We would like to acknowledge Professor Roger Hainsworth, who developed this technique. We are grateful to Mr. King Hang Chao and Mr. Wang-Joe Woo for their assistance with photography.
This work is supported by Simon Fraser University and the Heart and Stroke Foundation of Canada.

Throughout testing, continuous beat-to-beat blood pressure and electrocardiogram (ECG) monitoring is paramount. This ensures subject safety, and prompt termination of the test with the onset of presyncope. Beat-to-beat blood pressure recordings can be obtained through arterial catheterization, or finger plethysmography31-33. The latter is used in this protocol because it is non-invasive and can assess the onset of presyncope with the same accuracy as catherization31,34, without the detrimental impact of invasive monitoring on OT1. Using the Modelflow technique changes in stroke volume, cardiac output, and total peripheral resistance can be derived from the finger arterial pressure waveform35,36. Additional noninvasive measures that may aid the haemodynamic evaluation can also be conducted, and will be described here. Continuous end tidal oxygen (PETO2) and carbon dioxide (PETCO2) monitoring using a nasal cannula allows the evaluation of any contribution of hyperventilation to the subject's symptoms. Finally, monitoring both brachial and cerebral blood flow velocities using Doppler ultrasound can be undertaken to allow the determination of peripheral and cerebral vascular responses to orthostasis. In addition, measurements of venous pooling and capillary filtration could also be obtained using impedance plethysmography20. Ultimately, this protocol allows assessment of postural cardiovascular reflex control in a controlled and reproducible setting.
1. Equipment
<ol>
 <li>The manually adjustable tilt table is capable of moving from -15 to 60 &deg; in 10 &deg; increments (Figure 1). It includes an adjustable right-arm rest with an adjustable holder for a brachial ultrasound probe (Figure 1D), an adjustable footplate for the subject to stand on, and a seat belt to secure the subject's legs (Figure 1E).</li>
 <li>An LBNP chamber with an attached pressure gauge fits in a wooden groove filled with neoprene onto the bottom half of the tilt table and is secured in place by four adjustable straps (Figure 1). A clear Plexiglas material can be employed for the chamber if desired, to enable visualization of the subjects' legs, and thus visual monitoring of any skeletal muscle pumping activity.</li>
 <li>A wooden waist board is attached to the top of the chamber with a neoprene surround at the level of the subject's iliac crest to provide an air-tight seal with the chamber (Figure 1B). The level of the iliac crest is chosen because it avoids the application of LBNP to the abdomen, which can be uncomfortable, but ensures a standardized stimulus for individuals of different statures.</li>
 <li>Perform continuous beat-to-beat data acquisition with a sampling frequency of 1 KHz using an analog-to-digital converter. Visualize all data simultaneously in real time throughout testing using LabChart (Powerlab 16/30, AD Instruments, Colorado Springs, CO).</li>
 <li>Conduct testing in a temperature-controlled room (20-22 &deg;C) to avoid the known effect of heat stress upon OT37. Tests are ideally conducted in the mornings because of the effect of diurnal rhythms on baroreflex control38. In some cases, a familiarization session is advised to minimize the influence of a "stress response" on the procedure.</li>
 <li>Instruct subjects to have only a light breakfast, to minimize possible confounders due to post-prandial hypotension, and to avoid caffeine and strenuous exercise on the morning of testing. Subjects should fast for 2 hr prior to testing. They should also abstain from drinking alcohol for 24 hr prior to testing, to eliminate the diuretic effect of alcohol and consequent reductions in plasma volume, which are known to reduce OT39.</li>
 <li>Figure 2 outlines the protocol. Stand the subject on the table foot plate and move them into a supine position (Figure 1). Once supine, align the iliac crest with the center of the table. This allows for ease of tilting, and ensures standardized positioning of the LBNP chamber. Adjust the footplate accordingly. A foot plate support is preferred because tables with saddle supports are associated with increased false positive responses, probably due to excess compression of leg and pelvic veins6.</li>
 <li>Loosley position a strap just above the knee to promote passive standing and provide postural support. Instruct subjects not to move their legs during testing. Position the subject's right arm on the arm rest, adjusted so that it is comfortably supported at heart level. Attach monitoring equipment.</li>
 <li>Conduct beat-to-beat blood pressure monitoring using the Finometer, according to the manufacturer's instructions40. Chose the finger cuff that fits appropriately onto the middle phalanx of the subject's right middle finger. Use a brachial cuff to internally calibrate the Finometer prior to data collection. Enter the subject's sex, age, height and weight into the Finometer to enable appropriate assumptions for the Modelflow algorithms35,36.</li>
 <li>Continuous ECG monitoring is important for the accurate determination of heart rate responses, and prompt identification of any cardiac arrhythmia, should they occur. Apply ECG electrodes in a modified lead II configuration, ensuring that the electrode sites do not interfere with positioning of the neoprene waist board.</li>
 <li>Determine peripheral vascular responses using Doppler ultrasound of the brachial artery of the right arm supported at heart level (to avoid the influence of hydrostatic pressure changes on blood pressure and velocity in the arm). Palpate the artery until the pulse is located. Apply ultrasound gel to this area and position an 8 MHz ultrasound probe so that a brachial artery velocity waveform is obtained (Figures 3A,B).</li>
