Citation
The Role of Ketamine in Hypoxic Hypoxia Induced Inflammation and Apoptosis in Fetal Ovine Brain and Kidneys

Material Information

Title:
The Role of Ketamine in Hypoxic Hypoxia Induced Inflammation and Apoptosis in Fetal Ovine Brain and Kidneys
Creator:
Chang, Eileen I
Publisher:
University of Florida
Publication Date:
Language:
English

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Medical Sciences
Physiology and Pharmacology (IDP)
Committee Chair:
WOOD,CHARLES E
Committee Co-Chair:
SCHEUER,DEBORAH
Committee Members:
KASAHARA,HIDEKO
BYRNE,BARRY JOHN
Graduation Date:
8/9/2014

Subjects

Subjects / Keywords:
Cytokines ( jstor )
Fetus ( jstor )
Genes ( jstor )
Hypoxia ( jstor )
Kidneys ( jstor )
Metabolism ( jstor )
N methyl D aspartate receptors ( jstor )
Oxygen ( jstor )
Regulator genes ( jstor )
Sheep ( jstor )
fetus
hypoxia

Notes

General Note:
The purpose of this dissertation is to elucidate the effects of ketamine on fetal neuroendocrine and hemodynamic response under acute hypoxic stress. Acute hypoxic hypoxia is a form of fetal stress where both maternal and fetal arterial partial pressure of oxygen is decreased, resulting in activation of fetal neuroendocrine stress responses. We hypothesized that hypoxia, a common fetal event that can cause far-reaching postnatal deficits, triggers glutamatergic dependent inflammatory and apoptotic pathways in the fetal brain, and ketamine, a noncompetitive N-methyl-D-aspartate receptor antagonist, would blunt these responses. At around gestational day 125, ovine fetuses and ewes were both chronically catheterized with femoral arterial and venous catheters; and tracheostomy was performed on the ewe for the induction of acute hypoxia. During the fetal hypoxic experiments, ketamine was administered intravenously to the fetus 10 min prior to the induction of acute hypoxia, which was induced by administering nitrogen gas directly to the ewe for 30 min. Throughout the hypoxia stimulus, blood samples were collected periodically to monitor maternal and fetal blood gases. Fetal brain and kidneys were collected at 24 hrs after the initial start of hypoxic insult. Blood plasma samples were used in hormone analysis for measuring plasma ACTH and cortisol levels. Snap frozen tissue samples were extracted for mRNA and used in molecular analysis for microarray and real-time qPCR validations. Formalin fixed and paraffin embedded tissues were used in histological and immunohistological analysis. We have shown that hypoxia increases both fetal plasma ACTH and cortisol, and pretreatment of the fetus with ketamine partially inhibits the ACTH response. In addition, our ovine microarray analysis indicates that hypoxic stress stimulates inflammatory, immune, and apoptotic genomic responses, and ketamine treatment reduces these hypoxia-induced signaling events in the fetal brain and kidneys. Our results suggest that ketamine, commonly used medically, could be administered in the neonatal intensive care unit as a preventative measure to reduce inflammatory responses in preterm infants and mitigate poor neurological outcomes.

Record Information

Source Institution:
UFRGP
Rights Management:
All applicable rights reserved by the source institution and holding location.
Embargo Date:
8/31/2016

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THE R OLE OF KETAMINE IN HYPOXIC HYPOXIA INDUCED INFLAMMATION AND APOPTOSIS IN FETAL OVINE BRAIN AND KIDNEYS By E ILEEN I LING CHANG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014

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© 2014 Eileen I Ling Chang

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To my parents, Leo and Mandy Chang, for their love, encouragement, and support for me to follow my dreams

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4 ACKNOWLEDGMENTS I have been blessed to encounter many people throughout my life that played important roles in shaping and developing me both scientifically and intellectually to the person I am today. First of all, I want to thank Dr. Charles E. Wood for being a wonderful mentor by providing a challenging and nurturing environment for me to dev elop as a researcher. Not only h ad he taught me valuable scientific and unique surgical knowledge, but had also showed me how to be a responsible , kind, and ethical scientist. His infectious positive attitude and professionalism in his interaction with h is peers and students alike is something I hope to apply throughout the rest of my professional career. I could not have asked for a better mentor to make my Ph.D. an exciting , memorable, and enriching experience. I would like to thank and acknowledge all my committee members: Dr. Barry J. Byrne, Dr. Deborah A. Scheuer , and Dr. Hideko Kasahara for all of their support and encouragement throughout the years . I want to thank Dr. Byrne for his valuable time and contribution to my doctoral research . His dedication, passion, and drive for his research and patients are characteristics I hope to master in my future. I want to thank Dr. Scheuer for her kindness and the invaluable suggestions and contribution s to my understanding of physiology . Her passion for teaching the next generation of medical professionals and researchers are the attributes I hope to acquire in the future. Lastly, I especially want to thank Dr. Kasahara for her constant encouragement s and friendship throughout the years . I am grateful for the opportunity to maintain and practice microsurgical skills on various projects; and I will a lways cherish our conversations and our time together .

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5 I would like to express my sincere appreciation to Dr. Maureen Keller Wood for her wisdom and encouragements to me as a young female scientist . I have gained a great deal of scientific knowledge, and learned valuable life lessons on balancing resea rch and family. The Woods are the ideal role model for me to strive towards beca use of their scientific accomplishments while maintaining a balanced lifestyle , loving family, and strong marriage . I want to give special thanks to Dr. Maria Belen Rabaglino . E ver since we started working together in the Wood L ab , she ha s been a positive influence in my life. Not only is she a wonderful friend, surgery partner, office mate, conference roommate, and mentor, but she is like a sister to me . I will never forget the many hours we spent in surgery and nursing our patients back to hea lth in th e sheep ICU , and all the veterinarian knowledge she has bestow ed on me. I truly appreciate her patience and willingness in explaining microarray analysis and statistics to me until I underst ood the process. I cannot imagine what m y graduate school experience would have been without her. Both m embers of Wood and Keller Wood L ab have equally played vital role s in my success throughout my graduate education. I especially want to thank Dr. Elaine Richards for teaching me the molecular techniques of pre paring samples for microarray hybridization and helping me with various experiments. I also want to thank Xiaoyang (Lisa) Fang for her wonderful assistance in various assays and experiments , and her happy and friendly personality is something I will alway s remember. Other members, both previous and current, who contributed to my success: Miguel A. Zárate, Andrew

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6 Antolic , Xiaodi Feng, Jarret McCartney , Heidi Straub , Tatiana Ramirez Hiller , and Kristina Steinfeldt . I would also like to acknowledge those who have been a great mentor, supporter, and friend in my decision in choosing to go towards a scientific research career: members of the Doyle Lab at Georgia Institute of Technology, and the Spinale Lab at Medical University of South Carolina. I want to sp ecially thank Dr. Bahareh Azizi, my first scientific mentor, for playing such a n important role in igniting and encouragin g me towards a scientific research career. She taught me many things, including molecular biology, biochemistry, and cell culture . O ver the years, she has been like an older sister and ha s contributed to both my professional and personal growth. I also want to thank Dr. Kenyetta A. Johnson for being a great friend and a role model. She encourag es and focuses on the positive side of things, and always make s me laugh. I want to thank D r. Priyanka Rohatgi besides research. Finally, I would like offer my sincere gratitude to my fam ily: my parents, Leo and Mandy, my sister and brother in law, Crystal and Patrick, and my niece and nephew , Preston and Jubilee . Even though there are thousands of miles between us, they are always on my mind, and I appreciate their support in my decision to pursue my dreams . Also, I want to thank Timothy D. Fritz for his friendship and support throughout the years. Lastly, I want to give a heartfelt appreciation to Dr. Miguel A. Zárate for his endless love, positive encouragement s , constructive suggesti ons, and continual support, especially during this past year and with the writing of this dissertation. His contagious passion for science, life, music, food, and of course, soccer, ha ve influenced

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7 me to be a more dedicated scientist and loving individual . Finally, I want to thank God for blessing me with an enriched life filled with amazing and rem arkable people , who believe that I am capable of more than I think I can ever achieve.

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8 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 11 LIST OF FIGURES ................................ ................................ ................................ ........ 12 LIST OF ABBREVIATIONS ................................ ................................ ........................... 13 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 16 Fetal Stress ................................ ................................ ................................ ............. 16 Definition of Fetal Stress ................................ ................................ .................. 16 Models of Fetal Hypoxia ................................ ................................ ................... 17 Consequences of Fetal Hypoxic Stress ................................ ............................ 18 Fetal Circulation ................................ ................................ ................................ ...... 1 9 Fetal Response to Hypoxic Stress ................................ ................................ .......... 20 Fetal Cardiovascular Response to Hypoxia ................................ ...................... 21 Fetal Neuroendocrine Response to Hypoxia ................................ .................... 22 Fetal Inflammatory Response to Hypoxia ................................ ......................... 23 Effects of Hypoxia on the Kidneys ................................ ................................ .... 24 Ketamine ................................ ................................ ................................ ................. 25 History of Ketamine ................................ ................................ .......................... 25 Pharmacology and Mechanism of Action ................................ ......................... 26 Neuroprotective and Anti inflammatory Effect of Ketamine .............................. 29 Main Objectives and Clinical Relevance of the Studies ................................ .......... 30 Main Objective of the Studies ................................ ................................ ........... 30 Clinical Relevance of the Studies ................................ ................................ ..... 32 2 KETAMINE ATTENUATES THE ACTH RESPONSE TO HYPOXIA IN LATE GESTATION OVINE FETUS ................................ ................................ .................. 34 Introduction ................................ ................................ ................................ ............. 34 Materials and Methods ................................ ................................ ............................ 35 Fetal Surgery ................................ ................................ ................................ .... 35 In Vivo Experimental Procedures ................................ ................................ ..... 36 Hormone Assays ................................ ................................ .............................. 37 Calculations and Statistical Analysis ................................ ................................ 38 Results ................................ ................................ ................................ .................... 38 Blood Gases ................................ ................................ ................................ ..... 38 Fetal Cardiovascular Variables ................................ ................................ ......... 39

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9 Endocrine Variables ................................ ................................ ......................... 39 Discussion ................................ ................................ ................................ .............. 40 3 KETAMINE DECREASE IMMUNE GENE EXPRESSION IN OVINE FETAL FRONTAL CORTEX EXPOSED TO ACUTE HYPOXIC HYPOXIA ........................ 47 Introduction ................................ ................................ ................................ ............. 47 Materials and Methods ................................ ................................ ............................ 48 Fetal Surg ery ................................ ................................ ................................ .... 48 In Vivo Experimental Procedures ................................ ................................ ..... 49 Microarray Procedures ................................ ................................ ..................... 49 Real Time PCR ................................ ................................ ................................ 50 Calculations and S tatistical Analysis ................................ ................................ 50 Results ................................ ................................ ................................ .................... 51 Discussion ................................ ................................ ................................ .............. 54 4 KETAMINE DECREASES IMMUNE, INFLAMMATORY, AND APOPTOTIC GENE EXPRESSION IN OVINE FETAL HIPPOCAMPUS EXPOSED TO HYPOXIC HYPOXIA ................................ ................................ ............................... 68 Introduction ................................ ................................ ................................ ............. 68 Materials and Methods ................................ ................................ ............................ 69 Fetal Surgery ................................ ................................ ................................ .... 69 In Vivo Experiment al Procedures ................................ ................................ ..... 69 Microarray Procedures ................................ ................................ ..................... 70 Real Time PCR ................................ ................................ ................................ 70 Calculations and Statistical Analysis ................................ ................................ 70 Results ................................ ................................ ................................ .................... 71 Discussion ................................ ................................ ................................ .............. 75 5 KETAMINE SUPRESSES INFLAMMATORY RESPONSE IN THE KIDNEY CORTEX OF LATE GESTATION OVINE FETUS EXPOSED TO ACUTE HYPOXIC STRESS ................................ ................................ ................................ 86 Introduction ................................ ................................ ................................ ............. 86 Materials and Method s ................................ ................................ ............................ 88 Fetal Surgery ................................ ................................ ................................ .... 88 In Vivo Experimental Procedures ................................ ................................ ..... 89 Microarray Procedures ................................ ................................ ..................... 89 Real Time PCR ................................ ................................ ................................ 90 Calculations and Statistical Analysis ................................ ................................ 90 Results ................................ ................................ ................................ .................... 91 Discussion ................................ ................................ ................................ .............. 93 6 CONCLUSIONS ................................ ................................ ................................ ... 104 Fetal Programming: Long Term Effect of Fetal Stress ................................ .......... 104 Future Directions ................................ ................................ ................................ .. 107

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10 LIST OF REFERENCES ................................ ................................ ............................. 108 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 126

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11 LIST OF TABLES Table page 3 1 Primers for real time PCR validation. ................................ ................................ . 59 3 2 Top 10 gene ontology biological processes that were significantly regulated in the frontal cortex during acute hypoxic stres s, with or without ketamine. ....... 61 3 3 Top 10 enriched KEGG pathways that were significantly regulated in the frontal cortex during acute hypoxic stress, with or without ketamine. ................. 62 3 4 Top 10 gene ontology biological processes and enriched KEGG pathways that we re significantly up regulated in the frontal cortex during acute hypoxic stress, but was down regulated with ketamine. ................................ .................. 64 3 5 Top 10 common pathways associated with the genes that were significantly up regulated in the frontal cortex during acute hypoxic stress, but was down regulated with ketamine. ................................ ................................ ..................... 65 3 6 Top 10 enriched diseases associated with the genes that were significantly up regulated in the frontal cortex during acute hypoxic stress, but was down regulated with ketamine. ................................ ................................ ..................... 66 4 1 Top 10 gene ontology biological processes that were significantly regulated in the hippocampus during acute hypoxic stress, with or without ketamine. ....... 81 4 2 Enriched KEGG pathways that were significantly regulated in the hippocampus during acute hypoxic stress, with or without ketamine. ................. 82 4 3 Top 10 gene ontology biological processes, enriched KEGG pathways, common pathways, and enriched diseases associated with ge nes that were significantly up regulated in the hippocampus during acute hypoxic stress, but was down regulated with ketamine. ................................ .............................. 84 5 1 Primers for real time PCR validation. ................................ ................................ . 97 5 2 Top 10 gene ontology biological processes that were significantly regulated in the kidney cortex during acute hypoxic stress, with or without ketamine. ....... 99 5 3 Top 10 enriched KEGG pathways that were significantly regulated in the kidney cortex during acute hypoxic stress, with or without ketamine. ............... 100 5 4 Top 10 gene ontology biological processes and enriched KEGG pathways that were significantly up regulated in the kidney cortex during acute hypoxic stress, but was down regulated with ketamine. ................................ ................ 102

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12 LIST OF FIGURES Figure page 2 1 Maternal and fetal arteria l blood gases and pH ................................ .................. 44 2 2 Fetal mean arterial pressure and heart rate ................................ ....................... 45 2 3 Fetal plasma concentrations of ACTH and cortisol, and maternal pla sma concentration of cortisol ................................ ................................ ...................... 46 3 1 Volcano plot illustrating the relationship of gene expression in fetal frontal cortex measured by log 2 of fold change ................................ .............................. 60 3 2 Venn diagram of the number of genes that were significantly regulated by hypoxia and hypoxia+ketamine ................................ ................................ .......... 63 3 3 mRNA gene expression was measured by real time qPCR for apoptotic, inflammatory, and immune related genes ................................ ........................... 67 4 1 Volcano plot illustrating the relationship of gene expression in fetal hippocampus measured by log 2 of fold change ................................ .................. 80 4 2 Venn diagram and network analysis of significant gene expression up regulated by hypoxia, but down regulated by ketamine ................................ ...... 83 4 3 mRNA gene expression was measured by real time qPCR for apoptotic, inflammatory, and immune related genes ................................ ........................... 85 5 1 Volcano plot illustrating the relationship of gene expression in fetal kidney cortex measured by log 2 of fold change ................................ .............................. 98 5 2 Venn diagram and network analysis of significant gene expression up regulated by hypoxia, but down regulated by ketamine ................................ .... 101 5 3 mRNA gene expression was measured by real time qPCR for inflammat ory and immune related genes ................................ ................................ ............... 103

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13 LIST OF ABBREVIATIONS ACTH Adrenocorticotropic hormone BCO Brachiocephalic artery occlusion BP Blood pressure COX 2 Cyclooxygenase 2 GO Gene ontology HH Hypoxic Hypoxia HPA Hypothalamus pituitary adrenal HR Heart rate KEGG Kyoto Encyclopedia of Genes and Genomes NMDA N methyl D aspartate NTS Nucleus tractus s olitaries MAP Mean arterial pressure P a CO 2 A rterial partial pressure of carbon dioxide P a O 2 A rterial partial pressure of oxygen PGHS 2 Cyclooxygenase 2 UCO Umbilical cord occlusion VPN Ventral posterior nucleus

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14 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE ROLE OF KETAMINE IN HYPOXIC HYPOXIA INDUCED INFLAMMATION AND APOPTOSIS IN FETAL OVINE BRAI N AND KIDNEYS By Eileen I Ling Chang August 2014 Chair: Charles E. Wood Major: Medical Sciences Physiology and Pharmacology The purpose of this dissertation is to elucidate the effects of ketamine on fetal neuroendocrine and hemodynamic response under acute hypoxic stress. Acute hypoxic hypoxia is a form of fetal stress where both maternal and fetal arterial partial pressure of ox ygen is decreased, resulting in activation of fetal neuroendocrine stress responses. We hypothesized that hypoxia, a common fetal event can cause far reaching postnatal deficits, triggers glutamatergic dependent inflammatory and apoptotic pathways in the fetal brain, and ketamine, a noncompetitive N methyl D aspartate receptor antagonist, would blunt these responses. At around g estational day 125, ovine fetuses and ewe s were both chronically catheterized with femoral arterial and venous catheters ; and t r acheostomy was performed on the ewe for the induction of acute hypoxia. During the fetal hypoxic experiment s , k etamine was administered intravenously to the fetus 10 min prior to the induction of acute hypoxia , which was induced by administering nitrogen gas directly to the ewe for 30 min. Throughout the hypoxia stimulus, blood samples were collected periodically to monitor maternal and fetal blood gases. Fetal brain and kidneys were collected at 24 hrs after the initial start of hypoxic insult . Blood plasma samples were

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15 used in hormone analysis for measuring plasma ACTH and cortisol levels. Snap frozen tissue samples were extracted for mRNA and used in molecular analysis for microarray and real time qPCR validations. Formalin fixed and paraff in embedded tissues were used in histological and immunohistological analysis. We have shown that hypoxia increases both fetal plasma ACTH and cortisol, and pretreatment of the fetus with ketamine partially inhibits the ACTH response. In addition, our ovi ne microarray analysis indicates that hypoxic stress stimulates inflammatory , immune, and apoptotic genomic responses, and ketamine treatment reduces these hypoxia induced signaling events in the fetal brain and kidneys . Our results suggest that ketamine, commonly used medically, could be administered in the neonatal intensive care unit as a preventative measure to reduce inflammatory responses in preterm infants and mitigate poor neurological outcomes.

