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Ischemic stroke occurs with no warning, and therefore, very little is known about hemodynamic perturbations in the brain immediately after stroke onset. Here, functional ultrasound imaging was used to monitor variations in relative cerebral blood volume (rCBV) compared to baseline. rCBV levels were analyzed brain-wide and continuously at high spatiotemporal resolution (100μm, 2Hz) until 70mins after stroke onset in rats. We compared two stroke models, with either a permanent occlusion of the middle cerebral artery (MCAo) or a tandem occlusion of both the common carotid and middle cerebral arteries (CCAo+MCAo). We observed a typical hemodynamic pattern, including a quick drop of the rCBV after MCAo, followed by spontaneous reperfusion of several brain regions located in the vicinity of the ischemic core. The severity and location of the ischemia were highly variable between animals. Still, both parameters were, on average, in good agreement with the final ischemic lesion volume measured 24hrs after stroke onset for the MCAo but not the CCAo+MCAo model. For the latter, we observed that the infarct was extended to regions that were initially not ischemic and located rostrally of the ischemic core. These regions strongly colocalize with the origin of transient hemodynamic events associated with spreading depolarizations.

Serum BDNF concentrations in 2053 participants of the Berlin Aging Study II (BASE-II; 1572 individuals from the older age group [60-85 years], 481 individuals from the younger-age reference group [22-37 years]) were studied. There was no effect of age, sex, body mass index, self-reported depression, or BDNF Val66Met variant on serum BDNF concentrations. Multiple linear regression analysis failed to detect significant relationships of Digit Symbol Substitution Test score and Consortium to Establish a Registry for Alzheimer's Disease memory score to BDNF levels. However, we detected a positive correlation between platelet counts and BDNF levels (r = 0.303, p < 0.001). Our findings do not support an effect of aging, self-reported depression, or the Val66Met variant on serum BDNF concentrations. The role of thrombocytes in the biology of serum BDNF merits further study.

Spreading depolarization is observed as a large negative shift of the direct current potential, swelling of neuronal somas, and dendritic beading in the brain’s gray matter and represents a state of a potentially reversible mass injury. Its hallmark is the abrupt, massive ion translocation between intraneuronal and extracellular compartment that causes water uptake (= cytotoxic edema) and massive glutamate release. Dependent on the tissue’s energy status, spreading depolarization can co-occur with different depression or silencing patterns of spontaneous activity. In adequately supplied tissue, spreading depolarization induces spreading depression of activity. In severely ischemic tissue, nonspreading depression of activity precedes spreading depolarization. The depression pattern determines the neurological deficit which is either spreading such as in migraine aura or migraine stroke or nonspreading such as in transient ischemic attack or typical stroke. Although a clinical distinction between spreading and nonspreading focal neurological deficits is useful because they are associated with different probabilities of permanent damage, it is important to note that spreading depolarization, the neuronal injury potential, occurs in all of these conditions. Here, we first review the scientific basis of the continuum of spreading depolarizations. Second, we highlight the transition zone of the continuum from reversibility to irreversibility using clinical cases of aneurysmal subarachnoid hemorrhage and cerebral amyloid angiopathy. These illustrate how modern neuroimaging and neuromonitoring technologies increasingly bridge the gap between basic sciences and clinic. For example, we provide direct electrophysiological evidence for the first time that spreading depolarization-induced spreading depression is the pathophysiological correlate of the migraine aura.

Vascular mechanisms underlying the adverse effects that depression and stress-related mental disorders have on stroke outcome are only partially understood. Identifying the transcriptomic signature of chronic stress in endothelium harvested from the ischemic brain is an important step towards elucidating the biological processes involved. Here, we subjected male 129S6/SvEv mice to a 28-day model of chronic stress. The ischemic lesion was quantified after 30 min filamentous middle cerebral artery occlusion (MCAo) and 48 h reperfusion by T2-weighted MRI. RNA sequencing was used to profile transcriptomic changes in cerebrovascular endothelial cells (ECs) from the infarct. Mice subjected to the stress procedure displayed reduced weight gain, increased adrenal gland weight, and increased hypothalamic FKBP5 mRNA and protein expression. Chronic stress conferred increased lesion volume upon MCAo. Stress-exposed mice showed a higher number of differentially expressed genes between ECs isolated from the ipsilateral and contralateral hemisphere than control mice. The genes in question are enriched for roles in biological processes closely linked to endothelial proliferation and neoangiogenesis. MicroRNA-34a was associated with nine of the top 10 biological process Gene Ontology terms selectively enriched in ECs from stressed mice. Moreover, expression of mature miR-34a-5p and miR-34a-3p in ischemic brain tissue was positively related to infarct size and negatively related to sirtuin 1 (Sirt1) mRNA transcription. In conclusion, this study represents the first EC-specific transcriptomic analysis of chronic stress in brain ischemia. The stress signature uncovered relates to worse stroke outcome and is directly relevant to endothelial mechanisms in the pathogenesis of stroke. Electronic supplementary material The online version of this article (10.1007/s12035-019-01822-3) contains supplementary material, which is available to authorized users.

In the adult hippocampal dentate gyrus (DG), the majority of newly generated cells are eliminated by apoptotic mechanisms. The apoptosis repressor with caspase recruitment domain (ARC), encoded by the Nol3 gene, is a potent and multifunctional death repressor that inhibits both death receptor and mitochondrial apoptotic signaling. The aim of the present study was to parse the role of ARC in the development of new granule cell neurons. Nol3 gene expression as revealed by in situ hybridization is present in the entire dentate granule cell layer. Moreover, a comparison of Nol3 expression between FACS-sorted Sox2-positive neural stem cells and Doublecortin (DCX)-positive immature neurons demonstrates upregulation of Nol3 during neurogenesis. Using ARC-deficient mice, we show that proliferation and survival of BrdU birth-dated cells are strongly reduced in the absence of ARC while neuronal-glial fate choice is not affected. Both the number of DCX-positive cells and the number of calretinin (CR)-positive immature postmitotic neurons are reduced in the hippocampus of ARC-/- mice. ARC knockout is not associated with increased numbers of microglia or with microglia activation. However, hippocampal brain-derived neurotrophic factor (BDNF) protein content is significantly increased in ARC-/- mice, possibly representing a compensatory response. Collectively, our results suggest that ARC plays a critical cell-autonomous role in preventing cell death during adult granule cell neogenesis.

