Radial Type Low-Intensity Extracorporeal Shockwave Therapy Enhances Penile Microvascular Perfusion in an Aging Rat Model: A Novel Interventional Strategy to Treat Erectile Dysfunction.

PURPOSE: Physiological aging is associated with microvascular dysfunction, including in the penis, and this may contribute to age-related erectile dysfunction (ED). Low-intensity extracorporeal shockwave therapy (Li-ESWT) is a non-invasive intervention for ED, but its effect on penile microvascular function, remains unclear. Our objectives are to (i) evaluate the effect of Li-ESWT (specifically radial type ESWT [rESWT]) on penile microvascular perfusion (PMP) in aging rats, (ii) elucidate a possible mechanism, and (iii) evaluate its impact on angiogenic and smooth muscle biomarkers in cavernosal tissue. MATERIALS AND METHODS: Male rats (n=9; 15-18 months) were anesthetized and subjected to rESWT while monitoring PMP. The nitric oxide (NO) pathway involvement was assessed by measuring the effect of rESWT on PMP following an intracavernosal injection of N(G)-nitroarginine methyl ester (L-NAME) (NO synthase inhibitor). To elucidate the cellular mechanism, another group of rats received repeated rESWT (n=4) or no treatment (n=4) three times/week for two weeks. Rats were euthanized at the end of the study and penile tissues were analyzed for angiogenic markers (vascular endothelial growth factor-A [VEGF-A], endothelial nitric oxide synthase [eNOS]) and smooth muscle content (α-actin) using immunostaining, Western blot, and quantitative polymerase chain reaction (qPCR). RESULTS: rESWT resulted in more than a 2-fold increase in PMP (from 68.5 arbitrary units; 163.7 AU). L-NAME injection produced a <40%-50% decrease (185.3 to 101.0 AU) in rESWT-induced PMP response. Immunostaining revealed increased α-actin, eNOS, and VEGF-A in the cavernosum and these findings were confirmed by qPCR and Western blot results. CONCLUSIONS: rESWT improved PMP, which may be mediated via increased VEGF expression, which stimulates the NO/cyclic guanosine monophosphate pathway, resulting in sustained PMP. rESWT devices could offer a safe, non-invasive treatment for age-related ED.

Non-Invasive Radiofrequency Hyperthermia Attenuates HMGB1/TLR4/NF-κB Inflammatory Axis in a Chronic Prostatitis/Chronic Pelvic Pain Syndrome Rat Model.

PURPOSE: The primary goal of this study is to evaluate the effect of the non-invasive radiofrequency hyperthermia (RFHT) device on chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) rat model and investigate the underlying mechanism. MATERIALS AND METHODS: In this study, Sprague-Dawley rats were randomly distributed into three groups: (1) normal control group, (2) CP/CPPS group, and (3) RFHT group. CP/CPPS rat models were induced by 17ß-estradiol and dihydrotestosterone for 4 weeks and RFHT was administered for 5 weeks after model establishment. During RFHT administration, core body temperatures were continuously monitored with a rectal probe. After administering RFHT, we assessed pain index for all groups and collected prostate tissues for Western blot analysis, immunofluorescence, and immunohistochemistry. We also collected adjacent organs to the prostate including urinary bladder, testes, and rectum for safety assessment via H&E staining along with a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. RESULTS: After administering RFHT, pain in rats was significantly alleviated compared to the CP/CPPS group. RFHT reduced high-mobility group box 1 (HMGB1) expression and improved inflammation by downregulating subsequent proinflammatory cytokines through inhibition of the toll-like receptor 4 (TLR4)-nuclear factor kappa B (NF-κB) pathway. In prostate-adjacent organs, no significant histological alteration or inflammatory infiltration was detected. The area of cell death also did not increase significantly after RFHT. CONCLUSIONS: In conclusion, RFHT demonstrated anti-inflammatory effects by inhibiting the HMGB1-TLR4-NF-κB pathway in CP/CPPS rat models. This suggests that RFHT could serve as a safe and promising therapeutic strategy for CP/CPPS.

Extracorporeal Shockwave Therapy Alleviates Inflammatory Pain by Down-Regulating NLRP3 Inflammasome in Experimental Chronic Prostatitis and Chronic Pelvic Pain Syndrome.

