Ovine Fetus Research Articles

Unexpected maturation of PI3K and MAPK-ERK signaling in fetal ovine cardiomyocytes.

In the first two-thirds of gestation, ovine fetal cardiomyocytes undergo mitosis to increase cardiac mass and accommodate fetal growth. Thereafter, some myocytes continue to proliferate while others mature and terminally differentiate into binucleated cells. At term (145 days gestational age; dGA) about 60% of cardiomyocytes become binucleated and exit the cell cycle under hormonal control. Rising thyroid hormone (T3) levels near term (135 dGA) inhibit proliferation and stimulate maturation. However, the degree to which intracellular signaling patterns change with age in response to T3 is unknown. We hypothesized that in vitro activation of ERK, Akt, and p70(S6K) by two regulators of cardiomyocyte cell cycle activity, T3 and insulin like growth factor-1 (IGF-1), would be similar in cardiomyocytes at gestational ages 100 and 135 dGA. IGF-1 and T3 each independently stimulated phosphorylation of ERK, Akt, and p70(S6K) in cells at both ages. In the younger mononucleated myocytes, the phosphorylation of ERK and Akt was reduced in the presence of IGF-1 and T3. However, the same hormone combination led to a dramatic twofold increase in the phosphorylation of these signaling proteins in the 135 dGA cardiomyocytes-even in cells that were not proliferating. In the older cells, both mono- and binucleated cells were affected. In conclusion, fetal ovine cardiomyocytes undergo profound maturation-related changes in signaling in response to T3 and IGF-1, but not to either factor alone. Differences in age-related response are likely to be related to milestones in fetal cardiac development as the myocardium prepares for ex utero life.

Developmental profiles of GFAP-positive astrocytes in sheep cerebellum.

Astroglial account for the largest glial population in the brain and play a variety of vital functions in the development of the central nervous system (CNS). An immunohistochemical study was performed in 19 ovine foetuses ranging from 2 to 5months of gestation, one newborn lamb and three adult sheep. Using the anit-glial fibrillary acidic protein (GFAP) marker, several variations were found in the degree of GFAP positive (GFAP+) astrocyte distribution between the different zones in the cerebellum of sheep during brain development. Our study indicates that the first appearance of astrocytes from restricted zones in the cerebellum occurs around the eighth week of gestation. Bergmann cells were found to be present from around the 15th week of gestation onwards. Our findings suggest that the maturation of astrocytes begins in the caudal parts of the cerebellum, developing from their initial ventral regions to spread first to dorsal regions radially within the white matter, then followed by the more rostral parts of the cerebellum. Astrocytes were also found to proliferate in the vermis before appearing in the cerebellar hemispheres.

Mineralocorticoid effects in the late gestation ovine fetal lung.

This study was designed to determine the effects of corticosteroids at MR in the late‐gestation fetal lung. Since both the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) are expressed at relatively high levels in the fetal lung, endogenous corticosteroids may act at MR as well as GR in the preterm fetal lung. The GR agonist, betamethasone, the MR agonist, aldosterone, or both were infused intravenously for 48 h in ovine fetuses of approximately 130 days gestation. Effects on airway pressures during stepwise inflation of the in situ lung, expression of ENaC alpha (SCNN1A), ENaC beta (SCNN1B), and Na,K ATPase (ATP1A1), and elastin and collagen content were determined after the infusions. We found that aldosterone significantly reduced the airway pressure measured during the initial step in inflation of the lung, although aldosterone had no overall effect on lung compliance, nor did aldosterone induce expression of ENaCα, ENaCβ or Na,K ATPaseα1. Betamethasone significantly increased expression of the epithelial sodium channel (ENaC) subunit mRNAs, and collagen and elastin content in the lungs, although this dose of betamethasone also had no effect on lung compliance. There was no synergy between effects of the MR and GR agonists. Transcriptomic analysis suggested that although aldosterone did not alter genes in pathways related to epithelial sodium transport, aldosterone did alter genes in pathways involved in cell proliferation in the lungs. The results are consistent with corticosteroid‐induced fluid reabsorption at birth through GR rather than MR, but suggest that MR facilitates lung maturation, and may contribute to inflation with the first breaths via mechanisms distinct from known aldosterone effects in other epithelia.

