Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Impact of repeated invasive procedures during neonatal intensive care on brain microstructure, growth,… Vinall, Jillian Frances 2015

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2015_november_vinall_jillian.pdf [ 5.35MB ]
Metadata
JSON: 24-1.0166783.json
JSON-LD: 24-1.0166783-ld.json
RDF/XML (Pretty): 24-1.0166783-rdf.xml
RDF/JSON: 24-1.0166783-rdf.json
Turtle: 24-1.0166783-turtle.txt
N-Triples: 24-1.0166783-rdf-ntriples.txt
Original Record: 24-1.0166783-source.json
Full Text
24-1.0166783-fulltext.txt
Citation
24-1.0166783.ris

Full Text

	   IMPACT OF REPEATED INVASIVE PROCEDURES DURING NEONATAL INTENSIVE CARE ON BRAIN MICROSTRUCTURE, GROWTH, NEURODEVELOPMENT AND BEHAVIOR IN CHILDREN BORN VERY PRETERM  by  Jillian Frances Vinall  B.A., York University, 2008    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  	  DOCTOR OF PHILOSOPHY  in  The Faculty of Graduate and Postdoctoral Studies  (Neuroscience)     THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)  October 2015    © Jillian Frances Vinall, 2015        	   ii ABSTRACT  Background As part of their lifesaving care in the neonatal intensive care unit (NICU), infants born very preterm (between 24 and 32 weeks gestation), undergo frequent invasive procedures that induce pain and stress, during a period of rapid brain development. We examined whether repeated exposure to invasive procedures was associated with altered brain development, and thereby poorer neurodevelopmental and behavioral outcome in children born very preterm. We also explored whether parent interaction moderates long-term effects of invasive procedures on child behavior.   Methods Data were collected from two prospective cohorts of infants born ≤32 weeks gestation between February 2001–July 2004 and March 2006–January 2009. Neonatal data were recorded from birth to term-equivalent age. Infants in the 2006–2009 cohort were scanned sequentially, once near birth and again at term-equivalent age. Infants in the 2001–2004 cohort were followed-up at 18 months corrected age (CA), and again at 7.5 years of age, when they underwent an MRI. At 18 months CA, parents of the 2001–2004 cohort completed questionnaires and participated in a recorded play session with their child, from which the parent-child interaction was later coded. All statistical analyses were adjusted for known neonatal and clinical confounders.  Results In a series of 4 studies, greater exposure to invasive procedures in the NICU was associated with slower postnatal body and head growth, and slower growth was associated with delayed cerebral cortical maturation. Among the preterm children exposed to a higher number of invasive procedures, more positive parental interaction was associated with fewer anxious/depressive behaviors reported at 18 months CA. Furthermore, greater exposure to invasive procedures was related to poorer white matter maturation at 7.5 years, and together these factors predicted lower IQ.  Conclusion Greater exposure to invasive procedures was associated with slower body and head growth, altered brain maturation and poorer outcomes, after adjustment for clinical confounders. It is necessary that pain management strategies be evaluated for the extent that they are brain protective, in order to minimize the long-term impact of ongoing pain/stress in the NICU. Furthermore, interventions should address the parent-child relationship in order to improve later outcomes.         	   iii PREFACE   This thesis entitled, “Impact of repeated invasive procedures during neonatal intensive care on brain microstructure, growth, neurodevelopment and behavior in children born very preterm,” summarizes the research I conducted during my 6 years in the Graduate Program in Neuroscience at the University of British Columbia, under the supervision of Professors Ruth E. Grunau and Steven P. Miller. The University of British Columbia/Children’s and Women’s Health Centre of British Columbia Research Ethics Board approved these studies (certificate numbers for each of the studies listed below: H08-00125, H05-70579, H01-70017, H06-03696, respectively), and parents provided written informed consent. Child assent was obtained when children were seen at 7.5 years of age. Neonatal brain imaging and neurodevelopmental outcome data was acquired from 2 prospective cohorts of infants admitted to the neonatal intensive care unit at the British Columbia’s Children’s and Women’s Hospitals between February 2001 – July 2004, and March 2006 – January 2009. Results from this data led to four first author publications.  A version of Chapter 2 has been published in Vinall J, Miller SP, Chau V, Brummelte S, Synnes A, Grunau RE (2012). Neonatal pain in relation to postnatal growth in infants born very preterm. Pain, 153(7):1374-81. I conducted the statistical analyses and drafted the manuscript. Professors Grunau and Miller conceptualized and designed the study. All authors contributed to the interpretation of the data and provided critical review of the manuscript for publication.   A version of Chapter 3 has been published in Vinall J, Grunau RE, Brant R, Chau V, Poskitt KJ, Synnes AR, Miller SP (2013). Slower postnatal growth is associated with delayed cerebral cortical maturation in preterm newborns. Science Translational Medicine, 5:168ra8. I identified and acquired diffusion parameters from 8 cortical regions of interest on the scans of 95 infants born very preterm that underwent MRI imaging at approximately 32 and 40 weeks postmenstrual age. In addition, I contributed to the statistical analyses, and drafted the manuscript. Professors Miller and Grunau conceptualized and designed the study. Drs. Poskitt and Chau contributed to the acquisition of data. Professor Rollin Brant conducted the linear mixed effect modeling. All authors contributed to the interpretation of the data and provided critical review of the manuscript for publication.   A version of Chapter 4 has been published in Vinall J, Miller SP, Synnes AR, Grunau RE (2013). Parent behaviors moderate the relationship between neonatal pain and internalizing behaviors at 18 months corrected age in children born very prematurely. Pain, 154(9):1831-39. I introduced the concept of Emotional Availability to the Grunau and Miller labs and applied the Emotional Availability Scale to 145, 5-minute recordings of parent-child interactions. I conducted the statistical analyses, and drafted the manuscript. Professor Grunau conceptualized and designed the overall study. I interpreted the results of the parent-child interaction data, and all of the authors provided critical review of the manuscript for publication.  	   iv A version of Chapter 5 has been published in Vinall J, Miller SP, Bjornson BH, Fitzpatrick KPV, Poskitt KJ, Brant R, Synnes AR, Cepeda IL, Grunau RE (2014). Invasive procedures in preterm children: brain and cognitive development at school age. Pediatrics,133(3):412-21. I identified and acquired diffusion parameters from 7 white matter regions of interest on the scans 50 children very preterm that underwent MRI imaging at 7 years of age. With the help of Professor Rollin Brant, I conducted the statistical analyses. Moreover, I drafted the manuscript. Professor Ruth Grunau generally conceptualized the study. The white matter regions of interest were acquired based on previous publications by Professor Steven Miller, and Professor Miller advised the analysis and interpretation of the brain imaging data. Drs. Bjornson and Poskitt, as well as Mr. Fitzpatrick contributed to the acquisition of data. All authors contributed to the interpretation of the data and provided critical review of the manuscript for publication.                                   	   v TABLE OF CONTENTS ABSTRACT ........................................................................................................................ ii PREFACE .......................................................................................................................... iii  TABLE OF CONTENTS .................................................................................................... v LIST OF TABLES ........................................................................................................... xiii LIST OF FIGURES .......................................................................................................... xv LIST OF ABBREVIATIONS .......................................................................................... xvi GLOSSARY ................................................................................................................... xvii ACKNOWLEDGEMENTS ........................................................................................... xviii DEDICATION ................................................................................................................. xix CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW .............................. 1 1.1 Preterm Birth ............................................................................................................. 1 1.2 Invasive Procedures during Neonatal Care ............................................................... 2 1.3 History of Pain Management in the NICU ............................................................... 2 1.4 Pain Transmission and Modulation .......................................................................... 3 1.4.1 Transduction, Transmission and Central Nociceptive Processing ..................... 4     1.5 Cortical Processing of Centrally Transmitted Nociceptive Activity ........................ 6 1.5.1 Imaging Responses to Noxious Stimuli in Adults ............................................. 7 1.5.2 Imaging Responses to Noxious Stimuli in Infants and Children ....................... 8     1.6 Supraspinal Descending Control Pathways ............................................................ 10 1.7 Pain Processing in Infants Born Very Preterm ....................................................... 11 1.8 Behavioural and Physiological Responses to Noxious Stimuli in the NICU ......... 12 1.8.1 Behavioral Responses to Invasive Procedures ................................................. 12 	   vi 1.8.2 Physiological Responses to Invasive Procedures ............................................ 13 1.9 Cortical Responses to Invasive Procedures in the NICU ........................................ 14 1.10 Vulnerability of the Developing Brain ................................................................. 15 1.11 Inflammation and Invasive Procedures in the NICU ............................................ 17 1.12 Oxidative Stress and Invasive Procedures in the NICU ....................................... 19 1.13 Brain Injury in Infants Born Very Preterm ........................................................... 20 1.13.1 Diffusion Tensor Imaging .............................................................................. 20 1.14 Invasive Procedures in the NICU and Stress ........................................................ 22 1.14.1 Stress Systems ................................................................................................ 22 1.15 Neonatal Stress and Growth .................................................................................. 23 1.16 Neonatal Stress and Immune Function ................................................................. 24 1.17 Neonatal Stress and Brain Development .............................................................. 25 1.18 Repeated Exposure to Invasive Procedures in the NICU and the Developing          Brain ...................................................................................................................... 27 1.19 Early Exposure to Stress and Cognitive, Motor and Behavioral Outcomes ......... 28 1.20 Caregivers and Stress Management ...................................................................... 29 1.20.1 Maternal Licking and Grooming Behavior in Rats ........................................ 29 1.20.2 Maternal Interaction in the NICU .................................................................. 30 1.21 Parenting Stress ..................................................................................................... 31 1.22 Maternal Interaction Post-Discharge from the NICU ........................................... 32 1.23 Caregiver Emotional Availability ......................................................................... 33 1.23.1 EA Caregiver Sensitivity ............................................................................... 34 1.23.2 EA Caregiver Structuring .............................................................................. 35 	   vii 1.23.3 EA Caregiver Nonintrusiveness ..................................................................... 35 1.23.4 EA Caregiver Nonhostility ............................................................................ 36 1.24 Maternal Emotional Availability in Infants Born Very Preterm .......................... 36 1.25 Dissertation Overview .......................................................................................... 37 CHAPTER 2 NEONATAL PAIN IN RELATION TO POSTNATAL GROWTH IN INFANTS BORN VERY PRETERM ........................................................................... 41 2.1 Introduction ............................................................................................................. 41 2.2 Methods................................................................................................................... 43 2.2.1 Study Overview ............................................................................................... 43 2.2.2 Participants ....................................................................................................... 44 2.2.3 Weight Percentiles ........................................................................................... 45 2.2.4 Neonatal Medical Chart Review ...................................................................... 45 2.2.5 Data Analyses .................................................................................................. 46 2.3 Results ..................................................................................................................... 47 2.3.1 Characteristics of the Cohort ........................................................................... 47 2.3.2 Early (Birth to 32 Weeks PMA) Neonatal Variables in Relation to Weight               Percentile at 32 Weeks PMA ........................................................................... 48 2.3.3 Early (Birth to 32 Weeks PMA) Neonatal Variables In Relation to Weight           Percentile at 40 Weeks PMA ........................................................................... 50 2.3.4 Later (32 to 40 Weeks PMA) Neonatal Variables in Relation to Weight           Percentile at 40 Weeks PMA ........................................................................... 51 2.3.5 Cumulative (Early and Late/Birth to 40 Weeks PMA) Neonatal Variables in           Relation to Weight Percentile at 40 Weeks PMA ............................................ 52 	   viii 2.3.6 Early (Birth to 32 Weeks PMA) Neonatal Variables in Relation to HC           Percentile at 32 Weeks PMA ........................................................................... 53 2.3.7 Early (Birth to 32 Weeks PMA) Neonatal Variables In Relation to HC           Percentile at 40 Weeks PMA ........................................................................... 55 2.3.8 Later (32 to 40 Weeks PMA) Neonatal Variables in Relation to HC Percentile           at 40 weeks PMA ............................................................................................. 56 2.3.9 Cumulative (Early and Late/Birth to 40 Weeks PMA) Neonatal Variables in           Relation to HC Percentile at 40 Weeks PMA .................................................. 56 2.4 Discussion ............................................................................................................... 56 CHAPTER 3 SLOWER POSTNATAL GROWTH IS ASSOCIATED WITH DELAYED CEREBRAL CORTICAL MATURATION IN PRETERM NEWBORNS ................................................................................................................... 61 3.1 Introduction ............................................................................................................. 61 3.2 Methods................................................................................................................... 63 3.2.1 Study Overview ............................................................................................... 63 3.2.2 Participants ....................................................................................................... 64 3.2.3 Magnetic Resonance Imaging .......................................................................... 65 3.2.4 Diffusion Tensor Imaging ................................................................................ 65 3.2.5 Reliability of the Cortical Gray Matter Regions of Interest ............................ 67 3.2.6 Reliability of the White Matter Regions of Interest ......................................... 68 3.2.7 Data Analyses .................................................................................................. 68 3.3 Results ..................................................................................................................... 70 3.3.1 Characteristics of the Cohort ........................................................................... 70 	   ix 3.3.2 Weight Change in Relation to Diffusion Parameters of the Cortical Gray              Matter ............................................................................................................... 73 3.3.3 Weight Change in Relation to FA of the White Matter ................................... 76 3.3.4 Weight Change in Relation to Diffusion Parameters of the Cortical Gray Matter Excluding Infants who Received Postnatal Corticosteroids ......................... 77 3.3.5 Length Change in Relation to Diffusion Parameters of the Cortical Gray           Matter ............................................................................................................... 78 3.3.6 Head Circumference Change in Relation to Diffusion Parameters of the Cortical Gray Matter ................................................................................................. 79 3.4 Discussion ............................................................................................................... 81 CHAPTER 4 PARENT BEHAVIORS MODERATE THE RELATIONSHIP BETWEEN NEONATAL INVASIVE PROCEDURES AND INTERNALIZING BEHAVIORS AT 18 MONTHS CORRECTED AGE IN CHILDREN BORN VERY PREMATURELY ........................................................................................................... 86 4.1 Introduction ............................................................................................................. 86 4.2 Methods................................................................................................................... 88 4.2.1 Study Overview ............................................................................................... 88 4.2.2 Participants ....................................................................................................... 89 4.2.3 Demographics .................................................................................................. 90 4.2.4 Neonatal Medical Chart Review ...................................................................... 90 4.2.5 Cognitive Development ................................................................................... 90 4.2.6 Parenting Stress ................................................................................................ 90 4.2.7 Child Internalizing Behavior ............................................................................ 91 	   x 4.2.8 Emotional Availability ..................................................................................... 92 4.2.9 Data Analyses .................................................................................................. 92 4.3 Results ..................................................................................................................... 94 4.3.1 Characteristics of the Cohort ........................................................................... 94 4.3.2 Parent and Child Behavior: Group by Gender Analyses ................................. 96 4.3.3 Correlations Among Neonatal Variables ......................................................... 96 4.3.4 Correlations Among Parent Variables ............................................................. 97 4.3.5 Invasive Procedures, EA Sensitivity, and Internalizing Behavior ................... 99 4.3.6 Invasive Procedures, EA Structuring, and Internalizing Behavior ................ 101 4.3.7 Invasive Procedures, EA Nonintrusiveness, and Internalizing Behavior ...... 102 4.3.8 Invasive Procedures, EA Nonhostility, and Internalizing Behavior .............. 102 4.3.9 Exclusion of Mothers Who Reported Drinking Alcohol During their           Pregnancy ....................................................................................................... 104 4.3.10 Parent Behavior and Stress in Relation to Internalizing Behavior in Children Born Full-Term ....................................................................................................... 104 4.4 Discussion ............................................................................................................. 104 CHAPTER 5 INVASIVE PROCEDURES IN PRETERM CHILDREN: BRAIN AND COGNITIVE DEVELOPMENT AT SCHOOL AGE ..................................... 109 5.1 Introduction ........................................................................................................... 109 5.2 Methods................................................................................................................. 111 5.2.1 Study Overview ............................................................................................. 111 5.2.2 Participants ..................................................................................................... 111 5.2.3 Neonatal Medical Chart Review .................................................................... 112 	   xi 5.2.4 Magnetic Resonance Imaging ........................................................................ 112 5.2.5 Diffusion Tensor Imaging .............................................................................. 112 5.2.6 Cognitive Testing ........................................................................................... 113 5.2.7 Data Analyses ................................................................................................ 114 5.3 Results ................................................................................................................... 115 5.3.1 Characteristics of the Cohort ......................................................................... 115 5.3.2 Number of Invasive Procedures in Relation to White Matter Microstructure at           Age 7 Years .................................................................................................... 120 5.3.3 Number of Invasive Procedures Interacts with the Superior White Matter to           Predict FSIQ at Age 7 Years .......................................................................... 122 5.3.4 Interaction Between Number of Invasive Procedures and White Matter Tracts           in Relation to FSIQ ........................................................................................ 125 5.3.5 Interaction between Number of Invasive Procedures and Superior White          Matter in relation to the WISC-IV Indices ..................................................... 125 5.4 Discussion ............................................................................................................. 127 CHAPTER 6 SUMMARY AND DISCUSSION OF RESEARCH FINDINGS ...... 132 6.1 Summary of Results .............................................................................................. 132 6.2 Timing of Exposure to Invasive Procedures ......................................................... 136 6.3 Mechanisms Linking Invasive Procedures, Growth, and Behavior ...................... 140 6.4 Pain/Stress Management in the NICU .................................................................. 142 6.4.1 Morphine ........................................................................................................ 143 6.4.2 Sucrose ........................................................................................................... 144 6.4.3 Swaddling, Facilitated Tucking, Non-nutritive Sucking, Kangaroo Care ..... 146 	   xii 6.5 Importance of Parent Involvement in the NICU ................................................... 148 6.6 Limitations ............................................................................................................ 149 6.7 Significance of this Research ................................................................................ 154 REFERENCES .............................................................................................................. 156 APPENDIX .................................................................................................................... 194 	                   	   xiii LIST OF TABLES	  Table 2.1: Infant Characteristics……………………………………………………….  48  Table 2.2: Early (Birth to 32 Weeks PMA) Neonatal Variables in Relation to Weight and                    HC Percentiles at 32 Weeks PMA………………………………………….  49  Table 2.3: Early (Birth to 32 Weeks PMA) Neonatal Variables in Relation to Weight and                   HC Percentile at 40 Weeks PMA…………………………………………...  51 Table 2.4: Later (32 to 40 Weeks PMA) Neonatal Variables in Relation to Weight and                   HC Percentile at 40 Weeks PMA…………………………………………...  52  Table 2.5: Cumulative (Early and Late/Birth to 40 Weeks PMA) Neonatal Variables in       Relation to Weight and HC Percentile at 40 Weeks PMA………………….  53 Table 3.1: Infant Characteristics……………………………………………………….   72 Table 3.2: Weight change in relation to mean FA values of 8 regions of interest in the                    cortical gray matter………………………………………………………....  74 Table 3.3 Weight Change in Relation to Mean λ2 and λ3 Values of 8 Regions of Interest                   in the Cortical Gray Matter…………………………………………………  75  Table 3.4: Weight Change in Relation to Mean λ1 Values of 8 Cortical Regions of Interest                    in the Cortical Gray Matter………………………………………………...  76 Table 3.5: Weight Change in Relation to Mean FA values of 7 Regions of Interest in the                   White Matter………………………………………………………………... 77  Table 3.6: Weight Change in Relation to Mean FA Values of 8 Regions of Interest in the                    Cortical Gray Matter Excluding Infants who Received Postnatal                    Corticosteroids……………………………………………………………..  78 Table 3.7: Length Change in Relation to Mean Fractional Anisotropy Values of 8    	   xiv                  Regions of Interest in the Cortical Gray Matter…………………………….  79 Table 3.8: Head Circumference Change in Relation to Mean FA Values of 8 Regions of                   Interest in the Cortical Gray Matter…………………………………………  81 Table 4.1: Characteristics of the Cohort………………………………………………..  95  Table 4.2: Pearson Correlations Among the Neonatal Variables of the Preterm                   Infants……………………………………………………………………….  97 Table 4.3: Pearson Correlations Among the Parent Variables for Preterm and Full-Term                   Groups………………………………………………………………………  98 Table 4.4: Greater Parent Sensitivity and Nonhostility were Associated with Fewer                    Internalizing Behaviors at 18 months CA among Preterm Children Exposed to                       a Higher Number of Invasive Procedures…………………………………. 100 Table 5.1: Characteristics of Children with Magnetic Resonance Imaging at Age       7 Years……………………………………………………………………... 117 Table 5.2: Characteristics of All the Children that Returned for Follow-Up at Age       7 Years………………………………………………………………………119 Table 5.3: Higher Numbers of Invasive Procedures was Associated with Lower                   Fractional Anisotropy at Age 7 Years………………………………………122 Table 5.4: Higher Number of Invasive Procedures and Lower Fractional Anisotropy of                   the Superior White Matter Predicts Lower IQ……………………………...124 Table 5.5: Higher Number of Invasive Procedures and Lower Fractional Anisotropy of the                   Superior White Matter Predicted Lower Verbal Comprehension and Working                   Memory……………………………………………………………………..126  	   xv LIST OF FIGURES  Figure 2.1: Early and Late Neonatal Windows…………………………………………  44 Figure 2.2: Weight Percentiles at 32 Weeks PMA in Relation to the Number of Invasive                    Procedures from Birth to 32 Weeks PMA………………………………….  50 Figure 2.3: Head Circumference Percentiles at 32 Weeks PMA in Relation to the Number                     of Skin Breaking Procedures from Birth to 32 Weeks PMA………………  55 Figure 3.1: Regions of Interest in the Cortical Gray Matter …………………………...  66 Figure 3.2: FA of the Cortical Gray Matter with PMA………………………………...  70 Figure 3.3: Postnatal Growth Restriction Delays Cortical Gray Matter Maturation…...  73 Figure 4.1: Parental Sensitivity Moderates the Relationship Between Invasive Procedures                    in the NICU and Internalizing Behavior in Children Born Very Preterm.... 101 Figure 4.2: Parental Nonhostility Moderates the Relationship Between Invasive                     Procedures in the NICU and Internalizing Behavior in Children Born Very                     Preterm…………………………………………………………………….. 103  Figure 5.1: Regions of Interest in the White Matter…………………………………... 113 Figure 5.2: Number of invasive procedures and brain microstructure predicts FSIQ.... 125        	   xvi LIST OF ABBREVIATIONS λ1 Eigenvalue 1 (Axial Diffusivity) λ2&3 Eigenvalue 2&3 (Radial Diffusivity) 5HT Serotonin AGA Appropriate Weight for Gestational Age ATP Adenosine Triphosphate  CA Corrected Age cAMP Cyclic Adenosine Monophosphate  CBCL Child Behavior Checklist CRF Corticotropin-Releasing Factor  DTI Diffusion Tensor Imaging EA Emotional Availability EEG Electroencephalography ELGA Extremely Low Gestational Age FA Fractional Anisotropy fMRI Functional Magnetic Resonance Imaging FSIQ Full Scale IQ GA Gestational Age GENLIN Generalized Linear Model HC Head Circumference HPA Hypothalamic-Pituitary Adrenal  IGF-1 Insulin-Like Growth Factor 1  IQ Intelligence Quotient IQR Interquartile Range IUGR Intrauterine Growth Restriction IVH Intraventricular Hemorrhage  LMEM Linear Mixed Effect Model MDI Mental Development Index MRI Magnetic Resonance Imaging NGF1-A Nerve Growth Factor 1-A  NMDA Glutamate N-Methyl-D-Aspartate  NICU Neonatal Intensive Care Unit PMA Postmenstrual Age PRI Perceptual Reasoning Index PRSI Processing Speed Index PSI Parenting Stress Index III PVHI Periventricular Hemhorragic Infarction PVL Periventricular Leukomalacia SES Socioeconomic Status SGA Small for Gestational Age SNAP-II Score for Neonatal Acute Physiology II VCI Verbal Comprehension Index VLGA Very Low Gestational Age WISC-IV Wechsler Intelligence Scale for Children– 4th Edition WMI White Matter Injury WRMI Working Memory Index 	   xvii GLOSSARY  Term  Definition CA The age of the child from the expected date of delivery  Chronological Age  Age calculated from birth  ELGA  Infants born from 24 to 28 weeks GA Full-term   Infants born from 39 to 41 weeks GA GA Age calculated from the first day of the last normal menstrual period and the day of delivery   PMA Age calculated from the first day of the last menstrual period and birth (GA) in addition to the age calculated from birth (chronological age)  Term-equivalent age The expected date of delivery (i.e. 40 weeks PMA)  Very preterm Infants born from 24 to 32 weeks GA   VLGA Infants born from 29 to 32 weeks GA            	   xviii ACKNOWLEDGEMENTS I would like to thank the children and their parents who participated in this study.  This work was supported by the Canadian Institutes of Health Research (CIHR MOP79262 to S.P.M. and MOP86489 to R.E.G.) and The Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01 HD39783 to R.E.G.). S.P.M. is currently Bloorview Children’s Hospital Chair in Paediatric Neuroscience and was supported by a Tier 2 Canadian Research Chair in Neonatal Neuroscience and the Michael Smith Foundation for Health Research Scholar Award. R.E.G. was supported by a Senior Scientist award from the Child and Family Research Institute (CFRI). I was awarded CIHR Frederick Banting and Charles Best Canada Scholarship Doctoral Award, CFRI Graduate Studentship and University of British Columbia’s Four Year Doctoral Fellowship.  My supervisors, Dr. Ruth Grunau and Dr. Steven Miller are my role models. There are not enough words to express my appreciation for all of their support over the last 6 years. I am a better student, researcher and person for having trained with the both of them.   I grateful for my supervisory committee members Dr. Joanne Weinberg and Dr. Charlotte Johnston for their immeasurable encouragement and advice throughout my degree. I am very thankful for the expertise and training I received from Dr. Kenneth Poskitt, Dr. Rollin Brant, Dr. Vann Chau and Dr. Anne Synnes. Moreover, I am further indebted to Dr. Rebecca Pillai Riddell, Dr. Susanne Brummelte, Dr. Manon Ranger, Dr. Liisa Holsti, Dr. John Peloza and Dr. Jill Zwicker for their outstanding mentorship. I also consider myself very fortunate to have been a member of the Pain in Child Health CIHR Strategic Training Initiative in Health Research, which provided many opportunities for development and networking among the world leaders of pediatric pain research.   This research would not have been possible if not for the additional support of Dr. Grunau and Dr. Miller’s colleagues (Dr. Bruce Bjornson and Dr. Deborah E. Giaschi) and staff (Dr. Ivan Cepeda, Mary Beckingham, Janet Rigney, Sandy Belanger, Mark Chalmers, Kevin Fitzpatrick, Gisela Gosse, Meisan Brownlum), and the neonatal follow-up staff of the BC Children’s and Women’s Hospital.   To all of my friends and family around the world (including, but not limited to: CR, GR, JW, TM, KD, AD, MV, BV) thank you for being my sounding board and cheer team. Of course, I would not be where I am today if it were not for Janet Miller and Angela Peloza.   Finally, I am most grateful to my fiancé Adam Miller. When I was 16 years old I told him that I wanted to become a doctor. With his unwavering support I have been able to make my dreams become a reality.      	   xix DEDICATION    To Adam Miller 	   1 CHAPTER 1  INTRODUCTION AND LITERATURE REVIEW 	  1.1 Preterm Birth Approximately 8% of all infants born in Canada are born less than 37 weeks gestational age (GA; Canadian Institute for Health Information 2012). Of these infants, approximately 15% are born very preterm (between 24 to 32 weeks GA; Canadian Institute for Health Information 2009). Advances in neonatal care have greatly improved infant survival, and reduced the number of severe disabilities (e.g. blindness, non-ambulatory cerebral palsy, developmental delay), particularly for infants born extremely low gestational age (between 24 to 28 weeks GA; Doyle et al. 2011; Moore et al. 2012). However, preterm children have more cognitive, motor and behavioral problems relative to children born full-term (~40 weeks GA) that appear early in life, and persist to adulthood (Anderson, Doyle, Victorian Infant Collaborative Study Group 2003; Doyle, Casalaz, Victorian Infant Collaborative Study Group 2001; Doyle and Anderson 2010; Grunau, Whitfield, Fay 2004; Johnson et al. 2009; Loe et al. 2011; Marlow et al. 2005; Marlow et al. 2007; Spittle et al. 2009). The birth of a preterm child causes substantial stress for families (Brummelte et al. 2011a; Garel, Dardennes, Blondel 2007; Glazebrook et al. 2007; Poehlmann and Fiese 2001; Singer et al. 2003; Thomas, Renaud, Depaul 2004), and puts considerable strain on health, educational and social systems (Johnston et al. 2014). In Canada, between 1996 and 2006, the cost to support children born preterm was estimated as $587.1 million (Johnston et al. 2014). To improve quality of life for the children and their families, it is imperative that we find ways to optimize the developmental outcomes of this vulnerable population of infants.  	   2  1.2 Invasive Procedures during Neonatal Care  The majority of infants born very preterm now survive as a result of advances in medical care, however, they must develop outside of the protective intrauterine environment during the third trimester of ‘‘fetal’’ life, which is a critical period of physiological immaturity and vulnerability. They are susceptible to a number of pathophysiological conditions for which invasive medical interventions are required. A recent North American survey of 14 Canadian neonatal intensive care units (NICU) revealed that in a single week, 580 neonates underwent over 17,500 painful/stressful procedures (Johnston et al. 2011). On average, infants undergoing neonatal intensive care required 4 to 14 procedures per day (Brummelte et al. 2012; Carbajal et al. 2008; Doesburg et al. 2013; Grunau et al. 2005; Johnston et al. 2011; Simons et al. 2003a). For infants born very preterm, repeated exposure to invasive procedures occurs during a period of rapid brain and stress system development (Brummelte et al. 2012; Brummelte et al. 2015; Doesburg et al. 2013; Grunau, Weinberg, Whitfield 2004; Grunau et al. 2005; Grunau et al. 2007; Ranger et al. 2013; Smith et al. 2011; Zwicker et al. 2013). There is an increasing amount of evidence which suggests that exposure to greater number of invasive procedures in the NICU leads to altered brain and neurodevelopmental outcomes in children born very preterm.   1.3 History of Pain Management in the NICU Concerns regarding the long-term effects of pain in very preterm infants are relatively recent. Prior to the 1980’s, infant surgery was routinely conducted with little to no anesthesia (Schechter, Allen, Hanson 1986). This was partly due to concerns about the safety of anesthesia (e.g. respiratory suppression), but also because it was believed that newborns could not feel pain, and that surgery could be safely accomplished using oxygen and a paralytic (Rodkey and Pillai 	   3 Riddell 2013). Therefore, preterm infants such as the famous Jeffery Lawson, born February 1985, at 26 weeks GA, were not anesthetized for surgery. Jeffery died 5 weeks after his surgery. The effect of this case was that Jill Lawson, and other parents began to advocate for the management of infant pain in the NICU (Rodkey and Pillai Riddell 2013). In 1987, Anand and colleagues published a seminal paper, which demonstrated for the first time that preterm infants treated with fentanyl for anesthesia, in addition to nitrous oxide and muscle relaxants, had decreased hormonal responses after surgery compared to the standard care (non-fentanyl treated) group (Anand, Sippell, Aynsley-Green 1987). This study by Anand and colleagues changed surgical practice in newborns. Concurrently, Grunau and Craig published the first quantified measure of infant behavior (Neonatal Facial Coding System), which greatly facilitated pain measurement for research in infants (Grunau and Craig 1987). Moreover, specific facial actions then became incorporated into multidimensional scales for clinical assessment (e.g. Stevens et al. 1996). Furthermore, Anand and Hickey published a critical literature review proposing that the human fetus has cortical and subcortical brain regions necessary for pain detection, as well as neurochemical systems associated with pain transmission (Anand and Hickey 1987). These events combined led to a rapid expansion in the field of pediatric pain research.  1.4 Pain Transmission and Modulation In order to understand the impact of repeated exposure to invasive procedures during neonatal intensive care on brain microstructure, growth, neurodevelopment and behavior in children born very preterm, it is important to address how the pain system operates in adults, and how it differs in infants born very preterm.  	  	   4   1.4.1 Transduction, Transmission and Central Nociceptive Processing   Peripheral tissue injury activates high-threshold sensory receptors of the somatosensory nervous system that are capable of transducing and encoding noxious stimuli along specialized nerve cells called nociceptive neurons (Fitzgerald 2005; Renn and Dorsey 2005; International Association for the Study of Pain 1979; Melzack and Wall 1965; Willis and Westlund 1997). Nociceptive neurons consist of small diameter A-delta (myelinated, fast transmission), and C-fibres (unmyelinated, slow transmission; Konietzny et al. 1981; Ochoa and Torebjork 1989). The likelihood of nociceptive neurons firing an action potential is influenced not only by adequent input (e.g. strong mechanical, thermal or chemical stimuli; Willis and Westlund 1997), but also by the numerous chemical agents produced or released at an injury site. Prostoglandins, leukotrienes, bradykinin, substance P, cytokines, and serotonin can both activate nociceptors, and sensitize nociceptors to subsequent stimuli (Woolf and Costigan 1999), which can result in allodynia (pain due to a stimulus that does not normally provoke pain; International Association for the Study of Pain 1979).   Sensory neuron cell bodies are located in the dorsal root ganglia (Bourne, Machado, Nagel 2014). These neurons project from the dorsal root ganglia to the spinal cord, entering laterally through Lissauer’s tract, and extending vertically in this tract for several spinal segments before synapsing onto second-order neurons (Earle 1952). The gray matter of the spinal cord consists of 10 laminae, 6 of which are in the dorsal horn. The laminae of the dorsal horn can be grouped into the superficial layers (laminae I and II) and the deep layers (laminae III-VI). A-delta fibers terminate lamina I, II and V, whereas C-fibers terminate in lamina II (Traub and Mendell 1988).  	   5 The dorsal horn is not simply a relay station, but rather is a network of neurons through which inputs from the periphery are transduced and modulated by local, and descending, excitatory and inhibitory mechanisms (Woolf and Salter 2000). Excitatory synaptic transmission is mediated by glutamate acting on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate ligand-gated ion channels. Dampening of this signal may occur through segmental and descending activation of inhibitory neurons, which co-release glycine and γ-aminobutyric acid (Chery and De Koninck 1999). Modulation of the nociceptive transmission involves activation of intracellular signaling cascades, which facilitate excitatory synaptic responses and depress inhibition (Woolf and Salter 2000). These sequences of cellular events can lead to the occurrence of central sensitization, which involves increased responsiveness of nociceptive neurons within the central nervous system to normal or subthreshold afferent input (International Association for the Study of Pain 1979; Woolf 2011). Unlike peripheral sensitization, central sensitization can increase sensitivity in non-inflamed tissue by changing the response elicited by normal input long after the initial stimulus has disappeared (Woolf 2011). The nociceptive transmission leaves the dorsal horn via second-order neurons, which cross the midline, and ascend through the ventrolateral quadrant of the contralateral half of the spinal cord to connect with nuclei within the thalamus. Lamina I spinothalamic tract neurons project to the posterolateral, ventral medial, ventral posterior, ventral posterior inferior and medial dorsal nuclei of the thalamus. (Craig 2003). Lamina V spinothalamic tract neurons axons terminate in ventral posterior, ventral posterior inferior, venterolateral and the intralaminar nuclei of the thalamus (Craig 2003).  Projections from the ventral posteromedial nucleus and ventroposterolateral nucleus synapse directly with the primary somatosensory cortex (Bourne, Machado and Nagel 2014; Gingold et al., 1991; Kenshalo et al., 1980). The primary somatosensory cortex is somatotopically 	   6 organized (Jones, Kulkarni, and Derbyshire 2003), and demonstrates a graded response according to intensity of noxious stimulus (Chudler et al., 1990; Kenshalo et al., 1988; Kenshalo and Isensee, 1983; Lamour et al., 1983). Therefore, the primary somatosensory cortex is thought to be involved in the location and discrimination of pain. The secondary somatosensory cortex receives input from the posteromedial, ventroposterolateral nuclei as well as the primary somatosensory cortex, and it also responds according to the magnitude of noxious stimuli (Bourne, Machado and Nagel 2014; Stevens et al. 1993). The insula receives input from the primary and secondary somatosensory cortex, the ventral posterolateral, dorsomedial and intralaminar nuclear groups (Bourne, Machado and Nagel 2014; Robinson 1997). In addition, it projects to limbic structures such as amygdala and perirhinal cortex (Bourne, Machado and Nagel 2014; Shi and Cassell, 1998). The insula has been shown to be involved in both the discriminatory and motivational-affective responses to pain (Bourne, Machado and Nagel 2014; Schnitzler and Ploner 2000). Finally, the anterior and middle cingulate cortices receive projections from the venterolateral, medial and intralaminar thalamic nuclei and are involved in the affective or motivational responses to pain (Baleydier and Mauguiere, 1980; Bourne, Machado and Nagel 2014). With the advancement of noninvasive neuroimaging techniques in humans, researchers are acquiring a better understanding of how sensory neurons, the spinal cord, and higher centers of the brain act in concert to contribute to the processing of nociceptive stimuli.   1.5 Cortical Processing of Centrally Transmitted Nociceptive Activity Functional magnetic resonance imaging (fMRI) and positron emission tomography have been used to study regional responses to noxious stimuli in the brain.  	   7   1.5.1 Imaging Responses to Noxious Stimuli in Adults Many researchers have attempted to identify ascending nociceptive systems and their central nervous system targets (e.g. primary and secondary somatosensory cortices, insula, anterior cingulate cortex, and thalamus; Apkarian et al. 2005; Bushnell, Ceko and Low 2013; Duerden and Albanese 2013; Iadarola and Coghill 1999; Willis and Westlund 1997), in addition to showing that these cortical and subcortical brain areas correlate with the modality and intensity of noxious input (Atlas et al. 2014; Apkarian et al. 2005; Coghill et al. 1999; Wager et al. 2013). For example, in a recent fMRI study involving 114 healthy adult participants, Wager and colleagues (2013) found a highly sensitive and specific neurologic signature of physical pain. It included both medial (e.g., “affective”; anterior cingulate cortex) and lateral (e.g., “sensory”; somatosensory cortices) “pain systems” that were consistent across individuals (Wager et al. 2013). Although pain has relatively well-defined primary targets, pain is a multidimensional experience, with sensory, cognitive, and evaluative aspects. Therefore, other networks may be simulataneously activated during invasive procedures, and may influence how the brain responds to noxious stimuli. The salience network comprising of the anterior insula, midcingulate cortex, temporoparietal junction, and dorsolateral prefrontal cortex is more strongly activated when an individual is paying attention to the noxious stimulus (Kucyi et al. 2012; Seeley et al. 2007). In contrast, the default mode network (posterior cingulate cortex/precuneus, medial prefrontal cortex, lateral parietal lobe, and areas within the medial temporal lobe) becomes activated when a person is at rest, and unaware of the noxious stimulus (Andrews-Hanna et al. 2014; Kucyi et al. 2013). Moreover, the fronto-striatal pathway, connecting ventromedial prefrontal cortex and nucleus accumbens, and the antinociceptive pathway, connecting the medial prefrontal cortex 	   8 and periaqueductal gray, are involved in the modulatory aspects of pain (Beccera 2008; Borsook et al. 2010; Petrovic et al. 2000; Tracey et al. 2002; Fields 2007; Valet et al. 2004). Therefore, in adults the processing of nociceptive activity involves a complex integration of brain networks involved in the cognitive, emotional, and sensorimotor aspects of pain.  1.5.2 Imaging Responses to Noxious Stimuli in Infants and Children In comparison to the extensive study of pain processing in adults, very few studies to date have examined the cortical processing of noxious stimuli in children and infants. Hohmeister and colleagues (2010) demonstrated that children born full-term and children born very preterm seen between the ages 11 to 16 years showed activation in several of the same brain regions known to be activated in response to noxious stimuli in adults.  