Neutrophil biology in injuries and diseases of the central and peripheral nervous systems.

The role of inflammation in nervous system injury and disease is attracting increased attention. Much of that research has focused on microglia in the central nervous system (CNS) and macrophages in the peripheral nervous system (PNS). Much less attention has been paid to the roles played by neutrophils. Neutrophils are part of the granulocyte subtype of myeloid cells. These cells, like macrophages, originate and differentiate in the bone marrow from which they enter the circulation. After tissue damage or infection, neutrophils are the first immune cells to infiltrate into tissues and are directed there by specific chemokines, which act on chemokine receptors on neutrophils. We have reviewed here the basic biology of these cells, including their differentiation, the types of granules they contain, the chemokines that act on them, the subpopulations of neutrophils that exist, and their functions. We also discuss tools available for identification and further study of neutrophils. We then turn to a review of what is known about the role of neutrophils in CNS and PNS diseases and injury, including stroke, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, spinal cord and traumatic brain injuries, CNS and PNS axon regeneration, and neuropathic pain. While in the past studies have focused on neutrophils deleterious effects, we will highlight new findings about their benefits. Studies on their actions should lead to identification of ways to modify neutrophil effects to improve health.

First in Human Subjects Testing of the UroMonitor: A Catheter-free Wireless Ambulatory Bladder Pressure Monitor.

PURPOSE: Urodynamics is the standard method of diagnosing bladder dysfunction, but involves catheters and retrograde bladder filling. With these artificial conditions, urodynamics cannot always reproduce patient complaints. We have developed a wireless, catheter-free intravesical pressure sensor, the UroMonitor, which enables catheter-free telemetric ambulatory bladder monitoring. The purpose of this study was twofold: to evaluate accuracy of UroMonitor pressure data, and assess safety and feasibility of use in humans. MATERIALS AND METHODS: Eleven adult female patients undergoing urodynamics for overactive bladder symptoms were enrolled. After baseline urodynamics, the UroMonitor was transurethrally inserted into the bladder and position was confirmed cystoscopically. A second urodynamics was then performed with the UroMonitor simultaneously transmitting bladder pressure. Following removal of urodynamics catheters, the UroMonitor transmitted bladder pressure during ambulation and voiding in private. Visual analogue pain scales (0-5) were used to assess patient discomfort. RESULTS: The UroMonitor did not significantly alter capacity, sensation, or flow during urodynamics. The UroMonitor was also easily inserted and removed in all subjects. The UroMonitor reproduced bladder pressure, capturing 98% (85/87) of voiding and nonvoiding urodynamic events. All subjects voided with only the UroMonitor in place with low post-void residual volume. Median ambulatory pain score with the UroMonitor was rated 0 (0-2). There were no post-procedural infections or changes to voiding behavior. CONCLUSIONS: The UroMonitor is the first device to enable catheter-free telemetric ambulatory bladder pressure monitoring in humans. The UroMonitor appears safe and well tolerated, does not impede lower urinary tract function, and can reliably identify bladder events compared to urodynamics.

Brain-Derived Neurotrophic Factor Is Indispensable to Continence Recovery after a Dual Nerve and Muscle Childbirth Injury Model.

In women, stress urinary incontinence (SUI), leakage of urine from increased abdominal pressure, is correlated with pudendal nerve (PN) injury during childbirth. Expression of brain-derived neurotrophic factor (BDNF) is dysregulated in a dual nerve and muscle injury model of childbirth. We aimed to use tyrosine kinase B (TrkB), the receptor of BDNF, to bind free BDNF and inhibit spontaneous regeneration in a rat model of SUI. We hypothesized that BDNF is essential for functional recovery from the dual nerve and muscle injuries that can lead to SUI. Female Sprague-Dawley rats underwent PN crush (PNC) and vaginal distension (VD) and were implanted with osmotic pumps containing saline (Injury) or TrkB (Injury + TrkB). Sham Injury rats received sham PNC + VD. Six weeks after injury, animals underwent leak-point-pressure (LPP) testing with simultaneous external urethral sphincter (EUS) electromyography recording. The urethra was dissected for histology and immunofluorescence. LPP after injury and TrkB was significantly decreased compared to Injury rats. TrkB treatment inhibited reinnervation of neuromuscular junctions in the EUS and promoted atrophy of the EUS. These results demonstrate that BDNF is essential to neuroregeneration and reinnervation of the EUS. Treatments aimed at increasing BDNF periurethrally could promote neuroregeneration to treat SUI.

