THE ANATOMICAL RECORD 227:111-123 (1990)
Development of the Conducting Airway Epithelium in Fetal Syrian Golden Hamsters During Normal and Diabetic Pregnancies ELIZABETH M. McDOWELL, WILLIAM P. COLEMAN, ANDREA M. DE SANTI, CARNELL NEWKIRK, AND JUDY M. STRUM Departments o f Pathology (E.M.M., W.P.C., A.M.D., and C.N.) and Anatomy (J.M.S.), University of Maryland School of Medicine and Maryland Institute for Emergency Medical Services Systems (W.P.C.), Baltimore, Maryland
ABSTRACT The conducting airway epithelium of fetal Syrian golden hamsters was studied from gestational day 12 to day 15, during normal and uncontrolled diabetic pregnancies. Diabetes was induced in the pregnant hamsters by injecting streptozotocin a t 60 mglkg body weight, subcutaneously, early on gestational day 10. Cells in S-phase were labelled immunochemically with bromodeoxyuridine (BrdU), and the day on which endocrine cells and ciliated cells first appeared was determined. In control fetuses, the BrdU-labelling indices (LI’s) of different anatomical airway levels were significantly different from one gestational day to the next. For example, the LI of the lobar bronchus was significantly different on each gestational day (P < .0001), and the same was true of the bronchioles. Moreover, the difference between LI’s of the lobar bronchus and bronchioles-terminal buds was highly significant on day 12 ( P < .0001), and on day 13 the differences between lobar bronchus and bronchioles, lobar bronchus and terminal buds, and bronchioles and terminal buds were also highly significant ( P < ,0001). However, on gestational days 14 and 15, the LI’s were reduced and were comparable at different airway levels. The BrdU-labelling indices were very consistent among fetuses of the same age, and the differences between the average LI’s for pups of different litters was numerically very small. Hyperglycemia (mild, moderate, severe) did not alter LI’s in the fetal airway epithelial cells. Furthermore, although glycogen was not depleted from the airway epithelium of the hyperglycemic fetuses as it was in the controls, the endocrine cells first appeared on gestational days 12,13, and 14, respectively, in the trachea, lobar bronchus and bronchioles, followed 1 day later by the ciliated cells, in the fetuses of control and diabetic mothers. In our experimental model, induction of diabetes in the pregnant hamsters on gestational day 10 did not appear to alter development or differentiation of the fetal conducting airway epithelium. Early during normal development of the airways of Syrian golden hamsters, the undifferentiated epithelial cells contain abundant glycogen at all airway levels. These glycogen stores are depleted progressively a s the endocrine cells make their primordial appearance in the trachea, lobar bronchus, and bronchioles (gestational days 12, 13, and 14, respectively). Preciliated and ciliated cells then begin to differentiate a t each airway level, just 1 day after the endocrine cells first appear. Meanwhile, glycogen reaccumulates in the cytoplasm of the presecretory cells until the day of birth (McDowell e t al., 1985; Sarikas et al., 1985a). Little is known about the factors that regulate growth, differentiation, and functional maturation of the epithelial cells in the conducting airways during fetal development, but this wave of glycogen depletion followed by repletion, along the length of the developing airways must have some biological significance. 0 1990 WILEY-LISS, INC.
The appearance of the first endocrine cells coincides with a loss of glycogen from all airway levels, suggesting that energy derived from glycogenolysis might be required for the differentiation of these cells. Moreover, the subsequent reaccumulation of glycogen in the presecretory cells may be used later to provide energy for their functional maturation. Insulin-dependent maternal diabetes is associated with a n increased risk of early growth retardation in human embryos (Pedersen and Mdsted-Pedersen, 1981) and small fetuses are common in the offspring of
Received April 25, 1989; accepted September 12, 1989. Address reprint requests to Elizabeth M. McDowell, Ph.D., Department of Pathology, University of Maryland School of Medicine, 10 South Pine Street, Baltimore, MD 21201.
