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Ischemic stroke is one of the leading causes of death worldwide and causes significant morbidity among survivors. Studies have shown correlations between small noncoding RNAs (microRNAs, miRNAs) and stroke. We have previously reported that miR-34a depletion decreases infarct volume and attenuates neurological deficits in stroke mice. To further investigate the role of miR-34a in stroke, we utilized inducible miR-34a overexpression (miR-34a TG ) mice. Transient middle cerebral artery occlusion (tMCAO) was performed on 6-month old doxycycline-induced miR-34a TG mice compared to wild-type (WT) controls. We confirmed that miR-34a TG mice had a ~5-fold increase of miR-34a in plasma at 24 hours post-stroke as detected by real-time PCR compared with WT naive controls (p < 0.0001) and WT-stroke controls (p < 0.0001). Following ischemia due to 60 min tMCAO, overexpression of miR-34a significantly exacerbated neurological deficit scores (p = 0.0035) and increased infarct volume compared to WT control mice at 24 hours of reperfusion (p = 0.002). Systemic delivery of a locked nucleic acid (LNA)-modified miR-34a inhibitor, antagomir-34a, in stroke mice substantially reduced infarct volume (p = 0.0021) and improved neurological deficits (p = 0.0057) at 24 hours post-stroke. Our post-stroke behavioral study revealed that antagomir-34a significantly improved functional outcomes including motor function assessed by accelerated rotarod tests at 2 weeks post-stroke (p = 0.0046) and memory deficits assessed by Morris Water Maze tests at 4 weeks post-stroke (p = 0.0012). These data demonstrate that miR-34a plays an important role in modulating acute stroke outcomes and suggest that miR-34a may be a promising therapeutic target for stroke therapy.

PURPOSE: Therapeutic strategies to treat ischemic stroke are limited due to the heterogeneity of cerebral ischemic injury and the mechanisms that contribute to the cell death. Since oxidative stress is one of the primary mechanisms that cause brain injury post-stroke, we hypothesized that therapeutic targets that modulate mitochondrial function could protect against reperfusion-injury after cerebral ischemia, with the focus here a mitochondrial protein, mitoNEET, that modulates cellular bioenergetics. METHOD: In this study, we evaluated the pharmacology of the mitoNEET ligand NL-1 in an in vivo therapeutic role for NL-1 in a C57Bl/6 murine model of ischemic stroke. RESULTS: NL-1 decreased hydrogen peroxide production with an IC(50) of 5.95 μM in neuronal cells (N2A). The in vivo activity of NL-1 was evaluated in a murine 1h transient middle cerebral artery occlusion (t-MCAO) model of ischemic stroke. We found that mice treated with NL-1 (10 mg/kg, i.p.) at time of reperfusion and allowed to recover for 24 hours showed a 43% reduction in infarct volume and 68% reduction in edema compared to sham-injured mice. Additionally, we found that when NL-1 was administered 15 min post-t-MCAO, the ischemia volume was reduced by 41%, and stroke-associated edema by 63%. CONCLUSION: As support of our hypothesis, as expected, NL-1 failed to reduce stroke infarct in a permanent photothrombotic occlusion model of stroke. This report demonstrates the potential therapeutic benefits of using mitoNEET ligands like NL-1 as novel mitoceuticals for treating reperfusion-injury with cerebral stroke.

Aneurysmal subarachnoid hemorrhage (aSAH) leads to a robust systemic inflammatory response. We hypothesized that an early systemic glycolytic shift occurs after aSAH, resulting in a unique metabolic signature and affecting systemic inflammation.Control patients and patients with aSAH were analyzed. Samples from patients with aSAH were collected within 24 h of aneurysmal rupture. Mass spectrometry-based metabolomics was performed to assess relative abundance of 16 metabolites involved in the tricarboxylic acid cycle, glycolysis, and pentose phosphate pathway. Principal component analysis was used to segregate control patients from patients with aSAH. Dendrograms were developed to depict correlations between metabolites and cytokines. Analytic models predicting functional outcomes were developed, and receiver operating curves were compared.A total of 122 patients with aSAH and 38 control patients were included. Patients with aSAH had higher levels of glycolytic metabolites (3-phosphoglycerate/2-phosphoglycerate, lactate) but lower levels of oxidative metabolites (succinate, malate, fumarate, and oxalate). Patients with higher clinical severity (Hunt-Hess Scale score ≥ 4) had higher levels of glyceraldehyde 3-phosphate and citrate but lower levels of α-ketoglutarate and glutamine. Principal component analysis readily segregated control patients from patients with aSAH. Correlation analysis revealed distinct clusters in control patients that were not observed in patients with aSAH. Higher levels of fumarate were associated with good functional outcomes at discharge (odds ratio [OR] 1.76, 95% confidence interval [CI] 1.15-2.82) in multivariable models, whereas higher levels of citrate were associated with poor functional outcomes at discharge (OR 0.36, 95% CI 0.16-0.73) and at 3 months (OR 0.35, 95% CI 0.14-0.81). No associations were found with delayed cerebral ischemia. Levels of α-ketoglutarate and glutamine correlated with lower levels of interleukin-8, whereas fumarate was associated with lower levels of tumor necrosis factor alpha.Aneurysmal subarachnoid hemorrhage results in a unique pattern of plasma metabolites, indicating a shift toward glycolysis. Higher levels of fumarate and lower levels of citrate were associated with better functional outcomes. These metabolites may represent targets to improve metabolism after aSAH.

