Neurotoxicologyand Teratology,Vol. 13, pp. 503-506. ©PergamonPress plc, 1991. Printedin the U.S.A.
0892-0362/91 $3.00 + .00
The Effect of Prenatal Cocaine Exposure on Umbilical Cord Length in Fetal Rats S U S A N B A R R O N , 1 J O H N A. F O S S A N D E D W A R D P. R I L E Y
Department of Psychology, San Diego State University, San Diego, CA 92182-0350 and Argus Research Laboratories, Inc. Received 10 D e c e m b e r 1990 BARRON, S., J. A. FOSS AND E. P. RILEY. The effect of prenatal cocaine exposure on umbilical cord length in fetal rats. NEUROTOXICOL TERATOL 13(5) 503-506, 1991.--Umbilicai cord length has been considered a reliable indicator of fetal movement. In this study, the effect of prenatal cocaine exposure on umbilical cord length was examined in rats. Pregnant rats were intubated with either 0 or 60 mg/kg cocaine hydrochloride daily from gestation day (GD) 14-21. Fetuses were removed via Caesarean section on GD 21 and umbilical cord length, placental weight and fetal body weight were measured. Fetuses exposed to cocaine in utero had significantly shorter umbilical cords than intubated controls, although there were no differences in placental or fetal body weights. These data suggest that prenatal cocaine exposure suppresses fetal movement, which could contribute to some of the long-term effects observed in cocaine-exposed offspring. Prenatal cocaine effects
Fetal movement
Umbilical cord
Teratology
cocaine during the late organogenic or postorganogenic period displayed hemorrhages and limb malformations typically associated with vasoconstriction and related hypoxia (42). Since hypoxia has also been shown to have behavioral teratogenic effects [see (19) for a review], it has been suggested that this may be one mechanism by which cocaine exerts its teratogehic actions
COCAINE abuse has increased dramatically in recent years and has become a major public health concern. Among women, the greatest number of cocaine abusers are within childbearing ages [e.g., (15,29)]. Consequently, there is serious concern regarding the effects of prenatal cocaine exposure on subsequent fetal development. It is somewhat surprising that until fairly recently, there were few studies examining the teratogenic potential of cocaine (11, 21, 22). Within the last five years, clinical reports have revealed a number of risks associated with maternal cocaine abuse during pregnancy. Cocaine use has been associated with an increased frequency of spontaneous abortion (6), abruptio placentae (2, 6, 18) and premature labor (17, 30, 32). Infants with prenatal cocaine histories display increased irritability, tremors and altered state lability (6, 7, 13, 20), as well as altered suckling (5), increased startle reactivity (3) and a variety of motor abnormalities (6). Within the animal literature, studies also suggest that prenatal cocaine exposure results in learning deficits (34,41). One potential mechanism by which cocaine exposure affects the fetus stems from cocaine-induced hypertension which could induce fetal hypoxia. In human studies, reports of retroplacental blood clots suggest that the hypertensive effects of cocaine affects blood flow to the fetus (2). Isolated human umbilical arteries also show a potentiated-contractile response to noradrenaline in the presence of cocaine, suggesting possible vasoactive effects and hypoxia for the human fetus (31). Similarly, in animal studies, cocaine administration to the pregnant ewe caused increased blood pressure in both the ewe and the fetus and significandy reduced uterine blood flow for 15 min after administration (26). Cocaine can also cause intermittent vasoconstriction of the placenta in mice, and the associated decreased blood flow to the fetus could result in brief periods of fetal hypoxia (22). Webster has shown that fetal rats exposed to
(11,26). In the present study, we examined the effects of prenatal cocaine exposure on fetal movement, in part, because fetal movement is affected by hypoxia. In rats, transient hypoxia produces a distinctive behavioral response which begins with an initial suppression of fetal movement, followed by a brief period of hyperactivity and a second period of behavioral suppression (38). To assess fetal movement, we examined an indirect measure of fetal movement, umbilical cord length. There is evidence from a number of sources suggesting that umbilical cord length is influenced by at least two factors; the incidence of fetal movement and the availability of intrauterine space. In humans, shortened umbilical cords have been reported in infants with structural or functional limb defects in which fetal movement was limited (23), in pregnancies with abnormally small volumes of amniotic fluid (oligohydramnios) which also reduces fetal movement (28), and in syndromes associated with decreased fetal movement such as Down's syndrome (25) and Pena-Shokeir Syndrome (9). In rodent studies, manipulations that decrease fetal movements, such as injections of curare (24) or alcohol exposure (4,36) decrease fetal movement and result in shorter umbilical cords. It should be noted that the role of umbilical cord length as an indicator of fetal movement has recently been questioned (12). However, there are a number of studies which support the use of umbilical cord length as an indicator of fetal activity.