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16 CHAPTER 1 INTR ODUCTION Fetal Stress D efinition of F etal S tress Fetal stress threatens the survival of the fetus and the w ell being of the newborn baby , and is defined as the response produced by the fetus to an internal or external agent that is able to influence the uterine environment and perturb homeostasis. Fetuses are vulnerable and sensitive to disruption of homeostasis, especially changes in fetal blood gas composition s ( 106 ) . A growing fetus is dependent on the appropriate maintenance of blood pressure (BP) , blood volume, and arterial blood g as es in order to survive and properly develop throughout the gestation period. There are several factors that contribute to fetal stress, and can be di vided into three categories: m aternal (diet, hypertension/hypotension, psychological stress, emergency surgeries), uterine/placental unit (abnormal development, infectious diseases, multiple fetuses, and preeclampsia), and fetal (umbilical cord occlusion, congenital diseases). Research has shown that all these factors , either chronic or acute, can influence fetal development ( 44 , 108 , 112 , 134 ) . In the past few decades, it was proposed that fetal stress may play a critical role on adult health and the development of chronic diseases, specifically the development of cardiovascular related diseases ( 52 ) . Among all, low levels of oxygen or hypoxic stress , is the most common and difficult fetal stressor for survival , especially on late gestation fetuses . During pregnancy, the normal amount of arterial partial pressure of oxygen (P a O 2 ) delivered from the mother to the fetus is normally low, with valu es around 20 mm Hg ( 64 ) . These relative low oxygen levels make the fetus vulnerable to small changes in fetal blood gas

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17 composition . The consequences of these stressors can influence fetal development, and may permanently alter the neuroendocrine system ( 176 ) causing i ntrauterine growth restriction , preterm birth, and fetal origins of adult disease ( 17 ) . Models of F etal H ypoxia Currently, our lab utilizes three different ty pes of animal models to study fetal hypoxic stress: hypoxic hypoxia, brachiocephalic occlusion, and umbilical cord occlusion. Hypoxic hypoxia (HH) . Hypoxic hypoxia is also known as ventilatory hypoxia . It is a mild form of fetal hypoxia model characterized by a reduction in maternal oxygen intake , thus, decreasing the maternal blood oxygen level s diffused to the fetus. This can be achieved by infusing nitrogen gas directly into a maternal tracheostomy tube to reduce maternal partial pressure of oxygen from appr oximately 100 mm Hg to 50 mm Hg. This will result in a decrease of fetal P a O 2 from 20 mm Hg to around 10 mmHg . Causes of fetal HH in humans are due to the influence of high altitude or the presence of chronic cardiopulmonary diseases in pregnant woman. Brachiocephalic occlusion (BCO) . This specific model of hypoxia targets the fetal brain by obstructing the brachiocephalic artery, the main blood supply contributor to both carotid arteries. A vascular occluder is placed into the brachiocephalic artery, and once the occluder is expanded , it induces a transient cerebral ischemia/reperfusion episode. BCO is characterized by a slight increase in fetal peripheral P a O 2 levels, an increase in partial pressure of carbon dioxide in blood (P a CO 2 ), and a reduction of arterial fetal pH. This type of fetal asphyxia is observed during obstructed labors (dystocia) with possible emergency surgical intervention.

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18 Umbilical cord occlusion (UCO) . U mbilical cord occlusion involves the obstruction of the umbilical cord vessels (2 arteries and 1 vein) by the action of a vascular occluder. This will lead to a dramatic decrease in blood supply from the maternal circulation to the fetus, resulting in a reduction of CO 2 transpor t back from the fetus to the maternal circulation for further elimination. This is a severe hypoxia induction model because it involves a systemic fetal response that can lead to a multiple organ failure if not controlled on time. In our model, fetuses a re targeted to have a P a O 2 reduction of around 50 %, resulting in an increase in P a CO 2 , and low arterial pH . UCO cases that occurred in Neonatal Intensive Care Units (NICUs) are part of umbilical cord complications such as cord knots, nuchal cords, and co rd stricture. Consequences of F etal H ypoxic S tress The level of injury from fetal hypoxia stress depends on the severity of the insult. The brain, particularly, shows more susceptibility to a hypoxic stimulus than other organs due to high demand for oxygen and the lack of energy reserves in the neurons ( 170 ) . The most common consequence from brain hypoxia is hypoxic ischemic encephalopathy, a pathology that is able to produce detrimental consequences for the future neonate such as com promised development ( 2 ) , cerebral palsy ( 42 ) , endocrine damage ( 128 ) , and disorder s that might appear later in lif e ( 17 , 20 ) . Hypoxic ischemic encephalopath y is highly correlated to premature birth as the leading cause of long term neurological disabilities in children. Recently, research has shown that even mild hypoxic insults could possibly lead to severe cardiovascular and metabolic diseases in the adult , a concept called fetal programming ( 52 ) .

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19 Fetal Circulation The fetal circulation is unique and different from adult circulation. The adult cardiovascular system is made up of two circuits, pulmonary and systemic, in a serial circulatory desi gn. The left ventricle pumps the blood from the heart to the body, returning the deoxygenated blood back to the right side of the heart, and then enters the lungs for reoxygenation. But the fetus is in an enclosed environment and the only way to receive oxygenated blood from the maternal side is through the placenta. Therefore, the fetus requires a parallel circulatory system that utilizes various anatomical shunts. The main function of the placenta is to supply the fetus with oxygen and nutrients , as well as eliminating waste products ; and the shunts are uniquely placed to supply oxygenated rich blood to the developing organs while preventing excessive blood flow and overwhelm the delicate organs ( 148 ) . The three unique fetal anatomical features are describe d as follow: the foramen ovale, an open ing between t he right and left atrium in order to maximize oxygen delivery to the fetal brain by by passing oxygenating blood through the left ventricle; the ductus venosus, a vessel connecting the hepatic portal veins with the inferior vena cava that allows the majori ty of the oxygenated blood to reach the fetal heart ; and t he ductus arteriosus, a vessel that transports the majority of the blood from the right ventricle directly to the aorta, thus decreasing the amount of blood from going directly to the developing lun gs ( 149 ) . O xygenated blood is transported from the mother to the fetus by the umbilical vein, passing through the developing liver, and reaching the fetal right atrium ( 121 ) . Under normal physio logical conditions, the oxygen saturation in the umbilical vein and the inferior vena cava are 80 % and 70 % , respectively. Reaching the heart, the blood from the inferior vena cava will preferentially flow from the right atrium , through the

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20 foramen ovale , towards the left atrium , and to the left ventricle. The blood in the left ventricle is more saturated with oxygen (65 %) because this supplies the heart and the brain with oxygenated blood ( 85 ) . The blood returning from the superior vena cava will empty into the r ight atrium, and into the right ventricle (55 % oxygen saturation) where it will go through the ductus arteriosus and into the aorta, where it joins the blood originate from the left ventricle ( 85 ) . The combined oxygen saturation systemically is 60 % ( 85 ) . The fetus relies on t he proper function of the circulatory system for the appropriate delivery of oxygenated blood to supply the growth and differentiation for each individual organ. The parallel circuitry system allows the fetus to have a high combined ventricular cardiac ou tput aiming at providing oxygen to all organ/tissue levels (around 400 500 ml kg 1 ) ( 142 ) . This is accomplished by increasing the fetal heart rate up to values around twic e as much as an adult. However, the fetus will adapt when threaten ed with various forms of hypoxic stress. Fetal R esponse to H ypoxic S tress When confronted with a stress stimulus, like hypoxia, the fetal defense mechanism integrates cardiovascular, endocr ine, and metabolic responses to facilitate fetal survival. Under hypoxic stress, the fetus is physiologically equipped to respond accordingly in order to protect the most vital organs ( 146 ) . In period s of reduced oxygen availability or hypoxia, fetal peripheral baroreceptor a nd chemoreceptor reflexes play vital role s in defending fetal cardiovascular circulation by increasing blood pressure, decreasing heart rate , and redistributi ng combined ventricular output towards the umbilical placental vascular bed ( 51 , 148 ) . On the other hand, the physiological response to central chemoreceptor activation by BCO and UCO is even more pronounced than the peripheral chemoreceptors, due to an in crease in carbon dioxide

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21 (or a decrease in pH). The fetal central chemoreceptors respond aggressively to increased H + concentration in the cerebrospinal fluid by increasing both fetal blood pressure and heart rate ( 185 ) . Fetal C ardiovascular R esponse to H ypoxia The activation of arterial chemoreceptors by hypoxia stimulates a robust sympathetic autonomic and neuroendocrine response in the fetus to balance and correct the cardiovasc ular and fluid deficit ( 182 , 184 ) . The combination of sympathetic autonomic response with increased plasma concentrations of angiotensin II and vasopressin results in redistribution of fetal combined ventricular output to shunt blood away from gastrointestinal tract, skin, kidneys, spleen, lungs, and skeletal muscle beds and maintain blood flow towards the brain, heart, adrenal glands, and maximize blood flow to the umbilical vascular beds ( 32 , 148 ) . When the fetal P a O 2 decreases by approximately 50 %, the fetal oxygen consumption will rapidly decrease to as low as 60 % of co ntrol, and is stable for up to 45 min and is reversible once the stress is eliminated ( 193 ) . This hypoxic response is accompanied by fetal bradycardia, a decrease of approximately 30 beats/min (from 170 beats/min to 14 0 beats/min ) , and an increase in fetal arterial blood pressure (approximate ly 54 mmHg to 61 mmHg) ( 193 ) . With umbilical cord occlusion, there will be a progressive fetal acidosis (fetal arterial pH 7.38 to 7.33 after 25 min ) ; however, this response is not accompanied when the hypoxic stress is due to maternal ventilatory hypoxia ( 193 ) . With a decrease in oxygen supply, the partially vasoconstri cted peripheral beds will switch to an anaerobic metabolism, inducing lactic acidosis accumulation thus resulting in metabolic acidosis. The se fetal responses to hypoxia, described above , are the initial and temporary initial compensatory mechanism for fe tal

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22 survival. For acute hypoxia, the fetus is able to match the decrease oxygen availability to oxygen consumption in vital organs by increasing blood flow. However, this short term solution of redistributing blood flow can only alleviate the cerebral an d myocardial oxygen consumption briefly. When the fetus is faced with severe hypoxia/asphyxia and is unable to match the oxygen expenditure of the heart and the brain, then the fetal cardiac output, arterial blood pressure, and blood flow will decrease ( 193 ) . Prolonged hypoxemia will cause t issue damage and fetal death ( 193 ) . Fetal N euroendocrine R esponse to H ypoxia The neuroendocrine pathway, stimulated by hypoxic stress , begins at the nucleus tractus solitaries (NTS) in the medullary brainstem, which is the location where bot h arterial chemoreceptors and baroreceptors synapse s . The neurons in the NTS project to other brain regions , like the ventrolateral me dulla and the hypothalamus, and activate the sympathetic nervous system and neuroendocrine response s ( 139 ) . During BCO, central chemoreceptors respond to decreased cerebral spinal fluid pH and stimulate fetal hypothalamus pituitary adrenal (HPA) axis, which results in homeostatic endocrine and neural responses ( 182 ) . The initial activation of HPA axis , under fetal stress stimulus, starts at the paraventricular nucleus (PVN) of the hypothalamus , which synthesize s and secrete s corticotrophin releasing hormone (CRH) and vasopressin. These two hormones are rel eased into the median eminence and are transpor ted to the anterior pituitary to stimulate the production and secretion of ACTH into the systemic cir culatory system . The rise in plasma ACTH will result in the release of cortisol by the adrenal cortex . The rise in circulating cortisol will negatively feedback to both the hypothalamus and pituitary gland to inhibit the synthesis and secretion of both CRH and ACTH ( 81 ) . The main

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23 negative feedback system is regulated by the glucocorticoid receptors located in the PVN ( 151 , 175 ) and hippocampus ( 75 ) . How ever, continuous exposure to a stressor, like hypoxia, will produce a failure of the negative feedback mechanism in the HPA axis. This action mainly occurs by the inhibition of binding between cortisol and the glucocorticoid receptor located in the brain. A study by Wang et al . , demonstrated that interleukin (IL) 1alpha, an inflammatory cytokine, may play a key role in the glucocorticoid receptors mediated feedback inhibition via the activation of p38 mitogen activated prote in kinase (MAPK) pathway ( 172 ) . T he HPA axis in late gestation fetuses is developed an d mature, and will initiate the normal physiological response to stress ( 50 ) , and consequently, the negative feedback response ( 181 , 183 ) . Therefore, t he physiological indications of fetal stress can be determined by the rise in fetal plasma ACTH and cortisol levels ( 136 ) . Fetal I nflammatory R esponse to H ypoxia Activ ation of chemoreceptor pathways, during fetal stress, causes a release of glutamate , an excitatory amino acid, into the neuronal extracellular space resulting in the release of intracellular calcium . The increase in elevated level s of intracellular calcium will trigger the activation of proinflammatory markers and cytokines, such as c y clooxygenase 2 (COX 2, PGHS 2) , interleukins, tumor necrosis factor (TNF) , and toll like receptors (TLR) . COX 2 is an enzyme that converts arachidonic acid to form prostaglandin H2 (PGH2), and PGH2 is then converted by cell specific synthases to prostaglandins (PG) and thromboxane A2. In response to tissue injury (e.g. hypoxia), cells synthesize PG as a component of inflammatory pathways ( 167 ) . A study by Weiner et al ., reported an increase in proinflammatory cytokines in the fetal brain regions damaged from chronic hypoxia using fetal guinea pigs ( 192 ) . Other stud ies

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24 demonstrated that in response to hypoxic/ischemic stress, the injured site is susceptible to infection and inflammatory signals mainly by the up regulation of TLR4 express ion ( 19 , 83 ) . In addition, newborns who suffered severe hypoxic ischemic encephalopathy injury and abnormal neurological outcomes have elevated levels of neuronal proinflammato ry biomarkers: IL1, IL6, IL 8, and TNF ( 22 ) . COX 2 is an important mediator of fetal HPA response to BCO: BCO upregulates COX 2 mRNA and protein expression, and blocking COX 2 prolongs gestation and blunts fetal cardiovascular and endocr ine stress responses ( 186 ) . Furthermore, p rostaglandin endoperoxide synthase 2 ( PGHS 2 ) activity partially mediates the fetal HPA axis response to NMDA mediated glutamatergic stimulation ( 86 ) . Prolonged stimulation of N methyl D aspartate (NMDA) receptor by glutamate results in excitotoxicity and neuronal degeneration ( 26 ) . Under hypoxic insults, the over stimulated NMDA receptors, located in the external membrane of the neurons causes an increase in intracellular calcium. The excessive influx of calcium activates cyto plasmic proteases (e.g. caspase 3) to initiate neurona l apoptosis ( 174 ) . Neuroinflammation is linked to apoptosis ( 120 ) , and t he activation of proinflammatory pathways can be seen in a wide range of neurodegerative diseases ( 33 ) , and as a consequence of cerebral ischemia and stroke ( 16 ) . Effects of H ypoxia on the K idneys NMDA receptors are not only located within the central nervous system, but are also present in extraneuronal tissues such as the kidneys , heart, aorta, and pulmon ary artery of the cardiovascular system ( 96 ) . When the fetus experiences hypoxic stress, the normal physiological response is to shunt combined ventricular output towards vital organs, such as the brain, heart, and adrenals, and away from peripheral organs like the kidneys, resulting in potential renal damage ( 4 , 107 ) . The decrease in renal blood

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25 flow and partial pressure of oxygen to the kidneys can cause hypoxic an d ischemic damage , especially in the renal tubules ( 45 , 48 ) . Consequently, more severe hypoxic or asphyxic events, suc h as p artial umbilical cord occlusion (60 min, pH <6.9) can cause renal tubular necrosis in near term fetuses ( 73 ) , and complete umbilical cord occlusion (30 min, 72 hrs post) can cause renal distal tubule apoptosis in 60% gestation fetuses ( 127 ) . An upregulation and activation of renal NMDA receptors were found in ischemia reperfusion and hypoxia reoxygenation induced acute renal injury ( 138 , 198 ) . Over activation of NMDA receptors can evoke significant bursts amounts of rea ctive oxygen species and calcium ion overload and damage renal tubules, resulting in induction of apoptosis and necrosis of tubular cells, which may contribute to renal dysfunction ( 198 ) . Ketamine We propose that ketamine, a non competitive NMDA receptor antagonist, will reduce brain inflammation and glutamatergic NMDA receptor associated apoptosis in late ges tation fetuses exposed to hypoxia . By using ketamine, we will be able to relate our findings more c losely to clinical application. History of K etamine In 1957, Woodbridge evaluated all the curr ently used anesthesia and analyzed each one accord ing to four categories: sensory block, motor block, blocking of reflex es (respiratory, circulatory, gastrointestinal), and mental block ( 188 ) . However, he concluded that there is a lack of available general anesthetic s that are able to block all four categories simultaneously to protect patients from anesthesia awareness, a condition where the patient remain conscious but paralyzed while under general anesthesia. The call for action result ed in a new cl ass of drugs called

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26 cyclohexylamines, and in 1959, pharmacologists synthesized phencyclidines (PCPs: CI 395 and CI 400), but the side effects were too severe ( 58 ) . In 1962, a PCP derivative, 2(O Chlorophenyl) 2 methylaminocyclohexanone or also known as ketamine ( CI 581 ) , was synthesized and possessed similar properties of ana lgesia and anesthesia, shorter duration of action, and generated less convulsions ( 40 , 115 ) . During the initial animal ( 115 ) and human testing s (from 6 weeks to 86 years old) ( 35 ) , researchers found that as an anesthetic, ketamine increased heart rate, both systolic and diastolic pressures, and only slightly decreased respirat ory rate . Due to the unique pharmacological propertie s as a potent analgesic and anesthetic while maintaining a safe hemodynamic and respiratory profile, ketamine was used on American soldiers during the Vietnam War, and was later approved for clinical pr actices by 1970 ( 155 ) . In a few cases, ketamine produced mild hallucinations and vivid ( 35 ) . In 1990, a landmark study reviewed 11,589 cases where ketamine was used for sedation, and established the efficacy and safety of ketamine for pediatric uses ( 56 ) . Currently, ketamine is used for surgical procedures in many fields of medicine: pediatric emergency medicine ( 119 ) , pediatric and neonatal intensive care units ( 98 ) , soldiers on the battlefield ( 145 ) , and veterinary medicine ( 189 ) . Pharmacology and M echanism of A ction Ketamine, 2 ( o chlorophe nyl) 2 methylamino cylohexanone, has a molecular weight of 238 g/mol and a melting point of 26 2 °C. Ketamine is produced as a racemic 50:50 mixture of S(+) and R( ) in aqueous solution , and both isomers have a short half life of 2 4 minutes ( 155 ) . T he S(+) enantiomer is app roximately three time as potent as the R( ) enantiomer, with faster recovery time and fewer psychoactive side effects ( 178 ,

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27 202 ) , favorable cardiovascular profile, and neuroprotective effects ( 65 ) . S ingle enantiomer compound is available in Europe, but not the United States. However, recently, the R( ) ketamine was discovered to have a longer potency and longer lasting antidepressant effect than the S(+) isomer ( 206 ) . Ketamine is versatile drug with many modes of administration ( 135 ) , but the main ones are intravenous, intramuscular, oral, nasal, and rectal ( 5 5 , 109 ) . The chemical structure of ketamine makes this drug both lipophilic and hydrophilic due to its benzene rings and amine group, respectively ( 89 ) . T hus , ketamine is rapidly taken up into highly perfused tissues and readily crosses the blood brain barrier ( 31 ) , and only 10 30 % are bound to plasma proteins ( 104 ) . In the liver, ketamine is meta bolized by enzymes: cytochrome P450 (CYP) 3A4, CYP2B6, and CYP2C9. These enzymes convert ketamine to norketamine, the primary active metabolite N demethylation. Other minor inactive metabolite, dehydroxynorketamine, is g enerated by direct oxidation ( 135 ) . The metabolism half life is 2.5 3 hrs, so even though n orketamine is approximately less than one sixth as potent as ketamine , it may provide prolonged analgesia effect ( 155 ) . K etamine is favored drug for sedation In the pediatric settings because of the fast onset ( 1 2 min intravenous or 5 min intramuscular), and short duration ( 45 min ), while supporting hemodynamic and maintaining respiratory stability ( 98 ) . In the brain, ketamine mainly blocks the NMDA receptor to produce an analgesic effect at low concentrations (0.125 0. 6 mg/kg intravenous or intramuscular) ( 3 , 111 ) and produce an anesthesia effect at higher concentrations (1 6 mg/kg intravenou s) ( 36 , 57 , 113 ) . interaction with an array of receptors and the complexity of the mechanism of action

