Bilateral Cavernous Nerve Injury Rats Research Articles

Nrf2 enhances the therapeutic efficiency of adipose-derived stem cells in the treatment of neurogenic erectile dysfunction in a rat model

BackgroundErectile dysfunction (ED) caused by intraoperative nerve injury is a major complication of pelvic surgery. Adipose-derived stem cells (ADSCs) have presented therapeutic potential in a rat model of bilateral cavernous nerve injury (BCNI), while inadequate in vivo viability has largely limited their application. Nuclear factor-E2-related Factor (Nrf2) is a key transcription factor that regulates cellular anti-oxidative stress. In this work, we investigated the effect of Nrf2 expression regulation on the viability of ADSCs, and explore its repair potential in a BCNI rat model.ResultsThe survival time of tert-Butylhydroquinone (tBHQ)-ADSCs in BCNI model increased obviously. In addition, the tBHQ-ADSCs group presented better restoration of major pelvic ganglion (MPG) nerve contents and fibers, better improvement of erectile function, and less penile fibrosis than the other groups. Moreover, the expression of Nrf2 and superoxide dismutase 1 (SOD1) were higher than those of other groups.ConclusionNrf2 could enhance the anti-oxidative stress ability of ADSCs, so as to improve the therapeutic effect of ADSCs on BCNI rat model.

Biodegradable nano black phosphorus based SDF1-α delivery system ameliorates Erectile Dysfunction in a cavernous nerve injury rat model by recruiting endogenous stem/progenitor cells

Stem cell (SC) therapy has been shown high prospects in erectile dysfunction (ED) treatment. Without ethical issues and risks of immune rejection and tumorigenesis of exogenous SC therapy, endogenous stem/progenitor cells (S/PCs) have a better potential for ED management, and their homing and redistribution are controlled by SDF1-α/CXCR4 axis. Considering black phosphorus nanosheet (BPNS) has emerged as an efficient and safe drug vehicle due to its large surface area, biodegradability, and the ability to retain and slowly release its loaded drugs, BPNS is utilized to load SDF1-α, a chemokine for S/PCs, to construct the BP@SDF1-α complex to efficiently recruit stem cells (SCs) by injury-site injection and thus ameliorate ED within the bilateral cavernous nerve injury (BCNI) rat models. We find that BP@SDF1-α can efficiently recruit exogenous SCs and endogenous S/PCs to corpus cavernosum and main pelvic ganglion (MPG) by local administration. Of note, ascribing to endogenous S/PCs recruitment, it also successfully alleviates ED in BCNI rat models by enhancing the protein expression levels of α-SMA, CD31, and nNOs, and eliciting less collagen deposition in the penis after its combined injection at corpus cavernosum and MPG. Thus, this study provides a new insight into the treatment of ED with endogenous S/PCs.Biodegradable Nano Black Phosphorus based SDF1-α delivery system ameliorates Erectile Dysfunction in a cavernous nerve Injury Rat Model by recruiting endogenous Stem/Progenitor cells

Dimethyl fumarate ameliorates erectile dysfunction in bilateral cavernous nerve injury rats by inhibiting oxidative stress and NLRP3 inflammasome-mediated pyroptosis of nerve via activation of Nrf2/HO-1 signaling pathway

ObjectiveTo investigate the therapeutic potential of dimethyl fumarate (DMF) in improving erectile function of bilateral cavernous nerve injury (BCNI) rats, along with elucidating its underlying mechanisms. MethodsA BCNI rat model was established by clamping bilateral cavernous nerve (CN). DMF was given by gavage at low (20 mg/kg/day) and high (40 mg/kg/day) dosages for a duration of 4 weeks. Erectile function was assessed by electrical stimulation of CN. Penis and CN tissues were collected for subsequent analysis. Additionally, PC-12 cell line was used to verify the mechanism of DMF in vitro. Nfe2l2 or Ho-1 gene knockdown PC-12 cell lines were constructed by lentiviral transfection, respectively. A damaged cell model was induced using H2O2. And then molecular biological methods were employed to analyze cellular molecules and proteins. ResultsDMF administration for 4 weeks led to improvements in erectile function, reduced fibrosis of penis corpus cavernosum in BCNI rats. The morphology of CN was improved and the number of nerve fibers increased. Furthermore, the levels of nNOS, NO, and cGMP were increased, while Ca2+ was decreased in penis corpus cavernosum. Notably, the levels of ROS, 3-NT and NLRP3 inflammasomes production were reduced, alongside increased expression of Nrf2 and HO-1 proteins in the dorsal penile nerve (DPN) and CN. In vitro, DMF increased cell viability, reduced ROS level, promoted SOD, diminished 3-NT, MDA and DNA damage markers, and inhibited the activation of NLRP3 inflammasomes in H2O2 induced PC-12 cells. Nfe2l2 knockdown and Ho-1 knockdown significantly attenuated the protective effect of DMF, respectively. Furthermore, inhibition of ROS production by N-acetylcysteine led to a reduction in NLRP3 inflammasome activation in H2O2 induced PC-12 cells. ConclusionsDMF improved erectile function of BCNI rats by protecting nerves through inhibiting oxidative stress and the activation of NLRP3 inflammasome-mediated pyroptosis via activation of Nrf2/HO-1 pathway.

Caveolin-1 scaffolding domain peptide prevents corpus cavernosum fibrosis and erectile dysfunction in bilateral cavernous nerve injury-induced rats.

