Recurrent infections drive persistent bladder dysfunction and pain via sensory nerve sprouting and mast cell activity.

Urinary tract infections (UTIs) account for almost 25% of infections in women. Many are recurrent (rUTI), with patients frequently experiencing chronic pelvic pain and urinary frequency despite clearance of bacteriuria after antibiotics. To elucidate the basis for these bacteria-independent bladder symptoms, we examined the bladders of patients with rUTI. We noticed a notable increase in neuropeptide content in the lamina propria and indications of enhanced nociceptive activity. In mice subjected to rUTI, we observed sensory nerve sprouting that was associated with nerve growth factor (NGF) produced by recruited monocytes and tissue-resident mast cells. Treatment of rUTI mice with an NGF-neutralizing antibody prevented sprouting and alleviated pelvic sensitivity, whereas instillation of native NGF into naïve mice bladders mimicked nerve sprouting and pain behavior. Nerve activation, pain, and urinary frequency were each linked to the presence of proximal mast cells, because mast cell deficiency or treatment with antagonists against receptors of several direct or indirect mast cell products was each effective therapeutically. Thus, our findings suggest that NGF-driven sensory sprouting in the bladder coupled with chronic mast cell activation represents an underlying mechanism driving bacteria-independent pain and voiding defects experienced by patients with rUTI.

In vitro high glucose increases apoptosis, decreases nerve outgrowth, and promotes survival of sympathetic pelvic neurons.

Background: Diabetes mellitus (DM) is a common cause of erectile dysfunction (ED), yet the molecular basis of DM neurogenic ED remains unknown. Aim: In this study we examined the impact of high glucose on survival and growth of primary cultured pelvic neurons in a rat model and assessed whether coculturing with healthy Schwann cells (SCs) can rescue pelvic neuron growth in patients with DM. Methods: Major pelvic ganglia (MPGs) from adult male Sprague Dawley rats (n = 8) were dissociated and plated on coverslips. Neurons were exposed to high glucose (45 mM) for 24 or 48 hours and compared to time-matched controls (25 mM). Neurons were stained for neuron-specific beta-tubulin, neuronal nitric oxide synthase, vesicular acetylcholine transferase, tyrosine hydroxylase, and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling) assay. Schwann cells were dissociated from MPGs of healthy male Sprague Dawley rats (n = 4) and grown to confluence. Additional Sprague Dawley rats were made diabetic with streptozotocin (50 mg/kg, n = 4), and 5 weeks later MPGs were collected from these rats, dissociated, and cocultured on healthy SCs. Neurons and SCs were stained with beta-tubulin and S100. Outcomes: Length, branching, and survival of nitrergic, parasympathetic, and sympathetic neurons was assessed in neurons exposed to normal or high glucose concentrations, and neuron length was measured in neuron-SC coculture. Results: The total number of neurons and the length and number of branches were significantly decreased after 24 and 48 hours of high glucose (P < .05). The percentage of nitrergic neurons decreased 10% after 24 hours and 50% after 48 hours of high glucose (P < .05). After 24 hours of high glucose, cholinergic-positive neurons were unchanged; however, these neurons decreased 30% after 48 hours (P < .05). The proportion of sympathetic neurons increased 25% after 48 hours of high glucose (P < .05). At both timepoints, there was a 2-fold increase in the total apoptotic neurons with high glucose (P < .05). Neurite outgrowth recovered to control lengths after coculture of diabetic neurons with healthy SCs (P < .05). Clinical Translation: Glucose can be used as a tool to investigate the direct effects of DM on neuritogenesis. Our data suggest that an effective treatment for DM ED protects and repairs the penile neuronal supply. Strengths and Limitations: Exposing MPG neurons to high glucose offers a quick and, inexpensive proxy for DM-related conditions. A limitation of our study is that our model reflects type 1 DM, whereas clinically, most diabetic ED patients have type 2 DM. Conclusion: Culturing pelvic neurons in high glucose can be used as a tool to elucidate how to protect proerectile neurons from cell death and may lead to new therapeutic strategies for diabetic men suffering from ED.

Cocultured Schwann Cells Rescue Irradiated Pelvic Neuron Outgrowth and Increase Survival.

