Atherosclerosis-induced chronic arterial insufficiency causes clitoral cavernosal fibrosis in the rabbit

In our previous studies we found that aging-associated fibrosis of clitoral cavernosal tissue correlated with the prevalence of cardiovascular disease in elderly women. The aim of this study was to determine specifically, arterial insufficiency-related structural changes of clitoral cavernosal tissue in a rabbit model. New Zealand white female rabbits were divided into clitoral cavernosal ischemia (CCI, n = 5) and control (n = 5) groups. The CCI group underwent balloon endothelial injury of the iliac arteries and received 0.5% cholesterol diet. The control group received a regular diet. After 16 weeks, arteriography was performed then the animals were sacrificed. The iliac arteries and the entire clitoris were removed. Cross-sections of the iliac arteries and clitoris were processed for histologic evaluation The percentage of smooth muscle and connective tissue in trichrome stained sections of clitoral cavernosal tissue was determined by computer-assisted histomorphometry. Arteriography revealed diffused occlusive disease in the common iliac, internal iliac and pudendal arteries in the CCI group. Histology showed that arterial occlusive disease spreads from the site of balloon injury to the smaller branches involving the clitoral cavernosal arteries. Diffuse fibrosis was observed in the clitoral cross-sections of the CCI group. The percentage of clitoral cavernosal smooth muscle (mean +/- standard error) in the CCI group (53% +/- 0.9%) was significantly decreased compared with the control group (62% +/- 0.8%) (P = 0.0001). Chronic clitoral cavernosal ischemia causes significant fibrosis and loss of smooth muscle in the clitoral cavernosal tissue. These findings suggest that chronic clitoral cavernosal arterial insufficiency may play a role in the pathophysiology of female sexual arousal disorders.     

Decreased circulating levels of estrogen alter vaginal and clitoral blood flow and structure in the rabbit.

Aging and menopause related decline in circulating levels of estrogen has been shown to adversely affect female sexual arousal function. Our aim was to study the effects of circulating levels of estrogen on the hemodynamic mechanism of vaginal and clitoral engorgement and on the structure of the vaginal and clitoral cavernosal tissue in the rabbit. New Zealand White female rabbits (3.5-4 kg) were randomly divided into three groups with five rabbits in each group: control; bilateral oophorectomy; bilateral oophorectomy undergoing subcutaneous injection of estrogen (40 microg/kg/day). After 6 weeks, the serum levels of 17 beta-estradiol were measured and systemic blood pressure was monitored. Vaginal and clitoral cavernosal blood flows were measured with laser Doppler flowmeter before and after pelvic nerve stimulation. Cross sections of the clitoris and vagina were processed for histologic examination and histomorphometric image analysis. Serum level of 17 beta-estradiol (pg/ml; mean+/-s.d.) revealed a significant decrease in the oophorectomy group (25.4+/-5.1) compared with the control (38.5+/-7.6) and estrogen replacement (115.9+/-57.3) groups (P<0.05). Nerve stimulation-induced peak vaginal and clitoral intracavernosal blood flows in the oophorectomy group (28.9+/-16.3 and 6.1+/-1.4, respectively) were significantly less than those recorded in the control (48.9+/-6.5 and 11.0+/-2.4, respectively) or estrogen replacement (48.7+/-12.2 and 10.1+/-2.8, respectively) group (P<0.05). In histology, marked thinning of the vaginal epithelial layers, decreased vaginal submucosal microvasculature, and diffuse clitoral cavernosal fibrosis were evident in the oophorectomy group but not in the estrogen supplement and control groups. In histomorphometry, the percentage of clitoral cavernosal smooth muscle in the oophorectomy group (49.6+/-6.2) was significantly decreased compared with the control (56.8+/-2.6) and estrogen replacement (58+/-3.0) groups (P<0.05). Our studies show that decline in circulating levels of estrogen impairs the hemodynamic mechanism of vaginal and clitoral engorgement and leads to histopathologic changes in the vagina and clitoral cavernosal tissue. These observations suggest that decreased circulating levels of estrogen, a physiologic change in the menopausal state, may play a role in the development of female sexual arousal dysfunction.     

