Sources of heterogeneity when studying the cardiovascular effects of fetal growth restriction: an overview of the issues.

Intrauterine growth restriction (IUGR) has been repeatedly identified as a risk factor for cardiovascular disease (CVD). A possible explanation for this association is the effect of IUGR on cardiovascular structure and function. However, the specific changes observed are not consistent among studies. In this paper, we analyze several sources of heterogeneity within and between studies related to exposure, outcome and co-variables. A broad IUGR definition might include different phenotypes, expressing heterogeneity as an outcome. Outcome heterogeneity may also be the result of the postnatal effect modification that can be explored within studies. In order to do so, it is important to move beyond mean differences between groups, for example using unsupervised, stratified or interaction analysis. Different definitions of IUGR and the inclusion of different postnatal variables as confounders are potential sources of heterogeneity between studies. Researchers should be aware that postnatal variables may play different roles throughout a person's life and are not limited to behave as confounders. Therefore, their inclusion in the statistical model needs to be carefully considered. We discuss when sources of heterogeneity need to be controlled, and when they need to be identified and shown as a result.

Neonatal Invasive Procedures Predict Pain Intensity at School Age in Children Born Very Preterm.

INTRODUCTION: Children born very preterm display altered pain thresholds. Little is known about the neonatal clinical and psychosocial factors associated with their later pain perception. OBJECTIVE: We aimed to examine whether the number of neonatal invasive procedures, adjusted for other clinical and psychosocial factors, was associated with self-ratings of pain during a blood collection procedure at school age in children born very preterm. MATERIALS AND METHODS: 56 children born very preterm (24 to 32 weeks gestational age), followed longitudinally from birth, and free of major neurodevelopmental impairments underwent a blood collection by venipuncture at age 7.5 years. The children's pain was self-reported using the Coloured Analog Scale and the Facial Affective Scale. Parents completed the Child Behavior Checklist and the State-Trait Anxiety Inventory. Pain exposure (the number of invasive procedures) and clinical factors from birth to term-equivalent age were obtained prospectively. Multiple linear regression was used to predict children's pain self-ratings from neonatal pain exposure after adjusting for neonatal clinical and concurrent psychosocial factors. RESULTS: A greater number of neonatal invasive procedures and higher parent trait-anxiety were associated with higher pain intensity ratings during venipuncture at age 7.5 years. Fewer surgeries and lower concurrent child externalizing behaviors were associated with a higher pain intensity. CONCLUSIONS: In very preterm children, exposure to neonatal pain was related to altered pain self-ratings at school age, independent of other neonatal factors. Neonatal surgeries and concurrent psychosocial factors were also associated with pain ratings.

[Formula: see text]Higher cortisol is associated with poorer executive functioning in preschool children: The role of parenting stress, parent coping and quality of daycare.

Child executive functions (cognitive flexibility, inhibitory control, working memory) are key to success in school. Cortisol, the primary stress hormone, is known to affect cognition; however, there is limited information about how child cortisol levels, parenting factors and child care context relate to executive functions in young children. The aim of this study was to examine relationships between child cortisol, parenting stress, parent coping, and daycare quality in relation to executive functions in children aged 3-5 years. We hypothesized that (1) poorer executive functioning would be related to higher child cortisol and higher parenting stress, and (2) positive daycare quality and positive parent coping style would buffer the effects of child cortisol and parenting stress on executive functions. A total of 101 children (53 girls, 48 boys, mean age 4.24 years ±0.74) with complete data on all measures were included. Three saliva samples to measure cortisol were collected at the child's daycare/preschool in one morning. Parents completed the Behavior Rating Inventory of Executive Function - Preschool Version (BRIEF-P), Parenting Stress Index (PSI), and Ways of Coping Questionnaire (WCQ). The Early Childhood Environment Rating Scale - Revised (ECERS-R) was used to measure the quality of daycare. It was found that children with poorer executive functioning had higher levels of salivary cortisol, and their parents reported higher parenting stress. However, parent coping style and quality of daycare did not modulate these relationships. Identifying ways to promote child executive functioning is an important direction for improving school readiness.

Stress in parents of children born very preterm is predicted by child externalising behaviour and parent coping at age 7†years.

