Neural supply to the clitoris: immunohistochemical study with three-dimensional reconstruction of cavernous nerve, spongious nerve, and dorsal clitoris nerve in human fetus

IntroductionLittle detailed information is available concerning autonomic and somatic nerve supply to the clitoris, potentially causing difficulties for nerve preservation during pelvic and perineal surgery.AimTo identify the location and type (nitrergic, adrenergic, cholinergic and sensory) of nerve fibers in the clitoris and to provide a three‐dimensional (3D) representation of their structural relationship in the human female fetus.MethodsSerial transverse sections were obtained from five human female fetuses (18–31 weeks of gestation) and subjected to histological and immunohistochemical investigations; digitized serial sections were used to construct a 3D representation of the pelvis.Main Outcome MeasuresPelvic‐perineal nerve location and type were evaluated qualitatively.ResultsThe female neurovascular bundle (NVB) is the anteroinferior terminal portion of the inferior hypogastric plexus that runs along the postero‐lateral then lateral face of the vagina and is rich in nNOS‐positive fibers. The cavernous nerve (CN) is a thin ventrocaudal collateral projection of the NVB, and this projection does not strictly follow the NVB course. The CN runs along the lateral surface of the vagina and urethra and penetrates the homolateral clitoral crus. The CN provides adrenergic, cholinergic, and nitrergic innervation to the clitoris, but not sensory innervation. The spongious nerve (SN) is the terminal and main projection of the NVB and provides nitrergic innervation to the vestibular bulbs. The dorsal clitoris nerve (DCN), somatic branche of the pudendal nerve, runs along the superior surface of the clitoral crus and body and has a segmental proerectile nitrergic activity related to communicating branches with the CN.Conclusions“Computer‐assisted anatomic dissection” allowed the identification of the precise location and distribution of the autonomic and somatic neural supply to female erectile bodies, providing an anatomical basis for nerve‐sparing surgical techniques, and participating to the understanding of neurogenic female sexual dysfunction. Moszkowicz D, Alsaid B, Bessede T, Zaitouna M, Penna C, Benoit G, and Peschaud F. Neural supply to the clitoris: Immunohistochemical study with 3D reconstruction of cavernous nerve, spongious nerve and dorsal clitoris nerve in human fetus.     

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.     

Comparative Study of the Neuropeptide-Y Sympathetic Nerves in Endometriotic Involved and Noninvolved Sacrouterine Ligaments in Women with Pelvic Endometriosis

Study ObjectiveTo show the relationship between the neuropeptide-Y pelvic sympathetic nerves and neoangiogenesis in the development of endometriosisDesignProspective study.SettingAcademic community teaching hospital.PatientsFifteen consecutive women with unilateral endometriotic infiltration of the sacrouterine ligaments.InterventionsA laparoscopic excision/biopsy of involved and noninvolved parts of the sacrouterine ligaments were taken. The sections were incubated with the neuronal marker rabbit polyclonal anti-protein gene product 9.5 and rabbit polyclonal anti-neuropeptide-Y. We made a comparative study on the distribution of nerve fibers and their relationship to the vessels on intact and endometriotic involved tissue.Measurements and Main ResultsThe results show that a large amount of nerves are present around the blood vessels in the endometriosis samples, and a large number of these nerves are neuropeptide-Y sympathetic nerves. Adrenergic fibers are also present in the intact control subjects, however, in significantly smaller amounts.ConclusionThis finding shows a strong relationship between the neuropeptide-Y sympathetic pelvic nerves and the neoangiogenesis required for the development of endometriosis.     

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.     

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.     

Impaired Cavernous Reinnervation after Penile Nerve Injury in Rats with Features of the Metabolic Syndrome

