tPA-induced Hemorrhagic Transformation Research Articles

Poria cocos Extract Alleviates tPA-Induced Hemorrhagic Transformation after Ischemic Stroke through Regulation of Microglia M1/M2 Phenotypes Polarization.

Delayed thrombolytic therapy with tissue plasminogen activator (tPA), the only FDA-approved drug for ischemic stroke, can cause catastrophic hemorrhagic transformation (HT) after ischemic stroke. However, it remains largely unknown how microglial polarization dynamically changes in HT. Poria cocos is a widely used functional edible fungus in Asia and has been used for more than 2000 years as a food and medicine in China. Our preliminary study found that P. cocos extract (PCE) significantly reduced the volume of cerebral infarction. We performed the effects of PCE on tPA-induced HT in rat models of autologous thromboembolism middle cerebral artery occlusion in vivo and BV-2 cells injured by oxygen-glucose deprivation/reperfusion in vitro. Hemorrhage test and triphenyltetrazolium chloride staining were performed to examine the efficiency of PCE. The expression level of proteins associated with microglia polarization was detected using Western blotting and immunofluorescence staining. Small interfering RNA transfection reveals the regulatory mechanism of PCE on microglia polarization. PCE plus tPA reduced hemorrhage and infarct volumes after ischemic stroke. During tPA-induced HT, M1 microglia increased over time from 3 days onward and remained high for at least 7 days, reaching the peak at 7 days, M2 microglia gradually increased after 3 days and continued to increase for at least 14 days. Furthermore, PCE inhibited the secretion of pro-inflammatory cytokines in M1 microglia and improved the secretion of anti-inflammatory cytokines in M2 microglia, which related to the regulation of the IRF5-IRF4 axis. This current study indicates that PCE alleviates tPA-induced HT after ischemic stroke by modulating microglia M1/M2 phenotype polarization.

Histidine-rich glycoprotein modulates neutrophils and thrombolysis-associated hemorrhagic transformation

Intravenous thrombolysis using recombinant tissue plasminogen activator (tPA) remains the primary treatment for patients with acute ischemic stroke (AIS). However, the mechanism of tPA-related hemorrhagic transformation (HT) remains poorly understood. Elevation of histidine-rich glycoprotein (HRG) expression was detected by nano-liquid chromatography tandem mass spectrometry at 1 h following tPA infusion as compared to baseline prior to tPA infusion (discovery cohort, n = 10), which was subsequently confirmed in a validation cohort (n = 157) by ELISA. Surprisingly, no elevation of HRG was detected in individuals who subsequently developed HT. During in vitro experiments, HRG reduced neutrophil NETosis, inflammatory cytokine production, and migration across the blood–brain barrier induced by tPA. In a photothrombotic murine AIS model, HRG administration ameliorated HT with delayed thrombolysis, by inhibiting neutrophil immune infiltration and downregulating pro-inflammatory signaling pathways. Neutrophil depletion or NETosis inhibition also alleviated HT, whereas HRG siRNA treatment exacerbated HT. In conclusion, fluctuations in HRG levels may reflect tPA therapy and its associated HT. The inhibitory effect of HRG on neutrophils may counteract tPA-induced immune abnormalities and HT in patients with AIS.

Effects of Lithium Ions on tPA-Induced Hemorrhagic Transformation under Stroke.

Thrombolytic therapy with the tissue plasminogen activator (tPA) is a therapeutic option for acute ischemic stroke. However, this approach is subject to several limitations, particularly the increased risk of hemorrhagic transformation (HT). Lithium salts show neuroprotective effects in stroke, but their effects on HT mechanisms are still unknown. In our study, we use the models of photothrombosis (PT)-induced brain ischemia and oxygen-glucose deprivation (OGD) to investigate the effect of Li+ on tPA-induced changes in brain and endothelial cell cultures. We found that tPA did not affect lesion volume or exacerbate neurological deficits but disrupted the blood-brain barrier. We demonstrate that poststroke treatment with Li+ improves neurological status and increases blood-brain barrier integrity after thrombolytic therapy. Under conditions of OGD, tPA treatment increased MMP-2/9 levels in endothelial cells, and preincubation with LiCl abolished this MMP activation. Moreover, we observed the effect of Li+ on glycolysis in tPA-treated endothelial cells, which we hypothesized to have an effect on MMP expression.

