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Specific Inflammatory Stimuli Lead to Distinct Platelet Responses in Mice and Humans.
PUBLISHED: 07-07-2015
Diverse and multi-factorial processes contribute to the progression of cardiovascular disease. These processes affect cells involved in the development of this disease in varying ways, ultimately leading to atherothrombosis. The goal of our study was to compare the differential effects of specific stimuli - two bacterial infections and a Western diet - on platelet responses in ApoE-/- mice, specifically examining inflammatory function and gene expression. Results from murine studies were verified using platelets from participants of the Framingham Heart Study (FHS; n = 1819 participants).
Authors: Keen Sung, Sanda Dolcos, Sophie Flor-Henry, Crystal Zhou, Claudia Gasior, Jennifer Argo, Florin Dolcos.
Published: 07-06-2011
The ability to gauge social interactions is crucial in the assessment of others’ intentions. Factors such as facial expressions and body language affect our decisions in personal and professional life alike 1. These "friend or foe" judgements are often based on first impressions, which in turn may affect our decisions to "approach or avoid". Previous studies investigating the neural correlates of social cognition tended to use static facial stimuli 2. Here, we illustrate an experimental design in which whole-body animated characters were used in conjunction with functional magnetic resonance imaging (fMRI) recordings. Fifteen participants were presented with short movie-clips of guest-host interactions in a business setting, while fMRI data were recorded; at the end of each movie, participants also provided ratings of the host behaviour. This design mimics more closely real-life situations, and hence may contribute to better understanding of the neural mechanisms of social interactions in healthy behaviour, and to gaining insight into possible causes of deficits in social behaviour in such clinical conditions as social anxiety and autism 3.
27 Related JoVE Articles!
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Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus
Authors: Daniel E. Venegas-Pino, Nicole Banko, Mohammed I. Khan, Yuanyuan Shi, Geoff H. Werstuck.
Institutions: McMaster University, McMaster University.
Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.
Medicine, Issue 82, atherosclerosis, atherosclerotic lesion, Mouse Model, aortic sinus, tissue preparation and sectioning, Immunohistochemistry
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Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities
Authors: Khadija Elhabazi, Safia Ayachi, Brigitte Ilien, Frédéric Simonin.
Institutions: Université de Strasbourg.
Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.
Neuroscience, Issue 89, mice, nociception, tail immersion test, tail pressure test, morphine, analgesia, opioid-induced hyperalgesia, tolerance
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Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Authors: Yves Molino, Françoise Jabès, Emmanuelle Lacassagne, Nicolas Gaudin, Michel Khrestchatisky.
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),
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Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets
Authors: Philip A. Kramer, Balu K. Chacko, Saranya Ravi, Michelle S. Johnson, Tanecia Mitchell, Victor M. Darley-Usmar.
Institutions: University of Alabama at Birmingham.
Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging. Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis. This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry. An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.
Immunology, Issue 85, bioenergetics, translational, mitochondria, oxidative stress, reserve capacity, leukocytes
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Inducing Myointimal Hyperplasia Versus Atherosclerosis in Mice: An Introduction of Two Valid Models
Authors: Mandy Stubbendorff, Xiaoqin Hua, Tobias Deuse, Ziad Ali, Hermann Reichenspurner, Lars Maegdefessel, Robert C. Robbins, Sonja Schrepfer.
Institutions: University Hospital Hamburg, Cardiovascular Research Center (CVRC) and DZHK University Hamburg, University Heart Center Hamburg, Columbia University, Cardiovascular Research Foundation, New York, Karolinska Institute, Stockholm, Stanford University School of Medicine, Falk Cardiovascular Research Center.
Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque formation and stenosis, as observed for example in ischemic heart disease. Two highly reproducible mouse models – both resulting in artery stenosis but each underlying a different pathway of development – are introduced here. The models represent the two most common causes of artery stenosis; namely one mouse model for each myointimal hyperplasia, and atherosclerosis are shown. To induce myointimal hyperplasia, a balloon catheter injury of the abdominal aorta is performed. For the development of atherosclerotic plaque, the ApoE -/- mouse model in combination with western fatty diet is used. Different model-adapted options for the measurement and evaluation of the results are named and described in this manuscript. The introduction and comparison of these two models provides information for scientists to choose the appropriate artery stenosis model in accordance to the scientific question asked.
