An acetylcholinesterase inhibitor donepezil currently is used to treat patients with Alzheimer disease. However, its direct effect on cerebral blood vessels has not been evaluated. The present study was designed to examine whether donepezil induces acute cerebral arteriolar dilation and whether neuronal nitric oxide synthase contributes to this vasodilator response.


Brain slices were obtained from neuronal nitric oxide synthase knock-out or C57BL/6J strain (control) mice as well as Wistar rats. Parenchymal arterioles were monitored using videomicroscopy. During constriction to prostaglandin F2alpha (5 x 10 m), donepezil (10-10 m) or acetylcholine (10-10 m) was added. In some experiments, brain slices were treated with a nonselective or a selective nitric oxide synthase inhibitor (N-nitro-L-arginine methyl ester [10 m] and S-methyl-L-thiocitrulline [10 m], respectively). An immunohistochemical analysis was performed using antibodies for neuronal nitric oxide synthase and acetylcholinesterase.


Acetylcholine concentration-dependently dilated rat parenchymal arterioles, while S-methyl-L-thiocitrulline as well as N-nitro-L-arginine methyl ester completely abolished this response. Donepezil produced arteriolar dilation, which was inhibited by S-methyl-L-thiocitrulline or N-nitro-L-arginine methyl ester. Donepezil failed to induce arteriolar dilation in the brain slice from the neuronal nitric oxide synthase knock-out mice. Immunohistochemical analysis revealed spatial relationship between neuronal nitric oxide synthase and acetylcholinesterase in the arteriolar wall.


Donepezil produces acute vasodilation induced by a selective activation of neuronal nitric oxide synthase in the cerebral parenchymal arterioles. This agent may be capable of enhancing this enzymatic activity directly or via acetylcholinesterase existing on the arteriolar wall.

ALZHEIMER disease, which is a leading cause of dementia in the elderly population, is well characterized by the impairment of central cholinergic neurotransmission.1Indeed, previous studies demonstrated the tight relationship between the cholinergic dysfunction in diverse brain areas and the severity of cognitive dysfunction.2,3Cerebral blood vessels reportedly are innervated with cholinergic nerve fibers in both humans and animals, resulting in augmentation of cerebral blood flow and/or vasodilation via  activation of neuronal nitric oxide synthase.4–8Importantly, in patients with Alzheimer disease, cortical cerebral blood vessels and nitric oxide synthase-containing neurons are deprived of the cholinergic control, indicating such neurovascular deficits are major pathogenic factors underlying the malfunction of cerebral blood flow regulation in this diseased state.9–12 

An acetylcholinesterase inhibitor, donepezil, is clinically used worldwide for patients with mild to severe Alzheimer disease.1,13,14Chronic treatment with donepezil is known to preserve regional cerebral blood flow in these patients.15,16However, there is no direct evidence showing the acute vasodilator effect of donepezil via  nitric oxide synthase on cerebral blood vessels. In addition, recent studies have demonstrated that cholinergic activation provoked by oral donepezil reduces hypersensitivity of the spinal cord in an animal model of neuropathic pain, indicating that this agent may be a useful tool in pain medicine.17,18These results led us to the idea that studies evaluating the role as well as the mechanism of this compound in the modulation of neurovascular function will allow us to obtain crucial information for donepezil use in the clinical practice.

Therefore, the aim of this study was to examine in the brain whether clinically relevant concentrations of donepezil induce acute arteriolar dilation, and if so, whether this vasodilator response is mediated by the selective activation of neuronal nitric oxide synthase.

The animal experiment was approved and conducted in accordance with local institutional guidelines for the care and use of laboratory animals in Wakayama Medical University (Wakayama, Japan). Male Wistar rats (300–400 g), neuronal nitric oxide knock-out mice or C57BL/6J mice as the control (40–55 g), were obtained from the Jackson Laboratory (Bar Harbor, ME) via  Charles River Japan (Yokohama, Japan). Rats and mice were housed in groups of three in cages under standard conditions for animal studies (20–21°C, 12 h of artificial lighting/day), were fed ad libitum , and had free access to filtered tap water. Animals were anesthetized by inhalation of 3% halothane in 100% oxygen (3 l/min) to perform a midline thoracotomy, and 50 ml of saline was infused intracardially into the left ventricle while a right atrial incision simultaneously was made for blood drainage. The animals were then decapitated, and the brains were rapidly removed and rinsed with artificial cerebrospinal fluid of the following composition (mm): NaCl 119, KCl 4.7, CaCl22.5, MgSO41.17, KH2PO41.18, NaHCO325, and glucose 11.

