The pathogenic roles of receptor-mediated sphingosine 1-phosphate (S1P) signaling in cerebral ischemia have been evidenced mainly through the efficacy of FTY720 that binds non-selectively to four of the five S1P receptors (S1P_1,3,4,5). Recently, S1P₁ and S1P₂ were identified as specific receptor subtypes that contribute to brain injury in cerebral ischemia; however, the possible involvement of other S1P receptors remains unknown. S1P₃ can be the candidate because of its upregulation in the ischemic brain, which was addressed in this study, along with underlying pathogenic mechanisms.

We used transient middle cerebral artery occlusion/reperfusion (tMCAO), a mouse model of transient focal cerebral ischemia. To identify S1P₃ as a pathogenic factor in cerebral ischemia, we employed a specific S1P₃ antagonist, CAY10444. Brain damages were assessed by brain infarction, neurological score, and neurodegeneration. Histological assessment was carried out to determine microglial activation, morphological transformation, and proliferation. M1/M2 polarization and relevant signaling pathways were determined by biochemical and immunohistochemical analysis.

Inhibiting S1P₃ immediately after reperfusion with CAY10444 significantly reduced tMCAO-induced brain infarction, neurological deficit, and neurodegeneration. When S1P₃ activity was inhibited, the number of activated microglia was markedly decreased in both the periischemic and ischemic core regions in the ischemic brain 1 and 3 days following tMCAO. Moreover, inhibiting S1P₃ significantly restored the microglial shape from amoeboid to ramified microglia in the ischemic core region 3 days after tMCAO, and it attenuated microglial proliferation in the ischemic brain. In addition to these changes, S1P₃ signaling influenced the proinflammatory M1 polarization, but not M2. The S1P₃-dependent regulation of M1 polarization was clearly shown in activated microglia, which was affirmed by determining the in vivo activation of microglial NF-κB signaling that is responsible for M1 and in vitro expression levels of proinflammatory cytokines in activated microglia. As downstream effector pathways in an ischemic brain, S1P₃ influenced phosphorylation of ERK1/2, p38 MAPK, and Akt.

This study identified S1P₃ as a pathogenic mediator in an ischemic brain along with underlying mechanisms, involving its modulation of microglial activation and M1 polarization, further suggesting that S1P₃ can be a therapeutic target for cerebral ischemia.