Time-dependent progress of lower urinary tract dysfunction in streptozotocin-induced diabetic rats with or without low-dose insulin treatment.

Objectives: To examine time-dependent functional and structural changes of the lower urinary tract in streptozotocin-induced diabetic rats with or without low-dose insulin treatment and explore the pathophysiological characteristics of insulin therapy on lower urinary tract dysfunction (LUTD) caused by diabetes mellitus (DM). Methods: Female Sprague-Dawley rats were divided into five groups: normal control (NC) group, 4 weeks insulin-treated DM (4-DI) group, 4 weeks DM (4-DM) group, 8 weeks insulin-treated DM (8-DI) group and 8 weeks DM (8-DM) group. DM was initially induced by i.p. injection of streptozotocin (65 mg/kg), and then the DI groups received subcutaneous implantation of insulin pellets under the mid dorsal skin. Voiding behavior was evaluated in metabolic cages. The function of bladder and urethra in vivo were evaluated by simultaneous recordings of the cystometrogram and urethral perfusion pressure (UPP) under urethane anesthesia. The function of bladder and urethra in vitro were tested by organ bath techniques. The morphologic changes of the bladder and urethra were investigated using Hematoxylin-Eosin and Masson's staining. Results: Both 4-and 8-weeks diabetic rats have altered micturition patterns, including increased 12-h urine volume, urinary frequency/12 hours and voided volume. In-vivo urodynamics showed the EUS bursting activity duration is longer in 4-DM group and shorter in 8-DM group compared to NC group. UPP change in 8-DM were significantly lower than NC group. While none of these changes were found between DI and NC groups. Organ bath showed the response to Carbachol and EFS in bladder smooth muscle per tissue weights was decreased significantly in 4- and 8-weeks DM groups compared with insulin-treated DM or NC groups. In contrast, the contraction of urethral muscle and maximum urethral muscle contraction per gram of the tissue to EFS stimulation were significantly increased in 4- and 8-weeks DM groups. The thickness of bladder smooth muscle was time-dependently increased, but the thickness of the urethral muscle had no difference. Conclusions: DM-induced LUTD is characterized by time-dependent functional and structural remodeling in the bladder and urethra, which shows the hypertrophy of the bladder smooth muscle, reduced urethral smooth muscle relaxation and EUS dysfunction. Low-dose insulin can protect against diuresis-induced bladder over-distention, preserve urethral relaxation and protect EUS bursting activity, which would be helpful to study the slow-onset, time-dependent progress of DM-induced LUTD.

Study on the Fingerprint Spectrum and the Spectrum-Effect Relationship of Analgesic and Anti-Inflammatory Effects of the Aqueous Extract from Dalbergia hancai Benth.

Dalbergia hancai Benth. (D. hancai) is one of the most frequently utilized traditional Chinese medicine in Zhuang medicine. Simultaneously, it has been included in the "Quality Standard of Zhuang medicine in Guangxi Zhuang Autonomous Region (Vol. 2)" and possessed outstanding pharmacological effects. However, the pharmacodynamic material basis of D. hancai still remains unclear. In this study, the high-performance liquid chromatography (HPLC) method had been employed to establish the fingerprint of 10 batches of aqueous extract of D. hancai originated from different parts of China. At the same time, similarity evaluation, cluster analysis, and principal component analysis (PCA) had also been conducted to evaluate the common peaks. The acetic acid-induced writhing in mice had been employed as an analgesic model, and the carrageenan-induced toe swelling in mice was utilized as an anti-inflammatory model for pharmacodynamic experiments. The gray relational analysis (GRA) and partial least squares regression (PLSR) were applied to correlate the fingerprint and pharmacodynamic data to thoroughly examine its spectrum-effect relationship, whereby its analgesic and anti-inflammatory material basis had been comprehensively explored. The results revealed that the HPLC fingerprint of the aqueous extract of D. hancai had successfully identified 12 common peaks whereby two of which were further identified as protocatechuic acid and vitexin. Subsequently, through the analysis of GRA and PLSR, the chromatographic peaks that possess a critical correlation degree with the analgesic and anti-inflammatory effects of D. hancai had also been successfully discovered. Ultimately, the analgesic and anti-inflammatory effects of the 10 batches of D. hancai aqueous extract had been conclusively proved, and it was evidently indicated that these effects were attributable to the synergistic interactions between various components. Therefore, this study aims to serve as an effective analytical method for screening and predicting the effective substances of traditional Chinese medicine on the basis of the spectrum-effect relationship.

