Bilateral Cavernous Nerve Injury Research Articles

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.

Comparison of the therapeutic effects of human umbilical cord blood-derived mesenchymal stem cells and adipose-derived stem cells on erectile dysfunction in a rat model of bilateral cavernous nerve injury.

Background: Cavernous nerve injury (CNI) is the leading cause of erectile dysfunction (ED) after radical prostatectomy and pelvic fracture. Transplantation of human adipose-derived stem cells (ASCs) has been widely used to restore erectile function in CNI-ED rats and patients. Umbilical cord blood-derived MSCs (CBMSCs) are similarly low immunogenic but much primitive compared to ASCs and more promising in large-scale commercial applications due to the extensive establishment of cord blood banks. However, whether CBMSCs and ASCs have differential therapeutic efficacy on CNI-ED and the underlying mechanisms are still not clear. Materials and methods: A bilateral cavernous nerve injury (BCNI) rat model was established by crushing the bilateral cavernous nerves. After crushing, ASCs and CBMSCs were intracavernously injected immediately. Erectile function, Masson staining, and immunofluorescence analyses of penile tissues were assessed at 4 and 12 weeks. PKH-26-labeled ASCs or CBMSCs were intracavernously injected to determine the presence and differentiation of ASCs or CBMSCs in the penis 3 days after injection. In vitro experiments including intracellular ROS detection, mitochondrial membrane potential assay, EdU cell proliferation staining, cell apoptosis assay, and protein chip assay were conducted to explore the underlying mechanism of CBMSC treatment compared with ASC treatment. Results: CBMSC injection significantly restored erectile function, rescued the loss of cavernous corporal smooth muscles, and increased the ratio of smooth muscle to collagen. PKH-26-labeled CBMSCs or ASCs did not colocalize with endothelial cells or smooth muscle cells in the corpus cavernosum. Moreover, the conditioned medium (CM) of CBMSCs could significantly inhibit the oxidative stress and elevate the mitochondria membrane potential and proliferation of Schwann cells. Better therapeutic effects were observed in the CBMSC group than the ASC group both in vivo and in vitro. In addition, the content of neurotrophic factors and matrix metalloproteinases in CBMSC-CM, especially NT4, VEGF, MMP1, and MMP3 was significantly higher than that of ASC-CM. Conclusion: Intracavernous injection of CBMSCs exhibited a better erectile function restoration than that of ASCs in CNI-ED rats owing to richer secretory factors, which can promote nerve regeneration and reduce extracellular matrix deposition. CBMSC transplantation would be a promising therapeutic strategy for CNI-ED regeneration in the future.

Co-overexpression of VEGF and Smad7 improved the therapeutic effects of adipose-derived stem cells on neurogenic erectile dysfunction in the rat model.

Cavernous nerve injury is the main cause of erectile dysfunction (ED) after radical prostatectomy (RP). In our previous study, injection of adipose-derived stem cells (ADSCs) into the cavernosum can repair damaged cavernosum nerves and ED can be restored to a certain extent. In order to improve these therapeutic effects, we evaluated the efficacy of ADSCs co-modified with VEGF and Smad7 in a rat model. SD rats were randomly divided into six groups: a sham surgery group, and the five bilateral cavernous nerve injury (BCNI) groups were injected with ADSC or ADSCs genetically modified by VEGF (ADSC-V), Smad7 (ADSC-S), or VEGF&Smad7 (ADSC-V&S) or phosphate-buffered saline (PBS). The results indicated that the erectile function of the ADSC-V, ADSC-S, and ADSC-V&S groups was significantly recovered, and the erectile function of the ADSC-V&S group was more distinctly recovered as compared to the other groups. The same results are shown in the expression of neuronal nitric oxide synthase and the smooth muscle/collagen ratio of penile tissue comparing the ADSC-V&S group to the ADSC-V and ADSC-S group. These experimental data suggest that ADSCs co-overexpressed with VEGF and Smad7 can significantly improve erectile function after BCNI. This study provides new therapeutic thoughts for ED following RP.

Buyang Huanwu Decoction Ameliorates Damage of Erectile Tissue and Function Following Bilateral Cavernous Nerve Injury.

