Intravenously engrafted human multilineage-differentiating stress-enduring (Muse) cells rescue erectile function after rat cavernous nerve injury.

OBJECTIVE: To evaluate the effect of intravenous administration of human multilineage-differentiating stress-enduring (Muse) cells on rat postoperative erectile dysfunction (ED) with cavernous nerve (CN) injury without an immunosuppressant. MATERIALS AND METHODS: Male Sprague-Dawley rats were randomised into three groups after CN crush injury. Either human-Muse cells, non-Muse mesenchymal stem cells (MSCs) (both 1.0 × 105 cells), or vehicle was infused intravenously at 3 h after CN injury without immunosuppressant. Erectile function was assessed by measuring intracavernous pressure (ICP) and arterial pressure (AP) during pelvic nerve electrostimulation 28 days after surgery. At 48 h and 28 days after intravenous infusion of Muse cells, the homing of Muse cells and non-Muse MSCs was evaluated in the major pelvic ganglion (MPG) after CN injury. In addition, expressions of C-X-C motif chemokine ligand (Cxcl12) and glial cell line-derived neurotrophic factor (Gdnf) in the MPG were examined by real-time polymerase chain reaction. Statistical analyses and comparisons among groups were performed using one-way analysis of variance followed by the Tukey test for parametric data and Kruskal-Wallis test followed by the Dunn-Bonferroni test for non-parametric data. RESULTS: The mean (SEM) ICP/AP values at 28 days were 0.51 (0.02) in the Muse cell group, 0.37 (0.03) in the non-Muse MSC group, and 0.36 (0.04) in the vehicle group, showing a significant positive response in the Muse cell group compared with the non-Muse and vehicle groups (P = 0.013 and P = 0.010, respectively). In the MPG, Muse cells were observed to be engrafted at 48 h and expressed Schwann cell markers S100 (~46%) and glial fibrillary acidic protein (~24%) at 28 days, while non-Muse MSCs were basically not engrafted at 48 h. Higher gene expression of Cxcl12 (P = 0.048) and Gdnf (P = 0.040) was found in the MPG of the Muse group than in the vehicle group 48 h after infusion. CONCLUSION: Intravenously engrafted human Muse cells recovered rat erectile function after CN injury in a rat model possibly by upregulating Cxcl12 and Gdnf.

BMP4 and GREM1 are targets of SHH signaling and downstream regulators of collagen in the penis.

BACKGROUND: Cavernous nerve (CN) injury, caused by prostatectomy and diabetes, initiates a remodeling process (smooth muscle apoptosis and increased collagen) in the corpora cavernosa of the penis of patients and animal models that is an underlying cause of erectile dysfunction (ED), and the Sonic hedgehog (SHH) pathway plays an essential role in the response of the penis to denervation, as collagen increases with SHH inhibition and decreases with SHH treatment. AIM: We examined if part of the mechanism of how SHH prevents penile remodeling and increased collagen with CN injury involves bone morphogenetic protein 4 (BMP4) and gremlin1 (GREM1) and examined the relationship between SHH, BMP4, GREM1, and collagen in penis of ED patients and rat models of CN injury, SHH inhibition, and SHH, BMP4, and GREM1 treatment. METHODS: Corpora cavernosa of Peyronie's disease (control), prostatectomy, and diabetic ED patients were obtained (N = 30). Adult Sprague Dawley rats (n = 90) underwent (1) CN crush (1-7 days) or sham surgery; (2) CN injury and BMP4, GREM1, or mouse serum albumin (control) treatment via Affi-Gel beads or peptide amphiphile (PA) for 14 days; (3) 5E1 SHH inhibitor, IgG, or phosphate-buffered saline (control) treatment for 2 to 4 days; or (4) CN crush with mouse serum albumin or SHH for 9 days. OUTCOMES: Immunohistochemical and Western analysis for BMP4 and GREM1, and collagen analysis by hydroxyproline and trichrome stain were performed. RESULTS: BMP4 and GREM1 proteins were identified in corpora cavernosa smooth muscle of prostatectomy, diabetic, and Peyronie's patients, and in rat smooth muscle, sympathetic nerve fibers, perineurium, blood vessels, and urethra. Collagen decreased 25.4% in rats with CN injury and BMP4 treatment (P = .02) and increased 61.3% with CN injury and GREM1 treatment (P = .005). Trichrome stain showed increased collagen in rats treated with GREM1. Western analysis identified increased BMP4 and GREM1 in corpora cavernosa of prostatectomy and diabetic patients, and after CN injury (1-2 days) in our rat model. Localization of BMP4 and GREM1 changed with SHH inhibition. SHH treatment increased the monomer form of BMP4 and GREM1, altering their range of signaling. CLINICAL IMPLICATIONS: A better understanding of penile remodeling and how fibrosis occurs with loss of innervation is essential for development of novel ED therapies. STRENGTHS AND LIMITATIONS: The relationship between SHH, BMP4, GREM1, and collagen is complex in the penis. CONCLUSION: BMP4 and GREM1 are downstream targets of SHH that impact collagen and may be useful in collaboration with SHH to prevent penile remodeling and ED.

