Embolic Middle Cerebral Artery Occlusion Research Articles

The Water Extract of Rhubarb Prevents Ischemic Stroke by Regulating Gut Bacteria and Metabolic Pathways.

Rhubarb (RR), Chinese name Dahuang, is commonly used in the treatment of ischemic stroke (IS). However, its potential mechanism is not fully elucidated. This study intended to verify the effect of RR on IS and investigate the possible mechanism of RR in preventing IS. IS in male rats was induced by embolic middle cerebral artery occlusion (MCAO) surgery, and drug administration was applied half an hour before surgery. RR dramatically decreased the neurological deficit scores, the cerebral infarct volume, and the cerebral edema rate, and improved the regional cerebral blood flow (rCBF) and histopathological changes in the brain of MCAO rats. The 16S rRNA analysis showed the harmful microbes such as Fournierella and Bilophila were decreased, and the beneficial microbes such as Enterorhabdus, Defluviitaleaceae, Christensenellaceae, and Lachnospira were significantly increased, after RR pretreatment. 1H-nuclear magnetic resonance (1H-NMR) was used to detect serum metabolomics, and RR treatment significantly changed the levels of metabolites such as isoleucine, valine, N6-acetyllysine, methionine, 3-aminoisobutyric acid, N, N-dimethylglycine, propylene glycol, trimethylamine N-oxide, myo-inositol, choline, betaine, lactate, glucose, and lipid, and the enrichment analysis of differential metabolites showed that RR may participate in the regulation of amino acid metabolism and energy metabolism. RR exerts the role of anti-IS via regulating gut bacteria and metabolic pathways.

An Ultrasmall Cu/Cu2O Nanoparticle-Based Diselenide-Bridged Nanoplatform Mediating Reactive Oxygen Species Scavenging and Neuronal Membrane Enhancement for Targeted Therapy of Ischemic Stroke.

Ischemic stroke is one of the major causes of death and disability worldwide, and an effective and timely treatment of ischemic stroke has been a challenge because of the narrow therapeutic window and the poor affinity with thrombus of the thrombolytic agent. In this study, rPZDCu, a multifunctional nanoparticle (NP) with the effects of thrombolysis, reactive oxygen species (ROS) scavenging, and neuroprotection, was synthesized based on an ultrasmall Cu4.6O NP, the thrombolytic agent rt-PA, and docosahexaenoic acid (DHA), which is a major component of the neuronal membrane. rPZDCu showed strong thrombus-targeting ability, which was achieved by the platelet cell membrane coating on the NP surface, and a good thrombolytic effect in both the common carotid artery clot model and embolic middle cerebral artery occlusion (MCAO) model of rats. Furthermore, rPZDCu exhibited a good escape from the phagocytosis of macrophages, effective promotion of the polarization of microglia, and efficient recovery of neurobiological and behavioral functions in the embolic MCAO model of rats. This is a heuristic report of (1) the Cu0/Cu+ NP for the treatments of brain diseases, (2) the integration of DHA and ROS scavengers for central nervous system therapies, and (3) diselenide-based ROS-responsive NPs for ischemic stroke treatments. This study also offers an example of cell membrane-camouflaged stimuli-responsive nanomedicine for brain-targeting drug delivery.

Application of Sofia Plus catheter tip shaping in the treatment of acute middle cerebral artery occlusion: A case control study.

To explore the safety and efficacy of Sofia Plus distal access catheter tip shaping for treatment of acute middle cerebral artery embolism. This single-center retrospective study involved patients eligible for acute embolic middle cerebral artery occlusion from January 2020 to October 2021. They were divided into a shaping and non-shaping group according to whether the Sofia Plus catheter tip was shaped intraoperatively. Baseline data, preoperative Alberta Stroke Program Early Computed Tomography (ASPECT) score, National Institutes of Health Stroke Scale (NIHSS) score, onset-to-admission time, admission-to-puncture time, Sofia Plus-clot time, puncture-to-reperfusion time, surgical approach, and use of a stent for rescue thrombectomy were compared between the 2 groups. Postoperative symptomatic intracerebral hemorrhage and the modified Rankin scale score at the 90-day follow-up were observed. In total, 54 patients were enrolled in this study (shaping group, 26 patients; non-shaping group, 28 patients). Their mean age was 64.8 ± 14.6 years, and the proportion of men was 68.5% (37/54). Successful recanalization was achieved in all patients. There were no differences in the baseline data (age, sex, history, pre-admission ASPECT score, or NIHSS score) between the shaping and non-shaping groups. Patients treated with a shaped Sofia Plus catheter had a shorter Sofia Plus-clot time [median (25th, 75th percentile: 4 (4, 7) vs 10.5 (5.25, 14) min, P = .006] and puncture-to-reperfusion time [16.5 (12, 30.5) vs 26 (16.25, 38.25) min, P = .036]. There were significant differences in the surgical approaches between the 2 groups. The rates of a favorable outcome (57.7% vs 64.3%, P = .62) and postoperative symptomatic intracerebral hemorrhage (7.7% vs 3.6%, P = .60) were not significantly different between the groups. Sofia Plus catheter tip shaping improved catheter trafficability and reduced the operative time. It was safe and effective for treatment of acute middle cerebral artery thrombotic occlusion.