BACKGROUND: Prostatic radiation therapy (RT) leads to erectile dysfunction by damaging peri-prostatic pro-erectile nerves of the pelvic ganglion. Schwann cells (SC) facilitate neuronal repair after mechanical injury, however, their role in repair of pelvic neurons post-radiation hasn't been explored. AIM: To determine if SCs cocultured with primary pelvic neurons can rescue neuronal survival and growth after ex vivo RT. METHODS: Major pelvic ganglia (MPG) were collected from adult male Sprague-Dawley rats (n = 12) to isolate SCs. SCs received RT (0 or 8 Gy), were plated on coated coverslips and grown to confluence before the addition of neurons. Additional MPGs were irradiated (0 or 8 Gy) and digested to isolate pelvic neurons. Dissociated neurons were plated alone or atop SC-coated coverslips to create 6 experimental groups (n = 3/grp): (i) Control (CON) MPG, (ii) RT MPG, (iii) CON SC + CON MPG, (iv) CONSC + RT MPG, (v) RT SC + CON MPG, and (iv) RT SC + RT MPG. After 72 hours, coverslips were fixed and stained for beta-tubulin (neuron marker), S100 (SC marker), neuronal nitric oxide synthase (nitrergic marker), tyrosine hydroxylase (sympathetic marker), and terminal deoxynucleotidyl transferase dUTP nick-end labeling. OUTCOMES: We measured neurite length, branching, specific neuron populations and apoptosis. RESULTS: Ex vivo RT decreased MPG neuron length, increased apoptosis and decreased nitrergic neurons in monoculture. Compared to all other groups, CON SC + RT MPG cocultures demonstrated increased neurite outgrowth (P < .001). Neurite branching was decreased in the RT MPG + RT SC coculture, but unchanged in other cocultures. Groups containing RT MPG neurons exhibited increased apoptosis, but coculture with CON SC reduced the degree of RT-induced apoptosis (P < .01). The number of tyrosine hydroxylase positive neurons was unchanged while nitrergic neurons were significantly lower in RT neurons and coculture with CON SCs was unable to prevent nitrergic loss. CLINICAL TRANSLATION: These findings suggest that SCs may be an important target in prostate cancer patients with radiation-induced pelvic neuropathy to promote MPG neuron survival and neuronal repair after RT. STRENGTHS AND LIMITATIONS: This is the first study to characterize the ex vivo ability of SCs to rescue pelvic nerve growth and survival. The study is limited by little supporting mechanistic molecular data and the need to confirm the ability of healthy SCs to promote pelvic neuron survival and repair following prostatic RT in vivo. CONCLUSION: Unirradiated SCs partially mitigated RT-induced MPG apoptosis but did not affect the loss of nitrergic neuron populations suggesting that SCs promote irradiated MPG neuron survival and facilitate intrinsic repair functions. Randolph JT, Pak ES, McMains JC, et al. Cocultured Schwann Cells Rescue Irradiated Pelvic Neuron Outgrowth and Increase Survival. J Sex Med 2022;19:1333-1342.

Cardiometabolic Diseases and Female Sexual Dysfunction: Animal Studies.

BACKGROUND: The association between erectile dysfunction and cardiometabolic disease is well characterized; men are often diagnosed with cardiovascular disease 2-5 years following the incidence of erectile dysfunction. There is evidence that this relationship may also exist for cardiometabolic diseases and female sexual dysfunction (FSD) - particularly sexual arousal disorders. AIM: To provide a summary of the preclinical literature related to the evidence of FSD in animal models of cardiometabolic diseases and indicate where further research is needed. METHODS: A detailed Medline search of peer-reviewed publications was performed on the associations between animal models of cardiometabolic diseases, FSD and underlying mechanisms. OUTCOMES: A summary of the preclinical evidence of FSD in animal models of cardiometabolic diseases. RESULTS: Common methods for assessing female sexual arousal and physiology in animal models include: 1) behavioral assessments (apomorphine-induced genital vasocongestive arousal; hormone-dependent lordosis), 2) nerve-mediated vaginal and clitoral blood flow, 3) pudendal artery, vaginal and clitoral smooth muscle physiology (vasoreactivity and molecular biology), 4) morphology of genital tissues. Twenty-eight studies examined female animal models of atherosclerosis, hypertension, diabetes (type 1 and 2) and obesity. They showed functional alterations, including decreased lordosis, lubrication, or vaginal and clitoral blood flow, and structural impairments, such as increased clitoral and vaginal fibrosis. Several possible mechanisms have been described including increased TGF-ß, renin angiotensin system and endothelin/rho-kinase signaling, increased reactive oxygen species, and decreased nitric oxide/cGMP signaling. CLINICAL TRANSLATION: In line with existing clinical studies, preclinical evidence supports that cardiometabolic diseases alter female genital tissue's function and structure leading to impaired sexual arousal. STRENGTHS AND LIMITATIONS: This masterclass paper gives an overview of the preclinical research assessing FSD in cardiometabolic disease. Limitations include the small number of studies that have assessed sexual function and arousal in female cardiometabolic animal models. CONCLUSION: Preclinical evidence exists showing cardiometabolic diseases alter the structure and function of female genital tissues. However, similar to clinical studies, there are few studies to draw from, particularly in models of type 2 diabetes, obesity and metabolic syndrome. More studies are required using optimized animal models and methodology to confirm the mechanisms underlying cardiometabolic disease-induced FSD. Angulo J, Hannan JL. Cardiometabolic Diseases and Female Sexual Dysfunction: Animal Studies. J Sex Med 2022;19:408-420.

High-fat diet induces obesity in adult mice but fails to develop pre-penile and penile vascular dysfunction.

