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Studies on the normal and abnormal lung growth in the human and in the rat with emphasis on the connective… Cherukupalli, Kamala 1989

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STUDIES ON THE NORMAL AND ABNORMAL LUNG GROWTH IN THE HUMAN AND IN THE RAT WITH EMPHASIS ON THE CONNECTIVE TISSUE FIBERS OF THE LUNG By Kamala Cherukupalli B. Sc (Biochemistry) Delhi University, India, 1980 M. Sc. (Biochemistry) M. S. University, Baroda, India 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES ACADEMIC PATHOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA (cT)Ms Kamala Cherukupalli, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of M-frT>£m>C P/PTTiOLQC^y The University of British Columbia Vancouver, Canada Date /WfrfZCH 2Jf^ / ? ? p DE-6 (2/88) Infants with bronchopulmonary dysplasia (BPD), showed impaired body growth when compared to control infants. In terms of changes in the biochemical composition of the lung, BPD infants had higher DNA, soluble protein, collagen and desmosine contents as well as increased concentrations of DNA, collagen and desmosine in their lungs when compared to the growth patterns obtained for the lungs of control infants. Pathologically BPD was classified into 4 grades. Grade I BPD, was a phase of acute lung injury, grades II and III were proliferative phases. In grade IV BPD, lung structure returned towards normal. Evidence of fibrosis was seen by a significant increase in collagen concentration in grades II and III while desmosine concentration was seen to increase in grades III and IV suggesting that the increase in collagen and desmosine contents in the lungs of BPD infants may be controlled by two different mechanisms. Collagen type I/III ratio was seen to decrease progressively from grade II to grade IV BPD in comparison to age matched controls, indicating a higher proportion of type III collagen in the lungs of infants with BPD. From the clinical analysis and the results obtained from discriminant analysis proce-dure, it was seen that there was a high degree of correlation between the continuation of the disease and collagen accumulation in the lungs suggesting that pulmonary fibrosis with excessive collagen accumulation is an integral part of BPD. This fibrotic process seemed to correlate significantly with assisted ventilation and high oxygen supplementa-tion received by the infants, but it was difficult to assess the individual contribution of the two treatments in the pathogenesis of BPD. Other variables such as severity of the initial disease and the length of survival of the infants, made the assessment of individual contribution much more difficult. Table of Contents Abstract ii List of Tables viii List of Figures xi Acknowledgement xv Dedication xvi 1 INTRODUCTION 1 1.1 INTRAUTERINE LUNG GROWTH 2 1.1.1 The lung at birth 3 1.2 POSTNATAL LUNG GROWTH 5 1.2.1 Animals 5 1.3 CONNECTIVE TISSUE OF LUNG 10 .1.3.1 Collagen 10 1.3.2 Types of Collagen in the Lung 11 1.3.3 Elastin in the lung 13 1.3.4 Elastin and Development of the Lung 15 1.4 POST-PNEUMONECTOMY LUNG GROWTH 15 1.5 FIBROTIC LUNG DISEASE 20 1.6 BRONCHOPULMONARY DYSPLASIA 23 1.6.1 Clinical Manifestations 25 iv 1.6.2 Radiological Manifestations 26 1.6.3 Pathologic Manifestations 27 1.7 RATIONALE 29 2 MATERIALS AND METHODS 32 2.1 CHEMICALS 32 2.2 ANIMAL EXPERIMENTATION 32 2.3 LUNG DEVELOPMENT STUDY 32 2.3.1 Experimental Design 33 2.4 THE BAPN- PNEUMONECTOMY STUDY OF MANIPULATION OF LUNG GROWTH 34 2.4.1 Experimental Design 34 2.4.2 Plotting of the Pressure-Volume Curves 36 2.4.3 Morphometry . 37 2.4.4 Biochemistry 40 2.5 STATISTICAL ANALYSIS 45 2.5.1 Lung Development Study 45 2.5.2 BAPN-Pneumonectomy Study 45 2.6 INFANT LUNG STUDY 47 2.6.1 Experimental Design 47 2.6.2 Clinical Studies 48 2.6.3 Histopathological Studies 48 2.6.4 Biochemical Studies 50 2.6.5 Extraction of the DNA, RNA and Protein 51 2.6.6 Estimation of content and synthesis of DNA 52 2.6.7 Protein Estimation 54 v 2.6.8 Extraction of Collagen and Elastin 55 2.6.9 Measurement of Hydroxyproline Content 56 2.6.10 Measurement of Elastin content 58 2.6.11 Radioimmunoassay for Desmosine 59 2.6.12 Measurement of Collagen Type I/III Ratio by SDS- polyacrylamide Gel Electrophoresis: 61 2.7 STATISTICAL ANALYSIS 63 2.7.1 Lung Growth Patterns 63 2.7.2 Disease Classification 64 3 RESULTS: PART I 66 3.1 LUNG GROWTH AND DEVELOPMENT IN AN ANIMAL MODEL (RAT) 66 4 RESULTS: PART II 81 4.1 EXPERIMENTAL ALTERATION OF LUNG GROWTH 81 4.1.1 Compensatory Growth following Pneumonectomy 81 4.1.2 Effect of BAPN on normal lung growth 92 4.1.3 Effect of BAPN on Post-pneumonectomy Lung Growth 99 5 RESULTS: PART TJI. 107 5.1 LUNG GROWTH AND DEVELOPMENT DURING GESTATION AND NEONATAL LIFE IN HUMAN 107 5.1.1 Biochemical Studies 107 5.2 LUNG GROWTH AND DEVELOPMENT IN INFANTS WITH BRON-CHOPULMONARY DYSPLASIA 112 5.2.1 Histopathology 116 vi 5.2.2 Biochemical Studies 123 5.2.3 Disease Classification 130 6 DISCUSSION 138 6.1 GENERAL POINTS 138 6.2 GROWTH AND DEVELOPMENT OF LUNG IN THE RAT 140 6.3 EXPERIMENTAL ALTERATION OF LUNG GROWTH 144 6.3.1 Post-pneumonectomy lung growth 144 6.3.2 Effect of Beta-aminopropionitrile on postnatal lung growth . . . . 147 6.3.3 Effect of Beta-aminopropionitrile on post-pneumonectomy lung growth 150 6.4 LUNG GROWTH AND DEVELOPMENT IN THE HUMAN INFANT . 151 6.5 GROWTH PATTERNS IN THE LUNGS OF INFANTS WITH BRON-CHOPULMONARY DYSPLASIA 157 7 CONCLUSIONS 174 7.1 RECOMMENDATIONS FOR FUTURE WORK 178 Bibliography 179 vii List of Tables 2.1 Morphometric calculations 43 2.2 Disease assessment reproducibility; Initial grading 50 2.3 Disease assessment reproducibility; Revised grading 51 3.4 Somatic and lung growth in the rat 67 3.5 Changes in DNA and soluble protein during growth in the rat 73 3.6 Soluble and insoluble contents and collagen concentration in the rat lung during growth 78 3.7 Soluble/insoluble collagen ratio and the desmosine contents in a growing rat lung 79 3.8 Collagen/protein, collagen/DNA and collagen/elastin ratios in a develop-ing rat lung 80 4.9 Results of the control and sham operated groups on days 7,14 and 21 post-pneumonectomy 82 4.10 Extent of compensatory lung growth in the pneumonectomy group when compared to sham/control group • . . 87 4.11 Results of the lung mechanics for the sham/control and pneumonectomy groups 91 4.12 Results of morphometric analysis of the sham/control and pneumonectomy groups at day 21 post-pneumonectomy 91 4.13 Results of lung mechanics in the BAPN group and the sham/control group 93 viii 4.14 Results of the biochemical variables obtained for BAPN administered and sham/control groups 96 4.15 Results of morphometric analysis of the BAPN administered and sham/control groups 98 4.16 Results of lung mechanics of the BAPN-pneumonectomy and pneumonec-tomy groups on day 21 post-pneumonectomy 99 4.17 Results obtained for the biochemical analysis of the lungs of BAPN+pneumonectomy and pneumonectomy groups 104 4.18 Results of morphometric analysis of the BAPN administered -fpneumonec-tomized group and the pneumonectomy group 106 5.19 Clinical history of normal infants 109 5.20 Clinical history of normal infants contd I l l 5.21 Clinical history and histology of ventilated infants 113 5.22 Chnical history and histology of ventilated infants contd 114 5.23 Mean values ±S.D. of the chnical and biochemical variables obtained for the lungs of control and ventilated infants 115 5.24 Summary of individual fits in non-linear statistical model 124 5.25 Summary of combined fits in non-linear statistical model 125 5.26 Classification Matrix for disease stages using ventilator days and days on O2 >60% as independent variables 131 5.27 Classification Matrix for the disease stages using ventilator days, days on FiO2>60%, conceptional age, collagen concentration and collagen I/III ratio as independent variables 132 5.28 Results of biochemical and chnical analysis in the different disease stages 133 ix 5.29 Results of the biochemical analysis in the lungs of ventilated infants and their age-matched controls List of Figures 1.1 Compensatory growth resulting after pneumonectomy 16 2.2 Test grid used for hght microscopic morphometry 41 2.3 Illustration of alveolar counting 42 2.4 Illustration of intercept counting 44 2.5 Extraction of soluble protein and DNA 53 2.6 Extraction of collagen and elastin 57 3.7 Body weight gain from late gestation to 7 weeks postnatal life in the rat 67 3.8 Results of the wet lung weight gain during growth in the rat 68 3.9 Dry lung weights in the rat during gestation and postnatal life 69 3.10 Ratio of wet lung/dry lung weights during growth in the rat 70 3.11 Increase in total DNA per lung during growth 71 3.12 Growth pattern of DNA concentration in the rat 72 3.13 Changes in the total protein per lung during growth in the rat 73 3.14 Growth pattern of protein concentration in the rat lung 74 3.15 Total collagen contents during growth of the rat lung 75 3.16 Growth pattern of collagen concentration in the rat lung 76 3.17 Ratio of soluble/insoluble collagen during growth of the rat lung 77 3.18 Changes in the total desmosine contents in the rat lung during growth . . 79 3.19 Growth pattern of desmosine concentration in the rat lung 80 xi 4.20 Results obtained for the right lung weights in the 5 groups on days 7, 14 and 21 post-pneumonectomy 83 4.21 Total lung weight results obtained for the 5 groups on days 7, 14 and 21 post-pneumonectomy 84 4.22 Total DNA contents obtained for the right lungs in the 5 groups on days 7, 14 and 21 post-pneumonectomy 85 4.23 DNA synthesis levels obtained for the lungs in the 5 groups on days 7, 14 and 21 post-pneumonectomy 86 4.24 DNA concentration in the lungs of the 5 groups on days 7, 14 and 21 post-pneumonectomy 88 4.25 Total soluble protein content in the right lungs of the 5 groups on days 7, 14 and 21 post-pneumonectomy 89 4.26 Pressure-volume curves obtained for the sham/control and pneumonec-tomy groups on day 21 post-pneumonectomy 90 4.27 Pressure-volume curves obtained for sham/control and BAPN groups on day 21 post-pneumonectomy 93 4.28 Protein concentrations obtained for the lungs of the 5 groups on days 7, 14 and 21 post-pneumonectomy 94 4.29 Total collagen contents obtained for the right lungs of the 5 groups on days 7, 14 and 21 post-pneumonectomy 95 4.30 Collagen concentrations obtained for the lungs of the 5 groups on days 7, 14 and 21 post-pneumonectomy 97 4.31 Comparison of the pressure-volume curves obtained for the BAPN+pneumonectomy group and the pneumonectomy group on day 21 post-pneumonectomy . . 100 4.32 Total desmosine contents in the lungs of the 5 groups on days 7, 14 and 21 post-pneumonectomy 101 xii 4.33 Desmosine concentrations in the lungs of the 5 groups on days 7, 14 and 21 post-pneumonectomy 102 5.34 Body weight gain with increasing age in control and ventilated infants . . 108 5.35 Changes in lung weight with age in control and ventilated infants . . . . 108 5.36 Case No. 85-055: Showing residual hyaline membranes and pulmonary interstitial emphysema; magnification40X 116 5.37 Case No. 85-055: Showing residual hyaline membranes and pulmonary interstitial emphysema; magnificationlOOX 117 5.38 Case No. 86-211: Stage I BPD; magnificationlOOX 117 5.39 Case No. 88-095: Stage II BPD; magnificationlOOX 118 5.40 Case No. 88-095: Stage II BPD; Thick interstitium with small air spaces; magnificationlOOX 119 5.41 Case No. 88-027: Stage III BPD; Terminal air ways enlarged, muscular hypertrophy, PMN's in air spaces; magnificationlOOX 120 5.42 Case No. 88-061: Stage III BPD; magnification40X 120 5.43 Case No. 87-115: Stage IV BPD; Enlarged irregular spaces; thinner walls, Muscular hypertrophy; magnification40X . 121 5.44 Case No. 86-067: Healed BPD stage; Small and large air spaces, thin interstitium, Minimal fibrosis; magnification40X 122 5.45 Pattern of DNA concentration in the lungs of normal and ventilated infants 124 5.46 Total DNA contents in the lungs of normal and ventilated infants . . . . 125 5.47 Total alkali soluble protein content of normal and ventilated lungs . . . . 126 5.48 Pattern of collagen concentration in the lungs of normal and ventilated infants 127 5.49 Total collagen contents in the lungs of normal and ventilated infants . . . 127 xiii 5.50 Changes in the collagen I/III ratio with increasing age in normal and ventilated infants 128 5.51 Pattern of desmosine concentration in the lungs of normal and ventilated infants 129 5.52 Total desmosine contents in the lungs of normal and ventilated infants . . 130 xiv Acknowledgement I wish to express my gratitude to my teacher and supervisor, Dr. W. M. Thurlbeck who despite his busy schedule always found time to guide me throughout my graduate studies. My sincere thanks to Dr. R. H. Pearce for his valuable guidance and suggestions for the problems I faced during this work. I would hke to thank my coUeagues Harman Sekhon, Hilary Brown, Felix Ofulue, Jan Larson, Susan Alburscheim, Mary Battell, Hassan Khadempour, Avi Rotschild, Emad Massoud and George Spurr for their friendship and encouragement. I wish to express my gratitude to my parents and my husband Sudhakar who inspired me to undertake this venture. The financial assistance of the Medical Research Council of Canada is gratefully acknowl-edged. xv Dedication Dedicated to my parents LAKSHMI and RAMAMOORTY and to SRI SATYA SAI BABA x v i Chapter 1 INTRODUCTION The human fetal lung at birth mediates gas exchange between blood and air. When the fetal-placental circulation is interrupted and the fetus makes a sudden transition from intrauterine to extrauterine life, adaptation to the extrauterine environment is crucial to the fetus and an inability to assume this function has life threatening consequences. The respiratory distress syndrome (RDS) of the newborn, is the leading cause of death in human newborns. A goal of many investigators is to determine methods for stimulating normal lung maturation either in-utero or after birth so as to prevent the development of RDS. In order to understand and interpret data concerning the experimental and therapeutic manipulations of lung maturation, it is necessary first to understand the normal process of pre- and post-natal lung growth and development. It is now believed that growth and maturation may progress separately in the lung. Growth is a quantitative phenomenon while maturation is qualitative and these terms can be applied to cells or tissues. The lung undergoes a series of complex structural changes during late-fetal and early post-natal fife, which are related to alveolar formation. These structural changes are accompanied by cell multiplication and cell maturation as well as by tissue growth (increase in lung wt) and tissue maturation (alveolar multiplication, thinning & restructuring of alveolar walls). 1 Chapter J. INTRODUCTION 2 1.1 INTRAUTERINE LUNG GROWTH The lung develops from a laryngotracheal groove in the endodermal tube. In humans, the ventral groove appears at 26 days of gestation and evaginates to form the lung bud which branches by 28 days gestational age. The human intrauterine lung growth is divided into five stages. The term, EMBRYONIC PERIOD is used for the first 6 weeks after conception and during this period the proximal part of the bronchial tree develops. 65-75% of bronchial branching occurs between the 10th & 14th week and is completed by the 16th week. At this stage, lung has a distinct glandular appearance with airways lined by columnar epithelium and separated from each other by poorly differentiated mesenchyme. At this stage the lung is said to be in the PSEUDO-GLANDULAR PERIOD. This is followed by CANALICULAR PERIOD which lasts from 16th to around 24th- 26th week and is characterized by the proliferation of mesenchyme and the development of a rich blood supply within it, together with flattening of epithelium that lines the airways. At about 24 weeks of gestation, the initially smooth walled saccules develop an ir-regular, slightly wavy configuration. This is the beginning of the TERMINAL SAC PERIOD and of secondary crest formation. At about 26 weeks and continuing to about 32 weeks, there is a further striking change, although vascularization and thinning of the epithelium proceed through out this period. The previously thick interstitium thins progressively and the air space walls become narrower and more compact. At the same time the secondary crests first noted at 24 weeks become progressively taller and thin-ner. The air space walls and those of the developing crests retain their earlier double capillary structure with separate capillary networks oriented beneath the epithelium of each surface. Recognizable alveoli make their appearance at 32 weeks of gestation, marking the beginning of ALVEOLAR PERIOD, and continue to increase in number to term and Chapter 1. INTRODUCTION 3 beyond. The alveoli can be difficult to distinguish initially from the spaces formed by growing secondary crests. However, they are distinguished by their thin walled, multi-faceted polygonal structure and their single capillary wall from the more rounded spaces formed by the double walled secondary crests. The first comprehensive morphometric study of the growth and development of the human lungs was done by Langston &; Thurlbeck [118], on a group of pre-term and term infants up to one month post-natal age. Although the lungs of pre-term infants and fetuses have developmental stages ap-proximating pseudoglandular, canalicular, terminal sac and alveolar phases, they are not as well defined with regard to the time of occurrence in the humans as in animals. In case of rats and mice, no further development occurs in utero after the saccular phase as their lungs lack alveoli at birth. 1.1.1 The lung at birth The degree of lung development at birth varies widely among species but surprisingly little precise information is available. It has been suggested that lung development parallels general body development [74j. As has been mentioned earlier, rats and mice have no alveoli at birth, whereas rabbits, kittens, calves and humans have a few alveoli at birth. Guinea pigs and other animals who lead an active life right from birth seem to have well alveolated lungs at birth. This indicates that considerable maturation of the lung occurs postnatally in most species. The number of alveoli at birth in humans varies considerably with a mean of 55 million and a range of 10 - 149 miUion [118]. The cause for this wide variation is not known and it does not seem to be due to inter-observer variation as considerable variation in the total alveolar number has been found by a single observer [118]. Wigglesworth & Desai [229] have stressed that lung development may be critically dependent on the amount of amniotic fluid and on intrauterine respiration and that these quantities may be influenced by such factors as catecholamines, smoking, Chapter 1. INTRODUCTION 4 maternal hypoglycemia, alcohol and barbiturates. Thus it is possible that intrauterine lung development may be very sensitive to external stimuli and that the wide variation in total alveolar number at birth reflects this sensitivity. Glucocorticoids [116],[44],[215], depending on the dose administered near term, result in differentiation of the alveolar epithelium and secretion of the surfactant at the expense of cell multiplication. Thyroxine administration to rabbit fetuses results in functional lung maturation and surfactant secretion, although lung weights are unchanged and the cell numbers unknown [233]. Intrauterine decapitation of fetal rats and rabbits resulted in immaturity of the alveolar wall cells, indicating a role for pituitary in the process of maturation of lung [140],[20]. One possible way that the pituitary may be effective is through growth hormone [34],[35] and since growth hormone only acts through somatomedins, it suggests that the lung is able to generate somatomedins for the process of maturation [195]. Maternal heroin addiction and alcohol administration are associated with a decrease in the incidence of respiratory distress syndrome; and an increase in lung surface activity. T h e mechanism is unknown: perhaps the effect may be mediated through adrenocortical hormones or through alteration in lipid metabolism. Experimental starvation, low calorie diet and low protein diet during pregnancy in rats are associated with decreased surface activity [78]. On the other hand, Naeye et al [143], showed that in humans, underweight mothers after 32 weeks gestation, with low weight gains during pregnancy, had infants whose lungs were more mature compared to infants of overnourished mothers. Clearly multiple factors seem to be involved in mediating alveolar wall maturation. Some of these may be mediated through hormonal control but there may be many as yet unidentified factors. Chapter 1. INTRODUCTION 5 1.2 POSTNATAL LUNG G R O W T H From the events in the intrauterine development of lung, it is seen that considerable maturation of the lung to achieve fully functional capacity occurs postnatally in most species. Postnatal lung growth has long been of interest to researchers and due to its unique expansile character it has been suggested in the past that the lung grows only or mainly by expansion. This theory has since then been proven not to be true as it has been shown that there is not only an increase in tissue weight as the lung grows, but also an increase in cell multiplication and hypertrophy. The lung differs from other organs in that its total volume increases more than its tissue mass, the net result is progressively higher air per gram of tissue throughout childhood [201],[207]. 1.2.1 Animals Postnatal lung growth in animals [38],[39],[3],[65], [180], particularly in the rat [38],[39], has been extensively studied by numerous investigators. Postnatal development of the rat lung is described as occurring in three phases -.LUNG EXPANSION; TISSUE PRO-LIFERATION; AND EQUILIBRATED GROWTH [38]. • LUNG EXPANSION : Days 1-4 comprise the phase of lung expansion. During this period, the lung grows primarily by expansion and little lung tissue is added. Lung volume increases less than bod}' weight and the volume proportion of air in the lung increases while the volume proportion of tissue decreases [39]. Cellular multiplication, as assessed by nuclear labeling using tritiated thymidine, is fairly low at this stage [61], suggesting that there may be little additional tissue added during this period. However, studies conducted in the mouse [3] using scanning electron microscopy, showed an increase in mass of lung tissue by showing an in-crease in the elongation of secondary crests and increased laying down of collagen Chapter 1. INTRODUCTION 6 and elastin fibers, and increased mitosis in interstitial cells by the 3rd & 4th day of postnatal fife. Other studies also conducted in the mouse [61], showed an increase in the labeling index during the first few days of life thus suggesting that some cell multiplication occurs in this phase. A characteristic feature of the wall of the primary saccule is the presence of a double capillary network. Progressive fusion of the two capillary layers occurs during postnatal development [191]. The wall of the saccule contains a relatively large amount of connective tissue flanked on each side by capillaries. The main component of the central connective tissue is the primary interstitial cell or the fibroblast which is also termed as myofibroblast, due to the presence of contractile filaments in the cell. The primary saccules are fined by type I & II pneumonocytes with the latter forming a relatively high proportion of the alveolar surface in fetal lungs as compared to adult lungs. • TISSUE PROLIFERATION : The second phase of lung growth in the rat has been designated as the phase of tissue proliferation and starts from 4th postnatal da}' and lasts until approximately day 14. This phase is characterised by the subdi-vision of the primary saccule by secondary crests and by formation of definitive alveoli [39]. The rate of lung growth is relatively faster than that of the previous phase. Lung volume increases faster than body weight., so that specific lung volume (vol./ gm.body wt.) actually increases. Alveolar surface area rapidly increases due to the process of septation and surface area per unit body weight increases. Surface area also increases to the 1.6 power of lung volume during this phase [226]. If the lung grew by expansion only, surface area would increase to the two-thirds power of the change in the lung volume. This greater increase in surface area indicates the marked increase in complexity of the lung and rapid proliferation of alveoli, so Chapter 1. INTRODUCTION 7 that the surface area per unit volume increases. • EQUILIBRATED GROWTH : This is the third phase of alveolar growth and begins at approximately 2 weeks of age [39]. This phase is characterised by slowing of the increase in lung volume, indicating a decrease in specific lung volume. The rate of cell proliferation falls and there is a consequent decrease in surface area per unit weight. New alveoli continue to be added, because alveolar surface area increases directly with lung volume rather than to the two-thirds power as would be anticipated on the basis of simple expansion. The secondar3* crests lengthen and only a single capillary layer can be found in the walls of the airspaces. • A fourth phase exists in some, if not most species:- the phase of SIMPLE EX-PANSION which starts when alveolar.multiplication ceases. It is not clear whether alveoli continue to multiply throughout life in rats, because continued somatic and lung growth is characteristic of this species [191] or whether alveolar multiplication is completed by 10 weeks of age [37:. There are certain controversies with regard to the time periods assigned to these stages of postnatal development of lung. Data obtained by Holmes & Thurlbeck [98] in the rat, indicate that the alveolar multiplication occurs between 4 and 10 weeks of age. While most investigators agree that the majority of alveoli appear in the postnatal period, the time at which alveolar multiplication ceases is much disputed [7],[53],[72],[210],[65],[98], [24]. The exact way or ways in which alveoli grow is uncertain, as is the relationship between the synthesis and deposition of connective tissue components of the lung and alveolar formation. Emery [72],[73] hypothesized that the elastic fiber network is the key to alveolar multiplication. An elastic fiber, together with collagen, is consistently found at the free margins of the secondary crests that subdivide primary saccules and Chapter 1. INTRODUCTION 8 around the mouths of alveoli. The elastin-collagen network forms an interconnecting net which has been referred to as the fishnet by investigators [129];[73]. These connective tissue fibers achieve this network by the formation of crosslinks between their respective macromolecules. These inter - and intra- molecular cross-links of collagen and elastin are formed with the help of the enzyme, lysyl oxidase, a C u + + requiring enzyme. This enzyme aids in the oxidation of the -amino group of lysine or hydroxylysine to the corre-sponding -semi aldehyde, allysine or hydroxyallysine. These semialdehydes then undergo a spontaneous extracellular condensation by an aldol condensation of two allysine alde-hydes forming allysine aldol (intramolecular), thus forming aldol type of crosslinks or by the shift" base reaction between allysine( or hydroxyallysine) and a lysyl ( or hydroxylysyl) residue of an adjacent polypeptide to give aldimine type of crosslinks (intermolecular) such as hydroxylysinonorleucine in collagen and desmosine and isodesmosine as present in elastin. These cross-links are necessary for fibril formation as well as for the normal ten-sile strength and biological function of connective tissue fibers. These functions are profoundly altered if the cross-linking enzyme, lysyl oxidase is inhibited [32]. This con-nective tissue defect can be produced experimentally by administering to the animals, beta-amino propionitrile (BAPN), a lathyrogen which is an irreversible inhibitor of lysyl oxidase and thus prevents the cross-linking of elastin and collagen. This results in an interference with alveolar multiplication and causes a reduction in alveolar multiplication in the lungs of BAPN-treated animals [112], [113]. These observations therefore lend support to the fishnet theory of Emery et al. [129], [73] and also show that cross-link formation is undoubtedly of great importance in determining lung structure and changes in cross-link formation may play a role in the molecular basis of development, aging and lung disease. The location, amount, stability, and changes in aging and disease of lung collagen cross-links has not been explored directly. Chapter 1. INTRODUCTION 9 Emery and Fagan [73] have formalized the description of alveolar growth and have hypothesized that there are three ways in which alveolar multiplication may occur. • Segmentation • Alveolarisation • Compoundment The first process, Segmentation, describes the way that the primitive alveolar saccules become subdivided into smaller units, finally developing alveoli. Loosli and Potter [129] and later on Emery and Fagan [73], stressed the importance of connective tissue in the process of alveolarisation. Emery and Fagan [73] describe that after birth, there is a progressive development of a fish-net type of elastic-collagen structure, the apertures of which form the mouths of alveoli. They regarded the elastic-collagen tissue formation as the primary event producing the smaller units. Emery and Fagan [73] thus conceive the appearance of alveoli in the developing lung as if a balloon with a very phant wall were inflated within and through a container formed by a semirigid mesh. The term Alveolarisation was used to describe the process by which alveoli develop in the non-alveolated walls of the airways whereby respiratory bronchioles are converted to alveolar ducts and terminal bronchioles to respiratory bronchioles. The relative im-portance of alveolarisation and the time of its occurrence, is uncertain. The term Compoundment was used to describe the growth of alveoli in inter-saccular walls (common wall between two saccules) as opposed to segmentation which involves a single wall. Elastic tissue fibers form and alveoli protrude between the elastic-collagen net, and the alveoh may grow into either of the saccules between which the wall lies. Chapter 1. INTRODUCTION 10 1.3 CONNECTIVE TISSUE OF LUNG Connective tissue comprises approximately 25% of the adult human lung and is an inti-mate part of all lung structures. In the lung, collagen Sz elastin form the major connective tissue elements with collagen representing 60- 65% and elastin about 20- 25% of all con-nective tissue, the rest being represented by proteoglycans and glycoproteins. 1.3.1 Collagen In the adult, collagen comprises 10 -20% of the dry weight of the lung and represents 60 to 65 percent of all lung connective tissue [28], [167]. There is some species variation in the relative abundance of lung collagen; for example, in the adult mouse it comprises 6% of dry weight [128] and in the human it comprises 20 % [28]. At the microscope level, collagen is found throughout the extracellular space of the lung. In the alveolar structures, collagen is present in the alveolar interstitium as well as the endothelial and epithelial basement membranes [131]. Although the content of collagen (amount/ dry wt.) in the tracheobronchial tree and pulmonary vasculature is greater than that in the parenchyma, the total mass of the parenchjrma is much larger and thus the bulk of the collagen in lung is that present in the alveolar structures. In the adult, most lung collagen is insoluble. It has been assumed that the relative insolubility of lung collagen results from the extracellular covalent interactions among collagen molecules [106] and between collagen and other components of the extracellular matrix that form soon after the newly synthesized collagen molecule has been secreted from the cell [91],[167]. Lysyl oxidase, the enzyme responsible for collagen cross-Unking, is present in the lung [32]and the lysyl oxidase derived cross- links, lysylnorleucine, hydroxy lysylnorleucine, dihydroxy lysylnorleucine as well as the newly discovered collagen cross-links namely, pyridine and hydroxy pyridine [77] have been isolated from hydrolysates of Chapter 1. INTRODUCTION 11 lung collagen. 1.3.2 Types of Collagen in the Lung Lung collagen is complex in that it is heterogenous in type and anatomical localization. The lung contains a spectrum of known collagen types. In the adult parenchyma, types 1 and III account for more than 90% of the collagen. From studies of the adult human lung, the relative amounts of Type I & III collagens have been shown to be in the ratio of 2 to 1 [187],[188]. Type I is present throughout the interstitium of the alveolar structures and like type I in other tissues, is thought to be the major collagen type comprising the banded fibers observed by electron microscopy [99]. Immunofluorescent studies with t3rpe-specific antibodies have shown that type III collagen is found along with type I throughout the interstitium [131],[132]. Collagen types I and III are also present in the connective tissue sheaths surrounding the tracheobronchial tree and the pulmonar}' arteries and veins as well as in the visceral pleura. Type II collagen in lung is confined to cartilage in the trachea and large bronchi. Type IV collagen has been localized to the epithehal and capillary basement mem-branes of the alveolar structures, where it represents about 5% of the parenchymal collagen [167],[131],[132]. Type V collagen is present in the alveolar structures but it is not clear whether it is limited to the endothelial and epithehal basement membranes or whether it is present in the interstitial matrix as well [131],[132]. Like type IV colla-gen, type Vis thought to represent approximately 5% of parenchymal lung collagen [167]. Its anatomic form is unknown. All available evidence suggests that the collagen types present in the lung are ge-netically identical or in other words their molecular structures are identical to their counterparts in other organs. In this regard, most attention has been focused on collagen types I <5J III. Cyanogen bromide (CnBr) peptides of alpha- chains from lung collagens Chapter 1. INTRODUCTION 12 I and III map on carboxymethyl cellulose and sodium- dodecyl sulphate acrylamide gels similarly to alpha 1 (I), alpha 2 and alpha 1 (III) peptides from other tissues [164]. As seen in other organs, changes in collagen content, extractability, types and produc-tion are associated with lung maturation. The most detailed studies concerning develop-mental changes in lung collagen content have been carried out in the rabbit [28]. They reported a dramatic increase (2-3 fold) in lung collagen content (collagen/ dry wt.) in the last trimester of fetal development. The location of this new collagen is uncertain because there is little recognizable interstitial collagen present in the distal lung parenchyma of humans or the rat during this period [218]. It has been shown [91] that the basement membrane collagen in the rat is complete beneath the large airway epithelium but is in-complete beneath the developing peripheral airways as late as the 18th day of gestation. Although epithelial basement membranes are well developed.in distal lung parenchyma during the later days of fetal life, endothelial basement membranes are incomplete and discontinuous. Thus collagen seems to appear early in fetal development in association with airway morphogenesis and later as the component of the interstitium and basement membranes during alveolar development. Bradley and coworkers [28] have shown in rabbits that the accumulation of lung colla-gen continues past fetal life into early postnatal life and then slows, although the amount of collagen expressed as percent of lung weight continues to increase throughout the first 3 years of the rabbit's life. It is unclear how much of the increased collagen mea-sured biochemically is interstitial and how much is airway, vessel, or basement membrane collagen. There are only fragmentary data concerning the changes in human lung collagen dur-ing lung growth. There is a 12-fold increase in parenchymal collagen concentration as the lung matures from the second trimester fetal to adult stage [30], but there is no infor-mation at intermediate times. It is not clear whether human lung collagen concentration Chapter 1. INTRODUCTION 13 remains stable once maturity is reached [158]. In general, major changes have not been found. Much of the data is based on older, less specific methods, and differences in site selection and sex [158] make comparisons between studies difficult. In tissues other than the lung, there is increasing evidence that there are age related changes in the types of collagen present. For example, in skin, the ratio of type III collagen to type I collagen decreases with age. The marked heterogeneity of lung collagen, together with the known age related differences in the development of lung structures, suggest that significant age related alterations in the quantity of each collagen type may be found. 