The vagus nerve as a modulator of intestinal inflammation - TI Pharma

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Vagal anti-inflammatory pathway mediated via JAK2-STAT3 activation transfection, cells were selected using neomycin (2.0 mg/mL; Sigma-Aldrich) for 16 h, washed, and treated with nicotine-LPS 24 h after transfection. Transfection was verified by immunoblotting using HRP-tagged anti-HA rabbit polyclonal antibodies (Abcam). For siRNA transfection, a siRNA oligonucleotide specific to Socs-3 (ID 160220; Ambion) was transfected using RNAiFect (Qiagen) according to the manufacturer’s instructions. A FITC labeled control random RNA oligo nucleotide (Ambion) was co-transfected to allow optimalisation of transfection efficiency. Immunoblotting. Cells were scraped in 50 µL of ice-cold lysis buffer containing 150 mM NaCl, 0.5% Triton X-100, 5 mM EDTA, and 0.1% SDS. Samples were taken up in 50 µL sample buffer (125 mM Tris-HCl pH 6.8, 2% SDS, 10% βz-mercaptoethanol, 10% glycerol, and 0.5 mg/mL bromophenol blue), loaded onto SDS-PAGE gels and blotted onto PVDF membranes (Millipore). Membranes were blocked in TBS/0.1% Tween- 20 (TBST) containing 5% non-fat dry milk and incubated overnight with appropriate antibodies in TBST/1% BSA. HRP-conjungated secondary antibodies were visualized using Lumilite plus (Boehringer-Mannheim, Germany). Immunoprecipitation. Peritoneal macrophages (1*10 6 per cm 2 ) were scraped in lysis buffer (20 mM Tris-HCl pH 7.6, 2.5 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% sodiumdeoxycholate, 10% glycerol, 1 mM Na 3 VO 4 , 50 mM NaF, 1 µg/mL aproptinine, 1 µg/mL leupeptine, and 1 mM PMSF), sonicated for 10 s and centrifuged at 14000 x g at 4 °C for 20 min. Lysates, preabsorbed with 20 µL Protein A/G (Sigma-Aldrich), were incubated overnight with the appropriate antibodies, and immunoprecipitated with 40 µL of Protein A/G. Alternatively, immunoprecipitation was carried out using the TrueBlot system (eBioscience, San Diego, CA) according to the manufacturer’s instructions. Immunoprecipitates were recovered by centrifugation, washed in icecold wash buffer (TBS, 0.1% Triton X-100, 1 mM PMSF), taken up in sample buffer (125 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, and 0.5 mg/mL bromophenol blue), and immunoblotted as described above. Surgical procedures. Mice (female BalB/C) were used at 15-20 weeks of age. IL-6 and IL-10 deficient mice and their respective C57Bl wild types were obtained from Jackson Laboratories (Maine, USA). LysM-Cre Stat-3fl/fl and Stat-3fl/fl mice were maintained at Osaka University, Japan. Abdominal surgery with intestinal manipulation was performed as described elsewhere 12 . Mice (n=10-12) were divided in 4 groups: 1-undergoing control surgery of only laparotomy (L), 2-laparotomy followed by intestinal manipulation (IM) combined with sham preparation of the cervical area, or 3-L- or 4-IM in combination with electrical stimulation of the vagus nerve. IM consisted of manipulation from the distal duodenum to the cecum during 5 min using sterile moist cotton applicators. At 3 or 24 h after surgery, mice were killed by cervical dislocation. Small intestine was removed, flushed, and fixed in ice-cold 100% ethanol 43
Chapter 3 for preparation of whole mounts. Small intestinal muscularis strips were prepared by pinning freshly isolated intestinal segments in ice-cold PBS and removal of mucosa facing upwards. Muscle strips were snap-frozen in liquid nitrogen until analysis. Electric stimulation of the vagal nerve. Stimulation of the vagus nerve was essentially performed as described previously 2 . The left cervical nerve was prepared free from the carotid artery and ligated with 6-0 silk suture. The distal part of the ligated nerve trunk was placed between a bipolar platinum electrode unit. In part of the experiments, the vagus nerve was transected, and the distal part stimulated. Voltage stimuli (5Hz, 2ms, 1 or 5 V) were applied for 5 min before-, and 15 min following the intestinal manipulation protocol described above. In sham VNS control mice the cervical skin was opened and left for 20 min. covered by moist gaze. Local hexamethonium application. Local blockade of nicotinic receptors in the ileum was performed as follows: in anaesthetized mice (n=7) a midline laparotomy was performed, and 6 cm of ileum proximal to the cecum was carefully externalized and placed in a sterile preheated tube. The segment was continuously flushed with a preheated (37 °C) solution of hexamethonium (10 -4 M in 0.9% NaCl), or vehicle for 20 min. Temperature of intestinal tissue was monitored using a thermal probe. Leakage of hexamethonium solution into the peritoneal cavity was strictly avoided. After incubation, the hexamethonium solution was removed, the ileal segment was washed three times with 0.9% NaCl, and included in the manipulation protocol. Measurement of gastric emptying. Gastric emptying of a semi-liquid, noncaloric test meal (0.5% methylcellulose) containing 10Mq 99 Tc was determined by scintigraphic imaging as described previously 42 . Quantification of leukocyte accumulation at the intestinal muscularis. Myeloperoxidase (MPO) activity in ileal muscularis tissue was assayed as a measure of leukocyte infiltration as described 12, 23 . Whole mounts of ethanol-fixed ileal muscularis were prepared and stained for MPO activity as described 12, 23 . RT-PCR. Total RNA from tissue was isolated using Trizol (Invitrogen, Carlsbad, CA), treated with Dnase, and reverse transcribed. The resulting cDNA (0.5 ng) was subjected to Light Cycler PCR (CYBR Green Fast start polymerase; Roche, Mannheim, Germany) for 40 cycles. Primers used were: TNF: As 5-AAAGCATGATCCGCGACGT-3 and Sen 5-TGCCACAAGCAGGAATGAGAA-3; MIP-2: As 5-AGTGAACTGCGCTGTCAATGC-3 and Sen 5-GCAAAC TTTTTGACCGCCCT-3; Socs-3 As 5-ACCTTTCTTATCCGCGACAG-3and Sen 5’-TGCACCAGCTTGAGTACACAG-3’; and GAPDH As 5’- ATGTGTCC GTCGTGGATCTGA-3’ and Sen 5’-ATGCCTGCTTCACCACCTTCT-3’. PCR products were quantified using a linear regression method on the Log(fluorescence) per cycle number data 43 , and expressed as percentage of GAPDH transcripts for 44
Page 1 and 2: The vagus nerve as a modulator ofin
Page 3 and 4: The vagus nerve as a modulator of i
Page 5 and 6: Promotiecommissie Promotores: Prof.
