Intrauterine growth restriction affects the maturation of myelin

Experimental Neurology 232 (2011) 53–65

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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / yex n r

Intrauterine growth restriction affects the maturation of myelin Mary Tolcos ⁎, Elizabeth Bateman, Rachael O'Dowd, Rachel Markwick, Krijn Vrijsen, Alexandra Rehn, Sandra Rees Department of Anatomy and Cell Biology, The University of Melbourne, Grattan Street, Parkville, Victoria, 3010, Australia

a r t i c l e

i n f o

Article history: Received 17 August 2010 Revised 21 May 2011 Accepted 8 August 2011 Available online 16 August 2011 Keywords: Cerebral palsy Chronic placental insufficiency Foetal brain development Hypoxia Oligodendrocyte lineage Myelin Myelin basic protein White matter

a b s t r a c t Intrauterine growth-restriction (IUGR) can lead to adverse neurodevelopmental sequelae in postnatal life. Our objective was to determine whether IUGR, induced by chronic placental insufficiency (CPI) in the guinea pig results in long-term deficits in brain myelination and could therefore contribute to altered neural function. CPI was induced by unilateral ligation of the uterine artery at mid-gestation (term ~ 67 days of gestation; dg), producing growth-restricted (GR) foetuses (60 dg), neonates (1 week) and young adults (8 week); controls were from the unligated horn or sham-operated animals. In GR foetuses (n = 8) and neonates (n = 7), white matter (WM) volume was reduced (p b 0.05); this reduction did not persist in young adults (n = 11) however the corpus callosum width was reduced (p b 0.05). Immunoreactivity (IR) for myelin basic protein (MBP), myelin-associated glycoprotein (MAG) and myelin proteolipid protein (PLP), all markers of myelinating oligodendrocytes (OL), was reduced in GR foetuses compared to controls. MBP was the most markedly affected with an abnormal retention of protein in the OL soma and a reduction of its incorporation into the myelin sheath. MAG-IR OL density was reduced (p b 0.05), while the density of OLs immunoreactive for Olig-2, a transcription factor expressed throughout the entire OL lineage, was increased (p b 0.05). MBP-, MAG- and PLP-IR recovered to control levels postnatally. These results suggest that IUGR transiently delays OL maturation and myelination in utero but that myelination and WM volume are restored to control levels postnatally. Long-term deficits in myelination are therefore unlikely to be the major factor underlying the altered neurological function which can be associated with IUGR. Crown Copyright © 2011 Published by Elsevier Inc. All rights reserved.

Introduction Intrauterine growth restriction (IUGR) can lead to infants being born small-for-gestational age (SGA) and is second to prematurity as the leading cause of perinatal morbidity and mortality (Ghidini, 1996); it affects 3–10% of all pregnancies (Pryor, 1997). IUGR can occur as a result of an inadequate supply of oxygen and nutrients to the foetus due, for example, to placental damage, physical occlusion of the umbilical cord (Mann, 1986) or nicotine-induced vasoconstriction of the uteroplacental blood vessels (Nash and Persaud, 1988). Each of these factors can lead to acute or chronic placental insufficiency (CPI) and Abbreviations: BDNF, brain-derived growth factor; CPI, chronic placental insufficiency; CRL, crown-rump length; dg, days of gestation; GCL, granule cell layer; GFAP, glial fibrillary acidic protein; GM, grey matter; GR, growth-restricted; Iba-1, ionised calcium binding adapter molecule 1; IGF, insulin-like growth factor; IR, immunoreactive/immunoreactivity; IUGR, intrauterine growth restriction; MAG, myelin-associated glycoprotein; MBP, myelin basic protein; ML, molecular layer; OD, optical density; OL, oligodendrocyte; Olig2, oligodendrocyte transcription factor 2; PB, phosphate buffer; PFA, paraformaldehyde; PLP, myelin proteolipid protein; preOL, preoligodendrocyte; PVL, periventricular leukomalacia; SD, standard deviation; SGA, small for gestational age; UAL, uterine artery ligation; WM, white matter. ⁎ Corresponding author. Monash University, Monash Institute of Medical Research, The Ritchie Centre, P.O. Box 5418, Clayton, Victoria 3168, Australia. E-mail address: [email protected] (M. Tolcos).

foetal hypoxemia. Neurodevelopmental sequelae of IUGR range from learning difficulties (Geva et al., 2006; Low et al., 1992) and decreased intelligence and cognition (de Bie et al., 2010; Geva et al., 2006) in childhood, to cerebral palsy (Blair and Stanley, 1990; Jacobsson and Hagberg, 2004); the outcome is likely to depend on the length, severity and timing of the intrauterine compromise. Previously, we have shown that CPI induced via uterine artery ligation (UAL) throughout the second half of gestation in the guinea pig, results in structural and functional deficits in the nervous system in the short- (Dieni and Rees, 2003; Mallard et al., 1999, 2000; Nitsos and Rees, 1990; Tolcos and Rees, 1997) and long-term (Bui et al., 2002; Rehn et al., 2002, 2004). To date our structural studies have focused on dendritic growth (Dieni and Rees, 2003), astroglial development (Nitsos and Rees, 1990; Tolcos and Rees, 1997) and neuronal survival (Loeliger et al., 2004; Mallard et al., 1999, 2000). We have also shown that CPI leads to a delay in the process of myelination in the brain and spinal cord of the foetal guinea pig (52 dg and 62 dg; term ~ 67 dg) (Nitsos and Rees, 1990), but have not investigated the postnatal and long-term effects on myelination, a vital aspect of neural maturation and function. An advantage of studying this process in guinea pig, compared to rodent models, is that myelination commences in utero, as it does in humans (Back et al., 2001) albeit at a more accelerated rate.

