Name: a real-world high-intensity interval training protocol for cardiorespiratory fitness improvement
Uploaded: 2022-02-22T14:00:00
Description: Watch this Scientific Journal Video about A Real-World High-Intensity Interval Training Protocol for Cardiorespiratory Fitness Improvement at JoVE.com

Thanks to the Centro Integrado de P&#243;s-Gradua&#231;&#227;o e Pesquisa em Sa&#250;de, (CIPq-Sa&#250;de) from the Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM) for providing equipment and technical support for experiments. Thanks to the Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de Minas Gerais (FAPEMIG) (finance codes APQ-00214-21, APQ-00583-21, APQ-00938-18, APQ-03855-16, APQ-01728-18), Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq) (finance code 438498/2018-6), and Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior (CAPES) (Finance code 001) for providing financial support.

This study was approved by the Federal University of the Jequitinhonha and Mucuri Valleys Ethics and Research Committee. All participants were informed of the study objectives and experimental procedures of the study and signed a written informed consent form before their participation.
1. Experimental design
<ol>
	<li>Select individuals who meet the inclusion criteria: nonsmoking healthy individuals aged between 30 and 50 years with body mass index (BMI) of &#8805;25 kg&#18...

<ol>
	<li>Gibala, M. J., Little, J. P., Macdonald, M. J., Hawley, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+adaptations+to+low-volume,+high-intensity+interval+training+in+health+and+disease.">Physiological adaptations to low-volume, high-intensity interval training in health and disease.</a> The Journal of Physiology. 590 (5), 1077-1084 (2012).</li><li>Gist, N. H., Fedewa, M. V., Dishman, R. K., Cureton, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sprint+interval+training+on+aerobic+capacity:+a+systematic+review+and+meta-analysis.">Sprint interval training on aerobic capacity: a systematic review and meta-analysis.</a> Sports Medicine. 44 (2), 269-279 (2014).</li><li>Gillen, J. B., Gibala, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+high-intensity+interval+training+a+time-efficient+exercise+strategy+to+improve+health+and+fitness.">Is high-intensity interval training a time-efficient exercise strategy to improve health and fitness.</a> Applied Physiology, Nutrition, and Metabolism. 39 (3), 409-412 (2014).</li><li>Fowles, J. R., O'Brien, M. W., Solmundson, K., Oh, P. I., Shields, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+is+Medicine+Canada+physical+activity+counselling+and+exercise+prescription+training+improves+counselling,+prescription,+and+referral+practices+among+physicians+across+Canada.">Exercise is Medicine Canada physical activity counselling and exercise prescription training improves counselling, prescription, and referral practices among physicians across Canada.</a> Applied Physiology, Nutrition, and Metabolism. 43 (5), 535-539 (2018).</li><li>Batacan, R. B., Duncan, M. J., Dalbo, V. J., Tucker, P. S., Fenning, A. S. <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+high-intensity+interval+training+on+cardiometabolic+health:+a+systematic+review+and+meta-analysis+of+intervention+studies.">Effects of high-intensity interval training on cardiometabolic health: a systematic review and meta-analysis of intervention studies.</a> British Journal of Sports Medicine. 51 (6), 494-503 (2017).</li><li>Gray, S. R., Ferguson, C., Birch, K., Forrest, L. J., Gill, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-intensity+interval+training:+key+data+needed+to+bridge+the+gap+from+laboratory+to+public+health+policy.">High-intensity interval training: key data needed to bridge the gap from laboratory to public health policy.</a> British Journal of Sports Medicine. 50 (20), 1231-1232 (2016).</li><li>Leger, L. A., Mercier, D., Gadoury, C., Lambert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multistage+20+metre+shuttle+run+test+for+aerobic+fitness.">The multistage 20 metre shuttle run test for aerobic fitness.