DOWNLOAD PDF SUSCEPTIBILITY OF THE IMMATURE LUNG TO OXIDATIVE AND MECHANICAL INJURY JAQUES BELIK

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1 Chapter 1 : The newborn lung UTS Library Select Chapter 5 - Susceptibility of the Immature Lung to Oxidative and Mechanical Injury Book chapter Full text access Chapter 5 - Susceptibility of the Immature Lung to Oxidative and Mechanical Injury. In this review article, we have attempted to analyze and consolidate current knowledge about the role played by persistent prenatal and postnatal inflammation in the pathogenesis of BPD. While some parameters of the early inflammatory response neutrophils, cytokines, etc. Introduction Bronchopulmonary dysplasia BPD is the most common chronic respiratory disease affecting infants wherein the developmental program of the lung is altered secondary to preterm birth of the baby 1. Lung development progresses in five distinct stages: Human preterm babies who develop BPD are born in the late canalicular or early saccular stage of lung development. The late canalicular stage is characterized by development of the primitive alveoli and the alveolar capillary barrier, and the differentiation of type I and type II pneumocytes. The early saccular stage is marked by initiation of surfactant production, pulmonary vascularization, and enlargement of terminal airways 2 â 5. Unique to lung development is the fact that unlike other organs, the lungs complete their development after birth up to 8 years of age 6. Alveolar sacs are formed by secondary septation of alveolar ducts. With preterm birth, this programed development is disrupted, and in the setting of inflammation [whether it is due to infection, mechanical ventilation MV, or hyperoxia] causes impaired alveolarization leading to BPD. We need to remember that while in sheep, baboons, and humans, the saccular stage occurs in utero; in rodent models, it begins at embryonic day 18 and continues through postnatal PN day 5 4, 5. In spite of many advances in neonatal medicine in the past few decades, like the introduction of better MV strategies and the use of surfactant and antenatal steroids, the incidence of BPD has not declined 7. Pulmonary and neurodevelopmental sequelae of this devastating disease extend even into adulthood 9. This on top of genetic susceptibility and prematurity leads to persistent inflammation leading to lung remodeling and evolution of BPD. Genetic predisposition and persistent inflammation due to environmental factors sepsis, invasive mechanical ventilation, and hyperoxia acting on the foundation of immature lung underlie the pathogenesis of BPD. We searched PubMed for articles limited to English language with the keywords: We focused on articles published over the last 10 years and used the most relevant ones for this review. Mediators of Inflammation in BPD Bronchopulmonary dysplasia has been linked to the development of an inflammatory response that can occur in absence of clinical infection. Systemic fetal inflammatory response 11 and neonatal leukemoid reactions 12 have been implicated as a risk factor for BPD. Pulmonary inflammation in BPD is characterized by the presence of inflammatory cells like neutrophils and monocytes, pro-inflammatory cytokines, and other mediators, including soluble adhesion molecules. The innate immunity and adaptive immunity reinforce each other and act in unison. Cells of the innate immune system secrete cytokines, which can prime lymphocytes thereby modulating adaptive immunity Exposure to a specific antigen causes these primed lymphocytes to have a more rapid and intense immune response 14, Upon activation, the T cells shed their surface CD62L molecules. CD54 intercellular adhesion molecule-1 or ICAM-1 is an adhesion molecule that mediates a co-stimulatory signal in T cell activation. CD54 expression is increased upon cell activation The premature lung is exposed to ongoing oxidative and cellular damage. In contrast, decreased levels of IL in serum and tracheal aspirates have been shown in studies of those infants who developed BPD. In addition to ILs, a large variety of other biomarkers have been detected and associated with the development of BPD in tracheal aspirates, as well as blood and urine samples of premature infants 9, The ones that have been implicated in the animal models include inflammatory cytokines, matrix proteins, growth factors, and vascular factors 9, 18 â Their role is illustrated in Table 1. Selected mediators of inflammation, their role, and corresponding expression in BPD. We will now describe the major environmental factors that contribute to inflammation, and its persistence, in the pathogenesis of BPD. These include prenatal influences chorioamnionitis and postnatal influences, namely early- and late- onset sepsis, invasive MV, and hyperoxia. Prenatal Factors Page 1

