Background and Purpose—
Recovery from stroke is limited in part by an inhibitory environment in the post-ischemic brain, but factors preventing successful remodeling are not well known. We sought to investigate if signaling from the axon guidance molecule semaphorin 3A (Sema3A) via eicosanoid second messengers can contribute to this inhibitory environment, and if blocking the Sema3A pathway can provide a benefit following experimental stroke.
Methods—
Cultured cortical neurons from mice were treated with recombinant Sema3A, or with the eicosanoids 12-HETE and 12-HPETE. Neurons from ALOX15 knockout mice, and a human brain endothelial cell line, were treated similarly. The filament model of MCAO was used to induce experimental stroke in mice, in some of which Sema3A was injected stereotactically into the striatum. The 12/15-LOX inhibitor LOXBlock-1 was injected intraperitoneally one week after MCAO.
Results—
Expression levels of 12/15-lipoxygenase (12/15-LOX) were increased within two hours after exposure of primary neurons to 90nM recombinant Sema3A. Either Sema3A, or the 12/15-lipoxygenase (12/15-LOX) metabolites 12-HETE and 12-HPETE at 300nM, blocked axon extension in neurons compared to solvent controls, and decreased tube formation in endothelial cells. The Sema3A effect was reversed by inhibiting 12/15-LOX, and neurons derived from 12/15-LOX knockout mice were insensitive to Sema3A. Following middle cerebral artery occlusion to induce stroke in mice, immunohistochemistry showed both Sema3A and 12/15-LOX are increased in the cortex up to two weeks. To determine if a Sema3A-dependent damage pathway is activated following ischemia, we injected recombinant Sema3A into the striatum. Sema3A alone did not cause injury in normal brains. But when injected into post-ischemic brains, Sema3A increased cortical damage by 79%, and again this effect was reversed by 12/15-LOX inhibition. Administration of the 12/15-LOX inhibitor LOXBlock-1 7 days after transient MCAO increased vascularization in the infarcted and peri-infarct area one week later.
Conclusions—
Our findings suggest that blocking the semaphorin pathway may provide a novel therapeutic strategy to improve stroke recovery.
Differential development and electrophysiological activity in cultured cortical neurons from the mouse and cynomolgus monkey
