Microenergy acoustic pulse therapy restores urethral wall integrity and continence in a rat model of female stress incontinence.

OBJECTIVE: To determine the outcomes and mechanisms of microenergy acoustic pulse (MAP) therapy in an irreversible rat model of female stress urinary incontinence. MATERIALS AND METHODS: Twenty-four female Sprague-Dawley rats were randomly assigned into four groups: sham control (sham), vaginal balloon dilation and ovariectomy (VBDO), VBDO + ß-aminopropionitrile (BAPN), and VBDO + ß-aminopropionitrile treated with MAP (MAP). MAP therapy was administered twice per week for 4 weeks. After a 1-week washout period, all 24 rats were evaluated with functional and histological studies. The urethral vascular plexus was examined by immunofluorescence staining with antibodies against collagen IV and von Willebrand factor (vWF). The urethral smooth muscle stem/progenitor cells (uSMPCs) were isolated and functionally studied in vivo and in vitro. RESULTS: Functional study with leak point pressure (LPP) measurement showed that the MAP group had significantly higher LPPs compared to VBDO and BAPN groups. MAP ameliorated the decline in urethral wall thickness and increased the amount of extracellular matrix within the urethral wall, especially in the urethral and vaginal elastic fibers. MAP also improved the disruption of the urethral vascular plexus in the treated animals. In addition, MAP enhanced the regeneration of urethral and vaginal smooth muscle, and uSMPCs could be induced by MAP to differentiate into smooth muscle and neuron-like cells in vitro. CONCLUSION: MAP appears to restore urethral wall integrity by increasing muscle content in the urethra and the vagina and by improving the urethral vascular plexus and the extracellular matrix.

Exosome Released From Schwann Cells May Be Involved in Microenergy Acoustic Pulse-Associated Cavernous Nerve Regeneration.

BACKGROUND: Neurogenic erectile dysfunction (ED) is often refractory to treatment because of insufficient functional nerve recovery after injury or insult. Noninvasive mechano-biological intervention, such as microenergy acoustic pulse (MAP), low-intensity pulsed ultrasound, and low-intensity extracorporeal shockwave treatment, is an optimal approach to stimulate nerve regeneration. AIM: To establish a new model in vitro to simulate nerve injury in neurogenic ED and to explore the mechanisms of MAP in vitro. METHODS: Sprague-Dawley rats were used to isolate Schwann cells (SCs), major pelvic ganglion (MPG), and cavernous nerve with MPG (CN/MPG). SCs were then treated with MAP (0.033 mJ/mm2, 1 Hz, 100 pulses), and SC exosomes were isolated. The MPG and CN/MPG were treated with MAP (0.033 mJ/mm2, 1 Hz) at different dosages (25, 50, 100, 200, or 300 pulses) or exosomes derived from MAP-treated SCs in vitro. OUTCOMES: Neurite growth from the MPG fragments and CN was photographed and measured. Expression of neurotropic factors (brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3) was checked. RESULTS: Neurite outgrowth from MPG and CN/MPG was enhanced by MAP in a dosage response manner, peaking at 100 pulses. MAP promoted SC proliferation, neurotropic factor (brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3) expression, and exosome secretion. SC-derived exosomes significantly enhanced neurite outgrowth from MPG in vitro. CLINICAL IMPLICATIONS: MAP may have utility in the treatment of neurogenic ED by SC-derived exosomes. STRENGTH & LIMITATIONS: We confirmed that MAP enhances penile nerve regeneration through exsomes. Limitations of this study include that our study did not explore the exact mechanisms of how MAP increases SC exosome secretion nor whether MAP modulates the content of exosomes. CONCLUSION: This study revealed that neurite outgrowth from MPG was enhanced by MAP and by SC-derived exosomes which were isolated after MAP treatment. Our findings indicate that one mechanism by which MAP induces nerve regeneration is by stimulation of SCs to secrete exosomes. Peng D, Reed-Maldonado AB, Zhou F, et al. Exosome Released From Schwann Cells May Be Involved in Microenergy Acoustic Pulse-Associated Cavernous Nerve Regeneration. J Sex Med 2020;17:1618-1628.

Smooth Muscle Differentiation of Penile Stem/Progenitor Cells Induced by Microenergy Acoustic Pulses In Vitro.

