International Urology and Nephrology
https://doi.org/10.1007/s11255-024-04136-z

UROLOGY – ORIGINAL PAPER

Effects of low‑intensity extracorporeal shock wave on bladderand urethral dysfunction in spinal cord injured rats
Kota Kawase1,2 · Tadanobu Chuyo Kamijo1
Minoru Miyazato1· Naohisa Kusakabe1 · Keita Nakane2· Takuya Koie2·

Received: 6 June 2024 / Accepted: 22 June 2024
© The Author(s), under exclusive licence to Springer Nature B.V. 2024

Abstract
Purpose To investigate the effects of low-intensity extracorporeal shock wave therapy (LiESWT) on bladder and urethral
dysfunction with detrusor overactivity and detrusor sphincter dyssynergia (DSD) resulting from spinal cord injury (SCI).
Methods At 3 weeks after Th9 spinal cord transection, LiESWT was performed on the bladder and urethra of adult female
Sprague Dawley rats with 300 shots of 2 Hz and an energy flux density of 0.12 mJ/mm2, repeated four times every 3 days,
totaling 1200 shots. Six weeks postoperatively, a single cystometrogram (CMG) and an external urethral sphincter electromyogram (EUS-EMG) were simultaneously recorded in awake animals, followed by histological evaluation.
Results Voiding efficiency significantly improved in the LiESWT group (71.2%) compared to that in the control group(51.8%). The reduced EUS activity ratio during voiding (duration of reduced EUS activity during voiding/EUS contraction
duration with voiding + duration of reduced EUS activity during voiding) was significantly higher in the LiESWT group(66.9%) compared to the control group (46.3%). Immunohistochemical examination revealed that fibrosis in the urethralmuscle layer was reduced, and S-100 stained-positive area, a Schwann cell marker, was significantly increased in the urethraof the LiESWT group.
Conclusion LiESWT targeting the urethra after SCI can restore the EUS-EMG tonic activity during voiding, thereby partiallyameliorating DSD. Therefore, LiESWT is a promising approach for treating bladder and urethral dysfunction following SCI.
Keywords Spinal cord injury (SCI) · Detrusor sphincter dyssynergia (DSD) · Low-intensity extracorporeal shock wave
therapy (LiESWT) · Urethra · External urethral sphincter-electromyogram (EUS-EMG) · Cystometrogram (CMG)

Introduction
Micturition is highly regulated by the central and peripheral nervous systems through sympathetic, parasympathetic,
and somatic nerves. During the storage phase, sympathetic
nerves originating from the thoracolumbar spinal cord are
excited to relax the bladder smooth muscles and contract theurethral smooth muscles, whereas the stimulation of somatic* Minoru Miyazato
miyaz929@med.u-ryukyu.ac.jp
Kota Kawase
kawase.kota.b5@f.gifu-u.ac.jp
1

Department of Systems Physiology, Graduate Schoolof Medicine, University of the Ryukyus, 207 Uehara,
Nishihara, Okinawa 903‑0215, Japan2

Department of Urology, Gifu University Graduate Schoolof Medicine, Gifu, Japan

nerves from Onuf’s nucleus in the sacral spinal cord resultsin the contraction of the external urethral sphincter (EUS).
During the voiding phase, the central nervous system,
including the pontine micturition center, excites the parasympathetic nerves and inhibits sympathetic and somatic
nerves [1, 2]. However, the coordinated action of the bladderand urethra is completely arrested following supranuclear
spinal cord injury (SCI).
After SCI, the voiding reflex disappears causing acuteurinary retention. In the chronic phase, the spinal voidingreflex emerges, inducing detrusor overactivity (DO) and detrusor sphincter dyssynergia (DSD) through the activation ofsilent C-fiber bladder afferents [3]. However, this irreversible
condition results in increased post-void residual volume [4,5], urinary tract infection, hydronephrosis, and renal function deterioration [1] Intermittent catheterization can prevent
urinary disturbances [6]; however, an alternative and effective treatment for SCI has not been established.
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International Urology and Nephrology

