Search

Recently it was shown that the hematophagous salivary gland protein agaphelin exhibits multiple antithrombotic effects without promoting the risk of bleeding. Agaphelin inhibits neutrophil elastase and thereby reduces cathepsin G-induced platelet aggregation. However, it is still unclear, whether pharmacological treatment with agaphelin in brain ischemia is protective and, regarding its bleeding risk, safe. To elucidate this issue, male C57BL/6 mice were subjected to 60 min of transient middle cerebral artery occlusion (tMCAO) and treated with 0.25 mg/kg agaphelin intravenously immediately after tMCAO. On day 1 and 7, infarct volume and functional neurological outcome were assessed by behavioural tests, histochemistry and magnetic resonance imaging. Thrombus formation, intracerebral bleeding risk, blood-brain barrier damage and the local inflammatory response were determined on day 1. This study shows for the first time a protective effect of agaphelin characterized by smaller infarct volume, reduced neurological deficits and reduced animal mortality. This protective effect was associated with reduced local thrombus formation, increased blood-brain barrier integrity and reduced brain inflammatory response. It is essential to mention that the protective effect of agaphelin was not linked to an increased risk of intracerebral bleeding. The promotion of brain tissue survival and inhibition of thromboinflammation identifies agaphelin as a promising treatment option in ischemic stroke, which considering the lack of bleeding risk should potentially be safe.

Platelet activation plays a critical role in thrombosis. Inhibition of platelet activation is a cornerstone in treatment of acute organ ischemia. Platelet ACKR3 surface expression is independently associated with all-cause mortality in CAD patients. In a novel genetic mouse strain, we show that megakaryocyte/platelet-specific deletion of ACKR3 results in enhanced platelet activation and thrombosis in vitro and in vivo. Further, we performed ischemia/reperfusion experiments (transient LAD-ligation and tMCAO) in mice to assess the impact of genetic ACKR3 deficiency in platelets on tissue injury in ischemic myocardium and brain. Loss of platelet ACKR3 enhances tissue injury in ischemic myocardium and brain and aggravates tissue inflammation. Activation of platelet-ACKR3 via specific ACKR3 agonists inhibits platelet activation and thrombus formation and attenuates tissue injury in ischemic myocardium and brain. Here we demonstrate that ACKR3 is a critical regulator of platelet activation, thrombus formation and organ injury following ischemia/reperfusion. CA extern

Ischemic disorders are the leading cause of death worldwide. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are thought to affect the outcome of ischemic stroke. However, it is under debate whether activation or inhibition of ERK1/2 is beneficial. In this study, we report that the ubiquitous overexpression of wild-type ERK2 in mice (ERK2wt) is detrimental after transient occlusion of the middle cerebral artery (tMCAO), as it led to a massive increase in infarct volume and neurological deficits by increasing blood–brain barrier (BBB) leakiness, inflammation, and the number of apoptotic neurons. To compare ERK1/2 activation and inhibition side-by-side, we also used mice with ubiquitous overexpression of the Raf-kinase inhibitor protein (RKIPwt) and its phosphorylation-deficient mutant RKIPS153A, known inhibitors of the ERK1/2 signaling cascade. RKIPwt and RKIPS153A attenuated ischemia-induced damages, in particular via anti-inflammatory signaling. Taken together, our data suggest that stimulation of the Raf/MEK/ERK1/2-cascade is severely detrimental and its inhibition is rather protective. Thus, a tight control of the ERK1/2 signaling is essential for the outcome in response to ischemic stroke.