~Requests for reprints should be addressed to Susan Bah'on, Department of Psychology, Kastle Hail, University of Kentucky, Lexington, KY 40506-0044. 503
504
BARRON, FOSS AND RILEY METHOD
36
Prenatal Exposure Parent animals were Long-Evans rats obtained from Blue Spruce Farms, Altamont, NY. Following a 1-week acclimation period to the laboratory, virgin females (approximately 100 days of age) were individually housed with a male rat each evening until a vaginal plug was noted, marking day 1 of pregnancy (GD 1). Females were weighed and individually housed in clear plastic breeding cages with hardwood bedding in a temperature controlled nursery with a 12-h light-dark cycle. Pregnant females were assigned to one of two treatment groups. From GD 14--GD 21, the C60 group ( n = 10) received 60 mg/kg cocaine hydrochloride daily in a single intragastric intubation between the hours of 1000 and 1300. The drug was dissolved in distilled H20 and administered in a 4.5 ml/kg volume. The 60 mg/kg dose was chosen based on findings from preliminary work indicating that this dose did not alter offspring survival or body weight, although maternal weight gain during pregnancy was reduced. The second treatment group (CO) (n = 9) received daily intubations of distilled water (4.5 ml/kg) following the same schedule as the C60 group.
z
34
:::E (.9 :7
w
-..1
52
123 0
~
3O
-.A .< .-.I
m
28
26 0 MG/KG
60 MO/KG
COCAINE FIG. 1. Mean umbilical cord length (SEM) as a function of prenatal treatment (0 mg/kg or 60 mg/kg cocaine hydrochloride respectively).
Sample Collection The dams were part of a preliminary study of plasma cocaine levels following intragastric administration. Blood samples were taken at one of 4 time intervals (5, 20, 35 or 50 min) following dosing on GD 21. Animals were anesthetized with a combination of ketamine and xylazine (10 mg/kg and 20 mg/kg respectively). Ten minutes later, blood was obtained by cardiac puncture (to which saturated sodium fluoride was added for a final NaF concentration of 0.4%), and then the dam was sacrificed by exposure to CO2. At this time, the fetuses were delivered via caesarean section and umbilical cords were measured.
Umbilical Cord Measure After the delivery via caesarean section, the fetuses were placed on a petri dish wet with saline. Each fetus was removed from its amniotic sac and lifted vertically until the placenta just touched the surface of the petri dish. Care was taken to ensure that the traction applied to each cord was kept constant. Umbilical cord lengths were measured (to the nearest ram) from the placental plate to the abdominal wall using a caliper. The caliper was then placed against a millimeter ruler to measure cord length. For each litter, one experimenter measured the cord length of each fetus and called the measurement out to a second experimenter. After cord lengths were recorded for the entire litter, a second measure was taken. These two measures rarely varied by more than 2 mm, and the average of these two measurements was the dependent variable. After umbilical cord lengths were examined, the umbilical cord was cut from the fetus and from the placenta. Placental weight, fetal body weight and fetal sex were then recorded. To control for potential experimenter bias, the experimenter making the measurements was blind to treatment condition until the conclusion of the experiment. This method has been used previously in our laboratory and provides reliable and consistent measurements (4). RESULTS
Maternal Toxicity Significant differences were observed in maternal weight gain during pregnancy between the two treatment groups (32.4% vs. 47.8% weight gain for the C60 vs. CO group, respectively). One
C60 dam that had originally been designated for this study had seizures following the sixth intubation and died shortly after the seventh intubation of cocaine. The mean (_+ SEM) plasma cocaine level for the C60 group was 4 3 0 _+ 160 ng/ml. Although samples were taken at 4 time intervals over a 45-min period, there were no reliable differences in plasma cocaine levels across the sampling intervals,
Fetal Data Umbilical cord lengths are presented in Fig. 1. Umbilical cords were significantly shorter in the C60 group relative to the CO intubated control group. The data were analyzed by taking the average cord length for each sex within a litter and using these means as the dependent measure to avoid potential litter bias (1). An analysis of variance (ANOVA) revealed a significant main effect of prenatal treatment, F(1,30) = 23.93, p
0892-0362/91 $3.00 + .00
The Effect of Prenatal Cocaine Exposure on Umbilical Cord Length in Fetal Rats S U S A N B A R R O N , 1 J O H N A. F O S S A N D E D W A R D P. R I L E Y
