Masters thesis of science intestinal permeability and functional properties of duodenal enteric neurons in a mouse model of autism

Intestinal Permeability and Functional Properties of Duodenal Enteric Neurons in a Mouse Model of Autism A thesis submitted in fulfilment of the requirements for the degree of Master of Science Joshua Kenneth Williams BSc in Biomedical science and Biotechnology (RMIT University, Melbourne) School of Health and Biomedical Science College of Science, Technology, Engineering and Math RMIT University August 2022 Thesis declaration I certify that except where due acknowledgement has been made, this research is that of the author alone; the content of this research submission is the result of work which has been carried out since the official commencement date of the approved research program; any editorial work, paid or unpaid, carried out by a third party is acknowledged; and ethics procedures and guidelines have been followed In addition, I certify that this submission contains no material previously submitted for award of any qualification at any other university or institution, unless approved for a joint-award with another institution, and acknowledge that no part of this work will, in the future, be used in a submission in my name, for any other qualification in any university or other tertiary institution without the prior approval of the University, and where applicable, any partner institution responsible for the jointaward of this degree I acknowledge that copyright of any published works contained within this thesis resides with the copyright holder(s) of those works I give permission for the digital version of my research submission to be made available on the web, via the University’s digital research repository, unless permission has been granted by the University to restrict access for a period of time I acknowledge the support I have received for my research through the provision of an Australian Government Research Training Program Scholarship Joshua Kenneth Williams 02 August 2022 i Acknowledgements I would like to express my deepest gratitude to my supervisors A/Prof Elisa Hill and Dr Suzanne Hosie Both Elisa and Suzanne were encouraging, inspiring and supportive throughout my candidature Thank you both for providing me with constructive criticism, along with skills in electrophysiology, permeability, scientific presentations, and scientific writing I would also like to acknowledge my lab members: Jackson, Tanya, Chalystha, Pasindu, Rachele, Samantha and Miti for providing me with invaluable feedback on my presentations and troubleshooting problems with experiments that arose during my candidature I would like to acknowledge my partner Latasha who provided constant emotional support and encouragement throughout my candidature ii Table of Contents Thesis declaration i Acknowledgements ii List of Figures vi List of Tables vii List of abbreviations and units vii Abstract Chapter 1: Introduction 1.0 Autism Spectrum Disorder (ASD) overview 1.1 Gastrointestinal disturbances in Autism patients 1.2 The gastrointestinal tract 1.3 The enteric nervous system 1.4 The submucosal plexus 1.5 The myenteric plexus 2.0 The gastrointestinal mucosal barrier 10 2.1 Intestinal epithelial cells 12 2.2 Apical junction protein complex .13 2.2.1 Tight junctions .14 2.2.2 Paracellular permeability 16 2.3 Gastrointestinal distress and ASD .17 2.4 Restoration of intestinal permeability 18 2.4.1 The impact of L-glutamine on intestinal permeability 20 2.4.2 The impact of caffeine on intestinal permeability 23 3.0 Classification of enteric neurons 26 3.1 Morphological classification of enteric neurons 26 3.2 Electrophysiological classification of enteric neurons 28 3.2.1 Synaptic-neurons (S-neurons) 28 3.2.2 AH (after-hyperpolarisation) neurons 28 3.3 Functional classification of enteric neurons 29 3.3.1 Intrinsic primary afferent neurons (IPANs) 30 3.3.2 Interneurons 31 3.3.3 Muscle motor neurons 32 3.4 Neurochemical classification of myenteric neurons 33 4.0 ASD genetic mutations 35 4.1 ASD and synaptic cell adhesion molecules 35 4.2 Neuroligins 36 4.3 Neuroligin-3 overview .37 4.4 Neuroligin functionality .37 4.5 NL3R451C mutation 38 4.6 Expression of Nlgn3 in the gastrointestinal tract 39 5.0 Project rationale 41 5.1 Assessing permeability in an autism mouse model .41 5.2 Action potential characteristics in duodenal myenteric neurons using an autism mouse model 42 5.3 Aims and hypotheses 43 Chapter 2: Measuring intestinal permeability in the Neuroligin-3R451C mouse model of autism 44 1.0 Introduction 44 2.0 Methods and materials 45 iii 2.1 Animals 45 2.2 Segmentation of the small and large intestine 45 2.3 Preparation of L-glutamine and