However, children born very preterm demonstrated greater activation within these regions compared to children born full-term (Hohmeister et al. 2010). Exposure to mild/moderately painful heat in children born very preterm also led to signaling in several regions that were not activated in full-term controls, including the thalamus, anterior cingulate cortex, cerebellum, basal ganglia, regions known to be involved in the sensory, affective, and cognitive aspects of pain. Repeated exposure to pain/stress in the first weeks of life may have led to increased afferent drive to CNS sites that respond to noxious input (LaPrairie and Murphy 2007; Ranger et al. 2013). Increased connectivity along pathways associated with pain/stress signaling (LaPrairie and Murphy 2007; Ranger et al. 2013) may in part explain why children and adolescents born very preterm showed greater brain activation compared to their term-born peers in response to noxious stimulation.   Recently, studies have used fMRI to study the responses of infants born full-term to noxious 	   9 stimuli (Goksan et al. 2015; Williams et al. 2014). First the feasibility of using fMRI to study nociceptive evoked brain activity in a healthy unsedated full-term newborn was established (Williams et al. 2014). Slater and colleagues then conducted a study to compare fMRI responses in healthy full-term newborns with those of adults. They found that full-term infants showed increases in activity in all but 2 (amygdala and the orbitalfrontal cortex) of the 20 regions that were active in adults in response to acute noxious stimulation (Goksan et al. 2015). The amygdala and orbitalfrontal cortex are involved in the emotional and cognitive processing of pain signals (Simons et al. 2014; Winston et al. 2014). Other brain structures known to be involved in emotional processing of pain, such as the anterior cingulate, were activated in both the full-term infants and the adults (Goksan et al. 2015). Therefore, it may be that no prior experience of pain is necessary to produce sensory and some emotional responses to pain/stress. While thalamocortical connectivity is present earlier, the period between 35 and 37 weeks PMA is dominated by the growth of interhemispheric connections, which continue to develop after term-equivalent age (Kostovic and Judas 2010). Therefore, the complex integrated perceptual, emotional and cognitive pain experience is not likely possible until later in infancy. Moreover, the cognitive component that requires experience to contextualize pain may develop later in childhood.   The prenatal development of thalamocortical and cortico-cortical connections is a prolonged process that involves afferents projecting towards the cortex and forming transient, functional circuits with subplate neurons before proliferating into the cortical layers. At 24 and 25 weeks postmenstrual age (PMA), thalamocortical afferents are ‘waiting,’ in the subplate zone, just beneath the cortical plate (Kanold 2009; Kostovic and Judas 2010). Thus, at 24 weeks PMA the subplate neurons can receive extrinsic input from sensory and associative thalamic nuclei 	   10 (Kostovic and Judas 2010). Between 26 and 28 weeks PMA, thalamocortical axons begin to penetrate the cortical plate (Kostovic and Judas 2010; Vasung et al. 2010), and evoked potentials can be recorded from the somatosensory cortex (Kostovic and Rakic 1990). However, fMRI has not been used to study activation in the brain in response to nociceptive stimulation in infants born very preterm. Given the immaturity of the brain and its connections, cognitive and affective processes associated with pain are unlikely to be developed at this early age. However, repeated exposure to invasive procedures during this critical period of brain development may lead to alterations in both structure and function of these developing regions.  1.6 Supraspinal Descending Control Pathways Pain inhibitory pathways have also been well characterized. Inhibition of pain is initiated by the activation of the periaqueductal gray, which forms connections with the prefrontal cortex, anterior cingulate cortex, hypothalamus, and central nucleus of the amygdala (Fields 2004; Schweinhardt and Bushnell 2010). The periaqueductal gray connects with the rostral ventroventral medulla and dorosolateral pontine tegmentum, and these two regions project through the spinal cord (dorsolateral funiculus), to selectively target the nociceptive relay neurons housed in lamina I, II and V of the spinal cord (Fields 2004; Koch and Fitzgerald 2014).  While in neonatal rats the anatomical connections for nociceptive modulation are present at birth, descending inhibitory controls are not functionally active until postnatal day 21, beyond term-equivalent age in humans (Hathway et al. 2009a; van Praag and Frenk 1991). Up to postnatal day 21 in the rat, the rostroventral medulla of the brainstem exclusively facilitates spinal pain transmission (Hathway et al. 2009a). After this age there is a shift to inhibition of the pain signal (Hathway et al. 2009a). The delayed maturation of descending inhibition may therefore contribute to increased vulnerability of the immature somatosensory system to repeated 	   11 exposure to invasive procedures in infants born very preterm.   1.7 Pain Processing in Infants Born Very Preterm  Preterm infants have the nociceptive circuitry required to process pain signals, however, this system is functionally immature (Fitzgerald 2005; Fitzgerald and Walker 2009). Cutaneous receptive fields are large in the neonate, and peripheral sensory fibers are sensitive to tissue injury and have reduced peak firing frequencies (Andrews and Fitzgerald 1994; Beggs et al. 2002; Fitzgerald and Walker 2009; Granmo, Petersson, Schouenborg 2008; Jennings and Fitzgerald 1998; Li et al. 2009). Receptors innervated by Aδ fibers (myelinated, fast transmitting) are distinguishable in the preterm neonate (Andrews and Fitzgerald 1994; Jennings and Fitzgerald 1998; Li et al. 2009), however, C-fibre (unmyelinated, slow transmitting) evoked activities are not observable until approximately 28 weeks PMA (Jennings and Fitzgerald 1998). Axon terminals temporarily overlap in lamina II of the spinal cord with low-threshold tactile inputs, making it more difficult for neonates to discriminate between noxious and non-noxious stimuli (Beggs et al. 2002; Granmo, Petersson, Schouenborg 2008). Peripheral injury triggers a long-lasting increase in the excitability of spinal cord neurons, which manifests as a reduction in threshold (allodynia), and increase in responsiveness (hyperalgesia; Woolf 2011). The amplification of neural signaling in the spinal cord that generates pain hypersensitivity is referred to as central sensitization (Woolf 2011). Prior to 35 weeks PMA, infants demonstrate central sensitization and therefore, react more strongly to subsequent noxious and non-noxious stimuli (Andrews and Fitzgerald 1994; Fitzgerald, Millard, McIntosh 1989; Grunau et al. 2001; Holsti et al. 2005; Holsti et al. 2006; Walker, Tochiki, Fitzgerald 2009). Moreover, descending inhibition of nociceptive activity develops later, beyond term equivalent age (Hathway et al. 	   12 2009a; van Praag and Frenk 1991). Given that infants born very preterm have reduced localization and specification to noxious stimuli, become sensitized to repeated noxious stimuli and lack descending inhibitory control, identifying, relieving, and preventing pain are very important aspects of NICU care.  1.8 Behavioural and Physiological Responses to Noxious Stimuli in the NICU A wide variety of behavioral and physiological responses (e.g., facial actions, body movements, cry, heart rate, respiratory rate, blood pressure, and oxygen saturation) are used to identify pain in nonverbal patients (Holsti et al. 2008; Stevens, Johnston, Horton 1993). However, these indicators are not specific to pain, and may also represent agitation or distress. While all pain is stressful, not all stress is painful. This ambiguity in identifying pain in the NICU presents a challenge to both clinicians and researchers. Therefore, throughout this dissertation pain in infants born very preterm will be broadly defined as pain/stress. Responses to invasive procedures vary based on a number of clinical factors, for example, GA, illness severity, and prior exposures to pain/stress. The following will highlight why variation in pain/stress responses make it difficult for clinicians to discriminate and appropriately manage pain/stress in infants born preterm.   1.8.1 Behavioral Responses to Invasive Procedures Infants born full-term demonstrate clear facial responses to pain/stress including eye squeeze, brow contraction, naso-labial furrow, taut tongue, and open mouth (Grunau and Craig 1987). Subsequently, these behavioral responses to pain/stress were confirmed in very preterm infants (Craig et al. 1993). However, infants born <28 weeks GA demonstrate fewer facial responses to invasive procedures (Craig et al. 1993; Gibbins et al. 2008; Holsti et al. 2006; Xia et al. 2002). 	   13 Greater PMA has been associated with an increase in the amplitude and likelihood of responding to an invasive procedure (Johnston and Stevens 1996; Johnston et al. 1999; Slater et al. 2009; Williams et al. 2009). In contrast, greater exposure to invasive procedures in the NICU has been associated with reduced behavioral responses in infants born preterm, after adjusting for GA (Grunau et al. 2001; Grunau et al. 2005). Moreover, the timing of the last invasive and/or noninvasive procedure appears to impact how the infant responds to pain/stress (Grunau et al. 2000; Holsti et al. 2005; Holsti et al. 2006; Johnston et al. 1999; Porter, Wolf, Miller 1998). Greater illness severity is also associated with dampened responses to invasive procedures (Evans et al. 2005; Johnston and Stevens 1996; Valeri et al. 2012; Williams et al. 2009). Sleep/awake state matters for pain/stress behaviors, such that full-term (Grunau & Craig 1987) and preterm infants who are awake during heel lance procedure are more likely to display facial change in response to the stimulus (Slater et al. 2009; Stevens, Johnston, Horton 1994). Therefore, there appear to be a large number of factors that lead to either increased or decreased behavioral expression in response to invasive procedures, particularly in infants born very preterm, undergoing neonatal intensive care.   1.8.2 Physiological Responses to Invasive Procedures Many studies have also examined the physiological responses to a heel lance procedure in infants born preterm. Similar to behavioral responses, there are several factors that may influence physiological responses to pain/stress. GA was found to be significantly associated with oxygen saturation and/or heart rate, such that oxygen saturation levels were lower and heart rate was higher in lower GA infants (Gibbins et al. 2008; Grunau et al. 2001). Greater exposure to invasive procedures in the NICU was associated with greater heart rate variability during heel lance procedures (Grunau et al. 2001). Although exposure to analgesia was found to reduce heart 	   14 rate variability, steroid exposure increased heart rate variability in infants born preterm (Grunau et al. 2001). Greater illness severity on the other hand, has been associated with both raising and lowering heart rate during invasive procedures (Morison et al. 2003; Valeri et al. 2012). Given that physiological responses are affected by clinical conditions such as infection, autonomic measures can be even more variable than facial responses and finger splaying in response to pain/stress in hospitalized neonates (Grunau et al. 2000; Holsti et al. 2005; Stevens et al. 2007). Therefore physiological responses have often been measured together with behavioral responses in multidimensional scales to assess pain/stress in infants born preterm.    1.9 Cortical Responses to Invasive Procedures in the NICU The use of neuroimaging at the bedside may also assist clinicians in identifying pain/stress responses in infants born very preterm. Using near-infrared spectroscopy, changes in cerebral oxygenation have been detected over the prefrontal and somatosensory cortex in response to noxious stimulation (routine heel lance) in infants between the ages of 25 and 45 weeks PMA (Bartocci et al. 2006; Ozawa et al. 2011; Slater et al. 2006). Responses within the somatosensory cortex were significantly greater in infants that were awake (Slater et al. 2006). Moreover, the strength of signal increased with PMA (Bartocci et al. 2006; Slater et al. 2006), and the latency to respond decreased with PMA (Slater et al. 2006). Neuroactivity in response to stimuli was also measured within the somatosensory cortex using electroencephalography (EEG) in infants between the ages of 28-45 weeks PMA (Fabrizi et al. 2011; Slater et al. 2010a). Prior to 35 weeks PMA, both touch (tapping of a tendon hammer again the heel) and routine heel lance lead to nonspecific neuronal bursts on EEG (Fabrizi et al. 2011). However, for infants between 35–37 weeks PMA, touch and heel lance evoked characteristic somatosensory potentials that differed both in timing and morphology for the two modalities of stimulation (Fabrizi et al. 	   15 2011). By 40 weeks PMA, infants born very preterm demonstrated larger evoked potentials on EEG following a routine heel lance compared to infants born full-term, but not for non-noxious (light tapping of a rubber bung) stimuli (Slater et al. 2010a). This difference in responding could not be explained by the infants’ age at birth or presence of brain injury (Slater et al. 2010a). Therefore, given that prior to 35 weeks PMA, the brain responds similarly to both invasive and non-invasive stimulation, and by 40 weeks PMA, neural responses to skin-breaks are greater for infants born very preterm, it is important that we consider the long-term impact of repeated invasive procedures on the developing brain.   1.10 Vulnerability of the Developing Brain Over the course of their NICU stay, infants born very preterm are repeatedly exposed to invasive procedures during a critical period of brain development, which is characterized by cell proliferation, migration, axonal growth, neuronal differentiation and synaptogenesis (Kostovic and Judas 2010). Repeated exposure to invasive procedures in the NICU appears to disrupt the maturation of the developing brain in infants born very preterm (Brummelte et al. 2012; Doesburg et al. 2013; Ranger et al. 2014; Zwicker et al. 2013). Two cell populations are particularly vulnerable to injury in the premature brain: subplate neurons and preoligodendrocytes (Back and Miller 2014; Volpe 2009).   Subplate neurons are among the first cells generated in the mammalian cerebral cortex, and are the first cortical neurons to receive excitatory synaptic inputs from thalamic axons, establishing a temporary link between thalamic axons and their final target in the cerebral cortex (Kostovic et al. 2002; Kostovic and Judas 2002; McQuillen and Ferriero 2005). Subplate neurons are particularly vulnerable to excitotoxic death, as was demonstrated by the selective ablation of 	   16 subplate neurons after the administration of glutamate agonist kainite into embryonic (embryonic day 42) and newborn kittens (Ghosh et al. 1990; Ghosh and Shatz 1992). Glutamate n-methyl-D-aspartate (NMDA) receptors are more active during early life because of the developmentally delayed expression of NR2A receptor subunits, relative to NR2B in the developmental course (Monyer et al. 1994; Sheng et al. 1994). Therefore, repeated excitation of subplate neurons can lead to an excessive release of glutamate, influx of calcium and apoptosis, (Deng et al. 2003; McDonald and Johnston 1990; Qu et al. 2003; Talos et al. 2006), thereby disrupting neuronal migration, synapse formation and dendritic pruning (Gambrill and Barria 2011; Luthi et al. 2001; Zhang, Peterson, Liu 2013).   Preoligodendrocytes, are cells that ensheath axons prior to differentiating into myelin-producing oligodendrocytes (Volpe 2009). There are four stages of oligodendroglial maturation, which include: 1) the oligodendroglial progenitor, 2) the pre-oligodendrocyte (or late oligodendroglial progenitor), 3) the immature oligodendrocyte, and 4) the mature myelin-producing oligodendrocyte. At 28 weeks GA preoligodendrocytes account for 90% of the total oligodendroglial population (Back et al. 2001). Between 28–40 weeks of gestation, preoligodendrocytes begin to differentiate into immature oligodendrocytes. Immature oligodendrocytes account for approximately 30% of the total oligodendrocyte population during the later premature period, and about 50% by term-equivalent age (Back et al. 2001). Disruption of the preoligodendrocytes and/or immature oligodendrocytes can lead to the arrest of their development, and alterations in myelination in preterm infants (Buser et al. 2012). Fewer cortical connections or poorer myelination of white matter tracts may underlie the cognitive, motor and behavior problems frequently observed in children born very preterm (Ball et al. 2015; Bora et al. 2014; Chau et al. 2013; Counsell et al. 2008; Doesburg et al. 2011; Estep et al. 2014; Mullen 	   17 et al. 2011; Thompson et al. 2014).   Both subplate neurons, preoligodendrocytes and immature oligodendrocytes are vulnerable to inflammation, and oxidative stress, which involves the overproduction of reactive oxygen, nitrogen species, and cytokines secreted by microglia (Adams et al. 2010; Back et al. 1998; Back et al. 2005; Buntinx et al. 2004; Chau et al. 2009; Chau et al. 2012; Dean et al. 2011; Glass et al. 2008; Haynes et al. 2003; McQuillen et al. 2003; Pang, Cai, Rhodes 2005; Sizonenko et al. 2003; Sizonenko et al. 2005; Wikstrom et al. 2008; Zwicker et al. 2013). Repeated exposure to pain/stress may lead to neuroinflammation or oxidative stress within the CNS, thereby disrupting the development of the subplate neurons or preoligodendrocytes, which may lead to long-term alterations in brain microstructure (Brummelte et al. 2012; Doesburg et al. 2013; Ranger et al. 2013; Zwicker et al. 2013), and adverse neurodevelopmental and behavioral outcomes in children very preterm.  1.11 Inflammation and Invasive Procedures in the NICU Tissue damage results in localized inflammation, hyperalgesia and allodynia (Fitzgerald and Beggs 2001). Inflammation associated with tissue damage results in the leak of intracellular contents into the extracellular fluid, the co-release of ions (hydrogen, potassium), amines (5-hydroxytryptamine, histamine), kinins (bradykinin), prostanoids (prostaglandin), purines (ATP), nitric oxide, cytokines (tumor necrosis factor-α, interleukin-1 and interleukin-6), growth factors (leukemia inhibitory factor, nerve growth factor), and further recruitment of inflammatory cells (Woolf and Costigan 1999). These may act directly with peripheral nociceptors, or act indirectly to sensitize nociceptors and alter their responses to subsequent stimuli (Woolf and Costigan 1999), increasing the likelihood of nociceptive neurons, A-delta and C-fibres firing an action 	   18 potential.  In adult rats, intense stimulation of peripheral C-fibres activates microglia within the spinal cord (Hathway et al., 2009). Microglial activation in the dorsal horn of the spinal cord results in the release of cytokines and growth factors, which excite nociceptive dorsal horn neurons, thereby contributing to the development of central sensitization and hyperalgesia (Trang et al., 2011). In preterm neonates C-fibre evoked activity is not observed until approximately 28 weeks PMA (Jennings and Fitzgerald 1998), and tissue injury-induced activation of spinal microglia is reduced in neonates compared to adults (Moss et al., 2007; Costigan et al., 2009). However, neoonatal tissue injuries may ‘prime’ the immune system so that it becomes more easily activated in later life. A neonatal skin incision in the hindpaw of a rat pup led to enhanced microglial reactivity when the same hindpaw was re-incised during adulthood (Beggs et al., 2012). Therefore, microglial activity may be enhaced by early exposure to invasive procedures.   Both subplate neurons and preoligodendrocytes are vulnerable to neuroinflammatory responses (Adams et al. 2010; Beggs et al. 2012; Buntinx et al. 2004; Chau et al. 2009; Chau et al. 2012; Dean et al. 2011; Glass et al. 2008; Pang, Cai, Rhodes 2005; Grunau et al. 2013; Strunk et al. 2014; Wikstrom et al. 2008; Zwicker et al. 2013). In animal models, inflammatory pain leads to increased cell death in the neonatal rat brain (Anand et al. 2007; Rovnaghi et al. 2008), and in preterm neonates inflammation/infection has been associated with alterations to the developing brain microstructure and function (Adams et al. 2010; Chau et al. 2009; Chau et al. 2012; Ellison et al. 2005; Glass et al. 2008; Strunk et al. 2014; Wikstrom et al. 2008; Zwicker et al. 2013). Taken together, these studies suggest that inflammatory factors may play a role in effects of pain/stress on the CNS.  	   19 1.12 Oxidative Stress and Invasive Procedures in the NICU Oxidative stress is another mechanism through which repeated exposure to invasive procedures may interfere with cortical maturation. Both cerebral oxygenation and cerebral blood volume are affected with changes in oxygen saturation (Pryds 1991; Yamamoto et al. 2003). Infants born preterm are known to exhibit lower mean arterial blood pressure and poorer cerebral perfusion (Menke et al. 1997; Pryds et al. 1989; Pryds 1991). Oxidative stress results from the production of reactive species or oxidants, and is a sequela of cerebral ischemia (shortage of oxygenated blood/reperfusion; Traystman, Kirsch, Koehler 1991). Previously it has been shown that exposure to invasive procedures leads to reduced oxygen saturation (Bauer et al. 2004; Gonsalves and Mercer 1993), and increased heart rate, indicative of increased energy expenditure and oxygen consumption (Bauer et al. 2004). A recent study examined markers of adenosine triphosphate (ATP) utilization and oxidative stress (uric acid and malondialdehyde concentration) in the plasma of preterm before and after a tape removal procedure during discontinuation of an indwelling central arterial or venous catheter. Slater et al. (2012) compared the results of preterm infants that underwent this tissue-damaging procedure to preterm infants that did not undergo this procedure. Although the markers for uric acid significantly decreased over time in the control group, these values remained stable for the infants that underwent the tape removal (Slater et al. 2012). Malondialdehyde levels, however, decreased over time for the control neonates, but increased for the preterm infants that underwent the tape removal (Slater et al. 2012). Moreover, their concentrations of malondialdehyde correlated with their pain scores on the Premature Infant Pain Profile (Slater et al. 2012). Taking these results together, it would appear that there is a relationship between exposure to invasive procedures in the NICU and oxidative stress in infants born very preterm. Given the selective vulnerability of subplate neurons and preoligodendrocytes to oxidative stress (Back et al. 1998; Baud et al. 2004; 	   20 McQuillen et al. 2003; Sizonenko et al. 2003; Sizonenko et al. 2005), it is possible that repeated exposure to invasive procedures may lead to disturbances in cortical connections and myelination in infants born very preterm (Brummelte et al. 2012; Zwicker et al. 2013).   1.13 Brain Injury in Infants Born Very Preterm  The immaturity of the very preterm brain leaves infants vulnerable to brain injuries (i.e. periventricular leukomalacia [PVL], intraventricular hemorrhage [IVH], white matter injury [WMI], ventriculomegaly). Although cystic PVL was previously the major form of WMI in preterm infants, major advances in perinatal care (e.g. use of antenatal corticosteroids and exogenous surfactant therapy) has led to a marked decline in the incidence of cystic PVL (<5% of cases; Counsell et al. 2003; Groenendaal et al. 2010; Hamrick et al. 2004; Inder et al. 2003; Maalouf et al. 2001; Miller et al. 2003). Advances in neuroimaging (e.g. Magnetic Resonance Imaging [MRI] versus ultrasound) have led to the identification of diffuse WMI, which is now the pattern of brain injury most frequently observed in infants born very preterm (Back and Miller 2014). The extent of diffuse WMI is still difficult to define using conventional neuroimaging; however, it is often identified on MRI scans as multifocal lesions, which are seen in approximately, 37% of infants born very preterm (Chau et al. 2009; Miller et al. 2005). Advances in MRI techniques have allowed us to also obtain quantifiable measurements of brain development in vivo (Duerden, Taylor, Miller 2013). Techniques, such as Diffusion Tensor Imaging (DTI), allow us to extract information from the scans at a microstructural level, thereby expanding our interpretation of scans beyond visible injuries to measure dysmaturation.  1.13.1 Diffusion Tensor Imaging DTI is an MRI sequence that allows for the robust and noninvasive capture of maturation-	   21 dependent changes of water diffusivity in the cortex of premature newborns, reflecting cortical microstructure. Specifically, DTI describes an ellipsoid space, where the size, shape, and orientation are given by eigenvalues (λ1, λ2 and λ3; Mukherjee et al. 2002). Fractional anisotropy (FA) reflects the variance of λ1, λ2 and λ3, and thereby describes overall directionality of the water diffusion. λ1 corresponds to axial diffusion (Song et al. 2002). This is the preferred diffusion direction because water readily diffuses along white matter tracks and radial glia of the developing cortical gray matter. In contrast, λ2 and λ3 correspond to radial diffusion (Song et al. 2002). In the cerebral cortex, FA decreases between 25 and 40 weeks PMA, corresponding with neuronal maturation, synaptogenesis and the disappearance of the radial glia (Deipolyi et al. 2005; Jespersen et al. 2012; Kroenke et al. 2007; McKinstry et al. 2002; Sizonenko et al. 2007). In the white matter, FA increases with maturation, corresponding to the maturation of the oligodendrocyte lineage and early events of myelination (Drobyshevsky et al. 2005; Huppi et al. 1998; Partridge et al. 2004; Song et al. 2002). Parameters obtained through DTI are highly sensitive, which allow for the quantitative measure of change in brain microstructure over time that are relevant to brain injury and dysmaturation in infants born preterm.   DTI has been shown to be particularly useful for detecting and quantifying microstructural changes associated with brain injuries in children born very preterm. Focal brain injuries can affect overall brain development (Chau et al. 2009; Dubois et al. 2008b; Inder et al. 1999), and lead to moderate to severe neurodevelopmental disability (Inder et al. 2005; Miller et al. 2005; Woodward et al. 2006). However, DTI measures of brain microstructure have been shown to be better predictors of outcomes in children born very preterm seen at 18 months corrected age (CA), than white matter injuries observable on MRI (Chau et al. 2013). Using DTI imaging, our group has found widespread alterations in microstructure that have been associated with the 	   22 infant’s clinical condition (Bonifacio et al. 2010; Chau et al. 2009; Chau et al. 2013; Deipolyi et al. 2005) and interventions (Brummelte et al. 2012; Zwicker et al. 2013) during neonatal care. Our group is using DTI to study the relationship between repeated exposure to invasive procedures in the NICU and brain development in children born very preterm.  1.14 Invasive Procedures in the NICU and Stress Exposure to invasive procedures activates the sympathetic adrenomedullary system, and the hypothalamic-pituitary adrenal (HPA) axis. However, in the NICU, stress from the invasive procedures cannot be differentiated from stress due to factors such as illness and maternal separation. Therefore, measures of stress hormones in the NICU are not specific to pain and may even be downregulated in the youngest, sickest infants due to repeated stimuli (Bolt et al. 2002; Grunau et al. 2005; Scott and Watterberg 1995).    1.14.1 Stress Systems The sympathetic adrenomedullary system is part of the sympathetic division of the autonomic nervous system, which increases circulating epinephrine to facilitate rapid mobilization of metabolic resources and activate the fight/flight response. Epinephrine produced by the adrenal medulla, and norepinephrine produced by the postganglionic sympathetic neurons act together to activate metabolic resources, increasing heart rate, dilating blood vessels in the muscles, and constricting blood vessels in the skin and gut to ensure adequate blood supply to vital organs and muscles (Gunnar and Quevedo 2007; Tsigos and Chrousos 2002). Epinephrine also stimulates glycogenesis of the liver resulting in and increase in serum glucose, while norepinephrine promotes vigilance, arousal, and attention, and activates the HPA system (Gunnar and Quevedo 2007).  	   23   Stress triggers a cascade of events activating the HPA axis, which results in the production of glucocorticoids by the adrenal cortex. Activation of the hypothalamus leads to the co-release of corticotropin-releasing factor (CRF) and arginine vasopressin (Francis et al. 1999b; Gunnar and Quevedo 2007; Heim and Nemeroff 2001; Peters 1998), which in turn stimulates the synthesis and release of adrenocorticotropin from the anterior pituitary. This influences the release of glucocorticoids (cortisol in humans) from the adrenal cortex into the general circulation (Francis et al. 1999b; Gunnar and Quevedo 2007; Heim and Nemeroff 2001; Peters 1998). Cortisol binds with glucocorticoid receptors in the hypothalamus, hippocampus and other brain regions to inhibit further production of cortisol (Francis et al. 1999b; Gunnar and Quevedo 2007). Importantly, glucocorticoids regulate the transcription of genes (Chrousos 2009). Given the widespread actions of cortisol on multiple systems, repeated exposure to invasive procedures could lead to the suppression of growth, dampening of the immune system, and changes to the developing brain.   1.15 Neonatal Stress and Growth Although approximately 80% of preterm infants born very preterm are born an appropriate weight for their GA, many preterm infants develop persistent growth deficits postnatally (Steward and Pridham 2002; Wilson et al. 1997). By discharge from the NICU the majority of preterm infants are considered growth restricted, that is, <10th percentile for their PMA (Steward and Pridham 2002; Wilson et al. 1997). Cortisol inhibits growth hormone secretion (Tsigos and Chrousos 2002). Growth hormones stimulate the production of insulin-like growth factor 1 (IGF-1) from the liver, which is important for the promotion of cell growth, and multiplication and inhibition of apoptosis in cells throughout the body. IGF-1 is lower in preterm infants (Cutfield 	   24 et al. 2004), and is associated with lower body weight, length and head circumference at birth (Lo et al. 2002). Moreover, poorer postnatal growth has been associated with lower IGF-1 levels (Ahmad et al. 2007). Postnatal growth failure in the NICU is associated with increased incidence of cerebral palsy and neurodevelopmental impairment, after accounting for prenatal growth, systemic illness, and brain injury (Ehrenkranz et al. 2006). Therefore, it is important to identify and ameliorate factors, which may impact postnatal growth in infants born very preterm.   Ongoing pain/stress in the NICU may contribute to suppression of postnatal growth, given that early environmental stressors in animals have been shown to induce slower body weight gain (Bhatnagar et al. 2006; Gamallo, Villanua, Beato 1986). Reduced weight gain has been reported in rat pups exposed to pain for the first 7 days of life (Anand et al. 1999). Invasive procedures, a common stressor in the NICU, have not been examined in relation to postnatal growth in infants born very preterm. However, very preterm infants who received massage therapy for three 15-minute periods over 5 consecutive days did gain more weight during hospitalization, and had higher IGF-1 levels (Field et al. 2008). These relationships could not be attributed to caloric intake (Field et al. 2008). It is possible that massage therapy led to the reduction of stress, and increase in vagal activity (Acolet et al. 1993; Diego, Field, Hernandez-Reif 2005; Hernandez-Reif, Diego, Field 2007), thereby allowing for the secretion of growth hormones, promoting growth in the preterm neonates. However, more studies are needed to identify whether pain and/or stress is one of the factors contributing to the persistent growth deficits in the NICU.  1.16 Neonatal Stress and Immune Function Another important factor to consider is the effect that ongoing neonatal pain/stress can have on immune function. Inflammatory cytokines, tumor necrosis factor-α, interleukin-1β and 	   25 interleukin-6 can activate the HPA axis alone, or in combination with each other (Chrousos 1995; Tsigos et al. 1997). Activation of the HPA axis has profound inhibitory effects on the inflammatory/immune response, as almost all the components of the immune response are inhibited by cortisol production (Chrousos 1995; Elenkov et al. 1999). Therefore, repeated exposure to invasive procedures in the NICU may lead to immune suppression. This is of concern given that infants born preterm are already at increased risk for numerous neonatal complications that are related to inflammation and immune function (e.g. infection, necrotizing enterocolitis, chronic lung disease; Rubens et al. 2014). Infants born earlier and sicker require more medical interventions to ensure their survival. Therefore, there are close relationships between infant GA at birth, illness severity, PMA, mechanical ventilation and invasive procedures. While at this time it is difficult to prevent premature birth (Rubens et al. 2014), reducing pain/stress in the NICU may allow clinicians the opportunity to improve neurodevelopmental outcomes among children born very preterm.    1.17 Neonatal Stress and Brain Development Glucocorticoids are involved in normal brain maturation and cell survival (Korte 2001; Meaney et al. 1996; Meyer 1983). However, either excess levels or too low levels of glucocorticoids can have deleterious effects on the developing brain. Glucocorticoids increases serotonin (5HT) transporter expression, thereby reducing 5HT availability both in the hippocampus and throughout the brain (Fumagalli et al. 1996; Slotkin et al. 1996). This is important because 5HT acts on ketanserin-sensitive 5HT7 receptor subtypes within the hippocampus to stimulate cyclic adenosine monophosphate (cAMP; Meaney et al. 2000; Yau et al. 1997). cAMP induces the expression of nerve growth factor 1-A (NGF1-A), which binds to the glucocorticoid receptor gene promoter (Meaney et al. 2000). Fewer glucocorticoid receptors within the hippocampus 	   26 leads to poorer negative feedback following glucocorticoid expression (Francis et al. 1999b; Gunnar and Quevedo 2007). Rat pups exposed to early in life stress, have fewer hippocampal glucocorticoid receptors, and higher corticotropin releasing factor, adrenocorticotropin and corticosterone production, during adulthood (Meaney et al. 1996). Similarly, rat pups exposed to inflammatory pain on day 1 of life have fewer hippocampal glucocorticoid receptors, and an attenuated stress response during adulthood (Victoria et al. 2013). Results from these animal studies suggest that there is the potential for repeated exposure to invasive procedures in the NICU to lead to poorer glucocorticoid feedback and altered brain maturation in infants born very preterm, which may persist in adulthood.   While very preterm infants are in the NICU, their cortisol levels are frequently lower than expected, considering the amount of pain/stress they are exposed to during hospitalization (Fernandez and Watterberg 2009; Grunau et al. 2005; Peters 1998). Downregulation of glucocorticoids may occure among these physiologically immature neonates due to multiple clinical factors such as illness and infection in this medical context. Greater exposure to invasive procedures in the NICU has been associated with lower cortisol responses to stress at 32 weeks PMA, after statistically adjusting for GA a birth, early illness severity and morphine exposure (Grunau et al. 2005). Futhermore, greater exposure to neonatal pain/stress after accounting for neonatal risk factors related to prematurity, is associated with an altered trajectory of cortisol expression from infancy through age 7 years (Brummelte et al. 2015; Grunau et al. 2005; Grunau et al. 2013; Grunau, Weinberg, Whitfield 2004). It would appear that repeated exposure to stress early in life programs the endocrine stress system to prepare for a stressful postnatal environment.   	   27 1.18 Repeated Exposure to Invasive Procedures in the NICU and the Developing Brain There is a major literature in preterm children describing the neurodevelopmental and behavioral differences between children born very preterm and full-term (e.g. Anderson, Doyle, Victorian Infant Collaborative Study Group 2003; Doyle, Casalaz, Victorian Infant Collaborative Study Group 2001; Doyle and Anderson 2010; Grunau, Whitfield, Fay 2004; Johnson et al. 2009; Loe et al. 2011; Marlow et al. 2005; Marlow et al. 2007; Spittle et al. 2009). It has also been well-established that individuals born preterm demonstrate altered brain maturation (e.g. reduced brain volumes) throughout infancy (Nguyen et al. 2009; Srinivasan et al. 2007; Thompson et al. 2007; Thompson et al. 2011), childhood (Kesler et al. 2004; Kesler et al. 2008; Lax et al. 2013; Lowe et al. 2012; Peterson et al. 2000; Yung et al. 2007), adolescence (Gimenez et al. 2006; Nagy, Lagercrantz, Hutton 2011; Nosarti et al. 2002; Nosarti et al. 2008; Nosarti et al. 2011), and young adulthood (Aanes et al. 2015; Bjuland et al. 2014; Lawrence et al. 2014; Nosarti et al. 2014), in comparison to individuals born full-term. Perinatal and neonatal risk factors such as postnatal infection have been identified as being linked to altered brain development (Chau et al. 2009; Chau et al. 2012; Glass et al. 2008; Leviton et al. 2010; Miller et al. 2005; Shah et al. 2008). Recent studies by Brummelte et al. (2012), and Smith et al. (2012) have demonstrated for the first time in very preterm infants that repeated exposure to invasive procedures in the NICU was associated with altered brain development from early in life to term-equivalent age, above and beyond known risk factors related to prematurity (Brummelte et al. 2012; Smith et al. 2011). These studies are supported by animal models, which have demonstrated that both inflammatory pain and repeated injections increase cell death in the neonatal rat brain (Anand et al. 2007; Duhrsen et al. 2013; Rovnaghi et al. 2008). Associations between the number of invasive procedures in the NICU and brain development also appear to extend beyond the relationships early life (Doesburg et al. 2013; Ranger et al. 2013). At 7 years of age, higher numbers of 	   28 invasive procedures in the NICU were associated with thinner cortical gray matter in 21 out of 66 cerebral regions assessed, predominately affecting the frontal and parietal lobes (Ranger et al. 2013). Moreover, among infants born <28 weeks GA, greater exposure to neonatal pain/stress was also associated with alterations in spontaneous neuromagnetic activity (Doesburg et al. 2013). Therefore, it appears that repeated exposure to invasive procedures in the NICU may be altering brain development and may account for some of the neurodevelopmental and behavioral differences observed between infants born preterm versus full-term.   1.19 Early Exposure to Stress and Cognitive, Motor and Behavioral Outcomes Given that ongoing exposure to stress early in life is associated with changes in hormones, brain structure and function, we would also expect to find relationships between exposures to neonatal pain/stress and neurodevelopmental outcomes. Early maternal separation in animal models causes significant distress and is commonly used as a neonatal stressor in basic research. Rat pups periodically separated from their mothers demonstrated more anxiety-like behaviors and cognitive impairments as adults, in comparison with pups that either remained with their mother or were briefly handled (Aisa et al. 2007; Kalinichev et al. 2002; Ogawa et al. 1994; Oomen et al. 2010; Wigger and Neumann 1999). Similarly peer-reared rhesus monkeys demonstrated reduced locomotion, fewer interactive behaviors, and increased stereotypical behaviors compared to the mother-reared monkeys (Feng et al. 2011). Therefore, animals models of early life stress have revealed that greater exposure to stress in the first week(s) of life results in poorer cognitive and behavior outcomes.  In recent years, studies have started to explore the potential long-term impact of neonatal pain/stress on neurodevelopmental outcomes of both humans and animals. Rat pups exposed to 	   29 either repeated skin-breaking procedures or inflammatory pain in the first week of life, demonstrated poorer cognitive, behavioral and motor outcomes during adulthood (Anand et al. 1999; Bhutta et al. 2001; Negrigo et al. 2011; Rovnaghi et al. 2008). In humans, higher numbers of invasive procedures in the NICU were also associated with poorer cognitive and motor outcomes at 8 and 18 months CA in children born very preterm, independent of early illness severity, morphine and postnatal corticosteroid exposure (Grunau et al. 2009). Moreover, greater exposure to invasive procedures was associated with more internalizing (anxious/depressive) behaviors at 7.5 years of age in non-ventilated children born very preterm, after accounting for neonatal confounders and concurrent parenting stress (Ranger et al. 2014). While previous research, particularly studies using animal models, has given us insight into how repeated exposure to invasive procedures may be lead to adverse outcomes in children born very preterm, more research is required in order for us to understand the mechanisms underlying these relationships.  1.20 Caregivers and Stress Management Parenting plays a central role in stress regulation and normal brain development in both humans and animals.   1.20.1 Maternal Licking and Grooming Behavior in Rats  Positive parental interaction is important not only for the relationship between the parent and child, but also, for the overall development of the child, particularly in at risk children, such as those born very preterm (Brummelte et al. 2011b; Crnic and Greenberg 1987; Tu et al. 2007). Animal models have given us insight into the mechanisms that underlie the importance of positive maternal interactions. Rat dams exhibit considerable variation in the amount they lick 	   30 and groom their pups (Champagne et al. 2003), and these behaviors remain stable across their litters. Variations in pup licking and grooming during the first week of life affected HPA and behavioral responses to stress, and were correlated with hippocampal glucocorticoid receptor expression in adulthood (Caldji et al. 1998; Francis et al. 1999a; Liu et al. 1997; Menard, Champagne, Meaney 2004; van Hasselt et al. 2012; Weaver et al. 2004; Zhang et al. 2006). The adult offspring of low licking and grooming mothers showed reduced hippocampal glucocorticoid receptor expression, poorer glucocorticoid feedback sensitivity, greater corticotrophin releasing factor and greater glucocorticoid production in comparison to pups reared by high licking and grooming mothers (Francis et al. 1999a; Liu et al. 1997). These changes in physiology were related to alterations in behavior as adults. Offspring of low licking and grooming mothers showed greater anxiety-like behavior and deficits in spatial learning and memory (Caldji et al. 1998; Liu et al. 2000; Pena et al. 2014; Starr-Phillips and Beery 2014; van Hasselt et al. 2012). Therefore, variations in maternal behavior are related to differences in HPA axis functioning, as well as alterations in learning and behavior in offspring. Given that the same pathways altered by repeated exposure to early life stress are programmed by maternal behavior, positive maternal behavior may ameliorate some of the negative effects of repeated exposure to early life stress on brain and stress system development, as well as neurodevelopmental and behavioral outcomes.     1.20.2 Maternal Interaction in the NICU The hospitalization of neonates born very preterm interferes with mother-infant bonding, given that these infants spend prolonged periods in an incubator, often separate from their mother. Kangaroo care, also known as skin-to-skin contact, involves resting a diapered infant on the caregiver's bare chest. The skin-to-skin contact is an efficacious method for reducing pain-	   31 related stress and improving pain regulation in infants born preterm (Pillai Riddell et al. 2011). Parent sensitivity training in the NICU is designed to help parents recognize signs of infant stress and teach them how to proactively soothe their infant without overwhelming them (Milgrom et al. 2010; Milgrom et al. 2013; Rauh et al. 1990). In a randomized control trial comparing mothers who received stress sensitivity training versus standard care, in just a few short sessions with a developmental specialist, mothers in the intervention group demonstrated greater sensitivity to infant cues, and were able to reduce infant stress behavior (Milgrom et al. 2013). Importantly, infants of mothers who received sensitivity training showed increased maturation of the white matter microstructure at term equivalent age (Milgrom et al. 2010), and more advanced communication development at 6 months CA, compared to controls (Milgrom et al. 2013). Participation in this training in the NICU, plus 4 training sessions at home was found to reduce parental concerns about the infant long after discharge from the NICU (Landsem et al. 2014). Therefore, supporting positive parent interactions in the NICU, and involving parents in infant care is important for minimizing neonatal pain/stress and improving outcomes after discharge from the NICU.   1.21 Parenting Stress Stress in families with preterm infants has been found to be high during infant hospitalization (Glazebrook et al. 2007; Poehlmann and Fiese 2001; Thomas, Renaud, Depaul 2004), and persists well beyond discharge from the NICU (Brummelte et al. 2011a; Garel, Dardennes, Blondel 2007; Singer et al. 2003; Treyvaud et al. 2014). Parenting stress may partly reflect realistic concerns regarding the child’s development (Brummelte et al. 2011a; Docherty, Miles, Holditch-Davis 2002). In infants born very preterm, decreasing cognitive scores between 8 and 18 months CA was associated with higher parenting stress (Brummelte et al. 2011a). However, 	   32 other factors, such as lower maternal education, a predictor of premature birth and a standard indicator of socioeconomic status, was also associated with greater parenting stress in mothers of infants born very preterm (Brummelte et al. 2011a; Docherty, Miles, Holditch-Davis 2002; Woodward et al. 2014). Lower parenting stress was associated fewer internalizing behaviors at school age in children born very preterm (Ranger et al. 2014). However, it is important to note that this relationship is bidirectional, such that greater child internalizing may have contributed to higher parenting stress. Lower parenting stress may modulate the adverse effects of repeated invasive procedures on negative reactivity at 8 months CA (Voigt et al. 2013), and cognitive outcomes at 18 months CA (Grunau et al. 2009). Furthermore, the relationship between maternal stress and outcomes may be modulated by maternal behavior. Mothers who reported low concurrent stress were more sensitive when interacting with their child post-discharge (Muller-Nix et al. 2004; Tu et al. 2007). However, among mothers who reported low concurrent stress, maternal behavior buffered the relationship between greater exposure to invasive procedures and poorer focused attention (Tu et al. 2007). It is recognized in the literature that the birth of a preterm child is stressful for parents and families, and that greater parenting stress is associated with poorer outcomes among children born very preterm. Importantly, involving parents in NICU care may lower parenting stress, improve parent-child interactions and contribute to the optimization of neurodevelopmental and behavioral outcomes in children born very preterm.   