Laminin and fibronectin in retinoid-induced keratolenticular dysgenesis.

Acute embryonic exposure to isotretinoin during gastrulation (gestational day 7) in the mouse results in delay or failure of separation of the lens vesicle from the surface ectoderm. During normal lens vesicle detachment, laminin is localized within the lens, keratolenticular stalk and adjacent surface ectoderm. The mesenchyme surrounding the stalk stains positively for fibronectin. In contrast, isotretinoin-exposed embryos at the same stage of gestation exhibit reduced staining for both extracellular matrix components. Persistent keratolenticular attachment observed later in gestation in the exposed embryos is associated with increased production of laminin by the keratolenticular stalk and anterior lens epithelium. A delay in the sequence of production of extracellular matrix may be causally associated with persistence of the keratolenticular stalk

Sequential Scanning Electron Microscopic Analyses of Normal and Spontaneously Occurring Abnormal Ocular Development in C57B1/6J Mice

Embryos of C57B1/6J mice were examined grossly, and by light and scanning electron microscopy on days 8 through 19 of gestation. Adult eyes were examined by slit lamp biomicroscopy and light microscopy. A spontaneous incidence of eye malformations including microphthalmia, microphakia, corneal opacity and anterior segment dysgenesis was observed at a rate of 13.2% in the adults and 10.8% in the day 14 embryos. Scanning electron microscopy demonstrates the complex series of coordinated changes in shape and tissue interrelationships observed in normal ocular development. Possible routes of abnormal ocular morphogenesis beginning as early as the time of optic vesicle formation are discussed

Parameters of Growth in the Embryonic and Neonatal Chick Basilar Papilla

The growth of the basilar papilla in the chick cochlear duct was studied utilizing light, scanning, and transmission electron microscopy. The ages of the cochleae investigated ranged from embryonic day 6 to post-hatching day 7. The changes in the length and width of the basilar papilla as well as the establishment of its spatula-like shape were correlated with the maturation of the hair cells\u27 apical surfaces and the changes in the cellular organization of the sensory epithelium. The histological reorganization of the distal hair cell nuclei was concomitant with the broadening of the distal region of the basilar papilla and occurred at a later stage than the reorganization of the proximal hair cell nuclei. Since the stereociliary bundles on all the hair cells are differentiated quite early, it appears that the delayed reorganization of the distal nuclei is associated with anatomical constraints on the cochlear duct, rather than a later differentiation of the distal sensory epithelium. A clear understanding of how growth of the cochlear duct influences both the distribution of hair cells on the basilar papilla\u27s surface and the cellular organization in the sensory epithelium is critical to future studies correlating ultrastructural development with functional maturation of the auditory system

Retinoic acid-induced spina bifida: evidence for a pathogenetic mechanism

Treatment of C57Bl/6J mice with three successive doses of all-trans retinoic acid (28 mg kg-1 body weight) on 8 day, 6 h (8d,6h), 8d,12h, and 8d,18h of gestation resulted in a high incidence (79%, 31/39 fetuses) of spina bifida with myeloschisis (spina bifida aperta) in near term fetuses. Twelve hours following the last maternal dose (9d,6h), the caudal aspects of treated embryos, were abnormal, with eversion of the neural plate at the posterior neuropore, as compared to its normal concavity in comparably staged control specimens. This eversion persisted in affected embryos through the time that the posterior neuropore should normally close. The distribution of cell death in control and experimental embryos was determined using vital staining with Nile blue sulphate and with routine histological techniques. Twelve hours following the maternal dosing regimen, experimental embryos showed evidence of excessive cell death, predominantly in the mesenchyme associated with the primitive streak and in the endoderm of the tail gut, both of which are readily identifiable sites of physiological cell death at this stage of development. In addition, the presumptive trunk neural crest cells located in the dorsal midline, cranial to the posterior neuropore, exhibited a marked amount of cell death in the experimental embryos. We propose that the major factor in the generation of spina bifida in this model is excessive cell death in the tail gut and mesenchyme ventral to the neuroepithelium of the posterior neuropore.(ABSTRACT TRUNCATED AT 250 WORDS