Various animal studies have been conducted in an attempt
to treat bladder and urethral dysfunction following SCI,
including intrathecal baclofen [7, 8], bladder wall injectionsof botulinum toxin A [9], and administration of anti-brainderived neurotrophic factor antibodies [10]. However, theeffects of these treatments are limited and they are relatively
invasive. Low-intensity extracorporeal shock wave therapy(LiESWT) has anti-inflammatory effects and promotes
angiogenesis, and it is already used as a simple, minimallyinvasive treatment method with an option for the treatmentof orthopedics, angina pectoris, and erectile dysfunction [11,12]. Moreover, LiESWT promotes tissue regeneration in thevagina and urethra, ameliorates stress urinary incontinence
in rats [13], and restores damaged nerve function in diabetic
rats [14]. Moreover, a recent review mentioned that LiESWT
could be involved in nerve regeneration, even in a spinal
cord contusion model, through the enhancement of neurotrophic factors and the activation of the neuroprotective
signal pathway [15]. Thus, we hypothesized that LiESWT
could reduce DSD after SCI, characterized by dramatic neurological changes.
Therefore, we aimed to elucidate the effects of LiESWT
on bladder and urethral dysfunction with DO and DSD
after SCI. Accordingly, we applied LiESWT to the lower
abdomen of rats with chronic SCI and assessed its effects
via cystometrography (CMG) and EUS electromyography(EUS-EMG), as well as histological studies of the bladderand urethra.

subcutaneously for 5 days. The bladders of spinalized rats
were emptied by abdominal compression two or three times
a day for 14 days until reflex voiding was restored postoperatively. We warmed animals on the day after spinalization
to minimize pain; the bed was changed in the cage daily, andcream ointment was applied to the lesion on the bedsore asneeded [16]. Rats with induced spinal injury were divided
into two groups: the LiESWT group (n = 9) and an untreatedcontrol group (n = 8). At the end of the experiment, rats
were euthanized with pentobarbital (100 mg/kg peritoneally)
then the bladders were removed and weighed.

LiESWT techniqueIn the LiESWT group, voiding was induced in awake animals after tail pinching and Crede maneuver to reduce
bladder distention. Thereafter, LiESWT was performed at
3 weeks after spinal cord transection with each 300 shots of
2 Hz and an energy flux density of 0.12 mJ/mm2, repeatedfour times every 3 days, i.e., 1200 shots in total, using the
ED-1000 device (Medispec, Ltd., Yehud, ISR) under isoflurane anesthesia, based on our recent acute model [16]and preliminary experiments. The shockwave applicator wasplaced on a gel pad (Yosojima Proceed Co., Ltd. Hyogo,
JPN) on the shaved skin of the rat’s lower abdomen using
ultrasound gel, and the angle was adjusted toward the pelvic
floor so that the targeting shock wave was involved in mostof the urethra (Fig. 1).

Materials and methods
Animals
All experimental procedures were performed using a protocol (A2021078) approved by the Institutional Animal Careand Use Committee of University of the Ryukyus, in compliance with the ARRIVE guidelines (https://​arriv​eguid​elines.​org/).

Spinal cord injury (SCI) model
Twenty-one female Sprague Dawley rats (6 weeks old,weighing 140–180 g) were used in this study. The EUSEMG activity was compared between four normal rats and
SCI rats. SCI was induced in rats by Th9 transection underanesthesia with 2% isoflurane and pentobarbital (30 mg/kg
subcutaneously) for analgesics on the day. The dura mater
was opened, the Th9-Th10 spinal cord was removed completely using a scalpel, and a sterile sponge (LTL PharmaCo., Ltd. Tokyo, JPN) was placed between the cut ends ofthe spinal cord. The muscle and skin layers were closed.
Postoperatively, cephazolin (100 mg/kg) was injected

Fig. 1  Illustration of LiESWT technique. The ED-1000 probe hasa therapeutic effect in a range of 9 mm in diameter and 135 mm indepth from the tip, of which a particularly effective range exists. In
this study, the gel pad was doubled (40 mm) and the probe position
and angle were adjusted from 50 mm cephalad from the pubic symphysis to the pelvic floor so that more urethra is in the most effective
range. LiESWT low-intensity extracorporeal shock wave therapy