1.22 Maternal Interaction Post-Discharge from the NICU The caregiver continues to be important for regulating the HPA axis in premature infants post-discharge. In humans, positive maternal interaction at 18 months CA was associated with better regulation of cortisol levels in children born very preterm (Brummelte et al. 2011b). In neonatal rats, cross-fostering the animals to high licking and grooming mothers, and enriching social 	   33 environments post-weaning, reverses the adult phenotype associated with poor quality maternal care (Champagne and Meaney 2007; Francis et al. 1999b). Based on these findings, through sensitive and responsive caregiving, parents of children born very preterm may be able to ameliorate the long-term effects early environmental pain/stress and maternal deprivation on the brain and neurodevelopmental and behavioral outcomes, post-discharge from the NICU.   Children born very preterm are particularly influenced by their environmental context and are more greatly affected by their interactions with their parents than their term-born peers (Brummelte et al. 2011b; Crnic and Greenberg 1987; Erickson et al. 2013; Forcada-Guex et al. 2006; Tu et al. 2007). There is debate as to whether the quality of interaction differs between parents of preterm versus full-term children, given that the differences between these two groups narrows, after accounting for factors such as maternal education, parenting stress and child IQ (Brummelte et al. 2011b; Forcada-Guex et al. 2006; Greenberg and Crnic 1988; Greene, Fox, Lewis 1983; Harrison 1990; Jaekel, Wolke, Chernova 2012; Muller-Nix et al. 2004; Potharst et al. 2012; Rahkonen et al. 2014; Tu et al. 2007). However, specifically among families with children born very preterm, higher quality parent-child interactions post-discharge were associated with better cognitive and behavioral outcomes (Beckwith, Rodning, Cohen 1992; Erickson et al. 2013; Magill-Evans and Harrison 2001; Rahkonen et al. 2014; Spittle et al. 2010; Tu et al. 2007). Therefore, evidence-based interventions focusing on the parent-child relationships may lead to improved outcomes in children born very preterm.    1.23 Caregiver Emotional Availability  A relatively new construct used to assess the parent-child relationship is emotional availability (EA). Attachment theory was one of the founding concepts of EA, due to its emphasis on 	   34 appropriate responding to infant cues and communications (Ainsworth et al. 1978; Bowlby 1969; Bowlby 1973). In addition to attachment theory, several other influences contributed to the conceptualization of EA. Mahler et al. (1975) first used the term “emotional availability” to describe a mother’s supportive presence and encouragement during child exploration (Mahler, Pine, Bergman 1975). Emde and Easterbrooks (1985) described emotional availability as being an affective barometer of the relationship. Finally, the systems view proposed by Guttman (1991) was important for its description of reciprocity of influence, where each person both contributes to the relationship, and is affected by the other person’s involvement in the relationship (Guttman 1991). Each of the above perspectives contributed to the conceptualization of EA and provided the foundation for the observation scales developed by Biringen and colleagues (Biringen, Robinson, Emde 1998; Biringen et al. 2014; Biringen 2000; Biringen 2008). These scales evaluate the caregiver and child as an interactive dyad, where each person in the relationship is capable of influencing and affecting how the other responds. Thus, the EA scale examines whether a caregiver is supportive of their child, and whether the caregiver possesses an authenticity of affect, appropriate responding (sensitivity/nonhostility/nonintrusiveness), and provision of guidance (structuring), which encourages the child to respond and/or involve the caregiver in their play or general activities (Biringen 2008). The EA scale is comprised of four parent dimensions, caregiver Sensitivity, Structuring, Nonintrusiveness and Nonhostility (Biringen 2008). The descriptions for each of these parent behaviors are listed below, and reflect the individual items addressed in each dimension included in the 4th edition of the EA scales (Biringen 2008).     1.23.1 EA Caregiver Sensitivity Sensitivity reflects expression of genuineness and appropriateness of affect. For example the 	   35 caregiver may generally be balanced and low-keyed. When appropriate, however, they may be more animated in their behavior, and demonstrate a clear enjoyment of the child. The caregiver should be aware of their child’s signals (i.e. approach, avoidance), and demonstrate a willingness to respond their needs. The caregiver should also be aware of their own timing (e.g. abruptness) while interacting with their child. To be considered sensitive the caregiver must also demonstrate flexibility and creativity during play with the child. Finally, a sensitive caregiver speaks and acts in respectful ways, is present within the interaction, and effectively moves conflicts towards a resolution.   1.23.2 EA Caregiver Structuring Structuring requires that the caregiver provides guidance, and creates an environment that holds the child’s attention. A caregiver is considered emotionally available in their structuring if they use subtle and varied suggestions, to bring the child’s learning to a higher level. Such scaffolding may include verbal and nonverbal cues, but should not overwhelm the child. Within a given task an emotionally available parent should set appropriate limits, remain committed to those boundaries, and maintain a controlled environment.    1.23.3 EA Caregiver Nonintrusiveness   Child exploration is a stepping-stone to acquiring autonomy. It is important that within the limits the caregiver sets for the child that the parent allow the child space to figure out tasks/challenges, without controlling, manipulating (physically or verbally) or completing the exercise for the child. An emotionally available, nonintrusive, caregiver will wait for the opportune time to enter the interaction and/or offer assistance, as opposed to interrupting the flow verbally or physically (except in cases of emergency). They will also use commands sparingly. Talking and teaching 	   36 should be an interactive form of communication between the parent and child.   1.23.4 EA Caregiver Nonhostility  Emotionally available nonhostile caregivers will have control over their emotions. They will attempt to avoid being overly negative or stressed when interacting with the child. This includes maintaining control over both verbal and nonverbal expressions. Caregivers will receive lower scores on the EA scale if they mock, ridicule, or express any kind of disrespectful statement or behavior (e.g. boredom, impatience). Hostility also includes using silence and/or leaving as a threat to gain control over their child, not just the use physical or verbal assaultive behaviors. Furthermore, a nonhostile caregiver will play in a manner that is appropriate to the context/materials and is not unnecessarily malevolent.  1.24 Maternal Emotional Availability in Infants Born Very Preterm Emotional Availability is a relatively new construct, and few studies have examined parental EA in families with children born very preterm. When infants were 5 months CA, mothers who reportedly perceived their preterm infant as being more vulnerable, behaved more intrusively, and displayed greater hostility while interacting with their child (Stern et al. 2006). This corresponds with earlier research, using other mother-infant interaction scales, which has shown that mothers of preterm infants may work harder to engage their infants, and display less positive affect during interactions with their infant compared to parents of infants born full-term (Brachfeld, Goldberg, Sloman 1980; Crnic et al. 1983). Higher maternal education and lower maternal anxiety in the NICU were associated with higher maternal sensitivity at 24 months CA, after adjustment for birth weight (Zelkowitz et al. 2009). Although Zelkowitz et al. (2011) reported significant correlations between maternal sensitivity and child cognitive, motor and 	   37 behavioral development at 24 months CA, the relationships between concurrent maternal behavior and outcomes were no longer significant after adjustment for GA, neonatal morbidity, maternal education and anxiety (Zelkowitz et al. 2011). Therefore, it is not clear to what extent parental EA may moderate the relationships between maternal and environmental stressors and child neurodevelopmental and behavioral outcomes. It would appear, however, that parental EA is an important factor to consider when examining the etiology of child developmental outcomes.   1.25 Dissertation Overview Infants born very preterm have the nociceptive circuitry required to perceive pain. However, tactile threshold is lower, descending inhibitory pathways are immature, and neonates become sensitized to repeated tactile and skin-breaking stimulation, leading to greater sensitivity to invasive procedures during this vulnerable period. As part of their life-saving care, infants born very preterm undergo repeated invasive procedures in the NICU. These procedures occur during a period of rapid brain and stress system development, and therefore, repeated exposure to invasive procedures in the NICU could lead to alterations in brain microstructure. Dysmaturation of the cerebral white and gray matter may underlie the persistent differences in cognitive, motor and behavioral outcomes observed between children born very preterm versus full-term. Importantly, positive parental interaction may partially ameliorate the long-term effects of repeated exposure to neonatal stress in the NICU, suggesting opportunities for intervention.    The purpose of this dissertation is to firstly examine whether repeated exposure to invasive procedures during neonatal intensive care is associated with altered brain microstructure, postnatal growth, and neurodevelopment in children born very preterm. A further aim was to examine for the first time the extent that parental emotional availability may either ameliorate or 	   38 exacerbate the relationships of invasive procedures with child behavior at 18 months CA. Studies presented in Chapters 2 and 3 include data from a a prospective cohort of infants recruited from the NICU at the BC Children’s and Women’s Hospital, born between March 2006 and January 2009, and followed to term equivalent age. Studies presented in Chapters 4 and 5 include data from an earlier prospective cohort of infants recruited from the same NICU, born between February 2001 and July 2004, and followed to age 7 years.   Chapter 2 is the first known study to examine whether exposure to invasive procedures in the NICU is related to postnatal body and head growth in infants born very preterm, after adjusting for age, prematurity, size at birth, and clinical factors. We found that greater exposure to invasive procedures in the NICU was associated with delayed early postnatal body and head growth, independent of medical confounders. This work has uniquely identified repeated exposure to pain/stress as one of the factors underlying early postnatal growth failure in the NICU in infants born very preterm.   Given our finding that repeated exposure to invasive procedures was associated with slower growth in the NICU, using data from the same cohort in Chapter 3 we examined whether postnatal growth indicated by change in weight, length and head circumference between approximately 32 and 40 weeks PMA is related to cortical brain development in infants born very preterm, after controlling for GA, size at birth, sex, PMA, brain injury, and systemic illness. We found that impaired postnatal growth (weight, length, and head circumference) was significantly associated with delayed cortical maturation, even after adjusting for neonatal and medical confounders. However, it was the cortical gray matter, rather than the white matter, which appeared to be most susceptible to impairments in postnatal growth. This study 	   39 demonstrated for the first time that postnatal growth, over and above prenatal growth, systemic illness and brain injury was associated with cerebral cortical maturation in children born very preterm.   Previously, in the cohort born between February 2001 and July 2004, our group found that in mothers with lower parenting stress, maternal interactive behavior buffered the relationship between greater pain/stress in the NICU and poorer attention at 8 months CA (Tu et al. 2007). When these same children were seen at 18 months CA, more positive mother-child interactions were associated with better cortisol regulation during cognitive testing, and fewer anxious/depressive behaviors in children born very preterm (Brummelte et al. 2011b).  My research extended these findings in the same cohort, by introducing the newer construct of emotional availability (EA) in parent-infant interaction. In Chapter 4 we examined whether parent EA (adjusted for parenting stress), moderates the relationship between pain/stress in the NICU and internalizing behaviors (adjusted for child cognition) in children born very preterm. Furthermore, we also examined the relationship between parent EA and internalizing behavior in healthy full-term controls who were born at the same hospital. Among the very preterm children, exposure to a higher number of invasive procedures (adjusted for confounding neonatal medical factors), greater parent sensitivity and nonhostility was associated with fewer internalizing behaviors at 18 months CA. In contrast, none of the parent factors was a significant predictor of internalizing behavior in children born full-term. Therefore, our group has robustly demonstrated using two different scales to assess parent behavior that positive parent-child interactions can moderate the adverse relationships between greater pain/stress and neurodevelopmental and behavioral outcomes in children born very preterm.   	   40 Previously, in the cohort born between March 2006 and January 2009, our group found a relationship between greater exposure to pain/stress and altered white matter maturation from early in life to term-equivalent age, after adjusting for neonatal clinical confounders (Brummelte et al. 2012; Zwicker et al. 2013). Using data from the other cohort of children born very preterm, born February 2001 and July 2004, in Chapter 5 we examined whether the extent of pain/stress during NICU care was associated with maturation of white matter microstructure at age 7 years, and whether pain/stress together with measures of brain microstructure were associated with cognitive outcome at school age in children born very preterm, after accounting for degree of prematurity, systemic illness, medications, and concurrent brain injury. Greater number of invasive procedures during neonatal care was associated with altered white matter microstructure at school age. Moreover, greater exposure to invasive procedures together with altered myelination of the white matter, predicted lower IQ among children born very preterm, even after adjustment for neonatal and medical confounders. The study in Chapter 5, built on our previous work, to now provide evidence from two independent cohorts, at two separate ages, that neonatal pain/stress is associated with altered brain development. Further, we provided the first link between greater pain/stress, altered brain microstructure and poorer cognitive development at school age.    Chapter 6 summarizes the results of this dissertation and discusses these findings within the context of the current theoretical field regarding the effects of pain on brain development, and parents as moderators of the long-term effects of stress in children born very preterm.      	   41 CHAPTER 2  NEONATAL PAIN IN RELATION TO POSTNATAL GROWTH IN INFANTS BORN VERY PRETERM 2.1 Introduction Invasive procedures are an inherent part of life-saving care in the NICU. However, chronic exposure to procedure-related pain/stress in the NICU may have detrimental effects on the growth and development of infants born very preterm. Infants born very preterm have a limited metabolic reserve (Polin, Fox, Abman 2003). Mounting a pain/stress response requires a substantial amount of energy. Greater numbers of invasive procedures in the NICU have been associated with a dampening of pain/stress and cortisol responses, and may lead to exhaustion and downregulation of resources among infants born very preterm (Grunau et al. 2001; Grunau et al. 2005).  Maintaining a consistent weight gain in infants born very preterm poses a major challenge for clinicians. Although approximately 80% of preterm infants are born at an appropriate weight (10th–90th percentile) for their GA and sex, during hospitalization the growth of the majority of preterm infants appears inadequate, such that by NICU discharge many are considered growth restricted (<10th percentile) (Ehrenkranz et al. 1999; Wilson et al. 1997). Prematurity and illness often prevent clinicians from being able to provide the daily-recommended dietary intake for newborns. Therefore, infants born very preterm develop a significant nutrient deficit over the first few weeks of life (Embleton, Pang, Cooke 2001). However, nutritional intake only accounts for approximately 45% of the variance in postnatal growth (Embleton, Pang, Cooke 2001). 1A version of Chapter 2 has been published in Vinall J, Miller SP, Chau V, Brummelte S, Synnes A, Grunau RE (2012). Pain, 153(7):1374-81. 	   42 Growth patterns can vary depending on GA, birth weight, days of respiratory support, and use of postnatal corticosteroids (Berry, Abrahamowicz, Usher 1997; Billeaud, Piedboeuf, Chessex 1992; Cockerill et al. 2006; Halliday, Ehrenkranz, Doyle 2010). Therefore, there are multiple factors to consider when evaluating predictors of postnatal growth. To the best of our knowledge, the relationship between invasive procedures in the NICU and postnatal growth has not been examined previously.  Growth hormones stimulate the production of IGF-1, a small peptide, which is primarily synthesized in the liver and is a key regulator of growth (Yamini et al. 2010). IGF-1 values are lower in preterm infants compared to infants born full-term (Cutfield et al. 2004), and are positively associated with body weight, length and head circumference at birth (Lo et al. 2002). IGF-1 values gradually increase after preterm birth and are correlated with postnatal weight gain (Kurtoglu et al. 2010; van de Lagemaat et al. 2013). Stress inhibits the production of growth hormones (Tsigos and Chrousos 2002). Reducing stress in the NICU may improve postnatal growth in infants born very preterm. In a randomized control trial after receiving a 3 µg/kg does of fentanyl, mechanically ventilated preterm infants showed increased IFG-1 and moderately lower cortisol values compared to infants that were given a placebo (Guinsburg et al. 1998).   In animal models, repeated exposure to stress is associated with slower body weight gain (Bhatnagar et al. 2006; Gamallo, Villanua, Beato 1986). Anand et al. (1999) exposed rat pups to either skin-breaking procedures or tactile stimulation daily for the first 7 days of life. Pups that underwent the painful procedures weighed less at postnatal day 8 and 15 (Anand et al. 1999). Moreover, repeated exposure to painful procedures was also associated with greater anxiety-like behaviors during adulthood (Anand et al. 1999). 	   43  Slower body weight gain and head growth in the NICU were associated with increased incidence of cerebral palsy and neurodevelopmental impairment (Ehrenkranz et al. 2006; Leppanen et al. 2014). Therefore, it is of the utmost importance that we understand the possible relationships between exposure to invasive procedures and postnatal growth, as this research may lead to evidence-based interventions to improve outcomes within this vulnerable population.   Hypothesis  We hypothesized that greater exposure to invasive procedures in the NICU would be associated with poorer growth in the first weeks of life and at term-equivalent age, independent of other neonatal and medical risk factors.   2.2 Methods 2.2.1 Study Overview Birth weight and birth head circumference (HC) were obtained from the neonatal medical chart review. Postnatal growth was assessed by recording weight and HC at approximately 32 weeks PMA (early weigh-in), and again at approximately 40 weeks PMA (later weigh-in). Age at the early and later weigh-ins were timed to coincide with other study examinations (Adams et al. 2010; Chau et al. 2009) and varied in cases where infants were unstable or were discharged prior to term-equivalent age. Therefore, PMA was included in statistical analyses to control for variability in age at each weigh-in. Variables obtained from chart review were recorded in ‘‘windows’’ based on birth, early and later weigh-ins (Figure 2.1). The early window included measures between birth and 32 weeks PMA (early weigh-in); the later window included measures between 32 (early weigh-in) and 40 weeks PMA (later weigh-in). Finally, we 	   44 combined the early and late windows to create one cumulative (early and late) neonatal window, which included measures between birth and 40 weeks PMA (later weigh-in).  Figure 2.1: Early and Late Neonatal Windows  Medical chart review was performed from birth to term-equivalent age or hospital discharge (whichever came first), and variables from chart review were divided into neonatal windows (early: birth to 32 weeks PMA; later: 32 to 40 weeks PMA; cumulative: early and late) to capture NICU events in relation to the ages infants were weighed and measured (i.e. birth, 32 and 40 weeks PMA).    2.2.2 Participants  A prospective cohort of 78 very preterm infants (≤32 weeks GA) born between March 2006 and January 2009 were recruited from the NICU at the British Columbia Children’s and Women’s Hospital, as part of a larger ongoing study of neonatal invasive procedures, brain and neurodevelopment (Adams et al. 2010; Chau et al. 2009; Grunau et al. 2005; Grunau et al. 2007; Grunau et al. 2009). Exclusions from the study were: a major congenital malformation or syndrome, antenatal infection, severe brain injury on neonatal ultrasound (i.e. large parenchymal 	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  40	  weeks	  (later	  weigh-­‐in)	  32	  weeks	  (early	  weigh-­‐in)	  Birth	  	  Early	  number	  of	  invasive	  procedures,	  days	  of	  ventilation,	  infection,	  morphine,	  hydrocortisone,	  dexamethasone	  exposure	  Later	  number	  of	  invasive	  procedures,	  days	  of	  ventilation,	  infection,	  morphine,	  hydrocortisone,	  dexamethasone	  exposure	  	  Illness	  severity	  day	  1	  	  Cumulative	  (early	  and	  late)	  number	  of	  invasive	  procedures,	  days	  of	  ventilation,	  infection,	  morphine,	  hydrocortisone,	  dexamethasone,	  exposure	  	   45 hemorrhagic infarction >2 cm) or missing neonatal data (e.g. due to transfer to hospitals outside of the Lower Mainland of British Columbia).    2.2.3 Weight Percentiles Infants were weighed and HC measured at birth (median GA 27 weeks; interquartile range [IQR] 25.9-29.7), within the first few weeks of life (early weigh-in: median PMA 32 weeks; IQR 30.7-33.6), and again at term-equivalent age or discharge (later weigh-in: median PMA 40 weeks; IQR 38.6-42.6). These time points were chosen to coincide with other study protocol measures and span the neonatal period (Adams et al. 2010; Chau et al. 2009). Values at all 3 time points were converted into percentiles from sex-specific British Columbia population-based data. Growth percentiles are normed for age and sex and provide a more meaningful description of postnatal growth than changes in raw values. For example, a change from the 8th (growth-restricted) to the 15th (appropriate weight for PMA) weight percentile is a more meaningful interpretation of growth than a change from 1500 to 3000 grams.   2.2.4 Neonatal Medical Chart Review A neonatal research nurse performed medical and nursing chart review from birth to term-equivalent age or discharge (whichever came first). Data included but were not limited to birth weight, GA at birth, illness severity on day 1 (Score for Neonatal Acute Physiology II [SNAP-II] (Richardson et al. 2001), number of invasive procedures (see Appendix Table A.1), days of mechanical ventilation, presence of infection, and exposure to morphine and corticosteroids (hydrocortisone, dexamethasone). Morphine, hydrocortisone and dexamethasone exposure were entered as binary variables reflecting whether or not these drugs were administered. A high score on the SNAP-II indicates greater illness severity. Postnatal infections were identified by positive 	   46 culture in the blood, urine, cerebral spinal fluid or if ≥4 white blood cells were found in the tracheal aspirates associated with clinical pneumonia. Given that these data were drawn from a longitudinal study designed to examine long-term effects of neonatal pain on brain and stress system development, data on nutrition were not collected.   2.2.5 Data Analyses Normality plots were examined and skewed variables (number of invasive procedures, birth weight percentile, weight and HC percentile at 32 and 40 weeks PMA) were log transformed. Analysis of variance was used to examine whether there were differences between the infants included and excluded in this study. Analysis of variance was also used to examine sex differences in the raw weights and HCs at birth, 32 and 40 weeks PMA, and to examine whether there were differences in the number of early, late and cumulative (early and late) invasive procedures performed on male and female preterm infants. Generalized linear models (GENLIN SPSS 18; IBM, Armonk, NY, USA) were used to examine the independent contributions of: 1) early neonatal variables (birth to 32 weeks PMA) to weight and HC percentile at 32 weeks PMA; 2) early neonatal variables (birth to 32 weeks PMA) to weight and HC percentile a 40 weeks PMA; 3) later neonatal variables (32 to 40 weeks PMA) to weight and HC percentile at 40 weeks PMA; and 4) cumulative (early and late/birth to 40 weeks PMA) variables to weight and HC percentile at 40 weeks PMA. Generalized linear modeling permits examination of potential confounders that are intercorrelated; predictors can be continuous, discrete, dichotomous, or a mix of these, as is the case with our data. This model uses the Wald test to examine the statistical significance of each coefficient B in the model.   	   47 2.3 Results 2.3.1 Characteristics of the Cohort There were no significant differences between the included and excluded infants on birth weight percentile, HC percentile or illness severity on day 1. Characteristics of the included infants are provided in Table 2.1. It is noteworthy that the median number of invasive procedures was more than double in the early neonatal window compared to after 32 weeks PMA. Male and female infants did not differ significantly in raw values of weight or HC at birth, 32 or 40 weeks PMA (each P> 0.25). Furthermore, there were no significant differences in the early, late or cumulative (early and late) number of invasive procedures performed on male and female infants (each P> 0.32). Sex-specific normative weight and HC percentiles were used in the generalized linear models for all data analyses, and sex was not considered further.             	  	   48  Table 2.1 Infant Characteristics Characteristics Neonatal Window  Birth to 32 weeks 32 to 40 weeks Birth to 40 weeks Birth weight (percentile), median (IQR)  34.5 (13.3-57.0) - - Birth HC (percentile), median (IQR) 40.0 (10.0-63.0) - - Sex, (% male) 50.0% - - Illness severity on day 1, median (IQR) 12.0 (5.0-24.3) - - PMA (weeks), median (IQR) 32.1 (30.7-33.6) 40.2 (38.6-42.6) - PMA weight (percentile), median (IQR) 9.0 (4.0-16.3) 18.5 (4.8-55.0) - PMA HC (percentile), median (IQR) 8.50 (4.0-17.0) 60.0 (26.3-87.8) - Number of invasive procedures, median (IQR) 64.5 (35.0-131.5) 30.5 (18.8-52.3) 113.0 (67.0-182.0) Mechanical ventilation (days), median (IQR) 19.0 (7.0-46.3) 17.5 (0.0-31.8) 42.5 (12.8-81.0) Morphine exposure, number (%) 47 (60) 11 (14) 50 (64) Hydrocortisone exposure, number (%) 11 (14) 5 (6) 14 (18) Dexamethasone exposure, number (%)  12 (15) 6 (8) 14 (18) Postnatal infection, number (%) 33 (42) 9 (12) 38 (49) IQR= Interquartile range; HC= Head circumference; PMA= Postmenstrual age  2.3.2 Early (Birth to 32 Weeks PMA) Neonatal Variables in Relation to Weight Percentile at 32 Weeks PMA Lower birth weight percentile, greater exposure to invasive procedures and dexamethasone exposure, were independently associated with decreased weight percentile at 32 weeks PMA, after adjusting for the number of days on mechanical ventilation, morphine exposure, hydrocortisone exposure, postnatal infection, illness severity on day 1, and PMA at weigh-in (Table 2.2). As shown in Fig. 2.2, invasive procedures prior to 32 weeks PMA accounted for approximately 21% of the variance in early body growth, and greater exposure to invasive 	   49 procedures was related to decreased weight percentiles at 32 weeks PMA, after accounting for multiple medical confounders.   Table 2.2 Early (Birth to 32 Weeks PMA) Neonatal Variables in Relation to Weight and HC Percentiles at 32 Weeks PMA Early Neonatal variables Weight Percentile at 32 weeks PMAa HC Percentile at 32 weeks PMAb  Wald χ2 P Wald χ2 P Birth weight percentile 124.45 0.001 - - Birth HC percentile - - 20.47 0.001 Number of invasive procedures 7.36 0.01 4.36 0.04 Days of mechanical ventilation 0.38 0.54 4.25 0.04 Morphine exposure 0.39 0.53 0.95 0.33 Hydrocortisone exposure 0.01 0.93 0.69 0.41 Dexamethasone exposure 4.83 0.03 0.05 0.82 Postnatal infection 0.43 0.51 1.43 0.23 Illness severity on day 1 2.06 0.15 1.02 0.31 PMA at 32 week weigh-in 0.38 0.55 15.36 0.001 HC= Head circumference; PMA= Postmenstrual age Generalized linear models revealed that between birth and 32 weeks PMA: alower birth weight percentile, greater number of invasive procedures, and dexamethasone exposure, independently predicted slower body growth after accounting for the other neonatal factors; blower HC birth percentile, greater neonatal number of invasive procedures and duration of mechanical ventilation, predicted slower head growth, after accounting for the other neonatal factors. Directions of relationships between variables were determined by B values (not shown).         	   50 Figure 2.2 Weight Percentiles at 32 Weeks PMA in Relation to the Number of Invasive Procedures from Birth to 32 Weeks PMA  Predicted values of early growth (weight percentile at 32 weeks PMA in relation to early number of invasive procedures from birth to 32 weeks PMA), adjusted for other neonatal variables (i.e. birth weight percentile, illness severity on day 1, PMA at 32-week weigh-in, days of mechanical ventilation, infection, and exposure to morphine and corticosteroids [hydrocortisone, dexamethasone]).     2.3.3 Early (Birth to 32 Weeks PMA) Neonatal Variables In Relation to Weight Percentile at 40 Weeks PMA  Lower birth weight percentile and hydrocortisone exposure, rather than the number of invasive procedures, were independently associated with decreased weight percentile at 40 weeks PMA, 	   51 after adjusting for morphine exposure, dexamethasone exposure, infection, illness severity on day 1, and PMA at weigh-in (Table 2.3). There was a trend for duration of mechanical ventilation from birth to 32 weeks PMA to be associated with lower weight percentile at 40 weeks PMA.  Table 2.3 Early (Birth to 32 Weeks PMA) Neonatal Variables in Relation to Weight and HC Percentile at 40 Weeks PMA Early Neonatal variables Weight Percentile at 40 weeks PMAa HC Percentile at 40 weeks PMAb  Wald χ2 P Wald χ2 P Birth weight percentile 19.91 0.001 - - Birth HC percentile - - 8.43 0.004 Number of invasive procedures 0.15 0.70 1.70 0.19 Days of mechanical ventilation 3.35 0.07 6.72 0.01 Morphine exposure 0.26 0.61 0.81 0.37 Hydrocortisone exposure 4.80 0.03 6.15 0.01 Dexamethasone exposure 0.08 0.76 2.60 0.11 Postnatal infection 0.14 0.71 0.01 0.92 Illness severity on day 1 2.26 0.13 0.19 0.67 PMA at 32 week weigh-in 2.10 0.15 4.76 0.03 HC= Head circumference; PMA= Postmenstrual age Generalized linear model revealed that between 32 and 40 weeks PMA: alower birth weight percentile and hydrocortisone exposure independently predicted slower body growth, after accounting for other neonatal factors; blower HC birth percentile and duration of mechanical ventilation predicted slower head growth, after accounting for the other neonatal factors. Directions of relationships between variables were determined by B values (not shown).    2.3.4 Later (32 to 40 Weeks PMA) Neonatal Variables in Relation to Weight Percentile at 40 Weeks PMA Lower weight percentile at 32 weeks PMA and later neonatal infection, rather than the number 	   52 of invasive procedures, were independently associated with decreased weight percentile at 40 weeks PMA, after adjusting for the number of days on mechanical ventilation, morphine, hydrocortisone and dexamethasone exposure, and PMA at weigh-in (Table 2.4).  Table 2.4 Later (32 to 40 Weeks PMA) Neonatal Variables in Relation to Weight and HC Percentile at 40 Weeks PMA  Later Neonatal variables Weight Percentile at 40 weeks PMAa HC Percentile at 40 weeks PMAb  Wald χ2 P Wald χ2 P Weight percentile at 32 weeks 63.58 0.001 - - HC percentile at 32 weeks - - 36.53 0.001 Number of invasive procedures 0.05 0.82 0.00 0.99 Days of mechanical ventilation 0.76 0.38 0.35 0.55 Morphine exposure 0.81 0.37 0.17 0.68 Hydrocortisone exposure 0.02 0.90 2.03 0.15 Dexamethasone exposure 0.39 0.53 3.79 0.05 Postnatal infection 5.09 0.02 0.85 0.36 PMA at 40 week weigh-in 50.54 0.001 39.71 0.001 HC= Head circumference; PMA= Postmenstrual age Generalized linear models revealed that between 32 and 40 weeks PMA: alower weight percentile at 32 weeks PMA and neonatal infection independently predicted slower body growth, after accounting for other later neonatal factors; blower HC percentile at 32 weeks PMA and dexamethasone exposure predicted slower head growth, after accounting for other later neonatal factors. Directions of relationships between variables were determined by B values (not shown).  2.3.5 Cumulative (Early and Late/Birth to 40 Weeks PMA) Neonatal Variables in Relation to Weight Percentile at 40 Weeks PMA Lower birth weight percentile, hydrocortisone exposure and infection, rather than the number of invasive procedures, were independently associated with lower weight percentile at 40 weeks PMA, after adjusting for the number of days on mechanical ventilation, morphine exposure, 	   53 dexamethasone exposure, illness severity on day 1, and PMA at weigh-in (Table 2.5).  Table 2.5 Cumulative (Early and Late/Birth to 40 Weeks PMA) Neonatal Variables in Relation to Weight and HC Percentile at 40 Weeks PMA Cumulative Neonatal Variables Weight Percentile at 40 weeks PMAa HC Percentile at 40 weeks PMAb  Wald χ2 P Wald χ2 P Birth weight percentile 23.19 0.001 - - Birth HC percentile - - 13.58 0.001 Neonatal pain (number of skin-breaking procedures) 1.64 0.20 0.92 0.34 Days of mechanical ventilation 0.001 0.98 0.54 0.46 Morphine exposure 1.52 0.22 0.10 0.76 Hydrocortisone exposure 4.23 0.04 15.43 0.001 Dexamethasone exposure 1.08 0.30 0.08 0.78 Postnatal infection 4.02 0.05 0.16 0.69 Illness severity on day 1 0.94 0.33 0.33 0.57 PMA at 40 week weigh-in 19.40 0.001 32.13 0.001 HC= Head circumference; PMA= Postmenstrual age  Generalized linear models revealed that between birth and 40 weeks PMA: alower birth weight percentile, hydrocortisone exposure and infection independently predicted slower body growth, after accounting for other neonatal factors; blower birth HC percentile and hydrocortisone exposure predicted slower head growth in the NICU, after accounting for other neonatal factors. Directions of relationships between variables were determined by B values (not shown).  2.3.6 Early (Birth to 32 Weeks PMA) Neonatal Variables in Relation to HC Percentile at 32 Weeks PMA Lower birth HC percentile, greater number of invasive procedures and longer duration of mechanical ventilation, were independently associated with decreased HC percentile at 32 weeks PMA, after adjusting for morphine, dexamethasone and hydrocortisone exposure, illness severity 	   54 on day 1, and PMA at weigh-in (Table 2.2). As shown in Figure 2.3, the number of invasive procedures prior to 32 weeks PMA accounted for approximately 12% of the variance in early head growth, and greater exposure to invasive procedures was related to decreased HC percentiles at 32 weeks PMA, after accounting for multiple medical confounders.                    	   55 Figure 2.3 Head Circumference Percentiles at 32 Weeks PMA in Relation to the Number of Skin Breaking Procedures from Birth to 32 Weeks PMA  Predicted values of early growth (weight percentile at 32 weeks PMA in relation to early neonatal pain (number of skin-breaking procedures from birth to 32 weeks PMA), adjusted for other neonatal variables (i.e. birth weight percentile, illness severity on day 1, PMA at 32-week weigh-in, days of mechanical ventilation, infection, and exposure to morphine and corticosteroids [hydrocortisone, dexamethasone]).  2.3.7 Early (Birth to 32 Weeks PMA) Neonatal Variables In Relation to HC Percentile at 40 Weeks PMA Lower birth HC percentile, longer duration of mechanical ventilation and hydrocortisone exposure, rather than the number of invasive procedures, were independently associated with decreased HC percentile at 40 weeks PMA, after adjusting for morphine exposure, 	   56 dexamethasone exposure, infection, illness severity on day 1, and PMA at weigh-in (Table 2.3).  2.3.8 Later (32 to 40 Weeks PMA) Neonatal Variables in Relation to HC Percentile at 40 weeks PMA Lower HC percentile at 32 weeks PMA and dexamethasone exposure, rather than the number of invasive procedures, were independently associated with decreased HC percentile at 40 weeks PMA, after adjusting for the number of days on mechanical ventilation, morphine exposure, hydrocortisone exposure, infection, and PMA at weigh-in (Table 2.4).  2.3.9 Cumulative (Early and Late/Birth to 40 Weeks PMA) Neonatal Variables in Relation to HC Percentile at 40 Weeks PMA Lower birth HC percentile and hydrocortisone exposure, rather than the number of invasive procedures, were independently associated with lower HC percentile at 40 weeks PMA, after adjusting for the number of days on mechanical ventilation, morphine exposure, dexamethasone exposure, infection, illness severity on day 1, and PMA at weigh-in (Table 2.5).  2.4 Discussion To our knowledge, this is the first study to examine the relationship between invasive procedures and postnatal growth in the NICU in infants born very preterm. Our results showed that the timing of invasive procedures and other neonatal interventions were important for postnatal body and head growth. Specifically, we demonstrated that greater exposure to invasive procedures was associated with delayed early postnatal body and head growth in the NICU, independent of other medical confounders. In contrast, reduced weight gain at term was associated with later neonatal infection. 	   57  Infants that are born earlier and sicker require more interventions to ensure their survival. Therefore, these factors are strongly associated with the number of invasive procedures in the NICU. In order to determine whether the number of invasive procedures in the NICU independently impacted postnatal growth, it was essential to statistically account for the multiple co-occurring factors. Invasive procedures rather than illness severity on day 1 of life was associated with postnatal growth in the NICU. This suggests that the rate at which infants grow in the NICU depends more on adversities encountered during the first weeks of life, as opposed to how sick the infants are at birth. By effectively managing pain/stress, clinicians may have the opportunity to improve postnatal growth in the NICU. However, despite best attempts to manage pain/stress in the NICU, this area of clinical care remains a challenge.    Prior to the 35 weeks PMA infants born very preterm cannot distinguish between nociceptive and mechanical stimulation (Andrews and Fitzgerald 1994; Fabrizi et al. 2011; Fitzgerald, Millard, McIntosh 1989; Grunau et al. 2001; Holsti et al. 2005; Holsti et al. 2006; Walker, Tochiki, Fitzgerald 2009). Therefore, infants born very preterm may experience pain/stress from both routine care and invasive procedures performed in the NICU. The majority of life-saving procedures are performed prior to 32 weeks PMA. While there are potential medical confounders (as in all clinical cohort studies) that can contribute to the number of invasive procedures performed in the NICU, we identified and statistically controlled for the key indicators. Even after accounting for early illness severity and markers of illness through the NICU stay, such as infection and duration of mechanical ventilation, the relationship between greater numbers of invasive procedures and impaired early growth persisted. The amount of energy expended in response to repeated stimulation, may have exhausted infants limited reserves, leading to slower 	   58 growth prior to 32 weeks PMA in infants born very preterm.   Lower pain/stress after 32 weeks PMA may have led to increased growth hormone production, and subsequent growth. At term age, variability in growth patterns increases among infants born very preterm (Yumani, Lafeber, van Weissenbruch 2015). For some infants, exposure to fewer invasive procedures after 32 weeks PMA may equate to rapid catch-up growth. However, environmental conditions must be conducive to postnatal growth. Therefore, even if the number of invasive procedures is reduced after 32 weeks PMA, infants born very preterm may continue to grow slowly if they are ill or continue to be exposed other environmental stressors (Yumani, Lafeber, van Weissenbruch 2015). This may explain why we found a relationship between later postnatal infection and slower postnatal body growth.   Unlike body growth, head growth appeared to be preserved after 32 weeks PMA. This is indicative of “brain protection,” as seen in growth-restricted fetuses, such that energy is preferentially directed to head growth (Peleg, Kennedy, Hunter 1998). However, head growth both before and after 32 weeks PMA may be impacted in infants with underdeveloped lungs. We found a positive association between days of mechanical ventilation prior to 32 weeks PMA and later head growth.   Corticosteroids are used to treat bronchopulmonary dysplasia, and consistent with previous reports, both dexamethasone and hydrocortisone exposure were associated with slower growth in the NICU (Doyle LW, Ehrenkranz RA, Halliday HL 2014a; Doyle LW, Ehrenkranz RA, Halliday HL 2014b). The administration of dexamethasone increases 5HT transporter expression, thereby reducing 5HT availability both in the hippocampus and throughout the brain 	   59 (Fumagalli et al. 1996; Slotkin et al. 1996). This is important because 5HT incites a downstream cascasde of molecular events within the hippocampus that induces the expression of NGF1-A, which binds to the glucocorticoid receptor gene promoter (Meaney et al. 2000; Yau et al. 1997). Therefore, exposure to corticosteroids results in fewer glucocorticoid receptors present within the hippocampus, which results in poorer negative feedback following glucocorticoid expression (Francis et al. 1999b; Gunnar and Quevedo 2007). Glucocorticoids (cortisol in humans) inhibit growth hormone secretion (Tsigos and Chrousos 2002), thereby resulting in slower growth. Cortisol is also expressed following neonatal pain/stress exposure. Importantly, even after statistically adjusting for corticosteroid exposure, a known predictor of reduced postnatal growth; neonatal pain/stress was still significantly associated with poorer growth early in the neonatal period.  Size at birth (weight and HC) continues to be a significant predictor of growth throughout infant hospitalization (Bhatnagar et al. 2006; Embleton, Pang, Cooke 2001). Typically infants born an appropriate size for GA require less medical support, and have better the growth trajectories in the NICU.    An important limitation of this study was that we did not have data on neonatal nutrition. Infants in this study were participants from a larger longitudinal study examining invasive procedures in relation to neurodevelopment and stress systems in infants born very preterm (Adams et al. 2010; Chau et al. 2009; Grunau et al. 2005; Grunau et al. 2007; Grunau et al. 2009). The original study was not designed with the intention of examining nutrition, caloric intake, and feeding of preterm infants. Future studies are needed to examine the role of nutrition in the relationship between invasive procedures and early postnatal growth in the NICU. Given that this was a 	   60 correlational study, future research is needed to confirm the mechanisms that underlie the relationship between invasive procedures and early postnatal growth in the NICU.  In conclusion, greater exposure to invasive procedures was associated with decreased early postnatal body and head growth in the NICU. Reducing pain/stress in the NICU may not only improve postnatal growth, but may also optimize long-term neurodevelopmental outcomes in infants born very prematurely.                   	   61 CHAPTER 3  SLOWER POSTNATAL GROWTH IS ASSOCIATED WITH DELAYED CEREBRAL CORTICAL MATURATION IN PRETERM NEWBORNS  3.1 Introduction Infants born very preterm have reduced brain volumes (Nguyen et al. 2009; Srinivasan et al. 2007; Thompson et al. 2007; Thompson et al. 2011), and poorer cognitive, motor and behavioral outcomes relative to children born full-term (Anderson, Doyle, Victorian Infant Collaborative Study Group 2003; Doyle, Casalaz, Victorian Infant Collaborative Study Group 2001; Doyle and Anderson 2010; Grunau, Whitfield, Fay 2004; Johnson et al. 2009; Loe et al. 2011; Marlow et al. 2005; Marlow et al. 2007; Spittle et al. 2009). As part of their life saving care infants born very preterm are repeatedly exposed to invasive procedures in the NICU. Greater exposure to invasive procedures is associated with poorer early body and head growth (Vinall et al. 2012) and altered white matter microstructure during NICU care and at term-equivalent age (Brummelte et al. 2012; Smith et al. 2011; Zwicker et al. 2013).   Infants born both earlier and smaller require more medical interventions given that they are at higher risk for neonatal comorbidities (Damodaram et al. 2011). Therefore, size at birth is a significant predictor of growth throughout infant hospitalization (Bhatnagar et al. 2006; Embleton, Pang, Cooke 2001; Vinall et al. 2012). Intrauterine growth restriction (IUGR) refers to infants whose birth weights are <10th percentile due to growth failure in utero. Premature  IUGR newborns demonstrate a pattern of discordant gyrification relative to preterm infants born an appropriate weight for gestation age (AGA: 10th to 90th percentile, such that their sulcation 1A version of Chapter 3 has been published in Vinall J, Grunau RE, Brant R, Chau V, Poskitt KJ, Synnes AR, Miller SP (2013). Sci Transl Med 5:168ra8. 	   62 index is high relative to cortical surface area (Dubois et al. 2008b). Preterm IUGR infants also demonstrate reduced cortical volumes and altered microstructure, relative to AGA preterm infants (Dubois et al. 2008b; Toft et al. 1995; Tolsa et al. 2004). Cortical gray matter appears to be more greatly impacted by growth restriction relative to white matter (Dubois et al. 2008b; Padilla et al. 2011; Toft et al. 1995; Tolsa et al. 2004). Moreover, abnormal cortical volumes in premature IUGR infants are associated with poorer neurodevelopmental outcomes at term and 18 months CA (Padilla et al. 2011; Tolsa et al. 2004).   The majority of preterm infants are born AGA, and develop persistent growth deficits postnatally (Steward and Pridham 2002; Wilson et al. 1997). Slower postnatal body and head growth in the NICU is associated with increased incidence of cerebral palsy and neurodevelopmental impairment, after accounting for prenatal growth, systemic illness, and brain injury (Ehrenkranz et al. 1999). It is not known whether alterations to brain microstructure mediate the relationship between postnatal growth and neurodevelopmental outcomes. One study has examined the relationship between postnatal growth and white matter microstructure at term (Lepomaki et al. 2013). Weight and length change from birth to term were not associated with white matter microstructure (Lepomaki et al. 2013). However, fast catch-up head growth between birth and term-equivalent age was associated less mature white matter microstructure at term age, after accounting for gestational age and birth weight (Lepomaki et al. 2013).  