Kathleen K. Sulik - US grants

The proposed studies are concerned with the use of an animal model we have developed for the study of malformations induced by isotretinoin (13-cis-retinoic acid, Accutane); malformations which are comparable to those induced by this drug in humans. Since its introduction in September 1982, isotretinoin has become a widely used drug which is very effective for the treatment of severe cystic acne. As is the case with other retinoids (vitamin A), isotretinoin was known to be teratogenic in experimental animals. Despite warnings against use during pregnancy, there have been between 200 and 300 known exposures. In the prenancies which have been permitted to go to term, high incidences of craniofacial, heart and thymic abnormalities have occurred. Malformations in our animal model are very similar to those seen in human newborns and it has been possible to show that interference with neural crest development is a major feature in their pathogenesis. In addition, we have evidence that a metabolite, 4-oxo-isotretinoin (which reaches concentrations 3-5 times that of the parent compound in the serum of patients undergoing treatment) is also teratogenic. In this application, we propose to complete descriptive morphological studies using scanning EM, light microscopy and transmission EM. An immunohistological procedure will be utilized to assist in the identification of neural crest cells. Pharmacokinetic studies of isotretinoin and 4-oxo-isotretinoin which have been initiated will be completed. Regimens for maintenance in mice of blood levels of both compounds which are similar to human therapeutic levels are being developed. Following maternal administration, the levels of these compounds in the embryos will also be determined. Further studies concerning the mechanisms by which isotretinoin alters the development of neural crest and other cell populations in this model are also proposed. These include the use of 3H-isotretinoin and frozen section autoradiography to determine whether the compound is concentrated in crest cells at certain stages of their development as suggested by other studies using 14C-tretinoin (14C-all-transretinoic acid). Alterations in cell proliferation will be studied using 3H-thymidine autoradiography. Alterations in fibronectin and cell adhesion molecule will also be examined.

A major component of the pathogenesis of alcohol-induced major malformations, as observed in animal models of alcohol related birth defects, is associated with cell death. We propose to examine the selective sensitivity of various cell populations in early mouse embryos to ethanol-induced cell death. This will involve in vivo as well as in vitro (whole embryo culture) studies of control and ethanol-exposed gestational day 7-9 1/2 mouse embryos. Morphological analyses utilizing a vital dye, Nile blue sulphate which comprehensively reveals patterns of cell death, as well as light, scanning and transmission electron microscopy will be performed. The patterns of ethanol-induced cell death will be compared to those of normal programmed cell death as well as to sites of intense mitotic activity, cell migration, etc. in control embryos and will be related to subsequent patterns of malformation. Dose-response and time- response studies utilizing the whole embryo culture system will be conducted using excessive cell death and other morphological abnormalities as the developmental end point. Electron microscopic analysis of these embryos to determine whether ethanol-induced cell death is apoptotic or necrotic will be carried out. The objective of this investigation is to generate data which will provide us with a better understanding of the mechanism of ethanol-induced cellular damage as well as the basis for the vulnerability of selected cell populations to this teratogenic agent.

We have been successful in identifying, in an acute-exposure mouse model, specific cell populations in early embryos (gestational days 7 - 9 1/2) that are selectively vulnerable to the cytotoxic effects of ethanol. The genesis of subsequent major malformations (including anencephaly, a number of facial malformations including cleft lip, limb reduction defects, and urinary tract abnormalities) can be explained based on the loss of the identified selectively vulnerable progenitor cell populations. We plan to extend our studies with particular attention to the developing brain. To evaluate the induced perturbations, we propose to: 1) comprehensively map vulnerable cell populations in the brains of later stage embryos; 2) compare, using fluorescence recovery after photobleaching (FRAP), membrane fluidity in vulnerable versus non-vulnerable neuronal cell populations within and between sensitive and resistant mouse strains in the presence or absence of ethanol; 3) attempt, using antioxidants, to ameliorate lipid peroxidation and cell death induced by ethanol in whole embryo culture and in vivo; and 4) determine the effect of excessive antioxidant (superoxide dismutase) activity in transgenic mice relative to ethanol-induced teratogenicity. The proposed studies will help us to understand (with special attention to mechanisms of action) the teratogenic effects of this drug of abuse. They will identify vulnerable neural populations, providing important information relative to expected outcomes in children born to women exposed to ethanol during the first trimester of pregnancy.