To verify the effect of Buyang Huanwu Decoction (BHD) in ameliorating erectile dysfunction (ED) after radical prostatectomy (RP). The composition of BHD was verified by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) analysis. Bilateral cavernous nerve crush injury (BCNI) in rats was used to mimic the neurovascular injury occurring after RP. By the envelope method, forty rats were randomly divided into 4 groups as follows: sham (cavernous nerves exposed only), model (BCNI), low-dosage BHD [LBHD, 12.8 g/(kg·d)], and high-dosage BHD [HBHD, 51.2 g/(kg·d)] groups, 10 rats in each group, feeding for 3 weeks respectively. Erectile function was evaluated by measuring intracavernosal pressure (ICP). Changes in the histopathology of corpus cavernosum (CC) were examined by hematoxylin-eosin staining. Meanwhile, the fibrosis of CC was measured by Masson's trichrome staining and Western blot was used to detect the expressions of collagen I, transforming growth factor beta 1 (TGF- β 1) and α-smooth muscle actin (α-SMA). Apoptosis index was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) and Western blot for determining the expressions of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax). The oxidative stress in the CC were assessed by the superoxide dismutase (SOD), malondialdehyde (MDA) and reactive oxygen species (ROS) levels. The proteins expression of c-Jun N-terminal kinase (JNK) and c-Jun were detected by Western blot. In addition, the expression of α-SMA and p-c-Jun in the CC was observed by double immunofluorescence staining. The UPLC-QTOF-MS/MS analysis showed that BHD contained calycosin-7-O- β -D-glucoside, ononin, calycosin and formononetin. Compared with the model group, LBHD and HBHD treatment improved the ICP and the circumference, area, and weight of CC (P<0.05 or P<0.01). Furthermore, LBHD and HBHD treatments increased CC smooth muscle content and decreased apoptosis index (P<0.05 or P<0.01). LBHD and HBHD also elevated SOD and expression level of α -SMA and Bcl-2, and reduced MDA and ROS levels, as well as expression of TGF- β 1, collagen I, Bax, p-c-JNK, p-JNK in the CC compared with the model group (P<0.05 or P<0.01). The double immunofluorescence staining showed that the fluorescence degree of p-c-Jun in both LBHD and HBHD treatment groups was significantly reduced, whereas the α -SMA expression increased (P<0.05 or P<0.01). BHD can improve ED of rats with BCNI, which is related to inhibiting fibrosis, apoptosis, and oxidative stress of CC. The ROS/JNK/c-Jun signaling pathway may play an important role in the process.

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.

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.

Recovery of erectile dysfunction after administration of hydrogen sulfide donor sodium hydrogen sulfide and transplantation of adipose-derived stem cells in rats with bilateral cavernous nerve injury

ABSTRACT Introduction Radical prostatectomy causes nerve injury-related erectile dysfunction (ED), which results in a poor response to phosphodiesterase 5 inhibitors. Hydrogen sulfide (H2S), an endogenous gasotransmitter, is produced by cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) and participates in erectile physiology. Adipose-derived stem cells (ADSCs) represent a promising cell source for cell replacement therapy in several degenerative diseases because of its self-renewal properties and multi-differentiation potential with low immunogenicity. Objective The current study was to evaluate the effects of H2S donor, sodium hydrogen sulfide (NaHS), and transplantation of ADSCs per se and their combination treatment on bilateral cavernous nerve injury (BCNI)-induced erectile dysfunction (ED). Methods A total of 35 Sprague-Dawley rats were divided into 5 groups, including sham-operated (Sham), BCNI (4-wk), BCNI treated with NaHS (5.6 mg/kg/day, i.p.), BCNI treated with ADSCs (1x106 cells by intracavernosal injection) and BCNI treated with NaHS and ADSCs groups. Rats in the ADSCs treatment groups were subjected to intracavernosal injection after BCNI. Erectile function was evaluated by the ratio of intracavernous pressure (ICP)/mean arterial pressure (MAP) and total ICP. The relaxant and contractile responses of corpus cavernosum (CC) were measured from in vitro studies. Protein expression and localization of nitric oxide synthases (NOS) and H2S synthesis enzymes were analyzed by Western blotting and immunohistochemistry. The smooth muscle/collagen ratio was determined using Masson trichrome staining. Results Rats with BCNI displayed significantly reduced ICP/MAP and total ICP (p&amp;lt;0.001 vs controls) when were partially restored by NaHS and ADSCs treatments alone. Interestingly, combined treatment completely enhanced decreased in vivo erectile responses (p&amp;lt;0.001 vs the NC group). Relaxation responses to acetylcholine and electrical field stimulation (EFS) as well as contractile responses to phenylephrine and EFS in the CN group were less than in controls (p&amp;lt; 0.05), which was normalized after all treatments groups. The combined treatment modulated changes of protein expressions and reduced ratio of smooth muscle to collagen in rats with BCNI. Conclusions The current data indicated that the combination treatment with NaHS and ADSCs is more successful than monotherapies in reversing BCNI-induced ED. H2S and cell-based therapy most probably may have a synergistic effect on erectile function via controlling molecular regulation and morphological penile alterations, which supports combined treatment for expanding clinical outcomes radical prostatectomy-induced ED. Disclosure Work supported by industry: no.

