IGF-1 infusion increases growth in fetal sheep when euinsulinemia is maintained.

Insulin-like growth factor 1 (IGF-1) is a critical fetal anabolic hormone. IGF-1 infusion to the normally growing sheep fetus increases the weight of some organs but does not consistently increase body weight. However, IGF-1 infusion profoundly decreases fetal plasma insulin concentrations, which may limit fetal growth potential. In this study, normally growing late gestation fetal sheep received an intravenous infusion of either: IGF-1 (IGF), IGF-1 with insulin and dextrose to maintain fetal euinsulinemia and euglycemia (IGF+INS), or vehicle control (CON) for one week. The fetus underwent a metabolic study immediately prior to infusion start and after one week of the infusion to measure uterine and umbilical uptake rates of nutrients and oxygen. IGF+INS fetuses were 23% heavier than CON (P=0.0081) and had heavier hearts, livers, and adrenal glands than IGF and CON (P<0.01). By design, final fetal insulin concentrations in IGF were 62% and 65% lower than IGF+INS and CON, respectively. Final glucose concentrations were similar in all groups. IGF+INS had lower final oxygen content than IGF and CON (P<0.0001) and lower final amino acid concentrations than CON (P=0.0002). Final umbilical oxygen uptake was higher in IGF+INS compared to IGF and CON (P<0.05). Final umbilical uptake of several essential amino acids was higher in IGF+INS compared to CON (P<0.05). In summary, maintaining euinsulinemia and euglycemia during fetal IGF-1 infusion is necessary to maximally support body growth. We speculate that IGF-1 and insulin stimulate placental nutrient transport to support fetal growth.

Mitochondrial respiration is lower in the intrauterine growth-restricted fetal sheep heart.

Fetuses affected by intrauterine growth restriction have an increased risk of developing heart disease and failure in adulthood. Compared with controls, late gestation intrauterine growth-restricted (IUGR) fetal sheep have fewer binucleated cardiomyocytes, reflecting a more immature heart, which may reduce mitochondrial capacity to oxidize substrates. We hypothesized that the late gestation IUGR fetal heart has a lower capacity for mitochondrial oxidative phosphorylation. Left (LV) and right (RV) ventricles from IUGR and control (CON) fetal sheep at 90% gestation were harvested. Mitochondrial respiration (states 1-3, LeakOmy, and maximal respiration) in response to carbohydrates and lipids, citrate synthase (CS) activity, protein expression levels of mitochondrial oxidative phosphorylation complexes (CI-CV), and mRNA expression levels of mitochondrial biosynthesis regulators were measured. The carbohydrate and lipid state 3 respiration rates were lower in IUGR than CON, and CS activity was lower in IUGR LV than CON LV. However, relative CII and CV protein levels were higher in IUGR than CON; CV expression level was higher in IUGR than CON. Genes involved in lipid metabolism had lower expression in IUGR than CON. In addition, the LV and RV demonstrated distinct differences in oxygen flux and gene expression levels, which were independent from CON and IUGR status. Low mitochondrial respiration and CS activity in the IUGR heart compared with CON are consistent with delayed cardiomyocyte maturation, and CII and CV protein expression levels may be upregulated to support ATP production. These insights will provide a better understanding of fetal heart development in an adverse in utero environment. KEY POINTS: Growth-restricted fetuses have a higher risk of developing and dying from cardiovascular diseases in adulthood. Mitochondria are the main supplier of energy for the heart. As the heart matures, the substrate preference of the mitochondria switches from carbohydrates to lipids. We used a sheep model of intrauterine growth restriction to study the capacity of the mitochondria in the heart to produce energy using either carbohydrate or lipid substrates by measuring how much oxygen was consumed. Our data show that the mitochondria respiration levels in the growth-restricted fetal heart were lower than in the normally growing fetuses, and the expression levels of genes involved in lipid metabolism were also lower. Differences between the right and left ventricles that are independent of the fetal growth restriction condition were identified. These results indicate an impaired metabolic maturation of the growth-restricted fetal heart associated with a decreased capacity to oxidize lipids postnatally.

Chronic Fetal Leucine Infusion Increases Rate of Leucine Oxidation but Not of Protein Synthesis in Late Gestation Fetal Sheep.

