Download Effects of Aniracetam on Bladder Overactivity in Rats with Cerebral

0022-3565/00/2933-0921$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
JPET 293:921–928, 2000 /2333/823035
Vol. 293, No. 3
Printed in U.S.A.
Effects of Aniracetam on Bladder Overactivity in Rats with
Cerebral Infarction
YASUSHI NAKADA,1 OSAMU YOKOYAMA, KAZUTO KOMATSU, KOICHI KODAMA, SATOSHI YOTSUYANAGI,
SUSUMU NIIKURA, YASUHIRO NAGASAKA, and MIKIO NAMIKI
Department of Urology, Kanazawa University School of Medicine, Kanazawa, Japan
Accepted for publication February 8, 2000
This paper is available online at http://www.jpet.org
Urinary frequency and incontinence are often observed in
patients with brain ischemia, because damage to the neural
circuitry in the forebrain can produce overactivity of the
urinary bladder and urinary incontinence (Tsuchida et al.,
1983; Kitada et al., 1991; Griffiths et al., 1994). It has been
reported in cats that decerebration above the inferior colliculus induces an overactive bladder (Tang and Ruch, 1956).
This overactivity has been attributed to the interruption of
inhibitory pathways from the forebrain to the micturition
center in the brainstem [i.e., the pontine micturition center
(PMC)]. An experimental model to study the effect of forebrain lesions on voiding function has recently been developed
in rat by occluding the middle cerebral artery (MCA) under
pentobarbital or halothane anesthesia (Ishiura, 1996;
Yokoyama et al., 1997, 1998, 1999). One-half hour after MCA
occlusion, bladder capacity in cerebral infarcted rats was
markedly reduced, indicating an overactive bladder
(Yokoyama et al., 1997).
In animals with an intact neuraxis, a supraspinal reflex
Received for publication November 29, 1999.
1
Present address: Research Laboratories, Toyama Chemical Co. Ltd.,
Toyama 930-8508, Japan.
cerebral infarcted rats but not in sham-operated rats. Aniracetam had no significant effect on bladder contraction pressure
or micturition threshold pressure in either sham-operated or
cerebral infarcted rats. Furthermore, i.c.v. administration of atropine (1 ␮g/rat), a muscarinic acetylcholine receptor antagonist, completely inhibited the enhancing effects of aniracetam
on bladder capacity in cerebral infarcted rats. The effects of
aniracetam on bladder overactivity are thought to be mediated
in part by activation of cholinergic inhibitory mechanisms in the
brain. These results indicate that aniracetam may improve the
neurogenic voiding dysfunction observed in patients with cerebrovascular disease.
pathway passing through the PMC in the brainstem initiates
micturition, and transmission in the PMC is modulated by
cortical-diencephalic mechanisms (Noto et al., 1989). Glutamate is the principal excitatory transmitter in the ascending
and descending limbs of the micturition reflex pathway (Yoshiyama et al., 1993). Other neurotransmitters that regulate
transmission in the micturition reflex pathway include
␥-aminobutyric acid (GABA), enkephalins, dopamine, and
acetylcholine (ACh). ACh has been found to have both excitatory and inhibitory effects on the micturition pathway (De
Groat, 1998). Anticholinergic (antimuscarinic) drugs are generally used for the treatment of urinary frequency and incontinence, but their influence on this pathway in the brain has
not been clarified. Recently, Yamamoto et al. (1995) reported
a decrease in bladder capacity in a rat model of cholinergic
denervation in the cerebral cortex. These results suggest that
the cholinergic pathway in the cerebral cortex plays an important role in the regulation of the micturition reflex. We
therefore speculated that the effect of a cholinergic activating
agent on the cerebral cortex would be capable of increasing
bladder capacity in cerebral infarcted rats.
Aniracetam is widely used for the treatment of a variety of
neuropsychiatric symptoms in patients suffering from cere-
ABBREVIATIONS: PMC, pontine micturition center; aCSF, artificial cerebrospinal fluid; AMPA/KA, ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate; CMG, cystometrography; ICA, internal carotid artery; ACh, acetylcholine; mAChR, muscarinic acetylcholine receptor; MCA,
middle cerebral artery; rCBF, regional cerebral blood flow.
921
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
ABSTRACT
Aniracetam has been used to improve the mental condition of
patients with cerebrovascular disease. Previous studies have
demonstrated that aniracetam activates the residual functions
of cholinergic neurons in damaged brain areas. In this study,
the effects of aniracetam on bladder overactivity after left middle cerebral artery occlusion were assessed through oral or
i.c.v. administration in sham-operated and cerebral infarcted
rats. Oral administration of aniracetam (100 and 300 mg/kg)
resulted in a significant and dose-dependent increase in bladder capacity in cerebral infarcted rats but had no effect on
bladder capacity in sham-operated rats. Intracerebroventricular
administration of aniracetam (0.25 and 2.5 ␮g/rat) resulted in a
significant and dose-dependent increase in bladder capacity in
922
Nakada et al.
Materials and Methods
Animals. The experiments were performed on 132 female
Sprague-Dawley rats weighing between 220 and 270 g (Japan SLC
Inc., Hamamatsu, Japan). They were housed at a constant temperature (23 ⫾ 2°C) and relative humidity (50 – 60%) under a regular
12-h light/dark schedule (lights on 7:00 AM–7:00 PM). Tap water and
standard rat chow were freely available. All experiments were performed in strict accordance with the guidelines of the Institutional
Animal Care and Use Committee (IACUC).
