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.

FK506 and Rapamycin Neuroprotect Erection and Involve Different Immunophilins in a Rat Model of Cavernous Nerve Injury

IntroductionImmunophilin ligands function by binding to receptor proteins such as FK506 binding proteins (FKBPs). FKBPs are studied for their roles in neuroprotection.AimCompare the effect of FK506 (FK) and rapamycin (RAP) on erectile function (EF) recovery and FKBP expressions in penis and major pelvic ganglion (MPG) after cavernous nerve (CN) injury.MethodsAdult male rats were divided into four groups: sham surgery (CN exposure only) + vehicle; bilateral CN injury (BCNI; bilateral crush, 3 minutes with hemostat clamp) + vehicle; BCNI + FK (5 mg/kg/day, 5 days, sc); and BCNI + RAP (2 mg/kg/day, 5 days, sc). At both 24 hours (Day 1) or 1 week (Day 7) after BCNI, EF was assessed by intracavernosal pressure measurement and FKBPs 12, 38, 52, and 65 expressions were evaluated by Western blot analysis in collected penises and MPGs.Main Outcome MeasuresEF and change in protein expressions of FKBPs in the rat penis and MPG after BCNI with and without immunophilin ligand treatment.ResultsBoth FK- and RAP-treated rats had preserved EF compared with vehicle-treated rats after BCNI. FKBPs changed variably following injury and treatment. In particular, in the penis at Day 1, FKBP 38 expression was decreased after BCNI and both FK and RAP attenuated this decrease. In MPG at Day 1, FKBP 38 expression was also decreased after BCNI and FK attenuated the decrease, while at Day 7, FKBP 38 expression was still decreased and RAP attenuated the decrease. Also, in the penis at Day 1, FKBP 65 expression decreased after BCNI and FK attenuated the decrease. In the MPG, FKBP 65 expression increased at both Days 1 and 7 with FK treatment.ConclusionsImproved EF after BCNI, as shown with RAP, further suggests a role of immunophilin ligands as a protective therapy of CN injury associated erectile dysfunction. Our findings also suggest that select FKBPs, such as FKBP 38 and FKBP 65, may mediate these effects. Lagoda G, Sezen SF, and Burnett AL. FK506 and rapamycin neuroprotect erection and involve different immunophilins in a rat model of cavernous nerve injury. J Sex Med 2009;6:1914–1923.     

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.     

Insulin-Like Growth Factor-1–Loaded Polymeric Poly(Lactic-Co-Glycolic) Acid Microspheres Improved Erectile Function in a Rat Model of Bilateral Cavernous Nerve Injury

Background

Previous studies have documented improvement in erectile function after bilateral cavernous nerve injury (BCNI) in rats with the use of pioglitazone. Our group determined this improvement to be mediated by the insulin-like growth factor-1 (IGF-1) pathway.

Inhibition of interleukin-6 attenuates erectile dysfunction in a rat model of nerve-sparing radical prostatectomy

IntroductionThe precise mechanisms underlying erectile dysfunction (ED) occurring after cavernous nerve (CN)‐sparing surgery remain to be determined.AimTo evaluate the expression of interleukin‐6 (IL‐6) and IL‐6 receptor (IL‐6R) after CN injury, and the effect of inhibiting IL‐6 bioactivity on nerve injury‐related ED.MethodsMale Sprague‐Dawley rats were divided into three groups: sham operation; bilateral CN dissection without crushing or cutting; and bilateral CN resection. In the interventional experiment, male rats underwent bilateral CN dissection, and anti‐rat IL‐6 antibody in phosphate‐buffered saline (PBS) or vehicle alone was injected intraperitoneally immediately and 24 hours after CN dissection.Main Outcome MeasuresOne, 3, 7, 28, and 56 days after surgery, the expression of IL‐6 and IL‐6R in the major pelvic ganglion (MPG) was examined by real‐time polymerase chain reaction. In the interventional experiment, erectile function was assessed by determining intracavernous pressure divided by arterial pressure (ICP/AP) during electrical pelvic nerve stimulation at 4 weeks after surgery in the anti‐IL‐6‐injected rats and PBS‐injected rats. The degree of nerve injury was also evaluated by retrograde dye tracing with Fluorogold.ResultsThe expression levels of IL‐6 and IL‐6R were increased in the early period of CN injury, as compared with the sham group. IL‐6 expression on day 1 was particularly enhanced. Four weeks after CN dissection, the anti‐IL‐6 group had greater ICP/AP and more FG‐positive cells than the PBS group.ConclusionsExpression levels of IL‐6 in the MPG were increased in the acute phase following CN injury. Inhibition of IL‐6 bioactivity attenuated ED following CN dissection. Thus, the suppression of excess inflammatory responses in the acute phase may lead to improvements in ED occurring after nerve‐sparing radical prostatectomy. Yamashita S, Kato R, Kobayashi K, Hisasue S, Arai Y, and Tsukamoto T. Inhibition of interleukin‐6 attenuates erectile dysfunction in a rat model of nerve‐sparing radical prostatectomy. J Sex Med 2011;8:1957–1964.     

