Spatiotemporal sphingosine-1-phosphate receptor 3 expression within the cerebral vasculature after ischemic stroke.

Sphingosine-1-phosphate receptors (S1PRs) are promising therapeutic targets in cardiovascular disease, including ischemic stroke. However, important spatiotemporal information for alterations of S1PR expression is lacking. Here, we investigated the role of S1PR3 in ischemic stroke in rodent models and patient samples. We show that S1PR3 is acutely upregulated in perilesional reactive astrocytes after stroke, and that stroke volume and behavioral deficits are improved in mice lacking S1PR3. Further, we find that administration of an S1PR3 antagonist at 4-h post-stroke, but not at later timepoints, improves stroke outcome. Lastly, we observed higher plasma S1PR3 concentrations in experimental stroke and in patients with ischemic stroke. Together, our results establish S1PR3 as a potential drug target and biomarker in ischemic stroke.

Plasma Galectin-4 Levels Are Increased after Stroke in Mice and Humans.

Epidemiological studies have associated plasma galectin-4 (Gal-4) levels with prevalent and incident diabetes, and with an increased risk of coronary artery disease. To date, data regarding possible associations between plasma Gal-4 and stroke are lacking. Using linear and logistic regression analyses, we tested Gal-4 association with prevalent stroke in a population-based cohort. Additionally, in mice fed a high-fat diet (HFD), we investigated whether plasma Gal-4 increases in response to ischemic stroke. Plasma Gal-4 was higher in subjects with prevalent ischemic stroke, and was associated with prevalent ischemic stroke (odds ratio 1.52; 95% confidence interval 1.01-2.30; p = 0.048) adjusted for age, sex, and covariates of cardiometabolic health. Plasma Gal-4 increased after experimental stroke in both controls and HFD-fed mice. HFD exposure was devoid of impact on Gal-4 levels. This study demonstrates higher plasma Gal-4 levels in both experimental stroke and in humans that experienced ischemic stroke.

Pregabalin improves axon regeneration and motor outcome in a rodent stroke model.

Ischaemic stroke remains a leading cause of death and disability worldwide. Surviving neurons in the peri-infarct area are able to establish novel axonal projections to juxtalesional regions, but this regeneration is curtailed by a growth-inhibitory environment induced by cells such as reactive astrocytes in the glial scar. Here, we found that the astroglial synaptogenic cue thrombospondin-1 is upregulated in the peri-infarct area, and hence tested the effects of the anticonvulsant pregabalin, a blocker of the neuronal thrombospondin-1 receptor Alpha2delta1/2, in a mouse model of cortical stroke. Studying axonal projections after cortical stroke in mice by three-dimensional imaging of cleared whole-brain preparations, we found that pregabalin, when administered systemically for 5 weeks after stroke, augments novel peri-infarct motor cortex projections and improves skilled forelimb motor function. Thus, the promotion of axon elongation across the glial scar by pregabalin represents a promising target beyond the acute phase after stroke to improve structural and functional recovery.

Not All Lectins Are Equally Suitable for Labeling Rodent Vasculature.

The vascular system is vital for all tissues and the interest in its visualization spans many fields. A number of different plant-derived lectins are used for detection of vasculature; however, studies performing direct comparison of the labeling efficacy of different lectins and techniques are lacking. In this study, we compared the labeling efficacy of three lectins: Griffonia simplicifolia isolectin B4 (IB4); wheat germ agglutinin (WGA), and Lycopersicon esculentum agglutinin (LEA). The LEA lectin was identified as being far superior to the IB4 and WGA lectins in histological labeling of blood vessels in brain sections. A similar signal-to-noise ratio was achieved with high concentrations of the WGA lectin injected during intracardial perfusion. Lectins were also suitable for labeling vasculature in other tissues, including spinal cord, dura mater, heart, skeletal muscle, kidney, and liver tissues. In uninjured tissues, the LEA lectin was as accurate as the Tie2-eGFP reporter mice and GLUT-1 immunohistochemistry for labeling the cerebral vasculature, validating its specificity and sensitivity. However, in pathological situations, e.g., in stroke, the sensitivity of the LEA lectin decreases dramatically, limiting its applicability in such studies. This work can be used for selecting the type of lectin and labeling method for various tissues.

Simvastatin therapy attenuates memory deficits that associate with brain monocyte infiltration in chronic hypercholesterolemia.

