The Postnatal Growth of the Rat Lung 11. AUTORADIOGRAPHY ' SHIRLEY L. KAUFFMAN,a PETER H. BURR13 AND EWALD R. WEIBEL3 a Department of Pathology, State University of New York, Downstate Medical Center, Brooklyn, New York and Department of Anatomy, University of Berne, Berne, Switzerland ABSTRACT Combined morphometric and autoradiographic methods were used to analyze the postnatal growth of rat lung from 1-21 days after birth. Each cell population had distinct growth patterns with an increase in the number of fibroblasts and capillary endothelial cells largely determining the increase in interstitial volume and capillary surface, respectively. The height of proliferation activity in mesodermally-derived cells was concurrent with the outgrowth of secondary alveolar septa (between days 4 and 13). Analysis of the location of labeled cells on day 7 showed that the higher labeling index on septal crests could be ascribed to the proliferative activity of fibroblasts and endothelial cells. Within the alveolar epithelium only the type I1 alveolar cells had a detectable labeling index. Over the first week, the number of type I epithelial cells steadily increased while the number of type I1 cells remained constant. Subsequently the number of type I1 cells increased rapidly, reached a peak on day 13 and then decreased, whereas type I cells continued to increase in number. These facts led us to consider that type I1 epithelial cells may represent the stem cell population of alveolar epithelium. The height of proliferative activity of type I1 cells on day 7 coincided with the outgrowth of septal crests and was followed by the steepest increase in number of type I and I1 cells. Between the 10th and 21st day labeling indices rapidly declined, cell production became undetectable after day 13.Increase in alveolar and capillary surface area however continued, resulting in a thinning of the interstitial layer and of the epithelial and endothelial sheets. The lung of the newborn rat is an immature organ which undergoes major postnatal morphogenetic changes during the first 21 days of life. Expansion of the air spaces, formation of the alveolar septa, thinning of the interstitium and remodeling of the capillary network, all act to transform the gas exchange apparatus from its late fetal to near mature form within these 3 weeks (Burri, '74). Although no previous studies have been specifically directed toward examination of the kinetics of the various cell populations involved in these changes, some data have been obtained which indicate a decline in 3H-thymidine labeling index in fetal rat lung as a whole between the 17th and 19th day of gestation and birth (Kury et al., '67) followed by a postnatal peak of cell proliferation (Crocker et al., '70; ANAT. REC..180: 63-76. OHare and Townes '70). The present autoradiographic investigation was directed towards evaluation of the cell kinetics of the populations involved in the growth and morphogenesis of the rat lung, and a correlation of this with the morphometric data described in the first paper of this series was attempted. MATERIALS AND METHODS A. Tissue preparation CFN-COBS strain rats ( 3 animals per group) were injected with 3H-thymidine, 1 pc/gram body weight (3.0 Ci/mM, New England Nuclear) at the following ages: 24 hours, 4, 7, 10, 13, 21 days. From Oct. 2. '73. Acceuted Feb. .5, '74. study was supported by grants from the Council for Tobacco Research U S A . and from the Swiss National Science Foundation 3.5.68 and 3.682.71. Received -. 1 This 63 64 S. L. KAUFFMAN, P. H. BURR1 A N D E. R. WEIBEL day 4 onwards the injections were made at the same daily hours, from 8 to 12 AM, following a strict time-table to avoid possible diurnal variations in cell division rates. These animals were part of the same population of rats as that used for the morphometric study reported i n the companion paper (Burri et al., '74); they were fixed simultaneously. Thirty-five minutes after 3H-thymidine injection, the rats were anesthetized with HypnormR, the dosage being 0.2 m1/100 gram body eight.