Podocyte endowment and the impact of adult body size on kidney health.

Low birth weight is a risk factor for chronic kidney disease, whereas adult podocyte depletion is a key event in the pathogenesis of glomerulosclerosis. However, whether low birth weight due to poor maternal nutrition is associated with low podocyte endowment and glomerulosclerosis in later life is not known. Female Sprague-Dawley rats were fed a normal-protein diet (NPD; 20%) or low-protein diet (LPD; 8%), to induce low birth weight, from 3 wk before mating until postnatal day 21 (PN21), when kidneys from some male offspring were taken for quantitation of podocyte number and density in whole glomeruli using immunolabeling, tissue clearing, and confocal microscopy. The remaining offspring were fed a normal- or high-fat diet until 6 mo to induce catch-up growth and excessive weight gain, respectively. At PN21, podocyte number per glomerulus was 15% lower in low birth weight (LPD) than normal birth weight (NPD) offspring, with this deficit greater in outer glomeruli. Surprisingly, podocyte number in LPD offspring increased in outer glomeruli between PN21 and 6 mo, although an overall 9% podocyte deficit persisted. Postnatal fat feeding to LPD offspring did not alter podometric indexes or result in glomerular pathology at 6 mo, whereas fat feeding in NPD offspring was associated with far greater body and fat mass as well as podocyte loss, reduced podocyte density, albuminuria, and glomerulosclerosis. This is the first report that maternal diet can influence podocyte endowment. Our findings provide new insights into the impact of low birth weight, podocyte endowment, and postnatal weight on podometrics and kidney health in adulthood.NEW & NOTEWORTHY The present study shows, for the first time, that low birth weight as a result of maternal nutrition is associated with low podocyte endowment. However, a mild podocyte deficit at birth did not result in glomerular pathology in adulthood. In contrast, postnatal podocyte loss in combination with excessive body weight led to albuminuria and glomerulosclerosis. Taken together, these findings provide new insights into the associations between birth weight, podocyte indexes, postnatal weight, and glomerular pathology.

Early impact of moderate preterm birth on the structure, function and gene expression of conduit arteries.

NEW FINDINGS: What is the central question of this study? What is the immediate impact of moderate preterm birth on the structure and function of major conduit arteries using a pre-clinical sheep model? What is the main finding and its importance? Postnatal changes in conduit arteries, including a significant decrease in collagen within the thoracic aortic wall (predominately males), narrowed carotid arteries, reduced aortic systolic blood flow, and upregulation of the mRNA expression of cell adhesion and inflammatory markers at 2 days of age in preterm lambs compared to controls, may increase the risk of cardiovascular impairment in later life. ABSTRACT: The aim of this work was to compare the structure and function of the conduit arteries of moderately preterm and term-born lambs and to determine whether vascular injury-associated genes were upregulated. Time-mated ewes were induced to deliver either preterm (132 ± 1 days of gestation; n = 11 females and n = 10 males) or at term (147 ± 1 days of gestation; n = 10 females and n = 5 males). Two days after birth, ultrasound imaging of the proximal ascending aorta, main, right and left pulmonary arteries, and right and left common carotid arteries was conducted in anaesthetized lambs. Lambs were then killed and segments of the thoracic aorta and left common carotid artery were either snap frozen for real-time PCR analyses or immersion-fixed for histological quantification of collagen, smooth muscle and elastin within the medial layer. Overall there were few differences in vascular structure between moderately preterm and term lambs. However, there was a significant decrease in the proportion of collagen within the thoracic aortic wall (predominantly in males), narrowing of the common carotid arteries and a reduction in peak aortic systolic blood flow in preterm lambs. In addition, there was increased mRNA expression of the cell adhesion marker P-selectin in the thoracic aortic wall and the pro-inflammatory marker IL-1ß in the left common carotid artery in preterm lambs, suggestive of postnatal vascular injury. Early postnatal differences in the function and structure of conduit arteries and evidence of vascular injury in moderately preterm offspring may place them at greater risk of cardiovascular impairment later in life.

Impact of High-Dose Caffeine on the Preterm Ovine Cerebrum and Cerebellum.

