Prenatal alcohol alters inflammatory signatures in enteric portal tissues following adult-onset cerebrovascular ischemic stroke.

Prenatal alcohol exposure (PAE) impairs recovery from cerebrovascular ischemic stroke in adult rodents. Since the gut becomes dysbiotic following stroke, we assessed links between PAE and enteric portal inflammation. Adult control and PAE rat offspring received a unilateral endothelin-1-induced occlusion of the middle cerebral artery. Post-stroke behavioral disabilities and brain cytokines were assessed. Mesenteric adipose and liver transcriptomes were assessed from stroke-exposed and stroke-naive offspring. We identified, in the liver of stroke-naive animals, a moderate correlation between PAE and a gene network for inflammatory necroptosis. PAE inhibited the acute-phase brain inflammatory cytokine response to stroke. Post-stroke neurological function was correlated with an adipose gene network associated with B-lymphocyte differentiation and nuclear factor κB (NF-κB) signaling and with a liver pro-inflammatory gene network. Collectively, PAE inhibits brain inflammation but results in an inflammatory signature in enteric portal tissues after stroke, suggesting that PAE persistently and adversely impacts the gut-brain axis following adult-onset disease.

The aging ovary impairs acute stroke outcomes.

In experimental stroke, ovariectomized (OVX) adult rats have larger infarct volumes and greater sensory-motor impairment as compared to ovary-intact females and is usually interpreted to indicate that ovarian hormones are neuroprotective for stroke. Previous work from our lab shows that middle-aged, acyclic reproductively senescent (RS) females have worse stroke outcomes as compared to adult (normally cycling) females. We hypothesized that if loss of ovarian estrogen is the critical determinant of stroke outcomes, then ovary-intact middle-aged acyclic females, who have reduced levels of estradiol, should have similar stroke outcomes as age-matched OVX. Instead, the data demonstrated that OVX RS animals showed better sensory-motor function after stroke and reduced infarct volume as compared to ovary-intact females. Inflammatory cytokines were decreased in the aging ovary after stroke as compared to non-stroke shams, which led to the hypothesis that immune cells may be extravasated from the ovaries post-stroke. Flow cytometry indicated reduced overall T cell populations in the aging ovary after middle cerebral artery occlusion (MCAo), with a paradoxical increase in regulatory T cells (Tregs) and M2-like macrophages. Moreover, in the brain, OVX RS animals showed increased Tregs, increased M2-like macrophages, and increased MHC II + cells as compared to intact RS animals, which have all been shown to be correlated with better prognosis after stroke. Depletion of ovary-resident immune cells after stroke suggests that there may be an exaggerated response to ischemia and possible increased burden of the inflammatory response via extravasation of these cells into circulation. Increased anti-inflammatory cells in the brain of OVX RS animals further supports this hypothesis. These data suggest that stroke severity in aging females may be exacerbated by the aging ovary and underscore the need to assess immunological changes in this organ after stroke.

Sex differences in cognitive impairment after focal ischemia in middle-aged rats and the effect of iv miR-20a-3p treatment.

Stroke is a major cause of death and disability worldwide and is also a leading cause of vascular dementia and Alzheimer's disease, with older women experiencing accelerated decline. Our previous studies show that intravenous (iv) injections of miR-20a-3p, a small noncoding RNA (miRNA) delivered after stroke improves acute stroke outcomes in middle-aged male and female rats. The present study tested whether mir-20a-3p treatment would also ameliorate stroke-induced cognitive decline in the chronic phase. Acyclic middle-aged females and age-matched male Sprague Dawley rats were subjected to middle cerebral artery occlusion using endothelin-1 or sham surgery, and treated iv with miR-20a-3p mimics or scrambled oligos at 4 hours, 24 hours, and 70 days post-stroke. Stroke resulted in a significant sensory motor deficit, while miR-20a-3p treatment reduced these deficits in both sexes. Cognitive impairment was assessed periodically for 3 months after stroke using contextual fear conditioning and the novel object recognition task. Overall, the tests of associative and episodic memory were affected by focal ischemia only in female rats, and miR-20a-3p ameliorated the rate of decline.

Assessing Depression and Cognitive Impairment Following Stroke and Neurotrauma: Behavioral Methods for Quantifying Impairment and Functional Recovery.

Rodent models of stroke and neural injury are reliable and useful tools for testing new interventions and therapeutics. In addition to physical (motor) impairment, cognitive deficits and depressive behaviors are often observed due to neurotrauma. Proper experimental design of pre- and post-assessments of these behaviors that reduce or minimize the confounding effects of motor impairment are essential for determining markers of progression of impairment or recovery. This chapter provides step-by-step laboratory protocols for assessing cognition using the Barnes maze and the novel object recognition test (NORT) and depressive-like behaviors using the sucrose preference test, the three-chambered sociability approach test, and the burrowing test.

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 Long-Term Consequences of Stroke.

