Thromboembolic Stroke Model Research Articles

Abstract 144: A Novel Multi-function Small Molecule for the Treatment of Acute Ischemic Stroke in Rodent and Non-human Primate

Background and rationale: DC007 is a synthetic small molecule with multiple pharmacological activities. It has been shown to have thrombolytic, anti-platelet and free radical scavenging activities in vitro. To explore its potential in treating acute ischemic stroke, we evaluated its safety and efficacy using a thromboembolic stroke model in rattus and in Cynomolgus macaque. Methods: Effects of DC007 (10mg/kg) were compared to intravenous tPA (10mg/kg in rat; 0.9mg/kg in monkey) using a model of focal embolic cerebral ischemia in rats and monkeys. Pre-formed blood clots were introduced into middle cerebral artery through catheter cannulated into internal carotid artery of the animals. Animals (12 rats per group; 4 monkeys per group) were randomized to receive intravenous infusion of saline, tPA or DC007 at 3 hours after embolization was confirmed. Results: DC007 reduced brain infarction and hemispheric swelling in the stroke rats more significantly than saline or tPA did, when treatments were given at 3 hours after stroke onset. At 24 hours after stroke, infraction volumes were 49.36%, 31.98%, and 40.66% for saline, DC007 and tPA group, respectively; while brain swelling rates were 17.67%, 13.20% and 22.58% for saline, DC007 and tPA group, respectively. In the thromboembolic stroke model in monkey, DC007 also showed a stronger beneficial effect on vessel patency (DC007: 46.3±10.3% vs. tPA: 22.2±12.8% in the 8 hours time period after stroke), brain infarction (DC007: 3361±354 mm 3 vs. tPA: 9072±2096 mm 3 measured by MRI at 24 hours after stroke) and neurobehavioral score measured at 72 hours after stroke (DC007: 46.3±10.3 vs. tPA: 60±9.8, where score 0 indicated normal, while score 77 indicated death). Conclusions: DC007 appears to be a more potent thrombolytic compound with superior safety profile than IV rt-PA when given at 3 hours after the stroke onset in rats and monkeys. Further investigation is warranted for its potential in the treatment of AIS clinically.

Abstract WP296: Short Term Acute Preconditioning Exercise Improves Stroke Outcomes and Reduces Neurovascular Injury via Increased Endothelial Nitric Oxide Synthase Activity

Physical exercise is becoming a prominent therapeutic strategy to improve stroke outcomes. It was previously shown that long term exercise reduces infarct volume, but this was never tested in acute short term preconditioning exercise in a thromboembolic model of stroke. Exercise induced shear stress improves vascular function through increased activity of endothelial nitric oxide synthase (eNOS) and its upstream principal activator, AMP-activated protein kinase (AMPK). However, this was not previously tested in a stroke setting. Accordingly, we tested the hypothesis that acute short term preconditioning exercise improves stroke outcomes through increased AMPK and eNOS activity. Methods: Male Wistar rats (300g) were subjected to treadmill exercise for four days (25 minutes/day), break for 2 days and then one acute bout for 30 minutes. Exercised animals were subjected to thromboembolic stroke at 1h, 6h or 24h after the last exercise session. At 24h, control (sedentary) and exercised rats were tested for neurological outcomes, infarct size and edema. Western Blotting was used to measure the expression of active eNOS (p-S1177-eNOS) and active AMPK (p-T172-AMPK). Results: Acute exercise significantly reduced infarct size and edema and improved functional outcomes compared to control. It also significantly increased the expression of peNOS and pAMPK in the brain, cerebral vessels and aorta (Table). Conclusion: Acute exercise preconditioning significantly reduced neurovascular injury and improved functional outcome after stroke through increased eNOS activity. Our findings are novel to point out the role of preconditioning exercise induced AMPK and eNOS activation in improving stroke outcomes.