Background Postoperative delirium (POD) describes a multifactorial disease process occurring after surgery. However, few studies have focused on patients undergoing brain tumor resection, and its influencing factors are unclear. Methods We performed a 1-year, single-center, cross-sectional, retrospective survey at Huashan Hospital. Patients were screened using the Confusion Assessment Method for the Intensive Care Unit (CAM-ICU), Confusion Assessment Method, and Richmond Agitation Sedation Scale by trained bedside nurses. Perioperative data were collected using demographic and disease-related questionnaires. The primary outcome measures were the incidence of POD and subtype of POD. Independent predictors of POD were estimated from multivariate logistic regression models, and receiver operating characteristic analysis was used to compare the predictive performance of the models. Results Of the 916 patients included in the study, 893 were analyzed. The overall incidence was 14.78%, 67 had hyperactive delirium (50.76%), 55 had hypoactive delirium (41.67%), and 10 had mixed delirium (7.57%). Age, sex, working status, tobacco use history, comorbidities, physical restraint, axillary temperature (>38.5°C), electrolyte disturbances, duration of anesthesia, pathologic diagnosis, tumor site, length of disease, and duration of operation were risk factors for POD. Conversely, saddle area mass was a protective factor. Age, tobacco use history, electrolyte disturbances, physical restraint, and duration of operation were included in the model. Conclusions POD is harmful to patients undergoing brain tumor resection, increasing length of stay in the intensive care unit and hospitalization costs. Intraoperative factors and postoperative factors, in addition to older age and tobacco use history, are associated with POD.

Introduction Stroke‐associated pneumonia (SAP) is a major cause of mortality in patients who have suffered from severe ischemic stroke. Although multifactorial in nature, stroke‐induced immunosuppression plays a key role in the development of SAP. Previous studies using a murine model of transient middle cerebral artery occlusion (tMCAO) have shown that focal ischemic stroke induction results in functional defects of lymphocytes in the spleen, thymus, and peripheral blood, leading to spontaneous bacterial infection in the lungs without inoculation. However, how ischemic stroke alters immune cell niche and the expression of cytokines and chemokines in the lungs has not been fully characterized. Methods Ischemic stroke was induced in mice by tMCAO. Immune cell profiles in the brain and the lungs at 24‐ and 72‐hour time points were compared by flow cytometric analysis. Cytokine and chemokine expression in the lungs were determined by multiplex bead arrays. Tissue damage and bacterial burden in the lungs following tMCAO were evaluated. Results Ischemic stroke increases the percentage of alveolar macrophages, neutrophils, and CD11b+ dendritic cells, but reduces the percentage of CD4+ T cells, CD8+ T cells, B cells, natural killer cells, and eosinophils in the lungs. The alteration of immune cell niche in the lungs coincides with a significant reduction in the levels of multiple chemokines in the lungs, including CCL3, CCL4, CCL5, CCL17, CCL20, CCL22, CXCL5, CXCL9, and CXCL10. Spontaneous bacterial infection and tissue damage following tMCAO, however, were not observed. Conclusion This is the first report to demonstrate a significant reduction of lymphocytes and multiple proinflammatory chemokines in the lungs following ischemic stroke in mice. These findings suggest that ischemic stroke directly impacts pulmonary immunity.

Acute stroke causes complex, pathological, and systemic responses that have not been treatable by any single medication. In this study, using a murine transient middle cerebral artery occlusion stroke model, a novel therapeutic strategy is proposed, where blood replacement (BR) robustly reduces infarctions and improves neurological deficits in mice. Our analyses of immune cell subsets suggest that BR therapy substantially decreases neutrophils in blood following a stroke. Electrochemiluminescence detection demonstrates that BR therapy reduces cytokine storm in plasma and ELISA demonstrates reduced levels of matrix metalloproteinase-9 (MMP-9) in the plasma and brains at different time points post-stroke. Further, we have demonstrated that the addition of MMP-9 to the blood diminishes the protective effect of the BR therapy. Our study is the first to show that BR therapy leads to profoundly improved stroke outcomes in mice and that the improved outcomes are mediated via MMP-9. These results offer new insights into the mechanisms of stroke damage.

Chronic brain hypoperfusion is the primary cause of vascular dementia and has been implicated in the development of white matter disease and lacunar infarcts. Cerebral hypoperfusion leads to a chronic state of brain inflammation with immune cell activation and production of pro-inflammatory cytokines, including IL-1β. In the present study, we induced chronic, progressive brain hypoperfusion in mice using ameroid constrictor, arterial stenosis (ACAS) surgery and tested the efficacy of an IL-1β antibody on the resulting brain damage. We observed that ACAS surgery causes a reduction in cerebral blood flow (CBF) of about 30% and grey and white matter damage in and around the hippocampus. The IL-1β antibody treatment did not significantly affect CBF but largely eliminated grey matter damage and reduced white matter damage caused by ACAS surgery. Over the course of hypoperfusion/injury, grip strength, coordination, and memory-related behavior were not significantly affected by ACAS surgery or antibody treatment. We conclude that antibody neutralization of IL-1β is protective from the brain damage caused by chronic, progressive brain hypoperfusion.

Globally, stroke is a leading cause of death and disability. Traditional risk factors like hypertension, diabetes, and obesity do not fully account for all stroke cases. Recent infection is regarded as changes in systemic immune signaling, which can increase thrombosis formation and other stroke risk factors. We have previously shown that administration of lipopolysaccharide (LPS) 30-minutes prior to stroke increases in infarct volume. In the current study, we found that animals intermittently exposed to LPS have larger cortical infarcts when compared to saline controls. To elucidate the mechanism behind this phenomenon, several avenues were investigated. We observed significant upregulation of tumor necrosis factor-alpha (TNF-α) mRNA, especially in the ipsilateral hemisphere of both saline and LPS exposed groups compared to sham surgery animals. We also observed significant reductions in expression of genes involved in autophagy in the ipsilateral hemisphere of LPS stroke animals. In addition, we assessed DNA methylation of autophagy genes and observed a significant increase in the ipsilateral hemisphere of LPS stroke animals. Intermittent exposure to LPS increases cortical infarct volume, downregulates autophagy genes, and induces hypermethylation of the corresponding CpG islands. These data suggest that intermittent immune activation may deregulate epigenetic mechanisms and promote neuropathological outcomes after stroke.

