Thank you to Jessica Ottmar for protocol suggestions and contributions to the representative data. Special thanks to Sabrina Blackledge, Lauren Kohoutek and Kerisa Shelton for demonstrating this protocol.

1. Set Up
<ol>
	<li>All research should be approved by the appropriate Institutional Review Board or human subjects review committee prior to data collection.</li>
	<li>Ideally, two rooms should be available to conduct the TSST. A comfortable waiting room should be available to participants prior to beginning the TSST and during recovery periods. A separate interview room should be used during the speech preparation, speech performance and math portions of the TSST to introduce novelty and uncontrolability.</li>
	<li>Arrange the interview room so that the participant will be facing confederate interviewers and position video or audio recording equipment in the visual field of the participant. Lab coats should be available for interviewers to wear during the TSST to increase stress during the TSST. Instruct interviewers to maintain eye contact with participants and refrain from making emotional facial expressions during the TSST.</li>
	<li>Carefully consider the research question(s) when selecting dependent measurements. Guidelines for the analysis and publication of various measurements of stress response are provided by several sources (guidelines for the publication and analysis of psychophysiological measures are provided by the Society for Psychophysiological Research: <a href="http://www.sprweb.org/journal/index.cfm#guidelines" target="_blank">http://www.sprweb.org/journal/index.cfm#guidelines</a>).5 Consultation of these resources during the design of experiments is recommended.</li>
	<li>Set up all equipment needed to record physiological, psychological or endocrine responses to the TSST. This equipment may include (but is not limited to) electrodes and electronic equipment and/or software to record heart rate, cardiovascular reactivity, skin conductance or response, eye blink, muscle contraction/tone, respiratory rate; paper and pencil or electronic versions of psychological instruments such as the State Trait Anxiety Inventory;6 tubes and other collection materials for urine, blood or saliva samples; or video recording equipment. For this demonstration, we will be using three-lead electrocardiogram (ECG) electrodes to measure heart rate during the TSST, and repeated administration of the State Trait Anxiety Inventory to measure psychological response before and after the stressor. We will utilize a basic recording system designed to measure physiological responses (BioPac MP36 system) to record heart rate.</li>
	<li>If blood samples (rather than saliva) will be collected during the experiment, efforts to minimize pain and distress during collection should be made. Blood collection procedures can introduce additional physical or psychological stress, confounding response to the TSST.</li>
	<li>When the participant arrives, welcome them to the lab. Obtain informed consent and seat the participant comfortably.</li>
	<li>Collect demographic information, screening information or additional arrival measures that are required by your experiment. Verify that the participant has not consumed food or beverage (other than water) in the past hour if collecting cortisol measurements. A short period of fasting is recommended if you will be collecting saliva samples for analysis. Changes in pH associated with acidic food or drink or visible contamination can interfere with accurate measurement of salivary markers. Urine toxicology screening may be required if measures of interest may be contaminated by medication or other metabolites. Information about factors that may affect stress response (e.g. medications, illness, sleep, recent stressful events, social phobia) should also be considered when screening participants.</li>
</ol>
2. Pre-stress Measurements
<ol>
	<li>Instruct the participant to wait comfortably for 45 minutes in the waiting room. You may choose to provide the participant with reading material, such as magazines containing emotionally-neutral content, or another emotionally-neutral activity during this period.</li>
	<li>After the 45-minute waiting period, administer the State Trait Anxiety Inventory to assess pre-stress anxiety.6 Alternatively, other instruments that are designed to evaluate state changes in stress, anxiety or relaxation may be administered (e.g. visual analogue scales).</li>