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28 ( 104 ) . The main receptor that ketamine binds to is the NMDA receptor, but ketamine also binds to other recepto rs: serotonin receptors ( 78 , 194 ) , dopamine receptors ( 78 ) , non NMDA glutamamte receptors, nicotinic and muscarinic cholinergic receptors, opioid receptors ( 88 ) , and weakly influences the GAB A A and glycine receptors ( 194 ) . The NMDA receptors are glutamate ligand gated and voltage dependent ion channels, which allow the movement of sodium, calcium, and potassium across the post synaptic membrane. The NMDA receptor forms tetrameric complexes consists of homologous subunits, and according to sequence homology, there are currently three subfamililes, and seven subunits. The different composit ion of subunits provides diversity in NMDA receptor composition, therefore resulting in an array of receptor signaling properties ( 132 ) . In the fetal brain, NR2B is the predominant subunit that makes up the NMDA receptors; however, in the early postna tal period, a developmental switch occurs in the somatosensory cortex and the ventroposterior nucleus of the thalamus , where the NR2B subunits will be outnumbered and replaced by the NR2A subunit ( 102 ) . The switch in subunits varies with brain region and may play a role in developmental synaptic plasticity by facilitating and regulating calcium entry ( 38 ) . The NMDA receptor is modulated by endogenous an d exogenous compounds: glutamate, glycine, zinc ions, polyamines, phenylethanolamines, and protons ( 70 ) . To activate the NMDA receptor and dislodge the magnesium blockade, three things are required: excitatory postsynaptic potential and the binding of both glutamate and glycine at their respective binding sites. When glutamate, a neurotransmitter, is secreted by the presynaptic neuron i nto the synaptic cleft, the glutamate will bind to receptors in the postsynaptic neuron. Both NMDA and non NMDA (AMPA and kainite) receptors at the

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29 postsynaptic neuron will be activated by glutamate, and they work synergistically to stimulate action poten tial. The release of magnesium blockade in the transmembrane pore of the NMDA receptor will allow the inf lux of calcium into the neuronal cell s and activate downstream pathways . Ketamine inhibits the NMDA receptor by two distinct mechanisms: blocking the open channel, which reduces channel mean open time, and binding the closed receptors via allosteric mechanism, which decreases channel opening frequency ( 130 ) . Neuroprotective and A nti inflammatory E ffect of K etamine K etamine have been shown to avoid exacerbated proinflammatory response by suppressing proinflammatory cytokine productions without affecting the production of anti inflammatory cytokines. The idea that ketamine interferes with the immune system was formed from clinical observations of improved outcomes in patients with septic shock ( 200 ) and animal models of septic shock ( 168 ) . This observation inspired many researchers to uncover the mechanisms behind the beneficial effect of ketamine. In later studies performed with in vitro , ex vivo , and in vivo models, ketamine was found t o stabilize neutrophil activation, reduce the release of inflammatory mediators : TNF , IL1 , IL6, and IL 8 ( 60 , 79 , 80 , 93 , 94 , 152 ) . A study by Taniguchi et al ., suggests that in the absence of an inflammatory stimulus, ketamine have no effect on the immune cells producing inflammatory cytokines, and the action of ketamine is more pr onounced when administered prior to the inflammatory challenge ( 166 ) . Ketamine also have neuroprote ctive effects, which is seen with in vitro and animal studies with duration of up to 7 days ( 65 ) . In either permanent or transient brain insults, such as cerebral artery occlusion ( 23 , 99 ) or ischemia/reperfusion models ( 154 ) ,

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30 ketamine was found to attenuate the degree of injury. Given systemically, k etamine can exert partial ne uroprotective action in hypoxic ischemic damaged brains of 7 day old rat pups ( 158 ) . In addition, NMDA receptor antagonists have shown to ra pidly reverse chronic stress induced structural and funct ional deficit of prefrontal cortex neurons ( 97 ) . However, there are clinical and animal evidences that suggest ketamine may have a neurotoxic effect and may lead to neuroapoptosis if exposed to the developing brain ( 196 , 197 ) . Interestingly, in the immature brain, repeated exposures to ketamine without surgical stimuli may be harmful and neurotoxic; however, when ketamine is used in conjunction with surgical procedures, then it have a neuroprotective effect ( 195 ) . Ketamine is a complicated drug with an array of complex mechanism of actions, and to determine if ketamine is neurotoxic or neuroprotective in the fetal or neonatal brain, one has to consider the following: dose, timing, frequency , and extent of surgical insult. Currently, the debate on whether if ketamine is neurotoxic or neuroprotective , to the developing brain , is still undetermined. Main Objectives and Clinical Relevance of the Studies Main O bjective of the S tudies Maintaining homeostasis is vital in fetal development and the most common fetal stressors are hypoxia and asphyxia. Fetuses detect changes in blood gas composition though central and peripheral chemoreceptors. Central chemoreceptors are sensitive to increases in arterial partial pressure of carbon dioxide (P a CO 2 ), while peripheral chemoreceptors are responsive to decreases in arterial partial pressure of oxygen (P a O 2 ). When a fetus is threatened with changes in P a CO 2 and P a O 2 , the fetus will compe nsate the deficit by responding with dramatic physiological cardiovascular and endocrine changes. We believe that exposing late gestation fetuses to acute hypoxic

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31 events will lead to activation of inflammatory pathways mediated by NMDA glutamatergic recep tors. Consequently, excessive stimulation of NMDA receptors can lead to neuronal apoptosis and other adverse neuroendocri ne and cardiovascular effects. We are interested in elucidating not only the physiological effect of fetal stress, but also uncovering the changes in genomic expression of neuronal inflammatory and apoptotic pathways that are associated with hypoxia. In order to understand the full physiological spectrum of hypoxia in fetuses, our experiments will incorporate whole animal, cellular, and molecular studies. The proposed aims are directed at elucidating the physiological, neuroendocrine, and genetic effects of ventilatory hypoxic hypoxia on 1) NMDA glutamate mediated inflammatory signaling in the hippocampus , frontal cortex , and kidney cor tex , and 2) whether blocking the NMDA glutamate pathway with ketamine will reduce cerebral and renal inflammation and apoptosis in late gestational fetal sheep . We propose that ketamine will decrease stress induced glutamatergic NMDA receptor activity, and have a neuroprotective effect in the fetal brain , and attenuate inflammatory responses in the kidneys . In our previous work, we have shown that ketamine is able to reduce plasma ACTH in late gestational fetal sheep stimulated with BCO ( 136 ) . Therefore, we know that a glutamatergic NMDA receptor pathway plays a role in fetal stress re sponse and ketamine is a candidate drug to reduce glutamatergic NMDA acti vity in the fetus . In addition, a study by Spand oua et al . d emonstrated that administering ketamine systemically can exert partial neuroprotective action in hypoxic ischemic damaged brains of 7 day old rat pups ( 158 ) .

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32 Clinical R elevance of the S tudies Our work is clinically relevant to acute interruption of oxygen to the fetus, as seen during prolonged labor or insufficient uterine blood flow that could have acute and long lasting effects which continue into adulthood. The benefits of the experiments lie in the combination of in utero monitoring and manipulation of the unanesthetized fetus with genomic techniques, and collectively, these techniques will allow us to understand the holistic integration of fetal stress and damage to the fetal brain and to identify a feasible countermeasure to hypoxia stimulated brain inflammation and apoptosis. By elucidating the link between the activity o f visceral sensory pathway and markers of brain inflammation, we can further understand and perhaps predict the outcome of common drugs given to pregnant women , late gestation fetuses, and even premature infants in the neonatal intensive care units . The c urrent pharmacologic interventions for improving neonatal survival are predominantly steroidal (for fetal lung maturation) and nonsteroidal anti inflammatory drugs (brain vasculature) . Although these drugs are administered for specific targets in prematur e infants, we now expect the se drugs to also reduce in flammatory markers in the brain and i mproved or spared brain function . The advantage of using late gestation ovine fetus es for our hypoxic animal model s is that fetal sheep correlate more closely to a human in fetal development , weight, and size than do rodent animal models . Our model is unique in the sense that we are able to manipulate and measure physiological changes of the fetus in utero without the influence of anesthesia or the added complicatio n of maternal and fetal stress response. Furthermore, the majority of rodent models in current fetal hypoxia research use postnatal pups that had already experienced the stress of parturition, and the additional environmental change from breathing amnioti c fluid to breathing

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33 atmospheric air. The drastic adaptations of these rodent pups to their new surroundings could induce profound changes in neuronal synaptic connectivity and gene expression in the brain. Therefore, the neuronal genomic results gathere d from these postnatal rodent pups, after exposure to hypoxia, might not correlate to the human fetal stress response in the brain and could obscure actual molec ular pathways induced by hypoxia in utero . Our experimental approach has the advantage in eluc idating pure fetal physiological stress response to hypoxia. Our ability to combine physiological, endocrine, neuroanatomical, molecular, and genomic techniques in individual fetuses is unique and innovative. G enomics and molecular pathway analysis will p rovide us with a ketamine modifiable physiological processes responding to hypoxia . Molecular analysis will allow us to visualize entire networks of interacting genes. These cerebral networks generated in response to fetal stre ss, will provide us with a mechanistic blueprint of fetal survival during changes in BP and blood gases. By un derstanding these mechanisms in the late gestation ovine fetus in an in utero environment, we can improve survival and reduce morbidity in premat ure infants in the neonatal intensive care units .

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34 CHAPTER 2 K ETAMINE ATTENUATES THE ACTH RESPONSE TO HYPOXIA IN LATE GESTATION OVINE FETUS Introduction Ketamine is a unique dissociative anesthetic drug with hypnotic, analgesic, and amnesic properties ( 10 ) . The pharmacological properties of ketamine include a fast onset and short duration, while it also preserves respiratory and hemodynamic profiles ( 156 ) . Currently, ketamine is used frequently in surgical procedures for inducing anesthesia in neonates, children, animals, and soldiers on the battlefield ( 10 ) . Ketamine is both lipid and water soluble ( 144 ) , thus enabling it to cross the blood brain barrier ( 61 ) . In the brain, ketamine mainly blocks the NMDA receptor to produce an analgesic effect at low concentrations and produce an anesthesia effect at higher concentrations ( 130 ) . Recently, ketamine have been shown to possess neuroprotective effects in both in vivo and in vitro studies ( 53 , 66 , 67 ) . We propose that ketamine will decrease hypoxi a induced glutamatergic NMDA receptor activity, and reduce the HPA response . In our previous work, we have shown that ketamine is able to red uce the fetal plasma ACTH response to brachiocephalic artery occlusion (BCO), a direct ischemic insult to the fetal brain ( 136 ) . The mechanism of r esponse to BCO is likely to be different than to the response to hypoxia, raising the question of whether ketamine suppresses the ACTH response to all hypoxic stimuli (i.e., hypoxia vs. ischemia vs. asphyxia). We therefore performed the present study to t est the hypothesis that ketamine reduces the HPA response to hypoxia, similar to its actions with regard to BCO.

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35 Materials and Methods These experiments were approved by the University of Florida Animal Care and Use Committee and were performed in accordan ce with the Guiding Principles for Use of Animals of the American Physiological Society. We studied 50 chronically catheterized fetal sheep between the gestational age of 12 6±5 (SD) days (term= 145 147 days ). The pregnant ewes were of mix ed breed s , and we re pregnant with either singleton (n=9) or twin (n=18) fetuses. Fetal Surgery All surgical procedures were performed between 115 130 days of gestation (full term is 145 147 days). Pre operation procedures for the ewes included: 24 hrs fast, shaving and cl eaning surgical site, and intramuscular injection of 750 mg of ampicillin (Polyflex®, Boehringer Ingelheim VetMedica, Inc). Before and during the surgery, the ewes were anesthetized and intubated with 0.5 2 % isoflurane with oxygen. The maternal blood pr essure, heart rate, electrocardiogram, end tidal oxygen and carbon dioxide, and rectal temperature were monitored closely throughout the surgery. The surgical procedures for chronic catheterization of fetal and maternal femoral arteries and veins were desc ribed in detail previously ( 136 , 185 ) . Briefly, using aseptic techniques, we delivered the fetal hindlimbs from the uterus and surgically placed vascular catheters in both femoral arteries and veins of the fetus, and the amniotic fluid pressure catheter was sutu red to the exterior of a hindlimb of the fetus. In the case of twins, both fetuses were catheterized. Before the uterus was sutured closed, we injected 500 mg of ampicillin into the amniotic fluid. The maternal linea alba and skin were sutured closed in separate layers. We then catheterized both maternal femoral arteries and veins. Polyvinylchloride catheters were used for fetal and maternal

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36 catheterization: fetal femoral arteries (0.030 in ID, 0.050 in OD), fetal femoral veins (0.040 in ID, 0.070 in OD ), fetal amniotic fluid (0.050 in ID, 0.090 in OD), maternal femoral arteries and veins (0.050 in ID, 0.090 in OD). All the fetal and maternal catheters were filled with heparin (1,000 units/ml; Elkins Sinn, Cherry Hill, New Jersey) and plugged closed. T he catheters were then routed subcutaneously to the flank, exited the ewe, and protected by a cloth pouch. To induce maternal hypoxic hypoxia, we surgically implanted a tracheostomy tube in the ewe using an intravenous extension tubing ( 114 ) . After the ewes recover from anesthesia, they were returned to their pens. The ewes received a minimum of 5 days post operative treatments before experimentation. Daily post operative care included two doses of ampicillin (750 mg, IM), two rectal temperatures, and monitored food consumption, infection, and signs of distress. The suture and catheter sites were monitored and cleaned with iodine solution if necessary. In Vivo Experimental Procedures During the in utero experiments, the ewes w ere conscious and freestanding in their pens, and were allowed free access to food. We subjected each fetus to only one experiment. The fetal arterial pressure, amniotic fluid pressure, and HR were measured and recorded continuously throughout the entire experiment using standard pressure transducers (Transpac IV, Hospira, Lake Forest, IL), an analog to digital converter (National Instruments, Austin, TX), and custom written software (LabView, National Instruments) ( 136 ) . The fetal HR was calculated from the phasic arterial pressure signal, and the fetal arterial pressures were corrected by subtraction of the amniotic fluid pressure.

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37 B lood pressure recording was initiated at least one minute prior to injection of ketamine. Ketamine (3 mg/kg) was administered intravenously or a sham injection was performed 10 min prior to 30 min of hypoxic hypoxia (HH). HH was commenced by infusing nit rogen gas directly into the maternal tracheostomy tube to decrease maternal partial pressure of oxygen (PaO2) from 100 mmHg to ~50 mmHg, which correspond to a decrease of fetal PaO2 from 20 mm Hg to ~10 mmHg. Fetal and maternal arterial blood samples (5 m L) were collected in chilled K2EDTA tubes (10.8 mg, Vacutainer, Becton Dickinson, Franklin Lakes, NJ) at six time points ( 10, 0, 5, 10, 20, and 30 min) for measurement of plasma ACTH and cortisol concentrations. Additional samples (1 mL) of fetal and mat ernal arterial blood were drawn anaerobically into heparinized syringes for measurement of blood gases (ABL80 Radiometer, Copenhagen, Denmark). All blood samples were kept on ice until measurement of blood gases or separation of plasma by centrifugation (3 ,000xg for 20 min at 4 °C). After centrifugation, the plasma was aliquoted into polypropylene tubes and stored at 20 °C until hormones were assayed. Hormone Assays Fetal plasma ACTH and cortisol concentrations were measured using commercially available a ssay kits. Plasma ACTH concentrations were measured using a radioimmunometric assay kit (Diasorin, Stillwater, MN , catalog #27130 ) according to . This assay has been previously validated for use in fetal plasma ( 123 ) . Plasma cortisol concentrations were measured using an enzyme immunoassay kit (Oxford Biomedical, Oxford, MI , catalog #EA65 ) a fter ethanol deproteinization . Both of these assays as performed in this laboratory have been completely described elsewhere ( 136 ) .

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38 Calculations and Statistical A nalysis Data are presented as mean values standard error of the mean (SEM) with consideration for statistical significance at P <0.05. Fetal femoral arterial blood pressures were corrected by subtraction of amniotic fluid pressure. Unless stated, HR, BP, blood gas, pH, and plasma hormone data was analyzed by tw o way ANOVA (time, ±HH; time, ±k etamine) with Mixed Procedure of SAS/STAT® 9.3 ; corrected for repeated measures in one dimension (time), and if significant, by Duncan post hoc test . Results Blood Gases Maternal blood gases and pH measurements are reported in F igure 2 1A 2 1C. Hypoxia decreased the maternal P a O 2 from 98±2 mmHg to 56±2 mmHg, while during normoxia values remained constant throughout the experiment ( P <0.0001, stimulus*time). Maternal P a CO 2 decreased from 37±0.5 mmHg to 32±1 mmHg during hypoxia, but was unchanged during normoxia ( P <0.0001, stimulus*time in two way ANOVA). The maternal plasma pH increased from 7.47±0.01 to 7.52±0.01, during hypoxia but did not change during normoxia ( P <0.0001, stimulus*time in two way ANOVA). Fetal blood gases and pH measurements were reported in F igure s 2 1D 2 1F . In both control and ketamine treated groups, hypoxia decreased fetal P a O 2 (17±0.7 mmHg to 10±0.5 mmHg for both groups, P <0.0001) , and decrease d P a CO 2 (54±0.7 mmHg to 52±0.4 mmHg) , resulting in a slight alkalosis. The changes in blood gases during hypoxia were statistically significant when analyzed by two way ANOVA (stimulus*t ime: P a O 2 , P <0.0001 ; P a CO 2 , P <0.0 01; pH , P <0. 00 01). There was no statistically significant effect of ketamine on the fetal blood gas response to hypoxia .

