|Abstract:||Intrauterine growth restriction (IUGR), a form of fetal malnutrition that leads to small for gestational age (SGA) infants, is a condition that affects 7.5 million children born worldwide. Infants affected by IUGR are more prone to lengthy hospital stays, repeated infections, and neurodevelopmental delays. These children also have an increased risk for chronic inflammation and psychiatric diseases later in life. Inflammation during early development is associated with psychiatric disease risk by disrupting normal neurodevelopmental processes. Due to the potentially devastating effects of IUGR, it is important to both characterize the physiological effects of IUGR and work to develop nutritional interventions that will combat the effects of fetal malnutrition and inflammation.
Domestic piglets (Sus scrofa) were used as models to conduct the following studies, as they offer advantages in their anatomical and physiological similarity to human infants. The first study sought to determine if a pre-digested fat (PDF) system developed by Abbott Nutrition would lead to increased white matter development and improved learning ability in the SGA piglet. Sex-matched, littermate pairs of SGA and appropriate for gestational age (AGA) piglets were randomly assigned to standard formula (CON) or PDF formula diets on postnatal day (PD) 2. On PD14, half were used for hippocampal RNA-sequencing; the other half began a spatial learning task. At approximately 4 weeks old, brain structure was assessed by magnetic resonance imaging (MRI) and brain tissues were submitted for fatty acid analysis. PDF diet did not increase growth rates in SGA piglets. Hippocampal gene expression changes and whole brain white matter microstructural deficits due to SGA status were not corrected by PDF. Hippocampal and cerebellar DHA levels were not affected by PDF. PDF diet did, however, increase relative white matter and several white matter associated structure volumes in both SGA and AGA piglets, as well as transiently increased performance of AGA piglets in the spatial learning task.
Since SGA infants are prone to infections and neurodevelopmental sequela, we next wanted to determine if SGA piglets would have an increased neuroinflammatory response to peripheral immune stimulation. Sex-matched, littermate pairs of SGA and AGA piglets were weaned on PD2, fed a commercial liquid milk replacer diet, and on PD14 given an intraperitoneal (i.p.) injection of 5 μg/kg lipopolysaccharide (LPS) or sterile saline. Piglets were monitored, and blood and tissue collection for gene expression and fatty acid analysis occurred 4 h post injection. Both SGA and AGA piglets had decreased feeding scores 2 h after LPS injection. SGA piglets had a lower lymphocyte count than AGA piglets along with decreased thymopoetic gene expression. SGA status was associated with downregulated expression of cytokine interleukin 6 (IL-6) regardless of LPS treatment. LPS decreased brain derived neurotrophic factor (BDNF), myelin basic protein (MBP), and glutamic acid decarboxylase (GAD1) expression regardless of birth weight. SGA piglets had an increased hippocampal total omega-6 to total omega-3 fatty acid ratio. These data show that SGA piglets had reduced lymphocyte populations, suggesting susceptibility to infections. We did not see cytokine expression differences between SGA and AGA piglets in the hippocampus with the exception of IL-6, however, the susceptibility of SGA infants to infections could lead to repeated insults, putting them at risk for reduced white matter development and behavioral disorders.
The final study sought to determine if supplementation with herring roe oil, a source of omega-3 fatty acids, would reduce neuroinflammation after peripheral immune stimulation in neonates. Full term AGA piglets were obtained from the University of Illinois Swine Farms on PD 2 and randomly assigned to herring roe oil (HRO) supplemented or control (CON) diet. HRO provided 40 mg/kg body weight/day of DHA and 12 mg/kg body weight/day of EPA. In study 1, piglets were sacrificed at PD14 and complete blood cell counts with differential, fatty acid profile, and fatty acid metabolite profiles of brain tissue were generated, and microglial cells were isolated and stimulated with 10 ng/mL LPS. In study 2, piglets were given a 10 µg/kg i.p. injection of LPS, and sacrificed 4 h later to assess markers of peripheral inflammation, neuroinflammation, and microglial cell activation. HRO did not attenuate the cytokine response of microglia ex vivo or in vivo, nor did it affect fever or circulating resolvin D1 levels. HRO may have created a more neuroprotective microglial phenotype in vivo, as C-X-C motif chemokine ligand 16 (CXCL16), transforming growth factor beta receptor 1 (TGFBR1), and complement component C1QA were upregulated. The biggest effects of HRO were in the periphery, as supplemented piglets consistently had decreased circulating neutrophils, and when injected with LPS, had decreased pro-inflammatory cytokine expression in the liver. HRO supplementation may have beneficial effects on inflammation in the periphery, and further studies are needed to elucidate the effects of omega-3 supplementation on microglial cell physiology in the neonate.
In conclusion, we found that PDF may enhance total white matter deposition in the neonatal brain, and effects were not limited to SGA piglets. More studies are needed to confirm increased myelination and assess long-term outcomes of PDF feeding. We also concluded that SGA piglets may not have an overactive neuroinflammatory response to infections, but their susceptibility to infections leaves them vulnerable to multiple hits and the neurodevelopmental disruption that can result. Dietary supplementation with omega-3 fatty acids showed some benefits in reducing pro-inflammatory cytokine production in AGA piglets, and also affected gene expression in microglial cells. More studies are needed to determine the effects of supplementation on microglial cell physiology and long term outcomes of omega-3 supplementation in neonates.