International Urology and Nephrology
https://doi.org/10.1007/s11255-023-03770-3

UROLOGY - ORIGINAL PAPER

Effects of low‑intensity extracorporeal shock wave therapy
on lipopolysaccharide cystitis in a rat model of interstitial cystitis/
bladder pain syndrome
Naohisa Kusakabe1,2 · Tadanobu Chuyo Kamijo1
Minoru Miyazato1· Naoki Wada3 · Hiroki Chiba2 · Nobuo Shinohara2 ·Received: 17 June 2023 / Accepted: 26 August 2023
© The Author(s), under exclusive licence to Springer Nature B.V. 2023

Abstract
Purpose To investigate the effect of low-intensity extracorporeal shock wave therapy (LiESWT) on lipopolysaccharide(LPS)-induced cystitis in an animal model of interstitial cystitis/bladder pain syndrome (IC/BPS).
Methods Sprague–Dawley rats were divided into three groups: control, cystitis (LPS group, intravesical injection of LPS(1 mg) twice), and cystitis with LiESWT (LiESWT group). On the third and fourth days, LiESWT was administered (0.12 mJ/
mm2, 300 shots each time) on the lower abdomen toward the bladder. On the seventh day, the rats underwent pain assessment
and a metabolic cage study. Subsequently, a continuous cystometrogram (CMG) was performed under urethane anaesthesia.
Immunohistochemical studies were also performed, including S-100 staining, an immunohistochemical marker of Schwann
cells in the bladder.
Results In the LPS group, the pain threshold in the lower abdomen was significantly lower than that in the control group.
In the metabolic cage study, the mean voided volume in the LPS group significantly increased. The CMG also revealed asignificant decrease in bladder contraction amplitude, compatible with detrusor underactivity in the LPS group. Immunohistochemical studies showed inflammatory changes in the submucosa, increased fibrosis, and decreased S-100 stain-positive
areas in the muscle layer of the LPS group. In the LiESWT group, tactile allodynia and bladder function were ameliorated,
and S-100 stain-positive areas were increased.
Conclusion By restoring nerve damage, LiESWT improved lower abdominal pain sensitivity and bladder function in an
LPS-induced cystitis rat model. This study suggests that LiESWT may be a new therapeutic modality for IC/BPS.
Keywords Low-intensity extracorporeal shockwave therapy · Interstitial cystitis · Bladder pain syndrome · Detrusor
underactivity · Nerve regeneration

Introduction* Minoru Miyazato
miyaz929@med.u-ryukyu.ac.jp
1

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

Department of Renal and Genitourinary Surgery, Graduate
School of Medicine, Hokkaido University, Sapporo, Japan3

Department of Pathology and Oncology, Graduate Schoolof Medicine, University of the Ryukyus, Okinawa, Japan

Interstitial cystitis/bladder pain syndrome (IC/BPS) is achronic inflammatory disease affecting the bladder wall.
IC/BPS symptoms are characterised by persistent pain in
the lower abdomen and pelvis, accompanied by urinary frequency and urgency [1], thus disturbing the patient’s quality of life (QOL) [2]. Furthermore, patients experiencing
IC/BPS incur higher healthcare costs compared to those
without the condition, thus posing a cost issue [3]. This disease is more prevalent in women than men; for instance,
2.7–6.5% of adult females in the US experience IC/BPS[4]. The subtypes are cystoscopically classified into Hunner and non-Hunner lesions [5]. However, a comprehensive

