Life Sciences 279 (2021) 119690

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Life Sciences
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Age-associated bladder and urethral coordination impairment and changes
in urethral oxidative stress in rats
Asato Otsubo a, Minoru Miyazato b, *, Takuma Oshiro c, Ryu Kimura c, Tomohiro Matsuo a,
Yasuyoshi Miyata a, Hideki Sakai aabc

Department of Urology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
Department of Systems Physiology, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
Department of Urology, Graduate School of Medicine, University of the Ryukyus, Okinawa, JapanA R T I C L E I N F O

A B S T R A C T

Keywords:
Nitric oxide (NO)Soluble guanylyl cyclase (sGC)
Oxidative stress marker
Ischemia
UrethraAims: We examined age-associated changes in bladder and urethral coordination involving the nitric oxide (NO)/soluble guanylyl cyclase (sGC) system, which induces urethral smooth muscle relaxation, and urethral ischemic/oxidative stress changes in rats.
Main methods: Sixteen female Sprague-Dawley rats were divided into young (3 months old) and middle-aged(12–15 months old) groups. Urethral activity was evaluated by simultaneously recording intravesical pressure
under isovolumetric conditions and urethral perfusion pressure (UPP) under urethane anesthesia. Sodium
nitroprusside (SNP, 0.1 mg/kg), an NO donor, and BAY 41-2272, a novel NO-independent stimulator of sGC (0.1mg/kg), were administered intravenously to both groups. N-nitro-L-arginine methyl ester hydrochloride (LNAME, 100 mg/kg) was also injected intravenously, to inhibit NO synthase activity in both groups. Staining forthe ischemic marker, hypoxia-inducible factor-1α (HIF-1α), and the oxidative stress markers, 8-hydroxy-2′ deoxyguanosine (8-OHdG) and malondialdehyde (MDA), was performed on tissue sections of the urethra, in bothgroups.
Key findings: Baseline UPP and UPP changes were significantly lower in middle-aged rats than in young rats. Afteradministration of SNP and BAY 41-2272, baseline UPP and UPP nadir were significantly decreased in bothgroups. After administration of L-NAME, UPP change/bladder contraction amplitude in young rats was still lower
than at baseline but was completely restored to control levels in middle-aged rats. Immunoreactivity of HIF-1α,
8-OHdG, and MDA was higher in middle-aged rats than in young rats.
Significance: Age-associated ischemic and oxidative stress in the urethra might be correlated with impairment of
the NO/sGC system and with coordination of the bladder and urethra.

1. Introduction
Voiding is performed by coordinated action between the bladder andurethra, which is voluntarily or involuntarily controlled by the spino­bulbospinal micturition reflex pathway via the pontine micturition
center [1,2]. Due to the intricacy of these systems, coordination of the
bladder and urethra can be damaged by numerous pathological condi­
tions, such as spinal cord injury [3] or diabetes [4]. Previous reports[5–7] have suggested that aging also impairs coordination, which cancause an underactive bladder with residual urine, ultimately reducing
the quality of life [8]. Moreover, incontinence is more prevalent in

women than men, especially after menopause. In Japan, it has beenreported that the prevalence rate of urge incontinence and stress in­continence for 1 or more times per week was 15.8% and 19.4%,
respectively, among women ages 40 years or older [9]. In our recent
studies [6,7], urethral smooth muscle-mediated relaxation during
bladder contractions was diminished in 12–24-month-old rats, as shownby simultaneously recording intravesical pressure and urethral perfu­
sion pressure (UPP). However, the precise mechanisms underlying thecoordinated action between the bladder and urethra, as a function ofage, are not well understood.
Acetylcholine from the parasympathetic nerves contracts the* Corresponding author at: Department of Systems Physiology, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 9030215, Japan.
E-mail address: miyaz929@med.u-ryukyu.ac.jp (M. Miyazato).
https://doi.org/10.1016/j.lfs.2021.119690
Received 3 March 2021; Received in revised form 22 May 2021; Accepted 30 May 2021
Available online 7 June 2021
0024-3205/© 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Life Sciences 279 (2021) 119690