Despite its decades' long therapeutic use in psychiatry, the biological mechanisms underlying lithium's mood-stabilizing effects have remained largely elusive. Here, we investigated the effect of lithium on tryptophan breakdown via the kynurenine pathway using immortalized human microglia cells, primary human microglia isolated from surgical specimens, and microglia-like cells differentiated from human induced pluripotent stem cells. Interferon (IFN)-gamma, but not lipopolysaccharide, was able to activate immortalized human microglia, inducing a robust increase in indoleamine-2,3-dioxygenase (IDO1) mRNA transcription, IDO1 protein expression, and activity. Further, chromatin immunoprecipitation verified enriched binding of both STAT1 and STAT3 to the IDO1 promoter. Lithium counteracted these effects, increasing inhibitory GSK3 beta(S9) phosphorylation and reducing STAT1(S727) and STAT3(Y705) phosphorylation levels in IFN-gamma treated cells. Studies in primary human microglia and hiPSC-derived microglia confirmed the anti-inflammatory effects of lithium, highlighting that IDO activity is reduced by GSK3 inhibitor SB-216763 and STAT inhibitor nifuroxazide via downregulation of P-STAT1(S727) and P-STAT3(Y705). Primary human microglia differed from immortalized human microglia and hiPSC derived microglia-like cells in their strong sensitivity to LPS, resulting in robust upregulation of IDO1 and anti-inflammatory cytokine IL-10. While lithium again decreased IDO1 activity in primary cells, it further increased release of IL-10 in response to LPS. Taken together, our study demonstrates that lithium inhibits the inflammatory kynurenine pathway in the microglia compartment of the human brain.

The ‘Stroke Complications after Traumatic Experiences and Stress’ (SATURN) study was designed to investigate the effects of a prior traumatic event on PTSD symptoms triggered by a subsequent stroke. First-ever ischemic stroke patients were surveyed 9–13 months after hospitalization at the Charite University Medical Center. Stroke-induced PTSD symptoms were measured using the Impact of Event Scale-revised (IES-R). Prior traumatization together with past PTSD symptoms was assessed retrospectively with the Brief Trauma Questionnaire (BTQ) and the 7-item Short Screening Scale for PTSD. Depressive symptoms were assessed with the Beck Depression Inventory (BDI-II). The Short Form (SF)-36 was used to evaluate physical and mental health outcomes. We received 258 responses from 636 eligible patients (~41%). Based on respondents' scores on the IES-R, the prevalence of probable PTSD due to the stroke event was 11% in our sample. Female sex and younger age were associated with more severe PTSD symptoms. Psychological endpoints did not differ between patients who denied prior trauma exposure and those who reported earlier trauma exposure but denied subsequent PTSD symptoms. However, a history of trauma exposure in tandem with endorsing subsequent PTSD symptoms was linked with significantly increased post-stroke PTSD and depressive symptoms together with decreased psychological well-being. Self-reported physical health did not differ across groups. Younger age, being a woman, and having developed PTSD symptoms in the aftermath of a prior trauma were associated with adverse psychological outcomes after stroke.

The beneficial effects of cortical activation for functional recovery after ischemic stroke have been well described. However, little is known about the role of early sensory stimulation, i.e. stimulation during first 6 h after stroke onset even during acute treatment. In recent years, various preclinical studies reported significant effects of acute sensory stimulation that range from entire neuroprotection to increased infarct volumes by 30–50%. Systematic knowledge about the effect of acute sensory stimulation on stroke outcome is highly relevant as stroke patients are subject to uncontrolled sensory stimulation during transport, acute treatment, and critical care. This article discusses the current stage of knowledge about acute sensory stimulation and provides directions for future experimental and clinical trials.

After stroke, macrophages in the ischemic brain may be derived from either resident microglia or infiltrating monocytes. Using bone marrow (BM)-chimerism and dual-reporter transgenic fate mapping, we here set out to delimit the responses of either cell type to mild brain ischemia in a mouse model of 30 min transient middle cerebral artery occlusion (MCAo). A discriminatory analysis of gene expression at 7 days post-event yielded 472 transcripts predominantly or exclusively expressed in blood-derived macrophages as well as 970 transcripts for microglia. The differentially regulated genes were further collated with oligodendrocyte, astrocyte, and neuron transcriptomes, resulting in a dataset of microglia- and monocyte-specific genes in the ischemic brain. Functional categories significantly enriched in monocytes included migration, proliferation, and calcium signaling, indicative of strong activation. Whole-cell patch-clamp analysis further confirmed this highly activated state by demonstrating delayed outward K+ currents selectively in invading cells. Although both cell types displayed a mixture of known phenotypes pointing to the significance of 'intermediate states' in vivo, blood-derived macrophages were generally more skewed toward an M2 neuroprotective phenotype. Finally, we found that decreased engraftment of blood-borne cells in the ischemic brain of chimeras reconstituted with BM from Selplg-/- mice resulted in increased lesions at 7 days and worse post-stroke sensorimotor performance. In aggregate, our study establishes crucial differences in activation state between resident microglia and invading macrophages after stroke and identifies unique genomic signatures for either cell type.

Microglia are the immune cells of the brain and become activated during any type of brain injury. In the middle cerebral artery occlusion (MCAo) model, a mouse model for ischemic stroke, we have previously shown that microglia and invaded monocytes upregulate the expression of the muscarinic acetylcholine receptor 3 (M3R) in the ischemic lesion. Here we tested whether this upregulation has an impact on the pathogenesis of MCAo. We depleted the m3R receptor in microglia, but not in circulating monocytes by giving tamoxifen to CX3CR1-CreERT+/+M3Rflox/flox (M3RKOmi) animals 3 weeks prior to MCAo. We found that M3RKOmi male mice had bigger lesions, more pronounced motor deficits after one week and cognitive deficits after about one month compared to control males. The density of Iba1+ cells was lower in the lesions of M3RKO male mice in the early, but not in the late disease phase. In females, these differences were not significant. By giving tamoxifen 1 week prior to MCAo, we depleted m3R in microglia and in circulating monocytes (M3RKOmi/mo). Male M3RKOmi/mo did not differ in lesion size, but had a lower survival rate, showed motor deficits and a reduced accumulation of Iba1+ positive cells into the lesion site. In conclusion, our data suggest that the upregulation of m3R in microglia and monocytes in stroke has a beneficial effect on the clinical outcome in male mice.

Die posttraumatische Belastungsstorung (PTBS) wurde bisher hauptsachlich mit Kriegstraumata, sexueller oder physischer Gewalteinwirkung in Verbindung gebracht. Tatsachlich kommt die PTBS jedoch auch haufig nach lebensbedrohlichen korperlichen Erkrankungen wie Herzinfarkt und Schlaganfall vor. Eine PTBS wird in der klinischen Praxis in diesem Kontext viel zu selten diagnostiziert, obwohl ein erheblicher Einfluss auf das klinische Outcome und die Mortalitat nach vaskularen Ereignissen besteht. Zudem bildet die PTBS einen wichtigen Risikofaktor fur die Entwicklung vaskularer Erkrankungen. Diese Ubersichtsarbeit erlautert den bidirektionalen Zusammenhang zwischen PTBS und vaskularen Ereignissen und fasst die bisherigen Forschungsergebnisse bezuglich Klinik, Pravalenz und Pathophysiologie zusammen.

