translational medicine: bench research & baby brain ...€¦ · translational medicine: bench...

Translational Medicine: Bench Research & Baby Brain Development to Clinical Care

Ruth Eckstein Grunau, PhD Neonatology Division, Pediatrics, University of British Columbia

Developmental Neurosciences & Child Health Child & Family Research Institute

Vancouver BC, Canada

AAAS Vancouver June 2014

BASIC RESEARCH

CLINICAL RESEARCH

HEALTH CARE

MISSION To promote human health by providing a forum for communication and cross-fertilization among basic, translational, and clinical research

DOI: 10.1126/scitranslmed.3004669, 168ra7 (2013);5 Sci Transl Med

et al.Justin M. DeanThrough Disturbances in Neuronal ArborizationPrenatal Cerebral Ischemia Disrupts MRI-Defined Cortical Microstructure

Editor's Summary

determine how differences in brain anatomy and MRI data translate into developmental and cognitive outcomes.developing brain to develop methods for preventing any resulting injury. In addition, long-term studies should help to

More studies are needed to understand how postnatal growth, nutrition, illness, and prenatal ischemia affect the

neurons.disturbances in the branching of neuronal dendrites and abnormal formation of synapse connections with other histological and structural aberrations. The growth impairment seen in the animals' brains by MRI corresponded toischemic episodes. Here, the authors also saw abnormalities in brain development by MRI and correlated them with

undergoafter an in utero ischemic event, and these data were compared to those of age-matched animals that did not gestation time. The lambs were analyzed both by MRI and by histological analysis of the brain at 1, 2, or 4 weeksstructures of fetal lambs that had experienced ischemia in utero at a time that corresponded to about two-thirds of full

took a different approach to studying premature brain development: they analyzed the brainet al.Dean

illnesses the infants may have experienced early in life.brain structure correlated with postnatal growth (and presumably nutrition) even after accounting for any otherof infections or other serious illnesses. A detailed analysis of the MRI scans showed that the development of normal

as well as data on other factors that could affect brain growth, including the presence−−weight, length, and head size−−dates and the other scan when they reached full term. The authors also tracked the infants' growth parameters

The authors performed two sets of MRI scans on these infants: one scan was done about 2 months before their due Vinall and coauthors examined 95 premature newborn babies who were born at 24 to 32 weeks of gestation.

angles using diffusion tensor magnetic resonance imaging (MRI) in human infants and newborn lambs.can be difficult to predict. Now, two sets of authors have obtained new data that approach this problem from differentcomplications. In particular, cognitive problems and developmental delays are common in this patient population and

Despite all of the recent advances in medical care for premature newborns, these infants still often experience

Early Start for Better Brains

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(print ISSN 1946-6234; online ISSN 1946-6242) is published weekly, except theScience Translational Medicine

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MULTIDISCIPLINARY

Full-term 37-41 weeks gestation

Very preterm 24-32 weeks gestation

Neonatal Intensive Care Unit (NICU)

Born 2-4 months early

Neonatal Intensive Care •  Mechanical ventilation •  Infections •  Illness •  Medications •  Surgery •  10 invasive procedures pre day

NEURODEVELOPMENT in Children Born Very Preterm §  Poorer attention, cognition, memory, executive

functions, motor, academics, behavior §  Little knowledge of mechanisms

Fetal Brain Development

(Cunningham FG, Leveno KL, Bloom SL, et al Williams Obstetrics, 22nd Ed)

Brain Development Differs in Infants/Children Born Preterm

§  White matter dysmaturation on MRI/DTI (e.g. Inder et al 1999, Miller et al 2003, Miller & Ferriero 2009, Volpe 2009, Smyser et al 2010)

§  Smaller brain volumes in multiple regions (e.g. Nosarti et al 1999, Peterson et al 2003)

§  Thinner cortex (e.g. Ranger et al 2013)

§  Altered connectivity & networks (e.g. Kostovic & Jovanov-Milosevic 2006, Uhlhaas et al 2009, Doesburg et al 2011)

PRETERM B IRTH

Slower Postnatal Growth Is Associated with DelayedCerebral Cortical Maturation in Preterm NewbornsJillian Vinall,1,2 Ruth E. Grunau,1,2,3,4 Rollin Brant,2,5 Vann Chau,2,3,4 Kenneth J. Poskitt,2,3,6

Anne R. Synnes,2,3,4 Steven P. Miller2,3,7*

Slower postnatal growth is an important predictor of adverse neurodevelopmental outcomes in infants bornpreterm. However, the relationship between postnatal growth and cortical development remains largely un-known. Therefore, we examined the association between neonatal growth and diffusion tensor imaging mea-sures of microstructural cortical development in infants born very preterm. Participants were 95 neonates bornbetween 24 and 32 weeks gestational age studied twice with diffusion tensor imaging: scan 1 at a median of32.1 weeks (interquartile range, 30.4 to 33.6) and scan 2 at a median of 40.3 weeks (interquartile range, 38.7 to42.7). Fractional anisotropy 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 betweenscans was examined in relation to diffusion tensor imaging measures at scans 1 and 2, accounting for gestationalage, birth weight, sex, postmenstrual age, known brain injury (white matter injury, intraventricular hemorrhage,and cerebellar hemorrhage), and neonatal illness (patent ductus arteriosus, days intubated, infection, and necro-tizing enterocolitis). Impaired weight, length, and head growth were associated with delayed microstructural devel-opment of the cortical gray matter (fractional anisotropy: P < 0.001), but not white matter (fractional anisotropy: P =0.529), after accounting for prenatal growth, neonatal illness, and brain injury. Avoiding growth impairment duringneonatal care may allow cortical development to proceed optimally and, ultimately, may provide an opportunity toreduce neurological disabilities related to preterm birth.

INTRODUCTIONSurvival rates of very preterm infants (!32 weeks gestational age) haverisen markedly owing to advances in obstetrical and neonatal intensivecare, but these improvements have not been accompanied by a reduc-tion in long-termmorbidity in this population (1–3). Lower gestationalage and birth weight increase the risk for neonatal comorbidities (forexample, infection and respiratory complications) (4), and these in turnare associated with adverse white matter development (5).

Intrauterine growth restriction (IUGR) refers to infants whose birthweights are <10th percentile because of growth failure in utero. Pre-mature IUGR newborns demonstrate a pattern of discordant gyrifica-tion (6) and reduced cortical volumes (7, 8) when compared to preterminfants born an appropriate weight for gestational age (10th to 90thpercentile) and/or full-term controls. Abnormal cortical volumes inpremature IUGR infants and in experimental models have been asso-ciated with poorer neurodevelopmental outcome (9, 10). Althoughgrowth in utero appears to be important for brain development, thema-jority of premature newborns are not born IUGR (11–13). Therefore,IUGR cannot fully account for the extent of abnormal neurodevelop-ment and brain (gray andwhitematter) injuries observable onmagneticresonance imaging (MRI) within the preterm population (14–16).However, many premature newborns develop persistent growth deficits

postnatally, such that by discharge from the neonatal intensive care unit(NICU), the majority of preterm infants are considered growth-restricted, that is, <10th percentile for their postmenstrual age (12, 13).Postnatal growth failure in the NICU is associated with increased inci-dence of cerebral palsy and neurodevelopmental impairment, afteraccounting for prenatal growth, systemic illness, and brain injury (17).Additionally, the rate of change in cortical surface area between 24and 44 weeks postmenstrual age predicts cognitive ability at 2 and6 years corrected age in children born very preterm (18). At age 7 years,children born preterm demonstrate altered cortical connectivity (19)and synchronization during cognitive tasks relative to full-term con-trols, even in the absence of major disability (20). However, the etiologyfor their altered cortical development and processing remains un-known. Given this, we set out to examine the extent to which poorerpostnatal growth in the NICU relates to diffusion tensor imaging mea-sures of microstructural development of the cerebral cortex in infantsborn very preterm.