bladder, simultaneously, the release of nitric oxide (NO) relaxes theurethral smooth muscle [9]. The external urethral sphincter (EUS) also
relaxes during voiding, and rhythmic contractions and relaxation of the
EUS during voiding occur to expel urine, which is speculated to generate
high-frequency oscillations (HFOs) in UPP, which is only identified inrats and dogs [10,11]. In both animal and human experiments, NO
synthesis in the bladder, urethra, and prostate and acetylcholine releasefrom pelvic nerve have been reported to decrease with age, whereas
non-neuronal acetylcholine release from the urothelium increased withage, suggesting reduced bladder contractility and increased urinary
frequency [12,13]. In our recent study, aging was also assumed toinduce dysfunction in NO-mediated urethral smooth muscle relaxation
during voiding; however, L-arginine, a NO substrate, could not restore
impaired urethral smooth muscle relaxation except urethral contraction
during UPP change [6]. NO activates soluble guanylyl cyclase (sGC),
resulting in an increase in intracellular cyclic guanosine monophosphate(cGMP), which relaxes urethral smooth muscle [13]. Thus, we hypoth­esized that the NO/sGC system, which induces urethral smooth muscle
relaxation, is impaired by aging.
Therefore, in this study, we used young and middle-aged rats toinvestigate urethral function by simultaneously recording intravesicalpressure and UPP, before and after intravenous injections of sodium
nitroprusside (SNP), a NO donor, BAY 41-2272 [14], a novel NOindependent stimulator of sGC, and N-nitro-L-arginine methyl ester hy­
drochloride (L-NAME), a NO inhibitor. To explore the impairment in the
NO/sGC mechanism, we also examined the ischemic and oxidative
status of the urethra by immunostaining for the oxidative stress markers,
hypoxia-inducible factor-1α (HIF-1α), 8-hydroxy-2-deoxyguanosine (8OHdG), and malondialdehyde (MDA).

equal volume. Saline was continuously infused into the urethral catheter
at a rate of 0.05 ml/min. PowerLab (AD Instruments Pty, Ltd., Castle
Hill, New South Wales, Australia) was used for data acquisition andmanipulation. The maximum amplitude of isovolumic contractions andthe intravesical pressure threshold to induce urethral relaxation were
measured when rhythmic bladder contractions were stable for at least
30 min. Urethral pressure was changed by contraction of the bladder.
Therefore, the following specific parameters of UPP were measured
during bladder contractions: UPP nadir during reflex urethral relaxationand the baseline UPP between reflex bladder contractions. The change in
UPP was calculated as the value just below the UPP nadir, minus thebaseline UPP. Negative changes in UPP indicate a bladder-urethralsynergistic pattern. Because urethral activity varies depending on
bladder activity, UPP changes and bladder contraction amplitude werealso evaluated. The mean velocity (Hz) and the amplitude of the urethral
striated muscle HFOs during reflex bladder contractions, were alsomeasured (Fig. 1). These parameters were averaged over 30 min andused as control (baseline) levels.
After a one-hour control period, SNP (Sigma-Aldrich Co., Tokyo,
Japan) was injected intravenously (0.1 mg/kg) to increase the level of
NO. After bladder contraction stabilized, SNP (0.1 mg/kg) and BAY412272 (0.1 mg/kg) (NAMIKI SHOJI Co., Tokyo, Japan) were simulta­
neously injected intravenously.
After SNP and BAY41-2272 treatment, rats were injected intrave­
nously with L-NAME (100 mg/kg; Sigma-Aldrich Co.) to inhibit NO
synthase activity. The amount of dosage was decided according to pre­vious reports [14] and our preliminary experiments. Bladder and ure­thral pressure parameters were measured after each intravenous drug
injection, were averaged, and then compared to the control values.

