This work is supported by funding from the United States Army, Medical Research and Materiel Command, Combat Casualty Care Program.&#160;We thank LTC Kevin S. Akers, MD and Ms. Kristen R. Lye for their assistance in making the video.

Prior to any human procedure, the institutional&#160;review board (IRB) must approve the protocol. The protocol used in this study was approved by the US Army Medical Research and Materiel Command IRB. The protocol is designed to demonstrate the physiological responses of compensation to a progressive reduction in central blood volume similar to that experienced by individuals during ongoing hemorrhage in a controlled and reproducible laboratory setting. The laboratory room temperature is controlled at 23 - 25 &#730;C.
1. Equipment Preparation
<ol>
	<li>Turn on equipment and devices requiring warm-up and calibration. 
	NOTE: Equipment and devices include a data acquisition system to record data at 1 Hz; two separate devices that provide noninvasive, continuous measurements of brachial artery blood pressure and arterial oxygen saturation (SpO2) using two separate infrared finger photoplethysmography cuff sensors9-11; a capnograph for measurement of end-tidal CO2 and respiratory rate; and a finger pulse oximeter to acquire peripheral pulsatile arterial waveforms for measuring Compensatory Reserve.</li>
	<li>Synchronize all of the instruments with internal clocks by adjusting the time stamp on each instrument to match a laboratory master clock that will be used to mark time during the experiment.</li>
</ol>
2. Subject Preparation
<ol>
	<li>Instruct the subject to avoid caffeine, alcohol, and strenuous exercise 24 h prior to testing, and to avoid eating at least 2 h prior to the protocol in the event that hemodynamic decompensation induces nausea.</li>
	<li>Prior to initiation of the protocol, have the physician perform a medical screening exam to ensure the subject meets minimal health requirements, and ensures the absence of exclusion criteria (nicotine use, hypertension, autonomic dysfunction, or history of syncopal episodes). Since pregnancy is an exclusion criterion for participation, require female participants to take a standard urine pregnancy test on the day of the study. 
	NOTE: For the safety of the subject, the study physician is certified in advanced life support, and is present during the study. A fully-equipped &#39;crash cart&#39; is immediately available to support the subject&#39;s airway, respiration, and circulation in the event of loss of consciousness or an acute cardiac arrhythmia taking place during the LBNP procedure.</li>
	<li>Inform the subject about the procedure, and obtain written consent to participate in the study. 
	NOTE: Explain to the subject that the goal of the study is to apply LBNP until the onset of cardiovascular decompensation (presyncope). Explain that there are cardiovascular parameters that define this point, and LBNP will be terminated when these cardiovascular parameters are observed. Inform the subject that they may also experience symptoms typically associated with presyncope during the LBNP procedure. Instruct the subject to notify the investigator if these symptoms occur and LBNP will immediately be terminated.</li>
	<li>Place the neoprene LBNP skirt on the subject. Ensure that the skirt is snug around the waist and torso in order to create an air-tight seal.</li>
	<li>Instruct the subject to lay supine on the bed of the LBNP chamber while straddling a stationary post to secure the torso in place during LBNP. Instruct the subject to relax the lower body during LBNP exposure. Secure the subject into the LBNP chamber by sliding the bed into the chamber and attaching the neoprene skirt to the chamber opening to create an air-tight seal. 
	NOTE: The LBNP chamber provides the capability of accurately (within 0.1 mmHg) controlling the internal pressure from 0 to -100 mmHg either manually or with a computerized profile. The chamber includes an adjustable saddle to secure the subject&#39;s body position. Clear plexiglass windows allow for visualization of the subject&#39;s legs. An adjustable aluminum waist board allows for an air-tight seal to be created by a neoprene skirt worn by the subject and the LBNP chamber at the level of the iliac crest (Figure 1).</li>
	<li>Place electrocardiogram (ECG) electrodes on the right and left humoral-clavicular joints, and on the right and left lower ribs (total of 4) in a modified lead II configuration (Figure 1) for continuous measurement of heart rate.</li>
	<li>Position the subject&#39;s arms on the arm rests, adjusted so that the hands are supported at heart level. Using appropriate size finger cuffs, place an infrared finger photoplethysmography&#160;device on the left and right middle fingers for continuous noninvasive beat-to-beat measurement of blood pressure.</li>
	<li>Attach the finger cuffs to the pressure monitors. Calibrate the devices and record blood pressure according to the manufacturer instructions.12 Enter subject information (age, sex, height, and weight) to enable the appropriate assumptions for calculation (estimation) of stroke volume, cardiac output and peripheral vascular resistance by the Modelflow algorithm if desired.13,14</li>
	<li>Place the finger pulse oximeter on the right index finger for continuous measurement of compensatory reserve1,12 (Figure 2).</li>
	<li>Place a nasal cannula on the subject and instruct the subject to breathe through the nose to assure sensitive reflections in inspiration and expiration. Nasal air sampling will allow the subject to talk freely for self-reporting of developing symptoms. Connect the nasal cannula to the capnograph for the continuous measurement of respiration and end tidal CO2.</li>
</ol>
3. Performing the LBNP Protocol
<ol>
	<li>Start data recording by clicking the &#34;Start&#34; button on the data acquisition system. Record baseline data for 5 min. Initiate the first level of central hypovolemia by turning on the vacuum motor and setting negative pressure to -15 mmHg, and hold this pressure for 5 min. Figure 3 outlines the protocol.</li>
	<li>Increase the LBNP to -30 mmHg, and hold this pressure for 5 min.</li>
	<li>Increase the LBNP to -45 mmHg, and hold this pressure for 5 min.</li>
	<li>Increase the LBNP to -60 mmHg, and hold this pressure for 5 min.</li>
	<li>Increase the LBNP to -70 mmHg, and hold this pressure for 5 min.</li>
	<li>Continue to increase LBNP levels by -10 mmHg every 5 min until the end of the protocol (5 min at -100 mmHg LBNP) or the point of hemodynamic decompensation. Terminate the LBNP by pressing the pressure release button on the LBNP chamber. 
	NOTE: Hemodynamic decompensation is identified by a precipitous fall in systolic arterial pressure below 80 mmHg, or the subject reporting presyncopal symptoms such as grey-out (loss of color vision), tunnel vision, sweating, nausea or dizziness (Figure 4).</li>
	<li>Continue recording data on the data acquisition system during 10 min after the cessation of LBNP (postLBNP recovery).</li>
	<li>Stop recording data at the end of the 10-min recovery period by clicking the &#34;Stop&#34; button on the data acquisition system.</li>
	<li>Detach all instrumentation from the subject and remove the subject from the LBNP chamber. Ask the subject to sit after stepping down from the LBNP platform to ensure they are symptom-free before leaving the laboratory. The study is now complete.</li>
	<li>Download data files from the acquisition system for extraction of the Compensatory Reserve Index (CRI), Mean Arterial Pressure (MAP), heart rate, and SpO2 values. 1,15,16</li>
</ol>

