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

BackgroundErectile 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.AimThe 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).MethodsSeventy 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.OutcomesGene 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.ResultsBCNI 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 TranslationTNF-α inhibition may prevent ED after prostatectomy.Strength/LimitationsTNF-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.ConclusionsTNF-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.     

Cocultured Schwann Cells Rescue Irradiated Pelvic Neuron Outgrowth and Increase Survival

BackgroundProstatic 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.AimTo determine if SCs cocultured with primary pelvic neurons can rescue neuronal survival and growth after ex vivo RT.MethodsMajor 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.OutcomesWe measured neurite length, branching, specific neuron populations and apoptosis.ResultsEx 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 TranslationThese 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 LimitationsThis 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.ConclusionUnirradiated 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.     

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.

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

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

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

IntroductionNeurogenic 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.AimTo examine whether bilateral cavernous nerve injury (BCNI) in a rat model of erectile dysfunction would increase recruitment of neurotoxic M1 macrophages to the MPG.MethodsMale 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 MeasuresCorpora 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.ResultsBCNI 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-1β) 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.ConclusionThese 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.     

Increased Expression of the Neuroregenerative Peptide Galanin in the Major Pelvic Ganglion Following Cavernous Nerve Injury

IntroductionErectile dysfunction (ED) remains a frequent complication of radical prostatectomy due to injury to the cavernous nerves (CNs). A recent microarray showed the neuropeptide galanin to be one of the most strikingly upregulated genes in the rat major pelvic ganglion (MPG) after bilateral CN crush injury (BCNI).AimThe aim of this study is to evaluate the temporal regulation of galanin in the MPG after BCNI and its relationship to functional nerve regeneration.MethodsChanges in galanin, galanin receptor (galR), and c‐JUN mRNA expression were assessed in Sprague‐Dawley rats after sham operation (n = 10) and at 48 hours (n = 10), 7 (n = 10), 14 (n = 5), 21 (n = 5), 30 (n = 5), and 60 (n = 5) days after BCNI using quantitative PCR. Erectile function was assessed by measuring intracavernous pressure (ICP) divided by mean arterial pressure (MAP) during CN electrostimulation. Immunohistochemistry was performed on the MPG in sham‐operated animals and 5 days after BCNI.Main Outcome MeasuresICP/MAP upon CN stimulation; galanin, galR1, ‐2, ‐3, and c‐JUN mRNA expression at various time points after BCNI; and nNOS, galanin, and galR distribution in the MPG of sham‐operated rats and after BCNI.ResultsAfter BCNI, ICP/MAP values quickly deteriorate, while after 60 days, spontaneous restoration of erectile responses to CN stimulation is observed, reflecting CN regeneration. Galanin mRNA in the MPG is up to 186‐fold upregulated compared with sham‐operated rats at 48 hours and 7 days after BCNI and gradually declines with increasing time from injury, whereas galanin receptor expressions decrease and c‐JUN gradually increases. Galanin expression shows a strong inverse correlation with erectile responses to CN stimulation with time from injury. Injured MPGs show a colocalization between galanin‐ and nNOS‐positive neuronal cell population in the MPG.ConclusionsGalanin is upregulated in the MPG in the early phase after CN injury after which it gradually decreases and is present in nNOS‐positive neurons of the ganglion. We hypothesize that galanin upregulation is an important factor in the endogenous neuroregenerative response to CN injury. Weyne E, Albersen M, Hannan JL, Castiglione F, Hedlund P, Verbist G, De Ridder D, Bivalacqua TJ, and Van der Aa F. Increased expression of the neuroregenerative peptide galanin in the major pelvic ganglion following cavernous nerve injury. J Sex Med 2014;11:1685–1693.     

