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MK-801 inhibits the micturition reflex in chronic bladder irritation caused by crystalluria in the rat
Autonomic Neuroscience: Basic and Clinical 105 (2003) 1 – 7 www.elsevier.com/locate/autneu
MK-801 inhibits the micturition reflex in chronic bladder irritation caused by crystalluria in the rat Fumitaka Ishida a,*, Tomoya Sato a,b, Michiru Imaizumi a, Norio Funayama c, Kenji Ikegami a, Toshie Nimura a, Masahito Kawatani a b
a Department of Physiology, School of Medicine, Akita University, 1-1-1 Hondo, Akita 010-8543, Japan Department of Urology, School of Medicine, Shinshuu University, 3-1-1 Asahimachi, Matsumoto 390-8621, Japan c POLA Chemical Industries Inc., 27-1 Takashimadai, Kanagawa-ku, Yokohama 221-0833, Japan
Received 10 April 2002; received in revised form 3 December 2002; accepted 5 December 2002
Abstract Urodynamic and pharmacological studies were performed to investigate the effect of crystalluria on the micturition reflex and the involvement of glutamatergic transmission. The rats, which were given LP-805 (100 mg/kg/day) orally for 12 days, voided crystalluria. The pH of these crystalluria (LP-805 urine) was the same as normal urine. The amount of crystals was 70 – 100/division magnified 400 . The end of the crystals was sharp. Intravesical administration of LP-805 urine induced hyperreflexia of the micturition reflex in normal rats. When the infusion solution was changed to LP-805 urine from saline, the latency was reduced to 57.6 F 2.1% of control in single cystometrogram (CMG) or was reduced to 51.4 F 0.9% of control in continuous CMG. The voiding volume was reduced to 52.1 F 3.6% of control in single CMG or was reduced to 62.5 F 0.8% of control in continuous CMG. These parameters were recovered after LP-805 urine was removed. Intravesical administration of acetic acid did not induce hyperreflexia of the micturition reflex in LP-805-treated rats. These data suggest that the chronic irritation by aculeate crystals might induce hyperreflexia of the micturition reflex, which increase afferent neuronal activity. Intravenous administration of MK-801 (0.001 to 1 mg/kg) inhibited the micturition reflex in a dose-dependent manner. The ID50 in LP805-treated rats (0.03 mg/kg i.v.) was lower than that in normal rats (0.56 mg/kg i.v.). After chronic irritation of the bladder epithelium, MK801 sensitivity was enhanced for the micturition reflex. These data suggested that crystalluria elicit hyperreflexia in the micturition reflex that mediated with NMDA glutamatergic receptors. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Chronic cystitis; Micturition reflex; Crystalluria; NMDA; MK-801; Visceral pain; Urinary bladder
1. Introduction It is well known that the most common symptoms of cystitis are hyperreflexia of the micturition reflex and vesical pain (Marsh, 1976). Afferent activity that produces distension of the urinary bladder during urine storage activates the brainstem micturition center for the initiation of the micturition reflex (de Groat et al., 1993). Hyperreflexia of the micturition reflex, including that in cystitis,
* Corresponding author. Tel.: +81-18-884-6073; fax: +81-18-8362605. E-mail address: [email protected] (F. Ishida).
might be produced by an increase in the afferent activity of urinary bladder. Afferent terminals of urinary bladder have been activated by irritant chemicals, different pH solutions or cold water (Ha¨bler et al., 1988, 1990; Morrison, 1999; Fall et al., 1990). A cystitis model has been established by intravesical administration of acetic acid, turpentine oil or acetone (Birder and de Groat, 1992a,b; McMahon, 1988; Kato et al., 1990). Intravesical administration of these irritants elicited severe inflammation and/or vesical pain. Frequent voiding reflexes and/or vesical pain were also noticed with vesical stones (Blandy, 1976). These symptoms were improved by the excretion of the vesical stones. Touch and pinprick of the bladder epithelium elicited vesical pain (Cervero, 1994). No systematic analysis has been performed
1566-0702/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S1566-0702(02)00289-8
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of chronic irritation of the bladder epithelium by vesical stones or pins. Glutamatergic mechanisms are involved in spinal nociceptive and non-nociceptive transmission from the pelvic viscera (Coderre et al., 1993; de Groat et al., 1998). Expression of c-fos in the spinal cord after bladder irritation involved NMDA receptors (Birder and de Groat, 1992a,b, 1993; Kakizaki et al., 1996). The hyperreflexia of the micturition reflex that was induced with turpentine oil was prevented by intrathecal administration of AP-5, an NMDA receptor antagonist (Rice and McMahon, 1994). These data suggest that NMDA receptors might be involved in hyperreflexia of the micturition reflex during acute cystitis. Another investigator has also indicated that spinal NMDA receptors were important for mediating persistent pain and hyperalgesia of the pelvic viscera (Cervero, 2000). However, the involvement of NMDA receptors in chronic irritation of the bladder epithelium has not been studied. We investigated the effect of crystalluria on the micturition reflex and the involvement of glutamatergic transmission after chronic irritation of the bladder epithelium by crystalluria.
