tPA Mobilizes Immune Cells That Exacerbate Hemorrhagic Transformation in Stroke.

RATIONALE: 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. OBJECTIVE: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Tissue plasminogen activator worsens experimental autoimmune encephalomyelitis by complementary actions on lymphoid and myeloid cell responses.

BACKGROUND: Tissue plasminogen activator (tPA) is a serine protease involved in fibrinolysis. It is released by endothelial cells, but also expressed by neurons and glial cells in the central nervous system (CNS). Interestingly, this enzyme also contributes to pathological processes in the CNS such as neuroinflammation by activating microglia and increasing blood-brain barrier permeability. Nevertheless, its role in the control of adaptive and innate immune response remains poorly understood. METHODS: tPA effects on myeloid and lymphoid cell response were studied in vivo in the mouse model of multiple sclerosis experimental autoimmune encephalomyelitis and in vitro in splenocytes. RESULTS: tPA-/- animals exhibited less severe experimental autoimmune encephalomyelitis than their wild-type counterparts. This was accompanied by a reduction in both lymphoid and myeloid cell populations in the spinal cord parenchyma. In parallel, tPA increased T cell activation and proliferation, as well as cytokine production by a protease-dependent mechanism and via plasmin generation. In addition, tPA directly raised the expression of MHC-II and the co-stimulatory molecules CD80 and CD86 at the surface of dendritic cells and macrophages by a direct action dependent of the activation of epidermal growth factor receptor. CONCLUSIONS: Our study provides new insights into the mechanisms responsible for the harmful functions of tPA in multiple sclerosis and its animal models: tPA promotes the proliferation and activation of both lymphoid and myeloid populations by distinct, though complementary, mechanisms.

Prothymosin alpha and its mimetic hexapeptide improve delayed tissue plasminogen activator-induced brain damage following cerebral ischemia.

Tissue plasminogen activator (tPA) administration beyond 4.5 h of stroke symptoms is beneficial for patients but has an increased risk of cerebral hemorrhage. Thus, increasing the therapeutic window of tPA is important for stroke recovery. We previously showed that prothymosin alpha (ProTα) or its mimetic hexapeptide (P6Q) has anti-ischemic activity. Here, we examined the beneficial effects of ProTα or P6Q against delayed tPA-induced brain damage following middle cerebral artery occlusion (MCAO) or photochemically induced thrombosis in mice. Brain hemorrhage was observed by tPA administration during reperfusion at 4.5 and 6 h after MCAO. Co-administration of ProTα with tPA at 4.5 h inhibited hemorrhage and motor dysfunction 2-4 days, but not 7 days after MCAO. ProTα administration at 2 and 4.5 h after MCAO significantly inhibited tPA (4.5 h)-induced motor dysfunction and death more than 7 days. Administration of tPA caused the loss of tight junction proteins, zona occulden-1 and occludin, and up-regulation of matrix metalloproteinase-2/9, in a ProTα-reversible manner. P6Q administration abolished tPA (4.5 h)-induced hemorrhage and reversed tPA (6 h)-induced vascular damage and matrix metalloproteinase-2 and 9 up-regulation. Twice administrations of P6Q at 2 h alone and 6 h with tPA significantly improved motor dysfunction more than 7 days. In photochemically induced thrombosis ischemia, similar vascular leakage and neuronal damage (infarction and motor dysfunction) by late tPA (4.5 or 6 h) were also inhibited by P6Q. Thus, these studies suggest that co-administration with ProTα or P6Q would be beneficial to inhibit delayed tPA-induced hemorrhagic mechanisms in acute ischemic stroke.

Assessment of recombinant tissue plasminogen activator (rtPA) toxicity in cultured neural cells and subsequent treatment with poly-arginine peptide R18D.