Caffeine is one of the few treatments available for infants with apnea of prematurity. As the recommended dosing regimen is not always sufficient to prevent apnea, higher doses may be prescribed. However, little is currently known about the impact of high-dose caffeine on the developing brain; thus, our aim was to investigate the consequences of a high-dose regimen on the immature ovine brain. High-dose caffeine (25 mg/kg caffeine base loading dose; 20 mg/kg daily maintenance dose; n = 9) or saline (n = 8) was administered to pregnant sheep from 105 to 118 days of gestation (DG; term = 147 days); this is broadly equivalent to 28-33 weeks of human gestation. At 119DG, the cerebral cortex, striatum, and cerebellum were assessed histologically and by immunohistochemistry. Compared with controls, caffeine-exposed fetuses showed (i) an increase in the density of Ctip2-positive layers V-VI projection neurons (p = 0.02), Tbr1-positive layers V-VI projection neurons (p < 0.0001), astrocytes (p = 0.03), and oligodendrocytes (p = 0.02) in the cerebral cortex, (ii) a decrease in the density of Cux1-positive layers II-IV projection neurons (p = 0.01) in the cerebral cortex, and (iii) a reduction in the area of Purkinje cell bodies in the cerebellum (p = 0.03). Comparing high-dose caffeine-exposed fetuses with controls, there was no difference (p > 0.05) in: (i) the volume of the cerebral cortex or striatum, (ii) the density of neurons (total and output projection neurons) in the striatum, (iii) dendritic spine density of layer V pyramidal cells, (iv) the density of cortical GABAergic interneurons, microglia, mature oligodendrocytes or proliferating cells, (v) total cerebellar area or dimensions of cerebellar layers, or (vi) the density of cerebellar white matter microglia, astrocytes, oligodendrocytes, or myelin. Daily exposure of the developing brain to high-dose caffeine affects some aspects of neuronal and glial development in the cerebral cortex and cerebellum in the short-term; the long-term structural and functional consequences of these alterations need to be investigated.

Maternal allergic asthma during pregnancy alters fetal lung and immune development in sheep: potential mechanisms for programming asthma and allergy.

KEY POINTS: Experimental maternal allergic asthma in sheep provides an experimental model in which to test impacts on progeny. Fetuses from allergic asthmatic ewes had fewer surfactant-producing cells in lungs. A greater proportion of lymphocytes from thymus were CD44 positive in fetuses from allergic asthmatic ewes than in controls. These changes to fetal development might contribute to poor neonatal lung function and increased risk of allergy seen in offspring of pregnancies complicated by asthma. ABSTRACT: Asthma is prevalent in pregnancy and increases the risk of disease in offspring, including neonatal respiratory distress and childhood asthma and allergy, but the mechanisms are not understood. We hypothesized that fetal lung structure and immune phenotype in late gestation fetal sheep would be impaired in our sheep model of maternal allergic asthma during pregnancy. Singleton-bearing ewes were either sensitized before pregnancy to house dust mite (HDM, allergic, n = 7) or were non-allergic (control, n = 5). The ewes were subsequently subjected to repeated airway challenges with HDM (allergic group) or saline (control group) throughout gestation. Tissues were collected at 140 ± 1 days gestational age (term, ∼147 days). The density of type II alveolar epithelial cells (surfactant protein C-immunostained) in the lungs was 30% lower in fetuses from allergic ewes than in controls (P < 0.001), but tissue-to-air space ratio and numbers of leucocytes and macrophages were not different between groups. The proportion of CD44+ lymphocytes in the fetal thymus was 3.5-fold higher in fetuses from allergic ewes than in control ewes (P = 0.043). Fewer surfactant-producing type II alveolar epithelial cells may contribute to the increased risk of neonatal respiratory distress in infants of asthmatic mothers, suggesting that interventions to promote lung maturation could improve their neonatal outcomes. If the elevated lymphocyte expression of CD44 persists postnatally, this would confer greater susceptibility to allergic diseases in progeny of asthmatic mothers, consistent with observations in humans. Further experiments are needed to evaluate postnatal phenotypes of progeny and investigate potential interventions.

The therapeutic effect of mesenchymal stem cells on pulmonary myeloid cells following neonatal hyperoxic lung injury in mice.