Background and Purpose— The selection of appropriate neurological scores and tests is crucial for the evaluation of stroke consequences. The validity and reliability of neurological deficit scores and tests has repeatedly been questioned in ischemic stroke models in the past. Methods— In 198 male mice exposed to transient intraluminal middle cerebral artery occlusion, we examined the validity and reliability of 11 neurological scores (Bederson score 0–3, Bederson score 0–4, Bederson score 0–5, modified neurological severity [0–14], subjective overall impression [0–10], or simple neurological tests: grip test, latency to move body length test, pole test, wire hanging test, negative geotaxis test, and elevated body swing test) in the acute stroke phase, that is, after 24 hours. Combinations of neurological scores or tests for predicting infarct volume were statistically analyzed. Results— Infarct volume was left skewed (median [Q1–Q3], 78.4 [54.8–101.3] mm 3 ). Among all tests, the Bederson (0–5; r=0.63, P P P P 3 ; r=0.33, P =0.027) and large (>50 mm 3 ; r=0.48, P Conclusions— Despite their simplicity, the Bederson (0–5) score, modified neurological severity score, and subjective overall impression have reasonable validity and reliability in the acute stroke phase. The Bederson (0–5) score equally distinguishes infarct volume in small and large infarcts. Visual Overview— An online visual overview is available for this article.

Ischemic stroke is a predominant cause of disability worldwide, with thrombolytic or mechanical removal of the occlusion being the only therapeutic option. Reperfusion bears the risk of an acute deleterious calcium-dependent breakdown of the blood-brain barrier. Its mechanism, however, is unknown. Here, we identified type 5 NADPH oxidase (NOX5), a calciumactivated, ROS-forming enzyme, as the missing link. Using a humanized knockin (KI) mouse model and in vitro organotypic cultures, we found that reoxygenation or calcium overload increased brain ROS levels in a NOX5-dependent manner. In vivo, postischemic ROS formation, infarct volume, and functional outcomes were worsened in NOX5-KI mice. Of clinical and therapeutic relevance, in a human blood-barrier model, pharmacological NOX inhibition also prevented acute reoxygenationinduced leakage. Our data support further evaluation of poststroke recanalization in the presence of NOX inhibition for limiting stroke-induced damage.

Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-)physiology has remained elusive. The present study explored the impact of the CK2 regulatory β-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2β (

Background and Purpose— T lymphocytes have recently been identified as key mediators of tissue damage in ischemic stroke. The interaction between very late antigen-4 (VLA-4) and vascular adhesion molecule-1 is crucial for the transvascular egress of T lymphocytes, and inhibition of this interaction by specific antibodies is a powerful strategy to combat autoimmune neuroinflammation. However, whether pharmacological blocking of T-lymphocyte trafficking is also protective during brain ischemia is still unclear. We investigated the efficacy of a monoclonal antibody directed against VLA-4 in mouse models of ischemic stroke. Methods— Transient and permanent middle cerebral artery occlusion was induced in male C57Bl/6 mice. Animals treated with a monoclonal anti-CD49d antibody (300 μg) 24 hours before or 3 hours after the onset of cerebral ischemia and stroke outcome, including infarct size, functional status, and mortality, were assessed between day 1 and day 7. The numbers of immune cells invading the ischemic brain were determined by immunocytochemistry and flow cytometry. Results— Blocking of VLA-4 significantly reduced the invasion of T lymphocytes and neutrophils on day 5 after middle cerebral artery occlusion and inhibited the upregulation of vascular adhesion molecule-1. However, the anti-CD49d antibody failed to influence stroke outcome positively irrespective of the model or the time point investigated. Conclusions— Pharmacological inhibition of the VLA-4/vascular adhesion molecule-1 axis in experimental stroke was ineffective in our hands. Our results cast doubt on the effectiveness of anti-CD49d as a stroke treatment. Further translational studies should be performed before testing anti–VLA-4 antibodies in patients with stroke.

