Endothelin A receptor in nociceptors is essential for persistent mechanical pain in a chronic pancreatitis of mouse model

Abstract

BACKGROUND

Chronic pancreatitis (CP) accompanied with persistent abdominal pain represents a major clinical challenge for the symptom management in patients. Although with clear involvement of neuropathy, the detailed mechanisms underlying pain hypersensitivity associated with CP are not totally clear. The endothelin system has been reported to contribute to chronic pain and chronic inflammatory settings, and is a potential therapeutic target for the treatment of chronic pain.

AIM

To evaluate the role of nociceptor-specific endothelin A receptor (ETAR) in pain hypersensitivity in a CP mouse model and its potential contributing mechanisms.

METHODS

Oral gavage delivery of dibutyltin dichloride (DBTC) was used to induce CP in mice. A conditional knockout (CKO) strain which specifically delete ETAR in dorsal root ganglion (DRG) nociceptive neurons was generated. Abdominal pain hypersensitivity associated with CP and other behaviors were evaluated. The size of mouse gallbladder was measured and pancreatic histopathology was examined to validate the CP model. Calcitonin gene-related peptide expression and immune cells in the innervated DRGs and spinal cord were also examined. Calcium imaging in dissociated DRG neurons was performed to investigate the excitability of affected nociceptive neurons.

RESULTS

Specific deletion of endothelin receptor type A gene in nociceptive DRG neurons did not affect basal abdominal thermal and mechanical pain threshold in mice. Abdominal mechanical pain hypersensitivity was persistent in DBTC-treated WT mice but was significantly reduced in DBTC-treated CKO mice. DBTC treatment did not affect mouse nociceptive responses to heat and cold stimuli, as well as motor functions and anxiety-like behaviors of mice. DBTC treatment induced severe pancreatic inflammation and obvious gallbladder enlargement in wild type (WT) mice, but less in CKO mice. DBTC-induced increase of calcitonin gene-related peptide- and induction of brown adipocytes 1-positive signals in the DRG and spinal cord in WT mice were remarkably attenuated in CKO mice. DRG neurons from CKO mice exhibited less excitability and sensitivity in response to endothelin-1 exposure than those from WT mice.

CONCLUSION

DBTC intragastric administration in mice produced a convenient and reliable animal model for studying abdominal pain associated with CP. ETAR-dependent endothelin signaling in nociceptors is important for the development of persistent abdominal mechanical hypersensitivity in mice.

Key Words: Chronic pancreatitis; Abdominal pain; Endothelin; Endothelin A receptor; Calcitonin gene-related peptide; Macrophage infiltration; Microglia activation

Core Tip: Currently the mechanism underlying abdominal pain associated with chronic pancreatitis (CP) is not well understood. Utilizing dibutyltin dichloride-induced CP mouse model, it was found that the nociceptor-expressing endothelin A receptor (ETAR) is essential for the development of mechanical hypersensitivity following dibutyltin dichloride treatment. ETAR may contribute to this through regulating calcitonin gene-related peptide expression and activating immune cells. Therefore, targeting ETAR-dependent endothelin signaling in nociceptive sensory neurons may be a potential therapeutic approach for treating CP patients.

INTRODUCTION

The prevalence of chronic pancreatitis (CP) is estimated to be up to 150 per 100000, with being abdominal pain the most common symptom[1-3]. More than 80% of patients experience abdominal pain during the clinical course of CP[2-5]. However, the management of pain in CP patients remains unsatisfactory, partially due to its complex and not fully understood mechanisms. The pancreas, as an important organ in the digestive and endocrine systems, is innervated by various nerves, which can be damaged following local inflammation. Consequently, the pathogenesis of abdominal pain in CP shares similarities with neuropathic pain disorders, involving peripheral neuropathy and neuroplastic changes, and central sensitization[6-11]. CP is characterized by recruitment of inflammatory cells, fibrogenesis, and cell injury[9,12]. leading to the release of many inflammatory molecules, including H⁺, adenosine triphosphate, bradykinin, trypsin, nerve growth factor, and transforming growth factor-β, which directly stimulate local nociceptor receptors, increasing the excitability of primary sensory neurons, and resulting in peripheral sensitization[3,8,13,14]. Such pain signaling cascade is also key to maintain central sensitization[8,14,15]. Although alcohol consumption and tobacco use are two main risk factors for CP, other environmental and genetic factors also play important role in its pathogenesis[12,16]. Therefore, detailed investigations using disease-relevant animal models are essential to further understand the etiologies of persistent pain in CP patients.

Secretagogue-induced pancreatitis models, like caerulein, are the most common models to study intracellular signaling events during pancreatitis[17]. However, the mild disease severity limits their utility[18,19]. Surgical ligation of the bile duct is also frequently used to induce pancreatitis, but it may not be relevant to human disease onset mechanism and needs more experimental skills[18,19]. Dibutyltin dichloride (DBTC) has been shown to induce pancreatitis[20] and is utilized to create a convenient CP model in animals[21]. Tail vein injection of DBTC in rats caused pancreatic inflammation, immune cell infiltration, and fibrosis, which could last until 2 months[21]. To avoid minor leakage-induced tail necrosis, DBTC was also administered by oral gavage in mice[22]. Chronic inflammation, fibrosis, and obvious pancreatic damage were observed in treated mice. Besides, these animals developed abdominal mechanical hypersensitivity, which provides a great model to study pain associated with CP[22].

