Gestational long-term hypoxia induces metabolomic reprogramming and phenotypic transformations in fetal sheep pulmonary arteries.

Gestational long-term hypoxia increases the risk of myriad diseases in infants including persistent pulmonary hypertension. Similar to humans, fetal lamb lung development is susceptible to long-term intrauterine hypoxia, with structural and functional changes associated with the development of pulmonary hypertension including pulmonary arterial medial wall thickening and dysregulation of arterial reactivity, which culminates in decreased right ventricular output. To further explore the mechanisms associated with hypoxia-induced aberrations in the fetal sheep lung, we examined the premise that metabolomic changes and functional phenotypic transformations occur due to intrauterine, long-term hypoxia. To address this, we performed electron microscopy, Western immunoblotting, calcium imaging, and metabolomic analyses on pulmonary arteries isolated from near-term fetal lambs that had been exposed to low- or high-altitude (3,801 m) hypoxia for the latter 110+ days of gestation. Our results demonstrate that the sarcoplasmic reticulum was swollen with high luminal width and distances to the plasma membrane in the hypoxic group. Hypoxic animals were presented with higher endoplasmic reticulum stress and suppressed calcium storage. Metabolically, hypoxia was associated with lower levels of multiple omega-3 polyunsaturated fatty acids and derived lipid mediators (e.g., eicosapentaenoic acid, docosahexaenoic acid, α-linolenic acid, 5-hydroxyeicosapentaenoic acid (5-HEPE), 12-HEPE, 15-HEPE, prostaglandin E3, and 19(20)-epoxy docosapentaenoic acid) and higher levels of some omega-6 metabolites (P < 0.02) including 15-keto prostaglandin E2 and linoleoylglycerol. Collectively, the results reveal broad evidence for long-term hypoxia-induced metabolic reprogramming and phenotypic transformations in the pulmonary arteries of fetal sheep, conditions that likely contribute to the development of persistent pulmonary hypertension.

Hypoxia-induced inhibition of mTORC1 activity in the developing lung: a possible mechanism for the developmental programming of pulmonary hypertension.

Perinatal hypoxia induces permanent structural and functional changes in the lung and its pulmonary circulation that are associated with the development of pulmonary hypertension (PH) in later life. The mechanistic target of the rapamycin (mTOR) pathway is vital for fetal lung development and is implicated in hypoxia-associated PH, yet its involvement in the developmental programming of PH remains unclear. Pregnant C57/BL6 dams were placed in hyperbaric (760 mmHg) or hypobaric chambers during gestation (505 mmHg, day 15 through postnatal day 4) or from weaning through adulthood (420 mmHg, postnatal day 21 through 8 wk). Pulmonary hemodynamics and right ventricular systolic pressure (RVSP) were measured at 8 wk. mTOR pathway proteins were assessed in fetal (day 18.5) and adult lung (8 wk). Perinatal hypoxia induced PH during adulthood, even in the absence of a sustained secondary hypoxic exposure, as indicated by reduced pulmonary artery acceleration time (PAAT) and peak flow velocity through the pulmonary valve, as well as greater RVSP, right ventricular (RV) wall thickness, and RV/left ventricular (LV) weight. Such effects were independent of increased blood viscosity. In fetal lung homogenates, hypoxia reduced the expression of critical downstream mTOR targets, most prominently total and phosphorylated translation repressor protein (4EBP1), as well as vascular endothelial growth factor, a central regulator of angiogenesis in the fetal lung. In contrast, adult offspring of hypoxic dams tended to have elevated p4EBP1 compared with controls. Our data suggest that inhibition of mTORC1 activity in the fetal lung as a result of gestational hypoxia may interrupt pulmonary vascular development and thereby contribute to the developmental programming of PH.NEW & NOTEWORTHY We describe the first study to evaluate a role for the mTOR pathway in the developmental programming of pulmonary hypertension. Our findings suggest that gestational hypoxia impairs mTORC1 activation in the fetal lung and may impede pulmonary vascular development, setting the stage for pulmonary vascular disease in later life.

Thalamic mediation of hypoxic respiratory depression in lambs.