Relative roles of cyclooxygenase and nitric oxide synthase pathways in ischemia-induced increased contraction of cavernosal smooth muscle

PURPOSE: To study the mechanism of chronic ischemia-induced increased cavernosal smooth muscle contraction in an animal model of vasculogenic erectile dysfunction. MATERIALS AND METHODS: New Zealand White rabbits were divided into control (n = 6, fed with a regular diet), hypercholesterolemic (n = 9, fed with a diet containing 0.5% cholesterol) and chronic cavernosal ischemia (CCI, n = 10, underwent balloon de-endothelialization of iliac arteries and received a diet containing 0.5% cholesterol) groups. After 16 weeks, the relationship between iliac artery blood flow and cavernosal smooth muscle contraction was studied. The roles of cyclooxygenase and nitric oxide (NO) pathways in chronic ischemia-induced increased smooth muscle contraction were also examined. RESULTS: Iliac artery blood flow in the CCI group was significantly reduced compared with the control and hypercholesterolemic groups. Hypercholesterolemia alone did not affect cavernosal smooth muscle contraction. Atherosclerosis-induced chronic cavernosal arterial insufficiency did not affect contraction to norepinephrine while causing a significant increase in electrical field stimulation-induced neurogenic contraction. Inhibition of the cyclooxygenase pathway by indomethacin decreased electrical field stimulation-induced contraction in all animals but failed to normalize the differences between CCI and control groups. In the presence of indomethacin, L-arginine decreased electrical field stimulation-induced contraction in the control and hypercholesterolemic groups but not in the CCI group. In the presence of indomethacin, treatment with nitric oxide synthase (NOS) inhibitor, N(G)-monomethyl-L-arginine (L-NMMA), increased electrical field stimulation-induced contraction in all groups. This effect of L-NMMA on smooth muscle contraction was significantly greater in the control and hypercholesterolemic groups compared with the CCI group. After tissue treatment with L-NMMA, the magnitude of contraction in cavernosal tissue from control and hypercholesterolemic groups was similar to those observed in the CCI group. CONCLUSIONS: Mechanism of chronic ischemia-induced increased cavernosal smooth muscle contraction involves increased output of constrictor eicosanoids and impairment of the inhibitory influence of NO pathway in cavernosal tissue.     

Intracavernosal injections of vascular endothelial growth factor protects endothelial dependent corpora cavernosal smooth muscle relaxation in the hypercholesterolemic rabbit: a preliminary study.

Atherosclerosis is a major risk factor for erectile dysfunction, and loss of endothelium-dependent vasodilation appears early in the development of this disorder. Nitric oxide (NO) appears to be the principle mediator of erectile function and is generated in part by the sinusoidal endothelium. Vascular endothelial growth factor (VEGF) is an angiogenic growth factor and an endothelial cell-specific mitogen and the actions of VEGF are coupled to NO. In this preliminary study, we investigated whether VEGF could be used to protect endothelial dependent cavernosal relaxation from the atherosclerotic injury induced by a hypercholesterolemic diet.Two groups of New Zealand white adult male rabbits received a 1% cholesterol diet for four weeks, and two groups consumed normal rabbit chow. Half of the rabbits consuming the 1% cholesterol diet received weekly penile injections of 0.3 mg VEGF (n=8), and half injections of normal saline (n=8). Rabbits fed normal chow followed a similar protocol, half received weekly penile injections of 0.3 mg VEGF (n=6) and half were given weekly penile injections of normal saline (n=6). Isometric tension studies (with norepinephrine, acetylcholine, sodium nitroprusside and histamine) were performed on isolated strips of corpora cavernosa. The degree of corporal smooth muscle relaxation in response to ACH and SNP administration was recorded and compared.Significant elevation in serum total cholesterol levels occurred in rabbits receiving 4 weeks of the 1% cholesterol diet (727+/-75.6 mg/dl vs 38.7+/-5.53 mg/dl) P<0.01. There were no significant differences in cavernosal contraction in any group, while cavernosal smooth muscle from rabbits on normal chow retained the ability to relax in response to ACH and SNP in tissue bath. The hypercholesterolemic rabbits receiving VEGF had a significantly higher maximal per-cent relaxation to ACH (111+/-28.9) compared to the hypercholesterolemic rabbits that received NS (77+/-23.1, P<0.001). This difference in percent maximal relaxation to SNP was also present for hypercholesterolemic/VEGF rabbits (129.4+/-24) versus the hypercholesterolemic/NS rabbits (115.0+/-18, P=0.033).In conclusion, intracavernosal injections of VEGF appear to protect corporal endothelium from hypercholesterolemia induced injury, thus preserving endothelial dependent corporal smooth muscle relaxation in hypercholesterolemic rabbit.     