Obesity can lead to cardiovascular disease, diabetes, and erectile dysfunction (ED), which decreases overall quality of life. Mechanisms responsible for obesity-induced ED are unknown. Current mouse models of high-fat diet (HFD)-induced obesity yield conflicting results. Genetic variants among common "wild type" strains may explain contradictory data. Adult male C57BL/6N and 6J mice were fed a 45% HFD for 12 weeks. Weekly food intake, weight gain, and body-fat percentage were measured. After 12 weeks, ex vivo vascular reactivity was measured in aortas, internal pudendal arteries, and penises. We assessed smooth muscle contractility, endothelial-dependent and -independent relaxation, and penile neurotransmitter-mediated relaxation. C57BL/6N mice developed greater obesity and glucose sensitivity compared to C57BL/6J mice. Aortas from both strains that fed a HFD had decreased contraction, yet contraction was unchanged in HFD pudendal arteries and penises. Interestingly, endothelial-dependent and -independent relaxation was unchanged in both systemic and penile vasculature. Likewise, HFD did not impair penile neurotransmitter-mediated relaxation. Both strains fed 12 weeks of HFD-developed obese phenotypes. However, HFD did not impair pre-penile or penile smooth muscle vasoreactivity as demonstrated in previous studies, suggesting that this preclinical model does not accurately represent the clinical phenotype of obesity-induced ED.

Ex vivo Akt inhibition reverses castration induced internal pudendal artery and penile endothelial dysfunction.

AIMS: Androgen deprivation therapy is a common prostate cancer treatment which causes men to have castrate levels of testosterone. Unfortunately, most testosterone deficient patients will suffer severe erectile dysfunction (ED) and have no effective ED treatment options. Testosterone deficiency causes endothelial dysfunction and impairs penile vasodilation necessary to maintain an erection. Recent evidence demonstrates testosterone activates androgen receptors (AR) and generates nitric oxide (NO) through the Akt-endothelial NO synthase (eNOS) pathway; however, it remains unknown how castration impacts this signaling pathway. MATERIALS AND METHODS: In this study, we used a surgically castrated rat model to determine how castration impacts ex vivo internal pudendal artery (IPA) and penile relaxation through the Akt-eNOS pathway. KEY FINDINGS: Unlike systemic vasculature, castration causes significant IPA and penis endothelial dysfunction associated with a 50% AR reduction. Though testosterone and acetylcholine (ACh) both phosphorylate Akt and eNOS, castration did not affect testosterone-mediated IPA and penile Akt or eNOS phosphorylation. Surprisingly, castration increases ACh-mediated Akt and eNOS phosphorylation but reduces the eNOS dimer to monomer ratio. Akt inhibition using 10DEBC preserves IPA eNOS dimers. Functionally, 10DEBC reverses castration induced ex vivo IPA and penile endothelial dysfunction. SIGNIFICANCE: These data demonstrate how castration uncouples eNOS and provide a novel strategy for improving endothelial-dependent relaxation necessary for an erection. Further studies are needed to determine if Akt inhibition may treat or even prevent ED in testosterone deficient prostate cancer survivors.

Dysfunctional voiding behavior and impaired muscle contractility in a rat model of detrusor underactivity.

AIMS: Detrusor underactivity (DU) is an understudied health concern with inadequate clinical management. The pathophysiology of DU is unclear, and current therapies fail to improve symptoms. The current studies characterized voiding function and contractility of bladder and urethral tissues in a novel rat model of DU. METHODS: Female obese prone (OP) and obese resistant (OR) rats were fed a 60 kcal% fat diet at 8 weeks old. A subset of rats (n = 4/strain) underwent uroflowmetry biweekly for 18 weeks in metabolic cages. At 40-56 weeks old, rats (n = 9-10/strain) underwent instrumented cystometry under urethane anesthesia. Following cystometry, bladder and urethral tissues (n = 8-9/strain) were harvested for in vitro assessments of contractility in response to carbachol, electric field stimulation, atropine, alpha, beta-methylene ATP, and caffeine. RESULTS: OP rats exhibited increased urinary frequency (p = 0.0031), decreased voided volume (p = 0.0093), and urine flow rate (p = 0.0064) compared to OR rats during uroflowmetry. Bethanechol (10 mg/kg) did not alter uroflowmetry parameters. During cystometry, OP rats exhibited decreased bladder emptying efficiency (p < 0.0001), decreased pressure to generate a void (p < 0.0001), and increased EUS activity during filling (p = 0.0011). Bladder contractility was decreased in OP rats when exposed to carbachol (p < 0.0003) and ATP (p = 0.0004), whereas middle urethral contractility was increased when exposed to carbachol (p = 0.0014), EFS (p = 0.0289), and caffeine (p = 0.0031). CONCLUSION: Impaired cholinergic and purinergic signaling in the bladder may contribute to poor voiding function in OP rats. In addition, increased urethral activity may engage a guarding reflex to augment continence and exacerbate incomplete emptying.

Increased Level of Tumor Necrosis Factor-Alpha (TNF-α) Leads to Downregulation of Nitrergic Neurons Following Bilateral Cavernous Nerve Injury and Modulates Penile Smooth Tone.

