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Introduction: Vascular cognitive impairment (VCI) is the second most common cause of clinical dementia after Alzheimer’s disease. VCI results from injury to the cerebral blood vessels. Cerebral perfusion is diminished in elderly individuals and additional reduction of cerebral blood flow increases the risk of developing VCI. These findings have been successfully modeled in mice with bilateral common carotid artery stenosis (BCAS). Age is associated with gut dysbiosis and transplantation of aged microbiome leads to cognitive decline in young animals. However, there are large gaps in our understanding of the molecular mechanisms induced by chronic hypoperfusion contributes to impaired cognitive function. Methods: C57Bl6 aged (~18m) male mice were subjected to a sham or a BCAS surgery using 0.18mm titanium coils placed on both common carotid arteries. Mice were followed for 90d after surgery to assess both gut microbial content and behavioral changes. 16S ribosomal RNA (rRNA) sequencing analysis was performed on fecal samples collected from aged baseline, sham and BCAS animals at 7 and 28 days. Cognition was assessed using Y-maze. Tissues were collected at the time of euthanasia for metabolomics and histological analysis. Results: BCAS resulted in significant reduction of cerebral blood flow, measured using laser speckle (p Conclusions: We found that chronic cerebral hypoperfusion is associated with significant changes in gut microbiome and cognitive impairment in aged mice. Importantly, shifts in the microbiome preceded cognitive decline. These findings suggest that targeting these detrimental changes in the gut microbiome might be a novel therapeutic strategy to delay or prevent progression of VCI.

Background: The kynurenine pathway (KP) is the major route of tryptophan catabolism in mammals. KP branches into two major pathways - one (dependent on the kynurenine aminotransferases) implicated in neuroprotection, the other (dependent on the enzyme kynurenine-3-monooxygenase (KMO) in neurotoxicity. Under physiological conditions, the neuroprotective branch is more active, but under inflammatory conditions, such as seen in stroke, metabolism is shifted through the KP via KMO to produce toxic metabolites, including quinolinic acid. Recent studies have demonstrated that stroke leads to the activation of the KP. Here we tested whether inhibiting KMO activity after stroke could prevent and/or minimize the neuronal death and improve outcomes. Methods: WT C57Bl6 aged male mice were subjected to a 60-minute reversible middle cerebral artery occlusion (MCAO). Tissues collected from a sub-cohort of these mice was used for metabolomics. Other sub-cohort of mice was used for pharmacological studies. For this, mice received either an inhibitor of KMO (Ro 61-8048, 40mg/kg/day i.p) or vehicle immediately after stroke, and infarct was evaluated at day 3 post-stroke. Neurological deficit scores and open field analysis was performed to assess recovery. A separate cohort of aged WT and KMO knockout (KMO-/-) mice was also subjected to MCAO and assessed for infarct and recovery at 3 days after stroke to further evaluate the involvement of KMO on outcome. Results: Stroke caused significant reduction in the aged brain kynurenic acid/quinolinic acid (KYNA/QUIN) ratio, suggesting activation of the neurotoxic/KMO branch of the KP pathway. KMOi treatment administered immediately after the stroke reduced ischemic infarct size (p Conclusions: Inhibition or deletion of KMO reduces neuronal death and improves motor function after stroke in aged male mice. Further studies are needed to ascertain if beneficial effects of KMOi on post-stroke recovery is not due to smaller injury, using aged animals of both sexes.

The establishment of macromolecular complexes by scaffolding proteins is key to the local production of cAMP by anchored adenylyl cyclase (AC) and the subsequent cAMP signaling necessary for cardiac functions. We identify a novel AC scaffold, the Popeye domain-containing (POPDC) protein. The POPDC family of proteins is important for cardiac pacemaking and conduction, due in part to their cAMP-dependent binding and regulation of TREK-1 potassium channels. We show that TREK-1 binds the AC9:POPDC1 complex and copurifies in a POPDC1-dependent manner with AC9 activity in heart. Although the AC9:POPDC1 interaction is cAMP-independent, TREK-1 association with AC9 and POPDC1 is reduced upon stimulation of the β-adrenergic receptor (βAR). AC9 activity is required for βAR reduction of TREK-1 complex formation with AC9:POPDC1 and in reversing POPDC1 enhancement of TREK-1 currents. Finally, deletion of the gene-encoding AC9 (Adcy9) gives rise to bradycardia at rest and stress-induced heart rate variability, a milder phenotype than the loss of Popdc1 but similar to the loss of Kcnk2 (TREK-1). Thus, POPDC1 represents a novel adaptor for AC9 interactions with TREK-1 to regulate heart rate control.

Background and Purpose: Stroke induces the expression of several long noncoding RNAs in the brain. However, their functional significance in poststroke outcome is poorly understood. We recently observed that a brain-specific long noncoding RNA called Fos downstream transcript (FosDT) is induced rapidly in the rodent brain following focal ischemia. Using FosDT knockout rats, we presently evaluated the role of FosDT in poststroke brain damage. Methods: FosDT knockout rats were generated using CRISPR-Cas9 genome editing on a Sprague-Dawley background. Male and female FosDT −/− and FosDT +/+ cohorts were subjected to transient middle cerebral artery occlusion. Postischemic sensorimotor deficits were evaluated between days 1 and 7 and lesion volume on day 7 of reperfusion. The developmental expression profile of FosDT was determined with real-time polymerase chain reaction and mechanistic implications of FosDT in the ischemic brain were conducted with RNA-sequencing analysis and immunostaining of pathological markers. Results: FosDT expression is developmentally regulated, with the adult cerebral cortex showing significantly higher FosDT expression than neonates. FosDT −/− rats did not show any anomalies in growth and development, fertility, brain cytoarchitecture, and cerebral vasculature. However, when subjected to transient focal ischemia, FosDT −/− rats of both sexes showed enhanced sensorimotor recovery and reduced brain damage. RNA-sequencing analysis showed that improved poststroke functional outcome in FosDT −/− rats is partially associated with curtailed induction of inflammatory genes, reduced apoptosis, mitochondrial dysfunction, and oxidative stress. Conclusions: Our study shows that FosDT is developmentally dispensable, mechanistically important, and a functionally promising target to reduce ischemic brain damage and facilitate neurological recovery.

Background Probiotic Lactobacillus reuteri DSM 17938 (LR 17938) is beneficial to infants with colic. To understand its mechanism of action, we assessed ultrasonic vocalizations (USV) and brain pain/stress genes in newborn mice exposed to maternal separation stress. Methods Pups were exposed to unpredictable maternal separation (MSU or SEP) or MSU combined with unpredictable maternal stress (MSU + MSUS or S + S), from postnatal days 5 to 14. USV calls and pain/stress/neuroinflammation-related genes in the brain were analyzed. Results We defined 10 different neonatal call patterns, none of which increased after MSU. Stress reduced overall USV calls. Orally feeding LR 17938 also did not change USV calls after MSU. However, LR 17938 markedly increased vocalizations in mice allowed to stay with their dams. Even though LR 17938 did not change MSU-related calls, LR 17938 modulated brain genes related to stress and pain. Up-regulated genes following LR 17938 treatment were opioid peptides, kappa-opioid receptor 1 genes, and CD200, important in anti-inflammatory signaling. LR 17938 down-regulated CCR2 transcripts, a chemokine receptor, in the stressed neonatal brain. Conclusions USV calls in newborn mice are interpreted as "physiological calls" instead of "cries." Feeding LR 17938 after MSU did not change USV calls but modulated cerebral genes favoring pain and stress reduction and anti-inflammatory signaling. Impact We defined mouse ultrasonic vocalization (USV) call patterns in this study, which will be important in guiding future studies in other mouse strains. Newborn mice with maternal separation stress have reduced USVs, compared to newborn mice without stress, indicating USV calls may represent "physiological calling" instead of "crying." Oral feeding of probiotic Lactobacillus reuteri DSM 17938 raised the number of calls when newborn mice continued to suckle on their dams, but not when mice were under stress. The probiotic bacteria had a dampening effect on monocyte activation and on epinephrine and glutamate-related stress gene expression in the mouse brain.

