Aging Microbiota-Gut-Brain Axis in Stroke Risk and Outcome.

The microbiota-gut-brain-axis (MGBA) is a bidirectional communication network between gut microbes and their host. Many environmental and host-related factors affect the gut microbiota. Dysbiosis is defined as compositional and functional alterations of the gut microbiota that contribute to the pathogenesis, progression and treatment responses to disease. Dysbiosis occurs when perturbations of microbiota composition and function exceed the ability of microbiota and its host to restore a symbiotic state. Dysbiosis leads to dysfunctional signaling of the MGBA, which regulates the development and the function of the host's immune, metabolic, and nervous systems. Dysbiosis-induced dysfunction of the MGBA is seen with aging and stroke, and is linked to the development of common stroke risk factors such as obesity, diabetes, and atherosclerosis. Changes in the gut microbiota are also seen in response to stroke, and may impair recovery after injury. This review will begin with an overview of the tools used to study the MGBA with a discussion on limitations and potential experimental confounders. Relevant MGBA components are introduced and summarized for a better understanding of age-related changes in MGBA signaling and its dysfunction after stroke. We will then focus on the relationship between the MGBA and aging, highlighting that all components of the MGBA undergo age-related alterations that can be influenced by or even driven by the gut microbiota. In the final section, the current clinical and preclinical evidence for the role of MGBA signaling in the development of stroke risk factors such as obesity, diabetes, hypertension, and frailty are summarized, as well as microbiota changes with stroke in experimental and clinical populations. We conclude by describing the current understanding of microbiota-based therapies for stroke including the use of pre-/pro-biotics and supplementations with bacterial metabolites. Ongoing progress in this new frontier of biomedical sciences will lead to an improved understanding of the MGBA's impact on human health and disease.

Restructuring the Gut Microbiota by Intermittent Fasting Lowers Blood Pressure.

[Figure: see text].

The gut microbiome contributes to blood-brain barrier disruption in spontaneously hypertensive stroke prone rats.

In recent years, it has become apparent that the gut microbiome can influence the functioning and pathological states of organs and systems throughout the body. In this study, we tested the hypothesis that the gut microbiome has a major role in the disruption of the blood-brain barrier (BBB) in the spontaneously hypertensive stroke prone rats (SHRSP), an animal model for hypertensive cerebral small vessel disease (CSVD). Loss of BBB is thought to be an early and initiating component to the full expression of CSVD in animal models and humans. To test this hypothesis, newly born SHRSP pups were placed with foster dams of the SHRSP strain or dams of the WKY strain, the control strain that does not demonstrate BBB dysfunction or develop hypertensive CSVD. Similarly, WKY pups were placed with foster dams of the same or opposite strain. The rationale for cross fostering is that the gut microbiomes are shaped by environmental bacteria of the foster dam and the nesting surroundings. Analysis of the bacterial genera in feces, using 16S rRNA analysis, demonstrated that the gut microbiome in the rat pups was influenced by the foster dam. SHRSP offspring fostered on WKY dams had systolic blood pressures (SBPs) that were significantly decreased by 26 mmHg (P < .001) from 16-20 weeks, compared to SHRSP offspring fostered on SHRSP dams. Similarly WKY offspring fostered on SHRSP dams had significantly increased SBP compared to WKY offspring fostered on WKY dams, although the magnitude of SBP change was not as robust. At ~20 weeks of age, rats fostered on SHRSP dams showed enhanced inflammation in distal ileum regardless of the strain of the offspring. Disruption of BBB integrity, an early marker of CSVD onset, was improved in SHRSPs that were fostered on WKY dams when compared to the SHRSP rats fostered on SHRSP dams. Although SHRSP is a genetic model for CSVD, environmental factors such as the gut microbiota of the foster dam have a major influence in the loss of BBB integrity.

Gut Microbiota-Derived Short-Chain Fatty Acids Promote Poststroke Recovery in Aged Mice.