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TABLE 1. Control hamsters'
Dam No. 19-2 19-3 21-16 18-3 19-7 21-2 18-10 21-1 21-14 21-6 21-8 21-12
Treatment on gestational day 10
A, B A, B N A, B A, B N A, B A, B N A, B A, €3 N
9 199 176 177 191 201 157 175 133 177 198 189 178
Blood glucose levels (mgidl) on gestational day 11 12 13 14 178 141K 146 126K 16gK 160 146K 155 126 127K 163K 154 130K 153 155 138 144 134K 12EiK 176 166 166 122 177 161 188 167
Total No. of 15 fetuses in litter 15 16 10 7 15 8 15 8 13 10IK 10 127K 9 116K 9
No. of dead fetuses in litter 0 0 0 0 0 0 0 0 0 0 0 0
Mean fetal weight (gm) ,201 k .034 ,244 ? ,029 .223 2 ,031 ,453 ,063 ,416 t ,052 ,492 2 ,068 1.001 ? ,110 1.032 2 ,094 1.115 2 0.92 1.705 2 .13 1.886 t ,173 1.785 +- ,198
*
'A = metaphane anesthesia; B = subcutaneous injection of 0.4 M citrate buffer, pH 4.2; K = killed; N = no treatment. 'Blood glucose levels according to results of the hexokinase method. The hamsters were anesthesized with metaphane every time a blood sample was taken from the orbital sinus. Blanks in the table indicate that no blood sample was taken at that time.
rodents with experimental diabetes (reviewed by phological changes occur each day in the developing Freinkel, 1988). The etiology of diabetic embryopathy lung a t different airway levels during the latter part of is complex and likely to be multifactorial, but hypergly- pregnancy (see Introduction). Changes in the epithelial cemia is one of the factors implicated (Freinkel et al., cells in the trachea precede similar changes in the lo1986; Eriksson et al., 1987; Sadler et al., 1989). More- bar bronchus and bronchioles by one and two days, over, the incidence of respiratory distress syndrome respectively (McDowell et al., 1985; Sarikas et al., (RDS), caused by delayed alveolar type I1 cell matura- 1985a). Thus, specific developmental changes can be tion and defective synthesis of surfactant, is increased observed at the different airway levels, and the normal in the offspring of some diabetics (Farrell and Avery, timing of these changes is very predictable. Timed-bred Syrian golden hamsters were purchased 1975; Robert et al., 1976). Glycogenolysis normally occurs in the type I1 cells near the end of gestation. This from Charles River (Newfield, NJ). The first gestaprocess is impaired in fetal lungs during experimental tional day was taken to be the day after mating. The diabetic pregnancies and fetal hyperglycemia is one of hamsters arrived a t our animal facility on the second the factors implicated (reviewed by Bourbon and Far- gestational day. They were caged individually, given rell, 1985). The under-utilization of glycogen in the de- food and water ad libitum, and maintained under stanveloping alveoli is thought to reduce the biosynthesis of dard laboratory conditions in a room with a 12-hour two major surfactant phospholipids, which impairs sur- lightil2-hour dark cycle. A total of sixteen pregnant hamsters served a s confactant production (reviewed by Tyden et al., 1986). Since impaired glycogenolysis appears to be linked trols. On gestational day 9, twelve of them were aneswith delayed maturation of the alveolar type I1 cells, thesized lightly with methoxyflurane (Metaphane, we postulated that the growth and differentiation of Pittman-Moore, Inc., Washington Crossing, NJ) and a the fetal conducting airway epithelium would likely be blood sample was taken from the orbital sinus (Pansky altered in the fetuses of mothers with diabetes. There- et al., 1961) to provide base-line blood glucose levels fore, our purpose in undertaking a study of fetal airway (Table 1). Early on gestational day 10, eight of these development in Syrian golden hamsters during normal hamsters were given a subcutaneous injection of 0.22 and uncontrolled diabetic pregnancies was threefold: 1) ml chilled 0.4 M citrate buffer (pH 4.2), under light to quantify epithelial cell growth using bromodeoxy- anesthesia, but four hamsters received no treatment uridine (BrdU)-immunochemical labelling; 2) to char- (Table 1). In addition, four control hamsters (not listed acterize the pattern and timing of glycogenolysis a t in Table 1)received no intervention during pregnancy. different anatomical levels; and 3) to characterize the Blood samples were not taken from these hamsters a t onset of cell differentiation. Our study will show that any time, nor were they anesthesized until they were the growth and differentiation of the fetal airway epi- killed. thelium was not impaired by maternal diabetes, a t Twelve pregnant hamsters served as experimental least under the conditions of our experiments. Never- animals, and diabetes was induced by injection of theless, our results with BrdU-labelling of the epithe- streptozotocin (STZ); the toxicity of this drug is dilial cells provide new data on the orderly progression of rected against the B cells of the pancreatic islets (refetal airway development. viewed by Rerup, 1970). On gestational day 9, the pregnant animals were anesthesized lightly and a blood MATERIALS AND METHODS sample was taken from the orbital sinus to provide We chose Syrian golden hamsters a s our experimen- base-line blood glucose levels (Table 2). Early on gestal model to study fetal airway development, as this tational day 10, the hamsters were anesthesized again species offers some unique advantages. The gestation and streptozotocin (Sigma Chemical Co., St. Louis, MO, period is unusually short for small mammals, about 16 USA) was injected subcutaneously a t 60 mg/kg, b.w., days (Bruce and Hindle, 1934).Moreover, specific mor- dissolved in 0.22 ml of 0.4 M citrate buffer, pH 4.2. The