IntroductionThe metabolic syndrome is a cluster of cardiovascular risk factors that predispose toward the development of diseases such as diabetes. Erectile dysfunction (ED) is common in men with metabolic syndrome, but its etiology is poorly understood. Pro‐erectile nitrergic nerves innervating penile erectile tissue are also susceptible to mechanical injury during pelvic surgical procedures, which can lead to sexual dysfunction.AimsThe aims of this article are: (i) to examine erectile function in an experimental model of metabolic syndrome, the phosphoenolpyruvate carboxykinase (PEPCK)‐overexpressing rat; and (ii) to study function and cavernous reinnervation after penile nerve crush injury, which permits regeneration, in transgenic rats.MethodsWe analyzed the density of noradrenergic and nitrergic nerves and performed organ bath pharmacology to assess neurogenic activity.Main Outcome MeasuresBy analyzing changes in neural structure, function, and pharmacologic responses of cavernous tissue after nerve crush injury, we were able to reveal neurologic deficits in rats with metabolic syndrome.ResultsAnimals with features of metabolic syndrome did not develop notable changes in cavernous autonomic nerve density or nerve‐evoked smooth muscle activity. However, regeneration of nitrergic nerves after crush injury in transgenic rats was impaired compared with injured controls. This was manifested as a deficit in axon regrowth and responses to axon activation. However, unlike injured controls, injured PEPCK‐overexpressing rats did not develop a reduced maximal response to the nitric oxide (NO) donor, sodium nitroprusside. This suggests preserved NO responsiveness in tissues from rats with metabolic syndrome, despite impaired regeneration and return of function.ConclusionsThis study revealed that rats with features of metabolic syndrome display impaired cavernous nerve regeneration after penile nerve injury, but the degree of functional impairment may be attenuated due to reduced plasticity of NO signaling. This reinnervation deficit may be of clinical relevance for understanding why ED persists in some (particularly aged) men after pelvic surgery. Nangle MR, Proietto J, and Keast JR. Impaired cavernous reinnervation after penile nerve injury in rats with features of the metabolic syndrome.     

The Visceromotor and Somatic Afferent Nerves of the Penis

IntroductionInnervation of the penis supports erectile and sensory functions.AimThis article aims to study the efferent autonomic (visceromotor) and afferent somatic (sensory) nervous systems of the penis and to investigate how these systems relate to vascular pathways.MethodsPenises obtained from five adult cadavers were studied via computer-assisted anatomic dissection (CAAD).Main Outcome MeasuresThe number of autonomic and somatic nerve fibers was compared using the Kruskal–Wallis test.ResultsProximally, penile innervation was mainly somatic in the extra-albugineal sector and mainly autonomic in the intracavernosal sector. Distally, both sectors were almost exclusively supplied by somatic nerve fibers, except the intrapenile vascular anastomoses that accompanied both somatic and autonomic (nitrergic) fibers. From this point, the neural immunolabeling within perivascular nerve fibers was mixed (somatic labeling and autonomic labeling). Accessory afferent, extra-albugineal pathways supplied the outer layers of the penis.ConclusionsThere is a major change in the functional type of innervation between the proximal and distal parts of the intracavernosal sector of the penis. In addition to the pelvis and the hilum of the penis, the intrapenile neurovascular routes are the third level where the efferent autonomic (visceromotor) and the afferent somatic (sensory) penile nerve fibers are close. Intrapenile neurovascular pathways define a proximal penile segment, which guarantees erectile rigidity, and a sensory distal segment. Diallo D, Zaitouna M, Alsaid B, Quillard J, Ba N, Allodji RS, Benoit G, Bedretdinova D, and Bessede T. The visceromotor and somatic afferent nerves of the penis. J Sex Med 2015;12:1120–1127.     

Innervation of the Labia Minora of Prepubertal Girls

IntroductionSurgical and histologic sources of information give little reference to innervation, vascular, and epithelial details of the labia minora. Little is known about areas of nerve density, epithelial qualities, and vascular compartments of the labial minora that contribute to sexual arousal and orgasm. Surgical procedure development and counsel about surgical risks related to labioplasty and surgical flaps created from labial tissue may be based on inadequate information.MethodsLabial samples from 10 normal girls (aged 2–9 years) who underwent surgery for labial fusion utilized waste tissue strips for immunohistochemical identification of S-100 and neuronal nitric oxide synthase (nNOS) in the labia minora.ResultsVascular and lymphatic plexus lie within the reticular dermis, which contains a dense mesh of nerve fibers with a higher concentration of nerve fiber at the level of the subepithelial plexus. Dense innervations are located at the epidermis, extending along the basal and spinous layers of the epithelium of labia minora. Nerve bundles in the papillary dermis are associated with sebaceous and eccrine glands and nerve terminals located throughout the epithelium. The introital epithelium of the labia minora is highly innervated with widespread and intense staining, detected in the introital border of the labia minora versus the external one. The dermis appeared to display S-100 and nNOS immunolabelling. S-100 was also immunopositive in the epidermis.ConclusionLabia minora is highly innervated along its entire edge. Related vascular compartment tissue involved in engorgement during sexual arousal makes this tissue important for sexual response. Labioplasty risks removal of tissue with an important contribution to sensory sexual arousal. Movement of labial tissue during genitoplasty may have different sensory outcomes dependent on which labial surface is used.     

