Postnatal Growth and Development of the Lung' WILLIAM M. THURLBECK
Contents
Introduction Growth and Development of the Fetal Lung Stages of Development of the Human Lung Maturation and Control of Development of the Alveolar Wall Control of Branching of the Fetal Lung Postnatal Growth and Development of the Lung Laboratory Animals Body, Organ, and Lung Growth Incorporation of Tritiated Thymidine into Lung Cell Nuclei Morphogenesis of the Acinus in Animal Lungs Growth of Other Parts of the Lung Growth of the Human Lung Alveolar Growth and Multiplication Morphogenesis of the Acinus, with Recapitulation of Embryology Growth of Other Structures in the Lung Airways Vascular System Differences between Tall and Short Subjects Alterations in Postnatal Lung Growth Introduction
There has been general interest in the postnatal development of the lung in the last 15 years, and more recently, particular interest in the way that lung growth can be manipulated. It is perhaps artificial to divide lung growth and development sharply into antenatal and postnatal phases. Birth really represents a minor event in the development of the lung of many species, but there are several reasons for concentrating this review primarily on postnatal growth. The first is that the antenatal development of the lung has been well studied and described and is 1 From the Midhurst Medical Research Institute, Midhurst, Sussex, England GU29 OBL.
Problems of Interpretation Resection of Lung Tissues Animals Control of Regeneration of Lung Tissue Changes in Human Lungs after Pneumonectomy Alterations in Chest Cage and Diminution of Lung Volume Animals Man Alterations in Ambient Oxygen and Pressure Hypoxia Animals Man High Altitude and Experimental Hypobaric Conditions Animals Man Hyperoxia Exercise, Oxygen Consumption, and Thyroid Function Growth Hormone Animals Experimental and Naturally Occurring Disease Summary generally well known. More important, it appears likely that most, if not all, of the alveoli develop after birth in many species; much of the information is recent, and reviews are not readily available. Postnatal growth has long been a subject of general interest to biologists, and the lung's unique expansile character has suggested to some in the past that the lung could grow only, or mainly, by expansion. Such a notion would be regarded as ludicrous when applied to other organs, such as the liver or kidney, and, as will be shown, cell multiplication and enlargement occur in the lung in much the same way as they do in many other organs. A simple observation stresses the extent to which the lung grows; in the human at birth, the lung weighs approxi-
AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME Ill, 1975
803
804
WILLIAM M. THURLBECK
mately 60 g, whereas it weighs 10 times this, or more, in the average adult. The lung differs from other organs in that its total volume increases more than its tissue mass, and thus it contains progressively more air per gram of tissue throughout childhood. For example, the human lung contains approximately 3 ml of air per g of tissue at full inflation at birth, and approximately 8 ml of air per g of tissue at 6 years of age (1). That the increase in the ratio of volume to tissue mass with age also occurs in rabbits and is just as much a feature of intrauterine life as postnatal life is illustrated in figure I. Postnatal growth of the lung should also not be viewed as a problem fundamentally different from growth in other organs; it is subject to control mechanisms common to many organs. Each organ presumably has its own specific features, and characteristics of the lung besides its expansile, air-filled nature make it different from other organs. The lung constitutes the major content of the thoracic cavity; hence, alteration of the size and shape of the chest wall might be anticipated to have significant effects on the lung. In general terms, an organ enlarges by increasing the number of its cells, the size of the cells, or both. Within the organ concerned, the cells may be arranged in units (such as the nephron) so that the units may enlarge, multiply, or be modified with growth and development. Such is the case of the lung; cell multiplication occurs, but it occurs in a particular way, so that the gasexchanging units (acini) undergo extensive remodeling in the postnatal period, and many new alveoli are formed. Postnatal growth and development are thus
0
only parts of the subject of lung growth, which, in turn, is only a facet of the wider fundamental biologic problem of development. Relevant, or newer aspects, of intrauterine development will be briefly discussed first, and these will be followed by a detailed description of the postnatal growth of the lung. Experimental manipulation or naturally occurring alterations of lung growth will be reviewed next; for both normal and abnormal growth, data from animals will be considered first, because the data are more complete, and they will be followed by information concerning humans. Growth and Development of the Fetal Lung
The degree of development of the lung at birth varies widely, but little is known in detail concerning the differences between species. It has been suggested that lung development parallels general body development at birth (2). The opossum lung is very primitive; rats and mice have lungs that have no alveoli at birth, and kittens, calves, and humans have few alveoli, whereas the lungs of lambs are quite well developed (2). In the latter species at birth, the lungs are considerably larger, heavier, and have greater alveolar surface area than the lungs of human infants (3). Presumably, African antelopes that must be able to run, often for their lives, soon after birth, and aquatic mammals that must swim immediately postnatally have lungs that are even better developed. Stages of Development of the Human Lung
The lung develops from a laryngotracheal groove in the endodermal tube, although in mice,
100
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75
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90
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Intra-uterine
AGE (days)
Fig. I. When the percentage of the lung that is tissue (iqterstitial tissue) is plotted against age in the rabbit, it is apparent that there is a decrease throughout intrauterine life that continues into the postnatal period until at least 3 months of age. Graphical illustrations of tabular data from
Short (53).
