune cells were isolated from the small intestine immediately after photoconversion and 2 days later. Percentage of immune cells (CD45+KikR+) that expressed the red variant of the protein (KikR) was higher immediately after photoconversion than 2 days later, indicating cell egress from the small intestine. To investigate whether intestinal immune cells traffic to the periphery and/or the central nervous system (CNS) after stroke, we analyzed KikR+ immune cells (2 days after photoconversion) in peripheral lymphoid organs, meninges and brain, 3 and 14 days after transient occlusion of the middle cerebral artery (tMCAo) or sham-surgery. Although migration was observed in naïve and sham animals, stroke induced a higher mobilization of gut KikR+ immune cells, especially at 3 days after stroke, to all the organs analyzed. Notably, we detected a significant migration of CD45hi immune cells from the gut to the brain and meninges at 3 days after stroke. Comparison of cell trafficking between organs revealed a significant preference of intestinal CD11c+ cells to migrate from the small intestine to brain and meninges after stroke. We conclude that stroke increases immune cell trafficking from the small intestine to peripheral lymphoid organs and the CNS where they might contribute to post-stroke inflammation.

Microbiota-derived short chain fatty acids modulate microglia and promote Aß plaque deposition.

Previous studies have identified a crucial role of the gut microbiome in modifying Alzheimer's disease (AD) progression. However, the mechanisms of microbiome-brain interaction in AD were so far unknown. Here, we identify microbiota-derived short chain fatty acids (SCFA) as microbial metabolites which promote Aß deposition. Germ-free (GF) AD mice exhibit a substantially reduced Aß plaque load and markedly reduced SCFA plasma concentrations; conversely, SCFA supplementation to GF AD mice increased the Aß plaque load to levels of conventionally colonized (specific pathogen-free [SPF]) animals and SCFA supplementation to SPF mice even further exacerbated plaque load. This was accompanied by the pronounced alterations in microglial transcriptomic profile, including upregulation of ApoE. Despite increased microglial recruitment to Aß plaques upon SCFA supplementation, microglia contained less intracellular Aß. Taken together, our results demonstrate that microbiota-derived SCFA are critical mediators along the gut-brain axis which promote Aß deposition likely via modulation of the microglial phenotype.

The Gut Ecosystem: A Critical Player in Stroke.

The intestinal microbiome is emerging as a critical factor in health and disease. The microbes, although spatially restricted to the gut, are communicating and modulating the function of distant organs such as the brain. Stroke and other neurological disorders are associated with a disrupted microbiota. In turn, stroke-induced dysbiosis has a major impact on the disease outcome by modulating the immune response. In this review, we present current knowledge on the role of the gut microbiome in stroke, one of the most devastating brain disorders worldwide with very limited therapeutic options, and we discuss novel insights into the gut-immune-brain axis after an ischemic insult. Understanding the nature of the gut bacteria-brain crosstalk may lead to microbiome-based therapeutic approaches that can improve patient recovery.

Distinct Commensal Bacterial Signature in the Gut Is Associated With Acute and Long-Term Protection From Ischemic Stroke.

Background and Purpose- Commensal gut bacteria have a profound impact on stroke pathophysiology. Here, we investigated whether modification of the microbiota influences acute and long-term outcome in mice subjected to stroke. Methods- C57BL/6 male mice received a cocktail of antibiotics or single antibiotic. After 4 weeks, fecal bacterial density of the 16S rRNA gene was quantitated by qPCR, and phylogenetic classification was obtained by 16S rRNA gene sequencing. Infarct volume and hemispheric volume loss were measured 3 days and 5 weeks after middle cerebral artery occlusion, respectively. Neurological deficits were tested by the Tape Test and the open field test. Results- Mice treated with a cocktail of antibiotics displayed a significant reduction of the infarct volume in the acute phase of stroke. The neuroprotective effect was abolished in mice recolonized with a wild-type microbiota. Single antibiotic treatment with either ampicillin or vancomycin, but not neomycin, was sufficient to reduce the infarct volume and improved motorsensory function 3 days after stroke. This neuroprotective effect was correlated with a specific microbial population rather than the total bacterial density. In particular, random forest analysis trained for the severity of the brain damage revealed that Bacteroidetes S24.7 and the enzymatic pathway for aromatic metabolism discriminate between large versus small infarct size. Additionally, the microbiota signature in the ampicillin-treated mice was associated with a reduced gut inflammation, long-term favorable outcome shown by an amelioration of the stereotypic behavior, and a reduction of brain tissue loss in comparison to control and was predictive of a regulation of short-chain fatty acids and tryptophan pathways. Conclusions- The findings highlight the importance of the intestinal microbiota in short- and long-term outcomes of ischemic stroke and raises the possibility that targeted modification of the microbiome associated with specific microbial enzymatic pathways may provide a preventive strategy in patients at high risk for stroke. Visual Overview- An online visual overview is available for this article.

