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Intracerebral hemorrhage (ICH), a devastating form of stroke, has a mortality of 40% and no available treatment. Inflammation contributes to the genesis and expansion of perihematomal edema (PHE) in ICH, thus driving secondary injury and subsequent neurological deterioration. This study was carried out to elucidate contributions of lymphocytes, specifically CD4 + cells in the formation of PHE and ICH-induced neural injury. Upon examination of brain sections of ICH patients, we observed abundant CD4 + T cells among the PHE-infiltrating immune cells as early as 24 hours post-ictus. Analogous findings in ICH mouse model induced with autologous blood or collagenase injection confirmed the phenomenon. Using anti-CD4 antibody to deplete CD4 + T cells, we find that brain-infiltrating CD4 + T cells exacerbates acute ICH injury in mice by reducing BBB integrity and promoting leukocytes recruitment with early and time-dependent CD4 + T cells activation. Brain infiltrating CD4 + T cells exhibit activated transcriptome signatures, promoting local inflammation via IL-17. In the hemorrhagic brain, CD4 + T cells induces CD31 + endothelial cell apoptosis via death receptor-ligands TRAIL-DR5 pathway; specific ablation of CD4 + T cells with depleting antibody results in reduced PHE volume and improved long-term neurological outcome. In all, these data sheds new lights on T cell-mediated immune responses contributing to PHE and brain injury in ICH.

Introduction: Type 2 diabetes mellitus (T2DM) is a major comorbidity exacerbating ischemic brain injury and impairing post-stroke recovery. Our previous study suggested that recombinant human fibroblast growth factor (rFGF) 21 might be a potent therapeutic targeting multiple aspects of pathophysiology in T2DM stroke. As a continuation of our previous work investigating the therapeutic effects of rFGF21 in T2DM stroke, this study aims to evaluate the potential effects of rFGF21 on cerebrovascular remodeling during a delayed phase after T2DM stroke. Methods: Permanent distal middle cerebral artery occlusion was performed in heterozygous non-diabetic db/+ and homozygous diabetic db/db mice. Daily rFGF21 administration was initiated one week after stroke induction and maintained for up to two weeks thereafter. Multiple markers associated with post-stroke recovery, including angiogenesis, oligodendrogenesis, white matter integrity and neurogenesis, were assessed up to three weeks after stroke. Results: Our results showed an impairment in post-stroke vascular remodeling under T2DM condition, reflected by the decreased expression of trophic factors in brain microvessels and impairments of angiogenesis. The defected cerebrovascular remodeling was accompanied by the decreased oligodendrogenesis and neurogenesis. However, delayed rFGF21 administration normalized post-stroke hyperglycemia and improved neurological outcomes, which may partially via the promotion of pro-angiogenic trophic factor expression in brain microvessels and cerebrovascular remodeling. The better cerebrovascular remodeling may also contribute to oligodendrogenesis, white matter integrity and neurogenesis after T2DM stroke. Conclusion: Delayed rFGF21 administration may improve neurological outcomes in T2DM stroke mice, at least in part by normalizing the metabolic abnormalities and promoting cerebrovascular remodeling and white matter repair.

Background and Purpose— Dual antiplatelet treatment poses a risk for increased hemorrhagic transformation (HT) following intravenous thrombolysis and mechanical thrombectomy. The aim of this study was to implement a model of experimental stroke with tissue-type plasminogen activator (tPA)-associated HT in mice on dual antiplatelet treatment to enable mechanistic studies and also to allow for an initial assessment of therapeutic approaches to limit HT. Methods— Male C57BL6 mice were fed with Aspirin and Clopidogrel via drinking water for 3 days. Subsequently, mice were subjected to 2-hour transient middle cerebral artery occlusion, and tPA was infused when indicated. HT was quantified by measuring hemorrhaged areas in brain sections with ImageJ. TTC staining was used to determine infarct size. Platelet function was tested in vitro using flow cytometry and in vivo with standard tail bleeding tests. Results— Both flow cytometry and tail bleeding volumes indicated significantly reduced platelet function following Aspirin and Clopidogrel treatment. While tPA administered 2 hours after onset of middle cerebral artery occlusion did not cause bleeding in control mice (0.51±0.13 mm2), HT significantly increased by 18.9±5.4 mm2 ( P =0.0045) in Aspirin and Clopidogrel mice treated with tPA. HT in aspirin and clopidogrel mice not treated with tPA was nonsignificantly elevated by 8.0±4.6 mm 2 ( P =0.3784) compared with controls. Infarct sizes did not differ between groups. The HT persisted when the tPA dosage was reduced. Conclusions— We successfully established a translational stroke model of tPA treatment under dual antiplatelet treatment. The impaired platelet function led to an increased risk for HT in tPA-treated mice. Reducing the dosage of tPA did not prevent this hemorrhagic complication.

Background and Purpose: Our previous studies demonstrated that recombinant human Fibroblast growth factor 21 (rFGF21), an endocrine member of the FGF family is potently beneficial for improving long-term neurological outcomes of type 2 diabetes (T2D) stroke mice. Here we tested hypothesis that rFGF21 protects against post-stroke BBB damage by PPARγ activation of cerebral micovascular endothelium. Methods: T2D db/db mice and their non-diabetic counterparts db/+ mice were subjected to focal stroke of dMCAO. Four experimental groups: 1) db/+ stroke, 2) db/db stroke, 3) db/db stroke+rFGF21, and 4) db/db stroke+rFGF21+GW9662. rFGF21 (1.5mg/kg, i.p.) was injected at 6 hours after stroke and PPARγ inhibitor GW9662 (4mg/kg, i.p.) was injected 30 min prior to rFGF21 treatment. At 24 hours post-stroke, we collected peri-infarct nuclear fraction for PPARγ-DNA binding activity assay using EMSA, microvascular isolation for RT-PCR analyzing mRNA levels of proteins constituting BBB junctional complex (occluding, clauding-5, VE-cadherin and ZO-1) and PPARγ targeted downstream genes (CD36 and FABP4) as indicators of PPARγ activity in microvasculature. BBB permeability was assessed by measuring 3kDa FITC-dextran or Evans blue extravasations at 48 hour post-stroke. Results: Ischemic stroke induced a significant decrease of PPARγ-DNA binding activity and mRNA levels of BBB junctional proteins in peri-infarct area, and a significant increase of BBB permeability in diabetic db/db stroke mice compared to db/+ stroke mice. Changes of mRNA levels of CD36 and FABP4 in brain microvascular isolation were consistent with changes of PPARγ-DNA binding activity. rFGF21 administration significantly increased PPARγ-DNA binding activity, elevated mRNA levels of BBB junctional complex proteins and ameliorated BBB leakage. However, pre-treatment of GW9662 partially abolished the post-stroke BBB protective effects of rFGF21. Conclusions: rFGF21 has strong protective effects in acute BBB leakage after stroke, and the underlying mechanisms is partially via increase in PPARγ-DNA binding activity and mRNA expression of BBB junctional complex proteins. Together with our previous investigations, rFGF21 might be a promising candidate for treating diabetic stroke.

