Introduction
A large proportion of intrauterine growth-restricted (IUGR) infants exhibit adverse long-term neurological outcomes such as sensory, learning and attention difficulties, behavioural issues, school failure, psychiatric disorders, epilepsy and cerebral palsy [
1‐
4]. Clinical imaging studies in IUGR infants demonstrate structural alterations and changes in grey and white matter volume [
5‐
7]. Structural changes that persist at 1 year of age are associated with developmental disabilities [
6,
8] that persist well into adulthood [
9]. However, few studies have focused on mechanisms of brain damage in the IUGR neonate. This being pertinent as no therapeutic interventions are currently available to prevent or treat brain damage in the IUGR newborn. Determining the underlying mechanisms behind grey and white matter damage in the IUGR infant will help guide the development and choice of suitable therapies to protect and promote healthy brain development in the vulnerable IUGR brain.
Neuronal and white matter impairment have been demonstrated in several animal models of IUGR [
10‐
13]. However, mechanisms of neurodevelopmental impairment in the IUGR neonate are complex and not well understood. A number of mechanisms proposed to be involved in mediating cellular damage in the IUGR brain include excitotoxicity, oxidative stress, necrotic and apoptotic neurodegeneration and neuroinflammation [
14,
15]. Neuroinflammation is regarded as a key mechanism in several neurodegenerative disorders and preliminary studies suggest it may also mediate abnormal brain development in the IUGR neonate [
16]. Activation of glial cells exacerbates neuroinflammation and brain injury. In the IUGR brain, evidence suggests that the major inflammatory mediators are activated microglia, reactive astrocytes and proinflammatory cytokines [
16]. In the preterm rodent model of hypoxic-ischemic (HI), the sustained presence of activated microglia and proinflammatory cytokines in the brain following an acute HI event has been associated with ongoing white matter and neuronal damage [
17], though few studies have examined the potential negative impact of neuroinflammation in IUGR animal models.
Of the limited studies investigating inflammation in the IUGR brain, varying results have been reported [
16]. Postnatal examination in different IUGR models has shown increased numbers of microglia and astrogliosis [
18‐
23] while others report little change [
20,
23,
24]. Only one study to date has examined the cytokine response in the IUGR brain [
25]. In the guinea pig hypoxemic IUGR model, inflammatory cytokines are up-regulated in the fetal brain at 64–65 days gestation (full gestation = ~ 65 days) and related to severity of brain injury as demonstrated by increased apoptosis and neuronal loss at this time point [
25]. These studies were largely carried out in small animal models. Given the conflicting results, it is essential to examine whether this potential inflammatory state is associated with ongoing neuronal and white matter impairment in a more translatable model. This information will be critical for the design and implementation of successful therapeutic interventions to protect the IUGR newborn brain. Evidence from fetal sheep studies has led to clinical trials of in utero interventions to reduce IUGR [
26,
27]. However, there are pros and cons with all animal models; i.e. varying degrees of brain maturation at birth. The piglet is regarded an appropriate animal to examine altered brain development arising from comprising perinatal events [
28‐
30]. The newborn piglet is similar to the human newborn infant in size, development, circulation, metabolism and cerebral maturation. The piglet brain is gyrencephalic and has a similar grey to white matter ratio [
31] as well as brain growth spurt in the perinatal period similar to the human [
32]. Furthermore, growth restriction in the piglet occurs spontaneously obviating the need for surgical induction of growth retardation. The piglet model of IUGR mimics many of the human pathophysiological outcomes associated with IUGR including asymmetrical growth restriction with brain sparing [
33]. Asymmetrical growth restriction is the most common form of growth restriction in humans and occurs in around 70–80% of all IUGR newborns [
34]. Disruption to fetal growth occurs mainly in the third trimester for asymmetric growth restriction with fetal circulatory redistribution occurring; that is blood flow is selectively redirected to the brain away from other peripheral organs to the brain resulting in ‘brain sparing’. Inadequate fetal growth in pigs is caused by alterations associated with placental insufficiency which is the most common cause of IUGR in the human population [
33]. Therefore, data obtained from the piglet model translates well to human IUGR pathology.