Enhanced relaxation of diabetic rabbit cavernosal smooth muscle in response to nitric oxide: potential relevance to erectile dysfunction

New Zealand white rabbit cavernosal smooth muscle strips (n=6) were mounted in organ baths. Relaxations to nitric oxide (10(-7)-10(-4) mol/l) were measured and the same procedure was repeated on strips from rabbits 6 months after alloxan-induced diabetes (n=6). Transverse cavernosal sections were obtained from the same penises. Low and high resolution autoradiographs were prepared using [(3)H]-L-N(G)-nitroarginine (an index of nitric oxide binding sites) and analysed densitometrically. Histochemical analysis was performed on adjacent sections using NADPH diaphorase (an index of nitric oxide synthase activity).Nitric oxide relaxed control rabbit cavernosal smooth muscle strips in a concentration-dependent manner. Diabetic rabbit cavernosal smooth muscle strips were significantly (P<0.03) more sensitive to nitric oxide (mean IC(50)=3.9 x 10(-6) mol/l). Nitric oxide synthase binding sites were localised to the cavernosal endothelium and smooth muscle. Nitric oxide synthase activity was increased in 6 month diabetic cavernosal smooth muscle. These findings suggest impairments in the L-arginine-nitric oxide pathway may play a role in the pathophysiology of diabetic erectile dysfunction.     

A quantitative histological analysis of the dilated ureter of childhood.

A quantitative histological study of the dilated ureter of childhood was performed on 26 ureters. The specimens were from 15 male and 11 female patients 10 days to 12 years old (mean age 2.0 years). A color image analysis system was used to examine and compare collagen and smooth muscle components of the muscularis layers to normal control ureters of similar age. In comparing primary obstructed (12) to primary refluxing (14) megaureters and control ureters (6), there was a statistically different collagen-to-smooth muscle ratio (p < 0.001) between the primary obstructed and primary refluxing megaureter groups. For patients with primary refluxing megaureter there was a 2-fold increase in the tissue matrix ratio of collagen-to-smooth muscle when compared to patients with primary obstructed megaureter. In the primary obstructed megaureters the amount of collagen and smooth muscle was not statistically different from controls (p > 0.01). The increased tissue matrix ratio of 2.0 +/- 0.35 (collagen-to-smooth muscle) in the refluxing megaureter group compared to 0.78 +/- 0.22 in the obstructed megaureter group and 0.52 +/- 0.12 in controls was found to be due not only to a marked increase in collagen but also a significant decrease in the smooth muscle component of the tissue. Primary obstructed and normal control ureters had similar quantitative amounts of smooth muscle with 60 +/- 5% and 61 +/- 6%, respectively, while refluxing megaureters had only 40 +/- 5% smooth muscle. The percentage collagen was 36 +/- 5 in the obstructed megaureter group and 30 +/- 5 in controls, with refluxing megaureters having 58 +/- 5% collagen on analysis. Our findings emphasize the significant differences in the structural components (collagen and smooth muscle) of the dilated ureter of childhood, and provide us with further insight into the pathological nature of these dilated ureters and their surgical repair.     

Diabetes induced alteration of clitoral hemodynamics and structure in the rabbit

PURPOSE: We investigated the effect of diabetes on clitoral hemodynamics and structures in the rabbit. MATERIALS AND METHODS: A total of 25 New Zealand White female rabbits weighing 3 to 3.5 kg. were divided into 2 groups, including 5 in the control and 20 in the experimental group. Experimental animals received intravenous injection of alloxan hydrochloride (Sigma Chemical Co., St. Louis, Missouri) (100 mg./kg.). The development of diabetes was verified by measuring body weight and blood glucose levels. After 12 weeks clitoral cavernous blood flow in ml. per minute per 100 gm. tissue was measured with a laser Doppler flowmeter. Cross sections of the clitoris were used for histochemistry and histomorphometric image analysis. RESULTS: After 12 weeks 5 animals were included in the diabetes group. Mean baseline flaccid and peak clitoral cavernous blood flow plus or minus standard deviation significantly decreased in the diabetic group compared with the control group (3.9 +/- 1.6 and 5.8 +/- 2.2 versus 7.2 +/- 2.5 and 12.9 +/- 5.8 ml. per minute per 100 gm. tissue, respectively, p <0.05). Histology revealed diffuse clitoral fibrosis in the diabetic group. On histomorphometry the mean proportion of clitoral cavernous smooth muscle in the diabetic group was significantly decreased compared with the control group (51.9% +/- 4.9% versus 62.3% +/- 3.1%, p < 0.05). CONCLUSIONS: These results show that diabetes mellitus produces significant adverse effects on the hemodynamic mechanism of clitoral engorgement and leads to diffuse clitoral cavernous fibrosis. It implies that decreased sexual arousal in diabetic women may result from structural changes in the clitoris.     

