Effects of opioid subtypes on detrusor overactivity in rats with cerebral infarction
Osamu Yokoyama md, phd, Department of Urology, University of Fukui, 23-3 Matsuokashimoaizuki, Eiheiji-cho, Yoshida-gun, Fukui 910-1193, Japan. Email: email@example.com
Aim: In order to determine the influence of different opioid receptor subtypes on detrusor overactivity after left middle cerebral artery (MCA) occlusion, cystometric recordings were obtained in conscious rats.
Methods: Female Sprague-Dawley rats were used in this study. Control cystometrography was followed by left MCA occlusion. The sham-operated (SO) rats underwent the same procedures except for MCA occlusion. [D-Ala2, Phe4, Gly5]-enkephalin (DAGO; μ-opioid agonist), [D-Pen2,5]-enkephalin (DPDPE; δ1-opioid agonist), deltorpin II (δ2-opioid agonist), and U-50488 (κ-opioid agonist) were administered intracerebroventricularly at graded doses. The bladder capacity, residual volume, micturition threshold pressure, and bladder contraction pressure were determined. Finally, the volume of the infarction was measured.
Results: The intracerebroventricular administration of DAGO and DPDPE significantly increased the bladder capacity in the cerebrally infarcted (CI) and SO rats, but differences in the changes in bladder capacity between the CI and SO rats were not significant. Deltorpin II did not produce any changes in the bladder capacity in the CI or SO rats at any dose examined. However, the intracerebroventricular administration of U-50488 significantly increased the bladder capacity in the CI rats but not in the SO rats. None of the drugs affected the residual volume, micturition threshold pressure or bladder contraction pressure at any dosage examined. The mean infarcted volumes were not significantly different from those in the vehicle-treated rats.
Conclusion: These results suggest that the opioid receptor subtypes, μ and δ1 in the brain, are related to the micturition reflex. Furthermore, the κ opioid agonist might be useful for the suppression of detrusor overactivity caused by cerebral infarction.
Damage to the neural circuitry in the forebrain can produce overactivity of the urinary bladder and urinary incontinence.1 Decerebration above the inferior colliculus has been shown to induce detrusor overactivity in cats.2 This overactivity has been attributed to the interruption of the inhibitory pathways from the forebrain to the micturition center in the brainstem (i.e. the pontine micturition center, PMC). However, the precise mechanisms, including the neurotransmitter systems, related to the development of detrusor overactivity are not known.
The opioid receptors mediate the analgesic and other pharmacological actions, such as respiratory and cardiovascular functions. However, opioid drugs are not currently used in the treatment of bladder overactivity because of their range of side-effects and because their actions are largely uncharacterized. Opioids exert their diverse physiological effects through three distinct membrane-bound receptor subtypes, mu, delta, and kappa, in the central nervous system and periphery. These three receptors are also differentially implicated in the mediation of various physiological and behavioral effects of opiates. In vitro and in vivo pharmacological studies have strongly substantiated a further subdivision of each of the receptor types into subtypes.3,4 The different receptors have diverse behavioral characteristics; for example, euphoria, physical dependence, and respiratory depression are mainly associated with the mu and delta receptors.5 In contrast, opioids acting on the kappa receptors produce dysphoric rather than euphoric effects, which limits their physical dependence liability.4
The spontaneous motor activity of the urinary bladder is known to be altered by opiates and opioid peptides. Within the central nervous system, opioid modulation of bladder activity can occur at the level of the brain or spinal cord. In this regard, supraspinal administration of morphine results in the sustained suppression of volume-induced bladder contractions; this effect is rapidly reversed or prevented by the opioid antagonist, naloxone.6,7 Further evidence suggests that the mu and delta opioid receptors are involved in the mediation of this inhibitory response at both supraspinal8 and at spinal9 sites. In contrast, previous studies indicated that urinary bladder activity is not influenced by the stimulation of kappa opiate receptors,7 but in recent years, it was found that kappa agonists injected into the sacral level in cats inhibited bladder contractions due to the stimulation of the sacral dorsal root.10
It remains unclear whether different opioid subtypes result in the suppression of detrusor overactivity after left middle cerebral artery (MCA) occlusion. Therefore, we evaluated the effects of different opioid subtypes on detrusor overactivity caused by left MCA occlusion.
