www.elsevier.com/locate/brainres

Brain Research 999 (2004) 53–61

Research report

Nilvadipine antagonizes both Ah vasoactivity in isolated arteries,

and the reduced cerebral blood flow in APPsw transgenic mice

Daniel Paris*, Amita Quadros, James Humphrey, Nikunj Patel, Robert Crescentini,Fiona Crawford, Michael Mullan

The Roskamp Institute, 2040 Whitfield Avenue, Sarasota, FL 34243, USA

Accepted 4 November 2003

Abstract

The development of Alzheimer’s disease (AD) is generally thought to correlate with cerebral accumulation of Ah. It has previously been

shown that Ah peptides enhance vasoconstriction in isolated arteries and oppose certain vasorelaxants. Moreover, exogenous application of

Ah peptides causes cerebral vasoconstriction in rodents and in transgenic mouse models of AD that overexpress Ah there is reduced cerebral

blood flow. In the present study, we investigated the effect of nilvadipine, a dihydropyridine-type calcium channel blocker, on Ah induced

vasoconstriction in isolated arteries and in vivo on cerebral blood flow (CBF) of an AD transgenic mouse model overexpressing Ah (Tg

APPsw line 2576). Nilvadipine completely inhibited the vasoactivity elicited by Ah in rat aortae and in human middle cerebral arteries. The

effect of a short treatment duration (2 weeks) with nilvadipine on regional CBF was investigated in 13-month-old Tg APPsw mice and

control littermates using a laser Doppler imager. Additionally, CBF was also measured in 20-month-old Tg APPsw mice and control

littermates that were chronically treated with nilvadipine for 7 months. Untreated Tg APPsw mice showed a reduction of regional CBF

compared to their untreated control littermates. Nilvadipine restored cortical perfusion levels in Tg APPsw to values similar to those observed

in control littermates without notably affecting the CBF of control mice. All together, these data suggest that nilvadipine might be useful for

the treatment of oligemia associated with AD.

D 2003 Elsevier B.V. All rights reserved.

Theme: Other systems of the CNS

Topic: Brain metabolism and blood flow

Keywords: Nilvadipine; Alzheimer; Abeta; Amyloid; Cerebral blood flow; Transgenic

1. Introduction gained increasing attention in recent years. Vascular pathol-

Alzheimer’s disease (AD) is the major cause of dementia

in the elderly in Western countries, and is characterized by

the progressive accumulation of intracellular neurofibrillary

tangles, extracellular parenchymal senile plaques and cere-

brovascular deposits [29]. The principal component of

senile plaques and cerebrovascular deposits is the 39–43

amino acid h-amyloid peptide (Ah), which is proteolyticallyderived from the amyloid precursor protein (APP) [20].

Morphological abnormalities of cerebral capillaries and

related deficient cerebral circulation observed in AD have

0006-8993/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2003.11.061

* Corresponding author. Tel.: +1-941-752-2949; fax: +1-941-752-

2948.

E-mail address: dparis@rfdn.org (D. Paris).

ogy is the norm in advanced cases of AD, with cerebral

amyloid angiopathy (CAA) being one of the commonest

abnormalities detected at autopsy [6].

The association of cerebral hypoperfusion and AD is

well established [1,13,39]. Functional imaging techniques

including positron emission tomography (PET) and single

photon emission computerized tomography (SPECT) have

also revealed the existence of hypoperfusion in individuals

prior to the clinical diagnosis of AD [12,18]. Recent data

have shown that cerebral hypoperfusion may generate not

only white matter changes but also cortical watershed

infarcts, which further worsen cognitive decline in AD [36].

Ah peptides have been suggested to play a critical role in

the pathobiology of AD since all the mutations associated

with familial form of AD result in the increased production of

these peptides [34]. We have shown previously that Ah

D. Paris et al. / Brain Research 999 (2004) 53–6154

peptides synergistically enhance endothelin-1-induced vaso-

constriction [24] and that in vivo Ah can act as a cerebral

vasoconstrictor [35], suggesting that the accumulation of Ahpeptides in the brain and around cerebrovessels of AD

patients might be a contributing factor to the cerebral olige-

mia suffered by these patients. Additionally, topical applica-

tion of synthetic Ah peptides has been reported to induce

vasoconstriction in the cerebrovasculature of rodents [21].