Immaturity of respiratory controllers in preterm infants dispose to recurrent apnea and oxygen deprivation. Accompanying reductions in brain oxygen tensions evoke respiratory depression, potentially exacerbating hypoxemia. Central respiratory depression during moderate hypoxia is revealed in the ventilatory decline following initial augmentation. This study determined whether the thalamic parafascicular nuclear (Pf) complex involved in adult nociception and sensorimotor regulation (Bentivoglio M, Balerecia G, Kruger L. Prog Brain Res 87: 53-80, 1991) also becomes a postnatal controller of hypoxic ventilatory decline. Respiratory responses to moderate isocapnic hypoxia were studied in conscious lambs. Hypoxic ventilatory decline was compared with peak augmentation. Pf and/or adjacent thalamic structures were destroyed by the neuron-specific toxin ibotenic acid (IB). IB lesions involving the thalamic Pf abolished hypoxic ventilatory decline. Lesions of adjacent thalamic nuclei that spared Pf and control injections of vehicle failed to blunt hypoxic respiratory depression. Our findings reveal that the thalamic Pf region is a critical controller of hypoxic ventilatory depression and thus a key target for exploring molecular concomitants of forebrain pathways regulating hypoxic ventilatory depression in early development.

Xanthine oxidase and the fetal cardiovascular defence to hypoxia in late gestation ovine pregnancy.

Hypoxia is a common challenge to the fetus, promoting a physiological defence to redistribute blood flow towards the brain and away from peripheral circulations. During acute hypoxia, reactive oxygen species (ROS) interact with nitric oxide (NO) to provide an oxidant tone. This contributes to the mechanisms redistributing the fetal cardiac output, although the source of ROS is unknown. Here, we investigated whether ROS derived from xanthine oxidase (XO) contribute to the fetal peripheral vasoconstrictor response to hypoxia via interaction with NO-dependent mechanisms. Pregnant ewes and their fetuses were surgically prepared for long-term recording at 118 days of gestation (term approximately 145 days). After 5 days of recovery, mothers were infused i.v. for 30 min with either vehicle (n = 11), low dose (30 mg kg(-1), n = 5) or high dose (150 mg kg(-1), n = 9) allopurinol, or high dose allopurinol with fetal NO blockade (n = 6). Following allopurinol treatment, fetal hypoxia was induced by reducing maternal inspired O2 such that fetal basal P aO 2 decreased approximately by 50% for 30 min. Allopurinol inhibited the increase in fetal plasma uric acid and suppressed the fetal femoral vasoconstrictor, glycaemic and lactate acidaemic responses during hypoxia (all P < 0.05), effects that were restored to control levels with fetal NO blockade. The data provide evidence for the activation of fetal XO in vivo during hypoxia and for XO-derived ROS in contributing to the fetal peripheral vasoconstriction, part of the fetal defence to hypoxia. The data are of significance to the understanding of the physiological control of the fetal cardiovascular system during hypoxic stress. The findings are also of clinical relevance in the context of obstetric trials in which allopurinol is being administered to pregnant women when the fetus shows signs of hypoxic distress.

Vitamin C prevents intrauterine programming of in vivo cardiovascular dysfunction in the rat.

BACKGROUND: Fetal hypoxia is common and in vitro evidence supports its role in the programming of adult cardiovascular dysfunction through the generation of oxidative stress. Whether fetal chronic hypoxia programmes alterations in cardiovascular control in vivo, and if these alterations can be prevented by antioxidant treatment, is unknown. This study investigated the effects of prenatal fetal hypoxia, with and without maternal supplementation with vitamin C, on basal and stimulated cardiovascular function in vivo in the adult offspring at 4 months of age in the rat. METHODS AND RESULTS: From days 6 to 20 of pregnancy, Wistar rats were subjected to Normoxia, Hypoxia (13% O2), Hypoxia+Vitamin C (5mg/ml in drinking water) or Normoxia+Vitamin C. At 4 months, male offspring were instrumented under urethane anaesthesia. Basal mean arterial blood pressure, heart rate and heart rate variability (HRV) were assessed, and stimulated baroreflex curves were generated with phenylephrine and sodium nitroprusside. Chronic fetal hypoxia increased the LF/HF HRV ratio and baroreflex gain, effects prevented by vitamin C administration during pregnancy. CONCLUSIONS: Chronic intrauterine hypoxia programmes cardiovascular dysfunction in vivo in adult rat offspring; effects ameliorated by maternal treatment with vitamin C. The data support a role for fetal chronic hypoxia programming cardiovascular dysfunction in the adult rat offspring in vivo through the generation of oxidative stress in utero.

A maternal diet supplemented with creatine from mid-pregnancy protects the newborn spiny mouse brain from birth hypoxia.

