DOCTORAL PROGRAM IN DRUG RESEARCH AND INNOVATIVE TREATMENTS CYCLE XXXII Coordinator: Prof. Elisabetta Teodori STUDY OF POST-INFLAMMATORY VISCERAL HYPERSENSITIVITY MECHANISMS AND INNOVATIVE TREATMENTS: EVIDENCE OF ADENOSINE A3 RECEPTOR AGONISTS EFFICACY AND MICROBIOTA RELEVANCE IN ABDOMINAL PAIN PERSISTENCE Scientific Disciplinary Sector: BIO/14 Doctoral Candidate Tutor Elena Lucarini Prof. Carla Ghelardini Coordinator Prof. Elisabetta Teodori Years 2016/2019 TABLE OF CONTENTS ABSTRACT ........................................................................................................................................ 5 INTRODUCTION .............................................................................................................................. 8 1. Visceral Pain definition and features ............................................................................................ 8 1.1. Pain .......................................................................................................................................... 8 1.2. Visceral pain ............................................................................................................................ 9 1.3. Chronic visceral pain ............................................................................................................. 10 1.4. Visceral pain in IBS ................................................................................................................ 10 1.5. Visceral pain in IBD ............................................................................................................... 11 1.6. Visceral pain comorbidities ................................................................................................... 13 2. Visceral Pain assessment in patients ........................................................................................... 14 2.1. Pain rating scales ................................................................................................................... 14 2.2. Perceptional response to mechanical stimuli (rectal distension) ........................................... 16 2.3. Perceptional response to non-mechanical stimuli ................................................................. 18 2.4. Brain Imaging and fMRI ........................................................................................................ 19 3. Visceral pain pathways ................................................................................................................. 22 3.1. Enteric nervous system ........................................................................................................... 22 3.2. Peripheral visceral neurotransmission .................................................................................. 24 3.3. Viscerosomatic convergence .................................................................................................. 26 3.4. Central modulation of visceral pain ....................................................................................... 27 3.5. Gut-Brain axis ........................................................................................................................ 28 4. Chronic visceral pain mechanisms .............................................................................................. 30 4.1. Increased gut permeability ..................................................................................................... 30 4.2. Abnormal serotonin availability ............................................................................................. 31 4.3. Dysbiosis in gut microbiota .................................................................................................... 34 4.4. Altered gut immune response ................................................................................................. 38 4.5. Altered enteric nervous signalling ......................................................................................... 42 4.6. Peripheral and central Glia involvement in visceral pain ..................................................... 44 4.7. Stress ...................................................................................................................................... 47 4.8. Genetic predisposition ............................................................................................................ 49 5. Visceral pain therapy.................................................................................................................... 50 5.1. Dietary interventions .............................................................................................................. 51 5.2. Probiotics supplementation .................................................................................................... 54 1 5.3. Non-absorbed antibiotic, rifaximin ........................................................................................ 57 5.4. Antispasmodics and prokinetics ............................................................................................. 57 5.5. Serotonin receptors agonist/antagonist.................................................................................. 59 5.6. Antidepressants ...................................................................................................................... 60 5.7. Secretagogues......................................................................................................................... 61 5.8. Peripheral Opioid Receptor Agonists/Antagonists ................................................................ 62 5.9. Histamine receptor antagonist ............................................................................................... 62 5.10. NK receptor antagonists ....................................................................................................... 63 5.11. Gabapentenoids .................................................................................................................... 64 6. New therapeutic opportunities ..................................................................................................... 65 6.1. Emerging pharmacological targets in visceral pain management ......................................... 65 6.2. Adenosine receptors ............................................................................................................... 68 7. Faecal Microbiota Transplantation (FMT) ................................................................................ 70 7.1. Faecal Microbiota Transplantation in IBS and IBD .............................................................. 70 7.2. Faecal Microbiota Transplantation procedure ...................................................................... 73 8. Animal models of visceral pain .................................................................................................... 76 8.1. Genetic/Spontaneous models of visceral hypersensitivity ...................................................... 77 8.2. Early Life Stress models of visceral pain ............................................................................... 78 8.3. Stress-Induced models of colonic hypersensitivity in adulthood ............................................ 79 8.4. Colonic irritation models of hypersensitivity ......................................................................... 80 8.5. Models of post-infection/inflammatory visceral pain ............................................................. 81 AIMS OF THE STUDY ................................................................................................................... 84 MATERIALS AND METHODS ..................................................................................................... 86 1. Animals ......................................................................................................................................... 86 2. Induction of colitis ....................................................................................................................... 86 3. Drug administrations ................................................................................................................... 86 4. Assessment of visceral sensitivity by Viscero Motor Response (VMR) ...................................... 87 5. Assessment of visceral sensitivity by Abdominal Withdrawal Reflex (AWR) ............................ 87 6. Assessment of depression-related behaviour by Forced Swim Test (FST) ................................ 88 7. Assessment of anxiety-related behaviour by Open Field Test (OFT) ......................................... 88 8. Assessment of anxiety-related behaviour by Elevated Plus Maze Test (EPMT) ........................ 88 9. Histological evaluation of colon damage .................................................................................... 89 10. Evaluation of collagen deposition and inflammatory cells in the colon wall .......................... 89 11. Immunodetection of SP-positive fibres and MHC-II-positive macrophages in the colon ....... 90 12. Image analysis for the histochemical and immunohistochemical staining of the colon ......... 90 13. Analysis of spinal cord Iba-1 and GFAP positive cells by immunofluorescence..................... 90 2 14. Adenosine A3 receptor agonists administration ........................................................................ 91 15. Faecal Microbiota Transplantation (FMT) .............................................................................