There are a number of additional factors to consider when evaluating the relationship between neonatal growth and cortical development in infants born very preterm. Systemic illness and medical interventions are important determinants of growth and brain development (Berry, Abrahamowicz, Usher 1997; Billeaud, Piedboeuf, Chessex 1992; Bonifacio et al. 2010; 	   63 Brummelte et al. 2012; Chau et al. 2012; Cockerill et al. 2006; Halliday, Ehrenkranz, Doyle 2010; Lodygensky et al. 2005; Vinall et al. 2012; Zwicker et al. 2013). Moreover, focal brain injuries have been found to affect overall brain development, leading to moderate to severe neurodevelopmental disability (Inder et al. 2005; Miller et al. 2005; Woodward et al. 2006). Therefore it is not only important to determine whether postnatal growth impacts brain development, but also whether this relationship exists independent of prematurity, illness severity, brain injury and exposure to medications in the NICU.   Hypothesis This study examines whether neonatal growth is related to microstructural development of the cerebral cortex in infants born very preterm. We hypothesized that poorer growth in the NICU would be associated with delayed cortical maturation, independent of prenatal growth, systemic illness, and brain injury.  3.2 Methods  3.2.1 Study Overview Infants born very preterm (between 24 and 32 weeks GA) were studied twice with DTI: scan 1 at a median of 32.1 weeks (IQR: 30.4 to 33.6) and scan 2 at a median of 40.3 weeks (IQR: 38.7 to 42.7). FA and eigenvalues were recorded from 15 anatomically defined cortical regions. Weight, head circumference, and length were recorded at birth and at the time of each scan. Growth between scans was examined in relation to DTI parameters at scans 1 and 2, accounting for GA, birth weight, sex, PMA, neonatal illness (patent ductus arteriosus, days intubated, infection, and necrotizing enterocolitis) and brain injury (WMI, IVH and cerebellar hemorrhage).  	   64 3.2.2 Participants Infants born very preterm (between 24 and 32 weeks GA) were admitted to the NICU at the British Columbia’s Women’s Hospital between March 2006 and January 2009. As in previous studies, infants from this cohort were excluded if they had a congenital malformation or syndrome, antenatal infection, or evidence on ultrasound of a parenchymal hemorrhagic infarction >2 cm (Adams et al. 2010; Chau et al. 2009; Papile et al. 1978). After parental informed consent was obtained, 98 infants were included in the present study. A neonatal research nurse performed medical and nursing chart review from birth to term-equivalent age or discharge (whichever came first). Data included but were not limited to GA, sex, birth weight, presence of patent ductus arteriosus, duration of intubation, infection, necrotizing enterocolitis, and corticosteroid (hydrocortisone and/or dexamethasone) exposure. Infants with clinical sepsis (who had negative cultures but were treated with antibiotics for ≥5 days) or with confirmed infections (positive cultures of the blood, urine, or cerebral spinal fluid, or ≥4 white blood cells found in tracheal aspirates associated with clinical pneumonia) were included in this study because these types of infections are associated with abnormal brain maturation (Chau et al. 2012). This approach is also consistent with the study by Stoll et al., which demonstrated that neonatal infections among extremely low birth weight infants are associated with poor neurodevelopmental outcome, even in the absence of positive cultures (Stoll et al. 2004). Infants were classified as having necrotizing enterocolitis if they met either stage 2 (clinical signs and symptoms, and pneumatosis intestinalis on x-ray) or stage 3 (critically ill, clinical signs and symptoms, and pneumatosis intestinalis on x-ray) of Bell’s criteria (Bell et al. 1978). Infants were assessed for neonatal growth (weight, length, and head circumference) at the time of each MRI scan: median of 32 weeks (IQR: 30.4 to 33.6; total range, 27.3 to 40.7) and 40 weeks (IQR: 38.7 to 42.7; total range, 33.4 to 46.4) PMA. 	   65 3.2.3 Magnetic Resonance Imaging Infants were scanned without pharmacological sedation when stable at median 32 (scan 1) and 40 weeks (scan 2) PMA. All newborns were scanned in an MRI-compatible isolette (Lammers Medical Technology) with a specialized neonatal head coil (Advanced Imaging Research). A Siemens 1.5-T Avanto magnet and VB 13A software were used to obtain the following sequences: three-dimensional coronal volumetric T1-weighted images (repetition time, 36; echo time, 9.2; field of view, 200 mm; slice thickness, 1 mm; no gap) and axial fast spin echo T2-weighted images (repetition time, 4610; echo time, 107; field of view, 160 mm; slice thickness, 4 mm; gap, 0.2 mm). Neuroradiologist K.J.P., blinded to infant medical history, assessed the images for cerebellar hemorrhage and the severity of WMI and IVH (Miller et al. 2005; Papile et al. 1978). Twenty random scans were rescored; intra-observer reliability of k > 0.9 was comparable with previous reported scores (Miller et al. 2005). In addition, K.J.P. identified seven subjects with white matter cysts typical of cystic PVL on at least one imaging study. Three neonates had mild PVL, with less than four cysts <2mm in diameter; four neonates demonstrated cysts >1 cm in diameter. A variable identifying infants with cystic PVL was not included in the statistical models, given the small number in each category. These infants were, however, included in this study and were identified as having moderate to severe WMI.  3.2.4 Diffusion Tensor Imaging DTI was acquired with a multirepetition, single-shot echo planar sequence with 12 gradient directions (repetition time, 4900; echo time, 104; field of view, 160 mm; slice thickness, 3 mm; no gap), three averages of two diffusion weightings of 600 and 700 s/mm2 (b values), and an image without diffusion weighting, resulting in an in plane resolution of 1.3 mm. DTI parameters of FA and λ1, λ2 and λ3 were collected bilaterally in 15 regions of interest by two 	   66 observers. Eight regions of interest in the cortical gray matter were identified by J.V. in 95 neonates (precentral gyrus, postcentral gyrus, secondary somatosensory cortex, superior frontal gyrus, dorsolateral prefrontal cortex, ventrolateral prefrontal cortex, anterior insula, and occipital gray matter; Figure 3.1), and seven regions of interest in the white matter were identified by V.C. in 97 neonates (anterior, middle, and posterior subcortical white matter, genu, and splenium of the corpus callosum; posterior limb of the internal capsule; and optic radiations), as described previously (Chau et al. 2009). Values from regions of interest on a degraded diffusion tensor image were not measured (7% of regions of interest).  Figure 3.1 Regions of Interest in the Cortical Gray Matter    (A and B) Diffusion tensor image-encoded anisotropy color maps of an infant born at 26.29 weeks gestation and scanned at 30 weeks postmenstrual age. The images demonstrate the relatively high FA of the cerebral cortex typical for this age. The color convention used to display the predominant diffusion direction has red representing right-left, green representing anterior-posterior, and blue representing superior-inferior anatomical directions (56, 58). Eight cerebral cortical regions of interest were examined, and values of each region were averaged bilaterally: (a) precentral gyrus, (b) postcentral gyrus, (c) secondary somatosensory cortex, (d) superior frontal gyrus, (e) dorsolateral prefrontal cortex, (f ) ventrolateral prefrontal cortex, (g) anterior insula, and (h) occipital gray matter. 	   67  3.2.5 Reliability of the Cortical Gray Matter Regions of Interest Many considerations were given to the size and placement of the region of interest voxel boxes in the cerebral cortex. First, it was important to determine the size of voxel box that could fit within the thin layer of cortical gray matter, which is about 2 mm thick in the newborn (Dubois et al. 2008b). We observed that 2 x 3 voxel boxes could fit within the boundaries of the cerebral cortex and surrounding structures.   Second, replication of the regions of interest over time was complicated by the fact that there is a marked change in the complexity of the cortex, with increasing sulcation and gyration between 32 and 40 weeks (Dubois et al. 2008a; Kroenke et al. 2007). However, we found that 2 x 3 voxel boxes could be reliably placed at the height of the gyrus in the cortical gray matter for both the first and second scans. Intra-rater reliability was calculated on 20% of the regions of interest in the cortical gray matter, by Bland Altman analyses (Bland and Altman 1986), and values were compared with those previously published in the literature (Adams et al. 2010): Scan 1 showed an FA mean difference of 0.001 (limits of agreement, −0.001 to 0.003), and scan 2 showed an FA mean difference of −0.002 (limits of agreement, −0.004 to 0.000).  Finally, reduction of cortical diffusion takes place according to an inside-out laminar gradient (Jespersen et al. 2012), thereby introducing the possibility for partial averaging within the measured regions of cortex. To address this issue, we considered whether the values of the top three voxels of the 2 x 3 voxel box compared favorably with the bottom three voxels of the 2 x 3 voxel box. FA mean differences for the top three and bottom three voxel boxes across all regions of interest were minor: At scan 1, the mean difference was 0.007 for all regions of interest, with 	   68 a mean difference range of −0.010 to 0.020 across individual regions of interest; at scan 2, the mean difference was 0.006 for all regions of interest, with a mean difference range of −0.010 to 0.020 across individual regions of interest. Given that there were no systematic differences between using 1 x 3 versus 2 x 3 voxel boxes, and that use of 2 x 3 voxel boxes improved reliability, we proceeded to use 2 x 3 voxel boxes to extract data from the regions of interest within the cerebral cortex.  3.2.6 Reliability of the White Matter Regions of Interest On the basis of the repeated analysis of 20% of the regions of interest in the white matter (Chau et al. 2012) by Bland Altman analyses, intra-rater reliability was considered high: FA mean difference of 0.001 (limits of agreement, −0.018 to 0.017).  3.2.7 Data Analyses Statistical analysis was performed with R version 2.13 (R Development Core Team 2011). Normality plots were examined, and skewed variables (DTI parameters [FA and λ1, λ2 and λ3]) and growth measures (change in weight [gram], head circumference [centimeter], and length [centimeter] between scan 1 and scan 2) were log-transformed. t tests were also used to examine whether there were differences in the cortical gray matter FA values for infants that were growth restricted (<10th weight percentile) compared to those with an appropriate weight for their PMA and sex in the NICU at scan 1 and scan 2, and whether these differences were affected by the exclusion of infants born small for their GA and sex. Then, linear mixed effects models (LMEMs) were used to examine longitudinal associations between change in weight and DTI parameters between scan 1 and scan 2 in the cortical gray matter and white matter. Included in the LMEMs were terms for multiple regions of interest (8 cortical gray matter regions or 7 white 	   69 matter regions) and interaction terms for region of interest and postmenstrual age. Splines (values produced by three smooth polynomial segments) were used to account for the nonlinearities between postmenstrual age and FA values (Figure 3.2), and growth over time relative to birth weight. The independent variables entered in the initial model were GA, birth weight, and sex. If weight change was a significant predictor of FA in the basic model (step 1), we extended the model to include brain injury (step 2: WMI, IVH, and cerebellar hemorrhage) and systemic illness (step 3: patent ductus arteriosus, days intubated, infection, and necrotizing enterocolitis). This model was then reapplied while excluding infants who had received postnatal corticosteroids. Steps 1, 2, and 3 were repeated to examine the relationship between weight change and radial (λ2 and λ3) and axial (λ1) diffusion axes. Moreover, if weight change was a significant predictor of FA, results were confirmed by repeating steps 1 to 3 for length change and head circumference change.             	   70  Figure 3.2 FA of the Cortical Gray Matter with PMA 	  FA of the cortical gray matter decreases nonlinearly with increasing PMA. Graphed are the raw, unadjusted FA values from the pre-central gyrus in relation to PMA. FA decreases rapidly in the cortical gray matter until ~36 weeks, when values reached the noise floor. Given the nonlinear relationship between diffusion tensor parameters and PMA, splines (3 smooth polynomial segments) were used to approximate this relationship, and their corresponding values were included in the statistical models to account for the contribution of PMA to cortical maturation.   3.3 Results 3.3.1 Characteristics of the Cohort The characteristics of the 98 included infants are provided in Table 3.1. Ninety-five newborns had diffusion tensor images of sufficient quality for cortical analyses. In univariate unadjusted analyses, infants with poor postnatal weight gain (n= 27) appeared to have higher cortical gray matter FA values at scan 2 (MRI at median 40.3 weeks postmenstrual age) compared to infants with appropriate weight for their postmenstrual age and sex in the NICU, but the difference was 	   71 not statistically significant [ventrolateral cortex: 95% confidence interval (CI)= −0.002 to 0.04; P= 0.069; Figure 3.3]. The magnitude of this difference was more pronounced and reached statistical significance when infants born small for their gestational age and sex (n= 19) were excluded (ventrolateral cortex: 95% CI= 0.002 to 0.05; P= 0.032; across all regions of interest: 95% CI= 0.001 to 0.02; P= 0.036). Infants born small for their gestational age and sex were therefore included in the longitudinal multivariable models to provide a more conservative estimate of the difference in cortical diffusion tensor imaging parameters related to postnatal growth restriction.                 	   72 Table 3.1 Infant Characteristics *12 infants were missing values for head circumference and 14 infants were missing values for length at birth; therefore, birth weight was used in the statistical models as a marker of prenatal growth †Measured from birth to term-equivalent age or discharge (whichever came first) IQR= Interquartile range; PVHI= periventricular hemhorragic infarction; Scan 1= magnetic resonance imaging at ~32 weeks postmenstrual age; Scan 2= magnetic resonance imaging at ~40 weeks postmenstrual age       Characteristics Birth Scan 1 Scan 2 Sex (male), number (%) 45.0 (45.9) - - Age (weeks), median (IQR) 27.4 (26.0-29.6) 32.1 (30.4-33.6) 40.3 (38.7-42.7) Weight percentile <10%, number (%) 19.0 (19.4) 54.0 (55.1) 30.0 (30.6) Weight (grams), median (IQR) 988 (803-1278) 1310 (1139-1601) 3160 (2543-3685) Head circumference (cm), median (IQR) 25.0 (23.5-27.0)* 27.6 (26.0-29.0) 35.0 (33.0-36.8) Length (cm), median (IQR) 36.0 (33.5-39.4)* 39.0 (37.0-41.0) 49.0 (45.0-51.5) Mild WMI (score), number (%)  - 11 (11.2) 5 (5.1) Moderate-severe WMI (score), number (%)  - 20 (20.4) 18 (18.4) IVH (Grade 1-2), number (%) - 36 (37.1) 26 (26.5) IVH (Grade 3 or PVHI), number (%) - 7 (7.1) 6 (6.1) Cerebellar hemorrhage, number (%) - 14 (14.3) 10 (10.2) Patent ductus arteriosus, number (%) - - 48 (49.0)† Days intubated, median (IQR) - - 5.5 (1.0-29.8)† Postnatal infection, number (%) - - 48 (49.0)† Necrotizing enterocolitis, number (%)   9 (9.2)† Corticosteroid exposure (hydrocortisone and/or dexamethasone), number (%) - - 32 (32.7)† 	   73 Figure 3.3 Postnatal Growth Restriction Delays Cortical Gray Matter Maturation  Preterm infants with postnatal growth restriction demonstrated delayed cortical gray matter maturation. Graphed are the raw, unadjusted FA values from the ventrolateral prefrontal cortex of very preterm infants that were growth restricted (<10th weight percentile) at scan 2 (MRI at median 40.3 weeks PMA, versus very preterm infants that were an appropriate weight for their postmenstrual age during neonatal intensive care. (A) Preterm infants that were growth restricted postnatally demonstrated moderately delayed cortical gray matter maturation compared to preterm infants who were an appropriate weight for their PMA. (B) After excluding infants born <10th weight percentile, preterm infants that were growth restricted postnatally demonstrated significantly delayed cortical gray matter maturation compared to preterm infants who were an appropriate weight for their PMA.   3.3.2 Weight Change in Relation to Diffusion Parameters of the Cortical Gray Matter Longitudinal models revealed that lower GA (effect size= −0.038; SE= 0.011; P< 0.001), birth weight (effect size< −0.001; SE< 0.001; P= 0.016), and slower weight gain [weight at scan 2 (MRI at ~40 weeks PMA) − weight at scan 1 (MRI at ~32 weeks PMA)] (effect size= −0.410; SE= 0.089; P< 0.001) were independently associated with higher FA values in the cortical gray matter, after adjusting for sex, brain injury [WMI, IVH, and cerebellar hemorrhage (brain injury model)], systemic illness [patent ductus arteriosus, days intubated, postnatal infection, and necrotizing enterocolitis (extended model)], and age at scan (Table 3.2). Therefore, neonatal growth was associated with cortical gray matter maturation in the NICU, independent of birth 	   74 weight, brain injury, and systemic illness. Change in FA reflected changes in the radial diffusion axes (λ2 and λ3; Table 3.3), but not the axial diffusion axis (λ1; Table 3.4), suggesting a delay in neuronal process formation and/or apoptosis in the cerebral cortices of infants who are born very preterm and have impaired growth.  Table 3.2 Weight change in relation to mean FA values of 8 regions of interest in the cortical gray matter  Basic Model n=95 Brain Injury Model n=95 Extended Model n=95  Effect size P  Effect size P  Effect size P GA -0.027 0.007 -0.028 0.006 -0.038 <0.001 Birth weight <-0.001 0.025 <-0.001 0.032 <-0.001 0.016 Male 0.037 0.101 0.034 0.131 0.031 0.189 Weight change -0.422 <0.001 -0.424 <0.001 -0.410 <0.001 WMI  - - -0.010 0.404 -0.009 0.942 IVH - - -0.002 0.840 -0.004 0.708 Cerebellar hemorrhage - - -0.009 0.790 0.004 0.917 Patent ductus arteriosus - - - - -0.036 0.242 Days intubated  - - - - -0.001 0.238 Infection - - - - -0.047 0.111 Necrotizing enterocolitis - - - - 0.040 0.394 Weight change= weight at scan 2 (DTI at ~40 weeks PMA) - weight at scan 1 (DTI at ~32 weeks PMA)     	   75 Table 3.3 Weight Change in Relation to Mean λ2 and λ3 Values of 8 Regions of Interest in the Cortical Gray Matter   Basic Model n= 95 Brain Injury Model n= 95 Extended Model n= 95  Effect size P  Effect size P Effect size P  GA 0.001 0.703 0.001 0.784 0.004 0.260 Birth weight <0.001 0.004 <0.001 0.007 <0.001 0.002 Male -0.009 0.243 -0.006 0.447 -0.008 0.334 Weight change 0.064 0.056 0.070 0.034 0.068 0.043 WMI - - 0.006 0.147 0.003 0.498 IVH - - -0.010 0.007 -0.009 0.013 Cerebellar hemorrhage - - <0.001 0.978 -0.004 0.762 Patent ductus arteriosus - - - - 0.017 0.099 Days intubated  - - - - <0.001 0.145 Infection - - - - 0.010 0.344 Necrotizing enterocolitis - - - - -0.010 0.518 λ2 & λ3 = radial diffusion axis; Weight change= weight at scan 2 (DTI at ~40 weeks PMA) - weight at scan 1 (DTI at ~32 weeks PMA)          	   76 Table 3.4 Weight Change in Relation to Mean λ1 Values of 8 Cortical Regions of Interest in the Cortical Gray Matter              Basic Model          n= 95  Effect size P  GA -0.012 <0.001 Birth weight <0.001 0.014 Male -0.005 0.456 Weight change 0.005 0.872 λ1= axial diffusion axis; Weight change= weight at scan 2 (DTI at ~40 weeks PMA) - weight at scan 1 (DTI at ~32 weeks PMA)   3.3.3 Weight Change in Relation to FA of the White Matter Weight change was not significantly associated with FA values in the white matter in the basic statistical model (effect size= −0.035; SE= 0.055; P= 0.529; Table 3.5). Therefore, white matter maturation appears to be relatively spared from the effects of postnatal growth restriction. Rather, postnatal infection (effect size= −0.057; SE= 0.020; P= 0.005) was independently associated with lower FA values in the white matter after adjusting for gestational age, birth weight, sex, brain injury, systemic illness, weight change, and age at scan.        	   77 Table 3.5 Weight Change in Relation to Mean FA values of 7 Regions of Interest in the White Matter         Basic Model     n= 97  Effect size P  GA 0.048 <0.001 Birth weight <0.001 0.392 Male 0.013 0.404 Weight change -0.035 0.529 Weight change= weight at scan 2 (DTI at ~40 weeks PMA) - weight at scan 1 (DTI at ~32 weeks PMA)   3.3.4 Weight Change in Relation to Diffusion Parameters of the Cortical Gray Matter Excluding Infants who Received Postnatal Corticosteroids Neither dexamethasone (effect size, −167.044; SE, 223.072; P= 0.454) nor hydrocortisone (effect size, −341.346; SE, 245.124; P= 0.164) was associated with weight change after adjusting for GA, birth weight, sex, brain injury, systemic illness, and age at scan. Nonetheless, as a sensitivity analysis, we examined weight change in relation to FA of the cortical gray matter excluding infants who received postnatal corticosteroids. In newborns who did not receive corticosteroids postnatally, lower GA (effect size −0.034; SE= 0.012; P= 0.005), birth weight (effect size< −0.001; SE= 0.001; P= 0.009), and slower weight gain (effect size, −0.512; SE= 0.114; P< 0.001) between scan 1 and scan 2 were independently associated with higher FA values in the cortical gray matter, in longitudinal models adjusting for gestational age, sex, brain injury, systemic illness, and age at scan (Table 3.6). Given that the relationship between weight change and FA values did not change meaningfully after the exclusion of infants who received postnatal corticosteroids (hydrocortisone and/or dexamethasone), exposed infants were 	   78 included in all other longitudinal models.  Table 3.6 Weight Change in Relation to Mean FA Values of 8 Regions of Interest in the Cortical Gray Matter Excluding Infants who Received Postnatal Corticosteroids  Basic Model n= 65* Brain Injury Model n= 65* Extended Model n= 65*  Effect size P  Effect size P  Effect size P GA -0.022 0.052 -0.024 0.037 -0.034 0.005 Birth weight <-0.001 0.035 <-0.001 0.045 <-0.001 0.009 Male 0.058 0.030 0.052 0.051 0.051 0.062 Weight change -0.541 <0.001 -0.528 <0.001 -0.512 <0.001 WMI  - - -0.012 0.371 0.002 0.866 IVH  - - -0.014 0.259 -0.002 0.901 Cerebellar hemorrhage - - -0.022 0.585 -0.031 0.448 Patent ductus arteriosus - - - - -0.077 0.026 Days intubated  - - - - -0.002 0.366 Infection - - - - -0.057 0.069 Necrotizing enterocolitis - - - - 0.016 0.837 *30 infants who had received corticosteroids (dexamethasone and/or hydrocortisone) were excluded from the analyses Weight change= weight at scan 2 (DTI at ~40 weeks PMA) - weight at scan 1 (DTI at ~32 weeks PMA)   3.3.5 Length Change in Relation to Diffusion Parameters of the Cortical Gray Matter Longitudinal models revealed that lower GA (effect size= −0.030; SE= 0.010; P= 0.002), confirmed necrotizing enterocolitis (effect size= 0.125; SE= 0.050; P= 0.012), and slower linear growth (effect size= −0.837; SE= 0.177; P< 0.001) between scan 1 and scan 2 were 	   79 independently associated with higher FA values in the cortical gray matter, after adjusting for birth weight, sex, brain injury, systemic illness, and age at scan (Table 3.7). Change in FA reflected changes in the radial diffusion axes (λ2 and λ3: effect size= 0.189; SE= 0.073; P= 0.010) and not the axial diffusion axis (λ1: effect size= −0.043; SE= 0.062; P= 0.488).  Table 3.7 Length Change in Relation to Mean Fractional Anisotropy Values of 8 Regions of Interest in the Cortical Gray Matter  Basic Model n= 89* Brain Injury Model n= 89* Extended Model n= 89*  Effect size P  Effect size P  Effect size P GA -0.025 0.009 -0.023 0.016 -0.030 0.002 Birth weight <-0.001 0.141 <-0.001 0.166 <-0.001 0.051 Male 0.024 0.269 0.021 0.355 0.009 0.689 Length change -0.796 <0.001 -0.817 <0.001 -0.837 <0.001 WMI  - - -0.021 0.078 -0.012 0.307 IVH - - -0.008 0.488 -0.008 0.474 Cerebellar hemorrhage - - <-0.001 0.992 -0.013 0.731 Patent ductus arteriosus - - - - -0.047 0.116 Days intubated  - - - - -0.001 0.233 Infection - - - - -0.031 0.262 Necrotizing enterocolitis - - - - 0.125 0.012 *Unable to obtain length measurements of 6 infants.   Length change= length at scan 2 (DTI at ~40 weeks PMA) – length at scan 1 (DTI at ~32 weeks PMA)   3.3.6 Head Circumference Change in Relation to Diffusion Parameters of the Cortical Gray Matter Longitudinal models revealed that lower gestational age (effect size= −0.030; SE= 0.010; P= 	   80 0.004) and slower head growth (effect size= −1.090; SE= 0.025, P< 0.001) between scan 1 and scan 2 were independently associated with higher FA values in the cortical gray matter, after adjusting for GA, birth weight, sex, brain injury, systemic illness, and age at scan (Table 3.8). Change in FA reflected change in the radial diffusion axes (λ2 and λ3: effect size= 0.265; SE= 0.098; P= 0.007) and not the axial diffusion axis (λ1: effect size= 0.058; SE= 0.086; P= 0.498). Results from these models are consistent with the models above examining the relationship between weight change and length with diffusion parameters, and therefore provide further support for the finding that neonatal growth over and above birth weight, brain injury, and systemic illness predicted cortical gray matter maturation in the NICU.                	   81 Table 3.8 Head Circumference Change in Relation to Mean FA Values of 8 Regions of Interest in the Cortical Gray Matter  Basic Model n= 94* Brain Injury Model n= 94* Extended Model n= 94*  Effect size P Effect size P Effect size P Gestational age -0.028 0.004 -0.027 0.007 -0.030 0.004 Birth weight <-0.001 0.180 <-0.001 0.176 <-0.001 0.073 Male 0.008 0.731 0.004 0.874 0.007 0.765 Head circumference change -1.030 <0.001 -1.050 <0.001 -1.09 <0.001 White matter injury - - -0.018 0.137 -0.014 0.251 Intraventricular hemorrhage - - -0.010 0.371 -0.011 0.355 Cerebellar hemorrhage - - 0.010 0.773 0.013 0.706 Patent ductus arteriosus - - - - -0.038 0.204 Days intubated  - - - - -0.001 0.199 Infection - - - - 0.017 0.553 Necrotizing enterocolitis - - - - -0.009 0.843 *Unable to obtain head circumference measurement of 1 infant. Head circumference change= head circumference at scan 2 (DTI at ~40 weeks PMA) – head circumference at scan 1 (DTI at ~32 weeks PMA  3.4 Discussion This study examined whether neonatal growth is related to microstructural development of the cortical gray and white matter in infants born very preterm. We found that impaired neonatal growth (weight, length, and head circumference) was significantly associated with delayed cortical maturation after accounting for GA, birth weight, sex, PMA, brain injury, and systemic illness. However, consistent with previous studies it was the cortical gray matter, rather than the white matter, which appeared to be most susceptible to impairments in growth (Dubois et al. 2008b; Padilla et al. 2011; Toft et al. 1995; Tolsa et al. 2004). 	   82  The finding that the cortical gray matter is more greatly influenced by postnatal growth relative to the white matter, builds on results from previous studies examining the relationship between IUGR and cortical volumes/microstructure early in life (Dubois et al. 2008b; Toft et al. 1995; Tolsa et al. 2004). Animal models of IUGR have demonstrated a transient delay in oligodendrocyte maturation and myelination (Tolcos et al. 2011). Although markers of myelinating oligodendrocytes were reduced in utero, white matter volumes returned to control levels postnatally, and persisted into adulthood (Tolcos et al. 2011). Thus, it has been suggested that the altered neurodevelopment associated with IUGR is likely not due to long-term deficits in myelination. Rather, it is the reduction of cerebral cortical volumes and altered microstructure associated with prenatal growth restriction that have been more pronounced, persistent, and associated with functional impairment (Eixarch et al. 2012; Padilla et al. 2011; Tolsa et al. 2004).  Microstructural integrity of the cortical gray matter can be inferred from diffusion parameters (Deipolyi et al. 2005; Jespersen et al. 2012; Kroenke et al. 2007; McKinstry et al. 2002; Sizonenko et al. 2007). Between 25 and 40 weeks PMA, FA decreases as the developing cortex increases in complexity (Deipolyi et al. 2005; McKinstry et al. 2002), with the arborization of the basal dendrites, formation of thalamocortical and cortico-cortical connections, and disappearance of the radial glia (Kostovic and Rakic 1990; Kostovic and Jovanov-Milosevic 2006; Marin-Padilla 1992; Mrzljak et al. 1988; Sidman and Rakic 1973). We found that higher FA was reflective of change in the radial diffusion axes (λ2 and λ3) between approximately 32 and 40 weeks PMA, indicative of alterations to the neuronal complexity in infants born very preterm with impaired growth. In a sheep model of premature birth, relative to controls, ischemic 	   83 sheep had poorer cortical growth and higher FA values associated with disturbances in the radial diffusion axis (Dean et al. 2013). The impaired decline in cortical FA was due to altered maturation of the basal dendritic arbor of cortical neurons, resulting in relatively high anisotropy compared to controls (Dean et al. 2013).  Lower GA and birth weight were associated with higher FA. However, cortical maturation was more strongly predicted by postnatal growth. This finding was supported by Smart et al., who used a rat model to demonstrate that nutritional deprivation during both gestation and neonatal periods had a greater influence on cortical development as opposed to deprivation during either one of these periods alone; however, the damage to the forebrain was largely determined by nutritional deprivation in the postnatal period (Smart et al. 1973). Studies of neonatal rats deprived of adequate postnatal nutrition have also provided evidence for altered neuronal activity (Mourek et al. 1967; Seidler, Bell, Slotkin 1990; Villescas et al. 1981). By 20 to 24 weeks GA, a large proportion of neurons have been produced in the ventricular and subventricular zones (Kostovic and Jovanov-Milosevic 2006). These precursor cells are vulnerable to nutrient insufficiency (Inder et al. 1999). Moreover, the nutritional demand of rapid brain growth, synaptogenesis, and sensory-driven activity between 24 to 42 weeks gestation leaves the neonatal cortex particularly vulnerable to nutritional insult (Georgieff 2007).   Substantial energy is also required to mount a response invasive/painful procedures in the NICU. Infants that are born earlier and sicker often require more invasive procedures, however, for these infants nutritional intakes are usually less, and the deficits in postnatal growth are greater (Embleton, Pang, Cooke 2001; Vinall et al. 2012). Importantly, stress inhibits the production of growth hormones, a regulator of IGF-1 (Tsigos and Chrousos 2002). IGF-1 levels positively 	   84 correlate with total brain, gray matter, unmyelinated white matter and cerebellar volumes at term, after adjustment for GA, mean protein and caloric intakes, gender, brain injury, and steroid exposure (Hansen-Pupp et al. 2011; Hansen-Pupp et al. 2013). Moreover, IGF-1 levels during infant hospitalization predict neurodevelopmental outcomes at 2 years of age (Hansen-Pupp et al. 2013).   We did not have data on neonatal nutrition, caloric intake, and feeding because the infants in this study were participants from a larger longitudinal study examining as part of a larger ongoing study of neonatal invasive procedures, brain and neurodevelopment (Adams et al. 2010; Chau et al. 2009; Grunau et al. 2005; Grunau et al. 2007; Grunau et al. 2009). The nutrition protocols in place during this study included: 1) starting parenteral nutrition upon admission to the NICU, and 2) encouraging the use of breast milk and early trophic feeds. The standard fluid intake was 150 ml/kg per day, with a goal of 120 calories/kg per day. Weight was measured daily unless the patient was too unstable. Fluid and caloric intake were assessed daily and adjusted to optimize nutrition and growth. Postnatal growth is affected by a multitude of factors, which include fluid management, nutritional and caloric intake, catabolic stressors associated with severity of illness, and endocrine, genetic, and environmental factors, including procedural pain/stress. The consistency in findings across measures of weight, length, and head circumference supports the hypothesis that the alterations in cortical development reflect growth rather than fluid management alone. Our study was able to account for several medical confounders, which were likely to affect both growth and brain development, although residual confounding remains possible. Future studies are needed to examine the specific roles of systemic illness and nutrition, and to determine the optimal postnatal growth for cortical maturation in the NICU.   	   85 The results of this study have important clinical implications. Neonatal growth over and above birth weight, brain injury, and systemic illness correlated with cortical gray matter maturation in the NICU. Therefore, by diagnosing, treating, and preventing poor postnatal growth, clinicians may have the opportunity to optimize conditions for cortical development to proceed normally in infants born very preterm.                    	   86 CHAPTER 4  PARENT BEHAVIORS MODERATE THE RELATIONSHIP BETWEEN NEONATAL INVASIVE PROCEDURES AND INTERNALIZING BEHAVIORS AT 18 MONTHS CORRECTED AGE IN CHILDREN BORN VERY PREMATURELY  4.1 Introduction Infants born very preterm are repeatedly exposed to invasive procedures in the NICU. Greater exposure to invasive procedures has been associated with slower postnatal growth (Vinall et al. 2012), and slower growth in the NICU is associated with altered cortical development between approximately 32 and 40 weeks PMA (Vinall et al. 2013b). Neonatal rats exposed to 2 to 4 heel pokes over the first 7 days of life, grew more slowly and had increased anxiety-like behaviors during adulthood compared to rats exposed to tactile stimulation (Anand et al. 1999). Greater internalizing (anxious/depressive) behaviors in preterm children compared to full-term controls have been reported as early 2 years CA, persist to late adolescence, and appear to be independent of cognitive ability (Aarnoudse-Moens et al. 2009; Anderson, Doyle, Victorian Infant Collaborative Study Group 2003; Bhutta et al. 2002; Grunau, Whitfield, Fay 2004; Loe et al. 2011; Spittle et al. 2009). A recent study by our group has demonstrated that among non-ventilated children born very preterm, greater exposure to invasive procedures in the NICU was associated with higher reported internalizing behavior at 7 years of age (Ranger et al. 2014).  Experimental animal models have also demonstrated that early stress can permanently reorganize hormonal, physiological and behavioral systems (Matthews 2002; Meaney, Szyf, Seckl 2007; Murgatroyd and Spengler 2011; Pryce and Feldon 2003). Greater exposure to  A version of Chapter 4 has been published in Vinall J, Miller SP, Synnes AR, Grunau RE (2013). Pain 154:1831-39. 	   87  invasive procedures in the NICU was associated with altered stress hormone (cortisol) regulation in extremely low gestational age (ELGA: 24 to 28 weeks) children (Grunau, Weinberg, Whitfield 2004; Grunau et al. 2007). Cortisol expression among ELGA children, and to a lesser degree very low gestational age (VLGA; 29 to 32 weeks) children, was associated with  internalizing behaviors at 18 months CA (Brummelte et al. 2011b). This research emphasizes the importance of managing pain/stress in the NICU, to prevent long-term effects on child behavior.   Parents also play a vital role in the management of stress and development of their infant (Gunnar 1998). However, the birth of a preterm infant is a highly stressful experience for parents (Meyer et al. 1995; Miles, Funk, Carlson 1993; Younger, Kendell, Pickler 1997). One of the most stressful experiences reported by parents of infants in the NICU is seeing their infant in pain (Gale et al. 2004; Miles, Funk, Carlson 1993; Miles and Holditch-Davis 1997). Neonatal intensive care-based interventions that increase parental involvement in infant pain management have been found to improve parents’ efficacy in supporting their infant post-discharge from the hospital (Franck et al. 2011). This is important given that parenting stress appears to persist well-beyond discharge from the NICU (Brummelte et al. 2011a; Garel, Dardennes, Blondel 2007; Holditch-Davis et al. 2009; Singer et al. 2003). Among preterm infants exposed to greater numbers of invasive procedures in the NICU, if parents reported having lower parenting stress, infants showed less negative reactivity at 12 months CA, compared to infants whose parents reported having higher parenting stress (Voigt et al. 2013). While parenting stress may be reflective of realistic parental concerns with their child's development (Brummelte et al. 2011a), higher parenting stress is predictive of child internalizing behavior (Zelkowitz et al. 2011), and is associated with decreased parent emotional availability at 2 years CA in preterm children 	   88 (Zelkowitz et al. 2009). Importantly, parent support to promote sensitive and responsive interactions during hospitalization appears to improve white matter maturation in infants born preterm (Milgrom et al. 2010). Greater maturation of the white matter at term age has been associated with better social-emotional outcomes at age 5 in children born preterm (Rogers et al. 2012). Although more positive parent interaction was found to buffer the relationship between invasive procedures in the NICU and poorer focused attention at 8 months CA (Tu et al. 2007), the extent that parental behavior moderates the relationship between invasive procedures in the NICU and internalizing behavior remains unknown.  Therefore, we examined whether the number of invasive procedures in the NICU (adjusted for neonatal and medical confounders) is related to parent report of internalizing behaviors at 18 months CA, and whether parental EA (adjusted for parenting stress), moderates the relationship between invasive procedures and internalizing behaviors (adjusted for child cognition) in children born very preterm. Further, we examined the relationship between parent EA and internalizing in children born full term.  Hypothesis   We hypothesized that greater parent EA would be associated with fewer internalizing behaviors at 18 months CA in children born very preterm exposed to greater numbers of invasive procedures.  4.2 Methods 4.2.1 Study Overview Very preterm infants were recruited from the NICU at the B.C. Children’s & Women’s 	   89 Hospitals. Full-term infants were born at the B.C. Women’s Hospital and were contacted through their pediatricians. Written consent was obtained from a parent. At 18 months CA, the children and their parent(s) returned to the center for the study visit. The Bayley Scales of Infant Development II was administered (Bayley 1993), followed by the videotaped semi-structured parent–child teaching session, later scored for parent EA (Biringen 2008). We examined whether parental behavior adjusted for parenting stress, parent’s years of education, number of children in the home, and parent age moderated the relationship between invasive procedures (adjusted for GA, illness severity on day 1, days on mechanical ventilation, and total morphine exposure) and internalizing behaviors (adjusted for gender and child cognition) at 18 months CA in children born very preterm.  4.2.2 Participants Ninety-six infants born very preterm (≤32 weeks GA) and 49 full-term control infants born at the B.C. Children’s & Women’s Hospitals between February 2001 and July 2004 were recruited as part of a larger ongoing study of the effects of neonatal pain on the neurodevelopment of infants born very preterm (Brummelte et al. 2011b; Grunau et al. 2005; Grunau et al. 2007; Tu et al. 2007). Infants were excluded if they were born small or large for GA; if they had a major congenital anomaly, major neurosensory impairment (legally blind, non-ambulatory cerebral palsy, sensory-neural hearing impairment), or severe brain injury evident on neonatal ultrasound (PVL and/or grade 3 or 4 IVH); or if the mother reported use of illicit drugs during pregnancy. All full-term infants in our study were born healthy, and none was under observation for medical complications. Ninety-four mothers and 2 fathers of children born very preterm, and 47 mothers and 2 fathers of children born full term participated in the study at 18 months CA.  	   90  4.2.3 Demographics Parent information was obtained by questionnaire. Because parent’s years of education is the most important socioeconomic status (SES) indicator in relation to child development (Bohm et al. 2002; Resnick et al. 1990), we used parent’s years of education as the index of SES for statistical analysis.   4.2.4 Neonatal Medical Chart Review A neonatal research nurse carried out medical and nursing chart review from birth to term-equivalent age, as described previously (Brummelte et al. 2011b; Grunau et al. 2007). Data included but were not limited to GA, gender, illness severity on day 1 (SNAP-II; Richardson et al. 2001), number of invasive procedures (see Appendix Table A.1), days of mechanical ventilation, and cumulative morphine exposure adjusted for weight.  4.2.5 Cognitive Development At 18 months CA, child development was assessed with the Bayley Scales of Infant Development II (Bayley 1993). We used the Bayley Mental Development Index (MDI) to adjust our statistical models for child cognitive function. The Bayley MDI measures language, memory and problem-solving abilities in infants and toddlers aged 1 to 42 months. The Bayley MDI is a standardized score for overall cognitive development, with a mean of 100 and standard deviation of 15.  4.2.6 Parenting Stress Parent’s completed the Parenting Stress Index III (PSI; Abidin 1995) , which includes 120 items rated on a 6-point Likert scale from 1 (strongly agree) to 6 (strongly disagree). The PSI yields a 	   91 Total Score and 2 domain scores: Child Domain (concern about the child) and Parent Domain (concern about their own parenting ability). Given that the Child Domain reflects parent’s concerns about the child’s behavior, including internalizing behaviors, we only included the Parent Domain in the statistical analysis because our focus was on how parental factors may influence child behavior.  4.2.7 Child Internalizing Behavior Parent’s rated their child’s behavior with the Child Behavior Checklist for children ages 1. to 5 years (CBCL; Achenbach and Rescorla 2000), a widely used questionnaire for identifying problem behaviors in children. Ninety-nine items are rated on a Likert scale ranging from 0 (not true) to 2 (very true or often true). Seven syndrome scales (Emotionally Reactive [e.g. moody, whining], Anxious/Depressed [e.g. nervous, sad], Somatic Complaints [e.g. does not eat well, stomachaches], Withdrawn [e.g. avoids eye contact, unresponsive to affection], Sleep Problems [e.g. nightmares, wakes often], Attention Problems [e.g. cannot concentrate, cannot sit still], and Aggressive Behavior [e.g. hits others, easily frustrated]) are empirically derived and form 2 broad domains, Internalizing and Externalizing Problems. The Internalizing scale encompasses the Emotionally Reactive, Anxious/Depressed, Somatic Complaints, and Withdrawn Behaviors, whereas the Externalizing scale includes Attention Problems and Aggressive Behaviors. However, only the Internalizing domain was used, given that Internalizing, not Externalizing problems are associated with prematurity (Aarnoudse-Moens et al. 2009; Grunau, Whitfield, Fay 2004). Reliability for the Internalizing subscale is high (test–retest Pearson r = 0.90; Cronbach’s alpha 0.92; Achenbach and Rescorla 2000).   	   92 4.2.8 Emotional Availability The primary caregiver participated in a 5-min videotaped semi-structured teaching scenario with their child. This involved the caregiver trying to teach her child to perform tasks of varying difficulty. The easier and more familiar task involved stacking or nesting colored cups of varying sizes. The novel and more difficult task involved sorting plastic pigs and cows into separate containers. Parent behavior during this interaction was later scored from videotape using the Emotional Availability Scale IV (Biringen 2008).  The EA scale captures 4 dimensions of parent behavior: Sensitivity (appropriateness/authenticity of affect), Structuring (provision of guidance), Nonintrusiveness (no overstimulation/overprotection), and Nonhostility (nonthreatening/non-frightening; Biringen 2008). Each EA dimension has 7 subscales, which are summed to provide a total score for the dimension. Scores range from 7 to 29, and higher scores denote emotionally available parenting. According to the clinical cutoffs for the EA scale, parents with scores ranging from 7 to 17 are considered to be Nonoptimally EA, 18 to 25 Inconsistently EA, and 26 to 29 Optimally EA (Biringen 2008). An average score, in the mid range, falls within the Inconsistent category. This represents a parent that has adequate EA, but may have moments during the interaction where they are less emotionally available to their child. Trained coders assessed EA from videotape: 1 primary coder and 2 reliability coders blinded to all other information about the participants. Inter-rater reliability assessed with intraclass correlation coefficients was 0.89, 0.86, 0.89, and 0.87 for Sensitivity, Structuring, Nonintrusiveness, and Nonhostility, respectively.  4.2.9 Data Analyses Predictive Analytics Software (PASW) Statistics 18.0.3 (IBM) was used. Normality plots were 	   93 examined and the number of invasive procedures was log transformed. Demographic characteristics of the preterm and full-term groups, and comparisons between infants included and excluded in this study were examined by t-tests or chi-square tests, when appropriate. Univariate analysis of variance (ANOVA) was performed to examine group (preterm and full term; ELGA and VLGA) by gender differences on EA and Internalizing scores at 18 months. Pearson correlations were used to examine associations among measures for both the preterm and full-term groups. Multivariate analyses were performed using GENLIN. For each EA dimension (Sensitivity, Structuring, Nonintrusiveness, Nonhostility), GENLIN modeling was used to examine whether parent EA adjusted for Parenting Stress, parent’s years of education, number of children in the home, and parent age moderated the relationship between the number of invasive procedures (adjusted for illness severity on day 1, days on mechanical ventilation, and total morphine exposure) and Internalizing behaviors (adjusted for gender and Bayley MDI) at 18 months CA in children born very preterm. In addition, we included a variable in our analysis to account for prematurity at birth (ELGA or VLGA), given that ELGA children exhibited greater associations between an altered pattern of cortisol expression and internalizing behaviors at 18 months CA relative to VLGA children (Brummelte et al. 2011b). An interaction term between EA and the number of invasive procedures was included in each of the models. Post hoc t-tests were used to further explore statistically significant interactions. The univariate ANOVAs, GENLIN models, and post hoc t-tests were repeated excluding the 3 mothers in our sample who reported drinking alcohol during their pregnancy. Finally, to better understand the etiology of the highly prevalent internalizing behaviors seen in children born prematurely relative to full-term control children, GENLIN models for each EA dimension (Sensitivity, Structuring, Nonintrusiveness, Nonhostility) were used to examine whether parent EA adjusted for parenting Stress, parent’s years of education, number of children in the home, and parent age 	   94 was associated with Internalizing behaviors.  