In this study, we used 76 female Sprague-Dawley rats (Japan SLC, Hamamatsu, Japan) weighing between 200 and 270 g. All experiments were performed in compliance with the guidelines of the Institutional Animal Care and Use Committee of the University of Kanazawa, Japan.
Surgical procedures for cystometrography, catheter implantation, and intracerebroventricular drug administration
We adopted the method of Yaksh et al. for cystometrography (CMG) in conscious rats.11 To implant the bladder catheter, the animals were anaesthetized with 1.5% halothane and the bladder was exposed via a midline incision in the abdomen. The bladder end of a size 4 polyethylene catheter with an internal diameter (ID) and outer diameter (OD) of 0.8 and 1.3 mm, respectively (Kunii Company, Tokyo, Japan) was softened by heating to create a collar and passed through a small incision at the apex of the bladder dome, after which a suture was tightened around the collar of the catheter. The other end of the catheter was passed through the subcutaneous tissue and turned through the skin at the back of the neck. After suturing the abdominal skin, the rats were positioned in a stereotactic frame (ST-7; Narishige Company, Tokyo, Japan). A scalp incision was made over the sagittal suture and a hole of ≈ 1.0 mm in diameter was drilled in the right parietal bone to expose the dural surface 1.0 mm lateral and 0.3 mm anterior from the bregma. A sterile, stainless steel cannula (OD = 0.6 mm, ID = 0.3 mm, length = 10.5 mm) was lowered 5.3 mm ventrally from the bregma with a micromanipulator. Using a small screw placed in the skull as an anchor, the cannula was fixed to the skull with dental acrylic. A sterile obturator (OD = 0.3 mm) was inserted into the cannula to maintain patency.
After suturing the scalp, the rats were placed in a restraining cage (Ballman KN-326 Type 3; Natsume Seisakusho Company, Tokyo, Japan) that was large enough to permit them to adopt a normal crouched posture, but narrow enough to prevent them from turning around. The cystometrography catheter was then connected to a pump (TE-311; Terumo Company, Tokyo, Japan) for continuous saline infusion and to a pressure transducer (TP-200T; Nihon-Kohden Company, Tokyo, Japan) by a polyethylene T-tube. The CMG was recorded by infusing physiological saline into the bladder at 0.04 mL/min, followed by the collection and measurement of saline voided from the urethral meatus to determine the voided volume. Evacuating the bladder through the CMG catheter enabled us to measure the postvoided residual urine volume after the micturition reflex. Four CMG parameters were determined from each CMG: (i) bladder capacity (sum of the voided and postvoided residual urine volume); (ii) residual urine volume; (iii) micturition threshold pressure (bladder pressure immediately prior to micturition); and (iv) bladder contraction pressure (the maximum bladder pressure during micturition). The bladder capacity of each rat before drug administration was assumed to represent 100% for that rat. The postadministration volume was expressed as a percentage of the preadministration volume and the micturition threshold pressure and bladder contraction pressure were expressed similarly.
Induction of cerebral infarction in rats
Cystometry was performed before occlusion of the left MCA in the unanaesthetized rats. Two hours after the CMG catheter implantation, the rats were anaesthetized with 1% halothane and the left carotid bifurcation was exposed through a midline incision in the neck. The left common carotid artery was then occluded and the branches of the left external carotid artery were dissected and divided. This was followed by the isolation and careful separation of the left internal carotid artery from the adjacent vagus nerve. The left pterygopalatine branch was identified and ligated close to its origin. A 4–0 monofilament nylon thread with the tip rounded by exposure to a flame was introduced into the left internal carotid artery and advanced 17 mm from the carotid bifurcation as far as the origin of the left MCA, where it occluded the blood flow and thus induced infarction on the left side of the brain. In the sham-operated (SO) rats, the left carotid bifurcation was exposed through a middle incision in the neck and the common and external carotid arteries were occluded. No further procedures were performed. All surgical procedures were performed within 30 min after induction of the halothane anaesthesia. After suturing the neck incision, the rats were placed in a restraining cage and allowed to recover from the halothane anaesthesia. Cystometry was again recorded after the recovery of consciousness was confirmed.
