Michael Gliem

Macrophage-derived osteopontin induces reactive astrocyte polarization and promotes re-establishment of the blood brain barrier after ischemic stroke

Infarcted regions of the brain after stroke are segregated from the intact brain by scar tissue comprising both fibrous and glial components. The extent and quality of scarring is influenced by inflammation. The matricellular glycoprotein osteopontin (OPN) is strongly induced in myeloid cells after stroke and may contribute to repair of ischemic brain lesions. To elucidate the role of OPN in scar formation, we induced photothrombotic brain infarction, characterized by circumscribed cortical infarctions with a well‐defined border zone toward the intact brain parenchyma. The cellular source and functional role of OPN was addressed by studies in OPN null (OPN‐/‐) mice, wild‐type mice depleted of hematogenous monocytes/macrophages by clodronate‐filled liposome treatment, and CCR2‐/‐ bone marrow chimeric mice characterized by impaired hematogenous macrophage influx into the infarctions. OPN was mainly produced by hematogenous macrophages infiltrating into the inner border zone of the infarcts whereas astrocyte activation occurred in the outer border zone. In OPN‐/‐ as well as macrophage‐depleted mice, reactive astrocytes failed to properly extend processes from the periphery toward the center of the infarctions. This was associated with incomplete coverage of neovessels by astrocytic endfeet and persistent leakiness of the damaged blood brain barrier. In conclusion, OPN produced by hematogenous macrophages induces astrocyte process extension toward the infarct border zone, which may contribute to repair of the ischemic neurovascular unit. GLIA 2015;63:2198–2207

IFNβ secreted by microglia mediates clearance of myelin debris in CNS autoimmunity

IntroductionMultiple sclerosis (MS) is a chronic demyelinating disorder of the central nervous system (CNS) leading to progressive neurological disability. Interferon β (IFNβ) represents a standard treatment for relapsing-remitting MS and exogenous administration of IFNβ exhibits protective effects in experimentally induced CNS autoimmunity. Also, genetic deletion of IFNβ in mice leads to an aggravation of disease symptoms in the MS model of experimental autoimmune encephalomyelitis (EAE). However, neither the underlying mechanisms mediating the beneficial effects nor the cellular source of IFNβ have been fully elucidated.ResultsIn this report, a subpopulation of activated microglia was identified as the major producers of IFNβ in the CNS at the peak of EAE using an IFNβ-fluorescence reporter mouse model. These IFNβ expressing microglia specifically localized to active CNS lesions and were associated with myelin debris in demyelinated cerebellar organotypic slice cultures (OSCs). In response to IFNβ microglia showed an enhanced capacity to phagocytose myelin in vitro and up-regulated the expression of phagocytosis-associated genes. IFNβ treatment was further sufficient to stimulate association of microglia with myelin debris in OSCs. Moreover, IFNβ-producing microglia mediated an enhanced removal of myelin debris when co-transplanted onto demyelinated OSCs as compared to IFNβ non-producing microglia.ConclusionsThese data identify activated microglia as the major producers of protective IFNβ at the peak of EAE and as orchestrators of IFNβ-induced clearance of myelin debris.

Protective features of peripheral monocytes/macrophages in stroke

Hematogenous recruitment of monocytes and macrophages has traditionally been viewed as a harmful process causing exacerbation of brain injury after stroke. However, emerging findings suggest equally important protective features. Inflammatory monocytes are rapidly recruited to ischemic brain via a CCR2-dependent pathway and undergo secondary differentiation in the target tissue towards non-inflammatory macrophages, mediating neuroprotection and repair of the ischemic neurovascular unit. In contrast, independent recruitment of non-inflammatory monocytes via CX3CR1 does not occur. Thus, protective features of hematogenous macrophages mainly depend on initial CCR2-dependent cell recruitment. Under therapeutic considerations, specific modulation of monocyte-derived macrophages will therefore be more appropriate than non-selectively blocking their hematogenous recruitment. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.