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.     

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.     

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.     

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.     

Valproic Acid Prevents Penile Fibrosis and Erectile Dysfunction in Cavernous Nerve‐Injured Rats

IntroductionBilateral 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.AimsThis study will characterize the molecular changes in penile HDAC after BCNI and determine if HDAC inhibition can prevent BCNI‐induced ED and penile fibrosis.MethodsFive 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‐β1 (TGF‐β1) 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 MeasuresWe measured ICP; HDAC3, HDAC4, fibronectin, and TGF‐β1 protein expression; penile fibrosis; penile α‐SMA content.ResultsThere 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‐β1 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).ConclusionHDAC‐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. Hannan JL, Kutlu O, Stopak BL, Liu X, Castiglione F, Hedlund P, Burnett AL, and Bivalacqua TJ. Valproic acid prevents penile fibrosis and erectile dysfunction in cavernous nerve‐injured rats. J Sex Med 2014;11:1442–1451.     

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.     

Therapeutic Effect of Adipose‐Derived Stem Cells and BDNF‐immobilized PLGA Membrane in a Rat Model of Cavernous Nerve Injury

IntroductionCavernous nerve injury is the main reason for post‐prostatectomy erectile dysfunction (ED). Stem cell and neuroprotection therapy are promising therapeutic strategy for ED.AimTo evaluate the therapeutic efficacy of adipose‐derived stem cells (ADSCs) and brain‐derived neurotrophic factor (BDNF) immobilized Poly‐Lactic‐Co‐Glycolic (PLGA) membrane on the cavernous nerve in a rat model of post‐prostatectomy ED.MethodsRats were randomly divided into five groups: normal group, bilateral cavernous nerve crush injury (BCNI) group, ADSC (BCNI group with ADSCs on cavernous nerve) group, BDNF‐membrane (BCNI group with BDNF/PLGA membrane on cavernous nerve) group, and ADSC/BDNF‐membrane (BCNI group with ADSCs covered with BDNF/PLGA membrane on cavernous nerve) group. BDNF was controlled‐released for a period of 4 weeks in a BDNF/PLGA porous membrane system.Main Outcome MeasuresFour weeks after the operation, erectile function was assessed by detecting the ratio of intra‐cavernous pressure (ICP)/mean arterial pressure (MAP). Smooth muscle and collagen content were determined by Masson's trichrome staining. Neuronal nitric oxide synthase (nNOS) expression in the dorsal penile nerve was detected by immunostaining. Phospho‐endothelial nitric oxide synthase (eNOS) protein expression and cyclic guanosine monophosphate (cGMP) level of the corpus cavernosum were quantified by Western blotting and cGMP assay, respectively.ResultsIn the ADSC/BDNF‐membrane group, erectile function was significantly elevated, compared with the BCNI and other treated groups. ADSC/BDNF‐membrane treatment significantly increased smooth muscle/collagen ratio, nNOS content, phospho‐eNOS protein expression, and cGMP level, compared with the BCNI and other treated groups.ConclusionsADSCs with BDNF‐membrane on the cavernous nerve can improve erectile function in a rat model of post‐prostatectomy ED, which may be used as a novel therapy for post‐prostatectomy ED. Piao S, Kim IG, Lee JY, Hong SH, Kim SW, Hwang T‐K., Heang S, Lee JH, Ra JC, and Lee JY. Therapeutic effect of adipose‐derived stem cells and BDNF‐immobilized PLGA membrane in a rat model of cavernous nerve injury. J Sex Med 2012;9:1968–1979.     

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.     