0014-4886/$ – see front matter. Crown Copyright © 2011 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2011.08.002

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M. Tolcos et al. / Experimental Neurology 232 (2011) 53–65

In rodents, foetal compromise induced via UAL in late gestation (Olivier et al., 2005) or by hypoxia throughout gestation (Baud et al., 2004) results in a reduction postnatally in the density of O4-IR preoligodendrocytes (preOLs) and MBP-IR in forebrain white matter (WM). However, the long-term effects of these insults appeared to differ; late gestation CPI resulted in sustained deficits in MBP-IR while MBP-IR was only transiently affected by chronic prenatal hypoxia. Despite these differences, both studies claimed that the deficits in myelination could be attributed to the innate vulnerability of preOLs to oxidative stress (Back, 2006; Volpe, 2001). More recent studies in a rodent model of perinatal hypoxia–ischemia (Segovia et al., 2008) and in human periventricular leukomalacia (PVL) (Billiards et al., 2008) have demonstrated that although there is a reduction in early myelinating OLs (Segovia et al., 2008), there is a concurrent increase, rather than a decrease, in the density of Olig2-IR OLs (Billiards et al., 2008) and preOLs (Segovia et al., 2008). Together, these results suggest that alterations in myelination in injured WM might be accounted for by an arrest in the maturation of the OL lineage. Here, our objective was to determine whether the prenatal delay in myelination previously identified in our model of CPI (Nitsos and Rees, 1990), manifests as a reduction in myelin/OL protein expression and WM volume in postnatal and young adult life and hence could contribute to altered neural function. To assess the effects of CPI on the entire pool of OLs, we used the oligodendrocyte transcription factor 2 (Olig2), which is expressed throughout the OL lineage (Ligon et al., 2004, 2006). Early myelinating OLs and the specific components of mature myelin were also assessed using myelin basic protein (MBP), myelin-associated glycoprotein (MAG), and myelin proteolipid protein (PLP). Furthermore, we examined the relationship between any deficits in myelination (WM volume and altered myelin/OL protein expression) and WM injury (astrogliosis and microgliosis). Experimental procedures Experiments were approved by the University of Melbourne Animal Experimentation and Ethics Committee and were carried out in accordance with the National Health and Medical Research Council of Australia and international guidelines. Surgery CPI was induced by unilateral UAL in date-mated Dunkin-Hartley guinea pigs (n = 33) as described previously (Nitsos and Rees, 1990). At 28–30 days of gestation (dg; term ~ 67 days) pregnant guinea pigs were anaesthetised (ketamine, 40 mg/kg; Ilium Laboratories, Victoria, Australia and xylazil, 6 mg/kg; Troy Laboratories, NSW, Australia, i.m.) and a midline incision made in the abdominal wall exposing the peritoneal cavity. The mesometrium of one uterine horn was located and the uterine artery was ligated near the cervical end of the arterial arcade; the ligature remained in place for the duration of the pregnancy. After suturing, the incision was treated with a chlorohex C/ethanol solution and a topical antibiotic powder (Cicatrin® powder; GlaxoSmithKline, Middlesex, UK). Foetal reabsorptions or abortion occurred in approximately 26% of dams following UAL. Foetuses (60 dg) from the unligated horn were used as controls. Controls for the postnatal cohorts were obtained from sham-operated animals (n = 14). Animals Foetuses were delivered by Caesarean section, under deep anaesthesia (Lethabarb; sodium pentobarbitone, 130 mg/kg; i.p.; Virbac Pty. Ltd., Australia), between 60 and 62 days of gestation. Foetuses and placentae were weighed and the crown-rump length (CRL) measured.

Pups were born naturally and housed with their mother and siblings. On the day of birth, each neonate was weighed and the CRL measured. At 1 and 8 weeks of age, neonates were killed with an overdose of Lethabarb (i.p.) and body weight and CRL recorded. Foetal and newborn guinea pigs were considered to be growth-restricted (GR) if 1) their mean body weight and CRL were 2 standard deviations (SD) below that of agematched controls and 2) their mean brain to body weight ratio was 2 SD above the mean ratio for age-matched controls (Jones et al., 1984). Perfusion and tissue preparation Foetal (60–62 dg; control: n = 8; GR: n = 8), neonatal (1 week; control: n = 7; GR: n = 7) and young adult (8 week; control: n = 12; GR: n = 11) animals were transcardially perfused with saline and 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB; pH 7.4). Whole brain and cerebellar weights were recorded and tissue postfixed in 4% PFA for 4 h at 4 °C. The right cerebral hemisphere and right half of the cerebellum were immersed in 20% sucrose in PB (0.1 M, pH 7.4) and then serial coronal (hemispheres) or sagittal (cerebellum) sections (40 μm) cut on a freezing microtome; every fifth section was stained with 0.01% thionin in acetate buffer (pH 4.5). Intervening sections were collected and stored at − 20 °C in cryoprotectant (15% sucrose in 0.01 M PB containing ethylene glycol, pH 7.2) until required for free-floating immunohistochemistry. The left cerebral hemisphere and left half of the cerebellum were embedded in paraffin, serially sectioned (8 μm) and every 10th section collected onto slides for immunohistochemistry. Immunohistochemistry Single-labelling immunohistochemistry All primary antibodies, dilutions, suppliers and regions stained are summarised in Table 1. Free-floating immunohistochemistry was performed to localise MBP, MAG, PLP, Olig2 and the neurofilament marker SMI 312 using techniques previously described (Tolcos and Rees, 1997). Immunoreactivity (IR) for glial fibrillary acidic protein (GFAP) and ionised calcium binding adapter molecule 1 (Iba-1) was localised on paraffin-embedded sections using techniques previously described (Duncan et al., 2002). When the primary antibody was replaced with phosphate buffered saline (pH 7.4), immunoreactivity failed to occur. For each antibody, sections from each age group were stained simultaneously to ensure uniform conditions for subsequent analysis. Olig2 and GFAP double-labelling immunofluorescence Free-floating sections from the cerebral hemispheres (frontal cortex) of control (n = 3) and GR (n = 3) foetuses were stained sequentially using rabbit anti-Olig2 (1:500) and Alexa-Fluour-488 goat anti-rabbit IgG (1:500; Molecular Probes, USA) followed by rabbit anti-GFAP (1:1000) and Rhodamine (TRITC) AffiniPure donkey antirabbit IgG (1:500; Jackson ImmunoResearch, PA, USA) as previously described (Munro et al., 2009). Sections were blocked in 10% normal rabbit serum in Tris buffered saline for 1 h following the completion of Olig2 and prior to GFAP immunofluorescence to prevent crossreactivity. Quantitative analysis Volumetric and immunohistochemical analyses were performed using a calibrated Image Pro Plus software package (Version 4.1; MediaCybernetics, MD, USA). Slides were coded in order to prevent experimenter bias and all measurements were made in randomly selected fields. Means were calculated for each animal and a mean of means for GR and control groups determined; all densities are expressed as cells/mm 2.