Background: Stroke is a major cause of death and disability worldwide. Among its complications, post-stroke depression (PSD) leads to a significant burden. The diagnosis of PSD is complex, and there are no biomarkers that can assist in its early identification and adequate management. Objective: The aim of the present study is to investigate peripheral biomarkers in the acute phase of stroke and their potential association with depressive symptoms. Methods: We evaluated 60 patients in the acute phase of stroke by using standardized instruments of psychiatric and neurological assessment (Mini International Neuropsychiatric Interview-Plus- MINI-Plus, Hospital Anxiety and Depression Scale-HADS, and National Institutes of Health Stroke Scale-NIHSS) and measured peripheral biomarkers. Results: In multivariate analysis, low peripheral levels of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) and higher NIHSS scores were associated with PSD. The severity of depressive symptoms was inversely correlated with sTREM-1 and glial cell-derived neurotrophic factor (GDNF) levels. Conclusion: This is the first study indicating an association between sTREM-1 and PSD. Our results may point to the involvement of glial mechanisms in the manifestation of depressive symptoms after stroke.

The establishment of macromolecular complexes by scaffolding proteins such as A-kinase anchoring proteins is key to the local production of cAMP by anchored adenylyl cyclase (AC) and the subsequent cAMP signaling necessary for many cardiac functions. We have identified herein a novel AC scaffold, the Popeye domain-containing (POPDC) protein. Unlike other AC scaffolding proteins, POPDC1 binds cAMP with high affinity. The POPDC family of proteins are important for cardiac pacemaking and conduction, due in part to their cAMP-dependent binding and regulation of TREK-1 potassium channels. TREK-1 binds the AC9:POPDC1 complex and co-purifies in a POPDC1-dependent manner with AC9-associated activity in heart. Although the interaction of AC9 and POPDC1 is cAMP independent, TREK-1 association with AC9 and POPDC1 is reduced in an isoproterenol-dependent manner, requiring an intact cAMP binding Popeye domain and AC activity within the complex. We show that deletion of Adcy9 (AC9) gives rise to bradycardia at rest and stress-induced heart rate variability. The phenotype for deletion of Adcy9 is milder than previously observed upon loss of Popdc1, but similar to loss of Kcnk2 (TREK-1). Thus, POPDC1 represents a novel scaffolding protein for AC9 to regulate heart rate control.ONE-SENTENCE SUMMARYAdenylyl cyclase type 9 binds in an isoproterenol-dependent manner to the POPDC1:TREK-1 complex regulating heart rate.

Inter-α inhibitor proteins (IAIPs) are a family of endogenous plasma and extracellular matrix molecules. IAIPs suppress proinflammatory cytokines, limit excess complement activation, and bind extracellular histones to form IAIP-histone complexes, leading to neutralization of histone-associated cytotoxicity in models of sepsis. Many of these detrimental processes also play critical roles in the pathophysiology of ischemic stroke. In this study, we first assessed the clinical relevance of IAIPs in stroke and then tested the therapeutic efficacy of exogenous IAIPs in several experimental stroke models. IAIP levels were reduced in both ischemic stroke patients and in mice subjected to experimental ischemic stroke when compared with controls. Post-stroke administration of IAIP significantly improved stroke outcomes across multiple stroke models, even when given 6 hours after stroke onset. Importantly, the beneficial effects of delayed IAIP treatment were observed in both young and aged mice. Using targeted gene expression analysis, we identified a receptor for complement activation, C5aR1, that was highly suppressed in both the blood and brain of IAIP-treated animals. Subsequent experiments using C5aR1-knockout mice demonstrated that the beneficial effects of IAIPs are mediated in part by C5aR1. These results indicate that IAIP is a potential therapeutic candidate for the treatment of ischemic stroke.

The role of oxytocin (OT) in social cognition of patients with Huntington’s disease (HD) has been studied, but its impact on executive functioning has not been explored yet. Healthy controls, premanifest HD, and manifest HD participants underwent executive functioning assessment and OT plasma measurement. There were no significant group differences in plasma OT levels. Higher OT levels were associated with better executive functioning in premanifest HD participants. Our findings revealed an association between OT levels and depressive symptoms in premanifest and manifest HD participants. The potential role of OT in HD deserves further investigation.

Stroke induces the expression of several long noncoding RNAs (lncRNAs) in the brain. However, their functional significance in post-stroke outcome is poorly understood. We recently observed that a brain-specific lncRNA called Fos downstream transcript (FosDT) is induced rapidly in rodent brain following focal ischemia. We here show that FosDT expression is developmentally regulated with adults specifically display higher expression in the cerebral cortex than neonates. To understand its significance in ischemic brain damage, we developed FosDT knockout rats using CRISPR-Cas9 genome editing. We found that FosDT knockout rats did not show any anomalies in growth and development, fertility, brain cytoarchitecture and cerebral vasculature. However, when subjected to transient focal ischemia, FosDT knockout rats of both sexes showed enhanced sensorimotor recovery and reduced brain damage. To further understand the mechanistic implications of FosDT in the ischemic brain, we conducted RNA-seq analysis. The result showed that improved post-stroke functional outcome in FosDT knockout rats is partially associated with curtailed post-ischemic induction of inflammatory genes. When rats subjected to transient focal ischemia were treated with FosDT siRNA, there was significant neuroprotection and better functional outcome irrespective of sex and age. FosDT siRNA was efficacious when administered peripherally and also in a delayed manner. Thus, preventing FosDT activation is beneficial for the post-stroke outcome.

Introduction: Acute ischemic stroke patients with cardiac complications have increased in-hospital mortality. Most of these cardiac complications have been attributed to pre-existing cardiac comorbidities. Aging is a known risk factor for both stroke and myocardial injury. It is being increasingly recognized that ischemic injury to the brain in itself may lead to cardiac injury. However, the post stroke brain- heart interaction is not well defined in the aged. This study was designed to fill this gap in our knowledge. Methods: C57BL6/J young (3 months) and aged (18 months) male mice were subjected to 60 minutes of middle cerebral artery occlusion (MCAO). Mice had electrocardiogram (EKG, using rodent surgical monitor) and Echocardiogram (using vevo 3100 imaging system) pre-stroke, post stroke day 3, 7, and 14. All mice were sacrificed at day 14. EKG and ECHO results were analyzed using generalized estimated equation method with post hoc group comparison test at each time point, Bonferroni correction. Results: There was a significant decrease in cardiac output after stroke in aged but not young mice at three days post stroke, as compared to baseline (young, baseline:18.5±1.6ml/min vs day 3:16.6±5.16 ml/min, n=8; aged, baseline 25.4±6.8 6 ml/min vs. day 3, 16.8±6.8 n=6, p=0.0038). The cardiac output normalized to baseline in young but remained significantly low in aged mice even at day 14. Interestingly, the Left ventricular (LV) mass decreased significantly after stroke in aged mice at day 3, 118.4±23.2 mg as compared to baseline, 160.7±22.9 mg. This remained significantly lower than baseline at post stroke day seven, 124.6±32.9 mg and fourteen, 129.5±37.2 mg, p0.05. Conclusion: Our data indicates that cardiac output decreases after stroke in aged mice. Interestingly, there is a significant decrease in left ventricular mass and ventricular wall thickness in aged mice after MCAO, indicating cardiac atrophy. We speculate this may be secondary to cardiac ischemia caused by stroke. Ongoing work in our laboratory is aimed at examining the histological changes in cardiomyocytes after MCAO.