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The authors declare that they have no competing financial interests. The views expressed herein are the private views of the authors and are not to be construed as representing those of the US Department of the Army or the US Department of Defense.

Using LBNP to cause progressive and continuous reductions in central blood volume, we were able to induce a typical response of hemodynamic decompensation in the subject, characterized by a sudden onset of hypotension and bradycardia (Figure 7). It is important to understand that the integrated compensatory response to hemorrhage is very complex,19 resulting in significant individual variability in the tolerance to blood loss.1 As such, some individuals have relatively responsive compensatory mechanisms while others do not compensate as effectively. Therefore, a critical step in the protocol is to conduct the experiment to the point of the onset of cardiovascular decompensation so that tolerance to hypovolemia can be accurately assessed. Premature termination of the experiment will not provide tolerance data. The experiments on more than 250 humans allowed us to classify individuals into two general populations1,15,20-23 &#8212; those with relatively high tolerance (completion of the -60 mmHg level of the LBNP protocol) to reduced central blood volume (i.e., good compensators) and those with low tolerance (poor compensators who failed to complete the -60 mmHg level of the LBNP protocol). One third (33%) of the humans we have tested has low tolerance, and two thirds (67%) of the subjects have high tolerance to hypovolemia. The subject tested in the presentation (Figure 7) would be classified as having high tolerance since he completed the -60 mmHg LBNP level.
LBNP is a well-established technique in the study of hypovolemia in humans, and troubleshooting is rarely necessary. However, using LBNP to assess TOLERANCE to hypovolemia requires that the experiment be conducted to the point of presyncope. A key factor in this experiment is maintaining a minimal risk of an adverse event (syncope) for the subject. As a result, all experiments are conducted in the presence of a study physician. In addition, all experiments are terminated immediately upon request of the subject or when systolic arterial pressure falls below 80 mmHg. The cessation of LBNP immediately redistributes blood volume to vital organs such as the brain and heart, subsequently restoring hemodynamic stability (Figure 4).
As can be expected, the airtight seal around the waist of the subject is a critical requirement to allow the progressive increases in negative pressure in the chamber. Occasionally, especially at higher LBNP levels, the airtight seal can be compromised. At this point, modifications can be made to reinforce the seal by tightening the laces on the neoprene skirt or placing foam pads between the subject&#39;s waist and LBNP table. The LBNP vacuum device can accommodate minor leaks in the seal without affecting the pressure in the chamber.
The hemodynamic responses to LBNP have been shown to mimic those observed during hemorrhage.8,17,24,25 We have used LBNP to study the compensatory responses to progressive bleeding in an effort to evaluate the body&#39;s integrative effort to maintain cardiovascular stability during blood loss (compensatory reserve) and to provide a measurement of compensatory reserve. While LBNP is a valid model for studying the compensatory responses to hemorrhage in humans, a limitation of this technique is the absence of other factors usually associated with hemorrhage such as trauma and pain. Clearly, the effects of these factors on the hemodynamic responses to hemorrhage cannot be assessed by LBNP induced hypovolemia in human volunteers.
Consistent with previously reported observations1,15,16 we used the LBNP model of hemorrhage to demonstrate that measurement of the compensatory reserve identifies a trajectory to hemodynamic instability (decompensation) well in advance of clinically significant changes in currently available vital signs. This is an important point to understand since earlier recognition of clinical urgency is critical to improving patient outcomes, particularly in the emergency medical setting.26-34 Existing methods for predicting cardiovascular decompensation rely on traditional vital signs that do not change until the onset of decompensation. The ability of the CRI algorithm to assess continuous changes in features of the arterial waveform allows machine-learning of the clinical status of the individual patient. In this regard, continuous real-time measurement of the compensatory reserve provides the most sensitive and specific technique to assess the tolerance of each individual to blood loss, and represents a significant improvement over existing methods for predicting hemorrhagic shock in the clinical setting.
