UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Effects of loss of amniotic fluid on lung growth and maturation in rat fetuses Blachford, Karen Grace 1985

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1985_A6_7 B53.pdf [ 4.17MB ]
Metadata
JSON: 831-1.0095997.json
JSON-LD: 831-1.0095997-ld.json
RDF/XML (Pretty): 831-1.0095997-rdf.xml
RDF/JSON: 831-1.0095997-rdf.json
Turtle: 831-1.0095997-turtle.txt
N-Triples: 831-1.0095997-rdf-ntriples.txt
Original Record: 831-1.0095997-source.json
Full Text
831-1.0095997-fulltext.txt
Citation
831-1.0095997.ris

Full Text

EFFECTS OF LOSS OF AMNIOTIC FLUID ON LUNG GROWTH AND MATURATION IN RAT FETUSES by KAREN GRACE BLACHFORD B.Sc, The University of Manitoba, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept this thesis as conforming to the Featured standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1985 © Karen Grace Blachford, 1985 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may"be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f ^nc±JhxJLo, The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 - i i -ABSTRACT This study was designed to examine the hypothesis that the amount of amniotic fluid present during gestation is critical to normal lung growth and maturation. On day 16 of gestation the amniotic sacs of the right or left uterine horns of timed pregnant Sprague-Dawley rats were punctured with a 20 gauge needle. The fetuses of the opposite horn served as controls. On day 21 of gestation (one day prior to natural delivery) the fetuses were delivered by Cesarean section. An unbalanced, mixed model analysis of variance was performed on the data collected from each fetus. Probability values of less than 0.05 between control and experimental animals were considered significant. Amniotic sac puncture resulted in a significant loss of amniotic fluid as indicated by reduced amniotic fluid volume on day 21. Experimental body weight was significantly reduced indicating fetal growth retardation. Lung growth was also retarded as indicated by significantly reduced lung weight to body weight ratios and lung volume to body weight ratios following amniotic sac puncture. There was a reduction in the amount of fluid present within the experimental lungs. There appeared to be no significant effect on the structural units of the lung as indicated by no significant difference between control and experimental fetal lungs in terms of cell number, cell size, total protein to body weight ratio, maturation of type II cells, volume fraction of saccular air, saccular wall, conducting air and nonparenchyma, airspace size, saccular surface area to body weight ratio and surface to volume ratio. Thus, loss of amniotic fluid significantly affected lung growth, more than i t affected overall body growth, without having an effect on lung maturation. - i i i -TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES vi LIST OF FIGURES v i i DEDICATION v i i i ACKNOWLEDGEMENTS ix ABBREVIATIONS x INTRODUCTION 1 I. Objective 1 II . Lung Development, Growth and Maturation 1 a) General introduction 1 b) Intrauterine lung development 2 c) Differentiation of respiratory epithelium 6 d) Lung growth and maturation 9 II I . Control of Lung Development 12 a) Early intrauterine 12 b) Late intrauterine 14 IV. Midtrimester Amniocentesis 22 V. Rationale for the Present Investigation 26 VI. Morphometry 27 - iv -MATERIALS AND METHODS 28 I. Materials 28 II . Methods 28 a) Experimental protocol 28 b) General research design 30 c) Extraction of non-connective tissue protein, DNA, phospholipid and disaturated phosphatidylcholine 30 d) Estimation of non-connective tissue protein content 36 e) Estimation of DNA content 37 f) Estimation of phospholipid content 38 g) Lung fixation 39 h) Estimation of fixed lung volume 40 i ) Lung sampling and processing 40 j) Estimation of shrinkage 41 k) Light microscopy morphometry 42 1) Light microscopy morphology 49 m) S t a t i s t i c a l analysis 50 RESULTS 50 I. General Characteristics 50 a) Expression of results 50 b) Amniotic f l u i d volume 51 c) Body weight 51 II . Biochemistry 52 a) Body weight 52 b) Wet lung weight 52 - V -c) Dry lung weight 52 d) DNA content 53 e) NCT protein content 54 f) Phospholipid and disaturated phosphatidylcholine content 54 III. Morphometry 58 a) Body weight 58 b) Fixed lung volume 58 c) Volume fraction and total volume of tissue components 59 d) Mean linear intercept 59 e) Saccular surface area 60 IV. Morphology 62 DISCUSSION 62 I. General Points 62 II. Summary of Results 64 III. Comparison with Related Work 68 IV. Relevance to Human Midtrimester Amniocentesis 71 BIBLIOGRAPHY 74 - vi -LIST OF TABLES TABLE PAGE I. Calculation of morphometric parameters of lung growth and maturation 48 II . Mean amniotic fl u i d volumes and mean body weights for control and experimental groups 51 I I I . Biochemistry results for control and experimental groups 57 IV. Morphometry results for control and experimental groups 61 - v i i -LIST OF FIGURES FIGURE PAGE 1. Extraction of NCT protein and DNA 33 2. Extraction of NCT protein, DNA, phospholipid and disaturated phosphatidylcholine 34 3. Test grid used for light microscopy morphometry 44 4. Illustration of intercept counting Rule 1 45 5. Illustration of intercept counting Rule 2 • 46 6. Illustration of intercept counting Rule 3 47 - v i i i -DEDICATION To my husband and friend, Bart, whose love, support and encouragement made this possible - ix -ACKNOWLEDGEMENTS A special thank-you to my supervisor, Dr. W.M. Thurlbeck, for believing in me and providing me with the opportunity to learn about research and to gain some valuable laboratory s k i l l s under the guidance of such a well-known and respected researcher. Your advice and constructive criticism helped me through every step of this study. My gratitude i s also extended to my committee members, Dr. J.L. Wright, Dr. J.C. Hogg, Dr. A. Churg and Dr. P. Hahn, for their constructive criticism, support and encouragement throughout this study. I would li k e to extend sincere thanks to my coworkers and friends, Kate Ross, Nicola O'Connor, Craig Smith, Mary Batt e l l , Norinder Thiara, Dr. Atsushi Nagai, Dr. Felix Ofulue, Dr. Larry Paul and Kim Simpson who have provided me with technical assistance and advice, and continual support and encouragement. A special thanks to Glenn Dong, a medical student, who performed the pilot work for this study, and to Anne Bishop and staff of the Academic Pathology General Office, University of British Columbia, for invaluable assistance in typing this thesis. - X -ABBREVIATIONS BSA = bovine serum albumin CC1. = carbon tetrachloride 4 CHC13 = chloroform CHJ3H = methanol DNA = deoxyribonucleic acid DSPC = disaturated phosphatidylcholine DWL = dry lung weight HCl = hydrochloric acid KC1 = potassium chloride = mean linear intercept L T = total projected length of grid test lines N 2 = nitrogen NaOH = sodium hydroxide NCT = non-connective tissue NH^ OH = ammonium hydroxide OsO^  = osmium tetroxide Pj. = inorganic phosphorus RNA = ribonucleic acid Sy w = gas exchanging surface to volume ratio S = saccular surface area w TCA = trichloroacetic acid V L = fixed lung volume VVa = v o ^ u m e fraction of saccular air VVb = vo-'-ume f a c t i o n of conducting air VVnp = vo-'-ume fraction of nonparenchyma V V w = volume fraction of saccular wall Wfa = body weight W. = wet lung weight - 1 -INTRODUCTION I. Objective The purpose of the research described in this thesis was to test the hypothesis that loss of amniotic flu i d on day 16 of gestation in the Sprague-Dawley rat causes pulmonary hypoplasia. Day 16 of gestation in the rat corresponds to the glandular stage of fetal lung development according to Burri and Weibel (1977) and Dr. Will Blackburn (Farrell, 1982). This finding i s in agreement with personal observations of day 16 gestation Sprague-Dawley rats. The relevance of the findings to human midtrimester amniocentesis w i l l be discussed. II . Lung Development, Growth and Maturation a) General introduction Studies of lung development to date have shown that, with certain exceptions, major aspects of lung development are similar in general terms in a l l mammals, although the timing of events varies from species to species. For example, rat fetuses enter the terminal sac period at 90-95% of gestation while the human shows respiratory saccules at 60% of term (Farrell, 1982). As well, both rats and man undergo a period of rapid alveolar formation, but these events, which take place during the f i r s t few weeks of l i f e in the rat (Burri et a l . , 1974; Burri, 1974) occur in late gestation and the f i r s t few years of l i f e in man (Dunnill, 1962; Thurlbeck, 1982). - 2 -b) Intrauterine lung development The lung develops from the laryngotracheal groove in the endodermal tube. In humans, the ventral groove appears at 26 days gestation and evaginates to form the lung bud. The lung bud, which is covered by a mesodermal layer, branches at 26-28 days gestation. Differentiation of the human respiratory system has recently been divided into five phases (Langston et al., 1984): (1) the embryonic phase, comprising the first 6 weeks after conception and leading to initiation of airways formation; (2) the pseudoglandular phase from 6 to 16 weeks of gestation, during which time formation of the conducting airways and pre-acinar arteries is completed; (3) the canalicular phase which begins at the 17th week, with development of the acinar region (future air sacs) and the acinar blood vessels, and continues to 28 weeks gestation; (4) the saccular phase from 28 to 36 weeks of gestation, during which time the saccules are subdivided by secondary crests and surfactant appears in the lung; (5) the alveolar phase, which is initiated at 36 weeks gestation and continues until approximately 2 years of age. Human gestation is approximately 40 weeks from the beginning of the last menstrual period, with a range between 37 and 42 weeks (Niswander, 1981). It should be noted that these five phases of lung differentiation are not sharply defined but overlap and vary somewhat in timing among individuals. Dunnill found that the number of alveoli increases over tenfold between birth and adult l i f e with the increase occurring mainly in the first eight years of l i f e (Dunnill, 1962). A similar pattern of postnatal alveolar multiplication was found by Davies and Reid (1970). Boyden feels that at birth one cannot estimate the number of alveoli because - 3 -they are too shallow and small to be counted by the intercept method. He feels Dunnill's (1962) estimate of 24 million alveoli (at birth) may in fact represent saccules, since saccules are the respiratory units of the next larger order of magnitude (Boyden, 1977). During the embryonic phase of lung development the proximal part of the bronchial tree, consisting of the main and lobar bronchi, is formed. The bronchial buds continue to subdivide by asymmetric dichotomy during the pseudoglandular phase. A study of intrasegmental bronchial tree development (beginning with the segmental bronchus and ending with the terminal bronchiole) revealed that 65-75% of the bronchial branching occurs between the 10th and 14th weeks of fetal l i f e and by 16 weeks bronchial and bronchiolar development are complete as far as the terminal bronchioles (Bucher and Reid, 1961). At this stage, histologic sections of the lung have a distinctly glandular appearance with the "glands" (in reality tubular airways) being lined by columnar, glycogen-containing endodermal cells and separated from each other by primitive mesenchyme. The pre-acinar blood vessels follow the development of the airways and although vascularization of the mesenchyme is not apparent, the pre-acinar artery and airway pattern is complete by the end of the pseudoglandular phase (Farrell, 1982). The canalicular phase, which follows the pseudoglandular phase, is characterized by further branching of the epithelium, thinning of the epithelium and a decrease in the relative amount of mesenchyme, together with vascularization. The vascularization process, which consists of capillaries forming within the mesenchyme surrounding the developing acinus, occurs in a centripetal fashion (from the periphery toward the - A -hilum), with the last phase being extension to the respiratory bronchioles (Farrell, 1982). The primitive acinus consists of the structure distal to a terminal bronchiole. As the endodermal branches beyond the terminal bronchioles grow to form the future air sacs (saccules) the epithelium is flattened and surrounding capillaries begin to protrude in many areas resulting in a close approximation of capillary lumen to saccular surface. This proceeds until most of the saccules are lined by flattened epithelium with only the very terminal spaces being lined by cuboidal epithelium. As the saccules approach each other and the interstitium thins, the walls of the saccules develop a double capillary layer, each derived from one saccule. The precise sequence of events at the periphery of the lung has not yet been established. Lamellar inclusions typical of type II cells were first observed by one group of investigators during the sixth month of gestation in the human (Campiche et al., 1963). Toward the end of the canalicular phase, at about 28 weeks gestation in the human, the epithelium is thin, both type I and type II cells can be recognized and gas exchange can be maintained. During the saccular phase, at 28-32 weeks gestation in the human, small low subdivisions begin to appear in the saccules and these correspond closely to the secondary crests or septa that are documented in animals. At this point in lung development, birth occurs in mice and rats, whose lungs lack alveoli at birth (Amy et al., 1977; Burri et al., 1974; Burri, 1974). The lungs of mice and rats at birth differ in • appearance from human lungs at about 28 weeks gestation in that the interstitium -is thicker and the saccules are shorter and stubbier in humans. -5 -The small low subdivisions elongate and produce small, multifaceted air spaces that may or may not have a double capillary layer. Structures resembling alveoli can be seen in some lungs as early as 30 to 32 weeks gestation and alveoli are clearly present in a l l lungs by 36 weeks gestation in the human. During the alveolar phase, alveoli develop in a centripetal fashion, first on the saccules and then on the respiratory bronchioles (Farrell, 1982). The number of alveoli at birth in the human is quite variable, with a mean of 50 million and a range of 10-149 million (Langston et al., 1984). Dunnill (1962) found 24 x 106 alveoli in one infant while Davies and Reid (1970) found 17 x 10^ alveoli in a stillborn infant. Hieronymi (1960) reported 110 x 106 alveoli and Thurlbeck and Angus (1975) found 71 x 106 alveoli in one infant. In a recent abstract, Hislop and Fairweather (1984) studied 23 cases aged from 29 weeks of gestation to 18 weeks postnatal and reported a variation between 49 and 82 million alveoli per lung by term. The cause of the wide variation in total alveolar number at birth is uncertain. Studies of rabbits, sheep, and rhesus monkeys, primarily using physiologic techniques, have indicated that lobar differences are present during the process of lung development (Farrell, 1982). Of greatest importance is the observation that upper or cephalad lobes show more advanced maturation than lower or caudal lobes. Whether or not human or rat lung differentiation follow this sequence of development remains to be determined. Studies of the embryonic and fetal development of rats have shown that the lung bud appears at approximately day 12 of gestation (Shepard, 1983). Following division of the lung bud, the two resulting - 6 -epithelial buds grow laterally into the surrounding mesenchyme. Through a number of successive branchings of the growing buds, a tree of epithelial tubes evolves, which lies embedded in mesenchyme, and around which a meshwork of blood vessels develops. This developmental stage, which corresponds to the embryonic and pseudoglandular phases in the human, has been termed the glandular phase of lung development in the rat (Burri and Weibel, 1977). It is believed to begin around day 12 and last until about day 18 after conception. This is followed by the canalicular phase on days 18-20 and the saccular phase on days 21 and 22 (term). A similar subdivision of lung development was made by Dr. Will Blackburn who examined fetal rat lungs on day 16 through day 22 of gestation and found the lungs to be in the glandular stage on days 16-18, in the canalicular stage on days 18-19 and in the terminal sac or saccular stage on days 20-22 (Farrell, 1982). The final stage, the alveolar stage, was . found to take place during the first few weeks of l i f e in the rat (Burri et al., 1974; Burri, 1974). It should be noted that the transition from one phase to the other is a gradual process and thus, subdivision of lung development into these four phases is to some extent artif i c i a l (Burri and Weibel, 1977). c) Differentiation of respiratory