International Urology and Nephrology

Cystometrogram (CMG) and external urethral
sphincter electromyogram (EUS‑EMG)
Six weeks after spinal cord transection, the rats were anesthetized with 2% isoflurane, and the abdomen was openedat the midline. Both ureters were cut, and the distal end
was subjected to ligature. A polyethylene catheter (PE-50;
Intramedic, Becton, Dickinson and Company, NJ, USA) was
inserted into the bladder dome. After abdominal closure,rats were restrained (Universal Rat Restrainer; Braintree
Scientific, Inc., MA, USA) and allowed to rest in the darkfor one hour. Rats were subjected to CMG while awake.
The intravesical catheter was connected to a pressure transducer and an infusion pump via a three-way valve. Saline
was infused at a rate of 3.0 mL/h to induce repetitive bladdercontractions and bladder activity was monitored. PowerLab(ADInstruments, Pty. Ltd., New South Wales, AUS) wasused for data acquisition. Single CMG was performed toevaluate and compare the voided volume, amplitude of voiding pressure, bladder contraction duration during voiding,and number and amplitude of non-voiding bladder contractions (NVCs) between both groups. NVCs were defined asrhythmic intravesical pressure increases greater than 7 cm
­H2O from the baseline pressure without voiding, and thenumber and amplitude of NVCs were measured 5 min before
micturition. Saline volume from the urethral meatus was collected and measured to determine the voided volume. Aftereach void, bladder infusion was stopped, and the intravesicalsolution was retrieved through the catheter by gravity, thevolume of which was considered the post-void residual volume. The voiding efficiency (VE) was calculated using theformula: VE (%) = [voided volume/(voided volume + postvoid residual volume) × 100].
EUS-EMG activity was recorded using two 500 μm polyurethane-coated stainless needle electrodes (Bio ResearchCenter Co., Ltd. Aichi, JPN). These were inserted on bothsides of the EUS device transperitoneally and connected
to a computer system with an analog-to-digital converter(PowerLab). EUS-EMG data were amplified and digitizedusing a computer system. In SCI rats, EUS-EMG was performed to assess the changes in DSD as EUS contraction
duration with voiding. Improvement in DSD was evaluatedas the reduced EUS activity ratio during voiding (duration of
reduced EUS activity during voiding/ EUS contraction duration with voiding + duration of reduced EUS activity duringvoiding) (Fig. 2). In addition, EUS-EMG was compared innormal rats (n = 4) and SCI rats.

Bladder and urethra morphology
The bladder and urethra were removed at the end of theexperiment to determine the histological changes caused by
LiESWT. The collected bladder and urethra were fixed inFig. 2  Illustration of cystometrogram (CMG) and external urethral
sphincter electromyogram (EUS-EMG) during voiding bladder contraction. EUS-EMG during voiding shows EUS contraction withtonic activity (i.e., detrusor sphincter dyssynergia) or reduced EUS
activity

4% paraformaldehyde for 24 h, and 4-micrometer-thick sections were cut using a microtome from paraffin-embedded
specimens and thawed on gelatin-coated slides. Following
deparaffinization, hematoxylin and eosin (H&E) staining(HAE-1-1FU, ScyTek Laboratories Inc., UT, USA) and
Masson’s trichrome staining (TRM-1-1FU, ScyTek Laboratories Inc. Lcc, UT, USA) were performed according tostandard protocols. In Masson’s trichrome staining, threerandomly selected fields of view (objective × 10 and × 20,
respectively) were analyzed to quantify the fibrosis area. The
percentage of the integrated area of fibrosis in each section was calculated using FIJI ImageJ software (ver. ImageJ
1.53q, Java 1.8.0_322 [64 bit], https://​imagej.n​ et/F
​ iji/​Downl​
oad).
For S-100, a Schwann cell marker, immunohistochemical examination was performed. The sections were thenincubated overnight at 4 °C with rabbit primary antibody
against S-100 (1:2000, GTX48819, GeneTex, Irvine, CA,
USA). Thereafter, the sections were incubated with horseanti-rabbit antibodies for 30 min. The reaction sites were
stained with diaminobenzidine (DAB) chromogenic agent
until the desired staining intensity was achieved, and thensections were counterstained with hematoxylin. The area of
the S-100 stain-positive range for each section was calculated using FIJI ImageJ software.