INTRODUCTION: Modulating tissue-resident stem and progenitor cells with a non-invasive, mechanobiological intervention is an optimal approach for tissue regeneration. Stem cell antigen-1 (Sca-1) has been identified as a stem cell marker within many organs but never within the penis. AIM: To localize and isolate penile stem/progenitor cells (PSPCs) and to evaluate cellular differentiation after exposure to induction medium and microenergy acoustic pulse (MAP) therapy. METHODS: Six male Sprague-Dawley rats were used to isolate PSPCs. Isolation was followed by stem cell characterization and differentiation assays. The PSPCs were then treated with MAP (0.033 mJ/mm2, 1 Hz) at various dosages (25, 50, 100, and 200 pulses) and for different durations (1, 2, 4, 6, or 8 hours) in vitro. MAIN OUTCOME MEASURE: The PSPCs (Sca-1-positive cells) were isolated using the magnetic-activated cell sorting system. PSPC cellular differentiation was assessed after induction with induction medium and with MAP in vitro. Wnt/ß-catenin signaling was also assayed. RESULTS: The PSPCs were successfully localized within the penile subtunic and perisinusoidal spaces, and they were successfully isolated using magnetic-activated cell sorting. The stemness of the cells was confirmed by stem cell marker characterization and by multiple differentiation into smooth muscle cells, endothelial cells, adipocytes, and neurons. MAP-induced PSPCs differentiated into smooth muscle cells by activating the Wnt/ß-catenin signaling pathway in a time- and dosage-dependent manner. CLINICAL IMPLICATIONS: By modulating resident PSPCs, MAP may have utility in the treatment of erectile dysfunction (ED). STRENGTHS & LIMITATIONS: This study provides solid evidence in support of microenergy therapies, including both MAP and low-intensity extracorporeal shock wave therapy, for the treatment of ED. Additional studies are needed and should include additional stem cells markers. Furthermore, studies exploring the underling mechanisms for PSPC activation and differentiation are required. CONCLUSION: PSPCs were successfully identified, localized, and isolated. Additionally, MAP provoked PSPCs to differentiate into smooth muscle cells via the Wnt/ß-catenin signaling pathway. As such, MAP provides a novel method for activating endogenous tissue-resident stem/progenitor cells and might facilitate stem cell regenerative therapy targeting ED. Peng D, Yuan H, Liu T, et al. Smooth Muscle Differentiation of Penile Stem/Progenitor Cells Induced by Microenergy Acoustic Pulses In Vitro. J Sex Med 2019; 16:1874-1884.

Delayed Low-Intensity Extracorporeal Shock Wave Therapy Ameliorates Impaired Penile Hemodynamics in Rats Subjected to Pelvic Neurovascular Injury.

BACKGROUND: Erectile dysfunction (ED) caused by pelvic neurovascular injury (PNVI) is often refractory to treatment. In many cases, erectogenic therapy is administered in a delayed fashion. AIM: To evaluate penile hemodynamic effects and histologic changes associated with delayed low-intensity extracorporeal shock wave therapy (Li-ESWT) after PNVI ED in a rat model. We visualized images using immunofluorescence and 3-dimensional imaging of solvent-cleared organs (3DISCO), a novel imaging technique. METHODS: A total of 32 Sprague-Dawley male rats aged 12 weeks were divided equally into 4 groups: sham surgery as normal controls (NC), PNVI controls (PC), PNVI with very-low-energy Li-ESWT (PVL), and PNVI with low-energy Li-ESWT (PL). Bilateral cavernous nerve crush and internal pudendal bundle ligation were performed in the 3 PNVI groups. Li-ESWT was administered twice a week for 4 weeks in the PL and PVL groups starting at 4 weeks after PNVI. OUTCOMES: Intracavernous pressure (ICP) studies (normalized to mean arterial pressure [MAP]) were conducted in all subject animals. After testing, tissue was harvested for immunofluorescence staining and 3DISCO analysis. RESULTS: Mean ICP/MAP was lower in PC animals compared with NC animals (0.37 ± 0.03 vs 0.91 ± 0.03, respectively; P = .001). The ICP/MAP ratio was significantly higher in PVL and PL animals (0.66 ± 0.07 and 0.82 ± 0.05, respectively) compared with PC animals (P = .002 and .001, respectively). Detailed microstructures and trajectories of nerves and vessels were identified with immunofluorescence and 3DISCO. The PC group had lower density of nerves, axons, neuronal nitric oxide synthase-positive nerves, and Schwann cells in the dorsal penis. Animals in the PL group had significantly higher expression of all of these markers compared with PC animals. CLINICAL IMPLICATIONS: Li-EWST may have utility in the management of severe ED related to PNVI from severe pelvic injury or radical pelvic surgeries, even when administered in a delayed fashion. STRENGTH & LIMITATIONS: This study of a severe ED phenotype involved treatment administered in a delayed fashion, which is more consistent with how therapy likely would be delivered in a real-world clinical context. Moreover, because the treatment commenced at 4 weeks after injury, when nerve and tissue atrophy have already occurred, the results imply that Li-ESWT can be used for regenerative therapy. Additional studies on dose optimization and treatment interval are needed to inform the design of human clinical trials. CONCLUSION: Li-ESWT ameliorates the negative functional and histologic effects of severe pelvic neurovascular injury in a rat model system. 3DISCO provides high-resolution images of neuroanatomy and neural regeneration. Wang HS, Ruan Y, Banie L, et al. Delayed Low-Intensity Extracorporeal Shock Wave Therapy Ameliorates Impaired Penile Hemodynamics in Rats Subjected to Pelvic Neurovascular Injury. J Sex Med 2019;16:17-26.

The effects of microenergy acoustic pulses on animal model of obesity-associated stress urinary incontinence. Part 2: In situ activation of pelvic floor and urethral striated muscle progenitor cells.