4.3 Results  4.3.1 Characteristics of the Cohort  Of the families we contacted, 120 of 159 (75%) of the parents of children born very preterm and 57 of 71 (80%) of the parents of children born full term returned for the 18-month follow-up. After exclusions, we included 96 very preterm and 49 full-term children in this study. Importantly, the 96 children born very preterm that were included in the present study did not differ in GA, birth weight, or sex from the original sample of infants recruited from the NICU at the B.C. Children’s & Women’s Hospitals between February 2001 and July 2004 (all P> .05). Similarly, the 49 full-term control children did not differ in GA or sex from the original sample of full-term infants recruited at birth (all P> .05). The full-term control children included in this study had a lower mean birth weight than the full-term infants in the original sample (t[96]=  -2.26, P= .03). This difference, however, was no longer significant after the 6 children born large for their GA were removed from the analysis (t[90]= -1.35, P= .18). As expected, GA and birth weight differed between infants born very preterm versus full term. The only significant difference between parents of preterm versus full-term infants was parent’s years of education; parents of infants born very preterm had fewer years of education than parents of infants born full term. Children born very preterm had lower Bayley MDI cognitive scores and demonstrated more Internalizing behaviors at 18 months CA than children born full term. Characteristics of the sample are listed in Table 4.1.    	   95 Table 4.1 Characteristics of the Cohort  Neonatal characteristics  Preterm n= 96 Full-term n= 49 P  GA (weeks), median (IQR) 29.4 (26.6-31.3)* 40.0 (39.4-40.6) 0.001 Birth weight (grams), median (IQR) 1222 (813-1641) 3475 (3240-3678) 0.001 Gender (male), number (%) 47 (49) 21 (43) 0.49 Illness severity on day 1 (score), median (IQR) 9 (5-19) - - Number of invasive procedures, median (IQR) 87 (49-176) - - Mechanical ventilation (days), median (IQR) 3 (0-18) - - Total morphine exposure (mg/kg),  median (IQR) 0.10 (0.00-1.28) - - Parent characteristics at 18 month visit    Age (years), median (IQR) 36 (31-39) 37 (33-40) 0.07 Marital status (married), number (%) 93 (97) 46 (94) 0.50 Ethnicity (Caucasian), number (%)  73 (76) 37 (76) 1.00 Parent education (years), median (IQR) 15 (13-17) 18 (15-19) 0.001 Children at home (number), median (IQR) 2 (1-3) 2 (1-2) 0.18 PSI Parenting stress (score), median (IQR) 109 (97-128) 115 (92-122) 0.24 EA Sensitivity (score), median (IQR) 19 (16-22) 21 (15-23) 0.72 EA Structuring (score), median (IQR) 20 (17-22) 19 (16-24) 0.37 EA Nonintrusiveness (score), median (IQR) 19 (16-22) 20 (16-22) 0.62 EA Nonhostility (score), median (IQR) 21 (19-24) 22 (19-24) 0.66 Child Characteristics    Bayley MDI (score), median (IQR) 91 (78-101) 97 (87-107) 0.01 CBCL Internalizing (t-score), median (IQR) 45 (41-51) 41 (33-49) 0.02 *44 children (46%) were born EGLA, 52 children (54%) were born VLGA CBCL, Child Behavior Checklist (Achenbach and Rescorla 2000); EA, Emotional Availability (Biringen 2000); IQR, interquartile range; Bayley MDI, Bayley Mental Development Index (Bayley 1993); PSI, Parenting Stress Index (Abidin 1995).  	   96 4.3.2 Parent and Child Behavior: Group by Gender Analyses Assumptions were met for the ANOVAs: Levene’s test for equality of variances was nonsignificant, and skewness was between -1 and 1 for the variables Internalizing, Sensitivity, Structuring, and Nonintrusiveness for the preterm, full-term, ELGA, and VLGA groups. Children born preterm demonstrated significantly more Internalizing behaviors at 18 months CA than children born full term (F[1,141]= 5.88, P= .02); gender was not significant, and there was no group-by-gender interaction (P= .76). Internalizing behaviors, however, did not differ between ELGA and VLGA children (P= .39). Internalizing behavior was not correlated with the Bayley MDI for the preterm (r= -0.19, P= .07) or full-term children (r= 0.06, P= .67). Parent EA (Sensitivity, Structuring, Nonintrusiveness, Nonhostility) did not differ significantly by group (preterm, full term) or by gender (all P> .36). However, parents of children born ELGA children provided less Structure than parents of VLGA children (F[1,92]= 4.68, P= .03).  4.3.3 Correlations Among Neonatal Variables Among the infants born very preterm, lower GA at birth was correlated with higher illness severity on day 1, greater number of invasive procedures, more days of mechanical ventilation, and more total morphine exposure (Table 4.2). Given that all correlations were r< 0.80, multicollinearity among the neonatal predictors was not considered to be problematic (Katz 2011).      	   97 Table 4.2 Pearson Correlations Among the Neonatal Variables of the Preterm Infants  Illness severity on day 1 # of invasive procedures  Days on mechanical ventilation Total morphine exposure GA group -0.59***           -0.77*** -0.64*** -0.39*** Illness severity on day 1 -            0.58*** 0.51*** 0.28*** # of invasive procedures -                - 0.80*** 0.52*** Days on mechanical ventilation -                - - 0.77*** *** P < 0.001  GA group (24-28 weeks GA or 29-32 weeks GA)    4.3.4 Correlations Among Parent Variables The correlations among the 4 EA dimensions for parents of preterm children ranged from r= 0.47 to r= 0.80, and for parents of full-term children ranged from r= 0.62 to r= 0.88 (Table 4.3). Each EA dimension was entered in a separate multivariate model. In the preterm group, more years of education was associated with higher parent age, Sensitivity, and Nonhostility. In the full-term group, more years of education was associated with higher parent age and lower Parenting Stress. Unlike the preterm group, higher parent age among parents of full-term children was associated with greater parent Sensitivity, Nonintrusiveness, and Nonhostility (Table 4.3).     	   98 Table 4.3 Pearson Correlations Among the Parent Variables for Preterm and Full-Term Groups  Preterm Structuring Non-intrusiveness Non-hostility Parenting Stress Parent’s years of education # of children in the home Parent age Sensitivity 0.70*** 0.70*** 0.80*** -0.05 0.21* 0.41 -0.02 Structuring - 0.37*** 0.47*** 0.30 0.07 0.05 -0.02 Nonintrusiveness - - 0.52*** -0.17 0.11 0.05 0.12 Nonhostility - - - 0.04 0.25* -0.05 0.003 Parenting Stress - - - - -0.10 0.08 0.04 Parent’s years of education - - - - - -0.14 0.29** # of children in the home - - - - - - 0.03  Full-term        Sensitivity 0.78*** 0.82*** 0.88*** -0.21 0.04 0.05 0.31* Structuring - 0.62*** 0.72*** -0.19 -0.01 0.14 0.27 Nonintrusiveness - - 0.78*** -0.21 0.09 0.13 0.30* Nonhostility - - - -0.27 0.09 0.07 0.31* Parenting Stress - - - - -0.30* 0.21 -0.18 Parent’s years of education - - - - - -0.05 0.34* # of children in the home - - - - - - 0.18 *   P < 0.05; ** P < 0.01; *** P < 0.001    	   99 4.3.5 Invasive Procedures, EA Sensitivity, and Internalizing Behavior In the GENLIN models, there was a significant interaction between parent Sensitivity and the number of invasive procedures in relation to Internalizing behavior at 18 months CA in children born very preterm (B= 1.30, P= .05; Table 4.4), after adjusting for neonatal medical confounders (GA group [ELGA or VLGA], illness severity on day 1, days of mechanical ventilation, cumulative morphine exposure), concurrent environmental stressors (Parenting Stress, parent’s years of education, number of children in the home), parent age, gender, and Bayley MDI. In order to understand this 2-way interaction, Internalizing scores were plotted by number of invasive procedures separately for subgroups of parent EA behavior: Nonoptimal Sensitivity (n= 37), Inconsistent Sensitivity (n= 54), and Optimal Sensitivity (n= 5) (Figure 4.1). Post hoc t tests revealed significant differences in Internalizing behavior between Nonoptimal and Inconsistently Sensitive parents; among preterm children exposed to a higher number of invasive procedures, greater parent sensitivity was associated with lower internalizing behaviors at 18 months CA (t[44]= 2.32, P= .03; Figure 4.1). Higher Parenting Stress (B= 0.15, P= .001), fewer years of education (B= -0.87, P= .008), and fewer children in the home (B= -2.46, P= .004) were independently associated with more Internalizing behaviors at 18 months CA in children born very preterm. 	        	   100 Table 4.4 Greater Parent Sensitivity and Nonhostility were Associated with Fewer Internalizing Behaviors at 18 months CA among Preterm Children Exposed to a Higher Number of Invasive Procedures  Child Internalizing  Sensitivity†  Model Structuring†  Model Nonintrusiveness†  Model Nonhostility†  Model Predictors B P B P B P B P EA† -2.32 0.07 -1.31 0.40 0.22 0.85 -4.01 0.02 Number of invasive procedures  -19.08 0.129 -9.69 0.50 8.15 0.50 -45.76 0.02 EA† x number of invasive procedures 1.30 0.05 0.74 0.32 -0.19 0.75 2.32 0.006 Days on mechanical ventilation -0.06 0.62 -0.05 0.64 -0.10 0.33 -0.05 0.59 Total morphine exposure 0.08 0.67 0.05 0.80 0.07 0.71 0.18 0.33 Illness severity on day 1 -0.004 0.96 -0.02 0.85 -0.01 0.89 -0.01 0.95 GA group 0.12 0.97 0.39 0.90 0.78 0.79 0.22 0.94 Gender -2.85 0.14 -3.15 0.12 -3.93 0.05 -2.43 0.20 Parenting stress 0.15 0.001 0.15 0.001 0.14 0.001 0.16 0.001 Parent’s years of education -0.87 0.008 -0.81 0.01 -0.74 0.03 -0.82 0.01 # of children in the home -2.46 0.004 -2.29 0.009 -2.17 0.01 -2.25 0.007 Parent age -0.13 0.49 -0.17 0.37 -0.23 0.21 -0.16 0.35 Bayley MDI -0.05 0.56 -0.05 0.44 -0.06 0.35 -0.07 0.22 Adjusted R2 0.26* 0.23* 0.22* 0.31* †The GENLIN was repeated four times: each time a different EA variable (Sensitivity, Structuring, Nonintrusiveness, Nonhostility) was entered into the model. *Computed using linear regression.  Parent Sensitivity and Nonhostility moderated the relationship between the number of invasive procedures and Internalizing in children born very preterm at 18 months CA after adjusting for neonatal medical confounders (GA group [24-28 weeks GA or 29-32 weeks GA]), illness severity on day 1, days of mechanical ventilation, cumulative morphine exposure), concurrent environmental stressors (Parenting Stress, parent’s years of education, number of children in the home), parent age, child gender and cognition (Bayley MDI). Higher Parenting Stress, fewer maternal years of education and fewer children in the home were independently associated with greater Internalizing at 18 months corrected age in children born very preterm.  	   101 Figure 4.1 Parental Sensitivity Moderates the Relationship Between Invasive Procedures in the NICU and Internalizing Behavior in Children Born Very Preterm    Predicted values of Internalizing behaviors (t-score) in relation to number of invasive procedures from birth to term-equivalent age adjusted for GA group (24 to 28 weeks GA or 29 to 32 weeks GA), illness severity on day 1, days of mechanical ventilation, cumulative morphine exposure, Parenting Stress, parent’s years of education, number of children in the home, parent age, child gender, and cognition. Differences in Internalizing scores were between nonoptimal and inconsistently sensitive parents, whose infants received a high number of invasive procedures. Among preterm children exposed to a higher number of invasive procedures, greater parent sensitivity was associated with lower Internalizing behaviors at 18 months corrected age.    4.3.6 Invasive Procedures, EA Structuring, and Internalizing Behavior In the GENLIN models, the interaction between parent Structuring and invasive procedures in relation to Internalizing behavior at 18 months CA in children born very preterm was not significant (B= 0.74, P= .32; Table 4.4), after adjusting for neonatal medical confounders (GA group, illness severity on day 1, days of mechanical ventilation, cumulative morphine exposure), concurrent environmental stressors (Parenting Stress, parent’s years of education, number of children in the home), parent age, gender, and Bayley MDI. However, higher Parenting Stress (B= 0.15, P= .001), fewer years of education (B = -0.81, P= .01), and fewer children in the home 	   102 (B = -2.29, P= .009) were independently associated with more Internalizing behaviors at 18 months CA in children born very preterm.  4.3.7 Invasive Procedures, EA Nonintrusiveness, and Internalizing Behavior In the GENLIN models, the interaction between parent Nonintrusiveness and neonatal pain in relation to Internalizing at 18 months CA in children born very preterm was not significant (B = -0.19, P= .75; Table 4.4), after adjusting for neonatal medical confounders (GA group, illness severity on day 1, days of mechanical ventilation, cumulative morphine exposure), concurrent environmental stressors (Parenting Stress, parent’s years of education, number of children in the home), parent age, gender, and Bayley MDI. However, higher Parenting Stress (B= 0.14, P= .001), fewer years of education (B= -0.74, P= .03), and fewer children in the home (B= -2.17, P= .01) were independently associated with more Internalizing behaviors at 18 months CA in children born very preterm.  4.3.8 Invasive Procedures, EA Nonhostility, and Internalizing Behavior In the GENLIN models, there was a significant interaction between parent Nonhostility and neonatal pain in relation to Internalizing behavior at 18 months CA in children born very preterm (B= 2.32, P= .006; Table 4.4), after adjusting for neonatal medical confounders (GA group, illness severity on day 1, days of mechanical ventilation, cumulative morphine exposure), concurrent environmental stressors (Parenting Stress, parent’s years of education, number of children in the home), parent age, gender, and Bayley MDI. In order to understand this 2-way interaction, Internalizing scores were plotted by number of skin-breaking procedures, separately for subgroups of parent EA behavior: Nonoptimal Nonhostile (n= 16), Inconsistent Nonhostile (n= 75), and Optimal Nonhostile (n= 5) (Figure 4.2). Although the relationship between 	   103 Nonoptimal and Inconsistently Nonhostile parenting and neonatal pain (Figure 4.1) was similar to the Nonoptimal and Inconsistently Sensitive parents whose infants received a high number of skin-breaking procedures (Figure 4.2), the group size was limited (t[45] = 0.57, P = .57). Consistent with the analyses above, higher Parenting Stress (B= 0.16, P= .001), fewer years of education (B= -0.82, P= .01), and fewer children in the home (B= -2.25, P= .007) were independently associated with more Internalizing behaviors at 18 months CA in children born very preterm.  Figure 4.2 Parental Nonhostility Moderates the Relationship Between Invasive Procedures in the NICU and Internalizing Behavior in Children Born Very Preterm   Predicted values of Internalizing behaviors (t-score) in relation to number of invasive procedures from birth to term-equivalent age adjusted for gestational age (GA) group (24 to 28 weeks GA or 29 to 32 weeks GA), illness severity on day 1, days of mechanical ventilation, total morphine exposure, Parenting Stress, parent’s years of education, number of children in the home, parent age, child gender, and cognition. Differences in Internalizing scores were between nonoptimal and inconsistently nonhostile parents, whose infants received a high number of invasive procedures. Among preterm children exposed to a higher number of invasive procedures, greater parent nonhostility appears to lower Internalizing behaviors at 18 months corrected age.    	   104 4.3.9 Exclusion of Mothers Who Reported Drinking Alcohol During their Pregnancy The univariate ANOVAs, GENLIN models, and post hoc t-tests were repeated excluding the 3 mothers in our sample who reported drinking alcohol during their pregnancy, and the results of our models remained unchanged.  4.3.10 Parent Behavior and Stress in Relation to Internalizing Behavior in Children Born Full-Term There were no significant associations between parent EA (Sensitivity, Structuring, Nonintrusiveness, Nonhostility) or stress (Parenting Stress, parent’s years of education, number of children in the home), and Internalizing behavior (adjusted for GA, parent age, gender, and Bayley MDI) at 18 months in children born full-term.  4.4 Discussion In this study, we examined whether parent emotional availability moderated the relationship between invasive procedures in the NICU and internalizing behavior at 18 months CA in children born very preterm. We found that among children born very preterm exposed to a higher number of invasive procedures (adjusted for confounding neonatal medical factors), greater parent sensitivity and nonhostility were associated with lower internalizing behaviors at 18 months CA. In addition, lower parenting stress, more years of education, and more children in the home were also independently associated with fewer internalizing behaviors in children born very preterm at 18 months CA. In contrast, none of the parent factors were a significant predictor of internalizing behavior in children born full term. Despite the similarities in parenting stress and parent behavior, children born very preterm were more influenced by interactions with their 	   105 parents compared to their term-born peers, consistent with previous findings from our group and others (Brummelte et al. 2011b; Crnic and Greenberg 1987; Tu et al. 2007).  Both in humans and animals, repeated exposure to invasive procedures is associated with increased anxious/depressive behaviors (Anand et al. 1999; Ranger et al. 2014). Repeated exposure to invasive procedures in the NICU is associated with the reprogramming of the HPA axis. At 32 weeks PMA, greater exposure to invasive procedures is associated with lower cortisol responses, independent GA, early illness severity and morphine exposure (Grunau et al. 2005). However, at 8 and 18 months CA, greater number of invasive procedures in the NICU was associated with higher levels of cortisol (Grunau, Weinberg, Whitfield 2004; Grunau et al. 2007). Among infants born ELGA, there is evidence for a shift from low basal cortisol levels at 3 months to relatively high levels at 8 and 18 months CA, which suggests a biological “resetting” of endocrine stress systems (Grunau et al. 2007). Cortisol levels at 18 months CA were associated with internalizing behaviors among both ELGA and VLGA children (Brummelte et al. 2011b).  Parental behavior also plays an important role in the programming stress response (Ahnert et al. 2004; Coplan et al. 1996; De Bellis 2005; Gunnar et al. 1996; Meaney and Szyf 2005; Pryce and Feldon 2003). In rats, high licking and grooming by the dam results in the hypomethylation of the NGFI-A transcription factor, thereby permitting binding of NGFI-A to the glucocorticoid receptor promoter (Weaver et al. 2004; Weaver et al. 2007). The adult offspring of high licking and grooming mothers show increased hippocampal glucocorticoid receptor expression, better glucocorticoid feedback sensitivity, less corticotrophin releasing factor and less glucocorticoid production compared to pups reared by low licking and grooming mothers (Francis et al. 1999a; 	   106 Liu et al. 1997). Rats with higher licking and grooming mothers have fewer anxiety-like behavior during adulthood, indicated by fewer startle responses, increased open-field exploration, greater social interaction and shorter latencies to eat in a novel environment (Caldji et al. 1998; Pena et al. 2014; Starr-Phillips and Beery 2014; van Hasselt et al. 2012). Similarly, in humans, sensitive maternal behavior was associated with lower cortisol levels, and fewer internalizing behaviors at 18 months CA in children born very preterm (Brummelte et al. 2011b). Among the preterm infants exposed to higher numbers of invasive procedures, greater maternal sensitivity and nonhostility were associated with fewer internalizing behaviors at 18 months CA.   Parent’s concern regarding their own parenting ability (parenting stress) was associated with child internalizing behaviors at 18 months CA. Parenting stress may interfere with how the parent interacts with their child. Mothers who reported higher stress in the NICU and/or higher concurrent stress were less sensitive/emotionally available when interacting with their child post-discharge (Muller-Nix et al. 2004; Tu et al. 2007; Zelkowitz et al. 2009). Higher parenting stress may also reflect trait anxiety, which has also been shown to be associated with increased internalizing in children born very preterm at 18 months CA, independent of maternal education and neonatal morbidity (Zelkowitz et al. 2011).   Fewer years of education, an indicator of lower SES, was associated with greater internalizing behavior at 18 months CA in children born very preterm. Importantly, the relationship between parent’s years of education and internalizing behavior remained significant after accounting for child cognition, which is associated with both parent’s years of education and child interactive behavior (Lowe, Erickson, MacLean 2010). Parents with fewer years of education were less sensitive and more hostile compared to parents with more years of education. Previous studies 	   107 have shown that the relationship between socioeconomic risk and behavior in preterm infants and children was mediated by maternal behavior (Candelaria, Teti, Black 2011; Linver, Brooks-Gunn, Kohen 2002). NICU-based programs designed to enhance the quality of interaction between low SES mothers and their infants have lead to improved home environments and better infant temperament at 4 and 8 months CA (Parker et al. 1992). It is noteworthy, however, that the level of parent’s education was relatively high in our cohort, with parents of preterm infants having a median of 15 years education, indicative of some postsecondary college attendance.  Fewer children in the home was also associated with greater internalizing behavior in children born very preterm. First-time parents may underestimate the personal impact of the birth of the infant (Evans, Whittingham, Boyd 2012); a life-altering change in combination with an unexpected preterm birth appears to increase the risk for a negative transition to parenthood (Harwood, McLean, Durkin 2007). Realistic expectations of parenthood may improve parent attachment/responsiveness and lessen parent psychological symptoms (Evans, Whittingham, Boyd 2012), factors that are in turn associated with fewer internalizing behaviors (Gravener et al. 2012; O'Connor et al. 2011). Moreover, siblings can also play an important role in behavioral development; support from siblings can be a buffer to feelings of loneliness and depression in contexts of decreased parental and/or peer support (East and Rook 1992; Milevsky and Levitt 2005). Adult siblings of children born preterm have retrospectively described their relationships with their brother or sister as both positive and protective (Gaal et al. 2010).  Given the correlational nature of this study and the bidirectional nature of parent–child interaction, it is important to note that greater child internalizing behavior may contribute to lower parent emotional availability. Parent sensitivity and nonhostility appear to moderate the relationship between neonatal pain and internalizing behavior in children born very 	   108 preterm, who are more sensitive to their environment than full-term control children. Future research is needed to determine whether early or concurrent emotional availability training can effectively prevent or reduce the long-term effects of neonatal pain on internalizing behavior in children born very preterm. NICU-based or follow-up programs designed to facilitate parent emotional availability may be able to effectively prevent or reduce internalizing behavior in children born very preterm. However, with limited resources for training, the results from this study suggest that parents who have fewer years of education, are highly stressed, or are first-time parents may be a priority for support. Helping parents to appropriately regulate pain/stress in their infant may also help to improve infant interactions, thereby reducing and/or preventing the development of internalizing behaviors in their preterm child.  Invasive procedures, inherent to life-saving care in the NICU, may contribute to the development of internalizing behavior in children born very preterm, consistent with the animal literature on early stress. Interventions focused on improving parent-child interaction and reducing parenting stress may help to ameliorate the negative long-term effects of invasive procedures on child behavior.         	   109 CHAPTER 5  INVASIVE PROCEDURES IN PRETERM CHILDREN: BRAIN AND COGNITIVE DEVELOPMENT AT SCHOOL AGE  5.1 Introduction Advances in neonatal medical care have greatly improved the chances of survival for infants born very preterm. However, cognitive impairment appears to have increased among children with birth weights ≤800 grams (Doyle et al. 2011; Moore et al. 2012; Synnes et al. 2010). Even in the absence of major disability (e.g., blindness, nonambulatory cerebral palsy, developmental delay), cognitive problems and school difficulties are common among children born very preterm (Grunau, Whitfield, Davis 2002; Grunau, Whitfield, Fay 2004; Grunau et al. 2009; Johnson et al. 2009; Larroque et al. 2008; Saigal and Doyle 2008).  Infants born very preterm are repeatedly exposed to invasive life-saving procedures during a sensitive and rapid period of brain development (Kostovic and Jovanov-Milosevic 2006; Volpe 2009). Two cell populations are particularly vulnerable to injury in the premature brain: subplate neurons and preoligodendrocytes (Back and Miller 2014; Volpe 2009). Subplate neurons and preoligodendrocytes are vulnerable to excitoxicity, reactive oxygen, nitrogen species and cytokines (Back et al. 1998; Back et al. 2005; Buntinx et al. 2004; Ghosh et al. 1990; Ghosh and Shatz 1992; Haynes et al. 2003; McQuillen and Ferriero 2005; Pang, Cai, Rhodes 2005). Greater exposure to invasive procedures has been shown to be both directly and indirectly associated  with altered brain development in the NICU and at term-equivalent age (Brummelte et al. 2012; Smith et al. 2011; Vinall et al. 2012; Vinall et al. 2013b; Zwicker et al. 2013). A version of Chapter 5 has been published in Vinall J, Miller SP, Bjornson BH, Fitzpatrick KPV, Poskitt KJ, Brant R, Synnes AR, Cepeda IL, Grunau RE (2014). Pediatrics,133(3):412-21. 	   110 These findings are supported by evidence from animal models that have demonstrated both inflammatory pain and repeated injections increase apoptosis in the neonatal rat brain (Anand et al. 2007; Duhrsen et al. 2013). Moreover, greater exposure to invasive procedures in the NICU was associated with poorer cognitive outcomes at 8 and 18 months CA in children born very preterm (Grunau et al. 2009).  Recent studies of our group have shown that the impact of invasive procedures on the brain and neurodevelopmental outcomes may extend beyond the neonatal period. At 7 years of age, greater numbers of invasive procedures in the NICU were associated with thinner cortical gray matter in 21 out of 66 cerebral regions assessed, predominately affecting the frontal and parietal lobes (Ranger et al. 2013). Moreover, among infants born ELGA, greater exposure to invasive procedures were also associated with alterations in spontaneous neuromagnetic activity (Doesburg et al. 2013). Lower synchronization of oscillatory activity was associated with poorer visual perceptual ability at school age (Doesburg et al. 2013). Therefore, repeated exposure to invasive procedures in the NICU appears to be associated with long-term alterations to cortical volumes and brain function. However, we still do not know from these studies how the brain microstructure underlying changes in volume and function is altered by repeated exposure to invasive procedures early in life.   The current study examined whether the number of invasive procedures during neonatal care was associated with white matter microstructure at age 7 years, and whether the number of invasive procedures together with measures of brain microstructure predicted cognitive outcome at school age in children born very preterm.  	   111 5.2 Methods 5.2.1 Study Overview Children born very preterm recruited from the NICU of the BC Children’s & Women’s Hospitals. Neonatal data were acquired from medical chart review performed from birth to term-equivalent age. At a median age of 7.6 years (interquartile range, 7.5– 7.7), children underwent MRI and cognitive testing. T1- and T2-weighted images were assessed for the severity of brain injury. Magnetic resonance diffusion tensor sequences were used to measure FA, an index of white matter maturation, from 7 anatomically defined white matter regions. Multivariate modeling was used to examine relationships between invasive procedures, brain microstructure, and cognition, adjusting for GA, small for gestational age (SGA: <10th weight percentile at birth), illness severity on day 1, days on mechanical ventilation, infection, gender, age at scan, brain injury, surgery, morphine, fentanyl, corticosteroids and midazolam.   5.2.2 Participants Fifty children born very preterm (≤32 weeks GA) recruited from the NICU of the BC Children’s & Women’s Hospitals between February 2001 and July 2004 underwent MRI at median age 7.6 years (interquartile range [IQR], 7.5–7.7) as part of an ongoing study on the effects of neonatal pain on neurodevelopment of children born very preterm (Doesburg et al. 2013; Grunau et al. 2007; Grunau et al. 2009; Ranger et al. 2013; Ranger et al. 2014; Vinall et al. 2013a). Children were excluded if they had a major congenital anomaly, major neurosensory impairment (legally blind, nonambulatory cerebral palsy, sensori-neural hearing impairment), or severe brain injury evident on neonatal ultrasound (PVL or grade 3 or 4 IVH).  	   112 5.2.3 Neonatal Medical Chart Review Neonatal data were acquired from medical chart review performed from birth to term-equivalent age or discharge (whichever came first) by a neonatal research nurse. We defined the number of invasive procedures as every attempt at a procedure as listed in Appendix Table A.1, from birth to term-equivalent age, adjusted for clinical confounders (e.g. illness severity on day 1 [SNAP-II; Richardson et al. 2001], days of mechanical ventilation, confirmed infection, morphine exposure).   5.2.4 Magnetic Resonance Imaging Children were scanned at a median age of 7.6 years (IQR, 7.5–7.7). A Siemens 1.5 Tesla Avanto magnet, standard 12-channel head coil, and VB 16 software were used to obtain the following sequences: 3-dimensional T1-weighted spoiled gradient recalled acquisition (repetition time [ms] 18/echo time [ms] 9.2/field of view [mm] 256/slice thickness [mm] 1/gap [mm] 0/matrix 256 x 256) and T2-weighted images axial fast spin echo (4030/90/220/3/1/512 x 354) and axial fluid attenuation inversion recovery (8900/87/220/5/1/256 x 154). Neuroradiologist K.J.P., blinded to the child’s medical history, assessed these images for brain injury (i.e. evidence of cerebellar hemorrhage, ventriculomegaly, or moderate to severe WMI, as described previously; Miller et al. 2005).   5.2.5 Diffusion Tensor Imaging DTI was acquired with a multirepetition, single-shot echo planar sequence with 12 gradient directions (7800/82/256/2/0/128 x 128), 3 averages of diffusion weighting 700 (b value). DTI parameters of FA, λ1, λ2, and λ3 were obtained from 7 bilateral regions of interest in the white matter (Figure 5.1), consistent with our neonatal studies (Brummelte et al. 2012; Chau et al. 	   113 2009). Intrarater reliability, based on the repeated analysis of a random 20% of regions of interest, was comparable with previously published findings (FA mean difference of -0.002 [Bland–Altman limits of agreement, -0.011 to 0.007]; Brummelte et al. 2012; Chau et al. 2009) .  Figure 5.1 Regions of Interest in the White Matter   A) Superior white matter: (a) anterior, (b) middle, and (c) posterior subcortical white matter.  B) white matter tracts: (d) genu of the corpus callosum, (e) posterior limb of the internal capsule, (f) splenium of the corpus callosum, and (g) optic radiations.    5.2.6 Cognitive Testing At age 7 years, IQ was measured by using the standardized Wechsler Intelligence Scale for Children– 4th Edition (WISC-IV; Wechsler 2003), which includes 4 index scores that make up the Full Scale IQ (FSIQ): Verbal Comprehension, Perceptual Reasoning, Working Memory, and Processing Speed. 	   114  5.2.7 Data Analyses Statistical analyses were performed by using Stata 9.2 (Stata Corp, College Station, TX). Normality plots were examined, and skewed variables (number of invasive procedures, days on mechanical ventilation, morphine exposure, FA values, and age at scan) were log transformed. IQ, GA, birth weight, and illness severity on day 1 of the included and excluded preterm infants were compared by using t-tests. Demographic characteristics of the preterm infants exposed to lower and higher numbers of invasive procedures were compared by using t-tests or χ2, when appropriate. Multivariate analyses were adjusted for confounders: GA, size at birth (small for gestational age versus appropriate for gestational age), illness severity on day 1, days of mechanical ventilation, morphine exposure, infection, gender, age at scan, and concurrent brain injury. A generalized estimating equation was used to examine whether the number of invasive procedures was associated with FA at age 7 years in an initial pain model. This model was repeated for the axial and radial axes. The pain model was extended to include variables for surgery and fentanyl exposure (surgery model), and corticosteroids and midazolam (steroid model). The regression coefficients for these models are reported as effect sizes. FA values were then grouped a priori into superior white matter (anterior, middle, and posterior subcortical white matter) and white matter tracts (genu and splenium of the corpus callosum, posterior limb of the internal capsule, optic radiations), and group means were used for analysis. Generalized linear modeling was used to examine whether the number of invasive procedures interacted with FA values from either the superior white matter or white matter tracts to predict FSIQ.  	  	   115 5.3 Results 5.3.1 Characteristics of the Cohort Of the 131 eligible children contacted for the 7-year follow-up, 22 refused to participate and 7 withdrew, so that 102/131 (78%) were seen at school age. One child diagnosed with autism was excluded, leaving 101 children in this study. Of the 101 who returned for follow-up (psychometric assessment) at median age 7.6 years (IQR, 7.5–7.8), 58 (57%) parents and children consented/assented to an MRI. Research scans were available only on weekdays after 4 PM, and booking limitations affected study consents for MRI. Scans were not completed for 3 of the participants, and 3 were of poor quality because of motion artifact. Moreover, 2 children were missing either neonatal or follow-up data. Therefore, data from 50 children born very preterm were included in the current study. Importantly, the FSIQ of the children included (n= 50) did not differ from that of the other 51 children who returned for 7-year follow-up (95% confidence interval [CI]: -7.18 – 3.86, P= .55). Moreover, children included in the current study did not differ in GA (95% CI: -1.40 – 0.44, P = .30), birth weight (95% CI: -187.33 – 147.52, P=.81), or early illness severity (95% CI: -2.81 – 5.73, P = .50) from the children who returned for follow-up or from the 81 infants in the original sample (95% CI: -1.10 – 0.63, P= .59; CI:  -134.99 – 194.17, P= .72; and 95% CI: -2.50 – 5.73, P= .44; respectively).  Among the 50 children with imaging data at age 7 years, exposure to higher numbers of invasive procedures (median 122; IQR, 81 – 210) was associated with lower GA, higher illness severity on day 1, more days on mechanical ventilation, and a greater exposure to surgery, infection, dexamethasone, and morphine compared with children exposed to lower numbers of procedures (median 46; IQR, 30 – 55) (Table 5.1). Among the 101 children born very preterm who returned for follow-up at 7 years, exposure to higher numbers of invasive procedures (median 127; IQR, 	   116 87 – 200) were also associated with increased exposure to midazolam and fentanyl and a significantly lower FSIQ compared with children exposed to lower numbers of procedures (median 43; IQR, 32 – 52) (Table 5.2).                      	   117 Table 5.1 Characteristics of Children with Magnetic Resonance Imaging at Age 7 Years   Lower # of Invasive Procedures Median 46 IQR 30-55 Higher # of Invasive Procedures Median 122 IQR 81-210  Neonatal Characteristics n=50 n=25 n=25 P Gestational age (weeks), median (IQR) 29.8 (28.1-31.9) 31.4 (29.7-32.3) 28.4 (26.9-30.4) <0.001 Small for gestational age, number (%) 6 (12) 2 (8) 4 (16) 0.13 Illness severity on day 1, median (IQR) 8 (0-18) 0 (0-9) 14 (5-23) 0.01 Invasive Procedures, median (IQR) 74 (46-124) - -  Surgery, number (%) 8 (16) 0 (0) 8 (32) <0.001 Infection, number (%) 11 (22) 1 (4) 10 (40) <0.001 Mechanical ventilation (days),  median (IQR) 2 (0-8) 0 (0-1) 7 (3-20) <0.001 Dexamethasone or hydrocortisone,  number exposed (%) 5 (10) 0 (0) 5 (20) <0.001 Morphine exposure (mg/kg), median (IQR)  number exposed (%) 0.0 (0.0-0.6) 24 (48) 0.0 (0.0-0.0) 4 (16) 0.5 (0.1-1.8) 20 (80) 0.03 Midazolam exposure (mg/kg), median (IQR)  number exposed (%) 0.0 (0.0-0.0) 3 (6)  0.0 (0.0-0.0) 0 (0) 0.0 (0.0-0.0) 3 (12) 0.12 Fentanyl exposure (µg/kg), median (IQR)  number exposed (%) 0.0 (0.0-0.0) 6 (12) 0.0 (0.0-0.0) 0 (0) 0.0 (0.0-3.0) 6 (24) 0.12  	   118      Child Characteristics      n=50 Lower # of Invasive Procedures Median 46 IQR 30-55 n=25 Higher # of Invasive Procedures Median 122 IQR 81-210 n=25      P Gender (male), number (%) 21 (42) 6 (24) 15 (60) <0.001 Age at scan (years), median (IQR) 7.6 (7.5-7.7) 7.6 (7.5-7.6) 7.6 (7.5-7.7) 0.82 Moderate to severe brain injury, number (%) 6 (12) 4 (16) 2 (8) 0.13 WISC-IV FSIQ, median (IQR) 102 (91-110) 103 (92-110) 95 (85-112) 0.30 WISC-IV VCI, median (IQR) 98 (93-109) 99 (94-109) 98 (89-109) 0.56 WISC-IV PRI, median (IQR) 104 (94-113) 104 (98-112) 100 (91-119) 0.68 WISC-IV WRMI, median (IQR) 98 (91-110) 97 (91-109) 99 (88-115) 0.80 WISC-IV PSI, median (IQR) 94 (86-108)a 100 (88-115) 91 (83-105) 0.09 FSIQ, full scale intelligence quotient; IQR, interquartile range; PRI, perceptual reasoning index; PRSI, processing speed index; VCI, verbal comprehension index; WISC-IV, Weschler Intelligence Scale IV(Weschler 2003); WRMI, working memory index. a2 children did not complete the PRSI.            	   119 Table 5.2 Characteristics of All the Children that Returned for Follow-Up at Age 7 Years    Lower # of Invasive Procedures Median 43 IQR 32-52 Higher # of Invasive Procedures Median 127 87-200  Neonatal Characteristics n=101 n=49 n=51 P Gestational age (weeks), median (IQR) 29.9 (27.5-31.7) 31.6 (29.9-32.4) 27.7 (26.3-29.3) <0.001 Small for gestational age, number (%) 10 (10)a 4 (8) 6 (12) 0.48 Illness severity on day 1, median (IQR) 9 (0-19)a 5 (0-9) 16 (8-25) <0.001 Invasive Procedures, median (IQR) 73 (43-129)a - -  Surgery, number (%) 17 (17)a 1 (2) 16 (31) <0.001 Infection, number (%) 24 (24)a 1 (2) 23 (45) <0.001 Mechanical ventilation (days),  median (IQR) 2 (0-10)a 0 (0-0) 9 (3-31) <0.001 Dexamethasone or hydrocortisone,  number exposed (%) 8 (8) 0 (0) 8 (16) <0.001 Morphine exposure (mg/kg), median (IQR)  number exposed (%) 0.0 (0.0-0.6)a 49 (49) 0.0 (0.0-0.0) 6 (12) 0.6 (0.1-1.8) 43 (84) 0.001 Midazolam exposure (mg/kg) median(IQR)  number exposed (%) 0.0 (0.0-0.0)a 8 (8) 0.0 (0.0-0.0) 0 (0) 0.0 (0.0-0.0) 8 (16) 0.02 Fentanyl exposure (µg/kg), median (IQR),  number exposed (%) 0.0 (0.0-0.0)a 12 (12) 0.0 (0.0-0.0) 0 (0) 0.0 (0.0-3.0) 12 (24) 0.04 	   120      Child Characteristics      n=101 Lower # of Invasive Procedures Median 43 IQR 32-52 n=49 Higher # of Invasive Procedures Median 127 87-200 n=51      P Gender (male), number (%) 50 (50) 19 (39) 31 (61) 0.003 Age at follow-up (years), median (IQR) 7.6 (7.5-7.8) 7.6 (7.5-7.7) 7.6 (7.5-7.8) 0.80 WISC-IV FSIQ, median (IQR) 100 (91-110)b 104 (94-114) 95 (87-108) 0.009 WISC-IV VCI, median (IQR) 98 (93-108)b 99 (95-114) 98 (89-104) 0.09 WISC-IV PRI, median (IQR) 100 (92-116)b 104 (98-117) 100 (88-110) 0.07 WISC-IV WRMI, median (IQR) 97 (88-110)b 99 (91-110) 94 (88-108) 0.10 WISC-IV PRSI, median (IQR) 94 (85-106)b 100 (90-113) 88 (83-99) 0.003 FSIQ,	  full	  scale	  intelligence	  quotient;	  IQR,	  interquartile	  range;	  PRI,	  perceptual	  reasoning	  index;	  PRSI,	  	  processing	  speed	  index;	  VCI,	  verbal	  comprehension	  index;	  WISC-­‐IV,	  Weschler	  Intelligence	  Scale	  IV19;	  WRMI,	  working	  memory	  index.	  a1	  child	  was	  missing	  neonatal	  data;	  2	  children	  did	  not	  have	  neonatal	  infection	  data.	  b4	  children	  did	  not	  complete	  the	  FSIQ;	  2	  children	  did	  not	  complete	  the	  VCI;	  1	  child	  did	  not	  complete	  the	  PRI;	  3	  children	  did	  not	  complete	  the	  WRMI;	  6	  children	  did	  not	  complete	  the	  PRSI.	   5.3.2 Number of Invasive Procedures in Relation to White Matter Microstructure at Age 7 Years Children born very preterm exposed to a greater number of invasive procedures in the NICU had lower FA values at age 7 years (effect size= -0.02, P= .01; CI: -0.04 – -0.005) after adjusting for confounders (GA, birth weight, illness severity on day 1, days of mechanical ventilation, morphine exposure, infection, gender, age at scan, and concurrent brain injury; pain model, Table 5.3). Infants who received the lowest number of invasive procedures (i.e. 10 invasive procedures) had 7% higher FA values than infants who underwent the highest number of invasive procedures (i.e. 267 invasive procedures). The relationship between the number of 	   121 invasive procedures and FA of the white matter was driven by the radial diffusion axes (λ2 and λ3: effect size= 0.05; CI: 0.01 – 0.09; P= .01), such that greater numbers of invasive procedures from birth to term-equivalent age were associated with higher radial diffusion values. In contrast, the number of invasive procedures was not associated with the axial diffusion axis (λ1: effect size= 20.05; CI: -0.15 – 0.06; P= .38). Neither adjustment for surgery and fentanyl nor corticosteroids and midazolam significantly changed the results of the pain model (surgery and steroid models, Table 5.3).                  	   122 Table 5.3 Higher Numbers of Invasive Procedures was Associated with Lower Fractional Anisotropy at Age 7 Years  5.3.3 Number of Invasive Procedures Interacts with the Superior White Matter to Predict FSIQ at Age 7 Years The interaction between number of invasive procedures and FA values of the superior white matter was significantly associated with FSIQ (B= 412.18; P= .02; CI: 55.59 – 768.77; adjusted  Fractional anisotropy  Pain model n=50 Surgery model n=50 Steroid model n=50 Predictors Effect Size P Effect Size P Effect size P Number of invasive procedures -0.02 0.01 -0.02 0.007 -0.02 0.01 Gestational age -0.001 0.71 -0.001 0.73 -0.001 0.76 Small for gestational age -0.003 0.68 -0.003 0.67 -0.005 0.52 Illness severity  <0.001 0.57 <0.001 0.65 <0.001 0.84 Mechanical ventilation -0.003 0.70 -0.002 0.78 <0.001 1.00 Postnatal infection 0.009 0.16 0.008 0.21 0.008 0.26 Gender -0.002 0.71 -0.002 0.72 -0.004 0.49 Age at scan -0.19 0.17 -0.20 0.17 -0.18 0.21 Brain injury -0.009 0.10 -0.009 0.10 -0.007 0.16 Surgery - - 0.002 0.84 -  - Morphine exposure 0.008 0.28 0.008 0.37 0.003 0.77 Fentanyl exposure - - <-0.001 0.24 -  - Corticosteroids - - - - -0.007 0.43 Midazolam - - - - 0.001 0.22 	   123 R2 = 0.22; Table 5.4), such that greater numbers of invasive procedures (adjusted for confounders) and lower FA of the superior white matter were associated with lower FSIQ at age 7.5 years in children born very preterm (Figure 5.2). To assist with the interpretation of this interaction, post hoc analyses were conducted. We used a cutoff of FSIQ< 100 versus FSIQ> 100, because children with major impairments had been excluded. Among children exposed to either higher or lower numbers of invasive procedures (median split), we examined whether a change in FA from the 75th percentile to the 25th percentile corresponded with a decrease in FSIQ> 2.60 (i.e. beyond the SE of measurement). Specifically, among the children with lower FSIQ (<100), exposed to higher numbers of invasive procedures (.74 invasive procedures), a change in FA in the posterior subcortical white matter from the 75th percentile (0.67) to the 25th percentile (0.58) corresponded to a 13.1 point decrease in FSIQ. In contrast, a change in FA from the 75th percentile to the 25th percentile for children exposed to lower numbers of invasive procedures (<74 invasive procedures) corresponded to a non-significant 0.86 point change in FSIQ, less than the SE of measurement for FSIQ.           	   124 Table 5.4 Higher Number of Invasive Procedures and Lower Fractional Anisotropy of the Superior White Matter Predicts Lower IQ   Full Scale IQ n=50  B P Number of invasive procedures x fractional anisotropy  412.18 0.02 Fractional anisotropy -735.63 0.03 Number of invasive procedures 83.35 0.005 Gestational age 0.88 0.57 Small for gestational age -1.64 0.80 Illness severity  -0.50 0.03 Mechanical ventilation -5.22 0.54 Morphine exposure 1.75 0.84 Postnatal infection 6.17 0.24 Gender 0.37 0.93 Age at scan -168.10 0.12 Brain injury -2.41 0.67          	   125  Figure 5.2 Number of invasive procedures and brain microstructure predicts FSIQ  	  Higher numbers of invasive procedures (above median: red) and lower FA values of the superior white matter were associated with lower FSIQ after adjustment for neonatal and clinical confounders, age at scan, and concurrent brain injury. 	  	  5.3.4 Interaction Between Number of Invasive Procedures and White Matter Tracts in Relation to FSIQ The interaction between the number of invasive procedures and FA values of the white matter tracts was not associated with FSIQ (B= -304.22; P= .46; CI: -1106.38 – 497.94).  5.3.5 Interaction between Number of Invasive Procedures and Superior White Matter in relation to the WISC-IV Indices The interaction between the number of invasive procedures and fractional anisotropy of the superior white matter to predict FSIQ, was driven by the Verbal (B= 402.41, P= 0.05, CI: -3.40 – 	   126 808.21) and Working Memory (B= 352.98, P= 0.04, CI: 24.86 – 681.11) (Table 5.5) components of the FSIQ, whereas Perceptual Reasoning (B= 296.20, P= 0.19, CI: -148.49 – 740.90) and Processing Speed (B= 266.48, P= 0.29, CI: -226.85 – 759.80) were not significantly associated with the interaction between the number of invasive procedures and FA of the superior white matter.   Table 5.5 Higher Number of Invasive Procedures and Lower Fractional Anisotropy of the Superior White Matter Predicted Lower Verbal Comprehension and Working Memory   Verbal Comprehension Index  n=50 Working Memory Index n=50  B P B P Number of invasive procedures x fractional anisotropy 402.41 0.05 352.98 0.04 Fractional anisotropy -675.44 0.09 -638.96 0.04 Number of invasive procedures 69.93 0.04 76.30 0.005 Gestational age -0.88 0.62 1.57 0.27 Small for gestational age 2.81 0.70 -7.14 0.23 Illness severity  -0.51 0.05 -0.31 0.15 Mechanical ventilation 0.007 1.00 1.47 0.85 Morphine exposure -6.68 0.51 -5.91 0.47 Postnatal infection 3.65 0.54 6.43 0.18 Gender 4.28 0.35 -3.62 0.33 Age at scan -175.89 0.16 -54.46 0.59 Brain injury -8.87 0.17 2.08 0.69  	   127 5.4 Discussion Greater numbers of invasive procedures during neonatal care were associated with altered white matter microstructure at school age in children born very preterm, after accounting for degree of prematurity, systemic illness, medications, and concurrent brain injury. Specifically, in 7-year-olds without severe brain injuries or major neurosensory impairment, a higher number of invasive procedures during NICU care was associated with an increase in radial diffusion values at age 7 years, which in animals models has been related to reduced myelation as opposed to axonal loss (Song et al. 2002; Griffith et al. 2012; Lodygensky et al. 2010). Greater numbers of invasive procedures and reduced myelination of the superior white matter were associated with lower IQ in children born very preterm at school age. The relationship between the number of invasive procedures, brain microstructure and IQ was driven by 2 frontoparietal functions, verbal comprehension and working memory, which share common neural substrates (Glascher et al. 2009).   Preoligodendrocytes are cells that differentiate into myelin-producing oligodendrocytes. These cells are abundant in the preterm brain (Back et al. 2001), and are particularly vulnerable to injury (Back and Miller 2014; Volpe 2009). Oxidative stress results from the production of reactive species or oxidants, is a sequela of cerebral ischemia (shortage of oxygenated blood/reperfusion) (Traystman, Kirsch, Koehler 1991). Reactive oxygen, nitrogen species, and cytokines arrest the development of the preoligodendrocytes (Back et al. 1998; Back et al. 2005; Buntinx et al. 2004; Haynes et al. 2003; Pang, Cai, Rhodes 2005), and lead to altered myelination in preterm infants (Buser et al. 2012).   