Proteomic analysis of the umbilical cord in fetal growth restriction and preeclampsia.

Fetal growth restriction (FGR) is associated with adverse perinatal outcomes. Pre-eclampsia (PreE) increases the associated perinatal morbidity and mortality. The structure of the umbilical cord in the setting of FGR and PreE is understudied. This study aimed to examine changes in the umbilical cord (UC) composition in pregnancies complicated by FGR and FGR with PreE. UC from gestational age-matched pregnancies with isolated FGR (n = 5), FGR+PreE (n = 5) and controls (n = 5) were collected, and a portion of the UC was processed for histologic and proteomic analysis. Manual segmentation analysis was performed to measure cross-section analysis of umbilical cord regions. Wharton's Jelly samples were analyzed on a tims-TOF Pro. Spectral count and ion abundance data were analyzed, creating an intersection dataset from multiple mass spectrometry search and inference engines. UCs from FGR and FGR with PreE had lower cross-sectional area and Wharton's Jelly area compared with control (p = 0.03). When comparing FGR to control, 28 proteins were significantly different in abundance analysis and 34 in spectral count analysis (p < 0.05). Differential expression analysis between PreE with FGR vs controls demonstrated that 48 proteins were significantly different in abundance and 5 in spectral count. The majority of changes occurred in proteins associated with extracellular matrix, cellular process, inflammatory, and angiogenesis pathways. The structure and composition of the UC is altered in pregnancies with FGR and FGR with PreE. Future work in validating these proteomic differences will enable identification of therapeutic targets for FGR and FGR with PreE.

Mu Opioid Splice Variant MOR-1K Contributes to the Development of Opioid-Induced Hyperalgesia.

BACKGROUND: A subset of the population receiving opioids for the treatment of acute and chronic clinical pain develops a paradoxical increase in pain sensitivity known as opioid-induced hyperalgesia. Given that opioid analgesics are one of few treatments available against clinical pain, it is critical to determine the key molecular mechanisms that drive opioid-induced hyperalgesia in order to reduce its prevalence. Recent evidence implicates a splice variant of the mu opioid receptor known as MOR-1K in the emergence of opioid-induced hyperalgesia. Results from human genetic association and cell signaling studies demonstrate that MOR-1K contributes to decreased opioid analgesic responses and produces increased cellular activity via Gs signaling. Here, we conducted the first study to directly test the role of MOR-1K in opioid-induced hyperalgesia. METHODS AND RESULTS: In order to examine the role of MOR-1K in opioid-induced hyperalgesia, we first assessed pain responses to mechanical and thermal stimuli prior to, during, and following chronic morphine administration. Results show that genetically diverse mouse strains (C57BL/6J, 129S6, and CXB7/ByJ) exhibited different morphine response profiles with corresponding changes in MOR-1K gene expression patterns. The 129S6 mice exhibited an analgesic response correlating to a measured decrease in MOR-1K gene expression levels, while CXB7/ByJ mice exhibited a hyperalgesic response correlating to a measured increase in MOR-1K gene expression levels. Furthermore, knockdown of MOR-1K in CXB7/ByJ mice via chronic intrathecal siRNA administration not only prevented the development of opioid-induced hyperalgesia, but also unmasked morphine analgesia. CONCLUSIONS: These findings suggest that MOR-1K is likely a necessary contributor to the development of opioid-induced hyperalgesia. With further research, MOR-1K could be exploited as a target for antagonists that reduce or prevent opioid-induced hyperalgesia.

Early postnatal respiratory viral infection induces structural and neurochemical changes in the neonatal piglet brain.

Infections that cause inflammation during the postnatal period are common, yet little is known about their impact on brain development in gyrencephalic species. To address this issue, we investigated brain development in domestic piglets which have brain growth and morphology similar to human infants, after experimentally infecting them with porcine reproductive and respiratory syndrome virus (PRRSV) to induce an interstitial pneumonia Piglets were inoculated with PRRSV on postnatal day (PD) 7 and magnetic resonance imaging (MRI) was used to assess brain macrostructure (voxel-based morphometry), microstructure (diffusion tensor imaging) and neurochemistry (MR-spectroscopy) at PD 29 or 30. PRRSV piglets exhibited signs of infection throughout the post-inoculation period and had elevated plasma levels of TNFα at the end of the study. PRRSV infection increased the volume of several components of the ventricular system including the cerebral aqueduct, fourth ventricle, and the lateral ventricles. Group comparisons between control and PRRSV piglets defined 8 areas where PRRSV piglets had less gray matter volume; 5 areas where PRRSV piglets had less white matter volume; and 4 relatively small areas where PRRSV piglets had more white matter. Of particular interest was a bilateral reduction in gray and white matter in the primary visual cortex. PRRSV piglets tended to have reduced fractional anisotropy in the corpus callosum. Additionally, N-acetylaspartate, creatine, and myo-inositol were decreased in the hippocampus of PRRSV piglets suggesting disrupted neuronal and glial health and energy imbalances. These findings show in a gyrencephalic species that early-life infection can affect brain growth and development.

