Adverse respiratory patterns in near-term spontaneously breathing newborn lambs with elevated airway liquid volumes at birth.

Introduction: Recent evidence indicates that respiratory distress (RD) in near-term infants is caused by elevated airway liquid (EL) volume at the beginning of air-breathing after birth. While the adverse effects EL volumes on newborn lung function are known, the effects on respiratory control and breathing patterns shortly after birth (<4 h) are unknown. We investigated the effects of EL volumes on cardiorespiratory function and breathing patterns in spontaneously breathing near-term newborn lambs in the first hours after birth. Methods: At 137-8 days gestation (2-3 days prior to delivery; term ∼147 days), sterile surgery was performed on fetal sheep (n = 17) to implant catheters and blood flow probes. At 140 days, lambs were delivered via caesarean section under spinal anaesthesia. Airway liquid volumes were adjusted to mimic the level expected following vaginal delivery (∼10 ml/kg; Controls; n = 7), or elective caesarean section (∼30 ml/kg; elevated airway liquid group; EL; n = 10). Spontaneous breathing and cardiorespiratory parameters were recorded over four hours after birth. Non-invasive respiratory support with supplemental oxygen was provided if required. Results: EL lambs required higher inspired oxygen levels (p = 0.0002), were less active (p = 0.026), fed less (p = 0.008) and had higher respiratory morbidity scores than Controls (p < 0.0001). EL lambs also displayed higher rates of breathing patterns associated with RD, such as expiratory braking and tachypnoea. These patterns were particularly evident in male EL lambs who displayed higher levels of severe respiratory morbidity (e.g., expiratory braking) than female EL lambs. Conclusion: The study demonstrates that EL volumes at birth trigger respiratory behaviour and breathing patterns that resemble clinically recognised features of RD in term infants.

Respiratory patterns in spontaneously breathing near-term lambs delivered by caesarean section under spinal anaesthesia.

Introduction: The transition to newborn life has typically been studied in intubated and mechanically ventilated newborn lambs delivered via caesarean section (CS) under general anaesthesia. As a result, little is known of the spontaneous breathing patterns in lambs at birth, particularly those at risk of developing respiratory distress (RD). We have developed a method for delivering spontaneously breathing near-term lambs to characterise their breathing patterns in the immediate newborn period. Methods: At 137-8 days gestation (2-3 days prior to delivery; term ∼147 days), fetal lambs (n = 7) were partially exteriorised for instrumentation (insertion of catheters and flow probes) before they were returned to the uterus. At 140 days, lambs were delivered via CS under light maternal sedation and spinal anaesthesia. Lambs were physically stimulated and when continuous breathing was established, the umbilical cord was clamped. Breathing patterns were assessed by measuring intrapleural and upper-tracheal pressures during the first four hours after birth. Results: Newborn lambs display significant heterogeneity in respiratory patterns in the immediate newborn period that change with time after birth. Seven distinct breathing patterns were identified including: (i) quiet (tidal) breathing, (ii) breathing during active periods, (iii) breathing during oral feeding, (iv) tachypnoea, (v) expiratory braking manoeuvres, (vi) expiratory pauses or holding, and (vii) step changes in ventilation. Conclusions: We have described normal respiratory behaviour in newborn lambs, in order to identify respiratory behaviours that are indicative of RD in term newborn infants.

Assessing lung aeration using ultrasound after birth in near-term lambs at risk of respiratory distress.