Treatment of stroke in aged male and female rats with Vepoloxamer and tPA reduces neurovascular damage.

Male and female Wistar rats at 18 months of age were subjected to embolic middle cerebral artery occlusion and treated either with monotherapy of tPA or Vepoloxamer, a combination of these two agents, or saline at 4 h after stroke onset. Neurological outcomes were evaluated with a battery of behavioral tests including adhesive removal, foot-fault, and modified neurological severity score tests at 1 and 7 days after stroke onset, followed by histopathological analysis of infarct volume. Residual clot size and vascular patency and integrity were analyzed. The combination treatment with Vepoloxamer and tPA significantly reduced infarct volume and neurological deficits in male and female rats compared to rats treated with saline and the monotherapies of tPA and Vepoloxamer. While Vepoloxamer monotherapy moderately reduced neurological deficits, monotherapies with tPA and Vepoloxamer failed to reduce infarct volume compared to saline treatment. Furthermore, the combination treatment with tPA and Vepoloxamer accelerated thrombolysis, reduced ischemia and tPA-potentiated microvascular disruption, and concomitantly improved cerebrovascular integrity and perfusion in the male ischemic rats. Combination treatment with tPA and Vepoloxamer at 4 h after stroke onset effectively reduces ischemic neurovascular damage by accelerating thrombolysis and reducing ischemia and tPA potentiated side effects in the aged rats. This funding suggests that the combination treatment with tPA and Vepoloxamer represents a promising strategy to potentially apply to the general population of stroke patients.

Enhanced Cerebroprotection of Xenon-Loaded Liposomes in Combination with rtPA Thrombolysis for Embolic Ischemic Stroke.

Xenon (Xe) has shown great potential as a stroke treatment due to its exceptional ability to protect brain tissue without inducing side effects. We have previously developed Xe-loaded liposomes for the ultrasound-activated delivery of Xe into the cerebral region and demonstrated their therapeutic efficacy. At present, the sole FDA-approved thrombolytic agent for stroke treatment is recombinant tissue plasminogen activator (rtPA). In this study, we aimed to investigate the potential of combining Xe-liposomes with an intravenous rtPA treatment in a clinically relevant embolic rat stroke model. We evaluated the combinational effect using an in vitro clot lysis model and an in vivo embolic middle cerebral artery occlusion (eMCAO) rat model. The treatment groups received intravenous administration of Xe-liposomes (20 mg/kg) at 2 h post-stroke onset, followed by the administration of rtPA (10 mg/kg) at either 2 or 4 h after the onset. Three days after the stroke, behavioral tests were conducted, and brain sections were collected for triphenyltetrazolium chloride (TTC) and TUNEL staining. Infarct size was determined as normalized infarct volume (%). Both in vitro and in vivo clot lysis experiments demonstrated that Xe-liposomes in combination with rtPA resulted in effective clot lysis comparable to the treatment with free rtPA alone. Animals treated with Xe-liposomes in combination with rtPA showed reduced TUNEL-positive cells and demonstrated improved neurological recovery. Importantly, Xe-liposomes in combination with late rtPA treatment reduced rtPA-induced hemorrhage, attributing to the reduction of MMP9 immunoreactivity. This study demonstrates that the combined therapy of Xe-liposomes and rtPA provides enhanced therapeutic efficacy, leading to decreased neuronal cell death and a potential to mitigate hemorrhagic side effects associated with late rtPA treatment.

Pharmacological Characterization of a Novel Neuroactive Steroid Prodrug, NTS-104, and Its Neuroprotective Activity in Experimental Stroke Models.