 <li>Once the signal is identified, optimize the depth and gain, and tighten the adjustable holder to keep the angle of insonation constant for the duration of the test (Figure 3B). This is important because, according to the Doppler shift equation, changes in velocity will be proportional to changes in flow if the angle of insonation and arterial diameter are constant19,21.</li>
 <li>Determine cerebral vascular responses similarly (Figures 4A,B). Secure the cerebral ultrasound probe (2 MHz) in place using a plastic headset or fabric headbands (Figure 4B) to ensure faithful signal detection and a constant angle of insonation throughout testing.</li>
 <li>Determine blood velocity continuously from the middle cerebral artery. This vessel is chosen because of its convenience of identification, large contribution to global cerebral perfusion, and constant diameter during orthostatic stress41, ensuring changes in velocity are proportional to changes in flow. Apply ultrasound gel to the subject's temple and locate the vessel (Figure 4A). Once identified, optimize the depth and gain settings.</li>
 <li>Attach the nasal cannula. Nasal sampling is preferred because it permits the subject to talk freely during testing (unlike with use of a mouthpiece). This is important to enable self-report and recognition of symptoms, while avoiding invasive blood sampling that may adversely impact OT. However, during speech, or when the subject is breathing through their mouth, accuracy of these readings may be affected. Encourage subjects to breathe through their nose.</li>
 <li>Place the LBNP chamber on the table and secure with the straps. Select a waist board that fits snugly so that an airtight seal with the neoprene can be achieved (Figure 1B). The wooden component of the waist board should not be touching the subject. Secure the waist board to the chamber. Connect the chamber to a negative pressure source, via a variable resistor (Figure 1C).</li>
</ol>
2. Data Collection
<ol>
 <li>Record data for 20 min in the supine position. Shorter rest periods are associated with greater falls in blood pressure when upright, presumably because the reabsorption of any fluid that has collected in the dependent limbs prior to lying down is not yet complete20.</li>
 <li>At the end of the supine phase maneuver the table to a tilt angle of 60 &deg;. This angle is preferred because it affords nearly 90% of the maximal vertical displacement, while allowing the subject to remain relaxed and supported against the tilt table (minimizing the confounding effect of skeletal muscle pumping activity with higher tilt angles). Less steep angles may increase the false negative rate; although the physiological effects of tilting are similar for angles &ge;60 &deg;, higher tilt angles are associated with a reduction in specificity6.</li>
 <li>Complete the transition to upright within thirty seconds. The transition time is not known to affect OT and it is more pleasant for the subject if tilting is not excessively rapid. However, very slow tilt maneuvers may be associated with lower activation of muscle sympathetic nerve activity and should be avoided42.</li>
 <li>Continuously monitor the subject's cardiovascular parameters as well as their subjective experience. The test should be stopped immediately, and the subject returned to a supine position, if any of the following end-point criteria are met: blood pressure is 80 mmHg or below; heart rate is lower than 50 beats per minute (bpm) or higher than 170 bpm; and the subject experiences symptoms such as dizziness, warmth, and requests to stop; or the protocol is completed.</li>
 <li>Unless there is a specific desire to initiate syncope, terminate the test rapidly at the onset of presyncopal signs and symptoms, avoiding frank syncope.</li>
 <li>After 20 min of tilting apply LBNP, while still tilted, at -20 mmHg for a further 10 min. It is important to inform the subject of the impending onset of LBNP to prevent a startle response to the sensation and sound of the LBNP.</li>
 <li>After 10 min, increase the LBNP to -40 mmHg for another 10 min.</li>
 <li>After 10 min, increase the LBNP to -60 mmHg and continue for another 10 min. At the end of this phase, turn off the LBNP and return the subject to the supine position.</li>
 <li>It is theoretically possible to increase the vacuum source to achieve -80 mmHg LBNP if a presyncopal end-point is not reached at the end of this phase. However, in practice this high level of LBNP is uncomfortable for the subject, and presyncope usually occurs at much lower levels of orthostatic stress, even in healthy controls. The largest OT we have recorded was 50 min (the end of -60 mmHg) and this was in a Peruvian high altitude resident with Chronic Mountain Sickness and an extremely high blood volume43.</li>
 <li>OT is defined as the time to presyncope in minutes from the onset of the head-up tilting phase.</li>
 <li>To ensure the rapid termination of symptoms and signs of presyncope, and so to minimize the likelihood of syncope or asystole, a rapid return to supine is desired (ideally ~1 sec). For this reason, a manual tilt table may be preferred to an autonomic one, which may not have the capability for such rapid transitions. Returning the table to a slightly head-down position (-15 &deg;) may promote faster resolution of the presyncopal event. Once all variables have returned to the supine levels and any symptoms are resolved, remove the monitoring equipment.</li>
 <li>After removing the LBNP chamber, remove the strap over the subject's legs and lower the footplate to its original position. Ensure the subject is positioned with their feet on the plate.</li>
 <li>Before returning the table to an upright position, instruct the subject to tense their leg muscles throughout the transition to avoid any symptoms from reoccurring as the table is tilted to facilitate them getting off44,45. Ask the subject to sit after stepping off the table to ensure they are symptom-free before leaving the laboratory.</li>
</ol>

