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1.  Diffusion Tractography and Neuromotor Outcome in Very Preterm Children with White Matter Abnormalities 
Pediatric research  2014;76(1):86-92.
Background
Moderate-severe white matter abnormality (WMA) in the newborn has been shown to produce persistent disruptions in cerebral connectivity, but does not universally result in neurodevelopmental disability in very preterm (VPT) children. The aims of this hypothesis driven study were to apply diffusion imaging to: 1) examine whether bilateral WMA detected in VPT children in the newborn period can predict microstructural organization at age 7; 2) compare corticospinal tract (CST) and corpus callosum (CC) measures in VPT children at age 7 with neonatal WMA with normal versus impaired motor functioning.
Methods
Diffusion parameters of the CST and CC were compared between VPT 7-year-olds with (n=20) and without (n=42) bilateral WMA detected in the newborn period. For those with WMA, diffusion parameters were further examined.
Results
Microstructural organization of CST and CC tracts at age 7 years were altered in VPT children with moderate-severe WMA detected at term equivalent age compared to those without injury. Furthermore, diffusion parameters differed in the CC for children with WMA categorized by motor outcome (N=8).
Conclusions
WMA on conventional MRI at term equivalent age is associated with altered microstructural organization of the CST and CC at 7 years of age.
doi:10.1038/pr.2014.45
PMCID: PMC4062577  PMID: 24713814
2.  Hippocampal Volume and Memory and Learning Outcomes at 7 Years in Children Born Very Preterm 
Using magnetic resonance imaging, this study compared hippocampal volume between 145 very preterm children and 34 children born full term at 7 years of age. The relationship between hippocampal volume and memory and learning impairments at 7 years was also investigated. Manual hippocampal segmentation and subsequent 3D volumetric analysis revealed reduced hippocampal volumes in very preterm children compared with term peers. However, this relationship did not remain after correcting for whole brain volume and neonatal brain abnormality. Contrary to expectations, hippocampal volume in the very preterm cohort was not related to memory and learning outcomes. Further research investigating the effects of very preterm birth on more extensive networks in the brain that support memory and learning in middle childhood is needed.
doi:10.1017/S1355617713000891
PMCID: PMC3964592  PMID: 23947431
Neonatal; Hippocampal Formation Segmentation; Memory and Learning
3.  Neonatal white matter abnormality predicts childhood motor impairment in very preterm children 
AIM
Children born very preterm are at risk for impaired motor performance ranging from cerebral palsy (CP) to milder abnormalities, such as developmental coordination disorder. White matter abnormalities (WMA) at term have been associated with CP in very preterm children; however, little is known about the impact of WMA on the range of motor impairments. The aim of this study was to assess whether WMA were predictive of all levels of motor impairments in very preterm children.
METHOD
Two hundred and twenty-seven very preterm infants (<30wks’ gestational age or birthweight <1250g) had brain magnetic resonance imaging at term-equivalent age to assess for WMA, which were categorized as nil, mild, or moderate to severe. At 5 years of age children were classified as having a moderate to severe motor impairment if they were below the 5th centile or mild to severe motor impairment if their score placed them no higher than the 15th centile on the Movement Assessment Battery for Children (MABC). WMA (nil vs mild and nil vs moderate–severe) were explored as predictors of motor impairment using logistic regression. Analyses were repeated adjusting for the effects of other perinatal variables and excluding children with CP.
RESULTS
Of the 193 very preterm children (97 males, 96 females) assessed with the MABC, 53 (27%) were classified as having a moderate to severe motor impairment and 96 (50%) a mild to severe motor impairment. WMA were predictive of motor impairment in very preterm children, with mild versus no WMA increasing the odds of moderate to severe motor impairment by over fivefold (odds ratio [OR] 5.6; 95% confidence interval [CI] 1.9–16.1; p=0.002) and mild to severe impairment by twofold (OR 2.2; 95% CI 1.1–4.2; p=0.02). Compared with no WMA, moderate to severe WMA increased the odds for moderate to severe impairment 19-fold (OR 19.4; 95% CI 5.6–66.7; p<0.001) and for mild to severe motor impairment ninefold (OR 9.4; 95% CI 3.2–28.1; p<0.001). Results remained similar after controlling for several potential confounders and after excluding 14 children who had CP at age 2 years.
INTERPRETATION
WMA predict motor impairment at 5 years, with rates of impairment increasing with more severe WMA. Very preterm children with any WMA at term require follow-up throughout childhood.
doi:10.1111/j.1469-8749.2011.04095.x
PMCID: PMC4040368  PMID: 22014319
4.  Neurobehaviour between birth and 40 weeks’ gestation in infants born <30 weeks’ gestation and parental psychological wellbeing: predictors of brain development and child outcomes 
BMC Pediatrics  2014;14:111.
Background
Infants born <30 weeks’ gestation are at increased risk of long term neurodevelopmental problems compared with term born peers. The predictive value of neurobehavioural examinations at term equivalent age in very preterm infants has been reported for subsequent impairment. Yet there is little knowledge surrounding earlier neurobehavioural development in preterm infants prior to term equivalent age, and how it relates to perinatal factors, cerebral structure, and later developmental outcomes. In addition, maternal psychological wellbeing has been associated with child development. Given the high rate of psychological distress reported by parents of preterm children, it is vital we understand maternal and paternal wellbeing in the early weeks and months after preterm birth and how this influences the parent–child relationship and children’s outcomes. Therefore this study aims to examine how 1) early neurobehaviour and 2) parental mental health relate to developmental outcomes for infants born preterm compared with infants born at term.
