Infarct Volume In Ischemic Stroke Research Articles

Abstract 138: Platelet RIPK1 Promotes Platelet Activation And Aggregation In Arteriolar Thrombosis And Ischemic Stroke

Receptor-interacting protein Ser/Thr kinase 1 (RIPK1) is a molecular switch for inflammation and cell death in nucleated cells, and its function is controlled by phosphorylation at the Ser166 residue. However, it is unknown whether RIPK1 regulates platelet function in arterial thrombosis. Intravital microscopy using megakaryocyte-specific Ripk1 conditional knockout (CKO) mice ( Ripk1 fl/fl;Pf4-cre ) revealed that platelet Ripk1 promoted platelet thrombus formation at sites of laser-induced cremaster arteriolar thrombosis. Deletion of platelet Ripk1 prolonged the time to occlusion in FeCl 3 -induced carotid arterial thrombosis without affecting tail bleeding times. Further, compared to wild-type (WT) mice, Ripk1 CKO mice exhibited improved neurological deficits and reduced infarct volume in ischemic stroke induced by transient middle cerebral artery occlusion. Flow cytometry and aggregation assays showed that deletion of platelet Ripk1 reduced P-selectin exposure, αIIbβ3 integrin activation, and platelet aggregation induced by thrombin, collagen-related peptides (CRP), and U46619, but not ADP. Pretreatment of Ripk1-null platelets with ADP rescued the aggregation defect. Treatment of platelets with Nec-1s, a RIPK1 inhibitor that blocks RIPK1-Ser166 phosphorylation, did not affect platelet activation and aggregation. Instead, RIPK1-Ser320/321 was rapidly phosphorylated in thrombin- or CRP-stimulated human and mouse platelets. Treatment of platelets with an inhibitor of phospholipase C, p38, or MAPK-activated protein kinase 2 (MK2), but not a protein kinase C inhibitor, decreased RIPK1-Ser320/321 phosphorylation after thrombin or CRP stimulation, implying that the p38-MK2 axis phosphorylates RIPK1-Ser320/321. Using a genetic approach in human megakaryoblastic MEG-01 cells, we found that RIPK1 knockdown reduced granule secretion from stimulated MEG-01 cells, which was rescued by overexpressing WT RIPK1, but not mutant RIPK1-Ser320Ala, in the RIPK1 knockdown MEG-01 cells. These results suggest that platelet RIPK1 enhances granule secretion during platelet activation through p38-MK2-mediated phosphorylation of RIPK1-Ser320/321, thereby contributing to the pathology of thrombotic disease.

Platelet ERO1α Is Crucial for Ca2+ Mobilization and Platelet Activation in Atherothrombosis

Endoplasmic reticulum oxidoreductase 1α (ERO1α) is a key oxidase of protein disulfide isomerase (PDI) during protein folding in the ER. Platelet ERO1α was reported to interact with PDI and αIIbβ3 integrin and regulate platelet function. However, the regulatory mechanism and pathophysiological role remain unknown. In the present study, we demonstrate that platelet ERO1α orchestrates intracellular Ca2+ signaling during platelet activation and contributes to thrombogenesis. Intravital microscopy using megakaryocyte-specific Ero1α conditional knockout (CKO) and KO mice reveals that platelet ERO1α is crucial for platelet thrombus formation in mouse models of laser-induced cremaster arteriolar thrombosis and FeCl3-induced carotid arterial thrombosis. Deletion of Ero1α does not affect bleeding times and blood loss at the site of tail amputation, implying that ERO1α is not required for hemostasis. Studies using Ero1α-null platelets, novel function-blocking antibodies, and small-molecule inhibitors reveal that intracellular, but not extracellular, ERO1α promotes P-selectin exposure, αIIbβ3 integrin activation, and platelet aggregation induced by multiple agonists, such as thrombin and collagen-related peptide. Importantly, the regulatory function of ERO1α is independent of PDI activity. Mass spectrometry and immunoprecipitation assays identify stromal interaction molecule 1 (STIM1) and sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2) as ERO1α binding partners. We have found that ERO1α-STIM1 binding is enhanced during platelet activation, whereas ERO1α-SERCA2 binding is reduced. Intriguingly, platelet ERO1α interacts with STIM1 through Cys49 and Cys56 residues and promotes its function by oxidizing the Cys49-Cys56 disulfide bond, contributing to Ca2+ store content and enhancing Ca2+ mobilization during platelet activation. Using a high throughput screen, we identify a small-molecule ERO1α inhibitor, B20, which inhibits ERO1α activity with an IC50 value of 10.0 μM but does not affect the activities of H2O2 and PDI. Treatment of platelets with B20 recapitulates the functional defects as seen in Ero1α-null platelets. Intravenous injection of B20 and eptifibatide, an αIIbβ3 integrin antagonist, into WT mice significantly inhibits platelet thrombus formation following laser- or FeCl3-induced vascular injury. While treatment of mice with eptifibatide markedly prolongs tail bleeding times and causes blood loss, B20 does not impair hemostatic function. Furthermore, treatment with B20 reduces the infarct volume in ischemic stroke. These results indicate that platelet ERO1α is a novel regulator of Ca2+ signaling and promotes platelet activation and aggregation in a PDI-independent manner, contributing to the pathogenesis of arterial thrombosis and thromboinflammation. Targeting ERO1α may be an attractive therapeutic strategy for treating patients with thrombotic disease.