Background: High blood pressure (BP) is a well recognized risk factor for ischemic stroke, and aggressive BP control is critical for secondary stroke prevention. However, hypertensive (HTN) outpatients are often found to have uncontrolled BP, in part due to poor compliance with daily at-home BP monitoring and consequent poor medication adherence. Here, we explore the association of patient home BP self-monitoring compliance with stroke recurrence. Method: Ischemic stroke patients with pre-existing HTN were prospectively recruited in accordance with IRB protocol for this observational study. All patients were trained on BP self-monitoring as per standard of care, and were asked to monitor BP at home daily consistently for 2 weeks. BP monitoring compliance, per completion of BP log, was assessed at each follow-up clinical visit by an investigator blinded to patient outcome. Stroke recurrence was followed for 5 years and independently adjudicated by two vascular neurologists. Result: In a total of 219 patients, only 36 patients (16.4%) were compliant in completing the home BP log as instructed. The compliant and non-compliant groups showed no baseline difference and no medication usage difference (p>0.05) (Table 1). Compliance with at-home BP monitoring was associated with well controlled systolic (p Conclusion: Regular home BP monitoring is associated with lower risk of stroke recurrence in HTN stroke patients. Study in expanded patient cohort is ongoing to leverage technology for better home BP monitoring and to create incentives for compliant behavior for better secondary stroke prevention.

Background: Hypertension (HTN) is a well-recognized risk factor for vascular cognitive decline. Poorly controlled HTN is common in the outpatient setting, with multi-factorial components including a lack of consistent, continuous and accurate reporting of blood pressure (BP) to help clinicians to tailor therapy. In the current prospective study, we explore the association of stroke patients’ at-home BP self-monitoring compliance with clinical and imaging risk markers of developing cognitive impairment. Method: 146 consecutive stroke outpatients with diagnosed HTN were recruited per IRB protocol. All patients received BP education at their first clinical visit and were encouraged to monitor BP at home on a daily basis for two weeks three times during the course of the 5-year study. Compliance with BP monitoring was evaluated by completed BP log and questionnaire in follow-up visits at fixed intervals. At the end of the study, white matter disease (WMD) severity was estimated by Fazekas and Sheltens scores. Cognitive impairment was assessed using Montreal Cognitive Assessment (MoCA) scales. Result: Only 16.4% (n=24) patients had “good compliance,” monitoring their home BP regularly and consistently as instructed. The compliant group had better controlled systolic and diastolic BP (Figure A, p Conclusion: Compliance with at-home BP monitoring is essential for better BP control in vascular cognitive impairment (VCI) patients post stroke. Consistent home BP monitoring resulted in decreased imaging-apparent small vessel disease and better long-term cognitive performance. Expanded study with a larger patient cohort is ongoing to account for other behavioral and psycho-social factors and to create improved home monitoring technology and treatment strategies.

Background and Purpose: DC007D is a novel small molecule designed to have thrombolytic and free radical scavenging activities. In this study, we aimed to test the potential neuroprotective effects of DC007D in embolic clot model of focal ischemic stroke in rats. Methods: All animals received intravenous infusion of either saline, 10 mg/kg DC007D or 10 mg/kg tPA at each experimental time point (1.5, 3 and 4.5 hrs after stroke, respectively). In first set of experiments, animals were treated at 1.5-hr after stroke. We performed a time course of multi-parametric MRI analysis including perfusion, diffusion and T2 relaxation. Brain infarction size and swelling rate were measured by TTC staining at 24 hrs after stroke. Next, in 3- and 4.5-hrs treatment experiments, we examined acute brain infarct volume, brain swelling rate, hemorrhagic transformation, neurological deficits. Additionally, we also tested potentially direct neuroprotection effects of DC007D in a mechanical filament pMCAO rat model. Lastly, the effect of DC007D in tPA activity in vitro, INR, and serum levels of tPA and PAI-1 activities in vivo at 24 hrs after stroke were assessed and compared. Results: Both tPA and DC007D treated at 1.5 hrs after stroke reduced hypoperfused area, diffusion, T2 lesion size and TTC-stained infarction. When treated at 3 hrs after stroke, DC007D had significantly better therapeutic effects in reducing brain infarction size, swelling rate and hemorrhagic transformation compared to conventional tPA treatment group. When treated at 4.5 hrs after stroke, both tPA and DC007D had no effects in brain infarction. However, tPA, but not DC007D, significantly increased brain swelling and intracerebral hemorrhagic transformation compared to saline controls. Our experimental results also showed that DC007D failed to have direct neuroprotection in the mechanical pMCAO rat model. Lastly, DC007D did not interfere tPA activity in vitro, and also did not significantly alter INR, serum levels of tPA and PAI-1 activities post stroke in vivo. Conclusions: DC007D is neuroprotective to embolic focal stroke of rats, likely via tPA/PAI-1 activity independent thrombolytic mechanism. It might be developed as a new and novel thrombolytics or adjunct for tPA thrombolytic reperfusion therapy.

Background: Although growing evidence has suggested that elderly stroke patients can benefit from intravenous tissue plasminogen activator (tPA), impaired functional outcome and increased risk of hemorrhagic transformation have also been frequently observed in those patients after tPA treatment. By cleaving von Willebrand factor (vWF), ADAMTS13 (A Disintegrin and Metalloproteinase with Thrombospondin motifs 13) is involved in arterial thrombosis and tPA regulation. Recent studies have identified decreased ADAMTS13 activity and increased vWF levels with aging and vascular cognitive impairment, which may put elderly individuals in a prothrombotic status at baseline. We therefore hypothesized that the ADAMTS13/vWF axis may also play a role in regulating the age effect on tPA response. Method: Consecutive tPA-treated ischemic stroke patients were prospectively recruited in accordance with IRB protocol. Peripheral venous blood was sampled at 12, 24 and 72hr post tPA administration. Result: A total of 173 patients were recruited, of whom 128 were younger than 70 years and 45 were older than 70 years. Impaired tPA response (mRankins > 2 at 3 months) was observed in patients ≥70 years compared to patients Conclusion: Our results suggest that the ADAMTS13/vWF axis may contribute to impaired tPA response in older stroke patients and that ADAMTS13 may be a potential adjunct therapy to enhance the tPA efficacy. This proof of concept data may pave the way for future clinical trials.

Introduction: Thrombolytic treatment under dual antiplatelet therapy (DAPT) is usually performed with great restrictions due to an increased risk of hemorrhagic transformation (HT). This translational study aimed to explore the risk of tPA-associated HT under DAPT with Aspirin and Clopidogrel (ASA+CPG), particularly focusing on the evaluation of recently developed clinical strategies to reduce HT. To monitor DAPT efficiency and to further investigate tPA effects on platelets, we successfully established a flow cytometry based test of platelet function. Hypothesis: The impaired platelet function after DAPT contributes to tPA associated HT following ischemic stroke. Methods: Male C57/BL6 mice were fed with ASA+CPG or placebo via drinking water for 3 days. Platelet function was assessed in vitro using flow cytometry. Animals were subjected to 2h MCAO, followed by tPA treatment when indicated. 24h later standard tail bleeding tests were conducted and HT was quantified by measuring hemorrhaged areas on the brain surface and in brain sections using ImageJ. Sample sizes varied between 8 to 15 mice per group. Results: In ASA+CPG treated mice, platelet function (indicated by the expression of CD62P) was significantly reduced by 40.55±4.23% (p Conclusions: Using our flow cytometry-based platelet function assay, we demonstrated that ASA+CPG treatment reduces CD62P expression rates, which is a reliable indicator of impaired platelet function. Investigating the in vivo effects of this impairment, we found that, ASA+CPG results in significant hemorrhage in mice given 2h MCAO and tPA administration, when using both low- or high-dose tPA.