It is important to recognize the CRI algorithm output as reflecting the integration of all physiological compensatory mechanisms involved in the compensation for a relative deficit in circulating blood volume. This notion is logical since the arterial waveform is made up of two distinct waves &#8212; the ejected wave (caused by contraction of the heart) and the reflected wave (caused by the arterial wave that reflects back from the arterial vasculature). All compensatory mechanisms that impact cardiac output (e.g., autonomic nerve activity, cardiac filling, respiration, cardiac medications, etc.) are contained within features of the ejected wave while all compensatory mechanisms that affect vascular resistance (e.g., sympathetic nerve activity, circulating catecholamines, arterial pH or CO2, arterial elasticity, muscle contractions, etc.) are represented by features of the reflected wave.1 As illustrated in Figure 8, the characteristic features change distinctly from an apparent single wave with a small notch in a normovolemic state (left panel) to two separated waves with smaller magnitudes of height and width in conditions of reduced central blood volume (right panel) such as occurs during hemorrhage. As such, changes in features of the arterial waveform in response to hemorrhage give a unique individual-specific predictive capability to assess one&#39;s capacity to compensate adequately for blood loss. Each individual&#39;s compensatory reserve is correctly estimated in real time because the machine-learning capability of the CRI algorithm accounts for compromised circulating blood volume as it &#34;learns&#34; and &#34;normalizes&#34; the totality of compensatory mechanisms based on the individual&#39;s arterial waveform features.1 In this regard, the compensatory reserve is a superior measure of the physiological status of a bleeding patient than any one or combination of vital signs.
CRI has also been estimated in case reports beyond the standard LBNP laboratory environment. Compensatory reserve measurements were obtained from humans with conditions of compromised tissue perfusion caused by controlled hemorrhage 16, trauma 1, trauma followed by sepsis 35, acute appendicitis 35, burn injury 35, massive hematemesis 35, childbirth 35, cardiac arrest 35, postural orthostatic tachycardia 35, progressive hypovolemia with heat stress 35, and Dengue hemorrhagic fever. 1 These results indicate that the measurement of compensatory reserve using the CRI algorithm has provided accurate patient diagnosis in clinical conditions of compromised tissue perfusion associated with pain and tissue injury, and in varying environmental challenges.
The ability to measure the compensatory changes associated with blood loss is critical to providing acute care in emergency situations in both military and civilian scenarios. The LBNP technique will continue to be used as a valid model of human hemorrhage to provide data for creating, testing and refining future algorithms and devices to measure Compensatory Reserve.

The most important function of the cardiovascular system is the control of adequate perfusion (blood flow and oxygen delivery) to all tissues of the body through homeostatic regulation of arterial blood pressure. Various mechanisms of compensation (e.g., autonomic nervous system activity, cardiac rate and contractility, venous return, vasoconstriction, respiration) contribute to maintain normal physiological levels of oxygen in the tissues.1 Reductions in circulating blood volume such as those caused by hemorrhage can compromise the ability of cardiovascular compensatory mechanisms and ultimately lead to low arterial blood pressure, serious tissue hypoxia, and circulatory shock that can be fatal.
Circulatory shock caused by severe bleeding (i.e., hemorrhagic shock) is a leading cause of death due to trauma.2 One of the most challenging aspects of preventing a patient from developing shock is our inability to recognize its early onset. Early and accurate assessment of the progression toward the development of shock is currently limited in the clinical setting by technologies (i.e., medical monitors) that provide measurements of vital signs that change very little in the early stages of blood loss because of the body&#39;s numerous compensatory mechanisms for regulating blood pressure.3-6 As such, the capability to measure the sum total of the body&#39;s reserve to compensate for blood loss represents the most accurate reflection of tissue perfusion state and the risk of developing shock.1 This reserve is called the compensatory reserve which can be accurately assessed by real-time measurements of changes in features of the arterial waveform.1 Depletion of the compensatory reserve replicates the terminal cardiovascular instability observed in critically ill patients with sudden onset of hypotension; a condition known as hemodynamic decompensation.7
The relationship between the utilization of the compensatory reserve and regulation of blood pressure during ongoing blood loss in humans can be demonstrated in the laboratory using a comprehensive set of physiological measurements (e.g., blood pressures, heart rate, arterial blood oxygen saturation, stroke volume, cardiac output, vascular resistance, respiration rate, pulse character, mental status, end-tidal CO2, tissue oxygen) provided by standard physiological monitoring during continuous progressive reductions in central blood volume similar to those that occur during hemorrhage. Lowered central blood volume can be induced noninvasively with progressive increases in Lower Body Negative Pressure (LBNP).8 Using this combination of physiological measurements and LBNP, the conceptual understanding of how to assess the body&#39;s ability to compensate for reduced central blood volume can easily be demonstrated. This study depicts the prelab preparation, the demonstration of compensatory response in relation to other physiological responses during simulated hemorrhage, and the postlab evaluation of results. The experimental techniques necessary for making measurements of compensatory reserve are demonstrated in a human volunteer.