Supplemental material, sj-pdf-1-asn-10.1177_1759091421991769 for Intermittent Lipopolysaccharide Exposure Significantly Increases Cortical Infarct Size and Impairs Autophagy by Ashley E. Russell, John Z. Cavendish, Ali Rai, Mya Vannoy, Ahmad H. Dakhlallah, Heng Hu, Xuefang Ren, Amal Amer, Candice M. Brown, Clay B. Marsh, James W. Simpkins and Duaa Dakhlallah in ASN Neuro

Acute stroke causes complex, pathological, and systemic responses that have not been treatable by any single medication. In this study, using a murine transient middle cerebral artery occlusion stroke model, a novel therapeutic strategy is proposed, where blood replacement (BR) robustly reduces infarctions and improves neurological deficits in mice. Our analyses of immune cell subsets suggest that BR therapy substantially decreases neutrophils in blood following a stroke. Electrochemiluminescence detection demonstrates that BR therapy reduces cytokine storm in plasma and ELISA demonstrates reduced levels of matrix metalloproteinase-9 (MMP-9) in the plasma and brains at different time points post-stroke. Further, we have demonstrated that the addition of MMP-9 to the blood diminishes the protective effect of the BR therapy. Our study is the first to show that BR therapy leads to profoundly improved stroke outcomes in mice and that the improved outcomes are mediated via MMP-9. These results offer new insights into the mechanisms of stroke damage., Acute stroke causes complex, pathological, and systemic responses which remain challenging to treat. Here, the authors show that substituting the blood of stroke model mice with whole-blood from naive healthy donor mice reduces infarct volume and improves neurological deficits.

As mitigation of brain aging continues to be a key public health priority, a wholistic and comprehensive consideration of the aging body has identified immunosenescence as a potential contributor to age-related brain injury and disease. Importantly, the nervous and immune systems engage in bidirectional communication and can exert profound influence on each other. Emerging evidence supports numerous impacts of innate, inflammatory immune responses and adaptive T cell-mediated immunity in neurological function and diseased or injured brain states, such as stroke. Indeed, a growing body of evidence supports key impacts of brain-resident immune cell activation and peripheral immune infiltration in both the post-stroke acute injury phase and the long-term recovery period. As such, modulation of the immune system is an attractive strategy for novel therapeutic interventions for a devastating age-related brain injury for which there are few readily available neuroprotective treatments or neurorestorative approaches. However, the role of B cells in the context of brain function, and specifically in response to stroke, has not been thoroughly elucidated and remains controversial, leaving our understanding of neuroimmune interactions incomplete. Importantly, emerging evidence suggests that B cells are not pathogenic contributors to stroke injury, and in fact may facilitate functional recovery, supporting their potential value as novel therapeutic targets. By summarizing the current knowledge of the role of B cells in stroke pathology and recovery and interpreting their role in the context of their interactions with other immune cells as well as the immunosenescence cascades that alter their function in aged populations, this review supports an increased understanding of the complex interplay between the nervous and immune systems in the context of brain aging, injury, and disease.

Recent literature implicates gut epithelia mucosa and intestinal microbiota as important players in post-stroke morbidity and mortality. As most studies have focused on the acute effects of stroke on gut dysbiosis, our study objective was to measure chronic, longitudinal changes in the gut microbiota and intestinal pathology following ischemic stroke. We hypothesized that mice with experimental ischemic stroke would exhibit chronic gut dysbiosis and intestinal pathology up to 36 days post-stroke compared to sham controls. Male C57BL/6J mice were subjected to 60 minutes of transient middle cerebral artery occlusion (tMCAO) or sham surgery. To determine the long-term effects of tMCAO on gut dysbiosis, fecal boli were collected pre- and post-tMCAO on days 0, 3, 14, and 28. Bioinformatics analysis demonstrate significant differences in abundance among Firmicutes and Bacteroidetes taxa at the phylum, family, and species levels in tMCAO compared to sham mice that persisted up to one month post-stroke. The most persistent changes in post-stroke microbial abundance were a decrease in bacteria family S24-7 and significant increases inRuminococcaceae. Overall, these changes resulted in a persistently increased Firmicutes:Bacteroidetes ratio in stroke animals. Intestinal histopathology showed evidence of chronic intestinal inflammation that included marked increases in immune cell infiltration with mild-moderate epithelial hyperplasia and villous blunting. Increased astrocyte and microglial activity were also detected one-month post-stroke. These results demonstrate that acute, post-stroke disruption of the gut-brain-microbiota axis progresses to chronic gut dysbiosis, intestinal inflammation, and chronic neuroinflammation.Clinical PerspectivesThe microbiota-gut-brain axis, recently implicated in several neurological disorders, remains largely unexplored at chronic time points post-tMCAO.Our results demonstrate chronic gut dysbiosis, prolonged behavioral deficits, and persistent cerebral and intestinal inflammation post-tMCAO in male C57BL/6J mice.These results suggest that manipulation of microbiota may help reduce poor outcomes after stroke and lead to improved post-stroke functional recovery.