Methods/Design
This prospective cohort study will describe the neurobehaviour of 150 infants born at <30 weeks’ gestational age from birth to term equivalent age, and explore how early neurobehavioural deficits relate to brain growth or injury determined by magnetic resonance imaging, perinatal factors, parental mental health and later developmental outcomes measured using standardised assessment tools at term, one and two years’ corrected age. A control group of 150 healthy term-born infants will also be recruited for comparison of outcomes. To examine the effects of parental mental health on developmental outcomes, both parents of preterm and term-born infants will complete standardised questionnaires related to symptoms of anxiety, depression and post-traumatic stress at regular intervals from the first week of their child’s birth until their child’s second birthday. The parent–child relationship will be assessed at one and two years’ corrected age.
Discussion
Detailing the trajectory of infant neurobehaviour and parental psychological distress following very preterm birth is important not only to identify infants most at risk, further understand the parental experience and highlight potential times for intervention for the infant and/or parent, but also to gain insight into the effect this has on parent–child interaction and child development.
doi:10.1186/1471-2431-14-111
PMCID: PMC4016657  PMID: 24758605
Preterm; Neurobehaviour; Magnetic resonance imaging; Neurodevelopment; Parent mental health; Parent–child interaction
5.  Hippocampal shape variations at term equivalent age in very preterm infants compared with term controls: perinatal predictors and functional significance at age 7 
NeuroImage  2013;70:278-287.
The hippocampus undergoes rapid growth and development in the perinatal months. Infants born very preterm (VPT) are vulnerable to hippocampal alterations, and can provide a model of disturbed early hippocampal development. Hippocampal shape alterations have previously been associated with memory impairment, but have never been investigated in infants. The aims of this study were to determine hippocampal shape differences between 184 VPT infants (<30 weeks’ gestation or <1250 g at birth) and 32 full-term infants, effects of perinatal factors, and associations between infant hippocampal shape and volume, and 7 year verbal and visual memory (California Verbal Learning Test- Children’s Version and Dot Locations). Infants underwent 1.5T magnetic resonance imaging at term equivalent age. Hippocampi were segmented, and spherical harmonics-point distribution model shape analysis was undertaken. VPT infants’ hippocampi were less infolded than full-term infants, being less curved toward the midline and less arched superior-inferiorly. Straighter hippocampi were associated with white matter injury and postnatal corticosteroid exposure. There were no significant associations between infant hippocampal shape and 7 year memory measures. However, larger infant hippocampal volumes were associated with better verbal memory scores. Altered hippocampal shape in VPT infants at term equivalent age may reflect delayed or disrupted development. This study provides further insight into early hippocampal development and the nature of hippocampal abnormalities in prematurity.
doi:10.1016/j.neuroimage.2012.12.053
PMCID: PMC3584256  PMID: 23296179
preterm infant; magnetic resonance imaging; hippocampus; morphology; memory
6.  Impaired language abilities and white matter abnormalities in children born very preterm and/or very low birth weight 
The Journal of pediatrics  2012;162(4):719-724.
Objectives
To investigate language abilities in children born very preterm (VPT; <32 weeks’ gestational age (GA)) or very low birth weight (VLBW; <1500 g) at 7 years of age and compare their performances with children born at term, and to determine whether group differences could be explained by cerebral white matter abnormality on neonatal MRI.
Study design
A cohort of 198 children born <30 weeks’ GA and/or <1250 g, and 70 term controls were examined. White matter abnormalities were rated quantitatively on brain MRI at term-equivalent age. Language was assessed at age 7 years using standardized language tests. Differences between groups were tested in the five language sub-domains of phonological awareness, semantics, grammar, discourse, and pragmatics. A mediation effect was tested between birth group, white matter abnormality, and language sub-domains.
Results
The VPT/VLBW group performed significantly worse than controls on all language sub-domains (all p <.001). White matter abnormality mediated the effect of group differences on phonological awareness, and partly mediated this effect for semantics, grammar and discourse. White matter abnormality was not significantly associated with pragmatics (p = .13).
Conclusions
Language is an important area of concern in children born VPT/VLBW. Neonatal white matter abnormality is an important predictor of outcome; however, different language abilities are differentially associated with neonatal white matter abnormality.
doi:10.1016/j.jpeds.2012.10.017
PMCID: PMC3600107  PMID: 23158026
Prematurity; development
7.  Association between Postnatal Dexamethasone for Treatment of Bronchopulmonary Dysplasia and Brain Volumes at Adolescence in Infants Born Very Preterm 
The Journal of Pediatrics  2014;164(4):737-743.e1.
Objectives
To compare brain volumes in adolescents who were born extremely preterm (<28 weeks gestation) who had received postnatal dexamethasone, and to determine if there was a postnatal dexamethasone dose–response effect on brain volumes.
Study design
Geographical cohort study of extremely preterm adolescents born in 1991-1992 in Victoria, Australia. T1-weighted magnetic resonance imaging was performed at 18 years of age. Segmented and parcellated brain volumes were calculated using an automated segmentation method (FreeSurfer) and compared between groups, with and without adjustment for potential confounders. The relationships between total postnatal dexamethasone dose and brain volumes were explored using linear regression.