Introduction: Ischemic strokes are frequently accompanied by subsequent hemorrhage, particularly in patient subpopulations treated with anticoagulants or platelet inhibitors. In an AHA-funded study, we are investigating how symptomatic hemorrhagic transformation (HT) occurs subsequent to experimental ischemic stroke in mice. Hypothesis: In various settings where HT contributes to stroke pathology, increased vascular 12/15-lipoxygenase (12/15-LOX) is central to a vascular injury program that causes HT. Methods: Mice were pretreated with warfarin for 24h, or with aspirin plus clopidogrel (ASA/CPG) for 72h, in both cases added to the drinking water. Control mice in both cases received regular drinking water. The filament model was used to induce MCAO in C57Bl6J mice for the times indicated. tPA was infused i.v. at the time of reperfusion, and the 12/15-LOX inhibitor ML351 or vehicle were injected i.p. Shortwave infrared (SWIR) imaging of i.v. injected fluorescent quantum dot nanoparticles was used in some mice to detect early HT in vivo, and used to determine the appropriate time point for sacrifice. Immunohistochemistry of brain sections was used to further analyze the hemorrhage and surrounding areas. Results: Pretreatment with either warfarin or with aspirin plus clopidogrel led to increased 12/15-LOX predominantly in the vasculature of mice subjected to experimental stroke. SWIR imaging of brain sections taken at the early stages of hemorrhage showed 12/15-LOX specifically increased near sites of hemorrhage. ASA/CPG pretreatment combined with tPA at the time of reperfusion led to increased HT, which was significantly reduced when the 12/15-LOX inhibitor ML351 was administered. Conclusions: 12/15-LOX is increased in the vasculature under conditions that favor HT, suggesting it may have a causal role. Inhibition of 12/15-LOX may be considered as a strategy to limit HT of ischemic strokes.

Background and Purpose— The complexity and heterogeneity of stroke, as well as the associated comorbidities, may render neuroprotective drugs less efficacious in clinical practice. Therefore, the development of targeted therapies to specific patient subsets has become a high priority in translational stroke research. Ischemic stroke with type 2 diabetes mellitus has a nearly double mortality rate and worse neurological outcomes. In the present study, we tested our hypothesis that rFGF21 (recombinant human fibroblast growth factor 21) administration is beneficial for improving neurological outcomes of ischemic stroke with type 2 diabetes mellitus. Methods— Type 2 diabetes mellitus db/db and nondiabetic genetic control db/+ mice were subjected into permanent focal ischemia of distal middle cerebral artery occlusion, we examined the effects of poststroke administration with rFGF21 in systemic metabolic disorders, inflammatory gatekeeper PPARγ (peroxisome proliferator-activated receptor γ) activity at 3 days, mRNA expression of inflammatory cytokines and microglia/macrophage activation at 7 days in the perilesion cortex, and last neurological function deficits, ischemic brain infarction, and white matter integrity up to 14 days after stroke of db/db mice. Results— After permanent focal ischemia, diabetic db/db mice presented confounding pathological features, including metabolic dysregulation, more severe brain damage, and neurological impairment, especially aggravated proinflammatory response and white matter integrity loss. However, daily rFGF21 treatment initiated at 6 hours after stroke for 14 days significantly normalized systemic metabolic disorders, rescued PPARγ activity decline, inhibited proinflammatory cytokine mRNA expression, and M1-like microglia/macrophage activation in the brain. Importantly, rFGF21 also significantly reduced white matter integrity loss, ischemic brain infarction, and neurological function deficits up to 14 days after stroke. The potential mechanisms of rFGF21 may in part consist of potent systematic metabolic regulation and PPARγ-activation promotion-associated antiproinflammatory roles in the brain. Conclusions— Taken together, these results suggest rFGF21 might be a novel and potent candidate of the disease-modifying strategy for treating ischemic stroke with type 2 diabetes mellitus.

Background: Hereditary hypercoagulation is an important risk factor in patent foramen ovale (PFO) related cryptogenic stroke (CS). Body mass index (BMI) has been associated with venous thrombosis by genetic and hereditary factors. Since the pathogenesis of PFO-related stroke is considered to be due to paradoxical thrombus from the venous system, it is imperative to explore the relationship between hereditary hypercoagulation and BMI. Methods: Eligible patients were consecutively and prospectively recruited in accordance with an IRB. All patients underwent full evaluation of stroke etiopathogenesis, including MRI/MRA, long-term (>30 days) cardiac telemetry, echocardiography, and extensive hypercoagulable screening (protein S, protein C, FV Leiden , PTGM, ATIII, APLAb, lupus anticoagulant, lipoprotein(a), homocysteine). Results: In 1,131 consecutively recruited acute ischemic stroke patients, 59 (17.1%) were diagnosed with PFO-related CS and hereditary hypercoagulable state. BMI was statistically significantly higher in patients with hereditary hypercoagulable state (36.01±8.88 vs. 29.14±6.58, P=0.023). Higher BMI (>=35.5) can independently predict hereditary hypercoagulable state in PFO CS with high specificity (90.9%, AUC 0.72, p=0.05) compared to other stroke risk factors. Conclusion: We found that hereditary hypercoagulation was more common in obese PFO-related CS patients, independent of DM status. While metabolic syndrome is well studied in diabetic patients with CVD, to our knowledge, this is first report of its importance in PFO-related CS. Further studies are needed to validate these initial findings.

Background: May-Thurner Syndrome (MTS) is a potential risk factor for cryptogenic stroke (CS). While retrospective reports have found important association with MTS in CS, prospective data has been lacking. In this study, we prospectively explored the epidemiology and clinical correlates of MTS patients with cryptogenic stroke. Method: Eligible patients were prospectively recruited in accordance with an IRB-approved protocol. All patients underwent full evaluation of etiopathogenesis of stroke such as MRI/MRA, long-term cardiac telemetry (>30 days), echocardiography, and extensive thrombophilia screen (AT3, FV Leiden, Proteins C and S; prothrombin gene mutations, APLAbs, lipoprotein(a), homocysteine) and 1.5T pelvic MRV. Results: We prospectively recruited 1,131 acute ischemic stroke patients, with 59 patients with PFO-related CS; 15 (40.5%) had May-Thurner anatomy (MTA), and 6 (16.2%) had MTS. MTS pts are younger (36.33±9.95 vs. 53.35±14.78, p=0.011), with a lower trend for BMI (24.40±4.87 V.S. 31.13±8.75, p=0.051, table 1). Lower BMI is predictive of MTS in CS ( Conclusion: In this prospective study, we found MTA and MTS to be very common in PFO CS patients - especially in younger patients with lower BMI and without traditional stroke risk factors. MTS is a novel risk factor that may aide in the diagnosis of PFO CS, and needs further study.