Secondary Intracerebral Hemorrhage Due to Early Initiation of Oral Anticoagulation After Ischemic Stroke An Experimental Study in Mice

Background and Purpose— The uncertain risk of secondary intracerebral hemorrhage (sICH) frequently keeps clinicians from initiating oral anticoagulation (OAC) early after ischemic cardioembolic stroke. The goal of this experimental study was to determine the risk of sICH depending on the timing of OAC initiation relative to stroke onset and to address the role of hematogenous macrophages for repair processes preventing OAC-associated sICH. Methods— C57BL/6 mice were subjected to transient middle cerebral artery occlusion. Subgroups were treated with either the vitamin K antagonist (VKA) phenprocoumon or the direct thrombin inhibitor dabigatran etexilate. Hematogenous macrophages were depleted using intraperitoneal injections of clodronate-filled liposomes. Results— Time to therapeutic OAC was 48 hours with VKA and 0.5 hours with dabigatran etexilate treatment. In VKA-treated mice, the risk of sICH was high if effective OAC was already present at stroke onset or achieved within 48 hours after ischemia. With more delayed OAC, the risk of sICH rapidly decreased. Compared with VKA treatment, effective anticoagulation with dabigatran etexilate was associated with a significantly reduced extent of sICH, either if present at stroke onset or if achieved 48 hours later. Partial depletion of macrophages greatly increased the extent of OAC-associated sICH in the subacute stage of 3 to 4 days after ischemia. Conclusions— Our findings suggest that repair mechanisms involving hematogenous macrophages rapidly decrease the risk of OAC-associated sICH in the first days after ischemic stroke. The lower risk of sICH under dabigatran etexilate compared with VKA treatment may facilitate early initiation of OAC after cardioembolic stroke.

Hyperglycemia and PPARγ Antagonistically Influence Macrophage Polarization and Infarct Healing After Ischemic Stroke

Background and Purpose— Secondary intracerebral hemorrhage (sICH) is a potentially serious complication of ischemic stroke, in particular under concomitant oral anticoagulation. Previous studies in murine stroke models defined a novel vascular repair function of hematogenous monocytes/macrophages (MO/MP), which proved essential for the prevention of oral anticoagulation–associated sICH. Here, we addressed the question whether hyperglycemia as a clinically relevant prohemorrhagic risk factor and peroxisome proliferator-activated receptor gamma (PPAR&ggr;) activation affect MO/MP differentiation and the risk of sICH after ischemic stroke. Methods— Oral anticoagulation–associated sICH was induced by phenprocoumon feeding to mice undergoing transient middle cerebral artery occlusion. Hyperglycemia was induced by streptozotocin treatment. The role of PPAR&ggr;-dependent MO/MP differentiation was addressed in mice with myeloid cell–specific PPAR&ggr;-knockout (LysM-PPAR&ggr;(KO)). Pharmacological PPAR&ggr; activation via pioglitazone was tested as a treatment option. Results— Hyperglycemic mice and normoglycemic LysM-PPAR&ggr;(KO) mice exhibited abnormal proinflammatory skewing of their hematogenous MO/MP response and abnormal vascular remodeling in the infarct border zone, leading to an increased rate of oral anticoagulation–associated sICH. Pharmacological PPAR&ggr; activation in hyperglycemic mice corrected the inflammatory response toward an anti-inflammatory profile, stabilized neovessels in the infarct border zone, and reduced the rate of sICH. This preventive effect was dependent on the presence of macrophages, but independent from effects on blood glucose levels. Conclusions— Hyperglycemia and macrophage-specific PPAR&ggr; activation exert opposing effects on MO/MP polarization in ischemic stroke lesions and, thereby, critically determine the risk of hemorrhagic infarct transformation.

