Funding was provided by the Natural Sciences and Engineering Research Council of Canada and The Heart and Stroke Foundation of Canada.

This protocol follows the guidelines of the human research ethics board at the University of Alberta and conforms to the standards set by the latest revision of the Declaration of Helsinki.
1. Graded Exercise Test (VO2peak)
<ol>
	<li>Obtain written, informed consent from the subject. Have the subject read and answer the questions listed on the Physical Activity Readiness Questionnaire+ (PAR-Q+) to determine their readiness for exercise24.</li>
	<li>Adjust the seat height of the cycle ergometer in accordance to subject preference. Place four electrocardiogram (ECG) electrodes on the back of the patient according to standard 3-lead ECG placement, with modified limb leads to measure the heart rate (HR)25.</li>
	<li>Insert the mouthpiece into the subject&#39;s mouth to measure the exhaled gas and ventilation throughout the test using a metabolic measurement system25. 
	NOTE: The metabolic system will measure real-time oxygen consumption (VO2), carbon dioxide production (VCO2), ventilation (VE), heart rate (HR), and end tidal CO2 (PETCO2).</li>
	<li>Following 2 min of collection of baseline data, instruct the subject to start cycling with an initial workload of 50 watt, to maintain a consistent cadence of&#160; &#8805;60 RPM. Increase the workload in 25 W steps every 2 min, until the subject reaches volitional exhaustion or requests to stop the test25.</li>
</ol>
2. Multiple Fraction of Inspired Oxygen (FIO2 ) Diffusing Capacity (DLCO ) Method7
<ol>
	<li>Calculate the workloads corresponding to 30%, 50%, 70%, and 90% of the VO2peak using the peak VO2 obtained in the graded exercise test. At least 48 h after the graded exercise test, have the subject return to the laboratory for DLCO maneuvers.</li>
	<li>Do not exceed 12 DLco tests per day, as carboxyhemoglobin (COHb) build-up can occur with repeated testing5. Therefore, perform testing on multiple days based on the number of exercise workloads to be conducted and the quality of the DLCO data.</li>
	<li>Prepare pre-breathing gases by attaching a tank of 100% O2 gas and a tank of medical-grade air (21% O2 and 79% N2) to an air blender system. Fill two 60 L non-diffusing Douglas bags, one containing 40% O2, and one containing 60% O2, using the air blender system.</li>
	<li>Set up two large-bore, three-way stopcock valves that will allow for the modulation of inhaled gas mixtures. These will be referred to as the &#34;pre-breath valves.&#34;</li>
	<li>Connect the Douglas bags to the valve system using flexible, non-compressible tubing. Connect the valve system to a two-way, T-shaped non-rebreathing valve connected to the test gas intake assembly of the mass flow sensor of the metabolic measurement system.</li>
	<li>For resting measurements, have the subject seated upright, with both feet flat on the floor. For exercise trials, ensure that the subject is in a steady state by monitoring HR using the ECG (HR &#177; 3 bpm for steady state). 
	NOTE: Steady state may not be reached at 90% of the VO2peak; thus, begin the measurement once the subject has reached the HR equivalent to 90% of the VO2peak on the graded exercise test.</li>
	<li>Collect a single drop of capillary blood via a finger prick and analyze it for hemoglobin concentration. Then, adjust all subsequent DLCO for [Hb] using the following equation26: 
	<img alt="Equation 1" src="/files/ftp_upload/54949/54949equation1.jpg" /></li>
	<li>Select an FIO2 (21%, 40%, or 60%) at random by switching the pre-breathe valves to the desired orientation. Choose the corresponding FIO2-DLCO gas by turning the DLCO gas valve selector (see Figure 1C).</li>
	<li>Instruct the subject to affix the nose clips and to breathe normally into the mouthpiece for five breaths from the Douglas bag corresponding to the respective FIO2.</li>
	<li>Instruct the subject to expire to residual volume. When the lung volume plateaus at residual volume, have the subject inhale the DLCO gas mixture to total lung capacity and hold their breath for 6 s before exhaling to residual volume.</li>
	<li>Monitor the methane tracing during the exhalation to ensure that the slope is horizontal, as this indicates that the CO test gas is well equilibrated in the lung. 
	NOTE: Alveolar volume (VA) and breath hold time are calculated automatically and reported by the metabolic measurement system.</li>
	<li>Ensure that the VA for each DLco maneuver is within 5% of previous trials. Similarly, breath hold time should be 6.0 &#177; 0.3 s. If not, repeat the maneuver.</li>
	<li>Wait 4 min to allow residual carbon monoxide to wash out, and then repeat steps 2.8 - 2.11 for each remaining FIO2 at rest.</li>
	<li>At least 48 h later, repeat steps 2.9 - 2.15 during steady state at each exercise intensity (30%, 50%, 70%, and 90% of the VO2peak) for each FIO2. Reduce the workload between the breath holds at 90% of the VO2peak workload to recover the subject.</li>
	<li>Wait 2 min between DLco tests during exercise to clear alveolar CO during exercise. Do not exceed 12 DLco tests per day to avoid carboxyhemoglobin (COHb) build-up5.</li>