Results
Of the 148 extremely preterm participants, 55 (37%) had received postnatal dexamethasone, with a cumulative mean dose of 7.7 mg/kg. Compared with participants who did not receive postnatal dexamethasone, those who did had smaller total brain tissue volumes (mean difference −3.6%, 95% CI [−7.0%, −0.3%], P value = .04) and smaller white matter, thalami, and basal ganglia volumes (all P < .05). There was a trend of smaller total brain and white matter volumes with increasing dose of postnatal dexamethasone (regression coefficient −7.7 [95% CI −16.2, 0.8] and −3.2 [−6.6, 0.2], respectively).
Conclusions
Extremely preterm adolescents who received postnatal dexamethasone in the newborn period had smaller total brain tissue volumes than those who did not receive postnatal dexamethasone, particularly white matter, thalami, and basal ganglia. Vulnerability of brain tissues or structures associated with postnatal dexamethasone varies by structure and persists into adolescence.
doi:10.1016/j.jpeds.2013.10.083
PMCID: PMC4029072  PMID: 24332820
BPD, Bronchopulmonary dysplasia; MRI, Magnetic resonance imaging; PCS, Postnatal corticosteroid
8.  Neural Correlates of Impaired Vision in Adolescents Born Extremely Preterm and/or Extremely Low Birthweight 
PLoS ONE  2014;9(3):e93188.
Background
Adolescents born extremely preterm (EP; <28 weeks' gestation) and/or extremely low birthweight (ELBW; <1000 g) experience high rates of visual impairments, however the potential neural correlates of visual impairments in EP/ELBW adolescents require further investigation. This study aimed to: 1) compare optic radiation and primary visual cortical structure between EP/ELBW adolescents and normal birthweight controls; 2) investigate associations between perinatal factors and optic radiation and primary visual cortical structure in EP/ELBW adolescents; 3) investigate associations between optic radiation and primary visual cortical structure in EP/ELBW adolescents and the odds of impaired vision.
Methods
196 EP/ELBW adolescents and 143 controls underwent magnetic resonance imaging at a mean age of 18 years. Optic radiations were delineated using constrained spherical deconvolution based probabilistic tractography. Primary visual cortices were segmented using FreeSurfer software. Diffusion tensor variables and tract volume of the optic radiations, as well as volume, surface area and thickness of the primary visual cortices, were estimated.
Results
Axial, radial and mean diffusivities within the optic radiations, and primary visual cortical thickness, were higher in the EP/ELBW adolescents than controls. Within EP/ELBW adolescents, postnatal corticosteroid exposure was associated with altered optic radiation diffusion values and lower tract volume, while decreasing gestational age at birth was associated with increased primary visual cortical volume, area and thickness. Furthermore, decreasing optic radiation fractional anisotropy and tract volume, and increasing optic radiation diffusivity in EP/ELBW adolescents were associated with increased odds of impaired vision, whereas primary visual cortical measures were not associated with the odds of impaired vision.
Conclusions
Optic radiation and primary visual cortical structure are altered in EP/ELBW adolescents compared with controls, with the greatest alterations seen in those exposed to postnatal corticosteroids and those born earliest. Structural alterations to the optic radiations may increase the risk of impaired vision in EP/ELBW adolescents.
doi:10.1371/journal.pone.0093188
PMCID: PMC3964000  PMID: 24663006
9.  Alterations in the optic radiations of very preterm children—Perinatal predictors and relationships with visual outcomes☆ 
NeuroImage : Clinical  2013;4:145-153.
Children born very preterm (VPT) are at risk for visual impairments, the main risk factors being retinopathy of prematurity and cerebral white matter injury, however these only partially account for visual impairments in VPT children. This study aimed to compare optic radiation microstructure and volume between VPT and term-born children, and to investigate associations between 1) perinatal variables and optic radiations; 2) optic radiations and visual function in VPT children. We hypothesized that optic radiation microstructure would be altered in VPT children, predicted by neonatal cerebral white matter abnormality and retinopathy of prematurity, and associated with visual impairments.
142 VPT children and 32 controls underwent diffusion-weighted magnetic resonance imaging at 7 years of age. Optic radiations were delineated using constrained spherical deconvolution tractography. Tract volume and average diffusion tensor values for the whole optic radiations and three sub-regions were compared between the VPT and control groups, and correlated with perinatal variables and 7-year visual outcome data.
Total tract volumes and average diffusion values were similar between VPT and control groups. On regional analysis of the optic radiation, mean and radial diffusivity were higher within the middle sub-regions in VPT compared with control children. Neonatal white matter abnormalities and retinopathy of prematurity were associated with optic radiation diffusion values. Lower fractional anisotropy in the anterior sub-regions was associated with poor visual acuity and increased likelihood of other visual defects.
This study presents evidence for microstructural alterations in the optic radiations of VPT children, which are largely predicted by white matter abnormality or severe retinopathy of prematurity, and may partially explain the higher rate of visual impairments in VPT children.