Objective Mitochondrial dysfunction is known to be implicated in stroke, but the complex mechanisms of stroke have led to few stroke therapies. The present study to disrupted mitochondrial oxidative phosphorylation through a known electron transport chain (ETC) uncoupler, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP). Analyzing the resulting neurological deficits as well as infarct volume could help determine the role of mitochondria in stroke outcome and determine whether uncoupling the ETC could potentially be a strategy for new stroke therapies. The objective of this study was to determine the effects of uncoupling electron flow on mitochondrial oxidative phosphorylation and stroke infarction. Methods Cerebral endovascular cells (CECs) were treated with various concentrations of FCCP, and bioenergetics were measured. For the stroke mouse model, FCCP (1 mg/kg, i.p) or vehicle was administered followed by 1-hour transient middle cerebral artery occlusion (tMCAO). Infarct volume was measured after a 23-hour reperfusion, and triphenyl tetrazolium chloride (TTC) staining was used to assess infarct volume. Results FCCP significantly decreased basal respiration, ATP turnover, maximal respiration, and spare capacity when the concentration of FCCP was greater than 1000 nM. The mice pretreated with FCCP had a significantly increased infarct volume within the cortex, striatum, and total hemisphere. Mice receiving FCCP had a significantly increased neurological deficit score compared to the vehicle. Conclusions FCCP compromised mitochondrial oxidative phosphorylation in CECs in a dose-dependent manner. Uncoupling the electron transport chain with FCCP prior to tMCAO exacerbated stroke infarction in mice.

Chronic cerebrovascular hypoperfusion results in vascular dementia and increases predisposition to lacunar infarcts. However, there are no suitable animal models. In this study, we developed a novel model for chronic irreversible cerebral hypoperfusion in mice. Briefly, an ameroid constrictor was placed on the right carotid artery to gradually occlude the vessel, while a microcoil was placed on the left carotid artery to prevent compensation of the blood flow. This procedure resulted in a gradual hypoperfusion developing over a period of 34 days with no cerebral blood flow recovery. Histological analysis of the brain revealed neuronal and axonal degeneration as well as necrotic lesions. The most severely affected regions were located in the hippocampus and the corpus callosum. Overall, our paradigm is a viable model to study brain pathology resulting from gradual cerebrovascular hypoperfusion.

Blood-brain barrier (BBB) dysfunction occurs in cerebrovascular diseases and neurodegenerative disorders such as stroke. Opening of the BBB during a stroke has a negative impact on acute outcomes. We have recently demonstrated that miR-34a regulates the BBB by targeting cytochrome c (CYC) in vitro. To investigate the role of miR-34a in a stroke, we purified primary cerebrovascular endothelial cells (pCECs) from mouse brains following 1 h transient middle cerebral artery occlusion (tMCAO) and measured real-time PCR to detect miR-34a levels. We demonstrate that the miR-34a levels are elevated in pCECs from tMCAO mice at the time point of BBB opening following 1 h tMCAO and reperfusion. Interestingly, knockout of miR-34a significantly reduces BBB permeability, alleviates disruption of tight junctions, and improves stroke outcomes compared to wild-type (WT) controls. CYC is decreased in the ischemic hemispheres and pCECs from WT but not in miR-34a−/− mice following stroke reperfusion. We further confirmed CYC is a target of miR-34a by a dural luciferase reporter gene assay in vitro. Our study provides the first description of miR-34a affecting stroke outcomes and may lead to discovery of new mechanisms and treatments for cerebrovascular and neurodegenerative diseases such as stroke.

Inverse packet sets is a set model with dynamic characteristics, which is got by introducing dynamic characteristics into Cantor set $$ X $$ and improving Cantor $$ X $$ . By using inverse packet sets to improve the concept of ordinary augmented matrix, the structure of internal inverse packet augmented matrix, outer inverse packet augmented matrix and inverse packet augmented matrix are given. Internal inverse packet augmented matrix reasoning, outer inverse packet augmented matrix reasoning, inverse packet augmented matrix reasoning and reasoning model are given. Finally intelligent dynamic separating-acquisition of information is given. By using these theoretical results, the application of intelligent dynamic separating-acquisition of educational information is given. Inverse packet matrix reasoning provides a new theory and new method for mining and acquisition of dynamic educational information. This is also a successful teaching demonstration on the research of matrix Teaching.

Stroke-associated pneumonia (SAP) is a major cause of mortality in patients who have suffered from severe ischemic stroke. Although multi-factorial in nature, stroke-induced immunosuppression plays a key role in the development of SAP. Previous studies of focal ischemic stroke induction, using a murine model of transient middle cerebral artery occlusion (tMCAO) have shown that severe brain damage results in massive apoptosis and functional defects of lymphocytes in the spleen, thymus, and peripheral blood. However, how immune alternations in remote tissues lead to a greater susceptibility to lung infections is not well-understood. Importantly, how ischemic stroke alters immune-cell fates, and the expression of cytokines and chemokines in the lungs that directly impact pulmonary immunity, has not been characterized. We report here that ischemic stroke increases the percentage of alveolar macrophages, neutrophils, and CD11b+ dendritic cells (DCs), but reduces the percentage of CD4+ T cells, CD8+ T cells, B cells, natural killer (NK) cells, and eosinophils in the lungs. The depletion of immune cells in the lungs is not caused by apoptosis, cell infiltration to the brain, or spontaneous pneumonia following ischemic stroke as previously described, but correlates with a significant reduction in the levels of multiple chemokines in the lungs, including: CCL3, CCL4, CCL5, CCL17, CCL20, CCL22, CXCL5, CXCL9, and CXCL10. These findings suggest that ischemic stroke negatively impacts pulmonary immunity to become more susceptible for SAP development. Further investigation into the mechanisms that control pulmonary immune alternations following ischemic stroke may identify novel diagnostic or therapeutic targets for SAP.