Diffusion tensor imaging, anMRI technique, can be used to charac-terize the spatial distribution of water diffusion in each voxel (three-dimensional pixel) of the image, providing ameasure of regional brainmicrostructural development (21). In the cerebral cortex, fractionalanisotropy (FA), a measure of the directionality of water diffusion, de-creases between 25 and 40 weeks postmenstrual age, coincident withthe disappearance of the radial glia and increasing complexity of thedeveloping cortex (22–28). In contrast, in the white matter, FA in-creases with maturation, coincident with maturation of the oligoden-drocyte lineage and early events of myelination (5, 29).

Systemic illness and medical interventions are important determi-nants of brain injury and abnormal brain development (5, 30, 31). More-over, focal brain injuries have been also found to affect overall braindevelopment (32–34), leading to moderate to severe neurodevelopmental

1Neuroscience, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.2Developmental Neurosciences & Child Health, Child & Family Research Institute, Vancou-ver, British Columbia V6H3V4, Canada. 3Pediatrics, University of British Columbia, Vancouver,British Columbia V6H 3V4, Canada. 4BC Children’s & Women’s Hospitals, Vancouver,British Columbia V6H3V4, Canada. 5Statistics, University of British Columbia, Vancouver, BritishColumbia V6T 1Z2, Canada. 6Radiology, University of British Columbia, Vancouver, BritishColumbia V5Z 4E3, Canada. 7Pediatrics, The Hospital for Sick Children and the University ofToronto, Toronto, Ontario M5G 1X8, Canada.*To whom correspondence should be addressed. E-mail: [email protected]

R E S EARCH ART I C L E

www.ScienceTranslationalMedicine.org 16 January 2013 Vol 5 Issue 168 168ra8 1

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et al.Jillian VinallMaturation in Preterm NewbornsSlower Postnatal Growth Is Associated with Delayed Cerebral Cortical

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Preterm Neonatal MRI Brain Imaging

Diffusion tensor imaging (DTI) Fractional Anisotropy (FA) MR spectroscopy imaging N-acetylaspartate (NAA)/choline

Scan 1: Shortly after birth

Scan 2: Term-equivalent age

Birth – Scan 1 Scan 1 – Scan 2 Number of invasive procedures & multiple clinical confounders

Growth (weight) change from early MRI to term equivalent is associated with maturation

of cortical gray matter

Head circumference change in relation to diffusionparameters of the cortical gray matterLongitudinal models revealed that lower gestational age (effect size,!0.030; SE, 0.010; P = 0.004) and slower head growth (effect size,!1.090; SE, 0.025, P < 0.001) between scan 1 and scan 2 were indepen-dently associated with higher FA values in the cortical graymatter, afteradjusting for gestational age, birth weight, sex, brain injury, systemicillness, and age at scan (Table 6). Change in FA reflected change inthe radial diffusion axes (l2 and l3: effect size, 0.265; SE, 0.098; P =0.007) and not the axial diffusion axis (l1: effect size, 0.058; SE, 0.086;P = 0.498). Results from these models are consistent with the modelsabove examining the relationship between weight change and lengthwith diffusion parameters, and therefore provide further support forthe finding that neonatal growth over and above birth weight, brain in-jury, and systemic illness predicted cortical gray matter maturation inthe NICU.

DISCUSSION

This study examined whether neonatal growth is related to micro-structural development of the cerebral cortex in infants born very pre-term. We found that impaired neonatal growth (weight, length, and

head circumference) was significantly associated with delayed corticalmaturation. This independent association of postnatal growth with ear-ly brain development persisted even after controlling for gestational age,birth weight, sex, postmenstrual age, brain injury, and systemic illness.However, it was the cortical gray matter, rather than the white matter,which appeared to be most susceptible to impairments in postnatalgrowth. Similar to our previous studies (31, 33, 37), abnormal develop-ment of the white matter, on the other hand, was more strongly asso-ciated with postnatal infection.

Our finding, which demonstrates vulnerability of the cortical graymatter rather thanwhitematter to the effects of neonatal growth, buildson results from previous studies examining the relationship betweenIUGR and cortical volumes/microstructure early in life (6–8). Animalmodels of IUGR have demonstrated a transient delay in oligodendro-cyte maturation and myelination (38). Although markers of myelinat-ing oligodendrocytes were reduced in utero, white matter volumesreturned to control levels postnatally and persisted into adulthood(38). Thus, it has been suggested that the altered neurodevelopmentassociated with IUGR is likely not due to long-term deficits in myeli-nation. Rather, it is the reduction of cerebral cortical volumes and mi-crostructure associated with prenatal growth restriction that have beenmore pronounced, persistent, and associated with functional impair-ment (8–10).

Microstructural integrity of the cortical gray matter can be inferredfrom the diffusion parameters (22, 23, 39–41). Between 25 and 40weekspostmenstrual age, FA decreases as the developing cortex increases incomplexity (22, 23) with arborization of the basal dendrites, formationof thalamocortical and cortical-cortical connections, and disappearanceof the radial glia (24–28). Therefore, given that higher FA was reflec-tive of change in the radial diffusion axes (l2 and l3) between ~32 and40 weeks postmenstrual age, there may be delayed expansion of neuro-nal process formations, synaptogenesis, and/or apoptosis in the cerebralcortices of infants who are born very preterm and have impairedgrowth. Similarly, in IUGR fetal sheep, the numerical densities ofsynapses in layer 1 of the visual cortex were reduced by 17% comparedto controls (42). Moreover, fewer cells were found in the cortical plate

Table 4. Weight change in relation to mean l1 values of eight corticalregions of interest in the cortical gray matter. l1, axial diffusion axis.

Basic model (n = 95)

Effect size SE P

Gestational age !0.012 0.003 <0.001

Birth weight <0.001 <0.001 0.014

Male !0.005 0.007 0.456

Weight change 0.005 0.031 0.872

Table 3. Weight change in relation to mean l2 and l3 values of eight regions of interest in the cortical gray matter. l2 and l3, radial diffusion axes.

Basic model (n = 95) Brain injury model (n = 95) Extended model (n = 95)

Effect size SE P Effect size SE P Effect size SE P

Gestational age 0.001 0.003 0.703 0.001 0.035 0.784 0.004 0.036 0.260

Birth weight <0.001 <0.001 0.004 <0.001 <0.001 0.007 <0.001 <0.001 0.002

Male !0.009 0.008 0.243 !0.006 0.008 0.447 !0.008 0.008 0.334

Weight change 0.064 0.033 0.056 0.070 0.033 0.034 0.068 0.034 0.043

White matter injury — — — 0.006 0.004 0.147 0.003 0.004 0.498

Intraventricular hemorrhage — — — !0.010 0.004 0.007 !0.009 0.004 0.013

Cerebellar hemorrhage — — — <0.001 0.012 0.978 !0.004 0.013 0.762

Patent ductus arteriosus — — — — — — 0.017 0.011 0.099

Days intubated — — — — — — <0.001 <0.001 0.145

Infection — — — — — — 0.010 0.010 0.344

Necrotizing enterocolitis — — — — — — !0.010 0.016 0.518



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(Vinall et al Sci Transl Med 2013)

Weight change in relation to FA of the white matterWeight change was not significantly associated with FA values in thewhite matter in the basic statistical model (effect size, !0.035; SE,0.055; P = 0.529; table S2). Therefore, white matter maturation appearsto 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 afteradjusting for gestational age, birth weight, sex, brain injury, systemicillness, weight change, and age at scan.

Weight change in relation to diffusion parameters of thecortical gray matter excluding infants who receivedpostnatal corticosteroidsNeither 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 gestationalage, birth weight, sex, brain injury, systemic illness, and age at scan.Nonetheless, as a sensitivity analysis, we examined weight change inrelation to FA of the cortical gray matter excluding infants who re-ceived postnatal corticosteroids. In newborns who did not receive cor-ticosteroids postnatally, lower gestational age (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 higherFA values in the cortical gray matter, in longitudinal models adjustingfor gestational age, sex, brain injury, systemic illness, and age at scan(table S3). Given that the relationship between weight change and FAvalues did not change meaningfully after the exclusion of infants whoreceived postnatal corticosteroids (hydrocortisone and/or dexamethasone),exposed infants were included in all other longitudinal models.