OBJECTIVE: To examine factors which predict parenting stress in a longitudinal cohort of children born very preterm, and seen at age 7 years. METHODS: We recruited 100 very preterm (≤32 weeks gestational age) child-parent dyads and a control group of 50 term-born dyads born between 2001 and 2004 with follow-up at 7 years. Parents completed the Parenting Stress Index, Ways of Coping Questionnaire, Child Behavior Check List, Beck Depression Inventory and the State Trait Anxiety Inventory questionnaires. Child IQ was assessed using the Wechsler Intelligence Scale-IV. RESULTS: After controlling for maternal education, parents of preterm children (95% CI 111.1 to 121.4) scored higher (p=0.027) on the Parenting Stress Index than term-born controls (95% CI 97.8 to 113.2). Regression analyses showed that child externalising behaviour, sex and parent escape/avoidance coping style, predicted higher parenting stress in the preterm group. Parents of preterm girls expressed higher levels of stress than those of boys. CONCLUSIONS: Maladaptive coping strategies contribute to greater stress in parents of very preterm children. Our findings suggest that these parents need support for many years after birth of a very preterm infant.

Cortisol levels in former preterm children at school age are predicted by neonatal procedural pain-related stress.

Early life stress can alter hypothalamic pituitary adrenal (HPA) axis function. Differences in cortisol levels have been found in preterm infants exposed to substantial procedural stress during neonatal intensive care, compared to infants born full-term, but only a few studies investigated whether altered programming of the HPA axis persists past toddler age. Further, there is a dearth of knowledge of what may contribute to these changes in cortisol. This prospective cohort study examined the cortisol profiles in response to the stress of cognitive assessment, as well as the diurnal rhythm of cortisol, in children (n=129) born at varying levels of prematurity (24-32 weeks gestation) and at full-term (38-41 weeks gestation), at age 7 years. Further, we investigated the relationships among cortisol levels and neonatal procedural pain-related stress (controlling for multiple medical confounders), concurrent maternal factors (parenting stress, depressive and anxiety symptoms) and children's behavioral problems. For each aim we investigate acute cortisol response profiles to a cognitive challenge as well as diurnal cortisol patterns at home. We hypothesized that children born very preterm will differ in their pattern of cortisol secretion from children born full-term, possibly depended on concurrent child and maternal factors, and that exposure to neonatal pain-related stress would be associated with altered cortisol secretion in children born very preterm, possibly in a sex-dependent way. Saliva samples were collected from 7-year old children three times during a laboratory visit for assessment of cognitive and executive functions (pretest, mid-test, end-study day acute stress profile) and at four times over two consecutive non-school days at home (i.e. morning, mid-morning, afternoon and bedtime-diurnal rhythm profile). We found that cortisol profiles were similar in preterm and full-term children, albeit preterms had slightly higher cortisol at bedtime compared to full-term children. Importantly, in the preterm group, greater neonatal procedural pain-related stress (adjusted for morphine) was associated with lower cortisol levels on the study day (p=.044) and lower diurnal cortisol at home (p=.023), with effects found primarily in boys. In addition, child attention problems were negatively, and thought problems were positively, associated with the cortisol response during cognitive assessment on the study day in preterm children. Our findings suggest that neonatal pain/stress contributes to altered HPA axis function up to school-age in children born very preterm, and that sex may be an important factor.

Optimization of an in vitro regeneration system for colombian indica rice varieties / Optimización de un sistema in vitro de regeneración para variedades colombianas de arroz indica

In order to optimize an in vitro regeneration system for colombian rice varieties, the amount of plant regulators used for each step of the process was modified, together with other factors such as seed position and time of exposition to plant regulators. Modification of 2,4-D concentration during callus induction and coculture has a negative effect on regeneration potential. However, it is possible to increase the amount of produced calli by modifying the position in which the seed is placed on the culture medium. Moreover, a longer callus induction time decreased the regeneration percentage. During the regeneration step, for FEDEARROZ 2000 variety, a combination of 1,0 mg/l g/l mg/l of naftalenacetic acid + 4,0 mg/l g/l mg/l of kinetin increases the regeneration percentage, without increasing the amount of abnormal shoots and without decreasing their size. For FEDEARROZ 50 variety, the best combination of plant regulators is 0,5 mg/l g/l mg/l of naftalenacetic acid + 2,0 mg/l g/l mg/l of kinetin.

