| Accepted: 24 August 2022

DOI: 10.1002/nau.25043

BASIC SCIENCE ARTICLE

Influence of cerebral infarction on both bladder andurethral activities and changes after tramadol
administration in rats
Satoko Nagamine1| Tadanobu Chuyo Kamijo PhD1|
2
Asuka Ashikari MD, PhD| Minoru Miyazato MD, PhD1
1

Department of Systems Physiology,
Graduate School of Medicine, Universityof the Ryukyus, Nishihara, Okinawa,
Japan

2

Department of Urology, Graduate Schoolof Medicine, University of the Ryukyus,
Nishihara, Okinawa, Japan
Correspondence
Minoru Miyazato, MD, PhD, Department
of Systems Physiology, Graduate Schoolof Medicine, University of the Ryukyus,
207 Uehara, Nishihara 903‐0215,
Okinawa, Japan.
Email: miyaz929@med.u-ryukyu.ac.jp
Funding information
Japan Society for the Promotion of
Science

Abstract
Aims: We investigated the changes in bladder and urethral function after
cerebral infarction (CI) and the influence of tramadol on these functions.
Methods: Twenty‐eight female Sprague Dawley rats were divided into normal
and CI groups. In the awake condition, metabolic cage study and bloodpressure were evaluated. Under urethane anesthesia, the intravenous effect oftramadol (0.01–1 mg/kg), which has both µ‐opioid receptor stimulation andinhibition of norepinephrine and serotonin reuptake, on continuous
cystometry, and simultaneous measurements of bladder and urethralperfusion pressure (UPP) were recorded. Infarcted lesions were examined bystaining with triphenyltetrazolium chloride, a marker of mitochondrial
enzyme activity.
Results: CI rats showed impaired sympathetic activity with Horner's
syndrome and lower blood pressure. In metabolic cage study, urinary
frequency during the dark phase was increased in CI rats. On bladder activity,
in CI rats, the baseline pressure threshold for inducing bladder contractions
was significantly lower (p < 0.01), and the intercontraction interval was
prolonged after tramadol administration. On urethral activity, the baseline
UPP was significantly lower in CI rats than in normal rats and it did notchange after tramadol administration. Residual urine rate was significantly
increased in normal rats, but not in CI rats. CI rats showed brain infarction
including the cortex and hypothalamus, which is a center of the autonomic
nervous system.
Conclusions: CI‐induced ischemic brain damage results in impairment ofboth bladder and urethral functions, in addition to decreased sympathetic
activity. Bladder overactivity after CI can be improved by tramadol; however,urethral activity cannot be improved by it.
KEYWORDS

bladder, infarction, stress urinary incontinence, sympathetic activity, urethra

Neurourol Urodyn. 2022;41:1679–1691.

wileyonlinelibrary.com/journal/nau© 2022 Wiley Periodicals LLC.

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Received: 16 June 2022

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NAGAMINE ET AL.

INTRODUCTION

The micturition function consists of two phases: thestorage phase, in which urine is collected in the bladder,and the voiding phase, in which urine is drained throughthe urethra. These coordinated actions are intricately
controlled not only through the peripheral nervous
system (PNS, parasympathetic, sympathetic, andsomatic) but also through the central nervous system (CNS).1
Disorders in the urinary storage phase include
overactive bladder accompanied by detrusor overactivity(DO) and stress urinary incontinence (SUI), whereas in
the voiding phase, it includes an underactive bladderwith residual urine.2 These lower urinary tract symptoms
impair the quality of life.3,4 Numerous neurologicaldeficits, such as cerebral infarction (CI), damage neuralcontrol of the lower urinary tract.5 In our recent humanstudy, 23.5% of chronic stroke patients reported nocturia;
9.8%, urgency incontinence; and 17.6%, SUI.6 Moreover,previous studies have reported that half and one‐third ofacute and chronic CI patients, respectively, have overactive bladder symptoms.3,7 However, CI pathophysiology in the reciprocal function of the bladder andurethra has not been completely elucidated.
Glutamic acid, the major excitatory neurotransmitterin the CNS, facilitates bladder voiding function; whereas,
noradrenaline and serotonin enhance the urethralcontinence reflex.8–10 In our previous study using
microtip‐transducer catheter methods in CI rats, theurethral baseline pressure was lower than that in normal
rats, and it did not change after intravenous administration of duloxetine, a norepinephrine‐ and serotonin‐
reuptake inhibitor.11 Among the CI patients, 28%–79%
experienced urinary incontinence associated with DO.12
These results suggest that urinary incontinence after CI
has an SUI component (i.e., impaired urethral continence mechanism) and DO in relation to bladder storage
function. However, few studies have examined thechanges in urethra after CI. Recently, we reported that
intravenous administration of tramadol, which has dual
effects of µ‐opioid receptor stimulation and inhibition of
norepinephrine and serotonin reuptake, enhanced both
sympathetic innervated urethral smooth muscle andbrain stem‐derived external urethral sphincter continence reflexes in normal rats.13 Tramadol has an
inhibitory effect on DO in CI rats.14 Thus, tramadol has
dual effects on bladder and urethral functions. In thisstudy, we investigated the changes in bladder andurethral function by continuous cystometry and simultaneous recordings of these functions after CI and theintravenous administration of tramadol in both normal
and CI rats. In addition, to confirm the ischemic changes

