Intestinal epithelial stem cell transplants as a novel therapy for cerebrovascular stroke.

Almost 2/3rds of stroke survivors exhibit vascular cognitive impairment and a third of stroke patients will develop dementia 1-3 years after stroke. These dire consequences underscore the need for effective stroke therapies. In addition to its damaging effects on the brain, stroke rapidly dysregulates the intestinal epithelium, resulting in elevated blood levels of inflammatory cytokines and toxic gut metabolites due to a 'leaky' gut. We tested whether repairing the gut via intestinal epithelial stem cell (IESC) transplants would also improve stroke recovery. Organoids containing IESCs derived from young rats transplanted into older rats after stroke were incorporated into the gut, restored stroke-induced gut dysmorphology and decreased gut permeability, and reduced circulating levels of endotoxin LPS and the inflammatory cytokine IL-17A. Remarkably, IESC transplants also improved stroke-induced acute (4d) sensory-motor disability and chronic (30d) cognitive-affective function. Moreover, IESCs from older animals displayed senescent features and were not therapeutic for stroke. These data underscore the gut as a critical therapeutic target for stroke and demonstrate the effectiveness of gut stem cell therapy.

Sex differences in the diathetic effects of shift work schedules on circulating cytokine levels and pathological outcomes of ischemic stroke during middle age.

Shift work is associated with increased risk for vascular disease, including stroke- and cardiovascular-related mortality. However, evidence from these studies is inadequate to distinguish the effect of altered circadian rhythms in isolation from other risk factors for stroke associated with shift work (e.g., smoking, poor diet, lower socioeconomic status). Thus, the present study examined the diathetic effects of exposure to shifted LD cycles during early adulthood on circadian rhythmicity, inflammatory signaling and ischemic stroke pathology during middle age, when stroke risk is high and outcomes are more severe. Entrainment of circadian activity was stable in all animals maintained on a fixed light:dark 12:12 cycle but was severely disrupted during exposure to shifted LD cycles (12hr advance/5d). Following treatment, circadian entrainment in the shifted LD group was distinguished by increased daytime activity and decreased rhythm amplitude that persisted into middle-age. Circadian rhythm desynchronization in shifted LD males and females was accompanied by significant elevations in circulating levels of the inflammatory cytokine IL-17A and gut-derived inflammatory mediator lipopolysaccharide (LPS) during the post-treatment period. Middle-cerebral artery occlusion, 3 months after exposure to shifted LD cycles, resulted in greater post-stroke mortality in shifted LD females. In surviving subjects, sensorimotor performance, assessed 2- and 5-days post-stroke, was impaired in males of both treatment groups, whereas in females, recovery of function was observed in fixed but not shifted LD rats. Overall, these results indicate that early exposure to shifted LD cycles promotes an inflammatory phenotype that amplifies stroke impairments, specifically in females, later in life.

Prenatal alcohol-induced sex differences in immune, metabolic and neurobehavioral outcomes in adult rats.

Prenatal alcohol exposure (PAE) can result in neurobehavioral anomalies, that may be exacerbated by co-occurring metabolic and immune system deficits. To test the hypothesis that the peripheral inflammation in adult PAE offspring is linked to poor glucose metabolism and neurocognitive deficits, pregnant Sprague-Dawley rats were exposed to ethanol vapor or ambient air during the latter half of gestation. We assessed, in adult offspring of both sexes, performance on a battery of neurocognitive behaviors, glucose tolerance, circulating and splenic immune cells by flow-cytometry, and circulating and tissue (liver, mesenteric adipose, and spleen) cytokines by multiplexed assays. PAE reduced both the ratio of spleen to body weight and splenic regulatory T-cell (Treg) numbers. PAE males, but not females exhibited an increase in circulating monocytes. Overall, PAE males exhibited a suppression of cytokine levels, while PAE females exhibited elevated cytokines in mesenteric adipose tissue (IL-6 and IL1α) and liver (IFN-γ, IL-1ß, IL-13, IL-18, IL-12p70, and MCP-1), along with increased glucose intolerance. Behavioral analysis also showed sex-dependent PAE effects. PAE-males exhibited increased anxiety-like behavior while PAE-females showed decreased social interaction. PAE offspring of both sexes exhibited impaired recognition of novel objects. Multilinear regression modeling to predict the association between peripheral immune status, glucose intolerance and behavioral outcomes, showed that in PAE offspring, higher levels of adipose leptin and liver TNF- α predicted higher circulating glucose levels. Lower liver IL-1 α and higher plasma fractalkine predicted more time spent in the center of an open-field with sex being an additional predictor. Higher circulating and splenic Tregs predicted better social interaction in the PAE-offspring. Collectively, our data show that peripheral immune status is a persistent, sex-dependent predictor of glucose intolerance and neurobehavioral function in adult PAE offspring.

Sex differences in stroke outcome correspond to rapid and severe changes in gut permeability in adult Sprague-Dawley rats.