Para optimizar un sistema de regeneración in vitro de variedades colombianas de arroz, se modificaron las concentraciones de los reguladores utilizados a lo largo de cada etapa del proceso de cultivo, junto con otros factores como la posición de siembra de la semilla y el tiempo de exposición a los reguladores. Se encontró que la modificación de la concentración de 2,4-D para las etapas de inducción de callo y cocultivo repercute de forma negativa en la regeneración. Por otra parte, es posible incrementar el porcentaje de formación de callo cambiando la posición de siembra de la semilla. Adicionalmente, un mayor tiempo de formación de callo provocó una disminución del porcentaje de regeneración. En la etapa de regeneración, para la variedad FEDEARROZ 2000 se encontró que una combinación de 1,0 mg/l g/l mg/l de ácido naftalenacético + 4,0 mg/l g/l mg/l de kinetina aumenta el porcentaje de regeneración, sin incrementar la cantidad de brotes anormales y sin disminuir de forma considerable el tamaño de los brotes. Para la variedad FEDEARROZ 50, la mejor combinación de reguladores durante la regeneración es de 0,5 mg/l g/l mg/l de ácido natfalenacético + 2,0 mg/l g/l mg/l de kinetina.

Invasive procedures in preterm children: brain and cognitive development at school age.

BACKGROUND: Very preterm infants (born 24-32 weeks' gestation) undergo numerous invasive procedures during neonatal care. Repeated skin-breaking procedures in rodents cause neuronal cell death, and in human preterm neonates higher numbers of invasive procedures from birth to term-equivalent age are associated with abnormal brain development, even after controlling for other clinical risk factors. It is unknown whether higher numbers of invasive procedures are associated with long-term alterations in brain microstructure and cognitive outcome at school age in children born very preterm. METHODS: Fifty children born very preterm underwent MRI and cognitive testing at median age 7.6 years (interquartile range, 7.5-7.7). T1- and T2-weighted images were assessed for the severity of brain injury. Magnetic resonance diffusion tensor sequences were used to measure fractional anisotropy (FA), an index of white matter (WM) maturation, from 7 anatomically defined WM regions. Child cognition was assessed using the Wechsler Intelligence Scale for Children-IV. Multivariate modeling was used to examine relationships between invasive procedures, brain microstructure, and cognition, adjusting for clinical confounders (eg, infection, ventilation, brain injury). RESULTS: Greater numbers of invasive procedures were associated with lower FA values of the WM at age 7 years (P = .01). The interaction between the number of procedures and FA was associated with IQ (P = .02), such that greater numbers of invasive procedures and lower FA of the superior WM were related to lower IQ. CONCLUSIONS: Invasive procedures during neonatal care contribute to long-term abnormalities in WM microstructure and lower IQ.

Neonatal pain-related stress and NFKBIA genotype are associated with altered cortisol levels in preterm boys at school age.

Neonatal pain-related stress is associated with elevated salivary cortisol levels to age 18 months in children born very preterm, compared to full-term, suggesting early programming effects. Importantly, interactions between immune/inflammatory and neuroendocrine systems may underlie programming effects. We examined whether cortisol changes persist to school age, and if common genetic variants in the promoter region of the NFKBIA gene involved in regulation of immune and inflammatory responses, modify the association between early experience and later life stress as indexed by hair cortisol levels, which provide an integrated index of endogenous HPA axis activity. Cortisol was assayed in hair samples from 128 children (83 born preterm ≤ 32 weeks gestation and 45 born full-term) without major sensory, motor or cognitive impairments at age 7 years. We found that hair cortisol levels were lower in preterm compared to term-born children. Downregulation of the HPA axis in preterm children without major impairment, seen years after neonatal stress terminated, suggests persistent alteration of stress system programming. Importantly, the etiology was gender-specific such that in preterm boys but not girls, specifically those with the minor allele for NFKBIA rs2233409, lower hair cortisol was associated with greater neonatal pain (number of skin-breaking procedures from birth to term), independent of medical confounders. Moreover, the minor allele (CT or TT) of NFKBIA rs2233409 was associated with higher secretion of inflammatory cytokines, supporting the hypothesis that neonatal pain-related stress may act as a proinflammatory stimulus that induces long-term immune cell activation. These findings are the first evidence that a long-term association between early pain-related stress and cortisol may be mediated by a genetic variants that regulate the activity of NF-κB, suggesting possible involvement of stress/inflammatory mechanisms in HPA programming in boys born very preterm.