</a> Journal of Sports Sciences. 6 (2), 93-101 (1988).</li><li>Olds, T., Tomkinson, G., L&#233;ger, L., Cazorla, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worldwide+variation+in+the+performance+of+children+and+adolescents:+an+analysis+of+109+studies+of+the+20-m+shuttle+run+test+in+37+countries.">Worldwide variation in the performance of children and adolescents: an analysis of 109 studies of the 20-m shuttle run test in 37 countries.</a> Journal of Sports Sciences. 24 (10), 1025-1038 (2006).</li><li>Ainsworth, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compendium+of+physical+activities:+a+second+update+of+codes+and+MET+values.">Compendium of physical activities: a second update of codes and MET values.</a> Medicine and Science in Sports and Exercise. 43 (8), 1575-1581 (2011).</li><li>Roy, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIIT+in+the+real+world:+outcomes+from+a+12-month+intervention+in+overweight+adults.">HIIT in the real world: outcomes from a 12-month intervention in overweight adults.</a> Medicine and Science in Sports and Exercise. 50 (9), 1818-1826 (2018).</li><li>Medina-Mirapeix, F., Escolar-Reina, P., Gascon-Canovas, J. J., Montilla-Herrador, J., Collins, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personal+characteristics+influencing+patients'+adherence+to+home+exercise+during+chronic+pain:+a+qualitative+study.">Personal characteristics influencing patients' adherence to home exercise during chronic pain: a qualitative study.</a> Journal of Rehabilitation Medicine. 41 (5), 347-352 (2009).</li><li>Medina-Mirapeix, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictive+factors+of+adherence+to+frequency+and+duration+components+in+home+exercise+programs+for+neck+and+low+back+pain:+an+observational+study.">Predictive factors of adherence to frequency and duration components in home exercise programs for neck and low back pain: an observational study.</a> BMC Musculoskeletal Disorders. 10 (1), 1-9 (2009).</li><li>Palazzo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barriers+to+home-based+exercise+program+adherence+with+chronic+low+back+pain:+Patient+expectations+regarding+new+technologies.">Barriers to home-based exercise program adherence with chronic low back pain: Patient expectations regarding new technologies.</a> Annals of Physical and Rehabilitation Medicine. 59 (2), 107-113 (2016).</li><li>Lunt, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+intensity+interval+training+in+a+real+world+setting:+a+randomized+controlled+feasibility+study+in+overweight+inactive+adults,+measuring+change+in+maximal+oxygen+uptake.">High intensity interval training in a real world setting: a randomized controlled feasibility study in overweight inactive adults, measuring change in maximal oxygen uptake.</a> PloS One. 9 (1), 83256 (2014).</li><li>Jung, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiorespiratory+fitness+and+accelerometer-determined+physical+activity+following+one+year+of+free-living+high-intensity+interval+training+and+moderate-intensity+continuous+training:+a+randomized+trial.">Cardiorespiratory fitness and accelerometer-determined physical activity following one year of free-living high-intensity interval training and moderate-intensity continuous training: a randomized trial.</a> International Journal of Behavioral Nutrition and Physical Activity. 17 (1), 1-10 (2020).</li><li>Taylor, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+delivery+and+monitoring+of+high+intensity+interval+training+in+clinical+populations.">Guidelines for the delivery and monitoring of high intensity interval training in clinical populations.</a> Progress in Cardiovascular Diseases. 62 (2), 140-146 (2019).</li><li>Grip, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIIT+is+superior+than+MICT+on+cardiometabolic+health+during+training+and+detraining.">HIIT is superior than MICT on cardiometabolic health during training and detraining.</a> European Journal of Applied Physiology. 121 (1), 159-172 (2021).</li></ol>

