No influence of dabigatran anticoagulation on hemorrhagic transformation in an experimental model of ischemic stroke

Background: Dabigatran etexilate (DE) is a new oral direct thrombin inhibitor. Clinical trials point towards a favourable risk-to-benefit profile of DE compared to warfarin. In this study, we evaluated whether hemorrhagic transformation (HT) occurs after experimental stroke under DE treatment as we have shown for warfarin. Methods: 44 male C57BL/6 mice were pretreated orally with 37.5 mg/kg DE, 75 mg/kg DE or saline and diluted thrombin time (dTT) and DE plasma concentrations were monitored. Ischemic stroke was induced by transient middle cerebral artery occlusion (tMCAO) for 1 h or 3 h. We assessed functional outcome and HT blood volume 24 h and 72 h after tMCAO. Results: After 1 h tMCAO, HT blood volume did not differ significantly between mice pretreated with DE 37.5 mg/kg and controls (1.5±0.5 µl vs. 1.8±0.5 µl, p>0.05). After 3 h tMCAO, DE-anticoagulated mice did also not show an increase in HT, neither at the dose of 37.5 mg/kg equivalent to anticoagulant treatment in the therapeutic range (1.3±0.9 µl vs. control 2.3±0.5 µl, p>0.05) nor at 75 mg/kg, clearly representing supratherapeutic anticoagulation (1.8±0.8 µl, p>0.05). Furthermore, no significant increase in HT under continued anticoagulation with DE 75 mg/kg could be found at 72 h after tMCAO for 1 h (1.7±0.9 µl vs. control 1.6±0.4 µl, p>0.05). Conclusion: Our experimental data suggest that DE does not significantly increase hemorrhagic transformation after transient focal cerebral ischemia in mice. From a translational viewpoint, this indicates that a continuation of DE anticoagulation in case of an ischemic stroke might be safe, but clearly, clinical data on this question are warranted

FTY720 treatment in the convalescence period improves functional recovery and reduces reactive astrogliosis in photothrombotic stroke

Background: The Sphingosine-1-phosphate (S1P) signaling pathway is known to influence pathophysiological processes within the brain and the synthetic S1P analog FTY720 has been shown to provide neuroprotection in experimental models of acute stroke. However, the effects of a manipulation of S1P signaling at later time points after experimental stroke have not yet been investigated. We examined whether a relatively late initiation of a FTY720 treatment has a positive effect on long-term neurological outcome with a focus on reactive astrogliosis, synapses and neurotrophic factors. Methods: We induced photothrombotic stroke (PT) in adult C57BL/6J mice and allowed them to recover for three days. Starting on post-stroke day 3, mice were treated with FTY720 (1 mg/kg b.i.d.) for 5 days. Behavioral outcome was observed until day 31 after photothrombosis and periinfarct cortical tissue was analyzed using tandem mass-spectrometry, TaqMan®analysis and immunofluorescence. Results: FTY720 treatment results in a significantly better functional outcome persisting up to day 31 after PT. This is accompanied by a significant decrease in reactive astrogliosis and larger post-synaptic densities as well as changes in the expression of vascular endothelial growth factor α (VEGF α). Within the periinfarct cortex, S1P is significantly increased compared to healthy brain tissue. Conclusion: Besides its known neuroprotective effects in the acute phase of experimental stroke, the initiation of FTY720 treatment in the convalescence period has a positive impact on long-term functional outcome, probably mediated through reduced astrogliosis, a modulation in synaptic morphology and an increased expression of neurotrophic factors

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

Timeline diagram of the experimental procedures.

<p>Timeline diagram of the experimental procedures.</p

No difference in HT and neurological outcome under continuous anticoagulation 72 h after 1 h tMCAO.

<p>A) After 72 h of continuous anticoagulation after 1 h tMCAO, HT blood volume in DE 75 mg/kg pretreated mice (n = 6) and in control mice (n = 7) showed no significant difference. Haemoglobin assay was used for HT blood volume measurement. Results are given in µl presented in a box and whiskers plot depicting mean values, extreme values and the 25 to 75 percent interquartile range. B) Neurological function was evaluated on a 14 point scale (mNSS) after 72 h. Mice which died during the observation period were given 14 points as the worst outcome on the mNSS scale. The values of single mice and the medians are depicted in a dot plot. C) The number of surviving mice per group is given besides their mean body weight in gram.</p

DE leads to a prolongation of the dTT in linear correlation with DE plasma concentrations (Hemoclot™ assay).

<p>A) dTT was measured with the Hemoclot™ assay in anticoagulation-naïve mice (n = 4), mice receiving 3×37.5 mg/kg over a 24 h feeding period (n = 3) and mice receiving 3×75 mg/kg (n = 3). Statistical significance was assessed with one-way ANOVA and Bonferroni correction. B) Calibration of the coagulometer with lyophilized standard DE plasma gives the DE concentration from the dTT values. Statistical significance was assessed with one-way ANOVA and Bonferroni correction. ** p<0.01; *** p<0.001.</p

Inclusion – exclusion criteria for MCAO experiments.

<p>Inclusion – exclusion criteria for MCAO experiments.</p

DE pretreatment does not lead to symptomatic hemorrhagic transformation after tMCAO.

<p>A) Neurological function after 1 h and 3 h tMCAO was assessed on a 14 point scale (mNSS) directly prior to reperfusion and at B) 24 h. The values of single mice and the medians are depicted in a dot plot. Statistical significance was assessed with a Mann-Whitney Test for two groups and a Kruskal-Wallis-Test with Dunn’s correction for three or more. * p<0.05.</p

Mice pretreated with DE did not show a greater risk of HT after tMCAO than control mice.

<p>The left two bars show HT volume after 1 h MCAO in control mice (n = 5) and DE-pre-treated mice 37,5 mg/kg (n = 6). Shown on the right is the HT volume after 3 h MCAO in control mice (n = 6), DE-pre-treated mice 37,5 mg/kg (n = 5) and DE-pre-treated mice 75 mg/kg (n = 5). Haemoglobin assay was used for HT blood volume measurement and results are given in µl. Results are showed in a box and whiskers plot depicting mean values, extreme values and the 25 to 75 percent interquartile range. Statistical significance was assessed with a one-way ANOVA with Bonferroni correction. No significant differences were detected.</p

S1P is increased in the periinfarct cortex, not explained by changes in the expression of enzymes of S1P metabolism.

<p>a) S1P, FTY720 and pFTY720 were measured at day 4 by tandem mass spectrometry. b) SK1, SK2 and SGPL1 were measured at day 4 by RT-PCR. Differences between treatment groups were analyzed using Student’s unpaired two-tailed t-test. Data are presented as means ± S.D. **, <i>P</i>≤0.01; *, <i>P</i>≤0.05; n.s., non-significant; <i>n</i> = 10/group.</p