M. Tolcos et al. / Experimental Neurology 232 (2011) 53–65

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Table 1 Immunohistochemistry: primary and secondary antibodies. 1° antibody and dilution

Localises

Supplier

2° antibody and dilution

Brain region and age

Rabbit anti-GFAP 1:500 Rabbit anti-Iba-1a 1:1500 Mouse anti-MAG 1:1000 Rat anti-MBP 1:500

Astrocytes

ZO334; DAKO, CA, USA

Biotinylated anti-rabbit IgG; 1:200

Microglia

019-19741; Wako Pure Chemical Industries, Osaka, Japan MAB1567; Millipore, MA, USA

Biotinylated anti-rabbit IgG; 1:200

MAB395; Millipore, MA, USA

Biotinylated anti-rat IgG; 1:200

Rabbit anti-Olig2 1:1000 Mouse anti-PLP 1:1000 Mouse anti-SMI-312 1:2000

Oligodendrocytes

AB9610; Millipore, MA, USA

Biotinylated anti-rabbit IgG; 1:200

Myelinated fibres

MAB388; Millipore, MA, USA

Biotinylated anti-mouse IgG; 1:200

Axons

Sternberger Monoclonal Inc. MD, USA

Biotinylated anti-mouse IgG; 1:200

Cerebral hemispheres and cerebellum: 60 dg and 1 week Cerebral hemispheres and cerebellum: 60 dg and 1 week Cerebral hemispheres and cerebellum: 60 dg and 1 week Cerebral hemispheres: 60 dg, 1 week, 8 weeks Cerebellum: 60 dg and 1 week Cerebral hemispheres and cerebellum: 60 dg and 1 week Cerebral hemispheres and cerebellum: 60 dg and 1 week Cerebral hemispheres and cerebellum: 60 dg

a

Myelinated fibres and cell bodies Myelinated fibres and cell bodies

Biotinylated anti-mouse IgG; 1:200

Pre-treatment using citrate buffer (pH 6) and microwaving.

Structural analysis Cross-sectional areas of regions of interest were measured in every 10th section with a digitizer interfaced to the software. Total regional volumes were calculated according to the Cavalieri principle using the formula V = Σ APt, where V is the total volume, Σ A is the sum of the areas measured, P is the inverse of the sampling fraction and t is the section thickness (Gundersen and Jensen, 1987). At 60 dg and 1 week, WM (including the corpus callosum and external capsule) and neocortical (superior to the rhinal fissure) volumes were measured from the most rostral extent of the corpus callosum to the caudal extremity of the dentate gyrus. In the cerebellum, the volume of the cerebellar WM and cortex (molecular layer (ML) and granule cell layer (GCL)) was measured. The WM to cortex ratios in both regions were determined. At 8 weeks, WM volume (including the corpus callosum and external capsule) was measured from the most rostral level of the corpus callosum to the caudal extent of the hippocampus. The width of the corpus callosum was measured at the midline in each section in which it was present and a mean taken for each animal. Figs. 1(A–F) illustrate these regions of the cerebral hemispheres and cerebellum. Immunohistochemical analysis For each brain, two sections at three levels of the cerebral hemisphere (frontal cortex, rostral extent of the hippocampus and mid-hippocampus) and two sections at the level of the cerebellar vermis were assessed. In the cerebellum, IR was assessed in the deep WM and in lobules I or X and VII or VIII. Projection of MAG- and PLP-IR fibres into the cerebral cortex at 60 dg and 1 week. The full cortical depth and the extent to which myelinated axons projected into the cortex were measured at three points of levelmatched sections; the percentage of cortex occupied by myelinated axons was then determined. MAG- and Olig2-IR OL cell densities at 60 dg and 1 week. MAG- and Olig2-IR OLs were counted in 3 fields/section in the WM (Olig2; field, 0.019 mm2) and cerebral cortex layer VI (MAG; field, 0.09 mm2), in the deep cerebellar WM (Olig2; field, 0.019 mm2), and in 6 fields/section in the GCL (MAG and Olig2; field, 0.019 mm2). MAG-IR OLs were not counted in the cerebral or cerebellar WM as the intense staining of MAG-IR fibres made it difficult to identify MAG-IR cell bodies accurately. Optical density (OD) of MAG- and PLP-IR at 60 dg and 1 week. The OD of MAG- and PLP-IR was assessed in 4 fields within both the WM and cortical layer VI, in 3 fields in the GCL and in 5 fields in the cerebellar WM as previously described (Mallard et al., 1999).

GFAP- and Iba-1-IR cell densities at 60 dg and 1 week. GFAP- (field, 0.05 mm 2) and Iba-1-IR (field, 0.04 mm 2) cell counts were performed in 6 regions of the WM (GFAP and Iba-1) and cerebral cortex layer VI (Iba-1 only). In the cerebellum, GFAP- (field, 0.04 mm 2) and Iba-1-IR (field, 0.06 mm 2) cell counts were performed in 6 regions of deep WM. Iba-1-IR cell counts were also performed in 3 regions of the GCL and ML (field, 0.04 mm 2). Olig2 and GFAP co-localisation. Sections of frontal cortex were examined using confocal microscopy (Zeiss Meta confocal laser scanning system). Fluorophores were visualised using a 488 nm excitation filter and 522/535 nm emission filter for Alexa 488, and 568 nm excitation and 605/632 nm emission filter for Rhodamine (TRITC). Optical images were acquired using a 40× oil immersion lens. For each control and GR animal, 4 slices from each of 3 areas of WM were stacked and projected using LSM Image Browser (Zeiss). The number of cells co-localised for Olig2 and GFAP was counted in stacked images (field, 0.05 mm 2) and expressed as a percentage of Olig2-IR cells. Qualitative analysis Sections from the cerebral hemispheres and cerebellum (section level and number as for quantitative analysis) of control and GR foetuses, neonates and young adults (MBP only) were assessed qualitatively for alterations in MBP- and SMI-312-IR. Western blotting An additional cohort of foetal (60 dg; control n = 2 and GR n = 1) and 1 week (control n = 4 and GR n = 4) animals was anaesthetised with an overdose of Lethobarb (i.p.) and the cerebral cortex and underlying WM (right frontal/parietal lobe) and cerebellum (right vermis/lateral hemisphere) were dissected. This tissue was used for both western blot analysis and quantitative real-time PCR (see Quantitative real-time PCR). Tissue was homogenised in lysis buffer (50 mM Tris–HCl (ph 7.4), 150 mM NaCl, 1 mM EDTA, proteinase inhibitor cocktail (Complete, Mini; Roche Applied Sciences, IN, USA)) and incubated with 1% (w/v) triton-X100 on ice for 30 min. Following the determination of protein concentration, lysates were electrophoretically separated and transferred, and membranes prepared for the analysis of MBP protein expression (1:250; rat anti-MBP; MAB395; Millipore, MA, USA) as previously described (Munro et al., 2009). The OD of MBP protein bands (20 kDa) was quantified using Analyze 7.0 software (Mayo Clinic, MN, USA).