Fig. 1. (A) Effect of nilvadipine on Ah1 – 40 vasoactivity in isolated rat aortae. Ao

nilvadipine, nilvadipine +Ah1 – 40, or untreated (control) 5 min before the addition

ET-1 dose ( P < 0.001), Ah ( P < 0.001) and of nilvadipine ( P< 0.001). There w

( P< 0.001) or nilvadipine ( P < 0.001) and among ET-1 dose, Ah and nilvadipi

between-group differences ( P< 0.001), and post hoc testing showed significant dif

( P< 0.001), but not between control and Ah + nilvadipine ( P= 0.697), control an

The symbol * on the bar graph indicates a statistically significant difference with th

in isolated human middle cerebral arteries. Human cerebral artery rings were t

nilvadipine +Ah1 – 40, 1 AM of scrambled Ah, or untreated (control) 5 min before

ANOVA of ET-1 dose ( P < 0.001), Ah ( P< 0.001) and nilvadipine ( P < 0.001) b

significant interactive terms between ET-1 dose and either Ah ( P < 0.02) or nilvad

between-groups differences ( P< 0.001), and post hoc testing showed significant

( P< 0.009) and Ah and Ah + nilvadipine ( P < 0.001), but not between control and

The symbol * on the bar graph indicates a statistically significant difference with

Ah overexpression in young (2–3 months) transgenic animal

models of AD has been shown to reduce the increased CBF

produced by activation of the vibrissae in the somatosensory

cortex, suggesting that increased Ah levels produces a

potentially deleterious mismatch between substrate delivery

and energy demands imposed by neural activity [8,11,22].

We wished to determine whether calcium channel block-

ers could oppose Ah’s vasoconstriction enhancing effects

rtic rings were treated with 1 AM of freshly solubilized Ah1 – 40, 100 nM of

of a dose range of ET-1. There were significant main effects by ANOVA of

ere also significant interactive terms between ET-1 dose and either Ahne ( P < 0.001). One-way ANOVA across ET-1 doses revealed significant

ferences between control and Ah ( P < 0.001), and Ah and Ah+ nilvadipine

d nilvadipine ( P= 0.979), or nilvadipine and nilvadipine +Ah ( P= 0.468).

e vehicle treatment ( P< 0.05). (B) Effect of nilvadipine on Ah vasoactivity

reated with 1 AM of freshly solubilized Ah1 – 40, 100 nM of nilvadipine,

the addition of a dose range of ET-1. There were significant main effects by

ut no significant main effect of scrambled Ah ( P= 0.549). There were also

ipine ( P< 0.001). One-way ANOVA across ET-1 doses revealed significant

differences between control and Ah ( P< 0.007), control and nilvadipine

scrambled Ah ( P= 0.999), or nilvadipine and nilvadipine +Ah ( P= 0.999).

the vehicle treatment ( P < 0.05).

D. Paris et al. / Brain Research 999 (2004) 53–61 55

and the reduced CBF in transgenic APPsw mice. Experi-

ments were performed ex vivo by recording isometric

tension in isolated rat aortae and human middle cerebral

arteries, and in vivo by measuring regional variation of CBF

in Tg APPsw mice and control littermates using a laser

scanner Doppler imager.

Fig. 2. Effect of nilvadipine on mean arterial blood pressure in 13-month-

old control and Tg APPsw mice. ANOVA revealed a significant main effect

of nilvadipine ( P < 0.001) but no significant main effect of genotype

( P= 0.999). Post hoc analysis showed significant differences between

control mice treated with vehicle and control mice treated with nilvadipine

( P< 0.03), Tg APPsw mice treated with vehicle and Tg APPsw treated with

nilvadipine ( P < 0.01) but no significant difference between control and Tg

APPsw mice treated with vehicle ( P= 0.999) and between control and Tg

APPsw mice treated with nilvadipine ( P= 0.998).

