Intravesical resiniferatoxin decreases spinal c-fos expression and increases bladder volume to reflex micturition in rats with chronic inflamed urinary bladders

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Intravesical resiniferatoxin decreases spinal c-fos expression and increases bladder volume to reflex micturition in rats with chronic inflamed urinary bladders
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  ©   2004 BJU INTERNATIONAL | 94, 153–157 | doi:10.1111/j.1464-410X.2004.04855.x 153 l lINTRAVESICAL RESINIFERATOXIN IN CHRONIC INFLAMED URINARY BLADDERSDINIS ET AL. Intravesical resiniferatoxin decreases spinal c-fos expression and increases bladder volume to reflex micturition in rats with chronic inflamed urinary bladders PAULO DINIS*, ANA CHARRUA*†, ANTÓNIO AVELINO*† and FRANCISCO CRUZ*† *Department of Urology, Hospital São João, Porto and Faculty of Medicine of Porto, Institute of Histology and Embryology, Porto, †IBMC of University of Porto, Porto, Portugal  Accepted for publication 9 March 2004 urethane anaesthesia, a needle was inserted in the bladder dome and saline infused at 6 mL/h for 2 h. Finally the animals were perfusion-fixed through the ascending aorta with 4% paraformaldehyde. Transverse sections cut from L6 spinal cord segments were immunoreacted for Fos protein and positive cells in the dorsal horn counted. In a further set of equal groups the bladders were prepared in the same way under urethane anaesthesia and after 30-min of stabilization, saline was infused at 6 mL/h and the volume evoking reflex micturition determined. RESULTS The mean ( SD ) number of positive c- fos   cells per spinal cord section was 85 (21), 42 (9) ( P    =  0.002) and 55 (10) in groups 1 to 3, respectively; the values for group 2 and 3 were similar ( P    =  0.22) and statistically less than that of group 1 ( P    =  0.02). Reflex micturition occurred at, respectively, 0.26 (0.09), 0.49 (0.18) and 0.52 (0.11) mL, being similar in group 2 and 3 ( P    =  0.74) but lower in group 1 ( P    =  0.003). CONCLUSION Intravesical resiniferatoxin decreases c-fos   expression and increases bladder capacity in chronically inflamed rat bladders. These findings suggest that desensitizing the vanilloid receptor type 1 by intravesical resiniferatoxin is relevant to the treatment of pain and voiding frequency in patients with chronic inflammatory bladder conditions. KEYWORDS bladder, inflammation, pain, hyperactivity, resiniferatoxin OBJECTIVE To evaluate the effect of intravesical resiniferatoxin on spinal c  - fos   expression and bladder volume at reflex micturition in rats with chronic urinary bladder inflammation. MATERIALS AND METHODS Of three groups of female Wistar rats, group 1 received cyclophosphamide (75 mg/kg body weight) intraperitoneally every third day (cyclophosphamide is an antitumoral agent that induces bladder inflammation after urinary excretion of its metabolite, acrolein); group 2 comprised sham-inflamed rats that received saline instead of cyclophosphamide, and group 3 received cyclophosphamide, as group 1, every third day but plus 10 nmol/L resiniferatoxin intravesically, through a urethral catheter, at 7 days. At 8 days, under INTRODUCTION Chronic inflammation of the urinary bladder reduces the threshold for excitation of type C bladder sensory fibres [1]. As these fibres encode for nociceptive stimuli and drive an involuntary spinal micturition reflex, such a change may enhance pain perception, leading to allodynia and hyperalgesia, and facilitate a micturition reflex causing frequency, urgency and urge incontinence [1,2]. The inactivation of C-fibres innervating inflamed bladders may therefore constitute a useful strategy for treating patients with chronic inflammatory bladder conditions, including interstitial cystitis. Recent studies in a rat model of chronic bladder inflammation suggested that, at least for allodynia and hyperalgesia, the strategy could be effective. Indeed, sensory desensitization by high systemic doses of capsaicin prevented the spinal expression of c -fos   brought about at the L6 spinal cord segment by innocuous distension of the urinary bladder [3]. This segment is the area of termination of most bladder afferents [4] and c-fos   is an immediate early gene that, in the spinal cord, is predominantly activated by nociceptive sensory input [5].Severe respiratory and CNS toxicity precludes the systemic administration of capsaicin to humans [6]. Topical bladder application is also hindered by the extreme pungency of the compound. Nevertheless, a few clinical trials in patients with neurogenic detrusor overactivity of spinal srcin suggested that intravesical capsaicin could reduce frequency, urgency and urge incontinence after the inactivation of an involuntary C-fibre-driven spinal micturition reflex [7–9]. The effect of intravesical capsaicin on bladder pain in patients with interstitial cystitis, despite some anecdotal reports [8,10] remains essentially undetermined.Resiniferatoxin, a less pungent capsaicin