RATIONALE: The elderly experience profound systemic responses after stroke, which contribute to higher mortality and more severe long-term disability. Recent studies have revealed that stroke outcomes can be influenced by the composition of gut microbiome. However, the potential benefits of manipulating the gut microbiome after injury is unknown. OBJECTIVE: To determine if restoring youthful gut microbiota after stroke aids in recovery in aged subjects, we altered the gut microbiome through young fecal transplant gavage in aged mice after experimental stroke. Further, the effect of direct enrichment of selective bacteria producing short-chain fatty acids (SCFAs) was tested as a more targeted and refined microbiome therapy. METHODS AND RESULTS: Aged male mice (18-20 months) were subjected to ischemic stroke by middle cerebral artery occlusion. We performed fecal transplant gavage 3 days after middle cerebral artery occlusion using young donor biome (2-3 months) or aged biome (18-20 months). At day 14 after stroke, aged stroke mice receiving young fecal transplant gavage had less behavioral impairment, and reduced brain and gut inflammation. Based on data from microbial sequencing and metabolomics analysis demonstrating that young fecal transplants contained much higher SCFA levels and related bacterial strains, we selected 4 SCFA-producers (Bifidobacterium longum, Clostridium symbiosum, Faecalibacterium prausnitzii, and Lactobacillus fermentum) for transplantation. These SCFA-producers alleviated poststroke neurological deficits and inflammation, and elevated gut, brain and plasma SCFA concentrations in aged stroke mice. CONCLUSIONS: This is the first study suggesting that the poor stroke recovery in aged mice can be reversed via poststroke bacteriotherapy following the replenishment of youthful gut microbiome via modulation of immunologic, microbial, and metabolomic profiles in the host.

Peripherally-sourced myeloid antigen presenting cells increase with advanced aging.

Aging is associated with dysfunction of the gut microbiota-immune-brain axis, a major regulatory axis in both brain health and in central nervous system (CNS) diseases. Antigen presenting cells (APCs) play a major role in sensing changes in the gut microbiota and regulation of innate and adaptive immune responses. APCs have also been implicated in various chronic inflammatory conditions, including age-related neurodegenerative diseases. The increase in chronic low-level inflammation seen with aging has also been linked to behavioral decline. Despite their acknowledged importance along the gut microbiota-immune-brain axis, there is limited evidence on how APCs change with aging. In this study, we examined age-related changes in myeloid APCs in the gut, spleen, and brain as well as changes in the gut microbiota and behavioral phenotype in mice ranging in age from 2 months up to 32 months of both sexes. Our data show that the number of peripherally-sourced myeloid APCs significantly increases with advanced aging in the brain. In addition, our data showed that age-related changes in APCs are subset-specific in the gut and sexually dimorphic in the spleen. Our work highlights the importance of studying myeloid APCs in an age-, tissue-, and sex-specific manner.

Age-related changes in the gut microbiota influence systemic inflammation and stroke outcome.

OBJECTIVE: Chronic systemic inflammation contributes to the pathogenesis of many age-related diseases. Although not well understood, alterations in the gut microbiota, or dysbiosis, may be responsible for age-related inflammation. METHODS: Using stroke as a disease model, we tested the hypothesis that a youthful microbiota, when established in aged mice, produces positive outcomes following ischemic stroke. Conversely, an aged microbiota, when established in young mice, produces negative outcomes after stroke. Young and aged male mice had either a young or an aged microbiota established by fecal transplant gavage (FTG). Mice were subjected to ischemic stroke (middle cerebral artery occlusion; MCAO) or sham surgery. During the subsequent weeks, mice underwent behavioral testing and fecal samples were collected for 16S ribosomal RNA analysis of bacterial content. RESULTS: We found that the microbiota is altered after experimental stroke in young mice and resembles the biome of uninjured aged mice. In aged mice, the ratio of Firmicutes to Bacteroidetes (F:B), two main bacterial phyla in gut microbiota, increased ∼9-fold (p < 0.001) compared to young. This increased F:B ratio in aged mice is indicative of dysbiosis. Altering the microbiota in young by fecal gavage to resemble that of aged mice (∼6-fold increase in F:B ratio, p < 0.001) increased mortality following MCAO, decreased performance in behavioral testing, and increased cytokine levels. Conversely, altering the microbiota in aged to resemble that of young (∼9-fold decrease in F:B ratio, p < 0.001) increased survival and improved recovery following MCAO. INTERPRETATION: Aged biome increased the levels of systemic proinflammatory cytokines. We conclude that the gut microbiota can be modified to positively impact outcomes from age-related diseases. Ann Neurol 2018;83:23-36.

