Intrauterine growth restriction alters the activity of drug metabolising enzymes in the maternal-placental-fetal unit.

PURPOSE: Ten percent of pregnancies are affected by intrauterine growth restriction (IUGR), and evidence suggests that affected neonates have reduced activity of hepatic cytochrome P450 (CYP) drug metabolising enzymes. Given that almost all pregnant individuals take medications and additional medications are often required during an IUGR pregnancy, we aimed to determine the impact of IUGR on hepatic CYP activity in sheep fetuses and pregnant ewes. METHODS: Specific probes were used to determine the impact of IUGR on the activity of several CYP isoenzymes (CYP1A2, CYP2C19, CYP2D6 and CYP3A) in sheep fetuses and pregnant ewes. Probes were administered intravenously to the ewe at 132 days (d) gestation (term 150 d), followed by blood sampling from the maternal and fetal circulation over 24 h. Maternal and fetal liver tissue was collected at 139-140 d gestation, from which microsomes were isolated and incubated with probes. Metabolite and maternal plasma cortisol concentrations were measured using Liquid Chromatography - tandem mass spectrometry (LC-MS/MS). RESULTS: Maternal plasma cortisol concentration and maternal hepatic CYP1A2 and CYP3A activity was significantly higher in IUGR pregnancies. Maternal hepatic CYP activity was higher than fetal hepatic CYP activity for all CYPs tested, and there was minimal CYP1A2 or CYP3A activity in the late gestation fetus when assessed using in vitro methods. CONCLUSIONS: The physiological changes to the maternal-placental-fetal unit in an IUGR pregnancy have significant effects on maternal drug metabolism, suggesting changes in medications and/or doses may be required to optimise maternal and fetal health.

Development of a method to determine cytochrome P450 1A2, 2C9, 2D6 and 3A4 activity sheep hepatic microsomes.

INTRODUCTION: Ex vivo studies of human fetal hepatic drug metabolism are uncommon as it requires access to functional liver tissue and therefore raises practical and ethical concerns. Large animal models provide an alternative opportunity to study changes in cytochrome P450 (CYP) activity in the mother and fetus during pregnancy. We aimed to develop methods to determine the activity of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in sheep hepatic microsomes. METHODS: We identified optimal conditions to determine the activity of CYP1A2 (using the probe drug phenacetin), CYP2C9 (diclofenac), CYP2D6 (dextromethorphan) and CYP3A4 (midazolam) by varying techniques for microsome extraction, probe drug concentration, incubation time and microsome concentration. The specificity of each probe drug was assessed by determining the rate of metabolism when specific CYP enzyme inhibitors were included in the reaction. RESULTS: The optimum incubation time and probe drug concentration was six hours with 5 µM phenacetin (CYP1A2), four hours with 10 µM diclofenac (CYP2C9), 30 min with 1 µM of midazolam (CYP3A4) and 10 min with 1 µM dextromethorphan (CYP2D6). For both CYP2D6 and CYP3A4 reactions required 20 µg of microsomal protein, whereas for CYP1A2 and CYP2C9, reactions required 40 µg of microsomal protein. Metabolism of phenacetin, dextromethorphan and midazolam was reduced by specific enzyme inhibitors, but the specific CYP2C9 inhibitor sulfaphenazole did not substantially inhibit diclofenac metabolism. DISCUSSION: This study identifies the optimal conditions for determining CYP activity in maternal sheep hepatic microsomes. In doing so, we have developed a standardised protocol for assessment of microsomal activity of CYP3A4, CYP1A2 and CYP2D6, but we were unable to optimise conditions for assessment of CYP2C9. This approach can be applied to investigate the impact of pregnancy complications on maternal and fetal hepatic drug metabolism.

The impact of intrauterine growth restriction on cytochrome P450 enzyme expression and activity.