epithelium The link between surfactant surface tension lowering properties, lung maturity and the ability to successfully negotiate the transition between liquid and airbreathing at birth was made in 1959 by Avery and Mead. The sequence of events associated with differentiation of fetal alveolar type 2 cells and formation of the lamellar body containing - 7 -surfactant lipoprotein as well as the developmental patterns for disaturated phosphatidylcholine in lung tissue and alveoli has been described in several species and appears identical in the species studied to date (Burri and Weibel, 1977; Clements and Tooley, 1977). Between gestational days 17 and 19 in the rat, undifferentiated epithelial cells begin to accumulate glycogen such that there is a two-fold increase in glycogen per cell as measured in fetal lung homogenates (Williams and Mason, 1977). Between gestational days 19 and 21, epithelial cells begin to differentiate and cell proliferation slows. The future type 2 cells diminish in size, glycogen is depleted and lamellar bodies appear in the cytoplasm. At the base of differentiating cells, phospholipid bilayers (lamellae) first appear within membrane-bound dense granules. The content of the dense granules is believed to be largely protein and i t has been shown that the enzyme acid phosphatase is present. These granules develop into lamellar bodies by the addition of phospholipid. How phospholipid is initial l y deposited within the dense granule and is subsequently added to the growing granule is uncertain. Later in development, growing lamellar bodies fuse with multivesicular bodies which contain both lysosomal enzymes and phospholipid. The lamellar body enlarges, moves toward the apex of the cell and is secreted into the alveolar lumen (Williams and Mason, 1977). An inverse relationship exists between glycogen and phospholipid with lung glycogen per deoxyribonucleic acid (DNA) falling 60% and lung phospholipid per DNA rising 60% between days 19 and 21. By day 21, many type 2 cells have acquired adult type 2 cell characteristics, epithelial type 1 cells have begun to evolve and the tubular myelin figures of secreted lamellar - 8 -bodies begin to appear in the saccules. ' The surfactant complex which is responsible for lowering surface tension and thus establishing saccular and alveolar stability and the level of end expiration at birth, contains 85-88% lipid of which 82-88% is phospholipid (Farrell and Avery, 1975; Perelman et al., 1981; Smith and Bogues, 1982). These phospholipids are primarily highly saturated phosphatidylcholine (lecithin) and lipids unique to the lung, phosphatidylinositol and phosphatidylglycerol (Hallman et al., 1980; Hill et al., 1983). Palmitate, a saturated 16 carbon fatty acid, makes up 71% of the fatty acid component of phosphatidylcholine (Farrell and Avery, 1975). Approximately 10% of surfactant consists of unique apoproteins which together with the phospholipid form a lipoprotein complex. Synthesis of pulmonary lecithin is primarily through a cytidine 5'-diphosphate choline pathway although there is an alternate methylation pathway through phosphatidylethanolamine. Synthesis of phosphatidylinositol and phosphatidylglycerol is through a cytidine 5'-diphosphate diacylglycerol pathway (Hallman et al., 1980; Perelman et al., 1981). Phosphatidylinositol in the amniotic fluid characteristically increases up to 36 to 38 weeks gestation in the human. Thereafter i t decreases inversely to the increase in phosphatidylglycerol. Phosphatidylglycerol first appears in the amniotic fluid at 35-36 weeks gestation in the human and i t increases to term. Newborn babies with phosphatidylglycerol do not have respiratory distress syndrome whereas the absence of phosphatidylglycerol in the newborn almost inevitably means respiratory distress (Hallman et al., 1980). The actual mechanism by which the alpha-saturated, beta-unsaturated - 9 -phosphatidylcholine, phosphatidylinositol and phosphatidylglycerol are remodelled to form the disaturated molecules present in surfactant lipoprotein is unclear. In a l l species studied, surfactant synthesis increases in late gestation and this increase in synthesis is associated with dramatic changes in the enzymes associated with phospholipid synthesis. It has been shown that the specific activity of amniotic fluid phosphatidic acid phosphohydrolase, a key enzyme in phosphatidylcholine synthesis, increases approximately one week before the increase in the lecithin/sphingomyelin ratio in the surface active lipid extract of the amniotic fluid (Herbert et al., 1978; Jimenez and Johnston, 1976). d) Lung growth and maturation Lung growth and maturation are terms which can be applied either to cells or to tissues. During late fetal and early postnatal l i f e the human lung undergoes a series of complex structural changes which are related to the formation of alveoli. These structural changes are accompanied by cell multiplication (increase in numbers of interstitial, epithelial and endothelial cells), and by cell maturation (differentiation of epithelial and interstitial cells), as well as by tissue growth (increase in lung weight and/or lung volume) and by tissue maturation (alveolar multiplication, thinning and restructuring of the alveolar wall). Growth implies an increase in size which in the lung, may be brought about by an increase in the number of cells (hyperplasia), an increase in the size of cells (hypertrophy) and/or by increasing the air that the lung can contain per gram of tissue. At birth, lungs - 10 -contain about 3 ml of air per gram of lung tissue and this increases until age 6 years when the adult value of 8 ml per gram is found (Stigol et al., 1972). It is only now becoming apparent that growth (a quantitative phenomenon) and maturation (a qualitative phenomenon) may progress separately in the lung. For example, administration of glucocorticoids in late gestation results in maturation of the epithelial cells of the saccule walls so that there is an increased proportion of cells containing lamellar bodies and an increased proportion of flattened cells (Wang et al., 1971). Functionally, there is increased surface activity and deflation stability (Kotas and Avery, 1971; Liggins, 1969). This may also be accompanied by a decrease in the number of cells and in lung weight (Carson et al., 1973; Kotas et al., 1974) depending on the dose of glucocorticoid administered. It has been suggested that low dose steroids lead to maximal airspace development and intermediate dose steroids accelerate maturation of the type II cell without suppression of lung growth or body growth (as indicated by weight) while high doses suppress tissue growth (Kauffman, 1977). Thus, lungs may be mature but hypoplastic. Tracheal ligation in the developing fetal lamb resulted in increased lung tissue mass, but type II cells were infrequent and they contained scanty lamellar bodies (Alcorn et al., 1977). This is an example of hyperplastic immature lungs. Recently a suggestion has been made that human lungs may be hypoplastic and immature or hypoplastic and mature (Wigglesworth and Desai, 1982). Many papers discussing pulmonary hypoplasia have been published. The diagnosis of pulmonary hypoplasia has been made on the basis of a - 11 -number of different criteria. The criteria used by many physicians has been a reduced lung weight to body weight ratio (deLorimier et al., 1967; Finegold et al., 1971; Perlman and Levin, 1974; Perlman et al., 1976; Potter, 1946; Reale and Esterly, 1973; Swischuk et al., 1979; Symchych and Winchester, 1978). Others have used decreased lung weight (Goldstein and Reid, 1980; Liberman et al., 1969) or decreased lung size for a given gestational age (Kohler et al., 1970; Thomas and Smith, 1974). Another criterion has been decreased lung volume (Kitagawa et al., 1971). Thus, a universally accepted definition of pulmonary hypoplasia does not exist. In a recent paper i t was stated that a low wet weight and organ/body weight ratio are" only suggestive of hypoplasia and that the finding of decreased total lung DNA, used as an index of cell number, is diagnostic of pulmonary hypoplasia, particularly in association with a lower lung DNA/body weight ratio (Moessinger et al., 1983a). This definition of pulmonary hypoplasia will be used in this thesis. While measurement of DNA is an expected procedure in experimental studies, this is usually not feasible in a clinical setting. More realistically, lung weight to body weight ratio is used because lungs are routinely weighed at autopsy. This may not be the method of choice since there are obvious potential errors in that lung weight may be increased due to lung fluid, congestion, edema or infection, thus underestimating the frequency of hypoplasia. Perhaps lung volume would be a better clinical predictor of pulmonary hypoplasia. Lung development requires carefully regulated coordination of anatomic, physiologic, and biochemical processes. The result of these maturational events must be an organ having an adequate surface area, - 1? -sufficient vascularization., and the metabolic capability to sustain oxygenation and ventilation during the neonatal period. Tissue maturity may be morphometrically assessed by the volume proportion of airspaces, mean linear intercept and gas exchanging surface area, with the volume proportion of airspaces and gas exchanging surface area increasing and the mean linear intercept decreasing as the lung matures. Radial counts, which are a measure of the number of alveoli or saccules between respiratory bronchioles and the periphery of the acinus, provide an assessment of acinar complexity. Epithelial type II cell synthesis of surface active phospholipids, which are necessary in establishing normal lung function after birth, may be assessed biochemically by directly measuring the phospholipid content of the lung. III. Control of Lung Development a) Early intrauterine Two factors important in controlling lung development in the early part of gestation have been established. These include mesenchymal-epithelial interactions and collagen. Mesoderm is required for the branching of airways in culture as well as for determining the way the branching occurs. For example, chick lung mesenchyme applied to mouse lung epithelium produces the branching pattern of the mouse initial l y , followed by the branching pattern of the chick (Taderera, 1967). The origin of the mesenchyme also influences the way the endoderm will be affected. Gut mesoderm can bring about bronchial budding, but bronchial branching requires bronchial mesoderm. Bronchial and gut - 13 -mesoderm w i l l induce budding in tracheal epithelium, but tracheal mesoderm inhibits both bronchial budding and branching (Wessells, 1970). An "epithelial growth factor" found in the mesenchyme has an affect on epithelial growth in the developing lung (Alescio and DiMichele, 1968). Whether this relationship depends on c e l l to c e l l contacts between epithelium and mesenchyme which have been described (Bluemink et a l . , 1976) or on a secreted factor, such as the fibroblast-pneumonocyte factor (Smith, 1979; Tanswell and Smith, 1979), i s uncertain. The developing bronchus has been described by Hutchins and coworkers (1981) as a cylinder of epithelium, with a well-defined basement membrane which encases a l l but the advancing t i p , which pushes into and compresses the investing mesenchyme. They have suggested that as the bronchial tip grows, resistance to i t s further advance increases progressively as a result of compression by the mesenchymal tissues at the t i p , approach to the pleural surface, or approach to neighboring bronchi. At some c r i t i c a l level of resistance, forward growth i s arrested because i t becomes easier for the bronchus to branch in the region immediately behind the tip where the basement membrane has not yet formed and the mesenchyme i s not consolidated by compression. The branches so formed then grow until they in turn bifurcate as a result of encountering similar resistance to further forward progression. By this process, the bronchial tree proliferates with repeated subdivisions to f i l l the volume provided by the continued proliferation of mesenchyme while the mesenchymal elements and vasculature become gradually compressed between the growing bronchial branches (Hutchins et a l . , 1981). Collagen also affects the development of airways since the administration of a proline - IA -analogue which interferes with collagen synthesis and secretion, results in diminished numbers of bronchial buds (Alescio, 1973; Spooner and Faubion, 1980). b) Late intrauterine A number of factors have recently been established, both clinically and experimentally, as being important in controlling lung development in the latter part of gestation. These factors include distortion of the lungs by external pressure, the amount of amniotic and lung fluid and intrauterine respiration. Lung development may be disturbed by compression (or lack of stretch) of the lung. Pulmonary hypoplasia has occurred in association with diaphragmatic hernia both clinically (Kitagawa et al., 1971; Reale and Esterly, 1973) and experimentally (deLorimier et al., 1967), with thoracic dystrophy (Finegold et al., 1971), and in examples of membranous diaphragm (Briggs et al., 1973; Goldstein and Reid, 1980). ;The importance of the amount of lung fluid has been stressed in recent experiments (Alcorn et al., 1977). These authors found that tracheal ligation in sheep produced abnormally large lungs due to an increase in fluid and to increased tissue, with decreased cellular differentiation. Chronic tracheal drainage resulted in the reverse changes in the lungs: abnormally small lungs due to a decrease in fluid and tissue, with increased cellular differentiation. Oligohydramnios, a deficiency in the amount of amniotic fluid, may be regarded as being somewhat similar to the tracheal drainage experiment and lung hypoplasia is a well recognized complication of this condition (Bain and Scott, - 15 -1960; Blanc et al., 1962; Kohler et al., 1970; Liberman et al., 1969; Perlman and Levin, 1974; Perlman et al., 1976; Potter, 1946; Renert et al., 1972; Thomas and Smith, 1974). In early pregnancy amniotic fluid is i believed to be mainly derived from the fetal and maternal blood by diffusion across the fetal skin and membranes because the amniotic fluid is roughly equivalent to isotonic extracellular fluid (Abramovich, 1981; Seeds, 1980). Thomas Lind feels that during this phase of fetal development, the concentrations of major solutes in amniotic fluid are more closely related to those in fetal than maternal serum, and that amniotic fluid may be regarded as part of the fetal extracellular fluid space (Lind, 1981). In the latter half of pregnancy an increasing proportion of amniotic fluid is provided by production of fetal urine (Abramovich, 1981; Seeds, 1965; Seeds, 1980) and fetal lung fluid (Biggs et al., 1973; Carmel et al., 1965; Hill et al., 1983; Lanman et al., 1971; Towers, 1959). Approximately 500 ml of hypotonic low-sodium, high-urea content urine and 100-200 ml of lung liquid enters the amniotic cavity per day, by term in the human (Whitfield, 1978). A mean swallowing rate of 198 ml per day at 38-40 weeks gestation in the human i has been reported (Abramovich et al., 1979). The volume of amniotic fluid is maintained by an equilibrium between the amount of amniotic fluid swallowed by the fetus and the amount of lung fluid and urine entering the amniotic cavity, with the excess fluid being transported to the mother by diffusion across the fetal surface of the placenta (Gitlin et al., 1972; Whitfield, 1978). This movement of water from the amniotic sac to the maternal compartment may occur in the third trimester, mediated by the chemical potential gradient between hypctonic amniotic - 16 -fluid and isotonic maternal fluids (Seeds, 1980). It has been suggested that the amniochorion is another site of water transport (Abramovich, 1981; Minh et al., 1980) but Seeds (1980) believes this is highly improbable in view of the sparse vascularity in this region. The result of a l l this movement of amniotic fluid is that there is complete replacement of fluid every three hours (Graham and Sanders, 1982). It has been shown that amniotic fluid gradually increases in pregnancy from a mean volume of 35 ml at 12 weeks, to 250 ml at 17 to 18 weeks, and 1,100 ml at term. In the third trimester, the amniotic fluid volume decreases in the last 2 to 3 weeks of gestation. The term oligohydramnios is applied when there is l i t t l e or no amniotic fluid (less than 400 ml at term or less than 2 standard deviations below the mean prior to term). Oligohydramnios may be caused by renal disease, urinary (outflow) tract obstruction or chronic leakage of amniotic fluid. It has been suggested that the non-renal features of Potter's syndrome - pulmonary hypoplasia, facial (and ear) anomalies, fetal growth deficiency and limb-positioning defects - should be referred to as the "oligohydramnios tetrad" since these features may a l l be