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

BackgroundProstatic radiation therapy (RT) often causes erectile dysfunction (ED) and the mechanisms governing RT-induced ED are unclear with a lack of therapeutic strategies.AimTo determine the effects of ex vivo RT on major pelvic ganglion (MPG) neuron survival, and neurite growth in whole vs dissociated culture.MethodsMPGs 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.OutcomesAfter 72 hours, neurite length, branching, autonomic neuron density, and apoptosis were assessed, and gene expression was measured.ResultsRT 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 TranslationProstatic RT leads to increased neuronal cell death and less erectogenic nitrergic neurons contributing to ED.Strengths & LimitationsThe 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.ConclusionThe 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.     

Useful Pelvic Retroperitoneal Neuroanatomy for Benign Gynecologic Surgery: A Cadaveric Dissection

ObjectiveKnowledge of the retroperitoneal anatomy is particularly important to facilitate surgical procedure and reduce the number of complications. The objective of this video is to demonstrate pelvic neuroanatomic structures and their relationships in the pelvic sidewall and the presacral space in a laparoscopic cadaveric dissection.DesignCase report (anatomic study).SettingMedical training center (AdventHealth Nicholson Center, Orlando, FL).InterventionsThe dissection started with the mobilization of the iliac vessels from the pelvic sidewall to identify the obturator nerve. The peritoneum of the ovarian fossa was opened, and the ureter was dissected up to the level of the uterine artery. The hypogastric nerve was identified. The close relationship between the ovarian fossa and the obturator nerve could be demonstrated. The deep dissection of the obturator fossa allowed for the identification of the lumbosacral trunk, S1, the sciatic nerve, S2, S3, S4, and the splanchnic nerves. Then, the ischial spine and the sacrospinous ligament were identified. The pudendal nerve and vessels could be observed passing below the sacrospinous ligament, entering the pudendal canal (Alcock's canal). The presacral space was dissected, and the hypogastric fascia was opened. S1 to S4 were identified coming from the sacral foramens. The laparoscopic dissection, using the cadaveric model, allowed for the development of the entire retroperitoneal anatomy, focusing on the dissection of the pelvic innervation. Anatomic relationships among the ureter, the hypogastric nerve, the uterosacral ligament, the splanchnic nerves, the inferior hypogastric plexus, and the organs (bowel, vagina, uterus, and bladder) could be demonstrated.ConclusionA laparoscopic cadaveric dissection can be used as a resource to demonstrate and educate surgeons about the neurologic retroperitoneal structures and their relationships.     

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.     

Laparoscopic Dissection and Anatomy of Sacral Nerve Roots and Pelvic Splanchnic Nerves