Thrombolytic tPA-induced hemorrhagic transformation of ischemic stroke is mediated by PKCβ phosphorylation of occludin

AbstractThe current standard of care for moderate to severe ischemic stroke is thrombolytic therapy with tissue plasminogen activator (tPA). Treatment with tPA can significantly improve neurologic outcomes; however, thrombolytic therapy is associated with an increased risk of intracerebral hemorrhage (ICH). The risk of hemorrhage significantly limits the use of thrombolytic therapy, and identifying pathways induced by tPA that increase this risk could provide new therapeutic options to extend thrombolytic therapy to a wider patient population. Here, we investigate the role of protein kinase Cβ (PKCβ) phosphorylation of the tight junction protein occludin during ischemic stroke and its role in cerebrovascular permeability. We show that activation of this pathway by tPA is associated with an increased risk of ICH. Middle cerebral artery occlusion (MCAO) increased phosphorylation of occludin serine 490 (S490) in the ischemic penumbra in a tPA-dependent manner, as tPA−/− mice were significantly protected from MCAO-induced occludin phosphorylation. Intraventricular injection of tPA in the absence of ischemia was sufficient to induce occludin phosphorylation and vascular permeability in a PKCβ-dependent manner. Blocking occludin phosphorylation, either by targeted expression of a non-phosphorylatable form of occludin (S490A) or by pharmacologic inhibition of PKCβ, reduced MCAO-induced permeability and improved functional outcome. Furthermore, inhibiting PKCβ after MCAO prevented ICH associated with delayed thrombolysis. These results show that PKCβ phosphorylation of occludin is a downstream mediator of tPA-induced cerebrovascular permeability and suggest that PKCβ inhibitors could improve stroke outcome and prevent ICH associated with delayed thrombolysis, potentially extending the window for thrombolytic therapy in stroke.

The Protective Role of Immunomodulators on Tissue-Type Plasminogen Activator-Induced Hemorrhagic Transformation in Experimental Stroke: A Systematic Review and Meta-Analysis.

Background: Recanalization with tissue plasminogen activator (tPA) is the only approved agent available for acute ischemic stroke. But delayed treatment of tPA may lead to lethal intracerebral hemorrhagic transformation (HT). Numerous studies have reported that immunomodulators have good efficacy on tPA-induced HT in ischemic stroke models. The benefits of immunomodulators on tPA-associated HT are not clearly defined. Here, we sought to conduct a systematic review and meta-analysis of preclinical studies to further evaluate the efficacy of immunomodulators. Methods: The PubMed, Web of Science, and Scopus electronic databases were searched for studies. Studies that reported the efficacy of immunomodulators on tPA-induced HT in animal models of stroke were included. Animals were divided into two groups: immunomodulators plus tPA (intervention group) or tPA alone (control group). The primary outcome was intracerebral hemorrhage, and the secondary outcomes included infarct volume and neurobehavioral score. Study quality was assessed by the checklist of CAMARADES. We used standardized mean difference (SMD) to assess the impact of interventions. Regression analysis and subgroup analysis were performed to identify potential sources of heterogeneity and evaluate the impact of the study characteristics. The evidence of publication bias was evaluated using trim and fill method and Egger’s test. Results: We identified 22 studies that met our inclusion criteria involving 516 animals and 42 different comparisons. The median quality checklist score was seven of a possible 10 (interquartile range, 6–8). Immunomodulators improved cerebral hemorrhage (1.31 SMD, 1.09–1.52); infarct volume (1.35 SMD, 0.95–1.76), and neurobehavioral outcome (0.9 SMD, 0.67–1.13) in experimental stroke. Regression analysis and subgroup analysis indicated that control of temperature and time of assessment were important factors that influencing the efficacy of immunomodulators. Conclusion: Our findings suggested that immunomodulators had a favorable effect on tPA-associated intracerebral hemorrhage, cerebral infarction, and neurobehavioral impairments in animal models of ischemic stroke.