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

BACKGROUND: 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. AIM: 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. METHODS: 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 3 weeks postoperatively. In addition, the fibrotic model of CCSMCs was used to further explore the mechanism of CSD peptide action in vitro. OUTCOMES: The assessments of EF, SMC/collagen ratio, α-smooth muscle actin, caveolin-1 (CAV1), and profibrotic indicators expressions were conducted. RESULTS: 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)-ß1/Smad2 activity. However, impaired EF, activated CC fibrosis, and Smad2 signaling were attenuated after 3 weeks of CSD peptide treatment in BCNI rats. In vitro, TGF-ß1-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. CLINICAL IMPLICATIONS: 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. STRENGTHS AND LIMITATIONS: 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. CONCLUSION: Administration of CSD peptide significantly retarded collagen synthesis in CCSMCs, limited CC fibrosis, and prevented ED via confrontation of TGF-ß1/Smad signaling in BCNI rats.

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.

[Application of Rat Model of Neurotic Erectile Dysfunction in Experiment].

Erectile dysfunction (ED) refers to the persistent inability to achieve and/or maintain a sufficient erection of the penis to obtain a satisfactory sexual life,which affects the quality of life of the patients and their sexual partners.To decipher the pathophysiological mechanism of ED,researchers have established a variety of animal models and achieved a series of progress.The cavernous nerve (CN) of rodents,anatomically similar to that of humans,is cost-effective,thick,and easy to be identified,which has gradually become the mainstream of animal models.In this paper,we reviewed the modeling methods of the neurological ED caused by bilateral CN injury in rats in recent years,summarized the model evaluation indicators,and discussed the application and progress of ED models in basic experimental research.

An update on the current status and future prospects of erectile dysfunction following radical prostatectomy.

BACKGROUND: Radical prostatectomy (RP) and radiation treatment are standard options for localized prostate cancer. Even though nerve-sparing techniques have been increasingly utilized in RP, erectile dysfunction (ED) due to neuropraxia remains a frequent complication. Erectile function recovery rates after RP remain unsatisfactory, and many men still suffer despite the availability of various therapies. OBJECTIVE: This systematic review aims to summarize the current treatments for post-RP-ED, assess the underlying pathological mechanisms, and emphasize promising therapeutic strategies based on the evidence from basic research. METHOD: Evaluation and review of articles on the relevant topic published between 2010 and 2021, which are indexed and listed in the PubMed database. RESULTS: Phosphodiesterase type 5 inhibitors, intracavernosal and intraurethral injections, vacuum erection devices, pelvic muscle training, and surgical procedures are utilized for penile rehabilitation. Clinical trials evaluating the efficacy of erectogenic drugs in this setting are conflicting and far from being conclusive. The use of androgen deprivation therapy in certain scenarios after RP further exacerbates the already problematic situation and emphasizes the need for effective treatment strategies. CONCLUSION: This article is a detailed overview focusing on the pathophysiology and mechanism of the nerve injury developed during RP and a compilation of various strategies to induce cavernous nerve regeneration to improve erectile function (EF). These strategies include stem cell therapy, gene therapy, growth factors, low-intensity extracorporeal shockwave therapy, immunophilins, and various pharmacological approaches that have induced improvements in EF in experimental models of cavernous nerve injury. Many of the mentioned strategies can improve EF following RP if transformed into clinically applicable safe, and effective techniques with reproducible outcomes.

Erectile dysfunction resulting from pelvic surgery is associated with changes in cavernosal gene expression indicative of cavernous nerve injury.

