Neurotoxicologyand Teratology,VoL 14, pp. 313-319, 1992
0892-0362/92 $5.00 + .00 Copyright©1992PergamonPressLtd.
Printed in the U.S.A. All rightsreserved.
Prenatal Cocaine Exposure in the Laboratory Mouse: Effects on Maternal Water Consumption and Offspring Outcome MICHAEL
W . C H U R C H .1 A N D H E L E N E
C. R A U C H t
*Department o f Obstetrics~Gynecology and tDepartment o f Immunology~Microbiology, Wayne State University School o f Medicine, Detroit, Nil 48201 Received 27 S e p t e m b e r 1991; A c c e p t e d 18 M a y 1992 CHURCH, M. W. AND H. C. RAUCH. Prenatalcocaine exposure in the laboratory mouse: Effects of maternal water consumption and offspring outcome. NEUROTOXICOL TERATOL 14(5) 313-319, 1992-Pregnant mice were given 50 mg/kg cocaine HCI (1070solution, sc) once daily from gestation days 7 through 18 (sperm positive ffi day 0; term = day 19). Pair-fed and untreated control groups were also used. The pregnant cocaine-treated females showed normal weight gain and food consumption but had significantly increased water consumption. The cocaine-treated group had a significant increase in embryonic resorptions but no significant effects on stillbirths or postnatal mortality. The offspring of cocaine-treated females had significantly reduced birth weights and postnatal weight gains up to the age of 28 days. There was also a delay in their c a r opening but not in other maturational milestones. Increased water consumption following cocaine treatment has been reported by other studies. We speculate that cocaine has a diuretic effect. We discuss the impfications of this effect during pregnancy. Cocaine Pregnancy
Dinresis Maternal water consumption Prenatal
Mouse
COCAINE consumption by women during pregnancy is assod a t e d with a variety of adverse gestational effects such as placental abruption, hypertension, preterm and precipitous labor, and poor maternal weight gain. Prenatal cocaine exposure also causes various morbidities in the offspring including fetal distress, postnatal mortalities, decreased birth weights, cerebral hemorrhages, delayed maturation, various birth defects, and neurobehavioral and sensory deficits (4). In two previous cocaine studies that used pregnant rats, we observed an unexpected increase in maternal water consumption (1,8). Data published by another research group similarly suggest that cocaine-treated female rats increase their water consumption (10). This article reports that the same phenomenon is seen in mice and that the phenomenon in our mice was dependent on the state o f pregnancy. We also report on other aspects o f maternal and offspring outcome following cocaine treatment in the laboratory mouse.
Polydipsia
Postnatal maturation
cause their offspring are useful for immunologic studies. The immunologic deficiencies of our prenatally exposed mouse offspring will be described at another time. After mating, females were housed individually in polycarbonate cages with wood chip bedding material. The animal room was temperature (22 ° ± 1°C) and humidity (40% 50%) controlled with a timed light cycle of 12 h per day (7:00 a.m. to 7:00 p.m.). Animals were assigned to an untreated ad lib fed control group (ADL), a pair-fed control group (PFC), or a cocaine-treated group (COC). The PFC group served two purposes. First, it helped to control for the effects of undernutrition that sometimes accompanies cocaine treatment (1-3). Second, the PFC group also received vehicle (saline) injections to assess the influence of handling stress. The morning of mating was designated gestation day 0 (GD0). Starting on GD7 and continuing until GDI8 (term = GD19), the mated females in the COC group received subcutaneous injections o f 50 mg/kg cocaine hydrochloride dissolved in normal saline (2e/0 solution). Dose selection and route of administration were based on previous research (1-3,8,11,18). Injections were given once daily between 9:00 a.m. and 10:00 a.m. Similarly, the PFC group received isovolumetric injections (SC) o f saline solution. The dams were weighed daily between 8:00 a.m. to 9:00
METHOD Female B A L B / C mice were impregnated by SJL males. Mating was restricted to the hour between 8 a.m. and 9 a.m. Mating was conf'Lrmed by observing a sperm plug in the vagina. The B A L B / C females were mated with SJL males be-
'Requests for reprints should be addressed to Michael W. Church, Ph.D., Fetal Alcohol Research Center, 275 East Hancock Avenue, Detroit, MI 48201. 313
314
CHURCH AND R A U C H