BACKGROUND: Leucine increases protein synthesis rates in postnatal animals and adults. Whether supplemental leucine has similar effects in the fetus has not been determined. OBJECTIVE: To determine the effect of a chronic leucine infusion on whole-body leucine oxidation and protein metabolic rates, muscle mass, and regulators of muscle protein synthesis in late gestation fetal sheep. METHODS: Catheterized fetal sheep at ∼126 d of gestation (term = 147 d) received infusions of saline (CON, n = 11) or leucine (LEU; n = 9) adjusted to increase fetal plasma leucine concentrations by 50%-100% for 9 d. Umbilical substrate net uptake rates and protein metabolic rates were determined using a 1-13C leucine tracer. Myofiber myosin heavy chain (MHC) type and area, expression of amino acid transporters, and abundance of protein synthesis regulators were measured in fetal skeletal muscle. Groups were compared using unpaired t tests. RESULTS: Plasma leucine concentrations were 75% higher in LEU fetuses compared with CON by the end of the infusion period (P < 0.0001). Umbilical blood flow and uptake rates of most amino acids, lactate, and oxygen were similar between groups. Fetal whole-body leucine oxidation was 90% higher in LEU (P < 0.0005) but protein synthesis and breakdown rates were similar. Fetal and muscle weights and myofiber areas were similar between groups, however, there were fewer MHC type IIa fibers (P < 0.05), greater mRNA expression levels of amino acid transporters (P < 0.01), and a higher abundance of signaling proteins that regulate protein synthesis (P < 0.05) in muscle from LEU fetuses. CONCLUSIONS: A direct leucine infusion for 9 d in late gestation fetal sheep does not increase protein synthesis rates but results in higher leucine oxidation rates and fewer glycolytic myofibers. Increasing leucine concentrations in the fetus stimulates its own oxidation but also increases amino acid transporter expression and primes protein synthetic pathways in skeletal muscle.

Fetal Sex Does Not Impact Placental Blood Flow or Placental Amino Acid Transfer in Late Gestation Pregnant Sheep With or Without Placental Insufficiency.

Pregnant sheep have been used to model complications of human pregnancies including placental insufficiency and intrauterine growth restriction. Some of the hallmarks of placental insufficiency are slower uterine and umbilical blood flow rates, impaired placental transport of oxygen and amino acids, and lower fetal arterial concentrations of anabolic growth factors. An impact of fetal sex on these outcomes has not been identified in either human or sheep pregnancies. This is likely because most studies measuring these outcomes have used small numbers of subjects or animals. We undertook a secondary analysis of previously published data generated by our laboratory in late-gestation (gestational age of 133 ± 0 days gestational age) control sheep (n = 29 male fetuses; n = 26 female fetuses; n = 3 sex not recorded) and sheep exposed to elevated ambient temperatures to cause experimental placental insufficiency (n = 23 male fetuses; n = 17 female fetuses; n = 1 sex not recorded). The primary goal was to determine how fetal sex modifies the effect of the experimental insult on outcomes related to placental blood flow, amino acid and oxygen transport, and fetal hormones. Of the 112 outcomes measured, we only found an interaction between fetal sex and experimental insult for the uterine uptake rates of isoleucine, phenylalanine, and arginine. Additionally, most outcomes measured did not show a difference based on fetal sex when adjusting for the impact of placental insufficiency. Exceptions included fetal norepinephrine and cortisol concentrations, which were higher in female compared to male fetuses. For the parameters measured in the current analysis, the impact of fetal sex was not widespread.

Lower citrate synthase activity, mitochondrial complex expression, and fewer oxidative myofibers characterize skeletal muscle from growth-restricted fetal sheep.

Skeletal muscle from the late gestation sheep fetus with intrauterine growth restriction (IUGR) has evidence of reduced oxidative metabolism. Using a sheep model of placental insufficiency and IUGR, we tested the hypothesis that by late gestation, IUGR fetal skeletal muscle has reduced capacity for oxidative phosphorylation because of intrinsic deficits in mitochondrial respiration. We measured mitochondrial respiration in permeabilized muscle fibers from biceps femoris (BF) and soleus (SOL) from control and IUGR fetal sheep. Using muscles including BF, SOL, tibialis anterior (TA), and flexor digitorum superficialis (FDS), we measured citrate synthase (CS) activity, mitochondrial complex subunit abundance, fiber type distribution, and gene expression of regulators of mitochondrial biosynthesis. Ex vivo mitochondrial respiration was similar in control and IUGR muscle. However, CS activity was lower in IUGR BF and TA, indicating lower mitochondrial content, and protein expression of individual mitochondrial complex subunits was lower in IUGR TA and BF in a muscle-specific pattern. IUGR TA, BF, and FDS also had lower expression of type I oxidative fibers. Fiber-type shifts that support glycolytic instead of oxidative metabolism may be advantageous for the IUGR fetus in a hypoxic and nutrient-deficient environment, whereas these adaptions may be maladaptive in postnatal life.