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M. Tolcos et al. / Experimental Neurology 232 (2011) 53–65

Fig. 1. Representation of brain regions analysed. Coronal, thionin-stained hemisections of the cerebral hemisphere at the level of the (A) frontal cortex, (B) rostral extent of the hippocampus and (C) mid-hippocampus. White matter (black dashed line) and the neocortex (region extending between the black dotted line and the boundary of the WM) were examined for volumetric analyses. (D) High-power image of a thionin-stained section of the cerebral hemisphere defining cortical laminae I–VI and the WM. Analysis of immunohistochemical staining was performed in cortical layer VI and the WM. (E) Sagittal, thionin-stained section through the vermis of the cerebellum. Cerebellar WM (black dashed line) and cortex (region extending from the black dotted line to the boundary of the WM) were examined for volumetric analysis. Immunohistochemical measurements were obtained in the WM including the deep WM, GCL and ML of early developing lobules I/X and late developing lobules VII/VIII. (F) High-power image of a thionin-stained section illustrating layers of the cerebellum. I–VI, cortical layers 1–6; I, VII, VIII, X, cerebellar lobules, 1, 7, 8, 10; CA3, Cornu Ammonis region 3; cc, corpus callosum; cg, cingulum; CPu, caudate putamen; cx, cortex; dg, dentate gyrus; dWM, deep white matter, GCL, granule cell layer; HiF, hippocampal formation; LV, lateral ventricle; MB, midbrain; ML, molecular layer; PCL, Purkinje cell layer; rf, rhinal fissue; Th, thalamus; WM, white matter. Scale bars: D = 400 μm; F = 200 μm.

Quantitative real-time PCR Total RNA was isolated from the cortex and underlying WM of the cerebral hemispheres (right frontal/parietal lobe) and cerebellum (right

vermis/lateral hemisphere) of foetal (60 dg; control n = 2 and GR n = 2) and 1 week (control n = 5 and GR n = 4) animals (QIAGEN RNeasy Minikit, QIAGEN Inc., CA, USA). cDNA was synthesised with a reverse transcriptase reaction using a standard technique (Superscript™ First

M. Tolcos et al. / Experimental Neurology 232 (2011) 53–65

Strand Synthesis System for RT-PCR, Invitrogen Corp, CA, USA) with random hexamers, deoxy-NTPs, and total RNA extracted from control and GR animals. To assess genomic DNA contamination, controls without reverse transcriptase were included. Real-time PCR (Bustin, 2000) was performed using the Rotorgene 3000 (Corbett Life Science, NSW, Australia) using primers for SYBR green real-time PCR (MBP: Fwd primer, 5′-CCAAGATGAAAACCCTGT and Rev primer, 5′-CTCTGCCTCCATAGCCAAAT; GAPDH: Fwd primer, 5′-CACTGCCACCCAGAAGAC and Rev primer, 5′-CCACAACCGACACATTAGG). PCR reactions (20 mL) were carried out consisting of 10 mL SensiMix, 0.4 mL UNG, 0.4 mL SYBR green solution (Bioline, MA, USA), 300 nM Fwd primer, 300 nM Rev primer, 6.4 mL of RNase free water and 16 nM cDNA. Reactions for MBP and GAPDH were carried out in separate tubes in duplicate. A standard curve method was used to quantify gene expression of MBP relative to GAPDH.

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at 60 dg revealed a reduction in WM volume in GR foetuses compared to controls (p b 0.01); there was no difference in the neocortical volume (p N 0.05; Table 3). The ratio of WM to cortex was reduced in GR foetuses compared to controls (p b 0.05; Table 3). At 1 week postnatal, there was a decrease in both WM (p b 0.01) and neocortical volumes (p b 0.05) and a reduction in WM to neocortex ratio (p b 0.01; Table 3) in GR neonates compared to controls. Eight weeks after birth, the corpus callosum width was decreased in GR animals compared to controls (control: 434.89± 14.11 μm vs. GR: 388.49 ± 12.3 μm, p b 0.05, Figs. 2E vs. F); there was no change between groups in WM volume (control: 54.82 ± 3.28 mm3 vs. GR: 54.30± 4.22 mm 3, p N 0.05).

Body and brain weights

MBP-IR and mRNA Intensely stained MBP-IR fibres were present in the WM and projected up to layer II/III of the cerebral cortex in control animals at 60 dg (Fig. 2A). In GR foetuses (Fig. 2B) there was a profound reduction in MBP-IR fibres in the cortex, but IR cell bodies were present in layers III–VI and in the WM. This pattern of staining was evident in 5/7 GR foetuses. At 1 (Figs. 2C and D) and 8 weeks (Figs. 2E and F), the intensity of MBP-IR fibres in layers III–VI of the cerebral cortex (Figs. 2C and D) and the WM (Figs. 2C and D), including the corpus callosum (Figs. 2E and F), appeared similar in control and GR animals; MBP-IR cell bodies were not evident. At 60 dg, there was a reduction in the level of MBP mRNA (Fig. 2G) and protein (control: 1.28 ± 0.24 vs. GR: 0.73 ± 0; Fig. 2H) expression in GR foetuses compared to controls. There was no difference in mRNA (Fig. 2G) or protein (control: 0.99 ± 0.02 vs. GR: 1.1 ± 0.04; Fig. 2H) expression at 1 week.

At 60 dg, there was a decrease in body weight (pb 0.001), CRL (pb 0.001), whole brain (pb 0.001) and cerebellar (pb 0.001) weights in GR foetuses (n= 8) compared to controls (n= 8; Table 2). The ratio of brain to body weight was increased (pb 0.001) in GR foetuses compared to controls (Table 2), reflecting the relative sparing of the brain. In the 1 week cohort, there was a decrease in body weight at birth (pb 0.001), body weight at post-mortem (pb 0.001), CRL (pb 0.001), brain (pb 0.001) and cerebellar weights (pb 0.001) and an increase in the brain to body weight ratio (pb 0.05) in GR neonates (n = 7) compared to controls (n= 7; Table 2). In the 8 week cohort, there was a decrease in body weight at birth (pb 0.001), body weight at postmortem (pb 0.001), CRL (pb 0.05), brain weight (pb 0.001), cerebellar weight (pb 0.001) and brain to body weight ratio (pb 0.001) in GR animals (n= 12) in comparison to controls (n= 11; Table 2).

MAG-IR In controls at 60 dg, dense MAG-IR (Fig. 3A) was evident in WM. Intensely stained MAG-IR fibres and cells extended to layer II/III; some fibres were also evident in layer I (Fig. 3A). The percentage of cortex occupied by MAG-IR fibres was reduced (illustrated by comparing Figs. 3A (control) and B (GR); p b 0.05, Fig. 3I) in GR foetuses compared to controls; this did not persist at 1 week (illustrated by comparing Figs. 3C (control) and D (GR); p N 0.05, Fig. 3I). At 60 dg, the OD of MAG-IR within the WM and layer VI of the cortex was not different between control and GR foetuses (p N 0.05, Fig. 3J), although there was a reduction in the density of MAG-IR OLs in cortical layer VI (p b 0.05, Fig. 3K); this reduction did not persist at 1 week (p N 0.05 Fig. 3K).

