brain research 1545 (2014) 1–11
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Research Report
Differential vulnerability of gray matter and white matter to intrauterine growth restriction in preterm infants at 12 months corrected age Nelly Padillaa,f,g,n, Carme Junque´e,f, Francesc Figuerasa,f,g, Magdalena Sanz-Cortesa,f,g, Nu´ria Bargallo´c,f, Angela Arranza,f,g, Antonio Donaired, Josep Figuerasb, Eduard Gratacosa,f,g a
Department of Maternal–Fetal Medicine, ICGON, Hospital Clínic, Universidad de Barcelona, C/Sabino de Arana 1, Helios III, 08028 Barcelona, Spain b Department of Neonatology, ICGON, Hospital Clínic, Universidad de Barcelona, C/Sabino de Arana 1, 08028, Barcelona, Spain c Department of Radiology, Centre de Diagnòstic per la Imatge (CDIC), Hospital Clínic, Universidad de Barcelona, C/Villarroel 170, 08036 Barcelona, Spain d Department of Neurology, Institute of Neuroscience, Hospital Clínic, Universidad de Barcelona, C/ Villarroel 170, 08036 Barcelona, Spain e Department of Psychiatry and Clinical Psychobiology, Faculty of Medicine, Universidad de Barcelona, C/ Casanova 143, 08036 Barcelona, Spain f Institut D’investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), C/ Villarroel 170, 08036 Barcelona, Spain g Centro de Investigación Biomédica en Enfermedades Raras (CIBERER), Corporació Sanitària Clínic, C/ Villarroel 170, 08036 Barcelona, Spain
ab st rac t
art i cle i nfo Article history:
Intrauterine growth restriction (IUGR) is associated with a high risk of abnormal neurode-
Accepted 6 December 2013 Available online 17 December 2013
velopment. Underlying neuroanatomical substrates are partially documented. We hypothesized that at 12 months preterm infants would evidence specific white-matter
Keywords:
microstructure alterations and gray-matter differences induced by severe IUGR. Twenty
Prematurity
preterm infants with IUGR (26–34 weeks of gestation) were compared with 20 term-born
Brain development
infants and 20 appropriate for gestational age preterm infants of similar gestational age.
Gray matter development
Preterm groups showed no evidence of brain abnormalities. At 12 months, infants were
White matter development
scanned sleeping naturally. Gray-matter volumes were studied with voxel-based morpho-
Voxel-based morphometry
metry. White-matter microstructure was examined using tract-based spatial statistics.
Diffusion tensor imaging
The relationship between diffusivity indices in white matter, gray matter volumes, and
Abbreviations: AGA,
Appropriate for gestational age; AD,
Anatomical Registration through Exponentiated Lie Algebra; DTI, FWE,
family-wise error; GM,
imaging; RD,
gray matter; IUGR,
radial diffusivity; SGA,
Axial diffusivity; CA,
Corrected age; DARTEL,
diffusion tensor imaging; FA,
intrauterine growth restriction; MD,
small for gestational age; TBSS,
mean diffusivity; MRI,
magnetic resonance
tract-based spatial statistics; VBM, voxel-based morphometry;
WM, white matter n Corresponding author at: Universidad de Barcelona, Department of Maternal–Fetal Medicine, ICGON, Hospital Clínic, Sabino de Arana 1 – Helios III, 08028 Barcelona, Spain. E-mail addresses: [email protected], [email protected] (N. Padilla). 0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.12.007
Diffeomorphic
fractional anisotropy;
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perinatal data was also investigated. Gray-matter decrements attributable to IUGR comprised amygdala, basal ganglia, thalamus and insula bilaterally, left occipital and parietal lobes, and right perirolandic area. Gray-matter volumes positively correlated with birth weight exclusively. Preterm infants had reduced FA in the corpus callosum, and increased FA in the anterior corona radiata. Additionally, IUGR infants had increased FA in the forceps minor, internal and external capsules, uncinate and fronto-occipital white matter tracts. Increased axial diffusivity was observed in several white matter tracts. Fractional anisotropy positively correlated with birth weight and gestational age at birth. These data suggest that IUGR differentially affects gray and white matter development preferentially affecting gray matter. At 12 months IUGR is associated with a specific set of structural gray-matter decrements. White matter follows an unusual developmental pattern, and is apparently affected by IUGR and prematurity combined. & 2013 Elsevier B.V. All rights reserved.
1.
Introduction
Intrauterine growth restriction (IUGR) is one of the common reasons indicated for preterm delivery. This condition, due to placental insufficiency, affects 5–10% of all pregnancies and is associated with chronic hypoxia and under-nutrition during fetal life. The combination of preterm birth and IUGR results in a higher rate of perinatal complications and consequently worse long-term outcomes (Geva et al., 2006; Guellec et al., 2011; Leitner et al., 2007). The structural alterations underlying the effects of IUGR on the preterm brain are only partially documented. In this respect, studies have been hampered by the widespread practice of using the terms small for gestational age (SGA) and IUGR synonymously. Defining SGA as a birth weight (BW) below a given threshold compared to appropriate for gestational age (AGA) groups, SGA children had smaller brain volumes (De Bie et al., 2011; Martinussen et al., 2009; Xydis et al., 2013) and altered white matter (WM) microstructure (Eikenes et al., 2012; Lepomaki et al., 2013). In contrast, comparisons between preterm neonates
with and without intrauterine growth restriction, defined as BW below the 10th percentile and abnormal Doppler values within the umbilical artery, showed that IUGR is associated with reduced volumes of the cortical gray matter (GM; Tolsa et al., 2004), decreased volumes of the hippocampus (Lodygensky et al., 2008) and a discordant pattern of gyrification (Dubois et al., 2008) related to behavioral alterations. At 12 months, comparisons between preterm infants with and without growth restriction and term-born controls suggested that the most pronounced differences in the IUGR preterm group were related to a different distribution of the GM and WM (Padilla et al., 2011) as well as a different cortical brain complexity (Esteban et al., 2010), both associated to neurodevelopmental difficulties. However, the GM alterations induced by IUGR, and the existence of microstructural WM differences, in preterm infants at 12 months, excluding the influence of prematurity itself, have not been investigated. The aims of this study were (1) to detect specific regional GM volume changes as a consequence of IUGR in preterm infants at 12 months corrected age (CA); (2) to explore whether IUGR induces specific WM microstructure alterations
Fig. 1 – Study population. IUGR, intrauterine growth restriction; AGA, appropriate for gestational age; PVL, periventricular leukomalacia; US, ultrasound; DTI, diffusion tensor imaging; MRI, magnetic resonance imaging.
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in the same population; and (3) to correlate these changes with birth weight and gestational age at birth. For this, we included a control group of preterm infants appropriate for gestational age (AGA) to exclude the effect of prematurity per se.
2.
Results
A total of 57 infants with IUGR, 70 preterm AGA infants and 72 term-born infants were initially included. The final sample included 20 infants with IUGR, 20 preterm AGA infants and
20 term-born infants (Fig. 1). Perinatal, demographic, and anthropometric characteristics of the infants are detailed in Table 1. There were no significant differences in perinatal data between the subjects scanned and those who were not scanned. As expected, full-term children differed significantly in neonatal data compared with both preterm groups. During the neonatal period, preterm infants with IUGR differed significantly in the rate of cesarean section, birth weight, and umbilical cord arterial pH value compared to the AGA preterm group (Table 2). Of note, IUGR infants had significantly lower weight, length and head circumference compared with preterm AGA infants and term infants at 12 months CA.