Brains were cut freehand into blocks containing the neocortex, followed by immediate sectioning into slices (125 mm thick) with a mechanical tissue slicer (Vibratomes 1000; Ted Pella, Redding, CA). Throughout the slicing procedure, brain blocks were continuously bathed in the perfusion solution bubbled with 95% O2and 5% CO2at 4°C. Individually, slices were then transferred to a recording chamber filled with the artificial cerebrospinal fluid, which was mounted on an inverted microscope (Olympus IX70; Shinjuku-ku, Tokyo, Japan). The recording system consisted of a recording chamber (3 ml) and a tubing compartment (7 ml) including the perfusion chamber. The slices were continuously superfused with perfusion fluid at the flow rate of 1.5 ml/min, bubbled with mixture gas of oxygen and carbon dioxide (Pco2= 40 mmHg, pH = 7.4, 37°C in the recording chamber). An intraparenchymal arteriole (4.0–8.0 μm in internal diameter,  11.0–16.5 μm in external diameter) was located within the neuronal tissue, and its internal diameter was continuously monitored with the live computerized videomicroscopy.19The videomicroscopy equipment consisted of an inverted microscope, a 40× objective (Olympus), and a 2.25× video projection lens (Olympus). The image of a parenchymal arteriole was transmitted to a video camera (C6790-81, Olympus) and displayed on a computer via  a media converter (Physio-Tech; Chiyoda-ku, Tokyo, Japan).

The differentiation between the arteriole and the venule in the brain was based on previous studies documenting that, in the brain, a layer of smooth muscle cells should be identified in the arteriole and that the venule resembles a large capillary with no more to its walls than endothelial cells resting on a basal lamina.20,21 

We calculated the ratio of internal to external diameter of the vessel, and a vessel demonstrating a ratio less than 0.5 was used as an arteriole for the following experiments. We defined the internal diameter as the length between the internal margins of arteriolar walls. Changes of internal diameter in cerebral microvessels were recorded on computer image files and then analyzed using the image analysis software with a sensitivity to 0.01 μm (Physio-Tech).19,22Microvessel diameters were derived as an average of four measurements taken along approximately 20 μm of vessel length.

Each slice was equilibrated for at least 30 min before the start of the experimental protocols. All experiments were performed during constriction in response to prostaglandin F(5 × 10−7m), a concentration that produces submaximal vasoconstriction of approximately 70% compared with maximal contraction induced by prostaglandin F(10−5m) in the cerebral parenchymal arterioles.19A nonselective nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (10−4m) or a selective neuronal nitric oxide synthase inhibitor S-methyl-L-thiocitrulline (10−5m) was applied 15 min before the addition of prostaglandin F(5 × 10−7m). Concentration-responses to donepezil (10−9–10−8m), acetylcholine (10−6–10−4m), or sodium nitroprusside (3 × 10−8to 3 × 10−6m) were obtained in a cumulative fashion. Only one concentration-response was made for each slice. The amount of dilation of the cerebral arteriole induced by vasodilators was normalized using the constriction produced by prostaglandin F(5 × 10−7m) in each arteriole. Therefore, the percent dilation was calculated by the following equation: % dilation = 100 × (the diameter after administration of the vasodilator minus the diameter after administration of prostaglandin F[5 × 10−7m]) / (the diameter of control condition minus the diameter after administration of prostaglandin F[5 × 10−7m]). Percentage of constriction to prostaglandin F= 100 × (the diameter after administration of prostaglandin F[5 × 10−7m] minus the diameter of control condition) / the diameter of control condition.