found in cases of oligohydramnios without renal anomalies (Thomas and Smith, 1974). This suggestion is further supported by the observation that the oligohydramnios tetrad is absent in those rare examples of renal agenesis or other renal abnormalities not associated with oligohydramnios (Bain and Scott, 1960; Kohler, 1972; Marras et al., 1983; Thomas and Smith, 1974). When amniotic sac puncture was performed on gestational days 16 or 17 in the rat, a marked reduction in the fetal lung weight and lung weight/body weight ratio was observed at term (Symchych and Winchester, - 17 -1978). These results suggest lung hypoplasia, although no consistent qualitative histologic difference in the lungs from control and experimental animals was found. When minimum (0.1 ml) or maximum volume (0.5 ml +) amniocentesis was performed on day 17 of gestation in the rat, a reduction in the weight of the lung was found at term following maximum volume amniocentesis only (Blackburn et al., 1978-abstract only). Since the DNA content of the lung was not affected, Blackburn et al. concluded that large volume amniocentesis reduces lung size by obliterating the fetal lung space rather than inhibiting lung cell proliferation. Moessinger et al. reported selective growth retardation of the lung following experimental oligohydramnios in the rat and guinea-pig. Fetal membranes were punctured and/or drainage was performed and the fetuses were allowed to develop until term. Weights and measurements of the fetus, placenta and various organs revealed a significant reduction in lung weight and lung weight to body weight ratio only (Moessinger et al., 1978-abstract only). Single needle puncture of the fetal rat membranes on day 15 of gestation resulted in significantly reduced lung weight, lung weight to body weight ratio, DNA per lung, DNA per gram of fetal weight and lung protein/DNA ratio indicating lung hypoplasia and hypotrophy (reduction in cell size) (Moessinger et al., 1983a). When short term oligohydramnios was produced by creating amnio-peritoneal fistulas in the fetal guinea-pig on day 45 (term: 59-75 days with a mean of 68 days) there was a significant decrease in lung weight and total lung DNA content on day 50 with values not significantly greater than day 45 controls indicating a near arrest in lung growth following oligohydramnios (Moessinger et al., 1983b - abstract only). The time - 18 -period used by these, investigators was believed to correspond to the late canalicular-early terminal sac stages of lung development (20 to 28 weeks of gestation in man). These investigators went on to vary the timing of onset and the duration of oligohydramnios and found the earlier the onset of oligohydramnios and the longer its duration, the greater the impact on lung growth in terms of lung DNA per gram of fetal body weight (Moessinger et al., 198A - abstract only). When oligohydramnios was produced by shunting amniotic fluid into the maternal abdominal cavity from day 24 or 25 of gestation to day 31 (term) or by occluding the bladder outlet of the fetal rabbit on day 25 of gestation, there was a significant reduction in lung weight and lung weight/body weight ratio on day 31. If along with occluding the bladder outlet, the fetus received a constant infusion of normal saline into the amniotic cavity or the fetus had its abdomen opened to allow its viscera to herniate and thus avoid thoracic compression from a diaphragm elevated by a dilated urinary tract and ascites, i t was found that the lung weight/body weight ratio at term was significantly higher than those of the rabbits with occlusion of the bladder outlet only. These experiments suggest that mechanical restriction to lung growth plays a role in the development of pulmonary hypoplasia associated with oligohydramnios, and that pulmonary hypoplasia may be partially reversible by procedures which reduce thoracic compression (Nakayama et al., 1983). The mechanism of the prenatal secretion of lung fluid in the lamb has been investigated by Strang and his associates (Strang, 1977). The fetal lamb lung contains about 30 ml of liquid per kg body weight, a volume similar to the functional residual capacity of a normal human neonate. This fluid forms at the rate of 2-3 - 19 -ml/hr/kg during the latter part of fetal l i f e . The fluid leaves the lungs via the trachea and is then swallowed by the fetus or i t appears in the amniotic fluid. This lung liquid is not an ultrafiltrate of plasma and it.differs from amniotic fluid as well. Its constitution is best explained by the presence of epithelial pumps which actively transport chloride and potassium in one direction and bicarbonate in the other. Thus chloride is transported from plasma to the airspace fluid in excess of a reverse bicarbonate flux; sodium follows the electrochemical gradient set up by chloride movement and net water flow into the airspaces is attributed to the osmotic force of sodium chloride. Study of tracer movement into lung liquid has established that the epithelium acts as a rather tight barrier for metabolically inert polar non-electrolytes with a functional pore radius of 0.55 - 0.60 nm (Normand et al., 1971). There appear to be separate functional channels for nonpolar (lipid soluble) and polar substances with the nonpolar substances permeating the epithelium much more readily than polar solutes. The exact site of the epithelial pump(s) and the mechanism of macromolecular transport are uncertain. The importance of fetal respiration in lung development has recently become apparent. High spinal cord section in fetal rabbits resulted in a 43% reduction in wet lung weight/body weight (fluid plus tissue) and a 16% reduction in total lung DNA/body weight (cell number) (Wigglesworth et al., 1977). In a subsequent paper, intrauterine cervical cord section at C1-C3 in fetal rabbits was shown to reduce lung growth by 70% while section at C5-C8 reduced lung growth by 40% (Wigglesworth and Desai, 1979). The authors attributed this difference - 20 -to the greater effect on fetal respiratory movement when the sectioning occurred above the origin of the phrenic nerve (C4-C5), which innervates the diaphragm. Daily transuterine injections of curare, a skeletal muscle relaxant, from day 18 of gestation until term (day 21), into fetal rats resulted in pulmonary hypoplasia as indicated by a significantly decreased DNA content when compared to sham-operated littermate controls (Moessinger, 1983). Bilateral phrenic nerve section in fetal sheep resulted in cessation of subsequent lung growth and structural development, although epithelial differentiation appeared normal (Alcorn et al., 1980). Interestingly, the effect of sectioning the cord above the phrenic nerve origin in rabbits could be reversed by ligating the trachea (Wigglesworth and Desai, 1979). Rudimentary respiratory centers are present in the medulla and capable of function as early as 40-50 days gestation in the fetal lamb, but at 60 days they come under inhibition from pontine centers (Strang, 1977). Fetal breathing movements are almost entirely confined to periods of active REM sleep. This association means that the respiratory center can respond to stimuli arising above the brain stem, thus suggesting that the pathways required for the behavioral response are working, but the link between breathing and metabolism is not yet established. There is evidence that both central medullary chemoreceptors, which are responsive to rising PCO2 and falling pH, and peripheral chemoreceptors (carotid body and aortic body), which are responsive to hypoxia, are depressed in the fetal lamb (Strang, 1977). Respiratory movements have been detected in the undisturbed human fetus with the use of a high-speed ultrasound imaging system (Stephens and Birnholz, 1978). - 21 -It has been proposed' that fetal lung growth may depend on lung distention whereas lung maturation is less dependent on distension and more dependent on the hormonal environment in utero (Liggins and Kitterman, 1981). Measurements of tracheal pressure during periods without fetal breathing movements in chronically cannulated fetal lambs have shown that i t is approximately 3 Torr higher than amniotic pressure. This finding is consistent with the hypothesis that lung volume is controlled by resistance to the outflow of tracheal fluid. The presence of a sphincter mechanism at the laryngeal outlet controlling the outflow of fetal lung fluid has been demonstrated in the fetal lamb (Adams et al., 1967). Tonic intrathoracic negative pressure generated by diaphragmatic tone and phasic intrathoracic negative pressure generated by contractions of the diaphragm during fetal breathing activity could contribute to the maintenance of lung volume and thus to lung growth (Liggins and Kitterman, 1981). In a recent study, published in abstract form only, designed to test the hypothesis that fetal breathing movements affect lung growth by transiently increasing the total volume of fluid in the potential airways and airspaces, i t was found in each of 13 fetal sheep that the volume of fluid collected from the lungs after a period of fetal breathing movement exceeded that collected after a period of no fetal breathing movement, thus supporting the hypothesis (Murai et al., 1983). It has been suggested that the hormones mainly responsible for controlling the various aspects of maturation probably include Cortisol, iodothyronines and catecholamines but the interrelationships of these hormones and the extent of involvement of other hormones is uncertain (Liggins and Kitterman, 1981). - 22 -The control of late f e t a l type 2 c e l l d i f f e r e n t i a t i o n and of the accelerated synthesis and discharge of lung surfactant i s presently unclear although i t i s a topic of considerable investigation. A variety of hormones such as glucocorticoids, 11-oxosteroids, thyroid hormone, thyrotropin releasing hormone, i n s u l i n , p r o l a c t i n , and l o c a l mediators such as c y c l i c AMP, methylxanthines, fibroblast-pneumonocyte factor and e p i t h e l i a l growth factor, have been noted to influence the alveolar wall and e p i t h e l i a l c e l l maturation and the release of surfactant from type II c e l l s i n the f e t a l lung (Smith and Bogues, 1982). In a recent abstract i t has been suggested that Vitamin B^, i n association with glucocorticoids, may play a role i n f e t a l lung maturation (Newman and Masters, 1984). In a study designed to investigate f e t a l c o r t icosteroid metabolism and i t s r e l a t i o n to lung maturation i t was observed that between the 34-35 and 36-37 weeks of gestation i n the human the r a t i o of amniotic f l u i d progesterone to amniotic f l u i d deoxycorticosterone decreased s i g n i f i c a n t l y suggesting the induction of the steroid 21-hydroxylase enzyme system. This was followed by a s i g n i f i c a n t r i s e i n amniotic f l u i d C o r t i s o l l evels between the 36-37 and 38-39 weeks of gestation. The greatest r i s e of amniotic f l u i d palmitate, used as an index of f e t a l lung surfactant, did not occur u n t i l a week l a t e r , presumably after the period of f e t a l lung maturation ( P e t t i t and Fry, 1978). IV. Midtrimester Amniocentesis In the second trimester, the majority of amniocenteses are carried out for chromosome analysis on fetuses at r i s k as a result of maternal - 23 -age (Bennett, 1981). The risk of a l l chromosome abnormalities rises with advancing maternal age, becoming auite steep after the age of AO years. At present a l l known morphological chromosomal abnormalities and more than A5 inborn errors of metabolism can be identified by amniocentesis. Since the discovery that amniotic fluid levels of alpha-fetoprotein are elevated in the presence of open neural tube defects, the measurement of amniotic alpha-fetoprotein has rapidly become the second most freguent indication for amniocentesis and the most common investigation performed on amniotic fluid. Results for alpha-fetoprotein levels may be available 2 weeks after amniocentesis but karyotype results reauire A weeks. The remaining l i s t of indications for which the procedure may be performed accounts in the United Kingdom for no more than 25% of a l l second trimester amniocenteses (Bennett, 1981). Three reports of studies of large numbers of amniocenteses have been published. These include a Canadian study of 1,223 amniocenteses (Simpson et al., 1976), a United States study of 1,0A0 amniocenteses (National Institute of Child Health and Human Development National Registry for Amniocentesis Study Group, 1976) and a United Kingdom study of 2,A28 amniocenteses (Medical Research Council Working Party on Amniocentesis, 1978). These reports suggest that the fetal complications following amniocentesis can be divided into two seemingly separate groups: (1) the risk of fetal loss (spontaneous abortion, fetal death in utero, and stillbirth) and (2) the risk of perinatal complications. Subjects undergoing a second trimester amniocentesis have a slightly higher incidence of fetal loss, less than 1% above controls (Bennett, 1981; Roberts et al., 1983; Turnbull and MacKenzie, 1983). Leakage of - 24 -amniotic fluid occurs in about 1% of midtrimester amniocenteses. Unlike the Canadian and United States studies, the United Kingdom study revealed a number of perinatal problems following amniocentesis at 16 to 20 weeks gestation. Both the United Kingdom and United States studies indicated an increased incidence of respiratory distress in the amniocentesis group of infants, but this increase was statistically significant only in the United Kingdom study. The United Kingdom study also indicated an increased incidence of major orthopedic postural deformities (severe clubfoot, dislocation and subluxation of the hip) and severe antepartum hemorrhage, with a significant excess of placental abruptions following amniocentesis. It was also noted that amniocentesis carries a risk of Rhesus isoimmunization which can be eliminated i f every rhesus-negative woman without anti-D antibodies receives a dose of anti-D immunoglobulin after undergoing an amniocentesis. A more recent study has indicated that complications are minimal and culture cell growth excellent when 18 to 20 ml of amniotic fluid is withdrawn in pregnancies beyond 15 weeks of gestation (Johnson, 1982). The mean total volume of amniotic fluid at 16 weeks is 200 ml and at 20 weeks, 400 ml. In a study of maternal, fetal and neonatal results and complications following 923 genetic amniocenteses performed during the fourteenth week of gestation, the only neonatal complication believed to be associated with amniocentesis was a marked increase in the incidence of lower-extremity orthopedic abnormalities such as metatarsus adductus, dislocated hip and clubfoot (Cruikshank et al., 1983). These investigators did not feel that midtrimester amniocentesis resulted in an increased incidence of spontaneous abortion or neonatal respiratory problems. - 25 -Following pediatric and neurodevelopmental examinations of 122 children between the ages of 5 and 7 years (62 children had undergone amniocentesis and 60 children were controls) no difference between the two groups was revealed in respect of pediatric and neurodevelopmental disorders, orthopedic abnormalities, or respiratory problems during the neonatal period indicating that the risk for developmental complications is not increased in children born after amniocentesis in the second trimester (Gillberg et al., 1982). Turnbull and MacKenzie feel that clinical audit of amniocentesis is likely to demonstrate a greater hazard than is really due to the test because patients referred to it are probably at an increased risk of spontaneous abortion (Turnbull and MacKenzie, 1983). But based on the very large difference in the incidence of unexplained respiratory difficulty in infants delivered between 34 and 37 weeks gestation (8.2% in amniocentesis subjects and 0.9% in control subjects) reported in the United Kingdom study, Turnbull and MacKenzie feel that respiratory distress is a true complication of amniocentesis. In a recent study, published in a letter to the editor, in which cynomolgus monkeys underwent amniocentesis between 47 and 64 days from the date of mating (equivalent to 14-17 weeks of human gestation), i t was found that at term (around 165 days) there was a significant reduction in birth weight, fixed lung volume, total alveolar number and number of generations of respiratory bronchioles to the left lower lobe of the lung when compared to a control group of infant monkeys (Hislop and Fairweather, 1982). As well, there was an increase in alveolar size. These results suggest that removal of amniotic fluid or its continued leakage after amniocentesis - 26 -may interfere with normal lung development. Hislop and Fairweather stated the long-term effect of reduced numbers of enlarged alveoli is not known but they suggested i t may increase the risk of emphysema in adulthood. In a subsequent study, published in abstract form only, in which the amount of amniotic