caffeine stock solutions 46 2.4 Injection of FITC-Dextran 47 2.5 Time course permeability experiment 47 2.6 Construction of standard curve using log serial dilutions 49 2.7 Statistical analysis 50 3.0 Results 51 3.1 Small intestinal permeability in non-fasted wild-type and mutant mice 51 3.2 Permeability effects in wild-type and mutant fasted mice .53 3.3 Effects of fasting on NL3R451C mice 54 3.4 Effects of fasting on wild-type mice 56 3.5 Effects of L-glutamine on fasted NL3R451C mice 58 3.6 Effects of L-glutamine on fasted wild-type mice .60 3.7 Effects of caffeine on fasted NL3R451C mice .62 3.8 Effects of caffeine on fasted wild-type mice .64 4.0 Discussion 66 4.1 Understanding how NL3R451C mutation and feeding conditions effect paracellular permeability 66 4.2 Understanding how L-glutamine and caffeine impact paracellular permeability 70 5.0 Conclusion 77 Chapter 3: Optimisation of the patch-clamp recording technique in the enteric nervous system to examine action potential characteristics in mouse duodenal myenteric neurons 78 1.0 Introduction 78 2.0 Methods and materials 80 2.1 Animals 80 2.2 General perfusion/dissecting Krebs solution 80 2.3 Microdissection 81 2.4 Identification of myenteric ganglion 83 2.5 Protease solution .83 2.6 Whole-cell patch recording .83 2.7 External patching solution 84 2.8 Current clamp recording protocol .85 2.9 Statistical analysis 87 3.0 Results 88 3.1 Comparison of success rate of neuronal recordings for older versus younger mice 88 3.2 Recording action potentials in younger mice .90 3.2.1 Comparison of firing properties in myenteric neurons .92 3.2.2 Action potential characteristics in younger mice 92 3.3 Action potential characteristics observed in Neurons 1, and .93 3.4 Neuronal profiles in younger mice 97 3.4.1 Comparison of action potentials full trace 98 3.4.2 Comparison of current-voltage (IV) curves 100 3.4.3 Comparison of action potentials and current curves 100 3.5 Discussion 102 3.5.1 Success rate of whole-cell patch clamp recording in older versus younger mice 102 3.5.2 Future directions for patch-clamp electrophysiology in the ENS .104 3.6 Conclusion .107 References 108 Appendices 118 Appendix: Ethics approval letter 118 Appendix: Current clamp protocol used for whole-cell patch clamping 118 iv Electrophysiology appendices 119 Appendix Table 119 Appendix Table 119 Appendix Table 120 Appendix Table 120 Permeability appendices 121 Appendix Table 121 Appendix Table 121 Appendix Table 121 Appendix Table 122 Appendix Table 122 Appendix Table 10 122 Appendix Table 11 122 Appendix Table 12 123 Appendix Table 13 123 Appendix Table 14 123 Appendix Table 15 123 Appendix Table 16 124 Appendix Table 17 124 Appendix Table 18 124 Appendix Table 19 125 Appendix Table 20 126 Appendix Table 21 127 Appendix Table 22 128 Appendix Table 23 129 Appendix Table 24 130 Appendix Table 25 131 Appendix Table 26 132 Appendix Table 27 133 Appendix Table 28 134 Appendix Table 29 135 Appendix Table 30 136 Appendix Table 31 136 Appendix Table 32 137 Appendix Table 33 137 Appendix table 34 137 v List of Figures Figure 1: Organization of the mouse gastrointestinal tract Figure 2: The organization of the enteric nervous system Figure 3: The physiology of the mucus layer in the colon is different to the small intestine 11 Figure 4: Cross-sectional image of small intestine depicting the major cell types 12 Figure 5: Tight junctions, desmosomes and adherens junctions, make up apical junctional complexes 13 Figure 6: Intestinal epithelial cells utilise both transcellular and paracellular pathways 16 Figure 7: Potential mechanisms for L-glutamine restoring paracellular permeability in IEC’s 21 Figure 8: Overeating and duration of food deprivation influences intestinal mucus composition 24 Figure 9: Three major functional classes of enteric neurons 28 Figure 10: Setup for measuring the permeability of FITC through the paracellular route 46 Figure 11: Standard curve of known concentrations of FITC with absorbance .47 Figure 12: Small intestinal paracellular permeability in non-fasted NL3R451C and WT mice 49 Figure 13: Small and large intestinal paracellular permeability fasted NL3R451C and WT mice 51 Figure 14: Small intestinal paracellular permeability for fasted and non-fasted NL3R451C mice 52 Figure 15: Effect of fasting and non-fasting on intestinal paracellular permeability in WT mice 54 Figure 16: L-Glutamine restored paracellular permeability in NL3R451C mice to WT levels in small and large intestinal regions 56 Figure 17: Small and large intestinal paracellular permeability on wild-type mice treated with L-glutamine .58 Figure 18: Caffeine restored paracellular permeability in NL3R451C mice to WT concentrations in small and large intestinal