Immunofluorescence Analysis

In some experiments, the brain slices were fixed with 10% formalin buffered with phosphate buffer solution (pH 7.2) and embedded in paraffin, followed by the preparation of 4-6–μm sections. After the deparaffinization, sections were incubated with phosphate buffer solution containing 1% normal donkey serum and 1% bovine serum albumin to reduce nonspecific reactions. The sections were further incubated with the combination of rabbit antineuronal nitric oxide synthase and goat antiacetylcholineesterase polyclonal antibodies at the concentration of 1 μg/ml at 4°C overnight. After incubation with Cy3-conjugated donkey antirabbit immunoglobulin G polyclonal antibodies and fluorescein isothiocyanate-conjugated donkey antigoat immunoglobulin G polyclonal antibodies (10 μg/ml) at room temperature for 30 min, the sections were observed by fluorescence microscopy and fluorescence images were digitally merged. As negative controls, specimens were incubated with normal rabbit and goat immunoglobulin G or in the absence of the primary antibodies.


The following pharmacologic agents were used: acetylcholine, dimethyl sulfoxide, NG-nitro-L-arginine methyl ester, S-methyl-L-thiocitrulline, sodium nitroprusside, and prostaglandin F(Sigma Aldrich, St. Louis, MO). The rabbit antineuronal nitric oxide synthase and goat antiacetylcholineesterase polyclonal antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Donepezil was a generous gift from Eizai Pharmaceutical (Tokyo, Japan). Drugs except donepezil were dissolved in distilled water such that volumes of less than 60 μl were added to the perfusion system. The stock solution of donepezil (10−6m) was prepared in dimethyl sulfoxide (3 × 10−4m). Drugs were dissolved in distilled water such that volumes of less than 60 ml were added to the perfusion system. The concentrations of drugs are expressed as final molar concentrations.

Statistical Analysis

The data were expressed as means ± SD; n refers to the number of rats or mice from which brain slices were taken. Power calculations were done with the inhibitory effect of S-methyl-L-thiocitrulline on the vasodilation in response to donepezil as the primary end point, and we calculated that a sample size of five gave 80% power to detect a change of 16.1% in vasoconstriction at a significance level of 0.05 (SD = 8%). Statistical analysis was performed by StatView® 5.0 (SAS Institute, Cary, NC) or Sample Power® 2.0 (SSPS Japan, Tokyo, Japan). Data were evaluated using repeated-measures of analysis of variance or a factorial analysis of variance as appropriate, followed by Student-Newman-Keuls test as a post hoc  analysis. Differences were considered to be statistically significant when P < 0.05.

Figure 1shows the representative example of the arteriole and the venule with similar external diameters (16.5 or 17.0 μm for the arteriole and the venule, respectively) in the brain parenchyma. The arteriole, which has a smooth muscle layer, has a smaller internal diameter (5.5 μm) compared with that of the venule (11.4 μm).

During submaximal constriction in response to prostaglandin F(5 × 10−7m), acetylcholine dilated rat parenchymal arterioles in a concentration-dependent fashion, while NG-nitro-L-arginine methyl ester completely abolished this response (fig. 2A). Donepezil (3 × 10−9and 10−8m) produced arteriolar dilation, which was completely abolished by S-methyl-L-thiocitrulline as well as NG-nitro-L-arginine methyl ester (fig. 2B).

Donepezil (3 × 10−9and 10−8m) induced cerebral arteriolar dilation in the brain slice from control mice (C57BL/6J strain mice), while it did not induce any dilator responses in that from the neuronal nitric oxide synthase gene knock-out mice (fig. 3A). In contrast, sodium nitroprusside similarly produced arteriolar dilation in the brain slices from both strains (fig. 3B).

A double-color immunofluorescence analysis revealed that in the brain parenchyma, neuronal nitric oxide synthase and acetylcholinesterase are similarly located in the arterioles as well as neuronal cells (fig. 4).

Previous studies including ours suggest that cholinergic stimuli augment cerebral blood flow and/or vasodilation via  activation of neuronal nitric oxide synthase.6–8These results are supported by studies showing that choline acetyltransferase-containing neurons project to neurons in which neuronal nitric oxide synthase is included, suggesting that augmentation of cholinergic neurotransmission contributes to increased cerebral blood flow mediated by neuronal nitric oxide synthase.23Histochemical evaluation also documented the existence of acetylcholinesterase in nerve fibers distributing to cerebral blood vessels, indicating the modulation of cerebral vasomotor function by the reduction of cholinergic neurotransmission.4,5In the current study, we have first revealed the spatial relationship between acetylcholinesterase and neuronal nitric oxide synthase in the brain structures, including neuronal cells and cerebral arterioles. Therefore, our results together with previous studies draw the conclusion that the modulation of cholinergic neurotransmission via  changes in acetylcholine levels plays a role in the regulation of cerebral blood flow and/or vasodilation mediated by enzymatic activity of neuronal nitric oxide synthase.24 

