feasibility of fetal cardiac access using a percutaneous transhepatic approach in the mid-gestati... more feasibility of fetal cardiac access using a percutaneous transhepatic approach in the mid-gestational fetal lamb, as a model for human fetus. Method Eight fetal lambs of 95-97 days gestation (term 147 days) were studied. Under ultrasound guidance, the fetal right hepatic vein was punctured percutaneously, using a 16GA intravenous cannula (BD Angiocath). A coronary catheter (FineCross TM MG, Terumo) was inserted into the 16 GA cannula, over a 0.014inch guidewire, and advanced into the inferior vena cava and the right atrium. Contrast was injected to document position of the catheter. Three fetal lambs were to be euthanized at the end of the procedure to evaluate blood loss. The rest were to deliver vaginally at term, and euthanized for postmortem examination. Results Percutaneous fetal cardiac access was successful in 7 out of the 8 fetuses. All 4 heart chambers were catheterized in the last 3 fetuses. One fetus died during the procedure, post-mortem showed pericardial and peritoneal haemorrhage. The other two fetuses dedicated for immediate post-mortem, after having survived the procedure, had small haemoperitoneum. Averaged fetal weight was 1027±153g. All other lambs were born normally at term. Conclusion Ultrasound-guided fetal cardiac catheterization through a percutaneous transhepatic approach is feasible. Our experience provides a potentially safer route for human fetal cardiac intervention at midgestational age.
Background: Delaying umbilical cord clamping until after aeration of the lung (physiological-base... more Background: Delaying umbilical cord clamping until after aeration of the lung (physiological-based cord clamping; PBCC) maintains cardiac output and oxygenation in preterm lambs at birth, however, its efficacy after intrauterine inflammation is not known. Given the high incidence of chorioamnionitis in preterm infants, we investigated whether PBCC conferred any benefits compared to immediate cord clamping (ICC) in preterm lambs exposed antenatally to 7 days of intrauterine inflammation. Methods: Ultrasound guided intraamniotic injection of 20 mg Lipopolysaccharide (from E. coli:055:B5) was administered to pregnant ewes at 0.8 gestation. Seven days later, ewes were anesthetized, preterm fetuses exteriorised via cesarean section, and instrumented for continuous measurement of pulmonary, systemic and cerebral pressures and flows, and systemic, and cerebral oxygenation. Lambs were then randomized to either PBCC, whereupon ventilation was initiated and maintained for 3 min prior to umbilical cord clamping, or ICC where the umbilical cord was cut and ventilation initiated 30 s later. Ventilation was maintained for 30 min. Results: ICC caused a rapid fall in systemic (by 25%) and cerebral (by 11%) oxygen saturation in ICC lambs, concurrent with a rapid increase in carotid arterial pressure and heart rate. The overshoot in carotid arterial pressure was sustained in ICC lambs for the first 20 min of the study. PBCC maintained cardiac output and prevented the fall in cerebral oxygen delivery at birth. PBCC lambs had lower respiratory compliance and higher respiratory requirements throughout the study. PBCC mitigated the adverse effects of ICC on oxygenation and cardiac output, and therefore could be more beneficial in preterm babies exposed to antenatal inflammation as it maintains cardiac output and oxygen delivery. The increased respiratory requirements require further investigation in this sub-group of preterm infants.
Translational Investigation nature publishing group Background: Preterm infants often receive mec... more Translational Investigation nature publishing group Background: Preterm infants often receive mechanical ventilation and oxygen at birth. exposure to large tidal volumes (V T s) at birth causes lung inflammation, and oxygen may amplify the injury. We hypothesized that normal V T ventilation at birth causes lung injury that is exacerbated by 95% oxygen. Methods: The head and chest of anesthetized preterm fetal sheep (129 ± 1 d gestation) were surgically exteriorized while maintaining the placental circulation. Fetuses were randomized to four groups with either V T ventilation to 6 ml/kg or continuous positive airway pressure of 5 cm h 2 O, and either 95%O 2 /5%cO 2 or 95%N 2 /5%cO 2 . age-matched fetuses were used as controls. after a 15-min intervention, the fetal lamb was returned to the uterus for 1 h 45 min. results: In ventilated lambs, V T was 6.2 ± 0.4 ml/kg at 15 min. Ventilation increased proinflammatory cytokines as compared with controls and lambs on continuous positive airway pressure, with recruitment of primarily monocytes to bronchoalveolar lavage fluid. early response protein 1 was activated around the bronchioles in V T -ventilated animals. The 15-min oxygen exposure did not change inflammatory mediators or other markers of lung and oxidative stress. conclusion: a V T of 6-7 ml/kg at birth increased early markers of injury and lung inflammation. Brief exposure to 95% oxygen did not alter lung inflammation.
American Journal of Obstetrics and Gynecology, 2010
Objectives-We hypothesized that fetal LPS exposures to the chorioamnion, lung or gut would induce... more Objectives-We hypothesized that fetal LPS exposures to the chorioamnion, lung or gut would induce distinct systemic inflammatory responses. Study Design-Groups of 5-7 time-mated ewes were used to surgically isolate the fetal respiratory and the gastrointestinal systems from the amniotic compartment. Outcomes were assessed at 124d gestational age, 2d and 7 d after LPS (10 mg, E.coli 055:B5) or saline infusions into the fetal airways or amniotic fluid. Results-LPS induced systemic inflammatory changes in all groups in the blood, lung, liver, and thymic lymphocytes. Changes in lymphocytes in the posterior mediastinal lymph node draining lung and gut, occurred only after direct contact of LPS with the fetal lung or gut. Conclusions-Fetal systemic inflammatory responses occurred after chorioamnion, lung or gut exposures to LPS. The organ responses differed based on route of the fetal exposure.