YiQiFuMai Lyophilized Injection ameliorates tPA-induced hemorrhagic transformation by inhibiting cytoskeletal rearrangement associated with ROCK1 and NF-κB signaling pathways

Ethnopharmacological relevanceThrombolytic therapy with tissue plasminogen activator (tPA) after ischemic stroke exacerbates blood–brain barrier (BBB) breakdown and leads to hemorrhagic transformation (HT). YiQiFuMai Lyophilized Injection (YQFM) is a modern preparation derived from Sheng-mai San (a traditional Chinese medicine). YQFM attenuates the BBB dysfunction induced by cerebral ischemia–reperfusion injury. However, whether YQFM can suppress tPA-induced HT remains unknown. Aim of the studyWe investigated the therapeutic effect of YQFM on tPA-induced HT and explored the underlying mechanisms in vivo and in vitro to improve the safety of tPA use against stroke. MethodsMale C57BL/6J mice were subjected to 45 min of ischemia and 24 h of reperfusion. tPA (10 mg/kg) were infused 2 h after occlusion and YQFM (0.671 g/kg) was injected 2.5 h after occlusion. The in vitro effect of YQFM (100, 200, 400 μg/mL) on tPA (60 μg/mL)-induced dysfunction of the microvascular endothelial barrier in the brain following oxygen-glucose deprivation/reoxygenation (OGD/R) was observed in bEnd.3 cells. ResultsYQFM suppressed tPA-induced high hemoglobin level in the brain, mortality, neurologic severity score, BBB permeability, expression and activation of matrix metalloproteinase (MMP)-9 and MMP-2, and degradation of tight-junction proteins. Furthermore, YQFM significantly blocked tPA-induced brain microvascular endothelial permeability and phosphorylation of Rho-associated kinase (ROCK)1, myosin light chain (MLC), cofilin and p65 in vivo and in vitro. ConclusionYQFM suppressed tPA-induced HT by inhibiting cytoskeletal rearrangement linked with ROCK-cofilin/MLC pathways and inhibiting the nuclear factor-kappa B pathway to ameliorate BBB damage caused by tPA.

Abstract WP312: Rosiglitazone Ameliorates Tissue Plasminogen Activator-Induced Hemorrhagic Transformation After Stroke via Promoting Phagocytic Activity of Microglia

Objective: Delayed thrombolytic treatment with recombinant tissue plasminogen activator (tPA) may lead to lethal hemorrhagic transformation (HT) after ischemic stroke. Rosiglitazone(RSG) is one of the PPAR-γ agonists and has been reported to protect against cerebral ischemia. However, whether RSG can alleviate HT after tPA treatment still remains unknown. In this study, we sort to examine the role of RSG on tPA-induced HT. Methods and results: We used the murine suture middle cerebral artery occlusion (MCAO) models of stroke to investigate the therapeutic potential of RSG against tPA-induced HT. Delayed administration of tPA (10mg/kg, 2 hours after reperfusion) resulted in HT in the ischemic territory 1 day after ischemia. When RSG(6mg/kg) were intraperitoneally administered 1 hour before MCAO , HT was significantly decreased. In addition, we found the BBB disruption and tight junction damage in tPA-treated MCAO mice can be mitigated after RSG treatment. Using flow cytometry and immunostaining, we confirmed that the expression of CD206 was significantly upregulated while the expression of iNOS was down-regulated in microglia of the tPA and RSG treated MCAO mice. Simultaneously, the expression of arg-1, CD36, CD68 and TREM2 were upregulated in tPA and RSG treated stroke mice. To further address our hypothesis that RSG could exert protection against the BBB disruption via phenotypic change into enhanced phagocytosis activity, we intend to use the two-photon live imaging to elucidate that CX3CR1-GFP + microglia could phagocytose more exogenously injected rhodamine-dextran while treated with RSG and tPA compared with those treated with tPA alone. In addition, we plan to inject pH-sensitive beads through the lateral ventricle to demonstrate an increased engulfment of pH-sensitive beads after co-treatment with RSG and tPA. In vitro, phagocytic efficiency in primary microglia and BV2 cells will be verified through adding latex beads after co-administrating RSG and tPA. Conclusions: In conclusion, our data demonstrates that RSG treatment ameliorates HT in delayed tPA-treated stroke mice. Enhanced phagocytic activity of microglia may be responsible for the RSG afforded protection against the BBB disruption in the tPA-induced HT after stroke.

