- Email: [email protected]
Interstitial Cystitis
Ice Water Testing Reveals Hypersensitivity in Adult Rats That Experienced Neonatal Bladder Inflammation: Implications for Painful Bladder Syndrome/Interstitial Cystitis Alan Randich,* Hannah Mebane and Timothy J. Ness From the Departments of Psychology and Anesthesiology (TJN), University of Alabama at Birmingham, Birmingham, Alabama
Purpose: We determined whether clinical observations of hypersensitivity to ice water testing, that is infusion of ice-cold saline into the bladder, in patients with painful bladder syndrome/interstitial cystitis have a parallel in a rat model of bladder hypersensitivity produced by neonatal inflammation. Materials and Methods: Rat pups were anesthetized as neonates (postnatal days 14 to 16). In some pups the bladder was inflamed by intravesical zymosan treatment. As adults, the rats were re-anesthetized and tested for abdominal muscle contractions to ice water testing, measured on electromyogram. Various neonatally treated groups of rats underwent bladder re-inflammation/no reinflammation and/or bladder distention before ice water testing. Other control rats were treated only in adulthood. Results: Rats that underwent bladder inflammation as neonates manifested bladder hypersensitivity in adulthood, as indexed by significantly greater mean electromyogram responses during ice water testing. This bladder hypersensitivity did not require adult re-inflammation to manifest. Hypersensitivity was also observed with or without prior bladder distention, although the magnitude of electromyogram responses during ice water testing significantly correlated with the magnitude of electromyogram responses to bladder distention. Neonatally induced effects were not significantly related to estrous cycle phase. Exposure to menthol did not significantly enhance the overall magnitude of the electromyogram response to ice water testing in neonatally treated rats. Conclusions: Current results parallel those in a recent study showing that most patients with painful bladder syndrome/interstitial cystitis experience pain when undergoing ice water testing after previous urodynamic testing. These findings suggest that this animal model may be useful for understanding the etiology of and treatment for painful bladder syndrome/interstitial cystitis. Key Words: urinary bladder; rats, Sprague-Dawley; cystitis, interstitial; animals, newborn; hypersensitivity IN a recent study Mukerji et al found that 76.5% of female patients with PBS/IC reported pain during IWT involving intravesical instillation of icecold saline after undergoing routine urodynamic tests involving UBD.1 Fe-
males with IDO or NDO and controls did not report pain during IWT. These investigators suggested that pain during IWT in patients with PBS/IC may be a useful marker for differentiating patients with PBS/IC from those with
0022-5347/09/1821-0337/0 THE JOURNAL OF UROLOGY® Copyright © 2009 by AMERICAN UROLOGICAL ASSOCIATION
Vol. 182, 337-342, July 2009 Printed in U.S.A. DOI:10.1016/j.juro.2009.02.107
Abbreviations and Acronyms A ⫽ anesthesia BCR ⫽ bladder cooling reflex EMG ⫽ electromyogram IDO ⫽ idiopathic detrusor overactivity IWT ⫽ ice water testing NDO ⫽ neurogenic detrusor overactivity P ⫽ postnatal day PBS/IC ⫽ painful bladder syndrome/interstitial cystitis TRP ⫽ transient receptor potential cation channel TRPA1 ⫽ TRP subfamily A, member 1 TRPM8 ⫽ TRP subfamily M, member 8 UBD ⫽ bladder distention Z ⫽ zymosan Submitted for publication October 14, 2008. Study received approval from the University of Alabama at Birmingham animal care and use committee. Supported by Grants NIH R01-DK051413, NIH R01-DK073218 and NIH R01-DK078655 (AR). * Correspondence: Department of Psychology, University of Alabama at Birmingham, Birmingham, Alabama 35294 (e-mail: [email protected]).
www.jurology.com
337
338
HYPERSENSITIVITY AND NEONATAL BLADDER INFLAMMATION
other overactive bladder disorders and for identifying patients with PBS/IC for clinical trials. It has been hypothesized that neonatal exposure to bladder inflammation may predispose some individuals to PBS/IC in adulthood.2– 4 In animal studies addressing this hypothesis neonatal exposure to bladder inflammation increased micturition frequency and decreased the micturition reflex threshold on cystometric testing in adulthood.4 Neonatal exposure to bladder inflammation also resulted in an enhanced abdominal EMG response to UBD if the animals also underwent repeat exposure to bladder inflammation in adulthood.2 These data support the view that neonatal exposure to bladder inflammation predisposes an organism to global bladder hypersensitivity. The increased micturition frequency, decreased micturition reflex threshold and enhanced EMG responses to graded UBD observed in animal studies parallel the symptoms of frequency, urgency and pain seen in humans with PBS/IC. To further characterize the animal model described we examined whether rats that underwent neonatal bladder inflammation would show enhanced EMG responses during IWT. We also examined whether prior testing with UBD or adult reexposure to bladder inflammation would be necessary to observe an enhanced response to IWT. Possible sensitization of the response was tested by intravesical menthol administration.
