Received 2001-12-13 Accepted 2002-04-12

This project was supported by the National Natural Science Foundation of China (No. 39970772).

Corresponding author. Tel: +86-24-23261454; Fax: +86-24-23254417; E-mail: hwei100@yahoo.com.cn

Acta Physiologica Sinica

Aug. 2002, 54 (4), 342～348

Research Paper

Effects of pregnancy cocaine exposure on the mother and fetus:a murine model

SONG Jun1, GUAN Xiao-Wei1, REN Jia-Qian2, HE Wei1,

1Department of Histoembryology, China Medical University, Shenyang 110001, China; 2Department of Neurology, Massachusetts General Hospital, Charlestown, MA 02129, USA

Abstract: The aim of the experiments was to develop and characterize a murine model for investigating the effects of prenatal cocaine exposure on the mother and fetus. Pregnant mice were separated into three groups: group 1 was treated with cocaine HCl at 10 mg/kg twice daily (COC); group 2 was treated with saline at 10 ml/kg twice daily (SAL); and group 3 was pair-fed with the COC dams and was injected with saline following the same schedule (SPF) from embryonic day (E) 8 to 17. We utilized high-pressure liquid chromatography (HPLC) with UV detector and electrochemical detector to test the concentrations of cocaine, dopamine and serotonin, as well as HE staining to observe morphological alterations of liver and placenta. Though less food intake and lower weight gain were observed in COC and SPF groups but not in SAL dams, lower fetal body weight and brain weight were only seen in COC offspring. Pharmacological analysis revealed that cocaine was found in fetal plasma at 15 min following intraperitoneal administration on E17, accompanied with elevated concentrations of dopamine (DA) and serotonin (5-HT) in fetal brain. We also observed morphological changes in liver and placenta of cocaine-exposed fetuses. The present study indicates that pregnancy cocaine exposure can lead to maternal undernutrition and developmental abnormality of the fetal brain, liver and placenta. It is suggested that the developmental abnormality of the fetuses induced by cocaine is due to the toxicological effect of cocaine but not to maternal undernutrition.

Key words: cocaine; pregnancy; maternal exposure; fetus; neurotransmitters; fetal development; chromatography; high pressure liquid

妊娠期给予可卡因对母体和胎儿的影响: 小鼠动物模型

宋君1，关晓伟1，任嘉谦2, 何威1,

1中国医科大学组织胚胎学教研室, 沈阳 110001；

2哈佛大学麻省总医院神经科研究室, 美国

摘要: 探讨妊娠期给予可卡因对母体和胎儿的影响。妊娠小鼠分为3组：可卡因注射组(每日两次注射盐酸可卡因10 mg/kg, COC); 盐水对照组（每日两次注射生理盐水10 ml/kg, SAL）; 饮食对照组(每日两次注射生理盐水10 ml/kg, 饮食参考可卡因给药组, SPF)。用高压液相色谱分析法检测胎鼠血中可卡因浓度及纹状体中神经递质多巴胺和5-羟色胺的含量, 并结合HE染色观察胎鼠肝脏和胎盘的形态学改变。尽管COC和SPF组母鼠摄食量和体重增长量均降低, 但是仅仅COC组胎鼠的体重和脑重减少。高压液相色谱分析结果显示, 在COC组胎鼠血浆中可检测出可卡因, 并伴有纹状体神经递质含量的异常增高。同时, 也观察到了COC组胎盘和肝脏的形态学变化。本研究表明, 妊娠期给予可卡因能引起妊娠母体营养不良, 子代脑、肝脏和胎盘发育异常; 可卡因引起的胎儿发育异常是由可卡因的毒性作用而不是母体营养不良产生的。

关键词: 可卡因; 妊娠期; 母体给予; 胎儿; 神经递质; 胎儿发育; 高压液相色谱

学科分类号: Q954.6; R332; R992

The impact of cocaine on adults has been well documented over the past few years, but the mechanisms by which cocaine acts on the fetuses are still largely unknown[1]. In adult animals, cocaine affects the concentrations of dopamine (DA), norepinephrine (NE) and serotonin (5-HT)[2]. Similar processes may operate developmentally. To better understand the effects of cocaine on the developing body, we have pursued a transplacental model of cocaine exposure on the fetuses. This model permits a control over the route of administration, gestational timing and frequency of exposure, as well as the performance of prenatal pair-fed (to control the undernutrition induced by cocaine administration).

