Teruhiko Yokoyama

BOTULINUM TOXIN URETHRAL SPHINCTER INJECTION TO RESTORE BLADDER EMPTYING IN MEN AND WOMEN WITH VOIDING DYSFUNCTION

PURPOSEnBotulinum toxin injection into the external urinary sphincter in spinal cord injured men with detrusor-sphincter dyssynergia has been reported. We expand the clinical use of botulinum toxin for a variety of bladder outlet obstructions and to decrease outlet resistance in patients with acontractile detrusor but who wish to void by the Valsalva maneuver.nnnMATERIALS AND METHODSnProspective treatment was performed for voiding dysfunction in 8 men and 13 women 34 to 74 years old. The reasons for voiding dysfunction included neurogenic detrusor-sphincter dyssynergia in 12 cases, pelvic floor spasticity in 8 and acontractile detrusor in 1 patient with multiple sclerosis who wished to void by the Valsalva maneuver. Using a rigid cystoscope and a collagen injection needle, a total of 80 to 100 units of botulinum A toxin (Botox) were injected into the external sphincter at the 3, 6, 9 and 12 oclock positions.nnnRESULTSnPreoperatively 19 of 21 patients were on indwelling or intermittent catheterization. After botulinum A injection all but 1 patient were able to void without catheterization. No acute complications, such as general paralysis or respiratory depression, occurred and none of the patients had dribbling or stress urinary incontinence. Postoperative post-void residual decreased by 71% and voiding pressures decreased on average 38%. Of the 21 patients 14 (67%) reported significant subjective improvement in voiding. Followup ranges from 3 to 16 months, with a maximum of 3 botulinum A injections in some patients.nnnCONCLUSIONSnUrethral sphincter botulinum injection should be considered for complex voiding dysfunction. Encouraging improvement without complications were seen in most of our patients. We have expanded the use of botulinum toxin to treat pelvic floor spasticity and also women.

Preliminary results of myoblast injection into the urethra and bladder wall: A possible method for the treatment of stress urinary incontinence and impaired detrusor contractility

The purpose of this study is to explore the feasibility of myoblasts, the precursors of muscle fibers, injected periurethrally as a potential treatment of stress urinary incontinence. We also studied myoblast injection into the bladder wall to potentially improve detrusor contractility. A myoblast cell line was transduced with adenovirus carrying the expression of the β‐galactosidase reporter gene while in culture. The cells were incubated with fluorescent latex microspheres (FLMs) to follow the outcome of the injected cells. The tissue was harvested 3–4 days after injection; sectioned, fixed, assayed for β‐galactosidase expression, and counterstained with H+E. Photographs of the slides were taken under light and fluorescence microscopy. We have noted a large number of cells expressing β‐galactosidase and containing FLMs in the urethral and bladder walls under fluorescent microscopy (8 animals). Many regenerative myofibers expressing β‐galactosidase were also seen in the urethral and bladder walls. The fusion of injected myoblasts to form myotubes was seen in both the urethral and bladder walls. The introduction of myoblasts into the urethral and bladder wall is feasible and results in formation of myotubes and myofibers in the smooth muscle layers of the lower urinary tract. We hypothesize that myoblast injections can be used as a non‐allergenic agent to enhance urethral closure and bladder function. Neurourol. Urodynam. 19:279–287, 2000.

PERSISTENCE AND SURVIVAL OF AUTOLOGOUS MUSCLE DERIVED CELLS VERSUS BOVINE COLLAGEN AS POTENTIAL TREATMENT OF STRESS URINARY INCONTINENCE