MATERIALS AND METHODS Animal Subjects and Neonatal Treatments All studies were approved by the University of Alabama at Birmingham animal care and use committee, and they conformed to National Institutes of Health guidelines for the care and use of laboratory animals. Female rat pups were obtained from timed pregnant female Sprague-Dawley rats (Harlan™) and had documented birth dates. Pups were anesthetized for 30 minutes with inhaled halothane (2% to 5%) in oxygen for 3 consecutive days from ages P14 to P16. On each day the external urethra was swabbed with 10% povidone-iodine solution. In groups exposed to bladder inflammation a 24 gauge angiocatheter was inserted into the bladder via the urethra. Intravesical Z (0.1 ml 1% solution in saline) was administered and left to dwell for 30 minutes before being drained. Control groups were only administered A for 30 minutes. All rat pups were administered ampicillin (100 mg/kg subcutaneously) on each day, awakened and returned to their mother. Rat pups remained housed with their mothers until weaning at P21. They were raised to adulthood and tested between ages 12 and 16 weeks. Additional adult female SpragueDawley rats were obtained and evaluated at ages 12 to 16 weeks.
Adult Testing Procedures Since the estrous cycle may affect responses to UBD, daily vaginal lavage for a minimum of 2 complete cycles was
performed to determine the stage of estrus. Adult pretreatments were performed in rats grouped based on being in the metestrus/diestrus or proestrus/estrus phase of the estrous cycle. The stage of estrus was determined immediately before pretreatment and also on the day of testing. On the day before testing all rats were anesthetized (2% to 5% isoflurane in oxygen) and received intravesical Z (0.5 ml 1% solution with a 30-minute dwell time via a 22 gauge transurethral angiocatheter) or A only as adult pretreatment. All animals were administered ampicillin (100 mg/kg subcutaneously). At 24 hours later rats were re-anesthetized with isoflurane (2% to 5% in oxygen). The trachea was surgically cannulated for artificial ventilation. An intravesical 22 gauge angiocatheter was placed transurethrally and held in place by a tight suture around the distal urethral orifice. Platinum wires were surgically placed into the left external oblique musculature of each animal for differential amplification of EMG activity. Isoflurane A was decreased to 0.75% and maintained at this level for the duration of testing.
Treatment Groups One set of neonatal Z (13) and A (11) treated rats was re-exposed to Z as adults, designated condition 1 Z/Z and A/Z, respectively, reflecting neonatal/adult treatments. These rats underwent graded UBD (10 to 80 mm Hg 20-second distentions at 3-minute intertrial intervals) immediately before cold saline infusion to mimic the urodynamic conditions reported in the study by Mukerji et al.1 EMG recordings performed during IWT consisted of a 1-minute baseline period, followed by 10 minutes of intravesical infusion of ice-cold saline (1 ml per minute). A dual concentric catheter system allowed the infused ice-cold saline to drain continuously from a T-port after bladder lavage. To determine whether bladder contractions occurred during ice water testing, intravesical pressure was monitored via an in-line pressure transducer. To test whether experience with the UBD procedure was critical for obtaining a response during IWT the same procedure was performed in another 3 groups of rats that received Z and/or A as neonates and adults, called condition 2 Z/Z (9), A/Z (9) and Z/A (7). However, these rats underwent no graded UBD before IWT. After the initial IWT condition 2 animals also received intravesical administration of a 0.6 mM solution of menthol for 5 minutes, followed by repeat IWT. This was done to determine whether menthol would sensitize the response to IWT. In a separate group of control rats (condition 3) IWT was performed in normal adult rats with no neonatal pretreatment. These rats only underwent adult exposure to Z or A, termed groups ⫺/Z (12) and ⫺/A (8), respectively, 24 hours before IWT. Some of these rats underwent the UBD procedure before IWT and some did not (in condition 3 –/Z 8 of 12 rats were distended and in condition 3 ⫺/A 4 of 8 rats were distended. However, distended and not distended data were combined since subsequent results showed that prior UBD did not alter responses to IWT. The table lists all conditions and treatments.
Data Collection and Statistics In studies of graded UBD the EMG response was defined using the equation, (rectified EMG activity during UBD ⫺ rectified baseline EMG before UBD)/rectified baseline
HYPERSENSITIVITY AND NEONATAL BLADDER INFLAMMATION
Conditions and treatments
Groups Condition 1: Z/Z A/Z Condition 2: Z/Z A/Z Z/A Condition 3: ⫺/A ⫺/A ⫺/Z ⫺/Z
Adult Test
No.
P14–P16 Treatment
Adult Pretreatment
1
13 11
Z A
Z Z
UBD UBD
Cold Cold
9 9 7
Z A Z
Z Z A
Cold Cold Cold
Menthol/cold Menthol/cold Menthol/cold
8 4 4 4
Z Z A A
Z Z Z Z
UBD — UBD —
Cold Cold Cold Cold
2
339
baseline period. Latency to the first EMG response to exceed twice the baseline activity for a minimum of 10 seconds was determined and converted to a temperature threshold using a time-temperature curve measured using an intravesical thermistor during preliminary studies. Intravesical temperature decreased from approximately 30C at the start of infusion to 14C at 10 minutes. All data are presented as the group mean ⫾ SEM. They were analyzed using 1-way ANOVA.
