Culture of major pelvic ganglion neurons from adult rat.

Successful culturing of neurons from adult animals has been historically difficult for a relatively long time. In this study, we reported the development of a novel method for the isolation and the culture of major pelvic ganglion (MPG) neurons from adult rat. The cultured cells were identified by neuron morphology and staining with neuronal marker (neurofilament-200, NF-200). The results demonstrate that the new protocol we used was reliable in obtaining a relatively high yield of MPG neurons. Furthermore, it improves the speed and simplicity in neuronal isolation. The viability of neurons can be maintained for about 2 weeks, which should be sufficient for investigating physiological and pathological processes occurring in mature major pelvic ganglia. And this may provide a useful assessment to currently available techniques for the culture of adult neurons.

Long-term safety, tolerability, and efficacy of α1-adrenergic blocker in young men with primary bladder neck obstruction: results from a single centre in China.

OBJECTIVE: Primary bladder neck obstruction (PBNO) is a nonneurogenic voiding disorder and frequently overlooked in young men. Prior studies have reported the efficacy of α-blockers only in the short-term for male patients with PBNO. We hereby report our long-term results using α1-blocker therapy in young men with PBNO. METHODS: Between January 2005 and December 2009, PBNO was diagnosed in 30 young men (mean age 27.3 years, range 18-35) at our institution. Doxazosin 4 mg once daily was administered for at least 12 months. Safety and tolerability were assessed, and efficacy was evaluated from International Prostate Symptom Score (I-PSS), Quality of Life (QOL), uroflowmetry, and post-void residual following 3- and 12-month treatment. Successful treatment was defined as at least 3 ml per second increase in the maximum flow rate and more than a 40% decrease in I-PSS. RESULTS: In all 30 patients, Mean symptom duration was 26.4 (3-65) months. The most common symptoms were hesitancy (93.3%), weak stream (76.7%), and frequency (66.7%). A total of 24 patients (80%, 24/30) successfully completed the 12 month of treatment. The medication period was 15.2 months, and follow-up duration was 16.3 months. Doxazosin was safe and well tolerated. The efficacy of doxazosin was maintained over the 12-month treatment period. Relative to baseline, there were reductions in the number of mean I-PSS (from 17.7 ± 4.2 to 10.4 ± 4.8), mean QOL (from 4.2 ± 1.1 to 2.4 ± 1.3), and mean post-void residual urine (from 79.3 ± 33.4 to 47.1 ± 21.3), and an increase in mean maximum flow rate (from 11.4 ± 2.9 to 15.1 ± 3.2 ml) after 12-month treatment. Treatment was successful in 16 patients (66.7%, 16/24) according to the improvement in both symptoms and maximum urine flow. CONCLUSIONS: α1-blocker therapy displayed a favorable safety, tolerability, and efficacy profile during 12-month treatment in young male patients with PBNO.

[Endourological treatment of aged high-risk patients with benign prostate hyperplasia: a report of 283 cases].

OBJECTIVE: To evaluate the safety and effectiveness of endourological techniques in the treatment of benign prostate hyperplasia (BPH) in aged high-risk patients. METHODS: We used endourological techniques in the treatment of 283 BPH patients aged over 70 years and complicated with hydronephrosis, renal failure, heart failure, cerebral infarction, respiratory dysfunction, anemia, diabetes, bladder tumor, or prostate weight over 80 g, TURP (transurethral resection of the prostate) for 112 cases and PKRP (transurethral plasmakinetic resection of the prostate) for the other 171. All the patients were followed up for 1-30 months. RESULTS: In the TURP group, the scores on IPSS and QOL were decreased from 27.5 +/- 2.8, 5.5 +/- 1.0 to 5.8 +/- 1.2, 1.0 +/- 0.5, and the residual urine volume (RUV) from (75.0 +/- 20.0) ml to (8.0 +/- 3.0) ml, but the maximal flow rate (Qmax) increased from (6.5 +/- 2.0) ml/s to (18.5 +/- 1.5) ml/s (P < 0.05), while in the PKRP group, the scores on IPSS and QOL were decreased from 28.2 +/- 2.2, 5.5 +/- 1.0 to 5.4 +/- 1.6, 1.0 +/- 0.5, and RUV from (80.0 +/- 20.0) ml to (7.0 +/- 3.0) ml, and Qmax increased from (6.8 +/- 2.1) ml/s to (20.0 +/- 1.5) ml/s (P < 0.05). There were no statistically significant differences in IPSS, QOL, Qmax and RUV after treatment between the two groups (P > 0.05), but significantly less complications were found in the PKRP than in the TURP group (P < 0.05). CONCLUSION: Endourological treatment, especially PKRP, with comprehensive perioperative preparations, unerring operative skills, well-controlled operation time, and intensive postoperative monitoring and nursing, has the advantages of high safety, less bleeding, fewer complications and definite effectiveness for aged high-risk BPH patients.

