Effects of sodium valproate and oxygen on the CD-1 mouse fetus

This study reports the effects of valproic acid (VA) on the CD-1 mouse fetus when the drug is administered continuously via osmotic minipumps at human therapeutic drug plasma levels. Two VA-filled Alzet osmotic minipumps were implanted subcutaneously on gestation day 5 for continuous exposure of a total daily dosage of 850 mg/kg on gestation days 5-12. Dams were then exposed continuously to either normoxic (21% oxygen), hyperoxic (50% oxygen), or hypoxic (12% oxygen) controlled environments during gestation days 5-12, in order to determine if hyperoxic maternal conditions offered a protective environment for the fetus, and conversely, if hypoxia exacerbated teratogenicity. Dams were sacrificed on gestation day 18, and litter and fetal data were collected. It was determined in separate groups under normoxic conditions that the osmotic minipump system maintained VA plasma levels corresponding to human therapeutic levels. Sodium valproate was found to induce developmental toxicity in the CD-1 mouse fetus at human therapeutic drug plasma levels. Fetal weights were reduced, and the number of resorptions, deaths, and hematomas was increased. While hypoxia exacerbated the toxic effect on the fetus, hyperoxia failed to ameliorate the outcome.     

Teratogenic effects on the neuroepithelium of the CD-1 mouse embryo exposed in utero to sodium valproate

A causal association has now been recognized between the use of the anticonvulsant drug sodium valproate during pregnancy and the increased incidence of spina bifida in the human population. The objective of this study was to investigate the teratogenic effects of sodium valproate on the cephalic 1) neuroepithelium, 2) extracellular matrix, and 3) embryonic protein content in the CD-1 mouse embryo. Nulliparous female CD-1 mice were dosed intraperitoneally on day 8 of gestation with 340 mg/kg of sodium valproate. On day 10 of gestation, females were killed by cervical dislocation, and all live embryos were assigned to one of the following groups and processed accordingly for: 1) head measurements, 2) scanning electron microscopy, 3) total protein determination, 4) two-dimensional polyacrylamide gel electrophoresis, 5) immunohistochemistry, and 6) light microscopy. Exposure to sodium valproate at the selected dosage resulted in a 30% incidence of neural tube defects in the cranial region of these embryos. Treated embryos showed a significant reduction in head size, indicating a drug-induced microcephaly. No major differences were seen in the total embryonic protein patterns between control and treated embryos. Immunoreactivity to laminin and fibronectin showed a similar distribution in control and treated embryos except in the vasculature pattern of the hindbrain neuroepithelium. The neuroepithelium of the treated embryos showed marked disorganization when it was examined histologically, particularly in the forebrain region. Cells were disoriented, and there was a noticeable loss of intercellular adhesion in the juxtaluminal region. Increased cellular blebbing was apparent at the ependymal surface, and large protrusions of cells were seen invading the neural tube lumen. The lumen was distorted in shape and frequently contained blood cells. Irregularities and gaps were observed in the underlying basal lamina. These results suggest that treatment with sodium valproate during a critical time in neurogenesis in the CD-1 mouse embryo alters the normal architecture of the neuroepithelium, with a loss of integrity at both the basal and apical surfaces. The alterations seen in the neuroepithelium at any of these sites in this animal model could help explain the increased incidence of spina bifida seen in children of epileptic mothers receiving sodium valproate.     

Teratogenic effects of dosage levels and time of administration of carbamazepine, sodium valproate, and diphenylhydantoin on craniofacial development in the CD-1 mouse fetus