Short-Chain Fatty Acids Improve Poststroke Recovery via Immunological Mechanisms.

Recovery after stroke is a multicellular process encompassing neurons, resident immune cells, and brain-invading cells. Stroke alters the gut microbiome, which in turn has considerable impact on stroke outcome. However, the mechanisms underlying gut-brain interaction and implications for long-term recovery are largely elusive. Here, we tested the hypothesis that short-chain fatty acids (SCFAs), key bioactive microbial metabolites, are the missing link along the gut-brain axis and might be able to modulate recovery after experimental stroke. SCFA supplementation in the drinking water of male mice significantly improved recovery of affected limb motor function. Using in vivo wide-field calcium imaging, we observed that SCFAs induced altered contralesional cortex connectivity. This was associated with SCFA-dependent changes in spine and synapse densities. RNA sequencing of the forebrain cortex indicated a potential involvement of microglial cells in contributing to the structural and functional remodeling. Further analyses confirmed a substantial impact of SCFAs on microglial activation, which depended on the recruitment of T cells to the infarcted brain. Our findings identified that microbiota-derived SCFAs modulate poststroke recovery via effects on systemic and brain resident immune cells.SIGNIFICANCE STATEMENT Previous studies have shown a bidirectional communication along the gut-brain axis after stroke. Stroke alters the gut microbiota composition, and in turn, microbiota dysbiosis has a substantial impact on stroke outcome by modulating the immune response. However, until now, the mediators derived from the gut microbiome affecting the gut-immune-brain axis and the molecular mechanisms involved in this process were unknown. Here, we demonstrate that short-chain fatty acids, fermentation products of the gut microbiome, are potent and proregenerative modulators of poststroke neuronal plasticity at various structural levels. We identified that this effect was mediated via circulating lymphocytes on microglial activation. These results identify short-chain fatty acids as a missing link along the gut-brain axis and as a potential therapeutic to improve recovery after stroke.

The microbiome-gut-brain axis in acute and chronic brain diseases.

The gut microbiome - the largest reservoir of microorganisms of the human body - is emerging as an important player in neurodevelopment and ageing as well as in brain diseases including stroke, Alzheimer's disease and Parkinson's disease. The growing knowledge on mediators and triggered pathways has advanced our understanding of the interactions along the gut-brain axis. Gut bacteria produce neuroactive compounds and can modulate neuronal function, plasticity and behavior. Furthermore, intestinal microorganisms impact the host's metabolism and immune status which in turn affect neuronal pathways in the enteric and central nervous systems. Here, we discuss the recent insights from human studies and animal models on the bi-directional communication along the microbiome-gut-brain axis in both acute and chronic brain diseases.

AGO CLIP Reveals an Activated Network for Acute Regulation of Brain Glutamate Homeostasis in Ischemic Stroke.