Length change in relation to diffusion parameters of thecortical gray matterLongitudinal models revealed that lower gestational age (effect size,!0.030; SE, 0.010; P = 0.002), confirmed necrotizing enterocolitis (effectsize, 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 indepen-dently associated with higher FA values in the cortical graymatter, afteradjusting for birth weight, sex, brain injury, systemic illness, and age atscan (Table 5). Change in FA reflected changes in the radial diffusionaxes (l2 and l3: effect size, 0.189; SE, 0.073; P= 0.010) and not the axialdiffusion axis (l1: effect size, !0.043; SE, 0.062; P = 0.488).

Table 2.Weight change in relation to mean FA values of eight regions of interest in the cortical gray matter. Weight change = weight at scan 2 (MRIat ~40 weeks postmenstrual age) ! weight at scan 1 (MRI at ~32 weeks postmenstrual age).

Basic model (n = 95) Brain injury model (n = 95) Extended model (n = 95)

Effect size SE P Effect size SE P Effect size SE P

Gestational age !0.027 0.010 0.007 !0.028 0.010 0.006 !0.038 0.011 <0.001

Birth weight <!0.001 <0.001 0.025 <!0.001 <0.001 0.032 <!0.001 <0.001 0.016

Male 0.037 0.022 0.101 0.034 0.023 0.131 0.031 0.023 0.189

Weight change !0.422 0.087 <0.001 !0.424 0.088 <0.001 !0.410 0.089 <0.001

White matter injury — — — !0.010 0.012 0.404 !0.009 0.012 0.942

Intraventricular hemorrhage — — — !0.002 0.010 0.840 !0.004 0.010 0.708

Cerebellar hemorrhage — — — !0.009 0.033 0.790 0.004 0.034 0.917

Patent ductus arteriosus — — — — — — !0.036 0.030 0.242

Days intubated — — — — — — !0.001 0.001 0.238

Infection — — — — — — !0.047 0.029 0.111

Necrotizing enterocolitis — — — — — — 0.040 0.046 0.394

Fig. 1. Description of the regions of interest obtained within the corti-cal gray matter. (A and B) Diffusion tensor image-encoded anisotropycolor maps of an infant born at 26.29 weeks gestation and scannedat 30 weeks postmenstrual age. The images demonstrate the relativelyhigh FA of the cerebral cortex typical for this age. The color conventionused to display the predominant diffusion direction has red represent-ing right-left, green representing anterior-posterior, and blue represent-ing superior-inferior anatomical directions (56, 58). Eight cerebralcortical regions of interest were examined, and values of each regionwere averaged bilaterally: (a) precentral gyrus, (b) postcentral gyrus,(c) secondary somatosensory cortex, (d) superior frontal gyrus, (e) dor-solateral prefrontal cortex, (f ) ventrolateral prefrontal cortex, (g) anteriorinsula, and (h) occipital gray matter.



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PRETERM B IRTH

Prenatal Cerebral Ischemia Disrupts MRI-DefinedCortical Microstructure Through Disturbancesin Neuronal ArborizationJustin M. Dean,1*† Evelyn McClendon,1† Kelly Hansen,1 Aryan Azimi-Zonooz,1 Kevin Chen,1

Art Riddle,1 Xi Gong,1 Elica Sharifnia,1 Matthew Hagen,1 Tahir Ahmad,1 Lindsey A. Leigland,2,3

A. Roger Hohimer,4‡ Christopher D. Kroenke,2,3‡ Stephen A. Back1,5§

Children who survive preterm birth exhibit persistent unexplained disturbances in cerebral cortical growth with as-sociated cognitive and learning disabilities. Themechanisms underlying these deficits remain elusive.We used ex vivodiffusionmagnetic resonance imaging todemonstrate in a preterm large-animalmodel that cerebral ischemia impairscortical growth and the normal maturational decline in cortical fractional anisotropy (FA). Analysis of pyramidal neu-rons revealed that cortical deficits were associated with impaired expansion of the dendritic arbor and reduced syn-aptic density. Together, these findings suggest a link between abnormal cortical FA and disturbances of neuronalmorphological development. To experimentally investigate this possibility, wemeasured the orientation distributionof dendritic branches and observed that it corresponds with the theoretically predicted pattern of increased anisot-ropywithin cases that exhibited elevated cortical FA after ischemia.We conclude that cortical growth impairments areassociated with diffuse disturbances in the dendritic arbor and synapse formation of cortical neurons, which mayunderlie the cognitive and learning disabilities in survivors of preterm birth. Further, measurement of cortical FAmay be useful for noninvasively detecting neurological disorders affecting cortical development.

INTRODUCTIONThe leading causes of chronic neurological impairment in survivors ofpremature birth include a spectrum of cerebral palsy and intellectualdisabilities (1, 2). Although preterm infants most commonly sustainwhite matter injury (WMI), there has been a recent decline in its overallseverity (3). Nevertheless, up to 25 to 50% of preterm survivors continueto display awide range of unexplained cognitive and learning disabilities,attentional deficits, and impaired social interactions (4), which suggestsa role for graymatter pathology. Indeed, several largemagnetic resonanceimaging (MRI) studies have identified significant reductions in cerebralcortical and subcortical growth in survivors of preterm birth (5–10). Thisimpairment in cortical growth correlates with cognitive, but not motor,outcomes in early childhood (11).

The underlying cellular mechanisms related to impaired corticalgrowth in contemporary cohorts of preterm survivors continue to becontroversial. Loss of cortical neurons and axonal degeneration werereported in preterm human autopsy cases with significant necroticWMI (12, 13). However, no neuronal loss was observed in cases withnonnecrotic diffuse WMI (12), which is the predominant type of WMIseen with modern medical management of premature infants (3, 14).Because a mechanism of cortical neuronal degeneration appearsinconsistent with the patterns of WMI currently observed in pretermsurvivors, we considered an alternative mechanism in which cerebral

ischemia disrupts cortical development via disturbances in neuronalmaturation.

During the last trimester of fetal development, before the onset ofmyelination, a marked expansion of the cerebral cortex occurs (15) thatis associated with prolific axonal growth, dendritic sprouting, and syn-apse formation (16, 17). This highly active period of neuronal elabora-tion may be particularly sensitive to insults that disrupt normal corticalmaturation. There is, however, no defined role for reduced neuronalcomplexity as a cause of cortical volume loss and cognitive deficits insurvivors of preterm birth. Diffusion tensor imaging (DTI) studies inhumans (18, 19) and other species (20–22) demonstrated that normalcorticalmaturation is associatedwith a decline inMRImeasurements offractional anisotropy (FA), which is hypothesized to arise from in-creasing morphological complexity of cortical neurons (18, 21, 23). Re-cently, we demonstrated that water diffusion anisotropy is directlyrelated to characteristics of neuronal morphology, including the orien-tation and maturation of the arbor of neuronal cell processes (24), andthat FA measurements have sufficient sensitivity to discriminate be-tween normal and abnormal cortical differentiation (25). Thus, mea-surement of cortical FA may be useful for detecting neurologicaldisorders affecting cortical development.

Hence, we tested the hypothesis that cerebral ischemia disrupts cor-tical maturation by disturbing dendrite and spine formation, but not bycausing neuronal loss, and that these changes disrupt cortical micro-structure as defined by MRI measurements of FA. We used a well-established preterm fetal sheep cerebral ischemia model (26–29) thatgenerates a spectrum of moderate WMI, which closely reproducesthe patterns seen in contemporary cohorts of human preterm survivors(3). We used high-field ex vivo MRI diffusion-weighted data combinedwith a Golgi impregnation analysis of neuronal complexity to providean explanation for cognitive disturbances in preterm survivors that in-volves diffuse abnormalities in the dendritic arbor and synapse forma-tion of cortical neurons. By applying a recently developedmathematical

1Department of Pediatrics, Oregon Health & Science University, Portland, OR 97239, USA.2Advanced ImagingResearchCenter, OregonHealth& ScienceUniversity, Portland, OR 97239,USA. 3Department of Behavioral Neuroscience, Oregon Health & Science University, Portland,OR 97239, USA. 4Department of Obstetrics and Gynecology, Oregon Health & ScienceUniversity, Portland, OR 97239, USA. 5Department of Neurology, Oregon Health & ScienceUniversity, Portland, OR 97239, USA.*Present address: Department of Physiology, Faculty of Medical and Health Sciences,University of Auckland, Auckland 1142, New Zealand.†These authors contributed equally to this work.‡These senior investigators contributed equally to this work.§To whom correspondence should be addressed. E-mail: [email protected]



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arbor and associated spines could explain the disturbances in corticalgrowth and cognition that are now the major cause of disability in sur-vivors of premature birth.

