Effects Of Neonatal Repetitive Procedural Painonbehavioral Development And Its Mechanism Through The Regulation Of Hypothalamic-pituitary-adrenal Axis

Pain is referred as an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage. With medical and technological progress over the past two decades, the survival of infants born extremely premature has increased substantially. As part of their life-saving care, these infants are often exposed to unhandled repetitive invasive procedures, causing acute pain in the neonatal period. Exposing to repetitive and prolonged procedure pain can not only alter pain responsiveness, impair the behavioral development, cause emotional and cognitive abnormity, but also can become heavy burden to the family and society. So it has been a great challenge to explore the impacts of neonatal repetitive procedural pain on children health and underlying mechanism as well as improve the perceptions of pain managementamong pediatrician and nurses, thus to establish neonatal pain management guideline in China.In children population, the behavioral development parallels with the neuropsychological development. Since the behavioraldevelopmentis a reflection of maturation of central nervous system especially brain, the structure and function of brain can be affected by various biological and environmentalfactors. Early period of life (usually the first1000days from a embryo) is a critical window for brain development, which is of great plasticity. The central nervous system is more vulnerable for preterm infants. Early repetitive procedural pain can alter the early programming of hypothalamic-pituitary-adrenal axis (HPA), cause basal and stress hormones like glucocorticoid level permanently increased or decreased. The glucocorticoid receptor (GR) in hippocampus, which plays an important role in cognition, learning and memory, regulates as a central negative feedback of HPA axis. As a result, the programming of HPA axis is coupled with the abnormal development of behavior, cognition, stress and endocrine system, which is proved by the evidence that early child abuse, maternal separation, low maternal care and perinatal stress cause permanent modification of phenotype in adulthood. However, the effect of neonatalrepetitive procedural pain on the GR expression and HPA regulation is not yet clear.Given a large amount of neonatalprocedural pain occurs in the critical time of brain development, in our study, we aim to investigate the effects of neonatal pain on children behavioral development and underlying mechanism. We observed the impacts of neonatal procedural pain on the infants pain sensitivity, behavioral development and circulating glucocorticoid levels and also used the animal model to analyze the behavioral alternation and programming of HPA axis after neonatal repetitive needlestick pain exposure in pubertal and adult rats. The findings help to enhance the medical personnel’s perceptions regarding long-term consequences of pain in human neonates, thus providing scientific evidences for early prevention and intervene of neonatal pain. Section OneEffects of neonatal repetitive procedural painonchildren behavioral development and cortisol levelPart Ⅰ Effects of neonatal repetitive procedural painonbehavioral development and cortisol level in preterm neonatesObjectiveTo examine the effects of early pain experience on the subsequent pain responses and plasma cortisol levels in preterm infants.MethodsWe prospectively collected data of all painful procedures performed on the total57preterm neonates (male27, female30) from their admission to discharge in a neonatal intensive care unit (NICU) of a university-affiliated hospital in China. Facial and behavioral responses during painful procedures were observed by digital video recording and cardiac activities were obtained by bedside monitor. The pain scores were rated by Neonatal Facial Coding System (NFCS) and Premature Infant Pain Profile (PIPP) respectively.Morning plasma cortisol levels werecompared at admission and two weeks later. Results1. During hospitalization, each preterm was subjected to a median of104.0painful procedures. After exposure to above40times of needlestick procedures, the pain responsivenessduring the procedure for preterm infant were significantly enhanced rated by PIPP and NFCS scores compared to first time of needlestick (PIPP: P40=0.035; NFCS:P40=0.046), accompanied by significantly elevated NFCS scores at preparation phases (P40=0.003).2. The plasma cortisol levels were significantly decreased after two-weeks’ hospitalization (166.3nmol/L,34.6-595.3nmol/L) than admission (306.2nmol/L,39.1-1750.0nmol/L,P=0.021) and were negatively associated with cumulative pain experience during hospitalization (r=-0.756, P<0.001).ConclusionRepetitive procedural pain in NICUcauses irritation during the preparing phase preceding pain and hyperalgesia during subsequent painful procedures and predicts lowerplasma cortisol level in preterm neonates during hospitalization. Part Ⅱ Effects of neonatal repetitive procedural pain on behavioral development and cortisol response in preterm toddlersObjectiveTo observethe behavioral development and saliva cortisol patterns in preterm toddler with former NICU pain experience, as well as the interaction of parenting environment.MethodsParticipants were selectedfrom preterm neonates who admitted to NICU in Nanjing Children Hospital affiliated to Nanjing Medical University after birth, seen as Preterm Group (n=25). Full-term Group (n=38) was chosen from term neonates who were health born at the same period compared with Preterm Group and without hospitalization experience. When follow-up at2years postconception age (PCA), the parents fill in the questionnaire made by us, including characteristics of children and parents, Parenting Stress Index-short form (PSI) and Child Behavior Check List (CBCL); while the toddler were underwent physical measurement, von Frey mechanical test, Gesell development Schedule(GDS) and cortisol