Table 1 – Characteristics of the study groups. IUGR n ¼ 20
Preterm n ¼20
Term n ¼ 20
Perinatal Gestational age at delivery range Cesarean section Umbilical artery pH Apgar 5 min Gender (M/F) Birth weight (g) Range Length at birth (cm) Range Head circumference at birth (cm) Range
30.9971.86n 26.50–34.0 17/20 (85%) † 7.18 (0.15)† 9.10 (1.21) 10/10 1049.757237.47n 600–1420 38.2073.65n 32–43 27.1772.65n 22–29
30.0472.35n 26.00–34.00 8/20 (40%) 7.29 (0.06) 9.30 (0.80) 11/9 1531.757402.94n 1000–2500 39.8573.97n 33–46 28.2772.85n 24–32
39.82 (1.40) 37.1–41.5 – 7.26 (0.08) 9.90 (0.24) 10/10 32727496.78 3150–4100 49.6071.75 46–52 34.0071.13 33–36
Demographic Mother0 s age (years) Range Mother0 s education less thanhigh school n (%) Breastfeeding 44 months n (%) Use of early intervention services Corrected age at scan (months)
33.0073.78 27–40 7/20 (35.0%) 16/20 (80.0%) 9/20 (45.0%) 12.9071.07
30.5074.38 22–37 6/20 (30.0%) 17/20 (85.0%) 6/20 (30.0%) 12.4070.68
32.1274.54 20–37 2/20 (10.0%) 16/20 (80.0%) – 13.0070.86
Anthropometric data at 12 months CA Weight at 12 months (kg) Range Height at 12 months (cm) Range Head circumference at 12 months (cm) Range
8.3570.98n, † 6.97–11.00 70.2373.05n, † 64.00–85.00 45.3771.62 † 42.00–47.50
9.6871.45 7.20–13.00 74.8574.45 70.00–85.00 46.3270.99 44.50–48.00
9.7471.02 8.00–12.10 73.9572.86 69.00–85.50 45.9971.16 43.00–47.50
Whole-brain measurements Gray matter (cm3) White matter (cm3) Total brain volume (cm3) Mean FA values
638.75751.23n, 201.77725.30 840.50775.66n, 0.33970.01n
691.80757.07 220.63732.15 912.42785.62 0.33470.09
683.14747.30 215.44728.09 898.59768.84 0.32970.01
†
†
SNAP-PE, score for neonatal acute physiology-perinatal extension; NICU, neonatal intensive care unit. po0.05 compared with term infants. † po 0.05 compared with preterm infants. Data are mean7SD. n
Table 2 – Neonatal characteristics. Characteristic
IUGR (20)
Preterm (20)
Statistics (p value)
Prenatal corticosteroids Cesarean section Umbilical artery pH Apgar 5 min
14/20 (70%) 17/20 (85%) 7.18 (0.15) 9.1 (1.21)
17/20 (85%) 8/20 (40%) 7.29 (0.06) 9.30 (0.80)
FET (0.451) FET (0.004) t¼ 2.99 (0.06) t¼ 0.616 (0.64)
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Table 2 (continued ) Characteristic
IUGR (20)
Preterm (20)
Statistics (p value)
SNAP-PE Days in NICU Mechanical ventilation Postnatal corticosteroids Late-onset neonatal sepsis Hypoglycemia Hyperglycemia
17/20 [23.9 (14.6)] 43.10 (27.94) 9/20 (45%) 0 5/20 (25%) 8/20 (40%) 7/20 (46.7%)
19/20 [16.3 (14.4)] 42.75 (20.40) 13/20 (65%) 2/20 (10%) 1/20 (5%) 7/20 (35%) 8/20 (40%)
t ¼ 1.824 (0.08) t ¼ 0.045 (0.96) FET (0.341) FET (0.487) FET (0.342) FET (1.0) FET (1.0)
IUGR, intrauterine growth restricted infants; SNAP-PE, score for neonatal acutephysiology—perinatal extension; NICU, neonatal intensve care unit: FET, Fisher0 s exact test.
Fig. 2 – Regions of gray-matter volume decrease (gray to white colors) in preterm IUGR infants compared with term-born infants (a) and preterm infants without IUGR (b). The bar represents the t scores. Numbers represent the corresponding slice. Right¼ left.
2.1.
Conventional MRI findings
Visual inspection of the MRI studies revealed mildly altered structural scans in 7 infants with IUGR (5 with slightly dilated lateral ventricles and 2 with a thinning of the body of the corpus callosum) and 5 preterm AGA (3 with slightly dilated lateral ventricles and 2 with a thinning of the body of the corpus callosum). The term-born group had no brain abnormalities.
3. Voxel-based morphometry DARTEL: GM results 3.1.
gyrus bilaterally, left occipital and parietal lobes, and right perirolandic area (Fig. 2b). The areas and significance levels of GM differences between groups are shown in Table 3.
3.2.
Appropriate for gestational age group
Compared to term-born infants, the preterm AGA group showed 1 region of GM decrements in the right temporal lobe.
4.
Diffusion-tensor imaging: WM results
4.1.
Intrauterine growth restricted group
Intrauterine growth restricted group
Infants with IUGR had significantly reduced absolute global GM volumes compared to preterm AGA and term-born infants. However, relative volumes were not significantly different between groups (Table 1). Compared with term-born infants, those with IUGR had GM reductions involving predominantly the temporal lobe bilaterally, the hippocampus, the amygdala and the right perirolandic area. Additional areas of GM volume reduction were found in the right frontal lobe, left parietal lobe, perirolandic area and basal ganglia (Fig. 2a). Compared with the preterm AGA group, infants with IUGR showed reduced GM volume in the amygdala, basal ganglia, thalami, insula, angular
Compared with the preterm AGA group, infants with IUGR had clusters of decreased FA predominantly localized in the splenium of the corpus callosum, and increased FA in the anterior corona radiata (Fig. 3a, b). Additionally, IUGR infants showed clusters of increased AD comprising the forceps minor and anterior corona radiata (Fig. 3c). No significant differences were identified in any other diffusivity parameter. The analyses were repeated at different thresholds; however, no between-group differences emerged. Compared with their term counterparts, infants with IUGR exhibited clusters of decreased FA comprising the splenium of the corpus callosum (Fig. 4a). Intrauterine growth restricted
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Table 3 – Significant differences in gray-matter volumes in the study groups. Anatomic region
Hemisphere R ¼right, L¼ left
Contrast
Cluster (mm3)
Cluster level p corrected
t
Superior temporal gyrus posterior part Middle and inferior temporal gyrus Fusiform gyrus Anterior temporal lobe lateral part Insula, putamen caudado, pallidum Parahippocampal and ambient gyri Amygdala Hippocampus posterior part Inferior frontal gyrus
R
IUGRoterm
5673
o0.001
7.17
Superior temporal gyrus posterior part Middle and inferior temporal gyrus Posterior temporal lobe Precentral gyrus Parahippocampal and ambient gyri
L
IUGRoterm
2046
o0.001
4.93
Putamen Caudate Pallidum
L L L
IUGRoterm
574
o0.001
5.14
Post-central gyrus
L
IUGRoterm
1968
o0.001
5.42
Inferior–lateral remainder parietal lobe Precuneus and cuneus
L L and R
IUGRopreterm
212
o0.001
4.49
Post-central and supramarginal gyri Parietal inferior gyrus Temporal superior gyrus Precuneus Pre-central gyrus
R
IUGRopreterm
3322
o0.001
5.1
Lateral reminder occipital lobe Lingual gyrus cuneus Post-central and pre-central gyrus
L
IUGRopreterm
4137
o0.001
4.2
L
IUGRopreterm
535
0,008
4.16
Putamen; pallidum; insula; caudate; amygdala Cingulate gyrus posterior part
L
IUGRopreterm
2336
o0.001
5.17
Putamen; insula; pallidum; caudate; amygdala Superior temporal gyrus Middle and inferior temporal gyrus
R
IUGRopreterm
1457
o0.001
5.21
R
pretermoterm
1506
o0.001
6.57
Lateral reminder occipital lobe Precuneus Cuneus
L L and R R
preterm4term
3287
o0.001
5.47
IUGR, intrauterine growth restricted infants.
infants, also had greater FA in several clusters (Fig. 4b), especially in the anterior regions involving commissural fibers (genu, the body, and forceps minor of the corpus callosum); projection fibers (anterior, superior and posterior corona radiata, the left anterior limb of the internal capsule, and posterior limb of the internal capsule bilaterally) and association tracts (right frontal course of the uncinate fasciculum and inferior fronto-occipital fasciculum, and left external capsule including the superior longitudinal fasciculum). Furthermore, intrauterine growth restricted infants, compared to term infants, showed higher AD in several
regions (Fig. 4c). No MD or RD differences were identified between IUGR and term infants.
4.2.
Appropriate for gestational age group
Compared to the term-born infants the preterm AGA group had clusters of decreased FA in the splenium and body of the corpus callosum, and increased FA in the forceps minor (Fig. 5a, b). No significant differences were seen in any other diffusivity parameter.
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Fig. 3 – White matter tracts showing regions (in black) of decreased (a) and increased (b) fractional anisotropy (FA), and increased axial diffusivity (AD) (c) between intrauterine growth restricted infants (IUGR) and preterm appropriate for gestational age infants (AGA). Numbers represent the corresponding slice. Right¼ left.
4.3. Relationship between GM volumes, diffusivity parameters, and perinatal data in the IUGR group In GM, significant correlations with the birth weight were observed comprising the right parahippocampal gyrus, hippocampus and right cerebellar hemisphere (cluster¼ 1043; t¼4.56; p¼ 0.02, FWE corrected). No significant correlations were found between regional GM volumes and gestational age at birth. Fractional anisotropy showed widespread positive linear associations with gestational age at birth and birth weight (Supplementary material, Fig. 2). No correlations were found between perinatal data and any other diffusion parameter.
5.