</ol>
3. Calculating Pulmonary Capillary Blood Volume and Membrane Diffusing Capacity
<ol>
	<li>Calculate the alveolar partial pressure of O2 (PAO2) using the following equation 
	<img alt="Equation 2" src="/files/ftp_upload/54949/54949equation2.jpg" /> 
	NOTE: FIO2 is the fraction of inspired O2, PBAR is the atmospheric pressure, PH2O is the water vapor pressure, PaCO2 is the pressure of arterial CO2, and RER is the respiratory exchange ratio.</li>
	<li>Estimate the RER and PaCO2 using the measured 30-s average PETCO2 and RER for the respective exercise intensity from the data obtained in the previous graded exercise test.</li>
	<li>Calculate &#952;CO using the following equation7. <img alt="Equation 3" src="/files/ftp_upload/54949/54949equation3.jpg" /></li>
	<li>Graph the relationship between 1/DLcoadj and 1/&#952;CO for each FIO2 and calculate the regression equation. 
	NOTE: The minimum acceptable r2 value is 0.95, and DLCO maneuvers should be repeated when r2 values are outside of this range21.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54949/54949fig2.jpg" /> 
Figure 2: Representative Graph of 1/DLCO versus 1/&#952;CO at Peak Exercise. The relationship between 1/DLCO and 1/&#952;CO is plotted for three breath holds at the various FIO2 (21%, 40%, and 60%). The calculation of Vc and Dm are derived from the regression equation for the relationship above. The inverse of the slope (1/0.00796) of the line gives the value for Vc (125.5 mL), and the inverse of the y-intercept (1/0.00869) gives the value for Dm (115.0 mL&#183;min-1&#183;mmHg-1). <a href="http://cloudfront.jove.com/files/ftp_upload/54949/54949fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Calculate Vc by taking the inverse of the slope of the regression equation between 1/DLCO and 1/&#952;CO. Calculate Dm by taking the inverse of the y-intercept of the equation.</li>
</ol>
4. Intrapulmonary Arteriovenous Anastomosis Recruitment
<ol>
	<li>On a separate day from the DLCO data collection, insert a 20-gauge intravenous (IV) catheter into an antecubital vein and attach it to a three-way stopcock via a 6-in IV extension tube for the injection of agitated saline for contrast echocardiography11,17.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/54949/54949fig3.jpg" /> 
Figure 3: Agitated Saline Contrast Setup. An IV catheter is placed in the antecubital space and is connected to a three-way stopcock via a 6-in extension. Two 10 mL syringes are attached to the stopcock to create the contrast solution, which contains 10 mL of saline and 0.5 mL of room air. <a href="http://cloudfront.jove.com/files/ftp_upload/54949/54949fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Connect two 10 mL syringes to the three-way stopcock. Combine 10 mL of 0.9% sterile saline with 0.5 mL of air, and forcefully agitate it through the three-way stopcock, back and forth between the two syringes, to form fine, suspended bubbles until the sonographer is ready for contrast.</li>
	<li>Have an experienced sonographer or cardiologist obtain a standard apical four-chamber view of the heart. At rest, have the echocardiographer evaluate the intra-atrial septum and ventricular septum for an intra-cardiac shunt with standard echocardiographic and color Doppler imaging.
	<ol>
		<li>If no intra-cardiac shunt is detected, instruct the subject to perform a Valsalva maneuver during the contrast injection to evaluate for a patent foramen ovale (PFO)11,17. Repeat the measurement during non-Valsalva.</li>
	</ol>
	</li>
	<li>Inject the contrast while the sonographer maintains the four-chamber view. Record 15 cardiac cycles following the detection of contrast in the right ventricle.</li>
	<li>Repeat the contrast-enhanced imaging during steady-state exercise at 30%, 50%, and 70% of the VO2peak. As steady state cannot be reached at 90% of the VO2peak, begin the imaging once the target HR, identified by the HR at 90% of the VO2peak during the graded exercise test, is reached. 
	NOTE: The time between exercise intensities depends on the clearance of contrast from both ventricles, &#8805; 2 min.</li>
	<li>Have an echocardiographer who is blinded to experimental conditions interpret the agitated saline contrast echocardiograms according to a previously-described scoring system17,27. 
	NOTE: Scoring is based on the maximum number of contrast bubbles visible within the left ventricle (LV) in a single echocardiographic frame, as follows: no contrast bubbles in the LV = 0, &#8804;3 bubbles = 1, 4 - 12 bubbles = 2, &#62; 12 bubbles = 3. 
	NOTE: The appearance of contrast in the left ventricle after five cardiac cycles suggests an IPAVA. An intracardiac shunt is graded by the appearance of contrast in less than five cardiac cycles27.</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/54949/54949fig4.jpg" /> 
Figure 4: Representative Images for IPAVA Scoring. The scale is 5 cm (solid white line). (A) Pre contrast injection. (B) IPAVA score = 0. (C) IPAVA score = 1. (D) IPAVA score = 3. <a href="http://cloudfront.jove.com/files/ftp_upload/54949/54949fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors declare that they have no competing financial interests.