Ischemic stroke affects about 700 000 patients per year in the United States, and to date, there are no effective pharmacological agents that promote recovery. Here, we studied the pharmacokinetics, pharmacodynamics, and efficacy of NTS-105, a novel neuroactive steroid, and NTS-104, a prodrug of NTS-105, in 2 models of ischemic stroke. The pharmacodynamics and pharmacokinetics of NTS-104/105 were investigated in naive and stroke rats, and models of embolic and transient middle cerebral artery occlusion were used to investigate the dose-related effects of NTS-104. All rats were randomly assigned into the experimental groups, and all outcome measurements were performed blindly. Blood plasma and brain pharmacokinetic analysis revealed that NTS-104 rapidly converted to NTS-105, which reached peak concentration at ≈1 hour after dosing and distributed similarly to normal and ischemic brains. NTS-104 administration 4 hours after embolic middle cerebral artery occlusion led to a dose-dependent improvement of neurological outcomes and a dose-dependent reduction of infarct volumes relative to vehicle-treated animals. A single dose level study confirmed that NTS-104 administered 4 hours after transient middle cerebral artery occlusion was also neuroprotective. Quantitative ELISA revealed that NTS-104 treatment resulted in time- and dose-dependent changes in AKT activation and cytokine levels within the ischemic brain, which included reductions of IL-6, VEGF, ICAM-1, IL-1β, MCP-1, RAGE, and GM-CSF. Time- and dose-dependent reductions in IL-6 and GM-CSF were also observed in the plasma along with an elevation of galectin-1. NTS-104 is a novel prodrug that converts to a novel neuroactive steroid, NTS-105, which improves functional outcomes in experimental ischemic stroke models.

J147 Reduces tPA-Induced Brain Hemorrhage in Acute Experimental Stroke in Rats.

Background and purposeJ147, a novel neurotrophic compound, was originally developed to treat aging-associated neurological diseases. Based on the broad spectrum of cytoprotective effects exhibited by this compound, we investigated whether J147 has cerebroprotection for acute ischemic stroke and whether it can enhance the effectiveness of thrombolytic therapy with tissue plasminogen activator (tPA).MethodsRats were subjected to transient occlusion of the middle cerebral artery (tMCAO) by insertion of an intraluminal suture or embolic middle cerebral artery occlusion (eMCAO), and treated intravenously with J147 alone or in combination with tPA.ResultsWe found that J147 treatment significantly reduced infarct volume when administered at 2 h after stroke onset in the tMCAO model, but had no effect in eMCAO without tPA. However, combination treatment with J147 plus tPA at 4 h after stroke onset significantly reduced infarct volume and neurological deficits at 72 h after stroke compared with saline or tPA alone groups in the eMCAO model. Importantly, the combination treatment significantly reduced delayed tPA-associated brain hemorrhage and secondary microvascular thrombosis. These protective effects were associated with J147-mediated inhibition of matrix metalloproteinase-9 (MMP9), 15-lipoxygenase-1, and plasminogen activator inhibitor (PAI) expression in the ischemic hemispheres (predominantly in ischemic cerebral endothelium). Moreover, the combination treatment significantly reduced circulating platelet activation and platelet-leukocyte aggregation compared with saline or tPA alone groups at 24 h after stroke, which might also contribute to reduced microvascular thrombosis and neuroinflammation (as demonstrated by reduced neutrophil brain infiltration and microglial activation).ConclusionOur results demonstrate that J147 treatment alone exerts cerebral cytoprotective effects in a suture model of acute ischemic stroke, while in an embolic stroke model co-administration of J147 with tPA reduces delayed tPA-induced intracerebral hemorrhage and confers cerebroprotection. These findings suggest that J147-tPA combination therapy could be a promising approach to improving the treatment of ischemic stroke.

167 Reversible VWF Inhibitor Lyses MCA Occlusion 6 Hours after Large Vessel Occlusion Stroke

INTRODUCTION: Acute ischemic stroke (AIS) is the leading cause of combined morbidity and mortality worldwide. Recombinant tissue plasminogen activator (rtPA) represents the only approved pharmacological treatment for AIS but is limited to treating those within 4.5 hours of stroke onset because of the risk of intracranial hemorrhage. Moreover, it is ineffective in treating large vessel occlusion (LVO) stroke. For this population, endovascular mechanical thrombectomy (MT) effectively recanalizes the occlusion but is limited to highly-specialized hospitals, leaving the vast majority without timely acute treatment. METHODS: Utilizing a canine embolic middle cerebral artery occlusion (eMCAO) model of LVO stroke, we assessed DTRI-031 on vessel recanalization after 6 hours of LVO stroke by digital subtraction angiography (DSA). We also measured the effect of recanalization on infarct volume (efficacy) and hemorrhage (safety) using magnetic resonance imaging (MRI). Finally, we assessed the ability of DTRI-025, an oligonucleotide that binds DTRI-031 to reverse DTRI-031 activity. RESULTS: DTRI-031 administration after 6 hours of LVO stroke resulted in >TICI 2A in 62.5% and >TICI 2B in 50% of canines (n = 8). Negative control group demonstrated no revascularization (n = 7). Recanalization resulted in reduced infarct volume compared to negative control (p < 0.05, unpaired t-test). None of the animals that received DTRI-031 had intracranial hemorrhage on MRI. Finally, DTRI-025 completely reversed the activity of DTRI-031 within 5 minutes of administration in both whole blood impedance aggregometry (WBA) and PFA-100. CONCLUSION: DTRI-031 effectively recanalizes LVO after 6 hours of stroke onset with reduced infarct volume and no incidence of hemorrhage. DTRI-025 rapidly reverses DTRI-031. This drug/reversal agent combination represents a robust yet safe approach to treat AIS.