HIIT has become a time-efficient alternative to the traditional MICT. This studypresents a low-cost, easy-to-implement HIIT protocol for a real-world setting. Most studies have proven the health benefits of HIIT using laboratory-based HIIT protocols6,10, and, recently, few studies have investigated the effects of real-world HIIT protocols in overweight untrained individuals10,14.
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Robust evidence has shown that High-Intensity Interval Training (HIIT) induces similar or even superior positive physiological adaptations than a traditional long-duration Moderate-Intensity Continuous Training (MICT)1,2,3. A HIIT session is composed of short bouts of high-intensity exercise interspersed with low-intensity exercise (active recovery) or rest (passive recovery). While a daily session with a MICT protocol lasts 30 to 60 min, on average, a daily session with HIIT may take half the time or less from a MICT session. Then, considering that sedentary individuals have indicated lack of time as the main barrier to engaging in a regular physical exercise program4, HIIT may be an interesting time-efficient approach to increase exercise adherence and improve health5.
However, despite the growing evidence pointing out the health benefits of HIIT, most studies have designed HIIT protocols for well-controlled laboratory environments using high-cost specialized equipment, such as treadmills and cycle ergometers. In the last 5 years, some studies have emphasized the importance of new studies confirming the health benefits of HIIT using exercise protocols for the real world, e.g., HIIT protocols performed in outdoor spaces without specialized equipment6. However, the difficulty in designing well-controlled studies to test HIIT protocols in non-laboratory environments has been the main challenge for researchers in this field.
In response to this challenge, a real-world HIIT protocol was developed here for scientific research and its efficiency in cardiorespiratory fitness was tested. A training protocol was developed using the shuttle test proposed by Leger et al.7 (named as Beep Training), and the effects of HIIT and MICT regiments based on this Beep training on VO2 max were compared in overweight untrained men. Briefly, although the duration of daily sessions with HIIT was almost half of the duration of MICT protocol, the Beep Training with HIIT was superior to the Beep Training with MICT in increasing VO2 max. Thus, Beep training with HIIT is a time-efficient and feasible approach to improve cardiorespiratory fitness in apparently healthy overweight/obese individuals. Moreover, general people may easily practice the beep training protocol as it is a low-cost and easy-to-implement physical training in a real-world scenario.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Beep Test software&#160;</td>
			<td>Bitworks</td>
			<td>N/A</td>
			<td>version 2.0</td>
		</tr>
		<tr>
			<td>Exercise Physiology Measurement &#38; Analysis System</td>
			<td>ADI INSTRUMENT</td>
			<td>PL3508B80</td>
			<td>PowerLab 8/35 and LabChart Pro software (which includes the Metabolic Module for calculating metabolic parameters such as VCO2, VO2, respiratory exchange ratio (RER) and minute ventilation) 
			Bio Amp 
			Gas Analyzer 
			Gas Mixing Chamber 
			Spirometer 
			Thermistor Pod 
			Exercise Physiology Accessory Kit</td>
		</tr>
		<tr>
			<td>GraphPad Software</td>
			<td>GraphPad Prism</td>
			<td>N/A</td>
			<td>version 7.00</td>
		</tr>
		<tr>
			<td>Heart Rate monitor</td>
			<td>Polar</td>
			<td>N/A</td>
			<td>RS800 Running Computer: The running computer displays and records your heartrate and other exercise data during exercise. 2. Polar WearLink W.I.N.D. transmitter: The transmitter sends the heart rate signal to the running computer. The transmitterconsists of a connector and a strap.</td>
		</tr>
		<tr>
			<td>Sound Forge PRO software</td>
			<td>Sound Forge</td>
			<td>N/A</td>
			<td>version 14.00</td>
		</tr>
		<tr>
			<td>Treadmill</td>
			<td>IMBRASPORT</td>
			<td>N/A</td>
			<td>Speed from 0 to 24 km/h. 
			Elevation from 0 to 26%. 
			Weight capacity for users up to 220 kg. 
			4 hp motor (220 v). 
			Automatic lubrication system. 
			With Safety Key and Emergency Stop Button. 
			Runs 14 preset protocols: Bruce, Modified Bruce, mini Bruce, Naughton Ellestad, Balke, Balke-Ware, Astrand, Cooper, Kattus, Male Mader, Female Mader, Stanford and Modified Stanford. 
			Run RAMP PROTOCOL.</td>
		</tr>
	</tbody>
</table>

a real-world high-intensity interval training protocol for cardiorespiratory fitness improvement