	<li>Collect a "pre-stress" saliva sample. Instruct the participant to rinse their mouth with water. Give the participant a 5-10 cm section of drinking straw and a clean, disposable 2.0 ml polypropylene vial. Instruct the participant to tilt their head forward to allow saliva to pool on the floor of the mouth then pass the saliva through the straw into the vial. Collect approximately 1.5-2.0 ml of saliva from the participant. Alternatively, saliva can be collected by using absorbent dental wick (or similar material) and instructing the participant to chew or saturate the material with saliva. If possible, leave the room to provide the participant with privacy. Label and store all saliva samples at -20° C as soon as possible.</li>
	<li>Record 5 minutes of pre-stress physiological measurements.</li>
</ol>
3. The TSST
<ol>
	<li>If you will be using a separate interview room (to increase the stress effects of the TSST), bring the participant from the waiting room into the interview room.</li>
	<li>To begin the TSST speech preparation period, read the following script to the participant: "This is the speech preparation portion of the task; you are to mentally prepare a five-minute speech describing why you would be a good candidate for your ideal job. Your speech will be videotaped and reviewed by a panel of judges trained in public speaking. You have ten minutes to prepare and your time begins now."</li>
	<li>Record physiological measurements during the 10-minute speech preparation period. Set a digital timer for 10 minutes and leave the room.</li>
	<li>After 10 minutes, return to the room wearing a lab coat. Read the following script to the participant: "This is the speech portion of the task. You are to deliver a speech describing why you would be a good candidate for your ideal job. You should speak for the entire the five-minute time period. Your time begins now." The prop video camera should be turned on now, to increase evaluative/performance stress.</li>
	<li>Record physiological measurements during the 5-minute speech performance period. If the participant stops talking during the speech, allow him or her to remain silent for 20 seconds. If he or she does not resume speaking, prompt the participant to continue speaking by instructing them: "You still have time remaining."</li>
	<li>At the end of the 5-minute speech performance period, read the following script to the participant: "During the final five-minute math portion of this task you will be asked to sequentially subtract the number 13 from 1,022. You will verbally report your answers aloud, and be asked to start over from 1,022 if a mistake is made. Your time begins now." If the participant makes a mistake, prompt them with: "That is incorrect, please start over from 1,022." Set a digital timer for 5 minutes.</li>
	<li>Record physiological measurements during the 5-minute math performance period.</li>
	<li>At the end of the math performance period of the TSST, collect a "post-stress" saliva sample.</li>
	<li>Administer post-stress State Trait Anxiety Inventory.</li>
</ol>
4. Post-stress Recovery Measurements
<ol>
	<li>Return participants to the original waiting room. During the first recovery period, instruct the participant to wait comfortably for 20 minutes. Set a digital timer for 20 minutes and leave the room.</li>
	<li>Return to the waiting room after 20 minutes and collect a "recovery 1" saliva sample.</li>
	<li>During the second recovery period, instruct the participant to again wait comfortably for 20 minutes. Set a digital timer for 20 minutes and leave the room.</li>
	<li>Return to the waiting room after 20 minutes and collect a "recovery 2" saliva sample.</li>
	<li>Instruct the participant to complete the "recovery" State Trait Anxiety Inventory.</li>
	<li>Debrief the participant as to the true nature of the experiment. Inform the participant that their performance was not recorded and that no analysis of their speech or math performance will be conducted. Explain to the participant that the tasks with which they were presented were unreasonably difficult and do not reflect upon the participant's aptitude or ability.</li>
</ol>
5. Data Analysis
<ol>
	<li>To analyze psychological measures, score each State Trait Anxiety Inventory from the pre-stress, post-stress and recovery time points.6</li>
	<li>To analyze physiological measures, compute a mean heart rate score for each of the 5-minute "pre stress," "speech preparation," "speech performance" and "math" periods of the TSST. Alternatively, compute mean scores for each minute of the TSST or measure heart rate at discrete time points during the TSST.</li>