fluid removed varied from 100% of the total present to nil (procedure only) i t was found that no significant relationship existed between the amount of fluid removed at amniocentesis and the features studied (Hislop et al., 1984). The results were the same as in the first study but when the experimental subjects were compared with weight matched controls there was a signficant decrease only in number of alveoli and generations of respiratory bronchioles. There was no difference in fixed lung volume or alveolar size. It was thus felt that the i n i t i a l insult of amniocentesis affected the branching of respiratory bronchioles which in turn lead to a reduction in alveolar number. A study of the lung capacity of healthy, term babies born to mothers who had undergone midtrimester amniocentesis (between 15 and 24 weeks gestation) revealed a reduction in the crying vital capacity to body weight ratio of these infants compared to control babies after normal pregnancies without amniocentesis (Vyas et al., 1982). V. Rationale for the Present Investigation The objective of this research was to determine whether or not a loss of amniotic fluid near the end of the glandular stage of fetal lung development in the rat causes pulmonary hypoplasia. All the studies to date have described an association between pulmonary hypoplasia (generally defined as a low lung weight for a given body weight) and - 27 -oligohydramnios. Lung weight has recently been regarded as an unreliable determinant of the size of fetal and infant lungs since i t may vary considerably according to the quantity of blood, edema fluid or lung fluid present. Pathologists may thus have underestimated the frequency of pulmonary hypoplasia. In a recent paper i t was stated that a low wet weight and organ/body weight ratio are only suggestive of hypoplasia and that the finding of decreased total lung DNA, used as an index of cell number, is diagnostic of pulmonary hypoplasia, particularly in association with a lower lung DNA/body weight ratio (Moessinger et al., 1983a). The definition of pulmonary hypoplasia used in this research was a reduction in total lung DNA for a given body weight. VI. Morphometry Morphometry is a body of methods used to obtain numerical information about anatomical structure, macrcscopic or microscopic, in terms of such quantities as volume, surface area, number of components, and size of components. Information about three-dimensional structure is derived from maneuvers carried out on two-dimensional images or sections. Most morphometric methods entail the use of test grids, superimposed repeatedly and randomly on the structure in question. Test qrids consist of a set of lines for estimating surface area, a lattice of test points for estimating volume fraction and a discrete test area for counting actual components. By measuring the total volume of the organ in question i t is possible to calculate the total volume of a particular substructure. The methods of morphometry are thus indirect, as no quantity is measured directly, and estimates are based on probabilistic considerations (Aherne and Dunnill, 1982). - 28 -MATERIALS AND METHODS I. Materials Timed pregnant Sprague-Dawley rats were purchased from Canadian Breeding Farm and Laboratory in Montreal, Canada. They arrived one week pregnant and were placed in individual cages in the Department Animal Room where they were fed a standard Purina rat chow and were supplied with unlimited water. All reagents used in this study were of analytical grade and were purchased from either Sigma Chemical Company (St. Louis, Missouri, USA), Western Scientific (Vancouver Representative, Vancouver, B.C.) or Fisher Scientific Limited (Vancouver Representative, Vancouver, B.C.), unless otherwise stated. II. Methods a) Experimental protocol On day 16 of gestation each rat was anesthetized briefly with anesthetic grade ether (Mallinckrodt Chemical Works product) and while slightly sedated an intraperitoneal injection of sodium pentabarbitone (purchased from BDH Chemicals Canada Limited, Vancouver, B.C.) was administered (1 cc/kg body weight of a 50 mg/ml solution). When the animal was anesthetized, after approximately 20 minutes, one horn of the bicornuate uterus was fully exposed following a midline incision in the abdominal wall. Either the left or the right uterine horn was randomly chosen to be the experimental horn, in which each individual amniotic sac was punctured transuterine with a sterile 20-gauge needle and amniotic fluid leakage was noted. The opposite horn in each case served as the control horn. The uterus was returned to its original position within the abdominal cavity and the abdominal wall was sutured in two layers (muscle and skin). Following surgery, each animal was returned to its cage in a room with the temperature set at 21°C. On day 21 of gestation (the last day prior to natural delivery) the rat was again anesthetized by the method used on day 16. A cesarean hysterectomy was performed and the mother was killed by sectioning the abdominal aorta. The uterus was cut along its longitudinal axis to expose one amniotic sac at a time. For the fetuses in the latter half of this study, amniotic fluid was aspirated from each amniotic sac with an 18-gauge needle and a 1 cc Tuberculin syringe. The amniotic fluid volume was recorded as an estimate of the amount of amniotic fluid present on day 21. The fetal membranes were removed and the umbilical cord was sectioned close to the fetus. Body weight was measured, with a Delta Range Mettler PC 440 scale. The fetuses were placed in a petri dish with 0.9% saline (sodium chloride) or Hank's balanced salt solution (purchased from Media Preparation Service, Cancer Control Agency of British Columbia, Vancouver, B.C.). The fetuses were considered to be alive at birth i f a muscular reflex was elicited by a sudden jolt to the petri dish. Only live fetuses were used for the study to ensure cell autolysis had not taken place. Following amniotic sac puncture on day 16, some fetuses were totally resorbed at day 21 with only the placenta remaining (degenerated buttons) while others were partially resorbed, white, limp fetuses at day 21. These fetuses were not used for the study as they had died in utero. - 30 -b) General research design Lungs from each of the experimental and control fetuses were used for either one of two types of experiments: 1) biochemical analysis of NCT protein, DNA and phospholipid content, or 2) morphometric analysis of the structural components of the lung. c) Extraction of non-connective tissue protein, DNA, phospholipid and  disaturated phosphatidylcholine Lungs from the control and experimental fetuses were dissected out with the use of a stereomicroscope, and weighed as wet tissue. During this procedure the remaining fetuses were placed in the refrigerator and thus maintained at 3°C. A Delta Range Mettler PC 440 scale or Sartorius 2004 MP6 semi-micro balance was used for the weighing. Following dissection, the lungs were stored at -13°C and were later lyophilized in a VirTis Preservator, Model 10-PR, until a constant dry weight was obtained. The dry lung weight was then recorded. For the extraction of non-connective tissue (NCT) protein and DNA, a modified Schmidt-Thannhauser method was used to obtain an acid soluble RNA fraction and a precipitate of protein and DNA (Schmidt and Thannhauser, 1945). The lipid solvent step used to obtain a lipid fraction was omitted. Schneider's method of hot acid extraction was used on the protein and DNA precipitate to obtain an acid soluble DNA fraction and protein precipitate (Schneider, 1945). The exact procedure used was as follows. Each dried lung was cut with a razor blade into paper thin pieces and transferred to pyrex homogenizers. Tissue was homogenized by - 31 -hand in 1.0 ml ice-cold 0.145 M saline or phosphate buffered saline (Dulbecco's Formula - purchased from Media Preparation Service, Cancer Control Agency of British Columbia, Vancouver, B.C.). A 1.0 ml aliquot of ice-cold 30% trichloroacetic acid (TCA) was added to the homogenate. The samples were vortexed (mixed in a vortex mixer) and then centrifuged at 1000 x g for 4 minutes. The supernatant containing acid soluble small molecules such as free nucleotides, nucleotide coenzymes, sugars, polysaccharides, inorganic phosphate and organic phosphorus compounds of low molecular weight was discarded. The precipitate, containing acid insoluble protein, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), was then washed with 3.0 ml ice-cold 15% TCA, vortexed and centrifuged at 1000 x g for 4 minutes. The supernatant was discarded and to the precipitate was added 2.0 ml prewarmed (37°C) 1 M sodium hydroxide (NaOH). The sample was then vortexed and incubated for 1 hour at 37°C. Following incubation, the samples were cooled on ice, vortexed, and a 0.250 ml aliquot was removed from each sample for alkali soluble non-connective tissue protein estimation. The remaining solution was neutralized with 0.4 ml ice-cold 6 M hydrochloric acid (HCl) and the acid insoluble protein and DNA were precipitated by the addition of 2.0 ml ice-cold 5% TCA. Following centrifugation at 1000 x g for 4 minutes the supernatant containing acid soluble RNA was discarded. The precipitate containing acid insoluble protein and DNA was heated at 90°C for 20 minutes in a water bath following the addition of 4.0 ml 10% TCA to each sample. The samples were cooled on ice and centrifuged at 1000 x g for 4 minutes. The supernatant containing acid soluble DNA was decanted into a test tube which was then placed on ice. The - 32 -procedure was repeated on the remaining precipitate (refer to the flow chart depicted in Figure 1). The decanted supernatants were pooled for each sample and the volume was measured with a 10 ml graduated cylinder. This fraction was used for the estimation of DNA content. The NCT protein and DNA samples thus collected were stored at -13°C until a number of samples were collected and the estimation of NCT protein and DNA content was then performed. For the samples which underwent phospholipid and disaturated phosphatidylcholine (DSPC) extraction, two 0.25 ml aliquots were removed from the original 1.0 ml homogenate (refer to the flow chart illustrated in Figure 2). For these samples, NCT protein and DNA were extracted from the remaining 0.50 ml of homogenate following the procedure described above with the volumes of the reagents reduced by one-half since the volume of the homogenate was reduced by this amount. Total lipids were extracted from the two 0.25 ml aliquots of lung homogenate (Folch et al., 1957) and this fraction was divided into a neutral lipid and a phospholipid fraction (King and Clements, 1972). One phospholipid fraction per fetus was allowed to react with osmium tetroxide (OsO^) in order to isolate and collect a DSPC fraction (Mason et al., 1976). The exact procedure used was as follows. For the extraction of total lipid, 5.0 ml chloroform:methanol (2:1, v/v) was added to each sample in stoppered graduated tubes. The tubes were inverted several times and the samples were allowed to stand overnight at room temperature. The following morning 1.0 ml 0.05 M potassium chloride (KC1) was added to each tube and the tubes were inverted several times. The tubes were then vortexed and centrifuged at 160 x g for 5 minutes. A biphasic system - 33 -lung paper thin pieces | 1 ml cold 0.145 M saline homogenize I 1 ml cold 30% TCA; vortex centrifuge at 1000 xg for 4 minutes discard supernatant pellet I 3 ml cold 15% TCA; vortex centrifuge at 1000 xg for 4 minutes discard supernatant | invert and drain tubes well pellet |2 ml prewarmed 1 M NaOH; vortex incubate 1 hr. at 37°C; cool tubes on ice 0.250 ml aliquot for. NCT PROTEIN ESTIMATION I vortex .solution 0.4 ml 6 M HCl 2.0 ml cold 5% TCA centrifuge at 1000 xg for 4 minutes discard supernatant jfellet 4 ml 10% TCA; vortex heat at 90°C for 20 min in a water bath; cool tubes on ice centrifuge at 1000 xg for 4 minutes decant supernatant (DNA)-place tube on ice pellet 4 ml 10% TCA; vortex heat at 90°C for 20 min in a water bath; cool tubes on ice centrifuge at 1000 xg for 4 minutes discard pellet decant supernatant (DNA) pool supernatants (DNA) measure volume I DNA ESTIMATION Figure 1. Extraction of NCT protein and DNA. - 34 -lung paper thin pieces I 1 ml cold phosphate buffered saline homogenize 0.50 ml homogenate process as in Figure 1 with the volumes of the reagents reduced by one-half I aliquots for NCT PROTEIN and DNA ESTIMATION discard (neutral lipids)-discard (neutral lipids)-I 0.25 ml aliquot 0.25 ml aliquot | 5 ml CHC13:CH30H (2:l,v/v)| invert stoppered graduated tubes several times and allow tubes to stand overnight at room temperature | 1 ml 0.05 M KC1; vortex centrifuge at 160 xg for 5 minutes pipet out upper aqueous phase and protein interphase and discard wash (twice) with 2.5 ml CHC13:CH30H:1•76% KC1(3:48:47,v/v); centrifuge at 160 xg for 5 minutes and pipet out the washing fluid and discard I I concentrate to dryness under N2 gas I 1 ml CHCl3:CH30H(2:l,v/v) | apply to s i l i c i c acid column and allow to stand for 2 minutes —I 2.5 ml CHC13 rinse column with 2.5 ml CH30H I 2.5 ml CH30H | collect eluted phospholipids in a Ipyrex tube glass hydrolysis tube concentrate to dryness under N2 gas 0.5 ml (0.1 gm/ml) I 0s0A:CCl4 allow to stand for 15 minutes I concentrate to dryness I 1 ml CHC13:CH30H (20:1,v/v) apply to aluminum oxide column 10 ml CHC13:CH30H (20:1,v/v) PHOSPHOLIPID ESTIMATION 5 ml CHC1 3:CH 30H:7 M N H 4 O H (70:30:2,v/v) collect eluted DSPC in glass hydrolysis tubes I concentrate to dryness under N2 gas DSPC ESTIMATION Figure 2. Extraction of NCT protein, DNA, phospholipid and disaturated phosphatidylcholine. - 35 -developed in each tube with an upper aqueous phase and a lower chloroform phase which contained the lipids. The upper aqueous phase and protein interphase was then pipetted out as completely as possible and discarded. The samples were washed twice with 2.5 ml chloroform:methanol: 1.76% potassium chloride (3:48:47, v/v), followed by centrifugation at 160 x g for 5 minutes, and the washing fluid was pipetted out and discarded. The samples were concentrated to dryness under nitrogen (N^) gas (purchased from Medigas Pacific, Vancouver, B.C.); redissolved in 1.0 ml chloroform:methanol (2:1, v/v); applied to a s i l i c i c acid column, formed by placing s i l i c i c acid on glass wool plugs in the necks of disposable 9 inch pasteur pipets, and allowed to stand for 2 minutes. Neutral lipids were eluted with 2.5 ml chloroform (CHCl^). The column was rinsed with 2.5 ml methanol (CH.J3H). Phospholipids were eluted with 2.5 ml methanol. The sample for total phospholipid estimation was collected in a glass hydrolysis tube while the sample for DSPC estimation was collected in a standard pyrex tube with a teflon-lined screw cap. The phospholipid samples were then concentrated to dryness under nitrogen gas. The sample residue collected for DSPC estimation was redissolved in 0.5 ml osmium tetroxide:carbon tetrachloride (0.1 gm/ml) and allowed to stand for 15 minutes at room temperature. To facilitate rapid evaporation the solution was then evaporated under light negative pressure in a fume hood. The sample residue was redissolved in 1.0 ml chloroform:methanol (20:1, v/v) and . applied to a neutral aluminum oxide column formed by placing 80-200 mesh aluminum oxide on glass wool plugs in the necks of disposable 9 inch pasteur pipets. Any remaining neutral lipids were eluted with 10 ml - 36 -chloroform:methanol (20:1, v/v) and discarded. Disaturated phosphatidylcholine was eluted with 5 ml chloroform:methanol:7 M ammonium hydroxide (70:30:2, v/v) and collected in glass hydrolysis tubes. The samples were then concentrated to dryness under nitrogen gas and the residues were used for the estimation of disaturated phosphatidylcholine content. d) Estimation of non-connective tissue protein content Alkali soluble NCT protein was measured by the method of Lowry et al. (1951) using bovine serum albumin (BSA)(Sigma A-4378) dissolved in 0.1 M NaOH as standard. The standard (500 ug BSA/ml) was prepared, aliquoted and stored at -13°C until i t was used in an assay. For each assay, one aliquot was thawed and pipetted out according to the method of preparation of the standard curve (0,10,50,75,100,125,150,200 ug BSA), with a l l tubes in duplicate. The protein samples, dissolved in 1 M NaOH, were diluted with distilled water so that they could be read on the standard curve. Aliquots of 100 u l of the diluted sample were used for the analysis, a l l samples in duplicate. A final volume of 500 u1 was reached for the standard curve and sample tubes by the addition of 0.1 M NaOH.. Five ml of the colour reagent (copper tartrate/sodium carbonate) was added to each tube which was then vortexed and allowed .to stand at room temperature for 15 minutes. The colour reagent was freshly prepared for each assay by adding 1.0 ml of 1% copper sulfate (freshly prepared for each assay) and 1.0 ml of 2% sodium potassium tartrate to 100 ml of 2% sodium