Waltraud Pfeilschifter

The perfect crime? CCSVI not leaving a trace in MS

Background Multiple sclerosis (MS) is a chronic, inflammatory demyelinating disease of the central nervous system, believed to be triggered by an autoimmune reaction to myelin. Recently, a fundamentally different pathomechanism termed ‘chronic cerebrospinal venous insufficiency’ (CCSVI) was proposed, provoking significant attention in the media and scientific community. Methods Twenty MS patients (mean age 42.2±13.3 years; median Extended Disability Status Scale 3.0, range 0–6.5) were compared with 20 healthy controls. Extra- and intracranial venous flow direction was assessed by colour-coded duplex sonography, and extracranial venous cross-sectional area (VCSA) of the internal jugular and vertebral veins (IJV/VV) was measured in B-mode to assess the five previously proposed CCSVI criteria. IJV-VCSA≤0.3 cm2 indicated ‘stenosis,’ and IJV-VCSA decrease from supine to upright position ‘reverted postural control.’ The sonographer, data analyser and statistician were blinded to the patient/control status of the participants. Results No participant showed retrograde flow of cervical or intracranial veins. IJV-VCSA≤0.3 cm2 was found in 13 MS patients versus 16 controls (p=0.48). A decrease in IJV-VCSA from supine to upright position was observed in all participants, but this denotes a physiological finding. No MS patient and one control had undetectable IJV flow despite deep inspiration (p=0.49). Only one healthy control and no MS patients fulfilled at least two criteria for CCSVI. Conclusions This triple-blinded extra- and transcranial duplex sonographic assessment of cervical and cerebral veins does not provide supportive evidence for the presence of CCSVI in MS patients. The findings cast serious doubt on the concept of CCSVI in MS.

FTY720 Ameliorates Acute Ischemic Stroke in Mice by Reducing Thrombo-Inflammation but Not by Direct Neuroprotection

Background and Purpose— Lymphocytes are important players in the pathophysiology of acute ischemic stroke. The interaction of lymphocytes with endothelial cells and platelets, termed thrombo-inflammation, fosters microvascular dysfunction and secondary infarct growth. FTY720, a sphingosine-1-phosphate receptor modulator, blocks the egress of lymphocytes from lymphoid organs and has been shown to reduce ischemic neurodegeneration; however, the underlying mechanisms are unclear. We investigated the mode of FTY720 action in models of cerebral ischemia. Methods— Transient middle cerebral artery occlusion (tMCAO) was induced in wild-type and lymphocyte-deficient Rag1−/− mice treated with FTY720 (1 mg/kg) or vehicle immediately before reperfusion. Stroke outcome was assessed 24 hours later. Immune cells in the blood and brain were counted by flow cytometry. The integrity of the blood–brain barrier was analyzed using Evans Blue dye. Thrombus formation was determined by immunohistochemistry and Western blot, and was correlated with cerebral perfusion. Results— FTY720 significantly reduced stroke size and improved functional outcome in wild-type mice on day 1 and day 3 after transient middle cerebral artery occlusion. This protective effect was lost in lymphocyte-deficient Rag1−/− mice and in cultured neurons subjected to hypoxia. Less lymphocytes were present in the cerebral vasculature of FTY720-treated wild-type mice, which in turn reduced thrombosis and increased cerebral perfusion. In contrast, FTY720 was unable to prevent blood–brain barrier breakdown and transendothelial immune cell trafficking after transient middle cerebral artery occlusion. Conclusions— Induction of lymphocytopenia and concomitant reduction of microvascular thrombosis are key modes of FTY720 action in stroke. In contrast, our findings in Rag1−/− mice and cultured neurons argue against direct neuroprotective effects of FTY720.