Oxygen saturation decreases following exposure to invasive procedures (Bauer et al. 2004; 	   128 Gonsalves and Mercer 1993; Mainous and Looney 2007). Preterm infants who underwent tape removal during a discontinuation of an indwelling central arterial or venous catheter, had markers of adenosine triphosphate (ATP) utilization and oxidative stress (uric acid and malondialdehyde concentration) in their plasma (Slater et al. 2012). The levels of these markers either remained stable or increased, following the tape removal, correlating with their pain intensity score (Slater et al. 2012). Therefore, the mechanisms underlying the relationship we found between repeated exposure to invasive procedures in the NICU and altered myelination of the superior white matter may be similar to those observed in models of cerebral ischemia (Back et al. 1998).  Myelination first occurs within the central white matter tracts, therefore the superior white matter may have been more enriched in progenitor stages of the oligodendrocyte lineage in contrast with the white matter tracts. Alternatively, the superior and central white matter tracts may have been similarly affected, but the latter had greater potential for recovery.  Alternatively, the superior white matter may have been indirectly affected by elevations in stress hormones. Repeated exposure to invasive procedures is associated with the reprogramming of the HPA axis (Grunau, Weinberg, Whitfield 2004; Grunau et al. 2007). Greater numbers of invasive procedures in the NICU are associated with lower stress hormone cortisol responses at 32 weeks PMA and higher levels at 8 and 18 months CA (Grunau, Weinberg, Whitfield 2004; Grunau et al. 2007). Brain regions rich in glucocorticoid receptors (e.g. prefrontal cortex) are particularly vulnerable to the effects of ongoing stress (McEwen 2004; Meaney et al. 1996). Alterations to the cortical gray matter may have led to alterations to the connecting subcortical white matter regions, as it has been shown that cortical gray and adjacent white matter 	   129 demonstrate synchronous maturation in very preterm infants (Smyser et al. 2015).  This may also explain why greater number of invasive procedures in the NICU was associated with alterations of the superior white matter, rather than the white matter tracts.   The HPA axis is not the only system changed by repeated exposure to invasive procedures in the NICU. Descending modulation of pain requires the activation of the periaqueductal gray and rostroventral medulla, regions responsible for the release of opioids within the spinal cord. Repeated exposure of invasive procedures in the NICU may lead to hyperinnervation of the periaqueductal gray and rostroventral medulla, thereby altering its functional integrity (LaPrairie and Murphy 2010). Inflammatory pain in neonatal rats has been found to increase the adult endogenous opioid tone (Laprairie and Murphy 2009). Therefore, repeated exposure to invasive procedures in the NICU may lead to chronically elevated opiate peptides, affecting the integrity of the subcortical white matter, which connects to the periaquaductal gray, a region rich in opioid receptors.  We previously reported that postnatal infections were significantly associated with 8% lower overall FA in infants born very preterm (Chau et al. 2012). After accounting for clinical confounders, the magnitude of change observed in this study relative to the number of invasive procedures was comparable (i.e. 7% lower FA in infants exposed to higher numbers of invasive procedures). The difference in FA between those exposed to higher and lower numbers of procedures was significantly related to IQ at 7 years in children born very preterm. This study suggests that the number of invasive procedures infants undergo in the NICU may one of the underlying factors explaining the variation in brain volumes, microstructure, and function, which are associated with cognitive outcomes in children and adults born preterm (Allin et al. 2011; 	   130 Ball et al. 2015; Chau et al. 2013; Counsell et al. 2008; Doesburg et al. 2011; Feldman et al. 2012; Fischi-Gomez et al. 2014; Peterson et al. 2000; Thompson et al. 2012; Van Kooij et al. 2012) .  The study sample was limited; therefore, residual confounding for clinical condition associated with invasive procedures may remain. We were also not able to include concurrent factors such as parenting stress and education in the analysis. Furthermore, the post hoc analyses should be interpreted with caution. FA values reach the noise floor in the cortical gray matter by approximately 36 weeks PMA (McKinstry et al. 2002; Vinall et al. 2013b), coinciding with neuronal maturation, synaptogenesis, and the disappearance of radial glial cells (Benders et al. 2014; Deipolyi et al. 2005; Jespersen et al. 2012; Kroenke et al. 2007; McKinstry et al. 2002; Sizonenko et al. 2007). Therefore, we could not examine the long-term relationships between the number of invasive procedures and DTI measures of cortical gray matter on cognitive outomes in children born very preterm. More studies using alternative methods for quantifying neuronal integrity (e.g. cortical thickness, volumetrics) are needed. The results of this work are a first step in understanding the relationship between the number of invasive procedures in the NICU, brain microstructure, and neurodevelopmental outcomes at school age.  In this cohort of preterm children without severe brain injury or major neurosensory/motor/cognitive impairment, we demonstrated that after accounting for degree of prematurity, systemic illness, medication exposures, and concurrent brain injury, greater numbers of invasive procedures together with alterations in white matter microstructure predicted lower IQ at school age. This research emphasizes the importance of finding ways to manage procedural stress in the NICU in order to optimize brain and neurodevelopmental 	   131 outcomes in this vulnerable population.                        	   132 CHAPTER 6  SUMMARY AND DISCUSSION OF RESEARCH FINDINGS  6.1 Summary of Results In two prospective cohorts of infants born very preterm we examined the relationships between invasive procedures in the NICU, postnatal growth, brain development, neurodevelopmental and behavior. Moreover, we investigated the extent that positive parental caregiving can moderate the relationship between invasive procedures and internalizing (anxiety/depressive) behaviors in children born very preterm. We found that over and above prematurity, systemic illness, and brain injury, greater exposure to invasive procedures was associated with poorer growth, altered brain development, and poorer cognitive and behavioral outcomes. Importantly, we also found that among children exposed to greater numbers of invasive procedures in the NICU, higher parent sensitivity and nonhostility were related to less internalizing behavior at 18 months CA in children born very preterm.   Postnatal growth is an indicator of how well an infant is thriving (Ehrenkranz et al. 1999). In the first study we examined whether repeated exposure to pain/stress was related to slower growth in the NICU, independent of size at birth, illness severity on day 1, days of mechanical ventilation, infection, PMA, morphine and corticosteroid exposure. Greater exposure to invasive procedures was associated with slower postnatal body and head growth until approximately 32 weeks PMA. However, neither early (prior to approximately 32 weeks PMA) nor later (after approximately 32 weeks PMA) exposure to invasive procedures was associated with weight and head circumference percentiles at term-equivalent age. Similarly, cumulative exposure to invasive procedures was not associated with weight and head circumference percentiles at term-	   133 equivalent age. Therefore, it is only during the first weeks of life when infants require more procedures, and are subject to significant energy and nutrient deficits (Ehrenkranz et al. 1999; Embleton, Pang, Cooke 2001), that we see a relationship between the number of invasive procedures in the NICU and postnatal growth.   Given that greater number of invasive procedures in the NICU is associated with slower postnatal growth, and slower growth in NICU is associated with poorer neurodevelopmental outcomes (Ehrenkranz et al. 2006), it was important to discern whether postnatal body and head growth was associated with cortical development after accounting for clinical confounders associated with growth and brain development (GA, size at birth, sex, WMI, IVH, cerebellar hemorrhage, patent ductus arteriosus, days on mechanical ventilation, postnatal infection, necrotizing enterocolitis). We found that the change in weight, length and head circumference between approximately 32 and 40 weeks PMA was associated with altered maturation of the gray matter, but not the white matter. More specifically, the direction of change within the cortical gray matter suggested that slower postnatal growth was associated with poorer maturation of the basal dendritic arbor of cortical neurons, consistent with findings of Dean et al., (2013). Corticosteroids were not associated with weight change, and excluding the children who received corticosteroids did not change the results. Therefore, by reducing and better managing the number of invasive procedures in the NICU, and reducing calorie and energy deficits, clinicians may have an opportunity to improve postnatal growth and perhaps to optimize cortical brain development in children born very preterm.   We then considered the role of the parent in moderating the long-term effects of repeated exposure to invasive procedures. Repeated exposure to invasive procedures in the NICU is 	   134 associated with increased internalizing (anxiety/depressive) behaviors at school age in children born very preterm (Ranger et al. 2014). As early as 2 years CA, children born very preterm show more internalizing behaviors compared to children born full-term (Spittle et al. 2009). Therefore, we explored whether greater exposure to invasive procedures is associated with internalizing behaviors at 18 months CA, and whether parent behavior (adjusted for parenting stress, number of children in the home, years of education, and age) was able to buffer the relationship between invasive procedures (adjusted for GA, illness severity on day 1, morphine exposure, days on mechanical ventilation), and internalizing behavior (adjusted for cognition and gender) in children born very preterm. Children born very preterm demonstrated more internalizing behaviors compared to children born full-term at 18 months CA, consistent with previous findings at age 2 (Brummelte et al. 2011b; Tu et al. 2007). Greater numbers of invasive procedures in the NICU were associated with higher parental report of internalizing behaviors in children born very preterm. Among children exposed to a higher number of invasive procedures, they demonstrated less internalizing behaviors at 18 months CA if their parents were emotionally available (i.e. sensitive and nonhostile). Additionally, higher parenting stress, more children in the home and more years of parent education were independently associated with fewer internalizing behaviors in children born very preterm. Importantly, in children born full-term, parent factors did not predict internalizing behavior. Our findings are consistent with previous findings from our group and others that found children born very preterm are more sensitive to interactions with their parents compared to children born full-term (Brummelte et al. 2011b; Crnic and Greenberg 1987; Tu et al. 2007). The parent is an important moderator of neonatal procedural stress. By helping caregivers, particularly those who are first time parents, concerned about their parenting ability, and/or are low SES, improve their emotional availability toward the child we may be able to reduce the prevalence of anxious/depressive behaviors among children 	   135 exposed to a higher number of invasive procedures in the NICU.    The last study examined whether repeated exposure to invasive procedures in the NICU was associated with altered brain development and cognition in children born very preterm at 7 years of age. Previously, our group has shown that greater number of invasive procedures in the NICU were associated with altered brain microstructure in the NICU and at term equivalent age (Brummelte et al. 2012; Zwicker et al. 2013). These results were consistent with Smith et al. (2011), that also found more stressors in the NICU were associated with decreased frontal and parietal brain width, altered diffusion measures and functional connectivity in the temporal lobes. Our group has also found evidence that these relationships persist beyond early life as greater number of invasive procedures in the NICU were associated with reduced cortical volumes and altered function at school age (Doesburg et al. 2013; Ranger et al. 2013). Moreover, greater exposure to invasive procedures in the NICU was associated with poorer cognitive outcomes at 8 and 18 months CA (Grunau et al. 2009). However, it was not known whether repeated exposure to invasive procedures together with alterations in brain microstructure leads to poorer cognitive outcomes in children born very preterm. Therefore, we examined whether greater exposure to invasive procedures was associated with altered superior white matter or white matter tracts, adjusting for GA, SGA, illness severity on day 1, days of mechanical ventilation, postnatal infection, age at scan, brain injury. In separate models the effects of surgery and corticosteroid exposure were also considered. We also examined whether the number of invasive procedures and the integrity of the white matter were associated with IQ at age 7.5 in children born very preterm, after adjusting for clinical confounders. We found that greater numbers of invasive procedures in the NICU was associated with altered superior white matter, but not the white matter tracts, after adjustment for clinical confounders. Neither 	   136 surgeries nor steroid exposure changed the results. The direction of change in the superior white matter suggested that the difference between those exposed to higher numbers of invasive procedures and lower numbers of invasive procedures was the amount of myelination in the superior white matter, as opposed to the number or complexity neural cells, compatible with histopathological correlates of MRI abnormalities using animal models (Song et al. 2002; Griffith et al. 2012; Lodygensky et al. 2010). Higher number of invasive procedures and reduced myelination of the superior white matter were associated with lower IQ at 7.5 years of age in children born very preterm. Therefore, the impact of repeated exposure to invasive procedures extends well beyond the NICU stay, and is related to both altered brain development and lower IQ in children at school age, over and above other clinical confounders. It is important that we find ways to alleviate pain/stress that are brain protective in order to improve the lives and outcomes of this vulnerable population.   6.2 Timing of Exposure to Invasive Procedures The development of the thalamocortical connections in the late fetus and preterm infant provides the necessary framework for sensory-driven organization, and cortical processing of noxious stimuli. Between 24 and 25 weeks PMA, thalamocortical afferents accumulate in the subplate (Kostovic and Rakic 1990; Kostovic and Judas 2002; Kostovic and Judas 2010). These afferents form transient, functional circuits with subplate neurons, before proliferating into the cortical layers between 26 and 28 weeks PMA (Ayoub and Kostovic 2009; Kostovic and Judas 2010). It is at this time that evoked potentials can be recorded from the somatosensory cortex.   Perlman and Volpe (1983) were the first to examine the relationships between pain/stress on brain activity. They used transcutaneous Doppler to measure blood flow in the anterior cerebral 	   137 arteries before, during, and 5 minutes after the cessation of routine suctioning in 35 intubated infants, between 26 to 35 weeks PMA (Perlman and Volpe 1983). They found an increase in cerebral flow velocity during the suctioning procedures, which corresponded with an increase in blood pressure (Perlman and Volpe 1983). The results of this early work have since been extended by studies using EEG and near-infrared spectroscopy technology (Bartocci et al. 2006; Fabrizi et al. 2011; Slater et al. 2006; Slater et al. 2010a).   As early as 24 weeks PMA, nociceptive-specific and sensory potentials have been recorded from the somatosensory cortex (Slater et al. 2010a). However, prior to 35-37 PMA, EEG responses touch and heel lance appear as dispersed neuronal bursts, in contrast to the modality-specific, localized, evoked potentials seen at term-equivalent age (Fabrizi et al. 2011). These changes in the EEG recordings correspond to neuronal maturation, synaptogenesis and disappearance of the radial glial cells in the cortex (Benders et al. 2014; Deipolyi et al. 2005; Jespersen et al. 2012; Kroenke et al. 2007; McKinstry et al. 2002; Sizonenko et al. 2007). Therefore, prior to 35 weeks PMA, given the immaturity of thalamocortical and corticocortical connections, infants are less capable of distinguishing between tactile from nociceptive stimulation. They also demonstrate a significant lowering of threshold or "sensitization" to repeated stimulation (Andrews and Fitzgerald 1994; Fabrizi et al. 2011; Fitzgerald, Millard, McIntosh 1989; Holsti et al. 2005; Holsti et al. 2006). Due to the vulnerability of the developing cortical circuitry (Back and Miller 2014) their inability to differentiate between tactile and noxious stimulation (Fabrizi et al. 2011), and sensitization to repeated stimulation (Andrews and Fitzgerald 1994; Fabrizi et al. 2011; Fitzgerald, Millard, McIntosh 1989; Holsti et al. 2005; Holsti et al. 2006), infants <35 weeks PMA may be particularly vulnerable to repeated procedural pain/stress. Grunau, Miller and colleagues have provided the first evidence that repeated exposure to invasive procedures is 	   138 associated with altered brain development, and poorer cognitive motor and behavioral outcomes in infants born very preterm, even after accounting for prematurity and systemic illness in the NICU (Brummelte et al. 2012; Doesburg et al. 2013; Grunau et al. 2009; Ranger et al. 2013; Ranger et al. 2014; Vinall et al. 2013a; Zwicker et al. 2013). However, this research highlights that it is not just the quantity and duration of exposure to invasive procedures that is important to the development of infants born very preterm, but also the timing of exposure.  Lower tactile threshold together with sensitization to repeated touch in preterm neonates, may lead to an exhaustion of resources in infants <35 weeks PMA, which is detrimental to the developing cortex. Subplate neurons are vulnerable to nutrient insufficiency, which can lead to focal or widespread white matter injury, as well as reduced cortical gray matter (Inder et al. 1999). Infants born very preterm have a limited metabolic reserve, and in the first weeks of life, they accumulate a significant nutrient and energy deficit (Embleton, Pang, Cooke 2001; Polin, Fox, Abman 2003). We have demonstrated for the first time that greater exposure to invasive procedures early in life (approximately <32 weeks PMA) is associated with slower body and head growth early in the NICU rather than at term-equivalent age. Furthermore, we found that slower growth in the NICU is associated with reduced maturation of the cortical gray matter.    Repeated exposure to invasive procedures in the NICU appears to interfere with cortical maturation. Subplate neurons and preoligodendrocytes are particularly vulnerable to excitotoxicity, oxidative stress, and inflammation (Back and Miller 2014; Volpe 2009), which can result from repeated exposure to invasive procedures (Anand et al. 2007; Brummelte et al. 2012; Duhrsen et al. 2013; Hansson 2006; Slater et al. 2012). Evidence for the disruption of the subplate neurons as a result of early exposure (birth to approximately 32 weeks PMA) to 	   139 pain/stress comes from our preliminary work examining the relationship between invasive procedures and altered maturation of the cortical gray matter (Vinall et al. 2014a). Moreover, our group has found that greater exposure to invasive procedures in the NICU was associated with reduced cortical volumes and altered cortical function at 7 years of age (Doesburg et al. 2013; Ranger et al. 2013). Preoligodendrocytes are cells that ensheath axons prior to differentiating into myelin-producing oligodendrocytes (Volpe 2009). Disturbances in myelination as seen with diffuse WMI, the most extensive lesions in preterm neonates, are due to the selective vulnerability of preoligodendrocytes, which account for approximately 90% of the oligodendrocyte population at 28 weeks PMA (Back et al. 2001; Back and Miller 2014; Buser et al. 2012). Previously, our group has shown that greater exposure to invasive procedures prior to 32 weeks PMA was associated with reduced brain maturation in the NICU, and at term-equivalent age (Brummelte et al. 2012). Early exposure to invasive procedures (birth to approximately 32 weeks PMA) rather than later (approximately 32 to 40 weeks PMA) was more detrimental to the developing white matter (Brummelte et al. 2012). Moreover, this relationship appeared to persist beyond term-equivalent age, as currently we found that greater exposure to invasive procedures in the NICU was associated with altered myelination of the superior white matter at age 7 years in children born very preterm (Vinall et al. 2014a). Alterations in myelination as a result of repeated exposure to invasive procedures was related to poorer cognitive outcomes at 7 years of age. This is important given that children born very preterm have more cognitive problems relative to children born full-term (Doyle, Casalaz, Victorian Infant Collaborative Study Group 2001; Grunau, Whitfield, Fay 2004; Johnson et al. 2009; Larroque et al. 2008; Lind et al. 2011; Marlow et al. 2005). This research suggests that the etiology of neurodevelopmental problems in very preterm infants, who escape major brain injury, is at least in part explained by disturbances in brain development through early exposures 	   140 to invasive procedures in the NICU.   6.3 Mechanisms Linking Invasive Procedures, Growth, and Behavior Exposure to invasive procedures activates the HPA axis results in the release of cortisol, epinephrine and neuroepinephrine, which stimulates a physiological response to nociceptive stimulation (i.e. increased heart rate, oxygen consumption, and blood pressure). This leads to changes in cerebral oxygenation and cerebral blood volume (Pryds 1991; Yamamoto et al. 2003), which can cause episodes of ischemia and/or reperfusion, affecting the preoligodendrocytes (Back et al. 1998; Baud et al. 2004). This physiological response also requires a substantial amount of energy, which is taxing for infants with limited resources (Ranger, Johnston, Anand 2007). The importance of adequate nutrition for optimal brain development is certainly recognized, though it may be difficult to achieve (Embleton, Pang, Cooke 2001; Keunen et al. 2012). Therefore, infants exposed to a higher number of invasive procedures may deplete energy needed to fully support growth and cortical gray matter development.   Ongoing stress in the NICU may suppress the production of growth hormones (Tsigos and Chrousos 2002). IGF-1 values are associated with postnatal weight gain (Kurtoglu et al. 2010; van de Lagemaat et al. 2013), brain volumes (Hansen-Pupp et al. 2011; Hansen-Pupp et al. 2013), and neurodevelopmental outcomes at 2 years (Hansen-Pupp et al. 2013). Greater exposure to invasive procedures early in life may lead to the reduction in IGF-1 values, thereby contributing to slower growth and altered cortical development in infants born very preterm.   Chronic stress also suppresses immune function (Chrousos 1995; Elenkov et al. 1999; Tsigos 	   141 and Chrousos 2002). Infants exposed to greater numbers of invasive procedures early in life were more likely to have an infection after 32 weeks PMA (Vinall et al. 2012). The presence of infection after 32 weeks PMA was associated with slower body growth (Vinall et al. 2012). Neonatal infection leads to systemic inflammation, and is often associated with reduced cerebral blood flow (Keunen et al. 2012). These factors can lead to the activation of microglia and release of free radicals and pro-inflammatory cytokines (Back et al. 2001; Volpe 2009), arresting the development of preoligodendrocytes (Back and Miller 2014; Buser et al. 2012) and altering the development of the white matter (Adams et al. 2010; Chau et al. 2009; Chau et al. 2012; Vinall et al. 2013b; Zwicker et al. 2013).   Programming of the HPA axis occurs during fetal and neonatal development (Matthews 2002). Repeated exposure to invasive procedures in the NICU is associated with dampened cortisol expression in the NICU, heightened cortisol expression during early childhood, and reduced cortisol expression at school age (Brummelte et al. 2015; Grunau, Weinberg, Whitfield 2004; Grunau et al. 2007; Grunau et al. 2013). It would appear that repeated exposure to invasive procedures in the NICU programs the HPA axis for a stressful postnatal environment. However, evidence from animal models suggest that positive parental interaction may be able to prevent and/or ameliorate the effects of invasive procedures on stress system development. Variations in pup licking and grooming during the first week of life affects HPA and behavioral responses to stress, and is correlated with hippocampal glucocorticoid receptor expression in adulthood (Caldji et al. 1998; Francis et al. 1999a; Liu et al. 1997; Menard, Champagne, Meaney 2004; van Hasselt et al. 2012; Weaver et al. 2004; Zhang et al. 2006). In animals models, adult offspring of high licking and grooming mothers had greater hippocampal glucocorticoid receptor expression, better glucocorticoid feedback sensitivity, reduced corticotrophin releasing factor and less 	   142 corticosteroid production in comparison to pups reared by low licking and grooming mothers (Francis et al. 1999a; Liu et al. 1997). Offspring of high-licking and grooming dams also demonstrate fewer anxiety-like behaviors as adults (Caldji et al. 1998; Pena et al. 2014; Starr-Phillips and Beery 2014; van Hasselt et al. 2012). Previously, our group has demonstrated that positive maternal interaction was associated with lower cortisol levels at 18 months CA (Brummelte et al. 2011b). Moreover, in the same study lower cortisol expression at 18 months CA was associated with less internalizing behavior (Brummelte et al. 2011b). Among the present reported studies, we found that among children exposed to a higher number of invasive procedures in the NICU, if parents were more sensitive and nonhostile, their children also showed less internalizing behavior at 18 months CA (Vinall et al. 2013a). Therefore, it would appear that positive parental interaction moderates the effect of repeated exposure to invasive procedures on the development of stress-sensitive anxiety or depressive behaviors in children born very preterm.    In summary, repeated exposure to invasive procedures in the NICU is associated with alterations in postnatal growth, brain development and neurodevelopmental outcomes, even after accounting for prematurity, systemic illness and brain injury. However, more research using animal models of prematurity is needed, in order to better understand the mechanisms underlying each of these relationships. In particular, this research highlights the importance of managing procedural pain/stress in the NICU in order to improve longitudinal outcomes in this vulnerable population.    6.4 Pain/Stress Management in the NICU Although clinicians recognize that there is a physiologic rationale to manage procedural pain, 	   143 there are major challenges related to both pharmacological and non-pharmacological interventions.  	  6.4.1 Morphine Morphine has been the most commonly used opiate for analgesia in the NICU (Anand 2007). In each of the three studies examining impact of invasive procedures, morphine exposure neither ameliorated nor exacerbated the effects of invasive procedures on postnatal growth, brain microstructure, cognitive or behavioral outcomes (Vinall et al. 2012; Vinall et al. 2013a; Vinall et al. 2014b). Similarly, Grunau and colleagues found no effect of morphine exposure cumulatively from birth to term-equivalent age on white or subcortical gray matter, cortisol levels, or cognitive development (Brummelte et al. 2012; Doesburg et al. 2013; Grunau et al. 2005; Grunau et al. 2007; Grunau et al. 2009). They did, however, find that greater morphine exposure was associated with altered cerebellar maturation early in life and at term-equivalent age (Zwicker et al. 2012), and poorer motor development at 8 months, but not 18 months CA (Grunau et al. 2009). Among ventilated infants born very preterm, greater morphine exposure was associated with increased internalizing behaviors at 7 years of age (Ranger et al. 2014).   The risks and benefits of continuous use of analgesics and anesthetics in very preterm infants are unclear (McPherson and Grunau 2014). In two large randomized controlled trials of effects of morphine ventilated preterm neonates, continuous morphine infusions did not reduce infant pain scores relative to placebo during an acute painful procedure (i.e suctioning) (Anand et al. 2004; Simons et al. 2003b). Moreover, providing morphine as a loading dose prior to continuous infusion also did not lower preterm infant’s pain scores in response to a heel lance procedure (Carbajal et al. 2005). Although there was evidence that morphine reduced pain/stress from 	   144 mechanical ventilation, it did not appear to provide adequate analgesia for acute procedural pain among preterm infants (Anand et al. 2004; Simons et al. 2003b).  Results from these two trials also raised concerns regarding the short and long-term effects of morphine. In the short-term continuous morphine infusions lead to longer duration of mechanical ventilation and longer time to reach enteral feeding (Anand et al. 2004). Following a small of subset of these children (N=19), at age 5-7 years children in the morphine-exposed group weighed less, had smaller head circumferences, impaired short-term memory, and according to parent report, they had more difficulty establishing friendships compared to children in the placebo-treated group (Ferguson et al. 2012). Morphine exposed children from the other trial were found to have poorer visual processing at age 5 years, and greater internalizing behaviors according to teacher report at age 8 to 9 years relative to the placebo-treated children (de Graaf et al. 2011). Although in present cohort studies morphine exposure neither ameliorated nor exacerbated the effects of invasive procedures on outcomes, given the lack of efficacy for acute pain management and risk of short and long-term effects on neurodevelopmental outcomes, it is recommended that opiates be used sparingly in the NICU for nonsurgical pain management of ventilated preterm neonates.  6.4.2 Sucrose Sucrose is the most widely used non-pharmacologic intervention for the treatment of minor procedures in preterm infants (Taddio et al. 2009). However, the BC Children’s and Women’s Hospital is one of the few remaining hospitals in Canada that does not use sucrose for the management of acute procedural pain in preterm infants, due to lack of studies of long-term 	   145 effects beyond NICU discharge. At this time very little is known about the mechanisms of action of sucrose in human infants, and whether there are long-term effects of repeated use of sucrose in the NICU on brain, metabolism or neurodevelopmental outcomes (Holsti and Grunau 2010). In preterm infants, administration of 24% sucrose (0.01 to 0.02 g), 2 minutes prior to minor procedures is efficacious in reducing crying, facial grimacing, and motor activity, therefore, it reduces pain scores in infants (Stevens et al. 2013). However,	  its effectiveness in modifying physiological indices (e.g. heart rate, heart rate variability, oxygen saturation) varies (Stevens et al. 2013), and it has been suggested that sucrose may act as a sedative rather than an analgesic (Fitzgerald 2009; Holsti and Grunau 2010). One study to date has examined the effects of sucrose on neurodevelopmental outcomes at 36 and 40 weeks PMA, and found that greater exposure to sucrose (>10 doses in 24 hours) was associated with poorer attention and motor outcomes (Johnston et al. 2002; Johnston et al. 2007). Infants born very preterm can receive as many as 15 invasive procedures per day in the first few weeks of life (Carbajal et al. 2008; Stevens et al. 2003). The most recent national review of sucrose use revealed that infants in the NICU may receive as many as 24 doses in one day (Taddio et al. 2009). While there is consistent support for the use of sucrose for acute painful procedures (Stevens et al. 2013), much more research is needed with regards to long-term effects, given the potential for high cumulative exposure to sucrose over the course of the NICU stay (Holsti and Grunau 2010; Stevens et al. 2013).  Most relevant to the work presented here was the finding that although sucrose reduces behavioral and sometimes physiological responses (Stevens et al. 2013), it does not dampen EEG responses to invasive procedures (Slater et al. 2010b). Therefore, sucrose does not appear to protect the brain from repeated stimulation. Furthermore, it was recently reported that very 	   146 preterm infants given a single dose of oral sucrose, prior to heel lance demonstrated significantly greater adenosine triphosphate (ATP) use and oxidative stress (Asmerom et al. 2013), increasing the likelihood that the intervention given to protect the infant from the adversity may have unintended consequences. This research highlights the importance of evaluating our current pain management strategies in the NICU for the extent that they are brain protective.  6.4.3 Swaddling, Facilitated Tucking, Non-nutritive Sucking, Kangaroo Care Sucrose reduces behavioral responses by 16% and 28% on pain-assessment scales (Johnston et al. 1997; Stevens et al. 2005). However, this can also be achieved by using other environmentally supportive interventions such as swaddling, facilitated tucking, non-nutritive sucking and kangaroo care (Axelin, Salantera, Lehtonen 2006; Carbajal et al. 1999; Castral et al. 2008; Ferber and Makhoul 2008; Ludington-Hoe, Hosseini, Torowicz 2005; Pillai Riddell et al. 2011). Swaddled infants are securely wrapped in a blanket to prevent the infant's limbs from moving around excessively. Facilitated tucking involves holding the infant, keeping the arms and legs in a flexed position, close to the trunk. For non-nutritive sucking, a pacifier is placed into an infant's mouth to stimulate sucking behavior. During kangaroo care the infant is placed on the caregiver’s bare chest for skin-to-skin contact. At the BC Children’s and Women’s Hospital these non-pharmacological interventions are the standard of care for acute painful/stressful procedures in very preterm infants. To the best of our knowledge, no studies to date have examined whether swaddling or non-nutritive sucking influence brain responses or long-term outcomes in infants born very preterm.   One study examined whether sucrose, facilitated tucking, or sucrose together with facilitated tucking influenced heart rate, oxygen saturation, and/or cortical responses following a heel lance 	   147 (Gerull et al. 2013). Despite increases in heart rate in all three groups, Gerull et al. (2013) did not find changes in oxygen saturation or near-infrared spectroscopy measures following heel lance. The lack of placebo group in this study makes it difficult to discern whether facilitated tucking and sucrose provided adequate analgesia. No studies to date have looked at whether the use of facilitated tucking in the NICU leads to better longitudinal outcomes in very preterm infants.   Effects of kangaroo care on brain function has been examine previously, however these analyses were not performed in conjunction with an invasive procedure. One study found that 30-min of skin-to-skin contact with mothers lowers infant heart rate and improves peripheral oxygen saturation and cerebral blood flow (Korraa et al. 2014). Another compared preterm infants who underwent 8 weeks of kangaroo care to two cohorts of premature and full-term neonates that did not undergo skin-to-skin intervention (Kaffashi et al. 2013). They found that the kangaroo care group had more complex EEG signaling, indicative of greater brain maturation (Kaffashi et al. 2013). The pattern of signaling at 40 weeks PMA was comparable to that of full-term neonates (Kaffashi et al. 2013). These changes to brain maturation and function may have led to long-term improvements in neurodevelopmental outcomes. Infants that received 1 hour of kangaroo care for 14 consecutive days had attenuated stress responses, more mature autonomic functioning, better organized sleep, more cognitive control, and greater mother–child reciprocity at 10 years of age (Feldman, Rosenthal, Eidelman 2014). Although these studies did not examine directly the extent that kangaroo care protects the brain from aversive stimuli, there is growing evidence of its effectiveness in stress reduction, emphasizing the importance of parental involvement in the management of stress in the NICU.    Therefore, to the best of our knowledge no studies to date have examined whether swaddling, 	   148 facilitated tucking, non-nutritive sucking or kangaroo care ameliorates the relationship between repeated exposure to invasive procedures in the NICU and poorer neurodevelopmental outcomes in infants born very preterm. Moreover, there is very little evidence to suggest that these procedures protect the brain from repeated exposure to aversive stimuli. Currently, these pain management strategies are the standard of care at the BC Children’s and Women’s Hospital for acute painful/stressful procedures. Despite the routine management of pain in the NICU, we still find relationships between greater exposure to invasive procedures and altered growth, brain development, and poorer neurodevelopmental and behavioral outcomes in 2 cohorts of children born very preterm (Vinall et al. 2012; Vinall et al. 2013b; Vinall et al. 2014b). Future research is needed to evaluate the extent that current pain management strategies reduce the long-term effects of repeated exposure of invasive procedures on developing pain/stress systems.   6.5 Importance of Parent Involvement in the NICU Supporting positive parent interactions in the NICU, and involving parents in infant care is important for minimizing neonatal pain/stress and improving outcomes after discharge from the NICU. Just a few short training sessions with a developmental specialist could help to minimize infant stress and improve white matter microstructure at term equivalent age in infants born very preterm (Milgrom et al. 2013). There is mounting evidence that caring for infants in single-family rooms, as opposed to the traditional open-bay model, will improve parent involvement and outcomes of infants born very preterm (Shahheidari and Homer 2012). Therefore, there has been widespread adoption of the single family room model of care. Recently, it was found that very preterm infants in the single family room NICU weighed more at discharge, had a greater rate of weight gain, required fewer medical procedures, had a lower PMA at full enteral feed, less sepsis, showed better attention, less physiologic stress, less hypertonicity, less lethargy, and 	   149 less pain (Lester et al. 2014). Differences in weight at discharge, and rate of weight gain were mediated by increased developmental support, whereas differences in stress and pain were mediated by maternal involvement (Lester et al. 2014). However, in private NICU rooms if families are less involved, than at term-equivalent age, infants born very preterm had alterations in brain structure and function, which may have contributed to their lower motor and language scores at 2 years of age (Pineda et al. 2014). Therefore, encouraging parental involvement in the NICU continues to be an important part of infant care, given that positive parent interaction may moderate pain/stress in children born very preterm (Vinall et al. 2013a), thereby improving behavioral outcomes.   6.6 Limitations There are several limitations to the papers presented in this dissertation. Overall, the goal of this work was to understand the impact of repeated exposure to invasive procedures during neonatal intensive care on brain microstructure, growth, neurodevelopment and behavior of children born very preterm. However, in clinical cohort studies, cause and effect cannot be inferred, given that only associations among variables can be examined. Therefore, it is important that our findings are consistent with the basic animal studies in this field.   As is a limitation with all clinical studies, we could not account for all of the factors that may impact the outcome variables in question. The data for these papers were drawn from longitudinal cohort studies designed to examine long-term effects of invasive procedures on brain and neurodevelopment. Examination of physical growth, for example, was not included in the original aims of these studies. Therefore, data on nutrition were not collected. However, it has been shown previously that birth weight and nutritional intake accounts for approximately 	   150 52% of the variance in postnatal growth (Embleton, Pang, Cooke 2001). Therefore, a portion of the unexplained variance in postnatal growth may be accounted for by repeated exposure to invasive procedures. However, there still remains antenatal (e.g. maternal nutrition, smoking) and neonatal stress factors (e.g. noise, light, maternal deprivation) not accounted for by our study, which may also impact postnatal growth, brain and neurodevelopment.   Standard care in the NICU at the BC Children’s and Women’s Hospital for acute painful procedures includes the use of either facilitated tucking, non-nutritive sucking, swaddling and/or kangaroo care, which may have reduced infant stress during procedures. Moreover, morphine was often provided for infants undergoing mechanical ventilation. In our data analyses, we adjusted the statistical models for cumulative morphine exposure, but we could not account for what was happening when the medication was given or how well timed it was to the painful/stressful procedure. Moreover, we could not account for the specific pain management that may or may not have been provided during each procedure. The efficacy of morphine for alleviating procedural pain/stress is likely not the same for every procedure in the NICU (Anand et al. 2004; Carbajal et al. 2005; Simons et al. 2003b). Therefore, it is notable, that in the context of our routine care that we still found an association of invasive procedures with growth, brain, neurodevelopment, and behavior in preterm infants and children, after adjusting for neonatal, clinical, and social confounders.   Pain and stress are difficult to discriminate in infants born very preterm undergoing neonatal intensive care. Preterm infant responses to invasive procedures vary depending on GA, sleep-wake state, illness severity, medications, previous exposures to pain and NICU care (Gibbins et al. 2008; Grunau et al. 2001; Holsti et al. 2005; Holsti et al. 2006; Holsti et al. 2008; Johnston 	   151 and Stevens 1996; Johnston et al. 1999; Slater et al. 2009; Stevens, Johnston, Horton 1994; Valeri et al. 2012). Although it is unlikely that every procedure elicits the same amount of pain/stress, the extent of reactivity is not simply a function of the type of procedure. Thus, we counted each procedure and/or attempt at a procedure listed in Appendix Table A.1, as 1 invasive procedure, without assigning any sort of ranking to the various types of procedures included in this list. It was not feasible to measure the pain/stress reactivity of neonates who receive on average 4 to 14 invasive procedures per day, across the NICU stay (Brummelte et al. 2012; Carbajal et al. 2008; Doesburg et al. 2013; Grunau, Weinberg, Whitfield 2004; Johnston et al. 2011; Simons et al. 2003a). Moreover, handling, diaper changes, bathing, and other procedures that are not inherently stressful to full-term newborns, can also induce considerable stress in this fragile population. During the “recovery” phase (first 4 min after the last contact by the technician), infants born very preterm continued to demonstrate stress cues following clustered care (changing the diaper, measuring the abdominal girth, taking the axillary temperature, and cleaning the mouth with gauze and sterile water) (Holsti et al. 2005). Prior to 35 weeks PMA, after repeated stimulation infants show lower threshold and sensitization to procedures (Andrews and Fitzgerald 1994). Consistent with this, our group found that infants between the ages 30 and 32 weeks PMA had heightened facial responses to a heel lance procedure when it was preceded by clustered nursing care (Holsti et al. 2006). Although we did not include procedures such as handling in our measure of cumulative procedural stress, another study, which did include a wider range of stressful procedures in the NICU (e.g. diaper and position changes), reported similar findings as our group, with regards to stress and brain development at term-equivalent age (Smith et al. 2011). Therefore, quantification of the number of invasive procedures in the NICU is a useful measure of neonatal pain/stress and is a modifiable risk factor for altered brain and neurodevelopment in children born very preterm.     	   152  There are limitations to manual acquisition of data from DTI. “Region of interest” based analysis of DTI can be limited by reproducibility of voxel sampling between scans, and by the risk of partial averaging To improve accuracy of our measurements and replication between scans, we compared different sizes and positions for our region of interest voxel boxes, and determined the optimal size and placement for them. Future advances in MRI acquisition and analysis that allow for automatic segmentation and quantification of cortical FA from early in life to term-equivalent age may refine our ability to detect differences in cortical maturation related to growth and outcome (Ball et al. 2013; Brown et al. 2014).   In chapter 5, regions of interest were averaged together based on their structural similarity. If regions of interest had been averaged together either by functionally relatedness or using data driven methods, our results may have differed. More studies are needed to determine whether associations between the number of invasive procedures in the NICU and brain development are global, regionally, or functionally specific, particularly in relation to outcome. In addition, cortical gray matter FA values reach the noise floor by 36 weeks PMA (McKinstry et al. 2002; Vinall et al. 2013b). Therefore, we could not examine the long-term effects of the number of invasive procedures on the DTI measures of cortical gray matter. Using alternative neuroimaging methods, our group has demonstrated that greater exposure to invasive procedures in the NICU is associated with reduced cortical volumes and altered cortical function at 7 years of age (Doesburg et al. 2013; Ranger et al. 2013). Animal models may help us to understand the long-term impact of repeated exposures to invasive procedures on the cerebral microstructure of infants born very preterm.  	   153 Clinically important, is the threshold at which exposure to invasive procedures may impact growth, brain and neurodevelopmental outcomes. Previously, Grunau et al. (2001) reported that at 32 weeks PMA, prior exposure to 20 invasive procedures appeared to be the point at which diminished behavioral expressions and autonomic responses to heel lance procedures were evident. With regards to the present studies, we were hesitant to propose a threshold for the number of invasive procedures during neonatal care, given the variability in GA, PMA and the number of invasive procedures. Moreover, it is unlikely that all procedures listed in Table A.1 are equally noxious. Therefore, recommending a cut-off for a number of invasive procedures that would not be deleterious for either brain development or neurodevelopmental outcomes is not possible at this stage of knowledge. Although identifying a threshold for the number of invasive procedures during neonatal care is clinically relevant, more research is required before this kind of recommendation can be made. The focus currently is to find ways to reduce the number of procedures performed in the NICU, and to find ways to manage pain/stress that are neuroprotective.   