Gene expression profiling of an arteriogenic impotence model.

Penile arterial insufficiency is one of the most common causes of ED. We have established a traumatic arteriogenic insufficiency rat model by the ligation of the pudendal arteries. To simulate both acute and chronic traumatic injuries, five ligation periods (6 h, 3 days, 7 days, 3 weeks, and 6 weeks) were chosen. By electrostimulation of the cavernous nerve, the intracavernous pressure was determined to be between 20 and 40-cm H(2)O for the ligated rats compared to around 100-cm H(2)O for the control rats. The erectile tissue in the corpus cavernosum of these rats was then subjected to microarray analysis, in which an array that contains cDNA fragments representing 1176 rat genes was used. The results demonstrated that normal rat corpus cavernosum expressed approximately 200 genes at detectable levels and that ligation produced differential expression of approximately 25 genes, depending on the duration of ligation. The most highly ligation-induced gene was apolipoprotein D (ApoD), with peak expression in the 3- and 7-day ligated rats. Three of the insulin-like growth factor binding proteins (IGFBP-1, 3, and 5) were upregulated in all ligated rats. IGFBP-6, which was one of the most highly expressed genes in the normal corpus cavernosum, was down-regulated in all ligated rats. Cysteine proteases of the cathepsin family were also differentially expressed between control and ligated rats, with cathepsin K being down-regulated most. A few genes were upregulated only in the 6-week ligated rats, including angiotensin-converting enzyme. Finally, VEGF, whose induction has been identified in many other ischemic tissues, was not induced in corpus cavernous tissue of ligated rats.     

Mesenchymal stem cell‐derived exosomes ameliorate erection by reducing oxidative stress damage of corpus cavernosum in a rat model of artery injury

Erectile dysfunction (ED) is a common ageing male's disease, and vascular ED accounts for the largest proportion of all types of ED. One of the mechanisms of vascular ED in the clinic is arterial insufficiency, which mainly caused by atherosclerosis, trauma and surgical. Moreover, oxidative stress damage after tissue ischemia usually aggravated the progress of ED. As a new way of acellular therapy, mesenchymal stem cell‐derived exosomes (MSC‐Exos) have great potential in ED treatment. In the current study, we have explored the mechanism of MSC‐Exos therapy in a rat model of internal iliac artery injury‐induced ED. Compared with intracavernous (IC) injection of phosphate‐buffered saline after artery injury, of note, we observed that both mesenchymal stem cells (MSCs) and MSC‐Exos through IC injection could improve the erectile function to varying degrees. More specifically, IC injection MSC‐Exos could promote cavernous sinus endothelial formation, reduce the organization oxidative stress damage, and improve the nitric oxide synthase and smooth muscle content in the corpus cavernosum. With similar potency compared with the stem cell therapy and other unique advantages, IC injection of MSC‐ Exos could be an effective treatment to ameliorate erectile function in a rat model of arterial injury.     

Effects of male silkworm pupa powder on the erectile dysfunction by chronic ethanol consumption in rats

Erectile dysfunction (ED) is a highly prevalent disorder that affects millions of men worldwide. ED is now considered an early manifestation of atherosclerosis, and consequently, a precursor of systemic vascular disease. This study was designed to investigate the effects of male silkworm pupa powder (SWP) on the levels of nitric oxide synthase (NOS) expression, nitrite, and glutathione (GSH); lipid peroxidation; libido; and erectile response of the corpus cavernosum of the rat penis. We induced ED in the study animals by oral administration of 20% ethanol over 8 weeks. The SWP-treated male rats were divided into 3 groups that were orally administered 200, 400, and 800 mg/kg. The libido of the SWP-administered male rats was higher than that of the ethanol control group. In addition, the erectile response of the corpus cavernosum was restored in males on SWP administration, to a level similar to that of the normal group without ED. The testosterone concentration did not increase significantly. The lipid peroxidation in the corpus cavernosum of the male rats administered SWP decreased significantly. In contrast, compared to the ethanol group, SWP-administered male rats showed increased GSH levels in the corpus cavernosum. The level of nitrite and NOS expression in the corpus cavernosum of SWP-administered male rats increased significantly. These results indicated that SWP effectively restored ethanol-induced ED in male rats.     