Abstract WMP80: Micro RNA-Therapy in Murine Thromboembolic Stroke

Background: Micro RNAs (miRNAs) could target multiple mRNAs, repressing the protein translation. We report acute changes in humoral miRNAome in a murine thromboembolic stroke model (eMCAo), and demonstrate the benefits of miRNA therapy in improving cerebral blood flow (CBF). Methods: Non-biased micro RNA (miRNA) array and bioinformatics analysis was performed in plasma collected at 4h post-eMCAo from male mice (C57/B6, 16-weeks). Individual PCR for miRNAs was also performed in brain tissues at 24h post-eMCAo. Moreover, frozen human plasma samples collected at ~4.5h post-stroke were also used for miRNA analysis. Finally, the miRNA mimic that was predicted to target genes of our interest was also tested in vivo and in vitro . Results: Principal component analysis (PCA) of the miRNA-array showed ~68% variance in the humoral miRNAome 4h after eMCAo in mice, and a significant change in Stroke vs. Sham groups (Cut off value >2 fold; p<0.05). Of interest, the hairpin precursor of miR-449b was downregulated (~2.35 fold, p<0.05) at 4h post-eMCAo, while the mature miR-449b was also significantly reduced at 24h post-eMCAo. Mature miR-449b was significantly reduced in human stroke plasma, too. In human brain endothelial cells, miR-449b mimic downregulated gene expressions of both plasminogen activator inhibitor (PAI-1) and alpha 2- antiplasmin (α-AP) only in hypoxia but not during normoxia. Therefore, we finally tested the cholesterol-conjugated miR-449b mimic in the murine eMCAo model. Pre-treatment with miR-449b mimic (8 mg/kg bwt) increased the absolute CBF and reduced edema (as determined by MRI), and also improved the neurological outcomes and reduced % infarct volume (p<0.05). Results: The miR-449b mimic could be a possible therapy to suppress aberrant gene expressions of PAI-1 and α-AP, which will allow more spontaneous reperfusion and benefits from low dose tPA.

Up-regulation of neurofilament light chains is associated with diminished immunoreactivities for MAP2 and tau after ischemic stroke in rodents and in a human case

As stroke therapies are still limited to a minority of patients, efforts have been intensified to an improved understanding of pathophysiological processes during ischemia formation, potentially allowing the development of specific therapeutic interventions. In this context, cytoskeletal elements became evident as key players during the transition process towards long-lasting tissue damage. This study focused on ischemia-related alterations of the cytoskeleton with a special focus on microtubule-associated proteins and neurofilament light chains (NF-L). Immunohistochemical analyses were applied to brain sections of mice and rats after experimental stroke and to autoptic samples from a stroke patient. To consider translational aspects, a thromboembolic model of stroke in rats, closely mimicking the human situation, was used in addition to the filament-based model of focal cerebral ischemia in mice. One day after ischemia onset, immunoreactivity of microtubule-associated protein tau and microtubule-associated protein-2 (MAP2) was reduced in ischemic areas. These findings were consistently present in the ischemia-affected striatum and the neocortex. In a quite opposite fashion, ischemic areas displayed NF-L-immunoreactivity in neuropathologically altered fibers, local agglomerations probably related to degraded cell bodies and neocortical pyramidal cells. Notably, up-regulation of NF-L was also confirmed in infarcted tissue from a human brain sample. Furthermore, analyses of rodent brain tissue revealed corkscrew curl-like fibers as a special feature of MAP2 in the ischemia-affected hippocampus. In conclusion, this study provides evidence for an opposite reaction of microtubule-associated proteins and neurofilaments after focal cerebral ischemia. Accordingly, cytoskeletal elements appear as a promising target for stroke treatment.

Plasminogen and stroke: more is better

Plasminogen and stroke: more is better

Endovascular ischemic stroke models of adult rhesus monkeys: a comparison of two endovascular methods.

To further investigate and improve upon current stroke models in nonhuman primates, infarct size, neurologic function and survival were evaluated in two endovascular ischemic models in sixteen rhesus monkeys. The first method utilized a micro-catheter or an inflatable balloon to occlude the M1 segment in six monkeys. In the second model, an autologous clot was injected via a micro-catheter into the M1 segment in ten monkeys. MRI scanning was performed on all monkeys both at baseline and 3 hours after the onset of ischemia. Spetzler neurologic functions were assessed post-operatively, and selective perfusion deficits were confirmed by DSA and MRI in all monkeys. Animals undergoing micro-catheter or balloon occlusion demonstrated more profound hemiparesis, larger infarct sizes, lower Spetzler neurologic scores and increased mortality compared to the thrombus occlusion group. In animals injected with the clot, there was no evidence of dissolution, and the thrombus was either near the injection site (M1) or flushed into the superior division of the MCA (M2). All animals survived the M2 occlusion. M1 occlusion with thrombus generated 50% mortality. This study highlighted clinically important differences in these two models, providing a platform for further study of a translational thromboembolic model of acute ischemic stroke.