BACKGROUND: Erectile dysfunction (ED) after injury to peripheral cavernous nerve (CN) is partly a result of inflammation in pelvic ganglia, suggesting that ED may be prevented by inhibiting neuroinflammation. AIM: The aim of this study is to examine temporal changes of TNF-α, after bilateral CN injury (BCNI), to evaluate effect of exogenous TNF-α on neurite outgrowth from major pelvic ganglion (MPG), and to investigate effect of TNF-α signal inhibition to evaluate effects of TNF-α on penile tone with TNF-α receptor knockout mice (TNFRKO). METHODS: Seventy Sprague-Dawley rats were randomized to undergo BCNI or sham surgery. Sham rats' MPGs were harvested after 48 hours, whereas BCNI groups' MPGs were at 6, 12, 24, 48 hours, 7, or 14 days after surgery. qPCR was used to evaluate gene expression of markers for neuroinflammation in MPGs. Western blot was performed to evaluate TNF-α protein amount in MPGs. MPGs were harvested from healthy rats and cultured in Matrigel with TNF-α. Neurite outgrowth from MPGs was measured after 3 days, and TH and nNOS immunofluorescence was assessed. Wild type (WT) and TNFRKO mice were used to examine effect of TNF-α inhibition on smooth muscle function after BCNI. MPGs were harvested 48 hours after sham or BCNI surgery to evaluate gene expression of nNOS and TH. OUTCOMES: Gene expression of TNF-α signaling pathway, Schwann cell and macrophage markers, protein expression of TNF-α in MPGs, and penile smooth muscle function to electrical field stimulation (EFS) were evaluated. RESULTS: BCNI increased gene and protein expression of TNF-α in MPGs. Exogenous TNF-α inhibited MPG neurite outgrowth. MPGs cultured with TNF-α had decreased gene expression of nNOS (P < .05). MPGs cultured with TNF-α had shorter nNOS+ neurites than TH+ neurites (P < .01). Gene expression of nNOS was enhanced in TNFRKO mice compared to WT mice (P < .01). WT mice showed enhanced smooth muscle contraction of penises of WT mice was enhanced to EFS, compared to TNFKO (P < .01). Penile smooth-muscle relaxation to EFS was greater in TNFKO mice compared to WT (P < .01). CLINICAL TRANSLATION: TNF-α inhibition may prevent ED after prostatectomy. STRENGTH/LIMITATIONS: TNF-α inhibition might prevent loss of nitrergic nerve apoptosis after BCNI and preserve corporal smooth muscle function but further investigation is required to evaluate protein expression of nNOS in MPGs of TNFKO mice. CONCLUSIONS: TNF-α inhibited neurite outgrowth from MPGs by downregulating gene expression of nNOS and TNFRKO mice showed enhanced gene expression of nNOS and enhanced penile smooth-muscle relaxation. Matsui H, Sopko NA, Campbell JD, et al. Increased Level of Tumor Necrosis Factor-Alpha (TNF-α) Leads to Downregulation of Nitrergic Neurons Following Bilateral Cavernous Nerve Injury and Modulates Penile Smooth Tone. J Sex Med 2021;18:1181-1190.

Impact of prostatic radiation therapy on bladder contractility and innervation.

AIMS: To determine the effect of prostatic radiation therapy (RT) on bladder contractility and morphology, and axon, or neuron profiles within the detrusor and major pelvic ganglia (MPG) in male rats. METHODS: Male Sprague-Dawley rats (8 weeks) received a single dose of prostatic RT (0 or 22 Gy). Bladders and MPG were collected 2- and 10-weeks post-RT. Detrusor contractile responses to carbachol and electrical field stimulation (EFS) were measured. Bladders were stained with Masson's trichrome, and antibodies for nonspecific neuronal marker, cholinergic nerve marker choline acetyltransferase (ChAT), and alpha-smooth muscle actin. MPG gene expression was assessed by quantitative polymerase chain reaction for ubiquitin carboxy-terminal hydrolase L1 (Uchl1) and Chat. RESULTS: At 2 weeks post-RT, bladder smooth muscle, detrusor cholinergic axon profiles, and MPG Chat gene expression were increased (p < .05), while carbachol and EFS-mediated contractions were decreased (p < .05). In contrast, at 10 weeks post-RT, nerve-mediated contractions were increased compared with control (p < .05), while bladder smooth muscle, detrusor cholinergic axon profiles, MPG Chat expression, and carbachol contractions had normalized. At both 2- and 10-weeks post-RT, there was no change in detrusor nonspecific axon profiles and MPG Uchl1 expression. CONCLUSION: In a rat model, RT of the prostate and MPG was associated with early changes in MPG Chat gene expression, and bladder cholinergic axon profiles and smooth muscle content which resolved over time. After RT recovery, bladder contractility decreased early and increased by 10 weeks. Long-term changes to the MPG and increased bladder cholinergic axons may contribute to RT-induced bladder dysfunction in prostate cancer survivors.

Ex Vivo Radiation Leads to Opposing Neurite Growth in Whole Ganglia vs Dissociated Cultured Pelvic Neurons.

