Activation of sphingosine 1-phosphate receptor-1 by FTY720 is neuroprotective after ischemic stroke in rats

Abstract

Background and purpose: FTY720 is a known sphingosine 1-phosphate receptor agonist. In the present study, we investigated the neuroprotective effect of postischemic administration of FTY720 in rats with 2 hours transient middle cerebral artery occlusion (MCAO).

Methods: One hundred eleven male rats were randomly assigned to sham-operated and MCAO treated with vehicle, 0.25 mg/kg and 1 mg/kg of FTY720, another selective sphingosine 1-phosphate receptor-1 agonist SEW2871 (5 mg/kg), or 0.25 mg/kg of FTY720 plus a sphingosine 1-phosphate antagonist, VPC23019 (0.5 mg/kg). Drugs were injected intraperitoneally immediately after reperfusion. Neurological score and infarct volume were assessed at 24 and 72 hours after MCAO. Western blotting, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling were conducted at 24 hours after MCAO.

Results: FTY720 significantly reduced infarct volume and improved neurological score at 24 and 72 hours after MCAO compared with the vehicle group. SEW2871 showed similar neuroprotective effects to FTY720, whereas VPC 20319 abolished the neuroprotective effects of FTY720. FTY720 significantly retained Akt and extracellular signal-regulated kinase phosphorylation and Bcl-2 expression and decreased cleaved caspase-3 expression and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling-positive neurons at 24 hours after MCAO. VPC23019 blocked the antiapoptotic effects of FTY720.

Conclusions: These data suggest that activation of sphingosine 1-phosphate-1 by FTY720 reduces neuronal death after transient MCAO.

Figure 1

Quantitative analysis of total infarct volume (A) and neurological score (B) in groups treated with vehicle (n=8), 0.25mg/kg of FTY720 (FTY; n=8), 1mg/kg of FTY720 (FTY-high; n=8), 5mg/kg of SEW2871 (SEW; n=8), 0.5mg/kg of VPC23019+0.25mg/kg of FTY720 (VPC+FTY; n=8), and/or sham-operation (sham; n=5) at 24 hours after MCAO. Values are the mean±SD; †P<0.05 vs. sham, *P<0.05 vs. vehicle, #P<0.05 vs. VPC+FTY, ANOVA.

Figure 2

Quantitative analysis of infarct volume (A, total; B, cortex; C, subcortex) and neurological score (D) in groups treated with vehicle (n=9), 0.25mg/kg of FTY720 (FTY; n=9), and/or sham-operation (sham; n=5) at 72 hours after MCAO. Values are the mean±SD; †P<0.05 vs. sham, *P<0.05 vs. vehicle, unpaired t test or ANOVA.

Figure 3

Changes in Akt and ERK phosphorylation, and Bcl-2 and cleaved caspase-3 expression in the infarct MCA region at 24 hours after MCAO (n=5). Representative ischemic brain (A) and Western blots (B), and quantitative analysis of Akt-Ser-473 (phosphor-Akt; C) and ERK-1 (phospho-ERK-1; D) phosphorylation and Bcl-2 (E) and 17kDa caspase-3 (cleaved caspase-3; F) expression in groups treated with vehicle, 0.25mg/kg FTY720 (FTY), 0.5mg/kg VPC23019+0.25mg/kg FTY720 (VPC+FTY) and preoperation (pre-ope). The band density values were calculated as a ratio of that of β-actin, and the values from the preoperation were used as 100%. Values are the mean±SD; †P<0.05 vs. sham, *P<0.05 vs. vehicle, #P<0.05 vs. VPC+FTY, ANOVA

Figure 4

Representative ischemic brain and the area which is defined as periinfarct cortex (A) (stained by NeuN, I, infarct area; N, non-infarct area). Immunohistochemical colocalization of sphingosine-1-phosphate receptor-1 (S1P1; red, B) or phosphorylated Akt (phospho-Akt; red, C) with NeuN (green) positive cells (arrow head) in the periinfarct cortex at 24 hours after MCAO (n=5). Scale bar: 50μm

Figure 5

Evaluation of neuronal cell death in the periinfarct cortex at 24 hours after MCAO (n=5). A: Colocalization of TUNEL (green) and NeuN (red) positive cells (arrow head). B: Quantitative analysis of TUNEL positive neurons in the periinfarct cortex. Scale bar: 50μm, *P<0.05 vs. vehicle, #P<0.05 vs. VPC+FTY, ANOVA