Abstract WP315: Neuroprotective Effects of Endogenous Adropin in Experimental Ischemic Stroke

Adropin is an endogenous peptide highly expressed in the brain and is encoded by the energy homeostasis-associated gene, Enho . We recently found that treatment with synthetic adropin peptide reduces infarct volume in ischemic stroke. This protection by exogenous adropin is associated with a significant increase in endothelial nitric oxide synthase (eNOS) phosphorylation (Ser 1176 ) and reduction of blood-brain barrier (BBB) permeability. However, it is not known whether endogenous adropin modulates ischemic brain damage. We hypothesize that lack of brain adropin increases stroke damage by exacerbating neurovascular dysfunction, while overexpression of this peptide decreases ischemic brain injury. We measured infarct size and BBB damage in male and female adropin overexpressing transgenics (AdrTg), adropin knockout ( Enho -/- ), and corresponding wild-type (WT) control mice subjected to permanent middle cerebral artery occlusion (pMCAO). At 48h after stroke, infarct volume was significantly smaller in AdrTg mice of both sexes compared to WT controls, while stroke resulted in a much larger infarction in both male and female Enho -/- compared to Enho +/+ mice. Brain levels of IgG and albumin, two sensitive markers of BBB disruption, were significantly reduced in AdrTg mice compared to the WT group, while the levels of these two plasma proteins in the ischemic brain of Enho -/- mice were dramatically increased compared to Enho +/+ animals after stroke. Latex vessel casting showed no differences in cerebrovascular anatomy or the diameter of the main brain arteries between Enho -/- , AdrTg and their respective WT controls. In AdrTg mice, we found a positive correlation between brain adropin levels and eNOS phosphorylation. These data suggest that increased eNOS phosphorylation by adropin is a potential mechanism underlying its protective effects in stroke. Consistent with this hypothesis, the protective effects of adropin were completely abolished in eNOS knockout (eNOS -/- ) mice subjected to pMCAO. Taken together, our findings show for the first time that endogenous adropin is a neuroprotective peptide in ischemic stroke. Understanding adropin signaling and function could offer a novel therapeutic strategy for the treatment of ischemic brain damage.

Single-Nucleotide Polymorphism 309T>G in the MDM2 Promoter Determines Functional Outcome After Stroke.

Background and Purpose- The E3 ubiquitin ligase MDM2 (murine double minute 2) is the main negative regulator of the p53 protein-a key player in neuronal apoptosis after ischemia. A functional single-nucleotide polymorphism in the human MDM2 gene promoter (rs2279744) regulates MDM2 protein expression. We investigated whether the MDM2 SNP309, by controlling p53-mediated apoptosis, determines functional outcome after stroke. Methods- Primary cortical neurons were subjected to oxygen and glucose deprivation. Mice were subjected to ischemic (transient middle cerebral artery occlusion) or hemorrhagic (collagenase injection) stroke models. Protein and mRNA levels of MDM2 and p53 were measured in both neuronal and brain extracts. The interaction of MDM2 with p53 was disrupted by neuronal treatment with nutlin-3a. siRNA was used to knockdown MDM2 expression. We analyzed the link between the MDM2 SNP309 and functional outcome, measured by the modified Rankin Scale scores, in 2 independent hospital-based stroke cohorts: ischemic stroke cohort (408 patients) and intracerebral hemorrhage cohort (128 patients). Results- Experimental stroke and oxygen and glucose deprivation induced the expression of MDM2 in the brain and neurons, respectively. Moreover, oxygen and glucose deprivation promoted MDM2 binding with p53 in neurons. Disruption of the MDM2-p53 interaction with nutlin-3a, or MDM2 knockdown by siRNA, triggered p53 accumulation, which increased neuronal susceptibility to oxygen and glucose deprivation-induced apoptosis. Finally, we showed that patients harboring the G allele in the MDM2 promoter had higher MDM2 protein levels and showed better functional outcome after stroke than those harboring the T/T genotype. The T/T genotype was also associated with large infarct volume in ischemic stroke and increased lesion volume in patients with intracerebral hemorrhage. Conclusions- Our results reveal a novel role for the MDM2-p53 interaction in neuronal apoptosis after ischemia and show that the MDM2 SNP309 determines the functional outcome of patients after stroke.