Effect of the artificial somato-autonomic neuroanastomosis on defecation after spinal cord injury and its underlying mechanisms.

A new artificial somatic-autonomic neuroanastomosis has been established in male rats with spinal cord injury (SCI). Anorectal manometry and neural retrograde tracing were conducted in this animal model to analyze the mechanisms and the effects on recovery of anorectal function. The left L4 ventral root (L4VR) was intradurally micro-anastomosed to the L6 ventral root (L6VR) to establish the new regenerated neural pathway. Three months later the spinal cord was completely transected at the T9-10 level. Eight weeks later the model rats were randomly divided into two groups. The rats in the group 1 (n=8) were applied for anorectal manometry, and those in the group 2 (n=4) were used for neural retrograde tracing study with fluorogold (FG) and dextran tetramethylrhodamine (TMR). The results of anorectal manometry showed the new reflex pathway could induce rectum to contract and simultaneously electric activity of external anal sphincter (EAS) to become weak or disappearing (indicating synergetic relaxation of EAS). FG and TMR dual labeled neurons with round and elliptical shape were mainly observed in L4 angulus anterior of model rats. The regenerated neural pathways were effective to improve the rectum external sphincter synergetic status and restore the anorectal function.

Nogo-A expresses on neural stem cell surface.

Nogo-A, as a myelin-associated molecule to inhibit axon regeneration in the injured adult central nervous system (CNS), has been detected to be enriched in numerous populations of cells in CNS. In this study, we found that Nogo-A was also expressed on the surface of neural stem cells (NSCs). The possible effects of NSCs-expressed Nogo-A on the NSC transplantation for CNS repair were discussed.

Effect of the Artificial Somato-Autonomic Neuroanastomosis on Defecation after Spinal Cord Injury and Its Underlying Mechanisms / 华中科技大学学报（医学）（英德文版）

A new artificial somatic-autonomic neuroanastomosis has been established in male rats with spinal cord injury(SCI).Anorectal manometry and neural retrograde tracing were conducted in this animal model to analyze the mechanisms and the effects on recovery of anorectal function.The left L4 ventral root(L4VR)was intradurally micro-anastomosed to the L6 ventral root(L6VR)to establishthe new regenerated neural pathway.Three months later the spinal cord was completely transected at the T9-10 level.Eight weeks later the model rats were randomly divided into two groups.The rats in the group 1(n=8)were applied for anorectal manometry,and those in the group 2(n=4)were used for neural retrograde tracing study with fluorogold(FG)and dextran tetramethylrhodamine(TMR).The results of anorectal manometry showed the new reflex pathway could induce rectum to contract and simultaneously electric activity of external anal sphincter(EAS)to become weak or disappearing (indicating synergetic relaxation of EAS).FG and TMR dual labeled neurons with round and elliptical shape were mainly observed in L4 angulus anterior of model rats.The regenerated neural pathways were effective to improve the rectum external sphincter synergetie status and restore the anorectal function.

Culture of motor neurons from newborn rat spinal cord.

A protocol for the isolation, purification and culture of motor neurons from newborn rat spinal cord was described and the effect of glial cell line-derived neurotrophic factor (GDNF) on the growth of neurite of motor neurons was investigated in vitro. Spinal motor neurons (SMNs) were dissociated from ventral spinal cord of postnatal day 1 rats. The culture system for SMNs was established by density gradient centrifugation, differential adhesion, and use of serum-free defined media and addition of exogenous GDNF. After 72-h culture, the cells displayed the characteristic morphology of motor neurons, exhibited extensive neuritic processes and were positive for choline acetyltransferase (ChAT) expression. The neurite length of SMNs in GDNF groups was significantly longer than that in control group (P<0.05). This protocol can be adapted for various postnatal motor neurons studies.

Culture of Motor Neurons from Newborn Rat Spinal Cord / 华中科技大学学报（医学）（英德文版）

ificantly longer than that in control group (P<0.05). This protocol can be adapted for various postnatal motor neurons studies.