Peroxisome proliferator-activated receptors (PPARs) control the expression of genes involved in glucose homeostasis, lipid metabolism, inflammation, and cell differentiation. Here, we analyzed the effects of aleglitazar, a dual PPARα and PPARγ agonist with balanced affinity for either subtype, on subacute stroke outcome. Healthy young adult mice were subjected to transient 30 min middle cerebral artery occlusion (MCAo)/reperfusion. Daily treatment with aleglitazar was begun on the day of MCAo and continued until sacrifice. Blood glucose measurements and lipid profile did not differ between mice receiving aleglitazar and mice receiving vehicle after MCAo. Aleglitazar reduced the size of the ischemic lesion as assessed using NeuN immunohistochemistry on day 7. Sensorimotor performance on the rotarod was impaired during the first week after MCAo, an effect that was significantly attenuated by treatment with aleglitazar. Smaller lesion volume in mice treated with aleglitazar was accompanied by a decrease in mRNA transcription of IL-1β, Vcam-1, and Icam-1, suggesting that reduced proinflammatory signaling and reduced vascular inflammation in the ischemic hemisphere contribute to the beneficial effects of aleglitazar during the first week after stroke. Further experiments in primary murine microglia confirmed that aleglitazar reduces key aspects of microglia activation including NO production, release of proinflammatory cytokines, migration, and phagocytosis. In aggregate, a brief course of PPARα/γ agonist aleglitazar initiated post-event affords stroke protection and functional recovery in a model of mild brain ischemia. Our data underscores the theme of delayed injury processes such as neuroinflammation as promising therapeutic targets in stroke. Key messages PPARα/γ agonist aleglitazar improves stroke outcome after transient brain ischemia.Aleglitazar attenuates inflammatory responses in post-ischemic brain.Aleglitazar reduces microglia migration, phagocytosis, and release of cytokines.Beneficial effects of aleglitazar independent of glucose regulation.Aleglitazar provides extended window of opportunity for stroke treatment.

To the Editor: We read with great interest the article by Weber et al. (1) in Biological Psychiatry describing the effects of microglia elimination and repopulation on stress sensitization induced by repeated social defeat (RSD). The article highlights brain-immune interactions and, in particular, the importance of stress-primed microglia for monocyte accumulation in the brain of RSD-sensitized mice following acute stress. The transcriptomic analysis of microglia 24 days after RSD could be very useful to other researchers, so the authors may wish to make this information accessible to the community by depositing it to an appropriate data repository.

To describe retinal lesion development in Susac syndrome during acute, postacute, and late phases of the disease.Cross-sectional study of four patients with Susac syndrome and longitudinal short-interval case study of one additional patient. Retinal changes were analyzed with high-resolution spectral domain optical coherence tomography and retinal fluorescein angiography.Retinal Susac syndrome lesions comprise four different lesion sections, which can be distinguished in acute and postacute phases of the disease: a primary section at the site of branch retinal artery occlusion, which spans more layers than supplied by the affected vessel; hypoxic sections from superficial and deep capillary networks; and an axonal damage section with degenerating axons from perished ganglion cells in the main and hypoxic sections. In the later stages, main and hypoxic lesion sections can no longer be distinguished, and both show degeneration from outer plexiform to retinal nerve fiber layers.The dynamics of lesion development and morphologically distinct lesion sections suggest more complex mechanisms of lesion evolution beyond an isolated endothelial immune reaction and subsequent hypoxic tissue damage. The characteristic lesion morphology assists in differentiating the diagnosis of acute visual loss in neuroinflammatory disease. Specificity of the identified changes has to be determined in future studies also including patients with other retinal vascular diseases.

The pattern of vascular remodelling in relation to recovery after stroke remains largely unclear. We used steady-state contrast-enhanced magnetic resonance imaging to assess the development of cerebral blood volume and microvascular density in perilesional and exofocal areas from (sub)acutely to chronically after transient stroke in rats. Microvascular density was verified histologically after infusion with Evans Blue dye. At day 1, microvascular cerebral blood volume and microvascular density were reduced in and around the ischemic lesion (intralesional borderzone: microvascular cerebral blood volume = 72 ± 8%; microvascular density = 76 ± 8%) (P

Evidence for a critical pathophysiological role of aberrant cytoskeletal dynamics is being uncovered in a growing number of neuropsychiatric syndromes. A sedentary lifestyle as well as overt psychopathology is prevalent in patients with the metabolic syndrome. Using mice deficient in gelsolin (Gsn-/-), a crucial actin-severing protein, we here investigated reduced actin turnover as a potential common driver of metabolic disturbances, sedentary behavior, and an anxious/depressive phenotype. Gelsolin deficiency resulted in reduced lifespan. As compared to wildtype controls, Gsn-/- mice (~ 9 weeks) fed a high-fat diet (HFD) over a span of 12 weeks showed increased body weight gain, fat mass, hepatic steatosis, and adipocyte hypertrophy as well as a significantly reduced respiratory quotient. Moreover, increased rigidity of the actin cytoskeleton in mice on HFD induced mRNA expression of Acc1, Acc2, Fasn, and Lipe, key genes involved in fatty acid metabolism in the liver. Glucose tolerance and insulin sensitivity were worsened in Gsn-/- HFD relative to Gsn+/+ HFD mice. Hypertension in Gsn-/- mice was associated with reduced endothelial NO synthase (eNOS) mRNA expression and reduced eNOS protein trafficking to the plasma membrane. Furthermore, acetylcholine-induced cGMP production and relaxation of aortic rings were impaired by actin filament stabilization. Gsn-/- mice on HFD displayed reduced corticosterone concentrations and reduced energy expenditure as compared to Gsn+/+ HFD mice. Moreover, Gsn-/- HFD mice displayed an overall pattern of hypoactive and anxious/depressive-like behavior. In aggregate, our results demonstrate that impaired actin filament dynamics promote the development of key behavioral and physiological aspects of the metabolic syndrome.