Medicine, Issue 87, vascular diseases, atherosclerosis, coronary stenosis, neointima, myointimal hyperplasia, mice, denudation model, ApoE -/-, balloon injury, western diet, analysis
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A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Authors: Daniel T. Claiborne, Jessica L. Prince, Eric Hunter.
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
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A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites
Authors: George Papadopoulos, Carolyn D. Kramer, Connie S. Slocum, Ellen O. Weinberg, Ning Hua, Cynthia V. Gudino, James A. Hamilton, Caroline A. Genco.
Institutions: Boston University School of Medicine, Boston University School of Medicine.
Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation. Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.
Immunology, Issue 90, Pathogen-Induced Chronic Inflammation; Porphyromonas gingivalis; Oral Bone Loss; Periodontal Disease; Atherosclerosis; Chronic Inflammation; Host-Pathogen Interaction; microCT; MRI
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Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat
Authors: Daniel E. Bradford, Katherine P. Magruder, Rachel A. Korhumel, John J. Curtin.
Institutions: University of Wisconsin-Madison.
Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical components. Both anxiety and fear can be studied in the laboratory by measuring the potentiation of the startle reflex. The startle reflex is a defensive reflex that is potentiated when an organism is threatened and the need for defense is high. The startle reflex is assessed via electromyography (EMG) in the orbicularis oculi muscle elicited by brief, intense, bursts of acoustic white noise (i.e., “startle probes”). Startle potentiation is calculated as the increase in startle response magnitude during presentation of sets of visual threat cues that signal delivery of mild electric shock relative to sets of matched cues that signal the absence of shock (no-threat cues). In the Threat Probability Task, fear is measured via startle potentiation to high probability (100% cue-contingent shock; certain) threat cues whereas anxiety is measured via startle potentiation to low probability (20% cue-contingent shock; uncertain) threat cues. Measurement of startle potentiation during the Threat Probability Task provides an objective and easily implemented alternative to assessment of negative affect via self-report or other methods (e.g., neuroimaging) that may be inappropriate or impractical for some researchers. Startle potentiation has been studied rigorously in both animals (e.g., rodents, non-human primates) and humans which facilitates animal-to-human translational research. Startle potentiation during certain and uncertain threat provides an objective measure of negative affective and distinct emotional states (fear, anxiety) to use in research on psychopathology, substance use/abuse and broadly in affective science. As such, it has been used extensively by clinical scientists interested in psychopathology etiology and by affective scientists interested in individual differences in emotion.
Behavior, Issue 91, Startle; electromyography; shock; addiction; uncertainty; fear; anxiety; humans; psychophysiology; translational
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Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research
Authors: Hugh Alley, Christopher D. Owens, Warren J. Gasper, S. Marlene Grenon.
Institutions: University of California, San Francisco, Veterans Affairs Medical Center, San Francisco, Veterans Affairs Medical Center, San Francisco.
The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The endothelium acts as a barrier, preventing leukocyte adhesion and aggregation, as well as controlling permeability to plasma components. Functionally, the endothelium affects vessel tone. Endothelial dysfunction is an imbalance between the chemical species which regulate vessel tone, thombroresistance, cellular proliferation and mitosis. It is the first step in atherosclerosis and is associated with coronary artery disease, peripheral artery disease, heart failure, hypertension, and hyperlipidemia. The first demonstration of endothelial dysfunction involved direct infusion of acetylcholine and quantitative coronary angiography. Acetylcholine binds to muscarinic receptors on the endothelial cell surface, leading to an increase of intracellular calcium and increased nitric oxide (NO) production. In subjects with an intact endothelium, vasodilation was observed while subjects with endothelial damage experienced paradoxical vasoconstriction. There exists a non-invasive, in vivo method for measuring endothelial function in peripheral arteries using high-resolution B-mode ultrasound. The endothelial function of peripheral arteries is closely related to coronary artery function. This technique measures the percent diameter change in the brachial artery during a period of reactive hyperemia following limb ischemia. This technique, known as endothelium-dependent, flow-mediated vasodilation (FMD) has value in clinical research settings. However, a number of physiological and technical issues can affect the accuracy of the results and appropriate guidelines for the technique have been published. Despite the guidelines, FMD remains heavily operator dependent and presents a steep learning curve. This article presents a standardized method for measuring FMD in the brachial artery on the upper arm and offers suggestions to reduce intra-operator variability.
