Special thanks to all volunteers who participated in the original study. The work of L. Eichhorn was supported through a scholarship of the Else-Kr&#246;ner-Fresenius Foundation. The authors would like to thank Springer, Part of Springer Science+Business Media, for copyright clearance (License Number 3894660871180) and the kind permission of reusing previously published data.

Ethics statement 
All procedures performed in studies involving human participants were in accordance with the ethical standards of the 1964 Helsinki declaration and its later amendments. The design of this study was approved by the local ethics committee of the University Hospital of Bonn, Germany.
NOTE: Ensure that subjects are in good and healthy condition, free of any anti-hypertensive medicine and at least 24 hours free of catecholamine inducing agents like caffeine or equal substances.
1. Preparation of the Test Subject
<ol>
	<li>Clean the skin of the forehead with 70% alcohol to degrease the skin prior to NIRS electrode positioning.</li>
	<li>Place the NIRS electrode on the right forehead above the eyebrow and to the right of the midsagittal sulcus (locus frontopolar 2) to measure cerebral (=central) tissue oxygenation.</li>
	<li>Evaluate the stability of the signal. The rSO2-signal should be constant (&#177; 3%) for at least 5 min.</li>
	<li>For measuring peripheral tissue oxygenation with NIRS (NIRStissue-electrode), place one electrode above the middle of the musculus quadriceps femoris (alternatively on the forearm). Do not place the electrode above a venous plexus or an artery.</li>
	<li>Place ECG-electrodes on the hair free chest. The ECG leads are marked with different letters. Place &#34;R&#34; on the sternocostal head of pectoralis major right, &#34;L&#34; on the sternocostal head of pectoralis major left, &#34;C&#34; on the fifth intercostal space middle of the medioclavicular line, &#34;F&#34; on the left lower rib edge, &#34;N&#34; on the right lower rib edge.</li>
	<li>Measure peripheral pulse oximetry (SpO2) on a fingertip on the same extremity and side where the NIRStissue-electrode is placed.</li>
	<li>Measure noninvasive blood pressure (NIBP) by using a blood pressure cuff. Use the contralateral extremity that allows peripheral pulse oximetry to be measured. In order to get a high time-resolution in blood pressure results, choose a one-minute interval for measuring. Choose NIBP by touching the screen and selecting &#34;settings&#34;.</li>
	<li>At least 20 min before the apnea, establish an intravenous line into the medial cubital vein of the right or left arm to draw blood samples at individual time points during and after apnea.
	<ol>
		<li>Clean the skin with 70% alcohol.</li>
		<li>Use a tourniquet to help the veins become more prominent.</li>
		<li>Use skin-disinfection to avoid infections and insert the needle through the skin.</li>
		<li>Reduce the insertion angle after blood flashback at the catheter hub. Push the catheter into the vein.</li>
		<li>Remove the needle and flush catheter with sterile saline (NaCl 0.9%).</li>
	</ol>
	</li>
</ol>
2. Data Collection
<ol>
	<li>Calibrate the internal clock of all monitors in order to synchronize measurements for later processing.
	<ol>
		<li>Click the bottom-right clock icon on the desktop, and tap &#34;change date and time settings&#34; in the pop-up window.</li>
		<li>Press the Settings menu button on the NIRS devise and change date and time via the menu.</li>
	</ol>
	</li>
	<li>To store physiological data for offline analysis, insert the monitor device into the docking station and connect it to the computer via the network cable. Ensure that the IP address and subnet mask of the docking station is correct in the network settings in order to get a connection. Contact device provider in order to get this information.</li>
	<li>Use a monitor device specific software to save measurements on the computer. Click &#34;start&#34; to begin recordings and save the results after the end of the measurement. 
	Note: In some devices, data has to be saved live during the measurement. 
	Note: For trouble-shooting take care of the following steps: If the variability of NIRStissue signals is too high, re-evaluate the position of the electrode (avoid bigger venous plexus or arteries directly under the electrodes). High variability of NIRScerebral signals can also be an indirect marker for hyperventilation of divers to reduce partial CO2. Instruct the subject to breath slower and with lower tidal-volumes and re-evaluate the signal. Subjects are allowed to take 3 deep inspirations before final apnea. Avoid including this period into the evaluation of baseline values. The first 30 sec after maximal inspiration are characterized by variable values. Do not use them for analysis.</li>
</ol>
3. Apnea
<ol>
	<li>Have the subjects rest for at least 15 min in a lying position to avoid stress induced changes in blood circulation due to vasoconstriction. Have subjects breathe normally to avoid influences of hyperventilation caused vasoconstriction. Limit the breathing frequency to &#8804; 15 breaths/min.</li>
	<li>Draw blood samples for baseline analysis. Discard the first 5 ml of drawn blood to avoid measurement uncertainty. Flush the catheter after each venous blood collection with sterile saline to prevent clotting.</li>
	<li>Ensure that monitor values are invisible to subjects to avoid visual influences to their apneic performance.</li>
	<li>Check each device for functionality and signal quality. Ensure that electrodes cannot be removed by involuntary movements of the test subject at the end of apnea.</li>
	<li>Conclude with clear agreements. Give a countdown of the last 2 min verbally. Subjects should breathe normally during this preparation time. Prior to the final breath 3 deep inspirations are allowed. Ask the subject to indicate the last inhalation by finger sign. Apnea should be performed as long as possible. 
	Note: The end of the final breath indicates the start of apnea. The end of apnea is defined as the first inspiration after apnea.</li>
	<li>Mark important events (i.e., beginning and end of apnea) electronically to avoid inaccuracies in further time analysis by pressing the &#34;Event Mark Button&#34; on the NIRS device. 
	Note: Movements of the chest and stomach induced by involuntary diaphragm activities are common in the second half of apnea and indicate the struggle phase.</li>
	<li>Draw blood samples at different time-points depending on the aim of the study.</li>
	<li>Centrifuge blood samples at 1,500 x g for 10 min. Take the supernatant and store it at -80 &#176;C for future analysis.</li>
</ol>
4. Processing Data
<ol>
	<li>Processing data from the monitor device:
	<ol>
		<li>Open the saved file on the computer and press &#34;start&#34; to analyze data.</li>
		<li>Click &#34;review&#34; to get access to the trend monitor and select &#34;options&#34; and then &#34;tools&#34; in the MENU submask. Time interval can be changed via &#34;trend interval&#34; if necessary.</li>
		<li>Select the mask &#34;trends&#34; and save. Open file &#34;trends&#34; in a spreadsheet program for further processing.</li>
	</ol>
	</li>
	<li>Processing data from NIRS device:
	<ol>
		<li>Open the software on the computer and connect the NIRS device via WIFI.</li>
		<li>Transfer the data from the NIRS device to the computer.</li>
		<li>Save the data in CSV-format.</li>
		<li>Open file in a spreadsheet program for further processing.</li>
	</ol>
	</li>
</ol>
5. Analyze Values
<ol>
	<li>Create a spreadsheet with both datasets to compare the values. Identify a time interval of at least 30 sec where NIRS-values and SpO2 are constant (&#177; 3%). Take an average of these values to define a baseline-level. 
	Note: Heart rate is known to change considerably prior to apnea. In order to conduct further analysis, a baseline heart rate is defined at a time point 30 sec after initiation of apnea.</li>
	<li>Find the start point of monotonic decrease in rSO2 and SpO2 during apnea by looking for a decrease of values &#62; 2% compared to baseline-levels. This time point is defined as &#34;begin of desaturation&#34;.</li>
	<li>Identify the start point of rSO2 and SpO2 increase at the end of apnea as a monotonic increase of values after termination of apnea. This point is defined as &#34;begin of re-saturation&#34;.</li>
	<li>Calculate the time difference between &#34;start of apnea&#34; and &#34;begin of desaturation&#34; and the time differences between &#34;end of apnea&#34; and &#34;begin of re-saturation&#34; for NIRScerebral, NIRStissue and SpO2. Save each difference in seconds on a separate spreadsheet.</li>
	<li>Optional: Calculate heart rate variability of each participant during the second and the last minute of apnea. This may reveal information about the sympathetic/parasympathetic balance during this stressful phase.</li>
</ol>
6. Statistical Processing
<ol>
	<li>Compare the time differences between &#34;beginning of desaturation&#34; of SpO2, NIRScerebral, and NIRStissue values. Test for Gaussian distribution of the measurement differences (e.g., using Shapiro-Wilk normality test for sample sizes smaller than 50).</li>
	<li>If the distribution of the measurement differences is significantly different from normal distribution, use Wilcoxon signed rank test. If normal distribution can be assumed, consider using paired t-test.</li>
</ol>

<ol>
	<li>Drager, L. F., Polotsky, V. Y., O'Donnell, C. P., Cravo, S. L., Lorenzi-Filho, G., Machado, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+approaches+to+understanding+metabolic+dysfunction+and+cardiovascular+consequences+of+obstructive+sleep+apnea.">Translational approaches to understanding metabolic dysfunction and cardiovascular consequences of obstructive sleep apnea.</a> Am J Physiol Heart Circ Physiol. 309 (7), 1101-1111 (2015).</li><li>Shah, N., Trivedi, N. K., Clack, S. L., Shah, M., Shah, P. P., Barker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+hypoxemia+on+the+performance+of+cerebral+oximeter+in+volunteer+subjects.">Impact of hypoxemia on the performance of cerebral oximeter in volunteer subjects.</a> J Neurosurg Anesthesiol. 12 (3), 201-209 (2000).</li><li>Ricci, M., Lombardi, P., 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=Near-infrared+spectroscopy+to+monitor+cerebral+oxygen+saturation+in+single-ventricle+physiology.">Near-infrared spectroscopy to monitor cerebral oxygen saturation in single-ventricle physiology.</a> J Thorac Cardiovasc Surg. 131 (2), 395-402 (2006).</li><li>Kusaka, T., Isobe, K., 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=Quantification+of+cerebral+oxygenation+by+full-spectrum+near-infrared+spectroscopy+using+a+two-point+method.">Quantification of cerebral oxygenation by full-spectrum near-infrared spectroscopy using a two-point method.</a> Comp Biochem Physiol A Mol Integr Physiol. 132 (1), 121-132 (2002).</li><li>Nishimura, N., Iwasaki, K., Ogawa, Y., Shibata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+administration,+cerebral+blood+flow+velocity,+and+dynamic+cerebral+autoregulation.">Oxygen administration, cerebral blood flow velocity, and dynamic cerebral autoregulation.</a> Aviat Space Environ Med. 78 (12), 1121-1127 (2007).</li><li>Wilson, M. H., Newman, S., Imray, C. 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+cerebral+effects+of+ascent+to+high+altitudes.">The cerebral effects of ascent to high altitudes.</a> Lancet Neurol. 8 (2), 175-191 (2009).</li><li>Sanborn, M. R., Edsell, 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=Cerebral+hemodynamics+at+altitude:+effects+of+hyperventilation+and+acclimatization+on+cerebral+blood+flow+and+oxygenation.">Cerebral hemodynamics at altitude: effects of hyperventilation and acclimatization on cerebral blood flow and oxygenation.</a> Wilderness Environ Med. 26 (2), 133-141 (2015).</li><li>Reynolds, J. C., Salcido, 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=Tissue+oximetry+by+near-infrared+spectroscopy+in+a+porcine+model+of+out-of-hospital+cardiac+arrest+and+resuscitation.">Tissue oximetry by near-infrared spectroscopy in a porcine model of out-of-hospital cardiac arrest and resuscitation.</a> Resuscitation. 84 (6), 843-847 (2013).</li><li>Andersson, J. P. A., Evaggelidis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+oxygen+saturation+and+diving+response+during+dynamic+apneas+in+breath-hold+divers.">Arterial oxygen saturation and diving response during dynamic apneas in breath-hold divers.</a> Scand J Med Sci Sports. 19 (1), 87-91 (2009).</li><li>Overgaard, K., Friis, S., Pedersen, R. B., Lykkeboe, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+lung+volume,+glossopharyngeal+inhalation+and+P(ET)+O2+and+P(ET)+CO2+on+apnea+performance+in+trained+breath-hold+divers.">Influence of lung volume, glossopharyngeal inhalation and P(ET) O2 and P(ET) CO2 on apnea performance in trained breath-hold divers.</a> Eur J Appl Physiol. 97 (2), 158-164 (2006).</li><li>Ferretti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extreme+human+breath-hold+diving.">Extreme human breath-hold diving.</a> Eur J Appl Physiol. 84 (4), 254-271 (2001).</li><li>Eichhorn, L., Erdfelder, 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=Evaluation+of+near-infrared+spectroscopy+under+apnea-dependent+hypoxia+in+humans.">Evaluation of near-infrared spectroscopy under apnea-dependent hypoxia in humans.</a> J Clin Monit Comput. 29 (6), 749-757 (2015).</li><li>Eichhorn, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulse+oximetry+as+a+standard+of+practice+in+anesthesia.">Pulse oximetry as a standard of practice in anesthesia.</a> Anesthesiology. 78 (3), 423-426 (1993).</li><li>Schewe, J. -. C., Thudium, M. O., 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=Monitoring+of+cerebral+oxygen+saturation+during+resuscitation+in+out-of-hospital+cardiac+arrest:+a+feasibility+study+in+a+physician+staffed+emergency+medical+system.">Monitoring of cerebral oxygen saturation during resuscitation in out-of-hospital cardiac arrest: a feasibility study in a physician staffed emergency medical system.</a> Scand J Trauma Resusc Emerg Med. 22, 58 (2014).</li><li>Ahn, A., Nasir, A., Malik, H., D'Orazi, F., Parnia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pilot+study+examining+the+role+of+regional+cerebral+oxygen+saturation+monitoring+as+a+marker+of+return+of+spontaneous+circulation+in+shockable+(VF/VT)+and+non-shockable+(PEA/Asystole)+causes+of+cardiac+arrest.">A pilot study examining the role of regional cerebral oxygen saturation monitoring as a marker of return of spontaneous circulation in shockable (VF/VT) and non-shockable (PEA/Asystole) causes of cardiac arrest.</a> Resuscitation. 84 (12), 1713-1716 (2013).</li><li>Moritz, S., Kasprzak, P., Arlt, M., Taeger, K., Metz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+cerebral+monitoring+in+detecting+cerebral+ischemia+during+carotid+endarterectomy:+a+comparison+of+transcranial+Doppler+sonography,+near-infrared+spectroscopy,+stump+pressure,+and+somatosensory+evoked+potentials.">Accuracy of cerebral monitoring in detecting cerebral ischemia during carotid endarterectomy: a comparison of transcranial Doppler sonography, near-infrared spectroscopy, stump pressure, and somatosensory evoked potentials.</a> Anesthesiology. 107 (4), 563-569 (2007).</li><li>Beilman, G. J., Groehler, K. E., Lazaron, V., Ortner, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+spectroscopy+measurement+of+regional+tissue+oxyhemoglobin+saturation+during+hemorrhagic+shock.">Near-infrared spectroscopy measurement of regional tissue oxyhemoglobin saturation during hemorrhagic shock.</a> Shock. 12 (3), 196-200 (1999).</li><li>Rhee, P., Langdale, L., Mock, C., Gentilello, 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=Near-infrared+spectroscopy:+continuous+measurement+of+cytochrome+oxidation+during+hemorrhagic+shock.">Near-infrared spectroscopy: continuous measurement of cytochrome oxidation during hemorrhagic shock.</a> Crit Care Med. 25 (1), 166-170 (1997).</li><li>Zweifel, C., Castellani, G., 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=Continuous+assessment+of+cerebral+autoregulation+with+near-infrared+spectroscopy+in+adults+after+subarachnoid+hemorrhage.">Continuous assessment of cerebral autoregulation with near-infrared spectroscopy in adults after subarachnoid hemorrhage.</a> Stroke. 41 (9), 1963-1968 (2010).</li><li>Scheeren, T. W. L., Schober, P., Schwarte, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+tissue+oxygenation+by+near+infrared+spectroscopy+(NIRS):+background+and+current+applications.">Monitoring tissue oxygenation by near infrared spectroscopy (NIRS): background and current applications.</a> J Clin Monit Comput. 26 (4), 279-287 (2012).</li><li>Boushel, R., Langberg, H., Olesen, J., Gonzales-Alonzo, J., B&#252;low, J., Kjaer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+tissue+oxygen+availability+with+near+infrared+spectroscopy+(NIRS)+in+health+and+disease.">Monitoring tissue oxygen availability with near infrared spectroscopy (NIRS) in health and disease.</a> Scand J Med Sci Sports. 11 (4), 213-222 (2001).</li><li>Aaslid, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+autoregulation+and+vasomotor+reactivity.">Cerebral autoregulation and vasomotor reactivity.</a> Front Neurol Neurosci. 21, 216-228 (2006).</li><li>Palada, I., Obad, A., Bakovic, D., Valic, Z., Ivancev, V., Dujic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+and+peripheral+hemodynamics+and+oxygenation+during+maximal+dry+breath-holds.">Cerebral and peripheral hemodynamics and oxygenation during maximal dry breath-holds.</a> Respir Physiol Neurobiol. 157 (2-3), 374-381 (2007).</li><li>Heusser, K., Dzamonja, G., 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=Cardiovascular+regulation+during+apnea+in+elite+divers.">Cardiovascular regulation during apnea in elite divers.</a> Hypertension. 53 (4), 719-724 (2009).</li><li>Joulia, F., Lemaitre, F., Fontanari, P., Mille, M. L., Barthelemy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulatory+effects+of+apnoea+in+elite+breath-hold+divers.">Circulatory effects of apnoea in elite breath-hold divers.</a> Acta Physiol (Oxf). 197 (1), 75-82 (2009).</li><li>Costalat, G., Coquart, J., Castres, I., Tourny, C., Lemaitre, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+adjustments+during+breath-holding+in+trained+divers.">Hemodynamic adjustments during breath-holding in trained divers.</a> Eur J Appl Physiol. 113 (10), 2523-2529 (2013).</li><li>Busch, D. R., Lynch, J. 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=Cerebral+Blood+Flow+Response+to+Hypercapnia+in+Children+with+Obstructive+Sleep+Apnea+Syndrome.">Cerebral Blood Flow Response to Hypercapnia in Children with Obstructive Sleep Apnea Syndrome.</a> Sleep. 39 (1), 209-216 (2016).</li><li>Alex, R., Bhave, G., 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=An+investigation+of+simultaneous+variations+in+cerebral+blood+flow+velocity+and+arterial+blood+pressure+during+sleep+apnea.">An investigation of simultaneous variations in cerebral blood flow velocity and arterial blood pressure during sleep apnea.</a> Conf Proc IEEE Eng Med Biol Soc. , 5634-5637 (2012).</li><li>Eichhorn, L., Erdfelder, 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=Influence+of+Apnea-induced+Hypoxia+on+Catecholamine+Release+and+Cardiovascular+Dynamics.">Influence of Apnea-induced Hypoxia on Catecholamine Release and Cardiovascular Dynamics.</a> Int J Sports Med. , (2016).</li></ol>

The authors have nothing to disclose.

The total apnea time is mainly caused by lung size and oxygen consumption per minute and influenced by an individuals&#39; ability to withstand the breathing reflex caused by increasing pCO2 or decreasing pO2. Apnea divers are trained to maximize their breath-hold duration and are used to doing so in maximal inspiration. Therefore, the time until hypoxia is detectable differs between individuals and depends on the subject&#39;s physical condition and training status and might even vary by their daily state and willingness to withstand the breathing reflex. The subject&#39;s stress levels can be reduced by detailed education of protocol steps and a calm ambient environment.
There are many factors that influence total apnea time, which means that the testing environment should be standardized in order to get results that are reliable and repeatable. If researchers are interested in studying the catecholamine increase or sympathetic nerve activity, substances influencing both (i.e., caffeine, nicotine, food like bananas, nuts, or any medical substances like monoamine oxidase (MAO) inhibitors, etc.) should be avoided. Also the intravenous line should be established at least 20 min before apnea. A subjects&#39; stress level will mainly influence catecholamine-levels and could falsify researchers&#39; results of blood analysis. In general, researchers should create baseline levels of each subject to normalize the results because of the large inter-individual differences.
Non-invasive measurements of tissue oxygenation by NIRS technology uses semi-quantitative changes in oxygenated and deoxygenated hemoglobin 21. The use of NIRS is constantly growing 20 and it can detect saturation of cerebral and peripheral tissue, independent of pulsatile blood flow. NIRS values depend on the amount of venous and arterial vessels placed under the NIRS-electrodes. NIRS values can therefore differ significantly depending on the amount of venous versus arterial vessels under the electrode. Also, placement and contact pressure will influence the reliability of values. Values should be checked for stability before starting measurement. If NIRS signals vary during baseline measurements, replace the electrodes or check for total skin contact. For interpretation of the NIRS results, relative de- or increase of values compared to baseline values should be used (not absolute).
Due to the physical burden of a maximal breath-hold, the number of apneas per subject is limited. The preparation protocols should be equal for each subject and all devices should be double-checked before they are used. Do not modify the protocol in one cohort. Standardized setups are mandatory to create results that are reproducible. Although hyperventilation before maximal breath hold lowers arterial CO2 levels and delays the breathing stimulus, it also affects cerebral autoregulation and vasomotor reactivity22. Active hyperventilation should be avoided to minimize disruptive effects by the subject.
The overall goal of this model is to simulate hypoxia in humans by breath hold. Therefore, additional measurement devices can be established to get more detailed information about blood pressure (i.e., invasive blood pressure measurement) or sympathetic nerve activity. Blood pressure measurements can be used to estimate the burden of prolonged apnea to the vessel system. ECG signals can be used to calculate beat-to-beat variability in R-R interval or to detect cardiac arrhythmia. Furthermore, cortisol-levels in saliva or catecholamine-levels 29 in blood-samples can be measured at different time points during and after apnea. The kinetics of these values opens up a number of possible study opportunities. Still, a reliable detection of hypoxia is necessary to ensure hypoxic conditions caused by apnea. Values measured by different devices but in the same apneic session can be compared directly. Time differences (for instance, until blood pressure increase, desaturation starts, etc.) from different individuals should be normalized to total apnea time.
The respiratory reflex is one of the strongest stimulus of the human body. Acute hypoxia and hypercapnia is therefore only seen in patients with pathologies (i.e., OSA, emergency situations, laryngospasm, CPR, etc.). Mostly unforeseen, hypoxia is difficult to detect, always influenced by a triggering event and difficult to evaluate because of a subjects&#39; comorbidities. Although total apnea time of divers and patients undergoing hypoxia should not be compared because of the completely different starting conditions, human compensatory mechanisms to avoid damage to the brain in case of hypoxia are identical 23-28. An extended voluntary breath-hold also empties the body&#39;s oxygen-storage and increases a subject&#39;s pCO2 29. Apneic divers were shown to generate reliable results during simulation of dynamic hypoxia in humans 12. We measured a minimum cerebral saturation only slightly higher than values seen in patients during cardiac arrest (42.2 &#177; 10.7%&#160;15 and 37.2 &#177; 17.0% 14). This indicates that our model is able to mimic clinically relevant hypoxia. Although hypoxia causes serious health problems, the underling physiological mechanisms are yet not completely understood 1 and till now no relevant clinical human model existed to simulate acute hypoxia in humans. Using healthy apneic divers as a clinical relevant model to simulate hypoxia and hypercapnia in humans holds large potential for future investigations. This model allows scientists to study the compensatory mechanism to avoid hypoxic damage in a reproducible human model. It allows a clinically relevant simulation of hypoxic emergency situations such as laryngospasm or &#34;cannot ventilate - cannot intubate&#34;. It might be used to prove the feasibility of new invasive or non-invasive tools for measuring human hypoxia. Furthermore, this model may help to understand the correlation of increased endogenous catecholamines and their impact on cardiac function (i.e., heart rate variability, cardiac output, etc.). By using different and new devices to observe hypoxia in apneic divers new parameters may be explored and may extend our understanding of hypoxia in the future.

Clinically relevant acute hypoxia and concomitant hypercapnia is mostly seen in patients with obstructive sleep apnea syndrome (OSAS), acute airway obstruction or during cardiopulmonary resuscitation. Major limitations in the field of OSAS and other hypoxemic conditions include the limited transferable knowledge about the pathophysiology derived from animal studies and that human models are non-existent 1. To mimic hypoxia in humans, hypoxic gas mixtures have so far been used 2-7. However, these conditions are more representative of high altitude surroundings than of clinical situations where hypoxia, in general, is accompanied by hypercapnia. To monitor tissue oxygenation during cardiac arrest and resuscitation, animal studies have been performed 8 to investigate physiological compensatory mechanisms.
Apneic divers are healthy athletes capable of depressing the breathing impulse that is evoked by low arterial oxygen saturation 9 and an increased pCO2 10,11. We investigated apneic divers in order to mimic clinical situations of acute hypoxia and concomitant hypercapnia 12. This model can be used to evaluate clinical setups, improve the pathophysiological understanding of patients with OSAS or pathological breathing disorders, and reveal new possibilities for studying a potential counter balancing mechanism in cases of apnea. Furthermore, different techniques to detect hypoxia in humans can be tested for feasibility and accuracy in the case of dynamic hypoxia that is present in emergency situations (i.e., airway obstructions, laryngospasm or cannot intubate, cannot ventilate situations) or to simulate intermittent hypoxia in patients with OSAS.
Noninvasive techniques to detect hypoxia in humans are limited. Peripheral pulse oximetry (SpO2) is an approved tool in pre-hospital and hospital settings to detect hypoxia 13. The method is based on light absorption of hemoglobin. However, SpO2 measurement is limited to peripheral arterial oxygenation and cannot be used in cases of pulseless electrical activity (PEA) or centralized minimal circulation 14. In contrast, Near-Infrared Spectroscopy can be used to evaluate cerebral tissue oxygen saturation (rSO2) in real-time during PEA, during hemorrhagic shock or following subarachnoid hemorrhage 15-19. Its use is constantly growing 20 and methodological studies have revealed a positive correlation between SpO2 and rSO2 3,4.
In this study, we provide a model to simulate clinically relevant hypoxia in humans and present a step-by-step methodology to compare peripheral pulse oximetry and NIRS in case of de- and re-saturation. By analyzing physiological data in case of apnea, our understanding of counter balancing mechanisms can be improved.

<table><tbody><tr><td>SpO&lt;sub&gt;2&lt;/sub&gt;</td><td>Dr&amp;auml;ger Medical AG&amp;amp;CO.KG</td><td>SHP ACC MCABLE-Masimo Set</td><td>peripheral SpO&lt;sub&gt;2&lt;/sub&gt;-Monitoring</td></tr><tr><td>Non Invasive Blood Pressure (NIBP)</td><td>Dr&amp;auml;ger Medical AG&amp;amp;CO.KG</td><td>NIBP cuff M+,&amp;nbsp; MP00916&amp;nbsp;</td><td></td></tr><tr><td>Electrocardiographic (ECG) &amp;nbsp;</td><td>Dr&amp;auml;ger Medical AG&amp;amp;CO.KG</td><td>Infinity M540 Monitor</td><td>ECG monitoring</td></tr><tr><td>Docking station</td><td>Dr&amp;auml;ger Medical AG&amp;amp;CO.KG</td><td>M500 Docking Station</td><td>connection of M540 to laptop</td></tr><tr><td>NIRS</td><td>NONIN Medical&amp;rsquo;s EQUANOX</td><td>Model 7600 Regional Oximeter System</td><td>measuring of cerebral and&amp;nbsp; tissue oxygenation</td></tr><tr><td>NIRS diodes</td><td>EQUANOX Advance Sensor</td><td>Model 8004CA</td><td>suited for measuring cerebral and somatic oxygen-saturation</td></tr><tr><td>&lt;strong&gt;Laptop&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DataGrabber</td><td>Dr&amp;auml;ger Medical AG&amp;amp;CO.KG</td><td>DataGrabber v2005.10.16</td><td>software to synchronize M540 with laptop</td></tr><tr><td>eVision</td><td>Nonin Medical. Inc.</td><td>Version 1.3.0.0</td><td>software to synchronize NONIN with laptop</td></tr></tbody></table>

a model to simulate clinically relevant hypoxia in humans

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

Figure 1 displays simultaneous recordings of SpO2 and NIRS values (NIRScerebral and NIRStissue) during apnea in one patient. Total apnea time was 363 sec. Following apnea NIRS and SpO2 values remained stable for approximately 140 sec. A decrease in SpO2 was detected after 204 sec by peripheral SpO2 whereas a decrease of NIRScerebral was detected after 238 sec. The lowest measured SpO2 following apnea was 58% and lowest measured NIRScerebral was 46%. At the end of apnea NIRScerebral increased after a time delay of 12 sec whereas SpO2 increased after a time delay of 30 sec.
In a recent study of ten apneic divers we showed a significant decrease in NIRScerebral values from 71% (range 85 - 55) to 54% (range 74 - 24) 12. Median SpO2 decreased from 98% (range 100 - 98) to 81% (range 94 - 67). Figure 2 displays the mean time delays between the beginning of apnea and decrease in NIRScerebral versus SpO2 values of these ten divers. Oxygen saturation measured by NIRScerebral decreased significantly later than oxygen saturation on the fingertip measured by SpO2 [175 sec; SD = 50 sec versus 134 sec; SD = 29 sec; (t(9) = 2.865, p = 0.019, r2 = 0.477)]. This can be taken as a sign for elevated cerebral blood flow and preferential oxygen supply of cerebral tissue during apnea.
After restart of respiration (Figure 2c), values of NIRScerebral increased significantly earlier than SpO2 values [10 sec; SD = 4 sec versus 21 sec; SD = 4 sec (t(9) = 7.703, p &#60; 0.001, r2 = 0.868)]. Figures 2b and d display de- and re-saturation measured on the fingertip (SpO2) and above the musculus quadriceps femoris (NIRStissue) during apnea. NIRStissue values decreased significantly earlier than SpO2 values [39 s; SD = 13 sec versus a delay of 125 sec; SD = 36 sec (t(6) = 4.869, p = 0.003, r2 = 0.798)]. This time delay might show that peripheral vasoconstriction leads to a decrease in tissue oxygenation, even before a decrease in arterial oxygen saturation &#8212; visualized by SpO2 &#8212; is measureable. There was no difference in time delay after restart of respiration between NIRStissue and SpO2 [NIRStissue 30 sec; SD = 16 sec versus SpO2 27 sec; SD = 7 sec (t(6) = 0.631, p = 0.551, r2 = 0.062)]. This indicates, that the observed time delay is not caused by the different devices themselves.
To compare de- and re-saturation during individual apnea durations, we normalized SpO2-, NIRStissue- and NIRScerebral-baseline values to 100% (Figure 3). To compare individual apnea duration, total apnea duration of each subject was also set to 100%. 12
<img alt="Figure 1" src="/files/ftp_upload/54933/54933fig1.jpg" /> 
Figure 1: Time-course of NIRS, SpO2, and Heart Rate (HR) during Apnea. Raw data of one participant is displayed. Total apnea-time was 363 sec. Subject exhibited an earlier decrease in SpO2 than in cerebral rSO2. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54933/54933fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54933/54933fig2.jpg" /> 
Figure 2: Time Delays during Apnea and Restart of Respiration. a) Mean time delay between beginning of apnea and decrease of NIRScerebral versus SpO2 values; b) Mean time delay between beginning of apnea and decrease of NIRStissue versus SpO2 values; c) Mean time delay between restart of respiration and an increase of NIRScerebral versus SpO2 values; d) Mean time delay between restart of respiration and an increase of NIRStissue versus SpO2 values. Error bars indicate standard error of the mean. Data and figure from Eichhorn et al. 2015 12. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54933/54933fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54933/54933fig3.jpg" /> 
Figure 3: Temporal Progression of Normalized SpO2, NIRScerebral and NIRStissue Values: To equilibrate individual variations in apnea time, all apnea times were standardized to 100%. Thus the variations in the three plotted parameters are assigned to the relative apnea times. Baseline values measured prior to apnea were defined as 100%. Error bars indicate standard error of the mean. Data and figure from Eichhorn et al. 2015&#160;12. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54933/54933fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Model to Simulate Clinically Relevant Hypoxia in Humans at JoVE.com

A Model to Simulate Clinically Relevant Hypoxia in Humans

Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2).

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...

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8.7K Views.  University Hospital of Bonn. Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2).

Video: A Model to Simulate Clinically Relevant Hypoxia in Humans

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

Cancer Research

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to adequate numbers prior to transplantation. Identifying approaches to refine this process, to augment the production of all CNS cell types (i.e., neurons), and to possibly contribute to therapeutic cell therapy protocols is a high research priority. This report describes an easily applied in vivo &#8220;pre-conditioning&#8221; stimulus which can be delivered to awake, non-anesthetized animals. Thus, it is a non-invasive and non-stressful procedure. Specifically described are the procedures for exposing mouse or rat pups (aged postnatal day 1-8) to a brief (40-80 min) period of intermittent hypoxia (AIH). The procedures included in this video protocol include calibration of the whole-body plethysmography chamber in which pups are placed during AIH and the technical details of AIH exposure. The efficacy of this approach to elicit tissue-level changes in the awake animal is demonstrated through the enhancement of subsequent in vitro expansion and neuronal differentiation in cells harvested from the subventricular zone (SVZ). These results support the notion that tissue level changes across multiple systems could be observed following AIH, and support the continued optimization and establishment of AIH as a priming or conditioning modality for therapeutic cell populations.

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

This article describes the methodology for administering short periods of intermittent hypoxia to postnatal day 1-8 mouse or rat pups. This approach effectively elicits a robust tissue level &#8220;priming effect&#8221; on cultured neural progenitor cells that are harvested within 30 min of hypoxia exposure.

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to ...

Developmental Biology

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Neuroscience

Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by international guidelines for patients suffering from cardiovascular diseases, more specifically, lower extremity artery diseases, where the patients' walking capacity is considerably altered, affecting their quality of life. 
Traditionally, both low continuous exercise and interval training have been used. Recently, supramaximal training has also been shown to improve athletes' performances via vascular adaptations, amongst other mechanisms. The combination of this type of training with hypoxia could bring an additional and/or synergic effect, which could be of interest for certain pathologies. Here, we describe how to perform supramaximal intensity training sessions in hypoxia on healthy mice at 150% of their maximal speed, using a motorized treadmill and a hypoxic box. We also show how to dissect the mouse in order to retrieve organs of interest, particularly the pulmonary artery, the abdominal aorta, and the iliac artery. Finally, we show how to perform ex vivo vascular function assessment on the retrieved vessels, using isometric tension studies.

High-intensity training in hypoxia is a protocol that has been proven to induce vascular adaptations potentially beneficial in some patients and to improve athletes&#39; repeated sprint ability. Here, we test the feasibility of training mice using that protocol and identify those vascular adaptations using ex vivo vascular function assessment.

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by ...

Behavior

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Biology

Translational 3D-Cell Culture Model to Assess Hyperoxia Effects on Human Neonatal Airway Epithelial Cells

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes oxygen (O2) tension higher than the ambient atmosphere - termed hyperoxia (&gt;21% O2). The effect of hyperoxia on the airway depends on various factors, including the developmental stage of the airway, the degree of hyperoxia, and the duration of exposure, with variable exposures potentially leading to unique phenotypes. While there has been extensive research on the effect of hyperoxia on neonatal lung alveolarization and airway hyperreactivity, little is known about the short and long-term underlying effect of hyperoxia on human neonatal airway epithelial cells. A major reason for this is the scarcity of an effective in vitro model to study human neonatal airway epithelial development and function. Here, we describe a method for isolating and expanding human neonatal tracheal airway epithelial cells (nTAECs) utilizing human neonatal tracheal aspirates and culturing these cells in air-liquid interface (ALI) culture. We demonstrate that nTAECs form a mature polarized cell-monolayer in ALI culture and undergo mucociliary differentiation. We also present a method for moderate hyperoxia exposure of the cell monolayer in ALI culture using a specialized incubator. Additionally, we describe an assay to measure cellular oxidative stress following hyperoxia exposure in ALI culture using fluorescent quantification, which confirms that moderate hyperoxia exposure induces cellular oxidative stress but does not cause significant cell membrane damage or apoptosis. This model can potentially be used to simulate clinically relevant hyperoxia exposure encountered by neonatal airways in the Neonatal Intensive Care Unit (NICU) and used to study the short and long-lasting effects of O2 on neonatal airway epithelial programming. Studies using this model could be utilized to explore ways to mitigate early-life oxidative injury to developing airways, which is implicated in the development of long-term airway diseases in former premature infants.

We describe a protocol to establish an air-liquid interface (ALI) culture model utilizing neonatal tracheal airway epithelial cells (nTAEC) and perform physiologically relevant hyperoxia exposure to study the effect of atmospheric-induced oxidative stress on cells derived from the developing neonatal airway surface epithelium.

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes ...

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by right heart&#160;catheterization. A broad spectrum of diseases can lead to PH, differing in their etiology, histopathology, clinical presentation, prognosis, and response to&#160;treatment. Despite significant progress in the last years, PH remains an uncured disease. Understanding the underlying mechanisms can pave the way for the development of new therapies. Animal models are important research tools to achieve this goal. Currently, there are several models available for recapitulating PH. This protocol describes a two-hit mouse PH model. The stimuli for PH development are hypoxia and the injection of SU5416, a vascular endothelial growth factor (VEGF) receptor antagonist. Three weeks after initiation of Hypoxia/SU5416, animals develop pulmonary vascular remodeling imitating the histopathological changes observed in human PH (predominantly Group 1). Vascular remodeling in the pulmonary circulation results in the remodeling of the right&#160;ventricle (RV). The procedures for measuring RV pressures (using the open chest method), the morphometrical analyses of the RV (by dissecting and weighing both cardiac ventricles) and the histological assessments of the remodeling (both pulmonary by assessing vascular remodeling and cardiac by assessing RV cardiomyocyte hypertrophy and fibrosis) are described in detail. The advantages of this protocol are the possibility of the application both in wild type and in genetically modified mice, the relatively easy and low-cost implementation, and the quick development of the disease of interest (3 weeks). Limitations of this method are that mice do not develop a severe phenotype and PH is reversible upon return to normoxia. Prevention, as well as therapy studies, can easily be implemented in this model, without the necessity of advanced skills (as opposed to surgical rodent models).

This protocol describes the induction of pulmonary hypertension (PH) in mice based on the exposure to hypoxia and the injection of a VEGF receptor antagonist. The animals develop PH and right ventricular (RV) hypertrophy 3 weeks after the initiation of the protocol. The functional and morphometrical characterization of the model is also presented.

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by ...

Multiple Intravenous Bolus Dosing and Invasive Hemodynamic Assessment in a Hypoxia-Induced Mouse Pulmonary Artery Hypertension Model

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, there is no cure for PAH. It is important to discover new compounds that can be used to treat PAH. The mouse hypoxia-induced PAH model is a widely used model for PAH research. This model recapitulates human clinical manifestations of PAH Group 3 disease and is an important research tool to evaluate the effectiveness of new experimental therapies for PAH. Research using this model often requires the administration of compounds in mice. For a compound that needs to be given directly into the bloodstream, optimizing intravenous (IV) administration is a key part of the experimental procedures. Ideally, the IV injection system should permit multiple injections over a set time course. Although the mouse hypoxia-induced PAH model is very popular in many laboratories, it is technically challenging to perform multiple IV bolus dosing and invasive hemodynamic assessment in this model. In this protocol, we present step-by-step instructions on how to carry out multiple IV bolus dosing via mouse jugular vein and perform arterial and right ventricle catheterization for hemodynamic assessment in mouse hypoxia-induced PAH model.

This protocol provides a step-by-step procedure for executing multiple intravenous bolus dose administration and invasive hemodynamic monitoring in mice. Investigators can use this protocol for future therapeutic compound screening for pulmonary artery hypertension.

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, ...

Experimental Approach to Examine Leptin Signaling in the Carotid Bodies and its Effects on Control of Breathing

An adipocyte-produced hormone leptin is a potent respiratory stimulant, which may play an important role in defending respiratory function in obesity. The carotid bodies (CB), a key organ of peripheral hypoxic sensitivity, express the long functional isoform of leptin receptor (LepRb) but the role of leptin signaling in control of breathing has not been fully elucidated. We examined the hypoxic ventilatory response (HVR) (1) in C57BL/6J mice before and after leptin infusion at baseline and after CB denervation; (2) in LepRb-deficient obese db/db mice at baseline and after LepRb overexpression in CBs. In C57BL/6J mice, leptin increased HVR and effects of leptin on HVR were abolished by CB denervation. In db/db mice, LepRb expression in CB augmented the HVR. Therefore, we conclude that leptin acts in CB to augment responses to hypoxia.

Our study focuses on the effects of leptin signaling in carotid body (CB) on the hypoxic ventilatory response (HVR). We performed &#39;loss of function&#39; experiments measuring the effect of leptin on HVR after CB denervation and &#39;gain of function&#39; experiments measuring HVR after overexpression of the leptin receptor in CB.

An adipocyte-produced hormone leptin is a potent respiratory stimulant, which may play an important role in defending respiratory function in obesity. The ...

A Model to Simulate Clinically Relevant Hypoxia in Humans

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In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

A Swine Model of Neonatal Asphyxia

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice

Induction and Testing of Hypoxia in Cell Culture

Translational 3D-Cell Culture Model to Assess Hyperoxia Effects on Human Neonatal Airway Epithelial Cells

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model

Multiple Intravenous Bolus Dosing and Invasive Hemodynamic Assessment in a Hypoxia-Induced Mouse Pulmonary Artery Hypertension Model

Experimental Approach to Examine Leptin Signaling in the Carotid Bodies and its Effects on Control of Breathing