2. Material and methods

2.4. Immunohistochemistry

2.1. Animal model

The bladder and urethra were removed from euthanized rats, fixed inphosphate-buffered formalin (10%), embedded in paraffin, and cut into
4 μm-thick sections.
Immunohistochemical analysis was performed using formalin-fixed,
paraffin-embedded sections. Primary antibodies for 8-OHdG (Santa Cruz
Biotechnology, TX, USA), MDA (Abcam, UK), and HIF-1α (Novus Bi­
ologicals, USA) were applied to the samples. All the sections weredeparaffinized in xylene and rehydrated using graded ethanol solutions.
Antigen retrieval was performed using 0.01 M sodium citrate buffer (pH6.0) at 95 ◦ C for 40 min. All sections were then immersed in 3%hydrogen peroxide for 15 min to block endogenous peroxidase activity.
The sections were incubated overnight with the primary antibody (8OHdG 1:500; MDA 1:100; HIF-1α 1:40) at 4 ◦ C, and then washed with
0.05% Tween-20 in phosphate-buffered saline (PBS). The sections wereincubated for 60 min in peroxidase using the labeled polymer method,with Dako EnVision+™ Peroxidase (Dako, Carpinteria, CA). The
peroxidase reaction was visualized using a liquid 3,3′ -diaminobenzidine
tetrahydrochloride (DAB) substrate kit (Muto Pure Chemicals Co.,Tokyo, Japan). Sections were counterstained using hematoxylin, dehy­
drated through graded alcohol solutions, cleared using xylene, andmounted with Poly-Mount mounting medium (Polysciences, Inc., War­rington, PA, USA). Masson's trichrome staining was also performed tocompare the morphological differences in the bladder between young
and middle-aged rats.

A total of 16 female Sprague-–Dawley rats (young: 3 months old, n =8; middle-aged: 12–15 months, n = 8) were used in this study. We used
rats aged 12–15 months because they are equivalent to middle-aged
humans who are mostly affected by lower urinary tract dysfunction.
Rats were housed at a constant room temperature and maintained on astandard diet (Nihonkurea, Tokyo, Japan) with free access to water. The
Institutional Animal Care and Use Committee of the University of Ryu­
kyus approved this study protocol.
2.2. Surgical procedure
Under isoflurane anesthesia, a polyethylene catheter (PE-10; Clay
Adams, Parsippany, NJ, USA) was inserted into the jugular vein for drug
injection. The bladder and ureter were exposed using a lower midline
abdominal incision. The ureter was cut from both sides and the distal
end was tied. The bladder neck was tied just below the ureterovesicaljunction to separate the functions of the bladder and urethra. A PE-90
catheter (Clay Adams) was inserted into the bladder from above thebladder wall to record intravesical pressure, and a PE-50 catheter (Clay
Adams) was inserted from the urethra to record the UPP. These catheters
were connected to a pressure transducer (Transbridge 4 M; World Pre­cision Instruments, Sarasota, FL, USA) and an infusion pump via a threeway stopcock. Following surgery, isoflurane anesthesia was terminatedand replaced with urethane anesthesia (1.2 g/kg, intraperitoneal andsubcutaneous; Sigma Chemical Company, St. Louis, MO). The abdomen
was then closed using sutures.

2.5. Statistical analysis
Data are presented as the mean ± standard error of the mean, except
for in the evaluation of HIF-1α. Statistical comparisons were performed
using paired or unpaired t-tests, where appropriate. The Mann-Whitney
U test was used to evaluate the immunohistochemically stained cells.
Statistical significance was set at p < 0.05. All statistical analyses were
two-sided and performed using StatView for Windows (version 5.0;
Abacus Concepts, Inc., Berkeley, CA, USA).

2.3. Simultaneous recordings of intravesical pressure under isovolumetricconditions and UPP in young and middle-aged rats
Saline was infused at a rate of 0.05 ml/min into the bladder to exceed
the threshold volume (0.4–1.2 ml) and induce rhythmic contractions of2

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Life Sciences 279 (2021) 119690

Fig. 1. Simultaneous recording of intravesical pressure and UPP under isovolumetric conditions. UPP, urethral perfusion pressure; HFOs, high-frequency oscillations.

3. Results

inducing urethral relaxation was not significantly changed in young rats,
however, was significantly decreased in middle-aged rats (p = 0.008,
from 20.4 ± 1.8 cmH2O to 16.8 ± 2.4 cmH2O). UPP change/bladdercontraction amplitude was significantly decreased in both young andmiddle-aged rats (p = 0.035 and p = 0.048, respectively).
In middle-aged rats, HFO amplitude was significantly decreased (p =0.008), but the mean rate of HFOs did not change in either young or
middle-aged rats.

