Effective Treatment For Ischemic Stroke Research Articles

Transdermal System Based on Solid Cilostazol Nanoparticles Attenuates Ischemia/Reperfusion-Induced Brain Injury in Mice.

Cilostazol (CIL) exerted a protective effect by promoting blood–brain barrier integrity as well as improving the status of neurological dysfunctions following cerebral ischemia/reperfusion (I/R) injury. We attempted to design a 0.5% CIL carbopol gel using solid nanoparticles (CIL-Ngel), and then investigated the relationships between energy-dependent endocytosis and the skin penetration of CIL-Ngel in this study. In addition, we evaluated whether the CIL-Ngel attenuated I/R-induced brain injury in a middle cerebral artery occlusion (MCAO)/reperfusion model mouse. The particle size of CIL was decreased using a bead mill, and the CIL particles (14.9 × 1014 particles/0.3 g) in the CIL-Ngel were approximately 50–180 nm. The release of CIL in the CIL-Ngel was higher than that in gel containing CIL powder (CIL-Mgel), and the CIL particles were released from the CIL-Ngel as nanoparticles. In addition, the percutaneous absorption of CIL from the CIL-Ngel was higher in comparison with that from CIL-Mgel, and clathrin-dependent endocytosis and caveolae-dependent endocytosis were related to the enhanced skin penetration of CIL-NPs. In the traditional (oral administration of CIL powder, 3 mg/kg) and transdermal administration (CIL-Ngel, 0.3 g) for 3 days (once a day), the area under the plasma CIL concentration–time curves (AUC) was similar, although the CIL supplied to the blood by the CIL-Ngel was more sustained than that via oral administration of CIL powder. Furthermore, the CIL-Ngel attenuated the ischemic stroke. In conclusion, we designed a gel using solid CIL-NPs, and we showed that the sustained release of CIL by CIL-Ngel provided an effective treatment for ischemic stroke in MCAO/reperfusion model mice. These findings induce the possibilities of developing novel applications of CIL solid nanoparticles.

Study Protocol for a Randomized, Double-Blind, Placebo-Controlled, Phase-II Trial: AdrenoMedullin for Ischemic Stroke Study

Adrenomedullin (AM), a vasoactive peptide, has strong anti-inflammatory and angiogenic properties, which have been reported to ameliorate the consequences of ischemic stroke in several animal models. After a phase I study in healthy volunteers, two phase II trials of AM for inflammatory bowel diseases have been recently completed. The current AdrenoMedullin For Ischemic Stroke (AMFIS) study aims to assess the safety and efficacy of AM in patients with acute ischemic stroke. The AMFIS study is an investigator-initiated, randomized, double-blind, phase-II trial. AM or placebo will be administered to patients with non-cardioembolic ischemic stroke within 24h after stroke onset. In the first cohort of the AMFIS study, patients will be randomly allocated to the investigation treatment A (30μg/kg of AM in total for 7 days, n=20) or placebo group (n=10). In the second cohort, patients will be assigned to the investigation treatment B (56μg/kg of AM in total for 7 days, n=20) or placebo group (n=10). Serious adverse events related to the protocol treatment will be evaluated as the primary outcome. All adverse events will be analyzed as the secondary outcome. Regarding efficacy endpoints, the change in National Institutes of Health Stroke Scale and modified Rankin Scale scores will be compared between investigation treatment and placebo groups. AM is expected to be a safe and effective treatment for ischemic stroke.

