Alvaro Lucioni, Department of Surgery, Section of Urology, University of Chicago Pritzker School of Medicine, 5841S. Maryland Avenue, MC 6038, Chicago, IL 60637, USA. e-mail: email@example.com
To determine the effect of botulinum toxin type A (BTX-A) on the release of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from isolated bladder preparations after acute injury with HCl and the induction of cyclophosphamide (CYP)-induced cystitis, as neurogenic inflammation has been increasingly identified in urological disorders such as interstitial cystitis.
MATERIALS AND METHODS
Adult rats had either an intraperitoneal injection with CYP or saline over a 10-day period to induce chronic bladder inflammation, after which the bladder was harvested, or normal bladder explants were injured acutely with incubation (20 s) in HCl (0.4 m). To measure the effect of BTX-A on the release of neurotransmitters, harvested bladders were incubated in an organ bath containing BTX-A (10 U) or vehicle. Bladders were transferred to a subsequent bath (physiological saline) and incubated for 15 min, and the bathing medium analysed to measure neurotransmitter release, as determined by radioimmunoassay. Bladder specimens from sham treatment, controls and experimental rats were compared histologically.
Acute injury with HCl caused a significantly greater release of both CGRP and SP release (1235 and 1655 pg/g, respectively) than in controls (183 and 449 pg/g, respectively; P < 0.001). This increase in neurotransmitter release was partly inhibited by exposure to BTX-A (870 and 1033 pg/g (P < 0.05 and <0.01). CYP-induced chronic inflammation caused significantly greater release of SP than in the controls (1060 and 605 pg/g, respectively; P < 0.005). Exposure to BTX-A partly inhibited the release of SP after CYP-induced cystitis (709 pg/g, P < 0.05).
The application of BTX-A inhibits the release of sensory neurotransmitters from isolated bladder preparations in rat bladder models of both acute injury and chronic inflammation, suggesting a potential clinical benefit of BTX-A in the treatment of neurogenic inflammation.
Bladder inflammation is known to induce significant neurochemical, electrophysiological, and organisational changes in afferent bladder pathways . The afferent action of the bladder represents a complex network involving unmyelinated and myelinated sensory neurones, neuropeptides, and interactions of supporting cells, including interstitial and inflammatory cell types. Studies using models of inflammation show changes in the expression of sensory neuropeptides and neuropeptide receptors in bladder tissue, and in lumbosacral micturition pathways . Clinically, such alterations might result in generalized manifestations like a reduced pain threshold (allodynia) and amplification of pain sensation (hyperalgesia) , or in more specific bladder symptoms, including increased voiding frequency .
As part of the sensory response to inflammation, the role of the sensory neurotransmitters substance P (SP) and calcitonin gene-related peptide (CGRP) has been evaluated. SP is a potent tachykinin that is found within peripheral afferent neurones and produces a significant inflammatory response through activation of distinct neurokinin (NK) receptor types . Based on these actions, investigations have identified the involvement of SP in the many responses to bladder inflammation, e.g. plasma extravasation and immune cell activation . Similarly, CGRP is a sensory neuropeptide found in capsaicin-sensitive neurones of the bladder, and is significant in the neurogenic inflammatory response through actions involving vasodilatation, leukocyte adhesion and tissue oedema .
During the last decade, intradetrusor injection with botulinum toxin type A (BTX-A) has emerged as a successful treatment for neurogenic and non-neurogenic detrusor overactivity (DOA) . The use of BTX-A was initiated based on its ability to block the neuronal release of acetylcholine and therefore to inhibit the abnormal smooth muscle contraction thought to underlie these disorders . However, accumulating evidence suggests that abnormalities in the afferent actions of the bladder also characterize DOA, as well as additional inflammatory bladder conditions such as interstitial cystitis (IC) . Concurrently, significant evidence also suggests that BTX-A has additional effects on sensory nerve function. Accordingly, BTX-A has been shown to decrease sensory neuropeptide release from dorsal root ganglion (DRG) neurones, whole bladder samples, and cultured urothelial cells [4,6,7]. In addition, sensory neuropeptide receptor expression is reduced after treatment with BTX-A .
Given the mounting evidence that afferent neurone actions and neurogenic inflammation might be involved in the pathophysiology of various urological disorders, we devised the present study. Our objective was to determine if acute HCl injury and/or chronic cyclophosphamide (CYP)-induced inflammation increased the release of sensory neurotransmitters in whole bladder specimens, and whether BTX-A inhibits this neuropeptide release.
MATERIALS AND METHODS
The experiments were conducted using adult male Sprague-Dawley rats (300–350 g), housed two per cage and with food and water freely available. The housing units were maintained at a constant temperature (36–37 °C), and animal care and experimentation complied with the University of Chicago Institutional Animal Care and Use Committee guidelines.