Background: Optimizing respiratory support after birth requires real-time feedback on lung aeration. We hypothesized that lung ultrasound (LUS) can accurately monitor the extent and progression of lung aeration after birth and is closely associated with oxygenation. Methods: Near-term (140 days gestation, term ∼147 days), spontaneously breathing lambs with normal (controls; n = 10) or elevated lung liquid levels (EL; n= 9) were delivered by Caesarean section and monitored for four hours after birth. LUS (Phillips CX50, L3-12 transducer) images and arterial blood gases were taken every 5-20 min. LUS images were analyzed both qualitatively (grading) and quantitatively (using the coefficient of variation of pixel intensity (CoV) to estimate the degree of lung aeration), which was correlated with the oxygen exchange capacity of the lungs (Alveolar-arterial difference in oxygen; AaDO2). Results: Lung aeration, measured using LUS, and the AaDO2 improved over the first 4 h after birth. The increase in lung aeration measured using CoV of pixel intensity, but not LUS grade, was significantly reduced in EL lambs compared to controls (p = 0.02). The gradual decrease in AaDO2 after birth was significantly correlated with increased lung aeration in both control (grade, r2 = 0.60, p < 0.0001; CoV, r2 = 0.54, p < 0.0001) and EL lambs (grade, r2 = 0.51, p < 0.0001; CoV, r2 = 0.44, p < 0.0001). Conclusions: LUS can monitor lung aeration and liquid clearance after birth in spontaneously breathing near-term lambs. Image analysis techniques (CoV) may be able detect small to moderate differences in lung aeration in conditions with lung liquid retention which are not readily identified using qualitative LUS grading.

Description of a method for inducing fetal growth restriction in the spiny mouse.

Intrauterine or fetal growth restriction (IUGR) is a major complication of pregnancy and leads to significant perinatal morbidities and mortality. Typically, induction of IUGR in animals involves the complete occlusion or ablation of vessels to the uterus or placenta, acutely impairing blood flow and fetal growth, usually with high fetal loss. We aimed to produce a model of reduced fetal growth in the spiny mouse with minimal fetal loss. At 27 days gestational age (term is 38-39 days), a piece of silastic tubing was placed around the left uterine artery to prevent the further increase of uterine blood flow with advancing gestation to induce IUGR (occluded). Controls were generated from sham surgeries without placement of the tubing. Dams were humanely euthanized at 37 days gestational age and all fetuses and placentas were weighed and collected. Of the 17 dams that underwent surgery, 15 carried their pregnancies to 37 days gestational age and 95% of fetuses survived to this time. The difference in fetal body weight between occluded and control was ~21% for fetuses in the left uterus side: there were no differences for fetuses in the right uterus side. Offspring from the occluded group had significantly lower brain, liver, lung, kidney and carcass weights compared with shams. Preventing the gestation-related increase of uterine blood flow induced significant growth restriction in the fetal spiny mouse, with minimal fetal loss. This technique could be readily adapted for other small animal.

Dopamine treatment during acute hypoxia is neuroprotective in the developing sheep brain.

Dopamine is often used to treat hypotension in preterm infants; these infants are at risk of developing brain injury due to impaired autoregulation and cerebral hypoperfusion. However the effects of dopamine on the immature brain under conditions of cerebral hypoxia are not known. We hypothesized that pretreatment with dopamine would protect the immature brain from injury caused by cerebral hypoxia. Preterm fetal sheep were used to determine the effects of intravenous dopamine on hypoxia-induced brain injury. In 16 pregnant sheep at 90days of gestation (0.6 of term, term=147days) catheters were implanted aseptically into the fetal carotid artery and jugular vein; an inflatable occluder was placed loosely around the umbilical cord for later induction of fetal hypoxemia. At 5days after surgery, dopamine (10µg/kg/min, n=7 fetuses) or saline (n=9 fetuses) was infused for 74h. Two hours after commencing the dopamine/saline infusion, we induced umbilical cord occlusion (UCO) for up to 25min to produce fetal asphyxia. Fetuses were allowed to recover, and brains were collected 72h later for assessment of neuropathology. Un-operated twin fetuses were used as age-matched non-UCO controls (n=8). In UCO+saline fetuses, microglial and apoptotic cell density in the subcortical and periventricular white matter, caudate nucleus and hippocampus was greater than that in age-matched controls; oxidative stress was elevated in the subcortical and periventricular white matter and caudate nucleus compared to that in age-matched controls. In UCO+dopamine fetuses microglial density and oxidative stress in the cerebral white matter and caudate nucleus were not different to that of age-matched controls. Apoptotic cell death was decreased in the cerebral white matter of UCO+dopamine brains, relative to UCO+saline brains. We conclude that pretreatment with dopamine does not exacerbate hypoxia-induced injury in the immature brain and may be neuroprotective because it led to decreased apoptosis, oxidative stress and neuroinflammation in the cerebral white matter and decreased neuroinflammation in the caudate nucleus.