Restoration of early deficiency of axonal guidance signaling by guanxinning injection as a novel therapeutic option for acute ischemic stroke

Despite of its high morbidity and mortality, there is still a lack of effective treatment for ischemic stroke in part due to our incomplete understanding of molecular mechanisms of its pathogenesis. In this study, we demonstrate that SHH-PTCH1-GLI1-mediated axonal guidance signaling and its related neurogenesis, a central pathway for neuronal development, also plays a critical role in early stage of an acute stroke model. Specifically, in vivo, we evaluated the effect of GXNI on ischemic stroke mice via using the middle cerebral artery embolization model, and found that GXNI significantly alleviated cerebral ischemic reperfusion (I/R) injury by reducing the volume of cerebral infarction, neurological deficit score and cerebral edema, reversing the BBB permeability and histopathological changes. A combined approach of RNA-seq and network pharmacology analysis was used to reveal the underlying mechanisms of GXNI followed by RT-PCR, immunohistochemistry and western blotting validation. It was pointed out that axon guidance signaling pathway played the most prominent role in GXNI action with Shh, Ptch1, and Gli1 genes as the critical contributors in brain protection. In addition, GXNI markedly prevented primary cortical neuron cells from oxygen-glucose deprivation/reoxygenation damage in vitro, and promoted axon growth and synaptogenesis of damaged neurons, which further confirmed the results of in vivo experiments. Moreover, due to the inhibition of the SHH-PTCH1-GLI1 signaling pathway by cyclopropylamine, the effect of GXNI was significantly weakened. Hence, our study provides a novel option for the clinical treatment of acute ischemic stroke by GXNI via SHH-PTCH1-GLI1-mediated axonal guidance signaling, a neuronal development pathway previously considered for after-stroke recovery.

Rapid and improved oral absorption of N-butylphthalide by sodium cholate-appended liposomes for efficient ischemic stroke therapy

As a multi-target drug to treat ischemic stroke, N-butylphthalide (NBP) is extremely water-insoluble and exhibits limited oral bioavailability, impeding its wide oral application. Effective treatment of ischemic stroke by NBP requires timely and efficient drug exposure, necessitating the development of new oral formulations. Herein, liposomes containing biosurfactant sodium cholate (CA-liposomes) were systemically investigated as an oral NBP delivery platform because of its high biocompatibility and great potential for clinical applications. The optimized liposomes have a uniform hydrodynamic size of 104.30 ± 1.60 nm and excellent encapsulation efficiency (93.91 ± 1.10%). Intriguingly, NBP-loaded CA-liposomes produced rapid drug release and the cumulative release was up to 88.09 ± 4.04% during 12 h while that for NBP group was only 6.79 ± 0.99%. Caco-2 cell monolayer assay demonstrated the superior cell uptake and transport efficiency of NBP-loaded CA-liposomes than free NBP, which was mediated by passive diffusion via transcellular and paracellular routes. After oral administration to rats, NBP-loaded CA-liposomes exhibited rapid and almost complete drug absorption, with a tmax of 0.70 ± 0.14 h and an absolute bioavailability of 92.65% while NBP suspension demonstrated relatively low bioavailability (21.7%). Meanwhile, NBP-loaded CA-liposomes produced 18.30-fold drug concentration in the brain at 5 min compared with NBP suspension, and the brain bioavailability increased by 2.48-fold. As expected, NBP-loaded CA-liposomes demonstrated significant therapeutic efficacy in a middle cerebral artery occlusion rat model. Our study provides new insights for engineering oral formulations of NBP with fast and sufficient drug exposure against ischemic stroke in the clinic.

Major histocompatibility complex Class II-based therapy for stroke.

This review discusses the potential of major histocompatibility complex (MHC) Class II constructs as stroke therapeutics. We focus on the delivery of MHC Class II construct, DRmQ, as a safe and effective treatment for ischemic stroke. DRmQ was observed to attenuate behavioral deficits and decrease microglia activation and proinflammatory cytokines, illustrating its ability to mitigate the secondary cell death following stroke. Similar anti-neuroinflammation treatments, such as transplantation of mesenchymal stem cells and mitochondrial transfers, are briefly discussed to provide further support that sequestration of inflammation stands as a robust therapeutic target for stroke.

