The authors would like to acknowledge Dr A. Meneses, whose previous work contributed to the refinement of the protocol described herein. Additionally, the authors would like to thank all of the research participants who have donated their time to enable protocols such as this to be developed in order to further clinical and scientific understanding.

All methods described here have been approved by the human research ethics committee of the University of the Sunshine Coast. Furthermore, all participants gave their written informed consent to participate in the measurements outlined in this protocol. Please note, vascular occlusion testing in the lower limb is contra-indicated in individuals who have previously had a revascularization procedure involving a vascular graft or stenting of the femoral or popliteal arteries. After preparing the equipment, the participant is instructed to rest in a supine position for 10 min. At this point, NIRS data collection commences, with an initial 2 min period, allowing for stability of NIRS signals to be achieved. Baseline data are then collected for 1 min, at which point a cuff located at the thigh is promptly inflated to achieve arterial occlusion. Occlusion is maintained for 5 min before the cuff is rapidly deflated. Data collection continues throughout the reactive hyperemia period until signals have recovered to baseline. Figure 1 depicts an overview of the reactive hyperemia protocol, and the detailed steps are provided below. The equipment used for the study are listed in the Table of Materials.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66511/66511fig01.jpg" /> 
Figure 1: Schematic outlining NIRS reactive hyperemia measurement protocol and timings. NIRS: near-infrared spectroscopy. <a href="https://www.jove.com/files/ftp_upload/66511/66511fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Equipment preparation
NOTE: Various NIRS, cuff inflation/occlusion, and data collection systems can be used to obtain the representative results outlined below. It is important that investigators consult their own specific user manuals and are aware of unique software, calibration, ambient light, and participant/cohort-specific considerations.
<ol>
	<li>Ensure that all measurements are performed in a quiet, temperature-controlled room (21-23 &#176;C).</li>
	<li>Confirm that all equipment and materials (see Table of Materials) are available.</li>
	<li>Ensure that the CW-NIRS device, and the computer that the device is transmitting to, are fully charged and powered on.</li>
	<li>Check that the NIRS device is paired with the computer displaying the NIRS data via Bluetooth and that measurement parameters of the NIRS unit are defined according to the experimental site and study design (an example: define the differential pathlength factor (DPF) of the near-infrared light). 
	NOTE: Most of the NIRS protocol steps and system settings are based on a combination of manufacturer&#39;s recommendations, researchers&#39; experience, and expert/consensus opinion. Be aware that the values generated during measurement depend on the particular device and probes used as well as the settings chosen in the device software. There is also potential for a high degree of inter-individual variability in the signals collected. Furthermore, to allow for improved comparison between studies, it is important to report NIRS instrumentation particulars (e.g., include probe design, source-detector separation distances and wavelengths used), system settings, probe position/orientation in relation to muscle geometry and the analysis parameters/data treatment when publishing results from these measurements.</li>
	<li>Enter the participant&#39;s data according to NIRS device software particulars.</li>
	<li>Connect the rapid cuff inflator to the air and power sources.</li>
</ol>
2. Participant preparation
<ol>
	<li>Ensure that the participant has read the participant information statement and consent form and has given their consent to participate in the measurement prior to commencement.</li>
	<li>Explain to the participant what to expect during the measurement while they remove their shoes and socks and stand stationary for relevant anthropometric measurements.</li>
	<li>Take a skinfold measurement (in triplicate) over the planned NIRS measurement site (e.g., on the medial aspect at the point of maximal calf circumference). 