We emphasized how parents can play a vital role in the regulation of stress and development of their infant. Prevsiouly it has been shown that mothers who reported lower concurrent stress relative to mothers who reported higher concurrent stress were more sensitive when interacting with their child post-discharge (Muller- Nix et al. 2004; Tu et al. 2007). However, greater parenting stress was related to declining cognitive development between 8 and 18 months CA (Brummelte et al. 2011a; Docherty, Miles, Holditch-Davis 2002). Therefore, parenting stress may in part reflect realistic concerns about their child. A child that displays more internalizing behavior may also influence their parent’s level of stress and quality of responding. Therefore, it is important to keep in mind that although parental sensitivity/nonhostility appears to moderate 	   154 the relationship between the number of invasive procedures and internalizing behavior in children born very preterm, infants exposed to higher number of procedures that are reported as having greater internalizing behavior may contribute to parent’s reduced emotional availability.  6.7 Significance of this Research More than half of children born very preterm will develop cognitive, motor and/or behavioral problems that persist to at least to early adulthood (Aarnoudse-Moens et al. 2009; Anderson, Doyle, Victorian Infant Collaborative Study Group 2003; Doyle, Casalaz, Victorian Infant Collaborative Study Group 2001; Doyle and Anderson 2010; Grunau, Whitfield, Fay 2004; Johnson et al. 2009; Loe et al. 2011; Marlow et al. 2005; Marlow et al. 2007; Spittle et al. 2009; Synnes et al. 2010). This can affect their quality of life, and cause considerable stress and burden for their families. Support for these individuals also puts a substantial strain on medical, educational and social systems.   A major body of literature has focused on describing the differences between children born preterm and full-term. However, few studies have focused on the mechanisms underlying these differences. This collection of works goes beyond the descriptive literature to show that it is not just the matter of being born very preterm, or the illness course in the NICU that leads to differences between these two groups. These infants undergo repeated exposure to invasive procedures, during a sensitive period of brain and stress system development, when they would normally be developing within the protective intrauterine environment. Greater exposure to invasive procedures in the NICU, over and above adjustment for prematurity, systemic illness, brain injury, and other clinical confounders was related to poorer growth, altered brain 	   155 development and poorer neurodevelopment and behavior in children born very preterm. Therefore, by reducing frequency and improving management of invasive procedures in the NICU, clinicians may have the opportunity to improve later outcomes of this population.   This work also points to the important role of the parent for managing stress in the premature infant. Infants born very preterm are sensitive to interactions with their parents. We found that even among children exposed to a higher number of invasive procedures in the NICU, if their parent was more emotionally available they had fewer internalizing behaviors in early childhood. We know from previous work that NICU-based interventions that increase parental involvement in pain/stress management have been found to improve parents’ efficacy in supporting their infant post discharge from the NICU (Franck et al. 2011). Moreover, parent sensitivity training in the NICU has been shown to improve white matter maturation at term (Milgrom et al. 2010). Therefore, including parents in the pain/stress management plan of infants born very preterm should help not only to improve the quality of interaction between the parent and child, but also improve neurobehavioral outcomes within this population.   Altogether, the results of this research provides a foundation for beginning to understand the role of neonatal pain/stress in the etiology of neurodevelopmental and behavioral problems in children born very preterm, and provides insight into the potential for early parent intervention strategies to optimize development in this vulnerable population.    	   156 REFERENCES Aanes S, Bjuland KJ, Skranes J, Lohaugen GC. 2015. Memory function and hippocampal volumes in preterm born very-low-birth-weight (VLBW) young adults. Neuroimage 105:76-83. Aarnoudse-Moens CS, Weisglas-Kuperus N, van Goudoever JB, Oosterlaan J. 2009. Meta-analysis of neurobehavioral outcomes in very preterm and/or very low birth weight children. Pediatrics 124(2):717-28. Abidin RR. 1995. Parenting stress index. 3rd ed. Odessa, FL: Psychological Assessment Resources, Inc. Achenbach T and Rescorla L. 2000. Manual for the ASEBA preschool forms and profiles. Burlington, VT: University of Vermont, Research Center for Children, Youth, and Families. Acolet D, Modi N, Giannakoulopoulos X, Bond C, Weg W, Clow A, Glover V. 1993. Changes in plasma cortisol and catecholamine concentrations in response to massage in preterm infants. Arch Dis Child 68(1 Spec No):29-31. Adams E, Chau V, Poskitt KJ, Grunau RE, Synnes A, Miller SP. 2010. Tractography-based quantitation of corticospinal tract development in premature newborns. J Pediatr 156(6):882,8, 888.e1. Ahmad I, Zaldivar F, Iwanaga K, Koeppel R, Grochow D, Nemet D, Waffarn F, Eliakim A, Leu SY, Cooper DM. 2007. Inflammatory and growth mediators in growing preterm infants. J Pediatr Endocrinol Metab 20(3):387-96. Ahnert L, Gunnar MR, Lamb ME, Barthel M. 2004. Transition to child care: Associations with infant-mother attachment, infant negative emotion, and cortisol elevations. Child Dev 75(3):639-50. Ainsworth MDS, Blehar M, Walters E, Wall S. 1978. Patterns of attachment: A psychological study of the strange situation. Hillsdale, New Jersey: Erlbaum. Aisa B, Tordera R, Lasheras B, Del Rio J, Ramirez MJ. 2007. Cognitive impairment associated to HPA axis hyperactivity after maternal separation in rats. Psychoneuroendocrinology 32(3):256-66. Allin MP, Kontis D, Walshe M, Wyatt J, Barker GJ, Kanaan RA, McGuire P, Rifkin L, Murray RM, Nosarti C. 2011. White matter and cognition in adults who were born preterm. PLoS One 6(10):e24525. Anand KJ. 2007. Pain assessment in preterm neonates. Pediatrics 119:605-7. Anand KJ and Hickey PR. 1987. Pain and its effects in the human neonate and fetus. N Engl J Med 317(21):1321-9. 	   157 Anand KJ, Sippell WG, Aynsley-Green A. 1987. Randomised trial of fentanyl anaesthesia in preterm babies undergoing surgery: Effects on the stress response. Lancet 1(8527):243-8. Anand KJ, Coskun V, Thrivikraman KV, Nemeroff CB, Plotsky PM. 1999. Long-term behavioral effects of repetitive pain in neonatal rat pups. Physiol Behav 66(4):627-37. Anand KJ, Garg S, Rovnaghi CR, Narsinghani U, Bhutta AT, Hall RW. 2007. Ketamine reduces the cell death following inflammatory pain in newborn rat brain. Pediatr Res 62(3):283-90. Anand KJ, Hall RW, Desai N, Shephard B, Bergqvist LL, Young TE, Boyle EM, Carbajal R, Bhutani VK, Moore MB, et al. 2004. Effects of morphine analgesia in ventilated preterm neonates: Primary outcomes from the NEOPAIN randomised trial. Lancet 363(9422):1673-82. Anderson P, Doyle LW, Victorian Infant Collaborative Study Group. 2003. Neurobehavioral outcomes of school-age children born extremely low birth weight or very preterm in the 1990s. JAMA 289(24):3264-72. Andrews-Hanna JR, Smallwood J, Spreng RN. 2014. The default network and self-generated thought: Component process, dynamic control, and clinical revalence. Ann NY Acad Sci 1316:29-52. Andrews K and Fitzgerald M. 1994. The cutaneous withdrawal reflex in human neonates: Sensitization, receptive fields, and the effects of contralateral stimulation. Pain 56(1):95-101. Apkarian AV, Bushnell MC, Treede RD, Zubieta JK. 2005. Human brain mechanisms of pain perception and regulation in health and disease. Eur J Pain 9(4):463-84. Asmerom Y, Slater L, Boskovic DS, Bahjri K, Holden MS, Phillips R, Deming D, Ashwal S, Fayard E, Angeles DM. 2013. Oral sucrose for heel lance increases adenosine triphosphate use and oxidative stress in preterm neonates. J Pediatr 163(1):29,35.e1. Atlas LY, Lindquist MA, Bolger N, Wager TD. 2014. Brain mediators of the effects of noxious heat on pain. Pain 155(8):1632-48. Axelin A, Salantera S, Lehtonen L. 2006. 'Facilitated tucking by parents' in pain management of preterm infants-a randomized crossover trial. Early Hum Dev 82(4):241-7. Ayoub AE and Kostovic I. 2009. New horizons for the subplate zone and its pioneering neurons. Cereb Cortex 19(8):1705-7. Back SA and Miller SP. 2014. Brain injury in premature neonates: A primary cerebral dysmaturation disorder? Ann Neurol 75(4):469-86. Back SA, Gan X, Li Y, Rosenberg PA, Volpe JJ. 1998. Maturation-dependent vulnerability of oligodendrocytes to oxidative stress-induced death caused by glutathione depletion. J 	   158 Neurosci 18(16):6241-53. Back SA, Luo NL, Borenstein NS, Levine JM, Volpe JJ, Kinney HC. 2001. Late oligodendrocyte progenitors coincide with the developmental window of vulnerability for human perinatal white matter injury. J Neurosci 21(4):1302-12. Back SA, Luo NL, Mallinson RA, O'Malley JP, Wallen LD, Frei B, Morrow JD, Petito CK, Roberts CT,Jr, Murdoch GH, et al. 2005. Selective vulnerability of preterm white matter to oxidative damage defined by F2-isoprostanes. Ann Neurol 58(1):108-20. Baleydier C, Mauguiere F. 1980. The duality of the cingulate gyrus in monkey. Neuroanatomical study and functional hypothesis. Brain 103(3):525–54. Ball G, Srinivasan L, Aljabar P, Counsell SJ, Durighel G, Hajnal JV, Rutherford MA, Edwards AD. 2013. Development of cortical microstructure in the preterm human brain. Proc Natl Acad Sci U S A 110(23):9541-6. Ball G, Pazderova L, Chew A, Tusor N, Merchant N, Arichi T, Allsop JM, Cowan FM, Edwards AD, Counsell SJ. 2015. Thalamocortical connectivity predicts cognition in children born preterm. Cereb Cortex . Bartocci M, Bergqvist LL, Lagercrantz H, Anand KJ. 2006. Pain activates cortical areas in the preterm newborn brain. Pain 122(1-2):109-17. Baud O, Greene AE, Li J, Wang H, Volpe JJ, Rosenberg PA. 2004. Glutathione peroxidase-catalase cooperativity is required for resistance to hydrogen peroxide by mature rat oligodendrocytes. J Neurosci 24(7):1531-40. Bauer K, Ketteler J, Hellwig M, Laurenz M, Versmold H. 2004. Oral glucose before venepuncture relieves neonates of pain, but stress is still evidenced by increase in oxygen consumption, energy expenditure, and heart rate. Pediatr Res 55(4):695-700. Bayley N. 1993. Bayley scales of infant development. 2nd ed. San Antonio, TX: TX-Psychological Corporation. Becerra L, Borsook D. 2008. Signal valence in the nucleus accumbens to pain onset and offset. Eur J Pain 12:866–9. Beckwith L, Rodning C, Cohen S. 1992. Preterm children at early adolescence and continuity and discontinuity in maternal responsiveness from infancy. Child Dev 63(5):1198-208. Beggs S, Currie G, Salter MW, Fitzgerald M, Walker SM. 2012. Priming of adult pain responses by neonatal pain experience: maintenance by central neuroimmune activity. Brain 135(Pt 2):404-17.  Beggs S, Torsney C, Drew LJ, Fitzgerald M. 2002. The postnatal reorganization of primary afferent input and dorsal horn cell receptive fields in the rat spinal cord is an activity-	   159 dependent process. Eur J Neurosci 16(7):1249-58. Bell MJ, Ternberg JL, Feigin RD, Keating JP, Marshall R, Barton L, Brotherton T. 1978. Neonatal necrotizing enterocolitis. therapeutic decisions based upon clinical staging. Ann Surg 187(1):1-7. Benders MJ, Palmu K, Menache C, Borradori-Tolsa C, Lazeyras F, Sizonenko S, Dubois J, Vanhatalo S, Huppi PS. 2014. Early brain activity relates to subsequent brain growth in premature infants. Cereb Cortex . Berry MA, Abrahamowicz M, Usher RH. 1997. Factors associated with growth of extremely premature infants during initial hospitalization. Pediatrics 100(4):640-6. Bhatnagar S, Vining C, Iyer V, Kinni V. 2006. Changes in hypothalamic-pituitary-adrenal function, body temperature, body weight and food intake with repeated social stress exposure in rats. J Neuroendocrinol 18(1):13-24. Bhutta AT, Cleves MA, Casey PH, Cradock MM, Anand KJ. 2002. Cognitive and behavioral outcomes of school-aged children who were born preterm: A meta-analysis. JAMA 288(6):728-37. Bhutta AT, Rovnaghi C, Simpson PM, Gossett JM, Scalzo FM, Anand KJ. 2001. Interactions of inflammatory pain and morphine in infant rats: Long-term behavioral effects. Physiol Behav 73(1-2):51-8. Billeaud C, Piedboeuf B, Chessex P. 1992. Energy expenditure and severity of respiratory disease in very low birth weight infants receiving long-term ventilatory support. J Pediatr 120(3):461-4. Biringen Z, Robinson J, Emde R. 1998. Emotional availability scales. 3rd ed. Boulder, Colorado: http://www.emotionalavailability.com. Biringen Z, Derscheid D, Vliegen N, Closson L, Easterbrooks MA. 2014. Emotional availability (EA): Theoretical background, empirical research using the EA scales, and clinical applications. Dev Rev 34(2):114-67. Biringen Z. 2008. The emotional availability (EA) scales and the emotional attachment & emotional availability (EA2) clinical screener: Infancy/early childhood version; middle childhood/youth versions; therapist/interventionist manual; couple relationship manual. 4th ed. Boulder, Colorado: http://emotionalavailabilty.com. Biringen Z. 2000. Emotional availability: Conceptualization and research findings. Am J Orthopsychiatry 70(1):104-14. Bjuland KJ, Rimol LM, Lohaugen GC, Skranes J. 2014. Brain volumes and cognitive function in very-low-birth-weight (VLBW) young adults. Eur J Paediatr Neurol 18(5):578-90. Bland JM and Altman DG. 1986. Statistical methods for assessing agreement between two 	   160 methods of clinical measurement. Lancet 1(8476):307-10. Bohm B, Katz-Salamon M, Institute K, Smedler AC, Lagercrantz H, Forssberg H.  Developmental risks and protective factors for influencing cognitive outcome at 5 1/2 years of age in very-low-birthweight children. Dev Med Child Neurol 2002;44:508–16. Bolt RJ, Van Weissenbruch MM, Popp-Snijders C, Sweep FG, Lafeber HN, Delemarre-van de Waal HA. 2002. Maturity of the adrenal cortex in very preterm infants is related to gestational age. Pediatr Res 52(3):405-10. Bonifacio SL, Glass HC, Chau V, Berman JI, Xu D, Brant R, Barkovich AJ, Poskitt KJ, Miller SP, Ferriero DM. 2010. Extreme premature birth is not associated with impaired development of brain microstructure. J Pediatr 157(5):726,32.e1. Bora S, Pritchard VE, Chen Z, Inder TE, Woodward LJ. 2014. Neonatal cerebral morphometry and later risk of persistent inattention/hyperactivity in children born very preterm. J Child Psychol Psychiatry 55(7):828-38. Borsook D, Upadhyay J, Chudler EH, Becerra L. 2010. A key role of the basal ganglia in pain and analgesia - insights gained through human functional imaging. Mol Pain 6:27. Bourne S, Machado AG, Nagel SJ. 2014. Basic anatomy and physiology of pain pathways. Neurosurg Clin N Am 25(4):629-38. Bowlby J. 1973. Attachment and loss: Separation. New York, New York: Basic Books. Bowlby J. 1969. Attachment and loss: Attachment. New York, New York: Basic Books. Brachfeld S, Goldberg S, Sloman J. 1980. Parent-infant interaction in free play at 8 and 12 months: Effects of prematurity and immaturity. Infant Beh Dev 3:289-305. Brown CJ, Miller SP, Booth BG, Andrews S, Chau V, Poskitt KJ, Hamarneh G. 2014. Structural network analysis of brain development in young preterm neonates. Neuroimage 101:667-80. Brummelte S, Grunau RE, Synnes AR, Whitfield MF, Petrie-Thomas J. 2011a. Declining cognitive development from 8 to 18 months in preterm children predicts persisting higher parenting stress. Early Hum Dev 87(4):273-80. Brummelte S, Grunau RE, Zaidman-Zait A, Weinberg J, Nordstokke D, Cepeda IL. 2011b. Cortisol levels in relation to maternal interaction and child internalizing behavior in preterm and full-term children at 18 months corrected age. Dev Psychobiol 53(2):184-95. Brummelte S, Chau CM, Cepeda IL, Degenhardt A, Weinberg J, Synnes AR, Grunau RE. 2015. Cortisol levels in former preterm children at school age are predicted by neonatal procedural pain-related stress. Psychoneuroendocrinology 51:151-63. Brummelte S, Grunau RE, Chau V, Poskitt KJ, Brant R, Vinall J, Gover A, Synnes AR, Miller SP. 2012. Procedural pain and brain development in premature newborns. Ann Neurol 	   161 71(3):385-96. Buntinx M, Moreels M, Vandenabeele F, Lambrichts I, Raus J, Steels P, Stinissen P, Ameloot M. 2004. Cytokine-induced cell death in human oligodendroglial cell lines: I. synergistic effects of IFN-gamma and TNF-alpha on apoptosis. J Neurosci Res 76(6):834-45. Buser JR, Maire J, Riddle A, Gong X, Nguyen T, Nelson K, Luo NL, Ren J, Struve J, Sherman LS, et al. 2012. Arrested preoligodendrocyte maturation contributes to myelination failure in premature infants. Ann Neurol 71(1):93-109. Bushnell MC, Ceko M, Low LA. 2013. Cognitive and emotional control of pain and its disruption in chronic pain. Nat Rev Neurosci 14:502–511. Caldji C, Tannenbaum B, Sharma S, Francis D, Plotsky PM, Meaney MJ. 1998. Maternal care during infancy regulates the development of neural systems mediating the expression of fearfulness in the rat. Proc Natl Acad Sci U S A 95(9):5335-40. Canadian Institute for Health Information. Highlights of 2010–2011 selected indicators describing the birthing process in canada. CIHI [Internet]. [revised June 21, 2012;cited March 27, 2015] Available from https://secure.cihi.ca/free_products/Childbirth_Highlights_2010-11_EN.pdf. Canadian Institute for Health Information. Too early, too small: A profile of small babies across canada. CIHI [Internet]. [revised January 29, 2009;cited March 27, 2015] Available from https://secure.cihi.ca/free_products/too_early_too_small_en.pdf. Candelaria M, Teti DM, Black MM. 2011. Multi-risk infants: Predicting attachment security from sociodemographic, psychosocial, and health risk among african-american preterm infants. J Child Psychol Psychiatry 52(8):870-7. Carbajal R, Chauvet X, Couderc S, Olivier-Martin M. 1999. Randomised trial of analgesic effects of sucrose, glucose, and pacifiers in term neonates. BMJ 319(7222):1393-7. Carbajal R, Lenclen R, Jugie M, Paupe A, Barton BA, Anand KJ. 2005. Morphine does not provide adequate analgesia for acute procedural pain among preterm neonates. Pediatrics 115(6):1494-500. Carbajal R, Rousset A, Danan C, Coquery S, Nolent P, Ducrocq S, Saizou C, Lapillonne A, Granier M, Durand P, et al. 2008. Epidemiology and treatment of painful procedures in neonates in intensive care units. JAMA 300(1):60-70. Castral TC, Warnock F, Leite AM, Haas VJ, Scochi CG. 2008. The effects of skin-to-skin contact during acute pain in preterm newborns. Eur J Pain 12(4):464-71. Champagne FA and Meaney MJ. 2007. Transgenerational effects of social environment on variations in maternal care and behavioral response to novelty. Behav Neurosci 121(6):1353-63. 	   162 Champagne FA, Francis DD, Mar A, Meaney MJ. 2003. Variations in maternal care in the rat as a mediating influence for the effects of environment on development. Physiol Behav 79(3):359-71. Chau V, Synnes A, Grunau RE, Poskitt KJ, Brant R, Miller SP. 2013. Abnormal brain maturation in preterm neonates associated with adverse developmental outcomes. Neurology 81(24):2082-9. Chau V, Brant R, Poskitt KJ, Tam EW, Synnes A, Miller SP. 2012. Postnatal infection is associated with widespread abnormalities of brain development in premature newborns. Pediatr Res 71(3):274-9. Chau V, Poskitt KJ, McFadden DE, Bowen-Roberts T, Synnes A, Brant R, Sargent MA, Soulikias W, Miller SP. 2009. Effect of chorioamnionitis on brain development and injury in premature newborns. Ann Neurol 66(2):155-64. Chery N and De Koninck Y. 1999. Junctional versus extrajunctional glycine and GABAA receptor-mediated IPSCs in identified lamina I neurons of the adult rat spinal cord. J Neurosci1 9(17):7342-55. Chrousos GP. 2009. Stress and disorders of the stress system. Nat Rev Endocrinol 5(7):374-81. Chrousos GP. 1995. The hypothalamic-pituitary-adrenal axis and immune-mediated inflammation. N Engl J Med 332(20):1351-62. Chudler EH, Dubner AF, Kenshalo DR Jr. 1990. Responses of nociceptive SI neurons in monkeys and pain sensation in humans elicited by noxious thermal stimulation: effect of interstimulus interval. J Neurophysiol 63(3):559-69. Cockerill J, Uthaya S, Dore CJ, Modi N. 2006. Accelerated postnatal head growth follows preterm birth. Arch Dis Child Fetal Neonatal Ed 91(3):F184-7. Coghill RC, Sang CN, Maisog JM, Iadarola MJ. 1999. Pain intensity processing within the human brain: a bilateral, distributed mechanism. J Neurophysiol 82:1934–1943. Coplan JD, Andrews MW, Rosenblum LA, Owens MJ, Friedman S, Gorman JM, Nemeroff CB. 1996. Persistent elevations of cerebrospinal fluid concentrations of corticotropin-releasing factor in adult nonhuman primates exposed to early-life stressors: Implications for the pathophysiology of mood and anxiety disorders. Proc Natl Acad Sci U S A 93(4):1619-23. Costigan M, Moss A, Latremoliere A, Johnston C, Verma-Gandhu M, Herbert TA, Barrett L, Brenner GJ, Vardeh D, Woolf CJ, Fitzgerald M. 2009. T-cell infiltration and signaling in the adult dorsal spinal cord is a major contributor to neuropathic pain-like hypersensitivity. J Neurosci 29:14415–22. Counsell SJ, Allsop JM, Harrison MC, Larkman DJ, Kennea NL, Kapellou O, Cowan FM, Hajnal JV, Edwards AD, Rutherford MA. 2003. Diffusion-weighted imaging of the brain in 	   163 preterm infants with focal and diffuse white matter abnormality. Pediatrics 112(1 Pt 1):1-7. Counsell SJ, Edwards AD, Chew AT, Anjari M, Dyet LE, Srinivasan L, Boardman JP, Allsop JM, Hajnal JV, Rutherford MA, et al. 2008. Specific relations between neurodevelopmental abilities and white matter microstructure in children born preterm. Brain 131(Pt 12):3201-8. Craig AD. 2003. Pain mechanisms: labeled lines versus convergence in central processing. Annu Rev Neurosci 26:1-30. Craig KD, Whitfield MF, Grunau RV, Linton J, Hadjistavropoulos HD. 1993. Pain in the preterm neonate: Behavioural and physiological indices. Pain 52(3):287-99. Crnic KA and Greenberg MT. 1987. Transactional relationships between perceived family style, risk status, and mother-child interactions in two-year-olds. J Pediatr Psychol 12(3):343-62. Crnic KA, Ragozin AS, Greenberg MT, Robinson NM, Basham RB. 1983. Social interaction and developmental competence of preterm and full-term infants during the first year of life. Child Dev 54(5):1199-210. Cutfield WS, Regan FA, Jackson WE, Jefferies CA, Robinson EM, Harris M, Hofman PL. 2004. The endocrine consequences for very low birth weight premature infants. Growth Horm IGF Res 14 Suppl A:S130-5. Damodaram M, Story L, Kulinskaya E, Rutherford M, Kumar S. 2011. Early adverse perinatal complications in preterm growth-restricted fetuses. Aust N Z J Obstet Gynaecol 51(3):204-9. De Bellis MD. 2005. The psychobiology of neglect. Child Maltreat 10(2):150-72. de Graaf J, van Lingen RA, Simons SH, Anand KJ, Duivenvoorden HJ, Weisglas-Kuperus N, Roofthooft DW, Groot Jebbink LJ, Veenstra RR, Tibboel D, et al. 2011. Long-term effects of routine morphine infusion in mechanically ventilated neonates on children's functioning: Five-year follow-up of a randomized controlled trial. Pain 152(6):1391-7. Dean JM, van de Looij Y, Sizonenko SV, Lodygensky GA, Lazeyras F, Bolouri H, Kjellmer I, Huppi PS, Hagberg H, Mallard C. 2011. Delayed cortical impairment following lipopolysaccharide exposure in preterm fetal sheep. Ann Neurol 70(5):846-56. Dean JM, McClendon E, Hansen K, Azimi-Zonooz A, Chen K, Riddle A, Gong X, Sharifnia E, Hagen M, Ahmad T, et al. 2013. Prenatal cerebral ischemia disrupts MRI-defined cortical microstructure through disturbances in neuronal arborization. Sci Transl Med 5(168):168ra7. Deipolyi AR, Mukherjee P, Gill K, Henry RG, Partridge SC, Veeraraghavan S, Jin H, Lu Y, Miller SP, Ferriero DM, et al. 2005. Comparing microstructural and macrostructural development of the cerebral cortex in premature newborns: Diffusion tensor imaging versus cortical gyration. Neuroimage 27(3):579-86. 	   164 Deng W, Rosenberg PA, Volpe JJ, Jensen FE. 2003. Calcium-permeable AMPA/kainate receptors mediate toxicity and preconditioning by oxygen-glucose deprivation in oligodendrocyte precursors. Proc Natl Acad Sci U S A 100(11):6801-6. Diego MA, Field T, Hernandez-Reif M. 2005. Vagal activity, gastric motility, and weight gain in massaged preterm neonates. J Pediatr 147(1):50-5. Dieleman GC, Huizink AC, Tulen JH, Utens EM, Creemers HE, van der Ende J, Verhulst FC. 2015. Alterations in HPA-axis and autonomic nervous system functioning in childhood anxiety disorders point to a chronic stress hypothesis. Psychoneuroendocrinology 51:135-50. Docherty SL, Miles MS, Holditch-Davis D. 2002. Worry about child health in mothers of hospitalized medically fragile infants. Adv Neonatal Care 2(2):84-92. Doesburg SM, Ribary U, Herdman AT, Miller SP, Poskitt KJ, Moiseev A, Whifield MF, Synnes A, Grunau RE. 2011. Altered long-range alpha-band synchronization during visual short-term memory retention in children born very preterm. NeuroImage 54(3):2330-9. Doesburg SM, Chau CM, Cheung TP, Moiseev A, Ribary U, Herdman AT, Miller SP, Cepeda IL, Synnes A, Grunau RE. 2013. Neonatal pain-related stress, functional cortical activity and visual-perceptual abilities in school-age children born at extremely low gestational age. Pain 154(10):1946-52. Doyle LW and Anderson PJ. 2010. Adult outcome of extremely preterm infants. Pediatrics 126(2):342-51. Doyle LW, Casalaz D, Victorian Infant Collaborative Study Group. 2001. Outcome at 14 years of extremely low birthweight infants: A regional study. Arch Dis Child Fetal Neonatal Ed 85(3):F159-64. Doyle LW, Ehrenkranz RA, Halliday HL. 2014a. Late (> 7 days) postnatal corticosteroids for chronic lung disease in preterm infants. Cocrhane Database Syst Rev 5:CD001145. Doyle LW, Ehrenkranz RA, Halliday HL. 2014b. Early (< 8 days) postnatal corticosteroids for preventing chronic lung disease in preterm infants. Cocrhane Database Syst Rev 5:CD001146. Doyle LW, Roberts G, Anderson PJ, Victorian Infant Collaborative Study Group. 2011. Changing long-term outcomes for infants 500-999 g birth weight in victoria, 1979-2005. Arch Dis Child Fetal Neonatal Ed 96(6):F443-7. Drobyshevsky A, Song SK, Gamkrelidze G, Wyrwicz AM, Derrick M, Meng F, Li L, Ji X, Trommer B, Beardsley DJ, et al. 2005. Developmental changes in diffusion anisotropy coincide with immature oligodendrocyte progression and maturation of compound action potential. J Neurosci 25(25):5988-97. Dubois J, Benders M, Cachia A, Lazeyras F, Ha-Vinh Leuchter R, Sizonenko SV, Borradori-	   165 Tolsa C, Mangin JF, Huppi PS. 2008a. Mapping the early cortical folding process in the preterm newborn brain. Cereb Cortex 18(6):1444-54. Dubois J, Benders M, Borradori-Tolsa C, Cachia A, Lazeyras F, Ha-Vinh Leuchter R, Sizonenko SV, Warfield SK, Mangin JF, Huppi PS. 2008b. Primary cortical folding in the human newborn: An early marker of later functional development. Brain 131(Pt 8):2028-41. Duerden EG and Albanese MC. 2013. Localization of pain-related brain activation: A meta-analysis of neuroimaging data. Hum Brain Mapp 34(1):109-49. Duerden EG, Taylor MJ, Miller SP. 2013. Brain development in infants born preterm: Looking beyond injury. Semin Pediatr Neurol 20(2):65-74. Duhrsen L, Simons SH, Dzietko M, Genz K, Bendix I, Boos V, Sifringer M, Tibboel D, Felderhoff-Mueser U. 2013. Effects of repetitive exposure to pain and morphine treatment on the neonatal rat brain. Neonatology 103(1):35-43. Earle KM. 1952. The tract of Lissauer and its possible relation to the pain pathway. J Comp Neurol 96(1):93–111. East PL and Rook KS. 1992. Compensatory patterns of support among children's peer relationships: A test using school friends, nonschool friends, and siblings. Dev  Psychol 28(1):163-72. Ehrenkranz RA, Dusick AM, Vohr BR, Wright LL, Wrage LA, Poole WK. 2006. Growth in the neonatal intensive care unit influences neurodevelopmental and growth outcomes of extremely low birth weight infants. Pediatrics 117(4):1253-61. Ehrenkranz RA, Younes N, Lemons JA, Fanaroff AA, Donovan EF, Wright LL, Katsikiotis V, Tyson JE, Oh W, Shankaran S, et al. 1999. Longitudinal growth of hospitalized very low birth weight infants. Pediatrics 104(2 Pt 1):280-9. Eixarch E, Batalle D, Illa M, Munoz-Moreno E, Arbat-Plana A, Amat-Roldan I, Figueras F, Gratacos E. 2012. Neonatal neurobehavior and diffusion MRI changes in brain reorganization due to intrauterine growth restriction in a rabbit model. PLoS One 7(2):e31497. Elenkov IJ, Webster EL, Torpy DJ, Chrousos GP. 1999. Stress, corticotropin-releasing hormone, glucocorticoids, and the immune/inflammatory response: Acute and chronic effects. Ann N Y Acad Sci 876:1,11; discussion 11-3. Embleton NE, Pang N, Cooke RJ. 2001. Postnatal malnutrition and growth retardation: An inevitable consequence of current recommendations in preterm infants? Pediatrics 107(2):270-3. Engle WA and American Academy of Pediatrics Committee on Fetus and Newborn. 2004. Age terminology during the perinatal period. Pediatrics 114(5):1362-4. 	   166 Erickson SJ, Duvall SW, Fuller J, Schrader R, MacLean P, Lowe JR. 2013. Differential associations between maternal scaffolding and toddler emotion regulation in toddlers born preterm and full term. Early Hum Dev 89(9):699-704. Estep ME, Smyser CD, Anderson PJ, Ortinau CM, Wallendorf M, Katzman CS, Doyle LW, Thompson DK, Neil JJ, Inder TE, et al. 2014. Diffusion tractography and neuromotor outcome in very preterm children with white matter abnormalities. Pediatr Res 76(1):86-92. Evans JC, McCartney EM, Lawhon G, Galloway J. 2005. Longitudinal comparison of preterm pain responses to repeated heelsticks. Pediatr Nurs 31(3):216-21. Evans T, Whittingham K, Boyd R. 2012. What helps the mother of a preterm infant become securely attached, responsive and well-adjusted? Infant Behav Dev 35(1):1-11. Fabrizi L, Slater R, Worley A, Meek J, Boyd S, Olhede S, Fitzgerald M. 2011. A shift in sensory processing that enables the developing human brain to discriminate touch from pain. Curr Biol 21(18):1552-8. Feldman HM, Lee ES, Loe IM, Yeom KW, Grill-Spector K, Luna B. 2012. White matter microstructure on diffusion tensor imaging is associated with conventional magnetic resonance imaging findings and cognitive function in adolescents born preterm. Dev Med Child Neurol 54(9):809-14. Feldman R, Rosenthal Z, Eidelman AI. 2014. Maternal-preterm skin-to-skin contact enhances child physiologic organization and cognitive control across the first 10 years of life. Biol Psychiatry 75(1):56-64. Feng X, Wang L, Yang S, Qin D, Wang J, Li C, Lv L, Ma Y, Hu X. 2011. Maternal separation produces lasting changes in cortisol and behavior in rhesus monkeys. Proc Natl Acad Sci U S A 108(34):14312-7. Ferber SG and Makhoul IR. 2008. Neurobehavioural assessment of skin-to-skin effects on reaction to pain in preterm infants: A randomized, controlled within-subject trial. Acta Paediatr 97(2):171-6. Ferguson SA, Ward WL, Paule MG, Hall RW, Anand KJ. 2012. A pilot study of preemptive morphine analgesia in preterm neonates: Effects on head circumference, social behavior, and response latencies in early childhood. Neurotoxicol Teratol 34(1):47-55. Fernandez EF and Watterberg KL. 2009. Relative adrenal insufficiency in the preterm and term infant. J Perinatol 29:S44-9.  Field T, Diego M, Hernandez-Reif M, Dieter JN, Kumar AM, Schanberg S, Kuhn C. 2008. Insulin and insulin-like growth factor-1 increased in preterm neonates following massage therapy. J Dev Behav Pediatr 29(6):463-6. Fields H. 2004. State-dependent opioid control of pain. Nat Rev Neurosci 5(7):565-75. 	   167 Fields HL. 2007. Understanding how opioids contribute to reward and analgesia. Region Anesth Pain M 32:242–6. Fischi-Gomez E, Vasung L, Meskaldji DE, Lazeyras F, Borradori-Tolsa C, Hagmann P, Barisnikov K, Thiran JP, Huppi PS. 2014. Structural brain connectivity in school-age preterm infants provides evidence for impaired networks relevant for higher order cognitive skills and social cognition. Cereb Cortex . Fitzgerald M. 2009. When is an analgesic not an analgesic? Pain 144(1-2):9. Fitzgerald M. 2005. The development of nociceptive circuits. Nat Rev Neurosci 6(7):507-20. Fitzgerald M and Beggs S. 2001. The neurobiology of pain: developmental aspects. Neuroscientist 7(3):246-57. Fitzgerald M and Walker SM. 2009. Infant pain management: A developmental neurobiological approach. Nat Clin Pract Neurol 5(1):35-50. Fitzgerald M, Millard C, McIntosh N. 1989. Cutaneous hypersensitivity following peripheral tissue damage in newborn infants and its reversal with topical anaesthesia. Pain 39(1):31-6. Forcada-Guex M, Pierrehumbert B, Borghini A, Moessinger A, Muller-Nix C. 2006. Early dyadic patterns of mother-infant interactions and outcomes of prematurity at 18 months. Pediatrics 118(1):e107-14. Francis D, Diorio J, Liu D, Meaney MJ. 1999a. Nongenomic transmission across generations of maternal behavior and stress responses in the rat. Science 286(5442):1155-8. Francis DD, Champagne FA, Liu D, Meaney MJ. 1999b. Maternal care, gene expression, and the development of individual differences in stress reactivity. Ann N Y Acad Sci 896:66-84. Franck LS, Oulton K, Nderitu S, Lim M, Fang S, Kaiser A. 2011. Parent involvement in pain management for NICU infants: A randomized controlled trial. Pediatrics 128(3):510-8. Fumagalli F, Jones SR, Caron MG, Seidler FJ, Slotkin TA. 1996. Expression of mRNA coding for the seretonin transporter in aged vs. young rat brain: Differential effects of glucocorticoids. Brain Res 6(719(1-2)):225-8. Gaal BJ, Pinelli J, Crooks D, Saigal S, Streiner DL, Boyle M. 2010. Outside looking in: The lived experience of adults with prematurely born siblings. Qual Health Res 20(11):1532-45. Gale G, Franck LS, Kools S, Lynch M. 2004. Parents' perceptions of their infant's pain experience in the NICU. Int J Nurs Stud 41(1):51-8. Gamallo A, Villanua A, Beato MJ. 1986. Body weight gain and food intake alterations in crowd-reared rats. Physiol Behav 36(5):835-7. Gambrill AC and Barria A. 2011. NMDA receptor subunit composition controls synaptogenesis 	   168 and synapse stabilization. Proc Natl Acad Sci U S A 108(14):5855-60. Garel M, Dardennes M, Blondel B. 2007. Mothers' psychological distress 1 year after very preterm childbirth. results of the EPIPAGE qualitative study. Child Care Health Dev 33(2):137-43. Georgieff MK. 2007. Nutrition and the developing brain: Nutrient priorities and measurement. Am J Clin Nutr 85(2):614S-20S. Gerull R, Cignacco E, Stoffel L, Sellam G, Nelle M. 2013. Physiological parameters after nonpharmacological analgesia in preterm infants: A randomized trial. Acta Paediatr 102(8):e368-73. Ghosh A and Shatz CJ. 1992. Involvement of subplate neurons in the formation of ocular dominance columns. Science 255(5050):1441-3. Ghosh A, Antonini A, McConnell SK, Shatz CJ. 1990. Requirement for subplate neurons in the formation of thalamocortical connections. Nature 347(6289):179-81. Gibbins S, Stevens B, McGrath PJ, Yamada J, Beyene J, Breau L, Camfield C, Finley A, Franck L, Johnston C, et al. 2008. Comparison of pain responses in infants of different gestational ages. Neonatology 93(1):10-8. Gimenez M, Junque C, Narberhaus A, Bargallo N, Botet F, Mercader JM. 2006. White matter volume and concentration reductions in adolescents with history of very preterm birth: A voxel-based morphometry study. Neuroimage 32(4):1485-98. Gingold SI, Greenspan JD, Apkarian AV. 1991. Anatomic evidence of nociceptive inputs to primary somatosensory cortex: relationship between spinothalamic terminals and thalamocortical cells in squirrel monkeys. J Comp Neurol 308(3):467-90. Glascher J, Tranel D, Paul LK, Rudrauf D, Rorden C, Hornaday A, Grabowski T, Damasio H, Adolphs R. 2009. Lesion mapping of cognitive abilities linked to intelligence. Neuron 61(5):681-91. Glass HC, Bonifacio SL, Chau V, Glidden D, Poskitt K, Barkovich AJ, Ferriero DM, Miller SP. 2008. Recurrent postnatal infections are associated with progressive white matter injury in premature infants. Pediatrics 122(2):299-305. Glazebrook C, Marlow N, Israel C, Croudace T, Johnson S, White IR, Whitelaw A. 2007. Randomised trial of a parenting intervention during neonatal intensive care. Arch Dis Child Fetal Neonatal Ed 92(6):F438-43. Goksan S, Hartley C, Emery F, Cockrill N, Poorun R, Moultrie F, Rogers R, Campbell J, Sanders M, Adams E, et al. 2015. fMRI reveals neural activity overlap between adult and infant pain. Elife 4:10.7554/eLife.06356. Gonsalves S and Mercer J. 1993. Physiological correlates of painful stimulation in preterm 	   169 infants. Clin J Pain 9(2):88-93. Granmo M, Petersson P, Schouenborg J. 2008. Action-based body maps in the spinal cord emerge from a transitory floating organization. J Neurosci 28(21):5494-503. Gravener JA, Rogosch FA, Oshri A, Narayan AJ, Cicchetti D, Toth SL. 2012. The relations among maternal depressive disorder, maternal expressed emotion, and toddler behavior problems and attachment. J Abnorm Child Psychol 40(5):803-13. Greenberg MT and Crnic KA. 1988. Longitudinal predictors of developmental status and social interaction in premature and full-term infants at age two. Child Dev 59(3):554-70. Greene JG, Fox NA, Lewis M. 1983. The relationship between neonatal characteristics and three-month mother-infant interaction in high-risk infants. Child Dev 54(5):1286-96. Griffith JL, Shimony JS, Cousins SA, Rees SE, McCurnin DC, Inder TE, Neil JJ. 2012. MR imaging correlates of white-matter pathology in a preterm baboon model. Pediatr Res 71(2):185-91. Groenendaal F, Termote JU, van der Heide-Jalving M, van Haastert IC, de Vries LS. 2010. Complications affecting preterm neonates from 1991 to 2006: What have we gained? Acta Paediatr 99(3):354-8. Grunau RE, Whitfield MF, Fay TB. 2004. Psychosocial and academic characteristics of extremely low birth weight (< or =800 g) adolescents who are free of major impairment compared with term-born control subjects. Pediatrics 114(6):e725-32. Grunau RE, Weinberg J, Whitfield MF. 2004. Neonatal procedural pain and preterm infant cortisol response to novelty at 8 months. Pediatrics 114(1):e77-84. Grunau RE, Whitfield MF, Davis C. 2002. Pattern of learning disabilities in children with extremely low birth weight and broadly average intelligence. Arch Pediatr Adolesc Med 156(6):615-20. Grunau RE, Holsti L, Whitfield MF, Ling E. 2000. Are twitches, startles, and body movements pain indicators in extremely low birth weight infants? Clin J Pain 16(1):37-45. Grunau RE, Oberlander TF, Whitfield MF, Fitzgerald C, Lee SK. 2001. Demographic and therapeutic determinants of pain reactivity in very low birth weight neonates at 32 weeks' postconceptional age. Pediatrics 107(1):105-12. Grunau RE, Haley DW, Whitfield MF, Weinberg J, Yu W, Thiessen P. 2007. Altered basal cortisol levels at 3, 6, 8 and 18 months in infants born at extremely low gestational age. J Pediatr 150(2):151-6. Grunau RE, Holsti L, Haley DW, Oberlander T, Weinberg J, Solimano A, Whitfield MF, Fitzgerald C, Yu W. 2005. Neonatal procedural pain exposure predicts lower cortisol and 	   170 behavioral reactivity in preterm infants in the NICU. Pain 113(3):293-300. Grunau RE, Whitfield MF, Petrie-Thomas J, Synnes AR, Cepeda IL, Keidar A, Rogers M, Mackay M, Hubber-Richard P, Johannesen D. 2009. Neonatal pain, parenting stress and interaction, in relation to cognitive and motor development at 8 and 18 months in preterm infants. Pain 143(1-2):138-46. Grunau RE, Cepeda IL, Chau CM, Brummelte S, Weinberg J, Lavoie PM, Ladd M, Hirschfeld AF, Russell E, Koren G, et al. 2013. Neonatal pain-related stress and NFKBIA genotype are associated with altered cortisol levels in preterm boys at school age. PLoS One 8(9):e73926. Grunau RV and Craig KD. 1987. Pain expression in neonates: Facial action and cry. Pain 28(3):395-410. Guinsburg R, Kopelman BI, Anand KJ, de Almeida MF, Peres Cde A, Miyoshi MH. 1998. Physiological, hormonal, and behavioral responses to a single fentanyl dose in intubated and ventilated preterm neonates. J Pediatr 132(6):954-9. Gunnar M and Quevedo K. 2007. The neurobiology of stress and development. Annu Rev Psychol 58:145-73. Gunnar MR. 1998. Quality of early care and buffering of neuroendocrine stress reactions: Potential effects on the developing human brain. Prev Med 27(2):208-11. Gunnar MR, Brodersen L, Krueger K, Rigatuso J. 1996. Dampening of adrenocortical responses during infancy: Normative changes and individual differences. Child Dev 67(3):877-89. Guttman HA. 1991. Systems theory, cybernetics, and epistemology. In: Handbook of family therapy. Gurman AS and Kniskern DP, editors. Philadelphia, Pennsylvania ed. Brunner/Mazel. 715 p. Halliday HL, Ehrenkranz RA, Doyle LW. 2010. Early (< 8 days) postnatal corticosteroids for preventing chronic lung disease in preterm infants. Cochrane Database Syst Rev (1)(1):CD001146. Hamrick SE, Miller SP, Leonard C, Glidden DV, Goldstein R, Ramaswamy V, Piecuch R, Ferriero DM. 2004. Trends in severe brain injury and neurodevelopmental outcome in premature newborn infants: The role of cystic periventricular leukomalacia. J Pediatr 145(5):593-9. Hansen-Pupp I, Hovel H, Lofqvist C, Hellstrom-Westas L, Fellman V, Huppi PS, Hellstrom A, Ley D. 2013. Circulatory insulin-like growth factor-I and brain volumes in relation to neurodevelopmental outcome in very preterm infants. Pediatr Res 74(5):564-9. Hansen-Pupp I, Hovel H, Hellstrom A, Hellstrom-Westas L, Lofqvist C, Larsson EM, Lazeyras F, Fellman V, Huppi PS, Ley D. 2011. Postnatal decrease in circulating insulin-like growth factor-I and low brain volumes in very preterm infants. J Clin Endocrinol Metab 	   171 96(4):1129-35. Hansson E. 2006. Could chronic pain and spread of pain sensation be induced and maintained by glial activation? Acta Physiol (Oxf) 187(1-2):321-7. Harrison MJ. 1990. A comparison of parental interactions with term and preterm infants. Res Nurs Health 13(3):173-9. Harwood K, McLean N, Durkin K. 2007. First-time mothers' expectations of parenthood: What happens when optimistic expectations are not matched by later experiences? Dev Psychol 43(1):1-12. Hathway GJ, Koch S, Low L, Fitzgerald M. 2009a. The changing balance of brainstem-spinal cord modulation of pain processing over the first weeks of rat postnatal life. J Physiol 587(Pt 12):2927-35. Hathway GJ, Vega-Avelaira D, Moss A, Ingram R. Fitzgerald M. 2009b. Brief, low frequency stimulation of rat peripheral C-fibres evokes prolonged microglial-induced central sensitization in adults but not in neonates. Pain 144:110–118. Haynes RL, Folkerth RD, Keefe RJ, Sung I, Swzeda LI, Rosenberg PA, Volpe JJ, Kinney HC. 2003. Nitrosative and oxidative injury to premyelinating oligodendrocytes in periventricular leukomalacia. J Neuropathol Exp Neurol 62(5):441-50. Heim C and Nemeroff CB. 2001. The role of childhood trauma in the neurobiology of mood and anxiety disorders: Preclinical and clinical studies. Biol Psychiatry 49(12):1023-39. Hernandez-Reif M, Diego M, Field T. 2007. Preterm infants show reduced stress behaviors and activity after 5 days of massage therapy. Infant Behav Dev 30(4):557-61. Hohmeister J, Kroll A, Wollgarten-Hadamek I, Zohsel K, Demirakca S, Flor H, Hermann C. 2010. Cerebral processing of pain in school-aged children with neonatal nociceptive input: An exploratory fMRI study. Pain 150(2):257-67. Holditch-Davis D, Miles MS, Weaver MA, Black B, Beeber L, Thoyre S, Engelke S. 2009. Patterns of distress in african-american mothers of preterm infants. J Dev Behav Pediatr 30(3):193-205. Holsti L and Grunau RE. 2010. Considerations for using sucrose to reduce procedural pain in preterm infants. Pediatrics 125(5):1042-7. Holsti L, Grunau RE, Oberlander TF, Osiovich H. 2008. Is it painful or not? discriminant validity of the behavioral indicators of infant pain (BIIP) scale. Clin J Pain 24(1):83-8. Holsti L, Grunau RE, Oberlander TF, Whitfield MF. 2005. Prior pain induces heightened motor responses during clustered care in preterm infants in the NICU. Early Hum Dev 81(3):293-302. 	   172 Holsti L, Grunau RE, Whifield MF, Oberlander TF, Lindh V. 2006. Behavioral responses to pain are heightened after clustered care in preterm infants born between 30 and 32 weeks gestational age. Clin J Pain 22(9):757-64. Huang WL, Harper CG, Evans SF, Newnham JP, Dunlop SA. 2001. Repeated prenatal corticosteroid administration delays astrocyte and capillary tight junction maturation in fetal sheep. Int J Dev Neurosci 19(5):487-93. Huang WL, Beazley LD, Quinlivan JA, Evans SF, Newnham JP, Dunlop SA. 1999. Effect of corticosteroids on brain growth in fetal sheep. Obstet Gynecol 94(2):213-8. Huppi PS, Maier SE, Peled S, Zientara GP, Barnes PD, Jolesz FA, Volpe JJ. 1998. Microstructural development of human newborn cerebral white matter assessed in vivo by diffusion tensor magnetic resonance imaging. Pediatr Res 44(4):584-90. Iadarola MJ and Coghill RC. 1999. Imaging of pain: Recent developments. Curr Opin Anaesthesiol 12(5):583-9. Inder TE, Warfield SK, Wang H, Huppi PS, Volpe JJ. 2005. Abnormal cerebral structure is present at term in premature infants. Pediatrics 115(2):286-94. Inder TE, Anderson NJ, Spencer C, Wells S, Volpe JJ. 2003. White matter injury in the premature infant: A comparison between serial cranial sonographic and MR findings at term. AJNR Am J Neuroradiol 24(5):805-9. Inder TE, Huppi PS, Warfield S, Kikinis R, Zientara GP, Barnes PD, Jolesz F, Volpe JJ. 1999. Periventricular white matter injury in the premature infant is followed by reduced cerebral cortical gray matter volume at term. Ann Neurol 46(5):755-60. International Association for the Study of Pain. 1979. Pain terms: A list with definitions and notes on usage. Pain 6:249. Jaekel J, Wolke D, Chernova J. 2012. Mother and child behaviour in very preterm and term dyads at 6 and 8 years. Dev Med Child Neurol 54(8):716-23. Jennings E and Fitzgerald M. 1998. Postnatal changes in responses of rat dorsal horn cells to afferent stimulation: A fibre-induced sensitization. J Physiol 509 ( Pt 3)(Pt 3):859-68. Jespersen SN, Leigland LA, Cornea A, Kroenke CD. 2012. Determination of axonal and dendritic orientation distributions within the developing cerebral cortex by diffusion tensor imaging. IEEE Trans Med Imaging 31(1):16-32. Johnson S, Hennessy E, Smith R, Trikic R, Wolke D, Marlow N. 2009. Academic attainment and special educational needs in extremely preterm children at 11 years of age: The EPICure study. Arch Dis Child Fetal Neonatal Ed 94(4):F283-9. Johnston C, Barrington KJ, Taddio A, Carbajal R, Filion F. 2011. Pain in canadian NICUs: Have 	   173 we improved over the past 12 years? Clin J Pain 27(3):225-32. Johnston CC and Stevens BJ. 1996. Experience in a neonatal intensive care unit affects pain response. Pediatrics 98(5):925-30. Johnston CC, Stremler RL, Stevens BJ, Horton LJ. 1997. Effectiveness of oral sucrose and simulated rocking on pain response in preterm neonates. Pain 72(1-2):193-9. Johnston CC, Stevens BJ, Franck LS, Jack A, Stremler R, Platt R. 1999. Factors explaining lack of response to heel stick in preterm newborns. J Obstet Gynecol Neonatal Nurs 28(6):587-94. Johnston CC, Filion F, Snider L, Limperopoulos C, Majnemer A, Pelausa E, Cake H, Stone S, Sherrard A, Boyer K. 2007. How much sucrose is too much sucrose? Pediatrics 119(1):226. Johnston CC, Filion F, Snider L, Majnemer A, Limperopoulos C, Walker CD, Veilleux A, Pelausa E, Cake H, Stone S, et al. 2002. Routine sucrose analgesia during the first week of life in neonates younger than 31 weeks' postconceptional age. Pediatrics 110(3):523-8. Johnston KM, Gooch K, Korol E, Vo P, Eyawo O, Bradt P, Levy A. 2014. The economic burden of prematurity in canada. BMC Pediatr 14:93,2431-14-93. Jones AK, Kulkarni B, Derbyshire SW. 2003. Pain mechanisms and their disorders. Br Med Bull 65:83-93. Kaffashi F, Scher MS, Ludington-Hoe SM, Loparo KA. 2013. An analysis of the kangaroo care intervention using neonatal EEG complexity: A preliminary study. Clin Neurophysiol 124(2):238-46. Kalinichev M, Easterling KW, Plotsky PM, Holtzman SG. 2002. Long-lasting changes in stress-induced corticosterone response and anxiety-like behaviors as a consequence of neonatal maternal separation in long-evans rats. Pharmacol Biochem Behav 73(1):131-40. Kanold PO. 2009. Subplate neurons: Crucial regulators of cortical development and plasticity. Front Neuroanat 3:16. Katz MH. 2011. Multivariable analysis a practical guide for clinical and public health researchers third edition. New York, USA: Cambridge University Press. Kenshalo DR Jr, Chudler EH, Anton F, Dubner R. 1988. SI nociceptive neurons participate in the encoding process by which monkeys perceive the intensity of noxious thermal stimulation. Brain Res 454(1-2):378–82. Kenshalo DR Jr, Giesler GH Jr, Leonard RB, Willis WD. 1980. Responses of neurons in primate ventral posterior lateral nucleus to noxious stimuli. J Neurophysiol. 