Neurourology and Urodynamics 35:464–470 (2016)
Spinal Neuronal Cannabinoid Receptors Mediate Urodynamic Effects of Systemic Fatty Acid Amide Hydrolase (FAAH) Inhibition in Rats € llhase,1,2* Andrea Schreiber,2,3 Armin Giese,4 Michael Schmidt,3 Francesco Montorsi,5,6 Claudius Fu Christian Gratzke,3 Giovanni La Croce,5,7 Fabio Castiglione,6,7 Christian Stief,3 and Petter Hedlund5,8 1
Department of Urology, University of Rostock, Rostock, Germany Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians-University, Munich, Germany 3 Department of Urology, Ludwig-Maximilians-University, Munich, Germany 4 Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Munich, Germany 5 Division of Oncology/Unit of Urology, Urological Research Institute, IRCCS Ospedale San Raffaele, Milan, Italy 6 Universita Vita San Raffaele, Milan, Italy 7 Department of Clinical and Experimental Pharmacology, Lund University, Lund, Sweden 8 Department of Clinical Pharmacology, Linko€ping University, Linko€ping, Sweden 2
Aims: To test if urodynamic effects from systemic Fatty Acid Amide Hydrolase (FAAH) inhibition involve sacral spinal cannabinoid type 1 (CB1) or type 2 (CB2) receptors. Methods: Male rats with or without partial urethral obstruction were used for cystometry or immunohistochemistry. Urodynamic effects of intravenous (IV) 0.3 mg/kg Oleoyl Ethyl Amide (OEtA; FAAH inhibitor), and intrathecal (IT) 5 mg rimonabant (CB1 antagonist) or 5 mg SR144528 (CB2 antagonist) were studied in awake rats. Results: After administration of rimonabant or SR144528, non-obstructed rats with normal bladder function developed bladder overactivity (BO), which was counteracted by OEtA. OEtA also counteracted BO in obstructed rats. SR144528 did not affect bladder function in obstructed rats but counteracted the urodynamic effects of OEtA. Surprisingly, rimonabant (and AM251, another CB1 antagonist) reduced BO in obstructed rats, whereafter OEtA produced no additional urodynamic effects. CB1 expression increased in the sacral spinal cord of obstructed rats whereas no changes were observed for CB2 or FAAH. Conclusions: Urodynamic effects of systemic FAAH inhibition involve activities at spinal neuronal CB1 and CB2 receptors in normal and obstructed rats. Endogenous spinal CB receptor ligands seem to regulate normal micturition and BO. Altered spinal CB receptor functions may be involved in the pathogenesis of obstruction-induced BO. Neurourol. Urodynam. 35:464–470, 2016. # 2015 Wiley Periodicals, Inc. Key words: AM251; bladder; endocannabinoid; obstruction; oleoyl ethyl amide; overactivity; rimonabant; SR144528
INTRODUCTION
The endocannabinoid system represents a putative drugtarget to treat bladder dysfunctions.1 Cannabinoid receptor agonists reduced bladder overactivity (BO) in various models and relieved bladder symptoms in humans.1 However, psychotropic effects may accompany the therapeutic benefits of exogenous cannabinoids.1,2 By contrast, endocannabinoids are produced ‘‘on demand’’ inside the body, and inhibiting their breakdown only affects cells with ongoing endocannabinoid activity.1,2 Fatty acid amide hydrolase (FAAH), which degrades endocannabinoids and fatty acid amides, is present in the bladder and micturition control centers.1,3–5 Both acute or chronic FAAH inhibition reduced micturition frequency more in rats with partial urethral obstruction (PUO), prostaglandin E2-induced BO, or in spontaneously hypertensive rats with BO than in controls.3,6,7 Intrathecal (IT) administration of the FAAH inhibitor Oleoyl Ethyl Amide (OEtA) produced urodynamic effects in normal and obstructed rats.3 Considering that IT drug delivery tests whether a drug acts on nerves in general, effects may be either on proximal axonal nerve branches that supply the central nervous system inside the blood–brain barrier (BBB), or on distal axonal nerve branches that innervate the peripheral nervous system outside of the BBB.8 Aizawa and colleagues provided further evidence that the urodynamic effects of #
2015 Wiley Periodicals, Inc.
FAAH inhibition are based on a neuronal site of action: URB937, a FAAH inhibitor that cannot cross the BBB, was shown to reduce afferent nerve firing of primary bladder afferent Ad- and C-fibers.9 It is not known if the urodynamic effects of FAAH inhibitors, which seem to be at least partially mediated via neuronal sites of action, are based on effects of endocannabinoids on neuronal CB type 1 (CB1) or CB type 2 (CB2) receptors. This study aimed to assess if urodynamic effects of intravenous OEtA, which passes the BBB, are inhibited by IT CB1 or CB2 antagonists.
[This article was corrected on 2 April 2015 after initial online publication. The family and given names of the authors were inverted.] Lori Birder led the peer-review process as the Associate Editor responsible for the paper. Potential conflicts of interest: Nothing to disclose. Grant sponsor: German Research Foundation (DFG); Grant number: GR3333/2-1; Grant sponsor: Gester Foundation, Sweden Correspondence to: Claudius F€ ullhase, M.D., Ph.D., Department of Urology, University of Rostock, Ernst-Heydemann-Street 6, 18055 Rostock, Germany. E-mail: [email protected] Received 24 November 2014; Accepted 21 January 2015 Published online 18 March 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/nau.22753
Spinal Cannabinoid Receptors and FAAH in Rat Bladder Dysfunction MATERIALS AND METHODS Animals
Male Sprague–Dawley rats (n ¼ 90) were housed at the Walter Brendel Centre of Experimental Medicine, Munich, or at the San Raffaele Scientific Institute, Milan. All experiments were approved by the local Animal Ethics Committees. Animals were kept and housed according to standard conditions in a 12:12 light–dark cycle with food and water ad libidum. Thirty-eight rats served as controls. Following partial urethral obstruction (PUO; n ¼ 52), nine rats were euthanized because of acute urinary retention. After IT catheterization, seven rats were excluded because of paralysis. Three rats were excluded because of excessive movement artefacts. The final groups for cystometry included 32 PUO rats and 27 controls, and for immunohistochemistry, six PUO rats and six controls.
465
Saline dilutions were made on the day of experiment. After baseline cystometry (45 min), rimonabant, SR144528, or vehicle was injected intrathecally (5 mg in 10 ml) for 1 min, and cystometry continued for an additional 45 min. Animals then received OEtA (IV, 0.3 mg/kg), and cystometry continued for a further 45 min. Vehicles had no effect. Statistical Analysis
Results are given as mean values standard error of the mean (SEM). Student’s t-test was used for comparison between two groups. For comparisons within groups, one-way repeatedmeasures analysis of variance (ANOVA), followed by Student– Newman–Keuls test, were used. SigmaPlot 11.0 software (Systat, Inc., Chicago, IL) was used to analyze statistics. A value of P < 0.05 was considered statistically significant. RESULTS
Partial Urethral Obstruction (PUO) 10
PUO was performed as previously described. The urethra was accessed through a perineal incision, isolated from the corpora cavernosa, and partially ligated with a 3–0 suture. Rats were used in experiments 14 days later. Immunohistochemistry
Spinal cord tissue (L6–S2)11 was harvested, processed, and cut into transversal sections. Immunohistochemistry for FAAH, CB1, and CB2 receptors was performed on a Ventana Benchmark staining-machine (Ventana Medical Systems, AZ) using polyclonal antibodies for CB1, CB2, and FAAH (Cayman Chemical, Ann Arbor, MI) at dilutions of 1:50, 1:300, and 1:50. These antibodies have been characterized before.5,6 The percent area of the spinal cord occupied by stainings was evaluated in Image J, v.1.45s (NIH, Bethesda, MD). Functional Experiments
Bladder and IT catheterizations were performed as previously described.12,13 A PE-50 polyethylene catheter was inserted into the bladder via laparotomy and secured with a purse-string suture (5–0). The catheter was tunneled under the skin to the neck and anchored. Next, the atlanto–occipital membrane was exposed and punctured. A 32-gauge IT catheter (CS-1, ReCathCo, Allison Park, PA) was inserted and its tip positioned two fingers above the hip joints. Catheter locations (L6–S2)13 were confirmed during necropsy after experiments. Cystometry
Cystometries were performed 3 days after catheter implantations.12 The parameters investigated were Intermicturition Interval (IMI), Bladder Capacity (BCap ¼ infusion rate per minute IMI), micturition volume (MV), residual volume (RV ¼ BCap MV), basal bladder pressure (BP ¼ minimum bladder pressure between two micturitions), intermicturition pressure (IMP ¼ mean bladder pressure between two micturitions), threshold pressure (TP ¼ bladder pressure at onset of the micturition contraction curve/peak), maximum micturition pressure (MMP), spontaneous bladder activity (SA ¼ IMP – BP), and bladder compliance (BCom ¼ BCap/(TPBP). Drug Administration
OEtA, rimonabant (SR141716), AM251, and SR144528 (Cayman Chemical) were dissolved in DMSO and stored at 808C. Neurourology and Urodynamics DOI 10.1002/nau
Immunohistochemistry
No stainings were detected in the spinal cord white matter. The gray matter exhibited diffuse FAAH staining (Fig. 1). Intense CB2 staining (Fig. 1) was found in the outer laminae (substantia gelatinosa) of the dorsal horns. No clear difference in staining patterns for CB2 or FAAH was observed between PUO rats and non-obstructed controls. By contrast, increases in CB1 stainings were seen in PUO rats in the intermediolateral zone, the sacral parasympathetic nucleus, the dorsal horns, and the dorsal commissure (Figs. 1 and 2). Cerebellar cortex control tissue exhibited expected staining patterns for CB1, CB2, and FAAH (Fig. 1). Functional Experiments Effect of PUO. Compared to controls, PUO rats had a shorter IMI (61 4%) and correspondingly higher micturition frequency (MF), showed a reduced BCap (60 7%), MV (44 6%), and BCom (73 4%), had higher intravesical pressures (BP þ76 9%, IMP þ114 8%, TP þ68 7%, and MMP þ76 6%), and a higher SA (þ196 21%), all P < 0.05 (Table I). Obstructed rats’ bladders weighed more than controls’ (1178 55 mg vs. 449 93 mg, P < 0.05), despite the groups’ similar body weights. Drug effects in non-obstructed control rats. In controls, OEtA (IV) increased IMI (þ31 9%), BCap (31 8%), MV (þ29 7%), and BCom (þ25 9%), all P < 0.05, but produced no further urodynamic effects (Table II). Intrathecal SR144528 reduced IMI (17 9%), and BCap (19 6%), and increased BP (þ20 9%), all P < 0.05 (Group 2A). OEtA (IV) reversed SR144528 (IT) effects to baseline values (Group 2B). Intrathecal rimonabant reduced IMI (22 8%), BCap (19 6%), MV (18 6%), and BCom (29 8%), and increased BP (þ58 11%), IMP (þ46 14%), TP (þ33 9%), MMP (þ15 7%), and SA (þ72 22%), all P < 0.05 (Group 2C). OEtA (IV) reversed rimonabant (IT) effects to baseline values (Group 2C). Drug effects in PUO rats. In obstructed rats, OEtA (IV) increased IMI (þ59 8%), BCap (þ80 10%), MV (þ42 14%), and BCom (þ188 21%), and reduced IMP (33 5%), TP (29 8%), MMP (30 8%), and SA (75 9%), all P < 0.05 (Table III) (Group 3A). Intrathecal SR144528 produced no urodynamic effect (Group 3B). Administering OEtA (IV) after SR144528 (IT) had
466
€ llhase et al. Fu
Fig. 1. Immunohistochemistry. Representative photomicrographs of the distribution of CB1 receptor stainings in (A) cerebellar cortex control tissue, and sacral spinal cords of (B) non-obstructed control rats and rats with (C) partial urethral obstruction (PUO); CB2 stainings in (D) cerebellar cortex control tissue, and sacral spinal cords of (E) non-obstructed controls and rats with (F) PUO; stainings of FAAH in (G) cerebellar cortex control tissue, and sacral spinal cords of (H) non-obstructed controls and rats with (I) PUO. DH, dorsal horn; DC, dorsal commissure; SPN, sacral parasympathetic nucleus.