2. Materials and methods Experiments were performed on female Wistar rats (n = 45) weighing 220 –360 g. 2.1. Collection of urine Animals (n = 18) were divided to two groups, control rats (n = 6) and drug-treated rats (n = 12) that were given LP-805 (100 mg/kg/day) (POLA, Tokyo, Japan) orally for 12 days (LP-805-treated rats). The animals were kept in individual cages in a 12-h light/12-h dark light schedule (7 a.m. to 7 p.m., light cycle) at 20 –25 jC. All animals had free access to water and food. Collection of the urine was performed from days 7 to 12. Each animal was placed in a collecting cage from 10 a.m. to 4 p.m. (6 h) every day. The urine was frozen immediately and kept in a freezer until use. Prior to the bladder infusion, the urine was defrosted at room temperature for pH measurement and microscopic examination (BH-2, Olympus, Tokyo, Japan). Microscopic images were obtained with a digitized cooled CCD camera (CoolSNAPcf, Photometrics, USA), and were analyzed with a digital image analysis program (Lumina Vision, Mitani, Tokyo, Japan). 2.2. Micturition reflex with LP-805 urine infusion in normal rats Normal animals (n = 13) were anesthetized with urethane (0.8 –1.2 g/kg, i.p.), and the trachea was cannulated with a
polyethylene tube (PE 205) to facilitate respiration. After a lower abdominal midline incision, the urinary bladder was isolated and a polyethylene tube (PE 50) was inserted into the apex of the bladder dome. The tube was connected to an infusion pump (Model 55-1111, Harvard Apparatus, Massachusetts, USA) and a pressure transducer. Bladder pressure was monitored with a polygraph (AD-600, Nihonkoden, Tokyo, Japan) and recorded on a chart graph (R-62, Rikadenki, Tokyo, Japan). A cystometrogram (CMG) in the normal rat was performed with the urine that corrected from LP-805-treated rats (LP-805 urine). Infusion of the bladder was performed at 3 ml/h. A single CMG was performed in normal rats (n = 4) infused with saline (pH 6.8) or LP-805 urine. The pump infusion was stopped when urine was visually identified from the urethral orifice. The amount of urine that came out was measured. The residual urine was sucked from the bladder and measured. Restart of a single CMG was set for 3 min after the previous CMG ended. Three micturition reflexes just before the change of the infusion solution were used as the control for each study. A continuous CMG was performed in normal rats (n = 9) infused with saline or LP-805 urine. Initially, the urinary bladder was infused with saline for the control study. After a stable CMG was recorded, the infusion solution was changed to LP-805 urine. The infusion of LP-805 urine was performed for 30 min and then the infusion solution was changed to saline. Three micturition reflexes just before the change of the infusion solution were used as the control for each study. The following parameters were studied: the latency, the threshold pressure, the peak amplitude of bladder contraction, and the voiding volume. 2.3. Micturition reflex with 0.1% acetic acid infusion in LP805-treated rats A single CMG was performed in LP-805-treated rats that were given LP-805 (100 mg/kg/day p.o. for 12 days) (n = 6) infused with saline or 0.1% acetic acid (pH 3.3). The animal preparation and methods were as described in the previous paragraph (Section 2.2). The following parameters were studied: the latency, the threshold pressure, the peak amplitude of bladder contraction, the voiding volume, and the residual urine volume. 2.4. Micturition reflex with intravenous administration of MK-801 in LP-805-treated rats Intravenous administration of MK-801 (Sigma, St. Louis, USA) was performed in normal rats (n = 4) and LP-805treated rats that were given LP-805 (100 mg/kg/day p.o. for 12 days) (n = 4). In addition to the animal preparation described in Section 2.2, the following surgical procedure was performed. The jugular vein was cannulated with a polyethylene tube (PE 50) for the intravenous administration
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of MK-801. A continuous CMG infused with saline was performed in these animals. The dose – response curves were plotted as the cumulative dose of MK-801 given at 15-min intervals. The peak amplitude of bladder contraction and the volume threshold were studied. 2.5. Statistical analysis The results were expressed as the mean F standard error of the mean. Student’s non-paired t-test was used to analyze differences in the amounts of urine, pH and the effects of MK-801 administration on the parameters of the micturition reflex. Mann –Whitney U-test was used to analyze differences in the parameters of the micturition reflex. A probability level of < 0.05 was considered significant. The protocols for animal experimentation described in this paper were previously approved by the Animal Committee, Akita University, and The ‘‘Guidelines for Animal Experimentation’’ of the University were strictly adhered to in all animal experiments.
Fig. 2. Examples of single CMG with saline infusion (A) and with LP-805 urine infusion (B) in normal rats (n = 4). The arrow head indicated the start of infusion. The bar graphs summarize single CMG data for the latency (C) and the voiding urine volume (D). The effects of LP-805 urine were compared to the control using Mann – Whitney U-test (**p < 0.02).
3. Results 3.1. Examination of LP-805 urine The pH of LP-805 urine was 6.5 F 0.3, and that of normal urine was 6.7 F 0.2. Thus, there was no significant difference in the urinary pH. Aculeate crystals were observed in LP-805 urine (12/12) (Fig. 1). The amount of crystals was 70 – 100/division magnified 400 . The lengths ranged from 10 to 150 Am. The diameters ranged from 3 to 5 Am. The end of the crystals
was sharp. Several crystals gathered and formed a pillar mass. No crystals were observed in the normal urine (6/6). 3.2. The effect of LP-805 urine infusion on the micturition reflex in normal rats In single CMG with saline infusion (control study) (Fig. 2A), the latency was 1.2 F 0.3 min, the threshold pressure was 10.2 F 0.8 cm H20, the peak amplitude of bladder contraction was 25.8 F 3.7 cm H20, the voiding volume
Fig. 1. Crystal(s) in the urine from LP-805-treated rats (LP-805 urine). Magnification: 25 (A), and 150 (B, C).