Systemic Thrombolysis and Post-stroke Epilepsy

Systemic thrombolysis is a highly effective treatment for ischemic stroke. The effect of thrombolysis on the development of post-stroke epilepsy (PSE) remains a subject of scientific debate. The aim of this study was to clarify the clinical and neuroimaging features of PSE in patients with ischemic stroke, who underwent systemic thrombolysis. . A total of 46 patients after ischemic stroke were examined, including 33 men and 13 women aged 28 to 80 years. Group I consisted of 9 patients with PSE after systemic thrombolysis with rtPA (alteplase) at a dose of 0.9 mg/kg. Group II included 8 patients without PSE after systemic thrombolysis. Group III consisted of 29 patients with PSE, who received standard drug therapy. Comparative group analysis took into account the medical history, clinical and neuroimaging data. . Patients with PSE were characterized by the absence of clinical effect after systemic thrombolysis (p = 0.026); infarcts extending to the frontal and insular lobes (p = 0.038), insular lobe and basal nuclei (p = 0.026); development of a hemorrhagic component in the cerebral cortex (p = 0.005), as well as in the territory of the deep and terminal branches of the middle cerebral artery (MCA) (p = 0.006). Development of PSE is associated with ineffective systemic thrombolysis and the appearance of extensive and large cerebral infarcts with a hemorrhagic component.

Fibroblast growth factor 2 contributes to the effect of salidroside on dendritic and synaptic plasticity after cerebral ischemia/reperfusion injury.

Ischemic stroke, a serious neurological disease, is associated with cell death, axonal and dendritic plasticity, and other activities. Anti-inflammatory, anti-apoptotic, promote dendritic and synaptic plasticity are critical therapeutic targets after ischemic stroke. Fibroblast growth factor-2 (FGF2), which is involved in the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/CAMP response element (CRE)-binding protein (CREB) pathway, has been shown to facilitate dendritic and synaptic plasticity. Salidroside (Sal) has been reported to have anti-inflammatory, anti-oxidative, and anti-apoptotic effects; however, the underlying mechanisms of Sal in promoting dendritic and synaptic plasticity remain unclear. Here, the anti-inflammatory, anti-apoptotic, dendritic and synaptic plasticity effects of Sal were investigated in vitro in PC12 cells under oxygen-glucose deprivation/reoxygenation (OGD/R) conditions and in vivo in rats with middle cerebral artery occlusion/reperfusion (MCAO/R). We investigated the role of Sal in promoting dendritic and synaptic plasticity in the ischemic penumbra and whether the FGF2-mediated cAMP/PKA/CREB pathway was involved in this process. The present study demonstrated that Sal could significantly inhibit inflammation and apoptosis, and promote dendritic and synaptic plasticity. Overall, our study suggests that Sal is an effective treatment for ischemic stroke that functions via the FGF2-mediated cAMP/PKA/CREB pathway to promote dendritic and synaptic plasticity.

012 Mechanical thrombectomy in pediatric stroke: systematic review, individual patient-data meta-analysis, and case series

IntroductionIn adults, there is strong evidence demonstrating the superiority of mechanical thrombectomy (MT) plus intravenous thrombolysis over thrombolysis alone for the treatment of acute ischemic stroke due to large vessel occlusion (LVO). The role of MT in the paediatric stroke population is less clear. Here we present an updated systematic review addressing the use of MT in paediatric patients, including three cases from our centre in Sydney, Australia. We have also completed an individual participant data (IPD) meta-analysis of clinical and angiographic outcomes based on these results.MethodOur systematic review and IPD meta-analysis was performed according to PRISMA-IPD (Preferred Reporting Items for Systematic Reviews and Meta-Analyses: Individual Participant Data) guidelines. Primary outcomes measures were change in NIHSS (National Institute of Health Stroke Scale) score following MT, and mRS (modified Rankin Scale) score at final reported follow-up. The secondary outcome measure was final angiographic result using the mTICI (modified Treatment in Cerebral Ischemia) scale.ResultsMT resulted in good long-term neurological outcomes (mRS 0–2) in 60/67 cases (89.6%;follow-up timing µ=4.1 months: 95%CI 2.9–5.3), good short-term neurological outcomes (reduction in NIHSS by 8 or more points or post-MT NIHSS of 0–1) in 37/52 cases (71.2%), and successful recanalization (mTICI 2b/3) in 57/67 cases (85.1%).ConclusionsIn paediatric patients, MT is an effective treatment for ischaemic stroke due to LVO. In the absence of a dedicated prospective registry and with randomized control trails unfeasible, this report represents the best available evidence for the use of MT in the paediatric setting.