This method enables the evaluation of the pulmonary diffusing capacity and intrapulmonary arteriovenous anastomosis recruitment during exercise.
Critical steps within the protocol
Although the DLCO breath hold is relatively simple at rest, breath holding during exercise presents a unique challenge to the subject, as it is counter-intuitive, and subjects have a high drive to breathe during exercise. Thus, a good-quality determination of Vc and Dm relies on the rapport and clear communication between the tester and the subject. The tester&#39;s technical ability can be quantified with the variability of the alveolar volume (&#177; 5% of previous trials) and a breath-hold time (BHT) of 6.0 &#177; 0.3 s.
Modifications and troubleshooting
At the conclusion of a Vc/Dm measurement, the tester should quickly graph the three DLCO maneuvers to determine the best-fit line of the data points; the DLCO measured with 21% FIO2 should always be greater than that with 40%, which should be greater than that with 60%. If not, it is recommended to check if the valve switch corresponds to the correct testing gas. Similarly, check that the pre-breathing bags are filled with the correct FIO2 gas corresponding to the testing gas (Figure 1B-1D). Caution should be taken when testing a participant who is a smoker, as elevated COHb levels may underestimate DLco.
For the IPAVA recruitment assessment, the position of the subject is critical to ensure high-quality image acquisition. It is possible to replace the upright cycle ergometer with a recumbent cycle ergometer to minimize the movement of the subject. However, recumbent cycle exercise will elicit a different metabolic response for a given work rate, and thus the graded exercise test should be repeated on the recumbent cycle ergometer. Scanning of the upper chest may be uncomfortable to some women; in this case, a female sonographer is recommended. Finally, the recommended exercise protocol is designed for a young, healthy individual; accordingly, the exercise protocol can be modified for a different target population.
Limitations of the technique
The principal limitations of the multiple FIO2 DLCO technique are the skill of the tester and the ability of the subject to follow commands and to remain calm during the breath hold, as Valsalva or M&#252;llerian maneuvers will affect the measurements. Secondly, the number of breath holds in one session should be limited to 12, due to an increase in CO backpressure, which may affect the Vc and Dm measurement5,30 and pose a health risk to the subject. Depending on the research design, it may be necessary to complete the testing across multiple sessions to allow for the clearance of CO and to limit participant fatigue. With good participant coaching and good technical ability, we have determined a satisfactory coefficient of variation between trials for DLco, Vc, and Dm to be 7%, 8%, and 15%, respectively.
The multiple FIO2 DLCO technique assumes that the alveolar O2 is the same as the capillary O2, and thus, caution should be exercised when interpreting the data in individuals with known gas exchange impairment.
Agitated saline contrast echocardiographic imaging is limited by the technical ability of the sonographer and the ability of the subject to minimize thoracic movement while exercising. It is also critical that the interpreter of the images be familiar with the scale for scoring IPAVA recruitment according to established procedures (Figure 4)27. The significance of a positive saline contrast echocardiography during exercise remains a topic of debate15,16, and there is some discussion that a positive agitated saline contrast in the left ventricle may be secondary to capillary distention, and not IPAVA recruitment. Ongoing work is attempting to resolve this issue.
Significance of the technique with respect to existing/alternative methods
By utilizing these physiological techniques, it is possible to assess the pulmonary vasculature during exercise in a variety of conditions, including in health, in disease, and in drug interventions. Although the quality relies with the ability of the tester, these skills are easily and quickly acquired with proper mentorship and training. The multiple FIO2 DLCO method is considered the &#34;gold standard&#34; in the measurement of Dm and Vc31. While these measures are not calculated clinically, the values could be used to determine the mechanisms for hypoxemia and exercise intolerance, to predict patient outcomes, and to further characterize diagnosis31,32. Likewise, the agitated saline echocardiography technique is the most widely-used method in determining the recruitment of IPAVAs.
Future applications or directions after mastering this technique
These techniques are applicable for use in a range of experimental conditions and interventions. We demonstrate these techniques during exercise, but they can easily be modified to measure pulmonary vascular responses during a drug infusion, such as dobutamine or dopamine, inotropes known to increase cardiac output17. Furthermore, it is possible to use these techniques in clinical populations, such as in those with heart failure34 or chronic obstructive pulmonary disease (COPD), in which the DLCO is lower compared to age-matched control subjects35.

