Effects of caffeine and nicotine administration on growth and ossification of the ICR mouse fetus.

The objective of this study was to determine how fetal effects are altered when nicotine (N) and caffeine (CA) are administered concurrently at dosages that individually produce minimal effects to the fetus. Female ICR mice were bred overnight and were assigned to four groups: CA (125 mg/kg), N (12mg/kg), CA plus N (125 mg/kg plus 12 mg/kg, respectively) treated, and control (distilled water) groups. Dams were intubated with these dosages three times daily during gestational days (GD) 6-18 and were euthanized on GD 18. Live fetuses were sexed, weighed, and examined for external malformations. One-half of the fetuses were fixed in 10% formalin and examined for internal malformations using Wilson's method. The remaining half was fixed in 95% ethanol (ETOH), stained, and cleared (Inouye's method) for skeletal examinations. Ossification was assessed by staging and measuring craniofacial bones, and counting ossification centra in sternbrae and in cervical and sacrococcygeal vertebrae. Data were analyzed by analysis of variance (ANOVA) followed by Student-Newman-Keuls post-hoc tests set at p < .05 significance level. The litter was used as the unit of measure and the ANOVA main effects were CA, N, and an interaction term (CA+N). In comparison to controls, CA treatment resulted in reduced bone measurements or reduced ossification scores in 5 of the 19 parameters examined, whereas for N only five parameters were significant. The main effects for interaction of CA+N were significant for seven parameters measured. Although it is difficult to assign the specific type of drug interaction that occurred because results were not completely consistent for all parameters measured, it may be concluded that in most parameters measured both CA and CA+N were different from controls, but CA was not different from CA+N. Under the experimental conditions of this study, we found that of the two drugs, caffeine had a significantly greater effect on fetal growth and ossification than nicotine.

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.

Effects of sodium valproate and oxygen on the craniofacial skeletal pattern in the CD-1 mouse embryo.

Growth retardation is a consistent finding in animal studies on the effect of sodium valproate (NaVP) in the embryo. Apart from fetal weight, the state of ossification in the embryo may be regarded as an indication of growth. The present study was to determine what effect sodium valproate at human therapeutic drug plasma levels had on the craniofacial skeletal pattern in the CD-1 mouse embryo relative to oxygen conditions, drug treatment or the interaction of the two. Two NaVP-filled Alzet osmotic minipumps were implanted subcutaneously on day 5 of gestation for continuous delivery of a total daily dosage of 850 mg/kg for 7 days. During this same time period the dams were also exposed to either normoxic (21% oxygen), hyperoxic (50% oxygen), or hypoxic (12% oxygen) controlled environments. Dams were removed from the oxygen chambers on day 12 and killed on day 18 of gestation. The fetuses were then processed for skeletal evaluation of the craniofacial region. Ossification centers were present in all but six of the skeletal elements studied. The primary ossification delay was in the tympanic bony labyrinth. In addition, there was a decrease in maxillary and mandibular length and cranial base measurements. The greatest toxic effect on the fetus for all skeletal components studied was in the NaVP/hypoxia treated group. This finding suggests that fetal skeletal maturation may be affected by a combination of intrauterine as well as external factors.

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.

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.

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.

Relationship between ossification and body weight of the CD-1 mouse fetus exposed in utero to anticonvulsant drugs.

The anticonvulsant drugs carbamazepine, Na valproate, and phenytoin have been suspected as a cause of human congenital defects. The malformations produced may include cleft lip and/or palate, heart defects, skeletal defects, and low body weights. Since the toxic effects of these anticonvulsant drugs manifest themselves in terms of fetal growth retardation, evaluation of the state of ossification attained in the fetus is important. In the present study, pregnant CD-1 mice received on gestational days 8-16 an oral dose of 375, 563, 938 mg/kg of carbamazepine; or 225, 338, 563 mg/kg of Na valproate; or 50, 75, 125 mg/kg of phenytoin. These groups were compared to two control groups. On day 17, the dams were killed by cervical dislocation and one-third of the live fetuses were weighed and fixed for skeletal examination. Photographs were taken of the fore- and hindlimb skeletons. From these photographs, the length and width measurement of ossified regions of the humerus and femur were determined using a Zeiss Video-Plan Morphometrics Computer. Of the three anticonvulsant drugs studied, the greatest correlation between reduced fetal weights and retarded ossification of the long bones was phenytoin at the 125 mg/kg dosage. Our results also showed that long bone ossification, when compared to the fetal weight, indicated that 59-66% of variability in weight is predictable by bone measurements.

Gastrointestinal malformations associated with prune belly syndrome: three cases and a review of the literature.

Prune belly syndrome (PBS), a triad consisting of abdominal musculature hypoplasia, urinary tract malformations, and cryptorchidism, is frequently associated with other congenital malformations. Although it is acknowledged that gastrointestinal (GI) malrotation and mesenteric anomalies are frequent in PBS, other GI anomalies are generally considered to be exceedingly rare. Here we describe 3 autopsy cases with severe malformations of both midgut and hindgut derivatives and review the world literature to evaluate the spectrum of GI malformations associated with this syndrome. The relatively high frequency of distal stenoses and atresias suggests that the anomalous mesenteric attachments may predispose to prenatal volvulus and subsequent anatomic bowel obstruction. Postnatal volvulus is also occasionally observed. Infants with PBS also appear to be at a higher risk for persistence of the common fetal cloaca.

Teratogenic effects of valproate in the CD-1 mouse fetus.

Valproate sodium has been implicated in the production of spina bifida in humans; this article reports an animal model. Teratogenicity of valproate sodium was studied by oral administration of single doses of 225, 340, and 560 mg/kg to pregnant CD-1 mice on days 7 through 12 of gestation. All fetuses were examined on day 17. Treated fetuses demonstrated external malformations and a decrease in weight. The incidence of malformations was greater at the higher dosage levels of 340 mg/kg and 560 mg/kg, with a predominance of exencephaly, open eyelids, and gross skeletal defects. There was a significant increase in the resorption rate of the fetuses in the treated groups. There was also a significant increase in the malformations observed per litter and per live fetus population when compared with controls.

Scanning electron microscope study of tongue development in the CD-1 mouse fetus.

The objective of this study was to examine three dimensionally the embryonic and fetal stages of tongue development with scanning electron microscopy. Time-bred CD-1 mice were sacrificed at quarter-day intervals on days 10-13, and at half-day intervals on days 13.5-16.5 of gestation. Fetal tongues were dissected and fixed in s-collidine buffered 4% glutaraldehyde at pH7.4, and subsequently processed for SEM viewing. Tongue development was initiated on the 11th day by the appearance of the tuberculum impar and the two lateral lingual swellings on arch I. This was followed by the elevation of the hypobranchial eminence, which unites arches III and IV in the ventral midline, and overgrows arch II anteriorly. During the 12th day, remodeling occurred in areas of arches II and III, forming the root of the tongue. A cone-shaped midline swelling, the epiglottis, appeared in the ventral midline of arches III and IV. By the 13th day, the general proportions of the tongue, occupied by the body, root, and epiglottis, were established. The single circumvallate papilla and fungiform papillae were initiated during the early part of the 13th day, followed on the 15th day by differentiation of filiform and foliate papillae and raised nodules of lingual tonsilar tissue. The SEM study documented the temporal and morphological sequence of events during mouse tongue development. The tuberculum impar persisted to the late fetal stages and may therefore contribute largely to the dorsum of the tongue anterior to the circumvallate papilla.

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.