With the increased prevalence of non-communicable disease and availability of medications to treat these and other conditions, a pregnancy free from prescribed medication exposure is rare. Up to 99% of women take at least one medication during pregnancy. These medications can be divided into those used to improve maternal health and wellbeing (e.g., analgesics, antidepressants, antidiabetics, antiasthmatics), and those used to promote the baby's wellbeing in either fetal (e.g., anti-arrhythmics) or postnatal life (e.g., antenatal glucocorticoids). These medications are needed for pre-existing or coincidental illnesses in the mother, maternal conditions induced by the pregnancy itself through to conditions that arise in the fetus or that will be encountered by the newborn. Thus, medications administered to the mother may be used to treat the mother, the fetus or both. Metabolism of medications is regulated by a range of physiological processes that change during pregnancy. Other pathological processes such as placental insufficiency can in turn have both immediate and lifelong adverse health consequences for babies. Individuals born growth restricted are more likely to require medications but may also have an altered ability to metabolise these medications in fetal and postnatal life. This review aims to determine the effect of suboptimal fetal growth on the fetal expression of the drug metabolising enzymes (DMEs) that convert medications into active or inactive metabolites, and the transporters that remove both these medications and their metabolites from the fetal compartment.

Identification of Novel miRNAs Involved in Cardiac Repair Following Infarction in Fetal and Adolescent Sheep Hearts.

AIMS: Animal models have been used to show that there are critical molecular mechanisms that can be activated to induce myocardial repair at specific times in development. For example, specific miRNAs are critical for regulating the response to myocardial infarction (MI) and improving the response to injury. Manipulating these miRNAs in small animal models provides beneficial effects post-MI; however it is not known if these miRNAs are regulated similarly in large mammals. Studying a large animal where the timing of heart development in relation to birth is similar to humans may provide insights to better understand the capacity to repair a developing mammalian heart and its application to the adult heart. METHODS: We used a sheep model of MI that included permanent ligation of the left anterior descending (LAD) coronary artery. Surgery was performed on fetuses (at 105 days gestation when all cardiomyocytes are mononucleated and proliferative) and adolescent sheep (at 6 months of age when all cardiomyocytes contribute to heart growth by hypertrophy). A microarray was utilized to determine the expression of known miRNAs within the damaged and undamaged tissue regions in fetal and adolescent hearts after MI. RESULTS: 73 miRNAs were up-regulated and 58 miRNAs were down-regulated significantly within the fetal infarct compared to remote cardiac samples. From adolescent hearts 69 non-redundant miRNAs were up-regulated and 63 miRNAs were down-regulated significantly in the infarct area compared to remote samples. Opposite differential expression profiles of 10 miRNAs within tissue regions (Infarct area, Border zone and Remote area of the left ventricle) occurred between the fetuses and adolescent sheep. These included miR-558 and miR-1538, which when suppressed using LNA anti-miRNAs in cell culture, increased cardiomyoblast proliferation. CONCLUSION: There were significant differences in miRNA responses in fetal and adolescent sheep hearts following a MI, suggesting that the modulation of novel miRNA expression may have therapeutic potential, by promoting proliferation or repair in a damaged heart.

Methamphetamine administration increases hepatic CYP1A2 but not CYP3A activity in female guinea pigs.

Methamphetamine use has increased over the past decade and the first use of methamphetamine is most often when women are of reproductive age. Methamphetamine accumulates in the liver; however, little is known about the effect of methamphetamine use on hepatic drug metabolism. Methamphetamine was administered on 3 occassions to female Dunkin Hartley guinea pigs of reproductive age, mimicking recreational drug use. Low doses of test drugs caffeine and midazolam were administered after the third dose of methamphetamine to assess the functional activity of cytochrome P450 1A2 and 3A, respectively. Real-time quantitative polymerase chain reaction was used to quantify the mRNA expression of factors involved in glucocorticoid signalling, inflammation, oxidative stress and drug transporters. This study showed that methamphetamine administration decreased hepatic CYP1A2 mRNA expression, but increased CYP1A2 enzyme activity. Methamphetamine had no effect on CYP3A enzyme activity. In addition, we found that methamphetamine may also result in changes in glucocorticoid bioavailability, as we found a decrease in 11ß-hydroxysteroid dehydrogenase 1 mRNA expression, which converts inactive cortisone into active cortisol. This study has shown that methamphetamine administration has the potential to alter drug metabolism via the CYP1A2 metabolic pathway in female guinea pigs. This may have clinical implications for drug dosing in female methamphetamine users of reproductive age.