In the rat, the dopaminergic nigrostriatal system develops earlier compared with other neurotransmitter systems. Dopaminergic neurons undergo mitosis in the ventral mesencephalon from embryonic day 11 to 15 and first extend axons into the striatal anlage on E14[3,4]. So we chose E17 dopaminergic nigrostriatal system as the researching target, and undertook the cocaine exposure from E8 to E17.

To quantitate the extent, to which cocaine administered to pregnant mice reaching the fetus, we examined the concentration of cocaine in fetal plasma by high-pressure liquid chromatography (HPLC) with UV detection. Since many of the behavioral effects of cocaine, including its reinforcing and addictive properties, have particularly been attributed to its influence on the DA and 5-HT system, and striatum is the main projecting area of dopamine system, we investigated the changes in dopamine and serotonin in fetal striatum by HPLC with electrochemical detector to study the effect of prenatal cocaine exposure on the neurotransmitters of fetal brain. We additionally characterized the morphological changes by using hematoxylin and eosin staining to investigate the toxicological effect of cocaine on other systems.

1 MATERIALS AND METHODS

1.1 Animal and group Female Kun-Ming Mice at 49 d of age were obtained from Animal Laboratories of China Medical University. They were housed at 21±2℃ and on a 12 h light:dark cycle (lights on at 07:00). Females were placed with males at 17:00. The presence of a vaginal sperm plug on the following morning defined the beginning of pregnancy (E0). Mice with sperm plug were weighed and caged individually. Pregnant dams of comparable weight were assigned to one of three groups: group 1 was treated with cocaine HCl (Qinghai Pharmaceutical Company, China) (COC, dams n=18); group 2 was treated with saline (SAL, dams n=19); and group 3 was treated with saline and pair-fed with cocaine group (SPF, dams n=19). From E1 to E7, all dams were treated identically, with food and water available ad libitum. COC and SAL groups were allowed free access to the diet from E8 to term. But each dam in the SPF group was only allowed to the same amount of food consumed on the same day of paired animals of COC group[5].

1.2 Injection Dams in the COC group received SC injections (in the region of the nape of the neck back) with cocaine HCl at 10 mg/kg twice daily, at 07:00 and 19:00, from E8 to E16 inclusive. SPF and SAL mice received injections of a 0.9% physiological saline solution (20 ml/kg·d－１) following the same schedule. On E17 all dams of three groups were injected intraperitoneally 15 min before sacrifice. Cocaine was dissolved in physiological saline at a concentration of 1 mg/ml. The cocaine dose was initially determined by review of the literature: prior studies in rodent[6].

1.3 Preparation of samples On E17, dams and their fetuses were sacrificed 15 min after administration of their morning cocaine or saline doses. Dams were anesthetized and fetuses were rapidly removed and weighed. After decapitation on ice, fetal brains were obtained, and blood samples were collected in tubes containing NaF (2.5 mg/ml) on ice and then centrifuged to separate plasma. Brain samples were weighed and striatum were separated and weighed. Striatum and plasma samples were frozen and stored at －70℃ till analysis. Some fetal hepatic and placental tissues were collected for histological procedures.

1.4 Physical variables All dams were weighed daily from E8 to E17. Diet consumption was recorded daily for the COC and SAL groups. Gestational length and abortion rate were recorded for each dam. On P0, the litter size, the number of live pups per litter and the number of displastic and dead pups were recorded. Pup weight, sex and biparietal diameter (measured with a micrometer placed in front of the external auditory meatus, BPD) were recorded on P1.

1.5 HE staining After observation for gross pathology, placenta and liver of fetuses were dissected and fixed in 4% buffered paraformaldehyde. Following dehydration, the tissues were embedded in paraffin, sectioned at 5 μm, mounted and stained with hematoxylin and eosin. The histologies were read blind to route of cocaine administration.

