Getting the guts to expand stroke treatment: The potential for microbiome targeted therapies

Abstract Aims This review focuses on the recent literature regarding the role of the gut‐brain axis (GBA) following ischemic stroke. Discussion Stroke is the 5th leading cause of death and disability in the United States; however, few therapies have been developed to improve prognoses. There is a plethora of evidence suggesting peripheral inflammatory responses play a large role in the pathogenesis of stroke. Additionally, hyperglycemic conditions may play a significant role in worsening stroke outcomes due to microbiome dysbiosis. Conclusion Recent research has illuminated the vital role of the GBA in propagating poor clinical outcomes, such as hemorrhagic transformation, following ischemic stroke. Considering this detrimental consequence of stroke, and the apparent role of the GBA role, future therapeutics should aim to mitigate this peripheral contribution to stroke complications.


| IS IT BUT TERFLIE S OR MI CROB I OTA?
The gut contains enormous immune cell reserve within its Peyer's patches, lamina propria immune cells, intraepithelial lymphocytes, and mesenteric lymph nodes, 14 accounting for more than 70% of the body's immune system. 17 When in need of an immune response, the CNS sends parasympathetic and sympathetic signals to the gut, ultimately regulating gut motility, secretions, permeability, microbiota, and immune cell activity. 18 Following these signals, the gut responds by sending vagal signals and microbiota signals, such as endotoxins, neurotransmitters, short chain fatty acids, indoles, or bile acids. 18 This dynamic communication has been deemed the gut-brain axis (GBA), 19 and has been hypothesized to play a dominant role in many neurological pathologies, such as Alzheimer's disease, general cognitive decline, stroke, emotional dysregulation, and memory. [20][21][22] As a result of stroke-induced sympathetic signaling, decreased microbiota diversity, altered microbiota populations, and inflammatory T-cell infiltration are noted within the gut and correlate with poor stroke outcomes, such as hemorrhagic transformation (HT). [23][24][25][26] HT is a deadly outcome of ischemic stroke possibly related to oxidative stress, inflammation, hyperglycemia, and other blood brain barrier (BBB) weakening factors. [11][12][13]27 The functioning BBB prohibits extracerebral components from entering the CNS, such as immune cells and pro-inflammatory signals. 28,29 After ischemic incidences, however, damage-associated molecular proteins (DAMPs), cytokines, and chemokines related to stroke can escape the CNS, activate systemic inflammation, and recruit peripheral macrophages, neutrophils, and lymphocytes to surpass the weakened BBB. 30 Recently, studies have proposed that the gut is a major contributor to bidirectional inflammatory signaling in stroke. Yet another critical contribution of the gut to stroke pathology includes its role in HT. 31 This review focuses on the microbiota changes observed after stroke, along with the role of glucose in modulating GBA-induced HT.

| G UT-WREN CHING CONS EQUEN CE S OF THE G BA
Understanding the role of the gut microbiota in stroke pathophysiology, accumulating evidence has aimed to determine whether the GBA can influence the deadly stroke consequence of HT. A major hypothesized contributor to altered GBA communications prompting HT involves glucose metabolism. Glucose has been shown to increase bacteria capable of breaking down the guts' mucus barrier, ultimately increasing inflammatory contributions. 32 Thus, HG rat models can aid in analyzing the role of glucose in microbiota makeup and stroke outcomes. 31 This is a novel and innovative idea, especially considering the high risk for stroke in patients with diabetes. 33,34 After undergoing middle cerebral artery occlusion, HG rats show worse outcomes, greater infarct volumes, and greater mortality compared to normoglycemic (NG) rats. 31 Since a large amount of glucose absorption occurs in the gut, facilitated by microbiota, 35 it is logical that HG models offer a great deal of insight to microbiota changes noted in stroke patients.