Anticoagulation With the Oral Direct Thrombin Inhibitor Dabigatran Does Not Enlarge Hematoma Volume in Experimental Intracerebral Hemorrhage

Background— The direct thrombin inhibitor dabigatran etexilate (DE) may constitute a future replacement of vitamin K antagonists for long-term anticoagulation. Whereas warfarin pretreatment is associated with greater hematoma expansion after intracerebral hemorrhage (ICH), it remains unclear what effect direct thrombin inhibitors would have. Using different experimental models of ICH, this study compared hematoma volume among DE-treated mice, warfarin-treated mice, and controls. Methods and Results— CD-1 mice were fed with DE or warfarin. Sham-treated mice served as controls. At the time point of ICH induction, DE mice revealed an increased activated partial thromboplastin time compared with controls (mean±SD 46.1±5.0 versus 18.0±1.5 seconds; P=0.022), whereas warfarin pretreatment resulted in a prothrombin time prolongation (51.4±17.9 versus 10.4±0.3 seconds; P<0.001). Twenty-four hours after collagenase-induced ICH formation, hematoma volume was 3.8±2.9 &mgr;L in controls, 4.8±2.7 &mgr;L in DE mice, and 14.5±11.8 &mgr;L in warfarin mice (n=16; Welch ANOVA between-group differences P=0.007; posthoc analysis with the Dunnett method: DE versus controls, P=0.899; warfarin versus controls, P<0.001; DE versus warfarin, P=0.001). In addition, a model of laser-induced cerebral microhemorrhage was applied, and the distances that red blood cells and blood plasma were pushed into the brain were quantified. Warfarin mice showed enlarged red blood cell and blood plasma diameters compared to controls, but no difference was found between DE mice and controls. Conclusions— In contrast with warfarin, pretreatment with DE did not increase hematoma volume in 2 different experimental models of ICH. In terms of safety, this observation may represent a potential advantage of anticoagulation with DE over warfarin.

Fingolimod for the treatment of neurological diseases—state of play and future perspectives

Sphingolipids are a fascinating class of signaling molecules derived from the membrane lipid sphingomyelin. They show abundant expression in the brain. Complex sphingolipids such as glycosphingolipids (gangliosides and cerebrosides) regulate vesicular transport and lysosomal degradation and their dysregulation can lead to storage diseases with a neurological phenotype. More recently, simple sphingolipids such ceramide, sphingosine and sphingosine 1-phosphate (S1P) were discovered to signal in response to many extracellular stimuli. Forming an intricate signaling network, the balance of these readily interchangeable mediators is decisive for cell fate under stressful conditions. The immunomodulator fingolimod is the prodrug of an S1P receptor agonist. Following receptor activation, the drug leads to downregulation of the S1P1 receptor inducing functional antagonism. As the first drug to modulate the sphingolipid signaling pathway, it was marketed in 2010 for the treatment of multiple sclerosis (MS). At that time, immunomodulation was widely accepted as the key mechanism of fingolimod’s efficacy in MS. But given the excellent passage of this lipophilic compound into the brain and its massive brain accumulation as well as the abundant expression of S1P receptors on brain cells, it is conceivable that fingolimod also affects brain cells directly. Indeed, a seminal study showed that the protective effect of fingolimod in experimental autoimmune encephalitis (EAE), a murine MS model, is lost in mice lacking the S1P1 receptor on astrocytes, arguing for a specific role of astrocytic S1P signaling in MS. In this review, we discuss the role of sphingolipid mediators and their metabolizing enzymes in neurologic diseases and putative therapeutic strategies arising thereof.

Intracerebral haemorrhage associated with antithrombotic treatment: translational insights from experimental studies

Little is known about the pathophysiology of intracerebral haemorrhage that occurs during anticoagulant treatment. In observational studies, investigators have reported larger haematoma volumes and worse functional outcome in these patients than in those with intracerebral haemorrhage and a normal coagulation status. The need to prevent extensive haematoma enlargement by rapid reversal of the anticoagulation seems intuitive, although no evidence is available from randomised clinical trials. New oral anticoagulants, such as the direct thrombin inhibitor dabigatran and the factor Xa inhibitor rivaroxaban, have been approved recently; however, intracerebral haemorrhage during dabigatran or rivaroxaban anticoagulation has not been characterised, and whether anticoagulation reversal can be beneficial in this scenario is unknown. In a translational approach, new experimental models have been developed to study anticoagulation-associated intracerebral haemorrhage in more detail and to test treatment strategies. Vitamin k antagonists enlarge haematoma volumes and worsen functional outcome in animal models. Rapid reversal of anticoagulation in the experimental setting prevents prolonged haematoma expansion and improves outcome. The new oral anticoagulants increase intracerbral haemorrhage volumes less than does warfarin. Haemostatic approaches that have been used for vitamin k-associated intracerebral haemorrhage also seem to be effective in intracerebral haemorrhage associated with the new anticoagulants. These experimental studies are valuable for filling gaps in knowledge, but the results need careful translation into routine clinical practice.

