Anatomical and functional characterization of a duodeno-pancreatic neural reflex that can induce acute pancreatitis

Abstract

Neural cross talk between visceral organs may play a role in mediating inflammation and pain remote from the site of the insult. We hypothesized such a cross talk exists between the duodenum and pancreas, and further it induces pancreatitis in response to intraduodenal toxins. A dichotomous spinal innervation serving both the duodenum and pancreas was examined, and splanchnic nerve responses to mechanical stimulation of these organs were detected. This pathway was then excited on the duodenal side by exposure to ethanol followed by luminal mustard oil to activate transient receptor potential subfamily A, member 1 (TRPA1). Ninety minutes later, pancreatic inflammation was examined. Ablation of duodenal afferents by resiniferatoxin (RTX) or blocking TRPA1 by Chembridge (CHEM)-5861528 was used to further investigate the duodeno-pancreatic neural reflex via TRPA1. ~40% of dorsal root ganglia (DRG) from the spinal cord originated from both duodenum and pancreas via dichotomous peripheral branches; ~50% splanchnic nerve single units responded to mechanical stimulation of both organs. Ethanol sensitized TRPA1 currents in cultured DRG neurons. Pancreatic edema and myeloperoxidase activity significantly increased after intraduodenal ethanol followed by mustard oil (but not capsaicin) but significantly decreased after ablation of duodenal afferents by using RTX or blocking TRPA1 by CHEM-5861528. We found the existence of a neural cross talk between the duodenum and pancreas that can promote acute pancreatitis in response to intraduodenal chemicals. It also proves a previously unexamined mechanism by which alcohol can induce pancreatitis, which is novel both in terms of the site (duodenum), process (neurogenic), and receptor (TRPA1).

Fig. 1.

Dichotomizing duodenal-pancreatic nerves at thoracic T₆–T₁₃ segments. A: dorsal root ganglia (DRG) neurons originated from pancreas (pancreatic-specific neurons shown in red). B: DRG neurons originated from duodenum (duodenal-specific neurons shown in green). C: overlay of A and B (double-labeled neurons shown in yellow). Magnification of these pictures is ×200 (object lens ×20, eyepiece ×10). D: percentage of double-labeled DRG of total no. of duodenal- and pancreatic-labeled DRG neurons at various segments.

Fig. 2.

Immunofluorescent staining with transient receptor potential (TRP) subfamily V, member 1 (V1) and/or TRP subfamily A, member 1 (TRPA1) antibody. A: one DRG section from normal rats double staining with TRPV1 and TRPA1 (TRPV1-positive neurons shown in green, TRPA1-positive neurons shown in red, and double-stained neurons shown in yellow, magnification is ×400). B: CTB-labeled DRG neurons staining with TRPA1 (pancreatic-specific neurons shown in red, duodenal-specific neurons shown in green, TRPA1-positive neurons shown in pseudocolored blue, and triple-stained neurons shown in purple, magnification is ×200). C: CTB-labeled DRG neurons staining with TRPV1 (pancreatic-specific neurons shown in red, duodenal-specific neurons shown in green, TRPV1-positive neurons shown in pseudocolored blue, and triple-stained neurons shown in purple, magnification is ×200).

Fig. 3.

Splanchnic nerve single unit response to gentle traction of the pancreatic duct and duodenum. A: representative recording of greater splanchnic nerve (GSN) fiber response to gentle traction of organs. B: splanchnic nerve single unit response to gentle traction of pancreatic duct and duodenum. A total of 51 splanchnic nerve single units were recorded. Of these, 4 (7.84%) responded to traction of the pancreatic duct and 8 (15.69%) responded to traction of the duodenum; 26 (50.98%) responded to traction of both organs, whereas 13 units (25.49%) had response to neither organ.

Fig. 4.

The TRPA channel inhibitor Chembridge (CHEM) inhibits ethanol- and mustard oil-sensitive inward currents. A: whole cell patch recording of current from corn oil in combination with 3% ethanol (EtOH). B: whole cell patch recording of current from mustard oil in combination with 3% EtOH. C: whole cell amplitudes activated by EtOH + corn oil and EtOH + mustard oil (n = 5 each group). D–I: single channel currents from a representative cell patch (c in panels represents closed state). D: single channel currents from 3% EtOH. E: current from corn oil. F: single channel current from mustard oil. G: single channel currents activated by mustard oil in combination with 3% EtOH. H: CHEM inhibition of single channel currents invoked by mustard oil in combination with 3% EtOH. I: single channel currents reactivated by mustard oil in combination with 3% EtOH after CHEM washout. J: single channel open probabilities in the presence of EtOH and mustard oil and the effects of CHEM (n = 5 each group). *P < 0.05 and **P < 0.01. Arrows denote time of application of agents [CHEM was perfused for 30 s before exposure to EtOH + mustard oil (MO)].

Fig. 5.

Effects of ethanol on pancreatic inflammation induced by intraduodenal TRPA1 activation. A: pancreatic edema. B: pancreatic histology severity score. C: serum amylase level. D: serum lipase level. E: myeloperoxidase (MPO) activity. Results were expressed as means ± SE (n = 3 for saline + corn oil, n = 4 for 40% EtOH + corn oil, and n = 9 each for saline + mustard oil and 40% EtOH + mustard oil). *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 6.

Effects of intraduodenal resiniferatoxin (RTX) on pancreatic inflammation induced by intraduodenal ethanol and mustard oil. A: pancreatic edema. B: pancreatic histology severity score. C: serum amylase level. D: serum lipase level. E: MPO activity. Results were expressed as means ± SE (n = 13 each for RTX + 40% EtOH + mustard oil and saline + 40% EtOH + mustard oil). *P < 0.05.

Fig. 7.

The role of the TRPA1 channel antagonist CHEM on pancreatic inflammation induced by intraduodenal ethanol and mustard oil. A: pancreatic edema. B: pancreatic histology severity score. C: serum amylase level. D: serum lipase level. E: MPO activity. Results were expressed as means ± SE (n = 6 for 40% EtOH + mustard oil + vehicle and n = 5 for 40% EtOH + mustard oil + CHEM). *P < 0.05.

Fig. 8.

The effects of intraduodenal capsaicin, with or without prior intraduodenal ethanol on pancreatic inflammation. A: pancreatic edema. B: MPO activity. C: serum amylase level. D: serum lipase level. Pancreatic histology severity score of all these groups was zero. Results were expressed as means ± SE (n = 3 each for capsaicin and saline group; n = 6 each for saline + capcaicin and 40% EtOH + capcaicin).

Fig. 9.

Representative images of pancreatic histology severity score. A: saline + corn oil. B: 40% EtOH + corn oil. C: saline + mustard oil. D: 40% EtOH + mustard oil. E: RTX + 40% EtOH + mustard oil. F: saline + 40% EtOH + mustard oil. G: CHEM + 40% EtOH + mustard oil. H: vehicle + 40% EtOH + mustard oil. I: saline. J: capsaicin. K: saline + capsaicin. L: 40% EtOH + capsaicin.