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1 Present address: University of Alabama at Birmingham, Department of Anesthesiology, Birmingham, AL.
Affiliations
Department of Neurobiology, University of Pittsburgh, Pittsburgh, PennsylvaniaPittsburgh Center for Pain Research, University of Pittsburgh, Pittsburgh, Pennsylvania
Department of Neurobiology, University of Pittsburgh, Pittsburgh, PennsylvaniaPittsburgh Center for Pain Research, University of Pittsburgh, Pittsburgh, Pennsylvania
Department of Neurobiology, University of Pittsburgh, Pittsburgh, PennsylvaniaPittsburgh Center for Pain Research, University of Pittsburgh, Pittsburgh, PennsylvaniaCenter for Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania
Department of Neurobiology, University of Pittsburgh, Pittsburgh, PennsylvaniaPittsburgh Center for Pain Research, University of Pittsburgh, Pittsburgh, PennsylvaniaCenter for Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania
1 Present address: University of Alabama at Birmingham, Department of Anesthesiology, Birmingham, AL. 2 Present address: University of New England, College of Osteopathic Medicine, Biddeford, ME.
We determined whether treatment with an artemin sequestering antibody (α-ARTN) affected cyclophosphamide-induced bladder hyperalgesia and bladder primary afferent function.
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α-ARTN effectively blocked the development of bladder hyperalgesia and reversed hyperalgesia once it was established.
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α-ARTN normalized noxious bladder distension–induced spinal expression of phospho-ERK1/2 immunoreactivity.
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Calcium imaging experiments of bladder afferents using mustard oil indicate that ARTN may regulates neuronal function via TRPA1.
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α-ARTN may be an effective therapeutic strategy for individuals suffering from interstitial cystitis–related bladder pain.
Abstract
Injury- or disease-induced artemin (ARTN) signaling can sensitize primary afferents and contribute to persistent pain. We demonstrate that administration of an ARTN neutralizing antibody, anti-artemin (α-ARTN), can block the development of, and reverse already established, bladder hyperalgesia associated with cyclophosphamide-induced cystitis in mice. We further demonstrate that α-ARTN therapy blocks upregulation of TRPA1, an ion channel contributing to persistent bladder pain during cyclophosphamide-induced cystitis, and decreases phospho-ERK1/2 immunoreactivity in regions of the spinal cord receiving bladder afferent input. Thus, α-ARTN is a promising novel therapeutic approach for treatment of bladder hyperalgesia that may be associated with interstitial cystitis/painful bladder syndrome, as well as cystitis associated with antitumor or immunosuppressive cyclophosphamide therapy.
Perspective
α-ARTN therapy effectively prevented and reversed ongoing bladder hyperalgesia in an animal model of cystitis, indicating its potential as an efficacious treatment strategy for ongoing bladder pain associated with interstitial cystitis/painful bladder syndrome.
Pelvic/suprapubic pain is a cardinal symptom of interstitial cystitis/painful bladder syndrome (IC/PBS), a chronic condition affecting 2.7 to 6.5% of women in the United States.
In contrast to bacterial cystitis, IC/PBS characteristically occurs in the absence of ongoing infection but may be accompanied by varying degrees of inflammation.
There is no single defining etiology or pathogenesis of IC/PBS; thus, there is no consistently effective pain management strategy to improve quality of life in individuals with the disease.
Animal studies have shown that visceral injury and inflammation are accompanied by increased peripheral growth factor expression, which in turn can drive hyperalgesia.
Studies of individuals with IC/PBS have reported increases in nerve growth factor (NGF) and glial cell line–derived neurotrophic factor (GDNF) in urethral and bladder tissue.
Elevated tryptase, nerve growth factor, neurotrophin-3 and glial cell line-derived neurotrophic factor levels in the urine of interstitial cystitis and bladder cancer patients.
We have previously shown in animal models of skin and colon inflammation that target-derived mRNA for artemin (ARTN), a member of the GDNF family, increases up to 5-fold more than the mRNAs for either NGF or GDNF.