Discussion
The results of the current study suggest that in preterm infants with IUGR at 12 months CA without apparent brain lesions, the GM and WM are differentially affected. In terms of GM, the current results expand on our previous studies that have reported GM brain alterations in infants with IUGR at 12 months (Padilla et al., 2011; Esteban et al., 2010) and further demonstrate that IUGR is associated with a specific set of
structural GM decrements comprising the amygdala, basal ganglia, thalami, left occipital and parietal lobes, and the right perirolandic area. Gray-matter volume decrements positively correlated with birth weight but not with gestational age at birth. In terms of white matter, it follows an unusual developmental pattern involving predominantly the anterior brain regions. These microstructural alterations are positively correlated with gestational age at birth and birth weight.
5.1.
Voxel-based morphometry DARTEL: GM results
The results from this study suggest that GM reductions ultimately attributable to IUGR comprised mainly the amygdala, basal ganglia, thalami, left occipital and parietal lobes, and the right perirolandic area. In fact, as previously reported for the neonatal period (Tolsa et al., 2004), IUGR predominantly affects cerebral cortical development, reflected in a decreased total brain volume, which is consistent with our results. Of particular note is the involvement of the insula, amygdala, and basal ganglia, all of which share a close anatomical and functional relationship and have a key role in the control of behavior, cognitive processing, and autonomic activity (Fukushima et al., 2011). These data are of
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Fig. 4 – White matter tracts showing regions (in black) of decreased (a) and increased (b) fractional anisotropy (FA), and increased axial diffusivity (AD) (c) between intrauterine growth restricted infants (IUGR) and term infants. Numbers represent the corresponding slice. Right¼ left.
particular interest, since IUGR is associated with altered developmental activity of the autonomic nervous system (Jones, 2011), and social, emotional, and behavioral disorders (Leitner et al., 2007; Tolsa et al., 2004). In the light of previously reported language problems in children with IUGR (Geva et al., 2006), the decreased GM in the angular gyrus bilaterally is of particular interest, since this structure is described as a core component of a distributed language network (Constable et al., 2008). Here, the additional involvement of the sensorimotor area, the insula, and the occipital cortex is consistent with some previous reports exploring language processing in preterm and term subjects (Schafer et al., 2009). Our study adds new data concerning the possible anatomical substrates related to language difficulties in preterm infants with IUGR at school age. Interestingly the precuneus showed specific vulnerability to IUGR (Padilla et al., 2011). The posterior left precuneus was affected notably, and the occipital area on the same side. These structures are known to be involved in visual–spatial
information processing. Our results complement previous findings suggesting a regional vulnerability of the occipital cortex to the IUGR (Thompson et al., 2007) and may point to an abnormal connection between the precuneus and the occipital cortex, which likely relate to the visual–spatial difficulties reported in IUGR children (Geva et al., 2006).
5.2.
Diffusion-tensor imaging: WM results
In this study, compared to term and preterm AGA groups, the IUGR infants presented clusters of decreased FA comprising the splenium of the corpus callosum. It should be noted that the preterm AGA group also showed decreased FA in the body and splenium of the corpus callosum when compared to term infants. These results are consistent with those of other studies that have examined FA in preterm and SGA subjects. A previous study including young adults born preterm showed decreased FA in several areas of the corpus callosum (Eikenes et al., 2011). Also, the FA in this structure has been
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Fig. 5 – White matter tracts showing regions (in black) of decreased (a) and increased (b) fractional anisotropy (FA) between preterm appropriate for gestational age (AGA) infants and term infants. Numbers represent the corresponding slice. Right¼left.
negatively correlated with total IQ in SGA subjects (Eikenes et al., 2012) suggesting a compensatory rewiring of brain white matter circuitry in the context of intrauterine growth restriction. Of note, the corpus callosum integrates sensory, motor, cognitive, and emotional functions from both hemispheres and could be adversely affected by prematurity (Constable et al., 2008; Rose et al., 2008) and specifically IUGR (Anderson et al., 2006; Tolcos et al., 2011). Contrary to the expectations, in comparisons between preterm IUGR and term infants, changes in WM microstructure were predominantly reflected by increased FA and AD comprising commissural, projection, and associative tracts. Notably, smaller clusters of increased FA were also found in preterm IUGR infants compared to the AGA group. In the preterm IUGR infants, not all tracts were equally affected. In general, the most pronounced changes were found in the anterior brain regions that are likely to be more vulnerable to insults that could be reflected in developmental difficulties (Guellec et al., 2011; Leitner et al., 2007). The most severely affected tract was the forceps minor, a WM tract connecting medial and lateral prefrontal regions via the genu of the corpus callosum. This structure is involved in higher cognitive functions as well as emotional and behavioral control. The increased FA found in this area has not been previously reported in preterm infants. Further confirmation of this finding is important since structural changes may be involved in the developmental disabilities that have been reported in children with IUGR (Geva et al., 2006; Leitner et al., 2007). Here, increased FA also comprised association fibers (inferior fronto-occipital fasciculum, inferior longitudinal fasciculum, superior longitudinal fasciculum, and uncinate fasciculum) that connect frontal and parieto-temporal–occipital areas. In a previous study using a standard VBM approach, we reported regional GM volume differences between preterm infants with IUGR and term-born infants involving the frontal, parietal, and occipital lobes (Padilla et al., 2011). Thus, our findings here complement these earlier results, suggesting
microstructural abnormalities of long-distance tracts that may be due to an aberrant fiber organization in preterm infants with IUGR. It bears mentioning that increased FA in associative tracts has also been reported in autism (Cheng et al., 2010), suggesting maturational disturbances. We also detected increased FA in the corona radiata and the posterior limb of the internal capsule. The involvement of these structures may contribute to prefrontal dysfunctions and differences in the motor skills reported in children and adolescents born with IUGR (Guellec et al., 2011; Padilla et al., 2011). The significance of increased FA in this study is uncertain. Similar FA increases have been reportedly associated with preterm birth (Allin et al., 2011; Eikenes et al., 2011) and developmental disorders (Cheng et al., 2010). In this respect, this apparently “increased” maturation in WM does not mean, “better” (Eikenes et al., 2012; Hoeft et al., 2007). Regions of increased FA in the preterm IUGR group might reflect an early deviation in the developmental trajectory of the WM. Compensatory changes as part of neuronal remodeling and the plastic reorganization of the brain secondary to adverse in utero-environment could be also involved. Furthermore, environmental factors that may influence the normal development during the first year of life should be considered. To aid the interpretation of FA findings, we measured RD and AD. Radial diffusivity and AD have been linked to myelin integrity (Song et al., 2002) and fiber organization (Takahashi et al., 2000), respectively. Here, changes affected exclusively AD and even at a low statistical threshold, we found no such area of altered RD. Consequently, myelination was not the main factor contributing to the cases of altered diffusivity in the present study. Thus, greater FA associated with widespread increase of AD as found here could result from changes in fiber organization related to reduced tortuosity to straighter fibers (Takahashi et al., 2000). This result differs from results in preterm subjects reporting increased RD associated with decreased FA (Eikenes et al., 2011) or no alteration in the organization of white matter fibers. These findings may relate
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to development in infancy and may suggest early aberration in the developmental trajectory of WM in preterm infants with IUGR at 12 months of life. The fact that we found no differences in MD does not rule out that MD alterations could occur later in development—a point which needs to be elucidated in future studies.
6.
Correlation analysis
The correlations between FA and perinatal data suggest that increasing prematurity at birth and lower birth weight have negative effects on WM microstructure that are evident at 12 months in preterm infants with IUGR. A similar finding has been suggested in studies assessing the repercussions of prematurity in older subjects (Allin et al., 2011; Eikenes et al., 2011). In regional GM volumes, correlations were found with birth weight but not with gestational age at birth. This finding highlights the impact of IUGR on GM development with documented reductions on regional GM volumes at 12 months CA. Our results are consistent with those reported during the neonatal period (Tolsa et al., 2004). From this perspective, the specific effect of the IUGR on GM development could be related in part with the neuroendocrine consequences of this condition associated with increased cortisol levels that might be associated with reduction in the cortical GM (Modi et al., 2001). Further studies are needed to test this hypothesis. Strengths of this study include a well-defined cohort characterized prenatally and followed prospectively, and the definition of preterm IUGR included antenatal Doppler findings. Another strength is the age of assessment. Twelve months constitutes a critical period in human development, where the knowledge regarding GM and WM development in preterm infants with IUGR is quite limited. In addition, this study combines 2 MRI analyses (VBM in GM and TBSS in WM) assessing different aspects of GM and WM, and consequently providing complementary information. A limitation of this study is the relatively small sample size, which may have prevented statistical differences from being observed in some comparisons.
7.
