British Journal of Urology (1997), 79, 285–287

P OI N T OF T E C H NI QU E

A modified in vitro technique to evaluate human detrusormuscleD .J. SMI TH and C. R. CHAPP LEDepartment of Urology, Royal Hallamshire Hospital, Sheffield, UK

The biopsy was taken to the laboratory, dissected inIndicationsTyrode’s solution under a dissecting microscope, and aloop of 6/0 polyglactin tied at each end. The specimensTo study human bladder function, most workers use

large strips of human bladder material obtained at open produced were about 4×2 mm and the muscle stripsamples were then mounted in a 5 mL tissue bath ofoperation [1] but there are numerous difficulties. The

amount of tissue available for these in vitro studies is standard design.The specimen was mounted at one end to a staticlimited and often the tissue collected is of poor quality

and viability. Therefore, most in vitro studies of the hook and at the other was tied to an isometric tensiontransducer (UFI 1030). Using a simple micro-bladder are performed on animals and the results

obtained extrapolated to dysfunctions in humans. There manipulator, the muscle strip was placed under a tensionof 1 g and allowed to equilibrate. The bath was filledare many species differences apparent between animals

and man [2] and any interpretation of human disease with Tyrode’s solution at 37°C and gassed continuouslyby 95% O

2and 5% CO

2.from animals must be viewed with caution.

Palfrey et al. [3] reported a technique that used Isometric contractions were recorded with a tensiontransducer (UFI 1030), the output being amplified anddetrusor muscle samples obtained during routine endos-

copic cold-cup biopsy. This excellent and simple tech- collected on a MacLab 8 software system using a Mac Sii.Standard cumulative agonist/antagonist dose-resp-nique was intended to increase the total availability of

tissue using smaller preparations and to improve onse curves were obtained to acetyl choline (AcH)/atrop-ine and, using simple parallel platinum electrodes, electr-the physiological diffusion of substances, with better

viability. ical field stimulation could also be applied to the tissues.After an initial pilot study to validate the field stimula-However, this technique has not been widely accepted

because the preparations could only be studied using a tion technique, trains of wave pulses were delivered viathe platinum electrodes (at 30 Hz, 20 V and 0.5 mssuperfusion technique and specialized tissue baths have

to be constructed. Thus, for routine physiological studies duration) to stimulate the tissue. Field stimulation of thebladder biopsies was achieved using these values within human bladder, workers have continued to look at

larger muscle strips. square-train pulses of 2 ms; this appeared to causemaximal stimulation sensitive to inactivation by tetrodo-We report a modification of the technique of Palfrey

et al. that allows standard designs of tissue bath to be toxin, a nerve poison that is used to differentiate betweenneurogenic and directly stimulated smooth muscleused which should increase the acceptance of this in

vitro technique. responses. All neurogenically mediated responses shouldbe eliminated by the addition of tetrodotoxin, and if anycontractile responses remain they are due to directMaterials and methodsmuscle stimulation.

Full informed consent was obtained to allow the collec-tion of human tissue at endoscopy, with local ethical Resultscommittee approval. All muscle samples were obtainedat routine cystoscopy using Storz cold-cup biopsy forceps. The success or viability rate of the tissues studied was

high. In most cases there were at least two viable biopsiesThe overlying mucosa was biopsied first and the samplesent for histological analysis, and then a further biopsy from the same patient to allow a comparison and to

produce repeatable results. Mostly, 4–6 biopsies wereof the underlying muscle was taken from the same ‘bitesite’. The specimen obtained was immediately immersed taken and about 70% of these samples gave reliable,

reproducible responses to the agonists used. Oncein modified Tyrode’s solution at room temperature [3].

285© 1997 British Journal of Urology

286 POI NT O F TECH NI QUE

confirmed as viable, most tissues could then elicit 3–4 (1×10−8 to 1×10−5 mol/L, Fig. 2). Atropine shiftedthe control response curve to the right, with no reductioncumulative dose-response curves. The tissues maintained

viability for 5–8 h in the organ bath. in the maximum response gained; this is compatiblewith a competitive antagonist.The bladder samples were used within 18 h of collec-

tion, with 70% used within 2 h of sampling. Some tissue There was a contraction in response to electricalstimulation; it was neurogenic (Fig. 3) and was elimin-was stored at 4°C overnight with no detrimental effect

on function; this usually enhanced viability, for which ated with tetrodotoxin. Figure 3 also shows the increas-ing response plotted as a percentage of the maximumwe have no explanation.