Effects of Intracavernous Injection of Adipose-Derived Stem Cells on Cavernous Nerve Regeneration in a Rat Model

The aim of this study was to investigate effects of intracavernous injection of adipose-derived stem cells (ADSCs) on cavernous nerve (CN) regeneration and functional status in a nerve-crush rat model. Thirty Sprague–Dawley male rats were randomly divided into three equal groups: one group underwent sham operation, while two groups underwent bilateral CN crush. Crush-injury group was treated at the time of injury with intracavernous injection of ADSCs, or injured control group with no further intervention. Erectile function was assessed by CN electrostimulation after 3 months. Penile tissue and crushed nerves were collected for histology. Three months after surgery, in the group that underwent bilateral nerve crushing with no further intervention, the functional evaluation showed a lower mean maximal intracavernous pressure (ICP) and maximal ICP per mean arterial pressure (MAP) with CN stimulation than those in the sham group. In the group with an immediate intracavernous injection of ADSCs, the mean maximal ICP and maximal ICP/MAP were significantly higher than those in the injured control group. Histologically, the group with the intracavernous injection of ADSCs had more myelinated axons of CNs and more NADPH-diaphorase-positive nerve fibers than the injured control group but fewer than the sham group. Intracavernous injection of ADSCs treatment had beneficial effects on the smooth muscle/collagen ratio in the corpus cavernosum. These results show that the intracavernous injection of ADSCs to the site of CN-crush injury facilitates nerve regeneration and recovery of erectile function. Our research indicates that penile injection of ADSCs can improve recovery of erectile function in a rat model of neurogenic ED.     

Alterations in the phosphodiesterase type 5 pathway and oxidative stress correlate with erectile function in spontaneously hypertensive rats

To explore how alterations in the phosphodiesterase type 5 (PDE5) signalling pathway and oxidative stress correlate with changes in the expression of relaxation and contraction molecules and erectile dysfunction (ED) in the corpus cavernosum smooth muscle (CCSM) of spontaneously hypertensive rats (SHR). In this study, SHR and Wistar‐Kyoto (WKY) rats were used. Erectile function was determined by apomorphine test and electrical stimulation (ES) of cavernous nerve. Masson''s trichrome staining and confocal microscopy were performed. Nitric oxide synthase (NOS), PDE5, phosphorylated‐PDE5 and α1‐adrenergic receptor (α1AR) were determined by RT‐PCR and Western blotting while oxidative stress in CC was determined by colorimetric analysis. SHR exhibited obvious ED. CC of SHR showed less SM but more collagen fibres. The expression of NOS isoforms in SHR was significantly decreased while all α1AR isoforms were increased. In addition, PDE5 and phosphorylated‐PDE5 were down‐regulated and its activity attenuated in the hypertensive rats. Meanwhile, the SHR group suffered oxidative stress, which may be modulated by endoplasmic reticulum stress and NADPH oxidase up‐regulation. Dysregulation of NOS and α1AR, histological changes and oxidative stress in CC may be associated with the pathophysiology of hypertension‐induced ED. In addition, PDE5 down‐regulation may lead to the decreased efficacy of PDE5 inhibitors in some hypertensive ED patients and treatment of oxidative stress could be used as a new therapeutic target for this type of ED.     

Effects of chelator treatment on aorta and corpus cavernosum from diabetic rats.

Transition-metal catalyzed reactions contribute to oxidative stress, which has been implicated in the pathogenesis of diabetic complications. The aim was to evaluate the effects of treatment with the transition metal chelator trientine on endothelium-dependent relaxation of aorta and corpus cavernosum from streptozotocin-induced diabetes of 8 weeks duration in rats. Effects on cavernosum autonomic innervation were also examined. Diabetes caused a 30.1 +/- 3.8% reduction in maximum aorta endothelium-dependent relaxation to acetylcholine (ACh), which was markedly attenuated (72.7 +/- 10.6%) by trientine treatment. Reversal treatment (4 weeks untreated diabetes, 4 weeks trientine) did not effect endothelium-dependent relaxation compared with aortas from rats with 4 weeks of diabetes, however, there was a 22.5 +/- 6.2% improvement compared with 8 weeks of diabetes. Eight weeks of diabetes caused a 41.5 +/- 6.6% reduction in corpus cavernosum endothelium-dependent maximum relaxation to ACh that was 70.1 +/- 16.9% prevented by trientine. Cavernosum nonadrenergic, noncholinergic (NANC) nerve stimulation caused frequency-dependent relaxation to a maximum of 40.9 +/- 2.4%, which was reduced by diabetes to 24.2 +/- 2.1%. Trientine partially prevented this deficit, maximum relaxation being 31.9 +/- 2.3%. Thus, metal chelator treatment has beneficial effects on aorta and cavernosum endothelium-dependent relaxation and on cavernosum NANC innervation.     