Background: Social isolation (SI) and loneliness are risk factors for stroke. Epidemiological studies have shown that women tend to have a higher risk of stroke at later age and elderly women are more likely to be isolated. The mechanisms underlying the detrimental effects of SI have not been well studied in older females. We hypothesized that SI in aged female mice would lead to impaired post-stroke recovery and could lead to differential regulation of microRNAs (miRNAs). Methods: In this study, aged C57BL/6N female mice were subjected to a 60-minute middle cerebral artery occlusion (MCAO) and were randomly assigned to either single housing (SI) or continued pair housing (PH) immediately after stroke for 15 days. Infarct size, mortality and recovery was assessed using open field, the adhesive-tape removal task and the Y-maze test. MiRNAs were comprehensively analyzed by miRNAome analysis on stroke brain, and changes in hippocampal cell proliferation was assessed from perfused brain sections. Results: Importantly, SI immediately after stroke led to significantly larger tissue loss and higher mortality in aged females, it also significantly delayed motor/sensory recovery in the adhesive removal test and impaired overall locomotor activity. In addition, these mice also demonstrated worse post-stroke cognitive function. In parallel, brains of these mice showed reduced miR-297a-3p expression and increased miR-18a-3p and miR-200c-3p expression with SI compared to PH cohort and reduced hippocampal cell proliferation. Conclusion: The results from this study suggest that SI after stroke can increase mortality and significantly impair post-stroke recovery in aged female mice. These worse outcomes are in parallel to the significant changes in several miRNAs and reduced hippocampal cell proliferation.

Social isolation and loneliness are risk factors for stroke. Elderly women are more likely to be isolated. Census data shows that in homeowners over the age of 65, women are much more likely to live alone. However, the underlying mechanisms of the detrimental effects of isolation have not been well studied in older females. In this study, we hypothesized that isolation impairs post-stroke recovery in aged female mice, leading to dysregulated microRNAs (miRNAs) in the brain, including those previously shown to be involved in response to social isolation (SI). Aged C57BL/6 female mice were subjected to a 60-min middle cerebral artery occlusion and were randomly assigned to either single housing (SI) or continued pair housing (PH) immediately after stroke for 15 days. SI immediately after stroke led to significantly more brain tissue loss after stroke and higher mortality. Furthermore, SI significantly delayed motor and sensory recovery and worsened cognitive function, compared to PH. A decrease in cell proliferation was seen in the dentate gyrus of SI mice assessed by bromodeoxyuridine (BrdU) labeling. miRNAome data analysis revealed changes in several miRNAs in the brain, such as miR-297a-3p and miR-200c-3p, which are known to regulate pathways involved in cell proliferation. In conclusion, our data suggest that SI can lead to a poor post-stroke recovery in aged females and dysregulation of miRNAs and reduced hippocampal cell proliferation.

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.

Introduction: Stroke is a sexually dimorphic disease. Notably, emerging evidence suggests that stroke leads to gut dysbiosis and pathological shifts in the gut microbiota. However, little is known about how the gut epithelial surface maintains homeostasis after stroke. In this study, we isolated intraepithelial lymphocytes (IELs), which are mainly CD8 + T cells and do not need priming against antigens, as well as intestinal epithelial cells (IECs). We examined their responses to stroke-induced gut epithelial damage. Methods: C57BL/6 (2-3 months) mice of both sexes were subjected to either 60-minute middle cerebral artery occlusion or sham surgery. At day 7 after stroke, IECs and IELs, which are intercalated between IECs, were isolated from mouse colonic tissues using Percoll density gradients. RNA was isolated from the cells and amplified to quantitate expression levels of cell-specific genes for antibacterial proteins and chemotactic cytokines. Tissues were then used for histological analysis by hematoxylin and eosin staining and bacterial fluorescence in situ hybridization (FISH). Results: Stroke caused significant colonic damage in male mice, compared to female mice. Bacterial FISH demonstrated microbial encroachment into the host gut epithelium in males after stroke; whereas female mice maintained spatial segregation between the host gut and bacteria. Stroke increased expression levels of pro-inflammatory cytokine genes in male IELs including IL-1β (28.3 fold increase, P P P P Conclusions: This is the first study that demonstrates robust sex differences in lymphocyte responses at the gut mucosal surface between males and females following stroke. The closer proximity of the gut microbiota with the host may direct gene expression related to microbial responses in the injured epithelium after brain injury. Our study provides novel insight into host-microbial homeostasis in stroke.

Introduction: Oxytocin (OXT) is a highly evolutionary conserved neuropeptide and an important modulator of inflammation in response to stressors such as ischemia. Recent studies have implicated OXT signaling in neuroprotection in young mice after ischemic injury, but it is not known if OXT is efficacious in aged animals. In our ongoing studies, we found that OXT levels decline after stroke and with age. Therefore, we tested if exogenous OXT administration could provide beneficial effects and improve recovery in aged animals. Methods: Aged (18-20m) C57BL/6 male mice were purchased from Jax Labs and aged in house. Stroke was induced by a right MCAO-60min. Mice were randomly assigned to receive OXT or Atosiban, an OXT antagonist (OXTA) (0.5mg/kg) or vehicle. In the first set of experiments, aged mice were pre-treated with OXT, vehicle, or OXTA for 10 days before stroke. Infarcts were quantified 7-days after stroke (n=6-9/grp). In a second set, we tested the efficacy of delayed treatment, at 3hrs after stroke, and OXT was administered intranasally or i.p.. Functional and behavioral recovery and brain infarct size was assessed 7-day post-stroke. OXT levels 24hrs after stroke in human plasma were measured. Results: OXT treatment (0.5mg/kg/i.p. QD) prior to stroke led to significant neuroprotection in aged male mice, whereas OXTA treatment significantly increased infarct (p Conclusions: We found that OXT treatment either before or after stroke is neuroprotective in aged mice and both i.p. and nasal OXT administration improves recovery in parallel to reductions in brain injury. Given that intranasal formulations of OXT are approved for human use, our proposed strategy of supplementing OXT is highly translatable.

Introduction: Epidemiological and clinical studies strongly support that social isolation (SI) is associated with higher mortality after stroke. SI has been identified as a risk factor in a wide variety of diseases, including stroke. However, few studies have attempted to identify the underlying mechanisms contributing to the detrimental effect of SI. To better understand systemic and global regulation networks governed by social factors, and in an attempt to identify viable biomarkers, we profiled changes in circulating microRNA (miRNA) expression in aged stroke mice after SI. Methods: Aged C57BL/6 mice (18-20 months) of both sexes were pair-housed (PH) or socially isolated (SI) for two weeks prior to sham or a 60-minute middle cerebral artery occlusion surgery. Mice were sacrificed two weeks after surgery and blood samples were obtained for profiling of plasma miRNAs. Using whole miRNAome analysis, differentially expressed miRNAs by isolation, stroke and sex differences were comprehensively identified and compared using bioinformatics. We further used mimic and antagomiR treatments to confirm a potential role of the identified miRNAs. Results: Stroke significantly changed 8 miRNAs including miR-467d-3p ( P =0.0014), miR-376b-3p ( P =0.013) and miR-297c-5p ( P =0.016) in PH groups (sham vs. stroke). In a comparison between PH vs. SI in stroke mice, stroke SI mice showed a differential miRNA profile (11 miRNAs) including let-7 family potentially targeting Dicer protein, a key enzyme for miRNA biogenesis. Interestingly, compared to sham SI mice, stroke SI mice had a 2-fold increase in the number of altered miRNAs (21 miRNAs) including miR-129-1-3p ( P =0.0075), miR-28a-3p ( P =0.0091) and miR-204-5p ( P =0.016). Finally, we found a strong sex difference in miRNA expression in response to SI. Conclusions: We identified several key miRNAs as potential biomarkers for 1) stroke, 2) SI and 3) SI/stroke. These miRNA signatures are highly unique and create a complex gene-regulatory network that depends on the social environment. Our data provide potential biomarkers and identify several miRNAs that could lead to significant improvements in diagnosis of elderly patients at elevated risk for the detrimental effects of loneliness.

