THE ANATOMICAL RECORD 229:495-498 (1991)
Pulmonary Development in the Fetal Lamb: Morphometric Study of the Alveolar Phase STEVEN G. DOCIMO, ROBERT K. CRONE, PAUL DAVIES, LYNNE REID, ALAN B. RETIK, AND JAMES MANDELL Department of Surgery, Division of Urology (S.G.D., A.B.R., J.M.), and Departments of Anesthesia (R.K.C.) and Pathology (P.D., L.R.), Children’s Hospital and Harvard Medical School, Boston, Massachusetts
ABSTRACT The alveolar stage of intra-uterine lung development was analyzed morphometrically in 18 fetal lambs a t 112, 124, 142, or 148 (term) days of gestation. The right lungs were fixed a t uniform distending pressure and the tissue embedded in Epon for light microscopy. Over the whole period, right lung volume (VL) increased more rapidly than body weight to reach a mean value of 185 cm3 at term. The increase in VL was greatest between 112 and 124 days gestation. Throughout the period of study the distal lung consisted of alveolar-like air-spaces lined by walls with a single capillary system. Despite this mature morphology, further differentiation took place as established by two independent measurements. First, interalveolar wall thickness decreased from 4.12 .03 pm a t 112 days to 2.55 t .02 pm a t term. Secondly, alveolar surface area increased at a greater rate than lung volume (as VL ‘ . 0 6 ) , suggesting a moderate increase in surface complexity, but not a subdivision of existing air-spaces. This latter finding was supported by numerical density values which remained fairly constant. We conclude that throughout this period active growth involves the addition of alveolar units of basically similar size. This is a similar process to the one noted previously in postpneumonectomy compensatory growth.
*
As in other mammalian species, lung development in the fetal lamb can be divided into several distinct phases: embryonic, pseudoglandular, canalicular, saccular, and alveolar. What distinguishes the lamb from the human and the intensively investigated rat is the fact that the lung is already fully alveolar a t birth. In humans and still more in rats, alveolar formation is a feature of postnatal development. The fetal lamb is often used to study the effects on lung development of various surgical manipulations in utero (Kitterman, 1984) and several studies have demonstrated the adaptability of the lung to such perturbations, particularly at later gestational ages. Since the lung a t these ages is already in a comparatively differentiated phase, we wished to know if its adaptation and maturation could be quantitatively described in a consistent fashion. In the postnatal lamb, lung development was recently described in a n extensive morphometric study (Davies et al., 1988). In the fetus, however, morphometry has not been applied to as large a series of animals nor in as rigorous a way. Thus, while Alcorn et al. provided the most complete survey of fetal lamb lung development to date (1981), they did not employ a standardized inflation fixation, making volumetric comparisons impossible. Our purpose was to define morphometric parameters of normal pulmonary development in the late fetal lamb, using lungs that were inflation-fixed in a reproducible manner. 0 1991 WILEY-LISS, INC
MATERIALS AND METHODS
Pulmonary tissue was obtained from 18 fetal lambs: four at 112 and 142 days gestation and five at 124 and 148 days (term). With a pregnant ewe of predetermined mating date under anesthesia, the fetal lamb was delivered by hysterotomy and anesthetized via the umbilical artery. Fetal respiration was prevented by covering the head of the anesthetized lamb with a rubber glove. Upon delivery, the fetus was weighed. The left mainstem bronchus was exposed and ligated. The trachea was cannulated and the right lung was inflated with a solution of 2% glutaraldehyde in 0.05M cacodylate buffer, pH 7.2, containing 24 giL sucrose, at 25 cm H,O pressure. Once the lung was fully inflated, the trachea was ligated and the lung removed from the chest. It was placed in the same fixative for 2-3 hours. At the end of this time the volume of the lung was determined by water displacement. The left lungs were frozen for use in biochemical and arterial perfusion studies to be described elsewhere. Gross lung sections from the approximate midpoint of the right upper and lower lobes obtained by transferse section were analyzed by a point-counting method
Received May 23, 1990; accepted August 16, 1990. Address reprint requests to James Mandell, MD, Division of Urology, Children’s Hospital, Longwood Avenue, Boston, MA 02115.