A Two-Week Insulin Infusion in Intrauterine Growth Restricted Fetal Sheep at 75% Gestation Increases Skeletal Myoblast Replication but Did Not Restore Muscle Mass or Increase Fiber Number.

Intrauterine growth restricted (IUGR) fetuses are born with lower skeletal muscle mass, fewer proliferating myoblasts, and fewer myofibers compared to normally growing fetuses. Plasma concentrations of insulin, a myogenic growth factor, are lower in IUGR fetuses. We hypothesized that a two-week insulin infusion at 75% gestation would increase myoblast proliferation and fiber number in IUGR fetal sheep. Catheterized control fetuses received saline (CON-S, n=6), and the IUGR fetuses received either saline (IUGR-S, n=7) or insulin (IUGR-I, 0.014 ± 0.001 units/kg/hr, n=11) for 14 days. Fetal arterial blood gases and plasma amino acid levels were measured. Fetal skeletal muscles (biceps femoris, BF; and flexor digitorum superficialis, FDS) and pancreases were collected at necropsy (126 ± 2 dGA) for immunochemistry analysis, real-time qPCR, or flow cytometry. Insulin concentrations in IUGR-I and IUGR-S were lower vs. CON-S (P ≤ 0.05, group). Fetal arterial PaO2, O2 content, and glucose concentrations were lower in IUGR-I vs. CON-S (P ≤ 0.01) throughout the infusion period. IGF-1 concentrations tended to be higher in IUGR-I vs. IUGR-S (P=0.06), but both were lower vs. CON-S (P ≤ 0.0001, group). More myoblasts were in S/G2 cell cycle stage in IUGR-I vs. both IUGR-S and CON-S (145% and 113%, respectively, P ≤ 0.01). IUGR-I FDS muscle weighed 40% less and had 40% lower fiber number vs. CON-S (P ≤ 0.05) but were not different from IUGR-S. Myonuclear number per fiber and the mRNA expression levels of muscle regulatory factors were not different between groups. While the pancreatic ß-cell mass was lower in both IUGR-I and IUGR-S compared to CON-S, the IUGR groups were not different from each other indicating that feedback inhibition by endogenous insulin did not reduce ß-cell mass. A two-week insulin infusion at 75% gestation promoted myoblast proliferation in the IUGR fetus but did not increase fiber or myonuclear number. Myoblasts in the IUGR fetus retain the capacity to proliferate in response to mitogenic stimuli, but intrinsic defects in the fetal myoblast by 75% gestation may limit the capacity to restore fiber number.

Reduced glucose-stimulated insulin secretion following a 1-wk IGF-1 infusion in late gestation fetal sheep is due to an intrinsic islet defect.

Insulin and insulin-like growth factor-1 (IGF-1) are fetal hormones critical to establishing normal fetal growth. Experimentally elevated IGF-1 concentrations during late gestation increase fetal weight but lower fetal plasma insulin concentrations. We therefore hypothesized that infusion of an IGF-1 analog for 1 wk into late gestation fetal sheep would attenuate fetal glucose-stimulated insulin secretion (GSIS) and insulin secretion in islets isolated from these fetuses. Late gestation fetal sheep received infusions with IGF-1 LR3 (IGF-1, n = 8), an analog of IGF-1 with low affinity for the IGF binding proteins and high affinity for the IGF-1 receptor, or vehicle control (CON, n = 9). Fetal GSIS was measured with a hyperglycemic clamp (IGF-1, n = 8; CON, n = 7). Fetal islets were isolated, and insulin secretion was assayed in static incubations (IGF-1, n = 8; CON, n = 7). Plasma insulin and glucose concentrations in IGF-1 fetuses were lower compared with CON (P = 0.0135 and P = 0.0012, respectively). During the GSIS study, IGF-1 fetuses had lower insulin secretion compared with CON (P = 0.0453). In vitro, glucose-stimulated insulin secretion remained lower in islets isolated from IGF-1 fetuses (P = 0.0447). In summary, IGF-1 LR3 infusion for 1 wk into fetal sheep lowers insulin concentrations and reduces fetal GSIS. Impaired insulin secretion persists in isolated fetal islets indicating an intrinsic islet defect in insulin release when exposed to IGF-1 LR3 infusion for 1 wk. We speculate this alteration in the insulin/IGF-1 axis contributes to the long-term reduction in ß-cell function in neonates born with elevated IGF-1 concentrations following pregnancies complicated by diabetes or other conditions associated with fetal overgrowth.NEW & NOTEWORTHY After a 1-wk infusion of IGF-1 LR3, late gestation fetal sheep had lower plasma insulin and glucose concentrations, reduced fetal glucose-stimulated insulin secretion, and decreased fractional insulin secretion from isolated fetal islets without differences in pancreatic insulin content.