Ureaplasma infection of the amniotic cavity is associated with adverse postnatal intestinal outco... more 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.
Corticosteroids are routinely administered to women at risk for preterm delivery to induce fetal ... more Corticosteroids are routinely administered to women at risk for preterm delivery to induce fetal lung maturation. Antenatal corticosteroids have immunomodulatory effects on fetal immune cells that are poorly understood. We hypothesized that maternal betamethasone would alter in fetal monocytes both the initiation of inflammation in response to pro-inflammatory stimuli and the resolution of inflammation by phagocytosis of apoptotic neutrophils. Preterm lambs at 124 d gestation were delivered 15 h, 1 d, 2 d, or 7 d after 0.5 mg/kg maternal intramuscular betamethasone. Monocytes from cord blood were isolated and cultured and results were compared with monocytes from preterm lambs exposed to maternal saline or monocytes from adult sheep. Phagocytosis of Escherichia coli was not changed, however, phagocytosis of apoptotic neutrophils was low in fetal monocytes but increased after 7 d exposure to maternal betamethasone to the level found in adult monocytes. Hydrogen peroxide production after endotoxin stimulus was significantly reduced to 7.1 Ϯ 2.2 mol at 5 h, 8.7 Ϯ 2.9 mol at 24 h, and 4.1 Ϯ 1.9 mol at 48 h versus 16.4 Ϯ 3.6 mol in control animals; at 7 d, the hydrogen peroxide production increased to 74.3 Ϯ 19.7 mol (p Ͻ 0.05, per 10 6 monocytes). IL-6 production was reduced at 15 h after maternal betamethasone but at no other time point. Maternal betamethasone initially suppressed several fetal monocyte functions, however, at 7 d, measurements of initiation and resolution of inflammation were increased to levels similar to monocytes from adult sheep. The time-dependent changes in maternal betamethasone modulation of the responses of fetal monocytes may influence immune function of the preterm lamb after delivery. (Pediatr Res 55: 764-768, 2004) Abbreviations BPD, bronchopulmonary dysplasia GA, gestational age
American Journal of Obstetrics and Gynecology, 2008
Objective-To determine effects in late gestation of U. parvum serovar 3 colonization, and effects... more Objective-To determine effects in late gestation of U. parvum serovar 3 colonization, and effects, preterm, of U. parvum serovar 6. Study design-Ewes received an intra-amniotic (IA) injection of U. parvum serovar 6 (20x10 6 cfu; n=9), U. parvum serovar 3 (20x10 3 cfu; n=6), vehicle (n=10) or saline (n=4) on day 80 of pregnancy (d). Lambs were delivered at 125d (ureaplasma, n=9; saline or media controls, n=9) or 145d (ureaplasma, n=6; media controls, n=5) for assessment of inflammation and lung maturation. Results-IA ureaplasmas caused histologic chorioamnionitis but not preterm delivery. Fetal lung epithelium was colonized with ureaplasmas at both gestational ages and pulmonary IL-8 levels had doubled in the ureaplasma-colonized animals compared to controls at 145d. Surfactant levels in bronchoalveolar lavage fluid had increased 8 fold and 2.5 fold at 125 and 145d, respectively after ureaplasma injection. Conclusions-Fetal lung inflammation and altered development accompanies ureaplasma colonization regardless of age at delivery.
We investigated the effects of intra-amniotic (IA) betamethasone or budesonide injections on lung... more We investigated the effects of intra-amniotic (IA) betamethasone or budesonide injections on lung structure 2 or 7 d after treatment in preterm fetal sheep. Pregnant ewes received intra-amniotic betamethasone (2 mg/kg fetal weight), budesonide (2 mg/kg), saline or maternal intramuscular betamethasone (0.5 mg/kg maternal weight), or saline. Lambs were delivered 2 or 7 d later at 124 d of gestation. Morphometric analysis was conducted on the right upper lung lobe. Intra-amniotic corticosteroids, 2 or 7 d before delivery, resulted in higher average alveolar volumes than controls (2 d: 25%-35%, 7d: 15%-25%). All corticosteroid treatments resulted in thinning of alveolar walls 7 d after treatment and a higher proportion of alveolar ducts and a lower alveolar wall fraction relative to controls 2 or 7 d after treatment. The changes in structural lung indices correlated with the improved lung function at 2 d. Structural lung indices and increased surfactant correlated with the improved lung function at 7 d. Similar structural changes induced by intra-amniotic corticosteroids and maternal intramuscular betamethasone were associated with improvements in lung function at 2 and 7 d. (Pediatr Res 62: 32-36, 2007
Inflammation is a defensive process by which the body responds to both localized and systemic tis... more Inflammation is a defensive process by which the body responds to both localized and systemic tissue damage by the induction of innate and adaptive immunity. Literature from human and animal studies links inappropriate in utero inflammation to preterm parturition and fetal injury. The pathways by which such inflammation may cause labor, however, are not fully understood. Any proinflammatory agonist in the amniotic fluid will contact the fetal skin, in its entirety, but a potential role of the fetal skin in the pathways to labor have not previously been explored. We hypothesized that the fetal skin would respond robustly to the presence of intra-amniotic lipopolysaccharide (LPS) in our ovine model of in utero inflammation. In vitro and in utero exposure of fetal ovine keratinocytes or fetal skin to Escherichia coli LPS reliably induced significant increases in interleukin 1b (IL-1b), IL-6, tumor necrosis factor a (TNF-a), and IL-8 expression. We demonstrate that, in utero, this expression requires direct exposure with LPS suggesting that the inflammation is triggered directly in the skin itself, rather than as a secondary response to a systemic stimuli and that inflammation involves Toll-like receptor (TLR) regulation and neutrophil chemotaxis in concordance with an acute inflammatory reaction. We show that this response involves multiple inflammatory mediators, TLR regulation, and localized inflammatory cell influx characteristic of an acute inflammatory reaction. These novel data strongly suggests that the fetal skin acts as an important mediator of the fetal inflammatory response and as such may contribute to preterm birth.