no additional effect besides increasing BCom (þ40 17%) (Group 3B). Intrathecal rimonabant increased IMI (þ39 16%), BCap (þ50 17%), MV (þ28 14%), and BCom (134 20%), and decreased BP (29 11%), IMP (44 6), TP (40 7%), MMP (20 7%), and SA (67 12%), all P < 0.05 (Group 3C). Administering OEtA (IV) after rimonabant (IT) had no additional effect (Group 3C).
and controls. In controls, IV rimonabant induced the same urodynamic changes as IT rimonabant (Tables II and V). Intravenous OEtA then reversed the changes induced by IV rimonabant similarly as it had with IT rimonabant. In obstructed animals with BO, IV rimonabant induced the same urodynamic changes as IT rimonabant (Tables III and V). When IV OEtA was given after IV rimonabant, no further effects were observed.
Extended investigations of CB1 receptor functions. To explore the different rimonabant results in PUO rats and controls, effects by another CB1 receptor antagonist, AM251, were studied in PUO rats. Here, IT administration of AM251 produced similar results as with rimonabant (Table IV). Furthermore, effects of IV rimonabant (0.3 mg/kg) prior to administering IV OEtA (0.3 mg/kg) were evaluated in PUO rats
This study shows that the urodynamic effects of systemic FAAH inhibition involve cannabinoid receptors in sacral spinal cord neurons. In control rats, IT delivery of CB1 or CB2 receptor antagonists prevented systemic FAAH inhibitor-induced changes in bladder function similarly. Interestingly, CB1 and
Neurourology and Urodynamics DOI 10.1002/nau
DISCUSSION
Spinal Cannabinoid Receptors and FAAH in Rat Bladder Dysfunction
467
Fig. 2. Analysis of stainings for CB1 immunohistochemistry in spinal cords from controls and rats with partial urethral obstruction (PUO). (A) Area occupied by CB1 stainings in the dorsal horns (DH); (B) area occupied by CB1 stainings in the sacral parasympathetic nucleus (SPN); (C) area occupied by CB1 stainings in the dorsal commissure (DC). Measurements were performed with Image J (n ¼ 4 for each group). Values are given as mean standard error of the mean for area occupied. Students t-test: **P < 0.01, ***P < 0.001.
CB2 receptor antagonists had per se effects in controls, suggesting that endogenous ligands at spinal neuron receptors regulate normal micturition. In obstructed rats, IT CB2, but not CB1, prevented the effects of IV OEtA, leading us to propose that neuronal CB2 receptors mediate urodynamic effects of systemic FAAH inhibitors in obstructed rats. The present urodynamic data and findings of increased expression of CB1 receptors in the sacral spinal cord areas of obstructed rats indicate that obstruction alters spinal neuronal CB1-receptor activity. In controls, rimonabant (IT) increased micturition frequency, bladder pressures, and spontaneous bladder activity, and decreased bladder capacity and micturition volume. Supporting these data, a role for the CB1 receptor in micturition has been proposed for CB1 knockout mice with increased micturition frequencies, and injecting rimonabant intraperitoneally in wild-type mice has been found to decrease IMI, BCap, and MV.14 Inversely, various CB1 agonists have increased IMI, BCap, and MV in different models.1 Similar to rimonabant, the CB2 receptor antagonist SR144528 (IT) decreased IMI and BCap in control rats, but did not affect intravesical pressures. AM630, another CB2 antagonist, has also been found to reduce IMI and BCap in normal mice.1 Other authors have reported that CB2 agonists increase IMI and BCap, further indicating a role for the CB2 receptor in micturition control.1,15–17 CB1- and CB2-receptor active compounds have been shown to affect bladder function. But this is, to the best of our knowledge, the first report describing the effects of IT CB1 or CB2 antagonists on bladder function that also suggests that spinal endogenous CB signals regulate normal micturition.
Supporting this, OEtA (IV) counteracted the urodynamic changes by IT delivery of rimonabant or SR144528. This effect of systemic OEtA administration suggests that FAAH substrates may act at neurons connected to the sacral spinal cord. Promoting the idea that one or more endogenous ligands tonically recruit neuronal CB1 or CB2 receptors to regulate bladder function, Aizawa et al. described that systemic administration of rimonabant or SR144528 per se modified the activities of primary bladder sensory-nerve fibers.9 This correlates well with the currently reported effects of both rimonabant and SR144528 on voiding frequency (as a surrogate parameter for sensory bladder functions), and may also relate to the current location of CB1 and CB2 in the sacral dorsal horns, which receive sensory information from the lower urinary tract (LUT).18 The effects by rimonabant on intravesical pressures and the histochemical location of CB1 in the sacral parasympathetic nucleus18,19 may reflect neuronal CB1-mediated regulation of bladder motor signals. Effects by IV OEtA on IMI, MV, and BCap were qualitatively similar in non-obstructed and obstructed rats, but effects were elevated 30–50% in the latter group. These findings correspond to previous reports of increased OEtA responses in BO models compared to normal animals, and suggest that FAAH substrates exhibit increased activities in bladder dysfunction.3,6,7 Unlike in control rats, OEtA also reduced bladder pressures and spontaneous bladder activity in obstructed rats. As IT SR144256 prevented effects by IV OEtA in obstructed rats, the CB2 receptor may play a critical role in mediating effects by FAAH substrates in PUO-induced BO.
TABLE I. Urodynamic Parameters of Non-Obstructed Control Rats Versus Obstructed Rats IMI (min)
BCap (ml)
MV (ml)
RV (ml)
BP (cmH2O)
IMP (cmH2O)
TP (cmH2O)
MMP (cmH2O)
SA (cmH2O)
BCom (ml/cmH2O)
Non-obstructed 9.12 0.32 1.5 0.1 1.6 0.1 0.0 0.0 4.7 0.2 7.2 0.3 14.2 0.4 42.7 1.7 2.4 0.2 0.162 0.007 (n ¼ 27) ** ** ** ** Obstructed 3.55 0.20 0.6 0.0 0.7 0.0 0.0 0.0 8.3 0.4 15.4 0.6** 23.9 1.0** 75.3 2.4** 7.1 0.5** 0.045 0.004** (n ¼ 32)
Bladder weight (mg) 449 93 1178 55**
IMI, intermicturition interval; BCap, bladder capacity; MV, micturition volume; RV, residual volume; BP, basal bladder pressure; IMP, intermicturition pressure; TP, threshold pressure; MMP, maximum micturition pressure; SA, spontaneous bladder activity; BCom, bladder compliance. Student’s t-test, *P < 0.05, **P < 0.001. Bold means significant.