IM-12 activates the Wnt-β-catenin signaling pathway and attenuates rtPA-induced hemorrhagic transformation in rats after acute ischemic stroke.

Hemorrhagic transformation (HT) is a devastating complication for patients with acute ischemic stroke (AIS) who are treated with tissue plasminogen activator (tPA). HT is associated with high morbidity and mortality, but no effective treatments are currently available to reduce the risk of HT. Therefore, methods to prevent HT are urgently needed. In this study, we used IM-12, an inhibitor of glycogen synthase kinase 3β (GSK-3β), to evaluate the role of the Wnt-β-catenin signaling pathway in recombinant tPA (rtPA)-induced HT. Sprague-Dawley rats were subjected to a middle cerebral artery occlusion (MCAO) model of ischemic stroke, and then were either administered rtPA, rtPA combined with IM-12, or the vehicle at 4 h after stroke was induced. Our results indicate that rats subjected to HT had more severe neurological deficits, brain edema, and blood-brain barrier (BBB) breakdown, and had a greater infarction volume than the control group. Rats treated with IM-12 had improved outcomes compared with those of rats treated with rtPA alone. Moreover, IM-12 increased the protein expression of β-catenin and downstream proteins while suppressing the expression of GSK-3β. These results suggest that IM-12 reduces rtPA-induced HT and attenuates BBB disruption, possibly through activation of the Wnt-β-catenin signaling pathway, and provides a potential therapeutic strategy for preventing tPA-induced HT after AIS.

Rosiglitazone ameliorates tissue plasminogen activator‐induced brain hemorrhage after stroke

ObjectiveDelayed thrombolytic therapy with recombinant tissue plasminogen activator (tPA) may exacerbate blood‐brain barrier (BBB) breakdown after ischemic stroke and lead to catastrophic hemorrhagic transformation (HT). Rosiglitazone(RSG), a widely used antidiabetic drug that activates peroxisome proliferator‐activated receptor‐γ (PPAR‐γ), has been shown to protect against cerebral ischemia through promoting poststroke microglial polarization toward the beneficial anti‐inflammatory phenotype. However, whether RSG can alleviate HT after delayed tPA treatment remains unknown. In this study, we sort to examine the role of RSG on tPA‐induced HT after stroke.Methods and resultsWe used the murine suture middle cerebral artery occlusion (MCAO) models of stroke followed by delayed administration of tPA (10 mg/kg, 2 hours after suture occlusion) to investigate the therapeutic potential of RSG against tPA‐induced HT. When RSG(6 mg/kg) was intraperitoneally administered 1 hour before MCAO in tPA‐treated MCAO mice, HT in the ischemic territory was significantly attenuated 1 day after stroke. In the tPA‐treated MCAO mice, we found RSG significantly mitigated BBB disruption and hemorrhage development compared to tPA‐alone‐treated stroke mice. Using flow cytometry and immunostaining, we confirmed that the expression of CD206 was significantly upregulated while the expression of iNOS was down‐regulated in microglia of the RSG‐treated mice. We further found that the expression of Arg‐1 was also upregulated in those tPA and RSG‐treated stroke mice and the protection against tPA‐induced HT and BBB disruption in these mice were abolished in the presence of PPAR‐γ antagonist GW9662 (4 mg/kg, 1 hour before dMCAO through intraperitoneal injection).ConclusionsRSG treatment protects against BBB damage and ameliorates HT in delayed tPA‐treated stroke mice by activating PPAR‐γ and favoring microglial polarization toward anti‐inflammatory phenotype.