1.3.3 Elastin in the lung Elastic fibers are present in virtually all vertebrate tissues, although it is within a few tissues, such as arteries, some ligaments, and the lung, that elastin comprises a substantial percentage of the total protein. Elastin is widely distributed in the tissues of the lung, although estimates of elastin content have varied widely, ranging from as low as 2% in rodents up to 25% in man. After careful lung dissection into separate septa, pleura, non-respiratory bronchioles, and larger vessels, Pierce and Ebert [158] found the highest concentration of elastin in the lung parenchyma (20-30% of the connective tissue dry weight). The elastic fiber is composed of two distinct components. The elastin, or amorphous component, is the major fraction, comprising some 90% of mature fiber. The second is the microfibrillar component, a glycoprotein, which appears as small fibrils 10-12nm in diameter. The association of the elastin with microfibrils occurs extracellularly where the microfibrils are thought to function as nucleation sites for future elastin deposition and initiate elastic fiber formation [173],[91]. Elastin is secreted from the cell as a 72000 molecular weight precursor (tropoelastin) Chapter 1. INTRODUCTION 14 which cross-links with other tropoelastin molecules with the aid of lysyl oxidase, the cross-linking enzyme to form an insoluble polymeric protein [177]. It is this highly cross-linked form that functions as an elastomer. Lysyl oxidase functions similarly here as with collagen, and initiates cross-link formation by catalyzing the oxidative deamination of the specific lysyl residues on the elastin precursor molecule converting them into allysines. The relatively reactive residues spontaneously condense to form various Schiff bases and aldol condensation cross-links. Within a few days most of the cross-links have isomerized into stable quaternary .pyridinium ring structures of desmosine and isodesmosine. These cross-links are unique to elastin and can be used as specific markers for the mature insoluble protein. Elastic fibers are ubiquitous in the lung, and as with other organs they are closely associated with collagen and proteoglycans. The elastic fibers can stretch to 140% of their resting length before breaking, whereas collagen which contributes tensile strength to the lung, can stretch only about 2%. The connective tissue of the lung represents a continuum which can be viewed as being made up of three functional components: the "axial connective tissue" of the bronchi, bronchioles and pulmonary vessels, the "peripheral connective tissue" of the pleura and the interlobar and interlobular septa and the "parenchymatous connective tissue" of the alveolar septa [224]. The axial connective tissue represents the immobile center and the peripheral connective tissue, the oscillating part of the system. The parenchymatous connective tissue forms the elastic link between the oscillating and immobile part. This gives a continuum of fibers throughout the lung such that any force exerted on the parenchyma is distributed throughout the lung. According to the Setnikar-Mead model [186],[137], collagen and elastin in the lung operate in parallel, and independently of each other. At low lung volumes the elastin fibers are readily extendable, giving the steep part of the volume-pressure curve. As the lung volume increases, the coiled collagen fibers are straightened and restrict further Chapter 1. INTRODUCTION 15 expansion, producing a decrease in the compliance of the combined collagen and elastin networks and increasing the stiffness of the lungs at maximum lung volumes. 1.3.4 Elastin and Development of the Lung The chronological appearance of the elastin in mammalian lung is similar to that seen in studies conducted in other tissues [138]. Generally, it has been observed that around the third trimester of pregnancy microfibrillar components appear, followed within a few days or weeks, depending on species, by deposition of amorphous elastin which steadily increases in amount until birth [111]. Recent studies conducted on fetal sheep lung [179] shows that there is a steep rise in elastin concentration during the canalicular period of lung development. In the rat, this event appears to commence later, during the period of alveolarization [180], [38]. Recent work of Desai and Wigglesworth [63] indicated that desmosine can first be .detected in the human lung at 22 weeks gestation and increases linearly up to about 15 weeks postnatal age. 1.4 POST-PNEUMONECTOMY L U N G G R O W T H Lung growth has been studied by various experimental manipulations to the lungs of animals; one is pneumonectomy, the process of removing one lung or portions of a lung. Pneumonectomy has provided useful information about lung growth. Studies show that following unilateral pneumonectomy or resection of lung tissue, the remaining lung tissue generally increases in weight, volume, connective tissue content and number of nuclei to equal approximately that of both lungs of control animals [208],[209],[172],[162]." The extent of this regenerative response has been seen to be different in different species and is said to have an age-dependent time course in various species. Chapter 1. INTRODUCTION 16 2 0 0 or o CONTROL s> <R*U^,'/ p N X ( R ) CONTROL (RJ 0 2 7 14 Figure 1.1: Compensatory growth resulting after pneumonectomy PNX=pneumonectomy, R=right lung, L=left lung In rats subjected to pneumonectomy, the following sequence of events occur [162]. In the Fig 1.1, right lung mass is compared to that of the right and both lungs of unoperated controls. Compensatory changes in right lung mass are not detected until after the second postoperative day; following this lag, dry lung mass increases rapidly until it reaches a value equal to that of both lungs in the control group. In animals of 85 gm. body weight (approximately 3 - 4 weeks of age) at the time of surgery, a normal total lung mass is restored at postoperative day 7. Right lung growth then slows to a rate appropriate to both lungs of controls. In older rats, however, the interval required for restoration of normal total lung mass increased by more than two-fold [162]. These observations are Chapter 1. INTRODUCTION 17 consistent with the conclusions of others that compensatory replacement of the resected tissue is more rapid in young than in adult rats [37],[145],[98]. The general features of this time course are retained independent of the age or gender of the animal on which the surgery is performed [162],[37],[145]. The lag preceding initi-ation of the rapid phase of growth is apparently constant, whereas the interval required to complete the response depends on both species and age [208],[37], [145],[98], and is related to the basal rate of lung growth. In case of other species like rabbits, cats and dogs, the resected lung mass is replaced more slowly and/or to an intermediate extent [54],[62],[230]. In the case of cats, adult animals that have reached sexual maturity lose the ability to initiate compensatory growth held by their immature counterparts [31]. Similarly, both human infants and young children have been reported to replace resected lung [202],[136], but the response appears to require ongoing growth of the tissue. In this regard, adult humans in whom both body and lung growth have ceased are incapable of tissue restoration and respond to lobectomy primarily by overdistention of the remaining air spaces [184]. This transition in the ability of humans to compensate for lung resection appears to correlate with cessation of alveolar growth and may begin as early as 2 years of age [209], [120]. Morphometric studies of the compensatory process in animals indicate that the growth is predominantly hyperplastic resulting in functional restoration of the tissue. The most comprehensive morphometric studies were conducted by Burrie and colleagues [40],[41],[17],[220], in which both young ( 50g body.wt.) and older (245g) rats, sub-jected to resection of the upper and middle lobes of the right lung, were studied 45 days after surgery. Quantitative electron microscopy showed in both groups of animals that total lung air space, capillary, and tissue volumes were restored completely and that both alveolar and capillary surface areas as well as diffusion barrier thickness returned within normal limits [40],[220]. These studies were carried out following a postoperative interval Chapter 1. INTRODUCTION 18 sufficiently long to, allow for completion of the process by which lung mass and volume were returned to normal over different time courses [98],[36]. Other studies [206] reported morphometric data for lungs fixed at approximately the midpoint of compensatory lung growth. Both surface area and volume of alveolar type II cells increased more extensively than predicted by the increase in lung mass. These observations suggest that a redistri-bution of lung cell populations may accompany ongoing compensatory growth and that the type II epithelium may be important in this process. Whether compensatory growth of lung following pneumonectomy involves formation of new alveoli is highly controversial, even within species. Publications which have postulated the formation of new alveoli [31], [50],[172] are counterbalanced by others denying this mechanism [37],[189]. Not all of these papers are based on solid quan-titative analysis, since the appropriate techniques were not available. However, even the more recent publications using modern morphometric techniques yield contradicting findings. This may be partly due to the problems inherent to the alveolar counting pro-cedures [211],[145],[21],[37] ,[189]. It is extremely difficult to properly define alveoli in histological sections and the counting procedure becomes more unreliable if alveoli alter their shape [98],[34],[211] which certainly happens during alveolar formation. The prob-lem is further complicated because different regions of the pulmonary parenchyma may present dissimilar patterns of reaction [98],[212]. Nattie and coworkers [145] described enlarging of central alveoli and multiplication of subpleural ones after a left-sided pneu-monectomy. This controversy is more than just of an academic interest, since presumably, alveolar multiplication might result in better restoration of function of the organ. Also, if alveolar multiplication occurs, lung resection may be a model to study mechanisms controlling alveolar multiplication in particular, as well as lung growth in general. Scanning electron microscopy showed that after transient widening of the air-spaces Chapter 1. INTRODUCTION 19 there was no difference in parenchymal morphology between operated animals and con-trols, 9 days after a bilobectomy [41]. Furthermore, scanning electron microscopy pro-vided no evidence of alveolar formation. Little is known about the biochemical events following pneumonectomy. There seems to be an increase in the adenylate cyclase/ cyclic AMP ratio in the first day following pneumonectomy before cellular multiplication occurs, suggesting an immediate hormone release [147]. The activity of the enzyme lysyl oxidase level, increases initially, then returns to normal by 2nd to 3rd day post-pneumonectomy [32]. Collagen synthesis in-creases substantially and it has been observed that following pneumonectomy, there is a shift in the protein synthesizing activity towards collagen synthesis [54]. As collagen accumulates and reaches a stable state, the protein synthesizing machinery goes back to synthesizing collagen at a normal rate. It is not clear what mechanism is involved in this sudden switching on and switching off of the connective tissue synthesis. If indeed connective tissue involvement in alveolar formation as hypothesized by Emery et al [73] in lung growth is true, then post-pneumonectomy lung growth may be accompanied by increased connective tissue synthesis. Assuming that post-pneumonectomy lung growth is similar to normal lung growth this would then result in alveolarization. In order to test the hypothesis of connective tissue involvement in alveolar formation, one could inject a set of the pneumonectomy performed animals with a lathyrogen such as beta-amino propionitrile (BAPN), and observe its effect on the connective tissue that is being newly synthesized and in turn test its effect on the process of alveolarisation. There-fore, the three questions that are being examined here are: a) Is post-pneumonectomy lung growth similar to normal lung growth? b) Does the post-pneumonectomy lung growth involve alveoli formation? c) Are the connective tissue components in the lung parenchyma involved in alveolarization? In view of this, the effect of BAPN on the lung compensator}' growth following pneumonectomy, may add considerable insight into the Chapter 1. INTRODUCTION 20 role of connective tissue in alveolar formation and compensatory lung growth. 1.5 FIBROTIC LUNG DISEASE Diffuse interstitial fibrosis is the final result of a multitude of diverse causes. It is a dis-order that follows a wide variety of lung injuries which may be environmental, infectious, immunologic, toxic or pharmacologic [82],[122],[12],[155]. In some patients, the cause is obscure and there is no associated condition or a specific agent and the disease is referred to as "idiopathic pulmonary fibrosis" (IPF) [58]. Regardless of the cause of lung fibrosis, the disease is characterized by diminished lung volume, decreased lung compliance, and abnormal gas exchange [216]. Clinically, the condition may progress rapidly or remain relatively stable [217]. The interstitium of fibrotic lung is thickened with an apparent in-crease in collagen and elastin, and intra-alveolar scarring may also be prominent features in some histologic sections [199]. Alveolar type II cells are prominent, especially early in the course, and an inflammatory exudate is present in the interstitium and alveolar -spaces. Due to limitations of analysis of human material, much of the study on lung fibrosis in the past was done in animals with experimentally induced pulmonary fibrosis. Pulmonary fibrosis can be induced by a number of agents including radiation [155], paraquat [88], diquat [133], silica [90], oxygen [170], and drugs such as bleomycin [197]. Although the precise nature of the injury is not well defined, fibrosis caused by bleomycin has been studied as a "nonspecific" form of pulmonary fibrosis. The importance of oxidant injur}', toxicology and industrial disease in pulmonary medicine has led to interest in other models such as those induced by "oxygen, ozone [123], paraquat or silica [88],[90]. While many of these models bear little relationship to the human fibrotic state, these models have enabled investigators to trace the development of pulmonary fibrosis in Chapter 1. INTRODUCTION 21 morphological, physiological, biochemical, and immunological terms. Lung 'fibrosis' can be described as a disordering of the cellular architecture of the lung parenchyma associated with characteristic alterations of the interstitial connective tissue matrix. This includes localized accumulations of collagen [88], alterations in the types of collagen [187], and derangement of the form of collagen [12]. While the local accumulation of interstitial collagen seen morphologically in fibrotic lung disease is obvious to even the casual observer, it is very difficult to define and quan-tify biochemically. The attempt to correlate histologic and mechanical properties with connective tissue levels in humans has been hampered by methodologic problems. Fulmer et al [82] were not able to document differences between collagen concentration in the lungs of nine patients with idiopathic pulmonary fibrosis and those of control patients. Furthermore, they could not demonstrate any correlation between collagen concentration and the degree of fibrosis. Zapol et al [235], examined human lungs after severe respi-ratory failure. Utilizing an extensive lung sampling technique from different lobes, they demonstrated increased total collagen content (two-fold normal levels within 3 weeks of the onset of the illness) in the lungs, although the collagen concentration appeared within the normal range. Data derived from a number of animal models have confirmed these observations and have shown that total lung collagen can be elevated in pulmonary fibro-sis [57],[91],[167],[88] (i.e., suggesting a net increase in collagen production or a decrease in collagen degradation) [125]. However, in some animal models [91],[167] and in human biopsies, collagen content(actually collagen concentration i.e., collagen content divided by tissue dry wt. or its DNA content) in fibrotic disease has been found to be nor-mal. [82], [52]. There are several possible explanations for this: (l)Total lung collagen has increased (ie., a net accumulation of collagen takes place) but the mass of the lung is also increased ie., the lung becomes hypercellular; thus, the content (collagen/dry wt.) will remain the same, if both total lung collagen and cell mass are increased proportionally; Chapter!. INTRODUCTION 22 (2) The local accumulations of collagen seen morphologically in some regions of the lung are balanced by local decreases of collagen in other regions of the lung; (3) What is seen morphologically as collagen is probably mostly type I collagen [91],[167],[187] and thus shifts towards more type I collagen would be recognized morphologically as an accumula-tion of collagen even though total collagen content may not have changed. Nevertheless, in spite of the failure of current biochemical data to confirm the morphological findings, most investigators generally accept the morphological finding of local accumulation of collagen in the alveolar interstitium in these disorders. Studies conducted by Clark et al [46],[47] and Phan et al [153], [154] on lung explant cultures, showed an increase in lung collagen due to greatly increased collagen synthesis rates occurring within days after instillation of bleomycin. Similar increases in synthesis are seen after paraquat administration [88]-,[89],radiation injury [155], and ozone exposure [123]. Although synthesis of non collagen protein is also increased,the increase in collagen synthesis is greater,, indicating a relatively selective process [47],[153]. Relatively little is known about other connective tissue components in pulmonary fibrosis. Changes in lung elastin synthesis have not been described, although elastin content approximately parallels increases in collagen content in bleomycin induced fibro-sis [87]. Total lung glycosaminoglycan content is also increased after bleomycin, and the increase is distributed among all types [109]. Qualitative changes in lung connective tissue occur in addition to quantitative changes. Although there have been few studies of collagen types during pulmonary fibrosis, they have been examined in fibrotic lesions in other organs [187],[199]. The results are, how-ever, inconsistent. In most fibrotic tissues, there seems to be a shift in the major collagens present such that there is more type I relative to type III. This seems to be true in lung as well, at least for idiopathic pulmonary fibrosis [188]. However, recent studies of the lung in progressive systemic sclerosis have shown that the ratio of type I to type III in the Chapter 1. INTRODUCTION 23 parenchyma is similar to that in the normals, suggesting that an increased type I/III ratio may not be a universal part of the fibrosis of the interstitial lung disorders [187]. Non-quantitative immunofluorescent studies of lung biopsies from patients with established fibrotic disease are consistent with the concept that there is increased amounts of type I collagen and decreased type III in the fibrotic parenchyma [131],[228]. Immunofluores-cent studies conducted in 1981 by Bateman et al [14] suggest that the relative proportions of types I and III are constantly changing as the disease progresses. Early in the disease, there appears to be an increase in type III, but with time, as the disease progresses, the type III fluorescence decreases and type I gradually increases. They confirmed the results of previous studies [228] in that areas of mature scar contain almost exclusively type I collagen. In addition, another report also suggests that biochemically the ratio of type III to type I collagen is initially increased in IPF [114] Thus, the role of different collagen types in fibrotic lung disease will probably vary with both the category and stage of each disease. The biological significance of these observations is not yet clear but it is known that native tj^ pe III collagen binds more avidly with fibronectin than other native collagens. In vivo, such an interaction could influence cell migration and connective tissue organization during periods of structural remodelling. Also, the protease susceptibility of collagen types varies, and this could be an important factor in the remodelling process. 1.6 B R O N C H O P U L M O N A R Y D Y S P L A S I A One type of fibrotic lung disorder occuring in infants, has received very little interest until the recent past. This fibrotic disease was noticed in newborn infants (mainly premature), when use of assisted ventilation became widespread during the 1960's and when chronic pulmonary disease in the nursery population became more common and more severe. The Chapter 1. INTRODUCTION 24 descriptive term "Bronchopulmonary dysplasia" was first applied by Northway, Rosan and Potter [148] to a disease process occurring in infants during respiratory therapy for acute hyaline membrane disease (HMD). Instead of improving rapidly after the initial acute disease, these infants went on to develop chronic pulmonary changes. With the advent of improved survival of the very low birth weight population in the special care nurseries, there is an increase in the incidence of chronic lung disease which may result from prolonged mechanical ventilation [175]. Although, all morphological studies of bron-chopulmonary^  dysplasia(BPD) have been made on autopsy specimens which represent the severe end of the disease spectrum, it is generally thought that BPD progressed from an initial exudative phase of diffuse alveolar damage to one of chronic repair. The diagnosis of BPD is based upon clinical findings as well as physiologic, radiologic and pathologic changes. As the disease progresses in severity, the diagnosis becomes increasingly evident, but in its initial stages of development, it is difficult to distinguish from the underlying respiratory difficulty. The diagnostic criteria for BPD have been recently formalized and most pediatricians would consider the following criteria of Ban-calari et~al [9] as being the most useful. • Intermittent positive pressure ventilation during the first week of life for at least 3 days. • Clinical signs of chronic respiratory disease for more than 28 days. • Supplemental oxygen for more than 28 days to maintain an arterial p02oi 50 mmHg. • Chest X-ray pictures showing persistent strands of radiodensity and areas of in-creased lucency that may coalesce into large bullae. There are many infants who do not fulfill the clinical criteria for BPD, but have abnormal lung function. It may be that either prematurity or neonatal ventilation is the precursor Chapter 1. INTRODUCTION 25 of long term abnormalities. The problem of definition of the disease makes the assessment of its incidence all the more difficult; it has been reported as being anywhere between 2.4 to 68% of all infants ventilated for prematurity [68],[234],[9]. The diagnosis of the disease is made by clinical, radiographic and with certainty from cytologic material obtained at autopsy or by biopsy. Owing to the similarity of radiologic findings in various forms of neonatal chronic pulmonary disease, the relative contributions of immaturity, residua of acute disease and BPD, cannot be easily determined in surviving infants.The diagnosis of BPD is made more difficult by a changing patient population with increasing immaturity in the infants ventilated, and changing techniques of ventilation. Physiologically, as the arterial oxygen tension decreases with progressive respiratory distress, the infants are treated with higher and higher concentrations of supplemental oxygen. With CO2 retention, there is dead space ventilation and this can be remedied by ventilating these infants particularly with increasing peak inspiratory pressures. However, occasionally hypoxia and severe respiratory acidosis due to CO2 retention may not be treatable in which case death may ensue in the early stages of the the disease. 1.6.1 Clinical Manifestations The initial chnical presentation is usually that of a premature infant with severe respi-ratory distress (RDS), although, the presenting respiratory difficulty may also be due to aspiration pneumonia, congenital heart disease or apnea, as well as Wilson-Mikity Syndrome. Serious respiratory insufficiency appears to be the common denominator. Hyaline membrane disease (HMD) is therefore not the only acute lung injury that can evolve into BPD but is by far the most common [161],[10],[168]. Typically, the infant is prematurely born, develops HMD, and requires assisted ven-tilation and supplemental oxygen to sustain life. The HMD is initiated by a relative deficiency of surfactant at the alveolar air-liquid interface that leads to stiff lungs; it Chapter 1. INTRODUCTION 26 is clinically characterized by signs of respiratory distress and hypoxemia despite sup-plemental oxygen. Infants who have typical uncomplicated HMD, then undergo a rapid improvement of lung function with the result that at one week of age they require minimal or no supplemental oxygen. In contrast, those infants destined to develop BPD, require prolonged ventilatory support and frequently experience pulmonary air leaks (intersti-tial emphysema, pneumothorax, or pneumomediastinum) [161],[10]. Pulmonary edema with a large left to right shunt through a patent ductus arteriosus can occur early in the development of the disease [84]. As the disease progresses, the infant shows increasing respiratory insufficiency with increased dependency on oxygen and artificial ventilation. Cor pulmonale and recurrent bouts of congestive heart failure may occur. The infant with BPD frequently succumbs after a prolonged course of several months of severe car-diopulmonary failure. 1.6.2 Radiological Manifestations Radiographic progression of BPD was originally described by Northway and colleagues [148] Radiographicallj', the disease is characterized as progressing through four stages. In stage I the initial chest radiograph shows bilateral fine granular pulmonary densities and air bronchograms indistinguishable from a severe case of RDS. Stage II occurs with further progression of the disease between 4 and 10 days of age and the X-ray changes were originally described as showing nearly complete opacification of both lung fields. This radiographic feature is now seen less frequently, presumably because of improvements in therapy. Further progression of the disease leads to stage HI which occurs at 10- 20 days of age by which time the radiograph reflects the alternating areas of atelectasis and emphysema in that it shows a striking picture of small rounded areas of radiolu-cency alternating with strands of radiopacity distributed throughout both lungs. If the infant survives to 30 days of age, radiographic stage IV is attained at which time the Chapter 1. INTRODUCTION 27 X-ray examination of the chest shows enlargement of the rounded lucent areas in the lungs alternating with thinner strands of radiodensity, increasing hyperexpansion and cardiomegaly. Episodes of segmental atelectasis may occur episodically in either or both lungs. Interstitial air leak, pneumomediastinum, and pneumothorax may complicate the radiographic picture in the early stages of the disease. The orderly progression from stage I to IV BPD as originally described [148] is now only rarely seen [149]; [69],[234]. Stages I and II are difficult to distinguish from HMD [149], and there is poor correlation between the pathologic and radiologic stages for stages I and II [70]. Although typical radiologic stage IV BPD still occurs, infants are increasingly seen with significant obstructive pulmonary disease and have hyperinflation as the predominant radiographic abnormality. These more recent observations demon-strate the difficulty in attempting to diagnose BPD purely by the original radiographic criteria of Northway et al [167]. A more useful approach would be to develop a staging system for the progression of the disease using additional parameters. 1.6.3 Pathologic Manifestations In the early development of the disease, the histopathology is that of the underlying respiratory difficulty. Since most of the infants have RDS, the initial pathology of the disease is usually indistinguishable from any severe case of RDS. Except for the underlying lung immaturity, these pathologic findings are quite similar to other forms of severe acute lung injury, e.g. adult respiratory distress syndrome (ARDS) and oxygen toxicity. As the disease progresses, there is increasing necrosis of the bronchial and bronchio-lar mucosa with replacement of the bronchial and bronchiolar epithelium by squamous epithelium. The lumens of the air passages become filled with a mixture of inflammatory cells, epithehal cell debris and accumulations of mucinous secretions. Small to medium size pulmonary arteries and arterioles undergo progressive changes with hyperplasia of Chapter 1. INTRODUCTION 28 the endothelial cells and intimal thickening and medial hypertrophy leading to a signifi-cant narrowing of the lumen. A progressive widening of the alveolar septa due to edema, hypercellularity and fibrosis occurs with marked damage of the gas exchanging portion of the lungs. There are large areas of atelectatic and distorted alveolae alternating with groups of enlarged air spaces creating a honeycomb pattern. The alveolar lining cells progressively change from type I to type II pneumocytes. In infants in whom the overall pulmonary damage is most severe, there is marked septal fibrosis and an increase in the number of septal cells, resulting in a marked thickening of the alveolar septum with focal complete obliteration of the alveolar lumens. The histologic appearance of the lung differs in infants dying at a younger age (less than 3 months post-natal age) from BPD than in older infants. While there is extensive bronchial and bronchiolar involvement with areas of thickened fibrotic alveolar septae alternating with areas of less fibrosis and enlarged airspaces [205],[23], in infants dying at a younger age with BPD, the changes are different in those infants dying at an'older age. In the older infants, there are fewer and less severe changes in the bronchi, with little squamous metaplasia of the epithelium and there is septal fibrosis in virtually every case [23], with areas of expansion with dilated and emphysematous alveoli. Studies have shown decreased lung growth as evidenced by a decrease in total alveolar number, and a 3-4 fold increase in the diameter of the alveoli, interpreted as emphysematous destruction of the acinar structure [196],[141]. Fibrosis may result from excessive deposition of collagen fibers in the interstitium or due to a change in the t}-pe of collagen being laid down. Lung collagen contents are reported to be elevated in BPD [95]. Both the mechanisms and the characteristics of this fibrotic pattern in BPD are not known. It is tempting to compare the fibrosis that occurs in BPD to that of interstitial lung disease in adults. However, the mechanisms may differ because fibrosis in BPD occurs when lung collagen content is increasing 2-fold in a period of four months in normals, during the process of Chapter 1. INTRODUCTION 29 lung growth [180] and also at a time when the normal ratio of the main parenchymal collagens (I and til) may not have reached adult values. 1.7 RATIONALE Previous studies of B P D have been primarily limited to morphologic observations at postmortem examination and chnical trials. As mentioned earlier, damage occurs to the lung during the fetal and early neonatal period when there is already a rapid accumulation of connective tissue, seen as fibrosis under a light microscope. Although evidence of fibrosis has been shown by morphological studies, the amount, type and extent of fibrosis has not been measured biochemically along with a prospective cohort study. Until the recent past,the biochemistry of B P D was poorly understood. Despite the increasing numbers of survivors of neonatal intensive care and the high frequency of respiratory problems in this group of infants, there has been no detailed biochemical study of this disease as well as the effects of high oxygen levels administered and the assisted ventilation received by the infants, both of which are additional potential mechanisms of lung injury for the premature infant. In order to see the changes caused by the disease on lung growth, it is first neces-sary to obtain a biochemical pattern of lung growth in a control infant of comparable age. Once the normal pattern of lung growth is determined, it would provide a baseline against which the growth of the lungs of infants with respiratory disease can be assessed. Although considerable research has been directed towards problems of lung development few studies have considered the quantitative aspects of lung growth. Therefore, in order to understand the processes which underlie such modifications of lung growth as B P D , it is necessary first to obtain knowledge of normal quantitative lung growth throughout fetal and neonatal period. Chapter 1. INTRODUCTION 30 However, there-are certain special problems encountered in a human study such as this. First of all, the specimens are obtained at the time of autopsy which in case of diseased lungs represent the severe end of the disease spectrum and hence make it difficult to study the sequence of changes in the lung growth brought about by the disease. Secondly, the lung sample taken for biochemical analysis represents only a small fraction of the lung and given the inhomogeneity of the fibrotic process within the lung, may not be representative of the whole lung. Because of the limitations with human material, experimentation with animals has become increasingly important in studying the sequence of events in various disease processes. First, the biochemical pattern of lung growth in a normal animal for example, the rat, should be obtained and can be compared with the lung growth occurring in human infants. Once that is achieved, then by experimentally manipulating lungs of rats to mimic various human diseases, sufficient knowledge with regard to sequence of changes in the growth of diseased lungs can be obtained. The objectives of the present research were: • To obtain a biochemical pattern of lung growth and development in a normally growing human infant in fetal and early life. • To study the fibrotic lung disease such as Bronchopulmonary Dysplasia and the consequent clinical treatment (mechanical ventilation; oxygen supplement) on nor-mal lung growth of infants. • To study the biochemical aspects of growth and development of lung in normal rats from gestational age to adult life. • To study postpneumonectomy lung growth in rats and compare it to normal lung growth in the same species. Chapter 1. INTRODUCTION 31 • To study the involvement of connective tissue fibers in the process of alveolarization during normal lung growth as well as in compensatory lung growth. Chapter 2 MATERIALS AND METHODS 2.1 CHEMICALS A l l reagents used in this study were of analytical grade and purchased, unless otherwise stated, from either Sigma Chemical Company [St. Louis, Miss., USA] or Fisher Scientific Limited [Fairlawn, New Jersey, USA]. A l l chemicals used for electrophoresis study were purchased from Bio-Rad Laboratories. Type I and III collagens of greater than 95% purity, used in this study were obtained from Calbiochem Laboratories. Elastin and desmosine standards were obtained from Elastin Products Limited [Pacific, M O . , USA]. Iodinated Bolton-Hunter Reagent used in desmosine Radioimmuno Assay was obtained from New England Nuclear Labs [Boston, Mass., USA] The desmosine antibody raised in rabbit for use in RIA. was a kind gift from Dr.S.Y.Yu, Veterans Administration Hospital, St.Louis, MO. , USA. 2.2 ANIMAL EXPERIMENTATION 2.3 LUNG D E V E L O P M E N T STUDY Timed pregnant Sprague-Dawley rats were purchased from Charles River Laboratories. They arrived one week pregnant and were placed in individual cages in the Shaughnessey Research Center (Vancouver, B.C.) animal unit where they were fed a standard Purina rat chow and were supplied ad libitum with water. 