Brain-Derived Neurotrophic Factor Is an Important Therapeutic Factor in Mesenchymal Stem Cell Secretions for Treatment of Traumatic Peripheral Pelvic Injuries.

Traumatic neuromuscular injury to the pudendal nerve and urethra during childbirth does not regenerate well and contributes to stress urinary incontinence in women. Mesenchymal stem cells (MSCs) can improve neuroregeneration via their secretions, or secretome, which includes brain-derived neurotrophic factor (BDNF). In this study, we investigated whether BDNF is a key factor in the secretome of MSCs for the facilitation of functional recovery following a dual simulated childbirth injury. BDNF knockdown (KD) MSCs were created using an anti-BDNF shRNA lentivirus vector. A scrambled sequence was used as a transduction control (scrambled). Cells were cultured for 24 h before media was concentrated 50x to create concentrated conditioned media (CCM) containing MSC secretome. CCM of unmanipulated MSCs was screened for high BDNF expression (high BDNF CCM). Concentrated control media (CM) was created by concentrating media not conditioned by cells. Female Sprague-Dawley rats underwent bilateral pudendal nerve crush and vaginal distension (Injury) or sham injury. One hour and 1 week after injury, sham injured rats received CM, and injured rats received CM, high BDNF CCM, KD CCM, or scrambled CCM (300 µl intraperitoneally). Three weeks after injury, rats underwent leak point pressure (LPP) and pudendal nerve sensory branch potential (PNSBP) recordings. The urethra and pudendal nerve were harvested for anatomical assessment. ANOVA followed by the Student-Newman-Keuls test determined significant differences between groups (p < 0.05). BDNF KD CCM had significantly decreased BDNF concentration compared to scrambled CCM, while the concentration in high BDNF CCM was significantly increased. LPP was significantly decreased in CM and KD CCM treated animals compared to sham injury, but not with scrambled or high BDNF CCM. PNSBP firing rate showed a significant decrease with CM treatment compared to sham injury. Neuromuscular junctions in the urethral sphincter in KD CCM, scrambled CCM, and high BDNF CCM were healthier than CM treated rats. While anatomical and nerve function tests demonstrate regeneration of the pudendal nerve with any CCM treatment, LPP results suggest it takes longer to recover continence with reduced BDNF in CCM. BDNF in MSC CCM is an important factor for the acceleration of recovery from a dual nerve and muscle injury.

Brain derived neurotrophic factor mediates accelerated recovery of regenerative electrical stimulation in an animal model of stress urinary incontinence.