Neural Stem Cells Transfected with Reactive Oxygen Species-Responsive Polyplexes for Effective Treatment of Ischemic Stroke.

Neural stem cells (NSCs), capable of ischemia-homing, regeneration, and differentiation, exert strong therapeutic potentials in treating ischemic stroke, but the curative effect is limited in the harsh microenvironment of ischemic regions rich in reactive oxygen species (ROS). Gene transfection to make NSCs overexpress brain-derived neurotrophic factor (BDNF) can enhance their therapeutic efficacy; however, viral vectors must be used because current nonviral vectors are unable to efficiently transfect NSCs. The first polymeric vector, ROS-responsive charge-reversal poly[(2-acryloyl)ethyl(p-boronic acid benzyl)diethylammonium bromide] (B-PDEA), is shown here, that mediates efficient gene transfection of NSCs and greatly enhances their therapeutics in ischemic stroke treatment. The cationic B-PDEA/DNA polyplexes can effectively transfect NSCs; in the cytosol, the B-PDEA is oxidized by intracellular ROS into negatively charged polyacrylic acid, quickly releasing the BDNF plasmids for efficient transcription and secreting a high level of BDNF. After i.v. injection in ischemic stroke mice, the transfected NSCs (BDNF-NSCs) can home to ischemic regions as efficiently as the pristine NSCs but more efficiently produce BDNF, leading to significantly augmented BDNF levels, which in turn enhances the mouse survival rate to 60%, from 0% (nontreated mice) or ≈20% (NSC-treated mice), and enables more rapid and superior functional reconstruction.

Would antioxidant-loaded nanoparticles present an effective treatment for ischemic stroke?

Ischemic stroke is a leading cause of death and disability worldwide and is in urgent need of new treatment options. The only approved treatment for stroke restores blood flow to the brain, but much of the tissue damage occurs during the subsequent reperfusion. Antioxidant therapies that directly address ischemia-reperfusion injury have shown promise in preclinical results. In this review, we discuss that reformulating antioxidant therapies as nanomedicine can potentially overcome the barriers that have kept these therapies from succeeding in the clinic. We begin by reviewing the pathophysiology of ischemic stroke with a focus on the effects of reperfusion injury. Next, we review nanotherapeutic systems designed to treat the disease with a focus on those addressing reperfusion injury. Mechanisms of passive and active transport required to traverse a blood-brain barrier are discussed. Finally, we conclude by outlining design parameters for potentially successful nanomedicines as front-line therapeutics for ischemic stroke.

Dexamethasone-Loaded, PEGylated, Vertically Aligned, Multiwalled Carbon Nanotubes for Potential Ischemic Stroke Intervention

The complete synthesis, optimization, purification, functionalization and evaluation of vertically aligned multiwalled carbon nanotubes (VA-MWCNTs) was reported for potential application in dexamethasone delivery to the ischemic brain tissue. The conditions for high yield were optimized and carbon nanotubes functionalized and PEGylated prior to dexamethasone loading. Morphological changes were confirmed by SEM and TEM. Addition of functional groups to MWCNTs was demonstrated by FTIR. Thermal stability reduced following MWCNTs functionalization as demonstrated in TGA. The presence of carbon at 2θ of 25° and iron at 2θ of 45° in MWCNTs was illustrated by XRD. Polydispersive index and zeta potential were found to be 0.261 and −15.0 mV, respectively. Dexamethasone release increased by 55%, 65% and 95% in pH of 7.4, 6.5 and 5.5 respectively as evaluated by UV-VIS. The functionalized VA-MWCNTs were demonstrated to be less toxic in PC-12 cells in the concentration range from 20 to 20,000 µg/mL. These findings have demonstrated the potential of VA-MWCNTs in the enhancement of fast and prolonged release of dexamethasone which could lead to the effective treatment of ischemic stroke. More work is under way for targeting ischemic sites using atrial natriuretic peptide antibody in stroke rats.

