Cilostazol, a phosphodiesterase 3 inhibitor, protects mice against acute and late ischemic brain injuries

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Abstract

Cilostazol, a selective inhibitor of phosphodiesterase 3, exerts neuroprotective effects on acute brain injury after cerebral ischemia in rats. However, it is unknown whether cilostazol affects the subacute or chronic ischemic injury. In the present study, we evaluated the dose- and time-dependent effects of cilostazol on acute ischemic brain injury and the long-lasting effect on the late (subacute/chronic) injury in mice with focal cerebral ischemia induced by transient middle cerebral artery occlusion. We found that pre-treatment of cilostazol (injected i.p. at 30 min before ischemia) significantly ameliorated the acute injury 24 h after ischemia, and the effective doses were 3–10 mg/kg. The post-treatment of cilostazol (10 mg/kg) was effective on the acute injury when it was injected 1 and 2 h after ischemia. In addition, for the late injury, post-treatment of cilostazol (10 mg/kg, i.p., for 7 consecutive days after ischemia) attenuated neurological dysfunctions, brain atrophy and infarct volume. It also inhibited astrocyte proliferation/glial scar formation and accelerated the angiogenesis in the ischemic boundary zone 7 and 28 days after ischemia. Thus, we conclude that cilostazol protects against not only the acute injury, but also the late injury in mice with focal cerebral ischemia; especially it can modify brain remodeling, astrogliosis and angiogenesis.

Introduction

Cilostazol, 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxyl]-3,4-dihydro-2-(1H)-quinolinone, increases intracellular cyclic adenosine monophosphate (cAMP) level through inhibiting the activity of phosphodiesterase 3 (PDE3) (Kimura et al., 1985, Lugnier, 2006). Cilostazol is currently used in the treatment of intermittent claudication in diabetic patients (Dawson et al., 1998, Jacoby and Mohler, 2004, Omi et al., 2004) and peripheral vascular occlusive diseases (Kwon et al., 2005, Lee et al., 2005). Cilostazol has also been shown the efficacy in prevention of silent brain infarction in Japanese patients with type 2 diabetes (Shinoda-Tagawa et al., 2002) and artherosclerosis (Ahn et al., 2004), which is considered as the results from its anti-platelet, vasodilation, and anti-proliferative actions.

Recently, it has been reported that cilostazol exerts neuroprotective effects on acute ischemic brain injury. Cilostazol decreased ischemic brain infarction, inhibited apoptotic and oxidative cell death (Choi et al., 2002), and attenuated gray and white matter damage 24 h after focal cerebral ischemia in rats (Honda et al., 2006); it also significantly reduced the brain ischemic infarction and edema as measured by magnetic resonance imaging (MRI) in rats (Lee et al., 2003). The protective effects of cilostazol are related to enhancing casein kinase 2 phosphorylation in rat brain after focal cerebral ischemia (Lee et al., 2004), suppressing tumor necrosis factor-α (TNF-α)-induced increased phosphatase and tensin homolog deleted from chromosome 10 (PTEN) phosphorylation in human neuroblastoma SK-N-SH cells (Kim et al., 2004, Lee et al., 2004), and activating cAMP-dependent protein kinase-mediated maxi-K ion channel (Park et al., 2006) and endothelial nitric oxide synthase (Hashimoto et al., 2006) in human endothelial cells. However, the effects of cilostazol on acute brain ischemic injury should be confirmed in mice, especially the dose-dependency and therapeutic window as well as the effect on the disruption of blood–brain barrier.

Moreover, it is still unknown whether cilostazol attenuates subacute/chronic ischemic brain injuries. Ischemic brain injury can be separated into 3 serial phases: acute phase (metabolic stress and excitotoxicity, minutes to hours), subacute phase (inflammation and apoptosis, hours to days), and chronic phase (repair and regeneration, days to months) (Dirnagl et al., 2003). The neurogenesis, gliosis/glial scar formation and angiogenesis are the morphologic changes (brain remodeling) in the chronic phase after ischemic or other brain injuries (Bramlett and Dietrich, 2004). Among these changes, the formation of a glial scar that results from reactive gliosis (Logan and Berry, 2002) (mainly consisted of proliferated astrocytes) may be a physical and biochemical barrier for the regeneration of axons (Silver and Miller, 2004). The angiogenesis can increase the cerebral blood flow and improve brain tissue recovery and functional outcome (Krupinski et al., 1994). Since cilostazol suppressed the activation of astrocytes after chronic cerebral hypoperfusion in rats (Lee et al., 2006), it may affect astrocyte proliferation (astrogliosis) and resultant glial scar formation. It is important to elucidate the effects of cilostazol on subacute/chronic ischemic injuries and the resultant brain remodeling.

Therefore, in the present study we observed the dose- and time-dependent effect of cilostazol on acute injury 24 h after focal cerebral ischemia in mice at first. Then using the dosing regimen based on the acute experiments, we further evaluated the effect of cilostazol on the brain injuries in the late phase (7 and 28 days after ischemia, including subacute and chronic phases). A selective cysteinyl leukotriene receptor-1 antagonist, pranlukast (ONO-1078), was used as a positive control because its neuroprotective effects on acute and late ischemic brain injuries had been proven in our previous studies (Yu et al., 2005a, Zhang and Wei, 2003, Zhang et al., 2002a).

Section snippets

Animals and chemicals

Male Institute of Cancer Research (ICR) mice weighting 25–30 g (Experimental Animal Center of Zhejiang Academy of Medical Sciences, Hangzhou, China, Certificate No. 20030001) were used in this study. All experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. The animals were housed under a controlled temperature, 12-h light/dark cycle and allowed free access to food and water.

Cilostazol was kindly gifted by Otsuka

Physiological variables and mortality

Mean arterial blood pressure, PaO2, PaCO2, arterial blood pH and glucose did not change between 30 min before and 1 h after ischemia and between the mice treated with sham operation and ischemia (with or without cilostazol 10 mg/kg) (Table 1).

The mortality of ischemic mice was about 20% (16.7–23.1%) in each group 24 h after ischemia, and there were no significant differences among groups (P > 0.05, χ2 test; data not shown). The mortality was 56.5% (13/23), 55.6% (15/27) and 57.7% (15/26) 7 days

Discussion

In the present study, on one hand, we confirm that cilostazol protects mice against the acute injury after focal cerebral ischemia in dose- and time-dependent manners as reported elsewhere (Choi et al., 2002, Lee et al., 2003). Evidence is that cilostazol attenuated neurological dysfunction, neuron degeneration, and blood–brain barrier disruption. The effective doses are ranged at 3 and 10 mg/kg, and the therapeutic window is within 2 h after ischemia. On the other hand, we found that

Acknowledgements

This study was supported by the National Natural Science Foundation of China (No. 30500613) and the Scientific Foundation of Education Ministry of China (20050335105). We thank Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan, for providing cilostazol and Dr. Masami Tsuboshima, Ono Pharmaceutical Co. Ltd., Osaka, Japan, for providing pranlukast.

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