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was 0.05 F 0.01 ml, and the residual urine volume was 0.02 F 0.01 ml. When the infusion solution was changed to LP-805 urine (Fig. 2B), the latency was reduced to 0.7 F 0.4 min (57.6 F 2.1% of control, p < 0.02) (Fig. 2C). The voiding volume was reduced to 0.03 F 0.01 ml (52.1 F3.6% of control, p < 0.02) (Fig. 2D). There was no significant difference in the threshold pressure between the control and LP-805 urine (10.8 F 0.6 cm H 2 0, 99.5 F 4.1% of control). Similarly, the peak amplitude of bladder contraction between the control and LP-805 urine (26.4 F 2.8 cm H20, 102.4 F 5.2% of the control), and the residual urine (0.02 F 0.01 ml, 101.3 F 2.6% of control) were not significantly different. In continuous CMG with saline infusion (control study) (Fig. 3A), the latency was 2.8 F 0.9 min, the threshold pressure was 9.8 F 1.1 cm H20, the peak amplitude of bladder contraction was 29.1 F 4.2 cm H 2 0, and the voiding volume was 0.2 F 0.05 ml. When the infusion solution was changed to LP-805 urine (Fig. 3B), the latency was reduced to
Fig. 4. Examples of single CMG with saline infusion (A) and with 0.1% acetic acid infusion (B) in LP-805-treated rats (n = 6). The arrow head indicated the start of infusion. The bar graphs summarize single CMG data for the latency (C) and the voiding urine volume (D). The effects of 0.1% acetic acid were compared to the control using the Mann – Whitney U-test (no significant difference).
1.4 F 0.4 min, (51.4 F 0.9% of control, p < 0.02) (Fig. 3D). The voiding volume was reduced to 0.12 F 0.02 ml (62.5 F 0.8% of control, p < 0.02). There were no significant differences in the threshold pressure (9.5 F 0.9 cm H20, 96.9 F 0.3% of control), or the peak amplitude of bladder contraction (28.7 F 5.2 cm H20, 97.6 F 0.6% of control) (Fig. 3E). When the infusion solution was changed to saline from LP-805 urine, the duration before bladder contraction and the voiding volume recovered to the control levels (2.5 F 0.6 min and 0.2 F 0.06 ml, respectively) (Fig. 3C). 3.3. The effects of acetic acid infusion on the micturition reflex in LP-805-treated rats There were no significant differences between saline and 0.1% acetic acid in the latency (4.1 F 0.6 and 4.7 F 0.6 min), the threshold pressure (8.2 F 0.4 and 7.1 F 0.5 cm H20), the peak amplitude of bladder contraction (24.8 F 0.3 and 25.1 F 0.8 cm H20), the voiding volume (0.24 F 0.02 and 0.26 F 0.03 ml), or the residual urine volume (0.01 F 0.02 and 0.02 F 0.01 ml) (Fig. 4). 3.4. The effects of MK-801 on the micturition reflex in LP805-treated rats
Fig. 3. Examples of continuous CMG with saline infusion (A), and with LP-805 urine infusion (B) and saline infusion recovery (C) in normal rats (n = 9). The bar graphs summarize continuous CMG data for the latency (D) and the voiding urine volume (E). The effects of LP-805 urine were compared to the control using the Mann – Whitney U-test (**p < 0.02).
The peak amplitude of bladder contraction was reduced in a dose-dependent manner after the administration of increasing doses of MK-801 (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1 mg/kg i.v.) in normal rats (ID50 = 0.56 mg/kg i.v., 105.6 F 1.2%, 102.5 F 0.8%, 97.8 F 9.6%, 98.6 F 11.2%, 63.9 F 9.3%, 52.1 F 5.7%, 39.4 F 3.8%, respectively) and
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in LP-805-treated rats (ID50 = 0.03 mg/kg i.v., 98.0 F 0.6%, 95.8 F 1.4%, 83.3 F 2.1%, 60.3 F 8.2%, 40.6 F 11.5%, 31.8 F 2.2%, 26.8 F 2.8%, respectively) (Fig. 5A). There were significant differences between the normal and LP805-treated rats at 0.03 mg/kg i.v. ( p < 0.05) and 0.3 mg/ kg i.v. ( p < 0.05). The volume threshold of the micturition reflex increased in a dose-dependent manner after the administration of increasing doses of MK-801 (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1 mg/kg i.v.) in normal rats (101.2 F7.7%, 86.7 F12.4%, 87.5 F12.1%, 11 2 . 3 F 1 0 . 3 % , 1 0 5 . 1 F 11 . 7 % , 1 9 8 . 3 F 4 2 . 4 % , 253.8 F 38.2%, respectively) and in LP-805-treated rats (105.4 F 10.2%, 122.5 F 20.8%, 125.8 F 14.6%, 255.2 F 79.4%, 390.2 F 84.3%, 439.1 F 75.7%, 462.4 F 90.8%, respectively) (Fig. 5B). There were significant differences between the normal and LP-805-treated rats at 0.01 mg/kg i.v. ( p < 0.05), 0.1 mg/kg i.v. ( p < 0.05) and 0.3 mg/kg i.v. ( p < 0.05).
Fig. 5. Log dose – response curves showing the effects of increasing MK801 during continuous CMG in normal rats (n = 4) (o) and in LP-805treated rats (n = 9) (E) on the peak amplitude of bladder contraction (A) and the volume threshold (B). The effects of MK-801 (i.v.) on LP-805treated rats were compared to those on normal rats using Student’s nonpaired t-test (*p < 0.05).