Moreover, a single injection of ARTN into uninflamed skin increases the duration of painlike behavior in vivo and potentiates nociceptor function in vitro.
Recent studies from our laboratories revealed that bladder afferent TRPA1 contributes to persistent hyperalgesia in a mouse model of cyclophosphamide (CYP)–induced cystitis.
Because of the previously demonstrated relationship between ARTN and TRPA1 expression, we hypothesized that treatment with an ARTN-neutralizing antibody (α-ARTN) might be effective in blocking bladder hyperalgesia.
To test our hypothesis, we used a mouse model of cystitis shown previously to elicit changes in TRPA1 expression and function
and quantified urinary bladder expression of growth factor messenger RNAs (mRNAs), bladder primary afferent TRPA1 expression and function, nociceptive behavior, and spinal phospho-ERK1/2 (pERK) immunoreactivity. A single injection of α-ARTN, at either the initiation or the conclusion of CYP treatment, effectively blocked or reversed, respectively, bladder hyperalgesia. α-ARTN prevented CYP-induced upregulation of bladder afferent TRPA1 and reduced the number of activated second-order neurons in the spinal cord that receive bladder primary afferent input. These results suggest that α-ARTN treatment could be effective for people experiencing bladder pain associated with IC/PBS.
Methods
Animals
Experiments were performed on female 8- to 12-week-old C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) housed in the Division of Laboratory Animal Resources at the University of Pittsburgh Medical Center. Mice received food and water ad libitum. All procedures conformed to National Institutes of Health guidelines and were in accordance with those of the University of Pittsburgh Institutional Animal Care and Use Committee and the Committee for Research and Ethical Issues of the International Association for the Study of Pain.
CYP-Induced Cystitis and α-ARTN Treatment
Cystitis was induced by intraperitoneal injection of CYP (100 mg/kg; Sigma-Aldrich, St. Louis, MO) every other day for a period of 5 days (3 injections total). A control group was administered intraperitoneal sterile saline injections on the same schedule. Some mice additionally received an intraperitoneal injection of α-ARTN (10 mg/kg; R&D Systems, Minneapolis, MN) or immunoglobulin G (IgG, 10 mg/kg; R&D Systems) 30 minutes prior to the first CYP or saline injection on day 1; a final group received α-ARTN 30 minutes following the final CYP injection on day 5 (Fig 1). Mice were randomly assigned to all groups. Endpoint measurements were assessed at 1 or 7 days following the final CYP injection.
Figure 1The experimental time line was as follows: cystitis was induced by injection of CYP (100 mg/kg, intraperitoneal) on days 1, 3, and 5; an ARTN neutralizing antibody (α-ARTN; 10 mg/kg, intraperitoneal) was administered to one group of mice 30 minutes prior to CYP on day 1, and to another group of mice 30 minutes following CYP on day 5. Experimental endpoints were collected from these groups on days 6 and 13, respectively.
Extracted RNA (5 μg) was treated with DNase (Invitrogen, Carlsbad, CA) to remove genomic DNA; then, 1 μg was reverse transcribed using Superscript II (Invitrogen). SYBR Green PCR amplification was performed using a 7000 real-time thermal cycler (Applied Biosystems, Waltham, MA). Each sample was run in duplicate, and the threshold cycle (Ct) values were recorded as a measure of initial template concentration of NGF, GDNF, and ARTN and were normalized to Ct values for β-actin. A relative change in mRNA expression was calculated as a ratio of the control group mean for each gene using the Pfaffl method.
Primer sequences used were NGF (F-TCCAATCCTGTTGAGAGTGG, R-CAGGCTGTGTCTATGCGGAT), GDNF (F-AAGGTCACCAGATAAACAAGCGG, R-TCACAGGAGCCGCTGCAATATC), ARTN (F-CTCAGTCTCCTCAGCCCG, R-TCCACGGTCCTCCAGGTG), β-actin (F-AGAGGGAAATCGTGCGTGAC, R-CAATAGTGATGACCTGGCCGT).