Spectrum-Effect Relationship between HPLC Fingerprint and Anti-Inflammatory and Analgesic Activities of Chloranthus fortunei (A. Gray) Solms-Laub.

The predominant objective of the research is to establish the anti-inflammatory and analgesic spectrum-effect relationship of Chloranthus fortunei (A. Gray) Solms-Laub (CF), to reveal the pharmacodynamic basis of the anti-inflammatory and analgesic effects of CF. The fingerprints of ten batches of CF from various origins were established by high-performance liquid chromatography (HPLC) and evaluated for similarity, hierarchical cluster analysis (HCA), and principal component analysis (PCA). The anti-inflammatory and analgesic effects of CF were evaluated with the xylene-induced ear swelling in mice and the acetic acid torsion test, while the anti-inflammatory and analgesic spectrum-effect relationship of CF was evaluated by gray relational analysis (GRA) and partial least squares regression analysis (PLSR) to effectively elucidate the anti-inflammatory and analgesic substance basis of CF. The ten batches of CF HPLC fingerprints established in this work successfully identified a total of 13 common peaks that refer to 4 components, with peak 1 being neochlorogenic acid, peak 3 being chlorogenic acid, peak 5 being cryptochlorogenic acid, and peak 10 being rosmarinic acid. The HCA results presented that the ten batches of CF samples were clustered into 3 categories, which was consistent with the PCA results. Simultaneously, the results of the spectrum-effect relationship also indicated that neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and rosmarinic acid were the possible anti-inflammatory and analgesic substances of CF. In order to better understand the anti-inflammatory and analgesic substance basis of CF, this experiment established the anti-inflammatory and analgesic spectrum-effect relationship of CF, which can provide a scientific foundation for the quality evaluation and further research as well as the usage of CF herbs.

Activation of the adenosine A1 receptor in the lumbosacral spinal cord improves bladder overactivity in rats with cystitis induced by cyclophosphamide.

PURPOSE: To investigate the effect of intrathecal administration of CCPA, an adenosine A1 receptor agonist, on voiding function in rats with cystitis induced by cyclophosphamide (CYP). METHODS: Thirty 8-week-old Sprague Dawley rats were randomly divided into a control group (n = 15) and a cystitis group (n = 15). Cystitis was induced by a single intraperitoneal injection of CYP (200 mg/kg, dissolved in physiological saline) in rats. Control rats were injected intraperitoneally with physiological saline. The PE10 catheter reached the level of L6-S1 spinal cord through L3-4 intervertebral space for intrathecal injection. Forty-eight hours after intraperitoneal injection, urodynamic tests were conducted to observe the effect of intrathecal administration of 10% dimethylsulfoxide (vehicle) and 1 nmol CCPA on micturition parameters, including basal pressure (BP), threshold pressure (TP), maximal voiding pressure (MVP), intercontraction interval (ICI), voided volume (VV), residual volume (RV), bladder capacity (BC), and voiding efficiency (VE). Histological changes of the bladder of cystitis rats were studied through hematoxylin-eosin staining (HE staining). Moreover, Western blot and immunofluorescence were used to study the expression of adenosine A1 receptor in the L6-S1 dorsal spinal cord in both groups of rats. RESULTS: HE staining revealed submucosal hemorrhage, edema, and inflammatory cell infiltration in the bladder wall of cystitis rats. The urodynamic test showed significant increase in BP, TP, MVP and RV in cystitis rats, while ICI, VV, BC and VE decreased significantly, indicating bladder overactivity. CCPA inhibited the micturition reflex in both control and cystitis rats, and significantly increased TP, ICI, VV, BC, and VE, but had no significant effect on BP, MVP and RV. Western blot and immunofluorescence showed that there was no significant difference in the expression of adenosine A1 receptor in the L6-S1 dorsal spinal cord between the control and cystitis rats. CONCLUSION: The findings of this study suggest that intrathecal administration of the adenosine A1 receptor agonist CCPA alleviates CYP-induced bladder overactivity. Furthermore, our results indicate that the adenosine A1 receptor in the lumbosacral spinal cord may be a promising target for treatment of bladder overactivity.

The potential involvement of MRP5 pump in urethral dysfunction in streptozotocin-induced diabetic rats.