No conflicts of interest declared.

This technique is highly reproducible, has the ability to discriminate normal and abnormal responses with high sensitivity and specificity, and can provoke presyncope in all subjects, allowing for symptom recognition in patients with recurrent syncope. In a clinical setting, different types of syncope can be distinguished, allowing tailored treatment and management approaches. The impact of interventions can readily be assessed. With additional cardiovascular monitoring, reflex responses can also be evaluated.
In order to differentiate certain sub-types of syncope in patient populations, the analysis of plasma catecholamine responses during orthostatic stress may be useful. However, the invasive nature of this procedure may interfere with the accurate determination of OT1.
This protocol can easily be modified to account for specific clinical or research questions, or the needs of specific populations. The angle of the table can be altered in situations when 60 &deg; may be difficult to sustain, for example when evaluating orthostatic intolerance in spinal cord injured populations. In such cases, tilting to 30-35 &deg; may be more appropriate and tolerable54-56. If desired, the protocol may be shortened in specific populations by stopping after completion of LBNP at -20 mmHg - individuals who reach the end of this phase essentially have a normal OT (Table 1). This may be applicable if the goal is to determine whether the response is abnormal or not, but not necessarily to define the precise OT. This may be particularly pertinent in paediatric populations.
Often with an automatic table, the transition back to supine is slow and this can increase the likelihood of profound bradycardia or asystole, and brief loss of consciousness, occurring coincident with a vasovagal response57. As such, the benefit of the manual table is that the pathophysiological and symptomatic responses associated with presyncope are quickly recovered.
An experienced physiologist, technician or nurse must be present during testing. There is a lack of agreement as to whether a medical doctor should also be present or available (on call) in case of asystole or other complications3,6,58. The laboratory should have access to atropine should asystole occur, as well as a defibrillator. Serious adverse events are rare and no deaths have been reported58. Brief periods of profound bradycardia or asystole may accompany vasovagal responses4,24,46, but they typically recover spontaneously with the return to a supine posture. Seizure-like activity may accompany syncope59, but can be avoided with termination of the test at presyncope. There is one report of cardiopulmonary resuscitation being required in the case of tilt-induced coronary vasospasm in a patient with known coronary vasospastic angina60.