Retrograde Labeling of Bladder Afferents
Mice were anesthetized using isoflurane, and the urinary bladder was exposed via laparotomy. Three injections (12 μL total volume) of Alexa Fluor–conjugated cholera toxin-β (CTβ) (Life Technologies, Carlsbad, CA) were made into the bladder wall, and abdominal incisions were sutured. Mice were returned to their home cages and allowed to recover for at least 3 days before initiation of CYP injections.
Single-Cell RT-PCR
Mice were deeply anesthetized with isoflurane and transcardially perfused with cold Ca2+/Mg2+-free Hank's balanced salt solution (HBSS; Invitrogen). Bilateral L5-S1 dorsal root ganglia were dissected into cold HBSS and dissociated.
Cells were plated in Dulbecco's modified Eagle medium–nutrient mixture F-12 media (Invitrogen) containing 10% fetal bovine serum and antibiotics (penicillin/streptomycin, 50 U/mL). Coverslips were flooded with media 2 hours after plating and used the same day.
Individual CTβ+ neurons were collected with large-bore (50 μm) glass pipettes and expelled into microcentrifuge tubes containing RT mix (Invitrogen). For each preparation of isolated cells on which single-cell PCR was performed, 2 negative controls were included: 1 omitting RT and 1 using a cell-free mix as template. The first-strand cDNA from CTβ+ neurons was used as template in a reaction containing 1 × GoTaq reaction buffer (Promega, Fitchburg, WI), 20 μM external primers, .2 M deoxynucleoside triphosphates, and .2 μL GoTaq DNA polymerase (Promega). Each initial PCR product served as template in a subsequent reaction using a nested (internal) primer pair. Products were electrophoresed on 2% agarose-ethidium bromide gels and photographed. Only samples with detectable GAPDH were analyzed. Primer sequences used were TRPA1 (EXT F-CTTCCTGGATTACAACAATGCTCTG, R-ATGTCCCCAACCGCCAAGC; INT F-CAGTGGCAATGTGGAGCAATAG, R-AAGGAAAGCAATGGGGTGC), TRPV1 (EXT F-GGGAAGAATAACTCACTGCCTGTG, R-TCATCCACCCTGAAGCACCAC; INT F-GGCGAGACTGTCAACAAGATTGC, R-TCATCCACCCTGAAGCACCAC), GAPDH (F-GCTGAGTATGTCGTGGAGTCTA, R-CATACTTGGCAGGTTTCTCCAG).
Calcium Imaging
Ca2+ imaging was performed on retrogradely labeled bladder afferent neurons that were isolated as described above. Neurons were incubated in the Ca2+ indicator Fura-2 AM ester (2 μM; Invitrogen) with .02% Pluronic F-127 (Invitrogen) in normal bath solution (in millimoles: 130 NaCl, 5 KCl, 1.5 CaCl2, .9 MgCl2, 20 HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], 5.5 glucose, .5 KH2PO4, .5 Na2HPO4, pH 7.4, osmolality = 325 mOsm) containing 5 mg/mL bovine serum albumin (Sigma-Aldrich) for 20 minutes at 37°C. Coverslips were mounted on an inverted microscope stage (Olympus, Tokyo, Japan) and continuously perfused with normal bath solution. Perfusion rate (5 mL/min) was controlled with a gravity flow and rapid-switching local perfusion system (Warner Instruments, Hamden, CT). Solutions were maintained at 30°C using a heated stage and in-line heating system (Warner Instruments). Firmly attached, refractile CTβ+ cells were identified as regions of interest. A ratio (R) of fluorescence emission at 510 nm in response to excitations at 340 and 380 nm was acquired at 1 Hz (Lambda DG-4 and 10-B SmartShutter; Sutter Instruments, Novato, CA) via camera (ORCA-ER; Hamamatsu Corporation, Bridgewater, NJ) and saved to a computer using HCImage (Hamamatsu Corporation). The intracellular concentration of calcium ([Ca2+]i) was determined following in situ calibration experiments according to the following equation: where Kd is the dissociation constant for Fura-2, Sf2/Sb2 is the fluorescence ratio of the emission intensity excited by 380 nm signal in the absence of Ca2+, and Rmin and Rmax are the minimal and maximal fluorescence ratios, respectively. The procedure for determination of these variables has been described previously.