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

The peripheral immune system plays a critical role in aging and in the response to brain injury. Emerging data suggest inflammatory responses are exacerbated in older animals following ischemic stroke; however, our understanding of these age-related changes is poor. In this work, we demonstrate marked differences in the composition of circulating and infiltrating leukocytes recruited to the ischemic brain of old male mice after stroke compared to young male mice. Blood neutrophilia and neutrophil invasion into the brain were increased in aged animals. Relative to infiltrating monocyte populations, brain-invading neutrophils had reduced phagocytic potential, and produced higher levels of reactive oxygen species and extracellular matrix-degrading enzymes (i.e., MMP-9), which were further exacerbated with age. Hemorrhagic transformation was more pronounced in aged versus young mice relative to infarct size. High numbers of myeloperoxidase-positive neutrophils were found in postmortem human brain samples of old (> 71 years) acute ischemic stroke subjects compared to non-ischemic controls. Many of these neutrophils were found in the brain parenchyma. A large proportion of these neutrophils expressed MMP-9 and positively correlated with hemorrhage and hyperemia. MMP-9 expression and hemorrhagic transformation after stroke increased with age. These changes in the myeloid response to stroke with age led us to hypothesize that the bone marrow response to stroke is altered with age, which could be important for the development of effective therapies targeting the immune response. We generated heterochronic bone marrow chimeras as a tool to determine the contribution of peripheral immune senescence to age- and stroke-induced inflammation. Old hosts that received young bone marrow (i.e., Young → Old) had attenuation of age-related reductions in bFGF and VEGF and showed improved locomotor activity and gait dynamics compared to isochronic (Old → Old) controls. Microglia in young heterochronic mice (Old → Young) developed a senescent-like phenotype. After stroke, aged animals reconstituted with young marrow had reduced behavioral deficits compared to isochronic controls, and had significantly fewer brain-infiltrating neutrophils. Increased rates of hemorrhagic transformation were seen in young mice reconstituted with aged bone marrow. This work suggests that age alters the immunological response to stroke, and that this can be reversed by manipulation of the peripheral immune cells in the bone marrow. Electronic supplementary material The online version of this article (10.1007/s00401-018-1859-2) contains supplementary material, which is available to authorized users.

Ischemic stroke triggers a series of complex pathophysiological processes including autophagy. Differential activation of autophagy occurs in neurons derived from males versus females after stressors such as nutrient deprivation. Whether autophagy displays sexual dimorphism after ischemic stroke is unknown. We used a cerebral ischemia mouse model (middle cerebral artery occlusion, MCAO) to evaluate the effects of inhibiting autophagy in ischemic brain pathology. We observed that inhibiting autophagy reduced infarct volume in males and ovariectomized females. However, autophagy inhibition enhanced infarct size in females and in ovariectomized females supplemented with estrogen compared to control mice. We also observed that males had increased levels of Beclin1 and LC3 and decreased levels of pULK1 and p62 at 24 h, while females had decreased levels of Beclin1 and increased levels of ATG7. Furthermore, the levels of autophagy markers were increased under basal conditions and after oxygen and glucose deprivation in male neurons compared with female neurons in vitro. E2 supplementation significantly inhibited autophagy only in male neurons, and was beneficial for cell survival only in female neurons. This study shows that autophagy in the ischemic brain differs between the sexes, and that autophagy regulators have different effects in a sex-dependent manner in neurons.

Background: Stroke is the second leading cause of death after ischemic heart disease and the third leading cause of disability-adjusted life-years lost worldwide. There is a great need for developing more effective strategies to treat stroke and its resulting impairments. Among several neuroprotective strategies tested so far, the kynurenine pathway (KP) seems to be promising, but the evidence is still sparse. Methods: Here, we performed a systematic review of preclinical and clinical studies evaluating the involvement of KP in stroke. We searched for the keywords: (“kynurenine” or “kynurenic acid” or “quinolinic acid”) AND (“ischemia” or “stroke” or “occlusion) in the electronic databases PubMed, Scopus, and Embase. A total of 1,130 papers was initially retrieved. Results: After careful screening, forty-five studies were included in this systematic review, being 39 pre-clinical and six clinical studies. Despite different experimental models of cerebral ischemia, the results are concordant in implicating the KP in the pathophysiology of stroke. Preclinical evidence also suggests that treatment with kynurenine and KMO inhibitors decrease infarct size and improve behavioral and cognitive outcomes. Few studies have investigated the KP in human stroke, and results are consistent with the experimental findings that the KP is activated after stroke. Conclusion: Well-designed preclinical studies addressing the expression of KP enzymes and metabolites in specific cell types and their potential effects at cellular levels alongside more clinical studies are warranted to confirm the translational potential of this pathway as a pharmacological target for stroke and related complications.

Introduction: Infections are a common cause of post-stroke morbidity and mortality. Recent studies suggest that gut microbiota are a major source of post-stroke infection. Gut mucus layers are a major area of communication between gut microbiota and host immune cells. To our knowledge there have been no studies evaluating the role of colonic epithelial mucins, where most of the gut bacteria reside, after ischemic stroke. Hypothesis: Epithelial mucin plays a protective role in stroke-induced colonic disruption. Methods: Mice were subjected to either sham or 60-minute middle cerebral artery occlusion (MCAO). Neurological deficit score (NDS) and levels of lipopolysaccharide-binding protein (LBP) in plasma were assessed at day 7 after stroke. Mice were sacrificed at day 7 after stroke and colonic epithelial cells (ECs) were isolated for a gene expression study. Tissues were used for histology and bacterial in situ hybridization. Fecal samples were collected for 16S sequencing and IgA ELISA. Results: Young female mice had lower NDS and LBP levels, with intact colonic structures (n=4/group, P P P Akkermansia bacteria and IgA in the feces after stroke (n=4/group, P Conclusions: Our data demonstrates that 1) stroke provokes a colonic epithelial response and 2) mucin is critical for colonic protection in stroke. In conclusion, mucin should be considered as a novel target to improve post-stroke recovery as well as an important element in maintaining colonic structure.

Introduction: Stroke is a sexually dimorphic disease. There is growing evidence that there are sex differences in the response to drugs, both in experimental and in clinical studies. Increase in BBB permeability occurs after stroke, but little is known if BBB disruption and permeability differs in males and females. In our ongoing studies with Inter-alpha Inhibitor Protein (IAIP), we found that although IAIP is significantly efficacious in both sexes, it is more effective in males. Therefore, here we tested if the sex-specific differences in efficacy of IAIP is due to BBB permeability. Methods: Aged male and female mice (18-20 months; C57BL/6), were subjected to right middle cerebral artery occlusion (MCAO-60min) and randomly assigned to a vehicle/IAIP (30mg/kg) group. Infarcts were quantified at 7-days after stroke. Transport of IAIP across the BBB in vivo was directly investigated using 131 I-IAIP and 125 I-albumin (n=4/gp) at 6h & 24h after injection; Radioactivity was determined by a gamma counter. Brain and tissue uptake of 131 I-IAIP was determined by comparing to 125 I-albumin. Results: Aged males receiving IAIP had significantly reduced infarcts compared to vehicle treated mice in cortex (p=0.01), striatum (p=0.01) and in total hemispheric infarct (p=0.018) (n=8-10/grp). Female mice treated with IAIP also had a significant reduction in total infarct (p=0.04), but not in cortex or striatum. IAIP treated males had significantly lower mortality (p131 I-IAIP and 125 I-albumin in aged males was significantly higher than in shams, confirming brain entry. However, uptake of IAIP was only seen in males (P Conclusions: Our data demonstrates that 1) IAIP treatment is neuroprotective in both sexes, but more efficacious in males and 2) IAIP do not significantly cross the BBB early after stroke/reperfusion (6h), but crosses in a disruption-dependent manner at 24h, but only in males. This may be one explanation for the more robust neuroprotective effects seen in aged males vs. females. These studies may have far-reaching implications for other drug development strategies.