32 Chapter 2. MATERIALS AND METHODS 33 2.3.1 Experimental Design On days 16,17,18,19 and 21 of gestation, each rat was anesthetized with anesthetic grade ether( Mallinckrodt chemical works product) and while sedated, an intraperitoneal in-jection of sodium pentabarbitone (BDH chemicals,Canada Ltd., Vancouver B.C.) was administered (5mg/100 g b.wt). A caesarean section was performed and the mother was exanguinated by sectioning the abdominal aorta. The uterus was cut along its longitu-dinal axis to expose one amniotic sac at a time. The fetal membranes were removed and the umbilical cord was sectioned close to the fetus. Body weights were measured. The fetuses were placed in a petri dish with normal saline and the lungs were removed within a few minutes. The fresh lung weights were recorded. Similarly, rats at one day of and 1,2,3,4,5,6 and 7 weeks of postnatal age were injected intraperitoneally with sodium pentababitone. Body weights of individual rats were mea-sured. The}1 were then sacrificed by sectioning the abdominal aorta in order to reduce the pulmonary blood volume. The peritoneal cavity was opened and the diaphragm was sectioned in order to collapse the lungs. The rib cage was cut open to expose the lungs. After the lungs were flushed with saline in order to remove most of the blood present in the pulmonary blood vessels, they were removed from the animal, dissected free of the main airways, patted dry on gauze cloth and their fresh weights were noted. The lungs were then stored in liquid nitrogen for future biochemical analyses. Lungs were then lyophilised to a constant weights and their dry weights were noted. In case of the fetuses, due to the small size and weights of their lungs, the lungs from fetuses of the same age (from 2-3 mothers) were pooled. This was done in order to get sufficient amount of dry lung samples for the biochemical analyses. The results obtained were then divided by total number of lungs pooled in order to express the results per fetus. After the dry weights were determined the lung samples were then analysed for Chapter 2. MATERIALS AND METHODS 34 DNA, total alkali soluble protein, collagen and elastin contents by methods that are described in later sections of this chapter. 2.4 T H E B A P N - P N E U M O N E C T O M Y S T U D Y O F M A N I P U L A T I O N O F L U N G G R O W T H Male Sprague-Dawley rats were used for this stud}-. All animals were purchased from Charles River breeding laboratories [Guelph, Ontario, Canada]. They arrived at three weeks of age in five batches of fort}' rats each, and were housed in the departmental animal care unit. They were given standard purina rat chow and water ad libitum. 2.4.1 Experimental Design The animal litters matched for body weight ( ± 7g) were randomly segregated into five groups a few days before they were four weeks of age. t Group 1: BAPN administered. • Group 2: BAPN administered and pneumonectomized. • Group 3: Pneumonectomy performed. • Group 4: Sham operated. • Group 5: Untouched controls A day prior to four weeks of age, animals from groups 1 &: 2 were administered BAPN solution [2 g/10 ml saline] intraperitoneally, at the amount of 5 p\ per gram body weight (lmg BAPN/gm body weight). This concentration of BAPN solution was administered ever}- two days for the next three weeks to the experimental animals of groups 1 and 2 . Similar amount of saline solution was administered to animals in group 4 for the same Chapter 2. MATERIALS AND METHODS 35 number of days. Lungs removed from the experimental animals were used for two types of experiments: 1. The quantitation of the in vivo changes by biochemical analyses. 2. The quantitation of the in vivo changes by morphometric analysis. Surgery At four weeks of age, the animals from groups 2, 3 &: 4 were intramuscularly given atropine sulphate solution (Rafter 8 products, Calgary, Canada), (0.4 mg/100 g body weight), approximately 30 minutes before surgery, in order to lessen bronchial secretions. Animals were then put in a halothane (Halocarbon laboratories Inc. New Jerse)-, USA) vapor saturated chamber until anaesthetized. During surgery, the rats were continuously anaesthetized with halothane inhalation with intermittent oxygen administration. Initial body weight of the rat was measured(Delta range Mettler P E 360) The left side of the chest was shaved clean and wiped with alcohol and iodine. A skin incision, approxi-mately an inch~ long, was made. The skin was separated from the muscular layer and the muscular layer was then incised.The thoracic cavity was opened by cutting through the fifth intercostal space. The hilum of the left lung was clamped and the left lung was removed. The hilum was ligated with 2-0 silk thread. The right lung was gently inflated with oxygen. The ribs were approximated and the thoracic cavity was closed with 4-0 polypropylene sutures. The muscular layer and the skin were closed in two layers with continuous sutures. A n intramuscular injection of penicillin-streptomycin was adminis-tered (0.1 ml/100 g body weight) to minimize the possibility of postoperative infection. Injection of Demerol (Winthrop Laboratories, Aurora, Ontario, Canada) was given (0.15 mg/100 g body weight) intramuscularly to relieve postoperative pain. Sham operations were similarly performed except that the thoracic cavity was not opened. Chapter 2. MATERIALS AND METHODS 36 The animals regained conciousness soon after the surgery. Post operative mortality rate was less than 4%. Animals were allowed access to food and water ad libitum. At 1, 2 and 3 weeks, post-pneumonectomy, at the age of 5, 6 and 7 weeks respectively the animals from all the groups were injected with sodium pentabarbitone (5mg/100g body weight) intra-peritonially, and sacrificed as above. For biochemical analysis, two hours prior to the sacrifice of the animals, they were injected intraperitoneally with a single 0.5 ml dose of [3H]thymidine (1 uci/ g body weight). After sacrificing the animals, the lungs were flushed with 10 ml of sahne in order to remove most of the blood present in the pulmonary blood vessels. The lungs were then removed, dissected free of airway branches, weighed to record fresh lung weight, then stored in liquid nitrogen for future biochemical analyses. For morphometric analysis, the animals were given 100% oxygen before exanguination, to degas their lungs. After exaguination the animals were sacrificed, the trachea was cannulated and the lungs were removed from the thoracic cavity along with the heart and the pulmonary tissue. 2.4.2 Plotting of the Pressure-Volume Curves The cannulated trachea was attached to a connector in a pressure-volume curve chamber(P-V Chamber), designed for excised lungs and connected to a pre-calibrated pressure vol-ume curve plotting equipment (Validyne, Model MC; Hewlett Packard, Model 7041A X-Y recorder). After 10 minutes the lungs were inflated with a step-wise motor driven air syringe to a trans-pulmonary pressure of 25 cm water and then deflated to a trans-pulmonary pressure of 0 cm water. For each animal, two pressure-volume curves were plotted and the second deflation hmb was used for analysis. Total lung capacity (TLC) was defined as the amount of air in the lung at a trans-pulmonary pressure of 25 cm water at the end of the second inflation. Recoil pressures were recorded at 10 percentiles of the TLC. According to the method of Colebatch et al. [49],all the data points over Chapter 2. MATERIALS AND METHODS 37 30% of TLC were analysed by fitting a single exponential to the pressure-volume curve, using a digital computer. The single exponential expression is: where, V is the volume at pressure p, VMAX is the theoretical volume of air at infinite trans-pulmonary pressure, b is the difference between the Vmax and the intercept on the volume axis and A" is a constant which describes the shape of the curve. 2.4.3 Morphometry a. Lung fixation and Lung volume determination After plotting the pressure-volume curve, the lungs were removed from the P-V chamber and subsequently inflated with 20% phosphate buffered formalin at a constant transpul-monary pressure of 25cm water for 3 days, with the lungs submerged in the fixative. After fixation, the trachea was clamped and the lung was removed from the fixative. The hilum was ligated and the trachea was removed. The fixed lung volume (V j r ) was determined by water displacement as described by Scherle [182]. A beaker is partially filled with distilled water and placed on a balance. The reading on the balance was adjusted to zero. The fixed lung was then suspended by a thread and completely sub-merged in water without touching either the sides or the bottom of the beaker. VL in cm 3 was estimated as equal to the weight in grams indicated on the balance. According to Archimedes Principle which states: (2.1) A body partially or totally submerged in a body of fluid experiences a bouyant force(FB) equal to the weight of the fluid displaced by the volume of that body. Weight of fluid displaced = Bouyant Force(FB) (2.2) Chapter 2. MATERIALS AND METHODS 38 ' Volume of lung(Vl) = — ^ — — - (2.3) specific gravity of fluid b. Lung sampling and tissue processing. A mid-sagittal block of each lobe was made. Blocks were photographed and contact prints were made from the negatives. Blocks were embedded in paraffin. Two 5pm thick sections were cut and stained with haematoxyliri and eosin for light microscopic studies. c. Tissue shrinkage factor. The area of the block was measured by a computer assisted digitizer, from the contact prints of the blocks taken for tissue processing and embedding. The area of the stained tissue section on the microscope shde, was also measured by the same technique men-tioned above. The shrinkage factor of the tissue was determined by dividing the area of the tissue sections by the area of the pre-processed lung tissue. Area shrinkage and linear shrinkage factors were calculated as follows: Area of the post — processed tissue Area Shrinkage factor (asf) = — — — : (2-4) Area of the pre — processed tissue /Area of the post — .processed tissue Linear Shrinkage factor (lsf) = W— — —: (2-0) V Area of the pre — processed tissue No corrections were made for the thickness of the section. d. Light Microscopic Morphometric Measurements. All the slides were coded to abolish chances of bias in morphometric measurements. A microscope with manual stage (Leitz, Telepromar) was used for morphometry. Each shde was projected on to a screen with a square grid with 42 equidistant test points and the two diagonal cross hairs test lines. Chapter 2. MATERIALS AND METHODS 39 Using a 40X objective, the pre-determined area of the grid was 0.0625 mm2 and the total length of the two diagonal cross test lines was 0.64mm. After leaving 1mm margin, the rest of the area was used to choose 20 pre-determined equi-distant fields using a 4x5 matrix. A field of more than about 25% of non-parechymatous tissue (blood vessels, wall of the conducting airways, connective tissue septi) was ignored and the next adjacent field was used for measurements. Each of the 42 test points were counted according to their placement on histological structures, ie., alveolar air, duct air, alveolar wall, bronchial air and non-parenchymatous tissue. The smallest discrete structure surrounded by alveolar walls was considered as alveoli. The alveolar duct air was considered as the cylindrical core of air within the alveolar ducts and sacs, internal to the mouth of the alveoli. Conducting airway air was referred to as the bronchial air. The counting principle used in measuring random sections was also based on Delesse principle which states: Area proportions are equivalent to volumetric proportions and the plani-metric fractions of a section occupied by sections of a given component cor-respond to the fraction of the tissue volume occupied by this component. Thus, number of test points for each of the tissue components were expressed as a proportion of the total number of test points. This represents volume proportion of the tissue structure as: Number of test points falling on a structure X (2 6) v x 42 or total number of test points Intercepts either counted as wall intercepts (Iw) or duct intercepts (Id) and the sum of the two (Iw-j-Id) represented total number of intercepts (la). If the wall of the alveoli fell across the test line, two intercepts were counted (2 air exchanging surfaces), but if the Chapter 2. MATERIALS AND METHODS 40 alveolar wall touched the upper or the right side of the test lines or the end of the test hne lodged in the wall of the alveolus or in any other tissue structure, it was counted as one intercept. For the duct intercepts, (Id), an interception of the test hne by an imaginary hne across the mouth of the alveolus was counted as two intercepts. From these morphometric values, mean hnear intercept or average interalveolar wall distance (Lm), mean chord length of alveoli (laiv) and alveolar surface area (SWT) were calculated. The total length of the test hne on the grid was corrected for tissue shrinkage in order to project on to the fixed tissue. The mean hnear intercepts were calculated by dividing the total projected length of the test lines by the number of the wall intercepts. The number of the alveoli that lay within the grid on the upper left and right sides of the grid were counted (see table 2.1). The known area (A) of the grid was corrected for tissue shrinkage during processing. The counted number of alveoli were used to calculate the number of alveoli per unit area (N^), number of alveoli per unit volume (Nv), total number of alveoli (NQ7-), and the average volume of an alveolus (Vaiv). The distribution constant of the characteristic hnear dimension of alveoh (J) was considered as one and shape constant (/3) of alveolus was taken as 1.55 [Weibel, 1963] [225]. ... . . .. , J x \/NA3 .. _, .Number of alveoh per unit volume = — (2.7) P * V * va 2.4.4 Biochemistry Lungs for biochemical analyses were dissected free of large airways and blood vessels and the wet lung weights were measured. Lungs were then frozen in liquid nitrogen and stored at — 70 °C until future use. The frozen lung samples were lyophihsed until constant weights were achieved. Their dry weights were measured. The lungs were thinly sliced and placed in 2 ml of phosphate-buffered saline (PBS). They were then homogenized and Chapter 2. MATERIALS AND METHODS 41 Chapter 2. MATERIALS AND METHODS Figure 2.3: Illustration of alveolar counting Chapter 2. MATERIALS AND METHODS 43 Parameter Calculation Wall intercepts = Iw Duct intercepts = Id Total intercepts (la) = Iw + Id Volume proportion of: Alveolar air = vu a Duct air = v0<i Alveolar wall — ^ VW Bronchial air = v t t i Non-parenchymatous = V o np Number of alveoli in a field = N Total length of the test line (corrected for linear shrinkage (Isf)) = L r Area of the grid (corrected for shrinkage (asf)) = A - 2Lr Iw Mean linear intercept (L m ) Mean chord length of alveoli ([<,/„) 2L r x V „ » la Alveolar surface area (Swx) 4xVI - MLI Alveoli per unit area (NA) N _ A Number of alveoli per unit volume _ J x V N A 3 ~ 0 X y/\T~ Distribution constant (J) = 1 Shape constant (/?) = 1.55 Total number of alveoli (Nar) Average alveolar volume (Voiv) = NvxVI _ VlxV T , n N a r Table 2.1: Morphometric calculations Chapter 2. MATERIALS AND METHODS 44 Figure 2.4: Illustration of intercept counting Chapter 2. MATERIALS AND METHODS 45 the volume made to 4 ml with PBS. The resultant lung homogenates were used for the extraction and determination of DNA, RNA, protein, collagen and elastin. The methods of extractions as well as the estimations are described in detail in the section dealing with human studies. 2.5 S T A T I S T I C A L A N A L Y S I S 2.5.1 Lung Development Study-Group means and the standard error of the means (SEM) were calculated for all the biochemical variables at all ages. 2.5.2 BAPN-Pneumonectomy Study The experiment was conducted using a three way factorial design and analysed using analysis of variance. The experimental set-up was such that there were five different treatment groups and three days- ( 7,14 and 21 days post-pneumonectomy) at which period the biochemical analysis was performed. There were 10 replicates per (day, treat-ment) cell and 20 variables were measured for each animal. The different treatment groups were: • TI Sham surgery (Sham controls) • T2 No surgery, No BAPN injection (Untouched controls) • T3 No surgery, with BAPN injection (BAPN group) • T4 Surgery, No BAPN injection (Pneumonectomy group) • T5 Surgery, with BAPN injection (BAPN+Pneumonectomy group) Chapter 2. MATERIALS AND METHODS 46 If there is no difference between T l and T2 then these categories can be merged and the data are appropriately laid out for a factorial analysis of variance with factors: day, surgery and injection. However to accomodate all possibilities within a common analytical framework, the second level factors, surgery and injection, were examined in terms of contrasts of the treatments T l to T5, and two-way analysis of variance was performed. For example, if there was no evidence of a difference in levels between T l and T2, then the effect of surgery was examined by considering a linear contrast between T l , T2, T3 (no surgery) and T4 and T5 (surgery). So, to investigate the difference between T l and T2, a two way analysis of variance, with factors day and treatment and their interactions, was first performed. Where an interaction between day and treatment was detected for a variable, all future analyses was performed seperately for each day, that is, one way analysis of variance was used to compare the daily treatments. With either anal}rsis, linear contrasts were used to compare the treatment effects as follows: First, T l was compared to T2. If no difference was found then T l was merged with T2 and usual linear contrasts were formed to examine the effects of surgery (pneumonectomy), BAPN administration and their interaction. Alternatively, if there was a difference between T l and T2, then then the following linear contrasts were used to compare treatments: • C l : Pneumonectomy vs sham surgery (T4 vs Tl) (i.e. 10 0-10) • C2 : BAPN vs control (T3 vs T2) (i.e. 0 1-10 0) • C3 : BAPN vs BAPN+pneumonectomy (T3 vs T5) (i.e. 0 0 1-10) • C4 : Pneumonectomy vs BAPN-fpneumonectomy (T5 vs T4) (i.e. 0 0 0 1 -1) • C5 : BAPN-l-pneumonectomy vs sham surgery (T5 vs Tl) (i.e. 1 0 0 0 -1) Chapter 2. MATERIALS AND METHODS 47 2.6 INFANT L U N G STUDY 2.6.1 Experimental Design Lung samples were obtained from infant lungs at the time of autopsy at the British Columbia's Children's Hospital, Vancouver, British Columbia, Canada. Lung samples for the developmental studies were obtained from control infants dying due to non-respiratory causes and who had normal somatic growth. Lung samples of infants dying from BPD or from HMD, were also used for biochemical studies and the results obtained were compared with the developmental pattern obtained from the control lungs. Lungs were collected over a three year period from infants ranging from 20 weeks gestational age to 7 years of postnatal age in case of control lungs,and from 24 weeks gestation to 2 years of age in case of the BPD lungs. Samples from the peripheral part from the right lung were taken for biochemical analysis. This was done in order to obtain mostly the lung parenchyma and to avoid the airway branches. In case of control lungs, the piece of the right lung parenchyma that looked most "normal" was taken and in the case of the diseased lungs, the piece that looked typically abnormal was taken for the analysis. They were frozen and stored at -70 °C. An adjacent section was taken from the right lung to assess disease in the biochemical sample and to compare with the left lung section. The entire left lung was fixed-inflated for at least 24 hours at 25 cm H 20 in Kamofsky's solution. Random sections were cut from the left lung and were taken for histological examination in order to ascertain the morphological normality in case of control lungs and for the assessment of disease in case of BPD lungs. The same sections were later used for the grading of the fight microscopically observed pulmonary histopathologic changes in bronchopulmonary dysplasia, into different grades. Chapter 2. MATERIALS AND METHODS 48 2.6.2 Clinical Studies For all the infants who died of BPD, hospital records were reviewed retrospectively for the duration of mechanical ventilation, the duration of supplementation of oxygen at concentrations 60% or greater, the presense of patent ductus arteriosus as well as any airway infections. Care was taken to see that none of the infants included in the control group had any history of mechanical ventilation or oxygen supplementation. For all the control and BPD infants, their gestational and postnatal ages were taken into account. A new parameter was used in this stud}r, called "conceptional Age" which is the infant's gestational and postnatal ages added together. The birth and death weights for all the infants in the study were noted. 2.6.3 Histopathological Studies From all the BPD and HMD cases, lung samples that were taken for biochemical analysis, a histological examination was conducted on H&E stained light microscopic slides derived from inflated left lung and uninflated right lung of each infant. This examination was conducted on coded samples without the knowledge of their clinical histories, autopsy records or the results of the biochemical analyses. The histopathological changes were initially graded into 6 grades: 1. HMD : Immature lung; Hyaline membranes present in the air-spaces. 2. BPD I : Hyaline membranes are still present in air- spaces. There is early, degen-erative changes in airway epithelium; increased fibroblast proliferation and very minimal fibrosis in the interstitium. 3. BPD II : Increased degenerative changes in the airway epithelium; the interstitium is highly cellular; increased fibrosis; thick interstitial walls and small air-spaces Chapter 2. MATERIALS AND METHODS 49 present. 4. BPD III : Interstitium with variably thickened walls is present; air-spaces are larger than in grade II of the disease; there is less active proliferation of fibroblasts; there is also the presence of alternating areas of greatly enlarged spaces with thin walls and fibrosis. 5. BPD IV : Interstitial walls are much thinner. Air- spaces are mostly large. There is minimal fibrosis and only few fibroblasts are present in the interstitium. 6. Healed BPD : Interstitial walls are quite thin, almost reaching normalcy. Interstitial fibrosis is slight, if present, and focal. Squamous cell metaplasia may still be present. The lung overall looks quite normal. Each shde after the examination was assigned one of the 6 grades. The shdes were examined for the second time after recoding them and were once again independently categorized into the grades. This was done in order to check the consistency in the pathological assessment of the disease by the pathologist(Dr.W.M.Thurlbeck). The two independent grading processes were highly consistent and the percentage of reproducibil-ity was approximately 80% (see Table 2.2). The shdes on which the grading was not consistent, a third independent examination and grading was done, and the two coinci-dental grades out of the three were chosen as the category for that shde to be placed in (see Table 2.2). Then the number of categories of the histopathological grading were reduced from 6 to 4 categories by merging the first two categories into grade I group and merged the last two categories into grade IV group. This was done to simplify the corre-lation analysis between the chnical, biochemical and pathological findings of the diseased lungs. It also increased the number of samples in each of the categories which made the statistical comparisons easier. This process also resulted in increasing the reproducibility Chapter 2. MATERIALS AND METHODS 50 B P D T V / H E A L E D 5 B P D IV 1 1 2 BPD III 6 10 1 o JKIN BPD II 9 <£. BPD I 5 P I E / H M D 11 1 P I E / H M D BPD I BPD II B P D III B P D IV B P D I V / H E A L E D RANKING # 2 Table 2.2: Disease assessment reproducibility; Initial grading between the two independent pathological examinations to 88% (see Table 2.3). The H&E slides taken from the fixed left lung samples were also compared with, the slides made from corresponding fresh right lung samples for both the control and BPD lungs. Sections were cut from the portions of the lung parenchyma adjacent to the piece that was taken for biochemical analysis. The examination of both left and right lung H&E slides, indicated that the abnormality noted in the lungs of BPD infants was uniform in both, lungs and therefore, any assessment made on the left lung could be extrapolated to the whole lung. Same was the case with the normal lungs. 2.6.4 Biochemical Studies In all, 80 control and 52 BPD, HMD lung samples were analysed. All biochemical analyses "were performed with coded samples without the knowledge of the diagnosis in each case. The right lung pieces were removed from the -70 °C freezer and lyophilised to constant weight. They were reduced to a fine powder with a mortar and pestle. Weighed samples Chapter 2: MATERIALS AND METHODS 51 < BPD IV 1 8 BPD III 6 10 1 BPD II 9 BPD I 17 BPD I BPD II BPD III BPD IV R A N K I N G # 2 Table 2.3: Disease assessment reproducibility; Revised grading each representative of the whole were taken for the extraction and measurements of DNA, protein, collagen and elastin. This approach was preferred to sampling the homogenized lungs because in the latter method(i.e. homogenates of lungs) it was difficult to sample the particulate material accurately as it tends to sediment from the aqueous phase. 2.6.5 Extraction of the D N A , R N A and Protein The procedure of Schmidt-Thaunhauser [181] as modified by Wannemacher [221] was used for extraction of DNA, RNA and protein. 2.0ml of phosphate-buffered saline (PBS) was added to powdered lung sample (pre-weighed). The samples were homogenized in PBS, and the final volume of the homogenate was made to 4ml with PBS. To this homogenate, 50% trichloroacetic acid (TCA) was added to give a final concentration of 15% T C A in the 4ml homogenate. The TCA precipitated lung samples were vortexed and kept in a refrigerator at 4 °C for lhour. The lung samples were then centrifuged at 2000g (Beckman model J-68) for 10 minutes at 4 °C. The supernatents were discarded. The T C A precipitates were then washed 3 times with sodium acetate saturated ethanol. The T C A precipitates were digested with 2.0ml of 1.0M sodium hydroxide (NaOH) in a water bath at 37 °C for 90 minutes. After centrifugation at 2000g for 10 minutes, the Chapter 2. MATERIALS AND METHODS 52 the supernatants were, decanted into clean labelled tubes and the remaining precipitate was digested one or more times with 2.0ml of 1.0M NaOH at 37 °C for 90 minutes. The supernatants from both the digestions were pooled. 1ml aliquot was taken from this pooled supernatants for protein estimation. Cold (4 °C) 6.0ml perchloric acid (PCA) was added to the remainder of the NaOH digest, to give give a final concentration of 0.3M PCA. The samples were vortexed and kept at 4 °C for 45 minutes. The PCA treated samples were then vortexed and centrifuged at 2000g for 10 minutes and the supernatants were removed into clean tubes and were used for RNA analysis. 1.0ml of 1.0M PCA was added to all the precipitates and digested at 90 °C for 20 minutes. The digests were removed from the 90 °C waterbath, cooled and centrifuged. The supernatants were poured into clean tubes. The remaining precipitates were redigested in a similar fashion and centrifuged. The supernatants were pooled and analysed for the amount and synthesis of DNA. 2.6.6 Estimation of content and synthesis of DNA DNA content was measured according to the method described by Burton [42] using calf thymus DNA (Sigma D-1501) as standard. The standard stock solution (0.4 mg/ml was prepared and aliquoted in 1.0ml tubes and stored at -70 °C ). For an assay an aliquot was thawed, diluted 1:2 with 1.7M PCA and incubated at 90 °C for 10 minutes. After cooling, ahquots were pipetted in duplicate, into assay tubes, obtaining standard concentrations of 0, 16, 32, 48 to 120 pg of DNA. 0.2 and 0.4 ml volumes of the sample were taken in duphcates for the assay. All standard and sample volumes were made up to 1.0ml with 1.7M PCA. 2.0ml of diphenylamine reagent (1 g.diphenylamine, 100 ml. glacial acetic acid,2.75 ml. of concentrated sulphuric acid) prepared fresh, was added and vortexed. The assaj' tubes were then incubated in a water bath at 90 °C for 10 minutes. After cooling, the absorbance of the blue colour obtained was read at 600nm in Chapter 2. MATERIALS AND METHODS 53 Supernatant — r ~ Discard Freeze Dried Lung homogenized in 4 ml PBS Lung Homogenate Precipitation with 50% TCA Vortex: keep @ 4 C; 1 hr. centrifuge @ 2000 rpm, 10 min. Supernatant Discard PROTEIN (Lowry's method) 1 Precipitate T Ethanol Extraction with sodium acetate saturated ethanol Digestion with 1M NaOH @37 C, 3hrs. Acidification with 0.3 M PCA Vortex, keep @ 4 C , 45min. centrifuge @ 2000 rpm, 10 min. Supernatant I Discard Precipitate I Digestion with 1M PCA @95 C, 40 mins. centrifuge <§> 2000 rpm, 10 min. Supernatant DNA Burton's Diphenylamine method Figure 2.5: Extraction of soluble protein and DNA Chapter 2. MATERIALS AND METHODS 54 a spectrophotometer (Philips Pye Unicam SP6-550 UV/VIS). The standard curve used for DNA estimation was linear and all the samples fell within the limits of the standard curve. The DNA content per lung was calculated as follows: XT . ._ . . ug DNA calculated from standard curve x DF DNA/Lung(mg) = — / &\ B) 1 0 Q 0 _., . r Total volume of supernatant after PCA incubation Dilution factor = ———— :  Vol. of fraction taken from DNA assay Total volume of NaOH supernatant 4 . Vol. taken from DNA precipitation 3 Specific radioactivity of DNA was estimated by adding 1 ml of the DNA extracts to 9.0ml of scintillation medium contained in standard scintillation vials and counting the !3Hj activity. The incorporation of [3Hjthymidine into DNA (dpm/ug DNA) was considered to be synonymous with the net rate of DNA synthesis. 2.6.7 Protein Estimation Protein content was measured as per the method of Lowry et al [130]. Bovine serum albumin (BSA, Sigma A4378) dissolved in 0.1M NaOH was used as standard stock solu-tion (500/xg BSA/ml). Stock solution was aliquoted into small tubes and frozen at -70 °C . For the assay one aliquot was thawed and pipetted in duplicates into assay tubes to obtain standard concentrations ranging from 10-200 pg of BSA. 40/xl of the samples were used in the assay in duplicate. Total volume of the samples and standards was made to 500/il with 0.1M NaOH. 5ml of freshly prepared Lowry's solution (2% sodium carbonate in 0.1 M sodium hydroxide solution, 1% copper sulphate solution, 2% sodium tartarate solution, all in a ratio of 100 : 1 : 1) was added to the tubes, vortexed and allowed to sit at room temperature for 15 to 20 minutes. 500/il of freshly prepared 1.0M Folin's reagent (2N Folin -Coicalteau phenol reagent diluted 1 : 2 with distilled water and pH adjusted to 1.8 with 1.0M sodium hydroxide solution) was then added to each Chapter 2. MATERIALS AND METHODS 55 assay tube, vortexed and kept at room temperature for half hour. The absorbance of the blue colour obtained was read at 660nm in a spectrophotometer. The readings obtained for the samples were well within the range of the standard curve. The Protein content in the lungs was calculated as follows: 2.6.8 Extraction of Collagen and Elastin Collagen and Elastin were extracted according to the method described by Laurent et al [125] in which cyanogen bromide (CNBr) essentially solubilized all the lung collagen leaving behind an insoluble residue with an amino-acid content similar to elastin. 3ml of lung homogenates were centrifuged at 5000rpm for 10 minutes and the supernatants retained. The residues were washed twice with 1.5ml of phosphate buffered saline and centrifuged. The washings were repeated twice with 1.5ml of 2% sodium dodecyl sul-phate(SDS) solution and centrifuged. Finally, the residues were washed one more time with 1.5ml of PBS in order to remove excess SDS added. All the supernatants were pooled and retained for hydroxyprohne estimation. The residues were then washed with 2.0ml acetone and left overnight in the fumehood to dry. The residues were rehomogenized in 2.0ml of 70% (w/v) formic acid. 0.5ml of a O.lg/ml solution of cyanogen bromide (CNBr) in 70% formic acid, was added to the residue to produce a final CNBr concentration of 20mg/ml. The solutions were vortexed and N2 gas was bubbled through the samples for 15 seconds. The tubes were then sealed and incubated in a shaking waterbath at 37 °C and the reaction was allowed to proceed for 24 hours. The resulting digests were centrifuged at 5000 rpm for 20 minutes. The supernatants were decanted into 50.0ml wide mouthed tubes. The residues were washed four times with 2.0ml of distilled water. The supernatents were pooled in the 50ml tubes and the final volumes were made upto Protein/lung(mg) = Protein pg from standard curve x Dilution factor (2.9) 1000 Chapter 2. MATERIALS AND METHODS 56 20.0ml with distilled water. Both the supernatants and the residues were frozen at -70 °C overnight and freeze-dried till constant weight was reached. 2.6.9 Measurement of Hydroxyproline Content Samples recovered from the SDS/PBS extraction representing soluble collagen as well as from CNBr solubilized samples which represent insoluble crosshnked collagen, were hydrolyzed in 6.ON hydrochloric acid ( HCL) , in order to liberate hydroxyproline from peptide hnkage. Hydrolysis was performed in hydrolysis tubes with teflon lined screw caps at 110 °C for 18 hours. After hydrolysis two drops of 0.02% methyl red indicator were added to the hydrolyzates and then 2.4ml of 2.5N sodium hydroxide ( NaOH) for each ml of 6.ON HC1 until a faint straw colour was obtained, mixing was done either by the vortex mixer or by tube inversion. The hydrolysates were then filtered with Whatman number 1 filter paper and made to 15.0ml with distilled water. Hydroxyproline was measured in the hydrolyzates using the spectrophotometric method described by Woessner [232]. Series of standards containing 0-10//g L-hydroxyprohne in 2.0ml total volume of distilled water as well as 2.0ml aliquots of hydrolysates were placed in assay tubes. 