The Frequency-Dependence of Pre- and Postganglionic Nerve Stimulation of Pig and Rat Bladder.

PURPOSE: The urinary bladder generates phasic contractions via action potentials generated in pre- and then postganglionic neurons. Whilst the frequency-dependence of postganglionic neurons to generate contractions has been quantified, the dynamic range of preganglionic neurons is less clear and if intramural ganglia exert frequency-dependent modulation of transmission between pre- and postganglionic neurons. The phosphodiesterase type 5 inhibitor sildenafil reduces neurotransmitter release from postganglionic fibres to detrusor smooth muscle and an additional question was if there was also a preganglionic action. This study aimed to compare the frequency range of bladder contractile activation by pre- and postganglionic stimulation in pig and rat bladders and if sildenafil exerted additional preganglionic actions. METHODS: An arterially-perfused ex vivo pig bladder preparation was used for preganglionic (pelvic nerve) and mixed pre-and postganglionic (direct bladder wall) stimulation at 36°C and postganglionic mediated contractions achieved by field-stimulation of in vitro isolated detrusor strips. With rats, pelvic nerve stimulation was carried out in vivo and postganglionic stimulation also with isolated detrusor strips. RESULTS: All contractions were abolished by 2% lignocaine indicating they are nerve-mediated. Stimulation targets were verified with hexamethonium that completely abolished pelvic nerve responses by had no effect on detrusor strips; responses to mixed bladder wall stimulation were partially reduced. The frequency-dependence of contractile activation was similar whether by pre- or postganglionic stimulation in both pigs and rats. Sildenafil reduced contractions to preganglionic stimulation significantly more than to postganglionic stimulation. Mixed pre- and postganglionic stimulation were reduced by an intermediate extent. CONCLUSION: Intramural ganglia offer no frequency-dependent modulation under the experimental conditions used here and the sildenafil data are consistent with multiple sites of action underlying generation of bladder contractions. A translational aspect of these findings is discussed in terms of setting stimulation parameters for neuromodulation protocols.

Stretch- and carbachol-induced ATP release from bladder wall preparations of young and aged mice.

AIMS: Bladder wall stretch increases tissue tension and releases adenosine 5'-triphosphate (ATP) as part of a transduction process to sense bladder filling. Aging is associated with bladder fibrosis to produce a stiffer bladder wall: this may augment ATP release and contribute to age-dependent urgency. Muscarinic agonists also release ATP and present a potential target for antimuscarinic agents, but its age-dependency is unknown. This study aimed, in young and old mice, to: (a) quantify the relationship between bladder wall stiffness and stretch-dependent ATP release and; (b) characterize muscarinic agonist-dependent release. METHODS: ATP release from young (9-12 weeks) and aged (24 months) mouse bladder wall was measured in vitro, with a luciferin-luciferase assay, after stretch or carbachol exposure. Bladder wall stiffness, measured simultaneously during stretch, was compared to histological proportions of connective tissue and detrusor muscle. RESULTS: With young mice, stretch-activated ATP release required an intact mucosa and was positively associated with wall stiffness. ATP release by carbachol was about four-fold greater compared to stretch. With aged mice: ATP release varied a hundred-fold and no association with stiffness; carbachol release diminished; connective tissue and mucosa thickness increased. CONCLUSIONS: With young mice, stretch, or muscarinic agonists potently induce bladder wall ATP release. Stretch-dependent release is proportional to bladder wall stiffness, independent of the extent of stretch. With aged mice dependence of stretch-activated ATP release with stiffness was lost. The huge variability of release suggests that aged mice do not form a homogenous cohort and may underlie the heterogeneity in bladder filling sensations.

The calculation and comparison of the Detrusor Contractility Parameter and Watts Factor.

AIMS: To test the significance of association between a validated index of detrusor contractility, vCE , the Watts Factor, and the Detrusor Contractility Parameter, t20-80 ; and to test whether t20-80 depends on outflow tract resistance as indicated by Bladder Outlet Obstruction Index (BOOI). METHODS: Thirty-seven pressure-flow traces from 20 male and 17 female patients were analyzed and forms of the Watts Factor, t20-80 and BOOI were compared with vCE . RESULTS: The Detrusor Contractility Parameter, t20-80 , is significantly associated with vCE for both women and men without a high degree of bladder outlet obstruction. The Watts Factor only had a significant association with vCE at the point of maximum flow in women. CONCLUSIONS: The Detrusor Contractility Parameter (DCP) (t20-80 ), can be measured easily from the pressure flow curves of a urodynamic test. The Watts Factor at maximum urine flow, WFQmax , can be readily calculated, but is only applicable to women. In both women and men without a high degree of bladder outlet obstruction, DCP is better associated with true detrusor contractility than any Watts Factor analysis.

Mild external heating and reduction in spontaneous contractions of the bladder.