Deficits in dendritic branching and spine formation are a feature ofseveral human neurodevelopmental disorders associated with mentalretardation, including Down and Rett syndromes [for review, see(31)]. Further, infants with extensive necrotic WMI and axonopathyor with direct cortical lesions exhibit marked alterations in neuronaldendritic development (32, 33). Currently, there is no direct evidencefor a similar impairment of cortical circuitry in preterm survivors withcognitive disabilities. Nevertheless, preterm infants can exhibit dysma-ture electroencephalogram (EEG) patterns at 6 weeks of life (34) andlate adolescence (35), which could be a result of abnormal corticalcircuitry. As observed here for fetal ovine cerebral development, humancortical neurons display amarked increase in dendritic arborization andsynapse formation during the latter half of gestation (16, 17) thatparallels a marked expansion in cortical volume (15, 36). During thisperiod in development, ischemia may disrupt cortical growth and

connectivity. Unexpectedly, we found that moderate cerebral ischemiain sheep was not associated with early or delayed neuronal loss orapoptosis in the cortex, but did trigger significant disruptions in den-dritic maturation and connectivity. By contrast, in the same animals,moderate cerebral ischemia was sufficient to cause diffuse but largelynonnecrotic WMI with selective degeneration of pre-oligodendrocytes(27). Thus, an ischemia-reperfusion insult that we have shown to besimilar in magnitude in the cortex and white matter (29) appears totrigger very different neuronal versus glial responses. Cortical neuronswere markedly more resistant to degeneration but were susceptible toglobal disturbances in dendritic maturation independent of neuronalsupragranular or infragranular location. Similarly, neurons in the pre-termhuman cerebral cortex weremarkedlymore resistant to damage incases where the cerebral white matter sustained significant nonnecroticWMI and glial degeneration (37). Cortical growth is a strong predictorof later neurocognitive outcome in preterm survivors because neuro-cognitive testing at 2 and 6 years of age correlated with the rate of ce-rebral cortical growth between 24 and 44 weeks postmenstrual age

(11). Widespread disturbances in matu-ration of the dendritic arbormay providea structural explanation for the decreasein cortical growth observed in survivorsof premature birth.

The normal maturational decrease incortical FA that occurs in several speciesincluding human (18–22) is hypothesizedto relate to morphological differentiationof the cortical neuropil (18, 21).We foundthat the fetal ovine cortex also demon-strated a progressive developmental de-crease in cortical FA, which was disruptedat 4 weeks after preterm ischemia. Con-sistent with the notion that normal de-velopmental changes in FA are related toincreasing structural complexity of neu-rons, we observed a progressive increase incortical volume and dendritic arbor com-plexity during the 4-week period of ourstudy. It has been proposed that in theimmature cortex, water diffusion is highlyanisotropic because of the predominantalignment of cellular processes perpen-dicular to the pial surface (18, 38), thusrestricting water diffusion in a directionparallel to the pial surface. With neuronaldifferentiation and elaboration of the den-dritic arbor, the distribution of orienta-tions of phospholipid bilayer structuresthat restrict water diffusion is believed tobecomemore isotropic, causing cortical FAto progressively decrease (21). In support,we recently quantified the orientation dis-tributions of processes ofGolgi-impregnatedcortical neurons in a model of bilateralenucleation in neonatal ferrets (25) andfound that impairment in the develop-mental decline in cortical FA was relatedto a reduced complexity of dendritic arbors

Fig. 4. Abnormal development of basal dendritic arborization of cortical pyramidal neuronswas seen in thecerebral cortex at 4 weeks after ischemia. (A) Example of Golgi-stained pyramidal neurons in the controlcortex. Scale bar, 20 mm. (B) Example of computer-assisted reconstructions of representative neurons inthe control (Con) and ischemia (HI) groups. (C to E) Total number of basal dendritic branches (C), total basaldendritic length (D), and total number of nodes (E) in control (black bars) and ischemia (white bars) groups.(F) Sholl analysis of the number of basal dendritic intersections in control (closed circles) and ischemia (opencircles) groups. (G to I) Branch order analysis of total number of basal dendritic branches (G), total basaldendritic length (H), and total number of nodes (I) in control (black bars) and ischemia (white bars) groups.Analyseswere performed independent of cortical location. n= 100 cells per group. Data aremeans ± SEM.*P < 0.05, **P < 0.01, ***P < 0.001.



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Normal Control Hypoxic Ischemia

Dean et al Sci Transl Med 2013

Sheep brain development

Sheep brain development

•  Reduced –  total dendri8c length –  number of branches –  branch endings

•  Notably, the dendri8c arbor was most simplified closer to the cell body, where synap8c integra8on occurs

neurons confirmed that there was no chronic loss of neu-rons. Cortical volume loss was thus accompanied by anincreased packing density of neurons. This unexpectedresult was explained by detailed analysis of the matura-tion of the dendritic arbor of pyramidal neurons, themajor population of cortical projection neurons. Duringnormal development, pyramidal neurons are highlyimmature in the preterm cerebral cortex, but in near-term animals the dendritic arbor becomes highly arbor-ized, which coincides with a marked increase in corticalvolume (Fig 4). In response to preterm ischemia, corticalgrowth impairment was accompanied by a significantreduction in the complexity of the dendritic arbor, con-sistent with the notion that neuronal maturation was dis-rupted in the setting of cerebral ischemia. Compared tocontrols, the ischemic animals displayed neuronal dysma-turation that was reflected in a reduction in the totaldendritic length as well as the number of branches,branch endings, and branch points. Notably, the dendri-tic arbor was most simplified closer to the cell body,where synaptic integration occurs.

This neuronal dysmaturation response was notrestricted to the cerebral cortex but has been observed inother brain regions. Disturbances in dendritic arboriza-tion have also been reported in CA1 pyramidal neuronsin the hippocampus in a near-term rodent model ofhypoxia–ischemia in which dendritic arborization distur-bances occurred as a response to significant necrotic cere-bral injury and neuronal loss.169 The caudate nucleusalso displayed reduced growth in response to ischemiabut with no apparent loss of c-aminobutyric acidergic(GABAergic) medium spiny projection neurons or inter-neurons.166 Deletion of GABAergic interneurons hasbeen proposed to occur during their migration throughhuman white matter during the period of high risk forWMI.170 However, reduced growth of the caudate wasnot explained by loss of GABAergic neurons, but ratherby a significant disruption in the dendritic arbor of cau-date projection neurons. Neuronal dysmaturation wasdefined by disrupted maturation of the dendritic arbor.Hence, widespread disturbances in maturation of corticaland caudate projection neurons occurred in associationwith nondestructive cerebral lesions that had diffuseWMI but lacked significant neuronal degeneration.

Neuronal Dysmaturation and Disturbancesin Synaptic ActivityA role for neuronal dysmaturation in cognitive andbehavioral disturbances in preterm survivors is suggestedby analysis of dendritic spines, the key sites for synapticactivity. In response to ischemia, reduced numbers ofspines were observed on the dysmature dendrites of pro-

FIGURE 4: The preterm brain is enriched in immature neu-rons that do not degenerate in response to ischemia, butare highly susceptible to impaired maturation that manifestsas a less mature dendritic arbor with reduced spine density.(A) A typical pyramidal neuron from the preterm cerebralcortex of a control fetal sheep. Note the paucity of proc-esses in contrast to the highly complex dendritic arbor of apyramidal neuron from a near-term animal (B).168 (C, D) Inresponse to preterm ischemia, cortical pyramidal neuronsdisplay disrupted maturation. Note that the typical controlcell (C) is more highly arborized in contrast to the responseto transient cerebral ischemia that resulted in a more simpli-fied dendritic arbor (D). The relative complexity of the cellscan be appreciated from the overlay of the red concentricScholl rings, which illustrates that the processes of the dys-mature neurons intersect less frequently with the rings. Theyellow, white, pink, green, and blue lines represent first-,second-, third-, fourth-, and fifth-order branches, respec-tively, from the soma. Note the overall reduction in the sizeand complexity of the branching pattern in D. (E) Reduc-tions in cortical growth also manifest as disturbances incortical anisotropy. Note the normal progressive decline infractional anisotropy (FA) in controls (blue) between pre-term and near-term cortical development, as adapted fromDean et al.168 In response to ischemia, higher cortical ani-sotropy (more restricted water diffusion) was observed inresponse to ischemia (red) relative to control (blue), whichwas related to the reduced complexity of the dendriticarbor of the ischemic neurons (eg, in D) versus controls (eg,in C). Scale bars 5 20nm. HI 5 hypoxia–ischemia.