Dipyrone comedication in aspirin treated stroke patients impairs outcome

BACKGROUND >50% of stroke patients rely on analgesic medication to control pain. Aspirin is the mainstay of medical treatment of stroke patients; however analgesic medication with dipyrone impairs aspirin antiplatelet effects ex-vivo. The clinical impact of this impairment is unknown. Therefore, we aimed to determine aspirin antiplatelet effects and neurological outcome in stroke patients with aspirin and dipyrone comedication. METHODS We conducted a prospective cohort study in 41 patients with stroke. Primary outcome was pharmacodynamic response to aspirin in dipyrone treated stroke patients. Secondary outcome was neurological recovery after stroke. Pharmacodynamic response to aspirin was measured using arachidonic acid induced aggregation in light-transmission aggregometry. Neurological outcome was determined three months after stroke onset by telephone interview. RESULTS Patients characteristics were similar in the aspirin-alone group and the aspirin+dipyrone group. Impaired pharmacodynamic response to aspirin occurred in 62% (14/21) of patients with aspirin and dipyrone co-medication. Only 10% (2/20) of aspirin treated patients without analgesic comedication displayed residual platelet reactivity (P=0.001; odds ratio [OR], 18 [95% CI, 3.2-100]). Excellent neurological recovery (measured by three months follow-up modified Rankin Scale<2) was observed in 80% (16/20) of patients in the aspirin-alone group and 48% (10/21) of patients in the aspirin+dipyrone group (P=0.037; OR, 4.4 [95% CI, 1.1-17.7]). CONCLUSIONS Dipyrone comedication in patients with stroke impairs pharmacodynamic response to aspirin. This is associated with worse clinical outcome. Therefore dipyrone should be used with caution in aspirin treated stroke patients. CLINICAL TRIAL REGISTRATION https://clinicaltrials.gov/show/NCT02148939; Identifier: NCT02148939.

Differential patterns of local gene regulation in crush lesions of the rat optic and sciatic nerve: relevance to posttraumatic regeneration.

Axon regrowth after nerve injury can occur in the peripheral but fails in the central nervous system. Cellular reactions at the lesion site affect axonal regrowth. We compared gene regulation in optic nerve (ON) and sciatic nerve (SN) crush lesions in adult rats by cDNA array analysis, quantitative RT-PCR and immunohistochemistry, focusing on the primary lesion site rather than the proximal or distal nerve stump. Four days after injury, identical gene regulation in ON and SN lesions was found for 19/1185 genes (15 up, 4 down). In contrast, tissue-specific regulations were identified for 48 genes in ON and 50 genes in SN crush lesions. Among these, in the ON many genes were downregulated (23 up, 25 down) whereas upregulation predominated in SN lesions (43 up, 7 down), especially for signaling, metabolism, and translation/transcription-related genes. In ON lesions aquaporin 4 downregulation corresponded to a transient loss of astrocytes. Tissue-type plasminogen activator was upregulated in the lesion and distal stump of SN while the urokinase-type plasminogen activator was upregulated only in the ON lesion indicating differences in local proteolysis between the systems. Typical neuronal genes were regulated at the crush site comprising neurotransmitter genes in ON and actin cytoskeleton-related genes in the SN. The differential orchestration of local gene regulation has implications for axonal regeneration in central nervous system lesions.

Outcome and Treatment Effects in Stroke Associated with Acute Cervical ICA Occlusion

Background Endovascular therapy (EVT) with stent retrievers in addition to i.v. thrombolysis (IVT) has proven effective in acute stroke patients with middle cerebral artery (MCA, M1 segment) and distal internal carotid artery (ICA) occlusion. Limited data exist concerning acute cervical ICA occlusion, either alone or in combination with intracranial ICA occlusion (tandem occlusion). Therefore we analyzed outcome and treatment effects in stroke associated with cervical ICA occlusion, with specific focus on the impact of intracranial ICA or M1 patency. Methods Seventy-eight patients with cervical ICA occlusion from our local stroke unit registry were analyzed retrospectively. Thrombolysis in Cerebral Infarction (TICI) classification, infarct size, modified Rankin scale (mRS), symptomatic intracerebral hemorrhage (ICH), and death were assessed as outcome parameters. Results Forty-three patients had isolated cervical ICA occlusion whereas 35 patients presented with extra-/intracranial tandem occlusion. Patients underwent IVT alone (n = 23), combined IVT/EVT (n = 28) or no treatment (n = 27). Treated and untreated patients with tandem occlusion had a worse outcome after 90 days compared to isolated cervical occlusion (OR for moderate outcome 0.29, 0.27–0.88, p = 0.01). Additional EVT improved outcome in patients with tandem occlusion (OR for moderate outcome: 15.43, 1.60–148.90, p = 0.008) but not isolated cervical occlusion (OR 1.33, 0.38–11.60, NS). Conclusions In contrast to tandem occlusion, stroke outcome in patients with isolated cervical ICA occlusion was generally more benign and not improved by combined IVT/EVT compared to IVT alone. Intracranial vessel patency may be critical for treatment decision in acute cervical ICA occlusion.