Modulation by the Noble Gas Helium of Tissue Plasminogen Activator: Effects in a Rat Model of Thromboembolic Stroke.

Helium has been shown to provide neuroprotection in mechanical model of acute ischemic stroke by inducing hypothermia, a condition shown by itself to reduce the thrombolytic and proteolytic properties of tissue plasminogen activator. However, whether or not helium interacts with the thrombolytic drug tissue plasminogen activator, the only approved therapy of acute ischemic stroke still remains unknown. This point is not trivial since previous data have shown the critical importance of the time at which the neuroprotective noble gases xenon and argon should be administered, during or after ischemia, in order not to block tissue plasminogen activator-induced thrombolysis and to obtain neuroprotection and inhibition of tissue plasminogen activator-induced brain hemorrhages. We show that helium of 25-75 vol% inhibits in a concentration-dependent fashion the catalytic and thrombolytic activity of tissue plasminogen activator in vitro and ex vivo. In vivo, in rats subjected to thromboembolic brain ischemia, we found that intraischemic helium at 75 vol% inhibits tissue plasminogen activator-induced thrombolysis and subsequent reduction of ischemic brain damage and that postischemic helium at 75 vol% reduces ischemic brain damage and brain hemorrhages. In a clinical perspective for the treatment of acute ischemic stroke, these data suggest that helium 1) should not be administered before or together with tissue plasminogen activator therapy due to the risk of inhibiting the benefit of tissue plasminogen activator-induced thrombolysis; and 2) could be an efficient neuroprotective agent if given after tissue plasminogen activator-induced reperfusion.

Therapeutic Effect of Ligustilide-Stimulated Adipose-Derived Stem Cells in a Mouse Thromboembolic Stroke Model.

Stroke is a result of cerebral ischemia that triggers a cascade of both physiological and biochemical events. No effective treatment is available for stroke; however, stem cells have the potential to rescue tissue from the effects of stroke. Adipose-derived stem cells (ADSCs) are an abundant source of adult stem cells; therefore, ADSC therapy can be considered as a future strategy for regenerative medicine. However, more research is required to improve the effectiveness of transplanted ADSCs as a treatment for stroke in the mouse stroke model. Ligustilide, isolated from the herb Angelica sinensis, exhibits a protective effect on neurons and inhibits inflammation. We also demonstrated that ligustilide treatment increases the expression levels of homing factors such as SDF-1 and CXCR4. In the present study, we evaluated the therapeutic effects of ADSC transplantation and ligustilide treatment in a mouse thromboembolic stroke model by behavioral tests, including beam walking, locomotor activity, and rotarod analysis. ADSCs pretreated with ligustilide were transplanted into the brains of stroke mice. The results showed that the therapeutic effect of ADSCs pretreated with ligustilide was better than that of ADSCs without ligustilide pretreatment. There was no difference between the recovery of mice treated by ADSC transplantation combined with subcutaneous ligustilide injection and that of mice treated only with ADSCs. The TUNEL assay showed fewer apoptotic cells in the brains of mice transplanted with ADSCs pretreated with ligustilide as well as in those without pretreatment. In summary, pretreatment of ADSCs with ligustilide improves the therapeutic efficacy of ADSC transplantation. The results of this study will help improve stem cell therapies being developed for future clinical applications.

Efficacy of Alteplase in a Mouse Model of Acute Ischemic Stroke: A Retrospective Pooled Analysis.