carbonate. Following the 15 minute reaction period 0.5 ml 1 N Folin Reagent was added to the mixture, vortexing each tube immediately - 37 -after addition. The 1 N Folin Reagent was prepared immediately before use by diluting 10 ml Phenol Reagent Solution 2N (Folin-Ciocalteau) with 10 ml distilled water. Following addition of Folin Reagent the tubes were allowed to stand at room temperature for 90 minutes. The colour which developed was read at 660 nm in a Philips Pye Unicam SP6-550 UV/VIS spectrophotometer. The standard curve was graphed and found to be linear in the range of 10 to 200 ug BSA. From the duplicate tubes an average optical density was calculated for each sample and the amount of NCT protein present (assay value) was determined according to the standard curve. The total NCT protein per lung was calculated as follows: total NCT protein (mg) = assay value ( yg) x sample volume (ml) x 1 mg dilution factor assay volume (ml) 1000 yg e) Estimation of DNA content DNA content was determined by measuring the deoxypentose content according to the method of Dische (1955) using calf thymus deoxyribonucleic acid (Sigma D-1501) dissolved in 0.005 M NaOH as standard. The original standard (400yg DNA/ml) was prepared, aliquoted and stored at -13°C until i t was required. For each assay, one 4.0 ml aliquot was thawed, diluted (1:2) with 4.0 ml 10% TCA and incubated in a 90°C water bath for 20 minutes. The final standard (200 yg DNA/ml) was then cooled on ice and pipetted out according to the method of preparation of the standard curve (0,40,80,100,120,160,200 ug DNA), with a l l tubes in duplicate. The final volume of each standard curve tube was made up to 1.0 ml by the addition of 10% TCA. The samples were pipetted in 1.0 ml aliquots, each sample in duplicate. To each tube was added - 38 -2.0 ml diphenylamine reagent which was freshly prepared for each assay by dissolving 1 gm diphenylamine (J.T. Baker Chemical Company product) in 100 ml glacial acetic acid and adding 2.75 ml concentrated sulfuric 0 acid. All tubes were vortexed, capped loosely with glass marbles and heated for 10 minutes in a boiling water bath. The tubes were then cooled on ice and the colour which developed was read at 600 nm in a Philips Pye Unicam SP6-550 UV/VIS spectrophotometer. The standard curve was graphed and found to be linear in the range of AO to 200 yg DNA. From the duplicate tubes an average optical density was calculated for each sample and the amount of DNA present (assay value) was determined according to the standard curve. The total DNA content per lung was calculated as follows: total DNA (mg) = assay value (ug) x sample volume (ml) x 1 mg x c assay volume (ml) 1000 ug c = sample volume before removal of aliquot for protein assay sample volume after removal of aliquot for protein assay f) Estimation of phospholipid content Total phospholipid and DSPC content was determined by measuring the phosphorus content according to the method of Fiske and Subbarow (1925) using an inorganic phosphorus (P^) standard (Western P-1A7A09; Pierce product). The sample residues were redissolved in 0.7 ml perchloric acid (72%, w/v) and the mixture was digested at 100°C for 3 1/2 hours. The tubes were then cooled on ice. The standard (50 ug Pj/ml) was pipetted out according to the method of preparation of the standard curve (0,5,10,20 ug P.) and to each standard curve tube was added 0.7 ml - 39 -perchloric acid (72%, w/v). To each of the standard curve and sample tubes was added distilled water (to make up a final volume of 4.7 ml), 0.2 ml 5% ammonium molybdate and 0.2 ml reducing agent. The reducing agent was freshly prepared by making one packet l-Amino-2-Naphthol-4-Sulfonic Acid dry mixture (containing 14.62 gm sodium bisulfite, 0.5 gm sodium sulfite and 0.25 gm l-amino-2-naphthol-4-sulfonic acid) up to 100 ml with hot distilled water. The solution was allowed to cool at room temperature and was then filtered. The filtered solution was stored in an amber bottle. The sample and standard curve tubes were then heated in a boiling water bath for 20 minutes and cooled on ice. The colour which developed was read at 660 nm in a Philips Pye Unicam SP6-550 UV/VIS spectrophotometer. The standard curve was graphed and found to be linear in the range of 5 to 10 yg P^ . For the calculations the optical density at 5 yg P^  was used. Total phospholipid and total DSPC per lung were calculated as follows: yg Pj/extract. = sample optical density  5 yg Pj[ standard optical density/5 y g P^  standard total phospholipid (mg) = yg Pj/extract x sample volume (ml) x 1 mg x 25 aliquot volume (ml) 1000 y g assuming the quantity of phospholipid in lung samples was 25 times the phosphorus content ( yg phospholipid = yg P. x 25). g) Lung fixation The trachea of five left horn- and five right horn-experimental - 40 -fetuses along with five right horn- and five left horn-control fetuses (control and experimental fetuses from the same mother and matched for the closest position in the uterine horn possible) were isolated and cut just below the larynx. A bevelled tuberculin needle was used to intubate the trachea. The anterior portion of the thoracic cage was carefully dissected away to allow the lungs to inflate during fixation with 2.5% glutaraldehyde (Kodak product) in 0.1 M sodium cacodylate buffer (purchased from B.D.H. Chemicals Canada Limited, Vancouver, B.C.). The lungs were fixed at 25 cm water transpulmonary pressure for 24 hours. h) Estimation of fixed lung volume The tuberculin needle was removed from the lung, the trachea was tied off with suture and the lungs were dissected out of the fetus. Fixed lung volume was measured by suspending the fixed lung from a laboratory stand and just submerging i t in a beaker full of distilled water placed on a balance (Delta Range Mettler PC 440) that had been previously tared to zero (Scherle, 1970). The weight reading on the balance was equal to the volume of the lung by Archimedes Principle which states: "a body partially or totally submerged in a body of fluid, experiences a buoyant force equal to the weight of the fluid displaced by the volume of that body." i) Lung sampling and processing The lungs were bisected into a left and a right lung. The left lung was processed for embedding in Spurr. Five 3 micron-thick sagittal - Al -sections were cut equidistantly through the l e f t lung and stained with t o l u i d i n e blue for l i g h t microscopy. The l e f t lung of the rat i s made up of one lobe (Greene, 1959). j ) Estimation of shrinkage Tissue shrinkage due to processing and sectioning was estimated by the following procedure. The length of the fixed l e f t lung was measured from apex to base with the use of a computer controlled d i g i t i z e r . The lung was embedded i n Spurr with the l a t e r a l side of the lung positioned along the surface of the block. The length of the embedded lung was then measured from apex to base. Linear shrinkage factor-^ was calculated as follows: l i n e a r shrinkage factori = length of embedded lung (fixed to processed) length of fixed lung Five 3 micron-thick step s e r i a l sections were cut equidistantly through the lung and for blocks number 1,6,11,16 and 20 the length and width of the sectioned lung ( s l i d e ) and embedded lung (block - immediately following a section) were measured with c a l i p e r s . Linear shrinkage f a c t o ^ was calculated as follows: shrinkage factor for length = length of sectioned lung length of embedded lung shrinkage factor for width = width of sectioned lung width of embedded lung l i n e a r shrinkage factor? = (processed to sectioned) \ shrinkage factor X shrinkage factor for length for width The l i n e a r shrinkage factor for the fixed to the sectioned state was then calculated as follows: - 1x2 -Linear Shrinkage Factor = l i n e a r shrinkage X mean li n e a r shrinkage factor^ f a c t o r 2 This factor was calculated for each lung sample and was used to correct the t o t a l projected length of gr i d test l i n e s (L-j.) from the sectioned to the fixed state of the lung tissue, and thus correct for tissue shrinkage due to processing and sectioning. Ly (fixed tissue) = t o t a l length of grid test l i n e s on sectioned tissue Linear Shrinkage Factor When the measurements and calculations were completed, i t was found that the mean shrinkage due to processing was 5%, with a range of 0-9%, and that there was no measurable shrinkage due to sectioning. k) Light microscopy morphometry A l l s l i d e s were coded and measured morphometrically (Aherne and Dunnill, 1982; Kawakami, 1984; Weibel, 1979), without knowing i f the sl i d e belonged to a control or experimental fetus. Each s l i d e was placed on the automatic stage of a Wild M501 microscope and viewed through a sauare test grid located on the screen of the microscope, using a 25X objective. The test grid i l l u s t r a t e d i n Figure 3, consisted of a square 2 of known area (0.450 mm ) which contained two diagonal cross h a i r l i n e s of known length (0.58 mm each) and 42 eguidistant test crosses. The t o t a l number of f i e l d s i n each lung section was determined and numbered in a systematic fashion. Twenty f i e l d s were randomly selected with the use of a Hewlett Packard 11C calculator random number generator. Specific x-y co-ordinates were determined for each f i e l d i n order to - A3 -ensure there was no overlapping of counted f i e l d s and therefore no redundant measurements. The use of co-ordinates enabled a f i e l d to be relocated at a l a t e r date i f i t required recounting. The various structures upon which one-half or more of each test cross (point) of the test g r i d f e l l were recorded for each f i e l d . The four categories counted included saccular a i r , saccular w a l l , conducting a i r (bronchiole a i r and bronchial a i r ) and nonparenchyma (blood vessels, excluding c a p i l l a r i e s , and walls of conducting airways). The number of test points from 100 fields/case (20 f i e l d s / s l i d e x 5 slides/case) were summed for each of the tissue components and th e i r volume fraction was expressed as a fraction of the t o t a l number of points (A2 p o i n t s / f i e l d x 100 fields/ c a s e ) . No correction was made for tissue thickness, which was presumed to be 3 microns. - 44 -Figure 3. Test grid used for light microscopy morphometry. The number of times a grid test line crossed a gas-exchanging surface in a field was counted. In the fetal lung during the saccular phase of lung development, the border between saccule wall epithelium and saccule air was considered a gas^exchanging surface. To avoid overcounting, the following intercept counting rules were followed: - 45 -If one of the grid lines crosses a saccular wall count 1, because 1 gas-exchanging surface has been intercepted by the line (Figure 4). Figure 4. Illustration of intercept counting Rule 1. - 46 -2) If the top of the north-south grid line touches but does not cross a saccular wall count 1. If the bottom of the grid line touches but does not cross a saccular wall count 0 (figure 5). Figure 5. Illustration of intercept counting Rule 2. Similarly, i f the right end of the east-west grid line touches but does not cross a saccular wall count 1. If the left end of the grid line touches but does not cross a saccular wall count 0 (Figure 5). - 47 -3) I f the north-south grid l i n e touches but does not transect a saccular wall count 1 i f the saccular wall epithelium touches the right side of the g r i d l i n e . I f the saccular wall epithelium touches the l e f t side of the grid l i n e count 0 (Figure 6 ) . Figure 6 . I l l u s t r a t i o n of intercept counting Rule 3 . S i m i l a r l y , i f the east-west grid l i n e touches but does not transect a saccular wall count 1 i f the saccular epithelium touches the top of the grid l i n e . I f the saccular epithelium touches the bottom of the g r i d l i n e count 0 (Figure 6 ) . - 48 -Using the point counts and intercept counts, calculations were performed as in Table I to obtain the morphometric values of volume fraction (Vy) of lung components, mean linear intercept (l-m)> saccular surface area (Sw) and gas exchanging surface to volume ratio (Sy ) which is a measure of the saccular surface area per unit volume of saccular air. Table I. Calculation of Morphometric Parameters of Lung Growth and Maturation. Parameters Abbreviations Calculations Fixed lung volume V|_ Volume fraction of saccular air Vya saccular wall Vyw conducting air Vy[-, nonparenchyma Vynp Total volume of saccular air Va VL x V V a saccular wall Vw VL x V V w conducting air VD VL x Vy^ nonparenchyma Vnp VL x V V n p Total projected length of grid test lines Lj* Mean linear intercept ( um) L^ , 2Lj/I^ Saccular surface area (cm2) Sy^ ^VL/Lm Surface to volume ratio (cm2/ml) Syw SW/VL x Vva * corrected for shrinkage I w = total number of intercepts - 49 -The counting principle used in measuring lung parameters on random sections was based on the Delesse principle (Delesse, 1848) which states: "area proportions are equivalent to volumetric proportions" and "the planimetric fractions of a section occupied by sections of a given component correspond to the fraction of the tissue volume occupied by this component." Thus, i t is possible to calculate three-dimensional parameters including volume fractions from two-dimensional area measurements as follows: Vy = volume fraction Vvx = number of test points falling on structure x total number of test points* *4200 points/case (42 points/field x 20 fields/slide x 5 slides/case) 1) Light microscopy morphology When the morphometry was completed the slides were again examined with the light microscope, this time knowing the lung was from a control or experimental fetus, to check for qualitative differences in the lungs. After studying the twenty lungs, a number of slides were picked at random and an attempt was made at deciding i f the slide was a control or experimental lung. - 50 -m) S t a t i s t i c a l analysis Control and experimental means _+ standard errors of the mean (SEM) were calculated for a l l variables and the probability values for the mother, treatment and mother-treatment interaction effects were given when the raw data was analysed by the UBC Genlin program. This program performed tests i n an unbalanced, mixed model (mothers random; treatment fixed - meaning the treatment was s p e c i f i c a l l y chosen) analysis of variance (Zar, 1974). The data was unbalanced because there was an unequal number of control and experimental fetuses per mother. Differences between control and experimental means (treatment effect) were considered s i g n i f i c a n t at probability l e v e l s of P less than 0.05 (95% confidence i n t e r v a l ) . Differences between control and experimental means with P less than 0.10 (90% confidence i n t e r v a l ) were also noted since the probability values were close to the arbitrary l e v e l of s t a t i s t i c a l significance chosen for t h i s work. RESULTS I. General Characteristics a) Expression of results Results were expressed i n absolute terms as well as per gram dry lung weight and per gram body weight for the biochemical parameters. The measures of organ growth, lung weight and lung volume, as well as of lung maturation, saccular surface area, were also expressed per gram body weight. This was necessary to d i f f e r e n t i a t e an absolute a l t e r a t i o n i n - 51 -the lung from a relative alteration due to growth retardation of the experimental fetuses. b) Amniotic fluid volume Mean amniotic fluid volumes (Vf) +. SEM's are shown in Table II below. The experimental fetuses were surrounded by significantly less amniotic fluid on day 21, 42% of the control amniotic fluid volume (P less than 0.01), indicating persistent oligohydramnios following amniotic sac puncture. c) Body weight Mean fetal body weights (Wb) +_ SEM's are shown in Table II below. The experimental fetuses weighed significantly less, 97% of the weight of controls (P = 0.02), indicating fetal growth retardation. Table II. Mean Amniotic Fluid Volumes and Mean Body Weights for Control and Experimental Groups. Variable Control Group N Experimental Group N P V a f (ml)+ 0.24+0.01 45 0.10+0.01 37 P<.01 Wb (qm)* 3.189 + 0.025 105 3.088 + 0.029 80 P=.02 Values are means +_ SEM N = number of fetuses P = probability value for the treatment effect *P < 0.05 +P < 0.01 V gf = amniotic fluid volume W^  = body weight - 52 -II. Biochemistry a) Body weight The experimental mean body weight for the fetuses used for biochemistry (2.981 _+ 0.035) was 96% of the control mean body weight (3.090 +_ 0.033). Though the difference was not statistically significant the probability value (P = 0.06) was close to the arbitrary level of statistical significance chosen for this work. b) Wet lung weight Mean wet lung weights (W^ ) and mean wet lung weight to body weight ratios (WL/W^ ) +_ SEM's are shown in Table III on page 57. Wet lung weight, a measure of lung tissue and fluid mass, was used as an index of lung growth. The experimental