Antioxidative mechanism of Lycium barbarum polysaccharides promotes repair and regeneration following cavernous nerve injury

Polysaccharides extracted from Lycium barbarum exhibit antioxidant properties.We hypothesized that these polysaccharides resist oxidative stress-induced neuronal damage following cavernous nerve injury.In this study,rat models were intragastrically administered Lycium barbarum polysaccharides for 2 weeks at 1,7,and 14 days after cavernous nerve injury.Serum superoxide dismutase and glutathione peroxidase activities significantly increased at 1 and 2 weeks post-injury.Serum malondialdehyde levels decreased at 2 and 4 weeks.At 12 weeks,peak intracavernous pressure,the number of myelinated axons and nicotinamide adenine dinucleotide phosphate-diaphorase-positive nerve fibers,levels of phospho-endothelial nitric oxide synthase protein and 3-nitrotyrosine were higher in rats administered at 1 day post-injury compared with rats administered at 7 and 14 days post-injury.These findings suggest that application of Lycium barbarum polysaccharides following cavernous nerve crush injury effectively promotes nerve regeneration and erectile functional recovery.This neuroregenerative effect was most effective in rats orally administered Lycium barbarum polysaccharides at 1 day after cavernous nerve crush injury.

Fibrosis of corpus cavernosum in animals following cavernous nerve ablation

Aim: To investigate alterations of smooth muscle celis and collagen fibers in corpus cavernosum following cavernous neurectomy and its relation to the expression of transforming growth factor-β1 (TGF-β1). Methods: Ten adult male SD rats (neurectomy group) were subject to a bilateral cavernous nerve (CN) resection aseptically under an operating microscope, with 6 sham-operated rats as the control. Fifteen weeks after the operation, the penile speci mens were collected and prepared for quantitative-analyzing of ratio of smooth muscle to collagen fibers in corpus cavernosum with confocal microscopy, and for detecting the expression of TGF-β1 by RT-PCR and western-blot. Resulte: Smooth muscle celis that show red color after fluorescent-labeling with tetramethylrhodamine isothiocyanate phalloidin and collagen fibers that produce green autofluorescence after paraformaldehyde fixation were clearly iden tified under the confocal microscope. Quantification of fluorescent intensity showed that the ratio of smooth muscle to collagen fibers in corpus cavernosum in neurectomy group was 0.265±0.125, which was significantly lower than that in sham-operated group (0.760±0.196, P<0.01). RT-PCR and western-blot analyses revealed a significantly higher expression of TGF-β1 in the penile tissues of the neurectomy animals than that in sham-operated group. Conclusion: Bilateral ablation of CN can lead to fibrosis of corpus cavernosum, which may be related to an increased expression of TGF-β1 induced by hypoxia in cavernous tissue after denervation.

Nanotechnology-assisted adipose-derived stem cell （ADSC） therapy for erectile dysfunction of cavernous nerve injury： In vivo cell tracking, optimized injection dosage, and functional evaluation

Stem cell therapy is a potentially promising option for erectile dysfunction; however, its risk of tumorigenicity is a clinical hurdle and the risk is positively related to the number of injected cells. Our previous study showed that nanotechnology improved adipose-derived stem cell （ADSC） therapy for erectile dysfunction of cavernous nerve injury （CNI） by attracting cells in the corpus cavernosum. These results indicated the possibility of using a reduced dosage of ADSCs for intracavernous injection. In this exploratory study, we used lower dosage （2 × 105 cells） of ADSCs for intracavernous injection （ICI） and the nanotechnology approach. Intracavernous pressure and mean arterial pressure were measured at day 28 to assess erectile function. The low-dose ADSC therapy group showed favorable treatment effects, and nanotechnology further improved these effects. In vivo imaging of ICI cells revealed that the fluorescein signals of NanoShuttle-bound ADSCs （NanoADSCs） were much stronger than those of ADSCs at days 0, 1, and 3. Both immunofluorescence and Western blot analysis showed a significant increase in smooth muscle, endothelium, and nerve tissue in the ADSC group compared to that in the CNI group; further improvement was achieved with assisted nanotechnology. These findings demonstrate that nanotechnology can be used to further improve the effect of small dosage of ADSCs to improve erectile function. Abundant NanoADSCs remain in the corpus cavernosum in vivo for at least 3 days. The mechanism of erectile function improvement may be related to the regeneration of the smooth muscle, endothelium, and nerve tissues.