BACKGROUND: Exposure to high levels of oxygen (hyperoxia) after birth leads to lung injury. Our aims were to investigate the modulation of myeloid cell sub-populations and the reduction of fibrosis in the lungs following administration of human mesenchymal stem cells (hMSC) to neonatal mice exposed to hyperoxia. METHOD: Newborn mice were exposed to 90% O2 (hyperoxia) or 21% O2 (normoxia) from postnatal days 0-4. A sub-group of hyperoxia mice were injected intratracheally with 2.5X105 hMSCs. Using flow cytometry we assessed pulmonary immune cells at postnatal days 0, 4, 7 and 14. The following markers were chosen to identify these cells: CD45+ (leukocytes), Ly6C+Ly6G+ (granulocytes), CD11b+CD11c+ (macrophages); macrophage polarisation was assessed by F4/80 and CD206 expression. hMSCs expressing enhanced green fluorescent protein (eGFP) and firefly luciferase (fluc) were administered via the trachea at day 4. Lung macrophages in all groups were profiled using next generation sequencing (NGS) to assess alterations in macrophage phenotype. Pulmonary collagen deposition and morphometry were assessed at days 14 and 56 respectively. RESULTS: At day 4, hyperoxia increased the number of pulmonary Ly6C+Ly6G+ granulocytes and F4/80lowCD206low macrophages but decreased F4/80highCD206high macrophages. At days 7 and 14, hyperoxia increased numbers of CD45+ leukocytes, CD11b+CD11c+ alveolar macrophages and F4/80lowCD206low macrophages but decreased F4/80highCD206high macrophages. hMSCs administration ameliorated these effects of hyperoxia, notably reducing numbers of CD11b+CD11c+ and F4/80lowCD206low macrophages; in contrast, F4/80highCD206high macrophages were increased. Genes characteristic of anti-inflammatory 'M2' macrophages (Arg1, Stat6, Retnla, Mrc1, Il27ra, Chil3, and Il12b) were up-regulated, and pro-inflammatory 'M1' macrophages (Cd86, Stat1, Socs3, Slamf1, Tnf, Fcgr1, Il12b, Il6, Il1b, and Il27ra) were downregulated in isolated lung macrophages from hyperoxia-exposed mice administered hMSCs, compared to mice without hMSCs. Hydroxyproline assay at day 14 showed that the 2-fold increase in lung collagen following hyperoxia was reduced to control levels in mice administered hMSCs. By day 56 (early adulthood), hMSC administration had attenuated structural changes in hyperoxia-exposed lungs. CONCLUSIONS: Our findings suggest that hMSCs reduce neonatal lung injury caused by hyperoxia by modulation of macrophage phenotype. Not only did our cell-based therapy using hMSC induce structural repair, it limited the progression of pulmonary fibrosis.

Moderate preterm birth affects right ventricular structure and function and pulmonary artery blood flow in adult sheep.

KEY POINTS: Preterm birth occurs when the heart muscle is immature and ill-prepared for the changes in heart and lung function at birth. MRI imaging studies show differences in the growth and function of the heart of young adults born preterm, with the effects more pronounced in the right ventricle. The findings of this study, conducted in sheep, showed that following moderate preterm birth the right ventricular wall was thinner in adulthood, with a reduction in the number and size of the heart muscle cells; in addition, there was impaired blood flow in the main artery leading from the right ventricle to the lungs. The findings indicate that being born only a few weeks early adversely affects the cellular structure of the right ventricle and blood flow to the lungs in adulthood. The reduced number of heart muscle cells has the potential to deleteriously affect right ventricular growth potential and function. ABSTRACT: Preterm birth prematurely exposes the immature heart to the haemodynamic transition at birth, which has the potential to induce abnormal cardiac remodelling. Magnetic resonance imaging studies in young adults born preterm have shown abnormalities in the gross structure of the ventricles (particularly the right ventricle; RV), but the cellular basis of these alterations is unknown. The aim of this study, conducted in sheep, was to determine the effect of moderate preterm birth on RV cellular structure and function in early adulthood. Male singleton lambs were delivered moderately preterm (132 ± 1 days; n = 7) or at term (147 ± 1 days; n = 7). At 14.5 months of age, intra-arterial blood pressure and heart rate were measured. Pulmonary artery diameter and peak systolic blood flow were determined using ultrasound imaging, and RV stroke volume and output calculated. Cardiomyocyte number, size, nuclearity and levels of cardiac fibrosis were subsequently assessed in perfusion-fixed hearts using image analysis and stereological methods. Blood pressure (systolic, diastolic and mean), heart rate, levels of myocardial fibrosis and RV stroke volume and output were not different between groups. There was, however, a significant reduction in RV wall thickness in preterm sheep, and this was accompanied by a significant reduction in peak systolic blood flow in the pulmonary artery and in RV cardiomyocyte number. Cellular changes in the RV wall and reduced pulmonary artery blood flow following preterm birth have the potential to adversely affect cardiac and respiratory haemodynamics, especially when the cardiovascular system is physiologically or pathologically challenged.

