Richard Harding

Monash University (Australia), Melbourne, Victoria, Australia

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Publications (271)660.65 Total impact

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    ABSTRACT: Infants born very preterm are usually exposed to high oxygen concentrations but this may impair lung function in survivors in later life. However, the precise changes involved are poorly understood. We determined how neonatal hyperoxia alters lung function at mid-adulthood in mice. Neonatal C57BL/6J mice inhaled 65% oxygen (HE group) from birth for 7 days. They then breathed room air until 11 months of age (P11mo); these mice experienced growth restriction. Controls breathed only room air. To exclude the effects of growth restriction, a group of dams was rotated between hyperoxia and normoxia during the exposure period (HE+DR group). Lung function was measured at P11mo. HE mice had increased inspiratory capacity, work of breathing and tissue damping. HE+DR mice had further increases in inspiratory capacity and work of breathing, and reduced FEV100/FVC. Total lung capacity was increased in HE+DR males. HE males had elevated responses to methacholine. Neonatal hyperoxia alters lung function at mid-adulthood, especially in males. Copyright © 2015. Published by Elsevier B.V.
    Respiratory Physiology & Neurobiology 07/2015; DOI:10.1016/j.resp.2015.07.004 · 1.97 Impact Factor
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    ABSTRACT: Preterm infants often require supplemental oxygen due to lung immaturity, but hyperoxia can contribute to an increased risk of respiratory illness later in life. Our aim was to compare the effects of mild and moderate levels of neonatal hyperoxia on markers of pulmonary oxidative stress and inflammation, and on lung architecture; both immediate and persistent effects were assessed. Neonatal mice (C57BL6/J) were raised in either room air (21% O2), mild (40% O2), or moderate (65% O2) hyperoxia from birth until postnatal day 7 (P7d). The mice were killed at either P7d (immediate effects), or lived in air until adulthood (P56d, persistent effects). We enumerated macrophages in lung tissue at P7d and immune cells in bronchoalveolar lavage fluid (BALF) at P56d. At P7d and P56d, we assessed pulmonary oxidative stress (heme oxygenase-1 (HO-1) and nitrotyrosine staining) and lung architecture. The data were interrogated for sex differences. At P7d, HO-1 gene expression was greater in the 65% O2 group than in the 21% O2 group. At P56d, the area of nitrotyrosine staining and number of immune cells were greater in the 40% O2 and 65% O2 groups relative to the 21% O2 group. Exposure to 65% O2, but not 40% O2, led to larger alveoli and lower tissue fraction in the short-term and to persistently fewer bronchiolar-alveolar attachments. Exposure to 40% O2 or 65% O2 causes persistent increases in pulmonary oxidative stress and immune cells, suggesting chronic inflammation within the adult lung. Unlike 65% O2, 40% O2 does not affect lung architecture. Copyright © 2014, American Journal of Physiology - Lung Cellular and Molecular Physiology.
    AJP Lung Cellular and Molecular Physiology 07/2015; DOI:10.1152/ajplung.00359.2014 · 4.04 Impact Factor
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    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.
    Respiratory Research 09/2014; 15(1):110. DOI:10.1186/s12931-014-0110-5 · 3.13 Impact Factor
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    41st Fetal and Neonatal Physiological Society, Saint Vincent, Italy; 08/2014
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    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.
    Clinical Science 05/2014; DOI:10.1042/CS20140097 · 5.63 Impact Factor
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    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.
    The Association for Research in Vision and Ophthalmology, Orlando, USA; 05/2014
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    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.
    The Association for Research in Vision and Ophthalmology, Orlando, USA; 05/2014
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    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 mice.
    Paediatric Academic Societies, Vancouver, Canada; 05/2014
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    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.Methods:High-dose caffeine (25mg/kg caffeine base loading dose; 20mg/kg daily maintenance dose; n=9) or saline (n=8) were administered to pregnant sheep from 0.7-0.8 of term, equivalent to approximately 27-34 weeks in humans. At 0.8 of term, the white and grey matter were assessed histologically and immunohistochemically.Results:Daily caffeine administration led to peak caffeine concentration of 32mg/L in fetal plasma at 1h, 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.Pediatric Research (2014); doi:10.1038/pr.2014.55.