Highlights
•This study compares optic radiations between very preterm and control 7-year-olds.•There are microstructural alterations in the optic radiations of VPT children.•The main risk factors are retinopathy of prematurity and white matter injury.•Microstructural alterations associate with poor visual acuity and visual defects.•This study elucidates neuroanatomical correlates of visual impairment in prematurity.
doi:10.1016/j.nicl.2013.11.007
PMCID: PMC3871291  PMID: 24371797
AD, Axial diffusivity; BWSDS, Birth weight standard deviation score; CI, Confidence interval; CSD, Constrained spherical deconvolution; FA, Fractional anisotropy; GA, Gestational age; MRI, Magnetic resonance imaging; MD, Mean diffusivity; RD, Radial diffusivity; ROP, Retinopathy of prematurity; VPT, Very preterm; Prematurity; Visual system; Tractography; Magnetic resonance imaging; Diffusion weighted imaging
10.  Psychiatric outcomes at age seven for very preterm children: rates and predictors 
Background
Uncertainty remains about the rate of specific psychiatric disorders and associated predictive factors for very preterm (VPT) children. The aims of this study were to document rates of psychiatric disorders in VPT children aged 7 years compared with term born children, and to examine potential predictive factors for psychiatric diagnoses in VPT children.
Methods
Participants were 177 VPT and 65 term born children. Perinatal medical data were collected, which included brain abnormalities detected using magnetic resonance imaging. The Infant-Toddler Social-Emotional Assessment (ITSEA) and Strengths and Difficulties Questionnaire (SDQ) were administered at 2 and 5 years respectively. At 7 years of age, the Developmental and Well-being Assessment (DAWBA) was used to indicate psychiatric diagnoses.
Results
Compared with term born children, VPT children had three times the odds of meeting criteria for any psychiatric diagnosis at age 7 years (odds ratio 3.03; 95% confidence interval 1.23, 7.47, p = .02). The most common diagnoses were anxiety disorders (11% VPT, 8% term), attention-deficit/hyperactivity disorder (10% VPT, 3% term) and autism spectrum disorder (4.5% VPT, 0% term). For VPT children, those with severe global brain abnormalities (p = .02), those who displayed social-emotional problems at age 5 (p = .000) and those with higher social risk at age 7 (p = .001) were more likely to meet criteria for a psychiatric illness at age 7.
Conclusions
Compared with term born children, VPT children have higher rates of psychiatric diagnoses at early school age, predicted by neonatal brain abnormalities, prior social-emotional problems and social factors.
doi:10.1111/jcpp.12040
PMCID: PMC3821531  PMID: 23347471
Preterm; psychiatric disorder; brain abnormality; predictor; mental health
11.  Contribution of Brain Size to IQ and Educational Underperformance in Extremely Preterm Adolescents 
PLoS ONE  2013;8(10):e77475.
Objectives
Extremely preterm (EP) survivors have smaller brains, lower IQ, and worse educational achievement than their term-born peers. The contribution of smaller brain size to the IQ and educational disadvantages of EP is unknown. This study aimed (i) to compare brain volumes from multiple brain tissues and structures between EP-born (<28weeks) and term-born (≥37weeks) control adolescents, (ii) to explore the relationships of brain tissue volumes with IQ and basic educational skills and whether this differed by group, and (iii) to explore how much total brain tissue volume explains the underperformance of EP adolescents compared with controls.
Methods
Longitudinal cohort study of 148 EP and 132 term controls born in Victoria, Australia in 1991-92. At age 18, magnetic resonance imaging-determined brain volumes of multiple tissues and structures were calculated. IQ and educational skills were measured using the Wechsler Abbreviated Scale of Intelligence (WASI) and the Wide Range Achievement Test(WRAT-4), respectively.
Results
Brain volumes were smaller in EP adolescents compared with controls (mean difference [95% confidence interval] of -5.9% [-8.0, -3.7%] for total brain tissue volume). The largest relative differences were noted in the thalamus and hippocampus. The EP group had lower IQs(-11.9 [-15.4, -8.5]), spelling(-8.0 [-11.5, -4.6]), math computation(-10.3 [-13.7, -6.9]) and word reading(-5.6 [-8.8, -2.4]) scores than controls; all p-values<0.001. Volumes of total brain tissue and other brain tissues and structures correlated positively with IQ and educational skills, a relationship that was similar for both the EP and controls. Total brain tissue volume explained between 20-40% of the IQ and educational outcome differences between EP and controls.
Conclusions
EP adolescents had smaller brain volumes, lower IQs and poorer educational performance than controls. Brain volumes of multiple tissues and structures are related to IQ and educational outcomes. Smaller total brain tissue volume is an important contributor to the cognitive and educational underperformance of adolescents born EP.
doi:10.1371/journal.pone.0077475
PMCID: PMC3793949  PMID: 24130887
12.  Preventing academic difficulties in preterm children: a randomised controlled trial of an adaptive working memory training intervention – IMPRINT study 
BMC Pediatrics  2013;13:144.
Background
Very preterm children exhibit difficulties in working memory, a key cognitive ability vital to learning information and the development of academic skills. Previous research suggests that an adaptive working memory training intervention (Cogmed) may improve working memory and other cognitive and behavioural domains, although further randomised controlled trials employing long-term outcomes are needed, and with populations at risk for working memory deficits, such as children born preterm.
In a cohort of extremely preterm (<28 weeks’ gestation)/extremely low birthweight (<1000 g) 7-year-olds, we will assess the effectiveness of Cogmed in improving academic functioning 2 years’ post-intervention. Secondary objectives are to assess the effectiveness of Cogmed in improving working memory and attention 2 weeks’, 12 months’ and 24 months’ post-intervention, and to investigate training related neuroplasticity in working memory neural networks 2 weeks’ post-intervention.