Study ObjectiveTo demonstrate the technique of laparoscopic dissection for identification of sacral nerve roots and pelvic splanchnic nerves.DesignCase report (Canadian Task Force classification III).SettingPrivate practice hospital in São Paulo, Brazil.PatientA 31-year-old woman with suspected iatrogenic and/or compression of sacral nerve roots. She reported debilitating pelvic, gluteal, and perineal unilateral left-sided pain (score 8 on a pain scale of 0–10), and had primary infertility with 1 previous failed attempt at in vitro fertilization. Surgical history included laparoscopic excision of endometriosis 10 months before the procedure and left oophoroplasty during adolescence because of a benign neoplasm.InterventionsStandard 4-puncture laparoscopy was performed. The peritoneum of the left pelvic sidewall was resected to preclude eventual residual endometriosis. This also enabled identification of uterine vessels including the deep uterine vein, which is the limit between the pars vascularis superiorly and the pars nervosa inferiorly in the uterine broad ligament. Surgery was using the laparoscopic neuro-navigation (LANN) technique, previously described by one of us (M. P.). For identification of the sacral roots, dissection was begun medial to the ureter and lateral to the uterosacral ligament. The Okabayashi pararectal space was entered as deep as possible via blunt dissection in avascular spaces. Hemostasis was performed using 5-mm bipolar forceps, and harmonic energy was not used. The hypogastric fascia was entered from medial to lateral, and the piriformis muscle was identified. The sacral nerve root S1 was identified lying over it. Dissection then proceeded caudally, and sacral roots S2 and S3 were sequentially identified. Small and delicate fibers forming the pelvic splanchnic nerves were isolated emerging from sacral roots S2 and S3. Other nerve fibers were identified caudally, probably representing pelvic splanchnic nerves emerging from S4.Measurements and Main ResultsThe surgical operative time was 70 minutes, and bleeding was minimal. No suspected compression or iatrogenic injury was identified. The patient was discharged on the day after the procedure. At 8-month follow-up, she had partial resolution of pain (score 5, pain scale 0–10), and another failed attempt at in vitro fertilization was attributed to unsatisfactory quality of the embryos. There were no symptoms or dysfunctions attributable to manipulation of the nerves.ConclusionLaparoscopy is a useful tool for identification of sacral roots and pelvic splanchnic nerves in suspected diseases. Its application in the field of neuropelveology can be expanded with proper knowledge and training.     

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.

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.     

Erectile Function Restoration After Repair of Excised Cavernous Nerves by Autologous Vein Graft in Rats

IntroductionCavernous nerves (CNs) injury is the main cause of erectile dysfunction (ED) following radical prostatectomy. Its restoration remains challenging.AimTo investigate the feasibility of erectile function recovery by autologous vein graft after bilateral CNs being excised in a rat model.MethodsA total of 36 adult male Sprague-Dawley rats were randomized into three groups. A 5 mm segment of CN was excised bilaterally in group B and C. In group B, a 7-mm segment of autologous saphenous vein was interposed at the defect site bilaterally, with two nerve stumps inserted into the vein lumen. Group C underwent no repair. Group A was accepted a sham operation. 4 months later, apomorphine tests were performed on each rat, followed by injection of 4% fluorogold into bilateral corpus cavernous. 5 days later, after monitoring intracorporal pressure (ICP) changes induced by electrostimulation of CN, rats were sacrificed and their bilateral major pelvic ganglions were obtained for detection of fluorogold, and penile tissues of middle shaft were obtained for detecting nitric oxide synthase-containing nerve fibers in penile dorsal nerves.Main Outcome MeasuresErectile function was assessed by apomorphine test and ICP monitoring. CN regeneration was judged by fluoroglod tracing and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining.ResultsApomorphine tests resulted in 58% rats with erectile responses in group B, whereas no erection was observed in group C. ICP monitoring also demonstrated a significant recovery in erectile function in group B compared with group C. Much more and brighter fluorogold coloring cells were examined in major pelvic ganglions of group B than those of group C. NADPH-diaphorase staining also showed much more positive fibers were detected in penile dorsal nerves in group B than in group C.ConclusionAutologous vein graft could provide a guide channel to induce CN regeneration and successfully restore autonomic erectile function after CNs being excised in rats. Hu W, Cheng B, Liu T, Li S, and Tian Y. Erectile function restoration after repair of excised cavernous nerves by autologous vein graft in rats.     

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.     