Pelvic surgery, even without direct cavernous nerve injury, carries a high risk of post-operative erectile dysfunction. The present studies were aimed at identifying molecular mechanisms by which pelvic surgery results in erectile dysfunction. As a model of pelvic surgery, male Sprague-Dawley rats underwent pelvic laparotomy, avoiding direct cavernous nerve injury. A second group of animals, serving as a model of direct cavernous nerve injury, underwent bilateral transection of the cavernous nerve. Cavernosometry demonstrated, that even in the absence of direct nerve injury, the pelvic surgery model exhibited significant erectile dysfunction 3 days post-operatively. Gene expression profiling also demonstrated that even in this animal model of nerve-sparing pelvic surgery, the profile of differentially expressed genes in cavernosal tissue was indicative of cavernous nerve injury. In addition, although 6 hr after surgery there were significant changes in circulating cytokine/chemokine levels, an inflammatory response in the major pelvic ganglion, cavernous nerve and cavernosal tissue was only observed 3 days post-surgery. Our results validate a rat model of pelvic surgery exhibiting erectile dysfunction and suggest systemic release of cytokines/chemokines following surgical trauma might mediate a pathological inflammatory response in tissues distal to the site of surgical trauma, indirectly resulting in cavernous nerve injury and erectile dysfunction.

Dual effect of chitosan activated platelet rich plasma (cPRP) improved erectile function after cavernous nerve injury.

BACKGROUND: The intracavernosal (IC) injection of chitosan activated platelet rich plasma (cPRP) has shown to improve the erectile dysfunction in cavernous nerve injury rat model. However, the action target of PRP in improving neurogenic erectile dysfunction remains unclear. We aimed to determine the effect of cPRP action at early stage that further mediates its effect on erectile function (EF) recovery in the bilateral cavernous nerve crushing (BCNC) injury rat model. METHODS: Fifty-four rats were randomly divided into two equal groups: intracavernosal ( IC) injection of saline after BCNC (group 1) and IC injection of cPRP after BCNC (group 2). Five animals in each group were euthanized at 3, 7 and 14 day (d) post-injection, and the tissues were harvested to conduct transmission electron microscopy and histological assays. Six animals in each group were used to determine the recovery of EF at 14 and 28 d post-injury. RESULTS: IC injections of cPRP increased all EF parameters at 28 d and 14 d post-injury (p < 0.05). cPRP injections simultaneously prevented the loss of neuronal nitric oxide synthase-positive neurons (p < 0.05) and nerve fibers (p < 0.05) in the major pelvic ganglion and cavernous nerve (CN), respectively, compared with saline injections. This simultaneous accelerated the regeneration of myelinated axons of the CN, reduced apoptosis, and enhanced the proliferation of the corporal smooth muscle cells at an earlier stage. CONCLUSION: These results suggest that the application of cPRP was beneficial to restore EF via neuroprotective and tissue-protective effects at early stage.

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-ß-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.

Caspase Signaling in ED Patients and Animal Models.