1.0ml of freshl}7 prepared chloroamine-T solution was added to each tube vortexed and allowed to stand at room temperature for 20 minutes. Then 1.0ml of 3.15 M P C A was added to the tubes in the same order, in order to destroy excess chloroamine-T and stop the oxidizing reaction. The tubes were then vortexed and left to stand for another 5 minutes. Finally, 1.0ml of Ehrlich's reagent (20g of p-dimethylaminobenzaldehyde in 100.0ml of n-propanol) was added, vortexed vigorously and heated for 20 minutes in a 60 °C waterbath. The tubes were cooled under tap water for 5 minutes and the absorbance of the samples were read in the spectrophotometer at 561nm. The standard curve prepared was hnear to o/ig hydroxyproline , and the absorbance of the lung samples were all within the hnear range. The hydroxyproline contents of the lung samples were estimated directly from the Chapter 2. MATERIALS AND METHODS 57 Freeze Dried Lung homogenized in 4 ml PBS Lung Homogenate £ Supernatant 1 TCA precipitation Supernatant (discarded) ~1 Precipitate SOLUBLE PROTEINS (for HyPro Assay) centrifuge @ 5000 rpm, 10 min. Precipitate — r ~ 2% SDS washes x 2 centrifuge @ 5000 rpm, 10 min. Supernatant Residue T homogenize, 70% formic acid I CNBr Digestion 0.5ml CNBr soln. (0.1g/ml formic acid) @ 37 C, 24 hrs. i CNBr Digest centrifuge @ 5000 rpm, 15 min, I Supernatant COLLAGEN Precipitate ELASTIN Figure 2.6: Extraction of Collagen and Elastin Chapter 2. MATERIALS AND METHODS 58 standard curve. Collagen contents were calculated from the hydroxyproline estimates of the PBS-SDS soluble extracts (soluble collagen) as well as the CNBr-solubilized lung samples (Insoluble collagen or mature collagen). Laurent et al [125] have indicated that lung collagen contains 12.2% (w/w) hydroxyproline based on amino acid analysis of collagen standards and lung tissue collagen extracted in CNBr. Thus, the quantity of collagen was determined accordingly to the formula: pg collagen = 8.2 x pg hydroxyproline (2-10) 2.6.10 Measurement of Elastin content Measurement of the elastin content in the lung was done by measuring the amount of desmosine amino acid present in the elastin fraction of the lung extracts, as desmosine is unique to elastin. Desmosine was measured by a radioimmunoassay procedure. As peptide-bound desmosine does not react with the antibody, the samples must .first be hydrolyzed in 6.ON hydrochloric acid (HC1) in order to liberate the bound desmosine. Hydrolysis of lung Elastin samples The CNBr insoluble residue was hydrolyzed in 6.ON HC1 at 110 °C for 48 hours in order to liberate desmosine amino acid from peptide linkage. The 72 hour hydrolysis at 100 °C , as reported in earlier studies was found to be unnecessary as maximum }4eld of desmosine was obtained in 48 hours of hydrolysis. An aliquot of 200p\ was removed from the hydrolyzates and evaporated to dryness and made up to 1.0ml with pH 7.7, sodium phosphate buffer. The final pH of the solution was 7.2. These solutions were further diluted 15 times with pH7.2 sodium phosphate buffer. 100p\ aliquots of these samples were used directly for the quantitation of desmosine by radioimmunoassay. Chapter 2. MATERIALS AND METHODS 59 2.6.11 Radioimmunoassay for Desmosine A . Preparation of the radioactive probe CONJUGATION OF DESMOSINE WITH IODINATED BOLTON HUNTER REAGENT [BH-I-125]: In order to prepare a labelled desmosine molecule for the assay, it is first conjugated with a radioactive iodinated reagent. This conjugation process is required to be conducted as soon as the iodinated reagent is received, since the BH-I-125 is unstable if kept for more than 48 hours. But once the conjugation with desmosine is completed, the labelled conjugate is stable at 4 C for 1- 2 months. The whole procedure was conducted in a well ventilated hood in accordance with ra-diation safety precautions. The di-iodo [1-125] Bolton Hunter Reagent with total activity of 0.5 mCi was received from New England Nuclear NEN Research Products, Boston MA. in anhydrous benzene. The bottle of BH-I-125 was placed in an ice bath and the benzene was carefully evaporated to dryness by passing a gentle stream of nitrogen gas. 20 p\ of 1 mg/ml desmosine solution was transferred into the BH-I-125 bottle. The reaction was carried out overnight at 4 C (cold room) with constant gentle shaking. The conjugation reaction des-BH-I-125 was stopped by adding 0.5ml of 0.2 M glycine solution. The radioactive conjugate was separated from free BH-I-125 by passing the mixture through a Bio-Gel P2 column, and eluted with 0.1M acetic acid. 54 vials of 3.0ml fractions were collected. 2 pi of solution from each vial was removed and placed in marked tubes, for counting the radioactivity in the tubes with the help of an LKB gamma counter. Once the peak with the desmosine conjugate was determined all the fractions from that peak were pooled and aliquoted into 50 pi fractions in small vials and stored at 4 C for further use. For each assay with 100 tubes one 50 pi vial was sufficient. Chapter 2. MATERIALS AND METHODS 60 B. Preparation, of Desmosine Standards Standard desmosine solution of 1 pg/ml concentration was prepared in distilled water. The solution was aliquoted into 5.0ml tubes and stored at -70 C until needed. One 5.0ml tube was sufficient for one assay. Serial dilutions of the standard from the initial concentration of 1 /xg/ml are made at the time of assay, with 7.2 pH phosphate buffer. The dilutions range from 5 to 1000 ng/ml. For each asay internal standard preparations from an elastin hydrolysate derived from rat lung, was used. C. Assay Procedure Desmosine in the lung tissue hydrolyzates was assayed by a modification of the method of Harel et al [93]. 100/xl aliquot of the sample as well as serially diluted Desmosine standards (ranging from 5-1000ng/ml) were added to the assay tubes. They were diluted with 100/il of 7.2pH phosphate buffer to give a total volume of 200/xl. The solution was mixed with 100/il of rabbit anti-desmosine serum (1:100 dilution with 7.2pH phosphate buffer). The mixture was gently vortexed and then incubated at 30 °C for half hour( This allows for the Ag-Ab complexes to form between desmosine from the lung sample and the antidesmosine antibody). 50 p\ of the radioactive desmosine (labelled with Bolton-Hunter125- I reagent; containing 20,000 cpm in 50pl of phosphate buffer) was then added to the tubes.The tubes were gently vortexed and further incubated for 1 hour at 30 °C (This process allows for the competitive binding of the radioactive probe to the desmosine antibody in the reaction mixture. As a result the reaction mixture at the end of the second incubation contains Ab-Des complexes, Ab-Des[I-125] complexes, free desmosine and free desmosine[I-125].). 50 p\ of 10%(w/v) solution of Pansorbin cells was added to the tubes and gently vortexed and incubated at 30 °C for another hour.(Pansorbin cells adsorb on the surface of the Ab which is already bound to the Antigen(des or des[I-125]) and Chapter 2. MATERIALS AND METHODS 61 precipitates the Ag-Ab complexes while the free desmosine and radioactive desmosine remain in the supernatant.) The tubes were then centrifuged at 4 °C for 30minutes at 3500rpm. The supernatant was aspirated out carefully. Radioactivity of the precipitate was measured in an L K B gamma counter. Al l the assays were carried out in triplicates. From the results obtained, a standard curve was plotted on a semi logarithmic paper. X-axis was the standard concentration and the Y-axis was the radioactivity obtained in counts per minute(cpm). The sample concentrations were calculated directly from the standard curve. The sensitivity of the desmosine RIA was optimal and linear between 50 and 500 ng. Isodesmosine was only weakly cross-reactive (< 0.1%). In order to check the nonspe-cific interference from other proteins, varying amounts of hydrolysates of collagen and bovine serum albumin (BSA) wrere added to a premeasured amount of desmosine. There was no significant interference in the assay by any of the hydrolysates up to 200 pg in concentration. The dilution of the lung hydrolysate used for the assays had desmosine concentrations of less than 100 pg. 2.6.12 Measurement of Collagen Type I/LTJ R a t i o by S D S - polyacrylamide G e l Electrophoresis: C N B r solubilized collagen from the lung samples was freeze dried to a constant weight. A small amount of the freeze dried sample was then dissolved in sample buffer (0.01M phosphate, 7.2pH, 1% SDS) in preparation for polyacrylamide gel electrophoresis. The amounts to be loaded were determined from hydroxyproline content of the lyophilised collagen. Polyacrylamide gel electrophoresis was performed according to the method by Laemmli [117] and was performed on slab gels with stacking gel and running gel acrylamide concentra-tions of 3 and 12% respectively. The gel dimensions were 16cm(L) X 9cm(H) X 1.5mm(D) Chapter 2. MATERIALS AND METHODS 62 with 10 sample wells each of 4mm width. Collagen type I and III of greater than 95% purity and characterized by SDS-PAGE, were used as the standards and were obtained from Calbiochem laboratories. These collagen standards were subjected to similar pro-cess of extraction with CNBr as the samples to be analysed. 0.02% bromophenol blue was added to the sample and standard solutions. The tubes were then heated at 90 °C for 4 minutes in the presence of O.OlmM dithiothreitol to disrupt any sulphide hnkages. The tubes were cooled at room temperature for a few minutes. 10.0 pi volumes of samples and standards were loaded into the wells using a microsyringe. Electrophoresis was performed at a constant voltage (200V). Once the bromophenol blue marker reached the bottom of the gel, the electrophoresis was stopped. The gel was then removed and stained in 250.0ml of an aqueous solution containing 0.2% (w/v) Coomassie Brilliant Blue R-250, 10% Acetic acid and 40%(v/v) methanol by shaking gently for one hour. The gel was destained for 3 hours with continuous shaking in several changes of destaining solution (10% (v/v) Acetic acid, 40% (v/v) methanol). All staining and destaining solutions as well as gel buffers, were filtered to minimize background absorption during subsequent scanning. Gel scanning Gel scanning was performed at 530nm with a Gilford 250 spectrophotometer equipped with a hnear transport device using a 5 x 0.1mm slit. Peaks were recorded on Gilford 600 recorder with a chart speed of lOcm/min. The peptides Q 1(I)CB-7 and a^III) CBS were chosen as marker peptides because : 1. They are sufficiently large peptides to minimize diffusion losses during staining and destaining of electrophoresis gels. 2. They are not known to be involved in inter or intramolecular cross-linking and the two peptides are derived from homologous regions of the parent alpha-chains. 3. Both are well separated from adjacent peptides for up to 25 ug of collagen loaded to the gel [92]. The relative Chapter 2.. MATERIALS AND METHODS 63 ratios of Type I and III collagen were quantitated by measuring the area under the peaks representing the two marker peptides. In calculating collagen ratios we have assumed that the colour yield from each marker peptide is directly proportional to its quantity within the collagen molecule. Since in type I collagen only two of the 3 a-chains are Qi, there are two aa(I)CB-7 per type I molecule. The area of this peak was therefore multiplied by 3/2. No correction was required for type III peptides, since this collagen type has three identical chains. Since, aj(I)CB-7 is reported to have 263 amino acids as compared with 223 for Qi(III)CB-5, the area of Q!(I)CB-7 was further corrected by the factor 0.848 (223/263) [92]. The amount of type III collagen may therefore be calculated as a percent of total by the expression: % T >' p e 1 1 1 = A(I - 7) x 3 / ^ 8 4 8 ^(111-5) * 1 0 0 ( 2 U ) where A(I-7) and A(III-5) are relative areas corresponding to the marker peptides. 2.7 S T A T I S T I C A L A N A L Y S I S 2.7.1 L u n g G r o w t h Patterns Growth patterns for normal lung development were derived for all the biochemical pa-rameters analyzed. The patterns obtained from the normal lungs were then compared to the growth patterns obtained from the diseased lungs. Linear regression analysis was done for each of the biochemical parameters, using conceptional age ( gestational-f postnatal age) as the independent variable. The normal growth curves were then compared to the growth curves of the diseased lungs by compar-ing the parameter estimates of the two growth curves, as well as their intercepts ( if the two curves happen to be parallel) using analysis of covariance. Statistical calculations were done using P C SAS P R O C R E G . Chapter 2. MATERIALS AND METHODS 64 Where the relationship between the biochemical parameter and conceptional age was not adequately described by a hnear model, a nonhnear model was used. The relationship was modelled by a double exponential curve of the following form: y = Poc + error (2-12) The term "error" in the above expression is assumed to be normally distributed, indepen-dent between subjects and have the same variance for each subject. The coefficient po, in the above equation can be interpreted as the limiting concentration as conceptional age becomes large and the quantity (3Qe^1^ can be interpreted as the "initial"(conceptional age=0) concentration. A model of the above form was applied separately to normal subjects and those with BPD. A combined model of the form y=(p0-r-p3*group)e^i -t error (2.1 was also applied to the data. The quantity "group" is an indicator variable coded 1 for normal subjects and 0 for subjects with BPD. This model allows a formal hypothesis test whether the two groups of subjects have identical limiting concentration and this is implemented by testing the hypothesis (3$ = 0 in the combined model. Estimation of the parameters in both of the above models was carried out using PC SAS PROC NLIN. "Outliers" which had a large effect on parameter estimates were deleted for the purposes of statistical inference. 2.7.2 Disease Classification In order to see if pathological grading was related to biochemical and chnical findings of BPD, discriminant analysis was applied using the NCSS(Number Cruncher Statistical System) package. This procedure was preferred to using the multiple regression analysis, Chapter 2. MATERIALS AND METHODS 65 because the latter procedure assumes that the dependent variable is continuous. When the dependent variable (in this case, disease classification) is categorical, the appropri-ate tool to be used is dicriminant analysis. This procedure assumes that the dependent variable represents a classification variable(for example, BPD I,II,III and IV). The in-dependent variables are the clinical and biochemical parameters. The object is to find equations that, for a given set of independent variable values, predict the appropriate category. In other words, based on independent variables, both from clinical as well as biochemical parameters, we want to see which variables can best predict the BPD cate-gory that each infant included in the study belongs to, and how close this prediction is to the actual pathological classification of the infants as classified by the pathologist. Because of probable age effects confounding above results, data was analysed further as follows: For each BPD grade, a set of all similarly aged control infants were selected and various measures were compared using two-sample t-tests. Chapter 3 RESULTS: PART I 3.1 L U N G GROWTH AND D E V E L O P M E N T LN A N ANIMAL MODEL (RAT) Normal lung growth in the rat was studied by measuring the individual body weights, total lung weights (both absolute and specific lung weights). The other variables analysed to measure the extent of lung growth were wet lung/drj' lung weight ratio, total DNA in the.organ, DNA per unit dry weight of the lung, total protein, protein per unit dry tissue weight, total lung collagen and desmosine in the organ and their concentrations. All measurements were expressed as their means ± SEM. Body weight (gm) showed an exponential increase from day 16 of gestation to 7 weeks post natal age (Fig. 3.7). Mean body weight at day 17 was almost double the body weight obtained for day 16, and by birth, it had increased 8-fold. Both wet and dry lung weights increased sharply from day 16 of gestation until 3 weeks of postnatal life (43 days conceptional age)(see Fig. 3.8), after which the increase was more gradual until 7 weeks of age(see Fig. 3.9). When the specific lung weight was measured (per lOOgm body weight) it remained fairly constant from 16 days until 21 days of gestation and then showed a sudden decrease at birth (22 days). Again from 1 week to two weeks of age it remained constant and then decreased gradually until 71 daj's conceptional age (table. 3.4). The water content of the lung also varied with age. Wet lung weight/dry lung weight ratio showed a sharp decrease until birth after 66 67 Chapter 3. RESULTS: PART I 300' E 2 CO o Ld >-Q O CD 250-200-150-100-50-J / / / / / / - r -64 71 78 15 22 29 36 43 50 57 CONCEPTIONAL AGE [days] Figure 3.7: Body weight gain from late gestation to 7 weeks postnatal life in the rat Age n Body Wt. Wet lung Drj* lung Wctlungwt . , n n V\etlunp*-t. Bodyweight X 1 U U Drlungw eight (days) (g) (mg) (mg) (g) 16 11 0.526 ± 0.001 13.78 ± 0.24 1.72 ± 0.031 2.62 ± 0.043 8.01 ± 0.123 17 9 0.844 ± 0.0015 22.60 ± 0.72 3.01 ± 0.086 2.68 ± 0.081 7.51 ± 0.079 18 9 1.236 ± 0.012 31.66 ± 0.46 4.39 ± 0.074 2.56 ± 0.016 7.21 ± 0.022 19 9 2.26 ± 0.07 55.37 ± 2.12 8.19 ± 0.139 2.45 ± 0.032 6.74 ± 0.15 21 10 4.35 ± 0.073 104.71 ± 3.78 16.39 ± 0.503 2.4 ± 0.051 6.39 ± 0.123 22 9 6.39 ± 0.05 115.18 ± 1.78 18.78 ± 0.477 1.8 ± 0.014 6.15 ± 0.072 29 9 18.68 ± 0.54 306.61 ± 5.12 55.33 ± 1.211 1.65 ± 0.024 5.57 ± 0.19 36 10 31.82 ± 0.69 481.89 ± 4.19 89.69 ± 1.406 1.52 ± 0.02 5.39 ± 0.13 43 10 60.5 ± 0.75 561.26 ± 2.0 106.63 ± 1.982 0.93 ± 0.008 5.28 ± 0.11 50 10 89.84 ± 0.96 669.23 ± 7.5 121.87 ± 1.467 0.74 ± 0.003 5.49 ± 0.02 57 10 130.6 ±1 .17 760.54 ± 4.33 152.07 ± 2.148 0.58 ± 0.002 5.012 ± 0.089 64 10 182.43 ± 1.54 904.76 ± 5.86 173.58 ± 4.39 0.5 ± 0.001 5.238 ± 0.117 71 10 253.45 ± 1.84 1085.78 ± 5.68 214.27 ± 5.85 0.43 ± 0.002 5.159 ± 0.156 Table 3.4: Somatic and lung growth in the rat Chapter 3. RESULTS: PART I 1500 i — o UJ o 3 i — UJ 1000 H 500 H / /* / / / / /10 20 30 40 50 60 CONCEPTIONAL AGE [DAYS] 70 80 Figure 3.8: Results of the wet lung weight gain during growth in the Chapter 3. RESULTS: PART I 69 300 0-T 10 20 30 40 50 60 CONCEPTIONAL AGE [days] 70 80 Figure 3.9: Dry lung weights in the rat during gestation and postnatal hfe i Chapter 3. RESULTS: PART I 70 9 - i — i O CONCEPTIONAL AGE [days] Figure 3.10: Ratio of wet lung/dry lung weights during growth in the rat which the decrease in the ratio was more gradual until 2 weeks of age after which the ratio remained more or less constant until 7 weeks of postnatal age which was the last age studied(see Fig 3.10). Early in hfe, in utero, as well as in early postnatal life, lung grows primarily by cell multiphcation. This is shown by a rapid increase in total DNA from day 16 of gestation until 2 weeks of postnatal hfe. After that the rate of increase in DNA was seen- to slow down for the next three weeks until 5 weeks of postnatal hfe. This period occurred around the time of weaning in rats ( 3weeks postnatal hfe). The amount of DNA then showed a sharp increase until 7 weeks of postnatal hfe (see Fig. 3.11). DNA concentration during lung growth in rats showed an interesting pattern. After an initial rapid decrease in the DNA concentration ( pg/mg dry tissue) from day 16 till Chapter 3. RESULTS: PART I 71 6-1 I 41 < r< o OH 10 / / 2 0 3 0 4 0 5 0 T— 6 0 70 CONCEPTIONAL AGE [days] 8 0 Figure 3.11: Increase in total DNA per lung during growth Chapter 3. RESULTS: PART I 40 V) 72 35-=1 O fee o z o o < 80 30 40 CONCEPTIONAL AGE [days] Figure 3.12: Growth pattern of DNA concentration in the rat day 19 of gestation, there is a brief increase in concentration just one day before birth and then fell sharply again from the day of birth until 5 weeks of postnatal life and thereafter remained constant(see Fig 3.12). Total alkali soluble protein(mg) in the lung increased from day 16 of gestation until 7 weeks postnatal life (table 3.5). The increase was rapid until 2 weeks of postnatal age after which the increase was more gradual from 3 weeks to 7 weeks of age (see Fig 3.13). Protein concentration (mg per dry tissue weight) on the other hand showed a rapid de-cline from 16 days gestation until birth, after which the concentration remained constant until 7 weeks postnatal age (see Fig 3.14). Collagen in the developing rat lung was detectable biochemically, only from the 18th Chapter 3. RESULTS: PART I 73 150 CO E. o o_ i_j i CO ZD I o < i< o 1 0 0 -5 0 0 -/ I / / X / / / / / / 10 20 i 30 r 40 50 60 i 70 80 Figure CONCEPTIONAL AGE [days] 3.13: Changes in the total protein per lung during growth in the rat Age n Total DNA DNA Concentration Total Soluble Protein Concentration (days) "(mg) (/ig/mg) Protein (mg) (xng/mg) 16 11 0.065 ± 0.003 37.55 ± 0.91 1.041 ± 0.022 0.604 ± 0.009 17 9 0.098 ± 0.004 32.4 ± 0.45 1.734 ± 0.046 0.576 ± 0.003 18 9 0.129 ± 0.002 29.48 ± 0.36 2.269 ± 0.077 0.515 ± 0.009 19 9 0.22 ± 0.01 26.75 ± 0.74 4.086 ± 0.121 0.498 ± 0.007 21 10 0.455 ± 0.012 27.84 ± 0.56 7.294 ± 0.139 0.447 ± 0.008 22 9 0.558 ± 0.006 29.8 ± 0.46 7.90 ± 0.094 0.422 ± 0.006 29 9 1.49 ± 0.06 26.78 ± 0.57 23.463 ± 0.713 0.424 ± 0.006 36 10 2.234 ± 0.101 24.82 ± 0.78 38.153 ± 0.753 0.425 ± 0.002 43 10 2.534 ± 0.075 23.72 ± 0.26 45.391 ± 0.981 0.425 ± 0.0013 50 10 2.732 ± 0.068 22.4 ± 0.34 53.21 ± 1.053 0.436 ± 0.005 57 10 3.143 ± 0.063 20.7 ± 0.20 65.861 ± 1.589 0.433 ± 0.004 64 10 3.607 ± 0.108 20.8 ± 0.42 81.049 ± 3.144 0.466 ± 0.007 71 9 4.613 ± 0.113 21.5 ± 0.27 101.813 ± 4.573 0.473 ± 0.009 Table 3.5: Changes in DNA and soluble protein during growth in the rat Chapter 3-. RESULTS: PART I 0.65-1 0.60 issue] -t— 0.55-CD JL z: i. i 0.50-PROTI 0.45 0.40 A 10 JL / 20 30 40 50 60 70 CONCEPTIONAL AGE [days] 80 Figure 3.14: Growth pattern of protein concentration in the rat lung Chapter 3. RESULTS: PART I 15 75 CD U J O < O o 29 35 43 50 57 64 71 CONCEPTIONAL AGE [days] ' Figure 3.15: Total collagen contents during growth of the rat lung day of gestation. Total collagen (mg) in the lung increased steadily from day 18 of gestation to 7 weeks postnatal age. By birth, the collagen amounts in the lung had increased almost 7 times. From birth until 4 weeks of age, collagen contents had increased 12 times and more than doubled from 4 to 7 weeks of age (see Fig 3.15). Collagen concentration ( /ig/mg dry tissue) had increased 1.5 times from 18 days gestation until term. From birth to fourth week of life,there was a further two-fold increase in the concentration followed by a slower further rise until 7 weeks postnatal age (see Fig. 3.16). The amounts of insoluble collagen or mature collagen present in the developing lung, was also measured and was found to follow the same pattern of development as the total lung collagen (table 3.6). While both soluble and insoluble compartments showed an er 3. RESULTS: PART I O.OB 0.06 0.04 H 0.02 0.00-71 / r / / 10 20 30 40 50 60 70 CONCEPTIONAL AGE [days] 80 Figure 3.16: Growth pattern of collagen concentration in the rat Chapter 3. RESULTS: PART I 77 0.61 : , • O < o o w m 3 O w 0.4-PQ 3 O CO 0.2-0.0 1 10 80 20 30 40 50 60 70 CONCEPTIONAL AGE [days] Figure 3.17: Ratio of soluble/insoluble collagen during growth of the rat lung increase during lung development, the amount of insoluble coUagen increased at a faster rate than the soluble form, reflecting the speed at which newly synthesized collagen was being converted to mature, functional collagen. The ratio of soluble/insoluble collagen showed a sharp decrease from 18 days gestation until birth and there after showed a gradual decrease until 3 weeks of postnatal hfe, then the ratio remained constant until 7 weeks (see Fig. 3-.17). Desmosine in the lung parenchyma which is indicative of the amount of mature elastin, was detectable by radioimmunoassay procedure at 21 days of gestation in the rat. The amount of desmosine (pg) in the developing lung increased gradually until birth. There-after, showed a rapid increase until the 5th postnatal week. From birth to 1 week of age, the total amount increased by 7-fold and it was followed by a 13-fold increase between 1 Chapter 3. RESULTS: PART I 78 Age n Soluble Insoluble Total Collagen Concentration (days) Collagen (mg) Collagen (mg) Collagen (mg) (mg/mg) 18 9 0.019 ± 0.0006 0.045 ± 0.002 .064 ± 0.002 0.0145 ± 0.0004 19 9 0.036 ± 0.0007 0.087 ± 0.002 0.123 ± 0.002 0.015 ± 0001 21 10 0.074 ± 0.003 0.206 ± 0.008 0.279 ± 0.01 0.017 ± 0.0002 22 9 0.076 ± 0.006 0.344 ± 0.013 0.42 ± 0.018 0.022 ± 0.0004 29 9 0.188 ± 0.0095 1.187 ± 0.017 1.376 ± 0.023 0.025 ± 0.0004 36 10 0.265 ± 0.022 2.556 ± 0.114 2.821 ± 0.135 0.031 ± 0.001 43 10 0.196 ± 0.008 4.043 ± 0.102 4.239 ± 0.106 0.0397 ± 0.0004 50 10 0.170 ± 0.008 4.877 + 0.043 5.047 ± 0.039 0.0414 ± 0.0002 57 10 0.354 ± 0.06 7.336 ± 0.088 7.691 ± 0.113 0.051 ± 0.0002 64 10 0.473 ± 0.05 9.42 ± 0.167 9.893 ± 0.15 0.057 ± 0.0006 71 9 0.481 ± 0.06 13.703 ± 0.274 14.184 ± 0.285 0.066 ± 0.0006 Table 3.6: Soluble and insoluble contents and collagen concentration in the rat lung during growth and 5 postnatal weeks. After that, the increase was more gradual until the 7th postnatal week (see Fig. 3.18). Desmosine concentration ( pg/mg dry tissue) showed a gradual increase from day 21 of gestation until 1 week postnatal age. Thereafter, until 3 weeks, there was sharp increase in the concentration, and the increase was noted to be almost 4-fold. From the third week until the 4th week there was a slow further rise in concentration(table 3.7). From the 4th week onwards the concentration remained fairly constant (see Fig. 3.19). An increase in the extracellular components in comparison to the cellular constituents of the lung, was indicated by the increase in collagen/DNA ratio. The ratio increased slowly until birth. From birth until 4 weeks of age, the ratio increased 2.5 times, there after increased gradually until 7 weeks. Collagen/protein ratio also showed a steady increase from 19 days of gestation until 5 weeks of postnatal hfe. When the ratio of hydroxyproline/desmosine was analysed, it was noted to decrease steadily from 21 dajrs gestation until 4 weeks of postnatal age, after which it showed a gradual increase until 7 weeks of hfe (table 3.8). Chapter 3. RESULTS: PART I 79 60 CD 40 3. CO O CO L d Q 20-O - T 20 / 2. / 30 40 50 60 CONCEPTIONAL AGE [DAYS] i 70 80 Figure 3.18: Changes in the total desmosine contents in the rat lung during growth Age n Soluble/Insoluble Desmosine Desmosine Concentration (days) Collagen (mg/mg) (Mg) (//g/mg) 18 9 0.428 ± 0.008 19 9 0.419 ± 0.004 21 10 0.357 ± 0.005 0.187 ± 0.014 0.0114 ± 0.0007 22 9 0.219 ± 0.0105 0.354 ± 0.018 0.019 ± 0.0007 29 9 0.158 * 0.007 2.38 ± 0.263 0.0433 ± 0.005 36 10 0.102" ± 0.004 9.23 ± 0.749 0.103 ± 0.085 43 10 0.049 ± 0.002 17.32 ± 0.911 0.162 ± 0.083 50 10 0.035 ± 0.002 23.84 ± 0.655 0.196 ± 0.0041 57 10 0.048 ± 0.008 31.96 ± 1.761 0.209 ± 0.009 64 10 0.0507 ± 0.006 33.95 ± 0.700 0.196 ± 0.001 71 9 0.0351 ± 0.004 42.77 ± 1.796 0.199 ± 0.003 Table 3.7: Soluble/insoluble collagen ratio and the desmosine contents in a growing rat lung Chapter 3. RESULTS: PART I 80 0.3 OO 00 2 0.2 H K E-u I S o o CO o CO w Q 0.1 0.0 / 3: / / / / X 20 30 40 50 60 70 CONCEPTIONAL AGE [days] 80 Figure 3.19: Growth pattern of desmosine concentration in the rat lung Age n Collagen/DNA CoUagen/Protein Hydroxy proline (days) (mg) (mg) /Desmosine(ug) 18 9 0.493 ± 0.012 0.0283 ± 0.0009 19 9 0.563 ± 0.016 0.0301 ± 0.0004 21 10 0.614 ± 0.016 0.0382 ± 0.001 181.95 ± 6.86 22 9 0.751 ± 0.024 0.053 ± 0.0016 144.69 ± 5.43 29 9 0.933 ± 0.026 0.0589 ± 0.001 70.5 ± 4.39 36 10 1.253 ± 0.02 0.0736 ± 0.0022 37.3 ± 2.12 43 10 1.675 ± 0.015 0.0933 ± 0.007 29.84 ± 2.55 50 10 1.854 ± 0.032 0.0951 ± 0.0015 25.82 ± 1.89 57 10 2.45 ± 0.024 0.117 ± 0.0013 29.35 ± 2.83 64 10 2.754 ± 0.043 0.123 ± 0.003 35.53 ± 3.02 71 9 3.078 ± 0.015 0.141 ± 0.004 40.44 ± 3.65 Table 3.8: Collagen/protein, collagen/DNA and collagen/elastin ratios in a developing rat lung Chapter 4 RESULTS: PART H 4.1 EXPERIMENTAL ALTERATION OF L U N G G R O W T H As mentioned earlier in chapter 2 with regards to the statistical analyses conducted, initially, an application of two way analysis of variance was applied to all response vari-ables. Whenever, an interaction between day and treatment were noted for a variable, a one-way anal}rsis of variance was conducted between the different groups separately for each day. First, the control and sham operated groups were compared (table 4.9). As statistically significant (p<0.01) differences were not found between them, the two groups were merged (and will now be referred to as the sham/control group) and hnear contrasts were formed to examine the effects of pneumonectomy on lung growth, the effects of BAPN administration on normal lung growth as well as on lung growth that occurred after pneumonectomy. 4.1.1 Compensatory Growth following Pneumonectomy From the results obtained (table 4.10) it is evident that pneumonectomy did not affect body weight gain of the animals. They continued to gain weight at a rate similar to sham/control up to three weeks after pneumonectomy. By day 7 post-pneumonectomy, right lung weights were increased (p<0.0001) by 140% of the weight of the right lung of sham/control rats. When the mean right lung weight of the pneumonectomy group was compared to the total lung weight of the sham/control 81 8 H P £ c-t-EL pf o> o O P P> P P-cn P -P> B O n> •i w m o P 13 cn O P CL. cn P Daya p o » t - p n o u m o n e c t o m y P A R A M E T E R S 7 Control Sham 1 Control 1 Sham 3 Control L Sham Body Weight 133.73 ± 5 . 0 7 132.23 ± 6 . 2 4 183.427 ± 6 . 2 1 183.198 ± 6 . 8 5 238.076 ± 6 . 6 1 236.947 ±6 . 64 Total Dry Lung Weight (mg) 1D2.1 ± 2 . 1 4 168.1 ± 2 . 3 4 173.58 ± 4 . 3 9 177.1 ± 6 . 0 5 214.27 ± 6 . 8 5 213 ± 2 . 0 3 Right Long Dry Weight (mg) 100.7 ± 1 . 4 1 104.3 ± 1 . 6 113.36 ±1 . 55 117 ± 1 . 0 3 137.9 ± 1 . 4 8 140.14 ± 1 . 8 DNA /Right long (mg) 2.081 ±.1138 2.129 ± . 0 8 3 2.354 ± . 0 4 4 2.348 ± . 0 4 7 2.97 ± . 0 2 3.055 ± . 0 5 1 DNA ( M g/mg dry tinne) 30.066 ± . 1 7 6 20.479 ± . 9 2 1 20.755 ± . 1 2 8 20.06 ± . 2 4 21.646 ± . 1 0 9 21.821 ± . 2 5 1 DNA Synlheiit (dpm /MS DNA) 5.1 ± . 4 1 5.1 ± . 5 7 4.3 ± . 1 1 2 4.73 ± . 1 9 3 6.1 ± . 0 5 3 4.8 ± . 1 3 4 Protein/Right Long (mg) 43.604 ± 1 . 0 3 44.87 ± . 7 1 62.854 ± 1 . 4 3 7 62.808 ± . 6 7 6 65.343 ± 1 . 8 3 1 68.286 ± 3 . 1 3 6 Piolein ( M E / m E dry tinne) 432.56 ± 4 . 4 430.2 ± 3 . 7 8 465.49 ± 6 . 5 2 4 451.25 ± 2 . 7 9 473.1 ± 8 . 5 2 472.16 ± 1 6 . 0 1 Soluble Collages /Right long (mg) 0.236 ± . 0 6 0.338 ± . 0 6 2 0.310 ± . 0 3 9 0.326 ± . 0 4 2 .309 ± . 1 4 6 0.348 ± . 1 5 1 Iniolnble Collagen /Right long (mg) 4.857 ± . 0 5 1 4.765 ± . 0 7 6.157 ± . 0 4 3 6.071 ± . 0 2 2 8.829 ± . 2 0 7 8.956 ± . 1 9 Total Collagen /Right lnng (mg) 6.002 ± . 0 7 3 6.103 ± . 0 7 9 6.467 ± . 0 2 1 6.397 ± . 0 1 7 9.138 ± . 2 3 9.304 ± . 1 9 2 Collagen (fig/nig dry tinne) 50.573 ± . 1 6 3 49.008 ± . 1 6 2 67.126 ± . 6 0 6 64.699 ± . 3 3 9 66.326 ± . 6 0 6 64.627 ± . 7 0 6 Detmoiine/Righ! lnng ( M E ) 21.17 ± 1 . 1 7 6 32.42 ± . 9 2 8 22.16 ± . 1 9 3 22.46 ± . 2 6 8 27.46 ± . 3 0 6 27.72 ± . 4 5 6 Deimoiine (ng/ mg dry tlitoe) 209.2 ± 8 . 7 1 216.19 ± 6 . 6 2 196.81 ± 1 . 0 9 4 191.9 ± . 5 6 2 199.13 ± 1 . 0 8 3 197.86 ± . 7 2 2 •8 la s CO 5 8 Biochemical parameters have been expressed per dry lung weight. Values expressed as mean ±SEM's o oo to Chapter 4. RESULTS: PART II 83 200 180 CO JL JE g> cn c 160-120 100 80--r ^ y / / r X y / A / Legend • Normal O S h a m 2 - " " ' V BAPN O BAPN+Pn»umo 0 Pnaumo 14 21 28 Days postpneumonectomy Figure 4.20: Results obtained for the right lung weights in the 5 groups on days 7, 14 and 21 post-pneumonectomy group, it was found that, the compensatory response was not complete and amounted to 90% of the total weight of the sham/control lungs.The right lung weight of pneumonec-tomy group was significantly higher on days 14 and 21 compared to the right lung weights of the sham/ control(P <0.0001)(see Fig. 4.20), but the compensatory response was not complete by day 21 postpneumonectomy, and was still 90% of the total lung weight of the sham controls(P <0.001) (see Fig. 4.21). Biochemical Changes Total DNA, total alkali soluble protein, collagen and desmosine in the right lung were significantly increased by day 7 post-pneumonectomy compared to the sham/control group. These biochemical variables continued to be significantly higher by days 14 and Chapter 4. RESULTS: PART II 84 01 240 220 200 JZ * 180 Ol c 3 .O 160 H 140 H 120-Legend • normol  O Sham  V BAPN O BAPN+Pneumo • Pnsumo 7 14 21 Days postpneumonectomy 28 Figure 4.21: Total lung weight results obtained for the 5 groups on days 7, 14 and 21 postpneumonectomy Chapter 4. RESULTS: PART II 85 CD JL 4 < 2 Q Legend • Normol  O Shorn  V BAPN O BAPN+Pn«umo # Pnaumo 2 — -14 21 Days postpneumonectomy 28 Figure 4.22: Total DNA contents obtained for the right lungs in the 5 groups on days 7, 14 and 21 post-pneumonectomy 21 post-pneumonectomy (p<0.0001). In case of the variables, total DNA, soluble protein, collagen and desmosine of the right lung, the compensatory response was completed by day 21 post-pneumonectomy and no significant differences were found when these variables were compared to the values obtained for both lungs of the sham/control group (see Fig. 4.22). Although DNA synthesis was sharply increased compared to the sham operated group by day 7 post-pneumonectomy (p<0.0001), DNA concentration remained the same as in the control group. By day 14 post-pneumonectomy, the DNA synthesis levels in the pneumonectomy group had decreased to the levels found in the sham/control group, but the DNA concentration had significantly increased (p<0.0001). By day 21, the synthetic levels continued to be the same as in the sham/ controls and the DNA concentration Chapter 4. RESULTS: PART II 86 i i 1 i 0 7 U 21 28 Days postpneumonectomy Figure 4.23: D N A synthesis levels obtained for the lungs in the 5 groups on days 7, 14 and 21 post-pneumonectomy continued to be significantly higher (see Fig. 4.23). Soluble protein concentration increased significantly by day 7 post-pneumonectomy (p<0.0001), while collagen and desmosine concentrations were not different from sham/controls. Protein concentration continued to be higher even at days 14 and 21 post-pneumonectomy (p<0.0001). Collagen concentration was not significantly different from that seen in sham/controls at 14 days post-pneumonectomy, but by day 21, it had increased sig-nificantly when compared to the concentrations for sham controls (p<0.0001) (see Ta-ble 4.10). By day 14 post-pneumonectomy, desmosine concentration in the pneumonectomy group was significantly higher(p<0.0001) than in sham control group and continued to be higher even on day 21 post-pneumonectomy (p<0.0001). Daya poal-pneumorieciomy V» ri* Mr i Sham 7 T, a rncomo 1 Sham 4 Pncnino A 21 Sham Pnenmoa Dody weight (Km) 132.23 ±1.24 132.87 ±2.08 183.2 ±2.86 182.9 ±2.01 230.95 ±1.04 230.77 ±1.72 Total Dry Lung Weight (mg) 168.1 ±2.34 H3.0 ±1.08 177.1 ±5.05 171.11 ±2.81 213.0 ±2.03 180.84 ±1.54 Right Lnng Dry Weight (mg) 11)4.3 ±1.0(1 143.0 ±1.08 117.0 ±1.03 171.11 ±2.81 140.14 ±1.8 180.84 ±1.64 DNA /Mghl lung (m) 2.12B ±.