mean wet lung weight was significantly less, 87% of the weight of control lungs, with a probability value less than 0.01. When the lung weights were corrected for body size, the experimental mean wet lung weight to body weight ratio was also found to be significantly less, 91% of control mean wet lung weight to body weight, P again less than 0.01. c) Dry lung weight Mean dry lung weights (DWL) and mean dry lung weight to body weight ratios (DWL/Wb) _+ SEM's are shown in Table III on page 57. Dry lung weight, a measure of lung tissue mass, was also used as an index of lung growth. The experimental mean dry lung weight was significantly less, 89% of control mean dry lung weight, P less than 0.01. When dry lung - 53 -weight was corrected for body size, the experimental mean DW^ /Wh w a s also significantly less, 92% of the control mean nWL/Wb, P = 0.03. These findings indicated that not only were the experimental lungs smaller than the control lungs, they were disproportionately smaller than expected for their given body weights. Mean wet lung weight to dry lung weight ratios (WL/DWL) are shown in Table III on page 57. The experimental mean wet .lung weight to dry lung weight ratio was 97% of control mean wet lung weight to dry lung weight ratio. Though the difference was not statistically significant the probability value (P = 0.07) was close to the arbitrary level of statistical significance chosen for this work suggesting a reduction in the fluid content of the experimental lungs. d) DNA content Mean total DNA (DNA), mean total DNA to dry lung weight ratio (DNA/DWL) and mean total DNA to body weight ratio (DNA/Wfa) + SEM's are shown in Table III on page 57. DNA content was measured as an index of cell number. The experimental mean total DNA was 91% of control mean total DNA. Though the difference was not statistically significant the probability value (P = 0.07) was close to the arbitrary level of statistical significance chosen for this work. When total DNA was corrected for body size, the experimental mean DNA/W^  was 93% of control mean DNA/W^  but the difference was not statistically significant, P = 0.16. When total DNA was corrected for lung size, the control and experimental mean total DNA to dry lung weight ratios were nearly identical with the control mean 99.5% of the experimental mean, P = 0.98. - 54 -e) NCT protein content Mean total NCT protein (Protein), mean total NCT protein to dry lung weight ratio (Protein/DWL), mean total NCT protein to body weight ratio (Protein/Wb) and mean total NCT protein to total DNA ratio (Protein/DNA) _+ SEM's are shown in Table III on page 57. The experimental mean total NCT protein was significantly less, 91% of control mean total NCT protein, P = 0.03. When corrected for body size, the experimental mean total NCT protein to body weight ratio was 92% of control mean Protein/W^. Though the difference was not statistically significant the probability value (P < 0.10) was close to the arbitrary level of statistical significance chosen for this work. When corrected for lung size, the control and experimental mean total NCT protein to dry lung weight ratios were nearly identical with the experimental mean 99% of the control mean, P = 0.91. When expressed relative to total DNA the control and experimental means were very similar,,with the control mean total NCT protein to total DNA ratio 99% of the experimental mean (P = 0.58), indicating the size of control and experimental lung cells were very similar. f) Phospholipid and disaturated phosphatidylcholine content Total phospholipid and disaturated phosphatidylcholine contents were measured as an index of the maturation of pulmonary type II cells. Mean total phospholipid (Phospholipid), mean total phospholipid to dry lung weight ratio (Phospholipid/DWL) and mean total phospholipid to body weight ratio (Phospholipid/^) +_ SEM's are shown in Table III on page 57. The experimental mean total phospholipid was significantly less, 89% - 55 -of control mean total phospholipid, P less than 0.05. When corrected for body size, the experimental mean total phospholipid to body weight ratio was also less, 91% of control mean Phospholipid/W^. Though the difference was not s t a t i s t i c a l l y significant the probability value (P = 0.08) was close to the arbitrary level of s t a t i s t i c a l significance chosen for this work. When corrected for lung size, the control and experimental mean total phospholipid to dry lung weight ratios were similar with the experimental mean slightly less, 97% of the control mean, P = 0.43. Mean total disaturated phosphatidylcholine (DSPC), mean total DSPC to dry lung weight ratio (DSPC/DWL), mean total DSPC to body weight ratio (DSPC/W. ), mean total DSPC to total DNA ratio (DSPC/DNA), mean total b DSPC to total NCT protein ratio (DSPC/Protein), and mean total DSPC to total phospholipid ratio (DSPC/Phospholipid) _+ SEM's are shown in Table III on page 57. The experimental mean total DSPC was substantially less, 81% of control mean total DSPC. Though the difference was not st a t i s t i c a l l y significant the probability value (P = 0.06) was close to the arbitrary level of s t a t i s t i c a l significance chosen for this work. When corrected for body size, the experimental mean total DSPC to body weight ratio was 84% of control mean DSPC/W^  but the difference was not st a t i s t i c a l l y significant, P=0.12. When corrected for lung size, the experimental mean was 89% of control mean total DSPC to dry lung weight ratio but the difference was not s t a t i s t i c a l l y significant, P=0.22. When expressed relative to total DNA, the experimental mean total DSPC to total DNA ratio was 87% of control mean DSPC/DNA but the difference was not s t a t i s t i c a l l y significant, P=0.34. When expressed relative to total - 56 -NCT protein, the experimental mean was 86% of control mean t o t a l DSPC to t o t a l NCT protein r a t i o but the difference was not s t a t i s t i c a l l y s i g n i f i c a n t , P=0.13. When expressed r e l a t i v e to t o t a l phospholipid, the experimental mean was 95% of control mean t o t a l DSPC to t o t a l phospholipid r a t i o but the difference was not s t a t i s t i c a l l y s i g n i f i c a n t , P=0.61. - 57 -Table III. Biochemistry Results for Control and Experimental Groups. Variable Control Group N Experimental Group N P WL(mg)+ 111+2 6A 97+2 56 P< .01 WL/Wb(mg/gm)+ 35.2+0.A 6A 31.9+0. 5 56 P< .01 DWL(mg)+ IA.92+0.31 A5 13.21+0. 32 A2 P< .01 DWL/Wb(mq/gm)* A.856+0.088 A5 A.A5A+0. 091 A2 P= .03 WL/DWL(mg/mg) 7.A6+0.12 A5 7.25+0. 12 A2 P= .07 DNA(mg) 0.61A+0.015 A3 0.559+0. 016 AO P= .07 DNA/Wb(mg/gm) 0.201+0.005 A3 0.187+0. 005 AO P= .16 DNA/DWL(mg/gm) A2.0+0.8 A3 A2.2+0. 8 AO P= .98 Protein(mg)* 7.18+0.16 A3 6.50+0. 16 AO P= .03 Protein/Wb(mg/gm) 2.3A9+0.0A3 A3 2.172+0. 0A5 AO P< .10 Protein/DWL(mg/gm) A93.1+ 7.A A3 A88.5+7. 7 AO P= .91 Protein/DNA(mg/mg) 11.93+0.2A A3 12.02+0. 25 AO P= .58 Phospholipid(mg)* 0.132+0.00A 30 0.117+0. 00A. 25 P< .05 Phospholipid/Wb(mg/gm) 0.0A28+0.0011 30 0.0391+0. 0012 25 P= .08 Phospholipid/DW^(mg/gm) 8.63+0.22 30 8.40+0. 2A 25 P= .A3 DSPC(mg) 0.053+0.002 29 0.0A3+0. 002 27 P= .06 D5PC/Wb(mg/gm) 0.0176+0.0007 29 0.01A7+0. 0007 27 P= .12 DSPC/DWL(mg/gm) 3.53+0.13 29 3.13+0. IA 27 P= .22 DSPC/DNA(mg/mg) 0.093+0.006 29 0.081+0. 006 27 P= .3A DSPC/Protein(mg/mg) 0.0077+0.0003 29 0.0066_+0. 000A 27 P= .13 DSPC/Phospholipid(mg/mg) 0.A2+0.02 29 0.A0+0. 02 2A P= .61 Values are means +_ SEM P = probability value for the treatment effect Abbreviations - see text *P < 0.05 +P < 0.01 I I I . Morphometry - 58 -a) Body weight No s i g n i f i c a n t difference was found between control and experimental body weights for the fetuses used for morphometry. The control mean body weight was 3.255 _+ 0.091 grams and the experimental mean body weight was 3.250 _+ 0.091 grams, P = 0.95. There were 10 control fetuses and 10 experimental fetuses. The cases were selected from lungs which were successfully i n f l a t e d , with control and experimental fetuses from the same mother and matched for the closest position i n the uterine horn possible. To obtain the twenty lungs for morphometry I attempted to i n f l a t e 38 experimental lungs and 59 control lungs. At the beginning I dissected out 14 experimental and 18 control lungs, weighed them and then i n f l a t e d them. This technique was not successful due to v i s c e r a l pleura damage. I then i n f l a t e d 24 experimental and 41 control lungs i n s i t u (as described i n the methods section) and from these I obtained the lungs for morphometry. The body weights of the 38 experimental and 59 control fetuses used for morphometry were not s i g n i f i c a n t l y d i f f e r e n t with a control mean body weight of 3.266 _+ 0.038 and an experimental mean body weight of 3.205 +_ 0.047, P = 0.25. I t should be noted that the ten experimental fetuses used for morphometry were i n the upper range of the experimental body weights. b) Fixed lung volume Mean fixed lung volume (V. ) and mean fixed lung volume to body - 59 -weight ratio (V^W^) ± SEM's are shown in Table IV on page 61. Lung volume was also used as an index of lung growth. The experimental mean fixed lung volume was significantly less, 91% of control mean fixed lung volume, P=0.02. When corrected for body size, the experimental mean VL^ Wb w a s a ^ S 0 s i 9 n i f i c a r , t l y less, 91% of control mean fixed lung volume to body weight ratio, P less than 0.01. Retardation of lung growth was further supported by these results. c) Volume fraction and total volume of tissue components Table IV on page 61, shows the mean volume fraction of saccular air (Vy g), saccular wall ( v V w)> conducting air (Vyb) and nonparenchyma (V V n p) for the control and experimental fetuses. No significant differences were found. The probability values were as follows: Vya (P = 0.77), V V w (P = 0.59), V^ (P = 0.66) and V y n p (P = 0.49). When the volume fraction of tissue components were multiplied by the fixed lung volume of each fetus the total volume of each lung substructure was determined. The mean total volume of saccular air (V ), saccular wall (V ), conducting air (V. ) and nonparenchyma 3 W D (V ) for the control and experimental fetuses are shown in Table IV on page 61. The total volume of each of the lung substructures was decreased with the decrease in total volume of saccular wall having a probability value close to the arbitrary level of statistical significance chosen for this work. d) Mean linear intercept The mean linear intercept, a morphometric estimate of the mean size - 60 -of peripheral airspaces at maximum i n f l a t i o n , i s shown for the control and experimental fetuses i n Table IV on page 61. The experimental mean li n e a r intercept was 97% of the control mean l i n e a r intercept but the difference was not s t a t i s t i c a l l y s i g n i f i c a n t , P = 0.72. e) Saccular surface area Saccular surface area was measured as an index of the area of the lung available for gas exchange. Mean saccular surface area ( S w ) , mean gas exchanging surface to volume r a t i o (Sy w) and mean saccular surface area to body weight r a t i o ( s w/ w^) a r e also shown i n Table IV. The experimental and control mean saccular surface areas were nearly i d e n t i c a l with the experimental mean 99% of the control mean S^, P = 0.85. When corrected for body s i z e , the experimental mean was 97% of control mean saccular surface area to body weight r a t i o but the difference was not s t a t i s t i c a l l y s i g n i f i c a n t , P = 0.64. When expressed r e l a t i v e to the volume of saccular a i r , the control mean gas exchanging surface to volume r a t i o was 95% of the experimental mean S V w but the difference was not s t a t i s t i c a l l y s i g n i f i c a n t , P = 0.52. - 61 -Table IV. Morphometry Results for Control and Experimental Groups. Variable Control Group N Experimental Group N p V L(ml)* 0.156 + 0.003 10 0.142 + 0.003 10 P=.02 VL/Wb(ml/gm)+ 0.0476 + 0.0006 10 0.0435 + 0.0006 10 P<.01 Vva 0.4635 + 0.0122 10 0.4564 + 0.0122 10 P=.77 VVw 0.3455 + 0.0075 10 0.3538 + 0.0075 10 P=.59 Vvb 0.0919 + 0.0060 10 0.0981 + 0.0060 10 P=.66 vVnp 0.0990 + 0.0047 10 0.0917 + 0.0047 10 P=.49 V a(ml) 0.073 + 0.003 10 0.066 + 0.003 10 P=.19 V w(ml) 0.053 + 0.002 10 0.050 + 0.002 10 P<.10 V b(ml) 0.015 + 0.001 10 0.013 + 0.001 10 P=.59 V n p(ml) 0.015 + 0.001 10 0.013 + 0.001 10 P=.21 L m (um) 129 + 9 10 125 + 9 10 P=.72 S w (cm2) 48.3 + 2.4 10 47.6 + 2.4 10 P=.85 Sw/Wb(cm2/gm) 14.9 + 0.7 10 14.4 + 0.7 10 P=.64 S V w(cm 2/ml) 689 + 23 10 725 + 23 10 P=.52 Values are means +_ SEM Abbreviations - see text P = probability value for the treatment effect *P < 0.05 +P < 0.01 - 62 -IV. Morphology No consistent qualitative histologic differences were found between the experimental and control lungs. All lungs were in the saccular stage of lung development. The acini of these lungs consisted of relatively smooth walled channels called saccules. Thick secondary crests were beginning to appear in the saccules. When slides were picked at random and examined, i t was not possible to sort out the control lungs from the experimental lungs. DISCUSSION I. General Points It should be noted that the fetuses used for biochemistry were derived from different mothers than the fetuses used for morphometry and morphology. The body weights of the fetuses used for biochemistry were less than the body weights of the fetuses used for morphometry. There was an average of 8 fetuses per mother in both the biochemistry and morphometry groups with a range of 2-13 fetuses per mother in the biochemistry group and 5-11 fetuses per mother in the morphometry group. In addition, the experimental mean body weight for the fetuses used for biochemistry was less than the control mean while the experimental and control mean body weights for the fetuses used for morphometry were similar. Seventy percent of the mothers whose fetuses were used for morphometry had fewer experimental fetuses than control fetuses while this was the case for only 33% of the mothers whose fetuses were used for biochemistry. Thus i t may be that the experimental fetuses used for - 63 -morphometry had a greater nutritional supply as well as more space within the uterine horn, compared to their littermate controls, than the experimental fetuses used for biochemistry, when compared to their littermate controls. This may account for the differences noted in body weight. Thus i t may be that the biochemistry results cannot be extrapolated to the fetuses used for morphometry and vice versa. In this study, 80 experimental and 105 control fetuses were used with 42 experimental and 46 control fetuses used for biochemistry and 38 experimental and 59 control fetuses used for morphometry. Thirty-seven experimental and 45 control amniotic fluid volumes were measured in the latter half of the study after this measurement had been suggested at one of my presentations. Fifty-six experimental and 64 control wet lung weights were measured. Of these wet lung weights, 42 experimental and 46 control weights were from the fetuses used for biochemistry and 14 experimental and 18 control weights were from fetuses used for morphometry. Of the fetuses used for biochemistry one control fetal lung was misplaced. Thus, there were 45 dry lung weights. In addition, 2 experimental and 2 control lung samples were spilled during the extraction procedure and therefore there was 40 experimental and 43 control DNA and NCT protein measurements. Approximately one-third of the way through the biochemistry analysis I decided to study type II cell maturation and thus 27 experimental and 30 control samples were analysed for total phospholipid and DSPC content. One control DSPC and two experimental total phospholipid samples were spilled during processing. - 64 -II. Summary of Results Loss of amniotic fluid on day 16 of gestation in the Sprague-Dawley rat resulted in fetal growth retardation as indicated by a significant reduction in body weight on day 21 of gestation. Lung growth retardation also occurred as indicated by a significant reduction in wet lung weight, dry lung weight, fixed lung volume and total NCT protein content with a reduction in total DNA content (cell number) which was not statistically significant but which had a probability value close to the arbitrary level of statistical significance chosen for this work. When the parameters of lung growth were corrected for body size, the wet lung weight to body weight ratio, dry lung weight to body weight ratio and fixed lung volume to body weight ratio were significantly reduced with a reduction in the total NCT protein to body weight ratio which had a probability value close to P less than 0.05. Since the reduction in total DNA to body weight ratio was not statistically significant, pulmonary hypoplasia did not occur. These findings indicate that the decreased size of the experimental lungs was greater than expected for the decrease in body size and thus loss of amniotic fluid had a direct effect