[Nicotine- and tar-free cigarette smoke extract reduces the penile erectile function of rats].

OBJECTIVE: To evaluate the impact of nicotine- and tar-free cigarette smoke extract (fCSE) on the serum testosterone (T) level and erectile function of male rats. METHODS: We randomized 30 male SD rats to three groups of equal number to receive subcutaneous injection of PBS (1.0 ml / 300 g body weight per day), fCSE (1.0 ml/300 g body weight per day), and reduced glutathione hormone (GSH, 200 mg per kg body weight per day) in addition to fCSE (fCSE + GSH), respectively, all for 8 weeks. Then we evaluated the erectile function of the rats by measuring the maximal intracavernous pressure (MICP), mean arterial pressure (MAP), ICP/MAP ratio, time of stimulation to MICP (Tmax), and cavernosal filling fate (CFR). We determined the serum T level, the activities of superoxide dismutase (SOD) , malondialdehyde (MDA), and nitric oxide synthase (NOS) in the cavernosal tissue, and also observed the morphological changes of the corpus cavernosum. RESULTS: Compared with the controls, the rats of the fCSE group showed obvious decreases in the levels of serum T ([5.37 ± 1.43] vs [3.22 ± 1.11] µg/L), NOS ([2.90 ± 0.27] vs [1.67 ± 0.18] U/mg) , and SOD ([18.41 ± 1.09] vs [13.36 ± 1.18] U/mg prot) and erectile function-related indexes MICP ([85.92 ± 6.36] vs [58.99 ± 10.76] mmHg), MICP/MAP (0.86 ± 0.09 vs [0.56 ± 0.08]), and CFR (2.14 ± 0.44 vs 0.89 ± 0.44), but markedly increased Tmax ([29.90 ± 5.78] vs [42.90 ± 8.56]s), with a positive correlation between the serum T level and CFR (r = 0. 364, P < 0.05). Masson staining revealed a lower ratio of the corpus cavernosum smooth muscle tissue to collagen fiber in the fCSE group (0.27 ± 0.04) than in the control (0.98 ± 0.12). Compared with the fCSE group, the fCSE + GSH group exhibited significantly improved MICP ([58.99 ± 10.76 ] vs [77.95 ± 7.71] mmHg), MICP/MAP (0.56 ± 0.08 vs 0.77 ± 0.09), and CFR (0.89 ± 0.44] vs 1.76 ± 0.42) and shortened Tmax ([42.90 ± 8.56 ] vs [32.10 ± 5.84 ] s). The ratio of the corpus cavernosum smooth muscle tissue to collagen fiber was higher in the fCSE + GSH than in the fCSE group (0.77 ± 0.09 vs 0.27 ± 0.04) but still lower than in the control (0.98 ± 0.12). CONCLUSION: Nicotine- and tar-free cigarette smoke extract reduces the serum T level and erectile function of rats, which is related to oxidative stress. Antioxidant therapy can improve erectile function but has a limited value for morphological protection of the penile tissue.

Tissue-engineered conduit using bladder acellular matrix and bladder epithelial cells for urinary diversion in rabbits.

BACKGROUND: For muscle invasive bladder cancer, radical cystectomy is the most effective treatment now and urinary diversion is often necessary. The use of intestinal tissue for urinary diversion is frequently associated with complications. In this study, we aimed to make a tissue-engineered conduit (TEC) using bladder epithelial cells and bladder acellular matrix (BAM) for urinary diversion in rabbits. METHODS: Bladder epithelial cells of rabbit were cultivated and expanded in vitro, then seeded on BAM, and cultured for 7 days. Then cell-seeded graft was used to make TEC. In the experimental group, most of bladder of the rabbit was removed while bladder trigone was retained. The proximal end of TEC was anastomosed with bladder trigone and the distal end was anastomosed with the abdominal stoma. In the control group, TEC was made using unseeded BAM. Haematoxylin and eosin staining was conducted, respectively, at 1, 2, 4, and 8 weeks postoperatively. Immunohistochemistry was performed 8 weeks postoperatively. Intravenous urography, retrograde pyelography, and cystoscopy of TEC were made at 12 weeks postoperatively. RESULTS: All animals were alive in the experimental group. Haematoxylin and eosin staining showed epithelial coverage in TEC. Immunohistochemistry showed anti-cytokeratin AE(1)/AE(3) antibody and anti-ZO1 antibody positive, confirming there were mature and functional epithelial cells on the lumen of TEC. Retrograde pyelography and intravenous urography showed that TEC developed well and that there was no obstruction. In the control group, four rabbits were dead within 2 weeks and scar formation, atresia, and severe hydronephrosis were found. CONCLUSIONS: We successfully made TEC using BAM and bladder epithelial cells for urinary diversion in rabbits. The lumen of this new TEC covered mature epithelial cells and could prevent urinary extravasation.

