Experimental Biology and Medicine 230:389-393 (2005)
© 2005 Society for Experimental Biology and Medicine
SYMPOSIA
Animal Model Systems for the Study of Alcohol Teratology
Timothy A. Cudd1
Department of Veterinary Physiology and Pharmacology and the Michael E. DeBakey Institute, Texas A&M University, College Station, Texas 77843-4466
1To whom requests for reprints should be addressed at Department of Veterinary Physiology and Pharmacology and the Michael E. DeBakey Institute, Texas A&M University, Highway 60, Building VMA, Room 332, College Station, TX 77843-4466. E-mail: tcudd{at}catamu.edu
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Abstract
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The incidence of fetal alcohol syndrome has not been declining even though alcohol has been established as a teratogen and significant efforts have been made to educate women not to abuse alcohol during pregnancy. In addition to further educational efforts, strategies to prevent or mitigate the damages of prenatal alcohol exposure are now under development. Animal models will play a significant role in the effort to develop these strategies. Because prenatal alcohol exposure causes damage by multiple mechanisms, depending on dose, pattern, and timing of exposure, and because no species of animal is the same as the human, the choice of which animal model to use is complicated. To choose the best animal model, it is necessary to consider the specific scientific question that is being addressed and which model system is best able to addressthe question. Animal models that are currently in use include nonhuman primates, rodents (rats, mice, guinea pigs), large animal models (pig and sheep), the chick, and simple animals, including fish, insects, and round worms. Each model system has strengths and weaknesses, depending on the question being addressed. Simple animal models are useful in exploring basic science questions that relate to molecular biology and genetics that cannot be explored in higher-order animals, whereas higher-order animal models are useful in studying complex behaviors and validating basic science findings in an animal that is more like the human. Substantial progress in this field will require the judicious use of multiple scientific approaches that use different animal model systems.
Key Words: fetal alcohol syndrome • alcohol teratology • animal models • ethanol • birth defects • prenatal alcohol exposure
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Introduction
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Alcohol has only recently become widely accepted by the scientific community and the public as a teratogen. Prenatal alcohol exposure is now acknowledged as the leading cause of mental retardation in the Western world (1). Yet even with this knowledge and after extensive efforts have been made to educate women about the dangers of drinking during pregnancy, the incidence of fetal alcohol syndrome (FAS) remains essentially unchanged. Currently, FAS in the United States occurs much less often as a consequence of ignorance and more often as a consequence of compulsive abuse of alcohol during pregnancy. Although convincing women to abstain from alcohol abuse during pregnancy is clearly the best solution, it is not always possible. Therefore, in addition to education, ways to prevent or mitigate the effects of prenatal alcohol exposure must be explored.
To pursue the development of effective preventive or mitigating strategies other than abstinence, the precise mechanism(s) by which alcohol mediates the neurodevelopmental injuries must be determined. Extensive evidence supports the conclusion that alcohol acts by different mechanisms, depending on the dose, pattern of exposure, and timing of exposure relative to fetal development and the target structure in question. The study of FAS in affected children has yielded important insights. However, human studies are severely limited because of ethical constraints. Therefore, it is necessary to use animal models to effectively address these questions. Animal models have already provided major contributions to our understanding of FAS. When the first scientific reports were published that identified alcohol as a teratogen, based on clinical observations coupled with histories of heavy maternal alcohol abuse during pregnancy, there were those who were skeptical. Many thought "after all, people have been drinking alcohol for thousands of years ... how can that be so?" The first animal studies were used to demonstrate unequivocally that alcohol was a teratogen. Later animal studies identified the specific targets of prenatal alcohol exposure, which was important because of the small number of available autopsy findings from FAS children. These findings were also important in directing future studies, both clinical and those conducted in animals. Animal models are now being used extensively to identify the mechanisms of damage and to devise protective and mitigating strategies.