We have been successful in identifying, in an acute-exposure mouse model, specific cell populations in early embryos (gestational days 7 - 9 1/2) that are selectively vulnerable to the cytotoxic effects of ethanol. The genesis of subsequent major malformations (including anencephaly, a number of facial malformations including cleft lip, limb reduction defects, and urinary tract abnormalities) can be explained based on the loss of the identified selectively vulnerable progenitor cell populations. We plan to extend our studies with particular attention to the developing brain. To evaluate the induced perturbations, we propose to: 1) comprehensively map vulnerable cell populations in the brains of later stage embryos; 2) compare, using fluorescence recovery after photobleaching (FRAP), membrane fluidity in vulnerable versus non-vulnerable neuronal cell populations within and between sensitive and resistant mouse strains in the presence or absence of ethanol; 3) attempt, using antioxidants, to ameliorate lipid peroxidation and cell death induced by ethanol in whole embryo culture and in vivo; and 4) determine the effect of excessive antioxidant (superoxide dismutase) activity in transgenic mice relative to ethanol-induced teratogenicity. The proposed studies will help us to understand (with special attention to mechanisms of action) the teratogenic effects of this drug of abuse. They will identify vulnerable neural populations, providing important information relative to expected outcomes in children born to women exposed to ethanol during the first trimester of pregnancy.

DESCRIPTION (provided by applicant): This research project is designed to increase our understanding of the mechanisms and molecular pathology underlying alcohol-induced dysmorphogenesis. It follows up on our previous identification of selected regions of the brain, otic, and optic primordial as targets of ethanol-induced apoptosis and subsequent birth defects, results of our ongoing microarray analyses, and significant contributions to this field by others. Focusing on Developmental stages in mice that correspond to weeks 3-6 of human embryogenesis and employing in situ hybridization, we will test the hypothesis that within hours following exposure to ethanol, abnormal expression of patterning genes occurs in the embryonic brain, eye and inner ear; changes that presage subsequent dysmorphogenesis. To elucidate pathogenic sequences, the time by which altered patterning is detectable will be compared to that for which apoptosis can initially be identified. Analyses of temporally and regionally-specific alterations in mouse embryogenesis, will be facilitated by utilization of an acute ethanol exposure paradigm, whole embryo culture, and laser confocal imaging. Recognizing that ethanol exposure can interfere with retinoid metabolism and that retinoic acid (RA) regulates gene expression; we will also examine the hypothesis that diminishing RA-dependent gene signaling underlies ethanol's teratogenicity. For this work, we will compare gene expression patterns and patterns of apoptosis in the developing brain, eye and innerear of retinoid-deficient (BMS493-treated)and ethanol-exposed mouse embryos and test RA's ameliorative potential. Additionally, we propose to conduct in vitro and in vivo investigations to test the hypothesis that altered genetic (esp. sonic hedgehog) signaling stemming from ethanol-induced RA deficiency will exacerbate diminished signaling subsequent to cholesterol reduction. These experiments will utilize both pharmacologically (AY9944) and genetically (7 dehydrocholestrol reductase gene modification) - induced cholesterol deficiency, both of which yield malformations consistent with those caused by ethanol. This work is expected to provide important new data relative to factors that may influence sensitivity to ethanol-induced teratogenesis in human populations. It will also establish a foundation for similar analyses to be utilized in following up on other developmental stages and vulnerable tissues.

DESCRIPTION (provided by applicant): This application requests funding for partial support of the 2005-2009 Fetal Alcohol Syndrome Study Group (FASSG) meetings. These are held annually as a satellite conference preceding the Research Society on Alcoholism (RSA) meeting. The study group meeting represents a national annual gathering of basic, social, and clinical scientists whose work focuses on the topic of Fetal Alcohol Spectrum Disorders (FASD). The meeting provides a forum for presentation of topics of priority interest to the FASD research community, provides opportunities for discussion of collaborative research, and fosters interactions between established investigators and new investigators, including students. Although the meetings, to date, have been stimulating and well attended, a lack of adequate external financial support has limited attendees to those with adequate grant support to cover their meeting costs, and speakers to "local talent" willing to provide their time at no cost. Clearly, future meetings of the FASSG would greatly benefit with the ability of this group to provide funding to defray meeting costs (especially for travel and accomodations) for promising students and other new investigators who may be unable to attend, otherwise. Additionally, the membership could benefit from presentations by distinguished invited speakers who have interests that overlap with those of the FASSG, but that may be somewhat peripheral to the focus of the group; individuals who would not normally attend the meeting. Publication of a meeting synopsis, as proposed herein, will document the annual proceedings and afford availabilty of the meeting content to the scientific community. This application requests funding for travel, registration fees and partial living expenses for 5 new investigators (including students), and all meeting related expenses plus a modest honorarium for 3 invited speakers each year for a period of five (5) years.