Objective: To determine the effect of intrauterine inflammation on fetal responses to umbilical c... more Objective: To determine the effect of intrauterine inflammation on fetal responses to umbilical cord occlusion (UCO). Study Design: In pregnant sheep, lipopolysaccharide (LPS) or saline (SAL) was infused intra-amniotically for 4 weeks from 80 days of gestation (d). At 110 d, fetuses were instrumented for UCOs (5 Â 2-minutes, 30-minute intervals: LPS þ UCO, n ¼ 6; SAL þ UCO, n ¼ 8) or no UCO (sham, n ¼ 6) on 117 and 118 d. Tissues were collected at 126 d. Results: Fetal physiological responses to UCO were similar between LPS þ UCO and SAL þ UCO. Histologic chorioamnionitis and increased amniotic fluid interleukin 8 (IL-8) were observed in LPS þ UCO pregnancies (versus SAL þ UCO, P < .05). CNPase-positive oligodendrocyte number in the cerebral white matter was lower in LPS þ UCO and SAL þ UCO than sham (P < .05); there was no effect on astrocytes or activated microglia/macrophages. Two of the SAL þ UCO fetuses had white matter lesions; none were observed in LPS þ UCO or sham. Conclusion: Chronic pre-existing intrauterine inflammation did not exacerbate fetal brain injury induced by intermittent UCO.
Erythromycin is the standard antibiotic used for treatment of infection with Ureaplasma spp. duri... more Erythromycin is the standard antibiotic used for treatment of infection with Ureaplasma spp. during pregnancy; however, maternally administered erythromycin may be ineffective at eliminating intra-amniotic ureaplasma infections. We examined whether erythromycin would eradicate intra-amniotic ureaplasma infections in pregnant sheep. At Gestational Day (GD) 50 (term, GD 150), pregnant ewes received intra-amniotic injections of erythromycin-sensitive Ureaplasma parvum serovar 3 (n ¼ 16) or 10B medium (n ¼ 16). At GD 100, amniocentesis was performed; five fetal losses (ureaplasma group, n ¼ 4; 10B group, n ¼ 1) had occurred by this time. Remaining ewes were allocated into treatment subgroups: medium only (n ¼ 7), medium and erythromycin (n ¼ 8), ureaplasma only (Up; n ¼ 6), or ureaplasma and erythromycin (Up/E; n ¼ 6). Erythromycin was administered intramuscularly (500 mg) every 8 h for 4 days (GDs 100-104). Amniotic fluid samples were collected at GD 105. At GD 125, preterm fetuses were surgically delivered, and specimens were collected for culture and histology. Erythromycin was quantified in amniotic fluid by liquid chromatographymass spectrometry. Ureaplasmas were isolated from the amniotic fluid, chorioamnion, and fetal lung of animals from the Up and Up/E groups, however, the numbers of U. parvum recovered were not different between these groups. Inflammation in the chorioamnion, cord, and fetal lung was increased in ureaplasma-exposed animals compared to controls but was not different between the Up and Up/E groups. Erythromycin was detected in amniotic fluid samples, although concentrations were low (,10-76 ng/ml). This study demonstrates that maternally administered erythromycin does not eradicate chronic, intra-amniotic ureaplasma infections or improve fetal outcomes in an ovine model, potentially because of the poor placental passage of erythromycin.