Methods/Design
This double-blind, placebo-controlled, randomised controlled trial aims to recruit 126 extremely preterm/extremely low birthweight 7-year-old children. Children attending mainstream school without major intellectual, sensory or physical impairments will be eligible. Participating children will undergo an extensive baseline cognitive assessment before being randomised to either an adaptive or placebo (non-adaptive) version of Cogmed. Cogmed is a computerised working memory training program consisting of 25 sessions completed over a 5 to 7 week period. Each training session takes approximately 35 minutes and will be completed in the child’s home. Structural, diffusion and functional Magnetic Resonance Imaging, which is optional for participants, will be completed prior to and 2 weeks following the training period. Follow-up assessments focusing on academic skills (primary outcome), working memory and attention (secondary outcomes) will be conducted at 2 weeks’, 12 months’ and 24 months’ post-intervention.
Discussion
To our knowledge, this study will be the first randomised controlled trial to (a) assess the effectiveness of Cogmed in school-aged extremely preterm/extremely low birthweight children, while incorporating advanced imaging techniques to investigate neural changes associated with adaptive working memory training, and (b) employ long-term follow-up to assess the potential benefit of improved working memory on academic functioning. If effective, Cogmed would serve as a valuable, available intervention for improving developmental outcomes for this population.
Trial registration
Australian New Zealand Clinical Trials Registry ACTRN12612000124831.
doi:10.1186/1471-2431-13-144
PMCID: PMC3848656  PMID: 24041245
Preterm; Extremely low birth weight; Cogmed; Cognitive training; Working memory; Academic outcomes; Neuroplasticity; Magnetic Resonance Imaging; Randomised controlled trial
13.  Corpus callosum alterations in very preterm infants: perinatal correlates and 2 year neurodevelopmental outcomes 
Neuroimage  2011;59(4):3571-3581.
The aim of this study was to relate altered corpus callosum (CC) integrity in 106 very preterm (VPT) infants (<30 weeks’ gestational age or <1250 g birth weight) at term equivalent to perinatal predictors and neurodevelopmental outcomes at two years. T1 and diffusion magnetic resonance images were obtained. The CC was traced, and divided into six sub-regions for cross-sectional area and shape analyses. Fractional anisotropy, mean, axial and radial diffusivity was sampled within the CC, and probabilistic tractography performed. Perinatal predictors were explored. The Bayley Scales of Infant Development (BSID-II) was administered at two years. Intraventricular hemorrhage was associated with a smaller genu and altered diffusion values within the anterior and posterior CC of VPT infants. White matter injury was associated with widespread alterations to callosal diffusion values, especially posteriorly, and radial diffusivity was particularly elevated, indicating altered myelination. Reduced CC tract volume related to lower gestational age, particularly posteriorly. Reduced posterior callosal skew was associated with postnatal corticosteroid exposure. This more circular CC was associated with delayed cognitive development. Higher diffusivity, particularly in splenium tracts, was associated with impaired motor development. This study elucidates perinatal predictors and adverse neurodevelopmental outcomes associated with altered callosal integrity in VPT infants.
doi:10.1016/j.neuroimage.2011.11.057
PMCID: PMC3288421  PMID: 22154956
Brain; Prematurity; Neonate; Magnetic resonance imaging; Diffusion tensor imaging; Tractography
14.  Regional Cerebral Development at Term Relates to School-Age Social-Emotional Development in Very Preterm Children 
Objective
Preterm children are at risk for social-emotional difficulties, including autism and attention deficit hyperactivity disorder. We assessed the relationship of regional brain development in preterm children, evaluated via MRI at term-equivalent postmenstrual age (TEA), to later social-emotional difficulties.
Method
MR images obtained at TEA from 184 very preterm infants (gestation <30 weeks or birthweight <1250 g) were analyzed for white matter abnormalities, hippocampal volume, and brain metrics. 111 infants underwent diffusion tensor imaging, which provided values for fractional anisotropy (FA) and apparent diffusion coefficient (ADC). Social-emotional development was assessed with the Infant Toddler Social and Emotional Assessment (ITSEA) at age 2 and the Strengths and Difficulties Questionnaire (SDQ) at age 5.
Results
Higher ADC in the right orbitofrontal cortex was associated with social-emotional problems at age 5 (peer problems, p<0.01). In females, smaller hippocampal volume was associated with increased hyperactivity (p<0.01), peer problems (p<0.05) and SDQ total score (p<0.01). In males, a smaller frontal region was associated with poorer prosocial (p<0.05) scores. Many of the hippocampal findings remained significant after adjusting for birthweight z score, intelligence, social risk, immaturity at birth, and parental mental health. These associations were present in children who had social-emotional problems in similar domains at age 2 and those who did not.
Conclusions
Early alterations in regional cerebral development in very preterm infants relate to specific deficits in social-emotional performance by school-age. These results vary by gender. Our results provide further evidence for a neuroanatomical basis for behavioral challenges found in very preterm children.
doi:10.1016/j.jaac.2011.11.009
PMCID: PMC3411187  PMID: 22265364
Preterm infant; Neurodevelopment; social-emotional development; orbitofrontal cortex; Hippocampus
15.  Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3 
BMC Biotechnology  2012;12:45.