and the possibility of impaired projection to thesympathetic pathway and lower urinary tract after CI,
2,3,5‐triphenyltetrazoliumchloride (TTC; a marker ofmitochondrial enzyme activity) staining in the brain
and heart rate and blood pressure as sympathetic activity
were evaluated.

2 | MATERIALS AND METHODS
2.1 | Animals
A total of 28 female Sprague Dawley rats (12‐week‐old
normal and CI rats) were used in this study. Experimental protocols were approved by the Institutional
Animal Care and Use Committee of the University of the
Ryukyus (Protocol No. A2019234) in compliance with
ARRIVE guideline (https://arriveguidelines.org/).

2.2 | Cerebral infarction
CI was performed as previously described.10 Briefly,
under 2% isoflurane anesthesia, the right carotid
bifurcation was exposed through a paramedian incisionon the neck. The distal side of the common carotid arteryand external carotid artery close to bifurcation wereligated. A 4‐0 monofilament nylon thread with the tip
rounded by a flame was introduced into the internal
carotid artery and advanced 17 mm from the carotid
bifurcation to the arterial circle of Willis. Experiment 1
was performed 2 days after CI induction, followed byexperiments 2 and 3 days after CI induction.

2.3 | Surgical procedures
In experiments 2 and 3 with the rat under 2% isoflurane,a polyethylene catheter (PE‐10, Clay Adams) was
inserted in the jugular vein for tramadol injection.
Through an abdominal incision, the ureters were incisedbilaterally; then, a PE‐90 catheter (Clay Adams) was
inserted into the bladder through the dome. In experiment 3, the bladder neck was tied to allow functional
separation of bladder and urethral activities. A PE‐50
catheter (Clay Adams) was inserted from the urethra
to record urethral perfusion pressure (UPP). Afterthese procedures, isoflurane anesthesia was turned
off and replaced with 1.0 g/kg of intraperitoneal andsubcutaneous urethane (Sigma‐Aldrich Co.). After
abdominal closure, catheters were connected to apressure transducer (Transbridge 4M; World Precision
Instruments).

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2.4 | Experiment 1: Metabolic cagestudy and measurements of blood pressure
Six normal and six CI rats were placed in a metabolic
cage (MATABOLICA, Sugiyama‐gen Co. Ltd.) for 24 h toevaluate their voiding behavior. The room was maintained at standard temperature (24°C–26°C) and humidity (37%–40%) with a 12:12‐h light‐dark cycle (lighting at
8 a.m.). Food and water were provided ad libitum, andeach animal was free to move around in the cage. The
mean urine volume and the number of voidings wererecorded in light and dark cycles separately. Twenty‐
four‐hour urine output was estimated. Mean urine
volume was calculated as [total urine volume (g)/numberof voidings]. Furthermore, noninvasive measurements ofheart rate and systolic and diastolic blood pressures were
performed using the tail‐cuff method (MK‐2000MU,
Muromachi Kikai Co., Ltd.) before and after metabolic
caging.

2.5 | Experiment 2: Effects of
intravenous tramadol on continuous
cystometry in normal and CI rats
In eight normal and nine CI rats (including those inexperiment 1), bladder activity was monitored by the
intravesical PE‐90 catheter under urethane anesthesia.
Physiological saline at room temperature was infused
into the bladder at a rate of 0.05 ml/min. A Power Lab(AD Instruments Pty, Ltd.) was used for data acquisition
and manipulation. Cystometry was continued for at least
120 min, and the maximum voiding contraction (MVC),
intercontraction interval (ICI), bladder pressure forthreshold (PT), and baseline pressure (BP) were evaluated during the final 30 min as the predrug control values(Figure 1A).
The number and amplitude of nonvoiding contractions (NVCs) were measured at 1–2 min before eachvoiding contraction (Figure 1A, shaded area). NVCs were
defined as contractions of >4 cmH2O from BP, occurring
during the filling phase. Thereafter, tramadol was
injected intravenously and cumulatively administeredat doses of 0.01, 0.1, and 1 mg/kg. Bladder activity wasrecorded for approximately 30 min after each concentration of drug was administered and compared to controls.
Additionally, the voiding volume (VV) before and after
each drug administration was measured, and the residual
urine rate (%) was calculated as [residual urine volume(ml)/VV (ml) + residual urine volume (ml)] × 100 after
single cystometry at the final dose.