Experiments that use animal models have the advantage that the conditions can be precisely controlled. Human studies depend on unreliable self-reporting, and most heavy drinkers also use tobacco or other drugs that alone or together with alcohol may interfere with fetal development. Other variables that are difficult to control in human studies include nutrition and genetics. In addition, animal studies allow for the collection of a greater array of dependent variables compared with humans. However, it must be appreciated that no species is the same as humans. All nonhuman animal species have differences with respect to mental function and neurodevelopment. In some cases these differences in neurodevelopment are small, whereas in others they are profound. Investigators must be aware of these differences if they are to successfully design an experiment that can effectively use an animal model to investigate FAS.
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The Challenges of Animal Models
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It is important to consider the differences in brain development between humans and a species under consideration for use in modeling alcohol-induced teratology, because brain structures develop at different times during gestation and the rate of brain growth varies at different times during gestation (2). Structures that are rapidly growing or first emerging or developing are far more vulnerable to alcohol than at later times when development has slowed or is complete. Therefore, the following factors are critically important to consider when selecting which species of animal to use: the timing of the development of structures of interest, the time during development to administer alcohol, and the stage of development to perform measurements. For example, the maximum velocity of brain growth occurs at the time of parturition in the human, whereas in rats and mice it occurs postnatally (Fig. 1
). Rats and mice are born even less precocious than are human infants, because brain development occurs proportionately later in these species. Therefore, if one is interested in using rats or mice to study the effects of alcohol on structures that develop during the third trimester of pregnancy in humans, then one would have to dose with alcohol and assess the responses postnatally. On the other hand, in nonhuman primates, sheep, and guinea pigs, brain development occurs proportionately earlier in gestation than that in humans. Nevertheless, careful thought and planning with regard to the relative timing of brain development can make it possible to reasonably extrapolate from animals to humans.
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Figure 1. The brain growth spurts of seven mammalian species expressed as first-order velocity curves of the increase in weight with age. The units of time for each species are as follows: guinea pig, days; rhesus monkey, 4 days; sheep, 5 days; pig, weeks; humans, months; rabbit, 2 days; and rat, days. Rates are expressed as weight gain as a percentage of adult weight for each unit of time. Reprinted from Ref. 2.
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Another challenge in using animal models to study FAS is that no single animal model has been shown to exhibit all the diagnostic criteria of FAS (facial dysmorphologic features, growth retardation, and neurodevelopmental defects). Mice have been used to investigate the facial dysmorphologic features of FAS, but these animals do not survive and maintain these deficits beyond parturition. Other animal models have been useful in the study of neuro-developmental deficits but have not expressed the facial dysmorphologic features, and some of these do not exhibit growth retardation. Consequently, one must carefully consider the focus of study when choosing an animal model to ensure that the species is an appropriate model.
An additional major challenge in using animal models to study FAS is that no species exhibits the same intelligence and behaviors as humans. However, there are animals that exhibit many behaviors analogous to those of humans. Yet with all of the challenges, investigators have found that good correspondence exists between the effects of prenatal alcohol exposure in humans and those in appropriate animal models (3). For example, the hippocampus is known to be affected in humans with FAS, and rats that receive prenatal alcohol exposure also exhibit reductions in hippocampal pyramidal cell numbers (4). Delays in neural migration and abnormalities in midline structures have been found in animal models (5) as occurs in humans with FAS. One of the brain structures most sensitive to prenatal alcohol exposure is the cerebellum. The cerebellum also is extremely sensitive in animal models (6–8). The development of the cerebral cortex (9), myelin, and glia (10–14) is affected to a lesser extent in both humans and other animals.
Correspondence in behavioral deficits of prenatal human exposure to alcohol has also been demonstrated in animal models. Documented behavioral abnormalities in children with FAS include abnormal activity, reactivity, and hyperactivity; attention deficits; lack of inhibition; impaired learning; reduced habituation; feeding difficulties; gait abnormalities; developmentally delayed and impaired motor skills; hearing abnormalities; and poor state regulation (sleep, jitteriness, and arousal abnormalities). These same deficits have been documented in animal studies (3).