Intracavernosal injection of vascular endothelial growth factor improves erectile function in aged rats

OBJECTIVES: To investigate whether intracavernosal injection of vascular endothelial growth factor (VEGF) can restore erectile function in the aging rat. MATERIALS AND METHODS: Ten young (4-5 months) and 30 old (24 months) Sprague-Dawley male rats were used. The old rats were divided into 3 groups: vehicle-only (phosphate buffered saline plus 0.1% bovine serum albumin; n = 10), VEGF 1 microg/kg (n = 10), and VEGF 10 microg/kg (n = 10). At 2 and 4 weeks after treatment, erectile function and histology were evaluated by hemodynamic study, histomorphometric analysis, and immunohistochemistry. RESULTS: After 4 weeks of treatment, the ratio of peak intracavernosal pressure to systemic arterial blood pressure in response to neurostimulation was significantly higher in both the VEGF 1 microg/kg (79.9 +/- 7.7%) and the VEGF 10 microg/kg group (76.8 +/- 5.8%) compared to the vehicle-only group (63.1 +/- 8.5%; p < 0.05). The percentage of cavernosal smooth muscle was significantly higher in the VEGF 10 microg/kg group (16.1 +/- 1.4%) compared to the vehicle-only group (12.8 +/- 2.2%; p = 0.047). VEGF treatment in old rats increased e-NOS and VEGF expression in both treatment groups. CONCLUSION: Intracavernosal injection of VEGF appears to restore smooth muscle integrity and improve erectile function in aged rats.     

Fetal cell transplantation: a comparison of three cell types.

OBJECTIVE: We have previously reported that fetal cardiomyocyte transplantation into myocardial scar improves heart function. The mechanism by which this occurs, however, has not been elucidated. To investigate possible mechanisms by which cell transplantation may improve heart function, we compared cardiac function after transplantation of 3 different fetal cell types: cardiomyocytes, smooth muscle cells (nonstriated muscle cells), and fibroblasts (noncontractile cells). METHODS: A left ventricular scar was created by cryoinjury in adult rats. Four weeks after injury, cultured fetal ventricular cardiomyocytes (n = 13), enteric smooth muscle cells (n = 10), skin fibroblasts (n = 10), or culture medium (control, n = 15 total) were injected into the myocardial scar. All rats received cyclosporine A (INN: ciclosporin). Four weeks after transplantation, left ventricular function was evaluated in a Langendorff preparation. RESULTS: The implanted cells were identified histologically. All transplanted cell types formed tissue within the myocardial scar. At an end-diastolic volume of 0.2 mL, developed pressures in cardiomyocytes group were significantly greater than smooth muscle cells and skin fibroblasts groups (cardiomyocytes, 134% +/- 22% of control; smooth muscle cells, 108% +/- 14% of control; skin fibroblasts, 106% +/- 17% of control; P =.0001), as were +dP/dt(max) (cardiomyocytes, 119% +/- 37% of control; smooth muscle cells, 98% +/- 18% of control; skin fibroblasts, 92% +/- 11% of control; P =. 0001) and -dP/dt(max) (cardiomyocytes, 126% +/- 29% of control; smooth muscle cells, 108% +/- 19% of control; skin fibroblasts, 99% +/- 16% control; P =.0001). CONCLUSIONS: Fetal cardiomyocytes transplanted into myocardial scar provided greater contractility and relaxation than fetal smooth muscle cells or fetal fibroblasts. The contractile and elastic properties of transplanted cells determine the degree of improvement in ventricular function achievable with cell transplantation.     

