Implications of Janus Kinase 2 Mutation in Embolic Stroke of Unknown Source.

The role of genetic mutations in cerebral ischemia is not completely understood. Among these genetic variations, Philadelphia-negative gain-of-function mutation in the janus kinase 2 (JAK2) protein leads to overexpression of the genes involved in cell growth and proliferation, and has been linked to development of hematological malignancies, specifically, myeloproliferative neoplasms (MPNs; essential thrombocythemia [ET], polycythemia vera [PV], and primary myelofibrosis). Overt ET and PV are known to induce a prothrombotic state that leads to development of vascular complications, including cerebral arterial or venous thrombosis. Thromboembolism can precede overt presentation of an MPN by 2-3 years. As such, for the selected cases of embolic stroke or cerebrovascular sinus thrombosis with otherwise undetermined source and persistent thrombocytosis or polycythemia, in the absence of a confirmed MPN diagnosis, screening for JAK2 mutation may be reasonable, as early diagnosis and appropriate treatment can influence outcome by preventing recurrent thrombotic events. In this article, we review the literature on the genetics, pathogenesis, clinical manifestations, and treatment of JAK2-associated thrombosis, and present 2 cases of JAK2-associated cerebral arterial infarction and cerebral and systemic venous thromboembolism with otherwise negative etiology workup for stroke.

Risk of cerebral embolism after interventional closure of symptomatic patent foramen ovale or atrial septal defect: a diffusion-weighted MRI and neuron-specific enolase-based study.

OBJECTIVES: The aim of this single-center prospective study is to investigate the silent and clinically apparent cerebral embolic events after transcatheter closure of atrial septal defect (ASD) and patent foramen ovale (PFO). BACKGROUND: Although transcatheter closure of ASD and PFO is a widely accepted technique and has been proven to be safe and effective with different kinds of devices, there are few studies in the literature that report the peri-interventional cerebral embolism risk and neurological complications. In this study, we investigated the peri-interventional cerebral embolism incidence with diffusion-weighted magnetic resonance imaging (DW-MRI) and its relation to patients' clinical neurologic examination findings and plasma neuron-specific enolase (NSE) levels. METHODS: Sixteen patients with hemodynamically significant ASD and 14 symptomatic PFO patients underwent transcatheter closure procedures with new-generation PFO or ASD occluder devices. All cases were examined with DW-MRI before and after the transcatheter closure procedure. Patients were clinically examined for any signs of neurologic deficit at the time of MRI studies. Blood samples for NSE, a marker of brain tissue damage involved in an ischemic event, were taken before the procedure and at 12 and 24 hours after the procedure. RESULTS: Successful transcatheter closure of PFO or ASD was achieved in all patients. In the DW-MRI exam following the procedure, a new microembolic lesion was found in only 1 of 30 patients (3.3%). None of the patients had positive clinical neurological exam findings. NSE levels after the procedure were found to be not correlated with presence of DWMRI lesion and intervention times. CONCLUSION: With the new-generation ASD and PFO occluder devices, the incidence of clinically silent peri-interventional cerebral embolic lesions after transcatheter closure of ASD and PFO is low. Plasma NSE levels offered no additional benefit for monitoring ischemic events after ASD and PFO transcatheter closure procedures.

Combination treatment with VELCADE and low-dose tissue plasminogen activator provides potent neuroprotection in aged rats after embolic focal ischemia.

BACKGROUND AND PURPOSE: Treatment with a selective proteasome inhibitor, VELCADE, in combination with tissue plasminogen activator (tPA) extended the therapeutic window to 6 hours in young rats after stroke. However, stroke is a major cause of death and disability in the elderly. The present study investigated the effect of VELCADE in combination with a low-dose tPA on aged rats after embolic stroke. METHODS: Male Wistar rats at the age of 18 to 20 months were treated with VELCADE (0.2 mg/kg) alone, a low-dose tPA (5 mg/kg) alone, combination of VELCADE and tPA, or saline 2 hours after embolic middle cerebral artery occlusion. To test the contribution of endothelial nitric oxide synthase to VELCADE-mediated neuroprotection, endothelial nitric oxide synthase knockout and wild-type mice were treated with VELCADE (0.5 mg/kg) 2 hours after embolic stroke. RESULTS: Treatment with VELCADE significantly reduced infarct volume, whereas tPA alone did not reduce infarct volume and aggravated blood-brain barrier disruption in aged rats compared with saline-treated rats. However, the combination treatment significantly enhanced the reduction of infarct volume, which was associated with an increase in endothelial nitric oxide synthase activity compared with saline-treated rats. Additionally, the combination treatment promoted thrombolysis and did not increase the incidence of hemorrhage transformation. VELCADE significantly reduced lesion volume in wild-type mice but failed to significantly reduce lesion volume in endothelial nitric oxide synthase knockout mice. CONCLUSIONS: Treatment with VELCADE exerts a neuroprotective effect in aged rats after stroke. The combination of VELCADE with the low-dose tPA further amplifies the neuroprotective effect. Endothelial nitric oxide synthase at least partly contributes to VELCADE-mediated neuroprotection after stroke.