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113
TABLE 2. Diabetic hamsters' Treatment on DamNo. gestationalday 10 9 19-4 A,B-S 160 19-12 A,B-S 146 20-5 A,B-S 198 18-8 A,B-S 153 18-1 A,B-S 195 20-11 A,B-S 177 20-6 A,B-S 206 20-7 A,B-S 192 20-13 A,B-S 170 20-3 A,B-S 151 20-12 A,B-S 174 20-9 A.B-S 171
Blood glucose levels (mg/dl) on gestational day 11 12 13 14 319 247K 380 338K 377 395K 308 233K 367 374K 327 440 41gK 350 277 213 151RK 331 288 423 380K 443 397 387 413K 430 296 394 370 264 427 395 468 397 348 405 449
No. of Total No. of dead fetuses 15 fetuses in litter in litter 13 0 15 0 10 1 12 0 12 0 12 0 12 0 8 0 8 1 0 183RK 12 11 0 424K 507K 8 0
Mean fetal weight (gm) ,210? .025 .2242 ,035 ,1782 ,028 ,5422 .060 ,5052 ,054 ,4632 ,052 1.1182 ,101 1.312? ,117 .802 t ,128 1.688 t .143 1.641 2 .21 1.831 ? ,067
'A = metaphane anesthesia; B-S = subcutaneous injection of streptozotocin (60 mg/kg, b.w.) dissolved in 0.4 M citrate buffer, pH 4.2; K = killed; RK = hyperglycemia had reverted by time of killing. 'Blood glucose levels according to results of the hexokinase method. The hamsters were anesthesized with metaphane every time a blood sample was taken from the orbital sinus. Blanks in the table indicate that no blood sample was taken a t that time. The base-line blood glucose levels are shown on gestational day 9.
buffer was chilled and the STZ was dissolved in i t immediately before the injection was made. In hamster fetuses, organogenesis occurs between gestational days 8 and 9%, and the tracheobronchial rudiment becomes independent of the esophageal duct at 9% gestational days (Boyer, 1953). Therefore, by injecting STZ into the dams early on gestational day 10, we were able to study the effect of maternal diabetes on airway development after the completion of organogenesis, from the time of independence of the tracheobronchial rudiment. Moreover, since the B cells of the fetal pancreatic islets do not differentiate and produce detectable insulin until gestational day 11 (Grillo, 1964), the direct toxicity of STZ to the fetal B cells was avoided. Blood glucose levels were monitored throughout pregnancy (Tables 1, 2). The blood samples were collected into heparinized capillary tubes. Each time a blood sample was taken, drops of blood were also placed on a glucose oxidase reagent strip (Chemstrip BG, Boehringer-Mannheim Biochemicals, Indianapolis, IN) for immediate determination of blood glucose (mg/dl) with a n Accu-check I1 reflectance meter (Boehringer-Mannheim Diagnostics, Indianapolis, IN). Blood in the capillary tubes was separated by centrifugation for 5 minutes and stored at -20°C until the plasma glucose was determined spectrophotometrically based on the reactions catalyzed by hexokinase and glucose-6-phosphate dehydrogenase, with the use of Dri-STAT@ glucoseHK Endpoint Reagent (Beckman Instruments, Inc., Carlsbad, CAI. On each of gestational days 12, 13, 14, and 15, four pregnant control hamsters and three pregnant diabetic hamsters were killed. Of the controls, two had received a n injection of citrate buffer on day 10 and had undergone several anesthesic and bleeding events, and one had been anesthesized and bled on day 9 (Table 1).In addition, one control dam, which had received no intervention during pregnancy, was killed on each gestational day. The range of hyperglycemia induced by STZ in the pregnant diabetic hamsters was wide, so whenever possible, the three diabetic hamsters killed on each gestational day were chosen to span that
range. In two cases of mild hyperglycemia, the blood glucose levels had reverted to control levels by the time the hamsters were killed, on gestational days 14 and 15 (Table 2). Two hours before being killed all pregnant hamsters listed in Tables 1 and 2 were anesthesized lightly and a small incision was made in the skin of the back. A tablet containing 25 mg 5-bromo-2'-deoxyuridine (Boehringer-Mannheim Biochemicals, Indianapolis, IN) was inserted subcutaneously. The skin was apposed with a suture. All hamsters were killed during the morning hours. At the time of killing, each pregnant hamster was anesthesized, the abdomen was opened, and the fetuses were rapidly removed from the gravid uterus and fixed by immersion in 4% formaldehyde-1% glutaraldehyde in a 200 mOsm phosphate buffer (McDowell and Trump, 1976). Fetuses taken from the control and diabetic dams listed in Tables l and 2 were fixed individually in preweighed vials containing fixative. This way, the fixation process was not delayed and the weight of each fetus could be determined later. On gestational days 12 and 13, fixation was achieved by immersion of the entire undamaged fetus and on days 14 and 15, the fetal abdomen was opened prior to immersion of the fetus in the fixative. The fetuses were fixed for at least 2 days a t room temperature and washed in 300 mOsm phosphate buffer for 2 days, prior to longitudinal embedment. Three fetuses from each of the 12 control dams listed in Table 1, and three fetuses from each of the diabetic dams listed in Table 2 (36 fetuses from controls; 36 fetuses from diabetics) were embedded in paraffin on their left sides. Longitudinal serial sections (5 pm thick) of the left lung were obtained. In addition, left lungs of fetuses from the four control dams which had received no intervention during pregnancy, were embedded in glycol methacrylate (Ruddel, 1967; Sorokin and Hoyt, 1978) and longitudinal serial sections (2 pm thick) were made. Sections (paraffin and glycol methacrylate) of lung which contained the lobar bronchus cut along its length, were selected for further study.