Corpus cavernosum (CC) fibrosis significantly contributes to post-radical prostatectomy erectile dysfunction (pRP-ED). Caveolin-1 scaffolding domain (CSD)-derived peptide has gained significant concern as a potent antagonist of tissue fibrosis. However, applying CSD peptide on bilateral cavernous nerve injury (BCNI)-induced rats remains uninvestigated. The aim was to explore the therapeutic outcome and underlying mechanism of CSD peptide for preventing ED in BCNI rats according to the hypothesis that CSD peptide may exert beneficial effects on erectile tissue and function following BCNI through limiting collagen synthesis in CC smooth muscle cells (CCSMCs) and CC fibrosis. After completing a random assignment of male Sprague Dawley rats (10 weeks of age), BCNI rats received either saline or CSD peptide treatment, as opposed to sham-operated rats. The evaluations of erectile function (EF) and succedent collection and histological and molecular biological examinations of penile tissue were accomplished 3weeks postoperatively. In addition, the fibrotic model of CCSMCs was used to further explore the mechanism of CSD peptide action in vitro. The assessments of EF, SMC/collagen ratio, α-smooth muscle actin, caveolin-1 (CAV1), and profibrotic indicators expressions were conducted. BCNI rats exhibited significant decreases in EF, SMC/collagen ratio, α-SMA, and CAV1 levels, and increases in collagen content together with transforming growth factor (TGF)-β1/Smad2 activity. However, impaired EF, activated CC fibrosis, and Smad2 signaling were attenuated after 3weeks of CSD peptide treatment in BCNI rats. In vitro, TGF-β1-induced CCSMCs underwent fibrogenetic transformation characterized by lower expression of CAV1, higher collagen composition, and phosphorylation of Smad2; then, the delivery of CSD peptide could significantly block CCSMC fibrosis by inactivating Smad2 signaling. Based on available evidence of CSD peptide in the prevention of ED in BCNI rats, this study can aid in the development and clinical application of CSD peptide targeting pRP-ED. This study provides data to suggest that CSD peptide protects against BCNI-induced deleterious alterations in EF and CC tissues. However, the available evidence still does not fully clarify the detailed mechanism of action of CSD peptide. Administration of CSD peptide significantly retarded collagen synthesis in CCSMCs, limited CC fibrosis, and prevented ED via confrontation of TGF-β1/Smad signaling in BCNI rats.

Implantation of skin-derived precursor Schwann cells improves erectile function in a bilateral cavernous nerve injury rat model

BackgroundThis study was conducted to investigate the therapeutic potential of the skin-derived precursor Schwann cells for the treatment of erectile dysfunction in a rat model of bilateral cavernous nerve injury.ResultsThe skin-derived precursor Schwann cells-treatment significantly restored erectile functions, accelerated the recovery of endothelial and smooth muscle tissues in the penis, and promoted nerve repair. The expression of p-Smad2/3 decreased after the treatment, which indicated significantly reduced fibrosis in the corpus cavernosum.ConclusionsImplantation of skin-derived precursor Schwann cells is an effective therapeutic strategy for treating erectile dysfunction induced by bilateral cavernous nerve injury.

Efficacy of a Red-Light Controllable Nitric Oxide Releaser for Neurogenic Erectile Dysfunction: A Study Using a Rat Model of Cavernous Nerve Injury.

Neurogenic erectile dysfunction (ED) is a common side effect of radical prostatectomy (RP) because of cavernous nerve damage. In these patients, the production of nitric oxide (NO), which is important for erection, is decreased in the corpus cavernosum. Therefore, NO donors are useful for post-RP ED. However, short half-life and systemic side effects are problems of NO application in ED therapy. To avert these problems, we developed a red-light controllable NO releaser, NORD-1. This study aimed to investigate the effect of NORD-1 and red-light irradiation on neurogenic ED using a rat model of bilateral cavernous nerve injury (BCNI). BCNI and sham operations were conducted on 8-week-old rats. After 4 weeks, erectile function was evaluated using changes in intracavernous pressure (ICP) during electrostimulation of the cavernous nerve. ICP was measured under three conditions; without NORD-1 and red-light irradiation, with NORD-1 and without red-light irradiation, and with NORD-1 and red-light irradiation. SiR650 which absorbs red-light but does not release NO was used for the negative control. After the experiment, localization of NORD-1 was observed using a microscope. Erectile function in a BCNI rat model was significantly decreased compared to sham-operated rats (p<0.05). After injecting NORD-1 into the penis, erectile function did not change without red-light irradiation. However, the combination of NORD-1 and red-light irradiation significantly improved erectile function (p<0.05) without affecting systemic arterial pressure. In contrast, when SiR650 was used, erectile function did not change in all three conditions. NORD-1 was detected only in the corpus cavernosum and not in the urethra and dorsal vein. NORD-1 combined with red-light irradiation is effective for ED induced by cavernous nerve injury. This treatment may have low risks of hypotension and urinary incontinence, and it can replace the current treatment for post-RP ED.

Injectable thermo-sensitive hydrogel containing ADSC-derived exosomes for the treatment of cavernous nerve injury

Radical pelvic surgery is commonly accompanied by the risk of postoperative erectile dysfunction induced by cavernous nerve injury (CNI-ED). The strategy of using adipose mesenchymal stem cell-derived exosomes (ADSC-Exo) to treat neurodegenerative diseases has shown promising results. However, it remains challenging to prolong the retention of unbound ADSC-Exo in damaged tissues to exert therapeutic effects. Herein, we develop a novel injectable thermo-sensitive hydroxyethyl chitosan/sodium β-glycerophosphate hydrogel (HG) encapsulating ADSC-Exo (HG@Exo) to manage CNI-ED. The HG exhibits excellent injectability, structural stability, and body temperature sensitivity. In vivo assessment demonstrates that the designed ADSC-Exo-loaded HG hydrogel enhances the retention of ADSC-Exo and displays a slow release. Furthermore, when HG@Exo is applied to the site of nerve injury, erectile function in the bilateral cavernous nerve injury rat model is significantly improved. Thus, our finding indicates that the developed bioactive hydrogel presents a promising strategy for the effective management of CNI-ED.