PURPOSE: To examine the effects of i.v. administration of MK-571, a MRP4/5 pump inhibitor, on urethral function in the urethane-anesthetized rat, and the changes of urethral multidrug resistance protein 5 (MRP5) pump in streptozotocin (STZ)-induced diabetes mellitus (DM) rats. METHODS: Isovolumetric cystometry and urethral perfusion pressure (UPP) measurements were carried out in normal control (NC) group and 8week DM groups under urethane anesthesia. When stable rhythmic bladder contractions were showed, UPP parameters were recorded after successive administration of various dose of MK-571. Additionally, urethral cyclic guanosine monophosphate (cGMP) protein level was evaluated by ELISA, and changes of MRP5 pump and neurogenic nitric oxide synthase (nNOs) in the urethra were examined with immunohistochemical staining and Western blot analysis. RESULTS: In NC group, UPPnadir was significantly decreased but UPP change increased after administration of MK-571, while no significant differences in UPP parameters were observed in 8-week DM group. Furthermore, urethral MRP5 protein level was up-regulated, whereas urethral cGMP and nNOS protein levels were down-regulated in 8-week DM group. CONCLUSIONS: MK-571 could not restore NO-mediated urethral relaxation dysfunction in DM rats, which may be attributed to the up-regulation of urethral MRP5 pump, and thus decrease of intracellular cGMP concentration in the urethra. These novel results would be useful for a better understanding of DM-related lower urinary tract dysfunction LUT (LUTD). Also, they could be helpful to study the importance of MRP pumps in the control of urethral relaxation mechanisms under physiological and pathological states.

Spectrum-effect relationship between hplc fingerprint and anti-inflammatory activity of n-butanol parts of Tetrastigma planicaule (Hook) Gagnep.

The present study aimed to evaluate the spectrum-effect relationships between high-performance liquid chromatography fingerprints and anti-inflammatory effects of Tetrastigma planicaule(Hook.)Gagnep. Chemical fingerprints of ten batches of Tetrastigma planicaule from various sources were obtained by HPLC. The anti-inflammatory activity was investigated by a model of ear swelling in mice caused by xylene and a model of cotton pellet granuloma. Hierarchical cluster analysis (HCA) results showed that all the samples were clustered into four categories, which was basically consistent with the principal component analysis (PCA) results. The results of the joint grey relational analysis (GRA) and partial least squares regression analysis (PLSR) showed that peaks 1, 2 and 12 were positively correlated with the anti-acute inflammatory effect (ear swelling) in mice, and peaks 3, 5, 6 and 11 were positively correlated with the anti-chronic inflammatory effect (cotton pellet granuloma) in mice. The anti-inflammatory effect of Tetrastigma planicaule is the result of the synergistic effect of multiple components, which provides a basis for further exploring the anti-inflammatory substances and quality evaluation of the herb.

Improvement of urethral dysfunction by 5-HT1A receptor agonist NLX-112 in diabetic rats.

OBJECTIVE: To examine the effects of the selective 5-HT1A receptor agonist, NLX-112, on urethral function in streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Female Sprague-Dawley rats (n = 32) were divided into two groups: rats with type 1 diabetes mellitus (T1DM) and age-matched normal control rats (NC). T1DM was induced by intraperitoneal injection of streptozotocin (65 mg/kg). Isovolumetric cystometry and urethral perfusion pressure (UPP) were evaluated 10 weeks postinjection in rats (n = 9 per group). The selective 5-HT1A receptor antagonist, WAY-100635 maleate salt, was administered after NLX-112 hydrochloride dose-response curve was generated (intravenously). The remaining rats were used for immunofluorescence and Western blot assays. RESULTS: Compared to controls, type 1 diabetic rats (T1D rats) had lower maximal intravesical pressure (IP max) and UPP changes. In T1D rats, NLX-112 hydrochloride (0.003-1.0 mg/kg) induced dose-dependent decreases in UPP nadir, IP max, high-frequency oscillations (HFOs) rate; and increases in UPP change and HFOs amplitude. WAY-100635 maleate salt (0.3 mg/kg) partially or completely reversed the NLX-112-induced changes. Immunofluorescence revealed that 5-HT1A receptors were found in the L6-S1 spinal cord dorsolateral nucleus, but the expression was significantly higher in the T1D rats. Additionally, Western blot showed there were significantly more 5-HT1A receptors in the ventral L6-S1 spinal cord of T1D rats. CONCLUSIONS: Urethral dysfunction in T1D rats was improved by NLX-112. 5-HT1A receptors were upregulated in the dorsolateral nucleus of L6-S1 spinal cord in T1D rats. These findings suggest that NLX-112 may constitute a novel therapeutic strategy to treat diabetic urethral dysfunction.