PURPOSE: To evaluate the anti-inflammatory and antioxidative effects of extracorporeal shockwave therapy (ESWT) on prostatitis and explore the mechanism of alleviating pain. MATERIALS AND METHODS: For in vitro testing, RWPE-1 cells were randomly divided into 5 groups: (1) RWPE-1 group (normal control), (2) LPS group (lipopolysaccharide inducing inflammation), (3) 0.1ESWT group (treated by 0.1 mJ/mm² energy level), (4) 0.2ESWT group (treated by 0.2 mJ/mm² energy level), and (5) 0.3ESWT group (treated by 0.3 mJ/mm² energy level). After ESWT was administered, cells and supernatant were collected for ELISA and western blot. For in vivo testing, Sprague-Dawley male rats were randomly divided into 3 groups: (1) normal group, (2) prostatitis group, and (3) ESWT group (n=12 for each). Prostatitis was induced by 17 beta-estradiol and dihydrotestosterone (DHT) administration. Four weeks after ESWT, the pain index was assessed for all groups and prostate tissues were collected for immunohistochemistry, immunofluorescence, apoptosis analysis and, western blot. RESULTS: Our in vitro studies showed that the optimal energy flux density of ESWT was 0.2 mJ/mm². In vivo, ESWT ameliorated discomfort in rats with prostatitis and inflammation symptoms were improved. Compared to normal rats, overexpressed NLRP3 inflammasomes triggered apoptosis in rats with prostatitis and this was improved by ESWT. TLR4-NFκB pathway was overactive after experimental prostatitis, compared to normal and ESWT groups, and prostatitis induced alterations in BAX/BAK pathway were inhibited by ESWT. CONCLUSIONS: ESWT improved CP/CPPS by reducing NLRP3 inflammasome and ameliorated apoptosis via inhibiting BAX/BAK pathway in a rat model. TLR4 may play a key role in bonding NLRP3 inflammasome and BAX/BAK pathways. ESWT might be a promising approach for the treatment of CP/CPPS.

Effect of Li-ESWT on Testicular Tissue and Function in Androgen-Deficient Rat Model.

Low-intensity extracorporeal shockwave therapy (Li-ESWT), as a microenergy therapy, has the effects of inhibiting oxidative stress, antiapoptosis, and tissue repair, which is increasingly applied to a variety of diseases. Our research aims to explore the protective effects of Li-ESWT in the aging rat model and its possible molecular mechanism through in vivo and in vitro experiments. In vitro, TM3 Leydig cells incubated with H2O2 were treated with Li-ESWT at 4 energy levels (0.01, 0.05, 0.1, and 0.2 mJ/mm2). In vivo, we employed an androgen-deficient rat model to simulate male aging and treated it with Li-ESWT at three different energy levels (0.01, 0.05, and 0.2 mJ/mm2). Li-ESWT increased the expression of vascular endothelial growth factor (VEGF) in TM3 cells, improved antioxidant capacity, and reduced apoptosis, with the effect being most significant at 0.05 mJ/mm2 energy level. In androgen-deficient rat model, LI-ESWT can improve sperm count, motility, and serum testosterone level, enhancing tissue antioxidant capacity and antiapoptotic ability, and the effect is most significant at 0.05 mJ/mm2 energy level. Therefore, Li-ESWT at an appropriate energy level can improve sperm count, motility, and serum testosterone levels in androgen-deficient rat models, reduce oxidative stress in the testis, and increase antioxidant capacity and antiapoptotic abilities. The mechanism of this condition might be related to the increased VEGF expression in Leydig cells by Li-ESWT.

Transpelvic Magnetic Stimulation Enhances Penile Microvascular Perfusion in a Rat Model: A Novel Interventional Strategy to Prevent Penile Fibrosis after Cavernosal Nerve Injury.

