1. TRPA1 and Substance P Mediate Colitis in Mice
Matthias A. Engel, Andreas Leffler, Florian Niedermirtl, Alexandru Babes, Katharina Zimmermann, Miloš R. Filipović, Iwona Izydorczyk, Mirjam Eberhardt, Tatjana I. Kichko, Sonja M. Mueller–Tribbensee, Mohammad Khalil, Norbert Siklosi, Carla Nau, Ivana Ivanović– Burmazović, Winfried L. Neuhuber, Christoph Becker, Markus F. Neurath, Peter W. Reeh 
Gastroenterology 
Volume 141, Issue 4, Pages (October 2011)
DOI: /j.gastro
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2. Figure 1
TNBS and AITC induced neuropeptide release from isolated mouse colon. (A and B) TNBS and AITC induced CGRP/SP release from the colons of WT mice in a concentration-dependent manner. (C) CGRP release by TNBS (1 mmol/L) was dependent on extracellular calcium ions (**P < .01, Mann–Whitney U test) and (D) was lacking in colon preparations from TRPA1−/− (**P < .01) but not TRPV1−/− mice. (E) In the presence of the TRPA1 antagonist HC (50 μmol/L), TNBS-induced colonic CGRP release was abolished (*P < .05). DSS (2%) did not induce colonic CGRP release. Baseline release: (A) CGRP, 115 ± 6 pg/mL; SP, 69 ± 6 pg/mL; (B) CGRP, 112 ± 11 pg/mL; (C) CGRP, 81 ± 7 pg/mL; (D) CGRP TRPA1+/+, 108 ± 7 pg/mL; TRPA1−/−, 122 ± 24 pg/mL; and TRPV1−/−, 117 ± 30 pg/mL; and (E) CGRP, 115 ± 12 pg/mL.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

3. Figure 2
TNBS activates irritant-sensing colonic DRG neurons during calcium-imaging. (A) Bright-field (left), DiI-fluorescence (middle), and merged (right) images of cultured DRG neurons. Colon-specific neurons are shown in red in the merged image. (B) Representative experiment in a single colon-specific DRG neuron showing calcium transients induced by TNBS (1 mmol/L) and AITC (100 μmol/L) in a capsaicin (Caps 300 nmol/L)-sensitive neuron from TRPA1+/+ (left) and TRPA1−/− (right) mice. (C) Fractions of cells responding to TNBS (1 mmol/L, left; 10 mmol/L, right), AITC, capsaicin, and KCl among colon-specific DRG neurons from TRPA1+/+ mice. KCl-positive (whole pie), capsaicin-responsive (white segment), AITC (light gray), and TNBS-positive (dark gray) cell fractions. In TRPA1−/− mice less than 1% of colonic DRGs showed small TNBS (at 1 and 10 mmol/L) or AITC responses, whereas 56% were capsaicin-positive.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
TNBS activates TRPA1 in patch-clamp experiments. (A) Representative current trace of a TNBS-induced inward current in a DRG neuron (Vm, −60 mV) which was reversibly blocked by the TRPA1 antagonist HC (100 μmol/L). (B) Representative current traces of TNBS and AITC induced inward currents in rTRPA1-expressing HEK-293t cells; cells were held at −60 mV and TNBS (1 mmol/L) or AITC (100 μmol/L) was applied. HC (100 μmol/L) also blocked TNBS-induced inward currents in these cells. Comparison of mean inward currents. (C) TNBS (1 mmol/L) induced inward currents in HEK293t cells expressing hTRPA1-WT, hTRPA13C, or hTRPA1-3CK. Mean inward currents in HEK293t cells expressing WT or mutant hTRPA1 (each experiment, n = 5 or 8 cells). (D) Representative current traces of rTRPV1, rTRPV2, rTRPV4, and transient receptor potential ion channel of the melastatin type 8 r(TRPM8) expressed in HEK293t cells. Each TRP subtype was probed with TNBS (1 mmol/L); protein expression was controlled by administration of established agonists (capsaicin, 2-aminoethoxydiphenyl-borate [2-APB], 4-alpha-phorbol-12,13-didecanoate [4α-PDD], or menthol).