| Hyperglycemic stroke induces inflammatory microbiome shifts
HG models demonstrate shifts in inflammatory microbiota composition. 31 In total, HG rats show 35 variations (from phylum to genus levels) from NG rats and 30 taxa are notably increased in abundancy compared to control rats. These include a plethora of pro-inflammatory taxons, such as Actinobacteria, Proteobacteria, Verrucomicrobia, Synergistetes, Erysipelotrichaceae, Butyricimonas, and Desulfovibrio. 36 In depth microbiota analysis revels significantly higher Firmicutes/Bacteroidetes (F/B) in NG rats compared to control groups. HG rats show even higher F/B levels, differing significantly from the NG rats. HG rats also show elevations in anaerobic Proteobacteria and Actinobacteria, 31 suggesting that hyperglycemia may promote anaerobic proliferation by shutting down oxygen formation in the gut. 37 Importantly, the phylum Proteobacteria includes Enterobacteriaceae, which have been shown to amplify inflammation after infarction. 25 In a mouse model of stroke, Enterobacteriaceae expansion was related to brain and gut ischemia induced nitrate formation. Systemic inflammation due to microbiota signals were amplified, worsening stroke outcomes. 25 Furthermore, F/B and Actinobacteria are also pro-inflammatory and known to proliferate in stroke patients. 38 This inflammation induced by dysbiosis may contribute to HT after stroke by weakening the BBB.
The GBA communicates largely through inflammatory mediators, which may be novel targets for altering GBA contributions to stroke pathogenesis. 16 Aberrant signals from the gut after stroke utilize BBB disruption to relay information to the brain. MMP-9 contributes to BBB weakening 39 and is higher in rats with HT. 31 Higher MMP-9 correlates with lower SFCA, propanoic acid concentration, Holdemania and Collinsella and correlates with higher Allobaculum, Erysipelotrichi, Erysipelotrichales, and Erysipelotrichaceae. 31 Notably, Holdemania induces anxiety, neuroinflammation, and negatively impacts metabolism. 40,41 In addition to contributing to BBB permeability, inflammatory cytokines TNFα, IL-17, and IL-1β are higher in HG and NG rats compared to controls. These cytokines are also significantly more elevated in HG rats compared to NG rats. IL-10, an anti-inflammatory cytokine is lower in HG and NG rats compared to controls. This inflammatory phenotype is also related to microbiota composition after stroke in murine models, with c_Erysipelotrichi, o_Erysipelotrichales, and f_Erysipelotrichaceae positively correlated to high levels of inflammatory cytokines. Contrarily, g_Holdemania, g_Collinsella, f_Lachnospiraceae, and g_Blautia negatively correlate to increases in inflammatory cytokines. Elevated g_Escherichia and p_proteobacteria directly relate to increased TNFα levels in stroke models. Regarding SFCAs, lower acetic acid and propionic acid are associated with higher IL-17 levels. In addition to altered inflammatory signaling, microbiota composition shifts also result in abnormal metabolic pathways, with HG groups showing greater carbohydrate metabolism, energy production, and energy conversion compared to NG. 31 There is an apparent relationship between the microbiota composition, inflammation, and BBB permeability, solidifying the role of the GBA in stroke pathology (Figure 1).

| Altered mucosal membranes in the setting of hyperglycemic stroke
HG may also dampen mucosal barriers by increasing proliferation of mucolytic bacteria. Mucolytic bacteria, such as Prevotella, Akkermansia, Streptococcus, and Helicobacter are increased in HG rats, suggesting a mechanism for GBA dysregulation. 31,32,36,42 Glucose has been shown to weaken the guts' mucosal barrier, 32 and permeable gut barriers can be seen in NG rat models of stroke as well. 36 Thus, a combination of HG and stroke may exacerbate the inflammatory GBA communication via increased barrier permeability and facilitated communication. Freed microbiota can enter the body and cause several detrimental consequences, which worsen stroke outcome, such as endotoxemia and infection. 36,43 Subsequent immune reactions may further amplify the body's inflammatory responses to stroke, weakening the BBB further, and increasing the risk of HT and poor prognosis.