PURPOSE: Penile microvascular dysfunction is a known contributor to erectile dysfunction (ED) and penile fibrosis has been shown to impair microvascular perfusion (MVP). Our objectives were to: (i) determine beneficial effects of TPMS to modulate penile MVP, (ii) determine its mechanism, (iii) evaluate impact of cavernosal nerve injury (CNI) on penile MVP, and (iv) determine time-course of cavernosal tissue elastin changes after CNI in rats. MATERIALS AND METHODS: Adult male rats (n=5) were anesthetized and subjected to TPMS (13%, 15%, and 17%) and MVP changes were recorded using laser speckle contrast imaging (LSCI). Another group of male rats were subjected to either bilateral cavernosal nerve injury (CNI; n=7) or sham surgery (n=7). After recovery, animals were monitored for MVP using LSCI before and after TPMS. Rat penile tissues were harvested and analyzed for fibrosis using a marker for elastin. RESULTS: Rat TPMS resulted in a stimulus dependent increase in MVP; maximal perfusion was observed at 17%. L-N(G)-Nitroarginine methyl ester (L-NAME) resulted in a marked decrease in TPMS induced MVP increase (393.33 AU vs. 210.67 AU). CNI resulted in 40% to 50% decrease in MVP. CNI produced a remarkable increase in elastin deposits that are noticeable throughout the cavernosal tissues post injury. CONCLUSIONS: TPMS is a novel and non-invasive intervention to improve penile MVP after CNI. Potential application includes treatment of ED and sexual function preservation following cancer treatment, possibly through improved penile hemodynamics that might help prevent penile hypoxia and fibrosis.

Preclinical applications of high-definition manometry system to investigate pelvic floor muscle contribution to continence mechanisms in a rabbit model.

External anal sphincter (EAS), external urethral sphincters, and puborectalis muscle (PRM) have important roles in the genesis of anal and urethral closure pressures. In the present study, we defined the contribution of these muscles alone and in combination with the sphincter closure function using a rabbit model and a high-definition manometry (HDM) system. A total of 12 female rabbits were anesthetized and prepared to measure anal, urethral, and vaginal canal pressures using a HDM system. Pressure was recorded at rest and during electrical stimulation of the EAS and PRM. A few rabbits (n = 6) were subjected to EAS injury and the impact of EAS injury on the closure pressure profile was also evaluated. Anal, urethral, and vaginal canal pressures recorded at rest and during electrical stimulation of EAS and PRM demonstrated distinct pressure profiles. EAS stimulation induced anal canal pressure increase, whereas PRM stimulation increased the pressures in all the three orifices. Electrical stimulation of EAS after injury resulted in about 19% decrease in anal canal pressure. Simultaneous electrical stimulation of EAS and PRM resulted in an insignificant increase of individual anal canal pressures when compared with pressures recorded after EAS or PRM stimulations alone. Our data confirm that HDM is a viable system to measure dynamic pressure changes within the three orifices and to define the role of each muscle in the development of closure pressures within these orifices in preclinical studies.NEW & NOTEWORTHY We anticipate that with this new HDM technology, physiological changes within these orifices may be redefined using the extensive data that are generated from 96 sensors. When compared with conventional methods, HDM offers the advantages of an increased response rate, as well as the utilization of 96 circumferential sensors to simultaneously measure pressure along the three orifices. Our findings suggest a potential use of this technology to better define urinary leak point pressure.

Novel Emerging Therapies for Erectile Dysfunction.

Currently, several treatments exist for the improvement of erectile dysfunction (ED). These include medical therapies such as phosphodiesterase type 5 inhibitors (PDE5-Is), invasive methods such as intracavernosal injection therapy of vaso-active substances, vacuum erection devices, and penile prosthesis implants. However, the percentage of patients that are unresponsive to available treatments and who drop out from treatments remains high. Current evidence reveals that the pathogenesis of ED is related to multiple factors including underlying comorbidities, previous surgery, and psychological factors. Diverse approaches using novel molecular pathways or new technologies have been tested as potential therapeutic options for difficultto-treat ED populations. Melanocortin receptor agonist, a centrally acting agent, showed promising results by initiating erection without sexual stimulation in non-responders to PDE5-Is. Recent clinical and pre-clinical studies using human tissues suggested that new peripherally acting agents including the Max-K channel activator, guanylate cyclase activator, and nitric oxide donor could be potential therapies either as a monotherapy or in combination with PDE5-Is in ED patients. According to several clinical trials, regeneration therapy using stem cells showed favorable data in men with diabetic or post-prostatectomy ED. Low-intensity shock wave therapy also demonstrated promising results in patients with vasculogenic ED. There are growing evidences which suggest the efficacy of these emerging therapies, though most of the therapies still need to be validated by well-designed clinical trials. It is expected that, should their long-term safety and efficacy be proven, the emerging treatments can meet the needs of patients hitherto unresponsive to or unsatisfied by current therapies for ED.