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4. Discussion Intravesical administration of LP-805 urine induced hyperreflexia of the micturition reflex in normal rats. Urinary pH, mechanical stimulation, and chemical irritants have been proposed as important factors for the facilitation of the micturition reflex (Ha¨bler et al., 1988, 1990; Morrison, 1999). The pH of LP-805 urine showed no difference to normal urine. As indicated in Fig. 1, many crystals were observed in the LP-805 urine. We hypothesize that the crystals were causing the hyperreflexia of the micturition reflex. Chemical analysis indicated few LP-805 metabolites in the urine and LP-805 might produce the crystals (unpublished observation). Since the hyperreflexia of the micturition reflex was recovered to the control level when the LP805 urine was removed, the crystals were major irritants causing the hyperreflexia of the micturition reflex. It has been reported that chemical irritants produced hyperreflexia of the micturition reflex (de Groat et al., 1993; Morrison, 1999). The crystals might cause the production and release of chemical irritants in the urinary bladder. In the present study, the latency was 1.2 F 0.5 min in normal rats infused with saline, but this was shorter than in LP-805-treated rats infused with saline (4.1 F 0.6 min). Similarly, the voiding volume of normal rats infused with saline (0.05 F 0.01 ml) was smaller than that of LP-805treated rats infused with saline (0.24 F 0.02 ml). These data suggest that chronic LP-805-treated animals have a larger volume threshold and longer duration before bladder contraction than normal animals. In contrast, intravesical administration of LP-805 urine reduced the latency (57.6 F 2.1% of control) and the voiding volume (52.1 F 3.6% of control) in normal rats. Similar data were observed for intravesical administration of chemical irritants. The latency and the voiding volume were initially reduced (Birder and de Groat, 1992b; McMahon, 1988), and these parameters were enhanced or undetectable several hours later. Histological experiments revealed that severe inflammatory changes had occurred in the xylene or acetic acid-treated bladder tissues (Maggi et al., 1988; Birder and de Groat, 1992b). In LP-805-treated rats, intravesical administration of acetic acid did not elicit hyperreflexia of the micturition reflex. This result might be due to neuronal activity elicited by intravesical administration of acetic acid that was already activated by chronic irritation of the bladder epithelium. Patch clamp studies revealed that the threshold for action potentials of dorsal root ganglia cells in LP-805-treated rats was lower than the threshold in normal rats (Imaizumi et al., 1997). An increase in primary afferent neuronal activity following bladder inflammation has been described (Ha¨bler et al., 1988, 1990). In addition to this observation, central sensitization of the dorsal horn neurons during urinary bladder inflammation has been proposed (McMahon, 1988). The cyclophosphamide (CPA)-induced cystitis model is well established as a chronic chemical cystitis model and is useful for the investigation of chronic visceral pain (Lante´ri-
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Minet et al., 1995; Ishigooka et al., 2001). After CPA administration, the volume threshold was reduced and micturition frequency was increased (Lecci et al., 1994; Ozawa et al., 1999). Patch clamp studies revealed that the threshold for action potentials of the dorsal root ganglia cells in CPA-treated rats was lower than the threshold in normal rats (Yoshimura and de Groat, 1999). These results were similar to our data. The present study indicates that LP-805treated rats are invaluable for the investigation of chronic irritation of the bladder epithelium. The present findings showed that MK-801 exerted an inhibitory effect on the micturition reflex in normal and/or LP-805-treated rats. Glutamate has been widely accepted as the major excitatory transmitter in the micturition reflex. Glutamate or its analogues injected into certain brain stem areas facilitated bladder activity in the cat and rat (de Groat et al., 1993). Administration of NMDA receptor antagonists inhibited the micturition reflex (Maggi et al., 1990; Yoshiyama et al., 1991, 1993a,b, 1994, 1995). Intrathecal administration of NMDA receptor antagonists reduced the amplitude of the action potentials in the dorsal part of the rostral pons, which were evoked by pelvic nerve stimulation (Kakizaki et al., 1998). Intravenous administration of NMDA receptor antagonists blocked the bladder contractions elicited by pontine micturition center stimulation (Matsumoto et al., 1995). These studies indicate that NMDA glutamatergic excitatory transmission is involved in the ascending and descending limbs of the spinobulbospinal micturition reflex. The present study demonstrated that MK-801 reduced the peak amplitude of bladder contraction and increased the volume threshold in normal rats. The effects were first detected with 0.03 mg/kg i.v. of MK-801 and the effects were increased in a dose-dependent manner. Complete abolishment of the micturition reflex was observed at 1 mg/kg i.v. of MK-801. These effective doses are very similar to the previous investigations (Maggi et al., 1990; Yoshiyama et al., 1991, 1993a,b). In LP-805-treated rats, MK-801 inhibited the micturition reflex in a similar manner to normal rats. NMDA receptor antagonists have been reported to block chronic cystitis. (Me´en et al., 2002). The ID50 in normal rats was 0.56 mg/ kg i.v., and that in LP-805-treated rats was 0.03 mg/kg i.v. This might indicate that lower doses of MK-801 have more effect on the micturition reflex in LP-805-treated rats. The volume threshold in LP-805-treated rats increased more than that in normal rats with the same dose of MK-801 i.v. These findings suggested that the more afferent inputs in LP-805-treated rats might need to evoke micturition reflex with MK-801 i.v. Thus, in LP-805-treated rats, NMDA glutamatergic excitatory transmission might be more important in micturition reflex. There is considerable evidence that spinal NMDA receptors are involved in central sensitization following somatic noxious stimuli (Coderre et al., 1993; McMahon et al., 1993; Neugebauer et al., 1994). We have provided evidence
that spinal NMDA receptors have a similar role following chronic visceral irritation. We suggest that spinal NMDA receptors should be explored for therapeutic potential with respect to conditions such as interstitial cystitis.