	<li>To analyze neuroendocrine measures, assay salivary samples for hormone concentration using commercially available EIA kits.</li>
</ol>
6. Optional: Additional Salivary Cortisol Assay Protocol Information (reproduced from Salimetrics salivary cortisol assay kit insert)
<ol>
	<li>Bring all salivary assay kit reagents to room temperature.</li>
	<li>Prepare wash buffer according to kit instructions.</li>
	<li>Determine plate layout and pipette 25 μL of standards and controls into appropriate wells in duplicate.</li>
	<li>Thaw saliva samples, vortex and centrifuge at 1,500 X G (@3,000 RPM) for 15 minutes.</li>
	<li>Pipette 25 μL of clear liquid from centrifuged samples into appropriate wells in plate (in duplicate).</li>
	<li>Prepare enzyme conjugate and pipette 200 μL into all wells.</li>
	<li>Mix plate for 5 minutes at 500 rpm and incubate for an additional 55 minutes.</li>
	<li>Wash and blot the plate 4 times with wash buffer.</li>
	<li>Add 200 μL tetramethylbenzidine (TMB) solution to each well using a multichannel pipette.</li>
	<li>Mix the plate for 5 minutes at 500 rpm and incubate in the dark for an additional 25 minutes.</li>
	<li>Add 50 μL stop solution to each well and mix for 3 minutes at 500 rpm to stop the reactions.</li>
	<li>Wipe the plate bottom clean and read the plate in a plate reader at 450 nm within 10 minutes of adding stop solution.</li>
	<li>To calculate cortisol concentration, compute the average optical density for all duplicate wells. Subtract the average optical density of the non-specific binding wells from the average optical density of the zero, standard, control and sample wells.</li>
	<li>Calculate the percent bound (B/Bo) for each standard, control and sample by dividing the average optical density of these wells (B) by the average optical density of the zero wells (Bo). Determine the concentration of the controls and samples by interpolation using software capable of logistics.</li>
</ol>
7. Representative Results:
The representative data presented here is compiled from a review of the literature and two studies that were conducted in our lab during the afternoon with healthy adult male participants. These data are intended to approximately represent the results that might be found using the protocol presented in this article.
This TSST protocol (Figure 1) induces increases in self-reported anxiety (as measured by the State Trait Anxiety Inventory; Figure 2), heart rate (Figure 3), and salivary cortisol concentrations (Figure 4). The experimental protocol illustrated in Figure 1 is meant to provide one example of how various measurements might be made during a data collection session with the TSST. The timing of measurements and the inclusion of different measures will be dictated by the specific research question. If using an intervention that is intended to reduce anxiety and/or stress response, blunted or attenuated increases in these measures are predicted.
<img alt="TSST diagram: timeline of stress test phases, including heart rate and saliva sampling measurements." src="/files/ftp_upload/3238/3238fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Experimental protocol. Note: Trier Social Stress Test (TSST); State Trait Anxiety Inventory (STAI); 5-minute Pre Stress period (PS); 10-minute Anticipatory Stress period (speech preparation) (AS); 5-minute Speech period (S); 5-minute Math period (M).
<img alt="State anxiety scores over time; TSST stress response; line graph; psychological study results." src="/files/ftp_upload/3238/3238fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Representative state anxiety scores. Note: State Anxiety score calculated from 20-iten state anxiety measure of State Trait Anxiety inventory (STAI). Representative scores reflect data collected in our lab and published in literature.
<img alt="Heart rate variation chart during TSST phases; stress response analysis; BPM vs. tasks graph." src="/files/ftp_upload/3238/3238fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Representative heart rate data. Note: Heart rate in beats per minute (BPM). Representative values reflect data collected in our lab and published in literature.
<img alt="Cortisol level graph; TSST impact on stress recovery; hormone analysis chart." src="/files/ftp_upload/3238/3238fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Representative salivary cortisol data. Note: Cortisol concentration in uL/dL. Representative values reflect data collected in our lab and published in literature.