Thrombolytic therapy with recombinant tissue plasminogen activator (rtPA) in ischaemic stroke has been associated with neurotoxicity, blood brain barrier (BBB) disruption and intra-cerebral hemorrhage. To examine rtPA cellular toxicity we investigated the effects of rtPA on cell viability in neuronal, astrocyte and brain endothelial cell (bEnd.3) cultures with and without prior exposure to oxygen-glucose deprivation (OGD). In addition, the neuroprotective peptide poly-arginine-18 (R18D; 18-mer of D-arginine) was examined for its ability to reduce rtPA toxicity. Studies demonstrated that a 4- or 24-h exposure of rtPA was toxic, affecting neuronal cell viability at ≥ 2 µM, and astrocyte and bEnd.3 cells viability at ≥ 5 µM. In addition, a 4-h exposure to rtPA after a period of OGD (OGD/rtPA) exacerbated toxicity, affecting neuronal, astrocyte and bEnd.3 cell viability at rtPA concentrations as low as 0.1 µM. Treatment of cells with low concentrations of R18D (0.5 and 1 µM) reduced the toxic effects of rtPA and OGD/rtPA, while on some occasions a higher 2 µM R18D concentrations exacerbated neuronal and bEnd.3 cell toxicity in OGD/rtPA exposed cultures. In exploratory studies we also demonstrated that OGD activates matrix metalloproteinase-9 (MMP-9) release into the supernatant of astrocyte and bEnd.3 cell cultures, but not neuronal cultures, and that OGD/rtPA increases MMP-9 activation. Furthermore, R18D decreased MMP-9 activation in OGD/rtPA treated astrocyte and bEnd.3 cell cultures. In summary, the findings show that rtPA can be toxic to neural cells and that OGD exacerbates toxicity, while R18D has the capacity to reduce rtPA neural cellular toxicity and reduce MMP-9 activation in astrocytes and bEnd.3. Poly-arginine-18 peptides, which are being developed as neuroprotective therapeutics for ischaemic stroke, therefore have the additional potential of reducing cytotoxic effects associated with rtPA thrombolysis in the treatment of ischaemic stroke.

Thrombolysis with rt-PA under Rivaroxaban Anticoagulation in a Hypertensive Rat Model of Intraluminal Middle Cerebral Artery Occlusion.

BACKGROUND: The aim of this study was to assess the risk and the threshold of hemorrhagic transformation (HT) after treatment with recombinant tissue plasminogen activator (rtPA) under the novel oral anticoagulant, rivaroxaban. METHODS: Fifty-three spontaneous hypertensive rats were used in this study. We performed transient middle cerebral artery occlusion for 270 minutes. Placebo, 10 mg/kg or 20 mg/kg rivaroxaban were administered via a stomach tube 180 minutes after induction of ischemia, and rtPA (10 mg/kg) was administered just before reperfusion. Ninety minutes after rivaroxaban administration we measured the rivaroxaban plasma concentration and prothrombin time (PT). HT volume was assessed by hemoglobin spectrophotometry. Additionally, infarct volume, IgG leakage volume, and neurological outcome were assessed. RESULTS: Rivaroxaban plasma concentration and PT increased in a dose dependent manner but were lower than human peak levels after a once-daily dose of 20 mg rivaroxaban. HT volume increased after treatment with 20 mg/kg rivaroxaban compared with placebo treated controls or those treated with 10 mg/kg rivaroxaban (26.5 ± 5.4, 26.8 ± 8.7, and 41.4 ± 12.6 µL in placebo, 10 mg/kg, and 20 mg/kg treated groups, respectively; P < .05). CONCLUSIONS: Our results suggest that even at therapeutic plasma concentrations, rivaroxaban may increase the risk of HT after thrombolysis in some conditions, such as hypertension and/or a prolonged ischemic period.

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.

Tissue plasminogen activator potently stimulates pleural effusion via a monocyte chemotactic protein-1-dependent mechanism.

Pleural infection is common. Evacuation of infected pleural fluid is essential for successful treatment, but it is often difficult because of adhesions/loculations within the effusion and the viscosity of the fluid. Intrapleural delivery of tissue plasminogen activator (tPA) (to break the adhesions) and deoxyribonuclease (DNase) (to reduce fluid viscosity) has recently been shown to improve clinical outcomes in a large randomized study of pleural infection. Clinical studies of intrapleural fibrinolytic therapy have consistently shown subsequent production of large effusions, the mechanism(s) of which are unknown. We aimed to determine the mechanism by which tPA induces exudative fluid formation. Intrapleural tPA, with or without DNase, significantly induced pleural fluid accumulation in CD1 mice (tPA alone: median [interquartile range], 53.5 [30-355] µl) compared with DNase alone or vehicle controls (both, 0.0 [0.0-0.0] µl) after 6 hours. Fluid induction was reproduced after intrapleural delivery of streptokinase and urokinase, indicating a class effect. Pleural fluid monocyte chemotactic protein (MCP)-1 levels strongly correlated with effusion volume (r = 0.7302; P = 0.003), and were significantly higher than MCP-1 levels in corresponding sera. Mice treated with anti-MCP-1 antibody (P < 0.0001) or MCP-1 receptor antagonist (P = 0.0049) demonstrated a significant decrease in tPA-induced pleural fluid formation (by up to 85%). Our data implicate MCP-1 as the key molecule governing tPA-induced fluid accumulation. The role of MCP-1 in the development of other exudative effusions warrants examination.