Introduction: Peyer’s patches (PP) are lymphatic tissue in the small intestine. Tissue-resident specialized epithelial cells [microfold (M) cells] are essential for immune surveillance in the gut by sensing pathogens and eliciting immune responses by lymphocytes. Emerging evidence highlights the importance of gut microbiota as an epicenter for post-stroke infection, however the role of PP and M cells in stroke is still undefined. Hypothesis: Ischemic stroke influences the antigen-sampling systems in intestinal PPs. Methods: Young (8-10 wks) and aged (18-20 months) C57BL/6N male mice were subjected to either sham or 60-minute middle cerebral artery occlusion (MCAO) followed by reperfusion. Mice were sacrificed at day 3 after stroke. The small intestines were isolated and PP tissue was obtained. The numbers and structure of the PP and M cells were assessed using whole-mounts and confocal microscopy. Gene expression in PP was examined using real-time qPCR. Finally, immune cells in PPs were evaluated using flow cytometry. Results: The number of PPs was dramatically reduced at day 3 after stroke. Sham mice had 7 to 10 PPs in their small intestines, whereas stroke mice only retained 2 to 5 PPs (n=4/group, P P + WGA - M cells as well as the structure of the PP. Stroke induced the loss and shrinkage of villus and decreased both villus M cells and fucosylated epithelial cells in the distal ileum. Additionally, stroke abrogated the mRNA expression of Gp2 and RANKL pathway genes such as Tnfsf11 (RANK) and Tnfsf11a (RANKL) that are responsible for M cell maturation and function, respectively (1.95, 1.31 and 2.67 fold change in Gp2, Tnfsf11 and Tnfsf11a, respectively, n=4/group, P Conclusions: The response of PP in stroke was highly epithelial M cell-specific. Although the understanding of interactions between luminal antigens including bacteria and PP is still needed, our data provides us with a new insight into how brain damage can alter antigen sampling and shape adaptive immunity in the host.

Published manuscript

Ketamine has been used safely in clinics for decades for analgesia and anesthesia. It is increasingly popular in clinical practice due to its new uses and importance for emergency procedures. It is known that ketamine is sequestered in the bone marrow and the major receptors for ketamine, noncompetitive N-methyl-d-aspartate receptors (NMDARs), are expressed in osteoclasts (OCs) and osteoblasts. However, the impact of ketamine on OCs or osteoblasts is unknown. In this study, we investigated the effects of ketamine on osteoclastogenesis and regulation of NMDARs expression in vitro. Bone marrows (BMs) or bone marrow macrophages (BMMs) were cultured in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) with or without ketamine for up to 6 days. OC formation peaked at day 5. On day 5 of culture, ketamine inhibited OC formation from both BM and BMM cultures at clinically relevant concentrations (3-200 µM). Ketamine inhibited RANKL-induced expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) in BMM cultures. Inhibition of ketamine on RANKL-induced osteoclastogenesis is associated with down-regulation of NMDARs. In addition, ketamine significantly inhibited the M-CSF induced migration of BMMs, inhibited cell fusion and significantly increased mature OC apoptosis. We conclude that clinically relevant concentrations of ketamine inhibit OC formation in both BM and BMM cultures in vitro through inhibiting migration and fusion process and enhancing mature OC apoptosis. It is likely that ketamine regulates osteoclastogenesis, at least in part, via its effects on NMDAR expression. J. Cell. Biochem. 118: 914-923, 2017. © 2016 Wiley Periodicals, Inc.

Aging is an important risk factor for post-stroke infection, which accounts for a large proportion of stroke-associated mortality. Despite this, studies evaluating post-stroke infection rates in aged animal models are limited. In addition, few studies have assessed gut microbes as a potential source of infection following stroke. Therefore we investigated the effects of age and the role of bacterial translocation from the gut in post-stroke infection in young (8-12 weeks) and aged (18-20 months) C57Bl/6 male mice following transient middle cerebral artery occlusion (MCAO) or sham surgery. Gut permeability was examined and peripheral organs were assessed for the presence of gut-derived bacteria following stroke. Furthermore, sickness parameters and components of innate and adaptive immunity were examined. We found that while stroke induced gut permeability and bacterial translocation in both young and aged mice, only young mice were able to resolve infection. Bacterial species seeding peripheral organs also differed between young (Escherichia) and aged (Enterobacter) mice. Consequently, aged mice developed a septic response marked by persistent and exacerbated hypothermia, weight loss, and immune dysfunction compared to young mice following stroke.

Introduction: Age is a non-modifiable risk factor for stroke. The elderly have high mortality and delayed recovery after a stroke compared to younger patients, a finding that is recapitulated in murine models. Young animals that received aged biome after stroke had lower short chain fatty acids (SCFAs) in their feces and delayed recovery compared to young mice receiving young biome. SCFAs, generated primarily by gut bacteria are important signaling molecules for gut homeostasis. We hypothesize that fecal transplant from young donor/ SCFA producing bacteria into aged mice will improve recovery compared to mice that received aged biome. Methods: Aged C57BL/6 male mice (18-20 months) were subjected to middle cerebral artery occlusion and randomly assigned to one of two groups, either received: 1. aged biome or 2. young biome. In addition, a separate cohort of aged mice were randomized as above, and divided into two groups: 1. received a cocktail of SCFA producers + Inulin as substrate or 2. received vehicle control group. Mice were tested for motor and cognitive recovery for 14 days. Fecal samples were subjected to 16s sequencing and metabolomics. Tissues were collected at sacrifice and used for flow cytometry and IHC. Results: Aged mice that received young fecal transplants after stroke expressed higher SCFAs in their fecal samples (p Conclusions: Metabolites produced by young microbiome improve post-stroke recovery in aged mice, enhance SCFAs, and reduce inflammation. Although SCFA production appears to be a key contributor to recovery, more studies are needed to elucidate the underlying mechanisms in order to develop a therapeutic strategy for the treatment of stroke patients.

Introduction: Stroke triggers a robust inflammatory response, allowing leukocytes to cross the blood brain barrier. T cells infiltrate in an antigen-independent manner early after injury, leading to additional neuronal injury. The pro-inflammatory cytokine, interferon-gamma (IFNγ), upregulates programed death ligand-1 (PD-L1) expression. Engagement of PD-L1 and programed death-1 (PD-1) “calls off” the cytolytic activity of infiltrated T cells. The immunosuppressive activity of PD-L1 is modulated by ubiquitination and glycosylation. Glycogen synthase kinase 3β (GSK3β) interacts with PD-L1 and induces degradation of PD-L1. To date, the role of PD-L1 in endothelial cells (ECs) remains elusive. Hypothesis: PD-L1 in EC reduces neuroinflammation via protection of microvascular integrity. Methods: Human brain ECs, HBEC-5i, were subjected to IFNγ treatment in normal or oxygen-glucose deprivation (OGD) conditions. Western blot, Fluorescence-activated cell sorting analysis, PD-1 binding assays and an in vitro T cell killing assay were performed to investigate EC viability and PD-L1 function. Male mice (12 weeks) were subjected to a 60-minute middle cerebral artery occlusion (MCAO) and given either vehicle or GSK3β inhibitor VIII (4 mg/kg). We examined PD-L1 expression, blood vessel integrity and T cell activation in brain tissue using anti-PD-L1, p-GSK3β, CD31, and Granzyme B antibodies for immunohistochemistry (IHC). Results: IFNγ strongly induced PD-L1 expression in ECs. However, when ECs were exposed to OGD, IFNγ-mediated PD-L1 upregulation was compromised (from 48.9% to 31.4%). PD-L1 binding affinity of EC was also reduced after OGD (from 33.1% to 1.3%). Consistently, human (n=22) and mice (n=6) brain IHC staining found lower levels of PD-L1 and p-GSK3β in ECs and higher expression of Granzyme B in stroke samples compared to controls (p Conclusion: This study reveals a protective role of PD-L1 in ECs during early brain injury. Activation of GSK3β by stroke destabilized endothelial PD-L1. Thus, stabilizing PD-L1 or preventing downregulation of PD-L1 may maintain microvascular integrity and reduce neuroinflammation after stroke.