Impact of preterm birth on the developing myocardium of the neonate.

BackgroundGlobally, ∼10% of infants are born before full term. Preterm birth exposes the heart to the demands of postnatal cardiovascular function before cardiac development is complete. Our aim was to examine, in hearts collected from infants at autopsy, the effects of preterm birth on myocardial structure and on cardiomyocyte development.Methods and resultsHeart tissue was collected at perinatal autopsies of 16 infants who died following preterm birth between 23 and 36 weeks of gestation, and survived for 1-42 days; the hearts of 37 appropriately grown stillborn infants, aged 20-40 weeks of gestation, were used for comparison. Using confocal microscopy and image analysis, cardiomyocyte proliferation, maturation, ploidy, and size were quantified, and interstitial collagen and myocardial capillarization were measured. Preterm birth resulted in a marked reduction in the proliferation of cardiomyocytes relative to age-matched stillborn infant controls (preterm vs. control P<0.0001). In contrast, preterm birth did not affect heart weight, capillarization, interstitial collagen or cardiomyocyte maturation, ploidy, and size.ConclusionsPreterm birth appears to lead to an abrupt reduction in cardiomyocyte cell division. This reduced cardiomyocyte proliferation in preterm infants may adversely impact upon the final number of cardiomyocytes which may reduce cardiac functional reserve, and impair the reparative capacity of the myocardium.

Erythropoietin Protects Against Lipopolysaccharide-Induced Microgliosis and Abnormal Granule Cell Development in the Ovine Fetal Cerebellum.

Erythropoietin (EPO) ameliorates inflammation-induced injury in cerebral white matter (WM). However, effects of inflammation on the cerebellum and neuroprotective effects of EPO are unknown. Our aims were to determine: (i) whether lipopolysaccharide (LPS)-induced intrauterine inflammation causes injury to, and/or impairs development of the cerebellum; and (ii) whether recombinant human EPO (rhEPO) mitigates these changes. At 107 ± 1 days gestational age (DGA; ~0.7 of term), fetal sheep received LPS (~0.9 µg/kg; i.v.) or an equivalent volume of saline, followed 1 h later with 5000 IU/kg rhEPO (i.v.) or an equivalent volume of saline (i.v.). This generated the following experimental groups: control (saline + saline; n = 6), LPS (LPS + saline, n = 8) and LPS + rhEPO (n = 8). At necropsy (116 ± 1 DGA; ~0.8 of term) the brain was perfusion-fixed and stained histologically (H&E) and immunostained to identify granule cells (Neuronal Nuclei, NeuN), granule cell proliferation (Ki67), Bergmann glia (glial fibrillary acidic protein, GFAP), astrogliosis (GFAP) and microgliosis (Iba-1). In comparison to controls, LPS fetuses had an increased density of Iba-1-positive microglia (p < 0.005) in the lobular WM; rhEPO prevented this increase (p < 0.05). The thickness of both the proliferative (Ki67-positive) and post-mitotic zones (Ki67-negative) of the EGL were increased in LPS-exposed fetuses compared to controls (p < 0.05), but were not different between controls and LPS + rhEPO fetuses. LPS also increased (p < 0.001) the density of granule cells (NeuN-positive) in the internal granule layer (IGL); rhEPO prevented the increase (p < 0.01). There was no difference between groups in the areas of the vermis (total cross-section), molecular layer (ML), IGL or WM, the density of NeuN-positive granule cells in the ML, the linear density of Bergmann glial fibers, the areal density or somal area of the Purkinje cells, the areal coverage of GFAP-positive astrocytes in the lobular and deep WM, the density of Iba-1-positive microglia in the deep WM or the density of apopotic cells in the cerebellum. LPS-induced intrauterine inflammation caused microgliosis and abnormal development of granule cells. rhEPO ameliorated these changes, suggesting that it is neuroprotective against LPS-induced inflammatory effects in the cerebellum.

Does maternal-fetal transfer of creatine occur in pregnant sheep?