43(6):1594-614. Kenshalo DR Jr, Isensee O. 1983. Responses of primate SI cortical neurons to noxious stimuli. J 	   174 Neurophysiol 50(6):1479-96/ Kesler SR, Ment LR, Vohr B, Pajot SK, Schneider KC, Katz KH, Ebbitt TB, Duncan CC, Makuch RW, Reiss AL. 2004. Volumetric analysis of regional cerebral development in preterm children. Pediatr Neurol 31(5):318-25. Kesler SR, Reiss AL, Vohr B, Watson C, Schneider KC, Katz KH, Maller-Kesselman J, Silbereis J, Constable RT, Makuch RW, et al. 2008. Brain volume reductions within multiple cognitive systems in male preterm children at age twelve. J Pediatr 152(4):513,20, 520.e1. Keunen K, Kersbergen KJ, Groenendaal F, Isgum I, de Vries LS, Benders MJ. 2012. Brain tissue volumes in preterm infants: Prematurity, perinatal risk factors and neurodevelopmental outcome: A systematic review. J Matern Fetal Neonatal Med 25 Suppl 1:89-100. Koch SC and Fitzgerald M. 2014. The selectivity of rostroventral medulla descending control of spinal sensory inputs shifts postnatally from A fibre to C fibre evoked activity. J Physiol 592(Pt 7):1535-44. Konietzny F, Perl ER, Trevino D, Light A, Hensel H. 1981. Sensory experiences in man evoked by intraneural electrical stimulation of intact cutaneous afferent fibers. Exp Brain Res 42(2):219-22. Korraa AA, El Nagger AA, Mohamed R, Helmy NM. 2014. Impact of kangaroo mother care on cerebral blood flow of preterm infants. Ital J Pediatr 40(1):83. Korte SM. 2001. Corticosteroids in relation to fear, anxiety and psychopathology. Neurosci Biobehav Rev 25(2):117-42. Kostovic I and Judas M. 2010. The development of the subplate and thalamocortical connections in the human foetal brain. Acta Paediatr 99(8):1119-27. Kostovic I and Jovanov-Milosevic N. 2006. The development of cerebral connections during the first 20-45 weeks' gestation. Semin Fetal Neonatal Med 11(6):415-22. Kostovic I and Judas M. 2002. Correlation between the sequential ingrowth of afferents and transient patterns of cortical lamination in preterm infants. Anat Rec 267(1):1-6. Kostovic I and Rakic P. 1990. Developmental history of the transient subplate zone in the visual and somatosensory cortex of the macaque monkey and human brain. J Comp Neurol 297(3):441-70. Kostovic I, Judas M, Rados M, Hrabac P. 2002. Laminar organization of the human fetal cerebrum revealed by histochemical markers and magnetic resonance imaging. Cereb Cortex 12(5):536-44. Kroenke CD, Van Essen DC, Inder TE, Rees S, Bretthorst GL, Neil JJ. 2007. Microstructural changes of the baboon cerebral cortex during gestational development reflected in magnetic 	   175 resonance imaging diffusion anisotropy. J Neurosci 27(46):12506-15. Kurtoglu S, Kondolot M, Mazicioglu MM, Hatipoglu N, Akin MA, Akyildiz B. 2010. Growth hormone, insulin like growth factor-1, and insulin-like growth factor-binding protein-3 levels in the neonatal period: A preliminary study. J Pediatr Endocrinol Metab 23(9):885-9. Kucyi A, Hodaie M, Davis KD. 2012. Lateralization in instrinsic functional connectivity of the temperoparietal junction with salience- and attention-related brain networks. J Neurophysiol 108(12):3382-92.  Kucyi A, Salomons TV, Davis KD. 2013. Mind wandering away from pain dynamically engages antinociceptive and default mode brain networks. PNAS 110(46):18692-7.  Lamour Y, Willer JC, Guilbaud G. 1983. Rat somatosensory (SmI) cortex: I. Characteristics of neuronal responses to noxious stimulation and comparison with responses to non-noxious stimulation. Exp Brain Res 49(1):35–45. Landsem IP, Handegard BH, Tunby J, Ulvund SE, Ronning JA. 2014. Early intervention program reduces stress in parents of preterms during childhood, a randomized controlled trial. Trials 15:387,6215-15-387. Laplante P, Diorio J, Meaney MJ. 2002. Serotonin regulates hippocampal glucocorticoid receptor expression via a 5-HT7 receptor. Brain Res Dev Brain Res 139(2):199-203. LaPrairie JL and Murphy AZ. 2010. Long-term impact of neonatal injury in male and female rats: Sex differences, mechanisms and clinical implications. Front Neuroendocrinol 31(2):193-202. Laprairie JL and Murphy AZ. 2009. Neonatal injury alters adult pain sensitivity by increasing opioid tone in the periaqueductal gray. Front Behav Neurosci 3:31. LaPrairie JL and Murphy AZ. 2007. Female rats are more vulnerable to the long-term consequences of neonatal inflammatory injury. Pain 132 Suppl 1:S124-33. Larroque B, Ancel PY, Marret S, Marchand L, Andre M, Arnaud C, Pierrat V, Roze JC, Messer J, Thiriez G, et al. 2008. Neurodevelopmental disabilities and special care of 5-year-old children born before 33 weeks of gestation (the EPIPAGE study): A longitudinal cohort study. Lancet 371(9615):813-20. Lawrence EJ, Froudist-Walsh S, Neilan R, Nam KW, Giampietro V, McGuire P, Murray RM, Nosarti C. 2014. Motor fMRI and cortical grey matter volume in adults born very preterm. Dev Cogn Neurosci 10:1-9. Lax ID, Duerden EG, Lin SY, Mallar Chakravarty M, Donner EJ, Lerch JP, Taylor MJ. 2013. Neuroanatomical consequences of very preterm birth in middle childhood. Brain Struct Funct 218(2):575-85. Lepomaki V, Leppanen M, Matomaki J, Lapinleimu H, Lehtonen L, Haataja L, Komu M, 	   176 Rautava P, Parkkola R, PIPARI study group. 2013. Preterm infants' early growth and brain white matter maturation at term age. Pediatr Radiol 43(10):1357-64. Leppanen M, Lapinleimu H, Lind A, Matomaki J, Lehtonen L, Haataja L, Rautava P. 2014. Antenatal and postnatal growth and 5-year cognitive outcome in very preterm infants. Duodecim 130(7):738. Lenz FA, Weiss N, Ohara S, Lawson C, Greenspan JD. 2004. The role of the thalamus in pain. Suppl Clin Neurophysiol. 57:50-61. Lester BM, Hawes K, Abar B, Sullivan M, Miller R, Bigsby R, Laptook A, Salisbury A, Taub M, Lagasse LL, et al. 2014. Single-family room care and neurobehavioral and medical outcomes in preterm infants. Pediatrics 134(4):754-60. Leviton A, Allred EN, Kuban KC, Hecht JL, Onderdonk AB, O’Shea TM, Paneth N. 2010. Microbiologic and histologic characteristics of the extremely preterm infant’s placenta predict white matter damage and later cerebral palsy. the ELGAN study. Pediatr Res 67:95-101. Li J, Walker SM, Fitzgerald M, Baccei ML. 2009. Activity-dependent modulation of glutamatergic signaling in the developing rat dorsal horn by early tissue injury. J Neurophysiol 102(4):2208-19. Lind A, Korkman M, Lehtonen L, Lapinleimu H, Parkkola R, Matomaki J, Haataja L, PIPARI Study Group. 2011. Cognitive and neuropsychological outcomes at 5 years of age in preterm children born in the 2000s. Dev Med Child Neurol 53(3):256-62. Linver MR, Brooks-Gunn J, Kohen DE. 2002. Family processes as pathways from income to young children's development. Dev Psychol 38(5):719-34. Liu D, Diorio J, Day JC, Francis DD, Meaney MJ. 2000. Maternal care, hippocampal synaptogenesis and cognitive development in rats. Nat Neurosci 3(8):799-806. Liu D, Diorio J, Tannenbaum B, Caldji C, Francis D, Freedman A, Sharma S, Pearson D, Plotsky PM, Meaney MJ. 1997. Maternal care, hippocampal glucocorticoid receptors, and hypothalamic-pituitary-adrenal responses to stress. Science 277(5332):1659-62. Lo HC, Tsao LY, Hsu WY, Chen HN, Yu WK, Chi CY. 2002. Relation of cord serum levels of growth hormone, insulin-like growth factors, insulin-like growth factor binding proteins, leptin, and interleukin-6 with birth weight, birth length, and head circumference in term and preterm neonates. Nutrition 18(7-8):604-8. Lodygensky GA, Rademaker K, Zimine S, Gex-Fabry M, Lieftink AF, Lazeyras F, Groenendaal F, de Vries LS, Huppi PS. 2005. Structural and functional brain development after hydrocortisone treatment for neonatal chronic lung disease. Pediatrics 116(1):1-7. Lodygensky GA, West T, Stump M, Holtzman DM, Inder TE, Neil JJ. 2010. In vivo MRI analysis of an inflammatory injury in the developing brain. Brain Behav Immun 24(5):759-	   177 67. Loe IM, Lee ES, Luna B, Feldman HM. 2011. Behavior problems of 9-16 year old preterm children: Biological, sociodemographic, and intellectual contributions. Early Hum Dev 87(4):247-52. Lowe J, Erickson SJ, MacLean P. 2010. Cognitive correlates in toddlers born very low birth weight and full-term. Infant Behav Dev 33(4):629-34. Lowe JR, Maclean PC, Caprihan A, Ohls RK, Qualls C, Vanmeter J, Phillips JP. 2012. Comparison of cerebral volume in children aged 18-22 and 36-47 months born preterm and term. J Child Neurol 27(2):172-7. Ludington-Hoe SM, Hosseini R, Torowicz DL. 2005. Skin-to-skin contact (kangaroo care) analgesia for preterm infant heel stick. AACN Clin Issues 16(3):373-87. Luthi A, Schwyzer L, Mateos JM, Gahwiler BH, McKinney RA. 2001. NMDA receptor activation limits the number of synaptic connections during hippocampal development. Nat Neurosci 4(11):1102-7. Maalouf EF, Duggan PJ, Counsell SJ, Rutherford MA, Cowan F, Azzopardi D, Edwards AD. 2001. Comparison of findings on cranial ultrasound and magnetic resonance imaging in preterm infants. Pediatrics 107(4):719-27. Magill-Evans J and Harrison MJ. 2001. Parent-child interactions, parenting stress, and developmental outcomes at 4 years. Child Health Care 30(2):135-50. Mahler M, Pine F, Bergman A. 1975. The psychological birth of the human infant. New York, New York: Basic Books. Mainous RO and Looney S. 2007. A pilot study of changes in cerebral blood flow velocity, resistance, and vital signs following a painful stimulus in the premature infant. Adv Neonatal Care 7(2):88-104. Marin-Padilla M. 1992. Ontogenesis of the pyramidal cell of the mammalian neocortex and developmental cytoarchitectonics: A unifying theory. J Comp Neurol 321(2):223-40. Marlow N, Hennessy EM, Bracewell MA, Wolke D, EPICure Study Group. 2007. Motor and executive function at 6 years of age after extremely preterm birth. Pediatrics 120(4):793-804. Marlow N, Wolke D, Bracewell MA, Samara M, EPICure Study Group. 2005. Neurologic and developmental disability at six years of age after extremely preterm birth. N Engl J Med 352(1):9-19. Matthews SG. 2002. Early programming of the hypothalamo-pituitary-adrenal axis. Trends Endocrinol Metab 13(9):373-80. 	   178 Maunu J, Parkkola R, Rikalainen H, Lehtonen L, Haataja L, Lapinleimu H, PIPARI Group. 2009. Brain and ventricles in very low birth weight infants at term: A comparison among head circumference, ultrasound, and magnetic resonance imaging. Pediatrics 123(2):617-26. McDonald JW and Johnston MV. 1990. Physiological and pathophysiological roles of excitatory amino acids during central nervous system development. Brain Res Brain Res Rev 15(1):41-70. McEwen BS. 2004. Protection and damage from acute and chronic stress: Allostasis and allostatic overload and relevance to the pathophysiology of psychiatric disorders. Ann N Y Acad Sci 1032:1-7. McGowan PO, Sasaki A, D'Alessio AC, Dymov S, Labonte B, Szyf M, Turecki G, Meaney MJ. 2009. Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse. Nat Neurosci 12(3):342-8. McKinstry RC, Mathur A, Miller JH, Ozcan A, Snyder AZ, Schefft GL, Almli CR, Shiran SI, Conturo TE, Neil JJ. 2002. Radial organization of developing preterm human cerebral cortex revealed by non-invasive water diffusion anisotropy MRI. Cereb Cortex 12(12):1237-43. McPherson C and Grunau RE. 2014. Neonatal pain control and neurologic effects of anesthetics and sedatives in preterm infants. Clin Perinatol 41(1):209-27. McQuillen PS and Ferriero DM. 2005. Perinatal subplate neuron injury: Implications for cortical development and plasticity. Brain Pathol 15(3):250-60. McQuillen PS, Sheldon RA, Shatz CJ, Ferriero DM. 2003. Selective vulnerability of subplate neurons after early neonatal hypoxia-ischemia. J Neurosci 23(8):3308-15. Meaney MJ and Szyf M. 2005. Environmental programming of stress responses through DNA methylation: Life at the interface between a dynamic environment and a fixed genome. Dialogues Clin Neurosci 7(2):103-23. Meaney MJ, Szyf M, Seckl JR. 2007. Epigenetic mechanisms of perinatal programming of hypothalamic-pituitary-adrenal function and health. Trends Mol Med 13(7):269-77. Meaney MJ, Diorio J, Francis D, Weaver S, Yau J, Chapman K, Seckl JR. 2000. Postnatal handling increases the expression of cAMP-inducible transcription factors in the rat hippocampus: The effects of thyroid hormones and serotonin. J Neurosci 20(10):3926-35. Meaney MJ, Diorio J, Francis D, Widdowson J, LaPlante P, Caldji C, Sharma S, Seckl JR, Plotsky PM. 1996. Early environmental regulation of forebrain glucocorticoid receptor gene expression: Implications for adrenocortical responses to stress. Dev Neurosci 18(1-2):49-72. Melzack R and Wall PD. 2008. The challenge of pain. 2nd ed. London, England: Penguin Books. 	   179 Melzack R and Wall PD. 1965. Pain mechanisms: A new theory. Science 150(3699):971-9. Menard JL, Champagne DL, Meaney MJ. 2004. Variations of maternal care differentially influence 'fear' reactivity and regional patterns of cFos immunoreactivity in response to the shock-probe burying test. Neuroscience 129(2):297-308. Menke J, Michel E, Hillebrand S, von Twickel J, Jorch G. 1997. Cross-spectral analysis of cerebral autoregulation dynamics in high risk preterm infants during the perinatal period. Pediatr Res 42(5):690-9. Meyer EC, Garcia Coll CT, Seifer R, Ramos A, Kilis E, Oh W. 1995. Psychological distress in mothers of preterm infants. J Dev Behav Pediatr 16(6):412-7. Meyer JS. 1983. Early adrenalectomy stimulates subsequent growth and development of the rat brain. Exp Neurol 82(2):432-46. Miles MS and Holditch-Davis D. 1997. Parenting the prematurely born child: Pathways of influence. Semin Perinatol 21(3):254-66. Miles MS, Funk SG, Carlson J. 1993. Parental stressor scale: Neonatal intensive care unit. Nurs Res 42(3):148-52. Milevsky A and Levitt MJ. 2005. Sibling support in early adolescence: Buffering and compensation across relationships. Eur  J  Dev  Psychol 2(3):299-320. Milgrom J, Newnham C, Anderson PJ, Doyle LW, Gemmill AW, Lee K, Hunt RW, Bear M, Inder T. 2010. Early sensitivity training for parents of preterm infants: Impact on the developing brain. Pediatr Res 67(3):330-5. Milgrom J, Newnham C, Martin PR, Anderson PJ, Doyle LW, Hunt RW, Achenbach TM, Ferretti C, Holt CJ, Inder TE, et al. 2013. Early communication in preterm infants following intervention in the NICU. Early Hum Dev 89(9):755-62. Miller SP, Cozzio CC, Goldstein RB, Ferriero DM, Partridge JC, Vigneron DB, Barkovich AJ. 2003. Comparing the diagnosis of white matter injury in premature newborns with serial MR imaging and transfontanel ultrasonography findings. AJNR Am J Neuroradiol 24(8):1661-9. Miller SP, Ferriero DM, Leonard C, Piecuch R, Glidden DV, Partridge JC, Perez M, Mukherjee P, Vigneron DB, Barkovich AJ. 2005. Early brain injury in premature newborns detected with magnetic resonance imaging is associated with adverse early neurodevelopmental outcome. J Pediatr 147(5):609-16. Mitchell JB, Iny LJ, Meaney MJ. 1990. The role of serotonin in the development and environmental regulation of type II corticosteroid receptor binding in rat hippocampus. Brain Res Dev Brain Res 55(2):231-5. Mitchell JB, Betito K, Rowe W, Boksa P, Meaney MJ. 1992. Serotonergic regulation of type II 	   180 corticosteroid receptor binding in hippocampal cell cultures: Evidence for the importance of serotonin-induced changes in cAMP levels. Neuroscience 48(3):631-9. Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH. 1994. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12(3):529-40. Moore T, Hennessy EM, Myles J, Johnson SJ, Draper ES, Costeloe KL, Marlow N. 2012. Neurological and developmental outcome in extremely preterm children born in england in 1995 and 2006: The EPICure studies. BMJ 345:e7961. Morison SJ, Holsti L, Grunau RE, Whitfield MF, Oberlander TF, Chan HW, Williams L. 2003. Are there developmentally distinct motor indicators of pain in preterm infants? Early Hum Dev 72(2):131-46. Moss A, Beggs S, Vega-Avelaira D, Costigan M, Hathway GJ, Salter MW, Fitzgerald M. 2007. Spinal microglia and neuropathic pain in young rats. Pain.128:215–224. Mourek J, Himwich WA, Myslivecek J, Callison DA. 1967. The role of nutrition in the development of evoked cortical responses in rat. Brain Res 6(2):241-51. Mrzljak L, Uylings HB, Kostovic I, Van Eden CG. 1988. Prenatal development of neurons in the human prefrontal cortex: I. A qualitative golgi study. J Comp Neurol 271(3):355-86. Mukherjee P, Miller JH, Shimony JS, Philip JV, Nehra D, Snyder AZ, Conturo TE, Neil JJ, McKinstry RC. 2002. Diffusion-tensor MR imaging of gray and white matter development during normal human brain maturation. AJNR Am J Neuroradiol 23(9):1445-56. Mullen KM, Vohr BR, Katz KH, Schneider KC, Lacadie C, Hampson M, Makuch RW, Reiss AL, Constable RT, Ment LR. 2011. Preterm birth results in alterations in neural connectivity at age 16 years. Neuroimage 54(4):2563-70. Muller-Nix C, Forcada-Guex M, Pierrehumbert B, Jaunin L, Borghini A, Ansermet F. 2004. Prematurity, maternal stress and mother-child interactions. Early Hum Dev 79(2):145-58. Murgatroyd C and Spengler D. 2011. Epigenetic programming of the HPA axis: Early life decides. Stress 14(6):581-9. Nagy Z, Lagercrantz H, Hutton C. 2011. Effects of preterm birth on cortical thickness measured in adolescence. Cereb Cortex 21(2):300-6. Negrigo A, Medeiros M, Guinsburg R, Covolan L. 2011. Long-term gender behavioral vulnerability after nociceptive neonatal formalin stimulation in rats. Neurosci Lett 490(3):196-9. Nguyen The Tich S, Anderson PJ, Shimony JS, Hunt RW, Doyle LW, Inder TE. 2009. A novel quantitative simple brain metric using MR imaging for preterm infants. AJNR Am J 	   181 Neuroradiol 30(1):125-31. Nosarti C, Al-Asady MH, Frangou S, Stewart AL, Rifkin L, Murray RM. 2002. Adolescents who were born very preterm have decreased brain volumes. Brain 125(Pt 7):1616-23. Nosarti C, Nam KW, Walshe M, Murray RM, Cuddy M, Rifkin L, Allin MP. 2014. Preterm birth and structural brain alterations in early adulthood. Neuroimage Clin 6:180-91. Nosarti C, Mechelli A, Herrera A, Walshe M, Shergill SS, Murray RM, Rifkin L, Allin MP. 2011. Structural covariance in the cortex of very preterm adolescents: A voxel-based morphometry study. Hum Brain Mapp 32(10):1615-25. Nosarti C, Giouroukou E, Healy E, Rifkin L, Walshe M, Reichenberg A, Chitnis X, Williams SC, Murray RM. 2008. Grey and white matter distribution in very preterm adolescents mediates neurodevelopmental outcome. Brain 131(Pt 1):205-17. Ochoa J and Torebjork E. 1989. Sensations evoked by intraneural microstimulation of C nociceptor fibres in human skin nerves. J Physiol 415:583-99. O'Connor E, Bureau JF, McCartney K, Lyons-Ruth K. 2011. Risks and outcomes associated with disorganized/controlling patterns of attachment at age three in the NICHD study of early child care and youth development. Infant Ment Health J 32(4):450-72. Ogawa T, Mikuni M, Kuroda Y, Muneoka K, Mori KJ, Takahashi K. 1994. Periodic maternal deprivation alters stress response in adult offspring: Potentiates the negative feedback regulation of restraint stress-induced adrenocortical response and reduces the frequencies of open field-induced behaviors. Pharmacol Biochem Behav 49(4):961-7. Oomen CA, Soeters H, Audureau N, Vermunt L, van Hasselt FN, Manders EM, Joels M, Lucassen PJ, Krugers H. 2010. Severe early life stress hampers spatial learning and neurogenesis, but improves hippocampal synaptic plasticity and emotional learning under high-stress conditions in adulthood. J Neurosci 30(19):6635-45. Ozawa M, Kanda K, Hirata M, Kusakawa I, Suzuki C. 2011. Influence of repeated painful procedures on prefrontal cortical pain responses in newborns. Acta Paediatr 100(2):198-203. Padilla N, Falcon C, Sanz-Cortes M, Figueras F, Bargallo N, Crispi F, Eixarch E, Arranz A, Botet F, Gratacos E. 2011. Differential effects of intrauterine growth restriction on brain structure and development in preterm infants: A magnetic resonance imaging study. Brain Res 1382:98-108. Pang Y, Cai Z, Rhodes PG. 2005. Effect of tumor necrosis factor-alpha on developing optic nerve oligodendrocytes in culture. J Neurosci Res 80(2):226-34. Papile LA, Burstein J, Burstein R, Koffler H. 1978. Incidence and evolution of subependymal and intraventricular hemorrhage: A study of infants with birth weights less than 1,500 gm. J Pediatr 92(4):529-34. 	   182 Parikh NA, Lasky RE, Kennedy KA, Moya FR, Hochhauser L, Romo S, Tyson JE. 2007. Postnatal dexamethasone therapy and cerebral tissue volumes in extremely low birth weight infants. Pediatrics 119(2):265-72. Parker SJ, Zahr LK, Cole JG, Brecht ML. 1992. Outcome after developmental intervention in the neonatal intensive care unit for mothers of preterm infants with low socioeconomic status. J Pediatr 120(5):780-5. Partridge SC, Mukherjee P, Henry RG, Miller SP, Berman JI, Jin H, Lu Y, Glenn OA, Ferriero DM, Barkovich AJ, et al. 2004. Diffusion tensor imaging: Serial quantitation of white matter tract maturity in premature newborns. Neuroimage 22(3):1302-14. Peleg D, Kennedy CM, Hunter SK. 1998. Intrauterine growth restriction: Identification and management. Am Fam Physician 58(2):453,60, 466-7. Pena CJ, Neugut YD, Calarco CA, Champagne FA. 2014. Effects of maternal care on the development of midbrain dopamine pathways and reward-directed behavior in female offspring. Eur J Neurosci 39(6):946-56. Perlman JM and Volpe JJ. 1983. Suctioning in the preterm infant: Effects on cerebral blood flow velocity, intracranial pressure, and arterial blood pressure. Pediatrics 72(3):329-34. Peters KL. 1998. Neonatal stress reactivity and cortisol. J Perinat Neonatal Nurs 11(4):45-59. Peterson BS, Vohr B, Staib LH, Cannistraci CJ, Dolberg A, Schneider KC, Katz KH, Westerveld M, Sparrow S, Anderson AW, et al. 2000. Regional brain volume abnormalities and long-term cognitive outcome in preterm infants. JAMA 284(15):1939-47. Petrovic P, Petersson KM, Ghatan PH, Stone-Elander S, Ingvar M. 2000. Pain-related cerebral activation is altered by a distracting cognitive task. Pain 85(1-2):19-30.  Pillai Riddell RR, Racine NM, Turcotte K, Uman LS, Horton RE, Din Osmun L, Ahola Kohut S, Hillgrove Stuart J, Stevens B, Gerwitz-Stern A. 2011. Non-pharmacological management of infant and young child procedural pain. Cochrane Database Syst Rev (10):CD006275. doi(10):CD006275. Pineda RG, Neil J, Dierker D, Smyser CD, Wallendorf M, Kidokoro H, Reynolds LC, Walker S, Rogers C, Mathur AM, et al. 2014. Alterations in brain structure and neurodevelopmental outcome in preterm infants hospitalized in different neonatal intensive care unit environments. J Pediatr 164(1):52,60.e2. Poehlmann J and Fiese BH. 2001. The interaction of maternal and infant vulnerabilities on developing attachment relationships. Dev Psychopathol 13(1):1-11. Polin RA, Fox WW, Abman SH. 2003. Fetal and neonatal physiology. Philadelphia, PA: Saunders. Porter FL, Wolf CM, Miller JP. 1998. The effect of handling and immobilization on the response 	   183 to acute pain in newborn infants. Pediatrics 102(6):1383-9. Potharst ES, Schuengel C, Last BF, van Wassenaer AG, Kok JH, Houtzager BA. 2012. Difference in mother-child interaction between preterm- and term-born preschoolers with and without disabilities. Acta Paediatr 101(6):597-603. Pryce CR and Feldon J. 2003. Long-term neurobehavioural impact of the postnatal environment in rats: Manipulations, effects and mediating mechanisms. Neurosci Biobehav Rev 27(1-2):57-71. Pryds O. 1991. Control of cerebral circulation in the high-risk neonate. Ann Neurol 30(3):321-9. Pryds O, Greisen G, Lou H, Friis-Hansen B. 1989. Heterogeneity of cerebral vasoreactivity in preterm infants supported by mechanical ventilation. J Pediatr 115(4):638-45. Qu Y, Vadivelu S, Choi L, Liu S, Lu A, Lewis B, Girgis R, Lee CS, Snider BJ, Gottlieb DI, et al. 2003. Neurons derived from embryonic stem (ES) cells resemble normal neurons in their vulnerability to excitotoxic death. Exp Neurol 184(1):326-36. Rahkonen P, Heinonen K, Pesonen A, Lano A, Autti T, Puosi R, Huhtala E, Andersson S, Metsaranta M, Raikkonen K. 2014. Mother-child interaction is associated with neurocognitive outcome in extremely low gestational age children. Scan J Pscychol 55:311-8. Ranger M, Johnston CC, Anand KJ. 2007. Current controversies regarding pain assessment in neonates. Semin Perinatol 31(5):283-8. Ranger M, Synnes AR, Vinall J, Grunau RE. 2014. Internalizing behaviours in school-age children born very preterm are predicted by neonatal pain and morphine exposure. Eur J Pain 18(6):844-52. Ranger M, Chau CM, Garg A, Woodward TS, Beg MF, Bjornson B, Poskitt K, Fitzpatrick K, Synnes AR, Miller SP, et al. 2013. Neonatal pain-related stress predicts cortical thickness at age 7 years in children born very preterm. PLoS One 8(10):e76702. Rauh VA, Nurcombe B, Achenbach T, Howell C. 1990. The mother-infant transaction program. the content and implications of an intervention for the mothers of low-birthweight infants. Clin Perinatol 17(1):31-45. Renn CL and Dorsey SG. 2005. The physiology and processing of pain: A review. AACN Clin Issues 16(3):277,90; quiz 413-5. Resnick MB, Stralka K, Carter RL, Ariet M, Bucciarelli RL, Furlough RR, Evans JH, Curran JS,  Ausbon WW. Effects of birth weight and sociodemographic variables on mental development of neonatal intensive care unit survivors. Am J Obstet Gynecol 1990;162:374–8. Richardson DK, Corcoran JD, Escobar GJ, Lee SK. 2001. SNAP-II and SNAPPE-II: Simplified 	   184 newborn illness severity and mortality risk scores. J Pediatr 138(1):92-100. Robinson AJ. 1997. Central nervous system pathways for pain transmission and pain control: issues relavent to the practicing clinican. J Hand Ther 10(2):64-77. Rodkey EN and Pillai Riddell R. 2013. The infancy of infant pain research: The experimental origins of infant pain denial. J Pain 14(4):338-50. Rogers CE, Anderson PJ, Thompson DK, Kidokoro H, Wallendorf M, Treyvaud K, Roberts G, Doyle LW, Neil JJ, Inder TE. 2012. Regional cerebral development at term relates to school-age social-emotional development in very preterm children. J Am Acad Child Adolesc Psychiatry 51(2):181-91. Rovnaghi CR, Garg S, Hall RW, Bhutta AT, Anand KJ. 2008. Ketamine analgesia for inflammatory pain in neonatal rats: A factorial randomized trial examining long-term effects. Behav Brain Funct 4:35,9081-4-35. Rubens CE, Sadovsky Y, Muglia L, Gravett MG, Lackritz E, Gravett C. 2014. Prevention of preterm birth: Harnessing science to address the global epidemic. Sci Transl Med 6(262):262sr5. Saigal S and Doyle LW. 2008. An overview of mortality and sequelae of preterm birth from infancy to adulthood. Lancet 371(9608):261-9. Seeley WW, Menon V, Schatzberg, Keller J, Glover GH, Kenna H, Reiss AL, Greicius MD. 2007. Disociable instrinsic connectivity networks for salience processing and executive control. J Neurosci 27(9):2349-56. Schechter NL, Allen DA, Hanson K. 1986. Status of pediatric pain control: A comparison of hospital analgesic usage in children and adults. Pediatrics 77(1):11-5. Schnitzler A, Ploner M. 2000. Neurophysiology and functional neuroanatomy of pain perception. J Clin Neurophysiol 17(6):592-603. Schweinhardt P and Bushnell MC. 2010. Pain imaging in health and disease--how far have we come? J Clin Invest 120(11):3788-97. Shi CJ, Cassell MD. 1998. Cortical, thalamic, and amygdaloid connections of the anterior and posterior insular cortices. J Comp Neurol 399(4):440–68. Scott SM and Watterberg KL. 1995. Effect of gestational age, postnatal age, and illness on plasma cortisol concentrations in premature infants. Pediatr Res 37(1):112-6. Seidler FJ, Bell JM, Slotkin TA. 1990. Undernutrition and overnutrition in the neonatal rat: Long-term effects on noradrenergic pathways in brain regions. Pediatr Res 27(2):191-7. Shah DK, Doyle LW, Anderson PJ, Bear M, Daley AJ, Hunt RW, Inder TE. 2008. Adverse neurodevelopment in preterm infants with postnatal sepsis or necrotizing enterocolitis is 	   185 mediated by white matter abnormalities on magnetic resonance imaging at term. J Pediatr 153:170–5. Shahheidari M and Homer C. 2012. Impact of the design of neonatal intensive care units on neonates, staff, and families: A systematic literature review. J Perinat Neonatal Nurs 26(3):260,6; quiz 267-8. Sheng M, Cummings J, Roldan LA, Jan YN, Jan LY. 1994. Changing subunit composition of heteromeric NMDA receptors during development of rat cortex. Nature 368(6467):144-7. Sidman RL and Rakic P. 1973. Neuronal migration, with special reference to developing human brain: A review. Brain Res 62(1):1-35. Simons LE, Moulton EA, Linnman C, Carpino E, Becerra L, Borsook D. 2014. The human amygdala and pain: Evidence from neuroimaging. Hum Brain Mapp 35(2):527-38. Simons SH, van Dijk M, Anand KS, Roofthooft D, van Lingen RA, Tibboel D. 2003a. Do we still hurt newborn babies? A prospective study of procedural pain and analgesia in neonates. Arch Pediatr Adolesc Med 157(11):1058-64. Simons SH, van Dijk M, van Lingen RA, Roofthooft D, Duivenvoorden HJ, Jongeneel N, Bunkers C, Smink E, Anand KJ, van den Anker JN, et al. 2003b. Routine morphine infusion in preterm newborns who received ventilatory support: A randomized controlled trial. JAMA 290(18):2419-27. Singer LT, Fulton S, Davillier M, Koshy D, Salvator A, Baley JE. 2003. Effects of infant risk status and maternal psychological distress on maternal-infant interactions during the first year of life. J Dev Behav Pediatr 24(4):233-41. Sizonenko SV, Kiss JZ, Inder T, Gluckman PD, Williams CE. 2005. Distinctive neuropathologic alterations in the deep layers of the parietal cortex after moderate ischemic-hypoxic injury in the P3 immature rat brain. Pediatr Res 57(6):865-72. Sizonenko SV, Sirimanne E, Mayall Y, Gluckman PD, Inder T, Williams C. 2003. Selective cortical alteration after hypoxic-ischemic injury in the very immature rat brain. Pediatr Res 54(2):263-9. Sizonenko SV, Camm EJ, Garbow JR, Maier SE, Inder TE, Williams CE, Neil JJ, Huppi PS. 2007. Developmental changes and injury induced disruption of the radial organization of the cortex in the immature rat brain revealed by in vivo diffusion tensor MRI. Cereb Cortex 17(11):2609-17. Slater L, Asmerom Y, Boskovic DS, Bahjri K, Plank MS, Angeles KR, Phillips R, Deming D, Ashwal S, Hougland K, et al. 2012. Procedural pain and oxidative stress in premature neonates. J Pain 13(6):590-7. Slater R, Fabrizi L, Worley A, Meek J, Boyd S, Fitzgerald M. 2010a. Premature infants display increased noxious-evoked neuronal activity in the brain compared to healthy age-matched 	   186 term-born infants. Neuroimage 52(2):583-9. Slater R, Cantarella A, Yoxen J, Patten D, Potts H, Meek J, Fitzgerald M. 2009. Latency to facial expression change following noxious stimulation in infants is dependent on postmenstrual age. Pain 146(1-2):177-82. Slater R, Cantarella A, Gallella S, Worley A, Boyd S, Meek J, Fitzgerald M. 2006. Cortical pain responses in human infants. J Neurosci 26(14):3662-6. Slater R, Cornelissen L, Fabrizi L, Patten D, Yoxen J, Worley A, Boyd S, Meek J, Fitzgerald M. 2010b. Oral sucrose as an analgesic drug for procedural pain in newborn infants: A randomised controlled trial. Lancet 376(9748):1225-32. Slotkin TA, Barnes GA, McCook EC, Seidler FJ. 1996. Programming of brainstem seretonin transporter development by prenatal glucocorticoids. Brain Res Dev Brain Res 93(1-2):155-61. Smart JL, Dobbing J, Adlard BP, Lynch A, Sands J. 1973. Vulnerability of developing brain: Relative effects of growth restriction during the fetal and suckling periods on behavior and brain composition of adult rats. J Nutr 103(9):1327-38. Smith GC, Gutovich J, Smyser C, Pineda R, Newnham C, Tjoeng TH, Vavasseur C, Wallendorf M, Neil J, Inder T. 2011. Neonatal intensive care unit stress is associated with brain development in preterm infants. Ann Neurol 70(4):541-9. Smyser TA, Smyser CD, Rogers CE, Gillespie SK, Inder TE, Neil JJ. 2015. Cortical Gray and Adjacent White Matter Demonstrate Synchronous Maturation in Very Preterm Infants. Cereb Cortex. pii: bhv164.  Song SK, Sun SW, Ramsbottom MJ, Chang C, Russell J, Cross AH. 2002. Dysmyelination revealed through MRI as increased radial (but unchanged axial) diffusion of water. Neuroimage 17(3):1429-36. Spittle AJ, Anderson PJ, Lee KJ, Ferretti C, Eeles A, Orton J, Boyd RN, Inder T, Doyle LW. 2010. Preventive care at home for very preterm infants improves infant and caregiver outcomes at 2 years. Pediatrics 126(1):e171-8. Spittle AJ, Treyvaud K, Doyle LW, Roberts G, Lee KJ, Inder TE, Cheong JL, Hunt RW, Newnham CA, Anderson PJ. 2009. Early emergence of behavior and social-emotional problems in very preterm infants. J Am Acad Child Adolesc Psychiatry 48(9):909-18. Srinivasan L, Dutta R, Counsell SJ, Allsop JM, Boardman JP, Rutherford MA, Edwards AD. 2007. Quantification of deep gray matter in preterm infants at term-equivalent age using manual volumetry of 3-tesla magnetic resonance images. Pediatrics 119(4):759-65. Starr-Phillips EJ and Beery AK. 2014. Natural variation in maternal care shapes adult social behavior in rats. Dev Psychobiol 56(5):1017-26. 	   187 Stern M, Karraker K, McIntosh B, Moritzen S, Olexa M. 2006. Prematurity stereotyping and mothers' interactions with their premature and full-term infants during the first year. J Pediatr Psychol 31(6):597-607. Stevens B, Yamada J, Lee GY, Ohlsson A. 2013. Sucrose for analgesia in newborn infants undergoing painful procedures. Cochrane Database Syst Rev 1:CD001069. Stevens B, Yamada J, Beyene J, Gibbins S, Petryshen P, Stinson J, Narciso J. 2005. Consistent management of repeated procedural pain with sucrose in preterm neonates: Is it effective and safe for repeated use over time? Clin J Pain 21(6):543-8. Stevens B, Franck L, Gibbins S, McGrath PJ, Dupuis A, Yamada J, Beyene J, Camfield C, Finley GA, Johnston C, et al. 2007. Determining the structure of acute pain responses in vulnerable neonates. Can J Nurs Res 39(2):32-47. Stevens B, McGrath P, Gibbins S, Beyene J, Breau L, Camfield C, Finley A, Franck L, Howlett A, McKeever P, et al. 2003. Procedural pain in newborns at risk for neurologic impairment. Pain 105(1-2):27-35. Stevens B, Johnston CC, Petryshen P, Taddio A. 1996. Premature infant pain profile: development and initial validation. Clin J Pain 12:13–22. Stevens BJ, Johnston CC, Horton L. 1994. Factors that influence the behavioral pain responses of premature infants. Pain 59(1):101-9. Stevens RT, London SM, Apkarian AV. 1993. Spinothalamocortical projections to the secondary somatosensory cortex (SII) in squirrel monkey. Brain Res 631(2): 241–6. Steward DK and Pridham KF. 2002. Growth patterns of extremely low-birth-weight hospitalized preterm infants. J Obstet Gynecol Neonatal Nurs 31(1):57-65. Stoll BJ, Hansen NI, Adams-Chapman I, Fanaroff AA, Hintz SR, Vohr B, Higgins RD, National Institute of Child Health and Human Development Neonatal Research Network. 2004. Neurodevelopmental and growth impairment among extremely low-birth-weight infants with neonatal infection. JAMA 292(19):2357-65. Strunk T, Inder T, Wang X, Burgner D, Mallard C, Levy O. 2014. Infection-induced inflammation and cerebral injury in preterm infants. Lancet Infect Dis 14(8):751-62. Synnes AR, Anson S, Arkesteijn A, Butt A, Grunau RE, Rogers M, Whitfield MF. 2010. School entry age outcomes for infants with birth weight </= 800 grams. J Pediatr 157(6):989,994.e1. Taddio A, Yiu A, Smith RW, Katz J, McNair C, Shah V. 2009. Variability in clinical practice guidelines for sweetening agents in newborn infants undergoing painful procedures. Clin J Pain 25(2):153-5. Talos DM, Follett PL, Folkerth RD, Fishman RE, Trachtenberg FL, Volpe JJ, Jensen FE. 2006. 	   188 Developmental regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit expression in forebrain and relationship to regional susceptibility to hypoxic/ischemic injury. II. human cerebral white matter and cortex. J Comp Neurol 497(1):61-77. Tam EW, Chau V, Ferriero DM, Barkovich AJ, Poskitt KJ, Studholme C, Fok ED, Grunau RE, Glidden DV, Miller SP. 2011. Preterm cerebellar growth impairment after postnatal exposure to glucocorticoids. Sci Transl Med 3(105):105ra105. Thomas KA, Renaud MT, Depaul D. 2004. Use of the parenting stress index in mothers of preterm infants. Adv Neonatal Care 4(1):33-41. Thompson DK, Lee KJ, Egan GF, Warfield SK, Doyle LW, Anderson PJ, Inder TE. 2014. Regional white matter microstructure in very preterm infants: Predictors and 7 year outcomes. Cortex 52:60-74. Thompson DK, Inder TE, Faggian N, Warfield SK, Anderson PJ, Doyle LW, Egan GF. 2012. Corpus callosum alterations in very preterm infants: Perinatal correlates and 2 year neurodevelopmental outcomes. Neuroimage 59(4):3571-81. Thompson DK, Inder TE, Faggian N, Johnston L, Warfield SK, Anderson PJ, Doyle LW, Egan GF. 2011. Characterization of the corpus callosum in very preterm and full-term infants utilizing MRI. Neuroimage 55(2):479-90. Thompson DK, Warfield SK, Carlin JB, Pavlovic M, Wang HX, Bear M, Kean MJ, Doyle LW, Egan GF, Inder TE. 2007. Perinatal risk factors altering regional brain structure in the preterm infant. Brain 130(Pt 3):667-77. Toft PB, Leth H, Ring PB, Peitersen B, Lou HC, Henriksen O. 1995. Volumetric analysis of the normal infant brain and in intrauterine growth retardation. Early Hum Dev 43(1):15-29. Tolcos M, Bateman E, O'Dowd R, Markwick R, Vrijsen K, Rehn A, Rees S. 2011. Intrauterine growth restriction affects the maturation of myelin. Exp Neurol 232(1):53-65. Tolsa CB, Zimine S, Warfield SK, Freschi M, Sancho Rossignol A, Lazeyras F, Hanquinet S, Pfizenmaier M, Huppi PS. 2004. Early alteration of structural and functional brain development in premature infants born with intrauterine growth restriction. Pediatr Res 56(1):132-8. Tracey I, Ploghaus A, Gati JS, Clare S, Smith S, Menon RS, Matthews PM. 2002. Imaging attentional modulation of pain in the periaqueductal gray in humans. J Neurosci 22(7):2748-52. Trang T, Beggs S, Salter MW. 2011. Brain-derived neurotrophic factor from microglia: a molecular substrate for neuropathic pain. Neuron Glia Biol 7:99–108. Traub RJ, Mendell LM. 1988. The spinal projection of individual identified A-delta- and C-	   189 fibers. J Neurophysiol 59(1):41-55.  Traystman RJ, Kirsch JR, Koehler RC. 1991. Oxygen radical mechanisms of brain injury following ischemia and reperfusion. J Appl Physiol (1985) 71(4):1185-95. Treyvaud K, Lee KJ, Doyle LW, Anderson PJ. 2014. Very preterm birth influences parental mental health and family outcomes seven years after birth. J Pediatr 164(3):515-21. Tsigos C and Chrousos GP. 2002. Hypothalamic-pituitary-adrenal axis, neuroendocrine factors and stress. J Psychosom Res 53(4):865-71. Tsigos C, Papanicolaou DA, Defensor R, Mitsiadis CS, Kyrou I, Chrousos GP. 1997. Dose effects of recombinant human interleukin-6 on pituitary hormone secretion and energy expenditure. Neuroendocrinology 66(1):54-62. Tu MT, Grunau RE, Petrie-Thomas J, Haley DW, Weinberg J, Whitfield MF. 2007. Maternal stress and behavior modulate relationships between neonatal stress, attention, and basal cortisol at 8 months in preterm infants. Dev Psychobiol 49(2):150-64. Uno H, Lohmiller L, Thieme C, Kemnitz JW, Engle MJ, Roecker EB, Farrell PM. 1990. Brain damage induced by prenatal exposure to dexamethasone in fetal rhesus macaques. I. hippocampus. Brain Res Dev Brain Res 53(2):157-67. Valeri BO, Gaspardo CM, Martinez FE, Linhares MB. 2012. Does the neonatal clinical risk for illness severity influence pain reactivity and recovery in preterm infants? Eur J Pain 16(5):727-36. Valet M, Sprenger T, Boecker H, Willoch F, Rummeny E, Conrad B, Erhard P, Tolle TR. 2004. Distraction modulates connectivity of the cingulo-frontal cortex and the midbrain during pain – An fMRI analysis. Pain 109(3):399-408. van de Lagemaat M, Rotteveel J, Heijboer AC, Lafeber HN, van Weissenbruch MM. 2013. Growth in preterm infants until six months postterm: The role of insulin and IGF-I. Horm Res Paediatr 80(2):92-9. van Hasselt FN, Cornelisse S, Zhang TY, Meaney MJ, Velzing EH, Krugers HJ, Joels M. 2012. Adult hippocampal glucocorticoid receptor expression and dentate synaptic plasticity correlate with maternal care received by individuals early in life. Hippocampus 22(2):255-66. Van Kooij BJ, Benders MJ, Anbeek P, Van Haastert IC, De Vries LS, Groenendaal F. 2012. Cerebellar volume and proton magnetic resonance spectroscopy at term, and neurodevelopment at 2 years of age in preterm infants. Dev Med Child Neurol 54(3):260-6. van Praag H and Frenk H. 1991. The development of stimulation-produced analgesia (SPA) in the rat. Brain Res Dev Brain Res 64(1-2):71-6. Vasung L, Huang H, Jovanov-Milosevic N, Pletikos M, Mori S, Kostovic I. 2010. Development 	   190 of axonal pathways in the human fetal fronto-limbic brain: Histochemical characterization and diffusion tensor imaging. J Anat 217(4):400-17. Victoria NC, Inoue K, Young LJ, Murphy AZ. 2013. Long-term dysregulation of brain corticotrophin and glucocorticoid receptors and stress reactivity by single early-life pain experience in male and female rats. Psychoneuroendocrinology 38(12):3015-28. Villescas R, Ostwald R, Morimoto H, Bennett EL. 1981. Effects of neonatal undernutrition and cold stress on behavior and biochemical brain parameters in rats. J Nutr 111(6):1103-10. Vinall J, Miller SP, Chau V, Brummelte S, Synnes AR, Grunau RE. 2012. Neonatal pain in relation to postnatal growth in infants born very preterm. 153(7):1374-81. Vinall J, Zwicker JG, Chau V, Poskitt KJ, Brant R, Synnes AR, Miller SP, Grunau RE. 2014a. Early neonatal pain exposure and brain microstructure interact to predict neurodevelopmental outcomes at 18 months corrected age in children born very preterm. Pediatric Academic Societies (E-PAS2014:2855.2). Vinall J, Miller SP, Synnes AR, Grunau RE. 2013a. Parent behaviors moderate the relationship between neonatal pain and internalizing behaviors at 18 months corrected age in children born very prematurely. Pain 154(9):1831-9. Vinall J, Grunau RE, Brant R, Chau V, Poskitt KJ, Synnes AR, Miller SP. 2013b. Slower postnatal growth is associated with delayed cerebral cortical maturation in preterm newborns. Sci Transl Med 5(168):168ra8. Vinall J, Miller SP, Bjornson BH, Fitzpatrick KP, Poskitt KJ, Brant R, Synnes AR, Cepeda IL, Grunau RE. 2014b. Invasive procedures in preterm children: Brain and cognitive development at school age. Pediatrics 133(3):412-21. Voigt B, Brandl A, Pietz J, Pauen S, Kliegel M, Reuner G. 2013. Negative reactivity in toddlers born prematurely: Indirect and moderated pathways considering self-regulation, neonatal distress and parenting stress. Infant Behav Dev 36(1):124-38. Volpe JJ. 2009. Brain injury in premature infants: A complex amalgam of destructive and developmental disturbances. Lancet Neurol 8(1):110-24. Wager TD, Atlas LY, Lindquist MA, Roy M, Woo CW, Kross E. 2013. An fMRI-based neurologic signature of physical pain. N Engl J Med 368(15):1388-97. Walker SM, Tochiki KK, Fitzgerald M. 2009. Hindpaw incision in early life increases the hyperalgesic response to repeat surgical injury: Critical period and dependence on initial afferent activity. Pain 147(1-3):99-106. Watkins LR and Maier SF. 2003. Glia: A novel drug discovery target for clinical pain. Nat Rev Drug Discov 2(12):973-85. Weaver IC, D'Alessio AC, Brown SE, Hellstrom IC, Dymov S, Sharma S, Szyf M, Meaney MJ. 	   191 2007. The transcription factor nerve growth factor-inducible protein a mediates epigenetic programming: Altering epigenetic marks by immediate-early genes. J Neurosci 27(7):1756-68. Weaver IC, Cervoni N, Champagne FA, D'Alessio AC, Sharma S, Seckl JR, Dymov S, Szyf M, Meaney MJ. 2004. Epigenetic programming by maternal behavior. Nat Neurosci 7(8):847-54. Weschler D. 2003. WISC-IV: Administration and scoring manual. San Antonio, TX: Psychological Corporation. Wigger A and Neumann ID. 1999. Periodic maternal deprivation induces gender-dependent alterations in behavioral and neuroendocrine responses to emotional stress in adult rats. Physiol Behav 66(2):293-302. Wikstrom S, Ley D, Hansen-Pupp I, Rosen I, Hellstrom-Westas L. 2008. Early amplitude-integrated EEG correlates with cord TNF-alpha and brain injury in very preterm infants. Acta Paediatr 97(7):915-9. Williams AL, Khattak AZ, Garza CN, Lasky RE. 2009. The behavioral pain response to heelstick in preterm neonates studied longitudinally: Description, development, determinants, and components. Early Hum Dev 85(6):369-74. Williams G, Fabrizi L, Meek J, Jackson D, Tracey I, Robertson N, Slater R, Fitzgerald M. 2014. Functional magnetic resonance imaging can be used to explore tactile and nociceptive processing in the infant brain. Acta Paediatr . Willis WD and Westlund KN. 1997. Neuroanatomy of the pain system and of the pathways that modulate pain. J Clin Neurophysiol 14(1):2-31. Wilson DC, Cairns P, Halliday HL, Reid M, McClure G, Dodge JA. 1997. Randomised controlled trial of an aggressive nutritional regimen in sick very low birthweight infants. Arch Dis Child Fetal Neonatal Ed 77(1):F4-11. Winston JS, Vlaev I, Seymour B, Chater N, Dolan RJ. 2014. Relative valuation of pain in human orbitofrontal cortex. J Neurosci 34(44):14526-35. Woodward LJ, Anderson PJ, Austin NC, Howard K, Inder TE. 2006. Neonatal MRI to predict neurodevelopmental outcomes in preterm infants. N Engl J Med 355(7):685-94. Woodward LJ, Bora S, Clark CA, Montgomery-Honger A, Pritchard VE, Spencer C, Austin NC. 2014. Very preterm birth: Maternal experiences of the neonatal intensive care environment. J Perinatol 34(7):555-61. Woolf CJ. 2011. Central sensitization: Implications for the diagnosis and treatment of pain. Pain 152(3 Suppl):S2-15. Woolf CJ, Costigan M. 1999. Transcriptional and posttranslational plasticity and the generation 	   192 of inflammatory pain. PNAS 96(14):7723-30. Woolf CJ, Salter MW. 2000. Neuronal plasticity: increasing the gain to pain. Science 288:1765-9. Xia C, Yang L, Zhao P, Zhang X. 2002. Response to pain by different gestational age neonates. J Huazhong Univ Sci Technolog Med Sci 22(1):84-6. Yamamoto A, Yokoyama N, Yonetan M, Uetani Y, Nakamura H, Nakao H. 2003. Evaluation of change of cerebral circulation by SpO2 on preterm infants with apneic episodes using near infrared spectroscopy. Pediatr Int 45:661-4. Yamini R, Chau V, Brant R, Synnes A, Belanger S, Poskitt KJ, Miller SP. 2010. Measures of growth in premature newborns: Relationship with brain development and white matter injury. Pediatric Academic Societies E-PAS2010:3746.498. Yau JL, Noble J, Widdowson J, Seckl JR. 1997. Impact of adrenalectomy on 5-HT6 and 5-HT7 receptor gene expression in the rat hippocampus. Brain Res Mol Brain Res 45(1):182-6. Younger JB, Kendell MJ, Pickler RH. 1997. Mastery of stress in mothers of preterm infants. J Soc Pediatr Nurs 2(1):29-35. Yumani DF, Lafeber HN, van Weissenbruch MM. 2015. Dietary proteins and IGF I levels in preterm infants: Determinants of growth, body composition, and neurodevelopment. Pediatr Res 77(1-2):156-63. Yung A, Poon G, Qiu DQ, Chu J, Lam B, Leung C, Goh W, Khong PL. 2007. White matter volume and anisotropy in preterm children: A pilot study of neurocognitive correlates. Pediatr Res 61(6):732-6. Zelkowitz P, Papageorgiou A, Bardin C, Wang T. 2009. Persistent maternal anxiety affects the interaction between mothers and their very low birthweight children at 24 months. Early Hum Dev 85(1):51-8. Zelkowitz P, Na S, Wang T, Bardin C, Papageorgiou A. 2011. Early maternal anxiety predicts cognitive and behavioural outcomes of VLBW children at 24 months corrected age. Acta Paediatr 100(5):700-4. Zhang TY, Bagot R, Parent C, Nesbitt C, Bredy TW, Caldji C, Fish E, Anisman H, Szyf M, Meaney MJ. 2006. Maternal programming of defensive responses through sustained effects on gene expression. Biol Psychol 73(1):72-89. Zhang ZW, Peterson M, Liu H. 2013. Essential role of postsynaptic NMDA receptors in developmental refinement of excitatory synapses. Proc Natl Acad Sci U S A 110(3):1095-100. Zwicker JG, Miller SP, Grunau RG, Chau V, Synnes A, Poskitt KJ, Tam EW. 2012. Morphine exposure is associated with altered cerebellar growth in premature newborns. Pediatric 	   193 Academic Societies E-PAS2012:1060. Zwicker JG, Grunau RE, Adams E, Chau V, Brant R, Poskitt KJ, Synnes A, Miller SP. 2013. Score for neonatal acute physiology-II and neonatal pain predict corticospinal tract development in premature newborns. Pediatr Neurol 48(2):123,129.e1.           	    	   194 APPENDIX Table A.1 Invasive Procedures in the Neonatal Intensive Care Unit   Injections* Umbilical artery catheter insertion Chest tube insertion* Umbilical venous catheter insertion Pleural tap* Lumbar puncture reservoir tap* Peripheral artery line insertion* Brainz needle insertion* Peripherally inserted central line insertion/removal* Heel poke (including glucometer pokes)* Penrose insertion/removal* Suprapubic bladder tap* Abscess drained* Catheter insertion for urine collection Peripheral intravenous sited or re-sited* Venous blood collection* Endotracheal tube prong change or re-taping Glycerin suppository Nasogastric tube insertion Orogastric tube insertion Healon/wydase for intravenous burns Insuflon device site change* Pericentesis* Endotracheal or nasopharyngeal intubation* Eye exam  Each attempt was counted  *Skin-breaking procedures  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.24.1-0166783/manifest

Comment

Related Items