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understanding of the pathogenesis of this disease remains
elusive, and no curative treatment exists.
Several IC/BPS animal models have been proposed[6]. Nonetheless, a standard model has yet to beestablished. Intraperitoneally or intravesically administeredlipopolysaccharide (LPS) has been used to induce cystitis
in animal models, which have been used as IC/BPS models
because they mimic the pathogenesis of IC/BPS and sustain
bladder inflammation over an extended period [6, 7]. In
addition, pathohistological studies of these models revealed
inflammatory cell infiltration and increased fibrosis in the
bladder, resembling the histopathological features observedin patients with IC/BPS [6–8]. Intravesical injection of LPS
also induces urinary disturbances and heightened sensitivity
to lower abdomen pain [7, 9]. Therefore, the intravesicalLPS-induced cystitis model may effectively replicate lower
urinary tract dysfunction, such as IC/BPS, thereby serving
as a valuable model for evaluating this disease.
Low-intensity extracorporeal shock wave therapy(LiESWT) is currently used clinically in orthopaedic,
angina pectoris, and erectile dysfunction therapy withminimal adverse effects [10–12]. Preclinical studies havedemonstrated its anti-inflammatory and angiogenic effects[13, 14]. Previous studies have indicated that LiESWT iseffective in models of lower urinary tract symptoms. In a rat
model of capsaicin-induced prostatitis, LiESWT suppressed
cytokine-positive cells in the prostate and improved scrotal
pain sensitivity [15]. Furthermore, LiESWT facilitatedbladder nerve regeneration and improved bladdercontractility in a diabetic bladder dysfunction rat model [16].
Thus, we hypothesised that LiESWT could effectively treat
IC/BPS, characterised by multifocal unknown pathogeneses
such as inflammation, fibrosis, and nerve damage.
Therefore, in the present study, we investigatedvoiding and pain behaviour, bladder function, and bladdermorphology and immunostaining for a marker of Schwann
cells, S-100 stain [17], to explore the therapeutic effect andmechanism of LiESWT in an LPS-induced IC/BPS modelin rats.

Fig. 1  Study protocol. In the LPS and LiESWT groups, LPS was
injected into the bladder on days zero and one, respectively. LiESWT
was administered to rats in the LiESWT group on days three and four.

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Materials and methods
Animals
A total of 24 adult female Sprague–Dawley rats (204–307 g)(Kyudo, Saga, Japan) were used. These rats were housed
under 12 h artificial light/dark cycles with free access to foodand water. The animals were randomly divided into threegroups: a control group (n = 8), a cystitis group exposed to
LPS (LPS group) (n = 8), and a cystitis group treated with
LiESWT (LiESWT group) (n = 8).

Study protocols
The study protocol is presented in Fig. 1. In the LPS and
LiESWT groups, LPS was injected into the bladder on days
zero and one, respectively. On days three and four, LiESWT
was administered to the LiESWT group. On day six, all rats
underwent the von Frey test and metabolic cage study, andthe next day (day 7), they underwent a continuous cystometrogram (CMG) and histological study.

Induction of LPS‑induced cystitis
The rats were anaesthetised with an intraperitoneal
injection of ketamine hydrochloride (90 mg/kg; Daiichi
Sankyo Propharma Co. Ltd., Tokyo, Japan) and xylazinehydrochloride (10 mg/kg; Elanco Japan Inc., Tokyo, Japan).
A polyethylene catheter (BD Intramedic™ Polyethene
Tubing PE-50; Becton, Dickinson and Company, NJ, USA)was inserted into the bladder via the urethra. First, thebladder was emptied, followed by intravesical infusion of
0.2 ml of LPS from Escherichia coli O55:B5 (Sigma-Aldrich
Co., LLC, MO, USA) at a concentration of 5 mg/mL insterilised saline. The LPS solution was allowed to remain in
the bladder for 60 min. After LPS exposure, the bladder wasallowed to drain freely through the open catheter end. ThisOn day six, all rats underwent the von Frey test and metabolic cagestudy, and the next day (day seven), they underwent a continuous cystometrogram (CMG) and histological study

International Urology and Nephrology

treatment was repeated every 24 h for two days to induce
cystitis.

LiESWT procedure
The shock waves were produced using an ED-1000 (Medispec, Yehud, Israel). The rats were anaesthetised with isoflurane and placed in a supine position, and their lower abdomen was shaved. An ultrasound gel pad (Yasojima Proceed
Co. Ltd., Hyogo, Japan) was applied between the probe andthe skin of the lower abdomen to ensure optimal coupling(Fig. 2). The probe was specifically designed to focus on the
rat bladder. Subsequently, LiESWT was applied to the pelvis
targeting the bladder with an energy flux of 0.12 mJ/mm2
at a frequency of 2 Hz, and 300 shots were modified fromprevious reports [18, 19].

