- Email: [email protected]
Vigor of visceromotor responses to urinary bladder distension in rats increases with repeated trials and stimulus intensity
Neuroscience Letters 306 (2001) 97±100
www.elsevier.com/locate/neulet
Vigor of visceromotor responses to urinary bladder distension in rats increases with repeated trials and stimulus intensity Pablo Castroman, T.J. Ness* Department of Anesthesiology, ZRB 940, University of Alabama at Birmingham, 1530 Third Avenue South, Birmingham, AL 35294-0006, USA Received 22 February 2001; received in revised form 20 April 2001; accepted 1 May 2001
Abstract This methodological study characterized visceromotor responses (VMRs) as abdominal contractile responses to urinary bladder distension (UBD) in the female rat. Electromyographic activity of the abdominal musculature was used as a measure of the VMR. Similar to previously characterized cardiovascular responses to UBD, VMRs to UBD demonstrated an initial sensitization period whereby repeated presentation of UBD stimuli led to increase vigor of the VMR. Graded UBD produced graded VMRs, therefore stimulus±response functions could be constructed. The intravenous administration of the opioid fentanyl produced a reduced vigor of the VMR in a fashion consistent with its analgesic effect. The present report supports the utility of this model for studies of urinary bladder nociception. q 2001 Published by Elsevier Science Ireland Ltd. Keywords: Visceral Pain; Visceromotor; Nociception; Bladder Distension; Sensitization; Analgesics
Pain arising from the internal organs of the body is of great clinical signi®cance, as it constitutes the primary symptomatology of a majority of the disease processes treated by internists. Our models of visceral pain have improved over the last 10 years with the validation of models using colonic distension, arti®cial uterocalcinosis and numerous visceral stimuli [9]. Visceromotor responses (VMRs), the re¯ex contraction of the abdominal musculature in response to a visceral stimulus, has been used classically as one measure of visceral sensation. Initially characterized by Sherrington [15] and subsequently used by numerous investigators [3,4,10], the absence or presence of a VMR has served as an indicator of the interruption or maintenance of visceral pain pathways and provided support for the `noxious' nature of various visceral stimuli. Quanti®cation of the visceromotor response has taken three forms: (1) the simple determination of presence or absence in response to a ®xed visceral stimulus of suf®cient intensity that it is accepted as `noxious'; (2) the determination of a visceromotor threshold (VMT) in which the intensity of the visceral stimulus is varied and the lowest tested intensity of stimulation evoking a VMR de®ned as the VMT; * Corresponding author. Tel.: 11-205-975-9643; fax: 11-205934-7437. E-mail address: [email protected] (T.J. Ness).
and (3) determination of the vigor of the muscular contraction in response to the visceral stimulus using force transducers or implanted electrodes and electrophysiological analysis. Spinal ventral root activity has been used as a correlate to the VMR [5], allowing for some quanti®cation of the vigor of the VMR to a visceral stimulus, which in these cases was the electrical stimulation of a visceral nerve. Using this approach the effect of the analgesic indomethacin has been demonstrated [5]. Using the stimulus of colorectal distension and an abdominal electromyographic activity as a measure of the VMR, quantitative effects of visceral in¯ammation and the intrathecal administration of pharmacological agents on the vigor of the VMRs has also been studied [2,7]. Our laboratory has characterized re¯ex responses to the visceral stimulus of urinary bladder distension (UBD) which includes a VMR [13]. This VMR consisted of the contraction of the external oblique abdominal musculature as measured by direct vision or by using implanted wires to generate an electromyogram. It was demonstrated that the occurrence of the VMR became more reliable and more vigorous with repeated presentations of the UBD stimulus and with increasing intensity of UBD. In our initial analysis, VMR was quanti®ed by the determination of a VMT. Subsequent additions to our electronic equipment has allowed for a more sophisticated analysis of myoelectrical activity. The following study was undertaken to determine whether our
0304-3940/01/$ - see front matter q 2001 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 1) 01 88 6- 9
98
P. Castroman, T.J. Ness / Neuroscience Letters 306 (2001) 97±100