regions 60 Figure 19: Caffeine decreased paracellular permeability in WT mice .62 Figure 20: Visual representation of obtaining an LMMP preparation .78 Figure 21: Visual representation of the general set up of patch-clamping 81 Figure 22: Custom-made hair cell attached to a glass micropipette .81 Figure 23: A myenteric neuron action potential with measured characteristics .83 Figure 24: Number of times whole cell seal configuration was obtained 84 Figure 25: Number of times whole cell seal obtained and/or AP firing recorded in myenteric neurons using younger and older mice 85 Figure 26: Three duodenal myenteric neurons exhibited action potentials in response to current steps .87 Figure 27: Action potential characteristics of three neurons 92 Figure 28: Characterisation of action potential profiles recorded throughout the full current injection protocol for three duodenal myenteric neurons 93 vi List of Tables Table 1: Myenteric neurons are classified according to their neurochemical coding Table 2: Known concentrations of FITC and absorbance produced Table 3: Action potential parameters for three duodenal myenteric neurons List of abbreviations and units AH: After-hyperpolarisation AHP: After-hyperpolarisation potential ASD: Autism Spectrum Disorder ATP: Adenosine triphosphate BA: Bile acid cAMP: cyclic adenosine monophosphate ChAT: Choline acetyltransferase CM: Circular muscle Cm: Membrane capacitance CNS: Central Nervous System DMEM: Dulbecco’s modified eagle medium EEC: Enteroendocrine cell EGF: Epidermal growth factor ENS: Enteric Nervous System ER: Endoplasmic reticulum mTOR: Mechanistic target of rapamycin FEPSPs: Fast excitatory post-synaptic potential FITC: Fluorescein Isothiocyanate GABA: Gamma-Aminobutyric acid vii 34 50 93 GI: Gastrointestinal HFD: High-fat diet HSP: Heat shock proteins HSF: Heat shock factor ICC: Interstitial cells of Cajal IEC: Intestinal epithelial cell IGF: Insulin-like growth factor IPAN: Intrinsic primary afferent neurons ISN: Intrinsic sensory neuron I-V curve: Current – Volt curve JAM: Junctional adhesion protein KO: Knock-out LM: Longitudinal-muscle LMMP: Longitudinal-muscle-myenteric-plexus LP: Lamina propria LPS Lipopolysaccharide MAPK: Mitogen-activated protein kinases ML: Mucosa layer MM: Muscularis mucosae MP: Myenteric plexus NO: Nitric oxide Neuroligin: NLGN3, Nlgn3 or NL3 NRXN: Neuroexin NOS: Nitric Oxide Synthase viii NF-B: nuclear factor-B pA: Picoamp PYY: Peptide YY Ra: Access resistance Rm: Membrane resistance RMP: Resting membrane potential SCFA: Short chain fatty acids S-neurons: Synaptic-neurons SMP: Submucosal plexus Tau: Membrane time constant TGF: Transforming growth factor TGF- α -: Transforming growth factor α TJ: Tight junction TMAO: trimethylamine N-oxide VAchT: Vesicular acetylcholine transporter VIP: Vaso-intestinal peptide WT: Wild-type ZO: Zonulin ix Appendix Table 12 Statistical parameters for colon permeability for NL3R451C and WT fasting mice utilising a two-way ANOVA with a Sidak multiple comparisons test Šídák's multiple comparisons test Colon (fasting) NL3 n=9 - WT n=8 minute minute 30 minutes 60 minutes 90 minutes 120 minutes Mean Diff 95.00% CI of diff Summary Adjusted P Value -3.6e-005 -6.0e-005 0.00010 -0.00041 -0.00090 -0.0014 -0.0013 to 0.0013 -0.0014 to 0.0012 -0.0012 to 0.0014 -0.0017 to 0.00092 -0.0022 to 0.00043 -0.0027 to -8.2e-005 ns ns ns ns ns * >0.99 >0.99 >0.99 0.95 0.36 0.03 Appendix Table 13 Statistical parameters for duodenal permeability for NL3R451C non-fasting and fasting mice utilising a two-way ANOVA with a Sidak multiple comparisons test Šídák's multiple comparisons test Duodenum NL3 n=10 (Fasting) - NL3 n=9 (non-fasting) minute minute 30 minutes 60 minutes 90 minutes 120 minutes Mean Diff 95.00% CI of diff Summary Adjusted P Value 0.00035 0.00019 0.00062 0.0020 0.0036 0.0033 -0.0037 to 0.0044 -0.0039to 0.0043 -0.0035 to 0.0047 -0.0021 to 0.0061 -0.00044 to 0.0078 -0.00083 to 0.0074 ns ns ns ns ns ns >0.99 >0.99 0.99 0.71 0.11 0.18 Appendix Table 14 Statistical parameters for jejunum permeability for NL3R451C non-fasting and fasting mice utilising a two-way ANOVA with a Sidak multiple comparisons test Šídák's multiple comparisons test Jejunum NL3 n=10 (Fasting) - NL3 n=12 (non-fasting) minute minute 30 minutes 60 minutes 90 minutes 120 minutes Mean Diff 95.00% CI of diff Summary Adjusted P Value 0.00043 0.00040 0.00086 0.0023 0.0035 0.0041 -0.0031 to 0.0040 -0.0031 to 0.0040 -0.0027 to 0.0044 -0.0012 to 0.0059 -8.1e-005 to 0.0071 0.00051 to 0.0077 ns ns ns ns ns * 0.99 0.99 0.98 0.42 0.06 0.02 Appendix Table 15 Statistical parameters for distal ileum permeability for NL3R451C non-fasting and fasting mice utilising a two-way ANOVA with a Sidak multiple comparisons