Supported by National Institutes of Health grants R01-AA015614, R01-AA014973, T32-AA014127 and K12-GM088021. The authors thank Samantha L. Blomquist for technical assistance and Drs. Kevin Caldwell and Donald Partridge for critically evaluating the manuscript and video.

All Animal procedures were approved by the University of New Mexico-Health Sciences Center Institutional Animal Care and Use Committee.
1. Vapor Chamber Assembly
<ol>
	<li>Cut polycarbonate sheets with a circular saw or jigsaw to the dimensions provided in the video for the top, bottom, front, back, sides, and door (Figure 1 and Table 1).</li>
	<li>With a circular saw or jigsaw, cut an opening 8 inches high by 16 inches wide in the middle of the front panel....

The authors have nothing to disclose.

Here, we describe in detail the methods for the construction of vapor inhalation chambers. The materials and tools required to build the chambers are readily available from a number of commercial suppliers and the steps for the construction of the chambers are relatively straightforward. The system that we describe here does not contain any in-line check valves to prevent back-flow and mixing. We were unable to measure any detectable ethanol in the air only chambers suggesting that we do not have any mixing or back flow ...

Drinking during pregnancy can harm the fetus, causing persistent alterations in many organs and systems that significantly decrease the quality of life for the affected individuals and their families. It is estimated that approximately 10-30% of women drink during pregnancy in the U.S., with 1-8% drinking in a binge pattern1,2. The range of effects produced by ethanol exposure during fetal development is collectively known as fetal alcohol spectrum disorders (FASDs). Recent estimates indicate that FASDs are a major public health problem with a prevalence as high as 2-5% in the U.S.3. The more severe manifestation of FASDs is Fetal Alcohol Syndrome (FAS), which is characterized by growth retardation, craniofacial abnormalities, and neurobehavioral deficits, including learning disabilities. The prevalence of FAS has been estimated to be 0.2-0.7% in the U.S.3. The currently available treatments for FASDs are only partially effective and development of more effective treatments is limited by the poor understanding of the cellular and molecular underpinnings of this complex spectrum of disorders.
Data from the National Birth Defects Prevention Study (NBDPS) indicate that pregnant women most commonly drink during the 1st trimester, before pregnancy has been detected, followed by abstinence during later stages of gestation 2. The NBDPS also found that the second most common pattern of ethanol consumption during gestation involves drinking throughout all trimesters of pregnancy2. The reasons for this include lack of awareness about the potentially harmful effects of fetal ethanol exposure (even at low doses), limited access to prenatal care, positive history for neuropsychiatric disorders, and abuse of or dependence on ethanol4. Interestingly, the NBDPS reported that the third most common pattern of consumption involved abstinence during the 1st and 2nd trimesters followed by consumption during the 3rd trimester, when it is often assumed that drinking is safe because organogenesis has been mostly completed. However, the 3rd trimester is a period of high susceptibility to ethanol-induced nervous system damage because this is a period when neuronal circuits undergo profound refinement2. The NBDPS also identified other, less frequent patterns of alcohol consumption that occur during pregnancy, including consumption throughout the 1st and 2nd trimesters followed by abstinence during the 3rd trimester2.
In an attempt to model the different patterns of ethanol consumption observed in pregnant women, a number of developmental ethanol exposure paradigms have been established using diverse animal species, with rats and mice being most common5,6. The duration of pregnancy in these animals typically lasts approximately 3 weeks, which corresponds to the 1st and 2nd trimesters of human pregnancy. Many rodent studies have assessed the impact of various doses and patterns of ethanol exposure during this period. Examples of the methods frequently used to administer ethanol to pregnant mice and rats include administration via liquid diets7,8, addition of ethanol to the drinking water9,10, voluntary drinking of saccharin-sweetened solutions11, gastric gavage12, vapor inhalation13, and subcutaneous or intraperitoneal injection14. Results of these studies have recapitulated several of the deficits observed in humans with FASDs, demonstrating that exposure during early stages of pregnancy is sufficient to damage neuronal circuits across the brain (reviewed in 6,15).
Experiments with rodents have also demonstrated that exposure during the equivalent to the 3rd trimester of human pregnancy, which approximately corresponds to the first 1-2 weeks of postnatal life in rats and mice, can significantly impair brain development. Exposure during this period has been modeled by administering ethanol to neonatal rats or mice. Ethanol has been administered to these animals using a variety of methods, including feeding via gastrostomy in artificially-reared animals16-18, intragastric intubation19, subcutaneous injection20, and vapor inhalation21,22. These studies have convincingly demonstrated that the brain growth spurt is a period of high vulnerability to the developmental effects of ethanol6.
As mentioned above, drinking during all trimesters of pregnancy is a common pattern of ethanol consumption in women2. However, comparatively few studies have assessed the impact of this pattern of exposure using animal models. Some of these studies have taken advantage of large animals where the 3rd trimester-equivalent occurs in utero rather than the neonatal period as in the case of rats and mice. These animal models include non-human primates23,24 and sheep25-27. However, these animal models have not been widely used in FASDs research, in part, because of high cost and the need for specialized care facilities. Rodents have been more commonly used to characterize the effect of all-trimester ethanol exposure on fetal development5. Guinea pigs have been particularly advantageous in this regard given their extensive prenatal development and similarities in brain maturation to that of humans28,29. With guinea pigs, it has been possible to characterize the effect of ethanol exposure in utero that includes the equivalent development period of the human 3rd trimester. The comparatively high cost of these animals, as well as the relatively long duration of their pregnancy (~67 days), has limited their use to a few laboratories working on FASDs research.