Neurourology and Urodynamics DOI 10.1002/nau
468
€ llhase et al. Fu
TABLE II. Urodynamic Parameters of Non-Obstructed Rats (n ¼ 21) IMI (min) Group 2A (n ¼ 5) Baseline (before drug) 7.84 0.34 After IV OEtA 10.27 0.67* Group 2B (n ¼ 8) Baseline (before drug) 9.30 0.84 After IT SR144528 7.77 0.77* After IV OEtA 9.27 0.92** Group 2C (n ¼ 8) Baseline (before drug) 9.51 0.49 After IT rimonabant 7.51 0.36* After IV OEtA 9.55 0.31**
BCap (ml)
1.3 0.1 1.7 0.1
MV (ml)
*
1.4 0.1 1.8 0.1
1.6 0.1 1.6 0.1 1.3 0.1* 1.4 0.2 ** 1.5 0.2 1.6 0.2
RV (ml)
*
0.0 0.0 0.1 0.1
BP (cmH2O)
IMP (cmH2O)
TP (cmH2O)
MMP (cmH2O)
SA (cmH2O)
BCom (ml/cmH2O)
4.6 0.3 4.4 0.2
6.5 0.5 5.8 0.5
12.9 0.7 13.2 0.6
41.5 2.6 41.0 3.8
2.0 0.4 1.4 0.3
0.15 0.009 0.197 0.014*
7.4 0.5 7.9 0.6 7.2 0.6
13.8 0.9 14.9 1.1 15.9 1.7
41.4 3.1 43.4 3.2 43.2 3.9
2.9 0.4 0.165 0.015 3.4 0.4 0.143 0.017 ** 2.2 0.2 0.147 0.014
0.0 0.0 4.5 0.4 0.0 0.0 5.4 0.4* 0.0 0.0 4.6 0.5**
1.6 0.1 1.7 0.1 0.0 0.0 5.3 0.3 8.1 0.5 14.0 0.7 49.1 2.5 2.5 0.3 1.3 0.1* 1.4 0.1* 0.0 0.0 8.4 0.6* 11.8 1.1* 18.6 1.2* 56.2 3.6* 4.3 0.6* ** ** ** ** ** 1.6 0.1 1.6 0.1 0.1 0.0 5.3 0.4** 7.8 0.5 14.7 1.2 46.9 3.2 2.6 0.4
0.183 0.014 0.131 0.012* 0.176 0.015**
OEtA, oleoyl ethyl amide (FAAH inhibitor); IMI, intermicturition interval; BCap, bladder capacity; MV, micturition volume; RV, residual volume; BP, basal bladder pressure; IMP, intermicturition pressure; TP, threshold pressure; MMP, maximum micturition pressure; SA, spontaneous bladder activity; BCom, bladder compliance. Group 2A before (baseline) and after intravenous (IV) OEtA (0.3 mg/kg) administration. Group 2B before and after intrathecal (IT) administration of 5 mg of SR144528 (CB2 antagonist) and after IV OEtA administration (after previous SR144528 IT delivery), and group 2 C (lower rows) before and after IT administration of 5 mg of rimonabant (CB1 antagonist) and after OEtA (0.3 mg/kg, IV) after previous rimonabant (IT). One-way ANOVA of repeated measurements. Bold means significant. *
P < 0.05 versus baseline.
**
P < 0.05 versus intrathecal values (medium rows).
Based on our findings in non-obstructed rats, we expected IT rimonabant to either have no effect or to increase urodynamic signs of BO in obstructed rats. Surprisingly, IT rimonabant instead increased IMI and decreased bladder pressures. These results were verified by showing that IV rimonabant, and IT AM251 also caused similar urodynamic effects. Hence, the data suggest that CB1 receptors mediate different effects in normal bladder function and obstruction-induced BO. This in turn indicates that obstruction alters CB1-mediated signals somewhere in the pathways that regulate micturition. We previously described by Western blot that a 70% increase in CB1 receptor expression occurs in the sacral spinal cord of obstructed rats.3 In the present study, increased CB1 expression was found in
regions of the lumbosacral spinal cord known to contain neurons and interneurons that receive and relay information from LUT sensory dorsal-root ganglion cells.18 These findings form a morphological basis for CB1-receptor plasticity in altering processing of sensory signals from the obstructed LUT, and we speculate that this explains our contrasting results with rimonabant. Similarly, Martin et al. described in a rat painmodel that IT rimonabant differently affected nociception and spinal expression patterns of c-fos (a nerve-activity marker) depending on the presence of chronic inflammation.20 It was concluded that cannabinoids likely act at CB1 receptors pre- and postsynaptically to modulate spinal cord activity under different conditions.20 Analagously, Sales-Carbonell et al. determined that
TABLE III. Urodynamic Parameters of Obstructed Rats (n ¼ 21)
Group 3A (n ¼ 5) Baseline (before drug) After IV OEtA Group 3B (n ¼ 8) Baseline (before drug) After IT SR144528 After IV OEtA Group 3C (n ¼ 8) Baseline (before drug) After IT rimonabant After IV OEtA
BP (cmH2O)
IMP (cmH2O)
TP (cmH2O)
MMP (cmH2O)
SA (cmH2O)
BCom (ml/cmH2O)
0.0 0.0 0.0 0.0
10.4 0.7 10.3 0.6
17.1 0.9 12.0 0.5*
28.6 2.3 20.5 1.4*
92.4 7.0 65.5 6.1*
6.7 0.6 1.7 0.5*
0.033 0.007 0.095 0.016*
0.7 0.1 0.7 0.1 0.8 0.2
0.0 0.0 0.0 0.0 0.0 0.0
8.1 1.2 7.0 1.5 8.0 1.7
15.8 2.4 13.9 2.0 13.8 2.5
24.2 3.0 20.1 3.0 19.2 3.5
71.5 5.1 65.1 6.8 63.3 9.1
7.7 1.5 5.6 1.3 5.8 1.4
0.047 0.011 0.052 0.006 0.073 0.009*y
0.7 0.1 0.9 0.1* 0.8 0.1*
0.1 0.0 0.0 0.0 0.1 0.0
8.2 0.9 5.9 0.8* 6.7 0.9
14.1 0.9 7.9 0.9* 8.4 1.2*
23.6 1.7 14.2 0.9* 13.4 0.9*
71.7 5.2 57.9 4.5* 57.6 4.4*
5.9 0.7 2.0 0.3* 1.7 0.4*
0.050 0.009 0.117 0.015* 0.138 0.022*
IMI (min)
BCap (ml)
MV (ml)
RV (ml)
3.23 0.27 5.15 0.25*
0.5 0.0 0.9 0.0*
0.7 0.1 1.0 0.1*
3.82 0.67 3.88 0.56 4.59 0.93
0.6 0.1 0.6 0.1 0.8 0.2
3.71 0.52 5.17 0.61* 4.99 0.76*
0.6 0.1 0.9 0.1* 0.8 0.1*
OEtA, oleoyl ethyl amide (FAAH inhibitor); IMI, intermicturition interval; BCap, bladder capacity; MV, micturition volume; RV, residual volume; BP, basal bladder pressure; IMP, intermicturition pressure; TP, threshold pressure; MMP, maximum micturition pressure; SA, spontaneous bladder activity; BCom, bladder compliance. Group 3A before (baseline) and after intravenous (IV) administration of OEtA (0.3 mg/kg), group 3B before and after intrathecal (IT) administration of 5 mg of SR144528 (CB2 antagonist) and after OEtA (IV, 0.3 mg/kg) after previous SR144528 (IT), and group 3C (lower rows) before and after IT administration of 5 mg of rimonabant (CB1 antagonist) and after OEtA (IV, 0.3 mg/kg) after previous rimonabant (IT). One-way ANOVA of repeated measurements. Bold means significant. *
P < 0.05 versus baseline.
y
P < 0.05 versus intrathecal values (medium rows).
Neurourology and Urodynamics DOI 10.1002/nau
Spinal Cannabinoid Receptors and FAAH in Rat Bladder Dysfunction
469
TABLE IV. Urodynamic Parameters of Obstructed Rats (n ¼ 5); Before and After Intrathecal (IT) Administration of 5 mg of AM251 (CB1 antagonist) and After Intravenous (IV) Administration of OEtA (0.3 mg/kg) After Previous AM251 (IT)
Group 4 (n ¼ 5) Baseline (before drug) After IT AM251 After IV OEtA
IMI (min)
BCap (ml)
MV (ml)
RV (ml)
BP (cmH2O)
IMP (cmH2O)
TP (cmH2O)
MMP (cmH2O)
SA (cmH2O)
BCom (ml/cmH2O)
3.42 0.41 4.58 0.59 5.54 1.06*
0.6 0.1 0.8 0.1 0.9 0.2*
0.8 0.1 1.0 0.1* 1.1 0.2*
0.0 0.0 0.0 0.0 0.0 0.0
8.2 0.8 6.1 0.9 5.2 0.6
13.6 0.5 8.6 1.0* 7.4 0.9*
17.9 0.9 12.6 1.0* 12.3 1.0*
70.8 3.2 57.2 4.5* 57.4 3.8*
5.4 1.0 2.5 0.5* 2.2 0.3*
0.063 0.009 0.130 0.027* 0.131 0.021*
OEtA, oleoyl ethyl amide (FAAH inhibitor); IMI, intermicturition interval; BCap, bladder capacity; MV, micturition volume; RV, residual volume; BP, basal bladder pressure; IMP, intermicturition pressure; TP, threshold pressure; MMP, maximum micturition pressure; SA, spontaneous bladder activity; BCom, bladder compliance. Before and after intrathecal (IT) administration of 5 mg of AM251 (CB1 antagonist) and after intravenous (IV) administration of OEtA (0.3 mg/kg) after previous AM251 (IT). One-way ANOVA of repeated measurements. Bold means significant. yP < 0.05 versus intrathecal values (medium rows). P < 0.05 versus baseline.