糖尿病大鼠海绵体神经诱发电位的实验研究

目的 :观察糖尿病 (DM)性大鼠海绵体的神经病变。　方法 :3 5只SD大鼠随机分为造模组 ( 2 5只 )和空白对照组 ( 10只 )。造模组应用腹腔内单次注射链脲佐菌素 ( 63mg/kg)制造DM模型。单波刺激海绵体神经 ,测定反射潜伏期及肌电位。　结果 :海绵体神经 海绵体平滑肌传导潜伏期中 ,DM组所需时间明显长于其他各组 (P <0 .0 1)。同时 ,与其他各组相比 ,其海绵体平滑肌电位明显升高 (P <0 .0 1)。　结论 :提示DM大鼠海绵体神经病变可能是导致勃起功能障碍的重要原因之一     

Factors effecting mortality in urban vertical free falls: evaluation of 180 cases.

AIM: The aim of this retrospective study was to evaluate the factors on mortality in urban free vertical falls. PATIENTS AND METHODS: A total of 180 urban vertical free fall victims who survived transport to the emergency room between the period of 1980-1998 were evaluated. Minor bruises, abrasions, haematomas, and soft tissue injuries were not encountered. Serious injuries such as bone fractures, liver lacerations, epi-subdural haematomas, haemothorax, haemomediastinum, retroperitoneal haematomas were evaluated. RESULTS: Of the total, 23% (n = 41) of patients were female and 73% (n = 139) were male. The mean age was 22.3 years (4-75 years). Extremity fractures were found in 6.7% (n = 12), cranial trauma in 14.4% (n = 26), thoracic trauma in 2.2% (n = 4) retroperitoneal trauma in 2.8% (n = 5), vertebral column trauma in 1.7% (n = 3) of cases. The overall number of the pathologies was 59. In-hospital mortality was 8.9% (n = 16). The injury severity scores (ISSs) of non-survivors and survivors were 33 +/- 4, and 5 +/- 0.6, respectively (P = 0.0001). The heights fallen were 8.6 +/- 2.3 m for non-survivors and 5.2 +/- 0.2 m for survivors (P = 0.022). The mean ages of non-survivors and survivors were 41.6 +/- 5.9 years and 20.4 +/- 1.2 years, respectively (P = 0.003). Serious cranial trauma was found in 68.7% (n = 11) and 9.1% (n = 5) of non-survivors and survivors, respectively (P = 0.0001). Extremity trauma was encountered in 31.2% (n = 5) and 4.2% (n = 7) of non-survivors and survivors, respectively (P = 0.0015). The ISSs were 6.8 +/- 1.0 and 8.9 +/- 1.1 for cases under the age of 6 years and others, respectively (P = 0.15). Using logistic regression analysis, ISS, height and age were found to be significant factors in mortality. CONCLUSION: Vertical deceleration injury represents a distinct form of trauma. With the results of this study, it can be concluded that ISS, height and age are significant factors in determining the severity of trauma.     

Studies on corpus cavernosal nerve evoked potential in male diabetic rats

Aim: To observe the cavernosal nerve function in male diabetic rats. Methods: Thirty-five male adult SD rats were divided randomly into the diabetic group (n=15) and the control group (n=10). In the diabetic group, diabetes was produced by the administration of streptozotocin (63 mg/kg) at a single intraperitoneal dosing. The latent period of reaction and the corpus cavernosal smooth muscle myopotential were observed after giving a single stimulus to the corpus cavernosal nerve. Results: Compared with the controls, the diabetic rats had a longer latent period and a higher myopotential (both P<0.01). Conclusion: Corpus cavernosal nerve dysfunction may play a role in the erectile dysfunction of diabetic rats.     

The effect of chronic L-arginine administration on vascular recovery following cold cardioplegic arrest in rats.

OBJECTIVE: Acute administration of L-arginine (LA), the physiological substrate of nitric oxide, has been used as a strategy for myocardial protection during ischemia-reperfusion. The aim of this study was to assess the effects of chronic oral LA administration on vascular functions and morphology after prolonged cold cardioplegic arrest. METHODS: Adult male Sprague-Dawley rats (600-650 g) were divided into control and LA groups, which received LA (4 mg/ml) for 6 weeks. Two experimental protocols were carried out. (1) Isolated rat heart perfusion was performed and hearts were subjected to ischemia for 4 h at 4 degrees C using cold crystalloid cardioplegia (n=8 in LA, n=7 in control). Endothelial and vascular smooth muscle functions were assessed through observations of pre- and post-ischemic coronary flow response to 5-hydroxytryptamine (5-HT) and glyceryl trinitrate (GTN) (%5-HT and %GTN, respectively). (2) Semi-quantitative assessment of tissue morphology was conducted after the same ischemia-reperfusion protocol (n=4 in each group). RESULTS: The LA group showed significantly better recovery (post-/pre-ischemic value) of %5-HT (97.0+/-65.6 versus 21.5+/-25.7%, P=0.015) and %GTN (124.5+/-117.6 versus 47.7+/-16.6%, P=0.021). The histological assessment showed no significant differences between the two groups. CONCLUSIONS: Chronic oral administration of LA significantly ameliorated the postischemic recovery of endothelial and vascular smooth muscle functions after cold cardioplegic arrest in rats.     