Only cells responsive to application of 50 mM K+ were used. Following a 5-minute recovery from 50 mM K+ stimulation, the percentage of cells exhibiting TRPA1-mediated Ca2+ influx was assessed by application of mustard oil (MO, 100 μM; Sigma). MO was applied 1 to 3 additional times (10-minute interstimulus interval) to determine whether desensitization (tachyphylaxis) occurred. Then, ARTN was applied for a duration of 7 minutes, and responses to MO were tested again. In neurons in which MO did not evoke Ca2+ influx, ARTN was applied for 7 minutes, and responses to MO were tested again. MO at 100 mM in 1-methyl-2-pyrrolidinone was used as a stock solution; 100 μM MO was made fresh daily in HBSS. Artemin (R&D Systems) was aliquoted at 10 μg/mL in HBSS, stored at −20°C, and diluted in HBSS to a final concentration of 100 ng/mL immediately before use. The magnitude of evoked Ca2+ transients was determined by peak evoked change in intracellular Ca2+ concentration (Δ[Ca2+]i).
Visceral Nociceptive Behavior
Mice were anesthetized via inhaled isoflurane in oxygen (4% induction, 2% maintenance), and a 24-gauge angiocatheter was passed through the urethra into the urinary bladder for delivery of compressed air. Silver wires were inserted into the left abdominal oblique musculature superior to the inguinal ligament for measurement of electromyographic (EMG) responses. Anesthesia was reduced until flexion reflexes were present in the absence of spontaneous escape behavior (∼1% isoflurane). Two 60-mmHg urinary bladder distensions were administered to overcome an initial period of sensitization
and were followed by graded distensions at pressures of 10 to 60 mmHg (10-second duration; 2-minute interstimulus interval). EMG data were recorded, rectified, and saved to a computer using Spike2 software (Cambridge Electronic Design Ltd, Cambridge, UK). Distension-evoked EMG responses were normalized to baseline EMG as follows: EMGevoked – EMGbaseline/EMGbaseline. Normalized responses are presented as a percentage of the maximal response of the sterile saline-treated control group.
Immunohistochemical Analysis of pERK Expression
Mice were deeply anesthetized with isoflurane and perfused with 4% paraformaldehyde. Lumbosacral spinal cord segments were dissected, embedded in gelatin (10% in .1 M phosphate buffer), postfixed for 4 hours in 4% paraformaldehyde, and cryoprotected overnight (30% sucrose) at 4°C. Transverse spinal cord sections (50 μm) were cut on a microtome. Following three 5-minute washes in .1 M phosphate buffer, floating spinal cord sections were incubated for 2 hours at room temperature in a blocking reagent (5% normal horse serum and .1% Triton X-100 in .1 M phosphate buffer), then overnight at room temperature in rabbit anti-human phospho-ERK1/2 antibody (1:4000 in blocking reagent; Cell Signaling Technology, Inc, Danvers, MA). Sections were washed, incubated for 2 hours at room temperature in Cy3-conjugated anti-rabbit IgG secondary antibody (1:500), washed, mounted, and coverslipped. Photographs were taken at ×20 using Leica Application Suite (Leica Microsystems Inc, Buffalo Grove, IL) software. The total number of pERK-positive cells in regions receiving input from bladder afferent neurons (ie, superficial dorsal horn, sacral parasympathetic nucleus, and dorsal commissure
The distribution of visceral primary afferents from the pelvic nerve to Lissauer's tract and the spinal gray matter and its relationship to the sacral parasympathetic nucleus.
) was counted in 9 to 13 sections per mouse by an experimenter (B.K.D., J.J.D.) blinded to treatment. The average for each mouse was calculated and used for statistical analysis.
Statistical Analysis
Statistical analyses were performed using Systat (Systat Software, Inc, Richmond, CA). EMG responses to graded bladder distension were compared using repeated measures analysis of variance followed by post hoc t-tests with Holm's correction.