Normal human and sheep fetal vessel oxygen saturations by T2 magnetic resonance imaging.

KEY POINTS: Human fetal Doppler ultrasound and invasive blood gas measurements obtained by cordocentesis or at the time of delivery reveal similarities with sheep (an extensively used model for human fetal cardiovascular physiology). Oxygen saturation (SO2 ) measurements in human fetuses have been limited to the umbilical and scalp vessels, providing little information about normal regional SO2 differences in the fetus. Blood T2 MRI relaxometry presents a non-invasive measure of SO2 in the major fetal vessels. This study presents the first in vivo validation of fetal vessel T2 oximetry against the in vitro T2-SO2 relationship using catheterized sheep fetuses and compares the normal SO2 in the major vessels between the human and sheep fetal circulations. Human fetal vessel SO2 by T2 MRI confirms many similarities with the sheep fetal circulation and is able to demonstrate regional differences in SO2 ; in particular the significantly higher SO2 in the left versus right heart. ABSTRACT: Blood T2 magnetic resonance imaging (MRI) relaxometry non-invasively measures oxygen saturation (SO2 ) in major vessels but has not been validated in fetuses in vivo. We compared the blood T2-SO2 relationship in vitro (tubes) and in vivo (vessels) in sheep, and measured SO2 across the normal human and sheep fetal circulations by T2. Singleton pregnant ewes underwent surgery to implant vascular catheters. In vitro and in vivo sheep blood T2 measurements were related to corresponding SO2 measured using a blood gas analyser, as well as relating T2 and SO2 of human fetal blood in vitro. MRI oximetry was performed in the major vessels of 30 human fetuses at 36 weeks (term, 40 weeks) and 10 fetal sheep (125 days; term, 150 days). The fidelity of in vivo fetal T2 oximetry was confirmed through comparison of in vitro and in vivo sheep blood T2-SO2 relationships (P = 0.1). SO2 was similar between human and sheep fetuses, as was the fetal oxygen extraction fraction (human, 33 ± 11%; sheep, 34 ± 7%; P = 0.798). The presence of streaming in the human fetal circulation was demonstrated by the SO2 gradient between the ascending aorta (68 ± 10%) and the main pulmonary artery (49 ± 9%; P < 0.001). Human and sheep fetal vessel MRI oximetry based on T2 is a validated approach that confirms the presence of streaming of umbilical venous blood towards the heart and brain. Streaming is important in ensuring oxygen delivery to these organs and its disruption may have important implications for organ development, especially in conditions such as congenital heart disease and fetal growth restriction.

Differential gene responses 3 days following infarction in the fetal and adolescent sheep heart.

There are critical molecular mechanisms that can be activated to induce myocardial repair, and in humans this is most efficient during fetal development. The timing of heart development in relation to birth and the size/electrophysiology of the heart are similar in humans and sheep, providing a model to investigate the repair capacity of the mammalian heart and how this can be applied to adult heart repair. Myocardial infarction was induced by ligation of the left anterior descending coronary artery in fetal (105 days gestation when cardiomyocytes are proliferative) and adolescent sheep (6 mo of age when all cardiomyocytes have switched to an adult phenotype). An ovine gene microarray was used to compare gene expression in sham and infarcted (remote, border and infarct areas) cardiac tissue from fetal and adolescent hearts. The gene response to myocardial infarction was less pronounced in fetal compared with adolescent sheep hearts and there were unique gene responses at each age. There were also region-specific changes in gene expression between each age, in the infarct tissue, tissue bordering the infarct, and tissue remote from the infarction. In total, there were 880 genes that responded to MI uniquely in the adolescent samples compared with 170 genes in the fetal response, as well as 742 overlap genes that showed concordant direction of change responses to infarction at both ages. In response to myocardial infarction, there were specific changes in genes within pathways of mitochondrial oxidation, muscle contraction, and hematopoietic cell lineages, suggesting that the control of energy utilization and immune function are critical for effective heart repair. The more restricted gene response in the fetus may be an important factor in its enhanced capacity for cardiac repair.