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The selected paraffin sections were stained with Alcian blue (pH 2.5)-periodic acid-Schiff (Mowry and Winkler, 1956), with and without diastase digestion. They were also stained immunochemically to detect BrdU-labelled cells. For the demonstration of BrdU, the sections were mounted on slides coated with chrom alum. This prevented the sections from floating off the slides during incubation in pepsin. Pepsin digestion was required because of the aldehydes in the fixative (Schutte et al., 1987). The sections were hydrated and endogenous peroxidase was blocked by incubation in 1%H,O, in methanol for 30 min. After washing in PBS, the sections were incubated in pepsin (Boehringer-Mannheim, Indianapolis, IN; 0.4 mgiml in 0.1 N HC1) for 30 min a t room temperature. They were then washed twice (5 min each) in 0.5% Tween 20 (Sigma Chemical Co., St. Louis, MO) in PBS, followed by incubation in 4 N HC1 for 20 min a t room temperature. Following two washes in 0.1 M borax buffer, and three washes in PBS, the sections were incubated overnight at 4°C with monoclonal antibody, anti-BrdU (Becton Dickinson Immunocytometry Systems, Mountain View, CA), diluted 1:40 with PBS. Then the sections were washed in PBS and incubated for 30 min a t room temperature with biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA), diluted 1:200 with PBS. Immunochemical localization was achieved by the avidin biotin peroxidase complex method using a VectastainB ABC Kit (Vector Laboratories, Burlingame, CAI. The sections were incubated in the ABC complex at room temperature for 45 min, followed by incubation in 0.01% diaminobenzidine.4HCl and 0.02% H,Oz in 0.5 M Tris buffer (pH 7.6) for 7 min. Finally the sections were counterstained with Gill’s No. 1 hematoxylin solution (Sigma Chemical Co.) for 5 seconds. To derive labelling indices on gestational days 12 and 13, 500 epithelial cells were counted a t each airway level, from each fetus. To derive labelling indices on gestational days 14 and 15, one thousand epithelial cells were counted at each airway level, from each fetus. Nuclei that were stained brown (lightly, medium or darkly-stained) were considered labelled. The labelling indices are expressed a s percentages. The selected glycol methacrylate sections were stained by Alcian blue (pH 2.5) PAS-lead hematoxylin (AB-PAS-PbH) as previously described (McDowell et al., 1985).
