Neuroprotective effects of cilostazol are mediated by multiple mechanisms in a mouse model of permanent focal ischemia

• Cilostazol reduces infarct volumes in mice permanent focal ischemia.
• Cilostazol reduces oxidative stresses in neurons around the infarct area.
• Cilostazol reduces NADPH oxidase (NOX) 2-positive neurons.
• Cilostazol keeps more pCREB-positive neurons after ischemia.
• Cilostazol has multiple mechanisms of action to ameliorate ischemic damage.

The phosphodiesterase (PDE) 3 inhibitor cilostazol, used as an anti-platelet drug, reportedly can also ameliorate ischemic brain injury. Here, we investigated the effects of cilostazol in a permanent focal ischemia mice model. Male Balb/c mice were subjected to permanent middle cerebral artery occlusion. Mice were then treated with either cilostazol (10 or 20 mg/kg) or vehicle administered at 30 min and 24 h post-ischemia, and infarct volumes were assessed at 48 h post-ischemia. Mice treated with 20 mg/kg of cilostazol or vehicle were sacrificed at 6 h or 24 h post-ischemia and immunohistochemistry was used for brain sections. Treatment with 20 mg/kg of cilostazol significantly reduced infarct volumes to 70.1% of those with vehicle treatment. Immunohistochemistry results for 8-hydroxydeoxyguanosine (OHdG) expression showed that some neurons underwent oxidative stress around the ischemic boundary zone at 6 h post-ischemia. Cilostazol treatment significantly reduced the percentage of 8-OHdG-positive neurons (65.8±33.5% with vehicle and 21.3±9.9% with cilostazol). Moreover, NADPH oxidase (NOX) 2-positive neurons were significantly reduced with cilostazol treatment. In contrast, immunohistochemistry results for phosphorylated cyclic-AMP response element binding protein (pCREB) showed that there were significantly more pCREB-positive neurons around the ischemic boundary zone of cilostazol-treated mice than in those of vehicle-treated mice at 24 h post-ischemia. These results suggested that cilostazol might have multiple mechanisms of action to ameliorate ischemic tissue damage, by attenuating oxidative stress mediated by suppressing NOX2 expression by ischemic neurons and an anti-apoptotic effect mediated through the pCREB pathway.