Evidence associates cardiovascular risk factors with unfavorable systemic and neuro-inflammation and cognitive decline in the elderly. Cardiovascular therapeutics (e.g., statins and anti-hypertensives) possess immune-modulatory functions in parallel to their cholesterol- or blood pressure (BP)-lowering properties. How their ability to modify immune responses affects cognitive function is unknown. Here, we examined the effect of chronic hypercholesterolemia on inflammation and memory function in Apolipoprotein E (ApoE) knockout mice and normocholesterolemic wild-type mice. Chronic hypercholesterolemia that was accompanied by moderate blood pressure elevations associated with apparent immune system activation characterized by increases in circulating pro-inflammatory Ly6Chi monocytes in ApoE-/- mice. The persistent low-grade immune activation that is associated with chronic hypercholesterolemia facilitates the infiltration of pro-inflammatory Ly6Chi monocytes into the brain of aged ApoE-/- but not wild-type mice, and links to memory dysfunction. Therapeutic cholesterol-lowering through simvastatin reduced systemic and neuro-inflammation, and the occurrence of memory deficits in aged ApoE-/- mice with chronic hypercholesterolemia. BP-lowering therapy alone (i.e., hydralazine) attenuated some neuro-inflammatory signatures but not the occurrence of memory deficits. Our study suggests a link between chronic hypercholesterolemia, myeloid cell activation and neuro-inflammation with memory impairment and encourages cholesterol-lowering therapy as safe strategy to control hypercholesterolemia-associated memory decline during ageing.

Vagus nerve stimulation reduces spreading depolarization burden and cortical infarct volume in a rat model of stroke.

Cortical spreading depolarization (SD) waves negatively affect neuronal survival and outcome after ischemic stroke. We here aimed to investigate the effects of vagus nerve stimulation (VNS) on SDs in a rat model of focal ischemia. To this end, we delivered non-invasive VNS (nVNS) or invasive VNS (iVNS) during permanent middle cerebral artery occlusion (MCAO), and found that both interventions significantly reduced the frequency of SDs in the cortical peri-infarct area compared to sham VNS, without affecting relative blood flow changes, blood pressure, heart rate or breathing rate. In separate groups of rats subjected to transient MCAO, we found that cortical stroke volume was reduced 72 h after transient MCAO, whereas stroke volume in the basal ganglia remained unchanged. In rats treated with nVNS, motor outcome was improved 2 days after transient MCAO, but was similar to sham VNS animals 3 days after ischemia. We postulate that VNS may be a safe and efficient intervention to reduce the clinical burden of SD waves in stroke and other conditions.

Improving Cerebrovascular Function to Increase Neuronal Recovery in Neurodegeneration Associated to Cardiovascular Disease.

Mounting evidence indicates that the presence of cardiovascular disease (CVD) and risk factors elevates the incidence of cognitive impairment (CI) and dementia. CVD and associated decline in cardiovascular function can impair cerebral blood flow (CBF) regulation, leading to the disruption of oxygen and nutrient supply in the brain where limited intracellular energy storage capacity critically depends on CBF to sustain proper neuronal functioning. During hypertension and acute as well as chronic CVD, cerebral hypoperfusion and impaired cerebrovascular function are often associated with neurodegeneration and can lead to CI and dementia. Currently, all forms of neurodegeneration associated to CVD lack effective treatments, which highlights the need to better understand specific mechanisms linking cerebrovascular dysfunction and CBF deficits to neurodegeneration. In this review, we discuss vascular targets that have already shown attenuation of neurodegeneration or CI associated to hypertension, heart failure (HF) and stroke by improving cerebrovascular function or CBF deficits.

The emerging alliance of sphingosine-1-phosphate signalling and immune cells: from basic mechanisms to implications in hypertension.

The immune system plays a considerable role in hypertension. In particular, T-lymphocytes are recognized as important players in its pathogenesis. Despite substantial experimental efforts, the molecular mechanisms underlying the nature of T-cell activation contributing to an onset of hypertension or disease perpetuation are still elusive. Amongst other cell types, lymphocytes express distinct profiles of GPCRs for sphingosine-1-phosphate (S1P) - a bioactive phospholipid that is involved in many critical cell processes and most importantly majorly regulates T-cell development, lymphocyte recirculation, tissue-homing patterns and chemotactic responses. Recent findings have revealed a key role for S1P chemotaxis and T-cell mobilization for the onset of experimental hypertension, and elevated circulating S1P levels have been linked to several inflammation-associated diseases including hypertension in patients. In this article, we review the recent progress towards understanding how S1P and its receptors regulate immune cell trafficking and function and its potential relevance for the pathophysiology of hypertension. LINKED ARTICLES: This article is part of a themed section on Immune Targets in Hypertension. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.12/issuetoc.

Stroke target identification guided by astrocyte transcriptome analysis.

Astrocytes support normal brain function, but may also contribute to neurodegeneration when they become reactive under pathological conditions such as stroke. However, the molecular underpinnings of this context-dependent interplay between beneficial and detrimental properties in reactive astrogliosis have remained incompletely understood. Therefore, using the RiboTag technique, we immunopurified translating mRNAs specifically from astrocytes 72 hr after transient middle cerebral artery occlusion in mice (tMCAO), thereby generating a stroke-specific astroglial translatome database. We found that compared to control brains, reactive astrocytes after tMCAO show an enrichment of transcripts linked to the A2 phenotype, which has been associated with neuroprotection. However, we found that astrocytes also upregulate a large number of potentially neurotoxic genes. In total, we identified the differential expression of 1,003 genes and 38 transcription factors, of which Stat3, Sp1, and Spi1 were the most prominent. To further explore the effects of Stat3-mediated pathways on stroke pathogenesis, we subjected mice with an astrocyte-specific conditional deletion of Stat3 to tMCAO, and found that these mice have reduced stroke volume and improved motor outcome 72 hr after focal ischemia. Taken together, our study extends the emerging database of novel astrocyte-specific targets for stroke therapy, and supports the role of astrocytes as critical safeguards of brain function in health and disease.