hyperglycemia, were comparable on each gestational day (Tables 1,2). However, one dead fetus was found in each of two litters from diabetic mothers with blood glucose levels close to 400 mg/dl, on gestational days 12 and 14 (Table 2, Dams 20-5 and 20-13). Development of the Conducting Airway Epithelium in Normal and Diabetic Pregnancies BrdU-labelling indices
The airway epithelial cell labelling indices in fetuses of control dams are shown in Table 3. The most striking feature of these data is the consistency of the labelling indices a t any one airway level among pups of the same and different litters, sacrificed on the same gestational day. In most cases, the difference between the average labelling index for pups of the same gestational age, from different litters, was numerically very small. On gestational day 12, the difference between the labelling indices of the lobar bronchus and the bronchioles-terminal buds, was highly significant (P< .0001). On day 13, the differences between the lobar bronchus and bronchioles, between lobar bronchus and terminal buds, and between bronchioles and terminal buds, were also highly significant (P
Development of the Conducting Airway Epithelium in Fetal Syrian Golden Hamsters During Normal and Diabetic Pregnancies ELIZABETH M. McDOWELL, WILLIAM P. COLEMAN, ANDREA M. DE SANTI, CARNELL NEWKIRK, AND JUDY M. STRUM Departments o f Pathology (E.M.M., W.P.C., A.M.D., and C.N.) and Anatomy (J.M.S.), University of Maryland School of Medicine and Maryland Institute for Emergency Medical Services Systems (W.P.C.), Baltimore, Maryland
ABSTRACT The conducting airway epithelium of fetal Syrian golden hamsters was studied from gestational day 12 to day 15, during normal and uncontrolled diabetic pregnancies. Diabetes was induced in the pregnant hamsters by injecting streptozotocin a t 60 mglkg body weight, subcutaneously, early on gestational day 10. Cells in S-phase were labelled immunochemically with bromodeoxyuridine (BrdU), and the day on which endocrine cells and ciliated cells first appeared was determined. In control fetuses, the BrdU-labelling indices (LI’s) of different anatomical airway levels were significantly different from one gestational day to the next. For example, the LI of the lobar bronchus was significantly different on each gestational day (P < .0001), and the same was true of the bronchioles. Moreover, the difference between LI’s of the lobar bronchus and bronchioles-terminal buds was highly significant on day 12 ( P < .0001), and on day 13 the differences between lobar bronchus and bronchioles, lobar bronchus and terminal buds, and bronchioles and terminal buds were also highly significant ( P < ,0001). However, on gestational days 14 and 15, the LI’s were reduced and were comparable at different airway levels. The BrdU-labelling indices were very consistent among fetuses of the same age, and the differences between the average LI’s for pups of different litters was numerically very small. Hyperglycemia (mild, moderate, severe) did not alter LI’s in the fetal airway epithelial cells. Furthermore, although glycogen was not depleted from the airway epithelium of the hyperglycemic fetuses as it was in the controls, the endocrine cells first appeared on gestational days 12,13, and 14, respectively, in the trachea, lobar bronchus and bronchioles, followed 1 day later by the ciliated cells, in the fetuses of control and diabetic mothers. In our experimental model, induction of diabetes in the pregnant hamsters on gestational day 10 did not appear to alter development or differentiation of the fetal conducting airway epithelium. Early during normal development of the airways of Syrian golden hamsters, the undifferentiated epithelial cells contain abundant glycogen at all airway levels. These glycogen stores are depleted progressively a s the endocrine cells make their primordial appearance in the trachea, lobar bronchus, and bronchioles (gestational days 12, 13, and 14, respectively). Preciliated and ciliated cells then begin to differentiate a t each airway level, just 1 day after the endocrine cells first appear. Meanwhile, glycogen reaccumulates in the cytoplasm of the presecretory cells until the day of birth (McDowell e t al., 1985; Sarikas et al., 1985a). Little is known about the factors that regulate growth, differentiation, and functional maturation of the epithelial cells in the conducting airways during fetal development, but this wave of glycogen depletion followed by repletion, along the length of the developing airways must have some biological significance. 0 1990 WILEY-LISS, INC.
The appearance of the first endocrine cells coincides with a loss of glycogen from all airway levels, suggesting that energy derived from glycogenolysis might be required for the differentiation of these cells. Moreover, the subsequent reaccumulation of glycogen in the presecretory cells may be used later to provide energy for their functional maturation. Insulin-dependent maternal diabetes is associated with a n increased risk of early growth retardation in human embryos (Pedersen and Mdsted-Pedersen, 1981) and small fetuses are common in the offspring of
Received April 25, 1989; accepted September 12, 1989. Address reprint requests to Elizabeth M. McDowell, Ph.D., Department of Pathology, University of Maryland School of Medicine, 10 South Pine Street, Baltimore, MD 21201.
112
E.M. McDOWELL ET AL.
TABLE 1. Control hamsters'
Dam No. 19-2 19-3 21-16 18-3 19-7 21-2 18-10 21-1 21-14 21-6 21-8 21-12
Treatment on gestational day 10
A, B A, B N A, B A, B N A, B A, B N A, B A, €3 N
9 199 176 177 191 201 157 175 133 177 198 189 178
Blood glucose levels (mgidl) on gestational day 11 12 13 14 178 141K 146 126K 16gK 160 146K 155 126 127K 163K 154 130K 153 155 138 144 134K 12EiK 176 166 166 122 177 161 188 167
Total No. of 15 fetuses in litter 15 16 10 7 15 8 15 8 13 10IK 10 127K 9 116K 9
No. of dead fetuses in litter 0 0 0 0 0 0 0 0 0 0 0 0
Mean fetal weight (gm) ,201 k .034 ,244 ? ,029 .223 2 ,031 ,453 ,063 ,416 t ,052 ,492 2 ,068 1.001 ? ,110 1.032 2 ,094 1.115 2 0.92 1.705 2 .13 1.886 t ,173 1.785 +- ,198
*
'A = metaphane anesthesia; B = subcutaneous injection of 0.4 M citrate buffer, pH 4.2; K = killed; N = no treatment. 'Blood glucose levels according to results of the hexokinase method. The hamsters were anesthesized with metaphane every time a blood sample was taken from the orbital sinus. Blanks in the table indicate that no blood sample was taken at that time.