Conclusion
The current study reveals a differential effect of IUGR on the developing GM and WM with the GM exclusively being affected at 12 months CA. The GM decrements affected a specific set of structures and correlated with birth weight only. The WM in IUGR infants showed an unusual developmental pattern with increased FA and AD and correlated with birth weight and gestational age at birth. Findings in GM are consistent with and provide further support to previous results in this prenatal disorder. Findings in WM provide new insights into microstructural changes underlying brain reorganization under IUGR. Future studies including larger sample sizes are clearly needed to explore these issues in greater depth. To evaluate the clinical relevance of our findings, neurodevelopmental follow-up of the infants is ongoing.
8.
Experimental procedures
8.1.
Subjects
9
Between 2007 and 2009 a cohort of consecutive singleton infants was selected with the following inclusion criteria: (i) gestational age at birth less than 34 weeks; (ii) fetal weight below the 10th percentile for gestational age confirmed at birth, and (iii) umbilical artery Doppler pulsatility index 495th percentile in at least 2 consecutive examinations 24 h apart. The preterm and term control groups were singleton AGA infants, with a BW between the 10th and 90th customized centiles according to local reference standards (Figueras et al., 2008). The preterm group was matched with cases by gestational age at birth (71 week). Pregnancies were dated according to the first-trimester crown-rumplength measurements. Corrected age was calculated subtracting from chronological age the number of weeks born before 40 weeks of gestation. Exclusion criteria for both cases and controls were: (i) any intraventricular hemorrhage or periventricular leukomalacia on neonatal ultrasound or magnetic resonance imaging; (ii) placental histological criteria for chorioamnionitis; (iii) congenital malformations, including chromosomal abnormalities and infections. Prenatal and neonatal data were prospectively recorded. Written informed consent was obtained from all parents of the participating infants. The Institutional Ethics Committee approved the study protocol, recruitment, and scanning procedures.
8.2.
MRI acquisition
Infants in all groups, sleeping naturally, were scanned at 1272 months CA (Padilla et al., 2012). MRI was performed on a TIM TRIO 3.0T scanner (Siemens, Erlangen, Germany), by using a 32channel head coil. The acquisition time of the entire protocol was about 30 min. A set of high-resolution T1-weighted, 3D images was acquired using the magnetization prepared rapid acquisition gradient echo sequence (MPRAGE; TR/TE¼2050 ms/ 2.41 ms; TI¼ 1050 ms; FOV¼220 220 mm2 and 256 256 matrix; flip angle 901). The whole-brain data were acquired in a sagittal plane, yielding contiguous slices with isotropic voxel of 0.9 0.9 0.9 mm3. T2-weighted images in sagittal orientation (TR/TE¼ 4640 ms/102 ms; 122 122 matrix; flip angle 901, voxel size 0.62 0.62 3 mm3) were also acquired to detect any lesion noticeable on the T1-weighted images. Single-shot diffusion weighted EPI sequences were acquired in 30 non-collinear directions with a p-value¼ 1000 s/mm2 and 1 image without diffusion gradient (b¼ 0 s/mm2). TR/TE¼9300 ms/94 ms; 122 122 matrix; flip angle 901, slice thickness 3.0 mm; no interslice gap; slices¼ 30; voxel size 1.64 1.64 3 mm3. The MRI scans were reviewed by a neuroradiologist (NB) blinded to group membership.
8.3.
Image analysis
For voxel based-morphometry (VBM) procedure the SPM v5 software, Wellcome Department, University College (London, UK) running on MATLAB v7.5 (MathWorks, Natrick, MA, USA) was used. The steps followed were: (1) line determination of
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the anterior–posterior commissures and image reorienting; (2) whole-brain segmentation of the GM, WM, and cerebral spinal fluid of the T1 structural MRI images (Supplementary material, Fig. 1) by employing the VBM 5.1 toolbox (http:// dbm.neuro.uni-jena.de/vbm/) implemented in SPM v5 software, using infant brain tissue probability maps; (3) images to be analyzed were imported and the diffeomorphic anatomical registration through exponentiated lie algebra (DARTEL) algorithm (Ashburner, 2007) was applied (Supplementary material, Fig. 1b). Finally, all images were modulated and smoothed with a full width at half-maximum of 6 mm Gaussian kernel. Global GM and WM volumes, intracranial volume (GMþWMþCSF), and total brain volume (GMþWM) were calculated using the native-space tissue maps of each subject. Global GM and WM volumes were normalized by intracranial volume to calculate the relative GM and WM volumes. Diffusion tensor images were processed with the FMRIB software library, FSL v4.1, Oxford Centre for Functional MRI of the brain, (Oxford, UK). The volumes were eddy-current- and motion-corrected relative to the first (b ¼0) volume by using FMRIB0 s diffusion toolbox (FDT; Smith et al., 2004). Non-braintissue components were removed using the brain extraction tool (Smith, 2002). Tract-based spatial statistics v1.2 (TBSS; Smith et al., 2006) was applied to the fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) maps. Fractional anisotropy maps from all subjects were aligned to the most representative map using non-linear registration and upsampled to 1x1 1 mm3 voxel size. The mean of all aligned FA images was then created and thinned to generate a skeletonized mean FA image (threshold40.2) to reflect common tracts across all subjects (Supplementary material, Fig. 1c). Mean diffusivity, AD, and RD were processed similarly to FA data with the exception that FA images were used to drive the nonlinear registration and skeletonization stages.
8.4.
Statistical analyses
All variables studied were checked for normality before each analysis. Quantitative data were analyzed with Student0 s t-test or one-way analyses of variance (ANOVA) and qualitative data with Fisher0 s exact test. All statistical analyses were performed using SPSS 17.0 (SPSS Inc, Chicago, USA). A po0.05 was considered statistically significant. In the DTI and VBM analyses, 2 covariates—total brain volume and postmenstrual age at scan—were introduced into each statistical design. Additional analyses were conducted to assess the probable influence of gender as well as cesarean section and umbilical pH on the results. However, these variables had a negligible effect on the p values and thus were not included as covariates. For DTI analyses we used a permutation-based approach, this being Randomise v 2.1 (Nichols and Holmes, 2002). For GM analysis, we used the VBM approach with a cluster threshold set at 60 voxels; t-test group comparisons were performed to evaluate the GM volume changes between groups and a “simple regression analysis” to test the possible relationship between GM and perinatal data. In both studies, we analyzed the following contrasts: patients greater than controls and controls greater than patients. All the results
underwent a family-wise error (FWE) correction method for multiple comparisons (po0.05).
Acknowledgments Funding support was provided by grants from the Cerebra Foundation for the brain-injured child (Carmarthen-Wales, UK), The Thrasher Research Fund (Salt-Lake-City, USA). NP was supported by a Sara Borrell post-doctoral fellowship (CD09/00263), Instituto de Salud Carlos III, Spain. The authors would like to thank all the participating infants and the parents, Marta Garcia and Alba Camacho for contacting and booking the infants, and Cesar Garrido for imaging acquisition.
Appendix A.
Supporting information
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres. 2013.12.007.
r e f e r e n c e s
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Padilla, N., Falcon, C., Sanz-Cortes, M., Figueras, F., Bargallo, N., Crispi, F., Eixarch, E., Arranz, A., Botet, F., Gratacos, E., 2011. Differential effects of intrauterine growth restriction on brain structure and development in preterm infants: a magnetic resonance imaging study. Brain Res. 1382, 98–108. Padilla, N., Botet, F., Gratacos, E., 2012. MRI at 1272 months0 corrected age without sedation. Pediatr. Radiol. 42, 385. Rose, S.E., Hatzigeorgiou, X., Strudwick, M.W., Durbridge, G., Davies, P.S., Colditz, P.B., 2008. Altered white matter diffusion anisotropy in normal and preterm infants at term-equivalent age. Magn. Reson. Med. 60, 761–767. Schafer, R.J., Lacadie, C., Vohr, B., Kesler, S.R., Katz, K.H., Schneider, K.C., Pugh, K.R., Makuch, R.W., Reiss, A.L., Constable, R.T., Ment, L.R., 2009. Alterations in functional connectivity for language in prematurely born adolescents. Brain 132, 661–670. Smith, S.M., 2002. Fast robust automated brain extraction. Hum. Brain Mapp. 17, 143–155. Smith, S.M., Jenkinson, M., Woolrich, M.W., Beckmann, C.F., Behrens, T.E., Johansen-Berg, H., Bannister, P.R., De Luca, M., Drobnjak, I., Flitney, D.E., Niazy, R.K., Saunders, J., Vickers, J., Zhang, Y., De Stefano, N., Brady, J.M., Matthews, P.M., 2004. Advances in functional and structural MR image analysis and implementation as FSL. Neuroimage 23 (Suppl. 1), S208–S219. Smith, S.M., Jenkinson, M., Johansen-Berg, H., Rueckert, D., Nichols, T.E., Mackay, C.E., Watkins, K.E., Ciccarelli, O., Cader, M.Z., Matthews, P.M., Behrens, T.E., 2006. Tract-based spatial statistics: voxelwise analysis of multi-subject diffusion data. Neuroimage 31, 1487–1505. Song, S.K., Sun, S.W., Ramsbottom, M.J., Chang, C., Russell, J., Cross, A.H., 2002. Dysmyelination revealed through MRI as increased radial (but unchanged axial) diffusion of water. Neuroimage 17, 1429–1436. Takahashi, M., Ono, J., Harada, K., Maeda, M., Hackney, D.B., 2000. Diffusional anisotropy in cranial nerves with maturation: quantitative evaluation with diffusion MR imaging in rats. Radiology 216, 881–885. Thompson, D.K., Warfield, S.K., Carlin, J.B., Pavlovic, M., Wang, H.X., Bear, M., Kean, M.J., Doyle, L.W., Egan, G.F., Inder, T.E., 2007. Perinatal risk factors altering regional brain structure in the preterm infant. Brain 130, 667–677. Tolcos, M., Bateman, E., O’Dowd, R., Markwick, R., Vrijsen, K., Rehn, A., Rees, S., 2011. Intrauterine growth restriction affects the maturation of myelin. Exp. Neurol. 232, 53–65. Tolsa, C.B., Zimine, S., Warfield, S.K., Freschi, M., Sancho Rossignol, A., Lazeyras, F., Hanquinet, S., Pfizenmaier, M., Huppi, P.S., 2004. Early alteration of structural and functional brain development in premature infants born with intrauterine growth restriction. Pediatr. Res. 56, 132–138. Xydis, V., Drougia, A., Giapros, V., Argyropoulou, M., Andronikou, S., 2013. Brain growth in preterm infants is affected by the degree of growth restriction at birth. J. Matern. Fetal Neonatal Med. 26, 673–679.