Extension of the thrombolytic time window with minocycline in experimental stroke.

BACKGROUND AND PURPOSE: Thrombolysis with tPA is the only FDA-approved therapy for acute ischemic stroke. But its widespread application remains limited by narrow treatment time windows and the related risks of cerebral hemorrhage. In this study, we ask whether minocycline can prevent tPA-associated cerebral hemorrhage and extend the reperfusion window in an experimental stroke model in rats. METHODS: Spontaneously hypertensive rats were subjected to embolic focal ischemia using homologous clots and treated with: saline at 1 hour; early tPA at 1 hour, delayed tPA at 6 hours; minocycline at 4 hours; combined minocycline at 4 hours plus tPA at 6 hours. Infarct volumes and hemorrhagic transformation were quantified at 24 hours. Gelatin zymography was used to measure blood levels of circulating matrix metalloproteinase-9 (MMP-9). RESULTS: Early 1-hour thrombolysis restored perfusion and reduced infarction. Late 6-hour tPA did not decrease infarction but instead worsened hemorrhagic conversion. Combining minocycline with delayed 6-hour tPA decreased plasma MMP-9 levels, reduced infarction, and ameliorated brain hemorrhage. Blood levels of MMP-9 were also significantly correlated with volumes of infarction and hemorrhage. CONCLUSIONS: Combination therapy with minocycline may extend tPA treatment time windows in ischemic stroke.

Delayed matrix metalloproteinase inhibition reduces intracerebral hemorrhage after embolic stroke in rats.

Hemorrhagic transformation (HT) and brain edema are life-threatening complications of recombinant tissue plasminogen activator (rt-PA)-induced reperfusion after ischemic stroke. The risk of HT limits the therapeutic window for reperfusion to 3 h after stroke onset. Pre-treatment with matrix metalloproteinase (MMP) inhibitors reduces HT and cerebral edema in experimental stroke. However, whether a delayed therapeutic intervention would be beneficial is unknown. In this study, 215 male Sprague-Dawley rats were subjected to embolic stroke and 75 rats were included in the final analysis. The animals were treated with the MMP inhibitor p-aminobenzoyl-gly-pro-D-leu-D-ala-hydroxamate before or after 3 or 6 h of ischemia. Animals were monitored for reperfusion and received rt-PA 6 h after ischemia onset. The results at 24 h showed that MMP inhibition 3 h after ischemia significantly decreased the degree of brain edema (17% of hemispheric enlargement in the treated group versus 24% in controls, P=0.018), reduced the risk (OR=0.163; 95% CI: 0.029 to 0.953) and gravity (0.09 versus 0.19 mg of parenchymal hemoglobin, P=0.02) of intracerebral hemorrhage, and improved neurological outcome (20% of the treated animals had a slight deficit; all of the controls had a bad outcome, P<0.05). Delaying MMP inhibition to 6 h after ischemia restricted the beneficial role of the treatment to a reduction in the risk of parenchymal hemorrhage (OR=0.242; 95% CI: 0.060 to 0.989). Our results confirm the involvement of MMPs in HT and support the possibility of extending the therapeutic window for thrombolysis in stroke by administering a broad-spectrum MMP inhibitor after the onset of ischemia.

Tadalafil, a long-acting type 5 phosphodiesterase isoenzyme inhibitor, improves neurological functional recovery in a rat model of embolic stroke.