Adaptive brain shut-down counteracts neuroinflammation in the near-term ovine fetus.

Objective: Repetitive umbilical cord occlusions (UCOs) in ovine fetus leading to severe acidemia result in adaptive shut-down of electrocortical activity [electrocorticogram (ECoG)] as well as systemic and brain inflammation. We hypothesized that the fetuses with earlier ECoG shut-down as a neuroprotective mechanism in response to repetitive UCOs will show less brain inflammation and, moreover, that chronic hypoxia will impact this relationship.Methods: Near-term fetal sheep were chronically instrumented with ECoG leads, vascular catheters, and a cord occluder and then underwent repetitive UCOs for up to 4 h or until fetal arterial pH was <7.00. Eight animals, hypoxic prior to the UCOs (SaO2 <55%), were allowed to recover 24 h post insult, while 14 animals, 5 of whom also were chronically hypoxic, were allowed to recover 48 h post insult, after which brains were perfusion-fixed. Time of ECoG shut-down and corresponding pH were noted, as well as time to then reach pH <7.00 (ΔT). Microglia (MG) were counted as a measure of inflammation in gray matter layers 4–6 (GM4–6) where most ECoG activity is generated. Results are reported as mean ± SEM for p < 0.05.Results: Repetitive UCOs resulted in worsening acidosis over 3–4 h with arterial pH decreasing to 6.97 ± 0.02 all UCO groups’ animals, recovering to baseline by 24 h. ECoG shut-down occurred 52 ± 7 min before reaching pH <7.00 at pH 7.23 ± 0.02 across the animal groups. MG counts were inversely correlated to ΔT in 24 h recovery animals (R = −0.84), as expected. This was not the case in normoxic 48 h recovery animals, and, surprisingly, in hypoxic 48 h recovery animals, this relationship was reversed (R = 0.90).Conclusion: Adaptive brain shut-down during labor-like worsening acidemia counteracts neuroinflammation in a hypoxia- and time-dependent manner.

Intestinal fatty acid-binding protein: a possible marker for gut maturation.

Gut immaturity is linked with postnatal intestinal disorders. However, biomarkers to assess the intestinal developmental stage around birth are lacking. The aim of this study was to gain more insight on intestinal fatty acid-binding protein (I-FABP) as an indicator of gut maturity. Antenatal I-FABP distribution and release was investigated in extremely premature, moderately premature, and term lambs, and these findings were verified in human urinary samples. Ileal I-FABP distribution was confirmed in autopsy material within 24 h postnatally. Median (range) serum I-FABP levels were lower in extremely premature lambs compared with moderately premature lambs (156 (50.0-427) vs. 385 (100-1,387) pg/ml; P = 0.02). Contrarily, median early postnatal urine I-FABP levels in human infants were higher in extremely premature compared with moderately premature and term neonates (1,219 (203-15,044) vs. 256 (50-1,453) and 328 (96-1,749) pg/ml; P = 0.008 and P = 0.04, respectively). I-FABP expression was most prominent in nonvacuolated enterocytes and increased with rising gestational age (GA) in ovine and human tissue samples. The epithelial distribution pattern changed from a phenotype displaying I-FABP-positive enterocytes merely in the crypts early in gestation into a phenotype with I-FABP expressing cells exclusively present in the villus tips at term in ovine and human tissue. In this ovine and human study, increasing GA is accompanied by an increase in I-FABP tissue content. Cord I-FABP levels correlate with gestation in ovine fetuses, identifying I-FABP as a marker for gut maturation. Raised postnatal urine I-FABP levels in preterm human infants may indicate intestinal injury and/or inflammation in utero.