Characteristics of patients with fluid extravasation during retrograde ureteroscopic holmium laser lithotripsy for renal calculi.

OBJECTIVE: To explore the risk factors of fluid extravasation during retrograde ureteroscopic holmium laser lithotripsy for renal calculi. METHODS: Three hundred and twenty-seven patients with renal calculi ranging 10 to 20 mm received retrograde ureteroscopic holmium laser lithotripsy at Renmin Hospital of Wuhan University, Wuhan, China from January 2004 to December 2010. The clinical records were reviewed, and the correlation was studied between various clinical factors and fluid extravasation complications during operation. The clinical factors to be tested included patients' gender and age (<30, 30-50, and >50 years), hydronephrosis degree, previous intervention for renal calculi (none, shock-wave lithotripsy, and open surgery), upper urinary tract infection, ureteral access sheath placement, and procedure duration (<60, 60-120, and >120 mins). The data were processed by SPSS Version 16.0 statistical software, x2 test, and binary logistic regression were used for analysis. RESULTS: Fluid extravasation complications appeared in 35 patients. Patients` gender, age, and hydronephrosis degree were irrelevant to the occurrence of fluid extravasation, while having previous open surgery for renal calculi, without ureteral access sheath placement, upper urinary tract infection, and long procedure duration were all responsible for higher incidence of the complications. CONCLUSION: Reasonable selection of patients, effective control of upper urinary tract infection, routine ureteral access sheath placement, and controlling procedure duration help to decrease the incidence of fluid extravasation complications in retrograde ureteroscopic holmium laser lithotripsy for renal calculi.

Effects of buried penis on the structure and function of corpus cavernosum in a rat model.

BACKGROUND: While the abnormal appearance of the concealed penis has been well recognized, the effect of buried penis on the structure and function of corpus cavernosum has not been well studied. To explore this issue, we established a rat model and evaluated the effect of buried penis on cavernosum weight, contents and ultrastructure of tissue, and nitric oxide synthase (NOS) activity. METHODS: Two hundred and ten rats were randomly divided into 3 equal cohorts for 2, 4 and 6 months study (groups A, B and C). Each group was randomly divided into buried group (n = 40), control group (n = 15), and normal group (n = 15), respectively. Intra-purse-string suture of the root of the penis was used to establish the model. Macroscopic development was judged by measuring the weight of the corpus cavernosum. Masson's trichrome staining was performed for observing microstructure while a transmission electron microscope was used for observing ultrastructure. The NOS activity was detected by a NOS activity assay kit. RESULTS: Buried penis had no significant influence on the appearance and weight of the corpus cavernosum. Buried penis resulted in decreased smooth muscle content (P > 0.05 in group A, and P < 0.05 in groups B and C) and increased fibrous connective tissue content (P > 0.05 in groups A and B, and P < 0.05 in group C) compared with the normal and control groups. Ultrastructural abnormalities of corpus cavernosum were observed in the 6-month buried group. Moreover, there was decrease of NOS activity in groups B and C (P < 0.05 in group B and P < 0.01 in group C) when compared with the normal and control groups. CONCLUSION: Buried penis affects the structure and function of corpus cavernosum in rats and the effect is positively correlated with the buried time, but there is no significant effect on the macroscopic development.

[Murine model of busulfan-induced spermatogenesis regeneration: a quantitative evaluation].

