[Show abstract][Hide abstract]ABSTRACT: 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.
[Show abstract][Hide abstract]ABSTRACT: 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.
Materials 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.
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.
Preterm birth and its common antecedents (intra-uterine growth restriction, chorioamnionitis and antenatal corticosteroids) have the potential to adversely impact cardiac structure immediately following birth and in later life. This article is protected by copyright. All rights reserved.
No preview · Article · Feb 2016 · Acta Obstetricia Et Gynecologica Scandinavica
[Show abstract][Hide abstract]ABSTRACT: Background:
Male preterm infants are more likely to experience RDS 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.
Unanaesthetized lambs (9F, 8M) were delivered at 0.90 of term. Blood gases, metabolites, cardiovascular and respiratory parameters were monitored in spontaneously breathing lambs for 8h. Supplemental oxygen was administered via a face mask at 4 cmH2O CPAP. At 8h, lung compliance was determined and bronchoalveolar lavage fluid (BALF) analysed 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.
For 8h after delivery, males had significantly lower arterial pH, higher PaCO2 and a greater percentage of males were dependent on supplemental oxygen than females. Inspiratory effort was greater and lung compliance 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.
The use of CPAP did not eliminate the male disadvantage, which continues for up to 8 hours after preterm birth.Pediatric Research (2015); doi:10.1038/pr.2015.175.
No preview · Article · Sep 2015 · Pediatric Research
[Show abstract][Hide abstract]ABSTRACT: The developing lung is vulnerable to adverse conditions during prenatal and postnatal development; the peripheral lung, including alveoli and small conducting airways, is especially vulnerable. Owing to a limited capacity for repair, the functional effects of lung injury or altered development during fetal or early postnatal life may persist into later life, affecting lung function and disease vulnerability. A range of environmental factors can likely cause epigenetic alterations in key genes regulating lung development, thus contributing to persistent changes in lung structure and/or function. New experimental evidence supports the effectiveness of cell-based therapies to prevent or repair neonatal lung injury, and clinical trials are now underway in very preterm infants at risk of developing bronchopulmonary dysplasia.
[Show abstract][Hide abstract]ABSTRACT: Lung aeration triggers the cardiovascular transition at birth by decreasing pulmonary vascular resistance, thereby increasing ventricular preload. As left ventricular (LV) preload is primarily from the umbilical circulation before birth, cord clamping before lung aeration causes a large reduction in LV preload; lung aeration before cord clamping allows the source of LV preload to immediately switch from the umbilical to the pulmonary circulation, minimising changes in cardiac output at birth. If birth occurs as a result of caesarean section without labour, the absence of the intra-partum mechanisms for airway liquid clearance will likely increase the volume of liquid that must be cleared into lung tissue. The resulting increase in interstitial pressures must increase the tendency for liquid to re-enter the airways, potentially resulting in transient tachypnoea of the newborn. Many unanswered questions remain regarding physiological and molecular mechanisms underlying the cardio-pulmonary transition at birth.
[Show abstract][Hide abstract]ABSTRACT: Like other organs, the lung is influenced by its nutritional environment during development. As lung tissue has a limited capacity to remodel itself following altered development, nutritionally induced alterations in lung structure can persist throughout life. Epidemiological evidence indicates that sub-optional nutrition during early life, leading to fetal growth restriction or retarded postnatal growth, can lead to impaired lung function in later life. Restricted nutrient intake during development can impact upon alveolarization and the small conducting airways; tethering of small airways is diminished. Although multiple tissue components of the lung can be affected by nutritional impairment during development, those most likely to have persistent effects on lung function are structural proteins such as elastin and basement membrane proteins. The long-term persistence of alterations in lung function and structure may be a reflection of the effective cessation of alveolarization in early life and epigenetic changes induced by early life exposures.