2 Causing Inflammation â Chorioamnionitis As the name suggests, chorioamnionitis is inflammation of the chorion and amnion membranes of the placenta Chorioamnionitis has been classified as either histological or clinical. With histological chorioamnionitis, there is infiltration of polymorphonuclear leukocytes and other inflammatory cells like macrophages and T cells as seen microscopically 35 â It has been shown in in vitro studies that bacterial products like phospholipase A2, peptidoglycan polysaccharide, proteolytic enzymes, and endotoxins can initiate an inflammatory response. Inoculation of the amniotic cavity with E. Adverse perinatal outcomes are seen with intra-amniotic inflammation irrespective of the presence of intra-amniotic infection. Colonization per se, without inflammation is not associated with adverse outcomes The severity of the adverse outcomes is directly related to the severity of the intra-amniotic inflammation Maternal antibiotic use has been associated with decreased BPD To summarize, in experimental animals, antenatal inflammation causes lung maturation and some degrees of lung injury, which is modified by the not fully developed innate immune response, exposure to antenatal steroids, and noxious postnatal factors. Not surprisingly, given the variability in definition and impact of various confounding factors, the issue of antenatal inflammation causing BPD in human infants is controversial 42, 46 â Chorioamnionitis increases the incidence of preterm birth, and if accompanied by lung inflammation could result in surfactant dysfunction allowing for prolonged exposure to supplemental oxygen and invasive MV 11, In addition, persistence and non-resolution of lung inflammation lead to BPD by inhibiting secondary septation, alveolarization and normal vascular development, and the compromised ability of the lungs to heal. Postnatal Factors Causing Inflammation â Sepsis Preterm infants are more susceptible to infections since their immune defenses are not fully developed, have vulnerable skin barrier, and require multiple invasive procedures Chorioamnionitis increases the risk of early-onset neonatal sepsis, which sets off an inflammatory cascade Also, it has been shown that late-onset sepsis induces a pro-inflammatory and pro-fibrotic response in the preterm lung predisposing it to BPD Local intra-tracheal exposure to LPS bacterial endotoxin or dsrna a marker of viral replication in the neonatal rat led to acute cellular and cytokine inflammatory responses, which were associated with histologic features of impaired alveolar development 52, Pre-treatment with CXCR2-neutralizing antibody was able to reverse the effects in the developing lung In summary, exposure to either bacterial or viral agents in the rodent model led to features of inflammation, with pulmonary histology suggestive of BPD. Inflammatory response secondary to viral infections in early post natal stages could be worth considering in the evolution of BPD. Increased neutrophil accumulation, increased expression of CXCL-1 and its receptor CXCR2, and decreased lung alveolarization have been seen with intra-tracheal delivery of viral pro-inflammatory dsrna in day-old mouse model Lung injury from MV results due to volutrauma, barotrauma, or atelectrauma Even ventilation at low tidal volumes is deleterious because of the stretch injury it can induce by over-distending partially collapsed lungs. Sustained lung inflation SLI has been shown to increase levels of pro-inflammatory cytokines and BPD-like changes in the lungs of preterm lambs In a 7-day-old rat model, exposure to MV for 24 h in room air led to cell cycle arrest 63, suggesting a harbinger to alveolar simplification, the pathologic hallmark of BPD. In an invasive MV model in 2-week-old mice well into the alveolar phase of lung development for 1 h, IL-6 lung levels were increased in the high tidal volume ventilation group Inhibiting lung elastase activity by using recombinant human elafin or genetically modified mice that expressed elafin in the vascular endothelium was protective of the lung injury 67, Early studies using a chronically ventilated 3â 4 weeks preterm lamb model of BPD showed evidence of non-uniform inflation patterns and impaired alveolar formation with an abnormal abundance of elastin Inflammation was evident by the presence of inflammatory cells, namely alveolar macrophages, neutrophils, and mononuclear cells and edema In this model, there was also reduced lung expression of growth factors that regulate alveolarization and differential alteration of matrix proteins that regulate ELN assembly It has been seen in preterm fetal sheep that there is increased expression of early response gene-1 Egr-1 as well as pro- and anti-inflammatory cytokines and dynamic changes in heat shock protein 70 HSP70 This was accompanied by increased presence of inflammatory cells in the bronchoalveolar lavage fluid BALF with initial increases in neutrophils and monocytes by 1 h and a Page 2

3 transition to macrophages by 24 h In addition, increased matrix metalloproteinase-9 MMP-9 levels were associated with lung inflammation and edema seen in this invasive ventilation model Alteration of VEGF was also noted in the lungs of various baboon models 75, Bombesin is a amino acid peptide, initially detected in amphibian skin, but immunoreactive studies have shown the presence of bombesin-like peptide BLP in multiple organ systems in mammals In the lung, BLP have been shown to be released by pulmonary neuroendocrine cells BLP blockade improved alveolar septation and angiogenesis in the preterm baboon models 78, In the day baboon model, treatment with early nasal continuous positive airway pressure NCPAP for 28 days led to a pulmonary phenotype similar to days gestational control lungs, suggesting that this non-invasive approach could minimize lung injury The optimal mode of non-invasive ventilation for example: In addition, there is increased cell death and cell cycle arrest. Thus, it appears that an initial inflammatory cascade triggers the signaling of additional molecular mediators that lead to dysregulated vascularization and impaired alveolarization. Interestingly, non-invasive nasal ventilation approaches were protective of these responses. Postnatal Factors Causing