BACKGROUND: Current treatments for erectile dysfunction (ED) are ineffective in prostatectomy and diabetic patients due to cavernous nerve (CN) injury, which causes smooth muscle apoptosis, penile remodeling, and ED. Apoptosis can occur via the intrinsic (caspase 9) or extrinsic (caspase 8) pathway. AIM: We examined the mechanism of how apoptosis occurs in ED patients and CN injury rat models to determine points of intervention for therapy development. METHODS AND OUTCOMES: Immunohistochemical and western analyses for caspase 3-cleaved, caspase-8 and caspase-9 (pro and active forms) were performed in corpora cavernosal tissue from Peyronie's, prostatectomy and diabetic ED patients (n = 33), penis from adult Sprague Dawley rats that underwent CN crush (n = 24), BB/WOR diabetic and control rats (n = 8), and aged rats (n = 9). RESULTS: Caspase 3-cleaved was observed in corpora cavernosa from Peyronie's patients and at higher abundance in prostatectomy and diabetic tissues. Apoptosis takes place primarily through the extrinsic (caspase 8) pathway in penis tissue of ED patients. In the CN crushed rat, caspase 3-cleaved was abundant from 1-9 days after injury, and apoptosis takes place primarily via the intrinsic (caspase 9) pathway. Caspase 9 was first observed and most abundant in a layer under the tunica, and after several days was observed in the lining of and between the sinuses of the corpora cavernosa. Caspase 8 was initially observed at low abundance in the rat corpora cavernosa and was not observed at later time points after CN injury. Aged and diabetic rat penis primarily exhibited intrinsic mechanisms, with diabetic rats also exhibiting mild extrinsic activation. CLINICAL TRANSLATION: Knowing how and when to intervene to prevent the apoptotic response most effectively is critical for the development of drugs to prevent ED, morphological remodeling of the corpora cavernosa, and thus, disease management. STRENGTHS AND LIMITATIONS: Animal models may diverge from the signaling mechanisms observed in ED patients. While the rat utilizes primarily caspase 9, there is a significant flux through caspase 8 early on, making it a reasonable model, as long as the timing of apoptosis is considered after CN injury. CONCLUSIONS: Apoptosis takes place primarily through the extrinsic caspase 8 dependent pathway in ED patients and via the intrinsic caspase 9 dependent pathway in commonly used CN crush ED models. This is an important consideration for study design and interpretation that must be taken into account for therapy development and testing of drugs, and our therapeutic targets should ideally inhibit both apoptotic mechanisms. Martin S, Harrington DA, Ohlander S, et al. Caspase Signaling in ED Patients and Animal Models. J Sex Med 2021;18:711-722.

Oral Administration of the p75 Neurotrophin Receptor Modulator, LM11A-31, Improves Erectile Function in a Mouse Model of Cavernous Nerve Injury.

BACKGROUND: Radical prostatectomy for prostate cancer can not only induce cavernous nerve injury (CNI), but also causes cavernous hypoxia and cavernous structural changes, which lead to a poor response to phosphodiesterase 5 inhibitors. AIM: To investigate the therapeutic effect of oral administration of LM11A-31, a small molecule p75 neurotrophin receptor (p75NTR) ligand and proNGF antagonist, in a mouse model of bilateral CNI, which mimics nerve injury-induced erectile dysfunction after radical prostatectomy. METHODS: 8-week-old male C57BL/6 mice were divided into sham operation and CNI groups. Each group was divided into 2 subgroups: phosphate-buffered saline and LM11A-31 (50 mg/kg/day) being administered once daily starting 3 days before CNI via oral gavage. 2 weeks after CNI, we measured erectile function by electrical stimulation of the bilateral cavernous nerve. The penis was harvested for histologic examination and Western blot analysis. The major pelvic ganglia was harvested and cultured for assays of ex vivo neurite outgrowth. OUTCOMES: Intracavernous pressure, neurovascular regeneration in the penis, in vivo or ex vivo functional evaluation, and cell survival signaling were measured. RESULTS: Erectile function was decreased in the CNI group (44% of the sham operation group), while administration of LM11A-31 led to a significant improvement of erectile function (70% of the sham operation group) in association with increased neurovascular content, including cavernous endothelial cells, pericytes, and neuronal processes. Immunohistochemical and Western blot analyses showed significantly increased p75NTR expression in the dorsal nerve of CNI mice, which was attenuated by LM11A-31 treatment. Protein expression of active PI3K, AKT, and endothelial nitric oxide synthase was increased, and cell death and c-Jun N-terminal kinase signaling was significantly attenuated after LM11A-31 treatment. Furthermore, LM11A-31 promoted neurite sprouting in cultured major pelvic ganglia after lipopolysaccharide exposure. CLINICAL IMPLICATIONS: LM11A-31 may be used as a strategy to treat erectile dysfunction after radical prostatectomy or in men with neurovascular diseases. STRENGTHS & LIMITATIONS: Unlike biological therapeutics, such as proteins, gene therapies, or stem cells, the clinical application of LM11A-31 would likely be relatively less complex and low cost. Our study has some limitations. Future studies will assess the optimal dosing and duration of the compound. Given its plasma half-life of approximately 1 hour, it is possible that dosing more than once per day will provide added efficacy. CONCLUSION: Specific inhibition of the proNGF-p75NTR degenerative signaling via oral administration of LM11A-31 represents a novel therapeutic strategy for erectile dysfunction induced by nerve injury. Yin GN, Ock J, Limanjaya A, et al. Oral Administration of the p75 Neurotrophin Receptor Modulator, LM11A-31, Improves Erectile Function in a Mouse Model of Cavernous Nerve Injury. J Sex Med 2021;18:17-28.

