932 A NOVEL IN VITRO PIG BLADDER MUCOSA MODEL FOR EXPERIMENTAL RESEARCH

age 48 years) who had undergone male-to-female gender reassignment surgery. Using the organ bath technique, the effects of vinpocetine (PDE2-I), MEP1 (PDE2-I), rolipram (PDE4-I), Sildenafil, Tadalafil and Vardenafil (PDE5-Is) (0.01 µM - 10 µM) on the tension induced by norepinephrine (10 µM) of urethral smooth muscle strip preparations were investigated. After cumulative administration of the respective drugs, tissue strips were rapidly frozen, homogenized, cyclic AMP and cyclic GMP extracted and measured by means of radioimmunological assays. In the experiments, the adenylyl cyclase activator forskolin and NO donor drug sodium nitroprusside (SNP) served as reference drugs. Results: The adrenergic tension was reversed by the drugs with the following rank order of efficacy: SNP (-68 ± 19%) > rolipram (-39 ± 16%) ≥ Sildenafil (-35 ± 18%) > forskolin (-27 ± 18%) ≥ Vardenafil (-26 ± 6%) > Tadalafil (-20 ± 7%) > vinpocetine (-16 ± 12%). Only negligible effects of the PDE2-I MEP1 were observed (-2 ± 16%). The relaxing effects of the drugs were paralleled by a 1.4-fold to 9.6-fold increase in tissue levels of cyclic AMP and 3-fold to 111-fold elevation in cyclic GMP. Conclusions: Relaxant actions of specific inhibitors of PDE4 and PDE5 on urethral smooth muscle were registered. Inhibition of PDE isoenzymes might represent an interesting option to facilitate the relaxation mediated by cyclic nucleotides of the human outflow region.

932

A novel in vitro pig bladder mucosa model for experimental research

Janssen D.A.W.1, Geutjes P.J.1, Odenthal J.1, Van Kuppevelt T.H.2, Schalken J.A.1, Feitz W.F.J.1, Heesakkers J.P.F.A.1 1 Radboud University Nijmegen Medical Centre, Dept. of Urology, Nijmegen, The Netherlands, 2Radboud University Nijmegen Medical Centre, Dept. of Biochemistry, Nijmegen, The Netherlands Introduction & Objectives: Experimental research on the urinary bladder often requires the use of laboratory animals. Organ tissue (organotypic) culturing could be an alternative for in vivo experiments. With current cell and organ culture technologies (tissue engineering), it is possible to keep organ tissue viable under in vitro conditions for longer periods. The aim of this study is to develop an strait forward experimental bladder model that can be used to investigate functionality (physiology), toxicity and regeneration of this organ. Materials & Methods: We mechanically isolated mucosa from freshly dissected pig bladders (abattoir & University animal laboratory). Twenty sterile punch biopsies (0.5 mm Ø) were taken. First, we cultured the biopsies on 3 substrates, i.e. type I collagen scaffold, PET membrane (0.4 µm pore size) and metal roster), in a CO2incubator(37°C). Secondly, scaffolds were glued on transwell inserts(Millipore) for tissue culturing. Five different culture media were evaluated (Keratinocyte, Smooth muscle cell, DMEM®, RPMI®, Epilife®). Biopsies were evaluated at 0d, 2d, 1wk, 3wk, 6wk, fixated and embedded in paraffin. Tissue was histologically evaluated with standard HE, scanning electron microscope (SEM) and immunohistochemical staining, i.e. apoptosis (TUNEL-assay), proliferation (Ki67) and cell type (urothelial cells (UC), smooth muscle cells (SMC) and interstitial cells(IC). Tissue permeability was assessed with an Inulin-FITC assay.

Results: On type I collagen scaffolds the mucosa remained viable for >3wk, compared to PET membrane (