unsophisticated impressions regarding the effects of stimulus intensity and of repeated presentations of UBD were valid and to determine the reproducibility of the VMR over time in our preparation. This would allow for a comparison with other studies and would describe methodology that could be used in future pharmacologic studies related to visceral nociception and related analgesic processes. To test for the role of VMR as a measure of nociception, the effect of intravenous fentanyl on the vigor of the VMR was also brie¯y investigated. These studies were approved by our Institutional Review Board for non-human studies. Female Sprague±Dawley rats were anesthetized with halothane (2±5% induction by mask, 1±2% maintenance). Jugular venous, arterial carotid and tracheal cannulae were placed and the rats arti®cially ventilated using a volume-cycled respirator. Wires were sutured to the abdominal musculature allowing for differential ampli®cation of their myoelectrical activity. Following completion of the surgical preparation, the halothane anesthesia was reduced until ¯exion re¯ex responses could be evoked by pinch of the foot but without escape behaviors, typically 0.5±0.7% halothane. At these levels of anesthesia, no spontaneous or re¯exive contractile response to distension of the bladder is observed [13]. The rats were not restrained in any fashion. The myoelectrical activity of the external oblique musculature was differentially ampli®ed using standard methodology (Grass P511 AC Ampli®er, 100 £ amp.; Astro-Med, Inc. West Warwick, RI) and converted to digital information using a Micro 1401 processor and associated Spike-2 software (Cambridge Electronic Design, Cambridge, UK). Using an analysis routine kindly provided by G.F. Gebhart and used in ref. [2], the signal could be quanti®ed as the number of episodes of non-recti®ed electromyographic activity exceeding a preset `threshold' voltage limit. This threshold was individually adjusted above the baseline level of activity of the abdominal musculature prior to the administration of UBD. The same threshold level was maintained throughout the course of a given experiment. The number of episodes above the threshold level was determined for 10 s prior to UBD and during the 20 s of UBD, the former serving as a measure of spontaneous activity and the latter of total activity during UBD. Subtraction of spontaneous activity from total activity during UBD allowed for the calculation of UBD-evoked activity. Since the quanti®ed EMG responses of the abdominal musculature produced by the same stimulus vary in maximum response in relation to small differences in electrode placement, in the pharmacological study EMG activity for each experiment was normalized by the determination of a response ratio. This was calculated as the maximum response to UBD (60 mmHg, 20 s) after drug or saline divided by the mean of the three control responses determined in the same rat prior to drug or saline administration. A 22-gauge te¯on angiocatheter was placed into the urinary bladder via the urethra and held in place by a tight suture
around the distal urethral ori®ce. UBD was administered as phasic stimuli (rapid onset, rapid offset) using compressed air and a previously described distension control device [1]. Thirty ®ve rats received a series of ten 60 mmHg, 20 s phasic UBD stimuli at 4 min intervals followed by single trials of graded (10±80 mmHg, 20 s) phasic stimuli given in sequence. In six female rats, additional investigation into the effects of analgesics was performed. After three baseline VMRs to UBD (60 mmHg, 20 s) every 4 min and baseline VMRs to graded UBD (20±80 mmHg, 20 s) were obtained, intravenous (i.v.) doses of fentanyl citrate (3 mg/kg) were injected slowly (over a 1 min period) as 1 ml/kg boluses. Control rats (n 5) were injected i.v. with the same volume of normal saline. For evaluation of stimulus-response functions to graded UBD, second boluses of the same dose of fentanyl or the equivalent volume of normal saline were performed after 80% of the baseline responses were obtained. VMR responses to graded UBD (20±80 mmHg) given in sequence were recorded before and after pharmacological administration. Due to the short lasting effect observed after the ®rst dose of fentanyl, graded UBDs after a second dose administration were performed in sequence and counterbalanced order (ascending, n 3; descending, n 3), 12 min after injection. Statistics are presented as the mean ^ SEM, unless otherwise stated. In the pharmacological study, one-way ANOVA for repeated measures followed by post-hoc Fisher's test were used to evaluate statistical signi®cance. Differences were considered to be signi®cant at P values , 0:05. Strong VMRs could be