Point-of-care versus central laboratory testing of INR in acute stroke.

Effective anticoagulant therapy is a contraindication to thrombolysis, which is an effective treatment of ischemic stroke if given within 4.5hours of symptom onset. INR above 1.7 is generally considered a contraindication for thrombolysis. Rapid measurement of INR in warfarin-treated patients is therefore of major importance in order to be able to decide on thrombolysis or not. We asked whether INR measured on a point-of-care instrument would be as good as a central laboratory instrument. A total of 529 consecutive patients who arrived at the emergency department at a large urban teaching hospital with stroke symptoms were enrolled in the study. INR was measured with a CoaguChek and a Sysmex instrument. Basic clinical information such as age, sex, and diagnosis (if available) was recorded. INR from the instruments was compared using linear regression and Bland-Altman plot. Of 529 patients, 459 had INR results from both instruments. Among these, 3 patients were excluded as outliers. The rest (n=456) showed good correlation between the methods (R2 =0.97). In the current setting, CoaguChek was in median 63minutes faster than Sysmex. Our results indicate that point-of-care testing is a safe mean to rapidly acquire a patient's INR value in acute clinical situations.

Efficacy of Thrombolytic Therapy in Treatment of Patients with Ischemic Stroke

The purpose: to evaluate the effectiveness of thrombolytic therapy in patients with ischemic stroke according to radiological methods.Materials and methods. The study included 60 people who were divided into two groups. The main group included 34 (56.7%) patients with ischemic stroke (20 men and 14 women) aged 22 to 78 years (mean age 62 ± 8.6 years) who were treated in Republican research center of emergency medical care. The control group consisted of 26 (43.3%) patients to estimate the parameters of normeperidine in the middle cerebral artery (MCA): the maximum blood flow velocity, linear blood flow velocity, index Gosling (PI) and coefficient overshoot (KO). Evaluation of patients included: assessment of nevrostatus according NIHSS scale, computered tomograhy of brain to exclude hemorrhagic stroke, transcranial and extracranial arteries Duplex scanning, digital subtraction angiography. Thrombolysis was performed to all patients of main group with ischemic stroke: systemic thrombolysis with streptokinase – in 17 (50%) cases, selective thrombolysis of Actilyse – 17 (50%). Thrombolysis was performed in a period from 90 minutes to 4 hours after the manifestation of symptoms. Results. Significant positive changes were observed in 22 (64.7%) patients. Asymptomatic hemorrhagic transformation of ischemic lesion was observed in 4 (11.7%). In acute occlusion of the internal carotid artery recanalization was registered only in 11.7% of cases, while the M1 MCA occlusion segment – at 52.9%. Transcranial Doppler is manifested by registration of hyperperfusion flow after thrombolysis in the presence of residual flow to thrombolysis. Re-occlusion was observed in 2 (5.8%) patients. In these cases, before and after thrombolysis TCD fixed flow hypoperfusion. Mortality was 14.7%.Conclusions. Thrombolytic therapy is effective treatment for ischemic stroke, especially in the delivery of the patient to a hospital for the first 2 hours after the manifestation of clinical symptoms. The marker of effectiveness thrombolysis is registration on the CMA normoperfusion or hyperperfusion pattern. Good functional outcome marked when a patient achieved result ≤2 points on the mRS. Asymptomatic hemorrhagic transformation can also be considered as a marker of effective thrombolysis. The combination of a CT scan of the brain with transcranial Doppler allows to assess not only the size of the lesion of the brain, but also to investigate the nature of cerebral hemodynamics. Further development of a set of measures designed to raise awareness among the population and general practitioners about the symptoms of stroke and its effective treatment in the case of delivery of the patient to a specialized hospital in which is possible to conduct thrombolysis.