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2.6 | Experiment 3: Effects of
intravenous tramadol on simultaneousrecordings of intravesical pressure and
UPP in normal and CI rats
To confirm the urethral activity concomitant withbladder activity before and after CI/tramadol, simultaneous recordings of intravesical pressure and UPP were
performed. In five normal and six CI rats (including
those from experiment 1), the bladder was infused withphysiological saline at a rate of 0.05 ml/min to exceed thethreshold volume, inducing isovolumetric bladder contractions. A urethral catheter was continuously infusedwith 0.05 ml/min of physiological saline. Under stabilized bladder contractions, the amplitude of the isovolumetric contractions and the intravesical pressure threshold for inducing urethral relaxation were measured. UPP
nadir during reflex urethral relaxation, baseline UPP
between reflex bladder contractions, and UPP change,
calculated as the difference between the UPP nadir andthe baseline UPP, were measured. Moreover, the mean
rate and amplitude of high‐frequency oscillations (HFOs)of the urethral striated muscle during reflex bladdercontractions were measured (Figure 1B). These parameters were averaged over 30 min and used as controls.
Following a control period, the rats were injected
intravenously with tramadol (1 mg/kg) at the final doseused in experiment 1. Bladder and UPP parameters after
tramadol were averaged and compared with thepretramadol‐treatment control values. To evaluate
the effect of tramadol on the urethral basal tone, Δ
baseline UPP ratio, calculated as (baseline UPP after
tramadol minus baseline UPP before tramadol/baseline
UPP before tramadol), was estimated.

2.7 | Experiment 4: Evaluation of theischemic damage
After experiments 1 and 2, normal rats (n = 4) and CI rats
(n = 4) were anesthetized with additional intraperitoneal
pentobarbital (50 mg/kg; NAKALAI TESQUE, Inc.) and
immediately decapitated to remove brain tissue. The
brains were immediately frozen in ice‐cold acetone(FUJIFILM Wako Pure Chemical Co., Ltd.) at −80°C
until further processing. A 2‐mm coronal brain sliceusing brain matrices (EM Japan Co., Ltd.) was used tomake six sections for each tissue. Staining with 2% TTC(Sigma‐Aldrich), a marker of mitochondrial enzyme
activity, was conducted with 20‐min immersion at room
temperature. After washing, the TTC‐stained brain slices

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NAGAMINE ET AL.

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F I G U R E 1 (A) Evaluation of continuous cystometry. (B) Evaluation of simultaneous recording of intravesical pressure and UPP underisovolumetric conditions. HFOs, high‐frequency oscillations; NVCs, nonvoiding contractions; UPP, urethral perfusion pressure

were scanned for examination of the size and location of
the CI with an image analysis software (ImageJ, National
Institutes of Health, Bethesda, MD, USA).

saline to a concentration of 2%. Both drugs were diluted on
the day of the experiment or before it.

2.9 | Statistical analysis
2.8| Drugs and assessment of infarction

Tramadol was dissolved in physiological saline and administered at 0.01, 0.1, and 1 mg/kg at a volume of 0.1 ml/rat. The
dosage was determined based on our previous report14 andpreliminary experiments. TTC was diluted in physiologicalThe data were expressed as mean ± standard error,
which was calculated for each group of animals fromthe average value of each rat before or after tramadol.
Wilcoxon signed‐rank test was used to compare thesedata before and after tramadol administration. The

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nonparametric Mann–Whitney U test with Bonferroni
correction for multiple comparisons was used forcomparison of normal and CI rats, with threshold ofstatistical significance at p < 0.05. All statistical analyses
were two‐sided and performed using GraphPad Prism 8
for Windows (GraphPad Software).