evoked by UBD and could be measured as electromyographic activity (see examples in Figs. 1±3). Similar to cardiovascular responses to UBD, the VMRs to UBD demonstrated an accelerating response to the repeated presentation of the visceral stimulus (Fig. 1). VMRs to the initial presentation of UBD are neither vigorous nor reliable but after only 3±5 UBDs the VMRs became both vigorous and reliable. The VMR is also graded in response to graded intensities of UBD (Fig. 2) in a curvilinear fashion. The VMR remains reliable and as a group (Fig. 3 -normal saline data) there is reproducibility of the response, although individual rats may have signi®cant trialto-trial variability. Intravenous fentanyl (3 mg/kg) produced, in comparison with intravenous saline, a statistically significant reduction (P , 0:01) in the vigor of the VMR to UBD (60 mmHg, 20 s every 4 min.), an effect which lasted 16 to 20 min (Fig. 3A). This brief effect is consistent with its action as an analgesic that undergoes rapid redistribution [6]. The drug also produced a statistically signi®cant inhibition of VMRs to graded UBD at 40, 60 and 80 mmHg (Fig. 3B) in comparison with intravenous saline. The present study con®rmed impressions generated during a previous characterization of physiologic responses to UBD that the VMRs underwent an initial period of accel-
P. Castroman, T.J. Ness / Neuroscience Letters 306 (2001) 97±100
99
appears to require a sensitization process such as in¯ammation or abnormal patterns of input. The present study and that of previous psychophysical studies suggest that repeated distension serves to activate this sensitization process. The precise mechanism of these sensitization processes has not been determined, but a role for spinal glutamate receptor activation has been demonstrated in models of colorectal pain [2,7]. It is likely that similar mechanisms may apply to other pelvic organs but more peripheral mechanisms related to neurogenic and nonneurogenic processes may prove to be of similar relevance. For instance, the physical effect on the urothelium due to the Fig. 1. Effect of repeated UBD on VMRs. In (A), typical examples of EMG activity during repeated UBD trials are shown. Trial number is given at left of each EMG. In (B), effect of the presentation of ten UBD (60 mmHg 20 s, every 4 min.) measured as electromyographic activity (EMG activity) of the abdominal musculature in 35 female rats is observed. Evoked EMG activity is obtained by subtraction of spontaneous activity from total activity during UBD. The thicker line represents the mean responses of the sample.
eration and were graded in response to graded stimuli. This period of `sensitization' has been noted in both human [8] and non-human [12,11] studies using colorectal distension as a noxious visceral stimulus and psychophysical measures or neuronal/physiological measures as responses. In humans, the repeated presentation of the urinary bladder distending stimulus as cystometrograms results in increasing intensities of sensation, decreasing thresholds for pain and increased vigor of physiologic responses to the stimulus with a threshold for the evocation of pain of 20±40 mmHg [14]. In order to be evoked, the sensation of visceral pain
Fig. 2. Effect of graded UBD on VMRs. In (A), a typical example of the effect of graded UBD (10±80 mmHg, 20 s) is shown. Intensity of UBD is given at left of each EMG. In (B), the effect of 20±80 mmHg of UBD pressure on electromyographic activity (EMG activity) in 35 female rats is observed. Evoked EMG activity is obtained by subtraction of spontaneous activity from total activity during UBD. The thicker line represents the mean responses of the sample.
Fig. 3. Effect of single intravenous boluses of 3 mg/kg of fentanyl (circles, n 6) or the equivalent volume of normal saline (squares, n 5) on (A) electromyographic activity (EMG activity) response ratio evoked by UBD (60 mmHg, 20s, every 4 min). A statistically signi®cant (P , 0:01) reduction of evoked EMG activity to UBD is observed 4±16 min after the injection of fentanyl in comparison with normal saline. Inset in (A) are EMG recordings of a typical experiment treated with fentanyl, in baseline conditions and 4 and 20 min after the drug injection. In (B), effects of intravenous fentanyl (3 mg/kg, circles, n 6,) and normal saline (squares, n 5) on evoked EMG activity response ratio produced by graded UBD (20±80 mmHg, 20 s). A statistically signi®cant (P , 0:01) reduction in the evoked EMG activity to 40, 60 and 80 mmHg is observed in comparison with normal saline. Data is presented as mean ^ SEM. *Indicates signi®cant differences between groups, with P , 0:01.
