Concurrent administration of cilostazol with donepezil effectively improves cognitive dysfunction with increased neuroprotection after chronic cerebral hypoperfusion in rats

Abstract

In the present study, we assessed whether concurrent treatment with low doses of cilostazol and donepezil effectively improve memory deficits in association with amelioration of the pathological changes in the white matter of rats subjected to permanent ligation of bilateral common carotid arteries (BCCAL). The escape latency in Morris water maze test was significantly increased at 7, 14 and 21 days in rats subjected to BCCAL. At 21 days after ligation, the white matter lesions including vacuole formation with rarefaction were increased in the optic tract and corpus callosum accompanied by a large increase in glial fibrillary acidic protein (GFAP) immunoreactivity with significantly decreased CNPase-positive oligodendrocytes, all of which were significantly alleviated by the combination therapy with suboptimal doses of cilostazol (30 mg/kg orally) and donepezil (0.3 mg/kg intraperitoneally). The phosphorylated cyclic AMP response element-binding protein (p-CREB)- and Bcl-2-positive cells were significantly decreased following BCCAL, which were completely restored by the combination therapy, whereas the monotherapy with cilostazol or donepezil showed marginal effect. In conclusion, concurrent treatment with cilostazol and donepezil effectively prevented the occurrence of neuropathological alterations in the white matter by activation of p-CREB and Bcl-2, thereby resulting in improvement of spatial learning memory in rats subjected to chronic cerebral hypoperfusion.

Introduction

Severe reduction in cerebral blood flow that is critically implicated in the vascular dementia is responsible for the cognitive decline and gait disorders in the elderly population (Boone et al., 1992). The white matter lesions including demyelination and axonal damage have been experimentally identified by chronic cerebral ischemia induced by the permanent BCCAL in rat (Tanaka et al., 1996). This animal model has been proposed as a model for vascular dementia and cerebrovascular white matter lesions (Tomimoto et al., 1997, Wakita et al., 1994, Wakita et al., 1999). Oligodendroglial cell death was reported to occur following bilateral carotid artery occlusion in gerbil (Kurumatani et al., 1998) and transient global ischemia in rat (Petito et al., 1998). The progressive learning and memory impairments in the eight-arm radial maze task were reported in this animal model (Ni et al., 1994).

Cilostazol (6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2-(1H)-quinolinone) was introduced to increase the intracellular cyclic AMP level by inhibiting its hydrolysis by type III phosphodiesterase (Kimura et al., 1985) and permitted to use for treating intermittent claudication by the FDA (Dawson et al., 1998). Recently, evidence emerges that cilostazol (30 mg/kg two times at 5 min and 4 h), which elevates intracellular cyclic AMP levels by inhibiting type 3 phosphodiesterase, has suppressed the hemispheric infarct lesions that occurred in rats subjected to 2-h occlusion of middle cerebral artery and 24-h reperfusion through up-regulation of the Akt/PKB and p-CREB and increased Bcl-2 protein, consequently exerting potent anti-apoptotic actions (Hong et al., 2003, Lee et al., 2003, Lee et al., 2004). Lee et al. (2006) further observed cilostazol's (60 mg/kg/day, orally) effect in preventing the occurrence of vacuolation and rarefaction of the white matter in association with reduced apoptosis in rats subjected to BCCAL, in that cilostazol strongly suppressed activated microglial and astroglial cells and restored the diminished oligodendrocytes in the white matter. Most recently, Park et al. (2007) have demonstrated the beneficial synergistic effects of concurrent treatment with cilostazol and probucol against focal cerebral ischemic injury in rats.

On the other hand, donepezil ((±)-2-[(1-benzylpiperidin-4-yl)methyl]-5,6-dimethoxy-indan-1-one monohydrochloride), a piperidine derivative, is known as one of the second-generation cholinesterase inhibitors for the treatment of Alzheimer's disease. The drug was demonstrated to be a potent and selective inhibitor of brain acetylcholinesterase in comparison with physostigmine and tacrine (Sugimoto et al., 2000). Recent reports with donepezil have documented significant improvement in cognition, global function and activities of daily living in patients with vascular dementia (Ogura et al., 2000). In addition, donepezil was demonstrated to increase phosphorylated CREB-positive cells in the dentate gyrus (Kotani et al., 2006, Zhao et al., 2003), suggesting that CREB signaling is also modulated by the brain cholinergic system.

Both cilostazol and donepezil were approved safe and efficient in their respective therapeutic categories with different action mechanisms: cilostazol elicits the anti-inflammatory and anti-apoptotic effects, while donepezil improves the cognition and global function in patients with mild to moderate Alzheimer's disease. On the other hand, some untoward effects have been reported, in that donepezil-treated patients complain of fatigue, diarrhea, nausea/vomiting and muscle cramps at 5–10 mg/day (Rogers et al., 1998), thereby making a limit to its long-term use, while cilostazol exerts some side effects such as headache (Mallikaarjun et al., 1999). In light of these pharmacological aspects, it is likely predicted that the combination therapy with cilostazol and donepezil with low doses may provide more beneficial therapeutic windows with fewer side effects. Thus, in the present study, to assess the more effective therapeutic window in improving cognitive deficits (by donepezil) through amelioration of the pathological changes in the white matter (by cilostazol) induced by BCCAL, we examined the efficacy of the combination therapy with cilostazol and donepezil in comparison with the monotherapy of either drug.

Given that cilostazol in combination with donepezil strongly suppresses the occurrence of vacuolation and rarefaction in the white matter with activation of p-CREB and Bcl-2 expression in accordance with improvement of spatial learning memory in rats subjected to chronic cerebral hypoperfusion, it is likely predicted that the combined treatment with low doses of cilostazol and donepezil may exert a beneficial protection against chronic cerebral hypoperfusion injury.