An EPO-loaded multifunctional hydrogel synergizing with adipose-derived stem cells restores neurogenic erectile function via enhancing nerve regeneration and penile rehabilitation.

Neurogenic erectile dysfunction (nED) is one of the most common and intractable postoperative complications of rectal and prostate cancer surgery and sometimes accompanies patients lifelong. The transplantation of stem cells has been proved a promising way for treatment. However, the therapeutic efficacy is severely impaired by excessive cell loss and death and poor accumulation in the injury site along with the traditional implantation strategy. Herein, an EPO‐loaded multifunctional hydrogel was designed. The hydrogels' adhesive property and mechanical strength were enhanced by adding catechol‐catechol adducts, thus significantly improving adipose‐derived stem cells (ADSC) retention and rescuing cell loss in the injury site. Meanwhile, the sustained release of EPO effectively ameliorated the viability and paracrine activity of ADSC, leading to enhanced migration of Schwann cells and differentiation of PC12 cells in vivo. On a bilateral cavernous nerve injury rat model, the present stem cell‐EPO‐hydrogel implanted strategy could significantly alleviate erectile dysfunction. The higher expression of Tuj1 and lower expression of GFAP in the major pelvic ganglia (MPG) indicated the acceleration of neural differentiation while the suppressing development of astrocytes. Also, the combined therapy restored the expression levels of eNOs, nNOs, and α‐SMA in penile tissues, suggesting the rehabilitation of the penis. Further analysis of Masson trichrome staining and apoptosis evaluation of the corpus cavernosum showed the preservation of vascular endothelium content and the prevention of penile fibrosis after denervation. Overall, we believe that this combined strategy presents a promising way not only for restoring neurogenic erectile function but also for the clinical translation of stem cell therapy.

Acetyl-L-carnitine improves erectile function in bilateral cavernous nerve injury rats via promoting cavernous nerve regeneration

ABSTRACT Introduction Acetyl-L-carnitine (ALCAR), an acetyl derivative of L-carnitine, has been proven to promote nerve repair and regeneration in a variety of neurodegenerative diseases. Neurogenic erectile dysfunction (NED) caused by CN injury is a common complication after radical prostatectomy, which lacks efficient curative treatments. Objective Although attempts have been made to apply ALCAR to treatment of peripheral neuropathy, the effects of ALCAR on NED remain unknown. Therefore, we intend to explore the potential role of ALCAR in the treatment of NED. Methods A rat model of bilateral cavernous nerve injury (BCNI) was established by crushing the CN 5 millimeters distal to the major pelvic ganglion (MPG). Twenty-four BCNI rats were randomly divided into BCNI group, BCNI+ALCAR-LD (50 mg/kg/day) group, and BCNI+ALCAR-HD (100 mg/kg/day) group, whereas the remaining eight age-matched rats were sham-operated as controls. Fourteen days after intraperitoneal injection of ALCAR, erectile function was evaluated by measuring intracavernosal pressure and mean arterial pressure. The penile tissues and CNs at the distal damage point were collected from each group of rats for subsequent histological and molecular biological analysis. Rat Schwann cell line S16 was used for CCK-8 and wound healing assay, and the expressions of SC markers Pmp22 and nerve growth factor (NGF) were verified by PCR. Results First, we found that the erectile function of rats in the BCNI model group was severely impaired, which was improved considerably in both BCNI+ALCAR-LD and BCNI+ALCAR-HD groups. Then, we observed that the penis was fibrotic after denervation in the BCNI group, which was manifested as decreased smooth muscle content and increased collagen content in the corpus cavernosum, and high expression of fibrosis indicators including TGF-beta, CTGF, and Smad 2/3. The above changes were alleviated after the administration of low and high-dose ALCAR. Meanwhile, our results also showed that after ALCAR treatment, the NO/cGMP pathway was promoted and the RhoA/ROCK pathway was inhibited in the corpus cavernosum of BCNI rats. Further, rats treated with ALCAR had high expression of ATF3 and S100 in the distal nerve tissues of the CN extrusion site, indicating the repairing effect of ALCAR on the CN. In vitro experiments showed that ALCAR promoted the proliferation and migration of SC, and increased the expression of Pmp22 and NGF. Conclusions ALCAR could boost CN repair and regeneration, thereby suppressing penile fibrosis and improving penile erection. ALCAR could inhibit penile fibrosis and improve penile erection by promoting proliferation and migration of SC and secretion of NGF to promote nerve repair and regeneration. Our study provides new insight into the therapeutic effect of ALCAR and preclinical evidence for a potential treatment strategy for NED. Disclosure Work supported by industry: no.

Acetyl-L-carnitine improves erectile function in bilateral cavernous nerve injury rats via promoting cavernous nerve regeneration.