Cystitis was induced using the chronic CYP model described by Vizzard , which produces chemical cystitis through the action of the CYP metabolite, acrolein. CYP was administered by i.p. injection, 150 mg/kg every third day to a total of three doses, to achieve chronic inflammation. An additional group of rats had a sham injection using a corresponding volume of normal saline (0.9%). All rats were killed 24 h after the last CYP injection.
The experimental protocol was approved by the University of Chicago Hospitals Institutional Animal Care and Use Committee (ACUP 71526); care was taken to minimize stress and/or discomfort, and accordingly, the rats were assessed daily by both the facility and laboratory staff. General monitoring included an assessment for evidence of discomfort or stress, e.g. behavioural change, lack of mobility and poor oral intake. All rats were weighed at the time of each injection. In general, rats treated with CYP had a ≈10% total weight loss at the time of death. No rats were killed before the scheduled time.
Neuropeptide release was assessed as previously reported ; briefly, the rats were killed after the induction of cystitis by exposure to 100% CO2 and bilateral thoracotomy. The whole bladders were removed, cleaned of adherent connective tissue, emptied of urine and weighed. The bladders were bivalves and affixed (one bladder per experiment) to a polystyrene rod using synthetic absorbable sutures (glycolide, dioxanone and trimethylene carbonate; Biosyn, United States Surgical, Norwalk, CO, USA) . The bladders were washed (30 s per incubation) in four aerated (95% O2: 5% CO2, 22° C) tissue baths (total volume 1.0 mL) that contained physiological saline (PS) to remove tissue debris (composition of PS, mm: KCl 5, NaCl 144, MgCl2 1, CaCl2 2, HEPES 10, glucose 10, pH 7.4). After washing, the bladders were transferred to another bath (PS, 1.0 mL) and incubated (6 h) in an organ bath containing BTX-A (10 U) or vehicle. The bladders were then incubated in a subsequent tissue bath to equilibrate after drug exposure (1.0 mL, 15 min per incubation) in PS. Finally, the bladders were transferred to another bath (PS, 1.0 mL) and incubated (15 min). The entire volume of this organ bath was collected and divided into 100 µL aliquots to measure the release of neuropeptide.
To induce HCl injury and assess neuropeptide release, normal rat bladders were harvested and prepared for tissue incubation as described above (CYP study). After washing, bladders were incubated (10 s) in two consecutive baths (1.0 mL) containing HCl (0.4 m) or vehicle to allow for tissue injury. Bladders were then incubated (1 h) in PS (1.0 mL) to allow equilibration after injury, with a subsequent incubation (1 h) in BTX (10 U) or vehicle, after which the bladders were incubated (15 min) in PS (1.0 mL) to allow equilibration after drug exposure. Finally, the bladders were incubated (15 min) in a separate organ bath (PS, 1.0 mL), after which the volume of this bath was collected and divided into 100 µL aliquots to measure neuropeptide release.
Pharmaceutical grade CYP was obtained from Baxter (Deerfield, IL, USA); the purchasing, handling and preparation of CYP for experimental use were done exclusively by the Department of Pharmacy staff. CYP was prepared in distilled water and diluted to the final concentration of 10 mg/mL. The handling and administration of CYP was in strict accordance with the Department of Health guidelines for chemotherapeutic agents. BTX-A was purchased from Allergan Inc, Irvine, CA, USA, prepared in sterile, preservative-free saline and diluted to final concentration. BTX-A was added directly to the PS bath immediately before bladder incubation. All other compounds were obtained from Sigma Chemical Co, St Louis, MO, USA.
For the histological assessment, adult rats were killed and the bladder dissected as above; the experiments were as previously described and tissue fixed to correspond with the time that the neurotransmitter release was measured. In addition, whole-bladder specimens were harvested from normal rats to assess histological differences in the sham-treated, control and experimental bladders. Bladders were fixed in 10% neutral buffered formalin; the entire bladder was sectioned and submitted for standard processing and paraffin embedding. Tissue sections (5 µm) were stained with haematoxylin and eosin, and images captured using a light microscope with a digital camera.
CGRP and SP were measured using commercially available radioimmunoassay kits (Phoenix Pharmaceuticals Inc, Belmont, CA, USA). Standard curves were produced for each experiment, and the concentration of CGRP and SP expressed as peptide released per bladder tissue weight. The detection limits of the radioimmunoassay were 1 pg/mL. For the tissue baths and incubations, polystyrene tubes were used, to reduce nonspecific peptide binding.
Quantitative data are expressed as the mean (sem) and analysed statistically using Student’s t-test, with P < 0.05 considered to indicate statistical significance.