a.m. Daily records were also kept on maternal water and food consumption (Tekald 10% mouse pregnancy diet, Harlan Sprague-Dawley, Inc.). For dams not delivering pups, the presence or absence of pregnancy was determined by sacrificing the animal, removing the uteri, and staining with ammonium sulfate for implantation sites. For dams delivering pups, the uteri were not removed and stained until the pups were weaned on postnatal day 20. Implantation sites were still clearly visible at this time. In past experiments with rats, we assigned newborn pups to untreated surrogate dams (1,5-8) but in a recent experiment, we let some cocaine-treated dams retain some of their pups and found that these pups developed as well as those assigned to surrogate dams (8). Moreover, female mice do not accept surrogate mothering as readily as female rats. Consequently, we did not use surrogate fostering in the present study to conserve animal resources. The weight and sex of each pup were determined as soon as possible after delivery of the entire litter. Pups were thereafter weighed on a weekly basis and assessed for postnatal maturation and mortality. Most data were first evaluated by analyses of variance (ANOVAs) or analyses o f covariance (ANCOVAs). For repeated measures analyses, the probability values were determined according to the Greenhouse-Geisser method. When an ANOVA or ANCOVA indicated a significant effect, a post hoc test (Duncan's Multiple Range Test) was used to determine which groups differed significantly from each other. Although all dams were mated (i.e., they were sperm-positive), about half of these dams proved not to be pregnant as indicated by the absence of embryonic implantation sites in their uteri. This resulted in a two-way A N O V A design with the pregnancy factor having 2 levels (pregnant, nonpregnant) and the treatment factor having 3 levels (ADL, PFC, COC). In the ADL, PFC, and COC treatment groups, there were 6, 6, and 8 pregnant dams and 5, 7, and 13 nonpregnant dams, respectively. For all data analyses involving the offspring, the litter average was used as the unit of measure. RESULTS
Maternal Outcome No females died during treatment. Moreover, treatment had no influence on gestational length. The pregnant ADL,
PFC, and COC dams had mean ( + SD) gestational lengths o f 19.2 + 0.4, 19.2 + 0.4, and 19.4 + 0.5 days, respectively, F(2, 17) = 0.50,p = 0.61. The effects of the pregnancy and treatment conditions on maternal weight gain, food consumption, and water consumption during the treatment period (GD7 through GD18) are presented in Table 1 and Fig. 1. The data in Table 1 are the total weight gains, food consumption, and water consumption. The data in Fig. 1 show the day-to-day trends in these variables. The pregnant dams in the ADL, PFC, and COC groups gained more weight than their nonpregnant counterparts. Cocaine treatment did not cause a reduction in weight gain among the pregnant mice. The same was true among the nonpregnant mice. The pregnant dams in the ADL and COC groups consumed more food than their nonpregnant counterparts. Some of the pregnant PFC dams did not eat all the food allocated to them (see Table 1). Consequently, their food consumption was less than that eaten by the pregnant A D L and COC dams. For water consumption, (a) pregnant and nonpregnant PFC dams drank similar amounts o f water, (b) pregnant and nonpregnant A D L dams drank similar amounts of water, and (c) pregnant COC females drank significantly more water than their nonpregnant COC counterparts. Indeed, the pregnant COC females drank significantly more water than any of the other five groups (see Table 1). One reason the pregnant COC females may have drank the most water was that they had greater body mass and consequently needed more water to sustain their fluid balance. To evaluate this possibility, the daily water consumption data were analyzed by a repeated measure ANCOVA using daily maternal weight as the covariate. The results indicated that maternal weight had little influence in that the pregnant COC females' water consumption was still significantly greater than the other pregnant and nonpregnant groups. That is, the Dose x Pregnancy interaction was significant, F(2, 38) = 9.50, p < 0.001, and all comparisons with the pregnant COC females were significant (all univariate F values = 7.25 to 15.88, df = 1,38, p values < 0.01) with the exception of the nonpregnant A D L females where the difference approached significance ( F = 2.42, df = 1,38, p = 0.065, one-tailed test). Another reason the pregnant COC dams may have drank
TABLE 1 MATERNAL WEIGHT GAIN, FOOD CONSUMPTION, AND WATER CONSUMPTION FROM GESTATION DAYS 7 THROUGH 18 (MEAN + SD) Nonpregnant Variable Maternal weight gain (g) Food consumption (g) Water consumption (ml) n
PFC
ADL -0.1
Pregnant
p values
COC
PFC
ADL
COC
Treat (T)
Preg (P)
Tx P
ns
0892-0362/92 $5.00 + .00 Copyright©1992PergamonPressLtd.