rodents with experimental diabetes (reviewed by phological changes occur each day in the developing Freinkel, 1988). The etiology of diabetic embryopathy lung a t different airway levels during the latter part of is complex and likely to be multifactorial, but hypergly- pregnancy (see Introduction). Changes in the epithelial cemia is one of the factors implicated (Freinkel et al., cells in the trachea precede similar changes in the lo1986; Eriksson et al., 1987; Sadler et al., 1989). More- bar bronchus and bronchioles by one and two days, over, the incidence of respiratory distress syndrome respectively (McDowell et al., 1985; Sarikas et al., (RDS), caused by delayed alveolar type I1 cell matura- 1985a). Thus, specific developmental changes can be tion and defective synthesis of surfactant, is increased observed at the different airway levels, and the normal in the offspring of some diabetics (Farrell and Avery, timing of these changes is very predictable. Timed-bred Syrian golden hamsters were purchased 1975; Robert et al., 1976). Glycogenolysis normally occurs in the type I1 cells near the end of gestation. This from Charles River (Newfield, NJ). The first gestaprocess is impaired in fetal lungs during experimental tional day was taken to be the day after mating. The diabetic pregnancies and fetal hyperglycemia is one of hamsters arrived a t our animal facility on the second the factors implicated (reviewed by Bourbon and Far- gestational day. They were caged individually, given rell, 1985). The under-utilization of glycogen in the de- food and water ad libitum, and maintained under stanveloping alveoli is thought to reduce the biosynthesis of dard laboratory conditions in a room with a 12-hour two major surfactant phospholipids, which impairs sur- lightil2-hour dark cycle. A total of sixteen pregnant hamsters served a s confactant production (reviewed by Tyden et al., 1986). Since impaired glycogenolysis appears to be linked trols. On gestational day 9, twelve of them were aneswith delayed maturation of the alveolar type I1 cells, thesized lightly with methoxyflurane (Metaphane, we postulated that the growth and differentiation of Pittman-Moore, Inc., Washington Crossing, NJ) and a the fetal conducting airway epithelium would likely be blood sample was taken from the orbital sinus (Pansky altered in the fetuses of mothers with diabetes. There- et al., 1961) to provide base-line blood glucose levels fore, our purpose in undertaking a study of fetal airway (Table 1). Early on gestational day 10, eight of these development in Syrian golden hamsters during normal hamsters were given a subcutaneous injection of 0.22 and uncontrolled diabetic pregnancies was threefold: 1) ml chilled 0.4 M citrate buffer (pH 4.2), under light to quantify epithelial cell growth using bromodeoxy- anesthesia, but four hamsters received no treatment uridine (BrdU)-immunochemical labelling; 2) to char- (Table 1). In addition, four control hamsters (not listed acterize the pattern and timing of glycogenolysis a t in Table 1)received no intervention during pregnancy. different anatomical levels; and 3) to characterize the Blood samples were not taken from these hamsters a t onset of cell differentiation. Our study will show that any time, nor were they anesthesized until they were the growth and differentiation of the fetal airway epi- killed. thelium was not impaired by maternal diabetes, a t Twelve pregnant hamsters served as experimental least under the conditions of our experiments. Never- animals, and diabetes was induced by injection of theless, our results with BrdU-labelling of the epithe- streptozotocin (STZ); the toxicity of this drug is dilial cells provide new data on the orderly progression of rected against the B cells of the pancreatic islets (refetal airway development. viewed by Rerup, 1970). On gestational day 9, the pregnant animals were anesthesized lightly and a blood MATERIALS AND METHODS sample was taken from the orbital sinus to provide We chose Syrian golden hamsters a s our experimen- base-line blood glucose levels (Table 2). Early on gestal model to study fetal airway development, as this tational day 10, the hamsters were anesthesized again species offers some unique advantages. The gestation and streptozotocin (Sigma Chemical Co., St. Louis, MO, period is unusually short for small mammals, about 16 USA) was injected subcutaneously a t 60 mg/kg, b.w., days (Bruce and Hindle, 1934).Moreover, specific mor- dissolved in 0.22 ml of 0.4 M citrate buffer, pH 4.2. The
DEVELOPMENT OF AIRWAY EPITHELIUM IN FETAL HAMSTERS
113
TABLE 2. Diabetic hamsters' Treatment on DamNo. gestationalday 10 9 19-4 A,B-S 160 19-12 A,B-S 146 20-5 A,B-S 198 18-8 A,B-S 153 18-1 A,B-S 195 20-11 A,B-S 177 20-6 A,B-S 206 20-7 A,B-S 192 20-13 A,B-S 170 20-3 A,B-S 151 20-12 A,B-S 174 20-9 A.B-S 171
Blood glucose levels (mg/dl) on gestational day 11 12 13 14 319 247K 380 338K 377 395K 308 233K 367 374K 327 440 41gK 350 277 213 151RK 331 288 423 380K 443 397 387 413K 430 296 394 370 264 427 395 468 397 348 405 449
