Delayed plasma kallikrein inhibition fosters post-stroke recovery by reducing thrombo-inflammation.

Activation of the kallikrein-kinin system promotes vascular leakage, inflammation, and neurodegeneration in ischemic stroke. Inhibition of plasma kallikrein (PK) - a key component of the KKS - in the acute phase of ischemic stroke has been reported to reduce thrombosis, inflammation, and damage to the blood-brain barrier. However, the role of PK during the recovery phase after cerebral ischemia is unknown. To this end, we evaluated the effect of subacute PK inhibition starting from day 3 on the recovery process after transient middle artery occlusion (tMCAO). Our study demonstrated a protective effect of PK inhibition by reducing infarct volume and improving functional outcome at day 7 after tMCAO. In addition, we observed reduced thrombus formation in cerebral microvessels, fewer infiltrated immune cells, and an improvement in blood-brain barrier integrity. This protective effect was facilitated by promoting tight junction reintegration, reducing detrimental matrix metalloproteinases, and upregulating regenerative angiogenic markers. Our findings suggest that PK inhibition in the subacute phase might be a promising approach to accelerate the post-stroke recovery process.

Dimethyl Fumarate Attenuates Lymphocyte Infiltration and Reduces Infarct Size in Experimental Stroke.

Ischemic stroke is associated with exacerbated tissue damage caused by the activation of immune cells and the initiation of other inflammatory processes. Dimethyl fumarate (DMF) is known to modulate the immune response, activate antioxidative pathways, and improve the blood-brain barrier (BBB) after stroke. However, the specific impact of DMF on immune cells after cerebral ischemia remains unclear. In our study, male mice underwent transient middle cerebral artery occlusion (tMCAO) for 30 min and received oral DMF (15 mg/kg) or a vehicle immediately after tMCAO, followed by twice-daily administrations for 7 days. Infarct volume was assessed on T2-weighted magnetic resonance images on days 1 and 7 after tMCAO. Brain-infiltrating immune cells (lymphocytes, monocytes) and microglia were quantified using fluorescence-activated cell sorting. DMF treatment significantly reduced infarct volumes and brain edema. On day 1 after tMCAO, DMF-treated mice showed reduced lymphocyte infiltration compared to controls, which was not observed on day 7. Monocyte and microglial cell counts did not differ between groups on either day. In the acute phase of stroke, DMF administration attenuated lymphocyte infiltration, probably due to its stabilizing effect on the BBB. This highlights the potential of DMF as a therapeutic candidate for mitigating immune cell-driven damage in stroke.

Thromboinflammation in Brain Ischemia: Recent Updates and Future Perspectives.

Despite decades of promising preclinical validation and clinical translation, ischemic stroke still remains as one of the leading causes of death and disability worldwide. Within its complex pathophysiological signatures, thrombosis and inflammation, that is, thromboinflammation, are highly interconnected processes leading to cerebral vessel occlusion, inflammatory responses, and severe neuronal damage following the ischemic event. Hence, we here review the most recent updates on thromboinflammatory-dependent mediators relevant after stroke focusing on recent discoveries on platelet modulation, a potential regulation of the innate and adaptive immune system in thromboinflammation, utterly providing a thorough up-to-date overview of all therapeutic approaches currently undergoing clinical trial.

ERK1/2 Activity Is Critical for the Outcome of Ischemic Stroke.

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.

Elastase inhibitor agaphelin protects from acute ischemic stroke in mice by reducing thrombosis, blood-brain barrier damage, and inflammation.

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.

Validity and Reliability of Neurological Scores in Mice Exposed to Middle Cerebral Artery Occlusion.

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] mm3). Among all tests, the Bederson (0-5; r=0.63, P<0.001), modified neurological severity (r=0.80, P<0.001), and subjective overall impression (r=-0.63, P<0.001) scores had the highest test validities, using infarct volume as external reference. Subjective overall impression had the best agreement between 5 raters (Kendall W=0.11, P<0.001). The Bederson (0-5) score discriminated infarct volume in mice with small (≤50 mm3; r=0.33, P=0.027) and large (>50 mm3; r=0.48, P<0.001) brain infarcts, all other tests only in mice with large infarcts. Combining subjective overall impression with Bederson (0-5) score explained 47.6% of the variance of infarct volume. 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.

CK2ß regulates thrombopoiesis and Ca2+-triggered platelet activation in arterial thrombosis.

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 ß-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2ß (ck2ß-/- ) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2ß-/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2ß-/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-triphosphate receptor-dependent intracellular calcium (Ca2+) release. Presumably due to a blunted increase in the concentration of cytosolic Ca2+, activation-dependent increases of α and dense-granule secretion and integrin αIIbß3 activation, and aggregation were abrogated in ck2ß-/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo was significantly blunted in ck2ß-/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2ß-/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2ßfl/fl mice. The present observations reveal CK2ß as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo.

