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Effect of aminophylline on lung maturation in preterm rabbit fetuses
Effect of aminophylline on lung maturation in preterm rabbit fetuses E. v. Cosmi, c. Saitto, A. Barbati, F. Del Bolgia, G. C. Di Renzo, G. Grossmann, B. Lachmann, and B. Robertson Perugia, Italy, Stockholm, Sweden, and Rotterdam, The Netherlands Pregnant rabbit does were treated intravenously with aminophylline (6 mg/kg/day) from the twenty-fifth day after the day of mating, and the fetuses were delivered by hysterotomy on the twenty-eighth day. One group of neonates was breathing air, and another group 100% oxygen. Lung mechanics were evaluated in the newborn animals during spontaneous or artificial ventilation, and the lungs were studied histologically with particular reference to the alveolar volume density. In one series of experiments, the lungs were washed and the lavage fluid was analyzed for phosphatidylcholine and phosphatidylglycerol. Aminophylline-treated litters had greater body weights, an improved survival rate, and an increased amount of phosphatidylglycerol in lung lavage fluid. Respiratory frequency was increased in aminophyllinetreated animals breathing air, but data on lung compliance showed no significant difference between treated and control animals. In the present model, the beneficial effect of aminophylline can be attributed largely to a combination of accelerated fetal growth and improved postnatal regulation of breathing and less to a specific influence on the biochemical and functional maturation of the lung. (AM J OBSTET GVNECOL 1986;154:436-9.)
Key words: Aminophylline, fetal lung maturation, phosphatidylcholine, phosphatidylglycerol
Data from animal experiments 1. 2 indicate that lung adaptation can be facilitated in the premature neonate by maternal treatment with the phosphodiesterase inhibitor aminophylline and that the mechanisms by which this effect is obtained are in part similar to those operating after treatment with I)-adrenergic agents or glucocorticoids. These mechanisms include an increase of cyclic adenosine monophosphate in fetal lung tissue,' which may promote glycogenolysis: increase the content of tissue phospholipids,' and stimulate the production of surfactant phospholipids by the alveolar type II cells.6-9 Direct injection of aminophylline into the premature fetus may, according to some authors,1O induce a release of surfactant phospholipids into the alveolar spaces, although other investigators" have failed to confirm this effect. In view of the possibility that aminophylline might be used in the prophylaxis of the neonatal respiratory
From the 2nd Department of Obstetrics and Gynecology, University of Perugia, the Department of Pathology, St. Giiran's Hospital, Stockholm, and the Department of Anesthesiology, Erasmus University, Rotterdam. This work was supported by Consiglio Nazionale di Ricerche and the Ministry for Public Instruction, Italy, the Swedish Medical Research Council (Project No. 3351), the Swedish National Association against Heart and Chest Diseases, the Research Funds of the Karolinska Institute, the "Expressen" Prenatal Research Foundation, and Allmanna Barnbiirdshusets Minnesfond. Presented at the Thirty-first Annual Meeting of the Society for Gynecologic Investigation, San Francisco, California, March 21-24, 1984. Reprint requests: B. Robertson, Department of Pathology, St. Giiran's Hospital, Box 12500, S-112 81 Stockholm, Sweden.