The impairment of cholinergic neurotransmission in the brain is reportedly one of the leading causes of cognitive dysfunction in patients with Alzheimer disease.3,25,26This is a reason why acetylcholinesterase inhibitors have been expected to ameliorate symptoms in these patients. Importantly, in patients with Alzheimer disease, cortical cerebral blood vessels and nitric oxide synthase-containing neurons are deprived of cholinergic control, indicating such neurovascular deficits are major pathogenic factors underlying the malfunction of cerebral blood flow regulation in this diseased state.9–12Therefore, it appears reasonable to administer acetylcholinesterase inhibitors to patients with Alzheimer disease to restore their cerebral blood flow. However, there has been no direct evidence showing vasodilator responses of cerebral arterioles to an acetylcholinesterase inhibitor, although in vivo  studies using single-photon emission computed tomography documented that, in these patients, a highly selective acetylcholinesterase inhibitor donepezil enhances regional cerebral blood flow including that in the cerebral cortex.1,27,28In the current study, we have used brain slices from the cerebral cortex to evaluate the effects of donepezil, because the cortex receives cholinergic neurons and it is one of the most affected brain areas in Alzheimer disease.25We have found that donepezil induces cerebral parenchymal arteriolar dilation, which was completely abolished by a selective as well as a nonselective inhibitor of neuronal nitric oxide synthase.29,30More importantly, we have demonstrated that vasodilator responses to donepezil were absent in the parenchymal arterioles within the cerebral cortex slice from the neuronal nitric oxide synthase gene knock-out mice. Therefore, it is most likely that donepezil induces cerebral arteriolar dilation via  the selective activation of neuronal nitric oxide synthase. In the current study, vasodilation in response to a nitric oxide donor sodium nitroprusside was not different between the gene knock-out and control mice, suggesting that the vasodilator response toward nitric oxide is preserved even in the condition with the neuronal nitric oxide synthase gene knock out.

The peak plasma concentration of donepezil in humans reportedly reaches 2–10 ng/ml after a single oral dose and the plasma protein binding is about 89%, indicating the plasma-free concentration of donepezil in clinical practice would be up to 3 × 10−9m (drug information supplied by Eizai Pharmaceutical, Tokyo, Japan). Therefore, concentrations of this agent used in the current study appear to be clinically relevant. We have documented spatial relationship between neuronal nitric oxide synthase and acetylcholinesterase in the cerebral parenchyma. Our results suggest the possibility that donepezil increases the local concentration of acetylcholine around cerebral parenchymal arterioles, leading to the dilation via  the increased enzymatic activity of neuronal nitric oxide synthase. However, it is still unclear whether this agent is capable of enhancing the enzymatic activity directly or via  acetylcholinesterase distributing to the cerebral arteriolar wall, and whether the enhanced neuronal activity induced by donepezil acts on the increase in cerebral blood flow.

It is important to note that in our experimental system, brain slices are maintained in artificial conditions in which vessels are not perfused and do not have intraluminal flow and that our confocal microscopic findings could be different from those in patients with Alzheimer disease. Therefore, it may be difficult to extrapolate directly our results to the in vivo  diseased condition. However, as already pointed out by some researchers, it is also true that such preparations, in which neuronal-vascular interactions are preserved, offer an additional means to isolated microvessels and whole animal experiments for investigating the cerebral microcirculation.31 

Donepezil is a leading product to treat patients with Alzheimer disease, as supported by clinical studies demonstrating the marked effectiveness of this agent on cognitive dysfunction.1,32The donepezil-induced cerebral arteriolar dilation shown in the current study may at least partly account for the clinical significance of this agent in Alzheimer disease. Donepezil reportedly prevents apoptotic neuronal death induced by glutamate neurotoxicity in the animal model, suggesting protective effects of this agent against cerebral damage such as ischemic brain injury.33In addition to the beneficial effects on the brain function, recent animal studies have demonstrated that cholinergic activation provoked by donepezil reduces hypersensitivity of the spinal cord in neuropathic pain, indicating that this agent may be useful to control such pain in humans.17,18More importantly, this acetylcholinesterase inhibitor is undergoing clinical trials in the treatment of diabetic neuropathic pain.‡‡Therefore, the roles of donepezil in the regulation of the central nervous system should be increasingly crucial in the clinical practice including the perioperative period and pain medicine.