3 |RESUL TS

3.1

| Animals

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during light phase. The 24‐h urine volume did not differ
between normal and CI rats.
There was no significant difference in the heart rate
between the normal and CI rats (Table 1). However,T A B L E 1 Comparisons in voiding behaviors and sympathetic
activity between normal and CI rats
Normalrats (n = 6)

CI rats (n = 6)

24 h0.76 ± 0.06

0.54 ± 0.06

light phase

0.80 ± 0.08

0.60 ± 0.06

dark phase

0.75 ± 0.06

0.53 ± 0.10

24 h

22.6 ± 0.2

38.2 ± 0.0*

light phase

8.3 ± 0.8

12.8 ± 0.1

dark phase

14.4 ± 1.6

25.2 ± 0.2*

16.8 ± 4.7

20.2 ± 10.1

heart rate (bpm)

426.4 ± 7.9

420.8 ± 6.5

systolic bloodpressure (mmHg)

141.8 ± 3.6

112.3 ± 6.2**

diastolic bloodpressure (mmHg)

82.7 ± 3.4

58.0 ± 3.0**

Metabolic cage study
voiding volume per time (g)Ptosis and drooping of the right upper eyelid wereobserved in all the CI rats (Figure 2). CI rats leaned
towards the left paralytic side. No significant difference
in body weight was found between normal and CI rats
(248.4 ± 3.6 g vs. 239.8 ± 2.5 g, respectively; p = 0.054).

3.2 | Experiment 1: Voiding behaviors
and sympathetic activity

number of voiding (times)

24‐h urine output (g)In the metabolic cage study, the mean voided volume
had a tendency to decrease in CI rats compared withnormal rats during either the light (p = 0.113) or darkcycle (p = 0.106) (Table 1).
The number of voiding was significantly increasedin CI rats than in normal rats during dark phase(25.2 ± 0.2 vs. 14.4 ± 1.6 times, respectively, p = 0.014)
and during 24 h (38.2 ± 0.0 vs. 22.6 ± 0.2 times, respectively, p = 0.031). However, no differences were observed

Sympathetic activity

Note: *p < 0.05 and **p < 0.01 (normal vs. CI). Mean ± standard error.
Abbreviation: CI, cerebral infarction.

F I G U R E 2 Right ptosis (i.e., Horner's syndrome) was shown after creation of CI by occlusion of right middle cerebral artery fromfrontal (A), right lateral (B), and left lateral (C) view. CI, cerebral infarction

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3.4 | Experiment 3: Changes in bladderactivity and UPP after intravenous
tramadol in normal and CI rats

systolic and diastolic blood pressures in CI rats
(112.3 ± 6.2 mmHg and 58.0 ± 3.0 mmHg, respectively)
were significantly lower (p = 0.001 in both) than thosein normal rats (141.8 ± 3.6 and 82.7 ± 3.4 mmHg,
respectively).

The baseline UPP before tramadol administration was
significantly lower in CI rats than in normal rats
(25.0 ± 2.0 vs. 30.2 ± 0.9, p = 0.018). Other UPP parameters, such as UPP nadir, UPP changes, and HFOs, did not
differ between normal and CI rats (Table 3 and Figure 4).
After tramadol administration, there was a tendency of
decrease (p = 0.074) in Δ baseline UPP ratio of CI rats
compared with normal rats (−0.29 ± 0.15 vs. 0.00 ± 0.01).
Bladder contraction amplitude was significantly
decreased (p = 0.029) in CI rats, and it was further
decreased after tramadol treatment (p = 0.031). Pressure
threshold for bladder contraction was significantly
decreased (p = 0.031) in CI rats. ICI and other UPP
parameters did not change in either normal or CI rats
after tramadol treatment.

3.3 | Experiment 2: Changes in bladderactivity after intravenous tramadol innormal and CI rats
PT was lower in CI rats than in normal rats
(10.0 ± 0.8 cmH2O vs. 16.6 ± 1.8 cmH2O, p = 0.008). ICI
tended to be shorter in CI rats (3.4 ± 0.3 min) than innormal rats (4.7 ± 0.8 min), but not significant(p = 0.245). However, NVCs (i.e., DO) were only
observed in CI rats. In normal rats, after administrationof tramadol, ICI tended to be prolonged (6.1 ± 1.2 min) ata low concentration (0.01 mg/kg) than that at thebaseline (4.7 ± 0.8 min), whereas it returned to the pre‐dose level at higher concentrations (0.1–1 mg/kg)(Table 2 and Figure 3A). However, in CI rats, ICI was
prolonged in a dose‐dependent manner (p = 0.0195 and0.0098) after administration of tramadol at 0.1 and 1 mg/
kg (4.6 ± 0.7 min and 6.4 ± 1.0 min, respectively) (Table 2
and Figure 3B). After the final dose of tramadol(1 mg/kg) was administered, the residual urine rate washigher in normal rats than in CI rats (53.5%, p = 0.014).
In one out of eight normal rats, urinary retention wasobserved at the final dose. However, BP, MVC, and VV
did not differ between normal and CI rats and did notchange in either normal or CI rats after tramadol(0.01–1 mg/kg) administration.

