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Increased heart rate response to parasympathetic and beta adrenergic blockade in growth-retarded fetal lambs
Increased heart rate response to parasympathetic and beta adrenergic blockade in growth-retarded fetal lambs ANTBAL. JAMES
J.
R. GREEN,
ROBERT
Ii.
ABRAHAM Sntl
LLANOS.
M.D.
CREASY, M.
Fra~li~isco,
M.D.* M.D.
RUDOLPH.
M.D.
California
To determine the influence of the autonomic nervous system on the circulation of normal and growth-retarded fetal lambs we measured the responses of heart rate and arterial blood pressure to parasympathetic (atropine 0.2 mg/kg) and p-adrenergic (propranolol 1 .O mg/kg) blockade in the last quarter of gestation. The heart rate response to parasympathetic blockade increased with gestational age in both normal and growth-retarded fetuses but in the growth-retarded fetuses there was a significantly greater response to parasympathetic blockade than in control fetuses from 121 days’ gestation to term. The heart rate response to /3-adrenergic blockade did not change with gestational age in normal fetuses. From 131 days’ gestation to term the heart rate response to p-adrenergic blockade in growth-retarded fetuses was significantly higher than in normal fetuses. The systemic arterial blood pressure responses to parasympathetic or p-adrenergic blockade were similar in growth-retarded and normal fetuses. These results indicate an increased parasympathetic and p-adrenergic influence on the heart of fetuses whose growth has been retarded. It may also suggest a more generalized increase in parasympathetic and sympathetic tone affecting other organs and systems in the growth-retarded fetal lambs. (AM. J. OBSTET. GYNECOL. 136:808, 1980.)
‘THE: AUTONOMIC nervous system is involved in the regulation of‘ heart rate and arterial blood pressure.‘-” of cardiac output and its distribution,“-6 and in response to hypoxemia and other forms of stress’-” in fetal and newborn mammals. Newborn infants who have sustained intrauterine growth retardation secondary to a probable reduction in uteroplacental blood
FTO~ the Department of Obstetrics, Gynecology, and Reprodurtive Sciences, and the Cardiovascular Research Instttute, C’niversity of California San Franckco. Supported HDOfXl9.
by United
States Public
RPWWY~ jar publication Accepted May
February
Health
Service Grant
20, 1979
29, 1979
Repmzt rPqmstS: DT. Robert K. Cress!, Depatiment qf Obstetncr, Gynecology, and ReprodurttzIe Sciences, G’niversity of Ca&amia at San Francwco, Sari Francisco, Cal$mrrio 94143. *Reciptent oj a Postdoctoral Fellows/$ from the Bay Area Heart Research Association. Present addresh: Departamento de Medicina Experimental, Sede Santiago Oviente-Escuela & Medic% Universidad de Chile. Ca.tilln 16038, Santiago 9, Chile.
808
flow have an increased hematocrit and red cell mass”, I2 suggesting that they may have been subjected to chronic hypoxemia during fetal life. Also, clinical experience with growth-retarded fetuses and newborn infants indicates that they are very susceptible to acute hypoxemia or other forms of stress and frequently die in the perinatal period.‘” In fetal lambs with growth retardation induced by embolization of the uteroplacental vascular bed, the fetal arterial PO, is chronically decreased and they show a different distribution of cardiac output and organ blood flow when compared to normal fetuses.‘“, ‘L ?2 In addition, the hematocrit is increasedI in the growthretarded fetal lambs as it is observed in human newborn infant whose growth has been retarded. The direct and indirect signs of hypoxemia in growthretarded fetal lambs and their alterations in organ perfusion suggest that the activity of the autonomic nervous system is increased in this syndrome. We therefore investigated the activity of the autonomic nervous system by serially measuring the effect of parasympathetic and /I-adrenergic blockade on fetal 0002-9378/80/060808+06$00.60/0
0 1980 The C. V. Mosby
Co.
Volume Number
136 6
heart rate and arterial blood pressure during ing gestation in normal and growth-retarded lambs.
Heart
advancfetal
rate
response
to parasympathetic
Table I. Mean body weight weights in growth-retarded
Sixteen mixed-breed Western ewes with known gestational ages were surgically prepared when they were between 105 and 110 days’ gestation. Polyvinyl carheters were inserted into fetal carotid and femoral arteries and jugular and femoral veins, the amniotic cavity, and a maternal femoral artery and vein. In nine ewes a catheter also was placed in the uterine artery; through this catheter we embolized the maternal placental bed daily with 15 pm diameter nonradioactive microspheres as we have described previously.*4 Seven ewes without an arterial uterine catheter were used as control subjects. At 135 days’ gestation two of the control animals spontaneously delivered dead fetuses. The organ weights of these two fetuses were not considered in the results, but the arterial pH, blood gases and hematocrit, heart rate, and arterial blood pressure were in the normal range throughout the experiments” and these values were included in the results. All fetuses were studied at least five days after surgery while the unsedated ewe stood quiently in her cage. We measured fetal arterial pH, blood gases, and hematocrit daily, and before and after each drug injection. Fetal and maternal blood pressure and amniotic fluid pressure were monitored with Statham P23 Db pressure transducers and a direct-writing recorder (Beckman Dynograph R). Fetal heart rate was monitored by a cardiotachometer: but during the experiments, to be more accurate, the changes in fetal and maternal heart rate were measured directly from rapid recordings of blood pressure tracings. Fetal arterial pressure was referred to the amniotic fluid pressure as zero. We measured the fetal and maternal cardiovascular variables and amniotic fluid pressure for at least 60 minutes before each drug injection. Atropine, 0.2 mg/kg of estimated body weight,t6 was injected by a bolus injection into a fetal femoral vein to produce parasympathetic blockade. Completeness of the blockage was assessed by inhibition of the effects of acetylcholine (15 pg/kg) on fetal heart rate and arterial pressure. Propranolol (1 mg/kg) was injected as a bolus into a fetal femoral vein for /3-adrenergic blockade. The completeness of the blockade was demonstrated by inhibition of the effects of isoproterenol (0.1 &g/kg) on fetal heart rate and arterial pressure. Sodium chloride 0.9% at room temperature was injected as a control before each drug study. Heart rate and arterial pressure responses to a single intravenous injection of
data
Gestational age (days) Body weight (kg) Body length (cm) Adrenal gland (am) Brain (gm) Heart
Mean
(N = 5) 138 ” 0.90
Growth-retarded (N = 9)
50 + 0.38
138 2 0.60 3.2 + 0.12t 43 + 1.oot
0.51 f 0.02
0.52 2 0.01
3.9 t 0.80 50 2 2.00 33 2 0.90
fern)
Liver (&) Thymus (gm) Brain-to-liver weight ratio
blockade
132 a 6.00 19 f 2.00 0.38 ? 0.01
f SE; n = number
809
and length and organ fetal lambs
Control Clinical
Materials and methods
and p-adrenergic
47 2 2.00 23 f. 0.50* 90 +- 3.00t 6a I.OOt
% of Con&r01 100 82 86 102 94 70 68 32
0.52 ? 0.02*
of animals.