Toll-like receptors (TLRs) are pattern recognition molecules that initiate innate immune response... more Toll-like receptors (TLRs) are pattern recognition molecules that initiate innate immune responses. Intra-amniotic exposure of fetal sheep to pro-inflammatory stimuli causes pulmonary inflammation and induced lung maturation. We examined TLR ontogeny and fetal lung responsiveness to three different TLR agonists. We cloned ovine TLRs 2, 3, and 4 and found 83-88% homology between these ovine and human TLRs. Lung TLR2 and 4 mRNAs increased throughout late gestation to 50% of adult level in the term newborn lamb. Doses of 10 mg of PAMCysK 4 (TLR2 agonist), poly I:C dsRNA (TLR3 agonist), or E. coli O55:B5 lipopoysaccharide (LPS) (TLR4 agonist) were given by intra-amniotic injection 2 d or 7 d before operative delivery of preterm lambs at 123 d (n ϭ 4 -7/group). The TLR4 agonist induced lung inflammation and maturation, whereas the TLR2 agonist gave less consistent responses. Intra-amniotic LPS increased TLR2 mRNA expression primarily in the inflammatory cells and TLR4 mRNA diffusely in multiple cell types. The TLR3 agonist had no effects, and TLR3 mRNA in the fetal lung did not change after LPS exposure. We conclude that TLR2 and TLR4 mRNAs increase through gestation and expression of TLR2 and TLR4 are induced by LPS in the fetal sheep lung. (Pediatr Res 63: 388-393, 2008) C horioamnionitis is the most common risk factor associ- ated with extremely premature births (1). Clinically, fetal exposure to chorioamnionitis is associated with early lung maturation and increased survival (2,3), but also with an increased risk for bronchopulmonary dysplasia in some clinical studies (4). Chorioamnionitis at early gestation ages is usually caused by organisms with relatively low pathogenicity, such as Ureaplasma, Mycoplasma, and Gardenella species (1). The ascending infection may be chronic and not apparent clinically (5). This chronic fetal exposure to organisms/infection can induce fetal responses that range from minimal systemic fetal inflammatory responses to sepsis (3). How the fetus recognizes and responds to infection remains essentially unstudied. Toll-like receptors (TLR) are a family of extracellular and intracellular receptors that recognize specific pathogenassociated molecular motifs (6). TLRs are expressed by both
Background: Although foetal pulmonary trunk (PT) blood pressure may exceed aortic trunk (AoT) pre... more Background: Although foetal pulmonary trunk (PT) blood pressure may exceed aortic trunk (AoT) pressure, the specifi c mechanism(s) underlying this pressure difference remain undefi ned. Method: Nine anaesthetised foetal sheep were instrumented with PT and AoT micromanometer catheters to measure high-fi delity pressure (P) and transit-time fl ow probes to obtain blood velocity (U). The PT-AoT instantaneous pressure difference (IPD) was calculated from PT and AoT P profi les. PT and AoT wave intensity (WI) was derived from the product of P and U rates of change. Results: While diastolic pressures were near-identical, systolic PT pressure exceeded AoT pressure (P < 0.001), with a maximal IPD of 6.5 ± 2.5 mmHg. Comparison of IPD with wave-related PT and AoT pressure changes indicated that 1) a greater pressure-generating effect of the PT forwardrunning compression wave arising from impulsive right ventricular (RV) contraction in early and mid-systole accounted for 2.3 ± 2.3 mmHg (35%) of the maximal IPD, and 2) a larger pressure-generating effect of a large mid-systolic backward-running compression wave transmitted into the PT from the pulmonary vasculature contributed 4.0 ± 1.5 mmHg (∼60%) of the maximal IPD. Conclusions: A higher PT-than-AoT blood pressure in foetal lambs is a systolic phenomenon mainly related to a relatively higher level of RV pump function manifest in early and mid-systole, and a pressure-increasing energy wave arising from the foetal pulmonary vasculature in mid-systole.
BackgroundOptimizing respiratory support after birth requires real-time feedback on lung aeration... more BackgroundOptimizing 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.MethodsNear-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).ResultsLung aeration, measured using LUS, and the AaDO2 improved over the first 4 h after birth. The increas...
Prophylactic creatine treatment may reduce hypoxic brain injury due to its ability to sustain int... more Prophylactic creatine treatment may reduce hypoxic brain injury due to its ability to sustain intracellular ATP levels thereby reducing oxidative and metabolic stress responses during oxygen deprivation. Using microdialysis, we investigated the real‐time in vivo effects of fetal creatine supplementation on cerebral metabolism following acute in utero hypoxia caused by umbilical cord occlusion (UCO). Fetal sheep (118 days’ gestational age (dGA)) were implanted with an inflatable Silastic cuff around the umbilical cord and a microdialysis probe inserted into the right cerebral hemisphere for interstitial fluid sampling. Creatine (6 mg kg−1 h−1) or saline was continuously infused intravenously from 122 dGA. At 131 dGA, a 10 min UCO was induced. Hourly microdialysis samples were obtained from −24 to 72 h post‐UCO and analysed for percentage change of hydroxyl radicals (•OH) and interstitial metabolites (lactate, pyruvate, glutamate, glycerol, glycine). Histochemical markers of protein a...
Dopamine is often used to treat hypotension in preterm infants; these infants are at risk of deve... more 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 90 days of gestation (0.6 of term, term = 147 days) 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 5 days after surgery, dopamine (10 lg/kg/min, n = 7 fetuses) or saline (n = 9 fetuses) was infused for 74 h. Two hours after commencing the dopamine/saline infusion, we induced umbilical cord occlusion (UCO) for up to 25 min to produce fetal asphyxia. Fetuses were allowed to recover, and brains were collected 72 h later for assessment of neuropathology. Unoperated 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 agematched controls. In UCO + dopamine fetuses microglial density and oxidative stress in the cerebral white matter and caudate nucleus were not different to that of agematched 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 hypoxiainduced 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.