Sildenafil, a type 5 phosphodiesterase isoenzyme (PDE5) inhibitor with a short half-life, increases brain cyclic guanosine monophosphate (cGMP) levels and improves neurological functional recovery when administered after stroke. In the present study, we investigated the effects of tadalafil (Cialis), a long acting PDE5 inhibitor, on brain cGMP levels, neurogenesis, angiogenesis, and neurological function during stroke recovery in a rat model of embolic stroke. Male Wistar rats (n=28) were subjected to embolic middle cerebral artery (MCA) occlusion. Tadalafil was orally administered every 48 h at a dose of 2 mg/kg or 10 mg/kg for 6 consecutive days starting 24 h after stroke onset. Control animals received the equivalent volume of saline at the same time points. For mitotic labeling, bromodeoxyuridine (BrdU, 100 mg/kg) was administered twice a day at 5, 6, and 7 days after stroke. ELISA assays were performed to evaluate the specificity of the effect of tadalafil on cGMP. Treatment with tadalafil at a dose of 2 or 10 mg/kg significantly improved neurological functional recovery compared with saline-treated rats. In addition, tadalafil treatment increased cerebral vascular density and the percentage of BrdU-positive endothelial cells around the ischemic boundary compared with saline-treated rats. Moreover, tadalafil-treated rats showed greater ipsilateral SVZ cell proliferation than saline-treated rats. However, treatment with tadalafil did not reduce infarct volume when compared to the saline group. Tadalafil selectively increased cGMP but not cyclic adenosine monophosphate (cAMP) in brain. Our data demonstrate that treatment of ischemic stroke with tadalafil improved functional recovery, which was associated with increases of brain cGMP levels and enhancement of angiogenesis and neurogenesis.

Treatment of embolic stroke in rats with bortezomib and recombinant human tissue plasminogen activator.

Stroke elicits a progressive vascular dysfunction, which contributes to the evolution of brain injury. Thrombolysis with tissue plasminogen activator (tPA) promotes adverse vascular events that limit the therapeutic window of stroke to three hours. Proteasome inhibitors reduce vascular thrombotic and inflammatory events, and consequently protect vascular function. The present study evaluated the neuroprotective effect of bortezomib, a potent and selective inhibitor of the proteasome, alone and in combination with delayed thrombolytic therapy on a rat model of embolic focal cerebral ischemia. Treatment with bortezomib reduces adverse cerebrovascular events including secondary thrombosis, inflammatory responses, and blood brain barrier (BBB) disruption, and hence reduces infarct volume and neurological functional deficit when administrated within 4 h after stroke onset. Combination of bortezomib and tPA extends the thrombolytic window for stroke to 6 h, which is associated with the improvement of vascular patency and integrity. Real time RT-PCR of endothelial cells isolated by laser-capture microdissection from brain tissue and Western blot analysis showed that bortezomib upregulates endothelial nitric oxide synthase (eNOS) expression and blocks NF-kappaB activation. These results demonstrate that bortezomib promotes eNOS dependent vascular protection, and reduces NF-kappaB dependent vascular disruption, all of which may contribute to neuroprotection after stroke.

[Antioxidant enzymes in acute cerebral infarction]. / Enzimas antioxidantes en el infarto cerebral agudo.

BACKGROUND AND PURPOSE: Oxidative modification of DNA, protein and lipids by reactive oxygen peroxides plays an important role in acute cerebral infarction. The biological antioxidant defence system against oxidative stress is an integrated array of enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathion peroxidase (GPx). The purpose of this work was: 1. To investigate the evolution of SOD and CAT activities as possible markers of oxidative activity in patients with acute cerebral infarction. 2. To establish the relationship between the type of stroke and the activity of these two enzymes. 3. To analyze the relationship between the degree of neurological deficits of patients with stroke and SOD and CAT activities. PATIENTS AND METHODS: This is a prospective study comprising 45 patients with acute cerebral infarction (24 females and 21 males), aged from 48 to 96. The infarcts were classified in atherotrombotics and cardioembolics. SOD and CAT activities were measured on days 1, 3, 6 and 15. RESULTS: Activities of SOD and CAT in patients on admission were lower than those of controls and showed a gradual increase over time. The activity mean of SOD was significantly higher in cardioembolic infarctions than in atherotrombotic infarctions (p < 0.05). SOD activity was significantly lower for Mathew score > 60, while CAT activity was higher. CONCLUSIONS: Our findings suggest that there is a progressive increase in SOD and CAT activity during ictus evolution, a different activity of SOD and CAT in cardioembolic and atherotrombotic infarctions, and that there is a relationship between activity of antioxidant enzymes and Mathew's score.