<table><tbody><tr><td>Dynamic Research Evaluation Workstation (DREW) data acquisition syetem</td><td>NA</td><td>NA</td><td>Custom Built by ISR personnel. The DREW allows for time synchronization of both digital and analog signal data collection from up to 16 independent instruments with a sampling rate of 1,000 Hz.</td></tr><tr><td>Finometer</td><td>Finapress Medical Systems (FMS)</td><td>Model 1</td><td>Device that provides noninvasive, continuous measurements of brachial artery blood pressure and arterial oxygen saturation (SpO&lt;sub&gt;2&lt;/sub&gt;) using two separate infrared finger photophlethymography cuff sensors.</td></tr><tr><td>BCI Capnocheck Plus</td><td>Smith Medical PM Inc.</td><td>9004</td><td>Capnograph used to measure end tidal CO&lt;sub&gt;2&lt;/sub&gt; and respiration rate</td></tr><tr><td>CipherOX&amp;nbsp;</td><td>Flashback Technologies Inc.</td><td>R200</td><td>Investigational device used to calculate Compensatory Reserve Index (CRI)</td></tr><tr><td>Nonin 9560 Pulse Oximeter</td><td>Nonin</td><td>9560</td><td>finger pulse oximeter</td></tr><tr><td>Lower Body Negative Pressure Chamber (LBNP)</td><td>NASA</td><td>79K32632-1</td><td>Custom Chamber built by NASA</td></tr><tr><td>ECG Biotach</td><td>Gould</td><td>13-6615-65</td><td>Electrocardiograph for measuring ECG</td></tr><tr><td>Nasal CO&lt;sub&gt;2&lt;/sub&gt; Sample Line</td><td>Salter Labs</td><td>REF 4000</td><td>Latex free nasal cannula for sampling expired air</td></tr></tbody></table>

integrated compensatory responses in a human model of hemorrhage

Hemorrhage is the leading cause of trauma-related deaths, partly because early diagnosis of the severity of blood loss is difficult. Assessment of hemorrhaging patients is difficult because current clinical tools provide measures of vital signs that remain stable during the early stages of bleeding due to compensatory mechanisms. Consequently, there is a need to understand and measure the total integration of mechanisms that compensate for reduced circulating blood volume and how they change during ongoing progressive hemorrhage. The body&#39;s reserve to compensate for reduced circulating blood volume is called the &#39;compensatory reserve&#39;. The compensatory reserve can be accurately evaluated with real-time measurements of changes in the features of the arterial waveform measured with the use of a high-powered computer. Lower Body Negative Pressure (LBNP) has been shown to simulate many of the physiological responses in humans associated with hemorrhage, and is used to study the compensatory response to hemorrhage. The purpose of this study is to demonstrate how compensatory reserve is assessed during progressive reductions in central blood volume with LBNP as a simulation of hemorrhage.