Cystometrography (CMG) in Conscious Rats. The method of
Yaksh and coworkers (1986) for cystometry in conscious rats was
used. Animals were anesthetized with halothane (2%), and the bladder was exposed via a midline incision in the abdomen. One end of a
polyethylene tube (size 4; i.d. 0.8 mm, o.d. 1.3 mm; Kunii Co. Ltd.,
Tokyo, Japan) was softened by heating to create a collar and passed
through a small incision at the apex of the bladder dome. A suture
was tied around the collar of the catheter and the other end of the
catheter was passed through the s.c. tissue and extracted through
the skin at the back of the neck. After suturing of the abdominal
skin, the rats were placed in a restraining cage (Ballman Cage,
KN-326 Type 3; Natsume Seisakusho Co. Ltd., Tokyo, Japan) and
allowed to recover from halothane anesthesia. The CMG catheter
was connected to a pump (TE-311; Terumo Co. Ltd., Tokyo, Japan)
for the continuous infusion of saline and to a pressure transducer
(TP-200T; Nihon-Kohden Co. Ltd., Tokyo, Japan) by means of a
polyethylene T-tube. Cystometric recording was achieved by infusing
physiological saline at room temperature into the bladder at a rate of
0.04 ml/min and by collecting and measuring the saline voided from
the urethral meatus to determine the voided volume. Evacuating the
bladder through the CMG catheter enabled us to measure residual
volume after the micturition reflex. Three cystometric parameters
(bladder capacity, bladder contraction pressure, and micturition
threshold pressure) were determined from each cystometry. Bladder
capacity was defined as the sum of the voided and residual volumes.
The protocol for the experiments is shown in Fig. 1.
Induction of Cerebral Infarction in Rats. Immediately after
completion of the first cystometric recording, the rats were anesthetized with halothane, and the left carotid bifurcation was exposed
through a midline incision in the neck. After ligation of the left
common carotid artery, the left internal carotid artery (ICA) was
isolated and carefully separated from the adjacent vagus nerve. The
pterygopalatine branch of the left ICA was then ligated close to its
origin. A 4-0 monofilament nylon thread with the tip rounded by
exposure to a flame was inserted into the left ICA and advanced a
distance of 17 mm from the carotid bifurcation as far as the origin of
the left MCA, where it occluded the blood flow and thus induced an
infarction on the left side of the brain (Longa et al., 1989). In shamoperated animals, the left carotid bifurcation was exposed through a
midline incision in the neck, but no additional procedures were
performed. These surgeries were performed within one-half hour
from induction of halothane anesthesia. After suturing of the incision
Fig. 1. Protocol for the experiments. Hatched columns show the recovery period from halothane anesthesia. After surgical implantation of the CMG
catheter (group 1), rats were recovered from anesthesia within one-half hour. After surgical implantation of the CMG catheter and i.c.v. cannula
(groups 2 and 3), rats were recovered from anesthesia within 1 h. After MCA occlusion or sham operation, rats were recovered from anesthesia within
one-half hour. The first and second CMG recordings were performed after rats regained consciousness.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
brovascular or Alzheimer’s disease, and its therapeutic effects on behavioral abnormalities and emotional disturbances have been demonstrated in two double-blind studies
with patients with cerebral infarction (Otomo et al., 1987,
1991). This drug is classified as a cholinergic and glutamatergic modulator because it enhances ACh release from the
hippocampus and glutamatergic transmission via ␣-amino3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate
(KA) receptors (Gouliaev and Senning, 1994). The identified
effects of aniracetam are limited to the central nervous system, and its influence on peripheral cholinergic neurotransmission has not been investigated (Himori et al., 1986).
Aniracetam can ameliorate overactivity of the bladder as
reported in some clinical cases (Ueda, 1998); however, there
are no critical studies focusing on the pharmacological and
action mechanisms of this drug in an experimental model of
overactive bladder. We therefore initiated this study to evaluate the effect of oral and i.c.v. administration of aniracetam
on bladder overactivity induced by MCA occlusion and to
determine its site of action.
Vol. 293
2000
Effects of Aniracetam on Bladder Overactivity
Results
Effect of Left MCA Occlusion on Cystometrogram.
We examined the effect of the MCA occlusion or the sham
operation on bladder capacity in the awake rats. The bladder
capacity before and 2 h after the sham operation was 0.44 ⫾
0.05 and 0.45 ⫾ 0.07 ml, respectively, and the corresponding
values for the occlusion were 0.50 ⫾ 0.05 and 0.24 ⫾ 0.03 ml.
Cerebral infarcted rats thus showed a significant reduction
in bladder capacity (P ⬍ .01 by two-tailed paired Student’s t
test). Residual volumes in cerebral infarcted and sham-operated rats were very small and insignificant, and the micturition volume was nearly equivalent to bladder capacity.
Bladder contraction pressure was 34.2 ⫾ 5.3 cm of water
before and 29.1 ⫾ 3.5 cm of water 2 h after the sham operation compared with 36.0 ⫾ 6.8 and 26.6 ⫾ 4.0 cm for the
occlusion (not significant). Bladder contraction pressure was
therefore not affected by the MCA occlusion.