Statistical analysis Body and brain weight data were expressed as the mean of means ± SD; all other data are expressed as the mean of means ± SEM. All analysis was performed using a Student's t-test using SigmaPlot 12 Statistical Software (Systat Software Inc., San Jose, CA, USA) unless a test for normality failed in which case a Mann–Whitney U test was performed. Data were considered significant when p b 0.05. Results

Cerebral hemispheres Structural analysis Qualitative assessment of thionin-stained sections of the cerebral hemispheres from GR animals did not show any gross morphological abnormalities, lesions or infarcts at any age. Quantitative examination

PLP-IR In control foetuses at 60 dg, dense PLP-IR fibres were present in WM with a fine network of fibres projecting up to cortical layer III (Fig. 3E). At 60 dg, the percentage of cortex occupied by PLP-IR fibres was reduced (illustrated by comparing Figs. 3E (control) and F (GR); p b 0.005, Fig. 3I) in GR foetuses compared to controls; this did not

Table 2 Body and brain weights of control and GR animals in the 60 dg, 1 week and 8 week cohorts. 60 dg

Body weight at birth (g) Body weight at PM (g) CRL (cm) Brain weight (g) Cerebellar weight (g) Brain:body

1 week

8 week

Control (n = 8)

GR (n = 8)

Control (n = 7)

GR (n = 7)

Control (n = 12)

GR (n = 11)

– 101.59 ± 11.79 13.63 ± 0.53 2.65 ± 0.19 0.29 ± 0.05 0.03 ± 0.003

– 51.95 ± 12.36** 10.48 ± 1.06** 2.16 ± 0.18** 0.20 ± 0.01** 0.04 ± 0.009**

109.57 ± 7.04 163.66 ± 8.75 15.83 ± 1.18 3.13 ± 0.18 0.34 ± 0.01 0.019 ± 0.001

69.43 ± 10.45** 116.25 ± 23.46** 12.93 ± 1.06** 2.70 ± 0.07** 0.29 ± 0.03** 0.024 ± 0.005*

106.46 ± 17.83 545.67 ± 51.91 24.42 ± 1.68 4.1 ± 0.28 0.58 ± 0.06 0.008 ± 0.0007

61.97 ± 8.66** 464.55 ± 47.52** 23.14 ± 1.63* 3.6 ± 0.27** 0.48 ± 0.06** 0.007 ± 0.0006**

Values are expressed as mean ± SD. *p b 0.05; **p b 0.001 compared to age-matched controls. PM = post-mortem.

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M. Tolcos et al. / Experimental Neurology 232 (2011) 53–65

Table 3 Volumetric analysis of the cerebral hemisphere and cerebellum in control and GR animals at 60 dg and 1 week of age. 60 dg

Cerebral hemisphere White matter (mm3) Neocortex (mm3) White matter:cortex Cerebellum White matter (mm3) Cortex (mm3) White matter:cortex

1 week

Control (n = 7)

GR (n = 8)

Control (n = 7)

GR (n = 7)

45.8 ± 2.1 258.4 ± 12.2 0.18 ± 0.01

36.3 ± 2.0** 246.1 ± 13.1 0.15 ± 0.01*

64.0 ± 2.1 354.6 ± 10.2 0.18 ± 0.01

49.4 ± 2.1** 308.1 ± 14.3* 0.16 ± 0.01**

26.6 ± 1.5 79.6 ± 4.3 0.33 ± 0.01

19.02 ± 1.1*** 58.3 ± 4.6** 0.33 ± 0.01

33.0 ± 2.8 93.2 ± 7.7 0.35 ± 0.01

25.9 ± 1.0* 72.4 ± 5.0* 0.36 ± 0.02

Values are expressed as a mean ± SEM.*p b 0.05; **p b 0.01, ***p b 0.001, compared to age-matched controls.

persist at 1 week (illustrated by comparing Figs. 3G (control) and H (GR); p N 0.05, Fig. 3I). At 60 dg, there was a reduction (pb 0.05, Fig. 3J) in the OD of PLP-IR in cortical layer VI, but not in the WM of GR foetuses compared to controls (Fig. 3J). At 1 week, there appeared to be no difference in the intensity of PLP-IR in the WM or layer VI between groups (Figs. 3G and H).

SMI-312-IR To determine whether mature axons extended throughout the entire cortical depth, the distribution of SMI-312-IR fibres was examined at 60 dg. No observable variations were detected between control and GR animals, with SMI-312-IR extending through the entire width of the cortex (data not shown).

Fig. 2. Analysis of MBP in the cerebral hemispheres of control and GR animals. Compared to controls (A) there was a dramatic reduction in MBP-IR fibres throughout cortical layers III–VI in 5/7 GR foetuses at 60 dg (B); MBP-IR cell bodies were present in the cortex and WM in GR foetuses (B) but not in controls (A). At 1 week, the intensity and projection of MBP-IR fibres were similar in control (C) and GR (D) animals and there were no MBP-IR cell bodies (C, D). At 8 weeks of age, the intensity of MBP-IR fibres in the corpus callosum appeared similar in control (E) and GR (F) animals. In GR foetuses at 60 dg, there was a reduction in the level of MBP gene (G; data represents an average of 2 control and 2 GR animals) and protein (H) expression in the cerebrum; no difference in MBP gene (G) or protein (H) expression at 1 week. I–VI, cortical layers 1–6; WM, white matter; CC, corpus callosum; LV, lateral ventricle. Scale bars: A–D = 400 μm, E, F = 230 μm.

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Fig. 3. Analysis of MAG- (A–D) and PLP-IR (E–F) in the cerebral hemispheres of control and GR animals. At 60 dg there was a reduction in the projection of MAG-IR (B, GR vs. A, control) and PLP (F, GR vs. E, control) fibres in the cortex. At I week there was no difference for MAG-IR (D, GR vs. C, control) or PLP (H, GR vs. G, control). (I) At 60 dg but not at 1 week, there was a significant decrease in the percentage of cortex occupied by MAG- and PLP-IR myelinated fibres. (J) At 60 dg there was a significant decrease in PLP OD in layer VI but not in WM; no difference for MAG-IR in either region. (K) At 60 dg but not at 1 week, there was a significant decrease in the density (cells/mm2) of MAG-IR OLs in layer VI. *p b 0.05, **p b 0.005. I–VI, cortical layers 1–6; WM, white matter. Scale bar = 400 μm.

