Stroke Outcome In Mice Research Articles

Protective actions of des-acylated ghrelin on brain injury and blood-brain barrier disruption after stroke in mice.

The major ghrelin forms, acylated ghrelin and des-acylated ghrelin, are novel gastrointestinal hormones. Moreover, emerging evidence indicates that these peptides may have other functions including neuro- and vaso-protection. Here, we investigated whether post-stroke treatment with acylated ghrelin or des-acylated ghrelin could improve functional and histological endpoints of stroke outcome in mice after transient middle cerebral artery occlusion (tMCAo). We found that des-acylated ghrelin (1mg/kg) improved neurological and functional performance, reduced infarct and swelling, and decreased apoptosis. In addition, it reduced blood-brain barrier (BBB) disruption invivo and attenuated the hyper-permeability of mouse cerebral microvascular endothelial cells after oxygen glucose deprivation and reoxygenation (OGD + RO). By contrast, acylated ghrelin (1mg/kg or 5mg/kg) had no significant effect on these endpoints of stroke outcome. Next we found that des-acylated ghrelin's vasoprotective actions were associated with increased expression of tight junction proteins (occludin and claudin-5), and decreased cell death. Moreover, it attenuated superoxide production, Nox activity and expression of 3-nitrotyrosine. Collectively, these results demonstrate that post-stroke treatment with des-acylated ghrelin, but not acylated ghrelin, protects against ischaemia/reperfusion-induced brain injury and swelling, and BBB disruption, by reducing oxidative and/or nitrosative damage.

Tert-butylhydroquinone compromises survival in murine experimental stroke

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.

Prolonged Fasting Improves Endothelial Progenitor Cell-Mediated Ischemic Angiogenesis in Mice

Background/Aims: Prolonged fasting (PF) was shown to be of great potency to promote optimal health and reduce the risk of many chronic diseases. This study sought to determine the effect of PF on the endothelial progenitor cell (EPC)-mediated angiogenesis in the ischemic brain and cerebral ischemic injury in mice. Methods: Mice were subjected to PF or periodic PF after cerebral ischemia, and histological analysis and behavioral tests were performed. Mouse EPCs were isolated and examined, and the effects of EPC transplantation on cerebral ischemic injury were investigated in mice. Results: It was found that PF significantly increased the EPC functions and angiogenesis in the ischemic brain, and attenuated the cerebral ischemic injury in mice that was previously subjected to cerebral ischemia. Periodic PF might reduce cortical atrophy and improve long-term neurobehavioral outcomes after cerebral ischemia in mice. The eNOS and MnSOD expression and intracellular NO level were increased, and TSP-2 expression and intracellular O₂^- level were reduced in EPCs from PF-treated mice compared to control. In addition, transplanted EPCs might home into ischemic brain, and the EPCs from PF-treated mice had a stronger ability to promote angiogenesis in ischemic brain and reduce cerebral ischemic injury compared to the EPCs from control mice. The EPC-conditioned media from PF-treated mice exerted a stronger effect on cerebral ischemic injury reduction compared to that from control mice. Conclusion: Prolonged fasting promoted EPC-mediated ischemic angiogenesis and improved long-term stroke outcomes in mice. It is implied that prolonged fasting might potentially be an option to treat ischemic vascular diseases.

Post-ischaemic silencing of p66Shc reduces ischaemia/reperfusion brain injury and its expression correlates to clinical outcome in stroke.

Constitutive genetic deletion of the adaptor protein p66(Shc) was shown to protect from ischaemia/reperfusion injury. Here, we aimed at understanding the molecular mechanisms underlying this effect in stroke and studied p66(Shc) gene regulation in human ischaemic stroke. Ischaemia/reperfusion brain injury was induced by performing a transient middle cerebral artery occlusion surgery on wild-type mice. After the ischaemic episode and upon reperfusion, small interfering RNA targeting p66(Shc) was injected intravenously. We observed that post-ischaemic p66(Shc) knockdown preserved blood-brain barrier integrity that resulted in improved stroke outcome, as identified by smaller lesion volumes, decreased neurological deficits, and increased survival. Experiments on primary human brain microvascular endothelial cells demonstrated that silencing of the adaptor protein p66(Shc) preserves claudin-5 protein levels during hypoxia/reoxygenation by reducing nicotinamide adenine dinucleotide phosphate oxidase activity and reactive oxygen species production. Further, we found that in peripheral blood monocytes of acute ischaemic stroke patients p66(Shc) gene expression is transiently increased and that this increase correlates with short-term neurological outcome. Post-ischaemic silencing of p66(Shc) upon reperfusion improves stroke outcome in mice while the expression of p66(Shc) gene correlates with short-term outcome in patients with ischaemic stroke.