TABLE 2

3.5 | Experiment 4: Evaluation of theischemic damage
Images of brain slices spaced 2 mm apart were recorded
using ImageJ, and the area of infarct areas was measured.
The average infarct area (n = 4) at the coronal sectionof the level of pituitary gland (thalamus) was 92.9 ±56.9 mm2 (9.2 ± 6.0% to the total area of this section) andthe maximum infarct area 1230 mm2 (33% to the total
area of this section) in a severe CI rat (Figure 5). The
brains of normal rats did not show infarcted areas on
TTC staining.

Effects of intravenous tramadol on continuous cystometry in normal and CI rats
Normal rats (n = 8)

CI rats (n = 9)

Tramadol (mg/kg)

Tramadol (mg/kg)

Before

0.01

0.1

1

Before

0.01

0.1

1

BP (cmH2O)

9.2 ± 0.8

9.5 ± 0.7

10.4 ± 1.4

8.8 ± 0.8

7.6 ± 0.6

8.1 ± 0.7

7.4 ± 0.7

7.1 ± 0.7

PT (cmH2O)

16.6 ± 1.8

17.3 ± 1.7

18.3 ± 3.3

13.1 ± 1.5

10.0 ± 0.8**

13.1 ± 2.0

10.1 ± 1.1

10.8 ± 1.1

MVC (cmH2O)

23.6 ± 1.8

23.8 ± 1.8

24.6 ± 3.1

22.6 ± 1.1

26.3 ± 3.1

26.1 ± 2.5

23.5 ± 2.1

23.2 ± 1.8

ICI (min)

4.7 ± 0.8

6.1 ± 1.2

4.7 ± 0.9

5.1 ± 1.6

3.4 ± 0.3

5.9 ± 1.4

4.6 ± 0.7†

6.4 ± 1.0††

VV (ml)0.2 ± 0.02

0.2 ± 0.05

0.2 ± 0.07

0.3 ± 0.10

0.2 ± 0.02

0.3 ± 0.06

0.3 ± 0.08

0.3 ± 0.07

RUR (%)
NVCs (number)

53.5*
0

0

0

0

12.7
1.0 ± 0.5000Note: *p < 0.05 and **p < 0.01 (normal vs. CI). †p < 0.05 and ††p < 0.01 (before vs. after tramadol). Mean ± standard error.
Abbreviations: BP, baseline pressure; CI, cerebral infarction; ICI, intercontraction interval; MVC, maximum voiding contraction; NVCs, nonvoiding
contractions; PT, pressure threshold; RUR, residual urine rate; VV, voiding volume.

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F I G U R E 3 Continuous cystometry in a (A) normal and a (B) CI rat before and after intravenous tramadol (0.01–1 mg/kg). In a normalrat, the interval between bladder contraction did not change. However, in a CI rat, it was prolonged after final dose of tramadol (1 mg/kg)
was administered. NVCs only shown in CI rats (black arrow) were inhibited after tramadol administration. CI, cerebral infarction;
NVCs, nonvoiding contractions

T A B L E 3 Effects of intravenous
tramadol (1 mg/kg) on simultaneousrecordings of intravesical pressure underisovolumetric conditions and UPP innormal and CI rats

Normal rats (n = 5)

CI rats (n = 6)

BeforeAfter

BeforeAfter

bladder contraction amplitude(cmH2O)

49.0 ± 5.2

43.5 ± 6.0

31.7 ± 5.9*

17.4 ± 3.7†

pressure threshold (cmH2O)

26.1 ± 7.6

27.9 ± 7.1

17.2 ± 1.2

12.7 ± 2.8†

intercontraction interval (min)1.9 ± 0.3

2.8 ± 0.6

1.5 ± 0.3

10.6 ± 6.1

baseline (cmH2O)

30.2 ± 0.930.5 ± 1.1

25.0 ± 2.0*

18.0 ± 4.3

Δbaseline UPP ratio–

0.00 ± 0.01–

–0.29± 0.15

nadir (cmH2O)

17.5 ± 2.1

18.8 ± 1.7

13.7 ± 2.2

11.1 ± 3.7

change (cmH2O)

11.3 ± 0.7

11.6 ± 1.1

11.0 ± 1.6

7.2 ± 1.8

HFOs rate (Hz)4.8 ± 0.1

4.7 ± 0.1

4.6 ± 0.3

3.8 ± 0.8

HFOs amplitude (cmH2O)

3.0 ± 0.2

3.5 ± 0.3

2.8 ± 0.4

2.5 ± 0.6

Intravesical pressure

UPP

Note: *p < 0.05 (normal vs. CI), †p < 0.05 (before vs. after). Mean ± standard error
Abbreviations: CI, cerebral infarction; HFOs, high frequency oscillations; UPP, urethral perfusionpressure.