Abstract 45: Vwf Inhibitor Lyses Middle Cerebral Artery Occlusion After 6 Hours Of Large Vessel Occlusion Stroke

Introduction: Acute ischemic stroke (AIS) is the leading cause of combined morbidity and mortality worldwide. Recombinant tissue plasminogen activator (rtPA) is the only FDA-approved drug to treat AIS, however, it is limited to treating patients within 4.5 hours of stroke onset because of the risk of intracranial hemorrhage. This limits rtPA administration to ~6% of patients. Moreover, it poorly lyses large vessel occlusion (LVO) stroke. Endovascular mechanical thrombectomy (MT) effectively recanalizes LVO but is limited to highly specialized hospitals, leaving the majority without acute therapy. Hypothesis: We hypothesize that targeted von Willebrand Factor (VWF) inhibition by DTRI-031 will recanalize arterial thrombosis in a canine model of LVO stroke, and DTRI-031 activity will be rapidly reversed by DTRI-025, which is specifically designed to inhibit DTRI-031. Methods: Utilizing a canine embolic middle cerebral artery occlusion (eMCAO) model of LVO stroke, we assessed DTRI-031 on vessel recanalization after 6 hours of LVO stroke by digital subtraction angiography (DSA). We also measured the effect of recanalization on infarct volume (efficacy) and hemorrhage (safety) using magnetic resonance imaging (MRI). Finally, we assessed the ability of DTRI-025, an oligonucleotide that binds DTRI-031 to reverse DTRI-031 activity. Results: DTRI-031 administration after 6 hours of LVO stroke resulted in ≥TICI 2A in 62.5% and ≥TICI 2B in 50% of canines (n=8). The negative control group demonstrated no revascularization (n=7). Recanalization resulted in reduced infarct volume compared to the negative control (p<0.05, unpaired t-test). None of the animals that received DTRI-031 had an intracranial hemorrhage on MRI. Finally, DTRI-025 completely reversed the activity of DTRI-031 within 5 minutes of administration in both whole blood impedance aggregometry (WBA) and PFA-100. There was no difference in heart rate, blood pressure, or temperature among the 3 groups studied. Conclusion: DTRI-031 effectively recanalizes LVO after 6 hours of stroke onset with reduced infarct volume and no incidence of hemorrhage. DTRI-025 rapidly reverses DTRI-031. This drug/reversal agent combination represents a robust yet safe approach to treat AIS.

Abstract WMP9: Pharmacological Characterization Of The Water Soluble Neurosteroid NTS-104 In The Rat Embolic Stroke Model

Background: Our objective was to characterize the pharmacodynamic changes induced by NTS-104/105 within the brain and plasma in rats following embolic middle cerebral artery occlusion (eMCAO). Previous pharmacokinetic analysis indicated that NTS-104 is rapidly metabolized in the blood to form the active compound NTS-105, which is readily detected in the brain. In contrast, NTS-104 does not cross the blood-brain barrier. Methods: Adult, male Wistar rats were subjected to eMCAO. Stroke severity was defined by Neurological Severity Score (NSS) assessment. Only rats that scored >8 on a 13-point scale were included. Rats were dosed IM with vehicle, or NTS-104 (5mg/kg or 20mg/kg) at 6 hours after stroke. Brain and plasma samples rats were collected at 6, 12, or 24hrs after injection. Samples were screened for expression of AKT, pAKT and against a panel of 67 cytokines by quantitative ELISA. Results: We found that NTS-104 treatment induced a significant reduction in IL-6, GM-CSF, MCP-1, IL-1α, RAGE, VEGF, and ICAM-1 levels within the brains of rats following intramuscular injection of 20 mg/kg NTS-104 at 6-hours after eMCAO compared to vehicle treated control rats. In contrast, treatment with 5mg/kg NTS-104 did not induce similar time and dose dependent changes in these specific cytokines relative to vehicle treated controls. NTS-104 treatment also significantly reduced IL-6 and GM-CSF levels, while increasing galectin-1 levels, within the plasma of these same rats in a time and dose dependent manner similar to what was observed within the brain. We further detected a significant increase in the levels of phosphorylated AKT (pAKT) within the brains of stroked rats treated with 20 mg/kg NTS-104 but not with 5 mg/kg. Conclusion: These data demonstrate that treatment with NTS-104 results in the significant activation of the AKT cell survival signaling pathway and significantly reduces critical neuroinflammatory signaling after stroke in a time and dose dependent manner.