Background: Stroke caused by paroxysmal atrial fibrillation (PAF) can be more severe than other cryptogenic stroke (CS), with higher recurrence rates and requiring different prevention strategies. Therefore, it is important to identify PAF early. Studies showed that age and PR-interval predict PAF, but with only moderate reliability. Additional PAF risk predictors are direly needed to triage cost-effective workup in real time clinical care. Since studies show strong association between white matter lesions (WML) and AFib, we explore whether chronic WML found during acute stroke hospitalization can prospectively predict the risk of PAF after discharge in CS patients. Method: Eligible patients were prospectively recruited in accordance with IRB. All patients underwent full cryptogenic stroke workup including MRI/MRA, outpatient cardiac monitor (>30 days), cardiac echo, and hypercoagulable testing. Results: Of 1,131 acute ischemic stroke patients enrolled, 126 (11.1%) were diagnosed with first ever CS at discharge. 18 pts were diagnosed with PAF at 1 year after discharge. PAF pts were older, with more incidence of cancer and diabetes, and more severe WML burden (table ). Total Scheltens score independently predicts post-discharge PAF in CS with cut-off value >=6 (sensitivity 76.47%, specificity 62%, p=0.013). Conclusion: We found chronic WML burden can prospectively predict later PAF risk in CS stroke patients at acute hospital discharge. To better triage cost-effective outpatient PAF workup, readily available imaging characteristics from acute hospitalization may be an important adjunct for future PAF risk prediction. A larger cohort with additional risk factors is needed to improve prospective prognostic value of WML in the prediction of PAF.

Background and Purpose— Poststroke hyperglycemia is associated with resistance to tissue plasminogen activator (tPA) reperfusion, higher risk of intracerebral hemorrhage, and worse neurological outcomes. In this study, we asked whether minocycline combined with intravenous tPA may ameliorate inflammation and brain injury after focal embolic stroke in type 1 diabetic rats. Methods— Type 1 diabetic rats were subjected to a focal embolic stroke. Three treatment groups were used: (1) saline at 1.5 hours after stroke; (2) tPA alone at 1.5 hours after stroke; (3) combined minocycline (intravenously) at 1 hour plus tPA at 1.5 hours, and second treatment of minocycline (intraperitoneally) at 12 hours after stroke. Acute brain tissue damages were assessed at 24 hours after stroke. Inflammatory biomarkers interleukin-1β and matrix metalloproteinases 2 and 9 were examined in plasma. Neutrophil infiltration, microglia activation, matrix metalloproteinase activation, and degradation of the tight junction protein claudin-5 were examined in the brain. Results— Compared with saline or tPA alone treatments, minocycline plus tPA combination therapy significantly reduced brain infarction, intracerebral hemorrhage, and hemispheric swelling at 24 hours after stroke. The combination also significantly suppressed the elevated plasma levels of matrix metalloproteinase-9 and interleukin-1β up to 24 hours after stroke. At 16 hours after stroke, neutrophil infiltration, microglia activation, matrix metalloproteinase-9, and tight junction protein claudin-5 degradation in the peri-infarct brain tissues were also significantly attenuated by the combination therapy. Conclusions— Combination therapy with minocycline plus tPA may be beneficial in ameliorating inflammation and reducing infarction, brain swelling, and hemorrhage after ischemic stroke with diabetes mellitus/hyperglycemia.

Background and Purpose— Therapeutic effects of early insulin glycemic control for poststroke hyperglycemia in combination with tissue-type plasminogen activator (tPA) thrombolytic therapy have not yet been studied but are of great clinical interest. In this study, we tested the effects of insulin plus tPA combination in a model of focal embolic stroke in Type I diabetic rats. Methods— Streptozotocin was used to produce Type I diabetes in male Wistar rats for 6 weeks and then embolic focal strokes were induced. All rats were treated with insulin or saline at 1 hour followed by tPA or saline at 1.5 hour after stroke. Mortality, infarction, hemispheric swelling, hemorrhagic transformation, and perfusion defects were examined at 24 hours after stroke. Total plasma plasminogen activator inhibitor-1 antigen and activity levels were measured before stroke and 1.5, 3, and 6 hours after stroke by ELISA. Results— Early insulin glycemic control alone or tPA thrombolysis alone had no significant effects on ischemic infarction. However, early insulin glycemic control combined with tPA significantly reduced brain infarction and swelling, ameliorated tPA-associated hemorrhagic transformation, and improved plasma perfusion at 24 hours after stroke. We also found that the combination significantly decreased plasma plasminogen activator inhibitor-1 antigen level at 6 hours and plasminogen activator inhibitor-1 activity at 1.5 and 6 hours after stroke. Conclusions— Early insulin glycemic control may be beneficial in combination with tPA thrombolysis for ischemic stroke with diabetes mellitus or poststroke hyperglycemia.

Background and Purpose— Poststroke hyperglycemia and diabetes mellitus are associated with lower thrombolytic efficacy and an increased risk of postischemic cerebral hemorrhage. We aimed to develop a rodent model of thrombolysis in diabetic stroke that mimics the clinical situation. Method— Male 6-week Type I diabetic rats (14 weeks old) were subjected to embolic focal stroke and treated with tissue-type plasminogen activator at 1.5 hours. Reperfusion and 24-hour neurological outcomes were measured and compared with nondiabetic control rats. Results— Diabetic rats exhibited resistance to thrombolytic reperfusion, larger infarction volumes, and increased intracerebral hemorrhage. Conclusions— This animal model would be relevant to future studies investigating pathophysiological mechanisms and in developing new therapeutic approaches to enhance the efficacy of tissue-type plasminogen activator thrombolysis in stroke patients with diabetes or poststroke hyperglycemia.

Background and Purpose— Normobaric oxygen (NBO) therapy may be neuroprotective in acute ischemic stroke. However, how NBO may affect intracerebral hemorrhage is unclear. We tested NBO in a rat model of striatal intracerebral hemorrhage. Methods— Intracerebral hemorrhage was induced by stereotactic injection of collagenase Type VII (0.5 U) into the right striatum of male Sprague-Dawley rats. One hour later, rats were randomized into controls (n=13) versus NBO treatment (n=13). NBO was applied for 2 hours. Hemorrhagic blood volume, brain water content, and neurological outcomes (forelimb placement test, forelimb asymmetry, neuroscore) were quantified at 72 hours. Experiments were repeated in a second independent laboratory to assess reproducibility in neurological outcomes (n=10 per group). Results— NBO did not worsen hemorrhage severity or brain edema. There were no significant differences in hemorrhagic blood volumes (control, 6.4±0.9 μL versus NBO, 7.0±2.1 μL; P =0.18) or brain water content (control, 81.9%±1.1% versus NBO, 81.6%±0.5%; P =0.58). NBO did not affect any of the neurological outcome tests in the primary or secondary studies. Conclusions— NBO therapy may not worsen outcomes in intracerebral hemorrhage.

