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.

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.

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.

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.

M1 Macrophages Are Predominantly Recruited to the Major Pelvic Ganglion of the Rat Following Cavernous Nerve Injury.

INTRODUCTION: Neurogenic erectile dysfunction is a common sequela of radical prostatectomy. The etiology involves injury to the autonomic cavernous nerves, which arise from the major pelvic ganglion (MPG), and subsequent neuroinflammation, which leads to recruitment of macrophages to the injury site. Currently, two macrophage phenotypes are known: neurotoxic M1 macrophages and neuroprotective M2 macrophages. AIM: To examine whether bilateral cavernous nerve injury (BCNI) in a rat model of erectile dysfunction would increase recruitment of neurotoxic M1 macrophages to the MPG. METHODS: Male Sprague-Dawley rats underwent BCNI and the MPG was harvested at various time points after injury. The corpora cavernosa was used to evaluate tissue myographic responses to electrical field stimulation ex vivo. Quantitative real-time polymerase chain reaction was used to examine the gene expression of global macrophage markers, M1 macrophage markers, M2 macrophage markers, and cytokines and chemokines in the MPG. Mathematical calculation of the M1/M2 index was used to quantify macrophage changes temporally. Western blot of MPG tissues was used to evaluate the protein amount of M1 and M2 macrophage markers quantitatively. Immunohistochemistry staining of MPGs for CD68, CD86, and CD206 was used to characterize M1 and M2 macrophage infiltration. MAIN OUTCOME MEASURES: Corpora cavernosa responsiveness ex vivo; gene (quantitative real-time polymerase chain reaction) and protein (western blot) expressions of M1 and M2 markers, cytokines, and chemokines; and immunohistochemical localization of M1 and M2 macrophages. RESULTS: BCNI impaired the corporal parasympathetic-mediated relaxation response to electrical field stimulation and enhanced the contraction response to electrical field stimulation. Gene expression of proinflammatory (Il1b, Il16, Tnfa, Tgfb, Ccl2, Ccr2) and anti-inflammatory (Il10) cytokines was upregulated in the MPG 48 hours after injury. M1 markers (CD86, inducible nitric oxide synthase, interleukin-1ß) and M2 markers (CD206, arginase-1, interleukin-10) were increased after BCNI in the MPG, with the M1/M2 index above 1.0 indicating that more M1 than M2 macrophages were recruited to the MPG. Protein expression of the M1 macrophage marker (inducible nitric oxide synthase) was increased in MPGs after BCNI. However, the protein amount of M2 macrophage markers (arginase-1) remained unchanged. Immunohistochemical characterization demonstrated predominant increases in M1 (CD68+CD86+) macrophages in the MPG after BCNI. CONCLUSION: These results suggest that an increase in M1 macrophage infiltration of the MPG after BCNI is associated with impaired neurogenically mediated erectile tissue physiology ex vivo and thus has significant implications for cavernous nerve axonal repair. Future studies are needed to demonstrate that inhibition of M1 macrophage recruitment prevents erectile dysfunction after CNI.

Impaired contraction and decreased detrusor innervation in a female rat model of pelvic neuropraxia.

INTRODUCTION AND HYPOTHESIS: Bilateral pelvic nerve injury (BPNI) is a model of post-radical hysterectomy neuropraxia, a common sequela. This study assessed the time course of changes to detrusor autonomic innervation, smooth muscle (SM) content and cholinergic-mediated contraction post-BPNI. METHODS: Female Sprague-Dawley rats underwent BPNI or sham surgery and were evaluated 3, 7, 14, and 30 days post-BPNI (n = 8/group). Electrical field-stimulated (EFS) and carbachol-induced contractions were measured. Gene expression was assessed by qPCR for muscarinic receptor types 2 (M2) and 3 (M3), collagen type 1α1 and 3α1, and SM actin. Western blots measured M2 and M3 protein expression. Bladder sections were stained with Masson's trichrome for SM content and immunofluorescence staining for nerve terminals expressing vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), and neuronal nitric oxide synthase (nNOS). RESULTS: Bilateral pelvic nerve injury caused larger bladders with less SM content and increased collagen type 1α1 and 3α1 gene expression. At early time points, cholinergic-mediated contraction increased, whereas EFS-mediated contraction decreased and returned to baseline by 30 days. Protein and gene expression of M3 was decreased 3 and 7 days post-BPNI, whereas M2 was unchanged. TH nerve terminals surrounding the detrusor decreased in all BPNI groups, whereas VAChT and nNOS terminals decreased 14 and 30 days post-BPNI. CONCLUSIONS: Bilateral pelvic nerve injury increased bladder size, impaired contractility, and decreased SM and autonomic innervation. Therapeutic strategies preventing nerve injury-mediated decline in neuronal input and SM content may prevent the development of a neurogenic bladder and improve quality of life after invasive pelvic surgery.