OBJECTIVE: Stress urinary incontinence (SUI) is prevalent among older women and can result from insufficient regeneration of the pudendal nerve (PN). Electrical stimulation (ES) of the PN upregulates brain derived neurotrophic factor (BDNF) and accelerates regeneration. Using tyrosine kinase B (TrkB) to reduce the availability of free BDNF, the aim of this study was to determine if BDNF is necessary for accelerated recovery via ES in a model of SUI. METHODS: Our SUI model consists of Female Sprague-Dawley rats, whose PNs were crushed bilaterally twice for 30 s, followed by insertion of a modified Foley catheter into the vagina with balloon inflation for 4 h. These rats were divided into 4 groups: 1) Sham PN crush and sham vaginal distension without electrode implantation and with saline treatment (sham injury); 2) SUI with sham stimulation and saline treatment (SUI); 3) SUI and ES with saline treatment (SUI&ES); and 4) SUI and ES with TrkB treatment (SUI&ES&TrkB). Animals underwent ES or sham stimulation four times a week for two weeks. Four weeks after injury, animals underwent functional testing consisting of leak point pressure (LPP) with simultaneous external urethral sphincter (EUS) electromyography (EMG) and pudendal nerve recordings. Data was analyzed using ANOVA with Holm-Sidak posthoc test (p < 0.05). EUS and PN specimen were sectioned and stained to semi-quantitatively evaluate morphology, regeneration, and reinnervation. RESULTS: LPP and EUS EMG firing rate were significantly increased in the sham injury and SUI&ES groups compared to the SUI and SUI&ES&TrkB groups. EUS of SUI rats showed few innervated neuromuscular junctions compared to sham injured rats, while both treatment groups showed an increase in reinnervated neuromuscular junctions. CONCLUSION: ES accelerates functional recovery via a BDNF-mediated pathway in a model of SUI. These findings suggest ES could be used as a potential regenerative therapy for women with SUI.

The pudendal nerve motor branch regenerates via a brain derived neurotrophic factor mediated mechanism.

Peripheral nerve injuries can significantly reduce quality of life. While some recover, most do not recover fully, resulting in neuropathic pain and loss of sensation and motor function. Research on the mechanisms of peripheral nerve regeneration could elucidate poor patient outcomes and potential treatments. This study was designed to determine if brain derived neurotrophic factor (BDNF) is necessary for pudendal nerve regeneration and functional recovery. Peripheral administration of tyrosine kinase B functional chimera (TrkB) was used to inhibit the BDNF regenerative pathway. Female Sprague-Dawley rats received tyrosine kinase B functional chimera (TrkB) or saline after a pudendal nerve crush (PNC) or Sham PNC and were divided into three groups: Sham PNC, PNC + Saline, and PNC + TrkB. Seven days after injury, relative ßII tubulin expression (1.0 ± 0.2) was significantly decreased after PNC + TrkB compared to PNC + saline (2.9 ± 1.0). Three weeks after injury, BDNF plasma concentration (1320.8 ± 278.1 pg/ml) was significantly reduced in PNC + TrkB compared to PNC + saline rats (2053.4 ± 211.0 pg/ml). Pudendal nerve motor branch firing rate (54.0 ± 9.5 Hz) was significantly decreased in the PNC + TrkB group compared to the PNC + saline group (120.4 ± 17.1 Hz); while nerve firing rate of the PNC + saline group was not significantly different from sham PNC rats (121.8 ± 26.6 Hz). This study demonstrated that peripheral administration of TrkB bound free BDNF and inhibited the regenerative response after PNC. BDNF is necessary for normal PN motor branch recovery after PNC.

Combination phosphodiesterase type 4 inhibitor and phosphodiesterase type 5 inhibitor treatment reduces non-voiding contraction in a rat model of overactive bladder.

INTRODUCTION: Current treatments for overactive bladder (OAB) are often discontinued due to side effects or lack of efficacy. The goal of this study was to determine if combining a phosphodiesterase type 4 inhibitor (PDE4i); with a type 5 inhibitor (PDE5i); would have a beneficial effect on OAB symptoms and if a reduced dose of PDE4i in combination with PDE5i could also provide a beneficial effect in OAB. We hypothesized that PDE5i and PDE4i combination treatment could be utilized to reduce non-voiding contractions and smooth muscle disruption in a rat model of OAB. METHODS: Fifty-eight age-matched Sprague-Dawley rats underwent PBOO and daily gavage with PDE4i alone (roflumilast; 1mg/kg), PDE5i alone (tadalafil;10mg/kg), high dose combination (PDE4i 1mg/kg, PDE5i 10mg/kg), low dose combination (PDE4i 0.2mg/kg, PDE5i 10mg/kg), or vehicle for 28 days. Fourteen animals underwent sham PBOO with vehicle. Rats underwent conscious and anesthetized cystometry 28 days after PBOO and were euthanized for qualitative bladder histology. One-way ANOVA on ranks with a Dunn's post hoc test was used to indicate statistically significant differences between groups (p<0.05). RESULTS: Bladder & urethral weight was significantly increased after PBOO with vehicle, PDE4i alone, and PDE5i alone, but not with either combination treatment. Frequency of non-voiding contractions during both conscious and anesthetized cystometry increased significantly after PBOO with vehicle, but not after PDE4i or high dose combination treatments compared to sham PBOO. Threshold pressure for voiding was significantly decreased with high dose combination compared to vehicle. PBOO treated with PDE4i alone or high dose combination showed less bladder smooth muscle fibrosis than vehicle, PDE5i alone, or low dose combination treatments. CONCLUSION: A PDE4i and PDE5i combination treatment has potential benefit in reducing OAB symptoms, but future research is needed.