RESULTS
EMG, as described previously.2 For the correlation data reported in this article we used the trapezoid rule to determine a single AUC measure of EMG responses to UBD pressures between 40 and 80 mm Hg. During IWT the EMG response was defined using the equation, mean EMG activity during the 10-minute infusion period ⫺ mean EMG activity during the 1-minute
Neonatal bladder inflammation produced bladder hypersensitivity to the infusion of ice-cold saline but did not produce uninhibited detrusor contractions. Baseline intravesical pressure was between 0 and 1 mm Hg and bladder pressure never increased more than 1 to 2 mm Hg during ice-cold saline infusion. Figure 1 shows group mean EMG responses during IWT. Neonatal exposure to Z significantly enhanced the EMG response during IWT in rats that underwent prior distention and adult Z (condition 1 Z/Z vs condition 1 A/Z), no prior distention and adult Z (condition 2 Z/Z vs condition 2 A/Z), and no prior
Figure 1. Mean group EMG responses during cold water infusion into bladder. Conditions 1 and 2 groups A/Z, condition 3 group ⫺/Z and condition 3 group ⫺/A did not differ. Asterisk indicates condition 1 distended group Z/Z and condition 2 not distended group Z/Z significantly different from group A/Z (p ⬍0.01). Pound sign indicates condition 2 not distended group Z/A significantly different from group A/Z (p ⬍0.05). Inset, examples of raw EMG tracings in condition 2 Z/Z (top) and A/Z (bottom) individual rats. One-minute baseline period was followed by cold water infusion for 10 minutes. Arrow indicates start of infusion.
340
HYPERSENSITIVITY AND NEONATAL BLADDER INFLAMMATION
distention and no adult Z (condition 2 Z/A vs condition 2 A/Z). Thus, neonatal exposure to Z was a necessary and sufficient condition to cause the observation of an enhanced EMG response during IWT. There were no significant differences in the magnitude of EMG responses in rats treated only as adults with A or Z (conditions 3 ⫺/A and ⫺/Z), indicating that adult bladder inflammation alone had little effect on the response during IWT. Similarly groups that received A as neonates and Z as adults (conditions 1 and 2 A/Z) did not significantly differ from rats that only received treatments as adults (condition 3 ⫺/A or ⫺/Z). While prior UBD was not critical for the enhancement effect, analysis of data on animals that underwent UBD revealed a significant positive correlation between mean EMG responses during IWT and the mean EMG response to UBD in the condition 1 Z/Z group (r ⫽ 0.61, p ⬍0.01, fig. 2, A) but not in the condition 1 A/Z group (r ⫽ 0.44, p ⬎0.08, data not shown). The temperature threshold for the EMG response during IWT was significantly higher in condition 1 Z/Z rats than in condition 1 A/Z rats (21.85C ⫾ 1.24C vs 17.54C ⫾ 0.98C). However, condition 2 and 3 rats did not show a similar effect, suggesting that prior distention may have been responsible for increasing the temperature threshold for the EMG response to IWT. The magnitude of the EMG response during IWT was not significantly enhanced by menthol treatment (fig. 2, B), although the effect of neonatal Z treatment on enhancing the EMG response during
IWT was again observed upon the second infusion in menthol treated animals. Menthol treatment also decreased the variability in the temperature thresholds among these groups and all menthol treated groups had similar temperature thresholds. There was no significant relationship between the magnitude of group mean EMG responses during IWT and the stage of the estrous cycle on the day of pretreatment or the day of testing (metestrus/ diestrus vs proestrus/estrus). There were also no significant between group differences in bladder or body weight.
DISCUSSION To our knowledge the current study is the first demonstration of an increase in cold sensitivity produced by neonatal bladder manipulation in a preclinical model. Similar hypersensitivity has been noted in humans with a diagnosis of PBS/IC,1 which suggests that this animal model of bladder hypersensitivity may be useful as a translational model of PBS/IC. In the clinical study by Mukerji et al BCR (uninhibited detrusor contractions evoked by IWT after routine urodynamic testing) was identified only in patients with IDO or NDO.1 Neither patients with PBS/IC nor controls had a BCR evoked. However, 76.5% of patients with PBS/IC reported pain during IWT, similar to the pain that they had previously experienced during tests using UBD (cystometric testing). Normal control females and those with IDO or NDO reported no pain during IWT. These findings in humans directly correlate with
Figure 2. A, there was significant positive correlation between mean EMG response to IWT and mean EMG response to UBD in group 1 Z/Z (r ⫽ 0.61, p ⬍0.01). B, menthol treatment (CM) did not significantly increase response to cold water infusion (C), that is no menthol treatment mean values differed significantly from original cold water infusion mean values (fig. 1). Only 5 group Z/A rats were tested with menthol and cold water infusion data were adjusted to reflect this fact. Enhancement effect of neonatal Z treatment was maintained during infusion 2. Pound sign indicates that condition 2 groups Z/Z and Z/A with cold water infusion were significantly different from group A/Z with cold water infusion (p ⬍0.05), as reported. Asterisk indicates condition 2 Z/Z and Z/A menthol treatment significantly different from condition 2 A/Z menthol treatment (p ⬍0.05).