Abstract T P223: Post-stroke Body Weight Loss Predicts Acute Stroke Outcome In Mice

Background and Purpose: Longitudinal imaging studies in stroke demonstrated that infarct development is a dynamic process and it matures over several days, even weeks depending on the severity of stroke. However, due to high cost and multiple exposure of anesthesia associated with repeated imaging, infarct volume has been typically measured at a fixed time point in preclinical studies, which prevent further assessment of behaviors during stroke recovery. In identifying a non-invasive physiological parameter that predicts the extent of stroke-induced brain injury, the study investigated whether acute body weight loss predicts the extent of brain injury and swelling. Methods: C57 male mice (10-12 wk old, 25-30 gr) were subjected to the proximal middle cerebral artery (MCA) occlusion for 30 min. Body weight was recorded daily. Infarct volume corrected with swelling (IVInd) and percent hemispheric swelling (%SW) were determined at 1, 3 and 7d after MCAO. In a retrospective study, we analyzed correlations between 3d %body weight reduction (%BWred) and stroke outcomes (IVInd & %SW) in C57 male animals that were subjected to MCAO by 3 different individuals over 8 years (n=91-96). Results: Stroke induced acute BW loss (%BWred; 1d, 88.9±3.3; 3d, 84.5±8.6; 5d, 86.8±10.6; 7d, 88.9±8.9). Correlation analyses in the post-ischemic time points showed IVind and %SW were significantly correlated with only at 3d BW reduction, but not 1d and 7d (Fig.1). A retrospective analysis in C57 mice also showed a significant correlation between 3d %BWred and IVInd & %SW (Fig.2). Conclusions: The study showed that acute post-stroke BWred is a simple and suitable index to predict the extent of stroke injury in C57 mice. Whether the prediction can be generalized across different strains and in comorbid conditions warrant further investigation.

Migraine prophylaxis, ischemic depolarizations, and stroke outcomes in mice.

Migraine with aura is an established stroke risk factor, and excitatory mechanisms such as spreading depression (SD) are implicated in the pathogenesis of both migraine and stroke. Spontaneous SD waves originate within the peri-infarct tissue and exacerbate the metabolic mismatch during focal cerebral ischemia. Genetically enhanced SD susceptibility facilitates anoxic depolarizations and peri-infarct SDs and accelerates infarct growth, suggesting that susceptibility to SD is a critical determinant of vulnerability to ischemic injury. Because chronic treatment with migraine prophylactic drugs suppresses SD susceptibility, we tested whether migraine prophylaxis can also suppress ischemic depolarizations and improve stroke outcome. We measured the cortical susceptibility to SD and ischemic depolarizations, and determined tissue and neurological outcomes after middle cerebral artery occlusion in wild-type and familial hemiplegic migraine type 1 knock-in mice treated with vehicle, topiramate or lamotrigine daily for 7 weeks or as a single dose shortly before testing. Chronic treatment with topiramate or lamotrigine reduced the susceptibility to KCl-induced or electric stimulation-induced SDs as well as ischemic depolarizations in both wild-type and familial hemiplegic migraine type 1 mutant mice. Consequently, both tissue and neurological outcomes were improved. Notably, treatment with a single dose of either drug was ineffective. These data underscore the importance of hyperexcitability as a mechanism for increased stroke risk in migraineurs, and suggest that migraine prophylaxis may not only prevent migraine attacks but also protect migraineurs against ischemic injury.

Microvascular thrombosis, fibrinolysis, ischemic injury, and death after cerebral thromboembolism are affected by levels of circulating α2-antiplasmin.

