222 INCREASED LEVELS OF SUPEROXIDE IONS AFFECTS BLADDER FUNCTION IN A NEW MUTANT MOUSE MODEL

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Urinary hydrogen peroxide: A probable marker of oxidative stress in interstitial cystitis

Increased levels of superoxide ions affects bladder function in a new mutant mouse model

Masuda H., Sakai S., Numao N., Komai Y., Tatokoro M., Kihara K.

Fullhase C.1, Soler R.2, Lu B.1, Bishop C.E.1, Andersson K.E.1, Wake Forest Institute for Regenerative Medicine

Tokyo Medical and Dental University, Dept. of Urology, Tokyo, Japan

1 2

Introduction & Objectives: Interstitial cystitis (IC) is a chronic inflammatory disease of the bladder. Urine hydrogen peroxide was postulated to be a biomarker of oxidative stress. We checked the endogenous hydrogen peroxide formation in the urine to investigate whether oxidative stress is involved in the etiology of IC. Overactive bladder (OAB) female patients were also investigated. Material & Methods: Urine specimens were collected from nine IC patients, ten OAB patients and 10 controls. All participants were female with mean ±SD age of 57±7 in IC group, 62±8 in OAB group and 59± 5 in control group. All IC patients had classic IC. Urinary tract infection was negative in all cases. Also, in IC patients, urine specimens were collected during remission period after treatment with hydrodistension, TUC or hyperberbic therapy. Urinary hydrogen peroxide was measured using FOX-2 methods. IC patients were asked questions about symptoms and related problems from O,Leary-Sant questionnaire. Results: Urine hydrogen peroxide concentration in the IC group (74.5±8.7 mmol/l) was significantly (p<0.05) higher than that in the control (12.6±4.3 mmol/l) or OAB group (21.2±5.8 mmol/l). There was no significant difference between groups of control and OAB. After treatment, both symptom and problem scores and total scores significantly (p<0.05) decreased along with significant reduction in urine hydrogen peroxide concentration. In 4 IC cases, urine hydrogen peroxide concentration again increased after appearance of severe symptoms. Conclusions: Oxidative stress formation in the bladder may be a part of pathogenesis in IC and clinical biomarker of IC.



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Wake Forest Institute for Regenerative Medicine, Winston-Salem, United States of America, Fedreal University of Sao Paulo, Dept. of Urology, Sao Paulo, Brazil

Introduction & Objectives: Nitric oxide (NO) released from postganglionic parasympathetic neurons mediates urethral smooth muscle relaxation during reflex micturition. Findings compatible with deficient urethral relaxation and increased outflow resistance were described in male nNOS-deficient mice. In a streptozotocin-induced diabetes mellitus model in female rats, a decreased NO responsiveness was associated with a decreased urethral smooth muscle relaxation. Using a transgenic insertional mutagenesis strategy, we generated a mouse with a mutation in the inner mitochondrial membrane peptidase 2-like (Immp2l) gene. This mutation leads to high superoxide ion levels, a consequent decrease in the bioavailability of NO and an increase in reactive oxygen species. We studied bladder function in vivo and in vitro in this model. Material & Methods: Using an ear biopsy, homozygote mutation for the Immp2l gene was demonstrated by PCR and Western blot analysis. Male mutants (4-6 months) (n=5) and healthy age-matched controls (wildtypes) (n=5) underwent bladder catheterization. A PE 20 tube was inserted into the dome of the bladder, anchored, and tunnelled subcutaneously to the skin of the neck. Three days later urodynamic evaluation was performed in conscious animals, with continuous bladder filling. Non-operated mutants (n=5) and wildtypes (n=5) were sacrificed and their bladders removed. Strips of smooth muscle were dissected and mounted in an oxygenated organ bath containing Krebs solution. After a resting period of one hour, strips were exposed to pharmacological and electrical stimulation. Results: Urodynamically the mutants showed significant lower micturition volumes (0.08±0.07 vs. 0.2±0.1ml, p<0.05) and higher residual volumes (0.17±0.03 vs. 0.04±0.03ml, p<0.001) than wildtypes. The mutants had pronounced difficulties in initiating micturition, and massive straining was seen before voiding. This could be objectified in measuring the time between the first raise in bladder pressure till the maximal pressure during micturition (77.9±29.3 vs. 24.1±15.1sec, p=0.007). However, no statistically significant differences could be shown between mutants and wildtypes neither in responses to carbachol in various concentrations nor to electrical stimulations at different frequencies. Conclusions: These results suggest that Immp2l mutant mice exhibit bladder dysfunction mainly characterized by emptying abnormalities, with a preserved detrusor function. This may be explained by a defect urethral relaxation secondary to a reduction in available NO. Since these are young animals, further evaluation of old animals will bring valuable information about chronic exposition to high superoxide and low NO levels. This makes these animals an interesting model to study diseases like diabetes and aging.