Stroke is the fifth leading cause of death and as healthcare intervention improves, the number of stroke survivors has also increased. Furthermore, there exists a subgroup of younger adults, who suffer stroke and survive. Given the overall improved survival rate, bettering our understanding of long-term stroke outcomes is critical. In this review we will explore the causes and challenges of known long-term consequences of stroke and if present, their corresponding sex differences in both old and young survivors. We have separated these long-term post-stroke consequences into three categories: mobility and muscle weakness, memory and cognitive deficits, and mental health and mood. Lastly, we discuss the potential of common preclinical stroke models to contribute to our understanding of long-term outcomes following stroke.

Prenatal alcohol exposure exacerbates acute sensorimotor deficits and impedes long-term behavioral recovery from the effects of an adult-onset cerebrovascular ischemic stroke.

BACKGROUND: Prenatal alcohol exposure (PAE) is a significant risk factor for developmental disability, although its health consequences across the lifespan are poorly understood. Here, we hypothesized that latent brain and systemic consequences of PAE influence resiliency to adult-onset neurological disease, specifically, cerebrovascular ischemic stroke. METHODS: Pregnant Sprague-Dawley rats were exposed episodically to ethanol during the fetal neurogenic period. Adult (5 months) male and female PAE and control offspring were subjected to endothelin-1-induced unilateral middle cerebral artery occlusion. In the acute injury phase outcomes including stroke volume and neurological, endocrine, and gut permeability markers were assessed. Because the effects of stroke in human populations evolve over months to years, we also assessed hippocampal- and amygdala-dependent memory function and social interaction preference up to 6 months following a stroke, in middle-aged offspring. RESULTS: Prenatal alcohol exposure did not alter infarct volume, but significantly increased neurological deficits in both sexes, and impaired interhemispheric sensorimotor integration in PAE females. The IGF-1/IGFBP3 ratio, a measure of bioavailable IGF-1, was significantly reduced, while circulating levels of bacterial lipopolysaccharide, an inflammagen, were significantly increased in PAE males. In PAE females, the circulating IGF-1/IGFBP3 ratio was significantly increased and estradiol-17b levels were significantly reduced. The intestinal fatty acid binding protein, a surrogate marker of gut permeability was also significantly increased in PAE females. Longer-term deficits in hippocampal-associated memory and social interactions were observed in PAE males, while deficits in amygdala-dependent memory were observed in PAE females. CONCLUSIONS: PAE contributes to adverse effects on brain health and decreased resiliency in response to a common adult-onset neurovascular disease, cerebrovascular ischemic stroke.

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.

Activation of G protein-coupled estrogen receptor fine-tunes age-related decreased vascular activities in the aortae of female and male rats.

BACKGROUND: Hormone replacement therapy was found to be effective in cardiovascular protection only in younger women, not in older women. In this study, we tested whether G protein-coupled estrogen receptor 1 (GPER) activation improves vascular activities in response to ET-1 and ACh in aging rats. METHODS: Isometric tension study was applied on aortic rings isolated from young adult (5-7 months) and reproductive senescent middle-aged (10-12 months) female Sprague Dawley rats and age matched males. RESULTS: The aortic contractile response to ET-1 and the relaxation response to ACh were reduced in the female middle-aged rats compared to the female young adult rats. The presence of G-1, the GPER agonist, normalized the reduced vascular activities. Cyclooxygenase inhibitor, meclofenamate, blocked the increased constriction effect of G-1, but further enhanced relaxation effect of G-1. There was no significant difference in aortic reactivity to either ET-1 or ACh between the male middle-aged and young adult rats. The contractile response to ET-1 was not different within the same age of the two sex groups, but there was a remarkable difference in relaxation response to ACh between young adult females and males with better response in females. GPER activation greatly improved the aortic relaxation of both young adult and middle-aged females, but not the males. CONCLUSIONS: Endothelial dysfunction occurs earlier in males, but in females, dysfunction delays until middle age. GPER activation improves the vascular activities in females, but not males. It is promising to employ GPER as a potential drug target in cardiovascular disease in women.

Impact of intestinal disorders on central and peripheral nervous system diseases.

Brain injuries and neurological diseases have a significant impact on the gut microbiome and the gut barrier. Reciprocally, gut disorders, such as Inflammatory Bowel Syndromes (IBS), can affect the development and pathology of neurodegenerative and neuropsychiatric diseases, although this aspect is less well studied and is the focus of this review. Inflammatory Bowel Syndrome (IBS) is a chronic and debilitating functional gastrointestinal disorder afflicting an estimated 9-23% of the world's population. A hallmark of this disease is leaky gut, a pathology in which the integrity of the gut blood barrier is compromised, causing gut contents such as immune cells and microbiota to enter the bloodstream leading to low-grade systemic inflammation. The increased levels of inflammation associated cytokines in circulation has the potential to affect all organs, including the brain. Although the brain is protected by the blood brain barrier, inflammation associated cytokines can damage the junctions in this barrier and allow brain infiltration of peripheral immune cells. Central inflammation in the brain is associated with various neurodegenerative disease such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and neuropsychiatric disorders, namely, depression, and anxiety. Neurodegenerative diseases are of particular concern due to the anticipated rise in the population of the elderly and consequently, the prevalence of these diseases. Additionally, depression and anxiety are the most common mental illnesses affecting roughly 18% of the American population. In this review, we will explore the mechanisms by which IBS can influence the risk and severity of neurological disease.