BACKGROUND: Prostatic radiation therapy (RT) often causes erectile dysfunction (ED) and the mechanisms governing RT-induced ED are unclear with a lack of therapeutic strategies. AIM: To determine the effects of ex vivo RT on major pelvic ganglion (MPG) neuron survival, and neurite growth in whole vs dissociated culture. METHODS: MPGs were removed and irradiated (0 or 8 Gy) from male Sprague Dawley rats. For dissociated culture, MPG neurons were digested in collagenase/dispase and cultured on coverslips. Immunofluorescent staining for beta-tubulin III (TUBB3; neuron marker), neuronal nitric oxide synthase (nNOS; nitrergic marker), tyrosine hydroxylase (TH; sympathetic marker), and terminal deoxynucleotidyl transferase dUTP nick end labeling assessed neurite length, branching, autonomic neuron density, and apoptosis. For whole organ culture, MPGs were grown in Matrigel. Gene expression of apoptotic markers (caspase 1, 3), TUBB3, nNOS, TH, and Schwann cells (Sox10, Krox20, glial fibrillary acid protein) was measured in whole organ cultured MPGs by quantitative polymerase chain reaction. OUTCOMES: After 72 hours, neurite length, branching, autonomic neuron density, and apoptosis were assessed, and gene expression was measured. RESULTS: RT increased apoptosis in dissociated neurons measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (P < .001) and whole MPG culture via upregulation of caspase 3 gene expression (P < .05). Nitrergic neurons were markedly decreased in irradiated dissociated culture (P < .05), while nNOS gene expression was upregulated in irradiated whole organ culture (P < .05). The proportion of dissociated sympathetic neurons and whole organ TH gene expression remained unchanged after RT. Interestingly, RT dissociated neurites were 22% shorter than controls, while RT whole organ neurites were 15% longer than controls (P < .01). MPG Schwann cells markers (Sox10, Krox20) were elevated after RT in whole organ culture. CLINICAL TRANSLATION: Prostatic RT leads to increased neuronal cell death and less erectogenic nitrergic neurons contributing to ED. STRENGTHS & LIMITATIONS: The advantages of dissociated neuron culture include distinct neurites which are easily measured for apoptosis, length/branching, and specific neuron types. In contrast, whole MPG culture is advantageous as it contains all the supporting cells present in vivo. CONCLUSION: The 2 different culture methods demonstrated opposing neurite growth after RT indicating the importance of supporting cell network to promote pelvic neuron neuritogenesis and survival following RT. Randolph JT, Pak ES, Koontz BF, et al. Ex Vivo Radiation Leads to Opposing Neurite Growth in Whole Ganglia vs Dissociated Cultured Pelvic Neurons. J Sex Med 2020;17:1423-1433.

Managing female pelvic floor disorders: a medical device review and appraisal.

Pelvic floor disorders (PFDs) will affect most women during their lifetime. Sequelae such as pelvic organ prolapse, stress urinary incontinence, chronic pain and dyspareunia significantly impact overall quality of life. Interventions to manage or eliminate symptoms from PFDs aim to restore support of the pelvic floor. Pessaries have been used to mechanically counteract PFDs for thousands of years, but do not offer a cure. By contrast, surgically implanted grafts or mesh offer patients a more permanent resolution but have been in wide use within the pelvis for less than 30 years. In this perspective review, we provide an overview of the main theories underpinning PFD pathogenesis and the animal models used to investigate it. We highlight the clinical outcomes of mesh and grafts before exploring studies performed to elucidate tissue level effects and bioengineering considerations. Considering recent turmoil surrounding transvaginal mesh, the role of pessaries, an impermanent method, is examined as a means to address patients with PFDs.

Chronic high-fat diet decreased detrusor mitochondrial respiration and increased nerve-mediated contractions.

AIMS: To assess the impact of chronic high-fat diet (HFD) on behavioral voiding patterns, detrusor contractility, and smooth muscle mitochondrial function in male mice. MATERIALS AND METHODS: Male C57BL/6J mice (6 weeks) were fed a control or HFD for 20 weeks. Bladder function was assessed by void spot assays. Bladders were collected and detrusor contractility to carbachol (10-9 -10-5 M), and electrical field stimulation (EFS, 0.5-32 Hz) in the presence and absence of atropine was measured. Homogenized detrusor samples were placed in oxygraphs to assess the rate of oxygen consumption of the mitochondria within the detrusor in the presence of different substrates. Mitochondrial hydrogen peroxide (H2 O2 ) emission was measured fluorometrically. Detrusor citrate synthase activity was measured via enzyme activity kit and Western blots assessed the electron transport chain (ETC) protein content. RESULTS: HFD significantly increased body weight, adiposity, and blood glucose levels. HFD mice demonstrated increased voiding frequency and increased EFS-induced detrusor contractility. There were no changes in detrusor relaxation or cholinergic-medicated contraction. Mitochondrial respiration was decreased with HFD and H2 O 2 emission was increased. The relative amount of mitochondria in the detrusor was similar between groups. However, ETC complexes V and III were increased following HFD. CONCLUSIONS: Chronic HFD increased adiposity, lead to more frequent voiding, and enhanced EFS-mediated detrusor contractions. Mitochondrial respiration was decreased and H2 O 2 emission increased following HFD. Further research is required to determine if alterations in mitochondrial function could play a role in the development of HFD-induced bladder dysfunction.

A free-choice high-fat, high-sucrose diet induces hyperphagia, obesity, and cardiovascular dysfunction in female cycling and pregnant rats.