The Quinazoline Otaplimastat (SP-8203) Reduces the Hemorrhagic Transformation and Mortality Aggravated after Delayed rtPA-Induced Thrombolysis in Cerebral Ischemia.

Acute ischemic stroke is the leading cause of morbidity and mortality worldwide. Recombinant tissue plasminogen activator (rtPA) is the only agent clinically approved by FDA for patients with acute ischemic stroke. However, delayed treatment of rtPA (e.g., more than 3 h after stroke onset) exacerbates ischemic brain damage by causing intracerebral hemorrhage and increasing neurotoxicity. In the present study, we investigated whether the neuroprotant otaplimastat reduced delayed rtPA treatment-evoked neurotoxicity in male Sprague Dawley rats subjected to embolic middle cerebral artery occlusion (eMCAO). Otaplimastat reduced cerebral infarct size and edema and improved neurobehavioral deficits. In particular, otaplimastat markedly reduced intracerebral hemorrhagic transformation and mortality triggered by delayed rtPA treatment, consequently extending the therapeutic time window of rtPA. We further found that ischemia-evoked extracellular matrix metalloproteases (MMPs) expression was closely correlated with cerebral hemorrhagic transformation and brain damage. In ischemic conditions, delayed rtPA treatment further increased brain injury via synergistic expression of MMPs in vascular endothelial cells. In oxygen-glucose-deprived endothelial cells, otaplimastat suppressed the activity rather than protein expression of MMPs by restoring the level of tissue inhibitor of metalloproteinase (TIMP) suppressed in ischemia, and consequently reduced vascular permeation. This paper shows that otaplimastat under clinical trials is a new drug which can inhibit stroke on its own and extend the therapeutic time window of rtPA, especially when administered in combination with rtPA.

Acute Hyperglycemia Exacerbates Hemorrhagic Transformation after Embolic Stroke and Reperfusion with tPA: A Possible Role of TXNIP-NLRP3 Inflammasome

ObjectivesAcute hyperglycemia (HG) exacerbates reperfusion injury after stroke. Our recent studies showed that acute HG upregulates thioredoxin-interacting protein (TXNIP) expression, which in turn induces inflammation and neurovascular damage in a suture model of ischemic stroke. The aim of the present study was to investigate the effect of acute HG on TXNIP-associated neurovascular damage, in a more clinically relevant murine model of embolic stroke and intravenous tissue plasminogen activator (IV-tPA) reperfusion. Materials and methodsHG was induced in adult male mice, by intraperitoneal injection of 20% glucose. This was followed by embolic middle cerebral artery occlusion (eMCAO), with or without IV-tPA (10 mg/kg) given 3 h post embolization. Brain infarction, edema, hemoglobin content, expression of matrix metalloproteinase (MMP-9), vascular endothelial growth factor A (VEGFA), tight junction proteins (claudin-5, occluding, and zonula occludens-1), TXNIP, and NOD‐like receptor protein3 (NLRP3)-inflammasome activation were evaluated at 24 h after eMCAO. ResultsHG alone significantly increased TXNIP in the brain after eMCAO, and this was associated with exacerbated hemorrhagic transformation (HT; as measured by hemoglobin content). IV-tPA in HG conditions showed a trend to decrease infarct volume, but worsened HT after eMCAO, suggesting that HG reduces the therapeutic efficacy of IV-tPA. Further, HG and tPA-reperfusion did not show significant differences in expression of MMP-9, VEGFA, junction proteins, and NLRP3 inflammasome activation between the groups. ConclusionThe current findings suggest a potential role for TXNIP in the occurrence of HT in hyperglycemic conditions following eMCAO. Further studies are needed to understand the precise role of vascular TXNIP on HG/tPA-induced neurovascular damage after stroke.

Endothelial Thioredoxin-Interacting Protein Depletion Reduces Hemorrhagic Transformation in Hyperglycemic Mice after Embolic Stroke and Thrombolytic Therapy.

We hypothesize that endothelial-specific thioredoxin-interacting protein knock-out (EC-TXNIP KO) mice will be more resistant to the neurovascular damage (hemorrhagic-transformation-HT) associated with hyperglycemia (HG) in embolic stroke. Adult-male EC-TXNIP KO and wild-type (WT) littermate mice were injected with-streptozotocin (40 mg/kg, i.p.) for five consecutive days to induce diabetes. Four-weeks after confirming HG, mice were subjected to embolic middle cerebral artery occlusion (eMCAO) followed by tissue plasminogen activator (tPA)-reperfusion (10 mg/kg at 3 h post-eMCAO). After the neurological assessment, animals were sacrificed at 24 h for neurovascular stroke outcomes. There were no differences in cerebrovascular anatomy between the strains. Infarct size, edema, and HT as indicated by hemoglobin (Hb)-the content was significantly higher in HG-WT mice, with or without tPA-reperfusion, compared to normoglycemic WT mice. Hyperglycemic EC-TXNIP KO mice treated with tPA tended to show lower Hb-content, edema, infarct area, and less hemorrhagic score compared to WT hyperglycemic mice. EC-TXNIP KO mice showed decreased expression of inflammatory mediators, apoptosis-associated proteins, and nitrotyrosine levels. Further, vascular endothelial growth factor-A and matrix-metalloproteinases (MMP-9/MMP-3), which degrade junction proteins and increase blood-brain-barrier permeability, were decreased in EC-TXNIP KO mice. Together, these findings suggest that vascular-TXNIP could be a novel therapeutic target for neurovascular damage after stroke.