4 |DISCUS SION

The voluntary function of the bladder and urethra isregulated by the spinobulbospinal reflex that passes
through the pontine micturition center and the brain.1
The present results indicate the following: (1) CI rats
show altered urinary frequency, as evidenced by reducedpressure threshold for inducing bladder contractions,presence of NVCs, and increased number of voidings in
the metabolic cage study compared with normal rats;(2) CI rats have lowered urethral basal tone, as evidenced
by the reduced baseline pressure of UPP and no change
after tramadol administration (i.e., no increase inresidual urine volume) compared with normal rats;(3) tramadol could inhibit bladder afferents after CI,
as evidenced by prolonged ICI after tramadol

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F I G U R E 4 Simultaneous recordings of isovolumetric bladder contractions and UPP of a normal (A) and a CI (B) rat before and after
intravenous tramadol (1 mg/kg). In a CI rat, baseline UPP (black arrow) was lower than that of a normal rat. After tramadol injection, thebladder activity and UPP did not change in normal and CI rats. CI, cerebral infarction; UPP, urethral perfusion pressure

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F I G U R E 5 TTC staining in a CI rat. Coronal sections of brain spaced 2 mm (a–f) are shown. A large lesion of cortex (A–E) was
infarcted. Infarction was involved in the striatum (B), hypothalamus (C), and lateral thalamus (D). Periaqueductal gray (PAG, F), ventral
tegmental area (VTA), and substantia nigra (SNR) were not involved in the infarction. CI, cerebral infarction; TTC, 2,3,5‐
triphenyltetrazoliumchloride

administration compared with normal rats; and (4) there
may be an injury of projection of the hypothalamus tosympathetic pathway after CI, as evidenced by ptosis,
involvement of infarction of the hypothalamus revealed
by TCC staining of brain, and decreased sympathetic
activity after CI.
The bladder and urethra function reciprocally.1 In thepresent study, tramadol (0.01–1 mg/kg) administration
did not alter ICI and MVC; however, it augmented
residual urine volume in normal rats in continuous
cystometry. In contrast, while tramadol prolonged ICI
without increased residual urine volume in CI rats. These
different reactions can be explained by different urethralchanges after tramadol treatment, prompting us to
examine the simultaneous recordings of bladder andurethral activity in experiment 3. The present study
showed that baseline UPP was 17% lower in CI rats thanthat in normal rats. Δ Baseline UPP ratio had a tendency
to be decreased in CI rats compared with normal rats(−0.29 ± 0.15 vs. 0.00 ± 0.01, p = 0.074). In our previous
study, microtip‐induced urethral baseline pressure andtilt leak point pressure were 43% and 29% lower,
respectively; in addition these did not increase afteradministration of duloxetine, a norepinephrine‐ andserotonin‐reuptake inhibitor, in CI rats.11 The results ofour present and previous11 studies suggest that

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sympathetically innervated urethral basal tone may be
impaired in CI rats. Microtip‐induced urethral measurements can measure the pin‐point area of the urethra,whereas UPP assesses the overall urethral resistance.
Thus, further studies are necessary to evaluate the direct
effect of CI‐induced urethral activity before and after
tramadol administration by microtip‐induced urethral
responses or leak point pressure.
Occlusion of the middle cerebral artery, which
supplies the basal ganglia, induces CI in rats.15,16 In thepresent study, CI rats showed TTC ischemic lesions in
the right cerebral hemisphere, including the prefrontal
cortex and hypothalamus. All CI rats exhibited droopingof the right upper eyelid. Horner's syndrome is aphenomenon characterized by ptosis, which arises from
dysfunction of the oculosympathetic pathway innervating the smooth muscle of the eyelids, Müller's muscle.17
The hypothalamus, located superior to the brain stem, isthe highest center of the autonomic nervous system.

NAGAMINE ET AL.