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Rodent Models
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One of the most widely used animal models for the study of FAS has been the rat. An advantage of using rats is the tremendous body of literature on a wide range of areas of scientific exploration in rats (e.g., anatomy, physiology, pharmacology, reproduction, and teratology). Rats also are especially well suited for behavioral studies, because so many behavioral measures have already been established in this species. However, an important consideration in using this species is that the highest velocity of brain growth occurs after parturition, in contrast to humans. Therefore, structures that develop in utero in humans during the third trimester, the cerebellum, for example, develop postnatally in rats. Investigators have used rats to study the effects of alcohol on the developing cerebellum where alcohol was administered postnatally during the period of high velocity of cerebellum growth. However, such experiments require the investigator to assume that parturition, the placenta, and the mother have no bearing on the effects of alcohol on the developing brain.
A singular advantage of the rat model is that established behavioral measures currently exist that can be used in both rats and humans. Stanton and Goodlett (15) have established eye-blink conditioning learning measures in both rats and humans, whereas Hamilton et al. (16) have established the use of a virtual Morris water maze in humans. The Morris water maze is a well-established technique to measure spacial learning and memory in rats. The development of the virtual water maze allows for the testing of spacial learning and memory in children who have poor motor function or are unable to swim (this is often the case in children with FAS).
Another rodent model that has been useful in the study of FAS is the mouse. The mouse shares several of the advantages of the rat. There is extensive literature on mice, and mice have a short gestation, are relatively inexpensive to acquire and maintain, and have more potential for genetic manipulation compared with the rat. The mouse genome has been sequenced, and there are many transgenic, knock-in, and knock-out strains available. The use of genetically altered mice has already advanced the alcohol field significantly (17). Another advantage is that the mouse model has been successfully used to study alcohol-induced facial dysmorphologic features (18), one of the few species where this has been accomplished. However, the highest velocity of brain growth in the mouse occurs postnatally as in the rat.
An additional rodent model that offers some different advantages is the guinea pig. The guinea pig has a much longer gestation (68 days) compared with rats and mice (21 days). This is a disadvantage from the perspective of efficiency but a potential advantage if studies involve targeting a precise time during development. Also, the guinea pig exhibits high velocity brain growth entirely during the prenatal period. Therefore, it is possible to study the effects of prenatal alcohol exposure on third trimester development in utero in the guinea pig. In such a model system, one does not have to assume that the mother, placenta, and events of parturition have no bearing on what is being studied. Investigators have demonstrated that oral administration of ethanol to mothers during the prenatal period results in reduced fetal brain and body weight (19), increased incidence in hyperactivity of offspring (20, 21), and deficits in the fetal cerebral cortex and other structures such as the hippocampus and cerebellum (22, 23). Taken together, these findings demonstrate that the guinea pig exhibits responses to prenatal alcohol exposure like those found in children and establishes the guinea pig as a useful model of FAS.
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Nonmammalian Models
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Nonmammalian models have also been successfully used to study prenatal ethanol exposure questions. The chick has proven to be a useful model to investigate the development of the face and how prenatal ethanol exposure results in facial dysmorphologic features (24). This model system has the advantages of economy, a short incubation, and no placenta. The placenta and mother are thus eliminated as interactions or confounding factors. Some of these advantages, such as short gestation and lack of placenta, can be limitations of this model system, depending on the question being addressed. Other nonmammalian model systems include the zebra fish (Brachydanio rerio), round worm (Caenorhabditis elegans), and fruit fly (Drosophila melanogaster). These model systems have simple nervous systems and short generation intervals. Moreover, these model systems are relatively inexpensive to use. Another important benefit of simple animal model systems is the ability to address basic questions that involve genetics and development. Concerns exist, however, because of the high alcohol concentrations necessary to create defects in these systems, raising the possibility that the mechanisms of damage in these animals at high alcohol concentrations might be different than the mechanisms that cause damage at lower alcohol concentration in higher-order animals. Findings determined using simple animals in many cases must be further investigated with more human-like species to ensure applicability to the human condition.