BJOG: An International Journal of Obstetrics & Gynaecology, 2007
Objective To determine the role of carotid sinus innervation on differential fetal organ growth ... more 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 posit...
feasibility of fetal cardiac access using a percutaneous transhepatic approach in the mid-gestati... more feasibility of fetal cardiac access using a percutaneous transhepatic approach in the mid-gestational fetal lamb, as a model for human fetus. Method Eight fetal lambs of 95-97 days gestation (term 147 days) were studied. Under ultrasound guidance, the fetal right hepatic vein was punctured percutaneously, using a 16GA intravenous cannula (BD Angiocath). A coronary catheter (FineCross TM MG, Terumo) was inserted into the 16 GA cannula, over a 0.014inch guidewire, and advanced into the inferior vena cava and the right atrium. Contrast was injected to document position of the catheter. Three fetal lambs were to be euthanized at the end of the procedure to evaluate blood loss. The rest were to deliver vaginally at term, and euthanized for postmortem examination. Results Percutaneous fetal cardiac access was successful in 7 out of the 8 fetuses. All 4 heart chambers were catheterized in the last 3 fetuses. One fetus died during the procedure, post-mortem showed pericardial and peritoneal haemorrhage. The other two fetuses dedicated for immediate post-mortem, after having survived the procedure, had small haemoperitoneum. Averaged fetal weight was 1027±153g. All other lambs were born normally at term. Conclusion Ultrasound-guided fetal cardiac catheterization through a percutaneous transhepatic approach is feasible. Our experience provides a potentially safer route for human fetal cardiac intervention at midgestational age.
Background: Delaying umbilical cord clamping until after aeration of the lung (physiological-base... more Background: Delaying umbilical cord clamping until after aeration of the lung (physiological-based cord clamping; PBCC) maintains cardiac output and oxygenation in preterm lambs at birth, however, its efficacy after intrauterine inflammation is not known. Given the high incidence of chorioamnionitis in preterm infants, we investigated whether PBCC conferred any benefits compared to immediate cord clamping (ICC) in preterm lambs exposed antenatally to 7 days of intrauterine inflammation. Methods: Ultrasound guided intraamniotic injection of 20 mg Lipopolysaccharide (from E. coli:055:B5) was administered to pregnant ewes at 0.8 gestation. Seven days later, ewes were anesthetized, preterm fetuses exteriorised via cesarean section, and instrumented for continuous measurement of pulmonary, systemic and cerebral pressures and flows, and systemic, and cerebral oxygenation. Lambs were then randomized to either PBCC, whereupon ventilation was initiated and maintained for 3 min prior to umbilical cord clamping, or ICC where the umbilical cord was cut and ventilation initiated 30 s later. Ventilation was maintained for 30 min. Results: ICC caused a rapid fall in systemic (by 25%) and cerebral (by 11%) oxygen saturation in ICC lambs, concurrent with a rapid increase in carotid arterial pressure and heart rate. The overshoot in carotid arterial pressure was sustained in ICC lambs for the first 20 min of the study. PBCC maintained cardiac output and prevented the fall in cerebral oxygen delivery at birth. PBCC lambs had lower respiratory compliance and higher respiratory requirements throughout the study. PBCC mitigated the adverse effects of ICC on oxygenation and cardiac output, and therefore could be more beneficial in preterm babies exposed to antenatal inflammation as it maintains cardiac output and oxygen delivery. The increased respiratory requirements require further investigation in this sub-group of preterm infants.
Translational Investigation nature publishing group Background: Preterm infants often receive mec... more Translational Investigation nature publishing group Background: Preterm infants often receive mechanical ventilation and oxygen at birth. exposure to large tidal volumes (V T s) at birth causes lung inflammation, and oxygen may amplify the injury. We hypothesized that normal V T ventilation at birth causes lung injury that is exacerbated by 95% oxygen. Methods: The head and chest of anesthetized preterm fetal sheep (129 ± 1 d gestation) were surgically exteriorized while maintaining the placental circulation. Fetuses were randomized to four groups with either V T ventilation to 6 ml/kg or continuous positive airway pressure of 5 cm h 2 O, and either 95%O 2 /5%cO 2 or 95%N 2 /5%cO 2 . age-matched fetuses were used as controls. after a 15-min intervention, the fetal lamb was returned to the uterus for 1 h 45 min. results: In ventilated lambs, V T was 6.2 ± 0.4 ml/kg at 15 min. Ventilation increased proinflammatory cytokines as compared with controls and lambs on continuous positive airway pressure, with recruitment of primarily monocytes to bronchoalveolar lavage fluid. early response protein 1 was activated around the bronchioles in V T -ventilated animals. The 15-min oxygen exposure did not change inflammatory mediators or other markers of lung and oxidative stress. conclusion: a V T of 6-7 ml/kg at birth increased early markers of injury and lung inflammation. Brief exposure to 95% oxygen did not alter lung inflammation.
American Journal of Obstetrics and Gynecology, 2010
Objectives-We hypothesized that fetal LPS exposures to the chorioamnion, lung or gut would induce... more Objectives-We hypothesized that fetal LPS exposures to the chorioamnion, lung or gut would induce distinct systemic inflammatory responses. Study Design-Groups of 5-7 time-mated ewes were used to surgically isolate the fetal respiratory and the gastrointestinal systems from the amniotic compartment. Outcomes were assessed at 124d gestational age, 2d and 7 d after LPS (10 mg, E.coli 055:B5) or saline infusions into the fetal airways or amniotic fluid. Results-LPS induced systemic inflammatory changes in all groups in the blood, lung, liver, and thymic lymphocytes. Changes in lymphocytes in the posterior mediastinal lymph node draining lung and gut, occurred only after direct contact of LPS with the fetal lung or gut. Conclusions-Fetal systemic inflammatory responses occurred after chorioamnion, lung or gut exposures to LPS. The organ responses differed based on route of the fetal exposure.