HYPERSENSITIVITY AND NEONATAL BLADDER INFLAMMATION
findings in the current study, in which none of the rats had an identifiable BCR but those that had undergone bladder inflammation as neonates demonstrated nociceptive reflex responses to IWT in the bladder. We have previously observed that neonatal exposure to bladder inflammation increases micturition frequency in adult rats, decreases the micturition reflex threshold and enhances EMG responses to graded UBD. Like the current findings, these outcomes are direct correlates to the symptoms of frequency, urgency and pain observed in patients with PBS/IC. The current study served as a test of reverse translation, in which a promising preclinical model of a human disorder was tested for correlation with the human disorder. Responses in the preclinical model system were found to parallel those of the disorder. Therefore, they further support use of the neonatal bladder inflammation model in studies of PBS/IC. Other models of hypersensitivity have used neonatal inflammatory insults as the source of altered neurosensory processing. Seminal studies by Ruda et al revealed that inflammation of the hind paw in the neonatal period resulted in an altered neuroanatomical substrate for hind paw sensation in adult rats.5 In their model there was an expansion of primary afferent neuron terminal sites in the spinal cord. In a model of gastrointestinal hypersensitivity Al-Chaer et al observed that neonatal colonic inflammation produced by mustard oil treatments (TRPA1 activation) led to altered spontaneous activity of the primary afferents responsible for colonic sensation as adults.6 If bladder afferents are similar to those in other model systems, one would expect a potential alteration in the excitability and function of primary afferents from the bladder following a neonatal inflammatory event. The current data are consistent with such an alteration. The bladder cold hypersensitivity noted in the current study did not require adult re-exposure to bladder inflammation. This indicates that neonatal exposure to bladder inflammation alone was a necessary and sufficient condition for the enhancement effect. The latter outcome is similar to those that we noted in tests of micturition frequency2 and micturition thresholds.4 Abdominal muscle contractions can be evoked during spontaneous voiding and voiding evoked by saline infusion within the physiological range of distending pressures.7 However, in our study the infusate was returned via the urethral catheter to a reservoir that was in line with a pressure transducer open to the atmosphere. This prevented any sustained increase in intravesical pressure during infusion. Transient fluctuations in pressure readings were not noted even when large abdominal contractions developed during IWT, indicating that uninhibited detrusor contractions were not occur-
341
ring. As noted, these animal data also parallel the data reported by Mukerji et al that BCR is absent in patients with PBS/IC.1 The temperature threshold for evoking the first response during IWT was significantly increased by neonatal exposure to Z but this only occurred in rats that underwent UBD before IWT. It is possible that prior bladder distention altered the urothelium and permitted more ready access of saline to cold receptors. Prior exposure to UBD was not a necessary condition for achieving the enhancement effect, although the magnitude of EMG responses to IWT significantly correlated with the magnitude of EMG responses to UBD in rats that received neonatal Z. This may mean that neonatal bladder inflammation induced similar changes in mechanical and cold sensitive afferents, although these data do not address the possibility that the same afferents mediate cold as well as mechanical sensitivity. TRPM8 and TRPA1 channels are now thought to be important for the perception of cold and noxious cold, respectively, although considerable controversy remains.8 –10 TRPM8 channels but not TRPA1 channels are sensitive to menthol but their role in nociceptive cold pain is still unclear.10 The number of TRPM8 immunoreactive nerve fibers is significantly increased in patients with PBS/IC and it positively correlates with patient pain scores.11 However, these increases were confined to suburothelial tissue and those investigators were unable to detect significant increases in urothelial TRPM8 levels relative to those in controls. However, since we observed no significant effect of menthol administration on the overall EMG response to cold water infusion in our animals, it seems unlikely that TRPM8 channels were affected by our neonatal treatment. Therefore, it is possible that neonatal inflammation treatments altered TRPA1 expressing neurons or other cold temperature sensing channels that remain to be identified. Unfortunately to our knowledge there are no data related to TRPA1 expressing neurons in the bladder tissue of patients with PBS/IC. This suggests a potential avenue for research related to novel pharmacological interventions for bladder pain that are related to the TRPA1 receptor channel.
CONCLUSIONS The current study demonstrated physiological responses in a preclinical model of bladder hypersensitivity that have direct correlates to PBS/IC symptoms. This suggests that this model of hypersensitivity, which is produced by bladder inflammation during a critical period of development (the neonatal period), may have usefulness in studies of novel therapy for PBS/IC.
342
HYPERSENSITIVITY AND NEONATAL BLADDER INFLAMMATION
REFERENCES 1. Mukerji G, Waters J, Chessell IP, Bountra C, Agarwal SK and Anand P: Pain during ice water test distinguishes clinical bladder hypersensitivity from overactivity disorders. BMC Urol 2006; 6: 31. 2. Randich A, Uzzell T, DeBerry JJ and Ness TJ: Neonatal urinary bladder inflammation produces adult bladder hypersensitivity. J Pain 2006; 7: 468. 3. DeBerry JJ, Ness TJ, Robbins M and Randich A: Inflammation-induced enhancement of the visceromotor reflex to urinary bladder distention: modulation by endogenous opioids and the effects of early-in-life experience with bladder inflammation. J Pain 2007; 8: 914.
4. DeBerry JJ, Uzzell T, Ness TJ and Randich A: Effects of early-in-life bladder inflammation on spontaneous micturition and micturition reflexes during cystometry. Soc Neurosci Abstr 2005. 5. Ruda MA, Ling QD, Hohmann AG, Peng YB and Tachibana T: Altered nociceptive neuronal circuits after neonatal peripheral inflammation. Science 2000; 289: 628. 6. Al-Chaer ED, Kawasaki M and Pasricha PJ: A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development. Gastroenterol 2000; 119: 1276. 7. Cruz Y and Downie JW: Abdominal muscle activity during voiding in female rats or irritated
bladder. Am J Physiol Regul Integr Comp Physiol 2005; 290: R1436. 8. Dhaka A, Viswanath V and Patapoutian A: TRP ion channels and temperature sensation. Ann Rev Neurosci 2006; 29: 135. 9. Story G: The emerging role of TRP channels in mechanisms of temperature and pain sensation. Curr Neuropharmacol 2006; 4: 183. 10. McKemy DD: How cold is it? TRPM8 and TRPA1 in the molecular logic of cold sensation. Molec Pain 2005; 1: 16. 11. Mukerji G, Yiangos Y, Corcoran SL, Selmer IS, Smith GD, Benham CD et al: Cool and menthol receptor TRPM8 in human urinary bladder disorders and clinical correlations. BMC Urol 2006; 6: 6.