IGF-1 infusion to fetal sheep increases organ growth but not by stimulating nutrient transfer to the fetus.

Insulin-like growth factor-1 (IGF-1) is an important fetal growth factor. However, the role of fetal IGF-1 in increasing placental blood flow, nutrient transfer, and nutrient availability to support fetal growth and protein accretion is not well understood. Catheterized fetuses from late gestation pregnant sheep received an intravenous infusion of LR3 IGF-1 (LR3 IGF-1; n = 8) or saline (SAL; n = 8) for 1 wk. Sheep then underwent a metabolic study to measure uterine and umbilical blood flow, nutrient uptake rates, and fetal protein kinetic rates. By the end of the infusion, fetal weights were not statistically different between groups (SAL: 3.260 ± 0.211 kg, LR3 IGF-1: 3.682 ± 0.183; P = 0.15). Fetal heart, adrenal gland, and spleen weights were higher (P < 0.05), and insulin was lower in LR3 IGF-1 (P < 0.05). Uterine and umbilical blood flow and umbilical uptake rates of glucose, lactate, and oxygen were similar between groups. Umbilical amino acid uptake rates were lower in LR3 IGF-1 (P < 0.05) as were fetal concentrations of multiple amino acids. Fetal protein kinetic rates were similar. LR3 IGF-1 skeletal muscle had higher myoblast proliferation (P < 0.05). In summary, LR3 IGF-1 infusion for 1 wk into late gestation fetal sheep increased the weight of some fetal organs. However, because umbilical amino acid uptake rates and fetal plasma amino acid concentrations were lower in the LR3 IGF-1 group, we speculate that animals treated with LR3 IGF-1 can efficiently utilize available nutrients to support organ-specific growth in the fetus rather than by stimulating placental blood flow or nutrient transfer to the fetus.NEW & NOTEWORTHY After a 1-wk infusion of LR3 IGF-1, late gestation fetal sheep had lower umbilical uptake rates of amino acids, lower fetal arterial amino acid and insulin concentrations, and lower fetal oxygen content; however, LR-3 IGF-1-treated fetuses were still able to effectively utilize the available nutrients and oxygen to support organ growth and myoblast proliferation.

Coronary vascular growth matches IGF-1-stimulated cardiac growth in fetal sheep.

As loss of contractile function in heart disease could often be mitigated by increased cardiomyocyte number, expansion of cardiomyocyte endowment paired with increased vascular supply is a desirable therapeutic goal. Insulin-like growth factor 1 (IGF-1) administration increases fetal cardiomyocyte proliferation and heart mass, but how fetal IGF-1 treatment affects coronary growth and function is unknown. Near-term fetal sheep underwent surgical instrumentation and were studied from 127 to 134 d gestation (term = 147 d), receiving either IGF-1 LR3 or vehicle. Coronary growth and function were interrogated using pressure-flow relationships, an episode of acute hypoxia with progressive blockade of adenosine receptors and nitric oxide synthase, and by modeling the determinants of coronary flow. The main findings were that coronary conductance was preserved on a per-gram basis following IGF-1 treatment, adenosine and nitric oxide contributed to hypoxia-mediated coronary vasodilation similarly in IGF-1-treated and Control fetuses, and the relationships between coronary flow and blood oxygen contents were similar between groups. We conclude that IGF-1-stimulated fetal myocardial growth is accompanied by appropriate expansion and function of the coronary vasculature. These findings support IGF-1 as a potential strategy to increase cardiac myocyte and coronary vascular endowment at birth.

Rates of myogenesis and myofiber numbers are reduced in late gestation IUGR fetal sheep.