1.6 Pharmacological analysis Only COC pups were used for pharmacological analysis. Fetal plasma cocaine concentration was determined by HPLC with UV detector. On the day of assay, 15 μl mazindol (internal standard, Sigma) and 15 μl HPLC-grade water were added to 30 μl volume plasma sample. They were extracted according to the procedure of Jatlow and Nadim[7]. The HPLC system consisted of a Kromasil 4.6 mm×20 cm C18 column (5 μm spheres, China), a Waters HPLC 6000A pump and a Waters 2487 UV detector. The mobile phase, consisting of one part of acetonitrile (HPLC grade) added to four parts of 50 mmol/L KH2PO4 buffer (pH 3.0) containing 0.43 mmol/L tetrabutylammonium dihydrogen phosphate (Fluka), was pumped at 1.0 ml/min at ambient temperature. Cocaine was detected at 235 nm and was quantified by comparison with the peak area of a cocaine chromatographic standard (Qinghai, China). Cocaine was expressed as nanograms of cocaine per milliliter of plasma.

1.7 Neurotransmitter analysis To determine the effects of pregnancy cocaine exposure on fetal neurotransmitters in vivo, concentrations of striatal DA and 5-HT were analyzed by HPLC with electrochemical detector. The same HPLC pump was used, along with a Waters 3.9 mm×15 cm Nova-pak C18 column (4 μm spheres) and a Waters 464 electrochemical detector. Prior to homogenization, 100 μl ice-cold homogenate buffer was added to the vial containing 100 mg striatal tissue samples. Homogenate buffer includes 0.16 mol/L perchloric acid containing 0.02% disodium EDTA and dihydroxybenzylamine (DHBA, Sigma) as the internal standard[8,9]. Homogenates were centrifuged at 15000×g for 30 min at 4℃. The supernatant was transferred to a new vial and used for HPLC assay. Mobile phase was prepared by adding one part of methanol (HPLC grade) to eight parts of HPLC-grade water containing 0.283 mmol/L EDTA, 20 mmol/L sodium citric acid, 50 mmol/L sodium acetic acid and 3.5 mmol/L dibutylamine, the pH adjusted to 3.7 with HCl. Mobile phase was pumped at ambient temperature at a rate of 0.7 ml/min and was recycled during use but made fresh each week. Peak areas were measured for each sample and referenced to the peak areas of various concentrations of the standards, which were chromatographed randomly during the assay of the tissue samples. DA and 5-HT were expressed as nanograms per gram of tissue wet weight for fetal brain.

1.8 Statistics analysis ANOVA and Student's t test were used to determine statistical significance. P<0.05 was considered significant. All data are expressed as means±SEM.

2 RESULTS

2.1 Maternal physical data

An ANOVA test indicated that there was a significant effect of prenatal treatment on gestational weight gain from E8 to E17, P<0.05. Table 1 indicates that SAL dams gained significantly more weight than SPF and COC dams; COC dams gained a little more weight than SPF dams. A t test revealed that SAL dams had a higher food intake value than COC dams through E17, P<0.01. We did not observe any significant differences across groups in gestational lengths and abortion rates, P>0.05.

Table　１. Summary of maternal data

Variable	COC	SPF	SAL
Gestational weight gain E8-E17 (g)	19.6±1.6*	19.5±2.1*	21.1±2.1
Total food intake E8-E17 (g)	59±4**		72±6
Gestational length (d)	18.5±0.4	18.8±0.5	19.0±0.3

Summary of maternal data from pregnant dams injected with cocaine (COC， n=18), injected with saline and pair-fed with the cocaine dams (SPF, n=19), and injected with saline and allowed access to food ad libitum (SAL, n=19).P<0.05 (SNK) COC, SPF vs SAL； P<0.01 (t test) COC vs SAL.