Inflammation â Hyperoxia Many studies have documented the injurious effects of perinatal supplemental oxygen on lung development. The morphologic changes of human BPD resemble hyperoxic lung injury in newborn animals Exposure to hyperoxia in the critical saccular stage of lung development replicates human BPD, with effects that are dose-dependent on the fraction of inspired oxygen FiO2 concentration; the effects last lifelong with increased susceptibility to respiratory tract infections 91 â Acute lung injury caused by hyperoxia Figure 2 occurs secondary to an inflammatory response, which causes destruction of the alveolarâ capillary barrier, vascular leak, influx of inflammatory mediators, pulmonary edema, and ultimately cell death With continued exposure to hyperoxia this inflammatory response and pulmonary edema improve initially but chronic pulmonary inflammation ensues in the following weeks At the cellular level, alveolar or interstitial macrophages express early response cytokines when exposed to hyperoxia, which in turn attract inflammatory cells to the lungs Note the alveolar exudates and presence of inflammatory cells in the hyperoxia-exposed lungs compared with litter-mate controls in room air. It has been shown that there exists a dose-dependent effect of hyperoxia on severity of BPD in the murine model. An oxygen dose-dependent inflammatory response to influenza-a viral infection in adult mice that had been exposed to hyperoxia as neonates has been reported Furthermore, this response was dependent upon the cumulative exposure to oxygen The specific role of individual inflammatory molecular mediators in the pathogenesis of BPD has been particularly well illustrated by utilizing lung-targeted overexpressing transgenic models, in room air, resulting in pulmonary phenotypes reminiscent of human BPD. A recent paper has reported that increased Cox-2 activity may contribute to proinflammatory responses in hyperoxia-exposed developing mouse lungs CINC-1 in premature rat lungs and newborn rabbits exposed to hyperoxia was upregulated. Also, IL-8 levels in tracheal aspirates of the premature baboon model of BPD have been shown to be increased Typically viewed as pro-inflammatory, these cytokines have been shown to be elevated very early in the respiratory course of the human preterm population that ultimately develops BPD Studies have found that serum and tracheal aspirate IL levels were decreased in those infants who developed BPD These include rosiglitazone,, hepatocyte growth factor HGF, B-naphthoflavone, arginyl-glutamine as well as docosahexaenoic acid, and a combination of vitamin A and retinoic acid To summarize, while variable initiation and duration of exposure to hyperoxia animal models have been reported as models of human BPD, exposure to hyperoxia for a relatively short PN1â 4 duration in mice, which is at the critical saccular stage of lung development, can result in an inflammatory response sufficient to create the BPD pulmonary phenotype. This can be recapitulated using transgenic mice models of the inflammatory mediators, but kept in room air. Importantly, exposure to 0. This would avoid the confounding variable of hyperoxia-induced alterations in multiple other molecular mediators, allowing delineation of targeted molecules in specific signaling pathways for maximal potential therapeutic relevance. Persistent Inflammation in BPD It is important to highlight the fact that for BPD to occur, it requires the known environmental factors to be exposed to the immature lung for a sustained duration, resulting in persistent inflammation. For the Page 3

4 relative short duration of exposure to invasive MV and hyperoxia in rodent models, 1 postnatal day in the saccular stage of lung development is equivalent to 3â 4 weeks in a human preterm infant. Page 4

5 Chapter 2 : ECU Libraries Catalog / Henry L. Halliday, Conner P. O'Neill --, Definitions and predictors of bronchopulmonary dysplasia / Michele C. Walsh -- New developments in the pathogenesis and management of neonatal pulmonary hypertension / Judy L. Aschner, Candice D. Fike -- Impact of perinatal lung injury in later life / Lex W. Doyle, Peter J. Anderson -- The oxygen. Remodeling of the extracellular matrix, apoptosis as well as altered growth factor signaling characterize the disease. The immediate consequences of these early insults have been studied in different animal models supported by results from in vitro approaches leading to the successful application of some findings to the clinical setting in the past. Nonetheless, existing information about long-term consequences of the identified early and most likely sustained changes to the developing lung is limited. Interesting results point towards a tremendous impact of these early injuries on the pulmonary repair capacity as well as aging related processes in the adult lung. The disease results from the impact of different risk factors on the undeveloped neonatal lung and is associated with a significantly increased risk for pulmonary and neurologic impairment persisting into adulthood in the cohort of formerly preterm infants 1. Even significant improvements in perinatal care including surfactant treatment, administration of antenatal corticosteroids, and improvement of invasive and non-invasive ventilation strategies could not significantly alter the incidence of long-term sequelae associated with the disease in the most immature infants 8. Clinically, this form of CLD presents with hypoxemia leading to the need for supplemental O2 as well as hypercapnia, reflecting