In conclusion, this is the first study demonstrating acute cerebral vasodilation induced by clinically relevant concentrations of donepezil. This compound appears to induce vasodilation via  a selective activation of neuronal nitric oxide synthase. Our results provide a piece of information regarding the role of donepezil in the cerebral circulation.

Sugimoto H, Ogura H, Arai Y, Imura Y, Yamanishi Y: Research and development of donepezil hydrochloride, a new type of acetylcholinesterase inhibitor. Jpn J Pharmacol 2002; 89:7–20
Bartus RT, Dean RL3rd, Beer B, Lippa AS. The cholinergic hypothesis of geriatric memory dysfunction. Science 1982;30: 408–14
Perry EK: The cholinergic hypothesis—ten years on. Br Med Bull 1986; 42:63–9
Benagiano V, Virgintino D, Rizzi A, Errede M, Bertossi V, Troccoli L, Roncali L, Ambrosi G: Cholinergic nerve fibres associated with the microvessels of the human cerebral cortex: a study based on monoclonal immunocytochemistry for choline acetyltransferase. Eur J Histochem 2000; 44:165–9
Saito A, Wu JY, Lee TJ: Evidence for the presence of cholinergic nerves in cerebral arteries: An immunohistochemical demonstration of choline acetyltransferase. J Cereb Blood Flow Metab 1985; 5:327–34
Lacombe P, Sercombe R, Vaucher E, Seylaz J: Reduced cortical vasodilatory response to stimulation of the nucleus basalis of Meynert in the aged rat and evidence for a control of the cerebral circulation. Ann NY Acad Sci 1997; 826:410–5
Toda N, Ayajiki K, Okamura T: Inhibition of nitroxidergic nerve function by neurogenic acetylcholine in monkey cerebral arteries. J Physiol 1997; 498:453–61
Nakahata K, Kinoshita H, Azma T, Matsuda N, Hama-Tomioka K, Haba M, Hatano Y: Propofol restores brain microvascular function impaired by high glucose via  the decrease in oxidative stress. Anesthesiology 2008; 108:269–75
Geaney DP, Soper N, Shepstone BJ, Cowen PJ: Effect of central cholinergic stimulation on regional cerebral blood flow in Alzheimer disease. Lancet 1990; 335:1484–7
Eberling JL, Jagust WJ, Reed BR, Baker MG: Reduced temporal lobe blood flow in Alzheimer's disease. Neurobiol Aging 1992; 13:483–91
Tong XK, Hamel E: Regional cholinergic denervation of cortical microvessels and nitric oxide synthase-containing neuron in Alzheimer's disease. Neuroscience 1999; 92:163–75
Postiglione A, Lassen NA, Holman BL: Cerebral blood flow in patients with dementia of Alzheimer's type. Aging 1993; 5:19–26
Kasa P, Papp H, Kasa P Jr, Torok I: Donepezil dose-dependently inhibits acethylcholinesterase activity in various areas and in the presynaptic cholinergic and the postsynaptic cholinoceptive enzyme-positive structures in the human and rat brain. Neuroscience 2000; 101:89–100
Winblad B, Kilander L, Eriksson S, Minthon L, Batsman S, Wetterholm AL, Jansson-Blixt C, Haglund A. Severe Alzheimer's Disease Study Group: Donepezil in patients with severe Alzheimer's disease: Double-blind, parallel-group, placebo-controlled study. Lancet 2006;367: 1057–65.
Nakano S, Asada T, Matsuda H, Uno M, Takasaki M: Donepezil hydrochloride preserves regional cerebral blood flow in patients with Alzheimer's disease. J Nucl Med 2001; 42:1441–5
Hanyu H, Shimizu T, Tanaka Y, Takasaki M, Koizumi K, Abe K: Regional cerebral blood flow patterns and response to donepezil treatment in patients with Alzheimer's disease. Dement Geriatr Cogn Disord 2003; 15:177–82
