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&deg; 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 &mu;L 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 &mu;L of clear liquid from centrifuged samples into appropriate wells in plate (in duplicate).</li>
	<li>Prepare enzyme conjugate and pipette 200 &mu;L 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 &mu;L 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 &mu;L 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="Figure 1" src="/files/ftp_upload/3238/3238fig1.jpg" /> 
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="Figure 2" src="/files/ftp_upload/3238/3238fig2.jpg" /> 
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="Figure 3" src="/files/ftp_upload/3238/3238fig3.jpg" /> 
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="Figure 4" src="/files/ftp_upload/3238/3238fig4.jpg" /> 
Figure 4. Representative salivary cortisol data. Note: Cortisol concentration in uL/dL. Representative values reflect data collected in our lab and published in literature.

<ol>
	<li>Kudielka, B. M., Hellhammer, D. H., Kirschbaum, C., Harmon-Jones, E., Winkielman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ten+years+of+research+with+the+Trier+Social+Stress+Test+-+Revisited.">Ten years of research with the Trier Social Stress Test - Revisited.</a> Social Neuroscience: Integrating Biological and Psychological Explanations. , (2007).</li><li>Dickerson, S. S., Kemeny, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+stressors+and+cortisol+responses:+A+theoretical+integration+and+synthesis+of+laboratory+research.">Acute stressors and cortisol responses: A theoretical integration and synthesis of laboratory research.</a> Psychol. Bull. 130, 355-391 (2004).</li><li>Kirschbaum, C., Pirke, K., Hellhammer, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+"Trier+social+stress+test"+-+a+tool+for+investigating+psychobiological+stress+response+in+a+laboratory+setting.">The "Trier social stress test" - a tool for investigating psychobiological stress response in a laboratory setting.</a> Neuropsychobiology. 28, 76-81 (1993).</li><li>Gruenewald, T. L., Kemeny, M. E., Aziz, N., Fahey, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+threat+to+the+social+self:+shame,+social+self-esteem,+and+cortisol+activity.">Acute threat to the social self: shame, social self-esteem, and cortisol activity.</a> Psychosom. Med. 66, 915-924 (2004).</li><li>Mendes, W. B., Harmon-Jones, E., Beer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+autonomic+nervous+system+activity.">Assessing autonomic nervous system activity.</a> Methods in Social Neuroscience. , 118-147 (2009).</li><li>Spielberger, C. D., Gorsuch, R. L., Lushene, P. R., Vagg, P. R., Jacobs, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual for the State-Trait Anxiety Inventory (Form Y). , (1983).</li><li>Dawans, B. v. o. n., Kirschbaum, C., Heinrichs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Trier+Social+Stress+Test+for+groups+(TSST-G):+A+new+research+tool+for+controlled+simultaneous+social+stress+exposure+in+a+group+format.">The Trier Social Stress Test for groups (TSST-G): A new research tool for controlled simultaneous social stress exposure in a group format.</a> Psychoneuroendocrinology. 36, 514-522 (2011).</li><li>Pace, T. W. W., Negi, L. T., Adame, D. D., Cole, S. P., Sivilli, T. I., Brown, T. D., Issa, M. J., Raison, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+compassion+meditation+on+neuroendocrine,+innate+immune+and+behavioral+responses+to+psychosocial+stress.">Effect of compassion meditation on neuroendocrine, innate immune and behavioral responses to psychosocial stress.</a> Psychoneuroendocrinology. 34, 87-98 (2010).</li><li>Kirschbaum, C., W&#252;rst, S., Hellhammer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consistent+sex+differences+in+cortisol+responses+to+psychological+stress.">Consistent sex differences in cortisol responses to psychological stress.</a> Psychosom. Med. 54, 648-657 (1992).</li><li>Therrien, F., Drapeau, V., Lalonde, J., Lupien, S. J., Beaulieu, S., Dore, J., Tremblay, A., Richard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortisol+response+to+the+Trier+Social+Stress+Test+in+obese+and+reduced+obese+individuals.">Cortisol response to the Trier Social Stress Test in obese and reduced obese individuals.</a> Biol. Psychol. 84, 325-329 (2010).</li><li>W&#252;st, S., Entringer, S., Federenko, I. S., Schlotz, W., Hellhammer, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birth+weight+is+associated+with+salivary+cortisol+responses+to+psychosocial+stress+in+adult+life.">Birth weight is associated with salivary cortisol responses to psychosocial stress in adult life.</a> Psychoneuroendocrinology. 30, 591-598 (2005).</li><li>Simeon, D., Yehuda, R., Cunill, R., Knutelska, M., Putnam, F. W., Smith, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+associated+with+resilience+in+healthy+adults.">Factors associated with resilience in healthy adults.</a> Psychoneuroendocrinology. 32, 1149-1152 (2007).</li><li>Quirin, M., Pruessner, J. C., Kuhl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPA+system+regulation+and+adult+attachment+anxiety:+Individual+differences+in+reactive+and+awakening+cortisol.">HPA system regulation and adult attachment anxiety: Individual differences in reactive and awakening cortisol.</a> Psychoneuroendocrinology. 33, 581-590 (2009).</li><li>Pierrehumbert, B., Torrisi, R., Glatz, N., Dimitrova, N., Heinrichs, M., Halfon, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+attachment+on+perceived+stress+and+cortisol+response+to+acute+stress+in+women+sexually+abused+in+childhood+or+adolescence.">The influence of attachment on perceived stress and cortisol response to acute stress in women sexually abused in childhood or adolescence.</a> Psychoneuroendocrinology. 34, 924-938 (2009).</li><li>Fiocco, A. J., Joober, R., Lupien, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Education+modulates+cortisol+reactivity+to+the+Trier+Social+Stress+Test+in+middle-aged+adults.">Education modulates cortisol reactivity to the Trier Social Stress Test in middle-aged adults.</a> Psychoneuroendocrinology. 32, 1158-1163 (2007).</li><li>Oswald, L. M., Zandi, P., Nestadt, G., Potash, J. B., Kalaydjian, A. E., Wand, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+cortisol+responses+to+stress+and+personality.">Relationship between cortisol responses to stress and personality.</a> Neuropsychopharmacology. 31, 1583-1591 (2006).</li><li>Fries, E., Hellhammer, D. H., Hellhammer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+of+the+hypothalamic-pituitary-adrenal+axis+responsivity+to+the+Trier+Social+Stress+Test+by+the+benzodiazepine+alprazolam.">Attenuation of the hypothalamic-pituitary-adrenal axis responsivity to the Trier Social Stress Test by the benzodiazepine alprazolam.</a> Psychoneuroendocrinology. 31, 1278-1288 (2006).</li><li>Kudielka, B. M., Fischer, J. 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. D., Roy, M., Peretz, I., Lupien, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+relaxing+music+on+salivary+cortisol+level+after+psychological+stress.">Effects of relaxing music on salivary cortisol level after psychological stress.</a> Ann. NY Acad. Sci. 999, 374-376 (2003).</li><li>Balodis, I. M., Wynne-Edwards, K. E., Olmstead, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+other+side+of+the+curve:+Examining+the+relationship+between+pre-stressor+physiological+responses+and+stress+reactivity.">The other side of the curve: Examining the relationship between pre-stressor physiological responses and stress reactivity.</a> Psychoneuroendocrinology. 35, 1363-1373 (2010).</li><li>Shultheiss, O. C., Stanton, S. J., Harmon-Jones, E., Beer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+salivary+hormones.">Assessment of salivary hormones.</a> Methods in Social Neuroscience. , 17-44 (2009).</li><li>Kudielka, B. M., Hellhammer, D. H., W&#252;st, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+we+respond+so+differently?+Reviewing+determinants+of+human+salivary+cortisol+responses+to+challenge.">Why do we respond so differently? Reviewing determinants of human salivary cortisol responses to challenge.</a> Psychoneuroendocrinology. 34, 2-18 (2009).</li><li>Buske-Kirschbaum, A., Jobst, S., Wustmans, A., Kirschbaum, C., Rauh, W., Hellhammer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuated+free+cortisol+response+to+psychosocial+stress+in+children+with+atopic+dermatitis.">Attenuated free cortisol response to psychosocial stress in children with atopic dermatitis.</a> Psychosom. Med. 59, 419-426 (1997).</li></ol>