436
distress syndrome as an alternative to glucocorticoids,12 we wanted to analyze whether maternal administration of this drug would improve in vivo lung mechanics and survival in prematurely delivered newborn rabbits. Since aminophylline may have a stimulatory effect on the central nervous system, including the units involved in the regulation of breathing,'3 we were interested in to what extent the effect would depend on whether the animals were breathing air or 100% oxygen. We also evaluated the effect of antenatal aminophylline treatment on the phospholipid profile of material sampled from the fetal air spaces by lung lavage. Material and methods The experiments were carried out on a total of 163 premature newborn rabbits, obtained from 19 New Zealand white does on the twenty-eighth day after the day of mating (term = the thirty-first day). Aminophylline was administered intravenously from the twenty-fifth to the twenty-seventh day in two daily injections, 6 mg/kg/day. Control rabbits received the same number of injections with normal saline solution. On the twenty-eighth day, the pregnant rabbits were killed by a rapid injection of 2 ml of pentobarbital sodium (40 mg/ml) and 5 ml of potassium chloride (I50 mg/ml). The fetuses were immediately obtained by hysterotomy. After delivery, the neonates were weighed and tracheotomy was performed. In one series of experiments (protocol 1), the animals were allowed to breathe air spontaneously for 2 hours; in another series (protocol 2), the animals breathed 100% oxygen for 1
Effect of aminophylline on lung maturation
Volume 154 Number 2
437
Table I. Body weight, survival rate, lung mechanics, and alveolar volume density in premature newborn rabbits after maternal treatment with aminophylline and in controls (protocol 1) Group
Parameters
Body weight (gm) (mean ± SEM) Survival rate (%) 30 min 60 min 120 min Respiratory frequency (breaths per min) ,(mean ± SEM) 30 min 60 min 120 min Cd," (mllcm H2 0/kg) (mean ± SEM) 30 min 60 min 120 min Vv (mean ± SEM)
Aminophylline (n = 33)
35 ± I
t
Controls (n = 35)
31 ±
P <0.005
67 33 30
46 9 9
NS <0.05 <0.05
40 ± 10 56 ± 13 99 ± 12
16 ± 6 69 ± 3 70 ± 14
=0.05 NS NS
0.43 ± 0.15 0.62 (n = I)
NS
0.56 0.96 0.81 0.41
± ± ± ±
0.11 0.16 0.38 0.03
*
0.39 ± 0.03
NS
Cdr" = Dynamic lung compliance; Vv = alveolar volume density. *No successful recordings.
hour and were then subjected to artificial ventilation for 1 hour. Protocol 1. The animals were kept in an incubator at 37° C. Lung mechanics during spontaneous ventilation were recorded 30, 60, and 120 minutes after birth, as described by Lachmann et al. 14 For this purpose, we used a fluid-filled esophageal catheter, a Fleisch tube connected to the tracheal cannula, a differential pressure transducer (EMT 32, SiemensElema, Solna, Sweden), an integrator unit (EMT 41, Siemens-Elema), and a recorder (Physiograph 6C, Narco Medical Systems, Dallas, Texas). Between the recordings the neonates were stimulated intermittently by gentle handling. Animals still making respiratory efforts 120 minutes after delivery were counted as survivors. At the end of the experiment, all animals received an overdose of intraperitoneal sodium pentobarbital. The lungs were fixed in situ by immersion of the unopened thorax in formalin. Paraffin sections from the lower lobes were studied histologically with particular reference to the expansion pattern. The volume density of the alveolar compartment was determined by pointcounting, using the total parenchyma as reference volume. Protocol 2. After tracheotomy the neonates were kept at 37° C in a system of multiple individual body plethysmographs, flushed with 100% oxygen. Respiratory frequency was evaluated 30 and 60 minutes after birth, by connecting the tracheal cannula to the Fleisch tube system described above. After the 60-minute recording, the animals were paralyzed with intraperitoneal pancuronium bromide (0.2 mg/ml, 0.1 ml) and connected in parallel to a pressureconstant ventilator system delivering 100% oxygen at a frequency of 40/min, and an inspiration: expiration
ratio of 1: 1. Tidal volumes were recorded pneumotachographically, by connecting the Fleisch tube to the body plethysmograph. The insufflation pressure was adjusted to maintain an average tidal volume of about 10 mil kg in each litter. In order to assess survival, recordings of electrocardiogram were obtained at the end of the experiment. The animals were then put to death with an overdose of intraperitoneal pentobarbital sodium. In 42 animals (21 aminophylline-treated, 21 controls), the lungs were fixed by vascular perfusion. A cannula was inserted into the pulmonary trunk, and the lungs were expanded by raising the intratracheal pressure to 30 cm H 2 0. This pressure was then lowered to 10 cm H 20 for 10 minutes, while the lungs were perfused via the pulmonary artery with a mixture of I % glutaraldehyde and 3.5% formaldehyde at a pressure of 65 cm H 20. The lungs were stored in 10% formaldehyde for at least 24 hours and embedded in paraffin. Histologic sections from the lower lobes were examined morphometrically as described above. In 39 animals (18 aminophylline-treated, 21 controls), the lungs were used for biochemical studies. These lungs were lavaged four times with 20 ml of saline per kilogram of body weight. Concentrations of phosphatidylcholine and phosphatidylglycerol were analyzed enzymatically according to Muneshige et al. " Statistical evaluation. The X2 test and the Wilcoxon two-sample (two-tailed) test were used for statistical evaluation of our results.