^ Following a tracheostomy, a bilateral pneumothorax was induced through the diaphragm via a n abdominal incision and the lungs immediately instilled with buffered osmium tetroxide through a tracheal cannula, under a constant instillation pressure of 20 cm of water (Burri and Weibel, '71). Instillations were done between 50 and 55 minutes after 'H-thymidine injection. The lungs were removed and fixed in toto for 2 hours i n the same solution. The lungs were dissected from the mediastinal contents and the lung volume determined by saline displacement. Multiple 1 m m slices were then made of the lungs and these were sectioned into 1 m m cubes. Ten sample blocks of each animal were embedded in Epon; of these, 4 blocks were cut with glass knives to produce 1.5 E". sections. Blocks were trimmed between sections to obtain 4 well spaced sections of each block. These were mounted on glass slides, dipped in NTBz emulsion and exposed in light proof boxes at 4°C for 30 days. After development in Dektol, the slides were stained with a n alkaline solution of toluidine blue. B. Autoradiography 1. Analysis of autoradiographs Autoradiographs were examined with a Leitz microscope fitted with a n eye piece enclosing a test area of 7225 The identity and the number of cells and the proportion of nuclei labeled for each cell type were determined in 60 test areas from each animal using the following criteria. Fibroblasts were identified by their location in the interstitium and their stellate intracytoplasmic lipid droplets (figs. 1-4). Capillary endothelial nuclei (fig. 3 ) were identified by their location, shape and attenuated cytoplasm adjacent to the nucleus, which helped to distinguish them from the more numerous intravascular mononuclear cells. Type I1 alveolar cells (figs. 1, 3 , 4 ) were identified by the combined occurrence of several of the following characteristics : location either on the alveolar surface or in the interstitium, presence of strongly osmiophilic, round cytoplasmic (lamellar) bodies and a fuzzy surface due to microvilli. The type I alveolar cell (figs. 3 , 4) was distinguished from the type I1 cell by its elongated nucleus and slim cytoplasm free of lamellar bodies, and from the capillary endothelial cell by its location. I n this context it must be stressed that, although the criteria worked out to differentiate the septal cells cannot be easily demonstrated i n micrographs, it does not cause too much difficulty for a n experienced investigator to identify the various cell types in the microscope. Furthermore, electron microscopic investigations confirmed the correctness of the criteria worked out above. 2. Labeling indices The 3H-thymidine labeling index (percent of nuclei in the DNA synthetic period) was obtained by counting a minimum of 1000 fibroblast and endothelial nuclei, and 500 type I1 nuclei from each animal, and recording the total number and the number labeled. A nucleus covered by 4 or more grains was considered labeled; the background was less than 2 grains per test area. 3. Location of labeled cells To determine whether labeled cells showed a preferential location on air space surface during the formation of secondary septa, the contours of air spaces were scanned; the identity of each cell encountered and its location was charted on graph paper. Cells were further characterized as either labeled or unlabeled and a s being either on primitive septa or on septal buds. In each of the three 7 days old animals four samples of about 500 cells each, i.e. over 2000 cells per animal were examined. The labeling index on septal buds, both 4 1 ml of HypnormR contains 0.2 mg Fentanyl and 10 mg Fluanison. AUTORADIOGRAPHY OF POSTNATAL LUNG GROWTH 65 Fig. 1 Telophase in type I1 alveolar epithelium (Tz);each cell contains large lamellar bodies. Notice large fibroblasts ( F ) with intracellular lipid. This and following photographs were taken of autoradiograms made from 1.5 p plastic section. Toluidine blue; oil immersion x 900. total and by cell type was compared with the overall labeling index. C. The total number of nuclei The number of nuclei per cm3 of lung tissue (numerical density, Nv) was calculated for each animal using the relation - Nv = NA/D, where Nv is the number of nuclei per unit volume, RAthe number of nuclei per unit section area, and is the mean nuclear caliper diameter (DeHoff and Rhines, '61 ). The mean caliper diameter of nuclei, is shape dependent and therefore varies between the cell types. For type I1 epithelial cells we assumed a spherical shape. In this case we calculated the true mean nuclear diameter b from the mean apparent diam- n, 66 S. L. KAUFFMAN, P. H. BURR1 AND E. R. WEIBEL Fig. 2 Septal bud containing labeled fibroblast (LF) adjacent to unlabeled fibroblast (F), both containing intracytoplasmic lipid. (a) eter of 100 nuclear profiles by the basic relationship : D=-d. - 4 i7 For shape of the fibroblast nuclei we assumed a prolate ellipsoid. Measurement of axial ratios of profiles revealed that the long axis was 1.8-2.0 times the small diameter. D was then calculated from the measurement of mean random chord length E on 100 