Ischemia/Reperfusion-induced neovascularization in the cerebral cortex of the ovine fetus.

Information on the effects of injury on neovascularization in the immature brain is limited. We investigated the effects of ischemia on cerebral cortex neovascularization after the exposure of fetuses to 30 minutes of cerebral ischemia followed by 48 hours of reperfusion (I/R-48), 30 minutes of cerebral ischemia followed by 72 hours of reperfusion (I/R-72), or sham control treatment (Non-I/R). Immunohistochemical and morphometric analyses of cerebral cortex sections included immunostaining for glial fibrillary acidic protein and collagen type IV (a molecular component of the vascular basal lamina) to determine the glial vascular network in fetal brains and Ki67 as a proliferation marker. Cerebral cortices from I/R-48 and I/R-72 fetuses exhibited general responses to ischemia, including reactive astrocyte morphology, which was not observed in Non-I/R fetuses. Cell bodies of reactive proliferating astrocytes, along with large end-feet, surrounded the walls of cerebral cortex microvessels in addition to the thick collagen type IV-enriched basal lamina. Morphometric analysis of the Non-I/R group with the I/R-48 and I/R-72 groups revealed increased collagen type IV density in I/R-72 cerebral cortex microvessels (p < 0.01), which also frequently displayed a sprouting appearance characterized by growing tip cells and activated pericytes. Increases in cerebral cortex basic fibroblast growth factor were associated with neovascularization. We conclude that increased neovascularization in fetal cerebral cortices occurs within 72 hours of ischemia.

Polymyxin B Agonist Capture Therapy for Intrauterine Inflammation: Proof-of-Principle in a Fetal Ovine Model

Intrauterine infection is a leading cause of preterm birth (PTB), most notably in deliveries occurring before 32 weeks gestation. Preterm infants exposed to intrauterine inflammation are more likely to have a host of neurological, respiratory, gastrointestinal, and visual pathologies. Preventing preterm delivery and protecting the fetus from injury is thus likely to require treatment of both intrauterine infection and inflammation. Polymyxin B (PMXB) is a cationic peptide antibiotic that binds Escherichia coli lipopolysaccharides (LPS) and prevents inflammatory activation. We hypothesized that intraamniotic administration of PMXB would selectively inhibit LPS-driven inflammation, serving as a proof-of-principle for targeted agonist capture therapy as a treatment for PTB and fetal injury. In vitro studies with primary fetal ovine keratinocytes demonstrated a significant and sustained reduction in tumor necrosis factor α and interleukin 8 messenger RNA expression after treatment with PMXB and LPS, relative to cells treated with LPS alone. In vivo studies with fetal sheep demonstrated a significant reduction in proinflammatory cytokines in the amniotic fluid and fetal lung (but not fetal skin or chorioamnion) in LPS + PMXB-treated animals, relative to those treated with LPS alone. These data are consistent with a partial resolution of LPS-driven intrauterine inflammation. They suggest the potential for agonist capture as a conceptual means of resolving the proparturition inflammation caused by infection of the amniotic cavity.

Proliferation suppression by thyroid hormone requires thyroid hormone receptor in fetal cardiomyocytes (850.1)