During exercise, cardiac output can increase up to six-fold above resting values1. Given that the lungs are the only organ to receive 100% of the cardiac output, exercise presents a considerable stress to the pulmonary system. With incremental exercise, pulmonary diffusing capacity (DLCO) must increase to meet the increased oxygen demand2. From rest to peak exercise, DLCO can increase to up to 150% of resting values without reaching an upper limit with respect to cardiac output3,4,5. The increase in diffusing capacity occurs as a result of increases in membrane diffusing capacity (Dm) and capillary blood volume (Vc), secondary to the recruitment and distension of pulmonary capillaries6.
Roughton and Forster (1957) developed a technique to partition Dm and Vc7 by modulating the fraction of inspired oxygen (FIO2) during a standard diffusion capacity for carbon monoxide test (DLCO). Oxygen and carbon monoxide (CO) competitively bind to heme sites on hemoglobin, such that increasing FIO2 will decrease the DLCO8,9. By modulating the FIO2 during a standard DLCO maneuver, this relationship can be exploited to measure Vc and Dm7. We have recently adapted this technique to be used during exercise5. Similar to previous work, we have found that DLCO continuously increases up to peak exercise secondarily to increases in both Vc and Dm5. Interestingly, we have found that in endurance-trained athletes who have a greater oxygen consumption and thus a greater need for diffusing capacity, there is an increase in the DLCO at peak exercise, secondary to an increased Dm, and not Vc, suggesting a potential adaptation in the pulmonary membrane of the athlete5.
The increases in Vc and Dm during exercise are accomplished by an increase in pulmonary artery pressure, which results in the recruitment and distension of pulmonary capillaries previously hypo-perfused at rest4,10. This results in an increase in the cross-sectional area of the pulmonary capillary network, thereby decreasing pulmonary vascular resistance and attenuating the increase in pulmonary artery pressure.
Studies using agitated saline contrast echocardiography have shown evidence of intrapulmonary arteriovenous anastomoses (IPAVA) recruitment during exercise11,12,13,14. The significance of IPAVA recruitment is not yet clear, and while some studies suggest that they may contribute to gas exchange impairment12,14 and may serve to unload the right ventricle11,12, the topic remains controversial15,16. Further, while the exact mechanism of IPAVA recruitment is not known, we have found that increasing cardiac output, as well as exogenous dopamine, causes IPAVA recruitment at rest17. An acutely-increasing pulmonary artery pressure18 or dopamine blockade does not appear to significantly affect IPAVA recruitment during exercise11. There is speculation that these larger-diameter IPAVA vessels may help to protect the pulmonary capillaries from the large increases in pulmonary artery pressure by reducing pulmonary vascular resistance12,17,19,20,21.
When combined with the evaluation of Vc and Dm, agitated saline contrast echocardiography is a valuable tool to examine the adaptation of the pulmonary circulation to the stress of exercise22,23.