Erectile Function Restoration After Repair of Resected Cavernous Nerves by Adipose-Derived Stem Cells Combined with Autologous Vein Graft in Rats

Cavernous nerve (CN) injury is the main cause of erectile dysfunction (ED) following radical prostatectomy. The recovery of erectile function following this procedure remains challenging. Here, we investigated the ability of adipose-derived stem cells (ADSCs) combined with autologous vein graft to improve erectile function in a rat model of bilateral long CN resection. Sprague–Dawley rats (n = 36) were randomized into four groups. Group A underwent sham operation. In Groups B, C, and D, an 8-mm segment of CN was excised bilaterally. In Group B and C, a 10-mm segment of autologous saphenous vein was interposed bilaterally at the site of injury, and the two nerve stumps were inserted into the vein lumen. 50 μL ADSCs were injected into each vein in Group B, and 50 μL of phosphate-buffered saline was injected in Group C. Group D underwent no repair. Erectile function assessed after 3 months by measuring intracavernosal pressure demonstrated significant recovery in erectile function in Group B with minimal recovery in Group C or D. Immunohistochemical staining showed that the nNOS-positive area was significantly larger in Group B than in Group D. ADSCs combined with autologous vein graft treatment had beneficial effects on the smooth muscle/collagen ratio in the corpus cavernosum. This procedure, therefore, provided a means of regenerating CN tissue and restoring autonomic erectile function after long bilateral CN resection (0.8 cm) in rats.     

Cavernous nerve reconstruction to preserve erectile function following non-nerve-sparing radical retropubic prostatectomy: a prospective study

Erectile dysfunction following radical prostatectomy for treatment of clinically localized prostate cancer remains a problem that deters many men from seeking surgical treatment. Sparing the cavernous nerves has been popularized as a method of preserving potency, but men with locally advanced disease may be at increased risk for positive margins with this technique. In this study, sural nerve grafting of the cavernous nerve bundles, to preserve postoperative potency while potentially maximizing cancer control, was examined. Thirty men were enrolled in this prospective phase I study and underwent non-nerve-sparing radical prostatectomy performed by one of two protocol surgeons. Preoperative erectile function was assessed both objectively, using a RigiScan (Timm Medical Technologies, Inc., Eden Prairie, Minn.), and subjectively. The cavernous nerves were identified and resected during the operation with the use of an intraoperative mapping device (CaverMap; Alliant Medical Technologies, Norwood, Mass.). Bilateral autologous sural nerve grafting to the cavernous nerve stumps was performed by one of two protocol plastic surgeons. Postoperative erectile dysfunction therapy, using intracorporeal injection, a vacuum pump, and/or oral sildenafil therapy, was instituted 6 weeks after the operation. Spontaneous erectile activity was subjectively and objectively measured every 3 months after the operation. Follow-up periods ranged from 13 to 33 months (mean, 23 months). Overall, 18 of 30 patients (60 percent) demonstrated both objective and subjective evidence of spontaneous erectile activity. Of those 18 men, 13 (72 percent) were able to have intercourse (seven unassisted and six with the aid of sildenafil). No disease or biochemical recurrences have been noted in this group of patients with locally advanced disease. In conclusion, autologous sural nerve grafting after non-nerve-sparing radical prostatectomy is an effective means of preserving spontaneous erectile activity after the operation while maximizing cancer control potential.     