100
P. Castroman, T.J. Ness / Neuroscience Letters 306 (2001) 97±100
repetitive UBD may produce histological damage, which could lead to in¯ammation and peripheral sensitization. The present model is amenable to the pharmacologic examination of visceral pain-related sensitization phenomena. The present demonstration of the inhibitory effects of intravenous fentanyl and the previous demonstration of similar effects of intravenous morphine and lidocaine on physiologic responses to UBD also support the use of this preparation as a model of urinary bladder nociception. The addition of a vigor measure to the quanti®cation of the VMR makes the model more sensitive to small changes in activity that may have been missed when using an all-or-none measure. Also, the use of a vigor measure is mechanistically simpler as it allows use of a consistent intensity of a visceral stimulus during a drug trial. In past studies of visceral sensation, we have had to perform separate experiments to allow adequate quanti®cation of cardiovascular and visceromotor components of re¯ex responses to a visceral stimulus [12]. The use of a vigor measure allows for the concurrent determination of both. In conclusion, this methodological study has demonstrated the utility of a quanti®ed EMG measure of the visceromotor response that is evoked by UBD in the female rat. Future studies will use this model to identify the mechanism of the sensitization process that occurs with the initial presentation of a visceral stimulus, using histological and pharmacological techniques. Further, novel pharmacological agents can be tested using this model to determine their potential use for the treatment of urinary bladder pain. Supported by the National Institutes of Health, DK 51413. The secretarial assistance of Sandra Roberts is particularly acknowledged. Graphics were generated by Amy Lewis Sides. [1] Anderson, R.H., Ness, T.J. and Gebhart, G.F., A distension control device useful for quantitative studies of hollow organ sensation, Physiol. Behav., 41 (1988) 635±638.
[2] Coutinho, S.V., Meller, S.T. and Gebhart, G.F., Intracolonic zymosan produces visceral hyperalgesia in the rat that is mediated by spinal NMDA and non-NMDA receptors, Brain Res., 736 (1996) 7±15. [3] Davis, L., The pathway for visceral afferent impulses within the spinal cord, Am. J. Physiol., 59 (1922) 381±393. [4] Downman, C.B.B., Skeletal muscle re¯exes of splanchnic and intercostal nerve origin in acute spinal and decerebrate cats, J. Neurophysiol., 18 (1955) 217±235. [5] Hu, X.H., Tang, H.W., Li, Q.S. and Huang, X.F., Central mechanism of indomethacin analgesia, Eur. J. Pharmacol., 263 (1944) 53±57. [6] Hug, C.C. and Murphy, M.R., Tissue redistribution of fentanyl and termination of its effects in rats, Anesthesiology, 55 (1981) 369±375. [7] Kolhekar, R. and Gebhart, G.F., NMDA and quisqualate modulation of visceral nociception in the rat, Brain Res., 651 (1994) 215±226. [8] Mayer, E.E., Munakata, J., Mertz, H., Lembo, T. and Bernstein, C.N., Visceral hyperalgesia and irritable bowel syndrome, In G.F. Gebhart (Ed.), Visceral Pain, Progress in Pain Research and Management, Vol. 5, IASP Press, Seattle, 1995, pp. 429±467. [9] Ness, T.J., Models of visceral nociception, Ins. Lab. Animal Res. J., 40 (1999) 119±128. [10] Ness, T.J. and Gebhart, G.F., Visceral pain: a review of experimental studies, Pain, 41 (1990) 167±234. [11] Ness, T.J. and Gebhart, G.F., Characterization of neurons responsive to noxious colorectal distension in the T13-L2 spinal cord of the rat, J. Neurophysiol., 60 (1988) 1419± 1438. [12] Ness, T.J. and Gebhart, G.F., Colorectal distension as a noxious visceral stimulus: physiologic and pharmacologic characterization of pseudaffective re¯exes in the rat, Brain Res., 450 (1988) 153±169. [13] Ness, T.J., Lewis-Sides, A. and Castroman, P.J., Characterization of pressor and visceromotor re¯ex responses to urinary bladder distension in the rat: sources of variability and effect of analgesics, J. Urol., 165 (2001) 968±974. [14] Ness, T.J., Ritcher, H.E., Varner, R.E. and Fillingim, R.B., A psychophysiological study of discomfort produced by repeated ®lling of the urinary bladder, Pain, 76 (1998) 61±69. [15] Woodworth, R.S. and Sherrington, C.S., A pseudaffective re¯ex and its spinal path, J. Physiol. (Lond.), 31 (1904) 234±243.