	NOTE: This allows for the depth of skin and adipose tissue (routinely referred to as adipose tissue thickness - ATT) to be confirmed in relation to NIRS signal penetration depth. Alternatively, ultrasound can be used to determine adipose tissue thickness. Always keep in mind that the depth of the NIRS signal/measurement is approximately half of the receiver-transmitter distance9.</li>
	<li>To maximize signal quality, check the planned site of the NIRS transmitter/receiver probe for any hair that may absorb light and remove hair by shaving if necessary. 
	NOTE: If the skin is compromised, for example, from cellulitis, or if edema is present, consider the appropriateness of measurement, as these issues relate to potentially reduced signal quality, signal penetration depth/tissue from which measurement is taken, as well as participant wound sterility.</li>
	<li>Take measurements of and/or mark the position of planned NIRS probe placement in relation to relevant anatomical landmarks (e.g., superior surface of the medial condyle) to allow for accurate placement of the NIRS probe between and within participants (dependent on study design and probe design/shape) as oxygenation responses can display large heterogeneity between different muscles or even within regions of the same muscle.</li>
	<li>Ask the participant to lie in a supine position on an examination plinth or bed. The participant then rests for 10 min.</li>
	<li>Place a cuff around the thigh, proximal to the knee, ensuring that the tubes do not come into contact with the calf or the NIRS device (Figure 2).</li>
	<li>Elevate the leg (~10 cm) with the foot and ankle on a foam support, leaving the lower leg stable and accessible for measurements (Figure 3). 
	NOTE: If the test is required to be conducted in both legs over multiple occasions, the test order is randomized (before initial testing), and the order of testing is maintained for subsequent assessments for each participant.</li>
	<li>Turn on the rapid cuff inflator module. 
	NOTE: Ensure that the mode and pressure settings of the rapid cuff inflator are consistent with the manufacturer&#39;s instructions and vascular occlusion protocol specifications.</li>
	<li>Turn on the cuff inflator air source, check that air can travel through the hose, and connect the hose to the thigh cuff.</li>
	<li>Ensure that the hose of the cuff inflator is still not in contact with the calf.</li>
	<li>Fix the NIRS transmitter/receiver probe securely on the skin overlying the measurement site(s) (routinely the medial aspect of the gastrocnemius; however, other sites such as the dorsum of the foot and tibialis anterior are also utilized depending on the study and participant specifics). 
	NOTE: Some NIRS systems allow for the possibility of multiple site measurements simultaneously.</li>
	<li>Cover the probe with black kinesiology tape or something similar, being careful to seal the edges to prevent ambient light from affecting the NIRS signal quality/values (Figure 3). 
	NOTE: When fixing the probe and the adhesive covering, be sure to eliminate probe movement, but avoid compressing skin/adipose tissue/muscle.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66511/66511fig02.jpg" /> 
Figure 2: Example of the occlusive cuff placement at the thigh. (A) From above. (B) From the side. <a href="https://www.jove.com/files/ftp_upload/66511/66511fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66511/66511fig03v3.jpg" /> 
Figure 3: Example of near-infrared spectroscopy probe position. (A) Probe attached to shaved skin at medial gastrocnemius. (B) Probe placement while ankle in foam support to allow access and ensure stability. (C) Ambient light shielding&#160;in place. <a href="https://www.jove.com/files/ftp_upload/66511/66511fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Baseline data collection
<ol>
	<li>Ask the participant to remain relaxed, refrain from talking, and keep their leg(s) still for the duration of the data collection.</li>
	<li>Start the NIRS device recording via the computer interface. 
	NOTE: If offline data collection is enabled and preferred, start the NIRS device directly.</li>
	<li>Allow for at least 2 min of data to be acquired prior to initiating the start of the measurement. This ensures steady-state signals are achieved prior to data acquisition.</li>
	<li>Monitor the computer screen to ensure data signal integrity and physiologically plausible values. For example, Data Acquisition (DAQ) values provide information regarding signal quality and the amount of ambient light detected by the NIRS device. They should be kept at an acceptable range throughout the measurement.</li>