Intraperitoneal medetomidine: a novel analgesic strategy for postoperative pain management in pregnant sheep.

The absorption of medetomidine released by continuous infusion from an osmotic pump in the abdominal cavity was studied in pregnant sheep during the 24 h postoperative period. Additionally pain and sedation was assessed. Eleven sheep were studied: six were treated with a medetomidine loaded osmotic pump delivering 10 µL/h (3 µg/kg/h medetomidine); and five with a saline loaded osmotic pump (control). Serial blood samples were taken and analysed to determine plasma medetomidine levels. Medetomidine was absorbed from the peritoneal cavity and a steady plasma concentration was achieved within 10 h, mean (SD) peak concentration was 2.87 (0.22) ng/mL. Sheep receiving medetomidine analgesia had significantly lower pain scores at 10 h than controls. Four control sheep required rescue analgesia, compared with 0 in the treatment group. Delivery of 3 µg/kg/h medetomidine by an intraperitoneal osmotic pump to pregnant sheep in the 24 h postoperative period provides adequate plasma concentrations of medetomidine for analgesia without sedation.

Effects of glucocorticoid treatment given in early or late gestation on growth and development in sheep.

Antenatal corticosteroids are used to augment fetal lung maturity in human pregnancy. Dexamethasone (DEX) is also used to treat congenital adrenal hyperplasia of the fetus in early pregnancy. We previously reported effects of synthetic corticosteroids given to sheep in early or late gestation on pregnancy length and fetal cortisol levels and glucocorticoids alter plasma insulin-like growth factor (IGF) and insulin-like growth factor binding protein (IGFBP) concentrations in late pregnancy and reduce fetal weight. The effects of administering DEX in early pregnancy on fetal organ weights and betamethasone (BET) given in late gestation on weights of fetal brain regions or organ development have not been reported. We hypothesized that BET or DEX administration at either stage of pregnancy would have deleterious effects on fetal development and associated hormones. In early pregnancy, DEX was administered as four injections at 12-hourly intervals over 48 h commencing at 40-42 days of gestation (dG). There was no consistent effect on fetal weight, or individual fetal organ weights, except in females at 7 months postnatal age. When BET was administered at 104, 111 and 118 dG, the previously reported reduction in total fetal weight was associated with significant reductions in weights of fetal brain, cerebellum, heart, kidney and liver. Fetal plasma insulin, leptin and triiodothyronine were also reduced at different times in fetal and postnatal life. We conclude that at the amounts given, the sheep fetus is sensitive to maternal administration of synthetic glucocorticoid in late gestation, with effects on growth and metabolic hormones that may persist into postnatal life.

The impact of maternal synthetic glucocorticoid administration in late pregnancy on fetal and early neonatal hypothalamic-pituitary-adrenal axes regulatory genes is dependent upon dose and gestational age at exposure.

In this study, we determined the gene and/or protein expression of hypothalamic-pituitary-adrenal (HPA) axis regulatory molecules following synthetic glucocorticoid exposures. Pregnant sheep received intramuscular saline or betamethasone (BET) injections at 104 (BET-1), 104 and 111(BET-2) or 104, 111 and 118 (BET-3) days of gestation (dG). Samples were collected at numerous time-points between 75 dG and 12 weeks postnatal age. In the BET-3 treatment group, fetal plasma cortisol levels were lower at 145 dG than controls and gestational length was lengthened significantly. The cortisol:adrenocorticotropic hormone (ACTH) ratio in fetal plasma of control and BET-3 fetuses rose significantly between132 and 145 dG, and remained elevated in lambs at 6 and 12 weeks of age; this rise was truncated at day 145 in fetuses of BET-3 treated mothers. After BET treatment, fetal and postnatal pituitary proopiomelanocortin mRNA levels were reduced from 109 dG to 12 weeks postnatal age; pituitary prohormone convertase 1 and 2 mRNA levels were reduced at 145 dG and postnatally; hypothalamic arginine vasopressin mRNA levels were lowered at all time-points, but corticotrophin-releasing hormone mRNA levels were reduced only in postnatal lambs. Maternal BET increased late fetal and/or postnatal adrenal mRNA levels of ACTH receptor and 3ß hydroxysteroid dehydrogenase but decreased steroidogenic acute regulatory protein and P450 17-α hydroxylase. The altered mRNA levels of key HPA axis regulatory proteins after maternal BET injections suggests processes that may subserve long-term changes in HPA activity in later life after prenatal exposure to synthetic glucocorticoids.