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39 Fetal C ardiovascular Variables Ana lysis of fetal blood pressure (F igures 2 2A 2 2D) and hea rt rate (F igures 2 2E 2 2H) by three way ANOVA corrected for repeated measures over time revealed a statistically significant effect of hypoxia and ketamine ( P <0.05 for three way interaction of time, ketamine, and hypoxia on blood pressure and P <0.05 for three wa y interaction for fetal heart rate). When individual groups were analyzed by one way ANOVA for repeated measures, blood pressure was increased by hypoxia ( P <0.001, F igure 2 2C) and decreased by ketamine alone ( P <0.0 5 , F igure 2 2 B ), but unchanged between the normoxia control and hypoxia ketamine group s ( F igures 2 2A , 2 2D ). It seems that k etamine blocked the increase in fetal blood pressure during hypoxia. In the fetuses subjected to hypoxia but not to ketamine, hypoxia appeared to reduce fetal heart rate from 161±10 beats per minute (bpm) to 141±9 bpm, although this was no t statistically significant (F igure 2 2G). Fetal heart rate was also not significantly changed in response to hyp oxia after ketamine treatment (F igure 2 2G). Administration of ketamine prior to hypoxia increased heart rate from 172±4 bpm and reached values of 206±10 bpm during the first 10 min of hypoxia ( P <0.05), returning to control in the last 20 min (F igure 2 2H). Ketamine also increased fetal heart rate in the group not subjected to hypoxia ( F igure 2 2F, P <0.05). Endocrine Variables Fetal plasma ACTH was increased by hypoxia in both control and ketamine groups ( P <0.0001, stimulus*time, F igure 2 3A ). Ketamine reduced the fetal ACTH response to hypoxia ( P <0. 005 , treatment*time) but did not have an effect on fetal ACTH in the normoxic groups. As shown in F igure 2 3B, f etal plasma cortisol was increased by hypoxia in both control and ketamine groups ( P <0.001, stimulus*time) . In contrast to

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40 its effect on fetal pl asma ACTH, ketamine did not attenuate fetal plasma cortisol levels when compared with the hypoxic controls. There was also a significant increase in maternal plasma cortisol concentrations during hypoxia but not during normoxia ( P <0.0001, F igure 2 3C). Di scussion We have previously reported that ketamine inhibits the fetal ACTH response to BCO, a model of transient brain ischemia and reperfusion in the fetal sheep ( 136 ) . The stimulus of hypoxic hypoxia, used in the present study, differs from BCO in that the stimulus results in a decrease in fetal P a CO 2 secondary to maternal hyperventilation where as BCO is an asphyxic stimulus. The results of the present study suggest that the ACTH response to BCO and hypoxia share a dependency on NMDA glutama tergic neurotransmission. Here we report that ketamine alters the fetal hemodynamic response to hypoxic hypoxia. Ketamine did not alter the magnitude of changes in fetal blood gases or pH, supporting the conclusion that the stimulus intensity was equal in both groups. The fetal blood pressure and heart rate responses to hypoxia were consistent with responses reported by us ( 141 ) and others ( 11 , 12 , 15 , 51 ) . Ketamine decreased fetal blood pressure and modified increases in blood pressure and decreases in heart rate during hypoxia. Whether the effect on blood pressure is purely the result of interruption of the chemoreflex secondary to NMDA blockade, or whether there are opposing but independent effects of hypoxia and ketamine; these changes cannot be discerned from the present experiments. Ho wever, interruption of chemoreflex pathways would be consistent with the effect of ketamine to reduce fetal blood pressure and heart rate

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41 responses to BCO ( 136 ) . It is also consistent with the known effect of ketamine on fetal chemoreflex control of heart rate ( 13 , 162 ) . Ketamine blunted the fetal plasma ACTH response to hypoxia, although less dramatically than the decrease in ACTH respon se to BCO ( 136 ) . In a general sense, the inhibition of HPA stress responses by ketamine is consistent with results of studies in adult animals. For example, in adult rats, both acute and chronic treatments of ketamine (15 mg/kg) reduced ACTH and corticosterone responses to chronic stress ( 47 ) . Ketamine was also shown to inhibit adrenocortical activation in rats undergoing laparotomy stress by decreasing noradren ergic signaling in the hypothalamus ( 125 ) . The inhibition of the ACTH response to hypoxia by ketamine suggests that ACTH responses to hypoxia are partially NMDA glutamate dependent. We know, from previous results in this laboratory, that NMDA stimulates fetal ACTH secretion ( 86 ) . However, it is also known that the fetal ACTH response to hypoxia is dependent on the carotid chemoreflex ( 136 , 167 , 180 ) , and that at least one report in the literature suggests that ketamine modulates fetal ovine cardiovascular responses to chemoreceptor stimulation ( 13 ) . NMDA receptors are located throughout the afferent s ympathetic pathway: nucleus tractus solitaries ( 131 ) , rostral ventrolateral medulla ( 1 ) , intermediolateral nucleus ( 201 ) , and paraventricular nucl eus ( 34 ) . Th e modification b y ketamine of the cardiovascular response to hypoxia in the present experiments supports the conclusion that the chemoreflex is sensitive to ketamine inhibition, and that blockade of at least a part of this pathway might be the mechanism of the inhibition of ACTH secretion.

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42 While ketamine is thought to work by inhibiting the NMDA receptor, it does have other actions, especially at higher concentrations. Aside from its major action a ntagonism of NMDA glutamatergic receptor ketamine may have an effect on oth er receptors: nicotinic acetylcholine receptors ( 194 ) , serotonin receptors ( 78 , 194 ) , dopamine receptors ( 78 ) , opioid receptors ( 72 , 157 ) , muscarinic acetylcholine receptors ( 72 ) , and weakly influencing the GABA A and glycine receptors ( 194 ) . However, most of these receptors have less affinity for ketamine than the NMDA re ceptors, and require a higher (10 20 fold) concentration of ketamine to obtain the same antagonist effect ( 88 ) . While we assume that the blunting of the ACTH response to hypoxia is secondary to blockade of NMDA receptors, we cannot rule out an action of the drug at other receptors. Ketamine can also have actions independent of the che moreflex response to hypoxia, and was reported to stimulate adrenocortical activity in adult rats ( 46 , 125 ) and humans ( 92 ) . The cortisol response to hypoxia was not attenuated by ketamine. We suspect that the cortisol response to hypoxia in both groups r epresented the maximal fetal adrenal response to ACTH ( 21 , 122 ) . In other words, we believe that while the fetal ACTH response to hypoxia was blunted by ketamine, it was still h igh enough to maximally stimulate the fetal adrenal transiently. This is consistent with our previous observation that ketamine did not reduce the fetal cortisol response to BCO ( 136 , 167 , 180 ) . In conclusion, we have demonstrated that ketamine blunts the fetal ACTH response to maternal hypoxia. The use of ketamine in late pregnancy or in neonates of similar developmental maturity could alter neurohormonal responses to transient

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43 hypoxia or brain ischemia, and therefore modify potentially benefi cial homeostatic responses. However, it is also possible that ketamine reduces glutamate excitotoxicity and that the reduction in ACTH response is an endocrine indicator of this action.

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44 Figure 2 1. Maternal and fetal arteria l blood gases and pH. Maternal and fetal (A, D) partial pressure of oxygen (PaO2), (B, E) partial pressure of carbon dioxide (PaCO2), and (C, F) pH were measured 10 min before and during 30 min of hypoxic hypoxia (HH) stimulation in the ewe, and hypoxic hy poxia stimulus±ketamine in the fetus (NC, normoxia control; NK, normoxia ketamine; HC, hypoxia control; HK, hypoxia ketamine). Results of Duncan test for pairwise comparison are expressed as: *vs. NC (P<0.05); ^HC vs. NC (P<0.05); avs. baselines ( 10 and 0 min; P<0.001); bHC vs. HC baseline ( 10 min; P<0.05); cHC vs. HC baseline (0 min; P<0.005); dHK vs. HK baseline (0 min; P<0.001); and eHK vs. HK baseline ( 10 min; P<0.05). Data are presented as means±SEM.

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45 Figure 2 2 . Fetal mean arterial pressure and heart rate. Fetal femoral arterial blood pressure (A D) and heart rate (E H) of fetal sheep 10 min before and during 30 min of hypoxic hypoxia (HH) stimulation. Results of Duncan test for pairwise comparison are expressed (P<0.05); #HK vs. NK (P<0.05); and * vs. baselines (average of 10 through 0 min; P<0.05). Abbreviations as in F igure 1. Fetal BP and HR values are reported in mean±SEM.

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46 Figure 2 3. Fetal plasma concentrations of ACTH and cortisol, and maternal plasma concentration of cortisol. Fetal plasma ACTH (A) and cortisol (B) concentrations 10 min before and during 30 min of HH stimulation±ketamine. (C) Maternal plasma concentrations of cortisol 10 m in before and during 30 min of HH stimulation. Results of Duncan pairwise comparisons are aHC vs. HC baseline ( 10 min; P<0.005); bHC vs. HC baseline (0 min; P<0.01); and cHK vs. HK b aselines ( 10 and 0 min; P<0.05). Abbreviations are as in F igure 1. All plasma ACTH and cortisol values are reported in mean±SEM.

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47 CHAPTER 3 K ETAMINE DECREASE IMMUNE GENE EXPRESSION IN OVINE FETAL FRONTAL CORTEX EXPOSED TO ACUTE HYPOXIC HYPOXIA Int roduction Transient fetal hypoxia is a common feature during fetal development without any pathological consequences. Through cardiovascular and neuroendocrine responses, the fetus redirects the combined ventricular output and increases blood flow toward s relevant organs such as brain, heart and adrenals. However, a severe or chronic hypoxic stimulus can pr oduce multiple organ damage as a result of metabolic acidosis. The main consequence of fetal hypoxia is brain damage due to the intolerance to low le vels of oxygen. Due to their large amount of neuronal bodies, the cerebral cortex, cerebellum, and hippocampus are very susceptible to a hypoxic stimulus ( 28 ) . Fetal brain damage occurs mainly due to the activation of inflammatory and apoptotic responses, a mechanism mediated by the overactivation of NMDA receptors. The fetal inflammatory response is characterized by an increase in proinflammatory mediators such as IL6 , IL expressed in activated immune cells in hypoxic brain regions ( 143 ) . A high degree of inflammation in the brain can lead to premature birth ( 143 ) , and later clinical disorders for the neonate such as cerebral palsy ( 124 ) , autism ( 84 ) , or schizophrenia ( 160 ) . NMDA recep tor antagonists have been extensively reported to be reduce brain damage induced by a hypoxic stimulus ( 49 , 62 , 179 ) . Among all, ketamine, an FDA approved non competitive NMDA receptor antagonist, has been shown to have anti inflammatory properties by reducing levels of IL 6 and TNF in early post o peratory human patients ( 9 ) and animals ( 93 , 152 ) . Previous studies also reported that ketamine

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48 has neuroprotective effects in vitro and in animal studies for up to 7 days ( 65 ) . We conduct ed a transcriptomics analysis to determine the effects of hypoxia on the fetal frontal cortex and the effects of ketamine in reducing the effects of acute hypoxic damage induced by the fet al inflammatory response. We hypothesized that ketamine administration reduces the expression of proinflammatory markers in the late gestation ovine fetal frontal cerebral cortex exposed to acute hypoxia. Materials and Methods These experiments were appro ved by the University of Florida Animal Care and Use Committee and were performed in accordance with the Guiding Principles for Use of Animals of the American Physiological Society. Sixteen chronically catheterized singleton or twin ovine fetuses were stu died between the gestational age of 122±5 days (full term=145 147 days). Fetal Surgery Ewes were fasted for 24 hours before surgery, and the fetal ovine surgical procedures were performed on 116±3 days of gestation. The fetal and maternal femoral arteries and veins chronic catheterization surgical procedures were described in detail previously ( 136 , 185 ) . Briefly, the ewes were given 750 mg ampicillin (Polyflex®, Boehringer Ingelheim 19 VetMedica, Inc., St. Joseph, MO, USA), then anesthetized and intubated with 0.5 2 % isofl urane with oxygen. A set of fetal femoral arterial and venous vascular catheter were surgically placed in the fetal hindlimbs, along with an amniotic fluid catheter. Before the uterus was sutured closed, 500 mg ampicillin was injected into the amniotic fl uid. The ewe received a set of femoral arterial and venous vascular catheter, and a tracheostomy tube to stimulate maternal induced ventilatory hypoxia or hypoxic hypoxia ( 114 ) . A minimum of 5 days of recovery were allowed befo re

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49 experimentation. Daily post operative care included two rectal temperatures, two doses of ampicillin (15 20 mg/kg, IM), and monitored for food consumption, infection, and signs of distress. In Vivo Experimental Procedures During the experiments, the ewes were conscious and freestanding in their pens with access to food. The sixteen fetuses were randomly assigned to one of the four groups (n=3 4/group): normoxic control, normoxia+ketamine, hypoxic control, and hypoxia+ketam ine. In ketamine treated groups, ketamine (3 mg/kg) was given intravenously, through the fetal femoral venous catheter, 10 min prior to the hypoxic stimulus (30 min). Hypoxia was induced by infusing nitrogen gas directly into the maternal tracheostomy tu be to suppress maternal pressure of oxygen (P a O 2 ) by 50 %. Consequently, the fetal P a O 2 mimicked the maternal response and was decreased by 50 % also ( C hapter 2 ). To closely monitor the changes in blood gas compositions (ABL80 Radiometer, Copenhagen, Den mark), both maternal and fetal arterial blood was drawn anaerobically (1 mL) every 10 minutes. The fetuses were sacrificed 24 hrs post initial stimulation of hypoxic stress, and various fetal tissues were snap frozen and stored at 80 °C until future anal ysis. Microarray Procedures The mRNA was extracted from fetal frontal cortex via RNeasy Plus Mini Kit (Qiagen, Valencia, CA), with mRNA integrity number (RIN) values between 7.7 9. 2 , were labeled with cyanine 3 CTP with the Quick Amp Labeling Kit (Cat# 519 0 0442, labeled cRNAs ranged from 1 2.9 to 1 to 14.4 . The cRNA samples were hybridized and processed for one channel Sheep Gene

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50 Expressi on Microarray (8 x15 K slide) 8 arrays with 15208 oligomers each (Cat# G4813A 019921, Agilent Technologies, Santa Clara, CA) as described earlier ( 140 , 187 ) . The slides were scanned with Microarray Scanner System (G2505 90021, Agilent) and the measured fluorescen ce was detected and converted using Agilent Interdisciplinary Center for Biotech Research. Cytoscape and various plug ins (GeneMANIA, ClusterONE, BiNGO) were used to an alyze gene network inference and clustering analysis ( 30 , 153 ) . The functional annotation of gene ontogeny for significantly up and down regulated genes were analyzed via DAVID Bioinformatics Resources 6.7 ( 68 , 69 ) and WEB based GEne SeT AnaLysis Toolkit (WebGestalt) ( 171 , 204 ) . Real T ime PCR The same set of mRNA used for the microarray was also used to for qPCR validation. The primers used were designed based on the known Ovis aries and Bos taurus genomes ( T able 1) for S YBR actin primers and probe were used ( 187 ) . The relative mRNA expression was calculated by the difference the triplicate mean Ct for each gene and the triplicate ac tin mRNA from the same sample. Calculations and Statistical Analysis Data are presented as mean values standard error of the mean (SEM) with consideration for statistical significance at P <0.05. The ovine Agilent 15.5k array results moderated t test using empirical Bayes method for small sample size per group ( P <0.05). Unless stated, the real time PCR data was analyzed by two way ANOVA

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51 (time, ±hypoxia; time, ±ketamine) with Mixed Procedure of SAS/STAT® 9.3; corrected for repeated measures in one dimension (time), and if significant, by Duncan post hoc test. Results In the fetal frontal cortex, acute fetal hypoxia stimulated the up regulation of 248 genes, and the down regulation of 480 genes (figure 3 1). With the pre treatment of ketamine, there were 570 genes that were significantly up regulated and 250 genes that were down regulated. In the fetuses that were treated with ketamine under normoxic conditions, only 50 genes were significantly up regulated and these did not provide any significant biological processes or pathways; however, there were 105 genes that we re down regulated in the frontal cortex and the significant biological processes included cellular potassium ion transmembrane transport (3 genes, adjusted P =3.78E 2), mitotic prometaphase (5 genes, adjusted P =3.53E 2), viral reproduction (14 genes, adjus ted P =2.15E 2), and interspecies interaction between organisms (10 genes, adjusted P =3.53E 2). The significantly regulated genes, either up or down regulated, were put through WebGestalt for analysis of gene ontology, KEGG pathways, common pathways, and a ssociated diseases. Twenty four hours after the onset of a 30 min acute hypoxic stimulus, the biological processes that were up regulated in the fetal frontal cortex were mainly associated with the regulation of immune system response, defense response, r esponse to stress, an d response to biotic stimulus (T able 3 2). The biological processes that were down regulated after the hypoxic insult were mainly involving in protein metabolic processes, negative regulation of MAP, ERK1, and ERK2 cascade. With the treatment of ketamine, the up regulated biological processes were organelle

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52 organization, cellular protein metabolic process, and macromolecule modification, and down regulated biological processes were defense response, response to stress, and regulation of immune response (T able 3 2). The enriched KEGG pathway analysis reveled that hypoxic stimulus activates the toll like receptor and chemokine signaling pathways, cytokine cytokine receptor interaction, and t he osteoclast differentiation (T able 3 3); and mainly down regulates metabolic pathways. With the presence of ketamine during hypoxic stimulus, the fetal frontal cortex have the opposite profile with an up regulation of metabolic pathways, and down regulation of toll like receptor and chemokines signa ling pathways. To determine the genomic effect of ketamine on the hypoxic fetal brain, the genes that were significantly up regulated by hypoxia, were compared with the genes that were significantly down regulated by hypoxia+ketamine (F igure3 2A). There w ere 126 genes that were uniquely up regulated by hypoxia, 128 genes that were down regulated by hypoxia+ketamine, and 122 common genes between the both groups. Using the gene ontology analysis, the 122 common genes yielded biological processes involving i n the regulation of immune, defense, and stress response ( T able 3 4). The main molecular functions are protein binding and cytokine receptor binding, and the main cellular components are extracellular region and plasma membrane. The enriched KEGG pathway analysis indicated the involvement of toll like receptor signaling, and chemokines signaling, and cytokine cytokine receptor interaction. The major common pathways associated with the 122 common genes were immune, inflammatory, apoptosis, metabolic, and cellular division (T able 3 5). The analysis of enriched disease

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53 associated genes resulted in inflammation, necrosis, immune sy stem, and autoimmune diseases (T able 3 6). Interestingly, when comparing the 166 common genes between the down regulated hypoxia versus the up regulated hypoxia+ketamine (F igure3 2B), there was only one significant gene ontology biological process, which was cellular protein metabolic process (50 g enes, adjusted P =3.34E 2). The KEGG pathway yielded a few significant pathways, with metabolic pathways as the main outcome (9 genes out of 166 common genes). The rest of the pathways involved very few genes, which varied between 1 3 % (2 5 genes out of 166 common genes). To validate the microarray and the analysis of pathways and networks, three genes were cho sen from the 122 common genes (F igure 3 2A), and real time qPCR was performed (F igure 3 3). The three genes, caspase 8 (CASP8), m yeloid differenti ation primary response 88 (MYD88), and nuclear factor kappa light chain enhancer of activated B cells (NF B), were selected to represent the apoptotic, inflammatory, and immune pathways that was up regulated by hypoxia, but down regulated by hypoxia+ketami ne. The results are shown as fold change from the normoxic control group. After 24 hrs following the initial acute hypoxic insult, all three genes were significantly increased. CASP 8 increased 2.01 fold, M YD88 increased 1.32 fold, and NF B increased 1. 4 fold. But the fetuses that were treated with ketamine, the CASP 8 only increased 0.98 fold, MYD88 increased 1.08 fold, and NF B increased 0.91 fold (F igure 3 3) There were no significant differences between the normoxic+ketamine versus the control fetu ses (CASP 8, 0.94 fold; HYD88, 1.06 fold; NF B, 0.76 fold).