AIM: To investigate the possibility and mechanism of microenergy acoustic pulses (MAP) for activating tissue resident stem/progenitor cells within pelvic and urethral muscle and possible mechanism. METHODS: The female Zucker Lean and Zucker Fatty rats were randomly divided into four groups: ZL control, ZLMAP, ZF control, and ZFMAP. MAP was applied at 0.033 mJ/mm2 , 3 Hz for 500 pulses, and the urethra and pelvic floor muscles of each rat was then harvested for cell isolation and flow cytometry assay. Freshly isolated cells were analyzed by flow cytometry for Pax-7, Int-7α, H3P, and EdU expression. Meanwhile, pelvic floor muscle-derived stem cells (MDSCs) were harvested through magnetic-activated cell sorting, MAP was then applied to MDSCs to assess the mechanism of stem cell activation. RESULTS: Obesity reduced EdU-label-retaining cells and satellite cells in both pelvic floor muscle and urethra, while MAP activated those cells and enhanced cell proliferation, which promoted regeneration of striated muscle cells of the pelvic floor and urethral sphincter. Activation of focal adhesion kinase (FAK)/AMP-activated protein kinase (AMPK) /Wnt/ß-catenin signaling pathways by MAP is the potential mechanism. CONCLUSIONS: MAP treatment activated tissue resident stem cells within pelvic floor and urethral muscle in situ via activating FAK-AMPK and Wnt/ß-catenin signaling pathway.

The effects of microenergy acoustic pulses on an animal model of obesity-associated stress urinary incontinence. Part 1: Functional and histologic studies.

AIM: Obesity is a strong independent risk factor for urinary incontinence. Effective therapeutic approaches for obesity-associated stress urinary incontinence (OA-SUI) are lacking as the mechanisms remain unclear. The aim of our study is to explore the impacts of microenergy acoustic pulse (MAP) therapy on urethral and pelvic floor muscle structure and function in female lean and fatty rats. METHODS: A total 24 Zucker fatty (ZF) and 24 Zucker lean (ZL) female 24-week-old rats were grouped into four groups: ZL control, ZLMAP, ZF control, and ZFMAP. For MAP treatment, 500 pulses were delivered at an energy level of 0.033 mJ/mm 2 and a frequency of 3 Hz and were applied twice a week for 4 weeks. After a 1-week washout, all rats underwent conscious cystometry and leak-point pressure (LPP) measurements followed by ex vivo organ-bath assay and histological study. RESULTS: ZF rats had lower LPP as compared to ZL rats, and MAP treatment significantly improved LPP in ZF rats (P < .05). Impaired muscle contractile activity (MCA) in organ-bath study was noted in ZF rats. MAP treatment significantly increased MCA in ZF rats (P < .05) and also increased the thickness of the striated muscle layer and the number of neuromuscular junctions (NMJs). In situ, MAP activated muscle satellite cells significantly (P < .05). CONCLUSIONS: Obesity impairs the function of both the urethral sphincter and the pelvic floor and leads to atrophy and distortion of the striated muscle in obese female rats. These issues contribute to OA-SUI. MAP improves continence by stimulating muscle regeneration and nerve innervation as well as by activating satellite cells.

The effect of low-intensity extracorporeal shockwave therapy in an obesity-associated erectile dysfunction rat model.

OBJECTIVES: To investigate the feasibility of the Zucker fatty (ZF) rat as a model for research in to obesity-associated erectile dysfunction (OAED) and to determine the effect of low-intensity extracorporeal shockwave therapy (Li-ESWT) on penile tissue and function in these rats. MATERIALS AND METHODS: Eight new-born male Zucker lean (ZL group) rats (ZUC-Leprfa 186) and 16 new-born male ZF rats (ZUC-Leprfa 185) were injected with 5-ethynyl-2'-deoxyuridine (EdU) at birth to identify and monitor endogenous stem cells. Insulin tolerance testing was performed at 10 weeks of age. Beginning at 12 weeks of age, eight ZF rats were kept as controls, and the remaining eight ZF rats were treated with Li-ESWT (0.02 mJ/mm2 , 3 Hz, 500 pulses; ZF + SW group) twice a week for 4 weeks. Following a 1-week washout period, erectile function was evaluated by measuring intracavernosal pressure (ICP) and mean arterial pressure (MAP). Penile tissues were then harvested for histological study to assess smooth muscle/collagen content and endothelium content in the corpora cavernosum. LipidTOX™ staining was used to evaluate lipid accumulation. EdU, as a marker of cell activation, and phosphorylated histone 3 (H3P), as a marker of cell mitosis, were also assessed. RESULTS: The ICP/MAP indicated that erectile function was severely impaired in the ZF group as compared with the ZL group. In the ZF + SW group, erectile function was significantly improved (P < 0.05). Muscle atrophy was seen in the ZF group, while Li-ESWT increased the muscle content in ZF + SW group. Moreover, the penile endothelium was damaged in the ZF group, and Li-ESWT enhanced the regeneration of endothelial cells (P < 0.01) in the ZF + SW group. Lipid accumulation was seen in the penile tissue of ZF rats. Li-ESWT significantly reduced both the amount and the distribution pattern of LipidTOX, suggesting decreased overall lipid infiltration. Furthermore, Li-ESWT increased EdU-positive cells and markedly enhanced the phosphorylation level of H3P at Ser-10 in the ZF + SW group. Most H3P-positive cells were located within smooth muscle cells, with some located in the endothelium suggesting that these tissues are the reservoirs of penile stem/progenitor cells. CONCLUSION: ZF rats can serve as an animal model in which to study OAED. This study reveals that obesity impairs erectile function by causing smooth muscle atrophy, endothelial dysfunction, and lipid accumulation in the corpus cavernosum. Li-ESWT restored penile haemodynamic parameters in the ZF rats by restoring smooth muscle and endothelium content and reducing lipid accumulation. The underlying mechanism of Li-ESWT appears to be activation of stem/progenitor cells, which prompts cellular proliferation and accelerates penile tissue regeneration. Our findings are of interest, not just as a validation of this emerging treatment for erectile dysfunction, but also as a novel and potentially significant method to modulate endogenous stem/progenitor cells in other disease processes.