Introduction: Recent literature implicates gut epithelia mucosa and intestinal microbiota as important players in post-stroke morbidity and mortality. As most studies have focused on the acute effects of stroke on gut dysbiosis, our study objective was to measure chronic, longitudinal changes in the gut microbiota and intestinal pathology following ischemic stroke. Hypothesis: We hypothesized that mice with experimental ischemic stroke would exhibit chronic gut dysbiosis and intestinal pathology at up to 35 days post-stroke compared to sham controls. Methods: Male C57BL/6J mice were subjected to 60 minutes of transient middle cerebral artery occlusion (tMCAO) or sham-surgery followed by reperfusion and euthanasia at day 35. To determine the long-term effects of tMCAO on gut dysbiosis, fecal boli were collected pre- and post-tMCAO on days 0, 3, 14, and 28. Following DNA extraction, amplification of the V3-V4 region of the 16s rRNA gene was performed via PCR, then library preparation and high-throughput sequencing on the HiSeq Ilumina platform. Longitudinal changes between tMCAO and sham-injured mice were analyzed with the DESeq2 pipeline and MicrobiomeAnalyst. Pathological indices of gut dysbiosis were assessed via Periodic acid-Schiff stain and immunolabeling of tight junction and mucin proteins. Results: Bioinformatics analysis revealed significant differences in abundance among taxa at the phylum, family, and species levels between baseline samples and post-injury fecal microbiome samples in tMCAO mice compared to sham mice that persisted up to 28 days post-stroke. The most persistent changes in post-stroke microbial abundance were decreases in beneficial bacteria families, Bifidobacteriaceae and Lactobacillaceae , and significant increases in the harmful bacteria families, Helicobacteraceae and Enterobacteriaceae . Intestinal histopathology at day 35 showed evidence of chronic intestinal inflammation that included marked increases in immune cell infiltration with mild-moderate epithelial hyperplasia and villous blunting. Conclusion: These results demonstrate that acute, post-stroke disruption of the gut-brain-microbiota axis progresses to chronic gut dysbiosis and intestinal inflammation.

It is generally accepted that gestational xenobiotic exposures result in systemic consequences in the adult F1 generation. However, data on detailed behavioral and cognitive consequences remain limited. Using our whole body nanoparticle inhalation facility, pregnant Sprague Dawley rats (gestational day 7) were exposed 4 days per week to either filtered air (control) or nanotitanium dioxide aerosols (nano-TiO2; count median aerodynamic diameter of 170.9±6.4 nm, 10.4±0.4 mg/m3, 5 hr/day) for 7.8±0.5 days of the remaining gestational period. All rats received their final exposure on GD 20 prior to delivery. The calculated daily maternal deposition was 13.9±0.5 μg. Subsequently, at 5 months of age, behavior and cognitive functions of these pups were evaluated employing a standard battery of locomotion, learning, and anxiety tests. These assessments revealed significant working impairments, especially under maximal mnemonic challenge, and possible deficits in initial motivation in male F1 adults. Evidence indicates that maternal engineered nanomaterial exposure during gestation produces psychological deficits that persist into adulthood in male rats.

Tert-butylhydroquinone (tBHQ), an Nrf2 signaling pathway inducer that is widely used as a food additive in the U.S., prevents oxidative stress-induced cytotoxicity in neurons. This study assesses the effects of tBHQ on ischemic stroke outcomes in mice. We measured infarct size, neurological deficits, and brain volume after tBHQ treatments in murine permanent middle cerebral artery occlusion (pMCAO) model in vivo. Further, we evaluated the regulation of tBHQ on mitochondrial function in cerebrovascular endothelial cells in vitro, which is critical to the blood-brain barrier (BBB) permeability. Our results demonstrated that tBHQ increased post-stroke mortality and worsened stroke outcomes. Mitochondrial function was suppressed by tBHQ treatment of cerebrovascular endothelial cells, and this suppression was potentiated by co-treatment with lipopolysaccharide (LPS), the bacterial mimic. These data indicate that tBHQ-exacerbated stroke damage might due to the compromised BBB permeability in permanent stroke.

Aging of the nervous system, and the occurrence of age-related brain diseases such as stroke, are associated with changes to a variety of cellular processes controlled by many distinct genes. MicroRNAs (miRNAs), short non-coding functional RNAs that can induce translational repression or site-specific cleavage of numerous target mRNAs, have recently emerged as important regulators of cellular senescence, aging, and the response to neurological insult. Here, we focused on the assessment of the role of miR-34a in stroke. We noted increases in miR-34a expression in the blood of stroke patients as well as in blood and brain of mice subjected to experimental stroke. Our methodical genetic manipulation of miR-34a expression substantially impacted stroke-associated preclinical outcomes and we have in vitro evidence that these changes may be driven at least in part by disruptions to blood brain barrier integrity and mitochondrial oxidative phosphorylation in endothelial cells. Finally, aging, independent of brain injury, appears to be associated with shifts in circulating miRNA profiles. Taken together, these data support a role for miRNAs, and specifically miR-34a, in brain aging and the physiological response to age-related neurological insult, and lay the groundwork for future investigation of this novel therapeutic target.

Introduction: Inducible nitric oxide synthase (iNOS) is a critical regulator of brain and systemic inflammatory responses in many neurological disorders. Intriguingly, iNOS null mice have produced equivocal outcomes in mouse models of ischemic stroke, a result that may be due, in part, to species-specific differences between the mouse and human iNOS genes. Due to numerous differences at the gene regulatory level, mouse iNOS naturally produces much higher levels of nitric oxide (NO) than the human gene. To resolve the functional differences between mouse and human iNOS in ischemic stroke, we employed a novel mouse that expresses human iNOS on a mouse iNOS null genetic background, termed the HN mouse. Our study objective was to compare functional and behavioral outcomes following ischemic stroke between wildtype (WT) and HN mice. Hypothesis: We hypothesized that post-stroke functional outcomes would be more severe in HN male mice than in WT mice. Methods: Male C57BL/6J (WT) and HN mice (4-6 months) were subjected to 60 minutes of transient middle cerebral artery occlusion (tMCAO) followed by reperfusion and behavioral assessments at seven-day intervals for 30 days. Laser-Doppler flowmetry pre- and post-tMCAO was used to assess the effect of iNOS on blood flow. A comprehensive neurological battery was used to assess sensorimotor ability, reflexes, and balance, followed by open field, wire hang, and rotarod testing to evaluate locomotor activity, limb strength, and coordination. Results and Conclusion: Blood flow was significantly decreased in the ipsilateral hemisphere of both WT and HN mice following acute ischemic stroke; however, HN mice exhibited a sustained global decrease in blood flow following reperfusion. HN mice also demonstrated decreased locomotor activity and coordination, as well as greater neurological deficits compared to WT mice. Overall, these results suggest that the HN mouse model recapitulates more neurological deficits associated with human ischemic stroke than WT mice. Thus, the HN mouse may represent a more “humanized” model of ischemic stroke than more commonly used mouse models that express the mouse iNOS gene.