[Show abstract][Hide abstract]ABSTRACT: Background
Lung immaturity due to preterm birth is a significant complication affecting neonatal health. Despite the detrimental effects of supplemental oxygen on alveolar formation, it remains an important treatment for infants with respiratory distress. Macrophages are traditionally associated with the propagation of inflammatory insults, however increased appreciation of their diversity has revealed essential functions in development and regeneration.Methods
Macrophage regulatory cytokine Colony-Stimulating Factor-1 (CSF-1) was investigated in a model of neonatal hyperoxia exposure, with the aim of promoting macrophages associated with alveologenesis to protect/rescue lung development and function. Neonatal mice were exposed to normoxia (21% oxygen) or hyperoxia (Hyp; 65% oxygen); and administered CSF-1 (0.5 ¿g/g, daily¿×¿5) or vehicle (PBS) in two treatment regimes; 1) after hyperoxia from postnatal day (P)7-11, or 2) concurrently with five days of hyperoxia from P1-5. Lung structure, function and macrophages were assessed using alveolar morphometry, barometric whole-body plethysmography and flow cytometry.Results and discussionSeven days of hyperoxia resulted in an 18% decrease in body weight and perturbation of lung structure and function. In regime 1, growth restriction persisted in the Hyp¿+¿PBS and Hyp¿+¿CSF-1 groups, although perturbations in respiratory function were resolved by P35. CSF-1 increased CSF-1R+/F4/80+ macrophage number by 34% at P11 compared to Hyp¿+¿PBS, but was not associated with growth or lung structural rescue. In regime 2, five days of hyperoxia did not cause initial growth restriction in the Hyp¿+¿PBS and Hyp¿+¿CSF-1 groups, although body weight was decreased at P35 with CSF-1. CSF-1 was not associated with increased macrophages, or with functional perturbation in the adult. Overall, CSF-1 did not rescue the growth and lung defects associated with hyperoxia in this model; however, an increase in CSF-1R+ macrophages was not associated with an exacerbation of lung injury. The trophic functions of macrophages in lung development requires further elucidation in order to explore macrophage modulation as a strategy for promoting lung maturation.
Full-text · Article · Sep 2014 · Respiratory Research
[Show abstract][Hide abstract]ABSTRACT: 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 left (LV) and right ventricles (RV). Responsiveness to isoprenaline and stop-flow/reperfusion was assessed. In other experiments, hearts were perfusion-fixed and cardiomyocyte nuclearity, volume and number determined. β-Adrenoceptor mRNA levels were determined in unfixed tissue. In hearts of LPS-exposed fetuses, contractility in LV and RV was suppressed by ~40% and cardiomyocyte numbers were reduced by ~25%. Immature mono-nucleated cardiomyocytes had lower volumes (~18%), while mature bi-nucleated cardiomyocyte volume was ~77% greater. While basal coronary flow was significantly increased by 21±7% in LPS-exposed hearts, following ischemia-reperfusion, end diastolic pressure was increased 2.4±0.3-fold and infarct area 3.2±0.6-fold versus in controls. Maximum responsiveness to isoprenaline was enhanced by LPS, without an increase in b-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 likely contribute to the increased vulnerability of organ perfusion in preterm neonates.
[Show abstract][Hide abstract]ABSTRACT: Purpose The incidence of retinopathy of prematurity (ROP) has increased globally due to advances in the care of very low weight premature infants. Neonates are now viable at earlier time points than was possible 20-30 years ago. In this context we sought to determine the short-term and long-term effects of earlier neonatal exposure to clinically relevant levels of oxygen (O2) than in the traditional mouse model of ROP.
Methods Neonatal C57BL/6J mice were raised in hyperoxic conditions (40% or 65% O2) from birth until postnatal day 7 (D0-D7) and then raised in normoxia until early adulthood (8 week old) or middle-age (40 week old). Control animals were raised in normoxia throughout the experiment. Brightfield fundus imaging and fluorescein angiography (Micron III) was performed at 8 and 40 weeks.
Results In vivo fundus examination revealed multiple retinal lesions and abnormal retinal and hyaloid vasculature. In animals exposed to 65% O2 we noted retinal vascular changes including hyperplasia, calibre changes (thinning) and increased tortuosity as well as persistent hyperplastic hyaloid vessels at 8 and 40 weeks of age. The average number of hyaloid vessels present in animals exposed to 65% O2 is significantly higher than normal (0.77±0.35), at both 8 weeks (5.45±0.37) and 40 weeks (4.9±0.17). Exposure to 40% O2 from D0-D7 caused altered retinal vasculature patterns but no retinal lesions were observed. Fundus appearance at 8 weeks was normal in animals exposed to normoxia.