Ischemic stroke is primarily attributable to thrombotic vascular occlusion. Elevated α2-antiplasmin (a2AP) levels correlate with increased stroke risk, but whether a2AP contributes to the pathogenesis of stroke is unknown. We examined how a2AP affects thrombosis, ischemic brain injury, and survival after experimental cerebral thromboembolism. We evaluated the effects of a2AP on stroke outcomes in mice with increased, normal, or no circulating a2AP, as well as in mice given an a2AP-inactivating antibody. Higher a2AP levels were correlated with greater ischemic brain injury (rs=0.88, P<0.001), brain swelling (rs=0.82, P<0.001), and reduced middle cerebral artery thrombus dissolution (rs=-0.93, P<0.001). In contrast, a2AP deficiency enhanced thrombus dissolution, increased cerebral blood flow, reduced brain infarction, and decreased brain swelling. By comparison to tissue plasminogen activator (TPA), a2AP inactivation hours after thromboembolism still reduced brain infarction (P<0.001) and hemorrhage (P<0.05). Microvascular thrombosis, a process that enhances brain ischemia, was markedly reduced in a2AP-deficient or a2AP-inactivated mice compared with TPA-treated mice or mice with increased a2AP levels (all P<0.001). Matrix metalloproteinase-9 expression, which contributes to acute brain injury, was profoundly decreased in a2AP-deficient or a2AP-inactivated mice versus TPA-treated mice or mice with increased a2AP levels (all P<0.001). a2AP inactivation markedly reduced stroke mortality versus TPA (P<0.0001). a2AP has profound, dose-related effects on ischemic brain injury, swelling, hemorrhage, and survival after cerebral thromboembolism. By comparison to TPA, the protective effects of a2AP deficiency or inactivation seem to be mediated through reductions in microvascular thrombosis and matrix metalloproteinase-9 expression.

Abstract T P206: Mkp-1 Inhibition Increases Stroke Injury via JNK Pathway Activation

Background: MKP-1, a negative modulator of several MAP kinases including JNK and p38, is a critical regulator of inflammation and apoptosis in the CNS. Inhibition of MKP-1 worsens stroke outcome in mice, suggesting that MKP-1 is an endogenous protective molecule, although the underlying mechanisms remain unclear. Here we investigated the downstream mediators of MKP-1’s effects in stroke. Methods: Stroke was induced by 60 min transient middle cerebral artery occlusion (tMCAO) in male mice. Levels of p-JNK and p-p38 in MKP-1 KO mice and WT littermates 6 hours after stroke were assessed by Western blots. In a second cohort, the selective JNK inhibitor (SP600125) was administered intraperitoneally to both MKP-1 KO and WT mice 30 min before stroke then infarcts were analyzed at 72 hours post ischemia. In a third cohort, WT mice were intraperitoneally administrated the inhibitor or vehicle 30 min before stroke and sacrificed at 6 hours for protein analysis. Results: MKP-1 deletion produced significantly higher levels of p-JNK 6 hours after stroke (0.71±0.12 WT vs. 1.32± 0.14 KO, p&lt;0.05, n= 3 p/g); this was not seen for p-p38 (1.06± 0.19 WT vs. 1.30± 0.34 KO, n=3 WT, 2 KO). Furthermore, MKP-1 deletion significantly increased expression of JNK pathway related molecules such as p-c-jun/c-jun (0.57± 0.17 WT vs. 3.65±1.19 KO, p&lt;0.05, n=3 p/g) and cleaved-caspase-3 (0.62 ± 0.09 WT vs. 1.81 ± 0.32 KO, p&lt;0.05, n=3 p/g), with no changes in cleaved-caspase-8 (1.11 ± 0.09 WT vs. 1.02 ± 0.04 KO, p&gt;0.05, n=3 p/g). There was no additive deleterious effects of pharmacological JNK inhibition in MKP-1 KO mice as measured by infarcts at 72 hours (cortex: 25.84±2.44%WT vs. 22.32±1.91%KO, striatum: 33.10±3.61% WT vs. 26.52±1.92% KO, total: 27.85±2.95% WT vs. 26.75±1.61% KO, p&gt;0.05, n=6 p/g). In WT mice, JNK inhibition significantly decreased p-JNK (0.99 ± 0.14 vehicle vs. 0.44 ± 0.13 drug, p&lt;0.05, n=3 p/g) 6 hours after stroke but had no effects on MKP-1 (2.36 ± 0.34 vehicle vs. 3.43 ± 0.45 drug, p&gt;0.05, n=3 p/g). Conclusions: JNK is an important downstream mediator of MKP-1’s effects in stroke. In the ischemic brain, activating MKP-1 may restrain JNK activity, thereby producing neuroprotective effects.