Background and Purpose— Thrombolytic therapy with tissue plasminogen activator (tPA) in ischemic stroke is limited by increased risks of cerebral hemorrhage and brain injury. In part, these phenomena may be related to neurovascular proteolysis mediated by matrix metalloproteinases (MMPs). Here, we used a combination of pharmacological and genetic approaches to show that tPA promotes MMP-9 levels in stroke in vivo. Methods— In the first experiment, spontaneously hypertensive rats were subjected to 3 hours of transient focal cerebral ischemia. The effects of tPA (10 mg/kg IV) on ischemic brain MMP-9 levels were assessed by zymography. In the second experiment, wild-type (WT) and tPA knockout mice were subjected to 2 hours of transient focal cerebral ischemia, and MMP-9 levels and brain edema during reperfusion were assessed. Phenotype rescue was performed by administering tPA to the tPA knockout mice. Results— In the first experiment, exogenous tPA did not change infarct size but amplified MMP-9 levels in ischemic rat brain at 24 hours. Coinfusion of the plasmin inhibitor tranexamic acid (300 mg/kg) did not ameliorate this effect, suggesting that it was independent of plasmin. In the second experiment, ischemic MMP-9 levels, infarct size, and brain edema in tPA knockouts were significantly lower than WT mice. Administration of exogenous tPA (10 mg/kg IV) did not alter infarction but reinstated the ischemic MMP-9 response back up to WT levels and correspondingly worsened edema. Conclusions— These data demonstrate that tPA upregulates brain MMP-9 levels in stroke in vivo, and suggest that combination therapies targeting MMPs may improve tPA therapy.

Reperfusion therapy with tissue plasminogen activator (tPA) is a rational therapy for acute ischemic stroke. Properly titrated use of tPA improves clinical outcomes. However, there is also an associated risk of hemorrhagic transformation after tPA therapy. Emerging data now suggest that some of these potentially neurotoxic side effects of tPA may be due to its signaling actions in the neurovascular unit. Besides its intended role in clot lysis, tPA is also an extracellular protease and signaling molecule in brain. tPA mediates matrix remodeling during brain development and plasticity. By interacting with the NMDA-type glutamate receptor, tPA may amplify potentially excitotoxic calcium currents. At selected concentrations, tPA may be vasoactive. Finally, by augmenting matrix metalloproteinase (MMP) dysregulation after stroke, tPA may degrade extracellular matrix integrity and increase risks of neurovascular cell death, blood–brain barrier leakage, edema, and hemorrhage. Understanding these pleiotropic actions of tPA may reveal new therapeutic opportunities for combination stroke therapy.

Background and Purpose — Apoptotic-like pathways may contribute to brain cell death after intracerebral hemorrhage. In this study, we used a simplified in vitro model of hemoglobin neurotoxicity to map the caspase cascades involved and to document the role of oxidative stress. Methods — Primary neuronal cultures were obtained from rat cerebral cortex and exposed to hemoglobin to induce cell death. Cytotoxicity was assessed via measurements of mitochondrial viability (MTT assay) and lactate dehydrogenase (LDH assay). Activation of caspase-3, -8, and -9 was measured by Western blot and enzyme activity assays. Various caspase inhibitors (zVADfmk, zDEVDfmk, zIETDfmk, and zLEHDfmk) were tested for neuroprotective efficacy. The role of oxidative stress was assessed with the use of U83836E as a potent scavenger of free radicals. Results — Exposure of primary cortical neurons to hemoglobin induced a dose- and time-dependent cytotoxicity. Western blots showed upregulation of cleaved caspase-3. Enzyme assays showed an increase in caspase-9–like and caspase-3–like activity. However, caspase inhibition did not result in neuroprotection. In contrast, the free radical scavenger U83836E significantly reduced hemoglobin-induced neuronal death. Combination treatment with both U83836E and the broad spectrum caspase inhibitor zVADfmk did not yield additional protection. Conclusions — Upstream and downstream caspases were upregulated after hemoglobin-induced neurotoxicity in vitro, but only an antioxidant approach with a potent free radical scavenger significantly improved neuronal survival. These data suggest that in addition to the activation of caspase cascades, parallel pathways of oxidative stress may predominate in this model of hemoglobin neurotoxicity.

Background and Purpose— We explored the hypothesis that injured neurons release lipocalin-2 as a help me signal. Methods— In vivo lipocalin-2 responses were assessed in rat focal cerebral ischemia and human stroke brain samples using a combination of ELISA and immunostaining. In vitro, microglia and astrocytes were exposed to lipocalin-2, and various markers and assays of glial activation were quantified. Functional relevance of neuron-to-glia lipocalin-2 signaling was examined by transferring conditioned media from lipocalin-2–activated microglia and astrocytes onto neurons to see whether activated glia could protect neurons against oxygen–glucose deprivation and promote neuroplasticity. Results— In human stroke samples and rat cerebral ischemia, neuronal expression of lipocalin-2 was significantly increased. In primary cell cultures, exposing microglia and astrocytes to lipocalin-2 resulted in glial activation. In microglia, lipocalin-2 converted resting ramified shapes into a long-rod morphology with reduced branching, increased interleukin-10 release, and enhanced phagocytosis. In astrocytes, lipocalin-2 upregulated glial fibrillary acid protein, brain-derived neurotropic factor, and thrombospondin-1. Conditioned media from lipocalin-2–treated astrocytes upregulated synaptotagmin, and conditioned media from lipocalin-2–treated microglia upregulated synaptophysin and post-synaptic density 95 (PSD95) and protected neurons against oxygen–glucose deprivation. Conclusions— These findings provide proof of concept that lipocalin-2 is released by injured neurons as a help me distress signal that activates microglia and astrocytes into potentially prorecovery phenotypes.

Aims: Plasminogen activator inhibitor-1 (PAI-1) is the main and potent endogenous tissue-type plasminogen activator (tPA) inhibitor, but an important question on whether PAI-1 in blood stream responds and interferes with the exogenously administered tPA remains unexplored. We for the first time investigated temporal profiles of PAI-1 concentration and activity in circulation after stroke and tPA administration in rats. Methods: Permanent MCAO focal stroke of rats were treated with saline or 10mg/kg tPA at 3 hours after stroke (n=10 per group). Plasma (platelet free) PAI-1 antigen and activity levels were measured by ELISA at before stroke, 3, 4.5 (1.5 hours after saline or tPA treatments) and 24 hours after stroke. Since vascular endothelial cells and platelets are two major cellular sources for PAI-1 in circulation, we measured releases of PAI-1 from cultured endothelial cells and isolated platelets after direct tPA (4 μg/ml) exposures for 60 min in vitro by ELISA (n=4 per group). Results: At 3 hours after stroke, both plasma PAI-1 antigen and activity were significantly increased (3.09±0.67, and 3.42±0.57 fold of before stroke baseline, respectively, all data are expressed as mean±SE). At 4.5 hours after stroke, intravenous tPA administration significantly further elevated PAI-1 antigen levels (5.26±1.24), while as expected that tPA neutralized most elevated PAI-1 activity (0.33±0.05). At 24 hours after stroke, PAI-1 antigen levels returned to the before baseline level, however, there was a significantly higher PAI-1 activity (2.51±0.53) in tPA treated rats. In vitro tPA exposures significantly increased PAI-1 releases into culture medium in cultured endothelial cells (1.65±0.08) and platelets (2.02±0.17). Conclution: Our experimental results suggest that tPA administration may further elevate stroke-increased blood PAI-1 concentration, but also increase PAI-1 activity at late 24 hours after stroke. The increased PAI-1 releases after tPA exposures in vitro suggest tPA may directly stimulate PAI-1 secretions from vascular walls and circulation platelets, which partially contributes to the PAI-1 elevation observed in focal stroke rats. The underlying regulation mechanisms and pathological consequence need further investigation.