Intracerebral transplantation of bone marrow stromal cells ameliorates tissue plasminogen activator-induced brain damage after cerebral ischemia in mice detected by in vivo and ex vivo optical imaging.

Detection and protection of the neurovascular unit (NVU) are essential for treatment of acute stroke patients, especially the use of tissue plasminogen activator (tPA). In the present study, we conducted in vivo and ex vivo optical imaging for detecting activation of matrix metalloproteinases (MMPs) and evaluated the protective effect of intracerebral transplantation of bone marrow stromal cells (BMSCs) obtained from green fluorescent protein (GFP) transgenic (Tg) mice on the NVU in tPA-mediated brain damage after transient middle cerebral artery occlusion (tMCAO) in mice. Compared with the tMCAO group, the tMCAO plus BMSC group showed significant reductions of in vivo and ex vivo fluorescent signals for MMPs at 48 hr after tMCAO, with a partial colocalization of BMSC-GFP signals. Intracerebrally transplanted BMSCs ameliorated MMP-9 activation by immunohistochemistry and Western blot with differentiation into microglial and astroglial cells. Double-immunofluorescence study revealed improved NVU disruption in the tMCAO plus BMSC group. The present study suggests that intracerebral BMSC transplantation reduced MMP activation and subsequent NVU disruption caused by tPA after tMCAO and that this MMP activation and BMSC effect were detectable with in vivo and ex vivo optical imaging.

Distinct effects of tissue-type plasminogen activator and SMTP-7 on cerebrovascular inflammation following thrombolytic reperfusion.

BACKGROUND AND PURPOSE: Thrombolysis therapy using tissue-type plasminogen activator (t-PA) is occasionally accompanied by harmful outcomes, including intracerebral hemorrhage. We have reported that Stachybotrys microspora triprenyl phenol-7 (SMTP-7), a candidate thrombolytic drug, has excellent therapeutic effect on cerebral infarction in embolic stroke model in mice; however, little is known regarding whether this agent influences cerebrovascular inflammation following thrombolytic reperfusion. The current study aimed to compare the effects of recombinant t-PA (rt-PA) and SMTP-7 on cerebrovascular inflammation. METHODS: The impact of rt-PA- and SMTP-7-induced thrombolytic reperfusion on leukocyte dynamics was investigated in a photochemically induced thrombotic middle cerebral artery occlusion (tMCAo) model in mice. RESULTS: Both rt-PA and SMTP-7 administration in tMCAo mice (each 10 mg/kg) resulted in thrombolytic reperfusion. The SMTP-7-administered mice showed relatively mild rolling and attachment of leukocytes to the vascular wall in the middle cerebral vein, with weak peroxynitrite reactions and proinflammatory gene expression (IL-1ß, TNF-α, ICAM-1, and VCAM-1); thus, a small infarct volume compared with rt-PA-administered mice. In vitro study suggested that rt-PA at 20 µg/mL, but not SMTP-7 at a similar concentration, promotes cytokine-induced reactive oxygen species generation in cultured endothelial cells; moreover, SMTP-7 suppressed cytokine-induced VCAM-1 induction in the cells and leukocyte/ endothelial cell adhesions. CONCLUSIONS: Relatively mild cerebrovascular inflammation and cerebral infarction in the SMTP-7 mice, compared with in rt-PA mice, is thought to be caused at least in part by direct antioxidative actions of SMTP-7 in ECs.

Tissue plasminogen activator neurovascular toxicity is controlled by activated protein C.