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Large Animal Models
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Large animal models have been used successfully to investigate prenatal alcohol exposure and have the shared advantages of having longer gestations and being more human-like. Nonhuman primates are the most intelligent of the animal species, exhibiting many of the same behaviors as humans. High velocity of brain growth in nonhuman primates occurs earlier than that of humans. Therefore, all of high velocity brain growth that occurs prenatally in humans, at the time of prenatal alcohol exposure, occurs prenatally in nonhuman primates. In nonhuman primates, it is possible to surgically manipulate and implant catheters (instrument) in the fetus. However, experiments performed in anesthetized animals may be confounded by anesthetics. Alcohol and almost any anesthetic agent interact, making it necessary that experiments that require instrumentation be conducted in chronically instrumented animals. However, the fetal loss rate following surgery has been high in nonhuman primate studies, and these studies are complicated by the need to restrain the subjects following instrumentation. Restraint stress thus becomes a potentially confounding issue. Further liabilities of using nonhuman primates include high cost, animal bites, and zoonotic diseases.
A few investigators have successfully used the pig to study prenatal alcohol exposure. This species has the distinct advantage that it is the only species, other than human, that has a preference for alcohol. Another advantage of the pig is that its brain growth velocity profile is the most similar to the human brain growth velocity profile, both of which peak at the time of parturition (Fig. 1
). The pig is highly intelligent, and although the number of studies that have used behavioral measures in this species is small, the potential exists to use the pig to investigate behavioral effects of prenatal ethanol exposure. Liabilities of this species are that they are relatively expensive to maintain and have large litters. In addition, the fetuses are difficult to instrument successfully on a long-term basis.
Sheep have also been used as a model system for the study of prenatal alcohol exposure. This species has been used extensively during the past 50 years to study basic issues of fetal physiology because of its robustness as a fetus preparation for long-term instrumentation. As a consequence, there is an extensive literature on fetal physiology and on long-term instrumentation techniques in this species, precluding the need for extensive preliminary studies to establish the viability of the model system. The fetal sheep preparation for long-term instrumentation has been used to address issues that include but are not limited to the actions of alcohol on brain metabolism (25); the disposition of alcohol in maternal and fetal compartments (26); and the actions of alcohol on brain activity (27), cerebral blood flow (25, 28), fetal brain neurotransmitter activity (29), the fetal pituitary-adrenal axis (30), and the pituitary-thyroid axis (31). As in nonhuman primates, the brain growth velocity profile is shifted earlier than in humans, and as such, all high velocity brain growth that occurs prenatally in humans also occurs prenatally in the sheep. This species, like nonhuman primates, exhibits a relatively long gestation (147 days), making it possible to better address specific temporal vulnerability issues and study a greater variety of patterns of alcohol exposure compared with animals with short gestations. The long gestation also poses a liability. Studies that use this species take more time and are considerably more costly compared with rodent models.
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Summary
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In summary, the study of FAS is complicated by the existence of multiple mechanisms by which alcohol mediates damage, depending on the dose and pattern of exposure and the timing of exposure relative to development. Because of the complexity of the problem and because no animal model system is exactly like the human, it is necessary to make carefully considered choices in which animal models are used to address these issues. When choosing an animal model to address a particular question, the differences between species should be carefully considered and whether or not the question at hand requires an experimental design that closely models human pregnancy and development. The consideration of lower cost as a rationale for choosing a model should only be made once it is certain that all choices under consideration are appropriate from a scientific standpoint; no savings are achieved if the ensuing study fails to be definitive because the model system was inappropriate. Animal models that possess the necessary strengths to address the particular question at hand should be chosen. No single animal model system is applicable to tackle all questions. Substantial progress toward the goal of finding and establishing effective means to prevent or mitigate the damage from prenatal alcohol exposure will require the judicious use of multiple scientific approaches that use different animal model systems.
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Footnotes
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This work was supported by the National Institute of Alcohol Abuse and Alcoholism grant RO1 AA10940.
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References
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Abstract
Introduction