2.2 Fetal and offspring data

An ANOVA test indicated a main effect of prenatal cocaine treatment on fetal body, brain and striatum weights, all with P<0.01 on E17. But there was no significant difference between SAL and SPF offspring, P>0.01(Table 2). We observed no significant difference between SAL, SPF and COC fetuses on the ratios of brain:body weight and of striatum : brain weight, P>0.05. In spite of the substantial reduction in weight gains of dams injected with cocaine, litter size, the number of live pups, displastic pups on P0 and sexes on P1 were found unaltered by prenatal cocaine exposure, P>0.05. On P1, we examined the body weight of all pups and found COC pups had the lowest body weight, only 1.73±0.08 g; and SAL pups were the heaviest, about 2.18±0.13 g. Separate one-way ANOVA indicated significant main effects of treatment on biparietal diameter. SAL pups had wider biparietal diameters than SPF and COC pups on P1 (Table 3).

Table　2. Summary of fetal data

Variable	COC	SPF	SAL
Fetus weights on E17 Body (g)	1.05±0.06*	1.22±0.10	1.21±0.08
Brain (mg)	65±7*	73±4	76±7
Striatum (mg)	8.7±0.5*	10.5±0.4	10.6±0.8
Body/brain weight ratio	16.5±1.4	16.8±1.3	16.2±1.0
Brain/striatum weight ratio	7.33±0.51	7.14±0.46	7.17±0.50

Summary of fetal data for COC (n=8 litters), SPF (n=9 litters ) and SAL (n=9 litters) groups. P<0.01 (SNK) COC vs SPF and SAL．

Table　3. Summary of offspring data

Variable	COC	SPF	SAL
Litter size at P0	9.6±0.6	10.0±0.4	10.2±0.7
Number of live pups per litter at P0	9.6±0.6	10.0±0.4	10.1±0.6
Offspring body weight at P1 (g)	1.73±0.08*	2.14±0.14	2.18±0.13
Offspring BPD at P1 (mm)	7.8±0.6*	9.2±0.3	9.2±0.6

Summary of offspring data for COC (n=10 litters), SPF (n=10 litters) and SAL (n=10 litters).P<0.01 (SNK), COC vs SPF and SAL．

2.3 HPLC analysis data

The cocaine concentration in plasma of COC group was 350±20 ng/ml. DA and 5-HT concentrations are subscribed in Fig.1. DA concentrations in the striatum of fetuses were increased by cocaine exposure; COC group fetuses also had higher concentration of 5-HT than SPF and SAL groups, P<0.01. And there was no significant difference between SPF and SAL groups, P>0.05.

Fig.1. Concentrations of DA and 5-HT in fetal striatum, *P<0.01.

Fig.2. HE stains of liver on E17. A: SAL group; B: SPF group; C: COC group. Bar, 25 μm. Hepatocytes are shown with arrows.

Fig.3. HE stains of placenta on E17. A: SAL group; B: SPF group; C: COC group. Bar, 50 μm. Trophoblastic cells are shown with arrows, villi shown with black triangles.

2.4 Morphological analysis

Examination revealed no overt gross pathology of any organ for mice exposed to cocaine. Histopathological examinations, however, distinguished COC fetuses from saline-injected mice. The most common hepatocellular pathology associated with cocaine was the deterioration of the architecture of the hepatic lobules and cords (see arrows in Fig.2). As shown in Fig.3, we also observed the pathological changes of placenta in COC group, dysplasia of villi (arrows).

3 DISCUSSION

The results of the experiment help characterize the effect of cocaine on pregnant mice and form the basis of a murine model to study the effects of maternal cocaine exposure on offspring. Knowing this information will facilitate the comparison of results obtained from using this model in future experiments to studies using other cocaine-exposure conditions and will bring about establishment of the generalization of our results to other species including humans.