Fetal cardiac catheterization using a percutaneous transhepatic access technique: preliminary experience in a lamb model.

OBJECTIVES: Human fetal cardiac intervention has hitherto typically involved a percutaneous transventricular approach. In fetal lambs, a transhepatic approach to access the fetal intra-abdominal veins after exteriorization of the uterus by laparotomy has been described. We aimed to develop a percutaneous transhepatic technique for catheterization of the fetal heart at mid-gestation that avoids maternal laparotomy. METHODS: In 10 fetal lambs (90-97 days' gestation), access to the fetal venous system was attempted by percutaneous puncture with a 5-F sheath into the umbilical vein (n = 1) or a 16-gauge IV-catheter into the hepatic vein (n = 9). This was followed by cardiac catheterization using a 1.8-2.6-F tapered coronary catheter. Euthanasia and postmortem examination were performed immediately postprocedure in two cases, or after normal term delivery in the remaining cases that survived the procedure. RESULTS: In one case fetal position precluded procedural attempts, and in another, the fetus, accessed by a 5-F sheath, died from umbilical hemorrhage. In eight cases, access to the fetal hepatic vein was achieved. In seven of these cases, the access catheter was advanced into the inferior vena cava, followed by catheterization of the right atrium (all cases) and four cardiac chambers (three cases). One fetus died during cardiac catheterization owing to right ventricle perforation, and the other seven fetuses were alive at the end of the procedure (87.5% survival). Immediate postmortem after euthanasia in two of the fetuses that survived the procedure detected intraperitoneal bleeding (4 mL and 20 mL), while postnatal postmortem examination following uneventful delivery at term in the remaining five fetuses revealed no vascular or cardiac trauma. CONCLUSIONS: Ultrasound-guided percutaneous transhepatic cardiac catheterization is feasible in mid-gestational fetal sheep. This technique has the potential for translation into human fetal cardiac and circulatory interventions.

Antenatal ureaplasma infection impairs development of the fetal ovine gut in an IL-1-dependent manner.

Ureaplasma infection of the amniotic cavity is associated with adverse postnatal intestinal outcomes. We tested whether interleukin-1 (IL-1) signaling underlies intestinal pathology following ureaplasma exposure in fetal sheep. Pregnant ewes received intra-amniotic injections of ureaplasma or culture media for controls at 3, 7, and 14 d before preterm delivery at 124 d gestation (term 150 d). Intra-amniotic injections of recombinant human interleukin IL-1 receptor antagonist (rhIL-1ra) or saline for controls were given 3 h before and every 2 d after Ureaplasma injection. Ureaplasma exposure caused fetal gut inflammation within 7 d with damaged villus epithelium and gut barrier loss. Proliferation, differentiation, and maturation of enterocytes were significantly reduced after 7 d of ureaplasma exposure, leading to severe villus atrophy at 14 d. Inflammation, impaired development and villus atrophy of the fetal gut was largely prevented by intra-uterine rhIL-1ra treatment. These data form the basis for a clinical understanding of the role of ureaplasma in postnatal intestinal pathologies.

Antenatal exposure to chorioamnionitis affects lipid metabolism in 7-week-old sheep.