The debate over the fact that experimental drugs proposed for the treatment of stroke fail in the translation to the clinical situation has attracted considerable attention in the literature. In this context, we present a retrospective pooled analysis of a large data set from preclinical studies, to examine the effects of early versus late administration of intravenous recombinant tissue-type plasminogen activator. We collected data from 26 individual studies from 9 international centers (13 researchers; 716 animals) that compared recombinant tissue-type plasminogen activator with controls, in a unique mouse model of thromboembolic stroke induced by an in situ injection of thrombin into the middle cerebral artery. Studies were classified into early (<3 hours) versus late (≥3 hours) drug administration. Final infarct volumes, assessed by histology or magnetic resonance imaging, were compared in each study, and the absolute differences were pooled in a random-effect meta-analysis. The influence of time of administration was tested. When compared with saline controls, early recombinant tissue-type plasminogen activator administration was associated with a significant benefit (absolute difference, -6.63 mm(3); 95% confidence interval, -9.08 to -4.17; I(2)=76%), whereas late recombinant tissue-type plasminogen activator treatment showed a deleterious effect (+5.06 mm(3); 95% confidence interval, +2.78 to +7.34; I(2)=42%; Pint<0.00001). Results remained unchanged after subgroup analyses. Our results provide the basis needed for the design of future preclinical studies on recanalization therapies using this model of thromboembolic stroke in mice. The power analysis reveals that a multicenter trial would require 123 animals per group instead of 40 for a single-center trial.

Kelussia odoratissima Mozaff attenuates thromboembolic brain injury, possibly due to its Z-ligustilide content

Primary objective: Essential oil (EO) of Kelussia odoratissima Mozaff, whose main composition is Z-ligustilide, has been shown to have strong antioxidant and anti-inflammatory effects and potent neuroprotective properties.Research design: This study examined whether or not the EO could ameliorate brain damage and behavioural dysfunction in a thromboembolic model of stroke in rats and compare its effects to that of the purified Z-ligustilide.Methods and procedures: Stroke was induced in rats by middle cerebral artery occlusion using an autologous pre-formed clot. EO (10 mg kg–1 and 45 mg kg–1) and Z-ligustilide (20 mg kg–1) were injected intraperitoneally 1 h prior to embolization. Behavioural scores, infarct size and brain oedema, as well as the level of tumour necrosis factor-alpha (TNF-α), malondialdehyde, glutathione, catalase and superoxide dismutase activity were determined in the ipsilateral cortex 24 hours following stroke induction.Main outcomes and results: EO (45 mg kg–1), statistically similar to Z-ligustilide (20 mg kg–1), curtailed brain infarction and oedema, improved behavioural scores and prevented enhanced oxidative stress and TNF-α level in the ischaemic brain tissues.Conclusions: The findings provide the first evidence of effectiveness of the extract in a thromboembolic model of stroke, whose action can be mediated, at least in part, by the antioxidative and anti-inflammatory mechanisms.

Abstract TP271: Improvement of Cerebral Blood Flow With DS-1040 in a Rat Thromboembolic Stroke Model

Introduction: DS-1040 is a novel inhibitor of the activated thrombin-activatable fibrinolysis inhibitor (TAFIa) and is in clinical development for the treatment of acute ischemic stroke (AIS). The objective of this study was to investigate the effect of DS-1040 on cerebral blood flow (CBF) in a rat thromboembolic stroke model. Methods: The CBF was transcranially and continuously monitored during the experiment by laser Doppler flowmetry system using a probe fixed to the skull above the territory of the right middle cerebral artery (MCA) of an anesthetized male SHR/Izm rat. After recording basal CBF, non-autologous whole blood clot was injected into the internal carotid artery. We conducted a 3-arm study. Saline (control) and DS-1040 (3.0 mg/kg) were intravenously injected as a bolus 5 min after the clot injection. The dosage of DS-1040 was set to achieve full inhibition of TAFIa during the experiment. Recombinant tissue plasminogen activator (rt-PA, 7.0 mg/kg; positive control) was intravenously injected 5 min after the clot injection by 10% bolus and 90% infusion for 60 min. The CBF(%) was defined as percent of the CBF at each time point to the mean basal CBF and the area under the curve of the CBF(%) after the drug injection, AUC 5-110min , was calculated. Results: The CBF(%) decreased after the clot injection, indicating the clot embolized the MCA. A significant increase of the AUC 5-110min was observed in the rt-PA group (8416.2 %·min ± 642.4 %·min, mean ± SE) compared to the control group (3728.7 %·min ± 684.2 %·min), indicating the model is sensitive to tPA. DS-1040 also significantly increased the AUC 5-110min (6645.3 %·min ± 861.9 %·min) compared to the control group. Conclusions: DS-1040 restored CBF in a rat thromboembolic stroke model suggesting DS-1040 is expected to be beneficial for the treatment of patients with acute ischemic stroke. To the authors’ knowledge, this is the first report of an inhibitor of TAFIa improving the CBF in a thromboembolic stroke model.