PURPOSEnWe explored the use of autologous muscle derived cells as a method of treating stress urinary incontinence. We determined whether urethral muscle derived cell injection is feasible and compared it with bovine collagen injection.nnnMATERIALS AND METHODSnMuscle derived cells isolated from female Sprague-Dawley rats were first transduced with retrovirus carrying the transgene for beta-galactosidase. We injected approximately 1 to 1.5 x 106 cells into the bladder wall and proximal urethra of 6 autologous animals. Tissue was harvested after 3 and 30 days, sectioned, stained for fast myosin heavy chain and assayed for beta-galactosidase. To compare muscle derived cell and bovine collagen injections 100 microl. of commercially available bovine collagen were also injected in Sprague-Dawley female rats. Tissue was harvested in 3 animals each after 3 and 30 days, sectioned and stained for trichrome. Subsequently, 3 adult SCID mice were used to compare the level of transgene expression at each time point after injecting 1.5 x 106 cells per injection, which were transduced with adenovirus carrying the transgene for beta-galactosidase.nnnRESULTSnA large number of cells expressing beta-galactosidase were observed in the bladder and urethral wall 3 and 30 days after autologous cell injection in Sprague-Dawley rats. The persistence of primary muscle derived cells at 3 days was similar to that of collagen. However, at 30 days there was significant cell persistence while only a minimal amount of injected bovine collagen was detectable. Approximately 88% of the beta-galactosidase expression at day 3 remained at day 30 in SCID mice.nnnCONCLUSIONSnWe present 2 new findings important for the emerging field of urological tissue engineering, including the feasibility of injecting autologous skeletal muscle derived cells into the lower urinary tract and the greater persistence of such injected cells versus injected bovine collagen. Therefore, autologous muscle derived cell injection may be an attractive alternative treatment option for stress urinary incontinence.

Muscle-derived cell-mediated ex vivo gene therapy for urological dysfunction.

We have tested the feasibility of muscle-based gene therapy and tissue engineering for urological dysfunction using highly purified muscle-derived cells (MDC) that display stem cell characteristics. We then explored the potential use of these MDC as an alternative therapy for the treatment of impaired detrusor contractility. The MDC were genetically engineered to express the gene encoding β-galactosidase and injected into the bladder walls of SCID mice. The injected bladders were harvested at various time-points after injection and assayed for β-galactosidase activity; the presence of myofibers within the injected tissue was determined by detection of fast myosin heavy chain isoform (MyHCs). We have demonstrated that the injected MDC are capable of not only surviving in the lower urinary tract, but also improving the contractility of the bladder following an induced injury. Two potential mechanisms can be used to explain this finding. First, we have observed that some of the β-galactosidase-expressing cells expressed α-smooth muscle actin, suggesting a differentiation into smooth muscle. Second, a stain for acetylcholine receptors (AChRs), which identifies the location of neuromuscular junctions, revealed that the myofibers derived from the doner cells became innervated into the bladder as early as 2 weeks after injection. These results suggest that gene therapy and tissue engineering based on MDC potentially can be used for urological dysfunction.

Effect of intravesical nitric oxide therapy on cyclophosphamide-induced cystitis

PURPOSEnThis study was conducted to examine effects of nitric oxide (NO) donors on bladder hyperactivity induced by cyclophosphamide (CYP)-induced cystitis.nnnMATERIALS AND METHODSnFemale Sprague-Dawley rats received a single intraperitoneal injection of CYP (100 mg./kg.), and then their micturition pattern including mean micturition volume and the number of micturitions during 24 hours was recorded in a metabolic cage before and after CYP treatment. Forty-eight hours after CYP injection, bladder function under urethane anesthesia was evaluated by cystometry with continuous saline infusion (0.04 ml. per minute) or under isovolumetric conditions (0.8 ml. bladder volume). NO donors, S-nitroso-N-acetyl-penicillamine (SNAP, 2 mM) or sodium nitroprusside (SNP, 1 mM), and an NO synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (L-NAME, 20 mM) were administered intravesically. Direct action of SNAP on bladder afferent neurons was also tested in a patch-clamp recording study.nnnRESULTSnThe number of micturitions significantly increased during the first 24 hours after CYP injection (19.0 +/- 0.88 versus 92.1 +/- 16.3 micturitions/24 hours, mean +/- SE, n = 25) (p <0.001). There was no significant difference in total micturition volume before (12.3 +/- 1.0 ml./24 hours) and after CYP treatment (15.6 +/- 1.5 ml./24 hours). During continuous infusion cystometry, intercontraction interval (ICI) was smaller in CYP-injected rats than in control rats. In CYP-injected animals, NO donors increased the ICI, but did not change the amplitude of bladder contractions. Continuous intravesical infusion of the NOS inhibitor did not alter the cystometric parameters. During cystometry under isovolumetric conditions, contraction frequency was decreased after NO donor administration. NO donors did not influence bladder activity in control rats. In patch clamp recordings, when SNAP (500 microM) was directly applied to dissociated afferent neurons innervating the urinary bladder, high-voltage-activated Ca2+ channel currents were suppressed by approximately 30%.nnnCONCLUSIONSnIntravesical NO donors can suppress CYP-induced bladder hyperactivity. We hypothesize that the effect of NO donors is not due to smooth muscle relaxation, but rather due to an inhibitory effect on bladder afferent pathways that was manifested by an increase in intercontraction interval without changes in contraction amplitude. NO donors may be considered as a possible treatment of CYP-induced and other types of bladder inflammation.

