Nerve growth factor combined with vascular endothelial growth factor enhances regeneration of bladder acellular matrix graft in spinal cord injury-induced neurogenic rat bladder.

OBJECTIVE: To determine the combined effects of nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) on regeneration of the bladder acellular matrix graft (BAMG) in spinal cord injury (SCI)-mediated neurogenic bladder in rats. MATERIALS AND METHODS: In all, 40 female Sprague-Dawley rats were used. At 8 weeks after spinalization surgery (neurogenic bladder), they were divided into five groups consisting of untreated controls and those whose bladders were injected with either no growth factor, NGF (2 microg/rat), VEGF (2 microg/rat) or both at partial BAMG replacement surgery. After 8 weeks, bladder function was assessed by urodynamic studies and the bladders were harvested for histological examination. Smooth muscle induction, collagen and nerve fibre regeneration were assessed immunohistochemically using antibodies to smooth muscle actin (alpha-actin), Masson's trichrome and protein gene product 9.5, respectively. RESULTS: Bladder capacity and compliance were significantly increased in all BAMG groups 8 weeks after surgery compared with that before bladder replacement surgery. Bladder capacity and compliance were much higher in the VEGF and NGF combined group than in the control, or NGF and VEGF alone groups. There was no significant difference in the residual volume ratio among all groups. CONCLUSIONS: This is the first report showing that NGF has a significant synergistic effect on the development, differentiation and functional restoration of the BAMG when administered with VEGF in neurogenic bladder. Our results indicate that NGF may be a useful cytokine for enhancing the regeneration of a functional bladder following acellular matrix grafting in a neurogenic rat model.

Effect of extended-term estrogen on voiding in a postpartum ovariectomized rat model.

INTRODUCTION: We tested the hypothesis that extended-term (5-week) estrogen therapy would negatively impact voiding function in a postpartum, ovariectomized rat model. METHODS: Immediately after delivery, 30 primiparous Sprague-Dawley rats underwent intravaginal balloon dilation, followed by ovariectomy 1 week later. Cystometry at postpartum week 2 determined normal or abnormal voiding patterns. After randomization, one-half the normal and abnormal voiding rats received 5 weeks of estrogen therapy, while the remainder received placebo. Estrogen effect was determined by repeat cystometry and immunohistochemical analysis of the urethra and vagina. RESULTS: Abnormal voiding increased from 60.0% to 73.3% in the estrogen- treated group and declined from 60% to 33% for the placebo group. Rats were then divided into 4 groups for comparison: normal voiding versus placebo (group 1), abnormal voiding versus placebo (group 2), normal voiding versus estrogen (group 3) and abnormal voiding versus estrogen (group 4). Bladder capacity, leak point pressure and maximum voiding pressure were most depressed in group 4. Estrogen treatment was associated with a significant downregulation of alpha(1A) and alpha(1D)-adrenoceptors in the urethral submucosa but an upregulation of nNOS in the urethral smooth muscle. CONCLUSION: Extended-term estrogen therapy in a rat model of simulated birth trauma and ovariectomy resulted in a higher rate of incontinence. Immunohistochemical examination demonstrated significant downregulation of urethral alpha(1A)- and alpha(1D)-adrenoceptors and upregulation of neuronal nitric oxide synthase (nNOS) in the urethra of estrogen-treated groups. These studies question the use of hormone replacement therapy in the treatment of postmenopausal incontinence.

An animal model to study lower urinary tract symptoms and erectile dysfunction: the hyperlipidaemic rat.

OBJECTIVE: To present evidence that rats fed a high-fat diet could serve as a useful animal model to study both lower urinary tract symptoms (LUTS) and erectile dysfunction (ED), as recent epidemiological studies have shown a strong association between LUTS and ED but the physiological basis behind this relationship is unknown. MATERIALS AND METHODS: In all, 24 male Sprague-Dawley rats were divided into two groups: nine controls were fed a 'normal' diet and 15 were fed a high-fat diet (hyperlipidaemic rats). After 6 months all the rats had bladder and erectile functions evaluated using awake cystometry and cavernosal nerve electrostimulation, respectively. After the functional studies were completed, the penis, prostate and bladder were collected for immunohistochemical analysis. RESULTS: The hyperlipidaemic rats had significantly higher serum cholesterol and low-density lipoprotein than the controls (P < 0.05). The hyperlipidaemic rats also had significantly worse erectile function (P = 0.004) and developed more bladder overactivity (P = 0.004) than the controls. In the hyperlipidaemic rats there was significant muscle hypertrophy in the peri-urethral lobe of the prostate (P < 0.001) and in the bladder (P < 0.05). There was also greater P2X(1) (purinoceptor) staining as well as other molecular changes in the bladder of the hyperlipidaemic rats. CONCLUSIONS: In this hyperlipidaemic rat model three abnormalities were consistently detected: prostatic enlargement, bladder overactivity, and ED. This rat model could be a useful research tool for understanding the common causes of LUTS and ED, as well as facilitating the development of preventive measures and better therapies to treat both conditions.