Introduction: Recent animal studies show rADAMTS13 (vWF cleaving protease) to block tPA-induced BBB opening, reducing tPA-related hemorrhage and augmenting tPA’s thrombolytic effect. From proteomic screening, we found ADAMTS13/vWF to be an important pathway in tPA responders. Thus, the vWF/ADAMTS13 (vWF cleaving protease) axis may be an important modifier of tPA response in the clinical setting. But the effect of tPA on the ADAMTS13/vWF balance has not been studied directly in stroke patients post-tPA. Here we measured vWF and ADAMTS13 in acute ischemic stroke patients post IV tPA to investigate their clinical utility to follow tPA response. Methods: Quantitative Mass Spectrometry (MS), latex immuno-turbidimetric assay, and ELISA were performed in acute ischemic stroke plasma over time, with clinical outcome measured at 3 months. Response was defined per NINDS trial as mRankins= 4 points improvement on NIHSS. Results: Post tPA (up to 72 hours), ADAMTS13 is persistently and significantly higher in tPA responders than in non-responders (Figure 1A). Conversely, within 72 hrs post tPA, the ratio of vWF/ADAMTS13 (a measure of clot burden vs clot lysis) is significantly higher in tPA patients who did not improve compared to those who did. tPA patients also had lower vWF/ADAMTS13 ratios than non-tPA strokes (Figure 1B). Conclusion: Through dynamic changes detected in the vWF/ADAMTS13 pathway, our data suggest the balance of clot burden vs lysis is crucial in tPA response as measured in patient plasma in real time. More vWF degradation in tPA patients via ADAMTS13 is correlated to better clinical outcome within the first 72 hours, and conversely, increased clot burden was found in tPA non-responders. Studying endogenous ADAMTS13 activity in tPA response is an initial step to obtain evidence for its potential clinical utility. And vWF/ADAMTS13 ratio may be a biologically relevant and potentially clinically useful marker of clotting/lytic status in early thrombolysis.

Background and Purpose— Tissue-type plasminogen activator (tPA) in combination with recombinant annexin A2 (rA2) is known to reduce acute brain damage after focal ischemia. Here, we ask whether tPA-plus-rA2 combination therapy can lead to sustained long-term neurological improvements as well. Methods— We compared the effects of intravenous high-dose tPA alone (10 mg/kg) versus a combination of low-dose tPA (5 mg/kg) plus 10 mg/kg rA2 in a model of focal embolic cerebral ischemia in rats. All rats were treated at 3 hours after embolization. Brain tissue and neurological outcomes were assessed at 1 month. Surrogate biomarkers for endogenous neurovascular remodeling in peri-infarct area were analyzed by immunohistochemistry. Results— Compared with high-dose tPA alone, low-dose tPA-plus-rA2 significantly decreased infarction and improved neurological function at 1-month poststroke. In peri-infarct areas, tPA-plus-rA2 combination therapy also significantly augmented microvessel density, vascular endothelial growth factor, and synaptophysin expression. Conclusions— Compared with conventional high-dose tPA alone, combination low-dose tPA plus rA2 therapy may provide a safe and effective way to improve long-term neurological outcomes after stroke.

Background: LLLT has been shown to be neurorprotective against cerebral ischemia in animals. Initial human clinical trials demonstrated safety and potential beneficial profiles. A pivotal clinical trial with TLT is ongoing for ischemic stroke. However, the underlying neuroprotective mechanisms require further definition. We previously showed LLLT protected primary neurons against OGD. In the current study, we tested the hypothesis that LLLT may attenuate OGD-induced nitric oxide (NO) and ROS production and associated cell survival signaling, causing reduced cell death in primary mouse cortical neurons. Method: At day 8 of culture, neurons were subjected to 4 hr OGD. LLLT was applied at 2 hr reoxygenation. NO level, nNOS expression, ROS level, and cell survival signaling were measured post-LLLT. Cell death was measured at 20 hr after OGD. Results were expressed as fold of normal control, mean ± SEM, n = 3-5 per group, P Results: OGD significantly increased NO level in primary neurons (2.12 ± 0.47, 2.67 ± 1.07, and 2 ± 0.77 at 5 min, 30 min and 1 hr post-treatment, respectively), but the increase was significantly reduced by LLLT (1.08 ± 0.44 at 5 min and 1.45 ± 0.73 at 30 min post-LLLT). OGD also significantly increased nNOS mRNA level (2.17 ± 0.42, 3.99 ± 1.7, and 2.60 ± 0.7 at 5 min, 30 min and 1 hr post-treatment, respectively), while the increase was significantly ameliorated by LLLT (0.50 ± 0.08 at 30 min and 0.34 ± 0.45 at 1 hr post-LLLT). Additionally, 50 uM NO donor SNAP exposure for 48 hr induced 23.6 ± 2.5% cell death, which was significantly ameliorated by LLLT (12.3 ± 4.2% cell death), indicating LLLT may directly protect NO-mediated neurotoxicity. Furthermore, LLLT significantly reduced OGD-induced ROS production at 5 min, 30 min and 1 hr post-LLLT. Finally, OGD suppressed neuron survival signaling p-Akt and Bcl-2 protein expressions, but they were significantly rescued by LLLT. Conclusion: These results suggest LLLT protection against OGD may be partially through reducing OGD-induced NO and ROS production, and restoring OGD-suppressed cell survival signaling.