In the current study, we used the spontaneously growth-restricted piglet as a model of human IUGR to examine neuropathology and neuroinflammatory mediators at birth (P1) and at postnatal day 4 (P4). We hypothesised that inflammation would be prevalent in the IUGR piglet brain not only on P1 but also on P4 and would be associated with ongoing neuronal and white matter disruption.
Materials and methods
Animals and tissue preparation
Large White piglets were obtained from The University of Queensland Gatton Piggery. Approval for this study was granted by The University of Queensland Animal Ethics Committee (MED/UQCCR/132/16/RBWH) and was carried out in accordance with National Health and Medical Research Council (NHMRC) guidelines (Australia) and ARRIVE guidelines.
Term piglets were born spontaneously and collected on first day of life (postnatal day 1 (P1); < 18 h). IUGR piglets were defined by birth bodyweight (< 10th percentile) [
35‐
37]. Litter-matched pairs were obtained from multiple sows (
n = 11). The IUGR piglet model is well-established and characterised by our group and others [
11,
33]. This model is caused by placental insufficiency [
33]; the most common cause of IUGR in the human population. On either P1 (NG
n = 6; IUGR
n = 6) or P4 (NG
n = 6; IUGR
n = 6) (equal males and females in each group), piglets were euthanased via an intracardiac injection of sodium pentobarbital (650 mg/kg; Lethabarb, Virbac, Australia). The brain was immediately removed, weighed, hemisected and coronally sliced. The right hemisphere sections were immersion fixed in 4% paraformaldehyde as previously described [
38]. The parietal cortex from the left hemisphere was snap frozen in liquid nitrogen and stored at − 80 °C for molecular studies.
Immunohistochemistry
Brain sections from the right hemisphere were embedded in paraffin and coronally sectioned 6 μm apart at the level of the brain containing the parietal cortex (Pig stereotaxic map, A 5.5 mm; Felix 1999). Sections were dewaxed and rehydrated using standard protocol. The rehydrated slides underwent heat-induced epitope retrieval in 10 mM citrate buffer (pH 6) at 80 C for 20 min before cooling to room temperature (RT).
Tissue sections were blocked with 5% donkey serum in phosphate-buffered saline (PBS) with 0.5% Triton-X 100 for 1 h at RT. Immunohistochemistry was performed on brain sections for visualisation of microglia (ionised calcium-binding adaptor molecule-1; Iba-1; 1:1000, ab5076; Abcam, Queensland, Australia), astrocytes (glial fibrillary astrocytic protein; GFAP; 1:1000, Z0334, Dako), neurons (NeuN; 1:1000, ab177487; Abcam), proliferating cells (Ki67; 1:200, ab15580; Abcam) and apoptotic cells (cleaved Caspase-3; 1:500, #9661; Cell Signalling). Co-localisation studies were also conducted for the inflammatory cytokines IL-1β (1:100, ab9722; Abcam), TNF-α (1:200, AF-410; R&D Systems, Minneapolis, MN), IL-18 (1:100, ab71495; Abcam), IL-4 (1:100, ab9622; Abcam) and nuclear factor-kappa beta (NF-kB p65: 1:500, ab16502; Abcam). Primary antibodies were incubated at 4 °C for 20 h. Slides were washed in tris-buffered saline followed by incubation with species-specific secondary fluorophores at RT for 1 h (Alexafluor 488, Alexafluor 594; 1:1000, Molecular Probes, Invitrogen Australia, Victoria, Australia). Tissue was then washed, counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted with Prolong Gold antifade (Molecular Probes, Invitrogen Australia, Victoria, Australia). Negative control sections without primary antibodies were processed in parallel. All staining was conducted in triplicate for each animal at each time point.
Luxol fast blue and Fluoro Jade C staining
We assessed the general myelination status of IUGR brains at P1 and P4 using Luxol fast blue (LFB) staining. Tissue sections underwent standard dewaxing and rehydration followed by overnight immersion in LFB solution at 57 °C. Sections were then immersed in 95% ethanol and differentiated in 0.05% lithium carbonate followed by 70% ethanol until grey and white matter could be distinguished and nuclei decolourized. Tissue was processed and stained simultaneously to minimise variability of LFB staining.