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 ...

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

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55.8K Views.  Northern Arizona University. 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.

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.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

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 ...

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 ...

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 ...

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal vascular occlusion, retinal detachment, and diabetic retinopathy. It was the object of this study to introduce a standardized stress model for future preclinical therapeutic testing.
Bovine retinas were prepared and perfused with an oxygen saturated standard solution, and the ERG was recorded. After recording stable b-waves, hypoxia (pure N2) or glutamate stress (250 &#181;m glutamate) was exerted for 45 min. To investigate the effects on photoreceptor function alone, 1 mM aspartate was added to obtain a-waves. ERG-recovery was monitored for 75 min.
For hypoxia, a decrease in a-wave amplitude of 87.0% was noted (p &#60;0.01) after an exposition time of 45 min (decrease of 36.5% after the end of the washout p = 0.03). Additionally, an initial decrease in b-wave amplitudes of 87.23% was recorded, that reached statistical significance (p &#60;0.01, decrease of 25.5% at the end of the washout, p = 0.03).
For 250 &#181;m glutamate, an initial 7.8% reduction of a-wave amplitudes (p &#62;0.05) followed by a reduction of 1.9% (p &#62;0.05). A reduction of 83.7% of b-wave amplitudes (p &#60;0.01) was noted; after a washout of 75 min the reduction was 2.3% (p = 0.62). In this study, a standardized stress model is presented that may be useful to identify possible neuroprotective effects in the future.

With this study, we introduce a standardized stress model for the isolated superfused bovine retina for future preclinical therapeutic testing. The effect of either hypoxia (pure N2) or glutamate stress (250 &#181;M glutamate) on retinal function represented by a- and b-wave amplitudes was evaluated.

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

A Revised Method for Inducing Secondary Lymphedema in the Hindlimb of Mice

Animal models are of paramount importance in the research of lymphedema in order to understand the pathophysiology of the disease but also to explore potential treatment options. This mouse model allows researchers to induce significant lymphedema lasting at least 8 weeks. Lymphedema is induced using a combination of fractioned radiotherapy and surgical ablation of lymphatics. This model requires that the mice get a dose of 10 Gray (Gy) radiation before and after surgery. The surgical part of the model involves ligation of three lymph vessels and extraction of two lymph nodes from the mouse hindlimb. Having access to microsurgical tools and a microscope is essential, due to the small anatomical structures of mice. The advantage of this model is that it results in statistically significant lymphedema, which provides a good basis for evaluating different treatment options. It is also a great and easily available option for microsurgical training. The limitation of this model is that the procedure can be time consuming, especially if not practiced in advance. The model results in objectively quantifiable lymphedema in mice, without causing severe morbidity and has been tested in three separate projects.

This animal model enables researchers to induce statistically significant secondary lymphedema in the hindlimb of mice, lasting at least 8 weeks. The model can be used to study the pathophysiology of lymphedema and to investigate novel treatment options.

Animal models are of paramount importance in the research of lymphedema in order to understand the pathophysiology of the disease but also to explore ...