Post-transcriptional regulation by microRNAs (miRNAs) is essential for complex molecular responses to physiological insult and disease. Although many disease-associated miRNAs are known, their global targets and culminating network effects on pathophysiology remain poorly understood. We applied Argonaute (AGO) crosslinking immunoprecipitation (CLIP) to systematically elucidate altered miRNA-target interactions in brain following ischemia and reperfusion (I/R) injury. Among 1,190 interactions identified, the most prominent was the cumulative loss of target regulation by miR-29 family members. Integration of translational and time-course RNA profiles revealed a dynamic mode of miR-29 target de-regulation, led by acute translational activation and a later increase in RNA levels, allowing rapid proteomic changes to take effect. These functional regulatory events rely on canonical and non-canonical miR-29 binding and engage glutamate reuptake signals, such as glial glutamate transporter (GLT-1), to control local glutamate levels. These results uncover a miRNA target network that acts acutely to maintain brain homeostasis after ischemic stroke.

Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections.

Analysis of entire transparent rodent bodies after clearing could provide holistic biological information in health and disease, but reliable imaging and quantification of fluorescent protein signals deep inside the tissues has remained a challenge. Here, we developed vDISCO, a pressure-driven, nanobody-based whole-body immunolabeling technology to enhance the signal of fluorescent proteins by up to two orders of magnitude. This allowed us to image and quantify subcellular details through bones, skin and highly autofluorescent tissues of intact transparent mice. For the first time, we visualized whole-body neuronal projections in adult mice. We assessed CNS trauma effects in the whole body and found degeneration of peripheral nerve terminals in the torso. Furthermore, vDISCO revealed short vascular connections between skull marrow and brain meninges, which were filled with immune cells upon stroke. Thus, our new approach enables unbiased comprehensive studies of the interactions between the nervous system and the rest of the body.

T cells in the post-ischemic brain: Troopers or paramedics?

The immune system is intricately involved in brain development and physiological neuronal function. The influence of the adaptive immune system on several brain diseases has been described in great detail. In ischemic stroke, numerous studies have particularly demonstrated a key role for T cells during the acute phase after the brain injury. Recently, a critical role for T cells has also become more evident for the chronic phase after stroke in modulating delayed neuronal (dys-) function and recovery. Here, T cells may also affect various non-immunological pathways by interacting with brain-resident immune cells and modulating mechanisms such as neurogenesis and angiogenesis. This novel concept suggests T cells as potential therapeutic targets to modulate post-stroke regeneration.

The meningeal and choroidal infiltration routes for leukocytes in stroke.

Stroke is a major health burden as it is a leading cause of morbidity and mortality worldwide. Blood flow restoration, through thrombolysis or endovascular thrombectomy, is the only effective treatment but is restricted to a limited proportion of patients due to time window constraint and accessibility to technology. Over the past two decades, research has investigated the basic mechanisms that lead to neuronal death following cerebral ischemia. However, the use of neuroprotective paradigms in stroke has been marked by failure in translation from experimental research to clinical practice. In the past few years, much attention has focused on the immune response to acute cerebral ischemia as a major factor to the development of brain lesions and neurological deficits. Key inflammatory processes after stroke include the activation of resident glial cells as well as the invasion of circulating leukocytes. Recent research on anti-inflammatory strategies for stroke has focused on limiting the transendothelial migration of peripheral immune cells from the compromised vasculature into the brain parenchyma. However, recent trials testing the blockage of cerebral leukocyte infiltration in patients reported inconsistent results. This emphasizes the need to better scrutinize how immune cells are regulated at the blood-brain interface and enter the brain parenchyma, and particularly to also consider alternative cerebral infiltration routes for leukocytes, including the meninges and the choroid plexus. Understanding how immune cells migrate to the brain via these alternative pathways has the potential to develop more effective approaches for anti-inflammatory stroke therapies.

Endogenous Protection from Ischemic Brain Injury by Preconditioned Monocytes.