impaired respiratory gas exchange and alveolar hypoventilation, resulting in a mismatch of ventilation and perfusion 9. Lung function is characterized by diminished compliance, tachypnea, increased minute ventilation, and work of breathing and can be accompanied by an increase in lung microvascular filtration pressure leading to interstitial pulmonary edema As a result of increased respiratory tract resistance and hyper-reactive airways, episodic bronchoconstriction and cyanosis can be observed 11, with early lung function impairment indicating more severe disease at term The increased lung vascular resistance, typically associated with impaired responsiveness to inhaled nitric oxide and other vasodilators, can progress to reversible or sustained pulmonary hypertension and right heart failure 13, As a trigger for the onset of these pathophysiologic processes, large clinical trials have identified important risk factors 15 â Besides postnatal infections, the requirement for prolonged assisted ventilation to treat acute respiratory failure caused by primary surfactant deficiency and the need for oxygen supplementation is known to injure the structural and functional immature lung 21 â Here, the use of large tidal volumes and high inflation pressures, in concert with the magnitude and duration of exposure to supplemental oxygen, are major risk factors for disease development 25, The occurence of pulmonary complications, e. With respect to postnatal growth and development, poor nutritional support, vitamin deficiency as well as insufficient adrenal and thyroid hormone release in the very premature infant is known to significantly contribute to adverse pulmonary outcome 27 â These postnatal stressors are known to act beyond the background of both prenatal as well as genetic risk factors influencing the capacity of the developing lung to respond to the indicated postnatal injuries: Intrauterine growth retardation increases the risk of BPD three to fourfold 30 â 34, most likely through impaired alveolar and vascular growth associated with altered growth factor signaling The prenatal impact of cytokines, in the presence, i. Several potential candidate genes have been associated with BPD, where genetic variants predisposing to BPD are usually polymorphisms, which are not causative, but have been shown to increase disease susceptibility. Genetic abnormalities include variations affecting the surfactant system or the innate immune response 38, Such conclusions are consistent with a genome-wide association study involving more than very low birth weight infants that was not able to link specific genomic loci or pathways with BPD Nonetheless, gestational age at birth still remains one of the most accurate ways to predict the incidence of BPD In addition, male preterm infants are at a higher risk for the development of long-term impairment, including BPD 43, and premature changes to hormonal regulation have been discussed as an underlying cause The effect of gender seems to be Page 5

6 different with respect to the adult population, as female adult BPD patients are more severely affected with respect to developing long-term pulmonary impairment Animal models were instrumental in elucidating some of the underlying mechanisms by which the indicated risk factors resulted in profound and durable structural changes in the developing lung and will be discussed in the pathophysiologic context. Pathophysiologic Characteristics With respect to histopathology, neonatal CLD is characterized by impaired alveolarization and vascularization 1. Alveolarization in humans begins during late fetal development and continues into early childhood Although continued growth of new alveoli was observed in rhesus monkeys up into adulthood 47, there is increasing evidence in both humans and rodents suggesting alveolar development to occur in two phases 46, 48, The majority of alveoli develops in the early phase and occurs as double micro-capillaries mature into a single capillary network. This phase is followed by ongoing alveolar growth arising from existing alveoli. Knowledge about these two distinct phases in alveolar development is critical to design therapeutic approaches aiming to promote alveolar development in the injured neonatal lung. As a well-known example, postnatal corticosteroids have been used to promote lung function, facilitating extubation and thereby trying to prevent or ameliorate BPD development. Although their anti-inflammatory effects are potent 50, dexamethasone administered to newborn rats has been shown to cause alveolar thinning and structural simplification, presumably by inhibiting the early phase of alveolar development Especially, the impact of postnatal steroid therapy on extracellular matrix ECM composition as observed in rats may account for long-term effects Not only is the window of lung development a critical variable for the decision on postnatal steroid therapy, but the increased risk for gastrointestinal bleeding, cardiovascular disease, and cerebral palsy in infants that had received steroid therapy 53, 54, strongly limits dose, time-point, and length of treatment. Besides the continuous changes in pre- and postnatal treatment strategies in preterm care, the altered histo- and pathophysiological picture of BPD over the last decades is mainly due to the degree of immaturity in the preterm cohort studied. Although the amount of interstitial fibrosis is substantially less in these infants and tends to be more diffuse when compared to histopathology in the pre-surfactant era, extensive ECM