Thus, in the present study, the hypothalamus and its
projections from the brain stem to the sympathetic
ganglion may be involved in the occurrence of ptosis
after CI, as seen in cats.17 Descending pathways throughthe raphe nuclei and locus coeruleus, which release
norepinephrine and serotonin, respectively, project toOnuf's nucleus of the pudendal nerve innervating theexternal urethral sphincter.1,18 Therefore, there may be
an interaction between the hypothalamus, oculosympathetic pathway, raphe nucleus, and locus coeruleus(Figure 6). Alternatively, there may be a projection fromthe hypothalamus to the pontine micturition center,
which is considered to directly project to the motor
neurons in Onuf's nucleus.1 Usually, patients after acutestroke are hypertensive as the body attempts to maintainblood circulation; this discrepancy in the present animal
study can be explained by the distinct central lesions
involved. In the present study, we confirmed no changes
in sympathetic innervated baseline pressure of UPP in CI

F I G U R E 6 Hypothesis of impairment of oculosympathetic pathway and urethral continence reflex after CI. The oculosympathetic
pathway, a projection from hypothalamus via thoracic (Th) and cervical (C) ganglion, innervates the smooth muscle of eyelids, Müller'smuscle. After CI (gray shade), this pathway may be interrupted, thus inducing ptosis and Horner's syndrome. The hypothalamus is also
considered to project to raphe nuclei and the locus coeruleus, releasing norepinephrine and serotonin, respectively, which terminates toOnuf's nucleus innervating the external urethral sphincter by the pudendal nerve (work as urethral continence reflex). CI, cerebral
infarction

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rats after tramadol, which has multiple pharmacologicaltargets,19 as well as norepinephrine and serotonin
reuptake inhibition. Thus, further studies are necessary
to examine the possibility of injured sympathetic activity
related to urethral activity by selective stimulation of thispathway, by other drugs (e.g., duloxetine, phenylephrine), optogenetic, or chemogenetic methods. Overall,this is the first study to show a potential mechanism of
impaired urethral continence reflex after CI.
Since the brain suppresses micturition,1 voluntary
control is disturbed after CI, resulting in an overactive
bladder. In the present study, the prefrontal cortex,
which inhibited bladder activity, was infarcted. The
increased number of voiding episodes in the dark phasein the metabolic cage study, reduced pressure thresholdfor inducing bladder contractions, and occurrence of
NVCs in CI rats suggest the presence of an overactive
bladder. In the present study, pressure threshold did notchange with prolonged ICI after tramadol administrationin CI rats in the continuous cystometry (experiment 2),
while it was decreased without prolonged ICI in
simultaneous recording of isovolumetric bladder contractions and UPP (experiment 3). These different responses
may be explained by different conditions of cystometry
and changes, as evidenced by decreased bladder contraction only in isovolumetric conditions after tramadol
administration, because urethral activity changes concomitant with bladder activity. Because a day‐nightdifference in voiding behavior exists in humans androdents,20 the dark phase refers to the awake period inrats. On continuous cystometry, in normal rats, ICI was
prolonged at a lower dose of tramadol (0.01 mg/kg), but
returned to the control level at a higher dose (0.1–1 mg/kg). In the CI rats, ICI was prolonged after tramadol(0.01–1 mg/kg) administration. NVCs were also inhibited
after tramadol. However, tramadol did not affect MVC ineither the normal or CI rats. Changes in ICI and MVC
seem to be related to the afferent and efferent activities ofmicturition reflex1; hence, tramadol may predominantly
inhibit the afferent activity, especially in CI rats. These
results are similar to those of a previous study on theinhibitory effect of tramadol on DO in CI rats.14 In thisstudy, urinary retention was observed only in normal rats
after tramadol administration. Overall, tramadol predominantly inhibited bladder afferents by failing toenhance urethral activity in CI rats.
Various neurotransmitters regulate the micturition reflex
in the CNS. Glutamic acid is a major excitatory neurotransmitter that facilitates bladder function, whereas glycine,
γ‐amino butyric acid, and opioids inhibit this function.8,9 It
has been reported that reduced bladder capacity was
inhibited by dizocilpine, a glutamate N‐methyl‐D‐aspartate(NMDA) receptor antagonist in CI rats.10 Glutamatergic