F I G U R E 1 This figure exemplifies
the impact of stroke on altering the gut microbiome, which ultimately impacts the CNS and stroke pathology. Potential treatment with stem cells may mitigate these deleterious effects

| Sugars or fats: hyperglycemic stroke alters SCFAs
While the microbiome is significantly different among HG rats, the SCFA makeup may also impact stroke outcomes. 44 Of note, utyric acid, propanoic acid, isobutyric acid, isovaleric acid, valeric acid, and hexanoic acid are decreased in HG rats compared to controls. 31 Valeric acid levels are decreased in HG rat feces compared to NG rat feces. Ultimately, HG rat feces have lower SCFA levels compared to control and NG rat feces, despite increased Ruminococcaceae and Lachnospiraceae in HG rats, both of which largely contribute to SCFA levels. 31 SCFA have been shown to improve stroke outcomes, as some SCFAs can induce neurogenesis and decrease inflammation. 45,46 Administration of SCFAs in mice after stroke alter microglial activation to enhance structural and functional remodeling. 45 Contrarily, reduction of SCFAs, such as butyrate, may detriment stroke outcomes, as shown in HG rats. 31 Butyrate is a key SCFA in energizing intestinal epithelial cells, regulating immune reactions, and modulating metabolic processes. 47,48 Lack of butyrate producing bacteria correlates with increased stroke risk, type II diabetes, obesity, and cardiovascular disease. [49][50][51][52] Lower butyrate producing bacteria are observed in stroke patients and correlate to higher infection rates following stroke. 44 Thus, replacing SFCA directly or via microbiome renewal may mitigate stroke damage. The wide range of microbiome changes suggests novel therapeutic targets within the GBA for stroke.

| MANAG ING THE MICROB IOME
Given the significant role of the GBA in stroke pathogenesis and prognosis, therapies should target this injurious dysbiosis. As noted, Enterobacteriaceae contribute to ischemic complications through LPS reactions with macrophages, ultimately inducing free radical formation. 25 To ameliorate this effect, aminoguanidine, superoxide dismutase, and tungstate treatments can be employed to lessen nitrate formation. More directly, Enterobacteriaceae concentrations can be reduced. 25 More drastic approaches to normalizing the microbiome involve recolonizing the gut to alter immune responses to stroke. In a germ-free rat model, fecal samples were taken from HG rats, which underwent HT. Microbiota transplantations from these samples were given to germ-free mice. MCAO was conducted 2 weeks later. Germ-free rats show improved infarct volumes, rates of HT and hemoglobin levels, inflammatory cytokine and MMP concentrations, and functionality compared to control rats. 31 Other results support these findings, showing that fecal transplants are beneficial in adapting a healthy microbiome following stroke. 24 In a similar model of germ-free mice undergoing stroke, amoxicillin can increase anti-inflammatory regulatory T-cell populations while decreasing pro-inflammatory Th-17 infiltration in the gut. Amoxicillin treatment demonstrates reduced infarct volumes and functional deficits. 53 Other methods targeting Treg differentiation from microbiome activation may be beneficial as well, such as IL-2/IL-2R antibody complexes. 54 Normalizing the microbiome may be a novel direction for research initiatives in regulating GBA communications following stroke.
Other potential therapeutic targets may involve microbiome products, such as TMAO or SFCA. Elevated TMAO is related to atherosclerosis by prompting foam cell formation, however, following stroke, levels of TMAO are decreased alongside microbiota shifts. 38,55,56 It is questionable whether down or up regulation of TMAO would be the most beneficial for stroke therapeutics. 38 Butyrate, previously discussed due to its neuroprotective properties, also demonstrates reduced infarct volumes and improved sensory motor function in MCAO rat models. 57 These effects may be due to the direct replenishment of butyrate, which is decreased after stroke. 44 As noted previously, SFCA administration after stroke in mice can improve motor function and enhanced neural connectivity via altered inflammatory signaling. 45,46 Restoring homeostatic levels of TMAO and SCFA can ameliorate harmful microbiome induced side effects, and potentially lessen aberrant inflammatory signals via the GBA.
Interventions targeting GBA permeability and communication capabilities may be beneficial, for example, targeting cytokines or MMPs. 58,59 Human bone marrow-derived NCS-01 cells offer therapeutic effects for ischemic stroke, likely by secreting fibroblast growth factor and IL-6. IL-6 is unique in its double-edged contributions to inflammation, acting as a pro-and anti-inflammatory cytokines. 60 While stem cell studies have shown increased IL-6 production mitigate stroke damage, 59 pharmaceutical interventions show the opposite. 61 While direct intervention in cytokine processing via stem cells may be beneficial in targeting the GBA, further research is needed to determine whether IL-6 acts as a pro-or anti-inflammatory mediator in GBA signaling. Combination therapy including Puerariae Lobatae Radix (PLR) and Chuanxiong Rhizoma (CXR), traditional Chinese medicines, have also been shown to lessen stroke damage by relieving dysbiosis and protecting the brain-gut mucosal barriers and preventing detrimental GBA communications. 36,62,63 Given the recent interest in the GBA and its role in stroke outcomes, 64 further research is needed to establish treatments targeting this bimodal inflammatory signaling and resultant dysbiosis.
Stem cells may also be a novel intervention to intercede the GBA signaling. Stem cells can directly impact stroke outcomes when targeting neural inflammation 11,65 ; however, there is also some evidence suggesting stem cells exert a bystander effect by ameliorating peripheral inflammation. 66 In fact, bone marrow mesenchymal cells can modulate dysbiosis, while also reducing neuronal death and functional deficits following stroke. 67 Furthermore, in Parkinson's disease, stem cells have been visualized traveling to the gut to lessen CNS and gut inflammation. 68 Thus, while not strongly established in the literature, future studies may discover a grand role for stem cells in improving stroke prognosis via interaction with the GBA.
Avoiding medical interventions, simple lifestyle adjustments may also be ideal microbiome normalizers. Ketogenic diets and vegetarian diets show reduced stroke damage and lessen dysbiosis, ultimately improving prognosis. 69,70 Dietary supplements, such as pro-or prebiotics can also reduce stroke severity and lessen the risk of further cardiovascular health crises. 70 Moreover, regular exercise diminishes stroke-related microbiome dysregulation, serving as a protective and low maintenance lifestyle adjustment. 71 Aerobic exercise paired with intermittent fasting can further improve microbiota composition, as measured by organic acid concentrations in feces. 72 In obese rats, intermittent fasting alone also ameliorates gut dysbiosis and lowers blood high-density lipoprotein and lowdensity lipoprotein. Considering the correlations between obesity and stroke, 73 intermittent fasting may be another intriguing stroke therapeutic targeting the microbiome. 74 Acupuncture has also demonstrated anti-inflammatory powers in neuroinflammatory and inflammatory gut diseases, such as Parkinson's disease and irritable bowel syndrome, by modulating vagal signals to the immune system and subsequent modulation of the GBA. 40,75,76 Thus, acupuncture may represent a more natural treatment consideration for stroke. 31 While there are a few potential pharmaceuticals or novel cell-based therapies for modulating GBA signaling, lifestyle adjustments may be the safest and most feasible approach for improving stroke prognosis by targeting the gut's contributions to peripheral inflammation.