Peptide amphiphile nanofiber hydrogel delivery of Sonic hedgehog protein to the penis and cavernous nerve suppresses intrinsic and extrinsic apoptotic signaling mechanisms, which are an underlying cause of erectile dysfunction.

Erectile dysfunction (ED) is a common and debilitating condition with high impact on quality of life. An underlying cause of ED is apoptosis of penile smooth muscle, which occurs with cavernous nerve injury, in prostatectomy, diabetic and aging patients. We are developing peptide amphiphile (PA) nanofiber hydrogels as an in vivo delivery vehicle for Sonic hedgehog protein to the penis and cavernous nerve to prevent the apoptotic response. We examine two important aspects required for clinical application of the biomaterials: if SHH PA suppresses intrinsic (caspase 9) and extrinsic (caspase 8) apoptotic mechanisms, and if suppressing one apoptotic mechanism forces apoptosis to occur via a different mechanism. We show that SHH PA suppresses both caspase 9 and 8 apoptotic mechanisms, and suppressing caspase 9 did not shift signaling to caspase 8. SHH PA has significant clinical potential as a preventative ED therapy, by management of intrinsic and extrinsic apoptotic mechanisms.

Enhanced effects of salidroside on erectile function and corpora cavernosa autophagy in a cavernous nerve injury rat model.

We explored the efficacy and mechanisms of salidroside treatment for erectile dysfunction induced by bilateral cavernous nerve injury (BCNI). Forty male rats were divided into four groups as follows: sham (cavernous nerves exposed only) (S); BCNI (M); BCNI + rapamycin (M + rapamycin); and BCNI + salidroside (M + salidroside). Erectile function in the rats was measured by intracavernosal pressure. Penile tissue was harvested for transmission electron microscopy, immunohistochemistry, immunofluorescence, Masson's trichrome staining, haematoxylin-eosin staining, TdT-mediated dUTP Nick End Labeling and western blotting. The M group exhibited a decrease in erectile responses and increased apoptosis and fibrosis compared to these in the S group. Meanwhile, nerve content and the penile atrophy index were also decreased in the M group. Treatment with salidroside and rapamycin for 3 weeks partially restored erectile function and significantly attenuated corporal apoptosis, fibrosis, nerve content and penile atrophy in the M group. Moreover, the autophagy level was further enhanced in the M + salidroside group, which was the same as that in the positive observation group (M + rapamycin). Salidroside treatment not only improved erectile function in rats with BCNI, but also inhibited apoptosis and fibrosis and ameliorated the loss of nerve content and endothelial and corpus cavernosum smooth muscle cells by promoting protective autophagy.

Comparative study of different transplantation methods of adipose tissue-derived stem cells in the treatment of erectile dysfunction caused by cavernous nerve injury.

We aimed to compare intracavernosal injection (ICI), tail vein injection (IV), and periprostatic injection (PPI) of adipose-derived stem cells (ADSCs) for their ability to improve erectile function in cavernous nerve injury-induced erectile dysfunction (CNIED) rats and to explore the possible mechanism. Eighty-four male SD rats were divided into the sham group (n = 6), BCNI group (bilateral CN crush injury, n = 6), PBS-ICI group (n = 6), PBS-IV group (n = 6), PBS-PPI group (n = 6), ADSC-ICI group (n = 18), ADSC-IV group (n = 18) and ADSC-PPI group (n = 18). ADSCs were labelled with 5-ethynyl-2'-deoxyuridine (EdU), and six rats each in the ADSC-ICI group, ADSC-IV group, and ADSC-PPI group were sacrificed 2, 7, and 28 days after injection. EdU-labelled ADSCs were tracked by immunofluorescence staining. The intracavernosal pressure (ICP)/mean arterial pressure (MAP) ratio, neuronal nitric oxide synthase (nNOS)-positive nerve fibres in the dorsal penile nerve and the smooth muscle/collagen ratio in the cavernosum between groups were also evaluated. ADSCs can significantly improve erectile function through ICI or IV. The two are similar in efficacy and superior to PPI. The mechanism may be that after CN injury, ADSCs are recruited to around the MPG and secrete a variety of neurotrophic factors that promote the repair of the CN, thereby improving erectile function.