The creatine-phosphocreatine shuttle is essential for the maintenance of cellular ATP, particularly under hypoxic conditions when respiration may become anaerobic. Using a model of intrapartum hypoxia in the precocial spiny mouse (Acomys cahirinus), the present study assessed the potential for maternal creatine supplementation during pregnancy to protect the developing brain from the effects of birth hypoxia. On day 38 of gestation (term is 39 days), the pregnant uterus was isolated and placed in a saline bath for 7.5 min, inducing global hypoxia. The pups were then removed, resuscitated, and cross-fostered to a nursing dam. Control offspring were delivered by caesarean section and recovered immediately after release from the uterus. At 24 h after birth hypoxia, the brains of offspring from dams fed a normal diet showed significant increases in lipid peroxidation as measured by the amount of malondialdehyde. In the cortical subplate, thalamus and piriform cortex there were significant increases in cellular expression of the pro-apoptotic protein BAX, cytoplasmic cytochrome c and caspase-3. When pregnant dams were fed the creatine supplemented diet, the increase in malondialdehyde, BAX, cytochrome c and caspase 3 were almost completely prevented, such that they were not different from control (caesarean-delivered) neonates. This study provides evidence that the neuroprotective capacity of creatine in the hypoxic perinatal brain involves abrogation of lipid peroxidation and apoptosis, possibly through the maintenance of mitochondrial function. Further investigation into these mechanisms of protection, and the long-term development and behavioural outcomes of such neonates is warranted.

Maternal creatine supplementation from mid-pregnancy protects the diaphragm of the newborn spiny mouse from intrapartum hypoxia-induced damage.

We hypothesized that maternal creatine supplementation from mid-pregnancy would protect the diaphragm of the newborn spiny mouse from the effects of intrapartum hypoxia. Pregnant mice were fed a control or 5% creatine-supplemented diet from mid-gestation. On the day before term, intrapartum hypoxia was induced by isolating the pregnant uterus in a saline bath for 7.5-8 min before releasing and resuscitating the fetuses. Surviving pups were placed with a cross-foster dam, and diaphragm tissue was collected at 24 h postnatal age. Hypoxia caused a significant decrease in the cross-sectional area (∼19%) and contractile function (26.6% decrease in maximum Ca2=-activated force) of diaphragm fibers. The mRNA levels of the muscle mass-regulating genes MuRF1 and myostatin were significantly increased (2-fold). Maternal creatine significantly attenuated hypoxia-induced fiber atrophy, contractile dysfunction, and changes in mRNA levels. This study demonstrates that creatine loading before birth significantly protects the diaphragm from hypoxia-induced damage at birth.

Fetal hypoxemia on a molecular level: adaptive changes in the hypothalamic-pituitary-adrenal (HPA) axis and the lungs.

The development of diseases in later life, such as diabetes type II, hypertension and cardiovascular disease, is linked to abnormal intrauterine conditions that reduce birth weight. Obviously, fetal development can be disturbed so profoundly, that fetal programming is changed permanently. We have examined the effects of hypoxia, or more precisely hypoxemia, on the fetal hypothalamic-pituitary-adrenal (HPA) axis and lungs using molecular biology techniques in order to elucidate the underlying mechanisms. Chronically catheterized fetal sheep were subjected to a hypoxemia (48 h) without change in arterial pH or paCO2. Major changes occurred, although the degree of hypoxemia was just moderate. There was a transient increase in the fetal plasma ACTH-concentrations with an upregulation of the cortisol-concentrations, which was more pronounced in the older, hypoxemic fetuses (134-136 days of gestation) than in the younger, hypoxemic animals (126-130 days of gestation; term is 145 days). There was an unique, differential regulation for pro-opiomelanocortin messenger RNA (mRNA), the precursor molecule of e.g. ACTH, in the pars distalis and pars intermedia of the pituitary gland. This finding supported the increased bioactivity besides the increased concentrations for ACTH. Simultaneously, there was an increase in the mRNAs of the ACTH-receptor and of the steroid-synthesizing enzymes in the fetal adrenal gland of the older, hypoxemic fetuses. No changes in the fetal plasma androstenedione-concentrations were observed. Clearly, there was a selective increase of the cortisol-synthesis. Growth and maturation of the fetal lung might also have been affected, because of the increase in surfactant-protein A mRNA in the older, hypoxemic animals and the decrease in the insulin-like growth factor-I and its binding protein-5 mRNA in the younger, hypoxemic fetuses. In summary, even a moderate degree of hypoxemia was shown to affect the different levels of fetal organism profoundly, offering a pathophysiological basis for changes in fetal development.

Effect of antalarmin, a novel corticotropin-releasing hormone antagonist, on the dynamic function of the preterm ovine fetal hypothalamo-pituitary-adrenal axis.