profiles; using the geo- metric formulae given by Hilliard ('67) we estimated that 1.7 X For endothelial and type I epithelial cells we proceeded similarly. The number of nuclei ( N ) of each cell type in the whole lung was calculated by multiplying the numerical density by the volume of lung parenchyma (i.e. the gas exchanging parts of the lung) estimated by light microscopic morphometry (Burri et al., '74). a= r. AUTORADIOGRAPHY OF POSTNATAL LUNG GROWTH 67 Fig. 3 Base of septal bud with labeled endothelial cell (LE), type I alveolar cell (Ti), type I1 (Tz)and fibroblast ( F ) also shown. D. Estimation of tissue volume The parenchymal volume of the lungs and the volume of epithelium, endothelium and interstitiurn were estimated by light and electron microscopic morphometry respectively on the animals used in a companion paper (Burn et al., '74). They were different animals, but were derived from the same population, and their lungs were fixed by instillation of glutaraldehyde instead of osmium tetroxide. to 13.25 X 10' on day 13 and then decreased si@ficantb to 8.9 lo' On day 21. The total number of fibroblasts (Nf = Nvf lung Parenchymal (fig. 5b) increased from 4.3 X 10' at 24 hours to 17.5 x 10' on the 13th day and then declined to 14.4 X 10' on the 21st day. The SH-labeling of fibroblasts (LIr) was 6.8% at 24 hours (fig. sa), increased and to a maximum of 14.2% on day then fell to less than 1% on day 13. No labeled fibroblasts were found on day 21. RESULTS Capillary endothelium The numerical density of endothelial nuclei (N",,,) was relatively constant, approximately 11-12 x 10' nuclei per cm' between days 1 and 10. Between days 10 and 13, there was a significant decrease in Nvento 8.1 X lo7 on day 13 and 7.1 X lo' on day 21. A. Proliferation pattern of t h e various cell types 1. Fibroblasts The numerical density of fibroblast nuclei (Nw) increased significantly over the growth period from 10.05 X lo' on day 1 2. 68 S. L. KAUFFMAN, P. H. BURR1 AND E. R. WEIBEL Fig. 4 Intravascular mitosis in septa1 bud (SB). Fibroblast ( F ) , type I1 (Tz) and type I (TI)alveolar epithelium are shown. The number of capillary endothelial cells (Nen;fig. 6b) in 24 hours rat lung was approximately equal to the number of fibroblasts (4.97 X lo7); the subsequent numerical increase was less, reaching 13.5 X 10‘ on the 10th day and then declining. The labeling index ( L L ) varied between 8 and 10% over the first 10 days and then fell rapidly between days 10 and 13 (fig. 6a). Labeled endothelial cells were not found on the 21st day. 3. Epithelium The numerical density of alveolar epithelial nuclei ( N Y e p I + I f ) decreased significantly from 4.3 X lo7to 2.6 X lo7between days 1 and 4. Numerical density of both type I and I1 decreased, but the largest AUTORADIOGRAPHY OF POSTNATAL LUNG GROWTH 69 FIBROBLASTS AND INTERSTITIUM L'f I 1 3----.J----j .20 / .16 i \ \ I "I I: 24- 2 k & .20- (1: W c - .16- LL 0 .12r 3 .080 > AGE IN DAYS Fig. 5 a. Labeling index of fibroblast (LIf) and total number of labeled fibroblasts (NIE)in pulmonary parenchymal interstitium. b. Total number of fibroblasts (Nr) and morphometrically determined absolute volume of interstitium (Vi.) (Burri et al., '74). For both figures values are means and brackets include k 1 standard error. 70 S. L. KAUFFMAN, P. H. BURR1 AND E. R. WEIBEL EN DOT HELIUM hen 106 2 /T / / +, ---d // z OO '\\ 71 \ // B5 YNI," \ \ m 6: \ \ \ 2 6 % \ r 0 m * v) F 0-, . a J en ~ e l l sc cm3 m* ,107 I4 3i .12--.24 / W I l0 n z / .10 .-.a3 // / / 1 -I \<Nen \ \.I __--_ -------e / SCV." / ./A 1 12 0 I- z 'O 0 71 89 U s 6 1 m !- 4 O P r v) 2 b Fig. 6 a. Labeling index of pulmonary capillary endothelium (Lien) and total number of labeled endothelial cells (NI~,,). b. Total number of endothelial cells (N.") and absolute volume of endothelium of pulmonary capillaries (Ve,,).Additionally absolute capillary surface area ( S c ) is shown. (Morphometric results from Burri et al. '74.) For both figures values are means and brackets include -C 1 standard error. 71 AUTORADIOGRAPHY OF POSTNATAL LUNG GROWTH component of this decrease was due to the type I1 alveolar epithelial cells. The total number of type I1 alveolar cells (NepI,) remained constant between days 1 and 7, and then increased rapidly from 13 X lo6 to 28 X 10' between day 7 and day 13 (fig. 7b). The number of type I alveolar cells (NepI ) increased more or less steadily over the entire period, from 4.3 X lo6 on day 1 to 12.8 X 10' on day 21 (fig. 7b), with only a slight acceleration between days 7 and 10. The proportion of labeled type I1 cells (LLP,,) increased from 0.45% to over 6.0% in the first week, and declined after the 10th day (fig. 7a). No labeled type I cells were found in any of the preparations (fig. 7a), although a total of approximately two thousands was screened over the course of the various tissue analyses. 