ANNALS of Neurology

476 Volume 75, No. 4



Editor's Summary














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P E R S P E C T I V E

www.ScienceTranslationalMedicine.org 16 January 2013 Vol 5 Issue 168 168ps2 1

Development of the brain’s cerebral cortex is regulated by a complex interplay between an unfolding genetic program and the en-vironment. Neuronal proliferation, migra-tion, di� erentiation, and circuit formation follow de� ned time scales, and their cho-reography is controlled by extrinsic factors mediated through blood-brain and placen-tal barriers (1). Premature birth can alter the normal developmental processes. If the preterm brain does not possess the ability to recover from these disruptions, then they may be associated with neurodevelopmental impairments, with some consequences not manifesting until years later. Understand-ing the timing and rationale for therapeutic intervention to prevent developmental ab-normalities in these infants requires the de-tailed monitoring of clinical parameters and the study of mechanistic aspects in animals.

Two papers published in this issue of Sci-ence Translational Medicine characterize ad-verse neurodevelopmental outcomes in pre-term newborn infants. Vinall and colleagues (2) examine the association between neona-tal growth (weight, length, and head size) and di� usion tensor imaging (DTI) measures of cortical development in infants born preterm (between 24 and 32 weeks gestational ages). Dean and colleagues (3) use a sheep model to examine the consequences of prenatal ce-rebral ischemia on cortical volume and relate magnetic resonance imaging (MRI)–de� ned cortical microstructure with brain histologi-cal analysis. Both of these translational stud-ies draw attention to grey matter damage and suggest that avoiding somatic growth impair-

ment during neonatal care may allow cortical development to proceed optimally and thus reduce neurological disabilities related to preterm birth.

DECIPHERING THE DEVELOPING CORTEXIt is well documented that survivors of pre-term birth show a reduction in cerebral cortical volume relative to infants born at term. Premature birth—even in the absence of overt hypoxic-ischemic injury—is associ-ated with loss of both cortical and subcorti-cal (thalamus and basal ganglia) grey-matter volume, and the more preterm the infant, the greater the reduction in volume (4). � is re-duction in the volumes of cortical and sub-cortical (thalamus, basal ganglia) grey mat-ter is not always associated with overt signs of white matter volume loss or injury. � ese observations are supported by the study by Vinall et al. (2), which showed that lower gestational age is associated with higher cor-tical fractional anisotropy (FA)—a di� usion imaging measure that re� ects axon density and diameter as well as extent of myelina-tion (white matter). FA values change during development of the cerebral cortex and are higher at early developmental stages because the majority of processes are radial—that is, structures mature and migrate from sites of neurogenesis (Fig. 1). Later, a� er completion of neurogenesis, radial glia progenitors dis-appear or are transformed to astrocytes (star-shaped glial cells that support neurons of the brain and contribute to the blood-brain barrier); cortical connections that transport nerve impulses from sense organs (a� erent) and to subcortical structures (e� erent) ma-ture; and neurons develop extensive branch-ing and arborization (treelike arrangement of processes). � ese processes are re� ected in a normal reduction in FA values with increas-ing age of the cortex.

� e Vinall et al. (2) study used a noninva-sive imaging method—di� usion tensor MRI [at 1.5 Tesla (T), a unit of measurement that

indicates the strength of a magnetic � eld]—to map the di� usion of water in the devel-oping brains of a large cohort (N = 95) of newborn preterm human infants (neonates). � e di� usion patterns of water provide an indirect measure of the existing macromo-lecular and structural elements—“obstacles” that alter � uid � ow in the organ being mea-sured, decreasing anisotropy—and thus yield information about the state (normal or abnormal) of the tissue architecture. � e authors investigated changes in the cortical microstructure of the preterm neonates over time; scan 1 was taken at ~32 weeks gesta-tion and scan 2 at ~40 weeks postmenstrual age (term equivalent). From these scans, Vi-nall et al. documented the expected decreas-es in the FA of cortical grey matter with the increasing postmenstrual age of the preterm neonates and then sought to establish which clinical parameters in� uenced cortical devel-opment a� er preterm delivery. Although the authors’ term-equivalent cortical data could be compared with data from term-born con-trol neonates, a complete understanding of the normality of the observed decreases in cortical FA over time a� er preterm delivery would ideally require a parallel study of nor-mal fetal cortical development in utero. Fetal di� usion studies have been performed, but in order to produce comparable data on cor-tical anisotropy, the imaging techniques re-quire considerable optimization to overcome the e� ects of maternal and fetal motion and of the inherently poor signal-to-noise ratio of the images.

MECHANISMS BEHIND THE MAYHEMAlthough it is accepted that cortical devel-opment is altered by preterm delivery, the causal mechanisms remain unclear. � ere are several alternative hypotheses. Accord-ing to the � rst hypothesis, cortical volume loss is a product of neuronal loss of function and death (primary neuronal degeneration), possibly accompanied by so-called second-ary retrograde neuro-axonal degeneration, which results from primary injury to imma-ture white matter and subplate, a common occurrence in preterm infants. � e second hypothesis is that cortical volume loss arises because of a failure of neuronal maturation rather than cell death or axonal degenera-tion. A third hypothesis—which implies that neither neuronal degeneration nor aborted maturation accounts for the brain volume decrease—suggests that thalamic areas of the brain (Fig. 1) are the primary sites that show altered growth in preterm infants (Fig. 2).

N E U R O L O G Y

Brain Maturation After Preterm BirthZoltán Molnár1* and Mary Rutherford2, 3

*Corresponding author. E-mail: [email protected]

1Department of Physiology, Anatomy and Genetics, University of Oxford, Le Gros Clark Building, South Parks Road, Oxford OX1 3QX, UK. 2Centre for the Developing Brain, Perinatal Imaging & Health, Imaging Sciences & Biomedical Engineering Division, King’s College Lon-don, London SE1 7EH, UK. 3Perinatal Imaging Group, Robert Steiner MR Unit, Medical Research Council (MRC) Clinical Sciences Centre, Imperial College, Ham-mersmith Hospital, London W12 OHS, UK.


Two translational studies—one in humans and one in sheep—suggest that (i) premature birth is associated with delayed maturation of grey matter in the cerebral cortex and (ii) medical care that prohibits impairment of growth in premature neonates may enhance cortical development and reduce neurological disabilities associated with preterm birth.

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www.ScienceTranslationalMedicine.org 16 January 2013 Vol 5 Issue 168 168ps2 1

Development of the brain’s cerebral cortex is regulated by a complex interplay between an unfolding genetic program and the en-vironment. Neuronal proliferation, migra-tion, di� erentiation, and circuit formation follow de� ned time scales, and their cho-reography is controlled by extrinsic factors mediated through blood-brain and placen-tal barriers (1). Premature birth can alter the normal developmental processes. If the preterm brain does not possess the ability to recover from these disruptions, then they may be associated with neurodevelopmental impairments, with some consequences not manifesting until years later. Understand-ing the timing and rationale for therapeutic intervention to prevent developmental ab-normalities in these infants requires the de-tailed monitoring of clinical parameters and the study of mechanistic aspects in animals.