Introduction: Infection is an important complication in the acute phase after stroke which effects up to 65% of stroke patients. These infections can in turn cause sepsis, which is a major contributor to hospital mortality. Emerging evidence indicates that these infections originate from the host commensal microbiota. We have found that stroke leads to increased gut permeability and bacterial translocation and that aged mice are more susceptible to post-stroke infections compared to young. We further investigated the possibility of how the response to infection differs between young and aged mice. Hypothesis: Impaired gut epithelial-specific response after stroke in aged mice leads to increased risk of infections and impaired recovery. Methods: Young (8-10 wks) and aged (18-20 mos) male mice (n=4-5/group) were subjected to either sham or 60-minute middle cerebral artery occlusion (MCAO) followed by reperfusion. Mice were sacrificed at day 8 after stroke. One cohort of mice (n=3/group) was evaluated for the histology and hypoxic barrier integrity. Finally, colonic epithelial cells (cECs) were isolated and changes of the gene expression in cECs were examined (n=4-5/group). Results: Epithelial dysfunction was observed in aged mice, including a decrease in goblet cells and mucus layer ( P P P P Conclusions: Our data indicates that 1) both aging and stroke impairs gut integrity and possibly leads to increase of infections and 2) epithelial cells could differentially respond to external stimuli after stroke, in an age-dependent manner. The current study provides the first insight into how intestinal epithelium is involved in the brain-gut axis in stroke.

Introduction: Inter-alpha Inhibitor Proteins (IAIP) are a family of endogenous plasma and extracellular matrix molecules. IAIPs have been shown to down-regulate inflammation, inhibit destructive serine proteases, and bind extracellular histones to neutralize cytotoxicity. Interestingly, many of these factors also regulate neuroprotection after stroke. In our initial studies, we found that IAIP treatment was neuroprotective in the middle cerebral artery occlusion model. Here, we analyzed if stroke alters IAIP using plasma samples from both mice and human stroke patients, and we investigated whether IAIP could provide neuroprotection using multiple stroke models. Methods: C57BL/6 mice were subjected to stroke with an autologous thromboembolic clot followed by reperfusion using t-PA. A separate cohort underwent the permanent occlusion model. Mice were randomized to receive IAIP or vehicle at 6h and 18h after stroke onset. Infarct was assessed 48h after reperfusion with t-PA, or 72h after permanent occlusion. Plasma samples from stroke patients and vascular risk factor matched controls were used for changes in IAIP levels using ELISA. Statistics were performed by two-sample t-test or Wilcoxon rank-sum test. P Results: IAIP treatment significantly reduced total injury size (t-PA alone 22±2% vs. t-PA+IAIP 13±3%; n=4-5/grp). Protection was also seen after permanent occlusion (vehicle 59.4±3.9% vs IAIP 39.5±5.9%; n=6-8/gp, p Conclusions: Our results show that delayed IAIP treatment can significantly reduce cell death in both permanent ischemia and/or in combination with t-PA. IAIP could potentially represent an important, novel treatment for stroke. Our studies in combination with t-PA further strengths this clinical translatability and to design personalized dosing based on IAIP levels.

Speech impairments affect one in four stroke survivors. However, animal models of post-ischemic vocalization deficits are limited. Male mice vocalize at ultrasonic frequencies when exposed to an estrous female mouse. In this study we assessed vocalization patterns and quantity in male mice after cerebral ischemia. FOXP2, a gene associated with verbal dyspraxia in humans, with known roles in neurogenesis and synaptic plasticity, was also examined after injury. Using a transient middle cerebral artery occlusion (MCAO) model, we assessed correlates of vocal impairment at several time-points after stroke. Further, to identify possible lateralization of vocalization deficits induced by left and right hemispheric strokes were compared. Significant differences in vocalization quantity were observed between stroke and sham animals that persisted for a month after injury. Injury to the left hemisphere reduced early vocalizations more profoundly than those to the right hemisphere. Nuclear expression of Foxp2 was elevated early after stroke (at 6 hours), but significantly decreased 24 hours after injury in both the nucleus and the cytoplasm. Neuronal Foxp2 expression increased in stroke mice compared to sham animals 4 weeks after injury. This study demonstrates that quantifiable deficits in ultrasonic vocalizations (USVs) are seen after stroke. USV may be a useful tool to assess chronic behavioral recovery in murine models of stroke.

Females show a varying degree of ischemic sensitivity throughout their lifespan, which is not fully explained by hormonal or genetic factors. Epidemiological data suggest that sex-specific life experiences such as pregnancy increase stroke risk. This work evaluated the role of parity on stroke outcome. Age-matched virgin (i.e., nulliparous) and multiparous mice were subjected to 60 min of reversible middle cerebral artery occlusion and evaluated for infarct volume, behavioral recovery, and inflammation. Using an established mating paradigm, fetal microchimeric cells present in maternal mice were also tracked after parturition and stroke. Parity was associated with sedentary behavior, weight gain, and higher triglyceride and cholesterol levels. The multiparous brain exhibited features of immune suppression, with dampened baseline microglial activity. After acute stroke, multiparous mice had smaller infarcts, less glial activation, and less behavioral impairment in the critical recovery window of 72 h. Behavioral recovery was significantly better in multiparous females compared with nulliparous mice 1 mo after stroke. This recovery was accompanied by an increase in poststroke angiogenesis that was correlated with improved performance on sensorimotor and cognitive tests. Multiparous mice had higher levels of VEGF, both at baseline and after stroke. GFP+ fetal cells were detected in the blood and migrated to areas of tissue injury where they adopted endothelial morphology 30 d after injury. Reproductive experience has profound and complex effects on neurovascular health and disease. Inclusion of female mice with reproductive experience in preclinical studies may better reflect the life-long patterning of ischemic stroke risk in women.

Introduction Acute ischemic injury leads to severe neuronal loss. One of the key mechanisms responsible for this effect is inflammation, which is characterized by the activation of myeloid cells, including resident microglia and infiltrating monocytes/macrophages. P2X4 receptors (P2X4Rs) present on these immune cells modulate the inflammatory response. For example, excessive release of adenosine triphosphate during acute ischemic stroke triggers stimulation of P2X4Rs, leading to myeloid cell activation and proliferation and further exacerbating post-ischemic inflammation. In contrast, during recovery P2X4Rs activation on microglia leads to the release of brain-derived neurotrophic factor (BDNF), which alleviate depression, maintain synaptic plasticity and hasten post-stroke behavioral recovery. Therefore, we hypothesized that deletion of the P2X4R specifically from myeloid cells would have differential effects on acute versus chronic recovery following stroke. Methods We subjected global or myeloid-specific (MS) P2X4R knock-out (KO) mice and wild-type littermates of both sexes to right middle cerebral artery occlusion (60 min). We performed histological, behavioral (sensorimotor and depressive), and biochemical (quantitative PCR and flow cytometry) analyses to determine the acute (three days after occlusion) and chronic (30 days after occlusion) effects of receptor deletion. Results Global P2X4R deletion led to reduced infarct size in both sexes. In MS P2X4R KO mice, only females showed reduced infarct size, an effect that did not change with ovariectomy. MS P2X4R KO mice of both sexes showed swift recovery from sensorimotor deficits during acute recovery but exhibited a more pronounced post-stroke depressive behavior phenotype that was independent of infarct size. Quantitative PCR analysis of whole cell lysate as well as flow-sorted myeloid cells from the perilesional cortex showed increased cellular interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) mRNA levels but reduced plasma levels of these cytokines in MS P2X4R KO mice after stroke. The expression levels of BDNF and other depression-associated genes were reduced in MS P2X4R KO mice after stroke. Conclusions P2X4R deletion protects against stroke acutely but predisposes to depression-like behavior chronically after stroke. Thus, a time-sensitive approach should be considered when targeting P2X4Rs after stroke.