The Neuroprotective Effect of Platelet‐rich Plasma on Erectile Function in Bilateral Cavernous Nerve Injury Rat Model

IntroductionNeurogenic erectile dysfunction resulting from cavernous nerve (CN) injury is a major complication caused by radical prostatectomy. The use of platelet‐rich plasma (PRP) on the nerve‐injured site has shown promising results for the nerve regeneration. However, the effects of PRP injection in corpus cavernosum after bilateral CN injury have never been investigated.AimTo assess the neuroprotective effect of PRP injection in corpus cavernosum after bilateral CN injury.MethodsMale Sprague‐Dawley rats were randomly divided into three groups: Group I underwent sham operation, while the remaining two groups underwent bilateral CN crush. Crush injury groups were treated at the time of injury with an application of PRP or normal saline only injection in the corpus cavernosum, respectively. Four weeks later, erectile function (EF) was assessed by CN electrosimulation, and CNs as well as penile tissue were collected for histology.Main Outcome MeasuresIntracavernous pressure (ICP) monitored during electrical stimulation of CNs; myelinated axons number of CNs and dorsal penile nerve; collagen type change, number of apoptotic cells, and mRNA expression of caspase‐3 and transforming growth factor‐β1 (TGF‐β1) in the corpus cavernosum.ResultsFour weeks after surgery, in the vehicle‐only group, the functional evaluation showed a lower mean maximal ICP than that in the sham group (P < 0.05). PRP treatments resulted in significant recovery of EF, as compared with the vehicle‐only group (P < 0.05). Histologically, the PRP‐treated group had a significant preservation of myelinated axons of CNs compared with the vehicle‐only group (P < 0.05) and reduced the apoptotic index. The mRNA expression of TGF‐β1 in the corpus cavernosum tissue was significantly decreased in the PRP group compared with the vehicle‐only group (P < 0.05).ConclusionsPRP injection in the corpus cavernosum increased the number of myelinated axons and facilitated recovery of EF in the bilateral CN injury rat model. Wu C‐C, Wu Y‐N, Ho H‐O, Chen K‐C, Sheu M‐T, and Chiang H‐S. The neuroprotective effect of platelet‐rich plasma on erectile function in bilateral cavernous nerve injury rat model. J Sex Med 2012;9:2838–2848.     

Involvement of Rho‐Kinase/LIM Kinase/Cofilin Signaling Pathway in Corporal Fibrosis after Cavernous Nerve Injury in Male Rats

IntroductionThe molecular mechanism of corporal fibrosis leading to erectile dysfunction (ED) following cavernous nerve (CN) injury is poorly understood.AimTo determine whether the LIMK2/cofilin pathway, the downstream effectors of ROCK1, was involved in ED and corporal fibrosis following bilateral CN injury in male rats.MethodsForty‐eight 10‐week‐old male Sprague‐Dawley rats were equally divided into three groups: sham surgery (S); bilateral CN crush injury (I); and bilateral CN resection (R). Within each groups, two subgroups were analyzed at 1 and 4 weeks postoperatively.Main Outcome MeasuresElectrostimulation was performed to assess erectile function by the ratio of maximal intracavernous pressure to mean arterial pressure (ICP/MAP) and areas under the ICP curve to MAP (AUC/MAP). Penile tissue was processed for Masson's trichrome staining, Western blot (ROCK1, total LIMK2, phospho‐LIMK2, total cofilin, phospho‐cofilin), immunohistochemistry (alpha‐SM actin [α‐SMA]), and double immunofluorescent staining (ROCK1, phospho‐LIMK2, vimentin).ResultsAt each time point, both I and R groups showed a significantly lower percent of ICP/MAP and AUC, and decreased SM cell/collagen ratio and expression of α‐SMA than S group. Densitometry revealed a significantly higher expression of ROCK1 in I and R groups compared with S group at all time points. The LIMK2 phosphorylation in I and R groups significantly increased at 1 week, but not at 4 weeks. The cofilin phosphorylation in R group significantly increased to that in S group starting at 1 week, while that in I group was increased significantly at 4 weeks. The double immunofluorescent staining noted that coexpression of vimentin with ROCK1 or phospho‐LIMK2 in I and R groups was significantly increased mainly in the subtunical area at 1 week but not at 4 weeks.ConclusionsThe ROCK1/LIMK2/cofilin pathway may be involved in ED related to corporal fibrosis, and it appears to be functional particularly in the early period after CN injury. Song SH, Park K, Kim SW, Paick J‐S, and Cho MC. Involvement of Rho‐kinase/LIM kinase/cofilin signaling pathway in corporal fibrosis after cavernous nerve injury in male rats. J Sex Med 2015;12:1522–1532.     

