Synergistic role of TRPV1 and TRPA1 in pancreatic pain and inflammation.

Pancreatic inflammation was induced by 8 hourly injections of caerulein (50 μg/kg). The extent of inflammation, pancreatic neuron TRP channel expression and function and excitability, and pain-related behaviors were evaluated over the course of the following week.

MATERIAL AND METHODS Animals

Experiments were performed on 8-week old male C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) housed in the AAALAC accredited Division of Laboratory Animal Resources at the University of Pittsburgh. Mice had access to water and food ad libitum. All protocols were approved by the Institutional Animal Care and Use Committee.

Surgical Procedures and Cell Labeling

All surgical procedures were performed under aseptic conditions in a designated surgery area. Anesthesia was maintained with 2% isoflurane. For back-labeling of pancreatic DRG and NG neurons, a laparotomy was performed to expose the pancreatic head into which three injections of Alexa Fluor 488-conjugated cholera toxin B (CTB; 2 mg/ml in sterile saline; Molecular Probes; Eugene, OR; total volume injected ~5 μl) were made 16 . CTB was employed because of previous studies indicating its efficacy for labeling pancreatic afferents 16 and because it appears to have no effect on afferent membrane properties including resting membrane potential, rheobase, or action potential characteristics, when compared to DiI-back-labeled afferents (data not shown). The abdominal muscles and overlying skin were sutured separately and mice were allowed to recover for at least 4 days. Acute Pancreatitis Acute pancreatitis was induced by repeated injections of the cholecystokinin analog caerulein (Sigma-Aldrich, St Louis, MO) dissolved in 0.01 M phosphate-buffered saline (PBS). Mice received intraperitoneal (i.p.) injections of either caerulein (50 μg/kg in 100 μl) or vehicle (PBS) hourly for 8 hrs. Mice were sacrificed 1, 3 or 7 days after the second hourly injection and pancreata, T9-12 DRG, and NG were harvested. Pancreata were processed immediately for myeloperoxidase activity or histology to assess the extent of inflammation. DRG and NG were acutely dissociated, cultured (see below) and employed for single cell PCR (expression of TRPV1 and TRPA1), Ca 2+ imaging or whole cell patch clamp. Myeloperoxidase (MPO) Assay and histology Briefly, mice were overdosed with inhaled isoflurane and pancreata were dissected, weighed, added to a beaker containing 1.0 mL 0.5% hexadecyltrimethylammonium bromide (HTAB; Sigma) and finely minced using spring scissors. 2mL HTAB was added and the sample was sonicated for 10 sec prior to homogenization for 30 sec. The samples underwent three freeze thaw cycles, were centrifuged twice, reacted with O-dianisidine dihydrochloride (Sigma) and read on a plate reader at 460nm. Histological assessments were made on paraffin-embedded cross-sections of pancreata stained with hematoxylin and eosin (H&E) in a blinded fashion. Cell Culture, Calcium Imaging and Whole Cell Recordings 1 or 3 days following vehicle or caerulein treatment, mice were overdosed with isoflurane, perfused with ice-cold Ca 2+ /Mg 2+ -free Hank’s Balanced Salt Solution (HBSS, Invitrogen). T9-12 DRG and NG were rapidly dissected and prepared for culture as described previously 17 . Dissociated cells were resuspended in F12 media (Invitrogen) containing 10% fetal calf serum and antibiotics (penicillin/streptomycin, 50 U/ml) and plated onto laminin (0.1mg/ml) and poly-D-lysine (5mg/ml) coated glass coverslips. No additional growth factors were added to the culture medium. Cells were incubated overnight at 37°C and only CTB-labeled pancreatic T9-12 DRG and NG neurons were either imaged or whole cell recordings were performed the following day as previously described 16 , 18 (see supplemental methods ). No unlabeled cells were investigated for the purposes of comparison to non-pancreatic afferents because it was possible that unlabeled cells were in fact pancreatic afferents that were not back-labeled by our limited dye injections. Single-Cell RT-PCR and qRT-PCR Individual CTB-labeled pancreatic neurons were collected with large-bore (~50 μm) glass pipettes and expelled into microcentrifuge tubes containing reverse-transcriptase (RT) mix (Invitrogen). . For each experiment, negative controls consisted of omitting reverse transcriptase or using a cell-free bath aspirate as template. The first-strand cDNA from a pancreatic sensory neuron was used as template in a PCR reaction containing 1× GoTaq reaction buffer (Promega Madison, WI), 20 mM outer primers, 0.2 mM dNTPs, and 0.2 mL GoTaq DNA polymerase (Promega); primer sequences are listed in supplemental Table 1 . Each initial PCR product served as template in a subsequent PCR reaction using a nested primer pair. Each initial PCR product served as template in a subsequent PCR reaction using a nested primer pair, the products of which were electrophoresed on 2% agarose– ethidium bromide gels and photographed. Only neurons producing detectable amplification of a housekeeping gene (GAPDH) were analyzed further. For qRT-PCR the first-strand cDNA of cells expressing the target genes was pre-amplified (26 cycles) using the PCR condition as described above. The products were used as template for real-time PCR using ABsolute ™ QPCR SYBR® Green ROX mix (ABgene, Rochester, NY) in an Applied Biosystems (Foster City, CA) 5700 real-time thermal cycler. Threshold cycle (Ct) values were recorded as a measure of initial template concentration and relative fold changes in RNA levels were calculated by the ΔΔCt method using GAPDH as a reference standard 19 TRP Receptor Antagonists Mice were treated with caerulein (n=9) according to the hourly protocol described above and either the TRPA1 antagonist HC-030031 (Hydra Bioscience, Cambridge, MA; 100 and 300 mg/kg b.w.), the TRPV1 antagonist AMG 9810 (100 and 300 mg/kg; TOCRIS, Chicago, IL) or vehicle (PBS) was administered twice i.p. 1 hr before the first caerulein injection and again 4 hr later. The interaction between TRPA1 and TRPV1 was studied by administering both antagonists together at doses of 50 or 100 mg/kg each following the same dosing schedule as above.