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			<td style="width:136px;">SensorMedics Inc.</td>
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			<td height="42" style="height:42px;width:259px;">Cycle Ergometer</td>
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			<td height="21" style="height:21px;width:259px;">60L Douglas Bags</td>
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			<td></td>
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			<td height="21" style="height:21px;width:259px;">Two-way T Valve</td>
			<td style="width:136px;">Hans Rudolph</td>
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			<td height="21" style="height:21px;width:259px;">Hemoglobin Measurement System</td>
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			<td height="21" style="height:21px;width:259px;">22-gauge Intravenous Catheter</td>
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			<td height="21" style="height:21px;width:259px;">Ultrasound&#160;</td>
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			<td style="width:119px;">ECHOpac</td>
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			<td height="50" style="height:50px;width:259px;">Compressed gas 21% O2, 0.3% CO, 0.3% CH4, balance nitrogen</td>
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			<td height="42" style="height:42px;width:259px;">Compressed gas 40% O2, 0.3% CO, 0.3% CH4, balance nitrogen</td>
			<td style="width:136px;">Praxair</td>
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			<td height="42" style="height:42px;width:259px;">Compressed gas 60% O2, 0.3% CO, 0.3% CH4, balance nitrogen</td>
			<td style="width:136px;">Praxair</td>
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			<td height="21" style="height:21px;width:259px;">Nose-clip</td>
			<td style="width:136px;">Vacu-Med</td>
			<td style="width:119px;">snuffer #1008</td>
			<td></td>
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assessment of pulmonary capillary blood volume, membrane diffusing capacity, and intrapulmonary arteriovenous anastomoses during exercise

Exercise is a stress to the pulmonary vasculature. With incremental exercise, the pulmonary diffusing capacity (DLCO) must increase to meet the increased oxygen demand; otherwise, a diffusion limitation may occur. The increase in DLCO with exercise is due to increased capillary blood volume (Vc) and membrane diffusing capacity (Dm). Vc and Dm increase secondary to the recruitment and distension of pulmonary capillaries, increasing the surface area for gas exchange and decreasing pulmonary vascular resistance, thereby attenuating the increase in pulmonary arterial pressure. At the same time, the recruitment of intrapulmonary arteriovenous anastomoses (IPAVA) during exercise may contribute to gas exchange impairment and/or prevent large increases in pulmonary artery pressure.
We describe two techniques to evaluate pulmonary diffusion and circulation at rest and during exercise. The first technique uses multiple-fraction of inspired oxygen (FIO2) DLCO breath holds to determine Vc and Dm at rest and during exercise. Additionally, echocardiography with intravenous agitated saline contrast is used to assess IPAVAs recruitment.
Representative data showed that the DLCO, Vc, and Dm increased with exercise intensity. Echocardiographic data showed no IPAVA recruitment at rest, while contrast bubbles were seen in the left ventricle with exercise, suggesting exercise-induced IPAVA recruitment.
The evaluation of pulmonary capillary blood volume, membrane diffusing capacity, and IPAVA recruitment using echocardiographic methods is useful to characterize the ability of the lung vasculature to adapt to the stress of exercise in health as well as in diseased groups, such as those with pulmonary arterial hypertension and chronic obstructive pulmonary disease.