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Differential vulnerability of gray matter and white matter to intrauterine growth restriction in preterm infants at 12 months corrected age Nelly Padillaa,f,g,n, Carme Junque´e,f, Francesc Figuerasa,f,g, Magdalena Sanz-Cortesa,f,g, Nu´ria Bargallo´c,f, Angela Arranza,f,g, Antonio Donaired, Josep Figuerasb, Eduard Gratacosa,f,g a
Department of Maternal–Fetal Medicine, ICGON, Hospital Clínic, Universidad de Barcelona, C/Sabino de Arana 1, Helios III, 08028 Barcelona, Spain b Department of Neonatology, ICGON, Hospital Clínic, Universidad de Barcelona, C/Sabino de Arana 1, 08028, Barcelona, Spain c Department of Radiology, Centre de Diagnòstic per la Imatge (CDIC), Hospital Clínic, Universidad de Barcelona, C/Villarroel 170, 08036 Barcelona, Spain d Department of Neurology, Institute of Neuroscience, Hospital Clínic, Universidad de Barcelona, C/ Villarroel 170, 08036 Barcelona, Spain e Department of Psychiatry and Clinical Psychobiology, Faculty of Medicine, Universidad de Barcelona, C/ Casanova 143, 08036 Barcelona, Spain f Institut D’investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), C/ Villarroel 170, 08036 Barcelona, Spain g Centro de Investigación Biomédica en Enfermedades Raras (CIBERER), Corporació Sanitària Clínic, C/ Villarroel 170, 08036 Barcelona, Spain
ab st rac t
art i cle i nfo Article history:
Intrauterine growth restriction (IUGR) is associated with a high risk of abnormal neurode-
Accepted 6 December 2013 Available online 17 December 2013
velopment. Underlying neuroanatomical substrates are partially documented. We hypothesized that at 12 months preterm infants would evidence specific white-matter
Keywords:
microstructure alterations and gray-matter differences induced by severe IUGR. Twenty
Prematurity
preterm infants with IUGR (26–34 weeks of gestation) were compared with 20 term-born
Brain development
infants and 20 appropriate for gestational age preterm infants of similar gestational age.
Gray matter development
Preterm groups showed no evidence of brain abnormalities. At 12 months, infants were
White matter development
scanned sleeping naturally. Gray-matter volumes were studied with voxel-based morpho-
Voxel-based morphometry
metry. White-matter microstructure was examined using tract-based spatial statistics.
Diffusion tensor imaging
The relationship between diffusivity indices in white matter, gray matter volumes, and
Abbreviations: AGA,
Appropriate for gestational age; AD,
Anatomical Registration through Exponentiated Lie Algebra; DTI, FWE,
family-wise error; GM,
imaging; RD,
gray matter; IUGR,
radial diffusivity; SGA,
Axial diffusivity; CA,
Corrected age; DARTEL,
diffusion tensor imaging; FA,
intrauterine growth restriction; MD,
small for gestational age; TBSS,
mean diffusivity; MRI,
magnetic resonance
tract-based spatial statistics; VBM, voxel-based morphometry;
WM, white matter n Corresponding author at: Universidad de Barcelona, Department of Maternal–Fetal Medicine, ICGON, Hospital Clínic, Sabino de Arana 1 – Helios III, 08028 Barcelona, Spain. E-mail addresses: [email protected], [email protected] (N. Padilla). 0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.12.007
Diffeomorphic
fractional anisotropy;
2
brain research 1545 (2014) 1–11
perinatal data was also investigated. Gray-matter decrements attributable to IUGR comprised amygdala, basal ganglia, thalamus and insula bilaterally, left occipital and parietal lobes, and right perirolandic area. Gray-matter volumes positively correlated with birth weight exclusively. Preterm infants had reduced FA in the corpus callosum, and increased FA in the anterior corona radiata. Additionally, IUGR infants had increased FA in the forceps minor, internal and external capsules, uncinate and fronto-occipital white matter tracts. Increased axial diffusivity was observed in several white matter tracts. Fractional anisotropy positively correlated with birth weight and gestational age at birth. These data suggest that IUGR differentially affects gray and white matter development preferentially affecting gray matter. At 12 months IUGR is associated with a specific set of structural gray-matter decrements. White matter follows an unusual developmental pattern, and is apparently affected by IUGR and prematurity combined. & 2013 Elsevier B.V. All rights reserved.
1.
Introduction
Intrauterine growth restriction (IUGR) is one of the common reasons indicated for preterm delivery. This condition, due to placental insufficiency, affects 5–10% of all pregnancies and is associated with chronic hypoxia and under-nutrition during fetal life. The combination of preterm birth and IUGR results in a higher rate of perinatal complications and consequently worse long-term outcomes (Geva et al., 2006; Guellec et al., 2011; Leitner et al., 2007). The structural alterations underlying the effects of IUGR on the preterm brain are only partially documented. In this respect, studies have been hampered by the widespread practice of using the terms small for gestational age (SGA) and IUGR synonymously. Defining SGA as a birth weight (BW) below a given threshold compared to appropriate for gestational age (AGA) groups, SGA children had smaller brain volumes (De Bie et al., 2011; Martinussen et al., 2009; Xydis et al., 2013) and altered white matter (WM) microstructure (Eikenes et al., 2012; Lepomaki et al., 2013). In contrast, comparisons between preterm neonates
with and without intrauterine growth restriction, defined as BW below the 10th percentile and abnormal Doppler values within the umbilical artery, showed that IUGR is associated with reduced volumes of the cortical gray matter (GM; Tolsa et al., 2004), decreased volumes of the hippocampus (Lodygensky et al., 2008) and a discordant pattern of gyrification (Dubois et al., 2008) related to behavioral alterations. At 12 months, comparisons between preterm infants with and without growth restriction and term-born controls suggested that the most pronounced differences in the IUGR preterm group were related to a different distribution of the GM and WM (Padilla et al., 2011) as well as a different cortical brain complexity (Esteban et al., 2010), both associated to neurodevelopmental difficulties. However, the GM alterations induced by IUGR, and the existence of microstructural WM differences, in preterm infants at 12 months, excluding the influence of prematurity itself, have not been investigated. The aims of this study were (1) to detect specific regional GM volume changes as a consequence of IUGR in preterm infants at 12 months corrected age (CA); (2) to explore whether IUGR induces specific WM microstructure alterations
Fig. 1 – Study population. IUGR, intrauterine growth restriction; AGA, appropriate for gestational age; PVL, periventricular leukomalacia; US, ultrasound; DTI, diffusion tensor imaging; MRI, magnetic resonance imaging.
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brain research 1545 (2014) 1–11
in the same population; and (3) to correlate these changes with birth weight and gestational age at birth. For this, we included a control group of preterm infants appropriate for gestational age (AGA) to exclude the effect of prematurity per se.
2.
