In vitro contractility studies

At the time o f post-mortem, fetal bladder samples, from mid-region o f bladders, were placed in Ca^^ free Hepes Tyrode’s solution (105 mM NaCl, 19.5 mM HEPES (N-[2- hydroxyethyl]piperazine-N’-[2-ethanesulphonic acid]), 3.6 mM KCl, 0.9 mM

MgCl2.H20, 3.6 mM NaH2P0 4-H2 0, 21.5 mM NaHCOs, 5.5 mM glucose, 4.5 mM Na

pyruvate, pH 7.1 adjusted with IM NaOH). Samples were transported to the laboratory at the Institute o f Urology and using a dissecting microscope (x 20), I removed the

urothelium and adventitia by dissection. I then excised a strip o f detrusor muscle

(denuded strip) attempting to keep the muscle strip diameter less than 1 mm to facilitate diffusion o f substrates into the preparation. The muscle strip was then placed in an experimental chamber which was mounted on a heavy metal base that, in turn, was mounted on a heavy metal table with metal legs embedded in sand (Figure 21). The

perspex experimental chamber contains a 3 mm wide trough that contains a static hook at one end. One end of the bladder strip was tied, using 8/0 silk, to this static hook; the other end o f the strip is tied to a hook attached to an isometric tension transducer (UC3, Gould Statham, GmBH, Seeheim-Ober Beerbach, Germany) that could be manipulated with a Prior micromanipulator that enabled alteration o f muscle strip resting length. Strips were

then superfused with normal Tyrode’s solution gassed with 95% O2 / 5% CO2, pH 7.4, 37

°C (Tyrode’s solution: 118 mM NaCl, 24 mM NaHCOg, 4.0 mM KCl, 1.0 mM

MgCl2.6H20, 0.4 mM NaH2?0 4.H2 0, 1.8 mM CaCb, 6.1 mM glucose and 5.0 mM Na

pyruvate). The sides o f the trough contained parallel platinum plate electrodes that delivered electrical field stimulation (EPS) to the strip using a Bioscience stimulator and programmer. This delivered 3 second trains o f 0.1 ms square wave pulses every 90 seconds with a pulse frequency ranging from 1-60 Hz. This sequence has been shown to generate phasic contractions by direct stimulation o f nerves embedded within the strip without direct muscular activation.

Pharmacological manipulation o f contractile function, with various agonists and

antagonists o f putative neurotransmitter action, was used to characterise those involved in regulating detrusor contractility. Control steady-state contractions were obtained at 8 Hz EPS. Nerve-mediated tension was taken as the difference between total and tetrodotoxin (TTX)-resistant force. The latter was calculated by generating a force-frequency

associated with its action potential and therefore, as a small element o f force remains after the addition o f TTX, this must be subtracted firom the EFS-generated force to determine true nerve-mediated force.

Muscarinic neurotransmission

In addition to EPS, contractures were elicited by carbachol, a muscarinic agonist (1 pM, 3 pM, 10 pM and 30 pM).

Purinergic neurotransmission

Strips were stimulated by EPS at 8 Hz in the presence o f atropine (1 pm), a competitive antagonist o f muscarinic receptors. Atropine-resistant contractions were taken as the difference between nerve-mediated responses in the absence or presence o f atropine and reflected the proportion o f nerve-mediated contraction elicited by acetyl choline release. The purinergic component o f contraction was obtained by eliciting contraction in the absence and presence o f 10 pm a-(3-methylene ATP (ABMA), a purinergic receptor (P2X) agonist that elicits a contracture in muscle strips. However, continued presence causes desensitisation o f this receptor to prevent any further activation by neurally released ATP. In addition, strips were stimulated at 8 Hz in the presence o f adenosine (1 mM) and, after steady-state baseline contractions at 8 Hz, contractures were elicited in unstimulated preparations by carbachol (1 pM), carbachol (1 pM) plus adenosine (1 mM), and then subsequently with carbachol (1 pM) alone. The latter experiments were performed to establish the effect of adenosine on pre-contracted strips; the mean force

generated by the carbachol contracture was determined from the pre- and post-adenosine experiment.