Early postnatal respiratory viral infection alters hippocampal neurogenesis, cell fate, and neuron morphology in the neonatal piglet.

Respiratory viral infections are common during the neonatal period in humans, but little is known about how early-life infection impacts brain development. The current study used a neonatal piglet model as piglets have a gyrencephalic brain with growth and development similar to human infants. Piglets were inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) to evaluate how chronic neuroinflammation affects hippocampal neurogenesis and neuron morphology. Piglets in the neurogenesis study received one bromodeoxyuridine injection on postnatal day (PD) 7 and then were inoculated with PRRSV. Piglets were sacrificed at PD 28 and the number of BrdU+ cells and cell fate were quantified in the dentate gyrus. PRRSV piglets showed a 24% reduction in the number of newly divided cells forming neurons. Approximately 15% of newly divided cells formed microglia, but this was not affected by sex or PRRSV. Additionally, there was a sexual dimorphism of new cell survival in the dentate gyrus where males had more cells than females, and PRRSV infection caused a decreased survival in males only. Golgi impregnation was used to characterize dentate granule cell morphology. Sholl analysis revealed that PRRSV caused a change in inner granule cell morphology where the first branch point was extended further from the cell body. Males had more complex dendritic arbors than females in the outer granule cell layer, but this was not affected by PRRSV. There were no changes to dendritic spine density or morphology distribution. These findings suggest that early-life viral infection can impact brain development.

The domestic piglet: an important model for investigating the neurodevelopmental consequences of early life insults.

Insults in the prenatal and early postnatal period increase the risk for behavioral problems later in life. One hypothesis is that pre- and postnatal stressors influence structural and functional brain plasticity. Understanding the mechanisms is important, but progress has lagged because certain studies in human infants are impossible, while others are extremely difficult. Furthermore, results from popular rodent models are difficult to translate to human infants owing to the substantial differences in brain development and morphology. Because it overcomes some of these obstacles, the domestic piglet has emerged as an important model. Piglets have a gyrencephalic brain that develops similar to the human brain and that can be assessed in vivo by using clinical-grade neuroimaging instruments. Furthermore, owing to their precocial nature, piglets can be weaned at birth and used in behavioral testing paradigms to assess cognitive behavior at an early age. Thus, the domestic piglet represents an important translational model for investigating the neurodevelopmental consequences of early life insults.

Early supplementation of phospholipids and gangliosides affects brain and cognitive development in neonatal piglets.

BACKGROUND: Because human breast milk is a rich source of phospholipids and gangliosides and breastfed infants have improved learning compared with formula-fed infants, the importance of dietary phospholipids and gangliosides for brain development is of interest. OBJECTIVE: We sought to determine the effects of phospholipids and gangliosides on brain and cognitive development. METHODS: Male and female piglets from multiple litters were artificially reared and fed formula containing 0% (control), 0.8%, or 2.5% Lacprodan PL-20 (PL-20; Arla Foods Ingredients), a phospholipid/ganglioside supplement, from postnatal day (PD) 2 to PD28. Beginning on PD14, performance in a spatial T-maze task was assessed. At PD28, brain MRI data were acquired and piglets were killed to obtain hippocampal tissue for metabolic profiling. RESULTS: Diet affected maze performance, with piglets that were fed 0.8% and 2.5% PL-20 making fewer errors than control piglets (80% vs. 75% correct on average; P < 0.05) and taking less time to make a choice (3 vs. 5 s/trial; P < 0.01). Mean brain weight was 5% higher for piglets fed 0.8% and 2.5% PL-20 (P < 0.05) than control piglets, and voxel-based morphometry revealed multiple brain areas with greater volumes and more gray and white matter in piglets fed 0.8% and 2.5% PL-20 than in control piglets. Metabolic profiling of hippocampal tissue revealed that multiple phosphatidylcholine-related metabolites were altered by diet. CONCLUSION: In summary, dietary phospholipids and gangliosides improved spatial learning and affected brain growth and composition in neonatal piglets.