Although thrombolytic effects of tissue plasminogen activator (tPA) are beneficial, its neurotoxicity is problematic. Here, we report that tPA potentiates apoptosis in ischemic human brain endothelium and in mouse cortical neurons treated with N-methyl-D-aspartate (NMDA) by shifting the apoptotic pathways from caspase-9 to caspase-8, which directly activates caspase-3 without amplification through the Bid-mediated mitochondrial pathway. In vivo, tPA-induced cerebral ischemic injury in mice was reduced by intracerebroventricular administration of caspase-8 inhibitor, but not by caspase-9 inhibitor, in contrast to controls in which caspase-9 inhibitor, but not caspase-8 inhibitor, was protective. Activated protein C (APC), a serine protease with anticoagulant, anti-inflammatory and antiapoptotic activities, which is neuroprotective during transient ischemia and promotes activation of antiapoptotic mechanisms in brain cells by acting directly on endothelium and neurons, blocked tPA vascular and neuronal toxicities in vitro and in vivo. APC inhibited tPA-induced caspase-8 activation of caspase-3 in endothelium and caspase-3-dependent nuclear translocation of apoptosis-inducing factor in NMDA-treated neurons and reduced tPA-mediated cerebral ischemic injury in mice. Data suggest that tPA shifts the apoptotic signal in stressed brain cells from the intrinsic to the extrinsic pathway which requires caspase-8. APC blocks tPA's neurovascular toxicity and may add substantially to the effectiveness of tPA therapy for stroke.

Cytotoxic effects of alteplase, a recombinant tissue plasminogen activator, on human retinal pigment epithelial cells.

PURPOSE: To evaluate the cytotoxic effects of alteplase, a recombinant tissue plasminogen activator, and its additives on human retinal pigment epithelial (hRPE) cells. STUDY DESIGN: Laboratory study. METHODS: We evaluated the cytotoxic effects of alteplase on human fetal RPE (hfRPE) cells, human induced pluripotent stem cell-derived RPE (hiPS-RPE), and ARPE-19 cells, as well as the cytotoxic effects of L-arginine and polysorbate 80, two additives of alteplase, on hfRPE cells. The effects of alteplase on the production of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) from hfRPE cells and the transepithelial resistance (TER) of hiPS-RPE cells were also assessed. The type of cell death induced by alteplase was investigated using ethidium homodimer III and FITC-Annexin V staining and terminal transferase deoxyuridine triphosphatase nick-end labeling. RESULTS: Alteplase reduced the viability of hfRPE cells significantly in a dose- and time-dependent manner. The reaction of hiPS-RPE and ARPE19 cells to alteplase was similar to that of hfRPE cells. Out of L-arginine and polysorbate 80, only treatment with L-arginine significantly reduced the viability of hfRPE cells. Alteplase (83 µg/ml, 6 h) had no significant effect on the production of VEGF and PEDF from hfRPE cells. Alteplase decreased the TER of hiPS-RPE cells in a dose- and time-dependent manner and induced necrosis as the type of cell death. CONCLUSION: Alteplase can be cytotoxic to human RPE cells in a concentration- and time-dependent manner, with L-arginine being a possible causative factor.

Verapamil as an Adjunct Therapy to Reduce tPA Toxicity in Hyperglycemic Stroke: Implication of TXNIP/NLRP3 Inflammasome.

Thrombolytic therapy has remained quite challenging in hyperglycemic patients for its association with poor prognosis and increased hemorrhagic conversions. We recently showed that tissue plasminogen activator (tPA)-induced cerebrovascular damage is associated with thioredoxin-interacting protein (TXNIP) upregulation, which has an established role in the detrimental effects of hyperglycemia. In the present work, we investigated whether verapamil, an established TXNIP inhibitor, may provide protection against hyperglycemic stroke and tPA-induced blood-brain barrier (BBB) disruption. Acute hyperglycemia was induced by intraperitoneal administration of 20% glucose, 15 min prior to transient middle cerebral artery occlusion (tMCAO). Verapamil (0.15 mg/kg) or saline was intravenously infused with tPA at hyperglycemic reperfusion, 1 h post tMCAO. After 24 h of ischemia/reperfusion (I/R), mice were assessed for neurobehavioral deficits followed by sacrifice and evaluation of brain infarct volume, edema, and microbleeding. Alterations in TXNIP, inflammatory mediators, and BBB markers were further analyzed using immunoblotting or immunostaining techniques. As adjunctive therapy, verapamil significantly reduced tPA-induced BBB leakage, matrix metalloproteinase 9 (MMP-9) upregulation, and tight junction protein deregulation, which resulted in lesser hemorrhagic conversions. Importantly, verapamil strongly reversed tPA-induced TXNIP/NLRP3 (NOD-like receptor pyrin domain-containing-3) inflammasome activation and reduced infarct volume. This concurred with a remarkable decrease in high-mobility group box protein 1 (HMGB-1) and nuclear factor kappa B (NF-κB) stimulation, leading to less priming of NLRP3 inflammasome. This preclinical study supports verapamil as a safe adjuvant that may complement thrombolytic therapy by inhibiting TXNIP's detrimental role in hyperglycemic stroke.