Angiopoietin-like 4 and ischaemic stroke: a promising start

This editorial refers to ‘Protective effects of angiopoietin-like 4 on cerebrovascular and functional damages in ischaemic stroke,[†][1]’ by C. Bouleti et al. , on page 3657 Stroke is a global cause of morbidity and mortality, ranking fourth among all causes of death.1 Although considerable progress has been made in developing effective tools for acute stroke treatment, at present the only drug approved is recombinant tissue plasminogen activator (rt-PA); thus, new strategies for its effective prevention and treatment are essential. Following ischaemia/reperfusion, the blood–brain barrier (BBB) becomes more permeable and, in doing so, it promotes an increased infiltration of pro-inflammatory cells, resulting in the so-called ‘reperfusion injury’.2 Given its key role in mediating ischaemia/reperfusion-related neuronal damage, the BBB is a central target for the development of novel therapeutical strategies. Bouleti and colleagues have provided an elegant study reporting a new target improving stroke outcome in mice.3 Their study shows that angiopoietin-like 4 (ANGPTL4) modulates endothelial permeability following ischaemia/reperfusion and thus represents a potential new therapeutical target for the treatment of stroke. ANGPTL4, first discovered in 2000, was originally classified as an adipokine playing roles in lipid metabolism.4 Over the last decade, ANGPTL4 has been recognized to play additional roles in tumorigenesis, angiogenesis, and redox regulation.4 Bouleti and colleagues investigated the role of ANGPTL4 in stroke by using a transient focal cerebral ischaemia murine model, where the middle cerebral artery was occluded for 1 h, followed by 24 h of reperfusion. Indeed, they could show that recombinant human ANGPTL4 (rhANGPTL4) treatment prior to the ischaemic episode greatly reduced the stroke size and consequent neurological deficit. To provide additional supporting evidence for the protective effects of ANGPTL4, Bouleti et al . analysed stroke outcome in ANGPTL4 knockout mice undergoing ischaemic stroke. Moreover, to translate their findings into a more … [1]: #fn-2

Effect of human amnion epithelial cells on stroke outcome in mice

We tested whether stem cells derived from the placenta (human amnion epithelial cells; hAECs) may represent a novel effective therapy for stroke. Male C57Bl6 mice were subjected to 0.5 h middle cerebral artery occlusion‐reperfusion, and injected with 1×106 fluorescently‐labelled hAECs or saline (vehicle) i.v. at either 1 h (acute treatment) or at 72 h (delayed treatment) post‐stroke and brains were removed after 3 or 14 d, respectively. Neurological and motor function as well as infarct damage were significantly improved in mice treated with hAECs vs. vehicle 1 h post‐stroke. Furthermore, immunofluorescence of the key marker of apoptosis, cleaved caspase‐3, revealed that less apoptosis occurred in the infarct core of mice treated with hAECs. Moreover, mice that received delayed post‐stroke hAEC treatment had improved survival to 14 d compared with vehicle‐treated mice, although functional outcomes did not differ between surviving animals from each treatment group. Thus, early post‐stroke i.v. delivery of hAECs reduces brain injury and functional deficit, and delayed post‐stroke hAEC treatment improves 14 d survival. hAECs could be a viable therapy for promoting recovery following stroke.

