Neurovascular Unit Alterations in the Growth-Restricted Newborn Are Improved Following Ibuprofen Treatment.

The developing brain is particularly vulnerable to foetal growth restriction (FGR) and abnormal neurodevelopment is common in the FGR infant ranging from behavioural and learning disorders to cerebral palsy. No treatment exists to protect the FGR newborn brain. Recent evidence suggests inflammation may play a key role in the mechanism responsible for the progression of brain impairment in the FGR newborn, including disruption to the neurovascular unit (NVU). We explored whether ibuprofen, an anti-inflammatory drug, could reduce NVU disruption and brain impairment in the FGR newborn. Using a preclinical FGR piglet model, ibuprofen was orally administered for 3 days from birth. FGR brains demonstrated a proinflammatory state, with changes to glial morphology (astrocytes and microglia), and blood-brain barrier disruption, assessed by IgG and albumin leakage into the brain parenchyma and a decrease in blood vessel density. Loss of interaction between astrocytic end-feet and blood vessels was evident where plasma protein leakage was present, suggestive of structural deficits to the NVU. T-cell infiltration was also evident in the parenchyma of FGR piglet brains. Ibuprofen treatment reduced the pro-inflammatory response in FGR piglets, reducing the number of activated microglia and enhancing astrocyte interaction with blood vessels. Ibuprofen also attenuated plasma protein leakage, regained astrocytic end-feet interaction around vessels, and decreased T-cell infiltration into the FGR brain. These findings suggest postnatal administration of ibuprofen modulates the inflammatory state, allowing for stronger interaction between vasculature and astrocytic end-feet to restore NVU integrity. Modulation of the NVU improves the FGR brain microenvironment and may be key to neuroprotection.

Seizures Are Associated with Blood-Brain Barrier Disruption in a Piglet Model of Neonatal Hypoxic-Ischaemic Encephalopathy.

Seizures in the neonatal period are most often symptomatic of central nervous system (CNS) dysfunction and the most common cause is hypoxic-ischaemic encephalopathy (HIE). Seizures are associated with poor long-term outcomes and increased neuropathology. Blood-brain barrier (BBB) disruption and inflammation may contribute to seizures and increased neuropathology but are incompletely understood in neonatal HIE. The aim of this study was to investigate the impact of seizures on BBB integrity in a preclinical model of neonatal hypoxic-ischaemic (HI) injury. Piglets (age: <24 h) were subjected to a 30-min HI insult followed by recovery to 72 h post-insult. Amplitude-integrated electroencephalography (aEEG) was performed and seizure burden and background aEEG pattern were analysed. BBB disruption was evaluated in the parietal cortex and hippocampus by means of immunohistochemistry and Western blot. mRNA and protein expression of tight-junction proteins (zonula-occludens 1 [ZO1], occludin [OCLN], and claudin-5 [CLDN5]) was assessed using quantitative polymerase chain reaction (qPCR) and Western blot. In addition, mRNA from genes associated with BBB disruption vascular endothelial growth factor (VEGF) and matrix metalloproteinase 2 (MMP2) as well as inflammatory cytokines and chemokines was assessed with qPCR. Piglets that developed seizures following HI (HI-Sz) had significantly greater injury, as demonstrated by poorer aEEG background pattern scores, lower neurobehavioural scores, and greater histopathology. HI-Sz animals had severe IgG extravasation into brain tissue and uptake into neurons as well as significantly greater levels of IgG in both brain regions as assessed by Western blot. IgG protein in both brain regions was significantly associated with seizure burden, aEEG pattern scores, and neurobehavioural scores. There was no difference in mRNA expression of the tight junctions, however a significant loss of ZO1 and OCLN protein was observed in the parietal cortex. The inflammatory genes TGFß, IL1ß, IL8, IL6, and TNFα were significantly upregulated in HI-Sz animals. MMP2 was significantly increased in animals with seizures compared with animals without seizures. Increasing our understanding of neuropathology associated with seizure is vital because of the association between seizure and poor outcomes. Investigating the BBB is a major untapped area of research and a potential avenue for novel treatments.

Developmental Changes in Expression of GABAA Receptor Subunits α1, α2, and α3 in the Pig Brain.

GABA is a major neurotransmitter in the mammalian brain. In the mature brain GABA exerts inhibitory actions via the GABAA receptor (GABAAR); however, in the immature brain GABA provides much of the excitatory drive. We examined the expression of 3 predominant GABAA α-subunit proteins in the pig brain at various pre- and postnatal ages. Brain tissue was collected from piglets born via caesarean section at preterm ages 91, 97, 100, and 104 days' gestational age (GA), at term equivalent (114 days' GA, caesarean section) and at term, postnatal day 0 (P0) (spontaneous delivery, term = 115 days). Tissue was obtained from piglets at P4 and P7. Adult tissue from sows was collected postmortem after caesarean section. In all cortical regions and basal ganglia (1) α3 exhibited a significant increase in protein expression at 100 days' GA, (2) α3 expression decreased with age after 100 days' GA, (3) α1 increased with age, with peak expression at P7 in cortices, hippocampus, and thalamus, and (4) α2 protein expression remained relatively constant across the ages examined. The subunit expression of α3 was most abundant at preterm ages, with α1 the predominant subunit expressed postnatally. Immunofluorescent labelling revealed α1 expression on the somatic membranes of pyramidal cells in the cortex and hippocampus, and in the cerebellar Purkinje cells. Positive α3 labelling was apparent on interneurones in the cortex and hippocampus. The switch between dominant α-subunits may coincide with the functional change in GABAergic neurotransmission from excitation to inhibition. Brain growth in the pig closely reflects that in the term human, making the pig a valuable non-primate model for studying development and the effects of insults on the perinatal brain.

Standard loading controls are not reliable for Western blot quantification across brain development or in pathological conditions.

A frequently utilized method of data quantification in Western blot analysis is comparison of the protein of interest with a house keeping gene or control protein. Commonly used proteins include ß-actin, glyceraldehyde 3 phosphate dehydrogenase (GAPDH), and α-tubulin. Various reliability issues have been raised when using this technique for data analysis-particularly when investigating protein expression changes during development and in disease states. In this study, we have demonstrated that ß-actin, GAPDH, and α-tubulin are not appropriate controls in the study of development and hypoxic-ischemic induced damage in the piglet brain. We have also shown that using an in-house pooled standard, loaded on all blots is a reliable method for controlling interassay variability and data normalization in protein expression analysis.