FTY720 Reduces Post-Ischemic Brain Lymphocyte Influx but Does Not Improve Outcome in Permanent Murine Cerebral Ischemia

BACKGROUND: The contribution of neuroinflammation and specifically brain lymphocyte invasion is increasingly recognised as a substantial pathophysiological mechanism after stroke. FTY720 is a potent treatment for primary neuroinflammatory diseases by inhibiting lymphocyte circulation and brain immigration. Previous studies using transient focal ischemia models showed a protective effect of FTY720 but did only partially characterize the involved pathways. We tested the neuroprotective properties of FTY720 in permanent and transient cortical ischemia and analyzed the underlying neuroimmunological mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: FTY720 treatment resulted in substantial reduction of circulating lymphocytes while blood monocyte counts were significantly increased. The number of histologically and flow cytometrically analyzed brain invading T- and B lymphocytes was significantly reduced in FTY720 treated mice. However, despite testing a variety of treatment protocols, infarct volume and behavioural dysfunction were not reduced 7d after permanent occlusion of the distal middle cerebral artery (MCAO). Additionally, we did not measure a significant reduction in infarct volume at 24 h after 60 min filament-induced MCAO, and did not see differences in brain edema between PBS and FTY720 treatment. Analysis of brain cytokine expression revealed complex effects of FTY720 on postischemic neuroinflammation comprising a substantial reduction of delayed proinflammatory cytokine expression at 3d but an early increase of IL-1β and IFN-γ at 24 h after MCAO. Also, serum cytokine levels of IL-6 and TNF-α were increased in FTY720 treated animals compared to controls. CONCLUSIONS/SIGNIFICANCE: In the present study we were able to detect a reduction of lymphocyte brain invasion by FTY720 but could not achieve a significant reduction of infarct volumes and behavioural dysfunction. This lack of neuroprotection despite effective lymphopenia might be attributed to a divergent impact of FTY720 on cytokine expression and possible activation of innate immune cells after brain ischemia

Hemostatic therapy in experimental intracerebral hemorrhage associated with the direct thrombin inhibitor dabigatran

Background and Purpose: Dabigatran-etexilate (DE) recently has been approved for stroke prevention in atrial fibrillation. However, lack of effective antagonists represents a major concern in the event of intracerebral hemorrhage (ICH). The aims of the present study were to establish a murine model of ICH associated with dabigatran, and to test the efficacy of different hemostatic factors in preventing hematoma growth. Methods: In C57BL/6 mice receiving DE (4.5 or 9.0 mg/kg), in vivo and in vitro coagulation assays and dabigatran plasma levels were measured repeatedly. Thirty minutes after inducing ICH by striatal collagenase injection, mice received an intravenous injection of saline, prothrombin complex concentrate (PCC; 100 U/kg), murine fresh-frozen plasma (200 μL), or recombinant human factor VIIa (8.0 mg/kg). ICH volume was quantified on brain cryosections 24 hours later. Results: DE substantially prolonged tail vein bleeding time and ecarin clotting time for 4 hours correspondi

Flow cytometric analysis of brain immune cells at 5d after permanent MCAO.

<p>Cells per one hemisphere (Mean±SD). N = 6 mice per group. Data of 2 individual experiments.</p

FTY720 changes serum cytokine levels.

<p>Serum cytokine concentrations of the pro-inflammatory cytokines IL-6, IFN-γ and TNF-α (<b>A–C</b>) and anti-inflammatory cytokines TGF-β and IL-10 (<b>D,E</b>) were measured in naïve mice and at 24 h and 5d after FTY720 or control treatment. Each assay was performed in duplicate (n = 5, serum sampling as 2–3 individual experiments, assays as one experiment). * P<0.05 between treatment groups at the respective time point.</p

FTY720 does not reduce infarct volume after cortical permanent ischemia.

<p>Animals in the treatment groups (FTY720) received daily administrations of 1 mg/kg FTY720 by oral gavage starting at 48 h before infarct induction.I Infarct volumes were determined (<b>A</b>) at 3d (n = 8 per group, p = 0.73, 2 individual experiments) and (<b>B</b>) at 7d (n = 17, 0.54, 4 individual experiments) after infarct induction. Control animals received daily PBS injections. (<b>C</b>) Animals were treated daily with either FTY720 or PBS, starting from 3 h after MCAO and infarct volumes were determined at 7d after brain ischemia (n = 10, p = 0.43, 2 individual experiments). (<b>D</b>) Mice received a single dose of FTY720 or PBS at 48 h before brain ischemia and infarct volumetry was performed at day 7 (n = 10, p = 0.27). Behavioural dysfunction and recovery after experimental stroke was assessed in FTY720 pretreated animals (daily treatment starting 48 h before MCAO) or in control animals by the (<b>E</b>) “cylinder test” (n = 12, 3 individual experiments) and the (<b>F</b>) “corner test” (n = 12, 3 individual experiments).</p

FTY720 treatment induces leukopenia.

<p>(<b>A</b>) Differential blood cell counting was performed in normal mice (Naive) and 6 h, 24 h, 3d and 7d after daily administration of FTY720. Mean values (n = 5 per time point) are depicted for total leukocytes, granulocytes, lymphocytes and monocytes as cells per µl whole blood. (<b>B</b>) Leukocyte subpopulations were further characterized by specific epitope markers for T cells (CD3, CD4, CD8), B cells (B220), regulatory T cells (Foxp3) and monocytes (CD11b) at the indicated time points in blood, spleen and mesenteric lymph nodes (n = 5 per group). Each experiment was performed 2–3 times.</p