analogue [6,11] is now being used instead of capsaicin to investigate the therapeutic role of bladder desensitization. Intravesical resiniferatoxin was shown to effectively improve frequency, urgency and urge incontinence in patients with neurogenic and non-neurogenic forms of detrusor overactivity [12,13]. Therefore, in theory, intravesical application of this compound should also be effective in reducing bladder pain and frequency in patients with interstitial cystitis. However, in a recent small placebo-controlled trial intravesical resiniferatoxin only had a very marginal effect on those symptoms [14]. The poor success of intravesical resiniferatoxin is surprising, considering the efficacy of systemic capsaicin  DINIS ET AL. 154 ©  2004 BJU INTERNATIONAL in reducing nociceptive input in chronic inflammatory bladder models [3]. Possible explanations are the low concentration of resiniferatoxin used [14] or the different effects of systemic capsaicin and topical resiniferatoxin on bladder sensory nerves. While systemic capsaicin induces a generalized degeneration of bladder sensory nerves within a few hours after administration, topical resiniferatoxin does not [15].While the present discrepancies between experimental data with systemic capsaicin and clinical results with intravesical resiniferatoxin are not re-appraised by a large controlled clinical study, experimental studies may contribute to clarifying the problem. In the present study we assessed the nociceptive input generated by an innocuous bladder distension in rats with chronic inflamed urinary bladders by measuring spinal c- fos   expression at the L6 spinal cord segment; we also measured the volume to reflex micturition. The effect of intravesical resiniferatoxin on both variables was then evaluated. Preliminary data were presented previously as an abstract [16]. MATERIALS AND METHODS Adult female Wistar rats from the Gulbenkian Foundation-derived colony (250 g) were used. Cyclophosphamide and resiniferatoxin were purchased from Asta Medica (Germany) and Sigma (Portugal), respectively. A 10 nmol/L resiniferatoxin solution (6.29 ng/mL) was prepared using 10% ethanol in saline as the vehicle. Experimental manipulations were approved by the Ethics Committee of our institution and followed the guidelines of the International Association for the Study of Pain [17], to minimize animal suffering.SPINAL C- FOS   EXPRESSIONChronic bladder inflammation was induced in five animals by intraperitoneal cyclophosphamide (75 mg/kg) every third day [3]. At 8 days the animals were anaesthetized with subcutaneous urethane (1.2 mg/kg). The body temperature was monitored by a rectal thermometer and maintained at 36–37 ∞ C with a heating pad. The urinary bladder was then exposed through a low midline abdominal incision and a 21 G needle inserted in the bladder dome. Saline at body temperature was infused at 6 mL/h for 2 h. Free outflow of saline through the urethra was possible during bladder contractions to avoid the occurrence of a noxious bladder overdistension. Finally animals were perfusion-fixed through the ascending aorta with 100 mL of 0.1 mol/L PBS followed by 1 L of 4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4 [11]. These animals constituted group 1. Another five rats (group 2) received saline every third day in a volume similar to that in group 1; at 8 days these animals were anaesthetized with subcutaneous urethane (1.2 mg/kg) and treated as described for group 1. In another group of five rats (group 3), bladder inflammation was induced as described for group 1 but at 7 days they were anaesthetized with halothane (4% for induction and 2% for maintenance) and their bladders instilled through a urethral catheter with 0.5 mL of 10 nmol/L resiniferatoxin solution for 30 min. At 8 days the animals were re-anaesthetized with subcutaneous urethane (1.2 mg/kg) and treated as described for group 1.After perfusion, L6 spinal cord segments were removed, immersed for 2 h in the same fixative and for 24 h in 30% sucrose in PBS, and cut in a freezing microtome into 40 m m transverse sections. One in every four consecutive sections was immersed and stored in a cryoprotective solution and kept at - 20 ∞ C [11]. When all animal experiments were concluded, the stored sections were washed with PBS and incubated in the same bath with a polyclonal antiserum against Fos protein raised in rabbit (Oncogene Science, Gaithersburg, USA) and diluted at 1 : 10.000. The immune reaction was visualized by the avidin-biotin method (Vector Labs, Burlingame, CA). Fos-immunoreactive cells in 10 sections taken at random from each animal were counted under light microscopy, as described previously [11].EVALUATION OF BLADDER VOLUME TO REFLEX MICTURITIONEight rats in each group were prepared as described above; after being anaesthetized with subcutaneous urethane (1.2 mg/kg) the urinary bladders were exposed through a low midline abdominal incision and a 21 G needle inserted in the bladder dome. The bladders were then left to stabilize