*p < 0.01. tp ‘Z 0.002. atropine and propranolol were tested in seven control and in nine growth-retarded fetuses between 110 and 120, 121 and 130, 131 and 140 days of gestation. Each animal was tested once with atropine and once with propranolol in each time period, so each animal had three studies of each drug throughout the whole study. The drugs were tested in random order and at least one day intervened between atropine and propranolol studies. We calculated the basal cardiovascular values before the injection of the drugs by averaging the measurements at one-minute intervals for 30 minutes before the injection. Fetal heart rate and arterial blood pressure values after atropine injection were calculated by averaging the measurements at one-minute intervals for ten minutes. Acetylcholine did not produce any modifications in fetal heart rate and arterial blood pressure during this time period. Fetal heart rate and arterial blood pressure values after propranolol injection were calculated by averaging the measurements at oneminute intervals for 30 minutes after the injection. Isoproterenol did not produce any modification in fetal heart rate and arterial blood pressure during this time period. The animals in both groups were killed at 140 days’ gestation, and fetal body weight and length and organ weights were measured.
Statistical analysis The Student unpaired t-test was used to compare the differences in body weight, length, organ weights, pH, blood gases, and hematocrit between control and embolized groups. A difference was considered significant when the p value was less than 0.05. The response to
810
Llanos
et al.
March Am. J. Obstet.
15, 1980 Gynecol.
Table II. Mean femoral arterial pH, blood gases, and hematocrit in growth-retarded fetal lambs from I31 to 140 days’ gestation
I Control I (N = 15)
PH
Pcop (torr) Po, (torr) Hematocrit (%)
7.39 + 0.007 44.7 + 0.59
Growth-retarded (N = 27) 7.39 k 0.005 46.6 rtr. 0.71
23.1 -c 0.67
18.5 * 0.45*
30.6
35.0
k 0.98
r 0.5t
Values are means & SE. “p < 0.02. tp < 0.01. GESTATIONAL
AGE (days)
Fig. 1. Mean it SE heart rate increase in control and emboliration fetal lambs from 110 to 140 days’ gestation after intravenous injection of 0.2 mg/kg of atropine. *p < 0.001 and *p c 0.005 when compared with control subjects of the same gestational age. *p < 0.02 when compared with control subjects at I IO to l?O days’ gestation.
atropine and propranolol throughout time was analyzed by the Student paired t-test utilizing the Bonferroni modification,” and a# disference was considered statistically significant when the value was less than 0.025. The response to atropine and propranolol between control and embolized animals at a given gestational age was analyzed by the Student unpaired t-test. A difference was considered statistically significant when the p value was less than 0.05. The results are expressed in the text as means t SE.
Results Fetal body weight and length and organ weights. Embolization of the placenta produced growth-retarded fetal lambs as indicated by the decrease in body weight and crown-to-rump length (Table I). The weights of the liver, thymus, and heart were reduced. The weights of the adrenal and the brain were similar to those of control subjects. The brain-to-liver weight ratio was significantly higher in the embolization group. Fetal arterial pH, blood gases, and hematocrit. The results were similar in both groups of fetuses early in gestation. Between 13 1 and 140 days’ gestation (Table II), the PO, decreased in the embolization group when compared to control subjects. The hematocrit increased in the embolization group, achieving a significant difference during the same time interval. Arterial blood pH or PcoZ were similar in both groups. Fetal basal heart rate and arterial blood pressure. Basal heart rate was similar in control and embolization
TabIe III. Basal fetal heart rate at different gestational ages Basal heart rate (beatslmin) Gestational Age (days)
(N = 7)
Embolised (N = 9)
110-120 121-130 131-140
194 -r- 3 175 + 7 156 t- 6*
187 t 4 177 + 3 153 ” 7*
Contl-ol
Mean 2 SE; N = number of animals. *p < 0.02; significantly lower when compared to f’etuses at gestational ages of 1 IO-120 days. fetuses at all gestational ages studied (Table III). In both groups of fetuses there was a significant decrease in the basal heart rate with age when the 13 1 to 140 day period was compared to the 110 to 120 day period (Table III). Fetal arterial blood pressure was similar in control and embolization groups at all gestational ages studied. Fetal responses to parasympathetic blockade. The heart rate response to parasympathetic blockade in control animals increased as gestation advanced. The absolute increase in heart rate at 131 to 140 days’ gestation was greater than that observed at 110 to 120 days’ gestation (Fig. 1). Parasympathetic blockade produced a similar increase in fetal heart rate in control and embolization groups at 110 to 120 days’ gestation (Fig. 1). After 121 days’ gestation the absolute increase in fetal heart rate was always greater in the embolization group than in control fetuses (Fig. I). Parasympathetic blockade produced a similar increase in the mean arterial blood pressure of both groups of fetuses at all the gestational ages studied. Parasympathetic blockade did not change fetal arterial pH, blood gases, or hematocrit, or maternal heart rate, arterial blood pressure, or amniotic fluid pressure. Fetal responses to p-adrenergic blockade. Heart
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Heart