test Šídák's multiple comparisons test Distal ileum NL3 n=9 (Fasting) - NL3 n=13 (non-fasting) minute minute 30 minutes 60 minutes 90 minutes 120 minutes Mean Diff 95.00% CI of diff Summary Adjusted P Value 0.00042 0.00039 0.0025 0.0032 0.0040 0.0044 -0.0036 to 0.0045 -0.0037 to 0.0044 -0.0015 to 0.0066 -0.00085 to 0.0073 3.8e-006 to 0.0081 0.00039 to 0.0085 ns ns ns ns * * 0.99 >0.99 0.47 0.19 0.040 0.02 123 Appendix Table 16 Statistical parameters for duodenal permeability for WT non-fasting and fasting mice utilising a two-way ANOVA with a Sidak multiple comparisons test Šídák's multiple comparisons test Duodenum WT n=8 (Fasting) - WT n=9 (non-fasting) minute minute 30 minutes 60 minutes 90 minutes 120 minutes Mean Diff 95.00% CI of diff Summary Adjusted P Value -0.00017 -0.00035 -0.0011 -0.0022 -0.0036 -0.0041 -0.0032 to 0.0029 -0.0034 to 0.0027 -0.0041 to 0.0019 -0.0052 to 0.00089 -0.0067 to -0.00054 -0.0071 to -0.0010 ns ns ns ns * ** >0.99 0.99 0.91 0.30 0.012 0.0035 Appendix Table 17 Statistical parameters for jejunum permeability for WT non-fasting and fasting mice utilising a two-way ANOVA with a Sidak multiple comparisons test Šídák's multiple comparisons test Jejunum WT n=11 (Fasting) - WT n=8 (non-fasting) minute minute 30 minutes 60 minutes 90 minutes 120 minutes Mean Diff 95.00% CI of diff Summary Adjusted P Value 0.00014 -1.0e-005 -0.0011 -0.0020 -0.0029 -0.0038 -0.0060 to 0.0063 -0.0062 to 0.0061 -0.0073 to 0.0050 -0.0082 to 0.0041 -0.0091 to 0.0031 -0.010 to 0.0023 ns ns ns ns ns ns >0.99 >0.99 0.99 0.93 0.73 0.45 Appendix Table 18 Statistical parameters for distal ileum permeability for WT non-fasting and fasting mice utilising a two-way ANOVA with a Sidak multiple comparisons test Šídák's multiple comparisons test Distal ileum WT n=8 (Fasting) - WT n=10 (non-fasting) minute minute 30 minutes 60 minutes 90 minutes 120 minutes Mean Diff 95.00% CI of diff Summary Adjusted P Value 0.00017 1.0e-005 0.0010 0.0022 0.0026 0.0027 -0.0024 to 0.0027 -0.0025 to 0.0026 -0.0015 to 0.0036 -0.00037 to 0.0048 4.3e-005 to 0.0052 0.00014 to 0.0053 ns ns ns ns * * >0.99 >0.99 0.88 0.13 0.044 0.033 124 Appendix Table 19 Statistical parameters for duodenal permeability for fasting NL3R451C mice versus fasting NL3R451C mice treated with L-glutamine utilising a two-way ANOVA with a Tukeys multiple comparisons test (Green fill represents glutamine restoring NL3 FITC concentrations to WT concentrations) Mean difference 95% CI of diff Summary Adjusted P Value -0.00021 -4.8e-006 4.4e-005 0.00021 0.00026 4.9e-005 -0.0031 to 0.0027 -0.0031 to 0.0031 -0.0030 to 0.0031 -0.0024 to 0.0028 -0.0022 to 0.0028 -0.0027 to 0.0028 ns ns ns ns ns ns 0.99 >0.99 >0.99 0.99 0.99 >0.99 minute NL3 + 30mM Glutamine n=5 vs NL3 n=10 NL3 + 30mM Glutamine n=5 vs NL3 + 90mM Glutamine n=7 NL3 + 30mM Glutamine n=5 vs WT n=8 NL3 n=10 vs NL3 + 90mM Glutamine n=7 NL3 n=10 vs WT n=8 NL3 + 90mM Glutamine n=7 vs WT n=8 -0.00022 -2.6e-006 5.0e-005 0.00021 0.00027 5.3e-005 -0.003162 to 0.0027 -0.003148 to 0.0031 -0.003012 to 0.0031 -0.002430 to 0.0028 -0.002277 to 0.0028 -0.002727 to 0.0028 ns ns ns ns ns ns 0.99 >0.99 >0.99 0.99 0.99 >0.99 30 minutes NL3 + 30mM Glutamine n=5 vs NL3 n=10 NL3 + 30mM Glutamine n=5 vs NL3 + 90mM Glutamine n=7 NL3 + 30mM Glutamine n=5 vs WT n=8 NL3 n=10 vs NL3 + 90mM Glutamine n=7 NL3 n=10 vs WT n=8 NL3 + 90mM Glutamine n=7 vs WT n=8 -0.00096 0.00059 0.00057 0.0015 0.0015 -2.4e-005 -0.0039 to 0.0019 -0.0025 to 0.0037 -0.0024 to 0.0036 -0.0010 to 0.0042 -0.0010 to 0.0040 -0.0028 to 0.0027 ns ns ns ns ns ns 0.83 0.96 0.96 0.42 0.40 >0.99 60 minutes NL3 + 30mM Glutamine n=5 vs NL3 n=10 NL3 + 30mM Glutamine n=5 vs NL3 + 90mM Glutamine n=7 NL3 + 30mM Glutamine n=5 vs WT n=8 NL3 n=10 vs NL3 + 90mM Glutamine n=7 NL3 n=10 vs WT n=8 NL3 + 90mM Glutamine n=7 vs WT n=8 -0.0029 0.00059 0.0014 0.0035 0.0043 0.00081 -0.0059 to -3.8e-005 -0.0025 to 0.0037 -0.0016 to 0.0044 0.00092 to 0.0062 0.0018 to 0.0069 -0.0019 to 0.0035 * ns ns ** **** ns 0.040 0.96 0.62 0.00 0.99 0.99 0.99 0.99 >0.99 minutes NL3 + 30mM Glutamine n=5 vs NL3 n=9 NL3 + 30mM Glutamine n=5 vs NL3 + 90mM Glutamine n=7 NL3 + 30mM Glutamine n=5 vs WT n=8 NL3 n=9 vs NL3 + 90mM Glutamine n=7 NL3 n=9 vs WT n=8 NL3 + 90mM Glutamine n=7 vs WT n=8 -0.00031 -2.0e-005 -6.5e-005 0.00029 0.00024 -4.4e-005 -0.0036 to 0.0030 -0.0035 to 0.0034 -0.0034 to 0.0033 -0.0027 to 0.0033 -0.0026 to 0.0031 -0.0031 to 0.0030 ns ns ns ns ns ns 0.99 >0.99 >0.99 0.99 0.99 >0.99 30 minutes NL3 + 30mM Glutamine n=5 vs NL3 n=9 NL3 + 30mM Glutamine n=5 vs NL3 + 90mM Glutamine n=7 NL3 + 30mM Glutamine n=5 vs WT n=8 NL3 n=9 vs NL3 + 90mM Glutamine n=7 NL3 n=9 vs WT n=8 NL3 + 90mM Glutamine n=7 vs WT n=8 -0.00093 0.00015 0.00076 0.0010 0.0017 