The LBNP procedure causes a reduction in air pressure around the lower torso and legs. As this vacuum is progressively increased, blood volume shifts from the head and upper torso to the lower body to create a state of central hypovolemia. The progressive reduction in central blood volume (i.e., LBNP) produces significant alterations in the features of the arterial waveform measured with the infrared finger photoplethysmograph (Figure 5). The Compensatory Reserve Index (CRI) is calculated from the recorded arterial pulse wave using a unique machine learning algorithm which analyzes changes in wave form characteristics to calculate an estimated compensatory reserve (Figure 6).1,15,16 Each continuous noninvasive photoplethysmograph waveform (represented as the monitored &#39;Patient&#39;s Arterial Waveform&#39;) is the input to calculate an estimate of an individual&#39;s compensatory reserve (represented as the &#39;CRI Estimate&#39;) based on comparison to a large &#39;library&#39; of reference waveforms (represented as the &#39;Algorithm Waveform Library&#39;) generated from progressive levels of central hypovolemia.
In this experiment, a subject was exposed to LBNP until the onset of hemodynamic decompensation which occurs when the body is no longer able to compensate for the hypovolemia. The values for mean arterial pressure, heart rate, SpO2, and CRI plotted against time (i.e., progressive reductions in central blood volume caused by increasing levels of LBNP) are shown in Figure 7. The results of the experiment show that changes in mean arterial pressure, heart rate, and SpO2 occur during the later phases of hemorrhage (i.e., &#62;15 min&#160;into the protocol for heart rate and &#62;25 min for mean arterial pressure and SpO2) while CRI decreases early and progressively throughout the multiple steps of LBNP.
Tolerance to reduced central blood volume is defined as the time from the start of the experiment to decompensation. In this example, tolerance was approximately 27.5 min at a level of -70 mmHg LBNP. Based on previous experiments that were designed to equate the magnitude of actual blood loss with LBNP,8 the equivalent blood loss that our subject was able to tolerate was estimated at approximately 1.2 L.
<img alt="Figure 1" src="/files/ftp_upload/54737/54737fig1.jpg" /> 
Figure 1: LBNP Chamber. A subject is shown in a supine position on the bed of the LBNP chamber. The neoprene skirt around the subject&#39;s waist is used to create an airtight seal within the LBNP chamber. Previously published in Cooke et al.17 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54737/54737fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54737/54737fig2.jpg" /> 
Figure 2: Compensatory Reserve Monitoring Device. The device consists of a noninvasive finger pulse oximeter that transmits pulse oximeter and waveform data via a USB connection to a compensatory reserve monitor. The monitor unit contains an algorithm which calculates a value for compensatory reserve known as the Compensatory Reserve Index&#160;(CRI)1,12. Data are recorded at each heart beat and displayed on the monitor and stored on a memory card. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54737/54737fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54737/54737fig3.jpg" /> 
Figure 3. Stepwise Changes in LBNP During Experiment. During the experimental protocol, LBNP (mmHg) is adjusted in a stepwise manner (5 min/level) to induce progressive central hypovolemia. This diagram shows LBNP increasing from 0&#160;to&#160;-100 mmHg during 40 min of an experimental protocol. Modified from Convertino et al.18 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54737/54737fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54737/54737fig4.jpg" /> 
Figure 4: Hemodynamic Decompensation. Sample blood pressure (mm Hg, yellow tracing) and lower body negative pressure (mmHg, white tracing) recordings are shown from a subject at the point of hemodynamic decompensation. At the point of decompensation, blood pressure is 78/55 mmHg, and lower body negative pressure is -60 mmHg. Blood pressure returns to normal after cessation of lower body negative pressure. Modified from Convertino et al.1 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54737/54737fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54737/54737fig5.jpg" /> 
Figure 5. Arterial Waveforms During LBNP. Sample recordings of arterial pressure waveforms are shown during baseline (upper tracing) and during -60 mmHg lower body negative pressure (LBNP, lower tracing). The changes in the characteristic features of the arterial waveforms are evaluated to estimate compensatory reserve. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54737/54737fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54737/54737fig6.jpg" /> 
Figure 6: How the CRI is Calculated. Diagram illustrating the process of the compensatory reserve index (CRI) algorithm that compares beat-to-beat arterial blood pressure waveform tracings over an interval of 30 heartbeats (A) to a &#39;library&#39; of waveforms (B) collected from humans exposed to progressive reductions in central blood volume for generation of an estimated CRI value (C). Reproduced from Convertino et al.15 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54737/54737fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54737/54737fig7.jpg" /> 
Figure 7. Sample Results of an LBNP Experiment. Values of Mean Arterial Pressure (MAP, mmHg), Heart Rate (HR, beats/min), arterial oxygen saturation (SpO2, %), Compensatory Reserve Index (CRI) and Lower Body Negative Pressure (LBNP, mmHg) are shown for one subject during an LBNP experiment. The dashed line represents the onset of cardiovascular decompensation, <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54737/54737fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/54737/54737fig8.jpg" /> 
Figure 8: Characteristic Features of the Arterial Waveform. Two wave forms are shown that demonstrate the characteristic features of the arterial ejected and reflected waveforms during normovolemia and hypovolemia. The red line indicates the integrated waveform that is recorded and observed in a tracing. Previously published in Convertino et al.1 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54737/54737fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Integrated Compensatory Responses in a Human Model of Hemorrhage at JoVE.com

Integrated Compensatory Responses in a Human Model of Hemorrhage

The purpose of this protocol is to demonstrate the techniques for measuring compensatory responses to reduced central blood volume using lower body negative pressure as a noninvasive experimental model of human hemorrhage which can be used to quantify the total integration of compensatory mechanisms to blood volume deficit in humans.

Hemorrhage is the leading cause of trauma-related deaths, partly because early diagnosis of the severity of blood loss is difficult. Assessment of ...

integrated-compensatory-responses-in-a-human-model-of-hemorrhage

Nanyang Technological University

Research

JoVE Journal

Medicine

12.4K Views.  JBSA Fort Sam Houston. The purpose of this protocol is to demonstrate the techniques for measuring compensatory responses to reduced central blood volume using lower body negative pressure as a noninvasive experimental model of human hemorrhage which can be used to quantify the total integration of compensatory mechanisms to blood volume deficit in humans.