Novel plasminogen activator inhibitor-1-derived peptide protects against impairment of cerebrovasodilation after photothrombosis through inhibition of JNK MAPK.

The sole FDA-approved treatment for acute stroke is recombinant tissue-type plasminogen activator (rtPA). However, rtPA aggravates the impairment of cerebrovasodilation induced by global hypoxia/ischemia; this impairment is attenuated by the preinjury treatment with the plasminogen activator inhibitor derivative EEIIMD. MAPK (a family of kinases, p38, and JNK) is upregulated after cerebral ischemia. In this study, we determined whether the novel plasminogen activator inhibitor-derived peptide, Ac-RMAPEEIIMDRPFLYVVR-amide, (PAI-1-DP) given 30 min before or 2 h after, focal central nervous system injury induced by photothrombosis would preserve responses to cerebrovasodilators and the role of p38 and JNK MAPK in such effects. Cerebrospinal fluid JNK and p38 levels were elevated by photothrombotic injury, an effect potentiated by rtPA. Cerebrovasodilation was blunted by photothrombosis and reversed to vasoconstriction by rtPA but restored to dilation by PAI-1-DP pre- and posttreatment. PAI-1-DP blocked JNK, but preserved p38 MAPK upregulation after photothrombosis. The JNK MAPK antagonist SP600125 prevented, and the p38 antagonist SB203580 potentiated, impaired cerebrovasodilation after photothrombosis. These data indicate that rtPA impairs cerebrovasodilation after injury by activating JNK, while p38 MAPK is protective, and that the novel peptide PAI-1-DP protects by inhibiting activation of JNK by rtPA. JNK MAPK inhibitors, including PAI-1-DP, may offer a novel approach to increase the benefit-to-risk ratio of thrombolytic therapy and enable its use in central nervous system ischemic disorders.

Safety of prolonged, repeated administration of a pulmonary formulation of tissue plasminogen activator in mice.

BACKGROUND: Disruption of fibrinolytic homeostasis participates in the pathogenesis of severe lung diseases like acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis (IPF) and plastic bronchitis. We have developed a pulmonary formulation of tissue plasminogen activator (pf-tPA) that withstands nebulization and reaches the lower airways. OBJECTIVE: Since treatment of ARDS, IPF and plastic bronchitis will require repeated administration of pf-tPA, the purpose of this study was to determine the safety of prolonged, repeated administration of pf-mouse tPA (pf-mtPA) to the lungs of healthy mice. METHODS: Male and female B6C3F1 mice received one of two intratracheal (IT) doses of either nebulized pf-mtPA or sterile saline twice daily for 28 days. Weekly blood samples were collected to estimate hematocrit. Following the dosing period, animals were sacrificed for gross necropsy, the acquisition of bronchoalveolar lavage fluid (BALF), and histological assessment of the lungs and other major organs. RESULTS: The low dose of pf-mtPA was well tolerated by both female and male mice. However, female and male mice that received the high dose experienced a 16% and 8% incidence, respectively, of fatal pulmonary hemorrhage. Although male mice had a lower incidence of bleeding, these events occurred at lower mean (+/-S.E.) doses (1.06+/-0.02mg/kg/d) of pf-mtPA compared with females (1.48+/-0.03mg/kg/d, p<0.001). In addition, male mice had higher BALF mtPA concentrations. Bleeding occurred six and 12 days in male and female mice, respectively, after the initiation of dosing suggesting that mtPA accumulated in the lungs. CONCLUSION: This study established a safe dose range and demonstrated the feasibility of prolonged, repeated dosing of pf-tPA. High doses (> or =1mg/kg/d) were associated with pulmonary hemorrhage that may be due, in part, to accumulation of drug in the lungs.