(183 3.121 ±.104 2.348 ±.04 7 3.868 ±.032 3.065 ±.051 4.673 ±.036 DNA (/if/mc dry tiitnc) 20.479 ±.021 21.732 ±.013 20.00 ±.24 22.570 ±.190 21.821 ±.251 24.006 ±.263 DNA Synlheiit (dpm/,»6 DNA) 6.1 ±.67 13.3 ±.74 4.73 ±.2 6.20 ±.140 4.8 ±.13 4.6 ±.9 r-rotetn/night Lnng (me) 44.S7 ±0.71 74.21 ±3.51 62.81 ±0.G8 90.84 ±2.28 06.293 ±3.14 109.01 ±2.29 Protein (/»g/mg dry tinne) 430.2 ±3.78 514.78 ±10.38 461.26 ±2.79 630.19 ±4.78 472.16 ±10.01 673.71 ±7.60 Solable Collagen /night lnng (mg) 0.338 ±.062 1.08 ±.080 0.326 ±.042 0.608 ±.082 .348 ±.151 0.765 ±.000 Intolnble Collagen /Right lnng nl. (mg) 4.706 ±.07 0.17 ±.074 0.071 ±.022 0.073 ±.077 8.960 ±.190 12.763 ±.062 Total Collagen /night lnng (mg) 6.103 ±.070 7.26 ±.104 0.307 ±.017 9.042 ±.009 9.304 ±.192 13.506 ±.023 Collagen (;ig/mg dry tinne) 49.01 ±.100 60.40 ±.15 64.7 ±.34 50.466 ±.8 04.627 ±.710 71.16 ±.62 Deimiiiine/nighl lnng ( f l ) 22.417 ±0.93 20.982 ±1.04 22.40 ±.268 34.601 ±.28 27,72 ±.46 40.69 ±.64 Deimotine (ng) mg dry tinne) 216.10 ±0.02 208.3 ±4.0 101.9 ±0.58 201.8 ±2.4 197.86 ±0.72 214.3 ±1.0 Biochemical parameters have been expressed per dry lung weight. Values expressed mean ±SEM's Chapter 4. RESULTS: PART II 88 , 3 , < Q 27-1 25 23 21 19-17 Legend • normal  O Shorn  V BAPN O BAPN+Pneumo $ Pneumo A " T " 14 21 Days postpneumonectomy 28 Figure 4.24: DNA concentration in the lungs of the 5 groups on days 7, 14 and 21 post-pneumonectomy The amount of insoluble collagen or mature, cross-linked collagen in the pneumonec-tomy group seemed to follow the same pattern as total collagen and was found to be significantly higher than in the sham/control group from day 7 to day 21 post-pneumonectomy. Similar pattern was also noted for soluble, newly synthesized col-lagen with the amount in the pneumonectomy group consistently higher than in the sham/control group (p<0.0001). The collagen/DNA ratio obtained for the pneumonectomy group was not different for that obtained for sham/control group and this ratio seemed to increase proportionately in both groups and no significant differences were noted even on days 14 and 21 post-pneumonectomy. When protein/DNA ratio (which is often used as an indicator of cellular hypertrophy) Chapter 4. RESULTS: PART II 89 i 1 1 1 i 0 7 14 21 28 Days postpneumonectomy Figure 4.25: Total soluble protein content in the right lungs of the 5 groups on days 7, 14 and 21 post-pneumonectomy was analysed for both groups, it was noted that in the pneumonectomy group the ratio was consistently higher than the sham/control group on all days (7, 14 and 21) but the increase was not significant on any of the days. Although the ratio of protein/DNA and RNA/DNA have been used in the past as cell size indicators, protein/DNA may not be a correct parameter to be used, as the alkali soluble protein fractions obtained from the lung may also contain some of the newly synthesized extracellular proteins. Lung Mechanics There was a total compensation with respect to lung volumes by day 21 post-pneumonectomy. Total lung capacity (TLC) and specific TLC increased to match the lungs of the sham/control Chapter 4. RESULTS: PART.II 90 100-80-3 30-20-10-0 Legend • Shorn Oparotad • Pneumonaclomy 10 15 PRESSURE [cm] OF WATER 20 25 Figure 4.26: Pressure-volume curves obtained for the sham/control and pneumonectomy groups on day 21 post-pneumonectomy group by day 21. The TLC/lung weight (ml/g total lung weight) was not different be-tween the two groups (see Table 4.11). When pressure- volume curves were analyzed for the two groups they did not differ except for a slight insignificant loss of elastic recoil in the mid-volume of the curve observed for the pneumonectomy group (see Fig. 4.26). The shape constant K of the pressure-volume curve was not seen to differ between the two groups (see table 4.11). Morphometric changes Mean hnear intercept (Lm) mean chord length of alveoh ((/a/v)),mean chord length of ducts (hi), total alveolar surface area (SHT ) , alveolar surface area for the right lung (Sw/i)) alveoli/unit area (Na), number of alveoli/unit volume (Nv), total alveolar number Chapter 4. RESULTS: PART II 91 Var iable Sham/Contro l Pneumonectomy F i x e d Lung Volume (ml) T L C (ml) TLC/ lOOg Body Weight(ml) T L C / Lung Weight (ml/g) Constant K 9.52 ± 0 . 2 1 8.6 ±0.33 3.63 ± 0 . 21 7.05 ±0 .32 0.121 ±0.005 9.85 ±0.63 8.5 ±0.47 3.59 ±0.17 6.96 ± 0 . 41 0.128 ±0.004 Table 4.11: Results of the lung mechanics for the sham/control and pneumonectomy groups Variable Sham Contro l Pneumonectomy L m (p,) 68.86 ±2.04 74.62 ±2.63 Ulv [p) N„ ( x lO 6 ) 40.24 ±0.87 42.45 ±0.82 3.632 ±0.117 2.822 ±0.156 N„ (x lO 6 ) SWR (m 2) 35.04 ±1.32 30.34 ±0.78 0.370 ±0.022 0.528 ±0.02 0.558 ±0.017 0.528 ±0.02 XAR ( x lO 6 ) NAT ( x l O 6 ) 23.13 ±0.85 27.78 ±1.06 36.58 ±1.15 27.78 ±1.06 V Q i l , ( x lO -8 ) 9.91 ±0.47 13.24 ±0.55 Table 4.12: Results of morphometric analysis of the sham/control and pneumonectomy groups at day 21 post-pneumonectomy (N^r), alveolar number in the right lung (NAR) and average volume of alveoh (Va;v) were calculated for each group at day 21 post-pneumonectomy. By day 21 post-pneumonectomy, there was a significant increase in alveolar surface area(S^J?) of the right lung (p<0.01) but was not significantly different when compared to both lungs of the sham/control group. There was no significant difference in the Lm and the (ZQj„)values between the groups. There was a significant increase in the total number of alveoh(Nx-R) in the pneumonectomy group as compared to the right lung of the sham/control group, but was less than that of both lungs of the sham/controls (P<0.01). There was a significant increase (p<0.001) in the average volume of the alveoh in the pneumonectomy group. Number of alveoli/unit area showed a significant decrease (p<0.01) and was accompanied by a significant decrease in number of alveoh/unit lung Chapter 4. RESULTS: PART II 92 volume(p<0.01)(see Table 4.12). In summary, there was complete compensation in the pneumonectomy group with regard to lung volume, total lung capacity, total DNA, soluble protein, collagen and desmosine contents, while there was incomplete compensation with regard to the lung weight. Results of the morphometric analysis at day 21 post-pneumonectomy. showed evidence of alveolar multiplication with increased alveolar surface area and increased total number of alveoh in the right lung. There was no difference in the mean hnear intercept and the mean chord length of the alveoh. There was also evidence of enlargement of existing structures as the number of alveoh per unit volume and per unit area showed a decrease in the pneumonectomy group along with an increase in the average alveolar volume. 4.1.2 Effect of B A P N on normal lung growth Although the body weights of BAPN treated animals were not significantly different from the sham/control group at the time periods studied (days 7, 14 and 21), they showed consistently lower values compared to the control group. Animals in the BAPN treated group had significantly lower lung weights by the first week (p<0.001) and continued to be significantly lower by the second (p<0.01) and the third week (p<0.01). Compared to the control group, the BAPN injected rats had significantly larger lung volumes (p<0.01) by day 21 and contained more air/gm of lung tissue (p<0.01). Their T L C had significantly increased (p<0.01) by day 21 compared to the control group (see Table 4.13). The air-filled pressure-volume curve of the BAPN injected rats was seen to shift significantly upwards and to the left compared to the non-injected animals (p<0.05) and the shift was most significant at mid-lung volume (p<0.01). The constant K was significantly higher in the BAPN treated group when compared to the control group(p<0.01) (see Fig. 4.27). Chapter 4. RESULTS: PART II 93 Legend • Control Group • BAPN Group 0-i , , , , 0 5 10 15 20 25 PRESSURE [cm] OF WATER Figure 4.27: Pressure-volume curves obtained for sham/control and BAPN groups on day 21 post-pneumonectomy Variable Sham/Control BAPN Injected Fixed Lung Volume(ml) TLC (ml) TLC/100 g Body Weight(ml) T L C / Lung Weight(ml/g) Constant K 9.52 ±0.21 8.6 ±0.33 3.63 ±0.21 7.05 ±0.32 0.121 ±0.005 10.31 ±0.16 10.2 ±0.45 4.5 ±0.24 8.95 ±0.53 0.144 ±0.005 Table 4.13: Results oflung mechanics in the BAPN group and the sham/control group. Chapter 4. RESULTS: PART II 94 600 550-O £ 500 " \ CO , 3 , o cr 450-400-350 O Shorn  V BAPN O 8APN+Pn«umo # Pneumo 7 14 21 Days postpneumonectomy 28 Figure 4.28: Protein concentrations obtained for the lungs of the 5 groups on days 7, 14 and 21 post-pneumonectomy Biochemical Changes Total DNA, total collagen and total desmosine in the lungs of BAPN injected rats were significantly lower by one week and continued the trend on day 14 and on day 21. Total alkali soluble protein content did not show any difference at one week but by the second week it was significantly lower (p<0.05) and continued to be lower by the third week. DNA synthesis did not differ significantly between the two groups on any of the days studied. DNA concentration on the other hand was significantly lower on day 7 and continued to be lower than the control group on day 14 (p<0.01) and day 21 (p<0.001). Similarly soluble protein concentrations were lower than the control group by day 7 (p<0.05) and continued to be lower (p<0.0001) by day 14 and by day 21 (p<0.05). Chapter 4. RESULTS: PART II 95 2 0 Legend • Normol O Shorn -1- ; P 7 M 21 Days postpneumonectomy Figure 4.29: Total collagen contents obtained for the right lungs of the 5 groups on days 7, 14 and 21 post-pneumonectomy Days post-pneumonectomy Vtriible 7 Control BAPN 1 Control 1 BAPN 2 Control 1 « BAPN Body weight (gm) 133.73 ±2.07 126.26 ±5.39 183.427 ±2.21 166.32 ±8.74 238.076 ±1.51 227.66 ±6.49 ToUl Dry Lung Weight (mg) 152.1 ±2.U 142.14 ±1.83 173.58 ±4.39 157.16 ±4.52 214.27 ±6.85 201.05 ±3.56 Right Lung Dry Weight (mg) 100.7 ±1.41 94.06 ±1.32 113.36 ±1.55 106.37 ±1.42 137.9 ±1.48 127.62 ±1.43 DNA /Right lung (m) 2.081 ±.038 1.742 ±.052 2.354 ±.044 2.063 ±.037 2.97 ±.02 2.639 ±.016 DNA (jig/mg dry tissue) 20.665 ±.175 18.434 ±.62 20.755 ±.128 19.405 ±.23 21.646 ±.109 19.903 ±.11 DNA Syntheiii (dpm/Mg DNA) B.l ±41 3.5 ±.44 4.3 ±.112 4.80 ±.1 4.8 ±.053 4.4 ±.06 Protein/Right Lung (mg) 43.604 ±1.03 40.48 ±.71 62.854 ±1.437 47.09 ±.96 65.343 ±1.831 56.184 ±1.398 Protein (Mg/mg dry tissue) 432.56 ±4.4 404.46 ±5.55 465.49 ±6.524 419.24 ±3.5 473.1 ±8.52 439.65 ±5.97 Soluble ColUgen /Right lung (mg) 0.236 ±.06 0.734 ±.071 0.310 ±.039 0.503 ±.049 .309 ±.146 0.50 ±.063 Insoluble Collagen /Right lung (mg) 4.857 ±.051 3.986 ±.104 6.157 ±.043 5.143 ±.079 8.829 ±.207 7.832 ±.033 Total ColUgen /Right lung (mg) 5.092 ±.073 4.72 ±073 6.467 ±021 5.646 ±.08 9.138 ±.017 8.332 ±.013 ColUgen (ng/mg dry tissue) 50.673 ±.183 50.268 ±.172 57.125 ±605 52.993 ±.67 66.325 ±.606 66.35 ±.632 Desmosine/Right lung iVg) 21.17 ±1.175 18.037 ±.779 22.18 ±.193 19.6 ±.30 27.46 ±.305 22.67 ±.2 Desmosine (ng/ mg dry tissue) 209.2 ±8.71 191.39 ±5.35 195.81 ±1.094 184.324 ±1.22 199.13 ±1.083 177.7 ±.61 Biochemical parameters have been expressed per dry lung weight. Values expressed as mean ±SEM's Chapter 4. RESULTS: PART II 97 90 80-CO 13, UJ o < o o 70 60 50-40 30-Legend • Normol  O Shorn  V BAPN O BAPN+Pneumo • Pneumo 14 21 Days postpneumonectomy 28 Figure 4.30: Collagen concentrations obtained for the lungs of the 5 groups on days 7, 14 and 21 post-pneumonectomy Collagen and desmosine concentration did not differ between the two groups at day 7, but by day 14 they were significantly lower in the BAPN group (p<0.001). Interestingly, by day 21, collagen concentration seemed to have returned to equal the control values in the BAPN injected group but desmosine concentration continued to be lower (p<0.001) (see Table 4.14). Soluble collagen concentrations were significantly higher in the BAPN group by 1 week(p<0.0001) and continued to be significantly higher by the 2nd(p<0.01) and 3rd weeks(p<0.05). Insoluble collagen on the other hand, was significantly lower in the BAPN group by the 1st week and continued to show significantly lower values on 2nd and 3rd weeks(p<0.0001). Chapter 4. RESULTS: PART II 98 Variable Sham/Control BAPN Lm (p.) 68.86 ±2.04 73.05 ±2.74 laiv (M) 40.24 ±0.87 41.19 ±0.71 Wt (M) 42.04 ±2.0 47.19 ±2.34 N v (xlO6) N a (xlO6) S;rr (m2) 3.632 ±0.117 3.446 ±0.180 35.04 ±1.32 33.2 ±0.83 0.558 ±0.017 0.540 ±0.044 N^r (xlO6) 36.58 ±1.14 33.78 ±2.04 V a h , (xlO-8) 9.91 ±0.47 10.33 ±0.42 Table 4.15: Results of morphometric analysis of the BAPN administered and sham/control groups Morphometric Changes In contrast to the biochemical analyses and lung mechanics results of morphometric analysis did not indicate dramatic differences between the BAPN and the control groups. Although insignificant, there was an increase in Lm, l a / v as well the average alveolar volume. There was slight decrease in alveolar surface area, total number of alveoli, alveoli/unit area and number of alveoli/unit volume (see Table 4.15). In summary, although there were no diffrences in body weight gain between the two groups, lung weights of BAPN group were significantly lower from 5 weeks to 7 weeks of age. Effect of BAPN on lung growth was seen biochemically as a decrease in total DNA, soluble protein, collagen and desmosine contents by 1 week of administration of the agent. There was a decrease in the concentrations of DNA and soluble protein by 1 week of administration and decrease in collagen and desmosine concentrations by 2 weeks of administration. Although collagen concentration levels returned to normal values by 3 weeks of administration, desmosine concentration remained low. Although morphometric analysis did not show significant differences between BAPN and sham/control groups, the lungs of the animals in BAPN group showed an increase in the mean linear intercept, decrease in alveolar surface area, total number of alveoli, Chapter 4.. RESULTS: PART II 99 Variable , BAPN+Pneumonectomy Pneumonectomy Fixed Lung Volume(ml) TLC (ml) TLC/100 g Body Weight (ml) TLC/ Lung Weight (ml/g) Constant K 10.72 ±0.35 9.7 ±0.26 4.16 ±0.19 8.1 ±0.65 0.134 ±0.006 9.85 ±0.63 8.5 ±0.47 3.59 ±0.17 6.96 ±0.41 0.128 ±0.004 Table 4.16: Results of lung mechanics of the BAPN-pneumonectomy and pneumonectomy groups on day 21 post-pneumonectomy alveoli/unit area and number of alveoli/unit volume, when compared to the lungs of the sham/control group. 4.1.3 Effect of B A P N on Post-pneumonectomy Lung Growth There was no significant difference in the body weights between the BAPN administered and pneumonectomy performed group (B-fP group) and the pneumonectomy group on all the 3 days studies (7, 14 and 21 days post-pneumonectomy). The right lung weights were reduced significantly in the B-fP group (p<0.0001) by day 7 and continued to show lower values compared to the pneumonectomy group on days 14 and 21 as well (p<0.0001). Lung Mechanics Compared to the pneumonectomy group the B ± P group had larger lung volumes by day 21 (p<0.05), and had a larger TLC (p<0.05) (see Table 4.16). The air-filled pressure-volume curve of the B+P group was seen to He between that obtained for BAPN group and pneumonectomy group but was not significantly different from either (see Fig. 4.31). Biochemical Changes Total DNA, total soluble protein, collagen and desmosine showed significantly lower values(p<0.001) in the B+P group on day 7 post-pneumonectomy, when compared to the Chapter 4. RESULTS: PART II 100 100 80-10 15 20 PRESSURE [cm] OF WATER 25 Figure 4.31: Comparison of the pressure-volume curves ob-tained for the BAPN+pneumonectomy group and the pneumonectomy group on day 21 post-pneumonectomy Chapter 4. RESULTS: PART II 101 cn 0> C "io o E w Q 50-45 40-35-30-25-20-15 Legend • Normol  O Shorn  V BAPN O BAPN+Pneumo • Pneumo 14 21 Days postpneumonectomy 28 Figure 4.32: Total desmosine contents in the lungs of the 5 groups on days 7, 14 and 21 post-pneumonectomy pneumonectomy group. While there was significantly lower total DNA values (p<0.001) on day 14, there was no difference in the total protein values between the two groups. Total collagen and desmosine were seen to be significantly lower(p<0.0001) in the B+P group on day 14. By day 21, there was no difference in the total DNA and protein value while total collagen and desmosine continued to be lower (p<0.0001). There was no difference in the DNA synthetic levels between the groups on 7, 14 or 21 days post-pneumonectomy. They seemed to follow the same pattern and both had significantly higher synthetic rates by day 7 when compared to the sham control and BAPN groups (p<0.0001). By day 14, the DNA synthesis levels in both groups had come down to the base levels of the control group and continued to be at this level on day 21. Chapter 4. RESULTS: PART II 102 260 240-"5) 220 H CD , C, £ 200 *55 o E CO OJ Q 180-160-140 Legend • Normal  O Sham  V BAPN O BAPN+Pneumo $ Pnaumo 14 21 28 Days postpneumonectomy Figure 4.33: Desmosine concentrations in the lungs of the 5 groups on days 7, 14 and 21 post-pneumonectomy DNA, soluble protein and collagen concentrations did not differ between the two groups on days 7 and 14 but the desmosine concentration showed significantly lower values in the B+P group (p<0.001) on day 7 and on day 14 and 21(p<0.0001). By day 21, the two groups showed no difference in the DNA and soluble protein concentrations, but the collagen concentration was lower in the B+P group (p<0.0001). Insoluble collagen amount in the lung followed the same pattern as the total collagen and were significantly lower in the B+P group on all the three days (p<0.0001). Soluble collagen values were higher in the B+P group when compared to the pneumonectomy group on all the three days (p<0.001). Chapter 4. RESULTS: PART II 103 Morphometeric, Changes Again, the significant biochemical differences between the groups were not validated by the morphometric data. Except for alveolar air proportion which was significantly higher in the pneumonec-tomy group compared to the B+P group (p<0.05) the rest of the morphometric variables were not found to be significantly different between the two groups. Comparison of B+P and B A P N Groups When the biochemical variables obtained for the B+P group were compared with the BAPN injected group, it was found that the right lung weight was significantly increased (p<0.0001) in the B-fP group by day 7 post-pneumonectomy when compared to the right lung of the BAPN group but when compared to both lungs of the BAPN group, it was significantly lower (p<0.0001). At day 14, the right lung weights of B+P groups matched the weights of both lungs of the BAPN group but by 21 days the right lung weights of the B+P group, although significantly higher than the right lung weight of BAPN group, did not match the total lung weight of BAPN rats and were significantly lower (p<0.0001). Total DNA, soluble protein and collagen were higher in the B+P group (p<0.0001) than the BAPN group on all the three days studied. Amounts of soluble collagen and insoluble collagen were also consistently higher in the B+P group on all three days. Total desmosine in the right lung was significantly high on day 7 in the B+P group (p<0.05) and continued to show higher levels in the B+P group on days 14 and 21 post-pneumonectomy (p<0.0001) (see Table 4.17). DNA and soluble protein concentrations were significantly higher in the B+P group on days 7, 14 and 21 post-pneumonectomy. Collagen concentration showed a slight Days post-pneumonectomy Variable 7 Pneumo° B + Pb H Pneumo° B + Pb 2 Pneumo0 1 > B4Pb Body weight (gm) 132.87 ±5.68 130 47 ±5.79 182.9 ±6.01 174.1 ±5.40 236.77 ±5.72 228.62 ±5.57 Right lung Weight (mg) 143.6 ±1.98 123.8 ±1.51 171.11 ±2.81 155.09 ±3.72 189.84 ±1.54 180.47 ±1.91 DNA (">g) 3 121 ±104 2.717 ±064 3.858 ±.032 3.622 ±026 4.673 ±.035 4.518 ±047 DNA (Mg/mg dry tiitue) 21.732 ±.613 21.949 ±.546 22.676 ±.196 22.784 ±.399 24.06 ±.263 25.041 ±.22 DNA Syntheiii (dpm//ig DNA) 13.3 ±.74 12.3 ±.86 5.20 ±135 5.10 ±114 4.5 ±45 4.6 ±13 Protein (mg) 74.21 ±3.51 62.97 ±1.83 90.836 ±2.276 84.99 ±3.46 109 01 ±2.29 105.45 ±3.40 Protein (fJg/mg dry titiue) 514.78 ±10.38 508.1 ±9.94 530.19 ±4.78 546.24 ±9.47 573.71 ±7.5 683.37 ±13.88 Soluble ColUgen (mg) 1.08 ±.086 1.44 ±.091 0.568 ±.082 1.296 ±067 .755 ±.066 1.072 ±064 Insoluble ColUgen (mg) 6.17 ±.074 6.023 ±.162 9.073 ±077 7.776 ±.071 12.763 ±.082 11.951 ±070 Total ColUgen (mg) 7.25 ±.104 6.463 ±069 9.642 ±069 9.072 ±074 13.508 ±.023 13.022 ±026 ColUgen (ng/mg dry tiiiue) 50 49 ±15 62.255 ±115 56.455 ±80 58.72 ±57 71.15 ±62 72.21 ±81 Deimoaine (Mg) 29.98 ±1.04 21.41 ±0 99 34.5 ±.28 24 71 ±.34 40.69 ±.54 30 68 ±26 Desmoiine (ng) mg dry tiuue) 208.3 ±4.60 172.5 ±5.9 201.B ±2 4 159.7 ±1.9 214.3 ±1.60 170.6 ±0.446 Biochemical parameters have been expressed per dry lung weight, a) Pneumonectomy b) BAPN injected + pneumonectomy. Values expressed as mean ±SEM's Chapter 4. RESULTS: PART II 105 difference between the groups (p=0.048), on day 7, with the values in the B+P group being higher. There was no difference in the desmosine concentration on day 7. By day 14, both collagen and desmosine concentrations showed a significant increase in the B+P group (p<0.0001) and this trend continued to hold true even on day 21 (p<0.001). When the morphometric results of the B+P group was compared to that of the BAPN group, it was noted that there were no significant differences in the Lm and (/a;u)values between the groups although the Lm showed shghtly higher values in the BAPN injected group. Mean chord length of the ducts and sacs showed higher values although not significant, in the BAPN group compared to the B+P group. Alveolar surface area was significantly higher in the B+P group (p<0.01) compared to the right lung of the BAPN group but was not different when compared to both lungs. There were no differences in the alveoh per unit area and per unit volume between the two groups. There was a significant increase in the total number of alveoh in the lungs of the B+P group (p<0.01) when compared to the right lung of the BAPN group but was not different when compared to both lungs. The proportion of duct air was higher in the BAPN group (p<0.01), while the alveolar wall proportion was significantly higher in the B+P group (p<0.01). In brief, the effect of BAPN on post-pneumonectomy lung growth resulted in a de-crease in lung weight, larger lung volumes and TLC, when compared to the pneumonec-tomy group. Although insignificant, there was a decrease in elastic recoil as indicated by the air-filled pressure-volume curves. Biochemically, the effect was seen as a decrease in collagen concentration, 3 weeks after administration of the chemical and a decrease in the desmosine concentration from 1 week of administration, when compared to the pneumonectomy group. There was also increased amounts of soluble collagen in the B+P group, indicating impaired cross-linking of newly synthesized collagen. Morpho-metric analysis did not show significant differences between the lungs of the two groups. Both groups had similar number of alveoli per unit area, per unit lung volume and total Chapter 4. RESULTS: PART II 106 Variable B+P Group Pneumonectomy Group Lm (p) 78.14 ±3.1 74.62 ±2.63 W {p) 41.95 ±1.07 42.45 ±0.82 Wt (M) 42.42 ±2.39 39.87 ±2.2 N „ (xlO6) 2.617 ±0.193 2.822 ±0.156 N Q (xlO6) -SWR (m2) 32.08 ±1.12 30.34 ±0.78 0.538 ±0.05 0.528 ±0.02 N A * (xlO6) 28.25 ±1.73 27.78 ±1.06 V ^ (xlO-8) 11.62 ±1.03 13.24 ±0.55 Table 4.18: Results of morphometric analysis of the BAPN administered +pneumonec-tomized group and the pneumonectomy group number of alveoli in the lungs. When the B-^P group was compared with the BAPN group, B+P group had sig-nificantly higher contents of DNA, soluble protein, collagen and desmosine. Desmosine concentration was higher in the B+P group by 2 weeks of administration of the chemical when compared to the BAPN group. Morphometric analysis showed that B+P group had larger surface area and total number of alveoli when compared to the right lung of the BAPN group but was not significantly different when compared to both lungs of the BAPN group. Chapter 5 RESULTS: P A R T HI. 5.1 L U N G G R O W T H A N D D E V E L O P M E N T D U R I N G G E S T A T I O N A N D N E O N A T A L L I F E IN H U M A N Infants in the control group had a mean gestational age of 31.24±0.98 weeks. The mean birth weight was 1394.2±187.9 gms, reflecting the nature of selection of the infants in this group. Birth weight was seen to increase hnearly with gestational age with an R2value of0.84 (p<0.0001). Of the 80 infants studied 4 infants were over 2 years of age. These four cases were excluded in the statistical analysis in order to keep the control group, age matched with the BPD group. Body weight increased with conceptional age as did the lung weights. The wet lung/dry lung weight ratio was seen to decrease with gestational age (R2=0.51) but in postnatal life, the linear correlation was lost with the ratio remaining stable with age(R2=0.05) 5.1.1 Biochemical Studies When biochemical variables were analysed, the results indicated that the DNA concentra-tion (/ig/mg dry tissue) during normal development of the lung, decreased quite rapidly from 20 weeks to about 30 weeeks of gestation after which the decrease in concentration was more gradual until birth and remained stable thereafter (Fig. 5.45). Total DNA in the lung on the other hand, showed a linear increase with conceptional age. When a regression analysis was performed, it showed a positive correlation between total DNA 107 Chapter 5. RESULTS: PART III. 108 10000 7500 to e O l 3= s ooo -'55 >» XI o CD 2 5 0 0 -o<5 • • o • Legend O NORMAL GROUP • BPD CROUP 0 5 0 1 0 0 1 5 0 Conceptional Age [weeks] Figure 5.34: Body weight gain with increasing age in control and ventilated infants 300-in E CT> CT) C 3 2 0 0 -o 0 Legend O NORMAL CROUP • BPDGROUP 100 — I — 150 Conceptional Age [weeks] Figure 5.35: Changes in lung weight with age in control and ventilated infants Chapter 5. RESULTS: PART III. 109 Case No. Gestational age Conceptional age Birth Wt. Cause (weeks) (weeks) (gms) of death 85-263 20 20 285 stillborn 85-310 20 20 357 stillborn 87-026 20 20 290.7 stillborn 85-156 20 20 402 stillborn 85-122 20 20 420 stillborn 87-350 20 20 425 stillborn 85-129 21 21 541 stillborn 87-315 21 21 280 stillborn 87-018 21 21 430 stillborn 86-409 21 21 305 stllborn 87-280 21 21 440 stillborn 85-183 21 21 365 stillborn 85-35 22 22 380 stillborn 85-141 22 22 425 stillborn 87-323 22 22 475 stillborn 87-009 22 22 482 stillborn 86-002 22 22 ' 450 stillborn 85-76 23 23 490 stillborn 85-73 23 23 490 stillborn 87-012 23 23 454 stillborn 85-218 23 23 886 stillborn 85-339 23 23 415 stillborn 85-77 23 23 590 stillborn 85-366 23 23 450 stillborn 85-338 23 23 520 stillborn 85-188 24 24 520 stillborn 88-082 24 24 485 perinatal death 85-389 24 24 575 stillborn 85-264 26 26 860 stillborn 85-212 28 28 1140 stillborn 88-098 29 29 830 stillborn 85-50 30 30 1420 stillborn 87-267 30 30 1230 stillborn 88-075 30 . 30 2625 stillborn 85-299 32 32 940 stillborn 85-136 32 32 1220 perinatal death 87-154 32 32 2150 stillborn 86-263 32 32 516 stillborn 86-82 33 33 1900 stillborn 88-070 34 34 1990 stillborn 87-219 35 35 2350 stillborn 85-94 36 36 2890 stillborn 86-94 36 36 3000 stillborn 86-40 38 38 3370 stillborn 88-105 38 38 2920 stillborn Table 5.19: Clinical history of normal infants Chapter 5. RESULTS: PART III. 110 values in the lungs and the conceptional age with an R2value of 0.82 (see Fig. 5.46). Total soluble protein concentration (mg/mg dry tissue) did not show any correlation with conceptional age and seemed to show a wide scatter through gestation and early postnatal life (R2=0.01). Total soluble protein in the lungs on the other hand, showed an increase from 20 weeks gestational age to early postnatal life. It showed a good correlation with conceptional age with an R2value of 0.88 (p<0.0001) (see Fig. 5.47). Collagen concentration (/xg/mg dry tissue) showed a steady and steep increase be-tween 20 weeks of gestation and birth with a more gradual increase postnatally (see Fig. 5.48). Total collagen in the lungs increased with age throughout gestation and early postnatal life. The R2value of 0.85 depicts an excellent correlation between total colla-gen and conceptional age (Fig. 5.49). Collagen type I/III ratio did not seem to change with age and remained more or less constant throughout gestational and early postna-tal life (see Fig. 5.50). The mean ratio was 1.76±0.16:1.0, indicating that in fetal and postnatal lungs there is a higher proportion of type III collagen compared to type I in the lung parenchyma. The R2value of the regression of the collagen type I/III ratio and conceptional age was 0.16. Desmosine concentration (pg/mg dry tissue) in the control infant lungs showed a steep increase from 20 weeks gestation to early postnatal life (Fig. 5.51). Total desmosine in the lungs of control infants showed a steady increase with conceptional age with an R2value of 0.96 indicative of an excellent correlation between total desmosine (mature elastin) and conceptional age (Fig. 5.52). Chapter 5. RESULTS: PART III. Ill Case # Gestational age Conceptional age Birthweight Cause (weeks) (weeks) (gms) of death 85-292 39 39 2400 stillborn 85-227 40 40 3100 stillborn 87-271 40 40 - stillborn 85-317 40 40 - stillborn 85-291 40 40 - stillborn 85-148 40 41 - stillborn 86-87 40 40 2700 stillborn 85-395 40 42 3670 stillborn 86-45 40 42 3170 disseminated herpes 87-230 40 47 - asphyxia 86-05 40 48 3300 aspirin OD. 87-272 40 - - 48 - SIDS 85-321 40 52 - cardiac arrest 87-270 40 52 - SIDS 87-317 38 54 - cardiac arrest 87-420 40 54 - cardiac arrest 85-147 40 56 - polycystic disease 87-296 40 58 - 'SIDS 85-70 40 64 3100 renal failure 87-292 40 66 - SIDS 85-173 40 76 - encephalopathy 86-41 40 72 - (L) ventric failure 86-038 40 76 - renal failure 85-300 40 96 - skuD fracture 86-001 40 100 - encephalitis 85-331 40 100 - skull fracture 87-314 40 104 - cardio-resp. arrest 88-110 40 184 - heart failure 86-350 40 6.5 yrs. - head injury 87-306 40 7 yrs. - head injury Table 5.20: Clinical history of normal infants contd. Chapter 5. RESULTS: PART III. 112 5.2 LUNG GROWTH AND D E V E L O P M E N T IN INFANTS WITH BRON-CHOPULMONARY DYSPLASIA Infants in the BPD group had a mean gestational age of 27.88±0.5 weeks while the normal group had a mean gestational age of 31.24±0.98 weeks, indicating that the infants in the BPD group were born at an earlier gestational age. Of the infants with BPD, 85% were below 30 weeks of gestation and mean birth weight was 1103.8± 91.63 gms. Out of all the infants studied in this group 45% had birth weights less than 1 kg and 34% had birth weights between 1 to 1.5kg. Body weights of BPD infants increased with conceptional age (fig.5.34) but beyond one year of age, the body weights of BPD infants were seen to be lower than the control infants of comparable age. Lung weights of these infants were also seen to increase with conceptional age (fig.5.35). The wet lung/dry lung weight ratios in the BPD infants showed a wide scatter throughout gestation and post-natal hfe, showing no hnear pattern with age. The R 2 value obtained for the regression analysis performed, between the ratio and age was 0.09. Out of the 52 infants in the diseased group, 49 infants started initially with hyaline membrane disease(HMD). Of the remaining three infants, one was diagnosed as having myotonic dystrophy, one was diagnosed as hydrops fetalis and was delivered by ceasarian section. The third was a mature infant who had meconium aspiration. Out of the 49 infants with a history of HMD, 11 infants had severe HMD and died despite intensive therapy. All the surviving infants with HMD went on to develop BPD to some degree. All these infants were ventilated and treated with oxygen levels of >60% concentration, for varying periods of their postnatal hfe. All the infants in the BPD group had chnical PDA (patent ductus arteriosus) for which they received the appropriate treatment. Chapter 5. RESULTS: PART III. 113 Case # Gest. Age Con. Age Birth wt. Vent. Days on Histology (weeks) (weeks) (gms) days 0 2 >60% 85-174 23 24 500 9 1 BPD I 85-284 24 24 580 0.5 0.5 BPD I 85-146 25 25 770 1 1 BPD I 88-56 25 25 500 1 1 BPD I 88-93 24 24 760 2 2 BPD I 85-190 26 26 1000 3 3 BPD I 87-321 25 26 560 4 4 BPD I 87-320 23 26 590 24 22 BPD III 85-169 26 27 660 3 3 BPD I 86-017 23 27 470 25 10 BPD II 85-309 27 27 1160 2 2 BPD I 86-136 26 27 840 10 10 BPD II 85-105 28 28 1320 1 1 BPD I 85-302 28 28 1060 2 2 BPD I 85-184 27 28 820 5 5 BPD I 86-184 26 58 880 224 220 BPD III 85-055 28 28 1200 1 1 BPD I 86-275 24 27 700 27 22 BPD III 86-211 28 28 1100 4 4 BPD I 88-097 29 29 1000 1 1 BPD I 85-304 29 30 1500 3 3 BPD I 86-315 27 30 640 18 18 BPD II 88-109 30 30 1275 1 1 BPD I 88-095 29 30.5 1500 10 10 BPD II 86-243 30 31 1460 8 8 BPD II Table 5.21: Clinical history and histology of ventilated infants 1. Gestational Age;2. Conceptional age; 3. Ventilator days Chapter 5. RESULTS: PART III. 114 Case # Gest. Age Con. Age Birth wt. Vent. Days on Histology (weeks) (weeks) (gms) days 0 2 >60% 86-359 28 32 940 32 32 BPD II 87-016 29 33 1340 26 22 BPD III 87-312 34 35 - 7 7 BPD II 88-061 30 35 1610 39 37 BPD III 88-060 32 36 2720 24 21 BPD II 85-211 27 37 840 77 68 BPD II 86-174 30 38 880 49 48 BPD III 86-051 32 44 1650 5 0 BPD IV 87-176 26 46 - 138 127 BPD III 87-115 30 42 1040 82 0 BPD IV 86-235 27 43 - 120 86 BPD III 85-219 27 44 1085 121 108 BPD III 87-198 26 45 670 130 100 BPD III 87-008 24 42 - 124 114 BPD III 85-305 40 46 3600 38 9 BPD III 86-067 29 47 1380 38 4 BPD IV 85-075 26 52 960 64 3 BPD IV 85-280 27 53 1000 176 165 BPD III 88-027 25 57 660 222 212 BPD III 85-261 31 54 1300 94 30 BPD IV 85-235 25 57 700 86 2 BPD IV 86-258 25 63 420 262 251 BPD III 88-055 24 62.5 700 232 260 BPD IV 85-234 33 78 1880 96 40 BPD III 85-390 35 101 2060 63 9 BPD IV 86-314 27 37.5 1160 73 73 BPD II 87-080 27 128 - 67 4 BPD IV Table 5.22: Chnical history and histology of ventilated infants contd. 1. Gestational Age;2. Conceptional age; 3. Ventilator days Chapter 5. RESULTS: PART III. 115 Variable Normal n=76 BPD n=52 Gest age (weeks) 31.24±0.98 27.88±0.52 Pnat age (weeks) 16.96±3.85 13.13±2.8 Con age (weeks) 48.77±7.15 40.81±2.91 Birth wt (grams) 1394.2±187.9 1103.18+91.6 Lung wt. (grams) 68.13±12.2 90.7±10.15 Vent days 50.54±9.0 0 2 >60% days 40.16±9.3 DNA cone (pg/mg) 17.7±0.51 17.83+0.53 Pro cone (mg/mg) 0.5±0.01 0.52±0.006 Coll cone (pg/mg) 69.84±5.52 87.49±5.82 Coll I/III ratio 1.71 ±0.05 1.48±0.09 Des cone (pg/mg) 0.66±0.065 0.65±0.05 Table 5.23: Mean values ±S.D. of the chnical and biochemical variables obtained for the lungs of control and ventilated infants NOTE: Ges age= Gestational age (weeks); Pnat age= Postnatal age (weeks); Con age=Conceptional age (in weeks); wt= Weight (gm); Tot lung wt.= Total lung weight (gms); Vent days= Ventilator days; Pro Conc.= Protein Concentration (mg/mg); DNA Conc= DNA concentration (pg/mg); Coll Con= Collagen concentration (pg/mg); Des Con.