    Pediatric Research 04/2014; DOI:10.1038/pr.2014.55 · 2.84 Impact Factor
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    ABSTRACT: Preterm infants who receive supplemental oxygen for prolonged periods are at increased risk of impaired lung function later in life. This suggests that neonatal hyperoxia induces persistent changes in small conducting airways (bronchioles). Although the effects of neonatal hyperoxia on alveolarization are well documented, little is known about its effects on developing bronchioles. We hypothesized that neonatal hyperoxia would remodel the bronchiolar walls, contributing to altered lung function in adulthood. We studied three groups of mice (C57BL/6J) to postnatal day 56 (P56; adulthood) when they either underwent lung function testing or necropsy for histological analysis of the bronchiolar wall. One group inhaled 65% O2 from birth until P7, after which they breathed room air; this group experienced growth restriction (HE+GR group). We also used a group in which hyperoxia-induced GR was prevented by dam rotation (HE group). A control group inhaled room air from birth. At P56, the bronchiolar epithelium of HE mice contained fewer Clara cells and more ciliated cells, and the bronchiolar wall contained ∼25% less collagen than controls; in HE+GR mice the bronchiolar walls had ∼13% more collagen than controls. Male HE and HE+GR mice had significantly thicker bronchiolar epithelium than control males and altered lung function (HE males: greater dynamic compliance; HE+GR males: lower dynamic compliance). We conclude that neonatal hyperoxia remodels the bronchiolar wall and, in adult males, affects lung function, but effects are altered by concomitant growth restriction. Our findings may partly explain the reports of poor lung function in ex-preterm children and adults. Anat Rec, 2014. © 2014 Wiley Periodicals, Inc.
    The Anatomical Record Advances in Integrative Anatomy and Evolutionary Biology 04/2014; 297(4). DOI:10.1002/ar.22867 · 1.53 Impact Factor
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    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
    The Journal of Physiology 04/2014; 592(12). DOI:10.1113/jphysiol.2013.262873 · 4.54 Impact Factor
  • Sheena Bouch, Foula Sozo, Richard Harding
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    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.
    Perinatal Society of Australia and New Zealand, Perth, Australia; 03/2014
  • 03/2014; 9(1):e153-e154. DOI:10.1016/j.gheart.2014.03.1775
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    ABSTRACT: ABSTRACT 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.
    Experimental Lung Research 12/2013; DOI:10.3109/01902148.2013.858197 · 1.75 Impact Factor
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    ABSTRACT: Creatine is an organic acid that contributes to the energy supply to the skeletal muscle. The basic substrates for creatine biosynthesis in the human body are semi-essential amino acids L-arginine, glycine and methionine. In this book, the authors discuss the biosynthesis, therapeutic uses and physiological effects of creatine supplementation. Topics include the role of creatine in the pathophysiology of depression and the possible mechanisms underlying its antidepressant effect; creatine treatment and positive effects on muscle performance, muscle mass gain, and the persistence and improvement of the quality of life in patients with chronic diseases; muscle ergogenic effects of creatine supplementation in resistance exercise training; experimental evidence that creatine supplementation during pregnancy is protective for the neonate; and creatine metabolism and role in sports physiology.
    Creatine: Biosynthesis, Therapeutic Uses and Physiological Effects of Supplementation, 978-1-62948-305-4 edited by Fernando D'Cruz, Victor Ribeiro, 12/2013: chapter Experimental Evidence that Creatine Supplementation during Pregnancy is Protective for the Neonate: pages 101-128; Nova Publishers.
  • Megan O'Reilly, Richard Harding, Foula Sozo
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    ABSTRACT: Background: Supplemental oxygen is necessary in the respiratory support of very preterm infants, but it may contribute to bronchopulmonary dysplasia and an increased risk of poor lung function in later life. It is well established that hyperoxia can inhibit alveolarization, but effects on the developing conducting airways, which are important determinants of lung function, are poorly understood. It is possible that prolonged exposure of the immature lung to hyperoxic gas alters the development of small conducting airways (bronchioles), and that these effects may persist throughout life. Objectives: To examine the effects of neonatal inhalation of hyperoxic gas on the bronchiolar walls in adulthood. Methods: Neonatal mice (C57BL/6J) born at term inhaled 65% O2 from birth until postnatal day 