Abstract 2673: C-reactive Protein Increases The Severity Of Stroke Outcome In Mice

C-reactive protein (CRP) is an acute-phase reactant that is elevated in a variety of chronic inflammatory conditions including obesity and autoimmune disorders. Elevated CRP is associated with greater incidence and severity of stroke. However, it is unknown whether CRP directly impacts the severity of stroke outcome. We previously demonstrated that CRP antagonizes endothelial NO synthase (eNOS) by attenuating eNOS serine 1176 phosphorylation via the activation of SHIP-1 in endothelium, which blunts signaling downstream of PI3 kinase. Furthermore, we have shown that modulation of the eNOS serine 1176 phosphorylation site affects vascular reactivity and determines stroke size in vivo. We therefore hypothesize that CRP increases stroke vulnerability in vivo by altering the phosphorylation state of eNOS. To test our hypothesis, we subjected wild-type (WT) and CRP transgenic mice (TG-CRP), which have modest elevations in CRP, to a stroke model of temporal focal ischemia with subsequent reperfusion. Mean blood pressure measured acutely from the common carotid artery under aerrane anesthesia (30 % oxygen, 70% nitrous oxide and 1.5% aerrane) was similar in TG-CRP mice (96 ± 8 mmHg, Mean ± SD, n=6) and WT mice (93 ± 9 mmHg, n=14). Using Laser Doppler flowmetry, we monitored cerebral blood flow (CBF) above the middle cerebral artery (MCA) during 30 minutes of MCA occlusion (MCAO) provoked by a filament and during the first 30 minutes of subsequent reperfusion. Although there was a directional trend, in TG-CRP mice CBF reperfusion after 30 minutes (55 ± 34 %, n=5) was not different from WT mice (80 ± 16 %, n=5, P=0.28). However, the TG-CRP mice demonstrated markedly larger infarct volumes as compared with WT mice after 30 minutes of MCAO and 48 hours of reperfusion (100 ± 26 mm3 in TG-CRP vs. 51 ± 21 mm3 in WT, n=5/group, p&lt; 0.03). Neurological deficit after 48 hours of reperfusion was also worse in TG-CRP mice (3.0 points) as compared with WT mice (1.8 point). These findings indicate that CRP increases the severity of stroke outcome in a mouse model of cerebral reperfusion. As such, future therapies aimed at CRP or its mechanism of action in stroke may potentially prevent or improve the stroke outcomes in patients with chronic inflammatory conditions such as obesity.

Abstract 3630: Function-Blocking Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) Antibody Injection is Neuroprotective after Focal Cerebral Ischemia

Introduction: Extracellular metalloproteinase inducer, EMMPRIN, is a cell surface glycoprotein found on endothelial cells and astrocytes and has a key role in the expression of MMPs. It was reported that EMMPRIN expression is upregulated in a mouse model of permanent cerebral ischemia and that EMMPRIN co-localized with MMP-9 in infarcted tissue. There is evidence to support that elevation of MMP activity contributes to injury after stroke and that inhibition of MMPs can reduce infarct volume in rats. In this study we hypothesized that inhibition of EMMPRIN with a function-blocking antibody would reduce infarct volume and improve stroke outcome in mice subjected to a MCAO stroke through reduction in MMP activation. Methods: Transient 90-minute middle cerebral artery occlusion (MCAO) was performed on male wild-type mice treated with vehicle, rat IgG2a (eBioscience), or with function blocking anti-EMMPRIN antibody (eBioscience) injected via tail vein. A single injection was given at stroke onset. Mice were sacrificed at 48 hours post reperfusion. Brains were harvested for TTC staining (n=4/group; significance p&lt;0.05). A second cohort of vehicle and drug treated mice (n=3 vehicle, n=7 drug) were subjected to transient 90-minute middle cerebral artery occlusion and were sacrificed at 72 hours. These mice were given the initial dose at stroke onset and an additional dose at 48 hours. Both cohorts were subjected to behavioral analysis using a modified 4 point scoring system. Results: Mice treated with the function blocking EMMPRIN antibody had smaller infarct volumes when compared with vehicle treated animals. This reduction in damaged tissue was consistent in the cortex, striatum and total hemisphere of each group. Drug treated mice had improved behavior scores when compared to vehicle treated mice at both the 48hr and 72hrs time points. Furthermore, vehicle treated mice had higher mortality rates (20% at 48hrs and 66% at 72hrs) than drug mice (0% at 48hrs and 14% at 72hrs) at both time points. Discussion: Animals treated with a function-blocking antibody to EMMPRIN presented with less infarct volume and better behavior scores than similar animals treated with vehicle. Thus, EMMPRIN inhibition is neuroprotective. Considering monoclonal antibody therapy has been successful in the treatment of other diseases, this data suggests that inhibition of EMMPRIN via administration of an antibody could have therapeutic potential for stroke patients.