= Desmosine concentration (pg/mg). Chapter 5. RESULTS: PART III. 116 Figure 5.36: Case No. 85-055: Showing residual hyaline membranes and pulmonary interstitial emphysema; magnification40X 5.2.1 Histopathology On histological examination, the infants in the BPD group had varying degrees of diffuse interstitial fibroplasia depending on the degree to which the disease had developed. BPD Grade I Infants in the first group (BPD I) as categorised by us were ones who had severe HMD and those who had progressed morphologically into the beginning stages of bronchopul-monary dysplasia often with hyaline membranes still present. The lungs showed early, degenerative epithehal airway changes, there were alternating sites of atelectasis and overinflation, there was increased fibroblast proliferation and very minimal connective tissue deposition. Pulmonary edema was present in most of the cases. Chapter 5. RESULTS: PART III. 117 Figure 5.38: Case No. 86-211: Stage I B P D ; magnificationlOOX Chapter 5. RESULTS: PART III. 118 Figure 5.39: Case No. 88-095: Stage II BPD; magnificationlOOX BPD Grade II The group of infants categorised by us as having BPD II showed widespread bronchial and bronchiolar mucosal lesions. This change was seen at the alveolar duct level as well. Airspaces seemed to be smaller in the lung parenchyma with a thickened interstitium separating adjacent air spaces. There was a predominance of type II cells in the alveolar epithelium and the alveolar structure in some areas of the lung showed irregularity, with alternating regions of over-inflation and atelectasis which was still prevalent in some areas of the lung. The presence of polymorphonucleocytes(PMN's) in the air spaces was also often noted during this stage. BPD Grade LTI The next set of BPD infants were grouped into the next category, BPD III. There was wide-spread squamous cell metaplasia at the bronchial and bronchiolar level in most of Chapter 5. RESULTS: PART III. 119 Figure 5.40: Case No. 88-095: Stage II BPD; Thick interstitium with small air spaces; magnificationlOOX the cases. There was a mixture of large and small air spaces when compared to the air spaces in stage II BPD. There were alternating areas of emphysema and fibrosis.The interstitium showed variably thickened walls with mature connective tissue fibers. Some infants showed quite severe peribronchiolar fibrosis. There was type II cell hyperplasia of the alveolar epithelium and the presence of PMN's in the air-spaces was often noted. There was thickening of the media of the pulmonary vessels. Some infants had focal mild edema and focal hemorrhage. The degree of pulmonary fibrosis varied greatly. The extent of fibrosis ranged from very severe to lesser amount of fibrosis with fairly thin walls dividing the airspaces with varying amount of fibrosis. Chapter 5. RESULTS: PART III. Figure 5.41: Case No. 88-027: Stage III BPD; Terminal air ways enlarged, muscular hypertrophy, PMN's in air spaces; magnification 100X Figure 5.42: Case No. 88-061: Stage III BPD; magnification40X Chapter 5. RESULTS: PART III. 121 Figure 5.43: Case No. 87-115: Stage IV BPD; Enlarged irregular spaces; thinner walls, Muscular hypertrophy; magnification40X BPD Grade IV The next category of the disease was called BPD IV and most of the infants grouped into this category still showed muscular hypertrophy of the pulmonary blood vessels. There was a mixture of small and large airspaces with relatively thin walls compared to stages II and III. There was less active fibrosis, with fewer fibroblasts. The interstitium was not very cellular but was also not thin enough to be called "normal". Some infants still showed pulmonary edema. Many of the infants in this stage showed evidence of pulmonary infection. Healed BPD We also had a separate category of infants who we have grouped to call them infants with "healed BPD". On histological examination, it was seen that the airspaces varied Chapter 5. RESULTS: PART III. 122 Figure 5.44: Case No. 86-067: Healed BPD stage; Small and large air spaces, thin interstitium, Minimal fibrosis; magnification40X from irregularity in sizes to a more regular appearance. The interstitial walls were much thinner. The interstitial fibrosis was generally slight and focal. Squamous cell metaplasia was still present in some areas. There were also a large number of macrophages present. Although we initially grouped the infants into 6 categories, namely,(l)HMD (2) BPD I (3) BPD H (4) BPD III (5) BPD TV and (6) Healed BPD; For statistical analysis, we decided to group (1) and (2) together & (5) and (6) together to get 4 categories. Although the histological examination was conducted as a blind study, it was interesting to note that the progression of the disease from HMD stage to the healed BPD stage showed a good correlation with conceptional age, with infants with HMD dying soon after birth and majority of those in the healed BPD stage having lived the longest (table 5.28). When the clinical histories and autopsy records of the infants in the "healed BPD" group were studied, it was noted that all these infants were diagnosed as having BPD early in life, Chapter 5. RESULTS: PART III. 123 and were given ventilation and supplemental oxygen treatments.They were then weaned from both and sent home. They were doing well except for intermittent hospitalization where they were again given ventilation and oxygen supplementation for short periods of time. They all died of other causes and not due to BPD. Out of these 7 infants, one died of acute pneumonia, one due to necrotizing enterocolitis. Two were diagnosed as SIDS, two died due to congenital heart disease and one due to cardiac failure. 5.2.2 Biochemical Studies The pattern of DNA concentration obtained from the normal lungs was compared with the pattern obtained for BPD infant lungs by a non-linear model. Table 5.24 gives the estimates of the coefficients (d0,/3i,82) from equation (2.12) for DNA concentration from the individual models for BPD and normal subjects. These fitted models are plotted in Fig 5.45. It shows that the pattern of DNA concentration obtained for the lungs of BPD infants, is similar to that of normal lungs, but from the results of an analysis of the combined nonhnear model (equation 2.13) for normal and BPD infants(table 5.25), it is seen that the hypothesis 83=0 is rejected and the two groups differed significantly (p=0.0006) by a limiting concentration of -1.17. This indicates that in the limit DNA concentrations on an average are 1.17 units(/xg/mg) lower in the normal infant lungs than the BPD infant lungs. Total DNA in the lungs of BPD infants did not show a good correlation with conceptional age with an R 2 value of 0.184 (see Fig. 5.46), with the values showing a wide scatter with age. Alkali soluble protein concentration showed a poor correlation with conceptional age in the BPD lungs with an R 2 of 0.12. The concentration remained more or less constant through out gestation and early postnatal life. Total-soluble protein per lung in the BPD lungs showed an increase with conceptional age, but there was not as strong a relationship with conceptional age as seen in the case Chapter 5. RESULTS: PART III. 124 i 1 CO 50 CO 40-i i O 30 "5 § 20 H u c o (J y = [14.15-1.17 x ^ p ] e ( 2 - 4 7 e ( - ° - , , 7 x c o n * 5 C ) Legend P = 0.0006 O _ BPD O Normal < Q 10-0 O O O i i i i i i i i i i i 15 25 35 45 55 65 75 85 95 105 115 Conceptional Age [weeks] Figure 5.45: Pattern of DNA concentration in the lungs of normal and ventilated infants Biochemical measurement Group code 00 0i 02 Collagen concentration 0=BPD(w/obs 127) 1=Normal 0=BPD (w/o2 obs 27) 257.43±72.67 133.65±16.76 170.54±46.44 -2.8±0.46 -2.87±0.41 -3.98+2.21 0.024±0.028 - 0.036±0.008 -0.049 ±0.007 Desmosine Pnnrpntrat i nn 0 = B P D 1=\Trvrm;il 1.775±0.29 9 9R3 ±n 99 -0.43x0.054 .n .?q ±n ntfi -0.0037±0.0008 -n nn9fi ± 0 nnn9 D N A Concentration 0 = B P D l=Normal (w/obs 67) l=Normal(w/o obs 67) 14.87±1.06 12.67±0.8 12.9±0.61 5.02±5.57 2.18±0.51 2.69±0.59 -0.1 ±0.05 -0.06±0.015 -0.07±0.013 Table 5.24: Summary of individual fits in non-linear statistical model 1. Indicates with observation. 2. Indicates without observation. Chapter 5. RESULTS: PART III. Biochemical measurement 00 0i 02 03 Collagen concentration 183.86±28.41 -3.25±0.63 0.04±0.01 -54.14±12.79 p<0.0001 Desmosine Concentration 2.41x0.23 -0.39±0.014 -0.0028±0.0002 -0.25±0.063 p<0.0001 DNA Concentration 14.15±0.8 2.47±0.67 -0.07±0.015 - 1.17±0.37 p=0.0006 Table 5.25: Summary of combined fits in non-linear statistical model 600-1 Legend O N O R M A L ( R 2 = 0.82) 1 I I i 1 I i I i 1 I 0 10 20 30 40 50 60 70 80 90 100 Conceptional Age [weeks] Figure 5.46: Total DNA contents in the lungs of normal and ventilated infants Chapter 5. RESULTS: PART III. 20 126 15-E i cn c ~o Q_ ~a 10 5-Legend O NORMAL (R2=0.88) O BPO (R2=0.54) 10 20 30 40 50 60 70 80 90 100 Conceptional Age [weeks] Figure 5.47: Total alkali soluble protein content of normal and ventilated lungs of normal lungs, showing an R2 value of 0.54 (see Fig 5.47). When the growth patterns obtained for control and BPD groups were compared by analysis of co variance, they were seen to be significantly different (p<0.0001) with the protein values in the BPD group being higher than the control values, with increasing age. Collagen concentration in the lungs of BPD infants showed an increase with concep-tional age and the increase is much steeper than that seen in the normal lungs (Fig. 5.48). Table 5.24 gives the estimates of the coemcients(/30,/3i and B2) for collagen concentration for normal and BPD subjects. When the two groups were compared by the combined nonlinear model (see Table 5.25), they were found to be significantly different (p<0.0001) and the hypothesis /33=0 was rejected. The limiting collagen concentration was on an average 54.14 units (/xg/mg) lower in normal infant lungs than in BPD lungs. Total collagen per lung in the BPD lungs showed a good positive correlation with conceptional age wi th an R 2 value of 0.88. When the two curves obtained for the control Chapter 5. RESULTS: PART III. 127 200 CO £ CD c o c o c o o c D> _o "o o 100-y = [183.86 - 54.14 x jroupje^ - 3- 2 5^- 0 0 4*"^') o © O O o -fa P < 0.0001 Legend o BPO O NORMAL 0 15 2 5 3 5 4 5 5 5 6 5 7 5 8 5 9 5 1 0 5 115 Conceptional Age [weeks] Figure 5.48: Pattern of collagen concentration in the lungs of normal and ventilated infants 6000-4 0 0 0 -J . c <D CD _o "o O 2 0 0 0 -"o L e g e n d O W Q * W A t <* imQ.tS\ & T i 1 : ; r 10 20 30 40 50 60 70 80 90 100 Conceptional Age [weeks] Figure 5.49: Total collagen contents in the lungs of normal and ventilated infants Chapter 5. RESULTS: PART III. 128 4 - | • — , O 0 10 20 30 40 50 60 70 80 90 100 110 Conceptional Age [weeks] Figure 5.50: Changes in the collagen I/III ratio with increasing age in normal and ven-tilated infants and BPD groups were compared by analysis of covariance, it was seen that they were significantly different(p<0.001) with different slopes (see Fig. 5.49). Collagen type I/III ratio in the BPD lungs showed a tendency towards a decrease with age, although there was only a weak correlation with conceptional age (R2=0.12). The mean ratio obtained was 1.48±0.09:1.0. When the growth patterns obtained for the control and BPD groups were compared, they were found to be significantly different with different slopes(p<0.001) (see Fig. 5.50). Desmosine concentration in the lungs of BPD infants showed a steady increase from 24 weeks gestation to 2 years of postnatal life. The pattern was similar to that obtained for the control lungs, but the values tended to be higher than the controls throughout gestation and early postnatal life. Table 5.24 gives the estimates of the coefficients 8 0 , Bx and 3 2 for desmosine concentrations from the individual models for BPD and Chapter 5. RESULTS: PART III. 2. 129 CD CD C o c OJ o c o CJ <D #c o Q 1 -O P < 0.0001 Legend © _JE3PD 0 _ N o r m a l y = [2.41 - 0.25 x 5rOitp]e(-°-39e(-0 007'>,co"t') 15 25 35 45 55 65 75 Conceptional Age [weeks] \ \ i 85 95 105 115 Figure 5.51: Pattern of desmosine concentration in the lungs of normal and ventilated infants normal infants. These fitted models are plotted in Fig.5.51 from which it can be seen that the desmosine concentration pattern in the lungs of infants in both groups are very similar. When the two populations were compared by a combined nonlinear model (see Table 5.25), they were found to be significantly different(p<0.0001) by a limiting difference in their concentration of 0.25(/ig/mg). Total desmosine indicative of total elastin per lung in the BPD infants, showed a steady increase with conceptional age, and there was a good correlation between total desmosine and conceptional age with an R 2 value of 0.93. The growth patterns obtained for the control and BPD groups were found to be significantly different in their slopes (p<0.003), with the values in the BPD group consistently higher than those of the normal lungs (see Fig 5.52). Chapter 5. RESULTS: PART III. 130 30-1 . Conceptional Age [weeks] Figure 5.52: Total desmosine contents In the lungs of normal and ventilated infants 5.2.3 Disease Classification Discriminant analysis was applied to determine predictors for the revised BPD grading. When one predictor variable at a time was used, the best predictor variable was days on ventilation. Using ventilator days alone, 63% of observations were classified correctly. When days on oxygen supplementation of > 60% concentration was taken as an individual predictor variable, 42% of observations were classified correctly. When both parameters were considered together, the prediction into the appropriate BPD category improved and 72% of observations were classified correctly(Table 5.26). With the discriminant coefficients obtained for each of these independent variables, equations can be derived, to give an estimate of the probability that a subject belongs to a particular BPD grade. For example, the equation derived with the discriminant coefficients obtained using days on ventilator as the independent variable are: Chapter 5. RESULTS: PART III. 131 P R E D I C T E D A n B P D I B P D II B P D III B P D IV C B P D I 16 15 1 0 0 T BPD II 11 5 5 1 0 U BPD III 15 1 4 9 1 A B P D IV 6 0 1 0 5 L Table 5.26: Classification Matrix for disease stages using ventilator days and days on O 2 >60% as independent variables • Prob(BPD I) = -0.001 + 0.001 vent.days • Prob(BPD II) = -0.2 + 0.014 vent.days • Prob(BPD III)= -4.08 + 0.062 vent.days • Prob(BPD IV) = -0.96 + 0.03 vent.days The equations derived with the discriminant coefficients obtained for the combined analysis of ventilator days and days on O 2 >60% are as follows: • Prob(BPD I) = -0.002 + 0.004 vent.days - 0.003 days on 0 2 >60% • Prob(BPD II) = -0.275 + 0.043 vent.days - 0.029 days on 0 2 >60% • Prob(BPD III)= -4.48 + 0.128 vent.days - 0.066 days on 0 2 >60% • Prob(BPD IV) = -9.57 + 0.34 vent.days - 0.31 days on 0 2 >60% When other independent variables were added to the analysis in a step-wise fashion, the overall best prediction occurred with three other variables. These variables were, Chapter 5. RESULTS: PART III. 132 P R E D I C T E D A n B P D I B P D II B P D III B P D IV C B P D I 16 15 1 0 0 T B P D II 11 1 9 1 0 U B P D III 15 3 2 10 0 A B P D IV 6 0 0 0 6 L Table 5.27: Classification Matrix for the disease stages using ventilator days, days on FiC>2>60%, conceptional age, collagen concentration and collagen I/III ratio as indepen-dent variables conceptional age(con.age), collagen concentration (col. con) and collagen type I/III ra-tio(colI/III). When these variables were added to ventilator days and days on 0 2 >60%, 82% of the observations were predicted accurately (Table 5.27) but the coefficients ob-tained for each of the variables included in the analysis were not easily interpretable. In summary, the results obtained from discriminant analysis, show that ventilator days and days on 0 2 >60% individually did not have good predictive power, although venti-lator days showed better prediction with 63% accuracy compared to 42% for 0 2 >60% days. Together they predicted the correct category more accurately, and this prediction further improved when other variables such as, conceptional age, collagen concentration and collagen I/III ratio were included in the analysis. When the four groups of B P D were compared with each other by analysis of variance, using the clinical and biochemical parameters that were studied, it was seen that the mean gestational ages of the infants in all four groups were not significantly different, while the mean postnatal ages varied considerably with the infants in B P D I category living only for a few hours to a few days, and the infants in B P D IV category having lived the Chapter 5. RESULTS: PART III. 133 Variable BPD I II III IV n=17 n=ll n=15 n=9 Gest age 2 6 . 5 ± 0 . 5 2 29.6x1.51 26 .53±0 .7 2 9 . 4 ± 1 . 1 8 Pnat age 0 .33±0 .076 3 .69±0 .93 18 .46±3 .33 3 5 . 3 8 ± 1 1 . 1 8 Con age 26 .88±0 .49 33 .35±1.87 4 5 . 0 5 ± 3 . 4 6 4 . 7 5 ± 1 1 . 1 8 Birth wt. 8 9 2 . 7 ± 9 2 . 4 1444.6x350.77 917.92x123.97 1298 .57±172 .5 Tot Lung wt 28.03x2.82 60 .84±12 .13 142.95 ± 1 4 . 4 3 153 .7±32 .53 Vent days 2.7+0.53 29.3x7.5 117 .6±20 .7 5 9 ± 1 0 . 7 4 0 2 >21% days 2 . 6 5 ± 0 . 5 4 29.45x7.5 126 .0±26 .2 80 .12±25 .12 0 2 >60% days 2 .07±0 .33 24 .2±7 .3 106 .4±21 .5 8 . 7 ± 4 . 4 DNA cone. 2 0 . 2 7 ± 0 . 8 2 18 .15±0.95 16 .55±0 .92 1 4 . 7 1 ± 1 . 1 6 Pro cone. 0.54x0.007 0.52x0.016 0 .53±0 .009 0 .49±0 .018 Coll Cone. 5 1 . 2 5 ± 3 . 8 6 93 .4±7 .98 103 .73±9 .59 120 .49±18 .36 CoU I/HI 1.65x0.08 1.45±0.05 1.33+0.09 1 .27±0 .04 Des con 0.39x0.009 0 .49±.05 0 .8± ,07 1 .16±0 .17 Tab le 5.28: Results of b i ochemica l and c l in ica l analys is i n the different disease stages NOTE: Values expressed as mean± S.D. Ges age= Gestational age (weeks); Pnat age= Postnatal age (weeks); Con age—Conceptional age (in weeks); wt= Weight (gm); Tot lung wt.— Total lung weight (gms); Vent days= Ventilator days; Pro Cone— Protein Concentration (mg/mg); DNA Conc= DNA concentration (pg/mg); Coll Con= Collagen concentration (pg/mg); Des Con.= Desmosine concentration (pg/mg). Chapter 5. RESULTS: PART III. 134 longest with a mean postnatal age of 35.4±11.2 weeks (see Table. 5.28). Postnatal age in infants in BPD groups III &: IV was significantly higher than groups I &; II (P<0.01). When the four groups were compared with respect to their birth weights, none of them differed significantly, interestingly though, the lowest birth weights were noted in BPD I (892.7±92.4 g) and BPD III (917.92±123.97 g) groups. The infants in BPD II category had a mean birth weight of 1445.6+350.8 g and those in BPD IV category had a mean birth weight of 1298.57± 172.5 g. There was a progressive increase in the total lung weights of infants with a mean weight of 28.03±2.8 g in BPD I group, to a mean weight of 153.7+32.5 g in the infants belonging to BPD IV group. BPD groups III & IV had significantly higher lung weight values than groups I & II (P<0.01). Infants in BPD I group had a low value for ventilator days (2.7+0.5 days) owing to the fact that they lived for a very short period of time postnatally and were mostly ventilated throughout their life. Requirement for ventilation in infants in BPD II group was higher with a mean value of 29.3+7.5 days. The mean ventilatory requirement days for infants in BPD III group was 117.6 + 21 days and was significantly higher than the requirements of groups I, II and IV (P<0.01). The mean ventilatory requirement days for infants in BPD IV group was 59+11 days, almost half the requirement for infants in BPD III group. Days of 0 2 >21% also followed the same pattern as the ventilator days with infants in BPD III group having the longest requirement with a mean value of 126+26 days which was significantly higher than the requirements for the I, II and IV groups(p<0.01). Owing to the short post-natal life, infants in BPD I group had a mean requirement of 2.7+0.5 days which was significantly lower than the other three groups(p<0.01). Infants in BPD IV group had a mean value of 80+25 days for their requirement of days on 0 2 >21%, while infants in BPD II group had a mean requirement of 29.45+7.5 days. Chapter 5. RESULTS: PART III. 135 When the four groups were compared for the requirements of oxygen supplementation of greater than 60% concentration, again the greatest requirement was found to be in infants categorised into BPD III (106.4±22 days) which was significantly higher than the values obtained for the other three groups(P<0.01). Infants in BPD IV group had a mean requirement of 8.7+4 days. DNA concentration ( pg/mg dry tissue) showed, a progressive decrease from a value of 20.27±0.82 ( pg/mg dry tissue) in infants of BPD I group to 14.71 ±1.16 ( pg/mg dry tissue), in BPD IV infants(P<0.01). Protein concentration(mg/mg dry tissue) did not differ among the four groups while collagen concentration ( pg/mg dry tissue) showed a progressive increase from infants in BPD I group (51.25±3.86) to infants in group BPD IV (120.49±18.36)(P<0.01). Mean collagen concentration in infants of BPD II was twice that in infants of BPD I group (93.4±7.98) and remained about the same in infants of BPD III group (103.73±9.59) (P<0.01). Collagen type I/III ratio varied considerably between the four BPD categories. The mean value for the type I/III ratio in infants of BPD I group was 1.65±0.08. The mean ratio showed a decrease for infants in the BPD II group, with a mean value of 1.45±0.25 and continued to be low in the infants of BPD III group which had a value of 1.33+0.09 indicating a decrease in the collagen I/III ratio as the disease advanced. The mean ratio was seen to be the lowest in infants of BPD IV group (1.27+0.04) and was seen to be significantly different from the ratio obtained for BPD I group (P<0.01). When desmosine concentration ( pg/mg dry tissue) in the lungs of infants was used for comparing the four groups, it showed a progressive increase form BPD I group (0.39 + 0.009) to the highest value seen in BPD IV group (1.16+0.17) (P<0.01). The mean value for desmosine concentration was seen to be doubled in BPD III group (0.8 + 0.07) when compared to the mean value obtained for infants in BPD II group (0.49 + 0.05) (P<0.01). Chapter 5. RESULTS: PART III. 136 From the results obtained from the discriminant analysis as well as from the results of the clinical analysis of the different disease groups, it was noted that there was a strong age effect on the disease progression as the infants in BPD I group lived the shortest time while the infants in BPD IV group on an average, lived the longest. In an attempt to remove this age effect, we compared the biochemical parameters obtained for the different disease groups to their age matched control groups using two sample t-tests. When the results of the biochemical analysis in each of the BPD grades (I-IV), were compared to their age matched controls, collagen concentrations in the BPD I group were not seen to differ significantly from their age matched controls. The two groups did not differ significantly in the collagen I/III ratios or their desmosine concentrations. The infants in the BPD II group differed significantly from their age-matched controls, in their collagen concentrations (p<0.05), with the infants in the BPD II group having higher amount of collagen concentration when compared to the age matched controls. The two groups did not differ significantly in the desmosine concentrations but differed in the collagen I/III ratios with the values in the BPD group being significantly lower (p<0.05) When the infants in the BPD III group were compared with their age matched con-trols, the}- were seen to differ significantly in their collagen and desmosine concentrations with the BPD III group having higher values for collagen and desmosine concentrations (p<0.05). The collagen I/III ratio differed significantly between the two groups as well, with the mean ratio in the BPD group being lower(p<0.05). Infants in BPD IV group did not differ significantly from their age-matched controls in their collagen and desmosine concentrations, but the collagen I/III ratio remained significantly lower in the BPD IV group when compared to its age matched control group (p<0.05). Chapter 5. RESULTS: PART III. 137 Group Mean Con age (weeks) Coll. Cone. (pg/mg) Coll I/III Des. Cone. (pg/mg) Control BPD I 26.75±0.92 26.88±0.49 48.1±1.69 51.25±3.86 1.72±0.18 1.65±0.08 0.366±0.027 0.39±0.009 Control BPD II 36.6±1.12 33.35±1.87 66.2±3.24 93.4±7.98* 1.70±0.007 1.45±0.05' 0.542±0.002 0.49±0.05 Control BPD III 41.48±1.8 45.03±3.40 69.81±2.98 103.73±9.59* 1.7±0.006 1.32±0.009* 0.63±0.003 0.8±0.07* Control BPD IV 63.11±4.49 64.75±11.18 94.56±6.21 120.49±18.36 1.9±0.006 1.27±0.04* 1.03±0.08 1.16±0.017 Table 5.29: Results of the biochemical analysis in the lungs of ventilated infants and their age-matched controls NOTE: ' p<0.05; con age= Conceptional age; coll. cone. =collagen concentration; des. cone. = desmosine concentration Chapter 6 DISCUSSION 6.1 G E N E R A L POINTS This thesis has dealt with the growth and development of the lung with particular em-phasis on the biochemical aspects of lung growth. Both intrauterine and postnatal lung growth was studied in an animal model( the rat ) as well as in the human infant. In this chapter we will not only discuss the biochemical aspects of lung growth in the two species, but will also attempt to compare the growth patterns obtained for the lungs of the two species. We also studied lung growth resulting after experimental manipulation of a normally growing lung, namely, pneumonectomy of the lung. It is of interest to study the growth occurring in the contralateral lung after removal of whole or portions of the lung, not just to satisfy a scientifically inquisitive mind but also with regard to its clinical relevance in removing diseased portion of a lung in humans with the hope of the lung resuming its growth and restoring the gas exchanging surface area back to normal. That the contralateral lung tissue undergoes a compensatory growth after pneumonectomy or re-section has never been questioned. However, the extent and the nature of the growth have been. Compensatory lung growth has raised important structural issues: does the lung parenchyma(gas-exchanging tissue) remodel itself to maintain a maximal gas-exchanging surface or does it compensate only by expansion? If the lung remodels itself, does it do so by alveolar multiplication or can the gas-exchanging surface area be restored by other 138 Chapter 6. DISCUSSION 139 means? Connective tissue forms a major structural component of the lung and plays an impor-tant role in normal lung development. Collagen and elastin which are major components of the connective tissue in the lung play a critical role in defining lung structure by playing a key role in the process of alveolarization. If indeed post-pneumonectomy compensatory lung growth is similar to normal lung growth, then by interrupting the process of mat-uration (cross-linking) of collagen and elastin by inhibiting the enzyme responsible for their maturation, we would expect both normal lung growth and compensatory growth in the contralateral lung to result in fewer but larger alveoli and fewer alveoli per unit volume of the lung. The final part of the thesis deals with abnormalities of lung growth resulting in a disease state. For this purpose we chose to study a fibrotic disease, bronchopulmonary dysplasia, occurring in neonates. It was of interest to study this particular disease as it occurs mostly in premature infants whose lungs are structurally and functionally still immature and are exposed to the insults imposed by the external environment when they are still not quite ready to cope with the sudden transition from intra- to extrauterine life. Our aim was to describe growth patterns of a normally developing infant lung and then compare these parameters in the diseased lungs that were studied. This way, we hoped to come one step further in understanding the factors causing the abnormality in lung growth resulting in the diseased state as well as understanding normal lung growth itself. A correlative study was also conducted of the histopathological, biochemical and clinical parameters obtained for the infants with BPD. Through the correlation of these parameters, we might begin to understand what might be the primary inciting agent causing the disease as well as understand the disease as a broad spectrum of changes in lung structure with resulting alteration in lung morphology and function. Chapter 6. DISCUSSION 140 6.2 GROWTH AND DEVELOPMENT OF LUNG IN THE RAT From the results obtained, it could be seen that the increase in the body weight and lung weight of the rat from day 16 of gestation until 7 weeks postnatal age was substantial. Major structural transformations of the lungs are seen to take place during this period. Analysis of morphometric data shows orderly changes with somatic growth and increasing gestational age [119]. Lung weight gain paralleled body weight gain during late gestation and the specific lung weight was seen to remain constant until birth. No significant increase in lung weight was seen on the first day of hfe although the average body weight increased 30% during this time. Therefore body weight increased faster than the lung weight lowering the lung weight/body weight ratio. This finding was consistent with the observation that nuclear labelling with tritiated thymidine is low on the first day of hfe in the lungs of mice [61]. Thus, the first phase of lung growth is primarily one of expansion with very little increase in tissue content, but this phase is very short in duration. Subsequently, lung weight doubled by 1 week of age and increased a further 1.5 times by 2 weeks of age. The most rapid rate of growth was therefore seen to occur in the first 2 weeks of life. During this phase, the lung weight seemed to increase in parallel with the bodj' weight and the ratio of lung wt./body wt. remained nearly constant from birth to 2 weeks of life. This probably reflects preparation by the lung for, and the beginning of "the explosive restructuring" of lung parenchyma and the formation of alveoli which starts in the first week of postnatal life in the rat. After the second week of hfe, the rate of lung tissue growth gradually fell and was slower than the general body growth, until a constant lung wt/body wt. ratio was reached by six weeks of age. The cell density and water content of the lung decreased as age increased, through gestation and postnatal life, which is illustrated by a progressive fall in DNA concen-tration and the wet lung/dry lung weight ratio. Fetal lungs were seen to contain more Chapter 6. DISCUSSION 141 water than lungs after birth. The decrease in wet lung/dry lung ratio with increasing age indicates a progressive increase in the lung tissue mass in comparison to the water con-tent of the lungs. This increase in tissue mass was also indicated by an increase in total DNA content which is indicative of increase in cellular content. Although this indicates an increase in cell number, it does not give any information as to which type of cells are proliferating. However, during late gestational period it is most likely that the epithelial cells of the lung parenchyma which are in a highly proliferative stage would contribute largely to the increase in DNA content during this period. The other cells which would show high proliferative rate during this period are the interstitial cells which synthesize the connective tissue proteins. Between day 21 of gestation and the day of birth, an 18% increase in DNA content was noted, indicating a slowing down in the cellular proliferation rate, 24 or more hours before birth which would probably be the period of pneumocyte cytodifferentiation. But between birth and two weeks of life, the greatest increase post-natally in DNA content occurred which again indicated the proliferative phase of lung development. A decrease in cell density during late gestation and postnatal life was noted which in-dicated an increase in tissue complexity. In other words, for a unit mass of the lung tissue there were more extracellular components being laid down, bringing the number of cells per unit mass of lung down. This finding is in agreement with the earlier reports [74],[180] which have shown the presence of extracellular components such as collagen and elastin by late gestation in the rat. The brief increase in DNA concentration, one day before birth, noted in our study cannot be explained. DNA concentration continued to decrease until 4-5 weeks postnatal life, after which it remained constant, long before lung tissue growth stopped. During the last phase of fetal maturation, the rate of soluble protein accumulation in the lung was more than DNA which resulted in an increase the protein/DNA ratio. Chapter 6. DISCUSSION 142 This would indicate a switch from increase in cell number during this phase to increasing cell size, as has been shown in earlier studies [231]. It should be mentioned here also that this increase in soluble protein is due at least partly, to the accumulation of newly synthesized connective tissue proteins, present both intra and extracellularly, which are soluble in alkali. The fetal lung appears to have httle importance in utero but assumes the vital function of gas exchange, immediately after birth. Maturational events such as deposition of extracellular components, preparing the lung for its postnatal role takes place in the latter part of gestation. The mechanical function of elastin and collagen after birth may be deduced in part from their rates of accumulation before birth. In our study, collagen was detected biochemically only from 18th day of gestation in the parenchyma of rat lung, which is approximately the early canalicular stage of development. Collagen content increased slowly during gestation but postnatally there was a substantial increase from birth until 7 weeks. The largest increase was seen to occur between the first and fourth postnatal week and then a slower further rise until 7 weeks. This large accumulation of collagen began at about the onset of alveolization in the rat and extended beyond the period of morphological maturation of the alveolar septa. In rabbit lung, a similar rapid increase in collagen accumulation between 10 days before birth and 20 days after birth has been found [54],[30]. Collagen concentration in the lung tissue increased steadily from day 18 of gestation indicating an increase in the extracellular mass of the tissue. This was further supported by a steady increase in the collagen/DNA ratio from gestation until adult hfe. There was an increase in the collagen/protein ratio with increasing age from late gestation well into postnatal life. This would probably indicate a shift in the protein synthesizing machinery towards more collagen production. There was also an indication of increasing maturity of collagen fibers with age, by a gradual decrease in the soluble/insoluble collagen ratio. Chapter 6. DISCUSSION 143 There was more soluble collagen or newly synthesized collagen present during fetal life compared to postnatal life and the decrease in the ratio was slow, but with the onset of birth, the rate of decrease in the ratio was much faster until 3 weeks of life indicating an increased proportion of cross-linked mature collagen in the lung. It has been reported by Brody et al [32] that the activity of the enzyme lysyl oxidase is substantially high in rabbit lung parenchyma during the first 3 weeks after birth. From the 3rd postnatal week, the soluble/insoluble collagen ratio remained constant indicating a dynamic equilibrium in collagen metabolism once adult levels were reached. This suggests that collagen synthesis and extracellular catabolism of older collagen are the decisive determinants of net lung tissue collagen content during growth [54]. Desmosine was detectable in the rat lung only at day 21 of gestation in this study, while others have measured its content in the lung at day 18 of gestation [180]. After an initial slow increase from late gestation until birth, there was a 7-fold increase in the desmosine content in the first week of life followed by a 13-fold increase until the fifth week. After the fifth week, the increase was more gradual and continued at least until 7 weeks of age. The maximum accumulation of desmosine occurred between the first and fifth week life during which period, rapid alveolar proliferation occurs. These results show that the newly synthesized tropoelastin was rapidly being converted to mature functional elastin soon after birth in the rat. Desmosine concentration(/xg/mg tissue) also showed an increase with age, with the highest rate of increase occurring in the first to third week of life. It increased gradually until 4 weeks of life bringing it to adult values after which it remained constant. It has been shown in sheep that desmosine concentration in the lung rises about 25% between 60 and 100 days of gestation and then increase 7-fold until term [179]. The timing of elastin accumulation and of alveolization is related to the degree of pulmonary and over-all maturity that is achieved at term in a given species [74]. In the sheep, which shows a Chapter 6. DISCUSSION 144 high degree of independence at birth, alveolarization [2] and rapid elastin accumulation are initiated in utero, whereas in the rat these events occur postnatally [180]. The onset of maximum rise of desmosine accumulation appears to precede the onset of alveolization in the sheep, occurring around the canahcular stage, while earlier reports in the rat [180] show the onset of the rise in desmosine accumulation occurs after the alveolar period. Our study however, showed that the maximum rise in desmosine occurs during the pe-riod of alveolarization. Throughout our study, we have reported our measurements of desmosine without converting them into elastin equivalents because there is evidence that the desmosine/isodesmosine ratio in the elastin of neonatal rat lung changes with age [160]. Collagen fibers seem to precede the appearance of mature elastic fibers, as collagen was found to be present by 18 days of gestation while desmosine was detected at 21 days of gestation. This does not mean that newly synthesized elastic tissue is not found earlier, it is just not cross-linked yet to give rise to the mature fibers. Collagen concentration in terms of units of hydroxy proline is 11-times greater than desmosine concentration on day 21 of gestation. The ratio hydroxy proline/desmosine decreased from day 21 of gestation until 4 weeks of postnatal age after which there was a gradual increase in the ratio. This indicated a rapid increase in the accumulation of desmosine during late gestation and early postnatal life, compared to the accumulation of collagen, but by 4 weeks of age, the accumulation of desmosine slowed down considerably and continued to increase at a slower rate than collagen thus increasing the ratio once again. 