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E., Metzenthin, P., Helfricht, S., Preckel, D., von K&#228;nel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+effect+of+5-day+treatment+with+acetylsalicylic+acid+(aspirin)+or+the+beta-blocker+propranolol+(Inderal)+on+free+cortisol+responses+to+acute+psychosocial+stress:+A+randomized+double-blind,+placebo-controlled+study.">No effect of 5-day treatment with acetylsalicylic acid (aspirin) or the beta-blocker propranolol (Inderal) on free cortisol responses to acute psychosocial stress: A randomized double-blind, placebo-controlled study.</a> Neuropsychobiology. 56, 159-166 (2007).</li><li>Gaab, J., Bl&#228;ttler, N., Menzl, T., Pabst, B., Stoyer, S., Ehlert, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Randomized+controlled+evaluation+of+the+effects+of+cognitive-behavioral+stress+management+on+cortisol+responses+to+acute+stress+in+healthy+subjects.">Randomized controlled evaluation of the effects of cognitive-behavioral stress management on cortisol responses to acute stress in healthy subjects.</a> Psychoneuroendocrinology. 28, 767-779 (2003).</li><li>Khalfa, S., Bella, S. 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No conflicts of interest declared.

In this article, we demonstrated how to conduct the Trier Social Stress Test in a healthy volunteer. The TSST is a standardized laboratory social stressor that induces robust and reliable increases in psychological, physiological and neuroendocrine measures. The TSST is a useful alternative to physical stressors such at the cold presser test or treadmill walking, and reproduces the more naturalistic psychological stress of performance in the presence of an evaluative audience.
Many aspects of the TSST can be modified to meet the needs of various research programs. The measurement time points demonstrated in this article represent a minimum of sampling. Many experiments may require additional sampling points and measurements to best capture changes in stress response. The length of the anticipatory, speech and math periods (generally ranging from 5-10 minutes), the inclusion or omission of recovery period(s), and the composition of the evaluative audience during the speech and math performances (ranging from a video camera to a panel of evaluators) can be modified. The TSST can be administered to groups as well as individuals.7 Additional measures of HPA- or SAM-axis activation, activity of the sympathetic and parasympathetic nervous system, or psychological self-report of stress can be included to assess response to the TSST. For example, some research groups may find it useful to measure heart rate variability, electrodermal response, blood pressure, startle or eye blink response, or changes in body temperature in response to the TSST. For a review of measures commonly used to assess autonomic nervous system activity, please see Mendes, 2009.7 Recently, some research groups have also begun to investigate changes in immune response to the TSST.8 Many options for assessing psychological response to the TSST are also available (e.g. the Profile of Mood States, visual analogue scales, or Likert-type ratings of stress, anxiety or relaxation). 
Differences in individual response to the TSST are an important concern when using this protocol. In healthy adult samples, the TSST has been reported to increase salivary cortisol levels two to three fold in approximately 70-80% of participants, however responses vary widely among individuals.1 Researchers have documented effects of age,1 gender,9, 10 birth weight,11 resilience/attachment style,12, 13 childhood abuse,14 education,15 personality traits,16 use of nicotine/alcohol/caffeine1 or use of medications (particularly sedatives, anxiolytics or hormone-based contraceptives),17, 18 cognitive behavioral therapy,19 compassion meditation,8 and the presence of music20 on TSST response in healthy populations. Recently, researchers have suggested measurements that are taken prior to a waiting period before the TSST are important and may reveal information about individual stress reactivity.21 For a more thorough discussion of individual differences in assessment of salivary hormones, and overview of the use of salivary hormone assessment in social neuroscience, see Schultheiss &amp; Stanton, 2009.22 For a more detailed presentation of individual differences in TSST performance, see Kudielka et al., 2009.23 
In addition to individual differences, cortisol is subject to circadian fluctuation, with high levels in the morning then decreasing through the day. If cortisol changes will be used as a measure in response to the TSST, it is important to collect samples at consistent time points for accurate analysis and to avoid confounding TSST-induced cortisol changes with the cortisol response that occurs upon awakening in the morning. 
Although many reports have focused on using the TSST to study stress response in healthy, adult populations, this protocol is appropriate for use in clinical populations and has been adapted for use with children.24 This protocol may contribute to important understanding about the role of various biological pathways involved in stress-related disorders or developmental variability of the stress response.

<table><tbody><tr><td>Name of the equipment/Supply </td><td>Company</td><td>Catalogue number</td><td></td></tr><tr><td>BSL Psychophysiology System, Mac OSIncluding MP36 Data Acquisition Unit and three lead electrocardiogram electrodes</td><td>Biopac Systems, Inc.</td><td>BSLPSY-M</td><td></td></tr><tr><td>State Trait Anxiety Inventory</td><td>Mind Garden, Inc.</td><td>STAID-B</td><td></td></tr><tr><td>Salivary Cortisol Enzyme Immuno Assay Kit</td><td>Salimetrics</td><td>1-3002</td><td></td></tr><tr><td>2.0 ml polypropylene vials</td><td>Fisher Scientific</td><td>05 40B 146</td><td></td></tr><tr><td>BioRad Microplate Reader, Model 680, with Microplate Manager software</td><td>Bio-Rad</td><td>Plate Reader: 168 1000Software: 1706800</td><td></td></tr></tbody></table>