Objectives: Ovine fetal cardiomyocyte (CM) maturation begins after 100 days gestation (dGA). At term (145dGA) ~60% of CMs are binucleated and cannot divide. Thyroid hormone (T3) rises near term and suppresses proliferation by insulin‐like growth factor‐1 (IGF‐1) in vitro. Despite T3 activation of extracellular regulatory kinase (ERK) and AKT we hypothesize that T3‐induced inhibition of proliferation is thyroid receptor (TR) mediated and reversed by the TR antagonist, NH3. Methods: Fetal CMs were isolated from 102±3 and 135±1dGA sheep (n=11/group). Cells were treated with T3 (0.37, 1.5nM), IGF‐1 (1ug/ml), NH3 (100nM) or a combination. Proliferation was quantified by BrdU uptake (10uM). Western blots measured ERK, AKT, TRa, and p21 expression. Results: IGF‐1 stimulated proliferation at both ages. T3 reduced this mitotic activity by ~50% in 100dGA and ~75% in 135dGA. At both ages T3 increased p21 (p&lt;0.001), and NH3 blocked the T3+IGF‐1 reduction of BrdU uptake without altering ERK or AKT. In 100dGA CMs NH3 did not reduce T3‐induced p21 expression but upregulated TRa (p&lt;0.05). In 135dGA CMs, NH3 alone reduced BrdU uptake as well as T3‐induced p21 (p&lt;0.05). Conclusion: Fetal ovine CMs are sensitive to the effects of NH3 differently depending on maturation state. NH3 inhibition of proliferation suggests that TRs are needed for normal proliferation in the developing myocardium and that p21 may be the main effector. Grant Funding Source: Supported by NIH NHLBI and NICHD

The effects of ketamine on the immune response in the fetal ovine brain in response to hypoxic hypoxia (877.8)

Fetal hypoxia is a reduction in oxygen supply to the fetus. HH occurs when maternal and fetal partial pressure of oxygen is decreased due to maternal partial ventilatory restriction. Recently, our lab reported that HH increased inflammatory genes in the fetal brain, and KET reduced the expression by microarray analysis. We hypothesized that HH causes an inflammatory reaction in the fetal ovine brain, and KET attenuates this response. We examined fetal ovine brain (n=3‐5/group) where mother and baby were chronically catheterized, and tracheostomy was performed on the ewe. A 50 % decrease in partial pressure of oxygen was induced by administering nitrogen gas directly to the ewe for 30 min. KET was administered intravenously to the fetus 10 min prior to the induction of HH. Brains were collected 24 hrs post HH stimulus, fixated in 4% paraformaldehyde, and then embedded in paraffin blocks for hematoxylin and eosin staining. After tissue examination under light microscope, lymphocytic infiltration, congestion in blood vessels, and the presence of proteinaceous substances were evaluated by a 5‐point scoring criteria in the HH brains. KET produced a decrease in inflammatory response in tissues subjected to HH, and the induction of HH produced a higher inflammatory response when compared to normoxia group (P&lt;0.05, Friedman test). We conclude that KET decreases the level of inflammation in the fetal ovine brain exposed to HH.Grant Funding Source: Supported by NIH grants HD033053 and HD05761 and by the APS STRIDE program.

Cyclic nucleotides down regulate PAF receptor mediated effects in newborn ovine pulmonary arterial smooth muscle: significance in oxygen therapy of persistent pulmonary hypertension of the newborn (LB605)

Platelet activating factor (PAF) is implicated in the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN). Baseline PAFr activity is higher in fetal ovine pulmonary artery smooth muscle cells (PASMC) and is augmented by hypoxia. In fetal PASMC we have shown evidence of crosstalk between PAFr‐mediated cell signaling and activity of cyclic nucleotides (Cnucs) via respective receptors PKG and PKA. Since these mechanisms are not well described in the newborn counterpart, we studied effects of Cnucs on PAFr protein expression in newborn PASMC. Because long term effects of oxygen therapy on newborn lung are not well understood, we included various oxygen tension study conditions. We hypothesize that PPHN results from failure of newborn lamb pulmonary system to down regulate PAFr activity or to up regulate vasodilatory Cnuc activity.PASMC harvested from ovine newborns 6‐12d old were incubated for 24h with culture media or media containing 10μM cGMP or cAMP analogs or 0.1μM of PAF in normoxia (pO2~100torr), hypoxia (pO2 &lt;40 torr) and hyperoxia (5% CO2, O2). Prostacyclin and TxA2 release were measured and compared to release by fetal PASMC. Proliferation studies were performed under the three conditions and PAFr, PKA and PKG expression were measured.Newborn PA baseline prostacyclin level was 9‐fold greater than in fetal PA, whereas baseline production of TxA2 by newborn PA was 7‐fold less. Overall, 24h hyperoxia attenuated cell proliferation across all treatments. Cnuc treatment attenuated PAF stimulation of proliferation and decreased PAFr expression.Elevated prostacyclin production over TxA2 suggests greater cyclooxygenase activity in newborn PA postnatally. Down regulation of PAFr effects by Cnucs indicates that normal newborn PA physiology favors vasodilatory pathways to minimize PAF‐induced hypertrophy or hyperplasia. Thus failure of newborn lung to anchor down regulation of vasoconstrictors with up regulation of vasodilators leads to pathogenesis of PPHN.

