A Model to Simulate Clinically Relevant Hypoxia in Humans

Summary

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).

Abstract

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.

Introduction

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.

Protocol

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

1. Clean the skin of the forehead with 70% alcohol to degrease the skin prior to NIRS electrode positioning.
2. 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.
3. Evaluate the stability of the signal. The rSO₂-signal should be constant (± 3%) for at least 5 min.
4. For measuring peripheral tissue oxygenation with NIRS (NIRS_tissue-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.
5. Place ECG-electrodes on the hair free chest. The ECG leads are marked with different letters. Place "R" on the sternocostal head of pectoralis major right, "L" on the sternocostal head of pectoralis major left, "C" on the fifth intercostal space middle of the medioclavicular line, "F" on the left lower rib edge, "N" on the right lower rib edge.
6. Measure peripheral pulse oximetry (SpO₂) on a fingertip on the same extremity and side where the NIRS_tissue-electrode is placed.
7. 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 "settings".
8. 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.
   1. Clean the skin with 70% alcohol.
   2. Use a tourniquet to help the veins become more prominent.
   3. Use skin-disinfection to avoid infections and insert the needle through the skin.
   4. Reduce the insertion angle after blood flashback at the catheter hub. Push the catheter into the vein.
   5. Remove the needle and flush catheter with sterile saline (NaCl 0.9%).

2. Data Collection

1. Calibrate the internal clock of all monitors in order to synchronize measurements for later processing.
   1. Click the bottom-right clock icon on the desktop, and tap "change date and time settings" in the pop-up window.
   2. Press the Settings menu button on the NIRS devise and change date and time via the menu.
2. 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.
3. Use a monitor device specific software to save measurements on the computer. Click "start" 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 NIRS_tissue signals is too high, re-evaluate the position of the electrode (avoid bigger venous plexus or arteries directly under the electrodes). High variability of NIRS_cerebral 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.

3. Apnea

1. 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 ≤ 15 breaths/min.
2. 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.
3. Ensure that monitor values are invisible to subjects to avoid visual influences to their apneic performance.
4. 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.
5. 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.
6. Mark important events (i.e., beginning and end of apnea) electronically to avoid inaccuracies in further time analysis by pressing the "Event Mark Button" 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.
7. Draw blood samples at different time-points depending on the aim of the study.
8. Centrifuge blood samples at 1,500 x g for 10 min. Take the supernatant and store it at -80 °C for future analysis.

4. Processing Data

1. Processing data from the monitor device:
   1. Open the saved file on the computer and press "start" to analyze data.
   2. Click "review" to get access to the trend monitor and select "options" and then "tools" in the MENU submask. Time interval can be changed via "trend interval" if necessary.
   3. Select the mask "trends" and save. Open file "trends" in a spreadsheet program for further processing.
2. Processing data from NIRS device:
   1. Open the software on the computer and connect the NIRS device via WIFI.
   2. Transfer the data from the NIRS device to the computer.
   3. Save the data in CSV-format.
   4. Open file in a spreadsheet program for further processing.

5. Analyze Values

1. 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 (± 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.
2. Find the start point of monotonic decrease in rSO₂ and SpO₂ during apnea by looking for a decrease of values > 2% compared to baseline-levels. This time point is defined as "begin of desaturation".
3. 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 "begin of re-saturation".
4. Calculate the time difference between "start of apnea" and "begin of desaturation" and the time differences between "end of apnea" and "begin of re-saturation" for NIRS_cerebral, NIRS_tissue and SpO₂. Save each difference in seconds on a separate spreadsheet.
5. 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.

6. Statistical Processing

1. Compare the time differences between "beginning of desaturation" of SpO₂, NIRS_cerebral, and NIRS_tissue values. Test for Gaussian distribution of the measurement differences (e.g., using Shapiro-Wilk normality test for sample sizes smaller than 50).
2. 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.

Results

Figure 1 displays simultaneous recordings of SpO₂ and NIRS values (NIRS_cerebral and NIRS_tissue) 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 NIRS_cerebral was detected after 238 sec. The lowest measured SpO₂ ...

Discussion

The total apnea time is mainly caused by lung size and oxygen consumption per minute and influenced by an individuals' 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's physical condition and training status and might even vary by their dail...

Materials

Name	Company	Catalog Number	Comments
SpO2	Dräger Medical AG&CO.KG	SHP ACC MCABLE-Masimo Set	peripheral SpO2-Monitoring
Non Invasive Blood Pressure (NIBP)	Dräger Medical AG&CO.KG	NIBP cuff M+,  MP00916
Electrocardiographic (ECG)	Dräger Medical AG&CO.KG	Infinity M540 Monitor	ECG monitoring
Docking station	Dräger Medical AG&CO.KG	M500 Docking Station	connection of M540 to laptop
NIRS	NONIN Medical’s EQUANOX	Model 7600 Regional Oximeter System	measuring of cerebral and  tissue oxygenation
NIRS diodes	EQUANOX Advance Sensor	Model 8004CA	suited for measuring cerebral and somatic oxygen-saturation
Laptop
DataGrabber	Dräger Medical AG&CO.KG	DataGrabber v2005.10.16	software to synchronize M540 with laptop
eVision	Nonin Medical. Inc.	Version 1.3.0.0	software to synchronize NONIN with laptop