Functional improvement in spinal cord injury-induced neurogenic bladder by bladder augmentation using bladder acellular matrix graft in the rat.

Spinal cord injury (SCI) rostral to the lumbosacral level causes bladder hyperreflexia and detrusor-sphincter dyssynergia (DSD), which are accompanied by bladder hypertrophy. We hypothesize that bladder augmentation using a bladder acellular matrix graft (BAMG) can improve the function of SCI-mediated neurogenic bladder. In female rats (n = 35), SCI was induced by transection of the spinal cord at the lower thoracic level. Eight weeks following spinalization, bladder augmentation using BAMG was performed after hemicystectomy of the hypertrophic bladder. Cystometrography was performed at 8 weeks after spinalization and again at 8 weeks after augmentation. Several urodynamic parameters were measured and the grafted bladder was histologically evaluated. Thirty one rats were alive 8 weeks after spinalization. Twenty two (71%) rats developed hyperreflexic bladders and nine (29%) rats had underactive bladders before bladder augmentation. Twenty six rats survived until 8 weeks after augmentation. Urodynamic parameters showed improvement in some bladder functions in both hyperreflexic and underactive bladders after augmentation. In addition, bladder compliance was increased in hyperreflexic bladders and decreased in underactive bladders. Bladder augmentation decreased bladder capacity in high-capacity rats and increased it in low-capacity rats. Histological evaluation showed complete regeneration of BAMG in SCI-induced neurogenic bladder at 8 weeks after augmentation. This is the first report suggesting that the voiding function in SCI-induced neurogenic bladder can be improved by augmentation using BAMG. Improved voiding function was accompanied by histological regeneration of BAMG.

The effect of FK1706 on erectile function following bilateral cavernous nerve crush injury in a rat model.

PURPOSE: We investigated the neurotrophic effect of FK1706 on erectile recovery following bilateral cavernous nerve crush injury in a rat model. MATERIALS AND METHODS: A total of 28 male Sprague-Dawley rats were randomly divided into 4 equal groups. Seven animals underwent sham operation and subcutaneous vehicle injection, whereas 21 underwent bilateral cavernous nerve crush injury followed by vehicle injection alone, or by low (0.1 mg/kg) or high (1.0 mg/kg) dose FK1706 treatment. Injections were continued 5 days weekly for 8 weeks. Erectile function was then assessed by cavernous nerve electrostimulation and penile tissue was evaluated immunohistochemically. RESULTS: No erectile dysfunction was identified in the sham treated group (mean maximal intracavernous pressure +/- SEM 106.8 +/- 6.4 cm H(2)O), whereas nerve injury significantly decreased ICP to 17.9 +/- 7.0 cm H(2)O. FK1706 facilitated neural and erectile recovery in a concentration dependent manner with a mean ICP in the high dose FK treatment group of 80.1 +/- 7.8 cm H(2)O compared with 44.1 +/- 12.9 cm H(2)O in the low dose group. Similar stepwise findings were observed using mean area under the curve data. Sham treated animals showed regular axon sizes and shapes with homogenous GAP-43 and neurofilament staining, whereas injured axons showed irregular shapes, sizes and staining patterns. FK1706 treatment restored axon shape and staining patterns. Injury significantly decreased nicotinamide adenine dinucleotide phosphate staining and FK1706 treatment showed a nonsignificant trend toward increased staining. CONCLUSIONS: Bilateral cavernous nerve crush causes reproducible erectile dysfunction, consistent with prior experiments. High dose subcutaneous FK1706 therapy promotes significant neuroregeneration and erectile function recovery.

Collagen I:III ratio in canine heterologous bladder acellular matrix grafts.

