Capsaicin mediates cell death in bladder cancer T24 cells through reactive oxygen species production and mitochondrial depolarization.

OBJECTIVES: To investigate the effects of capsaicin (CAP) on proliferation of bladder cancer T24 cells in vitro as well as on xenografts in nude mice in vivo. METHODS: T24 cells were assessed for cell viability and apoptosis by 3-(4, 5-dimethylthiazol-2-yl)-3, 5-diphenyltetrazolium bromide assay and flow cytometry analysis after incubation with different concentrations of CAP. To uncover the mechanism by which CAP affected the viability of T24 cells, intracellular production of reactive oxygen species (ROS) and mitochondrial membrane potential were assessed. To study the in vivo effects of CAP, T24 cells were grown as xenografts in nude mice and CAP (5 mg/kg by wt) was subcutaneously injected into nude mice with bladder tumors. RESULTS: CAP decreased the viability of T24 cells in a dose-dependent manner without marked apoptosis. CAP induced ROS production and mitochondrial membrane depolarization, thereby inducing cell death, not apoptosis, in T24 cells at a concentration of 100 microM or higher. Furthermore, these effects of CAP could be reversed by capsazepine, the antagonist of transient receptor potential vanilloid type 1 channel. In vivo experiment showed that CAP significantly slowed the growth of T24 bladder cancer xenografts as measured by size (661.80 +/- 62.03 vs 567.02 +/- 43.94 mm(3); P <.01). CONCLUSIONS: CAP mediates cell death in T24 cells through calcium entry-dependent ROS production and mitochondrial depolarization, and it may have a role in the management of bladder cancer.

Effects of TRPM8 on the proliferation and motility of prostate cancer PC-3 cells.

We investigated the effects of transient receptor potential M8 (TRPM8) channel on the proliferation and motility of androgen-independent prostate cancer PC-3 cells. After being permanently transfected with an empty vector and cDNA encoding the TRPM8 protein, cells were analysed for cell cycle distribution and motility using flow cytometry and scratch assay. Immunocytochemistry and Ca2+ imaging analysis revealed the overexpression of functional TRPM8 channel on both endoplasmic reticulum and plasma membrane of PC-3-TRPM8 cells. Cell cycle distribution and scratch assay analysis revealed that TRPM8 induced cell cycle arrest at the G0/G1 stage (P < 0.05) and facilitated the cell apoptosis induced by starvation (P < 0.05). Furthermore, TRPM8 inhibited the migration of PC-3-TRPM8 cells (P < 0.01) through the inactivation of focal-adhesion kinase. It appears that TRPM8 was not essential for the survival of PC-3 cells; however, the overexpression of TRPM8 had negative effects on the proliferation and migration of PC-3 cells. Thus, TRPM8 and its agonists may serve as important targets for the treatment of prostate cancer.

The effect of platelet-rich plasma on cavernous nerve regeneration in a rat model.

The aim of this study was to investigate the effect of platelet-rich plasma (PRP) on cavernous nerve (CN) regeneration and functional status in a nerve-crush rat model. Twenty-four Sprague-Dawley male rats were randomly divided into three equal groups: eight had a sham operation, eight underwent bilateral nerve crushing with no further intervention and eight underwent bilateral nerve crushing with an immediate application of PRP on the site of injury. Erectile function was assessed by CN electrostimulation at 3 months and nerve regeneration was assessed by toluidine blue staining of CN and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining of penile tissue. 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 application of PRP, the mean maximal ICP and maximal ICP/MAP were significantly higher than those in the injured control group. Histologically, the group with the application of PRP had more myelinated axons of CNs and more NADPH-diaphorase-positive nerve fibres than the injured control group but fewer than the sham group. These results show that the application of PRP to the site of CN-crush injury facilitates nerve regeneration and recovery of erectile function. Our research indicates that clinical application of PRP has potential repairing effect on CN and peripheral nerves.

Laparoscopic radical cystectomy with orthotopic gastric neobladder: technique and initial outcomes.

