Edited by: Zaleha Abdullah Mahdy, National University of Malaysia, Malaysia
Reviewed by: Pei Shan Lim, National University of Malaysia, Malaysia; Ubirajara Barroso Jr., Federal University of Bahia, Brazil
This article was submitted to Obstetrics and Gynecology, a section of the journal Frontiers in Medicine
†These authors share first authorship
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Acute urinary retention (AUR) is a troublesome urological disease, which causes various lower urinary tract symptoms. However, only few studies explored and evaluated the effective treatments to improve AUR. We aimed to find an effective approach to cure AUR through comparing the efficacy of existing classical low-frequency transcutaneous electrical nerve stimulation (TENS) and novel intravesical electrical stimulation (IVES). A total of 24 AUR female rats were divided into 3 groups as follows: control, TENS, and IVES groups. Rats in the control group had no fake stimulation. Rats in the TENS and IVES groups underwent transcutaneous or intravesical stimulation of a symmetrical biphasic rectangular current pulse with a frequency of 35 Hz, 30 min per day, for seven consecutive days. IVES significantly reduced the actin expression in the submucosal layer but increased its expression in the detrusor layer (
As a severe health problem, acute urinary retention (AUR) is often caused by obstruction of lower urinary tract. In clinical practice, AUR is alleviated by relieving the obstruction, and the most common treatment is catheterization. However, it takes a long time to completely regain the bladder function after catheterization, even 39% patients changing to chronic urinary retention with or without overactive bladder (OAB) (
Low-frequency electrical stimulation (LFES) has been widely used in the urological field in recent years. The most common approach to apply LFES is transcutaneous electrical nerve stimulation (TENS). Although it is convenient and less invasive, TENS has the disadvantage of uncertain electrical energy on the bladder, and its failure rate is relatively high (
In order to verify the advantages of IVES compare with TENS and to explore its underlying mechanism, we conducted this AUR rat model study by comparing the cystometry and pathological changes between the TENS and IVES groups.
Briefly, 12-week-old nulliparous female Sprague-Dawley rats weighing 280–320 g (
The experiments were performed on rats anesthetized using 2% alpha-chloralose (with a dosage of 62.5 mg/kg) (
The AUR modeled rats were randomly assigned into 3 groups as follows: (1) control group (
The electrical stimulation electrode in the TENS group was designed as clamps (
Electrical stimulation electrodes.
Before stimulation session, each anesthetized rat underwent a palpation and transcutaneous abdominal ultrasound examination to ensure the bladder was not empty, since the electrolyte was the rat urine. The skin electrodes were applied with conductive adhesive. The electrical parameters were determined according to our prospective clinical study. The two groups received the same parameters: a symmetrical biphasic rectangular current pulse with width of 200 μs, frequency of 35 Hz pulse, and an intensity of 8 mA was used until visible contraction could be witnessed by the observer without causing suffering to the animal. The time between the passage of current/muscle contraction and interruption/muscular rest (on/off) was 1-s pulse train rising edge, 4-s pulse train duration, and 1-s pulse train interval. The rats were manually placed in the supine position for the introduction of the electro-stimulator probe into the urethra and bladder. The probe was pre-lubricated and disinfected with iodophor. The rats were subjected to 30 min of electro-stimulation for one session per day in 7 consecutive days. After each session, the probes were cleaned with antiseptic soap and water and immersed in disinfectant container. During the experiment, all rats lied on an electric heating pad with a preset temperature of 37°C.
The cystometrograms were recorded by using AcqKnowledge 5.0 software (BIOPAC Systems, USA), which was connected with the catheter to the bladder as previously described (PE50). The recordings were collected both before and after AUR modeling. After TENS or IVES, the cystometrical evaluation was also conducted. The assessments of storage and voiding functions included the following parameters: (1) bladder hyperactivity; (2) threshold pressure (pressure at initiation of voiding contraction, Pthres); (3) maximum intravesical pressure (Pmax); (4) compliance; (5) voiding time; and (6) post-void intravesical pressure. The Pthres was the pressure at the end of the sharp increase of the trace slope. Bladder hyperactivity was defined as increases in intravesical pressure during the bladder filling. The cystometrogram has rising and falling branches (
Within 30 min after the last session of electrical stimulation, the rats were sacrificed, and bladders were immediately dissected. The specimen of the anterior bladder wall near the bladder base at the level of the trigone was obtained (8- to 10-mm width, 3- to 5-mm height), and the vesical fascia was preserved to minimize the damage on the integrity of the wall. Bladder tissues of 1 mm3 were immersed in 3% cacodylate buffered glutaraldehyde (0.1 M, pH 7.2) for transmission electron microscopy (TEM). Other specimens were fixed in 10% formaldehyde solution, dehydrated in alcohol, diaphanized in xylene, and embedded in paraffin. In order to get a comparable histological morphology and minimize the error and bias, the tissues' direction was adjusted to the same. Subsequently, 3-μm tissue sections were cut from these paraffin blocks. All of the bladder specimens were stained using H&E staining and Masson's trichrome. Images were captured using a NanoZoomer-S360 digital slide scanner (Hamamatsu, Hamamatsu City, Japan) with a 40× lens, and the quality of the images was manually checked before application of the digital algorithm. Morphometric analyses were performed using NDP.view 2.0 software (Hamamatsu, Hamamatsu City, Japan) and ImageJ software (National Institutes of Health, Bethesda, Maryland) by 2 authors (TC and BX).