Tactile allodynia assessment
The von Frey test was used to assess tactile sensitivity. Rats
were placed in a stimulation chamber with a wire meshbottom and left for 1 h to acclimatise to the environment.
Von Frey monofilaments (Stoelting Co., Wood Dale,
IL, USA) were applied to the lower abdomen close to

Fig. 2  LiESWT procedure. The shock waves were produced using
an ED-1000. The rats were anesthetized with isoflurane and placedsupine with their lower abdomen shaved. The ED-1000 has a therapeutic zone of 9 mm in diameter and 130 mm in depth from the apexof the probe, of which up to a depth of 60 mm from the contact area,
the therapeutic effect is weakened by interference between shockwaves. In this study, to prevent weakening therapeutic effect, an echogel pad 45 mm thick was applied between the skin and probe, and theprobe was applied at an angle toward the inside of the pelvis, assuming a rat bladder at a depth of about 60–90 mm from the apex of theprobe. LiESWT was applied at an energy flux of 0.12 mJ/mm2 at
2 Hz for 300 shots per treatment

the urinary bladder and hind paw. Positive responses tostimulation encompassed the following: (1) jumping, (2)rapid retraction from stimulation, and (3) licking of thestimulated area. The withdrawal threshold was calculatedusing the up-down method for 20 trials. The 50% withdrawal
threshold was formulated from the mean of the logarithmic
tabular values [­ log10 (force in g × ­104)] for the patterns ofpositive and negative responses [20].

Metabolic cage study
After the von Frey test, each rat was placed in a metabolic
cage (Metabolica™, Sugiyama-gen Co. Ltd., Tokyo, Japan)
while awake and allowed unrestricted movement for 24 h.
Urinary volume was continuously measured with PowerLab(ADInstruments Pty. Ltd., New South Wales, Australia). All
rats had free access to water and food during the recording.
The mean voided volume was calculated by dividing thetotal urinary volume (g) by the urinary frequency per 24 h.

Continuous cystometrogram (CMG)
The rats were anaesthetised with isoflurane, and catheter
implantation was performed. The right jugular vein was
exposed, and a polyethylene catheter (BD Intramedic™ Polyethene Tubing PE-10, Becton, Dickinson and Company, NJ,
USA) was inserted into the vessel for drug injection. The bladder and ureters were exposed through a lower midline abdominal incision. The bilateral ureters were cut, and the distal ends
were tied. A PE-50 polyethylene catheter with a cuff wasinserted into the bladder through the bladder dome to record
the intravesical pressure. The abdomen was closed with sutures.
Subsequently, isoflurane was turned off and replaced by onethird intraperitoneal urethane and the remaining two-thirds
subcutaneous urethane, a total of 1 g/kg (Sigma-Aldrich Co.,
LLC, MO, USA). The intravesical catheter was connected via
a three-way stopcock to a pressure transducer to record intravesical pressure, and a syringe pump was infused with saline.
The bladder was infused with physiological saline at a rateof 0.05 ml/min during the cystometry. PowerLab (ADInstruments Pty. Ltd., New South Wales, Australia) was used for data
amplification, acquisition, and manipulation. The intercontraction interval (ICI), pressure threshold (PT), baseline pressure,amplitude of voiding pressure, and number of non-voiding
contractions (NVCs) were measured when rhythmic bladdercontractions stabilised for at least 30 min. After the final contraction, bladder infusion was stopped, and the residual volume(RV) was measured by withdrawing intravesical fluid throughthe catheter by gravity. NVCs were defined as contractionsof > 7 ­cmH2O from the baseline pressure that occurred during the filling phase. After CMG, the animals were sacrificed,
and their bladders were harvested and weighed. Bladder weight
divided by body weight was compared among the three groups.