	<li>If, after 2 min, no fluctuations in the NIRS signal are noted (e.g., due to movement artifact), set the NIRS data baseline by pushing the corresponding button on the computer/device/syncing unit. This baseline reflects the comparative starting point of the measurement. The changes in oxygenated and deoxygenated hemoglobin will be interpreted relative to this baseline. 
	NOTE: During data collection, insert software-based event markers of specific milestones, namely start and end of baseline, start and end of cuff occlusion etc., to assist in data analysis.</li>
	<li>Collect at least 1 min of baseline data, again ensuring that no fluctuations or movement artifacts occur during this time. 
	NOTE: Fluctuations in baseline data will affect the interpretation of some potential variables, such as hyperemic reserve (see representative results section).</li>
</ol>
4. Vascular occlusion
<ol>
	<li>Set cuff pressure to 200 mmHg on the rapid cuff inflator before switching to cuff mode (or relevant setting on alternate systems). 
	NOTE: While some authors recommend inflation to 250 mmHg at the thigh9,19, in our experience, this is not well tolerated by some participants, leading to discontinuation of the measurement. Inflation to 200 mmHg is sufficient, in most participant groups, to occlude arterial inflow at rest while being tolerable and not resulting in bruising of the skin. To ensure effective occlusion is attained at 200 mmHg, our group routinely uses strain gauge plethysmography simultaneously with NIRS to confirm the absence of downstream blood flow during the period of cuff inflation.</li>
	<li>Advise the participant on what to expect (discomfort, tingling sensations, etc.) during the process of inflating the cuff to 200 mmHg for a period of 5 min, followed by a total cuff deflation. Remind the participant again to remain relaxed, refrain from talking, and keep their leg(s) immobile for the duration of the data collection. 
	NOTE: It is our experience that the first 30-60 s of the occlusion period is the least comfortable for participants. When participants let their legs relax, they tend to find the occlusion more tolerable.</li>
	<li>When ready to initiate cuff inflation, mark the end of the baseline period in NIRS software.</li>
	<li>Inflate the thigh cuff to a suprasystolic pressure of 200 mmHg, or alternatively to 220 mmHg in rare cases where 200 mmHg has proven ineffective.</li>
	<li>Monitor the computer or screen to ensure data integrity during the occlusion period.</li>
	<li>As the end of the 5 min of cuff occlusion approaches, prepare the participant by reminding them to keep their leg as still as possible and refrain from talking for approximately 3 min after cuff deflation (whilst the data from the vascular responses to reactive hyperemia are being collected). 
	NOTE: This reminder to the participant to remain still is important, as participants may be tempted to move the limb to relieve/ease discomfort as circulation is restored.</li>
</ol>
5. Reactive hyperemia
<ol>
	<li>After 5 min of occlusion has passed, rapidly deflate the thigh cuff completely (to 0 mmHg). Simultaneously, mark the end of the occlusive period in the NIRS software. The reactive hyperemia response, due to the resumption of blood flow and related factors, will be visible in the NIRS software display (Figure 4).</li>
	<li>After a minimum of 3 min post-occlusion, or alternatively, after the NIRS data has returned to baseline, mark the end of the recovery period in NIRS software and stop the measurement. 
	NOTE: The duration of recovery depends, in part, on the research or clinical question being investigated and, therefore, the specific NIRS parameters chosen for analysis, as well as the potential for concomitant measures (such as FMD) that may require a longer recovery duration.</li>
	<li>Initiate data saving and exportation of NIRS results for data treatment and analysis.</li>
</ol>
6. Follow up procedures
<ol>
	<li>Remove the NIRS device(s) and cuff from the participant.</li>
	<li>If required, clean the NIRS device (and occlusion cuff) following manufacturer&#39;s instructions and relevant hygiene standards.</li>
	<li>Inspect the NIRS device to ensure transmitter/receiver integrity and battery performance for future measurements.</li>
</ol>