Modulation of phosphatidylinositol 3-kinase signaling reduces intimal hyperplasia in aortocoronary saphenous vein grafts

OBJECTIVES: Fifty percent of human aortocoronary saphenous vein grafts are occluded after 10 years. Intimal hyperplasia is an initial step in graft occlusion and consists of vascular smooth muscle cell proliferation. Phosphatidylinositol 3-kinase and its downstream regulator, the inositol 3-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10), are important regulators of vascular smooth muscle cell proliferation, migration, and cell death. This study tests whether overexpression of PTEN in aortocoronary saphenous vein grafts can reduce intimal hyperplasia. METHODS: Adult dogs underwent aortocoronary bypass grafting to the left anterior descending artery by using the autologous saphenous vein. Saphenous vein grafts were treated with phosphate-buffered saline (n = 9), empty adenovirus (n = 8), or adenovirus encoding for PTEN (n = 8). Arteriography at 30 and 90 days assessed saphenous vein graft patency. A subset received saphenous vein grafts treated with a marker transgene (beta-galactosidase, n = 3), empty adenovirus (n = 4), or adenovirus encoding for PTEN (n = 4) and were killed on postoperative day 3 to confirm expression. Vascular smooth muscle cells were isolated from canine saphenous vein infected with adenovirus encoding for PTEN, and immunoblotting and proliferation assays were performed. RESULTS: Saphenous vein graft transgene expression was confirmed by means of immunohistochemistry, immunoblotting, and polymerase chain reaction. Arteriograms revealed all saphenous vein grafts to be patent. Saphenous vein grafts treated with adenovirus encoding for PTEN demonstrated reduced intimal area compared with those treated with empty adenovirus and phosphate-buffered saline (1.39 +/- 0.11 vs 2.35 +/- 0.3 and 2.57 +/- 0.4 mm 2 , P < .05), and the intima/media ratio was lower in saphenous vein grafts treated with adenovirus encoding for PTEN (0.50 +/- 0.05 vs 1.43 +/- 0.18 and 1.11 +/- 0.14, P < .005). PTEN overexpression in vascular smooth muscle cells inhibited platelet-derived growth factor-induced phosphorylation of Akt, a downstream effector of phosphatidylinositol 3-kinase. PTEN-treated vascular smooth muscle cells demonstrated decreased basal, platelet-derived growth factor-stimulated, and serum-stimulated proliferation. CONCLUSION: This study demonstrates that PTEN overexpression in aortocoronary saphenous vein grafts reduces intimal hyperplasia. The mechanism of this antiproliferative effect in vascular smooth muscle cells is likely due to inhibition of phosphatidylinositol 3-kinase signaling through Akt, with resultant decreases in vascular smooth muscle cell growth and survival. Therefore modulation of the phosphatidylinositol 3-kinase pathway through PTEN overexpression might represent a novel therapy to prevent saphenous vein graft intimal hyperplasia after coronary artery bypass grafting.     

Collagen-to-smooth muscle ratio helps prediction of prognosis after pyeloplasty

PURPOSE: We quantitatively evaluated the collagen-to-smooth muscle tissue matrix ratio in ureteropelvic junction obstruction, and compared the ratio with the degree of obstruction, patient age and postoperative renal recovery. MATERIALS AND METHODS: We analyzed histological sections from 65 patients with ureteropelvic junction obstruction and 6 normal controls. Morphological and functional grading systems were adapted to determine the degree of renal obstruction. To examine smooth muscle and collagen tissue, sections were stained using Masson's trichrome. Two distinct populations of collagen versus smooth muscle were identified and the tissue matrix ratio was calculated by color image analysis. RESULTS: The mean tissue matrix ratio plus or minus standard deviation was 1.32+/-0.79 in all patients with ureteropelvic junction obstruction but only 0.30+/-0.10 in normal controls (p <0.0001). It appeared that the lower the tissue matrix ratio, the better the improvement in postoperative hydronephrosis (r = -0.50, p = 0.0001). Better recovery of renal function after pyeloplasty was observed with a decrease in the tissue matrix ratio (r = -0.43, p = 0.0004). We divided patients according to the tissue matrix ratio into groups 1--ratio 1 or less, 2--greater than 1 to 1.5 and 3--greater than 1.5 to determine a more detailed and clinically applicable correlation of tissue matrix ratio with postoperative renal functional changes. Better improvement in postoperative renal function was observed in group 1 than in group 3 (p = 0.002). Also, the tissue matrix ratio increased with patient age (r = 0.33, p = 0.007). CONCLUSIONS: Since our data represent an objective and quantitative parameter associated with ureteropelvic junction obstruction, we believe that our findings may help to predict the prognosis after pyeloplasty.     