At least one-third of stroke survivors suffer from depression. The development of comorbid depression after stroke is clinically highly significant because post-stroke depression is associated with increased mortality, slows recovery and leads to worse functional outcomes. Here, we review the evidence that post-stroke depression can be effectively modelled in experimental rodents via a variety of approaches. This opens an exciting new window onto the neurobiology of depression and permits probing potential underlying mechanisms such as disturbed cellular plasticity, neuroendocrine dysregulation, neuroinflammation, and neurodegeneration in a novel context. From the point of view of translational stroke research, extending the scope of experimental investigations beyond the study of short-term end points and, in particular, acute lesion size, may help improve the relevance of preclinical results to human disease. Furthermore, accumulating evidence from both clinical and experimental studies offers the tantalizing prospect of 5-hydroxytryptaminergic antidepressants as the first pharmacological therapy for stroke that would be available during the subacute and chronic phases of recovery. Interdisciplinary neuropsychiatric research will be called on to dissect the mechanisms underpinning the beneficial effects of antidepressants on stroke recovery. Linked Articles This article is part of a themed section on Animal Models in Psychiatry Research. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-20

Adolescence is characterized by important molecular and anatomical changes with relevance for the maturation of brain circuitry and cognitive function. This time period is of critical importance in the emergence of several neuropsychiatric disorders accompanied by cognitive impairment, such as affective disorders and schizophrenia. The molecular mechanisms underlying these changes at neuronal level during this specific developmental stage remains however poorly understood. GluA1-containing AMPA receptors, which are located predominantly on hippocampal neurons, are the primary molecular determinants of synaptic plasticity. We investigated here the consequences of the inducible deletion of GluA1 AMPA receptors in glutamatergic neurons during late adolescence. We generated mutant mice with a tamoxifen-inducible deletion of GluA1 under the control of the CamKII promoter for temporally and spatially restricted gene manipulation. GluA1 ablation during late adolescence induced cognitive impairments, but also marked hyperlocomotion and sensorimotor gating deficits. Unlike the global genetic deletion of GluA1, inducible GluA1 ablation during late adolescence resulted in normal sociability. Deletion of GluA1 induced redistribution of GluA2 subunits, suggesting AMPA receptor trafficking deficits. Mutant animals showed increased hippocampal NMDA receptor expression and no change in striatal dopamine concentration. Our data provide new insight into the role of deficient AMPA receptors specifically during late adolescence in inducing several cognitive and behavioral alterations with possible relevance for neuropsychiatric disorders.

A growing body of evidence demonstrates that psychosocial stress is an important and often underestimated risk factor for cardiovascular disease such as myocardial infarction and stroke. In this article, we map out major biological interfaces between stress, stress-related psychiatric disorders, and stroke, placing special emphasis on the fact that stress and psychiatric disorders may be both cause and consequence of cardiovascular disease. Apart from high-risk lifestyle habits such as smoking and lack of exercise, neuroendocrine dysregulation, alterations of the hemostatic system, increased oxidative stress, and inflammatory changes have been implicated in stress-related endothelial dysfunction. Heart rate provides another useful and easily available measure that reflects the complex interplay of vascular morbidity and psychological distress. Importantly, heart rate is emerging as a valuable predictor of stroke outcome and, possibly, even a target for therapeutic intervention. Furthermore, we review recent findings highlighting the role of FK506-binding protein 51 (FKBP5), a co-chaperone of the glucocorticoid receptor, and of perturbations in telomere maintenance, as potential mediators between stress and vascular morbidity. Finally, psychiatric sequelae of cardiovascular events such as post-stroke depression or posttraumatic stress disorder are highly prevalent and may, in turn, exert far-reaching effects on recovery and outcome, quality of life, recurrent ischemic events, medication adherence, and mortality.

In the brain, retinoic acid (RA) concentrations are under tight spatio-temporal control. Here, we show that challenge of primary mouse microglia with lipopolysaccharide (LPS) results in increased release of nitric oxide (NO) and tumor necrosis factor-α (TNF-α). Co-administration of RA attenuated microglial activation. Similarly, pretreatment with RA-metabolism inhibitor liarozole potently reduced NO and TNF-α release. Conversely, activated microglia showed increased protein expression of RA-degrading cytochromes CYP26A1, CYP26B1, CYP3A4 and CYP2C. Correspondingly, RA catabolism by activated microglia was significantly increased. Our results indicate that RA reduces microglial activation, but also, conversely, that the activation state of microglia influences RA metabolism.

Evaluation of functional outcome over the course of several weeks after ischemia is a key component in improving the clinical relevance of experimental stroke studies. Using a battery of behavioral tests, we characterized functional outcome in mice over 4 weeks following 30min of proximal middle cerebral artery occlusion (MCAo). We evaluated rotarod, chimney, pole and cylinder tests to assess short term functional deficits in a transient stroke model which induces infarcts mainly in the striatum. The corner test, adhesive removal test, cylinder test, catwalk, paw preference test and novel tests of rotation were evaluated for long-term functional outcome. Rotarod detected deficits within the first week and pole test was reliable up to intermediate time points after MCAo. Corner test, adhesive removal test, catwalk and paw preference test detected deficits for up to 4 weeks, as did the novel corner rotation and bowl tests. Chimney and cylinder test did not prove useful in our model of mild stroke. In summary, we established the pole test and rotarod as useful tools to evaluate sensory motor deficits early after mild stroke, and corner test and adhesive removal test at later time-points. Alternatively, corner rotation may be a suitable test of long-term function. Test batteries may be further complemented by catwalk and paw preference test for clinically relevant deficits. There was no correlation of behavioral outcome with lesion size at 28 days, and determining whether these tests are useful for detecting a potential benefit of neuroprotective or regenerative therapies requires further testing.

Intravenous administration of iron oxide nanoparticles during the acute stage of experimental stroke can produce signal intensity changes in the ischemic region. This has been attributed, albeit controversially, to the infiltration of iron-laden blood-borne macrophages. The properties of nanoparticles that render them most suitable for phagocytosis is a matter of debate, as is the most relevant timepoint for administration. Both of these questions are examined in the present study. Imaging experiments were performed in mice with 30 minutes of middle cerebral artery occlusion (MCAO). Iron oxide nanoparticles with different charges and sizes were used, and mice received 300 μmol Fe/kg intravenously: either superparamagnetic iron oxide nanoparticles (SPIOs), ultrasmall SPIOs, or very small SPIOs. The particles were administered 7 days before MCAO, at the time of reperfusion, or 72 hours after MCAO. Interestingly, there was no observable signal change in the ischemic brains that could be attributed to iron. Furthermore, no Prussian blue-positive cells were found in the brains or blood leukocytes, despite intense staining in the livers and spleens. This implies that the nanoparticles selected for this study are not phagocytosed by blood-borne leukocytes and do not enter the ischemic mouse brain.