Page 7 and 8: Table of contents Chapter 1 Introdu
Page 9 and 10: Introduction and outline The aim of
Page 11 and 12: Introduction and outline Hence, the
Page 13 and 14: Introduction and outline 18. van de
Page 15 and 16: Chapter 2 ABSTRACT The cholinergic
Page 17 and 18: Chapter 2 neurotransmitter ACh with
Page 19 and 20: Chapter 2 THE VAGUS NERVE AND CHOLI
Page 21 and 22: Chapter 2 nicotine treatment impair
Page 23 and 24: Chapter 2 Figure 1. Hypothetical sc
Page 25 and 26: Chapter 2 unclear whether this hybr
Page 27 and 28: Chapter 2 reduction of NF-κB activ
Page 29 and 30: Chapter 2 CONCLUDING REMARKS The hy
Page 31 and 32: Chapter 2 34 17. The FO, Boeckxstae
Page 33 and 34: Chapter 2 55. Moser N, Mechawar N,
Page 35 and 36: Chapter 2 93. Shafique S, Dalsing M
Page 37 and 38: Chapter 3 ABSTRACT Acetylcholine re
Page 39: Chapter 3 by bowel manipulation 12
Page 43 and 44: Chapter 3 Figure 1. Nicotine attenu
Page 45 and 46: Chapter 3 Figure 2. Inhibition of m
Page 47 and 48: Chapter 3 macrophage cell lysates s
Page 49 and 50: Chapter 3 reports 2 . We next incub
Page 51 and 52: Chapter 3 Figure 5. Perioperative e
Page 53 and 54: Chapter 3 Figure 7. Stimulation of
Page 55 and 56: Chapter 3 Activation of the STAT3 c
Page 57 and 58: Chapter 3 REFERENCE LIST 60 1. Trac
Page 59 and 60: 4 CHAPTER Deciphering STAT3 signali
Page 61 and 62: INTRODUCTION Deciphering STAT3 sign
Page 63 and 64: Deciphering STAT3 signaling in chol
Page 69 and 70: DISCUSSION Deciphering STAT3 signal
Page 71 and 72: REFERENCES Deciphering STAT3 signal
Page 73 and 74: Chapter 5 ABSTRACT Background and A
Page 75 and 76: Chapter 5 METHODS Reagents and anti
Page 77 and 78: Chapter 5 described previously 6 .
Page 79 and 80: Chapter 5 Figure 1 Cholinergic agon
Page 81 and 82: Chapter 5 Figure 3 Cholinergic agon
Page 83 and 84: Chapter 5 we analyzed whether nicot
Page 85 and 86: Chapter 5 Zymosan induced formation
Page 87 and 88: Chapter 5 marker CD11 c (Fig. 7E),
Page 89 and 90: Chapter 5 Our data demonstrate that
Page 91 and 92:
Chapter 5 REFERENCE LIST 96 1. Boro
Page 93 and 94:
Thesis E.P.M. van der Zanden The va
Page 95 and 96:
Summary and conclusions The goal of
Page 97 and 98:
Summary and conclusions whether the
Page 99 and 100:
Summary and conclusions exposure co
Page 101 and 102:
REFERENCE LIST Summary and conclusi
Page 103 and 104:
Nederlandse samenvatting
Page 105 and 106:
Chapter 7 van STAT3 in de ‘cholin
Page 107 and 108:
Chapter 7 De bevinding dat nicotine
Page 109 and 110:
Chapter 7 macrofagen aan te tonen (
Page 111 and 112:
Chapter 7 18. Yu Z, Zhang W, Kone B
Page 113 and 114:
Shizuo Akira Department of Host Def
Page 115 and 116:
List of publications
Page 117 and 118:
Dankwoord
Page 119 and 120:
om de ene keer olijfolie te drinken
Page 121:
Curriculum Vitae Curriculum Vitae E
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