*
contrasting electrophysiological effects of cannabinoids in CNS cells depend upon regional and cell-specific differences in CB1 expression in pathophysiological situations.21 In addition, suggesting that spinal CB1 plasticity in obstruction-related BO includes altered neuromodulation of efferent motor signals to the bladder, we noted increased CB1 expression in the sacral spinal grey matter containing the sacral parasympathetic nucleus. Interestingly, this altered CB1 expression in spinal cords from obstructed rats with BO generally agree with the distribution of altered expression of GAP-43 (a marker for neuritogenesis and synapse formation) in rats with cystitis.19 The reorganization of micturition pathways reported to occur in the spinal cord of these animals may compare to the currently observed changes in CB1 receptor distribution patterns following outflow obstruction.19 Reflecting upon the different effects in PUO rats and controls by IT rimonabant per se, and the lack of effect by IT injection of SR144528 per se in obstructed rats when compared to controls, it seems that obstruction causes an imbalance of spinal CB1 and CB2 receptor activities. Based on the present results, a hypothetical model may be formed: compared to naive rats, a larger quantity of spinal CB1 receptors are available after obstruction, and these CB1 receptors are related to signals that induce BO. Tonic activity at these CB1 receptors may overwhelm and mask endogenous CB2 receptor-mediated signals in obstructed rats. When rimonabant was given intrathecally,
endogenous activities at the enlarged CB1 receptor pool seemed blocked, as a reduction of BO-induced changes in frequency, volumes, and pressures resulted. This may depict a removal of pathophysiological CB1 signals that promote obstructionrelated BO and allow endogenous CB2 receptor functions to remain active. Even though previous Western blot findings of a 20% increase in CB2 expression levels in the sacral spinal cords of obstructed rats could not be verified by immunohistochemistry in this study,3 it seems reasonable to speculate that endogenous activity at these receptors is already operating at maximum capacity: additional effects were not seen following OEtA administration, and effects by OEtA alone were substantially larger in obstructed rats than in controls. Other investigators have also reported changes in expression and function of spinal CB1 and CB2 receptors in diseased rodent models. In rats with experimental temporomandibular arthritis, different relative increases in mRNA for the CB1 and CB2 receptor of the spinal trigeminal tract were demonstrated; similar to findings by Burgos et al. in a model for orofacial pain, it was suggested that CB1 receptors participate in the antinociceptive response, whereas CB2 receptors seemed to be involved in anti-inflammatory effects.22,23 In mice subjected to brachial plexus avulsion, temporal variations in increases in mRNA and protein expression levels of CB1 and CB2 receptors at thoracocervikal spinal segments were correlated to differences in CB1 or CB2 receptor-mediated antinociceptive effects.24
TABLE V. Urodynamic Parameters Before and After Intravenous (IV) Rimonabant (0.3 mg/kg), and After IV Administration of OEtA (0.3 mg/kg) After Previous Rimonabant (IV) in Non-Obstructed (Group 5A) and Obstructed Rats (Group 5B) IMI (min) Group 5A Non-obstructed (n ¼ 6) Baseline (before drug) After IV rimonabant After IV OEtA Group 5B obstructed (n ¼ 6) Baseline (before drug) After IV Rimonabant After IV OEtA
BCap (ml)
MV (ml)
RV (ml)
BP (cmH2O)
IMP (cmH2O)
TP (cmH2O)
MMP (cmH2O)
SA (cmH2O)
BCom (ml/cmH2O)
9.43 0.49 1.6 0.1 1.7 0.1 0.0 0.0 4.1 0.4 6.3 0.5 16.0 0.9 36.8 3.4 2.2 0.2 0.132 0.012 6.97 0.60* 1.2 0.1* 1.3 0.1* 0.0 0.0 6.6 0.7* 10.5 1.1* 19.3 1.3* 44.7 3.4* 4.1 0.5* 0.096 0.012* 9.32 0.82y 1.6 0.1y 1.7 0.2y 0.0 0.0 5.3 0.5*y 7.9 0.7y 16.3 1.5y 40.2 3.5 2.6 0.4y 0.153 0.022y 3.10 0.21 0.5 0.0 0.7 0.0 0.0 0.0 8.2 0.7 5.28 0.50* 0.9 0.1* 1.0 0.1* 0.1 0.0 6.5 1.5 5.26 0.59* 0.9 0.1* 1.0 0.1* 0.0 0.0 5.5 0.9
16.9 1.0 23.8 1.2 76.4 3.8 8.7 0.7 0.037 0.005 11.1 1.4* 16.4 1.2* 59.3 1.4* 4.6 0.7* 0.093 0.012* 9.0 1.0* 14.4 1.5* 59.9 3.5* 3.5 0.5* 0.223 0.136*
OEtA, oleoyl ethyl amide (FAAH inhibitor); IMI, intermicturition interval; BCap, bladder capacity; MV, micturition volume; RV, residual volume; BP, basal bladder pressure; IMP, intermicturition pressure; TP, threshold pressure; MMP, maximum micturition pressure; SA, spontaneous bladder activity; BCom, bladder compliance. One-way ANOVA of repeated measurements. Bold means significant. P < 0.05 versus baseline.
*
y
P < 0.05 versus rimonabant values (medium rows).
Neurourology and Urodynamics DOI 10.1002/nau
470
€ llhase et al. Fu
This study focused on only one point in time, which limits it to report on findings obtained when PUO is considered to have changed micturition pathways and induced BO. The currently reported urodynamic changes in rats after PUO correspond with data from other groups that used the same model.25,26 Another limitation of this study is that the tissue levels of spinal endocannabinoids were not measured. Still, the effects of OEtA that were observed in normal and obstructed rats are similar to previous reports, and based on the pharmacology of the compound, may be assumed to reflect effects via elevated levels of endocannabioids and fatty acid amides.3,5–7 Also, potential changes in the function of the endocannabinoid system in peripheral LUT tissues or in supraspinal micturition pathways were not analyzed, and their potential impact on the outcome of this study cannot be excluded. CONCLUSION
This study supports a role for the spinal endocannabinoid system in the regulation of normal bladder function, and suggests that plasticity within this system is involved in the development of obstruction-induced BO. Effects on bladder function and obstruction-induced bladder dysfunction, resulting from systemic pharmacological inhibition of FAAH in awake rats, involve actions at neuronal CB1 and/or CB2 receptors at lumbosacral spinal levels. ACKNOWLEDGMENTS
This study was supported by the German Research Foundation (DFG) Grant no GR3333/2-1, and the Gester Foundation, Sweden. This work was awarded with the Swiss Continence Foundation Award at the 2nd International Neuro-Urology Meeting, 13–15 June 2013 in Zurich, Switzerland. REFERENCES 1. Hedlund P. Cannabinoids and the endocannabinoid system in lower urinary tract function and dysfunction. Neurourol Urodyn 2014;33:46–53. 2. Alvarez-Jaimes LJ, Palmer JA. The role of endocannabinoids in pain modulation and the therapeutic potential of inhibiting their enzymatic degradation. Curr Pharm Biotech 2011;12:1644–59. 3. Fullhase C, Russo A, Castiglione F, et al. Spinal cord FAAH in normal micturition control and bladder overactivity in awake rats. J Urol 2013;189:2364–70. 4. Giang DK, Cravatt BF. Molecular characterization of human and mouse fatty acid amide hydrolases. Proc Natl Acad Sci U S A 1997;94:2238–42. 5. Strittmatter F, Gandaglia G, Benigni F, et al. Expression of fatty acid amide hydrolase (FAAH) in human, mouse, and rat urinary bladder and effects of FAAH inhibition on bladder function in awake rats. Eur Urol 2012;61:98–106.
Neurourology and Urodynamics DOI 10.1002/nau
6. Gandaglia G, Strittmatter F, La Croce G, et al. The fatty acid amide hydrolase inhibitor oleoyl ethyl amide counteracts bladder overactivity in female rats. Neurourol Urodyn 2013;33:1251–8. 7. Gandaglia G, Benigni F, Buono R, et al. The peripheral active fatty acid amide hydrolase (FAAH) inhibitor URB937 counteracts detrusor overactivity in two different animal models. Eur Urol Suppl 2013;12: e69. 8. Nyberg-Hansen R. Innervation and nervous control of the urinary bladder. Anatomical aspects. Acta Neurol Scand 1966;42:7–24. 9. Aizawa N, Hedlund P, F€ ullhase C, et al. Inhibition of peripheral FAAH depresses activities of bladder mechanosensitive nerve fibers of the rat. J Urol 2014;192:956–63. 10. Melman A, Tar M, Boczko J, et al. Evaluation of two techniques of partial urethral obstruction in the male rat model of bladder outlet obstruction. Urology 2005;66:1127–33. 11. de Groat WC. Integrative control of the lower urinary tract: Preclinical perspective. Br J Pharmacol 2006;147(Suppl 2):S25–40. 12. Malmgren A, Sj€ ogren C, Uvelius B, et al. Cystometrical evaluation of bladder instability in rats with infravesical outflow obstruction. J Urol 1987;137: 1291–4. 13. Yaksh TL, Rudy TA. Chronic catheterization of the spinal subarachnoid space. Physiol Behav 1976;17:1031–6. 14. F€ ullhase C, Campeau L, Sibaev A, et al. Bladder function in a cannabinoid receptor type 1 knockout mouse. BJU Int 2014;113:144–51. 15. Campeau L, F€ ullhase C, Sawada N, et al. Characterization of bladder function in a cannabinoid receptor type 2 knockout mouse in vivo and in vitro. Neurourol Urodyn 2014;33:566–70. 16. Tambaro S, Casu MA, Mastinu A, et al. Evaluation of selective cannabinoid CB (1) and CB(2) receptor agonists in a mouse model of lipopolysaccharideinduced interstitial cystitis. Eur J Pharm 2014;729:67–74. 17. Wang ZY, Wang P, Bjorling DE. Treatment with a cannabinoid receptor 2 agonist decreases severity of established cystitis. J Urol 2014;191:153–8. 18. de Groat WC, Yoshimura N. Afferent nerve regulation of bladder function in health and disease. Handb Exp Pharmacol 2009;194:91–138. 19. Vizzard MA, Boyle MM. Increased expression of growth-associated protein (GAP-43) in lower urinary tract pathways following cyclophosphamide (CYP)induced cystitis. Brain Res 1999;844:174–87. 20. Martin WJ, Loo CM, Basbaum AI. Spinal cannabinoids are anti-allodynic in rats with persistent inflammation. Pain 1999;82:199–205. 21. Sales-Carbonell C, Rueda-Orozco PE, Soria-Gomez E, et al. Striatal GABAergic and cortical glutamatergic neurons mediate contrasting effects of cannabinoids on cortical network synchrony. Proc Natl Acad Sci U S A 2013;110: 719–24. 22. Burgos E, Pascual D, Martın MI, et al. Antinociceptive effect of the cannabinoid agonist, WIN 55,212–2, in the orofacial and temporomandibular formalin tests. Eur J Pain 2010;14:40–8. 23. Gondim DV, Ara ujo JC, Cavalcante AL, et al. CB1 and CB2 contribute to antinociceptive and anti-inflammatory effects of electroacupuncture on experimental arthritis of the rat temporomandibular joint. Can J Physiol Pharmacol 2012;90:1479–89. 24. Paszcuk AF, Dutra RC, da Silva KA, et al. Cannabinoid agonists inhibit neuropathic pain induced by brachial plexus avulsion in mice by affecting glial cells and MAP kinases. PLoS ONE 2011;6: e24034. 25. Berggren T, Uvelius B. Cystometrical evaluation of acute and chronic overdistension in the rat urinary bladder. Urol Res 1998;26:325–30. 26. Levin RM, Longhurst PA, Monson FC, et al. Effect of bladder outlet obstruction on the morphology, physiology, and pharmacology of the bladder. The Prostate Suppl 1990;3:9–26.