Printed in the U.S.A. All rightsreserved.
Prenatal Cocaine Exposure in the Laboratory Mouse: Effects on Maternal Water Consumption and Offspring Outcome MICHAEL
W . C H U R C H .1 A N D H E L E N E
C. R A U C H t
*Department o f Obstetrics~Gynecology and tDepartment o f Immunology~Microbiology, Wayne State University School o f Medicine, Detroit, Nil 48201 Received 27 S e p t e m b e r 1991; A c c e p t e d 18 M a y 1992 CHURCH, M. W. AND H. C. RAUCH. Prenatalcocaine exposure in the laboratory mouse: Effects of maternal water consumption and offspring outcome. NEUROTOXICOL TERATOL 14(5) 313-319, 1992-Pregnant mice were given 50 mg/kg cocaine HCI (1070solution, sc) once daily from gestation days 7 through 18 (sperm positive ffi day 0; term = day 19). Pair-fed and untreated control groups were also used. The pregnant cocaine-treated females showed normal weight gain and food consumption but had significantly increased water consumption. The cocaine-treated group had a significant increase in embryonic resorptions but no significant effects on stillbirths or postnatal mortality. The offspring of cocaine-treated females had significantly reduced birth weights and postnatal weight gains up to the age of 28 days. There was also a delay in their c a r opening but not in other maturational milestones. Increased water consumption following cocaine treatment has been reported by other studies. We speculate that cocaine has a diuretic effect. We discuss the impfications of this effect during pregnancy. Cocaine Pregnancy
Dinresis Maternal water consumption Prenatal
Mouse
COCAINE consumption by women during pregnancy is assod a t e d with a variety of adverse gestational effects such as placental abruption, hypertension, preterm and precipitous labor, and poor maternal weight gain. Prenatal cocaine exposure also causes various morbidities in the offspring including fetal distress, postnatal mortalities, decreased birth weights, cerebral hemorrhages, delayed maturation, various birth defects, and neurobehavioral and sensory deficits (4). In two previous cocaine studies that used pregnant rats, we observed an unexpected increase in maternal water consumption (1,8). Data published by another research group similarly suggest that cocaine-treated female rats increase their water consumption (10). This article reports that the same phenomenon is seen in mice and that the phenomenon in our mice was dependent on the state o f pregnancy. We also report on other aspects o f maternal and offspring outcome following cocaine treatment in the laboratory mouse.
Polydipsia
Postnatal maturation
cause their offspring are useful for immunologic studies. The immunologic deficiencies of our prenatally exposed mouse offspring will be described at another time. After mating, females were housed individually in polycarbonate cages with wood chip bedding material. The animal room was temperature (22 ° ± 1°C) and humidity (40% 50%) controlled with a timed light cycle of 12 h per day (7:00 a.m. to 7:00 p.m.). Animals were assigned to an untreated ad lib fed control group (ADL), a pair-fed control group (PFC), or a cocaine-treated group (COC). The PFC group served two purposes. First, it helped to control for the effects of undernutrition that sometimes accompanies cocaine treatment (1-3). Second, the PFC group also received vehicle (saline) injections to assess the influence of handling stress. The morning of mating was designated gestation day 0 (GD0). Starting on GD7 and continuing until GDI8 (term = GD19), the mated females in the COC group received subcutaneous injections o f 50 mg/kg cocaine hydrochloride dissolved in normal saline (2e/0 solution). Dose selection and route of administration were based on previous research (1-3,8,11,18). Injections were given once daily between 9:00 a.m. and 10:00 a.m. Similarly, the PFC group received isovolumetric injections (SC) o f saline solution. The dams were weighed daily between 8:00 a.m. to 9:00
METHOD Female B A L B / C mice were impregnated by SJL males. Mating was restricted to the hour between 8 a.m. and 9 a.m. Mating was conf'Lrmed by observing a sperm plug in the vagina. The B A L B / C females were mated with SJL males be-
'Requests for reprints should be addressed to Michael W. Church, Ph.D., Fetal Alcohol Research Center, 275 East Hancock Avenue, Detroit, MI 48201. 313
314
CHURCH AND R A U C H