For real-time PCR data, expression ratios for each target gene in bladder tissue were compared between groups using t-tests. Single-cell PCR data of bladder afferent gene expression were analyzed using χ2 tests and Fisher's exact tests. The total number of cells expressing pERK was compared by analysis of variance and post hoc t-tests. All data are expressed as mean ± standard error of the mean. P values of ≤.05 were considered significant.
Results
Bladder-Derived ARTN and NGF and Bladder Afferent TRPA1 Expression Are Increased Following CYP
We used real-time PCR to determine growth factor mRNA expression and single-cell PCR to determine transient receptor potential (TRP) channel mRNA expression in urinary bladder tissue and bladder afferent neurons, respectively. Expression of bladder-derived ARTN (F(2,18) = 11.26, P = .001) and NGF (F(2,18) = 7.157, P = .020) mRNAs were significantly upregulated 1 day post-CYP, with peak expression at >5-fold (P < .01) and 2.5-fold (P < .05) above control levels, respectively (Fig 2A; n = 7/group). ARTN and NGF returned to baseline levels by 7 days post-CYP. GDNF expression was unchanged (F(2,18) = 3.107, P = .069).
Figure 2CYP upregulates bladder-derived ARTN mRNA concurrent with upregulation and sensitization of bladder afferent TRPA1. (A) Bladder-derived ARTN and NGF mRNAs were significantly upregulated 1 day post-CYP. Expression peaked at >5-fold and 2.5-fold above control levels, respectively. Expression at 7 days post-CYP did not differ from control. GDNF mRNA expression was unchanged. (B) Bladder afferent TRPA1 mRNA expression was significantly increased at 1 day post-CYP and remained increased at 7 days post-CYP. In contrast, CYP had no effect on TRPV1 mRNA expression at either time point. (C) Isolated bladder afferents from naïve mice exhibited robust Ca2+ influx to application of 50 mM K+ (black dash). Approximately 15% of these responded to 100 μM MO (gray dash). In addition, 75% of MO-responsive afferents exhibited profound desensitization (tachyphylaxis) of the TRPA1 channel with repeated MO application (10-minute interstimulus interval). Rescue from tachyphylaxis by ARTN was observed in 28.5% of these neurons (no example shown). (D) ARTN exposure (gray bar; 7 minutes) was efficacious in recruiting de novo responses to MO in 45.8% of bladder afferents that were initially insensitive to MO. (E) As predicted by afferent TRPA1 mRNA expression, the percentage of bladder afferents exhibiting MO-evoked Ca2+ transients was significantly increased 1 day post-CYP relative to controls when mice were pretreated with IgG (the control for α-ARTN). This CYP-induced increase in MO-responsive afferents was prevented in mice pretreated with α-ARTN. *P < .05 versus control, #P < .05 α-ARTN versus IgG.
Differences in the expression of transient receptor potential channel V1, transient receptor potential channel A1 and mechanosensitive two pore-domain K+ channels between the lumbar splanchnic and pelvic nerve innervations of mouse urinary bladder and colon.
we found that bladder afferents arising from lumbosacral dorsal root ganglia express both TRPV1 and TRPA1 mRNA in normal conditions (Fig 2B; n = 18–25/group). After a 5-day series of CYP injections, TRPA1 mRNA expression was significantly increased (χ2(2) = 12.64, P < .01) in both the 1-day post-CYP (P < .01) and 7-day post-CYP (P < .05) groups. However, in contrast to studies of the colon showing significant upregulation of TRPV1 with inflammation (eg,
ARTN Sensitizes MO-Evoked Ca2+ Signaling in Bladder Afferents In Vitro
To determine the effect of ARTN on afferent TRPA1 function, we performed Ca2+ imaging experiments on dissociated bladder afferents (n = 45) from naïve mice. Progressive desensitization (ie, tachyphylaxis) to repeated application of the TRPA1 agonist, MO, was observed in 75% of MO-responsive bladder afferents (Fig 2C). In 28.5% of these neurons, application of ARTN restored MO-evoked responses (data not shown). Furthermore, ARTN induced de novo MO-evoked responses in 45.8% of bladder afferents; that is, nearly half of the afferents that were initially unresponsive to MO became responsive following exposure to ARTN (Fig 2D). On average (including the neurons that responded only after ARTN exposure), the MO-evoked change in [Ca2+]i after ARTN exposure was about 4 times greater than that seen prior to ARTN exposure (data not shown).