OBJECTIVE: To quantitatively evaluate the murine model of spermatogenesis regeneration induced by two-dose busulfan injection. METHODS: Fifty-four male mice were randomly divided into a control and two model groups of equal number, the former treated by two-dose intraperitoneal injection of 50% DMSO solution at 10 ml/kg, and the latter by that of busulfan at 10 mg/kg and 15 mg/kg respectively to establish spermatogenesis regeneration models, both at the interval of 24 days between the two doses. Spermatogenesis in seminiferous epithelia was evaluated by Johnsen score, and the expressions of GATA-4 and GDNF mRNA in Sertoli cells were detected by real time quantitative PCR at 3, 4 and 8 weeks after the treatment. RESULTS: Johnsen score kept stable in the control group at all stages (P > 0.05), but higher than in the model groups at 3 and 4 weeks (P < 0.01). It was lower in the 15 mg/kg than in the 10 mg/kg model group at 4 and 8 weeks (P < 0.01) , and than in the control group at 8 weeks (P < 0.05), but had no significant difference between the 10 mg/kg and the control groups (P > 0.05). Nor did the expression of GATA-4 mRNA in Sertoli cells show any significant difference among the three groups at different stages after the treatment (P > 0.05), and that of GDNF mRNA at different stages in the control group (P > 0.05). Compared with the controls, the level of GDNF mRNA in Sertoli cells was significantly higher at 3 weeks but lower at 4 weeks in the model groups (P < 0.01), and lower in the 15 mg/kg group (P < 0.01) and comparable in the 10 mg/kg group at 8 weeks (P > 0.05); and it was lower in the 15 mg/kg than in the 10 mg/kg group at all stages (P < 0.01). CONCLUSION: Two-dose intraperitoneal injection of 10 mg/kg busulfan at the interval of 24 days is an optimal option for the establishment of a murine model of spermatogenesis regeneration. Higher dose of busulfan may induce deficient expression of GDNF in Sertoli cells and result in incomplete restoration of spermatogenesis.

[Reconstruction of rabbit urethra using urethral extracellular matrix].

OBJECTIVE: To evaluate the feasibility of reconstruction of rabbit urethra using urethral extracellular matrix. METHODS: Extracellular matrix was obtained from the urethra of 20 donor New Zealand rabbits. In experimental group, 20 rabbits underwent segmental urethral resection (about 1.0 to 1.5 cm in length) and the defects were replaced by a tube of extracellular matrix. The serum TNFalpha was detected by ELISA to assess the immunity response preoperatively and 12, 24, 48 h postoperatively. The regenerated urethral segments were taken for histologic and pathologic study 10 days, 3 weeks, 6 weeks and 24 weeks after operation. The urodynamics, urethroscopy and urethrography were also performed. RESULTS: The serum TNFalpha in experiment group slightly rised, with no significant difference when compared with that in control group. 10 days after operation, epithelial cell migrated into the extracellular matrix from two ends, and small vessels were also found. 3 weeks later, several layers of urothelium covered the whole surface of the matrix tube. 6 weeks later, the irregularly arranged smooth muscle fibers were fist observed by Van Gieson staining. 24 weeks after operation, the smooth muscle cells increased, the appearance of the regenerated urethra segments were very similar to normal urethral wall components. The urethrography and urodynamic evaluation revealed no difference between the normal and the regenerated urethral tube. CONCLUSIONS: The urethral extracellular matrix might be an ideal replacement material for urethral defect.

[Long-term culture and identification of spermatogonial stem cells from BALB/c mice in vitro].

OBJECTIVE: To establish a long-term culture system for mouse spermatogonial stem cells (SSCs) and to discuss the key factor that supports mouse SSC self-renewal and proliferation. METHODS: Testis cells from 4-6 days postpartum male transgenic BALB/c mce were collected by a modified two-step enzymatic digestion method and plated on 0. 2% elatin-coated tissue culture plates. The germ cells were enriched by differential adherence selections after respectively incubated for 1, 5 and 24 h and then plated on the mitomycin C-inactivated mouse embryonic fibroblast (MEF) feeder layer. The basal culture medium was StemPro-34 SFM supplemented with other 15 nutrient factors. The 20 ng/ml Glial cell line-derived neurotrophic factor (GDNF), 10 ng/ml basic fibroblast growth factor (bFGF) and 200 ng/ml GDNF-family receptor alpha 1 (GFRalpha1) were added to the serum-free medium to promote SSC proliferation. Several important surface markers and special genes were examined by immunocytochemical staining and RT-PCR analysis. RESULTS: After 3-4 days culture on the MEF feeder, SSCs proliferated continuously and formed typical colonies. SSCs from the BALB/c mice could be cultured in a steady state for 3 months. Immunocytochemical staining showed that Oct4 was specifically expressed in the cultured SSC nucleus and GFRalpha1 strongly expressed on the surface of the membrane. RT-PCR confirmed that the cultured SSCs expressed Oct-4, GFRalpha1, Sox2 and several other special genes resembling undifferentiated spermatogonia. CONCLUSION: SSCs from BALB/c mice could be cultured in the improved culture system for 3 months. This culture system could help further understand the regulating mechanism of SSCs and might provide an opportunity for the treatment of male infertility by SSC transplantation.