Abstract WP113: Dose-response and Therapeutic Time-window of Compound 21: a Randomized Preclinical Trial in Rat Model of Thromboembolic Stroke

Background: We and others have previously shown promising results with the use of the angiotensin type 2 receptor agonist, compound 21 (C21), in experimental stroke. Here we aimed to determine the best dose and time window for C21 in a clinically relevant embolic stroke model. Methods: This study was conducted as a translational, randomized, controlled, blinded preclinical trial with the guidance of our biostatistician. Male Wistar rats (8-9 weeks old) were subjected to embolic middle cerebral artery occlusion (eMCAO). For the dose-response study, animals received C21 (0.01, 0.03 and 0.06 mg/kg/d) or saline (orally) for 5 days, with the first dose given IV at 3 h post-eMCAO. For the time-window study, the optimal dose of C21 was initiated at 3, 6 or 24 h post-eMCAO and daily thereafter, for 5 days. Behavioral outcomes (Bederson, paw grasp and grip strength) were blindly assessed at days, 1, 3, 5 and 7. Rats were then sacrificed and their brains collected for infarct size and vascular density analyses. Results (Table): In the dose-response study, repeated-measures ANOVA showed significant (p &lt; 0.05) behavioral improvement with the low dose (0.01 mg/kg). This dose was associated with higher vascularity in the ischemic penumbra, detected by laminin staining, compared to saline treated controls (65±6 vs 46±7 p &lt; 0.05). In the time-window study, the 0.01 mg/kg dose displayed non-statistically significant improvements when given 3 h and 6 h after eMCAO. There were no differences between doses or time-windows for either % infarct size or tissue loss. Conclusions: C21 given at 0.01 mg/kg/d was effective in improving behavioral dysfunction after embolic stroke when administered within 6 h. Compound C21 shows promise as a potential therapeutic agent and should be examined for safety and efficacy in clinical trials for ischemic stroke.

Combining Normobaric Oxygen with Ethanol or Hypothermia Prevents Brain Damage from Thromboembolic Stroke via PKC-Akt-NOX Modulation.

In a thromboembolic stroke model after reperfusion by recombinant tissue plasminogen activator (rt-PA), we aimed to determine whether therapeutic hypothermia (TH) and ethanol (EtOH) in combination with low concentration (60%) of normobaric oxygen (NBO) enhanced neuroprotection, as compared to using each of these agents alone. We further aimed to elucidate a potential role of the NADPH oxidase (NOX), phosphorylated protein kinase B (Akt), and protein kinase C-δ (PKC-δ) pathway in oxidative stress and neuroprotection. In Sprague-Dawley rats, a focal middle cerebral artery (MCA) occlusion was induced by an autologous embolus in the following experimental groups: rt-PA treatment alone, rt-PA + NBO treatment, rt-PA + TH at 33°C, rt-PA + EtOH, rt-PA + NBO + EtOH, rt-PA + NBO + TH, rt-PA + NOX inhibitor, rt-PA + EtOH + NOX inhibitor, or rt-PA + EtOH + Akt inhibitor. Control groups included sham-operated without stroke or stroke without treatment. Infarct volume and neurological deficit were assessed at 24h after rt-PA-induced reperfusion with or without treatments. ROS levels, NOX activity, and the protein expression of NOX subunits p22phox, p47phox, p67phox, gp91phox, as well as PKC-δ and phosphorylated Akt were measured at 3 and 24h after rt-PA-induced reperfusion. Following rt-PA in thromboembolic stroke rats, NBO combined with TH or EtOH more effectively decreased infarct volume and neurological deficit, as well as reactive oxygen species (ROS) production than with any of the used monotherapies. NOX activity and subunit expressions were downregulated and temporally associated with reduced PKC-δ and increased p-Akt expression. The present study demonstrated that combining NBO with either TH or EtOH conferred similar neuroprotection via modulation of NOX activation. The results suggest a role of Akt in NOX activation and implicate an upstream PKC-δ pathway in the Akt regulation of NOX. It is possible to substitute EtOH for TH, thus circumventing the difficulties in clinical application of TH through the comparatively easier usage of EtOH as a potential stroke management.