The objective of this study was to investigate the limitations of a heterologous bladder acellular matrix graft (BAMG) and the influence of the collagen ratio on functional regeneration in a large animal model. Ten female dogs underwent partial cystectomy; eight received BAMG (two homologous; six heterologous) and two partial cystectomy only. A cystometry was performed prior to surgery and 7 months postoperatively when all animals underwent sacral root stimulation. Tissue specimens were studied by histologic and immunohistochemical techniques and for collagen types. At month 7, all animals survived and bladder capacity in the grafted animals was increased. All grafts demonstrated all components of a normal bladder wall. Nerves were seen with the density decreasing with distance from the anastomosis. The BAMG processing and follow-up demonstrated no changes in the homologous tissue, whereas in the heterologous tissue, the amount of collagen changed with the processing during implantation. None of these heterologous specimens demonstrated a similar collagen ratio to the hosts'. The homologous BAMG undergoes more complete regeneration. In the heterologous BAMG, collagen seems not to be replaced. The amounts and ratio of collagen types I and III seem to influence smooth muscle regeneration.

The effect of intracavernosal growth differentiation factor-5 therapy in a rat model of cavernosal nerve injury.

OBJECTIVE: To determine whether the intracavernosal application of growth differentiation factor-5 (GDF-5) influences nerve regeneration and erectile function after cavernosal nerve injury in a rat model. MATERIALS AND METHODS: Thirty-two male Sprague-Dawley rats were randomly divided into four equal groups: eight had a sham operation (uninjured controls), while 24 had bilateral cavernosal nerve crush. The crush-injury groups were treated at the time of injury with an impregnated collagen sponge implanted into the right corpus cavernosum. The sponge contained no GDF-5 (injured controls), 2 microg (low concentration), or 20 microg GDF-5 (high concentration). Erectile function was assessed by cavernosal nerve electrostimulation at 8 weeks. Midshaft penile tissue samples were histochemically evaluated for neuronal nitric oxide synthase (nNOS)-containing fibres in the dorsal penile nerve. RESULTS: There was no erectile dysfunction in the uninjured control group, as shown by a mean (sem) maximal increase in intracavernosal pressure (ICP) of 149.5 (17.0) cmH(2)O on stimulation. By comparison, the ICP decreased in the injured control group, by 21.3 (6.7) cmH(2)O. After cavernosal nerve injury, the recovery of erectile function was greatest in the low-concentration GDF-5 group; the maximum ICP increase was 40.8 (13.3) cmH(2)O, vs 24.3 (5.9) cmH(2)O for 20 microg GDF-5. Histologically, the low-concentration group had significantly more nNOS-containing nerve fibres, at 163 (24.7), than the high-concentration group, at 76 (17.3), or injured controls, at 67 (23.8). By contrast, the uninjured controls had a mean of 538 (40.6) nerve fibres in the dorsal nerve. CONCLUSION: Bilateral cavernosal nerve crush resulted in erectile dysfunction with accompanying neurological changes in the rat. The intracavernosal application of GDF-5 enhanced the recovery of erectile function and n-NOS nerve preservation, with a 2-microg dose giving the most promising results.

Effect of vascular endothelial growth factor on regeneration of bladder acellular matrix graft: histologic and functional evaluation.

OBJECTIVES: To evaluate the effect of vascular endothelial growth factor (VEGF) on regeneration of the bladder acellular matrix graft (BAMG). METHODS: A total of 40 female rats were divided into two groups. In the experimental group (VEGF+), the BAMG was incubated in VEGF and VEGF was injected into the host bladder. In the control rats, sterile solution replaced VEGF. Urodynamic studies were performed at grafting and repeated at 2, 4, 8, and 12 weeks. The bladders were harvested for histologic examination. Immunostaining was used to monitor VEGF treatment by examining VEGF and VEGF-receptor 2 expression. Neovascularity, smooth muscle induction, and nerve regeneration were assessed immunohistochemically. RESULTS: Increased VEGF and VEGF-receptor 2 were present in the VEGF+ rats 2 weeks after grafting. The maximal intravesical pressure was significantly greater in the VEGF+ group at 2 weeks than in the controls, despite the same bladder capacity. At 4 weeks, a significant increase in bladder capacity and a decrease in the postvoid residual volume ratio were observed. After 8 weeks, no differences were noted in the urodynamic parameters between the two groups. Numerous alpha-actin-positive spindle cells were observed in the VEGF+ rats at 2 weeks, and the smooth muscle content was significantly greater at all points. Enhanced angiogenesis was noted at 2 and 4 weeks. Nerve fibers were observed at 4 weeks and nerve growth factor-positive cells were significantly greater at 2, 4, and 8 weeks in the VEGF+ rats. CONCLUSIONS: Administration of VEGF had significant effects on the BAMG at early periods after grafting. Our results have shown that VEGF enhances BAMG regeneration in rats as assessed by functional restoration and histologic examination.