Micturition threshold pressure was 4.56 ⫾ 0.60 cm of water before and 4.11 ⫾ 0.48 cm of water 2 h after the sham
operation compared with 5.33 ⫾ 0.83 and 3.10 ⫾ 0.48 cm for
the occlusion. The cerebral infarcted rats thus also showed a
significant decrease in micturition threshold pressure (P ⬍
.05 by two-tailed paired Student’s t test). On the other hand,
the micturition threshold pressure of the sham-operated rats
was not affected by the operation.
Effects of Oral Administration of Aniracetam on
Sham-Operated Rats and Cerebral Infarcted Rats.
Aniracetam increased bladder capacity in a dose-dependent
manner in cerebral infarcted rats (F3,24 ⫽ 7.92, P ⬍ .01 by
two-way ANOVA, Fig. 2B). Statistical analysis was based on
the percentage increase in bladder capacity at a dose of 300
mg/kg, which induced the maximum response. In the cerebral infarcted rats, the oral administration of aniracetam
significantly increased bladder capacity in comparison with
administration of the vehicle, with an increase in bladder
capacity of 34.3 ⫾ 15.3% for 100 mg/kg and of 40.9 ⫾ 11.6%
for 300 mg/kg 30 min after administration of aniracetam
doses (F3,24 ⫽ 4.81, P ⬍ .01 by two-way ANOVA, P ⬍ .01, and
P ⬍ .01 by Fischer’s protected least significant difference
test). In the sham-operated rats, the oral administration of
aniracetam did not significantly increase bladder capacity in
comparison with administration of the vehicle (F3,20 ⫽ 1.01,
P ⫽ 0.394 by two-way ANOVA, Fig. 2A). Any dose of aniracetam produced small and insignificant increases in the residual volume in both cerebral infarcted and sham-operated
rats.
In the sham-operated rats, the dose of 300 mg/kg aniracetam significantly reduced bladder contraction pressure
when compared with the effect of the vehicle (F3,20 ⫽ 6.49,
P ⬍ .01 by two-way ANOVA, P ⬍ .01 by Fischer’s protected
least significant difference test), but statistical analysis
based on percentage change in bladder contraction pressure
at a dose of 300 mg/kg did not show any significant change in
comparison with the bladder contraction pressure before the
administration (Fig. 2C). In the cerebral infarcted rats, the
dose of 300 mg/kg aniracetam showed similar results without
any significant change in comparison with the bladder contraction pressure before the oral administration (Fig. 2D).
Thus, aniracetam was found to have no influence on the
bladder contraction pressure in sham-operated and cerebral
infarcted rats. In neither the sham-operated nor the cerebral
infarcted rats did the effects of aniracetam on micturition
threshold pressures differ from those of the vehicle (Fig. 2, E
and F).
Effects of i.c.v. Administration of Aniracetam on
Sham-Operated Rats and Cerebral Infarcted Rats.
Aniracetam increased bladder capacity in a dose-dependent
manner in the cerebral infarcted rats (Table 1). Statistical
analysis was based on the percentage increase in bladder
capacity at a dose of 2.5 ␮g/rat, which induced the maximum
response. In the cerebral infarcted rats, the i.c.v. administra-
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
in the neck, the rats were placed in a restraining cage again and
allowed to recover from halothane anesthesia. The second cystometric recording was performed after identification of consciousness
recovery.
Intracerebroventricular Administration of Drug. The implantation of an injection tube into the intracerebroventricle was
performed immediately after implantation of the CMG catheter in
continuation of halothane anesthesia. The rats were positioned in a
stereotaxic frame (ST-7; Narishige Co. Ltd., Tokyo, Japan), the incisor bar was set at ⫺3.3 mm (Paxinos and Watson, 1986), a scalp
incision was made over the sagittal suture, and a hole (diameter
approximately 1.0 mm) was drilled in the right parietal bone to
expose the dural surface 1.0 mm lateral and 0 mm anterior from the
bregma (Paxinos and Watson, 1986). A sterile stainless steel cannula
(o.d. 0.6 mm, i.d. 0.3 mm, length 10.5 mm) was lowered 5.3 mm
ventrally from the skull surface with the end of a micromanipulator.
By using a small screw placed in the skull as an anchor, the cannula
was fixed to the skull with dental acrylic. The surgeries, including
implantation of the CMG catheter and injection tube into the intracerebroventricle, were performed within 1 h from induction of halothane anesthesia.
Evaluation of the Effects of Drug. The drug was administered
orally (5 ml/kg) or i.c.v. (5 ␮l/rat) as a single dose to the conscious
rats. Two hours after the left MCA occlusion or sham operation, the
effects of a single dose of aniracetam (30 –300 mg/kg p.o. or 0.025–2.5
␮g/rat i.c.v.), atropine [a muscarinic acetylcholine receptor (mAChR)
antagonist, 1 ␮g/rat i.c.v.], or vehicle [0.5% solution of methylcellulose or artificial cerebrospinal fluid (aCSF); 138.6 nM NaCl, 3.35 nM
KCl, 1.26 nM CaCl2, 1.16 nM MgCl2, 11.9 nM NaHCO3, pH 7.0 –7.2]
on bladder activity were examined in the awake rats after the control
cystometric recording. To examine the interactions between aniracetam and atropine, atropine (1 ␮g/rat) was i.c.v. administered 10 min
after the oral administration of aniracetam (100 mg/kg) in the cerebral infarcted rats. After the effects of the drugs had been evaluated,
the rats were euthanized by decapitation, and their brains were
removed. The forebrain was cut into five coronal slices, each 2-mm
thick, by using a Brain Matrix RBM-4000C (Activational Systems
Inc., Warren, MI), and immersed in a 2% solution of 2,3,5-triphenyltetrazolium chloride in saline at 37°C. After 15 to 30 min, the slices
were photographed and the area of ischemic lesion was quantified by
computer-assisted image analysis using NIH Image version 1.61.