Video: Integrated Compensatory Responses in a Human Model of Hemorrhage

Fixed Volume or Fixed Pressure: A Murine Model of Hemorrhagic Shock

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, 2, 3, 4, 5 These signaling events can also lead to sepsis and/or multiple organ dysfunction (MOD). 6, 7, 8, 9 It is critical then to investigate the causes of suppressed immune function and detrimental signaling cascades in order to develop more effective ways to help patients who suffer from traumatic injuries. 10 This fixed pressure Hemorrhagic Shock (HS) procedure, although technically challenging, is an excellent resource for investigation of these pathophysiologic conditions. 11, 12, 13 Advances in the assessment of biological systems, i.e. Systems Biology have enabled the scientific community to further understand complex physiologic networks and cellular communication patterns. 14 Hemorrhagic Shock has proven to be a vital tool for unveiling these cellular communication patterns as they relate to immune function. 15, 16, 17, 18 This procedure can be mastered! This procedure can also be used as either a fixed volume or fixed pressure approach. We adapted this technique in the murine model to enhance research in innate and adaptive immune function. 19, 20, 21 Due to their small size HS in mice presents unique challenges. However due to the many available mouse strains, this species represents an unparalleled resource for the study of the biologic responses. The HS model is an important model for studying cellular communication patterns and the responses of systems such as hormonal and inflammatory mediator systems, and danger signals, i.e. DAMP and PAMP upregulation as it elicits distinct responses that differ from other forms of shock. 22, 23, 24, 25 The development of transgenic murine strains and the induction of biologic agents to inhibit specific signaling have presented valuable opportunities to further elucidate our understanding of the up and down regulation of signal transduction after severe blood loss, i.e. HS and trauma 26, 27, 28, 29, 30. 
There are numerous resuscitation methods (R) in association with HS and trauma. 31, 32, 33, 34 A fixed volume resuscitation method of solely lactated ringer solution (LR), equal to three times the shed blood volume, is used in this model to study endogenous mechanisms such as remote organ injury and systemic inflammation. 35, 36, 38 This method of resuscitation is proven to be effective in evaluating the effects of HS and trauma 38, 39.

The Hemorrhagic Shock model has been a reliable and reproducible resource facilitating the identification and understanding of signaling cascades associated with inflammation and end-organ damage after trauma. This article provides a step-by-step description of surgical and mechanical aspects associated with the Hemorrhagic Shock experimental procedure in mice.

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, ...

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase 

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via a liver laceration that results in consistent blood loss, hemodynamic alterations, and survival.
Mice undergo a standardized resection of the left-middle lobe of the liver. They are allowed to bleed without mechanical intervention. Hemostatic agents can be administered as pre-treatment or rescue therapy depending on the interest of the investigator. During the time of hemorrhage, real-time hemodynamic monitoring via a left femoral arterial line is performed. Mice are then sacrificed, blood loss is quantified, blood is collected for further analysis, and organs are harvested for analysis of injury. Experimental design is described to allow for simultaneous testing of multiple animals.
Liver hemorrhage as a model of uncontrolled hemorrhage exists in the literature, primarily in rat and porcine models. Some of these models utilize hemodynamic monitoring or quantify blood loss but lack consistency. The present model incorporates quantification of blood loss, real-time hemodynamic monitoring in a murine model that offers the advantage of using transgenic lines and a high-throughput mechanism to further investigate the pathophysiologic mechanisms in uncontrolled hemorrhage.

Uncontrolled hemorrhage, an important cause of mortality among trauma patients, can be modeled using a standard liver laceration in a murine model. This model results in consistent blood loss, survival, and allows for testing hemostatic agents. This article provides the step-by-step process to perform this valuable model.

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via ...

Complete and Partial Aortic Occlusion for the Treatment of Hemorrhagic Shock in Swine

Hemorrhage remains the leading cause of preventable deaths in trauma. Endovascular management of non-compressible torso hemorrhage has been at the forefront of trauma care in recent years. Since complete aortic occlusion presents serious concerns, the concept of partial aortic occlusion has gained a growing attention. Here, we present a large animal model of hemorrhagic shock to investigate the effects of a novel partial aortic balloon occlusion catheter and compare it with a catheter that works on the principles of complete aortic occlusion. Swine are anesthetized and instrumented in order to conduct controlled fixed-volume hemorrhage, and hemodynamic and physiological parameters are monitored. Following hemorrhage, aortic balloon occlusion catheters are inserted and inflated in the supraceliac aorta for 60 min, during which the animals receive whole-blood resuscitation as 20% of the total blood volume (TBV). Following balloon deflation, the animals are monitored in a critical care setting for 4 h, during which they receive fluid resuscitation and vasopressors as needed. The partial aortic balloon occlusion demonstrated improved distal mean arterial pressures (MAPs) during the balloon inflation, decreased markers of ischemia, and decreased fluid resuscitation and vasopressor use. As swine physiology and homeostatic responses following hemorrhage have been well-documented and are like those in humans, a swine hemorrhagic shock model can be used to test various treatment strategies. In addition to treating hemorrhage, aortic balloon occlusion catheters have become popular for their role in cardiac arrest, cardiac and vascular surgery, and other high-risk elective surgical procedures.

