Hypothesis / aims of study
Cyclophosphamide is a commonly administered chemotherapeutic originally developed for cancer treatment. In more recent years it has also been used as an immunotherapeutic to treat a range of diseases such as rheumatoid arthritis, lupus nephritis, and interstitial lung disease. However, patients treated with cyclophosphamide often experience serious urotoxic side effects including urinary frequency, urgency, pain, nocturia through to haemorrhagic cystitis that significantly impact quality of life (1). Cyclophosphamide is also often used, with varying success, to create rodent models of cystitis (2). The urotoxic side effects of cyclophosphamide following cancer treatment are thought to be caused by the urinary metabolite acrolein, although the mechanism of action remains poorly understood. The aim of this study was to investigate the effects of intravesical acrolein (100µM) on bladder function, in an ex-vivo whole bladder preparation. It was hypothesised that acrolein would alter the contractility of the murine bladder and alter nerve-evoked responses.
Study design, materials and methods
Female C57/BL6J mice (12-13 weeks) were used in this study. Mice were euthanised, bladders were isolated and placed in 37°C Krebs-bicarbonate solution and aerated with carbogen (95% O2 and 5% CO2) gas. Whole bladder preparations were performed as previously described by our group (3). Briefly, a three-way catheter, equipped with perfusion pump, pressure transducer and outflow tap, was inserted via the urethra, and the bladder was distended at a rate of 30µL/minute. Changes in pressure were visualised using ADinstruments LabChart software. Bladders were distended either with 0.9% saline (controls) or acrolein (100µM in saline) to a pressure of 20mmHg and allowed to accommodate. Spontaneous activity, bladder compliance and accommodation, detrusor contractility and nerve-evoked responses were measured. Statistical analysis undertaken drew comparisons between control and treated groups were via one-tailed, unpaired Student’s test.
Intravesical acrolein did not affect bladder compliance (Figure 1A); however, a greater, though not significant, fall in intravesical pressure was observed in acrolein treated bladders during accommodation compared to controls (Figure 1B, P=0.06, one-tailed, unpaired Student's t-test). Frequency and amplitude of spontaneous contractions during filling and accommodation was not significantly affected by acrolein treatment. The muscarinic agonist carbachol produced concentration-dependant increase in intravesical pressure (figure 2A) in both control (n=6) and acrolein-infused (n=6) bladders. However, while potency of carbachol was unchanged by acrolein, maximum contractions were significantly enhanced when compared to the control (P<0.05, 31.82±3.12 mmHg and 23.82±4.00 mmHg respectively, one-tailed, unpaired Student's t-test). Similarly, depolarisation of efferent nerves by electrical field stimulation produced frequency-dependent increases in intravesical pressure, with a significant increase (P<0.01) observed at 5Hz in the acrolein group (n=6) when compared to the controls (n=6). Electrical field stimulation was also conducted in the presence of the muscarinic acetylcholine receptor antagonist atropine. Atropine decreased nerve-evoked contractions by 9.38 ±2.79% in control bladders, and 13.8 ±3.32% in acrolein treated bladders with no significant difference between groups. Similarly, desensitisation of purinergic receptors using alpha,beta-methylene ATP further reduced nerve evoked responses by 60.1 ±1.37% in the controls and 69.5 ±1.65% in acrolein infused bladders with no significant difference between each group. The beta-adrenoreceptor agonist isoprenaline produced concentration-dependant decrease in intravesical pressure in both control (n=6) and acrolein treated (n=6) bladders, with no significant difference between each group. Responses to purinergic agonist alpha,beta-methylene ATP (figure 2B) in acrolein-treated (n=6) bladders were also significantly increased (P<0.05) compared to controls (n=6, one-tailed, unpaired Student's t-test). Additionally, general detrusor contractility was assessed using potassium chloride and was unchanged by acrolein treatment (P=0.06, one-tailed, unpaired Student's t-test).
Interpretation of results
Bladder compliance, accommodation and spontaneous phasic activity were not significantly affected by acrolein treatment. However, intravesical acrolein increased pressure responses to muscarinic and purinergic receptor agonists. This may explain the generally enhanced responses observed following efferent nerve stimulation of acrolein-treated bladders.