Neonatal pain-related stress, functional cortical activity and visual-perceptual abilities in school-age children born at extremely low gestational age.

Children born very prematurely (< or =32 weeks) often exhibit visual-perceptual difficulties at school-age, even in the absence of major neurological impairment. The alterations in functional brain activity that give rise to such problems, as well as the relationship between adverse neonatal experience and neurodevelopment, remain poorly understood. Repeated procedural pain-related stress during neonatal intensive care has been proposed to contribute to altered neurocognitive development in these children. Due to critical periods in the development of thalamocortical systems, the immature brain of infants born at extremely low gestational age (ELGA; < or =28 weeks) may have heightened vulnerability to neonatal pain. In a cohort of school-age children followed since birth we assessed relations between functional brain activity measured using magnetoencephalogragy (MEG), visual-perceptual abilities and cumulative neonatal pain. We demonstrated alterations in the spectral structure of spontaneous cortical oscillatory activity in ELGA children at school-age. Cumulative neonatal pain-related stress was associated with changes in background cortical rhythmicity in these children, and these alterations in spontaneous brain oscillations were negatively correlated with visual-perceptual abilities at school-age, and were not driven by potentially confounding neonatal variables. These findings provide the first evidence linking neonatal pain-related stress, the development of functional brain activity, and school-age cognitive outcome in these vulnerable children.

Electroencephalographic activity in response to procedural pain in preterm infants born at 28 and 33 weeks gestational age.

OBJECTIVES: Preterm infants undergo frequent painful procedures in the neonatal intensive care unit. Electroencephalography (EEG) changes in reaction to invasive procedures have been reported in preterm and full-term neonates. Frontal EEG asymmetry as an index of emotion during tactile stimulation shows inconsistent findings in full-term infants, and has not been examined in the context of pain in preterm infants. Our aim was to examine whether heel lance for blood collection induces changes in right-left frontal asymmetry, suggesting negative emotional response, in preterm neonates at different gestational age (GA) at birth and different duration of stay in the neonatal intensive care unit. MATERIALS AND METHODS: Three groups of preterm infants were compared: set 1: group 1 (n=24), born and tested at 28 weeks GA; group 2 (n=22), born at 28 weeks GA and tested at 33 weeks; set 2: group 3 (n=25), born and tested at 33 weeks GA. EEG power was calculated for 30-second artifact-free periods, in standard frequency bandwidths, in 3 phases (baseline, up to 5 min after heel lance, 10 min after heel lance). RESULTS: No significant differences were found in right-left frontal asymmetry, or in ipsilateral or contralateral somatosensory response, across phases. In contrast, the Behavioral Indicators of Infant Pain scores changed across phase (P<0.0001). Infants in group 1 showed lower Behavioral Indicators of Infant Pain scores (P=0.039). DISCUSSION: There are technical challenges in recording EEG during procedures, as pain induces motor movements. More research is needed to determine the most sensitive approach to measure EEG signals within the context of pain in infancy.

Concentración mínima inhibitoria de higromicina B en callos embriogénicos de arroz -Oryza sativa L / Minimum inhibitory concentration of higromycin B in rice embryogenic calli -Oryza sativa L

Uno de los elementos imprescindibles en la ingeniería genética de plantas es un sistema de selección eficiente. El propósito de este trabajo fue evaluar la sensibilidad al marcador de selección higromicina B, de callos embriogénicos obtenidos a partir del escutelo de semilla de tres variedades colombianas de arroz (FEDEARROZ 2000, FEDEARROZ 50 y FEDEARROZ 369). Además, se validó la respuesta de estas variedades al protocolo de regeneración empleado. Se probaron cuatro concentraciones del antibiótico (25 mg/L, 50 mg/L, 75 mg/L y 100 mg/L) más un control sin higromicina B. Los resultados obtenidos mostraron que una concentración de 50 mg/L de antibiótico en el medio de regeneración es adecuada para la selección. Con esta concentración se impide la formación de brotes, aunque los callos no mueren completamente. Por otra parte, se estableció que el protocolo de regeneración utilizado es de baja eficiencia y, por consiguiente, es necesario optimizarlo para poder usarlo en procesos de ingeniería genética de cultivares colombianos de arroz.