2. Materials and methods

2.1. Vessel experiments

Human middle cerebral arteries (HMCA) were obtained

under IRB approval following the autopsy of 4 elderly

patients (average age: 80.3F 2.9 years and average post-

mortem delay including the time necessary to perform the

autopsy: 3 h 50 minF 23 min) with no recorded cerebro-

vascular disease, including history of stroke, transient is-

chemic attacks or cerebrovascular dementia. HMCA were

first rinsed in ice-cold HBSS containing 2� penicillin–

streptomycin and washed in 50 ml PBS. Rat aortic rings

were obtained from 9-month-old male Sprague–Dawley

rats (Zivic Miller, Zelienople, PA, USA). Arteries were

segmented into 3 mm rings and suspended in Kreb’s buffer

on hooks connected to a tensiometer linked to a MacLab

system. Artery rings were equilibrated for 2 h in 7 ml tissue

baths containing Kreb’s buffer (changed every 30 min)

oxygenated with 95% O2/CO2, and thermoregulated at 37

jC. A baseline tension of 2 g was applied to each ring, 2

min prior their treatment with either 1 AM Ah (purity

greater than 95%, Biosource, California), 100 nM nilvadi-

pine, or a combination of Ah and nilvadipine. Following 5

min of treatment, artery rings were submitted to a dose

range of endothelin-1 (ET-1, Sigma, Missouri) ranging from

1 to 5 nM. Each dose of ET-1 was added only after the

constriction response to the previous dose had reached a

plateau. The specificity of the Ah vasoactive effects was

tested by employing 1 AM of scrambled Ah (VIG-

KYHGMSNLVGRSFEVHQGKGAEVDAHGLF-

DIEAFVDV, Biosource) in isolated human middle cerebral

artery. Results were expressed as the meansF S.E. of the

tension (in g) obtained for each doses of ET-1 and for the

different treatments. Similar experiments were repeated with

100 nM amlodipine and 100 nM nitrendipine instead of

nilvadipine in rat aortae.

2.2. Determination of regional CBF by laser scanner

Doppler imaging

Mice expressing mutant APPK670N,M671L (Tg APPsw

line 2576) [13] and control littermates were studied at 13

months of age (control n = 11; Tg APPsw n = 11). Control

and Tg APPsw littermates were injected subcutaneously

with nilvadipine (1 mg/kg of body weight) or the vehicle

(50% DMSO in PBS) daily for 15 days. The dose of

nilvadipine used for this study was chosen according to

previously published pharmacokinetic data in rodents

[38]. Diastolic (DP) and systolic blood pressure (SP)

was measured in 13-month-old animals with a non-inva-

sive blood pressure analyzer (Columbus Instruments,

Ohio) 10 days after the beginning of the nilvadipine

treatment (20 h after the nilvadipine or vehicle injection).

Mean arterial blood pressure (MAP) was then calculated

using the following formula: MAP= diastolic blood pres-

sure + 1/3(systolic blood pressure� diastolic blood pres-

sure). Additionally, 13-month-old control and Tg APPsw

littermates were treated daily with a subcutaneous injec-

tion of nilvadipine (1 mg/kg of body weight) or the

vehicle (50% DMSO in PBS) for 7 months.

For CBF measurement, mice were anesthetized with a

gas mixture of 3% isofluorane, 0.9 l/min nitrous oxide and

0.5 l/min oxygen. CBF measurements were made within 6

h of the last dose of nilvadipine in the 15 days treated

animals and 10 days after the last injection in 7 months

treated animals. Animals were then immobilized on a mouse

stereotaxic table and maintained under anesthesia with a

mouse anesthetic mask (Kopf Instruments, Tunjunga, CA)

delivering 3% isofluorane, 0.5 l/min nitrous oxide and 0.3 l/

min oxygen. Rectal temperature was maintained at 37 jCusing a mouse homeothermic blanket system (Harvad Ap-

paratus, Holliston, MA). An incision was made through the

scalp and the skin retracted to expose the skull. The

periosteal connective tissue, which adheres to the skull,

was removed with a sterile cotton swab. Animals were

maintained on a mixture of 1.5% isofluorane, 0.5 l/min

D. Paris et al. / Brain Research 999 (2004) 53–6156

nitrous oxide and 0.3 l/min oxygen. Cortical perfusion was

measured with the Laser-Doppler Perfusion Imager from

Moor Instruments (Wilmington, DE) as previously de-

scribed [2]. A computer-controlled optical scanner directed

a low-powered He–Ne laser beam over the exposed cortex.