A single-cell analysis reveals multiple roles of oligodendroglial lineage cells during post-ischemic regeneration.

NG2 cells represent precursors of oligodendrocytes under physiological conditions; however, following cerebral ischemia they play an important role in glial scar formation. Here, we compared the expression profiles of oligodendroglial lineage cells, after focal cerebral ischemia (FCI) and in Alzheimer's-like pathology using transgenic mice, which enables genetic fate-mapping of Cspg4-positive NG2 cells and their progeny, based on the expression of red fluorescent protein tdTomato. tdTomato-positive cells possessed the expression profile of NG2 cells and oligodendrocytes; however, based on the expression of cell type-specific genes, we were able to distinguish between them. To shed light on the changes in the expression patterns caused by FCI, we employed self-organizing Kohonen maps, enabling the division of NG2 cells and oligodendrocytes into subpopulations based on similarities in the expression profiles of individual cells. We identified three subpopulations of NG2 cells emerging after FCI: proliferative; astrocyte-like and oligodendrocyte-like NG2 cells; such phenotypes were further confirmed by immunohistochemistry. Oligodendrocytes themselves formed four subpopulations, which reflected the process of oligodendrocytes maturation. Finally, we used 5-ethynyl-2' deoxyuridine (EdU) labeling to reveal that NG2 cells can differentiate directly into reactive astrocytes without preceding proliferation. In contrast, in Alzheimer's-like pathology we failed to identify these subpopulations. Collectively, here we identified several yet unknown differences between the expression profiles of NG2 cells and oligodendrocytes, and characterized specific genes contributing to oligodendrocyte maturation and phenotypical changes of NG2 cells after FCI. Moreover, our results suggest that, unlike in Alzheimer's-like pathology, NG2 cells acquire a multipotent phenotype following FCI.

Generation of reactive astrocytes from NG2 cells is regulated by sonic hedgehog.

NG2 cells, a fourth glial cell type in the adult mammalian central nervous system, produce oligodendrocytes in the healthy nervous tissue, and display wide differentiation potential under pathological conditions, where they could give rise to reactive astrocytes. The factors that control the differentiation of NG2 cells after focal cerebral ischemia (FCI) are largely unknown. Here, we used transgenic Cspg4-cre/Esr1/ROSA26Sortm14(CAG-tdTomato) mice, in which tamoxifen administration triggers the expression of red fluorescent protein (tomato) specifically in NG2 cells and cells derived therefrom. Differentiation potential (in vitro and in vivo) of tomato-positive NG2 cells from control or postischemic brains was determined using the immunohistochemistry, single cell RT-qPCR and patch-clamp method. The ischemic injury was induced by middle cerebral artery occlusion, a model of FCI. Using genetic fate-mapping method, we identified sonic hedgehog (Shh) as an important factor that influences differentiation of NG2 cells into astrocytes in vitro. We also manipulated Shh signaling in the adult mouse brain after FCI. Shh signaling activation significantly increased the number of astrocytes derived from NG2 cells in the glial scar around the ischemic lesion, while Shh signaling inhibition caused the opposite effect. Since Shh signaling modifications did not change the proliferation rate of NG2 cells, we can conclude that Shh has a direct influence on the differentiation of NG2 cells and therefore, on the formation and composition of a glial scar, which consequently affects the degree of the brain damage. GLIA 2016;64:1518-1531.

A Rat Model of Alzheimer's Disease Based on Abeta42 and Pro-oxidative Substances Exhibits Cognitive Deficit and Alterations in Glutamatergic and Cholinergic Neurotransmitter Systems.

Alzheimer's disease (AD) is one of the most serious human, medical, and socioeconomic burdens. Here we tested the hypothesis that a rat model of AD (Samaritan; Taconic Pharmaceuticals, USA) based on the application of amyloid beta42 (Abeta42) and the pro-oxidative substances ferrous sulfate heptahydrate and L-buthionine-(S, R)-sulfoximine, will exhibit cognitive deficits and disruption of the glutamatergic and cholinergic systems in the brain. Behavioral methods included the Morris water maze (MWM; long-term memory version) and the active allothetic place avoidance (AAPA) task (acquisition and reversal), testing spatial memory and different aspects of hippocampal function. Neurochemical methods included testing of the NR1/NR2A/NR2B subunits of NMDA receptors in the frontal cortex and CHT1 transporters in the hippocampus, in both cases in the right and left hemisphere separately. Our results show that Samaritan rats(™) exhibit marked impairment in both the MWM and active place avoidance tasks, suggesting a deficit of spatial learning and memory. Moreover, Samaritan rats exhibited significant changes in NR2A expression and CHT1 activity compared to controls rats, mimicking the situation in patients with early stage AD. Taken together, our results corroborate the hypothesis that Samaritan rats are a promising model of AD in its early stages.