<table border="1">
 <tbody>
 <tr>
 <td>Equipment</td>
 <td>Manufacturer</td>
 <td>Location</td>
 </tr>
 <tr>
 <td>Tilt Table</td>
 <td>Custom-build</td>
 <td>Leeds, United Kingdom</td>
 </tr>
 <tr>
 <td>Finometer</td>
 <td>Finapres Medical Systems</td>
 <td>Amsterdam, The Netherlands</td>
 </tr>
 <tr>
 <td>Doppler Box</td>
 <td>Compumedics</td>
 <td>Singen, Germany</td>
 </tr>
 <tr>
 <td>Doppler software</td>
 <td>The DWL Doppler Company</td>
 <td>Singen, Germany</td>
 </tr>
 <tr>
 <td>Aquasonic Ultrasound gel</td>
 <td>Parker Laboratories, Inc.</td>
 <td>Fairfield, USA</td>
 </tr>
 <tr>
 <td>Headbands</td>
 <td>Lululemon</td>
 <td>Burnaby, Canada</td>
 </tr>
 <tr>
 <td>Headset</td>
 <td>Canadian Tire</td>
 <td>Burnaby, Canada</td>
 </tr>
 <tr>
 <td>ECG</td>
 <td>Finapres ECG Module, Finapres Medical Systems</td>
 <td>Amsterdam, The Netherlands</td>
 </tr>
 <tr>
 <td>Electrodes</td>
 <td>Red Dot</td>
 <td>Ontario, Canada</td>
 </tr>
 <tr>
 <td>Antiseptic Isopropyl Alcohol Pads</td>
 <td>Lernapharm</td>
 <td>Quebec, Canada</td>
 </tr>
 <tr>
 <td>O2Cap-Oxygen Analyser</td>
 <td>Oxigraph Inc.</td>
 <td>California, USA</td>
 </tr>
 <tr>
 <td>Airlife Nasal Oxygen Cannula</td>
 <td>Cardinal Health</td>
 <td>Mountainview, USA</td>
 </tr>
 <tr>
 <td>Powerlab 16/30</td>
 <td>AD Instruments</td>
 <td>Colorado Springs, USA</td>
 </tr>
 </tbody>
</table>

tilt testing with combined lower body negative pressure: a "gold standard" for measuring orthostatic tolerance

Orthostatic tolerance (OT) refers to the ability to maintain cardiovascular stability when upright, against the hydrostatic effects of gravity, and hence to maintain cerebral perfusion and prevent syncope (fainting). Various techniques are available to assess OT and the effects of gravitational stress upon the circulation, typically by reproducing a presyncopal event (near-fainting episode) in a controlled laboratory environment. The time and/or degree of stress required to provoke this response provides the measure of OT. Any technique used to determine OT should: enable distinction between patients with orthostatic intolerance (of various causes) and asymptomatic control subjects; be highly reproducible, enabling evaluation of therapeutic interventions; avoid invasive procedures, which are known to impair OT1.
In the late 1980s head-upright tilt testing was first utilized for diagnosing syncope2. Since then it has been used to assess OT in patients with syncope of unknown cause, as well as in healthy subjects to study postural cardiovascular reflexes2-6. Tilting protocols comprise three categories: passive tilt; passive tilt accompanied by pharmacological provocation; and passive tilt with combined lower body negative pressure (LBNP). However, the effects of tilt testing (and other orthostatic stress testing modalities) are often poorly reproducible, with low sensitivity and specificity to diagnose orthostatic intolerance7.
Typically, a passive tilt includes 20-60 min of orthostatic stress continued until the onset of presyncope in patients2-6. However, the main drawback of this procedure is its inability to invoke presyncope in all individuals undergoing the test, and corresponding low sensitivity8,9. Thus, different methods were explored to increase the orthostatic stress and improve sensitivity.
Pharmacological provocation has been used to increase the orthostatic challenge, for example using isoprenaline4,7,10,11 or sublingual nitrate12,13. However, the main drawback of these approaches are increases in sensitivity at the cost of unacceptable decreases in specificity10,14, with a high positive response rate immediately after administration15. Furthermore, invasive procedures associated with some pharmacological provocations greatly increase the false positive rate1.
Another approach is to combine passive tilt testing with LBNP, providing a stronger orthostatic stress without invasive procedures or drug side-effects, using the technique pioneered by Professor Roger Hainsworth in the 1990s16-18. This approach provokes presyncope in almost all subjects (allowing for symptom recognition in patients with syncope), while discriminating between patients with syncope and healthy controls, with a specificity of 92%, sensitivity of 85%, and repeatability of 1.1&plusmn;0.6 min16,17. This allows not only diagnosis and pathophysiological assessment19-22, but also the evaluation of treatments for orthostatic intolerance due to its high repeatability23-30. For these reasons, we argue this should be the "gold standard" for orthostatic stress testing, and accordingly this will be the method described in this paper.