The scanner head was positioned parallel to the cerebral

cortex at a distance of 26 cm. The scanning procedure took

1 min 21 s for measurements of 5538 pixels covering an

area of 0.8� 0.8 cm. At each measuring site, the beam

illuminated the tissue to a depth of 0.6 mm. An image color-

coded to denote specific relative perfusion levels was

displayed on a video monitor. All images were acquired at

2-min intervals for a period of 30 min (15 images for each

animal). All images were stored in computer memory for

subsequent analysis. For each animal, a square area of 0.05

Fig. 3. (I) Two-dimensional color-coded microvascular flow maps of the brain of

Laser Doppler imaging flow data depicting the variation of regional CBF in the co

Tg APPsw mice treated with nilvadipine (D). (II) Effect of nilvadipine on the CBF

of genotype ( P< 0.001), of area of the brain examined ( P < 0.001), of nilvadip

( P< 0.002) and between genotype and nilvadipine ( P < 0.001). Post hoc analysis

and Tg APPsw ( P < 0.001), parietal cortex of control and Tg APPsw ( P < 0.00

differences were observed between control animals treated with the vehicle only

entire, parietal and occipital cortex ( P= 0.999; P= 0.992; P= 0.999) showing that

significant differences between Tg APPsw mice treated with the vehicle only and T

parietal and occipital cortex ( P < 0.003; P < 0.001; P< 0.001). However, no signif

with nilvadipine for the CBF measured in the entire, parietal and occipital cortex (

of Tg APPsw mice to values similar to those observed in control mice.

cm2 (360 pixels) equally distributed between the right and

left hemispheres was defined and applied to each image of

the series in order to measure the CBF in the parietal and

occipital cortex using the MoorLDI Image Processing V3.0h

software. CBF was also measured in the entire cortex by

manually delineating for each mouse the cortex area (0.51–

0.54 cm2 corresponding to 3504–3714 pixels). Relative

perfusion values for each area studied were normalized

against the CBF values obtained in untreated control mice

and expressed as a % of control CBF. At the end of the

procedure, arterial blood samples were collected from the

left carotid of the animals in order to assess physiological

parameters ( pCO2, pO2 and pH) using a PhOx2 blood gas

analyzer (Nova Biomedical, Waltham, MA). The pCO2 of

control 49.4F 3.4 and Tg APPsw 50.1F 3.9 mm Hg; the

13-month-old control and Tg APPsw recorded with a laser Doppler imager.

rtex of control (A), Tg APPsw (B), control treated with nilvadipine (C) and

of control and Tg APPsw mice. ANOVA revealed a significant main effect

ine ( P< 0.001) as well as an interactive term between genotype and area

revealed a significant difference between CBF in the entire cortex of control

1), occipital cortex of control and Tg APPsw ( P< 0.001). No significant

and control animals treated with nilvadipine for the CBF measured in the

nilvadipine does not affect CBF in control mice. Post hoc analysis showed

g APPsw mice treated with nilvadipine for the CBF measured in the entire,

icant difference were observed between control and Tg APPsw mice treated

P= 0.999; P= 0.999; P= 0.812), showing that nilvadipine increases the CBF

Fig. 3 (continued).

D. Paris et al. / Brain Research 999 (2004) 53–61 57

pO2 of control 95.3F 9.1 and Tg APPsw 101.8F 9.2 mm

Hg; the pH of control 7.20F 0.03 and Tg APPsw

7.22F 0.05 were comparable to regular physiological var-

iables usually recorded in mice [28].

2.3. Data analysis

Data are expressed as meansF S.E. Multiple compari-

sons were evaluated by analysis of variance and post hoc

comparisons performed with Scheffe’s method using SPSS

V11.0 for Windows. Probability values less than 5% were

considered statistically significant.

3. Results

The effect of nivaldipine on the vasoactivity elicited by

freshly solubilized Ah1–40 was investigated in isolated rat

aortae and human middle cerebral arteries. Nilvadipine

appears to potently inhibit the enhancement of endothelin-

1 (ET-1) induced vasoconstriction by Ah in isolated rat

aortae (Fig. 1). Nitrendipine and amlodipine (100 nM)

inhibited the vasoactive effect of Ah in isolated rat aortae

to an extent similar to nilvadipine (data not shown), further

suggesting that calcium channel blockers in general can

efficiently oppose the vasoconstrictive effect of Ah in rat

D. Paris et al. / Brain Research 999 (2004) 53–6158

aortae. Nilvadipine’s effect on Ah vasoactivity was further

tested in isolated human middle cerebral artery. We report

that Ah enhances ET-1-induced vasoconstriction in human

middle cerebral artery and that nilvadipine completely

prevents Ah vasoactivity in human cerebral artery (Fig. 1).