Results Protocol 1. In litters treated with aminophylline, the average body weight was increased by approximately 13% and the survival rate was significantly improved 60 and 120 minutes after birth (Table I). The rate of
438
Cosmi et al.
February, 1986 Am J Obstet Gynecol
Table II. Body weight, survival rate, lung mechanics, and alveolar volume density in premature newborn rabbits after maternal treatment with aminophylline and in controls (protocol 2) Group Aminophylline (n = 43)
Parameter Body weight (gm) (mean ± SEM) Survival rate (%) 30 min 60 min 120 min Respiratory frequency (breaths per min) (mean ± SEM) 30 min 60 min C (mllcm H,O/kg) (mean ± SEM) 60 min 120 min V" (mean ± SEM)
40 ± I
I
Controls (n = 52)
33 ± I
p <0,005 <0,05 NS NS
80 73 71
60 60 59
101 ± 12 98 ± 9
72 ± 12 88 ± 9
NS NS
1,00 ± 0,09 0,85 ± 0,10 0,65 ± 0.12
0.76 ± 0.09 0.64 ± 0.09 0.62 ± 0,11
NS NS NS
C = Lung-thorax compliance; V\'= alveolar volume density,
Table III. Phospholipids in lung lavage fluid (protocol 2) Group
I
Cantrall
p
15.2 ± 3.2
17.2 ± 3.8
NS
27.9 ± 6.8
9.7 ± 1.7
0.02
Phospholipids
Aminophylline
Phosphatidylcholine (nmollml) Phosphatidylglycerol (nmollml)
Values are given as mean ± SEM,
respirations among survivors was higher in the treated animals 30 minutes after birth, but values for dynamic lung compliance and alveolar volume density showed no statistically significant differences when compared with those of the control group (Table I), Protocol 2. Aminophylline-treated animals again had a higher body weight, and the survival rate was improved 30 minutes after birth (Table II). Respiratory frequency among survivors, lung compliance, alveolar volume density, and phosphatidylcholine in lung lavage fluid showed no statistically significant differences between treated animals and controls (Tables II and III), However, the concentration of phosphatidylglycerol in lavage fluid was increased in animals receiving aminophylline (Table III). There was no correlation between levels of phosphatidylglycerol in lung lavage fluid and respiratory frequency or between phosphatidylglycerol levels and lung-thorax compliance. Comparison and combination of data from protocol 1 and protocol 2. Survival rate after 60 minutes was significantly higher in the neonates breathing 100% oxygen (protocol 2) than in those breathing air (protocol 1); this holds [0/' both aminophylline-treated animals and controls (p < 0.001). If survival figures from the two protocols are combined, there is again a statis-
tically significant improvement among the aminophylline-treated animals at the 30- and 60-minute intervals, compared with the combined control groups (p < 0.02 and p < 0.05, respectively). Aminophylline-treated neonates used for protocol 2 had a higher body weight than those used for protocol I (p < 0,005); no such difference was found between the two control groups. The body weight remained significantly larger in the combined groups of aminophylline-treated animals than in the combined control groups, even if the individual litter was regarded as the sample: 38 ± I gm (n = 9) versus 34 ± 2 gm (n = 10) (mean ± SEM; p = 0.05), Differences in alveolar volume density between animals subjected to protocol I and protocol 2 can be attributed entirely to the fact that in the former the lungs were immersed in formalin without distending pressure, whereas in the latter group an expanding pressure was maintained during perfusion fixation. Comment