Thrombosis and inflammation are hallmarks of ischemic stroke still unamenable to therapeutic interventions. High-molecular-weight kininogen (KNG) is a central constituent of the contact-kinin system which represents an interface between thrombotic and inflammatory circuits and is critically involved in stroke development. Kng−/− mice are protected from thrombosis after artificial vessel wall injury and lack the proinflammatory mediator bradykinin. We investigated the consequences of KNG deficiency in models of ischemic stroke. Kng−/− mice of either sex subjected to transient middle cerebral artery occlusion developed dramatically smaller brain infarctions and less severe neurologic deficits without an increase in infarct-associated hemorrhage. This protective effect was preserved at later stages of infarction as well as in elderly mice. Targeting KNG reduced thrombus formation in ischemic vessels and improved cerebral blood flow, and reconstitution of KNG-deficient mice with human KNG or bradykinin restored clot deposition and infarct susceptibility. Moreover, mice deficient in KNG showed less severe blood-brain barrier damage and edema formation, and the local inflammatory response was reduced compared with controls. Because KNG appears to be instrumental in pathologic thrombus formation and inflammation but dispensable for hemostasis, KNG inhibition may offer a selective and safe strategy for combating stroke and other thromboembolic diseases.

Background and Purpose— Inflammation and thrombosis are pathophysiological hallmarks of ischemic stroke still unamenable to therapeutic interventions. The contact-kinin system represents an interface between inflammatory and thrombotic circuits and is involved in stroke development. C1-inhibitor counteracts activation of the contact-kinin system at multiple levels. We investigated the therapeutic potential of C1-inhibitor in models of ischemic stroke. Methods— Male and female C57Bl/6 mice and rats of different ages were subjected to middle cerebral artery occlusion and treated with C1-inhibitor after 1 hour or 6 hours. Infarct volumes and functional outcomes were assessed between day 1 and day 7, and findings were validated by magnetic resonance imaging. Blood–brain barrier damage, thrombus formation, and the local inflammatory response were determined poststroke. Results— Treatment with 15.0 U C1-inhibitor, but not 7.5 U, 1 hour after stroke reduced infarct volumes by ≈60% and improved clinical scores in mice of either sex on day 1. This protective effect was preserved at later stages of infarction as well as in elderly mice and in another species, ie, rats. Delayed C1-inhibitor treatment still improved clinical outcome. Blood–brain barrier damage, edema formation, and inflammation were significantly lower compared with controls. Moreover, C1-inhibitor showed strong antithrombotic effects. Conclusions— C1-inhibitor is a multifaceted antiinflammatory and antithrombotic compound that protects from ischemic neurodegeneration in clinically meaningful settings.

Recent evidence suggests that ischemic stroke is a thromboinflammatory disease. Plasma kallikrein (PK) cleaves high-molecular-weight kininogen to release bradykinin (BK) and is a key constituent of the proinflammatory contact-kinin system. In addition, PK can activate coagulation factor XII, the origin of the intrinsic coagulation cascade. Thus, PK triggers 2 important pathological pathways of stroke formation, thrombosis and inflammation.We investigated the consequences of PK inhibition in transient and permanent models of ischemic stroke.PK-deficient mice of either sex challenged with transient middle cerebral artery occlusion developed significantly smaller brain infarctions and less severe neurological deficits compared with controls without an increase in infarct-associated hemorrhage. This protective effect was preserved at later stages of infarctions as well as after permanent stroke. Reduced intracerebral thrombosis and improved cerebral blood flow could be identified as underlying mechanisms. Moreover, blood-brain barrier function was maintained in mice lacking PK, and the local inflammatory response was reduced. PK-deficient mice reconstituted with PK or BK again developed brain infarctions similar to wild-type mice. Important from a translational perspective, inhibition of PK in wild-type mice using a PK-specific antibody was likewise effective even when performed in a therapeutic setting up to 3 hours poststroke.PK drives thrombus formation and inflammation via activation of the intrinsic coagulation cascade and the release of BK but appears to be dispensable for hemostasis. Hence, PK inhibition may offer a safe strategy to combat thromboembolic disorders including ischemic stroke.