An in vivo three-dimensional magnetic resonance imaging-based averaged brain collection of the neonatal piglet (Sus scrofa).

Due to the fact that morphology and perinatal growth of the piglet brain is similar to humans, use of the piglet as a translational animal model for neurodevelopmental studies is increasing. Magnetic resonance imaging (MRI) can be a powerful tool to study neurodevelopment in piglets, but many of the MRI resources have been produced for adult humans. Here, we present an average in vivo MRI-based atlas specific for the 4-week-old piglet. In addition, we have developed probabilistic tissue classification maps. These tools can be used with brain mapping software packages (e.g. SPM and FSL) to aid in voxel-based morphometry and image analysis techniques. The atlas enables efficient study of neurodevelopment in a highly tractable translational animal with brain growth and development similar to humans.

A neonatal piglet model for investigating brain and cognitive development in small for gestational age human infants.

The piglet was investigated as a potential model for studying brain and cognitive deficits associated with being born small for gestational age (SGA). Naturally farrowed SGA (0.7-1.0 kg BW) and average for gestational age (AGA, 1.3-1.6 kg BW) piglets were obtained on postnatal day (PD) 2, placed in individual cages, and provided a nutritionally adequate milk replacer diet (285 ml/kg/d). Beginning at PD14, performance in a spatial T-maze task was assessed. At PD28, piglets were anesthetized for magnetic resonance (MR) imaging to assess brain structure (voxel-based morphometry), connectivity (diffusion-tensor imaging) and metabolites in the hippocampus and corpus callosum (proton MR spectroscopy). Piglets born SGA showed compensatory growth such that BW of SGA and AGA piglets was similar (P>0.05), by PD15. Birth weight affected maze performance, with SGA piglets taking longer to reach criterion than AGA piglets (p<0.01). Total brain volume of SGA and AGA piglets was similar (P<0.05), but overall, SGA piglets had less gray matter than AGA piglets (p<0.01) and tended to have a smaller internal capsule (p = 0.07). Group comparisons between SGA and AGA piglets defined 9 areas (≥ 20 clusters) where SGA piglets had less white matter (p<0.01); 2 areas where SGA piglets had more white matter (p<0.01); and 3 areas where SGA piglets had more gray matter (p<0.01). The impact of being born SGA on white matter was supported by a lower (p<0.04) fractional anisotropy value for SGA piglets, suggesting reduced white matter development and connectivity. None of the metabolites measured were different between groups. Collectively, the results show that SGA piglets have spatial learning deficits and abnormal development of white matter. As learning deficits and abnormalities in white matter are common in SGA human infants, the piglet is a tractable translational model that can be used to investigate SGA-associated cognitive deficits and potential interventions.

Respiratory viral infection in neonatal piglets causes marked microglia activation in the hippocampus and deficits in spatial learning.

Environmental insults during sensitive periods can affect hippocampal development and function, but little is known about peripheral infection, especially in humans and other animals whose brain is gyrencephalic and experiences major perinatal growth. Using a piglet model, the present study showed that inoculation on postnatal day 7 with the porcine reproductive and respiratory syndrome virus (PRRSV) caused microglial activation within the hippocampus with 82% and 43% of isolated microglia being MHC II(+) 13 and 20 d after inoculation, respectively. In control piglets, <5% of microglia isolated from the hippocampus were MHC II(+). PRRSV piglets were febrile (p < 0.0001), anorectic (p < 0.0001), and weighed less at the end of the study (p = 0.002) compared with control piglets. Increased inflammatory gene expression (e.g., IL-1ß, IL-6, TNF-α, and IFN-γ) was seen across multiple brain regions, including the hippocampus, whereas reductions in CD200, NGF, and MBP were evident. In a test of spatial learning, PRRSV piglets took longer to acquire the task, had a longer latency to choice, and had a higher total distance moved. Overall, these data demonstrate that viral respiratory infection is associated with a marked increase in activated microglia in the hippocampus, neuroinflammation, and impaired performance in a spatial cognitive task. As respiratory infections are common in human neonates and infants, approaches to regulate microglial cell activity are likely to be important.

Early life iron deficiency impairs spatial cognition in neonatal piglets.