No. of Total No. of dead fetuses 15 fetuses in litter in litter 13 0 15 0 10 1 12 0 12 0 12 0 12 0 8 0 8 1 0 183RK 12 11 0 424K 507K 8 0
Mean fetal weight (gm) ,210? .025 .2242 ,035 ,1782 ,028 ,5422 .060 ,5052 ,054 ,4632 ,052 1.1182 ,101 1.312? ,117 .802 t ,128 1.688 t .143 1.641 2 .21 1.831 ? ,067
'A = metaphane anesthesia; B-S = subcutaneous injection of streptozotocin (60 mg/kg, b.w.) dissolved in 0.4 M citrate buffer, pH 4.2; K = killed; RK = hyperglycemia had reverted by time of killing. 'Blood glucose levels according to results of the hexokinase method. The hamsters were anesthesized with metaphane every time a blood sample was taken from the orbital sinus. Blanks in the table indicate that no blood sample was taken a t that time. The base-line blood glucose levels are shown on gestational day 9.
buffer was chilled and the STZ was dissolved in i t immediately before the injection was made. In hamster fetuses, organogenesis occurs between gestational days 8 and 9%, and the tracheobronchial rudiment becomes independent of the esophageal duct at 9% gestational days (Boyer, 1953). Therefore, by injecting STZ into the dams early on gestational day 10, we were able to study the effect of maternal diabetes on airway development after the completion of organogenesis, from the time of independence of the tracheobronchial rudiment. Moreover, since the B cells of the fetal pancreatic islets do not differentiate and produce detectable insulin until gestational day 11 (Grillo, 1964), the direct toxicity of STZ to the fetal B cells was avoided. Blood glucose levels were monitored throughout pregnancy (Tables 1, 2). The blood samples were collected into heparinized capillary tubes. Each time a blood sample was taken, drops of blood were also placed on a glucose oxidase reagent strip (Chemstrip BG, Boehringer-Mannheim Biochemicals, Indianapolis, IN) for immediate determination of blood glucose (mg/dl) with a n Accu-check I1 reflectance meter (Boehringer-Mannheim Diagnostics, Indianapolis, IN). Blood in the capillary tubes was separated by centrifugation for 5 minutes and stored at -20°C until the plasma glucose was determined spectrophotometrically based on the reactions catalyzed by hexokinase and glucose-6-phosphate dehydrogenase, with the use of Dri-STAT@ glucoseHK Endpoint Reagent (Beckman Instruments, Inc., Carlsbad, CAI. On each of gestational days 12, 13, 14, and 15, four pregnant control hamsters and three pregnant diabetic hamsters were killed. Of the controls, two had received a n injection of citrate buffer on day 10 and had undergone several anesthesic and bleeding events, and one had been anesthesized and bled on day 9 (Table 1).In addition, one control dam, which had received no intervention during pregnancy, was killed on each gestational day. The range of hyperglycemia induced by STZ in the pregnant diabetic hamsters was wide, so whenever possible, the three diabetic hamsters killed on each gestational day were chosen to span that
range. In two cases of mild hyperglycemia, the blood glucose levels had reverted to control levels by the time the hamsters were killed, on gestational days 14 and 15 (Table 2). Two hours before being killed all pregnant hamsters listed in Tables 1 and 2 were anesthesized lightly and a small incision was made in the skin of the back. A tablet containing 25 mg 5-bromo-2'-deoxyuridine (Boehringer-Mannheim Biochemicals, Indianapolis, IN) was inserted subcutaneously. The skin was apposed with a suture. All hamsters were killed during the morning hours. At the time of killing, each pregnant hamster was anesthesized, the abdomen was opened, and the fetuses were rapidly removed from the gravid uterus and fixed by immersion in 4% formaldehyde-1% glutaraldehyde in a 200 mOsm phosphate buffer (McDowell and Trump, 1976). Fetuses taken from the control and diabetic dams listed in Tables l and 2 were fixed individually in preweighed vials containing fixative. This way, the fixation process was not delayed and the weight of each fetus could be determined later. On gestational days 12 and 13, fixation was achieved by immersion of the entire undamaged fetus and on days 14 and 15, the fetal abdomen was opened prior to immersion of the fetus in the fixative. The fetuses were fixed for at least 2 days a t room temperature and washed in 300 mOsm phosphate buffer for 2 days, prior to longitudinal embedment. Three fetuses from each of the 12 control dams listed in Table 1, and three fetuses from each of the diabetic dams listed in Table 2 (36 fetuses from controls; 36 fetuses from diabetics) were embedded in paraffin on their left sides. Longitudinal serial sections (5 pm thick) of the left lung were obtained. In addition, left lungs of fetuses from the four control dams which had received no intervention during pregnancy, were embedded in glycol methacrylate (Ruddel, 1967; Sorokin and Hoyt, 1978) and longitudinal serial sections (2 pm thick) were made. Sections (paraffin and glycol methacrylate) of lung which contained the lobar bronchus cut along its length, were selected for further study.