Detecting metabolic differences in fetal and adult sheep adipose and skeletal muscle tissues.

The primary metabolic pathway required to produce ATP differs as a result of tissue type, developmental stage and substrate availability. We utilized molecular and histological techniques to define the metabolic status in foetal and adult, adipose and skeletal muscle tissues. Redox ratios of these tissues were also determined optically by two-photon microscopy. Adult perirenal adipose tissue had a higher optical redox ratio than fetal perirenal adipose tissue, which aligned with glycolysis being used for ATP production; whereas adult skeletal muscle had a lower optical redox ratio than fetal skeletal muscle, which aligned with oxygen demanding oxidative phosphorylation activity being utilized for ATP production. We have compared traditional molecular and microscopy techniques of metabolic tissue characterization with optical redox ratios to provide a more comprehensive report on the dynamics of tissue metabolism.

Subcutaneous maternal resveratrol treatment increases uterine artery blood flow in the pregnant ewe and increases fetal but not cardiac growth.

KEY POINTS: Substrate restriction during critical developmental windows of gestation programmes offspring for a predisposition towards cardiovascular disease in adult life. This study aimed to determine the effect of maternal resveratrol (RSV) treatment in an animal model in which chronic fetal catheterisation is possible and the timing of organ maturation reflects that of the human. Maternal RSV treatment increased uterine artery blood flow, fetal oxygenation and fetal weight. RSV was not detectable in the fetal circulation, indicating that it may not cross the sheep placenta. This study highlights RSV as a possible intervention to restore fetal substrate supply in pregnancies affected by placental insufficiency. ABSTRACT: Suboptimal in utero environments with reduced substrate supply during critical developmental windows of gestation predispose offspring to non-communicable diseases such as cardiovascular disease (CVD). Improving fetal substrate supply in these pregnancies may ameliorate the predisposition these offspring have toward adult-onset CVD. This study aimed to determine the effect of maternal resveratrol (RSV) supplementation on uterine artery blood flow and the direct effects of RSV on the fetal heart in a chronically catheterised sheep model of human pregnancy. Maternal RSV treatment significantly increased uterine artery blood flow as measured by phase contrast magnetic resonance imaging, mean gestational fetal PaO2 and SaO2 as well as fetal weight. RSV was not detectable in the fetal circulation, and mRNA and protein expression of the histone/protein deacetylase SIRT1 did not differ between treatment groups. No effect of maternal RSV supplementation on AKT/mTOR or CAMKII signalling in the fetal left ventricle was observed. Maternal RSV supplementation is capable of increasing fetal oxygenation and growth in an animal model in which cardiac development parallels that of the human.

Differential Response to Injury in Fetal and Adolescent Sheep Hearts in the Immediate Post-myocardial Infarction Period.