In this large human cerebral artery, nilvadipine appears to

partially oppose the vasoconstriction induced by ET-1 alone,

an effect that was not observed in isolated rat aortae,

suggesting that nilvadipine displays a more profound vaso-

dilatator action in cerebral artery compared to peripheral

vessel. A scrambled Ah peptide was used to assess the

specificity of the Ah vasoactive effect and did not affect ET-

1-induced vasoconstriction.

Next, the effect of nilvadipine was investigated in vivo in

13-month-old Tg APPsw mice and control littermates. Mice

were treated for a period of 15 days with 1 mg/kg of body

weight of nilvadipine delivered subcutaneously. After 10

days of treatment, blood pressure was monitored and a

Fig. 4. Effect of chronic nilvadipine treatment in 20-month-old Tg APPsw

microvascular flow maps of the brain of 20-month-old control and Tg APPsw recor

the variation of regional CBF in the cortex of control (A), Tg APPsw (B), control tr

(II) Effect of chronic nilvadipine treatment on the CBF of 20-month-old control an

( P< 0.001), of area of the brain examined ( P< 0.001) and of nilvadipine ( P < 0.0

entire cortex of control and Tg APPsw ( P < 0.001). No significant differences were

animals treated with nilvadipine for the CBF measured in the entire and parietal co

between Tg APPsw mice treated with the vehicle only and Tg APPsw mice treat

significant reduction of blood pressure was noticed in the

group of animals treated with nilvadipine compared with

animals treated with vehicle (Fig. 2). No difference in blood

pressure was observed between Tg APPsw mice and control

littermates. Following 15 days of treatment with nilvadipine

or vehicle, cortical cerebral blood flow (CBF) was deter-

mined using a laser Doppler imager (LDI). A reduction of

CBF was observed globally (including occipital, parietal

and frontal cortical areas) in the cortex of Tg APPsw

compared to their control littermate and most of this

reduction was due to reduction of flow in the occipital

and parietal areas (Fig. 3). Nilvadipine treatment did not

significantly affect the CBF of control animals for the

different areas of the brain examined despite the observed

reduction of blood pressure. Interestingly, nilvadipine in-

creased the CBF of Tg APPsw mice to values similar to

those observed in control animals treated with nilvadipine or

vehicle alone for the different areas of the brain examined.

and control littermates. (I) Representative two-dimensional color-coded

ded with a laser Doppler imager. Laser Doppler imaging flow data depicting

eated with nilvadipine (C) and Tg APPsw mice treated with nilvadipine (D).

d Tg APPsw mice. ANOVA revealed a significant main effect of genotype

01). Post hoc analysis revealed a significant difference between CBF in the

observed between control animals treated with the vehicle only and control

rtex ( P= 0.992; P= 0.993). Post hoc analysis showed significant differences

ed with nilvadipine for the CBF measured in the entire cortex ( P < 0.04).

Fig. 4 (continued).

D. Paris et al. / Brain Research 999 (2004) 53–61 59

The effect of nilvadipine was also evaluated in older animals

(20 months) that were subjected to a chronic treatment for 7

months from the age of 13 months. Data analysis revealed

that a chronic treatment with nilvadipine reduces the CBF

deficits observed in the brain of 20-month-old Tg APPsw

mice without affecting notably the CBF of control animals

(Fig. 4).

4. Discussion

The contribution of the vasculature to the pathophysiol-

ogy of AD is becoming increasingly recognized [5,8,14,15].