Spinal Neuronal Cannabinoid Receptors Mediate Urodynamic Effects of Systemic Fatty Acid Amide Hydrolase (FAAH) Inhibition in Rats € llhase,1,2* Andrea Schreiber,2,3 Armin Giese,4 Michael Schmidt,3 Francesco Montorsi,5,6 Claudius Fu Christian Gratzke,3 Giovanni La Croce,5,7 Fabio Castiglione,6,7 Christian Stief,3 and Petter Hedlund5,8 1
Department of Urology, University of Rostock, Rostock, Germany Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians-University, Munich, Germany 3 Department of Urology, Ludwig-Maximilians-University, Munich, Germany 4 Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Munich, Germany 5 Division of Oncology/Unit of Urology, Urological Research Institute, IRCCS Ospedale San Raffaele, Milan, Italy 6 Universita Vita San Raffaele, Milan, Italy 7 Department of Clinical and Experimental Pharmacology, Lund University, Lund, Sweden 8 Department of Clinical Pharmacology, Linko€ping University, Linko€ping, Sweden 2
Aims: To test if urodynamic effects from systemic Fatty Acid Amide Hydrolase (FAAH) inhibition involve sacral spinal cannabinoid type 1 (CB1) or type 2 (CB2) receptors. Methods: Male rats with or without partial urethral obstruction were used for cystometry or immunohistochemistry. Urodynamic effects of intravenous (IV) 0.3 mg/kg Oleoyl Ethyl Amide (OEtA; FAAH inhibitor), and intrathecal (IT) 5 mg rimonabant (CB1 antagonist) or 5 mg SR144528 (CB2 antagonist) were studied in awake rats. Results: After administration of rimonabant or SR144528, non-obstructed rats with normal bladder function developed bladder overactivity (BO), which was counteracted by OEtA. OEtA also counteracted BO in obstructed rats. SR144528 did not affect bladder function in obstructed rats but counteracted the urodynamic effects of OEtA. Surprisingly, rimonabant (and AM251, another CB1 antagonist) reduced BO in obstructed rats, whereafter OEtA produced no additional urodynamic effects. CB1 expression increased in the sacral spinal cord of obstructed rats whereas no changes were observed for CB2 or FAAH. Conclusions: Urodynamic effects of systemic FAAH inhibition involve activities at spinal neuronal CB1 and CB2 receptors in normal and obstructed rats. Endogenous spinal CB receptor ligands seem to regulate normal micturition and BO. Altered spinal CB receptor functions may be involved in the pathogenesis of obstruction-induced BO. Neurourol. Urodynam. 35:464–470, 2016. # 2015 Wiley Periodicals, Inc. Key words: AM251; bladder; endocannabinoid; obstruction; oleoyl ethyl amide; overactivity; rimonabant; SR144528
INTRODUCTION
The endocannabinoid system represents a putative drugtarget to treat bladder dysfunctions.1 Cannabinoid receptor agonists reduced bladder overactivity (BO) in various models and relieved bladder symptoms in humans.1 However, psychotropic effects may accompany the therapeutic benefits of exogenous cannabinoids.1,2 By contrast, endocannabinoids are produced ‘‘on demand’’ inside the body, and inhibiting their breakdown only affects cells with ongoing endocannabinoid activity.1,2 Fatty acid amide hydrolase (FAAH), which degrades endocannabinoids and fatty acid amides, is present in the bladder and micturition control centers.1,3–5 Both acute or chronic FAAH inhibition reduced micturition frequency more in rats with partial urethral obstruction (PUO), prostaglandin E2-induced BO, or in spontaneously hypertensive rats with BO than in controls.3,6,7 Intrathecal (IT) administration of the FAAH inhibitor Oleoyl Ethyl Amide (OEtA) produced urodynamic effects in normal and obstructed rats.3 Considering that IT drug delivery tests whether a drug acts on nerves in general, effects may be either on proximal axonal nerve branches that supply the central nervous system inside the blood–brain barrier (BBB), or on distal axonal nerve branches that innervate the peripheral nervous system outside of the BBB.8 Aizawa and colleagues provided further evidence that the urodynamic effects of #
2015 Wiley Periodicals, Inc.
FAAH inhibition are based on a neuronal site of action: URB937, a FAAH inhibitor that cannot cross the BBB, was shown to reduce afferent nerve firing of primary bladder afferent Ad- and C-fibers.9 It is not known if the urodynamic effects of FAAH inhibitors, which seem to be at least partially mediated via neuronal sites of action, are based on effects of endocannabinoids on neuronal CB type 1 (CB1) or CB type 2 (CB2) receptors. This study aimed to assess if urodynamic effects of intravenous OEtA, which passes the BBB, are inhibited by IT CB1 or CB2 antagonists.
[This article was corrected on 2 April 2015 after initial online publication. The family and given names of the authors were inverted.] Lori Birder led the peer-review process as the Associate Editor responsible for the paper. Potential conflicts of interest: Nothing to disclose. Grant sponsor: German Research Foundation (DFG); Grant number: GR3333/2-1; Grant sponsor: Gester Foundation, Sweden Correspondence to: Claudius F€ ullhase, M.D., Ph.D., Department of Urology, University of Rostock, Ernst-Heydemann-Street 6, 18055 Rostock, Germany. E-mail: [email protected] Received 24 November 2014; Accepted 21 January 2015 Published online 18 March 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/nau.22753
Spinal Cannabinoid Receptors and FAAH in Rat Bladder Dysfunction MATERIALS AND METHODS Animals
Male Sprague–Dawley rats (n ¼ 90) were housed at the Walter Brendel Centre of Experimental Medicine, Munich, or at the San Raffaele Scientific Institute, Milan. All experiments were approved by the local Animal Ethics Committees. Animals were kept and housed according to standard conditions in a 12:12 light–dark cycle with food and water ad libidum. Thirty-eight rats served as controls. Following partial urethral obstruction (PUO; n ¼ 52), nine rats were euthanized because of acute urinary retention. After IT catheterization, seven rats were excluded because of paralysis. Three rats were excluded because of excessive movement artefacts. The final groups for cystometry included 32 PUO rats and 27 controls, and for immunohistochemistry, six PUO rats and six controls.
465
Saline dilutions were made on the day of experiment. After baseline cystometry (45 min), rimonabant, SR144528, or vehicle was injected intrathecally (5 mg in 10 ml) for 1 min, and cystometry continued for an additional 45 min. Animals then received OEtA (IV, 0.3 mg/kg), and cystometry continued for a further 45 min. Vehicles had no effect. Statistical Analysis
Results are given as mean values standard error of the mean (SEM). Student’s t-test was used for comparison between two groups. For comparisons within groups, one-way repeatedmeasures analysis of variance (ANOVA), followed by Student– Newman–Keuls test, were used. SigmaPlot 11.0 software (Systat, Inc., Chicago, IL) was used to analyze statistics. A value of P < 0.05 was considered statistically significant. RESULTS
Partial Urethral Obstruction (PUO) 10
PUO was performed as previously described. The urethra was accessed through a perineal incision, isolated from the corpora cavernosa, and partially ligated with a 3–0 suture. Rats were used in experiments 14 days later. Immunohistochemistry
Spinal cord tissue (L6–S2)11 was harvested, processed, and cut into transversal sections. Immunohistochemistry for FAAH, CB1, and CB2 receptors was performed on a Ventana Benchmark staining-machine (Ventana Medical Systems, AZ) using polyclonal antibodies for CB1, CB2, and FAAH (Cayman Chemical, Ann Arbor, MI) at dilutions of 1:50, 1:300, and 1:50. These antibodies have been characterized before.5,6 The percent area of the spinal cord occupied by stainings was evaluated in Image J, v.1.45s (NIH, Bethesda, MD). Functional Experiments
Bladder and IT catheterizations were performed as previously described.12,13 A PE-50 polyethylene catheter was inserted into the bladder via laparotomy and secured with a purse-string suture (5–0). The catheter was tunneled under the skin to the neck and anchored. Next, the atlanto–occipital membrane was exposed and punctured. A 32-gauge IT catheter (CS-1, ReCathCo, Allison Park, PA) was inserted and its tip positioned two fingers above the hip joints. Catheter locations (L6–S2)13 were confirmed during necropsy after experiments. Cystometry
Cystometries were performed 3 days after catheter implantations.12 The parameters investigated were Intermicturition Interval (IMI), Bladder Capacity (BCap ¼ infusion rate per minute IMI), micturition volume (MV), residual volume (RV ¼ BCap MV), basal bladder pressure (BP ¼ minimum bladder pressure between two micturitions), intermicturition pressure (IMP ¼ mean bladder pressure between two micturitions), threshold pressure (TP ¼ bladder pressure at onset of the micturition contraction curve/peak), maximum micturition pressure (MMP), spontaneous bladder activity (SA ¼ IMP – BP), and bladder compliance (BCom ¼ BCap/(TPBP). Drug Administration
OEtA, rimonabant (SR141716), AM251, and SR144528 (Cayman Chemical) were dissolved in DMSO and stored at 808C. Neurourology and Urodynamics DOI 10.1002/nau
Immunohistochemistry
No stainings were detected in the spinal cord white matter. The gray matter exhibited diffuse FAAH staining (Fig. 1). Intense CB2 staining (Fig. 1) was found in the outer laminae (substantia gelatinosa) of the dorsal horns. No clear difference in staining patterns for CB2 or FAAH was observed between PUO rats and non-obstructed controls. By contrast, increases in CB1 stainings were seen in PUO rats in the intermediolateral zone, the sacral parasympathetic nucleus, the dorsal horns, and the dorsal commissure (Figs. 1 and 2). Cerebellar cortex control tissue exhibited expected staining patterns for CB1, CB2, and FAAH (Fig. 1). Functional Experiments Effect of PUO. Compared to controls, PUO rats had a shorter IMI (61 4%) and correspondingly higher micturition frequency (MF), showed a reduced BCap (60 7%), MV (44 6%), and BCom (73 4%), had higher intravesical pressures (BP þ76 9%, IMP þ114 8%, TP þ68 7%, and MMP þ76 6%), and a higher SA (þ196 21%), all P < 0.05 (Table I). Obstructed rats’ bladders weighed more than controls’ (1178 55 mg vs. 449 93 mg, P < 0.05), despite the groups’ similar body weights. Drug effects in non-obstructed control rats. In controls, OEtA (IV) increased IMI (þ31 9%), BCap (31 8%), MV (þ29 7%), and BCom (þ25 9%), all P < 0.05, but produced no further urodynamic effects (Table II). Intrathecal SR144528 reduced IMI (17 9%), and BCap (19 6%), and increased BP (þ20 9%), all P < 0.05 (Group 2A). OEtA (IV) reversed SR144528 (IT) effects to baseline values (Group 2B). Intrathecal rimonabant reduced IMI (22 8%), BCap (19 6%), MV (18 6%), and BCom (29 8%), and increased BP (þ58 11%), IMP (þ46 14%), TP (þ33 9%), MMP (þ15 7%), and SA (þ72 22%), all P < 0.05 (Group 2C). OEtA (IV) reversed rimonabant (IT) effects to baseline values (Group 2C). Drug effects in PUO rats. In obstructed rats, OEtA (IV) increased IMI (þ59 8%), BCap (þ80 10%), MV (þ42 14%), and BCom (þ188 21%), and reduced IMP (33 5%), TP (29 8%), MMP (30 8%), and SA (75 9%), all P < 0.05 (Table III) (Group 3A). Intrathecal SR144528 produced no urodynamic effect (Group 3B). Administering OEtA (IV) after SR144528 (IT) had
466
€ llhase et al. Fu
Fig. 1. Immunohistochemistry. Representative photomicrographs of the distribution of CB1 receptor stainings in (A) cerebellar cortex control tissue, and sacral spinal cords of (B) non-obstructed control rats and rats with (C) partial urethral obstruction (PUO); CB2 stainings in (D) cerebellar cortex control tissue, and sacral spinal cords of (E) non-obstructed controls and rats with (F) PUO; stainings of FAAH in (G) cerebellar cortex control tissue, and sacral spinal cords of (H) non-obstructed controls and rats with (I) PUO. DH, dorsal horn; DC, dorsal commissure; SPN, sacral parasympathetic nucleus.
