Cilostazol attenuates kainic acid-induced hippocampal cell death

Abstract

Cilostazol is a selective inhibitor of type 3 phosphodiesterase (PDE3) and has been widely used as an antiplatelet agent. Cilostazol mediates this activity through effects on the cyclic adenosine monophosphate (cAMP) signaling cascade. Recently, it has attracted attention as a neuroprotective agent. However, little is known about cilostazol's effect on excitotoxicity induced neuronal cell death. Therefore, this study evaluated the neuroprotective effect of cilostazol treatment against hippocampal neuronal damage in a mouse model of kainic acid (KA)-induced neuronal loss. Cilostazol pretreatment reduced KA-induced seizure scores and hippocampal neuron death. In addition, cilostazol pretreatment increased cAMP response element-binding protein (CREB) phosphorylation and decreased neuroinflammation. These observations suggest that cilostazol may have beneficial therapeutic effects on seizure activity and other neurological diseases associated with excitotoxicity.

Keywords: Cilostazol; Hippocampus; Kainic acid; Neuroinflammation; Neuronal death.

Fig. 1. Effect of cilostazol pretreatment on seizure activity and hippocampal cell death in KA-treated mice.

(A) Behavioral seizure scores over time were monitored for 2 h after KA treatment. Data are presented as the mean±SEM. ^*p<0.05 versus CTL. (B) Representative microphotographs of Cresyl violet and TUNEL staining. Cresyl violet staining shows specific neuronal loss in the CA3 region of KA-treated mice. The areas in black squares in left panels were magnified on the central panels. TUNEL-positive cells indicate neuronal cell death in KA-treated hippocampus. Scale bar=100 µm.

Fig. 2. Effect of cilostazol pretreatment on phosphorylated CREB expression in KA-treated hippocampus.

Western blots of hippocampal pCREB and CREB. The mean densitometry values were obtained from three separate experiments (n=6 mice per group). Data are presented as the mean±SEM. ^*p<0.05 vs. CTL.

Fig. 3. Effect of cilostazol pretreatment on LCN2 and GFAP expression in KA-treated hippocampus.

Western blots and protein quantification of hippocampal LCN2 (A) and GFAP (B). The mean densitometry values were obtained from three separate experiments (n=6 mice per group). Data are presented as the mean±SEM. ^*p<0.05 vs. CTL. ^†p<0.05 vs. KA. (C) Representative images of triple-immunofluorescence staining for LCN2 (red), GFAP (purple), and Iba1 (green) in the CA3 region of hippocampus of CTL, KA, KA+Cilo, and Cilo mice. Scale bar=50 µm. (D) Representative images of LCN2 (red), GFAP (purple), and Iba1 (green)-positive cells in the CA3 region of hippocampus of KA-treated mice. Scale bar=10 µm.

Fig. 4. Effect of cilostazol pretreatment on COX-2 and TGF-β1 expression in KA-treated hippocampus.

Western blots and protein quantification of hippocampal COX-2 (A) and TGF-β1 (B). The mean values were obtained from three separate experiments (n=6 mice per group). Data are presented as the mean±SEM. ^*p<0.05 vs. CTL. ^†p<0.05 vs. KA.