Our aim was to determine the disposition of creatine in ovine pregnancy and whether creatine is transferred across the placenta from mother to fetus. Pregnant ewes received either 1) a continuous intravenous infusion of creatine monohydrate or saline from 122 to 131 days gestation, with maternal and fetal arterial blood and amniotic fluid samples collected daily for creatine analysis and fetal tissues collected at necropsy at 133 days for analysis of creatine content, or 2) a single systemic bolus injection of [13C]creatine monohydrate at 130 days of gestation, with maternal and fetal arterial blood, uterine vein blood, and amniotic fluid samples collected before and for 4 h after injection and analyzed for creatine, creatine isotopic enrichment, and guanidinoacetic acid (GAA; precursor of creatine) concentrations. Presence of the creatine transporter-1 (SLC6A8) and l-arginine:glycine amidinotransferase (AGAT; the enzyme synthesizing GAA) proteins were determined by Western blots of placental cotyledons. The 10-day creatine infusion increased maternal plasma creatine concentration three- to fourfold (P < 0.05) without significantly changing fetal arterial, amniotic fluid, fetal tissues, or placental creatine content. Maternal arterial 13C enrichment was increased (P < 0.05) after bolus [13C]creatine injection without change of fetal arterial 13C enrichment. SLC6A8 and AGAT proteins were identified in placental cotyledons, and GAA concentration was significantly higher in uterine vein than maternal artery plasma. Despite the presence of SLC6A8 protein in cotyledons, these results suggest that creatine is not transferred from mother to fetus in near-term sheep and that the ovine utero-placental unit releases GAA into the maternal circulation.

Experimentally Induced Preterm Birth in Sheep Following a Clinical Course of Antenatal Betamethasone: Effects on Growth and Long-Term Survival.

Preterm births account for approximately 10% of births worldwide, with the majority (∼80%) being moderate preterm. Our aim was to determine the effects of moderate preterm birth on survival and long-term growth of male and female offspring using an ovine model of preterm birth that was preceded by a clinically relevant dose of corticosteroids. Ewes were induced to deliver preterm or at term; those assigned to deliver preterm were administered antenatal betamethasone (11.4 mg, 2 doses, 24 hours apart). The growth (body weight and body dimensions) of offspring was monitored to adulthood (62 weeks) when the animals were humanely killed for organ collection. Survival in the immediate period following preterm birth was high (75% for both sexes). However, there were unexpected deaths between 5 and 12 weeks of age, as a result of vitamin E/selenium deficiency; this only occurred in preterm offspring. From birth until adolescence, preterm lambs were lighter than term lambs (controls). After this time, there was gradual catch-up in body weight in preterm females, whereas in preterm males, body weight remained lower than in controls. Preterm sheep were smaller in stature than controls throughout life. This clinically relevant model of preterm birth leads to equally high survival rates in both sexes and is an excellent animal model in which to examine the effects of moderate preterm birth on growth and development of organ systems into adulthood.

Caffeine for apnea of prematurity: Effects on the developing brain.

Caffeine is a methylxanthine that is widely used to treat apnea of prematurity (AOP). In preterm infants, caffeine reduces the duration of respiratory support, improves survival rates and lowers the incidence of cerebral palsy and cognitive delay. There is, however, little evidence relating to the immediate and long-term effects of caffeine on brain development, especially at the cellular and molecular levels. Experimental data are conflicting, with studies showing that caffeine can have either adverse or benefical effects in the developing brain. The aim of this article is to review current understanding of how caffeine ameliorates AOP, the cellular and molecular mechanisms by which caffeine exerts its effects and the effects of caffeine on brain development. A better knowledge of the effects of caffeine on the developing brain at the cellular and/or molecular level is essential in order to understand the basis for the impact of caffeine on postnatal outcome. The studies reviewed here suggest that while caffeine has respiratory benefits for preterm infants, it may have adverse molecular and cellular effects on the developing brain; indeed a majority of experimental studies suggest that regardless of dose or duration of administration, caffeine leads to detrimental changes within the developing brain. Thus there is an urgent need to assess the impact of caffeine, at a range of doses, on the structure and function of the developing brain in preclinical studies, particularly using clinically relevant animal models. Future studies should focus on determining the maximal dose of caffeine that is safe for the preterm brain.

Three-dimensional direct measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections.