Medicine, Issue 92, endothelial function, endothelial dysfunction, brachial artery, peripheral artery disease, ultrasound, vascular, endothelium, cardiovascular disease.
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Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease
Authors: Nathan Robbins, Allie Thompson, Adrien Mann, Andra L. Blomkalns.
Institutions: University of Cincinnati College of Medicine.
Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.
Medicine, Issue 93, Cardiovascular, aorta, murine, isolation, surgery, excision, anatomy
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Modulating Cognition Using Transcranial Direct Current Stimulation of the Cerebellum
Authors: Paul A. Pope.
Institutions: University of Birmingham.
Numerous studies have emerged recently that demonstrate the possibility of modulating, and in some cases enhancing, cognitive processes by exciting brain regions involved in working memory and attention using transcranial electrical brain stimulation. Some researchers now believe the cerebellum supports cognition, possibly via a remote neuromodulatory effect on the prefrontal cortex. This paper describes a procedure for investigating a role for the cerebellum in cognition using transcranial direct current stimulation (tDCS), and a selection of information-processing tasks of varying task difficulty, which have previously been shown to involve working memory, attention and cerebellar functioning. One task is called the Paced Auditory Serial Addition Task (PASAT) and the other a novel variant of this task called the Paced Auditory Serial Subtraction Task (PASST). A verb generation task and its two controls (noun and verb reading) were also investigated. All five tasks were performed by three separate groups of participants, before and after the modulation of cortico-cerebellar connectivity using anodal, cathodal or sham tDCS over the right cerebellar cortex. The procedure demonstrates how performance (accuracy, verbal response latency and variability) could be selectively improved after cathodal stimulation, but only during tasks that the participants rated as difficult, and not easy. Performance was unchanged by anodal or sham stimulation. These findings demonstrate a role for the cerebellum in cognition, whereby activity in the left prefrontal cortex is likely dis-inhibited by cathodal tDCS over the right cerebellar cortex. Transcranial brain stimulation is growing in popularity in various labs and clinics. However, the after-effects of tDCS are inconsistent between individuals and not always polarity-specific, and may even be task- or load-specific, all of which requires further study. Future efforts might also be guided towards neuro-enhancement in cerebellar patients presenting with cognitive impairment once a better understanding of brain stimulation mechanisms has emerged.
Behavior, Issue 96, Cognition, working memory, tDCS, cerebellum, brain stimulation, neuro-modulation, neuro-enhancement
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Operant Procedures for Assessing Behavioral Flexibility in Rats
Authors: Anne Marie Brady, Stan B. Floresco.
Institutions: St. Mary's College of Maryland, University of British Columbia.
Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these processes is the ability to adjust behavior in response to changes in one’s environment (i.e., behavioral flexibility). These processes are essential to normal human behavior, and may be disrupted in diverse neuropsychiatric conditions, including schizophrenia, alcoholism, depression, stroke, and Alzheimer’s disease. Understanding of the neurobiology of executive functions has been greatly advanced by the availability of animal tasks for assessing discrete components of behavioral flexibility, particularly strategy shifting and reversal learning. While several types of tasks have been developed, most are non-automated, labor intensive, and allow testing of only one animal at a time. The recent development of automated, operant-based tasks for assessing behavioral flexibility streamlines testing, standardizes stimulus presentation and data recording, and dramatically improves throughput. Here, we describe automated strategy shifting and reversal tasks, using operant chambers controlled by custom written software programs. Using these tasks, we have shown that the medial prefrontal cortex governs strategy shifting but not reversal learning in the rat, similar to the dissociation observed in humans. Moreover, animals with a neonatal hippocampal lesion, a neurodevelopmental model of schizophrenia, are selectively impaired on the strategy shifting task but not the reversal task. The strategy shifting task also allows the identification of separate types of performance errors, each of which is attributable to distinct neural substrates. The availability of these automated tasks, and the evidence supporting the dissociable contributions of separate prefrontal areas, makes them particularly well-suited assays for the investigation of basic neurobiological processes as well as drug discovery and screening in disease models.