REVIEW: Utilization of Pharmacotherapy for Erectile Dysfunction Following Treatment for Prostate Cancer

IntroductionPharmacotherapies improve sexual function following treatments for localized prostate cancer; however, patterns of care remain unknown.AimTo ascertain post-treatment utilization of pharmacotherapies for erectile dysfunction (ED) using a population-based approach.MethodsWe identified 38,958 men who underwent definitive treatment for localized prostate cancer during 2003–2006 from the MarketScan Medstat data.Main Outcome MeasuresWe compared the use of ED pharmacotherapy at baseline (up to 3 months prior) and up to 30 months following radical prostatectomy (RP) or radiotherapy (RT) for localized prostate cancer by utilizing National Drug Classification codes for phosphodiesterase-5 inhibitors (PDE5I), intracavernosal injectable therapies (IT), urethral suppositories and vacuum erection devices (VED). In adjusted analyses, we controlled for the effect of age, comorbidity, type of treatment, health plan and use of adjuvant hormone therapy on the use of pharmacotherapies.ResultsMen undergoing RP vs. RT were younger with less co-morbid conditions. Utilization of PDE5I was up to three times greater for men undergoing RP vs. RT, 25.6% vs. 8.8%, (P < 0.0001) in the first post-treatment year, and usage of these agents was greatest for men undergoing minimally-invasive RP procedures. A higher percentage of men also used IT, suppositories and VED after RP vs. RT (P < 0.001). However, more men in the RT group received adjuvant hormonal therapy (39.53% vs. 5.25% for RP, P < 0.01). In adjusted analyses, men undergoing RP vs. RT were more than two times likely (OR 2.1, 95% CI 1.98, 2.26) to use PDE5I post-treatment while men on adjuvant hormonal therapy were less likely to use PDE5I (OR 0.74, 95% CI 0.70–0.79, P < 0.0001).ConclusionMen undergoing RP vs. RT, particularly minimally-invasive RP, are more likely to employ IT, suppositories, VED, and PDE5I pharmacotherapy post-treatment. Prasad MM, Prasad SM, Hevlone ND, Gu X, Weinberg AC, Lipsitz SR, Palapattu GS, and Hu JC. Utilization of pharmacotherapy for erectile dysfunction following treatment for prostate cancer.     

Hydroxyl Fasudil,an Inhibitor of Rho Signaling,Improves Erectile Function in Diabetic Rats: A Role for Neuronal ROCK

IntroductionThe pathogenesis of diabetic erectile dysfunction (ED) includes neuropathy, but the molecular basis for neurogenic ED is incompletely understood. The RhoA/ROCK pathway has been implicated in diabetic neuropathy and in ED, but its role in diabetic neurogenic ED is not known.AimsThe aim of this study was to determine whether hydroxyl fasudil, a ROCK inhibitor, affects diabetic neuropathy‐related ED.MethodsType 1 diabetes mellitus was induced in male rats by streptozotocin (75 mg/kg, intraperitoneally). After 8 weeks, diabetic rats were administered hydroxyl fasudil, a selective ROCK inhibitor (10 mg/kg/day, intraperitoneally) or vehicle, for 4 weeks. Age‐matched control, nondiabetic, rats were treated intraperitoneally for 4 weeks with saline. At week 12, after a 2 day washout, neuro‐stimulated erectile function was evaluated. Major pelvic ganglia (MPG) were collected for Western blot analysis of RhoA, ROCK‐1, ROCK‐2, phospho (P)‐AKT (Ser⁴⁷³), and P‐phosphatase and tensin homolog (P‐PTEN) (Ser³⁸⁰/Thr^382/383).Main Outcome MeasuresEffect of ROCK inhibitor hydroxyl fasudil on erectile function and ROCK/P‐AKT/P‐PTEN pathway in the MPG of diabetic rats.ResultsErectile response was significantly (P < 0.05) reduced in diabetic rats compared with nondiabetic rats and was preserved (P < 0.05) in diabetic rats treated with hydroxyl fasudil. In diabetic rats, RhoA and ROCK‐2 protein expressions in MPG were increased (P < 0.05) and remained increased in hydroxyl fasudil‐treated rats. P‐AKT (Ser⁴⁷³) expression was decreased (P < 0.05), whereas P‐PTEN (Ser³⁸⁰/Thr^382/383) expression was increased (P < 0.05) in MPG of diabetic rats compared with nondiabetic rats, and both were reversed (P < 0.05) in diabetic rats treated with hydroxyl fasudil.ConclusionImproved erectile function and restored P‐AKT and P‐PTEN in the MPG with hydroxyl fasudil treatment suggest the role of Rho signaling via PTEN/AKT pathway in neurogenic diabetic ED. Sezen SF, Lagoda G, Musicki B, and Burnett AL. Hydroxyl fasudil, an inhibitor of Rho signaling, improves erectile function in diabetic rats: A role for neuronal ROCK. J Sex Med 2014;11:2164‐2171.     