Ureaplasma infection of the amniotic cavity is associated with adverse postnatal intestinal outco... more 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.
Corticosteroids are routinely administered to women at risk for preterm delivery to induce fetal ... more Corticosteroids are routinely administered to women at risk for preterm delivery to induce fetal lung maturation. Antenatal corticosteroids have immunomodulatory effects on fetal immune cells that are poorly understood. We hypothesized that maternal betamethasone would alter in fetal monocytes both the initiation of inflammation in response to pro-inflammatory stimuli and the resolution of inflammation by phagocytosis of apoptotic neutrophils. Preterm lambs at 124 d gestation were delivered 15 h, 1 d, 2 d, or 7 d after 0.5 mg/kg maternal intramuscular betamethasone. Monocytes from cord blood were isolated and cultured and results were compared with monocytes from preterm lambs exposed to maternal saline or monocytes from adult sheep. Phagocytosis of Escherichia coli was not changed, however, phagocytosis of apoptotic neutrophils was low in fetal monocytes but increased after 7 d exposure to maternal betamethasone to the level found in adult monocytes. Hydrogen peroxide production after endotoxin stimulus was significantly reduced to 7.1 Ϯ 2.2 mol at 5 h, 8.7 Ϯ 2.9 mol at 24 h, and 4.1 Ϯ 1.9 mol at 48 h versus 16.4 Ϯ 3.6 mol in control animals; at 7 d, the hydrogen peroxide production increased to 74.3 Ϯ 19.7 mol (p Ͻ 0.05, per 10 6 monocytes). IL-6 production was reduced at 15 h after maternal betamethasone but at no other time point. Maternal betamethasone initially suppressed several fetal monocyte functions, however, at 7 d, measurements of initiation and resolution of inflammation were increased to levels similar to monocytes from adult sheep. The time-dependent changes in maternal betamethasone modulation of the responses of fetal monocytes may influence immune function of the preterm lamb after delivery. (Pediatr Res 55: 764-768, 2004) Abbreviations BPD, bronchopulmonary dysplasia GA, gestational age
American Journal of Obstetrics and Gynecology, 2008
Objective-To determine effects in late gestation of U. parvum serovar 3 colonization, and effects... more Objective-To determine effects in late gestation of U. parvum serovar 3 colonization, and effects, preterm, of U. parvum serovar 6. Study design-Ewes received an intra-amniotic (IA) injection of U. parvum serovar 6 (20x10 6 cfu; n=9), U. parvum serovar 3 (20x10 3 cfu; n=6), vehicle (n=10) or saline (n=4) on day 80 of pregnancy (d). Lambs were delivered at 125d (ureaplasma, n=9; saline or media controls, n=9) or 145d (ureaplasma, n=6; media controls, n=5) for assessment of inflammation and lung maturation. Results-IA ureaplasmas caused histologic chorioamnionitis but not preterm delivery. Fetal lung epithelium was colonized with ureaplasmas at both gestational ages and pulmonary IL-8 levels had doubled in the ureaplasma-colonized animals compared to controls at 145d. Surfactant levels in bronchoalveolar lavage fluid had increased 8 fold and 2.5 fold at 125 and 145d, respectively after ureaplasma injection. Conclusions-Fetal lung inflammation and altered development accompanies ureaplasma colonization regardless of age at delivery.
We investigated the effects of intra-amniotic (IA) betamethasone or budesonide injections on lung... more We investigated the effects of intra-amniotic (IA) betamethasone or budesonide injections on lung structure 2 or 7 d after treatment in preterm fetal sheep. Pregnant ewes received intra-amniotic betamethasone (2 mg/kg fetal weight), budesonide (2 mg/kg), saline or maternal intramuscular betamethasone (0.5 mg/kg maternal weight), or saline. Lambs were delivered 2 or 7 d later at 124 d of gestation. Morphometric analysis was conducted on the right upper lung lobe. Intra-amniotic corticosteroids, 2 or 7 d before delivery, resulted in higher average alveolar volumes than controls (2 d: 25%-35%, 7d: 15%-25%). All corticosteroid treatments resulted in thinning of alveolar walls 7 d after treatment and a higher proportion of alveolar ducts and a lower alveolar wall fraction relative to controls 2 or 7 d after treatment. The changes in structural lung indices correlated with the improved lung function at 2 d. Structural lung indices and increased surfactant correlated with the improved lung function at 7 d. Similar structural changes induced by intra-amniotic corticosteroids and maternal intramuscular betamethasone were associated with improvements in lung function at 2 and 7 d. (Pediatr Res 62: 32-36, 2007
Inflammation is a defensive process by which the body responds to both localized and systemic tis... more Inflammation is a defensive process by which the body responds to both localized and systemic tissue damage by the induction of innate and adaptive immunity. Literature from human and animal studies links inappropriate in utero inflammation to preterm parturition and fetal injury. The pathways by which such inflammation may cause labor, however, are not fully understood. Any proinflammatory agonist in the amniotic fluid will contact the fetal skin, in its entirety, but a potential role of the fetal skin in the pathways to labor have not previously been explored. We hypothesized that the fetal skin would respond robustly to the presence of intra-amniotic lipopolysaccharide (LPS) in our ovine model of in utero inflammation. In vitro and in utero exposure of fetal ovine keratinocytes or fetal skin to Escherichia coli LPS reliably induced significant increases in interleukin 1b (IL-1b), IL-6, tumor necrosis factor a (TNF-a), and IL-8 expression. We demonstrate that, in utero, this expression requires direct exposure with LPS suggesting that the inflammation is triggered directly in the skin itself, rather than as a secondary response to a systemic stimuli and that inflammation involves Toll-like receptor (TLR) regulation and neutrophil chemotaxis in concordance with an acute inflammatory reaction. We show that this response involves multiple inflammatory mediators, TLR regulation, and localized inflammatory cell influx characteristic of an acute inflammatory reaction. These novel data strongly suggests that the fetal skin acts as an important mediator of the fetal inflammatory response and as such may contribute to preterm birth.