Conclusions The results demonstrated that neonatal exposure to 65% O2 produced marked clinical retinal changes that bore strong resemblance to some aspect of human ROP. This earlier exposure (D0-D7) to hyperoxia than the traditional model of ROP (D5-D12) also caused persistent hyperplastic hyaloid vasculature. To our knowledge the long term follow up of mice using this earlier exposure period to hyperoxia has not been reported previously and may be particularly relevant in an era where infants are surviving birth at earlier gestational ages.
[Show abstract][Hide abstract]ABSTRACT: Purpose Retinopathy of prematurity (ROP) is a significant cause of visual morbidity in very preterm infants (born < 32 weeks of gestation), with the incidence of ROP having increased globally as a result of advances in neonatal care. Due to the increased survival of neonates born after shorter lengths of gestation, the primary aim of this study was to determine the long-term ocular pathology following neonatal exposure to hyperoxia, starting earlier than in the traditional ROP mouse model.
Methods Neonatal (C57BL/6J) mice were raised in either 40% or 65% oxygen from birth until postnatal day 7 followed by room air until early adulthood (8 weeks) or middle-age (10 months). Control animals were raised in normoxia for the duration of the experiment. Eyes were collected for resin histology and immunofluorescence staining was performed on retinal whole mounts using anti-Iba-1 and anti-CD31 (PECAM) antibodies for the visualization of macrophages/microglia and vessels respectively. The density of vitreal hyalocytes and subretinal macrophages was calculated.
Results Histological examination of mice 8 weeks after exposure to 65% oxygen from day 0 to day 7 revealed marked disruption of the nuclear layers of the retina, with displacement of nuclei and photoreceptor loss, retinal thinning, retinal folds, the presence of pseudorosettes and neovascular tufts, and persistent hyaloid vasculature. The degree of pathology in the 40% oxygen group at 8 weeks was less severe than in the 65% oxygen group. Confocal microscopic analysis of retinal whole mounts at 8 weeks and 10 months revealed microglial activation and the accumulation of hyalocytes and subretinal macrophages in response to 40% and 65% oxygen. Analysis of CD31 staining in the 65% oxygen group revealed retinal vessel loss in the periphery at 8 weeks, which was persistent at 10 months of age.
Conclusions These data demonstrate that early neonatal exposure to hyperoxic conditions results in oxygen concentration-dependent pathology and long-term retinal injury, resembling various features of human ROP and hyperplastic vitreous. These novel findings may be particularly relevant for infants that are born at earlier gestational ages.
[Show abstract][Hide abstract]ABSTRACT: Background: We have recently shown that neonatal exposure to hyperoxic gas causes persistent alterations in
the structure of the small conducting airways (E-PAS2012: 3460.8); it also increases the number of immune
cells in bronchoalveolar lavage fluid (BALF) in adulthood.
Objective: Our objective was to determine if dietary supplementation with a rich source of antioxidants (tomato
juice, TJ) could protect the neonatal lung from hyperoxic exposure.
Design/Methods: Neonatal mice (C57BL6/J) were exposed to hyperoxia (65% O2) from birth until postnatal
day 7 (P7d); thereafter the mice were raised in room air until adulthood (P56d). Controls (CON) breathed room
air. In subsets of both of these groups drinking water was replaced with TJ (diluted 50:50 in water) ad libitum
from embryonic day 15 to necropsy. The lungs were collected at P7d (65% O2 n=31, CON n=27, 65% O2 + TJ
n=16, CON + TJ n=19) or P56d (65% O2 n=26, CON n=27, 65% O2 + TJ n=15, CON + TJ n=20) and lung
structure was morphometrically analysed. BALF was collected at P56d and immune cells counted.