Introduction: Cerebral small vessel disease (SVD) is a common finding in patients with stroke. It can manifest as white matter hyperintensities (WMH), microbleeds or lacunar strokes. Presence of SVD is an independent risk factor for stroke and is considered a poor prognostic factor in stroke recovery. In addition, SVD has been associated with vascular dementia, depression and gait disorder. There are no good animal models to study SVD. Hypothesis: The precise mechanism behind development of SVD is not clearly understood. It’s likely a result of multiple different processes including decreased blood flow, inflammation with blood brain barrier disruption, degeneration and myelin breakdown. We try to study the role of reduced blood flow and inflammation in the development of SVD with the eventual goal of developing an agent that may prevent the development or progression of SVD. Methods: We remodeled a novel murine SVD model - asymmetric common carotid artery surgery (ACAS), published recently. We placed an ameroid constrictor around the right common carotid artery (CCA) which gradually narrows and eventually occludes vessels by releasing hygroscopic casein material, and a microcoil around the left CCA in mice. This model causes gradual narrowing of the vessels and a significant reduction of cerebral blood flow as detected by Laser Speckle Imager. These mice were compared to sham control which underwent a sham surgery without any vessel occlusion (n=7 per group). Animals were monitored for 30 days. Results: In the ACAS group, the model caused morbidity in all mice and death in 2 of 7 mice. Histologically, we observed marked vacuoles in white matter evaluated by H&E staining. We devised a severity score based on the extent of white matter damage and presence of infarcts under microscope. Based on this score, the damage was significantly worse in the ACAS group as compared with sham controls. Conclusions: These findings demonstrate the role of gradual blood flow reduction in development of SVD. We plan to further study the mechanisms behind SVD and develop novel treatments with the goal of preventing SVD.

Background: The blood-brain barrier (BBB) is a crucial interface between the central nervous system (CNS) and the circulatory system that maintains cerebral homeostasis by selectively allowing entry of blood solutes into the CNS. BBB dysfunction has been observed in cerebrovascular diseases and neurodegenerative disorders such as stroke. The BBB is opened during stroke, having negative impacts on stroke outcomes. Mitochondria are key players in this abnormal opening of the BBB, and decreased cytochrome c (CYC) levels have been shown in the mitochondrial fraction in stroke. We have recently demonstrated miR-34a regulates the BBB by targeting CYC in vitro . Objective: To investigate the role of miR-34a in stroke and verify its target CYC in vivo . Methods and Results: Using a murine transient middle cerebral artery occlusion (tMCAO) model, we demonstrate elevated miR-34a expression in serum and primary cerebral endothelial cells (CECs) from stroke mice. We report that deficiency of miR-34a significantly reduces BBB permeability and improves stroke outcomes. CYC is decreased in the ischemic hemisphere from wild-type (WT) but not miR-34a -/- mice following stroke reperfusion. Uncoupling of electron flow by a pharmacological inhibitor Carbonyl-cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compromises mitochondrial oxidative phosphorylation (MOP) in cultured CECs and worsens infarction in stroke mice. Conclusions: Our study provides the first description of miR-34a and electron flow affecting stroke outcomes, which could lead to a revision of current miR-34a targets and may lead to discovery of new mechanisms and treatments for cerebrovascular and neurodegenerative diseases such as stroke and Alzheimer’s disease. Key words: Blood-brain barrier; Stroke; MiR-34a; Mitochondria; Cytochrome c. Non-standard Abbreviations and Acronyms: CEC: cerebral endothelial cell CYC: cytochrome c FCCP: Carbonyl-cyanide-4-(trifluoromethoxy)phenylhydrazone MOP: mitochondrial oxidative phosphorylation tMCAO: transient middle cerebral artery occlusion

The blood–brain barrier is composed of cerebrovascular endothelial cells and tight junctions, and maintaining its integrity is crucial for the homeostasis of the neuronal environment. Recently, we discovered that mitochondria play a critical role in maintaining blood–brain barrier integrity. We report for the first time a novel mechanism underlying blood–brain barrier integrity: miR-34a mediated regulation of blood–brain barrier through a mitochondrial mechanism. Bioinformatics analysis suggests miR-34a targets several mitochondria-associated gene candidates. We demonstrated that miR-34a triggers the breakdown of blood–brain barrier in cerebrovascular endothelial cell monolayer in vitro, paralleled by reduction of mitochondrial oxidative phosphorylation and adenosine triphosphate production, and decreased cytochrome c levels.