This study describes the effect of antalarmin on basal and stimulated activity of the hypothalamo-pituitary-adrenal (HPA) axis function in the late gestation ovine fetus. Fetuses received antalarmin (15 mg/h i.v.) or vehicle (cremophor El 50% in ethanol) from day 130 gestational age. Antalarmin infusion did not significantly affect immunoreactive corticotropin (ir-ACTH) concentrations, although there was a tendency for ir-ACTH to be lower and cortisol concentrations were lower in the antalarmin-treated fetuses (p < 0.01). The ir-ACTH response to corticotropin-releasing hormone (CRH) challenge was attenuated (p < 0.05) in the antalarmin-treated fetuses, but neither antalarmin- nor vehicle-treated fetuses had significant cortisol responses to CRH. The ir-ACTH response to hypoxia was diminished (p < 0.05) in the antalarmin-treated fetuses while the cortisol responses of antalarmin- and vehicle-treated fetuses were indistinguishable. Deconvolution analysis revealed no effect of antalarmin treatment on ir-ACTH secretory dynamics. In contrast, antalarmin decreased (p < 0.05) basal, mean and integrated cortisol. The plasma cortisol responses of antalarmin- and vehicle-treated fetuses to exogenous ACTH(1-24) were indistinguishable. These data indicate that, while antalarmin inhibits CRH- and stress-induced ir-ACTH secretion, basal ir-ACTH secretion may be less affected by antalarmin treatment. Paradoxically, cortisol secretion is impaired by antalarmin infusion, although adrenal responsiveness to ACTH is not impaired. These results confirm a role for CRH in stress-induced ACTH secretion in the ovine fetus, though its role in the regulation of basal ACTH and cortisol secretion is unclear.

Hyperthermia prevents metabolic and cerebral flow responses to hypoxia in the fetal sheep.

OBJECTIVE: Fetal brain temperature has been found to decrease during hypoxia, strongly suggesting a reduction in cerebral O2 consumption and increases in cerebral blood flow. These responses may protect the brain in part against hypoxic injury. This study was undertaken to examine whether these compensatory mechanisms are lost during fetal hyperthermia. METHODS: Intermittent fetal hypoxemia was induced by administering low-O2 gas mixtures to nine near-term ewes. Fetal brain and body core temperature responses were measured with and without fetal hyperthermia induced by circulating warm water through a plastic coil looped about the fetus in utero. RESULTS: In normothermic fetuses, fetal brain temperature relative to core decreased during a 30-minute period of hypoxia and then returned to normal during recovery. This response may be explained by a combination of cerebral hypometabolism and increased cerebral blood flow. However, in hyperthermic fetuses (intrauterine warming for 1 hour, raising body core and brain temperatures 0.66 +/- 0.06 and 0.61 +/- 0.10 C, respectively) a subsequent period of hypoxia no longer induced a reduction in brain temperature relative to body core. CONCLUSION: When temperature of the fetal sheep is elevated, as may occur with maternal fever, prolonged exercise, and elevated environmental temperatures, the fetal brain is less well protected against hypoxic injury.

Erythropoietin in obstetrics.

Our objective was to discuss the role of erythropoietin in fetal erythropoiesis and to review its clinical uses in perinatal medicine. All relevant articles compiled through a MEDLINE search (years 1986-1997) were reviewed. Erythropoietin is essential for fetal erythropoiesis and is produced in response to hypoxia and anemia. Cord blood erythropoietin is purely fetal and reflects tissue oxygenation. It has been found to be increased in many complicated pregnancies with underlying fetal hypoxia. Erythropoietin could be used as a marker of fetal hypoxia because its concentration rises rapidly by increased production in response to hypoxia. Its measurement might enable more accurate timing of hypoxic injury. In addition, erythropoietin levels have been well correlated with perinatal brain damage and may facilitate treatment of high risk neonates. Erythropoietin has also been used successfully in anemia of prematurity, decreasing the transfusion requirement. However, studies are still needed to determine the optimal doses of erythropoietin and iron supplementations required for maximizing the red blood cell response. Erythropoietin has been examined as potential maternal therapy in various disorders during pregnancy. These include end-stage renal disease, severe antepartum iron deficiency anemia, and postpartum anemia. Erythropoietin has been found to be effective and well tolerated in these conditions. An additional promising use lies in the optimization of maternal red blood cell mass to allow autologous blood donation. This may be critical in cases where a large amount of bleeding might be anticipated, as with placenta previa. This would also minimize the donor transfusion-related hazards. Erythropoietin with its wide clinical applications could improve maternal and neonatal outcome.