The endothelins (ETs) are 21-amino acid peptides, with G-protein coupled receptors, ET A receptor (ETAR) and B receptor (ETBR)[23,24]. Initially identified as a vasoconstricting factor secreted from endothelia cells[25], ETs play key roles in tissue development, fluid-electrolyte homeostasis, cardiovascular and neuronal function[23,26], and more recently are implicated in nociception and chronic pain[27]. ET-1 directly activates nociceptors[28] and is shown to be involved in inflammatory pain, neuropathic pain and cancer pain[27]. On the other hand, elevated local ET-1 expression was noticed in oleic acid-induced CP rats[29], as well as in CP patients[30]. Tobacco smoking also led to increased ET-1 level in CP patients[31]. These studies suggest the potential contribution of the ET system to the pathogenesis of abdominal pain associated with CP. In the dorsal root ganglion (DRG), ETAR is mainly localized in small and medium-sized nociceptors and their peripheral endings, while ETBR is exclusively expressed in satellite glia cells and Schwann cells[32]. Consistently, majority of the studies support that ET-1-induced nociceptive responses are mediated by ETAR, and possibly modulated by ETBR[27,33]. In this study, we generated nociceptor-specific ETAR knockout mice and utilized DBTC-induced CP model to investigate the role of ETAR-mediated ET signaling in abdominal pain in CP mice. Our results indicate that nociceptor-specific ETAR is crucial for persistent mechanical hypersensitivity in CP mice, potentially through the regulation of calcitonin gene-related peptide (CGRP) expression and immune cell activation, highlighting the involvement of the ET system in persistent abdominal pain in CP patients.

MATERIALS AND METHODS

Animals

All animal studies were performed based on relevant ethical guidelines of the International Association for the Study of Pain and approved by the Committees of the Use of Laboratory Animals of Nantong University (Approval No. S20220217-016). SNS-Cre (Nav1.8 promoter) and ETAR-flox transgenic mice were kindly provided by Professor Kuner R[34,35] and Professor Yanagisawa M[36], respectively. Both transgenic mouse lines were crossed to C57BL/6 mice to keep background consistency. ETAR-flox mice were crossed with SNS-Cre mice to maintain the breeding colonies[37,38]. For mouse genotyping, polymerase chain reaction was performed with genomic DNA isolated from the punched ear using a standard protocol. Primers used for genotyping SNS-Cre are as follows: Forward 5’-ATTTGCCTGCATTACCGGTC-3’; reverse 5’-GCATCAACGTTTTCTTTTCGG-3’. Primers for genotyping flox site of ET receptor type A are as follows: Forward 5’-CCTCAGGAAGGAAGTAGCAAGATTA-3’; reverse 5’- ACACAACCATGGTGTCGA-3’. After mating male SNS^cre/ETAR^f/f mice with female ETAR^f/f mice, the resulting SNS^cre/ETAR^f/f mice were used as conditional knockout (CKO) mice, and ETAR^f/f littermates [herein referred as wild type (WT)] were served as the control mice. Adult mice (8-10 weeks, 20-25 g) without obvious abnormalities (normal appearance and behaviors) were mainly employed for following experiments and maintained 3-5 mice per cage under a 12 hours/12 hours light-dark cycle at a temperature (22 ± 1 °C) and humidity (50%-65%)-controlled environment with free access to chow and water.

Chemicals and reagents

Paraformaldehyde (PFA), chloral hydrate and DBTC were supplied from Sigma-Aldrich (St. Louis., MO, United States). Acetone and sucrose were supplied from Sangon Biotech (Shanghai, China). Glycerol was supplied from Shenyang Hongqi Pharmaceutical Company (Shenyang, China). Hematoxylin and eosin stain kit (D006-1-1) was supplied from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Neutral balsam mounting medium (10004160) was supplied from Sinopharm company (Shanghai, China). ETAR primary antibody (PS08814, 1:800) was supplied from Abmart Shanghai Company (Shanghai, China). Neuronal nuclear protein (ab104224, 1:1000) and induction of ionized calcium-binding adapter molecule 1 (IBA-1, ab5076, 1:500) primary antibodies were supplied from Abcam (Cambridge, MA, United States). CGRP antibody (24112, 1:2000) was supplied from Immunostar (Hudson, WI, United States). The secondary antibodies (AF488, Cy5 or Cy3 conjugated) were supplied from Jackson ImmunoResearch Inc (West Grove, PA, United States). ET-1 was provided by R&D systems (Minneapolis, MN, United States).

DBTC-induced persistent CP

As reported previously[22], DBTC was dissolved in 950 mL/L ethanol and then mixed with glycerol (2:3 ratio). DBTC solution (10 mg/kg, 200 μL per mouse) was carefully administered to mice via oral gavage by 1-mL syringe with gavage needle #12. Vehicle group animals were treated with 950 mL/L ethanol and glycerol mixture (2:3 ratio). To induce persistent pancreatitis, mice received twice DBTC treatments within 2 weeks (1-week interval). Animals were monitored daily for appearance and activity. Body weight and water/food consumption were recorded regularly.