Evaluation of Age- and Radical-Prostatectomy Related Changes in Male Pelvic Floor Anatomy Based on Magnetic Resonance Imaging and 3-Dimensional Reconstruction.

PURPOSE: Puborectalis muscles (PRM) and ischiocavernosus muscles (ICM) play important roles in urinary continence and male erectile functions. Understanding of anatomy and surgical-injury related changes to these muscles is critical to monitor changes in continence or erectile function. Anatomical description of these muscles has undergone revisions because these conclusions were derived from cadavers. Our objectives were to: (i) elucidate male pelvic muscles by in-vivo magnetic resonance imaging (MRI) and 3-dimensional (3-D) reconstruction of these images and (ii) compare PRM and ICM thickness in healthy volunteers and symptomatic patients. MATERIALS AND METHODS: Healthy young male (mean age, 25 years; n=5), older male (age, 65-70 years; n=5), and post-prostatectomy patients with erectile dysfunction and urinary incontinence (age, 65-70 years; n=5) were scanned on a 3T-magnetic resonance scanner. Images were acquired from slices above urinary bladder base to urethra entry into penis. Pelvic bone, bladder/urethra, corpus cavernosum, ICM, PRM, and prostate were segmented. 3-D models of each structure were generated and assembled into composite images, and ICM and PRM thicknesses were calculated. RESULTS: We successfully reconstructed 3-D male pelvic floor anatomy including ICM, PRM, bladder, urethra, bulbospongiosus, corpus cavernosa, prostate and bones from the two groups. We documented significant reduction in PRM and ICM thickness in older men. CONCLUSIONS: This is perhaps the first 3-D reconstruction of male pelvic floor structures based on in-vivo MRI in healthy and symptomatic patients. Observed reduction in PRM and ICM thickness is possibly due to age-related atrophy.

Using machine learning of clinical data to diagnose COVID-19: a systematic review and meta-analysis.

BACKGROUND: The recent Coronavirus Disease 2019 (COVID-19) pandemic has placed severe stress on healthcare systems worldwide, which is amplified by the critical shortage of COVID-19 tests. METHODS: In this study, we propose to generate a more accurate diagnosis model of COVID-19 based on patient symptoms and routine test results by applying machine learning to reanalyzing COVID-19 data from 151 published studies. We aim to investigate correlations between clinical variables, cluster COVID-19 patients into subtypes, and generate a computational classification model for discriminating between COVID-19 patients and influenza patients based on clinical variables alone. RESULTS: We discovered several novel associations between clinical variables, including correlations between being male and having higher levels of serum lymphocytes and neutrophils. We found that COVID-19 patients could be clustered into subtypes based on serum levels of immune cells, gender, and reported symptoms. Finally, we trained an XGBoost model to achieve a sensitivity of 92.5% and a specificity of 97.9% in discriminating COVID-19 patients from influenza patients. CONCLUSIONS: We demonstrated that computational methods trained on large clinical datasets could yield ever more accurate COVID-19 diagnostic models to mitigate the impact of lack of testing. We also presented previously unknown COVID-19 clinical variable correlations and clinical subgroups.

Characterization of urethral fibrosis in a rabbit model: Potential roles of Wnt-ß catenin pathway and epithelial to mesenchymal transition.