Adjuvant therapies using normobaric oxygen with hypothermia or ethanol for reducing hyperglycolysis in thromboembolic cerebral ischemia

Background and purpose: Normobaric oxygen (NBO), ethanol (EtOH), and therapeutic hypothermia (TH) delivered alone or in combination have neuroprotective properties after acute stroke. We used an autologous thromboembolic rat stroke model to assess the additive effects of these treatments for reducing the deleterious effects of hyperglycolysis post-stroke in which reperfusion is induced with recombinant tissue plasminogen activator (rt-PA).Methods: Sprague–Dawley rats were subjected to middle cerebral artery (MCA) occlusion with an autologous embolus. One hour after occlusion, rt-PA was administered alone or with NBO (60%), EtOH (1.0g/kg), TH (33°C), either singly or in combination. Infarct volume and neurological deficit were assessed at 24h after rt-PA-induced reperfusion with or without other treatments. The extent of hyperglycolysis, as determined by cerebral glucose and lactate levels was evaluated at 3 and 24h after rt-PA administration. At the same time points, expressions of glucose transporter 1 (Glut1), glucose transporter 3 (Glut3), phosphofructokinase1 (PFK-1), and lactate dehydrogenase were (LDH) measured by Western blotting.Results: Following rt-PA in rats with thromboembolic stroke, NBO combined with TH or EtOH most effectively decreased infarct volume and neurological deficit. As compared to rt-PA alone, EtOH or TH but not NBO monotherapies significantly reduced post-stroke hyperglycolysis. The increased utilization of glucose and production of lactate post-stroke was prevented most effectively when NBO was combined with either EtOH or TH after reperfusion with rt-PA, as shown by the significantly decreased Glut1, Glut3, PFK-1, and LDH levels.Conclusions: In a rat thromboembolic stroke model, both EtOH and TH used individually offer neuroprotection after the administration of rt-PA. While NBO monotherapy does not appear to be effective, it significantly potentiates the efficacy of EtOH and TH. The similar neuroprotection and underlying mechanisms pertaining to the attenuation of hyperglycolysis provided by EtOH or TH in combination with NBO suggest a possibility of substituting EtOH for TH. Thus a combination of NBO and EtOH, which are widely available and easily used, could become a novel and effective neuroprotective strategy in the clinical setting.

Partial MHC Constructs Treat Thromboembolic Ischemic Stroke Characterized by Early Immune Expansion.

Inflammation and thrombosis are tightly linked, with inflammation contributing to thromboembolism and to stroke outcome. Thromboembolism is a frequent cause of ischemic stroke; yet, the most used occlusion mouse models of experimental stroke do not effectively replicate thromboembolism. Our group recently described a novel thromboembolic mouse model of stroke that successfully occludes the middle cerebral artery with high reproducibility. In the current study, we characterize the peripheral and local immune outcomes as well as the ischemic response to immune therapy in a clinically relevant mouse model of thromboembolic stroke. Brain and spleen tissues were harvested 24 h after thromboembolic stroke and cells immunophenotyped by flow cytometry. We observed a significant increase in neutrophils and early activated T cells in the spleen and an increase in neutrophils and activated monocytes/microglia in the ischemic cortex after thromboembolic stroke. Moreover, as was shown previously for transient MCAO models, treatment of thromboembolic stroke with partial MHC constructs significantly reduced ischemic damage indicating an equivalent effect of this immune-based therapy in the thromboembolic model that better mimics the pathophysiology of human stroke.