Role of lysyl oxidase like 1 in regulation of postpartum connective tissue metabolism in the mouse vagina†.

Pelvic organ prolapse (POP) in lysyl oxidase like-1 knockout (Loxl1 KO) mice occurs primarily in parous mice and is rare in nulliparous mice. We determined the effect of Loxl1 deficiency on postpartum regulation of connective tissue metabolism genes and degradative enzyme activity in the vagina at 20 days gestation or 4 h, 48 h, 7 days, 15 days, 25 days, 7 weeks, or 12 weeks postpartum. Nulliparous Loxl1 KO and wildtype (WT) mice aged 11, 18, or 23 weeks were controls. Gene expression and enzyme activity were assessed using real-time quantitative reverse transcription PCR and fluorescein conjugated gelatin zymography, respectively. Parity, but not aging, had a significant influence on gene expression both with time postpartum and between KO and WT mice. Mmp2, Timp1, Timp2, Timp3, Timp4, Col1a1, Col3a1, Acta2, and Bmp1 were differentially expressed between KO and WT mice. Correlational analysis of gene-gene pairs revealed 10 significant differences between parous KO and WT groups, 5 of which were due to lack of co-expression of Bmp1 in KO mice. The overall enzyme activity that could be attributed to MMPs was significantly higher in WT compared to KO mice both 25 days and 12 weeks postpartum, and MMP activity was significantly lower 15 days and 25 days postpartum compared to KO nulliparous controls, but not WT. These findings suggest that Loxl1 deficiency combined with parity has a significant impact on postpartum regulation of connective tissue metabolism, particularly as it relates to co-expression of Bmp1 and altered proteolytic activity.

Multiple doses of stem cells maintain urethral function in a model of neuromuscular injury resulting in stress urinary incontinence.

Stress urinary incontinence (SUI) is more prevalent among women who deliver vaginally than women who have had a cesarean section, suggesting that tissue repair after vaginal delivery is insufficient. A single dose of mesenchymal stem cells (MSCs) has been shown to partially restore urethral function in a model of SUI. The aim of the present study was to determine if increasing the number of doses of MSCs improves urethral and pudendal nerve function and anatomy. We hypothesized that increasing the number of MSC doses would accelerate recovery from SUI compared with vehicle treatment. Rats underwent pudendal nerve crush and vaginal distension or a sham injury and were treated intravenously with vehicle or one, two, or three doses of 2 × 106 MSCs at 1 h, 7 days, and 14 days after injury. Urethral leak point pressure testing with simultaneous external urethral sphincter electromyography and pudendal nerve electroneurography were performed 21 days after injury, and the urethrovaginal complex and pudendal nerve were harvested for semiquantitative morphometry of the external urethral sphincter, urethral elastin, and pudendal nerve. Two and three doses of MSCs significantly improved peak pressure; however, a single dose of MSCs did not. Single, as well as repeated, MSC doses improved urethral integrity by restoring urethral connective tissue composition and neuromuscular structures. MSC treatment improved elastogenesis, prevented disruption of the external urethral sphincter, and enhanced pudendal nerve morphology. These results suggest that MSC therapy for postpartum incontinence and SUI can be enhanced with multiple doses.