Olig-2-IR At 60 dg, but not 1 week of age, there was an increase in the density of Olig-2-IR OLs in the WM in GR foetuses compared to controls (p b 0.05, Fig. 4A); 2.8 ± 0.9% of Olig2-IR cells double-labelled for GFAP-IR in controls (Figs. 4B–D) and 4.1 ± 2.2% in GR foetuses (data not shown). GFAP- and Iba-1-IR At 60 dg, there was an increase (p b 0.05, Fig. 5A) in the density of GFAP-IR cells in the WM of GR foetuses compared to controls; this is illustrated by comparing Fig. 5C (control) with Fig. 5D (GR). There was no difference in the density of GFAP-IR cells in the WM at 1 week of age (Fig. 5A). At 60 dg, the density of Iba-1-IR cells in the WM was also increased (p b 0.01, Fig. 5B); this is illustrated by comparing Fig. 5E (control) with Fig. 5F (GR). There was no difference (p N 0.05) in the

density of Iba-1-IR cells in layer VI of the cerebral cortex at 60 dg or in the WM or layer VI at 1 week of age (Fig. 5D).

Cerebellum Structural analysis Qualitative assessment of thionin-stained sections of the cerebellum from GR animals did not show any gross morphological abnormalities, lesions or infarcts. In GR foetuses compared to controls, there was a decrease in the volume of the WM (pb 0.001) and cortex (pb 0.01) (Table 3), which persisted at 1 week (pb 0.05; Table 3). The ratio of WM to cortex was not different between control and GR foetuses or neonates (pN 0.05; Table 3), reflecting a similar vulnerability of these regions at these ages.

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Fig. 4. Analysis of Olig2-IR in the cerebral hemispheres of control and GR animals. (A) At 60 dg there was a significant increase in the density (cells/mm2) of Olig2-IR OLs in the WM in GR compared to control foetuses; there was no significant difference at 1 week of age. Double-labelling immunofluorescence in control and GR foetuses revealed that only a small percentage of Olig2-IR cells (arrowheads in B) were co-localised with GFAP (arrowheads in C); merged image (arrowheads in D). Images are taken from control foetuses. Scale bar = 50 μm. *p b 0.05.

Fig. 5. Analysis of GFAP- and Iba-1-IR cells in the cerebral hemispheres of control and GR animals. (A) The density (cells/mm2) of GFAP-IR cells was significantly greater in the WM of GR foetuses compared to controls; there was no difference at 1 week. This is illustrated by comparing images of GFAP-IR from control (C) and GR (D) foetuses. (B) The density (cells/ mm2) of Iba-1-IR cells was significantly increased in the WM of GR foetuses compared to controls at 60 dg but not 1 week of age; there was no difference in layer VI of the cerebral cortex or in either region at 1 week. In the WM, this is illustrated by comparing Iba-1-IR cells (arrowheads) in control (E) and GR (F) foetuses. GM, grey matter; WM, white matter. Scale bars: B, C = 100 μm, E, F = 45 μm. *p b 0.05, **p b 0.01.

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Fig. 6. Analysis of MBP in the cerebellum of control and GR animals. Compared to (A) controls at 60 dg, MBP-IR fibres were reduced in the GCL and MBP-IR cell bodies were present in the GCL and WM in GR foetuses (B). At 1 week, the distribution and intensity of MBP-IR were not different between (C) control and (D) GR animals. MBP (E) protein and (F) gene expression (data represents an average of 2 control and 2 GR animals) in the cerebellum were also reduced in GR foetuses at 60 dg; no difference in (E) protein or (F) gene expression at 1 week. GCL, granule cell layer; ML, molecular layer; PCL, Purkinje cell layer; WM, white matter. Scale bar = 250 μm.

MBP-IR and mRNA In control animals at 60 dg (Fig. 6A), MBP-IR fibres extended from the WM across the GCL to densely innervate Purkinje cells. In GR foetuses (5/7), there was a profound reduction in MBP-IR fibres in the GCL but MBP-IR cell bodies were present in both the GCL and WM (Fig. 6B). At 1 week, the distribution and intensity of MBP-IR fibres appeared similar in control and GR animals (Figs. 6C and D); MBP-IR cell bodies were not evident. At 60 dg, there was a reduction in the level of MBP protein (control: 1.72 ± 0.22 vs. GR: 0.64 ± 0; Fig. 6E) and mRNA expression (Fig. 6F) in GR foetuses compared to controls. There was no difference in protein (control: 0.67 ± 0.03 vs. GR: 0.91 ± 0.06; Fig. 6E) or mRNA expression (Fig. 6F) at 1 week. MAG-IR Control foetuses (Fig. 7A) and neonates (Fig. 7C) displayed intense MAG-IR within the cerebellar WM. Immunoreactivity for MAG extended the entire width of the GCL to the Purkinje cell layer in all control foetuses (Fig. 7A) but not in 2/7 GR foetuses (Fig. 7B). At 1 week, MAG-IR fibres projected to the Purkinje cell layer in all control (Fig. 7C) and GR (Fig. 7D) neonates. At 60 dg, the OD of MAG-IR was increased (p b 0.005) in the WM and decreased (p b 0.005) in the GCL (Fig. 7I) in GR compared to control foetuses; there was no difference (p N 0.05) at 1 week (Fig. 7I). The cell density of MAG-IR OLs was reduced in the GCL in GR foetuses compared to controls at 60 dg (p b 0.01; Fig. 7J); this did not persist at 1 week (p N 0.05, Fig. 7J). PLP-IR Intense PLP-IR was present within the cerebellar WM of control foetuses (Fig. 7E) and neonates (Fig. 7G). PLP-IR extended the entire width of the GCL to the Purkinje cell layer in all control foetuses

(Fig. 7E) but not in 6/7 GR foetuses (Fig. 7F). At 1 week, PLP-IR fibres projected to the Purkinje cell layer in all control (Fig. 7G) and GR (Fig. 7H) neonates. At 60 dg, the OD of PLP-IR was reduced (p b 0.001; Fig. 7K) in the GCL but not in the WM (p N 0.05; Fig. 7K) of GR foetuses compared to controls. At 1 week, there were no significant differences in either area (Fig. 7K).

SMI-312-IR Intensely stained SMI-312-IR fibres were observed projecting through the GCL to the Purkinje cells in both control and GR foetuses indicating normal afferent innervation of Purkinje cells (not shown).

Olig-2-IR The density of Olig-2-IR OLs was increased in the deep WM (pb 0.001, Fig. 8A) and the GCL (pb 0.05; Fig. 8A) in GR foetuses compared to controls. This is illustrated by comparing Fig. 8B (control) and Fig. 8C (GR). There was no difference in the deep WM or GCL at 1 week of age (Fig. 8A).