Aim: Characterizing the response to myocardial infarction (MI) in the regenerative sheep fetus heart compared to the post-natal non-regenerative adolescent heart may reveal key morphological and molecular differences that equate to the response to MI in humans. We hypothesized that the immediate response to injury in (a) infarct compared with sham, and (b) infarct, border, and remote tissue, in the fetal sheep heart would be fundamentally different to the adolescent, allowing for repair after damage. Methods: We used a sheep model of MI induced by ligating the left anterior descending coronary artery. Surgery was performed on fetuses (105 days) and adolescent sheep (6 months). Sheep were randomly separated into MI (n = 5) or Sham (n = 5) surgery groups at both ages. We used magnetic resonance imaging (MRI), histological/immunohistochemical staining, and qRT-PCR to assess the morphological and molecular differences between the different age groups in response to infarction. Results: Magnetic resonance imaging showed no difference in fetuses for key functional parameters; however there was a significant decrease in left ventricular ejection fraction and cardiac output in the adolescent sheep heart at 3 days post-infarction. There was no significant difference in functional parameters between MRI sessions at Day 0 and Day 3 after surgery. Expression of genes involved in glucose transport and fatty acid metabolism, inflammatory cytokines as well as growth factors and cell cycle regulators remained largely unchanged in the infarcted compared to sham ventricular tissue in the fetus, but were significantly dysregulated in the adolescent sheep. Different cardiac tissue region-specific gene expression profiles were observed between the fetal and adolescent sheep. Conclusion: Fetuses demonstrated a resistance to cardiac damage not observed in the adolescent animals. The manipulation of specific gene expression profiles to a fetal-like state may provide a therapeutic strategy to treat patients following an infarction.

Fetal hemodynamics and cardiac streaming assessed by 4D flow cardiovascular magnetic resonance in fetal sheep.

BACKGROUND: To date it has not been possible to obtain a comprehensive 3D assessment of fetal hemodynamics because of the technical challenges inherent in imaging small cardiac structures, movement of the fetus during data acquisition, and the difficulty of fusing data from multiple cardiac cycles when a cardiac gating signal is absent. Here we propose the combination of volumetric velocity-sensitive cardiovascular magnetic resonance imaging ("4D flow" CMR) and a specialized animal preparation (catheters to monitor fetal heart rate, anesthesia to immobilize mother and fetus) to examine fetal sheep cardiac hemodynamics in utero. METHODS: Ten pregnant Merino sheep underwent surgery to implant arterial catheters in the target fetuses. Anesthetized ewes underwent 4D flow CMR with acquisition at 3 T for fetal whole-heart coverage with 1.2-1.5 mm spatial resolution and 45-62 ms temporal resolution. Flow was measured in the heart and major vessels, and particle traces were used to visualize circulatory patterns in fetal cardiovascular shunts. Conservation of mass was used to test internal 4D flow consistency, and comparison to standard 2D phase contrast (PC) CMR was performed for validation. RESULTS: Streaming of blood from the ductus venosus through the foramen ovale was visualized. Flow waveforms in the major thoracic vessels and shunts displayed normal arterial and venous patterns. Combined ventricular output (CVO) was 546 mL/min per kg, and the distribution of flows (%CVO) were comparable to values obtained using other methods. Internal 4D flow consistency across 23 measurement locations was established with differences of 14.2 ± 12.1%. Compared with 2D PC CMR, 4D flow showed a strong correlation (R2 = 0.85) but underestimated flow (bias = - 21.88 mL/min per kg, p < 0.05). CONCLUSIONS: The combination of fetal surgical preparation and 4D flow CMR enables characterization and quantification of complex flow patterns in utero. Visualized streaming of blood through normal physiological shunts confirms the complex mechanism of substrate delivery to the fetal heart and brain. Besides offering insight into normal physiology, this technology has the potential to qualitatively characterize complex flow patterns in congenital heart disease phenotypes in a large animal model, which can support the development of new interventions to improve outcomes in this population.

Feasibility of phase-contrast cine magnetic resonance imaging for measuring blood flow in the sheep fetus.