Neurogenic erectile dysfunction (NED) caused by cavernous nerve (CN) injury is a typical complication after pelvic surgery, which lacks efficient treatments. Acetyl-L-carnitine (ALCAR) has been proven to promote nerve repair. To investigate the effect and potential mechanism of ALCAR in the treatment of NED. Thirty-two rats were randomly divided into bilateral CN injury (BCNI) group, BCNI + lower-dose ALCAR (50mg/kg/day) group, BCNI + higher-dose (100mg/kg/day) group, and sham-operated group. Erectile function was assessed 14 days after daily intraperitoneal injection of ALCAR or placebo. The penile tissues were gathered for subsequent histological and molecular biological analysis. Rat Schwann cell (SC) line S16 was used to verify the mechanism of ALCAR in vitro. We found that the erectile function of the rats in the BCNI group was severely impaired, which was improved considerably in both BCNI+ALCAR-LD and BCNI+ALCAR-HD groups. Also, we observed decreased smooth muscle and increased collagen content in the corpus cavernosum in the BCNI group. The expressions of fibrosis markers transforming growth factor-beta (TGF-β), connective tissue growth factor (CTGF), and Smad 2/3 were significantly up-regulated in the BCNI group. The above changes were alleviated after the administration of lower and higher-dose ALCAR. Meanwhile, the nitric oxide (NO)/cyclic guanosine monophosphate pathway (cGMP) was promoted and the Ras homolog gene family member A (RhoA)/Rho-associated protein kinase (ROCK) pathway was inhibited in the corpus cavernosum of BCNI rats after ALCAR treatment, accompanied by increased neuronal nitric oxide synthase (nNOS) and down-regulated tyrosine hydroxylase (TH). In vitro, ALCAR promoted the migration and proliferation of SC and increased the expression of 22-kD peripheral myelin protein and nerve growth factor (NGF). Further, rats treated with ALCAR had high expression of ATF3 and S100 in the distal nerve tissues of the CN extrusion site. ALCAR could promote nerve repair and regeneration, inhibit penile fibrosis, and improve penile erection by promoting the proliferation and migration of SC and the secretion of NGF. Our study confirms that ALCAR may be a potential treatment strategy for NED.

Lycium barbarum polysaccharides improves erectile function through suppression of inflammation and apoptosis in rats with bilateral cavernous nerve injury

ABSTRACT Introduction Neurogenic erectile dysfunction (NED) responds poorly to phosphodiesterase 5 (PDE5) inhibitors. Lycium barbarum polysaccharide (LBP) has broad-spectrum anti-inflammatory and anti-apoptotic effects, but whether it improves the erectile function of neuropathic ED through the above-mentioned mechanism is unclear. Objective We intend to explore the role of LBP in the treatment of NED. Methods A rat model of bilateral cavernous nerve injury (BCNI) was constructed by crushing the CN 5 mm distal to the major pelvic ganglion (MPG). Tweety-four BCNI rats were randomly divided into BCNI group, BCNI + LBP-LD (20 mg/kg/day) group and BCNI + LBP-HD (40 mg/kg/day) group, whereas another 8 age-matched rats formed the control group. Erectile function was evaluated by measuring intracavernosal pressure (ICP) and mean arterial pressure (MAP). The penile tissues, and MPG along with CN were collected from each group of rats for subsequent histological and molecular biological analysis. The rat Schwann cell line S16 was used for CCK-8 and wound healing assay, and the Schwann cell (SC) markers S100 and Pmp22, neuroinflammatory indicators IL-1β, TGF-β1, apoptosis-related proteins Caspase-3, Bax, Bcl-2 and nerve growth factor NGF were verified by PCR and WB. Results ICP/MAP indicated that the erectile function of rats in the BCNI model group was severely impaired, which was rescued in the BCNI+LBP-LD and BCNI+LBP-HD groups. Rats in the BCNI model group can highly express inflammatory indicators NF-kb, TNF-α and apoptosis-related proteins Bax, Caspase-3. TUNEL confirmed that the level of apoptosis in the penile tissue of BCNI rats increased. The above-mentioned penile inflammation and apoptosis changes can be inhibited by LBP. In addition, both immunofluorescence and WB confirmed that rats in the LBP-LD and LBP-HD groups highly expressed nNOS and down-regulated the expression of TH. In vitro experiments have shown that LBP can promote the proliferation and migration of SC, increase the expression of Pmp22 and NGF, and the inflammation and apoptosis-related proteins were down-regulated. Conclusion LBP can relieve the inflammation and apoptosis at the original site of nerve injury by promoting the proliferation and migration of SC, and secretion of NGF, thereby inhibiting the inflammation and apoptosis caused by hypoxia after denervation of penile tissue, and improving penile erection. Disclosure Work supported by industry: no.

Filtrated bone marrow-derived stem cell lysate may improve erectile function through nerve regeneration in a rat model of cavernous nerve injury