for 30 min while the body temperature was monitored and maintained at 36–37 ∞ C with a heating pad. The bladders were then emptied and saline at body temperature infused at 6 mL/h while the bladder pressure was constantly monitored with a pressure transducer. The infused volume that evoked reflex bladder contractions leading to urine expulsion through the urethra was measured, and taken as the bladder volume to reflex micturition.Results are presented as the mean ( SD ) number of Fos immunoreactive cells per spinal cord section and bladder volume to reflex micturition. Mean differences were compared by one-way ANOVA , followed by the posthoc Bonferroni test for multiple comparisons, with P    <  0.05 considered to indicate statistical significance. RESULTS At laparotomy, rats in groups 1 and 3 had inflamed bladders with abundant petechial haemorrhages on the serosal surface. In some of these animals the urine was bloody. The other abdominal organs of these animals were intact, because bladder inflammation was induced by the urinary excretion of acrolein, a hepatic metabolite of cyclophosphamide [18]. Rats in group 2 had healthy bladders and normal urine coloration.Continuous saline infusion into the bladder of group 2 rats induced a moderate bilateral c- fos   expression, mainly restricted to the most dorsal layer of the L6 spinal cord segment (lamina I), to the intermediolateral grey matter and to the dorsal commissure (Fig. 1A). The mean number of c- fos   cells per section in these animals was 42 (9). Saline infusion in the group 1 rats induced an intense bilateral Fos immunoreactivity; positive cells were particularly numerous in the intermediolateral grey matter and in the dorsal commissure (Fig. 1B). The mean number of c- fos   cells in these animals was 85 (21). In group 3 the number of Fos immunoreactive cells in the three spinal cord areas was similar to that found in group 2 (Fig. 1C), at 55 (10) and 42 (9), respectively ( P    =  0.22). Both these groups had less c- fos   cells than group 1 ( P    =  0.002, 1 vs 2; 0.02, 1 vs 3).Reflex micturition occurred at 0.26 (0.09), 0.49 (0.18) and 0.52 (0.11) mL in groups 1 to 3, respectively ( P    =  0.02, 1 vs 2; 0.74, 3 vs 2; 0.003, 1 vs 3). Examples of typical cystometrograms obtained in the three groups of animals are shown in Fig. 2.  INTRAVESICAL RESINIFERATOXIN IN CHRONIC INFLAMED URINARY BLADDERS ©  2004 BJU INTERNATIONAL 155 DISCUSSION This study showed that topical resiniferatoxin instilled into chronically inflamed rat bladders abolishes the spinal c- fos   overexpression evoked by innocuous bladder distension in a similar way to that previously shown for systemic capsaicin [3]. In addition, the study also showed that in the inflamed bladder intravesical resiniferatoxin restored the normal bladder volume to reflex micturition.C- fos   activation occurs in spinal neurones on the trans-synaptic arrival of noxious input conveyed by unmyelinated primary afferent fibres [5]. In accord, previous studies have shown that rats, intact or exposed to innocuous bladder stimuli, have a low spinal c- fos   reaction [11,19]. In contrast, rats exposed to noxious bladder stimuli have a strong increase of the gene expression in lamina I, intermediolateral grey matter and dorsal commissure at L6 spinal cord segment [11,19]. The present stimulus, a slow saline infusion through the bladder dome leading to periodic micturition through the urethra with no bladder over-distension, was innocuous. Indeed, saline infusion in humans is common during urodynamic testing and does not usually evoke pain. In accordance, spinal c- fos   expression in sham animals was low. Thus, the stronger c- fos   expression in the inflamed rats during the same innocuous stimuli should be taken as an indirect evidence of the occurrence of allodynia and hyperalgesia. Conversely, the decrease of c- fos   expression after intravesical resiniferatoxin in the inflamed bladders strongly suggests that this compound effectively reduces bladder pain perception.Resiniferatoxin is a specific ligand of the vanilloid receptor type 1 (VR1), also known as transient receptor potential vanilloid 1, largely expressed in bladder sensory fibres [20,21]. Thus the present results also suggest the involvement of VR1 in allodynia and hyperalgesia caused by chronic bladder inflammation. Previous data, although not obtained from the urinary bladder, already suggested such a hypothesis. In mice, genetic inactivation of the VR1 gene prevented the occurrence of thermal hyperalgesia in the carrageenan-inflamed hind-paw model [22]. In guinea pigs, capsazepine, a selective VR1 antagonist, prevented the development of mechanical hyperalgesia in the Freund’s complete adjuvant inflamed hind-paw model [23]. A nerve growth factor (NGF)-dependent  VR1 over-expression and VR1 sensitization may contribute to these phenomena in the inflamed skin [24,25]. Although an excess of  VR1 has yet to be reported in the inflamed bladder, this is probable considering that NGF increases in