Off-Target Effect of Sildenafil on Postsurgical Erectile Dysfunction: Alternate Pathways and Localized Delivery System.

INTRODUCTION: There is no consensus on the best oral phosphodiesterase type 5 inhibitor (PDE5I) for patients undergoing penile rehabilitation after surgical nerve injury. AIM: To determine the mechanism of PDE5I on cultured neuronal cells and the effectiveness of local drug delivery using nanospheres (NSPs) to sites of nerve injury in a rat model of bilateral cavernous nerve injury (BCNI). METHODS: The effects of sildenafil, tadalafil, and vardenafil on cyclic adenosine monophosphate, cyclic guanosine monophosphate, and cell survival after exposure to hypoxia and H2O2 were measured in PC12, SH-SY5Y, and NTERA-2 (NT2) cell cultures. The effects of phosphodiesterase type 4 inhibitor (PDE4I) and PDE5I on neuronal cell survival were evaluated. Male rats underwent BCNI and were untreated (BCNI), immediately treated with application of empty NSPs (BCNI + NSP), NSPs containing sildenafil (Sild + NSP), or NSPs containing rolipram (Rol + NSP). MAIN OUTCOME MEASURES: Viability of neuronal cells was measured. Intracavernous pressure changes after cavernous nerve electrostimulation and expression of neurofilament, nitric oxide synthase, and actin in mid-shaft of penis were analyzed 14 days after injury. RESULTS: Sildenafil and rolipram significantly decreased cell death after exposure to H2O2 and hypoxia in PC12, SH-SY5Y, and NT2 cells. PC12 cells did not express PDE5 and knockdown of PDE4 significantly increased cell viability in PC12, SH-SY5Y, and NT2 cells exposed to hypoxia. The ratio of intracavernous pressure to mean arterial pressure and expression of penile neurofilament, nitric oxide synthase, and actin were significantly higher in the Sild + NSP and Rol + NSP groups than in the BCNI and BCNI + NSP groups. Limitations included analysis in only two PDE families using only a single dose. CONCLUSION: Sildenafil showed the most profound neuroprotective effect compared with tadalafil and vardenafil. Sildenafil- or rolipram-loaded NSP delivery to the site of nerve injury prevented erectile dysfunction and led to increased neurofilament, nitric oxide synthase, smooth muscle content in rat penile tissue after BCNI.

Pathophysiology of erectile dysfunction.

Erectile dysfunction (ED) is a major health problem as the population ages. Basic science research for the last two decades has expanded the knowledge on ED and identified several key molecular changes associated with the pathogenesis of ED, including nitric oxide (NO) / cyclic guanosine monophosphate (cGMP) / protein kinase G (PKG) pathway, RhoA/Rho-associated protein kinase (ROCK) signaling pathway, reactive oxygen species (ROS), renin-angiotensin system (RAS) and tumor necrosis factor-alpha (TNF-α). The causes of ED are classified into aging, vasculogenic, neurogenic, endocrinological, drug-induced and psychogenic. ED is often associated with systemic diseases, such as diabetes and cardiovascular diseases. In this review, we will review the molecular mechanisms of ED and known mechanisms behind ED associated with systemic diseases.

Mini Review: Pathophysiology of Erectile Dysfunction.

Erectile dysfunction (ED) is a major health problem as the population ages. Basic science research for the last two decades has expanded the knowledge on ED and identified several key molecular changes associated with the pathogenesis of ED, including nitric oxide (NO) / cyclic guanosine monophosphate (cGMP) / protein kinase G (PKG) pathway, RhoA/Rho-associated protein kinase (ROCK) signaling pathway, reactive oxygen species (ROS), renin-angiotensin system (RAS) and tumor necrosis factor-alpha (TNF-α). The causes of ED are classified into aging, vasculogenic, neurogenic, endocrinological, drug-induced and psychogenic. ED is often associated with systemic diseases, such as diabetes and cardiovascular diseases. In this review, we will review the molecular mechanisms of ED and known mechanisms behind ED associated with systemic diseases.