OBJECTIVES: To report our operative technique and initial outcomes of laparoscopic radical cystectomy (LRC) and external orthoptic gastric neobladder. METHODS: Since 2003, nine patients have undergone laparoscopic radical cystectomy with orthotopic gastric neobladder at our institution. The specimen is extracted through a 6-cm vertical minilaparotomy incision above the umbilicus. The gastric neobladder was constructed as our open technique using the part of stomach body and antrum with the pedicle of gastroepiploic vascular bundle through the site of specimen retrieval. The operative data, complications and follow-up functional data were analyzed. RESULTS: The mean operative time was 365 min (300-450). Mean blood loss was 520 ml (200-1,500) and four patients (44.4%) required blood transfusion. In all cases no conversion to open surgery was necessary. The length of stay was 17 days and the total complication rate was 55.6% (five cases). All patients were free of recurrence at a mean follow-up of 22 months (3-48). The day and night incontinence rate was 11.1 and 44.4%, respectively. At 6 months after operation, urodynamic evaluations indicated a larger capacity, low pressure urinary reservoir. CONCLUSIONS: Laparoscopic radical cystectomy with orthotopic gastric neobladder is a feasible intervention. The external construction of the gastric neobladder using the part of stomach body and antrum is quick and safe. With precise and increased operative technique, the LRC with orthotopic gastric bladder may be a good choice for urinary diversion. However, the larger samples, long-term compared studies with bowel diversions are required to evaluate this new technique.

[Effect of platelet rich plasma on the regeneration of cavernous nerve: experiment with rats].

OBJECTIVE: To investigate the effect of platelet rich plasma (PRP) on the regeneration of injured cavernous nerve (CN). METHODS: Blood was collected from the hearts of 6 SD rats to prepare PRP. 24 male adult rats were randomly divided into 3 equal groups: pure suture group undergoing bilateral CN transaction and pure suture immediately, PRP group undergoing bilateral CN transaction + suture + PRP 200 microl to the site of suture, and sham operation group. 3 months later intracavernous pressure (ICP) was measured by CN electrostimulation and then samples of CN were obtained to undergo pathological examination. RESULTS: 3 months later after surgery, the ICP of the pure suture group was (46 +/- 8) cm H2O, significantly lower than that of the sham group [(109 +/- 13) cm H2O, P < 0.01], and that of the PRP group was (94 +/- 13) cm H2O, significantly higher than that of the pure sutured group (P < 0.01), however, still significantly lower than that of the sham operation group (P < 0.05). The number of axons of CN in the PRP group was (121 +/- 16), significantly higher than that of the pure sutured group (70 +/- 14, P < 0.01); however, still significantly lower than that of the sham operation group (181 +/- 21, P < 0.01). CONCLUSION: PRP can promote the regeneration of injured CN and the recovery of erectile function.

Improvement in erectile dysfunction after insulin-like growth factor-1 gene therapy in diabetic rats.

AIM: To determine whether adenoviral gene transfer of insulin like growth factor-1 (IGF-1) to the penis of streptozotocin (STZ)-induced diabetic rats could improve erectile capacity. METHODS: THE STZ diabetic rats were transfected with AdCMV-betagal or AdCMV-IGF-1. These rats underwent cavernous nerve stimulation to assess erectile function and their responses were compared with those of age-matched control rats 1 to 2 days after transfection. In control and transfected STZ diabetic rats, IGF-1 expression were examined by reverse transcription polymerase chain reaction (RT-PCR), Western blot and histology. The penis beta-galactosidase activity and localization of the STZ diabetic rats were also determined. RESULTS: One to two days after transfection, the beta-galactosidase was found in the smooth muscle cells of the diabetic rat penis transfected with AdCMV-betagal. One to 2 days after administration of AdCMV-IGF-1, the cavernosal pressure, as determined by the ratio of maximal intracavernous pressure-to-mean arterial pressure (ICP/MAP) and total intracavernous pressure (ICP), was increased in response to cavernous nerve stimulation. Transgene expression was confirmed by RT-PCR, Western blot and histology. CONCLUSION: Gene transfer of IGF-1 significantly increased erectile function in the STZ diabetic rats. These results suggest that in vivo gene transfer of IGF-1 might be a new therapeutic intervention for the treatment of erectile dysfunction (ED) in the STZ diabetic rats.