Evaluation of the effects of drugs
Two hours after the MCA occlusion or the sham operation and after control CMG recording, we examined the effects on bladder activity. A single drug was administered intracerebroventricularly to conscious rats at graded doses of 0.1–1000 ng. As the vehicle, we used artificial cerebrospinal fluid, composed of 138.6 nmol/L sodium chloride, 3.35 nmol/L potassium chloride, 1.26 nmol/L calcium chloride, 1.16 nmol/L magnesium chloride, and 11.9 nmol/L sodium hydroxide, with a pH of 7.0–7.2. The drugs were administered intracerebroventricularly in a single volume of 1 μL in the conscious rats. The drugs used in this study were [D-Ala2, Phe4, Gly5]-enkephalin (DAGO, μ-agonist; Research Biochemicals International, Natick, MA, USA), [D-Pen2,5]-enkephalin (DPDPE, δ1-agonist; Research Biochemicals International, Natick, MA, USA), deltorpin II (δ2-agonist; Research Biochemicals International, Natick, MA, USA), and U-50488 (κ-agonist; Research Biochemicals International, Natick, MA, USA). All drugs were dissolved in the artificial cerebrospinal fluid for intracerebroventricular administration. The drug dosage was increased at 60-min intervals. The bladder capacity, residual urine volume, micturition threshold pressure, and bladder contraction pressure were determined from each of the CMGs.
Evaluation of the corrected infarction volume
After evaluation of the effects of the drugs, the rat brain was stained by perfusion with 2% 2,3,5-triphenylterazolium chloride under urethane anaesthesia (TTC; Sigma, St Louis, MO, USA). We performed a thoracotomy, inserted a catheter into the ascending aorta via the left ventricle, perfused with heparinized saline, and incised the right atrium. After 2 min, the right atrium was clamped and 2% TTC in saline was infused over a period of 7 min. After completion of the perfusion, the brains were removed and the cerebral hemispheres were cut into five coronal slices, each 2 mm thick. The rostral surface of the TTC-stained sections was photographed with color slide film and the infarction volume was measured according to the method of Golanov and Reis.12
The data are expressed as the mean ± standard error of the mean. The statistical comparisons were performed by two-way repeated measures analysis of variance (anova) with subsequent individual comparisons by Fisher's protected least significant difference test. A level of P < 0.05 was considered to be statistically significant.
Effect of left middle cerebral artery occlusion on cystometrography
Just after the MCA occlusion, the rats showed the characteristic motor changes in which the forelimb contra-lateral to the side of the MCA occlusion was consistently flexed during suspension. The mean bladder capacity was 0.46 ± 0.07 mL and 0.49 ± 0.08 mL before and 2 h after the sham operation, respectively, indicating that the sham operation did not significantly alter bladder capacity. However, the mean bladder capacity was significantly reduced from 0.43 ± 0.08 mL to 0.20 ± 0.07 mL 2 h after the MCA occlusion (P < 0.01) and it remained consistently <0.35 mL for 10 h after occlusion. As the residual urine volumes in the cerebral infarction and sham operation groups were very small and insignificant, the micturition volume was almost equivalent to bladder capacity.
The mean micturition threshold pressure was 5.9 ± 0.6 cmH2O and 6.1 ± 0.7 cmH2O before and 2 h after the sham operation, respectively, compared with 5.4 ± 0.5 cmH2O and 6.8 ± 0.8 cmH2O before and 2 h after occlusion, respectively. The cerebral infarction group also showed a significant increase in the micturition threshold pressure (P < 0.01). However, the micturition threshold pressure of the SO rats was unaffected.