Behavior, Issue 96, executive function, behavioral flexibility, prefrontal cortex, strategy shifting, reversal learning, behavioral neuroscience, schizophrenia, operant
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Training Synesthetic Letter-color Associations by Reading in Color
Authors: Olympia Colizoli, Jaap M. J. Murre, Romke Rouw.
Institutions: University of Amsterdam.
Synesthesia is a rare condition in which a stimulus from one modality automatically and consistently triggers unusual sensations in the same and/or other modalities. A relatively common and well-studied type is grapheme-color synesthesia, defined as the consistent experience of color when viewing, hearing and thinking about letters, words and numbers. We describe our method for investigating to what extent synesthetic associations between letters and colors can be learned by reading in color in nonsynesthetes. Reading in color is a special method for training associations in the sense that the associations are learned implicitly while the reader reads text as he or she normally would and it does not require explicit computer-directed training methods. In this protocol, participants are given specially prepared books to read in which four high-frequency letters are paired with four high-frequency colors. Participants receive unique sets of letter-color pairs based on their pre-existing preferences for colored letters. A modified Stroop task is administered before and after reading in order to test for learned letter-color associations and changes in brain activation. In addition to objective testing, a reading experience questionnaire is administered that is designed to probe for differences in subjective experience. A subset of questions may predict how well an individual learned the associations from reading in color. Importantly, we are not claiming that this method will cause each individual to develop grapheme-color synesthesia, only that it is possible for certain individuals to form letter-color associations by reading in color and these associations are similar in some aspects to those seen in developmental grapheme-color synesthetes. The method is quite flexible and can be used to investigate different aspects and outcomes of training synesthetic associations, including learning-induced changes in brain function and structure.
Behavior, Issue 84, synesthesia, training, learning, reading, vision, memory, cognition
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Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System
Authors: Oren Levy, Priya Anandakumaran, Jessica Ngai, Rohit Karnik, Jeffrey M. Karp.
Institutions: Brigham and Women's Hospital, Brigham and Women's Hospital, Harvard University, Harvard University, Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology.
A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-α (TNF-α), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.
Bioengineering, Issue 80, Microfluidics, Endothelial Cells, Leukocyte Rolling, HL-60 cells, TNF-α, P-selectin, E-selectin
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Brain Imaging Investigation of the Neural Correlates of Emotion Regulation
Authors: Sanda Dolcos, Keen Sung, Ekaterina Denkova, Roger A. Dixon, Florin Dolcos.
Institutions: University of Illinois, Urbana-Champaign, University of Alberta, Edmonton, University of Alberta, Edmonton, University of Alberta, Edmonton, University of Alberta, Edmonton, University of Illinois, Urbana-Champaign, University of Illinois, Urbana-Champaign.
The ability to control/regulate emotions is an important coping mechanism in the face of emotionally stressful situations. Although significant progress has been made in understanding conscious/deliberate emotion regulation (ER), less is known about non-conscious/automatic ER and the associated neural correlates. This is in part due to the problems inherent in the unitary concepts of automatic and conscious processing1. Here, we present a protocol that allows investigation of the neural correlates of both deliberate and automatic ER using functional magnetic resonance imaging (fMRI). This protocol allows new avenues of inquiry into various aspects of ER. For instance, the experimental design allows manipulation of the goal to regulate emotion (conscious vs. non-conscious), as well as the intensity of the emotional challenge (high vs. low). Moreover, it allows investigation of both immediate (emotion perception) and long-term effects (emotional memory) of ER strategies on emotion processing. Therefore, this protocol may contribute to better understanding of the neural mechanisms of emotion regulation in healthy behaviour, and to gaining insight into possible causes of deficits in depression and anxiety disorders in which emotion dysregulation is often among the core debilitating features.
Neuroscience, Issue 54, Emotion Suppression, Automatic Emotion Control, Deliberate Emotion Control, Goal Induction, Neuroimaging
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Examining the Characteristics of Episodic Memory using Event-related Potentials in Patients with Alzheimer's Disease
Authors: Erin Hussey, Brandon Ally.
Institutions: Vanderbilt University.
Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic mild cognitive impairment (aMCI) and Alzheimer's disease (AD). Whereas behavioral data inform us about the patients' performance, ERPs allow us to record discrete changes in brain activity. Further, ERPs can give us insight into the onset, duration, and interaction of independent cognitive processes associated with memory retrieval. In patient populations, these types of studies are used to examine which aspects of memory are impaired and which remain relatively intact compared to a control population. The methodology for collecting ERP data from a vulnerable patient population while these participants perform a recognition memory task is reviewed. This protocol includes participant preparation, quality assurance, data acquisition, and data analysis. In addition to basic setup and acquisition, we will also demonstrate localization techniques to obtain greater spatial resolution and source localization using high-density (128 channel) electrode arrays.
Medicine, Issue 54, recognition memory, episodic memory, event-related potentials, dual process, Alzheimer's disease, amnestic mild cognitive impairment
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Investigating the Neural Mechanisms of Aware and Unaware Fear Memory with fMRI
Authors: David C. Knight, Kimberly H. Wood.
Institutions: University of Alabama at Birmingham.
Pavlovian fear conditioning is often used in combination with functional magnetic resonance imaging (fMRI) in humans to investigate the neural substrates of associative learning 1-5. In these studies, it is important to provide behavioral evidence of conditioning to verify that differences in brain activity are learning-related and correlated with human behavior. Fear conditioning studies often monitor autonomic responses (e.g. skin conductance response; SCR) as an index of learning and memory 6-8. In addition, other behavioral measures can provide valuable information about the learning process and/or other cognitive functions that influence conditioning. For example, the impact unconditioned stimulus (UCS) expectancies have on the expression of the conditioned response (CR) and unconditioned response (UCR) has been a topic of interest in several recent studies 9-14. SCR and UCS expectancy measures have recently been used in conjunction with fMRI to investigate the neural substrates of aware and unaware fear learning and memory processes 15. Although these cognitive processes can be evaluated to some degree following the conditioning session, post-conditioning assessments cannot measure expectations on a trial-to-trial basis and are susceptible to interference and forgetting, as well as other factors that may distort results 16,17 . Monitoring autonomic and behavioral responses simultaneously with fMRI provides a mechanism by which the neural substrates that mediate complex relationships between cognitive processes and behavioral/autonomic responses can be assessed. However, monitoring autonomic and behavioral responses in the MRI environment poses a number of practical problems. Specifically, 1) standard behavioral and physiological monitoring equipment is constructed of ferrous material that cannot be safely used near the MRI scanner, 2) when this equipment is placed outside of the MRI scanning chamber, the cables projecting to the subject can carry RF noise that produces artifacts in brain images, 3) artifacts can be produced within the skin conductance signal by switching gradients during scanning, 4) the fMRI signal produced by the motor demands of behavioral responses may need to be distinguished from activity related to the cognitive processes of interest. Each of these issues can be resolved with modifications to the setup of physiological monitoring equipment and additional data analysis procedures. Here we present a methodology to simultaneously monitor autonomic and behavioral responses during fMRI, and demonstrate the use of these methods to investigate aware and unaware memory processes during fear conditioning.
Neuroscience, Issue 56, fMRI, conditioning, learning, memory, fear, contingency awareness, neuroscience, skin conductance
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Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis
Authors: Joseph F.X. DeSouza, Shima Ovaysikia, Laura K. Pynn.
Institutions: Centre for Vision Research, York University, Centre for Vision Research, York University.
The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar tasks. Participants' behavior during task performance in an fMRI scanner can then be correlated to the brain activity using the blood-oxygen-level-dependent signal. We measure behavior to be able to sort correct trials, where the subject performed the task correctly and then be able to examine the brain signals related to correct performance. Conversely, if subjects do not perform the task correctly, and these trials are included in the same analysis with the correct trials we would introduce trials that were not only for correct performance. Thus, in many cases these errors can be used themselves to then correlate brain activity to them. We describe two complementary tasks that are used in our lab to examine the brain during suppression of an automatic responses: the stroop1 and anti-saccade tasks. The emotional stroop paradigm instructs participants to either report the superimposed emotional 'word' across the affective faces or the facial 'expressions' of the face stimuli1,2. When the word and the facial expression refer to different emotions, a conflict between what must be said and what is automatically read occurs. The participant has to resolve the conflict between two simultaneously competing processes of word reading and facial expression. Our urge to read out a word leads to strong 'stimulus-response (SR)' associations; hence inhibiting these strong SR's is difficult and participants are prone to making errors. Overcoming this conflict and directing attention away from the face or the word requires the subject to inhibit bottom up processes which typically directs attention to the more salient stimulus. Similarly, in the anti-saccade task3,4,5,6, where an instruction cue is used to direct only attention to a peripheral stimulus location but then the eye movement is made to the mirror opposite position. Yet again we measure behavior by recording the eye movements of participants which allows for the sorting of the behavioral responses into correct and error trials7 which then can be correlated to brain activity. Neuroimaging now allows researchers to measure different behaviors of correct and error trials that are indicative of different cognitive processes and pinpoint the different neural networks involved.