Intrauterine growth-restricted (IUGR) fetuses are born with reduced skeletal muscle mass. We hypothesized that reduced rates of myogenesis would contribute to fewer and smaller myofibers in IUGR fetal hindlimb muscle compared to the normally growing fetus. We tested this hypothesis in IUGR fetal sheep with progressive placental insufficiency produced by exposing pregnant ewes to elevated ambient temperatures from 38 to 116 days gestation (dGA; term = 147 dGA). Surgically catheterized control (CON, n = 8) and IUGR (n = 13) fetal sheep were injected with intravenous 5-bromo-2'-deoxyuridine (BrdU) prior to muscle collection (134 dGA). Rates of myogenesis, defined as the combined processes of myoblast proliferation, differentiation, and fusion into myofibers, were determined in biceps femoris (BF), tibialis anterior (TA), and flexor digitorum superficialis (FDS) muscles. Total myofiber number was determined for the entire cross-section of the FDS muscle. In IUGR fetuses, the number of BrdU+ myonuclei per myofiber cross-section was lower in BF, TA, and FDS (P < 0.05), total myonuclear number per myofiber cross-section was lower in BF and FDS (P < 0.05), and total myofiber number was lower in FDS (P < 0.005) compared to CON. mRNA expression levels of cyclins, cyclin-dependent protein kinases, and myogenic regulatory factors were lower (P < 0.05), and inhibitors of the cell cycle were higher (P < 0.05) in IUGR BF compared to CON. Markers of apoptosis were not different in IUGR BF muscle. These results show that in IUGR fetuses, reduced rates of myogenesis produce fewer numbers of myonuclei, which may limit hypertrophic myofiber growth. Fewer myofibers of smaller size contribute to smaller muscle mass in the IUGR fetus.

Skeletal muscle amino acid uptake is lower and alanine production is greater in late gestation intrauterine growth-restricted fetal sheep hindlimb.

In a sheep model of intrauterine growth restriction (IUGR) produced from placental insufficiency, late gestation fetuses had smaller skeletal muscle mass, myofiber area, and slower muscle protein accretion rates compared with normally growing fetuses. We hypothesized that IUGR fetal muscle develops adaptations that divert amino acids (AAs) from protein accretion and activate pathways that conserve substrates for other organs. We placed hindlimb arterial and venous catheters into late gestation IUGR (n = 10) and control (CON, n = 8) fetal sheep and included an external iliac artery flow probe to measure hindlimb AA uptake rates. Arterial and venous plasma samples and biceps femoris muscle were analyzed by mass spectrometry-based metabolomics. IUGR fetuses had greater abundance of metabolites enriched within the alanine, aspartate, and glutamate metabolism pathway compared with CON. Net uptake rates of branched-chain AA (BCAA) were lower by 42%-73%, and muscle ammoniagenic AAs (alanine, glycine, and glutamine) were lower by 107%-158% in IUGR hindlimbs versus CON. AA uptake rates correlated with hindlimb weight; the smallest hindlimbs showed net release of ammoniagenic AAs. Gene expression levels indicated a decrease in BCAA catabolism in IUGR muscle. Plasma purines were lower and plasma uric acid was higher in IUGR versus CON, possibly a reflection of ATP conservation. We conclude that IUGR skeletal muscle has lower BCAA uptake and develops adaptations that divert AAs away from protein accretion into alternative pathways that sustain global energy production and nitrogen disposal in the form of ammoniagenic AAs for metabolism in other organs.

A 1 week IGF-1 infusion decreases arterial insulin concentrations but increases pancreatic insulin content and islet vascularity in fetal sheep.

Fetal insulin is critical for regulation of growth. Insulin concentrations are partly determined by the amount of ß-cells present and their insulin content. Insulin-like growth factor-1 (IGF-1) is a fetal anabolic growth factor which also impacts ß-cell mass in models of ß-cell injury and diabetes. The extent to which circulating concentrations of IGF-1 impact fetal ß-cell mass and pancreatic insulin content is unknown. We hypothesized that an infusion of an IGF-1 analog for 1 week into the late gestation fetal sheep circulation would increase ß-cell mass, pancreatic islet size, and pancreatic insulin content. After the 1-week infusion, pancreatic insulin concentrations were 80% higher than control fetuses (P < 0.05), but there were no differences in ß-cell area, ß-cell mass, or pancreatic vascularity. However, pancreatic islet vascularity was 15% higher in IGF-1 fetuses and pancreatic VEGFA, HGF, IGF1, and IGF2 mRNA expressions were 70-90% higher in IGF-1 fetuses compared to control fetuses (P < 0.05). Plasma oxygen, glucose, and insulin concentrations were 25%, 22%, and 84% lower in IGF-1 fetuses, respectively (P < 0.05). The previously described role for IGF-1 as a ß-cell growth factor may be more relevant for local paracrine signaling in the pancreas compared to circulating endocrine signaling.