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activation through α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐
propionic acid (AMPA) receptors triggers the urethralclosure reflex during sneezing at the spinal level.21 These
results suggest that glutamate stimulates both bladder andurethral functions. In our previous study, we reported that
duloxetine, an inhibitor of norepinephrine‐ and serotonin‐
reuptake,22 and tramadol enhanced the sneeze‐induced
urethral continence reflex in normal rats.13 In the present
study, residual urine was increased after tramadol administration in normal rats. Thus, the activation of norepinephrine, serotonin, and µ‐opioid receptor stimulationin the spinal cord can work as storage (i.e., inhibition of the
bladder and stimulation of the urethra). The urethral basal
tone was lowered after CI, and it did not change after
tramadol administration in the present study, suggesting that
CI impair the noradrenergic, serotonergic, or opioid‐related
urethral activity.
The main lower urinary tract symptoms after CI are
wet or dry bladder overactivity.4 In our recent humanstudies, chronic stroke patients reported bladder‐related
urgency incontinence in 9.8% and urethra‐related SUI in
17.6%.6 Thus, CI patients further show SUI twice as often
as overactive bladder symptoms. Our present andprevious studies11 with lower urethral basal tone in CI
rats may support these clinical observations. Current
functional imaging studies, such as functional magnetic
resonance imaging, can help to understand that thefrontal lobe, superior frontal gyrus, insula, and periaqueductal gray are involved in exaggerated brain activity andlead to overactive bladder.23,24 Therefore, it can be
speculated that CI induces not only overactive bladder
but also SUI, which can account for mixed incontinencein CI patients. The clinical efficacy of tramadol for SUI
could be reduced in patients with CI. This pharmacologic
agent is useful for the treatment of overactive bladder.
Tramadol is a less addictive type analgesics, buttransported in the brain, and its concentration is fivetimes high than plasma.14 Therefore, it is important to
use the minimal dose for reducing side effects, such as
nausea, seizures, and slow breathing. Our dose iscompatible with human use.
This study had some limitations. First, we used female
rats after 2–3 days of CI and did not identify any differences
in sex hormones. Second, cystometry was performed underurethane anesthesia, which may have affected the bladderand urethral activities. Third, the bladder and urethral
activities were recorded separately by ligating the bladderneck. Fourth, there may be species differences in theneuroanatomical role of sympathetic activity. Despite theselimitations, this study can support the clinical findings after
stroke, which shows that urethral activity is weak andtramadol or duloxetine fails to enhance urethral activity.
Future studies are required to identify distinct neural

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NAGAMINE ET AL.

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NAGAMINE ET AL.

networks between the PNS and CNS, which control theurethral continence reflex.

5 |CONCLUSIONSThis study provides evidence that CI rats have impaired
bladder and urethral functions. After tramadol administration, µ‐opioid receptor stimulation, and inhibition of
norepinephrine and serotonin reuptake, ICI was prolonged
in continuous cystometry, whereas the reduced baseline
UPP did not change in CI rats. Infarction of the prefrontal
cortex and hypothalamus by TCC staining of the brain wasshown with ptosis and a decrease in blood pressure. This
supports our initial hypothesis that CI‐induced ischemicchanges in the brain and its projection to the spinal
sympathetic pathway may be correlated with impairmentof the urethral activity. Further, tramadol may be effective
for the treatment of overactive bladder after CI.
AUTHOR CONTRIBUTIONSSatoko Nagamine: investigation, data curation, andoriginal draft writing. Tadanobu Chuyo Kamijo:
investigation, data curation, and original draft writing
and reviewing. Asuka Ashikari: data curation andoriginal draft writing and reviewing. Minoru Miyazato:
conceptualization, methodology, supervision, and original draft writing, reviewing and editing.
ACKNOWLEDGMENTS
The authors thank Naohisa Kusakabe and Hiroyuki
Arakawa in our laboratory for technical assistance withexperiments and useful comments and Masanobu Yamazato
and Yoshitsugu Sunagawa in Cardiovascular Medicine,
Nephrology and Neurology, Graduate School of Medicine,
University of the Ryukyus, for assisting with blood pressure
measurements. Moreover, we thank Editage (www.editage.
com) for English language editing. This work was supported
by JSPS KAKENHI (Grant Numbers: JP 18K10749,
20K11158, 20K23256, and 22K11420).
C O N F LI C T S OF IN T E R E ST
The authors declare no conflicts of interest.
DATA A VAILABILITY S TATEMENT
The data of the present study are available from thecorresponding author on the request.
ETHI CS STATEMENT
Experimental protocols were approved by the Institutional Animal Care and Use Committee of the Universityof the Ryukyus (protocol no. A2019234) in compliancewith ARRIVE guideline (https://arriveguidelines.org/).

ORC IDhttps://orcid.org/0000-0001Satoko Nagamine
9332-1169
https://orcid.org/0000-0001Tadanobu Chuyo Kamijo
6587-7231
http://orcid.org/0000-0002-7064-3808
Asuka Ashikari
Minoru Miyazato
https://orcid.org/0000-00033669-4510
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How to cite this article: Nagamine S, Kamijo TC,
Ashikari A, Miyazato M. Influence of cerebral
infarction on both bladder and urethral activities
and changes after tramadol administration in rats.
Neurourol Urodyn. 2022;41:1679‐1691.
doi:10.1002/nau.25043

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