[Growth hormone enhances regeneration of cavernous nerves after their transplantation in rats].

OBJECTIVE: To investigate the effect of growth hormone (GH) on penile erection after reconstruction of cavernous nerves using sural nerve as an interposition nerve graft in rats. METHODS: Twenty-four male Sprague-Dawley rats (3-4 ms of age and 300-400 g in weight) were randomly divided into 2 groups: nerve graft group and GH group, each electrostimulated to determine the erectile potency 2 and 4 months after nerve graft (followed by hypodermic GH injection). The nNOS-positive nerve fibers in the corpora cavemosa were examined by streptavidin-peroxidase immunohistochemistry technique (SP method). Image analysis was used to calculate the area stained in pixel. RESULTS: Electrostimulation at 2 months produced 31.25% of erections in the GH group but none in the grafted rats. There was a significant difference in the erection rate produced by electrostimulation between the two groups at 2 months (P < 0.05). The pixel of the expression of nNOS-positive nerve fibers in the GH group (38971 +/- 7692) was also greater than that of the graft group (16538 +/- 3179, P < 0.05). At 4 months, 43.75% of the graft group and 75% of the GH group produced erections upon electrostimulation, with no significant difference between the two groups (P > 0.05). The pixels of the expression of nNOS-positive nerve fibers were 79276 +/- 12,021 and 91348 +/- 18965, respectively (P > 0.05). CONCLUSION: GH can accelerate the regeneration of cavernous nerves after bilateral nerve grafting, and GH administration may present a new physiological approach to the treatment of erectile dysfunction after radical pelvic surgery.

Effects of epidermal growth factor on sperm content and motility of rats with surgically induced varicoceles.

AIM: To investigate the effect of epidermal growth factor (EGF) on the sperm content and motility of the varicocelized rats. METHODS: Forty-eight male Wistar rats were randomly divided into five groups. Experimental varicocele was induced by partial ligation of the left renal vein in the varicocele, the varicocele repair, the varicocele with EGF and the varicocele repair with EGF groups, whereas the control group only received a sham induction of varicocele. Surgical repair of varicocele was performed 4 months later in the varicocele repair and varicocele repair with EGF groups. EGF administration was performed daily by s.c. injection in the varicocele with EGF and varicocele repair with EGF groups at the dose of 10 microg/(kg.day) from the next day of the second surgery. One month later, all animals were killed and bilateral cauda epididymal sperm counts and motility were evaluated. RESULTS: The mean sperm count and percentage of motile spermatozoa were significantly higher bilaterally in the varicocele with EGF group than in the varicocele group (P < 0.05). They were also significantly higher bilaterally in the varicocele repair with EGF group than in the varicocele repair and the varicocele with EGF groups (P < 0.05). CONCLUSION: EGF can improve bilateral epididymal sperm content and motility of the rat with surgically induced varicocele. The administration of EGF in combination with surgical repair is more effective than surgical repair or EGF administration alone. EGF might be useful for the treatment of infertility induced by varicocele.

[Vas-to-Epididymis antidromic injection of 30% ethanol reduces sperm motility in rats].