Effect of Prestroke Aspirin on Infarct Volume in Ischemic Stroke (S25.008)

Effect of Prestroke Aspirin on Infarct Volume in Ischemic Stroke (S25.008)

Inhibition of soluble epoxide hydrolase augments astrocyte release of vascular endothelial growth factor and neuronal recovery after oxygen-glucose deprivation.

Epoxyeicosatrienoic acids (EETs) are synthesized in astrocytes, and inhibitors of soluble epoxide hydrolase (sEH), which hydrolyzes EETs, reduce infarct volume in ischemic stroke. Astrocytes can release protective neurotrophic factors, such as vascular endothelial growth factor (VEGF). We found that addition of sEH inhibitors to rat cultured astrocytes immediately after oxygen-glucose deprivation (OGD) markedly increased VEGF concentration in the medium 48h later and the effect was blocked by an EET antagonist. The sEH inhibitors increased EET concentrations to levels capable of increasing VEGF. When the sEH inhibitors were removed from the medium at 48h, the increase in VEGF persisted for an additional 48h. Neurons exposed to OGD and subsequently to astrocyte medium previously conditioned with OGD plus sEH inhibitors showed increased phosphorylation of their VEGF receptor-2, less TUNEL staining, and increased phosphorylation of Akt, which was blocked by a VEGF receptor-2 antagonist. Our findings indicate that sEH inhibitors, applied to cultured astrocytes after an ischemia-like insult, can increase VEGF secretion. The released VEGF then enhances Akt-enabled cell survival signaling in neurons through activation of VEGF receptor-2 leading to less neuronal cell death. These results suggest a new strategy by which astrocytes can be leveraged to support neuroprotection.

Renal Impairment and Infarct Volume in Ischemic Stroke

Background The decreased estimated glomerular filtration rate (eGFR) levels at admission in patients with stroke have been associated with more severe clinical deficits and with worse outcomes; however, the relationship between eGFR levels and volumetric measurement of cerebral infarct size on neuroimaging has not been studied, to our knowledge. Methods Consecutive patients who presented within 48h of ischemic stroke and underwent brain diffusion-weighted imaging (DWI) over a 55-month period were studied. Patients with ischemic stroke of large artery atherosclerosis (LAA), small vessel occlusion (SVO), or cardioembolism (CE) etiologies were included. Those who were treated with thrombolysis and disabled before index stroke were excluded. Infarction volumes were log transformed to approximate normality. Chronic kidney disease (CKD) was defined as an eGFR < 60 mL/min per 1.73 m2. Subjects were categorized into two groups: CKD or no CKD. The relationship between CKD and DWI infarct volumes was examined using an analysis of covariance. Results Of the included 405 patients (mean age, 68 years; female, 60.2%; LAA, 60.0%; SVO, 16.5%; CE, 23.5%), 108 patients had CKD. Infarct volumes (mL, median [interquartile range]) were not significantly different between stroke patients with CKD and those without CKD in any stroke subtype (LAA, 0.77 [0.01-2.97] vs. 0.96 [0.01-5.22]; SVO, 0.18 [0.02-0.21] vs. 0.27 [0.01-0.52]; CE, 8.91 [0.04-28.54] vs. 5.22 [0.05-42.39]). Adjustment for possible confounders did not change the associations. Conclusion Renal impairment is not associated with cerebral infarct volume among acute ischemic stroke patients. Key Words: Renal impairment, Infarct volume

Abstract 529: A Chloride Intracellular Channel-4 (Clic4) and VEGF-A Signaling Pathway Regulates Maturation of the Collateral Circulation