Chronic Cigarette Smoking Impairs Erectile Function through Increased Oxidative Stress and Apoptosis,Decreased nNOS,Endothelial and Smooth Muscle Contents in a Rat Model

Cigarette use is an independent risk factor for the development of erectile dysfunction (ED). While the association between chronic smoking and ED is well established, the fundamental mechanism(s) of cigarette-related ED are incompletely understood, partly due to no reliable animal model of smoking-induced ED. The present study was designed to validate an in vivo rat model of chronic cigarette-induced ED. Forty 12-week old male Sprague-Dawley rats were divided into 4 groups. Ten rats served as control group and were exposed only to room air. The remaining 30 rats were passively exposed to cigarette smoke (CS) for 4 weeks (n = 10), 12 weeks (n = 10), and 24 weeks (n = 10). At the 24-week time point all rats were assessed with intracavernous pressure (ICP) during cavernous nerve electrostimulation. Blood and urine were collected to measure serum testosterone and oxidative stress, respectively. Corporal tissue was assessed by Western blot for neuronal nitric oxide synthase (nNOS). Penile tissues were subjected to immunohistochemistry for endothelial, smooth muscle, and apoptotic content. Mean arterial pressure (MAP) was significantly higher in 24-week cigarette exposed animals compared to the control animals. Mean ICP/MAP ratio and cavernosal smooth muscle/endothelial contents were significantly lower in the 12- and 24-week rats compared to control animals. Oxidative stress was significantly higher in the 24-week cigarette exposed group compared to control animals. Mean nNOS expression was significantly lower, and apoptotic index significantly higher, in CS-exposed animals compared to control animals. These findings indicate that the rat model exposure to CS increases apoptosis and oxidative stress and decreases nNOS, endothelial and smooth muscle contents, and ICP in a dose dependent fashion. The rat model is a useful tool for further study of the molecular and cellular mechanisms of CS-related ED.     

Phenotypic Modulation of Corpus Cavernosum Smooth Muscle Cells in a Rat Model of Cavernous Neurectomy

Background

Patients undergoing radical prostatectomy (RP) are at high risk for erectile dysfunction (ED) due to potential cavernous nerve (CN) damage during surgery. Penile hypoxia after RP is thought to significantly contribute to ED pathogenesis.

Aim

We previously showed that corpora cavernosum smooth muscle cells (CCSMCs) undergo phenotypic modulation under hypoxic conditions in vitro. Here, we studied such changes in an in vivo post-RP ED model by investigating CCSMCs in bilateral cavernous neurectomy (BCN) rats.

Methods

Sprague-Dawley rats underwent sham (n = 12) or BCN (n = 12) surgery. After 12 weeks, they were injected with apomorphine to determine erectile function. The penile tissues were harvested and assessed for fibrosis using Masson trichrome staining and for molecular markers of phenotypic modulation using immunohistochemistry and western blotting. CCSMC morphological structure was evaluated by hematoxylin-eosin (H&E) staining and transmission electron microscopy (TEM).

Results

Erectile function was significantly lower in BCN rats than in sham rats. BCN increased hypoxia-inducible factor-1α and collagen protein expression in corpora cavernous tissue. H&E staining and TEM showed that CCSMCs in BCN rats underwent hypertrophy and showed rough endoplasmic reticulum formation. The expression of CCSMC phenotypic markers, such as smooth muscle α-actin, smooth muscle myosin heavy chain, and desmin, was markedly lower, whereas vimentin protein expression was significantly higher in BCN rats than in control rats.

Conclusions

CCSMCs undergo phenotype modulation in rats with cavernous neurectomy. The results have unveiled physiological transformations that occur at the cellular and molecular levels and have helped characterize CN injury–induced ED.     

Effects of resveratrol on nerve functions, oxidative stress and DNA fragmentation in experimental diabetic neuropathy

Oxidative stress has been implicated in pathophysiology of diabetic neuropathy. All the pathways responsible for development of diabetic neuropathy are linked to oxidative stress in one way or the other. In the present study, we have targeted oxidative stress in diabetic neuropathy using resveratrol, a potent antioxidant. Eight weeks streptozotocin-diabetic rats developed neuropathy which was evident from significant reduction in motor nerve conduction velocity (MNCV), nerve blood flow (NBF) and increased thermal hyperalgesia. The 2-week treatment with resveratrol (10 and 20 mg/kg, i.p.) started 6 weeks after diabetes induction significantly ameliorated the alterations in MNCV, NBF, and hyperalgesia. Resveratrol also attenuated enhanced levels of malondialdehyde (MDA), peroxynitrite and produced increase in catalase levels in diabetic rats. There was marked reduction in DNA fragmentation observed after resveratrol treatment in diabetic rats as evident from decrease in Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells in sciatic nerve sections. Results of the present study suggest the potential of resveratrol in treatment of diabetic neuropathy and its protective effect may be mediated through reduction in oxidative stress and DNA fragmentation.     