AIM: To elucidate the precise cellular and molecular mechanisms that underlie urethral fibrogenesis. METHODS: Endoluminal electrocautery injury (using Karl Storz 10 Fr. Pediatric urethroscope) was employed in male rabbits (n = 6) to create mucosal injury. Retrograde urethrogram (RUG) and endoluminal ultrasound techniques were used to assess severity and changes in luminal cross-sectional area. Six control rabbits were subjected to sham injury, in which the electrocautery was inserted but not powered. Urethral tissues were harvested 30 days postinjury and subjected to RNA sequencing and quantitative polymerase chain reaction (qPCR) to determine changes in gene expression. Histological, immunostaining, and Western blot studies were used to determine changes in protein expression of known markers of fibrosis (eg, collagen, Integrinαv, GIV/Girdin, transforming growth factor-ß (TGF-ß), and pSMAD1,2,3). RESULTS: Trichrome staining confirmed increased connective tissue in urethral scar tissues. Immunostaining revealed a potential role for epithelial to mesenchymal cell transition (EMT) and positive labeling for all fibrotic markers (eg, collagen-1, Integrin αv, GIV/Girdin, transforming growth factor-ß (TGF-ß), and SMAD1,2,3). Western blot analysis confirmed increased protein levels of these fibrotic markers. CONCLUSION: Our RNA sequencing and qPCR studies, in conjunction with our protein data, suggest that urethral mucosal fibrogenesis may be mediated by novel fibrogenic signaling pathways involving Wnt-ß catenin, TGF-ß, GIV/Girdin, and EMT which lead to increased collagen deposition. Therapeutic strategies targeting these pathways may be beneficial in attenuating fibrogenesis and stricture progression.

A Novel Endoluminal Ultrasound Imaging Technique to Determine Urethral Luminal Cross-Sectional Area.

INTRODUCTION AND OBJECTIVES: Retrograde urethrogram (RUG) and voiding cystourethrogram (VCUG) are currently the gold standard imaging technique for diagnosis of urethral stricture and determination of stricture location. However, RUG and VCUG have multiple limitations. These techniques require exposure to ionizing radiation, the quality is operator and patient dependent, there is a moderate degree of invasiveness with urethral catheterization, can have artifacts because of patient positioning that underestimates stricture length. The development of novel imaging modalities without ionizing radiation to accurately evaluate the presence, location, length, and lumen cross-sectional area (CSA) of the urethral stricture would be of great value. The objective of this study was to develop a novel endoluminal ultrasound (ELUS) imaging technique that permits the accurate quantitation of urethral stricture. METHODS: Urethral strictures were created in rabbits (n = 5) by electrocautery and an ELUS technique was developed for subsequent luminal imaging. A 3.2F 40 MHz ultrasound (US) probe was introduced transurethrally and infused with US contrast agent. Images were recorded as the catheter was pulled back at a constant speed to acquire tomographic images. Lumen CSA over the entire urethral length was calculated using a custom methodology and validated in our laboratory. RESULTS: Urethral luminal CSA over the entire length of urethra before and after experimental stricture development was quantified including the length of stenosis. Intra- and interobserver variability (r = 0.99 for both) was excellent. CONCLUSIONS: Feasibility of ELUS as a quantitative technique to determine healthy urethral lumen and stricture CSA was demonstrated. The translational potential for a nonionizing imaging modality to better describe CSA, length, location, and uninvolved urethral CSA of the stricture is a significant improvement over current methodology.

Length-tension relationship of the external anal sphincter muscle: implications for the anal canal function.

The length at which a muscle operates in vivo (operational length) and the length at which it generates maximal force (optimal length) may be quite different. We studied active and passive length-tension characteristics of external anal sphincter (EAS) in vivo and in vitro to determine the optimal and operational length of rabbit EAS. For the in vitro studies, rings of EAS (n = 4) were prepared and studied in a muscle bath under isometric conditions. For in vivo studies, female rabbits (n = 19) were anesthetized and anal canal pressure was recorded by use of a sleeve sensor placed in the custom-designed catheter holders of 4.5-, 6-, and 9-mm diameters. Measurements were obtained at rest and during EAS electrical stimulation. Sarcomere length of EAS muscle was measured by laser diffraction technique with no probe and three probes in the anal canal. In vitro studies revealed 2,054 mN/cm(2) active tension at optimal length. In vivo studies revealed a probe size-dependent increase in anal canal pressure and tension. Maximal increase in anal canal tension with stimulation was recorded with the 9-mm probe. Increases in anal canal tension with increase in probe size were completely abolished by pancuronium bromide. EAS muscle sarcomere length without and with 9-mm probe in the anal canal were 2.11 +/- 0.08 and 2.99 +/- 0.07 microm, respectively. Optimal sarcomere length, based on the thin filament length measured by thin filament analysis, is 2.44 +/- 0.10 microm. These data show that the operational length of EAS is significantly shorter than its optimal length. Our findings provide insight into EAS function and we propose the possibility of increasing anal canal pressure by surgical manipulation of the EAS sarcomere length.