Antenatal exposure of the fetus to inflammation may alter postnatal organ development. In our previous work, we demonstrated that the fetal liver is involved in the systemic inflammation associated with chorioamnionitis, leading to metabolic changes. On the basis of these findings, we hypothesized that chorioamnionitis can lead to postnatal inflammation-related liver injury and disturbed lipid metabolism. Chorioamnionitis was induced in sheep by intra-amniotic injection of lipopolysaccharide (LPS) or saline at 90, 100 and 110 days of gestation. Liver homeostasis and lipid metabolism were analyzed at term and at 7 weeks of age. At term, hepatic T-lymphocytes and apoptotic hepatocytes were increased. In addition, hepatic cholesterol and triglyceride levels were decreased in LPS-exposed animals compared with controls. At 7 weeks of age, no hepatic inflammation could be detected. However, liver triglycerides and plasma cholesterol levels were increased in LPS-exposed animals relative to controls. The changes in lipid levels at 7 weeks of age were associated with increased leptin receptor mRNA levels, increased lipid peroxidation, increased expression of cytochrome c oxidase subunit 4 as a marker for mitochondrial function and increased circulating ceramide levels. These findings demonstrate that chorioamnionitis-mediated antenatal inflammation-related liver disturbances have long-lasting postnatal effects on lipid metabolism.

The cerebral critical oxygen threshold of ventilated preterm lambs and the influence of antenatal inflammation.

Perinatal inflammation is associated with adverse neurodevelopmental outcomes, which may be partly due to changes in the cerebral oxygen delivery/consumption relationship. We aimed to determine the critical oxygen delivery threshold of the brain of preterm, ventilated lambs and to determine whether the critical threshold is affected by exposure to inflammation in utero. Pregnant ewes received intra-amniotic injection of lipopolysaccharide or saline at 125 or 127 days of gestation. Pulmonary and systemic flow probes and catheters were surgically positioned in the fetus immediately before delivery at 129 days of gestation. After delivery, lambs were ventilated for 90 min using a positive end-expiratory pressure recruitment strategy. Cardio-respiratory variables and blood gases were measured regularly. Systemic and cerebral oxygen delivery, consumption (Fick), and extraction were calculated, and the relationship between cerebral delivery and consumption analyzed. Linear regression was used to define the transition or "critical" oxygen threshold as the point at which the slope of the oxygen delivery/consumption curve changed to be > 10°. Four subgroups were defined according to the calculated critical threshold. A total of 150 measurements were recorded in 18 lambs. Fetal cerebral oxygen consumption was increased by antenatal lipopolysaccharide (P < 0.05). The postnatal critical oxygen threshold was 3.6 ml·kg⁻¹·min⁻¹, corresponding to cerebral oxygen consumption of 0.73 ml·kg⁻¹·min⁻¹. High oxygen delivery and consumption were associated with increased pulmonary and carotid blood flow and systemic extraction compared with low oxygen delivery and consumption. No postnatal effect of antenatal inflammation was observed. Inflammation in utero increases fetal, but not postnatal, cerebral oxygen consumption. Adverse alterations to pulmonary blood flow can result in reduced cerebral blood flow, oxygen delivery, and consumption. Regardless of exposure to inflammation, there is a consistent postnatal relationship between cerebral oxygen delivery and consumption.

Differential appearance of placentomes and expression of prostaglandin H synthase type 2 in placentome subtypes after betamethasone treatment of sheep late in gestation.

UNLABELLED: Inappropriate fetal exposure to maternal glucocorticoid (GC) has been proposed as a mechanism for fetal programming where the effects of GC may be mediated by the placenta. However, the consequences of maternal GC on placental morphology and enzyme expression are unclear. OBJECTIVES: We used betamethasone (BET) to determine effects on placentome subtype distribution and expression of prostaglandin H synthase type 2 (PGHS-2) enzyme. METHODS: Pregnant sheep carrying male fetuses were randomized to receive injections of saline (n = 30) or one (104 days of gestation, (dG); n = 6), two (104, 111 dG; n = 6) or three (104, 111, 118 dG; n = 11) doses of BET (0.5 mg/kg). Placental tissue was collected prior to (75, 84, 101 dG), during (109, 116 dG) and after BET (122, 132, 146 dG). RESULTS: Total number of placentomes was not different between gestational ages. A- and B-subtypes were most affected by prenatal BET exposure; numbers of A-subtypes were increased and numbers of B-subtypes were decreased compared to controls at 116 dG. At term numbers of A-subtypes were lower after BET, but the weight range distribution was similar to controls. In controls, placental PGHS-2 protein levels increased with gestational age and PGHS-2 localized primarily to uninuclear trophoblast cells. After BET, PGHS-2 protein in C-subtypes at term was significantly increased compared to A-subtypes. CONCLUSIONS: Maternal BET treatment in late gestation affects the proportions of placentome subtypes and their differential expression of PGHS-2. Our data do not support previous hypotheses that A-subtypes develop into B-, C- and D-subtypes over the course of gestation.