Post‐traumatic stress disorder (PTSD) is a psychiatric disorder of high prevalence and major socioeconomic impact. Patients suffering from PTSD typically present intrusion and avoidance symptoms and alterations in arousal, mood and cognition that last for more than 1 month. Animal models are an indispensable tool to investigate underlying pathophysiological pathways and, in particular, the complex interplay of neuroendocrine, genetic and environmental factors that may be responsible for PTSD induction. Since the 1960s, numerous stress paradigms in rodents have been developed, based largely on Seligman's seminal formulation of ‘learned helplessness’ in canines. Rodent stress models make use of physiological or psychological stressors such as foot shock, underwater trauma, social defeat, early life stress or predator‐based stress. Apart from the brief exposure to an acute stressor, chronic stress models combining a succession of different stressors for a period of several weeks have also been developed. Chronic stress models in rats and mice may elicit characteristic PTSD‐like symptoms alongside, more broadly, depressive‐like behaviours. In this review, the major existing rodent models of PTSD are reviewed in terms of validity, advantages and limitations; moreover, significant results and implications for future research—such as the role of FKBP5, a mediator of the glucocorticoid stress response and promising target for therapeutic interventions—are discussed.

The aim of this study was to explore the signaling and neuroprotective effect of transactivator of transcription (TAT) protein transduction of the apoptosis repressor with CARD (ARC) in in vitro and in vivo models of cerebral ischemia in mice. In mice, transient focal cerebral ischemia reduced endogenous ARC protein in neurons in the ischemic striatum at early reperfusion time points, and in primary neuronal cultures, RNA interference resulted in greater neuronal susceptibility to oxygen glucose deprivation (OGD). TAT.ARC protein delivery led to a dose-dependent better survival after OGD. Infarct sizes 72 h after 60 min middle cerebral artery occlusion (MCAo) were on average 30 ± 8% (mean ± SD; p = 0.005; T2-weighted MRI) smaller in TAT.ARC-treated mice (1 μg intraventricularly during MCAo) compared with controls. TAT.ARC-treated mice showed better performance in the pole test compared with TAT.β-Gal-treated controls. Importantly, post-stroke treatment (3 h after MCAo) was still effective in affording reduced lesion volume by 20 ± 7% (mean ± SD; p < 0.05) and better functional outcome compared with controls. Delayed treatment in mice subjected to 30 min MCAo led to sustained neuroprotection and functional behavior benefits for at least 28 d. Functionally, TAT.ARC treatment inhibited DAXX–ASK1–JNK signaling in the ischemic brain. ARC interacts with DAXX in a CARD-dependent manner to block DAXX trafficking and ASK1–JNK activation. Our work identifies for the first time ARC–DAXX binding to block ASK1–JNK activation as an ARC-specific endogenous mechanism that interferes with neuronal cell death and ischemic brain injury. Delayed delivery of TAT.ARC may present a promising target for stroke therapy., The Journal of Neuroscience, 36 (31), ISSN:0270-6474, ISSN:1529-2401

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive neurostimulatory technique widely used in research, diagnostics, and neuro-psychiatric therapy. Despite its growing popularity, basic molecular mechanisms underlying the clinical effects of rTMS have remained largely under-researched. Here, we present a human-derived neuronal cell culture system responsive to rTMS effects. SH-SY5Y neuroblastoma cells were differentiated by retinoic acid treatment for 10 days, resulting in a neuronal phenotype characterized by upregulation of neuronal marker proteins and generation of an action potential in response to depolarizing current step injection. Repetitive magnetic stimulation of these cells resulted in increased intracellular cAMP levels and increased phosphorylation of transcription factor CREB. Pretreatment with ketamine (1 μM) potentiated, while pretreatment with lithium (2 mM) attenuated this cellular response to repetitive magnetic stimulation. In conclusion, we introduce here a novel in vitro system responding to rTMS at the level of second messenger signaling. The use of human-derived cells with neuron-like properties will prove useful for further studies on the cellular effects of rTMS.

Background and Purpose— Chronic stress is associated with increased stroke risk. However, the underlying pathophysiological mechanisms are poorly understood. We examined the effects of chronic stress on endothelial function and ischemic brain injury in a mouse model. Methods— 129/SV mice were treated with glucocorticoid receptor antagonist mifepristone (25 mg kg −1 /d) or vehicle and exposed to 28 days of chronic stress consisting of exposure to rat, restraint stress, and tail suspension. Heart rate and blood pressure were continuously recorded by telemetry. Endothelial nitric oxide synthase mRNA and protein expression as well as superoxide production and lipid hydroperoxides were quantified. Endothelium-dependent vasorelaxation was measured in aortic rings. Ischemic lesion volume was quantified after 30 minutes filamentous middle cerebral artery occlusion and 72 hours reperfusion. Results— Chronic stress caused a significant increase in heart rate, impaired endothelium-dependent vasorelaxation, increased superoxide production, and reduced aortic and brain endothelial nitric oxide synthase levels. Animals exposed to chronic stress showed major increases in ischemic lesion size. These deleterious effects of stress were completely reversed by treatment with mifepristone. Conclusions— Chronic stress increases stroke vulnerability likely through endothelial dysfunction, which can be reversed by a glucocorticoid receptor antagonist.

Death receptor (DR) signaling has a major impact on the outcome of numerous neurological diseases, including ischemic stroke. DRs mediate not only cell death signals, but also proinflammatory responses and cell proliferation. Identification of regulatory proteins that control the switch between apoptotic and alternative DR signaling opens new therapeutic opportunities. Fas apoptotic inhibitory molecule 2 (Faim2) is an evolutionary conserved, neuron-specific inhibitor of Fas/CD95-mediated apoptosis. To investigate its role during development and in disease models, we generated Faim2-deficient mice. The ubiquitous null mutation displayed a viable and fertile phenotype without overt deficiencies. However, lack of Faim2 caused an increase in susceptibility to combined oxygen–glucose deprivation in primary neuronsin vitroas well as in caspase-associated cell death, stroke volume, and neurological impairment after cerebral ischemiain vivo. These processes were rescued by lentiviral Faim2 gene transfer. In summary, we provide evidence that Faim2 is a novel neuroprotective molecule in the context of cerebral ischemia.