Study on the Spectrum-Effect Correlation of Anti-Inflammatory Active Extract of Sauropus spatulifolius Beille.

Sauropus spatulifolius Beille (S. spatulifolius) is a commonly used medicine of the Bourau and Yao nationalities. However, the composition of S. spatulifolius is complex, and simple chemical fingerprints cannot accurately evaluate the relationship between its composition and efficacy. In this study, high-performance liquid chromatography (HPLC) method was used to establish the fingerprint of the ethyl acetate extract of S. spatulifolius. Based on the evaluation of the similarity of chromatographic fingerprints of traditional Chinese medicine, combined with cluster analysis and principal component analysis (PCA), the common peaks of fingerprints were evaluated. The anti-inflammatory effect data were extracted through the dimethylbenzene-induced ear-swelling model in mice. The gray relational analysis (GRA) combined with partial least squares regression (PLSR) was used to study the spectrum-effect correlation of S. spatulifolius. As a result, the HPLC fingerprint of the ethyl acetate extract of S. spatulifolius was established, and 18 common peaks were identified. Except for S6, the other similarities are all above 0.915. The reference substance control method was used to identify two absorption peaks, namely, protocatechuic acid and caffeic acid. The cluster analysis results showed that 10 samples from different origins were grouped into four categories, which was consistent with the PCA results. Ethyl acetate extract of 10 batches of S. spatulifolius could significantly inhibit the ear swelling of mice (P < 0.01). Through GRA, the order of the contribution of each chemical component to the anti-inflammatory efficacy was obtained. The results of PLSR showed that the VIP values of peaks 3, 4, and 12 were greater than 1 and were positively correlated with anti-inflammatory activity. In this study, the HPLC fingerprint of the ethyl acetate extract of S. spatulifolius was established. Through the study of the spectrum-effect correlation, the anti-inflammatory active substance of the ethyl acetate extract of S. spatulifolius was obtained. The anti-inflammatory effect of S. spatulifolius was the result of the joint action of multiple ingredients. This research helps to quickly and accurately discover the active ingredient groups of traditional Chinese medicine and provides new ideas and methods for studying the effective substances of traditional Chinese medicine.

Urodynamic study findings and related influential factors in pediatric spastic cerebral palsy.

To investigate the urodynamic study (UDS) result in pediatric patients suffering from spastic cerebral palsy (CP). Medical records of patients diagnosed CP having pre-operative UDS results underwent selective dorsal rhizotomy (SDR) from Jan. 2020 to May. 2021 were retrospectively reviewed. Fifty-seven cases diagnosed spastic CP were included in the study (mean age, 6.73 ± 2.84 years), among which, 46 were ambulatory and 11 non-ambulatory. Average gross motor function measure-66 (GMFM-66) score was 62.16 ± 11.39. Reduced bladder capacity was seen in 49.12% of these cases and cases with lower GMFM-66 score possessed a higher incidence rate of having low bladder capacity (p < 0.01). Detrusor overactivity (DO) was shown in 33.33% of the patients. Cases with younger age presented a higher prevalence of DO (p < 0.05). Meanwhile, more non-ambulant patients suffered from DO (p < 0.05). Increased post-voiding residual (PVR) was seen in 21.05% of the cases. Those with higher average threshold in sphincter-associated input spinal nerve roots (rootlets) witnessed a higher rate of having abnormal PVR (p < 0.05). Abnormal UDS results were prevalent in pediatric patients suffering from CP. Motor function, age and threshold of their sphincter-associated spinal nerve roots laid corresponding effect on the abnormal UDS results.

Nerve growth factor-mediated Na+ channel plasticity of bladder afferent neurons in mice with spinal cord injury.