Iron deficiency is common throughout the world and has been linked to cognitive impairments. Using neonatal piglets to model human infants, we assessed the impact of iron deficiency on spatial learning and memory. Artificially reared piglets were fed 1 of 3 liquid diets with varying concentrations of iron: control (CON), mildly deficient (MID), or severely deficient (SID; 100, 25.0, or 10.0 mg iron/kg milk solids, respectively) for 4 wk. Relative to CON, SID and MID piglets had reduced hemoglobin (P < 0.05) as well as magenta skin color (P < 0.001), which correlated with hematocrit (R(2) = 0.76; P < 0.001). SID and MID hemoglobin differed at wk 3 and 4 (P < 0.05). In a hippocampal-dependent, spatial, T-maze task, SID piglets were unable to acquire the task (post hoc contrast: first vs. last day of acquisition), while MID piglets demonstrated deficits in reversal learning (P = 0.032). Iron concentrations in the liver (P < 0.001), serum (P = 0.003), and hippocampus (P = 0.004), but not prefrontal cortex, were lower in MID and SID compared with CON piglets. The level of the transferrin receptor mRNA (TFR) was greater in the prefrontal cortex of CON piglets than in MID and SID piglets (P = 0.001) but not the hippocampus. Gene expression of several neurotrophic factors and proinflammatory cytokines, as well as whole-brain and hippocampal volume, were not affected by dietary treatment. In conclusion, neonatal iron deficiency leads to cognitive impairment, which may be due in part to a reduced iron concentration in the hippocampus.

Brain growth of the domestic pig (Sus scrofa) from 2 to 24 weeks of age: a longitudinal MRI study.

An animal model with brain growth similar to humans, that can be used in MRI studies to investigate brain development, would be valuable. Our laboratory has developed and validated MRI methods for regional brain volume quantification in the neonatal piglet. The aim of this study was to utilize the MRI-based volume quantification technique in a longitudinal study to determine brain growth in domestic pigs from 2 to 24 weeks of age. MRI data were acquired from pigs 2-24 weeks of age using a 3-dimensional magnetization-prepared gradient echo sequence on a Magnetom Trio 3-tesla imager. Manual segmentation was performed for volume estimates of total brain, cortical, diencephalon, brainstem, cerebellar and hippocampal regions. Logistic modeling procedures were used to characterize brain growth. Total brain volume increased 130% (±12%) and 121% (±7%) from 2 to 24 weeks in males and females, respectively. The maximum increase in total brain volume occurred about the age of 4 weeks and 95% of whole brain growth occurred by the age of 21-23 weeks. Logistical modeling suggests there are sexually dimorphic effects on brain growth. For example, in females, the cortex was smaller (p = 0.04). Furthermore, the maximum growth of the hippocampus occurred about 5 weeks earlier in females than males, and the window for hippocampal growth was significantly shorter in females than males (p = 0.02, p = 0.002 respectively). These sexual dimorphisms are similar to what is seen in humans. In addition to providing important data on brain growth for pigs, this study shows pigs can be used to obtain longitudinal MRI data. The large increase in brain volume in the postnatal period is similar to that of human neonates and suggests pigs can be used to investigate brain development.

Magnetic resonance imaging of the neonatal piglet brain.

INTRODUCTION: Appeal for the domestic pig as a preclinical model for neurodevelopmental research is increasing. One limitation, however, is lack of magnetic resonance imaging (MRI) methods for brain volume quantification in the neonatal piglet. The purpose of this study was to develop and validate MRI methods for estimating brain volume in piglets. RESULTS: The results showed that MRI and manual segmentation reliably estimated the changes in volume of different brain regions in 2- and 5-wk-old piglets. Substantial increases in the volumes of all brain regions examined were evident during the 3-wk period. DISCUSSION: MRI can provide accurate estimates of brain region volume during the neonatal period in piglets. A piglet model that can be used in longitudinal studies may be useful for investigating how experimental (e.g., nutrition, infection) factors affect brain growth and development. METHODS: Anatomic MRI data (non-longitudinal) were acquired 2- and 5-wk-old piglets using a three--dimensional T1-weighted magnetization-prepared gradient echo (MPRAGE) sequence on a MAGNETOM Trio 3T imager. Manual segmentation was performed for volume estimates of total brain, cortical, diencephalon, brainstem, cerebellar, and -hippocampal regions. The MRI-based hippocampal volume estimates in 2- and 5-wk-old piglets were validated using histological techniques and the Cavalieri method.