Elsevier

Brain Research

Volume 1116, Issue 1, 20 October 2006, Pages 187-193
Brain Research

Research Report
Cilostazol reduces ischemic brain damage partly by inducing metallothionein-1 and -2

https://doi.org/10.1016/j.brainres.2006.07.125Get rights and content

Abstract

The neuroprotective effect of cilostazol, an antiplatelet drug, was examined after 24 h permanent middle cerebral artery (MCA) occlusion in mice, and explored the possible underlying mechanism by examining metallothionein (MT)-1 and -2 induction in vivo. Cilostazol (30 mg/kg) was intraperitoneally administered at 12 h before, 1 h before, and just after MCA occlusion. Mice were euthanized at 24 h after the occlusion, and the neuronal damage was evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Cilostazol significantly reduced the infarct area and volume, especially in the cortex. Real-time RT-PCR revealed increased mRNA expressions for MT-1 and -2 in the cortex of normal brains at 6 h after cilostazol treatment without MCA occlusion. MT-1 and -2 immunoreactivity was also increased in the cortex of such mice, and this immunoreactivity was observed in the ischemic hemisphere at 24 h after MCA occlusion (without cilostazol treatment). The strongest MT-1 and -2 immunoreactivity was detected in MCA-occlused mice treated with cilostazol [in the peri-infarct zone of the cortex (penumbral zone)]. These findings indicate that cilostazol has neuroprotective effects in vivo against permanent focal cerebral ischemia, especially in the penumbral zone in the cortex, and that MT-1 and -2 may be partly responsible for these neuroprotective effects.

Introduction

The metallothioneins (MTs) are a family of four low molecular weight, metal-binding proteins with a high cystein content, known as MT-1, MT-2, MT-3, and MT-4 (Hamer, 1986). In adult mice, MT-1 and MT-2 are found in all organs, MT-3 is expressed mainly in the brain (Palmiter et al., 1992), and MT-4 is most abundant in certain stratified squamous epithelial tissues (Quaife et al., 1994). Previous studies suggest that MT-1 and -2 serve as important regulators of metal homeostasis, and also as a source of zinc for incorporation into proteins, including transcription factors (Zeng et al., 1991, Palmiter, 1998). MT-1 and -2 are able to prevent zinc deficiency and toxicity in vivo (Dalton et al., 1996, Kelly et al., 1996, Lee et al., 1996), and have also been proposed to function as detoxifiers of other reactive metals and free radicals (Thornalley and Vasak, 1985, Liu et al., 1991, Liu et al., 1995). Interestingly, MT-1 isoform-overexpressing transgenic mice apparently possess a degree of protection against neuronal damage in a cerebral ischemia reperfusion model (Van Lookeren Campagne et al., 1999), and MT-1,2-knock-out mice have larger infarcts than wild-type mice after 2 h transient focal ischemia (Trendelenburg et al., 2002).

Cilostazol, an antiplatelet drug used to treat intermittent claudication, has been reported to increase the intracellular level of cyclic AMP by inhibiting its hydrolysis by type III phosphodiesterase. Its principal actions include inhibition of platelet aggregation (Kimura et al., 1985, Kohda et al., 1999), antithrombosis in feline cerebral ischemia, and vasodilation via mediation of increased cyclic AMP level (Tanaka et al., 1989). Recently, Lee et al. (2005) found that cilostazol has a neuroprotective effect against cerebral infarcts in rat brains subjected to middle cerebral artery (MCA) occlusion followed by 24 h reperfusion, and that this effect is exerted via antioxidant and antiapoptotic actions. Since Michael and Robert (1989) noted that dibutyryl cyclic AMP increased significantly liver MT-1 and -2, it seemed possible that cilostazol might reduce ischemic brain damage at least partly by inducing MT-1 and -2.

The purposes of this study, on mice, were therefore to examine (1) whether cilostazol induces MT-1 and -2 in the brain and (2) whether cilostazol reduces, via an MT-related mechanism, the brain damage occurring after permanent MCA occlusion.

Section snippets

Physiological variables

There was no significant difference between the vehicle and cilostazol groups in the following physiological variables: mean arterial blood pressure, blood pH, partial pressures of carbon dioxide (PaCO2) and oxygen (PaO2), and regional cerebral blood flow (rCBF) in the core area whether they were measured before or after 30 min ischemia (Table 1).

Effect of cilostazol on infarct size and volume

Animals injected i.p. with cilostazol at 30 mg/kg, at 12 h before, 1 h before, and just after the induction of focal ischemia showed no behavioral

Discussion

We examined the neuroprotective effects of cilostazol after 24 h permanent MCA occlusion in mice, and we explored the possible underlying mechanism by examining MT-1 and -2 induction in vivo. Cilostazol-treated mice showed significantly smaller infarct area and volume (vs. vehicle-treated mice) at 24 h after MCA occlusion, the trend being stronger in the cortex than in the subcortex (including the striatum). The expressions of MT-1 and -2 mRNAs were increased in the cortex of cilostazol-treated

Animal preparation

The experimental designs, and all procedures, were conducted in accordance with the Animal Care Guidelines of the Animal Experimental Committee of Gifu Pharmaceutical University. Male C57BL/6J type mice (body weight, 24–28 g) were housed at controlled room temperature (24.5–25.0 °C), with a 12/12-h light/dark cycle. Mouse food pellets and tap water were provided ad libitum.

Drugs

Cilostazol {6-[4-(1-cyclohexy-1H-tetrazol-5-yl)butoxyl]-3,4-dihydro-2-(1H)-quinolinone} was kindly gifted by Otsuka

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