Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.
23 Related JoVE Articles!
Spinal Cord Transection in the Larval Zebrafish
Institutions: University of Utah.
Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio
exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6 weeks post-injury. To take advantage of in vivo
analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2 days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.
Basic Protocol, Issue 87, zebrafish, larva, spinal cord, transection, injury, neurogenesis, regeneration, recovery
Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors
Institutions: Twincore, Centre for Experimental and Clinical Infection Research, The Rockefeller University, NY.
Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5
. HCV in vitro
research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8
. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans
-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans
-complementation, which is determined by measuring de novo
production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected naïve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9
) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10
. Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11
) and subsequently challenged with infectious HCV particles (HCVcc), de novo
production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans
-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.
Virology, Issue 65, Immunology, Molecular Biology, Genetics, cell fusion, HCV, restriction factor, heterokaryon, mouse, species-specificity
Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents
Institutions: Tufts University Cummings School of Veterinary Medicine, Manchester Metropolitan University.
Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals and humans. However, many animal models of depression were developed in males and are not applicable or effective in studies of postpartum females. Recent studies have reported significant effects of chronic social stress during lactation, an ethologically relevant and effective stressor, on maternal behavior, growth, and behavioral neuroendocrinology. This manuscript will describe this chronic social stress paradigm using repeated exposure of a lactating dam to a novel male intruder, and the assessment of the behavioral, physiological, and neuroendocrine effects of this model. Chronic social stress (CSS) is a valuable model for studying the effects of stress on the behavior and physiology of the dam as well as her offspring and future generations. The exposure of pups to CSS can also be used as an early life stress that has long term effects on behavior, physiology, and neuroendocrinology.
Behavior, Issue 76, Neuroscience, Neurobiology, Physiology, Anatomy, Medicine, Biomedical Engineering, Neurobehavioral Manifestations, Mental Health, Mood Disorders, Depressive Disorder, Anxiety Disorders, behavioral sciences, Behavior and Behavior Mechanisms, Mental Disorders, Stress, Depression, Anxiety, Postpartum, Maternal Behavior, Nursing, Growth, Transgenerational, animal model
Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat
Institutions: University College London, University of Gothenburg.
Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli
K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli
O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.
Infection, Issue 92, Bacterial infection, neonatal bacterial meningitis, bacteremia, sepsis, animal model, K1 polysaccharide, systemic infection, gastrointestinal tract, age dependency
Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology
Institutions: University of Pittsburgh School of Medicine, University of Pittsburgh School of Medicine.
We describe an in vitro
method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo
experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e.
smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo
. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro
smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.
Medicine, Issue 90, Krebs, species differences, in vitro, smooth muscle contractility, neural stimulation
DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat
Institutions: BC Children's Hospital.
Inflammatory Bowel Diseases (IBD), including Crohn's Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48 hr and rats fed SO by 72 hr. Weight gain resumed after 72 hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5 days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.
Medicine, Issue 84, Chemical colitis, Inflammatory Bowel Disease, intra rectal administration, intestinal inflammation, transmural inflammation, myeloperoxidase activity
Isolation of Neonatal Extrahepatic Cholangiocytes
Institutions: The Children's Hospital of Philadelphia, The Perelman School of Medicine at the University of Pennsylvania.
The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.
Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established1-4
. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.
This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.
Medicine, Issue 88, Bile Ducts, Bile Ducts, Extrahepatic, Common Bile Duct, Bile Duct Diseases, Cell culture, bile duct, biliary atresia, Liver, gallbladder, fibrosis
A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats
Institutions: Concordia University.
Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.
Behavior, Issue 91, Behavioral neuroscience, alcoholism, relapse, addiction, Pavlovian conditioning, ethanol, reinstatement, discrimination, conditioned approach
Isolation and Culture of Neonatal Mouse Cardiomyocytes
Institutions: King’s College London, University of California San Diego .
Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.
Cellular Biology, Issue 79, Biomedical Engineering, Bioengineering, Molecular Biology, Cell Culture Techniques, Primary Cell Culture, Cell Culture Techniques, Primary Cell Culture, Cell Culture Techniques, Primary Cell Culture, Cell Culture Techniques, Disease Models, Animal, Models, Cardiovascular, Cell Biology, neonatal mouse, cardiomyocytes, isolation, culture, primary cells, NMC, heart cells, animal model
Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction
Institutions: RWTH-Aachen University, Kyoto University .
Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2
, immunological responses 3,4
, hemodynamics 5,6
, and small-for-size syndrome 7
. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8
since the first model was described by Lee et al. 9
in 1973, the elimination of the hepatic arterial reconstruction 10
and the introduction of the cuff anastomosis technique by Kamada et al. 11
were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al.
, the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12
and the liver integrity 8,13,14
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo
liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.