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Histology and immunohistochemistry
Each harvested bladder was fixed in buffered 10%
formaldehyde, paraffin-embedded, and cut into 4-μm-thicksections. The sections were subjected to haematoxylin andeosin (H&E), Masson’s trichrome, and S-100 staining. All
the slides were dewaxed in xylene, dehydrated in gradedalcohol, and rehydrated in distilled water. H&E (HAE-11FU, ScyTek Laboratories Inc., UT, USA) and Masson’s
trichrome (TRM-1-1FU, ScyTek Laboratories Inc. Lcc, UT,USA) were performed using the respective kits, according
to the manufacturer’s instructions. Three randomly selected
high-power fields (objective × 20) surrounding the muscle
layer were analysed to quantify the fibrotic area to eliminate
potential submucosal collagenous tissue. The percentage of theintegrated area of fibrosis in each section was calculated using
FIJI ImageJ software (ver. ImageJ 1.53q, Java 1.8.0_322 [64bit], https://​imagej.​net/​Fiji/​Downl​oad).
For immunohistochemical examination, deparaffinised
sections were first treated with 3% ­H2O2 for 10 min to remove
endogenous peroxidase activity and then incubated with 2.5%
normal horse serum for 2 h at room temperature to block
nonspecific background. The sections were then incubatedwith primary rabbit antibodies against S-100 (1:2000,
GTX48819, GeneTex, Irvine, CA, USA) at 4 °C overnight.
Subsequently, the sections were incubated with a horse antirabbit antibody for 30 min. Finally, the reaction sites were
stained with diaminobenzidine (DAB) chromogen until thedesired stain intensity was developed, and the sections werethen counterstained with haematoxylin. A negative control
was performed to elucidate nonspecific immunostaining
without the primary antibody. Three randomly selected fields(objective × 10) were analysed to quantify peripheral nerves inthe bladder. The area of the S-100 stain-positive range in eachsection was calculated using FIJI ImageJ software.

Statical analysis
Data are presented as mean ± standard error (SE). A oneway analysis of variance followed by the Tukey–Kramer
test for post hoc comparisons was performed for intergroupcomparisons. Statistical significance was set at p < 0.05.

Results
Body weight and bladder weight among the threegroupsIn the LPS group (n = 8), bladder weight to body weight significantly increased up to 1.13 ± 0.26 mg/g compared to thecontrol group (n = 8) (Table 1, 0.44 ± 0.02 mg/g, p < 0.05),

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Table 1  Comparison of body and bladder weights among threegroupsBody weight (g)Bladder weight (mg)Weight ratio (bladderweight/body weight)

Control

LPS

LiESWT

221.1 ± 3.9
99.2 ± 3.4
0.44 ± 0.02

257.4 ± 13.0
285.4 ± 60.8*
1.13 ± 0.26*

247.0 ± 11.6
203.3 ± 17.2
0.79 ± 0.05

Data are expressed as the mean ± standard error*p < 0.05 vs. Control groupwhile it recovered in LiESWT group (n = 8) (Table 1,0.79 ± 0.05 mg/g).

Differences in pain threshold among the threegroupsIn the LPS group (n = 8), the pain threshold on the lower
abdomen was significantly lower than that in the control
group (n = 8) (Fig. 3A, 3.97 ± 0.43 vs. 5.62 ± 0.32; p < 0.01).
In contrast, the pain threshold in the LiESWT group (n = 8)
significantly higher than that in the LPS group (n = 8)
(Fig. 3A, 5.15 ± 0.16 vs. 3.97 ± 0.43; p < 0.05). The pain
threshold on the hind paw was not significantly different
among the three groups (Fig. 3B, 5.58 ± 0.21 vs. 4.92 ± 0.21
vs. 5.32 ± 0.17).

Differences in mean voided volume and urinary
frequency among the three groups
In the LPS group (n = 8), the mean voided volume in thelight and dark phases was significantly higher than thatin the control group (n = 8) (Table 2, in the light phase:
1.86 ± 0.41 ml vs. 0.89 ± 0.07 ml, p < 0.05; in the dark phase:
1.58 ± 0.34 ml vs. 0.70 ± 0.04 ml, p < 0.05). However, in
the LiESWT group (n = 8), it recovered to the level in thecontrol group (Table 2, in the light phase: 1.07 ± 0.09 ml vs.
0.89 ± 0.07 ml, p = 0.86; in the dark phase: 0.80 ± 0.10 ml
vs. 0.70 ± 0.04 ml, p = 0.94). There were no differences inurinary frequency in 24 h among the three groups (Table 2,
30.4 ± 1.6 vs. 28.6 ± 2.8 vs. 33.3 ± 1.6); however, that in thedark phase was significantly higher in the LiESWT groupthan in the LPS group (Table 2, 24.8 ± 2.0 vs. 17.9 ± 1.8,
p < 0.05).