BACKGROUND: Sex differences in experimental stroke outcomes are well documented, such that adult males have a greater infarct volume, increased stroke-induced mortality, and more severe sensory-motor impairment. Based on recent evidence that the gut is an early responder to stroke, the present study tested the hypothesis that sex differences in stroke severity will be accompanied by rapid and greater permeability of the gut-blood barrier and gut dysbiosis in males as compared to females. METHOD: Male and female Sprague-Dawley rats (5-7 months of age) were subject to endothelin (ET)-1-induced middle cerebral artery occlusion (MCAo). Sensory-motor tests were conducted pre- and 2 days after MCAo. Gut permeability was assessed in serum samples using biomarkers of gut permeability as well as functional assays using size-graded dextrans. Histological analysis of the gut was performed with H&E staining, periodic acid-Schiff for mucus, and immunohistochemistry for the tight junction protein, ZO-1. Fecal samples obtained pre- and post-stroke were analyzed for bacterial taxa and short-chain fatty acids (SCFAs). RESULTS: After stroke, males displayed greater mortality, worse sensory-motor deficit, and higher serum levels of proinflammatory cytokines IL-17A, MCP-1, and IL-5 as compared to females. MCAo-induced gut permeability was rapid and severe in males as indicated by dextran extravasation from the gut to the blood in the hyperacute (< 2 h) and early acute (2 days) phase of stroke. This was accompanied by dysmorphology of the gut villi and dysregulation of the tight junction protein ZO-1 in the acute phase. Fecal 16s sequencing showed no differences in bacterial diversity in the acute phase of stroke. Predictive modeling indicated that markers of gut permeability were associated with acute sensory-motor impairment and infarct volume. CONCLUSIONS: These data show that extensive leakiness of the gut barrier is associated with severe post-stroke disability and suggest that reinforcing this barrier may improve stroke outcomes.

Reproductive Senescence and Ischemic Stroke Remodel the Gut Microbiome and Modulate the Effects of Estrogen Treatment in Female Rats.

Our previous work has shown that reproductively senescent (or middle-aged; 10-12-month-old) Sprague-Dawley female rats, that are naturally estrogen-deficient, have worse stroke outcomes as compared to normally estrous-cycling adult (5-6-month-old) females. Paradoxically, estrogen replacement to this middle-aged group exacerbates stroke outcomes, while it is neuroprotective in adult females. Recent studies reveal an important role for the gut microbiome and gut metabolites in cardiovascular health, including stroke outcomes. To determine whether gut dysbiosis underlies stroke severity in reproductive senescent females, and underlies the anomalous effects of estrogen on stroke, we compared the gut microbiota and gut metabolites pre and post stroke in (a) gonadally intact adult and middle-aged females, (b) in ovariectomized and estrogen-treated (OVX+E) adult and OVX+E middle-aged females, and (c) in middle-aged OVX+E females after fecal microbiome transfer. Our data show significant gut dysbiosis in reproductive senescent females at baseline and after stroke as indicated by an elevated ratio of the major phyla, Firmicutes/Bacteroidetes (F:B), reduced alpha diversity, and significant shifts in beta diversity as compared with adult females. Specific bacterial families were also altered as a result of reproductive aging, as well as gut metabolites, including elevated serum endotoxin levels and decreased short-chain fatty acids (SCFAs), with a concomitant increase in IL-17A, indicating that reproductive senescence significantly affects gut communities under pathologic conditions. Despite the differences in gonadally intact adult and middle-aged females, estrogen-treated ovariectomized (OVX+E) females of either age group displayed no differences in the major phyla, but there was increased abundance in specific bacterial taxa, including Prevotella and Lactobacillus. The SCFA butyrate was significantly reduced at baseline in the middle-aged OVX+E females, while circulating endotoxin LPS were elevated in this group after stroke, suggesting that gut metabolites were differently affected by estrogen treatment in the two age groups. A fecal transfer from adult OVX+E females to middle-aged OVX+E females significantly reduced infarct volume, improved behavioral recovery and transiently reduced IL-17A expression. These data provide the first evidence that microbial gut communities and metabolites are altered by reproductive senescence in female rats at baseline and after stroke, and suggest that estrogen may impact stroke recovery differently in adult and reproductive senescent females due to an age-specific effect on gut microbiota and metabolites.

Mir363-3p attenuates post-stroke depressive-like behaviors in middle-aged female rats.

Women are more likely to develop Post Stroke Depression (PSD) than men and generally do not respond well to anti-depressants with age. This study investigated the effect of microRNA mir363-3p treatment on PSD using a physiologically-relevant animal model. Our previous work showed that mir363-3p treatment, delivered post-stroke, effectively reduces infarct volume in the acute phase of stroke in middle-aged females but not males. Middle-aged female Sprague Dawley rats were tested for baseline sensory motor function and depressive-like behaviors, and then subjected to ischemic stroke via middle cerebral artery occlusion (MCAo) or sham surgery. Animals received either control oligos (MCAo+scrambled, Sham+scrambled) or mir363-3p (MCAo+mir363-3p, Sham+mir363-3p) treatment 4 h later. Sensory motor function and depressive-like behaviors were reassessed up to 100 d after stroke, and circulating levels of IL-6, TNF-alpha and Brain-Derived Neurotrophic Factor (BDNF) were quantified at regular intervals. Prior to termination, Fluorogold was injected into the striatum to assess meso-striatal projections. MCAo+scrambled animals had impaired sensorimotor performance in the acute phase (5 days) of stroke and developed anhedonia, decreased sociability and increased helplessness in the chronic phase. MCAo+mir363-3p animals showed significantly less sensory motor impairment and fewer depressive-like behaviors. IL-6 and TNF-alpha were elevated transiently at 4 weeks after MCAo in both groups. BDNF levels decreased progressively after stroke in the MCAo+scrambled group, and this was attenuated in the mir363-3p group. The number of retrogradely-labeled SNc and VTA cells was reduced in the ischemic hemisphere of the MCAo+scrambled group. In contrast, there was no interhemispheric difference in the number of retrogradely-labeled SNc and VTA cells of MCAo+mir363-3p treated animals. Our results support a therapeutic role for mir363-3p for long-term stroke disability.