Medicine, Issue 73, Biomedical Engineering, Anatomy, Physiology, Immunology, Surgery, liver transplantation, liver, hepatic, partial, orthotopic, split, rat, graft, transplantation, microsurgery, procedure, clinical, technique, artery, arterialization, arterialized, anastomosis, reperfusion, rat, animal model
A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Institutions: University of Maine, Orono, University of Maine, Orono.
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g.
by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5
. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells6
, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1
. Originally published by Naal et al.1
, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11
, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2
. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280
= 4,200 L/M/cm)12
. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
Immunology, Issue 81, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells
Institutions: University of Houston.
Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ERα, and ERβ. ERα is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ERα's regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ERα-positive breast cancer cell lines were compared before and after estrogen-activation using both the µParaflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations, and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.
Medicine, Issue 84, breast cancer, microRNA, estrogen, estrogen receptor, microarray, qPCR
Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions
Institutions: Imperial College London, Institut Pasteur, Unité Macrophages et Développement de l'Immunité.
is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri
by compartmentalizing bacteria inside ‘septin cages’ and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro
using tissue culture cells and in vivo
using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.
Infection, Issue 91, ATG8/LC3, autophagy, cytoskeleton, HeLa cells, p62, septin, Shigella, zebrafish
Analysis of Oxidative Stress in Zebrafish Embryos
Institutions: University of Torino, Vesalius Research Center, VIB.
High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo
system to perform such studies and present a protocol to measure in vivo
oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo
: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.
Developmental Biology, Issue 89, Danio rerio, zebrafish embryos, endothelial cells, redox state analysis, oxidative stress detection, in vivo ROS measurements, FACS (fluorescence activated cell sorter), molecular probes
A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos
Institutions: University of Notre Dame.
Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ
hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.
Developmental Biology, Issue 93, zebrafish, chemical genetics, chemical screen, in vivo small molecule screen, drug discovery, whole mount in situ hybridization (WISH), high-throughput screening (HTS), high-content screening (HCS)
Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development
Institutions: University of New Mexico Health Sciences Center.
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.
Medicine, Issue 89, fetal, ethanol, exposure, paradigm, vapor, development, alcoholism, teratogenic, animal, mouse, model
Barnes Maze Testing Strategies with Small and Large Rodent Models
Institutions: University of Missouri, Food and Drug Administration.
Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.
bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.
distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.
random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.
shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.
Behavior, Issue 84, spatial navigation, rats, Peromyscus, mice, intra- and extra-maze cues, learning, memory, latency, search strategy, escape motivation
Strategies for Study of Neuroprotection from Cold-preconditioning
Institutions: The University of Chicago Medical Center.
Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro
model that closely reflects their in vivo
counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro
for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-α to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.
Neuroscience, Issue 43, innate immunity, hormesis, microglia, hippocampus, slice culture, immunohistochemistry, neural-immune, gene expression, real-time PCR
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos
Institutions: University of California, Irvine (UCI).
The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.
Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.
Developmental Biology, Issue 8, Neuroscience, Growth Factor, Heparin-Coated Beads, Chicken, Embryos
A Simple Way to Measure Ethanol Sensitivity in Flies
Institutions: University of Texas Southwestern Medical Center.
Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.
Neuroscience, Issue 48, Drosophila, behavior, alcohol, addiction
Detection of Protein Ubiquitination
Institutions: The Sanford Burnham Institute for Medical Research.
Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo
and test tubes in vitro
. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.
Cell Biology, Biochemistry, Issue 30, ubiquitination, cultured cell, in vitro system, immunoprecipitation, immunoblotting, ubiquitin, posttranslational modification
Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds
Institutions: Stanford University School of Medicine .
We describe a methodology by which we are able to collect and measure biochemical inflammatory and nociceptive mediators at the surgical wound site. Collecting site-specific biochemical markers is important to understand the relationship between levels in serum and surgical wound, determine any associations between mediator release, pain, analgesic use and other outcomes of interest, and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry. This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. We have measured wound exudate and serum mediators at the same time intervals as patient's pain scores and analgesics consumption for up to 48 hours post-cesarean delivery. Using this methodology we have been able to detect various biochemical mediators including nerve growth factor (NGF), prostaglandin E2 (PG-E2) substance P, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNFα, INFγ, G-CSF, GM-CSF, MCP-1 and MIP-1β. Studies applying this human surgical wound bioassay have found no correlations between wound and serum cytokine concentrations or their time-release profile (J Pain. 2008; 9(7):650-7).1
We also documented the utility of the technique to identify drug-mediated changes in wound cytokine content (Anesth Analg 2010; 111:1452-9).2
Medicine, Issue 68, Biochemistry, Anatomy, Physiology, Cytokines, Cesarean Section, Wound Healing, Wounds and Injuries, Surgical Procedures, Operative, Surgical wound, Exudate, cytokines, Substance P, Interleukin 10, Interleukin 6, Nerve growth factor, Prostaglandin E2, Cesarean, Analgesia