The blood-brain barrier (BBB) is an integral part of the neurovascular unit (NVU). The NVU is comprised of endothelial cells that are interconnected by tight junctions resting on a parenchymal basement membrane ensheathed by pericytes, smooth muscle cells and a layer of astrocyte end feet. Circulating blood cells, such as leukocytes, complete the NVU. BBB disruption is common in several neurological diseases, but the molecular mechanisms involved remain largely unknown. We analyzed the role of TWIK-related potassium channel-1 (TREK1, encoded by KCNK2) in human and mouse endothelial cells and the BBB. TREK1 was downregulated in endothelial cells by treatment with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Blocking TREK1 increased leukocyte transmigration, whereas TREK1 activation had the opposite effect. We identified altered mitogen-activated protein (MAP) kinase signaling, actin remodeling and upregulation of cellular adhesion molecules as potential mechanisms of increased migration in TREK1-deficient (Kcnk2(-/-)) cells. In Kcnk2(-/-) mice, brain endothelial cells showed an upregulation of the cellular adhesion molecules ICAM1, VCAM1 and PECAM1 and facilitated leukocyte trafficking into the CNS. Following the induction of experimental autoimmune encephalomyelitis (EAE) by immunization with a myelin oligodendrocyte protein (MOG)35-55 peptide, Kcnk2(-/-) mice showed higher EAE severity scores that were accompanied by increased cellular infiltrates in the central nervous system (CNS). The severity of EAE was attenuated in mice given the amyotrophic lateral sclerosis drug riluzole or fed a diet enriched with linseed oil (which contains the TREK-1 activating omega-3 fatty acid α-linolenic acid). These beneficial effects were reduced in Kcnk2(-/-) mice, suggesting TREK-1 activating compounds may be used therapeutically to treat diseases related to BBB dysfunction.

We have recently identified T cells as important mediators of ischemic brain damage, but the contribution of the different T-cell subsets is unclear. Forkhead box P3 (FoxP3)–positive regulatory T cells (Tregs) are generally regarded as prototypic anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. In the present study, we examined the role of Tregs after experimental brain ischemia/reperfusion injury. Selective depletion of Tregs in the DEREG mouse model dramatically reduced infarct size and improved neurologic function 24 hours after stroke and this protective effect was preserved at later stages of infarct development. The specificity of this detrimental Treg effect was confirmed by adoptive transfer experiments in wild-type mice and in Rag1−/− mice lacking lymphocytes. Mechanistically, Tregs induced microvascular dysfunction in vivo by increased interaction with the ischemic brain endothelium via the LFA-1/ICAM-1 pathway and platelets and these findings were confirmed in vitro. Ablation of Tregs reduced microvascular thrombus formation and improved cerebral reperfusion on stroke, as revealed by ultra-high-field magnetic resonance imaging at 17.6 Tesla. In contrast, established immunoregulatory characteristics of Tregs had no functional relevance. We define herein a novel and unexpected role of Tregs in a primary nonimmunologic disease state.

We assessed the neuroprotective potential of α2-adrenoceptors in ischemic stroke using mice with targeted deletions of individual α2-adrenoceptor subtypes (α 2A−/−, α 2B−/−, α 2C−/−, α 2A/C−/−). The effects of the α2-adrenoceptor agonist clonidine were studied in parallel. Focal cerebral ischemia was induced with or without clonidine pretreatment by transient middle cerebral artery occlusion. Neurologic outcome and infarct volumes were evaluated on day 1. Cerebral blood flow (CBF) and mean arterial pressure were determined. α2- Adrenoceptor null mice did not display larger infarct volumes compared with wild-type (WT) mice under basal conditions ( P>0.05). In line with this finding, pretreatment with clonidine did not protect from ischemic brain damage in WT mice or α 2A−/−, α 2B−/−, and α 2C−/− mice. Clonidine induced smaller infarct volumes only in α 2A/C−/− mice ( P < 0.05), but this did not translate into improved neurologic function ( P > 0.05). Importantly, while clonidine caused a significant decrease in arterial blood pressure in all groups, it had no blood pressure lowering effect in α 2A/C−/− mice, and this correlated with higher CBF and smaller infarct volumes in this group. In summary, we could not demonstrate a neuroprotective function of α2-adrenoceptors in focal cerebral ischemia. Careful controlling of physiological parameters relevant for stroke outcome is recommended in experimental stroke studies.