Diabetic Cystopathy Correlates With a Long-Term Decrease in Nerve Growth Factor Levels in The Bladder and Lumbosacral Dorsal Root Ganglia

PURPOSE It has been proposed that a deficiency in the axonal transport of nerve growth factor (NGF) may have an important role in inducing diabetic neuropathy, which contributes to diabetic cystopathy. Therefore, in streptozotocin (Sigma Chemical Co., St. Louis, Missouri) induced diabetic rats we investigated the relationship of bladder function with NGF levels in the bladder and lumbosacral dorsal root ganglia, which contain afferent neurons innervating the bladder. MATERIALS AND METHODS At 6 and 12 weeks after the induction of diabetes with streptozotocin (65 mg./kg. intraperitoneally) the effects of diabetes on Adelta afferent fiber dependent, conscious voiding were evaluated by metabolic cage measurements and awake cystometry. The effects of diabetes on C-fiber mediated bladder nociceptive responses were also investigated by cystometry with intravesical instillation of 0.25% acetic acid in the rats under urethane anesthesia. NGF levels in the bladder and L6 to S1 dorsal root ganglia were measured by enzyme-linked immunosorbent assay 3, 6, 9 and 12 weeks after streptozotocin injection. RESULTS In diabetic rats NGF levels in the bladder and L6 to S1 dorsal root ganglia were significantly decreased 12 weeks after streptozotocin injection (p <0.01). In cystometry and metabolic cage studies bladder capacity and post-void residual volume were significantly increased 12 weeks after streptozotocin injection (p <0.01). Bladder nociceptive responses revealed by a reduction in inter-contraction intervals after acetic acid infusion were significantly decreased in a time dependent manner 12 weeks after streptozotocin injection.CONCLUSIONS Rats with streptozotocin induced diabetes mellitus showed a significant time dependent decrease in NGF levels in the bladder and L6 to S1 dorsal root ganglia that was associated with voiding dysfunction attributable to defects in Adelta and C-fiber bladder afferents. Therefore, reduced production of NGF in the bladder and/or impaired transport of NGF to L6 to S1 dorsal root ganglia, which contain bladder afferent neurons, may be an important mechanism inducing diabetic cystopathy.

Herpes simplex virus mediated nerve growth factor expression in bladder and afferent neurons: Potential treatment for diabetic bladder dysfunction

PURPOSEnDiabetic cystopathy resulting from sensory neuropathy may potentially be treated by direct gene therapy. It has been suggested that nerve growth factor (NGF) has an ameliorative effect in preventing the death in diabetes of afferent dorsal root ganglion neurons, which control bladder function. We investigated NGF gene transfer to the bladder and bladder afferent pathways for treating diabetic cystopathy. We used replication competent and replication defective herpes simplex virus type 1 (HSV-1) vectors that express a functionally active form of the beta-subunit of mouse NGF (beta-NGF) to examine the level and duration of therapeutic gene expression after administration of the vectors.nnnMATERIALS AND METHODSnNGF expression during acute (3 days) and latent (21 days) infections was assessed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemical testing after the injection of 1 x 106 to 1 x 108 pfu HSV-NGF expression vectors into the bladder wall of adult rats.nnnRESULTSnHSV vectors with the strong human cytomegalovirus immediate early promoter used to drive beta-NGF gene expression exhibited increased NGF 3 days after infection in the bladder and L6 to S1 dorsal root ganglia, where bladder afferent neurons are located. ELISA analysis revealed that NGF in the bladder tissue and dorsal root ganglia was increased 7 to 9 and 2 to 4-fold, respectively, over the control vector. Increased NGF expression in L6 to S1 dorsal root ganglia neurons was also detected by immunohistochemical staining with antiNGF antibodies. Extended NGF expression was detected by ELISA 21 days after injection. Replication defective vectors containing HSV-1 latency promoter (LAP-2) driving NGF expressed NGF in the bladder and dorsal root ganglia 21 days after bladder injection. ELISA analysis confirmed an approximate 2 to 3-fold increase of NGF expression in the bladder and L6 to S1 dorsal root ganglia.nnnCONCLUSIONSnThe NGF gene may be transferred and expressed in the bladder and bladder afferent pathways using HSV vectors. To our knowledge our study represents the first demonstration of the effectiveness of gene therapy for altering neurotrophic expression in visceral sensory neurons. This technique of gene transfer may be useful for treating certain types of neurogenic bladder dysfunction, such as diabetic cystopathy, in which decreased NGF transport may be a causative factor.