The corrected infarction volume was then calculated with the
method of Golanov and Reis (1995).
Drugs. Aniracetam, 1-p-anisoyl-2-pyrrolidinone, was obtained
from Toyama Chemical Co., Ltd. (Tokyo, Japan). Atropine sulfate
was obtained from Sigma Chemical Co. (St. Louis, MO). These drugs
were of the highest purity commercially available. For the oral administration, aniracetam was suspended in 0.5% methylcellulose
solution. For the i.c.v. administration, the two drugs were dissolved
in distilled water and diluted with aCSF to adjust the concentration.
Data Analysis. Data are expressed as mean values ⫾ S.E. Statistical comparisons were performed by one-way or two-way ANOVA,
with subsequent individual comparisons by Fischer’s protected least
significant difference test. In some cases, two-tailed paired Student’s
t test was used. A level of P ⬍ .05 was considered statistically
significant.
923
924
Nakada et al.
tion of aniracetam significantly increased bladder capacity in
comparison with administration of the vehicle, with an increase in bladder capacity of 98.4 ⫾ 19.6% for 0.25 ␮g/rat and
of 122.4 ⫾ 41.1% for 2.5 ␮g/rat (F3,24 ⫽ 6.18, P ⬍ .01 by
one-way ANOVA, P ⬍ .05, and P ⬍ .01 by Fischer’s protected
least significant difference test). The bladder capacity with
i.c.v. administration of aniracetam reduced to the level of
vehicle-treated group at 60 min after administration. However, the percentage of control in bladder capacities of the
aniracetam-treated group was significantly increased at 120
min after administration for doses of 0.25 and 2.5 ␮g/rat
(F3,24 ⫽ 4.81, P ⬍ .01 by two-way ANOVA, P ⬍ .01, and P ⬍
.01 by Fischer’s protected least significant difference test,
Fig. 3B). In the sham-operated rats, the i.c.v. administration
of aniracetam did not significantly increase bladder capacity
in comparison with administration of the vehicle (F3,20 ⫽
0.77, P ⫽ .53 by one-way ANOVA, Table 1). The bladder
capacity with i.c.v. administration of aniracetam did not
show any response at 120 min after administration (F3,20 ⫽
0.67, P ⫽ .57 by two-way ANOVA, Fig. 3A). Effects of aniracetam on either bladder contraction pressure or micturition
threshold pressure in the sham-operated and cerebral infarcted rats did not differ from those of the vehicle (data not
shown).
Antagonistic Effects of Atropine on Aniracetam-Induced Increase in Bladder Capacity. To study the interactions between aniracetam and atropine, we administered a
fixed dose of atropine to construct a cumulative dose-response curve for aniracetam. This systemic administration
paradigm was necessary because atropine (1 ␮g/rat i.c.v.) at
the dose used here is known to have a protective effect on the
cholinergic neuronal activity in the brain. This dose was
selected because it did not cause any significant changes in
bladder capacity in the cerebral infarcted rats (Fig. 4). Single
doses of aniracetam (100 mg/kg) produced an increase in
bladder capacity that reached its maximum value within 30
min after oral administration and did not return to control
levels within 2 h (Fig. 2B).
For experiments designed to identify the pharmacological
interaction between aniracetam and atropine, atropine was
given 10 min after oral administration of aniracetam. Intracerebroventricular administration of aCSF after an oral administration of aniracetam resulted in a statistically significant increase in bladder capacity (F3,24 ⫽ 22.93, P ⬍ .01 by
two-way ANOVA, Fig. 4). In contrast, i.c.v. administration of
atropine completely nullified the aniracetam-induced increase in bladder capacity. There was a significant difference
in the percentage increase in bladder capacity between the
aniracetam ⫹ aCSF and the vehicle ⫹ aCSF groups (P ⬍ .01
by Fischer’s protected least significant difference test), and
between the aniracetam ⫹ aCSF and the aniracetam ⫹ atropine groups (P ⬍ .01 by Fischer’s protected least significant
difference test).
Effects of Aniracetam on Corrected Infarction Volume in Cerebral Infarcted Rats. As described by Longa et
al. (1989), MCA occlusion results in ischemic damage in the
frontal cortex, parietal cortex, striatum, and subcortical
basal ganglia. These areas showed the morphological characteristics of infarction during 7 h after MCA occlusion. Figure 5 shows the effect of aniracetam on the corrected infarction volume measured in terms of the area of ischemic
damage in the cerebral cortex and striatum at different stereotaxic levels. No significant differences in cerebral infarction volume were found between the vehicle- and aniracetamtreated groups, nor were any such differences found in the
study of the interactions between aniracetam and atropine.
Discussion
Focal neurogenic deficits often occur after an episode of
cerebral artery occlusion or hemorrhage, and voiding dysfunction is also common in patients with cerebrovascular
disease. The most common chronic expression of voiding dysfunction is a sense of urgency with or without urinary incontinence caused by an overactive bladder. Clinical investigations have also suggested that the frontal lobes and basal
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Fig. 2. Effects of oral administration of aniracetam on bladder capacity (A
and B), bladder contraction pressure (C and D), and micturition threshold
pressure (E and F) during continuous filling cystometry in sham-operated
(A, C, and E, n ⫽ 6 per group) and cerebral infarcted (B, D, and F, n ⫽ 7
per group) rats. “Pre” represents the control value recorded before the
MCA occlusion or sham operation (in the first CMG recording, see Fig. 1.).