Inhibitory Role of Gamma-Aminobutyric Receptors in Paraventricular Nucleus on Ejaculatory Responses in Rats

BackgroundAlthough abnormal sympathetic nerve system (SNS) activity has been demonstrated in the pathogenesis of ejaculation disorders, few data are available on its underlying mechanism.AimTo investigate whether differences in ejaculatory behavior of rats were associated with the state of SNS activity and gamma-aminobutyric (GABA) receptor expressions in the paraventricular nucleus (PVN) of the hypothalamus and the effects of GABA receptors in the PVN on ejaculatory behavior.MethodsBased on ejaculatory performance, Sprague-Dawley rats were divided into “sluggish,” “normal,” and “rapid” ejaculators. PVN microinjection was performed to evaluate the role of GABA receptors on sexual behavior.OutcomesThe outcomes include differences in expression and distribution of GABA receptors and norepinephrine level among the 3 groups and changes in copulation behavior parameters after PVN microinjection.ResultsCompared with “normal” rats, the “rapid” group ejaculated more times with shorter latency (P < .001, P < .001) and had lower expression and distribution of both GABA-A and GABA-B receptors, while the opposed results appeared in the “sluggish” group. The norepinephrine level was successively increased among “sluggish,” “normal,” and “rapid” rats (P < .001) and correlated with ejaculation frequency (r = 0.896, P < .001) and ejaculation latency (r = −0.835, P < .001). In addition, bilateral microinjection of the GABA-A and GABA-B receptor agonist (isoguvacine and baclofen) into the PVN both significantly prolonged the intromission latency and inhibited ejaculation, which could be blocked by antagonist gabazine and CGP-35348, respectively. Vigabatrin, the GABA-transaminase inhibitor, caused a significantly reduced ejaculation frequency and extended ejaculation latency in rats, which could be offset by simultaneous injections of gabazine and CGP-35348.Clinical ImplicationsOur findings provide new understanding about GABA receptors in the PVN on sexual behavior and enhance the comprehension of neurobiological mechanisms involved in premature ejaculation.Strengths & LimitationsOur results have indicated that GABA receptors in the PVN may inhibit ejaculation through restraining the activity of SNS. However, our study did not analyze the changes of GABA receptors in other brain areas, which needs further study.ConclusionEjaculation behaviors in male rats are associated with SNS activity and could be regulated by GABA receptors in the PVN, which may be of assistance in the treatment of ejaculation disorders in the future.Zhang QJ, Yang BB, Yang J, et al. Inhibitory Role of Gamma-Aminobutyric Receptors in Paraventricular Nucleus on Ejaculatory Responses in Rats. J Sex Med 2020;17:614–622.     

Low-Intensity Electrostimulation Enhances Neuroregeneration and Improves Erectile Function in a Rat Model of Cavernous Nerve Injury