remodeling together with increased smooth muscle in small pulmonary arteries and airways 56 remains a key finding in the diseased lung. Many studies have shown that characteristic inflammatory changes and altered growth factor signaling precede and accompany these changes to the pulmonary scaffold that may not only hinder physiologic lung development but also transfer its long-term effects into adulthood. The characteristic degradation of lung elastin that accompanies its pathologic distribution pattern in infants who later acquire BPD manifests in increased urinary excretion of desmosin, a breakdown product of the mature elastic fiber, preceded and paralleled by an associated increase in protease activity 57 â Nonetheless, studies in an inflammatory model of BPD have shown that a delicate balance of protease activity is critical for normal lung development, as complete matrix-metalloproteinase deficiency worsened lung injury Experimental studies in rodents recapitulated the changes to the ECM and allowed further insight into disease relevant pathophysiology, linking ECM remodeling to apoptosis, inflammation, and growth factor signaling 64 â First attempts were made in order to therapeutically prevent ECM degradation, thereby preserving lung growth in the presence of mechanical ventilation 68, Pulmonary inflammation induced by both non-infectious processes, such as positive-pressure ventilation or oxygen therapy aggravated by primary surfactant deficiency, or patent ductus arteriosus, as well as pre- or postnatal infections play an important role in the translation of different injury mechanisms to structural and functional changes as well as in the aggravation of ongoing pathologic processes in the developing lung 18, 22, 70, The characteristic initial influx of neutrophils into the lung is accompanied and followed by increased numbers of macrophages in the course of the disease 32, 72, Explaining the perpetuation of the inflammatory response, animal studies indicate that lung injury leading to ECM remodeling or early alveolar epithelial dysfunction further promotes lung inflammation 68, In contrast, inflammation in fetal sheep infected with Ureaplasma parvum, a common microorganism present in chorioaminionitis, did not affect lung development 75, suggesting that inflammation acts in concert with other risk factors to provoke the development of BPD. The release of Page 6

7 cytokines and disturbance of growth factor signaling, [e. The presence of dysmorphic capillaries is related to an altered pattern of angiogenic growth factors such as reduced expression of the vascular endothelial growth factor VEGF and its receptors 78 â 80 in the lung, accompanied by diminished endothelial nitric-oxide synthase enos and soluble guanylate cyclase sgc in lung blood vessels and airways 81, These changes contribute to subsequent development of pulmonary hypertension and impaired lung lymphatic drainage 13, The growing evidence that prenatal factors impact on the incidence of childhood asthma 83 further underlines the importance of studies focusing on the role of prenatal variables in respiratory development following premature birth including the broad issue of maternal health. Suggested by a study in newborn mice exposed to hyperoxia after maternal exposure to LPS during pregnancy, the enhancement of cardiovascular disease in the animals points to an impact of chorioamnionitis not only on surfactant production but also on cardiovascular health in children born premature undergoing early oxygen treatment Relative deficiencies of anti-oxidants and inhibitors of proteolytic enzymes render the very immature lung, especially vulnerable to the effects of toxic oxygen metabolites and proteases released by the ECM, resident lung cells, or activated neutrophils and macrophages 85 â Elevated urinary malondialdehyde concentrations in the first week of life, generated by peroxidation of lipid membranes after oxidant-mediated injury, were correlated with the risk for oxygen radical diseases including BPD The association of genetic polymorphisms of the superoxide dismutase with the development of BPD underlines the importance of a balanced redox system To understand how oxygen affects lung development, non-human primates, preterm sheep, newborn guinea pigs, and newborn rodents have been exposed to excessive levels of oxygen [for review in Ref. It also recapitulates many diseases in children who were born preterm, including altered host response to respiratory viral infections, mild cognitive changes, and cardiovascular disease. Despite the widespread use of hyperoxia as a tool to study BPD, a codified model of oxygen exposure has yet to be established, making the extrapolation of outcomes between different investigators using different doses and durations of hyperoxia on different developmental windows possible Today, preterm infants are often exposed to excess oxygen during the saccular stage of lung development, and discharged breathing room air when entering the first phase of completion in alveolarization. In order to mimic these clinically relevant conditions, the influence of oxygen on saccular stages in lung development, i. The process of organ maturation clearly is a modifier of the pulmonary response to oxygen exposure, as hyperoxia has been shown to reduce bone marrow, circulating and lung endothelial progenitor cells in the developing but not in the adult mouse lung Additionally, different oxygen concentrations affected lung development and the host response to influenza A virus in neonatal mice Hence, a