rate response to /l-adrenergic blockade in control animals was similar from 110 to 140 days’ gestation (Fig. 2). The percentage decrease in fetal heart rate was also similar during the same period (11 * 2; 9 +- 2; 12 ‘2% at 110 to 120, 121 to 130, and 131 to 140 days’ gestation, respectively). P-Adrenergic blockade produced a similar decrease in the heart rate in both groups of fetuses at 110 to 120 days’ gestation (Fig. 2). The percentage of decrease of the fetal heart rate under the basal value was also similar (11 ? 2% decrease in the control subjects versus 10 ? 2% in the embolization group). @Adrenergic blockade produced a similar decrease in fetal heart rate in both groups at 121 to 130 days (Fig. 2). However, the embolization group tended to have an increased absolute response during this gestational age period. The percentage decrease of the heart rate under the basal value was also similar with only a trend to have an increased response in the embolization group (9 f 2% decrease in the control and 16 * 3% in the embolization group; p > 0.05). At 131 to 140 days’ gestation /3-adrenergic blockade decreased heart rate more in the embolization than in control fetuses (Fig. 2). The percentage decrease of the fetal heart rate was also greater in the embolization group (19 + 1%) than in the control group (12 * 2%; p < 0.05). P-Adrenergic blockade administration to the fetus did not produce any change in the mean arterial pressure of both groups of fetuses at any of the gestational ages studied; it did not change fetal arterial pH, blood gases, or hematocrit and did not cause any changes in maternal heart rate and arterial blood pressure or in the amniotic fluid pressure. Sodium chloride 0.9% infusions did not change any of the fetal variables measured.
Comment We have previously reported that embolization of the uteroplacental vascular bed in pregnant sheep reduced fetal weight and length and increased fetal brain-to-liver weight ratio. The latter has been estimated as a good index of fetal growth retardation.18 The fetuses of the embolized sheep in this study had the organ weight pattern and the brain-to-liver weight ratio similar to our previous results.14* I5 In the last ten days of the study these fetuses also had a mild but significant decrease in fetal arterial PO, and an increase in hematocrit, also indicators of fetal growth retardation 147 15 The reactivity of the autonomic nervous system is increased in .the fetal growth-retardeh lambs from 120 days’ gestation to term. This increased reactivity is
rate response
to parasympathetic
and p-adrenergic
GESTATIONAL
blockade
811
AGE (days)
Fig. 2. Mean f SE heart rate decrease in control and embolization fetal lambs from 110 to 140 days’ gestation after intravenous injection of 1 mg/kg of propranolol. l p < 0.05 when compared with control subject of the same gestational age.
shown by the increase in heart rate responsiveness to parasympathetic and /3-adrenergic blockade when fetal growth-retarded lambs are compared to the control fetuses. We observed the increase in the heart rate responsiveness whether the results are expressed either as an absolute change in heart rate or as a percentage of change in heart rate over the basal value. The increased response in the heart rate to parasympathetic blockade observed in fetuses whose growth has been retarded may represent the accentuation and/or acceleration of a physiologic process that is taking place throughout gestation. This argument is supported by our own data, as well as the reports of Walker and associates2 and Nuwayhid and colleagues3 that showed a progressive increase in heart rate responsiveness to parasympathetic blockade during the last fifth of gestation. In contrast, Vapaavouri and coworkers’ reported that the heart rate response to parasympathetic blockade in normal fetuses did not increase after about 100 days of gestation. The progressive increase in response of heart rate to P-adrenergic blockade observed in growth-retarded fetuses in the last 10 days of the study represents a different pattern of response to /3-adrenergic blockade, since no changes in the degree of response are observed in the control fetuses throughout gestation. This argument is supported by our own control data land the data of Nuwayhid and associates3 which showed that the magnitude of the decrease of heart rate after p-adrenergic blockade does not change throughout gestation. Furthermore, Walker and colleagues* showed a decrease in the heart rate responsiveness to /3-adrenergic blockade as gestation advances in normal fetal lambs.
812
Llanos et al.
-I’he increased heart rate responsiveness to parasympathetic and P-adrenergic blockade may be triggered by mild chl-onic hypoxemia or other types of stress that growth-retarded fetuses may be subjected to, since an increased heart rate response to parasympathetic and p-adrenergic blockade has been wrell documented in sheep fttuses during severe acute hypoxemia.“. s In the latter situation there is actual bradycardia and also a substantial redistribution of blood How takes place which Favors heart. brain, and placenta.’ Our fetuses did develop a mild hypoxemia and they did exhibit an increased blood flow to the heart and brain.‘” Although they did not have hradycardia in basal conditions OUI current data showed an underlying increase in paras!-mpathetic and /%adrenergic modulation of the cardiovascular, svstem. The mechanism b! which the increase in responsiveness in the heart rate after- pal-asympathetic and p-adrenergic blockade occurs in the growth-retarded fetus is not known. It ma); reflect an increased vagal OI svmpathetic tone as an influence of’ the central nervous system, such ns acute hypoxia may elicit, OI- ma) reflect, local changes in the heart itself. These latter changes could be an increase in number or sensitivity of cholinergic and &adrenergic receptors, changes in uptake of catecholamines by the sympathetic nerve ending. or a diminution of the aret,lcholinesterase activitl
March Am. J. c-hstet.