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Distribution and neurochemistry of high affinity binding sites for Botulinum toxin type A in the urinary bladder

Suburothelial myofibroblasts in the human overactive bladder and the effect of Botulinum neurotoxin type a treatment

Coelho A.1, Dinis P.2, Pinto R.2, Gorgal T.2, Silva C.2, Andre A.2, Joao J.3, Cruz C.1, Cruz F.R.2, Avelino A.1

Roosen A.1, Apostolidis A.2, Stief C.G.1, Fowler C.J.2

Faculty of Medicine of Porto, Institute of Histology and Ibmc, Porto, Portugal, 2Hospital S Joao, Dept. of Urology, Porto, Portugal, 3Hospital S Joao, Dept. of Urology, Porto, Portugal

1

Introduction & Objectives: Botulinum neurotoxin type A (BoNT-A) administration has been successfully used in the treatment of symptoms associated with intractable overactive bladder. The specificity of BONT-A is due to the presence of membrane receptors (Synaptic Vesicle protein, SV2) that are exposed during neurotransmitter exocytosis. Once internalized, BONT-A undergoes a pH-dependent conformational change that causes the dissociation of the heavy and light chains. The latter has endopeptidase activity that cleaves specific sites of the SNAP-25 protein, preventing the assembly of the synaptic fusion complex SNARE and blocking neurotransmitter release. In the present study we studied the distribution of SV2 and SNAP-25 in three different species, including human. The neurochemistry of BONT-A sensitive structures was investigated using markers for parasympathetic sympathetic and sensory fibers. Material & Methods: Human bladders were obtained from cadaveric organ donors (19-65 years) after permission from the Ethics Committee of Hospital S. João. Rodent bladders were injected with 10U of BONT-A and were collected at 1, 3, 8 15 days and 1 month (rat) and 1, 3 and 8 days (guinea pig). Cryostat sections were processed for single or dual immunofluorescent staining using antibodies against SV2, SNAP-25 (cleaved and uncleaved), vesicular acetylcholine transporter (VAChT), tyrosine hydroxilase (TH) and calcitonin-gene related peptide (CGRP). Images were obtained in a Zeiss Apotome fluorescence microscope. Results: Immunoreactive (IR) fibers for SV2 and SNAP25 were found in the mucosa and muscular layers in all the species. In humans, IR- fibers were more abundant in the trigonal area and in the bladder of younger individuals. No staining was found in epithelial or muscular cells. Double labelling showed extensive colocalization of the two proteins. SV2 exhibited a high degree of colocalization with all types of nerve fibers. In contrast, SNAP25 exhibited a higher degree of colocalization with VAChT-IR fibers than with TH or CGRP-IR fibers. In the human bladder, a dense cholinergic plexus was found under the transitional epithelium that was not present in rodents. Immunostaining for cleaved SNAP25 in BONT-A treated animals showed sparse fibers at all time points studied in the rat (24h-1 month) and abundant fibers in the guinea pig muscular layer (24h-1 week). Cleaved SNAP25-IR was most abundant in VAChT-IR fibers. Conclusions: Our data show that: I) The targets presently known for BONT-A are exclusively nerve fibers. II) BONT-A targets and by-products of BONT-A action are more abundant in cholinergic, parasympathetic fibers. III) The amount of SNAP-25 cleavage in the bladder after a similar BONT-A dose is orders of magnitude more intense in the guinea-pig than in the rat. Supported by an unrestricted grant from Allergan.

Eur Urol Suppl 2009;8(4):176

1 Ludwig Maximilians University, Dept. of Urology, Munich, Germany, 2University College London, Dept. of Uro-Neurology, London, United Kingdom

Introduction & Objectives: An increasing body of evidence suggests a possible role of suburothelial myofibroblasts (MFs) in bladder mechanosensation and in the pathophysiology of detrusor overactivity (DO). To determine whether markers of MFs, including gap junction protein connexin43 (Cx43) and c-kit have altered immunohistochemical expression in the suburothelium of patients with neurogenic or idiopathic DO (NDO/IDO) and whether this is affected by successful treatment of DO with Botulinum neurotoxin type A (BoNT/A). Material & Methods: Patients with NDO (n=10) or IDO (n=11) were treated in a single-centre open-label study of intradetrusor BoNT/A injections. Control tissue was obtained from 10 patients undergoing pelvic floor repair procedures who had no OAB symptoms. Flexible cystoscopic bladder biopsies were obtained from NDO/IDO patients before and at 4 and 16wk after BoNT/A treatment, and from controls. They were studied with quantitative immunofluorescence using antibodies to Cx43, vimentin and c-kit. Differences in Cx43, vimentin and c-kit immunoreactivity (Fig. 1) between controls and NDO/IDO patients (primary outcome). Changes in NDO/IDO Cx43 and c-kit immunoreactivity after BoNT/A treatment (secondary outcome). Results: Cx43 immunoreactivity was increased in both IDO and NDO patients compared to controls, but remained unchanged after BoNT/A treatment. C-kit immunoreactivity was similar in NDO/IDO patients and controls and remained unchanged after BoNT/A treatment (Fig. 2). Conclusions: Increased gap junction formation in the suburothelium has been demonstrated in biopsies from humans with DO. It is hypothesised that this change could have a significant role in the pathogenesis of the detrusor abnormality. Successful treatment of NDO or IDO does not appear to be associated with changes in the expression of Cx43 or c-kit on suburothelial MFs.