Quantitative assessment of myocardial development and disease requires accurate measurement of cardiomyocyte volume, nuclearity (nuclei per cell), and ploidy (genome copies per cell). Current methods require enzymatically isolating cells, which excludes the use of archived tissue, or serial sectioning. We describe a method of analysis that permits the direct simultaneous measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections. To demonstrate the utility of our technique, heart tissue was obtained from four species (rat, mouse, rabbit, sheep) at up to three life stages: prenatal, weaning and adulthood. Thick (40 µm) paraffin sections were stained with Wheat Germ Agglutinin-Alexa Fluor 488 to visualise cell membranes, and DAPI (4',6-diamidino-2-phenylindole) to visualise nuclei and measure ploidy. Previous methods have been restricted to thin sections (2-10 µm) and offer an incomplete picture of cardiomyocytes. Using confocal microscopy and three-dimensional image analysis software (Imaris Version 8.2, Bitplane AG, Switzerland), cardiomyocyte volume, nuclearity, and ploidy were measured. This method of staining and analysis of cardiomyocytes enables accurate morphometric measurements in thick histological sections, thus unlocking the potential of archived tissue. Our novel time-efficient method permits the entire cardiomyocyte to be visualised directly in 3D, eliminating the need for precise alignment of serial sections.

The effects of preterm birth and its antecedents on the cardiovascular system.

INTRODUCTION: Preterm birth occurs in approximately 10% of all births worldwide. It prematurely exposes the developing cardiovascular system to the hemodynamic transition that occurs at birth and to the subsequent functional demands of life ex utero. This review describes the current knowledge of the effects of preterm birth, and some of its common antecedents (chorioamnionitis, intra-uterine growth restriction, and maternal antenatal corticosteroid administration), on the structure of the myocardium. MATERIAL AND METHODS: A thorough literature search was conducted for articles relating to how preterm birth, and its antecedents, affect development of the heart. Given that sheep are an excellent model for the studies of cardiac development, this review has focused on experimental studies in sheep as well as clinical findings. RESULTS: Our review of the literature demonstrates that individuals born preterm are at an increased risk of cardiovascular disease later in life, including increased mean arterial pressure, abnormally shaped and sub-optimally performing hearts and changes in the vasculature. The review highlights how antenatal corticosteroids, intra-uterine growth restriction, and exposure to chorioamnionitis also have the potential to impact cardiac growth in the preterm newborn. CONCLUSIONS: Preterm birth and its common antecedents (antenatal corticosteroids, intra-uterine growth restriction, and chorioamnionitis) have the potential to adversely impact cardiac structure immediately following birth and in later life.

Respiratory adaptation and surfactant composition of unanesthetized male and female lambs differ for up to 8 h after preterm birth.

BACKGROUND: Male preterm infants are more likely to experience respiratory distress syndrome than females. Our objectives were to determine if sex-related differences in physiological adaptation after preterm birth increase with time after birth and if the use of continuous positive airway pressure (CPAP) reduces these differences. METHODS: Unanesthetized lambs (9F, 8M) were delivered at 0.90 of term. Blood gases, metabolites, and cardiovascular and respiratory parameters were monitored in spontaneously breathing lambs for 8 h. Supplemental oxygen was administered via a face mask at 4 cmH2O CPAP. At 8 h, lung compliance was determined, and bronchoalveolar lavage fluid (BALF) was analyzed for total protein and surfactant phospholipids. Surfactant protein (SP) gene expression and protein expression of SP-A and pro-SP-C were determined in lung tissue. RESULTS: For 8 h after delivery, males had significantly lower arterial pH and higher Paco2, and a greater percentage of males were dependent on supplemental oxygen than females. Inspiratory effort was greater and lung compliance was lower in male lambs. Total protein concentration in BALF, SP gene expression, and SP-A protein levels were not different between sexes; pro-SP-C was 24% lower in males. CONCLUSION: The use of CPAP did not eliminate the male disadvantage, which continues for up to 8 h after preterm birth.

Development of an experimental model of maternal allergic asthma during pregnancy.