15. 1980 Gvnecol.
in the heart. Acetylcholinesterase activity increases with advancing gestational age in the fetal mouse.‘9 An eventual decrease in the cholinesterase activity in growth-retarded fetal lambs could be the reflection of an enzymatic deficiency or a delay in enzymatic maturation as has been described or suggested in studies of other enzymes in fetal growth retardatiom2”, ” A third possibility by which a change in sympathetic tone may occur is an increase in the catechoiamine concentration in the fetal blood stream. The normal adrenal weight and the increase in adrenal blood flow found in the growth-retarded fetal lamb?’ are consistent with this idea. Furthermore, it is known that during acute hypoxia the adrenal gland liberates catecholamines after 120 days’ gestation.2” We have demonstrafed an increased activity of the autonomic nervous system in the heart of the growthretarded fetal lamb as shown by the increased responsiveness to parasympathetic and /3-adrenergic blockade. This increase in cardiac responsiveness may be part of a more generalized increase in parasympathetic and sympathetic tone affecting other organs and systems as an adaptive response to a chronic stress. This possibility deserves further investigation. We thank Ms. Francoise Mauray and Mr. McWatters for their skillful technical assistance.
Carl
REFERENCES
1. Vapaavouri, E. K., Shinebourne, E. A., Williams, R. L., et al.: Development of cardiovascular responses to autonomic blockade in intact fetal and neonatal lambs. Biol. Neonate 22:177. 1973. 2. Walker. A. M., Cannata, J,, Dowling. M. H.. et al.: Sympathetic and parasympathetic control of heart rate in unanesthetized fetal and newborn lambs, Biol. Neonate 33: 135, 1978. Nuwayhid. B., Brinkman, C. E., SW. C., et al.: Derelopment of autonomic control of fetal circulation, Am. J. Physiol. 228:337, 1975. 4. Barrett. C. T.. Heymann, M. A.. and Rudolph, A. M.: Alpha and beta adrenergic receptor activity in fetal sheep. Au. J. OBSTET. GYNECOL. 112: 1114. 1972. 3. Siimes, A. S. I., Creasy. R. K., Heymann, M. A., et al.: Cardiac output and its distribution and organ flow in the fetal lamb during ritodrine administration, AM. J, OBSTET. GYNECOL. 132:42, 197% 6. Klopfenstein, S. H., and Rudolph, A. M.: Postnatal changes in the circulation and responses to volume loading in sheep, Circ. Res. 42:839, 197X. Cohn, H. E.. Sacks, E. J., Heymann. M. A., et al.: Car-diovascular responses to hypoxemia and acidemia in fetal lambs, AM. J. OBSTET. GYNECOL. 1 P&817, 1974. Parer, J. T.: Effect of atropine on heart rate and oxygen consumption of the hypoxic fetus, Gynecol. Invest. 8:50, 1977. (Abst.) Parer, J. T.: The effect of P-adrenergic blockade on the heart rate. umbilical blood flow, and oxygen consumption
IO. 11. 12.
13. 14. 15.
16.
17.
of the hypoxic fetus, Proc. lnt. Union. Physiol. Sciences, Paris. July, 1977. (Abst.) Toubas, P. L., Silverman, N. H., Heymann, M. A., et al.: Cardiovascular responses to acute hemorrhage in the fetal lambs, Circulation 48:38. 1973. (Abst.) Humbert, J. R., Abelson, H., Hathaway, W. E., et al.: Polycythemia in small for gestational age infants, J. Pediatr. 75:812, 1969. Llanos, A., Reyes. A., and Gallegos, R.: Red cell mass in intrauterine growth-retarded neonates. Proceedings Ninth Annual Meeting of the Latin American Society for Pediatric Research, Valdivia, Chile, 1969. Lugo, G., and Cassday, G.: Intrauterine growth retardation, AM. J. OBSTET. GYNECOL. 109:615, 1971. Greasy, R. K., Barrett, C. T., deSwiet, M., et al.: Experimental intrauterine growth retardation in the sheep, AM. J. OBSTET. GYNECOL. 112:566, 1972. Greasy, R. K., deSwiet, M.. KahanpP%, K. V.. et al.: Pathophysiological changes in the foetal lamb with growth retardation in foetal and neonatal physiology, in Comline, K. D., Cross, K. W., Dawes, G. S., and Nathaniels, P. W., editors: Sir Joseph Barcroft Centenary Symposium, Cambridge. 1973, Cambridge University Press, p. 39B. Stephenson, S. K.: Wool follicle development in the New Zealand Romney and N-type sheep. IV. Prenatal growth and changes in body proportions, Aust. J. Agric. Res. 10:433, 1959. Miller, R. G.: Simultaneous Statistical Inference, New York, 1966. McGraw-Hill Book Company. Inc.
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Heart
18. Dawkins, M. J. R.: In discussion of Lloyd, J. K. Diabetes Mellitus presenting as spontaneous hypoglycemia in childhood, April 24, Meeting of the Royal Society of Medicine, Hypoglycemia in Childhood, Proc. R. Sot. Med. 57:1063, 1964. 19. Maurer, M., Yuhas, D., and Miller, J.: Developmental factors contributing to susceptibility to bradycardia, Circulation 5G, Part II: 111-170, 1977. (Abst. No. 656.) 20. Yoshida, T., Metcoff, J., Morales, M., et al.: Human fetal growth retardation. Il. Energy metabolism in leukocytes, Pediatrics 50:55Q, 1972.
rate response
to parasympathetic
and p-adrenergic
blockade
813
21. Haymand, W. M., Karl, I. E., and Pagliara, A. S.: lncreased gluconeogenic substrates in the small-for-gestational age infant, N. Engl. J. Med. 291:322, 1974. 22. Llanos, A., Rose, J. C., Creasy, R. K., et al.: Plasma glucocorticoids and adrenocorticotropin concentrations measured serially in growth-retarded fetal lambs, Pediatr. Res. L&1089, 1979. 23. Jones, C. T., and Robinson, R. 0.: Plasma catecholamines in foetal and adult sheep, J. Physiol. 248: 15, 1975.
Information for authors Most of the provisions of the Copyright Act of 1976 became effective on January 1, 1978. Therefore, all manuscripts must be accompanied by the following written statement, signed by one author: “The undersigned author transfers all copyright ownership of the manuscript (title of article) to The C. V. Mosby Company in the event the work is published. The undersigned author warrants that the article is original, is not under consideration by another journal, and has not been previously published. I sign for and accept responsibility for releasing this material on behalf of any and all co-authors.” Authors will be consulted, when possible, regarding republication of their material.