Results
A total of 57 infants with IUGR, 70 preterm AGA infants and 72 term-born infants were initially included. The final sample included 20 infants with IUGR, 20 preterm AGA infants and
20 term-born infants (Fig. 1). Perinatal, demographic, and anthropometric characteristics of the infants are detailed in Table 1. There were no significant differences in perinatal data between the subjects scanned and those who were not scanned. As expected, full-term children differed significantly in neonatal data compared with both preterm groups. During the neonatal period, preterm infants with IUGR differed significantly in the rate of cesarean section, birth weight, and umbilical cord arterial pH value compared to the AGA preterm group (Table 2). Of note, IUGR infants had significantly lower weight, length and head circumference compared with preterm AGA infants and term infants at 12 months CA.
Table 1 – Characteristics of the study groups. IUGR n ¼ 20
Preterm n ¼20
Term n ¼ 20
Perinatal Gestational age at delivery range Cesarean section Umbilical artery pH Apgar 5 min Gender (M/F) Birth weight (g) Range Length at birth (cm) Range Head circumference at birth (cm) Range
30.9971.86n 26.50–34.0 17/20 (85%) † 7.18 (0.15)† 9.10 (1.21) 10/10 1049.757237.47n 600–1420 38.2073.65n 32–43 27.1772.65n 22–29
30.0472.35n 26.00–34.00 8/20 (40%) 7.29 (0.06) 9.30 (0.80) 11/9 1531.757402.94n 1000–2500 39.8573.97n 33–46 28.2772.85n 24–32
39.82 (1.40) 37.1–41.5 – 7.26 (0.08) 9.90 (0.24) 10/10 32727496.78 3150–4100 49.6071.75 46–52 34.0071.13 33–36
Demographic Mother0 s age (years) Range Mother0 s education less thanhigh school n (%) Breastfeeding 44 months n (%) Use of early intervention services Corrected age at scan (months)
33.0073.78 27–40 7/20 (35.0%) 16/20 (80.0%) 9/20 (45.0%) 12.9071.07
30.5074.38 22–37 6/20 (30.0%) 17/20 (85.0%) 6/20 (30.0%) 12.4070.68
32.1274.54 20–37 2/20 (10.0%) 16/20 (80.0%) – 13.0070.86
Anthropometric data at 12 months CA Weight at 12 months (kg) Range Height at 12 months (cm) Range Head circumference at 12 months (cm) Range
8.3570.98n, † 6.97–11.00 70.2373.05n, † 64.00–85.00 45.3771.62 † 42.00–47.50
9.6871.45 7.20–13.00 74.8574.45 70.00–85.00 46.3270.99 44.50–48.00
9.7471.02 8.00–12.10 73.9572.86 69.00–85.50 45.9971.16 43.00–47.50
Whole-brain measurements Gray matter (cm3) White matter (cm3) Total brain volume (cm3) Mean FA values
638.75751.23n, 201.77725.30 840.50775.66n, 0.33970.01n
691.80757.07 220.63732.15 912.42785.62 0.33470.09
683.14747.30 215.44728.09 898.59768.84 0.32970.01
†
†
SNAP-PE, score for neonatal acute physiology-perinatal extension; NICU, neonatal intensive care unit. po0.05 compared with term infants. † po 0.05 compared with preterm infants. Data are mean7SD. n
Table 2 – Neonatal characteristics. Characteristic
IUGR (20)
Preterm (20)
Statistics (p value)
Prenatal corticosteroids Cesarean section Umbilical artery pH Apgar 5 min
14/20 (70%) 17/20 (85%) 7.18 (0.15) 9.1 (1.21)
17/20 (85%) 8/20 (40%) 7.29 (0.06) 9.30 (0.80)
FET (0.451) FET (0.004) t¼ 2.99 (0.06) t¼ 0.616 (0.64)
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Table 2 (continued ) Characteristic
IUGR (20)
Preterm (20)
Statistics (p value)
SNAP-PE Days in NICU Mechanical ventilation Postnatal corticosteroids Late-onset neonatal sepsis Hypoglycemia Hyperglycemia
17/20 [23.9 (14.6)] 43.10 (27.94) 9/20 (45%) 0 5/20 (25%) 8/20 (40%) 7/20 (46.7%)
19/20 [16.3 (14.4)] 42.75 (20.40) 13/20 (65%) 2/20 (10%) 1/20 (5%) 7/20 (35%) 8/20 (40%)
t ¼ 1.824 (0.08) t ¼ 0.045 (0.96) FET (0.341) FET (0.487) FET (0.342) FET (1.0) FET (1.0)
IUGR, intrauterine growth restricted infants; SNAP-PE, score for neonatal acutephysiology—perinatal extension; NICU, neonatal intensve care unit: FET, Fisher0 s exact test.
Fig. 2 – Regions of gray-matter volume decrease (gray to white colors) in preterm IUGR infants compared with term-born infants (a) and preterm infants without IUGR (b). The bar represents the t scores. Numbers represent the corresponding slice. Right¼ left.
2.1.
Conventional MRI findings
Visual inspection of the MRI studies revealed mildly altered structural scans in 7 infants with IUGR (5 with slightly dilated lateral ventricles and 2 with a thinning of the body of the corpus callosum) and 5 preterm AGA (3 with slightly dilated lateral ventricles and 2 with a thinning of the body of the corpus callosum). The term-born group had no brain abnormalities.
3. Voxel-based morphometry DARTEL: GM results 3.1.
gyrus bilaterally, left occipital and parietal lobes, and right perirolandic area (Fig. 2b). The areas and significance levels of GM differences between groups are shown in Table 3.
3.2.
Appropriate for gestational age group
Compared to term-born infants, the preterm AGA group showed 1 region of GM decrements in the right temporal lobe.
4.
Diffusion-tensor imaging: WM results
4.1.
Intrauterine growth restricted group
Intrauterine growth restricted group
Infants with IUGR had significantly reduced absolute global GM volumes compared to preterm AGA and term-born infants. However, relative volumes were not significantly different between groups (Table 1). Compared with term-born infants, those with IUGR had GM reductions involving predominantly the temporal lobe bilaterally, the hippocampus, the amygdala and the right perirolandic area. Additional areas of GM volume reduction were found in the right frontal lobe, left parietal lobe, perirolandic area and basal ganglia (Fig. 2a). Compared with the preterm AGA group, infants with IUGR showed reduced GM volume in the amygdala, basal ganglia, thalami, insula, angular
Compared with the preterm AGA group, infants with IUGR had clusters of decreased FA predominantly localized in the splenium of the corpus callosum, and increased FA in the anterior corona radiata (Fig. 3a, b). Additionally, IUGR infants showed clusters of increased AD comprising the forceps minor and anterior corona radiata (Fig. 3c). No significant differences were identified in any other diffusivity parameter. The analyses were repeated at different thresholds; however, no between-group differences emerged. Compared with their term counterparts, infants with IUGR exhibited clusters of decreased FA comprising the splenium of the corpus callosum (Fig. 4a). Intrauterine growth restricted
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Table 3 – Significant differences in gray-matter volumes in the study groups. Anatomic region
Hemisphere R ¼right, L¼ left
Contrast
Cluster (mm3)
Cluster level p corrected
t
Superior temporal gyrus posterior part Middle and inferior temporal gyrus Fusiform gyrus Anterior temporal lobe lateral part Insula, putamen caudado, pallidum Parahippocampal and ambient gyri Amygdala Hippocampus posterior part Inferior frontal gyrus
R
IUGRoterm
5673
o0.001
7.17
Superior temporal gyrus posterior part Middle and inferior temporal gyrus Posterior temporal lobe Precentral gyrus Parahippocampal and ambient gyri
L
IUGRoterm
2046
o0.001
4.93
Putamen Caudate Pallidum
L L L
IUGRoterm
574
o0.001
5.14
Post-central gyrus
L
IUGRoterm
1968
o0.001
5.42
Inferior–lateral remainder parietal lobe Precuneus and cuneus
L L and R
IUGRopreterm
212
o0.001
4.49
Post-central and supramarginal gyri Parietal inferior gyrus Temporal superior gyrus Precuneus Pre-central gyrus
R
IUGRopreterm
3322
o0.001
5.1
Lateral reminder occipital lobe Lingual gyrus cuneus Post-central and pre-central gyrus
L
IUGRopreterm
4137
o0.001
4.2
L
IUGRopreterm
535
0,008
4.16
Putamen; pallidum; insula; caudate; amygdala Cingulate gyrus posterior part
L
IUGRopreterm
2336
o0.001
5.17
Putamen; insula; pallidum; caudate; amygdala Superior temporal gyrus Middle and inferior temporal gyrus
R
IUGRopreterm
1457
o0.001
5.21
R
pretermoterm
1506
o0.001
6.57
Lateral reminder occipital lobe Precuneus Cuneus
L L and R R
preterm4term
3287
o0.001
5.47
IUGR, intrauterine growth restricted infants.
infants, also had greater FA in several clusters (Fig. 4b), especially in the anterior regions involving commissural fibers (genu, the body, and forceps minor of the corpus callosum); projection fibers (anterior, superior and posterior corona radiata, the left anterior limb of the internal capsule, and posterior limb of the internal capsule bilaterally) and association tracts (right frontal course of the uncinate fasciculum and inferior fronto-occipital fasciculum, and left external capsule including the superior longitudinal fasciculum). Furthermore, intrauterine growth restricted infants, compared to term infants, showed higher AD in several
regions (Fig. 4c). No MD or RD differences were identified between IUGR and term infants.