Maternal asthma during pregnancy adversely affects pregnancy outcomes but identification of the cause/s, and the ability to evaluate interventions, is limited by the lack of an appropriate animal model. We therefore aimed to characterise maternal lung and cardiovascular responses and fetal-placental growth and lung surfactant levels in a sheep model of allergic asthma. Immune and airway functions were studied in singleton-bearing ewes, either sensitised before pregnancy to house dust mite (HDM, allergic, n = 7) or non-allergic (control, n = 5), and subjected to repeated airway challenges with HDM (allergic group) or saline (control group) throughout gestation. Maternal lung, fetal and placental phenotypes were characterised at 140 ± 1 days gestational age (term, ∼147 days). The eosinophil influx into lungs was greater after HDM challenge in allergic ewes than after saline challenge in control ewes before mating and in late gestation. Airway resistance increased throughout pregnancy in allergic but not control ewes, consistent with increased airway smooth muscle in allergic ewes. Maternal allergic asthma decreased relative fetal weight (-12%) and altered placental phenotype to a more mature form. Expression of surfactant protein B mRNA was 48% lower in fetuses from allergic ewes than controls, with a similar trend for surfactant protein D. Thus, allergic asthma in pregnant sheep modifies placental phenotype, and inhibits fetal growth and lung development consistent with observations from human pregnancies. Preconceptional allergen sensitisation and repeated airway challenges in pregnant sheep therefore provides an animal model to identify mechanisms of altered fetal development and adverse pregnancy outcomes caused by maternal asthma in pregnancy.

Exposure to intrauterine inflammation leads to impaired function and altered structure in the preterm heart of fetal sheep.

Intrauterine inflammation is a major contributor to preterm birth and has adverse effects on preterm neonatal cardiovascular physiology. Cardiomyocyte maturation occurs in late gestation in species such as humans and sheep. We tested the hypothesis that intrauterine inflammation has deleterious effects on cardiac function in preterm sheep which might be explained by altered cardiomyocyte proliferation and maturation. Pregnant ewes received an ultrasound-guided intra-amniotic injection of lipopolysaccharide (LPS) or saline 7 days prior to delivery at day 127 of pregnancy (term 147 days). Cardiac contractility was recorded in spontaneously beating hearts of the offspring, perfused in a Langendorff apparatus. Saline-filled latex balloons were inserted into the left ventricle (LV) and right ventricle (RV). Responsiveness to isoprenaline and stop-flow/reperfusion was assessed. In other experiments, hearts were perfusion-fixed, and cardiomyocyte nuclearity, volume and number were determined. ß-Adrenoceptor mRNA levels were determined in unfixed tissue. In hearts of LPS-exposed fetuses, contractility in the LV and RV was suppressed by ~40% and cardiomyocyte numbers were reduced by ~25%. Immature mono-nucleated cardiomyocytes had lower volumes (~18%), whereas mature bi-nucleated cardiomyocyte volume was ~77% greater. Although basal coronary flow was significantly increased by 21±7% in LPS-exposed hearts, following ischaemia/reperfusion (IR), end-diastolic pressure was increased 2.4±0.3-fold and infarct area was increased 3.2±0.6-fold compared with those in controls. Maximum responsiveness to isoprenaline was enhanced by LPS, without an increase in ß-adrenoceptor mRNA, suggesting altered second messenger signalling. Intrauterine inflammation altered cardiac growth, suppressed contractile function and enhanced responsiveness to stress. Although these effects may ensure immediate survival, they probably contribute to the increased vulnerability of organ perfusion in preterm neonates.

Impact of daily high-dose caffeine exposure on developing white matter of the immature ovine brain.

BACKGROUND: Caffeine is widely used to treat apnea of prematurity, but the standard dosing regimen is not always sufficient to prevent apnea. Before higher doses of caffeine can be used, their effects on the immature brain need to be carefully evaluated. Our aim was to determine the impact of daily high-dose caffeine administration on the developing white matter of the immature ovine brain. METHODS: High-dose caffeine (25 mg/kg caffeine base loading dose; 20 mg/kg daily maintenance dose; n = 9) or saline (n = 8) were administered to pregnant sheep from 0.7 to 0.8 of term, equivalent to approximately 27-34 wk in humans. At 0.8 of term, the white and gray matter were assessed histologically and immunohistochemically. RESULTS: Daily caffeine administration led to peak caffeine concentration of 32 mg/l in fetal plasma at 1 h, followed by a gradual decline, with no effects on mean arterial pressure and heart rate. Initial caffeine exposure led to transient, mild alkalosis in the fetus but did not alter oxygenation. At necropsy, there was no effect of daily high-dose caffeine on brain weight, oligodendrocyte density, myelination, axonal integrity, microgliosis, astrogliosis, apoptosis, or neuronal density. CONCLUSION: Daily high-dose caffeine administration does not appear to adversely affect the developing white matter at the microstructural level.

Does lung development differ in male and female fetuses?