Purpose: We determined whether clinical observations of hypersensitivity to ice water testing, that is infusion of ice-cold saline into the bladder, in patients with painful bladder syndrome/interstitial cystitis have a parallel in a rat model of bladder hypersensitivity produced by neonatal inflammation. Materials and Methods: Rat pups were anesthetized as neonates (postnatal days 14 to 16). In some pups the bladder was inflamed by intravesical zymosan treatment. As adults, the rats were re-anesthetized and tested for abdominal muscle contractions to ice water testing, measured on electromyogram. Various neonatally treated groups of rats underwent bladder re-inflammation/no reinflammation and/or bladder distention before ice water testing. Other control rats were treated only in adulthood. Results: Rats that underwent bladder inflammation as neonates manifested bladder hypersensitivity in adulthood, as indexed by significantly greater mean electromyogram responses during ice water testing. This bladder hypersensitivity did not require adult re-inflammation to manifest. Hypersensitivity was also observed with or without prior bladder distention, although the magnitude of electromyogram responses during ice water testing significantly correlated with the magnitude of electromyogram responses to bladder distention. Neonatally induced effects were not significantly related to estrous cycle phase. Exposure to menthol did not significantly enhance the overall magnitude of the electromyogram response to ice water testing in neonatally treated rats. Conclusions: Current results parallel those in a recent study showing that most patients with painful bladder syndrome/interstitial cystitis experience pain when undergoing ice water testing after previous urodynamic testing. These findings suggest that this animal model may be useful for understanding the etiology of and treatment for painful bladder syndrome/interstitial cystitis. Key Words: urinary bladder; rats, Sprague-Dawley; cystitis, interstitial; animals, newborn; hypersensitivity IN a recent study Mukerji et al found that 76.5% of female patients with PBS/IC reported pain during IWT involving intravesical instillation of icecold saline after undergoing routine urodynamic tests involving UBD.1 Fe-
males with IDO or NDO and controls did not report pain during IWT. These investigators suggested that pain during IWT in patients with PBS/IC may be a useful marker for differentiating patients with PBS/IC from those with
0022-5347/09/1821-0337/0 THE JOURNAL OF UROLOGY® Copyright © 2009 by AMERICAN UROLOGICAL ASSOCIATION
Vol. 182, 337-342, July 2009 Printed in U.S.A. DOI:10.1016/j.juro.2009.02.107
Abbreviations and Acronyms A ⫽ anesthesia BCR ⫽ bladder cooling reflex EMG ⫽ electromyogram IDO ⫽ idiopathic detrusor overactivity IWT ⫽ ice water testing NDO ⫽ neurogenic detrusor overactivity P ⫽ postnatal day PBS/IC ⫽ painful bladder syndrome/interstitial cystitis TRP ⫽ transient receptor potential cation channel TRPA1 ⫽ TRP subfamily A, member 1 TRPM8 ⫽ TRP subfamily M, member 8 UBD ⫽ bladder distention Z ⫽ zymosan Submitted for publication October 14, 2008. Study received approval from the University of Alabama at Birmingham animal care and use committee. Supported by Grants NIH R01-DK051413, NIH R01-DK073218 and NIH R01-DK078655 (AR). * Correspondence: Department of Psychology, University of Alabama at Birmingham, Birmingham, Alabama 35294 (e-mail: [email protected]).
www.jurology.com
337
338
HYPERSENSITIVITY AND NEONATAL BLADDER INFLAMMATION
other overactive bladder disorders and for identifying patients with PBS/IC for clinical trials. It has been hypothesized that neonatal exposure to bladder inflammation may predispose some individuals to PBS/IC in adulthood.2– 4 In animal studies addressing this hypothesis neonatal exposure to bladder inflammation increased micturition frequency and decreased the micturition reflex threshold on cystometric testing in adulthood.4 Neonatal exposure to bladder inflammation also resulted in an enhanced abdominal EMG response to UBD if the animals also underwent repeat exposure to bladder inflammation in adulthood.2 These data support the view that neonatal exposure to bladder inflammation predisposes an organism to global bladder hypersensitivity. The increased micturition frequency, decreased micturition reflex threshold and enhanced EMG responses to graded UBD observed in animal studies parallel the symptoms of frequency, urgency and pain seen in humans with PBS/IC. To further characterize the animal model described we examined whether rats that underwent neonatal bladder inflammation would show enhanced EMG responses during IWT. We also examined whether prior testing with UBD or adult reexposure to bladder inflammation would be necessary to observe an enhanced response to IWT. Possible sensitization of the response was tested by intravesical menthol administration.
MATERIALS AND METHODS Animal Subjects and Neonatal Treatments All studies were approved by the University of Alabama at Birmingham animal care and use committee, and they conformed to National Institutes of Health guidelines for the care and use of laboratory animals. Female rat pups were obtained from timed pregnant female Sprague-Dawley rats (Harlan™) and had documented birth dates. Pups were anesthetized for 30 minutes with inhaled halothane (2% to 5%) in oxygen for 3 consecutive days from ages P14 to P16. On each day the external urethra was swabbed with 10% povidone-iodine solution. In groups exposed to bladder inflammation a 24 gauge angiocatheter was inserted into the bladder via the urethra. Intravesical Z (0.1 ml 1% solution in saline) was administered and left to dwell for 30 minutes before being drained. Control groups were only administered A for 30 minutes. All rat pups were administered ampicillin (100 mg/kg subcutaneously) on each day, awakened and returned to their mother. Rat pups remained housed with their mothers until weaning at P21. They were raised to adulthood and tested between ages 12 and 16 weeks. Additional adult female SpragueDawley rats were obtained and evaluated at ages 12 to 16 weeks.