Dose-dependent teratogenicity of the synthetic cannabinoid CP-55,940 in mice

Potent synthetic cannabinoids (SCBs) are illegally distributed drugs of abuse that are frequently consumed in spite of their adverse consequences. This study was designed to determine if the toxicity observed in adults also extends to the prenatal period by examining the developmental toxicity/teratogenicity of one of these SCBs, CP-55,940, in a mammalian model. First, immunohistochemistry was employed for cannabinoid receptor 1 (CB1) localization within gestational day (GD) 8 mouse embryos; this receptor was identified in the cranial neural plate, suggesting that the endogenous cannabinoid system may be involved in normal development. Based on this information and on previous avian teratogenicity studies, the current investigation focused on cannabinoid exposure during neurulation. The treatment paradigm involved acute i.p. administration of vehicle, 0.0625, 0.125, 0.25, 0.5, 1.0, or 2.0 mg/kg CP-55,940 to time-mated C57Bl/6J mice on their 8th day of pregnancy (n > 10 litters per treatment group). On GD 17, litters were harvested and examined for numbers of live, dead, or resorbed fetuses, as well as for fetal weight, length, and gross morphological abnormalities. No effect on litter size, fetal weight, or crown rump length was seen at any of the CP-55,940 dosages tested. Major malformations involving the craniofacies and/or eyes were noted in all drug-treated groups. Selected fetuses with craniofacial malformations were histologically sectioned and stained, allowing investigation of brain anomalies. Observed craniofacial, ocular, and brain abnormalities in drug-treated fetuses included lateral and median facial clefts, cleft palate, microphthalmia, iridial coloboma, anophthalmia, exencephaly, holoprosencephaly, and cortical dysplasia. With the most commonly observed defects involving the eyes, the incidence and severity of readily identifiable ocular malformations were utilized as a basis for dose–response analyses. Ocular malformation ratings revealed dose-dependent CP-55,940 teratogenicity within the full range of dosages tested. While examination of additional critical periods and in depth mechanistic studies is warranted, the results of this investigation clearly show the dose-dependent teratogenicity of this SCB

The role of NOX enzymes in ethanol-induced oxidative stress and apoptosis in mouse embryos

Reactive oxygen species (ROS) play an important role in ethanol-induced apoptosis and teratogenesis. However, the major sources of ROS in ethanol-exposed embryos have remained undefined. This study was conducted to determine the role of NADPH oxidase (NOX) in ethanol-induced oxidative stress and apoptosis in mouse embryos. Analyses of mRNA expression indicated that ethanol treatment resulted in a significant increase in mRNA expression of NOX catalytic subunit Duox-1 in gestational day 9 (GD 9:0) mouse embryos. Ethanol exposure also resulted in significant increases in mRNA expression of NOX regulatory subunits, p22phox, p67phox, NOXA1 and NOXO1. In addition, a significant increase in NOX enzyme activity was found in the ethanol-exposed embryos as compared to controls. Co-treatment with the NOX inhibitor, diphenyleneiodonium (DPI), significantly prevented ethanol-induced increases in NOX enzyme activity, ROS generation and oxidative DNA damage in ethanol-exposed embryos. DPI treatment also resulted in a reduction in caspase-3 activation, decreased caspase-3 activity and diminished prevalence of apoptosis in ethanol-exposed embryos. These results support the hypothesis that NOX is a critical source of ROS in ethanol-exposed embryos and that it plays an important role in ethanol-induced oxidative stress and pathogenesis

Magnetic Resonance-Based Imaging in Animal Models of Fetal Alcohol Spectrum Disorder