ABSTRACT Introduction Oral phosphodiesterase-5 (PDE5) inhibitors and intracavernous injection of prostaglandin E1 (PGE1) are common therapies for erectile dysfunction (ED) following radical prostatectomy (RP). However, these treatments are not always effective, and the development of a novel and effective therapy for ED following RP is required. Recently, mesenchymal stem cell (MSC) transplantation was shown to induce nerve regeneration and improves erectile function in a rat model of bilateral cavernous nerve injury (BCNI), a routinely used animal model of RP. Thus, MSCs are expected to be a novel therapy for ED following RP. However, the known risks of stem cell therapy include induction of embolization and immunoreactions. To avert these problems, we explored the suitability of using filtrated stem cell lysate, which contains secreted factors but does not contain MSCs. Objective In this study, we investigated the effects of filtrated bone marrow-derived stem cells lysate (BSCL) on erectile function in a rat model of BCNI. We also investigated the neuroregenerative effects of BSCL using the major pelvic ganglia (MPG) of rats. Methods Bone marrow-derived stem cells were obtained from Wistar/ST rats. After culturing two or three passages, these cells were collected and diluted with phosphate buffered saline (PBS). We obtained BSCL by freeze fracturing and filtrating them. Two experiments were performed. Experiment I: Eight-week-old male Wistar/ST rats were divided into three groups: Sham+PBS (n=8), BCNI+PBS (n=8), and BCNI+BSCL (n=8). Rats in the Sham+PBS group underwent sham surgery as a control. Rats in the BCNI+PBS group underwent complete BCNI surgery. After both surgeries, PBS was injected into the corpus cavernosum. Rats in the BCNI+BSCL group also underwent BCNI surgery, but after surgery, BSCL was injected into the corpus cavernosum. After 4 weeks, erectile function was evaluated by measuring intracavernosal pressure (ICP)/mean arterial pressure (MAP) under stimulation of the cavernous nerve. Experiment II: the MPG were dissected from ten-week-old male Wistar/ST rats. The MPG were placed on Matrigel and incubated with PBS (n=4), BSCL (n=4), or vascular endothelial growth factor (VEGF) (n=4), which was used as a positive control. After 48 h, neurite outgrowth was measured using a z-stack microscope. Results ICP/MAP in the BCNI+PBS group was significantly lower than that in the Sham+PBS group (P&amp;lt;0.05), and ICP/MAP in the BCNI+BSCL group was significantly higher than that in the BCNI+PBS group (P&amp;lt;0.05). MPG treated with BSCL and VEGF had longer neurite outgrowth than those treated with PBS. Conclusions BSCL treatment improved erectile function in the BCNI rat model of RP. BSCL also exhibited neuroregenerative effects on rat MPG. This suggests that BSCL may regenerate a crushed cavernous nerve, which results in improved erectile function. Our findings suggest that BSCL may be a new approach for post-RP ED. Further study should be conducted to identify the active factors in BSCL. Disclosure Work supported by industry: no.

Tumor Necrosis Factor- α (TNF-α) and Rho-Associated Kinase 2 (ROCK2) Inhibitors Improve Erectile Function in a Bilateral Cavernous Nerve Injury Rat Model

ABSTRACT Introduction Prior studies suggest Rho-kinase (ROCK) upregulation and pro-inflammatory cytokines such as Tumor Necrosis Factor-α as causative factors of axonal degeneration, neuropathy, and penile fibrosis following peripheral cavernous nerve (CN) injury. We hypothesize, the combination therapy using inhibitors/antagonists of ROCK and TNF-α may prevent end-organ damage and neuroinflammation in the penis. The current study was designed to investigate selective inhibition of ROCK2 (SAR407899) and TNF-α (Infliximab) in a pre-clinical model of post-radical prostatectomy ED to prevent penile neuroinflammation, fibrosis, and preserve penile hemodynamics. Objective This study investigates the effect of FDA approved TNF- α inhibitor (Infliximab) in combination with ROCK2 selective inhibitor (SAR407899) on erectile function restoration in the bilateral crush nerve injury (BCNI) rat model. Methods Sprague Dawley rats were randomized to undergo either sham (6) or BCNI (28). The BCNI rats were further randomly stratified to receive saline (6), Infliximab (5 mg/kg once weekly) (6), low dose of SAR407899 (1.5 mg/kg QD) (5), high dose of SAR407899 (3 mg/kg QD) (5), or combination treatment SAR407899 (1.5 mg/kg QD) + Infliximab (5 mg/kg once weekly) (6). At 14 days after injury rats underwent cavernous nerve stimulation to determine erectile function. Penes were collected and immune infiltrate analyses were performed using multicolor flow-cytometry. Results Erectile response studies demonstrated that rodents treated with combination of Infliximab and SAR407899 had significantly improved (p&amp;lt;0.05) erectile function (ICP/MAP) at all voltages compared to BCNI alone (2V: 0.243 vs 0.051, 4V: 0.321 vs 0.0782, 6V: 0.456 vs 0.091, p &amp;lt;0.05). Peak ICP and total ICP were also significantly (p&amp;lt;0.05) improved compared to BCNI rats. Combination therapy, however, did not show a significant change compared to each treatment alone. Likewise, there was no significant difference between low and high dose SAR407899. Flow cytometry showed that Infliximab and SAR407899 significantly (p&amp;lt;0.05) decreased % of CD11b+ CD45+ monocytes in the penis. While combination therapy had no effect. The percent of CD11b+ MHCII+ CD45+ macrophages were increased (p&amp;lt;0.05) in the penes of rats treated with Infliximab and SAR407899 while combination therapy decreased penile macrophage population. We found no change in TNF- α+ monocytes and macrophages compared in penes from all groups investigated. Conclusions Our study suggests that treatment with commercially available TNF-α and ROCK2 inhibitors result in mitigation of erectile dysfunction in a BCNI rat model when administered at the time of nerve injury. The mechanism of the beneficial effects may involve reduction in macrophage population in the penis. Disclosure No

Dual Strategy with Intracavernosal Implantation of Adipose-derived Stem Cells and Daily Oral L-arginine Optimize Recovery of and Cavernosal Functions after Cavernous Nerve Injury in Rats