Aniracetam was orally administered at single doses of 30 (F), 100 (Œ), and
300 mg/kg (⽧) for each group at 0 min. The vehicle-treated group (E)
orally received a 0.5% solution of methylcellulose alone. Values represent
the mean ⫾ S.E. of six to seven determinations. *P ⬍ .05; **P ⬍ .01;
significantly different from the vehicle-treated group.
Vol. 293
2000
Effects of Aniracetam on Bladder Overactivity
925
TABLE 1
Acute effects of i.c.v. administration of aniracetam on sham-operated and cerebral infarcted rats
Values represent the mean ⫾ S.E. of six to seven determinations.
Bladder Capacity
Dose
After
Administration
Control
␮g/rat
Sham-operated rats
Vehicle
Aniracetam
Cerebral infarcted rats
Vehicle
Aniracetam
% of Control
ml
0
0.025
0.25
2.5
0.512 ⫾ 0.087
0.509 ⫾ 0.060
0.471 ⫾ 0.070
0.488 ⫾ 0.050
0.437 ⫾ 0.078
0.313 ⫾ 0.043a
0.353 ⫾ 0.046
0.460 ⫾ 0.092
88.51 ⫾ 14.22
68.41 ⫾ 15.71
77.91 ⫾ 7.16
91.93 ⫾ 9.72
0
0.025
0.25
2.5
0.257 ⫾ 0.016
0.242 ⫾ 0.026
0.194 ⫾ 0.014
0.224 ⫾ 0.035
0.313 ⫾ 0.031
0.230 ⫾ 0.044
0.374 ⫾ 0.030b
0.437 ⫾ 0.068a
123.94 ⫾ 14.00
91.73 ⫾ 13.12
198.40 ⫾ 19.60c
222.36 ⫾ 41.12d
P ⬍ .05 compared with bladder capacity in control (two-tailed paired Student’s t test).
P ⬍ .01 compared with bladder capacity in control (two-tailed paired Student’s t test).
P ⬍ .05 compared with percentage of control in vehicle-treated group (Fischer’s protected least significant difference test) after one-way ANOVA.
d
P ⬍ .01 compared with percentage of control in vehicle-treated group (Fischer’s protected least significant difference test) after one-way ANOVA.
a
b
c
ganglia are involved in the inhibitory regulation of bladder
activity (Kitada et al., 1991; Griffiths et al., 1994). Impairment of this regulatory system after cerebral artery occlusion
or hemorrhage is likely to be responsible for the development
of an overactive bladder (Tsuchida et al., 1983). Disruption of
the forebrain seems to be involved in the overactive bladder
associated with cerebrovascular disease. Patients with an
overactive bladder have been treated with anticholinergic
agents, which suppress the detrusor muscle at the peripheral
neuromuscular junction. Although anticholinergic agents are
useful for the suppression of overactivity of the bladder, the
effect is not always satisfactory and some patients suffer
from side effects such as dry mouth, flushing, and a small
urinary stream.
To study the mechanisms underlying neurogenic bladder
overactivity, we have developed an animal model of voiding
dysfunction induced by experimental cerebral infarction (Ishiura, 1996; Yokoyama et al., 1997). This model has proved to
be useful for the quantitative evaluation of drug actions on
micturition reflex. Aniracetam, a nootropic drug, can ameliorate urinary frequency and nocturia as reported in some clinical
cases (Ueda, 1998); however, there are no critical studies focusing on the pharmacological and action mechanism of this drug
in an experimental model of voiding dysfunction. Therefore, our
experimental model was used to investigate the effects of
aniracetam on the overactive bladder.
Our study showed that oral and i.c.v. administration of
aniracetam significantly increased the bladder capacity in
the cerebral infarcted rats. In the sham-operated rats, however, no significant change was seen in the bladder capacity
between the vehicle- and aniracetam-treated groups. The site
of action of aniracetam is believed to be the forebrain, which
influences the micturition reflex, because aniracetam appears to have no peripheral side effects in general pharmacological terms (Himori et al., 1986) and was found to specifically bind to the cortical membrane (Fallarino et al., 1995).
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Fig. 3. Effects of i.c.v. administration of aniracetam on bladder capacity in sham-operated (A, n ⫽ 6 per group) and cerebral infarcted (B, n ⫽ 7 per
group) rats. Aniracetam was i.c.v. administered at single doses of 0.025 (F), 0.25 (Œ), and 2.5 ␮g/rat (⽧) for each group at 0 min. The vehicle-treated
group (E) received i.c.v. administration of aCSF alone. Values represent the mean ⫾ S.E. of six to seven determinations. *P ⬍ .05; **P ⬍ .01;
significantly different from the vehicle-treated group.
926
Nakada et al.