Most of the cardiovascular risk factors (such as hyperten-

sion, hypercholesterolemia, atherosclerosis, APOEq4, dia-

betes, coronary or carotid artery disease) also constitute

independent risk factors for AD, suggesting that vascular

pathologies have a profound impact on the development of

AD. In fact, peripheral vascular diseases are believed to

alter the cerebral circulation resulting in chronic hypoper-

fusion or oligemia [14]. The clinical presentation of AD is

more severe in patients exhibiting evidence of cerebrovas-

cular disease (CVD) than patients without infarcts

[7,19,32]. There is increasing evidence that hypertension

may contribute to the development of dementia

[3,16,25,27]. Interestingly, some calcium channel blockers

D. Paris et al. / Brain Research 999 (2004) 53–6160

used for the treatment of hypertension have been shown to

reduce the incidence of stroke and dementia including AD

[9,26,33], although others such as nifedipine have been

associated with decreased cognition in the elderly [17]. We

therefore tested the effect of three different calcium channel

blockers (nilvadipine, nitrendipine and amlodipine) on the

enhancement of ET-1-induced vasoconstriction induced by

Ah in rat aortae. All these calcium channel blockers

completely antagonized Ah vasoactivity in rat aortae. In

isolated human middle cerebral artery, Ah also stimulated

ET-1-induced vasoconstriction and this effect was fully

prevented by nilvadipine.

Recently, nilvadipine has been shown to increase the

CBF in ischaemic regions of the brains of patients affected

with both hypertension and chronic major cerebral artery

occlusion [23]. Moreover, nilvadipine has been reported to

have a selective effect on the cerebral artery [37] contrary to

other dihydropyridine calcium channel blocker such as

nifedipine which acts as a vasodilating agent resulting in

blood pressure reduction without affecting regional blood

flow in the brain [31].

We thus investigated the effects of a 2-week treatment

regime with nilvadipine in a transgenic mouse model of

AD (Tg APPsw line 2576). The Tg APPsw mice used for

this study develop a partial AD-like phenotype including

learning and memory deficits and pathological findings of

amyloid plaque deposition, increased Ah1–40 and Ah1–42

levels, gliosis, inflammatory responses, phosphorylated tau

epitopes, but not neurofibrillary tangles or neuronal loss

[10]. Laser Doppler imaging revealed a regional decrease

in resting CBF in 13 months Tg APPsw mice compared to

control littermates. No change in cortical CBF was notice-

able between control mice treated with nilvadipine and

control animals injected with vehicle only. However,

nilvadipine treatment increased the CBF in Tg APPsw

animals to values similar to those observed in control

animals. Similar results were observed in 20-month-old

Tg APPsw mice that were treated chronically for 7 months

with nilvadipine. The effects in the chronically treated

group were not due to the immediate effects of nilvadipine

treatment, which was discontinued 10 days before CBF

measurements were made. Our observation showing that

nilvadipine increases the CBF of Tg APPsw mice suggests

that calcium channel blockers might be beneficial in

dementia by restoring CBF in these patients. Impaired

CBF is likely to impede the optimal delivery of nutrients

and oxygen to neurons and glial cells as well as the

efficient removal of metabolic wastes. In laboratory ani-

mals, chronic hypoperfusion has been shown to lead to

memory impairment and capillary degeneration [4]. A

growing number of clinical studies have shown that CBF

reduction and hypometabolism correlate positively with

cognitive decline in AD patients [30]. However, it is still

a matter of debate whether regional hypometabolism and

hypoperfusion result from or precede the neurodegenera-

tive changes observed in AD. Resting CBF is reduced in

Tg APPsw and in another transgenic mouse model of AD

(line 2123) before the appearance of Ah deposits and

cognitive deficits [22], suggesting that CBF reduction

might precede the pathologic changes observed in these

mouse models of AD. All together, these data suggest that

the accumulation of soluble and insoluble Ah species in

the brain of these animals is sufficient to impair CBF.

Although it is not clear whether nilvadipine directly effects

CBF via the cerebrovasculature or indirectly impacts CBF

via cerebral metabolism or even the peripheral vasculature,

our data suggest that nilvadipine may be potentially useful

in the treatment of oligemia affecting AD patients.

Acknowledgements

We are grateful to Dr. Minoru Ohtsuka from Medicinal

Science Research, Fujisawa Pharmaceutical, for helpful

discussion and to Fujisawa Pharmaceutical, for providing

financial support for the completion of this study. We wish

to thank Mr. Bob and Mrs. Diane Roskamp for providing

additional support, which helped to make this work

possible.

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