USPIO-SWI Shows Fingolimod Enhanced Alteplase Action on Angiographic Reperfusion in eMCAO Rats.

Noninvasive evaluation of the status of cerebral arteriole perfusion remains a practical challenge in murine stroke models, because conventional magnetic resonance imaging (MRI) is no longer capable of capturing these very small vessels. To investigate the feasibility of ultrasmall superparamagnetic iron oxide particles (USPIO)-based susceptibility weighted imaging (SWI)-MRI (USPIO-SWI) and T2* map-MRI (USPIO-T2* map) for monitoring angiographic perfusion in stroke rats. A preclinical randomized controlled trial. Normal rats (N= 9), embolic middle cerebral artery occlusion (eMCAO) rats (N= 66). 7 T; T2* map (multigradient echo), SWI (3D gradient echo). Experiment 1: To develop a method for angiographic reperfusion evaluation with USPIO-SWI. Normal rats were used to optimize the USPIO dosage (5.6, 16.8, and 56 mg/kg ferumoxytol) as well as scan time points for cerebral arterioles. Contrast-to-noise ratio (CNR) was measured. Stroke rats were further used and the number of visual cortical vessels were counted. Experiment 2: To examine whether fingolimod (lymphocytes inhibitor) enhances the action of tissue plasminogen activator (tPA) in eMCAO rats on cerebral angiographic reperfusion. Mann-Whitney test and two way-ANOVA were used. P< 0.05 was considered statistically significant. CNR values of cerebral cortical penetrating arteries in normal rats were significantly increased to 4.4 ± 0.5 (5.6 mg/kg), 6.1 ± 0.5 (16.8 mg/kg), and 3.4 ± 0.9 (56 mg/kg) after USPIO injection. The number of visual cortical vessels on USPIO-SWI images in ischemic regions was significantly less than in control regions (5 ± 2 vs. 56 ± 20) of eMCAO rats. Compared with eMCAO rats who received tPA only, eMCAO rats who received the combination of fingolimod and tPA exhibited significantly higher proportion of complete angiographic reperfusion (69% vs. 17%). This study supports the feasibility of angiographic perfusion evaluation with USPIO-SWI in stroke rats. 2 TECHNICAL EFFICACY STAGE: 1.

Pseudoginsenoside-F11 ameliorates thromboembolic stroke injury in rats by reducing thromboinflammation

Pseudoginsenoside-F11 (PF11), an ocotillol-type ginsenoside, has been reported to exert neuroprotective effects on ischemic stroke induced by permanent and transient middle cerebral artery occlusion in experimental animals. The aim of the present study was to investigate the effect of PF11 on thromboembolic stroke in rats and its possible mechanisms on thromboinflammation. PF11 (4, 12, 36 mg/kg) was injected intravenously (i.v.) once a day for 3 consecutive days to male Wistar rats followed by embolic middle cerebral artery occlusion (eMCAO). The results showed that PF11 significantly reduced the cerebral infarction volume, brain edema and neurological deficits induced by eMCAO. Meanwhile, the thromboinflammation in the ischemic hemisphere was observed at 24 h after eMCAO, as indicated by the increased number of microvascular thrombus and inflammatory response. Moreover, eMCAO resulted in the up-regulation of platelet glycoprotein Ibα (GPIbα) and VI (GPVI), as well as the activation of contact-kinin pathway. Notably, PF11 significantly reversed all these changes. Furthermore, PF11 prevented the eMCAO-induced loss of tight junction proteins and up-regulation of matrix metalloproteinase-9 (MMP-9), thus leading to the alleviation of blood-brain barrier (BBB) damage. In conclusion, the present study revealed that thromboinflammation was induced in the ischemic hemisphere of rats after eMCAO and PF11 exerted marked protective effects against thromboembolic stroke by attenuating thromboinflammation and preventing BBB damage. This research further identifies the potential therapeutic role of PF11 for ischemic stroke.