Isolation of primary endothelial and stromal cell cultures of the corpus cavernosum penis for basic research and tissue engineering

OBJECTIVES: Primary cell cultures derived from the corpus cavernosum are frequently used as in vitro models to define cellular mechanisms involved in erectile function. However, previous studies often lack detailed isolation protocols or a precise characterisation of the culture composition excluding especially contaminating fibroblasts. This study aimed at critically analysing and reproducing reported isolation methods, as well as establishing new procedures to receive highly pure and morphologically differentiated endothelial, smooth muscle and fibroblastic cells derived from the human penis. METHODS: We evaluated numerous isolation and enrichment techniques using cavernosal tissue from 57 patients. Assessment factors displayed the purity, cell yield, practicability and reproducibility. The purity in cultured cells was analysed using immunocytochemistry and Western blots. RESULTS: An enzymatic protocol was established for the isolation and cultivation of cavernosal endothelial cells with an impressive purity of 98.0+/-0.8%. In contrast, already published nearly pure smooth muscle cell cultures were not reproducible in our laboratory. Meaningful evidence for an overwhelming presence of fibroblasts in these widely accepted pure smooth muscle cell cultures is presented. CONCLUSION: Endothelial cell cultures derived from human corpora cavernosa are reproducible and reliable to serve for cell culture-based investigations of the endothelial dysfunction. The discrepancy in the purity of smooth muscle cell cultures might reflect laboratory and tissue source factors, lacking an exclusion of fibroblasts in other studies or changes in stromal phenotype under culture conditions. Further research is necessary to clarify a possible plasticity between smooth muscle cells and (myo)fibroblasts and assess functional properties.     

兔阴蒂海绵体平滑肌细胞的体外培养方法及生物学特性

目的：探索新西兰白兔阴蒂海绵体平滑肌细胞的体外培养方法及生物学特性。方法：取兔阴蒂海绵体，采用酶消化法进行平滑肌细胞体外培养；在倒置显微镜下观察培养细胞的生长状况、形态特征及贴壁过程；用计数法测定细胞贴壁率；用MTT法描绘原代培养细胞的生长曲线；利用逆转录聚合酶链反应(RT—PCR)方法检测平滑肌细胞特征性标记物α-actin表达。结果：培养的兔阴蒂海绵体平滑肌细胞具有典型的平滑肌细胞形态特征，为梭形，呈长轴平行排列，具有明显的方向性；体外贴壁快，生长迅速，体外培养的阴蒂海绵体平滑肌细胞在合适的传代条件和比例下能够生存并保持其稳定的生物学特性。结论：体外培养的兔阴蒂海绵体平滑肌细胞模型可用于进一步研究阴蒂组织细胞学、分子生物学机制以及受体介导的平滑肌收缩信号转导机制及药理学作用等。     

Loss of TGFbeta, Apoptosis, and Bcl-2 in Erectile Dysfunction and Upregulation of p53 and HIF-1alpha in Diabetes-Associated Erectile Dysfunction