Aminophylline exerts a wide range of pharmacologic actions, some of which are mediated by relaxation of the smooth muscles. For example, aminophylline inhibits uterine contractions and possibly increases uterine blood flow, I" This is one of the reasons why aminophylline is used for the treatment of preterm labor and eventually for the treatment of fetal distress due to uterine hyperactivity. I", 16 Another well-documented effect of aminophylline is bronchorelaxation, which makes it the traditional drug of choice for the treatment of asthma. Some of our results can be explained by the influence of aminophylline on the uterine tonus and the uteroplacental circulation. Enhanced uterine blood flow could lead to accelerated fetal growth, as indeed documented in the present series by increased average
Volume 154 "'umber 2
fetal body weight in both groups of aminophyllinetreated litters. After maternal treatment with aminophylline, the fetuses were also more vigorous, with improved figures for survival and respiratory frequency. These may be nonspecific effects, reflecting the general maturity level in these particular litters. However, the increased respiratory frequency observed shortly after birth in aminophylline-treated animals breathing air (protocol 1) can also be explained by a direct action of the drug on the central nervous system. Aminophylline is widely used for the treatment of the apnea of prematurity because it increases the central "respiratory drive" of the newborn infant. 17 Most previous studies on fetal rabbits have indicated that aminophylline accelerates the biochemical and functional maturation of the lung, as reflected by an increased production of surface-active phospholipids and improved pressure-volume characteristics. I. 2. 7,,1 In the present experiments, levels of phosphatidylglycerol were increased in lung lavage fluid from aminophylline-treated fetuses, suggesting accelerated lung maturity. We were unable to document a beneficial effect of aminophylline on the compliance of the lungs, either during spontaneous ventilation or during artificial ventilation. Average values for compliance were, in general, greater in aminophylline-treated animals than in controls, but there was a wide individual variation and the differences obtained with the present number of animals failed to reach statistical significance. To a large extent, this variation may reflect the normal situation in rabbit fetuses on the twenty-eighth day. This day represents a transitional stage of fetal maturation, between the apneic, surfactant-deficient state on the twenty-seventh day and the nearly mature state on the twenty-ninth. Large standard errors are to be expected in parameters of lung maturation recorded in fetuses delivered on the twenty-eighth day. We nevertheless chose this gestational age for our study, as we wanted to record lung mechanics during spontaneous ventilation, also in control animals. We conclude that the moderate beneficial effects of aminophylline, noted in the present study, can be attributed largely to a combination of accelerated fetal growth and improved postnatal regulation of breathing and only to a minor extent to a specific influence on biochemical and functional lung maturation.
REFERENCES I. Karotkin EH, Kido MK, Cashore WJ, et al. Acceleration of fetal lung maturation by aminophylline in pregnant rabbits. Pediatr Res 1976;10:722-4.