[unreadable] DESCRIPTION (provided by applicant): Fetal Alcohol Spectrum Disorders (FASD), significant components of which are Central Nervous System (CMS) and craniofacial abnormalities, are a major public health problem. While eliminating FASD is the ultimate goal for both clinical and basic FASD research, we recognize that in the near future, adverse effects from prenatal ethanol exposure will persist. To better diagnose and treat affected individuals, a more complete understanding of the full spectrum of the ethanol-induced abnormalities is needed. The proposed investigations are designed to integrate with those of other consortium members in meeting this need. For this work, both high resolution Magnetic Resonance Imaging (MRI), which can provide 29 micron (or less) isotropic scans and subsequent accurate 3-D reconstructions and segmental analyses, and Diffusion Tensor Imaging (DTI), which allows CMS fiber tract analyses, will be applied to the study of an FASD mouse model. Previous research utilizing this model has established critical exposure times that yield facial and CNS abnormalities that are consistent with full-blown Fetal Alcohol Syndrome, as well as other components of FASD. The proposed studies will employ this model and both acute and chronic ethanol treatment paradigms to test the overall hypothesis that in mice, ethanol induces structural abnormalities of the brain and face that are consistent with and informative for those in human FASD. To this end, utilizing MRI and DTI as high throughput screening platforms, we propose to address the following specific aims : 1) to provide comprehensive documentation and discovery of the ethanol-induced CNS dysmorphology that results from prenatal ethanol exposure at embryonic and early fetal stages of development; 2) to define the facial dysmorphology that results from prenatal ethanol exposure during embryonic and/or early fetal stages and to relate their character and severity to accompanying abnormalities of the brain; and 3) to identify regions other than the brain or face that may serve as diagnostic indicators of prenatal ethanol exposure. The results of the proposed studies will be compared to those of corresponding investigations by other consortium members. It is expected that the structural abnormalities of the brain and face that are induced by ethanol in mice will reflect the pattern of defects observed in children with FASD, will inform human diagnostic tests, and will provide new information that will be helpful in reducing the incidence of FASD. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The purpose of this project is to rapidly advance DTI technology and its application to discovery and documentation of alcohol's deleterious effects on the pre-and postnatal rodent brain. A major focus is on achieving faster imaging times than are currently possible. This will allow the extension of DTI analyses from ex vivo to in vivo specimens. To complement the advances in imaging, new methodologies including tools and software for data management and analyses are also being developed and applied to our analyses of the fetal and postnatal mouse brain. These techniques are being utilized to extend this laboratory's current investigation of fetal alcohol spectrum disorders (FASD) in a mouse model and to address the hypothesis that maternal alcohol (ethanol) administration limited to very early stages of prenatal development (corresponding to week 3 of human development) results in permanent central nervous system damage. To accomplish this, 3 specific aims are being addressed. Aim #1 is to develop new DTI methodologies that will allow faster imaging times than are currently feasible. This will be accomplished by the use of active staining techniques, 3D radial keyhole imaging, and a novel cryogenic radiofrequency coil applied to DTI. Aim #2 is to rapidly develop a software framework for a new, automated mouse brain data analysis based on an unbiased atlas generation. This analysis method will be used for voxel-wise analysis of DTI properties and connectivity patterns, as well as DTI fiber tractography-specific analysis. Aim #3 is to apply the methodologies advanced in the previous Aims to the examination of alcohol's effect on the pre- and postnatal mouse brain. For this, the brains of control mice and those exposed to alcohol on their 7th day of gestation, will be examined on their 17th prenatal day (GD 17), or their 45th and/or 90th postnatal day (PND 45, PND 90) for fiber tract morphology (including microstructural integrity) as well as for alterations in regional diffusion parameters such as measures of anisotropy (eg. fractional anisotropy [unreadable]FA and mean diffusivity [unreadable]MD). In addition to ex vivo imaging of the GD 17 and PND 45 and 90 brains, a subset of the PND 45 animals will be imaged in vivo. Since prenatal alcohol exposure is reportedly related to subsequent alcohol dependence in humans and also increases alcohol self administration and preference in animals, prior to imaging, the postnatal animals will be examined in a number of behavioral batteries designed to assess their reactions to acute alcohol challenge. It is expected that in addition to rapidly providing tools and approaches that can be directly applied to other rodent models and research questions, this Challenge grant project will provide important new information regarding the biological (both structural and functional) consequences of prenatal alcohol exposure.

