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Proquest Dissertations The role of milk-borne epidermal growth factor on hepatic development in artificially reared suckling rats Item Type text; Dissertation-Reproduction (electronic) Authors Baker, Gregory Lloyd Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 24/09/2021 21:56:40 Link to Item http://hdl.handle.net/10150/289229 INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis arxJ dissertation copies are in typewriter ^ce, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author dkj not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, t)eginning at the up>p>er left-hand comer and continuing from left to right in equal sections with small overiaps. Photographs included in the original manuscript have t)een reproduced xerographically in this copy. Higher quality 6' x 9' black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. Bell & Howell lnformatk>n and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 UMI THE ROLE OF MILK-BORNE EPIDERMAL GROWTH FACTOR ON HEPATIC DEVELOPMENT IN ARTIFICL\LLY REARED SUCKLING RATS by Gregory Lloyd Baker A Dissertation Submitted to the Faculty of the DEPARTMENT OF CELL BIOLOGY AND ANATOMY In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY WITH A MAJOR IN CELL BIOLOGY AND ANATOMY In the Graduate College THE UNIVERSITY OF ARIZONA 2000 UMI Number: 9992128 UMI UMI Microform 9992128 Copyright 2001 by Bell & Howell Information and Leaming Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17. United States Code. Bell & Howell Information and Leaming Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 2 THE UNIVERSITY OF ARIZONA ® GRADUATE COLLEGE As members of che Final Examinacion Commiccee, we cercify chat we have read che dissercacion prepared bv Gregory Lloyd Baker entitled The Role of Milk-Borne Epidermal Growth Factor on Hepatic Development in Artificially Reared Suckling Rats and recommend that it be accepted as fulfilling the dissercacion requirement for the Degree of Doctor of Philosophy ^K-9-7 lo/li/ov ^fiohuslav Dvorak,^h.D. Date /O //Sy/QCD R. Clark Lantz, Ph.C^g;^^ Date Kobert 5. McCuskey, Ph.D. Date /n /oo Lhaj^^jM^ee^ Dat4 ^ ;o / n/oo Raymond B. Runyan, Ph.D. Dat« Final approval and accepcance of chis dissercacion is concingenc upon the candidate's submission of che final copy of che dissercacion Co che Graduate College. I hereby certify Chat I have read this dissertation prepared under my direction and recomnend that it be accepted as fulfilling the dissertation requirement. /->—. /J Dissertation Director ' Date Robert S. McCuskey, Ph.D. 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rule of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of sources is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: 4 ACKNOWLEDGEMENTS I thank and acknowledge my dissertation advisor Dr. Robert S. McCuskey for the guidance, support, encouragement, training and advice he has given me over the years. Thank you also to Dr. R. Clark Lantz for his support, encouragement and advice. I also want to thank the members of my dissertation committee, Drs. Bohuslav Dvorak, Charlene McQueen and Raymond Runyan. My committee members have given me significant time, advice, training, access to equipment and reagents as well as advice on my dissertation. Thank you to Peggy McCuskey for all her help with the SEM samples and the many conversations about our feline friends. I especially wish to acknowledge and thank Ms. Nancy Machen for her hard work, encouragement, cheerful spirit and positive outlook, as well as Ms. Jermifer Wong for her hard work, and saying what was on her mind. I thank both of them for making our work environment a fun and enjoyable place. Thank you to the members of the Dvorak laboratory, Cathy Williams, Debrah McWilliam, Jim Grille, Jessica Dominguez and Emily Philipps for their work in making the feeding studies possible. I also want to extend a special thank you to Barbara Hall, Audrey Pallette, Susan Eastman, Jeannie Kucharczyk and Jacque Parker for going above and beyond the call of duty with all the help and assistance they have provided me over my years of graduate study. I also wish to acknowledge that this dissertation work was supported by NTH grant HD-26013 and by graduate fellowships and scholarships from The University of Arizona Center for Toxicology Flinn Foundation, and the Arizona Chapter of the ARCS Foundation. 