Abstract P487: Cerebral Endothelial Cell-Derived Small Extracellular Vesicles Enhance Neurovascular Function and Neurological Recovery in Rat Ischemic Stroke

Background: Treatment of patients with cerebral large vessel occlusion with thrombectomy and tissue plasminogen activator (tPA) often leads to incomplete reperfusion. There is a compelling need to develop therapies to enhance the perfusion and to improve neurological outcomes. Methods: Using rat models of embolic middle cerebral artery occlusion (eMCAO) and transient MCAO (tMCAO), we investigated the effect of small extracellular vesicles (sEVs) derived from healthy rat cerebral endothelial cells (CECs) administered intravenously in combination with tPA (CECs/tPA) and as an acute intraarterial adjunct therapy with mechanical reperfusion on stroke outcomes. Recanalization, cerebral blood flow (CBF), and blood-brain barrier (BBB) permeability were analyzed. MicroRNAs (miRs) and proteins were analyzed in CECs harvested from ischemic rats by RT-PCR and Western blots. Transmission electron microscopy was employed to analyze the brain distribution of CEC-sEVs. The effect of sEVs derived from clots acquired from patients undergone thrombectomy was tested on human CEC permeability. Results: CEC-sEVs/tPA given 4h after eMCAO or CEC-sEVs given upon reperfusion after 2h tMCAO significantly reduced infarct volume by ~36% and ~43%, respectively, and robustly improved neurological outcomes compared with tPA or ischemia/reperfusion alone (n=10 rats/group). CEC-sEVs/tPA and upon reperfusion after eMCAO or tMCAO, resepectively, significantly increased recanalization of the occluded MCA, enhanced CBF and reduced BBB leakage. CEC-sEVs/tPA substantially reduced a network of microRNAs and proteins that mediate thrombosis, coagulation and inflammation in CECs. Moreover, CEC-sEVs intravenously administered crossed the BBB and were internalized by CECs cells, astrocytes, and neurons. Stroke patient-clot derived exosomes impaired human CEC permeability and upregulated pro-inflammatory and -coagulatant proteins, which were blocked by CEC-sEVs. Conclusion: CEC-sEVs have a therapeutic effect on acute ischemic stroke in rats by reducing neurovascular damage. Suppressing the network of pro-thrombotic, -coagulant and -inflammatory microRNAs and proteins in CECs by CEC-sEVs likely contributes to the therapeutic effect of CEC-sEVs.

Effects of ML351 and tissue plasminogen activator combination therapy in a rat model of focal embolic stroke.

Thrombolytic stroke therapy with tissue plasminogen activator (tPA) is limited by risks of hemorrhagic transformation (HT). We have reported that a new 12/15-lipoxygenase (12/15-LOX) inhibitor ML351 reduced tPA related HT in mice subjected to experimental stroke under anticoagulation. In this study, we asked whether ML351 can ameliorate tPA induced HT in an embolic stroke model. Rats were subjected to embolic middle cerebral artery occlusion with 2 or 3hr ischemia and tPA infusion, with or without ML351. Regional cerebral blood flow was monitored 2hr after ischemia and continuously monitored for 1hr after treatment for determining reperfusion. Hemoglobin was determined in brain homogenates and infarct volume was quantified at 24hr after stroke.12/15-LOX, cluster of differentiation 68(CD68), immunoglobulin G (IgG), and tight junction proteins expression was detected by immunohistochemistry. ML351 significantly reduced tPA related hemorrhage after stroke without affecting its thrombolytic efficacy. ML351 also reduced blood-brain barrier disruption and improved preservation of junction proteins. ML351 and tPA combination improved neurological deficit of rats even though ML351 did not further reduce the infarct volume compared to tPA alone treated animals. Pro-inflammatory cytokines were suppressed by ML351 both in vivo and in vitro experiments. We further showed that ML351 suppressed the expression of c-Jun-N-terminal kinase (JNK) in brains and microglia cultures, whereas exogenous 12-HETE attenuated this effect in vitro. In conclusion, ML351 and tPA combination therapy is beneficial in ameliorating HT after ischemic stroke. This protective effect is probably because of 12/15-LOX inhibition and suppression of JNK-mediated microglia/macrophage activation.

TPA Mobilizes Immune Cells That Exacerbate Hemorrhagic Transformation in Stroke.