4.2.
Appropriate for gestational age group
Compared to the term-born infants the preterm AGA group had clusters of decreased FA in the splenium and body of the corpus callosum, and increased FA in the forceps minor (Fig. 5a, b). No significant differences were seen in any other diffusivity parameter.
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Fig. 3 – White matter tracts showing regions (in black) of decreased (a) and increased (b) fractional anisotropy (FA), and increased axial diffusivity (AD) (c) between intrauterine growth restricted infants (IUGR) and preterm appropriate for gestational age infants (AGA). Numbers represent the corresponding slice. Right¼ left.
4.3. Relationship between GM volumes, diffusivity parameters, and perinatal data in the IUGR group In GM, significant correlations with the birth weight were observed comprising the right parahippocampal gyrus, hippocampus and right cerebellar hemisphere (cluster¼ 1043; t¼4.56; p¼ 0.02, FWE corrected). No significant correlations were found between regional GM volumes and gestational age at birth. Fractional anisotropy showed widespread positive linear associations with gestational age at birth and birth weight (Supplementary material, Fig. 2). No correlations were found between perinatal data and any other diffusion parameter.
5.
Discussion
The results of the current study suggest that in preterm infants with IUGR at 12 months CA without apparent brain lesions, the GM and WM are differentially affected. In terms of GM, the current results expand on our previous studies that have reported GM brain alterations in infants with IUGR at 12 months (Padilla et al., 2011; Esteban et al., 2010) and further demonstrate that IUGR is associated with a specific set of
structural GM decrements comprising the amygdala, basal ganglia, thalami, left occipital and parietal lobes, and the right perirolandic area. Gray-matter volume decrements positively correlated with birth weight but not with gestational age at birth. In terms of white matter, it follows an unusual developmental pattern involving predominantly the anterior brain regions. These microstructural alterations are positively correlated with gestational age at birth and birth weight.
5.1.
Voxel-based morphometry DARTEL: GM results
The results from this study suggest that GM reductions ultimately attributable to IUGR comprised mainly the amygdala, basal ganglia, thalami, left occipital and parietal lobes, and the right perirolandic area. In fact, as previously reported for the neonatal period (Tolsa et al., 2004), IUGR predominantly affects cerebral cortical development, reflected in a decreased total brain volume, which is consistent with our results. Of particular note is the involvement of the insula, amygdala, and basal ganglia, all of which share a close anatomical and functional relationship and have a key role in the control of behavior, cognitive processing, and autonomic activity (Fukushima et al., 2011). These data are of
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Fig. 4 – White matter tracts showing regions (in black) of decreased (a) and increased (b) fractional anisotropy (FA), and increased axial diffusivity (AD) (c) between intrauterine growth restricted infants (IUGR) and term infants. Numbers represent the corresponding slice. Right¼ left.
particular interest, since IUGR is associated with altered developmental activity of the autonomic nervous system (Jones, 2011), and social, emotional, and behavioral disorders (Leitner et al., 2007; Tolsa et al., 2004). In the light of previously reported language problems in children with IUGR (Geva et al., 2006), the decreased GM in the angular gyrus bilaterally is of particular interest, since this structure is described as a core component of a distributed language network (Constable et al., 2008). Here, the additional involvement of the sensorimotor area, the insula, and the occipital cortex is consistent with some previous reports exploring language processing in preterm and term subjects (Schafer et al., 2009). Our study adds new data concerning the possible anatomical substrates related to language difficulties in preterm infants with IUGR at school age. Interestingly the precuneus showed specific vulnerability to IUGR (Padilla et al., 2011). The posterior left precuneus was affected notably, and the occipital area on the same side. These structures are known to be involved in visual–spatial
information processing. Our results complement previous findings suggesting a regional vulnerability of the occipital cortex to the IUGR (Thompson et al., 2007) and may point to an abnormal connection between the precuneus and the occipital cortex, which likely relate to the visual–spatial difficulties reported in IUGR children (Geva et al., 2006).
5.2.
Diffusion-tensor imaging: WM results
In this study, compared to term and preterm AGA groups, the IUGR infants presented clusters of decreased FA comprising the splenium of the corpus callosum. It should be noted that the preterm AGA group also showed decreased FA in the body and splenium of the corpus callosum when compared to term infants. These results are consistent with those of other studies that have examined FA in preterm and SGA subjects. A previous study including young adults born preterm showed decreased FA in several areas of the corpus callosum (Eikenes et al., 2011). Also, the FA in this structure has been
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Fig. 5 – White matter tracts showing regions (in black) of decreased (a) and increased (b) fractional anisotropy (FA) between preterm appropriate for gestational age (AGA) infants and term infants. Numbers represent the corresponding slice. Right¼left.
negatively correlated with total IQ in SGA subjects (Eikenes et al., 2012) suggesting a compensatory rewiring of brain white matter circuitry in the context of intrauterine growth restriction. Of note, the corpus callosum integrates sensory, motor, cognitive, and emotional functions from both hemispheres and could be adversely affected by prematurity (Constable et al., 2008; Rose et al., 2008) and specifically IUGR (Anderson et al., 2006; Tolcos et al., 2011). Contrary to the expectations, in comparisons between preterm IUGR and term infants, changes in WM microstructure were predominantly reflected by increased FA and AD comprising commissural, projection, and associative tracts. Notably, smaller clusters of increased FA were also found in preterm IUGR infants compared to the AGA group. In the preterm IUGR infants, not all tracts were equally affected. In general, the most pronounced changes were found in the anterior brain regions that are likely to be more vulnerable to insults that could be reflected in developmental difficulties (Guellec et al., 2011; Leitner et al., 2007). The most severely affected tract was the forceps minor, a WM tract connecting medial and lateral prefrontal regions via the genu of the corpus callosum. This structure is involved in higher cognitive functions as well as emotional and behavioral control. The increased FA found in this area has not been previously reported in preterm infants. Further confirmation of this finding is important since structural changes may be involved in the developmental disabilities that have been reported in children with IUGR (Geva et al., 2006; Leitner et al., 2007). Here, increased FA also comprised association fibers (inferior fronto-occipital fasciculum, inferior longitudinal fasciculum, superior longitudinal fasciculum, and uncinate fasciculum) that connect frontal and parieto-temporal–occipital areas. In a previous study using a standard VBM approach, we reported regional GM volume differences between preterm infants with IUGR and term-born infants involving the frontal, parietal, and occipital lobes (Padilla et al., 2011). Thus, our findings here complement these earlier results, suggesting
microstructural abnormalities of long-distance tracts that may be due to an aberrant fiber organization in preterm infants with IUGR. It bears mentioning that increased FA in associative tracts has also been reported in autism (Cheng et al., 2010), suggesting maturational disturbances. We also detected increased FA in the corona radiata and the posterior limb of the internal capsule. The involvement of these structures may contribute to prefrontal dysfunctions and differences in the motor skills reported in children and adolescents born with IUGR (Guellec et al., 2011; Padilla et al., 2011). The significance of increased FA in this study is uncertain. Similar FA increases have been reportedly associated with preterm birth (Allin et al., 2011; Eikenes et al., 2011) and developmental disorders (Cheng et al., 2010). In this respect, this apparently “increased” maturation in WM does not mean, “better” (Eikenes et al., 2012; Hoeft et al., 2007). Regions of increased FA in the preterm IUGR group might reflect an early deviation in the developmental trajectory of the WM. Compensatory changes as part of neuronal remodeling and the plastic reorganization of the brain secondary to adverse in utero-environment could be also involved. Furthermore, environmental factors that may influence the normal development during the first year of life should be considered. To aid the interpretation of FA findings, we measured RD and AD. Radial diffusivity and AD have been linked to myelin integrity (Song et al., 2002) and fiber organization (Takahashi et al., 2000), respectively. Here, changes affected exclusively AD and even at a low statistical threshold, we found no such area of altered RD. Consequently, myelination was not the main factor contributing to the cases of altered diffusivity in the present study. Thus, greater FA associated with widespread increase of AD as found here could result from changes in fiber organization related to reduced tortuosity to straighter fibers (Takahashi et al., 2000). This result differs from results in preterm subjects reporting increased RD associated with decreased FA (Eikenes et al., 2011) or no alteration in the organization of white matter fibers. These findings may relate
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to development in infancy and may suggest early aberration in the developmental trajectory of WM in preterm infants with IUGR at 12 months of life. The fact that we found no differences in MD does not rule out that MD alterations could occur later in development—a point which needs to be elucidated in future studies.