Department of Psychology, San Diego State University, San Diego, CA 92182-0350 and Argus Research Laboratories, Inc. Received 10 D e c e m b e r 1990 BARRON, S., J. A. FOSS AND E. P. RILEY. The effect of prenatal cocaine exposure on umbilical cord length in fetal rats. NEUROTOXICOL TERATOL 13(5) 503-506, 1991.--Umbilicai cord length has been considered a reliable indicator of fetal movement. In this study, the effect of prenatal cocaine exposure on umbilical cord length was examined in rats. Pregnant rats were intubated with either 0 or 60 mg/kg cocaine hydrochloride daily from gestation day (GD) 14-21. Fetuses were removed via Caesarean section on GD 21 and umbilical cord length, placental weight and fetal body weight were measured. Fetuses exposed to cocaine in utero had significantly shorter umbilical cords than intubated controls, although there were no differences in placental or fetal body weights. These data suggest that prenatal cocaine exposure suppresses fetal movement, which could contribute to some of the long-term effects observed in cocaine-exposed offspring. Prenatal cocaine effects
Fetal movement
Umbilical cord
Teratology
cocaine during the late organogenic or postorganogenic period displayed hemorrhages and limb malformations typically associated with vasoconstriction and related hypoxia (42). Since hypoxia has also been shown to have behavioral teratogenic effects [see (19) for a review], it has been suggested that this may be one mechanism by which cocaine exerts its teratogehic actions
COCAINE abuse has increased dramatically in recent years and has become a major public health concern. Among women, the greatest number of cocaine abusers are within childbearing ages [e.g., (15,29)]. Consequently, there is serious concern regarding the effects of prenatal cocaine exposure on subsequent fetal development. It is somewhat surprising that until fairly recently, there were few studies examining the teratogenic potential of cocaine (11, 21, 22). Within the last five years, clinical reports have revealed a number of risks associated with maternal cocaine abuse during pregnancy. Cocaine use has been associated with an increased frequency of spontaneous abortion (6), abruptio placentae (2, 6, 18) and premature labor (17, 30, 32). Infants with prenatal cocaine histories display increased irritability, tremors and altered state lability (6, 7, 13, 20), as well as altered suckling (5), increased startle reactivity (3) and a variety of motor abnormalities (6). Within the animal literature, studies also suggest that prenatal cocaine exposure results in learning deficits (34,41). One potential mechanism by which cocaine exposure affects the fetus stems from cocaine-induced hypertension which could induce fetal hypoxia. In human studies, reports of retroplacental blood clots suggest that the hypertensive effects of cocaine affects blood flow to the fetus (2). Isolated human umbilical arteries also show a potentiated-contractile response to noradrenaline in the presence of cocaine, suggesting possible vasoactive effects and hypoxia for the human fetus (31). Similarly, in animal studies, cocaine administration to the pregnant ewe caused increased blood pressure in both the ewe and the fetus and significandy reduced uterine blood flow for 15 min after administration (26). Cocaine can also cause intermittent vasoconstriction of the placenta in mice, and the associated decreased blood flow to the fetus could result in brief periods of fetal hypoxia (22). Webster has shown that fetal rats exposed to
(11,26). In the present study, we examined the effects of prenatal cocaine exposure on fetal movement, in part, because fetal movement is affected by hypoxia. In rats, transient hypoxia produces a distinctive behavioral response which begins with an initial suppression of fetal movement, followed by a brief period of hyperactivity and a second period of behavioral suppression (38). To assess fetal movement, we examined an indirect measure of fetal movement, umbilical cord length. There is evidence from a number of sources suggesting that umbilical cord length is influenced by at least two factors; the incidence of fetal movement and the availability of intrauterine space. In humans, shortened umbilical cords have been reported in infants with structural or functional limb defects in which fetal movement was limited (23), in pregnancies with abnormally small volumes of amniotic fluid (oligohydramnios) which also reduces fetal movement (28), and in syndromes associated with decreased fetal movement such as Down's syndrome (25) and Pena-Shokeir Syndrome (9). In rodent studies, manipulations that decrease fetal movements, such as injections of curare (24) or alcohol exposure (4,36) decrease fetal movement and result in shorter umbilical cords. It should be noted that the role of umbilical cord length as an indicator of fetal movement has recently been questioned (12). However, there are a number of studies which support the use of umbilical cord length as an indicator of fetal activity.