Adult Testing Procedures Since the estrous cycle may affect responses to UBD, daily vaginal lavage for a minimum of 2 complete cycles was
performed to determine the stage of estrus. Adult pretreatments were performed in rats grouped based on being in the metestrus/diestrus or proestrus/estrus phase of the estrous cycle. The stage of estrus was determined immediately before pretreatment and also on the day of testing. On the day before testing all rats were anesthetized (2% to 5% isoflurane in oxygen) and received intravesical Z (0.5 ml 1% solution with a 30-minute dwell time via a 22 gauge transurethral angiocatheter) or A only as adult pretreatment. All animals were administered ampicillin (100 mg/kg subcutaneously). At 24 hours later rats were re-anesthetized with isoflurane (2% to 5% in oxygen). The trachea was surgically cannulated for artificial ventilation. An intravesical 22 gauge angiocatheter was placed transurethrally and held in place by a tight suture around the distal urethral orifice. Platinum wires were surgically placed into the left external oblique musculature of each animal for differential amplification of EMG activity. Isoflurane A was decreased to 0.75% and maintained at this level for the duration of testing.
Treatment Groups One set of neonatal Z (13) and A (11) treated rats was re-exposed to Z as adults, designated condition 1 Z/Z and A/Z, respectively, reflecting neonatal/adult treatments. These rats underwent graded UBD (10 to 80 mm Hg 20-second distentions at 3-minute intertrial intervals) immediately before cold saline infusion to mimic the urodynamic conditions reported in the study by Mukerji et al.1 EMG recordings performed during IWT consisted of a 1-minute baseline period, followed by 10 minutes of intravesical infusion of ice-cold saline (1 ml per minute). A dual concentric catheter system allowed the infused ice-cold saline to drain continuously from a T-port after bladder lavage. To determine whether bladder contractions occurred during ice water testing, intravesical pressure was monitored via an in-line pressure transducer. To test whether experience with the UBD procedure was critical for obtaining a response during IWT the same procedure was performed in another 3 groups of rats that received Z and/or A as neonates and adults, called condition 2 Z/Z (9), A/Z (9) and Z/A (7). However, these rats underwent no graded UBD before IWT. After the initial IWT condition 2 animals also received intravesical administration of a 0.6 mM solution of menthol for 5 minutes, followed by repeat IWT. This was done to determine whether menthol would sensitize the response to IWT. In a separate group of control rats (condition 3) IWT was performed in normal adult rats with no neonatal pretreatment. These rats only underwent adult exposure to Z or A, termed groups ⫺/Z (12) and ⫺/A (8), respectively, 24 hours before IWT. Some of these rats underwent the UBD procedure before IWT and some did not (in condition 3 –/Z 8 of 12 rats were distended and in condition 3 ⫺/A 4 of 8 rats were distended. However, distended and not distended data were combined since subsequent results showed that prior UBD did not alter responses to IWT. The table lists all conditions and treatments.
Data Collection and Statistics In studies of graded UBD the EMG response was defined using the equation, (rectified EMG activity during UBD ⫺ rectified baseline EMG before UBD)/rectified baseline
HYPERSENSITIVITY AND NEONATAL BLADDER INFLAMMATION
Conditions and treatments
Groups Condition 1: Z/Z A/Z Condition 2: Z/Z A/Z Z/A Condition 3: ⫺/A ⫺/A ⫺/Z ⫺/Z
Adult Test
No.
P14–P16 Treatment
Adult Pretreatment
1
13 11
Z A
Z Z
UBD UBD
Cold Cold
9 9 7
Z A Z
Z Z A
Cold Cold Cold
Menthol/cold Menthol/cold Menthol/cold
8 4 4 4
Z Z A A
Z Z Z Z
UBD — UBD —
Cold Cold Cold Cold
2
339
baseline period. Latency to the first EMG response to exceed twice the baseline activity for a minimum of 10 seconds was determined and converted to a temperature threshold using a time-temperature curve measured using an intravesical thermistor during preliminary studies. Intravesical temperature decreased from approximately 30C at the start of infusion to 14C at 10 minutes. All data are presented as the group mean ⫾ SEM. They were analyzed using 1-way ANOVA.
RESULTS
EMG, as described previously.2 For the correlation data reported in this article we used the trapezoid rule to determine a single AUC measure of EMG responses to UBD pressures between 40 and 80 mm Hg. During IWT the EMG response was defined using the equation, mean EMG activity during the 10-minute infusion period ⫺ mean EMG activity during the 1-minute
Neonatal bladder inflammation produced bladder hypersensitivity to the infusion of ice-cold saline but did not produce uninhibited detrusor contractions. Baseline intravesical pressure was between 0 and 1 mm Hg and bladder pressure never increased more than 1 to 2 mm Hg during ice-cold saline infusion. Figure 1 shows group mean EMG responses during IWT. Neonatal exposure to Z significantly enhanced the EMG response during IWT in rats that underwent prior distention and adult Z (condition 1 Z/Z vs condition 1 A/Z), no prior distention and adult Z (condition 2 Z/Z vs condition 2 A/Z), and no prior
Figure 1. Mean group EMG responses during cold water infusion into bladder. Conditions 1 and 2 groups A/Z, condition 3 group ⫺/Z and condition 3 group ⫺/A did not differ. Asterisk indicates condition 1 distended group Z/Z and condition 2 not distended group Z/Z significantly different from group A/Z (p ⬍0.01). Pound sign indicates condition 2 not distended group Z/A significantly different from group A/Z (p ⬍0.05). Inset, examples of raw EMG tracings in condition 2 Z/Z (top) and A/Z (bottom) individual rats. One-minute baseline period was followed by cold water infusion for 10 minutes. Arrow indicates start of infusion.