Introduction: Earlier work by our laboratory and other groups has identified that aging leads to changes in both the immune system and the microbiome. The elderly have high mortality and more disability after a stroke, a finding that is recapitulated in murine model. Recently, pro-inflammatory γδ T cells have received increasing attention as a major contributor to gut immune responses. These cells may be a link in the bidirectional communication between the microbiome and the central nervous system. We hypothesize that fecal transplant of aged biome into young animals will enhance inflammation, γδ T cell numbers, and worsen functional recovery after stroke in young mice. Methods: Young C57BL/6 male mice, were randomized and subjected to sham surgery/right middle cerebral artery occlusion (MCAO-60min) followed by reperfusion. All mice received streptomycin treatment at 24h and 48h after MCAO. Subsequently, mice were gavaged with biome from either young or aged animals at 72 and 96 h post-stroke. Behavioral and functional outcomes were evaluated. Animals were sacrificed 15 days after stroke. Brain atrophy was quantified, and Flow Cytometry (FACS) and immunohistochemistry was performed on gut tissue and spleen to determine if stroke or the aged biome influence γδ T cells. Results: Young mice transplanted with aged biome take a longer time to regain their pre-stroke body weight. These mice have higher post-stroke hyperactivity compared with mice treated with young biome, as measured by average velocity (p Conclusion: Collectively our findings suggests that the gut microbiome plays an important role in post-stroke recovery. Understanding the underlying mechanisms may identify novel therapeutic targets for the treatment of stroke patients.

Epidemiologic studies have shown sex differences in ischemic stroke. The four core genotype (FCG) mouse model, in which the testes determining gene, Sry, has been moved from Y chromosome to an autosome, was used to dissociate the effects of sex hormones from sex chromosome in ischemic stroke outcome. Middle cerebral artery occlusion (MCAO) in gonad intact FCG mice revealed that gonadal males (XXM and XYM) had significantly higher infarct volumes as compared with gonadal females (XXF and XYF). Serum testosterone levels were equivalent in adult XXM and XYM, as was serum estrogen in XXF and XYF mice. To remove the effects of gonadal hormones, gonadectomized FCG mice were subjected to MCAO. Gonadectomy significantly increased infarct volumes in females, while no change was seen in gonadectomized males, indicating that estrogen loss increases ischemic sensitivity. Estradiol supplementation in gonadectomized FCG mice rescued this phenotype. Interestingly, FCG male mice were less sensitive to effects of hormones. This may be due to enhanced expression of the transgene Sry in brains of FCG male mice. Sex differences in ischemic stroke sensitivity appear to be shaped by organizational and activational effects of sex hormones, rather than sex chromosomal complement.

Social isolation in the pre-stroke environment leads to poorer outcomes after an ischemic injury in both animal and human studies. However, the impact of social isolation following stroke, which may be more clinically relevant as a target for therapeutic intervention, has yet to be examined. In this study, we investigated both the sub-acute (2 weeks) and chronic (7 weeks) effects of social isolation on post-stroke functional and histological outcome. Worsened histological damage from ischemic injury and an increase in depressive-like behavior was observed in isolated mice as compared to pair-housed mice. Mice isolated immediately after stroke showed a decrease in the levels of brain-derived neurotrophic factor (BDNF). These changes, both histological and behavioral, suggest an overall negative effect of social isolation on stroke outcome, potentially contributing to post-stroke depression and anxiety. Therefore, it is important to identify patients who have perceived isolation post-stroke to hopefully prevent this exacerbation of histological damage and subsequent depression.

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.

Neonatal hypoxic ischemia encephalopathy (HIE) leads to major deficits in language development. While clinically there is a known correlation in the degree of HIE injury and subsequent language disability, there are no treatments beyond speech and language therapy; therefore, experimental studies with a HIE animal model to test new interventions and therapeutics are warranted. Neonatal rodents normally ultrasonically vocalize at postnatal day 7 (PND 7) to PND 14 in response to removal from their mothers. At 6–8 weeks of age juvenile male rodents ultrasonically vocalize in response to exposure to a mature female mouse. Changes in ultrasonic vocalization (USV) production after neonatal brain injury, such as hypoxic ischemia (HI), have not been studied. This study examines the acute and long-term ultrasonic vocalization ability of mice after HI at PND 10. Pups were subjected to HI, sham, or naïve conditions; where in HI and sham surgeries the right common carotid artery was exposed, in the HI this artery was double ligated. The HI and sham pups were then exposed to 60 minutes of hypoxia. Naïve pups did not undergo surgery and were subjected to 60 minutes of room air. At 3 days following surgery, HI and sham pups vocalize less than nonsurgical naïve controls; yet “juvenile” mice of 6–8 weeks old that underwent HI at PND 10 vocalize less than sham and naïve mice. We conclude that HI injury has significant impact on later adult vocalization.

Middle cerebral artery occlusion(1) (MCAO) is a widely used experimental technique in rodents to model both the short-term pathological events and longer term neuroanatomical and functional damage associated with focal ischemia. Various neurobehavioral tasks have been developed to assess the motor and cognitive dysfunctions associated with MCAO in rodents, and these studies have revealed deficits related to long-term sensorimotor function, as well as retention of spatial memory. Assessment of auditory processing in a MCAO model has not been undertaken, despite findings suggesting an auditory processing deficit in humans with stroke induced-aphasia, a common post-stroke deficit. Using a modified pre-pulse inhibition paradigm, and other behavioral tasks thought to tap "language-related processing", adult male C57Bl/6 mice were subjected to 60 minute MCAO or Sham surgery and were behaviorally assessed from P58 to P124 (2 to 65 days post-surgery). Tasks were selected based on evidence that rapid auditory processing(2) (RAP) skills are associated with language processing indices in clinical populations. Cognitive and sensorimotor ability was evaluated using the Morris water maze, non-spatial water maze, and a post-injury rotarod task administered over multiple days (motor learning). Combined behavioral results from post-MCAO mice provide evidence of a RAP deficit as well as deficits in spatial, non-spatial, and motor learning. Overall results support a fuller characterization of behavioral deficits in auditory processing after MCAO.

Ketamine has been used safely in clinics for decades for analgesia and anesthesia. It is increasingly popular in clinical practice due to its new uses and importance for emergency procedures. It is known that ketamine is sequestered in the bone marrow and the major receptors for ketamine, noncompetitive N-methyl-d-aspartate receptors (NMDARs), are expressed in osteoclasts (OCs) and osteoblasts. However, the impact of ketamine on OCs or osteoblasts is unknown. In this study, we investigated the effects of ketamine on osteoclastogenesis and regulation of NMDARs expression in vitro. Bone marrows (BMs) or bone marrow macrophages (BMMs) were cultured in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) with or without ketamine for up to 6 days. OC formation peaked at day 5. On day 5 of culture, ketamine inhibited OC formation from both BM and BMM cultures at clinically relevant concentrations (3-200 µM). Ketamine inhibited RANKL-induced expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) in BMM cultures. Inhibition of ketamine on RANKL-induced osteoclastogenesis is associated with down-regulation of NMDARs. In addition, ketamine significantly inhibited the M-CSF induced migration of BMMs, inhibited cell fusion and significantly increased mature OC apoptosis. We conclude that clinically relevant concentrations of ketamine inhibit OC formation in both BM and BMM cultures in vitro through inhibiting migration and fusion process and enhancing mature OC apoptosis. It is likely that ketamine regulates osteoclastogenesis, at least in part, via its effects on NMDAR expression. J. Cell. Biochem. 118: 914-923, 2017. © 2016 Wiley Periodicals, Inc.