Curative effect and histocompatibility evaluation of reconstruction of traumatic defect of rabbit urethra using extracellular matrix.

OBJECTIVE: To investigate the curative effect and histocompatibility of reconstruction of traumatic urethral defect of rabbit using urethral extracellular matrix (ECM). METHODS: Urethral ECM was obtained by excision of the urethra in 20 donor rabbits. In experimental group, 20 rabbits were resected a 1.0 cm-1.5 cm segment of the urethra and artificially made a model of traumatic urethral defect, then reconstructed by the urethral extracellular matrix of the same length. The rabbit immunity response was assessed by lymphocyte transformation test and serum TNF-alpha level. The reconstructed urethral segments were stained with hematoxylin-eosin and Van Gieson stain and observed by histological examination postoperatively. The urethrography, urethroscopy and urodynamic examinations were performed. RESULTS: There was no significant difference in stimulative index of lymphocyte transformation between ECM group and control group. The serum TNF-alpha levels of ECM group slightly rose, but the increase was not significant as compared with control group. On postoperative day 10, epithelial cell had migrated from each side and small vessels were found in the extracellular matrix. In the 3rd week, several layers of urothelium covered the whole surface of the matrix tube. In the 6th week, the disorganized arrangements of smooth muscle fibers were firstly observed by Van Gieson staining. In the 24th week, the smooth muscle cells increased and the matrix tube appeared fairly similar to normal urethral wall components. The urethroscopy and urodynamic evaluation revealed that the surface of reconstructed urethra was smooth and emiction was unobstructed. CONCLUSION: The urethral extracellular matrix might be an ideal and safe biomaterial for the reconstruction of urethral traumatic defect.

[Preparation of amylopectin modified dipyridamole liposome and its tissue distribution in mice].

AIM: To prepare amylopectin anchored dipyridamole (DIP) liposome and to study its tissue distribution in mice. METHODS: The regular DIP liposomes were prepared by film-scatter method. The amphiphilic O-palmitoyl amylopectin was synthesized and added to modify the surface of liposome. The entrapping efficiency, zeta potential, mean diameter, span of modified and regular liposomes were assayed. The RP-HPLC was used for the determination of DIP concentration in mice tissue. RESULTS: After modification, the entrapping efficiency depressed, zeta potential was raised, mean diameter and span had no obvious change. The level of DIP in lung, liver and spleen for regular liposomes were higher than that of injections. Compared with regular liposomes, the modified liposomes increased the DIP level in lung, and decreased the DIP level in liver, spleen, moreover, lengthened the retention time of DIP in lung. CONCLUSION: The distribution of modified liposome in mice was markedly changed as compared with regular liposomes and injections. The modified liposomes had obvious lung targeting property.

Reconstruction of rabbit urethra using urethral extracellular matrix.

BACKGROUND: Urethral reconstruction for both congenital and acquired etiologies remains a challenge for most urologic surgeons. Tissue engineering has been proposed as a strategy for urethral reconstruction. The purpose of this study was to determine whether a naturally derived extracellular matrix substitute developed for urethral reconstruction would be suitable for urethral repair in an animal model. METHODS: A urethral segmental defect was created in 20 male rabbits. The urethral extracellular matrix, obtained and processed from rabbit urethral tissue, was trimmed and transplanted to repair the urethral defect. Then, the regenerated segment was studied histologically by haematoxylin-eosin staining and Van Gieson staining at 10 days, 3 weeks, 6 weeks, and 24 weeks postoperation. Retrograde urethrography was used to evaluate the function of the regenerated urethras of 4 rabbits 10 and 24 weeks after the operation. The urodynamics of 4 rabbits from the experimental group and control group I were assessed and compared. In addition, 4 experimental group rabbits were examined by a urethroscope 24 weeks after the operation. RESULTS: At 10 days after operation, epithelial cells had migrated from each side, and small vessels were observed in the extracellular matrix. The matrix and adjacent areas of the host tissue were infiltrated with inflammatory cells. The epithelium covered the extracellular matrix fully at 3 weeks postoperation. Well-formed smooth-muscle cells were first confirmed after 6 weeks, at which point the inflammatory cells had disappeared. At 24 weeks postoperation, the regenerated tissue was equivalent to the normal urethra. Urethrography and urodynamic evaluations showed that there was no difference between normal tissue and regenerated tissue. CONCLUSIONS: Urethral extracellular matrix appears to be a useful material for urethral repair in rabbits. The matrix can be processed easily and has good characteristics for tissue handling and urethral function.