340
HYPERSENSITIVITY AND NEONATAL BLADDER INFLAMMATION
distention and no adult Z (condition 2 Z/A vs condition 2 A/Z). Thus, neonatal exposure to Z was a necessary and sufficient condition to cause the observation of an enhanced EMG response during IWT. There were no significant differences in the magnitude of EMG responses in rats treated only as adults with A or Z (conditions 3 ⫺/A and ⫺/Z), indicating that adult bladder inflammation alone had little effect on the response during IWT. Similarly groups that received A as neonates and Z as adults (conditions 1 and 2 A/Z) did not significantly differ from rats that only received treatments as adults (condition 3 ⫺/A or ⫺/Z). While prior UBD was not critical for the enhancement effect, analysis of data on animals that underwent UBD revealed a significant positive correlation between mean EMG responses during IWT and the mean EMG response to UBD in the condition 1 Z/Z group (r ⫽ 0.61, p ⬍0.01, fig. 2, A) but not in the condition 1 A/Z group (r ⫽ 0.44, p ⬎0.08, data not shown). The temperature threshold for the EMG response during IWT was significantly higher in condition 1 Z/Z rats than in condition 1 A/Z rats (21.85C ⫾ 1.24C vs 17.54C ⫾ 0.98C). However, condition 2 and 3 rats did not show a similar effect, suggesting that prior distention may have been responsible for increasing the temperature threshold for the EMG response to IWT. The magnitude of the EMG response during IWT was not significantly enhanced by menthol treatment (fig. 2, B), although the effect of neonatal Z treatment on enhancing the EMG response during
IWT was again observed upon the second infusion in menthol treated animals. Menthol treatment also decreased the variability in the temperature thresholds among these groups and all menthol treated groups had similar temperature thresholds. There was no significant relationship between the magnitude of group mean EMG responses during IWT and the stage of the estrous cycle on the day of pretreatment or the day of testing (metestrus/ diestrus vs proestrus/estrus). There were also no significant between group differences in bladder or body weight.
DISCUSSION To our knowledge the current study is the first demonstration of an increase in cold sensitivity produced by neonatal bladder manipulation in a preclinical model. Similar hypersensitivity has been noted in humans with a diagnosis of PBS/IC,1 which suggests that this animal model of bladder hypersensitivity may be useful as a translational model of PBS/IC. In the clinical study by Mukerji et al BCR (uninhibited detrusor contractions evoked by IWT after routine urodynamic testing) was identified only in patients with IDO or NDO.1 Neither patients with PBS/IC nor controls had a BCR evoked. However, 76.5% of patients with PBS/IC reported pain during IWT, similar to the pain that they had previously experienced during tests using UBD (cystometric testing). Normal control females and those with IDO or NDO reported no pain during IWT. These findings in humans directly correlate with
Figure 2. A, there was significant positive correlation between mean EMG response to IWT and mean EMG response to UBD in group 1 Z/Z (r ⫽ 0.61, p ⬍0.01). B, menthol treatment (CM) did not significantly increase response to cold water infusion (C), that is no menthol treatment mean values differed significantly from original cold water infusion mean values (fig. 1). Only 5 group Z/A rats were tested with menthol and cold water infusion data were adjusted to reflect this fact. Enhancement effect of neonatal Z treatment was maintained during infusion 2. Pound sign indicates that condition 2 groups Z/Z and Z/A with cold water infusion were significantly different from group A/Z with cold water infusion (p ⬍0.05), as reported. Asterisk indicates condition 2 Z/Z and Z/A menthol treatment significantly different from condition 2 A/Z menthol treatment (p ⬍0.05).