Chronic hypoxia differentially modulates the response of fetal ovine middle cerebral arteries to endothelin‐1 (853.2)

To better understand how hypoxia‐induced vascular remodeling alters intracellular responses to ET‐1, we hypothesized that chronic hypoxia alters ET‐1 induced phenotypic transformation in arterial smooth muscle cells within the fetal cerebrovasculature. Endothelium denuded middle cerebral arteries (MCA) harvested from term fetuses of pregnant adult sheep kept at sea level or 3820m for 110 days were serum starved for 24h, then cultured 24h with ET‐1 and various kinase inhibitors. Fetal normoxic (FN) MCAs incubated with ET‐1 exhibited decreased colocalization between MLCK and α‐actin compared to control (starved), suggesting a shift of smooth muscle cells (SMCs) from a contractile to a more synthetic phenotype. This appeared to be mediated by multiple intracellular pathways, with PKC playing the largest role in normoxic arteries. Chronic hypoxia attenuated the ET‐1 induced decrease in MLCK and α‐actin colocalization, decreased the effects of PKC inhibitors, but increased the apparent effects of the CaMK‐dependent pathway, on colocalization. Given that plasma levels of ET‐1 were unaltered by chronic hypoxia, these results suggest that chronic hypoxia shifts the coupling of ET‐1 from PKC to CaMK to alter gene expression and thereby influence vascular remodeling.Grant Funding Source: Supported by National Institutes of Health Grants HD‐31266, HL‐64867, and NS‐076945

Preadipocyte and adipose tissue differentiation in meat animals: influence of species and anatomical location.

Early in porcine adipose tissue development, the stromal-vascular (SV) elements control and dictate the extent of adipogenesis in a depot-dependent manner. The vasculature and collagen matrix differentiate before overt adipocyte differentiation. In the fetal pig, subcutaneous (SQ) layer development is predictive of adipocyte development, as the outer, middle, and inner layers of dorsal SQ adipose tissue develop and maintain layered morphology throughout postnatal growth of SQ adipose tissue. Bovine and ovine fetuses contain brown adipose tissue but SQ white adipose tissue is poorly developed structurally. Fetal adipose tissue differentiation is associated with the precocious expression of several genes encoding secreted factors and key transcription factors like peroxisome proliferator activated receptor (PPAR)γ and CCAAT/-enhancer-binding protein. Identification of adipocyte-associated genes differentially expressed by age, depot, and species in vivo and in vitro has been achieved using single-gene analysis, microarrays, suppressive subtraction hybridization, and next-generation sequencing applications. Gene polymorphisms in PPARγ, cathepsins, and uncoupling protein 3 have been associated with back fat accumulation. Genome scans have mapped several quantitative trait loci (QTL) predictive of adipose tissue-deposition phenotypes in cattle and pigs.

Altitude, attitude and adaptation.