The objective of this investigation was to study the teratogenic effects of dosage levels and time of administration of three anticonvulsant drugs (carbamazepine [CMZ], sodium valproate [NaV], and diphenylhydantoin [DPH]) on craniofacial development in the CD-1 mouse fetus. Pregnant females were intubated on each of days 8-10, 11-13, 14-16, and 8-16 of gestation with the following dose levels for each drug: 375, 563, 938 mg/kg CMZ; 225, 338, 563 mg/kg NaV; 50, 75, 125 mg/kg DPH. Appropriate control groups were maintained for each drug. On gestation day 17, pregnant females were killed and implantation sites were recorded as live, dead, or resorbed. All live fetuses were examined for craniofacial defects. Results of examination of 1,398 fetuses indicated that CMZ, NaV, and DPH were teratogenic and embryotoxic at all dose levels. This study indicated that the observed decrease in mean fetal weight was drug-, dose-, and time-dependent. There was a drug-, dose-, and time-dependent increase observed in the number of dead fetuses, whereas the number of resorbed fetuses was observed to be only time-dependent. The observed frequencies of hydrocephalies, secondary palatal clefts, and submucous palatal clefts were significant for all three factors (drug, dose, and time) whereas the observed frequencies of hematomas and exencephalies were significant only for drug and time. Cleft lips were observed only in the highest dose level of DPH. Uterine horn distribution of defects indicated that fetuses located at the proximal end of the horns were less subject to major defects than those fetuses located at the distal end of the uterine horns. Fetuses with craniofacial hematomas were found in the proximal one-third of the uterine horn, resorbed fetuses, and fetuses with submucous palatal clefts in the middle one-third of the uterine horns and dead fetuses and fetuses with exencephalies, cleft lips, and secondary palatal clefts were localized in the distal one-third of the uterine horns. In comparing the effect of drug, dosage, and time on the development of craniofacial malformations in the CD-1 mouse fetus, CMZ was the least teratogenic and embryotoxic of the three anticonvulsant drugs employed in this study.     

Teratogenic effects on the CD-1 mouse embryo exposed to concurrent doses of ethanol and aspirin

Human fetal alcohol syndrome characteristics have been seen in the mouse fetus by several investigators who dosed the dam with only one or two doses of alcohol. The purpose of this study was to determine if the fetal effects of acute doses of alcohol (ethanol) are altered by aspirin. CD-1 mice were given two IP doses of a 25% v/v solution of 95% ethanol/saline (2.5 hours apart) and intubated with 250 mg/kg aspirin. The treatment regimen, begun at 8 days, 4 hours gestation, consisted of either aspirin pretreatment 1 hour before or posttreatment 1 hour after the ethanol. Control animals were treated similarly and included vehicle only, ethanol/vehicle, and aspirin/vehicle groups. One group was untreated. On gestational day 18, the dams were killed and the uterine horns were examined for live, dead, and resorbed fetuses. The live were weighed and examined for external malformations and either skeletal or visceral abnormalities. With the litter as the unit of analysis, no significant difference was found in the number of dead and resorbed among groups. There was a significant difference (P less than .01) in average fetal weight in the aspirin-pretreated group. When the total number of fetuses affected was considered, the aspirin pretreatment group showed significantly (P less than .05) more external and visceral malformations. The skeletal examination revealed a significant (P less than .05) difference in anomalies plus delayed ossification in both groups treated with the aspirin/ethanol combination. No significant differences were seen in any category in the groups receiving aspirin alone or ethanol alone. These results indicate an additive effect of aspirin and ethanol on the developing CD-1 mouse fetus.     

Morphometric analysis of the craniofacial development of the CD-1 mouse fetus exposed to alcohol on gestational day eight

Fetal alcohol syndrome (FAS) describes a pattern of dysmorphogenesis observed in some offspring of women who consumed alcohol during pregnancy; partial expression of this pattern are fetal alcohol effects (FAE). The purpose of this investigation was to measure selected craniofacial parameters in the CD-1 mouse fetus following exposure to alcohol on gestational day (D) 8. CD-1 mice were mated for 1 hr; D0.0 designated by the presence of a vaginal plug. On D8, 0 hr, and D8, 4 hr, 33 dams were injected intraperitoneally (IP) with 25% (v/v) alcohol in physiological saline solution (0.015 ml/gm maternal body weight). Appropriate controls were maintained. The animals were sacrificed every 12 hr from D12.0 through D17.0. Implantation sites were examined and recorded as live, dead, or resorbed fetuses. All live fetuses were weighed, examined for gross defects, and fixed in Bouin's solution. Twenty-three bilateral parameters were recorded for linear dimensions defining face and cranium. The fetal weights were statistically lower in treated as compared to control fetuses only on D16.0 through D17.0. Statistical analysis of the morphometrics identified five distinct growth patterns in treated mice as compared to controls. The anomalies induced in the CD-1 mouse fetus following exposure to alcohol on D8.0 resembled FAE rather than FAS. Morphometric analysis of the craniofacial region may be an important clinical tool for the quantitative identification of alcohol-related effects in the offspring of women who consumed alcohol while pregnant.     