Because of their cost-effectiveness and wide use in biomedical research, investigators have used rats to model exposure to ethanol during all trimesters of pregnancy. In initial studies, rats were exposed during pregnancy via liquid diets followed by administration of ethanol via gastrostomy to artificially reared neonates (postnatal days (P) 1-10) resulting in peak blood ethanol levels (BEC) in the dams of 0.08 g/dl&#160;and in the pups 0.16 g/dl. This paradigm caused long-lasting alterations in optic nerve myelination and reduced the number of Bergmann glia fibers in the cerebellum30-32. Similarly, Maier and collaborators using artificial rearing conditions administered ethanol to pregnant rat dams in a binge-like manner via intragastric intubation followed by neonatal administration during part of the 3rd trimester equivalent (P4-9)33,34. Peak maternal and pup BECs were 0.3 g/dl at both gestational day 20 and P6. This all-trimester exposure paradigm resulted in growth retardation that was significantly greater than that observed in pups exposed during selected periods of gestation33. In addition, rats exposed to ethanol during the equivalent to all trimesters exhibited a reduction in the number of cerebellar Purkinje and granule cells that was greater than that observed in animals exposed during other periods34. Reductions in hippocampal cell numbers were also reported with this paradigm, but these effects appear to be primarily a consequence of exposure during the 3rd trimester-equivalent35. A method that involves ethanol administration via intragastric gavage to both pregnant rats and neonatal mice has also been used to model all trimester exposure36. This method, which yielded BECs of 0.13 g/dl in the dams (gestational day 17) and 0.24 g/dl in P6 pups, induced long-lasting alterations in monoamine neurotransmitter levels in the hippocampus and hypothalamus, and increased expression of DNA methyltransferases and methyl CpG binding protein 2 in the hippocampus37,38. Using a similar exposure paradigm (BEC = 0.14-0.2 g/dl in dams and 0.2 g/dl in pups), Gil-Mohapel et al.39 detected an increase in the number of new immature neurons in the dentate gyrus of adult rats that may represent a compensatory mechanism to ethanol-induced neuronal damage or an alteration in the maturation of adult-born neurons. Investigators have also attempted to model all trimester ethanol exposure by exposing dams via liquid diets or drinking water during both pregnancy and lactation9,40. However, the utility of exposing the pups via their mother&#8217;s milk is limited because it typically results in low pup BECs (e.g., 0.002-0.05 g/dl; 41,42).
Mice have also been used extensively to characterize the effects of developmental ethanol exposure. This animal model shares many of the strengths described above for the rat animal model, with the additional advantage that many genetically modified mouse strains are available5. Mice have been successfully used to characterize the effects of ethanol during the 1st, 2nd or 3rd trimesters of pregnancy43,44. However, the impact of all trimester exposure on these animals has not been well characterized because it is technically more difficult to expose mice during the equivalent to all trimesters of human pregnancy. For instance, artificial rearing and gastric gavage, which have been used successfully in rats, require more specialized procedures in mice45. To the best of our knowledge, only one study to date has attempted to study the effect of all trimester ethanol exposure using mice; these animals were exposed to ethanol solution in drinking water during pregnancy and lactation46. Maternal BECs were 0.07 g/dl and pup BECs were not determined, but expected to be a fraction of those in dams.
Here, we describe a new model for all-trimester ethanol exposure of mice where alcohol is administered to both pregnant dams and neonates via vapor inhalation chambers. Vapor chambers were built based on a previous design47. We provide detailed instructions on how to build the inhalation chambers and carry out the exposure procedures. We also provide information on the BECs that can be achieved and the impact of exposure on pup survival and growth.

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construction of vapor chambers used to expose mice to alcohol during the equivalent of all three trimesters of human development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

Figure 2A shows that both pregnant mice and neonatal offspring were exposed to relatively stable ethanol vapor concentrations in the chambers. These ranged between 4-6 g/dl. Figure 2B shows the BECs achieved in the pregnant mice as a function of time. BECs were measured using a standard alcohol dehydrogenase based assay48. At G5, BECs quickly rose to ~60 mM 2 hr after the start of exposure and peaked at the end of the 4 hr exposure period. BECs gradually decreased to ~12 mM af...

Watch this Scientific Journal Video about Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development at JoVE.com

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3&#160;trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

construction-vapor-chambers-used-to-expose-mice-to-alcohol-during

Research

JoVE Journal

Medicine

14.5K visualizações.  University of New Mexico Health Sciences Center.  Nós demonstramos a construção de câmaras de vapor de álcool, utilizando materiais facilmente disponíveis que abrigam simultaneamente seis gaiolas de rato. Descrevemos ainda mais a sua utilização em um modelo do rato de exposição fetal ao álcool equivalente a todos os 3 trimestres de gestação humana. Este paradigma expõe animais durante a gestação e pós-natal dias 1-12. 