α-ARTN Reverses CYP-Induced Hyperalgesia In Vivo and Normalizes Expression of Bladder Afferent TRPA1 and Spinal pERK
We have previously shown that injections of CYP using the model described herein produces sustained bladder hyperalgesia.
In the current experiments, we quantified abdominal EMG responses to bladder distension at 2 time points following CYP injections (1 day post-CYP and 7 days post-CYP). We additionally treated some mice with α-ARTN to determine whether it exerted an antinociceptive effect on CYP-induced hyperalgesia. A repeated measures analysis of variance indicated a significant effect of treatment on abdominal EMG responses to bladder distension (F(5,32) = 5.438, P = .001). Similar to what we have previously reported, abdominal EMG responses to noxious bladder distension (40–60 mmHg) were significantly potentiated in CYP-treated mice at both 1 day post-CYP and 7 days post-CYP (all P values <.05; Fig 3A; n = 6–7/group). Remarkably, both preventive (pre-CYP) and palliative (post-CYP) treatment with α-ARTN blocked and reversed, respectively, CYP-induced potentiation of abdominal EMG responses (all P values >.05; Fig 3B). Mice treated with IgG (the control for α-ARTN) immediately before the first CYP injection exhibited responses like those of mice that were treated with CYP alone and that were significantly greater than control mice (P values <.05).
Figure 3Treatment with α-ARTN before or after CYP injections prevents and reverses, respectively, CYP-induced bladder hyperalgesia. (A) Relative to controls (open circles), mice treated with CYP exhibited augmented abdominal EMG responses to noxious distension pressure at 1 day post-CYP (black circles) that persisted at 7 days post-CYP (gray circles). *P < .05, 1 day post-CYP versus control; #P < .05, 7 days post-CYP versus control. (B) At 1 day post-CYP, mice pretreated with IgG (black triangles) exhibited abdominal EMG responses like those of mice treated with CYP alone (panel A). Pretreatment with α-ARTN prevented the development of hyperalgesia at 1 day post-CYP (black squares). Posttreatment with α-ARTN after CYP, when hyperalgesia was already established, reversed CYP-induced hyperalgesia at 7 days post-CYP (gray squares). +P < .05, 1 day post-CYP + IgG pretreatment versus control.
To determine whether bladder-derived ARTN regulates CYP-induced changes in afferent TRPA1 expression and function, we used Ca2+ imaging to measure TRPA1-mediated, MO-evoked Ca2+ influx. We quantified the proportion of retrogradely labeled bladder afferents from CYP-injected mice with and without α-ARTN treatment that were responsive to MO application (n = 36–78/group). Again, similar to what we have previously reported,
the percentage of MO-responsive afferents changed as a function of treatment group (χ2(2) = 11.12, P = .003). CYP administered in conjunction with IgG, the control for α-ARTN, significantly increased the percentage of MO-responsive afferents (P < .01 vs control; Fig 4A). This effect of CYP was absent in mice in which α-ARTN was administered in conjunction with CYP (P < .01 vs CYP + IgG).
Figure 4Compared to spinal neuronal pERK expression (white arrows) in control mice (A), pERK expression in response to noxious bladder distension was significantly increased 1 day post-CYP (B) in regions of the dorsal spinal cord receiving lumbosacral bladder afferent input. Pretreatment with α-ARTN before CYP (C) prevented upregulation of distension-evoked spinal pERK. Quantification of these data is shown in (D). SDH, superficial dorsal horn; DCM, dorsal commissure; CC, central canal; SPN, sacral parasympathetic nucleus. *P < .05, 1 day post-CYP versus naïve; +P < .05, α-ARTN versus 1 day post-CYP.