Objective: To determine the effect of intrauterine inflammation on fetal responses to umbilical c... more Objective: To determine the effect of intrauterine inflammation on fetal responses to umbilical cord occlusion (UCO). Study Design: In pregnant sheep, lipopolysaccharide (LPS) or saline (SAL) was infused intra-amniotically for 4 weeks from 80 days of gestation (d). At 110 d, fetuses were instrumented for UCOs (5 Â 2-minutes, 30-minute intervals: LPS þ UCO, n ¼ 6; SAL þ UCO, n ¼ 8) or no UCO (sham, n ¼ 6) on 117 and 118 d. Tissues were collected at 126 d. Results: Fetal physiological responses to UCO were similar between LPS þ UCO and SAL þ UCO. Histologic chorioamnionitis and increased amniotic fluid interleukin 8 (IL-8) were observed in LPS þ UCO pregnancies (versus SAL þ UCO, P < .05). CNPase-positive oligodendrocyte number in the cerebral white matter was lower in LPS þ UCO and SAL þ UCO than sham (P < .05); there was no effect on astrocytes or activated microglia/macrophages. Two of the SAL þ UCO fetuses had white matter lesions; none were observed in LPS þ UCO or sham. Conclusion: Chronic pre-existing intrauterine inflammation did not exacerbate fetal brain injury induced by intermittent UCO.
Erythromycin is the standard antibiotic used for treatment of infection with Ureaplasma spp. duri... more Erythromycin is the standard antibiotic used for treatment of infection with Ureaplasma spp. during pregnancy; however, maternally administered erythromycin may be ineffective at eliminating intra-amniotic ureaplasma infections. We examined whether erythromycin would eradicate intra-amniotic ureaplasma infections in pregnant sheep. At Gestational Day (GD) 50 (term, GD 150), pregnant ewes received intra-amniotic injections of erythromycin-sensitive Ureaplasma parvum serovar 3 (n ¼ 16) or 10B medium (n ¼ 16). At GD 100, amniocentesis was performed; five fetal losses (ureaplasma group, n ¼ 4; 10B group, n ¼ 1) had occurred by this time. Remaining ewes were allocated into treatment subgroups: medium only (n ¼ 7), medium and erythromycin (n ¼ 8), ureaplasma only (Up; n ¼ 6), or ureaplasma and erythromycin (Up/E; n ¼ 6). Erythromycin was administered intramuscularly (500 mg) every 8 h for 4 days (GDs 100-104). Amniotic fluid samples were collected at GD 105. At GD 125, preterm fetuses were surgically delivered, and specimens were collected for culture and histology. Erythromycin was quantified in amniotic fluid by liquid chromatographymass spectrometry. Ureaplasmas were isolated from the amniotic fluid, chorioamnion, and fetal lung of animals from the Up and Up/E groups, however, the numbers of U. parvum recovered were not different between these groups. Inflammation in the chorioamnion, cord, and fetal lung was increased in ureaplasma-exposed animals compared to controls but was not different between the Up and Up/E groups. Erythromycin was detected in amniotic fluid samples, although concentrations were low (,10-76 ng/ml). This study demonstrates that maternally administered erythromycin does not eradicate chronic, intra-amniotic ureaplasma infections or improve fetal outcomes in an ovine model, potentially because of the poor placental passage of erythromycin.