Daily bilateral pudendal nerve electrical stimulation improves recovery from stress urinary incontinence.

Stress urinary incontinence (SUI) in women is strongly associated with childbirth which injures the pudendal nerve (PN) and the external urethral sphincter (EUS) during delivery. Electrical stimulation (ES) can increase brain-derived neurotrophic factor (BDNF) expression in injured neurons, activate Schwann cells and promote neuroregeneration after nerve injury. The aim of this study was to determine if more frequent ES would increase recovery from SUI in a rat model. Forty female Sprague-Dawley rats underwent either sham injury or pudendal nerve crush (PNC) and vaginal distention (VD) to establish SUI. Immediately after injury, electrodes were implanted at the pudendal nerve bilaterally. Each injured animal underwent sham ES, twice per week ES (2/week), or daily ES of 1 h duration for two weeks. Urethral and nerve function were assessed with leak point pressure (LPP), EUS electromyography and pudendal nerve sensory branch potential (PNSBP) recordings two weeks after injury. LPP was significantly increased after daily ES compared to 2/week ES. EUS neuromuscular junction innervation was decreased after injury with sham ES, but improved after 2/week or daily ES. This study demonstrates that daily bilateral ES to the pudendal nerve can accelerate recovery from SUI. Daily ES improved urethral function more than 2/week ES.

Electrical stimulation for neuroregeneration in urology: a new therapeutic paradigm.

PURPOSE OF REVIEW: The present review highlights regenerative electrical stimulation (RES) as potential future treatment options for patients with nerve injuries leading to urological dysfunction, such as urinary incontinence, voiding dysfunction or erectile dysfunction. Additionally, it will highlight the mechanism of nerve injury and regeneration as well as similarities and differences between RES and current electrical stimulation treatments in urology, functional electrical stimulation (FES) and neuromodulation. RECENT FINDINGS: It has been demonstrated that RES upregulates brain-derived neurotrophic factor (BDNF) and its receptor to facilitate neuroregeneration, facilitating accurate reinnervation of muscles by motoneurons. Further, RES upregulates growth factors in glial cells. Within the past 2 years, RES of the pudendal nerve upregulated BDNF in Onuf's nucleus, the cell bodies of motoneurons that course through the pudendal nerve and accelerated functional recovery in an animal model of stress urinary incontinence. Additionally, electrical stimulation of the vaginal tissue in an animal model of stress urinary incontinence accelerated functional recovery. SUMMARY: RES has great potential but future research is needed to expand the potential beneficial effects of RES in the field of urology.

Electrical stimulation of the pudendal nerve promotes neuroregeneration and functional recovery from stress urinary incontinence in a rat model.

The pudendal nerve can be injured during vaginal delivery of children, and slowed pudendal nerve regeneration has been correlated with development of stress urinary incontinence (SUI). Simultaneous injury to the pudendal nerve and its target muscle, the external urethral sphincter (EUS), during delivery likely leads to slowed neuroregeneration. The goal of this study was to determine if repeat electrical stimulation of the pudendal nerve improves SUI recovery and promotes neuroregeneration in a dual muscle and nerve injury rat model of SUI. Rats received electrical stimulation or sham stimulation of the pudendal nerve twice weekly for up to 2 wk after injury. A separate cohort of rats received sham injury and sham stimulation. Expression of brain-derived neurotrophic factor (BDNF) and ßII-tubulin expression in Onuf's nucleus were measured 2, 7, and 14 days after injury. Urodynamics, leak point pressure (LPP), and EUS electromyography (EMG) were recorded 14 days after injury. Electrical stimulation significantly increased expression of BDNF at all time points and ßII-tubulin 1 and 2 wk after injury. Two weeks after injury, LPP and EUS EMG during voiding and LPP testing were significantly decreased compared with sham-injured animals. Electrical stimulation significantly increased EUS activity during voiding, although LPP did not fully recover. Repeat pudendal nerve stimulation promotes neuromuscular continence mechanism recovery possibly via a neuroregenerative response through BDNF upregulation in the pudendal motoneurons in this model of SUI. Electrical stimulation of the pudendal nerve may therefore improve recovery after childbirth and ameliorate symptoms of SUI by promoting neuroregeneration after injury.