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.

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.

Effects of the light-controlled nitric oxide donor “NORD-1” and light irradiation in rats with neurogenic erectile dysfunction

ABSTRACT Introduction Radical prostatectomy often causes erectile dysfunction (ED). This is considered to be due to surgery-induced nerve damage. Impairments in neurotransmission result in the release of nitric oxide (NO) in penile corpus cavernosum insufficiency. Therefore, phosphodiesterase-5 inhibitors are not necessarily effective for this type of ED. NO supplementation may be effective for this type of ED. However, NO acts on the whole body and thus may cause adverse effects. Recently, we have developed a light-controlled NO donor that enables spatially and temporally controlled NO release by light irradiation. Whether this type of NO donor is useful for neurogenic ED remains unknown. Objective We investigated whether this light-controlled NO donor “NORD-1” and light irradiation were effective for managing neurogenic ED in rats with bilateral cavernous nerve injury (BCNI). Methods We used eight-week-old male Wistar-ST rats. The rats were divided into four groups: Sham+SiR650 (control compound) (n=5), Sham+NORD-1 (n=5), BCNI+SiR650 (n=6), and BCNI+NORD-1 (n=6). Initially, sham or BCNI surgery was performed under anesthesia in each group. BCNI surgery was performed by crushing the bilateral cavernous nerves. Four weeks post-surgery, erectile function before and after the injection of each compound into the penile corpus cavernosum was evaluated based on the change in intracavernous pressure (ICP) under electrical stimulation of the cavernous nerve. The changes in ICP with and without light irradiation were also evaluated after the administration of each compound. Results Before administering each treatment without light irradiation, the ICP/mean arterial pressure (MAP) ratio under stimulation in the BCNI+SiR650 and BCNI+NORD-1 groups were lower than those in the Sham+SiR650 (P&amp;lt;0.01) and Sham+NORD-1 (P=0.06) groups. After the each treatment without light irradiation, the ICP/MAP ratio under stimulation in the Sham+NORD-1 group was slightly higher than that in the Sham+SiR650 group. However, the ICP/MAP ratio under stimulation in the BCNI+SiR650 and BCNI+NORD-1 groups remained significantly lower (P&amp;lt;0.05 and P&amp;lt;0.01, respectively). After the treatment with light irradiation, the ICP/MAP ratios under stimulation in the Sham+NORD-1 and BCNI+NORD-1 groups were significantly higher than those in the Sham+SiR650 (P&amp;lt;0.01) and BCNI+NORD-1 (P&amp;lt;0.01) groups, respectively. Conclusions The combination of NORD-1 and light irradiation may be useful for treating neurogenic ED, although its toxicity must be evaluated. Disclosure Work supported by industry: no.

MP49-11 FIDGETIN-LIKE 2 (FL2) DEPLETION AT THE SITE AND TIME OF CAVERNOUS NERVE INJURY IMPROVES BLADDER FUNCTIONAL OUTCOMES