Alterations in the gut microbiota can elicit hypertension in rats.

Gut dysbiosis has been linked to cardiovascular diseases including hypertension. We tested the hypothesis that hypertension could be induced in a normotensive strain of rats or attenuated in a hypertensive strain of rats by exchanging the gut microbiota between the two strains. Cecal contents from spontaneously hypertensive stroke prone rats (SHRSP) were pooled. Similarly, cecal contents from normotensive WKY rats were pooled. Four-week-old recipient WKY and SHR rats, previously treated with antibiotics to reduce the native microbiota, were gavaged with WKY or SHRSP microbiota, resulting in four groups; WKY with WKY microbiota (WKY g-WKY), WKY with SHRSP microbiota (WKY g-SHRSP), SHR with SHRSP microbiota (SHR g-SHRSP), and SHR with WKY microbiota (SHR g-WKY). Systolic blood pressure (SBP) was measured weekly using tail-cuff plethysmography. At 11.5 wk of age systolic blood pressure increased 26 mmHg in WKY g-SHRSP compared with that in WKY g-WKY (182 ± 8 vs. 156 ± 8 mmHg, P = 0.02). Although the SBP in SHR g-WKY tended to decrease compared with SHR g-SHRSP, the differences were not statistically significant. Fecal pellets were collected at 11.5 wk of age for identification of the microbiota by sequencing the 16S ribosomal RNA gene. We observed a significant increase in the Firmicutes:Bacteroidetes ratio in the hypertensive WKY g-SHRSP, as compared with the normotensive WKY g-WKY (P = 0.042). Relative abundance of multiple taxa correlated with SBP. We conclude that gut dysbiosis can directly affect SBP. Manipulation of the gut microbiota may represent an innovative treatment for hypertension.

Deficiency of the two-pore-domain potassium channel TREK-1 promotes hyperoxia-induced lung injury.

OBJECTIVES: We previously reported the expression of the two-pore-domain K channel TREK-1 in lung epithelial cells and proposed a role for this channel in the regulation of alveolar epithelial cytokine secretion. In this study, we focused on investigating the role of TREK-1 in vivo in the development of hyperoxia-induced lung injury. DESIGN: Laboratory animal experiments. SETTING: University research laboratory. SUBJECTS: Wild-type and TREK-1-deficient mice. INTERVENTIONS: Mice were anesthetized and exposed to 1) room air, no mechanical ventilation, 2) 95% hyperoxia for 24 hours, and 3) 95% hyperoxia for 24 hours followed by mechanical ventilation for 4 hours. MEASUREMENTS AND MAIN RESULTS: Hyperoxia exposure accentuated lung injury in TREK-1-deficient mice but not controls, resulting in increase in lung injury scores, bronchoalveolar lavage fluid cell numbers, and cellular apoptosis and a decrease in quasi-static lung compliance. Exposure to a combination of hyperoxia and injurious mechanical ventilation resulted in further morphological lung damage and increased lung injury scores and bronchoalveolar lavage fluid cell numbers in control but not TREK-1-deficient mice. At baseline and after hyperoxia exposure, bronchoalveolar lavage cytokine levels were unchanged in TREK-1-deficient mice compared with controls. Exposure to hyperoxia and mechanical ventilation resulted in an increase in bronchoalveolar lavage interleukin-6, monocyte chemotactic protein-1, and tumor necrosis factor-α levels in both mouse types, but the increase in interleukin-6 and monocyte chemotactic protein-1 levels was less prominent in TREK-1-deficient mice than in controls. Lung tissue macrophage inflammatory protein-2, keratinocyte-derived cytokine, and interleukin-1ß gene expression was not altered by hyperoxia in TREK-1-deficient mice compared with controls. Furthermore, we show for the first time TREK-1 expression on alveolar macrophages and unimpaired tumor necrosis factor-α secretion from TREK-1-deficient macrophages. CONCLUSIONS: TREK-1 deficiency resulted in increased sensitivity of lungs to hyperoxia, but this effect is less prominent if overwhelming injury is induced by the combination of hyperoxia and injurious mechanical ventilation. TREK-1 may constitute a new potential target for the development of novel treatment strategies against hyperoxia-induced lung injury.

A role for TREK1 in generating the slow afterhyperpolarization in developing starburst amacrine cells.