The fetus has the extraordinary capacity to respond to stress during development, which, in a large part, is mediated by the hypothalamo- pituitary-adrenal (HPA) axis. Hypoxia represents a significant risk to fetal homeostasis and can occur in a wide range of settings including maternal smoking, preeclampsia, preterm labor and high altitude. To study fetal adaptation to chronic, gestational hypoxia, we developed a model of high-altitude, long-term hypoxia (LTH) in pregnant sheep. We discuss the role of LTH on the HPA axis and potential programming of adaptive responses. LTH causes significant activation of the hypothalamic paraventricular nucleus (PVN) and anterior pituitary. In marked contrast, there is an adaptive inhibition in the adrenal, thus balancing the potentially maladaptive centrally mediated responses to LTH. Additionally, we discuss effects of LTH on adipose tissue development. LTH enhances leptin production, which in turn has a regulatory role on the adrenal cortex. Importantly, LTH also has a significant impact on programming of adipose tissue function. Together, our studies show that LTH induces a number of adaptive responses in the ovine fetus. Although they may be beneficial during fetal life, these adaptations could prove to be deleterious in the postnatal period and adulthood.

The effect of maternal arginine supplementation on the development of the thermogenic program in the ovine fetus

Brown adipose tissue (BAT) is a specialised fat store that is metabolised by the newborn lamb to ensure effective adaptation to the cold challenge of the extra-uterine environment. Increasing BAT reserves therefore has the potential to increase neonatal thermogenesis and survival. It is established that arginine supplementation can increase fetal BAT stores but the biological mechanisms involved are unclear. The objective of this study was to test the hypothesis that increased fetal BAT stores resulting from maternal arginine supplementation is mediated by activation of the thermogenic program. Brown adipose tissue was collected from fetuses of ewes supplemented with arginine from 100 to 140 days of gestation. Increased peri-renal fat stores in fetuses from arginine-supplemented ewes was associated with an increase in uncoupling protein 1 (UCP-1) and PRD1-BF-1-RIZ1 homologous domain containing protein-16 expression, but not proliferator-activated receptor gamma or proliferator-activated receptor gamma-co-activator-1α in BAT. The activity of UCP-1 is regulated by hormones including cortisol and thyroid hormones. Cortisol level in fetuses from supplemented sheep was 68% greater than those from control ewes, indicating that cortisol may control upregulation of UCP-1 expression in the ovine neonate. The DNA and RNA concentration in BAT of both groups suggest that increased peri-renal fat stores is not associated with an increase in cell number or number of ribosomes, but rather an increase in the size of individual fat cells. Collectively, these results indicate that maternal arginine supplementation during mid to late gestation improved the thermoregulatory ability of lambs and this could potentially increase their survival in early life.

Sampling rate of heart rate variability impacts the ability to detect acidemia in ovine fetuses near-term.

Background: To evaluate the impact of sampling rate on the predictive capability of continuous fetal heart rate (FHR) variability (fHRV) monitoring for detecting fetal acidemia during labor, we tested the performance of the root mean square of successive differences (RMSSD) in R–R intervals from the ECG when acquired with the sampling rate of 4 Hz currently available in FHR monitors, in comparison to the gold standard of 1000 Hz.Methods: Near-term ovine fetuses (N = 9) were chronically prepared with precordial electrodes for recording ECG, vascular catheters for blood sampling, and an umbilical cord occluder. For 1 min every 2.5 min, animals underwent mild partial umbilical cord occlusions (UCO) × 1 h, moderate partial UCO × 1 h, then complete UCO × 2 h, or until arterial pH reached <7.00. Arterial blood samples were drawn at baseline and every 20 min during the UCO series. RMSSD was calculated continuously in 5 min windows using an automated, standardized system (CIMVA.com). Results are presented as mean ± SEM with significance assumed for p < 0.05.Results: Repetitive UCO resulted in pH decreasing from 7.35 ± 0.01 to 7.00 ± 0.03. In all nine animals, RMSSD increased from 16.7 ± 1.0 ms at baseline to 44.4 ± 2.3 ms, 70 ± 15 min prior to reaching the pH nadir when sampled at 1000 Hz. When sampled at 4 Hz, RMSSD at baseline measured 36.1 ± 6.0 ms and showed no significant increase during the UCO series until the pH nadir was reached. Consequently, early detection of severe hypoxic-acidemia would have been missed in all fetuses.Conclusion: RMSSD as a measure of fHRV when calculated from FHR sampled at 1000 Hz allowed for the early detection of worsening hypoxic-acidemia in each fetus. However, when calculated at the low sampling rate of 4 Hz used clinically, RMSSD remained unchanged until terminally when the nadir pH was reached. For early detection of fetal acidemia during labor, more sensitive means of acquiring FHR are therefore recommended than currently deployed, e.g., trans-abdominal fetal ECG.