The main objective of these studies was to characterize metabolic, body composition, and cardiovascular responses to a free-choice high-fat, high-sucrose diet in female cycling and pregnant rats. In the nonpregnant state, female Sprague-Dawley rats offered a 3-wk free-choice high-fat, high-sucrose diet had greater energy intake, adiposity, serum leptin, and triglyceride concentrations compared with rats fed with standard chow and developed glucose intolerance. In addition, choice-diet-fed rats had larger cardiac ventricular weights, smaller kidney and pancreas weights, and higher blood pressure than chow-fed rats, but they did not exhibit resistance artery endothelial dysfunction. When the free-choice diet continued throughout pregnancy, rats remained hyperphagic, hyperleptinemic, and obese. Choice pregnant rats exhibited uterine artery endothelial dysfunction and had smaller fetuses compared with chow pregnant rats. Pregnancy normalized mean arterial blood pressure and pancreas weights in choice rats. These studies are the first to provide a comprehensive evaluation of free-choice high-fat, high-sucrose diet on metabolic and cardiovascular functions in female rats, extending the previous studies in males to female cycling and pregnant rodents. Free-choice diet may provide a new model of preconceptual maternal obesity to study the role of increased energy intake, individual food components, and preexisting maternal obesity on maternal and offspring physiological responses during pregnancy and after birth.

Prostate-Confined Radiation Decreased Pelvic Ganglia Neuronal Survival and Outgrowth.

BACKGROUND: Erectile dysfunction (ED) is common following radiation therapy (RT) for prostate cancer. Although the cause of RT-induced ED is unknown, damage to both the neuronal and vascular components supporting erections are often implicated. AIM: To determine the effects of prostatic RT on erections, penile vascular physiology, and major pelvic ganglia (MPG) neuron growth and survival in a rat model. METHODS: Male rats underwent 0 Gy or 22 Gy single fraction of prostate-confined, conformal RT. At 2 weeks or 10 weeks post-RT (n = 10/group), cavernous nerve stimulation was performed and erections were assessed. Tissue bath experiments were performed to assess both penile artery and internal pudendal artery (IPA) function. MPGs were dissociated and neurons grown in culture for 72 hours. Immunofluorescence staining was done to quantify neuron survival (terminal deoxynucleotidyl transferase nick-end labeling), outgrowth (beta-tubulin III), type (nitric oxide synthase [nNOS] and tyrosine hydroxylase [TH]), and nerve injury markers (small GTPase Rac1 and ninjurin-1 [Ninj-1]). Whole MPG real-time quantitative polymerase chain reaction (qPCR) was performed to measure expression of genes related to nerve type, neuron injury, repair, and myelination, such as Ninj-1, Rac1, ATF3, GAP43, GFAP, SOX10, and KROX20. OUTCOMES: Intracavernosal pressure (ICP) to mean arterial pressure (MAP) ratio, smooth muscle contractility and relaxation, gene expression, neuritogenesis, and apoptosis. RESULTS: Following RT, ICP/MAP was unchanged at 2 weeks or 10 weeks. Nerve-mediated penile contraction was increased at 2 weeks, whereas adrenergic contraction was reduced at 10 weeks. Penile relaxation and IPA vasoreactivity were unchanged. Neuronal apoptosis was more than doubled both early and late post-RT. RT caused a progressive decrease in neurite branching but an early increase and then late decrease in neurite lengthening. RT reduced the numbers of nNOS-positive neurons both early and late and also decreased MPG nitrergic gene expression. TH neurons and gene expression were unchanged at 2 weeks; however, both were decreased after 10 weeks. Although most markers of gene injury and repair were unaffected early post-RT, MPG expression of Ninj1 and GFAP increased. After 10 weeks, Ninj1 and GFAP remained elevated while markers of neuron injury (ATF3), outgrowth (GAP43 and Rac1), and myelin regulation (SOX10) were decreased. CLINICAL TRANSLATION: RT-induced ED may result from damage to the ganglia controlling erections. STRENGTHS & LIMITATIONS: This study used a clinically relevant, prostate-confined model to examine neurovascular structures not accessible in human studies. Unfortunately, rats did not exhibit ED at this time point. CONCLUSION: This is the first study to demonstrate impaired health and regeneration potential of dissociated MPG neurons following RT. Neuronal injury was apparent early post-RT and persisted or increased over time but was insufficient to cause ED at the time points examined. Powers SA, Odom MR, Pak ES, et al. Prostate-Confined Radiation Decreased Pelvic Ganglia Neuronal Survival and Outgrowth. J Sex Med 2019;16:27-41.

Clarifying the Relative Impacts of Vascular and Nerve Injury That Culminate in Erectile Dysfunction in a Pilot Study Using a Rat Model of Prostate Irradiation and a Thrombopoietin Mimetic.