Slow afterhyperpolarizations (sAHPs) play an important role in establishing the firing pattern of neurons that in turn influence network activity. sAHPs are mediated by calcium-activated potassium channels. However, the molecular identity of these channels and the mechanism linking calcium entry to their activation are still unknown. Here we present several lines of evidence suggesting that the sAHPs in developing starburst amacrine cells (SACs) are mediated by two-pore potassium channels. First, we use whole cell and perforated patch voltage clamp recordings to characterize the sAHP conductance under different pharmacological conditions. We find that this conductance was calcium dependent, reversed at EK, blocked by barium, insensitive to apamin and TEA, and activated by arachidonic acid. In addition, pharmacological inhibition of calcium-activated phosphodiesterase reduced the sAHP. Second, we performed gene profiling on isolated SACs and found that they showed strong preferential expression of the two-pore channel gene kcnk2 that encodes TREK1. Third, we demonstrated that TREK1 knockout animals exhibited an altered frequency of retinal waves, a frequency that is set by the sAHPs in SACs. With these results, we propose a model in which depolarization-induced decreases in cAMP lead to disinhibition of the two-pore potassium channels and in which the kinetics of this biochemical pathway dictate the slow activation and deactivation of the sAHP conductance. Our model offers a novel pathway for the activation of a conductance that is physiologically important.

Endothelium-dependent relaxations in the aorta from K(2p)6.1 knockout mice.

K2P6.1 or TWIK-2, a two-pore domain K channel, is an important regulator of cardiovascular function. K2P6.1 is highly expressed in vascular smooth muscle and endothelium. Mice (8-12 wk) lacking functional K2P6.1 (K2P6.1(-/-)) are hypertensive and have enhanced vascular contractility. It is not known whether the lack of functional K2P6.1 in endothelium has a role in the vascular dysfunction in K2P6.1(-/-) mice. We tested the hypothesis: K2P6.1(-/-) mice have impaired endothelium-dependent relaxations. K2P6.1(-/-) mice were ∼35 mmHg more hypertensive than WT mice at both 8-12 wk (young adult) and 20-24 wk (mature mice, P < 0.01; n = 8-10). Endothelium-dependent relaxations of the thoracic aorta were evaluated by isometric myography after contraction with phenylephrine (10(-6) M). Maximal ACh-dependent relaxations were increased from 65 ± 1% to 73 ± 1% in the aorta from young adult (P < 0.01; n = 6) and from 45 ± 1% to 74 ± 1% in the aorta from mature (P < 0.001; n = 5) K2P6.1(-/-) mice compared with K2P6.1(+/+) littermates. However, in the aorta from young adult and mature K2P6.1(+/+) mice, 10(-5) M indomethacin, a cyclooxygenase inhibitor, increased maximal ACh relaxations to knockout levels. Enhanced relaxation was also seen with ATP, a P2Y purinergic agonist, and A23187, a nonreceptor-based agonist in mature K2P6.1(-/-) mice. Mature adult aorta from K2P6.1(-/-) showed an attenuated ACh-mediated contraction in the presence of nitro-l-arginine methyl ester (l-NAME) and without precontraction of 0.97 mN vs. 7.5 mN in K2P6.1(-/-) and K2P6.1(+/+) (P < 0.001; n = 5). In summary, K2P6.1(-/-) mice, which are hypertensive, have enhanced endothelium-dependent relaxations in the aorta due to the suppression of an indomethacin-sensitive constrictor component.

A new rodent model for obstructive sleep apnea: effects on ATP-mediated dilations in cerebral arteries.