Toll-like receptors (TLRs) are pattern recognition molecules that initiate innate immune response... more Toll-like receptors (TLRs) are pattern recognition molecules that initiate innate immune responses. Intra-amniotic exposure of fetal sheep to pro-inflammatory stimuli causes pulmonary inflammation and induced lung maturation. We examined TLR ontogeny and fetal lung responsiveness to three different TLR agonists. We cloned ovine TLRs 2, 3, and 4 and found 83-88% homology between these ovine and human TLRs. Lung TLR2 and 4 mRNAs increased throughout late gestation to 50% of adult level in the term newborn lamb. Doses of 10 mg of PAMCysK 4 (TLR2 agonist), poly I:C dsRNA (TLR3 agonist), or E. coli O55:B5 lipopoysaccharide (LPS) (TLR4 agonist) were given by intra-amniotic injection 2 d or 7 d before operative delivery of preterm lambs at 123 d (n ϭ 4 -7/group). The TLR4 agonist induced lung inflammation and maturation, whereas the TLR2 agonist gave less consistent responses. Intra-amniotic LPS increased TLR2 mRNA expression primarily in the inflammatory cells and TLR4 mRNA diffusely in multiple cell types. The TLR3 agonist had no effects, and TLR3 mRNA in the fetal lung did not change after LPS exposure. We conclude that TLR2 and TLR4 mRNAs increase through gestation and expression of TLR2 and TLR4 are induced by LPS in the fetal sheep lung. (Pediatr Res 63: 388-393, 2008) C horioamnionitis is the most common risk factor associ- ated with extremely premature births (1). Clinically, fetal exposure to chorioamnionitis is associated with early lung maturation and increased survival (2,3), but also with an increased risk for bronchopulmonary dysplasia in some clinical studies (4). Chorioamnionitis at early gestation ages is usually caused by organisms with relatively low pathogenicity, such as Ureaplasma, Mycoplasma, and Gardenella species (1). The ascending infection may be chronic and not apparent clinically (5). This chronic fetal exposure to organisms/infection can induce fetal responses that range from minimal systemic fetal inflammatory responses to sepsis (3). How the fetus recognizes and responds to infection remains essentially unstudied. Toll-like receptors (TLR) are a family of extracellular and intracellular receptors that recognize specific pathogenassociated molecular motifs (6). TLRs are expressed by both
Background: Although foetal pulmonary trunk (PT) blood pressure may exceed aortic trunk (AoT) pre... more Background: Although foetal pulmonary trunk (PT) blood pressure may exceed aortic trunk (AoT) pressure, the specifi c mechanism(s) underlying this pressure difference remain undefi ned. Method: Nine anaesthetised foetal sheep were instrumented with PT and AoT micromanometer catheters to measure high-fi delity pressure (P) and transit-time fl ow probes to obtain blood velocity (U). The PT-AoT instantaneous pressure difference (IPD) was calculated from PT and AoT P profi les. PT and AoT wave intensity (WI) was derived from the product of P and U rates of change. Results: While diastolic pressures were near-identical, systolic PT pressure exceeded AoT pressure (P < 0.001), with a maximal IPD of 6.5 ± 2.5 mmHg. Comparison of IPD with wave-related PT and AoT pressure changes indicated that 1) a greater pressure-generating effect of the PT forwardrunning compression wave arising from impulsive right ventricular (RV) contraction in early and mid-systole accounted for 2.3 ± 2.3 mmHg (35%) of the maximal IPD, and 2) a larger pressure-generating effect of a large mid-systolic backward-running compression wave transmitted into the PT from the pulmonary vasculature contributed 4.0 ± 1.5 mmHg (∼60%) of the maximal IPD. Conclusions: A higher PT-than-AoT blood pressure in foetal lambs is a systolic phenomenon mainly related to a relatively higher level of RV pump function manifest in early and mid-systole, and a pressure-increasing energy wave arising from the foetal pulmonary vasculature in mid-systole.
BackgroundOptimizing respiratory support after birth requires real-time feedback on lung aeration... more BackgroundOptimizing 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.MethodsNear-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).ResultsLung aeration, measured using LUS, and the AaDO2 improved over the first 4 h after birth. The increas...
Prophylactic creatine treatment may reduce hypoxic brain injury due to its ability to sustain int... more Prophylactic creatine treatment may reduce hypoxic brain injury due to its ability to sustain intracellular ATP levels thereby reducing oxidative and metabolic stress responses during oxygen deprivation. Using microdialysis, we investigated the real‐time in vivo effects of fetal creatine supplementation on cerebral metabolism following acute in utero hypoxia caused by umbilical cord occlusion (UCO). Fetal sheep (118 days’ gestational age (dGA)) were implanted with an inflatable Silastic cuff around the umbilical cord and a microdialysis probe inserted into the right cerebral hemisphere for interstitial fluid sampling. Creatine (6 mg kg−1 h−1) or saline was continuously infused intravenously from 122 dGA. At 131 dGA, a 10 min UCO was induced. Hourly microdialysis samples were obtained from −24 to 72 h post‐UCO and analysed for percentage change of hydroxyl radicals (•OH) and interstitial metabolites (lactate, pyruvate, glutamate, glycerol, glycine). Histochemical markers of protein a...
Dopamine is often used to treat hypotension in preterm infants; these infants are at risk of deve... more 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 90 days of gestation (0.6 of term, term = 147 days) 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 5 days after surgery, dopamine (10 lg/kg/min, n = 7 fetuses) or saline (n = 9 fetuses) was infused for 74 h. Two hours after commencing the dopamine/saline infusion, we induced umbilical cord occlusion (UCO) for up to 25 min to produce fetal asphyxia. Fetuses were allowed to recover, and brains were collected 72 h later for assessment of neuropathology. Unoperated 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 agematched controls. In UCO + dopamine fetuses microglial density and oxidative stress in the cerebral white matter and caudate nucleus were not different to that of agematched 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 hypoxiainduced 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.
BJOG: An International Journal of Obstetrics & Gynaecology, 2007
Objective To determine the role of carotid sinus innervation on differential fetal organ growth ... more 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 posit...
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