Extracellular signal-regulated kinases regulate intracellular signal transduction, gene transcription, and posttranslational protein modifications. Lai et al
have shown that CYP-induced bladder hyperalgesia is positively correlated with increased neuronal pERK expression in regions of the dorsal horn receiving bladder sensory input, and that hyperalgesia can be reduced by spinal pERK inhibition. We therefore investigated whether α-ARTN treatment affected spinal pERK expression in CYP-treated mice (n = 3/group). As we anticipated, pERK immunoreactivity was significantly greater in the CYP plus bladder distension condition compared to distension alone in portions of the spinal cord receiving lumbosacral bladder input (t(4) = −2.503, P = .035; Fig 4B). In mice that were treated with α-ARTN prior to CYP, this effect was inhibited (t(4) = 2.835, P = .047; Fig 4C). As predicted by bladder afferent TRPA1 mRNA expression, the percentage of bladder afferents exhibiting MO-evoked Ca2+ transients was significantly different among mice treated with CYP with vs without α-ARTN (χ2(2) = 11.12, P = .003). Injections of CYP + IgG increased the percentage of MO-sensitive bladder afferents (P < .01 vs control), and this effect was prevented by α-ARTN (P < .01 vs CYP + IgG).
Discussion
Mature sensory neurons are sensitive to fluctuations in neurotrophic factor expression (eg, ARTN, NGF); in response, they exhibit plasticity with respect to anatomy (eg, sprouting
Artemin overexpression in skin enhances expression of TRPV1 and TRPA1 in cutaneous sensory neurons and leads to behavioral sensitivity to heat and cold.
Artemin overexpression in skin enhances expression of TRPV1 and TRPA1 in cutaneous sensory neurons and leads to behavioral sensitivity to heat and cold.
). Such plasticity provides multiple ways in which neurotrophic factors can modulate the quality and quantity of sensory information transmitted from the periphery to the spinal cord. In humans and in animal models of neuropathic and inflammatory pain, dramatic changes in neurotrophic factors have been correlated with both the development and resolution of hypersensitivity.
Artemin overexpression in skin enhances expression of TRPV1 and TRPA1 in cutaneous sensory neurons and leads to behavioral sensitivity to heat and cold.
and expression of neuropeptides overlaps largely with expression of the receptors for ARTN (GDNF family receptor α3 [GFRα3]) and NGF (tropomyosin receptor kinase A [TrkA]).
Differences in the expression of transient receptor potential channel V1, transient receptor potential channel A1 and mechanosensitive two pore-domain K+ channels between the lumbar splanchnic and pelvic nerve innervations of mouse urinary bladder and colon.
Thus, coexpression of the receptors for ARTN and NGF in the same neurons that express TRPA1 and/or TRPV1 makes these neurons particularly responsive to the inflammatory microenvironment. Interestingly, whereas a PubMed search of “Bladder AND NGF” yields almost 200 publications, there are no reports to our knowledge that examine the role of ARTN in bladder pain. Given that we observed an increase in ARTN mRNA that was more than twice greater than the increase in NGF mRNA, ARTN could be a very useful target for therapeutic interventions in bladder disease. It is worth noting that we conducted preliminary real-time PCR experiments using bladder tissue from CYP-treated mice to screen multiple reference genes. Although we found that β-actin was most resistant to CYP-induced changes in expression, it was increased in bladder tissue from the 1 day and 7 days post-CYP groups relative to the control group (data not shown). Although the robust increases in growth factor mRNA expression at 1 day post-CYP clearly overcome any confounding effect, it is likely that increased β-actin in the 7-day post-CYP group contributes to the decrease in normalized growth factor expression in this group relative to controls.
We have previously demonstrated that CYP-induced bladder hyperalgesia correlates with increased TRPA1 expression and function in bladder afferents and that a TRPA1 antagonist was effective at restoring normal sensitivity.