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
TNBS and DSS colitis scores and representative histologic slides of WT, TRPA1−/−, and TRPV1−/− mice. Asterisks indicate significance with regard to disease scores of WT mice with colitis (**P < .01, Mann–Whitney U test). (A) Acute TNBS colitis scores (3 days after enema) for the different genotypes and drug-treated mice. (B) H&E-stained histologic slides from distal colon sections showing severe acute colitis in TRPA1+/+ and TRPV1−/− mice and normal tissue in TRPA1−/− and HC-treated TRPA1+/+ mice. (C) Chronic TNBS colitis scores (21 days after enema) in TRPA1+/+ with and without treatment with the TRPA1 block and DSS colitis scores in TRPA1+/+ mice compared with those TRPA1−/− and TRPA1+/+ mice treated with HC from days 0–10. (D, upper panel) Body weights of TRPA1+/+ mice with chronic TNBS colitis decreased for 3 weeks after the TNBS enema. Treatment with HC during the last week led to considerable body weight gain. Arrows indicate HC treatment duration. The therapeutic effect of HC was significant from day 17 (*P < .05, **P < .01). Each point represents the mean body weight of 8 mice ± standard error of the mean. Statistical comparisons were made using analysis of variance followed by the Fisher LSD test. (D, lower panel) Body weight losses in DSS colitis in TRPA1+/+ mice compared with those in TRPA1−/−and TRPA1+/+ mice treated with HC. Representative histologic slides of TRPA1+/+ with or without HC treatment in (E) chronic TNBS and (F) both TRPA1 genotypes with DSS colitis. See Supplementary Material and Methods for the colitis scoring system and mortality rates.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
(A) 4-HNE-, 4-oxo-2-nonenal (4-ONE)–, and acrolein-induced neuropeptide release from isolated mouse colon (each n = 4). All 3 compounds induced CGRP and SP release in healthy WT but not TRPA1−/− mice (*P < .05, Mann–Whitney U test). Baseline release: WT CGRP, 101 ± 10 pg/mL; SP, 56 ± 6 pg/mL; TRPA1−/−, CGRP 102 ± 9 pg/mL; SP, 69 ± 6 pg/mL. (B) 4-HNE concentration in inflamed colons from both DSS- and TNBS-treated mice increased compared with that in healthy tissue (*P < .05; **P < .01). (C) TNBS-induced CGRP and SP release from isolated colon preparations from WTs with colitis. Values are normalized to baseline neurosecretion (each n = 8). On day 3 of acute TNBS colitis, TNBS (1 mmol/L)-induced colonic CGRP and SP release increased compared with that in mice receiving a vehicle enema. The response was abolished when HC (50 μmol/L) was co-applied with the TNBS stimulus. (D) On day 12, mice with DSS colitis also showed a drastic increase in stimulated neuropeptide release, which was prevented by the TRPA1 block HC. *Significant increase in colonic neuropeptide release compared with controls. +Abolished stimulated neuropeptide release from the inflamed colon by HC compared with controls. (*,+P<.05). Baseline release: (C) CGRP, 101 ± 8 pg/mL; SP, 79 ± 8 pg/mL; (D) CGRP, 119 ± 7 pg/mL; SP, 72 ± 10 pg/mL. (E) Chemical structure of a stable sulfinic acid transformation of the Cys-SH group by TNBS, as shown by mass spectrometry, which may contribute to sustained TRPA1 sensitization (see also Supplementary Figure 2).
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Acute TNBS colitis (A) sum scores and (B) representative histologic slides from WT, CGRP−/−, SP−/−, and double-null mutant mice; the latter 2 strains were completely protected. (C and D) After 8 days of DSS colitis, WT and CGRP−/− mice were seriously ill, whereas CGRP−/− mice treated with HC and SP−/− mice were completely protected, (E) which is reflected in reduced body weight loss, whereas the aggravation in CGRP−/− mice became evident as progressive wasting from day 2. Asterisks in the bar graphs indicate the significance of colitis scores for ill WT mice (*P < .05, **P < .01, Mann–Whitney U test). Statistical comparisons of the body weight course were made by analysis of variance followed by the Fisher LSD test (*P < .05, **P < .01).
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
(A) Representative photomicrographs of whole-mount preparations from the mouse distal colon at the level of the submucosal plexus. In WT mice intrinsic ganglionic neurons were surrounded by CGRP-positive (red) and CGRP/TRPV1 co-expressing (yellow) fiber baskets, however, they were neither CGRP nor TRPV1 immunoreactive. RTX-pretreated mice showed a complete loss of TRPV1 (green)-expressing sensory neurons. However, some intrinsic enteric CGRP immunoreactive fibers were still observed. (B) Capsaicin (30 nmol/L) and TNBS (1 mmol/L) failed to induce CGRP (left) and SP (right) release from the colons of RTX-pretreated mice but did so in controls (Con; each n = 8, *P < .05, Mann–Whitney U test). The baseline release of CGRP was 84 ± 8 pg/mL and that of SP was 55 ± 9 pg/mL. (C–E) RTX-pretreated WT mice were protected from TNBS and DSS colitis. (C, lower panel) The latter was reflected in the body weights during the 8-day period of DSS colitis (analysis of variance followed by the Fisher LSD test, *P < .05, **P < .01). In both colitis models, RTX-pretreated WT mice had preserved healthy mucosal architecture. (F) Chemically induced CGRP release from DRG neurons and MPNs. TNBS (1 mmol/L), AITC (100 μmol/L), and capsaicin (30 nmol/L) (each n = 8) induced profound CGRP release from cultured lumbosacral DRG neurons, which was not evident in MPNs (**P < .01). However, nonspecific depolarization with KCl (60 mmol/L) (n = 24) induced CGRP release from both classes of neurons.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions