Name: investigating stress-relaxation and failure responses in the trachea
Uploaded: 2022-10-18T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Investigating Stress-relaxation and Failure Responses in the Trachea at JoVE.com

Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under Award Number R15HD093024 and the National Science Foundation CAREER Award Number 1752513.

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	<li>Brand-Saberi, B. E. M., Sch&#228;fer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trachea:+Anatomy+and+physiology.">Trachea: Anatomy and physiology.</a> Thoracic Surgery Clinics. 24, 1-5 (2014).</li><li>Barnett, S. B., Nurmagambetov, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Costs+of+asthma+in+the+United+States:+2002-2007.">Costs of asthma in the United States: 2002-2007.</a> The Journal of Allergy and Clinical Immunology. 127 (1), 145-152 (2011).</li><li>Chronic Obstructive Pulmonary Disease (COPD). Centers for Disease Control and Prevention Available from: <a target="_blank" href="https://www.cdc.gov/copd/index.html">https://www.cdc.gov/copd/index.html</a> (2022)</li><li>Wilson, L., Devine, E. B., So, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+medical+costs+of+chronic+obstructive+pulmonary+disease:+chronic+bronchitis+and+emphysema).">Direct medical costs of chronic obstructive pulmonary disease: chronic bronchitis and emphysema).</a> Respiratory Medicine. 94 (3), 204-213 (2000).</li><li>Codd, S. L., Lambert, R. K., Alley, M. R., Pack, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensile+stiffness+of+ovine+tracheal+wall.">Tensile stiffness of ovine tracheal wall.</a> Journal of Applied Physiology. 76 (6), 2627-2635 (1994).</li><li>Noble, P. B., Sharma, A., McFawn, P. K., Mitchell, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+properties+of+the+bronchial+mucosa:+epithelial+unfolding+and+stretch+in+response+to+airway+inflation.">Elastic properties of the bronchial mucosa: epithelial unfolding and stretch in response to airway inflation.</a> Journal of Applied Physiology. 99 (6), 2061-2066 (2005).</li><li>Teng, Z., 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=Anisotropic+material+behaviours+of+soft+tissues+in+human+trachea:+An+experimental+study.">Anisotropic material behaviours of soft tissues in human trachea: An experimental study.</a> Journal of Biomechanics. 45 (9), 1717-1723 (2012).</li><li>Wang, 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=Mechanical+properties+of+the+tracheal+mucosal+membrane+in+the+rabbit.+I.+steady-state+stiffness+as+a+function+of+age.">Mechanical properties of the tracheal mucosal membrane in the rabbit. I. steady-state stiffness as a function of age.</a> Journal of Applied Physiology. 88 (3), 1014-1021 (2000).</li><li>Ambrosi, D., 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=Perspectives+on+biological+growth+and+remodeling.">Perspectives on biological growth and remodeling.</a> Journal of the Mechanics and Physics of Solids. 59 (4), 863-883 (2011).</li><li>Bai, T. R., Knight, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+changes+in+the+airways+in+asthma:+Observations+and+consequences.">Structural changes in the airways in asthma: Observations and consequences.</a> Clinical Science. 108 (6), 463-477 (2005).</li><li>Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extent+of+impaired+axoplasmic+transport+and+neurofilament+compaction+in+traumatically+injured+axon+at+various+strains+and+strain+rates.">Extent of impaired axoplasmic transport and neurofilament compaction in traumatically injured axon at various strains and strain rates.</a> Brain Injury. 31 (10), 1387-1395 (2017).</li><li>Singh, A., Kallakuri, S., Chen, C., Cavanaugh, 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=Structural+and+functional+changes+in+nerve+roots+due+to+tension+at+various+strains+and+strain+rates:+An+in-vivo+study.">Structural and functional changes in nerve roots due to tension at various strains and strain rates: An in-vivo study.</a> Journal of Neurotrauma. 26 (4), 627-640 (2009).</li><li>Singh, A., Lu, Y., Chen, C., Cavanaugh, 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=Mechanical+properties+of+spinal+nerve+roots+subjected+to+tension+at+different+strain+rates.">Mechanical properties of spinal nerve roots subjected to tension at different strain rates.</a> Journal of Biomechanics. 39 (9), 1669-1676 (2006).</li><li>Singh, A., Lu, Y., Chen, C., Kallakuri, S., Cavanaugh, 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=A+new+model+of+traumatic+axonal+injury+to+determine+the+effects+of+strain+and+displacement+rates.">A new model of traumatic axonal injury to determine the effects of strain and displacement rates.</a> Stapp Car Crash Journal. 50, 601-623 (2006).</li><li>Singh, A., Magee, R., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+in+vivo+biomechanical+testing+on+brachial+plexus+in+neonatal+piglets.">Methods for in vivo biomechanical testing on brachial plexus in neonatal piglets.</a> Journal of Visualized Experiments. (154), e59860 (2019).</li><li>Singh, A., Magee, R., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+cervical+spinal+cord+in+neonatal+piglet:+in+vitro.">Mechanical properties of cervical spinal cord in neonatal piglet: in vitro.</a> Neurology and Neurobiology. 3 (2), (2020).</li><li>Singh, A., Magee, R., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+study+to+investigate+biomechanical+responses+of+peripheral+nerves+in+hypoxic+neonatal+piglets.">An in vitro study to investigate biomechanical responses of peripheral nerves in hypoxic neonatal piglets.</a> Journal of Biomechanical Engineering. 143 (11), 114501 (2021).</li><li>Singh, A., Shaji, S., Delivoria-Papadopoulos, M., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+responses+of+neonatal+brachial+plexus+to+mechanical+stretch.">Biomechanical responses of neonatal brachial plexus to mechanical stretch.</a> Journal of Brachial Plexus and Peripheral Nerve Injury. 13, 8-14 (2018).</li><li>Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The effects of tensile loading on mechanical, neurophysiological and morphological changes in spinal nerve roots. , (2006).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Eskandari, M., Arvayo, A. L., Levenston, 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=Mechanical+properties+of+the+airway+tree:+Heterogeneous+and+anisotropic+pseudoelastic+and+viscoelastic+tissue+responses.">Mechanical properties of the airway tree: Heterogeneous and anisotropic pseudoelastic and viscoelastic tissue responses.</a> Journal of Applied Physiology. 125 (3), 878-888 (2018).</li><li>Toby, E. B., Rotramel, J., Jayaraman, G., Struthers, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+stress+relaxation+properties+of+peripheral+nerves+after+transection.">Changes in the stress relaxation properties of peripheral nerves after transection.</a> Journal of Hand Surgery. 24 (4), 694-699 (1999).</li><li>Safshekan, F., Tafazzoli-Shadpour, M., Abdouss, M., Shadmehr, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+properties+of+human+tracheal+tissues.">Viscoelastic properties of human tracheal tissues.</a> Journal of Biomechanical Engineering. 139 (1), (2017).</li><li>Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+teaching+biomechanics+through+active,+adaptive,+and+experiential+learning.">A new approach to teaching biomechanics through active, adaptive, and experiential learning.</a> Journal of Biomechanical Engineering. 139 (7), 0710011-0710017 (2017).</li><li>Singh, A., Ferry, D., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+clinical+simulation+based+training+in+biomedical+engineering+education.">Efficacy of clinical simulation based training in biomedical engineering education.</a> Journal of Biomechanical Engineering. 141 (12), 121011-121017 (2019).</li><li>Singh, A., Ferry, D., Mills, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+biomedical+engineering+education+through+continuity+in+adaptive,+experiential,+and+interdisciplinary+learning+environments.">Improving biomedical engineering education through continuity in adaptive, experiential, and interdisciplinary learning environments.</a> Journal of Biomechanical Engineering. 140 (8), 0810091-0810098 (2018).</li><li>Singh, A., Ferry, D., Ramakrishnan, A., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+virtual+reality+in+biomedical+engineering+education.">Using virtual reality in biomedical engineering education.</a> Journal of Biomechanical Engineering. 142 (11), 111013 (2020).</li><li>Majmudar, T., Balasubramanian, S., Magee, R., Gonik, B., Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vitro+stress+relaxation+response+of+neonatal+peripheral+nerves.">In-vitro stress relaxation response of neonatal peripheral nerves.</a> Journal of Biomechanical Engineering. 128, 110702 (2021).</li><li>Orozco, V., Magee, R., Balasubramanian, S., Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+the+tensile+biomechanical+properties+of+the+neonatal+brachial+plexus.">A systematic review of the tensile biomechanical properties of the neonatal brachial plexus.</a> Journal of Biomechanical Engineering. 143 (11), 110802 (2021).</li><li>Singh, A. M. T., Magee, R., Gonik, B., Balasubramanian, 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+pre-stretch+on+neonatal+peripheral+nerve:+An+in-vitro+study.">Effects of pre-stretch on neonatal peripheral nerve: An in-vitro study.</a> Journal of Brachial Plexus and Peripheral Nerve Injury. 17 (1), 1-9 (2022).</li><li>Balasubramanian, S., D'Andrea, C., Viraraghavan, G., Cahill, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+finite+element+model+of+the+pediatric+thoracic+and+lumbar+spine,+ribcage,+and+pelvis+with+orthotropic+region-specific+vertebral+growth.">Development of a finite element model of the pediatric thoracic and lumbar spine, ribcage, and pelvis with orthotropic region-specific vertebral growth.</a> Journal of Biomechanical Engineering. 144 (10), 101007 (2022).</li><li>Hadagali, P., Peters, J. R., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphing+the+feature-based+multi-blocks+of+normative/healthy+vertebral+geometries+to+scoliosis+vertebral+geometries:+Development+of+personalized+finite+element+models.">Morphing the feature-based multi-blocks of normative/healthy vertebral geometries to scoliosis vertebral geometries: Development of personalized finite element models.</a> Computer Methods in Biomechanics and Biomedical Engineering. 21 (4), 297-324 (2018).</li><li>Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Available+computational+and+physical+models+to+understand+the+mechanisms+of+neonatal+brachial+plexus+injury+during+shoulder+dystocia.">Available computational and physical models to understand the mechanisms of neonatal brachial plexus injury during shoulder dystocia.</a> Open Access Journal of Neurology and Neurosurgery. 9 (4), 555768 (2019).</li></ol>

The authors have nothing to disclose.

Very few studies have reported the stress-relaxation properties of the trachea21,23. Studies are needed to further strengthen our understanding of the time-dependent responses of the tracheal tissue. This study offers detailed steps to perform such investigations; however, the following critical steps within the protocol must be ensured for reliable testing: (1) proper tissue hydration, (2) similar tissue-type (number of cartilaginous rings and muscle) distributi...

Despite its critical role in pulmonary disease, the largest airway structure, the trachea, has limited studies detailing its viscoelastic properties1. An in-depth understanding of the time-dependent, viscoelastic behavior of the trachea is critical to pulmonary mechanics research since understanding the airway-specific material properties can help advance the science of injury prevention, diagnosis, and clinical intervention for pulmonary diseases, which are the third leading cause of death in the United States2,3,4.
Available tissue characterization studies have reported the stiffness properties of the trachea5,6,7,8. The time-dependent mechanical responses have been minimally investigated despite their importance in tissue remodeling, which is also altered by pathology9,10. Moreover, the lack of time-dependent response data also limits the predictive capabilities of the pulmonary mechanics computational models that currently resort to using the generic constitutive laws. There is a need to address this gap by performing stress-relaxation studies that can provide the required material characteristics to inform biophysical studies of the trachea. The current study offers details of testing methods, data acquisition, and data analyses to investigate the stress-relaxation behavior of the porcine trachea.

<table><tbody><tr><td>Disposable safety scalpels</td><td>Fine Science Tools Inc</td><td>10000-10</td><td></td></tr><tr><td>eXpert 7600</td><td>ADMET Inc.</td><td>N/A</td><td>Norwood, MA</td></tr><tr><td>Forceps</td><td>&amp;nbsp;Fine Science Tools Inc</td><td>11006-12 and 11027-12 or 11506-12</td><td></td></tr><tr><td>Gauge Safe</td><td>ADMET Inc.</td><td>N/A</td><td>Free Download</td></tr><tr><td>Image J</td><td>NIH</td><td>N/A</td><td>Open Source</td></tr><tr><td>Proramming Software - MATLAB&amp;nbsp;</td><td>Mathworks</td><td>N/A</td><td>version 2018A</td></tr><tr><td>Scissors</td><td>&amp;nbsp;Fine Science Tools Inc</td><td>14094-11 or 14060-09</td><td></td></tr><tr><td>Sterile phosphate buffer solution&amp;nbsp;</td><td>Millipore, Thomas Scientific</td><td>MFCD00131855</td><td></td></tr></tbody></table>

investigating stress-relaxation and failure responses in the trachea

The biomechanical properties of the trachea directly affect the airflow and contribute to the biological function of the respiratory system. Understanding these properties is critical to understanding the injury mechanism in this tissue. This protocol describes an experimental approach to study the stress-relaxation behavior of porcine trachea that were pre-stretched to 0% or 10% strain for 300 s, followed by mechanical tensile loading until failure. This study provides details of the experimental design, data acquisition, analyses, and preliminary results from the porcine tracheae biomechanical testing. Using the detailed steps provided in this protocol and the data analysis MATLAB code, future studies can investigate the time-dependent viscoelastic behavior of trachea tissue, which is critical to understanding its biomechanical responses during physiological, pathological, and traumatic conditions. Furthermore, in-depth studies of the biomechanical behavior of the trachea will critically aid in improving the design of related medical devices such as endotracheal implants that are widely used during surgeries.