Using this protocol, all subjects experience presyncope, and the definition of normal or abnormal responses is made largely based upon the time it takes to induce this reaction. OT is defined as the time to presyncope in minutes from the onset of upright tilting. Typical values for OT in healthy volunteers according to age and gender can be seen in Table 1. Patients with orthostatic intolerance exhibit presyncope earlier in the test, with 85% ending the test within the -20 mmHg phase compared to 23% of controls17. The threshold of normal is taken as the 20% incidence of syncope17.
At termination of the test the haemodynamic responses fall into essentially three categories: vasovagal syncope; postural tachycardia syndrome (POTS); or autonomic failure46. The vasovagal response is characterized as sudden onset hypotension and bradycardia, although the relative contributions of each component can vary greatly, and have been characterized according to the VASIS classification46,47. A representative response in an adult control is depicted in Figure 5. At tilt, the baroreflex compensates for blood pooling in the lower extremities through vasoconstriction, reflected in the reduced brachial blood flow and increased diastolic pressure, and tachycardia. This response is maintained until presyncope, at which point there is a sudden switch to vasodilatation and bradycardia. In this case the subject experienced a vasovagal response at presyncope, associated with symptoms of dizziness and warmth. This is the most common response to tilting in healthy controls.
In patients with autonomic failure there is an absence of adaptation of blood pressure to the upright position, with a slow and progressive decline in blood pressure, and small or absent heart rate response to orthostatic stress. Patients will develop symptoms once a critical level of blood pressure is reached (typically at or below a systolic pressure of 80 mmHg46, although many patients with autonomic failure tolerate much lower blood pressures than this) representing the lower limit for cerebral autoregulation. This response generally occurs only in older patients with syncope, who often present with other associated disorders46.
POTS is characterized by a heart rate rise of 30 bpm within 10 min of head-upright tilting, or with an upright heart in excess of 120 bpm48,49, associated with symptoms of presyncope. Blood pressure is usually well maintained. This disorder generally affects young females (&lt;40 years old), and typically presents with presyncope, and only occasionally with syncope48. Heart rate responses to orthostatic stress are brisk in children and adolescents, so alternative diagnostic criteria for POTS are recommended in this population50.
Regardless of the haemodynamic response, there is a drop in cerebral blood flow velocity at presyncope51. There may be impairments in cerebral autoregulation in patients with vasovagal syncope22 and POTS52.
In those with intact baroreflex control of vascular resistance there will be a progressive reduction in brachial blood flow velocity during the orthostatic stress, associated with increased forearm vascular resistance. In healthy controls a maximum vascular resistance response of +100&plusmn;12% is considered normal21. Smaller responses are indicative of impaired vascular resistance responses19,21, with 60% of patients with presyncope and poor OT having maximal vascular resistance responses below +80%, and 83% of controls having responses that exceed this value19. Patients with POTS often have particularly small peripheral vascular resistance responses21. There is a significant positive correlation between the maximum vascular resistance response and OT19.Typical heart rate responses in healthy controls at the end of the head up tilt phase and peak heart rates during the orthostatic stress are 71-82 bpm and 98-133 bpm respectively16,21.
Some degree of hyperventilation is common during orthostatic stress, with associated reductions in PETCO253. This tends to produce a hypocapnic cerebral vasoconstriction and peripheral vasodilation, and so contributes to the decreases in blood pressure and cerebral blood flow at presyncope. Some individuals are excessively sensitive to alterations in PETCO2 and this may be a factor in predisposing them to syncopal events53.
<img alt="Figure 1" src="/files/ftp_upload/4315/4315fig1.jpg" fo:content-width="4in" fo:src="/files/ftp_upload/4315/4315fig1highres.jpg"/> 