TRPM2 channels have been suggested to play a role in ischemic neuronal injury, specifically in males. A major hindrance to TRPM2 research has been the lack of specific TRPM2 inhibitors. The current study characterized the specificity and neuroprotective efficacy of a novel TRPM2 inhibitor.Fluorescent calcium imaging (Fluo5F) was used to determine inhibitor efficacy of the TRPM2 peptide inhibitor (tat-M2NX) in HEK293 cells stably expressing hTRPM2. Adult (2-3months) and aged (18-20months) mice were subjected to 60min middle cerebral artery occlusion (MCAO) and injected with tat-M2NX, control scrambled peptide (tat-SCR) or clotrimazole (CTZ) either 20min prior or 3h after reperfusion. Infarct size was assessed using TTC staining.TRPM2 inhibition by tat-M2NX was observed by decreased Ca(2+) influx following H2O2 exposure human TRPM2 expressing cells. Male mice pre-treated with tat-M2NX had smaller infarct volume compared to tat-SCR. No effect of tat-M2NX on infarct size was observed in female mice. Importantly, male TRPM2(-/-) mice were not further protected by tat-M2NX, demonstrating selectivity of tat-M2NX. Administration of tat-M2NX 3h after reperfusion provided significant protection to males when analyzed at 24h or 4days after MCAO. Finally, we observed that tat-M2NX reduced ischemic injury in aged male mice.These data demonstrate the development of a new peptide inhibitor of TRPM2 channels that provides protection from ischemic stroke in young adult and aged male animals with a clinically relevant therapeutic window.

Objective: Stroke is a sexually dimorphic disease. A growing body of literature from both clinical and experimental studies suggests that the outcomes after stroke differ in males versus females. Peripheral immune cells play a key role in stroke outcomes and also show sexual dimorphism. However, general immunosuppression has not shown an overall benefit in stroke patients. Therefore, identifying novel targets to improve stroke outcomes are urgently needed. P2X4 receptors (P2X4R) and P2X7 receptors (P2X7R) are the predominant subtypes expressed on immune and neural cells. The P2X7R and P2X4R interact and may physically associate with each other. Recent evidence demonstrates that P2X4R is necessary for the pro-inflammatory function of P2X7R. The goal of this study is to test the hypothesis that genetic deletion of P2X4R in myeloid immune cells impacts on stroke outcome and that such outcome differs in males vs. female mice. Methods: P2X4R myeloid knockout mice and wild type littermates of both sexes (∼20-25g; C57BL/6), were randomized and subjected to right middle cerebral artery occlusion (MCAO-90min) followed by 3 days of reperfusion. At 72h after stroke mice were perfused and infarcts were quantified from 30μicron cut cresyl violet stained sections. The effect of myeloid P2X4R deletion on chronic functional recovery was assessed with neurological scores, corner test, Open field and the novel object recognition test. Data are expressed as mean±sem. ANOVA was performed and a P < .05 was set for statistical significance. Results: A significant neuroprotective effect is seen in P2X4R KO compared to WT females (Cortex 36.1±1.5 vs 27.5±4.7; Striatum 67.4±1.5 vs 56.3±7.0; Total 34.8±1.6 vs 26.9±2.8. P Conclusions: There is a sex-specific effect of immune cell P2X4R deletion. Myeloid specific deletion of P2X4R protects females from ischemia/reperfusion injury but has no salutary effect in males. This is the first study to demonstrate a deleterious effect of myeloid P2X4R on stroke outcomes. These initial findings implicate myeloid P2X4 as a novel therapeutic approach to improve ischemic outcomes in females.

Ischemic preconditioning (IPC) induces endogenous neuroprotection from a subsequent ischemic injury. IPC involves downregulation of metabolic pathways. As adenosine 5'-monophosphate-activated protein kinase (AMPK) is a critical sensor of energy balance and plays a major role in cellular metabolism, its role in IPC was investigated. A brief 3-min middle cerebral artery occlusion (MCAO) was employed to induce IPC in male mice 72 h before 90-min MCAO. Levels of AMPK and phosphorylated AMPK (pAMPK), the active form of the kinase, were assessed after IPC. A pharmacological activator or inhibitor of AMPK was used to determine the dependence of IPC on AMPK signaling. Additionally, AMPK-α2 null mice were subjected to IPC, and subsequent infarct damage was assessed. IPC induced neuroprotection, enhanced heat shock protein-70 (HSP-70), and improved behavioral outcomes. These beneficial effects occurred in parallel with a significant inhibition of pAMPK protein expression. Although both pharmacological inhibition of AMPK or IPC led to neuroprotection, IPC offered no additional protective effects when co-administered with an AMPK inhibitor. Moreover, pharmacological activation of AMPK with metformin abolished the neuroprotective effects of IPC. AMPK-α2 null mice that lack the catalytic isoform of AMPK failed to demonstrate a preconditioning response. Regulation of AMPK plays an important role in IPC-mediated neuroprotection. AMPK may be a potential therapeutic target for the treatment of cerebral ischemia.

Depression is a frequent but often unrecognized and under treated complication of stroke that has scarcely been investigated in animal models particularly regarding treatment issues. Using the Forced Swim Test (FST) and testing spontaneous motor activity, we studied whether a transient focal cerebral ischemia modifies mice behaviours and antidepressant drug effects. We first evaluated whether FST realized 2 days or 1 week after brain reperfusion may be routinely used in male Swiss mice previously submitted to a 15, 30 or 60-min transient occlusion of the right middle cerebral artery. We then evaluated behavioural changes up to 5 weeks in mice previously submitted to a 15-min ischemia. Behaviours according to the administration of imipramine or fluvoxamine at 1 and 5 weeks after a 15-min ischemia were finally evaluated. Transient ischemia was associated with a decrease in immobility in the FST performed 2 days after reperfusion while no changes were observed in 1 and 5 weeks post-ischemia groups. Changes were related neither to brain ischemia duration nor to infarct volume. At both 1 and 5 weeks after brain ischemia, a dramatic decrease in the antidepressant response to imipramine related to a decrease in climbing behaviour was observed while the effects of fluvoxamine were improved through an increase in both climbing and swimming. Behaviours in the FST were unrelated to any spontaneous motor activity changes. Responses to anti-depressant drugs are strongly modified in mice previously submitted to brain ischemia. Present results underline that not all antidepressant drugs are appropriate after ischemic stroke.

Stroke is a serious global health care problem. It is now is the fourth leading cause of death and the primary cause of adult disability in the United States. Substantial evidence from both experimental and clinical studies has demonstrated that social isolation (SI) can increase stroke incidence and impair recovery. Epidemiological studies demonstrate that an increasing number of patients are living alone, and as the aging population increases, loneliness will only increase in prevalence. SI is increasingly identified as an independent risk factor for all-cause mortality. In contrast, individuals with high levels of social support exhibit more rapid and extensive functional and cognitive recovery after a wide variety of pathological insults, including stroke. Clinical data suggests that SI is an important risk factor for increased mortality and delayed functional recovery following ischemic stroke. Attesting to the importance of mortality and behavioral factors in stroke outcome is that these same effects can be reproduced in animal models of experimental stroke. This has allowed researchers to identify several mechanistic changes that occur with affiliative interactions. These include decreased systemic inflammation, elaboration of growth factors including brain derived neurotropic factor (BDNF), enhanced neurogenesis, and improved neuroimmune responsiveness in group housed animals. These may mediate the beneficial effects of social interaction on improving stroke recovery and reducing neuronal death. In this review we provide an overview of the effects of SI on ischemic injury and recovery and discuss their clinical and therapeutic implications.