Molecular characterization ofToxoplasma gondiiType II in sheep abortion in Sardinia, Italy

During 2009–2010, 161 tissue samples (142 placentas, 16 brains, and 3 livers) from aborted ovine fetuses on Sardinia Island, Italy, were tested for toxoplasmosis. Organs that showed a positive result by nested polymerase chain reaction (PCR) targeting the ITS1 region for Toxoplasma gondii were also amplified with 11 genetic markers (SAG1, 5′-SAG2, 3′-SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) and then subjected to PCR/RFLP for genetic typing. T. gondii DNA was found in 5 placentas, 14 brains, and 2 livers by PCR analysis and all isolates displayed Type II alleles at all 11 loci with all 11 markers. The results indicate that the Type II T. gondii is associated with ovine abortion.

Lamb model of respiratory syncytial virus-associated lung disease: insights to pathogenesis and novel treatments.

Preterm birth is a risk factor for respiratory syncytial virus (RSV) bronchiolitis and hospitalization. The pathogenesis underlying this is not fully understood, and in vivo studies are needed to better clarify essential cellular features and molecular mechanisms. Such studies include analysis of lung tissue from affected human infants and various animal models. The preterm and newborn lamb lung has developmental, structural, cellular, physiologic, and immunologic features similar to that of human infants. Also, the lamb lung is susceptible to various strains of RSV that infect infants and cause similar bronchiolar lesions. Studies in lambs suggest that viral replication in airways (especially bronchioles) is extensive by 4 days after infection, along with bronchiolitis characterized by degeneration and necrosis of epithelial cells, syncytial cell formation, neutrophil infiltration, epithelial cell hypertrophy and hyperplasia, and innate and adaptive immune responses. RSV bronchiolitis greatly affects airflow and gaseous exchange. RSV disease severity is increased in preterm lambs compared with full-term lambs; similar to human infants. The lamb is conducive to experimental assessment of novel, mechanistic therapeutic interventions such as delivery of vascular endothelial growth factor and enhancement of airway epithelial oxidative responses, Club (Clara) cell protein 10, and synthesized compounds such as nanobodies. In contrast, exposure of the fetal ovine lung in vivo to ethanol, a risk factor for preterm birth, reduces pulmonary alveolar development and surfactant protein A expression. Because the formalin-inactivated RSV vaccination enhances some inflammatory responses to RSV infection in lambs, this model has the potential to assess mechanisms of formalin-inactivated RSV enhanced disease as well as newly developed vaccines.

Antenatal Maternal Long-Term Hypoxia: Acclimatization Responses with Altered Gene Expression in Ovine Fetal Carotid Arteries

In humans and other species, long-term hypoxia (LTH) during pregnancy can lead to intrauterine growth restriction with reduced body/brain weight, dysregulation of cerebral blood flow (CBF), and other problems. To identify the signal transduction pathways and critical molecules, which may be involved in acclimatization to high altitude LTH, we conducted microarray with advanced bioinformatic analysis on carotid arteries (CA) from the normoxic near-term ovine fetus at sea-level and those acclimatized to high altitude for 110+ days during gestation. In response to LTH acclimatization, in fetal CA we identified mRNA from 38 genes upregulated >2 fold (P<0.05) and 9 genes downregulated >2-fold (P<0.05). The major genes with upregulated mRNA were SLC1A3, Insulin-like growth factor (IGF) binding protein 3, IGF type 2 receptor, transforming growth factor (TGF) Beta-3, and genes involved in the AKT and BCL2 signal transduction networks. Most genes with upregulated mRNA have a common motif for Pbx/Knotted homeobox in the promoter region, and Sox family binding sites in the 3′ un translated region (UTR). Genes with downregulated mRNA included those involved in the P53 pathway and 5-lipoxygenase activating proteins. The promoter region of all genes with downregulated mRNA, had a common 49 bp region with a binding site for DOT6 and TOD6, components of the RPD3 histone deacetylase complex RPD3C(L). We also identified miRNA complementary to a number of the altered genes. Thus, the present study identified molecules in the ovine fetus, which may play a role in the acclimatization response to high-altitude associated LTH.