Low-intensity extracorporeal shockwave therapy ameliorates diabetic underactive bladder in streptozotocin-induced diabetic rats.

OBJECTIVES: To evaluate the therapeutic effect of once-weekly low-intensity extracorporeal shock wave therapy (Li-ESWT) on underactive bladder (UAB) in the streptozotocin (STZ)-induced diabetic rat model. MATERIALS AND METHODS: In all, 36 female Sprague-Dawley rats were divided into three groups: normal control (NC), diabetes mellitus control (DMC), and DM with Li-ESWT (DM Li-ESWT). The two DM groups received an intraperitoneal 60 mg/kg STZ injection to induce DM. The Li-ESWT was applied toward the pelvis of the rats starting 4 weeks after STZ administration and lasting for 4 weeks. The Li-ESWT was given once weekly, with an energy flux density of 0.02 mJ/mm2 at 3 Hz for 400 pulses. All rats underwent conscious cystometry, leak-point pressure (LPP) assessment, ex vivo organ-bath study, histology, immunofluorescence, and Western Blot analysis. RESULTS: Conscious cystometry revealed voiding dysfunction in the DMC group, whereas the DM Li-ESWT group showed significantly improved voiding function, reflected in a reduced post-void residual urine volume and increased LPP compared to the DMC group. Ex vivo organ-bath studies showed that Li-ESWT enhanced muscle contractile activity of the bladder and urethra during electrical-field stimulation and drug stimulation. Histologically, Li-ESWT significantly restored bladder morphology, reflected by a reduction in the intravesical lumen area and increased muscle proportion of the bladder wall. Western Blot analysis showed higher smooth muscle actin expression in the bladder wall in the DM Li-ESWT group compared to the DMC group. Immunofluorescence showed decreased nerve-ending distribution, and destroyed and shortened nerve fibres in the DMC group, and recovery of neuronal integrity and innervation in the DM Li-ESWT group. CONCLUSIONS: In conclusion, Li-ESWT ameliorated UAB and urinary incontinence in the diabetic UAB rat model. The improvement appears to be the result of restoration of bladder and urethral structure and function by Li-ESWT. Li-ESWT is non-invasive and may become a better alternative therapy for UAB. Further investigations are warranted.

Low-intensity extracorporeal shock wave therapy promotes myogenesis through PERK/ATF4 pathway.

AIM: Stress urinary incontinence (SUI) is a significant health problem for women. Treatments employing muscle derived stem cells (MDSCs) may be a promising approach to this prevalent, bothersome condition, but these treatments are invasive and require collection of cells from one site for injection into another. It is also unknown whether or not these cells establish themselves and function as muscle cells in the target tissues. Alternatively, low-intensity extracorporeal shock wave therapy (Li-ESWT) is non-invasive and has shown positive outcomes in the treatment of multiple musculoskeletal disorders, but the biological effects responsible for clinical success are not yet well understood. The aim of this study is to explore the possibility of employing Li-ESWT for activation of MDSCs in situ and to further elucidate the underlying biological effects and mechanisms of action in urethral muscle. METHODS: Urethral muscle derived stem cells (uMDSCs) were harvest from Zucker Lean (ZUC-LEAN) (ZUC-Leprfa 186) rats and characterized with flow cytometry. Li-ESWT (0.02 mJ/mm2 , 3 Hz, 200 pulses) and GSK2656157, an inhibitor of PERK pathway, were applied to L6 rat myoblast cells. To assess for myotube formation, we used immunofluorescence staining and western blot analysis in uMDSCs and L6 cells. RESULTS: The results indicate that uMDSCs could form myotubes. Myotube formation was significantly increased by the Li-ESWT as was the expression of muscle heavy chain (MHC) and myogenic factor 5 (Myf5) in L6 cells in vitro. Li-ESWT activated protein kinase RNA-like ER kinase (PERK) pathway by increasing the phosphorylation levels of PERK and eukaryotic initiation factor 2a (eIF2α) and by increasing activating transcription factor 4 (ATF4). In addition, GSK2656157, an inhibitor of PERK, effectively inhibited the myotube formation in L6 rat myoblast cells. Furthermore, GSK2656157 also attenuated myotube formation induced by Li-ESWT. CONCLUSION: In conclusion, this experiment reveals that rat uMDSCs can be isolated successfully and can form myotubes in vitro. PERK/ATF4 pathway was involved in myotube formation, and L6 rat myoblast cells were activated by Li-ESWT to form myotubes. These findings suggest that PERK/ATF4 pathway is activated by Li-ESWT. This study elucidates one of the biochemical pathways responsible for the clinical improvements seen after Li-ESWT. It is possible that this information will help to establish Li-ESWT as an acceptable treatment modality and may help to further refine the use of Li-ESWT in the clinical practice of medicine.