3.1. Body weight differences between young and middle-aged rats
The body weight of middle-aged rats was significantly increased(553.5 ± 18.2 g, p < 0.01) compared to young rats (265.6 ± 14.4 g).
3.2. Differences between the control young and middle-aged rats

3.4. SNP and BAY 41-2272 treatment-induced changes in young andmiddle-aged rats

Under isovolumetric conditions, UPP measurements reflected ure­thral relaxation accompanied by HFOs when bladder contractions
occurred in young and middle-aged rats. Baseline UPP was significantly
lower in the middle-aged rats, compared to young rats (p = 0.022;
Table 1, Fig. 2). The mean UPP nadir during amplitude bladder con­tractions was not significantly different between young and middle-aged
rats (9.7 ± 0.7 and 9.1 ± 0.9 cmH2O, respectively; p > 0.05). However,
UPP changes during urethral relaxation were significantly smaller in
middle-aged rats compared with young rats (− 9.5 ± 0.9 vs − 14.8 ± 1.8
cmH2O, respectively; p = 0.021). The maximum amplitude of iso­volumetric contractions and the intravesical pressure threshold for
inducing urethral relaxation, and UPP change/bladder contraction am­
plitudes were not significantly different between the two groups (p >0.05). Additionally, the mean amplitude of HFOs was not significantly
different between young and middle-aged rats (4.3 ± 0.8 vs 4.5 ± 0.2
cmH2O, respectively; p > 0.05); however, the mean rate of HFOs was
smaller in middle-aged rats compared with young rats (4.4 ± 0.2 and
5.3 ± 0.2, respectively; p = 0.003).

After simultaneous intravenous injection of SNP (0.1 mg/kg) andBAY41-2272 (0.1 mg/kg), baseline UPP was significantly decreased inboth young and middle-aged rats (p = 0.003 and p < 0.001, respectively;
Figs. 3 and 4) compared with the UPP levels before injection (control).
UPP nadirs were significantly decreased in both young and middle-aged
rats (p = 0.002 and p < 0.001, respectively). In young rats, UPP nadir
after administration of SNP and BAY41-2272 was further decreased
compared to the UPP nadir after administration of SNP alone (p =0.044). UPP changes were significantly decreased in both young andmiddle-aged rats (p = 0.0.35, p = 0.013, respectively) compared tocontrol levels. The maximum amplitude of isovolumetric contractions
and the intravesical pressure threshold for inducing urethral relaxation
did not change in either young or middle-aged rats. After administrationof combined SNP and BAY41-2272, UPP change/bladder contraction
amplitude was significantly decreased in both young and middle-aged
rats (p = 0.009, p = 0.004) compared to control levels; however, thesechanges were not different from those induced by administration of SNP
alone, in both young and middle-aged rats.
The mean amplitude of HFOs was not significantly different before
and after the administration of combined SNP and BAY41-2272, in bothyoung and middle-aged rats. However, the mean rate of HFOs wasdecreased in both groups (p = 0.029 and p = 0.014, respectively),compared to the control measurement.

3.3. SNP treatment-induced changes in young and middle-aged rats
After SNP (0.1 mg/kg) administration, baseline UPP was signifi­
cantly decreased in both young and middle-aged rats (p = 0.029 and p =0.016, respectively; Figs. 3 and 4) compared with the UPP measured
before the SNP injection (control). The UPP nadir did not change ineither young or middle-aged rats. However, UPP changes were signifi­
cantly decreased in both young and middle-aged rats (p = 0.035 and p =0.013, respectively). The maximum bladder contraction amplitude wassignificantly decreased in both young and middle-aged rats (p = 0.017
and p = 0.011, respectively). The intravesical pressure threshold for3.5. L-NAME treatment-induced in young and middle-aged rats
When L-NAME (100 mg/kg) was intravenously administered after3

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Life Sciences 279 (2021) 119690

treatment.

Table 1
UPP and intravesical parameters before (control) or after SNP, SNP plus BAY 412272, and L-NAME.

Young rats (n = 8)
Max reflex bladdercontraction amplitude(cmH2O)Urethral relaxation
intravesical pressure
threshhold (cmH2O)
Baseline UPP(cmH2O)UPP nadir (cmH2O)
UPP change (cmH2O)
UPP change/bladdercontraction amplitude
HFO amplitude(cmH2O)HFO rate (Hz)Middle-aged rats (n = 8)
Max reflex bladdercontraction amplitude(cmH2O)Urethral relaxation
intravesical pressure
threshhold (cmH2O)
Baseline UPP(cmH2O)UPP nadir (cmH2O)
UPP change (cmH2O)
UPP change/bladdercontraction amplitude
HFO amplitude(cmH2O)HFO rate (Hz)Control(mean ±SE)