ADAMTS13-mediated thrombolysis of t-PA-resistant occlusions in ischemic stroke in mice.

Rapid vascular recanalization forms the basis for successful treatment of cerebral ischemia. Currently, tissue plasminogen activator (t-PA) is the only approved thrombolytic drug for ischemic stroke. However, t-PA does not always result in efficient thrombus dissolution and subsequent blood vessel recanalization. To better understand thrombus composition, we analyzed thrombi retrieved from ischemic stroke patients and found a distinct presence of von Willebrand factor (VWF) in various samples. Thrombi contained on average 20.3% ± 10.1% VWF, and this was inversely correlated with thrombus red blood cell content. We hypothesized that ADAMTS13 can exert a thrombolytic effect in VWF-containing thrombi in the setting of stroke. To test this, we generated occlusive VWF-rich thrombi in the middle cerebral artery (MCA) of mice. Infusion of t-PA did not dissolve these MCA occlusions. Interestingly, administration of ADAMTS13 5 minutes after occlusion dose-dependently dissolved these t-PA-resistant thrombi resulting in fast restoration of MCA patency and consequently reduced cerebral infarct sizes (P < .005). Delayed ADAMTS13 administration 60 minutes after occlusion was still effective but to a lesser extent (P < .05). These data show for the first time a potent thrombolytic activity of ADAMTS13 in the setting of stroke, which might become useful in treatment of acute ischemic stroke.

B cells do not have a major pathophysiologic role in acute ischemic stroke in mice.

BACKGROUND: Lymphocytes have been shown to play an important role in the pathophysiology of acute ischemic stroke, but the properties of B cells remain controversial. The aim of this study was to unravel the role of B cells during acute cerebral ischemia using pharmacologic B cell depletion, B cell transgenic mice, and adoptive B cell transfer experiments. METHODS: Transient middle cerebral artery occlusion (60 min) was induced in wild-type mice treated with an anti-CD20 antibody 24 h before stroke onset, JHD -/- mice and Rag1 -/- mice 24 h after adoptive B cell transfer. Stroke outcome was assessed at days 1 and 3. Infarct volumes were calculated from 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain sections, and neurological scores were evaluated. The local inflammatory response was determined by real-time PCR and immunohistochemistry. Apoptosis was analyzed by TUNEL staining, and astrocyte activation was revealed using immunohistochemistry and Western blot. RESULTS: Pharmacologic depletion of B cells did not influence infarct volumes and functional outcome at day 1 after stroke. Additionally, lack of circulating B cells in JHD -/- mice also failed to influence stroke outcome at days 1 and 3. Furthermore, reconstitution of Rag1 -/- mice with B cells had no influence on infarct volumes. CONCLUSION: Targeting B cells in experimental stroke did not influence lesion volume and functional outcome during the acute phase. Our findings argue against a major pathophysiologic role of B cells during acute ischemic stroke.

Blocking of plasma kallikrein ameliorates stroke by reducing thromboinflammation.

OBJECTIVE: 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. METHODS: We investigated the consequences of PK inhibition in transient and permanent models of ischemic stroke. RESULTS: 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. INTERPRETATION: 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 two-pore domain potassium channel KCNK5 deteriorates outcome in ischemic neurodegeneration.

Potassium channels can fulfill both beneficial and detrimental roles in neuronal damage during ischemic stroke. Earlier studies have characterized a neuroprotective role of the two-pore domain potassium channels KCNK2 (TREK1) and KCNK3 (TASK1). Protective neuronal hyperpolarization and prevention of intracellular Ca(2+) overload and glutamate excitotoxicity were suggested to be the underlying mechanisms. We here identify an unexpected role for the related KCNK5 channel in a mouse model of transient middle cerebral artery occlusion (tMCAO). KCNK5 is strongly upregulated on neurons upon cerebral ischemia, where it is most likely involved in the induction of neuronal apoptosis. Hypoxic conditions elevated neuronal expression levels of KCNK5 in acute brain slices and primary isolated neuronal cell cultures. In agreement, KCNK5 knockout mice had significantly reduced infarct volumes and improved neurologic function 24 h after 60 min of tMCAO and this protective effect was preserved at later stages of infarct development. KCNK5 deficiency resulted in a significantly reduced number of apoptotic neurons, a downregulation of pro-apoptotic and upregulation of anti-apoptotic factors. Results of adoptive transfer experiments of wild-type and Kcnk5 (-/-) immune cells into Rag1 (-/-) mice prior to tMCAO exclude a major role of KCNK5 in poststroke inflammatory reactions. In summary, KCNK5 expression is induced on neurons under ischemic conditions where it most likely exerts pro-apoptotic effects. Hence, pharmacological blockade of KCNK5 might have therapeutic potential in preventing ischemic neurodegeneration.