Cleavage pattern and emerging asymmetry of the mouse embryo

Early mammalian development is regulative - it is flexible and responsive to experimental intervention. This flexibility could be explained if embryogenesis were originally completely unbiased and disordered; order and determination of cells only arising later. Alternatively, regulative behaviour could be consistent with the embryo having some order or bias from the very beginning, with inflexibility and cell determination increasing steadily over time. Recent evidence supports the second view and indicates that the sequence and the orientations of cell divisions help to build the first asymmetries.     

Pre- and post-conceptional tobacco effects on the CD-1 mouse fetus

The objective of this study was to examine the effect of subchronic administration of an aqueous extract of smokeless tobacco (ST) on the development of the CD-1 mouse fetus. Mice were administered ST for approximately 5 weeks: for 2 weeks prior to breeding, during breeding, and during gestational days 0 to 17. Thus the initial peak nicotine levels occurred prior to breeding and not during the critical periods of gestation. Two ST dosages were administered by gastric intubation three times daily: ST/D-1, equivalent to a dose of 12 mg nicotine/kg of body weight, and ST/D-2, equivalent to 20 mg nicotine/kg body weight. Maternal plasma nicotine levels were determined 30 min after the second intubation during the pretreatment and gestational phases of treatment. At these ST dosages, the weight gain of ST-treated dams was not significantly affected in comparison to treated controls. The mean maternal plasma nicotine level for the low-dosage group was 363 ng/ml, and 481 ng/ml for the high-dosage group, with maternal lethality observed at 9.6% and 28.2%, respectively. No significant differences were seen between control and ST/D-1 maternal and/or fetal values, except for placental weights which were heaviest in the ST group (P less than 0.05). Several differences were noted between the ST/D-2 group and controls: fetal weights were reduced by 5.4% (P less than 0.05); decreased ossification was seen in femur measurements and in nine of ten characteristics measured (P less than 0.05); the frequency of resorptions (7.6%) was almost doubled (controls 4.2%); and the frequency of deaths and malformations was not affected. Under these experimental conditions, the low dose produced a negligible effect on the CD-1 mouse fetus and the dam. The high dose demonstrated growth retardation (P less than 0.05), increased embryotoxicity, and a significant decrease in ossification (P less than 0.05).     

Effects of anandamide on embryo implantation in the mouse

Anandamide (N-arachidonoylethanolamine), an arachidonic acid derivative, is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors. To investigate the possible effects of anandamide on embryo implantation in the mouse, we used a co-culture system in which mouse embryos are cultured with a monolayer of uterine epithelial cells. Our results indicate that 14 nM anandamide significantly promotes the attachment and outgrowth of the blastocysts on the monolayer of uterine epithelial cells, and those effects could be blocked by CB1-R antagonists SR141716A, but not by SR144528, a CB2-R antagonist. It suggests that the effects of anandamide on embryo attachment and outgrowth are mediated by CB1-R. However, 56 nM anandamide is capable of inhibiting the blastocyst attachment and outgrowth, we, therefore, conclude that anandamide may play an essential role at the outset of implantation.     