this organ during turpentine- [2] or bacterial- [26] induced inflammatory states. In addition it is conceivable that inflammation-induced local acidosis can modulate VR1 and decrease its threshold. In support of this hypothesis is that acidosis was already shown to decrease the VR1 heat threshold from 42 ∞ C to body temperature [27].Intravesical resiniferatoxin also increased the volume of reflex micturition in the inflamed bladders. A direct interaction of resiniferatoxin with VR1 to obtain this effect could be difficult to sustain in the light of the initial VR1 characterization. Indeed, VR1 was described as an ion channel responding to temperature and protons, but not to pressure [28]. Nevertheless, in a recent study, there was an increased bladder capacity to reflex micturition in VR1 ‘knockout’ mice [29] suggesting that in the bladder VR1 has, directly or indirectly, mechanosensitive properties. Therefore, VR1 overexpression [24] or sensitization [25] during inflammation might reduce its pressure threshold. Alternatively, resiniferatoxin might interact with other mechanosensitive receptors co-expressed with VR1. The P2X 3  receptor, a purinergic receptor subtype, was also shown to encode the degree of bladder filling, as ‘knockout’ mice for this receptor have larger bladder capacities than wild-type strains [30]. P2X 3  receptors can be activated by ATP released from urothelial cells [30]; these cells also express VR1 receptors, the activation of which was shown to release ATP [31]. Thus desensitization of VR1 receptors in urothelial cells could decrease ATP release and, consequently, P2X 3  receptor activation. However, attempts to desensitize VR1 receptors in urothelial cells with vanilloid FIG. 1. Photomicrographs of transverse sections from L6 spinal cord segments showing the distribution of Fos-immunoreactive cells in group 2 (A)  , 1 (B) and 3 (C) rats while micturition was induced by saline infusion through the bladder dome. Fos cells occur predominantly in lamina I (LI), intermediolateral grey matter (ILG) and in the dorsal commissure (DCM). There were more Fos cells in group 1, with similar numbers in 2 and 3. DCMILGILGLILI ABC FIG. 2. Typical cystometrograms obtained group 2 (A), (B) and 3 (C) rats while saline was infused through the bladder dome at 6 mL/h. Arrows indicate the first voiding contraction. Cm H 2 O10050010050010050010 minutes ABC  DINIS ET AL. 156 ©  2004 BJU INTERNATIONAL compounds were apparently unsuccessful [31].The mechanism by which intravesical resiniferatoxin inactivates VR1, generally known as desensitization, is still poorly understood but includes a marked decrease in the number of VR1 receptors present in the urinary bladder. Indeed, intravesical resiniferatoxin reduced VR1 immunoreactivity in the nerve fibres in that organ, both in rats [21] and in humans [32]. Nerve fibre degeneration, a well-known consequence of the systemic administration of capsaicin, was suggested as the main factor in such a decrease [33]. However, an ultrastructural study was unable to detect damaged nerve fibres in the rat bladder exposed to intravesical resiniferatoxin [15]. Hence, other explanations for the resiniferatoxin-induced  VR1 decrease in the bladder must be sought. Current possibilities include receptor internalization [34] or conformational modifications of its structure induced by ligand binding [35]. Another possibility to explain the VR1 decrease might be a prolonged down-regulation of VR1 synthesis.  VR1 gene translation is NGF-dependent [33] and intravesical resiniferatoxin might decrease the amount of that neurotrophin in bladder sensory neurones [36].In conclusion, the present study suggests that intravesical resiniferatoxin, like systemic capsaicin [3], can prevent allodynia, hyperalgesia and hyperactivity caused by chronic bladder inflammation. Thus, despite the poor results of the first attempts to treat patients with painful bladder syndromes using intravesical resiniferatoxin [14], a large prospective controlled trial should be undertaken in the future. Hopefully, resiniferatoxin concentrations and administration schedules different from those used to date might confirm the therapeutic properties of intravesical resiniferatoxin suggested by the present study. CONFLICT OF INTEREST None declared. Source of funding: FCT project POCTI 32466/NSE/2000 and a Boehringer Ingelheim research grant. REFERENCES 1  Yoshimura N, De Groat WC. Increased excitability of afferent nerves innervating rat urinary bladder after chronic bladder inflammation. J Neurosci   1999; 19 : 4644–532 Koltzenburg M, McMahon SB. Mechanically insensitive primary afferents innervating the urinary bladder. In Gebhart GF, ed. Progress in Pain Research and Management. Visceral Pain.   Vol. 5. Chapt 8. Seattle: IASP Press, 1995: 163–923  Vizzard MA. Alterations in spinal cord Fos protein expression induced by bladder stimulation following cystitis. Am J Physiol   2000; 278 : R1027–394 Nadelhaft I, Booth AM. 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