3-D; 3-Dimensional; Absolute ethanol; Acetylcysteine; Acetylin; Active Follow-up; Active Oxygen; Acute; Affect; Airbron; Albumins; Alcohol, Ethyl; Allen &Hanburys Brand of Acetylcysteine; Ammon Horn; Antioxidants; Apoptosis; Apoptosis Pathway; Approaches to prevention; Attention; Awareness; Awarenesses; Body Tissues; Brain; Brain Pathology; Brain Stem; Brain region; Brainstem; Bristol-Myers Squibb Brand of Acetylcysteine; Bristol-Myers Squibb Brand of Acetylcysteine Sodium Salt; Broncholysin; Brunac; Cell Communication and Signaling; Cell Death, Programmed; Cell Nucleus; Cell Signaling; Cell/Tissue, Immunohistochemistry; Cells; Central Nervous System; Cerebellum; Chimp; Chimpanzee; Chronic; Clinical Research; Clinical Study; Connector Neuron; Cornu Ammonis; Critical Period; Critical Period (Psychology); Cytokine Signal Transduction; Cytokine Signaling; Data; Defect; Development; Diagnostic; Dietary intake; Diffusion MRI; Diffusion Magnetic Resonance Imaging; Diffusion Weighted MRI; Disease model; 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In Vitro; Incidence; Inpharzam Brand of Acetylcysteine; Intercalary Neuron; Intercalated Neurons; Interneurons; Internuncial Cell; Internuncial Neuron; Intervention; Intervention Strategies; Intracellular Communication and Signaling; Investigation; L-Alpha-acetamido-beta-mercaptopropionic Acid; Label; Lead; MR Imaging; MR Tomography; MRI; Magnetic Resonance Imaging; Magnetic Resonance Imaging Scan; Mammals, Mice; Man (Taxonomy); Man, Modern; Medial; Mediating; Medical Imaging, Magnetic Resonance / Nuclear Magnetic Resonance; Mercapturic Acid; Method LOINC Axis 6; Methodology; Methods; Methods and Techniques; Methods, Other; Methylcarbinol; Mice; Mission; Molecular; Morbidity; Morbidity - disease rate; Muco Sanigen; Mucocedyl; Mucolator; Mucolyticum; Mucomyst; Mucosolvin; Mucret; Murine; Mus; N-Acetyl Cysteine; N-Acetyl-L-cysteine; N-Acetylcysteine; N-acetyl-3-mercaptoalanine; NAC; NAC Zambon; NF-kB; NF-kappa B; NF-kappaB; NFKB; NMR Imaging; NMR Tomography; Neo-Fluimucil; Nervous System, Brain; Nervous System, CNS; Neuraxis; Nuclear Factor kappa B; Nuclear Magnetic Resonance Imaging; Nuclear Transcription Factor NF-kB; Nucleus; Optipect Hustengetr??nk; Outcome; Oxidative Stress; Oxygen Radicals; Pan; Pan Genus; Pan Species; Partner in relationship; Parvolex; Pathogenesis; Pathology; Pathway interactions; Pattern; Pb element; Peptides; Play; Population; Pregnancy; Pregnancy Trimester, First; Prenatal Alcohol Exposure; Prenatal ETOH Exposure; Prenatal Ethanol Exposure; Prevention approach; Prevention strategy; Preventive strategy; Pro-Oxidants; Produpharm Lappe Brand of Acetylcysteine; Prosencephalon; Purpose; ROC Analysis; Range; Reactive Oxygen Species; Region, Septal; Reporter; Research; Resolution; Respaire; Rhombencephalon; Roberts Brand of Acetylcysteine; Role; Route; Screening procedure; Septal Region; Severities; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Source; Staging; Structure; Supplementation; Technics, Imaging; Techniques; Testing; Thiemann Brand of Acetylcysteine; Time; Time Study; Tissues; Tixair; Transcription Factor NF-kB; Trimester, First; UPSA Brand of Acetylcysteine; Urination; Visualization; Week; Woman; Work; Zambon Brand of Acetylcysteine; Zeugmatography; Zyma Brand of Acetylcysteine; alcohol exposed; alcohol exposure; alcohol-exposed pregnancy; alcoholic embryopathy; anti-oxidant; base; biological signal transduction; calbindin 2; calretinin; craniofacial; craniofacies; critical developmental period; day; dietary antioxidant; diffusion tensor imaging; disease prevention; disorder model; disorder prevention; dosage; embryofetal alcohol syndrome; embryofetal alcohol syndrome (EFAS); embryopathia alcoholica; ethanol exposed; ethanol exposure; experiment; experimental research; experimental study; exposed to alcohol; exposed to alcohol prenatally; exposure to alcohol; fetal; fetal alcohol syndrome (FAS); follow-up; gestation ETOH exposure; gestation alcohol exposure; gestation ethanol exposure; heavy metal Pb; heavy metal lead; high throughput screening; hindbrain; hippocampal; image evaluation; imaging; in situ Hybridization Staining Method; in vivo; interventional strategy; intraperitoneal; kappa B Enhancer Binding Protein; mate; micturition; mouse model; nuclear factor kappa beta; pathway; pregnancy ETOH exposure; pregnancy alcohol exposure; pregnancy ethanol exposure; prenatally alcohol exposed; prenatally exposed to alcohol; progenitor; reconstruction; research study; response; screening; screenings; social role; uRNA; voiding