AIM: To investigate the effect of nerve growth factor (NGF) neutralization on Na+ channel plasticity of bladder afferent neurons in mice with spinal cord injury (SCI). MAIN METHODS: Female C57/BL6 mice were randomly divided into spinal intact (SI) group, SCI group and SCI + NGF-Ab group. SCI was induced by spinal cord transection at the Th8/9 level. In SCI + NGF-Ab group, anti-NGF antibodies (10 µg·kg-1 per hour) were continuously administered for 2 weeks using osmotic pumps. Bladder afferent neurons were labelled with Fluoro­gold (FG) injected into the bladder wall. L6-S1 dorsal root ganglion (DRG) neurons were dissociated and whole-cell patch clamp recordings were performed on FG-labelled neurons. Expression of Nav1.7 and Nav1.8 was examined by immunofluorescent staining. KEY FINDINGS: Whole-cell patch clamp recordings showed that TTX only partially inhibited action potentials (AP) and Na+ currents of bladder afferent neurons in SI mice, but it almost completely inhibited them in SCI mice. Total and TTX-sensitive Na+ currents were increased and TTX-resistant currents were decreased in bladder afferent neurons from SCI mice vs. SI mice. These changes in SCI mice were significantly reversed by NGF-antibody treatment. Immunostaining results showed the increased and decreased levels of Nav1.7 and Nav1.8, respectively, in FG-labelled bladder afferent neurons in SCI mice vs. SI mice, which was significantly reversed in SCI + NGF-Ab mice. SIGNIFICANCE: NGF mediates the Na+ channel plasticity with a shift from TTX-resistant Nav1.8 to TTX-sensitive Nav1.7 in bladder afferent neurons, which could be a possible underlying mechanism of bladder afferent hyperexcitability and detrusor overactivity after SCI.

A Lipid-Nanosphere-Small MyoD Activating RNA-Bladder Acellular Matrix Graft Scaffold [NP(saMyoD)/BAMG] Facilitates Rat Injured Bladder Muscle Repair and Regeneration [NP(saMyoD)/BAMG].

BACKGROUND: Bladder tissue engineering is an excellent alternative to conventional gastrointestinal bladder enlargement in the treatment of various acquired and congenital bladder abnormalities. We constructed a nanosphere-small MyoD activating RNA-bladder acellular matrix graft scaffold NP(saMyoD)/BAMG inoculated with adipose-derived stem cells (ADSC) to explore its effect on smooth muscle regeneration and bladder repair function in a rat augmentation model. METHODS: We performed many biotechniques, such as reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, MTT assay, HE staining, masson staining, and immunohistochemistry in our study. Lipid nanospheres were transfected into rat ADSCs after encapsulate saRNA-MyoD as an introduction vector. Lipid nanospheres encapsulated with saRNA-MyoD were transfected into rat ADSCs. The functional transfected rat ADSCs were called ADSC-NP(saMyoD). Then, Rat models were divided into four groups: sham group, ADSC-BAMG group, ADSC-NP(saMyoD)/BAMG group, and ADSC-NP(saMyoD)/SF(VEGF)/BAMG group. Finally, we compared the bladder function of different models by detecting the bladder histology, bladder capacity, smooth muscle function in each group. RESULTS: RT-PCR and Western blot results showed that ADSCs transfected with NP(saMyoD) could induce high expression of α-SMA, SM22α, and Desmin. At the same time, MTT analysis showed that NP(saMyoD) did not affect the activity of ADSC cells, suggesting little toxicity. HE staining and immunohistochemistry indicated that the rat bladder repair effect (smooth muscle function, bladder capacities) was better in the ADSC-NP(saMyoD)/BAMG group, ADSC-NP(saMyoD)/SF(VEGF)/BAMG group than in the control group. CONCLUSIONS: Taken together, our results demonstrate that the NP(saMyoD)/SF(VEGF)/BAMG scaffold seeded with ADSCs could promote bladder morphological regeneration and improved bladder urinary function. This strategy of ADSC-NP(saMyoD)/SF(VEGF)/BAMG may has a potential to repair bladder defects in the future.

Urethral dysfunction and alterations of nitric oxide mechanisms in streptozotocin-induced diabetic rats with or without low-dose insulin treatment.