better understanding of how dose and duration of the respective harmful agent interfere with a certain developmental window is important to make progress in the development of treatment strategies that could improve pulmonary health in preterm infants. Neonatal hyperoxia disrupts postnatal alveolar development in the lung. Thickened alveolar septae thick arrow, inflammatory cells thin arrow, and simplified alveoli asterisks. Long-Term Consequences Minimizing long-term pulmonary impairment, and neurologic complications associated with BPD has become the main focus of perinatal care 97, Nonetheless, increasing evidence suggests that the early pathologic changes observed in BPD contribute to long-lasting consequences including premature aging of the lung. Although only some affected infants remain oxygen dependent beyond 2 years of age, oxygen dependency for months or years is frequently described 99, indicating the most severe lung disease. These infants require hospital readmission twice as often compared to infants who are not oxygen dependent. Here, infections with respiratory syncytial virus are the major cause for readmission among preterm infants regardless of BPD status Furthermore, patients with moderate or severe BPD suffer more frequently from episodes of wheezing and need for inhalation therapies The most severely affected children remain symptomatic into adulthood The described clinical symptoms are underlined by changes in pulmonary function as identified by clinical studies, suggesting that the presence of persistent airflow limitations and reduction in alveolar surface area as well as impaired lung growth, significantly lower peak oxygen consumption, forced expiratory volume at 1 s FEV1, and gas transfer at school age after premature birth are Page 7

8 accompanied by significantly lower peak workload, higher respiratory rates in combination with lower tidal volumes during peak exercise as well as lower lung volumes and decreased gas mixing efficiency during infancy in BPD patients, As young adult BPD survivors present with a reduced maximal airway function and some even show a trend towards an early and steeper decline in lung function with age, the concern was raised that BPD may be a precursor of a COPD-like phenotype later in life although the incidence has yet to be defined in this patient cohort With respect to the pathophysiologic concepts discussed, the effect of injury mechanism and treatment regimen has to be considered in the context of different stages in pulmonary development. Here, the inhibition of secondary alveolar formation in patients with BPD could result in premature lung structure degradation with age. Furthermore, the sustained and potentially irreversible reorganization of the ECM as observed after mechanical ventilation, oxygen exposure or steroid therapy may affect its function as a scaffold for lung development as well as its long-term repair potential, both resulting in long-term consequences. With respect to the lung vasculature, changes in angiogenic growth factor expression resemble the pattern observed in aged mice and are associated with reduced plasticity of the lung capillaries, potentially leading to sustained changes for lung development and injury response through life, In line with this, oxygen exposure in the first week of life FiO2 1. Again, maturational differences need to be considered and studied in more detail with respect to their initial impact and their consequences for aging related processes. Whereas oxygen enhances lung vascular and airway smooth muscle contraction and reduces nitric-oxide relaxation in the neonatal rat lung, the opposite occurs in the adult animal These changes are associated with altered lung function and right ventricular hypertrophy at 10 months of age, indicating significant pulmonary hypertension. At this late stage, bone morphogenetic protein signaling is altered and may contribute to the cardiovascular phenotype observed in the adult lung. Furthermore, recent studies suggest that long-term respiratory abnormalities after preterm birth may be associated with a sustained alteration of the oxidative stress response. Here, adolescent BPD patients show evidence of heightened oxidative stress in the airways Likewise, the early interference with different transcription factors disrupts normal lung morphogenesis in fetal life, subsequently resulting in pulmonary emphysema in adult mice preluded by severe chronic bronchial inflammation Studies focusing on the differential response to injury in the developing lung will help to broaden the understanding of identified targets and processes with a critical role in adult lung diseases. With respect to early treatment regimen, different findings indicate the need for a careful investigation of their potential to induce long-term effects. As an example, the prenatal administration of betamethasone, although widely used to enhance lung maturation thereby preventing respiratory distress and reducing BPD rates,, has been shown to be associated with an increase in lipid membrane peroxidation In addition, postnatal dexamethasone treatment in the neonate, besides its negative effects on neurologic and alveolar development, led to systolic dysfunction and reduced life expectancy in elderly rats, Furthermore, the broad use of antibiotic treatment in the mother at risk for premature delivery leads to a sustained alteration of the bacterial flora of the child, affecting immune function long-term as shown in neonatal in contrast to adult-germ free mice Some therapeutic agents