5. Uncited reference Williams, 1976
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ment of N-methyl-D-aspartate (NMDA) and non-NMDA receptors in the responsiveness of rat spinal neurons with input from the chronically inflamed ankle. Neurosci. Lett. 170, 237 – 240. Ozawa, H., Chancellor, M.B., Jung, S.-Y., Yokoyama, T., Fraser, M.O., Yu, Y., de Groat, W.C., Yoshimura, N., 1999. Effect of intravesical nitric oxide therapy on cyclophosphamide-induced cystitis. J. Urol. 162, 2211 – 2216. Rice, A.S.C., McMahon, S.B., 1994. Pre-emptive intrathecal administration of an NMDA receptor antagonist (AP-5) prevents hyper-reflexia in a model of persistent vesical pain. Pain 57, 335 – 340. Williams, R.E., 1976. Renal and ureteric calculi. In: Blandy, J. (Ed.), Urology, vol. I. Blackwell, London, pp. 291 – 347. Yoshimura, N., de Groat, W.C., 1999. Increased excitability of afferent neurons innervating rat urinary bladder after chronic bladder inflammation. J. Neurosci. 19, 4644 – 4653. Yoshiyama, M., Roppolo, J.R., Blastos, R.B., de Groat, W.C., 1991. The effects of MK-801, an NMDA receptor antagonist, on the micturition reflex in the rat. Neurosci. Lett. 126, 141 – 144. Yoshiyama, M., Roppolo, J.R., de Groat, W.C., 1993a. Effects of MK-801 on the micturition reflex in the rat-possible sites of action. J. Pharmacol. Exp. Ther. 265, 844 – 850. Yoshiyama, M., Roppolo, J.R., Thor, K.B., de Groat, W.C., 1993b. Effects of LY274614, a competitive NMDA receptor antagonist on the micturition reflex in the urethane-anesthetized rat. Br. J. Pharmacol. 110, 77 – 86. Yoshiyama, M., Roppolo, J.R., de Groat, W.C., 1994. Interactions between glutamatergic and monoaminergic systems controlling the micturition reflex in the urethane-anesthetized rat. Brain Res. 639, 300 – 308. Yoshiyama, M., Roppolo, J.R., de Groat, W.C., 1995. Interactions between NMDA and AMPA/kainate receptors in the control of micturition in the rat. Eur. J. Pharmacol. 287, 73 – 78.
MK-801 inhibits the micturition reflex in chronic bladder irritation caused by crystalluria in the rat Fumitaka Ishida a,*, Tomoya Sato a,b, Michiru Imaizumi a, Norio Funayama c, Kenji Ikegami a, Toshie Nimura a, Masahito Kawatani a b
a Department of Physiology, School of Medicine, Akita University, 1-1-1 Hondo, Akita 010-8543, Japan Department of Urology, School of Medicine, Shinshuu University, 3-1-1 Asahimachi, Matsumoto 390-8621, Japan c POLA Chemical Industries Inc., 27-1 Takashimadai, Kanagawa-ku, Yokohama 221-0833, Japan
Received 10 April 2002; received in revised form 3 December 2002; accepted 5 December 2002
Abstract Urodynamic and pharmacological studies were performed to investigate the effect of crystalluria on the micturition reflex and the involvement of glutamatergic transmission. The rats, which were given LP-805 (100 mg/kg/day) orally for 12 days, voided crystalluria. The pH of these crystalluria (LP-805 urine) was the same as normal urine. The amount of crystals was 70 – 100/division magnified 400 . The end of the crystals was sharp. Intravesical administration of LP-805 urine induced hyperreflexia of the micturition reflex in normal rats. When the infusion solution was changed to LP-805 urine from saline, the latency was reduced to 57.6 F 2.1% of control in single cystometrogram (CMG) or was reduced to 51.4 F 0.9% of control in continuous CMG. The voiding volume was reduced to 52.1 F 3.6% of control in single CMG or was reduced to 62.5 F 0.8% of control in continuous CMG. These parameters were recovered after LP-805 urine was removed. Intravesical administration of acetic acid did not induce hyperreflexia of the micturition reflex in LP-805-treated rats. These data suggest that the chronic irritation by aculeate crystals might induce hyperreflexia of the micturition reflex, which increase afferent neuronal activity. Intravenous administration of MK-801 (0.001 to 1 mg/kg) inhibited the micturition reflex in a dose-dependent manner. The ID50 in LP805-treated rats (0.03 mg/kg i.v.) was lower than that in normal rats (0.56 mg/kg i.v.). After chronic irritation of the bladder epithelium, MK801 sensitivity was enhanced for the micturition reflex. These data suggested that crystalluria elicit hyperreflexia in the micturition reflex that mediated with NMDA glutamatergic receptors. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Chronic cystitis; Micturition reflex; Crystalluria; NMDA; MK-801; Visceral pain; Urinary bladder
1. Introduction It is well known that the most common symptoms of cystitis are hyperreflexia of the micturition reflex and vesical pain (Marsh, 1976). Afferent activity that produces distension of the urinary bladder during urine storage activates the brainstem micturition center for the initiation of the micturition reflex (de Groat et al., 1993). Hyperreflexia of the micturition reflex, including that in cystitis,
* Corresponding author. Tel.: +81-18-884-6073; fax: +81-18-8362605. E-mail address: [email protected] (F. Ishida).
might be produced by an increase in the afferent activity of urinary bladder. Afferent terminals of urinary bladder have been activated by irritant chemicals, different pH solutions or cold water (Ha¨bler et al., 1988, 1990; Morrison, 1999; Fall et al., 1990). A cystitis model has been established by intravesical administration of acetic acid, turpentine oil or acetone (Birder and de Groat, 1992a,b; McMahon, 1988; Kato et al., 1990). Intravesical administration of these irritants elicited severe inflammation and/or vesical pain. Frequent voiding reflexes and/or vesical pain were also noticed with vesical stones (Blandy, 1976). These symptoms were improved by the excretion of the vesical stones. Touch and pinprick of the bladder epithelium elicited vesical pain (Cervero, 1994). No systematic analysis has been performed
1566-0702/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S1566-0702(02)00289-8
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of chronic irritation of the bladder epithelium by vesical stones or pins. Glutamatergic mechanisms are involved in spinal nociceptive and non-nociceptive transmission from the pelvic viscera (Coderre et al., 1993; de Groat et al., 1998). Expression of c-fos in the spinal cord after bladder irritation involved NMDA receptors (Birder and de Groat, 1992a,b, 1993; Kakizaki et al., 1996). The hyperreflexia of the micturition reflex that was induced with turpentine oil was prevented by intrathecal administration of AP-5, an NMDA receptor antagonist (Rice and McMahon, 1994). These data suggest that NMDA receptors might be involved in hyperreflexia of the micturition reflex during acute cystitis. Another investigator has also indicated that spinal NMDA receptors were important for mediating persistent pain and hyperalgesia of the pelvic viscera (Cervero, 2000). However, the involvement of NMDA receptors in chronic irritation of the bladder epithelium has not been studied. We investigated the effect of crystalluria on the micturition reflex and the involvement of glutamatergic transmission after chronic irritation of the bladder epithelium by crystalluria.