detection. Medical chart during NICU stay for Preterm Group were reviewed carefully for correlation analysis.Results1. Preterm Group has comparable height, body weight and head circumference with Full-term Group at2years PCA(P>0.05). However the von Frey mechanical withdraw threshold of Preterm Group was significantly lower than Full-term Group (P<0.05), although the parent rating of pain hypersensitivity were in no difference between the two groups at2years PCA (P=0.136).2. The PSI of Preterm group mother was significantly higher than Full-term Group mother, manifested by Parenting anxiety and Parent-child poor interaction(P<0.05).3. The development quotients (DQ) of Gross Motor, Fine Motor, Adaptive, LanguageandPersonal-Social ofGDS in Preterm Group were significantly decreased than Full-term Groupat2years PCA (P<0.05). Poor cognitive development of Preterm Group was related to the increasing number of NICU skin-breaking procedures they’ve been experience (P<0.05) and their mother’s PSI score (P<0.05) and decreased maternal education time (P<0.05).4. The internalizing behavior score manifested by Withdrawn Behaviors and Depression by parent rating CBCL were significantly increased in Preterm Group compared with Full-term Group at2years PCA (P<0.05). The alternation of internalizing behavior in Preterm Groupwas positively correlated with PSI (P<0.05), and negatively correlated with Fine Motor DQ (P<0.05).5. The saliva cortisol response pattern to novelty and cognition challenge in Preterm Group were significantly different from Full-term Group at2years PCA (P<0.05) and the alternation were related to their former NICU pain experience (P<0.05) rather than maternal factors (P>0.05).ConclusionCompared with full-term counterparts, the preterm born toddler exhibit mechanical hypersensitivity, lower cognitive development, increased internalizing behaviors and abnormal saliva cortisol response to novelty and cognition challenge at2years PCA. The alternation of cognition and cortisol pattern in preterm born toddlers are related to the number of skin-breaking procedures experienced during their NICU hospitalization. What’s more, longer time of maternal education can buffer the decrease of cognition for preterm toddlers. SectionTwoEffects of neonatal repetitive procedural pain on long-term behavioral alternation through dysregulation of the HPA axisObjectiveUse animal models to examine the effects of neonatal repetitive procedural pain onbehavioral development in prepubertal and adult rats and the regulation of HPAaxis function.MethodsSPF pregnant Sprague-Dawley (SD) rats were purchased. Male rat pups were randomly assigned to either group. Neonatal ratpups were stimulated four times each day onpostnatal days0-7(P0-P7), by either needlestick(Needle group) or cotton-tipped swab(Tactile group). Mechanical sensitivity was tested by the von Frey mechanical test. All subjects were tested on P24and P87for behavioral tests such as Morris water maze (MWM) for spatial learning and memory, as well as Open field (OF), Tail suspension test and Sucrose preference test for anxiety-and depression-like behavior. Biological, molecular and immunohistochemical test were analyzed on P24, P45,P59,and P87.Results1. No difference was found in body weight growth tendency between the Needle and Tactile groups during the experimental period (P>0.05).From P8to P85, mechanical hypersensitivity of the bilateral hindpaws were observed inthe Needle group than the Tactile group (P<0.001).2. The latency to hidden platform didn’t differ between the Needle and Tactile group either on P25-P29or on P88-P92inthe acquisition trials of MWM (P>0.05). However, the Needle group exhibited faster swim speed compared with the Tactile group on P25-P29(P<0.05). It took the Needle group more time to find the platform on the probe trial day than on the final day of training, as well as a decreased number of original platform site crossings(P<0.05) and poor search strategy when compared with the Tactile group on P30. There were no such differences between the two groups on probe trial day on P93.3. The distance in inner and outer area, the speed in outer area and number in inner area were significantly increased in theNeedle group than the Tactile group on P24in theOF (P<0.05). Also the duration and distance in inner area, number and speed both in inner and outer area were significantly increased in theNeedle group compared with the Tactile group on P87in the OF (P<0.05). What’s more, the immobility time for the Needle group was significantly longer than the Tactile group both on P24and P87in the Tail suspension test (P<0.05). However, there was no difference between the two groups for sucrose consumption portion in the Sucrose preference test either on P24or P87(P<0.05).4. Theserum corticosterone level after30minutes exposure to the OF stressor for the Tactile group were at peak during P24and P59than other age (P<0.05); while no such tendency were found in Needle group, with a decreased corticosterone response to stressor during this period (P<0.05), accompanied by the decreased serum adrenocorticotropic hormone (ACTH) response on P24(P<0.05). In contrast, the serum corticosterone responsiveness to stress was significantly increased in the Needle group compared with the Tactile group on P87(P<0.05).5. Compared to the Tactile group, the proportions of bilateral adrenals weight to body weight were significantly decreased in the Needle group on P59(P<0.05) and significantly increased on P87(P<0.01). 6. The hippocampal GRmRNA and protein expression as well as mean optical density of GR in CA1were significantly increased in the Needle group compared to the Tactile group on P24and P45(P<0.05), but decreased on P59and P87(P<0.05).ConclusionNeonatal repetitive procedural pain causes persistent mechanical hypersensitivity, impairs spatial learning ability, and persistent anxiety-and depression-like behavior from prepuberty to adulthood, along with early programming and dysregulation of the HPAaxis.