Neurobehavioural responses to hypergravity environment in the CD-1 mouse

Behavioural responses of CD-1 mice exposed to 2 g hypergravity (HG; 60 or 120 min) were investigated during adolescence and at adulthood. To characterise motion sickness (MS), pica behaviour, a reliable MS index measured through kaolin consumption, and spontaneous activity were evaluated before, during and after HG exposure. Short- and/or long-lasting effects on emotional responses, exploratory behaviour and spatial learning performances were also investigated and brain levels of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) assessed. An increased pica behaviour during post rotational days and a reduction in spontaneous activity during rotation indicated a mild sickness associated with HG, and susceptibility to MS was sex and age related. Short- and long-lasting effects of exposure were also observed, rotated mice showing altered emotional/anxiety behavioural profiles and impaired spatial learning performances. Moreover, central NGF levels were markedly increased after rotation, while minor changes were observed in BDNF levels.     

Alcohol and smokeless tobacco effects on the CD-1 mouse fetus.

Tobacco products and alcohol are commonly used as nonmedicinal drugs by pregnant women, and both are known to cause various effects on the fetus and the newborn. The objective of this study was to examine the fetal effects of both drugs when administered individually and simultaneously to pregnant CD-1 mice at moderate dosages. Specifically, we wanted to determine whether or not the effect on the fetus of these two biologically active substances was additive, ameliorative, or synergistic. A total of 65 CD-1 dams were divided among four groups receiving either ST equivalent to 8 mg/kg nicotine, ethanol (ETOH) 1.8 g/kg, a combination of ST+ETOH in the same dosages, or D-glucose (controls and ST alone) to supply calories equivalent to the dose of ethanol. Mice were dosed three times per day on gestational days 6-15. On gestational day 17 all dams were killed, fetal and placental weights recorded, and the number of resorbed, dead, and malformed fetuses noted. The mean maternal plasma drug levels were: nicotine-321 ng/ml and ethanol-0.105 g%. No significant differences were observed in maternal weight gain, litter size, or in the incidence of resorptions, deaths and/or malformations. Fetal weights were reduced in all three treatment groups (P less than 0.05), with the greatest reduction (13% decrease) recorded in the ST group, followed by a 9% decrease in the ETOH group, and a 7% decrease in the ST+ETOH group. Placentas of the ST group weighed significantly less (P less than 0.05) than controls. Ossification of the fetal skeleton, observed in ten sites, was affected to the greatest extent in the ST group, followed by the ETOH and ST+ETOH groups. Craniofacial measurements were significantly affected (P less than 0.05) in all three treatment groups, compared to controls. We conclude that under these experimental conditions, in terms of fetal growth and ossification, ST had the greatest effect, followed by ETOH and ST+ETOH. The interaction of ST+ETOH was neither additive, synergistic, nor ameliorative.     

Strain differences in the teratogenicity induced by sodium valproate in cultured mouse embryos

Strain differences in the teratogenicity of valproic acid (VPA) have been reported in mice. Finnell and Chernoff (Proc. Grnwd. Genet. Ctr. 5:162-163, 1985) showed that 300 mg/kg of VPA twice a day on days 6-8 of gestation induced exencephaly in 82% of SWV embryos but in 0% of C57BL/6J embryos. In the present experiment, we have collected similar results and investigated this strain difference using whole embryo culture in an attempt to determine whether maternal or embryonic factors are responsible for the difference. Mouse embryos were explanted on day 8.5 (plug day 0), and embryos at the 6-8-somite stage were cultured for 48 hours in rat serum containing various doses of sodium valproate (NaVP). All the embryos died within 24 hours with 4.5-mM and higher doses of NaVP in C57BL/6NCr1BR (C57) and with 3.0-mM and higher doses in SWV. Unfused brain folds were recognized in embryos treated with 3.0-mM and higher doses in C57, and with 1.0-mM and higher doses in SWV. Irregular somite formation was observed in many embryos treated with 1.6-mM and higher doses in C57 and with 1.0-mM and higher doses in SWV. These results indicate that SWV embryos have 1.5-3 times the sensitivity of C57 embryos to the embryolethal and teratogenic effects of NaVP. Furthermore, the results suggest that the basis of the strain difference resides within the embryo rather than the mother.     