no additional effect besides increasing BCom (þ40 17%) (Group 3B). Intrathecal rimonabant increased IMI (þ39 16%), BCap (þ50 17%), MV (þ28 14%), and BCom (134 20%), and decreased BP (29 11%), IMP (44 6), TP (40 7%), MMP (20 7%), and SA (67 12%), all P < 0.05 (Group 3C). Administering OEtA (IV) after rimonabant (IT) had no additional effect (Group 3C).
and controls. In controls, IV rimonabant induced the same urodynamic changes as IT rimonabant (Tables II and V). Intravenous OEtA then reversed the changes induced by IV rimonabant similarly as it had with IT rimonabant. In obstructed animals with BO, IV rimonabant induced the same urodynamic changes as IT rimonabant (Tables III and V). When IV OEtA was given after IV rimonabant, no further effects were observed.
Extended investigations of CB1 receptor functions. To explore the different rimonabant results in PUO rats and controls, effects by another CB1 receptor antagonist, AM251, were studied in PUO rats. Here, IT administration of AM251 produced similar results as with rimonabant (Table IV). Furthermore, effects of IV rimonabant (0.3 mg/kg) prior to administering IV OEtA (0.3 mg/kg) were evaluated in PUO rats
This study shows that the urodynamic effects of systemic FAAH inhibition involve cannabinoid receptors in sacral spinal cord neurons. In control rats, IT delivery of CB1 or CB2 receptor antagonists prevented systemic FAAH inhibitor-induced changes in bladder function similarly. Interestingly, CB1 and
Neurourology and Urodynamics DOI 10.1002/nau
DISCUSSION
Spinal Cannabinoid Receptors and FAAH in Rat Bladder Dysfunction
467
Fig. 2. Analysis of stainings for CB1 immunohistochemistry in spinal cords from controls and rats with partial urethral obstruction (PUO). (A) Area occupied by CB1 stainings in the dorsal horns (DH); (B) area occupied by CB1 stainings in the sacral parasympathetic nucleus (SPN); (C) area occupied by CB1 stainings in the dorsal commissure (DC). Measurements were performed with Image J (n ¼ 4 for each group). Values are given as mean standard error of the mean for area occupied. Students t-test: **P < 0.01, ***P < 0.001.
CB2 receptor antagonists had per se effects in controls, suggesting that endogenous ligands at spinal neuron receptors regulate normal micturition. In obstructed rats, IT CB2, but not CB1, prevented the effects of IV OEtA, leading us to propose that neuronal CB2 receptors mediate urodynamic effects of systemic FAAH inhibitors in obstructed rats. The present urodynamic data and findings of increased expression of CB1 receptors in the sacral spinal cord areas of obstructed rats indicate that obstruction alters spinal neuronal CB1-receptor activity. In controls, rimonabant (IT) increased micturition frequency, bladder pressures, and spontaneous bladder activity, and decreased bladder capacity and micturition volume. Supporting these data, a role for the CB1 receptor in micturition has been proposed for CB1 knockout mice with increased micturition frequencies, and injecting rimonabant intraperitoneally in wild-type mice has been found to decrease IMI, BCap, and MV.14 Inversely, various CB1 agonists have increased IMI, BCap, and MV in different models.1 Similar to rimonabant, the CB2 receptor antagonist SR144528 (IT) decreased IMI and BCap in control rats, but did not affect intravesical pressures. AM630, another CB2 antagonist, has also been found to reduce IMI and BCap in normal mice.1 Other authors have reported that CB2 agonists increase IMI and BCap, further indicating a role for the CB2 receptor in micturition control.1,15–17 CB1- and CB2-receptor active compounds have been shown to affect bladder function. But this is, to the best of our knowledge, the first report describing the effects of IT CB1 or CB2 antagonists on bladder function that also suggests that spinal endogenous CB signals regulate normal micturition.
Supporting this, OEtA (IV) counteracted the urodynamic changes by IT delivery of rimonabant or SR144528. This effect of systemic OEtA administration suggests that FAAH substrates may act at neurons connected to the sacral spinal cord. Promoting the idea that one or more endogenous ligands tonically recruit neuronal CB1 or CB2 receptors to regulate bladder function, Aizawa et al. described that systemic administration of rimonabant or SR144528 per se modified the activities of primary bladder sensory-nerve fibers.9 This correlates well with the currently reported effects of both rimonabant and SR144528 on voiding frequency (as a surrogate parameter for sensory bladder functions), and may also relate to the current location of CB1 and CB2 in the sacral dorsal horns, which receive sensory information from the lower urinary tract (LUT).18 The effects by rimonabant on intravesical pressures and the histochemical location of CB1 in the sacral parasympathetic nucleus18,19 may reflect neuronal CB1-mediated regulation of bladder motor signals. Effects by IV OEtA on IMI, MV, and BCap were qualitatively similar in non-obstructed and obstructed rats, but effects were elevated 30–50% in the latter group. These findings correspond to previous reports of increased OEtA responses in BO models compared to normal animals, and suggest that FAAH substrates exhibit increased activities in bladder dysfunction.3,6,7 Unlike in control rats, OEtA also reduced bladder pressures and spontaneous bladder activity in obstructed rats. As IT SR144256 prevented effects by IV OEtA in obstructed rats, the CB2 receptor may play a critical role in mediating effects by FAAH substrates in PUO-induced BO.
TABLE I. Urodynamic Parameters of Non-Obstructed Control Rats Versus Obstructed Rats IMI (min)
BCap (ml)
MV (ml)
RV (ml)
BP (cmH2O)
IMP (cmH2O)
TP (cmH2O)
MMP (cmH2O)
SA (cmH2O)
BCom (ml/cmH2O)
Non-obstructed 9.12 0.32 1.5 0.1 1.6 0.1 0.0 0.0 4.7 0.2 7.2 0.3 14.2 0.4 42.7 1.7 2.4 0.2 0.162 0.007 (n ¼ 27) ** ** ** ** Obstructed 3.55 0.20 0.6 0.0 0.7 0.0 0.0 0.0 8.3 0.4 15.4 0.6** 23.9 1.0** 75.3 2.4** 7.1 0.5** 0.045 0.004** (n ¼ 32)
Bladder weight (mg) 449 93 1178 55**
IMI, intermicturition interval; BCap, bladder capacity; MV, micturition volume; RV, residual volume; BP, basal bladder pressure; IMP, intermicturition pressure; TP, threshold pressure; MMP, maximum micturition pressure; SA, spontaneous bladder activity; BCom, bladder compliance. Student’s t-test, *P < 0.05, **P < 0.001. Bold means significant.