AIMS: To establish an animal model of diabetes mellitus (DM) with moderately elevated blood glucose levels, and to examine the nitric oxide (NO) mechanism controlling urethral function in streptozotocin (STZ)-induced DM rats. MAIN METHODS: Female Sprague-Dawley rats were used. DM was induced by intraperitoneal injection of STZ (65 mg/kg) and some of them received subcutaneous implantation of a low-dose insulin pellet. Voiding behavior was evaluated in metabolic cages. Isovolumetric cystometry and urethral perfusion pressure (UPP) were then evaluated under urethane anesthesia, during which L-arginine (100 mg/kg) and N-nitro-L-arginine methyl ester hydrochloride (L-NAME) (50 mg/kg) were administered intravenously. In vitro urethral activity was also tested by organ bath muscle strip studies. KEY FINDINGS: UPP changes and high-frequency oscillation (HFO) were significantly (P < 0.05) smaller in 8-weeks DM rats vs. normal control (NC) rats or insulin-treated DM rats, which showed reductions in urine overproduction and voided volume per micturition vs. untreated DM rats. UPP nadir was decreased by L-arginine in NC and insulin-treated DM groups, and decreased by L-NAME in all groups. Five of 6 untreated DM rats showed a detrusor-sphincter dyssynergia pattern after L-NAME. In in vitro studies, the relative ratio of L-NAME-induced reductions of urethral relaxation against pre-drug urethral relaxation was significantly smaller in DM vs. NC rats (P < 0.05). SIGNIFICANCE: Low-dose insulin-treated DM rats would be a useful model for studying natural progression of DM-induced lower urinary tract dysfunction. The impaired NO-mediated urethral relaxation mechanisms play an important role in DM-induced urethral dysfunction, which could contribute to DM-induced inefficient voiding.

Time-Dependent Changes of Urethral Function in Diabetes Mellitus: A Review.

This article reviewed the current knowledge on time-course manifestation of diabetic urethral dysfunction (DUD), and explored an early intervention target to prevent the contribution of DUD to the progression of diabetes-induced impairment of the lower urinary tract (LUT). In the literature search through PubMed, key words used included "diabetes mellitus," "diabetic urethral dysfunction," and "diabetic urethropathy." Polyuria and hyperglycemia induced by diabetes mellitus (DM) can cause the time-dependent changes in functional and morphological manifestations of DUD. In the early stage, it promotes urethral dysfunction characterized by increased urethral pressure during micturition. However, the detrusor muscle of the bladder tries to compensate for inducing complete voiding by increasing the duration and amplitude of bladder contractions. As the disease progresses, it can induce an impairment of coordinated micturition due to dyssynergic activity of external urethra sphincter, leading to detrusor-sphincter dyssynergia. The impairment of relaxation mechanisms of urethral smooth muscles (USMs) may additionally be attributable to decreased responsiveness to nitric oxide, as well as increased USM responsiveness to α1-adrenergic receptor stimulation. In the late stage, diabetic neuropathy may play an important role in inducing LUT dysfunction, showing that the decompensation of the bladder and urethra, which can cause the decrease of voiding efficiency and the reduced thickness of the urothelium and the atrophy of striated muscle bundles, possibly leading to the vicious cycle of the LUT dysfunction. Further studies to increase our understandings of the functional and molecular mechanisms of DUD are warranted to explore potential targets for therapeutic intervention of DM-induced LUT dysfunction.

Long-lasting bladder overactivity and bladder afferent hyperexcitability in rats with chemically-induced prostatic inflammation.

BACKGROUND: Benign prostatic hyperplasia (BPH) is one of the major causes of lower urinary tract symptoms (LUTS), including storage LUTS such as urinary frequency and urgency. Recently, a growing number of clinical studies indicate that prostatic inflammation could be an important pathophysiological mechanism inducing storage LUTS in patients with BPH. Here we aimed to investigate whether nonbacterial prostatic inflammation in a rat model induced by intraprostatic formalin injection can lead to long-lasting bladder overactivity and changes in bladder afferent neuron excitability. METHODS: Male Sprague-Dawley rats were divided into four groups (n = 12 each): normal control group, 1-week prostatic inflammation group, 4-week inflammation group, and 8-week inflammation group. Prostatic inflammation was induced by formalin (10%; 50 µL per lobe) injection into bilateral ventral lobes of the prostate. Voiding behavior was evaluated in metabolic cages for each group. Ventral lobes of the prostate and the bladder were then removed for hematoxylin and eosin (HE) staining to evaluate inflammation levels. Continuous cystometrograms (CMG) were recorded to measure intercontraction intervals (ICI) and voided volume per micturition. Whole-cell patch clamp recordings were performed on dissociated bladder afferent neurons labeled by fluorogold injected into the bladder wall, to examine the electrophysiological properties. RESULTS: Results of metabolic cage measurements showed that formalin-treated rats exhibited significantly (P < 0.05) increases in micturition episodes/12 hours and decrease in voided volume per micturition at every time point post injection. Continuous CMG illustrated the significant ( P < 0.05) higher number of nonvoiding contractions per void and shorter ICI in formalin-treated rats compared with control rats. HE staining showed significant prostatic inflammation, which declined gradually, in prostate tissues of formalin-induced rats. In patch clamp recordings, capsaicin-sensitive bladder afferent neurons from rats with prostatic inflammation had significantly ( P < 0.05) lower thresholds for spike activation and a "multiple" firing pattern compared with control rats at every time point post injection. CONCLUSIONS: Formalin-induced prostatic inflammation can lead to long-lasting bladder overactivity in association with bladder afferent neuron hyperexcitability. This long-lasting model could be a useful tool for the study of inflammation-related aspects of male LUTS pathophysiology.