POSTNATAL GROWTH AND DEVELOPMENT OF THE LUNG
the lung starts from 2 lateral buds that fuse cephalad to form the tracheal bud (4). In the human, the ventral groove appears when the embryo is 26 days of age and evaginates to form the lung bud, which branches at 26 to 28 days of age (5). A new terminology has been suggested for the phases of lung development (6). The new term, "the embryonic period," has been introduced to cover the first 5 weeks after ovulation, and it includes the earliest phases of lung development. Other new terms are the "pseudoglandular period" and the "terminal sac period," which replace the terms "glandular" and "alveolar," respectively. The bronchial buds continue to divide and subdivide by asymmetric dichotomy; 6.1) to 75 per cent of bronchial branching occurs between the tenth and fourteenth weeks, and bronchial development is complete by the sixteenth week (7). At this stage, the lung has a distinctly glandular appearance, with airways liped by columnar epithelium and separated from each other by poorly differentiated mesenchyme. The lung is obviously not truly glandular, and the term "pseudoglandular" is preferable. This is succeeded by the · "canalicular phase" (8-1 0), which lasts from the sixteenth to approximately the twenty-fourth to twenty-sixth week and is characterized by the proliferation of mesenchyme and the development of a rich blood supply within it, together with flattening of the epithelium that lines the airways. The epithelium becomes irregularly thinned, and cellular continuity between the cells may be lost at the margins of the cells, maintained only at their bases. Capillaries protrude into the epithelium, and occasional areas of thin blood-airway barrier, similar to those of the adult, appear. These become more frequent, and toward the end of the period, the various types of alveolar lining epithelium (Type I and Type II) can be seen, together with occasional osmiophilic bodies in the Type II cells (11). Until application of the electron microscope, it was widely speculated that the epithelial cells degenerated and desquamated, thus exposing capillaries directly to the lumen of the airways, without interposition of epithelium (9); however, no epithelial discontinuities were encountered when the lungs of developing mice were examined using the electron microscope (12), nor was evidence of epithelial degeneration found. Progressive thinning of epithelium and protrusion of capillaries leads to many more areas of close approximation of capillary lumen to airway surface, and the terminal generations of the airways are lined
805
only by flattened epithelium. Respiration can now be maintained. Alveoli are not present in the walls of these terminal sacs, so that this phase should not be referred to as the "alveolar period," but rather as the "terminal sac period"; this phase commences at approximately the twenty-fourth to twenty-sixth week in the human. Some species, notably the rat and the mouse, are born during this period, and alveoli are not present at birth (see below). The situation in the human is controversial and is also discussed further below (see pp. 826-828), but it seems likely that quite a considerable number of alveoli are present in humans at birth. Emery and Mithal (13) found twice as many alveolar wall intersections between respiratory bronchioles and the periphery of the acinus at term as at 24 to 27 weeks of gestation, and it was only at age 8 years that a further doubling of intersections was noted. Thus, in the human, at any rate, there may also be an intrauterine "alveolar period," but its time of onset is uncertain.