HYPERSENSITIVITY AND NEONATAL BLADDER INFLAMMATION
findings in the current study, in which none of the rats had an identifiable BCR but those that had undergone bladder inflammation as neonates demonstrated nociceptive reflex responses to IWT in the bladder. We have previously observed that neonatal exposure to bladder inflammation increases micturition frequency in adult rats, decreases the micturition reflex threshold and enhances EMG responses to graded UBD. Like the current findings, these outcomes are direct correlates to the symptoms of frequency, urgency and pain observed in patients with PBS/IC. The current study served as a test of reverse translation, in which a promising preclinical model of a human disorder was tested for correlation with the human disorder. Responses in the preclinical model system were found to parallel those of the disorder. Therefore, they further support use of the neonatal bladder inflammation model in studies of PBS/IC. Other models of hypersensitivity have used neonatal inflammatory insults as the source of altered neurosensory processing. Seminal studies by Ruda et al revealed that inflammation of the hind paw in the neonatal period resulted in an altered neuroanatomical substrate for hind paw sensation in adult rats.5 In their model there was an expansion of primary afferent neuron terminal sites in the spinal cord. In a model of gastrointestinal hypersensitivity Al-Chaer et al observed that neonatal colonic inflammation produced by mustard oil treatments (TRPA1 activation) led to altered spontaneous activity of the primary afferents responsible for colonic sensation as adults.6 If bladder afferents are similar to those in other model systems, one would expect a potential alteration in the excitability and function of primary afferents from the bladder following a neonatal inflammatory event. The current data are consistent with such an alteration. The bladder cold hypersensitivity noted in the current study did not require adult re-exposure to bladder inflammation. This indicates that neonatal exposure to bladder inflammation alone was a necessary and sufficient condition for the enhancement effect. The latter outcome is similar to those that we noted in tests of micturition frequency2 and micturition thresholds.4 Abdominal muscle contractions can be evoked during spontaneous voiding and voiding evoked by saline infusion within the physiological range of distending pressures.7 However, in our study the infusate was returned via the urethral catheter to a reservoir that was in line with a pressure transducer open to the atmosphere. This prevented any sustained increase in intravesical pressure during infusion. Transient fluctuations in pressure readings were not noted even when large abdominal contractions developed during IWT, indicating that uninhibited detrusor contractions were not occur-
341
ring. As noted, these animal data also parallel the data reported by Mukerji et al that BCR is absent in patients with PBS/IC.1 The temperature threshold for evoking the first response during IWT was significantly increased by neonatal exposure to Z but this only occurred in rats that underwent UBD before IWT. It is possible that prior bladder distention altered the urothelium and permitted more ready access of saline to cold receptors. Prior exposure to UBD was not a necessary condition for achieving the enhancement effect, although the magnitude of EMG responses to IWT significantly correlated with the magnitude of EMG responses to UBD in rats that received neonatal Z. This may mean that neonatal bladder inflammation induced similar changes in mechanical and cold sensitive afferents, although these data do not address the possibility that the same afferents mediate cold as well as mechanical sensitivity. TRPM8 and TRPA1 channels are now thought to be important for the perception of cold and noxious cold, respectively, although considerable controversy remains.8 –10 TRPM8 channels but not TRPA1 channels are sensitive to menthol but their role in nociceptive cold pain is still unclear.10 The number of TRPM8 immunoreactive nerve fibers is significantly increased in patients with PBS/IC and it positively correlates with patient pain scores.11 However, these increases were confined to suburothelial tissue and those investigators were unable to detect significant increases in urothelial TRPM8 levels relative to those in controls. However, since we observed no significant effect of menthol administration on the overall EMG response to cold water infusion in our animals, it seems unlikely that TRPM8 channels were affected by our neonatal treatment. Therefore, it is possible that neonatal inflammation treatments altered TRPA1 expressing neurons or other cold temperature sensing channels that remain to be identified. Unfortunately to our knowledge there are no data related to TRPA1 expressing neurons in the bladder tissue of patients with PBS/IC. This suggests a potential avenue for research related to novel pharmacological interventions for bladder pain that are related to the TRPA1 receptor channel.
CONCLUSIONS The current study demonstrated physiological responses in a preclinical model of bladder hypersensitivity that have direct correlates to PBS/IC symptoms. This suggests that this model of hypersensitivity, which is produced by bladder inflammation during a critical period of development (the neonatal period), may have usefulness in studies of novel therapy for PBS/IC.
342
HYPERSENSITIVITY AND NEONATAL BLADDER INFLAMMATION
REFERENCES 1. Mukerji G, Waters J, Chessell IP, Bountra C, Agarwal SK and Anand P: Pain during ice water test distinguishes clinical bladder hypersensitivity from overactivity disorders. BMC Urol 2006; 6: 31. 2. Randich A, Uzzell T, DeBerry JJ and Ness TJ: Neonatal urinary bladder inflammation produces adult bladder hypersensitivity. J Pain 2006; 7: 468. 3. DeBerry JJ, Ness TJ, Robbins M and Randich A: Inflammation-induced enhancement of the visceromotor reflex to urinary bladder distention: modulation by endogenous opioids and the effects of early-in-life experience with bladder inflammation. J Pain 2007; 8: 914.
4. DeBerry JJ, Uzzell T, Ness TJ and Randich A: Effects of early-in-life bladder inflammation on spontaneous micturition and micturition reflexes during cystometry. Soc Neurosci Abstr 2005. 5. Ruda MA, Ling QD, Hohmann AG, Peng YB and Tachibana T: Altered nociceptive neuronal circuits after neonatal peripheral inflammation. Science 2000; 289: 628. 6. Al-Chaer ED, Kawasaki M and Pasricha PJ: A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development. Gastroenterol 2000; 119: 1276. 7. Cruz Y and Downie JW: Abdominal muscle activity during voiding in female rats or irritated
bladder. Am J Physiol Regul Integr Comp Physiol 2005; 290: R1436. 8. Dhaka A, Viswanath V and Patapoutian A: TRP ion channels and temperature sensation. Ann Rev Neurosci 2006; 29: 135. 9. Story G: The emerging role of TRP channels in mechanisms of temperature and pain sensation. Curr Neuropharmacol 2006; 4: 183. 10. McKemy DD: How cold is it? TRPM8 and TRPA1 in the molecular logic of cold sensation. Molec Pain 2005; 1: 16. 11. Mukerji G, Yiangos Y, Corcoran SL, Selmer IS, Smith GD, Benham CD et al: Cool and menthol receptor TRPM8 in human urinary bladder disorders and clinical correlations. BMC Urol 2006; 6: 6.