with suggested immediate effects on the redox system, displayed their treatment potential more with respect to middle and long-term effects, Although improving oxygenation and pulmonary vascular development in preterm sheep, intra-tracheal administration of recombinant human CuZn superoxide dismutase did not diminish the incidence of BPD, but reduced respiratory morbidity at 1 year of age and the incidence of retinopathy ROP â As inflammatory cells recruited to the sites of oxidative damage or attracted during infections are very likely to contribute to disease development, a combination of anti-oxidant and anti-inflammatory therapies may be most efficacious for the treatment of the structural and functional immature lung in the preterm infant. Here, studies in newborn mice showed that blocking neutrophil influx using anti-cinc antibody in diminished hyperoxia-induced DNA damage and alveolar simplification, As oxygen is a leading risk factor but as well remains the main treatment option in infants suffering from impaired lung growth when diagnosed with BPD, the definition of the adequate dose for oxygen therapy will remain of critical importance. Several clinical trials have attempted to Page 8

9 identify adequate therapeutic oxygen concentrations providing maximal benefit with minimal harm. Early trials found that the incidence of ROP correlated with unrestricted use of oxygen and the benefits of oxygen-saturation targeting BOOST trial found that a higher oxygen-saturation range prolonged oxygen dependence Taken together, preterm infants are likely to be treated with high oxygen saturations despite the increased risk for ROP. With respect to its effect on lung function, infants with the highest quartile of oxygen exposure as a cumulative dose of inhaled gas over the first 3 days of life were two to three times more likely to experience symptomatic airway dysfunction than infants in the lowest quartile Page 9

10 Chapter 3 : - NLM Catalog Result The newborn lung UTS Library. Susceptibility of the immature lung to oxidative and mechanical injury / Jaques Belik. Humans and mature mice expressing the RG SNP exhibit increased cardiovascular disease but decreased lung disease. The impact of this SNP on the neonatal lung at baseline or with injury is unknown. Wild type and homozygous RG mice were injected with intraperitoneal bleomycin or phosphate buffered saline PBS three times weekly for three weeks and tissue harvested at 22 days of life. Vascular and alveolar development were evaluated by morphometric analysis and immunostaining of lung sections. Under control conditions, RG mice developed pulmonary vascular remodeling decreased vessel density and increased medial wall thickness and PH; alveolar development was similar between strains. After bleomycin injury, in contrast to WT, RG mice were protected from impaired alveolar development and their vascular abnormalities and PH did not worsen. RG neonatal mice demonstrate impaired vascular development and PH at baseline without alveolar simplification, yet are protected from bleomycin induced lung injury and worsening of pulmonary vascular remodeling and PH. Introduction Bronchopulmonary dysplasia BPD is a disorder of lung development affecting preterm infants resulting in respiratory morbidities that can persist through adulthood [ 1 ]. Despite advances in neonatal care, the incidence of BPD is increasing [ 2 ]. A subset of infants with BPD also develop pulmonary hypertension PH, characterized by impaired angiogenesis, pulmonary vascular remodeling and right ventricular failure, which significantly increases the morbidity and mortality [ 3, 4 ]. Interventions to prevent and treat BPD complicated by PH are limited, thus there is an urgent need to improve understanding of the mechanisms contributing to the development of these frequent and burdensome diseases of prematurity. Antioxidant enzymes and essential nutrients that contribute to antioxidant status increase prior to birth and prepare infants for the increased partial pressure of oxygen with post-natal room air breathing [ 6 ]. Premature neonates have decreased antioxidant defenses, and increased oxidative stress due to oxygen therapy, infection, and mechanical ventilation [ 6 ]. However, despite compelling evidence that an imbalance of oxidative stress contributes to BPD and PH, generalized antioxidant therapies have not proven efficacious to preventing or treating these diseases [ 6 ]. An enhanced understanding of how antioxidants impact regional redox signaling may lead to more effective targeted antioxidant therapies that improve clinical outcomes. SOD3 is typically tethered to the extracellular matrix ECM by a series of positively charged amino acids that bind to the negatively charged ligands in the ECM and glycocalyx of endothelial and epithelial cells [ 8, 9 ]. The rs SNP leads to a single amino acid substitution from arginine to glycine at position within the matrix binding region, lowering the matrix binding affinity of SOD3, and redistributing SOD3 from the lung parenchyma and vasculature into the plasma and epithelial lining fluid [ 7, 10, 11 ]. Adult mice harboring the same SNP are protected from intratracheal lipopolysaccharide LPS inflammation, bleomycin induced pulmonary fibrosis and PH, and ovalbumin OVA induced airway obstruction [ 7, 12, 14 ]. Conversely, this SNP is associated with worse outcomes in ischemic heart disease [ 15 ], and adult mice exhibit PH at baseline and exaggerated chronic hypoxia-induced PH [ 7, 14 ]. The divergent risk profiles imparted by the RG