DESCRIPTION (provided by applicant): This application addresses the broad Challenge Area (15): Translational Science and specific Challenge Topic, 15-AA-107: Refinements of Procedures for Diffusion Tensor Imaging (DTI) in Rodent Models of Alcohol Dependence. As set forth for this Challenge, the proposed effort is designed to rapidly advance DTI technology and its application to discovery and documentation of alcohol's deleterious effects on the pre-and postnatal rodent brain. A major focus will be on achieving faster imaging times than are currently possible. This will allow the extension of DTI analyses from ex vivo to in vivo specimens. To complement the advances in imaging, new methodologies including tools and software for data management and analyses will also be developed and applied to our analyses of the fetal and postnatal mouse brain. These techniques will be utilized to extend this laboratory's current investigation of fetal alcohol spectrum disorders (FASD) in a mouse model and to address the hypothesis that maternal alcohol (ethanol) administration limited to very early stages of prenatal development (corresponding to week 3 of human development) results in permanent central nervous system damage. Preliminary work from our laboratory utilizing DTI has shown significant alterations in fiber tract morphology in the mouse fetus following acute alcohol exposure. The proposed effort will allow faster imaging times than previously possible and will facilitate extension of our imaging analyses into the postnatal period. To this end, the following specific Aims will be addressed: Aim # 1 is to develop new DTI methodologies that will allow faster imaging times than are currently feasible. This will be accomplished by the use of active staining techniques, 3D radial keyhole imaging, and a novel cryogenic radiofrequency coil applied to DTI. Aim #2 is to rapidly develop a software framework for a new, automated mouse brain data analysis based on an unbiased atlas generation. This analysis method will be used for voxel-wise analysis of DTI properties and connectivity patterns, as well as DTI fiber tractography-specific analysis. Aim #3 is to apply the methodologies advanced in the previous Aims to the examination of alcohol's effect on the pre- and postnatal mouse brain. For this, the brains of control mice and those exposed to alcohol on their 7th day of gestation, will be examined on their 17th prenatal day (GD 17), or their 45th or 90th postnatal day (PND 45, PND 90) for fiber tract morphology (including microstructural integrity). In addition to ex vivo imaging of the GD 17 and PND 45 and 90 brains, a subset of the PND 45 animals will be imaged in vivo. Since prenatal alcohol exposure is reportedly related to subsequent alcohol dependence in humans and also increases alcohol self administration and preference in animals, prior to imaging, the postnatal animals will be examined in a number of behavioral batteries designed to assess their reactions to acute alcohol challenge. Promise for the successful completion of these Aims is provided by the expertise of the investigators in their respective fields (G.A Johnson in imaging technology development;M.A. Styner in image analysis software development;and K.K. Sulik in FASD research) and their previously productive collaborative efforts. It is expected that in addition to rapidly providing tools and approaches that can be directly applied to other rodent models and research questions, the proposed Challenge effort will provide important new information regarding the biological (both structural and functional) consequences of prenatal alcohol exposure. PUBLIC HEALTH RELEVANCE: The proposed work addresses the broad Challenge Area (15): Translational Science and specific Challenge Topic, 15-AA-107: Refinements of Procedures for Diffusion Tensor Imaging (DTI) in Rodent Models of Alcohol Dependence. As set forth for this Challenge, the proposed effort is designed to rapidly advance DTI technology and its application to discovery and documentation of alcohol's deleterious effects on the pre-and postnatal rodent brain. A major focus will be on achieving faster imaging times than are currently possible. This will allow the extension of DTI analyses from fixed to live specimens. To complement the advances in imaging, new methodologies including tools and software for data management and analyses will also be developed and applied to our study of alcohol-induced brain abnormalities.