Streptozotocin-induced diabetes causes upregulation of serotonin (5-HT)2A/C receptors in lumbosacral cord motoneurons and down regulation of serotonergic paraneurons in the urethra.

Lower urinary tract (LUT) dysfunction is the most common complication of diabetes mellitus (DM). An involvement of the 5-HT2A receptor in spinal micturition control has been demonstrated in urethane anaesthetized DM rats in which i.v. administration of the 5-HT2A/2C receptor agonist 2,5-methoxy-4-iodoamphetamine (DOI), stimulated high frequency oscillations (HFOs) and improved micturition. However, the mechanisms involved in these effects are not completely understood. The present work showed that 5-HT2A and -2C receptors were upregulated in lumbosacral cord motoneurons, and the number of serotonergic paraneurons were downregulated in the urethra in DM group. The importance of the downregulation of urethral paraneurons in DM remains to be elucidated but may be related to the reduced urethral sensation caused by the disease. We suggest that targets of 5-HT receptor agonists for improvement of voiding function may be found both in the LUT and lumbosacral spinal cord.

The serotonin (5-hydroxytryptamine) 5-HT7 receptor is up-regulated in Onuf's nucleus in rats with chronic spinal cord injury.

OBJECTIVES: To examine the effect of intrathecal (i.t.) serotonin (5-hydroxytryptamine) 5-HT7 agonist administration on voiding function in the urethane-anesthetised rat, and the change in 5-HT7 receptor (5-HT7 R) expression in the lumbosacral cord Onuf's nucleus after spinal cord injury (SCI). MATERIALS AND METHODS: In all, 32 female Sprague-Dawley (SD) rats were equally divided into a spinally intact (SI) group and SCI group (n = 16 each). At 8 weeks after transection, half of the rats underwent continuous cystometry under urethane anaesthesia, and the 5-HT7 R-selective agonist LP44 was given (i.t.). The remaining rats were used for pseudorabies (PRV) retrograde tracing, immunofluorescence, and Western Blot. RESULTS: LP44 administered i.t. had no effect in the SI rats. In SCI rats, LP44 (1-30 µg/kg) induced significant dose-dependent increases in micturition volume, voiding efficiency, number of high-frequency oscillations per micturition; and decreases in residual volume, bladder capacity, peak bladder pressure, threshold pressure and non-voiding contractions. The 5-HT7 R antagonist, SB-269970 (10 µg/kg), partially reversed LP44-induced changes. Using PRV retrograde tracing and immunofluorescence, 5-HT7 Rs were found in the L6-S1 spinal cord Onuf's nucleus in both SI and SCI rats, but the expression was significantly greater in the SCI rats. Western blot showed significantly more 5-HT7 Rs in the ventral L6-S1 spinal cord in SCI rats. CONCLUSION: A 5-HT7 R agonist, given i.t., improved voiding efficiency in urethane-anesthetised SCI rats, and the 5-HT7 R was significantly up-regulated in the lumbosacral cord Onuf's nucleus. If valid for humans, these findings suggest that the 5-HT7 R could be a target for therapeutic interventions.

Effect of 5-HT2A receptor antagonist ketanserin on micturition in male rats.