on lung growth without affecting cell proliferation or cell size as indicated by similar total NCT protein to total DNA ratios. There was a reduction in the amount of fluid present within the experimental lungs as indicated by a reduced wet lung weight to dry lung weight ratio. The fetal lung contains fluid within the airways and peripheral airspaces, blood, lung cells and intercellular spaces. Thus several factors may contribute to the reduction in fluid content. The lung cells were similar in size but there was a reduction in the absolute number of cells - 6 5 -in the experimental lungs. There was also a decrease in the total volume of saccular air and a slight reduction in the total volume of conducting air. Though these differences were not statistically significant, together they may account for the reduction in fluid content. The human fetal lung receives about 12% of the cardiac output. Blood is made up of red blood cells and plasma. If the hematocrit of the experimental fetuses was increased, and thus the blood within their lungs contained more red blood cells per unit blood volume and thus less fluid, this would also lead to a reduction in the wet lung weight to dry lung weight ratio. In addition, different amounts of blood may be trapped in the lung at the time of dissection and this could also contribute to differences in the fluid content. Maturation of the pulmonary type II cell appeared to be affected by the loss of amniotic fluid, as indicated by a significant reduction in total phospholipid content and a reduction in total disaturated phosphatidylcholine content with a probability value close to the arbitrary level of statistical significance chosen for this work. Tissue maturity was not affected as indicated by a similar volume fraction of saccular air, mean linear intercept (airspace size) and most importantly, gas exchanging surface area. Morphologically, the experimental lungs appeared to be as mature as the control lungs, with no consistent qualitative histologic differences noted. When the parameters of lung maturation were corrected for body size, there was a reduction in total phospholipid to body weight ratio with a probability value close to the arbitrary level of statistical significance chosen for this work but there was no significant decrease in total DSPC to body weight ratio. - 66 -The experimental saccular surface area to body weight ratio was not significantly different from the control value. When DNA, NCT protein, phospholipid and DSPC were corrected for lung size (dry lung weight), the control and experimental values for each were very similar. Thus, the amount of DNA, NCT protein, phospholipid and DSPC per lung was the appropriate amount per gram of tissue and the differences found in the absolute values were due more to lung growth retardation than body growth retardation. In support of this finding, no significant differences were found in total DSPC to total DNA (cell number), total DSPC to total NCT protein and total DSPC to total phospholipid ratios. It should be noted that while the dry lung weight to body weight ratio was significantly decreased the components of the lung which were measured (DNA, NCT protein and phospholipid), and corrected for body weight, were decreased but not significantly. The absolute values of DNA and NCT protein for the control group of fetuses was similar to that reported in the literature while the absolute values of phospholipid and DSPC were lower than expected. This was most likely due to a poor recovery of phospholipid and DSPC during the extraction procedure. A paper containing measurements of a l l the biochemical components of the 21 day gestation rat lung could not be found. Therefore, several papers were used to calculate the percentage of dry lung weight attributed to each component. According to the reported literature values, DNA, NCT protein and phospholipid account for approximately 70% of the dry lung weight with RNA, glycogen, lipids (other than phospholipid) and connective tissue (collagen, proteoglycans and elastin) accounting for - 67 -the remaining 30%. Thus, the decrease in DNA to body weight, NCT protein to body weight and phospholipid to body weight ratios, along with possible reductions in the other components of the lung, together may account for the significantly decreased dry lung weight to body weight ratio. Similarly, the fixed lung volume to body weight ratio was significantly decreased while the peripheral airspace size, saccular surface area to body weight ratio and total volumes of saccular air, saccular wall, conducting air and nonparenchyma were decreased but not significantly. The reduction in the total volume of saccular wall had a probability value close to the arbitrary level of statistical significance chosen for this work while the total volume of saccular air was decreased but not significantly perhaps due to the large standard deviation from the mean and small number of fetuses studied. The decreases in the total volume of each of the lung substructures together account for the significantly decreased fixed lung volume to body weight ratio. Thus, the lungs were smaller than expected for the size of the experimental fetuses, but they developed normally with no differences in terms of maturation. Of greatest importance is that the number and size of the cells, maturity of the type II cells and gas exchanging surface area of the experimental lungs were not significantly different from the control lungs. Perhaps in the lung there exists a hierarchy of what must be protected during development in adverse conditions, and i t makes sense teleologically, that DNA (cell) and DSPC (surfactant) synthesis and development of an adequate gas exchanging surface area would be protected above NCT protein synthesis and overall lung growth. - 68 -III. Comparison with Related Work These results are similar in some ways, but differ in others, from previously reported results. The protocol used in this study was based on work performed by Symchych and Winchester (1978), with this study including a more detailed examination of the lung. The results of this study were the same as that of the day 16 gestation group of Symchych and Winchester's study, with significantly decreased body weight, lung weight and lung weight to body weight ratio at term. As well, no consistent qualitative histologic differences were found. Symchych and Winchester reported a 33% fetal mortality rate due to puncture of the amniotic sac with an 18-gauge needle. In this study a 20-gauge needle was used with a 26% fetal mortality rate. Seventy-two percent of the dead fetuses were from the bottom half of the uterine horn with 28% from the top half. In abstract form only, Blackburn et al. reported a reduction in the weight of the lung at term following maximum volume amniocentesis only on day 17 of gestation in the rat, with P greater than 0.10 indicating the decrease was not statistically significant (Blackburn et al., 1978). This finding is different from that reported by Symchych and Winchester ' (1978). Blackburn et al. found that DNA content was not influenced by amniocentesis. This was found in the present study in which the loss of amniotic fluid occurred one day earlier, on day 16 of gestation. Amniocentesis did not alter the quantity of lung lipid at birth in the Blackburn et al. study. As well, the phospholipid fraction/lung was not reduced by minimum volume (0.1 ml) amniocentesis and the reduction following maximum volume (0.5 ml+) amniocentesis was not statistically significant, P greater than 0.05. In the present study a significant - 69 -reduction in total phospholipid was found following the loss of amniotic fluid one day earlier. When this was corrected for body size, and more importantly lung size, no significant differences were found. The lungs of the maximum volume amniocentesis group were found to have reduced alveolar space size and type II pneumocytes with few lamellar bodies and l i t t l e glycogen, based on light and electron microscope histology. The present study found no reduction in the size of the peripheral airspaces, based on light microscope observations and morphometric techniques which is less subjective than histologic observations alone. When amniotic sac puncture was performed one day earlier than in the present study, a significant reduction in DNA per.lung, DNA per gram of fetal weight and lung protein to DNA ratio were found at term as well as reduced body weight, lung weight and lung weight to body weight ratio (Moessinger et al., 1983a). Thus, not only was lung growth affected, cell proliferation and cell size were affected resulting in pulmonary hypoplasia and hypotrophy. DNA per gram of dry lung weight needs to be analysed to determine i f the decrease in DNA per lung and per gram of fetal weight is actually due to lung growth retardation as in the present study. Moessinger et al. varied the timing of onset and the duration of oligohydramnios, produced by creating amnio-peritoneal fistulas in the fetal guinea-pig, and found that the earlier the onset of oligohydramnios and the longer its duration, the greater the effect on lung growth in terms of lung DNA per gram of fetal body weight (Moessinger et al., 1984-abstract only). This study differs from the rat studies described above in that the beginning of the oligohydramnios occurred much later in - 70 -lung development in the guinea-pig study (late canalicular-early terminal sac stage) as compared to the rat studies (glandular stage). As well, the guinea-pigs were killed after only 5 or 10 days of oligohydramnios, 13 to 18 days before term (day 68), with term in the guinea-pig ranging from 59 to 75 days. To determine i f the lungs were capable of correcting the effect of oligohydramnios on cell proliferation some lungs should have been studied at term. In a study by Nakayama et al. (1983), as described in the introduction, oligohydramnios produced in the rabbit at 25-days gestation resulted in significantly decreased fetal body weight, lung weight and lung weight to body weight ratio at term (day 31). Lung histology was not affected, as in the present study. Nakayama et al. went on to test the hypothesis that mechanical restriction to lung growth plays a role in the development of pulmonary hypoplasia. They attempted to reduce the thoracic compression, which they believed occurred during oligohydramnios, and found that the lung weight to body weight ratio of these fetuses were significantly higher at term than the lung weight to body weight ratio of the fetuses subjected to oligohydramnios alone. This finding indicates that mechanical restriction, to lung growth plays a role in the development of lung growth retardation and possibly pulmonary hypoplasia (decreased cell number). In a recent abstract, discussed in the introduction, Hislop et al. published a study of the effect of amniocentesis and liquor drainage on lung development in cynomolgus monkeys (Hislop et al., 1984). Following amniocentesis, during a time period eguivalent to human midtrimester amniocentesis, the fetuses were delivered close to term. All animals - 71 -were f i t after delivery with no respiratory problems or skeletal abnormalities. Significant reductions in birth weight, fixed lung volume and number of alveoli and generations of respiratory bronchioles corrected for body weight, were found. In the present study, a significant reduction in body weight and fixed lung volume were also found, along with a significant reduction in the fixed lung volume to body weight ratio. In a t r i a l study, performed as a follow-up for this thesis, the fetuses of 3 Sprague-Dawley rats had their fetal membranes punctured on day 16 of gestation, as in the present study, and were then allowed to be born naturally. All 30 rats were f i t after delivery with no respiratory problems. Thus, the lungs were mature enough to permit adequate gas exchange and sustain l i f e . IV. Relevance to Human Midtrimester Amniocentesis In the present study, loss of amniotic fluid was produced near the end of the glandular stage of fetal lung development in the rat. Human midtrimester amniocentesis takes place at the end of the pseudoglandular phase - beginning of the canalicular phase of lung development. Thus, the present study may be somewhat relevant to human midtrimester amniocentesis. Of particular importance is the possibility of amniocentesis causing respiratory distress. A significantly increased incidence of unexplained respiratory difficulty following amniocentesis has been reported in only one study published to date, as discussed in the introduction. Respiratory distress is often associated with a lack of surfactant lining the peripheral airspaces. This suggests a delay in the - 72 -cellular maturation of the lung. In the present study, total phospholipid was significantly reduced and there was a decrease in total DSPC. These differences were found to be due to growth retardation of the lungs, and that the appropriate amounts of phospholipid and DSPC were present per gram of lung tissue. In the t r i a l study, performed after the experimental work for this thesis, no fetuses experienced respiratory distress at birth. As well, the cynomolgus monkeys, which underwent amniocentesis, did not experience any respiratory problems after delivery close to term (Hislop et al., 1984). The difference in results between the human and animal studies is most likely due to differences in the timing of birth with the animals being born at or near term and the humans being born early, between 34 and 37 weeks gestation. The finding of a significant reduction in the crying vital capacity to body weight ratio following amniocentesis (Vyas et al., 1982) may be due to growth retardation of the lung as was found in the present study. The author feels that loss of amniotic fluid has a significant effect on subsequent lung growth but i f enough time is allowed before birth, the lung will develop normally and provide adequate gas exchange at the time of birth. Thus the end result being a small but good lung. In summary, loss of amniotic fluid on day 16 of gestation in the Sprague-Dawley rat, followed by persistent oligohydramnios until term, resulted in fetal growth retardation and more importantly lung growth retardation. When the lungs were studied in detail they were found to be mature biochemically, morphometrically and morphologically. Thus, no significant effect on the structural units of the lungs were found. Since the fixed lung volume was significantly decreased, with a decrease - 73 -in the t o t a l volume of saccular a i r , while the s t r u c t u r a l units of the lung remained the same i t may be that the number of units i s too few. While t h i s may not be a problem i n terms of su r v i v a l at b i r t h , the long-term effect i s not known. - 74 -BIBLIOGRAPHY 1. Abramovich, D.R. Interrelation of fetus and amniotic fluid. Obstet. Gynecol. Annu. 10:27-43 (1981). 2. Abramovich, D.R., Garden, A., Jandial, L., and Page, K.R. Fetal swallowing and voiding in relation to hydramnios. Obstet. Gynecol. 54:15-20 (1979). 3. Adams, F.H., Desilets, D.T., and Towers, B. Control of flow of fetal lung fluid at the laryngeal outlet. Respir. Physiol. 2:302-309 (1967). 4. Aherne, W.A., and Dunnill, M.S. In Morphometry. Edited by W.A. Aherne and M.S. Dunnill. Edward Arnold Ltd., London. (1982). 5. Alcorn, D., Adamson, T.M., Lambert, T.F., Maloney, J.E., Ritchie, B.C., and Robinson, P.M. Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung. J. Anat. 123:649-660 (1977). 6. Alcorn, D., Adamson, T.M., Maloney, J.E., and Robinson, P.M. Morphological effects of chronic bilateral phrenectomy or vagotomy in the fetal lamb lung. J. Anat. 130:683-695 (1980). 7. Alescio, T. Effect of proline analogue, azetidine-2-carboxylic acid, on the morphogenesis in vitro of mouse embryonic lung. J.  Embryol. Exp. Morphol. 29:439-451 (1973). 8. Alescio, T., and DiMichele, M. Relationship of epithelial growth to mitotic rate in mouse embryonic lung developing in vitro. J.  Embryol. Exp. Morphol. 19:227-237 (1968). 9. Amy, R.W.M., Bowes, D., Burri, P.H., Haines, J., and Thurlbeck, W.M. Postnatal growth .of the mouse lung. J. Anat. 124:131-151 (1977). 10. Avery, M.E., and Mead, J. Surface properties in relation to atelectasis and hyaline membrane disease. Am. J. Pis. Child. 97:517-523 (1959). 11. Bain, A.D., and Scott, J.S. Renal agenesis and severe urinary tract dysplasia. A review of 50 cases, with particular reference to the associated anomalies. Br. Med. J. 1:841-846 (1960). 12. Bennett, M.J. Amniocentesis. In Amniotic Fluid and Its Clinical  Significance. Edited by M. Sandler. Marcel Dekker, Inc., New York. Pp. 27-36 (1981). 13. Biggs, J.S.G., Gaffney, T.J., and McGeary, H.M. Evidence that fetal lung fluid and phospholipids pass into amniotic fluid in late human pregnancy. J. Obstet. Gynaecol. Br. Commonw. 80:125-129 (1973). - 75 -IA. Blackburn, W.R., Logsdon, P.A., and Delli-Bovi, J. Fetal lung development after amniocentesis. Pediatr. Res. 12:515 (1978). 