Vacuum therapy ameliorates erectile dysfunction in bilateral cavernous nerve crush rats by inhibiting apoptosis and activating autophagy

Postprostatectomy erectile dysfunction(pPED)remains a current problem despite improvements in surgical techniques.Vacuum therapy is clinically confirmed as a type of pPED rehabilitation.However,its underlying mechanisms are incompletely understood.Recently,autophagy and apoptosis were extensively studied in erectile dysfunction resulting from diabetes,senescence,and androgen deprivation but not in the context of pPED and vacuum therapy.Therefore,this study was designed to investigate the roles of autophagy and apoptosis in pPED and vacuum therapy.Twenty-four adult male Sprague-Dawley rats were randomly divided into three groups:the control group,bilateral cavernous nerve crush(BCNC)group,and BCNC+vacuum group.After 4 weeks of treatment,intracavernosal pressure was used to evaluate erectile function.Real-time quantitative polymerase chain reaction,western blot,and immunohistochemistry were used to measure the molecular expression.TdT-mediated dUTP nick-end labeling staining was used to assess apoptosis.Transmission electron microscopy was used to observe autophagosomes.After treatment,compared with those of the BCNC group,erectile function and cavernosal hypoxia had statistically significantly improved(P<0.05).Apoptosis and the relative protein expression of B-cell lymphoma-2-associated X and cleaved Caspase3 were decreased(P<0.05).Autophagy-related molecules such as phosphorylated unc-51-like autophagy-activating kinase 1(Ser757)and p62 were decreased.Beclinl,microtubule-associated protein 1 light chain 3 A/B,and autophagosomes were increased(P<0.05).Besides,the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin signaling pathway,as a negative regulator of autophagy to some degree,was inhibited.This study revealed that vacuum therapy ameliorated pPED in BCNC rats by inhibiting apoptosis and activating autophagy.

Cavernous nerve reconstruction with autologous vein graft and platelet-derived growth factors

In this study, we investigated the feasibility of using autologous vein graft and platelet-derived growth factors to bridge transected cavernous nerve in a rat model. A short defect in the bilateral cavernous nerve was created and repaired with vein graft from the right jugular vein or vein graft plus platelet-derived growth factors. The 32 rats were divided into four groups, namely Group 1 - no repair as a negative control, Group 2 - vein graft alone, Group 3 - vein graft plus platelet-derived growth factors, and Group 4 - sham operation as a positive control. We evaluated nerve regeneration and functional recovery using retrograde tracing study with FluoroGold, Toluidine blue staining of cavernous nerve, and the intracavernous pressure at 3 months. Three months after surgery, rich FluoroGold-positive cells were observed in the sham and vein graft plus platelet-derived growth factors group, but very few were found in the no repair group. The number of myelinated axons of regenerated cavernous nerve and intracavernous pressure were increased obviously in the two vein graft groups, especially in the vein graft plus platelet-derived growth factors group. These findings confirm the feasibility of using autologous vein as guides for cavernous nerve regeneration, and the regeneration can be further enhanced when the vein is filled with Dlatelet-derived growth factors.

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

Objective To verify the effect of Buyang Huanwu Decoction(BHD)in ameliorating erectile dysfunction(ED)after radical prostatectomy(RP).Methods 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-β1)andα-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 ofα-SMA and p-c-Jun in the CC was observed by double immunofluorescence staining.Results 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 ofα-SMA and Bcl-2,and reduced MDA and ROS levels,as well as expression of TGF-β1,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 theα-SMA expression increased(P<0.05 or P<0.01).Conclusions 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.