Differences in uterine innervation at hysterectomy

OBJECTIVE: Our purpose was to identify patterns of uterine innervation in normal uteri and selected clinical conditions including adenomyosis and chronic pelvic pain. STUDY DESIGN: A retrospective survey was performed of stored uteri removed at hysterectomy for a variety of clinical conditions, including 8 uteri from nulliparous subjects (group 1, mean age 40.0 years, range 30-52 years), 21 uteri with no reported histologic abnormality from multiparous subjects (group 2, mean age 43.4 years, range 32-53 years; mean parity 2.0, range 1-4), 31 uteri reported with adenomyosis (group 3, mean age 42.4 years, range 29-54 years; parity 2.0, range 0-4), and 17 uteri from subjects with pelvic pain (group 4, mean age 39.1 years, range 30-52 years; parity 2.5, range 1-7). Sections were cut from paraffin blocks of the isthmus of stored uteri (in the majority of cases) and stained with protein gene product 9.5 to identify nerves. Sections of pancreas provided positive controls. Each section was reviewed by two unblinded observers. RESULTS: Group 1 (n = 8, nulliparous uteri) showed significant nerve bundles at the endometrial-myometrial interface and in the subserosal layers, with nerve fibers noted in intervening neurovascular bundles supplying the myometrial stroma. Group 2 (n = 21, histologically normal uteri from parous subjects) showed patterns of innervation similar to those of group 1 with the exception that 6 uteri demonstrated areas of nerve fiber proliferation (see below, group 4). In group 3 (n = 31, uteri with adenomyosis), 30 uteri (30/31) showed large areas of myometrium without nerves and absence of nerves in the neurovascular bundles supplying these areas. Five uteri showed areas of nerve fiber proliferation at the margins of the adenomyosis. Subserosal nerves were present in the majority of these uteri. In group 4 (n = 17), uteri were removed for chronic pelvic pain. Eleven uteri demonstrated proliferation of small-diameter nerve fibers throughout the myometrium; in 6 uteri there was asymmetry of nerve fiber proliferation. CONCLUSIONS: Variations in uterine innervation were noted in the isthmic region of uteri stored after hysterectomy. Uteri with adenomyosis frequently demonstrated large areas with absence of nerve fibers; uteri from subjects with chronic pelvic pain showed proliferation of small-diameter nerve fibers throughout the myometrial stroma. Nerve fiber proliferation was asymmetric in some of these specimens.     

Increased nerve fibers in placental bed myometrium in women with preeclampsia

Narrowing of the uterine spiral arterioles below the deciduomyometrial junction is 1 of the key pathophysiological changes in women with preeclampsia. The contribution of pelvic autonomic nerves to decidualization and impaired placentation in preeclampsia is not clear. Placental bed biopsies were obtained from 10 women with preeclampsia and 23 nornotensive women at caesarean section. We stained them with anti-S100 and CD34 antibodies to detect the presence of nerve fibers and blood vessels, respectively. We detected S100-immunoactive nerve fibers in the myometrium but not in the decidua in both groups of women. S100-immunoactive nerve fiber density in the placental bed myometrium was significantly increased in women with preeclampsia compared to normotensive women. There was no clear relationship between the densities of nerve fibers and CD34-positive blood vessels in these biopsies. These results suggest increased nerve fibers in the placental bed myometrium may play a role in the pathogenesis of the preeclampsia.     

Hyperinnervation in Intestinal Deep Infiltrating Endometriosis

Study ObjectiveTo investigate the extent and types of innervation of endometriotic lesions in various regions of the bowel.DesignRetrospective nonrandomized immunohistochemical study (Canadian Task Force classification II-3.SettingUniversity-based laboratory.PatientsThirty-six women undergoing laparoscopy or laparotomy because of deep infiltrating endometriosis in various regions of the bowel, including the sigmoid colon, appendix, and rectum.InterventionsImmunohistochemical staining of endometriotic specimens with antibodies against protein gene product 9.5, neurofilament, nerve growth factor, nerve growth factor receptors tyrosine kinase receptor A and p75, growth-associated protein 43, substance P, neuropeptide Y, and vasoactive intestinal peptide to demonstrate myelinated, unmyelinated, sensory, and autonomic nerve fibers.Measurements and Main ResultsThere were significantly more nerve fibers in intestinal deep infiltrating endometriosis (mean [SD] 172.6 [94.2]/mm²) than in other deep infiltrating endometriotic lesions (e.g., cul-de-sac and uterosacral ligament) (67.6 [65.1]/mm²; p < .01). Intestinal deep infiltrating endometriosis was innervated abundantly by sensory Aδ,sensory C, cholinergic, and adrenergic nerve fibers. Nerve growth factor, tyrosine kinase receptor A, and p75 were strongly expressed in endometriotic lesions, and growth-associated protein-43 was also strongly expressed in the endometriosis-associated nerve fibers.ConclusionThe hyperinnervation in intestinal deep infiltrating endometriosis may help to explain why patients with this type of lesion have more severe pain.