SNP in different diseases illustrates the importance of the site of oxidative stress and antioxidant defenses in disease pathogenesis. SOD3 is developmentally regulated, and SOD3 deficient neonatal mice exhibit impaired alveolar and pulmonary vascular development at baseline [ 16 ]. These findings provide evidence that SOD3 content is important in the normal developing lung and with neonatal lung injury, however the impact of the distribution of SOD3 on the developing lung and with injury is unknown. Bleomycin, a chemotherapeutic agent that induces significant lung fibrosis and inflammation in adults [ 22, 23 ], has been adapted into a model of experimental BPD and PH in neonatal rodents, characterized by alveolar simplification, decreased vascular development, and vascular remodeling similar to infants with BPD complicated by PH [ 16, 24, 25, 26 ]. Bleomycin was dosed per weight at each injection. Injections were started on day of life 2 and ended on day of life Since the lungs of newborn mice are in the saccular stage of lung development, the 2-day old Page 10

11 mouse at the onset of treatment models the stage of lung development of human infants born at 28â 32 weeks [ 27 ]. Mice were anesthetized with 1. Additionally, lung sections were stained with rabbit anti-vwf 1: Lung fields containing large vessels or airways were excluded, and a minimum of 8 fields were included per mouse. The width of the medial wall was measured in four perpendicular locations and an average was calculated. The external diameter was measured in two locations, this was then averaged and divided by two to calculate the average radius. The analysis was performed by an investigator blinded to the experiment group. Evaluation of Alveolar Development Radial alveolar counts RACs were calculated on hematoxylin and eosin stained lung sections by identifying the terminal bronchiole, drawing a perpendicular line to the lung periphery, and counting each intersection of lung alveoli [ 29 ]. Fields with large airways or vessels were excluded. After washing, blots were incubated for 1 hour at room temperature with the species-appropriate secondary IgG antibody 1: Images were obtained on FluorChemM camera system. Grossly hemolyzed samples were excluded to avoid elevated SOD activity resulting from the release of red blood cell SOD1. At 22 days, we observed significantly less SOD3 protein in the lungs of RG mice Figure 1 a and elevated serum SOD3 compared to wild type WT mice Figure 1 b, recapitulating what we reported previously in adult mice [ 7 ]. We next evaluated if the RG mice compensate for low lung SOD3 content by upregulating other key antioxidant enzymes in the neonatal lung. Page 11

12 Chapter 4 : Table of contents for Pulmonary The Newborn Lung Neonatology Questions and Controversies Chapter 5 - Susceptibility of the Immature Lung to Oxidative and Mechanical Injury. Jaques Belik. Pages. Table of contents for Pulmonary: Bibliographic record and links to related information available from the Library of Congress catalog. Contents data are machine generated based on pre-publication provided by the publisher. Contents may have variations from the printed book or be incomplete or contain other coding. Bancalari - Pulmonary The Newborn Lung: Questions and Controversies Section 1: Molecular basis for normal and abnormal lung development. Martin Post Martin Rutter 2. Hereditary Disorders of Alveolar Homeostasis in the Newborn. Jeffrey Whitsett Timothy Weaver 3. Injury in the Developing Lung 5. Susceptibility of the immature lung to oxidative and mechanical injury. Processed Jaques Belik 6. Carlton Lucky Jain 8. Role of inflammation in the pathogenesis of acute and chronic neonatal lung disease. Ilene Sosenko Eduardo Bancalari What is the evidence for drug therapy in the prevention and management of BPD. Definitions and Predictors of Bronchopulmonary Dysplasia. New developments in the pathogenesis and management of neonatal pulmonary hypertension. Judy Aschner Candice Fike Management of Respiratory Failure Unnumbered Section: Optimal levels of oxygenation in preterm infants: Impact on short and longterm outcomes. Mechanical Respiratory Support Non invasive respiratory support: Peter Davis Colin Morley Waldemar Carlo Ulrich Thome Respiratory Control and Apnea of Prematurity Neonatal Respiratory Control and Apnea of Prematurity. Strategies for prevention of apneic episodes in preterm infants: Are respiratory stimulants worth the risk? Chapter 5 : Publications Authored by Jaques Belik PubFacts Lung Fluid Balance During Development and in Neonatal Lung Disease - Processed Richard Bland David P. Carlton Lucky Jain 8. Role of inflammation in the pathogenesis of acute and chronic neonatal lung disease. Chapter 6 : Faculty Members Department of Physiology Note: Citations are based on reference standards. However, formatting rules can vary widely between applications and fields of interest or study. The specific requirements or preferences of your reviewing publisher, classroom teacher, institution or organization should be applied. Chapter 7 : Frontiers Bronchopulmonary Dysplasia Early Changes Leading to Long-Term Consequences Jaques Belik. Susceptibility of the Immature Lung to Oxidative and Mechanical Injury. Susceptibility of the Immature Lung to Oxidative and Mechanical Injury. The Newborn Lung, pages Chapter 8 : The Newborn Lung: Neonatology Questions and Controversies Matrix metalloproteinase-9 (MMP-9) is detected in lung tissues subjected to ventilator-induced injury and is involved in the process of lung injury. Page 12