 Figure 1. The manual, adjustable head-up tilt table and LBNP chamber. The manual design allows for rapid transitions between supine and tilt, ensuring the prompt recovery of presyncopal symptoms at test termination. A. The handle used to move the table into different positions. B. A pressure gauge monitors the level of LBNP during the protocol. C. A wooden waist board with neoprene is attached to the top of the chamber to ensure the chamber is air-tight. D. The adjustable arm rest with attached clamp for the brachial ultrasound probe. E. A seatbelt over the subject's legs minimizes skeletal muscle pumping activity during orthostatic stress. F. The lever used to adjust the table from -15 to 60 &deg;. G. The footplate can be adjusted for the subject's height once their iliac crest is positioned in the center of the table.
<img alt="Figure 2" src="/files/ftp_upload/4315/4315fig2.jpg" fo:content-width="4in" fo:src="/files/ftp_upload/4315/4315fig2highres.jpg"/> 
 Figure 2. Head-up tilt protocol. There are three different phases in the protocol: supine for 20 min; tilt for 20 min; and LBNP for 10 min each at -20 mmHg, -40 mmHg, and -60 mmHg.
<img alt="Figure 3" src="/files/ftp_upload/4315/4315fig3.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/4315/4315fig3highres.jpg"/> 
 Figure 3. Brachial blood flow velocity measurement. A. The brachial ultrasound probe is positioned overlying the brachial artery to enable measurements of forearm blood flow velocity, and the calculation of vascular resistance responses. B. Once in place, the probe is secured using an adjustable clamp to ensure the angle of insonation does not change throughout the test.
<img alt="Figure 4" src="/files/ftp_upload/4315/4315fig4.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/4315/4315fig4highres.jpg"/> 
 Figure 4. Middle cerebral artery blood flow velocity measurement. A. A 2MHz ultrasound probe is positioned on the transtemporal window overlying the middle cerebral artery. An example showing middle cerebral artery blood flow velocity is shown in the top panel. The birfurcation point, with the anterior cerebral artery waveform just visible as a negative deflection, enables confident identification of the middle cerebral artery. The bottom panel shows an example from the posterior cerebral artery. Although the flow profile is similar to the middle cerebral artery, the mean velocity is lower and insonation depth greater, enabling ease of discrimination between the two vessels. B. Once in place the probe is secured in position using either a plastic headset (upper) or fabric headbands (lower).
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/4315/4315fig5highres.jpg" src="/files/ftp_upload/4315/4315fig5.jpg" /> 
 Figure 5. Example tracing from an adult control volunteer. Beat-to-beat blood pressure (BP), electrocardiogram (ECG), brachial (BBFV) and cerebral (CBFV) blood flow velocity are shown. During the orthostatic stress a progressive baroreflex mediated tachycardia and vasoconstriction can be seen. At presyncope, the baroreflex begins to fail and there is a drop in blood pressure and cerebral blood flow velocity.
<table border="1">
 <tbody>
 <tr>
 <td>&nbsp;</td>
 <td colspan="6">Orthostatic Tolerance (min)</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td colspan="3">Males</td>
 <td colspan="3">Females</td>
 </tr>
 <tr>
 <td>Age (years)</td>
 <td>20-35</td>
 <td>36-50</td>
 <td>&gt;50</td>
 <td>20-35</td>
 <td>36-50</td>
 <td>&gt;50</td>
 </tr>
 <tr>
 <td>Mean</td>
 <td>35&plusmn;1.4*</td>
 <td>35&plusmn;1.7</td>
 <td>35&plusmn;1.7</td>
 <td>29&plusmn;1.5</td>
 <td>31&plusmn;1.8</td>
 <td>37&plusmn;2.3</td>
 </tr>
 <tr>
 <td>20% incidence</td>
 <td>30</td>
 <td>34</td>
 <td>32</td>
 <td>24</td>
 <td>28</td>
 <td>30</td>
 </tr>
 <tr>
 <td>50% incidence</td>
 <td>34</td>
 <td>37</td>
 <td>36</td>
 <td>29</td>
 <td>32</td>
 <td>38</td>
 </tr>
 </tbody>
</table>
Table 1. Predicted times to presyncope in healthy volunteers according to age and gender. Values are given for the mean orthostatic tolerance &plusmn; standard error, as well as the times at which 20% and 50% of individuals experienced presyncope. * denotes significant difference (p&lt;0.05) between males and females within the age group. There was a significant correlation between age and onset of presyncope in females (r=0.63; p=0.004), but not males. Combined data (n=63) from El-Bedawi and Hainsworth (1994), and Claydon, unpublished observations.