2. Materials and methods Experiments were performed on female Wistar rats (n = 45) weighing 220 –360 g. 2.1. Collection of urine Animals (n = 18) were divided to two groups, control rats (n = 6) and drug-treated rats (n = 12) that were given LP-805 (100 mg/kg/day) (POLA, Tokyo, Japan) orally for 12 days (LP-805-treated rats). The animals were kept in individual cages in a 12-h light/12-h dark light schedule (7 a.m. to 7 p.m., light cycle) at 20 –25 jC. All animals had free access to water and food. Collection of the urine was performed from days 7 to 12. Each animal was placed in a collecting cage from 10 a.m. to 4 p.m. (6 h) every day. The urine was frozen immediately and kept in a freezer until use. Prior to the bladder infusion, the urine was defrosted at room temperature for pH measurement and microscopic examination (BH-2, Olympus, Tokyo, Japan). Microscopic images were obtained with a digitized cooled CCD camera (CoolSNAPcf, Photometrics, USA), and were analyzed with a digital image analysis program (Lumina Vision, Mitani, Tokyo, Japan). 2.2. Micturition reflex with LP-805 urine infusion in normal rats Normal animals (n = 13) were anesthetized with urethane (0.8 –1.2 g/kg, i.p.), and the trachea was cannulated with a
polyethylene tube (PE 205) to facilitate respiration. After a lower abdominal midline incision, the urinary bladder was isolated and a polyethylene tube (PE 50) was inserted into the apex of the bladder dome. The tube was connected to an infusion pump (Model 55-1111, Harvard Apparatus, Massachusetts, USA) and a pressure transducer. Bladder pressure was monitored with a polygraph (AD-600, Nihonkoden, Tokyo, Japan) and recorded on a chart graph (R-62, Rikadenki, Tokyo, Japan). A cystometrogram (CMG) in the normal rat was performed with the urine that corrected from LP-805-treated rats (LP-805 urine). Infusion of the bladder was performed at 3 ml/h. A single CMG was performed in normal rats (n = 4) infused with saline (pH 6.8) or LP-805 urine. The pump infusion was stopped when urine was visually identified from the urethral orifice. The amount of urine that came out was measured. The residual urine was sucked from the bladder and measured. Restart of a single CMG was set for 3 min after the previous CMG ended. Three micturition reflexes just before the change of the infusion solution were used as the control for each study. A continuous CMG was performed in normal rats (n = 9) infused with saline or LP-805 urine. Initially, the urinary bladder was infused with saline for the control study. After a stable CMG was recorded, the infusion solution was changed to LP-805 urine. The infusion of LP-805 urine was performed for 30 min and then the infusion solution was changed to saline. Three micturition reflexes just before the change of the infusion solution were used as the control for each study. The following parameters were studied: the latency, the threshold pressure, the peak amplitude of bladder contraction, and the voiding volume. 2.3. Micturition reflex with 0.1% acetic acid infusion in LP805-treated rats A single CMG was performed in LP-805-treated rats that were given LP-805 (100 mg/kg/day p.o. for 12 days) (n = 6) infused with saline or 0.1% acetic acid (pH 3.3). The animal preparation and methods were as described in the previous paragraph (Section 2.2). The following parameters were studied: the latency, the threshold pressure, the peak amplitude of bladder contraction, the voiding volume, and the residual urine volume. 2.4. Micturition reflex with intravenous administration of MK-801 in LP-805-treated rats Intravenous administration of MK-801 (Sigma, St. Louis, USA) was performed in normal rats (n = 4) and LP-805treated rats that were given LP-805 (100 mg/kg/day p.o. for 12 days) (n = 4). In addition to the animal preparation described in Section 2.2, the following surgical procedure was performed. The jugular vein was cannulated with a polyethylene tube (PE 50) for the intravenous administration
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of MK-801. A continuous CMG infused with saline was performed in these animals. The dose – response curves were plotted as the cumulative dose of MK-801 given at 15-min intervals. The peak amplitude of bladder contraction and the volume threshold were studied. 2.5. Statistical analysis The results were expressed as the mean F standard error of the mean. Student’s non-paired t-test was used to analyze differences in the amounts of urine, pH and the effects of MK-801 administration on the parameters of the micturition reflex. Mann –Whitney U-test was used to analyze differences in the parameters of the micturition reflex. A probability level of < 0.05 was considered significant. The protocols for animal experimentation described in this paper were previously approved by the Animal Committee, Akita University, and The ‘‘Guidelines for Animal Experimentation’’ of the University were strictly adhered to in all animal experiments.
Fig. 2. Examples of single CMG with saline infusion (A) and with LP-805 urine infusion (B) in normal rats (n = 4). The arrow head indicated the start of infusion. The bar graphs summarize single CMG data for the latency (C) and the voiding urine volume (D). The effects of LP-805 urine were compared to the control using Mann – Whitney U-test (**p < 0.02).
3. Results 3.1. Examination of LP-805 urine The pH of LP-805 urine was 6.5 F 0.3, and that of normal urine was 6.7 F 0.2. Thus, there was no significant difference in the urinary pH. Aculeate crystals were observed in LP-805 urine (12/12) (Fig. 1). The amount of crystals was 70 – 100/division magnified 400 . The lengths ranged from 10 to 150 Am. The diameters ranged from 3 to 5 Am. The end of the crystals
was sharp. Several crystals gathered and formed a pillar mass. No crystals were observed in the normal urine (6/6). 3.2. The effect of LP-805 urine infusion on the micturition reflex in normal rats In single CMG with saline infusion (control study) (Fig. 2A), the latency was 1.2 F 0.3 min, the threshold pressure was 10.2 F 0.8 cm H20, the peak amplitude of bladder contraction was 25.8 F 3.7 cm H20, the voiding volume
Fig. 1. Crystal(s) in the urine from LP-805-treated rats (LP-805 urine). Magnification: 25 (A), and 150 (B, C).