Loss of Reelin protects mice against arterial thrombosis by impairing integrin activation and thrombus formation under high shear conditions

Reelin is a secreted glycoprotein and essential for brain development and plasticity. Recent studies provide evidence that Reelin modifies platelet actin cytoskeletal dynamics. In this study we sought to dissect the contribution of Reelin in arterial thrombus formation. Here we analyzed the impact of Reelin in arterial thrombosis ex vivo and in vivo using Reelin deficient (reeler) and wildtype mice. We found that Reelin is secreted upon platelet activation and mediates signaling via glycoprotein (GP)Ib, the amyloid precursor protein (APP) and apolipoprotein E receptor 2 (ApoER2) to induce activation of Akt, extracellular signal-regulated kinase (Erk), SYK and Phospholipase Cγ2. Moreover, our data identifies Reelin as first physiological ligand for platelet APP. Platelets from reeler mice displayed attenuated platelet adhesion and significantly reduced thrombus formation under high shear conditions indicating an important role for Reelin in GPIb-dependent integrin αIIbβ3 activation. Accordingly, adhesion to immobilized vWF as well as integrin activation and the phosphorylation of Erk and Akt after GPIb engagement was reduced in Reelin deficient platelets. Defective Reelin signaling translated into protection from arterial thrombosis and cerebral ischemia/reperfusion injury beside normal hemostasis. Furthermore, treatment with an antagonistic antibody specific for Reelin protects wildtype mice from occlusive thrombus formation. Mechanistically, GPIb co-localizes to the major Reelin receptor APP in platelets suggesting that Reelin-induced effects on GPIb signaling are mediated by APP-GPIb interaction. These results indicate that Reelin is an important regulator of GPIb-mediated platelet activation and may represent a new therapeutic target for the prevention and treatment of cardio- and cerebrovascular diseases.

High Incidence of Atrial Fibrillation After Embolic Stroke of Undetermined Source in Posterior Cerebral Artery Territory

Background Subclinical atrial fibrillation is one possible cause of embolic stroke of undetermined source (ESUS). It remains to be elucidated if a specific infarction site has a predictive value for detecting subclinical atrial fibrillation. We aimed to investigate the predictive value of infarction site in patients with ESUS for the detection of atrial tachyarrhythmia (AT) using an insertable cardiac monitor. Methods and Results Consecutive 146 patients (84 men; aged 62±12 years) underwent insertable cardiac monitor implantation after diagnosis of ESUS. The detection of AT >30 seconds was evaluated. The ESUS infarction sites were categorized into internal carotid artery and vertebral artery (VA) territories, with ophthalmic artery, anterior cerebral artery, and middle cerebral artery as internal carotid artery subterritories, and posterior cerebral artery and other vertebrobasilar arteries as VA subterritories. During a median follow‐up of 387 days, AT was detected in 33 patients (23%). Subclinical AT detection was significantly more frequent after VA territorial infarction opposed to internal carotid artery infarction (20/57 [35%] versus 13/89 [15%]; P=0.0039). Kaplan‐Meier analysis demonstrated a significantly higher AT detection rate after VA infarction (log‐rank, P=0.0076). Regression analysis revealed that VA territorial infarction, and particularly posterior cerebral artery area infarction, was an independent predictor of AT detection. Conclusions Patients with ESUS in the posterior cerebral artery territory had a higher rate of subclinical AT detection than those with other infarct localizations. Our data suggest that the possible usefulness of ESUS site to identify candidates for direct oral anticoagulation should be confirmed in future research.