Abstract 161: Age and Sex Influence the Neutrophil Response to Ischemic Stroke in Mice and Humans

Background and Purpose: Age and sex have important effects on stroke-induced inflammation. We conducted CBC analysis and RNA-sequencing of ischemic stroke patient blood, with follow-up studies in an experimental mouse model, to test the hypothesis that there are sex differences in neutrophil function and neutrophil-mediated damage after ischemic stroke. Methods: Blood from male and female stroke patients was collected 24 hours after stroke, with transient ischemic attack patients serving as controls. Absolute neutrophil count was quantified and RNA-sequencing was performed on an age and severity matched subset of patients. Results were analyzed by multivariate regression and differential expression analysis, respectively. For animal experiments, aged (18-20 month) and young (8-10 week) male and female mice were subjected to 60 minute middle cerebral artery occlusion and sacrificed at 24 hours. Neutrophils from blood, bone marrow, lung, brain and spleen were quantified and phenotyped by flow cytometry. Results were analyzed by student T-test and two way ANOVA. Results: Absolute neutrophil counts increased significantly after stroke in both sexes (p=.01). Interestingly, females had more differentially expressed genes after stroke than males, including matrix metalloproteinase 9 and TNFa. In animal experiments, aged animals were found to have higher percentages of neutrophils in the blood, spleen, lungs and bone marrow than young animals. In addition, aged female animals were found to have more activated neutrophils 24 hours after stroke than aged males (p=<.05). Conclusion: These results suggest that the immune response to ischemic stroke differs in aged males and females. Female neutrophils appear to exhibit enhanced activation, which may contribute to the poorer stroke outcomes seen in aged female patients. Understanding sex and age differences in the acute immune response is crucial to developing future immunomodulatory drugs for the safe and effective treatment of ischemic stroke in both sexes.

Electroacupuncture regulates the stress-injury-repair chain of events after cerebral ischemia/reperfusion injury.

Inflammation after stroke is the main cause of cerebral ischemia/reperfusion injury. Cascading events after injury can lead to cell death. Heat shock protein 70 and other endogenous injury-signaling molecules are released by damaged cells, which can lead to systemic stress reactions. Protecting the brain through repair begins with the stress-injury-repair signaling chain. This study aimed to verify whether acupuncture acts through this chain to facilitate effective treatment of ischemic stroke. Rat models of cerebral ischemia/reperfusion injury were established by Zea Longa's method, and injury sites were identified by assessing neurological function, 2,3,5-triphenyltetrazolium chloride staining, and hematoxylin-eosin staining. Electroacupuncture at acupoints Baihui (DU20) and Zusanli (ST36) was performed in the model rats with dilatational waves, delivered for 20 minutes a day at 2-100 Hz and an amplitude of 2 mA. We analyzed the blood serum from the rats and found that inflammatory cytokines affected the levels of adrenotrophin and heat shock protein 70, each of which followed a similar bimodal curve. Specifically, electroacupuncture lowered the peak levels of adrenocorticotrophic hormone and heat shock protein 70. Thus, electroacupuncture was able to inhibit excessive stress, reduce inflammation, and promote the repair of neurons, which facilitated healing of ischemic stroke.