the trier social stress test protocol for inducing psychological stress

This article demonstrates a psychological stress protocol for use in a laboratory setting. Protocols that allow researchers to study the biological pathways of the stress response in health and disease are fundamental to the progress of research in stress and anxiety.1 Although numerous protocols exist for inducing stress response in the laboratory, many neglect to provide a naturalistic context or to incorporate aspects of social and psychological stress. Of psychological stress protocols, meta-analysis suggests that the Trier Social Stress Test (TSST) is the most useful and appropriate standardized protocol for studies of stress hormone reactivity.2 In the original description of the TSST, researchers sought to design and evaluate a procedure capable of inducing a reliable stress response in the majority of healthy volunteers.3 These researchers found elevations in heart rate, blood pressure and several endocrine stress markers in response to the TSST (a psychological stressor) compared to a saline injection (a physical stressor).3 Although the TSST has been modified to meet the needs of various research groups, it generally consists of a waiting period upon arrival, anticipatory speech preparation, speech performance, and verbal arithmetic performance periods, followed by one or more recovery periods. The TSST requires participants to prepare and deliver a speech, and verbally respond to a challenging arithmetic problem in the presence of a socially evaluative audience.3 Social evaluation and uncontrollability have been identified as key components of stress induction by the TSST.4 In use for over a decade, the goal of the TSST is to systematically induce a stress response in order to measure differences in reactivity, anxiety and activation of the hypothalamic-pituitary-adrenal (HPA) or sympathetic-adrenal-medullary (SAM) axis during the task.1 Researchers generally assess changes in self-reported anxiety, physiological measures (e.g. heart rate), and/or neuroendocrine indices (e.g. the stress hormone cortisol) in response to the TSST. Many investigators have adopted salivary sampling for stress markers such as cortisol and alpha-amylase (a marker of autonomic nervous system activation) as an alternative to blood sampling to reduce the confounding stress of blood-collection techniques. In addition to changes experienced by an individual completing the TSST, researchers can compare changes between different treatment groups (e.g. clinical versus healthy control samples) or the effectiveness of stress-reducing interventions.1

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The Trier Social Stress Test Protocol for Inducing Psychological Stress

This article describes a protocol for inducing psychological stress in participants, which enables researchers to measure psychological, physiological and neuroendocrine responses to stress within single participants or between groups.

This article demonstrates a psychological stress protocol for use in a laboratory setting.  Protocols that allow researchers to study the biological ...

the-trier-social-stress-test-protocol-for-inducing-psychological

Nanyang Technological University

Research

JoVE Journal

Medicine

56.2K Views.  Northern Arizona University. Dit artikel beschrijft een protocol voor het induceren van psychologische stress bij de deelnemers, waarin onderzoekers in staat stelt om psychologische, fysiologische en neuro-endocriene respons op stress te meten binnen enkele deelnemers of tussen de groepen.

Video: The Trier Social Stress Test Protocol for Inducing Psychological Stress

Using Visual and Narrative Methods to Achieve Fair Process in Clinical Care

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is respect for patient's values, preferences, and expressed needs. Yet specific approaches to gaining this understanding and translating it to quality care in the clinical setting are lacking. From a patient perspective quality is not a simple concept but is best understood in terms of five dimensions: technical outcomes; decision-making efficiency; amenities and convenience; information and emotional support; and overall patient satisfaction. Failure to consider quality from this five-pronged perspective results in a focus on medical outcomes, without considering the processes central to quality from the patient's perspective and vital to achieving good outcomes. In this paper, we argue for applying the concept of fair process in clinical settings. Fair process involves using a collaborative approach to exploring diagnostic issues and treatments with patients, explaining the rationale for decisions, setting expectations about roles and responsibilities, and implementing a core plan and ongoing evaluation. Fair process opens the door to bringing patient expertise into the clinical setting and the work of developing health care goals and strategies. This paper provides a step by step illustration of an innovative visual approach, called photovoice or photo-elicitation, to achieve fair process in clinical work with acquired brain injury survivors and others living with chronic health conditions. Applying this visual tool and methodology in the clinical setting will enhance patient-provider communication; engage patients as partners in identifying challenges, strengths, goals, and strategies; and support evaluation of progress over time. Asking patients to bring visuals of their lives into the clinical interaction can help to illuminate gaps in clinical knowledge, forge better therapeutic relationships with patients living with chronic conditions such as brain injury, and identify patient-centered goals and possibilities for healing. The process illustrated here can be used by clinicians, (primary care physicians, rehabilitation therapists, neurologists, neuropsychologists, psychologists, and others) working with people living with chronic conditions such as acquired brain injury, mental illness, physical disabilities, HIV/AIDS, substance abuse, or post-traumatic stress, and by leaders of support groups for the types of patients described above and their family members or caregivers.