An efficient selection system is one of the most important elements of plant genetic engineering. The purpose of this study was to evaluate the sensitivity of scutellum-derived embriogenic calli obtained from three colombian rice varieties (FEDEARROZ 2000, FEDEARROZ 50 and FEDEARROZ 369), to the selection marker hygromycin B. Aditionally, the response of these varieties to the regeneration protocol was measured. Four antibiotic concentrations were tested (25 mg/L, 50 mg/L, 75 mg/L and 100 mg/L) plus one control without hygromycin B. The results show that 50 mg/L of antibiotic in the regeneration medium is adequate for selection. This concentration prevents the formation of shoots, though the calli do not die. It was also established that the regeneration protocol is a low-efficiency system and it needs to be improved, in order to use it for colombian rice genetic engineering.

Cortisol levels in relation to maternal interaction and child internalizing behavior in preterm and full-term children at 18 months corrected age.

Cortisol levels were compared in children born preterm at extremely low gestational age (ELGA; 24-28 weeks), very low gestational age (VGLA; 29-32 weeks), and full-term in response to cognitive assessment at 18 months corrected age (CA). Further, we investigated the relationship between maternal interactive behaviors and child internalizing behaviors (rated by the mother) in relation to child cortisol levels. EGLA children had higher "pretest" cortisol levels and a different pattern of cortisol response to cognitive assessment compared to VGLA and full-terms. Higher cortisol levels in ELGA, but not full-term, children were associated with less optimal mother interactive behavior. Moreover, the pattern of cortisol change was related to internalizing behaviors among ELGA, and to a lesser degree VLGA children. In conclusion, our findings suggest altered programming of the hypothalamic-pituitary-adrenal (HPA) axis in preterm children, as well as their greater sensitivity to environmental context such as maternal interactive behavior.

Neonatal pain, parenting stress and interaction, in relation to cognitive and motor development at 8 and 18 months in preterm infants.

Procedural pain in the neonatal intensive care unit triggers a cascade of physiological, behavioral and hormonal disruptions which may contribute to altered neurodevelopment in infants born very preterm, who undergo prolonged hospitalization at a time of physiological immaturity and rapid brain development. The aim of this study was to examine relationships between cumulative procedural pain (number of skin-breaking procedures from birth to term, adjusted for early illness severity and overall intravenous morphine exposure), and later cognitive, motor abilities and behavior in very preterm infants at 8 and 18 months corrected chronological age (CCA), and further, to evaluate the extent to which parenting factors modulate these relationships over time. Participants were N=211 infants (n=137 born preterm 32 weeks gestational age [GA] and n=74 full-term controls) followed prospectively since birth. Infants with significant neonatal brain injury (periventricular leucomalacia, grade 3 or 4 intraventricular hemorrhage) and/or major sensori-neural impairments, were excluded. Poorer cognition and motor function were associated with higher number of skin-breaking procedures, independent of early illness severity, overall intravenous morphine, and exposure to postnatal steroids. The number of skin-breaking procedures as a marker of neonatal pain was closely related to days on mechanical ventilation. In general, greater overall exposure to intravenous morphine was associated with poorer motor development at 8 months, but not at 18 months CCA, however, specific protocols for morphine administration were not evaluated. Lower parenting stress modulated effects of neonatal pain, only on cognitive outcome at 18 months.

Human retinal pigment epithelial cell implants ameliorate motor deficits in two rat models of Parkinson disease.

Intrastriatal transplantation of gelatin microcarrier-attached human retinal pigment epithelial cells (hRPE-GM) may represent an alternative source for cell therapy in Parkinson disease (PD). The use of human retinal pigment epithelial (hRPE) cells in PD relies on the capacity of these cells to produce l-dopa as an intermediate product in the eumelanin synthesis pathway. We investigated the behavioral effects of hRPE-GM implants on forelimb use asymmetries and hindlimb motor deficits in unilateral and bilateral 6-hydroxydopamine (6-OHDA) rat models of PD. We report that intrastriatal unilateral implantation of hRPE-GM in rats with 6-OHDA nigrostriatal lesions produce an amelioration of the contralateral forelimb disuse and the contralateral hindlimb deficits. These results further support the possibility that implantation of cultured hRPE cells may be a promising therapeutic option for patients with PD.