Postnatal effects of nicotine on first molar development in the CD-1 mouse

Young CD-1 mice, 4 days old, exposed to 0.1% nicotine sulfate on gestational days 6-20 were compared with untreated pups of the same age to determine its effect on the development of mandibular first molars. Pregnant mice were given intraperitoneal injections of nicotine at a dose of 1.67 mg/kg/day. Pups were then decapitated, their entire mandibles were excised, routinely prepared and embedded in paraffin, sectioned in the frontal plane and stained with hematoxylin and eosin for histological examination of developing lower first molars. The results demonstrated that the process of odontogenesis appears retarded in nicotine-treated animals while the molars of the control group revealed dentin and enamel formation. It was concluded that nicotine has a detrimental effect on molar development. Nicotine may interfere with cellular maturation of the tooth germ indicating that this effect is prenatal and extends postnatally.     

Effect of smokeless tobacco on the development of the CD-1 mouse fetus

The objective of this study was to examine the effect of an aqueous extract of smokeless tobacco (ST) on the development of the CD-1 mouse fetus. Three ST dosages were administered three times daily by gastric intubation during gestational days 1-17: 1 X ST equivalent to a dose of 4 mg nicotine/kg body weight, 3 X ST equivalent to 12 mg nicotine, and 5 X ST equivalent to 20 mg nicotine/kg body weight. Maternal plasma nicotine levels were determined 30 minutes after the second daily intubation at five different times during the gestational period. At these ST dosages, the weight gain of ST-treated dams was not significantly affected in comparison to treated controls, though the difference was significant (P less than .05) in comparison to untreated controls. The mean maternal plasma nicotine level for the low dosage (1 X) group was 99.0 ng/ml, which reasonably approximates human consumption levels. The 3 X ST and 5 X ST dosages produced higher nicotine plasma values, 398 ng/ml and 623 ng/ml, respectively, were considerably more toxic to the dams, and resulted in 18% and 31% maternal deaths. Fetal weights were reduced by 7.4% (P less than .001) in the highest ST dosage group (5 X), whereas at the 1 X and 3 X dosages fetal weight differences were not significantly different from treated controls. Resorptions increased in a dose-related manner (P less than .05), ranging from 4.7% in the 1 X, to 6.4% in the 3 X and 8.9% in the 5 X dosage compared to 3.2% in treated controls. External malformations were few and minor in extent. Internal malformations increased in a linear, dose-related manner (P less than .05). Placental weights were unaffected by ST. The results of skeletal examinations were inconclusive. Precocious ossification was seen in 60% and 70% of the parameters measured in the 1 X and 3 X dosage groups, respectively, in comparison to controls. In the 5 X ST group ossification levels were less than in controls for 30% of the parameters measured. Under these experimental conditions the lowest ST dosage (1 X) produced a negligible effect on the CD-1 mouse fetus and the dam. The highest ST dose (5 X) demonstrated embryotoxicity, growth retardation, few malformations, and maternal toxicity. The intermediate dose (3 X) showed a range of effects between the highest and lowest doses to both the fetus and the dam.     

Cytotoxic effect of oxygen on the skeletal muscle of mouse diaphragm

Incubation of the isolated mouse diaphragm with a high rate of oxygenation (10 ml s-1, 95% O2 + 5% CO2) causes a characteristic cellular damage with widely-separated myofibrils and swollen sarcotubular system within 10 min. This damage was ameliorated by inhibitors of the hydroxyl radical (.OH), desferrioxamine, dimethyl thiourea and 120 mM mannitol, and by incubation at 8 degrees C. It was not prevented either by inhibitors of the pathway leading to sarcolemma damage (nordihydroguaiaretic acid, alpha-tocopherol, butylated hydroxytoluene) nor by agents and treatments that inhibit the oxygen paradox of cardiac muscle (glucose, omission of extracellular calcium, incubation at 30 degrees C, superoxide dismutase and catalase). Nevertheless there are similarities between these two types of damage triggered by O2 and the possibility that in both an NAD(P)H oxidase is stimulated and cytotoxic oxygen radicals are generated is discussed.     