Optimizing NIRS for Assessing Lower Limb Vascular Function

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The authors have no disclosures or competing interests.

This article outlines standardized procedures for the assessment of lower limb reactive hyperemia using CW-NIRS TSI to evaluate microvascular function. This protocol has been refined by examination of cuff occlusion duration on response magnitude, NIRS test-retest reliability during reactive hyperemia, as well as the level of agreement between NIRS and other methods of microvascular evaluation such as contrast-enhanced ultrasound23,24. A longer cuff-occlusion dur...

Cardiovascular disease (CVD) is the leading contributor to global mortality1. While myocardial infarction and stroke are the most common manifestations of CVD, vascular diseases of the lower limbs, such as peripheral arterial disease (PAD) and diabetic foot disease, contribute substantially to the personal, social, and healthcare burden of CVD2,3,4. Importantly, these disease states are characterized by microvascular and macrovascular dysfunction5 that contribute to symptoms (e.g., intermittent claudication), functional impairment, poor mobility as well as social isolation and reduced quality of life6. Historically, upper-limb vascular assessment techniques have been used as a measure of systemic vascular function and associated cardiovascular risk; however, these methods are potentially not sensitive to local impairments in lower limb vascular function7,8. While there is currently a range of techniques used to assess vascular function in the lower limb, such as flow-mediated dilatation (FMD) and contrast-enhanced ultrasound, each method has disadvantages and limitations, such as equipment cost, operator skill, or the need for invasive venous access. For these reasons, there is a need for standardized and effective techniques to evaluate lower limb vascular (dys)function that can be more readily implemented in research and clinical settings.
Continuous wave near-infrared spectroscopy (CW-NIRS) is a non-invasive, low-cost, and portable method that quantifies the relative changes in hemoglobin oxygenation in vivo. As the NIRS oxygenated and deoxygenated hemoglobin signals are derived from the small (&#60;1 mm in diameter) vessels, local skeletal muscle metabolism and microvascular function are able to be evaluated9. Specifically, the tissue saturation index (TSI) [TSI = oxygenated hemoglobin/ (oxygenated hemoglobin + deoxygenated hemoglobin) x 100], provides a quantitative measure of tissue oxygenation9. When measured before, during, and after occlusion and reactive hyperemia, the changes in TSI indicate &#39;end-organ&#39; vascular responsiveness, relative to the pre-occlusion baseline. Importantly, this method is sensitive to alterations in muscle microvascular responsiveness and perfusion associated with ageing10, disease progression11, and clinical interventions (e.g., revascularization surgery12,13 or exercise rehabilitation14,15,16,17) in individuals with, or at risk of microvascular dysfunction.
The availability of NIRS systems has led to a rapid rise in the number of research studies reporting microvascular function18. However, differences in reactive hyperemia&#160;testing protocols, omission of detailed, repeatable NIRS methods, as well as a lack of uniformity in the description, presentation, and analysis of NIRS response parameters make comparisons across individual trials challenging. This limits the collation of data for meta-analysis and the formulation of clinical assessment recommendations9,15.
Therefore, in this article, we describe our laboratory&#39;s standardized NIRS and vascular occlusion testing protocols for the assessment of lower limb reactive hyperemia. By disseminating these methods, we aim to contribute to the improved standardization and repeatability of data collection procedures and harmonized reporting.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cuff Inflator Air Source</td>
			<td>Hokanson&#160;</td>
			<td>AG101 AIR SOURCE</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastic Cohesive Bandage</td>
			<td>MaxoWrap</td>
			<td>18228-BL</td>
			<td>For blocking out ambient light</td>
		</tr>
		<tr>
			<td>OxySoft</td>
			<td>Artinis</td>
			<td>3.3.341 x64</td>
			<td></td>
		</tr>
		<tr>
			<td>PortaLite (NIRS)</td>
			<td>Artinis</td>
			<td>0302-00019-00</td>
			<td></td>
		</tr>
		<tr>
			<td>PortaSync MKII (Remote)</td>
			<td>Artinis</td>
			<td>0702-00860-00</td>
			<td>For Marking milestones during measurement</td>
		</tr>
		<tr>
			<td>Rapid Cuff Inflator</td>
			<td>Hokanson&#160;</td>
			<td>E20 RAPID CUFF INFLATOR</td>
			<td></td>
		</tr>
		<tr>
			<td>Thigh Cuff</td>
			<td>Hokanson&#160;</td>
			<td>CC17</td>
			<td></td>
		</tr>
		<tr>
			<td>Transpore Surgical Tape</td>
			<td>3M</td>
			<td>1527-1</td>
			<td>For fixing probe to skin</td>
		</tr>
	</tbody>
</table>

near-infrared spectroscopy during reactive hyperemia for the assessment of lower limb vascular function 

Vascular diseases of the lower limb contribute substantially to the&#160;global burden of cardiovascular disease and comorbidities such as diabetes. Importantly, microvascular dysfunction can occur prior to, or alongside, macrovascular pathology, and both potentially contribute to patient symptoms and disease burden. Here, we describe a non-invasive approach using near-infrared spectroscopy (NIRS) during reactive hyperemia, which provides a standardized assessment of lower limb vascular (dys)function and a potential method to evaluate the efficacy of therapeutic interventions. Unlike alternative methods, such as contrast-enhanced ultrasound, this approach does not require venous access or sophisticated image analysis, and it is inexpensive and less operator-dependent. This description of the NIRS method includes representative results and standard terminology alongside the discussion of measurement considerations, limitations, and alternative methods. Future application of this work will improve standardization of vascular research design, data collection procedures, and harmonized reporting, thereby enhancing translational research outcomes in the areas of lower limb vascular (dys)function, disease, and treatment.

