http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_1 Acute alcohol administration has minimal effects on basal immune function. However, when the immune system is challenged, acute alcohol administration alters the immune system's response. In the first 3 h after infection or traumatic injury, the presence of alcohol is associated with a decreased inflammatory response. This defect lasts long after the alcohol is cleared. Conversely, by 48 h after traumatic injury, the presence of alcohol is associated with a heightened inflammatory response. Aside from its in vivo actions, systemic administration of alcohol also alters the ex vivo response of immune cells, resulting in a decreased production of multiple cytokines after stimulation by lipopolysaccharide, concanavilin A, zymosan, and CpG DNA. Here, we describe a standardized model of acute administration of ethanol to mice used to study both the invivo and ex vivo responses of immune cells to ethanol. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_10 In vivo studies are ideal for identifying the phenomenology of ethanol toxicity and teratology. They are limited in being able to explore cellular and molecular mechanisms of action. Two types of culture models have proven to be very instructive: monolayer primary cultures of dissociated cells and organotypic slice cultures. Dissociated cell preparations have the advantage of being enriched populations of cells, whereas the organotypic cultures have the advantage of providing normal cell associations. Details for the methods used to generate these preparations are described. As ethanol is a volatile liquid, the success of a culture model depends upon stabilizing the ethanol content in the culture medium. A method to maintain the ethanol concentration is described. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_11 CYP2E1, a member of the cytochrome P450 family, is induced by ethanol. CYP2E1 activates many hepatotoxins to their reactive toxic intermediate form, and generates reactive oxygen species (ROS) during its catalytic cycle. Induction of CYP2E1 plays an important role in ethanol-induced oxidant stress and ethanol toxicity. To study the biochemical and toxicological properties of CYP2E1, our laboratory developed a HepG2 cell line which constitutively expresses the human CYP2E1 form. These cells displayed elevated oxidative stress, loss of mitochondrial function and loss of viability when challenged with prooxidants such as ethanol, polyunsaturated fatty acids (PUFA) such as arachidonic acid, iron, or when depleted of the critical antioxidant glutathione, as compared with control HepG2 cells which do not express CYP2E1. In the sections below , protocols are described for use of these cell lines to assay for CYP2E1-dependent oxidant stress and toxicity. Methods are described as to how the cell lines were established and maintained, how CYP2E1 is assayed, how cellular viability, mitochondrial function and generation of oxidant stress are determined. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_12 During the second trimester period, neuroepithelial stem cells give birth to millions of new neuroblasts, which migrate away from their germinal zones to populate the developing brain and terminally differentiate into neurons. During this period, large numbers of cells are also eliminated by programmed cell death. Therefore, the second trimester constitutes an important critical period for neuronal proliferation, migration, differentiation and apoptosis. Substantial evidence indicates that teratogens like ethanol can interfere with neuronal maturation. However, there is a paucity of good model systems to study early, second trimester events. In vivo models are inherently interpretatively complex because cell proliferation, migration, differentiation, and death mechanisms occur concurrently in regions like the cerebral cortex. This temporal overlap of multiple developmental critical periods makes it difficult to evaluate the relative vulnerability of any individual critical period. Our laboratory has elected to utilize fetal rodent cerebral cortical-derived neurosphere cultures as an experimental model of the second-trimester ventricular neuroepithelium. This model has enabled us to use flow cytometric approaches to identify neuroepithelial stem cell and progenitor sub-populations and to show that ethanol accelerates the maturation of neural stem cells. We have also developed a simplified mitogen-withdrawal/matrix-adhesion paradigm to model the exit of neuroepithelial cells from the ventricular zone towards the subventricular zone and cortical plate, and their maturation into multipolar neurons. We can treat neurosphere cultures with ethanol to mimic exposure during the period of neuroepithelial proliferation and by using the step-wise maturation model, ask questions about the impact of prior ethanol exposure on the subsequent maturation of neurons as they migrate and undergo terminal differentiation. The combination of neurosphere culture and stepwise maturation models will enable us to dissect out the contributions of specific developmental critical periods to the overall teratology of a drug of abuse like ethanol. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_13 A significant body of evidence indicates that endotoxemia plays a crucial role in the pathogenesis of alcoholic liver disease. There are several possible factors that may be involved in inducing alcoholic endotoxemia, but increased intestinal permeability to enteric endotoxins appears to be the major contributing factor. In the normal gut, the epithelial barrier function prevents diffusion of toxins across the epithelium. However, the barrier is disrupted in patients with alcoholic liver disease. We showed that acetaldehyde disrupts intestinal epithelial tight junctions and increases paracellular permeability to endotoxins in Caco-2 cell monolayer, the extensively studied model of the differentiated intestinal epithelium. The mechanisms involved in acetaldehyde-induced increase in intestinal permeability to endotoxins can be elucidated in this model of the intestinal epithelium. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_14 Oxidative stress is increasingly suspected to contribute to the initiation and progression of many disease, including those caused by alcohol exposure. Two major products of reactive oxygen and nitrogen species formation are 4OH-nonenal and 3-nitrotyrosine protein adducts, both of which can be detected by immunohistochemistry. In the past, immunohistochemical techniques have served largely as qualitative measures of changes. However, coupled with digital capture and analysis of photomicrographs, one can now quantitate treatment-related changes with immunohistochemistry. This chapter summarizes techniques for immunohistochemical detection of these products of reactive oxygen and nitrogen species and subsequent image-analysis. Although the methods described herein are based on liver, these techniques have been employed successfully in most tissue types with minor modifications and are therefore broadly applicable. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_15 Chronic consumption of ethanol induces hepatic steatosis and inflammation, which can eventually lead to more severe liver injury, characterized by fibrosis and cirrhosis. Recruitment of neutrophils to the liver, as well as activation of Kupffer cells, mediates the inflammatory responses observed after chronic ethanol exposure. Kupffer cells, the resident macrophages of the liver, are critical to the onset of ethanol-induced liver injury. Activation of Kupffer cells leads to an increased production of proinflammatory cytokines, such as tumor necrosis factor-α and also reactive oxygen species, a process mediated in part by changes in lipopolysaccharide-induced TLR4-dependent signal transduction. The isolation and culture of Kupffer cells is an important technique with which one can elucidate the mechanisms that contribute to alcoholic liver injury. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_16 Dendritic cells (DCs) play a key role in the initiation of effective immune responses against infectious agents because they are unique in their ability to provide antigen-specific activation of naïve T cells. To do this, they must acquire antigen and migrate to spleen or lymph node to present the antigen to T cells in association with costimulatory molecules and cytokines. Murine models of chronic EtOH exposure have been developed for dissecting the mechanisms by which EtOH alters immune cell functions. This chapter details methods for assessing DC functions in such models. Methods are presented for 1) the identification and isolation of various DC subsets from spleen, epidermis, and lung, 2) measurement of LC migration out of epidermis and DC migration into peripheral and peribronchial lymph nodes, and 3) measurement of alloantigen presentation in vitro as well as transgenic T-cell activation in vitro and in vivo. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_17 Most ingested ethanol is eliminated from the body through oxidative metabolism in the liver. Alcohol dehydrogenase is the enzyme that is most important in the oxidation of ethanol to acetaldehyde. However, it has also been demonstrated that cytochrome P4502E1 also can contribute to this process. However, this is not the only aldehyde that is produced after chronic ethanol consumption because oxidative stress and lipid peroxidation can be induced in the liver, which results in the production of malondialdehyde and 4-hydroxy-2-nonenal. These aldehydes are highly reactive and have the ability to react with (adduct) many macromolecules to alter their structure and play a major role in the derangements of hepatic function. Therefore, the formation of these types of adducts in the liver has been proposed as key events leading to the development and/or progression of alcoholic liver disease. In this chapter, methods for the production and detection of these modified proteins will be discussed. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_18 Natural killer (NK) cells part of innate immunity. NK cells have been assigned numerous functions, including the ability to serve as a bridge between innate and adaptive immunity. In evaluating NK cell function, two pathways need to be examined: their ability to kill certain tumors spontaneously and their ability to secrete cytokines, interferon-gamma (IFN-γ), in particular. Although NK cells are distinct from T lymphocytes, a new lymphocyte subset, termed NKT cell, has been described. NKT cells express surface markers that are unique to NK cells (e.g., NK1.1) as well as markers that are unique to T cells (e.g., CD3). Most NKT cells recognize glycolipids and are thought to play an important immunoregulatory role. This chapter will detail the methodology needed for examination of NK and NKT cells in mice. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_19 Evaluation of the functional responses of T cells is of importance in determining the mechanism(s) of immunodeficiency resulting from chronic alcohol abuse and other conditions that lead to immune dysfunction. Mice that are chronically exposed to 20% (w/v) ethanol in water develop immunodeficiency and have T cells with abnormal activation profiles, reduced total numbers, increased CD4/CD8 ratios, and an increased memory/naïve phenotype ratio. These cells also have abnormal antigen-specific responses after inoculation of the ethanol mice with model infectious organisms. Study of the functional abnormalities of these cells requires a reliable system that can present appropriate activation stimuli in vitro for the generation of polyclonal or antigen-specific responses in enriched or purified T cells, free of the influence of previously ethanol exposed accessory cells. In this chapter, we describe protocols to assess the T cell response to polyclonal stimulation through the T cell receptor and the use of a model infectious disease bacterium, Listeria monocytogenes, that allows evaluation of the T-cell response to specific peptide epitopes of the bacterium after previous inoculation. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_2 The variety of animal models used in the study of alcoholic liver disease reflects the formidable task of developing a model that replicates the human disease. We show that oral feeding of fatty acids derived from fish oil and ethanol induces fatty liver, necrosis, inflammation, and fibrosis. Together with the study of oxidative and nitrosative stress markers, cytokines, proteasome function, and protein studies, this model has provided an inexpensive and technically simple method of establishing pathological alcoholic liver injury. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_20 Chronic alcohol abuse leads to multiple defects in the immune system, leading to an increased risk of infectious disease and malignancy. Immune lesions encompass both the innate and adaptive arms and include deficiencies in the B-cell compartment. Long-term alcoholics exhibit loss of B cells in the periphery and diminished ability to generate protective antibodies. To better mimic the chronic alcoholic patient, our group has used an ethanol-in-drinking-water mouse model. Mice consuming alcohol in this manner progressively develop a range of immune abnormalities, including defects in humoral immunity. To document and explore B-cell lesions in ethanol-consuming mice, our laboratory has used a broad panel of technologies. These include protocols to define the physical state of B cells in the bone marrow and periphery, in vitro approaches to test B-cell activation potential and in vivo experiments to document the humoral competence of the host. These key techniques are detailed in the present chapter. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_21 Bone is an important target tissue for alcohol. Moderate alcohol consumption may slow bone loss during aging, but alcohol consumption inhibits bone growth during adolescence, and alcohol abuse in adults is an important risk factor for osteoporosis. Various techniques have been applied for evaluating the impact of alcohol on bone, including densitometry for assessment of bone mass and density, computed tomography for evaluation of bone microarchitecture, serum biochemistry for measurement of markers of global bone resorption and formation, and histomorphometry for assessment of cellular activity. Of these methods, histomorphometry is the gold standard for assessing bone because it is the only method for the direct in situ analysis of bone cells and their activities. The procedures described in this chapter provide tools for the histomorphometric characterization of the effects of alcohol on cancellous and cortical bone growth and turnover. Specifically detailed are processes for embedding, cutting, staining, and evaluating histological bone specimens with a focus on rodent models. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_22 The development of alcoholic muscle disease, which affects both cardiac and skeletal muscle, leads to increased morbidity and mortality in patients who abuse alcohol. The disease pathology includes myocyte degeneration, loss of striations, and myofilament dissolution, which is consistent with alterations in structural and myofibrillar proteins. One explanation for the changes in myofibrillar architecture is that the expression of cellular proteins may be compromised by ethanol consumption. The dynamic balance of proteins in striated muscle is dependent upon rates of protein synthesis and protein degradation. We have shown that protein synthesis is depressed in striated muscle after either acute alcohol intoxication or chronic alcohol ingestion. The loss of myofibrillar proteins occurs prior to any detection of abnormal muscle function in vivo. It is therefore of major importance to evaluate the regulation of protein turnover after ethanol consumption. This review describes protocols to study protein synthesis either in vivo or under in vitro conditions. The methods can be modified for studies involving transgenic mice allowing mechanisms responsible for the defects in protein synthesis to be dissected. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_23 Chronic ethanol consumption dysregulates glucose and lipid homeostasis, is associated with insulin resistance, and alters serum levels of adipokines including adiponectin and tumor necrosis factor-α. However, the mechanisms involved in these chronic ethanol-induced pathologies are not fully understood. Adipose tissue has been implicated as an important contributor to chronic ethanol-induced disease states and, therefore, the effects of chronic ethanol feeding in rats on adipocytes has been investigated. Three major functions of the adipocyte include glucose transport, adipokine secretion, and triglyceride breakdown via lipolysis. Included in this chapter are protocols for studying the effect of chronic ethanol feeding on these adipocyte functions. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_24 Mitochondrial dysfunction is recognized as a contributing factor to a number of diseases, including chronic alcohol-induced hepatotoxicity. Although there is a detailed understanding of the metabolic pathways and proteins of the liver mitochondrion, little is known of how changes in the mitochondrial proteome contribute to the development of hepatic pathologies. In this short overview the insights gained from study of changes in the mitochondrial proteome in alcoholic liver disease will be described. Profiling the liver mitochondrial proteome has the potential to shed light on the alcohol-mediated molecular defects responsible for mitochondrial and cellular dysfunction. The methods presented herein demonstrate the power of using complementary proteomics approaches, that is, 2-D IEF/SDS-PAGE and BN-PAGE, to identify changes in the abundance of mitochondrial proteins after chronic alcohol consumption. These proteomic data can then be integrated into a logical and mechanistic framework to further our understanding of the role of mitochondrial dysfunction in the pathogenesis of alcohol-induced liver disease. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_25 Chronic alcohol consumption has been shown to severely compromise mitochondrial protein synthesis. Hepatic mitochondria isolated from alcoholic animals contain decreased levels of respiratory complexes and display depressed respiration rates when compared to pair-fed controls. One underlying mechanism for this involves ethanol-elicited alterations in the structural and functional integrity of the mitochondrial ribosome. Ethanol feeding results in ribosomal changes that include decreased sedimentation rates, larger hydrodynamic volumes, increased levels of unassociated subunits and changes in the levels of specific ribosomal proteins. The methods presented in this chapter detail how to isolate mitochondrial ribosomes, determine ribosomal activity, separate ribosomes into nucleic acid and protein, and perform two-dimensional nonequilibrium pH gradient electrophoretic polyacrylamide gel electrophoresis to separate and subsequently identify mitochondrial ribosomal proteins. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_26 DNA microarray studies offer a robust method for nonbiased analysis of whole genome messenger ribonucleic acid expression patterns. A growing number of studies have applied this experimental approach to studies on ethanol either in cell culture of animal models of ethanol exposure or self-administration. Expression profiling has identified novel gene networks responding to ethanol or differing across animal strains with differing responses to ethanol. Recent studies have shown benefit for meta-analysis of microarray data across different laboratories. Gene network analysis offers unique opportunities for understanding the molecular mechanisms of ethanol responses, toxicity and addiction. Eventually, such work may generate novel targets for future pharmacotherapy. To fully capitalize on the prom ise alluded to above, particularly in regard to meta-analysis of microarray data, it is critical that high quality standards are followed in the generation and analysis of microarray studies. This chapter will discuss experience of our laboratory in performing and analyzing microarray studies on ethanol, focusing discussion mainly on short oligonucleotide microarrays (Affymetrix). However, the general principals of technique and analysis that are discussed have broad applicability to other types of microarray platforms and experimental designs. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_3 Alcohol-associated life-style disease, as exemplified by alcoholic liver disease (ALD), is multifactorial with intricate interactions among genetic and environmental factors predicating individual predisposition. To experimentally dissect the interfaces of these interactions for better understanding of the pathogenesis, it is essential to have an animal model that provides maximal control over ethanol and dietary intake and that enables a precise addition or deletion analysis for a risk or protective factor of interest. Rodent intragastric ethanol infusion (IEI) model was developed two decades ago to meet this requirement. Work conducted with the model to date demonstrates the importance of both maximal ethanol intake and secondary risk factors in ALD. Mouse IEI model proved to be particularly useful for genetic analysis of the ALD pathogenesis and has the potential of producing synergistic pathologic outcome by combination of risk factors. The model is best used by alcohol researchers through a center-supported core facility and its tissue sharing program. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_4 Mice provide a useful model for the study of immune deficiency caused by chronic alcohol abuse. Their suitability is related to several factors, including in particular the extensive knowledge base in the immunology of mice already existing in the literature. Specific modeling of the immunodeficiency of the chronic human alcoholic requires that ethanol must be administered to the model for a significant portion of its life span. In mice, it has proven to be necessary to administer ethanol daily for up to 32 wk or longer to observe all the immune abnormalities that occur in middle-aged alcoholic humans. Such time spans are problematic with many of the common protocols for ethanol administration. It has been shown by others and confirmed by our group that the most practical way of accomplishing such long protocols is by administering ethanol in water as the only choice of water. Details of management of the chronic ethanol mouse colony are described here that are necessary for the success of such studies, including methods for initiating ethanol administration, maintenance of barrier protection, monitoring weight gain, strain differences and fetal alcohol exposure. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_5 It is largely accepted that vertebrates are more susceptible to chemical insult during the early life stage. It is implied that if a chemical such as ethanol is developmentally toxic, it must interfere with, or modulate, critical signaling pathways. The probable molecular explanation for increased embryonic susceptibility is that collectively there is no other period of an animal's lifespan when the full repertoire of molecular signaling is active. Understanding the mechanism by which ethanol exposure disrupts vertebrate embryonic development is enormously challenging; it requires a thorough understanding of the normal molecular program to understand how transient ethanol exposure disrupts signaling and results in detrimental long-lasting effects. During the past several years, investigators have recognized the advantages of the zebrafish model to discover the signaling events that choreograph embryonic development. External development coupled with the numerous molecular and genetic methods make this model a valuable tool to unravel the mechanisms by which ethanol disrupts embryonic development. In this chapter we describe procedures used to evaluate and define the morphological, cellular and molecular responses to ethanol in zebrafish. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_6 The avian embryo has proven utility for studying ethanol's damaging effects upon the embryo. Chicken and quail are long-established models for developmental biology research; much of what we know regarding limb, craniofacial, neural crest, hindbrain, and cardiac morphogenesis was first established with avian models. These models also are for popular mechanistic studies of teratogens, including ethanol. Avian models have been used to explore ethanol's effects on neurogenesis, cardiogenesis, intracellular signaling, neurobehavior, and apoptosis. Presented here are several of these methodologies for adaptation by interested researchers. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_7 Prenatal alcohol exposure disrupts development, leading to a range of effects referred to as fetal alcohol spectrum disorders (FASD). FASDs include physical, central nervous system, and behavioral alterations. Animal model systems are used to study the relationship between alcohol-related central nervous system damage and behavioral alterations, risk factors for FASD, mechanisms of alcohol-induced damage, as well as treatments and interventions. When using a rodent model, it is important to recognize that the timing of brain development relative to birth differs between humans and rodents. Thus, to model alcohol exposure during the third trimester equivalent, rats must be exposed during early postnatal development (postnatal days 4-9). Artificial rearing is one experimental paradigm that is used to expose neonatal rats to alcohol during this period of brain development. Neonatal rat pups are housed in an artificial rearing environment and automatically fed a milk diet substitute via an intragastric cannula to ensure adequate growth during the treatment period. Alcohol is delivered in the milk diet. This chapter provides a description of the methods needed for this administrative technique, including preparation of the artificial rearing environment, gastrostomy surgery, and care of artificially reared rat pups. 2008-05-02T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-242-7_8 Animal models of fetal alcohol spectrum disorder (FASD) have been instrumental in isolating alcohol as a teratogen and demonstrating behavioral and neural effects. There are a number of different models for rodents with various strengths and weaknesses. A three-trimester model of FASD is described here; the model uses intragastric intubation of both pregnant dams and pups to mimic alcohol exposure across all three trimesters in humans. The model does not use expensive equipment and is relatively easy to accomplish. The model allows excellent control of alcohol dose and uses an oral route of administration. There are no undernutrition effects with the doses used here. A drawback of the model is the stress of the intubation procedures and ways in which to minimize this stress are discussed. In addition, a method to measure blood alcohol levels is described. 2008-05-02T04:00:00Z