Background
Achieving efficient introduction of plasmid DNA into primary cultures of mammalian cells is a common problem in biomedical research. Human primary cranial suture cells are derived from the connective mesenchymal tissue between the bone forming regions at the edges of the calvarial plates of the skull. Typically they are referred to as suture mesenchymal cells and are a heterogeneous population responsible for driving the rapid skull growth that occurs in utero and postnatally. To better understand the molecular mechanisms involved in skull growth, and in abnormal growth conditions, such as craniosynostosis, caused by premature bony fusion, it is essential to be able to easily introduce genes into primary bone forming cells to study their function.
Results
A comparison of several lipid-based techniques with two electroporation-based techniques demonstrated that the electroporation method known as nucleofection produced the best transfection efficiency. The parameters of nucleofection, including cell number, amount of DNA and nucleofection program, were optimized for transfection efficiency and cell survival. Two different genes and two promoter reporter vectors were used to validate the nucleofection method and the responses of human primary suture mesenchymal cells by fluorescence microscopy, RT-PCR and the dual luciferase assay. Quantification of bone morphogenetic protein (BMP) signalling using luciferase reporters demonstrated robust responses of the cells to both osteogenic BMP2 and to the anti-osteogenic BMP3.
Conclusions
A nucleofection protocol has been developed that provides a simple and efficient, non-viral alternative method for in vitro studies of gene and protein function in human skull growth. Human primary suture mesenchymal cells exhibit robust responses to BMP2 and BMP3, and thus nucleofection can be a valuable method for studying the potential competing action of these two bone growth factors in a model system of cranial bone growth.
doi:10.1186/1472-6750-12-45
PMCID: PMC3431223  PMID: 22857382
Transfection; Nucleofection; Skull; Bone; Primary cell culture; Mesenchymal; BMP2; luciferase
16.  Bone to pick: the importance of evaluating reference genes for RT-qPCR quantification of gene expression in craniosynostosis and bone-related tissues and cells 
BMC Research Notes  2012;5:222.
Background
RT-qPCR is a common tool for quantification of gene expression, but its accuracy is dependent on the choice and stability (steady state expression levels) of the reference gene/s used for normalization. To date, in the bone field, there have been few studies to determine the most stable reference genes and, usually, RT-qPCR data is normalised to non-validated reference genes, most commonly GAPDH, ACTB and 18 S rRNA. Here we draw attention to the potential deleterious impact of using classical reference genes to normalise expression data for bone studies without prior validation of their stability.
Results
Using the geNorm and Normfinder programs, panels of mouse and human genes were assessed for their stability under three different experimental conditions: 1) disease progression of Crouzon syndrome (craniosynostosis) in a mouse model, 2) proliferative culture of cranial suture cells isolated from craniosynostosis patients and 3) osteogenesis of a mouse bone marrow stromal cell line. We demonstrate that classical reference genes are not always the most ‘stable’ genes and that gene ‘stability’ is highly dependent on experimental conditions. Selected stable genes, individually or in combination, were then used to normalise osteocalcin and alkaline phosphatase gene expression data during cranial suture fusion in the craniosynostosis mouse model and strategies compared. Strikingly, the expression trends of alkaline phosphatase and osteocalcin varied significantly when normalised to the least stable, the most stable or the three most stable genes.
Conclusion
To minimise errors in evaluating gene expression levels, analysis of a reference panel and subsequent normalization to several stable genes is strongly recommended over normalization to a single gene. In particular, we conclude that use of single, non-validated “housekeeping” genes such as GAPDH, ACTB and 18 S rRNA, currently a widespread practice by researchers in the bone field, is likely to produce data of questionable reliability when changes are 2 fold or less, and such data should be interpreted with due caution.
doi:10.1186/1756-0500-5-222
PMCID: PMC3476976  PMID: 22564426
Osteocalcin; Alkaline phosphatase; 18 S RNA; Gapdh; β-actin; geNorm; Normfinder; Craniosynostosis; Bone; Mineralization
17.  Characterization of the corpus callosum in very preterm and full-term infants utilizing MRI 
NeuroImage  2010;55(2):479-490.
The corpus callosum is the largest white matter tract, important for interhemispheric communication. The aim of this study was to investigate and compare corpus callosum size, shape and diffusion characteristics in 106 very preterm infants and 22 full-term infants. Structural and diffusion magnetic resonance images were obtained at term equivalent. The corpus callosum was segmented, cross-sectional areas were calculated, and shape was analyzed. Fractional anisotropy, mean, axial and radial diffusivity measures were obtained from within the corpus callosum, with additional probabilistic tractography analysis. Very preterm infants had significantly reduced callosal cross sectional area compared with term infants (p=0.004), particularly for the mid-body and posterior sub-regions. Very preterm callosi were more circular (p=0.01). Fractional anisotropy was lower (p=0.007) and mean (p=0.006) and radial (p=0.001) diffusivity values were higher in very preterm infants’ callosi, particularly at the anterior and posterior ends. The volume of tracts originating from the corpus callosum was reduced in very preterm infants (p=0.001), particularly for anterior mid-body (p=0.01) and isthmus tracts (p=0.04). This study characterizes callosal size, shape and diffusion in typically developing infants at term equivalent age, and reports macro- and micro-structural abnormalities as a result of prematurity.
doi:10.1016/j.neuroimage.2010.12.025
PMCID: PMC3035727  PMID: 21168519
Brain; Corpus callosum; Prematurity; Neonate; Infant; Magnetic resonance imaging; Diffusion tensor imaging
18.  Heterozygous Mutations of FREM1 Are Associated with an Increased Risk of Isolated Metopic Craniosynostosis in Humans and Mice 
PLoS Genetics  2011;7(9):e1002278.