All-trans retinoic acid and intra-amniotic endotoxin-mediated effects on fetal sheep lung.

All-trans retinoic acid (RA) is a potent modulator of lung development. Chorioamnionitis, which is frequently associated with preterm birth, causes fetal lung inflammation and improves lung function but also results in alveolar simplification and microvascular injury. Endotoxin-mediated chorioamnionitis reduces RA concentration in the fetal lung to 16% of control values. We hypothesized that administration of RA to the fetus before induction of chorioamnionitis would preserve septation of the distal airspaces. Time-mated ewes with singletons were assigned to receive a fetal intramuscular treatment with 20,000 IU of RA in olive oil (or olive oil only) 3 hr prior to intra-amniotic injection of endotoxin (20 mg, E. coli 055:B5) or saline, at 124-day gestational age and 7 days after the fetal treatment. The right cranial lung lobe was processed for morphometric analysis. RA treatment did not affect chorioamnionitis-induced fetal and systemic inflammation or interleukin-8 concentrations in lung tissue. RA administration alone did not alter lung structure. Relative to control lungs (5 +/- 3 mL/kg), lung volume increased similarly with endotoxin (22 +/- 4 mL/kg) or RA plus endotoxin (20 +/- 3 mL/kg; P < 0.05). Alveolar wall thickness was 4.2 +/- 0.3 mum after endotoxin-induced chorioamnionitis, 6.0 +/- 0.4 mum in controls (P < 0.05 versus endotoxin) and 5.5 +/- 0.2 mum after RA and endotoxin (P < 0.05 versus control, n.s. versus endotoxin). The ratio of airspace versus tissue was 4.6 +/- 0.3 in endotoxin-induced chorioamnionitis, 2.1 +/- 0.3 in controls and 4.1 +/- 0.5 after RA and endotoxin. We conclude that fetal treatment with RA did not prevent inflammation-induced alveolar simplification.

The carotid bodies influence growth responses to moderate maternal undernutrition in late-gestation fetal sheep.

OBJECTIVE: To determine the role of carotid sinus innervation on differential fetal organ growth during maternal nutrient restriction in late pregnancy. DESIGN: Randomised controlled study. SETTING: University research facility. SAMPLE: Thirty-nine Merino ewes. METHODS: At 113 days gestational age (dGA), fetuses were bilaterally carotid sinus denervated or sham denervated. From 118 dGA, the surgery groups were subdivided into two dietary groups, and their ewes were fed 100% of nutrient requirements or 50% until tissue collection at 140 dGA. This provided four groups (sham/control diet, sham/restricted diet, denervated/control diet and denervated/restricted diet). MAIN OUTCOME MEASURES: Fetal organ weights and hormone levels and maternal weight change during the dietary restriction. RESULTS: Adrenal glands were larger in sham/restricted diet fetuses than in sham/control diet or denervated/restricted diet fetuses (P < 0.05). Fetal adrenal weight and brain-to-liver weight ratio were positively related to maternal weight change during the nutritional challenge in sham fetuses only (P < 0.05). Fetal liver weight was negatively related to maternal weight change during nutritional challenge in sham fetuses only (P < 0.05). CONCLUSIONS: We have shown a reduction in liver growth but sparing of adrenal growth in response to moderate maternal undernutrition, which is dependent on intact carotid body innervation. This suggests a new role for the carotid bodies in the control of differential organ growth during such undernutrition.

Kynurenine production and catabolism in fetal sheep with embolized or nonembolized placentas.