PURPOSE: Radiation therapy (RT) offers an important and curative approach to treating prostate cancer, but it is associated with a high incidence of erectile dysfunction (ED). It is not clear whether the etiology of radiation-induced ED (RI-ED) is driven by RT-mediated injury to the vasculature, the nerves, or both. This pilot study sought to distinguish the effects of vascular and nerve injury in RI-ED by applying a vascular radioprotectant in a rat model of prostate RT. METHODS: A single dose of the thrombopoietin mimetic (TPOm; RWJ-800088), previously shown to mitigate radiation-induced vascular injury, was administered 10 minutes after single-fraction conformal prostate RT. Nine weeks after RT, rats were assessed for erectile and arterial function. Nerve markers were quantified with reverse transcriptase polymerase chain reaction. Immunofluorescent microscopy further characterized vascular effects of RT and TPOm. RESULTS: Sham animals and animals that received RT and TPOm showed significant arterial vasodilation in response to systemic hydralazine (24.1% ± 7.3% increase; P = .03 in paired t test). However, animals that received RT and vehicle were unable to mount a vasodilatory response (-7.4% ± 9.9% increase; P = .44 in paired t test). TPOm prevented RT-induced change in the penile artery cross-sectional area (P = .036), but it did not ameliorate cavernous nerve injury as evaluated by gene expression of neuronal injury markers. Despite significant structural and functional vascular protective effects and some trends for differences in nerve injury/recovery markers, TPOm did not prevent RI-ED at 9 weeks, as assessed by intracavernous pressure monitoring after cavernous nerve stimulation. CONCLUSIONS: These data suggest that vascular protection alone is not sufficient to prevent RI-ED and that cavernous nerve injury plays a key role in RI-ED. Further research is required to delineate the multifactorial nature of RI-ED and to determine if TPOm with modified dosing regimens can mitigate against nerve injury either through direct or vascular protective effects.

Galanin Administration Partially Restores Erectile Function After Cavernous Nerve Injury and Mediates Endogenous Nitrergic Nerve Outgrowth In Vitro.

BACKGROUND: Previously, we found that the neuropeptide galanin was strongly upregulated soon after bilateral cavernous nerve injury (BCNI) and that galanin and its receptors were expressed in nitrergic erectile innervation. Galanin has been observed to exert neuroregenerative effects in dorsal root ganglion neurons, but evidence for these effects in the major pelvic ganglion (MPG) after BCNI is lacking. AIM: To evaluate the neurotropic effects of galanin receptor agonists and antagonists in vitro in nitrergic neurons and MPG and in vivo in rats after BCNI. METHODS: Male Sprague-Dawley rats underwent BCNI and sham surgery. Organ culture and single-cell neuron culture of the MPG were performed. Osmotic pump treatment with the galanin agonist in vivo and measurement of erectile response to electrostimulation after BCNI, immunohistochemical localization of galanin and receptors in the human neurovascular bundle, and myographic analysis of rat corpus cavernosum smooth muscle relaxation to galanin receptor agonists were investigated. OUTCOMES: Neurite outgrowth in vitro and erectile response to electrostimulation after BCNI in vivo, immunohistochemical localization of galanin and receptors, and penile muscle relaxation in vitro. RESULTS: Galanin showed neurotrophic action in vitro and inhibition of endogenous galanin significantly impaired neurite outgrowth in nitrergic but not in sympathetic MPG neurons. In vivo administration of a selective galanin receptor-2 agonist, M1145, resulted in partial recovery of erectile function (EF) after BCNI. Galanin did not act as a direct vasodilator on corpus cavernosum muscle strips. CLINICAL TRANSLATION: Endogenous neurotrophins such as galanin could be used as a strategy to improve EF for patients after BCNI from radical prostatectomy. STRENGTHS AND LIMITATIONS: We evaluated the effect of galanin on nerve regeneration and EF recovery in vivo and in vitro. Limitations include the lack of washout period for the in vivo experiment and absence of differences in the expression of neuronal markers between treatment groups. CONCLUSIONS: We identified galanin as a potential endogenous mechanism for nerve regeneration after BCNI, which could play a physiologic role in EF recovery after radical prostatectomy. In vivo treatment with exogenous galanin was beneficial in enhancing EF recovery after BCNI, but further research is necessary to understand the underlying mechanisms. Weyne E, Hannan JL, Gevaert T, et al. Galanin Administration Partially Restores Erectile Function After Cavernous Nerve Injury and Mediates Endogenous Nitrergic Nerve Outgrowth In Vitro. J Sex Med 2018;15:480-491.

Specialized Pro-Resolving Lipid Mediators Regulate Ozone-Induced Pulmonary and Systemic Inflammation.

Exposure to ozone (O3) induces lung injury, pulmonary inflammation, and alters lipid metabolism. During tissue inflammation, specialized pro-resolving lipid mediators (SPMs) facilitate the resolution of inflammation. SPMs regulate the pulmonary immune response during infection and allergic asthma; however, the role of SPMs in O3-induced pulmonary injury and inflammation is unknown. We hypothesize that O3 exposure induces pulmonary inflammation by reducing SPMs. To evaluate this, male C57Bl/6J mice were exposed to filtered air (FA) or 1 ppm O3 for 3 h and necropsied 24 h after exposure. Pulmonary injury/inflammation was determined by bronchoalveolar lavage (BAL) differentials, protein, and lung tissue cytokine expression. SPMs were quantified by liquid chromatography tandem mass spectrometry and SPM receptors leukotriene B4 receptor 1 (BLT-1), formyl peptide receptor 2 (ALX/FPR2), chemokine-like receptor 1 (ChemR23), and SPM-generating enzyme (5-LOX and 12/15-LOX) expression were measured by real time PCR. 24 h post-O3 exposure, BAL PMNs and protein content were significantly increased compared to FA controls. O3-induced lung inflammation was associated with significant decreases in pulmonary SPM precursors (14-HDHA, 17-HDHA), the SPM PDX, and in pulmonary ALX/FPR2, ChemR23, and 12/15-LOX expression. Exogenous administration of 14-HDHA, 17-HDHA, and PDX 1 h prior to O3 exposure rescued pulmonary SPM precursors/SPMs, decreased proinflammatory cytokine and chemokine expression, and decreased BAL macrophages and PMNs. Taken together, these data indicate that O3-mediated SPM reductions may drive O3-induced pulmonary inflammation.