Results: At P7d, hyperoxia alone led to an increase in airspace and increase in the mean linear intercept within
the lung parenchyma; these changes were not ameliorated by TJ. At P56d, TJ supplementation corrected the
hyperoxia-induced increase in !-smooth muscle actin surrounding the bronchioles. However, it did not alter the
hyperoxia-induced increase in immune cell number in BALF at P56d.
Conclusions: TJ supplementation could potentially be beneficial in preventing airway smooth muscle
hypertrophy at P56d. However, it does not appear to attenuate the immune response to hyperoxia in adult
[Show abstract][Hide abstract]ABSTRACT: 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.
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.
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.
Daily high-dose caffeine administration does not appear to adversely affect the developing white matter at the microstructural level.
Full-text · Article · Apr 2014 · Pediatric Research
[Show abstract][Hide abstract]ABSTRACT: While the impact of alcohol consumption by pregnant women on fetal neurodevelopment has received much attention, the effects on the cardiovascular system are not well understood. We hypothesised that repeated exposure to alcohol (ethanol) in utero would alter fetal arterial reactivity and wall stiffness, key mechanisms leading to cardiovascular disease in adulthood. Ethanol (0.75 g per kg body weight) was infused intravenously into ewes over one hour daily for 39 days in late pregnancy (days 95–133 of pregnancy, term ∼147 days). Maternal and fetal plasma ethanol concentrations at the end of the hour were ∼115 mg dL−1, and then declined to apparent zero over 8 h. At necropsy (day 134), fetal body weight and fetal brain-body weight ratio were not affected by alcohol infusion. Small arteries (250–300 μm outside diameter) from coronary, renal, mesenteric, femoral (psoas) and cerebral beds were isolated. Endothelium-dependent vasodilation sensitivity was reduced 10-fold in coronary resistance arteries, associated with a reduction in endothelial nitric oxide synthase mRNA (P = 0.008). Conversely, vasodilation sensitivity was enhanced 10-fold in mesenteric and renal resistance arteries. Arterial stiffness was markedly increased (P = 0.0001) in all five vascular beds associated with an increase in elastic modulus and, in cerebral vessels, with an increase in collagen Iα mRNA. Thus, we show for the first time that fetal arteries undergo marked and regionally variable adaptations as a consequence of repeated alcohol exposure. These alcohol-induced vascular effects occurred in the apparent absence of fetal physical abnormalities or fetal growth restriction.This article is protected by copyright. All rights reserved
No preview · Article · Apr 2014 · The Journal of Physiology
[Show abstract][Hide abstract]ABSTRACT: Background: It has been previously shown that exposure of neonatal miceto 65% oxygen (O2) leads to persistent changes in lung structure; it alsoincreases immune cell numbers in broncho-alveolar lavage fluid (BALF) inadulthood. As the effects of lower O2 concentrations are relatively unclear,our aim was to compare the effects of neonatal exposure to 40% and 65%O2 on lung pathology.Methods: Mice (C57BL/6J) were raised in 40% or 65% O2from birth untilpostnatal day 7 (P7d). Controls (CON) breathed room air. Mice were eithereuthanized at P7d (CON n = 15, 40% O2 n = 20, 65% O2 n = 17), or maintained in room air until adulthood at P56d (CON n = 15, 40% O2 n = 19,65% O2 n = 17). At P7d and P56d, we assessed the structure of bronchiolarwalls and lung parenchyma, and nitrotyrosine staining (marker of oxidative stress) in lung tissue. At P56d, BALF was collected and immune cellscounted.Results: At P7d, the 65% O2 group had significantly larger alveoli, lowertissue fraction and fewer bronchiolar-alveolar attachments than controls (all p < 0.05). At P56d, there were fewer attachments in the 65% O2 group,and nitrotyrosine staining was significantly increased in both hyperoxiagroups. The BALF of mice exposed to 40% and 65% O2 showed significant 117% and 150% increases in immune cells, respectively. Conclusions: Although exposure to 65% O2 alters lung structure, exposure to 40% O2 has no apparent effect. Exposure to 40% and 65% O2 persistently increases pulmonary nitrotyrosine staining and immune cellnumbers, suggesting ongoing oxidative stress and inflammation.