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was 0.05 F 0.01 ml, and the residual urine volume was 0.02 F 0.01 ml. When the infusion solution was changed to LP-805 urine (Fig. 2B), the latency was reduced to 0.7 F 0.4 min (57.6 F 2.1% of control, p < 0.02) (Fig. 2C). The voiding volume was reduced to 0.03 F 0.01 ml (52.1 F3.6% of control, p < 0.02) (Fig. 2D). There was no significant difference in the threshold pressure between the control and LP-805 urine (10.8 F 0.6 cm H 2 0, 99.5 F 4.1% of control). Similarly, the peak amplitude of bladder contraction between the control and LP-805 urine (26.4 F 2.8 cm H20, 102.4 F 5.2% of the control), and the residual urine (0.02 F 0.01 ml, 101.3 F 2.6% of control) were not significantly different. In continuous CMG with saline infusion (control study) (Fig. 3A), the latency was 2.8 F 0.9 min, the threshold pressure was 9.8 F 1.1 cm H20, the peak amplitude of bladder contraction was 29.1 F 4.2 cm H 2 0, and the voiding volume was 0.2 F 0.05 ml. When the infusion solution was changed to LP-805 urine (Fig. 3B), the latency was reduced to
Fig. 4. Examples of single CMG with saline infusion (A) and with 0.1% acetic acid infusion (B) in LP-805-treated rats (n = 6). The arrow head indicated the start of infusion. The bar graphs summarize single CMG data for the latency (C) and the voiding urine volume (D). The effects of 0.1% acetic acid were compared to the control using the Mann – Whitney U-test (no significant difference).
1.4 F 0.4 min, (51.4 F 0.9% of control, p < 0.02) (Fig. 3D). The voiding volume was reduced to 0.12 F 0.02 ml (62.5 F 0.8% of control, p < 0.02). There were no significant differences in the threshold pressure (9.5 F 0.9 cm H20, 96.9 F 0.3% of control), or the peak amplitude of bladder contraction (28.7 F 5.2 cm H20, 97.6 F 0.6% of control) (Fig. 3E). When the infusion solution was changed to saline from LP-805 urine, the duration before bladder contraction and the voiding volume recovered to the control levels (2.5 F 0.6 min and 0.2 F 0.06 ml, respectively) (Fig. 3C). 3.3. The effects of acetic acid infusion on the micturition reflex in LP-805-treated rats There were no significant differences between saline and 0.1% acetic acid in the latency (4.1 F 0.6 and 4.7 F 0.6 min), the threshold pressure (8.2 F 0.4 and 7.1 F 0.5 cm H20), the peak amplitude of bladder contraction (24.8 F 0.3 and 25.1 F 0.8 cm H20), the voiding volume (0.24 F 0.02 and 0.26 F 0.03 ml), or the residual urine volume (0.01 F 0.02 and 0.02 F 0.01 ml) (Fig. 4). 3.4. The effects of MK-801 on the micturition reflex in LP805-treated rats
Fig. 3. Examples of continuous CMG with saline infusion (A), and with LP-805 urine infusion (B) and saline infusion recovery (C) in normal rats (n = 9). The bar graphs summarize continuous CMG data for the latency (D) and the voiding urine volume (E). The effects of LP-805 urine were compared to the control using the Mann – Whitney U-test (**p < 0.02).
The peak amplitude of bladder contraction was reduced in a dose-dependent manner after the administration of increasing doses of MK-801 (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1 mg/kg i.v.) in normal rats (ID50 = 0.56 mg/kg i.v., 105.6 F 1.2%, 102.5 F 0.8%, 97.8 F 9.6%, 98.6 F 11.2%, 63.9 F 9.3%, 52.1 F 5.7%, 39.4 F 3.8%, respectively) and
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in LP-805-treated rats (ID50 = 0.03 mg/kg i.v., 98.0 F 0.6%, 95.8 F 1.4%, 83.3 F 2.1%, 60.3 F 8.2%, 40.6 F 11.5%, 31.8 F 2.2%, 26.8 F 2.8%, respectively) (Fig. 5A). There were significant differences between the normal and LP805-treated rats at 0.03 mg/kg i.v. ( p < 0.05) and 0.3 mg/ kg i.v. ( p < 0.05). The volume threshold of the micturition reflex increased in a dose-dependent manner after the administration of increasing doses of MK-801 (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1 mg/kg i.v.) in normal rats (101.2 F7.7%, 86.7 F12.4%, 87.5 F12.1%, 11 2 . 3 F 1 0 . 3 % , 1 0 5 . 1 F 11 . 7 % , 1 9 8 . 3 F 4 2 . 4 % , 253.8 F 38.2%, respectively) and in LP-805-treated rats (105.4 F 10.2%, 122.5 F 20.8%, 125.8 F 14.6%, 255.2 F 79.4%, 390.2 F 84.3%, 439.1 F 75.7%, 462.4 F 90.8%, respectively) (Fig. 5B). There were significant differences between the normal and LP-805-treated rats at 0.01 mg/kg i.v. ( p < 0.05), 0.1 mg/kg i.v. ( p < 0.05) and 0.3 mg/kg i.v. ( p < 0.05).
Fig. 5. Log dose – response curves showing the effects of increasing MK801 during continuous CMG in normal rats (n = 4) (o) and in LP-805treated rats (n = 9) (E) on the peak amplitude of bladder contraction (A) and the volume threshold (B). The effects of MK-801 (i.v.) on LP-805treated rats were compared to those on normal rats using Student’s nonpaired t-test (*p < 0.05).