0.00061 -0.0042 to 0.0024 -0.0033 to 0.0036 -0.0026 to 0.0041 -0.0019 to 0.0041 -0.0012 to 0.0046 -0.0024 to 0.0037 ns ns ns ns ns ns 0.88 0.99 0.93 0.78 0.42 0.95 ns ns ns 0.23 0.98 >0.99 ns 0.06 ns ns 0.12 0.98 Tukey's multiple comparisons test Distal ileum minutes NL3 + 30mM Glutamine n=5 vs NL3 n=9 NL3 + 30mM Glutamine n=5 vs NL3 + 90mM Glutamine n=7 NL3 + 30mM Glutamine n=5 vs WT n=8 NL3 n=9 vs NL3 + 90mM Glutamine n=7 NL3 n=9 vs WT n=8 NL3 + 90mM Glutamine n=7 vs WT n=8 60 minutes NL3 + 30mM Glutamine n=5 vs NL3 n=9 NL3 + 30mM Glutamine n=5 vs NL3 + 90mM Glutamine n=7 NL3 + 30mM Glutamine n=5 vs WT n=8 -0.0024 0.00045 5.3e-005 NL3 n=9 vs NL3 + 90mM Glutamine n=7 0.0028 NL3 n=9 vs WT n=8 NL3 + 90mM Glutamine n=7 vs WT n=8 0.0024 -0.00040 -0.0057 to 0.00091 -0.0030 to 0.0039 -0.0033 to 0.0034 -0.00013 to 0.0059 -0.00043 to 0.0054 -0.0035 to 0.0027 NL3 + 30mM Glutamine n=5 vs NL3 n=9 NL3 + 30mM Glutamine n=5 vs NL3 + 90mM Glutamine n=7 NL3 + 30mM Glutamine n=5 vs WT n=8 NL3 n=9 vs NL3 + 90mM Glutamine n=7 NL3 n=9 vs WT n=8 NL3 + 90mM Glutamine n=7 vs WT n=8 -0.0041 0.00012 -0.00087 0.0042 0.0032 -0.00099 -0.0072 to 0.00076 -0.0033 to 0.0036 -0.0042 to 0.0025 0.0012 to 0.0072 0.00032 to 0.0061 -0.0041 to 0.0021 ** ns ns ** * ns 0.0092 0.99 0.91 0.0021 0.02 0.83 120 minutes NL3 + 30mM Glutamine n=5 vs NL3 n=9 NL3 + 30mM Glutamine n=5 vs NL3 + 90mM Glutamine n=7 NL3 + 30mM Glutamine n=5 vs WT n=8 NL3 n=9 vs NL3 + 90mM Glutamine n=7 NL3 n=9 vs WT n=8 NL3 + 90mM Glutamine n=7 vs WT n=8 -0.0047 0.00035 -0.00099 0.0051 0.0038 -0.0013 -0.0081 to -0.0014 -0.0031 to 0.0038 -0.0044 to 0.0024 0.0021 to 0.0081 0.00088 to 0.0067 -0.0044 to 0.0017 ** ns ns *** ** ns 0.0016 0.99 0.87 0.0001 0.0050 0.67 90 minutes 127 P Appendix Table 22 Statistical parameters for colon permeability for fasting NL3R451C mice versus fasting NL3R451C mice treated with L-glutamine utilising a two-way ANOVA with a Tukeys multiple comparisons test (Green fill represents glutamine restoring NL3 FITC concentrations to WT concentrations) Mean difference 95% CI of diff Summary Adjusted P Value -5.6e-006 7.2e-005 3.1e-005 7.8e-005 3.6e-005 -4.3e-005 -0.0018 to 0.0018 -0.0019 to 0.0020 -0.0018 to 0.0019 -0.0012 to 0.0013 -0.0011 to 0.0011 -0.0013 to 0.0013 ns ns ns ns ns ns >0.99 0.99 >0.99 0.99 0.99 0.99 minute NL3 + 30mM Glutamine n=2 vs NL3 n=9 NL3 + 30mM Glutamine n=2 vs NL3 + 90mM Glutamine n=5 NL3 + 30mM Glutamine n=2 vs WT n=8 NL3 n=9 vs NL3 + 90mM Glutamine n=5 NL3 n=9 vs WT n=8 NL3 + 90mM Glutamine n=5 vs WT n=8 -8.3e-006 9.3e-005 5.2e-005 0.00010 6.0e-005 -4.0e-005 -0.0018 to 0.0018 -0.0018 to 0.0020 -0.0018 to 0.0019 -0.0012 to 0.0014 -0.0010 to 0.0012 -0.0013 to 0.0013 ns ns ns ns ns ns >0.99 0.99 0.99 0.99 0.99 0.99 30 minutes NL3 + 30mM Glutamine n=2 vs NL3 n=9 NL3 + 30mM Glutamine n=2 vs NL3 + 90mM Glutamine n=5 NL3 + 30mM Glutamine n=2 vs WT n=8 NL3 n=9 vs NL3 + 90mM Glutamine n=5 NL3 n=9 vs WT n=8 NL3 + 90mM Glutamine n=5 vs WT n=8 -0.00039 -7.4e-006 -0.00049 0.00039 -0.00010 -0.00049 -0.0022 to 0.0014 -0.0019 to 0.0019 -0.0023 to 0.0013 -0.00092 to 0.0017 -0.0012 to 0.0010 -0.0018 to 0.00085 ns ns ns ns ns ns 0.94 >0.99 0.89 0.86 0.99 0.77 60 minutes NL3 + 30mM Glutamine n=2 vs NL3 n=9 NL3 + 30mM Glutamine n=2 vs NL3 + 90mM Glutamine n=5 NL3 + 30mM Glutamine n=2 vs WT n=8 NL3 n=9 vs NL3 + 90mM Glutamine n=5 NL3 n=9 vs WT n=8 NL3 + 90mM Glutamine n=5 vs WT n=8 -0.0010 -0.00011 -0.00065 0.00096 0.00041 -0.00054 -0.0029 to 0.00077 -0.0020 to 0.0018 -0.0025 to 0.0012 -0.00035 to 0.0022 -0.00073 to 0.0015 -0.0018 to 0.00080 ns ns ns ns ns ns 0.43 0.99 0.79 0.23 0.77 0.71 90 minutes NL3 + 30mM Glutamine n=2 vs NL3 n=9 NL3 + 30mM Glutamine n=2 vs NL3 + 90mM Glutamine n=5 NL3 + 30mM Glutamine n=2 vs WT n=8 NL3 n=9 vs NL3 + 90mM Glutamine n=5 NL3 n=9 vs WT n=8 NL3 + 90mM Glutamine n=5 vs WT n=8 -0.0015 -0.00011 -0.00068 0.0015 0.00090 -0.00057 -0.0034 to 0.00025 -0.0020 to 0.0018 -0.0025 to 0.0011 0.00016 to 0.0027 -0.00024 to 0.0020 -0.0019 to 0.00077 ns ns ns * ns ns 0.11 0.99 0.77 0.021 0.17 0.68 120 minutes NL3 + 30mM Glutamine n=2 vs NL3 n=9 NL3 + 30mM Glutamine n=2 vs NL3 + 90mM Glutamine n=5 NL3 + 30mM Glutamine n=2 vs WT n=8 NL3 n=9 vs NL3 + 90mM Glutamine n=5 NL3 n=9 vs WT n=8 NL3 + 90mM Glutamine n=5 vs WT n=8 -0.0021 2.7e-005 -0.00075 0.0022 0.0014 -0.00077 -0.0040 to -0.00032 -0.0019 to 0.0020 -0.0026 to 0.0011 0.00088 to 0.0035 0.00027 to 0.0025 -0.0021 to 0.00056 * ns ns *** ** ns 0.014 >0.99 0.72 0.0002 0.0087 0.43 Tukey's multiple comparisons test Colon minutes NL3 + 30mM Glutamine n=2 vs NL3 n=9 NL3 + 30mM Glutamine n=2 vs NL3 + 90mM Glutamine n=5 NL3 + 30mM Glutamine n=2 vs WT n=8 NL3 n=9 vs NL3 + 90mM Glutamine n=5 NL3 n=9 vs WT n=8 NL3 + 90mM Glutamine n=5 vs WT n=8 128 