ABSTRACT Introduction Radical prostatectomy for prostate cancer has a significant potential role in inducing cavernous nerve injury (CNI). Increasing hypoxia and fibrosis induces destructive changes within cavernous vasculature. Adipose-derived stem cells (ADSCs) can maintain self-renewal and enhanced multi-differentiation potential through the release of a variety of paracrine factors and extracellular vesicles, allowing to repair damaged tissues. Considerable attention has increasingly been paid to their application in tissue engineering and regeneration. In addition, ADSCs can be obtained with a minimally invasive procedure and easily expanded in vitro with low immunogenicity. Nitric oxide (NO) is synthesized from the amino acid L-arginine by a family of enzymes, the nitric oxide synthases (NOS). Orally administered L-arginine enhances the erectile response presumably by stimulating NO production by the corporal tissues increasing cGMP production. Objective To evaluate the efficacy and tolerability of ADSCs and daily oral L-arginine alone or combination in treating rats with bilateral CNI (BCNI)-induced ED. Methods Adult Sprague-Dawley rats (n = 35) were divided into five groups: 1) control; 2) BCNI (4-wk); 3) BCNI+ADSCs (1 × 106 cells by intracavernosal injection); 4) BCNI+ L-arginine (4wk,10 mg/kg/day, oral); and 5) combination treatment with ADSCs and L-arginine in BCNI. Rats in the ADSCs treatment groups were subjected to intracavernosal injection after BCNI. In vivo erectile response was measured by CN stimulation. The relaxant and contractile responses were also detected on in vitro preparations of corpus cavernosum (CC) strips. The expression and localization of neuronal (nNOS), endothelial NOS (eNOS) as well as hypoxia (hypoxia-inducible factor-1α, HIF-1α), fibrosis [transforming growth factor-beta 1 (TGF- β1)]and apoptosis (Bax and Bcl-2) markers were determined using Western blotting and immunohistochemistry. The relative area of smooth muscle to collagen was measured using Masson trichrome staining. Results Rats with CNI had the lowest ICP/MAP ratio at 63% and total ICP at 55% for 7.5 V (P &amp;lt; .001 vs controls) as well as in vitro electrical field stimulation-induced relaxant response at 10% (P &amp;lt; .05 vs controls) and endothelium-dependent acetylcholine-caused relaxation at 56% (P &amp;lt; .001 vs controls). The combined treatment showed more significantly improved erectile and relaxant responses more effectively than ADSCs or L-arginine treatment alone. Increased in the expressions of nNOS, HIF-1α, Bax, and Bcl-2 in the BCNI group were restored by all treatment regiments. eNOS and TGF- β1 protein expressions were similar among groups. Mono-treatment partially improved decreased smooth muscle mass while combined therapy completely recovered. Conclusions The intracavernous injection of ADSCs and oral administration of L-arginine, and, importantly, their combination could effectively protect against hypoxia-induced neurotoxicity, via their increasing endothelial cell function and smooth muscle content, anti-inflammatory, anti-apoptotic, anti-fibrotic properties in addition to reversing erectile function in the BCNI rat model. HIF-1α overexpression by the inhibition of the apoptotic pathway with the Bcl-2 family may have protective effects on the survival of cavernosal neurons. Disclosure No

Relaxin-2 Prevents Erectile Dysfunction by Cavernous Nerve, Endothelial and Histopathological Protection Effects in Rats with Bilateral Cavernous Nerve Injury.

Cavernous nerve injury induced erectile dysfunction (ED) is a refractory complication with high incidence in person under radical prostatectomy. Studies have shown that relaxin-2 (RLX-2) plays a vital role of endothelial protection, vasodilation, anti-fibrosis and neuroprotection in a variety of diseases. However, whether penile cavernous erection can benefit from RLX-2 remains unknown. The purpose of the experiment was to explore the effects of RLX-2 on ED in the rat suffering with bilateral cavernous nerve injury (BCNI). The rats were divided into three groups: Sham group was underwent sham operation, BCNI+RLX group or BCNI group was underwent bilateral cavernous nerve crush and then randomly treated with RLX-2 (0.4 mg/kg/d) or saline by continuous administration using a subcutaneously implanted micro pump for 4 weeks respectively. Then, erectile function was evaluated by electrical stimulation of cavernous nerves. Cavernous nerves and penile tissues and were collected for histological evaluation. Erectile function of rats with BCNI was partially improved after RLX-2 treatment. The BCNI group had lower expression of relaxin family peptide receptor (RXFP) 1, p-AKT/AKT, p-eNOS/eNOS ratios than sham operation rats, but RLX-2 could partially reversed these changes. Histologically, the BCNI+RLX group had a significant effect on preservation of neurofilament, neuronal glial antigen 2 of penile tissue and nNOS of cavernous nerves when compared with BCNI group. RLX-2 could inhibited the lever of BCNI induced corporal fibrosis and apoptosis via regulating TGFβ1-Smad2/3-CTGF pathway and the expression of Bax/Bcl-2 ratio, caspase3. RLX-2 could improve erectile function of BCNI rats by protecting cavernous nerve and endothelial function and suppressing corporal fibrosis and apoptosis via RXFP1 and AKT/eNOS pathway. Our findings may provide a promising treatment for refractory BCNI induced ED.

PD62-07 TUMOR NECROSIS FACTOR ALPHA (TNF- Α) INHIBITOR IMPROVED ERECTILE FUNCTION IN A BILATERAL CAVERNOUS NERVE INJURY RAT MODEL