Watch this Scientific Journal Video about Tilt Testing with Combined Lower Body Negative Pressure: a "Gold Standard" for Measuring Orthostatic Tolerance at JoVE.com

Tilt Testing with Combined Lower Body Negative Pressure: a "Gold Standard" for Measuring Orthostatic Tolerance

We describe a "gold standard" for evaluating orthostatic tolerance (OT) using tilt testing with combined lower body negative pressure (LBNP). This can be combined with non-invasive evaluations of cardiovascular reflex control. Normal and abnormal responses are defined.

Orthostatic tolerance (OT)  refers to the ability to maintain cardiovascular stability when upright,  against the hydrostatic effects of gravity, and ...

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20.6K Views.  Simon Fraser University. We describe a "gold standard" for evaluating orthostatic tolerance (OT) using tilt testing with combined lower body negative pressure (LBNP). This can be combined with non-invasive evaluations of cardiovascular reflex control. Normal and abnormal responses are defined.

Video: Tilt Testing with Combined Lower Body Negative Pressure: a "Gold Standard" for Measuring Orthostatic Tolerance

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Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable ...

Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical profiles, inclusive of blood glucose levels in both the fed and fasted state, HbA1c, insulin levels, clinical history of diet, body composition, and body weight status, an assessment of normal and abnormal glycemic control can be made. Interpretation of an IVGTT is done through measurement of changes in glucose and insulin levels over time in relation to the dextrose challenge. Critical components to be considered are: peak glucose and insulin levels reached in relation to T0 (end of glucose infusion), the glucose clearance rate K derived from the slope of rapid glucose clearance in the first 20 min (T1 to T20), the time to return to glucose baseline, and the area under the curve (AUC). These IVGTT measures will show characteristic changes as glucose homeostasis moves from a healthy to a diseased metabolic state5. Herein we will describe the characterization of nonhuman primates (Rhesus and Cynomolgus macaques), which are the most relevant animal model of Type II diabetes (T2D) in humans and the IVGTT and clinical profiles of these animals from a lean healthy, to obese dysmetabolic, and T2D state 8, 10, 11.

The goal of this protocol is to present a standard method to perform intravenous glucose tolerance tests (IVGTTs) to assess glycemic control in nonhuman primates and assess their metabolic status from healthy to dysmetabolic.

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical ...

The Lower Body Positive Pressure Treadmill for Knee Osteoarthritis Rehabilitation

Here, based on a clinician&rsquo;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

Here, based on a clinician&#8217;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

Here, based on a clinician&rsquo;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Implantation of Combined Telemetric ECG and Blood Pressure Transmitters to Determine Spontaneous Baroreflex Sensitivity in Conscious Mice

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. The baroreflex is a key homeostatic mechanism to counteract acute, short-term changes in arterial BP and to maintain BP in a relatively narrow physiological range. BP is sensed by baroreceptors located in the aortic arch and carotid sinus. When BP changes, signals are transmitted to the central nervous system and are then communicated to the parasympathetic and sympathetic branches of the autonomic nervous system to adjust HR. A rise in BP causes a reflex decrease in HR, a drop in BP causes a reflex increase in HR.
Baroreflex sensitivity (BRS) is the quantitative relationship between changes in arterial BP and corresponding changes in HR. Cardiovascular diseases are often associated with impaired baroreflex function. In various studies reduced BRS has been reported in e.g., heart failure, myocardial infarction, or coronary artery disease. 
Determination of BRS requires information from both BP and HR, which can be recorded simultaneously using telemetric devices. The surgical procedure is described beginning with the insertion of the pressure sensor into the left carotid artery and positioning of its tip in the aortic arch to monitor arterial pressure followed by the subcutaneous placement of the transmitter and ECG electrodes. We also describe postoperative intensive care and analgesic management. After a two-week period of post-surgery recovery long-term ECG and BP recordings are performed in conscious and unrestrained mice. Finally, we include examples of high-quality recordings and the analysis of spontaneous baroreceptor sensitivity using the sequence method.

The baroreflex is a heart-rate regulation mechanism by the autonomic nervous system in response to blood-pressure changes. We describe a surgical technique to implant telemetry transmitters for continuous and simultaneous measurement of electrocardiogram and blood pressure in mice. This can determine spontaneous baroreflex sensitivity, an important prognostic marker for cardiovascular disease.

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. ...

Surfactant Depletion Combined with Injurious Ventilation Results in a Reproducible Model of the Acute Respiratory Distress Syndrome (ARDS)

Various animal models exist to study the complex pathomechanisms of the acute respiratory distress syndrome (ARDS). These models include pulmo-arterial infusion of oleic acid, infusion of endotoxins or bacteria, cecal ligation and puncture, various pneumonia models, lung ischemia/reperfusion models and, of course, surfactant depletion models, among others. Surfactant depletion produces a rapid, reproducible deterioration of pulmonary gas exchange and hemodynamics and can be induced in anesthetized pigs using repeated lung lavages with 0.9% saline (35 mL/kg body weight, 37 &#176;C). The surfactant depletion model supports investigations with standard respiratory and hemodynamic monitoring with clinically applied devices. But the model suffers from a relatively high recruitability and ventilation with high airway pressures can immediately reduce the severity of the injury by reopening atelectatic lung areas. Thus, this model is not suitable for investigations of ventilator regimes that use high airway pressures. A combination of surfactant depletion and injurious ventilation with high tidal volume/low positive end-expiratory pressure (high Tv/low PEEP) to cause ventilator induced lung injury (VILI) will reduce the recruitability of the resulting lung injury. The advantages of a timely induction and the possibility to perform experimental research in a setting comparable to an intensive care unit are preserved.