BackgroundNeurogenic erectile dysfunction (ED) following radical prostatectomy (RP) is a frequent complication often leading to erectile tissue remodeling and permanent ED. Low-intensity electrostimulation (LIES) has been shown to enhance peripheral nerve regeneration, however, its application on cavernous nerves (CN) has never been investigated.AimsTo investigate whether LIES enhances CN regeneration, improves erectile function (EF) recovery, and prevents corpora cavernosal remodeling after CN injury, which is a principal factor for ED following RP.MethodsAdult male Sprague-Dawley rats were divided into Sham, Bilateral Cavernous Nerve Injury (BCNI), and BCNI + LIES (1V, 0.1ms, 12Hz, 1h/day). After 7days, EF was assessed (ICP measurement). Penes and CN were collected for molecular analyses of TGF-$\beta$1, Il-6, CRP, eNOS, ERK and AKT protein levels in corpus cavernosum (CC), and immunohistological analysis of DHE, total collagen and α-SMA in CC and S-100, Tub-III, DAPI, TUNEL, and nNOS in CN.OutcomesEffects of LIES on EF, erectile tissue remodeling and CN structure.ResultsEF was decreased (P < .05) 7 days after BCNI and increased (P < .05) by LIES. Intracavernosal reactive oxygen species (DHE) was increased (P < .05) after BCNI and normalized by LIES. Protein expressions of TGF-β1, IL-6, and CRP were increased in the penis (P < .05) after BCNI and normalized by LIES. The α-SMA and/or total collagen ratio was decreased (P < .05) after BCNI in the penis and normalized by LIES. Protein expression ratio of p-ERK/ERK and p-AKT/AKT did not change after BCNI but increased (P < .05) in LIES group. Myelination and number of nNOS positive cells in the CN were decreased (P < .05) after BCNI and normalized by LIES. The number of apoptotic nerve cells within the dorsal penile nerve was increased (P < .05) after BCNI and decreased (P < .05) by LIES compared to the BCNI group. There were no differences in eNOS expression in the penis between study groups.Clinical TranslationLIES may offer a potential new tool for penile rehabilitation and ED management following RP, potentially enhancing EF recovery and minimizing the side effects of this surgery.Strengths & LimitationsThis study provides evidence of the protective effect of LIES on EF and tissue remodeling following CN injury; nevertheless, this study has been conducted on animals and the translation to humans remains to be demonstrated. Further research to identify the underlying mechanisms of action is required.ConclusionThis study demonstrates that LIES of the CN after CN injury protects CN structure, enhances EF recovery, and prevents corpora cavernosal remodeling.Sturny M, Karakus S, Fraga-Silva R, et al. Low-Intensity Electrostimulation Enhances Neuroregeneration and Improves Erectile Function in a Rat Model of Cavernous Nerve Injury. J Sex Med 2022;19:686–696.     

Intravenous Preload of Mesenchymal Stem Cells Rescues Erectile Function in a Rat Model of Cavernous Nerve Injury

IntroductionWe evaluated the potential preventive effects and mechanisms of intravenously preloaded mesenchymal stem cells (MSCs) for erectile dysfunction (ED) in a cavernous nerve (CN) injury model.MethodsMale Sprague–Dawley (SD) rats were used for this study. Rats were randomized into two groups. One group was intravenously preloaded with MSCs (1.0 × 10⁶ cells in 1 mL total fluid volume) and the other was infused with medium alone (1 mL Dulbecco's modified Eagle's medium [DMEM]) for sham control, respectively. Crushed CN injury was induced immediately after infusion. The surgeon was blind to the experimental conditions (MSC or medium).Main Outcome MeasuresTo assess erectile function, we measured the intracavernous pressure (ICP) and arterial pressure (AP) at 1 hour and 2 weeks after CN injury. After measuring the initial ICP/AP of pre‐injury (normal) male SD rats, they were randomized into the two groups and infused with MSCs or medium. PKH26‐labelled MSCs were used for tracking. To investigate the mRNA expression levels of neurotrophins in the major pelvic ganglia (MPG), we performed real‐time quantitative real‐time polymerase chain reaction.ResultsThe reduction of ICP/AP and area under the curve of ICP (ICP‐AUC) in the MSC group was significantly lower than in the DMEM group (P < 0.05; P < 0.05) at 1 hour. The ICP/AP and ICP‐AUC at 2 weeks post‐injury in the MSC group was significantly higher than in the DMEM group (P < 0.01; P < 0.05). The preloaded PKH26‐labelled MSCs were detected in the MPG and CN using confocal microscopy indicating homing of the cells to the injured nerve and ganglia. Glia cell‐derived neurotrophic factor (GDNF) and neurturin, which are important neurotrophic factors for erection, had expression levels in MPG significantly higher in the MSC group than in the DMEM group (P < 0.01, 0.05).ConclusionIntravenous preload of MSCs before a CN injury may prevent or reduce experimental ED. Takayanagi A, Sasaki M, Kataoka‐Sasaki Y, Kobayashi K, Matsuda Y, Oka S, Masumori N, Kocsis JD and Honmou O. Intravenous preload of mesenchymal stem cells rescues erectile function in a rat model of cavernous nerve injury. J Sex Med 2015;12:1713–1721.     