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54 Discussion The transcriptomic analysis of the fetal frontal cortex reveled that twenty four hours post hypoxic stimulus, the genomic expression of immune, apoptotic, and stress responses were up regulated, and the cellular and protein metabolic processes were down regulated. However, the administration of ketamine before acute hypoxic insult was able to reverse these genomic responses. The results suggest that ketamine may have an anti inflammat ory action in response to fetal hypoxic stress. Hypoxic insult is the most common stressor encounter by the fetus during gestation, and may lead to mild or severe cerebral hypoxia. Hypoxia is a very strong insult for the central nervous system and injurie s can appear even in short amount of time ( 27 ) . The developing brain requires high levels of adenosine triphosphate (ATP) in order to support neurogenesis, synaptogenesis, and maintain normal physiological growth. When the fetus is threatened with a decrease in cerebral blood flow or oxygen supply, the brain is at risk of glutamate induced excitotoxity by the over act ivation of NMDA receptors ( 28 ) . The shortage of intracellular ATP will result in depolarization o f neuronal membrane, leading to the excessive release of glutamate into the synaptic cleft. In addition, the decrease of ATP will reduce the number of glutamate active transporters, resulting in a decrease of glutamate recycling by neuronal and glial cell s ( 82 ) . The influx of extracellular glutamate will over stimulate the NMDA receptors, leading to an increase level of intracellular calcium, which activates the inflammatory and apoptotic pathways ( 20 , 169 ) . In our study, we proposed the use of ketamine, a non competitive NMDA receptor antagonist, to attenuate the fetal hypoxic response and reduce neuronal excitotoxicity. In order to elucidate the effect of ketamine on the hy poxic fetal frontal

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55 cortex, we examined the common genes that were both significantly up regulated by hypoxia and significant ly down regulated by ketamine (F igure 3 2). The results indicated that with hypoxic hypoxia, inflammatory and apoptotic pathways w ere significantly up regulated; and cellular and protein metabolic processes were down regulated. With the administration of ketamine, we found an up regulation of metabolic processes, and a down regulation of inflam matory and apoptotic pathways (T able 3 2, 3 3). The inflammatory hypoxic stress response is characterized by an increase in cytokines, such as IL1, IL6, and TNF ( 29 , 63 , 163 ) . These proinflammatory cytokine, as well as NF B one of the main regulators of the inflammatory response via a hypoxia inducible factor mechanism ( 43 ) have been reported to have a direct cytotoxic effect on neurons and glial cells ( 170 ) . These inflammatory mediators promote the recruitment, activation, and infiltration of immune cells in the brain, and contribute to the severity of the hypoxic injury ( 20 , 76 ) . Hypoxic fetal stress response involves the activation of the HPA axis, which starts at the paraventricular nucleus (PVN) of the hypothalamus with the secretion of corticotropin releasing hormone (CRH). Besides its role in activating the pituitary to secrete ACTH, studies have shown that CRH has additional role as a proinflammatory mediator. In the brain, CRH activates microglial cells ( 129 ) , the local resident immune cell of the brain, whose excessive activation is linked to cytotoxicity, neurodegeneration , and necrosis ( 76 ) . The chronic activation of the HPA axis may induce negative feedback dysfunction, which occurs when glucocorticoid receptors, located in the fetal hypothalamus and hippocampus, are no longer affected by elevated levels of cortisol. Wang et al ., reported that the activation of

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56 the p38 mitogen activated protein kinase (MAPK) pathway by IL1 , may have a potential role in the inhibition of glucocorticoid receptor translocation ( 172 ) . Our results show a significant up regulation of inflammatory mediators men tioned above, and among others (IL13RA1, IL17RA, IL18BP, IL1R1, IL4R, IL6R, IL6ST, IL8, CCL2, CCL4L1, CCL5, CXCL10, CXCL16, CXCR7, TLR 2, CD1D, CD5, CD14, CD53, CD59, CD74, CD86, and CD109) as part of the fetal defense mechanism to hypoxic hypoxia. Apoptos is is one of the pathways that were significantly up regulated by hypoxia. Calcium influx into the neurons promotes neuronal apoptosis mainly via the caspase signaling pathway ( 169 ) . Several studies have reported that hypoxic stimulus elevate concentrations of the apoptotic factors caspase 3, 7, 8 and 9 ( 126 , 169 , 207 ) , and other adaptor proteins such as Bax/Bcl 2 ( 25 ) , Myd88 ( 173 ) , a nd NF ( 101 ) , under hypoxic stimulat ion. These observations correlate with our current findings: the up regulation of caspase mediated apoptosis in the fetal brain during hypoxic hypoxia. It is important to mention that the inflammatory cascade and apoptosis are closely related pathways si nce both share many mediators/messengers that were significantly up regulated in this study. Based on this, we can conclude that both events can happen at the same time and are important mechanisms of the fetal hypoxia stress response. Our result showed that ketamine was able to down regulate the caspase mediated apoptotic gene expression in the frontal cortex in response to acute hypoxia stress In this study, we found that maternal ventilatory hypoxia induced down regulation of cellular and protein metab olic processes. The lack of available ATP causes a metabolic shutdown of cellular functions that require a high energy expense such as protein biosynthesis ( 118 ) . Also, there was a down regul ation of mitochondrial

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57 ribosomal proteins (MRPL11, MRPL24, MRPL42, MRPL43, MRPS16, MRPS25, MRPS26, and MRPS6) , which assist in protein synthesis in the mitochondria. This indicates that one of the physiological responses to a hypoxic insult is a decrease in mitochondrial protein synthesis in the fetal brain to conserve energy. In addition, there was a down regulation of transcriptional regulators (zinc finger proteins : ZNF263, ZNF277, ZNF444, ZNF581, ZNF740, ZNHIT1, ZRANB2) and cell cycle regulators (ZAK, CCAR1). With the addition of ketamine, we found an up regulation of cellular metabolic processes, which is involved in the maintenance of the integrity and function of the neuronal cells, an important feature for normal fetal brain development. All the biological responses and cellular pathways discussed above were significantly influenced by the administration of ketamine prior to the hypoxic insult. In this study, ketamine was able to down regulate the main inflammatory mediators, such as IL1, IL6, TNF , and NF B, thus disrupting major fetal inflammatory pathways. Our findings are supported by others who reported on the anti inflammatory ability of ketamine in red ucing the levels of TNF , IL1 , IL6, and IL8, which are the main innate immune system modulators ( 60 , 79 , 80 , 93 , 94 , 152 ) . Other studies have also supported the role of ketamine as an anti inflammatory agent when used in both clinically ( 9 , 177 , 200 ) and experimentally ( 80 , 87 , 164 ) . Ketamine is known to bind an array of receptors, besides NMDA receptors: dopamine receptors ( 78 ) , non NMDA glutamamte receptors, opioid receptors, nicotinic and muscarinic cholinergic receptors ( 88 ) , serotonin receptors ( 78 , 194 ) , and weakly influencing the glycine and GABA A receptors ( 194 ) . We speculate that some of the fetal inflammatory components detected by the microarray may not solely due to local

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58 response, but might originate d from peripheral tissues such as thymu s, liver, spleen, bone marrow, or placenta. The reason is because the blood brain barrier is not completely developed in the late gestation fetal sheep ( 161 ) , and inflammatory markers produced by the brain can recruit immune cells from the periphery . Future research should focus on the mechanistic role of ketamin e in the regulation of local and systemic fetal immune response to a hypoxic stimulus. Ketamine might activate peripheral NMDA receptors, or possibly, play a role in reducing the systemic activation of innate immune response by binding to other receptors. The anti inflammatory and anti apoptotic effects of ketamine could potentially be beneficial for pre treating premature infants in the NICU undergoing surgical procedures.

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59 Table 3 1. Primers for real time PCR validation. Official Symbol Gene Name For ward Primer Reverse Primer Species NF B Nuclear factor kappa light chain enhancer of activated B cells TCCCACAGATGTTCACAA ACAGT GACGCTCAATCTTCATC TTGTGAT Ovis aries MYD88 Myeloid differentiation primary response 88 GCCTGAGTATTTTGATGC CTTCA GCTGCCGGATCATCTCA TG Ovis aries CASP8 Caspase 8 TGGCTGCCCTCAAGTTCC T GGAATAGCATCAAGGCA TCCTT Bos taurus

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60 Figure 3 1. Volcano plot illustrating the relationship of gene expression in fetal frontal cortex measured by log 2 of fold change. Significantly up regulated (blue) and down regulated (red) genes under hypoxia (A), hypoxia+ketamine (B), and normoxia+ketamine (C). The dotted line indicates whether the gene is statistically significant (above), P <0.05, or not significant (below). Non significant genes are indicated in black dots

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61 Table 3 2. Top 10 gene ontology biological processes that were significantly regulated in the frontal cortex during acute hypoxic stress, with or without ketamine. Groups Biological Processes # Genes involved Adjusted P values Hypoxia Up regulated Immune response 75 1.06E 27 Defense response 75 4.70E 27 Immune system process 94 5.61E 27 Response to stress 115 2.48E 23 Innate immune response 50 4.83E 23 Regulation of immune system process 60 1.20E 21 Regulation of immune response 46 4.49E 19 Positive regulation of immune system process 44 1.46E 17 Regulation of defense response 39 1.79E 17 Response to biotic stimulus 46 1.79E 17 Hypoxia Down regulated Cellular protein metabolic process 127 4.00E 04 Protein metabolic process 140 2.80E 03 Negative regulation of MAP cascade 11 2.80E 03 Negative regulation of ERK1 and ERK2 cascade 6 4.30E 03 Negative regulation of intracellular protein kinase cascade 13 8.00E 03 Water soluble vitamin metabolic process 9 1.09E 02 Negative regulation of MAP kinase activity 9 1.09E 02 Inactivation of MAPK activity 6 1.09E 02 Negative regulation of metabolic process 61 1.27E 02 Negative regulation of macromolecule metabolic process 58 1.27E 02 Hypoxia+ketamine Up regulated Organelle organization 97 8.50E 03 Cellular protein metabolic process 137 8.50E 03 Response to starvation 12 4.29E 02 Cellular metabolic process 297 4.29E 02 Protein modification by small protein conjugation or removal 37 4.29E 02 Cellular response to starvation 10 5.36E 02 Macromolecule modification 106 5.36E 02 Protein ubiquitination 32 5.36E 02 Cellular protein modification process 101 8.24E 02 Protein modification by small protein conjugation or removal 32 8.24E 02 Hypoxia+ketamine Down regulated Defense response 63 7.93E 17 Response to stress 104 3.34E 15 Regulation of immune response 42 6.46E 15 Immune response 58 9.22E 15 Regulation of immune system process 51 3.06E 14 Response to wounding 58 3.06E 14 Regulation of defense response 35 1.67E 13 Immune system process 74 1.70E 13 Innate immune response 39 1.70E 13 Regulation of response to stress 45 3.20E 13

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62 Table 3 3. Top 10 enriched KEGG pathways that were significantly regulated in the frontal cortex during acute hypoxic stress, with or without ketamine. Groups Pathway Names # Genes involved Adjusted P values Hypoxia Up regulated Chagas disease (American trypanosomiasis) 13 2.53E 12 Complement and coagulation cascades 11 7.75E 12 Osteoclast differentiation 13 1.29E 11 Toll like receptor signaling pathway 12 1.31E 11 Cytokine cytokine receptor interaction 16 4.97E 11 Staphylococcus aureus infection 9 3.29E 10 Pathways in cancer 16 7.88E 10 Chemokine signaling pathway 12 9.61E 09 Malaria 7 1.38E 07 Hematopoietic cell lineage 8 3.13E 07 Hypoxia Down regulated Vitamin digestion and absorption 5 3.00E 04 Metabolic pathways 28 1.30E 03 Pathways in cancer 13 1.30E 03 TGF beta signaling pathway 6 4.50E 03 Vibrio cholerae infection 5 4.50E 03 Wnt signaling pathway 7 1.38E 02 Oocyte meiosis 6 1.39E 02 Amino sugar and nucleotide sugar metabolism 4 1.59E 02 Cell cycle 6 1.75E 02 Collecting duct acid secretion 3 2.10E 02 Hypoxia+ketamine Up regulated Metabolic pathways 46 4.97E 10 mRNA surveillance pathway 9 6.34E 05 mTOR signaling pathway 7 2.00E 04 Tight junction 9 9.00E 04 Glycine, serine and threonine metabolism 5 9.00E 04 Spliceosome 9 9.00E 04 Insulin signaling pathway 9 1.20E 03 TGF beta signaling pathway 7 1.40E 03 Huntington's disease 10 1.40E 03 Vitamin digestion and absorption 4 1.90E 03 Hypoxia+ketamine Down regulated Complement and coagulation cascades 12 4.74E 13 Chagas disease (American trypanosomiasis) 13 1.50E 12 Staphylococcus aureus infection 10 1.88E 11 Malaria 9 2.17E 10 Osteoclast differentiation 12 2.17E 10 Pathways in cancer 15 1.07E 08 Leishmaniasis 8 8.78E 08 Toll like receptor signaling pathway 9 8.78E 08 Chemokine signaling pathway 11 1.24E 07 Amoebiasis 8 1.50E 06

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63 A. B. Figure 3 2. Venn diagram of the number of gene s that were significantly regulated by hypoxia and hypoxia+ketamine. (A) Venn diagram showing the number of significant genes that was up regulated by hypoxia (blue), down regulated with ketamine (red), and the common genes involved in both groups (purple). (B) Venn diagram of significant genes that was down regulated by h ypoxia (red), up regulated with ketamine (blue), and the common genes involved in both groups (purple).

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64 Table 3 4. Top 10 gene ontology biological processes and enriched KEGG pathways that were significantly up regulated in the frontal cortex during acute hypoxic stress, but was down regulated with ketamine. Analysis Pathways, processes, functions, components # Genes involved Adjusted P values Biological Process Immune response 42 7.39E 17 Immune system process 53 7.39E 17 Defense response 43 7.39E 17 Regulation of immune system process 38 1.12E 16 Regulation of immune response 30 4.30E 15 Response to wounding 40 6.90E 15 Positive regulation of immune system process 29 2.09E 14 Response to stress 63 2.09E 14 Innate immune response 28 9.60E 14 Response to lipid 28 6.08E 13 Molecular Function Protein binding 80 5.78E 07 Cytokine receptor binding 10 2.00E 04 Interleukin 6 receptor activity 2 1.30E 03 Receptor binding 22 1.30E 03 Chemokine receptor binding 5 1.30E 03 Interleukin 6 binding 2 1.30E 03 Cell surface binding 5 1.30E 03 Enzyme inhibitor activity 10 1.50E 03 Lipoteichoic acid binding 2 1.70E 03 Peptidase regulator activity 8 1.70E 03 Cellular Component Extracellular region part 38 2.64E 15 Extracellular space 32 9.79E 14 Extracellular region 44 2.03E 10 Plasma membrane 52 1.00E 04 Cell periphery 52 2.00E 04 Cytosol 34 4.00E 04 Cell surface 13 7.00E 04 Complement component C1 complex 2 8.00E 04 Interleukin 6 receptor complex 2 1.60E 03 Proteinaceous extracellular matrix 10 2.60E 03 KEGG Pathways Chagas disease (American trypanosomiasis) 11 2.78E 13 Complement and coagulation cascades 10 2.78E 13 Staphylococcus aureus infection 9 8.98E 13 Pathways in cancer 12 2.32E 09 Toll like receptor signaling pathway 8 6.40E 09 Osteoclast differentiation 8 3.27E 08 Cytokine cytokine receptor interaction 10 3.79E 08 Malaria 6 5.32E 08 Chemokine signaling pathway 8 4.61E 07 Systemic lupus erythematosus 7 7.49E 07

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65 Table 3 5. Top 10 common pathways associated with the genes that were significantly up regulated in the frontal cortex during acute hypoxic stress, but was down regulated with ketamine. Common Pathways # Genes involved Adjusted P values Immune System 27 6.57E 24 Innate Immune System 17 6.54E 19 Integrin family cell surface interactions 32 7.15E 19 TRAIL signaling pathway 31 2.09E 18 Glypican 1 network 30 1.01E 17 Signaling events mediated by Hepatocyte Growth Factor Receptor (c Met) 29 1.59E 17 Insulin Pathway 29 1.59E 17 S1P1 pathway 29 1.59E 17 Arf6 downstream pathway 29 1.59E 17 Arf6 signaling events 29 1.59E 17

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66 Table 3 6 . Top 10 enriched diseases associated with the genes that were significantly up regulated in the frontal cortex during acute hypoxic stress, but was down regulated with ketamine. Diseases # Genes involved Adjusted P values Inflammation 26 2.00E 24 Necrosis 21 2.44E 20 Immune System Diseases 26 5.81E 20 Autoimmune Diseases 20 4.53E 17 Chorioamnionitis 15 1.23E 16 Infection 20 2.16E 15 Preterm rupture of membranes 13 7.23E 14 Bronchiolitis 14 1.21E 13 Virus Diseases 18 1.50E 13 Bacterial Infections 13 1.64E 13

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67 Figure 3 3. mRNA gene expression was measured by real time qPCR for apoptotic, inflammatory, and immune related genes. Values are represented as a fold change from normoxic control group (NC, normoxia control; NK, normoxia ketamine; HC, hypoxia control; HK, hypoxia ketam ine). Statistically significant groups was declared at P <0.05 (two way ANOVA), and Duncan test was performed to determine differences between groups (a, b). Data are presented as means±SEM, and the y axis scale varies between plots.

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68 CHAPTER 4 KETAMINE DECREASES IMMUNE, INFLAMMATORY, AND APOPTOTIC GENE EXPRESSION IN OVINE FETAL HIPPOCAMPUS EXPOSED TO HYPOXIC HYPOXIA Introduction A developing fetus is very susceptible to changes in blood gasses, and solely depends on the maternal circulation for a consta nt supply of oxygen. When oxygen levels are reduced due to episodes of transient and/or mild hypoxia, the fetus has a mechanism of defense involving cardiovascular and neuroendocrine responses. However, when the hypoxic insult becomes more severe or chro nic, it can produce enormous damage and long term detrimental effects to the fetus . Clinically, h ypoxia and anoxia are among the most relevant causes of fetal death and morbidity. The fetal hippocampus, an important area of the brain involved in memory a nd spatial navigation, is largely affected by a hypoxic stimulus ( 150 ) . Recently, damage in the fetal hippocampus has been closely linked to the impairment of cognitive functions, memory, and schizophrenia ( 160 ) . Damage occurs mainly due to the activation of an inflammatory cascade response triggered by the overactiv ation of the NMDA receptors located in the neurons ( 28 ) . Ketamine, a non competitive NMDA receptor antagonist, has been reported to be neuroprotective and have anti inflammatory ef fects by suppressing the production of proinflammatory cytokines. In clinical trials, pre treatment with ketamine , as an aesthetic, has been shown to reduce serum levels of IL 6 and TNF in patients receiving liver transplantations and various cardiac surgical procedures ( 147 , 199 , 203 ) . For in vitro studies, ketamine has also been shown to decrease the gene expressio n of IL6, activated immune cells ( 165 , 190 ) .

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69 Our lab has recently developed transcriptomics methods to analyze the effects of hypoxia on the fetal brain. Pre viously, we have reported that an acute hypoxic stimulus induced an up regulation of inflammatory and immune mediators in the fetal hypothalamus ( 187 ) . Therefore, t he blockade of NMDA receptors and its activation in the fetal brain might contribute to reducing the adverse effects of acute hypoxic damage in the fetal hippocampus thus protecting its integrity, plasticity, and functionality. We hypothesized that ketamine can decrease the expression of proinflammatory markers in t he late gestation ovine fetal hippocampus exposed to acute hypoxia. Materials and Methods These experiments were approved by the University of Florida Animal Care and Use Committee and were performed in accordance with the Guiding Principles for Use of Ani mals of the American Physiological Society. Sixteen chronically catheterized singleton or twin ovine fetuses were studied between the gestational age of 122±5 days (full term=145 147 days). Fetal Surgery The surgical procedures for the chronic catheteriza tion of fetal and mate rnal femoral arteries and veins were described in C hapter 3 of this dissertation and in detail previously ( 136 , 185 ) . The procedures were performed on 116±3 days of gestation. In Vivo Experimental Procedures The detailed description s of the in vivo experiments were described previously in C hapter 2 and 3. The sixteen fetuses were randomly assigned to one of the following groups (n=3 4/group): normoxic control, normoxia+ketamine, hypoxic control, and hypoxia+ketamine .