Transgenic animal model for studying the mechanism of obesity-associated stress urinary incontinence.

OBJECTIVE: To study and compare the function and structure of the urethral sphincter in female Zucker lean (ZL) and Zucker fatty (ZF) rats and to assess the viability of ZF fats as a model for female obesity-associated stress urinary incontinence (SUI). MATERIALS AND METHODS: Two study arms were created: a ZL arm including 16-week-old female ZL rats (ZUC-Leprfa 186; n = 12) and a ZF arm including 16-week-old female ZF rats (ZUC-Leprfa 185; n = 12). I.p. insulin tolerance testing was carried out before functional study. Metabolic cages, conscious cystometry and leak point pressure (LPP) assessments were conducted. Urethral tissues were harvested for immunofluorescence staining to check intramyocellular lipid (IMCL) and sphincter muscle (smooth muscle and striated muscle) composition. RESULTS: The ZF rats had insulin resistance, a greater voiding frequency and lower LPP compared with ZL rats (P < 0.05), with more IMCL deposition localized in the urethral striated muscle fibres of the ZF rats (P < 0.05). The thickness of the striated muscle layer and the ratio of striated muscle to smooth muscle were lower in ZF than in ZL rats. CONCLUSION: Obesity impairs urethral sphincter function via IMCL deposition and leads to atrophy and distortion of urethral striated muscle. The ZF rats could be a consistent and reliable animal model in which to study obesity-associated SUI.

In Situ Activation of Penile Progenitor Cells With Low-Intensity Extracorporeal Shockwave Therapy.

BACKGROUND: We previously reported that progenitor cells, or stem cells, exist within penile tissue. We hypothesized that acoustic wave stimulation by low-intensity extracorporeal shockwave therapy (Li-ESWT) would activate local stem or progenitor cells within the penis, producing regenerative effects. AIMS: To study the feasibility of in situ penile progenitor cell activation by Li-ESWT. METHODS: We performed a cohort analysis of young and middle-age male Sprague-Dawley rats treated with 5-ethynyl-2'-deoxyuridine (EdU) pulse followed by Li-ESWT. In addition, Li-ESWT was applied to cultured Schwann cells and endothelial cells to study the molecular mechanism involved in cell proliferation. Thirty minutes before Li-ESWT, each rat received an intraperitoneal injection of EdU. Li-ESWT was applied to the penis at very low (0.02 mJ/mm2 at 3 Hz for 300 pulses) or low (0.057 mJ/mm2 at 3 Hz for 500 pulses) energy levels. The endothelial and Schwann cells were treated with very low energy (0.02 mJ/mm2 at 3 Hz for 300 pulses) in vitro. OUTCOMES: At 48 hours or 1 week after Li-ESWT, penile tissues were harvested for histologic study to assess EdU+ and Ki-67+ cells, and cell proliferation, Ki-67 expression, Erk1/2 phosphorylation, translocation, and angiogenesis were examined in cultured Schwann and endothelial cells after Li-ESWT. RESULTS: Li-ESWT significantly increased EdU+ cells within penile erectile tissues (P < .01) at 48 hours and 1 week. There were more cells activated in young animals than in middle-age animals, and the effect depended on dosage. Most activated cells were localized within subtunical spaces. In vitro studies indicated that Li-ESWT stimulated cell proliferation through increased phosphorylation of Erk1/2. CLINICAL TRANSLATION: The present results provide a possible explanation for the clinical benefits seen with Li-ESWT. STRENGTHS AND LIMITATIONS: The main limitation of the present project was the short period of study and the animal model used. Li-ESWT could be less effective in improving erectile function in old animals because of the decreased number and quality of penile stem or progenitor cells associated with aging. CONCLUSION: Li-ESWT activation of local penile progenitor cells might be one of the mechanisms that contribute to the beneficial effects of shockwave treatment for erectile dysfunction, which represents a non-invasive alternative to exogenous stem cell therapy. Lin G, Reed-Maldonado AB, Wang B, et al. In Situ Activation of Penile Progenitor Cells With Low-Intensity Extracorporeal Shockwave Therapy. J Sex Med 2017;14:493-501.

Impaired contractility of the circular striated urethral sphincter muscle may contribute to stress urinary incontinence in female zucker fatty rats.