Thrombolysis with recombinant tissue plasminogen activator under dabigatran anticoagulation in experimental stroke.

Anticoagulation with dabigatran etexilate (DE) has a favorable risk‐to‐benefit profile for the prevention of ischemic events in patients with atrial fibrillation compared to warfarin. Whereas warfarin constitutes a strong contraindication for thrombolysis, it is unclear whether patients anticoagulated with DE can be thrombolysed. We compared the risk of thrombolysis‐associated hemorrhagic transformation (HT) after pretreatment with DE or warfarin in a mouse model of ischemic stroke.

Increased Risk of Hemorrhagic Transformation in Ischemic Stroke Occurring During Warfarin Anticoagulation An Experimental Study in Mice

Background and Purpose— The prevalence of long-term oral anticoagulant therapy is rising. Treatment options for patients who have an ischemic stroke under oral anticoagulant therapy are limited, and clinical data on the risk of hemorrhagic transformation (HT) in this condition are scarce. We therefore aimed to establish a mouse model of ischemic stroke occurring during oral anticoagulant therapy to assess the frequency and characteristics of HT. Methods— C57BL/6 mice (n=59) were pretreated with warfarin. Untreated mice (n=32) served as controls. We performed a 3-hour transient filament occlusion of the right middle cerebral artery. In a first set of animals, ischemic lesion size and HT were evaluated macroscopically at 24 hours after middle cerebral artery occlusion. In a second set of mice, quantitative analysis of HT was performed at different time points after middle cerebral artery occlusion and in animals with different international normalized ratio levels using a photometric hemoglobin assay. Results— Oral anticoagulant therapy at the onset of ischemia led to HT in all anticoagulated mice, whereas only 14% of the control mice showed HT. Mean HT blood volume 24 hours after middle cerebral artery occlusion was 0.3±0.4 &mgr;L in controls, 4.2±1.7 &mgr;L in mice anticoagulated to a mean international normalized ratio of 1.9±0.5 (P<0.05 versus controls), and 5.2±2.7 &mgr;L in mice with an international normalized ratio of 2.9±0.9 (P<0.001 versus controls). Anticoagulated mice euthanized at the time point of reperfusion had less HT than mice euthanized after 21 hours of reperfusion (1.6±0.5 &mgr;L versus 5.9±3.6 &mgr;L, P<0.05). Conclusions— We present a mouse model of ischemic stroke occurring during oral anticoagulant therapy. Warfarin pretreatment dramatically increases the risk of HT 24 hours after middle cerebral artery occlusion. Reperfusion injury seems to be a critical component in this condition.

Ceramide Synthase 6 Plays a Critical Role in the Development of Experimental Autoimmune Encephalomyelitis

Ceramides are mediators of apoptosis and inflammatory processes. In an animal model of multiple sclerosis (MS), the experimental autoimmune encephalomyelitis (EAE) model, we observed a significant elevation of C16:0-Cer in the lumbar spinal cord of EAE mice. This was caused by a transiently increased expression of ceramide synthase (CerS) 6 in monocytes/macrophages and astroglia. Notably, this corresponds to the clinical finding that C16:0-Cer levels were increased 1.9-fold in cerebrospinal fluid of MS patients. NO and TNF-α secreted by IFN-γ–activated macrophages play an essential role in the development of MS. In murine peritoneal and mouse-derived RAW 264.7 macrophages, IFN-γ–mediated expression of inducible NO synthase (iNOS)/TNF-α and NO/TNF-α release depends on upregulation of CerS6/C16:0-Cer. Downregulation of CerS6 by RNA interference or endogenous upregulation of C16:0-Cer mediated by palmitic acid in RAW 264.7 macrophages led to a significant reduction or increase in NO/TNF-α release, respectively. EAE/IFN-γ knockout mice showed a significant delay in disease onset accompanied by a significantly less pronounced increase in CerS6/C16:0-Cer, iNOS, and TNF-α compared with EAE/wild-type mice. Treatment of EAE mice with l-cycloserine prevented the increase in C16:0-Cer and iNOS/TNF-α expression and caused a remission of the disease. In conclusion, CerS6 plays a critical role in the onset of MS, most likely by regulating NO and TNF-α synthesis. CerS6 may represent a new target for the inhibition of inflammatory processes promoting MS development.