Differences in bladder activity
In the LPS group (n = 8), the contraction amplitude wassignificantly lower than that in the control group (n = 8)
(Table 3, 13.95 ± 2.09 ­cmH2O vs. 19.51 ± 0.82 c­ mH2O;
p < 0.05) as presented in Fig. 4. The ICI in the LPS groupInternational Urology and Nephrology

Fig. 3  von Frey test. The logarithm thresholds for withdrawal
responses to filament stimulation on the (A) lower abdomen and(B) hind paws. A Pain threshold in the lower abdomen was significantly lower in the LPS group than in the control group. However,it was restored in the LiESWT group. B Pain threshold in the hind
paw was not significantly different among the three groups. Data areexpressed as mean ± standard error, and individual data are dot-plotted. *p < 0.05, **p < 0.01, n.s. not significant

Table 2  Comparison of bladder functions in metabolic cage study
among three groups

19.23 ± 1.21 ­cmH2O vs. 13.95 ± 2.09 c­ mH2O; p < 0.05) andreturned to the control level. The residual urine volume was
not significantly different among the three groups (Table 3.
0.11 ± 0.04 ml vs. 0.68 ± 0.52 ml vs. 0.12 ± 0.06 ml). However, in the LPS group, one rat (12.5%) experienced urinary
retention.

Control

LPS

LiESWT

24 h urine production (g) 22.83 ± 1.45 39.73 ± 4.96* 28.67 ± 3.98
Voided volume (g)Light phase
0.89 ± 0.07 1.86 ± 0.41* 1.07 ± 0.09
Dark phase
0.70 ± 0.04 1.58 ± 0.34* 0.80 ± 0.10†
24-h0.76 ± 0.04 1.60 ± 0.36* 0.85 ± 0.09
Urinary frequency (times)
Light phase10.3 ± 0.8
8.6 ± 1.3
8.5 ± 1.0
Dark phase19.9 ± 1.2
17.9 ± 1.8
24.8 ± 2.0†24-h30.4 ± 1.6
28.6 ± 2.8
33.3 ± 1.6
Data are expressed as the mean ± standard error*p < 0.05 vs. Control group, †p < 0.05 vs. LPS group

had a tendency to be prolonged compared to that in the control group (Table 3, 668.57 ± 97.48 s vs. 435.53 ± 44.05 s;
p = 0.0851); however, this was not statistically significant.
In the LiESWT group (n = 8), the contraction amplitude
was significantly higher than that in the LPS group (Table 3,
Table 3  Comparison of bladderfunctions in cystometry among
three groups

Histological and immunohistological response
Haematoxylin and eosin staining revealed submucosal
inflammatory changes in the LPS group (n = 8). However,these changes were ameliorated in the LiESWT-treatedgroup (n = 8). Masson’s trichrome staining showed that thepercentage of the fibrotic area in the LPS group (n = 8) was
significantly higher than that in the control group (n = 8)
(Fig. 5G, 17.65 ± 0.37% vs. 10.60 ± 1.08%; p < 0.01). In the
LiESWT group (n = 8), the extent of this area was decreased,
and the percentage was significantly decreased comparedto that in the LPS group (n = 8) (Fig. 5G, 13.90 ± 1.01%
vs. 17.65 ± 0.37%; p < 0.05). In S-100 staining, the positive area was significantly decreased in the LPS group(n = 8) compared with that in the control group (Fig. 5K,

Base line pressure (­ cmH2O)
Threshold pressure ­(cmH2O)
Amplitude of bladder contraction ­(cmH2O)
Intercontraction intervals (s)Non-voiding contractions (times)
Residual urine volume (ml)Control

LPS

LiESWT

5.27 ± 0.54
8.54 ± 0.58
19.51 ± 0.82
435.53 ± 44.05
0.00 ± 0.00
0.11 ± 0.04

7.83 ± 1.11
10.84 ± 1.60
13.95 ± 2.09*
668.57 ± 97.48
1.29 ± 0.75
0.68 ± 0.52

5.17 ± 0.57
8.76 ± 1.11
19.23 ± 1.21†
475.17 ± 71.41
1.13 ± 0.79
0.12 ± 0.06

Data are expressed as the mean ± standard error*p < 0.05 vs. Control group, †p < 0.05 vs. LPS group13