Appendix Table 23 Statistical parameters for duodenal permeability for fasting WT mice versus fasting WT mice treated with L-glutamine utilising a two-way ANOVA with a Sidak multiple comparisons test Mean difference 95% CI of diff Summary Adjusted P Value 0.00025 0.00015 -0.00010 -0.0027 to 0.0032 -0.0033 to 0.0036 -0.0032 to 0.0030 ns ns ns 0.97 0.99 0.99 minute WT + 90mM Glutamine n=5 vs WT n=8 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=8 vs WT + 30mM Glutamine n=4 0.00021 0.00018 -3.1e-005 -0.0027 to 0.0031 -0.0033 to 0.0036 -0.0032 to 0.0031 ns ns ns 0.98 0.99 0.99 30 minutes WT + 90mM Glutamine n=5 vs WT n=8 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=8 vs WT + 30mM Glutamine n=4 0.00087 -0.0013 -0.0022 -0.0020 to 0.0038 -0.0048 to 0.0021 -0.0054 to 0.00097 ns ns ns 0.76 0.63 0.22 60 minutes WT + 90mM Glutamine n=5 vs WT n=8 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=8 vs WT + 30mM Glutamine n=4 0.0012 -0.0027 -0.0039 -0.0016 to 0.0042 -0.0061 to 0.00078 -0.0071 to -0.00080 ns ns * 0.55 0.16 0.010 90 minutes WT + 90mM Glutamine n=5 vs WT n=8 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=8 vs WT + 30mM Glutamine n=4 0.0029 -0.0015 -0.0044 -2.7e-005 to 0.0059 -0.0050 to 0.0019 -0.0076 to -0.0012 ns ns ** 0.05 0.55 0.0035 120 minutes WT + 90mM Glutamine n=5 vs WT n=8 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=8 vs WT + 30mM Glutamine n=4 0.0028 -0.00083 -0.0037 -7.5e-005 to 0.0058 -0.0043 to 0.0026 -0.0069 to -0.00054 ns ns * 0.057 0.83 0.017 Sidak multiple comparisons test Duodenum minutes WT + 90mM Glutamine n=5 vs WT n=8 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=8 vs WT + 30mM Glutamine n=4 129 Appendix Table 24 Statistical parameters for jejunum permeability for fasting WT mice versus fasting WT mice treated with L-glutamine utilising a two-way ANOVA with a Sidak multiple comparisons test Sidak multiple comparisons test Jejunum minutes WT + 90mM Glutamine n=5 vs WT n=11 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=11 vs WT + 30mM Glutamine n=4 Mean difference 95% CI of diff Summary Adjusted P Value 2.0e-005 0.00010 8.1e-005 -0.0027 to 0.0027 -0.0033 to 0.0035 -0.0028 to 0.0030 ns ns ns 0.99 0.99 0.99 minute WT + 90mM Glutamine n=5 vs WT n=11 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=11 vs WT + 30mM Glutamine n=4 -3.6e-005 0.00014 0.00018 -0.0027 to 0.0027 -0.0032 to 0.0035 -0.0027 to 0.0031 ns ns ns 0.99 0.99 0.98 30 minutes WT + 90mM Glutamine n=5 vs WT n=11 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=11 vs WT + 30mM Glutamine n=4 -0.00018 -0.00014 3.4e-005 -0.0029 to 0.0025 -0.0035 to 0.0032 -0.0029 to 0.0030 ns ns ns 0.98 0.99 0.99 60 minutes WT + 90mM Glutamine n=5 vs WT n=11 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=11 vs WT + 30mM Glutamine n=4 0.00022 0.00064 0.00041 -0.0025 to 0.0029 -0.0027 to 0.0040 -0.0025 to 0.0033 ns ns ns 0.97 0.89 0.94 90 minutes WT + 90mM Glutamine n=5 vs WT n=11 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=11 vs WT + 30mM Glutamine n=4 0.00048 0.00091 0.00043 -0.0022 to 0.0032 -0.0024 to 0.0043 -0.0025 to 0.0034 ns ns ns 0.90 0.79 0.93 120 minutes WT + 90mM Glutamine n=5 vs WT n=11 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=11 vs WT + 30mM Glutamine n=4 0.00094 0.0013 0.00044 -0.0017 to 0.0036 -0.0020 to 0.0048 -0.0025 to 0.0034 ns ns ns 0.68 0.59 0.93 130 Appendix Table 25 Statistical parameters for distal ileum permeability for fasting WT mice versus fasting WT mice treated with L-glutamine utilising a two-way ANOVA with a Sidak multiple comparisons test Sidak multiple comparisons test Distal ileum minutes WT + 90mM Glutamine n=5 vs WT n=8 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=8 vs WT + 30mM Glutamine n=4 Mean difference 95% CI of diff Summary Adjusted P Value -0.00018 -0.00020 -1.2e-005 -0.0027 to 0.0024 -0.0032 to 0.0028 -0.0028 to 0.0027 ns ns ns 0.98 0.98 >0.99 minute WT + 90mM Glutamine n=5 vs WT n=8 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=8 vs WT + 30mM Glutamine n=4 -0.00019 -0.00013 6.1e-005 -0.0027 to 0.0024 -0.0031 to 0.0029 -0.0027 to 0.0028 ns ns ns 0.98 0.99 0.99 30 minutes WT + 90mM Glutamine n=5 vs WT n=8 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=8 vs WT + 30mM Glutamine n=4 -0.00037 -0.0015 -0.0011 -0.0029 to 0.0022 -0.0046 to 0.0015 -0.0039 to 0.0016 ns ns ns 0.93 0.45 0.57 60 minutes WT + 90mM Glutamine n=5 vs WT n=8 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=8 vs WT + 30mM Glutamine n=4 -0.0010 -0.0026 -0.0015 -0.0037 to 0.0015 -0.0056 to 0.00045 -0.0043 to 0.0012 ns ns ns 0.57 0.11 0.40 90 minutes WT + 90mM Glutamine n=5 vs WT n=8 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=8 vs WT + 30mM Glutamine n=4 -0.0014 -0.0028 -0.0014 -0.0040 to 0.0011 -0.0059 to 0.00017 -0.0042 to 0.0013 ns ns ns 0.38 0.068 0.44 120 minutes WT + 90mM Glutamine n=5 vs WT n=8 WT + 90mM Glutamine n=5 vs WT + 30mM Glutamine n=4 WT n=8 vs WT + 30mM Glutamine n=4 -0.0022 -0.0029 -0.00067 -0.0048 to 0.00034 -0.0059 to 0.00013 -0.0034 to 0.0021 ns ns ns 0.10 0.064 0.83 131 Appendix Table 26 Statistical parameters for colon permeability for fasting WT mice versus fasting WT mice treated with L-glutamine utilising a two-way ANOVA with a Sidak multiple comparisons test Sidak multiple comparisons test Colon minutes WT + 90 mM Glutamine n=5 vs WT n=9 WT + 90 mM Glutamine n=5 vs WT + 30mM Glutamine n=3 WT n=9 vs WT + 30mM Glutamine n=3 Mean difference 95% CI of diff Summary Adjusted P Value -2.4e-005 9.2e-005 0.00011 -0.0011 to 0.0011 -0.0013 to 0.0015 -0.0012 to 0.0014 ns ns ns 0.99 0.98 0.97 minute WT + 90 mM Glutamine n=5 vs WT n=9 WT + 90 mM Glutamine n=5 vs WT + 30mM Glutamine n=3 WT n=9 vs WT + 30mM Glutamine n=3 -2.6e-005 0.00013 0.00016 -0.0011 to 0.0011 -0.0013 to 0.0015 -0.0011 to 0.0015 ns ns ns 0.99 0.97 0.95 30 minutes WT + 90 mM Glutamine n=5 vs WT n=9 WT + 90 mM Glutamine n=5 vs WT + 30mM Glutamine n=3 WT n=9 vs WT + 30mM Glutamine n=3 -0.00055 0.00022 0.00077 -0.0016 to 0.00058 -0.0012 to 0.0016 -0.00056 to 0.0021 ns ns ns 0.47 0.92 0.35 60 minutes WT + 90 mM Glutamine n=5 vs WT n=9 WT + 90 mM Glutamine n=5 vs WT + 30mM Glutamine n=3 WT n=9 vs WT + 30mM Glutamine n=3 -0.00060 0.00035 0.00096 -0.0017 to 0.00052 -0.0011 to 0.0018 -0.00038 to 0.0023 ns ns ns 0.41 0.82 0.20 90 minutes WT + 90 mM Glutamine n=5 vs WT n=9 WT + 90 mM Glutamine n=5 vs WT + 30mM Glutamine n=3 WT n=9 vs WT + 30mM Glutamine n=3 -0.00074 0.00044 0.0011 -0.0018 to 0.00038 -0.0010 to 0.0018 -0.00015 to 0.0025 ns ns ns 0.26 0.74 0.092 120 minutes WT + 90 mM Glutamine n=5 vs WT n=9 WT + 90 mM Glutamine n=5 vs WT + 30mM Glutamine n=3 WT n=9 vs WT + 30mM Glutamine n=3 -0.00075 0.00067 0.0014 -0.0018 to 0.00038 -0.00077 to 0.0021 8.1e-005 to 0.0027 ns ns * 0.25 0.50 0.035 132 Appendix Table 27 Statistical parameters for duodenal permeability for fasting NL3 mice versus fasting NL3 mice treated with caffeine utilising a two-way ANOVA with a Sidak multiple comparisons test (Green fill represents caffeine restoring NL3 FITC concentrations to WT concentrations) Sidak multiple comparisons test Duodenum minutes NL3 (10mM caffeine) n=5 vs NL3 n=10 NL3 (10mM caffeine) n=5 vs WT n=8 NL3 n=10 vs WT n=8 Mean difference 95% CI of diff Summary Adjusted P Value -0.00018 7.5e-005 0.00026 -0.0038 to 0.0034 -0.0037 to 0.0038 -0.0028 to 0.0034 ns ns ns >0.99 >0.99 >0.99 minute NL3 (10mM caffeine) n=5 vs NL3 n=10 NL3 (10mM caffeine) n=5 vs WT n=8 NL3 n=10 vs WT n=8 -0.00021 5.3e-005 0.00027 -0.0038 to 0.0034 -0.0037 to 0.0038 -0.0028 to 0.0034 ns ns ns >0.99 >0.99 >0.99 30 minutes NL3 (10mM caffeine) n=5 vs NL3 n=10 NL3 (10mM caffeine) n=5 vs WT n=8 NL3 n=10 vs WT n=8 -0.0017 -0.00021 0.0015 -0.0053 to 0.0018 -0.0039 to 0.0035 -0.0016 to 0.0046 ns ns ns 0.93 >0.99 0.93 60 minutes NL3 (10mM caffeine) n=5 vs NL3 n=10 NL3 (10mM caffeine) n=5 vs WT n=8 NL3 n=10 vs WT n=8 -0.0048 -0.00048 0.0043 -0.0085 to -0.0012 -0.0042 to 0.0032 0.0012 to 0.0075 ** ns *** 0.0014 >0.99 0.0007 90 minutes NL3 (10mM caffeine) n=5 vs NL3 n=10 NL3 (10mM caffeine) n=5 vs WT n=8 NL3 n=10 vs WT n=8 -0.0067 -0.00033 0.0064 -0.010 to -0.0031 -0.0041 to 0.0034 0.0033 to 0.0095 **** ns **** 0.99 0.99 minute NL3 + 10mM caffeine n=5 vs NL3 n=9 NL3 + 10mM caffeine n=5 vs WT n=11 NL3 n=9 vs WT n=11 -0.00026 -0.00014 0.00012 -0.0041 to 0.0035 -0.0038 to 0.0035 -0.0029 to 0.0032 ns ns ns >0.99 >0.99 >0.99 30 minutes NL3 + 10mM caffeine n=5 vs NL3 n=9 NL3 + 10mM caffeine n=5 vs WT n=11 NL3 n=9 vs WT n=11 -0.0017 -0.0010 0.00068 -0.0056 to 0.0020 -0.0048 to 0.0026 -0.0024 to 0.0037 ns ns ns 0.96 0.99 >0.99 60 minutes NL3 + 10mM caffeine n=5 vs NL3 n=9 NL3 + 10mM caffeine n=5 vs WT n=11 NL3 n=9 vs WT n=11 -0.0045 -0.0025 0.0019 -0.0083 to -0.00067 -0.0063 to 0.0011 -0.0011 to 0.0050 ** ns ns 0.0088 0.49 0.66 90 minutes NL3 + 10mM caffeine n=5 vs NL3 n=9 NL3 + 10mM caffeine n=5 vs WT n=11 NL3 n=9 vs WT n=11 -0.0062 -0.0033 0.0028 -0.010 to -0.0023 -0.0070 to 0.00035 -0.00026 to 0.0059 **** ns ns 0.99 >0.99 minute NL3 + 10mM caffeine n=5 vs NL3 n=9 NL3 + 10mM caffeine n=5 vs WT n=8 NL3 n=9 vs WT n=8 -0.00015 8.7e-005 0.00024 -0.0033 to 0.0030 -0.0031 to 0.0033 -0.0025 to 0.0030 ns ns ns >0.99 >0.99 >0.99 30 minutes NL3 + 10mM caffeine n=5 vs NL3 n=9 NL3 + 10mM caffeine n=5 vs WT n=8 NL3 n=9 vs WT n=8 -0.0019 -0.00023 0.0017 -0.0051 to 0.0012 -0.0035 to 0.0030 -0.0010 to 0.0044 ns ns ns 0.70 >0.99 0.69 60 minutes NL3 + 10mM caffeine n=5 vs NL3 n=9 NL3 + 10mM caffeine n=5 vs WT