Characterization and survival of long-term implants of human retinal pigment epithelial cells attached to gelatin microcarriers in a model of Parkinson disease.

Previous studies have demonstrated that the intrastriatal implantation of human retinal pigment epithelial cells attached to gelatin microcarriers (hRPE-GM) ameliorates behavioral deficits in animal models of Parkinson disease. However, there are only sparse data on cell survival in the host. In this study, we characterized a variety of retinal pigment epithelial (RPE)-specific markers in vitro and used these markers to investigate the long-term survival of hRPE-GM implants. Sprague-Dawley rats (n = 22) were unilaterally lesioned with 6-hydroxydopamine (6-OHDA) and implanted with hRPE-GM without immunosuppression. Rats were euthanized at 48 hours, 7 days, 4 weeks, and 5 months postimplant and immunohistochemically processed using the following antibodies: 1) human-specific nuclear mitotic apparatus protein (NuMA-Ab2), 2) epithelial-specific extracellular matrix metalloproteinase inducer (EMMPRIN), 3) RPE cell-specific RPE65, and the inflammation markers 4) glial fibrillary acidic protein and 5) ED1 (rat CD68). Our analysis revealed NuMA-, EMMPRIN-, and RPE65-immunoreactive cells at different times postimplant. The morphologic features of hRPE cell implants (at 48 hours and 5 months) were confirmed by electron microscopy. Furthermore, despite evidence of chronic inflammation at the later time point, there is an appreciable number of surviving hRPE cells. This study suggests that hRPE-GM implants can survive in the absence of immunosuppression and can be potentially used as an alternative for treating Parkinson disease.

Evaluation of the integrity of the dopamine system in a rodent model of Parkinson's disease: small animal positron emission tomography compared to behavioral assessment and autoradiography.

PURPOSE: In the 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease (PD), it is important to determine lesion severity. This evaluation can be performed in vivo, through evaluation of dopamine (DA)-dependent motor function or with small animal positron emission tomography (microPET), or at postmortem, by examining markers for DA neurons. PROCEDURES: Rats were given mild or severe unilateral 6-OHDA lesions, scanned with the tracer [(11)C](+/-)dihydrotetrabenazine ([(11)C]DTBZ), and tested on a tapered/ledged beam-walking task. At postmortem, autoradiography was performed with [(11)C]DTBZ. RESULTS: Autoradiography was significantly correlated with microPET and behavioral scores, whereas the microPET and behavioral data were not significantly correlated. CONCLUSIONS: This study shows that behavioral analysis, microPET, and autoradiography are all good tools for measuring the integrity of the DA system, and demonstrates the utility of the tapered/ledged beam-walking test to screen for lesion severity, as well as the importance of including postmortem analysis after in vivo imaging studies.

Inducible enhancement of memory storage and synaptic plasticity in transgenic mice expressing an inhibitor of ATF4 (CREB-2) and C/EBP proteins.

To examine the role of C/EBP-related transcription factors in long-term synaptic plasticity and memory storage, we have used the tetracycline-regulated system and expressed in the forebrain of mice a broad dominant-negative inhibitor of C/EBP (EGFP-AZIP), which preferentially interacts with several inhibiting isoforms of C/EBP. EGFP-AZIP also reduces the expression of ATF4, a distant member of the C/EBP family of transcription factors that is homologous to the Aplysia memory suppressor gene ApCREB-2. Consistent with the removal of inhibitory constraints on transcription, we find an increase in the pattern of gene transcripts in the hippocampus of EGFP-AZIP transgenic mice and both a reversibly enhanced hippocampal-based spatial memory and LTP. These results suggest that several proteins within the C/EBP family including ATF4 (CREB-2) act to constrain long-term synaptic changes and memory formation. Relief of this inhibition lowers the threshold for hippocampal-dependent long-term synaptic potentiation and memory storage in mice.