Effect of Pregnancy and Delivery on Cytokine Expression in a Mouse Model of Pelvic Organ Prolapse.

OBJECTIVES: The aim of this study was to determine the effect of pregnancy and delivery mode on cytokine expression in the pelvic organs and serum of lysyl oxidase like-1 knockout (LOXL1 KO) mice, which develop pelvic organ prolapse after delivery. METHODS: Bladder, urethra, vagina, rectum, and blood were harvested from female LOXL1 KO mice during pregnancy, after vaginal or cesarean delivery, and from sham cesarean and unmanipulated controls. Pelvic organs and blood were also harvested from pregnant and vaginally delivered wild-type (WT) mice and from unmanipulated female virgin WT controls. Specimens were assessed using quantitative real-time reverse transcription polymerase chain reaction and/or enzyme-linked immunosorbent assay. RESULTS: Both CXCL12 and CCL7 mRNA were significantly up-regulated in the vagina, urethra, bladder, and rectum of pregnant LOXL1 KO mice compared with pregnant WT mice, suggesting systemic dysregulation of both of these cytokines in LOXL1 KO mice as a response to pregnancy.The differences in cytokine expression between LOXL1 KO and WT mice in pregnancy persisted after vaginal delivery. CCL7 gene expression increases faster and to a greater extent in LOXL1 KO mice, translating to longer lasting increases in CCL7 in serum of LOXL1 KO mice after vaginal delivery, compared with pregnant mice. CONCLUSIONS: Lysyl oxidase like-1 KO mice have an increased cytokine response to pregnancy perhaps because they are less able to reform and re-cross-link stretched elastin to accommodate pups, and this resultant tissue stretches during pregnancy. The up-regulation of CCL7 after delivery could provide an indicator of level of childbirth injury, to which the urethra and vagina seem to be particularly vulnerable.

Combination histamine and serotonin treatment after simulated childbirth injury improves stress urinary incontinence.

AIMS: Histamine and serotonin-related pharmaceuticals have the potential to modulate micturition and continence. The aim of this study was to determine if treatment with histamine and/or serotonin improves stress urinary incontinence (SUI) in female rats. METHODS: Twenty-six age-matched female rats underwent pudendal nerve crush and vaginal distension (PNC + VD), to produce SUI. One week after injury, rats were treated subcutaneously with saline, histamine (1.1 µg), serotonin (2µg), or the combination of both twice daily for another week. A sham injured group received sham PNC + VD and were treated with saline (n = 7). Leak point pressure (LPP) testing with simultaneous external urethral sphincter (EUS) electromyography (EMG) was conducted 2 weeks after injury. The urethra was harvested for qualitative and quantitative histology. Data were analyzed with a one-way ANOVA and Student-Newman-Keuls posthoc test with P < 0.05 indicating statistically significant differences between groups. RESULTS: Combination treatment significantly increased LPP after PNC + VD compared to injured sham treatment and treatment with either histamine or serotonin alone. Compared to injured sham treated rats, all three treatments significantly increased EUS EMG amplitude at both baseline and peak pressure and EUS EMG firing rate at peak pressure during LPP testing. There were more consistent urethral striated muscle fibers and thicker smooth and striated muscle with combination and histamine treatment. There was a statistically significant shift to a greater proportion of thicker collagen fibers in the urethra in serotonin and combination treated rats compared with injured sham treated rats. CONCLUSIONS: Combination treatment was the most effective and may provide an effective therapy for SUI. Neurourol. Urodynam. 35:703-710, 2016. © 2015 Wiley Periodicals, Inc.

Molecular Assessment of Neuroregenerative Response in the Pudendal Nerve: A Useful Tool in Regenerative Urology.