www.elsevier.com/locate/neulet
Vigor of visceromotor responses to urinary bladder distension in rats increases with repeated trials and stimulus intensity Pablo Castroman, T.J. Ness* Department of Anesthesiology, ZRB 940, University of Alabama at Birmingham, 1530 Third Avenue South, Birmingham, AL 35294-0006, USA Received 22 February 2001; received in revised form 20 April 2001; accepted 1 May 2001
Abstract This methodological study characterized visceromotor responses (VMRs) as abdominal contractile responses to urinary bladder distension (UBD) in the female rat. Electromyographic activity of the abdominal musculature was used as a measure of the VMR. Similar to previously characterized cardiovascular responses to UBD, VMRs to UBD demonstrated an initial sensitization period whereby repeated presentation of UBD stimuli led to increase vigor of the VMR. Graded UBD produced graded VMRs, therefore stimulus±response functions could be constructed. The intravenous administration of the opioid fentanyl produced a reduced vigor of the VMR in a fashion consistent with its analgesic effect. The present report supports the utility of this model for studies of urinary bladder nociception. q 2001 Published by Elsevier Science Ireland Ltd. Keywords: Visceral Pain; Visceromotor; Nociception; Bladder Distension; Sensitization; Analgesics
Pain arising from the internal organs of the body is of great clinical signi®cance, as it constitutes the primary symptomatology of a majority of the disease processes treated by internists. Our models of visceral pain have improved over the last 10 years with the validation of models using colonic distension, arti®cial uterocalcinosis and numerous visceral stimuli [9]. Visceromotor responses (VMRs), the re¯ex contraction of the abdominal musculature in response to a visceral stimulus, has been used classically as one measure of visceral sensation. Initially characterized by Sherrington [15] and subsequently used by numerous investigators [3,4,10], the absence or presence of a VMR has served as an indicator of the interruption or maintenance of visceral pain pathways and provided support for the `noxious' nature of various visceral stimuli. Quanti®cation of the visceromotor response has taken three forms: (1) the simple determination of presence or absence in response to a ®xed visceral stimulus of suf®cient intensity that it is accepted as `noxious'; (2) the determination of a visceromotor threshold (VMT) in which the intensity of the visceral stimulus is varied and the lowest tested intensity of stimulation evoking a VMR de®ned as the VMT; * Corresponding author. Tel.: 11-205-975-9643; fax: 11-205934-7437. E-mail address: [email protected] (T.J. Ness).
and (3) determination of the vigor of the muscular contraction in response to the visceral stimulus using force transducers or implanted electrodes and electrophysiological analysis. Spinal ventral root activity has been used as a correlate to the VMR [5], allowing for some quanti®cation of the vigor of the VMR to a visceral stimulus, which in these cases was the electrical stimulation of a visceral nerve. Using this approach the effect of the analgesic indomethacin has been demonstrated [5]. Using the stimulus of colorectal distension and an abdominal electromyographic activity as a measure of the VMR, quantitative effects of visceral in¯ammation and the intrathecal administration of pharmacological agents on the vigor of the VMRs has also been studied [2,7]. Our laboratory has characterized re¯ex responses to the visceral stimulus of urinary bladder distension (UBD) which includes a VMR [13]. This VMR consisted of the contraction of the external oblique abdominal musculature as measured by direct vision or by using implanted wires to generate an electromyogram. It was demonstrated that the occurrence of the VMR became more reliable and more vigorous with repeated presentations of the UBD stimulus and with increasing intensity of UBD. In our initial analysis, VMR was quanti®ed by the determination of a VMT. Subsequent additions to our electronic equipment has allowed for a more sophisticated analysis of myoelectrical activity. The following study was undertaken to determine whether our
0304-3940/01/$ - see front matter q 2001 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 1) 01 88 6- 9
98
P. Castroman, T.J. Ness / Neuroscience Letters 306 (2001) 97±100