SNP
(mean ±SE)

SNP +BAY41-2272(mean ± SE)

L-NAME

53.8 ± 3.0

48.3 ±
1.9*

49.4 ± 2.7

48.9 ± 3.8

27.9 ± 4.1

25.5 ±
3.6

26.8 ± 3.2

26.4 ± 3.8

24.4 ± 1.8

16.6 ± 1.5*

9.7 ± 0.7

18.5 ±
1.8*
8.5 ± 0.5

− 14.8 ±1.8
0.29 ±
0.04
4.3 ± 0.8

− 10.1 ±1.8*
0.21 ±
0.04
3.2 ± 0.4

− 9.4 ± 1.7*

3.8 ± 1.0

18.5 ±
2.2*
11.5 ±
0.9*, §
− 7.0 ±
1.9*
0.14 ±
0.03*
3.1 ± 0.4

5.3 ± 0.2

5.2 ± 0.1

5.0 ± 0.1*

5.2 ± 0.1

50.4 ± 4.6

44.6 ±
4.7*

45.0 ± 3.7

45.3 ± 3.4

20.4 ± 1.8

16.8 ±
2.4*

19.9 ± 2.3

20.9 ± 1.9

18.6 ± 1.4¶

13.2 ±
1.7*
7.4 ± 0.9
− 5.8 ±
1.2*
0.14 ±
0.03*
1.7 ±
0.3*
4.2 ± 0.2

10.6 ± 0.7*

0.10 ±
0.01*,¶
1.9 ± 0.4

15.8 ±
1.3§
9.8 ± 0.9§
− 6.0 ±
0.8*
0.13 ±
0.02
2.4 ± 0.4

3.9 ± 0.2*

4.4 ± 0.1§

9.1 ± 0.9
− 9.5 ±
0.9¶
0.20 ±
0.03
2.5 ± 0.2
4.4 ± 0.2¶

7.2 ± 0.4*, ‡0.19 ± 0.04*

6.2 ± 0.5*
− 4.4 ± 0.6*

3.6. Immunohistochemistry
As shown in Fig. 5A–D, immunoreactivity levels of 8-OHdG and MDA
in the urethra epithelium of middle-aged rats were significantly higher
than those of young rats (p < 0.001). Supplementary figure also showed
higher immunoreactivity levels of 8-OHdG and MDA in the bladderepithelium of middle-aged rats than those of young rats. On the other
hand, the ratio of HIF-1α-staining in urothelial cells (5.66 ± 0.17%,
median = 5.65, interquartile range = 5.28–6.08) was significantly
higher (p < 0.001) compared to that of young rats (2.25 ± 0.20%, me­dian = 2.4, interquartile range = 2.00–2.6; Fig. 5E and F). In Masson'strichrome staining, atrophy of the smooth muscles and fibrosis between
and around smooth muscles in middle-aged rats were stronger comparedto those in young rats (Fig. 5G and H).

(mean ±
SE)