LPS and TNF alpha modulate AMPA/NMDA receptor subunit expression and induce PGE2 and glutamate release in preterm fetal ovine mixed glial cultures

BackgroundWhite matter injury (WMI) is the major antecedent of cerebral palsy in premature infants, and is often associated with maternal infection and the fetal inflammatory response. The current study explores the therapeutic potential of glutamate receptor blockade or cyclooxygenase-2 (COX-2) inhibition for inflammatory WMI.MethodsUsing fetal ovine derived mixed glia cultures exposed to tumour necrosis factor-α (TNF-α) or lipopolysaccharide (LPS), the expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and N-methyl D-aspartate (NMDA) glutamate receptors and their contribution to inflammation mediated pre-oligodendrocyte (OL) death was evaluated. The functional significance of TNF-α and COX-2 signalling in glutamate release in association with TNF-α and LPS exposure was also assessed.ResultsAMPA and NMDA receptors were expressed in primary mixed glial cultures on developing OLs, the main cell-type present in fetal white matter at a period of high risk for WMI. We show that glutamate receptor expression and configuration are regulated by TNF-α and LPS exposure, but AMPA and NMDA blockade, either alone or in combination, did not reduce pre-OL death. Furthermore, we demonstrate that glutamate and prostaglandin E2 (PGE2) release following TNF-α or LPS are mediated by a TNF-α-COX-2 dependent mechanism.ConclusionsOverall, these findings suggest that glial-localised glutamate receptors likely play a limited role in OL demise associated with chronic inflammation, but supports the COX-2 pathway as a potential therapeutic target for infection/inflammatory-mediated WMI.

Altered canonical Wingless-Int signaling in the ovine fetal lung after exposure to intra-amniotic lipopolysaccharide and antenatal betamethasone

Antenatal inflammation and maternal corticosteroids induce fetal lung maturation but interfere with late lung development. Canonical Wingless-Int (Wnt) signaling directs lung development and repair. We showed that intra-amniotic (IA) lipopolysaccharide (LPS) exposure disrupted developmental signaling pathways in the preterm lamb lungs. Therefore, we hypothesized that pulmonary Wnt signaling was altered by exposure to IA LPS and/or antenatal corticosteroids. Ovine fetuses were exposed to IA LPS, maternal intramuscular betamethasone, a control saline injection, or a combination thereof at 107 and/or 114 d gestational age (term = 150 d gestational age) before delivery at 121 d gestational age. IA LPS exposure decreased the lung expression of lymphoid enhancer-binding factor 1 (LEF1), a major Wnt pathway effector. WNT1, WNT4, and downstream messenger β-catenin decreased after LPS exposure. WNT7b mRNA increased fourfold 14 d post-LPS exposure. Betamethasone treatment 7 d before LPS exposure prevented the reduction in LEF1 expression, whereas betamethasone administration after LPS normalized the LPS-induced increase in Wnt7b mRNA. IA LPS exposure decreased canonical Wnt signaling in the developing lung. Antenatal corticosteroids before or after IA inflammation had different effects on pulmonary Wnt signaling. This study provides new insights into possible mechanisms by which prenatal inflammation affects lung development and how corticosteroid can be beneficial in this setting.