The significance of pelvic floor support on the pelvic floor muscle functions of women with lower urinary tract symptoms

ObjectiveTo explore the significance of pelvic organ prolapse (POP) on pelvic floor muscle (PFM) function among women with lower urinary tract symptoms (LUTS).Materials and methodsFour-dimensional ultrasound data of 577 women with LUTS were retrospectively analyzed. The bladder neck and genital hiatus were assessed during resting, coughing, and squeezing. The bladder neck location, genitohiatal size, and genitohiatal location were evaluated with bladder neck distance (BNd) and bladder neck angle (BNa), genitohiatal dimension (GHd) and genitohiatal area (GHAR), and genitohiatal angle (GHa), respectively.ResultsCompared with women without POP (n = 306), women with POP (n = 271) exhibited higher rates of levator complete avulsion (6.5% vs. 40.2%, P < 0.001), shorter BNd (2.84 ± 1.56 cm vs. 2.45 ± 0.45 cm, P = 0.018), larger BNa (92 ± 15° vs. 101 ± 21°, P < 0.001), longer GHd (5.25 ± 0.72 cm vs. 5.60 ± 0.87 cm, P < 0.001), larger GHa (141 ± 10° vs. 145 ± 9°, P = 0.004), and larger GHAR (20.0 ± 4.7 cm² vs. 24.2 ± 5.6 cm², P < 0.001) during resting. Fewer women with POP were able to maintain stable bladder neck location (79.5% vs. 65.5%, P < 0.001), genitohiatal size (60.7% vs. 51.9%, P = 0.042), and genitohiatal location (61.6% vs. 52.8%, P = 0.044) following coughing. Fewer women with POP were capable of squeezing (77.8% vs. 58.3%, P < 0.001).ConclusionAmong women with LUTS, the presence of POP is associated with weaker resting, involuntary, and voluntary PFM functions.     

Diagnostic value of endometrial volume and flow parameters under 3D ultrasound acquisition in combination with serum CA125 in endometrial lesions

ObjectiveThis study aims to discuss the differential diagnosis value of endometrial volume and flow parameters in combination with serum carbohydrate antigen 125 (CA125) in endometrial benign and malignant lesions.Materials and methodsThe data of 250 patients with endometrial lesions were retrospectively analyzed. Carbohydrate antigen 125 (CA125) was determined before the operation. The morphology, hemodynamics, volume and flow parameters of the endometrium were measured by transvaginal three-dimensional-power Doppler angiography (3D-PDA). The endometrial volume (EV), 3D-PDA vascular index (VI), flow index (FI) and vascularization flow index (VFI) were calculated using the virtual organ computer-aided analysis software (VOCAL).ResultsAccording to the pathological results, 202 patients (80.8%) had benign endometrial lesions and 48 patients (19.2%) had endometrial cancer (EC). The endometrium of EC patients was thicker (15.64 ± 7.26 mm vs. 9.24 ± 5.06 mm, P < 0.001), the endometrial volume was larger (9.23 ± 4.08 ml vs. 2.26 ± 3.42 ml, P < 0.001), and the flow parameters VI, FI and VFI were higher, when compared to those of benign lesions (P < 0.001). The area under the receiver operating characteristic curve (AUROCC) of VI receptors was 0.86, while the AUC of endometrial thickness (ET) was only 0.66. Therefore, the best variable for distinguishing benign and malignant endometrial lesions was VI. The level of CA125 in the EC group significantly increased (40.57 ± 17.45 vs. 17.87 ± 7.64, P < 0.001), and the level of CA125 increased (P < 0.05) with the increase in clinical grade, degree of tumor differentiation, and pelvic lymph node metastasis (P < 0.05). However, the difference in myometrial invasion was not statistically significant (P > 0.05).ConclusionTransvaginal 3D-PDA can clearly show the morphological and hemodynamic characteristics of endometrial lesions, and assist in the detection of EC in combination with serum CA125. This may have important clinical application value.     

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.