Author Spotlight: Enhancing Vascular Function and Physical Capacity in Cardiovascular Disease Through Novel Interventions and NIRS Technology

Near-Infrared Spectroscopy During Reactive Hyperemia for the Assessment of Lower Limb Vascular Function 

Our research is focused on understanding how impairments in vascular function and blood flow contribute to the decline in physical function with age and age-related cardiovascular conditions. We also aim to develop novel therapies and exercise interventions that target vascular function and enhance physical capacity in quality of life in people living with cardiovascular disease. Differences in testing protocols, omission of detailed, repeatable NIRS methods, and a lack of uniformity in the description, presentation, and analysis of NIRS response parameters makes comparisons across individual trials challenging.This limits the collation of data for meta-analysis and the formulation of clinical assessment recommendations. Compared to plethysmography, NRS has high temporal resolution and directly evaluates changes in microvasculature. Compared to ultrasound, NIRS does not require sophisticated image analysis or intravenous contrast agents.The relative ease and affordability of NIRS means that users can be trained rapidly, enabling multicenter investigations with minimal inter operator variability. We're focused on improving skeletal muscle microvascular function through interventions like ischemic preconditioning and blood flow restriction exercise training. We're currently testing the effect of neuromuscular electrical stimulation as a therapy to promote vascular function and to enhance walking capacity in people with peripheral artery disease.

Near-infrared spectroscopy 
Continuous wave near-infrared spectroscopy devices measure relative changes in oxygenated (O2Hb) and deoxygenated (HHb) hemoglobin, which reflect local O2 delivery and utilization via light-emitting sources and photodetectors, set&#160;specific distances apart. Wavelengths of light between ~700 nm and 850 nm are emitted, corresponding with the peak absorbency of O2Hb and HHb. Once near-infrared light has penetrated skeletal muscle...

Read this Scientific Article Protocol about Author Spotlight: Enhancing Vascular Function and Physical Capacity in Cardiovascular Disease Through Novel Interventions and NIRS Technology at JoVE.com

Here, we describe a non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia in the lower limb. This protocol provides a standardized assessment of vascular and microvascular responsiveness that may be used to determine the presence of vascular dysfunction as well as the efficacy of therapeutic interventions.

Vascular diseases of the lower limb contribute substantially to the&#160;global burden of cardiovascular disease and comorbidities such as diabetes. ...

near-infrared-spectroscopy-during-reactive-hyperemia-for-assessment

Research

JoVE Journal

Biology

NIRS-Based Reactive Hyperemia Assessment in Lower Limb Vascular Studies

To begin, ask the participant to lie in a supine position on an examination plinth or bed. Place a cuff around the thigh, proximal to the knee, ensuring that the tubes do not come into contact with the calf. Elevate the leg with the foot and ankle on foam support, leaving the lower leg stable and accessible For measurements.Turn on the rapid cuff inflator module. Activate the cuff inflator air source. Verify air passage through the hose and connect the hose to the thigh cuff.Fix the near infrared spectroscopy or NIRS transmitter receiver probe on the skin overlying the measurement sites. Cover the probe with black kinesiology tape to block ambient light, preserving NIRS signal integrity. Instruct the participant to maintain a relaxed posture, avoid talking, and keep their legs still throughout the data collection period.Using the computer interface, start the NIRS device and acquire the data for a minimum of two minutes before starting the measurement. Monitor the computer screen to ensure data signal integrity and physiologically plausible values. If no fluctuations in the NIRS signal are noted, push the corresponding button on the software to set the NIRS data baseline.Collect at least one minute of baseline data ensuring no signal fluctuations or movement artifacts in recording. Set the cuff pressure to 200 millimeters of mercury on the rapid cuff inflator. before selecting the cuff mode.Inform the participant about potential sensations during the cuff's inflation to 200 millimeters of mercury for five minutes and subsequent total cuff deflation. When ready to initiate cuff inflation, mark the end of the baseline period in the recording. Inflate the thigh cuff to a supra systolic pressure of 200 millimeters of mercury.Monitor the computer or screen to ensure data integrity during the occlusion period. Just before the end of the five minute occlusion, remind the participant to keep their legs still, and refrain from talking for approximately three minutes after cuff deflation. After five minutes, rapidly deflate the thigh cuff to zero millimeters of mercury, and simultaneously mark the end of the occlusive period in the recording.After a three minute post-occlusion or after the NIRS data has returned to baseline, mark the end of the recovery period and stop the measurement. Finally, export the NIRS results and save the data treatment and analysis. NIRS distinguishes microvascular responsiveness between healthy individuals and those with peripheral arterial disease during reactive hyperemia testing.

654 Views.  University of the Sunshine Coast.  Here, we describe a non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia in the lower limb. This protocol provides a standardized assessment of vascular and microvascular responsiveness that may be used to determine the presence of vascular dysfunction as well as the efficacy of therapeutic interventions.

Optimizing NIRS for Assessing Lower Limb Vascular Function

Summary