Increased occurrence of PGP 9.5-immunoreactive epidermal Langerhans cells in rat plantar skin after sciatic nerve injury

This study examines the occurrence and distribution of epidermal dendritic cells (DCs) in cryostate sections from plantar skin in normal rats and in rats with a crush injury or neurotomy and suture of the sciatic nerve. The dendritic cells were visualized with antibodies against protein-gene product 9.5 (PGP 9.5). Counts under the fluorescence microscope showed that the occurrence of dendritic cells is increased and that the proportion of dendritic cells in the basal layer is elevated 3 months after sciatic neurotomy and suture but not after a crush lesion. The countings also revealed that the number of cells is elevated as soon as 1 week after neurotomy and suture. Labelling with specific antibodies showed that the dendritic cells examined represent Langerhans cells (LCs). These observations show that there is a neural influence on the occurrence and distribution of PGP 9.5-immunoreactive epidermal Langerhans cells. Whether this influence is direct or indirect remains to be ascertained.     

阴茎勃起及勃起功能障碍的研究进展

勃起功能障碍的基础研究须近十年来取得了较大进展,一氧化氮－cGMP(NO-cGMP)通路的发现使得阴茎平滑肌松驰的机制进一步阐明。一氧化氮合酶（NOS）、磷酸二酯酶（PDEs）的研究为勃起功能障碍的临床治疗提供了坚实的基础,进而促使了万艾可的问世。目前,勃起功能障碍的基因治疗停留在实验室阶段,但随着分子生物学的深入研究,转基因疗法可能成为临床上治疗勃起功能障碍的有效方法之一。     

Time-dependent alteration in cromakalim-induced relaxation of corpus cavernosum from streptozocin-induced diabetic rats

The purpose of the present study was to investigate the relaxant responses to the ATP-sensitive potassium (K(ATP)) channel opener cromakalim in corpus cavernosum strips from 1-, 2-, 4-, 6-, and 8-week streptozocin-induced diabetic rats. Cromakalim (1 nM-0.1 mM) produced concentration-dependent relaxation in phenylephrine (7.5 microM)-precontracted isolated rat corporal strips. Compared with age-matched control animals, a significant enhancement in cromakalim-induced relaxation of corpus cavernosum was observed in 2-week diabetic animals, whereas the relaxant responses to cromakalim were decreased in 6-and 8-week diabetic animals. However, the cromakalim-induced relaxation was not altered in either 1-week or 4-week rat corporal strips in comparison with corresponding age-matched non-diabetic groups. Preincubation with the K(ATP) channel blocker glibenclamide (10 microM) significantly inhibited the cromakalim-induced relaxation in both non-diabetic and diabetic rat corpus cavernosum, but neither the voltage-dependent K(+) channel (K(V)) antagonist 4-aminopyridine (1 mM) nor the calcium-activated K(+) channel (K(Ca)) antagonist charybdotoxin (0.1 microM) had significant effect on cromakalim-induced relaxation in both control and diabetic rat corporal strips. Relaxation responses to the nitric oxide donor sodium nitroprusside (1 nM-0.1 mM) in diabetic rat corpus cavernosum were similar to that of age-matched controls. These data demonstrated that the relaxant responses to cromakalim were altered in diabetic cavernosal strips in a time dependent manner, suggesting that the period of diabetes mellitus may play a key role in the K(ATP) channels function in rat corpus cavernosum.     