496
S.G. DOCIMO ET AL.
using 10 x 10 grid in a dissecting microscope a t 5 x magnification. Bronchi and blood vessels visible under the dissecting microscope were identified separately, and the rest of the lung was taken to be the respiratory region. Analysis consisted of calculating the percentage of points falling on each of these structures. This was carried out on at least two animals in each age group. Samples from apical and lower halves of the upper and lower lobes of the right lung were taken for histologic study. These blocks underwent four washes in buffer, and postfixation in 1%OsO, in 0.05M veronal acetate buffer, pH 7.2, for 2 hours a t room temperature followed by a brief wash in veronal. Staining was accomplished en bloc in 0.5% uranyl acetate in veronal buffer, pH 5.2, for 30 minutes. The samples were dehydrated in methanol and embedded in Polybed. Sections 1 pm thick were stained in Regaud‘s hematoxylin. Morphometric measurements were made on a Zeiss microscope with a projection head and an oil-immersion lens to provide a total magnification of 400 x . A multipurpose, coherent test lattice (Weibel, 1979) was incorporated into the projection head; it comprised 42 test points with an individual probe length of 42.84 Fm. The midpoint of the field was indicated by the intersection of perpendicular cross hairs. Ten fields were analyzed for each section, and a t least four sections were reviewed per animal. The analysis included counts of test points falling on air-spaces, alveolar wall tissue, and nonmuscular vessels and counts of intersections between alveolar surface and the test line system. Volume and surface densities were calculated relative to the reference compartment, respiratory lung. Alveolar number was estimated by counting alveolar profiles within the test area (NA).An alveolus was defined as an air-space either wholly enclosed by respiratory epithelium or partially enclosed, with the remaining boundary formed by an imaginary line connecting the ends of two septa (Hu et al., 1987). The numerical density (Nv) of alveoli in respiratory lung was calculated from the formula N, = K(NA)2’3p(v,)1’2, where K is the coefficient size distribution (taken to be 1) and p is the shape factor, in this case 1.55 (Weibel and Gomez, 1962; Weibel, 1963). The thickness of interalveolar septa was measured a t the point closest to the center of each field such that a line connecting the center of two adjacent alveoli transected the wall but did not pass through pre- and postcapillary blood vessels or extra-alveolar connective tissue. This technique has been previously described (Crone et al., 1983). Statistical analysis included Student’s T test and analysis of variance for inter-age-group differences. The paired T test was used to compare data from apical and diaphragmatic segments within groups. A P value less than 0.05 was considered statistically significant. RESULTS
Lung Volume and Body Weight
Mean fetal body weight increased from 1,530 g at 112 days to 3,930 g a t term. Over this same period, right lung volume increased from 52 cm3 t o 185 cm3, faster than the change in body weight. The allometric relationship of lung volume t o body weight shows that over
1
104 1000
1
10000
Body Weight (6)
Fig. 1. Allometric relationship between right lung volume and total body weight.
this period, lung volume grew as body weight to the power 1.08 (Fig. 1). Between 112 days and 124 days, right lung volume increased by 84% (P
Pulmonary Development in the Fetal Lamb: Morphometric Study of the Alveolar Phase STEVEN G. DOCIMO, ROBERT K. CRONE, PAUL DAVIES, LYNNE REID, ALAN B. RETIK, AND JAMES MANDELL Department of Surgery, Division of Urology (S.G.D., A.B.R., J.M.), and Departments of Anesthesia (R.K.C.) and Pathology (P.D., L.R.), Children’s Hospital and Harvard Medical School, Boston, Massachusetts
ABSTRACT The alveolar stage of intra-uterine lung development was analyzed morphometrically in 18 fetal lambs a t 112, 124, 142, or 148 (term) days of gestation. The right lungs were fixed a t uniform distending pressure and the tissue embedded in Epon for light microscopy. Over the whole period, right lung volume (VL) increased more rapidly than body weight to reach a mean value of 185 cm3 at term. The increase in VL was greatest between 112 and 124 days gestation. Throughout the period of study the distal lung consisted of alveolar-like air-spaces lined by walls with a single capillary system. Despite this mature morphology, further differentiation took place as established by two independent measurements. First, interalveolar wall thickness decreased from 4.12 .03 pm a t 112 days to 2.55 t .02 pm a t term. Secondly, alveolar surface area increased at a greater rate than lung volume (as VL ‘ . 0 6 ) , suggesting a moderate increase in surface complexity, but not a subdivision of existing air-spaces. This latter finding was supported by numerical density values which remained fairly constant. We conclude that throughout this period active growth involves the addition of alveolar units of basically similar size. This is a similar process to the one noted previously in postpneumonectomy compensatory growth.