Maturation and Control of Development of the Alveolar Wall The most recent studies of intrauterine lung development have demonstrated quite conclusively the endodermal origin of Type I and Type II epithelial cells of the alveolus and the mesodermal origin of the "primary interstitial cell" or "fibroblast" of the alveolar wall. The latter is characterized by the presence of numerous intracytoplasmic lipid inclusions, and it is an important cell in the postnatal development of alveoli in rats and mice. O'Hare and Sheridan (14) have shown that a distinct basal lamina is present at day 16 of gestation in rats and that this is never subsequently breached by cells or their extensions. In their study, an obvious morphologic transition existed between endodermal cells, bronchial cells, and Type I and Type II epithelial cells. These cells were always on the same side of the basal lamina and were connected by tight junctions. A similar obvious morphologic resemblance existed between mesodermal cells and the interstitial cells, which were always contained within the interstitium. Type I and Type II epithelial cells also showed the autoradiographic labeling characteristics of the endoderm, whereas the primary interstitial cell had the characteristics of mesoderm (15). O'Hare and co-workers (15) also stressed that glycogen occurred in the sites of rapid epithelial division, an observation first noted by Sorokin (16). Tritiated thymidine labeling in vivo from day 16 onward
806
WILLIAM M. THURLBECK
(15) and mitotic counts of developing lung in vitro show higher rates in endoderm than in mesoderm (16). The relationship of mitotic activity and the presence of glycogen link with 2 recent investigative thrusts concerning the developing lung. It is now well known that administration of steroids to fetuses accelerates the production of surfactant and induces premature delivery in fetal lambs (17-20); only glucocorticoids are effective. Steroid administration results in diminution of lung size, because there are too few cells in the lung, suggesting that maturation and surfactant secretion occur at the expense of lung growth (18-20). Conversely, intrauterine decapitation, which destroys the pituitary-adrenal-thyroid axis, retards cytodifferentiation and results in large lungs with cells that have excess quantities of glycogen (21). The complementary thrust has come from laboratories in Montreal and San Francisco (2228). These investigators have demonstrated the presence of specific glucocorticoid receptors in the fetal lungs of humans, rabbits, rats, and guinea pigs. Cytoplasmic macromolecules that form an 8S complex with steroids act as receptors to transfer the steroids to nuclear receptors. Only approximately 6,000 binding sites were found per nucleus in the fetal lung, and these were saturated with physiologic concentrations of cortisol. Glucocorticoid binding sites are more frequent in the lung than in other organs and 4 to 5 times more frequent than in the liver of rabbits and humans. Receptor activity reaches a maximum at days 28 to 30 of intrauterine life in rabbits. Rabbits are the only species in which receptors have been demonstrated in adults; in rats they disappear 2 days after birth. The hypothesis is that glucocorticoids act at a nuclear level via a receptor-mediated mechanism to trigger the synthesis of a specific ribonucleic acid (RNA) that codes for particular proteins. The proteins may be enzymes involved in the biosynthesis of surface-active phospholipids or enzymes involved in the breakdown of glycogen. Glycogen may also provide the energy or material for cell proliferation, accounting for its presence in the areas of rapid cell multiplication, as noted above. Sorokin (16) noted that cyanide, which destroys terminal oxidase activity, or malonate, which interferes with the Krebs tricarboxylic acid cycle activity, did not alter lung growth in culture. Fluoride, which prevents glycolysis, stops lung growth. Thus, it seems that steroids may divert biochemical activity derived from
glycogen from lung multiplication to surfactant secretion. As will be shown subsequently, elastic tissue appears to play a key role in postnatal alveolar development, and thus its intrauterine appearance is of interest. In the human (29, 30), elastic tissue begins to appear in arteries, trachea, and bronchi in the second and third months in utero and is quite well developed by the end of this period. In contrast, elastic tissue appears only in the peripheral part of the lung during the terminal sac period and at approximately 7.5 months of gestation. Elastic fibers are thin and limited to the mouths of alveoli. A recent paper has described the fine structure of elastogenesis in the rat (31). Elastic fibers become apparent in the walls of airways on the sixteenth day of gestation and seem to be secreted by myoblasts, because these are the only cells in the vicinity of the newly formed fibers. At days 20 and 21, mature fibroblasts appear in the "alveolar" (saccular) zone, and newly secreted elastic tissue is found adjacent to the fibroblasts and accompanies the appearance of the rudimentary alveoli. Control of Branching of the Fetal Lung
Recent experiments have stressed the importance of the interaction between mesoderm and endoderm in producing differentiation in the lung. It has long been known that the lung is self-differentiating in organ culture and that branching of the lung rudiment requires lung mesoderm (32). Interdependence of mesoderm and endoderm was shown by Taderera (32), who found that chick mesoderm would stimulate mouse endoderm to branch and that after a few days, the resultant lung resembled chicken lung, rather than mouse lung, in airway branching pattern. Conversely, epithelium induces mesodermal differentiation. Other experiments (4, 33) have shown that gut mesoderm will induce bronchial budding in the mouse, but that bronchial branching requires bronchial mesoderm, i.e., mesoderm from more mature embryos in which bronchial branching has started or is about to start. Bronchial or gut mesoderm will also induce budding in tracheal epithelium, but tracheal mesoderm will inhibit bronchial budding and branching. These experiments indicate the interdependence of mesenchyme and endoderm in differentiation, and it has also been postulated that mesenchyme contains an "epithelial growth factor" that can regulate the proliferation of epithelium (34).