OBJECTIVES: This study aimed to investigate the effects of ketanserin on micturition mediated via the 5-HT2A receptor in the motoneuron nucleus of the Lumbosacral cord, as reflected in high frequency oscillations (HFOs) of intravesical pressure and the external urethral sphincter electromyogram (EUS-EMG) in anesthetized male rats. METHODS：: Male Sprague-Dawley rats were used. Cystometry and EUS-EMG were performed in all rats under urethane anesthesia to examine the variations after successive intrathecal (i.t.) administration of various doses of ketanserin into the lumbosacral cord. Immunofluorescence staining and Western blotting were made to observe the distribution of 5-HT2 A and -2C receptors in the lumbosacral cord motor neurons. RESULTS: Compared to the controls, ketanserin-treated rats showed a declined trend of dose-dependent manner in the HFOs, in accordance with the variation of EUS-EMG, while decreased micturition volume, voiding efficiency, and increased post-void residual volume was only observed at the dose of 0.1 mg/kg. The effects of ketanserin on the HFO and EUS-EMG activity were partially or completely reversed by the 5-HT2A/2C receptor agonist, DOI. Meanwhile, immunofluorescence staining and Western blot analysis showed that immunoreactivity of 5-HT2A receptor was higher than that of 5-HT2C, labeling in the lumbosacral cord motoneurons. CONCLUSIONS: The intrathecally administrated 5-HT2A receptor antagonist ketanserin can weaken the EUS bursting activity, decrease HFOs, and reduce voiding efficiency as dose dependently. The effects of ketanserin on micturition may be mainly mediated via the 5-HT2A receptors in the motoneuron nucleus of the lumbosacral cord.

Prevascularized bladder acellular matrix hydrogel/silk fibroin composite scaffolds promote the regeneration of urethra in a rabbit model.

OBJECTIVE: To evaluate the effects of urethral regeneration with prevascularized bladder acellular matrix hydrogel (BAMH)/silk fibroin (SF) composite scaffolds in a rabbit model. MATERIALS AND METHODS: BAMH/SF and collagen Type I hydrogel/SF (CH/SF) scaffolds were prepared and the structure of the scaffolds was assessed using scanning electron microscopy. BAMH/SF, CH/SF and SF scaffolds were incubated in the omentum of male rabbits for two weeks and then harvested for repairing autologous urethral defects. Histological analysis of the incubated scaffolds was performed to evaluate the neovascularization capacity, and the outcomes of urethroplasty were evaluated at one and three months post-operatively. RESULTS: The composited scaffolds were composed of a highly porous BAMH or CH buttressed by compact SF outer layer. The histological analysis of the incubated BAMH/SF revealed a signifcant increase of the neovascularization among three groups after a two-week incubation. At three months, the urethra maintained wide caliber in the BAMH/SF group. Strictures were found in the CH/SF and SF groups. Histologically, at one month, intact and multilayer epithelium occurred in the BAMH/SF group, and one layer epithelium was found in the CH/SF and SF groups. However, there was similar epithelial regeneration in BAMH/SF and CH/SF groups at three months (p > 0.05). Comparisons of smooth muscle content and vessel density among the SF, CH/SF and BAMH/SF revealed a significant increase at each time point (p < 0.05). CONCLUSION: Our results demonstrate that incubated BAMH/SF promote neovascularization, and prevascularized BAMH/SF promote the regeneration of the urethral epithelium and smooth muscle, which indicates its potential for urethral reconstruction.

Therapeutic Potential of Human Adipose-Derived Stem Cell Exosomes in Stress Urinary Incontinence - An in Vitro and in Vivo Study.

BACKGROUND/AIMS: To evaluate whether local injection of exosomes derived from human adipose-derived stem cells (hADSCs) facilitates recovery of stress urinary incontinence (SUI) in a rat model. METHODS: For the in vitro study, a Cell Counting Kit-8 (CCK-8) array and proteomic analysis were performed. For the in vivo study, female rats were divided into four groups: sham, SUI, adipose-derived stem cell (ADSC), and exosomes (n = 12 each). The SUI model was generated by pudendal nerve transection and vaginal dilation. Vehicle, hADSCs, or exosomes were injected into the peripheral urethra. After 2, 4, and 8 weeks, the rats underwent cystometrography and leak point pressure (LPP) testing, and tissues were harvested for histochemical analyses. RESULTS: The CCK-8 experiment demonstrated that ADSC-derived exosomes could enhance the growth of skeletal muscle and Schwann cell lines in a dose-dependent manner. Proteomic analysis revealed that ADSC-derived exosomes contained various proteins of different signaling pathways. Some of these proteins are associated with the PI3K-Akt, Jak-STAT, and Wnt pathways, which are related to skeletal muscle and nerve regeneration and proliferation. In vivo experiments illustrated that rats of the exosome group had higher bladder capacity and LPP, and had more striated muscle fibers and peripheral nerve fibers in the urethra than rats of the SUI group. Both urethral function and histology of rats in the exosome group were slightly better than those in the ADSC group. CONCLUSIONS: Local injection of hADSC-derived exosomes improved functional and histological recovery after SUI.