To investigate the number of degenerating neurons, Fluoro Jade C (FJC) was used. This is a dye that specifically stains all degenerating neurons [
39]. Dewaxed and rehydrated slides were incubated in 0.006% potassium permanganate solution for 5 min. Slides were rinsed in PBS for 2 min then transferred to a 0.0001% solution of FJC (Merck Millipore, Germany) dissolved in 0.1% acetic acid vehicle, containing DAPI to counterstain the nuclei for 15 min. Slides were washed 2 × 1 min in PBS followed by air drying on a slide warmer at 50 °C for 5 min. Slides were cleared with xylene for 1 min before being coverslipped with DPX mounting media (Sigma-Aldrich).
Image analysis
Analysis of immunolabelled sections were performed using an Olympus BX41 light microscope with a DP70 camera as previously described [
40]. Photomicrographs (881.2 μm × 663.5 μm) of grey matter (parietal cortex) and white matter (intragyral white matter-IGWM; subcortical white matter-SCWM; periventricular white matter-PVWM) regions were captured for analysis. Four pictomicrographs were captured in each respective area in three sequential sections (50 μm apart) for each animal.
All tissue was imaged and analysed under blind conditions by KKC and JAW and manual counts for NeuN, FJC, Caspase-3, Ki67 and Iba-1 were performed. For LFB staining, slides were scanned using a Leica SCN400 Slide Scanner with a × 20 objective and analysed as previously reported [
41]. Briefly, the WM was automatically outlined using the wand tool in ImageJ software; FIJI (NIH Bethesda, USA). Incidental border regions, such as large blood vessels, were excluded from the analysis. Scanned images were converted to greyscale to determine the staining intensity from 0 to 127 (0, white; 255, black). This range was divided into quartiles and the percent area (% area) calculated for each quartile. The median grey level of each quartile (14.5, 46.0, 78.5 and 111.0) was then multiplied by % area/100 in each quartile, to give the total myelin index. Therefore, the ratio is defined as myelination index (%).
GFAP-positive astrocytes in the WMTs were quantified using densitometry by thresholding the intensity of GFAP labelling using ImageJ (Image Processing and Analysis in Java; National Institutes of Health, Bethesda, MD, USA). Areal density was expressed as a percentage of the whole white matter for each region covered.
Quantitative polymerase chain reaction
The Pig Inflammatory Cytokines & Receptors RT2 Profiler™ PCR Array (Qiagen, Hilden, Germany) profiles the expression of 84 key genes mediating the inflammatory response. Total RNA was purified using the RNeasy Tissue Mini Kit (Qiagen) from 30 mg of the parietal cortex. Total RNA concentration and quality was determined via UV spectroscopy using a NanoDrop (ND-1000 system). cDNA was synthesised from the purified total RNA via reverse transcription using the RT2 First Strand Kit (Qiagen). Synthesised cDNA were pooled for each group (total four groups: P1 NG n = 6, IUGR n = 6; P4 NG n = 6, IUGR n = 6), giving equal cDNA concentrations from each animal in the pooled sample. Pooled cDNA was added to the RT2SYBR Green Mastermix and pipetted into the Pig Inflammatory Cytokines & Receptors RT2 Profiler™ PCR Array. Polymerase chain reaction (PCR) was performed using a Rotor-Gene Q real-time cycler (Qiagen). The amplified transcripts were quantified with the comparative CT method using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression levels for normalisation. The same CT threshold value was used across all arrays to allow accurate comparison between runs.
Statistical analysis
Two-way ANOVA with the post-hoc Sidak analysis was used to determine differences between NG and IUGR animals at each age (Graph Pad Prism 5.0 software, San Diego, California, USA). Results were expressed as mean ± SEM with statistical significance accepted at p < 0.05.
Acknowledgements
We would like to thank Kishen Sukumar for some laboratory assistance. The authors acknowledge the Research Histology Facility from IHBI (QUT).