Effect of aminophylline on lung maturation
439
2. Barrett CT, Sevanian A, Phelps DL, Gilden C, Kaplan SA. Effects of cortisol and aminophylline upon survival, pulmonary mechanics, and secreted phosphatidyl choline of prematurely delivered rabbits. Pediatr Res 1978; 12:3842. 3. Barrett CT, Sevanian A, Lavin N, Kaplan SA. Role of adenosine 3',5' -monophosphate in maturation of fetal lungs. Pediatr Res 1976; 10:621-5. 4. Niiiar MS. Role of cyclic AMP and related enzymes in rat lung growth and development. Biochim Biophys Acta 1979;586:464-72. 5. Landers S, Corbet A, Cregan .I, Frink .I, Rudolph AJ. Effect of aminophylline and caffeine on total and surfactant phospholipid in fetal rabbit lung. Am Rev Respir Dis 1984; 130:204-8. 6. Gross I, Rooney SA. Aminophylline stimulates the incorporation of choline into phospholipid in explants of fetal rat lung in organ culture. Biochim Biophys Acta 1977: 488:263-9. 7. Sevanian A, Gilden C, Kaplan SA, Barrett CT. Enhancement of fetal lung surfactant production by aminophylline. Pediatr Res 1979; 13: 1336-40. 8. Ayromlooi J, Desiderio D, Tobias M, Steinberg H, Das DK. Effect of aminophylline on the synthesis of dipalmitoyl phosphatidyl choline in fetal rabbit lung. Pediatr Pharmacol 1981; I :209-14. 9. Aeberhard EE, Scott ML, Barrett CT, Kaplan SA. Effects of cyclic AMP analogues and phosphodiesterase inhibitors on phospholipid biosynthesis in fetal type II pneumocytes. Biochim Biophys Acta 1984;803:29-38. 10. Corbet Aj, Flax P, Alston C, Rudolph AJ. Effect of aminophylline and dexamethasone on secretion of pulmonary surfactant in fetal rabbits. Pediatr Res 1978; 12:797-9. II. Ekelund L, Burgoyne R, Brymer D, Enhorning G. Pulmonary surfactant release in fetal rabbits as affected by terbutaline and aminophylline. Scand .I Clin Lab Invest 1981;41:237-45. 12. Hadjigeorgiou E, Kitsiou S, Psaroudakis A, Segos C, Nicolo poulos D, Kaskarelis D. Antepartum aminophylline treatment for prevention of the respiratory distress syndrome in premature infants. A~1 .J OBSTET GYNECOL 1979; 135:257-60. 13. Cosmi EV. Drugs and anesthetics in the management of preterm labor and delivery. In: Cosmi EV, ed. Obstetric anesthesia and perinatology. New York: Appleton-Century-Crofts, 1981 :547-623. 14. Lachmann B, Grossmann G, Nilsson R, Robertson B. Lung mechanics during spontaneous ventilation in premature and fullterm rabbit neonates. Respir Physiol 1979;38:283-302. 15. Muneshige A, Okazaki T, QuirkJG, MacDonald PC, Nozaki M, Johnston JM. A rapid and specific enzymatic method for the quantification of phosphatidylcholine and phosphatidylglycerol. A~I .I OBSTET GV:\ECOL 1983; 145:474-80. 16. Liu DTY, Blackwell R.J. The value of a scoring system in predicting outcome of preterm labour and comparing the efficacy of treatment with aminophylline and salbutamol. Br .I Obstet Gynaecol 1978;85:418-24. 17. Gerhardt T, McCarthy J, Bancalari E. Effects of aminophylline on respiratory center and reflex activity in premature infants with apnea. Pediatr Res 1983; 17: 188-91.
Key words: Aminophylline, fetal lung maturation, phosphatidylcholine, phosphatidylglycerol
Data from animal experiments 1. 2 indicate that lung adaptation can be facilitated in the premature neonate by maternal treatment with the phosphodiesterase inhibitor aminophylline and that the mechanisms by which this effect is obtained are in part similar to those operating after treatment with I)-adrenergic agents or glucocorticoids. These mechanisms include an increase of cyclic adenosine monophosphate in fetal lung tissue,' which may promote glycogenolysis: increase the content of tissue phospholipids,' and stimulate the production of surfactant phospholipids by the alveolar type II cells.6-9 Direct injection of aminophylline into the premature fetus may, according to some authors,1O induce a release of surfactant phospholipids into the alveolar spaces, although other investigators" have failed to confirm this effect. In view of the possibility that aminophylline might be used in the prophylaxis of the neonatal respiratory
From the 2nd Department of Obstetrics and Gynecology, University of Perugia, the Department of Pathology, St. Giiran's Hospital, Stockholm, and the Department of Anesthesiology, Erasmus University, Rotterdam. This work was supported by Consiglio Nazionale di Ricerche and the Ministry for Public Instruction, Italy, the Swedish Medical Research Council (Project No. 3351), the Swedish National Association against Heart and Chest Diseases, the Research Funds of the Karolinska Institute, the "Expressen" Prenatal Research Foundation, and Allmanna Barnbiirdshusets Minnesfond. Presented at the Thirty-first Annual Meeting of the Society for Gynecologic Investigation, San Francisco, California, March 21-24, 1984. Reprint requests: B. Robertson, Department of Pathology, St. Giiran's Hospital, Box 12500, S-112 81 Stockholm, Sweden.