International Urology and Nephrology
Fig. 4  Representative traces ofcontinuous cystometrogram.
The control group exhibiteda regular and stable pattern(A). Two patterns of bladderactivity were observed in the
LPS group: unstable voiding
functions characterized by nonvoiding contractions (B); andprolonged micturition intervals
with low voiding amplitude (C).
The LiESWT group showed
improved voiding function characterized by regular micturitionintervals and restored voiding
amplitude (D). The scale bar
indicates 10 min

3124 ± 728 µm2 vs. 5737 ± 1040 µm2; p < 0.05). In the
LiESWT group (n = 8), the positive area was significantly
increased compared with that in the LPS group (n = 8)
(Fig. 5K, 6176 ± 1140 µm2 vs. 3124 ± 728 µm2; p < 0.05).

Discussion
The results of this study have several important implications.
First, the LPS-induced cystitis model mimics multifactorial
IC/BPS morphology, as evidenced by the histologicalchanges in the infiltration of inflammatory cells in theurothelium and an increase in fibrosis. Second, the LPSinduced cystitis model mimics the chronic model of
detrusor underactivity, as evidenced by an increase in thevoided volume in the metabolic cage study and decreased
bladder contractility in the CMG. Third, LiESWT improves
this unique model with pain and bladder dysfunction,
as evidenced by an amelioration of the pain thresholdon the lower abdomen, a decrease in the voided volume
in the metabolic cage study, and an increase in bladdercontractility in CMG. Fourth, LiESWT has a distinct
mechanism to improve the LPS-induced model, as evidenced
by the inhibition of fibrosis and restoration of the decreased
Schwann cell marker S-100.
In this study, we used an intravesical injection of LPS
to induce the disease model. LPS is a major component of13

the outer membrane of gram-negative bacteria that triggers innate immune responses in the human body [21].
LPS injected into the bladder has been reported to lower
the pain threshold in the lower abdomen in the von Freytest, shorten the voiding interval in CMG, and decrease thevoided volume, similar to interstitial cystitis [7, 9]. In addition, increased inflammatory cell infiltration and fibrosis
have been reported [7, 9]. In the present study, intravesical injection of LPS induced pain sensitivity in the lower
abdomen and histologically increased inflammatory cell
infiltration and fibrosis. These results are similar to those ofprevious studies [7, 9]. On the other hand, unlike previous
studies [7, 9], in continuous CMG and metabolic cage studies, the ICI had a tendency to be prolonged, and the voided
volume was increased. In addition, in the present study, bladder contractility was decreased, and one out of eight rats in
the LPS group developed urinary retention. Histologically,
S-100 staining, an immunohistochemical marker of Schwann
cells, in the muscle layer decreased in the LPS group. These
results suggest that myelinated nerves responsible for normal
bladder sensation/output, such as Aδ fibers, were strongly
injured in this study, which may have led to different results
for large bladder capacity with decreased bladder contractility. We have also reported that nitric oxide-mediatedurethral smooth muscle relaxation during voiding is important for efficient micturition and bladder contraction [22,23]. Another possibility is that our LPS model might haveInternational Urology and Nephrology

Fig. 5  Histology and Immunohistochemistry. A–C Illustrating the
microscopic finding (objective × 10) of H&E stain. u urothelium, mmuscle. D–F Illustrating the microscopic finding (objective × 20) of
Masson’s trichrome stain. All pictures show muscle layers. G The
percentage of the fibrotic area. H–J Illustrating the microscopic
finding (objective × 10) of S-100 stain. Black arrows (▲) indicate
positive S-100 staining of intermuscular nerves. K The area of the
S-100 stain-positive range. All scale bars represent 100 µm. Data areexpressed as the mean ± standard error. *p < 0.05, **p < 0.01. Hae-