7; thereafter, they were raised in room air until 10 months postnatal age (P10mo), which is advanced adulthood. Age-matched controls inhaled room air from birth. We investigated small conducting airways with a diameter between 105-310 µm. Results: At P10mo, bronchiolar walls of hyperoxia-exposed mice contained ∼18% more smooth muscle than controls (p < 0.05), although there was no effect on bronchiolar epithelium or collagen. Neonatal hyperoxia resulted in significantly fewer bronchiolar-alveolar attachments at P10mo (p < 0.05); this was accompanied by persistent simplification of the lung parenchyma, as indicated by greater mean linear intercept and less parenchymal tissue (p < 0.05). Conclusions: Neonatal exposure to hyperoxia induces remodeling of the bronchiolar walls and loss of bronchiolar-alveolar attachments in adulthood, both of which could contribute to impaired lung function and airway hyper-reactivity. © 2013 S. Karger AG, Basel.
    Neonatology 11/2013; 105(1):39-45. DOI:10.1159/000355641 · 2.37 Impact Factor
  • Sheena Bouch, Richard Harding, Foula Sozo
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    ABSTRACT: Background: Very preterm infants (born <32 weeks of gestation) usually require supplemental oxygen (O2) due to lung immaturity, and the hyperoxia may contribute to their greater risk of respiratory infections and asthma later in life. It is likely that this increased risk may be a result of persistent pulmonary oxidative stress and inflammation, and structural alterations to the bronchioles and alveolar region of the lung. Therefore, our aim was to examine the effects of neonatal exposure to clinically relevant levels of O2 on lung pathology, immune status and oxidative stress. Method: Neonatal mice (C57BL/6J) were raised in either 40% or 65% O2 (hyperoxia groups) from birth until postnatal day 7 (P7d). At birth, the lungs of mice are at a similar stage of development as very preterm babies. Controls (CON) breathed room air. Mice were either euthanized immediately following exposure at P7d (CON n=8, 40% O2 n=20, 65% O2 n=8), or kept in room air after the 7 days of hyperoxia until adulthood at P56d (CON n=8, 40% O2 n=19, 65% O2 n=9). At P7d and P56d, we analysed the wall composition of the bronchioles, the architecture of the alveolar region of the lung, and the area of nitrotyrosine staining in the alveolar region, as a marker of oxidative stress. At P7d, the proportion of macrophages in the alveolar region was determined using an immunofluorescent marker for galectin-3. At P56d, broncho-alveolar lavage fluid (BALF) was collected and the number of immune cells counted. Data were analysed using a one-way ANOVA. Results: At P7d, the 65% O2 group, but not the 40% O2 group, had less alveolar tissue, larger alveoli and fewer bronchiolar-alveolar attachments than controls. In the bronchioles of both hyperoxia groups, the epithelial area was not altered. At P7d, there was significantly less nitrotyrosine staining in the 40% O2 group compared to the CON and 65% O2 groups; however the proportion of macrophages was not significantly different between groups. At P56d, exposure to hyperoxia (40% and 65% O2) did not result in structural alterations. However, there was a tendency for the percentage of nitrotyrosine staining to be increased in the hyperoxia groups, and there was a significant increase in number of immune cells in the BALF from both the 40% O2 group (by 117%) and the 65% O2 group (by 150%) compared to controls. Conclusions: Although neonatal exposure to 65% O2 alters lung architecture at P7d, exposure to 40% O2 has no apparent effect. Exposure to 40% and 65% O2 causes a persistent increase in pulmonary immune cells and a tendency for the level of oxidative stress to be increased, suggesting ongoing inflammation and oxidative stress. Our findings may help explain why preterm infants are more susceptible to respiratory infections and asthma later in life.
    Developmental Origins of Health and Disease, Singapore, Singapore; 11/2013
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    ABSTRACT: Introduction: Retinopathy of prematurity (ROP) is a significant cause of visual morbidity in very preterm infants (born < 32 weeks of gestation) and usually follows prolonged exposure of these infants to hyperoxic gas. The incidence of ROP is 5-8% in developed countries and can reach up to 30% in developing countries. Mouse models are commonly utilised to investigate the disease processes underlying ROP. Previous murine studies of ROP have largely focused on the short-term effects of neonatal hyperoxic gas exposure on the developing eye. In this study our aim was to determine the long-term effects of neonatal exposure to clinically relevant levels of O2 exposure on the development of retinopathy in mice using live in vivo imaging and histological analysis. Method: Neonatal mice (C57BL/6J) were raised in either 40% or 65% O2 (hyperoxia groups) from birth