Statistical analysis
Data were analyzed using GraphPad Prism Version 8.4.3(GraphPad Software Inc, CA, USA) and expressed asmean ± standard error (SE) for normally distributed dataor medians and quartiles for non-normally distributed data.
Unpaired Student’s t-test was used for parametric data. Nonparametric data between-group comparisons were performed
using the Mann–Whitney U test (Jmp Pro, version 15.0.0

International Urology and Nephrology(SAS Institute, NC, USA). Differences between groups wereconsidered significant at p < 0.05.

Results
Comparisons of body and bladder weights
between SCI control and SCI LiESWT groups
The initial body weight was not significantly different in
the SCI control and SCI LiESWT groups (163.1 ± 4.7 g vs.
167.0 ± 2.9 g, p = 0.53). However, the final body weight
was significantly higher in the SCI LiESWT group thanin the SCI control group (284.3 ± 8.4 g vs. 257.1 ± 9.9 g,
p = 0.043). There were no differences in bladder weight
between the SCI control and SCI LiESWT groups (0.39(0.31–0.41) g vs. 0.32 (0.28–0.35) g, p = 0.66).

Fig. 3  Records of intravesical pressure from the start of saline infusion to voiding (*indicates a voiding contraction) in SCI rats treated
without LiESWT (a) or with LiESWT (b). a Prominent NVCs (NVCsduring 5 min are expressed as #) were observed early after the start

Table 1  Intravesical parametersin cystometrogram in the SCI
control and SCI LiESWT
groupsExperiment 1: comparison of CMG between SCI
control and SCI LiESWT groups
No significant differences were found regarding theamplitude of voiding pressure (53.0 ± 6.8 ­c mH 2O and44.8 ± 4.2 ­c mH 2O, p = 0.34), the amplitude of NVC(21.9 ± 3.0 ­c mH 2O and 20.1 ± 2.3 ­c mH 2O, p = 0.63),
the number of NVCs (7.8 (6.3–11.5)/5 min and 7.0(6.7–8.0)/5 min, p = 0.66, Fig. 3), the single voided volume (0.78 ± 0.19 mL and 1.09 ± 0.22 mL, p = 0.31),
or the post-void residual volume (1.18 ± 0.50 mL and
0.91 ± 0.48 mL, p = 0.63) between the SCI control
and SCI LiESWT groups, respectively. However, theVE was significantly higher in the SCI LiESWT group(71.2 ± 6.6%) than in the SCI control group (51.8 ± 6.7%,
p = 0.034, Table 1).

of saline infusion. b NVCs gradually appeared more frequently a few
minutes before voiding. SCI spinal cord injury, LiESWT low-intensity
extracorporeal shock wave therapy, NVC non-voiding bladder contraction

Amplitude of voiding pressure ­(cmH2O)
Amplitude of NVC ­(cmH2O)
Number of NVCs
Voided volume (mL)
Post-void residual volume (mL)
Voiding efficiency (%)

SCI control (n = 8)

SCI LiESWT (n = 9)p-value

53.0 ± 6.8
21.9 ± 3.0
7.8 (6.3–11.5)
0.78 ± 0.19
1.18 ± 0.50
51.8 ± 6.7

44.8 ± 4.2
20.1 ± 2.3
7.0 (6.7–8.0)
1.09 ± 0.22
0.91 ± 0.48
71.2 ± 6.6

0.34
0.63
0.66
0.31
0.63
0.034

Bold value indicates statistically significant (p-value < 0.05) than SCI control
Values are presented as mean ± standard error (SE) or median (interquartile range)
SCI spinal cord injury, LiESWT low-intensity extracorporeal shock wave therapy, NVC non-voiding bladdercontraction