Activation of sphingosine kinase 2 is an endogenous protective mechanism in cerebral ischemia

The two ubiquitously expressed sphingosine kinases (SphK) 1 and 2 are key regulators of the sphingolipid signaling pathway. Despite the formation of an identical messenger, i.e. sphingosine 1-phosphate (S1P), they exert strikingly different functions. Particularly, SphK2 is necessary for the phosphorylation of the sphingosine analog fingolimod (FTY720), which is protective in rodent stroke models. Using gene deficient mice lacking either SphK1 or SphK2, we investigated the role of the two lipid kinases in experimental stroke. We performed 2h transient middle cerebral artery occlusion (tMCAO) and analyzed lesion size and neurological function after 24h. Treatment groups received 1mg/kg FTY720. Neutrophil infiltration, microglia activation, mRNA and protein expression of SphK1, SphK2 and the S1P(1) receptor after tMCAO were studied. Genetic deletion of SphK2 but not SphK1 increased ischemic lesion size and worsened neurological function after tMCAO. The protective effect of FTY720 was conserved in SphK1(-/-) mice but not in SphK2(-/-) mice. This suggests that SphK2 activity is an important endogenous protective mechanism in cerebral ischemia and corroborates that the protective effect of FTY720 is mediated via phospho-FTY720.

Recanalization therapies in acute ischemic stroke patients: impact of prior treatment with novel oral anticoagulants on bleeding complications and outcome

Background— We explored the safety of intravenous thrombolysis (IVT) or intra-arterial treatment (IAT) in patients with ischemic stroke on non-vitamin K antagonist oral anticoagulants (NOACs, last intake <48 hours) in comparison with patients (1) taking vitamin K antagonists (VKAs) or (2) without previous anticoagulation (no-OAC). Methods and Results— This is a multicenter cohort pilot study. Primary outcome measures were (1) occurrence of intracranial hemorrhage (ICH) in 3 categories: any ICH (ICHany), symptomatic ICH according to the criteria of the European Cooperative Acute Stroke Study II (ECASS-II) (sICHECASS-II) and the National Institute of Neurological Disorders and Stroke (NINDS) thrombolysis trial (sICHNINDS); and (2) death (at 3 months). Cohorts were compared by using propensity score matching. Our NOAC cohort comprised 78 patients treated with IVT/IAT and the comparison groups of 441 VKA patients and 8938 no-OAC patients. The median time from last NOAC intake to IVT/IAT was 13 hours (interquartile range, 8–22 hours). In VKA patients, median pre-IVT/IAT international normalized ratio was 1.3 (interquartile range, 1.1–1.6). ICHany was observed in 18.4% NOAC patients versus 26.8% in VKA patients and 17.4% in no-OAC patients. sICHECASS-II and sICHNINDS occurred in 2.6%/3.9% NOAC patients, in comparison with 6.5%/9.3% of VKA patients and 5.0%/7.2% of no-OAC patients, respectively. At 3 months, 23.0% of NOAC patients in comparison with 26.9% of VKA patients and 13.9% of no-OAC patients had died. Propensity score matching revealed no statistically significant differences. Conclusions— IVT/IAT in selected patients with ischemic stroke under NOAC treatment has a safety profile similar to both IVT/IAT in patients on subtherapeutic VKA treatment or in those without previous anticoagulation. However, further prospective studies are needed, including the impact of specific coagulation tests.