Previous reports showed that aniracetam improved dysfunction of the cholinergic systems in the brain (Spignoli and
Pepeu, 1987; Toide, 1989). However, aniracetam did not interact, either as a receptor agonist or an antagonist, with
cholinergic receptors (Martin and Haefely, 1993). Aniracetam has a modulating effect on the presynaptic terminal of
the cholinergic neuron, resulting in an increase in ACh release (Giovannini et al., 1993). Based on the effect of aniracetam on cholinergic activation, it is used as a cognitive enhancer in animals (Cumin et al., 1982) and for human
neuropsychiatric disorders (Frostl and Maitre, 1989; Senin et
al., 1993). Aniracetam is also classified a glutamatergic modulator because it enhances glutamatergic transmission via
the AMPA/KA receptors (Gouliaev and Senning, 1994). Positive modulation of the AMPA/KA receptors by aniracetam
provides a molecular substrate, which explains the second
clinical effect of the drug, namely, as a memory and cognition
enhancer.
What accounts for the inhibitory effects of aniracetam on
the overactive bladder after cerebral infarction? In previous
reports we suggested that there were two glutamatergic
pathways that regulate bladder activity (Yokoyama et al.,
1997). The first is an inhibitory pathway that originates from
the forebrain, and the second is an excitatory pathway that is
located at a more caudal site, possibly in the brainstem or in
the spinal cord. Bladder overactivity after cerebral infarction
is reportedly related to the activation of glutamatergic excitatory input to the micturition reflex pathway mediated by
N-methyl-D-aspartate receptors (Yokoyama et al., 1997). Furthermore, glutamatergic mechanisms controlling the micturition reflex have been reported to be modulated by dopaminergic systems (Yoshiyama et al., 1994; Yokoyama et al.,
1999). Therefore, other neurotransmitter systems besides the
dopaminergic ones may interact with these glutamatergic
systems. Recent studies have shown that muscarinic activation inhibited the release of glutamate in the corticostriatal
pathway (Niittykoski et al., 1999). Cholinergic neurons may
thus influence the glutamatergic pathways that regulate the
micturition reflex in the brain.
We reported that a selective AMPA/KA receptor antagonist,
1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide, increased bladder capacity in cerebral
infarcted rats (Mizuno et al., 1999). These effects were the
opposite of the glutamatergic modulating effects of aniracetam. In this connection the AMPA/KA receptor modulating
effects by aniracetam needed a high concentration (0.1–5
mM) to become noticeable, according to previous reports
(Gouliaev and Senning, 1994). We interpret this as meaning
that the sensitivity of the AMPA/KA receptor-modulating
effects of aniracetam is lower than that of its cholinergic
activating effects. The cholinergic activating effects of aniracetam may play a more dominant role than the AMPA/KA
receptor-modulating effects in ameliorating bladder overactivity in cerebral infarcted rats.
In this study, the effect of aniracetam (100 mg/kg p.o.) on
bladder overactivity was completely suppressed by the i.c.v.
administration of atropine (1 ␮g/rat) at a dose that had no
effect on bladder activity in cerebral infarcted rats. This
indicates that aniracetam activates cortical ACh release, and
that cholinergic systems in the frontal cortex recover the
regulatory influence on the micturition reflex. Yamamoto et
al. (1995) reported a decrease in bladder capacity in a rat
model of cholinergic denervation in the central nervous system. This model was prepared by inducing severe neuronal
damage to the nucleus basalis magnocellularis in the basal
forebrain, which resulted in a reduction in choline-acetyltransferase activity in the prefrontal and sensorimotor cortices. Cholinergic systems in the cortex are thought to be one of
the inhibitory pathways that regulate bladder activity, so
that impairment of this regulatory system after cerebral
infarction is very likely responsible for the development of
the overactive bladder. The quantitative analysis of mAChR
and subtype mRNA in the acute phase of a MCA occlusion
model demonstrated a tendency toward a reduction in mRNA
levels in the acute ischemic region without a reduction in
mAChR (Kuji et al., 1997). These results indicate that the
cholinergic neurons in the ischemic regions are viable but
hypometabolic. Therefore, the ameliorating effects of aniracetam could be based on the activation of the cholinergic
inhibitory pathways in the frontal cortex.
Aniracetam has been reported to facilitate central cholinergic neurotransmission via an increase in ACh release (Giovannini et al., 1993). In other investigations, acetylcholinesterase inhibitors increased regional cerebral blood flow
(rCBF) in the bilateral cortices of rats subjected to unilateral
MCA occlusion (Scremin and Scremin, 1986). These findings
raise the possibility that aniracetam induces cerebral vasodilation by means of activation of cholinergic neurons. The
effect of vasodilator agents such as nifedipine to suppress
bladder overactivity after MCA occlusion implies the importance of rCBF restoration in the ischemic penumbra (Nakamura et al., 1999). However, the influence of aniracetam on
rCBF has not been studied in an ischemic animal model. It is
uncertain whether aniracetam ameliorates the rCBF in the
ischemic penumbra or not.
Other investigators indicated the neuroprotective property
of aniracetam by attenuating hydroxyl free-radical formation
in the ischemic brain (Himori et al., 1995). This study was
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Fig. 4. Antagonistic effects of atropine (ATR) on aniracetam-induced
increase in bladder capacity in cerebral infarcted rats (n ⫽ 7 per group).
ATR (1 ␮g/rat) or aCSF was i.c.v. administered 10 min after oral administration of aniracetam (100 mg/kg) or vehicle. This experiment involved
the following four groups: vehicle ⫹ aCSF (E), vehicle ⫹ ATR (F), aniracetam ⫹ aCSF (‚), and aniracetam ⫹ ATR (Œ). Values represent the
mean ⫾ S.E. of seven determinations. **P ⬍ .01; significantly different
from vehicle ⫹ aCSF group. ##P ⬍ .01; significantly different from aniracetam ⫹ ATR group.