Effects of ketamine on the fetal transcriptomic response to umbilical cord occlusion: comparison with hypoxic hypoxia in the cerebral cortex.

KEY POINTS: The cerebral response to fetal asphyxia is characterized by an upregulation of nucleic acid and chromatin modification processes, as well as a downregulation of metabolic processes at 1 h post-umbilical cord occlusion (UCO). Twenty-four hours post UCO, there was an upregulation of metabolic processes and protein modifications. UCO did not alter bacterial gene expression levels, nor did it produce a robust inflammatory response compared to maternal hypoxia. The administration of ketamine produced minimal effects on the fetal response to UCO in the cerebral cortex. ABSTRACT: Umbilical cord occlusion (UCO) is known to cause neurological disorders in the neonate. Previously, we have reported that hypoxic hypoxia (HH) stimulates the appearance of bacteria in the fetal brain and upregulates the expression of inflammatory markers in fetal cerebral cortex (CTX) and also that ketamine attenuates these responses. In the present study, we aimed to test the hypothesis that UCO, similar to HH, produces an inflammatory response in the fetal CTX and also that treatment with ketamine reduces these effects. In chronically instrumented fetal sheep (∼125 days), 30 min of partial UCO decreased fetal PaO2 levels by ∼50%. Half of the fetuses received ketamine (3 mg kg-1 ) 10 min prior to UCO (n = 4 per group). Fetal brains were collected 1 and 24 h after the experiment and mRNA was extracted and hybridized for microarray analyses. Differentially-expressed genes were analysed for significant association with gene ontologies and pathways. After 1 h, UCO upregulated nucleic acid processing and chromatin modification and downregulated metabolic processes compared to control. After 24 h, UCO upregulated metabolic and protein modification processes. Ketamine produced minimal effects. UCO did not alter the abundance of bacterial DNA in fetal brain, nor did it upregulate inflammation pathways compared to HH. We conclude that UCO produced time-dependent responses that did not include bacterial invasion or upregulation of inflammation pathways in fetal CTX. This contrasts with the response to HH, which resulted in the appearance of bacteria in the CTX and upregulated inflammation pathways. These responses in fetal CTX to oxygen deprivation are therefore modified by the maternal or placental response to the stimulus.

Ketamine Reduces Inflammation Pathways in the Hypothalamus and Hippocampus Following Transient Hypoxia in the Late-Gestation Fetal Sheep.

The physiological response to hypoxia in the fetus has been extensively studied with regard to redistribution of fetal combined ventricular output and sparing of oxygen delivery to fetal brain and heart. Previously, we have shown that the fetal brain is capable of mounting changes in gene expression that are consistent with tissue inflammation. The present study was designed to use transcriptomics and systems biology modeling to test the hypothesis that ketamine reduces or prevents the upregulation of inflammation-related pathways in hypothalamus and hippocampus after transient hypoxic hypoxia. Chronically catheterized fetal sheep (122 ± 5 days gestation) were subjected to 30 min hypoxia (relative reduction in PaO2∼50%) caused by infusion of nitrogen into the inspired gas of the pregnant ewe. RNA was isolated from fetal hypothalamus and hippocampus collected 24 h after hypoxia, and was analyzed for gene expression using the Agilent 15.5 k ovine microarray. Ketamine, injected 10 min prior to hypoxia, reduced the cerebral immune response activation to the hypoxia in both brain regions. Genes both upregulated by hypoxia and downregulated by ketamine after hypoxia were significantly associated with gene ontology terms and KEGG pathways that are, themselves, associated with the tissue response to exposure to bacteria. We conclude that the results are consistent with interruption of the cellular response to bacteria by ketamine.

Post-hypoxia Invasion of the fetal brain by multidrug resistant Staphylococcus.

Herein we describe an association between activation of inflammatory pathways following transient hypoxia and the appearance of the multidrug resistant bacteria Staphylococcus simulans in the fetal brain. Reduction of maternal arterial oxygen tension by 50% over 30 min resulted in a subseiuent significant over-expression of genes associated with immune responses 24 h later in the fetal brain. The activated genes were consistent with stimulation by bacterial lipopolysaccharide; an influx of macrophages and appearance of live bacteria were found in these fetal brains. S. simulans was the predominant bacterial species in fetal brain after hypoxia, but was found in placenta of all animals. Strains of S. simulans from the placenta and fetal brain were equally highly resistant to multiple antibiotics including methicillin and had identical genome sequences. These results suggest that bacteria from the placenta invade the fetal brain after maternal hypoxia.