The density of native (pre-existing) cerebral collaterals in healthy tissues varies widely and is a major determinant of the wide variation in infarct volume in ischemic stroke. Collateral number in adults has been shown to be dependent on 3 processes: formation in the embryo, maturation in the perinate (i.e. slight pruning, increase in diameterand muralization) and maintenance thereafter. However, the mechanisms underlying these processes are not well understood. We previously found that collaterogenesis is led by a unique “arteriolar” angiogenic sprouting-like mechanism involving the VEGF-Notch signaling pathway. We also observed that mice lacking chloride intracellular protein 4 (Clic4), a protein which appears to reside upstream of VEGF-A in hypoxia-induced signaling, had fewer pial collaterals on postnatal day (P)1 that underwent greater pruning by P21, to yield lower collateral number and more severe stroke in adults. Therefore, herein we hypothesized that Clic4 is required for both formation and maturation of the collateral circulation. Wildtype and Clic4 -/- mice formed similar numbers of collaterals through embryonic day (E)18.5. Thus, collaterogenesis does not require Clic4. However, perinatal pruning was strongly augmented in Clic4 -/- mice, resulting in reduced collateral number and larger infarct volume in adults after MCA occlusion. Pial diameter was also reduced, consistent with our previous findings. Overexpression of VEGF-A in Clic4 -/- mice by crossing them to Vegf hi/+ mice did not affect collaterogenesis but inhibited the excessive perinatal pruning, resulting in adults with the abundant collaterals and small infarct volume seen in wildtype mice. We conclude that Clic4 is not required for embryonic collaterogenesis but is essential for perinatal stabilization of nascent collaterals through a mechanism involving VEGF signaling.

The Relationship between Acute Stress Hyperglycemia and Infarct Volume in Ischemic Stroke (P07.032)

Objective: To investigate the relationship between acute infarct volume on diffusion-weighted imaging (DWI) and acute stress hyperglycemia (ASH) in a prospectively collected dataset. Background The published evidence on the relationship between ischemic lesion volume and ASH is conflicting. While some studies report higher infarct volumes in patients with ASH, others refute this by claiming that small infarcts such as lacunar infarcts are also associated with ASH. Small sample size, failure to use a consistent definition for ASH, inability to account for differences in timing of infarct volume and glucose measurements, and differences in infarct size determination contribute to the controversy. We sought to investigate the relationship between acute infarct volume and ASH in a prospectively collected dataset within the context of the Heart-Brain Interactions Study (HBS). Design/Methods: HBS is an ongoing study aiming to identify the neuroanatomic correlates of stroke-related cardiac injury and acute stress response. We performed blood glucose measurement within 36 hours and DWI within 72 hours of symptom onset in consecutive patients enrolled to the HBS. ASH was defined as elevated blood glucose (>144 mg/dl) in the absence of elevated Hemoglobin A1c ( Results: We identified 98 consecutive patients with ASH and 217 controls out of 1240. The median infarct volume was higher in patients than controls (11 cc vs. 4 cc, p Conclusions: The study shows that infarct volume is an independent predictor of ASH. The probability of ASH increases by increasing infarct volume. Disclosure: Dr. Helenius has nothing to disclose. Dr. Avery has nothing to disclose. Dr. Polnar has nothing to disclose. Dr. Caceres has nothing to disclose. Dr. Siket has nothing to disclose. Dr. AY has nothing to disclose.

Abstract 114: Neuroprotective Role of Brain Pericytes through PDGFRβ-Akt Signaling in Ischemic Stroke

Brain pericytes are a constituent of the neurovascular unit and play various important roles in brain functions, such as regulation of capillary blood flow, maintenance of blood-brain barrier and angiogenesis. Previous reports have elucidated that PDGF-B prevents neuronal cell death during ischemic insults in adult rodent models; however, the detailed mechanisms by which PDGF-B signaling protects neurons from ischemic damage are not fully understood. In the present study, we investigated whether brain pericytes play neuroprotective roles in brain ischemia, using a permanent middle cerebral artery occlusion stroke model (MCAO) and cultured human brain pericytes. Immunohistochemistry revealed that the expression of PDGF receptorβ(PDGFRβ) was induced predominantly in pericytes in peri-infarct areas. PDGF-B induced marked phosphorylation of Akt in cultured pericytes. Consistently, Akt was markedly phosphorylated in the PDGFRβ-expressing pericytes in peri-infarct areas. PDGF-B upregulated the expression of neurotrophins, such as neuronal growth factor (NGF) and neurotrophin-3 (NT-3), through Akt activation in the cultured pericytes. We subjected PDGFRβheterozygous knockout (PDGFRβ+/-) mice to MCAO. Infarct volume, as assessed by MAP2 immunostaining, was significantly greater in PDGFRβ+/- than wild-type mice ( 48% increase at day 7, p &lt; 0.01 , n=5). The number of TUNEL positive apoptotic cells was significantly greater in PDGFRβ+/- mice (54 % increase at day 4, p &lt; 0.001 , n=6). Production of NGF and NT-3 at mRNA and protein levels in infarct areas was significantly decreased in PDGFRβ+/- mice (NGF: 28% decrease, p&lt;0.05, NT-3: 22% decrease, p&lt;0.05). Since it has been established that neurotrophin receptors are induced in peri-infarct areas, the decreases in neurotrophin production may increase apoptotic neuronal cell death in the PDGFRβ+/- mice. In conclusion, brain pericytes may have a direct neuroprotective role through secreting neurotrophins via PDGFRβ-Akt signaling, thereby decreasing infarct volume in ischemic stroke.