Surfactant Depletion Combined with Injurious Ventilation Results in a Reproducible Model of the Acute Respiratory Distress Syndrome ARDS

A combination of surfactant washout using 0.9% saline (35 mL/kg body weight, 37 &#176;C) and high tidal volume ventilation with low PEEP to cause moderate ventilator induced lung injury (VILI) results in experimental acute respiratory distress syndrome (ARDS). This method provides a model of lung injury with low/limited recruitability to study the effect of various ventilation strategies for extended periods.

Various animal models exist to study the complex pathomechanisms of the acute respiratory distress syndrome (ARDS). These models include pulmo-arterial ...

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...

Evaluation of a Point-of-Care Testing Analyzer for Measuring Peripheral Blood Leukocytes

White blood cell (WBC) is an important indicator of inflammation in the body, and it can help distinguish between bacterial and viral infections. At present, most primary medical institutions in China have a poor percentage of adoption of blood-testing technology, and a hematology detection system with a high price to performance ratio and easy operation is urgently needed in primary healthcare centers. This paper introduces the principle and operation procedures of a point-of-care testing (POCT) card-based leukocyte analyzer (evaluated system), which was used to detect WBC indexes such as neutrophils, lymphocytes, and intermediate group cells (including eosinophils, basophils, and monocytes) in whole blood. The results from the evaluated system were compared to those from two commercial automatic hematology analyzers (reference system). The correlation and consistency between the evaluated system and the commercial reference systems were analyzed. The results showed that WBC count and number of granulocytes detected by the evaluated and reference systems showed a strong positive correlation (rs = 0.972 and 0.973, respectively), while the number of lymphocytes showed a relatively low correlation (rs = 0.851). A Bland-Altman plot showed that the major difference between the values detected by the evaluated system and the reference systems is within 95% limits of agreement (LoA), indicating that the two systems are in good agreement. In conclusion, the evaluated system has an excellent correlation, robust consistency, and a reliable comparison with the results of the widely used automatic hematology analyzers. It is ideal for WBC detection in primary medical institutions where a full-automatic five-category hematology analyzer is unavailable, especially during the COVID-19 normalized prevention and control period.

A protocol to measure peripheral blood leukocytes using a POCT card-based leukocyte analyzer is presented here. Same blood samples were tested by two automated hematology analyzers to evaluate the consistency and accuracy of the results. The results showed that the evaluated analyzer had a good correlation with the reference system.

White blood cell (WBC) is an important indicator of inflammation in the body, and it can help distinguish between bacterial and viral infections. At ...

Point-Of-Care Ultrasound Screening for Proximal Lower Extremity Deep Venous Thrombosis

Acute lower extremity deep venous thrombosis (DVT) is a serious vascular disorder that requires accurate and early diagnosis to prevent life-threatening sequelae. While whole leg compression ultrasound with color and spectral Doppler is commonly performed in radiology and vascular labs, point-of-care ultrasound (POCUS) is becoming more common in the acute care setting. Providers appropriately trained in focused POCUS can perform a rapid bedside examination with high sensitivity and specificity in critically ill patients. This paper describes a simplified yet validated approach to POCUS by describing a three-zone protocol for lower extremity DVT POCUS image acquisition. The protocol explains the steps in obtaining vascular images at six compression points in the lower extremity. Beginning at the level of the proximal thigh and moving distally to the popliteal space, the protocol guides the user through each of the compression points in a stepwise manner: from the common femoral vein to the femoral and deep femoral vein bifurcation, and, finally, to the popliteal vein. Further, a visual aid is provided that may assist providers during real-time image acquisition. The goal in presenting this protocol is to help make proximal lower extremity DVT exams more accessible and efficient for POCUS users at the patient&#39;s bedside.

Traditionally, lower extremity deep venous thrombosis (DVT) is diagnosed by radiology-performed venous duplex ultrasound. Providers appropriately trained in focused point-of-care ultrasound (POCUS) can perform a rapid bedside examination with high sensitivity and specificity in critically ill patients. We describe the scanning technique for focused POCUS DVT lower extremity examination.

Acute lower extremity deep venous thrombosis (DVT) is a serious vascular disorder that requires accurate and early diagnosis to prevent life-threatening ...