A novel nuclear factor erythroid 2-related factor 2 (Nrf2) activator RS9 attenuates brain injury after ischemia reperfusion in mice

Recanalization of occluded vessels leads to ischemia–reperfusion injury (IRI), with oxidative stress as one of the main causes of injury, despite the fact that recanalization therapy is the most effective treatment for ischemic stroke. The nuclear factor erythroid 2-related factor 2 (Nrf2) is one of the transcription factors which has an essential role in protection against oxidative stress. RS9 is a novel Nrf2 activator obtained from bardoxolone methyl (BARD), an Nrf2 activator that has already been tested in a clinical trial, using a biotransformation technique. RS9 has been reported to lead to higher Nrf2 activation and less cytotoxicity than BARD. In this study, we investigated the effects of RS9 on IRI. Mice were intraperitoneally treated immediately after 2h of transient middle cerebral artery occlusion (MCAO) with a vehicle solution or 0.2mg/kg of RS9. Post-onset treatment of RS9 attenuated the infarct volume and improved neurological deficits 22h after reperfusion. RS9 activated Nrf2 2 and 6h after reperfusion and activated heme oxygenase-1 at 6 and 22h after reperfusion. RS9 also attenuated the phosphorylation of NF-κB p65 2 and 6h after reperfusion. Finally, RS9 improved the survival rate and neurological deficits 7days after MCAO. Our results suggest that the activation of Nrf2 by RS9 has a neuroprotective effect, mediated by attenuating both oxidative stress and neuroinflammation, and that RS9 is an effective therapeutic candidate for the treatment of IRI.

Tissue Plasminogen Activator Neurotoxicity is Neutralized by Recombinant ADAMTS 13.

Tissue plasminogen activator (tPA) is an effective treatment for ischemic stroke, but its neurotoxicity is a significant problem. Here we tested the hypothesis that recombinant ADAMTS 13 (rADAMTS 13) would reduce tPA neurotoxicity in a mouse model of stroke. We show that treatment with rADAMTS 13 in combination with tPA significantly reduced infarct volume compared with mice treated with tPA alone 48 hours after stroke. The combination treatment significantly improved neurological deficits compared with mice treated with tPA or vehicle alone. These neuroprotective effects were associated with significant reductions in fibrin deposits in ischemic vessels and less severe cell death in ischemic brain. The effect of rADAMTS13 on tPA neurotoxicity was mimicked by the N-methyl-D-aspartate (NMDA) receptor antagonist M-801, and was abolished by injection of NMDA. Moreover, rADAMTS 13 prevents the neurotoxicity effect of tPA, by blocking its interaction with the NMDA receptor NR2B and the attendant phosphorylation of NR2B and activation of ERK1/2. Finally, the NR2B-specific NMDA receptor antagonist ifenprodil abolished tPA neurotoxicity and rADAMTS 13 treatment had no further beneficial effect. Our data suggest that the combination of rADAMTS 13 and tPA may provide a novel treatment of ischemic stroke by diminishing the neurotoxic effects of exogenous tPA.

Code stroke in Asturias

BackgroundIntravenous thrombolysis with alteplase is an effective treatment for ischaemic stroke when applied during the first 4.5hours, but less than 15% of patients have access to this technique. Mechanical thrombectomy is more frequently able to recanalise proximal occlusions in large vessels, but the infrastructure it requires makes it even less available. MethodsWe describe the implementation of code stroke in Asturias, as well as the process of adapting various existing resources for urgent stroke care in the region. By considering these resources, and the demographic and geographic circumstances of our region, we examine ways of reorganising the code stroke protocol that would optimise treatment times and provide the most appropriate treatment for each patient. ResultsWe distributed the 8 health districts in Asturias so as to permit referral of candidates for reperfusion therapies to either of the 2 hospitals with 24-hour stroke units and on-call neurologists and providing IV fibrinolysis. Hospitals were assigned according to proximity and stroke severity; the most severe cases were immediately referred to the hospital with on-call interventional neurology care. Patient triage was provided by pre-hospital emergency services according to the NIHSS score. ConclusionsModifications to code stroke in Asturias have allowed us to apply reperfusion therapies with good results, while emphasising equitable care and managing the severity-time ratio to offer the best and safest treatment for each patient as soon as possible.

