The sphingosine-1-phosphate analogue FTY720 is neuroprotective in a rodent model of brain ischemia

Sphingosine-1-phosphate (S1P) is a lipid mediator exerting pleitropic effects on a wide variety of cell types, including glial cells, neurons and endothelial cells. In particular, S1P is known to be a survival factor for various cell types and is a key player in protective mechanisms against hypoxia- or ischemia-mediated insults. FTY720 has shown significant protective effects after controlled bilateral kidney ischemia, and attenuates ischemia-reperfusion injury in both normal and cirrhotic livers by activation of cell survival Akt signaling. The purpose of this study was to evaluate the effect of FTY720 administration on in vivo and in vitro models of brain ischemia. Halothane-anesthetized C57Bl/6 mice (20-25 g) underwent 90-min focal cerebral ischemia induced with an 8.0 nylon monofilament coated with a silicone resin/hardener mixture, introduced into the left internal carotid artery up to the anterior cerebral artery. Regional cerebral blood flow was measured using laser-Doppler-flowmetry. Mice treated with 3 mg/kg FTY720 orally once a day for 3 days starting after reperfusion showed a significantly reduced infarct 3 days after reperfusion, as visualized by TTC staining (108 ± 14 vs. 68 ± 8 mm3 for vehicle-treated mice; p=0.04), and significantly attenuated neurological deficit (5-point scale) 1, 2 and 3 days after reperfusion (Q1, median, Q3: 1, 1, 1.5 vs. 2, 2, 3 on day 3; p=0.02). Intravenous 0.5 mg/kg FTY720 did not affect systemic blood pressure and CBF (assessed with laser speckle flowmetry) for up to 60 minutes. We then tested whether the effects of FTY720 on infarct size and neurological deficit were due to increased cerebral blood flow during ischemia using noninvasive laser speckle flowmetry. FTY720 (3 mg/kg) did not alter the CBF deficit when administered orally to isoflurane-anesthetized mice 1h before distal middle cerebral artery occlusion (dMCAO): the area of severely ischemic cortex (? 20% residual CBF) was 3.7±2.0mm2 in FTY-treated mice, compared to 4.2±0.9 mm2 in the saline-treated group. Because S1P is a potent anti-apoptotic factor, we then examined the effects of FTY720 in vitro, using a oxygen-glucose deprivation (OGD) cell death model. Mouse primary cortical neurons were exposed to 2-hr OGD/24-hr reoxygenation, decreasing neuron viability by more than 40% with respect to basal cell death (as assessed using Hoechst staining). 3 microM S1P and 100 nM FTY (added at the time of re-oxygenation) restored viability to control levels. Similarly, in rat brain primary endothelial cells subjected to 4-hr OGD/24hr recovery, 3 microM S1P and 30 nM FTY720 increased cell viability to levels observed in control cells. The presence of an Akt inhibitor (20 microM 1L-6-Hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate) or NOS inhibitor (100 microM L-NAME) reversed the protective effects of 3 microM S1P on rat brain endothelial cells following 4hr OGD/24hr re-oxygenation. Taken together, these results suggest that the S1P receptor/Akt/NOS pathway may play an important role in neuroprotection following brain ischemia.