*
As in other mammalian species, lung development in the fetal lamb can be divided into several distinct phases: embryonic, pseudoglandular, canalicular, saccular, and alveolar. What distinguishes the lamb from the human and the intensively investigated rat is the fact that the lung is already fully alveolar a t birth. In humans and still more in rats, alveolar formation is a feature of postnatal development. The fetal lamb is often used to study the effects on lung development of various surgical manipulations in utero (Kitterman, 1984) and several studies have demonstrated the adaptability of the lung to such perturbations, particularly at later gestational ages. Since the lung a t these ages is already in a comparatively differentiated phase, we wished to know if its adaptation and maturation could be quantitatively described in a consistent fashion. In the postnatal lamb, lung development was recently described in a n extensive morphometric study (Davies et al., 1988). In the fetus, however, morphometry has not been applied to as large a series of animals nor in as rigorous a way. Thus, while Alcorn et al. provided the most complete survey of fetal lamb lung development to date (1981), they did not employ a standardized inflation fixation, making volumetric comparisons impossible. Our purpose was to define morphometric parameters of normal pulmonary development in the late fetal lamb, using lungs that were inflation-fixed in a reproducible manner. 0 1991 WILEY-LISS, INC
MATERIALS AND METHODS
Pulmonary tissue was obtained from 18 fetal lambs: four at 112 and 142 days gestation and five at 124 and 148 days (term). With a pregnant ewe of predetermined mating date under anesthesia, the fetal lamb was delivered by hysterotomy and anesthetized via the umbilical artery. Fetal respiration was prevented by covering the head of the anesthetized lamb with a rubber glove. Upon delivery, the fetus was weighed. The left mainstem bronchus was exposed and ligated. The trachea was cannulated and the right lung was inflated with a solution of 2% glutaraldehyde in 0.05M cacodylate buffer, pH 7.2, containing 24 giL sucrose, at 25 cm H,O pressure. Once the lung was fully inflated, the trachea was ligated and the lung removed from the chest. It was placed in the same fixative for 2-3 hours. At the end of this time the volume of the lung was determined by water displacement. The left lungs were frozen for use in biochemical and arterial perfusion studies to be described elsewhere. Gross lung sections from the approximate midpoint of the right upper and lower lobes obtained by transferse section were analyzed by a point-counting method
Received May 23, 1990; accepted August 16, 1990. Address reprint requests to James Mandell, MD, Division of Urology, Children’s Hospital, Longwood Avenue, Boston, MA 02115.
496
S.G. DOCIMO ET AL.
using 10 x 10 grid in a dissecting microscope a t 5 x magnification. Bronchi and blood vessels visible under the dissecting microscope were identified separately, and the rest of the lung was taken to be the respiratory region. Analysis consisted of calculating the percentage of points falling on each of these structures. This was carried out on at least two animals in each age group. Samples from apical and lower halves of the upper and lower lobes of the right lung were taken for histologic study. These blocks underwent four washes in buffer, and postfixation in 1%OsO, in 0.05M veronal acetate buffer, pH 7.2, for 2 hours a t room temperature followed by a brief wash in veronal. Staining was accomplished en bloc in 0.5% uranyl acetate in veronal buffer, pH 5.2, for 30 minutes. The samples were dehydrated in methanol and embedded in Polybed. Sections 1 pm thick were stained in Regaud‘s hematoxylin. Morphometric measurements were made on a Zeiss microscope with a projection head and an oil-immersion lens to provide a total magnification of 400 x . A multipurpose, coherent test lattice (Weibel, 1979) was incorporated into the projection head; it comprised 42 test points with an individual probe length of 42.84 Fm. The midpoint of the field was indicated by the intersection of perpendicular cross hairs. Ten fields were analyzed for each section, and a t least four sections were reviewed per animal. The analysis included counts of test points falling on air-spaces, alveolar wall tissue, and nonmuscular vessels and counts of intersections between alveolar surface and the test line system. Volume and surface densities were calculated relative to the reference compartment, respiratory lung. Alveolar number was estimated by counting alveolar profiles within the test area (NA).An alveolus was defined as an air-space either wholly enclosed by respiratory epithelium or partially enclosed, with the remaining boundary formed by an imaginary line connecting the ends of two septa (Hu et al., 1987). The numerical density (Nv) of alveoli in respiratory lung was calculated from the formula N, = K(NA)2’3p(v,)1’2, where K is the coefficient size distribution (taken to be 1) and p is the shape factor, in this case 1.55 (Weibel and Gomez, 1962; Weibel, 1963). The thickness of interalveolar septa was measured a t the point closest to the center of each field such that a line connecting the center of two adjacent alveoli transected the wall but did not pass through pre- and postcapillary blood vessels or extra-alveolar connective tissue. This technique has been previously described (Crone et al., 1983). Statistical analysis included Student’s T test and analysis of variance for inter-age-group differences. The paired T test was used to compare data from apical and diaphragmatic segments within groups. A P value less than 0.05 was considered statistically significant. RESULTS
Lung Volume and Body Weight
Mean fetal body weight increased from 1,530 g at 112 days to 3,930 g a t term. Over this same period, right lung volume increased from 52 cm3 t o 185 cm3, faster than the change in body weight. The allometric relationship of lung volume t o body weight shows that over
1
104 1000
1
10000
Body Weight (6)
Fig. 1. Allometric relationship between right lung volume and total body weight.
this period, lung volume grew as body weight to the power 1.08 (Fig. 1). Between 112 days and 124 days, right lung volume increased by 84% (P