Here, we present a protocol demonstrating a hemorrhagic shock model in swine that uses aortic occlusion as a bridge to definitive care in trauma. This model has application in testing a wide range of surgical and pharmacological therapeutic strategies.

Hemorrhage remains the leading cause of preventable deaths in trauma. Endovascular management of non-compressible torso hemorrhage has been at the ...

Standardized Hemorrhagic Shock Induction Guided by Cerebral Oximetry and Extended Hemodynamic Monitoring in Pigs

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an insufficient oxygen supply and irreversible organ failure. The brain exerts only limited compensation capacities and is particularly at high risk of severe hypoxic damage.This article demonstrates the reproducible induction of life-threatening hemorrhagic shock in a porcine model by means of calculated blood withdrawal. We titrate shock induction guided by near-infrared spectroscopy&#160;and extended hemodynamic monitoring to display systemic circulatory failure, as well as cerebral microcirculatory oxygen depletion. In comparison to similar models that primarily focus on predefined removal volumes for shock induction, this approach highlights a titration by means of the resulting failure of macro- and microcirculation.

Hemorrhagic shock is a severe complication in seriously injured patients, which leads to life-threatening oxygen undersupply. We present a standardized method to induce hemorrhagic shock via blood withdrawal in pigs that is guided by hemodynamics and microcirculatory cerebral oxygenation.

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an ...

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional deficits. Numerous studies by our group and others have shown that mTBI stimulates cytokine expression and activates microglia, decreases cerebral blood flow and metabolism, and impairs cerebrovascular reactivity. Moreover, several works have reported an association between derangements in these neuroinflammatory and hemodynamic markers and cognitive impairments. Herein we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI in mice. Specifically, we describe how to perform a weight-drop model of mTBI, how to longitudinally measure cerebral blood flow using a non-invasive optical technique called diffuse correlation spectroscopy, and how to perform a Luminex multiplexed immunoassay on brain tissue samples to quantify cytokines and immunomodulatory phospho-proteins (e.g., within the MAPK and NF&#954;B pathways) that respond to and regulate activity of microglia and other neural immune cells. Finally, we detail how to integrate these data using a multivariate systems analysis approach to understand the relationships between all of these variables. Understanding the relationships between these physiologic and molecular variables will ultimately enable us to identify mechanisms responsible for mTBI.

This protocol presents methods to characterize the neuroinflammatory and hemodynamic response to mild traumatic brain injury and to integrate these data as part of a multivariate systems analysis using partial least squares regression.

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional ...

Neuroscience

A Preclinical Controlled Cortical Impact Model for Traumatic Hemorrhage Contusion and Neuroinflammation

Cerebral contusion is a severe medical problem affecting millions of people worldwide each year. There is an urgent need to understand the pathophysiological mechanism and to develop effective therapeutic strategy for this devastating neurological disorder. Intraparenchymal hemorrhage and post-traumatic inflammatory response induced by initial physical impact can aggravate microglia/macrophage activation and neuroinflammation which subsequently worsen brain pathology. We provide here a controlled cortical impact (CCI) protocol that can reproduce experimental cortical contusion in mice by using a pneumatic impactor system to deliver mechanical force with controllable magnitude and velocity onto the dural surface. This preclinical model allows researchers to induce moderately severe focal cerebral contusion in mice and to investigate a wide range of post-traumatic pathological progressions including hemorrhage contusion, microglia/macrophage activation, iron toxicity, axonal injury, as well as short-term and long-term neurobehavioral deficits. The present protocol can be useful for exploring the long-term effects of and potential interventions for cerebral contusion.

Intraparenchymal hemorrhage and neuroinflammation accompanied by cerebral contusion can trigger severe secondary brain injury. This protocol details a mouse controlled cortical impact (CCI) model allowing researchers to study hemorrhage contusion and post-traumatic immune responses and explore potential therapeutics.

Cerebral contusion is a severe medical problem affecting millions of people worldwide each year. There is an urgent need to understand the ...

Description of a Swine Infant Model of Volume-Controlled Hemorrhagic Shock

Hemorrhagic shock is a leading cause of morbidity and mortality in pediatric patients. Interpretation of the clinical indicators validated in adults to guide resuscitation and comparison between different therapies is difficult in children due to the inherent heterogeneity of this population. As a result, compared to adults, appropriate management of pediatric hemorrhagic shock is still not well established. In addition, the scarcity of pediatric patients with hemorrhagic shock precludes the development of clinically relevant studies. For this reason, an experimental pediatric animal model is necessary to study the effects of hemorrhage in children as well as their response to different therapies. We present an infant animal model of volume-controlled hemorrhagic shock in anesthetized young pigs. Hemorrhage is induced by withdrawing a previously calculated blood volume, and the pig is subsequently monitored and resuscitated with different therapies. Here, we describe a precise and highly reproducible model of hemorrhagic shock in immature swine. The model yields hemodynamic data that characterizes compensatory mechanisms that are activated in response to severe hemorrhage.