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The selected paraffin sections were stained with Alcian blue (pH 2.5)-periodic acid-Schiff (Mowry and Winkler, 1956), with and without diastase digestion. They were also stained immunochemically to detect BrdU-labelled cells. For the demonstration of BrdU, the sections were mounted on slides coated with chrom alum. This prevented the sections from floating off the slides during incubation in pepsin. Pepsin digestion was required because of the aldehydes in the fixative (Schutte et al., 1987). The sections were hydrated and endogenous peroxidase was blocked by incubation in 1%H,O, in methanol for 30 min. After washing in PBS, the sections were incubated in pepsin (Boehringer-Mannheim, Indianapolis, IN; 0.4 mgiml in 0.1 N HC1) for 30 min a t room temperature. They were then washed twice (5 min each) in 0.5% Tween 20 (Sigma Chemical Co., St. Louis, MO) in PBS, followed by incubation in 4 N HC1 for 20 min a t room temperature. Following two washes in 0.1 M borax buffer, and three washes in PBS, the sections were incubated overnight at 4°C with monoclonal antibody, anti-BrdU (Becton Dickinson Immunocytometry Systems, Mountain View, CA), diluted 1:40 with PBS. Then the sections were washed in PBS and incubated for 30 min a t room temperature with biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA), diluted 1:200 with PBS. Immunochemical localization was achieved by the avidin biotin peroxidase complex method using a VectastainB ABC Kit (Vector Laboratories, Burlingame, CAI. The sections were incubated in the ABC complex at room temperature for 45 min, followed by incubation in 0.01% diaminobenzidine.4HCl and 0.02% H,Oz in 0.5 M Tris buffer (pH 7.6) for 7 min. Finally the sections were counterstained with Gill’s No. 1 hematoxylin solution (Sigma Chemical Co.) for 5 seconds. To derive labelling indices on gestational days 12 and 13, 500 epithelial cells were counted a t each airway level, from each fetus. To derive labelling indices on gestational days 14 and 15, one thousand epithelial cells were counted at each airway level, from each fetus. Nuclei that were stained brown (lightly, medium or darkly-stained) were considered labelled. The labelling indices are expressed a s percentages. The selected glycol methacrylate sections were stained by Alcian blue (pH 2.5) PAS-lead hematoxylin (AB-PAS-PbH) as previously described (McDowell et al., 1985).
hyperglycemia, were comparable on each gestational day (Tables 1,2). However, one dead fetus was found in each of two litters from diabetic mothers with blood glucose levels close to 400 mg/dl, on gestational days 12 and 14 (Table 2, Dams 20-5 and 20-13). Development of the Conducting Airway Epithelium in Normal and Diabetic Pregnancies BrdU-labelling indices
The airway epithelial cell labelling indices in fetuses of control dams are shown in Table 3. The most striking feature of these data is the consistency of the labelling indices a t any one airway level among pups of the same and different litters, sacrificed on the same gestational day. In most cases, the difference between the average labelling index for pups of the same gestational age, from different litters, was numerically very small. On gestational day 12, the difference between the labelling indices of the lobar bronchus and the bronchioles-terminal buds, was highly significant (P< .0001). On day 13, the differences between the lobar bronchus and bronchioles, between lobar bronchus and terminal buds, and between bronchioles and terminal buds, were also highly significant (P