Vasa Nervorum in Rat Major Pelvic Ganglion are Innervated by Nitrergic Nerve Fibers

IntroductionThe vasa nervorum comprises a network of small diameter blood vessels that provide blood supply to nerves and ganglia. The cell bodies of autonomic nerves innervating the urogenital organs are housed in the major pelvic ganglia (MPG) in rats. The vasa nervorum of rat MPG have not been characterized previously, and it is not known whether these blood vessels are innervated by neuronal nitric oxide synthase (nNOS) containing nitrergic nerves.AimTo characterize the blood vessels in and around the rat MPG and to assess their nitrergic innervation.Main Outcome MeasuresCharacterization of small blood vessels in and around the rat MPG and expression of nNOS in nerve fibers around those blood vessels.MethodsMPG were obtained from healthy Sprague Dawley rats, fixed in paraformaldehyde, frozen and sectioned using a cryostat. The blood vessels and their nitrergic innervation were assessed with immunohistochemistry using antibodies against alpha‐smooth muscle actin (smooth muscle marker), CD31 (endothelial marker), collagen IV (basal membrane marker) and nNOS. The immunofluorescence was imaged using a laser scanning confocal microscope.ResultsThe neuronal cell bodies were contained within a capsule in the MPG. Blood vessels were observed within the capsule of the MPG as well as outside the capsule. The blood vessels inside the capsule were CD31‐positive capillaries with no smooth muscle staining. Outside the capsule capillaries, arterioles and venules were observed. The extra‐capsular arterioles and venules, but not the capillaries were innervated by nNOS‐positive nerve fibers.ConclusionsThis study, to our knowledge, is the first to demonstrate the blood vessel distribution pattern and their nitrergic innervation in the rat MPG. While similar studies in human pelvic plexus are warranted, these results suggest that the blood flow in the MPG may be regulated by nitrergic nerve fibers and reveal a reciprocal relationship between nerves and blood vessels. Beetson KA, Smith SF, Muneer A, Cameron NE, Cotter MA, and Cellek S. Vasa nervorum in rat major pelvic ganglion are innervated by nitrergic nerve fibers. J Sex Med 2013;10:2967–2974.     

14-3-3 tau (YWHAQ) gene promoter hypermethylation in human placenta of preeclampsia

IntroductionDisruption of the 14-3-3 tau (YWHAQ) gene has been shown to be involved in preeclampsia (PE). The YWHAQ promoter could be differentially regulated by methylation in severe PE patients.MethodsPlacental genomic DNA from patients with severe PE (n = 21) and controls who experienced a normal pregnancy (n = 16) was analyzed using dot-blot and immunohistochemistry. The placental methylation patterns of YWHAQ, expression of 14-3-3 tau and ten-eleven translocation (TET), were confirmed by bisulfite sequencing, immunohistochemistry, western blot and real-time PCR, respectively.ResultsGenomic 5 hmC (P < 0.001), expression of 14-3-3 tau (P < 0.01) and TET (P < 0.05) were down-regulated, whereas 5 mC was up-regulated (P < 0.001) in preeclamptic placentas. Significant hypermethylation of the YWHAQ promoter was detected in PE placentas compared with control samples (19.1% vs. 9.4%, P = 0.0095). PE-specific hypermethylation of CpG2 – 4, CpG9, CpG17, CpG19 was identified in PE patients compared with controls (CpG2: 13.3% vs. 2.5%, P < 0.0001; CpG3: 14.8% vs. 3.1%, P < 0.0001; CpG4: 19.5% vs. 5.0%, P < 0.0001; CpG9: 15.7% vs. 5.0%, P = 0.0018; CpG17: 16.2% vs. 6.3%, P = 0.0003; and CpG19: 78.1% vs. 59.4%, P < 0.0001).DiscussionThe observed participation of 14-3-3 tau in the regulation of the placental epigenome may participate in the molecular mechanisms that govern the pathological process of PE, although this requires further evaluation.     