unsophisticated impressions regarding the effects of stimulus intensity and of repeated presentations of UBD were valid and to determine the reproducibility of the VMR over time in our preparation. This would allow for a comparison with other studies and would describe methodology that could be used in future pharmacologic studies related to visceral nociception and related analgesic processes. To test for the role of VMR as a measure of nociception, the effect of intravenous fentanyl on the vigor of the VMR was also brie¯y investigated. These studies were approved by our Institutional Review Board for non-human studies. Female Sprague±Dawley rats were anesthetized with halothane (2±5% induction by mask, 1±2% maintenance). Jugular venous, arterial carotid and tracheal cannulae were placed and the rats arti®cially ventilated using a volume-cycled respirator. Wires were sutured to the abdominal musculature allowing for differential ampli®cation of their myoelectrical activity. Following completion of the surgical preparation, the halothane anesthesia was reduced until ¯exion re¯ex responses could be evoked by pinch of the foot but without escape behaviors, typically 0.5±0.7% halothane. At these levels of anesthesia, no spontaneous or re¯exive contractile response to distension of the bladder is observed [13]. The rats were not restrained in any fashion. The myoelectrical activity of the external oblique musculature was differentially ampli®ed using standard methodology (Grass P511 AC Ampli®er, 100 £ amp.; Astro-Med, Inc. West Warwick, RI) and converted to digital information using a Micro 1401 processor and associated Spike-2 software (Cambridge Electronic Design, Cambridge, UK). Using an analysis routine kindly provided by G.F. Gebhart and used in ref. [2], the signal could be quanti®ed as the number of episodes of non-recti®ed electromyographic activity exceeding a preset `threshold' voltage limit. This threshold was individually adjusted above the baseline level of activity of the abdominal musculature prior to the administration of UBD. The same threshold level was maintained throughout the course of a given experiment. The number of episodes above the threshold level was determined for 10 s prior to UBD and during the 20 s of UBD, the former serving as a measure of spontaneous activity and the latter of total activity during UBD. Subtraction of spontaneous activity from total activity during UBD allowed for the calculation of UBD-evoked activity. Since the quanti®ed EMG responses of the abdominal musculature produced by the same stimulus vary in maximum response in relation to small differences in electrode placement, in the pharmacological study EMG activity for each experiment was normalized by the determination of a response ratio. This was calculated as the maximum response to UBD (60 mmHg, 20 s) after drug or saline divided by the mean of the three control responses determined in the same rat prior to drug or saline administration. A 22-gauge te¯on angiocatheter was placed into the urinary bladder via the urethra and held in place by a tight suture
around the distal urethral ori®ce. UBD was administered as phasic stimuli (rapid onset, rapid offset) using compressed air and a previously described distension control device [1]. Thirty ®ve rats received a series of ten 60 mmHg, 20 s phasic UBD stimuli at 4 min intervals followed by single trials of graded (10±80 mmHg, 20 s) phasic stimuli given in sequence. In six female rats, additional investigation into the effects of analgesics was performed. After three baseline VMRs to UBD (60 mmHg, 20 s) every 4 min and baseline VMRs to graded UBD (20±80 mmHg, 20 s) were obtained, intravenous (i.v.) doses of fentanyl citrate (3 mg/kg) were injected slowly (over a 1 min period) as 1 ml/kg boluses. Control rats (n 5) were injected i.v. with the same volume of normal saline. For evaluation of stimulus-response functions to graded UBD, second boluses of the same dose of fentanyl or the equivalent volume of normal saline were performed after 80% of the baseline responses were obtained. VMR responses to graded UBD (20±80 mmHg) given in sequence were recorded before and after pharmacological administration. Due to the short lasting effect observed after the ®rst dose of fentanyl, graded UBDs after a second dose administration were performed in sequence and counterbalanced order (ascending, n 3; descending, n 3), 12 min after injection. Statistics are presented as the mean ^ SEM, unless otherwise stated. In the pharmacological study, one-way ANOVA for repeated measures followed by post-hoc Fisher's test were used to evaluate statistical signi®cance. Differences were considered to be signi®cant at P values , 0:05. Strong VMRs could be evoked by UBD and could be measured as electromyographic activity (see examples in Figs. 1±3). Similar to cardiovascular responses to UBD, the VMRs to UBD demonstrated an accelerating response to the repeated presentation of the visceral stimulus (Fig. 1). VMRs to the initial presentation of UBD are neither vigorous nor reliable but after only 3±5 UBDs the VMRs became both vigorous and reliable. The VMR is also graded in response to graded intensities of UBD (Fig. 2) in a curvilinear fashion. The VMR remains reliable and as a group (Fig. 3 -normal saline data) there is reproducibility of the response, although individual rats may have signi®cant trialto-trial variability. Intravenous fentanyl (3 mg/kg) produced, in comparison with intravenous saline, a statistically significant reduction (P , 0:01) in the vigor of the VMR to UBD (60 mmHg, 20 s every 4 min.), an effect which lasted 16 to 20 min (Fig. 3A). This brief effect is consistent with its action as an analgesic that undergoes rapid redistribution [6]. The drug also produced a statistically signi®cant inhibition of VMRs to graded UBD at 40, 60 and 80 mmHg (Fig. 3B) in comparison with intravenous saline. The present study con®rmed impressions generated during a previous characterization of physiologic responses to UBD that the VMRs underwent an initial period of accel-
P. Castroman, T.J. Ness / Neuroscience Letters 306 (2001) 97±100
99
appears to require a sensitization process such as in¯ammation or abnormal patterns of input. The present study and that of previous psychophysical studies suggest that repeated distension serves to activate this sensitization process. The precise mechanism of these sensitization processes has not been determined, but a role for spinal glutamate receptor activation has been demonstrated in models of colorectal pain [2,7]. It is likely that similar mechanisms may apply to other pelvic organs but more peripheral mechanisms related to neurogenic and nonneurogenic processes may prove to be of similar relevance. For instance, the physical effect on the urothelium due to the Fig. 1. Effect of repeated UBD on VMRs. In (A), typical examples of EMG activity during repeated UBD trials are shown. Trial number is given at left of each EMG. In (B), effect of the presentation of ten UBD (60 mmHg 20 s, every 4 min.) measured as electromyographic activity (EMG activity) of the abdominal musculature in 35 female rats is observed. Evoked EMG activity is obtained by subtraction of spontaneous activity from total activity during UBD. The thicker line represents the mean responses of the sample.
eration and were graded in response to graded stimuli. This period of `sensitization' has been noted in both human [8] and non-human [12,11] studies using colorectal distension as a noxious visceral stimulus and psychophysical measures or neuronal/physiological measures as responses. In humans, the repeated presentation of the urinary bladder distending stimulus as cystometrograms results in increasing intensities of sensation, decreasing thresholds for pain and increased vigor of physiologic responses to the stimulus with a threshold for the evocation of pain of 20±40 mmHg [14]. In order to be evoked, the sensation of visceral pain
Fig. 2. Effect of graded UBD on VMRs. In (A), a typical example of the effect of graded UBD (10±80 mmHg, 20 s) is shown. Intensity of UBD is given at left of each EMG. In (B), the effect of 20±80 mmHg of UBD pressure on electromyographic activity (EMG activity) in 35 female rats is observed. Evoked EMG activity is obtained by subtraction of spontaneous activity from total activity during UBD. The thicker line represents the mean responses of the sample.
Fig. 3. Effect of single intravenous boluses of 3 mg/kg of fentanyl (circles, n 6) or the equivalent volume of normal saline (squares, n 5) on (A) electromyographic activity (EMG activity) response ratio evoked by UBD (60 mmHg, 20s, every 4 min). A statistically signi®cant (P , 0:01) reduction of evoked EMG activity to UBD is observed 4±16 min after the injection of fentanyl in comparison with normal saline. Inset in (A) are EMG recordings of a typical experiment treated with fentanyl, in baseline conditions and 4 and 20 min after the drug injection. In (B), effects of intravenous fentanyl (3 mg/kg, circles, n 6,) and normal saline (squares, n 5) on evoked EMG activity response ratio produced by graded UBD (20±80 mmHg, 20 s). A statistically signi®cant (P , 0:01) reduction in the evoked EMG activity to 40, 60 and 80 mmHg is observed in comparison with normal saline. Data is presented as mean ^ SEM. *Indicates signi®cant differences between groups, with P , 0:01.