Hybrid synapse formation between spinal motoneurons and superior cervical ganglion neurons in vitro: a study of the functional reconstruction of visceral organs.

Spinal motoneurons (SMNs) were co-cultured with labeled superior cervical ganglion neurons (SCGNs) in complete medium on coverslips prepared by the microisland technique. "Hybrid" synapses formation between SMNs and SCGNs was examined by double immunostaining, reverse transcription-polymerase chain reaction (RT-PCR), and electrophysiological assays. The number of synaptic puncta was significantly higher in co-cultured cells compared to controls (P<0.01). Spontaneous synaptic currents (SSCs) were also significantly increased in SCGNs within the co-cultured population of cells (P<0.01). These SSCs could be blocked by a specific nicotinic receptor blocker, but not by a glutamate receptor antagonist. These observations clearly demonstrate the functional synapses formation increased in our co-culture system, suggesting that the "hybrid" synapse formation occurred between somatic and automatic neurons. Our data also indicate that acetylcholine and nicotinic receptors may be involved in mediating these processes.

Tumor cell-specific blockade of CXCR4/SDF-1 interactions in prostate cancer cells by hTERT promoter induced CXCR4 knockdown: A possible metastasis preventing and minimizing approach.

Stromal cell-derived factor-1 (SDF-1)/CXCR4 pathway has been shown to play an important role in prostate cancer (PCa) metastasis and siRNA expression using cell-specific promoters has been demonstrated to be a potential tool for targeted gene therapy. Here, we illustrate that human telomerase reverse transcriptase (hTERT) promoter-induced CXCR4 knockdown inhibits PCa bone metastasis. We first investigated CXCR4 expressions and interactions of CXCR4/SDF-1 in PCa cells, developed a retrovirus system that could stably express CXCR4 small hairpin RNA driven by hTERT promoter and then determined the inhibitory effects of cell-specific blockade of CXCR4/SDF-1 pathway on PCa metastasis. It was shown that both PCa tissues and cell lines expressed CXCR4 and the expression in PCa tissue had a positive correlation to clinical stages while not to Gleason scores or serum PSA level. PCa metastases most presenting human tissues expressed high levels of SDF-1. Exogenous SDF-1 enhanced in vitro adhesion, migration and invasion of PCa cells and these bioeffects were repressed by hTERT promoter-induced CXCR4-shRNA expression. This CXCR4 knockdown was also found to significantly inhibit bone metastasis in vivo. We conclude that CXCR4/SDF-1 pathway plays an important role in PCa bone metastasis. hTERT promoter-induced tumor cell-specific CXCR4 gene silencing may prevent in vitro invasiveness and in vivo bone metastasis of PCa. These findings may enable new avenues of prevention and treatment for PCa metastasis.

DBC2 gene is silenced by promoter methylation in bladder cancer.

Deletions at 8p are frequent in many human cancers and represent a genetic marker associated with a more aggressive tumor phenotype. Previous mutational analysis of DBC2 (deleted in breast cancer 2), a tumor suppressor gene located in the region of loss of heterozygosity (LOH) on 8p21, failed to show a high frequency of mutation linked to low expression in bladder cancer. Promoter hypermethylation may be an alternative mechanism of inactivation of the second allele. We detected the methylation status and expression of the DBC2 gene in 75 bladder cancer samples and 57 corresponding normal tissues. Aberrant methylation and down-regulation of DBC2 were observed preferentially in tumor samples (P < 0.05), and the expression changes were associated with methylation (P < 0.05). These findings, together with the previously mutation reports, suggest that aberrant methylation in DBC2 promoter may be responsible for the expression loss of DBC2 expression in bladder cancer and this hypermethylation event could play a crucial role in the early stage of bladder tumorigenesis.

[Efficacy and safety of vardenafil for kidney transplant recipients with erectile dysfunction].

OBJECTIVE: To evaluate the efficacy and safety of vardenafil in kidney transplant recipients with erectile dysfunction. METHODS: Thirty-nine kidney transplant recipients with erectile dysfunction (ED) and serum creatinine values <2 mg/dl were enrolled in a 4-week randomized, double blind, placebo-controlled study, 19 treated with placebo and 20 with vardenafil. Vardenafil efficacy was assessed with the IIEF questionnaire after 4 weeks of treatment, and its safety appraised by measuring serum creatinine levels, creatinine clearances and cyclosporine concentrations before and after the treatment. RESULTS: IIEF scores improved from 12.6 +/- 3.4 to 26.5 +/- 2.8 (P < 0.01), but renal function and cyclosporine concentrations remained unchanged in the vardenafil-treated patients. Adverse effects were observed in 4 patients: headache in 2, palpitation and flush in 1, and dyspepsia in the other. CONCLUSION: Oral vardenafil therapy has a high efficacy and a low incidence of adverse events for kidney transplant recipients with ED.