807
POSTNATAL GROWTH AND DEVELOPMENT OF THE LUNG
Postnatal Growth and Development of the Lung
Thus, the amount and quality of the connective tissue may also be limiting factors in growth and differentiation of the postnatal lung. Even subtler and closer relationships may exist, because transient gaps in the basal lamina have been reported, allowing epithelial-mesenchymal cell contact, in developing duodenum at the time of subtle epithelial differentiation (35). As mentioned above, the basal lamina of the lung has been carefully studied during gestational life and found continuous, so that these "epitheliolo-mesenchymal" contact sites may be of little relevance in the lung.
Laboratory Animals Lung growth must be considered in relation to growth of the whole organism and growth in other organs. It is not always recognized that the growth rate of the whole organism is most rapid immediately postnatally, declining gradually and steadily thereafter, other than for a brief reversal of this decline at the time of puberty (36). Even then the growth rate in humans is noticeably less than during the first year of life. The rate of increase of organ weight is similarly greatest soon after birth, but the behavior of individual
Whole Body
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Fig. 2. At birth, a rat has approximately 2 to 3 X I 09 nuclei (upper broken line) in its body and approximately 70 X 109 nuclei at 90 days of age. The solid line is the amount of deoxyribonucleic acid (D;-..rA) in the whole animal, and the body weight per nucleus is shown by the lower broken line (from figure I, reference 37, courtesy Dr. C. P. Leblond and the Editor, journal of Embryology and Experimental Morphology).
808
WILLIAM M. THURLBECK
.8
LUNG
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DAYS
Fig. 3. The amount of protein per nucleus (ordinate in m,ug. or ng) shows an increase with age in the whole body of the rat, indicating increasing cell size. This is mostly due to increase in cell size in tissues, such as fat and muscle, and in intercellular connective tissue. Lung and kidney ftrst in· crease by cell multiplication, so that whereas the total amount of deoxyribonucleic acid increases (figure 4), the amount of protein per nucleus remains the same. After approximately 2 weeks of age, cells enlarge as well as multiply, and the amount of protein per nucleus increases for approximately 2 months in the lung and approximately 6 weeks in the kidney. Cellular multiplication becomes slow in the lung by 2 weeks of age and in the kidney by approximately 1 month (figure 4). Spleen and thymus grow by cellular multiplication and renewal only, without cell enlargement (figure 4 from reference 38, courtesy of the authors and the Editor, Developmental Biology).
organs differs. Furthermore, their components, nuclei, cytoplasm, protein, and interstitial tissue, increase at different rates. The lung has the unique feature that its growth in -volume and weight may show considerable disassociation because of varying degrees of inflation. Body, Organ, and Lung Growth The details of organ growth and body growth have been well worked out in the rat (37, 38). At birth, there are approximately 2 to 3 X 109 nuclei in the whole animal, increasing to 67 X 109 at 95 days of age (figure 2). The weight of deoxyribonucleic acid (DNA), and hence the number of nuclei, usually does not quite match the increase in body weight even during the first 2 weeks of life, and thus, weight per nucleus tends to increase slowly. The amount of water in tissues and organs varies with age, so that relationships are better expressed as amount of protein per nucleus (figure 3). Body protein in-
creases faster than DNA, and thus protein per nucleus increases for most of the growing period. This is primarily due to increase in cell cytoplasm in tissues such as fat and muscle and increase in intercellular connective tissue. A similar, although slightly different, pattern is seen in most organs, as opposed to tissues and the whole body, and each organ, in turn, differs slightly from the others (figures 3-5). In general, organs show 3 phases. From birth to 14 to 17 days of age, organs grow primarily by cell multiplication; the amount of DNA increases rapidly, and the amount of protein per nucleus remains approximately the same. This is followed bv a phase in which cell multiplication is slower than protein synthesis; thus, whereas the amount of DNA and the number of nuclei still increase, there is an increase in the amount of protein per nucleus. This phase continues to approximately 5 to 7 weeks of age. In the final phase, cell proliferation stops, or is very slow,
809
POSTNATAL GROWTH AND DEVELOPMENT OF THE LUNG
128-
64-
32-
Contents
Introduction Growth and Development of the Fetal Lung Stages of Development of the Human Lung Maturation and Control of Development of the Alveolar Wall Control of Branching of the Fetal Lung Postnatal Growth and Development of the Lung Laboratory Animals Body, Organ, and Lung Growth Incorporation of Tritiated Thymidine into Lung Cell Nuclei Morphogenesis of the Acinus in Animal Lungs Growth of Other Parts of the Lung Growth of the Human Lung Alveolar Growth and Multiplication Morphogenesis of the Acinus, with Recapitulation of Embryology Growth of Other Structures in the Lung Airways Vascular System Differences between Tall and Short Subjects Alterations in Postnatal Lung Growth Introduction
There has been general interest in the postnatal development of the lung in the last 15 years, and more recently, particular interest in the way that lung growth can be manipulated. It is perhaps artificial to divide lung growth and development sharply into antenatal and postnatal phases. Birth really represents a minor event in the development of the lung of many species, but there are several reasons for concentrating this review primarily on postnatal growth. The first is that the antenatal development of the lung has been well studied and described and is 1 From the Midhurst Medical Research Institute, Midhurst, Sussex, England GU29 OBL.