Development of a novel RANKL-based peptide, microglial healing peptide1-AcN (MHP1-AcN), for treatment of ischemic stroke

Although the regulation of post-ischemic inflammation is an important strategy to treat ischemic stroke, all clinical trials have failed to show its efficacy. To solve the problem, we previously developed a novel partial peptide of RANKL, microglial healing peptide 1 (MHP1), which could reduce ischemic injury by inhibiting Toll-like receptor (TLR) induced inflammation. However, optimization of the peptide was necessary to increase the stability and efficacies for clinical use. According to information gathered through HPLC/MS in serum, we have newly designed a series of modified MHP1 peptides and have found that N-terminal acetylation and C-terminal amidation in MHP1 (MHP1-AcN), can strengthen its anti-inflammatory effects and increase its stability with anti-osteoclastogenic effects. Anti-TLR activity was reported to be reduced in MHP1 when incubated at 37 °C for 24 hrs, but MHP1-AcN could keep the activity under the same condition. The therapeutic effect of MHP1-AcN was observed in transient ischemic stroke model at lower dose than MHP1. Importantly, MHP1-AcN did not affect thrombolytic effects of tissue plasminogen activator (tPA) and inhibited tPA-induced hemorrhagic transformation. These findings indicated that MHP1-AcN was stable and effective anti-TLR signal peptide and could be a promising agent for treating stroke patients receiving tPA and endovascular therapy.

Extension of Tissue Plasminogen Activator Treatment Window by Granulocyte-Colony Stimulating Factor in a Thromboembolic Rat Model of Stroke.

When given beyond 4.5 h of stroke onset, tissue plasminogen activator (tPA) induces deleterious side effects in the ischemic brain, notably, hemorrhagic transformation (HT). We examined the efficacy of granulocyte-colony stimulating factor (G-CSF) in reducing delayed tPA-induced HT, cerebral infarction, and neurological deficits in a thromboembolic (TE) stroke model, and whether the effects of G-CSF were sustained for longer periods of recovery. After stroke induction, rats were given intravenous saline (control), tPA (10 mg/kg), or G-CSF (300 μg/kg) + tPA 6 h after stroke. We found that G-CSF reduced delayed tPA-associated HT by 47%, decreased infarct volumes by 33%, and improved motor and neurological deficits by 15% and 25%, respectively. It also prevented delayed tPA treatment-induced mortality by 46%. Immunohistochemistry showed 1.5- and 1.8-fold enrichment of the endothelial progenitor cell (EPC) markers CD34+ and VEGFR2 in the ischemic cortex and striatum, respectively, and 1.7- and 2.8-fold increases in the expression of the vasculogenesis marker von Willebrand factor (vWF) in the ischemic cortex and striatum, respectively, in G-CSF-treated rats compared with tPA-treated animals. Flow cytometry revealed increased mobilization of CD34+ cells in the peripheral blood of rats given G-CSF. These results corroborate the efficacy of G-CSF in enhancing the therapeutic time window of tPA for stroke treatment via EPC mobilization and enhancement of vasculogenesis.

Abstract TP350: Extension of tPA Treatment Window With Granulocyte-colony Stimulating Factor in a Thromboembolic Rat Model of Stroke

Background: When given beyond 4.5 hours of stroke onset, tissue plasminogen activator (tPA) produces deleterious side effects in the ischemic brain, notably, hemorrhagic transformation (HT). Extending the narrow time window of tPA will benefit a significant number of stroke patients. We previously showed that granulocyte-colony stimulating factor (G-CSF) attenuated delayed tPA (6 h post stroke)-induced HT in rats subjected to stroke via intraluminal filament occlusion of the middle cerebral artery. Here, we examined whether the drug reduces delayed tPA induced-HT, cerebral infarction and neurological deficits in rats subjected to a thromboembolic (TE) stroke, and whether efficacy of G-CSF treatment extends at longer periods of recovery. Stroke Therapy Academic Industry Roundtable (STAIR) guidelines indicate necessity of testing effects of a drug candidate in multiple ischemia models before it progresses to clinical development. Methods and Results: After stroke induction through the TE method, rats were given intravenous saline (control), tPA (10 mg/kg), or G-CSF (300 μg/kg) and tPA at 6 h post-stroke. Treatment with G-CSF decreased delayed tPA-associated HT in stroked rats as measured by the spectrophotometric hemoglobin assay. Reduction in HT was accompanied by a decrease in infarct volume and improvement in stroke-induced motor and neurological deficits at 7 days after stroke. Moreover, G-CSF treatment also reduced the mortality rate due to delayed tPA therapy by almost 50%. Flow cytometry showed significant increase in the population of CD34+/VEGFR2+ bone marrow stem cells in the blood of stroked rats subjected to the combination treatment compared with rats given saline alone or tPA. Double labeling of CD34+ and VEGFR2 revealed enrichment of CD34+/VEGFR2 expression in the ischemic cortex and striatum of rats subjected to G-CSF treatment, indicating involvement of endothelial progenitor cells in the therapeutic effects of G-CSF. Conclusion: These results further corroborate efficacy of G-CSF to reduce delayed tPA-induced HT and neurological deficits in experimental stroke models, and suggest potentiality of the drug to extend the therapeutic time window of tPA therapy for stroke.