You have accessJournal of UrologyCME1 May 2022MP49-11 FIDGETIN-LIKE 2 (FL2) DEPLETION AT THE SITE AND TIME OF CAVERNOUS NERVE INJURY IMPROVES BLADDER FUNCTIONAL OUTCOMES Yi Wang, Moses Tar, Marcia Urban-Maldonado, Lisa Baker, David Sharp, Sylvia Suadicani, and Kelvin Davies Yi WangYi Wang More articles by this author , Moses TarMoses Tar More articles by this author , Marcia Urban-MaldonadoMarcia Urban-Maldonado More articles by this author , Lisa BakerLisa Baker More articles by this author , David SharpDavid Sharp More articles by this author , Sylvia SuadicaniSylvia Suadicani More articles by this author , and Kelvin DaviesKelvin Davies More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002624.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Radical prostatectomy (RP) is the most common treatment for localized prostate cancer. Unfortunately, the procedure carries high risk for the development of lower urinary tract symptoms (LUTS). These urogenital complications are highly detrimental to the post-surgical well-being of men, for whom there are presently poor treatment options. We recently demonstrated in a rat model of nerve injury associated with RP, that Fidgetin-Like 2 (FL2) depletion through application of FL2-siRNA at the site and time of cavernous nerve injury (CNI), resulted in significantly enhanced functional nerve recovery, as determined by cavernosometry [1]. In the present studies, we determined if the same approach would also improve bladder function outcomes in the rat model of CNI. METHODS: Animals were submitted to bilateral CNI, or to CNI followed by treatment with either FL2-siRNA or control-siRNA at the time and site of injury (the nerve and major pelvic ganglia). Voiding function was evaluated 7-days post-injury using the voided stain on paper (VSOP) method. Changes in i) micturition frequency and ii) voided volume per micturition were compared to those of naïve (unoperated) rats. The decrease in micturition frequency and increase in voiding volume in the CNI, and CNI treatment groups (FL2-siRNA or control-siRNA groups) were compared to the naïve cohort. 6 rats were analyzed per group and significance determined by Student’s t-test. RESULTS: Voiding function was significantly altered following CNI injury; at 7-days post-CNI, when compared to naïve (unoperated) age-matched controls, CNI animals displayed significantly lower micturition frequency (0.4 versus 0.55 voids per hour, P=0.02), and more than 2-fold greater voided volume per micturition (P=0.055). Remarkably, FL2-siRNA treatment at the time and site of injury mitigated these side-effects of CNI on voiding function, whereas treatment with control-siRNA was ineffective. CONCLUSIONS: These findings indicate that in addition to promoting recovery of erectile function following CNI rats [1], FL2 depletion at the site and time of injury also leads to improved bladder function outcomes. However, given that our studies on the role of FL2 depletion in mitigating LUTS were conducted at 7-days rather than 2-weeks after injury (a time point too short for visible nerve regeneration), the primary mechanisms by which FL2 depletion improves these different urogenital conditions are likely to be different and are currently being investigated. 1] Baker, L., Tar, M., Villegas, G., Charafeddine, R.A., Kramer, A., Vafaeva, O., Nacharaju, P., Friedman, J., Davies, K.P., and Sharp, D.J. (2021) Fidgetin-like 2 is a novel negative regulator of axonal growth and can be targeted to promote functional nerve regeneration after injury. JCI Insight. 2021 May 10;6(9):138484. PMID: 33872220. Source of Funding: NIH R41 DK112476 (MPI: Baker, Davies, Sharp), NIH R01 DK109314 (MPI: Davies, Sharp) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e857 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yi Wang More articles by this author Moses Tar More articles by this author Marcia Urban-Maldonado More articles by this author Lisa Baker More articles by this author David Sharp More articles by this author Sylvia Suadicani More articles by this author Kelvin Davies More articles by this author Expand All Advertisement PDF DownloadLoading ...

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

Intracavernous Injection of Botulinum Toxin may Improve Erectile Function in a Bilateral Cavernous Nerve Injury Animal Model

ABSTRACT Introduction Erectile dysfunction (ED) is common after treatments for prostate cancer, such as radical prostatectomy (RP) and can negatively impact both patient and partner quality of life. Botulinum toxin (Botox) promotes smooth muscle relaxation and may augment erectile function by reducing basal sympathetic tone in the corpora cavernosa. Botox utilization has suggested a neuroprotective and nerve regenerative role after peripheral nerve injury. Objective We investigated Botox as a durable treatment option for ED in a bilateral cavernous nerve injury (BCNI) animal model. We evaluated the structural changes in penile smooth muscle after intracavernous injection of Botox in an animal model of post-RP ED. Methods Sixteen Sprague Dawley rats (4.5months; 450-550g) were randomized to 4 groups: (1) Sham + NS (2) BCNI + NS (3) Sham + Botox (4) BCNI + Botox. When cavernous nerves were identified during surgery, they were either crushed (BCNI) or left untouched (Sham). Next, either 10 units of Botox or 80umL of normal saline (NS) were injected into the corpora cavernosa. On day 14, erectile function was assessed via cavernous nerve electro-stimulation induced intracavernous pressure (ICP) changes. Penile tissue was harvested for immunohistochemistry and Western blot for neuronal nitric oxide synthase (nNOS) and smooth muscle α-actin (SMA). Results Fourteen animals were included for analysis; one animal in groups 2 and 3 were euthanized for weight loss and lethargy. The Botox treatment groups had a net weight loss whereas the NS groups had weight gain (p&amp;lt;0.05). Erectile function after BCNI recovered with Botox injections and both group 3 &amp; 4 had mean peak ICP and area under the curve values analogous to baseline (group 1) and statistically improved compared to control (group 2) (Figure 1). Western blot analysis revealed a recovery of both nNOS and SMA after Botox treatment (P=0.06). Conclusions This is the first animal study evaluating intracavernous Botox injections for treating ED after BCNI and compared to placebo, the intervention appears to recover erectile function. Further preclinical studies are needed to support this finding, but there may be a future role for using Botox to treat post-prostatectomy ED. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: boston scientific