OBJECTIVE: The effect of maternal tryptophan loading on fetal plasma and brain, kynurenic acid, and quinolinic acid concentrations was compared in late gestation fetal sheep with either chronically embolized or nonembolized placentas. STUDY DESIGN: The placentas of 4 ewes were embolized by daily injection of mucopolysaccharide microspheres into the umbilical artery from 120 days gestation in amounts sufficient to reduce the fetal arterial PO2 to < or = 12 mm Hg. Four fetuses with nonembolized placentas were the control group. At 135 to 138 days gestation, the ewe received an infusion of tryptophan (100 mg/kg, intravenously) or an equivalent volume of saline solution (100 mL) over 2 hours. Maternal and fetal arterial blood samples were obtained between 2 and 48 hours from the start of the infusion for the measurement of plasma tryptophan and kynurenine metabolites. Brains were then obtained from embolized and nonembolized fetuses 24 hours after a further maternal tryptophan loading experiment and from nonembolized non-tryptophan-treated fetuses for analysis of regional kynurenic acid and quinolinic acid content. RESULTS: Maternal tryptophan infusion resulted in a significant increase of kynurenine in fetal plasma, but this increase was significantly smaller in fetuses with an embolized placenta compared with a nonembolized placenta. Both kynurenic acid and quinolinic acid levels increased significantly in fetal plasma, with no differences between the groups. Kynurenic acid and quinolinic acid levels were increased in all regions of the fetal brain after maternal tryptophan loading, but these increases were greater in the fetuses with an embolized placenta, compared with a nonembolized placenta. CONCLUSION: Fetal tryptophan and kynurenine metabolism is significantly altered when placental function is chronically compromised in late gestation. The decreased production of kynurenine from tryptophan may result from the compromise of hepatic function in the fetus, whereas the increased production of kynurenic acid and quinolinic acid in the brain is likely to reflect alterations of metabolism of tryptophan and kynurenine to these neuroactive products by glial cells in the fetal brain.

Regional changes in kynurenic acid, quinolinic acid, and glial fibrillary acidic protein concentrations in the fetal sheep brain after experimentally induced placental insufficiency.

OBJECTIVE: This study was undertaken to examine the effects of chronic embolization of the umbilical circulation during late gestation on regional concentrations of quinolinic acid and kynurenic acid (neuroactive products of tryptophan catabolism) and of the astrocyte-associated glial fibrillary acidic protein in the fetal brain. STUDY DESIGN: Pregnant ewes bearing fetuses with long-term catheter placement were treated daily with injections of either saline solution (n = 4; control group) or mucopolysaccharide microspheres (n = 5; embolized group) into the umbilical circulation through a femoral artery catheter between 120 and 140 days' gestation. The fetuses in the embolized group received sufficient microspheres each day to reduce and maintain the femoral arterial PO2 at < or =12 mm Hg. Autopsies were performed at 140 days' gestation to obtain the fetal brain for chemical analysis. RESULTS: Umbilical embolization resulted in nonacidemic hypoxia and hypoglycemia at 140 days' gestation. Quinolinic acid concentrations in the embolized group were significantly increased in the medulla, pons, midbrain, hypothalamus, and hippocampus, whereas kynurenic acid concentrations in the embolized group were reduced in the hippocampus and hypothalamus. There were significant reductions in glial fibrillary acidic protein contents in the occipitoparietal cortex, hippocampus, and pons in the embolized group. CONCLUSION: Placental compromise during late pregnancy had effects on kynurenine metabolism and astrocyte function in some regions of the fetal sheep brain. We suggest that these changes increase the vulnerability of the brain to asphyxial injury during late gestation and the perinatal period.

Effect of hypoxia on respiratory activity in the foetus.