436
distress syndrome as an alternative to glucocorticoids,12 we wanted to analyze whether maternal administration of this drug would improve in vivo lung mechanics and survival in prematurely delivered newborn rabbits. Since aminophylline may have a stimulatory effect on the central nervous system, including the units involved in the regulation of breathing,'3 we were interested in to what extent the effect would depend on whether the animals were breathing air or 100% oxygen. We also evaluated the effect of antenatal aminophylline treatment on the phospholipid profile of material sampled from the fetal air spaces by lung lavage. Material and methods The experiments were carried out on a total of 163 premature newborn rabbits, obtained from 19 New Zealand white does on the twenty-eighth day after the day of mating (term = the thirty-first day). Aminophylline was administered intravenously from the twenty-fifth to the twenty-seventh day in two daily injections, 6 mg/kg/day. Control rabbits received the same number of injections with normal saline solution. On the twenty-eighth day, the pregnant rabbits were killed by a rapid injection of 2 ml of pentobarbital sodium (40 mg/ml) and 5 ml of potassium chloride (I50 mg/ml). The fetuses were immediately obtained by hysterotomy. After delivery, the neonates were weighed and tracheotomy was performed. In one series of experiments (protocol 1), the animals were allowed to breathe air spontaneously for 2 hours; in another series (protocol 2), the animals breathed 100% oxygen for 1
Effect of aminophylline on lung maturation
Volume 154 Number 2
437
Table I. Body weight, survival rate, lung mechanics, and alveolar volume density in premature newborn rabbits after maternal treatment with aminophylline and in controls (protocol 1) Group
Parameters
Body weight (gm) (mean ± SEM) Survival rate (%) 30 min 60 min 120 min Respiratory frequency (breaths per min) ,(mean ± SEM) 30 min 60 min 120 min Cd," (mllcm H2 0/kg) (mean ± SEM) 30 min 60 min 120 min Vv (mean ± SEM)
Aminophylline (n = 33)
35 ± I
t
Controls (n = 35)
31 ±
P <0.005
67 33 30
46 9 9
NS <0.05 <0.05
40 ± 10 56 ± 13 99 ± 12
16 ± 6 69 ± 3 70 ± 14
=0.05 NS NS
0.43 ± 0.15 0.62 (n = I)
NS
0.56 0.96 0.81 0.41
± ± ± ±
0.11 0.16 0.38 0.03
*
0.39 ± 0.03
NS
Cdr" = Dynamic lung compliance; Vv = alveolar volume density. *No successful recordings.
hour and were then subjected to artificial ventilation for 1 hour. Protocol 1. The animals were kept in an incubator at 37° C. Lung mechanics during spontaneous ventilation were recorded 30, 60, and 120 minutes after birth, as described by Lachmann et al. 14 For this purpose, we used a fluid-filled esophageal catheter, a Fleisch tube connected to the tracheal cannula, a differential pressure transducer (EMT 32, SiemensElema, Solna, Sweden), an integrator unit (EMT 41, Siemens-Elema), and a recorder (Physiograph 6C, Narco Medical Systems, Dallas, Texas). Between the recordings the neonates were stimulated intermittently by gentle handling. Animals still making respiratory efforts 120 minutes after delivery were counted as survivors. At the end of the experiment, all animals received an overdose of intraperitoneal sodium pentobarbital. The lungs were fixed in situ by immersion of the unopened thorax in formalin. Paraffin sections from the lower lobes were studied histologically with particular reference to the expansion pattern. The volume density of the alveolar compartment was determined by pointcounting, using the total parenchyma as reference volume. Protocol 2. After tracheotomy the neonates were kept at 37° C in a system of multiple individual body plethysmographs, flushed with 100% oxygen. Respiratory frequency was evaluated 30 and 60 minutes after birth, by connecting the tracheal cannula to the Fleisch tube system described above. After the 60-minute recording, the animals were paralyzed with intraperitoneal pancuronium bromide (0.2 mg/ml, 0.1 ml) and connected in parallel to a pressureconstant ventilator system delivering 100% oxygen at a frequency of 40/min, and an inspiration: expiration