High-Intensity Interval Training (HIIT) has emerged as an interesting time-efficient approach to increase exercise adherence and improve health. However, few studies have tested the efficiency of HIIT protocols in a &#34;real world&#34; setting, e.g., HIIT protocols designed for outdoor spaces without specialized equipment. This study presents a &#34;real world&#34; training protocol, named &#34;beep training&#34;, and compares the efficiency of a HIIT regiment versus a traditional long-duration Moderate-Intensity Continuous Training (MICT) regiment using this beep training protocol on VO2 max of overweight untrained men. Twenty-two subjects performed outdoor running with MICT (n = 11) or HIIT (n = 11). Cardiorespiratory fitness was assessed before and after training protocols using a metabolic analyzer. Both training protocols were performed 3 days a week for 8 weeks using the Beep Test results. The MICT group performed the exercise program at 60%-75% of the maximum speed of the 20 m shuttle test (Vmax) and with a progression of the distance of 3,500-5,000 m. The HIIT group performed the interval exercise with 7-10 bouts of 200 m at 85%-100% of the maximum speed of the 20 m shuttle test (Vmax), interspersed with 1 min of passive recovery. Although the HIIT group presented a significantly lower training volume than the MICT group (p &#60; 0.05) after 8 weeks of beep training, HIIT was superior to MICT in improving VO2 max (MICT: ~4.1%; HIIT: ~7.3%; p &#60; 0.05). The &#34;real world&#34; HIIT regiment based on beep training protocol is a time-efficient, low-cost, and easy-to-implement protocol for overweight untrained men.

Table 1 shows data of distance, speed, rest time, session duration, and mean heart rate from HIIT and MICT groups. During the 8 weeks of beep training, running distance and duration were higher in MICT than in the HIIT group (p &#60; 0.05), while running velocity and heart rate were higher in HIIT than in the MICT group (p &#60; 0.05). These data confirm the main differences between MICT and HIIT protocols, i.e., while MICT is characterized by long-duration moderate-intensity continuous exercises, HIIT i...

Watch this Scientific Journal Video about A Real-World High-Intensity Interval Training Protocol for Cardiorespiratory Fitness Improvement at JoVE.com

A Real-World High-Intensity Interval Training Protocol for Cardiorespiratory Fitness Improvement

This study presents a low-cost and easy-to-implement "real world" high-intensity interval training (HIIT) protocol for scientific research and discusses its efficiency for cardiorespiratory fitness.

High-Intensity Interval Training (HIIT) has emerged as an interesting time-efficient approach to increase exercise adherence and improve health. However, ...