a.m. Daily records were also kept on maternal water and food consumption (Tekald 10% mouse pregnancy diet, Harlan Sprague-Dawley, Inc.). For dams not delivering pups, the presence or absence of pregnancy was determined by sacrificing the animal, removing the uteri, and staining with ammonium sulfate for implantation sites. For dams delivering pups, the uteri were not removed and stained until the pups were weaned on postnatal day 20. Implantation sites were still clearly visible at this time. In past experiments with rats, we assigned newborn pups to untreated surrogate dams (1,5-8) but in a recent experiment, we let some cocaine-treated dams retain some of their pups and found that these pups developed as well as those assigned to surrogate dams (8). Moreover, female mice do not accept surrogate mothering as readily as female rats. Consequently, we did not use surrogate fostering in the present study to conserve animal resources. The weight and sex of each pup were determined as soon as possible after delivery of the entire litter. Pups were thereafter weighed on a weekly basis and assessed for postnatal maturation and mortality. Most data were first evaluated by analyses of variance (ANOVAs) or analyses o f covariance (ANCOVAs). For repeated measures analyses, the probability values were determined according to the Greenhouse-Geisser method. When an ANOVA or ANCOVA indicated a significant effect, a post hoc test (Duncan's Multiple Range Test) was used to determine which groups differed significantly from each other. Although all dams were mated (i.e., they were sperm-positive), about half of these dams proved not to be pregnant as indicated by the absence of embryonic implantation sites in their uteri. This resulted in a two-way A N O V A design with the pregnancy factor having 2 levels (pregnant, nonpregnant) and the treatment factor having 3 levels (ADL, PFC, COC). In the ADL, PFC, and COC treatment groups, there were 6, 6, and 8 pregnant dams and 5, 7, and 13 nonpregnant dams, respectively. For all data analyses involving the offspring, the litter average was used as the unit of measure. RESULTS
Maternal Outcome No females died during treatment. Moreover, treatment had no influence on gestational length. The pregnant ADL,
PFC, and COC dams had mean ( + SD) gestational lengths o f 19.2 + 0.4, 19.2 + 0.4, and 19.4 + 0.5 days, respectively, F(2, 17) = 0.50,p = 0.61. The effects of the pregnancy and treatment conditions on maternal weight gain, food consumption, and water consumption during the treatment period (GD7 through GD18) are presented in Table 1 and Fig. 1. The data in Table 1 are the total weight gains, food consumption, and water consumption. The data in Fig. 1 show the day-to-day trends in these variables. The pregnant dams in the ADL, PFC, and COC groups gained more weight than their nonpregnant counterparts. Cocaine treatment did not cause a reduction in weight gain among the pregnant mice. The same was true among the nonpregnant mice. The pregnant dams in the ADL and COC groups consumed more food than their nonpregnant counterparts. Some of the pregnant PFC dams did not eat all the food allocated to them (see Table 1). Consequently, their food consumption was less than that eaten by the pregnant A D L and COC dams. For water consumption, (a) pregnant and nonpregnant PFC dams drank similar amounts o f water, (b) pregnant and nonpregnant A D L dams drank similar amounts of water, and (c) pregnant COC females drank significantly more water than their nonpregnant COC counterparts. Indeed, the pregnant COC females drank significantly more water than any of the other five groups (see Table 1). One reason the pregnant COC females may have drank the most water was that they had greater body mass and consequently needed more water to sustain their fluid balance. To evaluate this possibility, the daily water consumption data were analyzed by a repeated measure ANCOVA using daily maternal weight as the covariate. The results indicated that maternal weight had little influence in that the pregnant COC females' water consumption was still significantly greater than the other pregnant and nonpregnant groups. That is, the Dose x Pregnancy interaction was significant, F(2, 38) = 9.50, p < 0.001, and all comparisons with the pregnant COC females were significant (all univariate F values = 7.25 to 15.88, df = 1,38, p values < 0.01) with the exception of the nonpregnant A D L females where the difference approached significance ( F = 2.42, df = 1,38, p = 0.065, one-tailed test). Another reason the pregnant COC dams may have drank
TABLE 1 MATERNAL WEIGHT GAIN, FOOD CONSUMPTION, AND WATER CONSUMPTION FROM GESTATION DAYS 7 THROUGH 18 (MEAN + SD) Nonpregnant Variable Maternal weight gain (g) Food consumption (g) Water consumption (ml) n
PFC
ADL -0.1
Pregnant
p values
COC
PFC
ADL
COC
Treat (T)
Preg (P)
Tx P
ns