Figure 1 shows the failed tissue near the clamping site and the presence of tissue within the clamp, confirming no slip during tensile testing. Figure 2 indicates various failure sites, including the top or bottom clamping sites or along the length of the tissue, that were observed during tensile testing among the tested samples. Data analysis results are summarized in Figures 3-4 and Tables...

Watch this Scientific Journal Video about Investigating Stress-relaxation and Failure Responses in the Trachea at JoVE.com

Investigating Stress-relaxation and Failure Responses in the Trachea

The present protocol determines the tensile stress-relaxation and failure properties of porcine tracheae. Results from such methods can help improve the understanding of the viscoelastic and failure thresholds of the trachea and help advance the capabilities of computational models of the pulmonary system.

The biomechanical properties of the trachea directly affect the airflow and contribute to the biological function of the respiratory system. Understanding ...

investigating-stress-relaxation-and-failure-responses-in-the-trachea

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

1.8K Views.  Widener University. The present protocol determines the tensile stress-relaxation and failure properties of porcine tracheae. Results from such methods can help improve the understanding of the viscoelastic and failure thresholds of the trachea and help advance the capabilities of computational models of the pulmonary system. 

Video: Investigating Stress-relaxation and Failure Responses in the Trachea

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 ...

Medicine

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively3,4,5. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7. M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10. These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle11. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")4,7. More detailed reviews on signaling pathways controlling airway constriction can be found4,12. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14, both well-known ASM modulators in humans and other species5,15.
Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+, Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood flow that results in a net influx of blood to key organs and an increase in core temperature. This stress-induced hyperthermia is used to assess stress. However, measuring core temperature is invasive. As blood flow is redirected to the core, the periphery of the body can cool. This paper describes a protocol where peripheral body temperature is measured non-invasively in wild blue tits (Cyanistes caeruleus) using infrared thermography. In the field we created a set-up bringing the birds to an ideal position in front of the camera by using a baited box. The camera takes a short thermal video recording of the undisturbed bird before applying a mild stressor (closing the box and therefore capturing the bird), and the bird&#8217;s response to being trapped is recorded. The bare skin of the eye-region is the warmest area in the image. This allows an automated extraction of the maximum eye-region temperature from each image frame, followed by further steps of manual data filtering removing the most common sources of errors (motion blur, blinking). This protocol provides a time series of eye-region temperature with a fine temporal resolution that allows us to study the dynamics of the stress response non-invasively. Further work needs to demonstrate the usefulness of the method to assess stress, for instance to investigate whether eye-region temperature response is proportional to the strength of the stressor. If this can be confirmed, it will provide a valuable alternative method of stress assessment in animals and will be useful to a wide range of researchers from ecologists, conservation biologists, physiologists to animal welfare researchers.

There is a need for a non-invasive assessment of stress. This paper describes a simple protocol using thermal imaging to detect a significant response in eye-region temperature in wild blue tits to a mild acute stressor.

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood ...

Neuroscience

Design of a Biocompatible Drug-Eluting Tracheal Stent in Mice with Laryngotracheal Stenosis

Laryngotracheal stenosis (LTS) is a pathologic narrowing of the subglottis and trachea leading to extrathoracic obstruction and significant shortness of breath. LTS results from mucosal injury from a foreign body in the trachea, leading to tissue damage and a local inflammatory response that goes awry, leading to the deposition of pathologic scar tissue. Treatment for LTS is surgical due to the lack of effective medical therapies. The purpose of this method is to construct a biocompatible stent that can be miniaturized to place into mice with LTS. We demonstrated that a PLLA-PCL (70% poly-L-lactide and 30% polycaprolactone) construct had optimal biomechanical strength, was biocompatible, practicable for an in vivo placement stent, and capable of eluting drug. This method provides a drug delivery system for testing various immunomodulatory agents to locally inhibit inflammation and reduce airway fibrosis. Manufacturing the stents takes 28&#8722;30 h and can be reproduced easily, allowing for experiments with large cohorts. Here we incorporated the drug rapamycin within the stent to test its effectiveness in reducing fibrosis and collagen deposition. Results revealed that PLLA-PCL tents showed reliable rapamycin release, were mechanically stable in physiological conditions, and were biocompatible, inducing little inflammatory response in the trachea. Further, the rapamycin-eluting PLLA-PCL stents reduced scar formation in the trachea in vivo.