Temporal changes in neurotrophic factors and neurite outgrowth in the major pelvic ganglion following cavernous nerve injury.

Despite nerve-sparing radical prostatectomy, nerve damage and erectile dysfunction (ED) prevail, and preventing neurodegeneration is of great importance. Neurotrophic factors and neurite outgrowth were characterized in major pelvic ganglia (MPG) following bilateral cavernous nerve injury (BCNI). Young male Sprague-Dawley rats underwent sham or BCNI surgery, and the intracavernosal pressure to mean arterial pressure ratio was measured 2, 7, 14, 21, 30, and 60 days following injury (n = 8/group). MPG gene expression (qPCR) and Western blot were performed for glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), neurturin, neurotrophin (NT)-3, NT4, brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor, and activating transcription factor 3 (ATF3). Additional rats were injured, and MPGs were removed 24 hr, 48 hr, 3 days, and 7 days following BCNI (n = 3/group). MPGs were cultured in Matrigel, and neurite outgrowth was measured. Erections were impaired early and improved by 60 days in BCNI rats. GDNF, NGF, BDNF, and ATF3 gene expression was significantly increased and NT3 was decreased in MPGs following BCNI (48 hr to 21 days, P < 0.05). GDNF and NGF protein levels were elevated in 48-hr BCNI rats. MPG neurite outgrowth from 24-hr and 48-hr BCNI was higher than sham (658 ± 19 µm, 607 ± 24 µm, 393 ± 23 µm, respectively, P < 0.05). Further studies examining the roles of neurotrophic factors in modulating signaling pathways may provide therapeutic avenues for neurogenically mediated ED.

Valproic acid prevents penile fibrosis and erectile dysfunction in cavernous nerve-injured rats.

INTRODUCTION: Bilateral cavernous nerve injury (BCNI) causes profound penile changes such as apoptosis and fibrosis leading to erectile dysfunction (ED). Histone deacetylase (HDAC) has been implicated in chronic fibrotic diseases. AIMS: This study will characterize the molecular changes in penile HDAC after BCNI and determine if HDAC inhibition can prevent BCNI-induced ED and penile fibrosis. METHODS: Five groups of rats (8-10 weeks, n = 10/group) were utilized: (i) sham; (ii and iii) BCNI 14 and 30 days following injury; and (iv and v) BCNI treated with HDAC inhibitor valproic acid (VPA 250 mg/kg; 14 and 30 days). All groups underwent cavernous nerve stimulation (CNS) to determine intracavernosal pressure (ICP). Penile HDAC3, HDAC4, fibronectin, and transforming growth factor-ß1 (TGF-ß1) protein expression (Western blot) were assessed. Trichrome staining and the fractional area of fibrosis were determined in penes from each group. Cavernous smooth muscle content was assessed by immunofluorescence to alpha smooth muscle actin (α-SMA) antibodies. MAIN OUTCOME MEASURES: We measured ICP; HDAC3, HDAC4, fibronectin, and TGF-ß1 protein expression; penile fibrosis; penile α-SMA content. RESULTS: There was a voltage-dependent decline (P < 0.05) in ICP to CNS 14 and 30 days after BCNI. Penile HDAC3, HDAC4, and fibronectin were significantly increased (P < 0.05) 14 days after BCNI. There was a slight increase in TGF-ß1 protein expression after BCNI. Histological analysis showed increased (P < 0.05) corporal fibrosis after BCNI at both time points. VPA treatment decreased (P < 0.05) penile HDAC3, HDAC4, and fibronectin protein expression as well as corporal fibrosis. There was no change in penile α-SMA between all groups. Furthermore, VPA-treated BCNI rats had improved erectile responses to CNS (P < 0.05). CONCLUSION: HDAC-induced pathological signaling in response to BCNI contributes to penile vascular dysfunction. Pharmacological inhibition of HDAC prevents penile fibrosis, normalizes fibronectin expression, and preserves erectile function. The HDAC pathway may represent a suitable target in preventing the progression of ED occurring post-radical prostatectomy.

Inhibition of Rho-kinase improves erectile function, increases nitric oxide signaling and decreases penile apoptosis in a rat model of cavernous nerve injury.