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70 Microarray Procedures The mRNA w as extracted from snap frozen fetal hippocampus via RNeasy Plus Mini Kit (Qiagen, Valencia, CA). The mRNA integrity number (RIN) values were between 8.0 9. 3 , and were labeled with cyanine 3 CTP with the Quick Amp Labeling Kit (Cat# 5190 0442, Agilent Tech nologies, Santa Clara, CA), according to the ranged from 1 1.7 14.5 and yielded 6.6 10.8 . The cRNA samples were hybridized and processed for one channel Sheep Gene Expres sion Microarray (8 x15 K slide) 8 arrays with 15208 oligomers each (Cat# G4813A 019921, Agilent Technologies, Santa Clara, CA) as described earlier ( 140 , 187 ) . The slides were scanned at the Genomics Division of the University of Flori ary Center for Biotech Research. The analysis of the microarray results were described in C hapter 3 . Real T ime PCR The same mRNA samples that were used to synthesize for the microarray were used to for qPCR validation also . The primers used were designed based on the known Ovis aries and Bos taurus genomes ( T able 3 1 ) for S YBR green. T actin primers and probe were used as a house keeping control ( 187 ) . The relative mRNA expression was cal actin mRNA from the same sample. Calculations and Statistical Analysis Data are presented as mean values standard error of th e mean (SEM) with consideration for statistical significance at P <0.05. The ovine Agilent 15.5k array results

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71 moderated t test using empirical Bayes method for small sample size per group ( P <0.05). Unless stated, the real time PCR data was analyzed by two way ANOVA (time, ±hypoxia; time, ±ketamine) with Mixed Procedure of SAS/STAT® 9.3; corrected for repeated measures in one dimension (time), and if significant, by Duncan post hoc test. Results In the fetal hippocampus, the microarray analysis revealed a total of 565 significantly regulated genes by acute hypoxic stress, and with an anesthetic dose of ketamine before the initial onset of hypoxic stimulus, a total of 255 significantly regulated genes were detected. Of the 565 significantly regulated genes by hypoxia, 266 were up regulated, while 299 were down regulated; and of the 255 significantly regulated genes by the addition of ketamine, 125 were up regulated and 130 were down regula ted (F igure 4 1). Various programs and software were used to analyze the large genomic datasets for determining gene ontology, KEGG pathways, and other functional annotation clustering and network analysis. All the significantly up and down regulated genes for each of the four groups we re analyzed for gene ontology (Table 4 1) and KEGG pathways (T able 4 2). With hypoxic stress, the main biological processes that were up regulated were immune, defense, stress, and cytokine stimulus respo nses, while the KEGG pathways showed an up regulation of cytokine cytokine receptor interaction, hematopoietic cell lineage, adipocytokine signaling pathway, chemokine signaling pathway, toll like receptor signaling pathway, JAK/STAT signaling pathway, B c ell receptor signaling pathway, MAPK signaling pathway, and apoptosis. The biological processes that were down regulated with hypoxia were the regulation of biological

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72 process, cellular metabolic process, and regulation of macromolecule metabolic processe s; and the KEGG analysis indicated a down regulation of pathways in cancer, protein processing in endoplasmic reticulum, splicesome, endocytosis, p53 signaling pathway, Wnt signaling pathways, RNA transport, calcium signaling pathway, focal adhesion, and M APK signaling pathway. However, with pre treatment of ketamine and acute hypoxic stress, the up regulated biological processes were cellular protein metabolic process, protein modification process, tubulin complex assembly, cellular protein modification pr ocess, and the acetylation of peptidyl lysine, histone, amino acid, and protein. The KEGG analysis showed an up regulation of vascular smooth muscle contraction, TGF beta signaling pathway, long term potentiation, gap junction, and amino sugar and nucleoti de sugar metabolism. On the other hand, hypoxia+ketamine indicated a down regulation of biological processes relating to the response to lipid, chemical stimulus, wounding, abiotic stimulus, organic substance, stress, inflammatory response, molecule of ba cterial origin, cytokine stimulus, and lipopolysaccharide. The KEGG pathway showed a down regulation of metabolic pathways, cytokine cytokine receptor interaction, MAPK signaling pathway, chemokine signaling pathway, toll like receptor signaling pathway, lysosome, pathways in cancer, and focal adhesion. The role of ketamine on the fetal hippocampus under hypoxic stress can be determined by comparing the common genes between the set of genes that were significantly up regulated by hypoxia versus the set of genes that were down regulated by hypoxic+ketamine (F igure 4 2). There were 194 up regulated genes due to hypoxic stress, 58 down regulated genes from the addition of ketamine, and 72 common genes

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73 between the both groups. Taking the set of common genes, various biological processes, pathways, and asso ciated diseases were analyzed (T able 4 3). The main biological processes were the response to lipid, organic substance, cytokine stimulus, biotic stimulus, chemical stimulus, organism, lipopolysaccharide, mo lecule of bacterial origin, external stimulus, and stress. The enriched KEGG pathway were cytokine cytokine receptor interaction, toll like receptor signaling pathway, MAPK signaling pathway, adipocytokine signaling pathway, chemokine signaling pathway, o steoclast differentiation, and JAK/STAT signaling pathway. The common pathway analysis indicated the involvement of integrin family cell surface interactions, GMCSF mediated signaling events, internalization of ErbB1, signaling events mediated by VEGFR1 a nd VEGFR2, IL3 mediated signaling events, PDGF receptor signaling network, Syndecan 1 mediated signaling events, Class I PI3K signaling events, plasma membrane estrogen receptor signaling, and PDGFR beta signaling pathway. The associated diseases from the 72 common genes were inflammation, necrosis, and immune system disease. The KEGG and gene ontogeny analysis of the 38 common genes between the significantly down regulated genes by hypoxia versus the significantly up regulated genes by hypoxia+ketamine di d not yield any significant biological processes or pathways ( F igure 4 2B, 4 2D). From the network analysis, these common genes were involved more heavily in pathways activated by the down regulation of hypoxia , then with the pathways stimulated by genes that were significantly up regulated in ketamine. The validation for the microarray and network analysis was performed with real time qPCR ( F igure 4 3). The genes were selected based on the results of significant biological pathways analyzed from the set of 72 common genes ( F igure 4 2). The three

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74 genes selected were caspase 8, myeloid differentiation primary response 88, and nuclear factor kappa light chain enhancer of activated B cells (T able 3 1). Twenty four hours after the initial stimulus of hypoxia , the fetal hippocampus increased gene expression of CASP8, MYD88, and NF B versus the normoxic control by 2.46 fold, 1.56 fold, and 1.46 fold, respectively. However, with the pre treatment of ketamine before hypoxic insult, the gene expression of CASP8 d ecreased from 2.46 to 1.39 fold, MYD88 decreased from 1.56 to 1.24 fold, and NF B decreased from 1.46 to 1.22 fold. When the normoxic fetuses were treated with ketamine, the gene expressions for CASP8, MYD88, and NF B were 1.06 fold, 1.01 fold, and 0.9 7 fold, respectively.

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75 Discussion Elucidating the mechanisms of fetal physiological responses to certain stimuli may require a global approach. Our lab has recently incorporated the transcriptomics approach to compliment our physiological approach in understanding late gestation fetal stress response due to acute hypoxic hypoxia. In previous studies, we found that hypoxia increased the level of fetal plasma ACTH and cortisol, and ketamine was able to partially attenuate the ACTH response (C hapter 2) . H owever, in this study, we are interested in the influence of ketamine on the local genomic response to acute hypoxia in the fetal hippocampus. We found that twenty four hours after the onset of a mild and acute hypoxic stimulus, the genomic response of inflammatory, defense, and stress activators and mediators were up regulated, and the regulation of cellular and metabolic processes were down regulated. However, with the pre treatment of ketamine prior to the hypoxic insult , the genomic responses of metabolic processes, protein modification, and histone acetylation were up regulated ; and the response to inflammatory , stress, and cytokine stimulus were down regulated. During gestation, one of the most common stressor the fetu s encounters is hypoxia. The fetus is able to defend the hypoxic stress for a short period of time by manipulating its neuroendocrine, hemodynamic, and genomic responses. The brain is a vital organ, and when encountering periods of hypoxia, the fetus wil l redistribute its combined ventricular output to maintain cell integrity and viability by conserving the energy require for the normal function of neuronal and glial cells. Due to the dense amount of cell bodies in the brain, and the lack of glucose rese rve, neuronal cells in hypoxic events will need to down regulate or completely shut down their metabolic processes in order to survive. The neurons may also utilize anaerobic metabolism to

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76 support the high energy demand, however an accumulation of lactic acid will cause the development of acidosis in the brain. Our previous study looked at the genomic response of the fetal hypothalamus, just one hour post acute hypoxic stress, and found there was an down regulation of metabolic processes that requires glu cose and oxygen, such as protein biosynthesis ( 187 ) . Similarl y, i n this study we also detected a down regulation of cellular metabolic processes due to hypoxic stress . In addition, we found an up regulation of genes belonging to the GLUT family (SLC2A1, SLC2A3, and SLC2A5). It is known that the fetal response to h ypoxia involves an immediate reduction in glucose uptake, thus, a decrease in glucose consumption. During the hypoxic insult, the glucose production was halted, however, immediately after the hypoxic insult, the glucose production increases ( 77 ) . We propose that during our acute hypoxic insult, the fetal hippocampus may experience a similar cellular response, but twenty four hours after the hypoxic insult, the neurons up regulate the glucose/fructose transporters in order to satisfy the energy demand. The administration of k etamine did not have an effect on the gene expressions of these GLUT transporters, s uggesting that this is a NMDA independent mechanism. T he physiological response to hypoxic stress is the activation of the HPA axis secretion of CRH by the PVN to the pituitary, then the release of ACTH in to the pheripheral circulation where the adrenal cortex secretes cortisol. Cortisol serves as a negativ e feedback mechanism to shut down this pathway. Chronic or frequent activation of the HPA axis may provoke negative feedback dysfunction . This happens when the glucocorticoid receptors, mainly located in the PVN of the hypothalamus and the hippocampus, a re no longer responding to the elevation cortisol levels . In addition,

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77 CRH have been shown to have a proinflammatory role in the activation of microglia cells in the brain , the local immune cells in the brain ( 129 ) . The over activation of microg lia l cells has been reported to induce cytotoxicity, neurodegeneration, and necrosis in ovine fetal hippocampus, periventricular, and subcortical white matter ( 76 ) . M ajor cytokines, such as IL1, TNF , IFN , involved in JAK/STAT, MAPK, ERK, and JNK pathways, may reduce or even inhibit the transcription and /or translocation of glucocorticoid receptors in the brain ( 133 , 172 ) . As a consequence, the negative feedback mechanism of the HPA axis is impaired leading to an elevated production of cortisol and ACTH, aggravating the health status of the fetus. Here, we report that fetal hypoxia up regulated the genomic response of inflammatory, defense, and stress pathways, involving toll like receptors, cytokine and chemokine signaling , MAPK , apoptotic , and JAK/STAT signaling pathway s. The fetal inflammatory response to hypoxic stress , in this study, feature d the up regulation of important inflammatory mediators, such as MYD88, CXCL10, CXCL16, CXCR7, CCL2, CCL4, IL6ST, IL4R, IL1, IL1 , IL17R, NF B IA , and TNF A IP6 , most of them are reported extensively in literature ( 29 , 63 , 163 ) . The up regulation of inflammatory facto rs, some of which were detected in this study, are attributed to have cytotoxic effects on neurons and glial cells ( 170 ) . Likewise, inflammatory cytokines promote the migration and activation of the innate immune cells in the brain, which might aggravate the extent of the hypoxic injury ( 20 , 76 ) . Furthermore, our enriched KEGG pathways analysis revealed a significant up regulation of apoptotic pathways, which include the following genes: CASP7, CASP9, MYD88, and NF B. The activation of apoptosis might be a direct effe ct of calcium influx into neuronal and glial cells, a process

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78 regulated mainly by the caspase pathway ( 169 ) . Several studies support our findings, with an additional up regulation of other important mediators of apoptosis: caspase 3 and Bax/Bcl 2 ( 25 , 101 , 126 , 169 , 173 , 207 ) . T he administration of ketamine , prior to the induction of maternal ventilatory hypoxia, produced a down regulation of many genomic expressions cited by this dissertation . Fetal hypoxic stress, mild or severe, may play a role in influencing the development of the brain. During hypoxic events, one of the most vulnerable region s of the brain is the hippocampus ( 150 ) , more speci fically, in the area s of Cornu A mmonis 1 and subiculum , where the regions are densely packed with pyramidal cells ( 137 ) . The hippocampus plays a role in cognitive functions and spatial navigation, and damage made t o this area of the brain may lead to clinical disorders . The inflammation and apoptotic pathways found in this study, as well as other reports, may induce neuronal damage and necrosis and affect the development of the hippocampus resulting in smaller siz e ( 100 ) . Children who experienced episodes of perinatal hypoxic stress may develop learning problems or neurodegenerative diseases. Clinical reports and meta analysis have demonstrated that there is a relationship between hypoxia and the risk of developing schizophrenia ( 18 ) . Also, in more severe cases like pregnancy and birth complications, the stress on the fetal hippocampus may result in schizophrenia ( 160 ) . In this C hapter, we have shown that ketamine may play a role in preventing cerebral inflammation and apoptosis, which may lead to neuronal damage . In recent studies, ketam ine have been shown to promote neuronal synaptic plasticity and neuronal growth of dendritic spines ( 97 ) . Currently, the debate on whether ketamine is neurotoxic or neuroprotective to a developing brain is still undetermined. Therefore, if

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79 ketamine is to be used clinically in preventing neurological disorders in preterm infants in the NICU, then future studies will need t o take the following factors into consideration: age, dose, and timing.

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80 Figure 4 1. Volcano plot illustrating the relationship of gene expression in fetal hippocampus measured by log 2 of fold change. Significantly up regulated (blue) and down regulated (red) genes under hypoxia (A), hypoxia+ketamine (B), and normoxia+ketamine (C). The dotted line indicates whether the gene is st atistically significant (above), P<0.05, or not significant (below). Non significant genes are indicated in black circles

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81 Table 4 1 . Top 10 gene ontology biological processes that were significantly regulated in the hippocampus during acute hypoxic st ress, with or without ketamine. Groups Biological Processes # Genes involved Adjusted P values Hypoxia Up regulated Immune response 54 1.54E 11 Defense response 55 1.54E 11 Response to organic substance 72 3.76E 11 Response to stress 96 6.68E 11 Response to cytokine stimulus 35 7.85E 11 Immune system process 70 9.62E 11 Response to chemical stimulus 90 2.39E 10 Innate immune response 33 7.06E 09 Response to lipid 34 1.09E 08 Cellular response to cytokine stimulus 28 1.79E 08 Hypoxia Down regulated Regulation of biological process 177 1.00E 04 Cellular metabolic process 186 1.00E 04 Regulation of cellular process 169 1.00E 04 Regulation of metabolic process 120 1.00E 04 Cellular macromolecule metabolic process 153 1.00E 04 Biological regulation 183 2.00E 04 Regulation of macromolecule metabolic process 106 2.00E 04 Organic substance metabolic process 188 3.00E 04 Macromolecule metabolic process 161 3.00E 04 Single organism metabolic process 190 4.00E 04 Hypoxia+ketamine Up regulated Cellular protein metabolic process 35 5.73E 02 Protein modification process 28 5.73E 02 Tubulin complex assembly 2 5.73E 02 Cellular protein modification process 28 5.73E 02 Peptidyl lysine acetylation 5 5.73E 02 Internal peptidyl lysine acetylation 5 5.73E 02 Histone acetylation 5 5.73E 02 Histone H3 acetylation 4 5.73E 02 Internal amino acid acetylation 5 5.94E 02 Protein acetylation 5 7.64E 02 Hypoxia+ketamine Down regulated Response to lipid 23 1.33E 07 Response to chemical stimulus 46 3.35E 05 Response to wounding 27 3.35E 05 Response to abiotic stimulus 22 6.08E 05 Response to organic substance 35 6.08E 05 Response to stress 46 1.00E 04 Inflammatory response 16 2.00E 04 Response to molecule of bacterial origin 11 2.00E 04 Response to cytokine stimulus 16 2.00E 04 Response to lipopolysaccharide 11 2.00E 04

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82 Table 4 2 . E nriched KEGG pathways that were significantly regulated in the hippocampus during acute hypoxic stress, with or without ketamine. Groups Pathway Names # Genes involved Adjusted P values Hypoxia Up regulated Cytokine cytokine receptor interaction 16 5.81E 10 Hematopoietic cell lineage 8 2.55E 06 Osteoclast differentiation 8 3.10E 05 Adipocytokine signaling pathway 6 5.83E 05 Metabolic pathways 20 2.00E 04 Chemokine signaling pathway 8 2.00E 04 Toll like receptor signaling pathway 6 3.00E 04 JAK/ STAT signaling pathway 7 3.00E 04 B cell receptor signaling pathway 5 4.00E 04 MAPK signaling pathway 8 8.00E 04 Apoptosis 5 8.00E 04 Hypoxia Down regulated Pathways in cancer 11 6.00E 04 Protein processing in endoplasmic reticulum 8 6.00E 04 Spliceosome 7 6.00E 04 Endocytosis 8 1.00E 03 p53 signaling pathway 5 1.00E 03 Long term potentiation 5 1.00E 03 Wnt signaling pathway 6 3.30E 03 RNA transport 6 3.40E 03 Calcium signaling pathway 6 5.60E 03 Focal adhesion 6 8.30E 03 MAPK signaling pathway 6 2.53E 02 Hypoxia+ketamine Up regulated Vascular smooth muscle contraction 4 6.90E 03 TGF beta signaling pathway 3 1.15E 02 Long term potentiation 3 1.15E 02 Gap junction 3 1.15E 02 Amino sugar and nucleotide sugar metabolism 2 3.14E 02 Wnt signaling pathway 3 3.14E 02 Calcium signaling pathway 3 4.24E 02 Long term depression 2 4.57E 02 Hypoxia+ketamine Down regulated Metabolic pathways 14 2.00E 04 Cytokine cytokine receptor interaction 7 2.00E 04 Malaria 4 2.00E 04 Renal cell carcinoma 4 4.00E 04 MAPK signaling pathway 6 7.00E 04 Chemokine signaling pathway 5 1.00E 03 Toll like receptor signaling pathway 4 1.00E 03 Lysosome 4 2.00E 03 Pathways in cancer 5 8.30E 03 Focal adhesion 4 8.30E 03

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83 A. B. C. D. Figure 4 2. Venn diagram and network analysis of significant gene expression up regulated by hypoxia, but down regulated by ketamine . (A , B ) Venn diagram s indicating the number of significant up regulated (blue), down regulated (red), and the common genes shared between both groups ( purple). (C, D ) Network analysi s of significantly regulated genes in each group, represented in the same corresponding colors as the Venn diagram above each network. (A, C) Comparison of hypoxia up regulated genes vs hypoxia+ketamine down regulated genes. (B,D) Comparison of hypoxia d own regulated genes vs. hypoxia+ketamine up regulated genes.

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84 Table 4 3 . Top 10 gen e ontology biological processes, enriched KEGG pathways , common pathways, and enriched diseases associated with genes that were significantly up regulated in the hippocampus during acute hypoxic stress, but was down regulated with ketamine. Analysis Pathways, processes, functions, components # Genes involved Adjusted P values Biological Process Response to lipid 18 4.49E 08 Response to organic substance 27 1.49E 06 Response to cytokine stimulus 15 1.49E 06 Response to biotic stimulus 16 1.53E 06 Response to chemical stimulus 33 1.53E 06 Response to other organism 15 4.53E 06 Response to lipopolysaccharide 10 4.53E 06 Response to molecule of bacterial origin 10 6.95E 06 Response to external stimulus 21 1.54E 05 Response to stress 32 1.61E 05 KEGG pathways Cytokine cytokine receptor interaction 7 8.15E 06 Malaria 4 2.32E 05 Toll like receptor signaling pathway 4 2.00E 04 MAPK signaling pathway 5 6.00E 04 Adipocytokine signaling pathway 3 1.20E 03 Chemokine signaling pathway 4 1.50E 03 Amoebiasis 3 3.10E 03 Osteoclast differentiation 3 4.60E 03 African trypanosomiasis 2 5.20E 03 Jak STAT signaling pathway 3 6.50E 03 Common Pathways Integrin family cell surface interactions 24 1.23E 16 GMCSF mediated signaling events 21 3.97E 15 Internalization of ErbB1 21 3.97E 15 Signaling events mediated by VEGFR1 and VEGFR2 21 3.97E 15 IL3 mediated signaling events 21 3.97E 15 PDGF receptor signaling network 21 3.97E 15 Syndecan 1 mediated signaling events 21 3.97E 15 Class I PI3K signaling events 21 3.97E 15 Plasma membrane estrogen receptor signaling 21 3.97E 15 PDGFR beta signaling pathway 21 3.97E 15 Associated Diseases Inflammation 15 1.33E 13 Necrosis 11 9.04E 10 Esophageal Diseases 7 4.14E 07 Stress 10 4.14E 07 Acute Phase Reaction 5 8.66E 07 Immune System Diseases 10 8.58E 06 Joint Diseases 7 1.32E 05 Crohn Disease 6 1.40E 05 Esophageal Neoplasms 6 1.64E 05 Arthritis 7 2.16E 05

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85 Figure 4 3. mRNA gene expression was measured by real time qPCR for apoptotic, inflammatory, and immune related genes. Values are represented as a fold change from normoxic control group (NC, normoxia control; NK, normoxia ketamine; HC, hypoxia control; HK, hypoxia ketamine). Statistically significant groups was declared at P <0.05 (two way ANOVA), and Duncan test was performed to determine differences between groups (a, b, c). Data are presen ted as means±SEM, and the y axis scale varies between plots.