The effect of increasing exercise intensity on oxygen consumption, diffusing capacity, pulmonary capillary blood volume, membrane diffusing capacity, and IPAVA score is shown in Table 1. VO2, DLCO, Vc, and Dm increase in response to increasing power output.
Figure 2 shows a representative calculation of Vc and Dm using the multiple FIO2-DLCO technique during exercise. DLCO decreases with increasing FIO2, and this relationship is exploited to partition Vc and Dm. Calculating the inverse of the slope of 1/DLCO versus 1/&#952;CO results in the Vc, and the inverse of the y-intercept yields the value for the Dm. As expected, both the Vc and Dm increase during exercise compared to resting values.
The results show that these techniques can be used to assess the pulmonary vasculature response during exercise. The multiple-FIO2 DLCO and agitated saline contrast echocardiography method provides investigators with more insight into the contributions of pulmonary capillary and membrane recruitment to the overall diffusion capacity and could supplement traditional pulmonary function testing in the clinical setting. Failure to increase Vc or Dm during exercise would lead to a diffusion limitation and hypoxemia. For example, a low DLCO secondary to a low Vc would indicate changes to the pulmonary capillaries; similarly, a decreased Dm would indicate changes to the pulmonary membrane.
Figure 4 shows representative tracings of four-chamber contrast echocardiographs. With increasing exercise intensity, the IPAVA score increases from 0 (i.e., no evidence of IPAVAs) at rest to 3 at the highest exercise intensity (Table 1). Previous work has shown that exercise increases the IPAVA score11,12,14, but there is no consensus as to how these IPAVAs are recruited. There is evidence that IPAVAs can be recruited pharmacologically at rest with dopamine17,28, as well as by increasing cardiac output with dobutamine17,28 and epinephrine28. Inotropes such as dopamine and epinephrine are of particular interest, as they increase endogenously during exercise29. Furthermore, there is some evidence that IPAVA recruitment may be important to exercise hemodynamics, in that the absence of IPAVAs appears to result in greater pulmonary artery pressure, decreased cardiac output, and decreased peak power output12. Thus, this technique may be used in studies examining individuals with pulmonary artery hypertension.
<img alt="Figure 1" src="/files/ftp_upload/54949/54949fig1.jpg" /> 
Figure 1: Multiple FIO2 DLCO Setup. (A) Setup overview. (B) Compressed-gas cylinders containing 21%, 40%, and 60% O2 with 0.3% CO, 0.3% methane, and balance nitrogen, as well as a supplemental oxygen compressed-gas cylinder. (C) Three-way valve selector for the three FIO2 DLCO tanks. (D) Valve switch for three-way valves in the series for the selection of FIO2 for pre-breathing. <a href="http://cloudflare.jove.com/files/ftp_upload/54949/54949fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/54949/54949table1.jpg" /> 
Table 1: Representative Data for One Subject at Rest and During Exercise at 30, 50, 70, and 90% of the VO2peak. VO2, volume of oxygen consumption relative to body mass; DLCO, diffusing capacity for carbon monoxide; Vc, pulmonary capillary blood volume; Dm, membrane diffusing capacity; IPAVA score, scoring of contrast appearance in the left ventricle after five cardiac cycles. Data modified from Tedjasaputra et al. 2016.

Watch this Scientific Journal Video about Assessment of Pulmonary Capillary Blood Volume, Membrane Diffusing Capacity, and Intrapulmonary Arteriovenous Anastomoses During Exercise at JoVE.com

Assessment of Pulmonary Capillary Blood Volume, Membrane Diffusing Capacity, and Intrapulmonary Arteriovenous Anastomoses During Exercise

To assess the pulmonary diffusion and vasculature responses to exercise, we describe the multiple-inspired oxygen diffusion capacity technique to determine capillary blood volume and membrane diffusing capacity, as well as agitated saline contrast echocardiography to assess the recruitment of intrapulmonary arteriovenous anastomoses.

Exercise is a stress to the pulmonary vasculature. With incremental exercise, the pulmonary diffusing capacity (DLCO) must increase to meet the increased ...

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13.0K Views.  University of Alberta. To assess the pulmonary diffusion and vasculature responses to exercise, we describe the multiple-inspired oxygen diffusion capacity technique to determine capillary blood volume and membrane diffusing capacity, as well as agitated saline contrast echocardiography to assess the recruitment of intrapulmonary arteriovenous anastomoses.

Video: Assessment of Pulmonary Capillary Blood Volume, Membrane Diffusing Capacity, and Intrapulmonary Arteriovenous Anastomoses During Exercise

Using Continuous Data Tracking Technology to Study Exercise Adherence in Pulmonary Rehabilitation

Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to exercise training recommendations. The study of exercise adherence is thus a key step towards the optimization of PR programs. To date, mostly indirect measures, such as rates of participation, completion, and attendance, have been used to determine adherence to PR. The purpose of the present protocol is to describe how continuous data tracking technology can be used to measure adherence to a prescribed aerobic training intensity on a second-by-second basis.
In our investigations, adherence has been defined as the percent time spent within a specified target heart rate range. As such, using a combination of hardware and software, heart rate is measured, tracked, and recorded during cycling second-by-second for each participant, for each exercise session. Using statistical software, the data is subsequently extracted and analyzed. The same protocol can be applied to determine adherence to other measures of exercise intensity, such as time spent at a specified wattage, level, or speed on the cycle ergometer. Furthermore, the hardware and software is also available to measure adherence to other modes of training, such as the treadmill, elliptical, stepper, and arm ergometer. The present protocol, therefore, has a vast applicability to directly measure adherence to aerobic exercise.