Exposure to low-dose lipopolysaccharide (LPS) before cerebral ischemia is neuroprotective in stroke models, a phenomenon termed preconditioning (PC). Although it is well established that LPS-PC induces central and peripheral immune responses, the cellular mechanisms modulating ischemic injury remain unclear. Here, we investigated the role of immune cells in the brain protection afforded by PC and tested whether monocytes may be reprogrammed by ex vivo LPS exposure, thus modulating inflammatory injury after cerebral ischemia in male mice. We found that systemic injection of low-dose LPS induces a Ly6Chi monocyte response that protects the brain after transient middle cerebral artery occlusion (MCAO) in mice. Remarkably, adoptive transfer of monocytes isolated from preconditioned mice into naive mice 7 h after transient MCAO reduced brain injury. Gene expression and functional studies showed that IL-10, inducible nitric oxide synthase, and CCR2 in monocytes are essential for neuroprotection. This protective activity was elicited even if mouse or human monocytes were exposed ex vivo to LPS and then injected into male mice after stroke. Cell-tracking studies showed that protective monocytes are mobilized from the spleen and reach the brain and meninges, where they suppress postischemic inflammation and neutrophil influx into the brain parenchyma. Our findings unveil a previously unrecognized subpopulation of splenic monocytes capable of protecting the brain with an extended therapeutic window and provide the rationale for cell therapies based on the delivery of autologous or allogeneic protective monocytes in patients after ischemic stroke.SIGNIFICANCE STATEMENT Inflammation is a key component of the pathophysiology of the brain in stroke, a leading cause of death and disability with limited therapeutic options. Here, we investigate endogenous mechanisms of protection against cerebral ischemia. Using lipopolysaccharide (LPS) preconditioning (PC) as an approach to induce ischemic tolerance in mice, we found generation of neuroprotective monocytes within the spleen, from which they traffic to the brain and meninges, suppressing postischemic inflammation. Importantly, systemic LPS-PC can be mimicked by adoptive transfer of in vitro-preconditioned mouse or human monocytes at translational relevant time points after stroke. This model of neuroprotection may facilitate clinical efforts to increase the efficacy of BM mononuclear cell treatments in acute neurological diseases such as cerebral ischemia.

Automated Morphological Analysis of Microglia After Stroke.

Microglia are the resident immune cells of the brain and react quickly to changes in their environment with transcriptional regulation and morphological changes. Brain tissue injury such as ischemic stroke induces a local inflammatory response encompassing microglial activation. The change in activation status of a microglia is reflected in its gradual morphological transformation from a highly ramified into a less ramified or amoeboid cell shape. For this reason, the morphological changes of microglia are widely utilized to quantify microglial activation and studying their involvement in virtually all brain diseases. However, the currently available methods, which are mainly based on manual rating of immunofluorescent microscopic images, are often inaccurate, rater biased, and highly time consuming. To address these issues, we created a fully automated image analysis tool, which enables the analysis of microglia morphology from a confocal Z-stack and providing up to 59 morphological features. We developed the algorithm on an exploratory dataset of microglial cells from a stroke mouse model and validated the findings on an independent data set. In both datasets, we could demonstrate the ability of the algorithm to sensitively discriminate between the microglia morphology in the peri-infarct and the contralateral, unaffected cortex. Dimensionality reduction by principal component analysis allowed to generate a highly sensitive compound score for microglial shape analysis. Finally, we tested for concordance of results between the novel automated analysis tool and the conventional manual analysis and found a high degree of correlation. In conclusion, our novel method for the fully automatized analysis of microglia morphology shows excellent accuracy and time efficacy compared to traditional analysis methods. This tool, which we make openly available, could find application to study microglia morphology using fluorescence imaging in a wide range of brain disease models.

The gut microbiome primes a cerebroprotective immune response after stroke.

Microbiome alterations have been shown to affect stroke outcome. However, to what extent the presence of a gut microbiome per se is affecting post-stroke neuroinflammation has not been tested. By comparing germfree mice with recolonized (Ex-GF) and conventional SPF mice, we were able to demonstrate that bacterial colonization reduces stroke volumes. Bacterial colonization increased cerebral expression of cytokines as well as microglia/macrophage cell counts in contrast to improved stroke outcome. Interestingly, the microbiome-mediated brain protection was absent in lymphocyte-deficient mice. These findings support the concept of lymphocyte-driven protective neuroinflammation after stroke under control of the microbiome.