~Requests for reprints should be addressed to Susan Bah'on, Department of Psychology, Kastle Hail, University of Kentucky, Lexington, KY 40506-0044. 503
504
BARRON, FOSS AND RILEY METHOD
36
Prenatal Exposure Parent animals were Long-Evans rats obtained from Blue Spruce Farms, Altamont, NY. Following a 1-week acclimation period to the laboratory, virgin females (approximately 100 days of age) were individually housed with a male rat each evening until a vaginal plug was noted, marking day 1 of pregnancy (GD 1). Females were weighed and individually housed in clear plastic breeding cages with hardwood bedding in a temperature controlled nursery with a 12-h light-dark cycle. Pregnant females were assigned to one of two treatment groups. From GD 14--GD 21, the C60 group ( n = 10) received 60 mg/kg cocaine hydrochloride daily in a single intragastric intubation between the hours of 1000 and 1300. The drug was dissolved in distilled H20 and administered in a 4.5 ml/kg volume. The 60 mg/kg dose was chosen based on findings from preliminary work indicating that this dose did not alter offspring survival or body weight, although maternal weight gain during pregnancy was reduced. The second treatment group (CO) (n = 9) received daily intubations of distilled water (4.5 ml/kg) following the same schedule as the C60 group.
z
34
:::E (.9 :7
w
-..1
52
123 0
~
3O
-.A .< .-.I
m
28
26 0 MG/KG
60 MO/KG
COCAINE FIG. 1. Mean umbilical cord length (SEM) as a function of prenatal treatment (0 mg/kg or 60 mg/kg cocaine hydrochloride respectively).
Sample Collection The dams were part of a preliminary study of plasma cocaine levels following intragastric administration. Blood samples were taken at one of 4 time intervals (5, 20, 35 or 50 min) following dosing on GD 21. Animals were anesthetized with a combination of ketamine and xylazine (10 mg/kg and 20 mg/kg respectively). Ten minutes later, blood was obtained by cardiac puncture (to which saturated sodium fluoride was added for a final NaF concentration of 0.4%), and then the dam was sacrificed by exposure to CO2. At this time, the fetuses were delivered via caesarean section and umbilical cords were measured.
Umbilical Cord Measure After the delivery via caesarean section, the fetuses were placed on a petri dish wet with saline. Each fetus was removed from its amniotic sac and lifted vertically until the placenta just touched the surface of the petri dish. Care was taken to ensure that the traction applied to each cord was kept constant. Umbilical cord lengths were measured (to the nearest ram) from the placental plate to the abdominal wall using a caliper. The caliper was then placed against a millimeter ruler to measure cord length. For each litter, one experimenter measured the cord length of each fetus and called the measurement out to a second experimenter. After cord lengths were recorded for the entire litter, a second measure was taken. These two measures rarely varied by more than 2 mm, and the average of these two measurements was the dependent variable. After umbilical cord lengths were examined, the umbilical cord was cut from the fetus and from the placenta. Placental weight, fetal body weight and fetal sex were then recorded. To control for potential experimenter bias, the experimenter making the measurements was blind to treatment condition until the conclusion of the experiment. This method has been used previously in our laboratory and provides reliable and consistent measurements (4). RESULTS
Maternal Toxicity Significant differences were observed in maternal weight gain during pregnancy between the two treatment groups (32.4% vs. 47.8% weight gain for the C60 vs. CO group, respectively). One
C60 dam that had originally been designated for this study had seizures following the sixth intubation and died shortly after the seventh intubation of cocaine. The mean (_+ SEM) plasma cocaine level for the C60 group was 4 3 0 _+ 160 ng/ml. Although samples were taken at 4 time intervals over a 45-min period, there were no reliable differences in plasma cocaine levels across the sampling intervals,
Fetal Data Umbilical cord lengths are presented in Fig. 1. Umbilical cords were significantly shorter in the C60 group relative to the CO intubated control group. The data were analyzed by taking the average cord length for each sex within a litter and using these means as the dependent measure to avoid potential litter bias (1). An analysis of variance (ANOVA) revealed a significant main effect of prenatal treatment, F(1,30) = 23.93, p