Effects of Intravenous Injection of Adipose‐Derived Stem Cells in a Rat Model of Radiation Therapy‐Induced Erectile Dysfunction

IntroductionRadiation therapy (RT) for prostate cancer is frequently associated with posttreatment erectile dysfunction (ED).AimTo investigate whether injection of adipose‐derived stem cells (ADSCs) can ameliorate RT‐associated ED.MethodsThirty male rats were divided into three groups. The control + phosphate‐buffered saline (PBS) group received tail‐vein injection of PBS. The radiation + PBS group received radiation over the prostate and tail‐vein injection of PBS. The radiation + ADSC group received radiation over the prostate and tail‐vein injection of ADSCs, which were labeled with 5‐ethynyl‐2‐deoxyuridine (EdU). Seventeen weeks later, erectile function was evaluated by intracavernous pressure (ICP) in response to electrostimulation of cavernous nerves (CNs). Penile tissue and major pelvic ganglia (MPG) were examined by immunofluorescence (IF) and EdU staining.Main Outcome MeasuresErectile function was measured by ICP. Protein expression was examined by IF, followed by image analysis and quantification.ResultsRadiation over the prostate caused a significant decrease in erectile function and in the expression of neuronal nitric oxide synthase (nNOS) in penis and MPG. Cavernous smooth muscle (CSM) but not endothelial content was also reduced. Injection of ADSCs significantly restored erectile function, nNOS expression, and CSM content in the irradiated rats. EdU‐positive cells were visible in MPG.ConclusionsRadiation appears to cause ED via CN injury. ADSC injection can restore erectile function via CN regeneration. Qiu X, Villalta J, Ferretti L, Fandel TM, Albersen M, Lin G, Dai Y, Lue TF, and Lin C‐S. Effects of intravenous injection of adipose‐derived stem cells in a rat model of radiation therapy‐induced erectile dysfunction. J Sex Med 2012;9:1851–1858.     

GGF2 Is Neuroprotective in a Rat Model of Cavernous Nerve Injury-Induced Erectile Dysfunction

IntroductionErectile dysfunction is a major complication of radical prostatectomy, commonly associated with penile neuropathy. In animal models of peripheral nerve injury, glial growth factor-2 (GGF2), a member of the neuregulin family of growth factors, has neuroprotective and neurorestorative properties, but this potential has not been established after cavernous nerve (CN) injury.AimsThe effectiveness of GGF2 in preserving axonal integrity and recovering erectile function in a rat model of radical prostatectomy-associated CN injury.MethodsAdult male Sprague-Dawley rats underwent bilateral CN crush injury (BCNI) or sham surgery. Rats were administered GGF2 (0.5, 5, or 15 mg/kg) or vehicle subcutaneously 24 hour pre and 24-hour post-BCNI, and once weekly for 5 weeks. Erectile function was assessed in response to electrical stimulation of the CN. CN survival was assessed by fluorogold retrograde axonal tracing in major pelvic ganglia (MPG). Unmyelinated axons in the CNs were quantitated by electron microscopy.Main Outcome MeasuresErectile function recovery, CN survival, and unmyelinated CN axon preservation in response to GGF2 treatment following BCNI.ResultsErectile function was decreased (P < 0.05) after BCNI, and it was improved (P < 0.05) by all doses of GGF2. The number of fluorogold-labeled cells in the MPG was reduced (P < 0.05) by BCNI and was increased (P < 0.05) by GGF2 (0.5 and 5 mg/kg). The percentage of denervated Schwann cells in the BCNI group was higher (P < 0.05) than that in the sham-treated group and was decreased (P < 0.05) in the GGF2-treated (5 mg/kg) BCNI group. In the BCNI + GGF2 (5 mg/kg) group, the unmyelinated fiber histogram demonstrated a rightward shift, indicating an increased number of unmyelinated axons per Schwann cell compared with the BCNI group.ConclusionsGGF2 promotes erectile function recovery following CN injury in conjunction with preserving unmyelinated CN fibers. Our findings suggest the clinical opportunity to develop GGF2 as a neuroprotective therapy for radical prostatectomy. Burnett AL, Sezen SF, Hoke A, Caggiano AO, Iaci J, Lagoda G, Musicki B, and Bella AJ. GGF2 is neuroprotective in a rat model of cavernous nerve injury-induced erectile dysfunction. J Sex Med 2015;12:897–905.     