J.
R. GREEN,
ROBERT
Ii.
ABRAHAM Sntl
LLANOS.
M.D.
CREASY, M.
Fra~li~isco,
M.D.* M.D.
RUDOLPH.
M.D.
California
To determine the influence of the autonomic nervous system on the circulation of normal and growth-retarded fetal lambs we measured the responses of heart rate and arterial blood pressure to parasympathetic (atropine 0.2 mg/kg) and p-adrenergic (propranolol 1 .O mg/kg) blockade in the last quarter of gestation. The heart rate response to parasympathetic blockade increased with gestational age in both normal and growth-retarded fetuses but in the growth-retarded fetuses there was a significantly greater response to parasympathetic blockade than in control fetuses from 121 days’ gestation to term. The heart rate response to /3-adrenergic blockade did not change with gestational age in normal fetuses. From 131 days’ gestation to term the heart rate response to p-adrenergic blockade in growth-retarded fetuses was significantly higher than in normal fetuses. The systemic arterial blood pressure responses to parasympathetic or p-adrenergic blockade were similar in growth-retarded and normal fetuses. These results indicate an increased parasympathetic and p-adrenergic influence on the heart of fetuses whose growth has been retarded. It may also suggest a more generalized increase in parasympathetic and sympathetic tone affecting other organs and systems in the growth-retarded fetal lambs. (AM. J. OBSTET. GYNECOL. 136:808, 1980.)
‘THE: AUTONOMIC nervous system is involved in the regulation of‘ heart rate and arterial blood pressure.‘-” of cardiac output and its distribution,“-6 and in response to hypoxemia and other forms of stress’-” in fetal and newborn mammals. Newborn infants who have sustained intrauterine growth retardation secondary to a probable reduction in uteroplacental blood
FTO~ the Department of Obstetrics, Gynecology, and Reprodurtive Sciences, and the Cardiovascular Research Instttute, C’niversity of California San Franckco. Supported HDOfXl9.
by United
States Public
RPWWY~ jar publication Accepted May
February
Health
Service Grant
20, 1979
29, 1979
Repmzt rPqmstS: DT. Robert K. Cress!, Depatiment qf Obstetncr, Gynecology, and ReprodurttzIe Sciences, G’niversity of Ca&amia at San Francwco, Sari Francisco, Cal$mrrio 94143. *Reciptent oj a Postdoctoral Fellows/$ from the Bay Area Heart Research Association. Present addresh: Departamento de Medicina Experimental, Sede Santiago Oviente-Escuela & Medic% Universidad de Chile. Ca.tilln 16038, Santiago 9, Chile.
808
flow have an increased hematocrit and red cell mass”, I2 suggesting that they may have been subjected to chronic hypoxemia during fetal life. Also, clinical experience with growth-retarded fetuses and newborn infants indicates that they are very susceptible to acute hypoxemia or other forms of stress and frequently die in the perinatal period.‘” In fetal lambs with growth retardation induced by embolization of the uteroplacental vascular bed, the fetal arterial PO, is chronically decreased and they show a different distribution of cardiac output and organ blood flow when compared to normal fetuses.‘“, ‘L ?2 In addition, the hematocrit is increasedI in the growthretarded fetal lambs as it is observed in human newborn infant whose growth has been retarded. The direct and indirect signs of hypoxemia in growthretarded fetal lambs and their alterations in organ perfusion suggest that the activity of the autonomic nervous system is increased in this syndrome. We therefore investigated the activity of the autonomic nervous system by serially measuring the effect of parasympathetic and /I-adrenergic blockade on fetal 0002-9378/80/060808+06$00.60/0
0 1980 The C. V. Mosby
Co.
Volume Number
136 6
heart rate and arterial blood pressure during ing gestation in normal and growth-retarded lambs.
Heart
advancfetal
rate
response
to parasympathetic
Table I. Mean body weight weights in growth-retarded
Sixteen mixed-breed Western ewes with known gestational ages were surgically prepared when they were between 105 and 110 days’ gestation. Polyvinyl carheters were inserted into fetal carotid and femoral arteries and jugular and femoral veins, the amniotic cavity, and a maternal femoral artery and vein. In nine ewes a catheter also was placed in the uterine artery; through this catheter we embolized the maternal placental bed daily with 15 pm diameter nonradioactive microspheres as we have described previously.*4 Seven ewes without an arterial uterine catheter were used as control subjects. At 135 days’ gestation two of the control animals spontaneously delivered dead fetuses. The organ weights of these two fetuses were not considered in the results, but the arterial pH, blood gases and hematocrit, heart rate, and arterial blood pressure were in the normal range throughout the experiments” and these values were included in the results. All fetuses were studied at least five days after surgery while the unsedated ewe stood quiently in her cage. We measured fetal arterial pH, blood gases, and hematocrit daily, and before and after each drug injection. Fetal and maternal blood pressure and amniotic fluid pressure were monitored with Statham P23 Db pressure transducers and a direct-writing recorder (Beckman Dynograph R). Fetal heart rate was monitored by a cardiotachometer: but during the experiments, to be more accurate, the changes in fetal and maternal heart rate were measured directly from rapid recordings of blood pressure tracings. Fetal arterial pressure was referred to the amniotic fluid pressure as zero. We measured the fetal and maternal cardiovascular variables and amniotic fluid pressure for at least 60 minutes before each drug injection. Atropine, 0.2 mg/kg of estimated body weight,t6 was injected by a bolus injection into a fetal femoral vein to produce parasympathetic blockade. Completeness of the blockage was assessed by inhibition of the effects of acetylcholine (15 pg/kg) on fetal heart rate and arterial pressure. Propranolol (1 mg/kg) was injected as a bolus into a fetal femoral vein for /3-adrenergic blockade. The completeness of the blockade was demonstrated by inhibition of the effects of isoproterenol (0.1 &g/kg) on fetal heart rate and arterial pressure. Sodium chloride 0.9% at room temperature was injected as a control before each drug study. Heart rate and arterial pressure responses to a single intravenous injection of
data
Gestational age (days) Body weight (kg) Body length (cm) Adrenal gland (am) Brain (gm) Heart
Mean
(N = 5) 138 ” 0.90
Growth-retarded (N = 9)
50 + 0.38
138 2 0.60 3.2 + 0.12t 43 + 1.oot
0.51 f 0.02
0.52 2 0.01
3.9 t 0.80 50 2 2.00 33 2 0.90
fern)
Liver (&) Thymus (gm) Brain-to-liver weight ratio
blockade
132 a 6.00 19 f 2.00 0.38 ? 0.01
f SE; n = number
809
and length and organ fetal lambs
Control Clinical
Materials and methods
and p-adrenergic
47 2 2.00 23 f. 0.50* 90 +- 3.00t 6a I.OOt
% of Con&r01 100 82 86 102 94 70 68 32
0.52 ? 0.02*
of animals.