Background and Purpose— The blood–brain barrier (BBB) is a selectively permeable cerebrovascular endothelial barrier that maintains homeostasis between the periphery and the central nervous system. BBB disruption is a consequence of ischemic stroke and BBB permeability can be altered by infection/inflammation, but the complex cellular and molecular changes that result in this BBB alteration need to be elucidated to determine mechanisms. Methods— Infection mimic (lipopolysaccharide) challenge on infarct volume, BBB permeability, infiltrated neutrophils, and functional outcomes after murine transient middle cerebral artery occlusion in vivo; mitochondrial evaluation of cerebrovascular endothelial cells challenged by lipopolysaccharide in vitro; pharmacological inhibition of mitochondria on BBB permeability in vitro and in vivo; the effects of mitochondrial inhibitor on BBB permeability, infarct volume, and functional outcomes after transient middle cerebral artery occlusion. Results— We report here that lipopolysaccharide worsens ischemic stroke outcome and increases BBB permeability after transient middle cerebral artery occlusion in mice. Furthermore, we elucidate a novel mechanism that compromised mitochondrial function accounts for increased BBB permeability as evidenced by: lipopolysaccharide-induced reductions in oxidative phosphorylation and subunit expression of respiratory chain complexes in cerebrovascular endothelial cells, a compromised BBB permeability induced by pharmacological inhibition of mitochondrial function in cerebrovascular endothelial cells in vitro and in an in vivo animal model, and worsened stroke outcomes in transient middle cerebral artery occlusion mice after inhibition of mitochondrial function. Conclusions— We concluded that mitochondria are key players in BBB permeability. These novel findings suggest a potential new therapeutic strategy for ischemic stroke by endothelial cell mitochondrial regulation.

Inflammatory mechanisms play a crucial role in the pathophysiologic processes after the onset of ischemic stroke (Vila et al. 2000). Ischemic brain injury is complex, and intracellular signaling that regulates innate and adaptive immunity, inflammation, cell death, angiogenesis, and repair processes plays an important role in the initiation, progression, and resolution of ischemia. Initiation of these critical intracellular regulators occurs through rapid cytokine signaling after ischemia, highlighting cytokines as key regulators of stroke damage (Hallenbeck 2002). Tumor necrosis factor alpha (TNF-α) is one of many pro-inflammatory cytokines associated with worsened clinical outcomes after stroke and exacerbations of infarct size in pre-clinical models (Nawashiro et al. 1997; Ormstad et al. 2011). However, our understanding of the pro-inflammatory effects of TNF-α is based primarily on studies of its peripheral actions. TNF-α mRNA increases within 1 h in the ischemic injury core, and the expression of its immunoreactive protein increases within 2–6 h after the onset of ischemia in preclinical models (Botchkina et al. 1997). TNF-α is increased in the serum of stroke patients between 6 and 12 h after symptom onset (Liu et al. 1994; Sotgiu et al. 2006). TNF-α signaling occurs through the TNF-α receptor (TNF-R), where ligand binding recruits adaptor proteins to a core signaling complex allowing for differences in signal transduction depending upon the stimulatory pattern (Wallach et al. 2002). The response to TNF-α is cell type dependent. In bovine and rat oligodendrocytes, TNF-α induces apoptosis, while in astrocytes, TNF-α enhances major histocompatibility complex class II and intracellular adhesion molecule 1 expression (Chao et al. 1995). In primary septo-hippocampal cultures, exposure to 30 ng/mL of TNF-α caused significant cytotoxicity but only with the addition of Actinomycin-D (Zhao et al. 2001). When rat primary cortical neurons are exposed to 10 ng/mL of TNF-α for 24 h, neurite retraction or formation of apoptotic bodies was not observed (Reimann-Philipp et al. 2001). However, in PC12 cells, TNF-α was cytotoxic (Reimann-Philipp et al. 2001). While these studies show neurotoxic effects of TNF-α, cultured embryonic rat hippocampal, septal, and cortical neurons are protected from glucose deprivation-induced injury and excitatory amino acid toxicity by exposure to TNF-α (Cheng et al. 1994). Furthermore, TNF-α can induce the expression of anti-apoptotic proteins B-cell lymphoma 2 and B-cell lymphoma-extra large (Bcl-xl) in hippocampal neurons (Tamatani et al. 1999). These few studies used high concentrations and long periods of exposure to TNF-α. Also, there is a single study of adipocytes that used high concentrations and 4-day exposures to assess mitochondrial function (Chen et al. 2010). In the present study, we tested the hypothesis that acute exposure to TNF-α concentrations seen in serum of stroke patients (Lambertsen et al. 2012; Nayak et al. 2012) causes neuronal cell death through rapid mitochondrial dysfunction. As such, we characterized the effect of acute exposure to low doses of TNF-α in a mouse hippocampal neuronal cell line (HT-22) and mouse primary cortical neurons. A rapid and profound mitochondrial dysfunction was observed after as little as 1.5 h of exposure to TNF-α which preceded cell death. These findings suggest that the damaging effect of TNF-α may be because of the impairment of neuronal mitochondrial function.

Chronic stroke is the phase of brain recovery and repair generally beginning 3 months after stroke onset. No pharmaceutical approach is currently available to enhance brain repair in chronic stroke. We have previously determined the therapeutic effects of stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) alone or in combination (SCF + G-CSF) in an animal model of chronic stroke and demonstrated that only SCF + G-CSF induces long-term functional recovery. However, the mechanism underlying the SCF + G-CSF-induced brain repair in chronic stroke remains largely elusive. In the present study, we determined the role of nuclear factor-kappa B (NF-κB) in neurovascular network remodeling and motor function improvement by SCF + G-CSF treatment in chronic stroke. SCF + G-CSF was subcutaneously administered for 7 days beginning 17 weeks after induction of experimental stroke. To inhibit NF-κB activation, NF-κB inhibitor was infused into the brain before SCF + G-CSF treatment. We observed that NF-κB inhibitor abolished the SCF + G-CSF-induced axonal sprouting, synaptogenesis and angiogenesis in the ipsilesional somatosensorimotor cortex. In addition, blockage of NF-κB activation resulted in elimination of the SCF + G-CSF-induced motor functional restoration in chronic stroke. These data suggest that NF-κB is required for the SCF + G-CSF-induced neuron-vascular network remodeling in the ipsilesional somatosensorimotor cortex and motor functional recovery in chronic stroke.