Blood-brain barrier disruption and matrix metalloproteinase-9 expression during reperfusion injury: mechanical versus embolic focal ischemia in spontaneously hypertensive rats.

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.

Selective reduction in type I adenylyl cyclase after microsphere embolism in rat brain.

Alterations of adenylyl cyclase (AC) subtypes after cerebral ischemia remain unclear. The purpose of the present study was to characterize alterations in AC after sustained cerebral ischemia. Sustained cerebral ischemia was induced by injection of 900 microspheres into the right (ipsilateral) internal carotid artery of rats. Microsphere embolism (ME) decreased the Ca(2+)/calmodulin-sensitive AC activity in the ipsilateral hippocampus examined up to 7 days after the embolism, whereas basal and 5'-guanylyl imidodiphosphate-sensitive AC activities were not altered. An immunoreactivity of type I adenylyl cyclase (AC-I) was decreased in the ipsilateral hippocampus during these periods, whereas type V/VI AC and VIII AC immunoreactivities were not altered. These results suggest that a selective reduction in the AC-I level and activity is induced by ME, which may lead to dysfunction of AC signal transduction.

Matrix metalloproteinase expression is related to hemorrhagic transformation after cardioembolic stroke.

BACKGROUND AND PURPOSE: In animal models of cerebral ischemia, matrix metalloproteinase (MMP) expression was significantly increased and related to blood-brain barrier disruption, edema formation, and hemorrhagic transformation (HT). MMP inhibitors reduce HT after embolic ischemia in tissue-type plasminogen activator-treated animals. We aimed to determine the relationship between MMPs and HT after human ischemic stroke. METHODS: Serial MMP-2 and MMP-9 determinations were performed by means of ELISA in 39 cardioembolic strokes in the middle cerebral artery territory. Hemorrhagic events were classified according to clinical and CT criteria (hemorrhagic infarction [HI] and parenchymal hematoma [PH]). HT was evaluated on CT at 48 hours (early HT) and again between day 5 and 7 (late HT). RESULTS: HT was present in 41% of the patients (43.75% early HI, 25% early PH and 31.25% late HI). MMP-2 values were within normal range and were unrelated to HT. Increased expression of MMP-9 (normal range <97 ng/mL) was found among patients with and without HT (159.3+/-82 versus 143.9+/-112.6 ng/mL; P=0.64). According to HT subtypes, the highest baseline MMP-9 levels corresponded to patients with late HI (240.4+/-111.2 versus 102.5+/-76.7 ng/mL for all other patients, P=0.002). Baseline MMP-9 was the only variable associated with late HI in the multiple logistic regression model (OR 9; CI 1.46, 55.24; P=0.010). Peak of MMP-9 at the 24-hour time point (250.6 ng/mL) was found before appearance of PH. CONCLUSIONS: MMPs are involved in some subtypes of HT after human cardioembolic stroke. Baseline MMP-9 level predicts late HI and a 24-hour peak precedes early PH.

Plasma MMP-9 - a marker of carotid plaque instability.

OBJECTIVES: to investigate whether peripheral blood levels of matrix metalloproteinases (MMPs) or their inhibitors are altered in patients with particulate cerebral embolisation. DESIGN: a prospective study. MATERIALS AND METHODS: using sandwich enzyme immunoassay, plasma levels of MMPs-1, -2, -3 and -9, plus TIMPs-1 and -2 were quantified in 70 consecutive patients undergoing carotid endarterectomy. Patients were monitored with transcranial Doppler (TCD) preoperatively and during the dissection phase of the operation to detect those with spontaneous particulate embolisation (n =21). RESULTS: the plasma level of MMP-9 was significantly higher in those patients with evidence of spontaneous embolisation compared to those without. There were no differences in other MMP levels, or plasma concentrations of TIMPs. CONCLUSIONS: plasma MMP-9 levels are elevated in patients with particulate cerebral embolisation, and this may represent a novel marker of atherosclerotic plaque instability.