This article aims to provide researchers with a detailed and accessible guide to set up an infant porcine model of hemorrhagic shock.

Hemorrhagic shock is a leading cause of morbidity and mortality in pediatric patients. Interpretation of the clinical indicators validated in adults to ...

Vascular Cannulation with Hemorrhagic Shock Induction in Rats

Developing a Clinically Relevant Hemorrhagic Shock Model in Rats

Over the recent decades, the development of animal models allowed us to better understand various pathologies and identify new treatments. Hemorrhagic shock, i.e., organ failure due to rapid loss of a large volume of blood, is associated with a highly complex pathophysiology involving several pathways. Numerous existing animal models of hemorrhagic shock strive to replicate what happens in humans, but these models have limits in terms of clinical relevance, reproducibility, or standardization. The aim of this study was to refine these models to develop a new model of hemorrhagic shock. Briefly, hemorrhagic shock was induced in male Wistar Han rats (11-13 weeks old) by a controlled exsanguination responsible for a drop in the mean arterial pressure. The next phase of 75 min was to maintain a low mean arterial blood pressure, between 32 mmHg and 38 mmHg, to trigger the pathophysiological pathways of hemorrhagic shock. The final phase of the protocol mimicked patient care with an administration of intravenous fluids, Ringer Lactate solution, to elevate the blood pressure. Lactate and behavioral scores were assessed 16 h after the protocol started, while hemodynamics parameters and plasmatic markers were evaluated 24 h after injury. Twenty-four hours post-hemorrhagic shock induction, the mean arterial and diastolic blood pressure were decreased in the hemorrhagic shock group (p&#160;&#60; 0.05). Heart rate and systolic blood pressure remained unchanged. All organ damage markers were increased with the hemorrhagic shock (p &#60; 0.05). The lactatemia and behavioral scores were increased compared to the sham group (p &#60; 0.05). In conclusion, we demonstrated that the protocol described here is a relevant model of hemorrhagic shock that can be used in subsequent studies, particularly to evaluate the therapeutic potential of new molecules.

Author Spotlight: Developing Innovative Therapeutic Strategies for Hemorrhagic Shock Research

Hemorrhagic shock kills 1.9 million people worldwide every year. Small animals are frequently used as hemorrhagic shock models but are associated with issues of standardization, reproducibility, and clinical significance, thus limiting their relevance. This article describes developing a new clinically relevant hemorrhagic shock model in rats.

Over the recent decades, the development of animal models allowed us to better understand various pathologies and identify new treatments. Hemorrhagic ...

Swine Axillary Artery Injury Model for Testing Pre-Hospital Hemostatic Products

A New Hybrid Quantitative Evaluation Model for Axillary Junctional Hemorrhage in Swine

In this study, we developed and validated a hybrid quantitative model for simulating upper extremity junctional hemorrhage in swine, aiming to advance the development of pre-hospital hemostatic products. Utilizing 12 healthy 8-month-old male Yorkshire swine, we demonstrated the feasibility of a swine axillary artery injury model for evaluating hemostatic efficacy. Animals were divided into three groups to undergo volume-controlled hemorrhage (VCH), mimicking Class I-III hemorrhagic shock by withdrawing blood at different rates. Subsequent external compression was applied using a novel device consisting of a mechanical arm and an inflatable hemostatic balloon, achieving controlled pressure to enhance clot formation. Hemodynamic parameters, including heart rate and blood pressure, were continuously monitored, highlighting the impact of controlled hemorrhage and external compression on physiological responses. The findings suggest that the combination of VCH with targeted external compression effectively simulates clinical scenarios of axillary artery injury, providing a valuable model for testing hemostatic interventions in a controlled, standardized manner. This study underscores the potential of the model in facilitating the development and evaluation of new hemostatic agents and devices for managing junctional hemorrhages.

This study presents a hybrid quantitative model for axillary junctional hemorrhage in swine, enhancing pre-hospital hemostatic intervention evaluation.

In this study, we developed and validated a hybrid quantitative model for simulating upper extremity junctional hemorrhage in swine, aiming to advance the ...

Integrated Compensatory Responses in a Human Model of Hemorrhage

Summary

Explore More Videos

Chapters in this video

Fixed Volume or Fixed Pressure: A Murine Model of Hemorrhagic Shock

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

Complete and Partial Aortic Occlusion for the Treatment of Hemorrhagic Shock in Swine

Standardized Hemorrhagic Shock Induction Guided by Cerebral Oximetry and Extended Hemodynamic Monitoring in Pigs

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

A Preclinical Controlled Cortical Impact Model for Traumatic Hemorrhage Contusion and Neuroinflammation

Description of a Swine Infant Model of Volume-Controlled Hemorrhagic Shock

Developing a Clinically Relevant Hemorrhagic Shock Model in Rats

A New Hybrid Quantitative Evaluation Model for Axillary Junctional Hemorrhage in Swine