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

Lipopolysaccharide-preconditioned allogeneic adipose-derived stem cells improve erectile function in a rat model of bilateral cavernous nerve injury

BackgroundErectile dysfunction (ED) often occurs due to cavernous nerve injury (CNI) after colorectal surgery. Cell-based therapies have great potential for the treatment of CNI-related ED; however, it needs to be optimised. In this study, we explored the therapeutic effects of lipopolysaccharide-preconditioned allogeneic adipose-derived stem cells (L-ADSCs) on CNI-induced ED in rats.ResultsThe results of this in vitro study revealed that low-dose lipopolysaccharide could increase the viability of ADSCs, inhibit caspase 3 activation induced by hydrogen peroxide and promote cell migration. Compared with the ADSC supernatant, the L-ADSC supernatant could better reduce fibrosis in the corpus cavernosum smooth muscle cells induced by transforming growth factor-beta 1 protein. In the in vivo study, it was compared to ADSCs therapy, where the L-ADSCs therapy indicated that could better improve erectile function by increasing smooth muscle content and alleviating penile fibrosis in rats 2 weeks after CNI. The outcome may be related to the increase in the hepatocyte growth factor content in the corpus cavernosum and myelin basic protein in the major pelvic ganglion.ConclusionsL-ADSC treatment may be a promising approach for restoring erectile function after CNI.

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

Neurogenic 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. To 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. Adult 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-β1, 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. Effects of LIES on EF, erectile tissue remodeling and CN structure. EF 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. LIES 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. This 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. This study demonstrates that LIES of the CN after CN injury protects CN structure, enhances EF recovery, and prevents corpora cavernosal remodeling.

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.

Is there a role for stem cell therapy in erectile dysfunction secondary to cavernous nerve injury? Network meta-analysis from animal studies and human trials.

Introduction:We carried out systematic review and network meta-analysis to investigate the role of stem cell therapy (SCT) in the management of erectile dysfunction (ED) secondary to cavernous nerve injury in rats and post-radical prostatectomy (RP) in humans.Patients and Methods:The protocol was registered with PROSPERO database. We searched studies analyzing the efficacy of SCT for ED due to bilateral cavernous nerve injury (BCNI) in rats using Healthcare Databases Advanced Search (HDAS) Export software (MEDLINE, EMBASE, Scopus) from inception to September 2020. The outcome measurements, for 29 animal studies, were intracavernosal pressure (ICP), ICP/MAP (mean arterial pressure) ratio, and histological/molecular changes. All three available human trials evaluating SCT in post-RP ED were assessed for International Index for Erectile Function (IIEF) Score and Erection Hardness Score (EHS).Results:For ICP measurement, animal studies were divided into adipose-derived stem cells (ADSCs) subgroup and bone marrow–derived stem cells (BMSCs) subgroup. Pooled analysis of these studies showed a beneficial effect of SCT in improving erectile function in rats with BCNI using network meta-analysis (95% confidence interval, CI; p < 0.001). There was an increase in ICP/MAP ratio in stem cell groups (including co-intervention) compared with control BCNI group. Histological and molecular evaluation of penile tissue revealed an increase in neuronal nitric oxide synthase (nNOS), smooth muscle content, and anti-apoptotic activity. Human trials revealed improved IIEF (70–150% from baseline at 6 months) and EHS (80–200% from baseline).Conclusion:Our results confirm that SCT does improve the erectile function in rats having cavernous nerve injury. Similarly, early human results have shown promising results.PROSPERO registration ID:CRD42020201343.

Effects of a Mixture of Three Extracts of Wolfberry, Chives and Graviola on the Erectile Dysfunction Induced by Bilateral Cavernous Nerve Injury in Rats

Effects of a Mixture of Three Extracts of Wolfberry, Chives and Graviola on the Erectile Dysfunction Induced by Bilateral Cavernous Nerve Injury in Rats