[CXCR4 combined with P504S or P63 in differential diagnosis of benign and malignant prostatic diseases].

OBJECTIVE: To evaluate the application of CXC chemokine receptor-4 (CXCR4) combined with alpha-methylacyl-CoA racemase (P504S) or P63 protein in the differential diagnosis of benign and malignant prostatic diseases. METHODS: The EnVision immunohistochemical method was used to detect the expressions of CXCR4, P504S and P63 protein in 40 specimens of PCa not treated by any anticancer therapy and 30 specimens of BPH tissues. The correlation was analyzed between CXCR4 expression and the characteristics of PCa metastasis. RESULTS: Of the 40 cases of PCa, 33 (82.5%) were stained positive for CXCR4, 37 (92.5%) for P504S and 2 (5%) for P63 protein. Of the 30 cases of BPH, 5 (16.6%) exhibited positivity for CXCR4, 1 for P504S and all for P63. P504S + P63 showed a higher rate of correct diagnosis of PCa than either CXCR4 + P63 or P504S + CXCR4. There was a statistically significant correlation between CXCR4 expression and cancer metastasis (P < 0.05). CONCLUSION: P504S, CXCR4 and P63 are useful tumor markers for the diagnosis and differentiation of benign and malignant prostatic diseases. CXCR4 gives a high rate of correct diagnosis when combined with P504S or P63, and has an important application value in the differential diagnosis of benign and malignant prostatic diseases.

Morphological changes of cholinergic nerve fibers in the urinary bladder after establishment of artificial somatic-autonomic reflex arc in rats.

OBJECTIVE: To establish an artificial somatic-autonomic reflex arc in rats and observe the following distributive changes of neural fibers in the bladder. METHODS: Adult Sprague-Dawley rats were randomly divided into three groups: control group, spinal cord injury (SCI) group, and reinnervation group. DiI retrograde tracing was used to verify establishment of the model and to investigate the transport function of the regenerated efferent axons in the new reflex arc. Choline acetyltransferase (ChAT) in the DiI-labeled neurons was detected by immunohistochemistry. Distribution of neural fibers in the bladder was observed by acetylcholine esterase staining. RESULTS: DiI-labeled neurons distributed mainly in the left ventral horn from L3 to L5, and some of them were also ChAT-positive. The neural fibers in the bladder detrusor reduced remarkably in the SCI group compared with the control (P < 0.05). After establishment of the somatic-autonomic reflex arc in the reinnervation group, the number of ipsilateral fibers in the bladder increased markedly compared with the SCI group (P < 0.05), though still much less than that in the control (P < 0.05). CONCLUSION: The efferent branches of the somatic nerves may grow and replace the parasympathetic preganglionic axons through axonal regeneration. Acetylcholine is still the major neurotransmitter of the new reflex arc. The controllability of detrusor may be promoted when it is reinnervated by the pelvic ganglia efferent somatic motor fibers from the postganglionic axons.

Altered expression of connexin-43 and impaired capacity of gap junctional intercellular communication in prostate cancer cells.

Connexin-43 (Cx43) expression in prostate cancer (PCa) cells and the potency of gap junctional intercellular communication (GJIC) in the cells were investigated, with an attempt to elucidate the reason why the so-called "bystander effect" mediated by thymidine kinase (TK) suicide gene therapy on PCa cells is not of significance and to explore the role of GJIC in PCa carcinogenesis. mRNA and protein expression of Cx43 in a PCa cell line PC-3m was detected by reverse-transcription polymerase chain reaction (RT-PCR) and strapt-avidin-biotin-enzyme complex (SABC) immunohistochemical staining, and inherent GJIC of PC-3m cells was assayed by scrape-loading and dye transfer (SLDT) assay. The expression of Cx43 in human normal and malignant prostate tissues was determined by SABC immunohistochemistry as well. It was found that Cx43 mRNA and protein expression in PC-3m cells was slightly reduced as compared with positive controls and the location of Cx43 protein was aberrant in cytoplasm rather than on membrane. Assessment of paraffin sections demonstrated that the expression of Cx43 protein in PCa cells was abnormally located and markedly diminished as compared with normal prostatic epithelial ones, displaying a negative correlation to the pathological grade (chi2=4.025, P<0.05). Additionally, capacity of inherent GJIC in PC-3m cells was disrupted, which was semi-quantified as (+) or (-). It was indicated that both down-regulated expression of Cx43 mRNA and aberrant location of Cx43 protein participated in the mechanisms leading to deficient GJIC in PC-3m cells. Lack of efficient GJIC is a molecular event, which may contribute not only to limited extent of "bystander effect", but also to initiation and progression of prostatic neoplasm.