AIMS: Assessing pudendal nerve neuroregenerative response provides valuable insight into injuries and regenerative treatments related to urinary incontinence. This project developed and validated a cost-effective, expedient, and adoptable method of assessing pudendal nerve neuroregenerative response. METHODS: Sprague Dawley rats underwent unilateral pudendal nerve crush prior to spinal cord harvest and laser microdissection for separate collection of the injured and uninjured Onuf's nuclei (pudendal motor neuron cell bodies). Commercially available kits were used to extract and isolate RNA, as well as reverse transcribe and amplify cDNA from cells. Utilizing standard quantitative polymerase chain reaction (Q-PCR), expression of ßII-Tubulin, a cytoskeletal protein indicative of nerve growth and neuroregenerative response, was determined in the injured side relative to the uninjured side 1 week after injury. RESULTS: Injury upregulated ßII-Tubulin 2.36±0.46 times via Q-PCR, which was not significantly (p=0.508) different from the 2.49±0.38 times increase noted with in-situ hybridization previously. Starting with tissue collection, results are available within 1 day using PCR, while in-situ hybridization requires 4-weeks. CONCLUSIONS: An easily adoptable PCR-based method of assessing the neuroregenerative response of the pudendal nerve successfully reproduced results obtained with a previous radioisotope-based in-situ hybridization technique.

Long-term effects of simulated childbirth injury on function and innervation of the urethra.

AIMS: Pudendal nerve and external urethral sphincter (EUS) injury during vaginal delivery are risk factors for stress urinary incontinence (SUI). Although most patients with short-term postpartum SUI regain continence within 1 year, they have a higher predisposition to develop recurrent SUI years later, suggesting a possible mechanistic relationship. In contrast, animal models generally recover spontaneously and have not been studied much in the long term. The aim of this study was to investigate the long-term effects of simulated childbirth injury in rats. METHODS: Thirty-four Sprague-Dawley female rats underwent sham injury or pudendal nerve crush and vaginal distension (PNC + VD), a simulated childbirth injury. Nine weeks later, leak point pressure (LPP) and EUS electromyography (EMG) were recorded simultaneously. The pudendal nerve was harvested for histological analysis. EUS neuromuscular junctions (NMJs) and their innervation were qualitatively assessed using immunofluorescence. A t-test was used to compare quantitative outcomes between groups, with P < 0.05 indicating a significant difference. RESULTS: There was no significant difference in LPP or EUS EMG amplitude or firing rate between the two groups. Nonetheless after PNC + VD, NMJs in the EUS were diffuse and were innervated by tortuous and multiple axons, demonstrating that reinnervation of the EUS was still in progress. CONCLUSIONS: Although continence function recovered 9 weeks after simulated childbirth injury, innervation of EUS was not complete at this time point, suggestive of persistent neurogenic deficiency which when compounded by the effects of aging may lead to a delayed recurrence of SUI in this animal model with increased age.

Somatomotor and sensory urethral control of micturition in female rats.

In rats, axons of external urethral sphincter (EUS) motoneurons travel through the anastomotic branch of the pudendal nerve (ABPD) and anastomotic branch of the lumbosacral trunk (ABLT) and converge in the motor branch of the sacral plexus (MBSP). The aim of the present study was to determine in female rats the contribution of these somatomotor pathways and urethral sensory innervation from the dorsal nerve of the clitoris on urinary continence and voiding. EUS electromyographic (EMG) activity during cystometry, leak point pressure (LPP), and voiding efficiency (VE) were assessed in anesthetized virgin Sprague-Dawley female rats before and after transection of the above nerve branches. Transection of the MBSP eliminated EUS EMG, decreased LPP by 50%, and significantly reduced bladder contraction duration, peak pressure, intercontraction interval, and VE. Transection of the ABPD or ABLT decreased EUS EMG discharge and LPP by 25% but did not affect VE. Transection of the dorsal nerve of the clitoris did not affect LPP but reduced contraction duration, peak pressure, intercontraction interval, and VE. We conclude that somatomotor control of micturition is provided by the MBSP with axons travelling through the ABPD and ABLT. Partial somatomotor urethral denervation induces mild urinary incontinence, whereas partial afferent denervation induces voiding dysfunction. ABPD and ABLT pathways could represent a safeguard ensuring innervation to the EUS in case of upper nerve damage. Detailed knowledge of neuroanatomy and functional innervation of the urethra will enable more accurate animal models of neural development, disease, and dysfunction in the future.