Regeneration of neuronal nitric oxide synthase (nNOS)-containing nerve fibers in rat corpus cavernosum

Aim: To investigate the effect of cavernous nerve injury on the nNOS-containing nerve fibers in rat corpus cavernosum.Methods: Thirty-three male SD rats were randomized into 3 groups: 5 rats underwent pelvic exploration without tran-section of cavernous nerve as the sham-operated controls, the unilateral injury group (14 rats) had the cavernous nerve cuton one side, and the bilateral injury group (14 rats) had the nerves cut on both sides. Corpora cavernosa were harvestedat the 3rd week and 6th month after surgery, nNOS-positive nerve fibers were examined with strepavidin peroxidase im-munohistochemistry techniques (SP method). Results: After bilateral ablation, the nNOS-positive nerve fibers weresignificantly decreased at both the 3rd week ( 17 ± 4) and the 6th month (16 ± 4). For the unilateral injury group, thenNOS-positive nerve fibers were similarly decreased on the side of the neurotomy at the 3rd week (18 ± 6), but by the 6thmonth, the number increased significantly (61±9) and approximated the level on the contralateral side (81 ± 13). Con-clusion: In rats after unilateral cavernous nerve ablation, nNOS-containing nerve fibers might regenerate 6 months afteroperation, but regeneration did not occur in animals with bilateral cavernous nerve injury. Results suggest that duringpelvic radical surgery, the cavernous nerve should be preserved at least on one side in order to accomplish adequate regen-eration. (Asian J Androl 1999 Sep ; 1: 135 - 138)

Regeneration of nNOS-containing nerve fibers in rat corpus cavernosum

Objective To investigate the effect of cavernous nerve injury on the nNOS containing nerve fibers in corpus cavernosum Methods Thirty three male Sprague Dawley (SD) rats were randomly divided into 3 groups: sham operated controls (n=5) underwent pelvic exploration without transection of the cavernous nerve; unilateral injury group (n=14) had their cavernous nerve cut on one side; and bilateral injury group (n=14) underwent neurotomy on both sides Corpora cavernosa were harvested at the 3rd week and 6th month after surgery nNOS positive nerve fibers were examined with streptavidin peroxidase immunohistochemistry techniques (SP method) Results After bilateral ablation, the nNOS positive nerve fibers were significantly decreased at the 3rd week (17±4) and remained so at the 6th month (16±4) For the unilateral injury group, the nNOS positive nerve fibers were similarly decreased on the side of the neurotomy at the 3rd week (18±6), but by the 6th month, the number increased significantly (61±9) and approximated the level on the contralateral side (81±13) Conclusion Following unilateral cavernous nerve ablation in rats, nNOS containing nerve fibers regenerate 6 months after surgery This regeneration process does not occur in animals with bilateral cavernous nerve injury, suggesting that during radical pelvic surgery, the cavernous nerve has to be preserved at least on one side in order to maintain the capacity for penile erection

Comparative study of intracavernous pressure and cavernous pathology after bilateral caver nous n erve crushing and resection in rats

This study aimed to compare the effects of bilateral cavernous nerve crushing(BCNC)and bilateral cavernous nerve resection(BCNR)on intracavernous pressure(ICP)and cavernous pathology in rats and to explore the optimal treatment time for the BCNC and BCNR models.Seventy-two male rats aged 12 weeks were randomly divided into three equal groups:Sham(both cavernous nerves exposed only),BCNC(BCN crushed for 2 min),and BCNR(5 mm of BCN resected).Erectile function was then measured at 1 week,3 weeks,and 5 weeks after nerve injury,and penile tissues were harvested for histological and molecular analyses by immunohistochemistry,immuno fluoresce nee,Western blot,and cytokine array.We found that erectile function parameters including the maximum,area,and slope of ICP/mean arterial pressure(MAP)significantly decreased after BCNR and BCNC at 1 week and 3 weeks.At 5 weeks,no significant differences were observed in ICP/MAP between the BCNC and Sham groups,whereas the ICP/MAP of the BCNR group remained significantly lower than that of the Sham group.After BCNC and BCNR,the amount of neuronal-nitric oxide synthase-positive fibers,smooth muscle cells,and endothelial cells decreased,whereas the amount of collage n III con tent increased.These pathological cha nges recovered over time,especially in the BCNC group.Our fin dings demonstrate that BCNC leads to acute and reversible erectile dysfunction,thus treatme nt time should be restricted to the first 3 weeks post-BCNC.In contrast,the self-healing ability of the BCNR model is poor,making it more suitable for long-term treatment research.