5 DEDICATION I dedicate this dissertation to my wife and family. Without their understanding and support none of this would be been possible. 6 TABLE OF CONTENTS LIST OF FIGURES 9 LIST OF TABLES 11 ABSTRACT 12 CHAPTER 1 - LITERATURE REVIEW 14 INTRODUCTION 14 THE LIVER 16 The Hepatic Functional Unit 20 Lobular Heterogeneity 25 Establishment of Lobular Heterogeneity 27 BREAT MILK 29 ARTIFICIAL MILK FORMULAS 30 EPIDERMAL GROWTH FACTOR (EGF) 31 Milk-bome EGF 32 EGF AND THE LIVER 34 HYPOTHESIS AND SPECIFIC AIMS FOR THIS DISSERTATION 35 CHAPTER 2 - DEVELOPMENT OF LIVER HETEROGENEITY IN THE LIVER OF SUCKLING AND WEANLING RATS 37 INTRODUCTION 37 MATERIALS AND METHODS 41 Animals 41 Collection of Tissue Samples 41 Light Microscopy 42 Glucose-6-Phosphatase Histochemistry 45 Immunohistochemistry 45 Scanning Electron Microscopy (SEM) 46 Intestinal Microbial Characterization 51 Measurement of Portal Venous Endotoxin 51 Reyerse-Transcription — Polymerase Chain Reaction rRT-PCR) for TNFa 51 Statistical Analysis 52 7 TABLE OF CONTENTS - Continued RESULTS 53 Body and Liver Weights 53 Light Microscopic Observations 60 Scanning Electron Microscopy 74 Microbial Characterization of Colon Contents 81 Portal Venous Endotoxin 84 RT-PCRforTNFa 87 DISCUSSION 90 Body Weights 90 Hepatocvtes 90 Sinusoidal Endothelial Cells 93 Kupffer Cells 94 Hepatic Stellate Cells 95 Hepatic Hematopoiesis 95 Colon Microflora and Endotoxin 96 Hepatic TNFa 97 Summary 98 CHAPTER 3 - DEVELOPMENT OF THE LIVER IN ARTIFICIALLY REARED SUCKLING RATS FED RAT MILK SUBSTITUTE SUPPLEMENTED WITH EPIDERMAL GROWTH FACTOR 100 INTRODUCTION 100 MATERIALS AND METHODS 102 Animals 102 Collection of Tissue Samples 106 Light Microscopy 107 Glucose-6-Phosphatase Histochemistry 107 Immunohistochemistrv 107 Scanning Electron Microscopy (SEM) 108 Intestinal Microbial Characterization 109 Measurement of Portal Venous Endotoxin 109 Reverse Transcription — Polymerase Chain Reaction (RT-PCR) for TNFa 109 cDNA Expression Array 110 Statistical Analysis Ill 8 TABLE OF CONTENTS - Continued RESULTS 112 Body and Liver Weights 112 Light Microscopy 119 SEM 135 Microbial Characterization of Colon Contents 142 Portal Venous Endotoxin 145 Hepatic TNFa 148 cDNA Expression Array 151 DISCUSSION 156 Body Weights 156 Hepatocytes 156 Sinusoidal Endothelial Cells 158 KupfTer Cells 159 Hepatic Stellate Cells 161 Liver Hematopoiesis 161 Colon Microflora and Endotoxin 162 Hepatic TNFa 162 cDNA expression array 163 Summary 163 CHAPTER 4 - SUMMARY 165 HEPATOCYTES 165 SINUSOIDAL ENDOTHELIAL CELLS 166 KUPFFER CELLS 166 HEPATIC STELLATE CELLS 167 HEMATOPOIETIC TISSUE 167 MICROBIAL CHARATERIZATION OF COLON CONTENTS. ENDOTOXIN AND TNFa 167 FUTURE EXPERIMENTS 168 SHOULD INFANT MILK FORMULA BE SUPPLEMENTED WIT EGF? 170 APPENDIX A. PCR COMPETITOR AND PCR PRODUCT cDNA SEQUENCES 171 APPENDIX B. cDNA EXPRESSION ARRAY RESULTS 173 APPENDIX C. UNIVERSITY OF ARIZONA lACUC APPROVAL FORM 196 REFERENCES 198 9 LIST OF FIGURES Figure 1. Models of the functional unit in the liver 23 Figure 2. Example of point counting grid over tissue section 43 Figure 3. Examples of immunohistochemically stained EDI, ED2 and Desmin positive liver cells in both the periportal and centrilobular hepatic regions 47 Figure 4. Example of SEM photomicrograph of liver sinusoidal endothelial cell wall 49 Figure 5. Suckling and weanling rats total body weights 54 Figure 6. Suckling and weanling rats liver weights 56 Figure 7. Liver as a percent of body weight in suckling and weanling rats 58 Figure 8. Percentage hepatocytes in single cell thick hepatocellular plates 62 Figure 9. Binucleated hepatocytes present in the liver 64 Figure 10. Quantification of liver hematopoietic cells 66 Figure 11. Example of developing gradient of glucose-6-phosphatase enzyme histochemistry in suckling rats 68 Figure 12. Moncytic derived cells (EDI positive cells) in the liver 70 Figure 13. Hepatic stellate cells (Desmin positive cells) in the liver 72 Figure 14. Diameters of sinusoidal endothelial cell fenestrae 75 Figure 15. Density of fenestrae per square micron endothelial cell surface area 77 Figure 16.
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