Abstract TP97: Experimental Stroke During Aging: Docosahexaenoic Acid Therapy Provides Robust Neuroprotection

Introduction: Recently we have shown that docosahexaenoic acid (DHA) is neuroprotective after experimental stroke in young rats. The purpose of this study was to determine whether treatment with DHA would be protective in aged rats after focal cerebral ischemia. Methods: Isoflurane/nitrous oxide-anesthetized normothermic (brain temperature 36-36.5 o C) male Sprague-Dawley aged rats (16-months old) received 2h middle cerebral artery occlusion (MCAo) by poly-L-lysine-coated intraluminal suture. The behavioral function was evaluated during occlusion (60 min), and on days 1, 3 and 7 after MCAo; a grading scale of 0-12 was employed (0=normal and 12=maximal deficit). The agent (DHA, 5 mg/kg; n=6) or vehicle (saline; n=7) was administered i.v. at 3h after MCAo onset. The composite neuroscore comprises two different neurological tests, the postural reflex test and the forelimb placing test, to measure visual, tactile and proprioceptive stimuli, which were evaluated on days 1, 2, 3 and 7. High resolution ex vivo MRI using an 11.7T Bruker was performed on day 7. The core and penumbra were automatically identified using the Hierarchical Region Splitting method for penumbra identification. Histopathology and immunostaining were conducted after completion of MRI on day 7. Results: DHA treatment improved neurological scores on days 1 (by 18%), 2 (by 25%), 3 (by 22%) and 7 (by 36%). Total, cortical and subcortical lesion volumes computed from T2WI were significantly reduced by DHA treatment (by 62-75%). DHA treatment also reduced cortical, subcortical and total brain infarction (by 65- 80%). In addition, DHA therapy decreased ED-1 positive microglia/microphages (by 62%), GFAP-positive astrocytes (by 21%), and NeuN-positive neurons (by34%), and it increased SMI-71-positive vessels (by 46%) in the ischemic penumbra. Conclusion: DHA therapy is highly neuroprotective in aged rats following focal cerebral ischemia and has potential for the effective treatment of ischemic stroke in aged individuals.

Abstract WP261: Sex Differences in Circulating Leukocyte Subtype and Activation Following Ischemic Stroke

Background and Purpose: Females exhibit a more robust immune response in many disease models, yet sex differences in the inflammatory response to ischemia remain largely unexplored. We conducted flow cytometry and RNA sequencing of blood from ischemic stroke patients, with follow-up studies in mice. Hypothesis: We assessed the hypothesis that there are sex differences in the acute immune response to ischemic stroke. Methods: Patient samples were drawn at 24 hours post-stroke for flow cytometry (n=6) and RNA sequencing Analysis (n=40). For murine studies, male and ovariectomized (OVX) female animals (n=14) were subjected to 90-minute middle cerebral artery occlusion or sham surgery and sacrificed at 24 hours. Murine blood was stained for leukocyte markers and CD62L (L-selectin). Results were analyzed by student T-test and two-way ANOVA. Results: RNA sequencing revealed that 24 hours after ischemic stroke, female patients had 79 significantly upregulated genes, compared to male patients with 6 significantly upregulated genes. Human flow cytometry revealed that male stroke patients had a significantly higher percentage of monocytes (p=.026), while females had a greater percentage of CD8+ T-cells (p=.023). Murine flow cytometry showed a post-stroke increase in peripheral myeloid cells at 24 hours in male mice only (p=.0046), whereas female mice had a higher CD8/CD4 T cell ratio (p=.0027). Neutrophils from male sham animals displayed greater L-selectin positivity, with stroke-induced shedding of L-selectin seen only in males (sex/stroke p=.0266). Male monocytes and lymphocytes also displayed higher L-selectin positivity (p=.0079, p=.0004). Conclusion: These results suggest that the immune response to ischemic stroke is different in male and female patients, a phenomenon that can be recapitulated in a mouse model of experimental stroke. Female immune cells exhibit a higher level of baseline activation (reduced L-selectin expression) and post-stroke activity, which may enable a quicker and more robust response to immune challenges. Understanding sex differences in the acute immune response is crucial to developing future immunomodulatory drugs for the safe and effective treatment of ischemic stroke in both sexes.