The thickness of the urothelium (200×) and detrusor muscle (80×) was analyzed by 10 random measurements. Briefly, 100 epithelium cells and spindle detrusor smooth muscle cells (DSMCs; 400×) were randomly selected to evaluate the size. To assess the results of Masson's trichrome staining, the mean proportion of collagen area was defined according to the following formula: (collagen)/(collagen + muscle). This technique was predicated on the area calculation of the smooth muscle, which was stained in red, and connective tissue, which was stained in blue.
The expressions of α-smooth muscle actin (α-SMA), cluster of differentiation factor 31 (CD31), and S100 protein in the bladder were assessed by immunohistochemistry. The slides were incubated with the primary antibodies against α-SMA (Abcam, 1:400), CD31 (Abcam, 1:2,000), and S100 (Abcam, 1:1,000). The proportion of α-SMA in the submucosal and detrusor muscle layers was, respectively, analyzed. The quantity and average diameter of vessel and nerve were evaluated.
Semi-thin sections (1 μm thick) from each block were stained with toluidine blue and examined by light microscopy to select the most appropriate blocks for thin sectioning. Thin sections were cut and mounted on uncoated copper grids. The determination of ultrastructure was performed with Tecnai TEM (FEI, Hillsboro, USA). Myofilaments, sarcoplasmic dense bodies, sarcoplasmic reticulum (SR), and mitochondria were observed.
Statistical analysis was performed using SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). All continuous variables were tested for normality with a Shapiro–Wilk test. The normally distributed variables were described as mean ± standard deviation (SD). The comparisons were detected by ANOVA, and Student–Newman–Keuls (SNK)
During storage period, the cystometry showed that no rat had bladder hyperactivity before modeling (
Cystometrograms.
The histological measurement showed that the epithelium was thicker in the IVES group (47.46 ± 11.98 μm) compared with the control group (34.35 ± 11.48 μm) or TENS group (29.40 ± 13.68 μm;
H&E staining and Masson's trichrome staining analysis.
Compared with the control group, IVES significantly reduced the α-SMA secretion in the submucosal layer (1.60 ± 0.57% vs. 2.47 ± 0.84%,
Immunohistochemical analysis.
At 7 days after AUR, the majority of DSMC appeared vacuolated due to dilation and disruption of cisternae of SR in the perinuclear region. Most DSMC displayed an extensive corrugation of the cell membrane with sarcoplasmic projections from 1 cell interlocking with deep infoldings of neighboring cells. After TENS or IVES treatment, myofilaments and folding of the cell membrane were regular (
TEM of detrusor muscle ultrastructure.
Our study revealed that AUR could affect the bladder function for more than 7 days. Cystometrical and histological results demonstrated that the effect of IVES treatment on AUR was significantly better than in the TENS. The intravesical probe material of titanium is safety and does not induce inflammation.
In cystometry study, AUR induced bladder hyperactivity and the bladder compliance became worse. We used post-void intravesical pressure and voiding time to determine the voiding function (
Some researchers observe inflammation in the submucosa 2 weeks after the induction of AUR in the rats' bladder (
Prolonged bladder overdistension has been deemed to induce stretch damage of the detrusor, denervation, and reduced contractility (
It is known that electrical stimulation acts directly not only on muscle fibers but also on nerve reflexes (
Holm et al. have pointed out that no significant ultrastructural features are noted in the detrusor muscle of 13 AUR patients (
The major strength of our study was the tetanic intravesical probe, and we used cystometrical and histological analyses to ensure its effect. However, there are also some limitations. The AUR rat model was not completely equal to the AUR patients, and much more animal models with lower urinary tract symptoms should be employed to confirm the efficacy of IVES. A sham group with catheter in the bladder and no stimulation would be added in further studies, which would control for histological changes of the bladder that may be secondary to the presence of the catheter and not by stimulation. There was an increase in the thickness of the detrusor in the IVES group, which might not be a desired event for patients with lower urinary tract dysfunction; thus, the high-resolution ultrasound will be added to observe and compare the
In conclusion, low-frequency IVES performed better than TENS in treating AUR. IVES relieved the bladder hyperactivity by reducing the submucosal fibroblasts, accelerated the epithelial recovery through increasing transitional cell layer and vascular amount, and strengthened the detrusor muscle by promoting α-SMA expression. As catheterization is still an adjuvant therapy for AUR patients, it is technically possible to place intravesical probe into the bladder through catheter without additional invasive intervention. Collectively, IVES could be a promising treatment for bladder dysfunction in clinical practice.
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.
The animal study was reviewed and approved by the Institutional Review Board (IRB) of the Peking University People's Hospital, Beijing, China (No. 2019PHE020).
TC, BX, and SY designed, performed, and interpreted the experiments. JiaqW offered the experimental materials. XY, BS, and XL participated in the experiments. TC and BX interpreted the data and edited the manuscript. XS and JianW finalized the manuscript. All authors contributed to the article and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.