Transgenic ablation of doublecortin-expressing cells suppresses adult neurogenesis and worsens stroke outcome in mice

Injury stimulates neurogenesis in the adult brain, but the role of injury-induced neurogenesis in brain repair and recovery is uncertain. One strategy for investigating this issue is to ablate neuronal precursors and thereby prevent neurogenesis, but this is difficult to achieve in a specific fashion. We produced transgenic mice that express herpes simplex virus thymidine kinase (TK) under control of the promoter for doublecortin (Dcx), a microtubule-associated protein expressed in newborn and migrating neurons. Treatment for 14 days with the antiviral drug ganciclovir (GCV) depleted Dcx-expressing and BrdU-labeled cells from the rostral subventricular zone and dentate gyrus, and abolished neurogenesis and associated neuromigration induced by focal cerebral ischemia. GCV treatment of Dcx-TK transgenic, but not WT, mice also increased infarct size and exacerbated postischemic sensorimotor behavioral deficits measured by rotarod, limb placing, and elevated body swing tests. These findings provide evidence that injury-induced neurogenesis contributes to stroke outcome and might therefore be a target for stroke therapy.

Endothelial progenitor cell transplantation improves long‐term stroke outcome in mice

Endothelial progenitor cells (EPCs) play an important role in tissue repairing and regeneration in ischemic organs, including the brain. However, the cause of EPC migration and the function of EPCs after ischemia are unclear. In this study, we demonstrated the effects of EPCs on ischemic brain injury in a mouse model of transient middle cerebral artery occlusion (tMCAO). Circulating human EPCs were characterized with immunofluorescent staining and flow cytometry. EPCs (1 x 10(6)) were injected into nude mice after 1 hour of tMCAO. Histological analysis and behavioral tests were performed from day 0 to 28 days after tMCAO. EPCs were detected in ischemic brain regions 24 hours after tMCAO. EPC transplantation significantly reduced ischemic infarct volume at 3 days after tMCAO compared with control animals (p < 0.05). CXCR4 was expressed in the majority of EPCs, and stromal-derived factor-1 (SDF-1) induced EPC migration, which was blocked by pretreated EPCs with AMD3100 in vitro. SDF-1 was upregulated in ischemic brain. Compared with control animals, injecting AMD3100-pretreated EPCs resulted in a larger infarct volume 3 days after tMCAO, suggesting that SDF-1-mediated signaling was involved in EPC-mediated neuroprotection. In addition, EPC transplantation reduced mouse cortex atrophy 4 weeks after tMCAO and improved neurobehavioral outcomes (p < 0.05). EPC injection potently increased angiogenesis in the peri-infarction area (p < 0.05). We conclude that systemic delivery of EPCs protects the brain against ischemic injury, promotes neurovascular repair, and improves long-term neurobehavioral outcomes. Our data suggest that SDF-1-mediated signaling plays a critical role in EPC-mediated neuroprotection.

Genetically-defined deficiency of mannose-binding lectin is associated with protection after experimental stroke in mice and outcome in human stroke.

BackgroundThe complement system is a major effector of innate immunity that has been involved in stroke brain damage. Complement activation occurs through the classical, alternative and lectin pathways. The latter is initiated by mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs). Here we investigated whether the lectin pathway contributes to stroke outcome in mice and humans.Methodology/Principal FindingsFocal cerebral ischemia/reperfusion in MBL-null mice induced smaller infarctions, better functional outcome, and diminished C3 deposition and neutrophil infiltration than in wild-type mice. Accordingly, reconstitution of MBL-null mice with recombinant human MBL (rhMBL) enhanced brain damage. In order to investigate the clinical relevance of these experimental observations, a study of MBL2 and MASP-2 gene polymorphism rendering the lectin pathway dysfunctional was performed in 135 stroke patients. In logistic regression adjusted for age, gender and initial stroke severity, unfavourable outcome at 3 months was associated with MBL-sufficient genotype (OR 10.85, p = 0.008) and circulating MBL levels (OR 1.29, p = 0.04). Individuals carrying MBL-low genotypes (17.8%) had lower C3, C4, and CRP levels, and the proinflammatory cytokine profile was attenuated versus MBL-sufficient genotypes.Conclusions/SignificanceIn conclusion, genetically defined MBL-deficiency is associated with a better outcome after acute stroke in mice and humans.