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86 CHAPTER 5 K ETAMINE SUPRESSES INFLAMMATORY RESPONSE IN THE KIDNEY CORTEX OF LATE GESTATION OVINE FETUS EXPOSED TO ACUTE HYPOXIC STRESS Introduction Throughout pregnancy, the fetus is at a constant demand of maintaining homeostasis in the uterine environment. Any amount of stress imposed to the fetus, acute or chronic, can impose programming effect s in the developing organs. Fetal stress occurs when there is an inadequate amount of blood flow or abnormal levels of blood gases to the fetus. Hypoxia is a common fetal stressor that the fetus encounters during gestation . In late gestation, the fetal organs are still maturing, and mild forms of hypoxia or asphyxia can possibly induce a long t erm programming effect, especially to the fetal kidneys. Recently, we have developed transcriptomics methods to elucidate the effect of hypoxia on the developing fetal brain. During acute hypoxia, we have seen an increase in inflammatory pathways in the f etal hypothalamus ( 187 ) , and with ketamine, the inflammatory response was decreased. Ketamine, an N Methyl D aspartate (NMDA) receptor antagonist, is commonly used in veterinary medicine, pediatric surgeries, and neona tal intensive care units (NICU) as an anesthetic and analgestic. Clinically, ketamine have been shown to be an anti inflammatory agent by decreasing serum levels of IL 6 and T NF in patients underwent cardiac surgeries and liver transplantations ( 147 , 199 , 203 ) . In addition, in vitro studies have shown that lipopolysaccharide (LPS) activated macrophages ( 190 ) and glial cells ( 165 ) have a decreased IL6, TNF , and IL1 gene expression when treated with ketamine. Consequently , if hypoxia induces inflammatory response in multiple peripheral fetal

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87 organs besides the brain, then is it possible that ketamine can potentially modulate the adverse inflammatory responses and prevent adverse fetal outcomes. The normal fetal physiologic response to fetal stress, such as umbilical cord occlusion and asphyxia, is shunting the combined ventricular output towards the brain, heart, and adrenals, and away from the kidneys, resulting in potential renal damage ( 4 , 5 , 107 ) . The combination of decrease partial pressure of oxygen and renal blood flow can cause hypoxic and ischemic damage to the kidneys, especially the renal tubules ( 45 , 48 ) . Consequently, a more severe fetal stressor, such as partial umbilical cord occlusion (60 min, pH <6.9) can cause renal tubular necrosis in near term fetuses ( 73 ) , and complete umbilical cord occlusion (30 min, 72 hrs post) can cause renal distal tubule apoptosis in 60% gestation fe tuses ( 127 ) . NMDA receptors are not only found throughout the nervous system, but are also present in various extraneuronal tissues such as the renal cortex and medulla, and in the atrium, ventricles, aorta, and pulmonary artery of the cardiovascular system ( 96 ) . An upregulatio n and activation of renal NMDA receptors were found in ischemia reperfusion and hypoxia reoxygenation induced acute renal injury ( 138 , 198 ) . Over activation of NMDA receptors can evoke significant bursts amounts of reactive oxygen species and calcium ion overload and damage renal tubules, resulting in induction of apoptosis and necrosis of tubular cells, which may contribute to renal dysfunction ( 198 ) . In these acute kidney injury models, various NMDA receptor antagonists like ketamine, were found to attenuate ischemia reperfusion induced injuries and significantly reduce oxidative stress ( 138 ) . Ketamine, by blocking NMDA receptors and their activation, may potentially be beneficial in decreasing the adverse effect of renal damage in the

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88 developing fetal kidneys. Therefore, we hypothesize that ketamine can reduce the expression of renal inflammatory genes in late gestation ovine fetus expose d to acute hypoxia. Materials and Methods These experiments were approved by the University of Florida Animal Care and Use Committee and were performed in accordance with the Guiding Principles for Use of Animals of the American Physiological Society. Six teen chronically catheterized singleton or twin ovine fetuses were studied between the gestational age of 122±5 days (full term=145 147 days). Fetal Surgery Ewes were fasted for 24 hours before surgery, and the fetal ovine surgical procedures were performe d on 116±3 days of gestation. The fetal and maternal femoral arteries and veins chronic catheterization surgical procedures were described in detail previously ( 136 , 185 ) . Briefly, the ewes were given 750 mg ampicillin (Polyflex®, Boehringer Ingelheim 19 VetMedica, Inc., St . Joseph, MO, USA), then anesthetized and intubated with 0.5 2 % isoflurane with oxygen. A set of fetal femoral arterial and venous vascular catheter were surgically placed in the fetal hindlimbs, along with an amniotic fluid catheter. Before the uterus w as sutured closed, 500 mg ampicillin was injected into the amniotic fluid. The ewe received a set of femoral arterial and venous vascular catheter, and a tracheostomy tube to stimulate maternal induced ventilatory hypoxia or hypoxic hypoxia ( 114 ) . A minimum of 5 days of recovery were allowed before experimentation. Daily post operative care included two rectal temperatures, two doses of ampicillin (15 20 mg/kg, IM), and monitored for food consumption, infection, and signs of di stress.

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89 In Vivo Experimental Procedures During the experiments, the ewes were conscious and freestanding in their pens with access to food. The sixteen fetuses were randomly assigned to one of the four groups (n=3 4/group): normoxic control, normoxia+keta mine, hypoxic control, and hypoxia+ketamine. In ketamine treated groups, ketamine (3 mg/kg) was given intravenously, through the fetal femoral venous catheter, 10 min prior to the hypoxic stimulus (30 min). Hypoxia was induced by infusing nitrogen gas di rectly into the maternal tracheostomy tube to suppress maternal pressure of oxygen (P a O 2 ) by 50 %. Consequently, the fetal P a O 2 mimicked the maternal response and was decrea sed by 50 % also (C hapter 2) . To closely monitor the changes in blood gas composi tions (ABL80 Radiometer, Copenhagen, Denmark), both maternal and fetal arterial blood was drawn anaerobically (1 mL) every 10 minutes. The fetuses were sacrificed 24 hrs post initial stimulation of hypoxic stress, and various fetal tissues were snap froze n and stored at 80 °C until future analysis. Microarray Procedures The mRNA was extracted from fetal kidney cortex via RNeasy Plus Mini Kit (Qiagen, Valencia, CA), with mRNA integrity number (RIN) values between 7.7 9.1, were labeled with cyanine 3 CTP wi th the Quick Amp Labeling Kit (Cat# 5190 0442, respectively. The cRNA samples were hybridiz ed and processed for one channel Sheep Gene Expression Microarray (8 x15 K slide) 8 arrays with 15208 oligomers each (Cat# G4813A 019921, Agilent Technologies, Santa Clara, CA) as described earlier ( 140 , 187 ) . The slides were sca nned with Microarray Scanner System (G2505 -

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90 90021, Agilent) and the measured fluorescence was detected and converted using Agilent Feature Extraction 9.1 software at the Genomics Division of the University of esearch. Cytoscape and various plug ins (GeneMANIA, ClusterONE, BiNGO) were used to analyze gene network inference and clustering analysis ( 30 , 153 ) . The functional annotation of gene ontogeny for significantly up and down regulated genes were analyzed via DAVID Bioinformatics Resources 6.7 ( 68 , 69 ) and WEB based GEne SeT AnaLysis Toolkit (WebGestalt) ( 171 , 204 ) . Real T ime PCR The mRNA used to syn thesize for the microarray were used to for qPCR validation. The primers used were designed based on the known Ovis aries and Bos taurus genomes ( T able 5 1) for SYBR actin primers and probe were used ( 187 ) . The relative mRNA expression was calculated by the actin mRNA from the same sample. Calculations and Statistical Analysis Data are presented as mean values standard error of the mean (SEM) with consideration for statistical significance at P <0.05. The ovine Agilent 15.5k array results moder ated t test using empirical Bayes method for small sample size per group ( P <0.05). Unless stated, the real time PCR data was analyzed by two way ANOVA (time, ±hypoxia; time, ±ketamine) with Mixed Procedure of SAS/STAT® 9.3; corrected for repeated measures in one dimension (time), and if significant, by Duncan post hoc test.

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91 Results Acute maternal ventilatory hypoxia induces significant changes in the regulation of gene expressions in the late gestation fetal ovine kidney cortex. From a total of 1475 signif icantly regulated genes, 460 were up regulated and 1015 were down regulated by hypoxia alone ( F igure 5 1). However, ketamine in the presence of hypoxic stimulus, 504 genes were significantly regulated, where 289 genes were up regulated, and 215 genes were down regulated. With ketamine alone, under normoxic condition, the 114 up regulated genes did not produce any statistically significant biological pathways, and the 107 down regulated genes were related with cellular transcription processes (data not sho wn). Analyzing the entire set of statistically regulated genes, the gene ontology analysis showed that hypoxia up regulated immune (108 genes, adj. P value=1.13E 31) and inflammatory (62 genes, adj. P value=1.82E 22) responses ( T able 5 2), and down regulated cellular metabolic pathways (461 genes, adj. P value=2.13E 09). The administration of ketamine under hypoxic stress up regulated metabolic processes (129 genes, adj. P value=2.44E 02), cellular repair, and replication; and down regu lated vascular development, angiogenesis, and response to bacterial and virulent factors. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis showed that hypoxia mainly up regulated pathways involving cytokine cytokine receptor interaction , toll like receptor signaling pathway, and chemokines signaling pathways ( T able 5 3). Hypoxia down regulated pathways involving cell proliferation and embryonic development, such as Wnt signaling cascade. The presence of ketamine under hypoxic stimulus resulted in the up regulation of metabolic pathways, and down -

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92 regulation of toll like receptor and MAPK signaling pathways, and cytokine cytokine receptor interaction. Comparing the hypoxia up regulated genes with the hypoxi a+ketamine down regulated genes will reveal the set of genes that were both sensitive to hypoxia and ketamine; there were 103 genes were found to be shared between both groups ( F igure 5 2A). The network analysis of each sets of genes (hypoxia: 357, hypoxia+ketamine: 112, common genes: 1 03), generated a closely related merged network ( F igure 5 2B). The set of common genes were further analyzed to determine gene ontology and KEGG pathways ( T able 5 4). The top statistically significant KEGG pathways were found to be involved in cytokine c ytokine receptor interaction, and toll like receptor, MAPK, and chemokines signaling pathways. The biological processes with higher significance were ones negatively involved in regulation of cellular processes. The top ranked molecular function terms we re cytokine receptor binding and activity, nucleic acid binding transcription factory activity, and DNA regulation. From the 103 common genes, most were located mainly in the cellular component of nucleus and membrane bounded organelle. To validate the mi croarry network and pathway analysis, real time qPCR was performed on statistically significant expressed genes in the 103 common genes shared by up regulated hypoxia and down regulated hypoxia+ketamine genes (Figure 5 3). The genes selected were involved in inflammatory and immune response pathways (TLR2, NF K B , IL6, IL1 , CSF1, CD14, TNF , BCL3, CCL4, CCL5, CXCL10, IRF1), and mRNA gene expressions were calculated by fold change from normoxia control group

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93 Discussion Utilizing broad systems modeling, we h ave found that 24 hrs post hypoxia, the genomic expressions of immune and inflammatory pathways in the ovine fetal kidney cortex were up regulated , and with the treatment of ketamine, the same genomic expre ssions were down regulated . Ketamine may play an anti inflammatory role, not only in the brain, but in other peripheral organs such as the kidneys, possibly due to secondary effects from the sympathetic neurotransmission mediated via NMDA pathway from the brain, or due to local anti inflammatory actions by local NMDA receptors. Hypoxia is a common event faced by the developing fetus, and in severe cases, it could induce growth retardation of the fetus and fetal organs due to mild ischemia/reperfusion to peripheral organs. In cases of hypoxemia and acidem ia, the redistribution of combined fetal cardiac output can result in decreasing fetal renal blood flow by 20 % ( 32 ) . Hypoxia has also been shown to play an adverse role in fetal renal development by stimulating inflammatory, immune, and even apoptotic response in the fetal kidneys ( 191 ) . In events s uch as h ypoxia /reoxygenation, ischemia/reperfusion, or even asphyxia/ u mbilical cord occlusion, renal damage could happen to fetal kidneys, which may have long term effects on the proper development of renal tubules or vasculature. The NMDA receptors are no t only located in neurons, but are also located in various parts of the renal nephron: glomerulus ( 205 ) , collecting ducts ( 159 ) , epithelial proximal tubular cells ( 14 ) , and podocytes ( 90 ) , which could indicate the importance of NMDA receptors in the regulation of proper renal function. Reports have shown that renal NMDA receptors increase ren al vasodilatation through nitric oxide pathway ( 39 ) ,

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94 thus, displaying the importance of NMDA receptors in its involvement in maintaining proper renal hemodynamics . In models of renal ischemia reperfusion and hypoxia reoxygenation , NMDA receptors were up regulat ed in the kidney cortex and the medulla ( 103 , 138 , 198 ) , and over activation of NMDA receptors is associated with increased ox idative stress and renal damage ( 138 ) . Excessive stimulation of NMDA receptors in the kidney can be toxic and cause apoptosis in proximal tubule like opossum kidney and distal tubule like madine darby caine kidney cells ( 95 ) . In podocytes, t he over stimulation of glutamatergic signaling pathway may weaken the integrity of the glomerular filtration barrier therefore causing glomerular damage ( 90 ) . However, with both in vitro and in vivo models, the adverse effect of excessive activation of NMDA receptors were attenuated with NMDA receptor antagonists ( 95 , 138 , 198 ) . It is known that ischemia/reperfusion causes apoptosis and inflammation in the kidneys. Kruger et. al. shows that during a renal ischemia/reperfusion event, activated caspases trigger an inflammatory response in the kidneys, by increasing IL1 and IL18, which leads to renal ischemia injury ( 116 , 117 ) . Our results show that hypoxia alone induces an up regulation of inflammatory mediators, such as, IL1 , IL18, IL1, IL6, IL8, TNF, TLR2, and TLR4. During hypoxic events, the proximal tubule epithelial cells stimulates toll like recepto r 4 (TLR4) mediated pathways which increases inflammatory ( TNF , IL8 ) , and apopto tic markers (Caspase 3, 8, 9) ( 103 ) . In another study, the increase in TLR4 gene and protein expressions has been shown to correlate wit h the degree of ischemia injury in the kidneys ( 91 ) . In a renal specific TLR4 knocked out mouse model, the endothelial adhesion molecules were not expressed (selectin E, ICAM1), suggesting that TLR4 is one of the main players in recruiting inflammatory

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95 cytokines ( 24 ) . Our data shows that hypoxia up regulates selectin P expression, which has an essential role in the recruitment of inflammatory cells. The result of selectin P expression might be a consequence of up regulation of toll like receptors. Here, we showed that ketamine, a non competitive NMDA receptor antagon ist, was able to decrease fetal renal inflammation and immune response to acute hypoxia. By finding the common set of genes that is both up regulated by hypoxia and down regulated by hypoxia+ketamine, we are able to elucidate the action of ketamine on the fetal renal cortex under hypoxic stimulus. With a list of 103 common genes (cytokines, chemokines, interleukins, glucose transporters), we analyzed common pathways and biological processes and found that the most significant pathways were involved in inf lammatory, immune, and metabolism. One study demonstrated that in normal conditions, the ovine fetal kidneys reabsorbs glucose and releases it into the bloodstream; however, under acute hypoxic conditions, the kidneys modifies the renal carbohydrate metab olism in order to utilize glucose in the local cells, due to the decrease in blood flow, thus, releasing lactate into the bloodstream ( 74 ) . Our finding also supports that during hypoxia, local glucose transporters (SLC2A1, SLC2A2) are up regulated, but with ketamine the expression was suppressed. Furthermore, we found that hypoxia increases the up regulat ion of SLC93A, gene responsible for the production of NHE3 (sodium/hydrogen exchanger), most likely due to the acidosis state of hypoxia, and with ketamine, this expression was down regulated. Ketamine seems to attenuate the severity of fetal response to hypoxic stimulus. By using ketamine and blocking NMDA receptors, we could reduce the consequences of hypoxic stress in the fetal kidneys . In humans, the kidneys are

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96 in crease throughout life. The potential impact of the optimal development of kidneys is important in the fetal adult life. The current trend in the rise of cardiovascular and metabolic diseases could be explained by the consequences of adverse fetal progra mming due to fetal stress, such as hypoxia. Hypoxic stress during gestation could alter local renal hemodynamic regulation, which could result in adverse programming effects on the cardiovascular system ( 54 ) . The renal system is an important regulator of blo od pressure, and poor neonatal outcomes due to kidney injury might be one of the causes resulting in the current increasing incidences of adult hypertension . O ur results strongly suggest that ketamine could be use clinically in the neonatal intensive care unit as a preventative measure to reduce adverse renal inflammatory response or damage in newborns undergoing surgical procedures or preterm infants.

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97 Table 5 1 . Primers for real time PCR validation. Official Symbol Gene Name Forward Primer Reverse Primer Species TLR2 Toll like receptor 2 GATTCTGCTGGAGC CCATTG TCATGATCTTCCGCA GCTTACA Ovis aries NF B Nuclear factor kappa light chain enhancer of activated B cells TCCCACAGATGTTCA CAAACAGT GACGCTCAATCTTCA TCTTGTGAT Ovis aries IL 6 Interleukin6 ATGCTTCCAATCTGG GTTCAA TCCAGAAGACCAGC AGTGGTT Ovis aries IL 1 Interleukin1 CGTGGCCATGGAGA AGCT GGTCATCATCACGG AAGACATGT Ovis aries CSF1 Macrophage Colony Stimulating Factor GACTGGAACATTTTC AGCAAGAACT TCAGGCTTGGTCAC CACATC Bos taurus CD14 Monocyte/macrophage surface antigens CCTAAAGGACTGCC GACCAA GCGGCTCCCTGCTT AGCT Ovis aries TNF Tumor Necrosis Factor CCCTTCCACCCCCTT GTT ATGTTGACCTTGGTC TGGTAGGA Ovis aries BCL3 B Cell CLL/Lymphoma 3 CATGGAACACCCCC TGTCA GGCGTATCTCCATCC TCATCA Ovis aries CCL4 Chemokine Ligand 4 TCTTACACCCTGCG GCAGAT GGCTGCTGGTCTCG TAGTAGTCA Ovis aries CCL5 Chemokine Ligand 5 CCAGCAGCAAGTGC TCCAT CGCACACCTGACGG TTCTT Ovis aries CXCL10 Chemokine Ligand 10 TTGAACTGATTCCTG CAAGTCA TTCCTTTTCATTGTG GCAATAATCT Ovis aries IRF1 Interferon Regulatory Factor 1 CCCAGGGCTGATCT GGATTA GCGTGCTTCCATGG GATCT Ovis aries

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98 Figure 5 1 . Volcano plot illustrating the relationship of gene expression in fetal kidney cortex measured by log 2 of fold change. Significantly up regulated (blue) and down regulated (red) genes under hypoxia (A), hypoxia+ketamine (B), and normoxia+ketamine (C). The dotted line indicates whether the gene is statistically significant (above), P <0.05, or not significant (below). Non significant genes are indicated in black circles .