The additive erectile recovery effect of brain-derived neurotrophic factor combined with vascular endothelial growth factor in a rat model of neurogenic impotence.

OBJECTIVE: To test the hypothesis that combined intracavernosal injection with vascular endothelial growth factor (VEGF) with adeno-associated virus-mediated brain-derived neurotrophic factor (AAV-BDNF) synergistically facilitates the neural regeneration and erectile function after cavernosal nerve injury. MATERIALS AND METHODS: Forty Sprague-Dawley male rats were randomly divided into five equal groups: eight had a sham operation while 32 had bilateral cavernosal nerve freezing followed by an immediate intracavernosal injection with either phosphate-buffered saline (PBS), VEGF, AAV-BDNF, or AAV-BDNF + VEGF. Erectile function was assessed by cavernosal nerve electrostimulation at 3 months, and samples of the major pelvic ganglia and penile tissue were evaluated histologically. RESULTS: In this animal model of impotence from nerve injury, the recovery of erectile function was greatest in those receiving AAV-BDNF + VEGF; the mean (sd) maximal intracavernosal pressure in this group was 87.2 (20.78) cmH2O, compared with 37.3 (11.39) for VEGF alone and 49.8 (29.58) for AAV-BDNF alone. No erectile dysfunction was identified in the sham group, with a pressure of 100.7 (22.70) cmH2O, while all treatment groups significantly outperformed the PBS (control) group, at 29.3 (13.52) cmH2O. Furthermore, all animals receiving monotherapy or combined treatment had more NADPH-diaphorase-positive nerve fibres than controls but less than in the sham group. CONCLUSION: Bilateral cavernosal nerve freezing causes erectile dysfunction with accompanying neurological changes. Intracavernosal injection with either VEGF or AAV-BDNF alone enhances nerve regeneration, with combined therapy (VEGF and AAV-BDNF) promoting neural and erectile recovery additively.

Long-term survival of autotransplanted major pelvic ganglion in the corpus cavernosum of adult rats.

PURPOSE: Neurogenic impotence is a common complication after radical pelvic surgery, irradiation or perineal trauma. Neuronal transplantation is a new frontier for treating neurological disorders. We investigated whether the major pelvic ganglion can survive and become functional after being implanted into the corpus cavernosum in adult rats. MATERIALS AND METHODS: Adult male rats (13) were divided into 3 groups and sacrificed at 3 time points, namely 30 (4), 60 (5) and 90 (4) days. All rats underwent excision of the right major pelvic ganglion and left cavernous nerve. The right ganglion was implanted into the right crus of the penis. Electrostimulation was applied to the left major pelvic ganglion and cavernous nerve (1.5 mA.) and right crus (10 mA.) at sacrifice. The crural region and left ganglion were then excised for immunostaining of neuronal nitric oxide synthase (nNOS), protein gene product 9.5 and growth associated protein 43. Image analysis was used to calculate the area stained in pixels. Electron microscopy of the implanted area was performed to assess neuronal survival. RESULTS: Although the degree varied, all neuronal implants survived after transplantation. The response to electrostimulation was insufficient to produce erection. No difference was noted among the areas of nNOS staining when specimens from the 3 time points were compared. The area of expression of nNOS, protein gene product 9.5 and growth associated protein 43 was larger in the implanted area than in the surrounding cavernous tissue. Under electron microscopy most surviving implants showed normal ultrastructure, although areas of fibrotic replacement were seen in several implants. CONCLUSIONS: Our results show that the autotransplanted major pelvic ganglion expresses nNOS, protein gene product 9.5 and growth associated protein 43, and survived up to 90 days after implantation into the corpus cavernosum. Further studies with fetal neuronal tissue seem warranted.

The effect of labor and/or ovariectomy on rodent continence mechanism--the neuronal changes.