4. Discussion
The coordination between the bladder and the urethra maintains
storage and facilitates the elimination of urine [1]. The results of thisstudy indicate that: (1) middle-aged rats have impaired urethral relax­
ation during voiding, as evidenced by reduced UPP changes during
urethral relaxation (similar to our previously published results) [6,7];(2) middle-aged rats show a decrease in the efficacy of their NO/sGC
system, as evidenced by a decrease in baseline UPP induced by com­bined SNP and BAY41-2272, and blockade of these reactions byadministration of L-NAME; (3) there are age-associated changes inoxidative stress and ischemia in the urethra which may be involved in
the impairment of coordination between the bladder and urethra, as
evidenced by immunostaining of oxidative stress markers (8-OHdG,
MDA) and ischemic markers (HIF-1α) in the urethra.
Urethral relaxation is important for efficient voiding. There are fewer
reports exploring the effects of aging on urethral function compared tothose on bladder function. Lluel et al. reported that anesthetized rats
with increased residual urine volume showed a decrease in resting
urethral pressure at the micturition threshold and a significant delay inurethral relaxation during micturition itself [5]. The present study
confirmed our previous work showing that UPP change was significantly
lower in middle-aged rats than in young rats [6,7]. Similarly, other work
done in rabbits has provided evidence for an age-related decrease in NOmediated relaxation and nitrinergic innervation of the prostate [15].
Additionally, NO release in human prostate tissue has also been shownto decrease with age [16]. These results suggest that age-associatedchanges affect NO synthesis in the lower urinary tract, resulting in
dysfunctional urethral smooth muscle relaxation. In our recent study,
decreased UPP change during reflex bladder contractions was notrestored in middle-aged rats after treatment with L-arginine, a NO sub­strate, except for in alterations of urethral contractions during UPPchange [6]. Studies using streptozotocin-induced diabetic rats have re­ported that impaired urethral relaxation was partially recovered by Larginine treatment [4]. However, in the present study, both baseline
UPP and nadir were significantly decreased by SNP alone or in combi­
nation with BAY41-2272, and these changes were completely restoredby L-NAME, in aged rats. These results suggest that the NO/sGC system,
which induces smooth muscle relaxation in the urethra, might be
impaired by age, and that SNP and BAY41-2272 could restore reduced
NO/sGC system more effectively than L-arginine. Because baseline UPP
reacted more than UPP nadir by SNP or combined SNP and BAY41-2272
in middle-aged rats, UPP change (as defined by baseline UPP minus UPP
nadir) might appear to be decreased. However, baseline UPP and UPP
nadir were also decreased in young rats, suggesting that additional NO
activation (induced by combined SNP and BAY41-2272) results inincreased intracellular cGMP, thus relaxing the urethral smooth muscle
in young rats. The change in UPP seems more prominent than bladderactivity with age because the amplitude of bladder contractions did not

SNP: Sodium nitroprusside, L-NAME: N-nitro-L-arginine methyl ester hydro­
chloride, UPP: urethral perfusion pressure, HFO: high frequency oscillations, SE:standard error.
SNP: 0.1 mg/kg, BAY41-2272: 0.1 mg/kg, L-NAME: 100 mg/kg.
*
P < 0.05 vs Control.
‡

P < 0.05 vs SNP.

§P < 0.05 vs SNP + BAY41-2272.

¶

P < 0.05 vs young.

combined SNP and BAY41-2272 treatment, baseline UPP was notaltered in young rats and was still lower (18.5 ± 2.2 cmH2O, p = 0.252;(Figs. 3 and 4)) compared to the levels before injection (control).
However, in middle-aged rats, baseline UPP was significantly increased
after L-NAME (15.8 ± 1.3 cmH2O, p = 0.004) administration, andrecovered to control levels. The UPP nadirs during reflex bladder con­tractions were significantly increased in both groups (young:11.5 ± 0.9,
middle-aged:9.8 ± 0.9 cmH2O). Changes in UPP were not significantly
altered by L-NAME in young and middle-aged rats (− 7.0 ± 1.9 cmH2O
and − 6.0 ± 0.8 cmH2O, respectively). The maximum amplitude of iso­volumetric contractions was not changed in young and middle-aged rats
(48.9 ± 3.8 and 45.3 ± 3.4 cmH2O, respectively). The UPP change/
bladder contraction amplitude was still smaller after L-NAME (0.14 ±
0.03, p < 0.001) compared with the control levels in young rats andrecovered to the control level in middle-aged rats (0.13 ± 0.02).
L-NAME treatment did not affect the mean amplitude of HFOs ineither group. However, the mean rate of HFOs was increased in middleaged rats (4.4 ± 0.1 Hz, p = 0.019) compared with SNP and BAY41-2272
4

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Life Sciences 279 (2021) 119690

Fig. 2. Representative traces of simultaneous recordings of intravesical pressure under isovolumetric conditions and in UPP of (A) young and (B) middle-aged rats. In
middle-aged rats, the UPP change was smaller compared with young rats. However, maximum bladder contraction amplitude did not differ between both groups.
UPP, urethral perfusion pressure.