Obstructive sleep apnea (OSA), a condition in which the upper airway collapses during sleep, is strongly associated with metabolic and cardiovascular diseases. Little is known how OSA affects the cerebral circulation. The goals of this study were 1) to develop a rat model of chronic OSA that involved apnea and 2) to test the hypothesis that 4 wk of apneas during the sleep cycle alters endothelium-mediated dilations in middle cerebral arteries (MCAs). An obstruction device, which was chronically implanted into the trachea of rats, inflated to obstruct the airway 30 times/h for 8 h during the sleep cycle. After 4 wk of apneas, MCAs were isolated, pressurized, and exposed to luminally applied ATP, an endothelial P2Y2 receptor agonist that dilates through endothelial-derived nitric oxide (NO) and endothelial-dependent hyperpolarization (EDH). Dilations to ATP were attenuated ~30% in MCAs from rats undergoing apneas compared with those from a sham control group (P < 0.04 group effect; n = 7 and 10, respectively). When the NO component of the dilation was blocked to isolate the EDH component, the response to ATP in MCAs from the sham and apnea groups was similar. This finding suggests that the attenuated dilation to ATP must occur through reduced NO. In summary, we have successfully developed a novel rat model for chronic OSA that incorporates apnea during the sleep cycle. Using this model, we demonstrate that endothelial dysfunction occurred by 4 wk of apnea, likely increasing the vulnerability of the brain to cerebrovascular related accidents.

Obstructive Sleep Apnea-Induced Hypertension Is Associated With Increased Gut and Neuroinflammation.

Background Obstructive sleep apnea (OSA) is an independent risk factor for the development of hypertension. We have demonstrated that OSA induces gut dysbiosis, and this dysbiotic microbiota contributes to hypertension. However, the mechanisms linking gut dysbiosis to blood pressure regulation remain unclear. Recent studies demonstrate that gut dysbiosis can induce a proinflammatory response of the host resulting in peripheral and neuroinflammation, key factors in the development of hypertension. We hypothesized that OSA induces inflammation in the gut that contributes to neuroinflammation and hypertension. Methods and Results OSA was induced in 8-week-old male rats. After 2 weeks of apneas, lymphocytes were isolated from aorta, brain, cecum, ileum, mesenteric lymph node, and spleen for flow cytometry. To examine the role of interleukin-17a, a monoclonal antibody was administered to neutralize interleukin-17a. Lymphocytes originating from the gut were tracked by labeling with carboxyfluorescein succinimidyl ester dye. OSA led to a significant decrease in T regulatory cells along with an increase in T helper (TH) 17 cells in the ileum, cecum, and brain. Interleukin-17a neutralization significantly reduced blood pressure, increased T regulatory cells, and decreased TH1 cells in the ileum, cecum, and brain of OSA rats. TH1, TH2, and TH17 cells from the gut were found to migrate to the mesenteric lymph node, spleen, and brain with increased frequency in rats with OSA. Conclusions OSA induces a proinflammatory response in the gut and brain that involves interleukin-17a signaling. Gut dysbiosis may serve as the trigger for gut and neuroinflammation, and treatments to prevent or reverse gut dysbiosis may prove useful in reducing neuroinflammation and hypertension.

Estradiol mediates colonic epithelial protection in aged mice after stroke and is associated with shifts in the gut microbiome.

The gut is a major source of bacteria and antigens that contribute to neuroinflammation after brain injury. Colonic epithelial cells (ECs) are responsible for secreting major cellular components of the innate defense system, including antimicrobial proteins (AMP) and mucins. These cells serve as a critical regulator of gut barrier function and maintain host-microbe homeostasis. In this study, we determined post-stroke host defense responses at the colonic epithelial surface in mice. We then tested if the enhancement of these epithelial protective mechanisms is beneficial in young and aged mice after stroke. AMPs were significantly increased in the colonic ECs of young males, but not in young females after experimental stroke. In contrast, mucin-related genes were enhanced in young females and contributed to mucus formation that maintains the distance between the host and gut bacteria. Bacterial community profiling was done using universal amplification of 16S rRNA gene sequences. The sex-specific colonic epithelial defense responses after stroke in young females were reversed with ovariectomy and led to a shift from a predominately mucin response to the enhanced AMP expression seen in males after stroke. Estradiol (E2) replacement prior to stroke in aged females increased mucin gene expression in the colonic ECs. Interestingly, we found that E2 treatment reduced stroke-associated neuronal hyperactivity in the insular cortex, a brain region that interacts with visceral organs such as the gut, in parallel to an increase in the composition of Lactobacillus and Bifidobacterium in the gut microbiota. This is the first study demonstrating sex differences in host defense mechanisms in the gut after brain injury.

Restoring a balanced pool of host-derived and microbiota-derived ligands of the aryl hydrocarbon receptor is beneficial after stroke.