Phase-contrast cine MRI (PC-MRI) is the gold-standard noninvasive technique for measuring vessel blood flow and has previously been applied in the human fetal circulation. We aimed to assess the feasibility of using PC-MRI to define the distribution of the fetal circulation in sheep. Fetuses were catheterized at 119-120 days of gestation (term, 150 days) and underwent MRI at ∼123 days of gestation under isoflurane anesthesia, ventilated at a FIO2 of 1.0. PC-MRI was performed using a fetal arterial blood pressure catheter signal for cardiac triggering. Blood flows were measured in the major fetal vessels, including the main pulmonary artery, ascending and descending aorta, superior vena cava, ductus arteriosus, left and right pulmonary arteries, umbilical vein, ductus venosus, and common carotid artery and were indexed to estimated fetal weight. The combined ventricular output, pulmonary blood flow, and flow across the foramen ovale were calculated from vessel flows. Intraobserver and interobserver agreement and reproducibility was assessed. Blood flow measurements were successfully obtained in 61 out of 74 vessels (82.4%) interrogated in 9 fetuses. There was good intraobserver [R = 0.998, P < 0.0001; intraclass correlation (ICC) = 0.997] and interobserver agreement (R = 0.996, P < 0.0001; ICC = 0.996). Repeated MRI measurements showed good reproducibility (R = 0.989, P = 0.0002; ICC = 0.990). We conclude that PC-MRI using fetal catheters for gating triggers is feasible in the major vessels of late gestation fetal sheep. This approach may provide a useful new tool for assessing the circulatory characteristics of fetal sheep models of human disease, including fetal growth restriction and congenital heart disease.

Improving pregnancy outcomes in humans through studies in sheep.

Experimental studies that are relevant to human pregnancy rely on the selection of appropriate animal models as an important element in experimental design. Consideration of the strengths and weaknesses of any animal model of human disease is fundamental to effective and meaningful translation of preclinical research. Studies in sheep have made significant contributions to our understanding of the normal and abnormal development of the fetus. As a model of human pregnancy, studies in sheep have enabled scientists and clinicians to answer questions about the etiology and treatment of poor maternal, placental, and fetal health and to provide an evidence base for translation of interventions to the clinic. The aim of this review is to highlight the advances in perinatal human medicine that have been achieved following translation of research using the pregnant sheep and fetus.

Intrauterine growth restriction may reduce hepatic drug metabolism in the early neonatal period.

The effects of intrauterine growth restriction (IUGR) extend well into postnatal life. IUGR is associated with an increased risk of adverse health outcomes, which often leads to greater medication usage. Many medications require hepatic metabolism for activation or clearance, but hepatic function may be altered in IUGR fetuses. Using a sheep model of IUGR, we determined the impact of IUGR on hepatic drug metabolism and drug transporter expression, both important mediators of fetal drug exposure, in late gestation and in neonatal life. In the late gestation fetus, IUGR decreased the gene expression of uptake drug transporter OATPC and increased P-glycoprotein protein expression in the liver, but there was no change in the activity of the drug metabolising enzymes CYP3A4 or CYP2D6. In contrast, at 3 weeks of age, CYP3A4 activity was reduced in the livers of lambs born with low birth weight (LBW), indicating that LBW results in changes to drug metabolising capacity in neonatal life. Together, these results suggest that IUGR may reduce hepatic drug metabolism in fetal and neonatal life through different mechanisms.

Mitochondrial imaging in live or fixed tissues using a luminescent iridium complex.

Mitochondrial morphology is important for the function of this critical organelle and, accordingly, altered mitochondrial structure is exhibited in many pathologies. Imaging of mitochondria can therefore provide important information about disease presence and progression. However, mitochondrial imaging is currently limited by the availability of agents that have the capacity to image mitochondrial morphology in both live and fixed samples. This can be particularly problematic in clinical studies or large, multi-centre cohort studies, where tissue archiving by fixation is often more practical. We previously reported the synthesis of an iridium coordination complex [Ir(ppy)2(MeTzPyPhCN)]+; where ppy is a cyclometalated 2-phenylpyridine and TzPyPhCN is the 5-(5-(4-cyanophen-1-yl)pyrid-2-yl)tetrazolate ligand; and showed that this complex (herein referred to as IraZolve-Mito) has a high specificity for mitochondria in live cells. Here we demonstrate that IraZolve-Mito can also effectively stain mitochondria in both live and fixed tissue samples. The staining protocol proposed is versatile, providing a universal procedure for cell biologists and pathologists to visualise mitochondria.

Does poor fetal growth influence the extent of fetal exposure to maternal medications?