This paper illustrates an innovative visual approach (photovoice or photo-elicitation) to achieve fair process in clinical care for patients living with chronic health conditions, illuminate gaps in clinical knowledge, forge better therapeutic relationships, and identify patient-centered goals and possibilities for healing.

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is ...

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

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.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

The Social Dimension of Stress: Experimental Manipulations of Social Support and Social Identity in the Trier Social Stress Test

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations supportive behavior of other people as well as positive social relationships can act as powerful resources to cope with stress. In order to study the interplay between these variables, this protocol describes two effective experimental manipulations of social relationships and supportive behavior in the laboratory. In the present article, these two manipulations are implemented in the Trier Social Stress Test (TSST)&#8212;a standard stress induction paradigm in which participants are subjected to a simulated job interview. More precisely, we propose (a) a manipulation of the relationship between different protagonists in the TSST by making a shared social identity salient and (b) a manipulation of the behavior of the TSST-selection committee, which acts either supportively or unsupportively. These two experimental manipulations are designed in a modular fashion and can be applied independently of each other but can also be combined. Moreover, these two manipulations can also be integrated into other stress protocols and into other standardized social interactions such as trust games, negotiation tasks, or other group tasks.

Previous research on the social dimension of stress has focused on two important variables: social identity and social support. This protocol introduces an effective experimental manipulation of these two social variables and describes their implementation in a standard stress induction paradigm (Trier Social Stress Test).

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations ...

Behavior

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the biological output produced by the Hypothalamic-Pituitary-Adrenal (HPA) axis in humans. While studies have explored how modifications to the TSST influence stress responsivity, few studies have created a modified TSST appropriate for vulnerable youth that elicits a significant cortisol stress response. Thus, the current study sought to modify or adjust the TSST in a culturally sensitive way for a vulnerable sample of 14 year-old adolescents. The present study took place within the context of a longitudinal birth cohort study of Mexican American families in California called the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS). The CHAMACOS sample was optimal to test the effectiveness of a modified culturally appropriate TSST, as it is comprised of Mexican American youth, who are often excluded from research. These youths also have experienced significant early life adversity. Example modifications included timed prompts, alternative math tasks, use of same-ethnicity peers as confederates, debriefing immediately after the conclusion of the TSST, and using an unknown youth examiner to administer the debrief. Saliva samples were collected at baseline (after a resting phase), and then again at 15, 30, and 45 min post-TSST onset to assess cortisol concentration. A pilot study of 50 participants (50% female) have been analyzed for cortisol reaction to the TSST. Results confirmed that this modified version of the TSST was successful at eliciting a significant cortisol reaction, with a wide range of variability likely due to individual differences. Goals for modifications and ethnicity considerations are discussed. This study provides the foundation for future research to utilize a modified TSST with vulnerable youth.

Here, we present a protocol that provoked cortisol reactivity in a vulnerable adolescent Mexican American sample utilizing a modified version of the Trier Social Stress Test (TSST). Saliva samples were collected at baseline, 15, 30, and 45 min post-TSST onset. Future research could utilize this modified TSST with vulnerable youth.

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the ...