Mouse behavioral tests

All mouse behavioral tests were conducted double-blindly and performed by the same experienced person. To evaluate nociceptive responses of mouse abdomen, the abdominal hair was removed by shaving one day ahead. Basal abdominal nociceptive thresholds were determined in WT and CKO mice. Then, animals were divided into three groups: WT-vehicle group, WT-DBTC group, and CKO-DBTC group. Each group contained 10 mice (half male and half female). Behavior tests of these mice were performed on indicated days. Before the testing, all animals were kept in the experimental room for at least 30 minutes to get familiar with the environment, which was kept quiet during the tests with consistent indirect light illumination at approximately 40 lux. The testing apparatuses were cleaned with 300 mL/L ethanol before and after each trial to remove olfactive cues.

Abdominal radiant heat test: Animals were placed in a transparent chamber on an elevated glass plate (2-mm thickness) for two hours before the tests, and the latency of any withdrawal responses (abdominal or whole-body withdrawal, licking of the irradiated skin area) was recorded when mouse upper abdominal skin was stimulated with the Hargreaves radiate heat apparatus (IITC Life Science, CA, United States). The intensity of radiant heat remains constant, with the cutoff of 25 seconds to avoid abdominal tissue damage.

Von Frey test: As previously reported[22,39], animals were placed in a transparent chamber on an elevated metal grid floor for two hours, and then a series of von Frey filaments (0.07, 0.16, 0.4, 0.6, 1.0 g; Stoelting, Wood Dale, IL) were applied to upper abdominal skin. The filaments were presented perpendicular to the skin surface for 1-2 seconds, and the appearance of any withdrawal responses (abdominal or whole-body withdrawal, licking of the stimulated skin area) was considered as positive nociceptive response. The testing interval was no less than 10 seconds. To determine the response threshold, each filament was tested for five times starting from 0.07 g and Dixon up-and-down method was used for the calculation. To determine the response frequency, 0.4-g von Frey filament was tested for ten times and the percentage of positive responses was calculated.

Acetone test: As reported previously[40], animals were tested with 15 μL of acetone.

Rotarod test: As reported previously[38], a rotarod system (IITC Life Science, CA, United States) was used to assess mouse motor functions. Mice were pre-trained for three days with 20 minutes each day. The training speed was accelerated from 0 to 10 rpm. After the training, mice were tested for three trails and the speed of rotation was accelerated from 4 to 40 rpm within 5 minutes. The falling latency was recorded and averaged.

Open field test: As mentioned previously[40], open field test was conducted. The bottom surface was evenly divided into 9 square areas by ANY-maze and the center grid was defined as “central zone”. Total travelled distance was measured to reflect the motor activity of mice. Time spent in the central zone was used to indicate anxiety-like behaviors of mice.

Elevated plus maze test: As mentioned previously[40], the elevated plus maze (EPM) test was performed. The analyses of the percentage of entries into open arms and time staying in the open arms were intended to reflect anxiety-like behaviors of mice.

Histological experiments

After the behavior tests, mice were anesthetized with 10 g/L pentobarbital sodium (100 mg/kg intraperitoneal administration). The gallbladder was gently dissected, and the long-axis length and short-axis length were measured using vernier scale. Mice were sequentially perfused with normal saline solution and 40 g/L PFA. Pancreas, the thoracic (T) 8-12 DRGs and spinal cord segments were harvested and post-fixed in 40 g/L PFA (pancreas for 4-6 hours, DRG for 2-3 hours and spinal cord for 8-12 hours). After dehydrated with 300 g/L sucrose in phosphate buffered saline (PBS) until sank to the bottom of the tube and embedded in optimal cutting temperature compound (Sakura, CA, United States), pancreas (14 μm), DRG (8-10 μm) and spinal cord (14-20 μm) sections were cut in a cryostat (-20 °C). For hematoxylin and eosin staining, pancreas sections were sequentially incubated with hematoxylin for 2 minutes and eosin for 5 minutes using PBS to wash away excess staining reagents. The sections were mounted with neutral balsam and images were captured with a wide-field microscope (Nanjing Jiangnan Novel Optics Co., Nanjing, China). For immunofluorescence staining, the sections were permeabilized and blocked in PBS with 100 mL/L donkey serum and 1 mL/L Trion X-100. The sections were incubated with primary antibodies (a guinea pig antibody against neuronal nuclear protein (1:2000, Millipore, Billerica, MA, United States), with a goat anti-IBA-1 antibody (1:300, Abcam) combined with a rabbit anti-CGRP antibody (1:2000, Immunostar Hudson, WI, United States)) overnight at 4 °C and secondary antibodies (1:500) for 2 hours at room temperature. After washing with PBS, the sections were mounted with antifade mounting medium, and images were captured using a confocal laser-scanning fluorescence microscope (Zeiss LSM 800, Carl Zeiss AG, Jena, Germany) with 20 objective lens (numerical aperture: 0.8) and sensitive GaAsP detectors. For quantification, Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, Maryland, United States) was used to count the number of positive cells and quantify the positive signal density. Images from three sections were analyzed for each mouse and 5 mice were used for each group.