Vol. 293
2000
Effects of Aniracetam on Bladder Overactivity
927
performed in the acute phase of focal ischemia, and the
neuroprotective effect of aniracetam could not be verified. No
significant differences in the volume of the cerebral infarct
were found between the vehicle- and aniracetam-treated
groups.
It has been reported that aniracetam has modulating effects on neurotransmitter systems and neuroprotective effects in the brain, but these effects have not been clarified to
be dependent on direct or indirect action (Gouliaev and Senning, 1994; Himori et al., 1995). Nakamura et al. (1998)
suggested that aniracetam indirectly induced the activation
of dopamine release in the striatum by interacting with glutamatergic systems. D1 dopaminergic mechanism has inhibitory influence on the micturition reflex (Yokoyama et al.,
1999). The possibility exists that aniracetam produces an
increase in bladder capacity in cerebral infarcted rats via
activation of D1 dopaminergic system.
In conclusion, oral or i.c.v. administration of aniracetam, a
cholinergic modulator, significantly increased bladder capacity in cerebral infarcted rats but not in sham-operated rats.
This enhancing effect was completely inhibited by mAChR
antagonist. It seems reasonable to conclude that the cholinergic pathway in the cerebral cortex has a role in this inhibitory influence on the micturition reflex. Aniracetam has no
side effects, such as dry mouth, flushing, and small urinary
stream, often seen in the clinical use of anticholinergic
agents for the overactive bladder. Therefore, aniracetam may
be expected to be beneficial for the treatment of the overactive bladder caused by cerebrovascular diseases.
References
Cumin R, Bandle EF, Gamzu E and Haefely WE (1982) Effects of novel compound
aniracetam (Ro-13-5057) upon impaired learning and memory in rodents. Psychopharmacology 78:104 –111.
De Groat WC (1998) Anatomy of the central neural pathways controlling the lower
urinary tract. Eur Urol 34 (Suppl 1):2–5.
Fallarino F, Genazzani AA, Silla S, L’Episcopo MR, Camici O, Corazzi L, Nicoletti F
and Fioretti MC (1995) [3H]Aniracetam binds to specific recognition sites in brain
membranes. J Neurochem 65:912–918.
Frostl W and Maitre L (1989) The families of cognition enhancers. Pharmacopsychiatry 22:54 –100.
Giovannini MG, Rodino P, Mutolo D and Pepeu G (1993) Oxiracetam and aniracetam
increase acetylcholine release from the rat hippocampus in vivo. Drug Dev Res
28:503–509.
Golanov EV and Reis DJ (1995) Contribution of cerebral edema to the neuronal
salvage elicited by stimulation of cerebellar fastigical nucleus after occlusion of the
middle cerebral artery in rat. J Cereb Blood Flow Metab 15:172–177.
Gouliaev AH and Senning A (1994) Piracetam and other structurally related nootropics. Brain Res Rev 19:180 –222.
Griffiths DJ, Mccracken PN, Harrison GM, Gormley EA, Moore K, Hooper R, Mcewan AJB and Triscott J (1994) Cerebral aetiology of urinary urge incontinence in
elderly people. Age Ageing 23:246 –250.
Himori N, Suzuki T and Ueno K (1995) Aniracetam, a pyrrolidinone-type cognition
enhancer, attenuates the hydroxyl free radical formation in the brain of mice with
brain ischemia. J Pharm Pharmacol 47:253–258.
Himori N, Watanabe H, Matsuura A, Umeda Y, Kuwahara T, Takemoto C, Takamiya M, Yajima T, Tanaka Y and Nakamura K (1986) General pharmacological
properties of aniracetam, a cerebral insufficiency improver. Jpn Pharmacol Ther
14 (Suppl 4):93–128.
Ishiura Y (1996) Experimental study of voiding dysfunction induced by cerebral
infarction in rats. Jpn J Urol 87:1221–1230.
Kitada S, Ikei Y, Hasui Y, Yamaguchi T, Nishi S and Osada Y (1991) Bladder
function in elderly males with subclinical MRI lesions. Neurourol Urodyn 10:436 –
437.
Kuji I, Matsuda H, Sumiya H, Taki J, Tsuji S, Kinuya K, Ichikawa A, Shiba K, Mori
H and Tonami N (1997) Discrepancy between blood flow and muscarinic receptor
distribution in rat brain after middle cerebral artery occlusion. Eur J Nucl Med
24:665– 669.
Longa EZ, Weinstein PR, Carlson S and Cummins R (1989) Reversible middle
cerebral artery occlusion without craniotomy in rats. Stroke 20:84 –91.
Martin JR and Haefely WE (1993) Pharmacology of aniracetam, a novel pyrrolidinone derivative with cognition enhancing activity. Drug Invest 5 (Suppl 1):4 – 49.
Mizuno H, Komatsu K, Ishiura Y, Yokoyama O and Namiki M (1999) Role of NMDA
and AMPA/kainate receptor channels in the prevention of bladder overactivity
after cerebral infarction in rats. J Urol 161 (Suppl):45.
Nakamura K, Kurasawa M and Tanaka Y (1998) Apomorphine-induced hypoattention in rats and reversal of the choice performance impairment by aniracetam. Eur
J Pharmacol 342:127–138.