The bladder contraction pressure was 30.6 ± 8.4 cmH2O and 32.0 ± 9.0 cmH2O before and 2 h after the sham operation, respectively, compared with 28.4 ± 9.4 cmH2O and 27.7 ± 6.7 cmH2O before and 2 h after occlusion, respectively. Therefore, the bladder contraction pressure was not significantly affected by the left MCA occlusion.
Effects of intracerebroventricular administration of drugs on cystometrography
The bladder capacity, bladder contraction pressure, and micturition threshold pressure before opioid or vehicle administration served as the control values. The percentage changes in these parameters were calculated with respect to these values and plotted in Figures 1–4. In both the sham operation and cerebral infarction groups, the intracerebroventricular administration of DAGO increased bladder capacity in a dose-dependent manner (Fig. 1). Lower doses of DAGO (0.1 and 1 ng) did not affect the bladder capacity compared with the vehicle-treated rats. However, higher doses of DAGO (10, 100, and 1000 ng) increased the bladder capacity in a dose-dependent manner in both groups (Fig. 1). The effects of DAGO on bladder capacity were not significantly different in the sham operation and cerebral infarction groups. At all the doses examined, DAGO produced small and insignificant increases in the postvoided residual urine volume in the sham operation and cerebral infarction groups (data not shown). At all the doses examined, DAGO produced insignificant increases in the bladder contraction pressure and micturition threshold pressure.
In both the sham operation and cerebral infarction groups, lower doses of DPDPE (0.1 and 1 ng) did not alter the bladder capacity compared with the vehicle-treated rats. However, higher doses of DPDPE (100 and 1000 ng) increased bladder capacity in a dose-dependent manner in both groups. But, the percentage increase in the bladder capacity in the cerebral infarction group at 10 ng of DPDPE was 84.4 ± 42.5%, which was significantly higher than that for the SO controls (P < 0.05). At all the doses examined, DPDPE produced insignificant increases in the bladder contraction pressure and micturition threshold pressure (Fig. 2).
In both the sham operation and cerebral infarction groups, deltorpin II did not significantly increase the bladder capacity compared with the vehicle alone and the effects of deltorpin II on the bladder contraction pressure and micturition threshold pressure did not differ from those in the vehicle-treated rats (Fig. 3).
In the SO rats, U-50488 did not significantly increase the bladder capacity compared with the vehicle and the effects of U-50488 on the bladder contraction pressure and micturition threshold pressure in the SO rats did not differ from those in the vehicle-treated rats. U-50488 increased the bladder capacity in a dose-dependent manner in the cerebral infarction group (Fig. 4). The bladder capacity increased just after administration of 10, 100, and 1000 ng of U-50488, while the dose of 1000 ng induced the maximum response in the cerebral infarction group. At all the doses examined, U-50488 produced insignificant increases in the postvoided residual urine volume (data not shown). Furthermore, the effects of U-50488 on the bladder contraction pressure and micturition threshold pressure in the cerebral infarction group did not differ from those of the vehicle alone.
The intracerebroventricular administration of opioid receptor subtypes did not have any effect on hemiparesis.
Influence of opioids on cerebral infarction volume
All of the rats used in this study had an infarction in the frontoparietal cortex and subcortical basal ganglia. The mean infarction volumes (DAGO: 202.1 ± 10.4 mm3; DPDPE: 201.9 ± 15.7 mm3; deltorpin II: 207.0 ± 12.9 mm3; U-50488: 203.1 ± 15.2 mm3) were not significantly different from those of the vehicle-treated rats (209.6 ± 13.2 mm3). No evidence of infarction was found in any of the SO rats.