CXCL5 Cytokine Is a Major Factor in Platelet-Rich Plasma's Preservation of Erectile Function in Rats After Bilateral Cavernous Nerve Injury

BackgroundThe neuro-protective and tissue-protective properties of platelet-rich plasma (PRP) have been demonstrated through treating bilateral cavernous nerve (CN) injury in rats, although the underlying mechanisms have not been fully clarified.AimTo determine factors released from PRP and explore their role in mediating preservation of erectile function (EF) in a rat model of CN injury.MethodsMale Sprague-Dawley rats (aged 10 weeks) were used in this study. 6 rats were used to obtain blood for PRP and whole plasma preparation. We probed samples using a cytokine antibody array and performed enzyme-linked immunosorbent assay (ELISA). We determined the expression patterns of C-X-C motif chemokine ligand 5 (CXCL5) and receptors in the major pelvic ganglion (MPG) and corpus cavernosum via immunostaining. 32 rats were divided into 4 groups based on the type of injection received: (i) sham, (ii) vehicle, (iii) 400 μL of PRP, and (iv) 30 ng/kg of CXCL5. Groups 2–4 were subjected to bilateral CN crush (BCNC) injury. 4 weeks later, EF was assessed by CN electrostimulation, and CNs and penile tissue were collected for histological analysis.OutcomeCytokine antibody array, ELISA, erectile response, and immunofluorescence staining readings.ResultsThe PRP contained high levels of CXCL5. MPG neurons expressed CXCL5 and CXCR2. PRP intracavernous injection stabilized CXCR2 and increased CXCL5 expression in the MPG after BCNC, thus enhancing neuroprotection. CXCL5 injection improved BCNC-induced erectile dysfunction by preventing smooth muscle atrophy.Clinical ImplicationsThe therapeutic efficacy of PRP in CN injury–induced erectile dysfunction may arise from the synergy among multiple biomolecules. Our study serves as a basis for future studies on PRP formulation to provide safe and effective medications for the maintenance of EF after radical prostatectomy in patients with prostate cancer.Strengths & LimitationsA strength of our study is that our model was able to isolate the role of cytokines, specifically CXCL5, as part of the mechanism responsible for PRP's protective properties. However, the rat cytokine array provided limited experimental targets. The rats used were not at the age corresponding to prostate cancer patients in clinical settings. Our study did not explore CXCL5 blocking in the PRP group. Finally, the main protein quantification results by western blotting were hampered because of small tissue samples.ConclusionsThis study provides evidence for the role of CXCL5 and CXCR2 as mediators of PRP effects in the preservation of EF after CN injury.Wu YN, Liao CH, Chen KC, et al. CXCL5 Cytokine Is a Major Factor in Platelet-Rich Plasma's Preservation of Erectile Function in Rats After Bilateral Cavernous Nerve Injury. J Sex Med 2021;18:698–710.     

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.     

FK506 neuroprotection after cavernous nerve injury is mediated by thioredoxin and glutathione redox systems

IntroductionImmunophilin ligands such as FK506 (FK) preserve erectile function (EF) following cavernous nerve injury (CNI), although the precise mechanisms are unclear. We examined whether the thioredoxin (Trx) and glutathione (GSH) redox systems mediate this effect after CNI.AimTo investigate the roles of Trx reductase 2 (TrxR2) and S‐Nitrosoglutathione reductase (GSNOR) as antioxidative/nitrosative and antiapoptotic mediators of the neuroprotective effect of FK in the penis after CNI.MethodsAdult male rats, wild‐type (WT) mice, and GSNOR deficient (GSNOR ‐/‐) mice were divided into four groups: sham surgery (CN [cavernous nerves] exposure only) + vehicle; sham surgery + FK (5 mg/kg/day/rat or 2 mg/kg/day/mouse, for 2 days, subcutaneous); CNI + vehicle; and CNI + FK. At day 4 after injury, electrically stimulated changes in intracavernosal pressure (ICP) were measured. Penises were collected for Western blot analysis of TrxR2, GSNOR, and Bcl‐2, and for immunolocalization of TrxR2 and GSNOR.Main Outcome MeasuresEF assessment represented by maximal ICP and total ICP in response to electrical stimulation. Evaluation of protein expression levels and distribution patterns of antioxidative/nitrosative and antiapoptotic factors in penile tissue.ResultsEF decreased after CNI compared with sham surgery values in both rats (P < 0.01) and WT and GSNOR ‐/‐ mice (P < 0.05). FK treatment preserved EF after CNI compared with vehicle treatment in rats (P < 0.01) and WT mice (P < 0.05) but not in GSNOR ‐/‐ mice. In rats, GSNOR (P < 0.01) and Bcl‐2 (P < 0.05) expressions were significantly decreased after CNI. FK treatment in CN‐injured rats restored expression of GSNOR and upregulated TrxR2 (P < 0.001) and Bcl‐2 (P < 0.001) expressions compared with vehicle treatment. Localizations of proteins in the penis were observed for TrxR2 (endothelium, smooth muscle) and for GSNOR (nerves, endothelium, smooth muscle).ConclusionsThe neuroprotective effect of FK in preserving EF after CNI involves antioxidative/nitrosative and antiapoptotic mechanisms mediated, to some extent, by Trx and GSH systems. Lagoda G, Xie Y, Sezen SF, Hurt KJ, Liu L, Musicki B, and Burnett AL. FK506 neuroprotection after cavernous nerve injury is mediated by thioredoxin and glutathione redox systems. J Sex Med **;**:**–**.     