Inhibition of TPA-induced hemorrhagic transformation by adenosine A2b receptor activation after cerebral ischemia

Background: The thrombolytic agent tissue plasminogen activator (tPA) is underutilized in ischemic stroke, in part because of its limited therapeutic window and hemorrhagic complications. Identifying an adjuvant drug that reduces the risk of hemorrhage could increase the applicability of tPA. Studies have shown that A2b receptor (A2bR) can regulate vascular leakage reduce MMP9 expression, so A2bR is the potential target.

Inhibition of tPA-induced hemorrhagic transformation involves adenosine A2b receptor activation after cerebral ischemia

Tissue plasminogen activator (tPA) is administered after ischemic stroke to dissolve intravascular clots, but its use can lead to hemorrhagic transformation (HT). Therapeutic strategies to reduce hemorrhagic complications of tPA might be of benefit for stroke patients. Adenosine A2b receptor (A2bR) plays pivotal roles in regulating vascular protection in peripheral organs. This study explored whether A2bR agonist BAY 60-6583 reduces hemorrhage risk after tPA usage. Using a rat transient middle cerebral artery occlusion model, we showed that mRNA and protein expression of A2bR increased to a greater extent after ischemia-reperfusion than did expression of the other three adenosine receptors (A1, A2a, and A3). tPA administration reduced A2bR expression in ischemic brain microvessels. Post-treatment with BAY 60-6583 (1mg/kg) at the start of reperfusion reduced lesion volume in the absence or presence of tPA (10mg/kg) and attenuated brain swelling, blood-brain barrier disruption, and tPA-exacerbated HT at 24h. Additionally, BAY 60-6583 mitigated sensorimotor deficits in the presence of tPA. BAY 60-6583 inhibited tPA-enhanced matrix metalloprotease-9 activation, probably through elevation of tissue inhibitor of matrix metalloproteinases-1 expression, and thereby reduced degradation of tight junction proteins. These effects would likely protect cerebrovascular integrity. A2bR agonists as an adjuvant to tPA could be a promising strategy for decreasing the risk of HT during treatment for ischemic stroke.

Strategies to Extend Thrombolytic Time Window for Ischemic Stroke Treatment: An Unmet Clinical Need.

To date, reperfusion with tissue plasminogen activator (tPA) remains the gold standard treatment for ischemic stroke. However, when tPA is given beyond 4.5 hours of stroke onset, deleterious effects of the drug ensue, especially, hemorrhagic transformation (HT), which causes the most significant morbidity and mortality in stroke patients. An important clinical problem at hand is to develop strategies that will enhance the therapeutic time window for tPA therapy and reduce the adverse effects (especially HT) of delayed tPA treatment. We reviewed the pharmacological agents which reduced the risk of HT associated with delayed (beyond 4.5 hours post-stroke) tPA treatment in preclinical studies, which we classified into those that putatively preserve the blood-brain barrier (e.g., minocycline, cilostazol, fasudil, candesartan, and bryostatin) and/or enhance vascularization and protect the cerebrovasculature (e.g., coumarin derivate IMM-H004 and granulocyte colony-stimulating factor). Recently, other new therapeutic modalities (e.g., oxygen transporters) have been reported which improved delayed tPA-associated outcomes by acting through other mechanisms. While the above-mentioned interventions unequivocally reduced delayed tPA-induced HT in stroke models, the long-term efficacy of these drugs are not yet established. Further optimization is required to expedite their future clinical application. The findings from this review indicate the need to explore the most ideal adjunctive interventions that will not only reduce delayed tPA–induced HT, but also preserve neurovascular functions. While waiting for the next breakthrough drug in acute stroke treatment, it is equally important to allocate considerable effort to find approaches to address the limitations of the only FDA-approved stroke therapy.