7-12 (120 hours). Type I alveolar cell population doubled between days 1 and 9, although no labeled cells were found. Labeling of cells on septal buds The question arises whether the labeled cells are evenly dispersed throughout the lung, or whether they occur with greater frequency on those parts of tissue which are in the process of forming interalveolar septa. We have therefore separately recorded the number of labeled cells on the so-called septal buds and on other parts of the air space wall. Lungs of 7 days old animals were chosen for this analysis because morphometric and electron microscopic studies showed this point corresponded to the most intense phase of septal outgrowth. Results of comparison of overall labeling index with labeling index of nuclei on septal buds B. Population doubling times are shown in table 1. Over the first 13 days, the population of I n 10 out of the twelve samples investifibroblasts increased by a factor of 4. The gated the ratio of the labeling index on first population doubling occurred between buds to the overall labeling index was days 1 and 6.2 (doubling time 126 hours) larger than 1, with a n average of 1.4. The and the second between 6.2 and 13 days 95% confidence interval being 0.18 it is (162 hours). This increase in doubling evident that the labeling index was sigtime can be related to the decline in the nificantly higher on septal buds than in fraction of labeled cells which began the parenchyma as a whole. Analysis of these 4th day. Capillary endothelial cells in- data with respect to the various cell types creased by a factor of 3 between days 1 present in the septa, indicated that both and 10; the initial population doubled be- endothelial cells and fibroblasts were retween days 1 and 8 (168 hours). The type sponsible for the higher labeling indices I1 alveolar cell population showed no nu- on crests, whereas labeled type I1 epithelial merical change until after the 7th day, and cells were evenly distributed over primitive then the population doubled between days septa and crests. C. TABLE 1 Comparison of overall septal labeling index w i t h labeling index on septal buds on day 7 Animal A B C Sample LI on septal buds (A) 1 2 3 4 1 2 3 4 1 2 3 4 Overall mean, 1.396; S.E., 0.130 0.131 0.160 0.185 0.057 0.071 0.107 0.087 0.088 0.146 0.096 0.097 LI on whole septal wall (B) 0.074 0.090 0.110 0.101 0.061 0.072 0.071 0.073 0.054 0.096 0.075 0.081 0.082; 95% confidence interval, f 0.180. A Ratio B 1.757 1.456 1.455 1.832 0.934 0.986 1.507 1.192 1.630 1.521 1.280 1.198 mean 1 S.E. 1.625 20.114 1.155 20.15 1.407 e0.117 72 S. L. KAUFFMAN, P. H. BURR1 AND E. R. WEIBEL DISCUSSION The combined analysis of autoradiographic and morphometric data from neonatal rat lungs suggests that the early postnatal growth period can be divided into 3 phases: Days 1-4: Mobilization of proliferative cells characterized by a high or rapidly increasing labeling index, an enlargement of the air space volume by 8 7 % , and only moderate tissue volume increase; Days 4-13: Period of intense cell production and outgrowth of secondary alveolar septa characterized by differential cell proliferation on septa1 buds and concomitant increase in alveolar and capillary surface area; Days 13-21: Period of differentiation with rapid decline in labeling index and cell production, and continued increase in alveolar and capillary surface area, resulting in a thinning of interstitium and mean barrier thickness. This period in which growth and maturation are concurrent, represents the beginning of the phase of equilibrated lung growth (Burri et al., '74). One feature of neonatal lung growth brought out in this study was the distinctly different proliferative pattern of the 3 main cell populations, as illustrated by their labeling indices (fig. 8). Fibroblast labeling index reached its peak on day 4. Despite the following rapid decline it remained relatively high (over 7% ) during the period of formation of secondary septa (days 4-10). Its decline was folIowed by an actual reduction in total cell number on day 21. Endothelial labeling index was high already at 24 hours and remained at the same level until day 10. There was a marked increase in the total number of endothelial cells and in the number of labeled cells after day 7, which was related to the proliferation of new capillaries in the