[The correction with nooglutil and L-pyroglutamyl-D-alanine amide of cognitive disorders in rats due to intrauterine hypoxia]. / Korrekstiia noogliutilom i amidom L-piroglutamil-D-alanina kognitivnykh narushenii u krys, vyzvannykh vnutriutrobnoi gipoksiei.

Novel nootropic compounds, nooglutyl (N-5-hydroxy(nicotinoyl)-L-glutamine acid, 25 mg/kg/day) and L-pyroglutamyl-D-alanine amide (1 mg/kg/day) administered intracutaneously from the 8th to 20th day of life prevent from movement hyperactivity in "open field", disturbances in ability to training and in memory in an alternate test and in tests of passive and active avoidance and normalize behavior of the adult mail rats (subjected to two-hour hypobaric hypoxia in on the 15-day of intrauterine life, vacuum corresponded to the height 8500 m) in-extrapolation avoidance test. Additionally, nooglutyl recovered the normal growth of rats in the first month of their life, prevented from deceleration of investigating behavior of adults animals and disturbances of the mink reflex in them.

Postnatal development of hippocampal and neocortical cholinergic and serotonergic innervation in rat: effects of nitrite-induced prenatal hypoxia and nimodipine treatment.

Postnatal development of ingrowing cholinergic and serotonergic fiber patterns were studied in the rat hippocampus and parietal cortex employing a histochemical procedure for acetylcholinesterase as a cholinergic fiber marker, and immunocytochemistry of serotonin for serotonergic fiber staining. The rat pups were killed at postnatal days 1, 3, 5, 7, 10, and 20. The development of cholinergic and serotonergic innervation was described and the fiber density quantified under normal conditions and after long-term prenatal anemic hypoxia induced by chronic exposure to sodium nitrite. Furthermore, a third group was studied in which the nitrite hypoxia was combined with a simultaneous treatment with the Ca(2+)-entry blocker nimodipine to test the neuroprotective potential of this drug. Quantitative measurement of fiber density from postnatal day 1 to day 20 yielded the following results: (i) both neurotransmitter systems revealed an age-dependent and an anatomically-organized developmental pattern; (ii) the serotonergic innervation of the dorsal hippocampus preceded that of cholinergic afferentation in postnatal days 1-3; (iii) prenatal hypoxia induced a transient delay in the innervation of parietal neocortex and dentate gyrus for both neurotransmitter systems, but left the innervation of the cornu ammonis unaffected; and (iv) the hypoxia-induced retardation of cholinergic and serotonergic fiber development was prevented by concomitant application of the Ca(2+)-antagonist nimodipine during the hypoxia. The results indicate that prenatal hypoxia evokes a temporary delay in the cholinergic and serotonergic fiber outgrowth in cortical target areas in a region-specific manner. The hypoxia-induced growth inhibition is prevented by the calcium antagonist nimodipine, which supports the importance of the intracellular Ca2+ homeostasis of cells and growth cones in regulating axonal proliferation.

[Sodium oxybutyrate normalizes the central nervous system functions in the progeny of rats subjected to hypobaric hypoxia during pregnancy]. / Natriia oksibutirat mormalizuet funktsii tsentral'noi nervnoi sistemy u potomstva krys, podvergshikhsia gipobaricheskoi gipoksii vo vremia beremennosti.

Two-hour hypobaric hypoxia of rats on day 15 of their pregnancy led to a reduction in weight gain of pups within 20 days after birth, disturbed memory in active and passive paradigms, changed adaptive behavior in the extrapolatory water avoidance test, and impaired sleep in adult animals. Postnatal treatment with sodium hydroxybutyrate given in a dose of 50 mg/kg/day on days 8 to 20 of life normalized mnestic functions of the brain, the process of falling asleep, and physical development which had been impaired by intrauterine hypoxia.

Pulmonary arterial hypertension induced by distention of the main pulmonary artery in conscious newborn, young, and adult sheep.

Distention of the main pulmonary artery by balloon inflation in sheep results in presumably reflex elevation of pulmonary arterial pressure and resistance distal to the balloon. This response to main pulmonary artery distention is significantly greater in newborn lambs than in older lambs or adult sheep. In several of the newborn lambs, pulmonary artery pressure was raised to suprasystemic levels. Further, in some of the newborn animals, these increases in pulmonary artery pressure and resistance were sustained after deflation of teh balloon for periods up to 2 hr. The functional significance of this pulmonary hypertension reflex was not elucidated. However, the data strongly suggest that this reflex may contribute to the maintenance of high pulmonary vascular resistance during fetal and early neonatal life.