Adult, female Sprague Dawley rats were obtained from Harlan Laboratories (Prattville, AL) and housed in group cages. Food and water were provided on an ad libitum basis. These studies were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee.
Experiment 1 – Time-course of inflammatory responses
This experiment determined the time-course of inflammation produced by the intravesical administration of zymosan into the urinary bladder. Inflammation was measured as a function of the amount of Evan's Blue plasma extravasation into the bladder using a slight variation on previously published methodology [10, 16–18]. Separate groups of rats (N = 5 for all groups) received either no treatment (naive group), or received intravesical administration of either saline (0.5 ml) or a 1% zymosan solution (0.5 ml). Rats receiving treatments were anesthetized with halothane (1–5%) by mask to provide immobility for the placement of a 22-gauge angiocatheter into the urinary bladder via the urethra without trauma. Solutions were then administered and left in place for 30 minutes. Bladders were then drained and rats recovered from anesthesia. To prevent the introduction of infection into the bladders a povidone surface preparation was performed prior to catheterization and each treated rat received 50–100 mg/kg of ampicillin (s.c.). Groups of rats receiving zymosan were tested 1, 4, 12, 24, or 48 hr after their treatment. The saline control group rats were tested 24 hr after their treatment. Saline administration was used to control for all aspects of the procedure excluding inflammation, e.g., placement of the transurethral catheter and bladder distension. At the time of testing, all rats were reanesthetized and administered Evan's Blue (50 mg/kg i.v. via external jugular) which was allowed to circulate for 15 min. Animals were euthanized with pentobarbital (100 mg/kg i.v.) and perfused intracardially with 500 ml of normal saline. Whole bladders were removed and placed in 10 ml of dimethylsulfoxide for subsequent analysis. The amount of Evan's Blue extracted from the bladder tissue was determined spectrophotometrically (620 nm) and data were expressed in μg/g of bladder weight.
Experiment 2 – Effect of Zymosan concentration
This experiment determined the effect of zymosan concentration on the degree of plasma extravasation of Evan's Blue in the bladder. The same general procedures were used as described in Experiment 1 except that separate groups of rats (N = 5 per group) received intravesical administration of differing concentrations of zymosan (0.1, 0.5, 1.0, and 2.0%) in normal saline and the Evan's Blue determination was performed 24 hr later. The 1% zymosan data were those derived from Experiment 1.
Experiment 3 – Time-course of effects on nociceptive reflexes
This experiment determined the time-course of the effects of intravesical administration of zymosan on abdominal EMG and systemic ABP responses to UBD. Separate groups of rats (N = 6–21/group) were treated similarly to those described in Experiment 1: rats received either no treatment (naive), or intravesical administration of either saline (0.5 ml) or a 1% zymosan solution (0.5 ml). Zymosan-treated rats were tested at 4, 12, 24, or 48 hr after their treatment and the saline control group was tested at 24 hr. Methodology is the same as that previously published . Briefly, at the time of testing, each rat was re-anesthetized with halothane and instrumented for cardiovascular and EMG measures. Carotid arterial and tracheal cannulae were inserted for recording of ABP and artificial respiration, respectively under deep halothane anesthesia (2–5%). The right vagus nerve is sometimes injured during carotid manipulation and so to provide consistency between preparations was formally transected. The left abdominal skin was incised and silver wires were sutured to the external oblique abdominal musculature allowing for differential amplification of EMG activity. A 22-gauge angiocatheter was placed into the urinary bladder via the urethra and held in place by a tight ligature around the distal urethral orifice.
Following completion of the surgical preparation, the halothane anesthesia was reduced until flexion reflex responses could be evoked by pinch of the foot but without spontaneous escape behaviors (typically 0.5–0.7% halothane). The rats were artificially ventilated and not restrained in any fashion. Body temperature was maintained using a heating pad and overhead radiant lights. All data were saved on computer using MacIntosh-based Spike-2 software and associated hardware (Micro 1401; CED, Cambridge, UK) and a similar MacLab system (Stoelting, Inc).
All rats received multiple distensions of the urinary bladder via the intravesical catheter. An in-line, pneumatically-linked, low volume pressure transducer was used to monitor intravesical pressure. Pressure within the bladder was controlled using a pressure control device . Three 60 mmHg, 20 second UBDs (4 min intertrial interval) were administered followed by 20 second, constant-pressure air distensions of the urinary bladder at pressures of 10, 20, 30, 40, 50, and 60 mmHg, respectively.
ABP was measured continuously via the pulse pressure signal from the arterial cannula. In tests of UBD, an ABP response was defined as the peak change in mean ABP (?mmHg) during UBD as compared with the average response during a baseline period immediately prior to UBD. The EMG response was electronically rectified and normalized to baseline electronic "noise" levels (the lowest EMG activity level recorded from any particular rat.) These signal-to-noise ratios were defined as the EMG response to UBD and consisted of the following: (mean rectified EMG activity during UBD – mean rectified baseline EMG measured immediately prior to UBD) / (mean rectified baseline EMG noise level).
Data are presented as the mean ± S.E.M. unless otherwise stated. ABP and EMG responses to UBD are presented as change from baseline measures. One way ANOVAs were used for Evan's Blue data and repeated measures ANOVAs were used for ABP and EMG. Statistical significance was defined as p values ≤ 0.05.