Background: Stroke is a major cause of morbidity and mortality, and its incidence increases with age. While acute therapies for stroke are currently limited to intravenous thrombolytics and endovascular thrombectomy, recent studies have implicated an important role for the gut microbiome in post-stroke neuroinflammation. After stroke, several immuno-regulatory pathways, including the aryl hydrocarbon receptor (AHR) pathway, become activated. AHR is a master regulatory pathway that mediates neuroinflammation. Among various cell types, microglia (MG), as the resident immune cells of the brain, play a vital role in regulating post-stroke neuroinflammation and antigen presentation. Activation of AHR is dependent on a dynamic balance between host-derived and microbiota-derived ligands. While previous studies have shown that activation of MG AHR by host-derived ligands, such as kynurenine, is detrimental after stroke, the effects of post-stroke changes in microbiota-derived ligands of AHR, such as indoles, is unknown. Our study builds on the concept that differential activation of MG AHR by host-derived versus microbiome-derived metabolites affects outcomes after ischemic stroke. We examined the link between stroke-induced dysbiosis and loss of essential microbiota-derived AHR ligands. We hypothesize that restoring the balance between host-derived (kynurenine) and microbiota-derived (indoles) ligands of AHR is beneficial after stroke, offering a new potential avenue for therapeutic intervention in post-stroke neuroinflammation. Method: We performed immunohistochemical analysis of brain samples from stroke patients to assess MG AHR expression after stroke. We used metabolomics analysis of plasma samples from stroke and non-stroke control patients with matched comorbidities to determine the levels of indole-based AHR ligands after stroke. We performed transient middle cerebral artery occlusion (MCAO) in aged (18 months) wild-type (WT) and germ-free (GF) mice to investigate the effects of post-stroke treatment with microbiota-derived indoles on outcome. To generate our results, we employed a range of methodologies, including flow cytometry, metabolomics, and 16S microbiome sequencing. Results: We found that MG AHR expression is increased in human brain after stroke and after ex vivo oxygen-glucose deprivation and reperfusion (OGD/R). Microbiota-derived ligands of AHR are decreased in the human plasma at 24 hours after ischemic stroke. Kynurenine and indoles exhibited differential effects on aged WT MG survival after ex vivoOGD/R. We found that specific indole-based ligands of AHR (indole-3-propionic acid and indole-3-aldehyde) were absent in GF mice, thus their production depends on the presence of a functional gut microbiota. Additionally, a time-dependent decrease in the concentration of these indole-based AHR ligands occurred in the brain within the first 24 hours after stroke in aged WT mice. Post-stroke treatment of GF mice with a cocktail of microbiota-derived indole-based ligands of AHR regulated MG-mediated neuroinflammation and molecules involved in antigen presentation (increased CD80, MHC-II, and CD11b). Post-stroke treatment of aged WT mice with microbiota-derived indole-based ligands of AHR reduced both infarct volume and neurological deficits at 24 hours. Conclusion: Our novel findings provide compelling evidence that the restoration of a well-balanced pool of host-derived kynurenine-based and microbiota-derived indole-based ligands of AHR holds considerable therapeutic potential for the treatment of ischemic stroke.

Alterations of the gut microbial community structure and function with aging in the spontaneously hypertensive stroke prone rat.

Gut dysbiosis, a pathological imbalance of bacteria, has been shown to contribute to the development of hypertension (HT), systemic- and neuro-inflammation, and blood-brain barrier (BBB) disruption in spontaneously hypertensive stroke prone rats (SHRSP). However, to date individual species that contribute to HT in the SHRSP model have not been identified. One potential reason, is that nearly all studies of the SHRSP gut microbiota have analyzed samples from rats with established HT. The goal of this study was to examine the SHRSP gut microbiota before, during, and after the onset of hypertension, and in normotensive WKY control rats over the same age range. We hypothesized that we could identify key microbes involved in the development of HT by comparing WKY and SHRSP microbiota during the pre-hypertensive state and longitudinally. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography and fecal microbiota analyzed by16S rRNA gene sequencing. SHRSP showed significant elevations in SBP, as compared to WKY, beginning at 8 weeks of age (p < 0.05 at each time point). Bacterial community structure was significantly different between WKY and SHRSP as early as 4 weeks of age, and remained different throughout the study (p = 0.001-0.01). At the phylum level we observed significantly reduced Firmicutes and Deferribacterota, and elevated Bacteroidota, Verrucomicrobiota, and Proteobacteria, in pre-hypertensive SHRSP, as compared to WKY. At the genus level we identified 18 bacteria whose relative abundance was significantly different in SHRSP versus WKY at the pre-hypertensive ages of 4 or 6 weeks. In an attempt to further refine bacterial candidates that might contribute to the SHRSP phenotype, we compared the functional capacity of WKY versus SHRSP microbial communities. We identified significant differences in amino acid metabolism. Using untargeted metabolomics we found significant reductions in metabolites of the tryptophan-kynurenine pathway and increased indole metabolites in SHRSP versus WKY plasma. Overall, we provide further evidence that gut dysbiosis contributes to hypertension in the SHRSP model, and suggest for the first time the potential involvement of tryptophan metabolizing microbes.