*p < 0.01. tp ‘Z 0.002. atropine and propranolol were tested in seven control and in nine growth-retarded fetuses between 110 and 120, 121 and 130, 131 and 140 days of gestation. Each animal was tested once with atropine and once with propranolol in each time period, so each animal had three studies of each drug throughout the whole study. The drugs were tested in random order and at least one day intervened between atropine and propranolol studies. We calculated the basal cardiovascular values before the injection of the drugs by averaging the measurements at one-minute intervals for 30 minutes before the injection. Fetal heart rate and arterial blood pressure values after atropine injection were calculated by averaging the measurements at one-minute intervals for ten minutes. Acetylcholine did not produce any modifications in fetal heart rate and arterial blood pressure during this time period. Fetal heart rate and arterial blood pressure values after propranolol injection were calculated by averaging the measurements at oneminute intervals for 30 minutes after the injection. Isoproterenol did not produce any modification in fetal heart rate and arterial blood pressure during this time period. The animals in both groups were killed at 140 days’ gestation, and fetal body weight and length and organ weights were measured.
Statistical analysis The Student unpaired t-test was used to compare the differences in body weight, length, organ weights, pH, blood gases, and hematocrit between control and embolized groups. A difference was considered significant when the p value was less than 0.05. The response to
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Table II. Mean femoral arterial pH, blood gases, and hematocrit in growth-retarded fetal lambs from I31 to 140 days’ gestation
I Control I (N = 15)
PH
Pcop (torr) Po, (torr) Hematocrit (%)
7.39 + 0.007 44.7 + 0.59
Growth-retarded (N = 27) 7.39 k 0.005 46.6 rtr. 0.71
23.1 -c 0.67
18.5 * 0.45*
30.6
35.0
k 0.98
r 0.5t
Values are means & SE. “p < 0.02. tp < 0.01. GESTATIONAL
AGE (days)
Fig. 1. Mean it SE heart rate increase in control and emboliration fetal lambs from 110 to 140 days’ gestation after intravenous injection of 0.2 mg/kg of atropine. *p < 0.001 and *p c 0.005 when compared with control subjects of the same gestational age. *p < 0.02 when compared with control subjects at I IO to l?O days’ gestation.
atropine and propranolol throughout time was analyzed by the Student paired t-test utilizing the Bonferroni modification,” and a# disference was considered statistically significant when the value was less than 0.025. The response to atropine and propranolol between control and embolized animals at a given gestational age was analyzed by the Student unpaired t-test. A difference was considered statistically significant when the p value was less than 0.05. The results are expressed in the text as means t SE.
Results Fetal body weight and length and organ weights. Embolization of the placenta produced growth-retarded fetal lambs as indicated by the decrease in body weight and crown-to-rump length (Table I). The weights of the liver, thymus, and heart were reduced. The weights of the adrenal and the brain were similar to those of control subjects. The brain-to-liver weight ratio was significantly higher in the embolization group. Fetal arterial pH, blood gases, and hematocrit. The results were similar in both groups of fetuses early in gestation. Between 13 1 and 140 days’ gestation (Table II), the PO, decreased in the embolization group when compared to control subjects. The hematocrit increased in the embolization group, achieving a significant difference during the same time interval. Arterial blood pH or PcoZ were similar in both groups. Fetal basal heart rate and arterial blood pressure. Basal heart rate was similar in control and embolization
TabIe III. Basal fetal heart rate at different gestational ages Basal heart rate (beatslmin) Gestational Age (days)
(N = 7)
Embolised (N = 9)
110-120 121-130 131-140
194 -r- 3 175 + 7 156 t- 6*
187 t 4 177 + 3 153 ” 7*
Contl-ol
Mean 2 SE; N = number of animals. *p < 0.02; significantly lower when compared to f’etuses at gestational ages of 1 IO-120 days. fetuses at all gestational ages studied (Table III). In both groups of fetuses there was a significant decrease in the basal heart rate with age when the 13 1 to 140 day period was compared to the 110 to 120 day period (Table III). Fetal arterial blood pressure was similar in control and embolization groups at all gestational ages studied. Fetal responses to parasympathetic blockade. The heart rate response to parasympathetic blockade in control animals increased as gestation advanced. The absolute increase in heart rate at 131 to 140 days’ gestation was greater than that observed at 110 to 120 days’ gestation (Fig. 1). Parasympathetic blockade produced a similar increase in fetal heart rate in control and embolization groups at 110 to 120 days’ gestation (Fig. 1). After 121 days’ gestation the absolute increase in fetal heart rate was always greater in the embolization group than in control fetuses (Fig. I). Parasympathetic blockade produced a similar increase in the mean arterial blood pressure of both groups of fetuses at all the gestational ages studied. Parasympathetic blockade did not change fetal arterial pH, blood gases, or hematocrit, or maternal heart rate, arterial blood pressure, or amniotic fluid pressure. Fetal responses to p-adrenergic blockade. Heart
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rate response to /l-adrenergic blockade in control animals was similar from 110 to 140 days’ gestation (Fig. 2). The percentage decrease in fetal heart rate was also similar during the same period (11 * 2; 9 +- 2; 12 ‘2% at 110 to 120, 121 to 130, and 131 to 140 days’ gestation, respectively). P-Adrenergic blockade produced a similar decrease in the heart rate in both groups of fetuses at 110 to 120 days’ gestation (Fig. 2). The percentage of decrease of the fetal heart rate under the basal value was also similar (11 ? 2% decrease in the control subjects versus 10 ? 2% in the embolization group). @Adrenergic blockade produced a similar decrease in fetal heart rate in both groups at 121 to 130 days (Fig. 2). However, the embolization group tended to have an increased absolute response during this gestational age period. The percentage decrease of the heart rate under the basal value was also similar with only a trend to have an increased response in the embolization group (9 f 2% decrease in the control and 16 * 3% in the embolization group; p > 0.05). At 131 to 140 days’ gestation /3-adrenergic blockade decreased heart rate more in the embolization than in control fetuses (Fig. 2). The percentage decrease of the fetal heart rate was also greater in the embolization group (19 + 1%) than in the control group (12 * 2%; p < 0.05). P-Adrenergic blockade administration to the fetus did not produce any change in the mean arterial pressure of both groups of fetuses at any of the gestational ages studied; it did not change fetal arterial pH, blood gases, or hematocrit and did not cause any changes in maternal heart rate and arterial blood pressure or in the amniotic fluid pressure. Sodium chloride 0.9% infusions did not change any of the fetal variables measured.