The premature fusion of the paired frontal bones results in metopic craniosynostosis (MC) and gives rise to the clinical phenotype of trigonocephaly. Deletions of chromosome 9p22.3 are well described as a cause of MC with variably penetrant midface hypoplasia. In order to identify the gene responsible for the trigonocephaly component of the 9p22.3 syndrome, a cohort of 109 patients were assessed by high-resolution arrays and MLPA for copy number variations (CNVs) involving 9p22. Five CNVs involving FREM1, all of which were de novo variants, were identified by array-based analyses. The remaining 104 patients with MC were then subjected to targeted FREM1 gene re-sequencing, which identified 3 further mutant alleles, one of which was de novo. Consistent with a pathogenic role, mouse Frem1 mRNA and protein expression was demonstrated in the metopic suture as well as in the pericranium and dura mater. Micro-computed tomography based analyses of the mouse posterior frontal (PF) suture, the human metopic suture equivalent, revealed advanced fusion in all mice homozygous for either of two different Frem1 mutant alleles, while heterozygotes exhibited variably penetrant PF suture anomalies. Gene dosage-related penetrance of midfacial hypoplasia was also evident in the Frem1 mutants. These data suggest that CNVs and mutations involving FREM1 can be identified in a significant percentage of people with MC with or without midface hypoplasia. Furthermore, we present Frem1 mutant mice as the first bona fide mouse model of human metopic craniosynostosis and a new model for midfacial hypoplasia.
Author Summary
Although twin and family studies have shown that genes play a critical role in the timing of fusion of skull bones, the identification of specific genes that may be involved has remained somewhat elusive except in the case of the dominantly inherited craniosynostosis syndromes. Metopic craniosynostosis (MC), the early fusion of the forehead (frontal) bones, accounts for 5%–15% of all craniosynostosis cases. This premature fusion of the frontal bones results in a characteristically altered skull shape, termed trigonocephaly, that usually requires surgical correction. Remarkably, the cause of the majority of cases of MC remains unknown (idiopathic). Here, we report genetic variants involving chromosome 9 which involve and interrupt the structure of the FREM1 gene in a large cohort of patients presenting with unisutural metopic craniosynostosis. Micro-computed tomographic (microCT) imaging and quantitative analysis of skull shape reveal both premature fusion of the PF suture (metopic equivalent) and also changes in frontal bone shape supportive of a role for Frem1 in regulation of the metopic suture. Taken together with Frem1 gene and protein expression findings, these data indicate that mutations in FREM1 can give rise to metopic craniosynostosis.
doi:10.1371/journal.pgen.1002278
PMCID: PMC3169541  PMID: 21931569
19.  Social Functioning in Children with Brain Insult 
Social dysfunction is commonly reported by survivors of brain insult, and is often rated as the most debilitating of all sequelae, impacting on many areas of daily life, as well as overall quality of life. Within the early brain insult (EBI) literature, physical and cognitive domains have been of primary interest and social skills have received scant attention. As a result it remains unclear how common these problems are, and whether factors predictive of recovery (insult severity, lesion location, age at insult, environment) in other functional domains (motor, speech, cognition) also contribute to social outcome. This study compared social outcomes for children sustaining EBI at different times from gestation to late childhood to determine whether EBI was associated with an increased risk of problems. Children with focal brain insults were categorized according to timing of brain insult: (i) Congenital (n = 38): EBI: first–second trimester; (ii) Perinatal (n = 33); EBI: third trimester to 1-month post-natal; (iii) Infancy (n = 23): EBI: 2 months–2 years post-birth; (iv) Preschool (n = 19): EBI: 3–6 years; (v) Middle Childhood (n = 31): EBI: 7–9 years; and (vi) Late Childhood (n = 19): EBI: after age 10. Children's teachers completed questionnaires measuring social function (Strengths and Difficulties Questionnaire, Walker–McConnell Scale of Social Competence and School Adjustment). Results showed that children with EBI were at increased risk for social impairment compared to normative expectations. EBI before age 2 years was associated with most significant social impairment, while children with EBI in the preschool years and in late childhood recorded scores closer to normal. Lesion location and laterality were not predictive of social outcome, and nor was social risk. In contrast, presence of disability (seizures) and family function were shown to contribute to aspects of social function.
doi:10.3389/fnhum.2010.00022
PMCID: PMC2904087  PMID: 20631858
social skills; childhood; infant; brain insult; plasticity; vulnerability
20.  Improving the outcome of infants born at <30 weeks' gestation - a randomized controlled trial of preventative care at home 
BMC Pediatrics  2009;9:73.
Background
Early developmental interventions to prevent the high rate of neurodevelopmental problems in very preterm children, including cognitive, motor and behavioral impairments, are urgently needed. These interventions should be multi-faceted and include modules for caregivers given their high rates of mental health problems.