Here, we demonstrate that one potential mechanism for these changes in TRPA1 is an increase in ARTN. In vitro application of ARTN to bladder afferents from naïve (not treated with CYP) mice rescued nearly one-third of MO-sensitive, TRPA1-expressing neurons from tachyphylaxis on repeated MO application. Application of ARTN also induced MO-evoked calcium transients in nearly half of bladder afferents that were initially not responsive to MO. Clearly, the latter must be the result of posttranslational modification such as membrane insertion of TRPA1 protein. However, despite that the majority of peptidergic visceral afferents express GFRα3, an elegant study by Forrest et al
characterized a microanatomical network, including a GFRα3-expressing, nonneuronal component, by which ARTN could influence bladder sensation. Whether the mechanism of ARTN regulation of TRPA1 we observed in vivo is direct or indirect remains unknown and will be addressed by future studies. Future studies will also address the roles of ARTN and TRPA1 in bladder function, which may differ mechanistically from bladder pain. Interactions between ARTN and TRPA1 could also explain a clinical feature of bladder pain: In response to intravesical instillation of cold saline, individuals with bladder hypersensitivity report pain.
Although it has previously been suggested that this cold-induced pain resulted from upregulation of the menthol receptor TRPM8, TRPA1 has also been linked to detection of noxious cold.
Artemin overexpression in skin enhances expression of TRPV1 and TRPA1 in cutaneous sensory neurons and leads to behavioral sensitivity to heat and cold.
and regulation of bladder afferent sensitivity by ARTN. Future studies aimed at empirically addressing the role of ARTN in regulation of TRPM8 will address this.
Behaviorally, CYP-induced cystitis facilitated responses to bladder stimulation within a noxious range of intravesical pressures (40–60 mmHg).
Hyperalgesia was accompanied by phosphorylation of ERK1/2 in regions of the lumbosacral spinal cord receiving bladder afferent input (ie, superficial dorsal horn, sacral parasympathetic nucleus, and dorsal commissure). Neural activation in these regions has been associated with noxious input (eg, superficial dorsal horn, dorsal commissure) and/or organ distension (eg, sacral parasympathetic nucleus, dorsal commissure).
Although α-ARTN treatment effectively prevented the development of hyperalgesia and prevented spinal ERK1/2 phosphorylation, it is not clear what effect α-ARTN therapy would have on allodynia. Regardless, that α-ARTN was effective after bladder hyperalgesia was established is extremely encouraging in terms of its potential for treating ongoing disease, and underscores the translational importance of this approach in treating bladder pain in IC/PBS patients. Moreover, the effectiveness of α-ARTN administration prior to development of hyperalgesia suggests that it could be used prophylactically in patients undergoing antitumor CYP therapy. A large number of biologics are currently used clinically, and most have a broader target profile than α-ARTN (eg, infliximab, which targets tumor necrosis factor-α). The receptors for ARTN (GFRα3 and the receptor tyrosine kinase-encoding proto-oncogene [RET]) have a relatively narrow distribution in the nervous system and other tissues that would limit off-target effects, potentially making α-ARTN a relatively safe biologic for use in humans.
References
Albers K.M.
Wright D.E.
Davis B.M.
Overexpression of nerve growth factor in epidermis of transgenic mice causes hypertrophy of the peripheral nervous system.
Artemin overexpression in skin enhances expression of TRPV1 and TRPA1 in cutaneous sensory neurons and leads to behavioral sensitivity to heat and cold.
Differences in the expression of transient receptor potential channel V1, transient receptor potential channel A1 and mechanosensitive two pore-domain K+ channels between the lumbar splanchnic and pelvic nerve innervations of mouse urinary bladder and colon.
The distribution of visceral primary afferents from the pelvic nerve to Lissauer's tract and the spinal gray matter and its relationship to the sacral parasympathetic nucleus.
Elevated tryptase, nerve growth factor, neurotrophin-3 and glial cell line-derived neurotrophic factor levels in the urine of interstitial cystitis and bladder cancer patients.
We would like to acknowledge the University of Pittsburgh Rodent Behavioral Analysis Core and our funding sources: NS0050758 (B.M.D.), DK094593 (J.J.D.), DKD101681 (J.J.D.), American Pain Society Future Leaders in Pain Research grant (J.J.D.), and NS033730 (K.M.A.).
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