It has been difficult to rule out the contribution of maternal undernutrition to the observed reduction in body and brain weights of cocaine-exposed fetuses; however, our experiments and other reports using rodent models[1] indicated that cocaine in the absence of other drugs could reduce body and brain weight. In our study, Cocaine 10 mg/kg twice daily during E8-E17 reduced food ingestion and weight gain, which was consistent with the findings of others[10-13]. In addition, the body, brain and striatum weights of the E17 fetuses of these groups were reduced in comparison with pair-fed group and saline controls. These facts showed that maternal undernutrition alone could not account for the reductions in fetal body and brain weight because such reductions were not observed for the fetuses of SPF dams which exhibited attenuated weight gains during pregnancy similar to that of cocaine-injected dams. The absence of fetal body and brain weight reductions for the SPF mice is not surprising because a number of reports have indicated that offspring development, particularly the brain, is frequently maintained at the expense of the dam under conditions of substantial undernutrition[14]. On the other hand, the brain: body weight ratio and the striatum:brain weight ratio were only slightly altered for COC fetuses in comparison with SAL and SPF fetuses, suggesting a symmetrical weight reduction with little sparing of the brain.

No significant differences were found in the mean litter size and the live pups per litter at term between groups, which was consistent with the previous study[5], showing no cannibalism in our experiments. Of note, there was no evidence of increased fetal wastage evident in pups of COC and SPF groups, suggesting that the regimen of cocaine that we followed was below the threshold for gross teratogenic effects.

In our model, COC offspring demonstrated significant lower brain weight and striatum weight on E17, and lower body weight and smaller biparietal diameters than SAL and SPF offspring on P1 (all with P<0.01). It is likely that cocaine-induction in BPD is the result of decreased striatum and cortical volume which represents the single largest contributor to this measure. We conclude that impairment in brain growth may be a marker of gestational cocaine exposure.

Our purpose of assessing concentrations of DA and 5-HT in the E17 fetal brain was to determine if the effects of cocaine on fetal catecholaminergic system were similar to its effects on the adult brain. Such evidence might in turn suggest possible mechanisms for the long-term effects of maternal cocaine on offspring brain development. The results of the present study indicate that pregnancy cocaine exposure can elevate the concentrations of DA and 5-HT in fetal striatum which was consistent with its effects on adult brain. It has been suggested that serotonin plays an important role in the normal development of the central nervous system. For instance, it induces neurogenesis and neuronal differentiation[15-17], affects migration of cranial neural crest cells, and inhibits growth cone motility and synaptogenesis[18-22]. Azmitia and his colleagues have recently shown that cocaine decreases the trophic factor S-100 β release from glial cells in the dentate gyrus of the rodent hippocampus[23,24]. Under physiological conditions, the release of this growth factor is mediated by 5-HT and 5-HT receptors. Based on these results as well as the fetal body and brain reduction, we anticipated that altering tightly regulated 5-HT levels might affect the release of S-100 β as well as synaptogenesis, which may lead to developmental delay of fetuses exposed to cocaine in utero, especially brain growth retardation. Previous study showed that nigrostriatal dopaminergic innervation and striatal D1 and D2 dopamine receptor binding sites developed early and to some extent correlatively[25-31], suggesting that dopamine may regulate some aspects of striatal neuronal development by coupling with DA receptors. The period of cocaine administration used in this study encompassed all of the major events in early dopamine ontogeny. Neurons within the substantia nigra and locus coeruleus are born from E10 to E15[4] and catecholamine synthesis in these cells can first be detected by E12-E13[31-34]. Hence, we infer that excessive dopamine induced by cocaine binds to its receptors, which prevents the development of striatum and leads to small volume of fetal brains with some unclear mechanisms.

In current research, we observed the deterioration of the architecture of the hepatic lobules and cords in COC group but not the regeneration reported by Pellinen and Stenback[35]. Perhaps there were different mechanisms between fetal and adult mice, which led to different results. Previously, few studies of placenta morphological alterations have been reported. In this study, we observed dysplasia of villi, which may be the toxicological effect of pregnant cocaine exposure.

In summary, the present study indicates that pregnancy cocaine exposure produced reductions in food ingestion and weight gains of mothers, reductions in fetal body, brain and striatum weights on E17 and reductions in body weight and biparietal diameter (BPD) on P1. Cocaine-induced developmental delay involves many systems including brain, liver and placenta. The developmental delay of brain may be the result of cocaine-induced increases in DA and 5-HT which are similar to the effect of cocaine on adults.

* Acknowledgements: The author would like to thank Mr. Sun Lin for technical assistance on the HPLC measurements.

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