Firstly by improving inverse P-sets, inverse P-data model is proposed, it consists of internal and external inverse P-data models, and demonstrates dynamic characteristics; then the expansion-contraction generations of attribute disjunctive normal form for inverse P-data model are shown, and inverse P-data reasoning is proposed; finally intelligent data separation-discovery is given, and verified by an application.

Introduction: The aim here is to elucidate the mechanisms involved in post-stroke cerebrovascular impairments in metabolic syndrome (MetS) that pre-dispose obese patients to more severe post-stroke deficits. Methods: 1 hr middle cerebral artery occlusion was performed in 17 week old lean (LZR) and obese Zucker (OZR: MetS model) rats. At 24 hr or 15 days post-stroke, the ipsilateral middle cerebral artery (MCA) was cannulated in an ex-vivo microvessel preparation to examine MCA reactivity. Cerebrovascular microvessel density (MVD) was examined 15 days post-stroke via IHC. To examine a potential role of Nox2 and oxidative stress, a specific inhibitor of Nox2 oxidase Nox2-dstat (i.p 10mg/kg) was given during the stroke procedure in OZR. Results: In LZR, MCA reactivity was impaired vs. non-stroke control at 24hr post-stroke and remained stable at 15 days post-stroke. In LZR, NO bioavailability was reduced at 24hrs vs. controls, which remained stable at 15 days. In OZR, the MCA impairment was greater at 24 hr vs. LZR, and worsened significantly at 15 days post stroke. NO bioavailability for OZR at 24 hr was similar to LZR stroke, but by 15 days had fallen. MVD was reduced 15 days post-stroke, with a larger loss in OZR vs. LZR. Stroke mortality and infarct size were higher for OZR. Nox2 inhibition restored MCA reactivity, improved MVD and NO levels, and limited stroke infarct size in OZR post-stroke. Discussion: Our data suggest that the MetS increases stroke severity and drives a progressive decline in cerebrovascular dysfunction following ischemic stroke. Inhibiting Nox2 production improved stroke outcome in MetS.

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Stroke is a complex vascular and neurological syndrome that can lead to death and disability. About 15 million people worldwide and 800,000 people in the United States suffer stroke each year . On average, in the U.S. every 40 seconds someone has a stroke and every 4 minutes someone dies from a stroke. This medical emergency has very limited treatments, and causes serious health and economic burdens in the United States and globally. Blood-brain barrier (BBB) is composed of highly specialized cerebrovascular endothelial cells, seals brain tissue from the circulating blood, and prevents blood, bacteria or toxins from reaching the central nervous system (CNS). In acute ischemic stroke, BBB is disrupted, and blood solutes penetrate into the CNS parenchymal extracellular space then cause cerebral edema . Although much has been observed in this stroke-induced brain edema, such as inflammatory infiltration, releasing of chemokines and cytokines, much less has the mechanism of BBB disruption been understood.

By improving function inverse P-sets, inverse P-information law model and its structures are proposed. Then attribute disjunctive expansion-contraction generations of inverse P-information law models are presented. Furthermore, inverse P-information reasoning and reasoning structures are proposed Finally intelligent generating theorems of information law are proposed, the reality-camouflage intelligent transformations of information image and their intelligent generations and applications are also given.

Stroke is a complex vascular and neurological syndrome that can lead to death and disability. The primary stroke injury is induced by the occlusion of directly supplied artery, and the secondary brain damage could be affected by the synaptic connections with the primary ischemic lesion. Tau protein is a microtubule associated cytoskeletal protein, expressed predominantly in neurons of the central nervous system. Tau protein plays a role in maintaining synaptic neuronal integrity and axonal transport, including promoting microtubule assembly and modulating stability of microtubules, which are regulated by tau phosphorylation state. Phosphorylated tau protein is induced by stroke. To investigate the role of human Tau protein in stroke, we employed female mice overexpressing normal human Tau (rTg(Tau)21221), female mice overexpressing P301L mutant human tau (rTg(TauP301L)4510, and age-matched female littermate wild type (WT) controls at 12 months old. Transient middle cerebral artery occlusion (tMCAO) was performed on diestrus as identified by virginal smears microscopically. Following a mild stroke injury of 30 min tMCAO, overexpression of normal human Tau significantly reduced infarction at 48 hours of reperfusion (P = 0.0157); following a moderate stroke injury of 60 min tMCAO, overexpression of normal human Tau also resulted in significantly reduced infarction compared to WT controls and mice expressing human tau with the P301L mutation (P = 0.0142). These novel data suggest that normal human Tau plays a protective role in acute stroke and support the view of targeting human tau as a new treatment for acute stroke.

Recent evidence suggests that Alzheimer’s disease is a vascular disorder that can be accelerated by ischemic stroke. We propose that the underlying pathophysiology accelerating degenerative changes is due to blood brain barrier disruption following ischemia. A transient middle cerebral artery occlusion suture model was used in triple-transgenic AD mice and C57/Bl6 wild type controls. A separate triple-transgenic AD mice group was used for sham control. The middle cerebral artery was occluded for 1 hour followed by reperfusion. Mice were evaluated for sickness behavior and received a modified neurologic score. 5 hours after reperfusion, blood brain barrier tracers (Texas red, IcG, and AIB1) were injected i.v. into the femoral vein and allowed to circulate for 10 minutes. H&E, Cresyl Violet, and Thioflavin were used to stain sections of the brain for areas of degeneration. Images were obtained by quantitative fluorescent and bright-field microscopy. One-way ANOVA with Tukey’s post-hoc comparison was used to compare between groups. P