| FOLLOW YOUR HE AD, HE ART… AND G UT
These studies gracefully demonstrate the profound role of the microbiome in modulating stroke pathogenesis. Researchers should aim to target stroke-induced dysbiosis to mitigate peripheral inflammation and, ultimately, improve stroke outcomes. Importantly, the role of HG in the GBA following stroke remains poorly understood but is highly clinically relevant due to the relationship between stroke, diet, and microbiome dysbiosis in HG stroke models. 31 Considering the brain's protective BBB, the gut may be a more feasible target for stroke therapeutics. Furthermore, the established communicative signaling between these two systems suggests promising discoveries will arise from further analysis of the GBA in stroke.

ACK N OWLED G M ENTS
We acknowledge the work on the artists, macrovector, brgfx, and starline, who designed and illustrated the vector images used for the figure. These vectors were downloaded from freep ik.com.

CO N FLI C T O F I NTE R E S T
C.V.B. was funded by the National Institutes of Health (NIH) R01NS090962, NIH R01NS102395, and NIH R21NS109575.
Additionally, C.V.B. was funded and received royalties and stock options from Astellas, Asterias, Sanbio, Athersys, KMPHC, and International Stem Cell Corporation and has also received consultant compensation from Chiesi Farmaceutici. C.V.B. also declares patents and patent applications related to stem cell therapy.

DATA AVA I L A B I L I T Y S TAT E M E N T
No data availability statement is applicable.