You have accessJournal of UrologySexual Function/Dysfunction: Basic Research & Pathophysiology (PD62)1 Sep 2021PD62-07 TUMOR NECROSIS FACTOR ALPHA (TNF- Α) INHIBITOR IMPROVED ERECTILE FUNCTION IN A BILATERAL CAVERNOUS NERVE INJURY RAT MODEL James Liu, Hotaka Matsui, Kara Lombardo, Serkan Karakus, Arthur Burnett, and Trinity Bivalacqua James LiuJames Liu More articles by this author , Hotaka MatsuiHotaka Matsui More articles by this author , Kara LombardoKara Lombardo More articles by this author , Serkan KarakusSerkan Karakus More articles by this author , Arthur BurnettArthur Burnett More articles by this author , and Trinity BivalacquaTrinity Bivalacqua More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002099.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Erectile dysfunction (ED) after injury to the peripheral cavernous nerves (CN) is partially the result of an increase in neuroinflammation in the pelvic ganglia. Inflammatory cytokines have been implicated in axonal degeneration and neuropathy. This study examines the role of the inflammatory cytokine, tumor necrosis factor –alpha (TNF- α), after bilateral CN injury (BCNI) in vivo. We also investigate the effect of commercially available TNF- α inhibitors (Infliximab) on erectile function restoration in the BCNI rodent model. METHODS: Seventy male Sprague Dawley rats were randomized to undergo either BCNI or sham surgery. Sham rats major pelvic ganglion (MPG) were harvested after 48 hours, whereas the BCNI groups had MPG harvested at 6 hours, 12 hours, 24 hours, 48 hours, 7 days, or 14 days after BCNI. MPGs were analyzed by qPCR to evaluate gene expression of TNF-α, TNF- αR1, TNF- αR2 and markers of macrophages (CD11b and CD68). Western blot of MPGs were also performed to confirm these finding. Additionally, 25 Sprague Dawley rats were randomized to three groups: sham surgery, BCNI, or BCNI treated with TNF- α inhibitor (Infliximab) (5mg/kg weekly) for two weeks and then underwent cavernous nerve stimulation and erectile parameters were measured (peak ICP, ICP/MAP, and total ICP). RESULTS: BCNI significantly increased (p<0.05) gene and protein expression of TNF- α and its receptors (R1, R2) in rat MPGS, with peak levels were observed at 2 and 7 days, respectively. Corresponding macrophage markers (CD11b and CD68) were significantly increased (p<0.05) following BCNI with maximal expression at 48 hours. Erectile response studies demonstrated that rodents treated with TNF- α inhibitors had significantly improved (p<0.05) erectile function (ICP/MAP) at all voltages compared to BCNI alone (2V: 0.203 vs 0.057, 4V: 0.279 vs 0.081, 6V: 0.317 vs 0.166. Control 2V: 0.347, 4V: 0.414, 6V: 0.486). Peak ICP and total ICP were also significantly (p<0.05) improved compared to BCNI rats. CONCLUSIONS: TNF- α is a key inflammatory cytokine which contributes to neuroinflammation following BCNI. Treatment with commercially available TNF- α inhibitor results in mitigation of erectile dysfunction in a BCNI rat model when administered at the time of nerve injury. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e1074-e1075 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information James Liu More articles by this author Hotaka Matsui More articles by this author Kara Lombardo More articles by this author Serkan Karakus More articles by this author Arthur Burnett More articles by this author Trinity Bivalacqua More articles by this author Expand All Advertisement Loading ...

Administration of H2S improves erectile dysfunction by inhibiting phenotypic modulation of corpus cavernosum smooth muscle in bilateral cavernous nerve injury rats

Phenotypic modulation of Corpus Cavernosum Smooth Muscle Cells (CCSMCs) is an important step in the development and progression of bilateral cavernous nerve injury induced erectile dysfunction (BCNI-ED). To investigate the effect of exogenous hydrogen sulfide (H2S) on the phenotypic modulation of CCSMCs in BCNI-ED rats, a total of 18 male Sprague-Dawley rats were equally divided into 3 groups, including sham-operated (Sham) group, BCNI group and BCNI treated with NaHS (BCNI + NaHS) group. The treated group received intraperitoneal injection of NaHS (100 μmol kg−1day−1) for 4 weeks starting day 1 postoperatively. Erectile function was measured by the ratio of intracavernous pressure (ICP)/mean arterial pressure (MAP), and relevant tissues were harvested for Immunohistochemistry, Hematoxylin and eosin (H&E), Masson's trichrome staining, H2S fluorescent probe WSP-1 and Western blot. The primary CCSMCs were isolated and pretreatment with NaHS before exposed to PDGF-BB (platelet-derived growth factor). Relative expression mRNA and protein of phenotypic biomarkers, RhoA, ROCK-1 and cell cycle proteins were detected. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), 3-mercaptopyruvate sulfurtransferase (3-MST) and H2S levels in penile tissue was significantly decreased in the BCNI group compared with the Sham group. Compared with the BCNI group, administration of NaHS significantly increased the ratio of ICP/MAP, ratio of smooth muscle to collagen, expressions of a-SMA, calponin and decreased the expression of OPN, collagen-I, RhoA, ROCK1 in the penile tissue. PDGF-BB-treated CCSMCs exhibited higher expression of OPN, RhoA, ROCK1, and lower α-SMA, calponin, which were attenuated by NaHS pretreatment. NaHS suppressed RhoA/ROCK activity and decreased the expression of CDK2, Cyclin E1, while increased the expression of P27kip1 induced by PDGF-BB in CCSMCs. Taken together, this study indicated that exogenous H2S inhibited the phenotypic modulation of CCSMCs by suppressing RhoA/ROCK1 signaling and affecting its downstream factor, CDK2, Cyclin E1, P27kip1, thereby improved BCNI rat erectile function.