Rat mesenchymal stem cell secretome promotes elastogenesis and facilitates recovery from simulated childbirth injury.

Vaginal delivery is a risk factor for stress urinary incontinence (SUI). Mesenchymal stem cells (MSCs) home to injured organs and can facilitate repair. The goal of this study was to determine if MSCs home to pelvic organs after simulated childbirth injury and facilitate recovery from SUI via paracrine factors. Three experiments were performed. Eighteen female rats received vaginal distension (VD) or sham VD and labeled intravenous (IV) MSCs to investigate if MSCs home to the pelvic organs. Whole-organ imaging and immunofluorescence were performed 1 week later. Thirty-four female rats received VD and IV MSCs, VD and IV saline, or sham VD and IV saline to investigate if MSCs accelerate recovery of continence. Twenty-nine female rats received VD and periurethral concentrated conditioned media (CCM), VD and periurethral control media, or sham VD and periurethral control media to investigate if factors secreted by MSCs accelerate recovery from VD. Urethral histology and function were assessed 1 week later. Significantly more MSCs were observed in the urethra, vagina, and spleen after VD compared to sham VD. Continence as measured by leak point pressure (LPP) was significantly reduced after VD in rats treated with saline or control media compared to sham VD but not in those given MSCs or CCM. External urethral sphincter (EUS) function as measured by electromyography (EMG) was not improved with MSCs or CCM. Rats treated with MSCs or CCM demonstrated an increase in elastin fibers near the EUS and urethral smooth muscle more similar to that of sham-injured animals than rats treated with saline or control media. MSCs homed to the urethra and vagina and facilitated recovery of continence most likely via secretion of paracrine factors. Both MSCs and CCM have promise as novel noninvasive therapies for SUI.

Neurotrophin therapy improves recovery of the neuromuscular continence mechanism following simulated birth injury in rats.

AIMS: Stress urinary incontinence (SUI) affects women both acutely and chronically after vaginal delivery. Current SUI treatments assume the neuromuscular continence mechanism, comprised of the pudendal nerve (PN) and external urethral sphincter (EUS), is either intact or irreparable. This study investigated the ability of neurotrophin therapy to facilitate recovery of the neuromuscular continence mechanism. METHODS: Virgin, Sprague Dawley rats received simulated childbirth injury or sham injury and treatment with continuous infusion of brain-derived neurotrophic factor (BDNF) or saline placebo to the site of PN injury. Continence was assessed by leak point pressure (LPP) and EUS electromyography (EMG) 14 and 21 days after injury. Structural recovery was assessed histologically. Molecular assessment of the muscular and neuroregenerative response was determined via measurement of EUS BDNF and PN ß(II) -tubulin expression respectively, 4, 8, and 12 days after injury. RESULTS: Following injury, LPP was significantly reduced with saline compared to either BDNF treatment or sham injury. Similarly, compared to sham injury, resting EUS EMG amplitude and firing rate, as well as amplitude during LPP were significantly reduced with saline but not BDNF treatment. Histology confirmed improved EUS recovery with BDNF treatment. EUS BDNF and PN ß(II)-tubulin expression demonstrated that BDNF treatment improved the neurogenerative response and may facilitate sphincteric recovery. CONCLUSIONS: Continuous targeted neurotrophin therapy accelerates continence recovery after simulated childbirth injury likely through stimulating neuroregeneration and facilitating EUS recovery and re-innervation. Neurotrophins or other therapies targeting neuromuscular regeneration may be useful for treating SUI related to failure of the neuromuscular continence mechanism.