Myoblast therapy for stress urinary incontinence and bladder dysfunction.

Abstract The field of tissue engineering and gene therapy has an exciting and promising future. During the past few years we have begun a comprehensive effort to investigate the use of myoblasts to improve and expand the treatment of stress urinary incontinence and bladder dysfunction. Moreover, we can expect the application of myoblast-mediated ex vivo gene transfer in the field of urology. In this paper we discuss the compositions of and methods involving the use of myogenic or muscle-derived cells for tissue engineering and cell-mediated gene therapy.

Intravesical liposome administration--a novel treatment for hyperactive bladder in the rat.

OBJECTIVESnTo examine the effect of intravesical administration of liposomes (LPs) on chemically induced bladder hyperactivity in the rat. It has been suggested that interstitial cystitis (IC) is associated with a dysfunctional or leaky epithelium. Thus, enhancement of epithelial barrier function might be useful in the treatment of IC. LPs are vesicles that are concentric phospholipid bilayers separated by an aqueous compartment and can fuse with cells to provide a molecular film that can promote wound healing.nnnMETHODSnThe intravesical pressure was recorded using a transurethral catheter in adult female Sprague-Dawley rats anesthetized with urethane (1.2 g/kg subcutaneously). Some animals were pretreated with capsaicin (125 mg/kg subcutaneously) 4 days before the experiments. Continuous cystometrograms were performed by slowly filling the bladder (0.04 mL/min) with solutions of varying compositions, including saline, acetic acid (AA, 0.1%), potassium chloride (KCl, 500 mM), protamine sulfate (PS, 10 mg/mL), LPs, PS/KCl, or LPs/KCl. The parameters measured included the intercontraction interval (ICI), amplitude of bladder contractions, compliance, and micturition pressure threshold.nnnRESULTSnThe ICI was decreased after exposure to AA (79.8% decrease) or PS/KCl (81% decrease); however, the ICI was not changed after LPs, PS, or KCl alone. The decreased ICI was partially reversed after infusion of LPs (172.8% increase) or LPs/KCl (63% increase), but was not significantly changed after switching to saline or KCl administration. Pretreatment with capsaicin delayed the onset of the irritative effects of AA by approximately 30 to 60 minutes, but had not changed the magnitude after 2 hours of infusion.nnnCONCLUSIONSnIntravesical administration of PS/KCl or AA activates capsaicin-sensitive and capsaicin-resistant afferents in a time-dependent sequence that is partially reversed by LP infusion. We hypothesize that LPs might enhance the barrier properties of a dysfunctional uroepithelium and increase resistance to irritant penetration. Thus, intravesical LP administration could be a novel treatment of patients with IC.

Muscle-derived cell transplantation and differentiation into lower urinary tract smooth muscle.

OBJECTIVESnTo explore the feasibility of primary skeletal muscle-derived cell (MDC)-based tissue engineering and gene transfer into the lower urinary tract and to explore whether the injected primary skeletal MDCs can persist and differentiate into myotubes and myofibers in the bladder wall.nnnMETHODSnPrimary MDCs isolated from normal mice were first transduced with adenovirus encoding the expression of the beta-galactosidase reporter gene. Adult severe combined immunodeficiency mice (n = 12) were used in this study. The MDCs were injected into the right and left lateral bladder walls with a 10-microL Hamilton microsyringe. The amount of injected MDCs ranged from 1 to 1.5 x 10(6) cells. The tissue was harvested after 5, 35, and 70 days, sectioned, stained for fast myosin heavy chain, and assayed for beta-galactosidase expression.nnnRESULTSnWe observed a large number of cells expressing beta-galactosidase in the bladder wall at each time point. Many myotubes and myofibers expressing beta-galactosidase and positively stained for fast myosin heavy chain were also seen in the bladder wall at 35 and 70 days after injection. Additionally, the size of the injected MDCs significantly increased during the course of the study (P <0.05).nnnCONCLUSIONSnWe have demonstrated the long-term survival and beta-galactosidase expression of MDCs injected into the bladder wall. Moreover, our results suggest that some injected MDCs can differentiate into myofibers. These results suggest that MDCs can be a desirable substance for tissue engineering and an ex vivo method for gene transfer into the lower urinary tract.