Abstract WP273: Focal Knockdown of Matrix Metalloprotease 3 Reduces Hemorrhagic Transformation and Improves Neurobehavioral Outcomes in Hyperglycemic Stroke

Matrix metalloprotease 3 (MMP3) is an endopeptidase with broad substrate specificity that can target and degrade the components of the neurovascular unit. MMP3 was shown to be a critical mediator of the tPA-induced hemorrhagic transformation (HT) after stroke. The role of MMP3 in mediating neurovascular injury in hyperglycemic stroke was not previously reported. Accordingly, this study tested the hypothesis that MMP3 focal knockdown in the brain reduces HT and improves functional outcomes in hyperglycemic stroke. Methods: MMP3 shRNA lentiviral particles or an empty vector (EV) was injected into both hemispheres to the lateral ventricles of male Wistar rats. Two weeks later, Control and mildly HG rats (140-200 mg/dl, achieved by 30% glucose injection (IP) 15 min prior to surgery, n=7-9/group) were subjected to 90 min middle cerebral artery occlusion and 24 h reperfusion. At 24 h, neurological deficit, infarct size, edema, Hb content in ischemic hemispheres and HT occurrence rate (HT index) were measured. In an additional group of animals, the spatial expression of MMP3 within the neurovascular unit was determined by immunohistochemistry. Results: MMP3 knockdown significantly reduced HT index, Hb content and edema with no effect on infarct size and this was associated with improved functional outcomes (Table). MMP3 expression was more evident in HG animals and co-localized with the neurons, blood vessels and pericytes but not astrocytes. Conclusion: MMP3 knockdown reduced the vascular injury and improved the functional outcomes. Our findings point out MMP3 as a potential therapeutic target in hyperglycemic stroke.

Abstract TMP43: Routine Labs may Help Predict tPA-related Hemorrhagic Transformation: A Role for Serial Monitoring of Prothrombin Time (PT) and Partial Thromboplastin Time (PTT) Post tPA

Background: Intravenous tissue plasminogen activator (tPA) is not routinely followed by blood work due to its reputed short half-life. While there has been a great deal of research on tPA’s extra-vascular effects on the neurovascular unit in the context of hemorrhagic complications, there is little clinical data on the intravascular efficacy of IV tPA - how and where it has its intended effect. Emerging data suggest that tPA may be most effective in the microvasculature and IV therapy may be a good adjunct to intra-arterial (IA) therapy. We previously found through proteomic profiling that the effects of IV tPA can last more than 72 hours. Now, we report that even routine blood labs can potentially help predict hemorrhagic transformation (HT). Method: 72 stroke patients treated with IV tPA were recruited post IRB approval. Clinical coagulation profiles (PT, PTT, fibrinogen and D-dimer) were performed at 12, 24, 72 hrs post IV tPA. Patients on medications (e.g. anticoagulants) or having other conditions (e.g. liver dysfunction, infection) that may affect these labs were excluded. Result: Compared to patients without HT, HT patients had significantly higher PT and PTT (Fig. 1 A,B) as early as 12 hrs post tPA administration and throughout the first 3 days of treatment. ROC analysis suggested PT and PTT at 12 and 24 hrs have potential to predict tPA-induced HT (Figure 1C,D. PT: AUC = 0.848, p = 0.001; PTT: AUC = 0.877; p = 0.003). Conclusion: Higher PT and PTT levels within 72 hours of IV tPA are early markers of HT post IV tPA in acute ischemic stroke. Whether these routine labs have value in symptomatic hemorrhage will require further study in a larger cohort. But this proof-of-concept study suggests that tPA efficacy can potentially be followed in real time, even with routine labs. The development of a reliable blood test would be of clinical utility to gauge thrombolytic efficacy in real time to guide and/or triage adjunct treatments.