AIM: Obesity has been an independent risk factor for female stress urinary incontinence (SUI), the mechanism of this association remains unknown. The aim of this study is to validate the hypothesis that urethral dysfunction is a possible contributor to SUI in obese women. METHODS: Ten Zucker Fatty (ZF) (ZUC-Leprfa 185) and 10 Zucker Lean (ZL) (ZUC-Leprfa 186) female rats at 12-week-old were used in this experiment. The urethral sphincter rings were harvested from the bladder neck through to the most proximal 2/3 regions. In the organ bath study, single pulses of electrical field stimulation (EFS) were applied. For the fatiguing stimulation, repeated multi-pulse EFS with 70 mA were applied at frequency of 5 Hz for 5 min. Caffeine-containing Krebs' solution was administrated to contract the urethra until the contraction began to reach a plateau for 10 min. We performed immunofluorescence staining of the urethra after the experiment was finished. RESULTS: Compared to ZL controls, ZF rats had significantly impaired muscle contractile activity (MCA) (P < 0.05). Also, ZF rats presented early fatiguing of MCA and had a significantly greater percentage of MCA decline from baseline in the fatiguing test (37.7% vs 25.6%, P < 0.05). The plateau of maximal MCA induced by caffeine in ZF rats was significantly lower than ZL controls (0.22 vs 0.36, P < 0.05). CONCLUSIONS: This novel study showed that obese female rats had significantly impaired contractile properties of striated urethral sphincter, suggesting urethral dysfunction could be an important contributor to SUI in obesity.

Low-Intensity Extracorporeal Shock Wave Therapy Enhances Brain-Derived Neurotrophic Factor Expression through PERK/ATF4 Signaling Pathway.

Low-intensity extracorporeal shock wave therapy (Li-ESWT) is used in the treatment of erectile dysfunction, but its mechanisms are not well understood. Previously, we found that Li-ESWT increased the expression of brain-derived neurotrophic factor (BDNF). Here we assessed the underlying signaling pathways in Schwann cells in vitro and in penis tissue in vivo after nerve injury. The result indicated that BDNF were significantly increased by the Li-ESWT after nerve injury, as well as the expression of BDNF in Schwann cells (SCs, RT4-D6P2T) in vitro. Li-ESWT activated the protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) pathway by increasing the phosphorylation levels of PERK and eukaryotic initiation factor 2a (eIF2α), and enhanced activating transcription factor 4 (ATF4) in an energy-dependent manner. In addition, GSK2656157-an inhibitor of PERK-effectively inhibited the effect of Li-ESWT on the phosphorylation of PERK, eIF2α, and the expression of ATF4. Furthermore, silencing ATF4 dramatically attenuated the effect of Li-ESWT on the expression of BDNF, but had no effect on hypoxia-inducible factor (HIF)1α or glial cell-derived neurotrophic factor (GDNF) in Schwann cells. In conclusion, our findings shed new light on the underlying mechanisms by which Li-ESWT may stimulate the expression of BDNF through activation of PERK/ATF4 signaling pathway. This information may help to refine the use of Li-ESWT to further improve its clinical efficacy.

Low-energy Shock Wave Therapy Ameliorates Erectile Dysfunction in a Pelvic Neurovascular Injuries Rat Model.

INTRODUCTION: Erectile dysfunction (ED) caused by pelvic injuries is a common complication of civil and battlefield trauma with multiple neurovascular factors involved, and no effective therapeutic approach is available. AIMS: To test the effect and mechanisms of low-energy shock wave (LESW) therapy in a rat ED model induced by pelvic neurovascular injuries. METHODS: Thirty-two male Sprague-Dawley rats injected with 5-ethynyl-2'-deoxyuridine (EdU) at newborn were divided into 4 groups: sham surgery (Sham), pelvic neurovascular injury by bilateral cavernous nerve injury and internal pudendal bundle injury (PVNI), PVNI treated with LESW at low energy (Low), and PVNI treated with LESW at high energy (High). After LESW treatment, rats underwent erectile function measurement and the tissues were harvested for histologic and molecular study. To examine the effect of LESW on Schwann cells, in vitro studies were conducted. MAIN OUTCOME MEASUREMENTS: The intracavernous pressure (ICP) measurement, histological examination, and Western blot (WB) were conducted. Cell cycle, Schwann cell activation-related markers were examined in in vitro experiments. RESULTS: LESW treatment improves erectile function in a rat model of pelvic neurovascular injury by leading to angiogenesis, tissue restoration, and nerve generation with more endogenous EdU(+) progenitor cells recruited to the damaged area and activation of Schwann cells. LESW facilitates more complete re-innervation of penile tissue with regeneration of neuronal nitric oxide synthase (nNOS)-positive nerves from the MPG to the penis. In vitro experiments demonstrated that LESW has a direct effect on Schwann cell proliferation. Schwann cell activation-related markers including p-Erk1/2 and p75 were upregulated after LESW treatment. CONCLUSION: LESW-induced endogenous progenitor cell recruitment and Schwann cell activation coincides with angiogenesis, tissue, and nerve generation in a rat model of pelvic neurovascular injuries.

Urethral musculature and innervation in the female rat.