Developmental Biology

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. 
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

Enactive Phenomenological Approach to the Trier Social Stress Test: A Mixed Methods Point of View

The single Trier Social Stress Test (TSST) and the TSST for groups (TSST-G) are the most used protocols to experimentally induce psychosocial stress. These tests are based on uncontrollability and social-evaluative threat, inducing psychological and physiological consequences (e.g., anxiety, emotional states, salivary cortisol increases). Many quantitative experimental studies have investigated these stress inducers and these consequences. But, as far as we know, this study is the first to provide a qualitative analysis to access the participants' voices so as to understand the dynamics of their experience throughout the TSST and the TSST-G. This paper outlines a mixed methods approach to the TSST. This approach can help to maximize the information that can be gained from the TSST, which researchers often use without looking more closely at what is qualitatively happening psychologically for participants during the stressor itself. In this way, this protocol is an example of mixed methods, showing the added value of using the enactive phenomenological approach to analyze experimental protocols more deeply. This kind of mixed methods is helpful to access the experience, to understand the actor's point of view, and to analyze in-depth the dynamics of cognitive processes like intentions, perceptions, enacted knowledge, and emotion. The discussion section shows the different uses of a mixed methods protocol, exploiting the enactive phenomenological approach to analyze a protocol or to give a cross vision of the same research subject. This section deals with different existing applications, pointing out some critical steps in this mixed methods approach.

Here, we present an original mixed method combining a quantitative and a qualitative approach to examine accurately participants&#39; experiences during the Trier Social Stress Test and the Trier Social Stress Test for groups.

The single Trier Social Stress Test (TSST) and the TSST for groups (TSST-G) are the most used protocols to experimentally induce psychosocial stress. ...

Threat-Safety Test for Stress Susceptibility or Resilience in Mice Models

Social Threat-Safety Test Uncovers Psychosocial Stress-Related Phenotypes

Social stress is a major cause of the development of mental disorders. To enhance the translational value of preclinical studies, social stress experience and its behavioral impact on mice should be comparable to humans. Chronic social defeat (CSD) utilizes a type of social stress involving physical attacks and sensory threats to induce mental dysfunctions resembling human affective disorders. To strengthen the psychosocial component of CSD, a 10-day CSD protocol was applied in which daily physical attacks are standardized to three 10 s episodes followed by a 24 h sensory phase. After the 10th sensory phase, the CSD protocol is followed by a refined behavioral assay called the social threat-safety test (STST). Post-stress behavioral assays need to determine how and to what extent the social stressor has influenced behavior. The STST allows chronically socially defeated male mice to interact with 2 novel male individuals (social targets): one social target from the attacking strain encountered during the CSD days and the other from a novel strain. Both are presented simultaneously in different compartments of a three-chambered test arena. The test enables a simultaneous assessment of social avoidance development to measure successful aversive conditioned learning and social threat-safety discrimination ability. The development of social avoidance towards both strains reflects a generalized aversive response and thus, a measurement of stress susceptibility. Meanwhile, the development of social avoidance towards only the attacking strain reflects threat-safety discrimination and thus, a measurement of stress resilience. Finally, the absence of social avoidance towards the attacking strain reflects impaired aversive conditioned learning. The protocol aims to refine the currently used mouse models of stress susceptibility/resilience by including translational criteria, specifically threat-safety discrimination and aversive response generalization, to categorize a single group of chronically socially defeated animals into resilient and susceptible subgroups, eventually advancing future translational approaches.

Author Spotlight: Unveiling Mechanisms of Stress Resilience - Significant Findings, Advancements, and Future Research

The social threat-safety test allows a simultaneous assessment of social avoidance development as a measurement of aversive conditioned learning and social threat-safety discrimination ability, both utilized to identify stress-susceptible and stress-resilient individuals within a single group of chronically socially defeated male mice.

Social stress is a major cause of the development of mental disorders. To enhance the translational value of preclinical studies, social stress experience ...

Modeling Social Stress

Stress negatively affects our quality of life. In particular, some individuals experience social stress when placed in a social environment that they are unfamiliar with or have difficulty adjusting to. Since it is hard to examine mechanisms of social stress in humans, modeling this condition in animals may help scientist in developing new therapies for treating this commonly encountered problem.
This science education video begins by discussing the known anatomy and physiology behind stress response. Then, we explain a well-established paradigm for modeling social stress in rodents, the Resident-Intruder task. In the applications section, we review some example studies in which response to stress is measured.

Physiology of Stress; Social Stress- Resident-Intruder Task

Stress negatively affects our quality of life. In particular, some individuals experience social stress when placed in a social environment that they are ...

The Trier Social Stress Test Protocol for Inducing Psychological Stress

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