The stem-cell marker CD93 (AA4.1/C1qRp) has been described as a potential complement C1q-receptor. Its exact molecular function, however, remains unknown. By using global expression profiling we showed that CD93-mRNA is highly induced after transient focal cerebral ischemia. CD93 protein is upregulated in endothelial cells, but also in selected macrophages and microglia. To elucidate the potential functional role of CD93 in postischemic brain damage, we used mice with a targeted deletion of the CD93 gene. After 30 min of occlusion of the middle cerebral artery and 3 d of reperfusion these mice displayed increased leukocyte infiltration into the brain, increased edema, and significantly larger infarct volumes (60.8 ± 52.2 versus 23.9 ± 16.6 mm3) when compared with wild-type (WT) mice. When the MCA was occluded for 60 min, after 2 d of reperfusion the CD93 knockout mice still showed more leukocytes in the brain, but the infarct volumes were not different from those seen in WT animals. To further explore CD93-dependent signaling pathways, we determined global transcription profiles and compared CD93-deficient and WT mice at various time points after induction of focal cerebral ischemia. We found a highly significant upregulation of the chemokine CCL21/Exodus-2 in untreated and treated CD93-deficient mice at all time points. Induction of CCL21 mRNA and protein was confirmed by PCR and immunohistochemistry. CCL21, which was formerly shown to be released by damaged neurons and to activate microglia, contributes to neurodegeneration. Thus, we speculate that CD93-neuroprotection is mediated via suppression of the neuroinflammatory response through downregulation of CCL21.

Rearrangement of the actin cytoskeleton is essential for dynamic cellular processes. Decreased actin turnover and rigidity of cytoskeletal structures have been associated with aging and cell death. Gelsolin is a Ca2+-activated actin-severing protein that is widely expressed throughout the adult mammalian brain. Here, we used gelsolin-deficient (Gsn−/−) mice as a model system for actin filament stabilization. InGsn−/−mice, emigration of newly generated cells from the subventricular zone into the olfactory bulb was slowed.In vitro, gelsolin deficiency did not affect proliferation or neuronal differentiation of adult neural progenitors cells (NPCs) but resulted in retarded migration. Surprisingly, hippocampal neurogenesis was robustly induced by gelsolin deficiency. The ability of NPCs to intrinsically sense excitatory activity and thereby implement coupling between network activity and neurogenesis has recently been established. Depolarization-induced [Ca2+]iincreases and exocytotic neurotransmitter release were enhanced inGsn−/−synaptosomes. Importantly, treatment ofGsn−/−synaptosomes with mycotoxin cytochalasin D, which, like gelsolin, produces actin disassembly, decreased enhanced Ca2+influx and subsequent exocytotic norepinephrine release to wild-type levels. Similarly, depolarization-induced glutamate release fromGsn−/−brain slices was increased. Furthermore, increased hippocampal neurogenesis inGsn−/−mice was associated with a special microenvironment characterized by enhanced density of perfused vessels, increased regional cerebral blood flow, and increased endothelial nitric oxide synthase (NOS-III) expression in hippocampus. Together, reduced filamentous actin turnover in presynaptic terminals causes increased Ca2+influx and, subsequently, elevated exocytotic neurotransmitter release acting on neural progenitors. Increased neurogenesis inGsn−/−hippocampus is associated with a special vascular niche for neurogenesis.

Background and Purpose— Although in vitro studies suggest that non-neurogenic regions of the adult central nervous system potentially contain multipotent parenchymal progenitors, neurons are clearly not replaced in most brain regions after injury. Here, in a well-established model of mild transient brain ischemia, we explored Olig2 antagonism and Pax6 overexpression as potential avenues to redirect endogenous progenitors proliferating in situ toward a neuronal fate. Methods— Retroviral vectors containing either Pax6 or a strong activator form of the repressor Olig2 (Olig2VP16), ie, a functionally dominant negative form of Olig2, were stereotaxically injected into the lateral striatum at 48 hours after 30 minutes middle cerebral artery occlusion (MCAo)/reperfusion. Results— Retroviral modulation of fate determinants resulted in a significant number of infected cells differentiating into Doublecortin (DCX)-expressing immature neurons that were not observed after injection of a control virus. Whole-cell patch-clamp recordings in acute brain slices showed that the percentage of virus-infected cells with Na + currents was increased by inhibition of the repressor function of Olig2 and by overexpression of Pax6. Furthermore, on retroviral transduction of fate determinants, we detected newly generated cells within the ischemic lesion that were capable of generating single action potentials and that received synaptic input. Conclusions— Taken together, these data show that resident glia in the striatum can be reprogrammed toward functional neuronal differentiation following brain injury.

It is common knowledge that ischemic stroke has major social and economic consequences. However, until now, translation of experimental studies into clinical reality has been sorely lacking. So far, most studies have focused on acute stroke outcome and early treatment paradigms affording neuroprotection. It is increasingly recognized that it will be necessary to harness the capacity of the brain for neuroregeneration to improve longer-term outcome. Endothelial nitric oxide synthase (eNOS) is emerging as a key target in molecular stroke research. eNOS ameliorates acute ischemic injury and promotes recovery following cerebral ischemia. This review summarizes the effects of eNOS on the regulation of cerebral blood flow, hemostasis, inflammation, angiogenesis as well as neurogenesis. The possible impact on stroke prevention as well as on strategies aimed at improving long-term stroke outcome are discussed.

Background and Purpose— Statins exert rapid cholesterol-independent vasoprotective effects. Here, we tested whether postevent treatment with intravenously (i.v.) administered rosuvastatin improves acute stroke outcome in mice. Methods— 129/SV wild-type mice were subjected to 1-hour filamentous middle cerebral artery occlusion (MCAo), followed by reperfusion, and were postevent treated with i.v. or intraperitoneal (i.p.) rosuvastatin given up to 6 hours after MCAo (dose range 0.02 to 20 mg kg −1 body weight). Results— Rosuvastatin, when administered i.v., significantly reduced lesion size when given up to 4 hours after MCAo and in doses as low as 0.2 mg kg −1 . In contrast, i.p. administration provided protection only when given directly on reperfusion at a dose of 20 mg kg −1 but not at lower doses or later time points. Lesion protection was evident as late as 5 days after brain ischemia and was associated with functional improvements in the pole-test and wire-hanging test (2.0 mg kg −1 dose). Neuroprotection with i.v. rosuvastatin was achieved with peak plasma concentrations −1 (ie, with 0.2 mg kg −1 ) and was associated with increased levels of phosphorylated Akt kinase and endothelial nitric oxide synthase in the vasculature. Conclusions— Rosuvastatin, given intravenously at pharmacologically relevant concentrations, protects from focal brain ischemia up to 4 hours after an event. In our opinion, the development of an intravenous statin formulation is warranted for acute stroke trials with statins in humans.