6.
Correlation analysis
The correlations between FA and perinatal data suggest that increasing prematurity at birth and lower birth weight have negative effects on WM microstructure that are evident at 12 months in preterm infants with IUGR. A similar finding has been suggested in studies assessing the repercussions of prematurity in older subjects (Allin et al., 2011; Eikenes et al., 2011). In regional GM volumes, correlations were found with birth weight but not with gestational age at birth. This finding highlights the impact of IUGR on GM development with documented reductions on regional GM volumes at 12 months CA. Our results are consistent with those reported during the neonatal period (Tolsa et al., 2004). From this perspective, the specific effect of the IUGR on GM development could be related in part with the neuroendocrine consequences of this condition associated with increased cortisol levels that might be associated with reduction in the cortical GM (Modi et al., 2001). Further studies are needed to test this hypothesis. Strengths of this study include a well-defined cohort characterized prenatally and followed prospectively, and the definition of preterm IUGR included antenatal Doppler findings. Another strength is the age of assessment. Twelve months constitutes a critical period in human development, where the knowledge regarding GM and WM development in preterm infants with IUGR is quite limited. In addition, this study combines 2 MRI analyses (VBM in GM and TBSS in WM) assessing different aspects of GM and WM, and consequently providing complementary information. A limitation of this study is the relatively small sample size, which may have prevented statistical differences from being observed in some comparisons.
7.
Conclusion
The current study reveals a differential effect of IUGR on the developing GM and WM with the GM exclusively being affected at 12 months CA. The GM decrements affected a specific set of structures and correlated with birth weight only. The WM in IUGR infants showed an unusual developmental pattern with increased FA and AD and correlated with birth weight and gestational age at birth. Findings in GM are consistent with and provide further support to previous results in this prenatal disorder. Findings in WM provide new insights into microstructural changes underlying brain reorganization under IUGR. Future studies including larger sample sizes are clearly needed to explore these issues in greater depth. To evaluate the clinical relevance of our findings, neurodevelopmental follow-up of the infants is ongoing.
8.
Experimental procedures
8.1.
Subjects
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Between 2007 and 2009 a cohort of consecutive singleton infants was selected with the following inclusion criteria: (i) gestational age at birth less than 34 weeks; (ii) fetal weight below the 10th percentile for gestational age confirmed at birth, and (iii) umbilical artery Doppler pulsatility index 495th percentile in at least 2 consecutive examinations 24 h apart. The preterm and term control groups were singleton AGA infants, with a BW between the 10th and 90th customized centiles according to local reference standards (Figueras et al., 2008). The preterm group was matched with cases by gestational age at birth (71 week). Pregnancies were dated according to the first-trimester crown-rumplength measurements. Corrected age was calculated subtracting from chronological age the number of weeks born before 40 weeks of gestation. Exclusion criteria for both cases and controls were: (i) any intraventricular hemorrhage or periventricular leukomalacia on neonatal ultrasound or magnetic resonance imaging; (ii) placental histological criteria for chorioamnionitis; (iii) congenital malformations, including chromosomal abnormalities and infections. Prenatal and neonatal data were prospectively recorded. Written informed consent was obtained from all parents of the participating infants. The Institutional Ethics Committee approved the study protocol, recruitment, and scanning procedures.
8.2.
MRI acquisition
Infants in all groups, sleeping naturally, were scanned at 1272 months CA (Padilla et al., 2012). MRI was performed on a TIM TRIO 3.0T scanner (Siemens, Erlangen, Germany), by using a 32channel head coil. The acquisition time of the entire protocol was about 30 min. A set of high-resolution T1-weighted, 3D images was acquired using the magnetization prepared rapid acquisition gradient echo sequence (MPRAGE; TR/TE¼2050 ms/ 2.41 ms; TI¼ 1050 ms; FOV¼220 220 mm2 and 256 256 matrix; flip angle 901). The whole-brain data were acquired in a sagittal plane, yielding contiguous slices with isotropic voxel of 0.9 0.9 0.9 mm3. T2-weighted images in sagittal orientation (TR/TE¼ 4640 ms/102 ms; 122 122 matrix; flip angle 901, voxel size 0.62 0.62 3 mm3) were also acquired to detect any lesion noticeable on the T1-weighted images. Single-shot diffusion weighted EPI sequences were acquired in 30 non-collinear directions with a p-value¼ 1000 s/mm2 and 1 image without diffusion gradient (b¼ 0 s/mm2). TR/TE¼9300 ms/94 ms; 122 122 matrix; flip angle 901, slice thickness 3.0 mm; no interslice gap; slices¼ 30; voxel size 1.64 1.64 3 mm3. The MRI scans were reviewed by a neuroradiologist (NB) blinded to group membership.
8.3.
Image analysis
For voxel based-morphometry (VBM) procedure the SPM v5 software, Wellcome Department, University College (London, UK) running on MATLAB v7.5 (MathWorks, Natrick, MA, USA) was used. The steps followed were: (1) line determination of
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the anterior–posterior commissures and image reorienting; (2) whole-brain segmentation of the GM, WM, and cerebral spinal fluid of the T1 structural MRI images (Supplementary material, Fig. 1) by employing the VBM 5.1 toolbox (http:// dbm.neuro.uni-jena.de/vbm/) implemented in SPM v5 software, using infant brain tissue probability maps; (3) images to be analyzed were imported and the diffeomorphic anatomical registration through exponentiated lie algebra (DARTEL) algorithm (Ashburner, 2007) was applied (Supplementary material, Fig. 1b). Finally, all images were modulated and smoothed with a full width at half-maximum of 6 mm Gaussian kernel. Global GM and WM volumes, intracranial volume (GMþWMþCSF), and total brain volume (GMþWM) were calculated using the native-space tissue maps of each subject. Global GM and WM volumes were normalized by intracranial volume to calculate the relative GM and WM volumes. Diffusion tensor images were processed with the FMRIB software library, FSL v4.1, Oxford Centre for Functional MRI of the brain, (Oxford, UK). The volumes were eddy-current- and motion-corrected relative to the first (b ¼0) volume by using FMRIB0 s diffusion toolbox (FDT; Smith et al., 2004). Non-braintissue components were removed using the brain extraction tool (Smith, 2002). Tract-based spatial statistics v1.2 (TBSS; Smith et al., 2006) was applied to the fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) maps. Fractional anisotropy maps from all subjects were aligned to the most representative map using non-linear registration and upsampled to 1x1 1 mm3 voxel size. The mean of all aligned FA images was then created and thinned to generate a skeletonized mean FA image (threshold40.2) to reflect common tracts across all subjects (Supplementary material, Fig. 1c). Mean diffusivity, AD, and RD were processed similarly to FA data with the exception that FA images were used to drive the nonlinear registration and skeletonization stages.
8.4.
Statistical analyses
All variables studied were checked for normality before each analysis. Quantitative data were analyzed with Student0 s t-test or one-way analyses of variance (ANOVA) and qualitative data with Fisher0 s exact test. All statistical analyses were performed using SPSS 17.0 (SPSS Inc, Chicago, USA). A po0.05 was considered statistically significant. In the DTI and VBM analyses, 2 covariates—total brain volume and postmenstrual age at scan—were introduced into each statistical design. Additional analyses were conducted to assess the probable influence of gender as well as cesarean section and umbilical pH on the results. However, these variables had a negligible effect on the p values and thus were not included as covariates. For DTI analyses we used a permutation-based approach, this being Randomise v 2.1 (Nichols and Holmes, 2002). For GM analysis, we used the VBM approach with a cluster threshold set at 60 voxels; t-test group comparisons were performed to evaluate the GM volume changes between groups and a “simple regression analysis” to test the possible relationship between GM and perinatal data. In both studies, we analyzed the following contrasts: patients greater than controls and controls greater than patients. All the results
underwent a family-wise error (FWE) correction method for multiple comparisons (po0.05).
Acknowledgments Funding support was provided by grants from the Cerebra Foundation for the brain-injured child (Carmarthen-Wales, UK), The Thrasher Research Fund (Salt-Lake-City, USA). NP was supported by a Sara Borrell post-doctoral fellowship (CD09/00263), Instituto de Salud Carlos III, Spain. The authors would like to thank all the participating infants and the parents, Marta Garcia and Alba Camacho for contacting and booking the infants, and Cesar Garrido for imaging acquisition.
Appendix A.
Supporting information
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres. 2013.12.007.
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