GFAP- and Iba-1-IR At 60 dg and 1 week of age, there was no difference in the density of GFAP-IR cells in the WM of control and GR foetuses (p N 0.05, Fig. 9A). This is illustrated by comparing Fig. 9B (60 dg, control) to Fig. 9C (60 dg, GR). The density of Iba-1-IR cells was increased (p b 0.005; Fig. 9D) in the WM in GR foetuses (Fig. 9E) compared to controls (Fig. 9F); there was no difference at 1 week of age. There was no difference between GR and controls in the GCL and ML at either age (p N 0.05, Fig. 9D).

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Fig. 7. Analysis of MAG- (A–D) and PLP-IR (E–H) in the cerebellum of control and GR animals. In GR foetuses at 60 dg but not at 1 week, there were (I) a significant increase in the OD of MAG in the WM, (I) a significant decrease in the GCL and (J) a significant decrease in the density (cells/mm2) of MAG-IR OLs in the GCL. (K) In GR foetuses at 60 dg but not at 1 week, there was a significant decrease in the OD of PLP in the GCL; no difference in the OD of PLP in the WM at either age. *p b 0.01, **p b 0.005, ***p b 0.001. GCL, granule cell layer; ML, molecular layer; PCL, Purkinje cell layer; WM, white matter. Scale bar = 250 μm.

Discussion The key findings of this study are firstly that IUGR, induced via CPI in the guinea pig, leads to a delay in the maturation of the OL cell lineage and hence myelination but that expression of myelin/OL proteins and myelination normalises to control levels postnatally. Secondly, that overall WM volume which is reduced in near-term and neonatal GR animals is at control levels by young adulthood; we acknowledge that there is a small but long-term deficit in the growth of a specific WM tract, the corpus callosum. Myelination is delayed in IUGR but restored postnatally The present study has confirmed our earlier hypothesis of a delay in myelination in the foetal IUGR guinea pig brain (Nitsos and Rees, 1990). Here we have identified a transient reduction in gene and protein expression and immunoreactivity for MBP in the cerebral cortex and cerebellum. In addition, we have shown an abnormal retention of MBP-IR in the OL soma and a lack of staining of processes. The processes are not lost or damaged however as they are evident in MAG-IR OLs. It is more likely that this phenomenon reflects a deficit or delay in the production and/or trafficking of sufficient MBP from the OL soma to its processes or in myelin sheath wrapping; formation of the myelin sheath involves the translocation of MBP mRNA from the OL cell body to its distal processes followed by wrapping and compaction of myelin (Pedraza et al., 1997). It is of interest that retention of MBP in the OL soma has also been observed in a mouse model of chronic perinatal hypoxia (Back et al., 2006) and in cases of human PVL (Billiards et al., 2008). Despite the dramatic effect on MBP-IR in the foetal brain, we found that MBP-IR was qualitatively restored to control levels in 1- and 8-week old GR

animals. Similarly, a transient reduction in MBP-IR has also been shown in a rodent model of chronic preterm hypoxia, where cerebral myelination was delayed during the first 2 postnatal weeks and returned to control levels by P21 (Baud et al., 2004). Our results however, differ from those of Olivier et al. (2005) who found a qualitative reduction in MBP-IR fibres in the corpus callosum, cingulum and overlying cortex throughout development and into adulthood following CPI in rats (Olivier et al., 2005). This discrepancy most likely reflects differences in the timing of the insult as well as the species used. Similarly to MBP, immunoreactivity for MAG and PLP was only transiently affected in our model of IUGR. At 60 dg, the projections of MAG- and PLP-IR fibres through the cerebral cortex and GCL in the cerebellum were reduced in GR foetuses. These results were paralleled by reductions in the optical density for immunoreactive product in these regions. By 1 week of age, myelination was restored to control levels with myelinated fibres projecting to layer II of the cerebral cortex and the entire width of the GCL in GR neonates. The reduced projection at 60 dg could not be attributed to a lack of axonal innervation, as SMI-312-IR fibres extended through the full width of the neocortex and GCL. Thus afferent and efferent innervation of the cortex and cerebellum appeared to be developing appropriately although axons might have been thinner and fewer in number as we have reported previously in CPI where we examined axons at the ultrastructural level (Nitsos and Rees, 1990). Thinner myelin sheaths could underlie the reduction in immunoreactivity for myelin proteins. Our data is in accord with studies that have shown a down-regulation in MAG and PLP genes (Curristin et al., 2002), and a reduction in MAGIR in the corpus callosum and other fibre pathways (Curristin et al., 2002; Weiss et al., 2004) in newborn mice subjected to chronic sublethal hypoxia.

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Abernethy, 1999; Nosarti et al., 2004) and a slowing in its growth rate after 2 weeks of age (Anderson et al., 2006). Thinning of the corpus callosum could be attributed to a reduction in axon number, axon diameter and/or myelin thickness; further ultrastructural analysis of the WM is required for clarification. Alterations to the corpus callosum can affect cerebral function. For example studies in preterm infants have shown an association between serious motor delay and cerebral palsy and significantly poor growth of the corpus callosum (Anderson et al., 2006). This study has also highlighted the need to analyse more than one marker of mature myelinating OLs. Here we show that although CPI and IUGR dramatically affects MBP-IR, with what may be interpreted as a complete loss of myelination, MAG- and PLP-IR, although reduced in GR foetuses, are still present. This supports the notion that myelination is still occurring albeit at a reduced level. We suggest that researchers should be circumspect when drawing conclusions about the state of myelination based on MBP-IR alone. Maturation of the OL lineage is delayed rather than permanently affected by IUGR

Fig. 8. Analysis of Olig2-IR in the cerebellum of control and GR animals. (A) At 60 dg there was a significant increase in the density (cells/mm2) of Olig2-IR OLs in the WM and GCL in GR compared to control foetuses; there was no difference at 1 week in either region. This is illustrated by comparing images of Olig2-IR in control (B) and GR (C). *p b 0.05, **p b 0.001. GCL, granule cell layer; ML, molecular layer; PCL, Purkinje cell layer; WM, white matter. Scale bar = 250 μm.