Neuroscience, Issue 64, fMRI, eyetracking, BOLD, attention, inhibition, Magnetic Resonance Imaging, MRI
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Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice
Authors: Michael T. Kuhlmann, Simon Cuhlmann, Irmgard Hoppe, Rob Krams, Paul C. Evans, Gustav J. Strijkers, Klaas Nicolay, Sven Hermann, Michael Schäfers.
Institutions: Westfälische Wilhelms-University Münster, Imperial College London , Imperial College London , Eindhoven University of Technology.
It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation. In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed. In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.
Medicine, Issue 59, atherosclerosis, mouse, cardiovascular disease, shear stress
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Habituation and Prepulse Inhibition of Acoustic Startle in Rodents
Authors: Bridget Valsamis, Susanne Schmid.
Institutions: University of Western Ontario.
The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3. We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4. Habituation is stimulus specific5. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used. Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10. Both habituation and PPI are disrupted in patients suffering from schizophrenia11, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15 Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.
Neuroscience, Issue 55, Startle responses, rat, mouse, sensory gating, sensory filtering, short-term habituation, long-term habituation, prepulse inhibition
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Behavioral Determination of Stimulus Pair Discrimination of Auditory Acoustic and Electrical Stimuli Using a Classical Conditioning and Heart-rate Approach
Authors: Simeon J. Morgan, Antonio G. Paolini.
Institutions: La Trobe University.
Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural auditory prostheses, such as auditory brainstem implants1-3 and auditory midbrain implants4,5. While acute experiments can give initial insight to the effectiveness of the implant, testing the chronically implanted and awake animals provides the advantage of examining the psychophysical properties of the sensations induced using implanted devices6,7. Several techniques such as reward-based operant conditioning6-8, conditioned avoidance9-11, or classical fear conditioning12 have been used to provide behavioral confirmation of detection of a relevant stimulus attribute. Selection of a technique involves balancing aspects including time efficiency (often poor in reward-based approaches), the ability to test a plurality of stimulus attributes simultaneously (limited in conditioned avoidance), and measure reliability of repeated stimuli (a potential constraint when physiological measures are employed). Here, a classical fear conditioning behavioral method is presented which may be used to simultaneously test both detection of a stimulus, and discrimination between two stimuli. Heart-rate is used as a measure of fear response, which reduces or eliminates the requirement for time-consuming video coding for freeze behaviour or other such measures (although such measures could be included to provide convergent evidence). Animals were conditioned using these techniques in three 2-hour conditioning sessions, each providing 48 stimulus trials. Subsequent 48-trial testing sessions were then used to test for detection of each stimulus in presented pairs, and test discrimination between the member stimuli of each pair. This behavioral method is presented in the context of its utilisation in auditory prosthetic research. The implantation of electrocardiogram telemetry devices is shown. Subsequent implantation of brain electrodes into the Cochlear Nucleus, guided by the monitoring of neural responses to acoustic stimuli, and the fixation of the electrode into place for chronic use is likewise shown.
Neuroscience, Issue 64, Physiology, auditory, hearing, brainstem, stimulation, rat, abi
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Perceptual and Category Processing of the Uncanny Valley Hypothesis' Dimension of Human Likeness: Some Methodological Issues
Authors: Marcus Cheetham, Lutz Jancke.
Institutions: University of Zurich.