OBJECTIVES: To measure the effect of external heating on bladder wall contractile function, histological structure and expression of proteins related to tissue protection and apoptosis. MATERIAL AND METHODS: In vitro preparations of bladder wall and ex vivo perfused pig bladders were heated from 37 to 42°C, 46 and 50°C for 15 min. Isolated preparations were heated by radiant energy and perfused bladders were heated by altering perfusate temperature. Spontaneous contractions or pressure variations were recorded, as well as responses to the muscarinic agonist carbachol or motor nerve excitation in vitro during heating. Tissue histology in control and after heating was analysed using haematoxylin and eosin staining and 4'-6-diamidino-2-phenylindole (DAPI) nuclear labelling. The effects of heating on protein expression levels of (i) heat shock proteins HSP27-pSer82 and inducible-HSP70 and (ii) caspase-3 and its downstream DNA-repair substrate poly-[ADP-ribose] polymerase (PARP) were measured. RESULTS: Heating to 42°C reduced spontaneous contractions or pressure variations by ~70%; effects were fully reversible. There were no effects on carbachol or nerve-mediated responses. Tissue histology was unaffected by heating, and expression of heat shock proteins as well as caspase-3 and PARP were also unaltered. A TRPV1 antagonist had no effect on the reduction of spontaneous activity. Heating to 46°C had a similar effect on spontaneous activity and also reduced the carbachol contracture. Urothelial structure was damaged, caspase-3 levels were increased and inducible-HSP70 levels declined. At 50°C evoked contractions were abolished, the urothelium was absent and heat shock proteins and PARP expression was reduced with raised caspase-3 expression. CONCLUSIONS: Heating to 42°C caused a profound, reversible and reproducible attenuation of spontaneous activity, with no tissue damage and no initiation of apoptosis pathways. Higher temperatures caused tissue damage and activation of apoptotic mechanisms. Mild heating offers a novel approach to reducing bladder spontaneous activity.

Estimation of bladder contractility from intravesical pressure-volume measurements.

AIMS: To describe parameters from urodynamic pressure recordings that describe urinary bladder contractility through the use of principles of muscle mechanics. METHODS: Subtracted detrusor pressure and voided flow were recorded from patients undergoing filling cystometry. The isovolumetric increase of detrusor pressure, P, of a voluntary bladder contraction before voiding was used to generate a plot of (dP/dt)/P versus P. Extrapolation of the plot to the y-axis and the x-axis generated a contractility parameter, vCE (the maximum rate of pressure development) and the maximum isovolumetric pressure, P0 , respectively. Similar curves were obtained in ex vivo pig bladders with different concentrations of the inotropic agent carbachol and shown in a supplement. RESULTS: Values of vCE , but not P0 , diminished with age in female subjects. vCE was most significantly associated with the 20-80% duration of isovolumetric contraction t20-80 ; and a weaker association with maximum flow rate and BCI in women. P0 was not associated with any urodynamic variable in women, but in men was with t20-80 and isovolumetric pressure indices. CONCLUSIONS: The rate of isovolumetric subtracted detrusor pressure (t20-80 ) increase shows a very significant association with indices of bladder contractility as derived from a derived force-velocity curve. We propose that t20-80 is a detrusor contractility parameter (DCP). Neurourol. Urodynam. 36:1009-1014, 2017. © 2016 Wiley Periodicals, Inc.

Contractile effects and receptor analysis of adenosine-receptors in human detrusor muscle from stable and neuropathic bladders.

To measure the relative transcription of adenosine receptor subtypes and the contractile effects of adenosine and selective receptor-subtype ligands on detrusor smooth muscle from patients with neuropathic overactive (NDO) and stable bladders and also from guinea-pigs. Contractile function was measured at 37°C in vitro from detrusor smooth muscle strips. Contractions were elicited by superfusate agonists or by electrical field stimulation. Adenosine-receptor (A1, A2A, A2B, A3) transcription was measured by RT-PCR. Adenosine attenuated nerve-mediated responses with equivalent efficacy in human and guinea-pig tissue (pIC50 3.65-3.86); the action was more effective at low (1-8 Hz) compared to high (20-40 Hz) stimulation frequencies in human NDO and guinea-pig tissue. With guinea-pig detrusor the action of adenosine was mirrored by the A1/A2-agonist N-ethylcarboxamidoadenosine (NECA), partly abolished in turn by the A2B-selectve antagonist alloxazine, as well as the A1-selective agonist N6- cyclopentyladenosine (CPA). With detrusor from stable human bladders the effects of NECA and CPA were much smaller than that of adenosine. Adenosine also attenuated carbachol contractures, but mirrored by NECA (in turn blocked by alloxazine) only in guinea-pig tissue. Adenosine receptor subtype transcription was measured in human detrusor and was similar in both groups, except reduced A2A levels in overactive bladder. Suppression of the carbachol contracture in human detrusor is independent of A-receptor activation, in contrast to an A2B-dependent action with guinea-pig tissue. Adenosine also reduced nerve-mediated contractions, by an A1- dependent action suppressing ATP neurotransmitter action.