Early Ultrafast Ultrasound Imaging of Cerebral Perfusion correlates with Ischemic Stroke outcomes and responses to treatment in Mice.

In the field of ischemic cerebral injury, precise characterization of neurovascular hemodynamic is required to select candidates for reperfusion treatments. It is thus admitted that advanced imaging-based approaches would be able to better diagnose and prognose those patients and would contribute to better clinical care. Current imaging modalities like MRI allow a precise diagnostic of cerebral injury but suffer from limited availability and transportability. The recently developed ultrafast ultrasound could be a powerful tool to perform emergency imaging and long term follow-up of cerebral perfusion, which could, in combination with MRI, improve imaging solutions for neuroradiologists. Methods: In this study, in a model of in situ thromboembolic stroke in mice, we compared a control group of non-treated mice (N=10) with a group receiving the gold standard pharmacological stroke therapy (N=9). We combined the established tool of magnetic resonance imaging (7T MRI) with two innovative ultrafast ultrasound methods, ultrafast Doppler and Ultrasound Localization Microscopy, to image the cerebral blood volumes at early and late times after stroke onset and compare with the formation of ischemic lesions.Results: Our study shows that ultrafast ultrasound can be used through the mouse skull to monitor cerebral perfusion during ischemic stroke. In our data, the monitoring of the reperfusion following thrombolytic within the first 2 h post stroke onset matches ischemic lesions measured 24 h. Moreover, similar results can be made with Ultrasound Localization Microscopy which could make it applicable to human patients in the future. Conclusion: We thus provide the proof of concept that in a mouse model of thromboembolic stroke with an intact skull, early ultrafast ultrasound can be indicative of responses to treatment and cerebral tissue fates following stroke. It brings new tools to study ischemic stroke in preclinical models and is the first step prior translation to the clinical settings.

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

ETHNOPHARMACOLOGICAL RELEVANCE: Thrombolytic 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 STUDY: We 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. METHODS: Male 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. RESULTS: YQFM 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. CONCLUSION: YQFM 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.

Whole body hypothermia extends tissue plasminogen activator treatment window in the rat model of embolic stroke.

Late treatment with tissue plasminogen activator (tPA) leads to reperfusion injury and poor outcome in ischemic stroke. We have recently shown the beneficial effects of local brain hypothermia after late thrombolysis. Herein, we investigated whether transient whole-body hypothermia was neuroprotective and could prevent the side effects of late tPA therapy at 5.5 h after embolic stroke. After induction of stroke, male rats were randomly assigned into four groups: Control, Hypothermia, tPA and Hypothermia+tPA. Hypothermia started at 5 h after embolic stroke and continued for 1 h. Thirty min after hypothermia, tPA was administrated. Infarct volume, brain edema, blood-brain barrier (BBB) and matrix metalloproteinase-9 (MMP-9) were assessed 48 h and neurological functions were assessed 24 and 48 hour post-stroke. Compared with the control or tPA groups, whole-body hypothermia decreased infarct volume (P < 0.01), BBB disruption (P < 0.05) and MMP-9 level (P < 0.05). However, compared with hypothermia alone a combination of hypothermia and tPA was more effective in reducing infarct volume. While hypothermia alone did not show any effect, its combination with tPA reduced brain edema (P < 0.05). Hypothermia alone or when combined with tPA decreased MMP-9 compared with control or tPA groups (P < 0.01). Although delayed tPA therapy exacerbated BBB integrity, general cooling hampered its leakage after late thrombolysis (P < 0.05). Moreover, only combination therapy significantly improved sensorimotor function as well as forelimb muscle strength at 24 or 48 h after stroke (P < 0.01). Transient whole-body hypothermia in combination with delayed thrombolysis therapy shows more neuroprotection and extends therapeutic time window of tPA up to 5.5 h.

Beneficial actions of prothymosin alpha-mimetic hexapeptide on central post-stroke pain, reduced social activity, learning-deficit and depression following cerebral ischemia in mice.