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4. Discussion Intravesical administration of LP-805 urine induced hyperreflexia of the micturition reflex in normal rats. Urinary pH, mechanical stimulation, and chemical irritants have been proposed as important factors for the facilitation of the micturition reflex (Ha¨bler et al., 1988, 1990; Morrison, 1999). The pH of LP-805 urine showed no difference to normal urine. As indicated in Fig. 1, many crystals were observed in the LP-805 urine. We hypothesize that the crystals were causing the hyperreflexia of the micturition reflex. Chemical analysis indicated few LP-805 metabolites in the urine and LP-805 might produce the crystals (unpublished observation). Since the hyperreflexia of the micturition reflex was recovered to the control level when the LP805 urine was removed, the crystals were major irritants causing the hyperreflexia of the micturition reflex. It has been reported that chemical irritants produced hyperreflexia of the micturition reflex (de Groat et al., 1993; Morrison, 1999). The crystals might cause the production and release of chemical irritants in the urinary bladder. In the present study, the latency was 1.2 F 0.5 min in normal rats infused with saline, but this was shorter than in LP-805-treated rats infused with saline (4.1 F 0.6 min). Similarly, the voiding volume of normal rats infused with saline (0.05 F 0.01 ml) was smaller than that of LP-805treated rats infused with saline (0.24 F 0.02 ml). These data suggest that chronic LP-805-treated animals have a larger volume threshold and longer duration before bladder contraction than normal animals. In contrast, intravesical administration of LP-805 urine reduced the latency (57.6 F 2.1% of control) and the voiding volume (52.1 F 3.6% of control) in normal rats. Similar data were observed for intravesical administration of chemical irritants. The latency and the voiding volume were initially reduced (Birder and de Groat, 1992b; McMahon, 1988), and these parameters were enhanced or undetectable several hours later. Histological experiments revealed that severe inflammatory changes had occurred in the xylene or acetic acid-treated bladder tissues (Maggi et al., 1988; Birder and de Groat, 1992b). In LP-805-treated rats, intravesical administration of acetic acid did not elicit hyperreflexia of the micturition reflex. This result might be due to neuronal activity elicited by intravesical administration of acetic acid that was already activated by chronic irritation of the bladder epithelium. Patch clamp studies revealed that the threshold for action potentials of dorsal root ganglia cells in LP-805-treated rats was lower than the threshold in normal rats (Imaizumi et al., 1997). An increase in primary afferent neuronal activity following bladder inflammation has been described (Ha¨bler et al., 1988, 1990). In addition to this observation, central sensitization of the dorsal horn neurons during urinary bladder inflammation has been proposed (McMahon, 1988). The cyclophosphamide (CPA)-induced cystitis model is well established as a chronic chemical cystitis model and is useful for the investigation of chronic visceral pain (Lante´ri-
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Minet et al., 1995; Ishigooka et al., 2001). After CPA administration, the volume threshold was reduced and micturition frequency was increased (Lecci et al., 1994; Ozawa et al., 1999). Patch clamp studies revealed that the threshold for action potentials of the dorsal root ganglia cells in CPA-treated rats was lower than the threshold in normal rats (Yoshimura and de Groat, 1999). These results were similar to our data. The present study indicates that LP-805treated rats are invaluable for the investigation of chronic irritation of the bladder epithelium. The present findings showed that MK-801 exerted an inhibitory effect on the micturition reflex in normal and/or LP-805-treated rats. Glutamate has been widely accepted as the major excitatory transmitter in the micturition reflex. Glutamate or its analogues injected into certain brain stem areas facilitated bladder activity in the cat and rat (de Groat et al., 1993). Administration of NMDA receptor antagonists inhibited the micturition reflex (Maggi et al., 1990; Yoshiyama et al., 1991, 1993a,b, 1994, 1995). Intrathecal administration of NMDA receptor antagonists reduced the amplitude of the action potentials in the dorsal part of the rostral pons, which were evoked by pelvic nerve stimulation (Kakizaki et al., 1998). Intravenous administration of NMDA receptor antagonists blocked the bladder contractions elicited by pontine micturition center stimulation (Matsumoto et al., 1995). These studies indicate that NMDA glutamatergic excitatory transmission is involved in the ascending and descending limbs of the spinobulbospinal micturition reflex. The present study demonstrated that MK-801 reduced the peak amplitude of bladder contraction and increased the volume threshold in normal rats. The effects were first detected with 0.03 mg/kg i.v. of MK-801 and the effects were increased in a dose-dependent manner. Complete abolishment of the micturition reflex was observed at 1 mg/kg i.v. of MK-801. These effective doses are very similar to the previous investigations (Maggi et al., 1990; Yoshiyama et al., 1991, 1993a,b). In LP-805-treated rats, MK-801 inhibited the micturition reflex in a similar manner to normal rats. NMDA receptor antagonists have been reported to block chronic cystitis. (Me´en et al., 2002). The ID50 in normal rats was 0.56 mg/ kg i.v., and that in LP-805-treated rats was 0.03 mg/kg i.v. This might indicate that lower doses of MK-801 have more effect on the micturition reflex in LP-805-treated rats. The volume threshold in LP-805-treated rats increased more than that in normal rats with the same dose of MK-801 i.v. These findings suggested that the more afferent inputs in LP-805-treated rats might need to evoke micturition reflex with MK-801 i.v. Thus, in LP-805-treated rats, NMDA glutamatergic excitatory transmission might be more important in micturition reflex. There is considerable evidence that spinal NMDA receptors are involved in central sensitization following somatic noxious stimuli (Coderre et al., 1993; McMahon et al., 1993; Neugebauer et al., 1994). We have provided evidence
that spinal NMDA receptors have a similar role following chronic visceral irritation. We suggest that spinal NMDA receptors should be explored for therapeutic potential with respect to conditions such as interstitial cystitis.
5. Uncited reference Williams, 1976
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