forming secondary septa. The labeling index of type I1 alveolar epithelial cells was low at birth and slowly rose to its peak on day 7, while type I cells showed no labeling at all. These patterns of cell production are related to those of growth in cell number and in volume of the various tissue components. Growth of the fibroblast population (Nf) was directly related to the pattern of change in the interstitial volume (Vi,,), as shown in figure 5b. Both curves rose to a peak on day 13, followed by a decline towards day 21. Whilst the highest number of capillary endothelial nuclei could be counted on day 10, the endothelial cell volume increased up to day 13 and leveled off until day 21. The capillary surface area, however, followed the volume increase until day 13, but then continued to increase steadily (fig. 6b). This difference between changes in cell number and volume on the one hand and surface growth on the other hand can be explained by a spreading out and thinning of the endothelial cytoplasm, as revealed by a decrease in the mean barrier thickness of endothelium (Burri et al., '74). The number of type I epithelial cells (NepI ) increased slowly from day 1 to 7 and from day 10 to 21 respectively. Its faster increase between days 7 and 10, occurred simultaneously with the steepest augmentation in the number of type I1 cells (NepII; fig. 7b). The period of days 13-21 showed the largest discrepancy between Sa and Vep on the one hand and N.,I on the other; this reflected the reduction of epithelial mean thickness, which was due to spreading out and thinning of the type I cytoplasmic processes. The changes in the number of type I1 cells (NepII)showed an independent pattern (fig. 7b). Particularly, they could not be related to either of the morphometric parameters V,, or S,. There are two reasons which could explain this devious proliferation pattern. On the one hand, the type I1 cell must be considered the secretory cell of the alveolar epithelium (Buckingham et al., '66; Weibel, '73; Gil and Reiss, '73); it is therefore not directly related to the formation of a large and thin Fig. 7 a. Labeling index of alveolar epithelium (LIep11) and total number of labeled epithelial cells (NleP11). Notice that only type I1 alveolar cells were labeled (LIepII and NlepI I ) , all the values for type I cells being 0. b. Number of type I (NepI ) and type I1 cells (NepI I ) , total cell number in alveolar epithelium (N, I and N, II), and absolute volume of alveolar epithelium (Vep), The absolute alveolar surface area (Sa) is also deyicted. (Morphometric results from Burri et al., 74). For both figures all the values represent group means and the brackets include -r- 1 standard error. 73 AUTORADIOGRAPHY OF POSTNATAL LUNG GROWTH Llep EPITHELIUM I Jlep 105 1 14 I2 z 0 71 \ A / :j W 0 10 \ / pl % ' *?J 4 I .12 2 ;; 6 0 rn r I- .08 4 w 1 2 I . 004 L a ' 1 vep Sa JeP 0 3 m2 P. 7' / / / -. NepI+Nepl[ ---/- .-. -0 / 106 35 2 -i 30 z 25 m 73 !O 7 I 15 P P r 10 5 I , 1 b 4 7 10 13 AGE IN DAYS Figure 7 21 74 S. L. KAUFFMAN, P. H. BURR1 AND E. R. WEIBEL AGE IN DAYS Fig. 8 For comparison the labeling indices of the various cell types in the parenchymal portion of the growing rat lung are shown in one graph. Each cell type displays its own pattern (see text): fibroblasts (f), endothelial cells ( e n ) and alveolar epithelial cells of type I1 (ep 11). For endothelium and fibroblasts no labeling could be detected on day 21. air-blood barrier, the main event of this growth period ( B d et al., '74; Burri, '74). On the other hand, this study suggests that the type I1 cell might represent the stem cell of the type I epithelial cell, because it showed that type I cells increased in number without the occurrence of any 3H-thymidinelabeling (DNA synthesis) in the nuclei of this cell type. Several independent observations related to the repair of damaged alveolar epithelium support our hypothesis. Firstly, in oxygen poisoning the squamous processes of type I alveolar epithelial ceIls become damaged and a repair ensues in the form of a lining of cuboidal cells, which have all the characteristics of type I1 cells; these cells are subsequently transformed into squamous type I cells (Kapanci et al., '69; Bowden and Adamson, '71; Gould et al., '72). Recently it could be shown that this is a characteristic pattern of alveolar epithelial repair, irrespective of the type of damage (Carrington and Green, '70; Bachofen and Weibel, '74). Secondly, Evans et al. ('73) have recently demonstrated in an autoradiographic study on tissue repair after NOz-damage, that only type I1 cells