until postnatal day 7 (P7d) and then raised in room air until early adulthood (P56d) or middle-age (10 months; P10m). Controls (CON) breathed room air for the duration of the experiment. At P56d (CON n=11, 40% O2 n=20, 65% O2 n=10) and P10m (CON n=15, 65% O2 n=15), in vivo brightfield and fluorescent angiographic imaging of the retinal fundus was performed with a Micron III camera under anaesthesia with subsequent recovery. At autopsy (P56d and P10m), eyes were also collected for histological examination and whole-mount retinal immunohistochemistry. Results: In vivo imaging of the fundus indicated that at P56d the 40% and 65% O2 groups and at P10m the 65% O2 group had a persistent hyaloid vasculature with an increased number of branches, multiple large retinal lesions and abnormal retinal vasculature compared to the CON groups. Histological examination of the 65% O2 group at P56d revealed thinning of the peripheral retina, multiple retinal folds, presence of pseudorosettes in the retina, and presence of hyaloid vessels in the vitreous extending to the posterior lens surface. The degree of pathology in the 40% O2 group was apparently less severe than in the 65% O2 group. Discussion: These preliminary data demonstrate for the first time that neonatal exposure to hyperoxic gas results in O2 concentration dependent pathology and long-term retinal injury resembling the pathology observed in human ROP following hyperoxia following very preterm birth. In addition, the use of in vivo fundal imaging enabled us to monitor and evaluate the condition of the eye without having to sacrifice the animal; this feature will likely be beneficial in evaluating the effectiveness of possible treatments for ROP. Further studies are required to understand the underlying mechanisms contributing to the abnormal retinal vasculature that we observed following neonatal hyperoxia exposure, as well as the persistence of hyaloid vessels, which would normally regress around P7d-P13d.
    Developmental Origins of Health and Disease, Singapore, Singapore; 11/2013
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    Fetal Alcohol Canadian Expertise (FACE) Research Association, St. John's Newfoundland; 09/2013
  • R. Harding, S. Bouch, F. Sozo
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    ABSTRACT: Background: In a previous study we found that neonatal exposure to 65% O2 leads to persistent changes in the structure of bronchioles and lung parenchyma in mice; it also increased immune cell numbers in bronchoalveolar lavage fluid (BALF). As the effects of lower oxygen concentrations are unknown, our present aim was to compare the effects of neonatal exposure to 40% and 65% oxygen. Method: Neonatal mice (C57BL/6J) were raised in either 40% or 65% O2 (hyperoxia groups, HYP) from birth until postnatal day 7 (P7d). Controls (CON) breathed room air. Mice were either euthanized at P7d (CON n=8, 40% HYP n=16, 65% HYP n=8), or kept in room air until adulthood at P56d (CON n=8, 40% HYP n=18, 65% HYP n=9). The structure of the bronchioles and lung parenchyma was morphometrically analysed at P7d. At P56d, BALF was collected and immune cells counted. Results: At P7d the 65%O2 HYP group, but not the 40%O2 HYP group, had less lung parenchymal tissue, larger alveoli and fewer bronchiolar-alveolar attachments than controls. In the bronchioles of both hyperoxia groups, there was no significant change in bronchiolar epithelial thickness. At P56d, there were significant increases in the number of immune cells in BALF of 40%O2 mice (95% increase) and 65%O2 mice (150% increase) compared to controls. Conclusions: Although neonatal exposure to 65% oxygen alters lung structure at P7d, exposure to 40% oxygen has no apparent effect. Neonatal exposure to both 40%O2 and 65% causes a persistent increase in pulmonary immune cells, suggesting ongoing inflammation.
    Paediatric respiratory reviews 07/2013; 14:S68. DOI:10.1016/S1526-0542(13)70103-9 · 2.22 Impact Factor

Publication Stats

5k Citations
660.65 Total Impact Points

Institutions

  • 1984–2015
    • Monash University (Australia)
      • • Department of Anatomy and Developmental Biology
      • • Department of Physiology
      • • Ritchie Centre for Baby Health Research
      Melbourne, Victoria, Australia
  • 2005–2014
    • University of Vic
      Vic, Catalonia, Spain
  • 1986–2006
    • University of Melbourne
      Melbourne, Victoria, Australia
  • 2002–2004
    • University of Western Australia
      • School of Women's and Infants' Health
      Perth, Western Australia, Australia
  • 2001
    • University of the Western Cape
      • Department of Physiological Sciences
      Cape Town, Province of the Western Cape, South Africa
  • 1992
    • Diabetes Australia, Victoria
      Melbourne, Victoria, Australia
  • 1991
    • University of Cambridge
      Cambridge, England, United Kingdom
  • 1981–1982
    • University of Queensland
      Brisbane, Queensland, Australia