Problems of Interpretation Resection of Lung Tissues Animals Control of Regeneration of Lung Tissue Changes in Human Lungs after Pneumonectomy Alterations in Chest Cage and Diminution of Lung Volume Animals Man Alterations in Ambient Oxygen and Pressure Hypoxia Animals Man High Altitude and Experimental Hypobaric Conditions Animals Man Hyperoxia Exercise, Oxygen Consumption, and Thyroid Function Growth Hormone Animals Experimental and Naturally Occurring Disease Summary generally well known. More important, it appears likely that most, if not all, of the alveoli develop after birth in many species; much of the information is recent, and reviews are not readily available. Postnatal growth has long been a subject of general interest to biologists, and the lung's unique expansile character has suggested to some in the past that the lung could grow only, or mainly, by expansion. Such a notion would be regarded as ludicrous when applied to other organs, such as the liver or kidney, and, as will be shown, cell multiplication and enlargement occur in the lung in much the same way as they do in many other organs. A simple observation stresses the extent to which the lung grows; in the human at birth, the lung weighs approxi-
AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME Ill, 1975
803
804
WILLIAM M. THURLBECK
mately 60 g, whereas it weighs 10 times this, or more, in the average adult. The lung differs from other organs in that its total volume increases more than its tissue mass, and thus it contains progressively more air per gram of tissue throughout childhood. For example, the human lung contains approximately 3 ml of air per g of tissue at full inflation at birth, and approximately 8 ml of air per g of tissue at 6 years of age (1). That the increase in the ratio of volume to tissue mass with age also occurs in rabbits and is just as much a feature of intrauterine life as postnatal life is illustrated in figure I. Postnatal growth of the lung should also not be viewed as a problem fundamentally different from growth in other organs; it is subject to control mechanisms common to many organs. Each organ presumably has its own specific features, and characteristics of the lung besides its expansile, air-filled nature make it different from other organs. The lung constitutes the major content of the thoracic cavity; hence, alteration of the size and shape of the chest wall might be anticipated to have significant effects on the lung. In general terms, an organ enlarges by increasing the number of its cells, the size of the cells, or both. Within the organ concerned, the cells may be arranged in units (such as the nephron) so that the units may enlarge, multiply, or be modified with growth and development. Such is the case of the lung; cell multiplication occurs, but it occurs in a particular way, so that the gasexchanging units (acini) undergo extensive remodeling in the postnatal period, and many new alveoli are formed. Postnatal growth and development are thus
0
only parts of the subject of lung growth, which, in turn, is only a facet of the wider fundamental biologic problem of development. Relevant, or newer aspects, of intrauterine development will be briefly discussed first, and these will be followed by a detailed description of the postnatal growth of the lung. Experimental manipulation or naturally occurring alterations of lung growth will be reviewed next; for both normal and abnormal growth, data from animals will be considered first, because the data are more complete, and they will be followed by information concerning humans. Growth and Development of the Fetal Lung
The degree of development of the lung at birth varies widely, but little is known in detail concerning the differences between species. It has been suggested that lung development parallels general body development at birth (2). The opossum lung is very primitive; rats and mice have lungs that have no alveoli at birth, and kittens, calves, and humans have few alveoli, whereas the lungs of lambs are quite well developed (2). In the latter species at birth, the lungs are considerably larger, heavier, and have greater alveolar surface area than the lungs of human infants (3). Presumably, African antelopes that must be able to run, often for their lives, soon after birth, and aquatic mammals that must swim immediately postnatally have lungs that are even better developed. Stages of Development of the Human Lung
The lung develops from a laryngotracheal groove in the endodermal tube, although in mice,
100
I
0
~
75
~
;;;
...-;; ~
50
~
*
25
~1--- 18
20
22
24 26
28
30
10
90
Adult
Post-natal
Intra-uterine
AGE (days)
Fig. I. When the percentage of the lung that is tissue (iqterstitial tissue) is plotted against age in the rabbit, it is apparent that there is a decrease throughout intrauterine life that continues into the postnatal period until at least 3 months of age. Graphical illustrations of tabular data from
Short (53).
