Transient receptor potential ankyrin 1 mediates chronic pancreatitis pain in mice

Abstract

Chronic pancreatitis (CP) is a devastating disease characterized by persistent and uncontrolled abdominal pain. Our lack of understanding is partially due to the lack of experimental models that mimic the human disease and also to the lack of validated behavioral measures of visceral pain. The ligand-gated cation channel transient receptor potential ankyrin 1 (TRPA1) mediates inflammation and pain in early experimental pancreatitis. It is unknown if TRPA1 causes fibrosis and sustained pancreatic pain. We induced CP by injecting the chemical agent trinitrobenzene sulfonic acid (TNBS), which causes severe acute pancreatitis, into the pancreatic duct of C57BL/6 trpa1(+/+) and trpa1(-/-) mice. Chronic inflammatory changes and pain behaviors were assessed after 2-3 wk. TNBS injection caused marked pancreatic fibrosis with increased collagen-staining intensity, atrophy, fatty replacement, monocyte infiltration, and pancreatic stellate cell activation, and these changes were reflected by increased histological damage scores. TNBS-injected animals showed mechanical hypersensitivity during von Frey filament probing of the abdomen, decreased daily voluntary wheel-running activity, and increased immobility scores during open-field testing. Pancreatic TNBS also reduced the threshold to hindpaw withdrawal to von Frey filament probing, suggesting central sensitization. Inflammatory changes and pain indexes were significantly reduced in trpa1(-/-) mice. In conclusion, we have characterized in mice a model of CP that resembles the human condition, with marked histological changes and behavioral measures of pain. We have demonstrated, using novel and objective pain measurements, that TRPA1 mediates inflammation and visceral hypersensitivity in CP and could be a therapeutic target for the treatment of sustained inflammatory abdominal pain.

Keywords: chronic pancreatitis; inflammation; pain; transient receptor potential ankyrin 1; trinitrobenzene sulfonic acid.

Fig. 1.

Trinitrobenzene sulfonic acid (TNBS)-induced chronic pancreatitis (CP) decreased body weight (A) and increased histological damage score (B and C) in mice. Mice with TNBS-induced CP lost more body weight (A) and had higher histological severity score (B and C) than did ethanol, saline, and sham-operated control groups. Values are means ± SE (n = 6–10). *P < 0.05, **P < 0.01, ****P < 0.0001 vs. sham. ####P < 0.0001 vs. saline. +P < 0.05, ++++P < 0.0001 vs. vehicle. C: representative hematoxylin-eosin (H & E)-stained histological sections of the pancreas 21 days after instillation of TNBS, vehicle, or saline into the pancreatic duct. Striking features of this CP model were fat replacement, fibrosis, and presence of inflammatory cells.

Fig. 2.

TNBS-induced CP reduced amylase activity (A) and pancreas size (B) in mice. Mice with TNBS-induced CP had significantly less amylase activity than control groups (A), consistent with loss of exocrine functionality of the pancreas. TNBS caused a significant decrease in pancreas size, as quantified (B and D) and shown in representative images (C), consistent with the presence of atrophy in the gland. Spleen size was similar among all experimental groups (D). Values are means ± SE (n = 5–15). **P < 0.05, ****P < 0.0001 vs. sham. ##P < 0.01, ###P < 0.001 vs. saline. +P < 0.05, +++P < 0.001, ++++P < 0.0001 vs. vehicle.

Fig. 3.

TNBS-induced CP increased pancreatic fibrosis in mice. Fibrosis was measured by immunohistochemistry and Western blotting: picrosirius red was used to stain collagen deposition (A and B), and Western blotting to α-smooth muscle actin (α-SMA) was used to measure activation of pancreatic stellate cells (PSCs, C), which have been shown top play a major role in promoting pancreatic fibrosis. Mice with TNBS-induced CP had robust pancreatic collagen deposition, which was absent in all control groups, as shown in representative images in A. EtOH, ethanol. Superficial area of stained collagen was significantly increased in mice with CP (B). Mice with TNBS-induced CP also had increased activation of PSCs compared with vehicle-treated controls (C). Values are means ± SE (n = 7–9). *P < 0.05, ***P < 0.001 vs. sham. #P < 0.05 vs. saline. +++P < 0.001 vs. vehicle.

Fig. 4.

TNBS-induced CP decreases voluntary wheel-running activity (A) and increases pain behaviors (B–D). In mice with TNBS-induced CP, voluntary wheel-running activity decreased dramatically within the first 2 wk compared with control groups (A). Mechanical sensitivity was measured with calibrated (0.07, 0.16, and 1 g) Von Frey filaments (VFFs). Mice were probed on the abdomen (B) and hindpaw (C). In both cases, mice with TNBS-induced CP had increased mechanical sensitivity compared with control groups. D: mice with CP also showed significantly more catalepsy (immobility measured over a 10-min period using the open-field apparatus) than sham-operated controls at all time points. Thus all the behavioral end points strongly support the presence of abdominal and generalized hypersensitivity in mice with TNBS-induced CP. Values are means ± SE (n = 6–10). *P < 0.5, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. sham-operated.

Fig. 5.

Transient receptor potential ankyrin 1 (TRPA1) mediates TNBS-induced CP. CP was induced by intraductal injection of TNBS in trpa1^+/+ and trpa1^-/- mice. HSS was decreased (A and D) and amylase activity was increased (B) in trpa1^-/- compared with trpa1^+/+ mice, suggesting that TRPA1 is involved in mediating inflammation in this CP model. C: no significant differences between the 2 genotypes were detected when pancreatic atrophy was measured. Values are means ± SE (n = 5–10). **P < 0.01 vs. trpa1^+/+ TNBS.

Fig. 6.

TRPA1 mediates TNBS-induced CP hypersensitivity. CP was induced by intraductal injection of TNBS in trpa1^+/+ and trpa1^-/- mice: trpa1 ^-/- mice showed no mechanical sensitivity, as suggested by lack of increase in referred abdominal (A) hypersensitivity and hindpaw sensitivity threshold (B) compared with trpa1^+/+ mice. These findings suggest that TRPA1 is involved in activating pain pathways in this model of CP. Values are means ± SE (n = 6–10). ****P < 0.0001 vs. trpa1^+/+ TNBS.