Mori's Uncanny Valley Hypothesis1,2 proposes that the perception of humanlike characters such as robots and, by extension, avatars (computer-generated characters) can evoke negative or positive affect (valence) depending on the object's degree of visual and behavioral realism along a dimension of human likeness (DHL) (Figure 1). But studies of affective valence of subjective responses to variously realistic non-human characters have produced inconsistent findings 3, 4, 5, 6. One of a number of reasons for this is that human likeness is not perceived as the hypothesis assumes. While the DHL can be defined following Mori's description as a smooth linear change in the degree of physical humanlike similarity, subjective perception of objects along the DHL can be understood in terms of the psychological effects of categorical perception (CP) 7. Further behavioral and neuroimaging investigations of category processing and CP along the DHL and of the potential influence of the dimension's underlying category structure on affective experience are needed. This protocol therefore focuses on the DHL and allows examination of CP. Based on the protocol presented in the video as an example, issues surrounding the methodology in the protocol and the use in "uncanny" research of stimuli drawn from morph continua to represent the DHL are discussed in the article that accompanies the video. The use of neuroimaging and morph stimuli to represent the DHL in order to disentangle brain regions neurally responsive to physical human-like similarity from those responsive to category change and category processing is briefly illustrated.
Behavior, Issue 76, Neuroscience, Neurobiology, Molecular Biology, Psychology, Neuropsychology, uncanny valley, functional magnetic resonance imaging, fMRI, categorical perception, virtual reality, avatar, human likeness, Mori, uncanny valley hypothesis, perception, magnetic resonance imaging, MRI, imaging, clinical techniques
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Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice
Authors: Kyung Ho Kim, Andrew Barazia, Jaehyung Cho.
Institutions: University of Illinois at Chicago , University of Illinois at Chicago .
Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil β2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-α-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.
Immunology, Issue 74, Medicine, Cellular Biology, Molecular Biology, Inflammation, Hematology, Neutrophils, Microscopy, Video, Thrombosis, Platelet Activation, Platelet Aggregation, Intravital microscopy, platelet, neutrophil, rolling, adhesion, vascular inflammation, thrombus formation, mice, animal model
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A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology
Authors: Christian Erbel, Deniz Okuyucu, Mohammadreza Akhavanpoor, Li Zhao, Susanne Wangler, Maani Hakimi, Andreas Doesch, Thomas J. Dengler, Hugo A. Katus, Christian A. Gleissner.
Institutions: University of Heidelberg, University of Heidelberg, SLK Hospital am Plattenwald.
Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.
Medicine, Issue 87, ex vivo model, human, tissue culture, atherosclerosis, immune response, inflammation, chronic inflammatory disease
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An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection
Authors: Marloes Vissers, Marrit N. Habets, Inge M. L. Ahout, Jop Jans, Marien I. de Jonge, Dimitri A. Diavatopoulos, Gerben Ferwerda.
Institutions: Radboud university medical center.
Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology. Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.
Immunology, Issue 82, Blood Cells, Respiratory Syncytial Virus, Human, Respiratory Tract Infections, Paramyxoviridae Infections, Models, Immunological, Immunity, HRSV culture, purification, quantification, PBMC isolation, stimulation, inflammatory pathways
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Using Eye Movements to Evaluate the Cognitive Processes Involved in Text Comprehension
Authors: Gary E. Raney, Spencer J. Campbell, Joanna C. Bovee.
Institutions: University of Illinois at Chicago.
The present article describes how to use eye tracking methodologies to study the cognitive processes involved in text comprehension. Measuring eye movements during reading is one of the most precise methods for measuring moment-by-moment (online) processing demands during text comprehension. Cognitive processing demands are reflected by several aspects of eye movement behavior, such as fixation duration, number of fixations, and number of regressions (returning to prior parts of a text). Important properties of eye tracking equipment that researchers need to consider are described, including how frequently the eye position is measured (sampling rate), accuracy of determining eye position, how much head movement is allowed, and ease of use. Also described are properties of stimuli that influence eye movements that need to be controlled in studies of text comprehension, such as the position, frequency, and length of target words. Procedural recommendations related to preparing the participant, setting up and calibrating the equipment, and running a study are given. Representative results are presented to illustrate how data can be evaluated. Although the methodology is described in terms of reading comprehension, much of the information presented can be applied to any study in which participants read verbal stimuli.
Behavior, Issue 83, Eye movements, Eye tracking, Text comprehension, Reading, Cognition
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Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry
Authors: Heather Inglis, Philip Norris, Ali Danesh.
Institutions: Blood Systems Research Institute, University of California, San Francisco, University of California, San Francisco.
Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal–to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.
Cellular Biology, Issue 97, microvesicles, flow cytometry, exosomes, extracellular vesicles, high throughput, microparticles
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