DRG neuron culture

DRG neurons were harvested and cultured as previously described[40-42].

Calcium imaging

After all behavioral tests, the T8-12 DRGs from 5 experimental mice per group were quickly excised, and DRG neurons were cultured. The calcium imaging was conducted and analyzed as described previously[40].

Bias control and outlier handling methods

The methods and experimental procedures outlined above have established rigorous protocols in our laboratory. Experimenters participated in operations after undergoing strict training. Behavioral tests were conducted as double-blind operation, and data analyses were carried out by individuals who have not involved in the experimental procedures. All animals were kept in a sound-attenuated, isolated holding facility in the laboratory one week prior to and throughout the duration of the behavioral assays to minimize stress. Consequently, all experiments have essentially eliminated bias. The dosage of DBTC used in the study was determined through multiple explorations conducted via gavage. It was discovered that this dosage effectively established the model without resulting in the death of experimental mice, thereby ensuring that there were no missing values in the study. Any values falling outside the range of mean ± 3 SD were considered abnormal and consequently deleted according to standard procedures. Importantly, it is worth noting that no outliers were identified throughout the study.

Statistical analysis

Data were shown as mean ± SEM and analyzed using GraphPad Prism 8.0 (La Jolla, CA, United States). For comparison between two groups, statistical significance was determined using unpaired t-test. For comparison among three groups, one-way analysis of variance followed by post hoc Bonferroni test was used. For the statistical analysis of pain behavior tests, two-way analysis of variance with post hoc Bonferroni test was performed. For comparison two constituent ratios, χ²-test was used. Data comparisons were considered statistically significant at P < 0.05.

RESULTS

Generation and characterization of SNS^cre/ETAR^f/f mice

To evaluate the role of nociceptive DRG neuron-expressing ETAR in abdominal pain associated with CP, we crossed SNS-Cre strain[34] and ETAR-floxp strain to generate CKO mice. In SNS-Cre mice, Cre recombinase activity is mainly restricted to nociceptive sensory neurons, with barely detected recombination in the brain, spinal cord and other peripheral tissues[34]. Polymerase chain reaction tests were used for the genotyping of transgenic mice to detect the presence of the Cre fragment (approximately 350 bp) (Figure 1A) or loxP site (with loxP, approximately 650 bp; without loxP, approximately 610 bp) (Figure 1B). Immunofluorescence staining was then performed to examine the expression of ETAR in mouse DRG neurons. Noticeably fewer ETAR-positive cells were observed in SNS^cre/ETAR^f/f CKO mouse DRGs comparing to ETAR^f/f (WT) mice (Figure 1C and D). Quantification analysis confirmed the reduction of the percentage of ETAR-positive neurons (in total DRG neurons) in CKO mice (24.97% ± 1.50% vs 56.28% ± 3.80%, T₍₅₎ = 6.699, P = 0.0011, Figure 1D), suggesting successful conditional deletion of ETAR.

Figure 1 Characterization of endothelin A receptor conditional knockout mouse line. A: Polymerase chain reaction genotyping of SNS-Cre mice (Cre band: Approximately 350 bp); B Polymerase chain reaction genotyping of endothelin A receptor (ETAR)-flox mice (with flox band: Approximately 650 bp; without flox band: Approximately 610 bp); C: Representative immunofluorescence images showing ETAR (green) signals in dorsal root ganglion neurons from ETAR conditional knockout (CKO) and littermate wild type (WT) mice; D: Analysis of ETAR-positive dorsal root ganglion neuron percentage in WT and CKO mice; E: Radiant heat test was used to examine thermal nociceptive response latency in WT and ETAR CKO mice; F: Response threshold to abdominal mechanical stimulation was measured in WT and ETAR CKO mice with von Frey filaments; G: Von Frey filaments with different forces were applied to the abdomen of WT and ETAR CKO mice. The response frequency of each filament was calculated; H: The experimental flowchart; I: The percentage of body weight change of experimental mice around 4 weeks after with dibutyltin dichloride or vehicle treatment. Scale bar, 50 μm. n = 3-20 mice. Data are shown as mean ± SEM. ^bP < 0.01, ^cP < 0.001, statistical comparisons were conducted with unpaired two-tailed t-test (D-F), two-way analysis of variance (G) or one-way analysis of variance with Sidak’s post hoc test (I). Ednra: Endothelin receptor type A gene; WT: Wild type; CKO: Conditional knockout; ETAR: Endothelin A type receptor; DBTC: Dibutyltin dichloride; EPM: Elevated plus maze.

Next, we assessed abdominal heat and mechanical nociceptive responses in WT and CKO mice to investigate whether ETAR in nociceptive neurons affects normal pain sensation in mouse abdomen under normal condition. The response latency to radiant heat stimuli of CKO mice was not significantly changed comparing to WT mice (T₍₂₈₎ = 1.853, P = 0.0744, Figure 1E). Using the von Frey test, we measured mechanical nociceptive threshold (T₍₂₈₎ = 0.0991, P = 0.9218, Figure 1F) and the response frequencies of different von Frey filaments (F_{(1, 28)} = 0.0139, P = 0.9072, Figure 1G) in mice, suggesting that both WT and CKO mice have similar and normal mechanical nociceptive responses. Therefore, loss of ETAR expression in nociceptive neurons does not affect normal abdominal pain sensation in naïve mice.