International Urology and Nephrology

Experiment 2: comparisons of EUS‑EMG between SCI
control and SCI LiESWT groups
In normal rats, EUS-EMG indicated bursting during voiding
at 97%; however, in SCI rats, bursting activity was reduced
at 4% during voiding (p < 0.001 versus the normal group)
instead of replacement by the EUS tonic activity (Fig. 4,
Table 2). There was no significant difference in the bursting activity ratio during voiding between the SCI control
group (4.0 ± 3.0%) and SCI LiESWT group (10.6 ± 7.5%,
p = 0.43). Simultaneous CMG and EUS-EMG recordings
showed no significant differences in the duration of reduced
EUS activity during voiding between the SCI control group(4.32 ± 0.59 s) and the SCI LiESWT group (5.05 ± 0.59 s,
p = 0.42). However, EUS contraction duration with voiding was significantly shorter (2.47 ± 0.50 s compared to
5.17 ± 0.78 s, p = 0.013), and reduced EUS activity ratio
during voiding was significantly higher (66.9 ± 5.1% compared to 46.3 ± 5.1%, p = 0.017) in the SCI LiESWT groupthan in the SCI control group (Table 2, Fig. 4).

Bladder and urethra morphology
As for the bladder, no significant structural differences could
be identified upon H&E staining, in addition, there was no
significant difference in fibrosis in Masson’s trichrome
between the SCI control (28.1 ± 1.8%) and SCI LiESWT
groups (24.3 ± 1.7%, p = 0.14, Fig. 5). In the urethra, H&E
staining showed no difference in hyperplasia of the urothelium, but the SCI LiESWT group showed significantly lower
fibrosis in Masson’s trichrome (35.8 ± 1.0%) than the SCI
control group (41.9 ± 0.5%, p = 0.023, Fig. 5). In S-100
immunostaining of the urethra, the positive area was significantly increased in the SCI LiESWT group than in the
SCI control group (3.0 ± 0.6% vs, 1.3 ± 0.2%, p = 0.016,
Fig. 5). The S-100 immunostaining of the bladder revealed
no significant differences between the SCI LiESWT and SCI
control groups (data not shown).

Discussion
The results of the present study indicate that the SCI
LiESWT group showed: (1) higher voiding efficiency, (2)higher reduced EUS-EMG activity during voiding, (3)a more prominent effect on the urethra, indicated by thelower fibrosis rate in the muscle layer of the urethra, (4)increased Schwann cell markers as indicated by increased
S-100 immunostaining positive areas in the urethra, and (5)higher voiding efficiency with lesser chance of urinary tract
infection, which affected general conditions, as indicatedby the significant increase in body weight, than those in thecontrol group. These results indicate that applying LiESWT