Implantation of Skin-Derived Precursors Derived Schwann Cells Improves Erectile Function in a Bilateral Cavernous Nerve Injury Rat Model

Objectives: In this study, we used skin-derived precursors derived Schwann cells (SKP-SCs) as a treatment to alleviate the erectile dysfunction resulting from bilateral cavernous nerve injury (BCNI) in rat models. And the effect was evaluated 2 weeks after treatment. Materials and Methods: SKP-SCs and primary Schwann cells(SCs) were extracted and incubated from 4-week-old male Sprague Dawley(SD) rats. The cells were used to co-culture with PC12 cells for measurement of neurites growth. Conditional medium of them was used for chemotaxis of M0 macrophages and ELISA assays. 24 male SD rats (10-week old) were randomly divided into 4 groups (n=6). One group was sham group and the other 3 groups underwent BCNI. The groups undergoing BCNI were treated with PBS, SKP-SCs, primary SCs respectively. We evaluated the therapeutic effects on BCNI rats by measuring the erectile function, histologic analysis and western blotting of penis and MPG tissues. Results: Treatment with SKP-SCs significantly restored the erectile function, accelerated the recovery of endothelial and smooth muscle tissues in the penis and promoted the nerve repair process. Expression of p-smad2 and p-smad3 decreased after treatment, indicating fibrosis in the corpus cavernosum was significantly blocked. Conclusion: Implantation of SKP-SCs is an effective and promising therapy for erectile dysfunction induced by BCNI. Funding Statement: Support of research grants from National Natural Science Foundation of China (No. 81971378) and Science and Technology Program of Guangzhou (No. 201903010027). Declaration of Interests: We claim that no support or financial relationships have been received from any organization that may have an interest in the submitted work, and there are no other relationships or activities that could appear to have influenced the submitted work. Ethics Approval Statement: The present study has been reviewed and permitted by the Institutional Animal Care and Use Subcommittee of the Third Affiliated Hospital of Sun Yat-sen University.

Stem Cell/Oxygen-Releasing Microparticle Enhances Erectile Function in a Cavernous Nerve Injury Model.

Erectile dysfunction caused by damage to the cavernous nerve is a common complication of radical prostatectomy for patients with localized prostate cancer. Various studies have investigated repair of damaged tissue and prevention of fibrosis in the corpus cavernosum using stem cell therapy. However, stem cell therapy has limitations, including insufficient nutrient and oxygen supply to transplanted stem cells. This study investigated whether stem cell/oxygen-releasing hollow microparticles (HPs) had therapeutic effect on erectile dysfunction in a rat model of bilateral cavernous nerve injury (BCNI). Therapeutic effects were observed in the BCNI model at 1, 2, and 4 weeks postcavernous nerve injury. Erectile function further improved after treatment with stem cell/oxygen-releasing HP system compared with treatment with only stem cells at 4 weeks. Stem cell/oxygen-releasing HP system increased cyclic guanosine monophosphate (cGMP) level and neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), α-smooth muscle actin (α-SMA), and muscarinic acetylcholine receptor 3 (M3) expression while decreasing fibrosis and apoptosis in the corpus cavernosum. Our results clearly show that stem cell survival increases around transplanted stem cell/oxygen-releasing hybrid system site. Taken together, an oxygen-releasing HP system supported prolonged stem cell survival, sustaining the paracrine effect of the stem cells, and consequently enhancing erectile function. These findings show promise with regard to prolonged stem cell survival in stem cell applications for various diseases and types of tissue damage. Impact statement In this study, we used an oxygen-releasing hollow microparticles (HPs) system with stem cells to attempt to overcome certain limitations of stem cell therapy, including insufficient nutrient and oxygen supplies for transplanted stem cells. Our results demonstrated that a stem cell/oxygen-releasing HP hybrid system could further improve erectile function, cyclic guanosine monophosphate (cGMP) level, and NOS level in a bilateral cavernous nerve injury rat model through prolonged stem cell survival. Our data suggest that a stem cell/oxygen-releasing HP system is a promising clinical treatment option for postprostatectomy erectile dysfunction. Furthermore, this system may be relevant in different disease therapies and regenerative medicine.

Icariside II facilitates the differentiation of ADSCs to schwann cells and restores erectile dysfunction through regulation of miR-33/GDNF axis

BackgroundAdipose derived stem cells (ADSCs) have the property to differentiate into neuron-like cells, which may provide a novel insight for the restoration of erectile dysfunction (ED) mainly induced by cavernous nerve injury. Icariside II (ICA II) has been reported to play a key role in the regulation of erectile function via stimulating the differentiation of ADSCs to Schwann Cells (SCs). However, the function and molecular mechanisms of ICA II in ED remains to be further clarified. MethodsThe expression of S100, P75, GDNF and miR-33 was detected by qRT-PCR. And the relative proteins expression was determined by western blot. Cell viability was measured by Cell Counting Kit-8 (CCK-8) assay. Bioinformatics, luciferase reporter and RNA immunoprecipitation (RIP) assays were performed to verify the interaction between miR-33 and GDNF. Intracavernosal pressure (ICP), the ratio of ICP and mean arterial pressure (MAP), as well as nNOS expression were examined to evaluate the erectile function of SD rats with bilateral cavernous nerve injury (BCNI). ResultsICA II and miR-33 respectively promoted and inhibited the differentiation of ADSCs to SCs. MiR-33 could negatively regulate P75 and GDNF expression. ICA II exerted promotion effects on differentiation of ADSCs to SCs via regulating miR-33. GDNF was identified to be a target of miR-33. MiR-33 overexpression abrogated the stimulatory effect of ICA II on ADSCs' differentiation, which was blocked by GDNF overexpression. treated with ICA II recovered the erectile function of BCNI model rats through regulation of miR-33. ConclusionICA II contributed to the differentiation of ADSCs to SCs viamiR-33/GDNF axis, contributing to the recovery of erectile function in BCNI rats.