n=8 NL3 n=9 vs WT n=8 -0.0033 -0.00084 0.0024 -0.0065 to -0.00013 -0.0041 to 0.0024 -0.00029 to 0.0052 * ns ns 0.033 >0.99 0.12 90 minutes NL3 + 10mM caffeine n=5 vs NL3 n=9 NL3 + 10mM caffeine n=5 vs WT n=8 NL3 n=9 vs WT n=8 -0.0043 -0.0010 0.0032 -0.0075 to -0.0011 -0.0043 to 0.0021 0.00045 to 0.0060 ** ns ** 0.0013 0.99 0.0099 120 minutes NL3 + 10mM caffeine n=5 vs NL3 n=9 NL3 + 10mM caffeine n=5 vs WT n=8 NL3 n=9 vs WT n=8 -0.0054 -0.0016 0.0038 -0.0086 to -0.0022 -0.0049 to 0.0016 0.0010 to 0.0065 **** ns ** 0.99 >0.99 >0.99 minute NL3 + 10mM caffeine n=3 vs NL3 n=9 NL3 + 10mM caffeine n=3 vs WTn=8 NL3 n=9 vs WTn=8 -0.00022 -0.00015 6.0e-005 -0.0021 to 0.001753 -0.0021 to 0.001844 -0.0013 to 0.001499 ns ns ns >0.99 >0.99 >0.99 30 minutes NL3 + 10mM caffeine n=3 vs NL3 n=9 NL3 + 10mM caffeine n=3 vs WTn=8 NL3 n=9 vs WTn=8 -0.00075 -0.00085 -0.00010 -0.0027 to 0.0012 -0.0028 to 0.0011 -0.0015 to 0.0013 ns ns ns 0.99 0.97 >0.99 60 minutes NL3 + 10mM caffeine n=3 vs NL3 n=9 NL3 + 10mM caffeine n=3 vs WTn=8 NL3 n=9 vs WTn=8 -0.0014 -0.0010 0.00041 -0.0033 to 0.00055 -0.0030 to 0.0010 -0.0010 to 0.0018 ns ns ns 0.41 0.91 0.99 90 minutes NL3 + 10mM caffeine n=3 vs NL3 n=9 NL3 + 10mM caffeine n=3 vs WTn=8 NL3 n=9 vs WTn=8 -0.0021 -0.0012 0.00090 -0.0041 to -0.00018 -0.0032 to 0.00074 -0.00053 to 0.0023 * ns ns 0.020 0.66 0.65 120 minutes NL3 + 10mM caffeine n=3 vs NL3 n=9 NL3 + 10mM caffeine n=3 vs WTn=8 NL3 n=9 vs WTn=8 -0.0030 -0.0015 0.0014 -0.0049 to -0.0010 -0.0035 to 0.00041 -1.6e-005 to 0.0028 *** ns ns 0.0002 0.27 0.055 Appendix Table 31 Statistical parameters for duodenal permeability for of fasting WT mice versus fasting WT mice treated with caffeine utilising a two-way ANOVA with a Sidak multiple comparisons test Šídák's multiple comparisons test Duodenum WT (10mM caffeine) n=5 - WT n=8 minute minute 30 minutes 60 minutes 90 minutes 120 minutes Mean Diff 95.00% CI of diff Summary Adjusted P Value 8.664e-005 7.340e-005 -0.0002189 -0.0002480 0.0007436 0.0007404 -0.002838 to 0.003012 -0.002852 to 0.002998 -0.003144 to 0.002706 -0.003173 to 0.002677 -0.002182 to 0.003669 -0.002185 to 0.003665 ns ns ns ns ns ns >0.9999 >0.9999 >0.9999 >0.9999 0.9830 0.9834 136 Appendix Table 32 Statistical parameters for jejunum permeability for fasting WT mice versus fasting WT mice treated with caffeine utilising a two-way ANOVA with a Sidak multiple comparisons test Šídák's multiple comparisons test Jejunum WT+ 10mM caffeine n=6 - WT n=11 minute minute 30 minutes 60 minutes 90 minutes 120 minutes Mean Diff 95.00% CI of diff Summary Adjusted P Value -0.0002265 -0.0002577 -0.001127 -0.002843 -0.003635 -0.003588 -0.003159 to 0.002706 -0.003190 to 0.002675 -0.004059 to 0.001805 -0.005775 to 8.947e-005 -0.006568 to -0.0007027 -0.006520 to -0.0006551 ns ns ns ns ** ** >0.9999 >0.9999 0.8863 0.0623 0.0074 0.0085 Appendix Table 33 Statistical parameters for distal ileum permeability for fasting WT mice versus fasting WT mice treated with caffeine utilising a two-way ANOVA with a Sidak multiple comparisons test Šídák's multiple comparisons test Distal ileum WT (10mM caffeine) n=6 - WT n=8 minute minute 30 minutes 60 minutes 90 minutes 120 minutes Mean Diff 95.00% CI of diff Summary Adjusted P Value -0.0001776 -0.0001928 -0.0004516 -0.001400 -0.001972 -0.003133 -0.003176 to 0.002821 -0.003192 to 0.002806 -0.003450 to 0.002547 -0.004398 to 0.001599 -0.004970 to 0.001027 -0.006132 to -0.0001342 ns ns ns ns ns * >0.9999 >0.9999 0.9990 0.7584 0.3917 0.0360 Appendix table 34 Statistical parameters for colon permeability for fasting WT mice versus fasting WT mice treated with caffeine utilising a two-way ANOVA with a Sidak multiple comparisons test Šídák's multiple comparisons test Colon WT + 10mM caffeine n=5 - WT n=9 minute minute 30 minutes 60 minutes 90 minutes 120 minutes Mean Diff 95.00% CI of diff Summary Adjusted P Value -0.0001743 -0.0001610 -0.0007668 -0.0009190 -0.001096 -0.001389 -0.001563 to 0.001215 -0.001550 to 0.001228 -0.002156 to 0.0006222 -0.002308 to 0.0004700 -0.002485 to 0.0002932 -0.002778 to 3.687e-007 ns ns ns ns ns ns 0.9997 0.9998 0.5929 0.3829 0.1979 0.0501 137 ... reserves are exhausted, the gastrointestinal lining becomes more susceptible to injury (Rao and Samak, 201 2a) Additionally, L-glutamine aids in the elimination of ammonia and in maintaining a healthy... zonulin-2, claudin-8, and zonulin-581, as well as increased claudin-15 expression, was observed in offspring of a maternal inflammatory activation mouse model of autism, similar to findings in patient... that may restore increased intestinal permeability in this population are understudied and correlations of increased GI permeability in different intestinal regions in animal models of ASD are