Behavioral Testing

To assess pain-related behaviors, mice were placed in Plexiglas boxes and exploratory behaviors were monitored photoelectrically for 15 min periods at 9, 10, 12, 14 and 24 hr post-treatment. Photoelectric beams were spaced 1.5 cm apart, providing 0.75 cm spatial resolution. TruScan software (Coulbourn Instruments; Whitehall, PA) analyzed time spent in different parts of the arena, path information, distance travelled and total movements simultaneously in the X-Y plane. The software also analyzed the amount of time each mouse spent in the “vertical plane” or standing position that required stretch of the abdominal muscles, a position that is assumed to be uncomfortable in the presence of abdominal hypersensitivity. Modulation of exploratory behavior was evaluated after combined TRPV1 and TRPA1 antagonist administration (100mg/kg each at a 4-hr interval as above) and, in different mice, also after morphine (2.5 mg/kg, subcutaneous) administered 30 min prior to the 24 hr post-treatment time point.

Data Presentation and Analysis

Data are presented as mean ± SEM and were analyzed using SigmaStat (Version 3.1, Systat Software). Statistical analyses for differences in changes over time in multiple groups were performed using two-way ANOVA followed by the Holm-Sidak post hoc test. Differences between groups were tested using Student’s t test (when comparing two groups) or one-way ANOVA (when comparing more than two groups) followed by post hoc Bonferroni-protected pairwise comparisons. Statistical significance was set at p ≤ 0.05.

Surgical Procedures and Cell Labeling

All surgical procedures were performed under aseptic conditions in a designated surgery area. Anesthesia was maintained with 2% isoflurane. For back-labeling of pancreatic DRG and NG neurons, a laparotomy was performed to expose the pancreatic head into which three injections of Alexa Fluor 488-conjugated cholera toxin B (CTB; 2 mg/ml in sterile saline; Molecular Probes; Eugene, OR; total volume injected ~5 μl) were made 16 . CTB was employed because of previous studies indicating its efficacy for labeling pancreatic afferents 16 and because it appears to have no effect on afferent membrane properties including resting membrane potential, rheobase, or action potential characteristics, when compared to DiI-back-labeled afferents (data not shown). The abdominal muscles and overlying skin were sutured separately and mice were allowed to recover for at least 4 days.

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