Establishment of DBTC-induced CP mouse model

In order to establish a reliable and reproducible CP model, we made slight changes to the DBTC treatment regimen based on the previous study[22]. Mice were given DBTC orally twice within a 2-week period and were closely monitored during and after the treatment (Figure 1H). A significant decrease in body weight (10%-15% decrease for DBTC-treated mice comparing to 5%-10% increase for vehicle-treated mice) (Figure 1I), as well as loss of coat shine and smooth texture, were observed in both WT and CKO mice around 4 weeks after DBTC treatment. However, there was no significant improvement in the above phenotypes in CKO mice compared to WT mice.

Less abdominal mechanical hypersensitivity in SNS^cre/ETAR^f/f mice after DBTC treatment

Although the mechanical threshold of both hind paw and abdomen was reported as decrease in DBTC-induced CP mice[22], our focus in this model was primary on mouse abdominal nociceptive responses for clinical relevance. We tested mechanical, heat and cold stimulations and found that DBTC-treated mice only exhibited significant hypersensitivity to mechanical stimuli (F_{(2, 27)} = 286.0, P < 0.0001; F_{(2, 27)} = 38.15, P < 0.0001; Figure 2A and B), with no changes in responses to heat (F_{(2, 27)} = 0.0183, P = 0.9819, Figure 2C) or cold stimuli (F_{(2, 27)} = 0.0847, P = 0.9191, Figure 2D). Abdominal mechanical hypersensitivity, including reduced mechanical threshold (Figure 2A) and an increased response frequency to a 0.4 g filament (Figure 2B), persisted for at least four weeks after DBTC treatment. Given elevated expression of ET-1 in CP animal models and patients[29-31], these results support the hypothesis that ETAR-dependent ET signaling in nociceptors play a crucial role in the development of persistent abdominal pain associated with CP.

Figure 2 Behavioral phenotypes of dibutyltin dichloride -induced chronic pancreatitis model. A: The response thresholds to abdominal mechanical stimulation were measured in experimental mice before and after chronic pancreatitis induction with von Frey filaments; B: Von Frey filament with 0.4-g force was applied to mouse abdomen and the response frequencies of mice were calculated; C: The abdominal response latencies to radiant heat were determined in mice from different groups; D: Acetone-induced nociceptive response events were counted to reflect mouse abdominal responses to cold stimuli; E: The fall latencies of mice were determined to reflect mouse motor abilities in rotarod test; F and G: The total travelled distance of mice was measured to reflect mouse motor activities (F) and the duration the central zone was recorded to reflect mouse mood alteration (G) in the open field test; H and I: The percentages of open arm entries (H) and duration in the open arm (I) were measured to reflect mouse anxiety-like behaviors in the EPM test. n = 10 mice. Data are shown as mean ± SEM. ^bP < 0.01, ^cP < 0.001, statistical comparisons were conducted with unpaired two-way analysis of variance (A-D) or one-way analysis of variance (E-I) with Sidak’s post hoc test. WT: Wild type; CKO: Conditional knockout; DBTC: Dibutyltin dichloride; EPM: Elevated plus maze.

To explore the possibility that the motor and anxiety conditions of DBTC-treated mice could impact their pain behavior, we assessed the motor functions and emotional changes in experimental mice. In the rotarod test, the falling latency remained unchanged in WT mice after DBTC treatment, as well as in DBTC-treated CKO mice (F_{(2, 27)} = 0.0004, P = 0.9996, Figure 2E). The open field test, which assesses both autonomic motor function (total travelled distance, Figure 2F) and anxiety-like behaviors (time spent in the central zone, Figure 2G), revealed no differences among three group mice (F_{(2, 27)} = 0.5541, P = 0.5810; F_{(2, 27)} = 0.2838, P = 0.7551; Figure 2F and G). Additionally, in the EPM test, the three groups of mice showed similar percentages of open arm entries (F_{(2, 27)} = 0.3220, P = 0.7274, Figure 2H) and duration in the open arms (F_{(2, 27)} = 0.3765, P = 0.6898, Figure 2I) following DBTC or vehicle treatment. These results suggest that nociceptor-expressing ETAR is crucial for the development of abdominal mechanical hypersensitivity induced by DBTC treatment in mice.

DBTC-induced CGRP upregulation and immune cell activation in the DRG and spinal cord were impaired in SNS^cre/ETAR^f/f mice

We investigated how the ET signaling contributes to abdominal pain associated with CP by exploring the potential underlying mechanisms. Previous studies have shown increased CGRP expression in CP animal models[43] and relief of pain behaviors with CGRP antagonist intrathecal treatment in rat CP models[44]. Therefore, we examined the distribution of CGRP in the T8-12 DRGs and spinal cord of WT and CKO mice after treatment with DBTC. In WT mice, DBTC treatment led to an increase in the percentages of CGRP-positive neurons in DRG (Figure 3A and C) and CGRP-positive fibers in the spinal cord dorsal horn (Figure 3B and D). However, CKO mice did not show a significant increase in CGRP expression in either DRG (F_{(2, 12)} = 32.92, P < 0.0001, Figure 3C) or spinal cord (F_{(2, 12)} = 16.18, P = 0.0004, Figure 3D), suggesting that deficiency of ETAR in nociceptors may reduce the elevation of CGRP expression in response to CP.

