Neuroprotective effects of zonisamide on cerebral ischemia injury via inhibition of neuronal apoptosis

Abstract

It is known that neuronal apoptosis contributes to pathology of cerebral ischemia injury. Zonisamide (ZNS) has shown anti-apoptosis effects in recent studies. The present study investigated whether the anti-apoptotic effect can account for the neuroprotective action of ZNS on cerebral ischemia. Neuronal cells were maintained under oxygen-glucose deprivation conditions to simulate cerebral ischemia and treated with ZNS simultaneously. The apoptosis of the cells and expression of apoptosis-related proteins were investigated by flow cytometry and western blot analysis, respectively. A cerebral ischemia mouse model was created via middle cerebral artery occlusion, and the mice were treated with ZNS. Neurological deficit scores and infarct volumes of the cerebral ischemia mice were measured. The apoptosis status of the neuronal cells was evaluated by TUNEL staining. In vitro, the ZNS treatment inhibited both the apoptosis of the neuronal cells and apoptosis-related protein expression (caspase-3, caspase-8, and calpain-1) induced by the oxygen-glucose deprivation. The anti-apoptosis effect of ZNS could occur through the blocking of reactive oxygen species. Moreover, ZNS treatment significantly ameliorated neurological deficits and reduced infarct volumes in the cerebral ischemia mice model. In this study, ZNS exerted neuroprotective effects by inhibition of apoptosis in neuronal cells in cerebral ischemia. Therefore, ZNS might be a promising therapy for cerebral ischemia.

Figures

Figure 1. HT22 cells were pretreated or not with zonisamide (ZNS), and then exposed to oxygen glucose deprivation (OGD) conditions for 2 h. A, The number of apoptotic (annexin V-positive) cells is indicated as the percentage of gated cells. Representative images and relative quantifications are shown. B, Apoptosis-related protein expression in cells under OGD. All the experiments were performed in triplicate, and the data are reported as means±SE (*P<0.05, **P<0.01 one-way ANOVA).

Figure 2. The cells received zonisamide (ZNS) or H2O2, for 24 h. A, Cells stained with annexin V and PI, showing the number of apoptotic (annexin V-positive) cells as the percentage of gated cells. B, Apoptosis-related protein expression in cells after different treatments via western blot (representative images and relative quantifications are shown). All the experiments were performed in triplicate, and the data are reported as means±SE (*P<0.05, **P<0.01 one-way ANOVA).

Figure 3. Effect of zonisamide (ZNS) on neurological deficit and infarct size in middle cerebral artery occlusion (MCAO) mice. A, Representative images of brain sections (from left to right: sham, MCAO, and ZNS; scale bar: 2 mm) and (B) statistical results. C, Mice with ZNS treatment showed lower neurological deficit scores. ZNS: middle cerebral artery occlusion surgery + ZNS treatment; sham: sham surgery. The data are reported as means±SE (*P<0.05, **P<0.01 one-way ANOVA).

Figure 4. Treatment with zonisamide (ZNS) inhibits apoptosis of neuronal cells and reactive oxygen species (ROS) production in the penumbra of middle cerebral artery occlusion (MCAO) mice. A, Representative images of TUNEL staining (400×, scale bar: 100 μm). B, Mice with ZNS treatment showed fewer TUNEL-positive cells in the brain slices and lower reactive oxygen species (ROS) levels, measured in DCF fluorescence, than the group without ZNS treatment. ZNS: middle cerebral artery occlusion surgery + ZNS treatment; sham: sham surgery. The data are reported as means±SE (*P<0.05, **P<0.01 one-way ANOVA).