to the pelvis of SCI rats may promote EUS relaxation andrestore the urethral nervous system, improving urinary conditions, primarily DSD.
In the chronic phase of SCI, the bladder becomes hyperreflexic due to an overactive spinal micturition reflex pathway, and the loss of coordination between the detrusor muscle and EUS leads to DSD and decreased VE [4, 5, 17, 18].
In the present study, the amplitude of voiding pressure wasrelatively lower in the LiESWT group than in the control
group. As DSD results in less efficient EUS relaxation andhigher voiding pressure [17], LiESWT may have affected
EUS function. In fact, applying LiESWT to the urethra of
rats with SCI increased the reduced EUS-EMG activity ratio
during voiding. In rats and dogs with a normal spiral cord,
tonic activity prior to the onset of voiding and bursting activity with clusters of high-frequency spikes during voiding
were detected using EUS-EMG as shown in Fig. 4. Thisbursting activity results in rhythmic contraction and relaxation of the EUS and contributes to synergic urethral pumping activity [5]. In humans, EUS-EMG during voiding issilenced and the EUS relaxes [19] which is relatively similar
to that in female mice [20]. The action of EUS during urination differs between species and sexes, and EUS bursting
occurs during voiding bladder contractions in spinal intact
and SCI rats [5, 19, 20]. In the present study, the rhythmic
bursting during voiding was observed along with significant
reduction to 4% in SCI rats instead of replacement by EUS
tonic activity. However, reduced EUS-EMG activity ratio
during voiding (duration of reduced EUS activity duringvoiding/EUS contraction duration with voiding + durationof reduced EUS activity during voiding) was higher in the
LiESWT group. LiESWT may have enhanced the cooperation of the bladder and urethral sphincter and partially contributed to improving DSD and VE. This is the first report todemonstrate the effect of LiESWT on DSD in SCI models.
In the chronic phase of SCI, the increase in NVCs can beprevented by capsaicin pretreatment; thus, C-fiber bladderafferent nerves are involved in NVCs in SCI [3, 21] However,the present study found no differences in NVCs between thecontrol and LiESWT groups. LiESWT improved DSD andreduced the fibrosis rate in the muscle layer, particularly inthe urethra. These findings indicate that applying LiESWT
to the pelvis may have affected the urethra rather than the
bladder. Tissue fibrosis is caused by excessive deposition of
extracellular matrix due to myofibroblast proliferation associated with chronic inflammation [22]. In rabbits, extracorporeal shock wave therapy reduced the number of collagenbundles by suppressing myofibroblast expression [23]. A
previous study has reported an increase in the number ofprogenitor cells in the urethral smooth muscle layer of vaginal balloon-dilated rats treated with LiESWT, resulting indramatic recovery of the smooth and striated muscle mass
[13]. In this study, there was no change in fibrosis in theInternational Urology and Nephrology
Fig. 4  Representative tracesof simultaneous recording ofcystometrogram and EUS-EMG
during voiding in normal rats(a) and SCI rats treated without
LiESWT (b) or with LiESWT(c). a EUS-EMG showed bursting activity during voiding. b
EUS-EMG showed long EUS
contraction duration and short
reduced EUS activity duringvoiding. c EUS-EMG showed
shorter EUS contraction duration and longer reduced EUS
activity with bursting activity
during voiding. SCI spinal cord
injury, LiESWT low-intensity
extracorporeal shock wave
therapy

International Urology and Nephrology
Table 2  Urethral parameters in EUS-EMG in the normal, SCI control, and SCI LiESWT groups

EUS contraction duration with voiding (s)Duration of reduced EUS activity during voiding (s)Reduced EUS activity ratio during voiding (%)
Bursting activity ratio during voiding (%)

Normal (n = 4)

SCI control (n = 8)

SCI LiESWT (n = 9)–––97.4 ± 1.8

5.17 ± 0.78
4.32 ± 0.59
46.3 ± 5.1
4.0 ± 3.0†

2.47 ± 0.50*
5.05 ± 0.59
66.9 ± 5.1*
10.6 ± 7.5†

Values are presented as mean ± standard error (SE)SCI spinal cord injury, LiESWT low-intensity extracorporeal shock wave therapy, EUS-EMG external urethral sphincter electromyography*†

p < 0.05 versus the SCI control groupp < 0.001 versus the normal group

Fig. 5  a, b, c In Masson’s trichrome staining of the bladder in the
SCI control group (a) and the SCI LiESWT group (b), the percentage of the fibrotic area of the bladder (c) was not different. d, e, f In
Masson’s trichrome staining of the urethra in the SCI control group(d) and SCI LiESWT group (e), the percentage of the fibrotic area ofthe urethra (f) was significantly decreased in the SCI LiESWT group.
g, h, i S-100 immunostaining of the urethra in the SCI control group(g) and the SCI LiESWT group (h), and the black arrows (▲) indicate positive S-100 staining of intermuscular nerves. The percentageof the S-100 positive area of the urethra (i) showed that the positive
area was significantly increased in the SCI LiESWT group. Data areexpressed as the mean ± standard error. *p < 0.05. LiESWT lowintensity extracorporeal shock wave therapy