Of more than 275 biopsies taken, only one caused a response with increasing frequency. Throughout theseexperiments there were no atropine-resistant responsesproblem; in one patient, a small bladder perforation was

recognized at operation, secondary to the biopsy, which (Fig. 4).required 2 extra days with an indwelling catheter butno long-term problem. Thus, the morbidity (perforation) Advantages and disadvantagesrate was <0.3%.

The quality of recordings was good, with reproducible Because of differences among species, any reliable infor-mation on the neurophysiology of the human bladderresponse curves obtained; although the magnitude of

response was relatively small (<1 g), it was possible to has been sparse and obtained by extrapolation or carefulcomparative studies. Using the present technique at theobtain adequate recordings after amplification. During

these experiments, the human tissue showed no spon- time of endoscopic surgery should ensure that sufficienttissue is collected to allow the further study of a largetaneous bladder contractions.

The concentration of agonist required to give half- range of different normal and abnormal human bladders.With more comprehensive studies of human detrusor,maximal response (EC

50) was used to indicate and

compare tissue sensitivity. With increasing cumulative the ambiguous data extrapolated from animal experi-ments can be verified more closely.doses of AcH, the tissues contracted reproducibly, with

a mean EC50

of 3.4×10−4 mol/L; a similar response Using small tissue samples in standard baths allowsmore experiments to be undertaken with better tissuewas found for potassium chloride (Fig. 1).

In most cases, the AcH agonist dose-response curve survival and good specimen perfusion. The technique ofPalfrey et al. [3] only allowed super perfusion experi-could then be repeated, using a varying dose of atropinements, but the present modification allows agonist/antagonist studies with no need for expensive specialized

120

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3 × 10–3

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Fig. 1. A comparison of the contractions achieved with KCl (red)and acetyl choline (green). Maximum contractions were attainedat lower doses of KCl; the EC

5Ofor acetyl choline was 3.4×10−4

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Fig. 2. Dose response curves to increasing concentrations of acetylmol/L and was 2.6×10−5 mol/L for KCl. The percentage maximumcontraction was compared against the maximum for KCl. choline and with atropine antagonism (green) n=6.

© 1997 British Journal of Urology 79, 285–287

POI NT O F T ECH NI QUE 287

the viability of the technique. The collection of specimensis simple and has little associated morbidity for thepatient; the potential risk of bladder perforation is verysmall.

Previous studies have found some spontaneous con-tractile response in human bladder muscle tissue. In thepresent study, there were no contractions during theequilibration period and none that were consideredspontaneous. It is not clear whether this was related tothe size of tissue sample or was a result of normalphysiology.

The technique gives reproducible results and allowsnerve-mediated and cholinergic agonist stimulation ofhuman detrusor to be examined in vitro. Both appear tobe sensitive to atropine and this lends further support tothe view that there is no significant non-cholinergic,atropine-resistant motor innervation of the functionallynormal bladder. The present results confirm the lack ofatropine resistance in normal bladders, in agreementwith the report of Palfrey et al. [3] and Kinder andMundy [4].

This technique provides safe, effective and ready accessto human bladder material, thereby allowing the in vitrostudy of the human detrusor muscle using existingpharmacological laboratory equipment.

120

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Fig. 3. a, The response curve to increasing frequency of electricalstimulation. Responses at >40 Hz were mainly by direct muscle Referencesstimulation and thus 30 Hz was chosen as the frequency applied

1 Eaton AC, Bates CP. An in vitro physiological study ofduring experiments. b, Chart recording obtained from muscle strips

normal and unstable human detrusor muscle. Br J Urolwith increasing frequency of stimulation. n=5.

1982; 54: 653–72 Hindmarsh JR, Idowu OA, Yeates WK, Zar MA.

Pharmacology of electrically evoked contractions of humanbladder. Br J Pharmacol 1977; 61: 115P

3 Palfrey ELH, Fry CH, Shuttleworth KED. A new in vitromicrosuperfusion technique for investigation of humandetrusor muscle. Br J Urol 1984; 56: 635–40

4 Kinder RB, Mundy AR. Atropine blockade of nerve-mediatedstimulation of the human detrusor. Br J Urol 1985;57: 418–21

0.2

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Atropine

10–4

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added

–0.2

–0.1Re

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Fig. 4. Chart recordings obtained during the addition of atropine,showing that there were no atropine resistant contractions in Authorshuman detrusor. D.J. Smith, MB, ChB, FRCS, Specialist Registrar in Urology.

C.R. Chapple, BSc, MD, FRCS (Urol), Consultant Urologist.tissue baths. Any laboratory using in vitro methods Correspondence: Mr D.J. Smith, Department of Urology, Royal

Hallamshire Hospital, Glossop Road, Sheffield, UK.should be able to collect the muscle specimens and assess

© 1997 British Journal of Urology 79, 285–287