NLRP3/IL-1ß mediates denervation during bladder outlet obstruction in rats.

AIMS: Denervation of the bladder is a detrimental consequence of bladder outlet obstruction (BOO). We have previously shown that, during BOO, inflammation triggered by the NLRP3 inflammasome in the urothelia mediates physiological bladder dysfunction and downstream fibrosis in rats. The aim of this study was to assess the effect of NLRP3-mediated inflammation on bladder denervation during BOO. METHODS: There were five groups of rats: (i) Control (no surgery); (ii) Sham-operated; (iii) BOO rats given vehicle; (iv) BOO rats given the NLRP3 inhibitor glyburide; and (v) BOO rats given the IL-1 receptor antagonist anakinra. BOO was constructed by ligating the urethra over a 1 mm catheter and removing the catheter. Medications were given prior to surgery and once daily for 12 days. Bladder sections were stained for PGP9.5, a pan-neuronal marker. Whole transverse sections were used to identify and count nerves while assessing cross-sectional area. For in vitro studies, pelvic ganglion neurons were isolated and treated with IL-1ß. After a 48 h incubation apoptosis, neurite length and branching were assessed. RESULTS: In obstructed bladders, the number of nerves decreased while total area increased, indicating a loss of cell number and/or branching. The decrease in nerve density was blocked by glyburide or anakinra, clearly implicating the NLRP3 pathway in denervation. In vitro analysis demonstrated that IL-1ß, a product of the inflammasome, induced apoptosis in pelvic ganglion neurons, suggesting one mechanism of BOO-induced denervation is NLRP3/IL-1ß triggered apoptosis. CONCLUSIONS: The NLRP3/IL-1ß-mediated inflammation pathway plays a significant role in denervation during BOO.

Enhanced Electrical Field Stimulated Nitrergic and Purinergic Vasoreactivity in Distal vs Proximal Internal Pudendal Arteries.

BACKGROUND: The internal pudendal arteries (IPAs) supply blood to the penis and are highly susceptible to vascular remodeling in rodent models of diabetes, hypertension, aging, and chronic kidney disease, thus contributing to erectile dysfunction. Interestingly, vascular remodeling primarily occurs in the distal and not in the proximal IPA, suggesting distinct local physiologic signaling differences within the IPA. AIM: To examine the role of purinergic signaling and neurotransmitter release by electrical field stimulation (EFS) in the regulation of proximal and distal IPA vascular tone. METHODS: Proximal and distal IPAs were mounted in wire myographs and vascular responses to phenylephrine, acetylcholine, and 2-(N,N-diethylamino)-diazenolate-2-oxide, diethyl-ammonium salt (DEA NONOate) were measured. EFS-mediated contraction and non-adrenergic non-cholinergic (NANC) relaxation were evaluated in the absence and presence of a nitric oxide synthase antagonist. Purinergic agonist and NANC relaxation responses were assessed in the presence and absence of P2X1 and P2Y1 antagonists. Protein expression of P2X1 and P2Y1 receptors was measured by western blot. MAIN OUTCOME MEASURES: Proximal and distal IPA contraction and relaxation were measured during increasing agonist administration and EFS in the presence and absence of antagonists. RESULTS: Proximal and distal IPA concentration response curves to phenylephrine, acetylcholine, and DEA NONOate did no differ. Interestingly, distal IPA exhibited greater EFS-mediated contraction and NANC relaxation compared with proximal IPA. Nitric oxide synthase inhibition completely inhibited distal IPA NANC relaxation but did not affect proximal IPA relaxation. P2X1 or P2Y1 receptor antagonism during NANC relaxation increased distal IPA relaxation but decreased proximal IPA relaxation. Combined P2X1 and P2Y1 receptor antagonism had no effect on proximal IPA relaxation but significantly increased distal IPA NANC relaxation. CLINICAL TRANSLATION: Understanding neurovascular regulation of IPA vascular tone through nitrergic and purinergic mechanisms could yield new therapeutic targets to improve IPA blood flow and treat vasculogenic erectile dysfunction. STRENGTHS AND LIMITATIONS: This study is the first to illustrate the differences in mechanisms responsible for regulating vascular tone in the proximal and distal IPAs. All presented findings are currently limited to ex vivo vascular function. CONCLUSION: The regulation of vascular tone differs regionally in the IPA. The distal IPA is controlled through neurotransmitter-mediated NO-dependent mechanisms and increased sensitivity to purinergic P2X1 and P2Y1 receptor inhibition. Odom MR, Pak ES, Brown DA, Hannan JL. Enhanced Electrical Field Stimulated Nitrergic and Purinergic Vasoreactivity in Distal vs Proximal Internal Pudendal Arteries. J Sex Med 2017;14:1285-1296.