6.3 E X P E R I M E N T A L A L T E R A T I O N OF L U N G G R O W T H 6.3.1 Post-pneumonectomy lung growth Our study was in agreement with previous work [37],[98],[172],[145],[162] on post-pneumonectomy compensatory growth in that there was a complete compensation for lung volume, total Chapter 6. DISCUSSION 145 lung capacity, total DNA, soluble protein, collagen and elastin contents. Lung weight showed an incomplete response when compared to both lungs of the control animal. Oc-currence of hyperplasia by day 7 post-pneumonectomy was indicated by a significant increase in DNA synthetic levels over those of controls. These levels fell to control levels by day 14, indicating that maximum cellular proliferation takes place within 2 weeks of pneumonectomy. This finding was in agreement with the report of Thet & Law [206] in which they found a post-pneumonectomy increase in numbers of type II pneumono-cytes and capillary endothelial cells in rats in their ultrastructural morphometric study. An autoradiography study in mice demonstrated increased DNA synthesis in pleural mesothelial cells by day 1 post-pneumonectomy, in the interstitium, endothelium, and peripheral alveoli by day 3, and the bulk of alveoli by day 6 [41]. A similar study in rabbits showed a significant increase in DNA synthesis in alveolar wall cells by day 5, which peaked on day 11 [62]. This stud}' showed an increase in tissue concentrations of collagen over control val-ues by day 21 post-pneumonectomy, and the tissue concentrations of desmosine by day 14. This result is in conflict with previous work [16],[222] which showed that pneu-monectomy had no effect on tissue concentrations of collagen and elastin and that both constituents increase in proportion to lung mass during compensatory growth. In our analysis, compensatory growth seems to mimic normal lung growth, in that, there seems to be an increased rate of deposition of the connective tissue components in comparison to the increase in the lung weight, resulting in an increase in their amounts per unit lung weight with increasing age. An increase in collagen synthesis in the pneumonectomy group was also indicated by a significant increase in the amounts of soluble collagen by day 7 post-pneumonectomy indicating increased amounts of newly synthesized colla-gen. Either formation of new alveoli' or enlargement of existing air spaces could account for the increase in cellular and extracellular components in post-pneumonectomy lung Chapter 6. DISCUSSION 146 growth. Over the decades, investigators have been divided evenly between these two mechanisms[208]. By enlargement of existing airspaces, it is generally meant that the alveoh and/or alveolar ducts grow in size with lengthening of their walls accompanied by cellular proliferation. On the other hand, with the help of morphometric techniques, some investigators advocate simple dilatation or distension of air spaces. From the results of the morphometric analysis, it was noted that by day 21 post-pneumonectomy, there was evidence of alveolar multiphcation with increased alveolar surface area and increased total number of alveoh in the lung. There was no difference in the mean linear intercept which again indicated an increase in internal complexity of the lung, caused by alveolar multiphcation. As indicated in earlier studies [208], an increase in internal complexity of the lung caused by extensive alveolar multiplication, the surface area of the lung would be directly proportional to the lung volume and the Lm would therefore remain constant. There was also evidence of enlargement of existing structures as the number of alveoli per unit volume and per unit area showed a decrease in the pneumonectomy group along with an increase in the average alveolar volume. Although post-pneumonectomy lung growth may have involved alveolar multiplica-tion early on after surgery, which was conducted when the rats were 4 weeks of age, certain amount of alveolar enlargement occurred in the remaining lung as the animal was given more time to recover from the surgery. This result could partly be due to the age of the animal at the time of surgery. Although there is evidence of growth of the lung and of alveolar multiphcation at this age, it is at a much slower rate than when the animal is younger, the highest rate of multiplication having been completed by the time the rat reaches 4 weeks of age. As a result, the compensatory response would show certain amount of alveolar multiplication since some amount of growth is still occurring at that age, but as the recovery period is prolonged, some amount of enlargement of existing structures is seen. This theory is supported by earlier observations that there Chapter 6. DISCUSSION 147 was alveolar multiplication in 4 week old rats [98], and evidence of alveolar enlargement in 8- and 12- week old rats after pneumonectomy. Similarly in another study, number of alveoli per unit volume was not significantly decreased after pneumonectomy in 10- and 18-week old rabbits, but it was significantly reduced in 26 week old rabbits [43]. Thus under certain circumstances, alveolar multiplication ensued, but in general, this was not complete and certain amount of enlargement of alveoli was seen. With respect to the physiological functions of the lung, it was seen that pneumonec-tomy did not alter lung recoil as evidenced by the similarity in the static pressure-volume curves between the two groups. This is consistent with volume restitution caused by alve-olar multiplication. There was also a complete response based on the values obtained for total lung capacity. In summary, with regards to the biochemical and physiological aspects of this study, post-pneumonectomy lung growth occurring after 4 weeks of age in the rat, appears to mimic normal lung growth but when the lung is looked at morphologically, it seems to differ from normal lung growth in that there appears to be enlargement of existing structures occurring as a response to the surgery along with some amount of alveolar multiplication. 6.3.2 Effect of Beta-aminopropionitrile on postnatal lung growth Beta-aminopropionitrile is a chemical that inhibits the enzyme lysyl oxidase which cat-alyzes the oxidative deamination of some peptidyl lysine and hydroxy lysine residues, and starts the cross-Unking process leading to the covalent crosshnking of both tropoelastin and collagen molecules. This cross-Unking process is an important step in the matura-tion of the two connective tissue components and inhibition of this process would inhibit the proper structural framework and ultimately the functional capacities of collagen and elastin. Chapter 6. DISCUSSION 148 In order to assess the importance of collagen-elastin network in alveolar acquisition, previous studies were conducted with administration of BAPN to rats in the first four weeks of hfe [112]. It was seen that BAPN did have an effect on lung growth and produced large lungs with diminished elastic recoil and too few and too large alveoh. Another study conducted by the same group of investigators [113] showed that when rats were administered BAPN for the first four weeks of hfe and then allowed a four week recovery period, the changes in the lungs caused by BAPN in the first four weeks were not reversible even after four weeks of recovery. It was not known whether BAPN continued its effect after cessation of administration. BAPN is known to bind irreversibly and inhibit lysyl oxidase. Whether or not it remained in situ or permanently inhibited lysyl oxidase is unknown. In our study, we administered BAPN to rats from 4 weeks of age until 7 weeks of age. With respect to lung mechanics, BAPN did show an effect by 7 weeks of age by producing lungs with larger volumes and diminished elastic recoil indicated by an increased compliance and a shift in the P-V curves upward and to the left as compared to the control lungs. From the biochemical results obtained by us, we can attribute these changes in lung mechanics to the alteration in tissue forces caused by, the impaired cross-linking of collagen and elastin. Although there were no differences in body weight gain from the control group, lung weights were significantly lower from 5 weeks to 7 weeks of age. Biochemical studies showed the effect of BAPN on lung growth, by showing decreased DNA and soluble protein concentrations by 1 week of administration of BAPN and decrease in collagen and desmosine concentrations by 2 weeks of administration. Although, collagen concentration levels returned to normal values by 3 weeks of administration, desmosine levels remained low. These results indicate that although BAPN affected both collagen and elastin cross-linking, the effect seemed to be more profound on elastin. Chapter 6. DISCUSSION 149 The differences seen in the biochemical analysis and lung mechanics was not indicated by morphometric analysis. This may be because biochemical analysis is a more sensitive technique to detect even small changes in the components of the lung which is not detected by an assessment with the naked eye, as done in morphometric analysis. Although morphologically there were no significant differences between BAPN and control groups, the lungs of the animals in BAPN group showed an increase in the mean linear intercept, decrease in alveolar surface area, total number of alveoli, alveoli/unit area and number of alveoli/unit volume, indicating a tendency towards enlargement of existing alveoli in the BAPN group. The results obtained by us are similar to the earlier studies [112],[113] in terms of lung mechanics but morphometrically, the results do not show the apparent differences between the groups as noted in the earlier studies [112],[113]. This apparent discrepancy could be attributed to the fact that older animals were used in this experiment. As indi-cated by our investigations of normal lung growth in rats, the most rapid accumulation of collagen and elastin occurs in the first four weeks of life, and by this time alveolar for-mation may be far advanced. Even though there is still some synthesis and accumulation of these proteins taking place in the lungs, it is at a much slower pace. When BAPN is administered to the rats at this age, minimal changes are to be anticipated as it would affect only the newly synthesized collagen and elastin and would have no effect on the mature already cross-linked collagen and elastin. Therefore when the lung is examined morphologically, there does not appear to be a significant difference between the control and BAPN groups, whereas the minimal changes in the collagen and elastin could be measured by biochemical analysis which is more sensitive compared to morphometry. Therefore, it is most likely that BAPN does have a significant effect on lung growth by inhibiting the structural framework of collagen and elastin of the lung, thereby inhibiting alveolar formation, when it is administered to an animal at its most rapid phase of lung Chapter 6. DISCUSSION 150 growth. Its effect seems to decrease if it is administered after the period of rapid growth, as by that time most of the connective tissue is laid down and BAPN does not have any degradative effect on mature, cross-linked connective tissue proteins. 6.3.3 Effect of Beta-aminopropionitrile on post-pneumonectomy lung growth The effect of BAPN on post-pneumonectomy lung growth in the B+P group was seen as a decrease in lung weight, larger lung volumes and total lung capacity, when compared to the pneumonectomy group. Although insignificant, there was a decrease in elastic recoil as indicated by the air- filled pressure-volume curves. The P-V curves obtained for the B+P group was seen to he between that obtained for BAPN and Pneumonectomy groups. Biochemically, the effect of BAPN was seen as a decrease in collagen concentration 3 weeks after administration of the chemical and a decrease in desmosine concentration from 1 week after administration, when compared to the pneumonectomy group. There was also increased amounts of soluble collagen indicating impaired cross-linking of newly synthesized collagen in the B+P group. Although the effect of BAPN could be seen biochemically by the decrease in the connective tissue components of the lung, morphologically there were no significant dif-ferences between the lungs of the two groups. Both groups had same number of alveoh per unit area, per unit lung volume and total number of alveoh in the lungs. When the B+P group was compared to the BAPN group, it was noted that the B+P group had significantly higher values for DNA, soluble protein and collagen concentrations within 1 week of surgery. Desmosine concentration was higher in the B+P group by 2 weeks of surgery. Morphologically, lungs of B+P group had larger alveolar surface area, and a significant increase in total number of alveoh in the lung, compared to the right lung of the BAPN group, indicating increase in alveolar multiphcation in the B+P group Chapter 6. DISCUSSION 151 compared to BAPN group. From the results obtained it can be seen that BAPN has an effect, although minimal, on post-pneumonectomy lung growth occurring at 4 weeks of age in the rat. This effect was seen biochemically as a decrease in connective tissue content as well as maturation. The effect was seen on lung mechanics as well, with an increase in lung volume and TLC. There was also a slight, insignificant shift upward and to the left in the P-V curve of the B+P group in comparison to the pneumonectomy group. Although anticipated by us, BAPN did not seem to effect the lung morphology as judged by light microscopy. This perhaps is due to the fact that pneumonectomy was performed at 4 weeks of age in these rats, by which time most of the alveoli may have already formed and the compensatory growth results in an enlargement of existing alveoli and incomplete restoration of the alveolar number. When BAPN is administered at this point, it would effect only the newly synthesized connective tissue proteins which may have only a slight effect on the general morphology of the lung parenchyma and would not be picked up by the naked eye during morphometric analysis. 6.4 L U N G G R O W T H A N D D E V E L O P M E N T IN T H E H U M A N I N F A N T Although fetal development has been studied extensively in humans, most of the data relating to the perinatal period have been obtained in animals. Little anatomical and bio-chemical information relating to early post-natal growth in humans is available. Because of the limitations with human material, studies of lung growth in normal and diseased lungs have been conducted extensively on animals and the results have been extrapolated to human lungs in the past. Such direct extrapolation from animals to man requires caution as there may be considerable interspecies variation in lung development. With this in mind, we compared the growth patterns of the lung obtained for human with that Chapter 6. DISCUSSION 152 of the rat. The body weights of the infants increased linearly with conceptional age as did their lung weights. The wet lung/dry lung weight ratio decreased with age indicating a decrease in the water content of the lung and an increase in lung tissue content. There was a sharp decrease in the wet lung/dry lung weight ratio from gestation until birth, after which the ratio remained constant. Somatic growth in the rat seemed to follow the same pattern as the human lung, with body weight increasing hnearly with conceptional age. Lung weight was also seen to increase hnearly with age. The pattern of wet lung/dry lung weight ratio in the rat was different from the pattern obtained for the human lungs in that, the decrease in the ratio continued beyond birth until 2 weeks of postnatal hfe after which, the ratio remained constant and did not change with increasing age. This pattern in the rat is perhaps due to the fact that, in the rat, alveolarization occurs entirely postnatally during which time, maximum amount of extracellular tissue is laid down. This increase in tissue mass would contribute to the dry weight of the lungs and would result in lowering of the wet/dry weight ratio of the lung. Since the rapid phase of alveolarization continues into second postnatal week, a decrease in the wet/dry ratio is seen. That the lung grew by cell multiphcation was indicated by a linear increase in total DNA content of the lung with age, both during gestation as well as early postnatal life in the human. Previous studies on the biochemical development of the lung in humans dealt with various stages of the first half of gestation from 6 to 20 weeks [108]. They indicated a progressive increase in the DNA concentration in the first half of pregnancy and the RNA/DNA and protein/DNA ratios remained stable during the same period. They related their data to the embryological observations that the fetal lung is relatively late in achieving its maximum development, and thus initial stages of gestation is devoted to extensive cell multiplication and very little differentiation and accumulation of the mesenchyme. Stable RNA/DNA and protein/DNA values also provide evidence to the Chapter 6. DISCUSSION 153 fact that the cells are not growing by hypertrophy but are multiplying. This study was conducted on the later stages of gestation (from 21 weeks) and into early postanatal life upto 2 years of age. The analysis of DNA concentration in the lungs from 21 weeks gestational age showed a progressive decrease until birth and thereafter remained constant in postnatal life. This study was conducted at the time when the lung was in the cananicular stage of development during gestation, which is a period during which extensive restructuring and differentiation in the lung interstitium occurs with the synthesis and accumulation of connective tissue proteins. At this period it would be expected that per unit of lung mass, the cellular content would decrease with respect to the amount of extracellular components being synthesized, hence the decrease in DNA concentration. The study on the rat lungs was also conducted from the canalicular period of development during gestation. Results obtained from this study showed a linear increase in the total DNA content of the lung with increase in conceptional age. On the other hand DNA concentration showed a decrease that continued beyond birth to about 5 weeks of postnatal age, indicating extensive connective tissue accumulation during this period. These results indicate that extensive accumulation of extracellular components would have started in utero by late gestation in the humans, suggesting the start of the process of alveolarisation during late gestation in the humans while in rats this process occurs postnatally. Soluble protein in the lung also increased linearly with age from gestation well into postnatal life showing a similar growth pattern as the rat model. Protein concentration in the human lungs on the other hand showed a wide scatter at all ages without any particular growth pattern unlike the -growth pattern obtained for the rat which showed a rapid decrease in protein concentration until birth after which it remained stable. Little is known about the rate of collagen accumulation in the human. The only avail-able data are from lungs of 12-17 weeks old fetuses in which, like the adult, approximately Chapter 6. DISCUSSION 154 4% of amino- acids that are incorporated into protein are incorporated into collagen [28]. Collagen concentration in this study was seen to increase sharply from 20 weeks gesta-tion and had increased 4-fold by birth, after which the increase was more gradual until 2 years of hfe. Total collagen in the lung also showed a hnear increase with age. These results indicate a rapid accumulation of collagen in the lungs during late gestation and early postnatal hfe in the humans. Studies concerning developmental changes of the lung collagen content in rabbits [30], show a 2-3 fold increase in lung collagen concentration in the last trimester of fetal development and a 2-fold increase from birth to maturity. Collagen concentration remains constant after the lung stops growing (at approximately 3 months) in the rabbit. Interestingly, during the period of collagen accumulation in the humans, the soluble protein concentration did not change with age. This resulted in an increase in the col-lagen/protein ratio which rose steadily with conceptional age (R2=0.68). This further emphasizes the striking changes in lung collagen accumulation and a shift in the pro-tein synthesizing machinery toward more collagen production. Decrease in extractabihty of collagen was seen with increasing age as the amount of insoluble "mature" collagen increased with conceptional age, consistent with the concept that lung maturation is associated with increased cross- linking in the collagen molecules. Rat lungs showed similar growth patterns as the human lung for total collagen con-tent. Collagen concentration seemed to increase rapidly with age well into adult hfe in the rat, unlike the pattern obtained for the human lung where the increase in concen-tration seemed to slow down by 2 years of age. Soluble protein concentration in the rat lung showed a rapid decrease from late gestation until term, after which there was no further change in the concentration with age. This resulted in a sharp increase in the collagen/protein ratio during gestation and a more gradual increase postnatally. In the adult lung parenchyma, types I and III collagen account for more than 90% of Chapter 6. DISCUSSION 155 the collagen. The best approximation of the relative amounts of these collagens comes from the studies on adult human lung suggesting type I collagen dominates type III in a ratio of 2:1 [187],[188]. In this study, the relative proportion of these two collagen types seem to differ in the fetal and early postnatal lungs in that there seems to be a higher proportion of type III collagen compared to type I with a mean ratio being 1.76±0.16:1. This result is in agreement with the immunohistochemical study conducted by Bateman et al [14] in human fetal lungs, and seems to follow the same pattern of development as seen in other fetal organs; for example, the skin [75]. With regards to the development of elastin in the lung, the appearance of elastic fibers in the mammalian lung was shown to occur early in prenatal and perinatal development. Fierer [80] showed that in the developing fetal lamb, the microfibrillar components are present at day 90 of gestation. Elastin appeared at day 110 and gradually increased relative to the amount of microfibrillar components until term which occurs at day 150. Our study on the rats showed the appearance of desmosine by day 21 of gestation and it continued to increase in concentration until 4 weeks of postnatal age after which it did not increase any more with age and remained constant. Before the advent of radioim-munoassays for desmosine [93] and isodesmosine [194], quantitative determinations of tissue elastin have been performed by gravimetry or amino acid analysis of the insoluble residue left after hot alkali extraction of the tissue. Tropoelastin and possibly incom-pletely crosslinked elastin are lost during the extraction with hot alkali and would have possibly contributed to the conflicting results reported by earlier investigators. Reported values for elastin content in the adult human lung vary from 12.5% [111] to 25% [104] of dry weight. Desmosine and isodesmosine were undetectable by amino acid analysis during fetal life in one study [111] and showed no significant change with gestational age in another [76]. Chapter 6. DISCUSSION 156 In general, during embryogenesis elastin synthesis in the lung is associated with spe-cific developmental periods. In this study, during the canalicular stage of lung develop-ment in the human, the desmosine concentration was low. By the period of alveolarisation which occurs in late gestation, the desmosine concentration had increased dramatically and continued to increase through the postnatal period up to 2 years and may even extend beyond this period. Total desmosine content of the lung also showed a steady increase from late gestation into early postnatal periods indicating the accumulation of elastin with the growth of the organ. In general, the lung growth patterns obtained for the human and rat lungs seemed to be similar with regard to an increase in lung weight, total DNA, soluble protein, collagen and desmosine with age. They differed in the following respects: 1. Wet/dry lung weight ratio in the humans decreased rapidly from 20 weeks gestation until birth after which, it remained constant. In the rat lungs, the decrease in the ratio continued beyond birth upto 2 weeks of postnatal hfe. 2. DNA concentration in the human lungs decreased with conceptional age until birth but remained stable thereafter, while in the rat lung, the decrease continued postnatally until 5 weeks of age. 3. Soluble protein concentration in the human lungs did not show any change with conceptional age, while in the rat lungs, it decreased rapidly during gestation until term, and remained constant thereafter until 5 weeks of age. 4. In the human lung, collagen concentration increased rapidly from 20 weeks ges-tation until birth, after which the increase was more gradual. In the lungs of the rat, collagen concentration increased rapidly from day 18 of gestation to adult life. 5. Desmosine concentration in the human lung showed a rapid increase from 20 weeks of gestation into early postnatal life(childhood). Desmosine concentration in the rat lungs increased rapidly from day 21 of gestation until 4 weeks of age after which, it did not Chapter 6. DISCUSSION 157 increase any further with age. The above observations indicate that both collagen and desmosine make their appear-ance at an earlier stage of development in the human lung suggesting a higher degree of maturity in the human lung by birth when compared to the rat lung. 6.5 GROWTH PATTERNS IN THE LUNGS OF INFANTS WITH BRON-CHOPULMONARY DYSPLASIA Abnormalities of growth and maturation of the fetal lung are major contributors to peri-natal mortality. Diseases like HMD carries a significant mortality and morbidity among preterm infants despite modern neonatal intensive care. The state of lung development is a major factor in determining the survival of the babies born prematurely and the re-action of the lung to intensive care treatment. Although considerable research effort has been directed to problems of lung development, few studies have considered the quanti-tative aspects of lung growth [65]. Until recently there has been incomplete knowledge of such elementary qualitative details of human structural lung development as the time of appearance of alveoli [94],[118],[96]. An understanding of the process which underlie the changes seen in lung growth and development as in BPD, requires a knowledge of normal quantitative lung growth through gestation and postnatal period and establish a baseline for human lung growth on the basis of which abnormal lung growth can be assessed. When the lungs of the infants with BPD were analysed quantitatively, it was noted that the slope of the regression line for the lung weights vs. conceptional age for the BPD group, was significantly different from that obtained for the control lungs(p<0.001), with an Revalue of 0.66. The lungs in the BPD group were heavier than in the control infants in the corresponding age group. Though the lung weight showed an increase with Chapter 6. DISCUSSION 158 conceptional age there was a wide scatter at all ages and this increase was not linear. This could be attributed to the variable amount of edema and inflammation that was seen to be present in the lungs of infants with BPD and the amount of edema seemed to depend upon the severity of the disease. As a result of the variation in the lung weights, no set pattern of wet lung/dry lung weight ratios could be obtained for the BPD group. The slope of the body weight against conceptional age in the BPD group was lower than that obtained for the controls, indicating impaired general body growth of the infants in the diseased group. It was seen that the infants in the BPD group initially had body weights in the same range as the control group, but beyond one year of age they could not keep up with the body weight gain of the controls and attained lower body weights in comparison to the control infants of same age. Total DNA content as well as DNA concentration was higher in the BPD group than in the normals. Since this diseases is primarily a parenchymal disease, we must take into consideration the four cell types (alveolar type I, alveolar type II, endothelial cells and fibroblasts) as well as the connective tissue interstitium. During injury and desquamation, the relative percentage of the cell types as well as the number of cells will probably change. The increase in cell number could be due to increased number of lymphocytes and macrophages that infiltrate the injured area, as well as an increase in the fibroblasts in the interstitium. Soluble protein content in the lungs of BPD infants was also higher which is under-standable since there is high amount of edema present in these lungs due to the increased permeability of pulmonary capillaries causing the plasma proteins to spill into the inter-stitium and air spaces, which would contribute to the total soluble protein content of the lung. Protein concentration as in the normal group showed wide variation at all ages and did not show anjr pattern of development with age. "Fibrosis" is a morphologic term that translated into biochemical nomenclature means "an increase in collagen". Total collagen contents of the lungs in BPD group were higher Chapter 6. DISCUSSION 159 than the normal group as was the collagen concentration. BPD is a fibrotic disease and increased amount of collagen has been noted histologically [148],[205],[23] and therefore increased collagen content is expected in the lung. This has been shown in other fi-brotic diseases in animal models [57]. However, in earlier studies conducted in animal models [91],[167] as well as on human biopsies, the collagen concentrations (amount/dry weight) were found to be normal. Although the results obtained in the present study are in conflict with the previous studies, the discrepancies may be attributed to the following; BPD is a disease occurring during the rapid growth phase in an human infant and also at the time of rapid alveolarisation when vast amounts of connective tissue components are being synthesized. Thus, an injury to the lung at this phase could cause an aberra-tion in normal collagen synthesis by the fibroblasts which then would respond by either increased collagen production rate per cell or increased recruitment of fibroblasts for col-lagen production. The collagen contents may therefore increase at a higher rate than the increase in cellular content of the lung thus bringing about an increase in collagen concentration. There have been conflicting reports with regards to characterization of collagen in the lung parenchyma of fibrotic lungs and the relative ratios of type I/III collagens present in the lungs. In adult respiratory distress syndrome as well as some idiopathic pulmonary fibrotic (IPF) diseases, there seems to be a shift in the major collagens present such that there is more type I relative to type III [188],[122]. This observation is consistent with the observations that of all collagen types, collagen type I forms the least distensible collagen fibers in vivo and that patients with IPF have decreased lung compliance. However, recent studies of the lung in progressive systemic sclerosis have shown that the ratio of type I/III in the parenchyma is similar to that in normals, suggesting that an increased I/III ratio may not be a universal part of fibrosis of the interstitial lung disorder [187]. Still other observers from their immunohistochemical studies of human lungs argued that Chapter 6. DISCUSSION 160 there is an increase in type III collagen compared to type I during active fibrosis [14]. From all these reports one comes to a conclusion that the relative proportions of type I and III collagens are constantly changing as the disease progresses. In sites of established mature fibrous tissue, there may be more type I collagen while in areas of active fibrosis type III collagen may be more abundant. Thus, the role of different collagen types in fibrotic lung disease will probably vary with both the category and stage of each disease. The relative ratio of type I/III collagen in the lungs of BPD infants in the present study, was seen to be less than the ratio obtained for the normal infants. The ratio seemed to have a tendency to decrease with increasing postnatal age although the R 2 value for this pattern is quite low (0.12). This result is in conflict with an earlier published result by Shoemaker et al [190] on lungs of ventilated infants with RDS, where the}' showed an increase in the type I/III ratio. The arguments that are put forth in support of the present results are as follows; 1. The insult to the lung is occurring during the neonatal period at which time the normal t}rpe I/III ratio is lower than the adult value showing therefore a higher propor-tion of type III collagen being synthesized by the lung. It is probable that in response to inflammation and other stimuh, "invading" or proliferating fibroblasts increasingly synthesize more type III collagen. When the injury is caused, perhaps those fibroblasts which are synthesizing type III collagen, may either increase in number or synthesize more collagen per cell thus lowering the ratio. 2. There is evidence that exogenous or locally produced growth factors play a role in the fibrotic changes in human adult and fetal lung diseases. The human alveolar macrophage appears to release mediators such as, fibronectin, platelet derived growth fac-tor, basic fibroblast derived growth factor and other macrophage derived growth factors, which contribute to the fibrosis seen in granulomatous lung disease [101]. Macrophage activation has been postulated as one of the mechanisms contributing to the development Chapter 6. DISCUSSION 161 of BPD [103]. Fibronectin activated by the macrophage,stimulates resting fibroblasts to replicate [19]. Fibronectin is also seen to bind more avidly to type III collagen than to type I [85]. Therefore, presence of increased fibronectin in the lung interstitium would probably result in increased type III collagen synthesis by the fibroblasts. 