2,2,2-trichloroethanol activates a nonclassical potassium channel in cerebrovascular smooth muscle and dilates the middle cerebral artery.

Trichloroacetaldehyde monohydrate [chloral hydrate (CH)] is a sedative/hypnotic that increases cerebral blood flow (CBF), and its active metabolite 2,2,2-trichloroethanol (TCE) is an agonist for the nonclassical two-pore domain K(+) (K(2P)) channels TREK-1 and TRAAK. We sought to determine whether TCE dilates cerebral arteries in vitro by activating nonclassical K(+) channels. TCE dilated pressurized and perfused rat middle cerebral arteries (MCAs) in a manner consistent with activation of nonclassical K(+) channels. Dilation to TCE was inhibited by elevated external K(+) but not by an inhibitory cocktail (IC) of classical K(+) channel blockers. Patch-clamp electrophysiology revealed that, in the presence of the IC, TCE increased whole-cell currents and hyperpolarized the membrane potential of isolated MCA smooth muscle cells. Heating increased TCE-sensitive currents, indicating that the activated channel was thermosensitive. Immunofluorescence in sections of the rat MCA demonstrated that, like TREK-1, TRAAK is expressed in the smooth muscle of cerebral arteries. Isoflurane did not, however, dilate the MCA, suggesting that TREK-1 was not functional. These data indicate that TCE activated a nonclassical K(+) channel with the characteristics of TRAAK in rat MCA smooth-muscle cells. Stimulation of K(+) channels such as TRAAK in cerebral arteries may therefore explain in part how CH/TCE increases CBF.

Cerebrovascular responses in mice deficient in the potassium channel, TREK-1.

We tested the hypothesis that TREK-1, a two-pore domain K channel, is involved with dilations in arteries. Because there are no selective activators or inhibitors of TREK-1, we generated a mouse line deficient in TREK-1. Endothelium-mediated dilations were not different in arteries from wild-type (WT) and TREK-1 knockout (KO) mice. This includes dilations of the middle cerebral artery to ATP, dilations of the basilar artery to ACh, and relaxations of the aorta to carbachol, a cholinergic agonist. The nitric oxide (NO) and endothelium-dependent hyperpolarizing factor components of ATP dilations were identical in the middle cerebral arteries of WT and TREK-1 KO mice. Furthermore, the NO and cyclooxygenase-dependent components were identical in the basilar arteries of the different genotypes. Dilations of the basilar artery to alpha-linolenic acid, an activator of TREK-1, were not affected by the absence of TREK-1. Whole cell currents recorded using patch-clamp techniques were similar in cerebrovascular smooth muscle cells (CVSMCs) from WT and TREK-1 KO mice. alpha-linolenic acid or arachidonic acid increased whole cell currents in CVSMCs from both WT and TREK-1 KO mice. The selective blockers of large-conductance Ca-activated K channels, penitrem A and iberiotoxin, blocked the increased currents elicited by either alpha-linolenic or arachidonic acid. In summary, dilations were similar in arteries from WT and TREK-1 KO mice. There was no sign of TREK-1-like currents in CVSMCs from WT mice, and there were no major differences in currents between the genotypes. We conclude that regulation of arterial diameter is not altered in mice lacking TREK-1.

Young versus aged microbiota transplants to germ-free mice: increased short-chain fatty acids and improved cognitive performance.

