Hypothesis / aims of study
Painful bladder syndrome/interstitial cystitis has a profound impact on quality of life. There is strong evidence that bladder inflammation is crucial in generating the symptoms of interstitial cystitis (1). Compared to healthy subjects, patients with interstitial cystitis have increased sensitivity to bladder distension and to the ice water test. However, it is not clear which types of bladder afferents are major contributors to bladder hypersensitivity in interstitial cystitis. We use zymosan (a ligand found on the surface of cell wall of yeast, which activate immune cells via Toll-like receptors) to cause a mild bladder inflammation in guinea pigs with lower cellular toxicity that is macroscopically similar to patients with non-ulcerative interstitial cystitis.
The main objective of the study was to validate the zymosan-induced cystitis model and identify the type of afferents which are sensitised in the bladder wall.
Study design, materials and methods
Zymosan-induced cystitis was induced by intravesical administration of zymosan (1ml, 10mg/ml for 1hr) after pre-treatment with protamine sulphate (1ml, 10mg/ml for 1hr) in isoflurane (2%) anaesthetised guinea pigs. The extent of the bladder inflammation was assessed next day by standard histological techniques. 24 hrs after zymosan treatment, micturition parameters were assessed in conscious or urethane-anaesthetised guinea pigs (1.2 g/kg, s.c.) by continuous cystometry infusing saline at 0.15 ml/min. Visceromotor responses were evoked by bladder distension with saline (10-60 mmHg for 20 s) in anaesthetised animals and recorded from external oblique abdominal wall muscles with electromyographic electrodes. Single unit extracellular recordings were made from fine nerve trunks of pelvic nerves entering the bladder ex vivo (2). Stretch-sensitivity of bladder afferents was determined by their responses to isotonic stretch by 1-60 g of increasing load.
Using the Hanker-Yates myeloperoxidase staining and hematoxylin and eosin staining (n=5), consistent infiltration of polymorphonuclear neutrophils predominantly in the lamina propria was revealed in protamine sulphate/zymosan treated animals (n=5) but not in controls (n=5). Guinea pigs with zymosan-induced cystitis showed significant reduction in voiding volume (by 34%, n=6, P<0.05) and increases in voiding frequency (by 28%, n=6, P<0.05) during continuous conscious cystometry. Interestingly, duration of micturition contractions was increase in animal with cystitis by 69% (n=6, P<0.05). In anaesthetised animals, administration of 0.5 M KCl during cystometry evoked a significant decrease in the inter-micturition interval (by 40%, n=4, P<0.05) in zymosan-treated guinea pigs but not in controls. Zymosan-induced cystitis induced significant hyperalgesia in guinea pigs, measured by an increase in visceromotor responses in response to 20-60 mmHg bladder distension (by 80% at 60 mmHg, n=6, P<0.05), compared to control animals.
Electrophysiological recordings from bladder ex vivo show that high threshold afferents were sensitised in zymosan-induced cystitis: their stretch-sensitive firing was increased in response to 20-60g load compared to control animals (by 90% at 60g load, n=5, P<0.05). However, stretch induced firing of low threshold afferents was unaffected in cystitis compared to controls (n=6).
Interpretation of results
Conscious voiding experiments demonstrate that zymosan-induced cystitis causes significant changes in micturition reflex pathways that mimic essential features of interstitial cystitis in humans. In anaesthetised animals, intravesical administration of hypertonic KCl evoked significant increase in micturition frequency in animals with zymosan-induced cystitis but not in control animals. This data suggests that permeability of the urothelium to urine solutes is increased in this model. Pain responses measured as visceromotor responses to bladder distention were significantly enhanced in zymosan-induced cystitis compared to controls. In contrast to low threshold mechanoreceptors, high threshold afferents showed significant increase in their excitability in cystitis.