POSTNATAL GROWTH AND DEVELOPMENT OF THE LUNG
the lung starts from 2 lateral buds that fuse cephalad to form the tracheal bud (4). In the human, the ventral groove appears when the embryo is 26 days of age and evaginates to form the lung bud, which branches at 26 to 28 days of age (5). A new terminology has been suggested for the phases of lung development (6). The new term, "the embryonic period," has been introduced to cover the first 5 weeks after ovulation, and it includes the earliest phases of lung development. Other new terms are the "pseudoglandular period" and the "terminal sac period," which replace the terms "glandular" and "alveolar," respectively. The bronchial buds continue to divide and subdivide by asymmetric dichotomy; 6.1) to 75 per cent of bronchial branching occurs between the tenth and fourteenth weeks, and bronchial development is complete by the sixteenth week (7). At this stage, the lung has a distinctly glandular appearance, with airways liped by columnar epithelium and separated from each other by poorly differentiated mesenchyme. The lung is obviously not truly glandular, and the term "pseudoglandular" is preferable. This is succeeded by the · "canalicular phase" (8-1 0), which lasts from the sixteenth to approximately the twenty-fourth to twenty-sixth week and is characterized by the proliferation of mesenchyme and the development of a rich blood supply within it, together with flattening of the epithelium that lines the airways. The epithelium becomes irregularly thinned, and cellular continuity between the cells may be lost at the margins of the cells, maintained only at their bases. Capillaries protrude into the epithelium, and occasional areas of thin blood-airway barrier, similar to those of the adult, appear. These become more frequent, and toward the end of the period, the various types of alveolar lining epithelium (Type I and Type II) can be seen, together with occasional osmiophilic bodies in the Type II cells (11). Until application of the electron microscope, it was widely speculated that the epithelial cells degenerated and desquamated, thus exposing capillaries directly to the lumen of the airways, without interposition of epithelium (9); however, no epithelial discontinuities were encountered when the lungs of developing mice were examined using the electron microscope (12), nor was evidence of epithelial degeneration found. Progressive thinning of epithelium and protrusion of capillaries leads to many more areas of close approximation of capillary lumen to airway surface, and the terminal generations of the airways are lined
805
only by flattened epithelium. Respiration can now be maintained. Alveoli are not present in the walls of these terminal sacs, so that this phase should not be referred to as the "alveolar period," but rather as the "terminal sac period"; this phase commences at approximately the twenty-fourth to twenty-sixth week in the human. Some species, notably the rat and the mouse, are born during this period, and alveoli are not present at birth (see below). The situation in the human is controversial and is also discussed further below (see pp. 826-828), but it seems likely that quite a considerable number of alveoli are present in humans at birth. Emery and Mithal (13) found twice as many alveolar wall intersections between respiratory bronchioles and the periphery of the acinus at term as at 24 to 27 weeks of gestation, and it was only at age 8 years that a further doubling of intersections was noted. Thus, in the human, at any rate, there may also be an intrauterine "alveolar period," but its time of onset is uncertain.