Aging is associated with cognitive decline and decreased concentrations of short-chain fatty acids (SCFAs) in the gut. SCFAs are significant in that they are protective to the gut and other organs. We tested the hypothesis that the aged gut microbiome alone is sufficient to decrease SCFAs in the host and produce cognitive decline. Fecal transplant gavages (FTGs) from aged (18-20 months) or young (2-3 months) male C57BL/6 mice into germ-free male C57BL/6 mice (N = 11 per group) were initiated at ~3 months of age. Fecal samples were collected and behavioral testing was performed over the study period. Bacterial community structures and relative abundances were measured in fecal samples by sequencing the bacterial 16S ribosomal RNA gene. Mice with aged and young microbiomes showed clear differences in bacterial ß diversity at 30, 60, and 90 d (P = .001 for each) after FTGs. The fecal SCFAs, acetate, propionate, and butyrate (microbiome effect, P < .01 for each) were decreased in mice with an aged microbiome. Mice with an aged microbiome demonstrated depressive-like behavior, impaired short-term memory, and impaired spatial memory over the 3 months following the initial FTG as assessed by the tail suspension (P = .008), the novel object recognition (P < .001), and the Barnes Maze (P = .030) tests, respectively. We conclude that an aged microbiome alone is sufficient to decrease SCFAs in the host and to produce cognitive decline.

Extracellular Vesicles Attenuate Nitrofen-Mediated Human Pulmonary Artery Endothelial Dysfunction: Implications for Congenital Diaphragmatic Hernia.

Congenital diaphragmatic hernia (CDH) leads to pathophysiologic pulmonary vasoreactivity. Previous studies show that mesenchymal stromal cell-derived extracellular vesicles (MSCEv) inhibit lung inflammation and vascular remodeling. We characterize MSCEv and human pulmonary artery endothelial cell (HPAEC) interaction, as well as the pulmonary artery (PA) response to MSCEv treatment. HPAECs were cultured with and without exposure to nitrofen (2,4-dichloro-phenyl-p-nitrophenylether) and treated with MSCEv. HPAEC viability, architecture, production of reactive oxygen species (ROS), endothelial dysfunction-associated protein levels (PPARγ, LOX-1, LOX-2, nuclear factor-κB [NF-κB], endothelial NO synthase [eNOS], ET-1 [endothelin 1]), and the nature of MSCEv-cellular interaction were assessed. Newborn rodents with and without CDH (nitrofen model and Sprague-Dawley) were treated with intravascular MSCEv or vehicle control, and their PAs were isolated. Contractility was assessed by wire myography. The contractile (KCL and ET-1) and relaxation (fasudil) responses were evaluated. HPAEC viability correlated inversely with nitrofen dose, while architectural compromise was directly proportional. There was a 2.1 × increase in ROS levels in nitrofen HPAECs (P < 0.001), and MSCEv treatment attenuated ROS levels by 1.5 × versus nitrofen HPAECs (P < 0.01). Nitrofen-induced alterations in endothelial dysfunction-associated proteins are shown, and exposure to MSCEv restored more physiologic expression. Nitrofen HPAEC displayed greater MSCEv uptake (80% increase, P < 0.05). Adenosine, a clathrin-mediated endocytosis inhibitor, decreased uptake by 46% (P < 0.05). CDH PA contraction was impaired with KCL (108.6% ± 1.4% vs. 112.0% ± 1.4%, P = 0.092) and ET-1 (121.7% ± 3.0% vs. 131.2% ± 1.8%, P < 0.01). CDH PA relaxation was impaired with fasudil (32.2% ± 1.9% vs. 42.1% ± 2.2%, P < 0.001). After MSCEv treatment, CDH PA contraction improved (125.9% ± 3.4% vs. 116.4 ± 3.5, P = 0.06), and relaxation was unchanged (32.5% ± 3.2% vs. 29.4% ± 3.1%, P = 0.496). HPAEC exposure to nitrofen led to changes consistent with vasculopathy in CDH, and MSCEv treatment led to a more physiologic cellular response. MSCEv were preferentially taken up by nitrofen-treated cells by clathrin-dependent endocytosis. In vivo, MSCEv exposure improved PA contractile response. These data reveal mechanisms of cellular and signaling alterations that characterize MSCEv-mediated attenuation of pulmonary vascular dysfunction in CDH-associated pulmonary hypertension.