Ketamine modulates fetal hemodynamic and endocrine responses to umbilical cord occlusion.

Umbilical cord occlusion (UCO) is a hypoxic insult that has been used to model birth asphyxia and umbilical cord compression in utero. UCO triggers vigorous neural and endocrine responses that include increased plasma ACTH and cortisol concentrations, increased blood pressure (BP), and decreased heart rate (HR). We have previously reported that ketamine, a noncompetitive N-methyl-D-aspartate receptor antagonist, can modify the fetal hemodynamic and ACTH responses to ventilatory hypoxia and cerebral ischemia-reperfusion. We performed the present experiments to test the hypothesis that ketamine has similar effects on the neuroendocrine and cardiovascular responses to UCO Fetal sheep were chronically catheterized at gestational day 125. Ketamine (3 mg/kg) was administered intravenously to the fetus 10 min prior to the insult. UCO was induced for 30 min by reducing the umbilical vein blood flow until fetal PaO2 levels were reduced from 17 ± 1 to 11 ± 1 mm Hg. UCO produced an initial increase on fetal BP in both control and ketamine groups (P = 0.018 time), followed by a decrease in the control group, but values remained higher with ketamine. HR decreased after UCO (P = 0.041 stimulus*time) in both groups, but the reduction was greater initially in control compared to ketamine groups. Fetal PaCO2 levels increased after UCO (P < 0.01 stimulus*time), but values were higher in the control versus ketamine groups. UCO significantly decreased fetal pH values (P < 0.01 stimulus*time) with a greater effect on the control versus ketamine group. Ketamine delayed the cortisol responses to UCO (P < 0.001 stimulus*time), and UCO produced a robust increase in ACTH levels from 19 ± 2 to 280 ± 27 pg/mL (P < 0.001 stimulus*time), but there were no differences in ACTH levels between UCO groups. We conclude that ketamine augmented the cardiovascular response to UCO, but did not alter the ACTH response to UCO.

Genomic Effect of Triclosan on the Fetal Hypothalamus: Evidence for Altered Neuropeptide Regulation.

Triclosan (TCS), an antibacterial compound commonly added to personal care products, could be an endocrine disruptor at low doses. Although TCS has been shown to alter fetal physiology, its effects in the developing fetal brain are unknown. We hypothesize that exposure to TCS during fetal life could affect fetal hypothalamic gene expression. The objective of this study was to use transcriptomics and systems analysis to identify significantly altered biological processes in the late gestation ovine fetal hypothalamus after direct or indirect exposure to low doses of TCS. For direct TCS exposure, chronically catheterized late gestation fetal sheep were infused with vehicle (n = 4) or TCS (250 µg/d; n = 4) iv. For indirect TCS exposure, TCS (100 µg/kg · d; n = 3) or vehicle (n = 3) was infused into the maternal circulation. Fetal hypothalami were collected after 2 days of infusion, and gene expression was measured through microarray. Hierarchical clustering of all samples according to gene expression profiles showed that samples from the TCS-treated animals clustered apart from the controls. Gene set enrichment analysis revealed that fetal hypothalamic genes stimulated by maternal and fetal TCS infusion were significantly enriching for cell cycle, reproductive process, and feeding behavior, whereas the inhibited genes were significantly enriching for chromatin modification and metabolism of steroids, lipoproteins, fatty acids, and glucose (P < .05). In conclusion, short-term infusion of TCS induces vigorous changes in the fetal hypothalamic transcriptomics, which are mainly related to food intake pathways and metabolism. If these changes persist to postnatal life, they could result in adverse consequences in adulthood.

Ketamine decreases inflammatory and immune pathways after transient hypoxia in late gestation fetal cerebral cortex.