Two papers published in this issue of Sci-ence Translational Medicine characterize ad-verse neurodevelopmental outcomes in pre-term newborn infants. Vinall and colleagues (2) examine the association between neona-tal growth (weight, length, and head size) and di� usion tensor imaging (DTI) measures of cortical development in infants born preterm (between 24 and 32 weeks gestational ages). Dean and colleagues (3) use a sheep model to examine the consequences of prenatal ce-rebral ischemia on cortical volume and relate magnetic resonance imaging (MRI)–de� ned cortical microstructure with brain histologi-cal analysis. Both of these translational stud-ies draw attention to grey matter damage and suggest that avoiding somatic growth impair-

ment during neonatal care may allow cortical development to proceed optimally and thus reduce neurological disabilities related to preterm birth.

DECIPHERING THE DEVELOPING CORTEXIt is well documented that survivors of pre-term birth show a reduction in cerebral cortical volume relative to infants born at term. Premature birth—even in the absence of overt hypoxic-ischemic injury—is associ-ated with loss of both cortical and subcorti-cal (thalamus and basal ganglia) grey-matter volume, and the more preterm the infant, the greater the reduction in volume (4). � is re-duction in the volumes of cortical and sub-cortical (thalamus, basal ganglia) grey mat-ter is not always associated with overt signs of white matter volume loss or injury. � ese observations are supported by the study by Vinall et al. (2), which showed that lower gestational age is associated with higher cor-tical fractional anisotropy (FA)—a di� usion imaging measure that re� ects axon density and diameter as well as extent of myelina-tion (white matter). FA values change during development of the cerebral cortex and are higher at early developmental stages because the majority of processes are radial—that is, structures mature and migrate from sites of neurogenesis (Fig. 1). Later, a� er completion of neurogenesis, radial glia progenitors dis-appear or are transformed to astrocytes (star-shaped glial cells that support neurons of the brain and contribute to the blood-brain barrier); cortical connections that transport nerve impulses from sense organs (a� erent) and to subcortical structures (e� erent) ma-ture; and neurons develop extensive branch-ing and arborization (treelike arrangement of processes). � ese processes are re� ected in a normal reduction in FA values with increas-ing age of the cortex.

� e Vinall et al. (2) study used a noninva-sive imaging method—di� usion tensor MRI [at 1.5 Tesla (T), a unit of measurement that

indicates the strength of a magnetic � eld]—to map the di� usion of water in the devel-oping brains of a large cohort (N = 95) of newborn preterm human infants (neonates). � e di� usion patterns of water provide an indirect measure of the existing macromo-lecular and structural elements—“obstacles” that alter � uid � ow in the organ being mea-sured, decreasing anisotropy—and thus yield information about the state (normal or abnormal) of the tissue architecture. � e authors investigated changes in the cortical microstructure of the preterm neonates over time; scan 1 was taken at ~32 weeks gesta-tion and scan 2 at ~40 weeks postmenstrual age (term equivalent). From these scans, Vi-nall et al. documented the expected decreas-es in the FA of cortical grey matter with the increasing postmenstrual age of the preterm neonates and then sought to establish which clinical parameters in� uenced cortical devel-opment a� er preterm delivery. Although the authors’ term-equivalent cortical data could be compared with data from term-born con-trol neonates, a complete understanding of the normality of the observed decreases in cortical FA over time a� er preterm delivery would ideally require a parallel study of nor-mal fetal cortical development in utero. Fetal di� usion studies have been performed, but in order to produce comparable data on cor-tical anisotropy, the imaging techniques re-quire considerable optimization to overcome the e� ects of maternal and fetal motion and of the inherently poor signal-to-noise ratio of the images.

MECHANISMS BEHIND THE MAYHEMAlthough it is accepted that cortical devel-opment is altered by preterm delivery, the causal mechanisms remain unclear. � ere are several alternative hypotheses. Accord-ing to the � rst hypothesis, cortical volume loss is a product of neuronal loss of function and death (primary neuronal degeneration), possibly accompanied by so-called second-ary retrograde neuro-axonal degeneration, which results from primary injury to imma-ture white matter and subplate, a common occurrence in preterm infants. � e second hypothesis is that cortical volume loss arises because of a failure of neuronal maturation rather than cell death or axonal degenera-tion. A third hypothesis—which implies that neither neuronal degeneration nor aborted maturation accounts for the brain volume decrease—suggests that thalamic areas of the brain (Fig. 1) are the primary sites that show altered growth in preterm infants (Fig. 2).

N E U R O L O G Y

Brain Maturation After Preterm BirthZoltán Molnár1* and Mary Rutherford2, 3

*Corresponding author. E-mail: [email protected]

1Department of Physiology, Anatomy and Genetics, University of Oxford, Le Gros Clark Building, South Parks Road, Oxford OX1 3QX, UK. 2Centre for the Developing Brain, Perinatal Imaging & Health, Imaging Sciences & Biomedical Engineering Division, King’s College Lon-don, London SE1 7EH, UK. 3Perinatal Imaging Group, Robert Steiner MR Unit, Medical Research Council (MRC) Clinical Sciences Centre, Imperial College, Ham-mersmith Hospital, London W12 OHS, UK.


Two translational studies—one in humans and one in sheep—suggest that (i) premature birth is associated with delayed maturation of grey matter in the cerebral cortex and (ii) medical care that prohibits impairment of growth in premature neonates may enhance cortical development and reduce neurological disabilities associated with preterm birth.

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The editors suggest the following Related Resources

Science Transla5onal Medicine

•  INTRACRANIAL HEMORRHAGE Overexpression of Vascular Endothelial Growth Factor in the

Germinal Matrix Induces Neurovascular Proteases and Intraventricular Hemorrhage Dianer Yang, Jessica M. Baumann, Yu-‐Yo Sun, Mianzhi Tang, Richard ScoQ Dunn, Ann L. Akeson, Steven G. Kernie, Suhas Kallapur, Diana M. Lindquist, Eric J. Huang, Stanley Steven PoQer, Hung-‐Chi Liang, and Chia-‐Yi Kuan Sci Transl Med 10 July 2013 5:193ra90

•  BRAIN DEVELOPMENT Cross-‐Hemispheric Func5onal Connec5vity in the Human Fetal Brain Moriah E. Thomason, Maya T. Dassanayake, Stephen Shen, Yashwanth Katkuri, Mitchell Alexis, Amy L. Anderson, Lami Yeo, Swa8 Mody, Edgar Hernandez-‐Andrade, Sonia S. Hassan, Colin Studholme, Jeong-‐Won Jeong, and Roberto Romero Sci Transl Med 20 February 2013 5:173ra24

•  PRETERM BIRTH A Small-‐Molecule Smoothened Agonist Prevents Glucocor5coid-‐Induced Neonatal Cerebellar Injury Vivi M. Heine, Amelie Griveau, Cheryl Chapin, Philip L. Ballard, James K. Chen, and David H. Rowitch Sci Transl Med 19 October 2011 3:105ra104

•  PRETERM BIRTH Preterm Cerebellar Growth Impairment AKer Postnatal Exposure to Glucocor5coids Emily W. Y. Tam, Vann Chau, Donna M. Ferriero, A. James Barkovich, Kenneth J. PoskiQ, Colin Studholme, Eric D.-‐Y. Fok, Ruth E. Grunau, David V. Glidden, and Steven P. Miller Sci Transl Med 19 October 2011 3:105ra105

•  PREMATURE INFANTS Integra5on of Early Physiological Responses Predicts Later Illness Severity in Preterm Infants Suchi Saria, Anand K. Rajani, Jeffrey Gould, Daphne Koller, and Anna A. Penn Sci Transl Med 8 September 2010 2:48ra65



Editor's Summary














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ALTERED BRAIN DEVELOPMENT IN PRETERM NEONATES

Sci Transl Med 16 January 2013: Vol. 5, Issue 168

•  Human preterm neonates (Vinall et al) •  Newborn lambs (Dean et al) •  Editor’s Summary (Hammond) •  Perspective Commentary (Molnar & Rutherford)

Animal Studies of Fetal Stress

(Matthews 2002)

Neonatal pain in relation to postnatal growth in infants born very preterm

Jillian Vinall a,b, Steven P. Miller b,c, Vann Chau b,c, Susanne Brummelte b,c, Anne R. Synnes b,c,Ruth E. Grunau a,b,!a Department of Neuroscience, University of British Columbia, Vancouver, BC, Canadab Developmental Neurosciences & Child Health, Child & Family Research Institute, Vancouver, BC, Canadac Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

a r t i c l e i n f o

Article history:Received 11 October 2011Received in revised form 17 December 2011Accepted 8 February 2012