Neurourology and Urodynamics DOI 10.1002/nau
468
€ llhase et al. Fu
TABLE II. Urodynamic Parameters of Non-Obstructed Rats (n ¼ 21) IMI (min) Group 2A (n ¼ 5) Baseline (before drug) 7.84 0.34 After IV OEtA 10.27 0.67* Group 2B (n ¼ 8) Baseline (before drug) 9.30 0.84 After IT SR144528 7.77 0.77* After IV OEtA 9.27 0.92** Group 2C (n ¼ 8) Baseline (before drug) 9.51 0.49 After IT rimonabant 7.51 0.36* After IV OEtA 9.55 0.31**
BCap (ml)
1.3 0.1 1.7 0.1
MV (ml)
*
1.4 0.1 1.8 0.1
1.6 0.1 1.6 0.1 1.3 0.1* 1.4 0.2 ** 1.5 0.2 1.6 0.2
RV (ml)
*
0.0 0.0 0.1 0.1
BP (cmH2O)
IMP (cmH2O)
TP (cmH2O)
MMP (cmH2O)
SA (cmH2O)
BCom (ml/cmH2O)
4.6 0.3 4.4 0.2
6.5 0.5 5.8 0.5
12.9 0.7 13.2 0.6
41.5 2.6 41.0 3.8
2.0 0.4 1.4 0.3
0.15 0.009 0.197 0.014*
7.4 0.5 7.9 0.6 7.2 0.6
13.8 0.9 14.9 1.1 15.9 1.7
41.4 3.1 43.4 3.2 43.2 3.9
2.9 0.4 0.165 0.015 3.4 0.4 0.143 0.017 ** 2.2 0.2 0.147 0.014
0.0 0.0 4.5 0.4 0.0 0.0 5.4 0.4* 0.0 0.0 4.6 0.5**
1.6 0.1 1.7 0.1 0.0 0.0 5.3 0.3 8.1 0.5 14.0 0.7 49.1 2.5 2.5 0.3 1.3 0.1* 1.4 0.1* 0.0 0.0 8.4 0.6* 11.8 1.1* 18.6 1.2* 56.2 3.6* 4.3 0.6* ** ** ** ** ** 1.6 0.1 1.6 0.1 0.1 0.0 5.3 0.4** 7.8 0.5 14.7 1.2 46.9 3.2 2.6 0.4
0.183 0.014 0.131 0.012* 0.176 0.015**
OEtA, oleoyl ethyl amide (FAAH inhibitor); IMI, intermicturition interval; BCap, bladder capacity; MV, micturition volume; RV, residual volume; BP, basal bladder pressure; IMP, intermicturition pressure; TP, threshold pressure; MMP, maximum micturition pressure; SA, spontaneous bladder activity; BCom, bladder compliance. Group 2A before (baseline) and after intravenous (IV) OEtA (0.3 mg/kg) administration. Group 2B before and after intrathecal (IT) administration of 5 mg of SR144528 (CB2 antagonist) and after IV OEtA administration (after previous SR144528 IT delivery), and group 2 C (lower rows) before and after IT administration of 5 mg of rimonabant (CB1 antagonist) and after OEtA (0.3 mg/kg, IV) after previous rimonabant (IT). One-way ANOVA of repeated measurements. Bold means significant. *
P < 0.05 versus baseline.
**
P < 0.05 versus intrathecal values (medium rows).
Based on our findings in non-obstructed rats, we expected IT rimonabant to either have no effect or to increase urodynamic signs of BO in obstructed rats. Surprisingly, IT rimonabant instead increased IMI and decreased bladder pressures. These results were verified by showing that IV rimonabant, and IT AM251 also caused similar urodynamic effects. Hence, the data suggest that CB1 receptors mediate different effects in normal bladder function and obstruction-induced BO. This in turn indicates that obstruction alters CB1-mediated signals somewhere in the pathways that regulate micturition. We previously described by Western blot that a 70% increase in CB1 receptor expression occurs in the sacral spinal cord of obstructed rats.3 In the present study, increased CB1 expression was found in
regions of the lumbosacral spinal cord known to contain neurons and interneurons that receive and relay information from LUT sensory dorsal-root ganglion cells.18 These findings form a morphological basis for CB1-receptor plasticity in altering processing of sensory signals from the obstructed LUT, and we speculate that this explains our contrasting results with rimonabant. Similarly, Martin et al. described in a rat painmodel that IT rimonabant differently affected nociception and spinal expression patterns of c-fos (a nerve-activity marker) depending on the presence of chronic inflammation.20 It was concluded that cannabinoids likely act at CB1 receptors pre- and postsynaptically to modulate spinal cord activity under different conditions.20 Analagously, Sales-Carbonell et al. determined that
TABLE III. Urodynamic Parameters of Obstructed Rats (n ¼ 21)
Group 3A (n ¼ 5) Baseline (before drug) After IV OEtA Group 3B (n ¼ 8) Baseline (before drug) After IT SR144528 After IV OEtA Group 3C (n ¼ 8) Baseline (before drug) After IT rimonabant After IV OEtA
BP (cmH2O)
IMP (cmH2O)
TP (cmH2O)
MMP (cmH2O)
SA (cmH2O)
BCom (ml/cmH2O)
0.0 0.0 0.0 0.0
10.4 0.7 10.3 0.6
17.1 0.9 12.0 0.5*
28.6 2.3 20.5 1.4*
92.4 7.0 65.5 6.1*
6.7 0.6 1.7 0.5*
0.033 0.007 0.095 0.016*
0.7 0.1 0.7 0.1 0.8 0.2
0.0 0.0 0.0 0.0 0.0 0.0
8.1 1.2 7.0 1.5 8.0 1.7
15.8 2.4 13.9 2.0 13.8 2.5
24.2 3.0 20.1 3.0 19.2 3.5
71.5 5.1 65.1 6.8 63.3 9.1
7.7 1.5 5.6 1.3 5.8 1.4
0.047 0.011 0.052 0.006 0.073 0.009*y
0.7 0.1 0.9 0.1* 0.8 0.1*
0.1 0.0 0.0 0.0 0.1 0.0
8.2 0.9 5.9 0.8* 6.7 0.9
14.1 0.9 7.9 0.9* 8.4 1.2*
23.6 1.7 14.2 0.9* 13.4 0.9*
71.7 5.2 57.9 4.5* 57.6 4.4*
5.9 0.7 2.0 0.3* 1.7 0.4*
0.050 0.009 0.117 0.015* 0.138 0.022*
IMI (min)
BCap (ml)
MV (ml)
RV (ml)
3.23 0.27 5.15 0.25*
0.5 0.0 0.9 0.0*
0.7 0.1 1.0 0.1*
3.82 0.67 3.88 0.56 4.59 0.93
0.6 0.1 0.6 0.1 0.8 0.2
3.71 0.52 5.17 0.61* 4.99 0.76*
0.6 0.1 0.9 0.1* 0.8 0.1*
OEtA, oleoyl ethyl amide (FAAH inhibitor); IMI, intermicturition interval; BCap, bladder capacity; MV, micturition volume; RV, residual volume; BP, basal bladder pressure; IMP, intermicturition pressure; TP, threshold pressure; MMP, maximum micturition pressure; SA, spontaneous bladder activity; BCom, bladder compliance. Group 3A before (baseline) and after intravenous (IV) administration of OEtA (0.3 mg/kg), group 3B before and after intrathecal (IT) administration of 5 mg of SR144528 (CB2 antagonist) and after OEtA (IV, 0.3 mg/kg) after previous SR144528 (IT), and group 3C (lower rows) before and after IT administration of 5 mg of rimonabant (CB1 antagonist) and after OEtA (IV, 0.3 mg/kg) after previous rimonabant (IT). One-way ANOVA of repeated measurements. Bold means significant. *
P < 0.05 versus baseline.
y
P < 0.05 versus intrathecal values (medium rows).
Neurourology and Urodynamics DOI 10.1002/nau
Spinal Cannabinoid Receptors and FAAH in Rat Bladder Dysfunction
469
TABLE IV. Urodynamic Parameters of Obstructed Rats (n ¼ 5); Before and After Intrathecal (IT) Administration of 5 mg of AM251 (CB1 antagonist) and After Intravenous (IV) Administration of OEtA (0.3 mg/kg) After Previous AM251 (IT)
Group 4 (n ¼ 5) Baseline (before drug) After IT AM251 After IV OEtA
IMI (min)
BCap (ml)
MV (ml)
RV (ml)
BP (cmH2O)
IMP (cmH2O)
TP (cmH2O)
MMP (cmH2O)
SA (cmH2O)
BCom (ml/cmH2O)
3.42 0.41 4.58 0.59 5.54 1.06*
0.6 0.1 0.8 0.1 0.9 0.2*
0.8 0.1 1.0 0.1* 1.1 0.2*
0.0 0.0 0.0 0.0 0.0 0.0
8.2 0.8 6.1 0.9 5.2 0.6
13.6 0.5 8.6 1.0* 7.4 0.9*
17.9 0.9 12.6 1.0* 12.3 1.0*
70.8 3.2 57.2 4.5* 57.4 3.8*
5.4 1.0 2.5 0.5* 2.2 0.3*
0.063 0.009 0.130 0.027* 0.131 0.021*
OEtA, oleoyl ethyl amide (FAAH inhibitor); IMI, intermicturition interval; BCap, bladder capacity; MV, micturition volume; RV, residual volume; BP, basal bladder pressure; IMP, intermicturition pressure; TP, threshold pressure; MMP, maximum micturition pressure; SA, spontaneous bladder activity; BCom, bladder compliance. Before and after intrathecal (IT) administration of 5 mg of AM251 (CB1 antagonist) and after intravenous (IV) administration of OEtA (0.3 mg/kg) after previous AM251 (IT). One-way ANOVA of repeated measurements. Bold means significant. yP < 0.05 versus intrathecal values (medium rows). P < 0.05 versus baseline.