OBJECTIVE: To investigate the reduction of sperm motility in rats induced by vas-to-epididymis antidromic injection of 30% ethanol and its mechanism. METHODS: Forty male adult Sprague-Dawley rats were randomized into 3 groups: bilateral vas injection (n = 15) , sham operation control (n = 15) and normal (n = 10). An aliquot of 0.5 ml of 30% ethanol was injected from vas to epididymis bilaterally. After 1 month, all the rats'vasa and epididymides were ablated for studies of the sperm motility, construction changes of the vas and contents of IL-6, IFN-gamma and carnitine of the epididymis. RESULTS: There was markedly significant difference in sperm motility in the injection group (P < 0.01). The number of sperms in the bilateral vas injection group was 31, while in the sham operation control and normal groups was 64 and 68, respectively. The contents of IL-6 and IFN-gamma increased, and the carnitine reduced significantly (P < 0.05). However, no significant differences were noted between the control and the normal groups (P > 0.05). The contents of IL-6, IFN-gamma and carnitine in the bilateral vas injection group were 772.7 pg/ml, 350.7 pg/ml and 491.1 mol/L. But the same indexes in the sham operation and normal groups were 308.5 pg/ml, 172. 2 pg/ml and 664. 6 mol/L and 287. 8 pg/ml, 163. 8 pg/ml and 605.5 mol/L. CONCLUSION: The antidromic injection of ethanol from vas to epididymis can not only interfere the environment for sperm maturation but also activate the immunologic cells that secrete many cytokines (CK) in the genital system. All the factors can induce the reduction of sperm motility.

Expression of transforming growth factor-beta1 in penile tissue from rats with bilateral cavernosal nerve ablation.

OBJECTIVE: To explore the expression of transforming growth factor-beta(1) (TGF-beta1) in penile tissue from rats after bilateral cavernosal nerve (CN) ablation, mimicking patients who have had no nerve-sparing during prostatectomy. MATERIALS AND METHODS: Ten adult male rats (neurectomy group) had a bilateral CN resection aseptically under an operating microscope, with six sham-operated rats as controls. Fifteen weeks after surgery an apomorphine test was used in all rats to assess penile erection. The penile specimens were then collected and prepared for detecting the expression of TGF-beta1 by reverse transcription-polymerase chain reaction (RT-PCR), western blot and immunohistochemistry, and for quantitative analysis of the ratio of smooth muscle to collagen fibres in the corpus cavernosum with confocal microscopy. RESULTS: All rats in the sham-operated group but none after neurectomy had an erectile response after subcutaneous injection with apomorphine (100 micro g/kg). Immunohistochemistry, RT-PCR and western blot analyses showed a significantly higher expression of TGF-beta1 in the penile tissues after neurectomy than after sham surgery. Smooth muscle cells (fluorescing red) and collagen fibres (green autofluorescence) after paraformaldehyde fixation, were clearly identified by confocal microscopy. The fluorescence intensity expressed as the mean (sem) ratio of smooth muscle to collagen fibres in the corpus cavernosum after neurectomy was 0.265 (0.125), significantly lower than that in the sham-operated group, at 0.760 (0.196) (P < 0.01). CONCLUSION: An increased expression of TGF-beta1 in penile tissue which promotes the synthesis of collagen may be one of the important factors for the erectile dysfunction caused by bilateral CN ablation. Similar pathophysiological processes may occur in the corpus cavernosum of patients after radical prostatectomy.

[Effects on erectile function of transplanted major pelvic ganglion into the corpus cavernosum of adult rats with bilateral cavernous nerve injury].

OBJECTIVE: To investigate the effects on erectile function of transplanted major pelvic ganglion into the corpus cavernosum of adult male rats undergoing transection of bilateral cavernous nerves. METHODS: Twenty-six male Sprague-Dawley rats (3 - 4 month-old and 300 - 400 g/each) were divided into 2 groups: experimental group (transection of bilateral cavernous nerves and transplantation of left ganglion into left crus of penis, n = 16) and control group (transection of bilateral cavernous nerves only, n = 10). Erectile function was measured by injecting APO, and intracavernous pressure was measured 1 and 3 months afterwards by electric-stimulating the right major pelvic ganglion or the left crus. Half animals in each group were sacrificed 1 and 3 months afterwards for detecting nNOS-containing nerve fibers of corpus cavernosum. Electron microscopy of the implanted area was performed to assess neuronal survival. RESULTS: Both of the two groups have no erectile response to APO injection. Electrostimulation on the right major pelvic ganglion and left crus failed to produce erection in experimental group. The mean pressure changes in the two groups, measured by stimulating the left crus, were (9.41 +/- 3.20) and (4.16 +/- 2.58) cmH(2)O 1 month afterwards, and (13.67 +/- 4.18) and (5.09 +/- 2.74) cmH(2)O 3 months afterwards, respectively (P < 0.05). An increased number of nNOS-containing nerve fibers in left crus was detected in experimental group 1 and 3 months later, compared with control one (218.7 +/- 24.5, 18.0 +/- 3.7; 183.2 +/- 19.7, 19.0 +/- 3.8; P < 0.05). Ultrastructure examination by transmission electron microscope confirmed the survival of the implanted ganglion. CONCLUSION: Major pelvic ganglion can survive in the corpus cavernosum, and it has significant effects on the number of nNOS-containing nerve fibers and the alteration of intracavernous pressure.

