Our knowledge of inflammation-induced changes in bladder tissue that result in bladder-related pain is limited. Bacterial, mycotic and viral infections of the urinary tract are all potential etiologies of bladder inflammation [1]. Our understanding of the neural substrates that are altered by infectious processes is virtually nonexistent. Recent studies have demonstrated that the injection of the substance lipopolysaccharide (LPS), derived from the cell wall of E. Coli bacteria, activates the innate immune mechanisms (Toll-like receptor 4-related) that are normally activated by infection [2]. Mycotic infections, such as those produced by Candida Albicans, activate similar, but distinctly different innate immune mechanisms [3, 4]. Zymosan is a mycotic equivalent to bacterial LPS in that it is yeast cell wall derivative which activates the Toll-like receptor 2 (TLR2) and the alternate complement pathway [4]. Zymosan can be viewed as a "natural" inflammogen which produces a receptor-mediated reactive cellular process as opposed to chemical irritants which result in frank, extensive tissue damage that leads to inflammation. Zymosan has been extensively characterized in behavioral, primary afferent, and dorsal horn neuron studies of cutaneous inflammation [e.g., [5, 6]]. A robust colitis also is produced by intracolonic administration of zymosan [7], but no one has reported on the use of zymosan in other visceral structures.

Only a few validated animal models of visceral nociception exist (for review, see [8]). Those that have been validated typically have used observations of animal posturing as well as reflex increases in arterial blood pressure (ABP) and electromyographic (EMG) activity of the abdominal muscles (visceromotor reflex) as response measures of visceral nociception. The presence of inflammation has been demonstrated to be a potent enhancer of responses to visceral stimuli. In particular, inflammation of the colorectal mucosa by the intracolonic application of turpentine [9, 10], acetic acid [11, 12], trinitrobenzene sulfonic acid [13] as well as zymosan [7] has been demonstrated to magnify reflex ABP and EMG responses to distension of the colon (CRD). In humans, inflammation also frequently leads to an enhancement of visceral pain sensations and so animal models demonstrating an enhancement of responses to a visceral stimulus produced by inflammation support the use of the models as behavioral indices of nociception.

Recently, we [14, 15] proposed using similar response measures evoked by urinary bladder distension (UBD) as an animal model of bladder pain. In our initial studies, we found these measures to be reliable and valid indices of bladder nociception in the rat with responses inhibited by analgesic manipulations. In the present studies, we sought to determine whether these response measures were affected by inflammation of the urinary bladder using zymosan as an inflammogen by determining the optimal concentration and time-course parameters for producing zymosan-related inflammation of the bladder as measured by Evan's Blue-related plasma extravasation and then correlating these inflammatory changes with nociceptive responses evoked by UBD of the inflamed bladder. Plasma extravasation of Evan's Blue dye has been validated as a measure of local inflammation in numerous previous studies [16–18].

The most important finding of the present study was that the yeast cell wall component, zymosan, which evokes innate immune responses in a fashion similar to mycotic infections, led to an inflamed bladder and magnified reflex responses to bladder distension. Specifically, the present studies parametrically characterized multiple variables associated with the zymosan-induced model of bladder inflammation including the effect of time and solution concentration. Within the parameters tested, a single 30 minute instillation of a 1% zymosan solution resulted in a significant inflammatory response in the bladder as early as 4 hr after administration. This response peaked at 24 hr and by 48 hr was returning to control levels. This study also showed that both 1 and 2% concentrations produced significantly greater responses than the 0.1 and 0.5% concentrations. These findings are important to future studies related to this form of bladder inflammation.

It is notable that in the present studies there was an apparent temporal dissociation between local tissue inflammatory changes and the enhancement of reflex responses to bladder stimulation: statistically significant evidence of inflammation occurred prior to (4 and 12 hr) the development of robust and statistically significant enhancement of ABP and EMG responses to UBD which only occurred 24 hr after administration of zymosan. This peak timepoint for reflex enhancement was the peak time of inflammation as measured by Evan's Blue extravasation and so there was, at the very least, a rough correlation between the two endpoints. This observation is similar to those noted in studies using the stimulus of colorectal distension in which turpentine was used as an inflammatory agent [10]. In that study, there were early indications of inflammation but a robust enhancement of nociceptive responses required a longer period of time (24 hr) to become manifest. It is possible that the underlying physiological changes produced by zymosan-induced inflammation requires time for expression; perhaps due to delays related to axonal transport from the periphery to the central nervous system of an inflammogen-induced signal. Alternatively, it may be the case that the nociceptive measures are less sensitive than the employed measures of inflammation.

The neurophysiological basis for the presently observed increase in sensitivity of the bladder due to zymosan pretreatment is, as yet, unknown although both central and peripheral mechanisms of sensitization associated with bladder inflammation have been proposed [20]. Leading theories include the recruitment of previously "silent" nociceptors by pathological processes such as inflammation [21] or the sensitization of spinal dorsal horn neurons by the release of neuropeptides or glutamate [22]. These processes are not mutually exclusive and each may have variable relevance to acute versus chronic visceral pain disorders. A combination of both peripheral and central phenomena is likely in most acute pathological disorders whereby the activation of a specific subset of afferent neurons may lead to alterations in central nervous system processing. This has been demonstrated in models of somatic inflammatory pain and extrapolations to visceral models have been proposed [20]. The present study provides important parametric analyses related to zymosan-induced inflammation and secondary reflex changes that may allow investigators to use this form of inflammatory change in correlative neurophysiological studies.

The relation of inflammatory changes induced by topically applied agents to chronic pain disorders is also yet to be determined. Subjects with the painful bladder disorder interstitial cystitis typically describe their painful sensations as being similar to a urinary tract infection with its associated inflammation-induced hypersensitivity. The similarity of sensory reports from interstitial cystitis and urinary tract infections implies a similarity in neurophysiological changes along the neural pathway associated with bladder sensation. The precise level at which this convergence occurs can only be defined with a precise definition of the pathways involved with at least one of the sensations. Psychophysical studies of subjects with interstitial cystitis recently reported by ourselves [23] demonstrated subjects with interstitial cystitis to be hypersensitive to bladder distension as well as other to other deep tissue stimuli. This suggests altered central processing of sensory data, a phenomenon known to occur following inflammation in somatic systems which is generally described in terms of hyperalgesia (magnified responses to painful stimuli) or allodynia (pain evoked by normally non-painful stimuli). In the present study, reflex responses to UBD were magnified by zymosan application at intensities of 20 mm Hg or greater. Others [e.g., [20]] have proposed that UBD becomes "noxious" to rats when distension pressures exceed 40 mm Hg. This would suggest that the augmentation of responses to UBD produced by zymosan are at the very least representative of a visceral hyperalgesia-related phenomena and may even be related to phenomena such as allodynia. The difficulties of interpretation inherent to cross-species comparisons of subjective sensations limits further speculation.

The present study parametrically examined responses to the intravesical administration of zymosan and demonstrated that this substance inflamed the bladder within 4 hr of administration and produced maximal inflammation 24 hr after topical administration. Statistically significant increases in ABP and EMG responses to UBD also occurred 24 hr post-zymosan administration but not at earlier timepoints. This suggests that any subsequent studies utilizing a single dose of intravesical zymosan to inflame the bladder would be optimized by a protocol in which pretreatment with a zymosan solution occurs 24 hr prior to subsequent measures.