3. Furthermore, the binding site of fibronectin on type III collagen molecule, cor-responds to the active site for mammalian collagenase, suggesting that the binding of fibronectin to type III collagen molecules may prevent its degradation by collagenase, thus causing an increase in the proportion of type III collagen in the lung interstitium. Total desmosine in the lungs of BPD infants as well as desmosine concentration were seen to increase sharply with conceptional age. They followed the same growth patterns as obtained in the normals except that the values in the BPD group were consistent!}' higher. This increase in desmosine could be due to an inadvertent increase in elastin synthesis by the same fibroblasts recruited for collagen synthesis, as the two synthetic processes are very similar. This increase in elastin is depicted by increased desmosine amounts which is indicative of mature cross linked elastin. The increased connective tissue components fit weD with the structural appearance of thick interalveolar septa and impaired alveolar formation in the disease group. Pathologically, the disease was seen to progress from an initial acute phase of HMD advancing into early BPD with some degenerative epithelial airway changes, increased fibroblast proliferation and minimal connective tissue deposition, on to an acute phase of the disease with widespread pulmonary fibrosis, and increased bronchial and bronchi-olar mucosal lesions leading to diminished alveolarisation and pulmonary dysfunction. The striking feature of this disease is that when an infant lives past the acute phase of the disease the lung apparently goes through a phase of regeneration and repair of the parenchyma. It may then go through two pathways:-1. It may go through a healing process to emerge as a normally functioning organ, Chapter 6. DISCUSSION 162 leading to the survival of the infant 2. The lung may be incapable of proper repair due to the extent of the injury, leading to the death of the infant. These two pathways were seen to occur in the infants in the BPD IV and healed BPD grades of our first pathological classification. The lungs of infants in BPD IV group showed evidence of decreased fibrosis, decreased number of fibroblasts and showed that the lungs were in the process of repair. Despite all this evidence of healing in the lungs, the infants in this group died due to lung injury, indicating perhaps, that the lungs were incapable of proper repair and restoration of normal architecture of the lung parenchyma, due to the extensive damage suffered by the lung. Infants in the healed BPD group, on the other hand, had lungs with relatively normal architecture of the parenchyma with the air spaces having more regular appearance and thin interstitial walls. One case in this group showed complete healing and was mistaken for a control lung by the examiner, when conducting a blind histological examination of the BPD and control cases. This indicates that the lungs are capable of complete healing after going through the phase of acute disease. It should be mentioned here again that none of the infants in the healed BPD group died due to BPD, but due to other causes. It should be emphasized here that in this study, all the morphological and biochemical analyses were conducted on autopsy specimens which represent the severe end of the disease spectrum. Therefore an infant who died in BPD I stages of the disease, died due to the severity of the HMD or other acute disease causing the respirator}' distress in the infant and thus represents the severe end of the BPD I stage. The same is the case with infants in the BPD II and III and IV groups. All these infants represent the severe end of their corresponding stages. It is not necessarily true that had the infants in the BPD I stage lived, the}' would have gone through all the other stages of BPD. How far the disease would progress in an infant is probably dependent on the degree of prematurity Chapter 6. DISCUSSION 163 of the infant; the severity of the initial disease and the agressiveness of the supportive care given to the infant. When a correlative study was performed using biochemical, clinical and morphological parameters, interesting results were obtained for the different grades of the disease. First of all, gestational age of the infant did not seem to predict how far the disease would progress in the infant as the mean gestational ages of infants in the four disease categories did not differ significantly. The disease progression seemed to show some dependence on the number of days the infant survived postnatally, although some infants in our study, showed BPD grade II and III type of lesions as early as 1 week of postnatal age, while one infant was in the healing phase of BPD by 9 weeks of postnatal age. DNA content per unit lung mass was highest in BPD I category indicating increased cellular infiltration and proliferation during this stage along with little connective tissue deposition. The DNA concentration decreased with progression of the disease indicating an increase in the extracellular components of the lung tissue. Soluble protein concentrations remained close to the normal values in all the stages of the disease which was surprising since one would expect to see higher soluble protein concentrations due to the high amount of edema as well as haemorrhage noted in the lungs of the BPD infants. Collagen concentrations were seen to double from BPD I to II stage indicating a rapid rate of collagen accumulation in stage II, indicative of fibrotic process and complements the morphologic appearance of the thickened interalveolar septa and thickened intersti-tium with connective tissue fibers. The collagen concentration remained about the same in BPD III and IV stages which indicates that maximum amount of accumulation of col-lagen occurred in stage II BPD and by stage III some amount of repair and regenerative processes may have begun resulting in no further increase in collagen. This possibility was indicated in the morphology of lungs in the BPD III stage which showed variable degree of fibrosis ranging from severe to very little with fairly thin walls dividing the air Chapter 6. DISCUSSION 164 spaces. When the infants in the four BPD groups were compared to their age matched con-trols, it was seen that collagen concentration was significantly higher in BPD II and III groups , indicating that infants in BPD III, still had abnormal amounts of collagen in their lungs compared to the control lungs, despite the fact that there was no significant increase in collagen amounts from BPD II to III grades. Lungs of infants in BPD IV group did not differ significantly in their collagen concentration when compared to their age matched controls, indicating a decrease in the abnormal deposition of collagen. Desmosine concentration was seen to increase slightly from BPD I to BPD II stage but was seen to almost double by BPD III stage. In general, when the entire BPD group was compared with the control group, the patterns obtained showed the BPD group as having higher demosine content which may be attributed to an inadvertent increase in elastin synthesis by fibroblasts which are synthesizing collagen in higher amounts. But when individual groups of BPD were compared with their age-matched controls an interesting pattern was obtained which showed that the increase seen in desmosine contents may not be a result of fibrotic processes occurring, as during the rapid accumulation of collagen in stage II, very little desmosine is accumulated. By stage III there is a rapid increase in elastin accumulation as indicated by the increase in desmosine amounts which might have occurred as a response to the reparative processes occurring in the lung and also due to a possible increase in alveolarization. Desmosine concentration was seen to increase 1.5 times by stage IV thus providing basis to our theory that elastin content was perhaps increasing in response to the healing processes of the lung and not due to fibrosis. When compared to their age matched controls, desmosine concentration in the lungs of infants in BPD I and II groups did not differ significantly. Infants in BPD III group had significantly higher amounts when compared to their age matched controls and this increase persisted in BPD IV. This lends credence to the theory that the increase in Chapter 6. DISCUSSION •165 desmosine concentration in BPD lungs is probably more due to the response by the lung to the reparative mechanisms, in its effort to structurally and functionally return to normal, and not due to the disease process itself. Ratio of collagen types I/III showed an interesting pattern with progression of the disease. Although the mean ratio obtained for infants in BPD I group was not signif-icantly different from the values obtained for the lungs of age matched control infants, they tended to be lower. As the disease progressed to stage II the ratio showed a signif-icant decrease and continued to show a significant decrease through stages III and IV. These results indicate a higher proportion of type III collagen in the lung parenchyma and this trend seemed to continue through all the stages of the disease including the stage of healing and repair (stage IV). In reference to the the earlier part of the discussion regarding the low mean values for I/III ratio obtained for the entire BPD group when compared to the normal group the various probable causes for this occurrence were:- 1) Increased recruitment of fibroblasts synthesizing type III collagen to the injured area. 2) Increased synthesis of type III collagen per cell. 3) Increased production of fibronectin during injury which binds to type III collagen resulting in decreased degradation of type III collagen by collagenase. As seen from the results obtained for the infants in stage II, while the collagen contents (per unit lung mass) had doubled in the lungs, the type I/III ratio was seen to decrease. This may indicate that there was an increased rate of type III collagen synthesis as compared to type I or there is a decrease in the rate of degradation of type III collagen, as the newly synthesized collagen are most susceptible to the degradative processes in the lung. In stage II BPD, clearly a high amount of synthesis must be occurring as indicated by an increased number of fibroblasts in the interstitium and increased collagen amounts. Here, probably an increased synthesis of type III collagen by fibroblasts is the cause of the decrease in the ratio. Chapter 6. DISCUSSION 166 An interesting possibility is that this fibrotic process occurring in the neonates may be a response by the lung tissue components to an injury to the lung caused by either an intrinsic or an extrinsic factor, much like the process of healing of a tissue when a wound is inflicted. The first response after a wound is inflicted is the formation of granulation tis-sue which is seen to consist of similar components as the fibrotic lung tissue in the neonates [8]. There are numerous reports of increased type III/I ratios in early granula-tion tissue [8],[48],[139]. Similar results were recently reported for acute post-traumatic pulmonary fibrosis (ARDS) where the authors found a higher proportion of type III col-lagen to type I in the lungs they examined [146]. They supported the theory that acute post-traumatic pulmonary fibrosis resembled a wound healing process in the lungs. In recent investigations, it has also been suggested that early active fibrosis consisted of enhanced type III collagen deposition [126],[114]. In our study, as the disease entered into grades III and IV BPD, the rate of collagen accumulation decreased but the ratio continued to be low. Perhaps, here the regenerative and repair processes of healing may have taken over, and the newly synthesized collagen produced in excess during stage II of the disease may have become susceptible to the degradative enzymes such as collagenese present in the lung tissue. This decrease in the fibrotic tissue can be witnessed morpho-logically in the lungs of stage III and IV BPD. Since fibronectin binds to the active site of collagenese on the type III collagen molecules, it protects the type III collagen from the degradative action of collagenase, while type I is left unprotected and may be degraded more in comparison to type III, and this process perhaps lowers the ratio further. Thus, from the morphological and biochemical point of view it is seen that the lungs of infants with BPD are severely compromised structurally and functionally. Since the time BPD was first discovered by Northway et al, in the newborns requiring assisted ventilation and oxygen therapy for RDS, a recurring question has been whether the Chapter 6. DISCUSSION 167 pulmonary changes observed in these infants are caused by the therapeutic use of high concentrations of oxygen or the mechanical ventilation that the infants are subjected to. Investigators have been roughly divided half and half in support of one of these two factors as the main causative agent in inciting the onset of the disease. Some evidence based on human and animal experimental studies has been accumu-lated over a few years indicating that oxygen and not artificial ventilation is the main cause of the morphologic alterations observed in the syndrome of BPD [23], [6]. In ex-perimental animals an increase in the number of fibroblasts and collagen has been shown electron microscopically in animals exposed to concentrations of oxygen over 80% for 7-12 days [107],[183]. In one of these studies on monkeys, a significant increase in fibrob-lasts was accompanied by an abundance of collagen deposition after 12 days of oxygen exposure [107]. In a recent study conducted on ventilated premature baboons and oxy-gen exposed adult hampsters [105], exposure of both the premature and adult lung to high concentrations of oxygen caused the elaboration of a growth factor that may induce fibroblast hyperplasia. This action may result in an increased production of connective tissue proteins and thereby contribute to the development of the fibrosis seen in BPD. Light and electron microscopic findings in human neonates [23] indicated that infants with severe RDS treated over a prolonged period of time with high concentrations of oxygen and mechanical ventilation eventually developed ciliary damage, bronchial and bronchiolar injury and widespread pulmonary fibrosis which severely compromised the cardiopulmonary status in the later life of the survivors. These changes were more pro-nounced in infants who survived the longest period of time and they developed symptoms and signs of cardiac atrial or ventricular stress including cor pulmonale, prior to their demise. These infants had the longest exposure to supplemental oxygen and showed histopathologically severe pulmonary fibrosis and emphysema. The recent development of newer ventilatory techniques for premature neonates, has Chapter 6. DISCUSSION 168 reduced the use of high concentrations (80 to 100%) of inspired oxygen as therapy for respiratory difficulty in the new born. With this reduction, there has been a concomitant increase in the use of concentrations of oxygen in the middle range (40-80%). The reduction in the inspired oxygen concentration however, seems to have resulted in little or no reduction in incidence of BPD which suggests the possibility that supplemental oxygen therapy may play a lesser role in the development of the disease than originahy postulated. Another point to consider is that compared to earher years the population receiving assisted ventilation presently consists of neonates with a higher degree of prematurity who may have an increased sensitivity to oxygen so that any benefit resulting from the lowering of inspired oxygen concentrations may be obscured. The toxicity of oxygen is known to be related to the concentrations of oxygen used, the duration of exposure and the susceptibihty of the subject. In the clinical situation, the duration of a given oxygen concentration is quite variable. The concentration used is varied according to the patient's need. How susceptibility to oxygen varies with degrees of immaturity is unknown. AU of these factors complicate any analysis of the role of oxygen toxicity in the development of BPD. Assisted ventilation is the other potential mechanism of lung injury for the premature infant. The response of developing immature lung components to filling with ambient inhaled gas and the implications of that response to the development of BPD have been the subject of discussion for many years. Indeed, there is evidence to support the hy-pothesis that assisted ventilation causes lung damage [205],[18],[168],[161]. One of these studies showed striking lesions in the bronchioles and terminal airways often leading to obliteration of the lumens in infants ventilated for 15 days [205]. The correlation between the presence of this type of florid lesions and the use of peak airway pressures was found to be highly significant. They suggested that the main factor initiating the process is distortion and disruption of terminal airways caused by the use of very high peak airway Chapter 6. DISCUSSION 169 pressures at a time when the air saccules are difficult or impossible to inflate because of surfactant deficiency. The immature lung is probably more subject to mechanical disruption than a ma-ture lung because the immature lung is a heterogeneous collection of airways and distal respiratory units [115]. This nonhomogeneity allows some units to be well ventilated initially, while other units remain unventilated. Those that are easily ventilated at the onset probably are exposed to relatively large volume and velocity changes, factors that probably cause injury. In the presence of natural surfactant, 27 day rabbits developed little small airway damage, whereas, rabbits ventilated without natural surfactant developed small airway damage within 10 minutes [115]. The marked damage seen one hour after ventilation was begun seemed to exclude oxygen toxicity as a cause. It was the investigators suggestion that in dealing with the very small premature infant who requires resuscitation, one may damage the lung by mechanical ventilation in the early period of life and then superimpose oxygen toxicity. In the present study, from the discriminant analysis conducted on the data collected it was seen that 60% of the infants were predicted correctly in the appropriate category of the disease when the days on ventilator was used as the independent variable, while only 40% were predicted correctly when days on oxygen >60% concentration was used as the independent variable. From these results alone, it is difficult to pinpoint ventilator days as the primary inciting agent in the pathogenesis of BPD, although it does predict the disease category with more accuracy than days on 0 2 >60%. When considered together in the analysis, it was seen that the accuracy of prediction increased to 70% indicating an interaction between the two treatments. From the clinical analysis it was seen that, the infants in BPD I.II and III groups are ventilated and oxygenated for most of their postnatal lives. Infants in BPD IV group on the other hand, were ventilated Chapter 6. DISCUSSION 170 for only 25% of their lives, suggesting that the infants in BPD IV group were subjected to less barotrauma in comparison to other groups. These infants were also subjected to high concentrations of oxygen for only 4% of their lives. These were the group of infants who were healed from BPD or were in the process of healing as indicated by their morphology.This raises an important question as to whether these infants' lungs were capable of healing because they were not subjected to continuous periods of therapeutic care for most of their lives as was the case with infants in the other three groups. If that is so, then which of the two treatments is more detrimental to the infant and causes the emergence of BPD? It is clear from the clinical data as well as from discriminant analysis, that the progression of the disease is to a certain extent dependent on the mechanical ventilation and oxygen supplementation that the infant is subjected to, but from the data we have, it is very difficult to assess the individual contributing roles of the two treatments as they are generally administered together to the infants. Although, it is difficult to pinpoint one of the two factors (oxygen toxicity, baro-trauma) as being solely responsible for the emergence of BPD, it is possible that the injury caused by barotrauma to the immature lung is augmented by the high concen-trations of oxygen administered. Perhaps, the mechanism is as follows: the ventilation of the immature lung causes injury to the lung due to barotrauma. The oxygen being administered may suppress cell proliferation and division so that repair mechanisms of the lung are altered. Such an effect is different from toxicity, but it permits the evidence of injury to accumulate because of poor repair. The fibroblast might have an opportunity to proliferate while other cells are suppressed. It has been shown that oxygen toxicity suppresses the endothelium while the interstitium appears to have considerable growth. Such an effect would result in pulmonary fibrosis [56]. A high degree of correlation between the disease progression, ventilator days, O2>60% days and the collagen concentration in the lungs was seen when the discriminant analysis Chapter 6. DISCUSSION 171 was performed with all the above variables as well as conceptional age of the infant. These variables when taken together for the analysis, improved the prediction of each case to the appropriate disease category and showed 83% accuracy in prediction. This shows that pulmonary fibrosis with excessive collagen accumulation is an integral part of this disease and this fibrotic process seems to correlate very significantly with assisted ventilation and (^supplementation, but more so with the assisted ventilation provided to the infant. Infants with "healed" BPD As mentioned in chapter 3, one set of infants were classified as "healed" BPD. These infants in general had lived the longest among all the infants in the BPD group. The air spaces in the lungs had a more regular appearance, the interstitial walls were much thinner and fibrosis was slight and focal. Some amount of squamous cell metaplasia was still present in some areas but in most areas the epithelium was normal(ciliated, pseudostratified). Thus the lungs seemed to be progressing towards normalcy through repair and restructuring. All these infants were diagnosed as having BPD early in life for which they were given supportive care. Thej' were then weaned from both assisted ventilation and (^supplementation sometime during their life but required intermittent hospitalization and supportive care. From their clinical histories it was noted that they were mostly given supplementary 0"2of concentrations between 21% and 60% and were not subjected to high concentrations of oxygen(>60%) for long periods of time. They all died of causes other than BPD. From this group of 7 infants only one showed histological evidence of pulmonary hypertension. Two infants were diagnosed as having congenital heart disease and died of congestive heart failure, but showed no evidence of pulmonary hypertension. Two infants died of extensive bronchopneumonia, one died of necrotizing enterocolitis, while two infants were diagnosed as SIDS (sudden infant death syndrome). Chapter 6. DISCUSSION 172 It is not certain why the lungs of these infants were able to undergo the healing process. Stocker et al [203], from their work on longstanding "healed" BPD, hypothesized that necrotizing bronchiolitis with occlusions of the brochiolar lumen by inflammatory debris, which occurs during acute stages of BPD, may play a role in determining the extent and distribution of the lesions seen in long standing healed BPD and "protecting" the parenchyma distal to the occlusion from the high ventilatory pressures as well as the direct toxic effects of the high oxygen tensions also used. If the infant survives the acute stages of BPD and resolution begins, the bronchioles become cleared and recanalised and the undamaged or less severely damaged "downstream" parenchyma can resume its role in oxygenation. These areas may represent the normal or hyperexpanded areas in which little or no septal fibrosis is present. This hypothesis has not been substantiated with experimental studies and further studies are required before we can understand the mechanisms involved in the healing processes following BPD in the lungs. Relationship between BPD and SIDS Lastly, it was interesting to find a few infants in the healed BPD group were diagnosed as having died of SIDS. An association between BPD and SIDS was suggested by Wertham-mer et al [227] who mentioned that "the incidence of sudden infant death syndrome was 7 times greater in infants with BPD when compared with a group of control infants without BPD". Werthammer et al while stating that all of the patients with SIDS-BPD showed histologic evidence of resolving BPD, based the diagnosis of SIDS on the fact that at autopsy "other known causes of death" were excluded. It is our feeling that the sudden or unexpected death in these infants is not really related to SIDS but perhaps reflects a change in the pulmonary status of the infants with a severely compromised pulmonary reserve. A mild viral or bacterial infection may be all that is necessary to produce sudden pulmonary or cardiac deterioration in these infants who often require Chapter 6. DISCUSSION 173 intermittent ventilation and supplemental C t^o maintain adequate oxygenation. If such an episode should occur during a sleeping period and is therefore unobserved, the death may appear to be similar to those resulting from SIDS. That these infants are prone to infections was evident by the deaths of two infants in this group being caused by bronchopneumonia. Chapter 7 CONCLUSIONS The biochemical aspects of intrauterine and postnatal growth and development of the lung have been studied in the rat as well in the human infant and attempts have been made to compare the growth patterns obtained in the two species. The lung growth patterns obtained for the human and rat lungs seem to be similar with regard to a substantial increase in body weight and lung weight from late gestation into postnatal hfe in both species as well as a substantial increase in the total lung con-tents of DNA, soluble protein, collagen and desmosine with age, and this trend continued well into postnatal hfe. The growth patterns in the two species differed in the following respects 1. Wet/dry lung weight ratio in the humans decreased rapidly from 20 weeks gestation until birth after which, it remained constant. In the rat lungs, the decrease in the ratio continued beyond birth up to 2 weeks of postnatal life. 2. DNA concentration in the human lungs decreased with conceptional age until birth but remained stable thereafter, while in the rat lung, the decrease continued postnatally until 5 weeks of age. 3. Soluble protein concentration in the human lungs did not show any change with conceptional age, while in the rat lungs, it decreased rapidly during gestation until term, and remained constant thereafter until 5 weeks of age. 4. In the human lung, collagen concentration increased rapidly from 20 weeks ges-tation until birth, after which the increase was more gradual. In the lungs of the rat, 174 Chapter 7. CONCLUSIONS 175 collagen concentration increased rapidly from day 18 of gestation to adult life. 5. Desmosine concentration in the human lung showed a rapid increase from 20 weeks gestation into early postnatal life(childhood). Desmosine concentration in the rat lungs increased rapidly from day 21 of gestation until 4 weeks of age after which, it did not increase any further with age. The above observations indicate that both collagen and desmosine make their ap-pearance at an earlier stage of development in the human lung in comparison to the rat lung, suggesting a higher degree of maturity in the human lung at the time of birth as compared to the rat lung. This was further substantiated by the observation that the decrease in DNA concentration as well as the wet lung/dry lung weight ratio with age in the human lung stabilized soon after birth. Study of postpneumonectomy compensatory lung growth following pneumonectomy in rats at 4 weeks of age provided two important observations; 1. With regards to the biochemical parameters (except for lung weight) and lung mechanics, there seemed to be total compensation for the loss of the left lung and this compensatory growth appeared to mimic normal lung growth. 2. However, when the lung was looked at morphologically it showed evidence of enlargement of existing structures as well as some amount of alveolar multiplication as a response to the surgerjr. Administration of BAPN to rats from 4 to 7 weeks of age resulted in lower lung weights and decreased DNA, soluble protein, collagen and desmosine contents in the lungs with the effect being more profound on the levels of desmosine when compared to other biochemical parameters. With regard to lung mechanics there was increase in lung volume and diminished elastic recoil which was concommitant to the decrease in desmosine and insoluble collagen levels. This suggsts that the mechanical changes observed in the lung may have been due to the alteration in tissue forces caused by the Chapter 7. CONCLUSIONS 176 impaired crosslinking of elastin and collagen. Results of morphometric analysis did not show significant differences between the BAPN administered and control groups. There seemed to be a minimal effect of BAPN on post-pneumonectomy lung growth. This effect was seen biochemically as a decrease in connective tissue content and matu-ration. The effect was seen in lung mechanics as well with an increase in lung volume and TLC. BAPN did not affect lung morphology as judged by morphometric analysis. This perhaps is due to the fact that pneumonectomy was performed at 4 weeks of age in the rats by which time the phase of rapid alveolarization would have been completed and the compensatory growth results in an enlargement of existing structures and incomplete restoration of the alveolar number. When BAPN is administered at this point it would affect only the newly synthesized connective tissue proteins which may have only a slight effect on the general morphology of the lung parenchyma and morphometric analysis may not be sensitive enough. The infants with BPD were divided into 4 groups. Group I had an average postnatal age of 3 days and represented the acute phase of lung injury. Groups II and III had average postnatal ages of 26 days and 18.5 weeks respectively and represented the pro-liferative phase of the disease. Group IV BPD had an average postnatal age of 36 weeks and in this group, the lungs tended to return to normal. It was seen that the infants with BPD who lived beyond 8 months of postnatal hfe, had impaired body growth when compared to control infants. In terms of biochemical changes, the lungs of BPD infants had higher DNA, soluble protein, collagen and desmosine contents as well as increased concentrations of DNA, collagen and desmosine in their lungs when compared to the growth patterns obtained for the lung of control infants. There was increased concen-tration of DNA in the lungs suggesting increased fibroblast proliferation, epithehal cell proliferation as well as infiltration of inflammatory cells into the lung tissue. Evidence of fibrosis was seen by a significant increase in collagen concentration in BPD II and III Chapter 7. CONCLUSIONS 177 groups when compared to age matched controls. Desmosine concentration oh the other hand, did not show an increase in BPD II group when compared to age matched controls but was seen to increase in groups III and IV suggesting that a different mechansim other than the one controlling collagen production, was involved in bringing about the increase in desmosine in the BPD lungs. Collagen type I/III ratio was seen to decrease in BPD II, III and IV groups in comparison to age matched controls, indicating a higher proportion of type III collagen in the lungs of infants with BPD. Clinical studies showed BPD infants as having heavier lungs compared to the controls which can be attributed to the large amounts of edema and variable amounts of haemor-rhage seen in the lungs of BPD infants. 50% of the BPD infants in our study had birth weights less than 1 kg, while 35% had birth weights between 1 and 1.5 kg. Infants in BPD I, II and III groups were ventilated and administered high oxygen concentrations for most of their lives, while the infants in BPD IV group which included the healed BPD infants, were ventilated for only 25% of their lives and given high oxygen for 4% of their lives. From the discriminant analysis and clinical data obtained, it was seen that the dis-ease progression was dependent on the two treatments (ventilation and high oxygen supplementation) rendered to the infants, but it was difficult to assess their individual contributions to the development of BPD. The other variables such as severity of the initial disease and the length of survival of the infant make the assessment of individual contributions that much more difficult. There seemed to be a high degree of correlation between the disease progression and collagen accumulation in the lungs indicating that pulmonary fibrosis with excessive collagen accumulation is an integral part of this disease and this fibrotic process seemed to correlate very significantly with assisted ventilation and high 0 2 supplementation administered to the infants. Chapter 7. CONCLUSIONS 178 7.1 RECOMMENDATIONS FOR F U T U R E WORK In this thesis we have presented preliminary data with regard to BPD and have perhaps come a step closer to understanding the pathogenesis of the disease. Based on these findings further studies are required to define the mechanisms involved in the emergence of the disease. Using the techniques of molecular and cell biology one can begin to understand the mechanism of control of collagen production in bronchopulmonary dysplasia. This can be done by (a) Determining if the amount of mRNA committed to type I and III collagen pro-duction changes during the disease process resulting in a decrease in the ratio of type I/III collagen that was observed in the lungs of these infants. (b) Determining if there is alteration in the transcription of procollagen in the fibrob-lasts fines derived from lungs of infants with BPD such that more of type III procollagen molecules are transcribed in comparison to type I procollagen peptides. _ . ~ (c) Determining if there is heterogeneity existing in the fibroblast lines derived from the lungs of infants with BPD; in other words, if there is a special hne of fibroblasts which has been recruited by the disease mechanisms to synthesize only type III collagen. (d) Studying the activity of the enzymes involved in the synthesis and degradation processes of type I and III collagens. Once these mechanisms are understood it should be possible to identify diugs which are specific in their site of action. This way, we may be able to induce those mechanisms which are involved in maintaining the lung parenchjrmal structure, to restore the lung to normal. Bibliography Ackerman N.B.Jr., Coalson J.J., Kuehl T.J., Stoddard R., Minnick L., Delamos R., Pulmonary interstitial emphysema in the premature baboon with hyaline membrane disease, Crit.Care.Med. 12 (6): pp512-516, 1984. Alcorn D., Adamson T.M. , Malonejr J.E., Robinson P.M., A morphological and morphometric analysis of fetal lung development in sheep, Anat. Rec. 201: pp655-667, 1981. Amy R.W.M., Bowes D., Burri P.H., Haines J., Thurlbeck W.M., Postnatal growth in the mouse lung. J.Anat. 124: 131-151, 1977. 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