A large proportion of women are prescribed a medication during pregnancy, and the conditions requiring treatment with these medicines are often also associated with placental dysfunction and abnormal fetal growth. For the fetus, exposure to maternal illness or medications can alter fetal growth trajectory, which is a key indicator of fetal and postnatal wellbeing. There is a large amount of human and animal evidence highlighting the hormonal and/or metabolic changes that occur in both the mother and the fetus as a result of maternal illness or either excessive or restricted fetal growth. These changes can affect the expression of drug metabolising enzymes and drug transporters in the both the mother and her fetus, and may ultimately alter fetal drug exposure. This review aims to explore the complex and multidirectional interplay between maternal illness, fetal growth trajectory, maternal drug treatment, and fetal drug exposure.

Feasibility of detecting myocardial infarction in the sheep fetus using late gadolinium enhancement CMR imaging.

BACKGROUND: Late gadolinium enhancement (LGE) cardiovascular magnetic resonance (CMR) imaging has enabled the accurate assessment of myocardial infarction (MI). However, LGE CMR has not been performed successfully in the fetus, where it could be useful for animal studies of interventions to promote cardiac regeneration. We believe that LGE imaging could allow us to document the presence, extent and effect of MI in utero and would thereby expand our capacity for conducting fetal sheep MI research. We therefore aimed to investigate the feasibility of using LGE to detect MI in sheep fetuses. METHODS: Six sheep fetuses underwent a thoracotomy and ligation of a left anterior descending (LAD) coronary artery branch; while two fetuses underwent a sham surgery. LGE CMR was performed in a subset of fetuses immediately after the surgery and three days later. Early gadolinium enhancement (EGE) CMR was also performed in a subset of fetuses on both days. Cine imaging of the heart was performed to measure ventricular function. RESULTS: The imaging performed immediately after LAD ligation revealed no evidence of infarct on LGE (n=3). Two of four infarcted fetuses (50%) showed hypoenhancement at the infarct site on the EGE images. Three days after the ligation, LGE images revealed a clear, hyper-enhanced infarct zone in four of the five infarcted fetuses (80%). No hyper-enhanced infarct zone was seen on the one sham fetus that underwent LGE CMR. No hypoenhancement could be seen in the EGE images in either the sham (n=1) or the infarcted fetus (n=1). No regional wall motion abnormalities were apparent in two of the five infarcted fetuses. CONCLUSION: LGE CMR detected the MI three days after LAD ligation, but not immediately after. Using available methods, EGE imaging was less useful for detecting deficits in perfusion. Our study provides evidence for the ability of a non-invasive tool to monitor the progression of cardiac repair and damage in fetuses with MI. However, further investigation into the optimal timing of LGE and EGE scans and improvement of the sequences should be pursued with the aim of expanding our capacity to monitor cardiac regeneration after MI in fetal sheep.

Normalisation of surfactant protein -A and -B expression in the lungs of low birth weight lambs by 21 days old.

Intrauterine growth restriction (IUGR) induced by placental restriction (PR) in the sheep negatively impacts lung and pulmonary surfactant development during fetal life. Using a sheep model of low birth weight (LBW), we found that there was an increase in mRNA expression of surfactant protein (SP)-A, -B and -C in the lung of LBW lambs but no difference in the protein expression of SP-A or -B. LBW also resulted in increased lysosome-associated membrane glycoprotein (LAMP)-3 mRNA expression, which may indicate an increase in either the density of type II Alveolar epithelial cells (AEC) or maturity of type II AECs. Although there was an increase in glucocorticoid receptor (GR) and 11ß-hydroxysteroid dehydrogenase (11ßHSD)-1 mRNA expression in the lung of LBW lambs, we found no change in the protein expression of these factors, suggesting that the increase in SP mRNA expression is not mediated by increased GC signalling in the lung. The increase in SP mRNA expression may, in part, be mediated by persistent alterations in hypoxia signalling as there was an increase in lung HIF-2α mRNA expression in the LBW lamb. The changes in the hypoxia signalling pathway that persist within the lung after birth may be involved in maintaining SP production in the LBW lamb.