Nakamura Y, Yokoyama O, Komatsu K, Mita E, Namiki M and Kontani H (1999)
Effects of nifedipine on bladder overactivity in rats with cerebral infarction. J Urol
162:1502–1507.
Niittykoski M, Ruotsalainen S, Haapalinna A, Larson J and Sirviö J (1999) Activation of muscarinic M3-like receptors and ␤-adrenoceptors, but not M2-like muscarinic receptors or ␣-adrenoceptors, directly modulates corticostriatal neurotransmission in vitro. Neuroscience 90:95–105.
Noto H, Roppolo JR, Steers WD and De Groat WC (1989) Excitatory and inhibitory
influences on bladder activity elicited by electrical stimulation in the pontine
micturition center in the rat. Brain Res 492:99 –115.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Fig. 5. Effects of aniracetam on corrected infarction volume in cerebral infarcted rats. Corrected infarction volume was calculated by measuring the
area of ischemic lesions in the coronal sections of the brain. Aniracetam was administered orally (A) or i.c.v. (B). The result of the experiment to study
the interactions between aniracetam and atropine (ATR) in cerebral infarcted rats is shown in C. ATR (1 ␮g/rat) or aCSF was i.c.v. administered 10
min after oral administration of aniracetam (100 mg/kg) or vehicle. This experiment involved the following four groups: vehicle ⫹ aCSF, vehicle ⫹
ATR, aniracetam ⫹ aCSF, and aniracetam ⫹ ATR. Values represent the mean ⫾ S.E. of seven determinations.
928
Nakada et al.
study of bladder function and effect of anesthetics. Am J Physiol 251:R1177–
R1185.
Yamamoto T, Koibuchi Y, Miura S, Sawada T, Ozaki R, Esumi K and Ohtsuka M
(1995) Effects of vamicamide on urinary bladder functions in conscious dog and rat
models of urinary frequency. J Urol 154:2174 –2178.
Yokoyama O, Ishiura Y, Komatsu K, Mita E, Nakamura Y, Kunimi K, Morikawa K
and Namiki M (1998) Effects of MK-801 on bladder overactivity in rats with
cerebral infarction. J Urol 159:571–576.
Yoshiyama M, Roppolo JR and De Groat WC (1993) Effects of MK-801 on the
micturition reflex in the rat–possible sites of action. J Pharmacol Exp Ther
265:844 – 850.
Yoshiyama M, Roppolo JR and De Groat WC (1994) Interactions between glutamatergic and monoaminergic systems controlling the micturition reflex in the urethane-anesthetized rat. Brain Res 639:300 –308.
Yokoyama O, Yoshikawa M, Namiki M and De Groat WC (1999) Glutamatergic and
dopaminergic contributions to rat bladder hyperactivity after cerebral artery occlusion. Am J Physiol 276:R935–R942.
Yokoyama O, Yoshiyama M, Namiki M and De Groat WC (1997) The influence of
anesthesia on bladder hyperactivity induced by middle cerebral artery occlusion in
the rat. Am J Physiol 273:R1900 –R1907.
Send reprint requests to: Yasushi Nakada, Dept. of Urology, Kanazawa
University School of Medicine, 13-1 Takara-machi Kanazawa Ishikawa 9208641 Japan. E-mail: [email protected]
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Otomo E, Hirai S, Hasegawa K, Araki G, Nishimura T and Inanaga K (1987)
Usefulness of aniracetam for cerebrovascular disorders: Double-blind trial against
Ca hopantenate. J Clin Exp Med (Igaku No Ayumi) 140:989 –1016.
Otomo E, Hirai S, Terashi A, Hasegawa K, Tazaki Y, Araki G, Itoh E, Nishimura T
and Furukawa T (1991) Clinical usefulness of aniracetam for psychiatric symptoms in patients with cerebrovascular disorders: Double-blind trial against placebo. Igaku No Ayumi 156:143–187.
Paxinos G and Watson C (1986) The Rat Brain in Stereotaxic Coordinates, 2nd ed.,
Academic Press, London.
Scremin OU and Scremin AME (1986) Physostigmine induced reversal of ischemia
following acute mild cerebral artery occlusion in the rat. Stroke 17:1004 –1009.
Senin U, Parnelti L, Cucinotta D, Criscuolo D, Longo A and Marini G (1993) Clinical
experience with aniracetam in the treatment of senile dementia of the Alzheimer’s
type and related disorders. Drug Invest 5 (Suppl 1):96 –105.
Spignoli G and Pepeu G (1987) Interactions between oxiracetam, aniracetam and
scopolamine on behavior and brain acetylcholine. Pharmacol Biochem Behav 27:
491– 495.
Tang PC and Ruch TC (1956) Localization of brain stem and diencephalic areas
controlling the micturition reflex. J Comp Neurol 106:213–245.
Toide K (1989) Effects of aniracetam on one-trial passive avoidance tests and cholinergic neurons in discrete brain regions of rats. Arch Int Pharmacodyn Ther
298:25–37.
Tsuchida S, Noto H, Yamaguchi O and Itoh M (1983) Urodynamic studies on
hemiplegic patients after cerebrovascular accident. Urology 21:315–318.
Ueda T (1998) Improvement of urinary frequency and nocturia by aniracetam in two
cases with cerebral infarction. Pharma Medica 16:129 –133.
Yaksh TL, Durant PAC and Brent CR (1986) Micturition in rats: A chronic model for
Vol. 293