Fig. 3. Representative traces of simultaneous recordings of intravesical pressure under isovolumetric conditions and UPP, in young rats. During control periods, large
negative UPP changes during bladder contractions were observed. After SNP treatment, baseline UPP and UPP negative change was decreased and were recovered
after about 1 min. After SNP plus BAY 41-2272 treatment, UPP nadir was further decreased (▾). After L-NAME, baseline UPP and nadir were increased, and UPPchange was partially recovered to the control level. UPP: urethral perfusion pressure; SNP: Sodium nitroprusside; L-NAME: N-nitro-L-arginine methyl esterhydrochloride.

differ between young and middle-aged rats in the present study, as wellas in our recent studies [6,7]. Overall, the decreased activity of the NO/sGC system might be a contributor to impairment of bladder and ure­thral smooth muscle coordination in aged rats (prominent in urethralrelaxation impairment).
Aging is associated with impaired vascular function which is pri­marily characterized by endothelial dysfunction [17]. The mechanisms
for age-dependent endothelial dysfunction include a reduction in NO
bioavailability due to decreased NO synthesis (NOS) or increased NO
scavenging and oxidative stress, leading to peroxynitrite formation[18,19]. In the present study, levels of HIF-1α (ischemic marker), 8OHdG, and MDA (lipid oxidative stress marker) in the urethral epithe­lium, which could affect NO synthesis, were also higher in middle-aged
rats than in young rats. In a rat model of chronic bladder ischemia,

impaired vascular occlusion has been reported to cause fibrosis andreduce bladder contractility [20]. Furthermore, Gotoh et al. [21] re­ported that within the urothelial layer of the bladder in diabetic rats,
expression of HIF-1α and 8-OHdG was stronger than in non-diabetic rats.
These results suggest that local ischemia and accumulation of oxidative
stress in the urethra may impair urethral and bladder function. In theurethra of sheep, neural NOS immunoreactivity was present in theintramural nerve and in the sarcolemma of some striated muscle fibers
and was denser at the neuromuscular junction [22]. NOS is expressed inendothelium lining the cavernous spaces and urethral epithelium of rats,and NOS protein is also decreased in diabetic rats [23]. The other pos­sibility is that low cyclical estrogen levels might have affected urethralischemia, especially in middle-aged female rats. Levin and co-workers
reported that ovariectomy resulted in lower bladder contractility and5

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Life Sciences 279 (2021) 119690

Fig. 4. Representative traces of simultaneous recordings of intravesical pressure under isovolumetric conditions and UPP in middle-aged rats. During the control
periods, a low UPP negative change during bladder contractions was observed. After SNP injection, the baseline UPP and UPP negative changes decreased, and they
recovered after about 2 min. After administration of combined SNP and BAY 41-2272, baseline UPP and UPP negative changes were decreased, similar to SNP alone.
After administration of L-NAME, baseline UPP and nadir were increased, and the UPP change completely recovered to control levels. UPP, urethral perfusionpressure; SNP, sodium nitroprusside; L-NAME, N-nitro-L-arginine methyl ester hydrochloride.

higher lipid peroxidation in vitro ischemia/perfusion experiments,
whereas estrogen supplementation protected the bladder against
oxidative stress [24,25]. Chronic stress related to urethral ischemia may
also impair urothelial mitochondria structure and function [26]. In thepresent study, atrophy of the smooth muscles and fibrosis in middleaged rats were significantly stronger compared to those in young rats.
Age-related ischemia and accumulation of oxidative stress in the bladdermight have caused atrophy of the smooth muscles and fibrosis. Based on
previous studies [20,21,23–26] and our present results, the specificcellular mechanisms involved with the aging process in the female
bladder/urethra could be hypothesized as follows: (i) blood flow to thebladder and urethral muscle and mucosa, which are under estrogenic
control, decreased as circulating estrogen decreased or when there isatherosclerosis of the pelvic arteries; (ii) decreased blood flow to thebladder and urethral smooth muscles and mucosa resulted in localized
tissue hypoxia and reactive oxygen species (ROS) and reactive nitrogenspecies (RNS) free radical generation (i.e., accumulation of oxidative
stress); and (iii) tissue hypoxia and oxidative stress resulted in neuronaland mitochondrial damage, impairment of NO/sGC system, as well as
increased connective tissue formation, smooth muscle atrophy, andbladder/urethra dysfunction. Our future study will aim to clarify
detailed mechanisms of chronic urethral ischemic influence on the NO/sGC system. This study is the first to report the accumulation of ischemic
and oxidative stress in the urethra of aged rats fed a standard diet.
HFOs are the pumping mechanism of the EUS, which increases theefficiency of voiding. In our previous work [6,7], the amplitude of HFOs
was lower in middle-aged rats than in young rats. In the present study,
the amplitude of HFOs in middle-aged rats tended to be lower than thatin young rats (p = 0.06). Additionally, using EUS-electromyography, we
have previously shown EUS bursting activity during voiding, accom­
panied by clear active and silent phases in young rats but unclear active
and silent phases in aged rats (i.e., detrusor-sphincter dyssynergia
pattern) [7]. In the present study, the amplitude of HFOs was decreased
after SNP administration, in accordance with the reaction to the urethralsmooth muscles in middle-aged rats, and was restored after L-NAME
administration. These results indicate that the urethral smooth muscle
and the EUS work together, and that aging induces both types of urethral
dysfunctions, which may lead to a persistent cycle of discoordination
between the bladder and urethra.
In the chronic phase of spinal cord injury, bladder-sphincter