Magnetic resonance imaging (MRI) techniques, such as magnetic resonance microscopy (MRM), diffusion tensor imaging (DTI), and magnetic resonance spectroscopy (MRS), have recently been applied to the study of both normal and abnormal structure and neurochemistry in small animals. Herein, findings from studies in which these methods have been used for the examination of animal models of Fetal Alcohol Spectrum Disorder (FASD) are discussed. Emphasis is placed on results of imaging studies in fetal and postnatal mice that have highlighted the developmental stage dependency of prenatal ethanol exposure-induced CNS defects. Consideration is also given to the promise of methodological advances to allow in vivo studies of aberrant brain and behavior relationships in model animals and to the translational nature of this work

Induction of the Nrf2-driven antioxidant response by tert-butylhydroquinone prevents ethanol-induced apoptosis in cranial neural crest cells

Previous studies have shown that ethanol exposure causes apoptosis in cranial neural crest cells (NCCs), an ethanol-sensitive cell population implicated in Fetal Alcohol Spectrum Disorders (FASD). Additionally, induction of endogenous antioxidants through activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) has been shown to prevent oxidative stress and apoptosis in ethanol-exposed mouse embryos. The objective of this study was to test whether tert-butylhydroquinone (tBHQ), an Nrf2 inducer, can protect NCCs against ethanol-induced apoptosis. Ethanol exposure was shown to cause a moderate increase in the protein expression of Nrf2 and its downstream antioxidants in the NCCs. Treatment of NCCs with tBHQ alone significantly increased the protein expression of Nrf2 and its downstream antioxidants and also significantly increased the activities of the antioxidant enzymes. In NCCs exposed to ethanol, the tBHQ-mediated antioxidant response prevented oxidative stress and apoptosis. These results clearly demonstrate that activation of Nrf2 signaling confers protection against ethanol-induced apoptosis in NCCs

Hypothalamic-pituitary-adrenal axis and behavioral dysfunction following early binge-like prenatal alcohol exposure in mice

The range of defects that fall within fetal alcohol spectrum disorder (FASD) includes persistent behavioral problems, with anxiety and depression being two of the more commonly reported issues. Previous studies of rodent FASD models suggest that interference with hypothalamic-pituitary-adrenal (HPA) axis structure and/or function may be the basis for some of the prenatal alcohol (ethanol) exposure (PAE)-induced behavioral abnormalities. Included among the previous investigations are those illustrating that maternal alcohol treatment limited to very early stages of pregnancy (i.e., gestational day [GD]7 in mice; equivalent to the third week post-fertilization in humans) can cause structural abnormalities in areas such as the hypothalamus, pituitary gland, and other forebrain regions integral to controlling stress and behavioral responses. The current investigation was designed to further examine the sequelae of prenatal alcohol insult at this early time period, with particular attention to HPA axis-associated functional changes in adult mice. The results of this study reveal that GD7 PAE in mice causes HPA axis dysfunction, with males and females showing elevated corticosterone (CORT) and adrenocorticotropic hormone (ACTH) levels, respectively, following a 15-min restraint stress exposure. Males also showed elevated CORT levels following an acute alcohol injection of 2.0 g/kg, while females displayed blunted ACTH levels. Furthermore, analysis showed that anxiety-like behavior was decreased after GD7 PAE in female mice, but was increased in male mice. Collectively, the results of this study show that early gestational alcohol exposure in mice alters long-term HPA axis activity and behavior in a sexually dimorphic manner

Dysmorphogenic Effects of First Trimester-Equivalent Ethanol Exposure in Mice: A Magnetic Resonance Microscopy-Based Study

The first trimester of human development and the equivalent developmental period in animal models is a time when teratogenic ethanol exposure induces the major structural birth defects that fall within Fetal Alcohol Spectrum Disorder (FASD). Previous FASD research employing an acute high dose maternal intraperitoneal ethanol treatment paradigm has identified sensitive periods for a number of these defects. Extending this work, this investigation utilized high resolution magnetic resonance imaging (MRM)-based analyses to examine the dysmorphology resulting from maternal dietary ethanol intake occurring during selected first trimester-equivalent time periods