Synthesis and phosphodiesterase 5 inhibitory activity of novel pyrido[1,2-e]purin-4(3H)-one derivatives

Synthesis and primary SAR of a novel series of 2-phenylpyrido[1,2-e]purin-4(3H)-one derivatives with piperazinyl sulfonamide substituents were described herein. As potential PDE5 inhibitors for erectile dysfunction (ED) treatment, representative compounds exhibit improved selectivity versus PDE1 and PDE6. Meanwhile, compound 3e demonstrated functional efficacy on rabbit corpus cavernosum strip in vitro.     

Histologic assessment of nerve regeneration in the rat

This study reports the degree of spontaneous regeneration that will occur in the sciatic nerve of a rat 5 months after complete resection of the nerve. In 30 animals, the sciatic nerve was excised. Histological assessment at 5 months revealed evidence of regeneration for a variable distance (mean 23.7 mm +/- 6.4 mm). Histological sections were studied at 1-cm intervals along the length of the nerve. Evidence of compartmentation with "minifascicle" formation was noted. The orientation of the nerve fibers was parallel to the long axis of the nerve. This study assessing spontaneous regeneration is meant to serve as a control for other studies evaluating the effect of factors that may influence nerve regeneration in the rat model.     

移植神经营养素-4基因工程细胞对脊髓前角神经元的保护作用

用重组逆转录病毒介导的体外神经营养素－４（ＮＴ－４）基因转移的方法,制备高表达ＮＴ－４基因工程细胞并移植大鼠坐骨神经离断处,尼氏染色和ＣｈＥ染色的结果表明,移植ＮＴ－４基因工程细胞对外周神经损伤所造成的运动神经元的退变有显著的改善作用,而且这种作用可以维持三个月。     

Regulation of cavernous nerve injury-induced apoptosis by sonic hedgehog

Thirty to eighty-seven percent of patients treated by radical prostatectomy experience erectile dysfunction (ED). The reduced efficacy of treatments in this population makes novel therapeutic approaches to treat ED essential. We propose that abundant apoptosis observed in penile smooth muscle when the cavernous nerve (CN) is cut (mimicking the neural injury which can result from prostatectomy) is a major contributing factor to ED development. We hypothesize that decreased Sonic hedgehog (SHH) signaling is a cause of ED in neurological models of impotence by increasing apoptosis in penile smooth muscle. We examined this hypothesis in a bilateral CN injury model of ED. We found that the active form of SHH protein was significantly decreased 1.2-fold following CN injury, that SHH inhibition causes a 12-fold increase in smooth muscle apoptosis in the penis, and that SHH treatment at the time of CN injury was able to decrease CN injury-induced apoptosis (1-3-fold) in a dose-dependent manner. These results show that SHH stabilizes the alterations of the corpora cavernosal smooth muscle following nerve injury.     

Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons

In situ hybridization histochemistry and RNA blot analysis were used to study expression of nerve growth factor receptor (NGF-R) mRNA in rat spinal cord motoneurons. The results show that NGF-R mRNA is expressed at high levels in rat spinal cord motoneurons at the time of naturally occurring cell death. This expression is sustained, but reduced, during synapse formation and is subsequently greatly reduced in the adult spinal cord. A unilateral crush lesion of the sciatic nerve resulted in an 8-fold increase in NGF-R mRNA in adult rat spinal cord motoneurons 3 days after lesion, compared with the nonlesioned side. NGF-R mRNA induction was even more pronounced 7 and 14 days after lesion, reaching levels 12 times higher than those on the nonlesioned side. However, 6 weeks after lesion, when the motor function of the leg was largely restored, NGF-R expression had decreased to levels similar to those on the contralateral side. We therefore suggest that NGF-R mediates a trophic or axonal guidance function for developing and regenerating spinal cord motoneurons.     

Neurohistological characteristics of regeneration of the ends of the injured nerve under measured traction]

A resection of sciatic nerve in 25 dogs was performed out and the ends of cut nerve were connected by sutures with special apparatus for quantified traction of nerve stumps. 5 days after resection traction was stented at the rate of 0.25 mm twice a day. A histological analysis performed in 5, 12, 19, 26, 33 days after resection an 2-12 months after nerve suture has shown that stress strain effects on intercalation of nerve structures (Ilizarov's effect). Newly formed Schwann chains free of products of axonal and myelin disruption have got a perfect longitudinal orientation and that's why can be considered as an optimum substratum for axonal regeneration.