Although immunoreactivity for myelin proteins was restored postnatally, we found that the reduction in cerebral and cerebellar WM volume in GR foetuses persisted at 1 week of age. Cerebral WM volume returned to control levels by 8 weeks, although a significant reduction in the width of the corpus callosum remained. This result is consistent with studies in models of chronic sublethal hypoxia (Ment et al., 1998) and CPI (Olivier et al., 2005) that have also shown a reduction in the area (Ment et al., 1998) and thickness (Olivier et al., 2005) of the corpus callosum in adult rats. Analysis in preterm infants has also revealed a thinning of the corpus callosum (Cooke and

A delay in the process of myelination in the IUGR brain is likely the result of alterations to the OL lineage. Indeed, in the present study, we found a significant but transient decrease in the density of early myelinating MAG-IR OLs in the cerebral hemispheres and cerebellum despite a significant increase in the density of Olig2-IR OLs. These results are consistent with recent studies that have found an alteration in the maturation of the OL lineage in human PVL cases (Billiards et al., 2008) and a rodent model of perinatal hypoxia–ischemia (Segovia et al., 2008). Similarly, they identified an increase in the density of Olig-2-IR OLs (Billiards et al., 2008) and preOLs (Segovia et al., 2008), and a reduction in early myelinating OLs (Segovia et al., 2008). In our model of IUGR where we were able to assess both foetal and postnatal ages we conclude that maturation of the OL lineage is delayed rather than permanently affected. The increased density of Olig2-IR OLs in foetal but not postnatal life, could be attributed to an increase in OL proliferation in response to CPI (Olivier et al., 2007). In a companion study, we have shown that very few cells are immuoreactive for the cell proliferation marker, Ki67, in the cerebral WM of control and GR

Fig. 9. Analysis of GFAP- and Iba-1-IR in the cerebellum of control and GR animals. (A) The density (cells/mm2) of GFAP-IR cells in the WM was similar in control and GR foetuses and neonates. This is illustrated by comparing images of GFAP-IR cells (arrowheads) in the WM of control (B) and GR (C) foetuses. (D) The density (cells/mm2) of Iba-1-IR cells was significantly increased in the WM of GR foetuses compared to controls; there was no difference in the GCL or ML at 60 dg or any region at 1 week. This is illustrated by comparing Iba-1-IR cells (arrowheads) in the WM of control (E) and GR (F) foetuses. *p b 0.005. GCL, granule cell layer; ML, molecular layer; WM, white matter. Scale bar = 100 μm.

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foetuses at 60 dg and that Olig2-IR cells are not co-localised with Ki67-IR in cells in the subventricular zone (M. Tolcos; unpublished observations). Earlier time-points will need to be examined as we may be missing the peak period of OL proliferation following CPI. WM injury occurs in association with deficits in myelination in IUGR In parallel with alterations to myelination we found a marked increase in the density of GFAP-IR astrocytes and Iba-1-IR microglia in the cerebral WM, and of Iba-1-IR microglia in the cerebellar WM in the GR foetal brain; these markers were not altered in the cerebral or cerebellar grey matter and were all at control levels in the WM in neonates. Previously in this model we have shown that GFAP-IR is transiently elevated in the foetal medulla (Tolcos and Rees, 1997) and adjacent to cortical blood vessels (Nitsos and Rees, 1990) but normalised postnatally (Tolcos et al., 2002). These results are in accord with studies that have shown astrogliosis and microgliosis accompanying a reduction in myelination in the cingulum and internal capsule of GR rat pups following CPI (Olivier et al., 2005, 2007). Currently we are unable to determine whether reactive astrogliosis and microgliosis within the WM of the GR foetal guinea pig brain is a consequence of the WM alterations reported here, and/or axonal loss (Nitsos and Rees, 1990), or whether their presence contributes to delayed OL maturation and impaired myelination via the release of factors such as nitric oxide (Kaur et al., 2006), glutamate (Matute et al., 2007) and hyaluronan (Back et al., 2005). Possible mechanisms leading to delayed WM development in IUGR The mechanisms underlying the effects of IUGR on WM development are likely to be multifactorial. Altered levels of oxygen, nutrients including glucose, hormones and growth factors are all known to affect the development of the oligodendroglial lineage and axonal growth, and could contribute to the deficits described here. We know from previous studies in this model that foetuses are chronically hypoxic, malnourished (Jones and Parer, 1983), hypoglycemic (Jones et al., 1984) and have an altered endocrine balance including reduced plasma levels of thyroid hormone (Jones et al., 1984) and insulin-like growth factor-I (IGF-1) (Jones et al., 1987). The brain is not ischemic as cerebral blood flow is actually elevated (Jensen et al., 1996) but as the foetuses are hypoxic it is likely that cerebral oxygen delivery is reduced. As discussed above, CPI, and foetal hypoxia, may have marked effects on the maturation of OLs, either directly, or indirectly via the up-regulation of astrocytes and microglia, thus leading to a delay in myelination. In vitro studies have shown that hypoglycemia inhibits OL development and myelination and induces apoptosis in cultured OL precursor cells (Yan and Rivkees, 2006). IGF-I (McMorris and Dubois-Dalcq, 1988), brain-derived growth factor (BDNF) (Djalali et al., 2005) and thyroid hormone (Ahlgren et al., 1997; Ibarrola and Rodriguez-Pena, 1997) have all been implicated in the regulation of OL proliferation, maturation and thus myelination. We know that BDNF expression is reduced in the forebrain of GR foetuses compared to controls (Dieni and Rees, 2005). Interestingly, a BDNF knockout study has revealed that BDNF is required for the expression of MBP (Djalali et al., 2005). However, a delay in the initiation of myelination may also be attributed to retardation in the radial growth of axons. We have previously shown that a critical diameter of 0.5 μm was necessary for the axonal acquisition of myelin, and that unmyelinated axons were 22% thinner in the corticospinal tract of GR foetuses (Nitsos and Rees, 1990). Although myelination reaches control levels postnatally, maturational delays might affect normal functional development of the nervous system. Myelin thickness influences conduction velocity (Waxman, 1980) and thus latency of action potentials in cerebral and cerebellar tracts; this could affect synchrony of neuronal firing. Synchronous firing of neurons is thought to be critical for the

refinement of connections during development (Meister et al., 1991). Recent studies of developing human (Hagmann et al., 2010) and mouse (Salami et al., 2003) cerebral hemispheres support the concept that appropriate development of myelinated axons plays an important role in establishing neuronal synchrony and thus connectivity. A delay in myelination at critical periods in development could conceivably affect connectivity and underlie long term behavioural, cognitive and functional activity.

Conclusion Using a model of IUGR induced via CPI in the guinea pig, we have shown that a delay in OL maturation and myelination observed in the foetal brain does not persist into postnatal life. Therefore although alterations to the process of myelination might play a role in maturational changes to brain development it is likely that other critical aspects of neural development such as axonal growth and connectivity, which we know are affected in IUGR (Dieni and Rees, 2003; Nitsos and Rees, 1990), are major contributing factors to adverse neurological, behavioural and cognitive outcomes.

Acknowledgments The authors wish to thank Mr Todd Briscoe for his surgical assistance, Ms Veronica Martin for her help with Olig2 analysis, Ms Amy Shields for her confocal microscopy expertise and Ms Aminath Azhan for her assistance with the immunohistochemistry. This study was supported by the National Health and Medical Research Council of Australia (project grant # 208937 and partially by 454536).

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