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99 Table 5 2 . Top 10 g ene ontology biological processes that were significantly regulated in the kidney cortex during acute hypoxic stress, with or without ketamine. Group s Biological Processes # Genes involved Adjusted P values Hypoxia Up regulated Immune response 108 1.13E 31 Immune system process 133 1.11E 26 Defense response 101 4.53E 26 Inflammatory response 62 1.82E 22 Regulation of immune system process 82 1.10E 21 Response to other organism 63 5.05E 19 Innate immune response 60 1.01E 18 Response to biotic stimulus 63 5.32E 18 Regulation of defense response 52 7.66E 18 Regulation of response to stimulus 136 8.40E 18 Hypoxia Down regulated Cellular macromolecule metabolic process 461 2.13 E 09 Macromolecule metabolic process 483 1.92 E 07 Nucleic acid metabolic process 322 1.92 E 07 Chromosome organization 78 1.92 E 07 Regulation of macromolecule metabolic process 305 1.83 E 07 Cellular component organization 302 1.83E 07 Cellular component organization at cellular level 256 1.68 E 07 Regulation of nitrogen compound metabolic process 266 1.68 E 07 Peptidyl lysine acetylation 26 1.68 E 07 Regulation of nucleobase containing compound metabolic process 263 1.12 E 07 Hypoxia+ketamine Up regulated Cellular macromolecule metabolic process 129 2.44 E 02 Response to DNA damage stimulus 23 2.44 E 02 Organelle organization 51 4.44 E 02 Chromosome organization 23 4.44 E 02 Double strand break repair 8 4.44 E 02 DNA dependent DNA replication 8 4.26 E 02 Cell cycle checkpoint 12 5.71 E 02 Cellular protein metabolic process 69 5.83 E 02 S phase 9 5.83 E 02 Proteasome regulatory particle assembly 2 5.83 E 02 Hypoxia+ketamine Down regulated Negative regulation of biological process 92 1.07 E 13 Negative regulation of cellular process 87 1.07 E 13 Vasculature development 34 2.59 E 12 Response to lipopolysaccharide 23 2.59 E 12 Blood vessel development 33 2.71 E 12 Blood vessel morphogenesis 31 2.80 E 12 Response to molecule of bacterial origin 23 5.43 E 12 Positive regulation of biological process 92 1.24 E 11 Angiogenesis 27 3.28 E 11 Response to chemical stimulus 79 5.06 E 11

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100 Table 5 3 . Top 10 enriched KEGG pathways that were significantly regulated in the kidney cortex during acute hypoxic stress, with or without ketamine. Groups Pathway Names # Genes involved Adjusted P values Hypoxia Up regulated Cytokine cytokine receptor interaction 30 3.39E 20 Osteoclast differentiation 23 3.39E 20 Toll like receptor signaling pathway 21 7.00E 20 Chagas disease (American trypanosomiasis) 18 1.08E 15 NOD like receptor signaling pathway 14 2.06E 14 Malaria 13 8.56E 14 MAPK signaling pathway 23 1.96E 13 Cytosolic DNA sensing pathway 13 2.43E 13 Rheumatoid arthritis 15 4.55E 13 Chemokine signaling pathway 19 1.69E 12 Hypoxia Down regulated Pathways in cancer 45 1.85E 20 Chronic myeloid leukemia 14 3.89E 08 Melanogenesis 16 3.89E 08 Wnt signaling pathway 19 3.93E 08 Pancreatic cancer 13 1.12E 07 TGF beta signaling pathway 14 1.12E 07 Colorectal cancer 12 1.84E 07 Ubiquitin mediated proteolysis 17 1.84E 07 Cell cycle 16 2.74E 07 Mismatch repair 8 3.03E 07 Hypoxia+ketamine Up regulated Metabolic pathways 23 4.89E 05 Nucleotide excision repair 4 6.20E 03 Mismatch repair 3 8.30E 03 DNA replication 3 1.86E 02 SNARE interactions in vesicular transport 3 1.86E 02 Drug metabolism other enzymes 3 2.89E 02 mTOR signaling pathway 3 2.89E 02 Pyrimidine metabolism 4 2.89E 02 Melanogenesis 4 2.89E 02 Calcium signaling pathway 5 3.16E 02 Hypoxia+ketamine Down regulated Osteoclast differentiation 15 8.21E 15 Pathways in cancer 19 1.54E 13 Leishmaniasis 11 1.74E 12 Rheumatoid arthritis 11 1.89E 11 Toll like receptor signaling pathway 11 4.52E 11 Malaria 9 4.52E 11 Chagas disease (American trypanosomiasis) 11 4.81E 11 MAPK signaling pathway 15 5.80E 11 NOD like receptor signaling pathway 8 3.77E 09 Cytokine cytokine receptor interaction 12 6.88E 08

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101 A. B. Figure 5 2. Venn diagram and network analysis of significant gene expression up regulated by hypoxia, but down regulated by ketamine. (A) Venn diagram showing the number of significant genes that was up regulated by hypoxia (blue), down regulated with ketamine (red), and the common genes involved in both groups (purple). (B) Network analysis of significantly regulated genes in each group are represented in the same corresponding colors (hypoxia blue, hypoxia+ketamine red, common genes purple).

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102 Table 5 4 . Top 10 g ene o ntology biological processes and enriched KEGG pathways that were significantly up regulated in the kidney cortex during acute hypoxic stress, but was down regulated with ketamine. Analysis Pathways, processes, functions, components # Genes involved Adjusted P values KEGG pathways Osteoclast differentiation 23 3.39E 20 Cytokine cytokine receptor interaction 30 3.39E 20 Toll like receptor signaling pathway 21 7.00E 20 Chagas disease (American trypanosomiasis) 18 1.08E 15 NOD like receptor signaling pathway 14 2.06E 14 Malaria 13 8.56E 14 MAPK signaling pathway 23 1.96E 13 Cytosolic DNA sensing pathway 13 2.43E 13 Rheumatoid arthritis 15 4.55E 13 Chemokine signaling pathway 19 1.69E 12 Biological Process Negative regulation of biological process 56 5.55E 13 Negative regulation of cell communication 25 1.31E 10 Negative regulation of cellular process 54 5.52E 13 Negative regulation of response to stimulus 28 2.03E 11 Negative regulation of signal transduction 24 2.25E 10 Negative regulation of signaling 25 1.31E 10 Regulation of cell communication 42 1.09E 10 Regulation of response to stimulus 46 3.32E 11 Regulation of signal transduction 39 1.31E 10 Regulation of signaling 42 1.09E 10 Molecular Function Protein binding 72 4.08E 07 Cytokine receptor binding 11 5.41E 06 Cytokine activity 9 2.00E 04 Nucleic acid binding transcription factor activity 19 3.00E 04 Sequence specific DNA binding transcription factor activity 19 3.00E 04 Regulatory region DNA binding 10 8.00E 04 Regulatory region nucleic acid binding 10 8.00E 04 Interleukin 1 receptor binding 3 1.00E 03 Sequence specific DNA binding 14 1.00E 03 RNA polymerase II core promoter proximal region sequence specific DNA binding transcription factor activity 5 2.70E 03 Cellular Component Cytosol 31 2.00E 04 I kappaB/NF kappaB complex 3 2.00E 04 Extracellular space 15 3.00E 03 Cell surface 10 9.80E 03 Extracellular region part 16 9.80E 03 External side of plasma membrane 6 2.03E 02 Nucleus 48 2.09E 02 Intracellular part 81 2.29E 02 Intracellular 82 2.98E 02 Membrane bounded organelle 67 3.05E 02

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103 Figure 5 3. mRNA gene expression was measured by real time qPCR for inflammatory and immune related genes. Values are represented as a fold change from normoxic control group (NC, normoxia control; NK, normoxia ketamine; HC, hypoxia control; HK, hypoxia ketamine). Stat istically significant groups was declared at P<0.05 (two way ANOVA), and Duncan test was performed to determine differences between groups (a, b, c). Data are presented as means±SEM, and the y axis scale varies between plots.

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104 CHAPTER 6 CONCLUSIONS The studies performed in this dissertation have elucidate d some of the beneficial roles of ketamine on late gestation ovine fetal brain and kidneys exposed to acute hypoxic stress. We incorporated whole animal, cellular, molecular, and genomic techniques to examine the changes in genomic profile and physiological effect of ketamine on fetal hypoxic stress. By utilizing a global approach , we uncovered major pathways associated with immune, inflamm ation, and apoptosis that are highly expressed w ith acute hypoxic hypoxia in both the fetal brain and kidneys. With the administration of ketamine, both the physiological and genomic profile of acute hypoxia was attenuated. The neuroprotective role of ketamine on the fetal brain may assist in improvin g adverse clinical outcomes in premature infants in the NICU. Fetal Programming : Long Term Effect of Fetal Stress In this dissertation, we have demonstrated that hypoxia is a mild, yet powerful stressor that induces a multi systemic fetal response. This g lobal fetal response is characterized by changes in hemodynamics, neuroendocrine, and genomic mechanisms. The hemodynamics response to a hypoxic insult, involves decrease in heart rate, and an increase in mean arterial blood pressure, which are mainly med iated by the chemo and baroreceptors. The neuroendocrine response consists of a rise in plasma ACTH and cortisol levels due to an activation of the fetal HPA axis. The major changes in genomic profile include an up regulation of inflammatory markers, and glucose/sodium transporters; and a down regulation of various cellular metabolic processes. We found that with pre treatment of ketamine, most of these responses were attenuated . H owever, due to the neurotoxic and adverse side effects of ketamine

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105 ( 195 ) , more work is needed to confirm these results if ketamine is to be use for therapeutic purposes on neonates. F etal stress during gestation has significant influence over the normal fetal development, and may permanently alter the metabolic and cardiovascular system s and promote fetal origins of adult disease ( 37 , 105 ) . In studies by Dr. David J. P. Barker , he found that there is a correla tion between low birth weight /poor nutrition to greater risks of developing cardiovascular diseases ( 6 , 7 ) . His work in fetal development and the epidemiology of cardiovascular diseases prompted a new idea in the field of fetal physiology s provided the scientific community a new angle and awareness to view fetal development as a probable cause of adult diseases, a concept called fetal programming. Hypoxia is a common fetal stressor, and based on the results gathered from this dissertation, it might have similar propensity to initiate origins of adult diseases, like what Dr. Barker found with poor nutrition and low birth weight. In our hypoxic hypoxia model, we found that the fetal brain and kidneys experiences dramatic immune response to acute hypoxia. The level of injury i n these tissues may be reflected later in life when confronted by a second challenge. The brain and the kidneys play a major role in the regulation of normal function of cardiovascular physiology . Damage to these organs, prematurely, may compromise the f uture health of the fetus. This hypothesis might explain why there is a high incidence of metabolic and cardiovascular diseases in the population nowadays. The current leading cause of death worldwide is cardiovascular disease resulting in 17 million dea ths, with ischemic heart disease as the

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106 main contributor resulting in seven million deaths, reported by the World Health Organization in July 2013. Fetal hypoxia might be the key to understanding why certain people are more likely to develop secondary hyp ertension, neurological , or even primary/ essential hypertension . Another aspect to consider is the inflammation initiated by hypoxic stress in the fetal brain, which might induce infant death or cause long term neurological disabilities. In both human an d animals, severe fetal stress is linked to abnormal cognitive, behavioral, and psychosocial outcomes ( 84 ) . There is an increase in children and teenagers diagnosed with autism and a ttention d eficit h yperactivity d isorder, reported by CDC. With an up regulation of inflammatory markers in the fetal brain, the damage induced by hypoxia might be the cause of these adverse neurological diseases. Ketamine might be a good candidate in reducing the neuronal inflammatory damage produced by hypoxia. However, more studies should be conducted in order to assess the health status of the neonate after a period of hypoxic insult after parturition. The fetal kidneys are also vulnerable to hypoxic stress. Our results suggest that inflammation induced by hypoxia could cause injury or apoptosis in the fetal glomeruli or the entire nephron. Hypoxic d amage to the kidneys , especially during the period of organogenesis , ( 59 ) , which is reduced formation and number of ne phron s . Since each individual is born with a set number of nephron, and the number may vary over a range of 10 fold, people with lower nephron number are more likely to develop renal diseases and develop hypertension ( 41 ) . It has been reported that there is a close association between low birth weight and glomerular numbers ( 71 , 110 ) , but the correlation to hypertension is yet to be confirmed ( 8 ) .

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107 Perhaps administration of ketamine to preterm infants, during hypoxic events may, decrease susceptib ility to kidney diseases, or cardiovas cular diseases. Future Directions Our results confirm the cons equences of acute hypoxia in late gestation fetuses. We have demonstrated that a hypoxic stimulus provokes a global fetal response involving hemodynamics, neuroendocrine, inflammatory, apoptotic, and metabolic mechanisms . The administration of ketamine p rior to the hypoxic stimulus was able to attenuate responses that might be detrimental to the fetus in later life. This dissertation only focused on the fetal frontal cortex, hippocampus, and kidney cortex; however, more studies should be done on other re levant fetal organs: liver, heart, spleen, bone marrow, pancreas, and thymus . This will lead to the understanding of local and peripheral immune response as a whole, embarked by the fetus under hypoxic stress, and to discover how ketamine is able to inf lu ence these responses. Fu ture work should be focused on a better understanding of the relationship between fetal hypoxic stress and the development of cardiovascular and metabolic disease s . T he long term consequences of hypoxic stress can be performed by analyzing the cognitive behaviors and physiological outcomes of newborn or juvenile lambs. For example, some of the cognitive behaviors we can observe are the time for a newborn lamb to stand and start nursing. In addition, telemetry devices can be impla nted in lambs to study postnatal cardiovascular parameters. Lastly, we can detect epigenetic outcomes by analyzing specific DNA methylation s, histone modifications, and non coding RNA associated genes. However, the ultimate goal for these studies is for researchers and clinicians to provide successful parturitions and increase the well being of preterm babies in later life.

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108 LIST OF REFERENCES 1. Amano M, Asari T, Kubo T , Excitatory amino acid recept ors in the rostral ventrolateral medulla mediate hypertension induced by carotid body chemoreceptor stimulation . Naunyn Schmiedebergs Arch Pharmacol 349: 549 554, 1994. 2. Anderson PJ , Neuropsychological outcomes of children born very preterm . Semin Fetal Neonatal Med 19: 90 96, 2014. 3. Aspinall RL, Mayor A , A prospective randomized controlled study of the efficacy of ketamine for postoperative pain relief in children after adenotonsillectomy . Paediatr Anaesth 11: 333 336, 2001. 4. Ball RH, Parer JT, Ca ldwell LE, Johnson J , Regional blood flow and metabolism in ovine fetuses during severe cord occlusion . Am J Obstet Gynecol 171: 1549 1555, 1994. 5. Ball RH, Espinoza MI, Parer JT, Alon E, Vertommen J, Johnson J , Regional blood flow in asphyxiated fetuses with seizures . Am J Obstet Gynecol 170: 156 161, 1994. 6. Barker DJ , The fetal origins of adult hypertension . J Hypertens Suppl 10: S39 44, 1992. 7. Barker DJ, Bull AR, Osmond C, Simmonds SJ , Fetal and placental size and risk of hypertension in adult li fe . BMJ 301: 259 262, 1990. 8. Barker DJ, Osmond C, Golding J, Kuh D, Wadsworth ME , Growth in utero, blood pressure in childhood and adult life, and mortality from cardiovascular disease . BMJ 298: 564 567, 1989. 9. Beilin B, Rusabrov Y, Shapira Y, Roytbl at L, Greemberg L, Yardeni IZ, Bessler H , Low dose ketamine affects immune responses in humans during the early postoperative period . Br J Anaesth 99: 522 527, 2007. 10. Bhutta AT , Ketamine: A controversial drug for neonates . Semin Perinatol 31: 303 308, 2007. 11. Blanco CE, Dawes GS, Hanson MA, McCooke HB , The response to hypoxia of arterial chemoreceptors in fetal sheep and new born lambs . J Physiol 351: 25 37, 1984. 12. Boddy K, Dawes GS, Fisher R, Pinter S, Robinson JS , Foetal respiratory movements, electrocortical and cardiovascular responses to hypoxaemia and hypercapnia in sheep . J Physiol 243: 599 618, 1974.

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126 BIOGRAPHICAL SKETCH Eileen I Ling Chang was born in Taipei, Taiwan. When she was five years old, her family moved to Rancho Palos Verdes, Califor nia, USA. She attended P oint Vicente Elementary S chool and Palos Verdes Intermediate School , where she was involved in many extracurricular activities including the Girl Scouts of America , club sports, and art classes. Her family also went on many mission trips to Mexico and Thailand. It was on a trip to the border of Thailand and Burma, also known as the den Triangle , where she witnessed wounded soldiers and child soldiers with missing limbs made her rea lize d the importance of making medicine available in rural areas. This experience impacted her world view and kindled a passion to study medicine and help communities in need. After five years of living by the Pacific Ocean, her family moved to Atlanta, Georgia . She attended Landmark Christian High School, where she excelled in both academics and athletics , and was involved in performing arts. During all four spring breaks, she traveled with medical mission teams to Belize and Venezuela , to serve those who require d medical attention but were unable to afford them. She then attended Georgia Institute of Technology, and decided to immerse herself in research. She joined her undergraduate career , where the primary goal of her project was to help develop a chemical complementation system in Saccharomyces cerevisiae , which would aid gene therapy in cancer research. She was awarded the Georgia Institute of Technology ward to conduct this research and attended regional and national conferences . Her mentor, Dr. Bahareh Azizi, and other graduate students in the laboratory encouraged her to pursue a career in research. She enjoyed

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127 and excelled in the challenging but rewarding research environment . After graduating with a degree in Bachelor of Science in c hemistry b iochemistry track, she worked as a technician for a year. She then decided to do a year of post baccalaureate premedical program at Drexel University in Philadelphia, Pennsy lvania where she fell in love with the subject of physiology and anatomy. She then accepted a position as a research specialist at Medical University of South Carolina with Dr. Francis G. Spinale , in the Division of Cardiothoracic Surgery. The main goal of the research group was to study the mechanism of heart failure and other forms of cardiovascular diseases. The experience proved to be valuable because she received multidisciplinary training by working in both murine physiology and biochemistry laboratories. T his unique opportunity allowed her to be independent and execute entire research protocols from setting up the animal models to statistical analyses on the data obtained from biochemical experiments. She quickly learned and mastered specialized techniques in murine cardiovascular surgeries, such as induction of myocardial infarction and myocardia l delivery of viral constructs. With encouragements from various faculties , mentors , and equipped with experiences in research, she decided to pursue a graduate degree in medical science s . For her doctoral graduate education, she applied and was accepted to the Interdisciplinary Program in Biomedical Sciences at the University of Florida Colle ge of Medicine. During her first year of laboratory rotations, she wanted to challenge herself and found a unique laboratory where she learned to work with large animals and mastered unique fetal surgical techniques. Her main doctoral project involved in elucidating the effects of ketamine on fetal neuroendocrine and hemodynamic

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128 response s under hypoxic stress. In the Wood l ab oratory , she gained valuable experiences and scientific foundation of knowledge in the field of fetal physiology and programming. Through these research experiences, she has developed pertinent skills in problem solving and critical thinking needed to be a productive postdoctoral researcher. With all her research experiences, she feels fortunate to be exposed to a wide variety of bi omedical disciplines including biochemistry, molecular biol ogy, genetics, and physiology. E ileen h a s Health and Science University for her post doctoral training. She hopes to further her knowledge and expe rtise in basic science and translational research in the field of fetal cardiovascular programming .