Vasculogenic erectile dysfunction (ED) is associated with collagen replacement of the cavernosal smooth muscle, mediated by an increase in transforming growth factor (TGF)-production secondary to hypoxemia. We tested the hypothesis that human ED is the result of an increase in apoptosis of the cavernosal smooth muscle cells with replacement by collagen, mediated by the TGFbeta upregulation. We also examined the tissue for proteins associated with apoptosis. Human cavernosal tissue was procured from impotent men at the time of prosthesis insertion. Normal corpous cavernosum served as a control. The TUNEL assay was used to assess apoptosis. Immunohistochemistry staining was used to detect TGFbeta and Bcl-2 expression, while Western blot analysis was used to detect expression of Bcl-2, p53, and hypoxia-inducible factor (HIF)-1a. Immunohistochemistry showed downregulation of TGFbeta protein expression in the corpus cavernosum of men with ED. Apoptotic nuclei were noted in cavernosal smooth muscle from a potent man but were not found in cavernosal tissue from men with ED. To gain insight into the possible mechanism of apoptosis in men with ED, the proto-oncogene Bcl-2, a potential inhibitor of apoptosis, was examined. Both immunohistochemistry and Western analysis revealed the presence of Bcl-2 in the cavernosal nerve of a potent man but its absence in cavernosal tissue from men with ED. Thus, loss of Bcl-2 expression correlated with the loss of apoptosis. In contrast, Western blotting demonstrated upregulation of p53 and HIF-1a expression in the cavernosal tissues from the men with ED and diabetes. Male ED follows an active process characterized by a loss of TGFb expression, apoptosis, and Bcl-2 expression. However, there is upregulation of p53 and HIF-1a in men with diabetes. These data support the possibility of hypoxia-mediated ED in diabetes via upregulation of p53 and HIF-1a but does not substantiate a role for TGFbeta in ED.     

Spontaneous activity of mouse detrusor smooth muscle and the effects of the urothelium

AIMS: To characterize the detrusor muscle of the mouse urinary bladder in order to understand more precisely spontaneous contractile behavior of this organ. This study examined the spontaneous electrical activity and Ca(2+) dynamics of the detrusor smooth muscle and investigated the role of the urothelium. MATERIALS AND METHODS: Detrusor smooth muscle strips were isolated from mouse bladders. The urothelium was either kept intact or removed. Changes in membrane potential were recorded using sharp electrode intracellular recording. To image Ca(2+) dynamics, tissue strips were exposed to 10 microM Oregon Green 488 BAPTA-1 AM for 70 min, and then image series were acquired with a laser-scanning confocal microscope. RESULTS: (1) Mouse detrusor smooth muscle cells (SMCs) generate nifedipine-sensitive spontaneous action potentials (sAPs) at a low frequency (1.3 +/- 0.9 min(-1), n = 11) in preparations with intact urothelium. This frequency increased when the urothelium was removed (7 +/- 8.3 min(-1), n = 17) (P < 0.05, Student's t test). (2) Frequent ATP-mediated spontaneous depolarizations were recorded in all cells. (3) The frequency of whole cell Ca(2+) flashes of detrusor smooth muscle cells was higher in preparations with the urothelium removed (median 1.2 min(-1), n = 7) than in urothelium denuded preparations (median 0.6 min(-1), n = 7) (P < 0.01, Mann-Whitney U-test). CONCLUSIONS: Spontaneous activity of the mouse detrusor smooth muscles was characterized enabling future comparative work on gene knock-out strains. Evidence suggesting release of an inhibitory factor by the urothelium was apparent.     

Opportunities and challenges in oral therapy

The major event in normal erectile function is relaxation of the smooth muscle both within the penile arterial system and the cavernosal tissue. The cavernosal smooth muscle is indistinguishable from peripheral vascular smooth muscle from a physiological point of view, ie both are mediated by the NO-cGMP pathway. In man, the major reason for erectile dysfunction (ED) is 'abnormal smooth muscle relaxation', where the smooth muscle of the cavernosa fails to trap blood within the cavernosa (trapping requires a high intracorporeal pressure). The failure of the cavernosal muscle to relax may be due to inadequate stimulation of the muscle or simply non-compliance of the smooth muscle itself. Therefore, attempts to reverse this abnormal smooth muscle relaxation should be directed at either increasing stimulation to the muscle and/or reversing the poor compliance of the muscle tissue itself. Because the major and most efficient pathway for smooth muscle relaxation is the NO-cGMP pathway, any medication that affects this system should prove to be more potent than other alternate pathways of corporal smooth muscle relaxation, as is evidenced from the clinical observations with current intracorporeal instilled drugs. Because the peripheral vascular smooth muscle is indistinguishable physiologically from the muscle of the cavernosal system, any oral drug must be able to differentiate the two smooth muscle compartments to prevent systemic side effects that could render such drugs clinically useless. At the present time, there are no methods to improve smooth muscle compliance but advances in tissue engineering may allow us to accomplish this in the future. International Journal of Impotence Research (2000) 12, Suppl 4, S59-S61.