[Hongjing-1 Recipe maintains the expression level of Connexin43 in the penile tissue of SD rats with bilateral cavernous nerve injury].

OBJECTIVE: To explore the effects of Hongjing-1 Recipe (HJ-1) on erectile function and the expression of the gap junction protein Connexin43 (Cx43) in the penile tissue in male rats with bilateral cavernous nerve injury (BCNI). METHODS: Fifty male SD rats were randomly divided into five groups of an equal number: sham operation, BCNI model control, and low-, medium- and high-dose HJ-1. The BCNI model was made in the latter four groups by clamping the bilateral cavernous nerves with hemostatic forceps. Three days after modeling, the rats in the sham operation and BCNI model control groups were treated intragastrically with pure water and those in the low-, medium- and high-dose HJ-1 groups with HJ-1 at 2.835, 5.67 and 11.34 g/kg/d, respectively, all for 28 successive days. Then, the animals were subjected to intracavernous pressure (ICP) measurement for evaluation of their erectile function and immunofluorescence staining and Western blot for determination of the Cx43 level in the penile tissue. RESULTS: The BCNI model controls, compared with the rats in the sham operation group, showed a dramatically decreased ratio of maximum ICP to mean arterial pressure (mICP/MAP) (0.40 ± 0.04 vs 0.83 ± 0.10, P < 0.01) and that of total ICP to MAP (tICP/MAP) (21.89 ± 2.16 vs 50.27 ± 4.45, P < 0.01), as well as a down-regulated expression of Cx43 in the penile tissue (P < 0.01). In comparison with the rats in the BCNI model control group, those in the medium- and high-dose HJ-1 groups exhibited significantly increased ratios of mICP/MAP (0.54 ± 0.05, P < 0.05; 0.61 ± 0.06, P < 0.01) and tICP/MAP (31.20 ± 3.85, P < 0.01; 37.82 ± 4.17, P < 0.01) and up-regulated expression of Cx43 (P<0.05 and P<0.01). CONCLUSIONS: Hongjing-1 Recipe can effectively improve ED in rats with bilateral cavernous nerve injury, which may be attributed to its effect of maintaining the expression level of the gap junction protein Cx43 in the penile tissue.

[Stem cell therapy for erectile dysfunction: An update].

Erectile dysfunction (ED) is a common andrological disorder, and traditional oral drugs often fail to achieve satisfactory therapeutic effects. As a new field of biomedicine, stem cell therapy (SCT) has seen a significantly increasing number of researches on its treatment of ED in recent years. Preclinical animal models for the study of ED mainly include the models of diabetes mellitus-, aging-, cavernous nerve injury-, and Peyronie's disease-related ED. Previous studies indicated that SCT improved erectile function through paracrine and was more effective when combined with other therapies than used alone in restoring ED-induced pathological changes. Although clinical trials on SCT have partially proved its safety and effectiveness for the treatment of ED, they were still in the early stages and with relatively small sample sizes. This article summarizes the latest advances in the treatment of ED by SCT.

Pericyte-Derived Extracellular Vesicle-Mimetic Nanovesicles Restore Erectile Function by Enhancing Neurovascular Regeneration in a Mouse Model of Cavernous Nerve Injury.