DESCRIPTION (provided by applicant): The objective of this proposal is to create learning resources that are designed to meet curriculum standards while addressing a vital societal need;promotion of Fetal Alcohol Spectrum Disorders (FASD) prevention. To this end, Science Learning Resources, Inc. (SLR, www.science-learning.com) and its collaborators will develop and distribute a classroom-ready, innovative high school health and science curriculum. An interactive DVD platform will be utilized to provide a menu of engaging activities for students as well as readily-employed resources for teachers. The student-centered components will include: 1) an age- appropriate short film that is designed to introduce the reality and tragedy of FASD and to stimulate discussion of prevention approaches;2) an exploration featuring micrographic images and computer animations that highlight the remarkable similarities between normal human development and that of an FASD animal model (the Medaka fish);3) a virtual experiment illustrating the effects of alcohol on brain development in the fish model system;4) a post- laboratory multimedia summary relating these experimental findings to the insult on human brain structure and function that prenatal alcohol exposure causes;and 5) a selection of proposed student-driven prevention activities. The resources for teachers will include: 1) instructive FASD background materials;2) sample lesson plans;3) student assessment instruments;and 4) alignment with National Education Standards. Each of the components of the curriculum, as well as the DVD itself, will be formally evaluated and the results will be used to guide final production. National distribution of this product will be facilitated via collaborative arrangements with well-respected science and health education companies (Realityworks, Inc. www.realityworks.com and Carolina Biological Supply Company www.carolina.com). This curriculum will fill a critical educational void and promises to aid in reducing common, yet preventable, birth defects. PUBLIC HEALTH RELEVANCE: Maternal alcohol consumption during pregnancy can result in a spectrum of birth defects estimated to affect 40,000 babies each year in our country. Each of these birth defects is entirely preventable if a woman avoids drinking during pregnancy. Educational resources focused on prevention of alcohol-induced birth defects are limited. The proposed science-based health curriculum for high school students will provide resources for teachers and students to heighten awareness about effects of alcohol on development of the brain and behavior, and thereby promote prevention of Fetal Alcohol Spectrum Disorders.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Fetal Alcohol Spectrum Disorders (FASD), significant components of which are Central Nervous System (CNS) and craniofacial abnormalities, are a major public health problem. While eliminating FASD is the ultimate goal for both clinical and basic FASD research, we recognize that in the near future, adverse effects from prenatal ethanol exposure will persist. To better diagnose and treat affected individuals, a more complete understanding of the full spectrum of the ethanol-induced abnormalities is needed. The proposed investigations are designed to contribute significantly toward meeting this need. For the proposed work, both high resolution Magnetic Resonance Imaging (MRI), which can provide 29 micron (or less) isotropic scans and subsequent accurate 3-D reconstructions and segmental analyses, and Diffusion Tensor Imaging (DTI), which allows CNS fiber tract analyses, will be applied to the study of an FASD mouse model. Previous research utilizing this model has established critical exposure times that yield facial and CNS abnormalities that are consistent with full-blown Fetal Alcohol Syndrome, as well as other components of FASD. The proposed studies will employ this model and both acute and chronic ethanol treatment paradigms to test the overall hypothesis that in mice, ethanol induces structural abnormalities of the brain and face that are consistent with and informative for those in human FASD. To this end, utilizing MRI and DTI as high throughput screening platforms, we propose to address the following specific aims : 1) to provide comprehensive documentation and discovery of the ethanol-induced CNS dysmorphology that results from prenatal ethanol exposure at embryonic and early fetal stages of development;2) to define the facial dysmorphology that results from prenatal ethanol exposure during embryonic and/or early fetal stages and to relate their character and severity to accompanying abnormaities of the brain;and 3) to identify regions other than the brain or face that may serve as diagnostic indicators of prenatal ethanol exposure. It is expected that the structural abnormalities of the brain and face that are induced by ethanol in mice will reflect the pattern of defects observed in children with FASD, will inform human diagnostic tests, and will provide new information that will be helpful in reducing the incidence of FASD.