*
contrasting electrophysiological effects of cannabinoids in CNS cells depend upon regional and cell-specific differences in CB1 expression in pathophysiological situations.21 In addition, suggesting that spinal CB1 plasticity in obstruction-related BO includes altered neuromodulation of efferent motor signals to the bladder, we noted increased CB1 expression in the sacral spinal grey matter containing the sacral parasympathetic nucleus. Interestingly, this altered CB1 expression in spinal cords from obstructed rats with BO generally agree with the distribution of altered expression of GAP-43 (a marker for neuritogenesis and synapse formation) in rats with cystitis.19 The reorganization of micturition pathways reported to occur in the spinal cord of these animals may compare to the currently observed changes in CB1 receptor distribution patterns following outflow obstruction.19 Reflecting upon the different effects in PUO rats and controls by IT rimonabant per se, and the lack of effect by IT injection of SR144528 per se in obstructed rats when compared to controls, it seems that obstruction causes an imbalance of spinal CB1 and CB2 receptor activities. Based on the present results, a hypothetical model may be formed: compared to naive rats, a larger quantity of spinal CB1 receptors are available after obstruction, and these CB1 receptors are related to signals that induce BO. Tonic activity at these CB1 receptors may overwhelm and mask endogenous CB2 receptor-mediated signals in obstructed rats. When rimonabant was given intrathecally,
endogenous activities at the enlarged CB1 receptor pool seemed blocked, as a reduction of BO-induced changes in frequency, volumes, and pressures resulted. This may depict a removal of pathophysiological CB1 signals that promote obstructionrelated BO and allow endogenous CB2 receptor functions to remain active. Even though previous Western blot findings of a 20% increase in CB2 expression levels in the sacral spinal cords of obstructed rats could not be verified by immunohistochemistry in this study,3 it seems reasonable to speculate that endogenous activity at these receptors is already operating at maximum capacity: additional effects were not seen following OEtA administration, and effects by OEtA alone were substantially larger in obstructed rats than in controls. Other investigators have also reported changes in expression and function of spinal CB1 and CB2 receptors in diseased rodent models. In rats with experimental temporomandibular arthritis, different relative increases in mRNA for the CB1 and CB2 receptor of the spinal trigeminal tract were demonstrated; similar to findings by Burgos et al. in a model for orofacial pain, it was suggested that CB1 receptors participate in the antinociceptive response, whereas CB2 receptors seemed to be involved in anti-inflammatory effects.22,23 In mice subjected to brachial plexus avulsion, temporal variations in increases in mRNA and protein expression levels of CB1 and CB2 receptors at thoracocervikal spinal segments were correlated to differences in CB1 or CB2 receptor-mediated antinociceptive effects.24
TABLE V. Urodynamic Parameters Before and After Intravenous (IV) Rimonabant (0.3 mg/kg), and After IV Administration of OEtA (0.3 mg/kg) After Previous Rimonabant (IV) in Non-Obstructed (Group 5A) and Obstructed Rats (Group 5B) IMI (min) Group 5A Non-obstructed (n ¼ 6) Baseline (before drug) After IV rimonabant After IV OEtA Group 5B obstructed (n ¼ 6) Baseline (before drug) After IV Rimonabant After IV OEtA
BCap (ml)
MV (ml)
RV (ml)
BP (cmH2O)
IMP (cmH2O)
TP (cmH2O)
MMP (cmH2O)
SA (cmH2O)
BCom (ml/cmH2O)
9.43 0.49 1.6 0.1 1.7 0.1 0.0 0.0 4.1 0.4 6.3 0.5 16.0 0.9 36.8 3.4 2.2 0.2 0.132 0.012 6.97 0.60* 1.2 0.1* 1.3 0.1* 0.0 0.0 6.6 0.7* 10.5 1.1* 19.3 1.3* 44.7 3.4* 4.1 0.5* 0.096 0.012* 9.32 0.82y 1.6 0.1y 1.7 0.2y 0.0 0.0 5.3 0.5*y 7.9 0.7y 16.3 1.5y 40.2 3.5 2.6 0.4y 0.153 0.022y 3.10 0.21 0.5 0.0 0.7 0.0 0.0 0.0 8.2 0.7 5.28 0.50* 0.9 0.1* 1.0 0.1* 0.1 0.0 6.5 1.5 5.26 0.59* 0.9 0.1* 1.0 0.1* 0.0 0.0 5.5 0.9
16.9 1.0 23.8 1.2 76.4 3.8 8.7 0.7 0.037 0.005 11.1 1.4* 16.4 1.2* 59.3 1.4* 4.6 0.7* 0.093 0.012* 9.0 1.0* 14.4 1.5* 59.9 3.5* 3.5 0.5* 0.223 0.136*
OEtA, oleoyl ethyl amide (FAAH inhibitor); IMI, intermicturition interval; BCap, bladder capacity; MV, micturition volume; RV, residual volume; BP, basal bladder pressure; IMP, intermicturition pressure; TP, threshold pressure; MMP, maximum micturition pressure; SA, spontaneous bladder activity; BCom, bladder compliance. One-way ANOVA of repeated measurements. Bold means significant. P < 0.05 versus baseline.
*
y
P < 0.05 versus rimonabant values (medium rows).
Neurourology and Urodynamics DOI 10.1002/nau
470
€ llhase et al. Fu
This study focused on only one point in time, which limits it to report on findings obtained when PUO is considered to have changed micturition pathways and induced BO. The currently reported urodynamic changes in rats after PUO correspond with data from other groups that used the same model.25,26 Another limitation of this study is that the tissue levels of spinal endocannabinoids were not measured. Still, the effects of OEtA that were observed in normal and obstructed rats are similar to previous reports, and based on the pharmacology of the compound, may be assumed to reflect effects via elevated levels of endocannabioids and fatty acid amides.3,5–7 Also, potential changes in the function of the endocannabinoid system in peripheral LUT tissues or in supraspinal micturition pathways were not analyzed, and their potential impact on the outcome of this study cannot be excluded. CONCLUSION
This study supports a role for the spinal endocannabinoid system in the regulation of normal bladder function, and suggests that plasticity within this system is involved in the development of obstruction-induced BO. Effects on bladder function and obstruction-induced bladder dysfunction, resulting from systemic pharmacological inhibition of FAAH in awake rats, involve actions at neuronal CB1 and/or CB2 receptors at lumbosacral spinal levels. ACKNOWLEDGMENTS
This study was supported by the German Research Foundation (DFG) Grant no GR3333/2-1, and the Gester Foundation, Sweden. This work was awarded with the Swiss Continence Foundation Award at the 2nd International Neuro-Urology Meeting, 13–15 June 2013 in Zurich, Switzerland. REFERENCES 1. Hedlund P. Cannabinoids and the endocannabinoid system in lower urinary tract function and dysfunction. Neurourol Urodyn 2014;33:46–53. 2. Alvarez-Jaimes LJ, Palmer JA. The role of endocannabinoids in pain modulation and the therapeutic potential of inhibiting their enzymatic degradation. Curr Pharm Biotech 2011;12:1644–59. 3. Fullhase C, Russo A, Castiglione F, et al. Spinal cord FAAH in normal micturition control and bladder overactivity in awake rats. J Urol 2013;189:2364–70. 4. Giang DK, Cravatt BF. Molecular characterization of human and mouse fatty acid amide hydrolases. Proc Natl Acad Sci U S A 1997;94:2238–42. 5. Strittmatter F, Gandaglia G, Benigni F, et al. Expression of fatty acid amide hydrolase (FAAH) in human, mouse, and rat urinary bladder and effects of FAAH inhibition on bladder function in awake rats. Eur Urol 2012;61:98–106.
Neurourology and Urodynamics DOI 10.1002/nau
6. Gandaglia G, Strittmatter F, La Croce G, et al. The fatty acid amide hydrolase inhibitor oleoyl ethyl amide counteracts bladder overactivity in female rats. Neurourol Urodyn 2013;33:1251–8. 7. Gandaglia G, Benigni F, Buono R, et al. The peripheral active fatty acid amide hydrolase (FAAH) inhibitor URB937 counteracts detrusor overactivity in two different animal models. Eur Urol Suppl 2013;12: e69. 8. Nyberg-Hansen R. Innervation and nervous control of the urinary bladder. Anatomical aspects. Acta Neurol Scand 1966;42:7–24. 9. Aizawa N, Hedlund P, F€ ullhase C, et al. Inhibition of peripheral FAAH depresses activities of bladder mechanosensitive nerve fibers of the rat. J Urol 2014;192:956–63. 10. Melman A, Tar M, Boczko J, et al. Evaluation of two techniques of partial urethral obstruction in the male rat model of bladder outlet obstruction. Urology 2005;66:1127–33. 11. de Groat WC. Integrative control of the lower urinary tract: Preclinical perspective. Br J Pharmacol 2006;147(Suppl 2):S25–40. 12. Malmgren A, Sj€ ogren C, Uvelius B, et al. Cystometrical evaluation of bladder instability in rats with infravesical outflow obstruction. J Urol 1987;137: 1291–4. 13. Yaksh TL, Rudy TA. Chronic catheterization of the spinal subarachnoid space. Physiol Behav 1976;17:1031–6. 14. F€ ullhase C, Campeau L, Sibaev A, et al. Bladder function in a cannabinoid receptor type 1 knockout mouse. BJU Int 2014;113:144–51. 15. Campeau L, F€ ullhase C, Sawada N, et al. Characterization of bladder function in a cannabinoid receptor type 2 knockout mouse in vivo and in vitro. Neurourol Urodyn 2014;33:566–70. 16. Tambaro S, Casu MA, Mastinu A, et al. Evaluation of selective cannabinoid CB (1) and CB(2) receptor agonists in a mouse model of lipopolysaccharideinduced interstitial cystitis. Eur J Pharm 2014;729:67–74. 17. Wang ZY, Wang P, Bjorling DE. Treatment with a cannabinoid receptor 2 agonist decreases severity of established cystitis. J Urol 2014;191:153–8. 18. de Groat WC, Yoshimura N. Afferent nerve regulation of bladder function in health and disease. Handb Exp Pharmacol 2009;194:91–138. 19. Vizzard MA, Boyle MM. Increased expression of growth-associated protein (GAP-43) in lower urinary tract pathways following cyclophosphamide (CYP)induced cystitis. Brain Res 1999;844:174–87. 20. Martin WJ, Loo CM, Basbaum AI. Spinal cannabinoids are anti-allodynic in rats with persistent inflammation. Pain 1999;82:199–205. 21. Sales-Carbonell C, Rueda-Orozco PE, Soria-Gomez E, et al. Striatal GABAergic and cortical glutamatergic neurons mediate contrasting effects of cannabinoids on cortical network synchrony. Proc Natl Acad Sci U S A 2013;110: 719–24. 22. Burgos E, Pascual D, Martın MI, et al. Antinociceptive effect of the cannabinoid agonist, WIN 55,212–2, in the orofacial and temporomandibular formalin tests. Eur J Pain 2010;14:40–8. 23. Gondim DV, Ara ujo JC, Cavalcante AL, et al. CB1 and CB2 contribute to antinociceptive and anti-inflammatory effects of electroacupuncture on experimental arthritis of the rat temporomandibular joint. Can J Physiol Pharmacol 2012;90:1479–89. 24. Paszcuk AF, Dutra RC, da Silva KA, et al. Cannabinoid agonists inhibit neuropathic pain induced by brachial plexus avulsion in mice by affecting glial cells and MAP kinases. PLoS ONE 2011;6: e24034. 25. Berggren T, Uvelius B. Cystometrical evaluation of acute and chronic overdistension in the rat urinary bladder. Urol Res 1998;26:325–30. 26. Levin RM, Longhurst PA, Monson FC, et al. Effect of bladder outlet obstruction on the morphology, physiology, and pharmacology of the bladder. The Prostate Suppl 1990;3:9–26.