BACKGROUND: Extracellular vesicle (EV)-mimetic nanovesicles (NVs) from embryonic stem cells have been observed to stimulate neurovascular regeneration in the streptozotocin-induced diabetic mouse. Pericytes play important roles in maintaining penile erection, yet no previous studies have explored the effects of pericyte-derived NVs (PC-NVs) in neurovascular regeneration in the context of erectile dysfunction. AIM: To investigate the potential effect of PC-NVs in neurovascular regeneration. METHODS: PC-NVs were isolated from mouse cavernous pericytes, and neurovascular regeneration was evaluated in an in vitro study. Twelve-week-old C57BL/6J mice were used to prepare cavernous nerve injury model. Erectile function evaluation, histologic examination of the penis, and Western blots were assessed 2 weeks after model creation and PC-NVs treatment. OUTCOMES: The main outcomes of this study are PC-NVs characterization, intracavernous pressure, neurovascular regeneration in the penis, and in vitro functional evaluation. RESULTS: The PC-NVs were extracted and characterized by cryotransmission electron microscopy and EV-positive (Alix, TSG101, CD81) and EV-negative (GM130) markers. In the in vivo studies, PC-NVs successfully improved erectile function in cavernous nerve injury mice (∼82% of control values). Immunofluorescence staining showed significant increases in pericytes, endothelial cell, and neuronal contents. In the in vitro studies, PC-NVs significantly increased mouse cavernous endothelial cells tube formation, Schwann cell migration, and dorsal root ganglion and major pelvic ganglion neurite sprouting. Finally, Western blot analysis revealed that PC-NVs upregulated cell survival signaling (Akt and eNOS) and induced the expression of neurotrophic factors (brain-derived neurotrophic factor, neurotrophin-3, and nerve growth factor). CLINICAL IMPLICATIONS: PC-NVs may be used as a strategy to treat erectile dysfunction after radical prostatectomy or in men with neurovascular diseases. STRENGTHS & LIMITATIONS: We evaluated the effect of PC-NVs in vitro and in a mouse nerve injury model, cavernous nerve injury. Additional studies are necessary to determine the detailed mechanisms of neurovascular improvement. Further study is needed to test whether PC-NVs are also effective when given weeks or months after nerve injury. CONCLUSION: PC-NVs significantly improved erectile function by enhancing neurovascular regeneration. Local treatment with PC-NVs may represent a promising therapeutic strategy for the treatment of neurovascular diseases. Yin GN, Park S-H, Ock J, et al. Pericyte-Derived Extracellular Vesicle-Mimetic Nanovesicles Restore Erectile Function by Enhancing Neurovascular Regeneration in a Mouse Model of Cavernous Nerve Injury. J Sex Med 2020;17:2118-2128.

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

BACKGROUND: Prostatic radiation therapy (RT) often causes erectile dysfunction (ED) and the mechanisms governing RT-induced ED are unclear with a lack of therapeutic strategies. AIM: To determine the effects of ex vivo RT on major pelvic ganglion (MPG) neuron survival, and neurite growth in whole vs dissociated culture. METHODS: MPGs were removed and irradiated (0 or 8 Gy) from male Sprague Dawley rats. For dissociated culture, MPG neurons were digested in collagenase/dispase and cultured on coverslips. Immunofluorescent staining for beta-tubulin III (TUBB3; neuron marker), neuronal nitric oxide synthase (nNOS; nitrergic marker), tyrosine hydroxylase (TH; sympathetic marker), and terminal deoxynucleotidyl transferase dUTP nick end labeling assessed neurite length, branching, autonomic neuron density, and apoptosis. For whole organ culture, MPGs were grown in Matrigel. Gene expression of apoptotic markers (caspase 1, 3), TUBB3, nNOS, TH, and Schwann cells (Sox10, Krox20, glial fibrillary acid protein) was measured in whole organ cultured MPGs by quantitative polymerase chain reaction. OUTCOMES: After 72 hours, neurite length, branching, autonomic neuron density, and apoptosis were assessed, and gene expression was measured. RESULTS: RT increased apoptosis in dissociated neurons measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (P < .001) and whole MPG culture via upregulation of caspase 3 gene expression (P < .05). Nitrergic neurons were markedly decreased in irradiated dissociated culture (P < .05), while nNOS gene expression was upregulated in irradiated whole organ culture (P < .05). The proportion of dissociated sympathetic neurons and whole organ TH gene expression remained unchanged after RT. Interestingly, RT dissociated neurites were 22% shorter than controls, while RT whole organ neurites were 15% longer than controls (P < .01). MPG Schwann cells markers (Sox10, Krox20) were elevated after RT in whole organ culture. CLINICAL TRANSLATION: Prostatic RT leads to increased neuronal cell death and less erectogenic nitrergic neurons contributing to ED. STRENGTHS & LIMITATIONS: The advantages of dissociated neuron culture include distinct neurites which are easily measured for apoptosis, length/branching, and specific neuron types. In contrast, whole MPG culture is advantageous as it contains all the supporting cells present in vivo. CONCLUSION: The 2 different culture methods demonstrated opposing neurite growth after RT indicating the importance of supporting cell network to promote pelvic neuron neuritogenesis and survival following RT. Randolph JT, Pak ES, Koontz BF, et al. Ex Vivo Radiation Leads to Opposing Neurite Growth in Whole Ganglia vs Dissociated Cultured Pelvic Neurons. J Sex Med 2020;17:1423-1433.