100
P. Castroman, T.J. Ness / Neuroscience Letters 306 (2001) 97±100
repetitive UBD may produce histological damage, which could lead to in¯ammation and peripheral sensitization. The present model is amenable to the pharmacologic examination of visceral pain-related sensitization phenomena. The present demonstration of the inhibitory effects of intravenous fentanyl and the previous demonstration of similar effects of intravenous morphine and lidocaine on physiologic responses to UBD also support the use of this preparation as a model of urinary bladder nociception. The addition of a vigor measure to the quanti®cation of the VMR makes the model more sensitive to small changes in activity that may have been missed when using an all-or-none measure. Also, the use of a vigor measure is mechanistically simpler as it allows use of a consistent intensity of a visceral stimulus during a drug trial. In past studies of visceral sensation, we have had to perform separate experiments to allow adequate quanti®cation of cardiovascular and visceromotor components of re¯ex responses to a visceral stimulus [12]. The use of a vigor measure allows for the concurrent determination of both. In conclusion, this methodological study has demonstrated the utility of a quanti®ed EMG measure of the visceromotor response that is evoked by UBD in the female rat. Future studies will use this model to identify the mechanism of the sensitization process that occurs with the initial presentation of a visceral stimulus, using histological and pharmacological techniques. Further, novel pharmacological agents can be tested using this model to determine their potential use for the treatment of urinary bladder pain. Supported by the National Institutes of Health, DK 51413. The secretarial assistance of Sandra Roberts is particularly acknowledged. Graphics were generated by Amy Lewis Sides. [1] Anderson, R.H., Ness, T.J. and Gebhart, G.F., A distension control device useful for quantitative studies of hollow organ sensation, Physiol. Behav., 41 (1988) 635±638.
[2] Coutinho, S.V., Meller, S.T. and Gebhart, G.F., Intracolonic zymosan produces visceral hyperalgesia in the rat that is mediated by spinal NMDA and non-NMDA receptors, Brain Res., 736 (1996) 7±15. [3] Davis, L., The pathway for visceral afferent impulses within the spinal cord, Am. J. Physiol., 59 (1922) 381±393. [4] Downman, C.B.B., Skeletal muscle re¯exes of splanchnic and intercostal nerve origin in acute spinal and decerebrate cats, J. Neurophysiol., 18 (1955) 217±235. [5] Hu, X.H., Tang, H.W., Li, Q.S. and Huang, X.F., Central mechanism of indomethacin analgesia, Eur. J. Pharmacol., 263 (1944) 53±57. [6] Hug, C.C. and Murphy, M.R., Tissue redistribution of fentanyl and termination of its effects in rats, Anesthesiology, 55 (1981) 369±375. [7] Kolhekar, R. and Gebhart, G.F., NMDA and quisqualate modulation of visceral nociception in the rat, Brain Res., 651 (1994) 215±226. [8] Mayer, E.E., Munakata, J., Mertz, H., Lembo, T. and Bernstein, C.N., Visceral hyperalgesia and irritable bowel syndrome, In G.F. Gebhart (Ed.), Visceral Pain, Progress in Pain Research and Management, Vol. 5, IASP Press, Seattle, 1995, pp. 429±467. [9] Ness, T.J., Models of visceral nociception, Ins. Lab. Animal Res. J., 40 (1999) 119±128. [10] Ness, T.J. and Gebhart, G.F., Visceral pain: a review of experimental studies, Pain, 41 (1990) 167±234. [11] Ness, T.J. and Gebhart, G.F., Characterization of neurons responsive to noxious colorectal distension in the T13-L2 spinal cord of the rat, J. Neurophysiol., 60 (1988) 1419± 1438. [12] Ness, T.J. and Gebhart, G.F., Colorectal distension as a noxious visceral stimulus: physiologic and pharmacologic characterization of pseudaffective re¯exes in the rat, Brain Res., 450 (1988) 153±169. [13] Ness, T.J., Lewis-Sides, A. and Castroman, P.J., Characterization of pressor and visceromotor re¯ex responses to urinary bladder distension in the rat: sources of variability and effect of analgesics, J. Urol., 165 (2001) 968±974. [14] Ness, T.J., Ritcher, H.E., Varner, R.E. and Fillingim, R.B., A psychophysiological study of discomfort produced by repeated ®lling of the urinary bladder, Pain, 76 (1998) 61±69. [15] Woodworth, R.S. and Sherrington, C.S., A pseudaffective re¯ex and its spinal path, J. Physiol. (Lond.), 31 (1904) 234±243.