AIMS: The urethral sphincter and urethral muscle innervation are critically involved in maintaining continence, especially in the female. However, the urethral muscle type and distribution, as well as the urethral nerves are far from being well documented. Our aim was to clearly identify the distribution of urethral striated muscle, smooth muscle, and urethral nerves. METHODS: In a cohort analysis of 3-month-old female Sprague-Dawley rats, cross and longitudinal sections of female rat urethra were extensively investigated using morphological techniques. Urethras were harvested to the sections, in order to provide both global and detailed visions of the urethra. H&E, Masson's Trichrome, phalloidin and immunoflourence stains were used. The cytoarchitecture, nitrergic, and cholinergic innervations were mainly investigated. Different layers of the segments of urethra were traced to draw curve graphs that represent the thickness of each muscle layer of urethral wall. RESULTS: The results showed that the primary peak of striated muscle is in the middle urethra. The inner layer close to mucosa was found to contain longitudinal smooth muscle. Near the bladder orifice, the circular smooth muscle dominates, which becomes thinner distally throughout the rest of urethra. In the middle urethra the vast majority of the urethral muscle are circularly oriented striated muscle cells. Typical nerve endings were present in high power images to show the different characteristic features of nerve innervation. CONCLUSIONS: This study has illustrated the detailed morphological structure and innervations of the normal female rat urethra and can serve as a basis for further study of stress urinary incontinence (SUI).

Comparison of Topical Hemostatic Agents in a Swine Model of Extremity Arterial Hemorrhage: BloodSTOP iX Battle Matrix vs. QuikClot Combat Gauze.

BloodSTOP iX Battle Matrix (BM) and QuikClot Combat Gauze (CG) have both been used to treat traumatic bleeding. The purpose of this study was to examine the efficacy and initial safety of both products in a swine extremity arterial hemorrhage model, which mimics combat injury. Swine (37.13 ± 0.56 kg, NBM = 11, NCG = 9) were anesthetized and splenectomized. We then isolated the femoral arteries and performed a 6 mm arteriotomy. After 45 s of free bleeding, either BM or CG was applied. Fluid resuscitation was provided to maintain a mean arterial pressure of 65 mmHg. Animals were observed for three hours or until death. Fluoroscopic angiography and wound stability challenge tests were performed on survivors. Tissue samples were collected for histologic examination. Stable hemostasis was achieved in 11/11 BM and 5/9 CG subjects, with recovery of mean arterial pressure and animal survival for three hours (p < 0.05, Odds Ratio (OR) = 18.82 (0.85-415.3)). Time to stable hemostasis was shorter for the BM-treated group (4.8 ± 2.5 min vs. 58 ± 20.1 min; Median = 2, Interquartile Range (IQR) = 0 min vs. Median = 60, IQR = 120 min; p < 0.05) and experienced longer total stable hemostasis (175.2 ± 2.5 min vs. 92.4 ± 29.9 min; Median = 178, IQR = 0 min vs. Median = 120, IQR = 178 min; p < 0.05). Post-treatment blood loss was lower with BM (9.5 ± 2.4 mL/kg, Median = 10.52, IQR = 13.63 mL/kg) compared to CG (29.9 ± 9.9 mL/kg, Median = 29.38, IQR = 62.44 mL/kg) (p = 0.2875). Standard BM products weighed less compared to CG (6.9 ± 0.03 g vs. 20.2 ± 0.4 g) (p < 0.05) and absorbed less blood (3.4 ± 0.8 g vs. 41.9 ± 12.3 g) (p < 0.05). Fluoroscopic angiography showed recanalization in 5/11 (BM) and 0/5 (CG) surviving animals (p = 0.07, OR = 9.3 (0.41-208.8)). The wound stability challenge test resulted in wound re-bleeding in 1/11 (BM) and 5/5 (CG) surviving animals (p < 0.05, OR = 0.013 (0.00045-0.375)). Histologic evidence indicated no wound site, distal limb or major organ damage in either group. BM is more effective and portable in treating arterial hemorrhage compared to CG. There was no histologic evidence of further damage in either group.

Estrogen attenuates TGF-ß1 induced elastogenesis in rat urethral smooth muscle cells by inhibiting Smad response elements.

PURPOSE: We investigated the effect and mechanism of estrogen on elastogenesis in urethral smooth muscle cells in vitro. MATERIALS AND METHODS: Urethral smooth muscle cells were isolated from normal adult female rats. For elastogenesis assay cells were treated with TGF-ß1, the potent TGF-ß1 receptor inhibitor SB431542 and estrogen for 2 weeks. Real-time polymerase chain reaction was performed to assay gene expression during this process. Activity of the TGF-ß1 responsive elements CAGA(12)-Luc and GCCG(12)-Luc were also assayed. Estrogen receptor and Smad2/3 interaction was evaluated by immunoprecipitation and Western blot. RESULTS: TGF-ß1 induced elastogenesis in rat urethral smooth muscle cells. This effect was partially blocked by estrogen and completely abrogated by SB431542. SB431542 completely inhibited activation of the Smad2/3 response element CAGA(12)-Luc and estrogen significantly inhibited activation. The Smad1/4 response element GCCG(12)-Luc was not affected by SB431542 treatment but estrogen partially inhibited the activation of GCCG(12)-Luc induced by TGF-ß1. Estrogen receptor bound to Smad 2 and 3 in vitro. CONCLUSIONS: Estrogen attenuated TGF-ß1 induced elastogenesis via binding of its activated receptor to Smad2/3 to inhibit the TGF-ß1 response element in rat urethral smooth muscle cells.