Dynamic Changes in Erectile Function and Histological Architecture After Intracorporal Injection of Human Placental Stem Cells in a Pelvic Neurovascular Injury Rat Model.

INTRODUCTION: The human placenta provides a bountiful and noncontroversial source of stem cells which have the potential for regeneration of injured tissue. These cells may restore erectile function after neurovascular tissue injury such as that seen in radical pelvic surgeries and pelvic trauma. AIM: To determine the effect of human placenta-derived stem cells on erectile function recovery and histological changes at various time points in a cavernous nerve injury rat model and to study the fate of injected stem cells throughout the regenerative process. METHODS: Human placental stem cells (PSCs) were dual labeled with monomeric Katushka far red fluorescent protein (mKATE)-renLUC using a lentivirus vector. A pelvic neurovascular injury-induced erectile dysfunction model was established in male, athymic rats by crushing the cavernous nerves and ligating the internal pudendal neurovascular bundles, bilaterally. At the time of defect creation, nonlabeled PSCs were injected into the corpus cavernosum at a concentration of 2.5 × 106 cells/0.2 mL. The phosphate-buffered saline-treated group served as the negative control group, and age-matched rats (age-matched controls) were used as the control group. Erectile function, histomorphological analyses, and Western blot were assessed at 1, 6, and 12 weeks after model creation. The distribution of implanted, dual-labeled PSCs was monitored using an in vivo imaging system (IVIS). Implanted cells were further tracked by detection of mKATE fluorescence in histological sections. MAIN OUTCOME MEASURE: The main outcome measure includes intracavernous pressure/mean arterial pressure ratio, neural, endothelial, smooth muscle cell regeneration, mKATE fluorescence, and IVIS imaging. RESULTS: The ratio of intracavernous pressure to mean arterial pressure significantly increased in PSC-injected rats compared with phosphate-buffered saline controls (P < 0.05) at the 6- and 12-week time points, reaching 72% and 68% of the age-matched control group, respectively. Immunofluorescence staining and Western blot analysis showed significant increases in markers of neurons (84.3%), endothelial cells (70.2%), and smooth muscle cells (70.3%) by 6 weeks in treatment groups compared with negative controls. These results were maintained through 12 weeks. IVIS analysis showed luminescence of implanted PSCs in the injected corpora immediately after injection and migration of cells to the sites of injury, including the incision site and periprostatic vasculature by day 1. mKATE fluorescence data revealed the presence of PSCs in the penile corpora and major pelvic ganglion at 1 and 3 days postoperatively. At 7 days, immunofluorescence of penile PSCs had disappeared and was diminished in the major pelvic ganglion. CLINICAL IMPLICATIONS: Placenta-derived stem cells may represent a future "off-the-shelf" treatment to mitigate against development of erectile dysfunction after radical prostatectomy or other forms of pelvic injury. STRENGTH & LIMITATIONS: Single dose injection of PSCs after injury resulted in maximal functional recovery and tissue regeneration at 6 weeks, and the results were maintained through 12 weeks. Strategies to optimize adult stem cell therapy might achieve more effective outcomes for human clinical trials. CONCLUSION: Human PSC therapy effectively restores the erectile tissue and function in this animal model. Thus, PSC therapy may provide an attractive modality to lessen the incidence of erectile dysfunction after pelvic neurovascular injury. Further improvement in tissue regeneration and functional recovery may be possible using multiple injections or systemic introduction of stem cells. Gu X, Thakker PU, Matz EL, et al. Dynamic Changes in Erectile Function and Histological Architecture After Intracorporal Injection of Human Placental Stem Cells in a Pelvic Neurovascular Injury Rat Model. J Sex Med 2020;17:400-411.

[Role of microRNAs in erectile dysfunction: An update].

MicroRNAs (miRNAs) are short non-coding RNAs consisting of approximately 19－23 nucleotides and involved in many pathological and physiological processes by regulating post-transcriptional gene expressions. ED is one of the common male sexual dysfunctions seriously affecting the patient's quality of life, for which there is currently a lack of effective treatments clinically. More and more experiments have demonstrated that miRNAs are involved in the pathological process of different types of ED. This article presents an overview of the progress in the studies of the pathogenic role of miRNAs in ED.