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Autopsies of children who have died of sudden infant death syndrome (SIDS) have identified deficits of the carotid body and the carotid arteries as one of the potential causative factors in their deaths. Likewise, epidemiological studies have identified prenatal ethanol exposure as another component involved in a certain subset of cases of SIDS. Finally, experiments from our laboratory have demonstrated ethanol-induced apoptotic cell death in regions of the developing mouse embryo that are crucial to the proper development of both the carotid body and carotid arteries. The experiments proposed here will test the hypotheses that early gestional ethanol exposure will result in hypoplasia of the carotid body, deficits in the normal respiratory response to hypoxia and abnormal vasculature of the head and neck, particularly the carotid arteries and their proximal branches.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Holoprosencephaly-spectrum defects can be induced by embryonic exposure to ethanol or Hedgehog signaling antagonists. Our first aim seeks to define the dose- and stage-dependent effects of Hedgehog signaling antagonist exposure through detailed characterization of CNS and craniofacial phenotype in affected fetal specimens. Our second aim is to apply MR microscopy in the study of facial morphometrics. Face shape is known to be useful in predicting both underlying brain morphology as well as predicting predisposition for congenital malformations. We will use MR acquired 3D facial images to identify morphological features of the face that are associated with specific brain malformations and to determine whether facial clefting can be predicted by subtle (non-clefting) morphometric changes. We intend for the first aim described above to be inter-relatable with work already performed in the Sulik laboratory defining the craniofacial and CNS phenotype of fetal mice exposed to ethanol embryonically. Thus, we will use the existing MRM protocol. We anticipate that the second research aim, utilizing MR for facial morphometric analysis, will be best served by modifying this protocol used for Aim1 by the addition of diffusion weighting to optimize the image quality of the facial surface.

DESCRIPTION (provided by applicant): The objective of the proposed basic research is to make clinically-relevant discoveries regarding prenatal alcohol (ethanol) exposure-induced pathology involving the brain and face. This proposal builds naturally on our CIFASD-supported basic research to date and continues to address the need for a more complete understanding of the spectrum and exposure stage-dependency of abnormalities caused by maternal alcohol use. Utilizing a well-established FASD mouse model, along with innovative technologies and approaches, and addressing 3 Specific Aims, we propose to test the overall hypothesis that alcohol induces structural abnormalities of the brain and face in mice that are consistent with and informative for those in human FASD. The Aim 1 studies compliment and extend the clinical research proposed by Drs. Foroud and Hammond. They employ Magnetic Resonance Imaging (MRI) and dense surface modeling (DSM) for experiments that are designed to identify exposure stage-dependent correlative abnormalities of the brain and face. The Aim 2 studies compliment and extend the Sowell group's neuroimaging-based clinical studies while following up on preliminary findings of cerebro-cortical thickness alterations in our mouse model. For this, MRI-based assessments of regional brain volumes and cerebro-cortical thickness changes, along with DTI-based investigations of fiber tract and structural connectivity alterations in adult animals are proposed. The Aim 3 studies are directed toward further defining the histopathology and genesis of early prenatal alcohol exposure- induced regional brain dysmorphology. They will employ routine histological methods, as well as immunohistochemistry, and stereology. Specimens selected for detailed histological analyses will include those postnatal brains that had previously been imaged and analyzed for Aim 2. Additional Aim 3 studies will focus on prenatal stages and will address the novel concept that early alcohol insult yields changes in Cajal-Retzius cell populations; changes that underlie subsequent cerebro-cortical lamination defects. The proposed work is consistent with the overall purpose/goals of the CIFASD in that it will facilitate diagnosis of the full range of birth defects associated with prenatal alcohol exposure, and it will aid in elucidating biological mechanisms that contribute to alcohol teratogenesis. The results of the proposed studies promise to fill a significant FASD research void, inform human clinical research, and continue to highlight the first trimester as a critical period for alcohol-induced defects of the face and brain.