Microglia is activated by brain injury. They migrate in response to ATP and although adenosine alone has no effect on wild type microglial migration, we show that inhibition of adenosine receptors impedes ATP triggered migration. CD39 is the dominant cellular ectonucleotidase that degrades nucleotides to nucleosides, including adenosine. Importantly, ATP fails to stimulate P2 receptor mediated migration in cd39(-/-) microglia. However, the effects of ATP on migration in cd39(-/-) microglia can be restored by co-stimulation with adenosine or by addition of a soluble ectonucleotidase. We also tested the impact of cd39-deletion in a model of ischemia, in an entorhinal cortex lesion and in the facial nucleus after facial nerve lesion. The accumulation of microglia at the pathological sites was markedly decreased in cd39(-/-) animals. We conclude that the co-stimulation of purinergic and adenosine receptors is a requirement for microglial migration and that the expression of cd39 controls the ATP/adenosine balance.

Background: VE-cadherin is the chief constituent of endothelial adherens junctions. However, the role of VE-cadherin in the pathogenesis of cerebrovascular diseases including brain ischemia has not yet been investigated.Methods: VE-cadherin heterozygous (VEC+/-) mice and wildtype controls were subjected to transient brain ischemia by 30 min filamentous middle cerebral artery occlusion (MCAo)/reperfusion.Results: Acute lesion sizes as assessed by MR-imaging on day 3 did not differ between genotypes. Unexpectedly, however, partial loss of VE-cadherin resulted in long-term stroke protection measured histologically on day 28. Equally surprisingly, VEC+/- mice displayed no differences in post-stroke angiogenesis compared to littermate controls, but showed increased absolute regional cerebral blood flow in ischemic striatum at four weeks. The early induction of VE-cadherin mRNA transcription after stroke was reduced in VEC+/- mice. By contrast, N-cadherin and β-catenin mRNA expression showed a delayed, but sustained, upregulation up to 28 days after MCAo, which was increased in VEC+/- mice. Furthermore, partial loss of VE-cadherin resulted in a pattern of elevated ischemia-triggered mRNA transcription of pericyte-related molecules α-smooth muscle actin (α-SMA), aminopeptidase N (CD13), and platelet-derived growth factor receptor β (PDGFR-β).Conclusions: Partial loss of VE-cadherin results in long term stroke protection. On the cellular and molecular level, this effect appears to be mediated by improved endothelial/pericyte interactions and the resultant increase in cerebral blood flow. Our study reinforces accumulating evidence that long-term stroke outcome depends critically on vascular mechanisms.

Serum insulin-like growth factor I (IGF-I), which is mostly produced by the liver, has recently been shown to have the unexpected ability to modulate normal brain function as well as brain response to injury. Moreover, serum IGF-I levels are modified in many brain diseases, including stroke. However, whether these modifications are related to the disease process remains uncertain. We now examined a potential relationship between serum IGF-I and ischemic brain injury after middle cerebral artery occlusion (MCAo) and reperfusion in mice with either high or low serum IGF-I levels prior to insult. Surprisingly, we found that chronic high serum IGF-I correlates with increased brain infarct size following MCAo, while low levels correlate with reduced lesion size. Immunocytochemistry and immunoblot analyses revealed that levels of phosphorylated (i.e., activated) MAPK, known to be associated with the severity of ischemic brain injury, were increased in IGF-I treated mice. No overall effect of IGF-I treatment on IGF family mRNA expression in the brain was observed. Altogether, these results indicate that serum IGF-I levels negatively correlate with stroke outcome. Therefore, lowering serum IGF-I levels in aging mammals, including humans, may be beneficial against the increased risk of stroke associated to old age. © 2007 Elsevier B.V. All rights reserved.

Actin is the most common intracellular protein in eukaryotic cells, with estimates for nonmuscle cells generally ranging from 1–5% of cellular protein (1). Until fairly recently, actin was widely regarded as a ubiquitous and rather humdrum molecule. However, accumulating research is increasingly pointing to a crucial role of impaired actin dynamics in central nervous system pathologies and, in particular, those related to aging and neurodegeneration. The study by Ojelade et al. (2) adds an exciting new angle to this emerging field. In a series of elegant experiments using mutant flies, the authors show that the gene-encoding Ras suppressor 1 (Rsu1) acts upstream of Rac1 and is required in the adult nervous system for regular ethanol sensitivity. Moreover, using macrophage-like Drosophila Schneider S2 cells, the authors show that knockdown of Rsu1 as well as constitutive overexpression of Rac1 causes robust decreases in the ratio of globular:filamentous actin. It may be worthwhile to ascertain whether a similar shift toward increased rigidity of the actin network also occurs in the nervous system of flies with a disruption of Rsu1. Of note, compensatory changes in other actin-binding proteins have been described in the brains of mice lacking actin-severing protein gelsolin (3). Interestingly, the mechanisms through which increased stability of the actin cytoskeleton promotes neurodegeneration and reduced sensitivity to ethanol-induced sedation likely show considerable overlap. We have previously demonstrated that an impairment in actin depolymerization interferes with the rundown of activated NMDA receptors and voltage-dependent Ca2+ channels, and thereby exacerbates neuronal injury following brain ischemia. Furthermore, actin filament stabilization in gelsolin-deficient synaptosomes increased depolarization-induced intracellular Ca2+ levels and enhanced exocytotic neurotransmitter release, which could be reversed by actin disruptor cytochalasin D (3, 4). Conversely, ethanol has been demonstrated to promote actin depolymerization in cerebellar granule neurons (5). In addition, neurons lacking Eps8, another important regulator of actin dynamics, show increased NMDA currents and enhanced NMDA receptor activity after ethanol exposure and Eps8-KO mice demonstrate increased ethanol consumption (5). Finally, Ojelade et al. report that, in humans, polymorphisms in Rsu1 are associated with brain activation in the ventral striatum during reward anticipation and with alcohol consumption (2). Taken together, these findings indicate that the dynamic state of the actin meshwork plays a key role in mediating ethanol preference. Accordingly, biological parameters related to actin dynamics deserve to be explored as potential markers of increased risk of alcohol use. The actin cytoskeleton may even prove useful as a novel target for pharmacological interventions in alcohol use disorders.

The interplay between BDNF signaling and the serotonergic system remains incompletely understood. Using a highly sensitive enzyme-linked immunosorbent assay, we studied BDNF concentrations in hippocampus and cortex of two mouse models of altered serotonin signaling: tryptophan hydroxylase (Tph)2-deficient (Tph2 (-/-)) mice lacking brain serotonin and serotonin transporter (SERT)-deficient (SERT(-/-)) mice lacking serotonin re-uptake. Surprisingly, hippocampal BDNF was significantly elevated in Tph2 (-/-) mice, whereas no significant changes were observed in SERT(-/-) mice. Furthermore, BDNF levels were increased in the prefrontal cortex of Tph2 (-/-) but not of SERT(-/-) mice. Our results emphasize the interaction between serotonin signaling and BDNF. Complete lack of brain serotonin induces BDNF expression.