Prothymosin alpha (ProTα)-mimetic hexapeptide (amino acid: NEVDQE, P6Q) inhibits cerebral or retinal ischemia-induced behavioral, electrophysiological and histological damage. P6Q also abolishes cerebral hemorrhage induced by ischemia with tissue plasminogen activator (tPA). In the present study we examined the beneficial effects of P6Q on other post-stroke prognostic psychology-related symptoms, which obstruct the motivation toward physical therapy. Intravenous (i.v.) administration with tPA (10 mg/kg) at 6 h after photochemically induced thrombosis (PIT) in mice resulted in bilateral central post-stroke pain in thermal and mechanical nociception tests and loss of social activity in the nest building test, both of which were significantly blocked by P6Q (30 mg/kg, i.v.) given at 5 h after PIT. P6Q (30 mg/kg, i.v.) also improved the memory-learning deficit in the step-through test and depression-like behavior in the tail suspension test when it was given 1 day after bilateral common carotid arteries occlusion (BCCAO) in mice. Thus, these studies suggest that P6Q could be a promising candidate to prevent negative prognostic psychological symptoms following focal and global ischemia.

Activated protein C promotes neovascularization and neurogenesis in postischemic brain via protease-activated receptor 1.

Activated protein C (APC) is a serine protease with anticoagulant and direct cytoprotective activities. Early postischemic APC application activates the cellular protein C pathway in brain endothelium and neurons, which is neuroprotective. Whether late APC administration after a transient ischemic attack is neuroprotective and whether APC influences brain repair is not known. Here, we determined safety and efficacy of late APC and tissue-plasminogen activator (tPA) administrations in a mouse model of transient brain ischemia. tPA given at 6 h after onset of ischemia killed all mice within 2 d, whereas APC given at 6 or 24 h after ischemia onset improved significantly functional outcome and reduced spread of the ischemic lesion. At 7 d postischemia, APC multiple dosing (0.8 mg/kg, i.p.) at 6-72 or 72-144 h enhanced comparably cerebral perfusion in the ischemic border by approximately 40% as shown by in vivo lectin-FITC angiography, blocked blood-brain barrier leakage of serum proteins, and increased the number of endothelial replicating cells by 4.5- to 4.7-fold. APC multidosing at 6-72 h or 72-144 h increased proliferation of neuronal progenitor cells in the subventricular zone (SVZ) by 40-50% and migration of newly formed neuroblasts from the SVZ toward the ischemic border by approximately twofold. The effects of APC on neovascularization and neurogenesis were mediated by protease-activated receptor 1 and were independent of the reduction by APC of infarction volume. Our data show that delayed APC administration is neuroprotective and mediates brain repair (i.e., neovascularization and neurogenesis), suggesting a significant extension of the therapeutic window for APC intervention in postischemic brain.

Antifibrinolytic agents reduce tissue plasminogen activator-mediated neuronal toxicity in vitro.

INTRODUCTION: Serine proteases and their inhibitors play an important role in physiological homeostasis including neuronal activity, hemostasis, and wound healing. Tissue plasminogen activator (tPA) is involved in normal neuronal plasticity and memory formation but can also be neurotoxic. We hypothesized that the serine protease inhibitor aprotinin confers neuronal protection by inhibiting tPA activity. METHODS: Using cultured rat dopaminergic neuroblasts (N27 line), tPA-induced cytotoxicity was quantitated by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and flow cytometry using propidium iodide DNA staining. The anti-apoptotic effects of aprotinin and other protease inhibitors were also evaluated using these systems. RESULTS: Treatment of cultured neuroblasts with tPA (10-20 microg/ml) caused a dose-dependent decrease in cell viability (71.3+/-2.4 at 10 microg/ml down to 52.7+/-2.5% at 20 microg/m tPA, 24-h treatment), which was potentiated in the absence of serum in the culture medium (59.5+/-6.3% at 10 microg/ml down to 47.9+/-4.7% at 20 microg/ml). Aprotinin was effective in ameliorating cell death when administered 30 min before tPA exposure as shown by increased cell viability (91.8+/-0.6% at tPA at 20 microg/ml), but this protection was significantly reduced when aprotinin was administered after tPA. The efficacy of aprotinin as a neuroprotectant was equivalent or superior to other direct tPA antagonist peptides Glu-Gly-Arg-chlormethylketone (EGRck) and Phe-Pro-Arg-chlormethylketone (FPRck) in this setting. CONCLUSION: These data suggest that one of the mechanisms of neuroprotection afforded by aprotinin may be inhibition of tPA-mediated neurotoxicity.