Admission international normalized ratio and acute infarct volume in ischemic stroke

The level of anticoagulation at the time of stroke onset may influence the size, composition, and dissolution rate of the occlusive clot. We explored the relation between admission international normalized ratio (INR) and acute infarct volume in patients with ischemic stroke. We studied 93 consecutive patients with preadmission warfarin use who had INR measurement and diffusion-weighted imaging performed within 24 hours of stroke onset. Ninety-three etiologic stroke subtype-matched patients without prior warfarin use served as control patients. Linear regression analysis was used to test for independence of INR as a predictor of infarct volume. In patients with preadmission warfarin use, admission INR was inversely correlated with lesion volume on diffusion-weighted imaging (r = -0.38). This relation was retained after adjustment for potential covariates (p = 0.014). INR less than 2.0 was associated with 3.5-fold (95% confidence interval, 2.9-4.2) greater lesion volume on diffusion-weighted imaging as compared with INR of 2.0 or more. Patients who were on therapeutic INR (>or=2.0) had smaller infarcts compared with patients without preadmission warfarin use (p = 0.001). Admission INR was inversely correlated with acute perfusion defect (r = -0.33), chronic infarct volume (r = -0.42), National Institutes of Health Stroke Scale score at admission (r = -0.27), and modified Rankin score at discharge (r = -0.28). These results suggest that preadmission warfarin use associated with therapeutic level of anticoagulation can offer a benefit in limiting the extent of ischemic injury in an event of acute stroke.

The Effect of Statin Pretreatment on Infarct Volume in Ischemic Stroke

Background: Treatment with statins reduces infarct volume in animal models of ischemic stroke, independently of the effect on cholesterol. This study examined this effect in humans by testing whether patients taking statins at onset of ischemic stroke had smaller infarct volumes than those not taking statins. Methods: The study design was a retrospective cohort analysis of all verified ischemic stroke patients admitted to the Medical University of South Carolina Hospital, in 2002–2006, with magnetic resonance diffusion-weighted imaging (DWI). Patients were grouped according to statin status at admission and compared relative to infarct volume, calculated (blinded to statin status) from DWI. Results: A smaller than median infarct volume following ischemic stroke was associated with the interaction of statin pretreatment and positive diabetes status. This association remained significant (p = 0.01) in multivariable analysis even after controlling for factors related to demographics, comorbidities and risk factors, clinical features on admission, use of other medications and stroke features. Conclusions: Among ischemic stroke patients in this study, infarct volume below the median was significantly associated with statin pretreatment among those with diabetes.

Prior antiplatelet use and infarct volume in ischemic stroke

Conflicting data exist on the role of antiplatelet agents in reducing incident ischemic stroke magnitude, but most prior studies used clinically-assessed neurologic deficit as the index of stroke extent rather than more precise volumetric measurements of infarct size. We assessed the relation of premorbid antiplatelet use to initial diffusion-weighted MRI (DWI) lesion volumes among acute ischemic stroke patients. Consecutive patients presenting within 24 h of ischemic stroke over an 18-month period were studied. DWI lesions were outlined using a semi-automated threshold technique. Subjects were categorized into two groups: antiplatelet (AP) or no antithrombotic (NA). The relationship between prestroke antithrombotic status and DWI infarct volumes was examined using multivariate quantile regression. One hundred sixty-six individuals met study criteria: 75 AP and 91 NA patients. Median DWI volume was lower in the AP group than in the NA group (1.5 cc vs. 5.4 cc, p=0.031). A multivariable model (adjusting for age, history of transient ischemic attack, admission temperature, admission blood pressure, admission serum glucose, stroke onset to imaging interval, stroke mechanism, premorbid statin and antihypertensive use) demonstrated smaller infarcts in the AP vs. NA group (adjusted volume difference: -1.3 cc, 95% CI=-0.09, -2.5, p=0.037). Prior statin use, no history of TIA, large vessel atherosclerosis and microvascular ischemic disease stroke mechanism were also independently associated with reduced infarct volume. Prior antiplatelet treatment is independently associated with reduced cerebral infarct volume among acute ischemic stroke patients. Premorbid statin use, TIA history and stroke mechanism also predict infarct volume in ischemic stroke.