International Urology and Nephrology

bladder, but a significant decrease in the urethra. The bladderis overdistended after SCI and the LiESWT partially affected
the bladder but had a greater effect on the urethra, possibly due to the ingenuity of an effective range of LiESWT,as shown in Fig. 1. Moreover, S-100 staining, an immunohistological marker of Schwann cells, was significantly
increased in the urethra of the LiESWT group, suggestingthat it may have promoted neural regeneration in the urethra.
LiESWT is also expected to restore the damaged nerve (i.e.,
normal Aδ afferents), evidenced by improvement in pain
in a rat model of interstitial cystitis through repair of damaged nerves [16], C-fiber degeneration, and the restorationof nerve expression in the urethral smooth muscle of diabetic
rats [14]. The pelvic nerve contains somatic fibers innervating the EUS and facilitates pumping activity [24]; myelinated parasympathetic, sympathetic, or somatic nerves may
work complementarily, especially in pathological conditions, and LiESWT may restore the damaged nerve involving
coordinated action of the bladder and urethra. In a previous
study [4] and in our previous studies [5, 7], desensitizationof C-fiber afferent pathways by subcutaneously administered capsaicin or intrathecal injection of inhibitory neurotransmitters such as glycine, GABA, or opioids improved
DSD. Therefore, another possibility is that the local effectof LiESWT on anti-inflammation, tissue, or nerve regeneration promoted neuromodulation in the interneurons of the
spinal reflex, such as during electrical stimulation [25] orreflex plasticity associated with changes in the properties ofneuropeptides, neurotrophic factors, or chemical receptorsof the bladder/urethra afferent neurons in SCI [3]. However,these predictions require further analysis. Overall, LiESWT
targeting the pelvic floor may restore peripheral nerve function around the urethra and reduce fibrosis. Future studies
are needed to evaluate the different and precise effects of
LiESWT on bladder and urethral activity.
The treatment options for SCI-related urinary disturbances are limited [7–14]. Treatments typically target thecentral nervous system and are often invasive and complicated. LiESWT is expected to be effective for DSD after
SCI by suppressing tissue fibrosis and having an effect on
peripheral nerves. Furthermore, it is minimally invasive,
convenient, and can be treated repeatedly. The action of EUS
during urination differs between species and sexes, and EUS
bursting occurs during voiding bladder contractions in ratsand dogs but not in humans [5, 19, 20]. However, LiESWT
shows potential as a new treatment option for urinary disturbances, as LiESWT targeting the pelvic floor may restore
peripheral nerve function around the urethra.
The current study has some limitations that warrant further consideration. First, we did not evaluate the long-term
voiding function after LiESWT. It is unclear whether thetherapeutic effects of LiESWT on CMG and EUS-EMG persist over time. Second, we did not monitor the activities of

the nerves innervating the EUS. Third, we measured EUSEMG activity in a small number of SCI rats, and all rats
were females. Third, our transabdominal approach might notaffect the urethra directly through the best therapeutic effects
compared with the perineal approach. Despite these limitations, the results of the present study indicate the efficacyof LiESWT in EUS and suggest that it may be a promisingtreatment option for urinary disturbances associated with
SCI.

Conclusion
This study provides evidence that LiESWT targeting the urethra after SCI can promote EUS relaxation and consequently
ameliorate DSD. LiESWT, which is more convenient andless invasive than other treatment methods, may be a promising approach for the treatment of urinary disturbances after
SCI, as shown in DSD models.
Acknowledgements The authors would like to thank Hitoshi Uehara
for helping in the preparation of the experiments and Taro Ikegami foroffering technical advice on histological examination.
Author contributions The contributions of each author are as follows:(1) Conception, experimental design, and investigation: KK, TCK, NK,
and MM. (2) Critical drafting and revision of the article for important intellectual content: KK, TCK, NK, KN, TK, and MM. (3) Finalapproval of the version to be published: KK, TCK, NK, KN, TK, and
MM. All contributors who do not meet these criteria for authorship are
listed in the Acknowledgements section.
Funding This work was supported by AMED, JP22gk0210025, Japan
Society for the Promotion of Science, 22K11420.
Data availability The data sets of the current study are available fromthe corresponding author upon reasonable request.

Declarations
Conflict of interests The authors have no competing interests to declare that are relevant to the content of this article.

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