Clayton BA, Hayashida K, Childers SR, Xiao R, Eisenach JC: Oral donepezil reduces hypersensitivity after nerve injury by a spinal muscarinic receptor mechanism. Anesthesiology 2007; 106:1019–25
Hayashida K, Parker R, Eisenach JC: Oral gabapentin activates spinal cholinergic circuits to reduce hypersensitivity after peripheral nerve injury and interacts synergistically with oral donepezil. Anesthesiology 2007; 106:1213–9
Nakahata K, Kinoshita H, Hirano Y, Kimoto Y, Iranami H, Hatano Y: Mild hypercapnia induces vasodilation via  adenosine triphosphate-sensitive K+channels in parenchymal microvessels of the rat cerebral cortex. Anesthesiology 2003; 99:1333–9
Peters A, Palay SL, Webster HF: Blood vessels, The Fine Structure of the Nervous System: Neurons and Their Supporting Cells, 3rd edition. Edited by Peters A, Palay SL, Webster HF. New York, Oxford University Press, 1991, pp 344–55Peters A, Palay SL, Webster HF
New York
Oxford University Press
Nakahata K, Kinoshita H, Tokinaga Y, Ishida Y, Kimoto Y, Dojo M, Mizumoto K, Ogawa K, Hatano Y: Vasodilation mediated by inward rectifier K+channels in cerebral microvessels of hypertensive and normotensive rats. Anesth Analg 2006; 102:571–6
Kinoshita H, Nakahata K, Dojo M, Kimoto Y, Hatano Y: Lidocaine impairs vasodilation mediated by adenosine triphosphate-sensitive K+channels but not by inward rectifier K+channels in rat cerebral microvessels. Anesth Analg 2004; 99:904–9
Vaucher E, Linville D, Hamel E: Cholinergic basal forebrain projections to nitric oxide synthase-containing neurons in the rat cerebral cortex. Neuroscience 1997; 79:827–36
Hamel E: Cholinergic modulation of the cortical microvascular bed. Prog Brain Res 2004; 145:171–8
Perry EK, Tomlinson BE, Blessed G, Bergman K, Gibson PH, Perry RH: Correlation of cholinergic abnormalities with senile plaques and mental test scores in senile dementia. BMJ 1978; 25:1457–9
Whitehouse PJ, Price DL, Struble RG, Clark AW, Coyle JT, Delong MR: Alzheimer's disease and senile dementia: Loss of neurons in the basal forebrain. Science 1982; 215:1237–9
Yoshida T, Ha-Kawa S, Yoshimura M, Nobuhara K, Kinoshita T, Sawada S: Effectiveness of treatment with donepezil hydrochloride and changes in regional cerebral blood flow in patients with Alzheimer's disease. Ann Nucl Med 2007; 21:257–65
Shimizu S, Hanyu H, Iwamoto T, Koizumi K, Abe K: SPECT follow-up study of cerebral blood flow changes during donepezil therapy in patients with Alzheimer's disease. J Neuroimaging 2006; 16:16–23
Kinoshita H, Katusic ZS: Nitric oxide and effects of cationic polypeptides in canine cerebral arteries. J Cereb Blood Flow Metab 1997; 17:470–80
Zanzinger J, Czachurski J, Seller H: Neuronal nitric oxide reduces sympathetic excitability by modulation of central glutamate effects in pigs. Circ Res 1997; 80:565–71
Hamel E: Perivascular nerves and the regulation of cerebrovascular tone. J Appl Physiol 2006; 100:1059–64
Rogers SL, Doody RS, Mohs RC, Friedhoff LT: Donepezil improves cognition and global function in Alzheimer disease: A 15-week, double-blind, placebo-controlled study. Donepezil Study Group. Arch Intern Med 1998; 158:1021–31
Takada Y, Yonezawa A, Kume T, Katsuki H, Kaneko S, Sugimoto H, Akaike A: Nicotinic acetylcholine receptor-mediated neuroprotection by donepezil against glutamate neurotoxicity in rat cortical neurons. J Pharmacol Exp Ther 2003; 306:772–7