matoxylin and eosin (H&E) staining showed inflammatory changes
in the submucosa (B), which were ameliorated in the LiESWT group(C). Masson’s trichrome staining showed that the area of fibrosis
was significantly increased in the LPS group (E) compared to thatin the control group (D), while it was significantly decreased in the
LiESWT group (F). S-100 staining showed that positive area wassignificantly decreased in the LPS group (I) compared to that in thecontrol group (H), while the area was significantly increased in the
LiESWT group (J)affected bladder and urethral function. Another possibility
is the differences in the number of LPS injections and theperiod between LPS injections and experiments when compared with previous studies [7, 9]. Overall, lower abdominal
pain and bladder dysfunction recovered after LiESWT, suggesting that this model was appropriate for examining theeffects of LiESWT.
Extracorporeal shock wave therapy (ESWT) was firstused to treat kidney stones in the 1980s [24] and remains astandard treatment for urinary stones. LiESWT, which is at
lower energy levels than that used in lithotripsy, has proventhat when shock waves hit tissue, they induce cavitationthat might generate a physical force as “shear stress” on
the cell membrane and ultimately exert biological effects[13]. In the present study, LiESWT significantly increased
the pain threshold in the lower abdomen, improved bladder
hypertrophy with fibrosis, and decreased inflammatory cell
infiltration into the bladder. We also found that the bladdercontraction amplitude was increased by 38% in the CMG,
and the mean voided volume was decreased by 47% in
the metabolic cage study in the LiESWT group comparedwith that in the LPS group. Bladder contractility is very

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important for efficient urination; a decrease in bladdercontractility leads to a low flow of urination and increased
residual urine volume [25, 26]. Chuang et al. reported
that LiESWT increased Ki-67 positive cells and restored
bladder contractility in a cryoinjury-induced myogenic
detrusor underactivity rat model, suggesting that LiESWT
regenerates muscle and improves voiding function [27].
In diabetic bladder dysfunction model rats, LiESWT has
also been reported to improve bladder contractility byactivating angiogenesis and bladder innervation [16, 28].
These findings suggest that LiESWT can restore the chronic
bladder dysfunction model through muscle restoration
and angiogenesis. In addition, LiESWT increased S-100
staining-positive area in the bladder wall in the present
study. LiESWT has been reported to significantly increaseneuronal nitric oxide synthase (nNOS) in urethral smoothmuscle and restore urethral function in diabetic bladderdysfunction model rats [28]. Furthermore, LiESWT
significantly elevated the number of nNOS-positive cells in
the penis of erectile dysfunction model rats and improved its
function [29]. Wang et al. reported a significant upregulationof brain-derived neurotrophic factor (BDNF) in erectile
dysfunction model rats with cavernous nerve injury,
achieved via activation of the PERK/AF4 pathway in vitro[30]. Considering the presumed relation of nNOS and BDNF
to nerve regeneration [31, 32], the increase in the S-100
positive area in the bladder may have been influenced bynNOS and BDNF synthesis. Overall, this is the first report
to describe the mechanisms of peripheral nerve regenerationin an IC/BPS animal model. Further studies are necessary
to clarify this point.
The primary symptom of IC/BPS is chronic lower
abdominal and pelvic pain, a serious problem that reduces apatient’s QOL [2]. In preclinical studies, LiESWT decreased
inflammatory molecules such as cyclooxygenase-2 (COX-2)
and tumour necrosis factor-α (TNF-α) in the prostate andameliorated tactile allodynia in capsaicin-induced prostatitis
model rats [15]. In addition, combination therapy withmelatonin and LiESWT in a rat model of neuropathic painwith chronic constriction injury to the sciatic nerve reduced
the expression of inflammatory proteins in dorsal ganglia
neurons. It increased the pain threshold of the hind paw
compared to that in rats treated with melatonin alone [33].
In the present study, LiESWT significantly increased thepain threshold only in the lower abdomen, indicating bladderinnervation and histologically decreased inflammatory cell
infiltration into the bladder, which is similar to previous
studies [15, 33]. Based on previous and present findings,
LiESWT could improve pain through anti-inflammatory
effects and restoration of damaged nerves.
The effects of LiESWT on other animal models of IC/
BPS have been reported. In a study by Wang et al. involving an HCl-induced cystitis model rat, it was noted that