Despite that growing evidence points out the health benefits of high-intensity interval training, most studies have designed its protocol for well-controlled laboratory environments using high-cost, specialized equipment such as treadmills and cyclometers. The high intensity interval training based on beep training is a feasible, time-efficient, low-cost, and easy-to-implement protocol designed to be practiced in a real world setting. The high intensity interval training based on beep training may help in the prevention and treatment of several cardiometabolic diseases such as diabetes, hypertension, and obesity.This is a real world exercise protocol developed to be used not only in scientific research in the field of exercise science, but also to be practiced for apparently healthy individuals. To practice this high intensity interval training protocol, it's essential to take advice from a professional of physical exercise. Begin by familiarizing the participant with the treadmill and mouthpiece used on the metabolic analyzer for at least 24 hours before testing.Calibrate the metabolic analyzer according to the manufacturer's recommendations. Use a heart rate monitor to measure the participant's heart rate. Place the non-rebreathing unilateral mouthpiece on the participant, and ensure that the mouth and nose are completely covered.Then turn on the treadmill, select the ramp protocol, and enter the subject's physical activity level to program the speed and degree of maximal metabolic equivalent increments. Press on"to start the ramp protocol. The program starts with a warmup of three minutes at five kilometers per hour.Record the heart rate and rating of perceived exertion every minute of the test using the Borg scale 6 to 20. After achieving the maximal oxygen uptake, return the treadmill speed to five kilometers per hour, and ask the participant to remain on the treadmill for an additional three minutes. Turn off the treadmill, and remove the mouthpiece from the participant.In the beep test, choose a flat surface to place two cones 20 meters apart. Orient the participant to run 20 meters within a predetermined period, signaled by a beep sound produced by a software developed for this test. Adjust the sound equipment connected to the computer with the software.After familiarizing the participant with the test, start the test. When the participant can no longer complete the 20 meter run within the time stipulated by the beep sound, end the test. For moderate intensity continuous training protocol, instruct the participant to maintain correct running speed and distance by periodically checking the clock speed and distance according to the GPS from the heart rate monitor.Ask the participant to complete a five-minute warmup by performing dynamic stretches and walking before each exercise session. Then put on a heart rate monitor equipped with GPS tracking and start the exercise session. Train the participants once a day, three times a week, either in the morning or afternoon.Initially, train the participants for two weeks at 60%of the individual maximum speed achieved during the 20-meter test, covering a distance of 3, 500 meters per session in the first week, and 4, 000 meters per session in the second week. Then, train the participants for four weeks at 65%of the maximum speed, covering a distance of 4, 000 meters per session in the third week, 4, 500 meters per session in the fourth and fifth week, and 5, 000 meters per session in the sixth week. In the seventh week, train the participants at 70%of maximum speed, covering a distance of 5, 000 meters per session.Then, in the eighth week, at 75%of maximum speed, covering a distance of 5, 000 meters per session. After each exercise session, transfer the data recorded by the heart rate monitor to a computer to verify if the prescribed distance and running speed were reached. Exclude the participants who do not complete all the training sessions in the week.Analyze the data to ensure that each participant performs the respective training regimen according to the prescribed distance and speed. For the high-intensity interval training protocol, mark a lane with cones every 20 meters. Then instruct the participant to follow the beep sound command to know the exact moment to reach the cone.After performing a five minute warmup, put on a heart rate monitor equipped with GPS, and start the exercise session. Train the participant once a day, three times a week, either in the morning or afternoon. In the first week, train the participant with seven sprints of 200 meters at 85%of maximum speed, interspersed by one minute of passive recovery between sprints.Train the participant with eight sprints in the second week. Then, train the participant with eight sprints in the third week, nine sprints in the fourth and fifth week, and 10 sprints in the sixth week, performed at 90%maximum speed for 200 meters, interspersed by one minute of passive recovery. In the seventh week, train the participants with 10 sprints of 200 meters at 95%maximum speed, and then 100%maximum speed with 10 sprints in the eighth week.After the exercise session, transfer the data recorded by the heart rate monitor to a computer, and exclude the participants who did not complete all the training sessions in the week. Finally, analyze the data to ensure that each participant performs the respective training regimen according to the prescribed distance and speed. In the representative analysis, the comparison of distance, speed, rest time, session duration, and mean heart rate from the moderate intensity continuous training and the high intensity interval training groups during eight weeks of training is shown.The running distance and duration were higher in moderate intensity continuous training than in the high intensity interval training group. In contrast, the running velocity and heart rate were higher in high intensity interval training than in the moderate intensity continuous training group. Before training, the maximal oxygen uptake was similar in the high intensity interval training and moderate intensity continuous training groups.After the beep training, the maximal oxygen uptake increased in both the groups. However, the increase was superior in high intensity interval training versus the moderate intensity continuous training group. The visual demonstration of this method is critical for a step-by-step comprehension of how a person must practice each daily exercise session of the beep training.Future studies must be performed to test the efficiency and feasibility of beep training in healthy and unhealthy individuals of different ages.

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Research

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Medicine

Vascular Occlusion Training for Inclusion Body Myositis: A Novel Therapeutic Approach

Inclusion body myositis (IBM) is a rare idiopathic inflammatory myopathy. It is known to produces remarkable muscle weakness and to greatly compromise function and quality of life. Moreover, clinical practice suggests that, unlike other inflammatory myopathies, the majority of IBM patients are not responsive to treatment with immunosuppressive or immunomodulatory drugs to counteract disease progression1. Additionally, conventional resistance training programs have been proven ineffective in restoring muscle function and muscle mass in these patients2,3. Nevertheless, we have recently observed that restricting muscle blood flow using tourniquet cuffs in association with moderate intensity resistance training in an IBM patient produced a significant gain in muscle mass and function, along with substantial benefits in quality of life4. Thus, a new non-pharmacological approach for IBM patients has been proposed. Herein, we describe the details of a proposed protocol for vascular occlusion associated with a resistance training program for this population.