Pharmacological investigations indicate that various neurotransmitters are involved in the central nervous system's control of micturition.13 Nishijima et al. reported that the glutamate level decreased in the cerebrum of the rats, while the glycine level decreased in the brainstem, cervicothoracic cord, and lumbosacral cord in cerebrally infarcted (CI) rats.14 The present study evaluated the contribution of opioidergic mechanisms to detrusor overactivity in conscious rats caused by cerebral infarction. Various pharmacological studies using intravenous, intrathecal, or intracerebroventricular administration of opioid agonists and antagonists suggested that opioids might be involved in the modulation of the micturition reflex.8,13 Intracerebroventricular or intrathecal administration of the highly selective µ-agonist, DAGO, and the selective δ1-receptor agonist, DPDPE, resulted in the suppression of bladder activity. Furthermore, the inhibitory effect of DPDPE, but not DAGO, was antagonized by prior administration of the selective delta antagonist, ICI 174 864.9 These findings suggest that distinct mu and delta receptors are involved in the modulation of bladder activity initiated at supraspinal and spinal sites. Furthermore, studies using several different types of opiate drugs indicated that delta opiate receptors are primarily responsible for opiate inhibition of micturition reflexes in the spinal cord, whereas mu and delta opiate receptors mediate inhibition in the brain.8,13 Based on the distributions of various dyes after intracerebroventricular and intracisternal administration of opiate drugs, it appears that supraspinal sites responsible for opioid-induced inhibition of micturition are located in the periventricular hindbrain15 and that the PMC is the most likely site for a periventricular hindbrain opioid action influencing micturition.16 We also evaluated the effects of DAGO, DPDPE, and deltorpin II on bladder activity in conscious SO and CI rats. The results of the present study indicated that selective opioid receptor agonists, such as DAGO and DPDPE, consistently inhibit bladder activity in SO rats, consistent with previous observations. The effects of DAGO and DPDPE on the micturition reflex in rats with cerebral infarction were similar to those in the SO rats. However, the δ2-agonist, deltorpin II, had no effect on bladder activity in the sham operation or cerebral infarction groups. These results indicate that urinary bladder activity is not influenced by stimulation of the δ2-opioid receptors, while the inhibition of the micturition reflex by delta opioid receptors is mainly controlled by δ1-opioid receptors.
It was reported that the kappa opioid receptors in the spinal cord were more important than those located supraspinally in relation to the antinociceptive effect of kappa agonists administered systemically.17 In recent years, it was found that the κ-agonist, U-50488, injected into the sacral level of cats, inhibited bladder contraction due to stimulation of the sacral dorsal root.10 Furthermore, Gotoh et al. reported that U-50488 inhibited the micturition reflex as well as other opioids and that the inhibition was due, at least in part, to the reduction of bladder sensation based on the activation of the descending monoaminergic systems through the spinal kappa opioid receptors.18 However, there have been no previous reports that supraspinal kappa opioid receptors mediate the micturition reflex. In our study, U-50488 was ineffective on bladder activity in SO rats, but increased the bladder capacity in rats with cerebral infarction. These differences in response to opioidergic agents between the sham operation and cerebral infarction groups seem to be related to the change in kappa opioid systems in the brain. The kappa opioid system in the brain is believed to exert a tonic inhibitory influence on the PMC. The down-regulation of these systems by cerebral infarction might increase the sensitivity of kappa agonists to the brain, influencing bladder activity. For this reason, it seems reasonable to speculate that cerebral infarction causes changes in kappa opioidergic mechanisms in the brain, resulting in detrusor overactivity.
The available opioid agonists relieve pain, but they usually have many adverse effects, such as constipation and central side-effects, including opioid dependence. Such adverse effects preclude their general use in clinical practise. In contrast, kappa opioid receptor agonists seem to be devoid of these side-effects. Tsuji et al. reported that selective kappa agonists are opioids that do not result in psychological dependence and their clinical application as analgesics is expected.19 Delgado-Aros et al. reported that asimadoline, a kappa agonist, increased acute nutrient intake and appeared to be safe in a clinical study.20
In conclusion, based on the increased sensitivity of infarcted rats to U-50488, it seems reasonable to conclude that this pathway is down-regulated after cerebral infarction and might contribute to the detrusor overactivity in rats with cerebral infarction. The κ-opioid agonist, U-50488, might be useful for the suppression of detrusor overactivity caused by cerebral infarction.