Regulatory T cells are strong promoters of acute ischemic stroke in mice by inducing dysfunction of the cerebral microvasculature.

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.

Blocking of α4 integrin does not protect from acute ischemic stroke in mice.

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.

Kininogen deficiency protects from ischemic neurodegeneration in mice by reducing thrombosis, blood-brain barrier damage, and inflammation.

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.

Impact of NKG2D Signaling on Natural Killer and T-Cell Function in Cerebral Ischemia.

Background Typically defined as a thromboinflammatory disease, ischemic stroke features early and delayed inflammatory responses, which determine the extent of ischemia-related brain damage. T and natural killer cells have been implicated in neuronal cytotoxicity and inflammation, but the precise mechanisms of immune cell-mediated stroke progression remain poorly understood. The activating immunoreceptor NKG2D is expressed on both natural killer and T cells and may be critically involved. Methods and Results An anti-NKG2D blocking antibody alleviated stroke outcome in terms of infarct volume and functional deficits, coinciding with reduced immune cell infiltration into the brain and improved survival in the animal model of cerebral ischemia. Using transgenic knockout models devoid of certain immune cell types and immunodeficient mice supplemented with different immune cell subsets, we dissected the functional contribution of NKG2D signaling by different NKG2D-expressing cells in stroke pathophysiology. The observed effect of NKG2D signaling in stroke progression was shown to be predominantly mediated by natural killer and CD8+ T cells. Transfer of T cells with monovariant T-cell receptors into immunodeficient mice with and without pharmacological blockade of NKG2D revealed activation of CD8+ T cells irrespective of antigen specificity. Detection of the NKG2D receptor and its ligands in brain samples of patients with stroke strengthens the relevance of preclinical observations in human disease. Conclusions Our findings provide a mechanistic insight into NKG2D-dependent natural killer- and T-cell-mediated effects in stroke pathophysiology.

C1-inhibitor protects from brain ischemia-reperfusion injury by combined antiinflammatory and antithrombotic mechanisms.

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.

Platelet depletion does not alter long-term functional outcome after cerebral ischaemia in mice.

Platelets are key mediators of thrombus formation and inflammation during the acute phase of ischaemic stroke. Particularly, the platelet glycoprotein (GP) receptors GPIbα and GPVI have been shown to mediate platelet adhesion and activation in the ischaemic brain. GPIbα and GPVI blockade could reduce infarct volumes and improve functional outcome in mouse models of acute ischaemic stroke, without concomitantly increasing intracerebral haemorrhage. However, the functional role of platelets during long-term stroke recovery has not been elucidated so far. Thus, we here examined the impact of platelet depletion on post-stroke recovery after transient middle cerebral artery occlusion (tMCAO) in adult male mice. Platelet depleting antibodies or isotype control were applied from day 3-28 after tMCAO in mice matched for infarct size. Long-term functional recovery was assessed over the course of 28 days by behavioural testing encompassing motor and sensorimotorical functions, as well as anxiety-like or spontaneous behaviour. Whole brain flow cytometry and light sheet fluorescent microscopy were used to identify resident and infiltrated immune cell types, and to determine the effects of platelet depletion on the cerebral vascular architecture, respectively. We found that delayed platelet depletion does not improve long-term functional outcome in the tMCAO stroke model. Immune cell abundance, the extent of thrombosis and the organisation of the cerebral vasculature were also comparable between platelet-depleted and control mice. Our study demonstrates that, despite their critical role in the acute stroke setting, platelets appear to contribute only marginally to tissue reorganisation and functional recovery at later stroke stages.

ACKR3 regulates platelet activation and ischemia-reperfusion tissue injury.

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.

Chemical shift sodium imaging in a mouse model of thromboembolic stroke at 9.4 T.

PURPOSE: To estimate changes in the (23)Na density and in the (23)Na relaxation time T(2) * in the anatomically small murine brain after stroke. MATERIALS AND METHODS: Three-dimensional acquisition weighted chemical shift imaging at a resolution of 0.6 × 0.6 × 1.2 mm(3) was used for sodium imaging and relaxation parameter mapping. In vivo measurements of the mouse brain (n = 4) were performed 24 hours after stroke, induced by microinjection of purified murine thrombin into the right middle cerebral artery. The measurement time was 14 minutes in one mouse and 65 minutes in the other three. An exponential fit estimation of the free induction decay was calculated for each voxel enabling the reconstruction of locally resolved relaxation parameter maps. RESULTS: The infarcted areas showed an increase in sodium density between 160% and 250%, while the T(2) * relaxation time increased by 5%-72% compared to unaffected contralateral brain tissue. CONCLUSION: (23)Na chemical shift imaging at a resolution of 0.6 × 0.6 × 1.2 mm(3) enabled sodium imaging of the anatomical small mouse brain and the acquired data allowed calculating relaxation parameter maps and hence a more exact evaluation of sodium signal changes after stroke.