Physical activity upregulates endothelial nitric oxide synthase (eNOS), improves endothelium function, and protects from vascular disease. Here, we tested whether voluntary running would enhance neovascularization and long-term recovery following mild brain ischemia. Wild-type mice were exposed to 30 minutes of middle-cerebral artery occlusion (MCAo) and reperfusion. Continuous voluntary running on wheels conferred long-term upregulation of eNOS in the vasculature and of endothelial progenitor cells (EPCs) in the spleen and bone marrow (BM). This was associated with higher numbers of circulating EPCs in the blood and enhanced neovascularization. Moreover, engraftment of TIE2/LacZ-positive BM-derived cells was increased in the ischemic brain. Four weeks after the insult, trained animals showed higher numbers of newly generated cells in vascular sites, increased density of perfused microvessels and sustained augmentation of cerebral blood flow within the ischemic striatum. Moreover, running conferred tissue sparing and improved functional outcome at 4 weeks. The protective effects of running on angiogenesis and outcome were completely abolished when animals were treated with a NOS inhibitor or the antiangiogenic compound endostatin after brain ischemia, and in animals lacking eNOS expression. Voluntary physical activity improves long-term stroke outcome by eNOS-dependent mechanisms related to improved angiogenesis and cerebral blood flow.

The evolution of cellular damage over time and the selective vulnerability of different neuronal subtypes was characterized in the striatum following 30‐minute middle cerebral artery occlusion and reperfusion in the mouse. Using autoradiography we found an increase in the density of [(3)H]PK11195 binding sites—likely reflecting microglial activation—in the lesion border at 3 days and in the whole striatum from 10 days to 6 weeks. This was accompanied by a distinct loss of [(3)H]flumazenil and [(3)H]CGP39653 binding sites from 10 days up to 6 weeks reflecting neuronal loss. Brain ischemia resulted in a substantial loss of medium spiny projection neurons as seen at three days by Nissl staining, TUNEL and immunocytochemistry using antibodies against microtubule‐associated protein (MAP2), NeuN, (μ‐opioid receptors, substance P, Lenkephalin, neurokinin B, choline acetyltransferase, parvalbumin, calretinin and somatostatin. Both patch and matrix compartments were involved in ischemic damage. In contrast, the numbers of cholinergic, GABAergic, and somatostatin‐containing interneurons in the ischemic striatum were not different from those in the contralateral hemisphere at 3 and 14 days. A low density of glutamate receptors, the ability to sequester calcium by calcium‐binding proteins and other hitherto unidentified factors may explain this relative resistance of interneurons to acute ischemia.

Background and Purpose— Folate deficiency and resultant hyperhomocysteinemia impair vascular function and increase stroke risk. We tested the hypothesis that folate deficiency and high homocysteine levels promote DNA damage and increase brain injury after cerebral ischemia/reperfusion. Methods— 129/Sv mice, uracil-DNA glycosylase–deficient ( Ung −/− ) mice, and Ung +/+ littermate mice were exposed to a folate-deficient diet for 3 months and then subjected to 30-minute middle cerebral artery (MCA) occlusion and reperfusion. Plasma homocysteine levels and physiological parameters were measured in selected animals. Outcome measures were neurological sensorimotor deficits, infarct size measured by computer-assisted volumetry, and oxidative DNA damage measured by a colorimetric assay. Results— Exposure to a folate-deficient diet for 3 months conferred ≈6- to 10-fold higher plasma homocysteine levels than those associated with a normal diet. Cerebral lesion volumes and neurological deficits after MCA occlusion and 72-hour reperfusion were significantly 2.1-fold increased in folate-deficient 129/SV wild-type mice compared with those associated with a normal diet, which could not be explained by obvious differences in physiological parameters. Abasic sites, hallmarks of oxidative DNA damage, were significantly increased in DNA from the ischemic brain of folate-deficient animals at early time points after MCA occlusion. Folate deficiency further increased brain lesion size in animals lacking uracil-DNA glycosylase compared with wild-type littermate mice. Conclusions— Folate deficiency and resultant hyperhomocysteinemia are not only associated with increased stroke risk but increase oxidative DNA damage and ischemic lesion size after MCA occlusion/reperfusion.

Regular physical activity is associated with a decrease of cerebrovascular and cardiovascular events, which may relate to enhanced endothelium-dependent vasodilation. Here, we provide evidence that physical activity protects against ischemic stroke via mechanisms related to the upregulation of endothelial nitric oxide synthase (eNOS) in the vasculature. Voluntary training on running wheels or exercise on a treadmill apparatus for 3 weeks, respectively, reduced cerebral infarct size and functional deficits, improved endothelium-dependent vasorelaxation, and augmented cerebral blood flow in wild-type mice. The neuroprotective effects of physical training were completely absent in eNOS-deficient mice, indicating that the enhanced eNOS activity by physical training was the predominant mechanism by which this modality protects against cerebral injury. Our results suggest that physical activity not only decreases stroke risk, but also provides a prophylactic treatment strategy for increasing blood flow and reducing brain injury during cerebral ischemia.

Background and Purpose— Statins (3-hydroxy-3-methylglutaryl–coenzyme A [HMG-CoA] reductase inhibitors) reduce stroke damage independent of lipid lowering by upregulation of endothelial nitric oxide synthase (eNOS). Acute withdrawal of statin treatment may suppress endothelial NO production and impair vascular function. Methods— To test this hypothesis, we treated 129/SV mice with atorvastatin (10 mg/kg) for 14 days and then withdrew treatment. Results— Treatment with atorvastatin conferred stroke protection by 40% after filamentous occlusion of the middle cerebral artery followed by reperfusion. Withdrawal of statin treatment, however, resulted in the loss of stroke protection after 2 and 4 days. In mouse aortas and brain vasculature, statins upregulated eNOS message 2.3- and 1.7-fold, respectively, as measured by reverse transcription–polymerase chain reaction. Withdrawal of statins resulted in 5- and 2.7-fold downregulation of eNOS in aorta and brain, respectively, after 2 days. Statin treatment decreased RhoA GTPase membrane expression to 48%, while withdrawal of statins resulted in 4-fold increase of RhoA in the membrane. Moreover, platelet factor 4 and β-thromboglobulin in plasma were significantly downregulated by statin treatment, but withdrawal of statins resulted in a 2.9- and 3.1-fold upregulation after 2 days, respectively. Thrombus formation induced by ligature of the inferior vena cava was significantly reduced by statin treatment. When statin treatment was withdrawn, however, protection was lost between 2 and 4 days. Conclusions— Acute termination of statin treatment results in a rapid loss of protection in mouse models of cerebral ischemia and thrombus formation independent of lipid lowering. In patients with acute or impending stroke, withdrawal of statins may impair outcome.