Background and Purpose-Oxidative stress contributes to ischemia/reperfusion neuronal damage in a consecutive 2-phase pattern: an immediate direct cytotoxic effect and subsequent redox-mediated inflammatory insult. The present study was designed to assess the neuroprotective mechanisms of edaravone, a novel free radical scavenger, through antioxidative and anti-inflammatory pathways, from the early period to up to 7 days after ischemia/reperfusion in mice. Methods-Mice were subjected to 60-minute ischemia followed by reperfusion. They were divided into the edaravone group (nϭ72; with different schedules for first administration) and the vehicle (control) group (nϭ36). Infarct volume and neurological deficit scores were evaluated at several time points after ischemia. Immunohistochemical analysis for 4-hydroxy-2-nonenal (HNE), 8-hydroxy-deoxyguanosine (8-OHdG), ionized calcium-binding adapter molecule 1 (Iba-1), inducible NO synthase (iNOS), and nitrotyrosine were performed at 24 hours, 72 hours, or 7 days after reperfusion. Result-Edaravone, even when administrated 6 hours after onset of ischemia/reperfusion, significantly reduced the infarct volume (68.10Ϯ6.24%; PϽ0.05) and improved the neurological deficit scores (PϽ0.05) at 24 hours after reperfusion. Edaravone markedly suppressed the accumulation of HNE-modified protein and 8-OHdG at the penumbra area during the early period after reperfusion (PϽ0.05) and reduced microglial activation, iNOS expression, and nitrotyrosine formation at the late period. Conclusion-Our results indicated that edaravone exerts an early neuroprotective effect through the early free radicals scavenging pathway and a late anti-inflammatory effect and suggested that edaravone is important for expansion of the therapeutic time window in stroke patients. (Stroke. 2005;36:2220-2225.)

Cerebral ischemia induces the expression of several growth factors and cytokines, which protect neurons against ischemic insults. Recent studies showed that granulocyte colony-stimulating factor (G-CSF) has a neuroprotective effect through the signaling pathway for the antiapoptotic cascade. The current study was designed to assess the neuroprotective mechanisms of G-CSF in ischemia/ reperfusion injury using bone marrow chimera mice known to express enhanced green fluorescent protein (EGFP). Mice were subjected to ischemia/reperfusion and divided into two groups: those treated with G-CSF (G-CSF group) and vehicle (control group) (n ¼ 35 in each group). Immunohistochemistry and immunoblotting for antiapoptotic protein, nitrotyrosine, and inducible nitrate oxide synthase (iNOS) were performed. G-CSF significantly reduced stroke volume (34%, Po0.006). G-CSF upregulated Stat3, pStat3, and Bcl-2 (Po0.05), and suppressed iNOS and nitrotyrosine expression. In EGFP chimera mice, G-CSF decreased the migration of Iba-1/EGFP-positive bone marrow-derived monocytes/macrophages and increased intrinsic microglia/macrophages at ischemic penumbra (Po0.05), suggesting that bone marrow-derived monocytes/macrophages are not involved in G-CSF-induced reduction of ischemic injury size. Our study indicated that G-CSF exerts a neuroprotective effect through the direct activation of antiapoptotic pathway, and suggested that G-CSF is important for expansion of the therapeutic time window in patients with cerebral ischemia.

Background and Purpose-White matter lesions contribute to cognitive impairment in poststroke patients. The present study was designed to assess the neuroprotective mechanisms of cilostazol, a potent inhibitor of type III phosphodiesterase, through signaling pathways that lead to activation of transcription factor cAMP-responsive element binding protein (CREB) phosphorylation using rat chronic cerebral hypoperfusion model. Methods-Rats underwent bilateral common carotid artery ligation. They were divided into the cilostazol group (nϭ80) and the vehicle (control) group (nϭ80). Performance at the Morris water maze task and immunohistochemistry for 4-hydroxy-2-nonenal (HNE), glutathione-S-transferase-pi (GST-pi), ionized calcium-binding adaptor molecule 1, phosphorylated CREB (p-CREB), Bcl-2, and cyclooxygenase-2 (COX-2) were analyzed at baseline and at 3, 7, 14, 21, and 28 days after hypoperfusion. Result-Cilostazol significantly improved spatial learning memory (6.8Ϯ2.3 seconds; PϽ0.05) at 7 days after hypoperfusion.Cilostazol markedly suppressed accumulation of HNE-modified protein and loss of GST-pi-positive oligodendrocytes in the cerebral white matter during the early period after hypoperfusion (PϽ0.05). Cilostazol upregulated p-CREB and Bcl-2 (PϽ0.05), increased COX-2 expression, and reduced microglial activation in the early period of hypoperfusion. Conclusion-Our results indicate that cilostazol exerts a brain-protective effect through the CREB phosphorylation pathway leading to upregulation of Bcl-2 and COX-2 expressions and suggest that cilostazol is potentially useful for the treatment of cognitive impairment in poststroke patients.

Extended Data Fig. 1 | Gating strategy for Luminex-based and Fc effector function assays. (a) Gating for Luminex-bead based antibody binding to spike-coated beads. (b) Gating for ADNP assay showing CD66 + neutrophils with opsinophagocytosed beads. (c) Gating for ADCP assay showing THP-1 monocytes and opsinophagocytosed beads. (d) Gating for ADCD assay showing complement deposition on spike and antibody coated beads. (e) Gating for NK cell activation assay showing CD107a expression.

Background Delayed cerebral ischemia remains a common and profound risk factor for poor outcome after subarachnoid hemorrhage (SAH). The aim of our current study is to define the role of endothelial nitric oxide synthase (eNOS) in isoflurane conditioning‐induced neurovascular protection after SAH. Methods and Results Ten‐ to 14‐week‐old male wild‐type mice (C57BL/6) as controls and eNOS knockout male mice (strain # 002684) were obtained for the study. Animals underwent either sham surgery, SAH surgery, or SAH with isoflurane conditioning. Anesthetic post conditioning was performed with isoflurane 2% for 1 hour, 1 hour after SAH. Normothermia was maintained with the homeothermic blanket. In a separate cohort, nitric oxide synthase was inhibited by a pan nitric oxide synthase inhibitor, L‐nitroarginine methyl ester. Vasospasm measurement was assessed 72 hours after SAH and neurological function was assessed daily. Isoflurane‐induced changes in the eNOS protein expression were measured. eNOS protein expression was significantly increased by isoflurane conditioning in naïve mice as well as mice subjected to SAH. Vasospasm of the middle cerebral artery and neurological deficits were evident following SAH versus sham surgery, both in wild‐type mice and eNOS knockout mice. Isoflurane conditioning attenuated vasospasm and neurological deficits in wild‐type mice. This delayed cerebral ischemia protection was lost in L‐nitroarginine methyl ester ‐administered mice and eNOS knockout mice. Conclusions Our data indicate isoflurane conditioning provides robust protection against SAH‐induced vasospasm and neurological deficits, and that this delayed cerebral ischemia protection is critically mediated via isoflurane‐induced augmentation of eNOS.