The objective of the proposed research Is to make novel discoveries regarding the functional and structural central nervous system (CNS) pathology that results from early prenatal alcohol exposure (PAE). Extending our previous research illustrating exposure stage-dependent patterns of brain dysmorphology in fetal mice, we propose to examine 3 distinct, but interrelated forebrain circuits that are expected to play key roles in the behavioral or functional manifestations of alcohol neuroteratogenesis in the postnatal animal. The proposed studies will utilize a well-established FASD mouse model, with acute, as well as dietary maternal alcohol administration at times equivalent to 3-6 weeks of human gestation. Innovative technologies and approaches will be employed to address 3 Aims. Aim 1 is designed to test the hypothesis that early PAE induces hippocampal pathology (esp. altered hippocampal volume and related fiber tract Integrity and circuitry) and deficiencies in the performance of hippocampus-mediated learning and memory tasks in adult mice. For this work, high-resolution ex vivo diffusion tensor imaging will be utilized to define structural deficiencies associated with PAE, while a behavioral test battery will be used to probe hippocampal-dependent learning and memory. Aim 2 is designed to test the hypothesis that early PAE causes defects in medial hypothalamic and pituitary circuitry as well as altered HPA axis regulation in adulthood. For this, immunohlstochemistry and in situ hybridization will be employed to characterize hypothalamic and pituitary pathology in fetal mice. HPA axis function will be examined in adults by measurement of adrenocorticotropic hormone and corticosterone plasma levels both prior and subsequent to an acute alcohol challenge or an acute restraint stress. Aim 3 is designed to test the hypothesis that early PAE results in deficient inhibitory GABAergic circuitry in corticostriatal circuits underlying reward perception and alcohol intake. For this, intracranial self-stimulation (ICSS), intermittent access drinking, and design-based stereology will be employed. Overall, the proposed work is in keeping with the binge alcohol pathology/neurocircuitry-directed central goal of the Alcohol Research Center (ARC) and will profit from ARC support and interaction. The results of the proposed studies promise to fill a significant FASD research void, inform human clinical research, and continue to highlight the first trimester as a critical period for alcohol-induced CNS defects.

The UNC Research Translation/Information Dissemination Component provides support for innovative efforts that disseminate information on alcohol to key groups to improve health. Translation and education on alcohol fetal toxicity and neurobiology in youth as well as health professional education on screening, interventions, and alcohol pathology create an exceptional NIAAA Comprehensive Center Core that expands its impact through partnerships with other educational groups. The lack of attention to alcohol pathology among medical professionals is addressed through medical student and continuing medical education conferences for health professionals. Aim 1 is focused on medical professionals and healthcare professionals-in-training on clinical issues of alcohol abuse, dependence and medical pathology. Specifically, we will continue the comprehensive substance abuse training in our medical school curriculum. We will also continue to educate physicians and other health providers via annual continuing medical education conferences. To improve public information. Aim 2 has a focus on youth. Within the ARC, we will train students, fellows and others in effective communication and engagement skills. In partnership with the North Carolina Museum of Life and Science, the North Carolina Association for Biomedical Research, and the UNC Morehead Planetarium and Science Center education on basic medical science as well as alcohol and health targets large groups of youth that visit these venues. Specific outreach activities include topical workshops and lab tours for teachers, interactive exhibits and booths for youth, and demonstrations/presentations for high school classes. The exceptional and informative activities within this Information Dessemination Core fit exactiy the goals of a Comprehensive NIAAA ARC.