[Expression of FasL in rat cryptorchidism].

OBJECTIVE: To investigate the expression of FasL in rat cryptorchidism and its significance. METHODS: Twenty-four male SD rats (22-day old) were randomly divided into two groups: unilateral cryptorchid group (n = 12) and pseudo-operation group (n = 12). When the rats were 110-day old, blood samples were taken and the rats were killed for analysis. Immunohistochemical method (SP) was used to detect FasL expression in testes and ELISA method to detect serum antisperm antibody (AsAb). RESULTS: The positive FasL expression rates in cryptorchid and contralateral testes were significantly higher than those in pseudo-operation group (P < 0.001). The serum AsAb positive rates in the cryptorchid group and the pseudo-operation group were 41.7% and 0, respectively, with significant difference(P < 0.01). CONCLUSIONS: FasL expression upregulating in both testes of the unilateral cryptorchid rat may be a protective response of the testis to autoimmunity.

[Germ cell apoptosis and expression of Bcl-2 and Bax following testicular torsion/detorsion in rats].

OBJECTIVES: To investigate the relationship between germ cell apoptosis and expression of Bcl-2 and Bax in experimental torsed/detorsed testes of the adult male rats. METHODS: Thirty healthy male Sprague-Dawley rats were divided into torsion group (n = 15) and control group (n = 15) randomly. Animals were submitted to unilateral 720 testicular torsion, then detorsion were done in two hours. Three days later, the evidence of germ cell apoptosis was detected by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) technique. The expression of Bcl-2 and Bax were detected by immunohistochemical method. RESULTS: In the torsed testes, the apoptosis index of germ cell and Bax expression significantly increased compared with that in the control group (P < 0.01) while Bcl-2 expression obviously decreased (P < 0.01). The apoptotic cells were mostly pechytene spermatocytes and round spermatides. CONCLUSIONS: The germ cell apoptosis is highly associated with expression of Bcl-2 and Bax in experimental testicular torsion. Bcl-2/Bax plays an important role in germ cell apoptosis.

[The rat model of erectile dysfunction caused by cavernous nerve injury].

OBJECTIVES: To identify rat cavernous nerve and establish a rat model of erectile dysfunction (ED) caused by injury of cavernous nerve. METHODS: Twenty rats were undergone dissections. Cavernous nerves were identified with the aid of operating microscope and confirmed by electrical stimulation. Then, 42 experimental rats were randomized into 3 groups, including sham operated controls, unilateral and bilateral cavernous nerve ablation groups. Three weeks after surgery, rat models were evaluated with Apomorphine Test. RESULTS: The major pelvic ganglion lies on either side of the dorsolateral lobes of the prostate. It includes 2 inflows, one called hypogastric nerve and another is pelvic nerve. The largest outflow is termed as cavernous nerve. Stimulus parameters which could induce obvious penile erection were 5 volts, frequency of 20 Hertz and duration of 5 milliseconds. Three weeks after surgery, apomorphine could induce penile erection of each rat in controls with mean (2.57 +/- 1.40) erections in 30 minutes, while there were no erections (0.00 +/- 0.00) either in unilateral or bilateral group. CONCLUSIONS: The rat of larger ganglion and its cavernous nerve can be easily identified, obvious response to electrical stimulation, low cost of animal purchase, easy housing and availability made rat as an ideal animal for establishing ED model caused by cavernous nerve injury. In addition, our study showed in the early period of cavernous nerve injury, either unilateral or bilateral, all rats lost their erectile function.