The present article presents the details pertaining to the application of resistance training associated to vascular occlusion in IBM patients.

Inclusion body myositis (IBM) is a rare idiopathic inflammatory myopathy. It is known to produces remarkable muscle weakness and to greatly compromise ...

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

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 ...

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Parabiosis in Mice: A Detailed Protocol

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 18641, the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival2. In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

Porcine As a Training Module for Head and Neck Microvascular Reconstruction

Live models that resemble surgical conditions of humans are needed for training free-flap harvesting and anastomosis. Animal models for training purposes have been available for years in many surgical fields. We used the female (because they are easy to handle for the procedure) Yorkshire pigs for the head and neck reconstruction by harvesting the deep inferior epigastric artery perforator or the superior epigastric artery perforator flap. The anastomosis site (neck skin defect or tracheal wall defect) was prepared via the dissection of the common carotid artery and the internal jugular vein, in which 3.5&#215; loupe magnification was used for anastomosis as we use on human cases in real life. This procedure demonstrates a new training method using a reliable learning model and provides a detailed anatomy in a live scenario. We focused on the ischemia time, harvesting, vessel anastomosis, and designing the flap to fit the defect site. This model improves tissue handling and with the use of proper instruments can be repeated many times so that the surgeon is fully confident before starting the surgery on humans.

Here we present a protocol for the use of the pig superior epigastric artery perforator flap as a learning module for head and neck microvascular reconstruction.

Live models that resemble surgical conditions of humans are needed for training free-flap harvesting and anastomosis. Animal models for training purposes ...

Transaxillary First Rib Resection for Treatment of the Thoracic Outlet Syndrome

Thoracic outlet syndrome (TOS) is a common disorder that causes a significant loss of productivity. The transaxillary first rib resection (TFRR) protocol has been used for the decompression of trapped neurovascular structures in the TOS. Among the other surgical procedures, the advantage of the TFRR is that it has the smallest rate of recurrence and better cosmetic outcomes. The disadvantage of TFRR is that it provides a narrow, and deep working corridor that makes obtaining vascular control challenging.

Here, we present a protocol of the transaxillary resection of the first rib for treatment of thoracic outlet syndrome caused by compression of the brachial plexus, subclavian vein and artery.

Thoracic outlet syndrome (TOS) is a common disorder that causes a significant loss of productivity. The transaxillary first rib resection (TFRR) protocol ...

A Training and Testing System for Performing Vascular Reconstruction In Vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has yet to be developed. In this study, we introduce an in vitro training and testing system using a magnetic anchoring technique with which a trainee can practice manual vascular reconstruction individually. Additionally, this system can also be used to test the quality of the reconstruction. The described system includes a vascular reconstruction training machine, magnetic tractors, and a magnetic suture puller. In this manuscript, we detail an end-to-end vein anastomosis using porcine right and left iliac veins. To identify the potential damage caused by a magnetic suture puller on the suture, we created three groups with six segments of 4-0 polypropylene sutures each: a control group with no intervention on the polypropylene suture, a group in which the polypropylene suture is manually pulled with sterile gloves 20x, and a magnetic puller group in which the magnetic puller pulled the polypropylene suture 20x. These groups were tested by light microscopy and breaking strength tests, and the effect of reconstruction was assessed. In the light microscopy test, the control group was less likely to be damaged (p &#60; 0.05) and the number of damaged points of the manual group and magnetic puller group were similar (p &#62; 0.05). The results of the breaking strength test were compared across groups and no significant difference was observed (p &#62; 0.05). The end-to-end anastomosis of the porcine iliac veins was successfully performed using this training system, and the reconstructed veins could undergo 2.0 kPa perfusion pressure. Using this training and testing system the trainee can practice manual vascular reconstruction in vitro individually with the aid of magnetic tractors and a magnetic suture puller, and the quality of the reconstruction can be tested.

Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has ...