Figure 3 Increased expression of calcitonin gene-related peptide in the dorsal root ganglion and spinal cord of mice with chronic pancreatitis. A and B: Representative images of immunofluorescence staining of calcitonin gene-related peptide (CGRP) on the T8-12 dorsal root ganglion (DRG) neurons (A) and spinal cord (B); C: Percentage of CGRP-positive DRG neurons; D: Relative CGRP-positive fiber density (normalized to wild type-vehicle group) in the dorsal spinal cord was analyzed for each group of mice. Scale bar, 50 μm (A) and 100 μm (B). n = 5 mice. Data are shown as mean ± SEM. ^bP < 0.01, ^cP < 0.001, statistical comparison were conducted with one-way analysis of variance with Sidak’s post hoc test. CGRP: Calcitonin gene-related peptide; WT: Wild type; CKO: Conditional knockout; DBTC: Dibutyltin dichloride; DRG: Dorsal root ganglion.

Peripheral and central immune cell activation, such as macrophages around DRG neurons and microglia in the spinal cord, play an important role in pain transmission and the development of chronic pain[45,46]. We further examined the involvement of macrophage infiltration and microglia activation in CP-associated pain hypersensitivity using IBA-1 immunostaining. After DBTC treatment, WT mice showed an increase in IBA-1-positive cells in both DRG (Figure 4A and C) and spinal cord (Figure 4B and D), while DBTC-treated CKO mice displayed fewer IBA-1-positive cells in these areas (F_{(2, 12)} = 35.75, P < 0.0001; F_{(2, 12)} = 16.76, P = 0.0003; Figure 4) comparing to treated WT mice. These data suggest that ETAR-dependent ET signaling contributes to neuroinflammation induced by DBTC, including macrophage infiltration into DRGs innervating the pancreas and microglia activation in the spinal cord innervating the pancreas.

Figure 4 Increased macrophage infiltration into the DRG and microglial activation in the spinal cord of mice with chronic pancreatitis. A and B: Representative images of immunofluorescence staining of ionized calcium-binding adapter molecule 1 (IBA-1) on the T8-12 dorsal root ganglion neurons (A) and spinal cord (B); C: The number of IBA-1-positive cells; D: Relative IBA-1-positive density (normalized to WT-vehicle group) in the dorsal spinal cord was analyzed for each group of mice. Scale bar, 50 μm (A) and 100 μm (B). n = 5 mice. Data are shown as mean ± SEM. ^bP < 0.01, ^cP < 0.001, statistical comparison were conducted with one-way analysis of variance with Sidak’s post hoc test. WT: Wild type; CKO: Conditional knockout; DBTC: Dibutyltin dichloride; DRG: Dorsal root ganglion; IBA-1: Ionized calcium binding adaptor molecule 1.

DRG neurons were less sensitive to ET-1 in SNS^cre/ETAR^f/f mice treatment with DBTC

To investigate the excitability of nociceptors in response to ET-1 during CP, we performed calcium imaging on dissociated T8-12 DRG neurons from both WT and SNS^cre/ETAR^f/f mice with CP around 4 weeks after DBTC treatment (Figure 1H). We found that acute exposure to ET-1 resulted in an increased intracellular calcium influx, which was more pronounced in neurons from DBTC-treated WT mice with than in those from vehicle control mice; however, this increased intracellular calcium influx was significantly reduced in DBTC-treated CKO mice (F_{(2, 49)} = 38.23, P < 0.0001, Figure 5A). Notably, the number of neurons responsive to ET-1 in DBTC-treated WT mice was significantly higher than in vehicle control mice (25 in total 36 vs 17 in total 38), while the number of responsive neurons was substantially lower in DBTC-treated CKO mice (10 in total 69, χ²₍₂₎ = 32.4, P < 0.0001, Figure 5B). These results suggest that DRG neurons from SNS^cre/ETAR^f/f mice with CP are less sensitive to ET-1 compared to those from WT mice with CP.

Figure 5 A higher sensitivity in primary sensory neurons and histological characterization of mice with chronic pancreatitis. A and B: Less sensitive to endothelin-1 in dissociated T8-12 dorsal root ganglion neurons from conditional knockout (CKO) mice with chronic pancreatitis than those from wild type (WT) mice with chronic pancreatitis measured by calcium imaging for the degree of calcium influx into the neurons (A) and the percentage of responsive cells from five mice (B). n = 36-69 neurons from 5 mice/group; C-E: Representative hematoxylin and eosin staining images of mouse pancreas sections from WT-vehicle group (C), WT-dibutyltin dichloride group (D), and CKO-dibutyltin dichloride group (E); F: The percentage of body weight change for experimental animals around 4 weeks. Scale bar, 50 μm (A) and 200 μm (C-E). n = 10 mice. Data are shown as mean ± SEM. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, statistical comparison were conducted with one-way analysis of variance with Sidak’s post hoc test (A and F) or χ²-test (B). WT: Wild type; CKO: Conditional knockout; D: Dibutyltin dichloride (DBTC); ET-1: endothelin-1; NS: Not significant; V: Vehicle.