Peptide Amphiphile Nanofiber Delivery of Sonic Hedgehog Protein to Reduce Smooth Muscle Apoptosis in the Penis after Cavernous Nerve Resection

IntroductionErectile dysfunction (ED) is a serious medical condition that affects 16–82% of prostate cancer patients treated by radical prostatectomy and current treatments are ineffective in 50–60% of prostatectomy patients. The reduced efficacy of treatments makes novel therapeutic approaches to treat ED essential. The secreted protein Sonic hedgehog (SHH) is a critical regulator of penile smooth muscle and apoptosis that is decreased in cavernous nerve (CN) injury and diabetic ED models. Past studies using Affi‐Gel beads have shown SHH protein to be effective in suppressing apoptosis caused by CN injury.AimWe hypothesize that SHH protein delivered via novel peptide amphiphile (PA) nanofibers will be effective in suppressing CN injury‐induced apoptosis.MethodsAdult Sprague Dawley rats (n = 50) were used to optimize PA injection in vivo. PA with SHH protein (n = 16) or bovine serum albumin (BSA) (control, n = 14) was injected into adult rats that underwent bilateral CN cut. Rats were sacrificed at 2, 4, and 7 days. Alexa Fluor‐labeled SHH protein was used to determine the target of SHH signaling (n = 3).Main Outcome MeasuresTerminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and semiquantitative immunohistochemical analysis for SHH protein and cluster differentiation protein three (CD3) were performed.ResultsSHH‐PA caused a 25% and 16% reduction in apoptosis at 4 and 7 days after CN injury and a 9.3% and 19% increase in SHH protein at 4 and 7 days after CN injury. CD3 protein was not observed in SHH‐PA‐treated penis. In vitro, 73% of SHH protein diffused from PA within 6 days. Labeled SHH was observed in smooth muscle.ConclusionsPA technology is effective in delivering SHH protein to the penis and SHH is effective in suppressing CN injury‐induced apoptosis. These results suggest substantial translational potential of this methodology and show that only a short duration of SHH treatment is required to impact the apoptotic index. Bond CW, Angeloni NL, Harrington DA, Stupp SI, McKenna KE, and Podlasek CA. Peptide amphiphile nanofiber delivery of Sonic hedgehog protein to reduce smooth muscle apoptosis in the penis after cavernous nerve resection. J Sex Med 2011;8:78–89.     

Sonic Hedgehog Is Neuroprotective in the Cavernous Nerve with Crush Injury

IntroductionThe cavernous nerve (CN) is commonly injured during prostatectomy, resulting in erectile dysfunction (ED). Although peripheral nerves have a limited ability to regenerate, a return of function typically does not occur due to irreversible down stream morphological changes in the penis that result from CN injury. We have shown in previous studies that sonic hedgehog (SHH) is critical for CN regeneration and improves erectile function after crush injury.AimsExamine a new direction, to determine if SHH is neuroprotective to the pelvic ganglia (PG)/CN after crush injury. A secondary focus is to examine if SHH signaling decreases with age in the PG/CN.MethodsSprague–Dawley rats underwent bilateral CN crush and SHH and glial fibrillary acidic protein were quantified by western analysis of the PG/CN (N = 6 rats at each time point) at 1, 2, 4, 7, and 14 days, and the apoptotic index was measured in the penis. SHH was quantified by western in the PG/CN with blockade of anterograde transport (N = 4 rats) in comparison to mouse IgG (N = 4 rats). If SHH is neuroprotective was examined at 4 (N = 14 rats) and 7 days (N = 16 rats) of treatment after CN crush. SHH protein was quantified in aging (P200‐300, N = 5 rats) PG/CN in comparison to normal adult (P115‐120, N = 3 rats) PG/CN.Main Outcome MeasuresSHH pathway was examined in PG via immunohistochemistry, in situ, western, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL).ResultsSHH is neuroprotective in the PG/CN with injury. SHH localization in the PG/CN suggests SHH interaction in neuronal/glial signaling. SHH protein is significantly decreased in the PG/CN after crush injury and in the aged PG/CN. Signals from the PG are required to maintain SHH in the CN.ConclusionsThere is a window of opportunity immediately after nerve insult in which manipulation of SHH signaling in the nerve microenvironment can affect long‐term regeneration outcome.