[Role of MAPK signal transduction pathway phosphorylation induced by granulocyte-macrophage-colony stimulating factor in the proliferation of hormone-independent prostate cancer].

OBJECTIVE: To investigate the phosphorylation intensity of MAPK pathway molecular Erk1/2 and the proliferation of prostate cancer cell line PC-3M. METHODS: Flow cytometry and RT-PCR were employed to study the ratio of different cell cycles and phases, respectively, before and after GM-CSF stimulation. Erk1/2 phosphorylation intensity was examined by Western blot simultaneously. RESULTS: The rate of PC-3M cells at S and G2/M stages and the expression intensity of Ki-67 increased after GM-CSF incubation in a dose-dependent manner. The phosphorylation intensity of Erk1/2 increased remarkably after stimulation with GM-CSF. CONCLUSION: The intensification of Erk1/2 phosphorylation is one important molecular mechanism of the proliferation of hormone-independent prostate cancer.

Anorectal functions in patients with lumbosacral spinal cord injury.

OBJECTIVE: To investigate the anorectal status in patients with lumbosacral spinal cord injury (SCI). METHODS: Twenty six patients (23 males, 3 females) with lumbosacral SCI and 13 normal volunteers were enrolled into this study as controls. The median age was 43.7 years (ranging 17-68 years) and the median time of patients since injury was 59.1 months (ranging 8 months-15 years). They were diagnosed as complete lumbosacral SCI (n =2, American Spinal Injury Association (ASIA) score A), or incomplete lumbosacral SCI (n=24, ASIA score B-D) with mixed symptoms of constipation and/or fecal incontinence, and were studied by anorectal manometry. None of the patients had any medical treatments for neurogenic bowel prior to this study. RESULTS: The maximum anal resting pressure in lumbosacral SCI patients group was slightly lower than that in control group (One-way ANOVA: P=0.939). During defecatory maneuvers, 23 of 26 (88.5%) patients with lumbosacral SCI and 1 of 13 (7.7%) in the control group showed pelvic floor dysfunction (PFD) (Fisher's exact test: P<0.0001). Rectoanal inhibitory reflex (RAIR) was identified in both patients with lumbosacral SCI and the controls. The rectal volume for sustained relaxation of the anal sphincter tone in lumbosacral SCI patients group was significantly higher than that in the control group (Independent-Samples t test: P<0.0001). The mean rectal volume to generate the first sensation was 92.7 ml+/-57.1 ml in SCI patients, 41.5 ml+/-13.4 ml in the control group (Independent-Samples t test: P<0.0001). CONCLUSIONS: Most of the patients with lumbosacral SCI show PFD during defecatory maneuvers and their rectal sensation functions are severely damaged. Some patients exhibit abnormal cough reflex. Anorectal manometry may be helpful to find the unidentified supraconal lesions. RAIR may be modulated by central nervous system (CNS).

Reinnervation for neurogenic bladder: historic review and introduction of a somatic-autonomic reflex pathway procedure for patients with spinal cord injury or spina bifida.

Neurogenic bladder caused by SCI or spina bifida is a major problem. Research in restoring functional micturition has mainly focused on electrical stimulation for many decades with good progress, but it is still not the definitive solution for majority of the SCI patients. An alternative approach has been to investigate restoring innervation to the lower urinary tract after spinal SCI. Different animal and clinical studies were reviewed historically in this article, focused on mainly cross over nerve surgery for reinnervation of the bladder. An artificial somatic-autonomic reflex pathway procedure and its mechanisms were introduced. Clinical application and the satisfactory results of the new procedure were reviewed in details in restoring voluntary bladder control in patients with SCI or spina bifida.