Erythrocyte (dis)aggregation in stroke model in rats

Introduction and aim of the study. Ischemic stroke develops in conjunction with interruption of blood flow in microvessels that depends on rheological blood properties. There is a lack of knowledge in hemorheological features of experimental stroke making more difficult to value the relevance of stroke models. The study aims investigation of microhemorheological parameters in two experimental stroke models - thromboembolic model and middle cerebral artery (MCA) ligation model. Methods. Male Wistar rats were subjected to focal brain ischemia in MCA ligation stroke model or thromboembolic stroke model. The neurological deficit, the size of ischemic brain lesion and hemorheological parameters (hematocrit, kinetics of red blood cells (RBC) (dis)aggregation and RBC deformability) were evaluated. Results. The neurological deficit was correlated with the size of brain ischemic lesion. The increased rate of RBC aggregate formation was detected in both stroke models. At the same time, the strength of RBC aggregates changed in a model-dependent manner, namely, it raised sharply in the MCA ligation stroke model, but was somewhat decreased in thromboembolic stroke model. Conclusion. The focal stroke models produce repeatable and neurologically significant lesions of brain followed with fundamental changes in the hemorheological parameters. The way of ischemia producing can be crucial for the direction of hemorheological changes.

Impact of Alcohol Consumption on the Outcome of Ischemic Stroke and Thrombolysis

Tissue-type plasminogen activator (tPA) is the only acute treatment for ischemic stroke. Unfortunately, the benefit of tPA-driven thrombolysis is not systematic, and understanding the reasons for this is mandatory. The balance between beneficial and detrimental effects of tPA might explain the limited overall efficiency of thrombolysis. Here, we investigated whether this balance could be influenced by excessive alcohol intake. We used a murine model of thromboembolic stroke, coupled to an array of biochemical assays, near-infrared or magnetic resonance imaging scans, 2-photon microscopy, hydrodynamic transfections, and immunohistological techniques. We found that 6 weeks of alcohol consumption (10% in drinking water) worsens ischemic lesions and cancels the beneficial effects of tPA-induced thrombolysis. We accumulate in vivo and in vitro evidence showing that this aggravation is correlated with a decrease in lipoprotein receptor-related protein 1-mediated hepatic clearance of tPA in alcohol-exposed mice. An efficient liver-driven clearance of tPA might influence the safety of thrombolysis after stroke.

Abstract T P232: Combination Therapy With Taurine Plus Tissue Plasminogen Activator in a Rat Model of Embolic Stroke

Background: Tissue plasminogen activator (tPA) is the only FDA-approved therapy for acute ischemic stroke, but it has narrow therapeutic window (within 4.5h) and increased risks of intracerebral hemorrhage. Taurine, an endogenous 2-aminoethansulfolic amino acid, exhibits a plethora of physiological functions. It has been found to protect against ischemic brain injury in animal models of stroke by diverse mechanisms. Strikingly, published data show that the therapeutic window of taurine was at least 8h after the onset of ischemic stroke, thus potentially allowing for delayed or more effective thrombolysis. In this study we investigated whether combination therapy with taurine plus tPA can block hemorrhagic transformation and thus extend the time window of tPA after delayed tPA treatment for ischemic stroke. Methods and Results: Male adult rats (330-380g) were subjected to embolic middle cerebral artery occlusion and then randomized into different treatment groups: saline injected at 4h; taurine (50 mg/kg) at 4h, tPA (10 mg/kg) at 6h, and combination therapy with taurine at 4h plus tPA at 6h after the onset of ischemia. Delayed 6-hour tPA did not decrease infarction but instead worsened brain hemorrhage. Combining taurine with delayed 6-hour tPA reduced tissue infarction, attenuated blood-brain barrier disruption by reduced degradation of tight junction proteins (claudin-5 and collagen IV), and ameliorated brain hemorrhage. These protective effects are correlated with decreased brain matrix metallopeptidase-9 activity possibly through inactivation of NF-kB and CD147 (a potent inducer of MMPs) in the postischemic brain, particularly in the cerebral microvessels (using structurally intact microvessels isolated from the brain). Conclusion: The present study demonstrates for the first time that combination therapy with taurine may attenuate delayed tPA-associated hemorrhagic transformation and extend tPA treatment time windows in a clinically relevant thromboembolic stroke model. Inhibition of MMP-9 in brain microvessels via inactivation of NF-κB and CD147 signaling may underlie the protective mechanisms of taurine.