Age-Related Changes in Urethral Structure and Responds to Injury: Single-Cell Atlas of a Rat Model of Vaginal Birth Injury induced Stress Urinary Incontinence.

Stress urinary incontinence (SUI) greatly affects the daily life of numerous women and is closely related to a history of vaginal delivery and aging. We used vaginal balloon dilation to simulate vaginal birth injury in young and middle-aged rats to produce a SUI animal model, and found that young rats restored urethral structure and function well, but not the middle-aged rats. To identify the characteristics of cellular and molecular changes in the urethral microenvironment during the repair process of SUI. We profiled 51,690 individual female rat urethra cells from 24 and 48 weeks old, with or without simulated vaginal birth injury. Cell interaction analysis showed that signal networks during repair process changed from resting to active, and aging altered the distribution but not the overall level of cell interaction in the repair process. Similarity analysis showed that muscle, fibroblasts, and immune cells underwent large transcriptional changes during aging and repair. In middle-aged rats, cell senescence occurs mainly in the superficial and middle urothelium due to cellular death and shedding, and the basal urothelium expressed many Senescence-Associated Secretory Phenotype (SASP) genes. In conclusion, we established the aging and vaginal balloon dilation (VBD) model of female urethral cell anatomy and the signal network landscape, which provides an insight into the normal or disordered urethra repair process and the scientific basis for developing novel SUI therapies.

Identification of active and quiescent adipose vascular stromal cells.

BACKGROUND AIMS: Recent studies have demonstrated the existence of both active and quiescent stem cells in bone marrow, hair follicle and intestine. We attempted to identify active and quiescent vascular stromal cells (VSC) in adipose tissue. METHODS: For identification of active VSC, adult rats were injected intraperitoneally with thymidine analog 5-ethynyl-2-deoxyuridine (EdU) and their subcutaneous tissue harvested 3 days later. For identification of quiescent VSC, newborn rats were injected intraperitoneally with EdU and their subcutaneous tissue harvested 9 weeks later. The harvested adipose tissues were examined for the co-localization of EdU with VSC marker CD34, smooth muscle marker SMA, endothelial marker RECA and pericyte marker CD140b. RESULTS: In adult rat adipose tissues harvested 3 days after EdU injection, there were 28.80 ± 8.70 (mean ± SD) EdU+ cells/100 × microscopic field, and approximately 6.2% of cell nuclei were labeled with EdU. The percentages of EdU+ cells expressing the following markers were approximately: 84 for CD34, 5.6 for RECA (rat endothelial marker), 3.7 for SMA and 14.8 for CD140b. In the adipose tissues of newborn rats that were harvested 9 weeks after EdU injection, the percentages of EdU+ cells expressing the following markers were approximately: 76 for CD34, 1.8 for RECA, 0 for SMA and 12.9 for CD140b. In both the short-term (active) and long-term (quiescent) EdU-labeled adipose tissues, the EdU label was consistently co-localized with CD34 and in the proximity of CD140b stain or in the adventitia. CONCLUSIONS: Both active and quiescent VSC expressed CD34 and localized to capillaries and the adventitia of larger blood vessels.

Microenergy acoustic pulse therapy restores function and structure of pelvic floor muscles after simulated birth injury.

Background: The mechanisms of the microenergy acoustic pulse (MAP) therapy on restoring structure and function of pelvic floor muscles (PFM) after simulated birth injury are not well understood. Methods: A total 24 female Sprague-Dawley rats were randomly grouped into sham control (sham), vaginal balloon dilation and ovariectomy (VBDO), VBDO + ß-aminopropionitrile (BAPN, an irreversible LOX inhibitor), and VBDO + BAPN and treated with MAP (n=6 in each group). The MAP therapy was administered 2 times per week for 4 weeks with 1-week washout, the functional and histological studies were conducted in all 24 rats. The viscoelastic behavior of the PFM, including iliococcygeus (IC) and pubococcygeus (PC), was examined with a biomechanical assay. The structure of the PFM was assessed by immunofluorescence and Masson's trichrome staining. Results: The leak point pressure (LPP) assay demonstrated that the MAP therapy group had higher LPPs compared to that of VBDO and BAPN groups. In the sham group, the muscular stiffness (K) of IC muscle was significantly higher than that of PC muscle while the pelvic floor muscle rebound activity (MRA) of PC muscle was stronger than that of IC muscle (291.26±45.33 and 241.18±14.23 N/cm2, respectively). Both VBDO and BAPN decreased the MRA and increased the K in both IC and PC. Histologic examination revealed increased fibrous tissue (collagen) and degeneration of muscle fibers in both VBDO and BAPN groups. MAP therapy significantly reduced the collagen content and improved the architecture of muscle fibers. Conclusions: MAP appears to restore the structure and function of PFM by regenerating muscular fibers and improving biomechanical properties in an animal model of simulated birth injury.