Hemorrhagic complications represent a major limitation of intravenous thrombolysis using tPA (tissue-type plasminogen activator) in patients with ischemic stroke. The expression of tPA receptors on immune cells raises the question of what effects tPA exerts on these cells and whether these effects contribute to thrombolysis-related hemorrhagic transformation. We aim to determine the impact of tPA on immune cells and investigate the association between observed immune alteration with hemorrhagic transformation in ischemic stroke patients and in a rat model of embolic stroke. Paired blood samples were collected before and 1 hour after tPA infusion from 71 patients with ischemic stroke. Control blood samples were collected from 27 ischemic stroke patients without tPA treatment. A rat embolic middle cerebral artery occlusion model was adopted to investigate the underlying mechanisms of hemorrhagic transformation. We report that tPA induces a swift surge of circulating neutrophils and T cells with profoundly altered molecular features in ischemic stroke patients and a rat model of focal embolic stroke. tPA exacerbates endothelial injury, increases adhesion and migration of neutrophils and T cells, which are associated with brain hemorrhage in rats subjected to embolic stroke. Genetic ablation of annexin A2 in neutrophils and T cells diminishes the effect of tPA on these cells. Decoupling the interaction between mobilized neutrophils/T cells and the neurovascular unit, achieved via a S1PR (sphingosine-1-phosphate receptor) 1 modulator RP101075 and a CCL2 (C-C motif chemokine ligand 2) synthesis inhibitor bindarit, which block lymphocyte egress and myeloid cell recruitment, respectively, attenuates hemorrhagic transformation and improves neurological function after tPA thrombolysis. Our findings suggest that immune invasion of the neurovascular unit represents a previously unrecognized mechanism underlying tPA-mediated brain hemorrhage, which can be overcome by precise immune modulation during thrombolytic therapy.

Delayed (21 Days) Post Stroke Treatment With RPh201, a Botany-Derived Compound, Improves Neurological Functional Recovery in a Rat Model of Embolic Stroke

BackgroundDespite the recent advances in the acute stroke care, treatment options for long-term disability are limited. RPh201 is a botany-derived bioactive compound that has been shown to exert beneficial effects in various experimental models of neural injury. The present study evaluated the effect of delayed RPh201 treatment on long term functional recovery after stroke.MethodsAdult male Wistar rats subjected to embolic middle cerebral artery occlusion (MCAO) were randomized into the following experimental groups (n = 20/group): (1) RPh201 treatment, and (2) Vehicle (cottonseed oil). RPh201 (20 μl) or Vehicle were subcutaneously administered twice a week for 16 consecutive weeks starting at 21 days after MCAO. An array of behavioral tests was performed up to120 days after MCAO.ResultsIschemic rats treated with RPh201 exhibited significant (p < 0.05) improvement of neurological function measured by adhesive removal test, foot-fault test, and modified neurological severity score at 90 and 120 days after MCAO. Immunohistochemistry analysis showed that RPh201 treatment robustly increased neurofilament heavy chain positive axons and myelin basic protein densities in the peri-infarct area by 61% and 31%, respectively, when compared to the Vehicle treatment, which were further confirmed by Western blot analysis. The RPh201 treatment did not reduce infarct volume.ConclusionOur data demonstrated that RPh201 has a therapeutic effect on improvement of functional recovery in male ischemic rats even when the treatment was initiated 21 days post stroke. Enhanced axonal and myelination densities by RPh201 in ischemic brain may contribute to improved stroke recovery.

Combined Treatment With 2-(2-Benzofu-Ranyl)-2-Imidazoline and Recombinant Tissue Plasminogen Activator Protects Blood-Brain Barrier Integrity in a Rat Model of Embolic Middle Cerebral Artery Occlusion.

Recombinant tissue plasminogen activator (rt-PA) is used to treat acute ischemic stroke but is only effective if administered within 4.5 h after stroke onset. Delayed rt-PA treatment causes blood-brain barrier (BBB) disruption and hemorrhagic transformation. The compound 2-(-2-benzofuranyl)-2-imidazoline (2-BFI), a newly discovered antagonist of high-affinity postsynaptic N-methyl-D-aspartate (NMDA) receptors, has been shown to have neuroprotective effects in ischemia. Here, we investigated whether combining 2-BFI and rt-PA can ameliorate BBB disruption and prolong the therapeutic window in a rat model of embolic middle cerebral artery occlusion (eMCAO). Ischemia was induced in male Sprague Dawley rats by eMCAO, after which they were treated with 2-BFI (3 mg/kg) at 0.5 h in combination with rt-PA (10 mg/kg) at 6 or 8 h. Control rats were treated with saline or 2-BFI or rt-PA. Combined therapy with 2-BFI and rt-PA (6 h) reduced the infarct volume, denatured cell index, BBB permeability, and brain edema. This was associated with increased expression of aquaporin 4 (AQP4) and tight junction proteins (occludin and ZO-1) and downregulation of intercellular adhesion molecule 1 (ICAM-1) and matrix metalloproteinases 2 and 9 (MMP2 and MMP9). We conclude that 2-BFI protects the BBB from damage caused by delayed rt-PA treatment in ischemia. 2-BFI may therefore extend the therapeutic window up to 6 h after stroke onset in rats and may be a promising therapeutic strategy for humans. However, mechanisms to explain the effects oberved in the present study are not yet elucidated.