ratio of 1: 1. Tidal volumes were recorded pneumotachographically, by connecting the Fleisch tube to the body plethysmograph. The insufflation pressure was adjusted to maintain an average tidal volume of about 10 mil kg in each litter. In order to assess survival, recordings of electrocardiogram were obtained at the end of the experiment. The animals were then put to death with an overdose of intraperitoneal pentobarbital sodium. In 42 animals (21 aminophylline-treated, 21 controls), the lungs were fixed by vascular perfusion. A cannula was inserted into the pulmonary trunk, and the lungs were expanded by raising the intratracheal pressure to 30 cm H 2 0. This pressure was then lowered to 10 cm H 20 for 10 minutes, while the lungs were perfused via the pulmonary artery with a mixture of I % glutaraldehyde and 3.5% formaldehyde at a pressure of 65 cm H 20. The lungs were stored in 10% formaldehyde for at least 24 hours and embedded in paraffin. Histologic sections from the lower lobes were examined morphometrically as described above. In 39 animals (18 aminophylline-treated, 21 controls), the lungs were used for biochemical studies. These lungs were lavaged four times with 20 ml of saline per kilogram of body weight. Concentrations of phosphatidylcholine and phosphatidylglycerol were analyzed enzymatically according to Muneshige et al. " Statistical evaluation. The X2 test and the Wilcoxon two-sample (two-tailed) test were used for statistical evaluation of our results.
Results Protocol 1. In litters treated with aminophylline, the average body weight was increased by approximately 13% and the survival rate was significantly improved 60 and 120 minutes after birth (Table I). The rate of
438
Cosmi et al.
February, 1986 Am J Obstet Gynecol
Table II. Body weight, survival rate, lung mechanics, and alveolar volume density in premature newborn rabbits after maternal treatment with aminophylline and in controls (protocol 2) Group Aminophylline (n = 43)
Parameter Body weight (gm) (mean ± SEM) Survival rate (%) 30 min 60 min 120 min Respiratory frequency (breaths per min) (mean ± SEM) 30 min 60 min C (mllcm H,O/kg) (mean ± SEM) 60 min 120 min V" (mean ± SEM)
40 ± I
I
Controls (n = 52)
33 ± I
p <0,005 <0,05 NS NS
80 73 71
60 60 59
101 ± 12 98 ± 9
72 ± 12 88 ± 9
NS NS
1,00 ± 0,09 0,85 ± 0,10 0,65 ± 0.12
0.76 ± 0.09 0.64 ± 0.09 0.62 ± 0,11
NS NS NS
C = Lung-thorax compliance; V\'= alveolar volume density,
Table III. Phospholipids in lung lavage fluid (protocol 2) Group
I
Cantrall
p
15.2 ± 3.2
17.2 ± 3.8
NS
27.9 ± 6.8
9.7 ± 1.7
0.02
Phospholipids
Aminophylline
Phosphatidylcholine (nmollml) Phosphatidylglycerol (nmollml)
Values are given as mean ± SEM,
respirations among survivors was higher in the treated animals 30 minutes after birth, but values for dynamic lung compliance and alveolar volume density showed no statistically significant differences when compared with those of the control group (Table I), Protocol 2. Aminophylline-treated animals again had a higher body weight, and the survival rate was improved 30 minutes after birth (Table II). Respiratory frequency among survivors, lung compliance, alveolar volume density, and phosphatidylcholine in lung lavage fluid showed no statistically significant differences between treated animals and controls (Tables II and III), However, the concentration of phosphatidylglycerol in lavage fluid was increased in animals receiving aminophylline (Table III). There was no correlation between levels of phosphatidylglycerol in lung lavage fluid and respiratory frequency or between phosphatidylglycerol levels and lung-thorax compliance. Comparison and combination of data from protocol 1 and protocol 2. Survival rate after 60 minutes was significantly higher in the neonates breathing 100% oxygen (protocol 2) than in those breathing air (protocol 1); this holds [0/' both aminophylline-treated animals and controls (p < 0.001). If survival figures from the two protocols are combined, there is again a statis-