Insulin Growth Factor-1 Gene Transfer Enhances Neurovascular Remodeling and Improves Long-Term Stroke Outcome in Mice

Insulin-like growth factor I (IGF-1) is a pleiotropic growth factor that has been demonstrated to protect against acute ischemic brain injury. Whether IGF-1 improves long-term functional outcome after ischemic stroke is not known. The aim of this study is to examine whether IGF-1 overexpression through adeno-associated virus (AAV) -mediated gene transfer enhances neurovascular remodeling and improves functional outcome in a mouse model of focal cerebral ischemia. Long-term cerebral IGF-1 overexpression was achieved with the AAV transduction system through stereotaxic injection. Control mice were injected with AAV-green fluorescent protein or saline. Three weeks after gene transfer, the mice underwent permanent distal middle cerebral artery occlusion. Histological and behavioral analyses were performed at day 21 after middle cerebral artery occlusion. IGF-1 gene transfer compared with control treatment significantly improved motor performance assessed by sensorimotor tests. The functional recovery was accompanied by reduced volume of cerebral infarction. Immunohistochemical analysis with endothelial cell marker CD31 revealed that IGF-1 gene transfer potently increased neovessel formation in the periinfarct and injection needle tract area compared with AAV-green fluorescent protein transduction. Increased vascular density was associated with increased local vascular perfusion. Additionally, AAV-IGF-1 treatment enhanced neurogenesis in the subventricular zone compared with AAV-green fluorescent protein treatment. These data demonstrate that IGF-1 overexpression promoted long-lasting functional recovery after cerebral infarction. The improved functional performance was paralleled by enhanced neovascularization and neurogenesis.

Intravenous Rosuvastatin for Acute Stroke Treatment

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. 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). 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 <0.5 ng ml(-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. 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.

Stroke Outcomes in Mice Lacking the Genes for Neuronal Heme Oxygenase-2 and Nitric Oxide Synthase

Heme oxygenase-2 (HO-2) has been suggested to be a cytoprotective enzyme in a variety of in vivo experimental models. HO-2, the constitutive isozyme, is enriched in neurons and, under normal conditions, accounts for nearly all of brain HO activity. HO-2 deletion (HO-2-/-) leads to increased neurotoxicity in cultured brain cells and increased damage following transient cerebral ischemia in mice. Moreover, pharmacologic inhibition of HO activity significantly augments focal ischemic damage in wildtype (WT) mice, but does not further exacerbate it in HO-2-/- mice. The HO system shares some similarities with nitric oxide synthase (NOS), notably their syntheses of carbon monoxide (CO) and nitric oxide (NO), respectively, which are diffusible gases with numerous biological actions, including neurotransmission and vasodilation. While deletion of HO-2 results in greater stroke damage, the pharmacologic inhibition of neuronal nitric oxide synthase (nNOS), or its gene deletion, confers neuroprotection in animal models of transient cerebral ischemia. To investigate the interactions, the outcome of focal cerebral ischemia-reperfusion in double knockout (HO-2-/- X nNOS-/-) mice lacking both genes was compared to control WT mice. Wildtype and double knockout male mice underwent intraluminal middle cerebral occlusion for 2 hours, followed by reperfusion for 22 hours. Outcomes in neurologic deficits and infarct size were determined. No difference was observed between WT and double knockout mice in the volume of infarction, neurologic signs, decrease in relative cerebral blood flow during ischemia, or core body temperature. The results suggest that the deleterious action of nNOS would counteract the role of HO-2 in neuroprotection.