Less tissue damage in SNS^cre/ETAR^f/f mice after DBTC treatment

Finally, we further estimated the severity of pancreatic tissues with histological analysis. The histopathology evaluation on mouse pancreas showed that DBTC treatment induced severe pancreatitis with the inflammatory cell infiltration, acinar atrophy, and abnormal duct structure compared to the normal pancreatic structure in vehicle control animals (Figure 5C-E), which is consistent with other group reports[20-22]; of note, these damages were reduced in CKO mice (Figure 5C-E). We also observed that the size of the gallbladder was dramatically expanded after DBTC treatment in WT mice, as indicated by increase in the lengths of both long axis (DBTC 9.77 ± 0.51 mm vs vehicle 3.97 ± 0.11 mm) and short axis (DBTC 5.74 ± 0.49 mm vs vehicle 1.97 ± 0.05 mm). This expanded size of the gallbladder was slightly reduced in DBTC-treated CKO mice (long axis, 8.04 ± 0.38 mm and short axis, 5.15 ± 0.28 mm; F_{(2, 27)} = 63.49, P < 0.0001; F_{(2, 27)} = 38.58, P < 0.0001; Figure 5F), suggesting that the bile duct stricture and cholestasis caused by pancreatic damage have been alleviated in CKO mice. These pathological phenotypes of DBTC-induced CP were mitigated in CKO mice, suggesting that nociceptor-specific ETAR is involved in the DBTC treatment-evoked CP. Together, these data suggest that ETAR in the peripheral nociceptive neurons is involved not only in pain sensitivity but also in pathological pancreatic damage.

No gender difference in SNS^cre/ETAR^f/f mice treatment with DBTC

Gender differences have been receiving increasing attention in pain mechanism research[47,48]. In our study, we investigated the gender difference in SNS^cre/ETAR^f/f mice after DBTC treatment and found that no significant differences were observed between males and females, including WT and CKO mice, in pain (Figure 6A-D), motor (Figure 6E and F) and (Figure 6F and G) emotion testing before or after DBTC treatment. Therefore, we did not pay attention to the gender differences at the histological and molecular levels in the following tests.

Figure 6 Gender differences in behavioral phenotypes of dibutyltin dichloride -induced chronic pancreatitis model. A and B: No gender difference was observed in mechanical pain sensitivity in experimental mice before and after chronic pancreatitis (CP); C and D: No gender differences were observed in heat pain sensitivity (C) and cold allodynia (D) in experimental mice before and after CP; E and F: No gender differences were observed in motor function (E) and autonomous activities (F) in experimental mice before and after CP; G: No gender differences were observed in emotional state in experimental mice before and after CP. n = 5 mice. Data are shown as mean ± SEM. Statistical comparisons were conducted with unpaired two-way analysis of variance (A-D) or one-way analysis of variance (E-G) with Sidak’s post hoc test. WT: Wild type; CKO: Conditional knockout; DBTC: Dibutyltin dichloride; EPM: Elevated plus maze.

DISCUSSION

Abdominal pain is the most common clinical symptom of CP with variable descriptions as a dull, sharp or nagging sensation in the upper abdomen with or without radiation to the back[1]. Pain assessment and management are challenging in patients due to its complicated and not well-characterized etiologies. In this study, we selectively deleted ETAR in mouse nociceptive neurons and utilized DBTC-induced CP model to evaluate the role of ETAR-dependent ET signaling in abdominal pain hypersensitivity associated with CP. Our results reveal that: (1) Naïve SNS^cre/ETAR^f/f mice exhibit normal abdominal nociceptive responses; (2) Loss of ETAR expression in nociceptive neurons remarkably attenuated abdominal mechanical hypersensitivity associated with DBTC-induced CP; (3) DBTC-induced increased expression of CGRP and immune cell activation in the DRG and spinal cord dorsal horn were also greatly reduced in SNS^cre/ETAR^f/f mice; and (4) DBTC-induced pancreatic damage was improved in SNS^cre/ETAR^f/f mice.

The pathogenesis of CP can be induced and influenced by various factors, including alcohol, smoking, obstructive lesions, other toxic agents, and genetic factors[4,18]. DBTC, an organotin compound, is commonly used in polyvinyl chloride plastic additive which may contaminate food and water. It was also reported to induce pancreatic injury in human beings when used in the paint component, highlighting its clinical relevance. LD50 of DBTC is 90.0 mg/kg in mice[49]. Systemic administration (intraperitoneal or intravenous injection) with DBTC (1.0 mg/kg, up to 8.0 mg/kg) caused acute pancreatitis (AP) in rats[20,21,50]. Oral gavage delivery of DBTC (10.0 mg/kg) twice within 10 days in mice induced severe pancreatitis, hepatic lesions and extensive fibrotic thickening in pancreatic and hepatic structures[22]. Treated animals also developed pain related secondary mechanical hypersensitivity[22]. We administered DBTC (10 mg/kg) mice for twice within two weeks to generate reproducible CP phenotypes, including pancreatic inflammation, gallbladder expansion and abdominal mechanical hypersensitivity (Figures 2 and 5). Notably, oral gavage delivery is not only more convenient than intravenous injection, but it also reduces the indirect effects of systemic administration on target organs, which is beneficial for establishing the CP model.