Background: The utilization of IV tPA continues to be low after more than a decade of FDA approval. We aim to widen the therapeutic window for tPA by understanding clinical thrombolysis response. Besides its intended role in clot lysis, tPA is also a pleiotrophic signaling protease in the blood, whose efficacy may potentially be monitored by proteomic profiles directly in stroke patients. Method/Results: To study thrombolysis response, we mapped quantitative profiles of all proteins pre and post IV tPA administration in plasma of stroke patients with good clinical outcome at 3 months. Plasma was sampled from stroke patients immediately before and 6-12hrs after tPA administration (n=28). Quantitative changes of proteins pre and post tPA were mapped by isotopic methods and analyzed on LC-MS. While both intra- and extracellular proteins were found, most were known plasma proteins, as seen on IPA analysis (Figure). Of 191 proteins found, 48 changed significantly post tPA (fold ratio in parenthesis). Compared to pre-tPA levels, we found significantly increased post-tPA levels of thrombolysis-related proteins such as fibrinogen gamma chain (↑11x), α-2-antiplasmin (↑4x), and a decrease in factor X (↓7x) etc. Changes were also found in factors outside of standard thrombolytic pathways, such as α-2-macroglobulin (↑2.3x); CD14 (↓2x) and GPX3 glutathione peroxidase 3 (↓2x) etc. Conclusion: In order to study thrombolysis response and monitor thrombolysis efficacy at the bedside, we mapped quantitative protein fold changes of IV tPA responders and found a coordinated change not only in reported thrombolysis pathways, but also in other enzymatic pathways in patient plasma. These early results are a step toward attempts to monitor clinical thrombolysis response in acute stroke patients in real time. Further studies are under way to compare plasma profiles of tPA-treated stroke patients with respect to different clinical outcome and imaging findings of reperfusion.

Alterations in thrombosis and fibrinolysis comprise important parts of stroke pathophysiology. A key step in the fibrinolytic process includes the tissue-type plasminogen activator (tPA)-mediated conversion of the proenzyme plasminogen into the active protease plasmin, which in turn degrades the fibrin structure of intravascular thrombi. There are a number of review articles well summarizing molecular mechanisms of the fibrinolytic system.1,2 Inhibition of the fibrinolytic system may occur at the level of plasminogen activation, mainly by a direct inhibition of tPA by plasminogen activator inhibitor 1 (PAI-1) or indirectly by thrombin-activatable fibrinolysis inhibitor and, at the level of plasmin, by α2-antiplasmin. The roles of these 3 inhibitors are complementary in thrombolysis.3 PAI-1 has become recognized as a central molecule linking pathogenesis and progression of thrombotic vascular events including stroke. As a main endogenous inhibitor of tPA, PAI-1 might be related to reperfusion efficacy and hemorrhagic risk of tPA thrombolytic therapy. Moreover, a clear association has been observed between elevated PAI-1 plasma levels and prothrombotic disease conditions such as hypertension, obesity, insulin resistance, and diabetes.4–7 Clinical and experimental studies show that PAI-1 deficiencies cause accelerated fibrinolysis and bleeding, whereas elevated PAI-1 plasma levels are associated with vascular thrombosis.8 Concentrations of active PAI-1 in newly formed thrombi can be several thousandfold greater than active PAI-1 concentrations in normal plasma9 and thus high enough to inhibit doses of tPA used for clinical thrombolysis. Furthermore, beyond its effects in modulating the activity of tPA and the functionally related urokinase-type plasminogen activator, which predominantly catalyzes plasmin formation in the extravascular space, PAI-1 also plays diverse roles in metabolic and vascular disease and may participate in the evolution of brain damage and recovery after stroke.4 …

Background: Low level light (or laser) therapy (LLLT) has been studied and practiced for promoting wound healing, reducing pain, inflammation, and ischemic tissue damage. Recently, a series of experimental and clinical investigations have suggested that LLLT may be a novel therapy against hypoxic/ischemic brain damage. A clinical trial of LLLT therapy for ischemic stroke is now on going. However, the molecular mechanism of LLLT-conferred neuroprotection remains poorly defined. In this study, we tested our hypothesis that LLLT may attenuate impairments of mitochondrial function induced by hypoxic/ischemic insults in primary cultured mouse cortical neurons. Method: At day 9 of culture, primary neurons were subjected to 4 hr OGD followed by reoxygenation. One 810-nm LLLT treatment was applied for 2 minutes at 2 hr after reoxygenation. Neurotoxicity was measured after 20 hr after reoxygenation by LDH release assay. We also measured MTT reduction and mitochondria membrane potential (MMP) at 2 hr after LLLT treatment as markers of mitochondrial function. Results: The neurotoxicity study showed that 4 hr OGD plus 20 hr reoxygenation caused 33.8± 3.4% neuronal cell death, while LLLT treatment significantly reduced the neuronal death rate to 23.6± 2.9% (30.2% reduction, n=6, p smaller than 0.05). Mitochondrial functional assays showed OGD decreased MTT reduction to 75.9± 2.68%, but LLLT treatment significantly rescued MTT reduction to 87.6±4.55% (15.4% improvement, n=6, p smaller than 0.05). Furthermore, after OGD, MMP was reduced to 48.9±4.39%, while LLLT treatment significantly ameliorated this reduction to 89.6± 13.9% (83% improvement, n=4, p smaller than 0.05) compared to normoxic controls. Conclusion: The present study suggests that LLLT treatment is protective against OGD-induced neurotoxicity of primary neurons and that this protection may be conferred through preservation or rescue of mitochondrial function.

Background: Accumulating evidences from our laboratory and others have demonstrated that neuroglobin (Ngb) is protective against hypoxic/ischemic brain injury, thus targeting Ngb may be a novel approach for endogenous neuroprotection, but the molecular mechanisms remain undefined. Mitochondrial permeability transition pore (mPTP) opening is a key mitochondrial response to hypoxia/ischemia, followed by release of mitochondrial factors such as Cyt c and activation of Caspase-dependent apoptosis pathways. In this study, we test our hypothesis that Ngb inhibits OGD-induced mPTP opening in primary neurons. Method: Primary mouse cortical neurons were transduced with AAV-Ngb to achieve Ngb over-expression and Ngb-shRNA to achieve Ngb knockdown. At day 9 of culture, primary neurons were subjected to 4 hr OGD followed by reoxygenation. Neurotoxicity was measured after 20 hr reoxygenation by LDH release assay. mPTP opening was measured at 4 hr after reoxygenation by testing mitochondria swelling, NAD release and Cyt c release. NAD release was also measured using isolated mitochondria treated with OGD after incubation with recombinant Ngb protein. Results: (1) Four hour OGD followed by 20 hr reoxygenation caused 40±4.5% neuron death; Ngb overexpression significantly reduced the death rate to 18±5.2% (n=5, p