Keywords:PainGrowthPretermPrematurityStress

a b s t r a c t

Procedural pain is associated with poorer neurodevelopment in infants born very preterm (632 weeksgestational age), however, the etiology is unclear. Animal studies have demonstrated that early environ-mental stress leads to slower postnatal growth; however, it is unknown whether neonatal pain-relatedstress affects postnatal growth in infants born very preterm. The aim of this study was to examinewhether greater neonatal pain (number of skin-breaking procedures adjusted for medical confounders)is related to decreased postnatal growth (weight and head circumference [HC] percentiles) early in lifeand at term-equivalent age in infants born very preterm. Participants were n = 78 preterm infants born632 weeks gestational age, followed prospectively since birth. Infants were weighed and HC measuredat birth, early in life (median: 32 weeks [interquartile range 30.7–33.6]) and at term-equivalent age(40 weeks [interquartile range 38.6–42.6]). Weight and HC percentiles were computed from sex-specificBritish Columbia population-based data. Greater neonatal pain predicted lower body weight (Waldv2 = 7.36, P = 0.01) and HC (Wald v2 = 4.36, P = 0.04) percentiles at 32 weeks postconceptional age, afteradjusting for birth weight percentile and postnatal risk factors of illness severity, duration of mechanicalventilation, infection, and morphine and corticosteroid exposure. However, later neonatal infection pre-dicted lower weight percentile at term (Wald v2 = 5.09, P = 0.02). Infants born very preterm undergorepetitive procedural pain during a period of physiological immaturity that appears to impact postnatalgrowth, and may activate a downstream cascade of stress signaling that affects later growth in the neo-natal intensive care unit.

! 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

1. Introduction

Infants born very preterm (632 weeks gestational age [GA])survive outside the protective intrauterine environment duringthe third trimester of ‘‘fetal’’ life, a time of physiological vulnerabil-ity. Very preterm infants are susceptible to a number of pathophys-iological conditions for which invasive medical interventions arerequired. Pain is an inherent part of life-saving care in the neonatalintensive care unit (NICU), and has been proposed as one criticalfactor that may impact regulatory functions during this sensitivedevelopmental period [3,24]. Peripheral and central nociceptivesystems are functionally active in infants born very preterm[7,18,21,25,35,36]. Tactile threshold is lower, descending inhibi-tory pathways are immature, and neonates become sensitized to

repeated tactile and skin-breaking stimulation, leading to greatersensitivity to pain during this vulnerable period [6,19,29,30]. Earlyenvironmental stressors in animals have been shown to induceslower body weight gain [9,20]. Importantly, reduced weight gainhas been reported in rat pups exposed to pain for the first 7 daysof life [4].

Although !80% of preterm infants are born at an appropriateweight (10th–90th percentile) for their gestational age and sex,during hospitalization the growth of the majority of preterm infantsappears inadequate, such that by NICU discharge many are consid-ered growth restricted (<10th percentile) [16,38]. Importantly, bothslower body weight gain and head growth in the NICU are associ-ated with increased incidence of cerebral palsy and neurodevelop-mental impairment [15]. Greater exposure to procedural pain in theNICU is also associated with poorer motor and cognitive develop-ment in very preterm infants [26]. In humans, inflammatory medi-ators involved in responses to stress are related to growth in infantsborn very preterm [2]. Neonatal pain, a common stressor in theNICU, has not been examined in relation to postnatal growth in

0304-3959/$36.00 ! 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.pain.2012.02.007

! Corresponding author. Address: Developmental Neurosciences & Child Health,Child & Family Research Institute, F605B-4480 Oak St. Vancouver, BC, Canada. Tel.:+1 604 875 2447.

E-mail address: [email protected] (R.E. Grunau).

w w w . e l s e v i e r . c o m / l o c a t e / p a i n

PAIN"

153 (2012) 1374–1381

Pain/stress is associated with early growth (weight) in the neonatal intensive care unit

(Vinall et al Pain 2013)

to examine the statistical significance of each coefficient (b) in themodel.

3. Results

3.1. Characteristics of the cohort

There were no significant differences between the included andexcluded infants on birth weight percentile, HC percentile or ill-ness severity on day 1 (SNAP II) [34]. Characteristics of the in-cluded infants are provided in Table 1. It is noteworthy that themedian number of skin-breaking procedures was more than dou-ble in the early neonatal window compared to after 32 weeks PCA.

Male and female infants did not differ significantly in raw weightor HC at birth, 32 or 40 weeks PCA (each P > 0.25). Furthermore,there were no significant differences in the early, late or cumulative(early and late) number of painful procedures performed on maleand female infants (each P > 0.32). Sex-specific normative weightand HC percentiles were used in the generalized linear models forall data analyses, and sex was not considered further.

3.2. Generalized linear models of measures for each neonatal windowand body weight

3.2.1. Early (birth to 32 weeks PCA) neonatal variables in relation toweight percentile at 32 weeks PCA

Lower birth weight percentile, more neonatal pain and dexa-methasone exposure,were independently associated with de-creased weight percentile at 32 weeks PCA, after adjusting forthe number of days on mechanical ventilation, morphine exposure,hydrocortisone exposure, postnatal infection, illness severity onday 1, and PCA at weigh-in (Table 2). As shown in Fig. 3, painfulprocedures prior to 32 weeks PCA accounted for approximately21% of the variance in early body growth, and greater exposureto repeated neonatal pain was related to decreased weight percen-tiles at 32 weeks PCA, after accounting for multiple medicalconfounders.

3.2.2. Early (birth to 32 weeks PCA) neonatal variables in relation toweight percentile at 40 weeks PCA

Lower birth weight percentile and hydrocortisone exposure,rather than neonatal pain, were independently associated with de-creased weight percentile at 40 weeks PCA, after adjusting for mor-phine exposure, dexamethasone exposure, infection, illnessseverity on day 1, and PCA at weigh-in (Table 3). There was a trend

for duration of mechanical ventilation from birth to 32 weeks PCAto be associated with lower weight percentile at 40 weeks PCA.

3.2.3. Later (32 to 40 weeks PCA) neonatal variables in relation toweight percentile at 40 weeks PCA

Lower weight percentile at 32 weeks PCA and later neonatalinfection,rather than neonatal pain, were independently associatedwith decreased weight percentile at 40 weeks PCA, after adjustingfor the number of days on mechanical ventilation, morphine,hydrocortisone and dexamethasone exposure, and PCA at weigh-in (Table 4).

3.2.4. Cumulative (early and late/birth to 40 weeks PCA) neonatalvariables in relation to weight percentile at 40 weeks PCA

Lower birth weight percentile, hydrocortisone exposure andinfection, rather than neonatal pain, were independently associ-ated with lower weight percentile at 40 weeks PCA, after adjustingfor the number of days on mechanical ventilation, morphine expo-sure, dexamethasone exposure, illness severity on day 1, and PCAat weigh-in (Table 5).

3.3. Generalized linear models of measures for each neonatal windowand head growth

3.3.1. Early (birth to 32 weeks PCA) neonatal variables in relation toHC percentile at 32 weeks PCA

Lower birth HC percentile, more neonatal pain and longer dura-tion of mechanical ventilation, were independently associated withdecreased HC percentile at 32 weeks PCA, after adjusting for mor-phine, dexamethasone and hydrocortisone exposure, illness sever-ity on day 1, and PCA at weigh-in (Table 2). As shown in Fig. 4,painful procedures prior to 32 weeks PCA accounted for approxi-mately 12% of the variance in early head growth, and greater expo-sure to repeated neonatal pain was related to decreased HCpercentiles at 32 weeks PCA, after accounting for multiple medicalconfounders.

3.3.2. Early (birth to 32 weeks PCA) neonatal variables in relation toHC percentile at 40 weeks PCA

Lower birth HC percentile, longer duration of mechanical venti-lation and hydrocortisone exposure, rather than neonatal pain,were independently associated with decreased HC percentile at40 weeks PCA, after adjusting for morphine exposure, dexametha-sone exposure, infection, illness severity on day 1, and PCA atweigh-in (Table 3).