coordination is impaired, leading to detrusor-sphincter dyssynergia [3].
Torimoto et al. [4] also reported that streptozotocin-induced diabetic
rats showed partial detrusor-sphincter dyssynergia changes. These lower
urinary tract dysfunctions then cause various problems, such as urinary
incontinence, recurrent urinary tract infection, and vesicoureteral
reflux, with or without upper urinary tract deterioration. The present
study also demonstrated bladder and urethral discoordination as afunction of age. Thus, the urethra might be a promising target to
improve lower urinary tract symptoms in the aging bladder.
This study has several limitations. First, we used 12–15-month oldrats as middle-aged rats, which might not be the best representative age
range for bladder dysfunction. Second, we only used female rats because
sex hormones influence nitrergic- or adrenergic-dependent urethralfunction [27]; in addition, middle-aged females are mostly affected bylower urinary tract dysfunction such as incontinence because of low
cyclical estrogen levels. Third, all experiments were performed underurethane anesthesia, which might have affected the results of this study,
given that urethane affects the NO pathway and urethral function[28,29]. Fourth, evidenced by an SNP-induced decrease in the maximum
amplitude of isovolumetric contractions and intravesical pressure
threshold for inducing urethral relaxation in middle-aged rats, we showthat the NO/sGC system also affects bladder function. Despite theselimitations, the outcomes of the present study may be of significance for
future studies on aging-related changes in the urethra.
5. Conclusion
This study provides evidence that aged rats have impaired bladderand urethral coordination. After combined administration of SNP, an NO
donor, and BAY41-2272, an sGC activator, the baseline UPP and nadir
were decreased, and the blockade of these reactions by L-NAME
administration, an NO inhibitor, restored all parameters to baseline
levels, only in middle-aged rats. This supports our initial hypothesis thatthe efficacy of the NO/sGC system is diminished by aging. Additionally,
the urethra of aged rats showed higher staining for HIF-1α, 8-OHdG, and
MDA levels. Therefore, age-associated ischemic and oxidative stress inthe urethra might be correlated with the occurrence of bladder andurethral coordination.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.lfs.2021.119690.
6

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Life Sciences 279 (2021) 119690

7(caption on next page)A. Otsubo et al.

Life Sciences 279 (2021) 119690

Fig. 5. 8-hydroxy-2-deoxyguanosine (8-OHdG) immunohistochemical staining in the urethra of young rats (A) and middle-aged rats (B). Malondialdehyde (MDA)
immunohistochemical staining in the urethra of young rats (C) and middle-aged rats (D). Hypoxia-inducible factor-1α (HIF-1α) immunohistochemical staining in theurethra of young rats (E) and middle-aged rats (F). Masson's trichrome staining in the bladder of young rats (G) and middle-aged rats (H). Expression levels of 8OHdG and MDA in the urethral epithelium in middle-aged rats were remarkably higher than those in young rats. Likewise, the frequency of HIF-1α-positive cells(arrows: nucleus in the urethral epithelium) in middle-aged rats was significantly higher compared to that in young rats. In Masson's trichrome staining, atrophy ofsmooth muscle and fibrosis in middle-aged rats were significantly stronger compared to those in young rats.

CRediT authorship contribution statement

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The contributions of each author are as follows: (1) Substantial
contributions to conception and designed experiments: AO, MM, TO. (2)Performing the experiments and analyzing data: AO, MM, TO, TM, YM.
(3) Drafting and revising the article critically for important intellectual
content: AO, MM, TO, RK, TM, and YM. (3) Final approval of the version
to be published: AO, MM, TO, RK, TM, YM, and HS.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported by JSPS KAKENHI Grant Numbers
18K09139, 18K10714, and 18K10749.
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