The effect of CYP-induced cystitis on SP and CGRP release was examined (Fig. 1); the mean bladder weight in the control and CYP-treated rats was 0.20 and 0.23 g, respectively. The release of SP in control rats was 605 (53) pg/g, but in the CYP-treated rats this increased to 1060 (125) pg/g, a 75% increase (P < 0.005). There was no statistically significant increase in CGRP release over the control in rats with CYP-induced cystitis. These results show that CYP successfully induced cystitis, as shown by the greater inflammatory neuropeptide release.
We then examined the effect of BTX-A on CYP-induced SP release (Fig. 1); the effect of BTX-A on CGRP release was not assessed, as preliminary studies failed to detect a statistically significant signal over baseline CGRP release after inducing cystitis with CYP. BTX-A reduced SP release in the CYP-treated group, to 709 (91) pg/g, less than a quarter of the increase caused by CYP treatment alone (P < 0.05). In addition, the SP release in BTX-A-treated rats was not statistically significantly different from that in control rats (P = 0.35). These results show that BTX-A can inhibit the release of sensory neuropeptide in a state of chronic inflammation.
The effect of acute HCl injury on CGRP and SP release was then assessed (Fig. 2); the mean bladder weight in control and HCl-treated rats was 0.11 and 0.10 g, respectively. The release of CGRP and SP in the control rats was 183 (59) and 449 (332) pg/g, respectively; CGRP and SP release in the HCl-treated group increased to 1235 (328) and 1655 (190) pg/g, respectively, a 6.7-fold and 3.7-fold increase in CGRP and SP release over the control, respectively (P < 0.001). These results show a significant increase in sensory neuropeptide release after acute injury with HCl.
BTX-A applied after injury reduced CGRP release in the HCl-treated group to 870 (175) pg/g, a 30% decrease compared with CYP-treated rats not receiving BTX-A (P < 0.05). BTX-A also reduced the release of SP by 38%, to 1033 (429) pg/g (P < 0.01). SP release in BTX-treated rats was not statistically different from the release in control rats (P = 0.35). These results show that BTX-A can partly inhibit the release of sensory neuropeptide after acute injury.
The histological assessment of sham-treated bladder specimens showed a normal urothelium, lamina propria and muscularis (Fig. 3). There were several abnormalities in CYP-treated and HCl-injured bladder specimens, with atrophied epithelium, focally consisting of a single cell layer. There was significant cytological atypia of the epithelial cells, with prominent ‘hob-nailing’ and large, hyperchromatic nuclei. The lamina propria was haemorrhagic. Furthermore there was significant tissue oedema compared with the controls. Treatment with BTX-A caused no histological changes in the bladder specimens.
We examined the effect of BTX-A on the release of sensory neurotransmitters after acute injury with HCl and chronic cystitis induced by CYP. There are two important conclusions: first, acute and chronic injury using established models produced significantly greater release of sensory neurotransmitters from whole bladder specimens than from sham-treated rats. Second, applying BTX-A after inducing inflammation significantly decreased the sensory neurotransmitter release, to levels consistent with those in control rats.
Studies with animal models show that both acute and chronic inflammation has numerous effects on the afferent neurone functions of the bladder. The present study used a well-characterized model of chronic inflammation reported by Hu et al.. Using this model, Vizzard  showed that CYP-induced inflammation produced significant changes in the neurochemical, electrophysiological and organisational properties of afferent pathways in the rat urinary bladder. Other animal models suggested that chronic inflammation can cause the recruitment of afferent pathways that are normally unresponsive to mechanical stimuli, and/or decrease the threshold of afferent nerves to noxious and benign stimuli [1,9]. There are similar but distinct abnormalities in models of acute injury, and these appear to represent a progressive reorganisation of micturition reflex pathways [1,2]. However, in both states these changes appear to be part of a complex immunosensory response to inflammation, involving afferent neurones, neurotransmitters (e.g. SP and CGRP), and inflammatory cell types (e.g. mast cells) .
As part of this immunosensory loop, the role of sensory neurotransmitters is important. Numerous studies in both animal and humans show changes in CGRP and SP activity in states of inflammation. Accordingly, there are changes in the expression of CGRP and SP in both the rat urinary bladder and in back-labelled DRG neurones after the induction of acute or chronic inflammation [1,11]. Bladder biopsies from patients with IC and DOA also show similar alterations in the expression of neuropeptide and neuropeptide receptors [12,13]. Based on these data, it is suggested that increases in a specific class of nerve fibres containing SP and CGRP might underlie the abnormalities in the afferent arm of the micturition cycle associated with DOA . Intrathecal injection of an SP antagonist reduced DOA caused by CYP-induced cystitis, further suggesting a direct role of SP in the inflammatory pathway .