Pelvic insufficiency fractures in patients with cervical and endometrial cancer treated with postoperative pelvic radiation

ObjectiveThere is an increased awareness of pelvic insufficiency fractures (PIF) as a potential morbidity of pelvic radiotherapy (RT). The purpose of this study was to determine the incidence of PIF and assess prognostic factors, including intensity-modulated RT (IMRT), in gynecologic oncology patients treated with postoperative pelvic RT.MethodsWe performed a retrospective review of all patients with endometrial or cervical carcinoma who received postoperative pelvic RT at our institution during 2000–2008. Patients who received definitive or palliative RT were excluded.ResultsA total of 222 patients were identified, of whom 11 (5%) developed PIF at a median time of 11.5 months (range, 5.5–87.3 months) from RT completion. The 5-year actuarial rate was 5.1% (95% CI 3.3–6.9). In patients with osteoporosis, the 5-year rate was 15.6% compared with 2.9% for those without (P = 0.01). Similarly, patients who were on prior hormone-replacement therapy (HRT) had a higher rate (14.8% vs 4.1%, P = 0.009). The median body-mass index (BMI) for patients who developed PIF was significantly lower than those who didn't (25.9 vs 27.2, P = 0.016). The rate of PIF was 4.9% whether patients received IMRT or conventional RT.ConclusionsThe 5-year risk of PIF for postoperative pelvic RT in cervical and endometrial cancer is 5.1%. Women with history of osteoporosis, prior HRT, or low BMI need to be counseled about the risk of PIF. The use of IMRT did not decrease PIF, but further studies are needed to determine if a dose/volume relationship exists between RT and PIF.     

Apocynin Improves Erectile Function in Diabetic Rats through Regulation of NADPH Oxidase Expression

IntroductionDiabetes is a risk factor for erectile dysfunction (ED). The proposed mechanisms responsible for diabetic ED are associated with an increase in reactive oxygen species (ROS) production, overactivity of RhoA/ROCK signaling pathway and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, as seen in experimental models of diabetic rats.AimThe aim of this study was to investigate whether NADPH oxidase inhibitor apocynin can ameliorate Streptozotocin (STZ)‐induced diabetes‐related ED by reducing the ROS production and inhibiting the activity of RhoA/ROCK signaling pathway.MethodsThe diabetic rats were treated with and without the NADPH oxidase inhibitor apocynin.Main Outcome MeasuresErectile responses were evaluated by determining mean arterial blood pressure (MAP) and intracavernosal pressure (ICP) with electrical stimulation of the cavernous nerve. Levels of mRNA expression were measured by real‐time polymerase chain reaction (RT‐PCR). Levels of protein expression were examined by Western Blot. ROS production was measured by dihydroethidium (DHE) staining and thiobarbituric acid reactive substances assay.ResultsThe ratio of Maximum ICP‐to‐MAP (MaxICP/MAP) was significantly decreased in diabetic ED rats, compared to that of age‐matched control rats (P < 0.05). Apocynin improved erectile function of diabetic rats (P < 0.05). Expression levels of RhoA (cytosol), nNOS and eNOS were reduced, compared to those of control rats (P < 0.05). Apocynin significantly elevated their expression levels in diabetic rats (P < 0.05). Expression levels of ROCK1, RhoA (membrane fraction), p‐MYPT1 and NADPH oxidase subunits p47^phox and p67^phox were increased in diabetic rats when compared to those of control rats (P < 0.05), and it was observed that apocynin significantly reduced their expression levels in diabetic rats (P < 0.05). ROS production was increased in diabetic rats when compared to that of control rats (P < 0.05), the effect of apocynin was a reduction in the ROS production in diabetic rats (P < 0.05).ConclusionNADPH oxidase inhibitor apocynin can ameliorate diabetes‐related ED by reducing the ROS production and inhibiting the activity of RhoA/ROCK signaling pathway. Li M, Zhuan L, Wang T, Rao K, Yang J, Yang J, Quan W, Liu J, and Ye Z. Apocynin improves erectile function in diabetic rats through regulation of NADPH oxidase expression. J Sex Med 2012;9:3041–3050.