Many women develop stress urinary incontinence (SUI) after childbirth, but the exact neuronal changes are largely unknown. This study is designed to identify the neuronal changes associated with pregnancy, delivery and ovariectomy. A total of 10 virgin and 48 pregnant rats were used. Cystometry and stress/sneeze tests were performed in the virgin once and the pregnant rats at certain time points. Postpartum the rats were equally grouped as follows: group I: delivery, group II: delivery + ballooning, group III: delivery + ovariectomy, group IV: delivery + ballooning + ovariectomy. Tissues from bladder, bladder neck, and urethra were analyzed by immunostaining for PGP 9.5, CGRP, SP, NPY, VIP, TH, n-NOS. We found complex innervation changes in the different tissue samples. Since the bladder neck and the mid-urethra play an important role in the continence mechanism the neuronal changes in these areas contribute to the observed functional changes.

Homologous acellular matrix graft for vaginal repair in rats: a pilot study for a new reconstructive approach.

The purpose of this paper is to investigate using an acellular matrix graft of vagina (VAMG) or bladder (BAMG) in vaginal reconstruction. In 18 rats, vaginal length was measured and a hysterectomy performed. In three control animals, the vaginal stump was closed. In eight rats, the vagina was augmented with a VAMG; in seven, a BAMG was used. After 2-12 weeks, the animals were sacrificed, the vaginal length was reevaluated, and the vaginas were prepared for histologic evaluation. In the controls, the vagina was markedly shorter postoperatively. In the grafted animals, vaginal length was not significantly less than preoperative values with either matrix. Epithelialization, vascularization, and alpha-actin expression in the grafts were consistently observed. Regeneration appeared to be slightly greater in the organ-specific vagina matrix. With either matrix, however, although the vaginal stump remained open, the grafts lost most of the lumen. Vaginal reconstruction with a vagina acellular matrix graft is technically feasible. If further experiments can address the problem of luminal collapse - with, for instance, tissue expanders in the matrix - this technique may offer an alternative to the complex therapeutic options currently available.

The effect of neural embryonic stem cell therapy in a rat model of cavernosal nerve injury.

OBJECTIVE: To isolate embryonic stem cells that have differentiated along the neuronal cell line, and to assess whether injecting these neural stem cells into the corpus cavernosum influences cavernosal nerve regeneration and functional status. MATERIALS AND METHODS: Embryonic neural stem cells were obtained; 26 male Sprague-Dawley rats were divided into four groups: five had a sham operation; eight (controls) had a bilateral cavernosal nerve crush and injection of culture medium into the corpora cavernosa; four had an injection of neural embryonic stem (NES) cells into the major pelvic ganglion (MPG); and nine had bilateral cavernosal nerve crush and injection of NES cells into the corpora cavernosa. Erectile response was assessed by cavernosal nerve electrostimulation at 3 months, and penile tissue samples were evaluated histochemically for nitric oxide synthase (NOS)-containing fibres, tyrosine hydroxylase and neurofilament staining. RESULTS: The groups injected with NES cells into the MPG and corpora cavernosa had significantly higher intracavernosal pressures than the control group. Immunohistochemical staining also revealed differences in the quality of the NOS-containing nerve fibres. Neurofilament staining was significantly better in the experimental groups injected with NES cells. CONCLUSION: We were able to isolate embryonic stem cells that had differentiated along the neural cell line and, using these NES cells intracavernosally, showed improved erectile function in a rat model of neurogenic impotence.

The bladder acellular matrix graft in a rat chemical cystitis model: functional and histologic evaluation.

PURPOSE: We investigated the feasibility of augmentation in a diseased bladder with a bladder acellular matrix graft. MATERIALS AND METHODS: In 50 female Sprague-Dawley rats chemical cystitis was induced by intravesical instillation of HCl repeated monthly to maintain chronic inflammation. Urodynamic studies were performed in all rats 1 week after the induction of chemical cystitis and repeated at sacrifice. The 29 rats in the experimental group underwent partial cystectomy (50% or greater), followed by bladder acellular matrix graft augmentation, while the 21 controls underwent monthly HCl instillation only. The rats were sacrificed at 2 weeks, 1, 2 and 3 months, respectively. The bladder was removed and examined for histological changes. RESULTS: Urodynamic studies showed that bladder capacity and compliance were significantly higher in the grafted than in the control group (p = 0.008 and 0.006, respectively, at 3 months). Histological studies revealed urothelial and smooth muscle regeneration within the bladder acellular matrix graft at 1 month and nerve regeneration at 3. The number of mast cells was significantly lower in the grafted region than in the host bladder of all grafted rats (p <0.001). CONCLUSIONS: In this rat chemical cystitis model bladder augmentation with a bladder acellular matrix graft led to functional and histological improvement over diseased host bladder.