tically significant improvement among the aminophylline-treated animals at the 30- and 60-minute intervals, compared with the combined control groups (p < 0.02 and p < 0.05, respectively). Aminophylline-treated neonates used for protocol 2 had a higher body weight than those used for protocol I (p < 0,005); no such difference was found between the two control groups. The body weight remained significantly larger in the combined groups of aminophylline-treated animals than in the combined control groups, even if the individual litter was regarded as the sample: 38 ± I gm (n = 9) versus 34 ± 2 gm (n = 10) (mean ± SEM; p = 0.05), Differences in alveolar volume density between animals subjected to protocol I and protocol 2 can be attributed entirely to the fact that in the former the lungs were immersed in formalin without distending pressure, whereas in the latter group an expanding pressure was maintained during perfusion fixation. Comment
Aminophylline exerts a wide range of pharmacologic actions, some of which are mediated by relaxation of the smooth muscles. For example, aminophylline inhibits uterine contractions and possibly increases uterine blood flow, I" This is one of the reasons why aminophylline is used for the treatment of preterm labor and eventually for the treatment of fetal distress due to uterine hyperactivity. I", 16 Another well-documented effect of aminophylline is bronchorelaxation, which makes it the traditional drug of choice for the treatment of asthma. Some of our results can be explained by the influence of aminophylline on the uterine tonus and the uteroplacental circulation. Enhanced uterine blood flow could lead to accelerated fetal growth, as indeed documented in the present series by increased average
Volume 154 "'umber 2
fetal body weight in both groups of aminophyllinetreated litters. After maternal treatment with aminophylline, the fetuses were also more vigorous, with improved figures for survival and respiratory frequency. These may be nonspecific effects, reflecting the general maturity level in these particular litters. However, the increased respiratory frequency observed shortly after birth in aminophylline-treated animals breathing air (protocol 1) can also be explained by a direct action of the drug on the central nervous system. Aminophylline is widely used for the treatment of the apnea of prematurity because it increases the central "respiratory drive" of the newborn infant. 17 Most previous studies on fetal rabbits have indicated that aminophylline accelerates the biochemical and functional maturation of the lung, as reflected by an increased production of surface-active phospholipids and improved pressure-volume characteristics. I. 2. 7,,1 In the present experiments, levels of phosphatidylglycerol were increased in lung lavage fluid from aminophylline-treated fetuses, suggesting accelerated lung maturity. We were unable to document a beneficial effect of aminophylline on the compliance of the lungs, either during spontaneous ventilation or during artificial ventilation. Average values for compliance were, in general, greater in aminophylline-treated animals than in controls, but there was a wide individual variation and the differences obtained with the present number of animals failed to reach statistical significance. To a large extent, this variation may reflect the normal situation in rabbit fetuses on the twenty-eighth day. This day represents a transitional stage of fetal maturation, between the apneic, surfactant-deficient state on the twenty-seventh day and the nearly mature state on the twenty-ninth. Large standard errors are to be expected in parameters of lung maturation recorded in fetuses delivered on the twenty-eighth day. We nevertheless chose this gestational age for our study, as we wanted to record lung mechanics during spontaneous ventilation, also in control animals. We conclude that the moderate beneficial effects of aminophylline, noted in the present study, can be attributed largely to a combination of accelerated fetal growth and improved postnatal regulation of breathing and only to a minor extent to a specific influence on biochemical and functional lung maturation.
REFERENCES I. Karotkin EH, Kido MK, Cashore WJ, et al. Acceleration of fetal lung maturation by aminophylline in pregnant rabbits. Pediatr Res 1976;10:722-4.
Effect of aminophylline on lung maturation
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