Platelet rich clots are resistant to lysis by thrombolytic therapy in a rat model of embolic stroke.

BackgroundEarly recanalization of occluded vessels in stroke is closely associated with improved clinical outcome. Microbubble-enhanced sonothrombolysis is a promising therapy to improve recanalization rates and reduce the time to recanalization. Testing any thrombolytic therapy requires a model of thromboembolic stroke, but to date these models have been highly variable with regards to clot stability. Here, we developed a model of thromboembolic stroke in rats with site-specific delivery of platelet-rich clots (PRC) to the main stem of the middle cerebral artery (MCA). This model was used in a subsequent study to test microbubble-enhanced sonothrombolysis.MethodsIn Study 1 we investigated spontaneous recanalization rates of PRC in vivo over 4 hours and measured infarct volumes at 24 hours. In Study 2 we investigated tPA-mediated thrombolysis and microbubble-enhanced sonothrombolysis in this model.ResultsStudy 1 demonstrated stable occlusion out to 4 hours in 5 of 7 rats. Two rats spontaneously recanalized at 40 and 70 minutes post-embolism. Infarct volumes were not significantly different in recanalized rats, 43.93 ± 15.44% of the ischemic hemisphere, compared to 48.93 ± 3.9% in non-recanalized animals (p = 0.7). In Study 2, recanalization was not observed in any of the groups post-treatment.ConclusionsSite specific delivery of platelet rich clots to the MCA origin resulted in high rates of MCA occlusion, low rates of spontaneous clot lysis and large infarction. These platelet rich clots were highly resistant to tPA with or without microbubble-enhanced sonothrombolysis. This resistance of platelet rich clots to enhanced thrombolysis may explain recanalization failures clinically and should be an impetus to better clot-type identification and alternative recanalization methods.Electronic supplementary materialThe online version of this article (doi:10.1186/s13231-014-0014-y) contains supplementary material, which is available to authorized users.

NURR1 involvement in recombinant tissue-type plasminogen activator treatment complications after ischemic stroke.

Despite the effectiveness of recombinant tissue-type plasminogen activator (r-tPA) during the acute phase of ischemic stroke, the therapy remains limited by a narrow time window and the occurrence of occasional vascular side effects, particularly symptomatic hemorrhages. Our aim was to investigate the mechanisms underlying the endothelial damage resulting from r-tPA treatment in ischemic-like conditions. Microarray analyses were performed on cerebral endothelial cells submitted to r-tPA treatment during oxygen and glucose deprivation to identify novel biomarker candidates. Validation was then performed in vivo in a mouse model of thromboembolic stroke and culminated in an analysis in a clinical cohort of patients with ischemic stroke treated with thrombolysis. The transcription factor NURR1 (NR4A2) was identified as a downstream target induced by r-tPA during oxygen and glucose deprivation. Silencing NURR1 expression reversed the endothelial-toxicity induced by the combined stimuli, a protective effect attributable to reduced levels of proinflammatory mediators, such as nuclear factor-kappa-beta 2 (NF-κ-B2), interleukin 1 alpha (IL1α), intercellular adhesion molecule 1 (ICAM1), SMAD family member 3 (SMAD3), colony stimulating factor 2 (granulocyte-macrophage; CSF2). The detrimental effect of delayed thrombolysis, in conditions in which NURR1 gene expression was enhanced, was confirmed in the preclinical stroke model. Finally, we determined that patients with stroke who had a symptomatic hemorrhagic transformation after r-tPA treatment exhibited higher baseline serum NURR1 levels than did patients with an asymptomatic or absence of cerebral bleedings. Our results suggest that NURR1 upregulation by r-tPA during ischemic stroke is associated with endothelial dysfunction and inflammation and the enhancement of hemorrhagic complications associated to thrombolysis.