1. Breathing movements stop soon after the induction of hypoxia in foetal animals, a response attributed to the active inhibition of the respiratory centres. Separate studies using punctate lesions have identified lateral pontine and thalamic sites in foetal sheep from which respiratory inhibition may arise, but whether either of these loci are actually oxygen sensitive or whether they receive input from other regions responsive to hypoxia, has not been established. 2. FOS immunocytochemistry was used to identify neuronal pools activated by hypoxia in the brain of late-gestation foetal and newborn sheep. FOS-positive cells were found in the pons in regions corresponding to the medial parabrachial and Kolliker-Fuse nuclei and were shown to be catecholaminergic. Neurons in this pontine region were also activated by the piperizine drug almitrine, which, like hypoxia, inhibits respiratory output in the foetus but is a respiratory stimulant in the newborn and adult. Because these pontine neurons do not express FOS protein after challenge with hypoxia or almitrine in the newborn lamb, we argue that they are crucial to the hypoxic inhibition of respiratory activity in foetal life. 3. The role of the placenta in determining these foetal responses and how this may change at birth is discussed.

Tryptophan metabolism in pregnant sheep: increased fetal kynurenine production in response to maternal tryptophan loading.

OBJECTIVE: The effects of a tryptophan load on the plasma concentration of kynurenine, the precursor for the production in the brain of the neuroactive products kynurenic acid and quinolinic acid, were determined in pregnant sheep at midgestation and late gestation and in nonpregnant sheep. STUDY DESIGN: Pregnant ewes were given an intravenous infusion of 100 mg/kg L-tryptophan during 2 hours at 95 to 98 days' gestation (n = 4) or 135 to 138 days' gestation (n = 10). Nonpregnant ewes (n = 6) were studied in late estrus. Arterial blood samples taken from 2 hours before to 48 hours after the start of the infusion were used for analysis of plasma tryptophan, kynurenine, and cortisol concentrations. RESULTS: Tryptophan loading at both gestational ages resulted in significantly greater increases in kynurenine concentrations in fetal plasma (at 95-98 days' gestation, from 5.7 +/- 1.2 micromol/L [baseline] to 247.9 +/- 86.7 micromol/L (peak); at 135-138 days' gestation, from 9.0 +/- 2.3 micromol/L [baseline] to 289.0 +/- 194.0 micromol/L [peak]) than in maternal plasma [at 95-98 days' gestation, from 4.6 +/- 0.8 micromol/L [baseline] to 118.0 +/- 79.7 micromol/L [peak]; at 135-138 days' gestation, from 4.8 +/- 2.9 micromol/L [baseline] to 98.3 +/- 67.8 micromol/L [peak]). It took longer for kynurenine concentrations to return to basal values in the fetus (24-30 hours) than in the ewe (8-12 hours). The kynurenine responses in pregnant and nonpregnant ewes were not different from each other. CONCLUSION: The production of kynurenine from tryptophan is significantly greater in the fetal lamb than in the pregnant or nonpregnant adult ewe.

Kynurenic acid in brain and cerebrospinal fluid of fetal, newborn, and adult sheep and effects of placental embolization.

Concentrations of the endogenous glutamate receptor antagonist kynurenic acid (KA) were measured in various brain regions and in cisternal cerebrospinal fluid of fetal, newborn, and adult sheep. KA concentrations were significantly higher in the fetal brain and cerebrospinal fluid at 90 and 140 d gestation compared with postnatal ages. In fetuses of 132-139 d gestation, KA concentrations in cerebrospinal fluid collected by drainage from an indwelling cisternal catheter increased significantly after infusion of the organic acid transport inhibitor probenecid (100 or 200 mg/kg, i.v.) indicating active transport of KA out of the fetal brain. In fetuses in which the umbilical circulation had been chronically restricted from 120 to 140 d gestation by partial embolization of the placenta, plasma concentrations of the KA precursor kynurenine were significantly lower than in control fetuses, and KA concentrations in the hypothalamus and hippocampus were significantly reduced; other brain regions were not affected. These results indicate that the production of KA is higher in the fetal brain compared with the newborn and adult brain. Because KA diminishes the risk of excitotoxic neuronal damage under hypoxic-ischemic conditions, the high levels of KA in the brain before birth may have a neuroprotective function. The decrease of KA concentrations in the hypothalamus and hippocampus after umbilical embolization suggests that, after chronic hypoxia in utero, these regions of the brain may become more vulnerable to subsequent episodes of acute hypoxia or ischemia encountered in late gestation or during parturition.