Comment We have previously reported that embolization of the uteroplacental vascular bed in pregnant sheep reduced fetal weight and length and increased fetal brain-to-liver weight ratio. The latter has been estimated as a good index of fetal growth retardation.18 The fetuses of the embolized sheep in this study had the organ weight pattern and the brain-to-liver weight ratio similar to our previous results.14* I5 In the last ten days of the study these fetuses also had a mild but significant decrease in fetal arterial PO, and an increase in hematocrit, also indicators of fetal growth retardation 147 15 The reactivity of the autonomic nervous system is increased in .the fetal growth-retardeh lambs from 120 days’ gestation to term. This increased reactivity is
rate response
to parasympathetic
and p-adrenergic
GESTATIONAL
blockade
811
AGE (days)
Fig. 2. Mean f SE heart rate decrease in control and embolization fetal lambs from 110 to 140 days’ gestation after intravenous injection of 1 mg/kg of propranolol. l p < 0.05 when compared with control subject of the same gestational age.
shown by the increase in heart rate responsiveness to parasympathetic and /3-adrenergic blockade when fetal growth-retarded lambs are compared to the control fetuses. We observed the increase in the heart rate responsiveness whether the results are expressed either as an absolute change in heart rate or as a percentage of change in heart rate over the basal value. The increased response in the heart rate to parasympathetic blockade observed in fetuses whose growth has been retarded may represent the accentuation and/or acceleration of a physiologic process that is taking place throughout gestation. This argument is supported by our own data, as well as the reports of Walker and associates2 and Nuwayhid and colleagues3 that showed a progressive increase in heart rate responsiveness to parasympathetic blockade during the last fifth of gestation. In contrast, Vapaavouri and coworkers’ reported that the heart rate response to parasympathetic blockade in normal fetuses did not increase after about 100 days of gestation. The progressive increase in response of heart rate to P-adrenergic blockade observed in growth-retarded fetuses in the last 10 days of the study represents a different pattern of response to /3-adrenergic blockade, since no changes in the degree of response are observed in the control fetuses throughout gestation. This argument is supported by our own control data land the data of Nuwayhid and associates3 which showed that the magnitude of the decrease of heart rate after p-adrenergic blockade does not change throughout gestation. Furthermore, Walker and colleagues* showed a decrease in the heart rate responsiveness to /3-adrenergic blockade as gestation advances in normal fetal lambs.
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-I’he increased heart rate responsiveness to parasympathetic and P-adrenergic blockade may be triggered by mild chl-onic hypoxemia or other types of stress that growth-retarded fetuses may be subjected to, since an increased heart rate response to parasympathetic and p-adrenergic blockade has been wrell documented in sheep fttuses during severe acute hypoxemia.“. s In the latter situation there is actual bradycardia and also a substantial redistribution of blood How takes place which Favors heart. brain, and placenta.’ Our fetuses did develop a mild hypoxemia and they did exhibit an increased blood flow to the heart and brain.‘” Although they did not have hradycardia in basal conditions OUI current data showed an underlying increase in paras!-mpathetic and /%adrenergic modulation of the cardiovascular, svstem. The mechanism b! which the increase in responsiveness in the heart rate after- pal-asympathetic and p-adrenergic blockade occurs in the growth-retarded fetus is not known. It ma); reflect an increased vagal OI svmpathetic tone as an influence of’ the central nervous system, such ns acute hypoxia may elicit, OI- ma) reflect, local changes in the heart itself. These latter changes could be an increase in number or sensitivity of cholinergic and &adrenergic receptors, changes in uptake of catecholamines by the sympathetic nerve ending. or a diminution of the aret,lcholinesterase activitl
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in the heart. Acetylcholinesterase activity increases with advancing gestational age in the fetal mouse.‘9 An eventual decrease in the cholinesterase activity in growth-retarded fetal lambs could be the reflection of an enzymatic deficiency or a delay in enzymatic maturation as has been described or suggested in studies of other enzymes in fetal growth retardatiom2”, ” A third possibility by which a change in sympathetic tone may occur is an increase in the catechoiamine concentration in the fetal blood stream. The normal adrenal weight and the increase in adrenal blood flow found in the growth-retarded fetal lamb?’ are consistent with this idea. Furthermore, it is known that during acute hypoxia the adrenal gland liberates catecholamines after 120 days’ gestation.2” We have demonstrafed an increased activity of the autonomic nervous system in the heart of the growthretarded fetal lamb as shown by the increased responsiveness to parasympathetic and /3-adrenergic blockade. This increase in cardiac responsiveness may be part of a more generalized increase in parasympathetic and sympathetic tone affecting other organs and systems as an adaptive response to a chronic stress. This possibility deserves further investigation. We thank Ms. Francoise Mauray and Mr. McWatters for their skillful technical assistance.