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LiESWT may reduce bladder inflammation by attenuating
the mitochondrial-dependent apoptotic pathway, resulting
in improved urinary frequency [34]. Similarly, in a study
investigating autoimmune cystitis induced by uroplakin 3A,
LiESWT was also reported to reduce inflammatory cells andinflammatory markers such as TNF-α and improve lower
abdominal pain and bladder function [35]. This study also
showed that LiESWT effectively relieves inflammation andimproves pain and bladder function with a neuro-regenerative effect.
The clinical benefits of LiESWT have been demonstrated.
Kikuchi et al. reported that LiESWT significantly improved
chest pain symptoms and cardiac function in patients withsevere angina pectoris without complications comparedto the sham group [11]. Furthermore, Kitrey et al.
indicated that in severe erectile dysfunction patients who
were phosphodiesterase type 5 inhibitor non-responders,
LiESWT significantly improved erectile rigidity withoutcomplications [12]. LiESWT may be helpful in many otherdiseases and clinical settings. In particular, the pathogenesis
of IC/BPS is not yet fully understood. Although stressrelief, oral medications, dimethyl sulfoxide intravesicalinstillation, and hydrodistention with coagulation havebeen used in clinical practice [36], their effects are notcurative, have a short duration, and are invasive. Chuang
et al. investigated the effect of LiESWT on patients with
IC/BPS in a clinical setting. They reported that LiESWT
did not improve O’Leary-Sant symptom scores or urinary
frequency compared to a placebo, but a significantly larger
number of patients recovered a visual analogue scale of 3 ormore without any adverse events [37], which suggests that
LiESWT can affect pain reduction in IC/BPS patients. In the
future, modifying the energy flux and frequency of LiESWT
may further enhance urinary function, making this treatment
a potential candidate for minimally invasive treatment inpatients with IC/BPS.
The present study has some limitations. First, LiESWT
was performed on days three and four after the first
intravesical investigation of LPS, when cystitis may not havebeen established. Therefore, this study indicates a preventive
rather than a therapeutic effect of LiESWT. Second, weused only female rats, possibly due to sex differences in themodel and the effects of LiESWT. However, IC/BPS is moreprevalent in women than men [4], so this study may havethe advantage of examining the effect of LiESWT on IC/
BPS. Third, the LiESWT protocol used in this study may
not have had an optimal shock wave dosage and frequency.
Fourth, the LPS cystitis model employed deviated from thetypical IC/BPS model characterised by urinary frequency;
rather, it exhibited a larger bladder capacity with decreased
bladder contractility. Despite these limitations, the results ofthe present study may be beneficial for future studies on theeffects of LiESWT on IC/BPS.

International Urology and Nephrology

Conclusions
We demonstrated that LiESWT significantly alleviated lower
abdominal pain and improved bladder function in an LPSinduced cystitis rat model. LiESWT may exert this effectby reducing inflammation and restoring peripheral nerve
damage.
Acknowledgements The authors express their gratitude to Hitoshi
Uehara for assisting in preparing our experiments, Hiroyuki Arakawa
for providing technical advice on pain assessment, and Ayako Nakaza
for offering technical advice on immunohistochemistry. Additionally,
we extend our appreciation to DOCTOR’s KITS Co., Ltd. for providing the ED-1000 and to Editage (www.​edita​ge.​com) for their English
language editing services.
Author contributions The contributions of each author are as follows:(1) Substantial contributions to conception, experimental design, andinvestigation: NK, TCK, and MM. (2) Drafting and revising the article
critically for important intellectual content: NK, TCK, NW, HC, NS,
and MM. (3) Final approval of the version to be published: NK, TCK,
NW, HC, NS, and MM. All contributors not meeting these criteria for
authorship should instead be listed in the acknowledgements section.
Funding This work was supported by AMED under Grant Number
JP22gk0210025 and JSPS KAKENHI Grant Number 22K11420.
Availability of data and material The data sets of the current study
are available from the corresponding author upon reasonable request.
Code availability Not applicable.

Declarations
Conflict of interest The authors declare no competing interests.
Ethics approval The study protocol was approved by the University of
Ryukyu Institutional Animal Care and Use Committee (protocol no.
A2020077, A2020019) in compliance with the ARRIVE guidelines.
Consent for publication Not applicable.

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