Laryngotracheal stenosis results from pathologic scar deposition that critically narrows the tracheal airway and lacks effective medical therapies. Using a PLLA-PCL (70% poly-L-lactide and 30% polycaprolactone) stent as a local drug delivery system, potential therapies aimed at decreasing scar proliferation in the trachea can be studied.

Laryngotracheal stenosis (LTS) is a pathologic narrowing of the subglottis and trachea leading to extrathoracic obstruction and significant shortness of ...

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. They are part of a family of chemosensory epithelial cells which include tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. Chemosensory cells in different epithelial compartments share key intracellular markers and a core transcriptional signature, but also display significant transcriptional heterogeneity, likely reflective of the local tissue environment. Isolation of tracheal brush cells from single cell suspensions is required to define the function of these rare epithelial cells in detail, but their isolation is challenging, potentially due to the close interaction between tracheal brush cells and nerve endings or due to airway-specific composition of tight and adherens junctions. Here, we describe a procedure for isolation of brush cells from mouse tracheal epithelium. The method is based on an initial separation of tracheal epithelium from the submucosa, allowing for a subsequent shorter incubation of the epithelial sheet with papain. This procedure offers a rapid and convenient solution for flow cytometric sorting and functional analysis of viable tracheal brush cells.

Brush cells are rare cholinergic chemosensory epithelial cells found in the na&#239;ve mouse trachea. Due to their limited numbers, ex vivo evaluation of their functional role in airway immunity and remodeling is challenging. We describe a method for isolation of tracheal brush cells by flow cytometry.

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. ...

Immunology and Infection

Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in na&iuml;ve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).

The  forced oscillation technique (FOT) is a powerful, integrative and translational  tool permitting the experimental assessment of lung function in mice ...

Biology

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Low-Dose Gamma Radiation Sterilization for Decellularized Tracheal Grafts

One of the main key aspects in ensuring that a transplant evolves correctly is the sterility of the medium. Decellularized tracheal transplantation involves implanting an organ that was originally in contact with the environment, thus not being sterile from the outset. While the decellularization protocol (through detergent exposition [2% sodium dodecyl sulfate], continuous stirring, and osmotic shocks) is conducted in line with aseptic measures, it does not provide sterilization. Therefore, one of the main challenges is ensuring sterility prior to in vivo implantation. Although there are established gamma radiation sterilization protocols for inorganic materials, there are no such measures for organic materials. Additionally, the protocols in place for inorganic materials cannot be applied to organic materials, as the established radiation dose (25 kGy) would completely destroy the implant. This paper studies the effect of an escalated radiation dose in a decellularized rabbit trachea. We maintained the dose range (kGy) and tested escalated doses until finding the minimal dose at which sterilization is achieved. After determining the dose, we studied effects of it on the organ, both histologically and biomechanically. We determined that while 0.5 kGy did not achieve sterility, doses of both 1 kGy and 2 kGy did, with 1 kGy, therefore, being the minimal dose necessary to achieve sterilization. Microscopic studies showed no relevant changes compared to non-sterilized organs. Axial biomechanical characteristics were not altered at all, and only a slight reduction in the force per unit of length that the organ can radially tolerate was observed. We can therefore conclude that 1 kGy achieves complete sterilization of decellularized rabbit trachea with a minimal, if any, effects on the organ.

Obtaining sterilization is essential for tracheal tissue transplant. Herein, we present a sterilization protocol using low-dose gamma irradiation that is fully tolerated by organs.

One of the main key aspects in ensuring that a transplant evolves correctly is the sterility of the medium. Decellularized tracheal transplantation ...

Investigating Stress-relaxation and Failure Responses in the Trachea

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Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds

Design of a Biocompatible Drug-Eluting Tracheal Stent in Mice with Laryngotracheal Stenosis

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

Low-Dose Gamma Radiation Sterilization for Decellularized Tracheal Grafts