Background: Stroke patients with diabetes mellitus or post-stroke hyperglycemia are associated with resistance to tPA reperfusion, higher risk of intracerebral hemorrhage and worse neurological outcomes. Our laboratory has established an embolic focal stroke model in type I diabetic rats that closely mimic the clinical situation with worse neurological outcomes observed after tPA thrombolysis. Clinically insulin intervention did not show significant benefit to acute ischemic brain injury, perhaps due to the relatively late infusion time after stroke onset. In this study for the first time we aimed to test effects of early insulin intervention combined with following tPA thrombolytic therapy in neurological outcomes of focal embolic stroke model in type I diabetic rats. Methods: Type I diabetic rats were induced by standard Streptozotocin i.p. injection. Six weeks type I diabetes rats were subjected into a focal embolic stroke. Four treatment groups: (1) saline at 1.5 hours after stroke; (2) standard rat dose 10 mg/kg of tPA at 1.5 hours after stroke; (3) insulin (i.h. 4 units and i.v. 2 units) at 1 hours plus saline at 1.5 hours; (4) insulin (i.h. 4 units and i.v. 2 units) at 1 hours plus 10 mg/kg tPA at 1.5 hours. Acute neurological outcomes were measured at 24 hours after stroke onset for brain infarction, hemispheric swelling, and hemorrhagic transformation. Results: Blood glucose level of diabetic rats was 484±92 mg/dl at before stroke. Insulin infusion significantly decreased blood glucose levels. At 2.5 hours after stroke, blood glucose levels were 515±94 mg/dl for saline, 509±98 mg/dl for tPA alone, 325±77mg/dl for insulin plus saline, and 295±64 mg/dl for insulin plus tPA, respectively. At 24 hours after stroke, tPA slightly reduced infarction volume than saline group. Insulin significantly reduced 24% of brain infarction in tPA group, but not in saline group (11% reduction) compare to non-insulin controls. Insulin significantly decreased hemispheric swelling in both saline (40% reduction) and tPA groups (43% reduction). tPA treatment significantly increased hemorrhagic volume (6.23μ l/hemisphere), but insulin intervention eliminated the increased hemorrhagic volume (4.45μ l/hemisphere) elevated by tPA thrombolytics. Discussion and Conclusion: Early insulin intervention may be safe and beneficial to tPA thrombolytics in reducing infarction volume, edema and intracerebral hemorrhage to ischemic stroke with diabetes mellitus or post-stroke hyperglycemia. Further investigations are needed for fully evaluating the early insulin intervention in preclinical settings and define underlying molecular mechanisms, in terms of optimizing its therapeutic effects in both safety and efficacy before clinical tests can be applied.

Hemorrhagic transformation, incomplete reperfusion, neurotoxicity, and the short treatment time window comprise major challenges for thrombolytic therapy. Improving tissue plasminogen activator therapy has become one of the highest priorities in the stroke field. Recent efforts have been aimed at identifying new strategies that might enhance the thrombolytic efficacy of tissue plasminogen activator at the same time as reducing its associated complications related to hemorrhage and neurotoxicity. We believe that the combination of low-dose tissue plasminogen activator with recombinant annexin A2 (a tissue plasminogen activator and plasminogen coreceptor) might constitute a promising approach. Our pilot study using a focal embolic stroke model in rats supports this hypothesis.

Response: We appreciate the comments by Dr Kelsen and colleagues regarding our recent article.1 Before addressing the specific comments in the letter, we would like to emphasize that by using our newly created Ngb transgenic (Ngb-Tg, nontissue specific) mouse, we studied the effects of Ngb overexpression in focal cerebral ischemia. So, this published study was not aimed to fundamentally define or interpret the role of endogenous Ngb. Instead, this was an “outcomes” study. Overexpression of Ngb resulted in a reduction of ischemic brain injury. Our results were broadly consistent with an earlier report …

Background and Purpose— Hemorrhagic conversion after tissue plasminogen activator (tPA) stroke therapy has been linked with elevations in matrix metalloproteinase-9 (MMP-9) at the neurovascular interface. Here, we test the idea that statins may directly ameliorate tPA-induced MMP-9 dysregulation. Methods— Recombinant human tPA (5 μg/mL) was added to primary rat cortical astrocytes. Zymography was used to quantify MMP-9 levels in conditioned media. Effects of simvastatin or the Rho kinase inhibitor Y-27632 were assessed by pretreating cells before tPA exposure. Results— Simvastatin (1 to 10 μmol/L) significantly reduced tPA-induced MMP-9 in cortical astrocytes. This effect may be mediated via the Rho kinase pathway because tPA-induced activation of Rho signaling was suppressed by simvastatin, and tPA-induced MMP-9 levels were similarly reduced by the Rho kinase inhibitor Y-27632 (1 to 10 μmol/L). Conclusions— Statins reduce tPA-induced MMP-9 dysregulation by inhibiting the Rho signaling pathway. Statins may ameliorate tPA-associated MMP imbalances in stroke.

Background and Purpose— Most experimental models of cerebral ischemia use mechanical methods of occlusion and reperfusion. However, differences between mechanical reperfusion versus clot thrombolysis may influence reperfusion injury profiles. In this study we compared blood flow recovery, blood-brain barrier (BBB) permeability, and matrix metalloproteinase-9 (MMP-9) expression in cortex after mechanical versus thrombolytic reperfusion in rat focal ischemia. Methods— Male spontaneously hypertensive rats were used. Mechanical ischemia/reperfusion was achieved with the use of an intraluminal filament to occlude the middle cerebral artery for 2 hours. Thrombolytic reperfusion was achieved by administering tissue plasminogen activator at 2 hours after embolic focal ischemia. Regional cortical blood flow was monitored by laser-Doppler flowmetry. BBB permeability in cortex was measured by Evans blue dye leakage. Cortical MMP-9 levels were assessed with zymography and immunohistochemistry. Results— Blood flow recovery during mechanical reperfusion was complete in both central and peripheral areas of ischemic cortex. However, after thrombolysis, reperfusion was incomplete, with moderate recovery in the periphery only. BBB permeability was mainly increased in the central regions of the ischemic cortex after mechanical reperfusion but was increased in both central and peripheral areas after thrombolysis. Overall, MMP-9 levels were higher after embolic versus mechanical ischemia/reperfusion, even though ischemic injury was similar in both models at 24 hours. Conclusions— There are significant differences in the profiles of blood flow recovery, BBB leakage, and MMP-9 upregulation in mechanical versus thrombolytic reperfusion after focal ischemia.

3 Background: The role of therapeutic oxygen in treatment of acute stroke is controversial. Oxygen improves cellular aerobic metabolism and can salvage ischemic tissue. However, oxygen free radicals can increase blood brain barrier (BBB) damage, and oxygen can induce vasoconstriction, which could worsen stroke outcome. We studied the effects of normobaric oxygen in cerebral ischemia and reperfusion. Methods: Rats were subjected to normobaric hyperoxia (FiO2 100%) or normoxia (FiO2 30%) during two hour filament occlusion and one hour reperfusion of the middle cerebral artery. Twenty-four hour infarct volumes, regional cerebral blood flow (rCBF) using laser Doppler flowmetry, and severity of BBB damage (assessed by quantifying Evan’s Blue dye (EB) leakage after one hour of reperfusion) were compared between groups. Results: Physiological parameters were similar in hyperoxic and control groups, except for paO2, which was expectedly higher in the hyperoxic group (pO2 484 mm Hg) as compared to controls (pO2 118 mm Hg). Mean rCBF dropped to 25% after onset of ischemia and recovered to 75–90% after arterial unocclusion, indicating successful reperfusion. Mean total (right hemispheric) infarct volume was 65 mm 3 in the hyperoxia group and 209 mm 3 in controls, p3 in hyperoxic rats and 129 mm 3 in controls (p3 in hyperoxic rats and 80 mm 3 in controls (pConclusion: Total and cortical infarct volumes can be significantly reduced with normobaric hyperoxia during transient cerebral ischemia and reperfusion. Hyperoxia does not decrease blood flow to ischemic brain, and its benefit in reducing infarct volume may outweigh any potential damage from BBB damage.