Maturation and Control of Development of the Alveolar Wall The most recent studies of intrauterine lung development have demonstrated quite conclusively the endodermal origin of Type I and Type II epithelial cells of the alveolus and the mesodermal origin of the "primary interstitial cell" or "fibroblast" of the alveolar wall. The latter is characterized by the presence of numerous intracytoplasmic lipid inclusions, and it is an important cell in the postnatal development of alveoli in rats and mice. O'Hare and Sheridan (14) have shown that a distinct basal lamina is present at day 16 of gestation in rats and that this is never subsequently breached by cells or their extensions. In their study, an obvious morphologic transition existed between endodermal cells, bronchial cells, and Type I and Type II epithelial cells. These cells were always on the same side of the basal lamina and were connected by tight junctions. A similar obvious morphologic resemblance existed between mesodermal cells and the interstitial cells, which were always contained within the interstitium. Type I and Type II epithelial cells also showed the autoradiographic labeling characteristics of the endoderm, whereas the primary interstitial cell had the characteristics of mesoderm (15). O'Hare and co-workers (15) also stressed that glycogen occurred in the sites of rapid epithelial division, an observation first noted by Sorokin (16). Tritiated thymidine labeling in vivo from day 16 onward
806
WILLIAM M. THURLBECK
(15) and mitotic counts of developing lung in vitro show higher rates in endoderm than in mesoderm (16). The relationship of mitotic activity and the presence of glycogen link with 2 recent investigative thrusts concerning the developing lung. It is now well known that administration of steroids to fetuses accelerates the production of surfactant and induces premature delivery in fetal lambs (17-20); only glucocorticoids are effective. Steroid administration results in diminution of lung size, because there are too few cells in the lung, suggesting that maturation and surfactant secretion occur at the expense of lung growth (18-20). Conversely, intrauterine decapitation, which destroys the pituitary-adrenal-thyroid axis, retards cytodifferentiation and results in large lungs with cells that have excess quantities of glycogen (21). The complementary thrust has come from laboratories in Montreal and San Francisco (2228). These investigators have demonstrated the presence of specific glucocorticoid receptors in the fetal lungs of humans, rabbits, rats, and guinea pigs. Cytoplasmic macromolecules that form an 8S complex with steroids act as receptors to transfer the steroids to nuclear receptors. Only approximately 6,000 binding sites were found per nucleus in the fetal lung, and these were saturated with physiologic concentrations of cortisol. Glucocorticoid binding sites are more frequent in the lung than in other organs and 4 to 5 times more frequent than in the liver of rabbits and humans. Receptor activity reaches a maximum at days 28 to 30 of intrauterine life in rabbits. Rabbits are the only species in which receptors have been demonstrated in adults; in rats they disappear 2 days after birth. The hypothesis is that glucocorticoids act at a nuclear level via a receptor-mediated mechanism to trigger the synthesis of a specific ribonucleic acid (RNA) that codes for particular proteins. The proteins may be enzymes involved in the biosynthesis of surface-active phospholipids or enzymes involved in the breakdown of glycogen. Glycogen may also provide the energy or material for cell proliferation, accounting for its presence in the areas of rapid cell multiplication, as noted above. Sorokin (16) noted that cyanide, which destroys terminal oxidase activity, or malonate, which interferes with the Krebs tricarboxylic acid cycle activity, did not alter lung growth in culture. Fluoride, which prevents glycolysis, stops lung growth. Thus, it seems that steroids may divert biochemical activity derived from
glycogen from lung multiplication to surfactant secretion. As will be shown subsequently, elastic tissue appears to play a key role in postnatal alveolar development, and thus its intrauterine appearance is of interest. In the human (29, 30), elastic tissue begins to appear in arteries, trachea, and bronchi in the second and third months in utero and is quite well developed by the end of this period. In contrast, elastic tissue appears only in the peripheral part of the lung during the terminal sac period and at approximately 7.5 months of gestation. Elastic fibers are thin and limited to the mouths of alveoli. A recent paper has described the fine structure of elastogenesis in the rat (31). Elastic fibers become apparent in the walls of airways on the sixteenth day of gestation and seem to be secreted by myoblasts, because these are the only cells in the vicinity of the newly formed fibers. At days 20 and 21, mature fibroblasts appear in the "alveolar" (saccular) zone, and newly secreted elastic tissue is found adjacent to the fibroblasts and accompanies the appearance of the rudimentary alveoli. Control of Branching of the Fetal Lung
Recent experiments have stressed the importance of the interaction between mesoderm and endoderm in producing differentiation in the lung. It has long been known that the lung is self-differentiating in organ culture and that branching of the lung rudiment requires lung mesoderm (32). Interdependence of mesoderm and endoderm was shown by Taderera (32), who found that chick mesoderm would stimulate mouse endoderm to branch and that after a few days, the resultant lung resembled chicken lung, rather than mouse lung, in airway branching pattern. Conversely, epithelium induces mesodermal differentiation. Other experiments (4, 33) have shown that gut mesoderm will induce bronchial budding in the mouse, but that bronchial branching requires bronchial mesoderm, i.e., mesoderm from more mature embryos in which bronchial branching has started or is about to start. Bronchial or gut mesoderm will also induce budding in tracheal epithelium, but tracheal mesoderm will inhibit bronchial budding and branching. These experiments indicate the interdependence of mesenchyme and endoderm in differentiation, and it has also been postulated that mesenchyme contains an "epithelial growth factor" that can regulate the proliferation of epithelium (34).