entered the division cycle by synthesising DNA, whereas type I cells did not; they also showed that labeled type I cells could be found within 48 hours presumably arising from labeled type I1 cells. The reason for the inability of type I cells to divide probably lies in their unusually high level of topological differentiation in that each cell forms multiple apical cytoplasmic plates (Weibel, '71, '74), a configuration which would make cytoplasmic division difficult. Our observations suggest that the alveolar epithelial cells of the growing lung proliferate in a very similar fashion to the adult epithelium during repair. OHare and Townes ('70) however found a relatively high incidence of labeled type I cells in newborn rats in a marker dilution study and in a daily label index study. In the marker dilution study postnatal labeling of AUTORADIOGRAPHY OF POSTNATAL LUNG GROWTH type I cells was obtained by injecting the mother with 3H-thymidine on the 16th day of gestation. Whereas the labeled type I cells found in this case could well have been derived from type I1 precursor cells, the results of the daily label index study, with labeling indices as high as 7% on the k s t and 13% on the third postnatal day are very much in contradiction with our present findings, This problem certainly needs further investigation, as it also remains to be proven whether in the growing lung newly divided type I1 cells can transform into type I cells. Another interesting observation with respect to alveolar epithelium deserves to be discussed in this context. The numerical density of type I1 nuclei was greatest at 24 hours and decreased by nearly 50% within 4 days. However, over this period of lung expansion the total number of type I1 cells remained constant. Several investigators (Short, '50; Woodside and Dalton, '58; Klika, '65) have described desquamation of type I1 alveolar cells in the neonatal lung and ascribed morphogenetic changes to this event. Loosli and Baker ('62) however, found no evidence of epithelial desquamation in newborn lung and believed the apparent decrease in epithelial nuclei was due to a flattening of alveolar lining cells. It seems unlikely from the present study that type I1 cells desquamated, because, in the presence of the low labeling index, desquamation of type I1 cells would have resulted in a decline in the number of cells, whereas the number remained constant. We must therefore conclude, that the reduction in numerical density of these cells is due essentially to an enlargement of the containing space rather than to a loss of cells. The most important morphogenetic event of the growth period investigated was the neoformation and outgrowth of secondary alveolar septa, which was accompanied by considerable transformations in the tissue (Burri, '74). We were led to ask whether the proliferating cells were randomly distributed throughout the tissue, or whether they showed a certain concentration in the region of the forming secondary septa. We therefore determined the labeling indices of the cells located in and at the base of septal crests at day 7 75 and found that the septal crests had a sigscantly higher labeling index than the tissue as a whole. This finding suggests that the formation of secondary septa is associated with differential proliferation, and is not simply due to cell migration or purely mechanical factors as postulated by Short ('50). Furthermore it appeared that the labeled type I1 cells were not preferentially located on crests, as was the case for both endothelium and fibroblasts. The growth of alveolar epithelium had hence once more a pattern different from that of cells of mesodermal origin. ACKNOWLEDGMENTS The authors wish to acknowledge the excellent technical assistance of Mr. Hiroshi Ozaki, Ms. Wally Wehle and Ms. Krishna Anand. The charts were drawn by Ms. Elizabeth Cuzzort and Mr. Karl Babl; the photomicrographs were made by Mr. Willy Kratil. LITERATURE CITED Bachofen, M., and E. R. Weibel 1974 Basic pattern of tissue repair in human lungs following unspecific injury. Chest, 65: 14s-19s. Bowden, D. H., and I. Y. R. Adamson 1971 Reparative change following pulmonary cell injury. Ultrastructural, cytodynamic and surfactant studies in mice after oxygen exposure. Arch. Path. 92: 279. Buckingham, S., H.0. Heinemann, S. C. Sommers and W. F. McNary 1966 Phopholipid synthesis in the large pulmonary alveolar cells. Am. J. Pathol., 48: 1027-1041. Burri, P. H. 1974 The postnatal growth of the rat lung. 111. Morphology. Anat. Rec., 180: 7798. Burri, P. H., J. 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