Concurrently, increasing evidence suggests that BTX-A inhibits the afferent actions of the bladder. There is inhibition of bladder urothelial ATP release after applying BTX-A, suggesting that BTX-A might inhibit sensory function through modulation of purinergic pathways . BTX-A is reported to inhibit both normal CGRP release and capsaicin-induced DOA in rat models, providing evidence of additional effects on TRPV1 pathways [7,16]. These proposed mechanisms are supported by the reduced expression of the TRPV1 and P2X3 receptors in patients with DOA, a finding that corresponded with the clinical benefit in these patients . Finally, BTX-A inhibits a calcium-dependent release of SP from rat DRG neurones in primary culture, supporting an additional mechanism of sensory neuromodulation through NK-mediated pathways . Evidence suggests that SP might share a common pathway, resulting in the activation of TRP channels, and that SP might potentiate the susceptibility of the P2X3 receptor to ATP activation [17,18]. These data would support the possibility that BTX-A-induced inhibition of purinergic and TRPV1-mediated pathways might occur through the additional actions of BTX-A on CGRP and SP release. Combined, these findings have led authors to propose that the effect of BTX-A on bladder sensory actions might result from a combined inhibition of sensory neurotransmitter release and neurotransmitter receptor expression, culminating in purinergic and TRPV1 pathway inhibition .
The present findings extend the work of previous authors by showing that BTX-A can inhibit the release of CGRP and SP from inflammatory whole-bladder specimens. We could find no other study directly quantifying bladder CGRP and SP release after inflammation and confirming the inhibition of release with BTX-A. Despite evidence showing a BTX-A-induced decrease in CGRP and SP expression of dorsal horn neurones, this finding was not corroborated in animal or human studies using bladder tissue . Further, studies using immunohistochemistry techniques are limited, as changes in expression might result from altered neuropeptide synthesis or breakdown, in addition to changes in release.
The exact mechanism of BTX-A-mediated inhibition of CGRP and SP release is unknown. Previous studies show that BTX-A-induced inhibition of sensory neurotransmitter release from bladder DRG neurones is associated with a concentration-dependent cleavage of synaptosome-associated protein (SNAP)-25 . This finding suggests the possibility that the sensory-specific effect of BTX-A might occur through inhibition of sensory neuropeptide vesicle release via a SNAP-mediated mechanism, similar to the blockade of acetylcholine release in motor neurones. However, other possible mechanisms of action cannot be excluded. SP exerts its biological actions through activation of the NK receptor class, which are present on mast cells and bladder myofibroblasts, as shown in animal studies [5,19]. As previously described, BTX-A is known to decrease bladder expression of other classes of sensory neuropeptide receptors, including purinergic and TRPV1 receptors . Accordingly, BTX-A-mediated inhibition of CGRP and SP receptor expression remains possible, and has been proposed as a possible mechanism of action . Investigations to better define the distribution of the expression of CGRP and SP receptors on bladder cellular types, and the effect of BTX-A on receptor expression and function, are needed.
The exact source of CGRP and SP detected in our assay remains unclear, due to the complex cellular architecture of a whole-bladder model. CGRP is primarily found in unmyelinated afferent neurones, suggesting a role of CGRP in nociception. Within the bladder, afferent neurones were shown to be the only cell type to contain high levels of CGRP . SP is a sensory neuropeptide found in myelinated Aä and unmyelinated C-fibres . However, immune cell types are also known to express and release SP . As such, whereas CGRP release detected in our assay is suspected to result primarily from actions on afferent fibres, additional release from immune cell populations might also be responsible for a portion of the SP release. Despite this issue, our model provides important information not previously examined. Other investigations evaluating the effect of BTX-A on sensory neuropeptide release used isolated DRG neurones, making difficult any conclusions about the site of action within the neurone. Using our model we sought to evaluate the effect of BTX-A in an end-organ, thereby isolating the neuronal nerve endings and local cellular populations. Given the present results, this model suggests that future study aimed at evaluating CGRP and SP receptor subtypes present on sensory nerve endings, urothelial cells, and other local cellular populations might provide more specific information on the possible sites and/or mechanisms of action of BTX-A.
In conclusion, the present study provides direct evidence that that both acute injury with HCl and CYP-induced cystitis increase the release of sensory neuropeptides in an isolated rat bladder model. Further, BTX-A partly but significantly inhibited the release of these neuropeptides after acute and chronic injury. These findings suggest a potential role of injections with BTX-A in urological disorders characterized by abnormal sensory activity and neuropeptide release, e.g. IC and DOA. Further study is needed to better delineate the effect of BTX-A on CGRP and SP receptor activity/expression, and is the focus of future study.
The authors acknowledge Katherine W. Turk for technical assistance.