Table 2Early (birth to 32 weeks PCA) neonatal variables in relation to weight and HC percentile at 32 weeks PCA.

Early neonatal variables Weight percentile at 32 weeks PCAa HC percentile at 32 weeks PCAb

Wald v2 P Value Wald v2 P Value

Birth weight percentile 124.45 0.001 - -Birth HC percentile - - 20.47 0.001Neonatal pain (number of skin-breaking procedures) 7.36 0.01 4.36 0.04Days of mechanical ventilation 0.38 0.54 4.25 0.04Morphine exposure 0.39 0.53 0.95 0.33Hydrocortisone exposure 0.01 0.93 0.69 0.41Dexamethasone exposure 4.83 0.03 0.05 0.82Postnatal infection 0.43 0.51 1.43 0.23Illness severity SNAP-II day 1 2.06 0.15 1.02 0.31PCA at 32-week weigh-in 0.38 0.55 15.36 0.001

HC, head circumference; PCA, postconceptional age; SNAP II, Score for Neonatal Acute Physiology II [34].Generalized linear models revealed that between birth and 32 weeks PCA:

a Lower birth weight percentile, greater neonatal pain, and dexamethasone exposure, independently predicted slower body growth after accounting for the other neonatal factors.b Lower HC birth percentile, greater neonatal pain 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).

J. Vinall et al. / PAIN!

153 (2012) 1374–1381 1377

Neonatal Pain-Related Stress Predicts Cortical Thicknessat Age 7 Years in Children Born Very PretermManon Ranger1,2, Cecil M. Y. Chau2,3, Amanmeet Garg4, Todd S. Woodward3,5, Mirza Faisal Beg4,

Bruce Bjornson1,2, Kenneth Poskitt2,6, Kevin Fitzpatrick2, Anne R. Synnes1,2,7, Steven P. Miller1,2,8,

Ruth E. Grunau1,2,7*

1 Pediatrics, University of British Columbia, Vancouver, British Columbia, Canada, 2 Developmental Neurosciences and Child Health, Child and Family Research Institute,

Vancouver, British Columbia, Canada, 3 BC Mental Health and Addictions Research Institute, Vancouver, British Columbia, Canada, 4 Engineering Science, Simon Fraser

University, Burnaby, British Columbia, Canada, 5 Psychiatry, University of British Columbia, Vancouver, British Columbia, Canada, 6 Radiology, University of British

Columbia, Vancouver, British Columbia, Canada, 7 BC Children’s and Women’s Hospitals, Vancouver, British Columbia, Canada, 8 Pediatrics, Hospital for Sick Children and

University of Toronto, Toronto, Ontario, Canada

Abstract

Background: Altered brain development is evident in children born very preterm (24–32 weeks gestational age), includingreduction in gray and white matter volumes, and thinner cortex, from infancy to adolescence compared to term-born peers.However, many questions remain regarding the etiology. Infants born very preterm are exposed to repeated proceduralpain-related stress during a period of very rapid brain development. In this vulnerable population, we have previously foundthat neonatal pain-related stress is associated with atypical brain development from birth to term-equivalent age. Ourpresent aim was to evaluate whether neonatal pain-related stress (adjusted for clinical confounders of prematurity) isassociated with altered cortical thickness in very preterm children at school age.

Methods: 42 right-handed children born very preterm (24–32 weeks gestational age) followed longitudinally from birthunderwent 3-D T1 MRI neuroimaging at mean age 7.9 yrs. Children with severe brain injury and major motor/sensory/cognitive impairment were excluded. Regional cortical thickness was calculated using custom developed software utilizingFreeSurfer segmentation data. The association between neonatal pain-related stress (defined as the number of skin-breaking procedures) accounting for clinical confounders (gestational age, illness severity, infection, mechanical ventilation,surgeries, and morphine exposure), was examined in relation to cortical thickness using constrained principal componentanalysis followed by generalized linear modeling.

Results: After correcting for multiple comparisons and adjusting for neonatal clinical factors, greater neonatal pain-relatedstress was associated with significantly thinner cortex in 21/66 cerebral regions (p-values ranged from 0.00001 to 0.014),predominately in the frontal and parietal lobes.

Conclusions: In very preterm children without major sensory, motor or cognitive impairments, neonatal pain-related stressappears to be associated with thinner cortex in multiple regions at school age, independent of other neonatal risk factors.

Citation: Ranger M, Chau CMY, Garg A, Woodward TS, Beg MF, et al. (2013) Neonatal Pain-Related Stress Predicts Cortical Thickness at Age 7 Years in ChildrenBorn Very Preterm. PLoS ONE 8(10): e76702. doi:10.1371/journal.pone.0076702

Editor: Yoko Hoshi, Tokyo Metropolitan Institute of Medical Science, Japan

Received June 3, 2013; Accepted August 25, 2013; Published October 18, 2013

Copyright: ! 2013 Ranger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This research was supported by the Eunice Kennedy Shriver Institute of Child Health and Human Development (NICHD/NIH) grant RO1 HD039783[R.E.G], the Canadian Institutes for Health Research (CIHR) grants MOP86489 [R.E.G.] and MOP79262 [R.E.G., S.P.M.]. Salary support includes a Senior Scientistaward, Child & Family Research Institute [R.E.G]; Bloorview Children’s Hospital Chair in Paediatric Neuroscience [S.P.M]; Post-doctoral Fellowships, CIHR and Pain InChild Health CIHR Strategic Training Initiative in Health Research [M.R.]. The funders had no role in study design, data collection and analysis, decision to publish,or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

Altered brain development is evident in children born verypreterm (24–32 weeks gestational age) in early infancy [1–3] andat school-age [4,5], including reduced gray and white mattervolumes in infancy, childhood and adolescence [6–10], comparedto term-born peers. Normal brain growth involves changes incortical thickness reflecting cellular maturational changes relatedto myelination and synaptic pruning [11]. Recent findings fromour group, in a separate cohort, showed delayed microstructural

development of the cortical gray matter in very preterm neonatesat term equivalent [12]. Children born preterm show alteredcortical thickness in childhood and adolescence [7,13–18].Specifically, thinner cortex has been reported in superior andtemporal, middle frontal, anterior cingulate cortex, supramaginal,precuneus, and post central regions when compared to term borncontrols at school age [7,14]. In adolescents born preterm, thinnercortex has been reported in the enthorhinal, temporal, andparietal regions [13,16,18]. Thicker cortex has been reported inpreterm children with periventricular leukomalacia, identified on

PLOS ONE | www.plosone.org 1 October 2013 | Volume 8 | Issue 10 | e76702

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Structural changes in mouse brain following neonatal pain

(Simon Beggs in preparation)

Sensitive parent

interaction may protect the developing

brain

(Milgrom et al Pediatric Research 2010)

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Editor's Summary














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Radiology Ken Poskitt

Brain Imaging Urs Ribary Tony Herdman Faisal Beg Alexander Moiseev Debbie Giaschi Hal Weinberg

Neonatology Anne Synnes Pascal Lavoie Alfonso Solimano Michael Whitfield Neurology Steven Miller Emily Tam Vann Chau Bruce Bjornson

Trainees Manon Ranger, Jillian Vinall, Cecil Chau, Beatriz Valeri, Sam Doesburg, Susanne Brummelte, Mai Thanh Tu, Teresa Cheung

Staff Ivan Cepeda, Mary Beckingham, Gisela Gosse, Katia Jitlina, Amanda Degenhart, Adi Keidar

Neuroscience Joanne Weinberg Adele Diamond Genetics Angela Devlin

Statistics Rollin Brant

Co-investigators & Collaborators

Occupational Therapy Liisa Holsti Child Development Julie Petrie-Thomas

Developmental Pediatrics Tim Oberlander

Immunology David Scheifele Stuart Turvey

Thank you to the families who par5cipate

Funding: NICHD R01 HD039783; CIHR MOP-86489, MOP-79262

translational medicine: bench research & baby brain ...€¦ · translational medicine: bench...

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