Carl
REFERENCES
1. Vapaavouri, E. K., Shinebourne, E. A., Williams, R. L., et al.: Development of cardiovascular responses to autonomic blockade in intact fetal and neonatal lambs. Biol. Neonate 22:177. 1973. 2. Walker. A. M., Cannata, J,, Dowling. M. H.. et al.: Sympathetic and parasympathetic control of heart rate in unanesthetized fetal and newborn lambs, Biol. Neonate 33: 135, 1978. Nuwayhid. B., Brinkman, C. E., SW. C., et al.: Derelopment of autonomic control of fetal circulation, Am. J. Physiol. 228:337, 1975. 4. Barrett. C. T.. Heymann, M. A.. and Rudolph, A. M.: Alpha and beta adrenergic receptor activity in fetal sheep. Au. J. OBSTET. GYNECOL. 112: 1114. 1972. 3. Siimes, A. S. I., Creasy. R. K., Heymann, M. A., et al.: Cardiac output and its distribution and organ flow in the fetal lamb during ritodrine administration, AM. J, OBSTET. GYNECOL. 132:42, 197% 6. Klopfenstein, S. H., and Rudolph, A. M.: Postnatal changes in the circulation and responses to volume loading in sheep, Circ. Res. 42:839, 197X. Cohn, H. E.. Sacks, E. J., Heymann. M. A., et al.: Car-diovascular responses to hypoxemia and acidemia in fetal lambs, AM. J. OBSTET. GYNECOL. 1 P&817, 1974. Parer, J. T.: Effect of atropine on heart rate and oxygen consumption of the hypoxic fetus, Gynecol. Invest. 8:50, 1977. (Abst.) Parer, J. T.: The effect of P-adrenergic blockade on the heart rate. umbilical blood flow, and oxygen consumption
IO. 11. 12.
13. 14. 15.
16.
17.
of the hypoxic fetus, Proc. lnt. Union. Physiol. Sciences, Paris. July, 1977. (Abst.) Toubas, P. L., Silverman, N. H., Heymann, M. A., et al.: Cardiovascular responses to acute hemorrhage in the fetal lambs, Circulation 48:38. 1973. (Abst.) Humbert, J. R., Abelson, H., Hathaway, W. E., et al.: Polycythemia in small for gestational age infants, J. Pediatr. 75:812, 1969. Llanos, A., Reyes. A., and Gallegos, R.: Red cell mass in intrauterine growth-retarded neonates. Proceedings Ninth Annual Meeting of the Latin American Society for Pediatric Research, Valdivia, Chile, 1969. Lugo, G., and Cassday, G.: Intrauterine growth retardation, AM. J. OBSTET. GYNECOL. 109:615, 1971. Greasy, R. K., Barrett, C. T., deSwiet, M., et al.: Experimental intrauterine growth retardation in the sheep, AM. J. OBSTET. GYNECOL. 112:566, 1972. Greasy, R. K., deSwiet, M.. KahanpP%, K. V.. et al.: Pathophysiological changes in the foetal lamb with growth retardation in foetal and neonatal physiology, in Comline, K. D., Cross, K. W., Dawes, G. S., and Nathaniels, P. W., editors: Sir Joseph Barcroft Centenary Symposium, Cambridge. 1973, Cambridge University Press, p. 39B. Stephenson, S. K.: Wool follicle development in the New Zealand Romney and N-type sheep. IV. Prenatal growth and changes in body proportions, Aust. J. Agric. Res. 10:433, 1959. Miller, R. G.: Simultaneous Statistical Inference, New York, 1966. McGraw-Hill Book Company. Inc.
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18. Dawkins, M. J. R.: In discussion of Lloyd, J. K. Diabetes Mellitus presenting as spontaneous hypoglycemia in childhood, April 24, Meeting of the Royal Society of Medicine, Hypoglycemia in Childhood, Proc. R. Sot. Med. 57:1063, 1964. 19. Maurer, M., Yuhas, D., and Miller, J.: Developmental factors contributing to susceptibility to bradycardia, Circulation 5G, Part II: 111-170, 1977. (Abst. No. 656.) 20. Yoshida, T., Metcoff, J., Morales, M., et al.: Human fetal growth retardation. Il. Energy metabolism in leukocytes, Pediatrics 50:55Q, 1972.
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21. Haymand, W. M., Karl, I. E., and Pagliara, A. S.: lncreased gluconeogenic substrates in the small-for-gestational age infant, N. Engl. J. Med. 291:322, 1974. 22. Llanos, A., Rose, J. C., Creasy, R. K., et al.: Plasma glucocorticoids and adrenocorticotropin concentrations measured serially in growth-retarded fetal lambs, Pediatr. Res. L&1089, 1979. 23. Jones, C. T., and Robinson, R. 0.: Plasma catecholamines in foetal and adult sheep, J. Physiol. 248: 15, 1975.
Information for authors Most of the provisions of the Copyright Act of 1976 became effective on January 1, 1978. Therefore, all manuscripts must be accompanied by the following written statement, signed by one author: “The undersigned author transfers all copyright ownership of the manuscript (title of article) to The C. V. Mosby Company in the event the work is published. The undersigned author warrants that the article is original, is not under consideration by another journal, and has not been previously published. I sign for and accept responsibility for releasing this material on behalf of any and all co-authors.” Authors will be consulted, when possible, regarding republication of their material.