Elevated local ET-1 expression was observed in the cytoplasm of vascular endothelial and ductal cells in oleic acid-induced CP model in rat, which may be associated with the morphological and hemodynamic changes of CP[29]. Another study using DBTC to induce AP in rats found that more than 2-fold increase of ET-1 mRNA in the DRG and spinal cord[50]. More interestingly, AP rats were intraperitoneally treated with ETAR antagonist (BQ123) or ETBR antagonist (BQ788)[50]. Both antagonists abolished DBTC-induced abdominal mechanical hypersensitivity, of which the effects of BQ123 (> 75 minutes) lasted longer than BQ788. Using hot plate test, they also observed DBTC-induced secondary thermal hypersensitivity which could be transiently normalized by both antagonists[50]. These results demonstrate the role of the ET system in AP-induced pain hypersensitivity. In our study, we focused on the role of nociceptor-specific ETAR-dependent ET signaling in abdominal pain associated with CP. Consistently, CP induction in WT mice caused abdominal mechanical hypersensitivity lasting around 4 weeks, while in CKO mice such hypersensitivity was significantly alleviated (Figure 2). However, we did not observe CP-induced abdominal hypersensitivity to heat and cold stimuli, which may be explained by the differences of animal species, DBTC treatment protocols, as well as testing methods and timepoints. Since ETAR is the main ET receptor expressed in DRG neurons[32], our results further clarify and emphasize the role of nociceptor-specific ETAR-dependent ET signaling in the development of abdominal pain hypersensitivity associated with CP.

It has been reported that CP-induced local release of chemicals and inflammatory molecules can directly activate peripheral sensory afferents and sensitize nociceptors, contributing to peripheral sensitization of persistent abdominal pain in CP patients. This process involves the regulation of many membrane proteins, including voltage-gated K⁺ channels[51], transient receptor potential (TRP) channels[52,53], proteinase activated receptor 2[54], and P2X receptors[55,56]. On the other hand, ET-1 was found to be able to potentiate TRPV1 activity through ETAR-dependent protein kinase C (PKC) pathway[57] and sensitize TRPA1 through ETAR-dependent PKA pathway[58] in both primary DRG neurons and HEK293 cells. Some studies found that ET-1 could also enhance P2X3 or acid-sensing ion channel currents via ETAR-PKC signaling[59,60]. Our study suggests that increased local release of ET-1 (autocrine or paracrine) in CP mice intensifies pain signaling through activation of ETAR and downstream membrane proteins, like TRPV1, in nociceptive DRG neurons. This hypothesis is strongly supported by our calcium imaging data (Figure 5A and B). The detailed mechanism still needs further investigations. It has been reported that ET-1 treatment could induce the release of CGRP[61], which is an important factor involved in various chronic pain conditions[62], including abdominal pain associated with CP[43,44]. Consistently, we observed an increase in CGRP expression in DRG neurons and the spinal cord after DBTC treatment, which was largely diminished in DBTC-treated ETAR CKO mice (Figure 3). CGRP is known to the bidirectionally releasing neuropeptide, which contributes pain processing in the CNS[63,64]; while exerts regulatory effects on immune in the PNS[65,66]. Accordingly, the reduced damage of pancreatic tissues, as well as less swelling in the gallbladder, in DBTC-treated CKO mice (Figure 5) might be related with the decrease of pancreatic secretion of neurogenic CGRP, although this is inconsistent with previous studies using ET receptor antagonists[50,67]. This is worthy of further investigation.

We found that DBTC caused macrophage infiltration in DRGs and microglia activation in spinal cord, which was also dependent on ETAR expression in nociceptive sensory neurons, consistent with microglia in the central nervous system involves pain processing[45,68]. These immune responses are important biomarkers and play critical roles in the pathogenesis of chronic pain[45,46]. Additionally, we did not observe the response from DBTC-treated mice to heat and cold stimuli, nor did it affect motor function and emotional state. These results may be due to various factors such as differences in animal species, DBTC treatment protocols, testing methods, and time points used in other studies. It is possible that oral administration of DBTC targets specific organs with less systemic toxicity. We supposed that oral administration of DBTC is much targeting organs and less systemic toxicity. In future study, we plan to conduct behavioral tests and histological evaluations at multiple and long-term time points to gain a comprehensive understanding of the progression and recovery of the disease. Overall, our study highlights the importance of ETAR-dependent ET signaling in nociceptors in persistent abdominal pain associated with CP.

CONCLUSION

In summary, nociceptor-expressing ETAR is necessary for the development of abdominal mechanical hypersensitivity in the DBTC-induced CP mouse model. ET signaling in nociceptors that is dependent on ETAR may play a role in the pain hypersensitivity linked to CP by controlling CGRP expression and immune cell activation. This finding suggests that targeting ETAR could be a potential therapeutic approach for managing abdominal pain in patients with CP.

ACKNOWLEDGEMENTS

We thank Professor Rohini Kuner from Heidelberg University and Professor Masashi Yanagisawa from University of Texas Southwestern Medical Center for kindly providing SNS-Cre and ETAR-flox mice, respectively.