Nitrergic neurotransmission

Strips were maximally stimulated with carbachol (10 pM) and after two test EPS responses, elicited at 8 Hz, a force fi'equency relation was determined. After return to control solutions and achieving steady-state baseline contractions at 8 Hz, a repeat carbachol contracture was elicited in the presence o f lH-[l,2,4]oxadiazolo[4,3-

a]quinoxalin-1 -one (ODQ), an agent that has been shown to reduce the relaxant effect of cyclic guanosine 3 ', 5 '-monophosphate (cGMP) that is generated by the release o f nitric oxide (NO) (Garthwaite et al, 1995).

At the end o f all experiments, the muscle was weighed and contractile force expressed in units o f mN.mg'^ wet weight o f tissue. Force-frequency relations and dose-response relations were fitted to the empirical equations (Bayliss et al, 1999):

T = Tmax. f T = Tmax [S]"

K i / 2 " + f E C 5 0 " + [ S f

where T is the tension, Tmax is the estimated maximum tension at high frequencies or high

concentrations,/is the frequency o f stimulation, [S] is the agonist concentration, K yj and

ECso are the frequency and concentration respectively required to achieve 71nax/2 and n is

The effect o f the mucosa

To determine the effect o f mucosa on detrusor muscle contractility, I also determined

force-frequency relations for full thickness bladder wall strips (mucosal strip) and

corrected force generated to the wet weight o f the detrusor in the preparation. The latter was calculated by histological study (see below). In addition, these strips were also stimulated in the presence o f ODQ (1 pM) to determine the role, if any, o f nitric oxide.

Detrusor thickness calculation

So that mucosal bladder strips could be corrected for wet weight o f muscle (to allow

direct comparison with denuded muscle strips), I calculated the percentage thickness of

the detrusor smooth muscle layer in bladder sections stained with Masson’s trichrome. Histology and Masson’s trichrome stain were performed as previously described in Section 4.2.4. Using the KS 300 computer program (Carl Zeiss), I calculated detrusor

thickness and whole wall thickness for bladders from sham and obstructed groups (n=5

both groups) to calculate the relative proportions.

Neuronal protein immunohistochemistry and western blot

To visualise nerves within fetal bladders, I performed immunohistochemistry o f two key neuronal proteins. S I00 is a calcium-binding protein, found in neuroepithelial and neural crest derived cells; it is present in all Schwann cells and in most neurons (Stefansson et al, 1982). Protein gene product 9.5 (PGP 9.5) is a cytosolic protein that is highly

expressed in neurons (Gulbenkian et al, 1987). Immuniohistochemistry was performed as previously described in section 4.2.6. In brief, the primary antibodies used were anti-

SlOO antibody (1:500; DAKO, Ely, UK) or anti-PGP 9.5 antibody (1:500; DAKO) and the secondary antibody used was biotinylated goat anti-rabbit antibody (1:200;

Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK).

To quantify any change o f innervation in the sham and obstructed fetal bladders, I

performed western blot o f PGP 9.5, as described in section 4.2.7. In brief, the primary antibody used was anti-PGP 9.5 antibody (1:1000; DAKO) or p-actin (1:10000), a ‘house-keeping’ protein and the secondary antibodies were either HRP-linked goat anti­ rabbit antibody (1:1000), for PGP 9.5 detection, or HRP-linked sheep anti-mouse

antibody (1:5000), for P-actin detection. Bands were detected by chemiluminescence, for 30 seconds (PGP 9.5) or 10 seconds (p-actin). SlOO protein binds calcium and as such, also reacts with calcium-binding proteins such as calmodulin and myosin light chain (Baudier, Glasser, & Gerard 1986) found in detrusor muscle; hence, it was appropriate only for immunohistochemistry.