Transient hypoxia in pregnancy stimulates a physiological reflex response that redistributes blood flow and defends oxygen delivery to the fetal brain. We designed the present experiment to test the hypotheses that transient hypoxia produces damage of the cerebral cortex and that ketamine, an antagonist ofNMDAreceptors and a known anti-inflammatory agent, reduces the damage. Late gestation, chronically catheterized fetal sheep were subjected to a 30-min period of ventilatory hypoxia that decreased fetal PaO2from 17 ± 1 to 10 ± 1 mmHg, or normoxia (PaO217 ± 1 mmHg), with or without pretreatment (10 min before hypoxia/normoxia) with ketamine (3 mg/kg, i.v.). One day (24 h) after hypoxia/normoxia, fetal cerebral cortex was removed andmRNAextracted for transcriptomics and systems biology analysis (n = 3-5 per group). Hypoxia stimulated a transcriptomic response consistent with a reduction in cellular metabolism and an increase in inflammation. Ketamine pretreatment reduced both of these responses. The inflammation response modeled with transcriptomic systems biology was validated by immunohistochemistry and showed increased abundance of microglia/macrophages after hypoxia in the cerebral cortical tissue that ketamine significantly reduced. We conclude that transient hypoxia produces inflammation of the fetal cerebral cortex and that ketamine, in a standard clinical dose, reduces the inflammation response.

Transcriptomics Modeling of the Late-Gestation Fetal Pituitary Response to Transient Hypoxia.

BACKGROUND: The late-gestation fetal sheep responds to hypoxia with physiological, neuroendocrine, and cellular responses that aid in fetal survival. The response of the fetus to hypoxia represents a coordinated effort to maximize oxygen transfer from the mother and minimize wasteful oxygen consumption by the fetus. While there have been many studies aimed at investigating the coordinated physiological and endocrine responses to hypoxia, and while immunohistochemical or in situ hybridization studies have revealed pathways supporting the endocrine function of the pituitary, there is little known about the coordinated cellular response of the pituitary to the hypoxia. RESULTS: Thirty min hypoxia (from 17.0±1.7 to 8.0±0.8 mm Hg, followed by 30 min normoxia) upregulated 595 and downregulated 790 genes in fetal pituitary (123-132 days' gestation; term = 147 days). Network inference of up- and down- regulated genes revealed a high degree of functional relatedness amongst the gene sets. Gene ontology analysis revealed upregulation of cellular metabolic processes (e.g., RNA synthesis, response to estrogens) and downregulation of protein phosphorylation, protein metabolism, and mitosis. Genes found to be at the center of the network of upregulated genes included genes important for purine binding and signaling. At the center of the downregulated network were genes involved in mRNA processing, DNA repair, sumoylation, and vesicular trafficking. Transcription factor analysis revealed that both up- and down-regulated gene sets are enriched for control by several transcription factors (e.g., SP1, MAZ, LEF1, NRF1, ELK1, NFAT, E12, PAX4) but not for HIF-1, which is known to be an important controller of genomic responses to hypoxia. CONCLUSIONS: The multiple analytical approaches used in this study suggests that the acute response to 30 min of transient hypoxia in the late-gestation fetus results in reduced cellular metabolism and a pattern of gene expression that is consistent with cellular oxygen and ATP starvation. In this early time point, we see a vigorous gene response. But, like the hypothalamus, the transcriptomic response is not consistent with mediation by HIF-1. If HIF-1 is a significant controller of gene expression in the fetal pituitary after hypoxia, it must be at a later time.

Ketamine suppresses hypoxia-induced inflammatory responses in the late-gestation ovine fetal kidney cortex.

Acute fetal hypoxia is a form of fetal stress that stimulates renal vasoconstriction and ischaemia as a consequence of the physiological redistribution of combined ventricular output. Because of the potential ischaemia-reperfusion injury to the kidney, we hypothesized that it would respond to hypoxia with an increase in the expression of inflammatory genes, and that ketamine (an N-methyl-D-aspartate receptor antagonist) would reduce or block this response. Hypoxia was induced for 30 min in chronically catheterized fetal sheep (125 ± 3 days), with or without ketamine (3 mg kg(-1)) administered intravenously to the fetus 10 min prior to hypoxia. Gene expression in fetal kidney cortex collected 24 h after the onset of hypoxia was analysed using ovine Agilent 15.5k array and validated with qPCR and immunohistochemistry in four groups of ewes: normoxic control, normoxia + ketamine, hypoxic control and hypoxia + ketamine (n = 3-4 per group). Significant differences in gene expression between groups were determined with t-statistics using the limma package for R (P ≤ 0.05). Enriched biological processes for the 427 upregulated genes were immune and inflammatory responses and for the 946 downregulated genes were metabolic processes. Ketamine countered the effects of hypoxia on upregulated immune/inflammatory responses as well as the downregulated metabolic responses. We conclude that our transcriptomics modelling predicts that hypoxia activates inflammatory pathways and reduces metabolism in the fetal kidney cortex, and ketamine blocks or ameliorates this response. The results suggest that ketamine may have therapeutic potential for protection from ischaemic renal damage.