Lactate dehydrogenase isoenzyme transitions during skeletal differentiation in the mouse embryo

(1) In the mouse embryo there are changes in lactate dehydrogenase activity and isoenzyme pattern during the differentiation of cartilage and bone. (2) The specific activity of lactate dehydrogenase rises during chondrogenesis and falls during osteogenesis. (3) Identical isoenzyme transitions occur in parallel in both tissues: undifferentiated limb bud mesenchyme contains isoenzymes 1-5 whereas in both the cartilaginous and bony portions of a long bone developing from the mesenchyme, there is a progressive shift towards a predominance of the 'anaerobic' isoenzymes 4 and 5.     

Cloning and expression pattern of a mouse homologue of drosophila sprouty in the mouse embryo.

Signaling molecules belonging to the Fibroblast growth factor (Fgf) family are necessary for directing bud outgrowth during tracheal development in Drosophila and lung development in mouse. A potential inhibitor of the Fgf signaling pathway, called Sprouty, has been identified in Drosophila. We have identified three potential mouse homologues of sprouty. One of them, called Sprouty4, exhibits a very restricted expression pattern. At 8.0 dpc (days post coitum) Sprouty4 is strongly expressed in the primitive streak region. At 9. 5 and 10.5 dpc, Sprouty4 is expressed in the nasal placode, the maxillary and mandibular processes, the otic vesicule, the second branchial arch, in the progress region of the limb buds and the presomitic mesoderm. Sprouty4 expression is also detected in the lateral region of the somites. In the developing lung, Sprouty4 is expressed broadly in the distal mesenchyme.     

Lrp5 and Lrp6 redundantly control skeletal development in the mouse embryo

The role of Wnt signaling in osteoblastogenesis in the embryo remains to be fully established. Although β-catenin, a multifunctional protein also mediating canonical Wnt signaling, is indispensable for embryonic osteoblast differentiation, the roles of the key Wnt co-receptors Lrp5 and Lrp6 are unclear. Indeed, global deletion of either Lrp5 or Lrp6 did not overtly affect osteoblast differentiation in the mouse embryo. Here, we generated mice lacking both receptors specifically in the embryonic mesenchyme and observed an absence of osteoblasts in the embryo. In addition, the double-deficient embryos developed supernumerary cartilage elements in the zeugopod, revealing an important role for mesenchymal Lrp5/6 signaling in limb patterning. Importantly, the phenotypes of the Lrp5/6 mutant closely resembled those of the β-catenin-deficient embryos. These phenotypes are likely independent of any effect on the adherens junction, as deletion of α-catenin, another component of the complex, did not cause similar defects. Thus, Lrp5 and 6 redundantly control embryonic skeletal development, likely through β-catenin signaling.     

Excitation-contraction uncoupling in the developing skeletal muscle of the muscular dysgenesis mouse embryo

The muscular dysgenesis recessive autosomal mutation is characterized by a total lack of muscular contraction and a myofibrillar non-organization. Many abnormalities involved in the excitation-contraction coupling are found in mdg/mdg myotubes: 1) the internal structural organization of the membrane coupling between the sarcoplasmic reticulum (SR) and the transverse (T)-tubule forming the triadic association is defective: the triad number is decreased in the muscle and there are a lack of periodic densities between the SR and T-tubule apposed membranes. 2) the voltage-dependent Ca2+ channel contents, identified by binding with the specific blocker PN 200-110, are decreased. The two fast (30 ms) and slow (100 ms) Ca2+ currents present in normal myotubes are absent in mdg/mdg myotubes in vitro. 3) the Ca2+-dependent K+ conductance triggering an action potential followed by a long lasting after hyperpolarization (ahp) is absent in mdg/mdg myotubes. This indicates a lack of the free intracellular Ca2+ increased by the action potential. These results suggest that: 1) the lack of differentiated triadic junctions is directly correlated with very low amounts of voltage-dependent Ca2+ channels; 2) the low amount of Ca2+ channels results directly in decreased Ca2+ currents; 3) the decreased Ca2+ currents are the consequence of the low intracellular Ca2+ concentration which is not sufficient to trigger a contraction. However, the addition of normal motoneurones to mdg/mdg myotubes in culture induces, few days later, an increase in Ca2+ currents.