PURPOSE: Bilateral cavernous nerve injury results in up-regulation of ROCK signaling in the penis. This is linked to erectile dysfunction in an animal model of post-prostatectomy erectile dysfunction. We evaluated whether daily treatment with the ROCK inhibitor Y-27632 (Tocris Bioscience, Ellisville, Missouri) would prevent erectile dysfunction in a rat model of bilateral cavernous nerve injury. MATERIALS AND METHODS: Sprague-Dawley® rats underwent surgery to create sham (14) or bilateral (27) cavernous nerve injury. In the injury group 13 rats received treatment with Y-27632 (5 mg/kg twice daily) and 14 received vehicle. At 14 days after injury, rats underwent cavernous nerve stimulation to determine erectile function. Penes were assessed for neuronal and nitric oxide synthase membrane-endothelial nitric oxide synthase. ROCK2 was assessed by Western blot. Cyclic guanosine monophosphate was determined by enzyme-linked immunosorbent assay. Cavernous homogenates were tested for ROCK and protein kinase G enzymatic activity. Penile apoptosis was evaluated using the Apostain technique (Alexis, San Diego, California). Data were analyzed on ROCK using ANOVA and the t test. RESULTS: While erectile function was decreased in rats with bilateral cavernous nerve injury, daily administration of Y-27632 improved erectile responses. Injury decreased neuronal and nitric oxide synthase membrane-endothelial nitric oxide synthase but ROCK2 was significantly increased. Y-27632 treatment restored neuronal nitric oxide synthase, nitric oxide synthase membrane-endothelial nitric oxide synthase and cyclic guanosine monophosphate levels, and protein kinase G activity. Treatment significantly decreased ROCK2 protein and ROCK activity. There were significantly fewer apoptotic cells after treatment than in injured controls. CONCLUSIONS: These results provide evidence for up-regulation of the RhoA/ROCK signaling pathway with detrimental effects on erectile function after bilateral cavernous nerve injury. ROCK inhibition improved erectile dysfunction associated with bilateral cavernous nerve injury by preserving penile nitric oxide bioavailability and decreasing penile apoptosis.

Internal pudendal artery from type 2 diabetic female rats demonstrate elevated endothelin-1-mediated constriction.

INTRODUCTION: Diabetes is a risk factor for female sexual dysfunction (FSD). FSD has several etiologies, including a vasculogenic component that could be exacerbated in diabetes. The internal pudendal artery supplies blood to the vagina and clitoris and diabetes-associated functional abnormalities in this vascular bed may contribute to FSD. AIM: The Goto-Kakizaki (GK) rat is a non-obese model of type 2 diabetes with elevated endothelin-1 (ET-1) activity. We hypothesize that female GK rats have diminished sexual responses and that the internal pudendal arteries demonstrate increased ET-1 constrictor sensitivity. METHODS: Female Wistar and GK rats were used. Apomorphine (APO)-mediated genital vasocongestive arousal (GVA) was measured. Functional contraction (ET-1 and phenylephrine) and relaxation (acetylcholine, ACh) in the presence or absence of the ETA receptor antagonist (ETA R; atrasentan) or Rho-kinase inhibitor (Y-27632) were assessed in the internal pudendal and mesenteric arteries. Protein expression of ET-1 and RhoA/Rho-kinase signaling pathway was determined in the internal pudendal and mesenteric arteries. MAIN OUTCOME MEASURE: APO-mediated GVAs; contraction and relaxation of internal pudendal and mesenteric arteries; ET-1/RhoA/Rho-kinase protein expression. RESULTS: GK rats demonstrated no APO-induced GVAs. Internal pudendal arteries, but not mesenteric arteries, from GK rats exhibited greater contractile sensitivity to ET-1 compared with Wistar arteries. ETA R blockade reduced ET-1-mediated constriction in GK internal pudendal and mesenteric arteries. Rho-kinase inhibition reduced ET-1-mediated constriction of GK internal pudendal but not mesenteric arteries; however, it had no effect on arteries from Wistar rats. RhoA protein expression was elevated in GK internal pudendal arteries. At the highest concentrations, ACh-mediated relaxation was greater in the GK internal pudendal artery; however, no difference was observed in the mesenteric artery. CONCLUSIONS: Female GK rats demonstrate decreased sexual responses that may be because of increased constrictor sensitivity to the ET-1/RhoA/Rho-kinase signaling in the internal pudendal artery.