Pulmonary rehabilitation is widely recognized in the management of respiratory diseases. A key component to successful pulmonary rehabilitation is adherence to the recommended exercise training. The purpose of the present protocol is to describe how continuous data tracking technology can be used to precisely measure adherence to a prescribed aerobic training intensity.

Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to ...

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic conditions. As part of a diagnostic assessment, the early detection of postural deficits is important so that appropriate and targeted interventions can be prescribed. The Sensory Organization Test (SOT) on the EquiTest determines an individual&#39;s use of the sensory systems (somatosensory, visual, and vestibular) that are responsible for postural control. Somatosensory and visual input are altered by the calibrated sway-referenced support surface and visual surround, which move in the anterior-posterior direction in response to the individual&#39;s postural sway. This creates a conflicting sensory experience. The Motor Control Test (MCT) challenges postural control by creating unexpected postural disturbances in the form of backwards and forwards translations. The translations are graded in magnitude and the time to recover from the perturbation is computed.
Intermittent claudication, the most common symptom of peripheral arterial disease, is characterized by a cramping pain in the lower limbs and caused by muscle ischemia secondary to reduced blood flow to working muscles during physical exertion. Claudicants often display poor balance, making them susceptible to falls and activity avoidance. The Ankle Brachial Pressure Index (ABPI) is a noninvasive method for indicating the presence of peripheral arterial disease and intermittent claudication, a common symptom in the lower extremities. ABPI is measured as the highest systolic pressure from either the dorsalis pedis or posterior tibial artery divided by the highest brachial artery systolic pressure from either arm. This paper will focus on the use of computerized dynamic posturography in the assessment of balance in claudicants.

Individuals with intermittent claudication exhibit poor balance compared to healthy controls. Computerized dynamic posturography is an objective method for measuring an individual's postural responses to balance disturbances. This provides an objective reflection of the person&#x2019;s ability to respond to situations under which sensory stimuli are altered and unexpected perturbations occur.

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic ...

A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in post-menopausal women has not been adequately studied. Accordingly, we investigated the impact of voluntary cage-wheel exercise and forced treadmill exercise on cardiac adaptation in menopausal mice. The most commonly used inducible model for mimicking menopause in women is the ovariectomized (OVX) rodent. However, the OVX model has a few dissimilarities from menopause in humans. In this study, we administered 4-vinylcyclohexene diepoxide (VCD) to female mice, which accelerates ovarian failure as an alternative menopause model to study the impact of exercise in menopausal mice. VCD selectively accelerates the loss of primary and primordial follicles resulting in an endocrine state that closely mimics the natural progression from pre- to peri- to post-menopause in humans. To determine the impact of exercise on exercise capacity and cardiac adaptation in VCD-treated female mice, two methods were used. First, we exposed a group of VCD-treated and untreated mice to a voluntary cage wheel. Second, we used forced treadmill exercise to determine exercise capacity in a separate group VCD-treated and untreated mice measured as a tolerance to exercise intensity and endurance.

Two exercise paradigms were tested on a newly developed chemically induced menopausal mouse model to examine the impact of menopause on exercise capacity and cardiac adaption to exercise.

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in ...

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

Ultrasound Assessment of Endothelial Function: A Technical Guideline of the Flow-mediated Dilation Test

Cardiovascular disease is the primary cause of mortality and a major cause of disability worldwide. The dysfunction of the vascular endothelium is a pathological condition characterized mainly by a disruption in the balance between vasodilator and vasoconstrictor substances and is proposed to play an important role in the development of atherosclerotic cardiovascular disease. Therefore, a precise evaluation of endothelial function in humans represents an important tool that could help better understand the etiology of multiple cardio-centric pathologies.
Over the past twenty-five years, many methodological approaches have been developed to provide an assessment of endothelial function in humans. Introduced in 1989, the FMD test incorporates a forearm occlusion and subsequent reactive hyperemia that promotes nitric oxide production and vasodilation of the brachial artery. The FMD test is now the most widely utilized, non-invasive, ultrasonic assessment of endothelial function in humans and has been associated with future cardiovascular events.
Although the FMD test could have clinical utility, it is a physiological assessment that has inherited several confounding factors that need to be considered. This article describes a standardized protocol for determining FMD including the recommended methodology to help minimize the physiological and technical issues and improve the precision and reproducibility of the assessment.