Abstract TP279: Hyperglycemia Mediates Matrix Metalloprotease 3 Activation Through Tyrosine Nitration After Stroke

Tissue plasminogen activator (tPA) induces hemorrhagic transformation (HT) after stroke and the incidence of hyperglycemia further exacerbates this injury. Matrix metalloprotease 3 (MMP3) amplifies the tPA-induced HT. The influence of the interaction between tPA and HG on activating MMP3 after stroke was not previously reported. Accordingly, we investigated the impact of tPA and HG on MMP3 activity and the mechanisms through which they induce MMP3 activation in hyperglycemic stroke. Methods. Control/normoglycemic and hyperglycemic (blood glucose: 140-200 mg/dl) male Wistar rats were subjected to either middle cerebral artery (MCA) suture occlusion for 90 minutes or thromboembolic occlusion and up to 24 h reperfusion, with or without tPA (1 mg/kg, IV, 2 h after stroke). MMP3 activity and MMP3 tyrosine nitration were evaluated in brain homogenates at 24 h. Brain vascular endothelial cells (BVEC) were subjected to 3 h hypoxia under either normal or high glucose conditions with or without tPA. MMP3 activity and MMP3 tyrosine nitration were assessed at 24 h. Results. HG and tPA significantly increased MMP3 activity in the brain after stroke and in BVECs after hypoxia/reoxygenation. HG significantly increased MMP3 tyrosine nitration in rats subjected to either suture or thromboembolic occlusion as well as in BVECs (Table). Conclusion. HG and tPA significantly increased MMP3 activity in the brain after stroke and this was associated with increased MMP3 nitration. Our findings suggest that tyrosine nitration may be the underlying mechanism through which MMP3 is activated in hyperglycemic stroke.

Oxygen or cooling, to make a decision after acute ischemia stroke.

The presence of a salvageable penumbra, a region of ischemic brain tissue with sufficient energy for short-term survival, has been widely agreed as the premise for thrombolytic therapy with tissue plasminogen activator (tPA), which remains the only United States Food and Drug Administration (FDA) approved treatment for acute ischemia stroke. However, the use of tPA has been profoundly constrained due to its narrow therapeutic time window and the increased risk of potentially deadly hemorrhagic transformation (HT). Blood brain barrier (BBB) damage within the thrombolytic time window is an indicator for tPA-induced HT and both normobaric hyperoxia (NBO) and hypothermia have been shown to protect the BBB from ischemia/reperfusion injury. Therefore, providing the O2 as soon as possible (NBO treatment), freezing the brain (hypothermia treatment) to slow down ischemia-induced BBB damage or their combined use may extend the time window for the treatment of tPA. In this review, we summarize the protective effects of NBO, hypothermia or their use combined with tPA on ischemia stroke, based on which, the combination of NBO and hypothermia may be an ideal early stroke treatment to preserve the ischemic penumbra. Given this, there is an urge for large randomized controlled trials to address the effect.

GSK-3β inhibitor TWS119 attenuates rtPA-induced hemorrhagic transformation and activates the Wnt/β-catenin signaling pathway after acute ischemic stroke in rats

Hemorrhagic transformation (HT) is a devastating complication for patients with acute ischemic stroke who are treated with tissue plasminogen activator (tPA). It is associated with high morbidity and mortality, but no effective treatments are currently available to reduce HT risk. Therefore, methods to prevent HT are urgently needed. In this study, we used TWS119, an inhibitor of glycogen synthase kinase 3β (GSK-3β), to evaluate the role of the Wnt/β-catenin signaling pathway in recombinant tPA (rtPA)-induced HT. Sprague-Dawley rats were subjected to a middle cerebral artery occlusion (MCAO) model of ischemic stroke and then were administered rtPA, rtPA combined with TWS119, or vehicle at 4h. The animals were sacrificed 24h after infarct induction. Rats treated with rtPA showed evident HT, had more severe neurologic deficit, brain edema, and blood-brain barrier breakdown, and had larger infarction volume than did the vehicle group. Rats treated with TWS119 had significantly improved outcomes compared with those of rats treated with rtPA alone. In addition, Western blot analysis showed that TWS119 increased the protein expression of β-catenin, claudin-3, and ZO-1 while suppressing the expression of GSK-3β. These results suggest that TWS119 reduces rtPA-induced HT and attenuates blood-brain barrier disruption, possibly through activation of the Wnt/β-catenin signaling pathway. This study provides a potential therapeutic strategy to prevent tPA-induced HT after acute ischemic stroke.