Methods/Design
We have designed a randomized controlled trial to assess the effectiveness of a preventative care program delivered at home over the first 12 months of life for infants born very preterm (<30 weeks of gestational age) and their families, compared with standard medical follow-up. The aim of the program, delivered over nine sessions by a team comprising a physiotherapist and psychologist, is to improve infant development (cognitive, motor and language), behavioral regulation, caregiver-child interactions and caregiver mental health at 24 months' corrected age. The infants will be stratified by severity of brain white matter injury (assessed by magnetic resonance imaging) at term equivalent age, and then randomized. At 12 months' corrected age interim outcome measures will include motor development assessed using the Alberta Infant Motor Scale and the Neurological Sensory Motor Developmental Assessment. Caregivers will also complete a questionnaire at this time to obtain information on behavior, parenting, caregiver mental health, and social support. The primary outcomes are at 24 months' corrected age and include cognitive, motor and language development assessed with the Bayley Scales of Infant and Toddler Development (Bayley-III). Secondary outcomes at 24 months include caregiver-child interaction measured using an observational task, and infant behavior, parenting, caregiver mental health and social support measured via standardized parental questionnaires.
Discussion
This paper presents the background, study design and protocol for a randomized controlled trial in very preterm infants utilizing a preventative care program in the first year after discharge home designed to improve cognitive, motor and behavioral outcomes of very preterm children and caregiver mental health at two-years' corrected age.
Clinical Trial Registration Number
ACTRN12605000492651
doi:10.1186/1471-2431-9-73
PMCID: PMC2797495  PMID: 19954550
21.  One Pathway Can Incorporate either Adenine or Dimethylbenzimidazole as an α-Axial Ligand of B12 Cofactors in Salmonella enterica▿  
Journal of Bacteriology  2007;190(4):1160-1171.
Corrinoid (vitamin B12-like) cofactors contain various α-axial ligands, including 5,6-dimethylbenzimidazole (DMB) or adenine. The bacterium Salmonella enterica produces the corrin ring only under anaerobic conditions, but it can form “complete” corrinoids aerobically by importing an “incomplete” corrinoid, such as cobinamide (Cbi), and adding appropriate α- and β-axial ligands. Under aerobic conditions, S. enterica performs the corrinoid-dependent degradation of ethanolamine if given vitamin B12, but it can make B12 from exogenous Cbi only if DMB is also provided. Mutants isolated for their ability to degrade ethanolamine without added DMB converted Cbi to pseudo-B12 cofactors (having adenine as an α-axial ligand). The mutations cause an increase in the level of free adenine and install adenine (instead of DMB) as an α-ligand. When DMB is provided to these mutants, synthesis of pseudo-B12 cofactors ceases and B12 cofactors are produced, suggesting that DMB regulates production or incorporation of free adenine as an α-ligand. Wild-type cells make pseudo-B12 cofactors during aerobic growth on propanediol plus Cbi and can use pseudo-vitamin B12 for all of their corrinoid-dependent enzymes. Synthesis of coenzyme pseudo-B12 cofactors requires the same enzymes (CobT, CobU, CobS, and CobC) that install DMB in the formation of coenzyme B12. Models are described for the mechanism and control of α-axial ligand installation.
doi:10.1128/JB.01386-07
PMCID: PMC2238215  PMID: 17981976
22.  Unravelling the molecular control of calvarial suture fusion in children with craniosynostosis 
BMC Genomics  2007;8:458.
Background
Craniosynostosis, the premature fusion of calvarial sutures, is a common craniofacial abnormality. Causative mutations in more than 10 genes have been identified, involving fibroblast growth factor, transforming growth factor beta, and Eph/ephrin signalling pathways. Mutations affect each human calvarial suture (coronal, sagittal, metopic, and lambdoid) differently, suggesting different gene expression patterns exist in each human suture. To better understand the molecular control of human suture morphogenesis we used microarray analysis to identify genes differentially expressed during suture fusion in children with craniosynostosis. Expression differences were also analysed between each unfused suture type, between sutures from syndromic and non-syndromic craniosynostosis patients, and between unfused sutures from individuals with and without craniosynostosis.
Results
We identified genes with increased expression in unfused sutures compared to fusing/fused sutures that may be pivotal to the maintenance of suture patency or in controlling early osteoblast differentiation (i.e. RBP4, GPC3, C1QTNF3, IL11RA, PTN, POSTN). In addition, we have identified genes with increased expression in fusing/fused suture tissue that we suggest could have a role in premature suture fusion (i.e. WIF1, ANXA3, CYFIP2). Proteins of two of these genes, glypican 3 and retinol binding protein 4, were investigated by immunohistochemistry and localised to the suture mesenchyme and osteogenic fronts of developing human calvaria, respectively, suggesting novel roles for these proteins in the maintenance of suture patency or in controlling early osteoblast differentiation. We show that there is limited difference in whole genome expression between sutures isolated from patients with syndromic and non-syndromic craniosynostosis and confirmed this by quantitative RT-PCR. Furthermore, distinct expression profiles for each unfused suture type were noted, with the metopic suture being most disparate. Finally, although calvarial bones are generally thought to grow without a cartilage precursor, we show histologically and by identification of cartilage-specific gene expression that cartilage may be involved in the morphogenesis of lambdoid and posterior sagittal sutures.
Conclusion
This study has provided further insight into the complex signalling network which controls human calvarial suture morphogenesis and craniosynostosis. Identified genes are candidates for targeted therapeutic development and to screen for craniosynostosis-causing mutations.
doi:10.1186/1471-2164-8-458
PMCID: PMC2222648  PMID: 18076769

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