Preterm male infants have a higher incidence of morbidity and mortality due to respiratory insufficiency than females of the same gestational age. This male disadvantage could be due to differences in lung architecture; however, few studies have compared lung architecture in male and female fetuses during late gestation. Our principal objectives were to compare the morphology of the fetal lung and the maturity of the surfactant system in preterm male and female fetuses. Lungs from male (n = 9) and female (n = 11) fetal sheep were collected at 0.9 of term (131 days of the 145-day gestation) for morphological and molecular analyses. In separate groups, tracheal liquid was obtained from male (n = 9) and female (n = 9) fetuses at 0.9 of term for determination of surfactant phospholipid composition. We found no sex-related differences in body weight, lung weight, right lung volume, lung tissue and airspace fractions, mean linear intercept, septal crest density, septal thickness, the proportion of proliferating and apoptotic cells, and the percentages of collagen or elastin. The gene expression of surfactant protein -A, -B, -C, and -D and tropoelastin was similar between sexes. There were no differences in the proportion of the major phospholipid classes in the tracheal liquid between sexes; however there was a significantly higher percentage of the phospholipid species phosphatidylinositol 38:5 in males. The greater morbidity and mortality in preterm male lambs do not appear to be related to differences in lung structure or surfactant phospholipid synthesis before birth, but may relate to physiological adaptation to air-breathing at birth.

Chronic intrauterine exposure to endotoxin does not alter fetal nephron number or glomerular size.

A reduced nephron endowment early in life adversely impacts on long-term functional reserve in the kidney. A recent study has shown that acute exposure to chorioamnionitis during late gestation can adversely impact on nephrogenesis. The present study aimed to examine the effects of chronic, low-dose endotoxin exposure in utero, during the period of nephrogenesis, on nephron number and glomerular size in preterm lambs. Ewes were administered either endotoxin (lipopolysaccharide; 1 mg/day) or saline at 110-133 days of gestation (term approximately 147 days) via surgically implanted osmotic minipumps within the amniotic cavity. The ewes were induced to deliver preterm at 133 days gestation and the kidneys of the lambs were analysed at 8 weeks after term-equivalent age. Nephron number per kidney was determined using a combined optical disector and fractionator stereological approach; renal corpuscle size was also measured stereologically. At 8 weeks after term-equivalent age there was no significant effect of in utero exposure to endotoxin on bodyweight or kidney weight and there were no significant differences in nephron number, nephron density or renal corpuscle volume between groups. We conclude that chronic intrauterine inflammation during the period of nephrogenesis may not adversely impact on the number of nephrons formed within the kidney or on the volume of the renal corpuscle.

Alcohol exposure during late ovine gestation alters fetal liver iron homeostasis without apparent dysmorphology.

High levels of alcohol (ethanol) exposure during fetal life can affect liver development and can increase susceptibility to infection after birth. Our aim was to determine the effects of a moderate level of ethanol exposure in late gestation on the morphology, iron status, and inflammatory status of the ovine fetal liver. Pregnant ewes were chronically catheterized at 91 days of gestation (DG; term ~145 DG) for daily intravenous infusion of ethanol (0.75 g/kg maternal body wt; n = 8) or saline (n = 7) over 1 h from 95 to 133 DG. At necropsy (134 DG), fetal livers were collected for analysis. Liver weight, general liver morphology, hepatic cell proliferation and apoptosis, perivascular collagen deposition, and interleukin (IL)-1ß, IL-6, or IL-8 mRNA levels were not different between groups. However, ethanol exposure led to significant decreases in hepatic content of ferric iron and gene expression of the iron-regulating hormone hepcidin and tumor necrosis factor (TNF)-α (all P < 0.05). In the placenta, there was no difference in transferrin receptor, divalent metal transporter 1, and ferritin mRNA levels; however, ferroportin mRNA levels were increased in ethanol-exposed animals (P < 0.05), and ferroportin protein tended to be increased (P = 0.054). Plasma iron concentration was not different between control and ethanol-exposed groups; control fetuses had significantly higher iron concentrations than their mothers, whereas maternal and fetal iron concentrations were similar in ethanol-exposed animals. We conclude that daily ethanol exposure during the third-trimester-equivalent in sheep does not alter fetal liver morphology; however, decreased fetal liver ferric iron content and altered hepcidin and ferroportin gene expression indicate that iron homeostasis is altered.