15. Blanc, W.A., Apperson, J.W., and McNally, J. Pathology of the newborn and of the placenta i n oligohydramnios. Bull. Sloane Hosp.  Women Columbia-Presbyt. Med. Cent. 8:51-6A (1962). 16. Bluemink, J.G., van Maurik, P., and Lawson, K.A. Intimate c e l l contact at the epithelial/mesenchymal interface in embryonic mouse lung. J. Ultrastruct. Res. 55:257-270 (1976). 17. Boyden, E.A. Development and growth of the airways. In Lung  Biology in Health and Disease, Volume 6, Development of the Lung. Edited by W.A. Hodson. Marcel Dekker, Inc., New York. Pp. 3-32 (1977). 18. Briggs, V.A., Rei l l y , B.J., and Loewig, K. Lung hypoplasia and membranous diaphragm in the congenital rubella syndrome - a rare case. J. Can. Assoc. Radiol. 2A:126-127 (1973). 19. Bucher, U., and Reid, L. Development of the intrasegmental bronchial tree: The pattern of branching and development of cartilage at various stages of intra-uterine l i f e . Thorax 16:207-218 (1961). 20. Burri, P.H. The postnatal growth of the rat lung. I I I . Morphology. Anat. Rec. 180:77-98 (197A). 21. Burri, P.H., Dbaly, J., and Weibel, E.R. The postnatal growth of the rat lung. I. Morphometry. Anat. Rec. 178:711-730 (197A). 22. Burri, P.H., and Weibel, E.R. Ultrastructure and morphometry of the developing lung. In Lung Biology in Health and Disease, Volume 6, Development of the Lung. Edited by W.A. Hodson. Marcel Dekker, Inc., New York. Pp. 215-227 (1977). 23. Campiche, M.A., Gautier, A., Hernandez, E.I., and Reymond, A. An electron microscope study of the fetal development of human lung. Pediatrics 32:976-99A (1963). 2A. Carmel, J.A., Friedman, F., and Adams, F.H. Fetal tracheal ligation and lung development. Am. J. Pis. Child. 109:A52-A56 (1965). 25. Carson, S.H., Taeusch, H.W., Jr., and Avery, M.E. Inhibition of lung c e l l division after hydrocortisone injection into fetal rabbits. J. Appl. Physiol. 3A:660-663 (1973). 26. Clements, J.A., and Tooley, W.H. Kinetics of surface-active material in the fetal lung. In Lung Biology in Health and Disease, Volume 6, Development of the Lung. Edited by W.A. Hodson. Marcel Dekker, Inc., New York. Pp. 3A9-366 (1977). - 76 -27. Cruikshank, D.P., Varner, M.W., Cruikshank, J.E., Grant, S.S., and Donnelly, E. Midtrimester amniocentesis. An analysis of 923 cases with neonatal follow-up. Am. J. Obstet. Gynecol. 146:204-211 (1983). 28. Davies, G., and Reid, L. Growth of the alveoli and pulmonary arteries in childhood. Thorax 25:669-681 (1970). 29. Delesse, A. Pour determiner la composition des roches. Annales des  Mines 13 (fourth series): 379-388 (1848). 30. deLorimier, A.A., Tierney, D.F. and Parker, H.R. Hypoplastic lungs in fetal lambs with surgically produced congenital diaphragmatic hernia. Surgery 62:12-17 (1967). 31. Dische, Z. Color reactions of nucleic acid components. In The  Nucleic Acids, Volume I. Edited by E. Chargaff and J.N. Davidson. Academic Press, New York. Pp. 285-305 (1955). 32. Dunnill, M.S. Postnatal growth of the lung. Thorax 17:329-333 (1962). 33. Farrell, P.M. Morphologic aspects of lung maturation. In Lung  Development: Biological and Clinical Perspectives, Volume 1, Biochemistry and Physiology. Edited by P.M. Farrell. Academic Press, New York. Pp. 13-25 (1982). 34. Farrell, P.M., and Avery, M.E. Hyaline membrane disease. Am. Rev.  Respir. Pis. 111:657-688 (1975). 35. Finegold, M.J., Katzew, H., Genieser, N.B., and Becker, M.H. Lung structure in thoracic dystrophy. Am. J. Pis. Child. 122:153-159 (1971). 36. Fiske, C.H., and Subbarow, Y. The colorimetric determination of phosphorus. J. Biol. Chem. 66:375-400 (1925). 37. Folch, J., Lees, M., and Sloane Stanley, G.H. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226:497-509 (1957). 38. Gillberg, C, Rasmussen, P., and Wahlstrom, J. Long-term follow-up of children born after amniocentesis. Clin. Genet. 21:69-73 (1982). 39. Gitlin, P., Kumate, J., Morales, C, Noriega, L., and Arevalo, N. The turnover of amniotic fluid protein in the human conceptus. Am.  J. Obstet. Gynecol. 113:632-645 (1972). 40. Goldstein, J.O., and Reid, L.M. Pulmonary hypoplasia resulting from phrenic nerve agenesis and diaphragmatic amyoplasia. Pediatrics 97:282-287 (1980). - 77 -Al. Graham, D., and Sanders, R.C. Amniotic fluid. Semin. Roentgenol. 17:210-218 (1982). A2. .Greene, E.C. Viscera. In Anatomy of the Rat, Volume 27. Noble Offset Printers Inc., New York. Pp. 89 (1959). A3. Hallman, M., Teramo, K., Kankaanpaa, K., Kulovich, M.V., and Gluck, L. Prevention of respiratory distress syndrome: Current view of fetal lung maturity studies. Ann. Clin. Res. 12:36-AA (1980). AA. Herbert, W.N.P., Johnston, J.M., MacDonald, P.C., and Jimenez, J.M. Fetal lung maturation. Human amniotic fluid phosphatidate phosphohydrolase activity through normal gestation and its relation to the lecithin/sphingomyelin ratio. Am. J. Obstet. Gynecol. 132:373-379 (1978). A5. Hieronymi, G. Veranderungen der Lungenstruktur in verschiedenen Lebensaltern. Verh. Dtsch. Ges. Pathol. AA:129-130 (1960). A6. Hill , L.M., Breckle, R., and Ellefson, R.D. The contribution of the fetal kidney to the amniotic fluid lung profile. Am. J. Obstet.  Gynecol. 1A6:709-710 (1983). A7. Hislop, A., and Fairweather, D.V.I. Amniocentesis and lung growth: An animal experiment with clinical implications. Lancet 2(8301-8313):1271-1272 (1982). A8. Hislop, A., and Fairweather, D.V.I. Alveolar number and size in the human fetal and neonatal lung. Am. Rev. Respir. Pis. 129:A208 (198A). A9. Hislop, A., Fairweather, P.V.I., Blackwell, R.J., and Howard, S. The effect of amniocentesis and liquor drainage on lung development in macaca fascicularis. Am. Rev. Respir. Pis. 129:A209 (198A). 50. Hutchins, G.M., Haupt, H.M., and Moore, W. A proposed mechanism for the early development of the human tracheobronchial tree. Anat.  Rec. 201:635-6A0 (1981). 51. Jimenez, J.M., and Johnston, J.M. Fetal lung maturation IV: The release of phosphatidic acid phosphohydrolase and phospholipids into the human amniotic fluid. Pediatr. Res. 10:767-769 (1976). 52. Johnson, M. Indications and techniques for genetic amniocentesis. J. Reprod. Med. 27:557-559 (1982). 53. Kauffman, S.L. Acceleration of canalicular development in lungs of fetal mice exposed transplacehtally to dexamethasone. Lab. Invest. 36:395-A01 (1977). 5A. Kawakami, M., Paul, J.L., and Thurlbeck, W.M. The effect of age on lung structure in male BALB/cNNia inbred mice. Am. J. Anat. 170:1-2 (198A). - 78 -55. King, R.J., and Clements, J.A. Surface active materials from dog lung. II. Composition and physiological correlations. Am. J.  Physiol. 223:715-726 (1972). 56. Kitagawa, M., Hislop, A., Boyden, E.A., and Reid, L. Lung hypoplasia in congenital diaphragmatic hernia. A quantitative study of airway, artery, and alveolar development. Br. J. Surg. 58:342-346 (1971). 57. Kohler, H.G. An unusual case of sirenomelia. Teratology 6:295-302 (1972). 58. Kohler, H.G., Peel, K.R., and Hoar, R.A. Extramembranous pregnancy and amniorrhoea. J. Obstet. Gynaecol. Br. Commonw. 77:809-812 (1970). 59. Kotas, R.V., and Avery, M.E. Accelerated appearance of pulmonary surfactant in the fetal rabbit. J. Appl. Physiol. 30:358-361.(1971). 60. Kotas, R.V., Mims, L.C., and Hart, L.K. Reversible inhibition of lung cell number after glucocorticoid injection into fetal rabbits to enhance surfactant appearance. Pediatrics 53:358-361 (1974). 61. Langston, C, Kida, K., Reed, M., and Thurlbeck, W.M. Human lung growth in late gestation and in the neonate. Am. Rev. Respir. Pis. 129:607-613 (1984). 62. Lanman, J.T., Schaffer, A., Herod, L., Ogawa, Y., and Catellanos, R. Distensibility of the fetal lung with fluid in sheep. Pediatr.  Res. 5:586-590 (1971). 63. Liberman, M.M., Abraham, J.M., and France, N.E. Association between pneumomediastinum and renal anomalies. Arch. Pis. Child. 44:471-475 (1969). 64. Liggins, G.C. Premature delivery of foetal lambs infused with glucocorticoids. J. Endocrinol. 45:515-523 (1969). 65. Liggins, G.C, and Kitterman, J.A. Pevelopment of the fetal lung. In The Fetus and Independent Life. Edited by K. Elliott and J. Whelan. Pitman, London. Pp. 308-330 (1981). 66. Lind, T. The biochemistry of amniotic fluid. In Amniotic Fluid and  its Clinical Significance. Edited by M. Sandler. Marcel Dekker, Inc., New York. Pp. 1-25 (1981). 67. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275 (1951). 68. Marras, A., Mereu, G., Dessi, C, and Macciotta, A. Oligohydramnios and extrarenal abnormalities in Potter syndrome. J. Pediatr. 102:597-598 (1983). - 79 -69. Mason, R.J., Nellenbogen, J., and Clements, J.A. Isolation of disaturated phosphatidylcholine with osmium tetroxide. J. Lipid  Res. 17:281-284 (1976). 70. Medical Research Council Working Party on Amniocentesis. An assessment of the hazards of amniocentesis. Br. J. Obstet.  Gynaecol. 85:Supplement 2 (1978). 71. Minh, H., Douvin, D. Smadja, A., and Orcel, L. Fetal membrane morphology and circulation of the liquor amnii. » Eur. J. Obstet.  Gynecol. Reprod. Biol. 10:213-223 (1980). 72. Moessinger, A.C. The fetal akinesia deformation sequence: An animal model. Pediatrics 72:857-863 (1983). 73. Moessinger, A., Ballantyne, G., James, L.S., and Blanc, W.A. Lung hypoplasia and selective growth retardation in experimental oligohydramnios. Fed. Proc. 37:854 (1978). 74. Moessinger, A.C, Bassi, G.A., Ballantyne, G., Collins, M.H., James, L.S., and Blanc, W.A. Experimental production of pulmonary hypoplasia following amniocentesis and oligohydramnios. Early Hum.  Dev. 8:343-350 (1983a). 75. Moessinger, A.C, Blanc, W.A., Bassi, J.A., Collins, M.H., Kleinerman, J., and James, L.S. Short term oligohydramnios and fetal lung growth. Pediatr. Res. 17:383A (1983b). 76. Moessinger, A.C, Collins, M.H., Blanc, W.A., and Kleinerman, J. Oligohydramnios-induced lung hypoplasia: The influence of timing and duration (Animal model). Lab. Invest. 50:8P (1984). 77. Murai, D.T., Lee, C.H., and Kitterman, J.A. Breathing movements transiently increase lung volumes in fetal sheep. Clin. Res. 31:916A (1983). 78. Nakayama, D.K., Glick, P.L., Harrison, M.R., Villa, R.L., and Noall, R. Experimental pulmonary hypoplasia due to oligohydramnios and its reversal by relieving thoracic compression. J. Pediatr. Surg. 18:347-353 (1983). 79. National Institute of Child Health and Human Development National Registry for Amniocentesis Study Group. Midtrimester amniocentesis for prenatal diagnosis. J. Am. Med. Assoc. 236:1471-1476 (1976). 80. Newman, L.M., and Masters, E.M. In vitro lung development: Effect of B£ and dexamethasone. Teratology 29:48A-49A (1984). 81. Niswander, K.R. Diagnosis and management of pregnancy. In Obstetrics, Essentials of Clinical Practice, Second Edition. Edited by K.R. Niswander. Little, Brown and Company, Boston. Pp. 51 (1981). - 80 -82. Normand, I.C.S., Olver, R.E., Reynolds, E.O.R., Strang, L.B. and Welch, K. Permeability of lung capillaries and alveoli to non-electrolytes in the foetal lamb. J. Physiol. Lond. 219:303-330 (1971) . 83. Perelman, R.H., Engle, M.J., and Farrell, P.M. Perspectives on fetal lung development. Lung 159:53-80 (1981). 84. Perlman, M., and Levin, M. Fetal pulmonary hypoplasia, anuria and oligohydramnios: Clinicopathologic observations and review of the literature. Am. J. Obstet. Gynecol. 118:1119-1123 (1974). 85. ' Perlman, M., Williams, J., and Hirsch, M. Neonatal pulmonary hypoplasia after prolonged leakage of amniotic fluid. Arch. Pis.  Child. 51:349-353 (1976). 86. Pettit, B.R., and Fry, P.E. Corticosteroids in amniotic fluid and their relationship to fetal lung maturation. J. Steroid Biochem. 9:1245-1249 (1978). 87. Potter, E.L. Bilateral renal agenesis. J. Pediatr. 29:68-76 (1946). 88. Reale, F.R., and Esterly, J.R. Pulmonary hypoplasia: A morphometric study of the lungs of infants with diaphragmatic hernia, anencephaly and renal malformations. Pediatrics 51:91-96 (1973). 89. Renert, W.A., Berdon, W.E., Baker, P.H., and Rose, J.S. Obstructive urologic malformations of the fetus and infant - relation to neonatal pneumomediastinum and pneumothorax. Radiology 105:97-105 (1972) . 90. Roberts, N.S., Dunn, L.K., Weiner, S., Godmilow, L., and Miller, R. Midtrimester amniocentesis. Indications, technique, risks and potential for prenatal diagnosis. J. Reprod. Med. 28:167-188 (1983). 91. Scherle, W. A simple method for volumetry of organs in quantitative stereology. Mikroskopie 26:57-60 (1970). 92. Schmidt, G., and Thannhauser, S.J. A method for the determination of desoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. J. Biol. Chem. 161:83-89 (1945). 93. Schneider, W.C. Phosphorus compounds in animal tissues. I. Extraction and estimation of desoxypentose nucleic acid and of pentose nucleic acid. J. Biol. Chem. 161:293-303 (1945). 94. Seeds, A.E. Water metabolism of the fetus. Am. J. Obstet.  Gynecol. 92:727-745 (1965). 95. Seeds, A.E. Current concepts of amniotic fluid dynamics. Am. J. Obstet. Gynecol. 138:575-586 (1980). - 81 -96. Shepard, T.H. Back cover of Catalog of Teratogenic Agents, Fourth Edition. Edited by T.H. Shepard. The Johns Hopkins University Press, Baltimore (1983). 97. Simpson, N.E., Dallaire, L., Miller, J.R., Siminovich, L., Hamerton, J.L., Miller, J., and McKeen, C. Prenatal diagnosis of genetic disease in Canada: Report of a collaborative study. Can. Med.  Assoc. J. 115:739-748 (1976). 98. Smith, B.T. Lung maturation in the fetal rat: Acceleration by injection of fibroblast-pneumonocyte factor. Science 204:1094-1095 (1979). 99. Smith, B.T., and Bogues, W.G. Effects of drugs and hormones on lung maturation in experimental animal and man. Pharmacol. Ther. 9:51-74 (1982). 100. Spooner, B.S., and Faubion, J.M. Collagen involvement in branching morphogenesis of embryonic lung and salivary gland. Dev. Biol. 77:84-102 (1980). 101. Stephens, J.D., and Birnholz, J.C. Noninvasive verification of fetal respiratory movements in normal pregnancy. J. Am. Med.  Assoc. 240:35-36 (1978). 102. Stigol, L.C., Vawter, G.F., and Mead, J. Studies on elastic recoil of the lung in a pediatric population. Am. Rev. Respir. Pis. 105:552-563 (1972). 103. Strang, L.B. Growth and development of the lung: Fetal and postnatal. Annu. Rev. Physiol. 39:253-276 (1977). 104. Swischuk, L.E., Richardson, C.J., Nichols, M.M., and Ingman, M.J. Primary pulmonary hypoplasia in the neonate. J. Pediatr. 95:573-577 (1979). 105. Symchych, P.S., and Winchester, P. Animal model of human disease. Potter's Syndrome. Animal model: Amniotic fluid deficiency and fetal lung growth in the rat. Am. J. Pathol. 90:779-782 (1978). 106. Taderera, J.V. Control of lung differentiation in vitro. Pev.  Biol. 16:489-512 (1967). 107. Tanswell, A.K., and Smith, B.T. Human fetal lung type II pneumonocytes in monolayer cell culture: The influence of oxidant stress, Cortisol environment, and soluble fibroblast factors. Pediatr. Res. 13:1097-1100 (1979). 108. Thomas, I.T., and Smith, P.W. Oligohydramnios, cause of the nonrenal features of Potter's syndrome, including pulmonary hypoplasia. Pediatrics 84:811-814 (1974). - 82 -109. Thurlbeck, W.M. Postnatal human lung growth. Thorax 37:564-571 (1982). 110. Thurlbeck, W.M., and Angus, G.E. Growth and aging of the normal human lung. Chest 67:3S-7S (1975). 111. Towers, B. Amniotic fluid and the foetal lung. Nature 183:1140-1141 (1959). 112. Turnbull, A.C, and MacKenzie, I.Z. Second-trimester amniocentesis and termination of pregnancy. Br. Med. Bull. 39:315-321 (1983). 113. Vyas, H., Milner, A.D., and Hopkin, I.E. Amniocentesis and fetal lung development. Arch. Pis. Child. 57:627-628 (1982). 114. Wang, N.S., Kotas, R.V., Avery, M.E., and Thurlbeck, W.M. Accelerated appearance of osmiophilic bodies in fetal lungs following steroid injection. J. Appl. Physiol. 30:362-365 (1971). 115. Weibel, E.R. In Stereological Methods, Volume 1, Practical Methods for Biological Morphometry. Edited by E.R. Weibel. Academic Press, London. (1979). 116. Wessells, N.K. Mammalian lung development: Interactions in formation and morphogenesis of tracheal buds. J. Exp. Zool. 175:455-466 (1970). 117. Whitfield, C.R. Amniotic fluid. In Scientific Basis of Obstetrics  and Gynaecology. Edited by R.R. Macdonald. Churchill Livingstone, Edinburgh. Pp. 1-25 (1978). 118. Wigglesworth, J.S., and Pesai, R. Effects on lung growth of cervical cord section in the rabbit fetus. Early Hum. Pev. 3:51-65 (1979). 119. Wigglesworth, J.S., and Desai, R. Use of DNA estimation for growth assessment in normal and hypoplastic fetal lungs. Arch. Pis. Child. -56:601-605 (1981). 120. Wigglesworth, J.S., and Pesai, R. Is fetal respiratory function a major determinant of perinatal survival? Lancet 1:264-267 (1982). 121. Wigglesworth, 3.5., Winston, R.M.L., and Bartlett, K. Influence of the central nervous system on fetal lung development. Arch. Pis.  Child. 52:965-967 (1977). 122. Williams, M.C, and Mason, R.J. Pevelopment of the type II cell in the fetal rat lung. Am. Rev. Respir. Pis. 115 (Part 2):37-47 (1977). 123. Zar, J.H. Two-factor analysis of variance. In Biostatistical  Analysis. Edited by W.P. McElroy and CP. Swanson. Prentice-Hall, Inc., Englewood Cliffs, New Jersey. Pp. 163-169 (1974). 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0095997/manifest

Comment

Related Items