The flow mediated dilation (FMD) test is the most commonly utilized, non-invasive, ultrasound assessment of endothelial function in humans. Although the FMD test has been related with the prediction of future cardiovascular disease and events, it is a physiological assessment with many inherent confounding factors that need to be considered.

Cardiovascular disease is the primary cause of mortality and a major cause of disability worldwide. The dysfunction of the vascular endothelium is a ...

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120&#176;/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.

This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...

'Boden Food Plate': Novel Interactive Web-based Method for the Assessment of Dietary Intake

Different methods can be used in research to assess dietary intake, many of which are still paper-based. Written estimated food diaries are often utilized in clinical trials, despite being a burden for both study participants and researchers. This method requires participant literacy, it is time consuming, labor intensive, and can easily lead to under-reporting. With advancements in technology, there is a growing interest in electronic diaries that automate the dietary assessment process. These are focused on improving accuracy, reducing both time and cost and providing users with a visual and more enjoyable experience. The methodology presented here aimed to validate the &#39;Boden Food Plate&#39;, a novel web-based platform for self-recording of food and drink items, compared to a traditional estimated food diary. The application was also rated on a satisfaction scale by study participants using a paper-based questionnaire. Sixty-seven participants completed the dietary measures on both the three-day electronic and paper food diaries. For the analysis, only dietary data completed at both study time points (baseline and week six) was utilized. Despite small mean differences between dietary data collection methods, Bland Altman analysis showed fairly wide 95% limits of agreement between the electronic platform and the written estimated food diary and there were few cases which did not fall within the 95% confidence intervals. Overall, participants found the electronic food diary to be more fun than the paper method and as easy to use as hard copy diaries. The new platform has potential as a self-recording tool for the collection of dietary data, particularly when utilized in clinical trial settings. However, further validation studies are needed to improve the validity of this novel electronic dietary data collection tool.

The &#39;Boden Food Plate&#39; is an electronic food diary designed to be an interactive and fun method to collect dietary intake using visual depictions. The purpose of this study was to validate the web-based application against a traditional three-day estimated food diary method.

Different methods can be used in research to assess dietary intake, many of which are still paper-based. Written estimated food diaries are often utilized ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Ultrasonographic Assessment During Cardiopulmonary Resuscitation

The US-CAB (Ultrasound, Circulation/Airway/Breathing) protocol integrates several sonographic techniques into a structured assessment of the circulation, airway, and breathing status of a patient during cardiopulmonary resuscitation (CPR) in an advanced life support-compliant manner. US-C provides a subxiphoid view of the heart, to look for potentially reversible causes of disease, such as pericardial effusion, pulmonary embolism, hypovolemia, and acute coronary thrombosis. Sonographic cardiac activity during CPR not only helps differentiate pseudo-pulseless electrical activity (PEA) from true PEA but also represents a higher chance of the return of spontaneous circulation (ROSC) and survival. Evaluation of the inferior vena cava (IVC) shows the fluid status of the patient and indicates the best methods to use for fluid resuscitation. If aortic dissection is suspected, a subxiphoid view of the aorta is suggested for identifying an intimal flap. Once intubation is done, tracheal ultrasound (US-A) at the suprasternal notch helps differentiate endotracheal intubation (one air-mucosal interface with one comet-tail) from esophageal intubation (double tract sign). Immediately following US-A, bilateral lung US (US-B) should be done to confirm proper bilateral ventilation using the lung sliding sign. In addition, US-C can be serially followed to see the dynamic changes in the cardiac chambers and IVC, or any cardiac contraction suggestive of ROSC. US-B can also detect coexisting lung or pleural pathologies without interfering with the performance of CPR. The main concern when implementing this method is maintaining high-quality CPR without delays in chest compressions when performing US-CAB. Rigorous training and continued practice are key to minimize any interruptions during resuscitation.

Here, we present a US-CAB (Ultrasound, Circulation/Airway/Breathing) protocol for use during cardiopulmonary resuscitation (CPR). US-C evaluates the subxiphoid view of the heart and inferior vena cava. After intubation, tracheal US (US-A) and lung US (US-B) help confirm endotracheal intubation and proper ventilation.

The US-CAB (Ultrasound, Circulation/Airway/Breathing) protocol integrates several sonographic techniques into a structured assessment of the circulation, ...

Assessment of Pulmonary Capillary Blood Volume, Membrane Diffusing Capacity, and Intrapulmonary Arteriovenous Anastomoses During Exercise

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