807
POSTNATAL GROWTH AND DEVELOPMENT OF THE LUNG
Postnatal Growth and Development of the Lung
Thus, the amount and quality of the connective tissue may also be limiting factors in growth and differentiation of the postnatal lung. Even subtler and closer relationships may exist, because transient gaps in the basal lamina have been reported, allowing epithelial-mesenchymal cell contact, in developing duodenum at the time of subtle epithelial differentiation (35). As mentioned above, the basal lamina of the lung has been carefully studied during gestational life and found continuous, so that these "epitheliolo-mesenchymal" contact sites may be of little relevance in the lung.
Laboratory Animals Lung growth must be considered in relation to growth of the whole organism and growth in other organs. It is not always recognized that the growth rate of the whole organism is most rapid immediately postnatally, declining gradually and steadily thereafter, other than for a brief reversal of this decline at the time of puberty (36). Even then the growth rate in humans is noticeably less than during the first year of life. The rate of increase of organ weight is similarly greatest soon after birth, but the behavior of individual
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Fig. 2. At birth, a rat has approximately 2 to 3 X I 09 nuclei (upper broken line) in its body and approximately 70 X 109 nuclei at 90 days of age. The solid line is the amount of deoxyribonucleic acid (D;-..rA) in the whole animal, and the body weight per nucleus is shown by the lower broken line (from figure I, reference 37, courtesy Dr. C. P. Leblond and the Editor, journal of Embryology and Experimental Morphology).
808
WILLIAM M. THURLBECK
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Fig. 3. The amount of protein per nucleus (ordinate in m,ug. or ng) shows an increase with age in the whole body of the rat, indicating increasing cell size. This is mostly due to increase in cell size in tissues, such as fat and muscle, and in intercellular connective tissue. Lung and kidney ftrst in· crease by cell multiplication, so that whereas the total amount of deoxyribonucleic acid increases (figure 4), the amount of protein per nucleus remains the same. After approximately 2 weeks of age, cells enlarge as well as multiply, and the amount of protein per nucleus increases for approximately 2 months in the lung and approximately 6 weeks in the kidney. Cellular multiplication becomes slow in the lung by 2 weeks of age and in the kidney by approximately 1 month (figure 4). Spleen and thymus grow by cellular multiplication and renewal only, without cell enlargement (figure 4 from reference 38, courtesy of the authors and the Editor, Developmental Biology).
organs differs. Furthermore, their components, nuclei, cytoplasm, protein, and interstitial tissue, increase at different rates. The lung has the unique feature that its growth in -volume and weight may show considerable disassociation because of varying degrees of inflation. Body, Organ, and Lung Growth The details of organ growth and body growth have been well worked out in the rat (37, 38). At birth, there are approximately 2 to 3 X 109 nuclei in the whole animal, increasing to 67 X 109 at 95 days of age (figure 2). The weight of deoxyribonucleic acid (DNA), and hence the number of nuclei, usually does not quite match the increase in body weight even during the first 2 weeks of life, and thus, weight per nucleus tends to increase slowly. The amount of water in tissues and organs varies with age, so that relationships are better expressed as amount of protein per nucleus (figure 3). Body protein in-
creases faster than DNA, and thus protein per nucleus increases for most of the growing period. This is primarily due to increase in cell cytoplasm in tissues such as fat and muscle and increase in intercellular connective tissue. A similar, although slightly different, pattern is seen in most organs, as opposed to tissues and the whole body, and each organ, in turn, differs slightly from the others (figures 3-5). In general, organs show 3 phases. From birth to 14 to 17 days of age, organs grow primarily by cell multiplication; the amount of DNA increases rapidly, and the amount of protein per nucleus remains approximately the same. This is followed bv a phase in which cell multiplication is slower than protein synthesis; thus, whereas the amount of DNA and the number of nuclei still increase, there is an increase in the amount of protein per nucleus. This phase continues to approximately 5 to 7 weeks of age. In the final phase, cell proliferation stops, or is very slow,
809
POSTNATAL GROWTH AND DEVELOPMENT OF THE LUNG
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